Immunosuppressive polypeptides and nucleic acids

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry, particularly to recombinant fused protein dimers intended to inhibit or suppress immune response in a mammal, which bind human CD80 or human CD86 or the extracellular domain of any thereof, and has higher capacity for suppressing immune response than a dimer of the fused protein LEA29Y-Ig. Also disclosed are nucleic acids which code said dimers, expression vectors containing said nucleic acids, as well as recombinant host cells containing said nucleic acids and/or said vectors. Disclosed are pharmaceutical compositions for inhibiting or suppressing immune response in a mammal, which contain said fused protein dimers, as well as use of said dimers to produce drugs for inhibiting or suppressing immune response in a mammal, treating diseases or disorders of the immune system or treating organ or tissue transplant rejection in a mammal. Methods of producing said fused protein dimers are also disclosed.

EFFECT: invention provides effective inhibition or suppression of immune response in a mammal.

9 cl, 15 dwg, 11 tbl, 12 ex

 

The technical field

This invention in General relates to polypeptides that bind CD80 and/or CD86, nucleic acid encoding such polypeptides, and methods of producing and using such polypeptides and nucleic acids.

The level of technology

T cells play a major role in the initiation and regulation of immune responses. For that to happen full activation of T cells required at least two separate signal events. The first signal is generated by the interaction of T-cell receptors (TCR)expressed on T-cells specific antigens (Ag), presented in the context of the molecules of the major histocompatibility complex (MHC)expressed on antigen-presenting cells (APC). The second (co-stimulatory signal is the result of interaction between co-stimulatory ligands expressed on APC, and their respective receptors expressed on T-cells. Dominant co-stimulatory path includes the interaction between CD80 (B7-1 or B7,1) and CD86 (B7-2 or B7,2) ligands expressed on APC with CD28 and CTLA-4 (also known as CD 152), expressed mainly on T cells. CTLA-4 (cytotoxic antigen 4T-lymphocyte) and CD28 serve as a receptor for CD80 and CD86 ligands.

The positive signal transmission is mediated through CD28 receptor. The CBE is ivanie CD80 and/or CD86 ligand(s) with CD28 lowers the threshold for T-cell activation by stimulating the formation of immunological synapses (Viola A. et al., Science 283:680-682 (1999)). Additionally, the CD28 costimulation activates or enhances the production factors targeted in T-cell proliferation and survival, such as interleukin-2 (IL-2), NF-κ (transcription factor Kappa b) and BcI-XL (Norton S. D. et at., J. Immunol. 149:1556-1561 (1992); A. T. Vella et al., J. Immunol. 158: 4714-4720 (1997)). In vivo CD28-deficient mice have severely depressed immunity and demonstrate weak antigen-specific T-cell responses (Green, J. M. et al., Immunity 1:501-508 (1994)). T-cell anergy or tolerance may occur when T cells are activated in the absence of co-stimulatory signal.

The negative signal transmission mediated by receptor CTLA-4. Each of CD80 and CD86 ligands binds with high avidity to CTLA-4 and balances immunoproliferative answers coming from CD28 signal. The potential mechanism of signal transmission CTLA-4 include the competitive binding of co-stimulatory molecules CD80/CD86 (Masteller, E. M. et al., J. Immunol. 164:5319 (2000)), inhibition of TCR signal transmission by engaging the phosphatase to immunogens (Lee K. M. et al., Science 282:2263 (1998)) and impaired immunological synapse (Pentcheva-Hoang T. et al., Immunity 21:401 (2004); Chikuma, S. et al., J. Exp. Med 197:129 (2003); H. Schneider et al., Science 313: 1972 (2006)). In vivo CTLA-4 deficient mice demonstrate a deep autoimmune phenotypes, characterized by extensive tissue infiltration and destruction of the bodies (Waterhouse P. et al., Science 270:985 (1995)).

Those who piticescu agents, designed to counteract the CD80/CD86 co-stimulatory transmission signal, such as a soluble human CTLA-4-Ig, perform obligations under the treatment of autoimmune diseases and disorders. This invention provides preferential molecules with improved ability to modulate or suppress signaling through CD80/CD86 co-stimulatory path and methods of using such molecules to selected and differential control T-cell responses. Such molecules are useful to use in various applications, discussed in detail below.

Brief description of the invention

In the first aspect, the invention provides isolated or recombinant polypeptide, CTLA-4, comprising the amino acid sequence that differs from the amino acid sequence of the extracellular domain of human CTLA-4, is presented in SEQ ID NO (identification number sequence): from 159 to 15 amino acid residues, where the isolated or recombinant polypeptide, CTLA-4 has the ability to bind CD80 or CD86 or an extracellular domain of both and/or has the ability to suppress or inhibit an immune response.

The polypeptide according to the first aspect of the present invention may have at least 90% sequence identity, or at the least is her least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:36. The polypeptide according to the first aspect of the present invention may include amino acid sequence of SEQ ID NO:36.

The polypeptide according to the first aspect of the present invention may have at least 90% sequence identity or at least 95%, at least 96%, at least 97%, at least 98%or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:50. The polypeptide according to the first aspect of the present invention may include amino acid sequence of SEQ ID NO:50.

The polypeptide according to the first aspect of the present invention may have the ability to bind human CD80 or human CD86 or their extracellular domain.

The polypeptide according to the first aspect of the present invention may include amino acid sequence with a length of 124 amino acid residue.

The polypeptide according to the first aspect of the present invention may include one amino acid substitution in amino acid position selected from the group comprising the amino acid position corresponding to position 50, 54, 55, 56, 64, 65, 70 or 85 relative to SEQ ID NO:159.

The polypeptide according to the first and is the aspect of the present invention may include two, three or four amino acid substitutions at amino acid positions selected from the group comprising the amino acid position corresponding to position 50, 54, 55, 56, 64, 65, 70 or 85 relative to SEQ ID NO:159.

The polypeptide according to the first aspect of the present invention may include the amino acid substitution at position 70 relative to SEQ ID NO:159, such as replacement S70F. The polypeptide according to the first aspect of the present invention may include amino acid replacement at position 104 relative to SEQ ID NO:159, such as replacement L104E.

The polypeptide according to the first aspect of the present invention may include amino acid replacement in position 30 relative to SEQ ID NO:159, such as replacement T30N/D/A or replacement T30N.

The polypeptide according to the first aspect of the present invention may include amino acid replacement at position 64 relative to SEQ ID NO:159, such as replacement S64P.

The polypeptide according to the first aspect of the present invention may include amino acid replacement at position 50 relative to SEQ ID NO:159, such as replacement AM.

The polypeptide according to the first aspect of the present invention may include amino acid replacement at position 54 relative to SEQ ID NO:159, such as replacement M54K/V or replacement M54K.

The polypeptide according to the first aspect of the present invention may include amino acid replacement at position 65 relative to SEQ ID NO:159, such as Zam is on I65S.

The polypeptide according to the first aspect of the present invention may include amino acid replacement in position 56 relative to SEQ ID NO:159, such as replacement N56D.

The polypeptide according to the first aspect of the present invention may include the amino acid substitution at position 55 relative to SEQ ID NO:159, such as replacement G55E.

The polypeptide according to the first aspect of the present invention may include amino acid substitution at positions 85 relative to SEQ ID NO:159, such as replacement MA.

The polypeptide according to the first aspect of the present invention may include amino acid replacement in position 24 relative to SEQ ID NO:159, such as replacement A24E/S or replacement AE.

The polypeptide according to the first aspect of the present invention may have an affinity binding to CD86 or an extracellular domain, which is almost equal to or greater than the binding affinity of Monomeric extracellular domain of human CTLA-4 extracellular domain or CD86 CD86.

The polypeptide according to the first aspect of the present invention may have an affinity binding to CD80 or its extracellular domain, which is greater than the binding affinity of Monomeric extracellular domain of human CTLA-4 extracellular domain of CD80 CD80 or.

The polypeptide according to the first aspect of the present invention may have the ability to suppress the immune response.

Polypep is D. according to the first aspect of the present invention may have the ability to inhibit T-cell activation or T-cell proliferation.

In the second aspect, the invention provides isolated or recombinant polypeptide multimer comprising at least two polypeptide according to the first aspect of the present invention.

In the third aspect, the invention provides isolated or recombinant protein, comprising (a) a polypeptide according to the first aspect of the present invention and (b) a second polypeptide, where the second polypeptide is an Ig Fc polypeptide, and where the protein has an ability to bind CD80 and/or CD86 or an extracellular domain of each or both and/or the ability to modulate or regulate the immune response.

In the fourth aspect, the invention provides isolated or recombinant dimeric protein comprising two Monomeric fused protein according to the third aspect of the present invention.

In the fifth aspect, the invention provides isolated or recombinant nucleic acid comprising a nucleotide sequence that encodes a polypeptide according to the first aspect of the present invention, multimer according to the second aspect of the present invention, a fused protein according to the third aspect of the present invention or dimeric protein according to the fourth aspect of the present invention.

In the sixth aspect, the invention provides a vector, in which with a nucleic acid according to the fifth aspect of the present invention.

In the seventh aspect, the invention provides isolated or recombinant cell host comprising the polypeptide according to the first aspect of the present invention, multimer according to the second aspect of the present invention, a fused protein according to the third aspect of the present invention, dimeric protein according to the fourth aspect of the present invention, the nucleic acid according to the fifth aspect of the present invention and/or a vector according to the sixth aspect of the present invention.

In the eighth aspect, the invention provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient, a pharmaceutically acceptable carrier or pharmaceutically acceptable solvent and one or more of the following: a polypeptide according to the first aspect of the present invention, multimer according to the second aspect of the present invention, a fused protein according to the third aspect of the present invention, dimeric protein according to the fourth aspect of the present invention, the nucleic acid according to the fifth aspect of the present invention, the vector according to the sixth aspect of the present invention and/or a host cell according to the seventh aspect of the present invention.

In the ninth aspect, the invention provides a method for suppressing the immune response, where the specified method, vkljuchajuwih the contact B7-positive cells with an effective amount at least one of: a polypeptide according to the first aspect of the present invention, multimer according to the second aspect of the present invention, a fused protein according to the third aspect of the present invention, dimeric protein according to the fourth aspect of the present invention, the nucleic acid according to the fifth aspect of the present invention, the vector according to the sixth aspect of the present invention and/or a host cell according to the seventh aspect of the present invention, suppression of the immune response, where the immune response, therefore, is suppressed.

In the tenth aspect, the invention provides a polypeptide according to the first aspect of the present invention, multimer according to the second aspect of the present invention, a fused protein according to the third aspect of the present invention, dimeric protein according to the fourth aspect of the present invention, the nucleic acid according to the fifth aspect of the present invention, the vector according to the sixth aspect of the present invention and/or the cell host according to the seventh aspect of the present invention for use in suppressing an immune response.

In the eleventh aspect, the invention provides use of a polypeptide according to the first aspect of the present invention, multimer according to the second aspect of the present invention, fused protein according to the third aspects is to this invention, dimer fused protein according to the fourth aspect of the present invention, the nucleic acid according to the fifth aspect of the present invention, the vector according to the sixth aspect of the present invention and/or a host cell according to the seventh aspect of the present invention in the manufacture of a medication to suppress the immune response.

In the twelfth aspect, the invention provides a conjugate comprising a polypeptide according to the first aspect of the present invention, multimer according to the second aspect of the present invention, a fused protein according to the third aspect of the present invention or dimeric protein according to the fourth aspect of the present invention, and polipeptidnoi part, covalently attached to such polypeptide, multimenu, fused protein or fused dimeric protein, where the specified conjugate has the ability to suppress the immune response.

Other aspects of the invention are described below.

In another aspect, the invention provides isolated or recombinant polypeptide comprising amino acid sequence having at least, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with at least one amino acid sequence selected from the group comprising SEQ ID NOS identification number sequences): 1-73, where the polypeptide binds CD80 CD86 or an extracellular domain (EVA) each, and/or has the ability to suppress or inhibit an immune response.

In another aspect, the invention provides isolated or recombinant polypeptide comprising amino acid sequence having at least, 95%, 96%, 97%, 98%, 99%), or 100% sequence identity with at least one amino acid sequence selected from the group comprising SEQ ID NOS:1-73, where the polypeptide has a binding affinity of human CD86 extracellular domain or the extracellular domain of human CD80, which is almost equal to or greater than the binding affinity of human CTLA-4 extracellular domain of human CD86 or an extracellular domain human CD80, respectively, and where the polypeptide does not necessarily have the ability to suppress the immune response. Some such polypeptides have an affinity binding to human CD86 extracellular domain, which is greater than the binding affinity of the extracellular domain of human CTLA-4 extracellular domain of human CD86. Some such polypeptides have an affinity binding to the extracellular domain of human CD80, which is greater than the binding affinity of the extracellular domain of human CTLA-4 extracellular domain of human CD80.

In another aspect, the invention provides isolated or recombinantly mutant polypeptide, CTLA-4, comprising the amino acid sequence which (a) differs from the amino acid sequence of the extracellular domain of human CTLA-4, is presented in SEQ ID NO:159 in no more than 10, 9, 8, 7, or 6 amino acid residues (for example, not more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues), and (b) includes at least one amino acid substitution in amino acid position corresponding to position 50, 54, 55, 56, 64, 65, 70 or 85 relative to SEQ ID NO:159, where the mutant polypeptide, CTLA-4 has the ability to bind CD80 or CD86 or an extracellular domain of each and/or has the ability to suppress or inhibit an immune response.

In another aspect, the invention provides isolated or recombinant polypeptide that includes an amino acid sequence comprising (i)at least, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence selected from the group comprising SEQ ID NOS:1-73 and (ii) the phenylalanine residue at amino acid position corresponding to position 70 of the specified amino acid sequence selected from the group comprising SEQ ID nos:1-73, where the polypeptide has an ability to bind CD80 and/or CD86 or an extracellular domain of each or both, and/or has the ability to suppress or inhibit an immune response.

In another aspect the invention of especial isolated or recombinant mutant polypeptide, CTLA-4, which binds CD80 and/or CD86 and/or the extracellular domain of each or both, and/or are able to suppress the immune response, where the specified polypeptide includes an amino acid sequence which (a) differs from the amino acid sequence of the polypeptide extracellular domain of human CTLA-4, is presented in SEQ ID NO:159 in no more than 10, 9, 8, 7, or 6 amino acid residues (for example, not more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues), and ((b) includes at least one amino acid substitution, where indicated, at least the amino acid substitution includes S70F, where the position of the amino acid residues are numbered according to SEQ ID NO:159.

In another aspect, the invention provides isolated or recombinant mutant polypeptide, CTLA-4, comprising the amino acid sequence which (a) differs from the amino acid sequence of the extracellular domain of human CTLA-4, is presented in SEQ ID NO:159 in no more than 11, 10, 9, 8, 7, or 6 amino acid residues (for example, not more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 amino acid residues), and (b) includes at least one the amino acid substitution at position amino acid residue corresponding to position 24, 30, 32, 50, 54, 55, 56, 64, 65, 70 or 85 relative to SEQ ID NO:159, where the mutant polypeptide, CTLA-4 has the ability to bind CD 80 or CD 86 or NR the cell each domain and/or has the ability to suppress or inhibit an immune response.

In another aspect, the invention provides isolated or recombinant polypeptide comprising amino acid sequence which (a) differs from the amino acid sequence represented in SEQ ID NO:31 is not more than 10, 9, 8, 7, or 6 amino acid residues (for example, not more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues), and (b) includes at least one of the following: a methionine residue at the position corresponding to position 50 of SEQ ID NO:31, a lysine residue at the position corresponding to position 54 of SEQ ID NO:31, a glutamic acid residue in the position corresponding to position 55 of SEQ ID NO:31, a Proline residue at a position corresponding to position 64 of SEQ ID NO:31), the serine residue at the position corresponding to position 65 of SEQ ID NO:31), the phenylalanine residue at the position corresponding to position 70 of SEQ ID NO:31, where the position of the amino acid residues are numbered according to SEQ ID NO:31, and the polypeptide binds CD80 and/or CD86 and/or EVA each or both and/or inhibits an immune response.

In another aspect, the invention provides isolated or recombinant dimer fused protein comprising two Monomeric fused protein coupled via at least one disulfide bond formed between two cysteine residues present in each Monomeric mutant fused boe is ke, where each Monomeric protein comprises (a) a polypeptide comprising amino acid sequence having at least, 95%, 96%, 97%, 98%, 99% or 100% identity with at least one amino acid sequence selected from the group comprising SEQ ID NOS:1-73 and (b) a polypeptide Ig Fc, where dimer fused protein has an ability to bind CD80 and/or CD86, and/or CD80-Ig and/or CD86-Ig, and/or has the ability to inhibit or to suppress the immune response.

In another aspect, the invention provides isolated or recombinant dimer fused protein comprising two Monomeric fused protein, each such Monomeric protein comprising amino acid sequence having at least, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with at least one amino acid sequence selected from the group comprising SEQ ID NOS:74-79, 197-200, 205-214, and 219-222, where the polypeptide binds CD80 and/or CD86, and/or its extracellular domain and/or suppresses immune response, or a complementary polynucleotide sequence.

In another aspect, the invention provides isolated or recombinant dimer fused protein comprising two Monomeric fused protein, where each Monomeric protein comprises: (1) a polypeptide comprising amino acid sequence that distinguishes the I from amino acid sequence, selected from the group comprising SEQ ID NOS:1-73 by not more than 10, 9, 8, 7, or 6 amino acid residues (for example, not more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues), and where the amino acid residue in the amino acid sequence in position 41, 50, 54, 55, 56, 64, 65, 70 or 85 is identical to the amino acid residue in the corresponding position of the specified selected amino acid sequence, and (2) a polypeptide Ig Fc, where dimer fused protein binds CD80 and/or CD86, and/or inhibits an immune response.

In another aspect, the invention provides isolated or recombinant dimer fused protein comprising two Monomeric fused protein, where each Monomeric protein comprises: (1) a mutant polypeptide extracellular domain of CTLA-4, comprising the amino acid sequence that (i) differs from the amino acid sequence of the extracellular domain of human CTLA-4, is presented in SEQ ID NO:159 in no more than 10, 9, 8, 7, or 6 amino acid residues (for example, not more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues), and (ii) includes at least one amino acid substitution in amino acid position corresponding to position 50, 54, 55, 56, 64, 65, 70 or 85 relative to the amino acid sequence of SEQ ID NO:159; and (2) a polypeptide Ig Fc, where dimer fused protein binds CD80 and/or CD86, and/or inhibits or inhibin is no immune response. Some of these dimers fused protein include one or more substitutions at amino acid positions relative to SEQ ID NO:159 selected from the group comprising AL, M54K, G55E, N56D, S64P, I65S and S70F; and (2) a polypeptide Ig Fc, and Ig Fc polypeptide is not necessarily IgG2 Fc polypeptide, where the mutant dimer CTLA-4-Ig binds hCD80 and/or hCD86 and/or suppresses or inhibits an immune response.

In another aspect, the invention provides isolated or recombinant polypeptide comprising amino acid sequence having at least, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence selected from the group comprising SEQ ID NO:26, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:50 and SEQ ID NO:56, where the polypeptide (i) binds CD80 and/or CD86 or an extracellular domain of each or both, and/or (ii) suppresses the immune response.

In another aspect, the invention provides isolated or recombinant polypeptide comprising amino acid sequence having at least, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence selected from the group comprising SEQ ID NOS:74-79, 197-200, 205-214, and 219-222, where the polypeptide (i) binds CD80 and/or CD86 or an extracellular domain of each or both, (ii) protein binds CD80-Ig and/or CD86 protein-Ig, and/or (iii) suppresses the immune response.

who also provides isolated or recombinant dimer fused protein, comprising two Monomeric fused protein coupled via at least one disulfide bond formed between two cysteine residues present in each Monomeric mutant fused protein, where each Monomeric protein comprises (a) a polypeptide comprising amino acid sequence having at least 95% identity with at least one amino acid sequence selected from the group comprising SEQ ID NO:79, SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200 and (b) a polypeptide Ig Fc, where dimer fused protein has (i) an ability to bind CD80 and/or CD86 and/or an extracellular domain of CD80 and/or CD86, (ii) the ability to bind CD80-Ig and/or CD86-Ig, and/or (iii) has the ability to inhibit or suppress an immune response.

In another aspect, the invention provides isolated or recombinant nucleic acid comprising a polynucleotide sequence that encodes a polypeptide comprising amino acid sequence having at least, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with at least one amino acid sequence selected from the group comprising SEQ ID NOS:1-79, 197-200, 205-214, and 219-222, where the polypeptide binds CD80 and/or CD86 and/or the extracellular domain of each or both, and/or has the ability to suppress the immune the answer is, or whom elementarnuu polynucleotide sequence.

In another aspect, the invention provides isolated or recombinant nucleic acid comprising a polynucleotide sequence that encodes a protein comprising amino acid sequence having at least, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with at least one amino acid sequence selected from the group comprising SEQ ID NOS:74-79, 197-200, 205-214, and 219-222, where the polypeptide binds CD80 and/or CD86, and/or its extracellular domain and/or suppresses an immune response, or its complementary polynucleotide sequence.

In another aspect, the invention provides isolated or recombinant nucleic acid comprising: (a) a polynucleotide sequence having at least, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with at least one polynucleotide sequence selected from the group comprising SEQ ID NOS:80-158, 201-204, 223, and 224; (b) a complementary polynucleotide sequence (a) or (C) a fragment of any of polynucleotide sequence (a) or (b)where the nucleic acid encodes a polypeptide that binds CD80 and/or CD86, and/or the extracellular domain of each or both, and/or has the ability to suppress or inhibit an immune response.

In another aspect of this image is giving provides an isolated or recombinant nucleic acid, comprising a polynucleotide sequence that encodes a polypeptide that includes the amino acid sequence of (a), which differs from the amino acid sequence selected from the group comprising SEQ ID NOS:1-73 by not more than 10, 9, 8, 7, or 6 amino acid residues (for example, not more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues), and (b) where the amino acid residue in the amino acid sequence in position 41, 50, 54, 55, 56, 64, 65, 70 or 85 is identical to the amino acid residue in the corresponding position of the specified amino acid sequence selected from the group comprising SEQ ID NOS:1-73, where the polypeptide binds CD80 and/or CD86, and/or the extracellular domain of each or both, and/or inhibits an immune response, or a complementary polynucleotide sequence.

In another aspect, the invention provides isolated or recombinant nucleic acid comprising a polynucleotide sequence that encodes a polypeptide comprising the amino acid sequence which (a) differs from the amino acid sequence of the extracellular domain of human CTLA-4, is presented in SEQ ID NO:159 in no more than 10, 9, 8, 7, or 6 amino acid residues (for example, not more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues), and (b) includes at least one amine is acid substitution at amino acid position corresponding to the position 50, 54, 55, 56, 64, 65, 70 or 85 relative to SEQ ID NO:159, where the specified polypeptide has an ability to bind CD80 and/or CD86 and/or the extracellular domain of each, and/or has the ability to suppress or inhibit an immune response, or a complementary polynucleotide sequence.

In another aspect, the invention provides isolated or recombinant nucleic acid comprising a polynucleotide sequence that encodes a polypeptide comprising amino acid sequence having (i)at least, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence selected from the group comprising SEQ ID NOS:1-73 and (ii) the phenylalanine residue at amino acid position corresponding to position 70 of the specified amino acid sequence selected from the group comprising SEQ ID nos:1-73, where the polypeptide binds hCD80 and/or hCD86 or EVA and/or inhibits an immune response, where the specified amino acid substitution includes optional S70F, or a complementary polynucleotide sequence.

In another aspect, the invention provides isolated or recombinant nucleic acid comprising a polynucleotide sequence that encodes a recombinant dimer of a polypeptide, comprising two polypeptide, where each t is such a polypeptide includes the amino acid sequence, with at least, 95%, 96%, 97%, 98%, 99% or 100% identity to a sequence selected from the group comprising SEQ ID NOS:1-73, where the dimer binds hCD80 and/or hCD86 and/or inhibits an immune response, or a complementary polynucleotide sequence.

In another aspect, the invention provides isolated or recombinant nucleic acid comprising a polynucleotide sequence that encodes a protein comprising (a) a polypeptide comprising amino acid sequence that has at least, 95%, 96%, 97%, 98%, 99% or 100% identity with at least one amino acid sequence selected from the group comprising SEQ ID NOS:1-73, and (b) Ig polypeptide, where the protein binds CD80 and/or CD86, and/or has the ability to suppress immune response, or a complementary polynucleotide sequence.

In another aspect, the invention provides isolated or recombinant nucleic acid comprising a polynucleotide sequence that encodes a polypeptide comprising the amino acid sequence which (a) differs from the amino acid sequence represented in SEQ ID NO:31 is not more than 10, 9, 8, 7, or 6 amino acid residues (for example, not more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues), and (b) includes at least one of the following: a methionine residue at the position corresponding to position 50 of SEQ ID NO:31, a lysine residue at the position corresponding to position 54 of SEQ ID NO:31, a glutamic acid residue in the position corresponding to position 55 of SEQ ID NO:31, a Proline residue at a position corresponding to position 64 of SEQ ID NO:31), the serine residue at the position corresponding to position 65 of SEQ ID NO:31), the phenylalanine residue at the position corresponding to position 70 of SEQ ID NO:31, where the position of the amino acid residues are numbered according to SEQ ID NO:31, and where the polypeptide binds CD80 and/or CD86, and/or inhibits an immune response, or a complementary polynucleotide sequence.

In another aspect, the invention provides an expression vector comprising: (i) a first polynucleotide sequence that encodes a first polypeptide comprising amino acid sequence having at least, 95%, 96%, 97%, 98%, 99% or 100% identity with at least one amino acid sequence selected from the group comprising SEQ ID NOS:1-73, where the first polypeptide binds human CD86 and/or human CD80 and/or the extracellular domain of each or both, and/or suppresses an immune response and (ii) a second polynucleotide sequence that encodes a second polypeptide comprising a hinge, the polypeptide CH2 domain in domain immunoglobulin (Ig), and Ig polypeptide is not necessarily human IgG2 Fc polypeptide.

In another aspect, the invention provides isolated or recombinant cell host, transfectional with nucleic acid that encodes a protein, and nucleic acid comprises: (i) a first nucleotide sequence encoding a first polypeptide comprising amino acid sequence having at least, 95%, 96%, 97%, 98%, 99% or 100% identity with at least one amino acid sequence selected from the group comprising SEQ ID NOS:1-73, where the first polypeptide has the ability to bind human CD86 and/or human CD80 and/or the extracellular domain of each or both, and/or has the ability to suppress the immune response; and (ii) a second nucleotide sequence encoding a second polypeptide comprising a hinge, the polypeptide CH2 domain and CH3 domain of the immunoglobulin (Ig)and Ig polypeptide is not necessarily human IgG2 Fc polypeptide, where a host cell can Express the protein.

In another aspect, the invention provides a method of suppressing immune response, where the method includes contact B7-positive cells with an effective amount of at least one polypeptide, conjugate, nucleic acid, vector or to EDI according to the invention, to suppress the immune response, where the immune response, therefore, is suppressed.

In another aspect, the invention provides a method of modulating the interaction of T cells expressing CD28 and/or CTLA-4 with B7-positive cells, where the method includes contact B7-positive cells with an effective amount of at least one polypeptide, conjugate, nucleic acid, vector or cell according to this invention, to modulate the interaction of B7-positive cells with CD28-positive T-cells and/or CTLA-4-positive T-cells, where it modulates the interaction between B7-positive cells with CD28-positive T-cells and/or CTLA-4-positive T-cells.

In another aspect, the invention provides a method of inhibiting the interaction of CD28-positive T cells and/or CTLA-4-positive T cells with B7 - positive cells, where the method includes contact B7-positive cells with an effective amount of at least one polypeptide, conjugate, nucleic acid, vector or cell according to the invention, where inhibited the interaction of CD28-positive T cells and/or CTLA-4-positive T cells with B7-positive cells.

In another aspect, the invention provides a method of inhibiting the interaction SV-positive T cells with B7-positive cells in the subject, where the method includes the introduction to the subject an effective amount of, at least one polypeptide, conjugate, nucleic acid, vector or cell according to the invention, where inhibited the interaction of endogenous CD28-positive T cells with endogenous B7-positive cells in the subject.

In another aspect, the invention provides a method of treatment of a subject having a disease or disorder of the immune system, mediated by interaction of endogenous T cells with endogenous cells expressing CD80 and/or CD86, where the method includes the administration to a subject in need of such treatment, a therapeutically effective amount of at least one polypeptide, conjugate, nucleic acid, vector or cell according to the invention, where the interaction(I) between endogenous T cells and endogenous cells expressing the indicated CD80 and/or specified CD86, inhibited, thus making treatment immune disease or disorder of a subject.

In another aspect, the invention provides a method of inhibiting rejection of a tissue or organ transplanted from a donor to a subject, recipient, where the method includes introducing to a subject, recipient, who is in need, a therapeutically effective amount of at least one polypeptide, conjugate, nucleic acid, vector or cell according to the image is the shadow, thus, inhibiting rejection of a tissue or organ that is transplanted to a subject, recipient.

In another aspect, the invention provides a method of obtaining a fused protein, where the method includes: (1) culturing a host cell transformed with nucleic acid in the cell environment, where the nucleic acid comprises (i) a first nucleotide sequence that encodes a polypeptide having at least, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of any of SEQ ID NOS:1-73, where the polypeptide binds CD86 and/or CD80, and/or the extracellular domain of each CD86 or CD80, and (ii) a second nucleotide sequence encoding Ig polypeptide comprising a hinge, CH2 domain and CH3 domain, where is expressed nucleic acid and is fused protein; and (2) the restoration of the fused protein.

It also provides a method of obtaining a polypeptide, comprising introducing into a population of cells a nucleic acid according to the invention, where the nucleic acid is functionally linked to a regulatory sequence effective to obtain a polypeptide encoded by a nucleic acid, and culturing the cells in the cellular environment to obtain the polypeptide; and secretion of the polypeptide from the cells or the cell environment.

It also provides compositions that include Molek is in accordance with the invention (for example, mutant molecule CTLA-4) and the filler, carrier or solvent. Also provided pharmaceutical compositions comprising a molecule according to the invention and a pharmaceutically acceptable excipient, carrier or solvent.

Additional aspects of the invention are described below.

Brief description of figures

Figure 1 is a schematic illustration of the plasmid expression vector kDNK mutant CTLA-4-Ig, which includes the nucleotide sequence encoding a mutant protein, CTLA-4-Ig. In figure 1 each mutant protein, CTLA-4-Ig includes a mutant polypeptide, CTLA-4 EVA according to the invention, fused at its C-end N-end of the human IgG2 (hIgG2) Fc polypeptide.

Figures 2A-2D are diagrams of examples of fused proteins hCD80-Ig, hCD86-Ig, LEA29Y-Ig, and hCTLA-4-IgG2, respectively. Signal peptide, extracellular domain (EVA), the linker (if any) and Ig Fc domain of each fused protein is shown schematically. Also shown is the amino acid residues present in the joints between the signal peptide, EVA linker (if any) and Ig Fc. The signal peptide of each fused protein is normally cleaved during processing and, thus, the indirect (Mature) protein usually does not contain the sequence of the signal peptide. Figure 2D is a scheme which eticheskoe image fused protein of human CTLA-4-IgG2 ("hCTLA-4-IgG2"), comprising the extracellular domain of human CTLA-4 ("hCTLA-4 EVA"), covalently fused at its C-end N-end polypeptide of human IgG2. The estimated amino acid sequence that is fused protein hCTLA-4-IgG2 shown in SEQ ID NO:161 and includes the following segments: signal peptide hCTLA-4 (amino acid residues 1-37), hCTLA-4 EVA polypeptide (amino acid residues 38-161) and the polypeptide of the human IgG2 Fc (amino acid residues 162-389). None of the linker (for example, none of the amino acid(s)) is not included between the end of the polypeptide hCTLA-4 EVA and N-end of the human IgG2 Fc. The polypeptide of the human IgG2 Fc includes the hinge region, CH2 domain and snz domain of human IgG2. Figure 2D shows the amino acid residues at the joints between these different segments. Specifically shows the last four amino acid residue signal peptide, the first five and the last five amino acid residue of the polypeptide hCTLA-4 EVA and the first five and the last five amino acid residue of the polypeptide of the human IgG2 Fc.

The signal peptide is usually cleaved during processing and, thus, the secretory protein (Mature protein) hCTLA-4-IgG2 usually does not contain the sequence of the signal peptide. Amino acid sequence of Mature or Sekretareva form of this fused protein hCTLA-4-IgG2 shown in SEQ ID NO:162. On sledovatelnot polypeptide hCTLA - 4 EVA includes amino acid residues 1-124 SEQ ID NO:162, and the sequence of the polypeptide of the human IgG2 Fc includes amino acid residues 125-352 SEQ ID NO:162. In another aspect of this Mature protein hCTLA-4-Ig does not include the C-terminal lysine (K) residue and, thus, includes amino acid residues 1-351 SEQ ID NO:162.

The Mature protein hCTLA-4-IgG2, which has a total of 352 amino acids, includes amino acid residues 38-389 amino acid sequence of the full-size protein DT hCTLA-4, is presented in SEQ ID NO:160, and begins with the amino acid sequence: methionine-histidine-valine-alanine. If necessary amino acids of the Mature form are numbered starting with Met (methionine) sequence Met-His-Val-Ala, defining the Met as the first residue (for example, the KJV includes amino acid residues are numbered 1-124), as in SEQ ID NO:162. Mature dimer hCTLA-4IgG2 is a form of fused protein, which is usually used in the analyses of the examples described below, unless otherwise stated. DNA (deoxyribonucleic acid) sequence encoding a protein hCTLA-4-IgG2, which includes hCTLA-4 EVA, merged with hIgG2 Fc polypeptide shown in SEQ ID NO:163.

Figure 3 shows SDS/PAGE (polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate) analyses the following proteins: molecular weight markers of different mass (kilodalton (kDa) (lane 1); illustrative mutant fused the protein CTLA - 4-Ig in this invention based on the clone D3 (namely, D3-IgG2) (lane 2); illustrative mutant protein, CTLA-4-Ig-based clone D4 (namely D4-IgG2) (lane 3); and Orencia® (Abatacept) protein (lane 4) (Bristol-Myers Squibb Co., Princeton, NJ).

Figure 4 represents the elution profile illustrative mutant fused protein, CTLA-4-Ig on this invention (namely D3-IgG2) of the SEC (size-exclusion chromatography) analysis, showing that the mutant fused proteins, CTLA-4-Ig in this invention are uniform in size after purification from randomly-transfection COS cells.

Figure 5 shows a typical Biacore™ analysis linking the following fused proteins with hCD86-Ig: Orencia® protein, LEA29Y-Ig, and D3-IgG2. Phase dissociation analysis begins at the point in time marked by the arrow. Orencia® protein formed by the polypeptide of the human wild-type CTLA-4 EVA, merged with the mutant polypeptide domain IgGI Fc, which effectively serves as the control of the human wild-type CTLA-4-Ig. The mutant protein, CTLA-4-Ig in this invention, such as D3 - IgG2, which has a higher avidity binding to CD86-Ig than protein Orencia®, has a slower dissociation rate from the CD86-Ig protein than Orencia®.

Figure 6 is a graphical representation of the results of the analysis of inhibition of proliferation of the IPC (peripheral blood mononuclear cells) (with the stimulation of antibodies against CD3), including Illustra the active mutant fused proteins, CTLA-4-Ig on this invention (D3-04-IgG2, D3-1 1-IgG2, D3-12-IgG2, D3-14-IgG2). These analyses show that the mutant fused proteins, CTLA-4-Ig in this invention is much stronger than Orencia®, LEA29Y-Ig in the inhibition of T-cell proliferation in vitro.

Figure 7 is a graphical representation of analysis of inhibition of CD4+T-cell proliferation (anti-CD3 stimulation and hB7,2-dependent costimulatory), including illustrative group of mutant fused proteins, CTLA-4-Ig in this invention. Slit proteins Orencia®, LEA29Y-Ig is included as a control for comparison.

Figure 8 is a graphical representation of analysis of inhibition of proliferation of the IPC (with PPD (purified protein derivative) antigenic stimulation), including illustrative group of mutant fused proteins, CTLA-4-Ig in this invention. Orencia®, LEA29Y-Ig included as controls for comparison.

Figure 9 is a graphical representation of analysis of inhibition of proliferation of unilateral reaction of the mixed culture of lymphocytes (SCR-reaction), including illustrative mutant protein, CTLA-4-Ig in this invention - D3-IgG2. Slit proteins Orencia®, LEA29Y-Ig included as controls for comparison.

Figure 10 is a schematic illustration showing the structure of an illustrative mutant fused protein, CTLA-4-Ig in this invention. Two identical Monomeric mutant fused protein, CTLA-4-Ig shown CX is automatically, each includes Mature mutant CTLA-4 EVA, fused at its C-end N-end of the polypeptide of the human IgG2 Fc. Each polypeptide of a human IgG2 includes IgG2 hinge region, CH2 domain and CH3 domain. Also shows illustrative amino acid residues present in the joints between EVA and Ig polypeptide F. Amino acid residues at the joints between these components may vary depending on the amino acid sequence of a mutant CTLA-4 EVA and/or amino acid sequence of Ig. Dimeric protein produced by forming at least one disulfide bond between cysteine residues in the same positions in the two monomers. Cysteine (C) residues that are potentially included in the formation of disulfide bonds between the two monomers is indicated by an asterisk. The signal peptide of each monomer fused protein is normally cleaved during processing, and therefore the indirect (Mature) protein usually does not contain the sequence of the signal peptide.

Figure 11 is a graphical representation of analysis of CD4+T-cell proliferation (anti-CD3 stimulation and hB7,2-dependent costimulatory), including fused proteins hCTLA-4-IgG2, Orencia®, LEA29Y-Ig.

Figures 12A-12F represent the alignment of the amino acid sequence of unikl the exact domain of wild-type human CTLA-4 (denoted in the figure as "hCTL4"), amino acid sequence of the polypeptide LEA29Y (denoted in the figure as "LEA29Y") and amino acid sequence illustrative of mutant polypeptides, CTLA-4 EVA on this invention. The names of the clones of these mutant polypeptides, CTLA-4 EVA in this invention is defined to the left. Amino acid residues that are identical to those in the human CTLA-4 EVA wild type, specified with a period (.).

Figure 13 is BLOSUM62 (proteins, with an average similarity of 62% in pairs) matrix.

Figures 14A-14D show an illustrative alignment and the alignment indicators defined by manual calculation for the two amino acid sequences.

Figures 15A-15C show the pharmacokinetic (PK) profiles for fused protein Orencia®, human CTLA-4-IgG2 and typical mutant fused proteins, CTLA-4-IgG2 according to this invention, is introduced at a dose of 1 mg/kg individual (A) intravenous (IV) bolus or subcutaneous (SC) injection in rats.

Detailed description of the invention

Definition

Also it should be clear that the terminology used in this description, is used only for the purpose of describing particular embodiments and is not meant to limit the scope of the invention. Except as specifically identified, all technical and scientific expressions used in this description have the same values, what is usually understood by the experts in the field, belongs to this invention.

The expression "nucleic acid" and "polynucleotide" are used interchangeably to denote a polymer residues of nucleic acids (e.g., deoxyribonucleotides or ribonucleotides) or in single - or double-stranded form. In addition to specifically limited, expression include nucleic acids containing known analogues of natural nucleotides that have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to nucleotides occurring in nature. Unless otherwise indicated, a particular nucleic acid sequence also includes its conservatively modified variants (for example, replacement of the degenerate codon) and a complementary nucleotide sequence, as a sequence of precisely defined. Specifically, substitution of degenerate codons can be achieved by the formation of sequences in which the third position of one or more selected (or all) codons is substituted by residues mixed, bases and/or deoxyinosine residues (Batzer et al., Nucleic acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605 2608 (1985); Cassol et al. (1992); Rossolini et al., Mol. Cell. Probes 8:91 98 (1994)). The expression of the nucleic acid or polynucleotide used in ahimsamismo with cDNA or mRNA, encoded by a gene.

The terms "gene" refers broadly to any segment of nucleic acid (e.g. DNA)associated with biological function. A gene may include the coding sequence and/or regulatory sequence required for its expression. A gene may also include expressively segment(s) of the nucleic acid DNA, which, for example, generates a sequence of recognition for another protein(s) (e.g., promoter, enhancer or other regulatory site). The gene can be obtained from various sources, including cloning from a source of interest, or the synthesis of the known or suspected source of information about the sequence, and may include one or more of the sequences are designed so that they have the necessary parameters.

The expression "polypeptide," "peptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues. Expressions apply to amino acid polymers in which one or more amino acid residues are synthetic chemical mimetic of a corresponding amino acids occurring in nature, and refers to polymers of amino acids occurring in nature and polymers of amino acids not found in nature. As used in the data description, expressions include amino acid chains of any length, including full-size proteins (i.e., antigens), where amino acid residues are linked by covalent peptide bonds.

The numbering of a given amino acid polymer or nucleic acid polymer "corresponds to" or "refers to" numbering the selected amino acid polymer or nucleic acid polymer, when the position of any component of this polymer (e.g., amino acid, nucleotide, which are also called in General "balance") shall be determined by reference to the same or an equivalent position in the selected amino acid or nucleic acid polymer, rather than the actual numerical position of the component in the polymer. Thus, for example, the numbering of a given amino acid position in this amino acid sequence corresponds to the same or an equivalent amino acid position in the selected amino acid sequence, which is used as a reference sequence.

"Equivalent position" (for example, "equivalent position of the amino acids", or "equivalent position of nucleic acid", or "equivalent position"completely") is defined here as a situation (such as the position of the amino acids, or the position of the nucleic acid, or position of rest) of the Academy of Sciences of laserwave amino acid (or analyzed polynucleotide) sequence which is aligned with the corresponding position of the reference amino acid (or reference polynucleotide) sequence when using aligned (mostly optimally aligned in the alignment algorithm, as described in this description. Don't need to equivalent amino acid position of the analyzed amino acid sequences have the same number of positions as the corresponding position of the analyzed polypeptide. Like the equivalent position of the nucleic acids analyzed polynucleotide sequence should not have the same number of positions as the corresponding position of the analyzed polynucleotide.

"Mutant" polypeptide includes an amino acid sequence that differs by one or more amino acid residues from the amino acid sequence source or reference polypeptide (such as, for example, amino acid sequence of wild-type (DT)). In one aspect, the mutant polypeptide comprises the amino acid sequence that differs from amino acid sequence source or reference polypeptide from about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 30% 40%, 50% or more of the total number of residues of the original or reference amino acid sequence. In another and is the aspect of the mutant polypeptide comprises the amino acid sequence, which has at least about 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence source or reference polypeptide. In another aspect, the mutant polypeptide comprises the amino acid sequence that differs from amino acid sequence source or reference polypeptide from 1 to 100, or more amino acid residues (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid residues). Mutant polypeptide can include the amino acid sequence that differs from amino acid sequence source or reference polypeptide by, for example, deletions, additions or substitutions of one or more amino acid residues (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid residues) source or reference polypeptide or any combination of such deletion(s), addition(s) and/or replacement(replacement). Reference source or polypeptide can be a mutant polypeptide.

"Mutant" nucleic acid includes a nucleotide sequence that differs by one or more residues of a nucleic acid from the nucleotide sequence source or reference nucleic acids (such as nucleic acid the acid DT). In one aspect, the mutant nucleic acid includes a nucleotide sequence that differs from the nucleotide sequence source or reference nucleic acid at from about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 30% 40%, 50% or more of the total number of residues of the original or reference nucleotide sequence. In another aspect, the mutant nucleic acid includes a nucleotide sequence that has at least about 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleotide sequence source or reference nucleic acid. In another aspect, the mutant nucleic acid includes a nucleotide sequence that differs from the nucleotide sequence source or reference nucleic acid for from 1 to 100 or more nucleotide residues (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotide residues). Mutant nucleic acid can include a nucleotide sequence that differs from such source or reference nucleic acid, for example, by deletion, addition or substitution of one or more nucleotide residues (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotide residues) of the source or Etalon the second nucleic acid, or any combination of such deletion(s), addition(s) and/or replacement(replacements). The mutation in the nucleic acid may also be the result of alternative splicing or cutting off of nucleotides or errors in the processing or removal of nucleotides. Reference or source nucleic acid may itself be a mutant nucleic acid.

"Found in nature"applied to the object means that the object can be found in nature that is different from what synthetically produced human. "Not occurring", applied to the object means that the object is not found in nature (namely, that the object cannot be found in nature). For example, is not a naturally occurring polypeptide refers to a polypeptide that is created by man, such as, for example, synthesized in vitro or prepared artificially.

"Subsequence" or "fragment" of a sequence, which is of interest, is any part of a sequence to a sequence that is of interest, but not including it.

Nucleic acid, protein or other component is "isolated"when it is partially or completely separated from components with which it is in normal conditions connected (other proteins, nucleic acids, cells, synthetic reagents, and the like). In praying the ments against isolated species are more numerous, than other species in the composition. For example, isolated species may include at least about 50%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (in molar ratio) of all present macromolecular species. Mostly the species of interest, clear to full homogeneity (i.e. polluting types should be defined in the compositions of the traditional ways of definitions). The purity and homogeneity can be determined using methods known in this technical field, such as electrophoresis in agarose or polyacrylamide gel sample protein or nucleic acid, with subsequent visualization by staining. If necessary for the purification material can be used in method with high resolution, such as high performance liquid chromatography (HPLC), or similar methods.

The expression "purified"applied to nucleic acids or polypeptides, in General, refers to nucleic acid or polypeptide, which is completely free from other components, as determined by analytical methods known in the art (for example, the purification of individual bands polypeptide or polynucleotide forms on an electrophoretic gel, the chromatographic eluate and/or by centrifugation in a density gradient). For example, nucleic acid and polypeptide, who dispersed only one band in an electrophoretic gel, are "purified." Purified nucleic acid or polypeptide have at least about 50% pure, usually at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,5%, 99,6%, 99,7%, 99,8% or more purity (for example, the percentage by weight in molar ratio).

In the same sense, the invention provides methods of enriching compositions of one or more molecules according to this invention, such as one or more polypeptides or polynucleotides according to this invention. Composition enriched molecule, when there is a significant increase in the concentration of molecules after the application of the method of purification or enrichment. Substantially pure polypeptide or polynucleotide will typically contain at least about 55%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98, 99%, 99,5% or more by weight (molar ratio) of all macromolecular species in a particular composition.

Nucleic acid or polypeptide is "recombinant"when he synthesized or constructed, or is derived from synthesized or engineered protein or nucleic acid.

The expression "recombinant", as used with respect to the cell, usually means that the cell replicates heterologous nucleic acid, or expresses polypeptide, encoded by heterologous nucleic acid. Recombinant cells include genes that are not found in native (non-recombinant) form of the cell. Recombinant cells include those cells that include genes that are found in the native form of the cell, but modified and re-built into the cell by artificial means. The expression also includes cells that include a nucleic acid endogenous to the cell, which modified without removing the nucleic acid from the cells; such modifications include those which are obtained by moving a gene, site-specific mutations and using similar methods known to experts in this field of technology. Recombinant DNA technique includes methods for the preparation of recombinant DNA in vitro and transferring recombinant DNA into cells, where it can be expressed or reproduced, thereby producing a recombinant polypeptide.

Cassette recombinant expression" or "expression cassette" is a nucleic acid construct, educated recombinante or synthetically, with elements of nucleic acid that is capable of expression of the structural gene in the tender hosts compatible with such sequences. The expression cassettes include at least the promoters and NeoMaster is but signals termination of transcription. Typically, the cartridge recombinant expression include nucleic acid that needs transcribing (for example, nucleic acid encoding the desired polypeptide and a promoter. Additional factors necessary or assist in the enforcement of expression, can also be used, as described in this description. For example, the expression cassette may also include nucleotide sequences that encode a signal sequence that directs the secretion of the expressed protein from the host cell. Signals termination of transcription, enhancers and other nucleic acid sequence that affect gene expression, can also be included in the expression cassette.

"Exogenous" nucleic acid", "exogenous DNA segment", "heterologous sequence" or "heterologous nucleic acid", as used herein, are those that have the origin of an alien with respect to specific cell-master, or, if they are from the same source, is modified from the original form. Thus, a heterologous gene in a cell-master includes a gene that is endogenous to the specific cell master, but modified. Modification of the heterologous sequence in the applications described is here usually done through the use of directed molecular evolution. Thus, the expression refers to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the nucleic acids of the host cell in which the element is not usual. The exogenous nucleic acid or exogenous DNA is expressed to obtain exogenous polypeptides.

"Vector" may be any agent capable of deliver or maintain the nucleic acid in the cell the owner and includes, for example, but not limited to the following: plasmid (e.g., DNA plasmids), naked nucleic acids, viral vectors, viruses, nucleic acids in combination with one or more polypeptides or other molecules, and nucleic acids immobilized on solid-phase particles. The vectors described in detail below. The vector can be used as an agent for delivery or retention of exogenous gene and/or protein in the cell host. The vector may be capable of transducible, transactivate or transform the cell, thereby causing the cell replicates or expresses a nucleic acid and/or proteins, non-native to the cell or method that is not native to the cell. The vector may include materials for CSP is to obstsalat penetration of the nucleic acid into the cell, such as a viral particle, a liposome, a protein shell or similar. Can be used with any method of transferring nucleic acid into the cell; if not specified, the expression vector does not imply any particular method of delivery of nucleic acid into the cell or imply that any particular cell type is the subject of transduction. This invention is not limited to any specific vector for delivery or retention of any nucleic acid according to this invention, including, for example, nucleic acid encoding a mutant polypeptide, CTLA-4 on a given invention or its fragment (e.g., mutant CTLA-4 EVA), which binds CD80 and/or CD86 or fragment (e.g., CD80 EVA or CD86 KJV).

The term "expression vector" generally refers to a nucleic acid construct or sequence formed recombinante or synthetically, with views of the specific elements of nucleic acid that permit transcription of a particular nucleic acid in a cell host. The expression vector typically includes a nucleic acid that need transcribing, functionally associated with the promoter. The term "expression" includes any step, included in the production of the polypeptide, including, but not limited to, transcription, post-transcriptional modification is s, translation, post-translational modification and/or secretion.

"Signal peptide" is a peptide (or amino acid) sequence, which usually precedes the polypeptide of interest, and is transmitted together with the polypeptide and directs or facilitates the access of the polypeptide to the secretory system. The signal peptide is typically covalently attached or fused to the amino end of the polypeptide of interest, and facilitates the secretion of the polypeptide of interest from the host cell. The signal peptide is typically cleaved from the polypeptide of interest, after the broadcast.

The terms "encoding" refers to the ability of the nucleotide sequence to encode one or more amino acids. The expression does not require a start or stop codon. Amino acid sequence can be encoded in any of six different reading frames secured polynucleotide sequence and its complement.

The expression "control sequence", as indicated in this description, includes all components which are necessary or advantageous for expression of the polypeptide according to this invention. Each control sequence may be native or alien with respect to nucleotide sequence that encodes a polypeptide. is this control sequence includes, but not limited to the following, a leader sequence, polyadenylation, propeptide sequence, the promoter sequence of the signal peptide and the terminator of transcription. Minimum control sequence includes a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers with the aim of embedding specific restriction sites that facilitate ligation of the control sequences with the coding area of the nucleotide sequence that encodes a polypeptide.

The terms "coding sequence" refers to nucleotide sequences that accurately indicates the amino acid sequence of its protein product. The boundaries of the coding sequence are determined using the open reading frames (ORFS), which could begin with the ATG start codon.

Nucleic acid is functionally linked" to another nucleic acid sequence when it is in functional cooperation with another nucleic acid sequence. For example, a promoter or enhancer functionally linked to the coding sequence if it directs transcription of the coding sequence. Functionally linked means that Ankoslitoflower, related are usually overlapping and, if you want to combine two sites, protein coding, overlapping and in reading frame. However, because the enhancers generally function when they are separated from the promoter of several thousand base pairs and intron sequences can be of varying lengths, some of polynucleotide elements may be functionally related, but not overlapping.

"A host cell" is any cell that is suitable for transformation with nucleic acid.

"In fact, the full length polynucleotide sequence" or "essentially the full length amino acid sequence" refers to at least about 50%, 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,5% or greater than the length polynucleotide sequence or amino acid sequence, respectively.

"Antigen" refers to a substance which reacts with the product(s) of the immune response stimulated by a specific immunogen. See, for example, JULIUS CRUSE ET AL., ATLAS OF IMMUNOLOGY 60 (1999); RICHARD COICO ET AL., IMMUNOLOGY: a SHORT COURSE 27-30 (5thed. 2003). The immune response may include humoral response and/or cell-mediated immune response (e.g., cytotoxic T lymphocytes (LTC)). The products of the immune response can include antibodies and/or LTC. Antigens - this is usually the macromolecule (for example, the polypeptides, nucleic acids, complex carbohydrates, phospholipids, polysaccharides)that are foreign to the host; the part of the antigen, which is known as antigenic determinant, is reacted with (e.g., binds to) the product(s) of the immune response, such as antibody or specific T-cell receptor on T lymphocytes. The antigen may be, but not necessarily, to induce an immune response and of reacting with the product(s) of the immune response. "Antigenicity" refers to the condition or quality of being antigenic - namely, to have the properties of the antigen. The specificity of the antigen can be demonstrated in respect of the antigen to its antibody, or Vice versa; the antigen is usually react with high specificity to its corresponding antibody and not with the same degree of specificity with other antibodies produced in response to the immunogen. "Antigenic amount" is the amount of antigen, which apparently reacts with the product(s) of the immune response stimulated(and) specific immunogen.

"Immunogen" is a substance that can induce an immune response rather than immunological tolerance. See, for example, JULIUS CRUSE ET AL., above at 60-61; RICHARD COICO, above 27-30. Immunogen also react with (e.g., contact) the product(s) caused by the immune response, which was caused specifically against them. Therefore clicks the zoom, all immunogene are antigens. "Immunogenicity" refers to the state or the property to be immunogenic and to have the properties of the immunogen. "Immunogenic amount" is an amount of immunogen, which is effective to cause visible immune response. The immunogen may elicit strong immune response in the subject, such as at least partial or complete protective immunity against at least one pathogen.

"Immunomodulator" or "immunomodulatory molecule, such as immunomodulatory polypeptide or nucleic acid, modulates the immune response. By "modulation" or "modulating" the immune response implies that the immune response is changed. For example, "modulation" or "modulating" immune response in the subject as a whole means that the immune response is stimulated is called, inhibited, reduced, inhibited, increased, enhanced or modified by another image of the subject. Such modulation of the immune response can be achieved by methods known to experts in the art, including those described below. "Immunosuppressive drug" or "immunosuppressant" is a molecule such as a polypeptide or nucleic acid that suppresses the immune response.

As used herein, "antibody" (abbreviated as "AT") refers to an immunoglobulin protein (abbreviated "I"), natural or wholly or partially synthetically derived. The expression includes all its derivatives, which support specific affinity for the antigen. The expression also includes any protein having a binding domain which is homologous or largely homologous to immunoglobulin binding domain. Such proteins can be obtained from natural sources, or may be partly or wholly synthetically derived. The antibody may be monoclonal or polyclonal. The antibody may be a member of any class of immunoglobulins, including any of the five human classes: IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (which includes subclasses IgG1, IgG2, IgG3 and IgG4 and IgM. Antibodies include paired heavy and light polypeptide chains, and each chain consists of individual immunoglobulin domains. Each circuit includes a constant (C) region and a variable (V) region. Typical structural unit of the antibody comprises the tetramer. Each tetramer contains two identical pairs of polypeptide chains, each pair has one "light" (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The heavy chain there are five main types (γ, µ, δ, α and ε) depending on the class of antibodies and contain about 450-600 amino acid residues. Light chains are of two main types (λ and κ) which contain approximately 230 amino acid residues. As an example, the IgG antibody is a tetramer protein, comprising two heavy chains and two light chains. Each IgG heavy chain contains four immunoglobulin domain, connected in the following order from N-Terminus to C-end: VH-CH1-CH2-CH3 (also sometimes abbreviated VH-CH 1-CH2-CH3). These abbreviations refer to the variable domain of the heavy chain constant domain 1 heavy chain constant domain 2 heavy chain and the constant domain 3 of the heavy chain), respectively. Heavy chain may also be referred to the class of antibodies, such as, for example, γ1, which represents the first constant domain gamma (γ) heavy chain IgG antibodies. Each IgG light chain comprises two immunoglobulin domain, connected in the following order from N - to C-end: VH-CLwhere VHand CLrefers to the variable domain light chain and the constant domain of the light chain, respectively.

Variable region of antibodies, which usually includes about 100-110 or more amino acids at the N end of each polypeptide comprises the antigen binding determinants and, thus, is primarily responsible for antigen recognition and specificity. The greatest degree of variation in amino acid sequence between the antibodies found in the variable regions. In the core the main sequence variability occurs in hypervariable sites (HLG), localized in the variable regions. In General, there are six HLG, three HLG in each heavy chain and three HLG in each light chain, which together form antigennegative website. HLG heavy chain are referred to as VHCDR1, VHCDR2, and VHCDR3, while HLG light chain are referred to as VLCDR1, VLCDR2 and VLCDR3. The area located outside of the group is called a frame (KO) region. Frame region different antibodies can vary in amino acid residues, but the degree of amino acid variation not greater than that which exists between the variable regions of different antibodies. In many cases, the frame region provide stable and permanent basis for amino acid diversity represented HLG.

The term "antibody fragment" refers to any derivative of an antibody that is less than a full-size antibody. Examples of fragments of antibodies include, but are not limited to the following, for example, antigen-binding fragment (Fab)that contains VH-CHI and VH-CLthe variable fragment (Fv)containing VHand VL, single-chain variable fragment (scFv)containing VHand VLlinked together in one chain, and other fragments of the V region, such as Fab', F(ab)2F(ab')2, dsFv diatel, Fc and Fd polypeptide fragments. Smo is ri Scott, T.A. and Mercer, E.I., CONCISE ENCYCLOPEDIA: BIOCHEMISTRY AND MOLECULAR BIOLOGY (de Gruyter, 3d ed. 1997), and Watson, J.D. et al, RECOMBINANT DNA (2d ed. 1992) (hereinafter called "Watson").

The antibody fragment may be obtained by any means known in the art. For example, the antibody fragment may be enzymatic or chemically obtained by fragmentation of an intact antibody, or it may be recombinante derived from a gene encoding a partial sequence of the antibody. For example, antibody fragments can be obtained by splitting peptidases. For example, pepsin digests an antibody below the disulfide bridges in the hinge to obtain F(ab')2, a dimer of Fab fragment, which is a light chain that is connected to VH-CHI by a disulfide bond. F(ab')2may be reduced under mild conditions to break the disulfide bridge at the hinge, thereby turning (Fab')2dimer in the Fab' monomer. Fab' monomer is essentially a Fab fragment with part of the hinge. See FUNDAMENTAL IMMUNOLOGY, W.E. Paul, ed., Raven Press, N.Y. (1993) for a more detailed description of the antibodies and fragments of antibodies. As various fragments of antibodies are indicated in expressions of intact antibodies, the person skilled in the art should understand that such Fab' fragments may be synthesized de novo either chemically, and using techniques of recombinant DNA. Thus, the expression t is the train includes fragments of antibodies, or obtained by the modification of whole antibodies or synthesized de novo using techniques of recombinant DNA.

Alternatively, the antibody fragment may be fully or partially obtained synthetically. The antibody fragment may optionally be single-stranded fragment of the antibody. Alternatively, the fragment can include multiple chains, linked together by a disulfide bridges. The fragment may also not necessarily be complex multimolecular lb. Functional fragment of the antibody typically comprises at least about 50 amino acids, and more typically will include at least about 200 amino acids.

The Fc region or domain of immunoglobulin molecules, or antibodies (also referred to as Ig Fc polypeptide or Fc polypeptide) corresponds largely constant region of the heavy chain of an immunoglobulin and is responsible for various functions, including the effector(s) function(s) of the antibody. For example, Ig Fc region of IgG molecules comprises immunoglobulin domains CH2 and CH3, and N-terminal hinge, leading to CH2. The hinge is the part of the heavy chain between Fc and SN, which contains a disulfide between the heavy chains and binds and gives flexibility to the molecule antibodies. The constant domains of an Fc region interact with cells of the immune system. Fc receptors are proteins that bind the Fc region of anti the L. One significant family of Fc receptors for IgG antibodies includes Fc gamma receptors (FcγR). Antibody binding to Fc receptors on cells mediates many functions of antibodies. Different subclasses of IgG are different affinity for Fc gamma receptors. In General, IgG1 and IgG3 bind receptors with greater affinity than IgG2 and IgG4. Fc receptors expressed on various cells, including, for example, In cells, monocytes, dendritic cells, neutrophils and some lymphocytes. Binding Ig Fc with its receptor brings these effector cells to sites of bound antigen, which eventually leads to signaling and immune responses, including cell activation, inflammatory responses, cytotoxic responses and phagocytic responses.

Merge Ig Fc is a molecule that includes one or more polypeptides (or one or more small molecules)that are functionally associated with the Fc region of the immunoglobulin or antibody. See, for example, Chamow et al., 1996, Trends Biotechnol. 14:52-60. Accordingly, Ig Fc protein is a molecule that includes one or more polypeptides that are functionally associated with Ig Fc region. Ig Fc protein may contain, for example, the Fc region of antibodies (which facilitates effector functions and pharmacokinetic characteristics) and linking the region or binding domain of a receptor protein or ligand of a protein, Il is another protein, or fragment. Linking the region or binding domain mediates recognition receptor target or ligand-target (comparable to those for the variable regions of the antibody for the antigen). Ig Fc region may be connected indirectly or directly with one or more polypeptides or small molecules (partners merge). Various linkers known in the art and are described in more detail hereinafter, can be used to connect Ig Fc with a merge partner to obtain Ig Fc fusion. Ig Fc protein typically involves the Ig Fc region, covalently linked directly or indirectly with at least one polypeptide, and the polypeptide normally binds the ligand-target or receptor target.

Can be used monoclonal or polyclonal antibodies can be prepared by any method known from the prior art (see, for example, Kohler &Milstein, Nature 256:495 497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Cole et al., pp.77 96 in Monoclonal Antibodies and Cancer Therapy (1985)). The procedures for the production of single-chain antibodies (U.S. Patent No. 4946778) can be adapted to generate antibodies to the polypeptides according to this invention. In addition, transgenic mice, or other organisms, including mammals, may be used for expression of humanized antibodies. The technique of phage display can be used for the detection of antibodies and Fab fragments, which specifically bind the selected antigens (see, for example, McCafferty et al. Nature 348:552 554 (1990); Marks et al, Biotechnology 10:779 783 (1992)).

The term "epitope" refers to an antigenic determinant capable of specifically bind to a part of the antibody. Epitopes usually consist of chemically active surface groupings of molecules such as side chains of amino acids or sugar, and usually have a specific 3-dimensional structural characteristics, as well as specific charging characteristics. Conformational and conformational epitopes differ in that the binding with the first and not the last is lost in the presence of denaturing solvents.

"The specific affinity of binding between two molecules, for example, ligand and receptor means the preferential binding of one molecule with another. The binding of molecules is usually considered as a specific, if the equilibrium binding constant of Association (namely, KA) is from about 1×102M-1to about 1×1013M-1or more, including about 104-1013M-1about 106-1012M-1about 108M-1-1011M-1or about 108-1010M-1. Values of KAfor the binding interaction between an antigen and an antibody usually lie in the range from about 105M- to about 1012M-1usually from about 107M-1to about 1011M-1and often from about 108M-1to about 1010M-1. KA(M-1) is determined by calculating the ka/kdwhere kais the rate constant of Association kdis the rate constant of dissociation. The units for kaand kdare the M-1s-1and s-1respectively. The equilibrium dissociation constant, KDis the return value of KA. KD=kd/ka. For the reaction + In <=> AT (which represents the binding of a single ligand with a separate protein that is of interest (e.g., receptor)), KDequal to ([A][V])/[AB]. Non-limiting examples of well-known techniques for measuring affinely binding and/or avednesday binding molecules include, for example, Biacore™ technology (GE Healthcare), as discussed herein, isothermal methods titrimetric microcalorimetry (MicroCal LLC, Northampton, MA USA), ELISA (enzyme-linked immunosorbent assays) and cell sorting with excitation fluorescence (FACS). For example, FACS or other sorting method can be used for the selection of populations of molecules (such as, for example, the ligands on the cell surface), which are specifically associated with an associated binding the m pair member (such as a receptor, for example, a soluble receptor). The complexes of the ligand-receptor can be detected and sorted, for example, using fluorescence (for example, by reaction of the complex with a fluorescent antibody that recognizes the complex). Molecules of interest that are associated with an associated binding pair member (e.g., a receptor) are combined and sorted again in the presence of lower concentrations of the receptor. By conducting multiple rounds of sorting in the presence of decreasing concentrations of receptor (illustrative concentration range from 10-6M to 10-13M, namely 1 micromoles (μm) to 1 nanomoles (nm)or less (for example, 10-11M or 10-12M), depending on the nature of the ligand-receptor interaction), can be selected population of molecules of interest, exhibiting specific affinity binding to the receptor.

The phrase "specifically (or selectively) binds" to an antibody or "specifically (or selectively) shows immunoreactivity to"when it refers to a protein, refers to a binding reaction which is determined in the presence of the protein in a heterogeneous population of proteins and other biological agents. Thus, under designated conditions, immunological analysis, specific antibodies bind to specific what Elcom, at least two times stronger than the background activity, and do not substantially bind in a sufficient amount to other proteins present in the sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein. Can be used in a variety of formats immunological analysis for selection of antibodies that specifically shows immunoreactivity to specific protein. For example, solid phase immunological ELISA assays are typically used to select antibodies that specifically show immunoreactivity for the protein (see, for example, Harlow &Lane, Antibodies, a Laboratory Manual (1988), for a description of the formats of immunoassay and conditions that can be used to determine specific immunoreactivity). Typically a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times above background.

The term "cytokine" includes, for example, but not limited to the following, interleukins, interferons (IFN), chemokines, hematopoietic factors, growth factors, tumor necrosis (TNF) and transforming growth factors. In General, proteins of small molecular weight that regulate the maturation, activation, proliferation and differentiate the cells of the immune system.

The term "screening" describes, in General, the process that determines the optimum molecule according to this invention, such as, for example, including the polypeptides according to this invention, and related fused proteins comprising them, and nucleic acids encoding these molecules. Some properties of the corresponding molecules can be used in the selection and screening, for example, the ability of the respective molecules to cause or modify the desired immune response in the test system or in vitro, ex vivo or in vivo applications. "Selection" is a form of screening, in which the definition and physical separation is achieved simultaneously with the expression of the selected marker, which, in some genetic circumstances, allows cells expressing the marker, to survive, while the other cells die (or Vice versa). Markers for screening include, for example, luciferase, beta-galactosidase and green fluorescent protein substrates for the reaction and the like. The selection markers include genes that are resistant to drugs and toxins and the like. Another way of selecting includes sorting based on defined events, such as the binding of ligand to the receptor, the reaction of the substrate with the enzyme, or any other physical process that can produce a detectable signal, either directly (for example, using chromogenic su the strata or ligand), or indirectly (for example, by reaction with a chromogenic secondary antibody). Selection by physical sorting can be done in various ways, including, but not limited to, for example, FACS in a cage or microanalysis.

Because of limitations in the study of primary immune responses in vitro, in vivo studies are especially important methods of screening. In some of these studies polynucleotide or polypeptide according to this invention, first built in the analyzed subject (e.g. a mammal, such as an animal), and further studied induced immune response by analyzing the type of immune response in an immunized animal (e.g., production of antibodies in the serum of an immunized animal, the proliferation of T-cells) or by examining the quality or strength of the induced immune response in an immunized animal (for example, caused the level of antibody titer).

The expression "subject", as used herein, includes, but is not limited to the following, the body or an animal, including mammals and insects memleketim. Mammal includes, for example, but not limited to the following, human, Primate, non-human (e.g., baboon, orangutan, monkey, gorilla, mouse, dog, pig, cow, goat, cat, rabbit, rat, Guinea pig, hamster, horse, sheep, or others who Sarafovo mammal, not a person. Namecapital includes, for example, but not limited to the following, invertebrate, non-mammal, and invertebrate non-mammals, such as poultry (such as chicken or duck) or a fish.

The phrase "pharmaceutical composition" refers to compositions suitable for pharmaceutical use in a subject, including an animal or human. A pharmaceutical composition generally comprises an effective amount of an active agent and a carrier, excipient or solvent. Carrier, filler or solvent are typically pharmaceutically acceptable carrier, excipient or solvent, respectively.

The expression "effective amount" refers to the dose (or dose) or the amount of chemical required to produce a desired result. The desired result may include an objective or subjective improvement in the recipient of the dosage or quantity. For example, the desired result may include measurable determined or verified induction, stimulation, enhancement, or modulation of the immune response in the subject, which introduced the dosage or amount of a particular antigen or immunogen (or composition). The dose (or dose) or immunogen, sufficient to provide this result, referred to as "immunogen who owned the dose (or dose) or number.

"Prophylactic treatment" is a treatment that is the subject that shows no signs or symptoms or exhibits only early signs or symptoms of pathology or disturbance, so that the treatment is administered to prevent or reduce the risk of diseases, pathologies or disorders. Prophylactic treatment functions as a preventive treatment against diseases, pathologies or disorders or as a treatment that inhibits or reduces the development or worsening of a disease, pathology or impairment. "Prophylactic activity" is an activity of the agent that, when administered to a subject who does not show signs or symptoms or exhibits only early signs or symptoms of pathology or disturbance, prevents or reduces the risk of development of a pathology, disease or impairment. "Prophylactically suitable agent (e.g., nucleic acid or polypeptide) refers to an agent which is suitable for the prevention of disease, pathology or violation of, or suitable for inhibiting or reducing the development or strengthening of disease, pathology or disturbance.

"Therapeutic treatment" is a treatment, which have a subject that exhibits symptoms or signs of pathology, diseases or disorders in which cured the e is administered to a subject to reduce or remove these signs or symptoms. "Therapeutic activity" is an activity of the agent, which removes or reduces the signs or symptoms of pathology, disease or impairment when administered to a subject suffering from such signs or symptoms. "Therapeutically suitable agent" means an agent that is suitable for the reduction, treatment or removal of signs or symptoms of disease, pathology or disturbance.

In General, the nomenclature used in this description, and many laboratory techniques in cell culture, molecular genetics, molecular biology, chemistry of nucleic acids and chemistry of proteins, described below, is well known to specialists in this field and are generally used by experts in the field of technology. Standard methods such as described in Sambrook et al., Molecular Cloning - a Laboratory Manual (2nd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989 (hereinafter designated as "Sambrook") and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, F. M. Ausubel et al., eds., Current Protocols in conjunction with Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (1994, revised edition 1999) (hereinafter designated as "Ausubel"), are used for recombinant methods relative to nucleic acids, nucleic acid synthesis, methods cell culture and transgenic including, for example, electroporation, injection, gene gun, penetrating through the skin, and lipofectin. In General, the stages of oligonucleotide synthesis and the sight of the surveillance are performed in accordance with the description of the invention. Techniques and procedures are generally performed according to the methods conventional in the art and various General references that are provided throughout the document. It is assumed that the procedures in this description are well known to specialists in this field and provided for the convenience of the reader.

Other expressions are defined or otherwise explained in this specification.

Molecules and methods according to the invention

This invention provides molecules and methods for treatment of diseases, disorders and conditions of the immune system, including, for example, those in which modulation of the immune system (for example, dependent T-cell immune responses) is desirable. The molecules of this invention (for example, the polypeptides according to this invention, the conjugates of the invention, soluble fused protein according to this invention, nucleic acids encoding such polypeptides or fused proteins) are suitable for the treatment of diseases, disorders and conditions of the immune system, in which immunosuppression is desirable, including, for example, but not limited to the following, the treatment of autoimmune diseases, disorders and conditions immunoproliferative disease, disorders associated with graft, and methods of treatment, including transplantation of tissues and cells, rhana or tissue or organ from donor to recipient, where suppression of the immune response of the recipient against the donor tissue, cell, organ or tissue or organ is desired.

In one aspect, the invention provides a new mutant molecules CTLA-4, which has improved properties compared with the molecule CTLA-4, such as the polypeptide of the wild type human CTLA-4 ("hCTLA-4") or its fragment that bind CD80 and/or CD86, such as the extracellular domain of human CTLA-4 ("hCTLA-4 EVA"). As discussed in more detail below, various strategies of mutagenesis and screening are used for education and identify new mutant molecules CTLA-4, which bind CD80 and/or CD86. In particular, such strategies are used for education and definitions of mutant molecules CTLA-4, which has improved the avidity of binding to CD80 (B7-1) and/or CD86 (B7-2) compared to human CTLA-4 ("hCTLA-4") and/or have enhanced binding affinity to CD80 and/or CD86 compared with the hCTLA-4 EVA. Mutant molecules CTLA-4 according to this invention, which bind endogenous ligands CD80 and/or CD86, expressed on antigen-presenting cells, effectively inhibit or block the interaction of these ligands with the endogenous receptor CD28, which is expressed on the surface of T cells. In the result of the co-stimulatory signal required for T-cell activation, which provides the I by the interaction of the receptor T-cell surface CD28 with B7 molecules (namely, CD80 and CD86), inhibited or blocked. Such T cells are activated and not enough have a reduced ability to proliferation.

When the signal transmission between the ligand CD80 or CD86 and CD28 receptor is blocked, the T-cells are not sufficiently stimulated to become active and, thus, insufficient induced to proliferate. Similar to this, when the signal transmission between the ligand CD80 or CD86 and CD28 receptor inhibited the activation and proliferation of T-cells is inhibited. In one aspect, the invention provides mutant molecules CTLA-4, which function as antagonists against CTLA-4 signaling. In another aspect, the invention provides mutant molecules CTLA-4, which function as antagonists to CD28 signaling, thereby inhibiting or blocking T-cell-dependent immune responses; these molecules function as immunosuppressive agents. In another aspect, the invention provides mutant molecules CTLA-4, which bind as CD80 and CD86, but who have the avidity of binding to CD86 than CD80, and thereby inhibit CD86-dependent costimulation more than CD80-dependent costimulation. Assume that all of these mutant molecules CTLA-4 according to this invention is suitable for treatment of disease, disorder or condition is to in which immunosuppression is desirable or would be preferred.

It is shown that the fused protein of human CTLA-4-Ig and specific mutant protein, CTLA-4-Ig - both developed by Bristol-Myers Squibb Co. (Princeton, NJ) is effective in the treatment of certain immunostained diseases or conditions. Protein Orencia® (also known as Abatacept ("AVA")) (Bristol-Myers Squibb Co. (Princeton, NJ)is soluble recombinant dimeric protein consisting of two identical Monomeric immunoglobulin (Ig) fused proteins, covalently bonded together by a disulfide bond formed between cysteine residue present in each Monomeric fused protein. ORENCIA is a registered trademark of Bristol-Myers Squibb Company. Each Monomeric Ig protein Orencia® dimer consists of the extracellular domain of human CTLA-4 (SEQ ID NO:159), fused at its C-end N-end specific mutant polypeptide IgG1 Fc (SEQ ID NO:186). Complete amino acid sequence of each such Monomeric fused protein shown in SEQ ID NO:164. Dimer Orencia® receive in the expression system of the mammal, and it has an approximate molecular weight of 92 kDa. Assume that two Monomeric Ig fused protein Orencia® dimer covalently linked together one by a disulfide bond formed between cysteine residue at position 120 ka the Dogo hCTLA-4-mutant IgG1 monomer, and that there are no disulfide bonds formed between two mutant IgG1 Fc polypeptides.

Dimer Orencia® is a selective co-stimulatory modulator that inhibits T-cell activation by binding to CD80 and CD86 and thereby blocking interaction with CD28. Now it has been confirmed the efficacy of the dimer Orencia® for the treatment of an adult suffering from rheumatoid arthritis (RA) from medium to severe. More information about the dimer Orencia® and its clinical assignments and efficiency provided on the world wide web site orencia.com and bms.com.

As noted above, each monomer of the dimer fused protein Orencia® contains the extracellular domain of human CTLA-4. Human CTLA-4 is a membrane protein, which incidentally is expressed on T-cells. Full protein sequence full-hCTLA-4 DT shown in SEQ ID NO:160, and the sequence of nucleic acids encoding a full-hCTLA-4 DT shown in SEQ ID NO:194. Human CTLA-4 includes a signal peptide (SP), extracellular domain (EVA), transmembrane domain (TD) and cytoplasmic domain (CD), covalently linked together in this order (for example, With the end of the JV covalently linked to the N-end of EVA, With the end of EVA covalently linked to the N-end of the AP, and With the end of the TD covalently linked to the N-end of the CD). Poly is eptid DT hCTLA-4 EVA usually includes balances 38-161 sequence of the full-size protein hCTLA-4 (SEQ ID NO:160) and is usually in the length of 124 amino acid residue. This amino acid sequence hCTLA-4 EVA shown in SEQ ID NO:159. Signal peptide (SP) full-size protein hCTLA-4, which typically includes amino acid residues 1-35 or 1-37 SEQ ID NO:160, cleaved during processing. See, e.g., Harper et al, J. Immunol. 147(3): 1037-1044 (1991). The sequence of human CTLA-4 signal peptide comprising amino acid residues 1-35 or 1-37 hCTLA-4 protein shown in SEQ ID NO:182 or SEQ ID NO:216, respectively. If the sequence of the signal peptide is presented in SEQ ID NO:182 or SEQ ID NO:216, when the signal peptide is cleaved, the Mature protein hCTLA-4 usually starts with a methionine residue at amino acid position 38 of the full sequence of the protein hCTLA-4, is presented in SEQ ID NO:160. Thus, even if the sequence of the signal peptide hCTLA-4 is SEQ ID NO:182, which includes amino acid residues 1-35 of protein hCTLA-4, the resulting Mature secretory protein hCTLA-4 starts with methionine, which is in position 38 full-hCTLA-4 protein. Residues lysine (K) and alanine (A) at positions 36 and 37, respectively, a full-sized protein hCTLA-4 not present in the Mature protein hCTLA-4, and it is assumed that they hatshepsuts from the Mature protein hCTLA-4 processing. Amino acid residues of the sequence of the Mature protein hCTLA-4, therefore, usually numbered, beginning the with methionine residue, present in position 38 full-sized protein hCTLA-4 as the first amino acid (namely, position 1); accordingly, his-tag residue is the amino acid position 2 of the Mature protein hCTLA-4, etc. Each monomer Orencia® dimer includes hCTLA-4 EVA amino acid sequence represented in SEQ ID NO:159. In the full-size protein DT hCTLA-4 signal peptide comprises amino acid residues 1-37, extracellular domain (EVA) includes amino acid residues 38-161, transmembrane domain (TD) includes amino acid residues 162-182 and cytoplasmic domain (CD) includes amino acid residues 183-223 SEQ ID NO:160. Mature domain (MD) protein hCTLA-4 typically comprises amino acid residues 36-223, or in some cases, amino acid residues 37-223 or 38-223 SEQ ID NO:160.

Nucleic acid sequence SEQ ID NO:194 includes a sequence of nucleic acids encoding the sequence of the signal peptide (nucleotide residues 1-111), the sequence of nucleic acids encoding a hCTLA-4 EVA (nucleotide residues 112-483), the sequence of nucleic acids encoding a hCTLA-4 transmembrane and cytoplasmic domains (nucleotide residues 484-669); the last 3 C-terminal nucleotide is a TGA stop codon.

Belatacept (also known as "LEA29Y-Ig," "LEA-Ig,or A29YL104E-Ig") (Bristol-Myers Squibb Co. (Princeton, NJ)is soluble is recombinantly dimeric protein, consisting of two identical fused Ig protein, covalently bonded together by a disulfide bond formed between cysteine residue in each monomer fused protein. Each Monomeric protein consists of a polypeptide mutant extracellular domain of CTLA-4 fused at its C-end N-end specific mutant IgG1 polypeptide. Amino acid sequence of a mutant CTLA-4 EVA differs from the amino acid sequence of human CTLA-4 EVA DT for two mutations, namely the replacement of tyrosine to alanine at position 29 (abbreviated replacement A29Y) and the substitution of glutamine for leucine at position 104 (abbreviated replacement L 104E), where amino acid residues in the human CTLA-4 EVA are numbered with methionine at N-end, representing the amino acid in position 1. Each monomer of Belatacept includes the amino acid sequence of a mutant IgG1 Fc presented in SEQ ID NO:186; this mutant polypeptide IgG1 Fc identical mutant polypeptide IgG1 Fc included in the protein Orencia®. Monomeric protein Belatacept, so different from each monomer fused protein Orencia® two amino acids. Amino acid sequence of each such Monomeric fused protein in Belatacept shown in SEQ ID NO:166. The name "LEA29Y-Ig", thus, reflects the fact that each Monomeric protein dimer Belataza the TA consists of a mutant CTLA-4 EVA which differs from the amino acid sequence of human CTLA-4 EVA on two mutations L104E and A29Y. It is shown that Belatacept links CD86 about 4 times stronger and binds CD80 about 2 times stronger than the Orencia® dimer (Larson et al., Amer. J. Transplat. 5:443-453, 444 (2005). It is shown that Belatacept is about 10 times stronger than the dimer Orencia® inhibition of T-cell activation in vitro, and has an improved in vivo immunosuppressive activity compared with protein Orencia®, as shown by its increased ability to inhibit T-cell-dependent antibody responses and enhanced extension of the viability of renal allograft in clinical trials in primates that are not human. For more information about Belatacept and its clinical assignments and efficacy presented at the world website bms.com.

In one aspect, the invention provides mutant molecules CTLA-4, including new soluble recombinant mutant fused proteins, CTLA-4-Ig, described here, which have the avidity of binding to CD86, which is greater than the avidity of binding of the dimer Orencia® (dimeric hCTLA-4-Ig)to CD86. The invention also provides mutant molecules CTLA-4, including new soluble recombinant dimeric mutant fused proteins, CTLA-4-Ig, which have the avidity of binding to CD80, which practically RA is on or more than the avidity of binding of the dimer Orencia® to CD80. In another aspect, the invention provides mutant molecules CTLA-4, including new soluble recombinant mutant fused proteins, CTLA-4-Ig, which have a greater ability to suppress one or more immune responses (e.g., T-cell-dependent immune responses)than Orencia® (Abatacept). Assume that the mutant molecule CTLA-4 according to this invention having one or more improved properties compared to the dimer Orencia®, are stronger and therefore more effective, suitable and advantageous than the dimer Orencia® in the treatment of diseases, disorders or conditions in which immunosuppression is desirable, including such diseases, disorders or conditions for which the effectiveness of the dimer Orencia® and/or it has been shown that he has a clinical effect, such as autoimmune diseases, including rheumatoid arthritis and psoriasis.

In another aspect, the invention provides mutant molecules CTLA-4, including new soluble recombinant mutant fused proteins, CTLA-4-Ig, described here, which have the avidity of binding to CD86, which is greater than the avidity of binding of Belatacept (LEA29Y-Ig) to CD86. The invention also provides mutant molecules CTLA-4, including new soluble recombinant is utente fused proteins, CTLA-4-Ig. Described here are the avidity of binding to CD86, which is greater than the avidity of binding of Belatacept to CD86. In another aspect, the invention provides mutant molecules CTLA-4, including new soluble recombinant mutant fused proteins, CTLA-4-Ig, described here, which have a greater ability to suppress one or more immune responses (e.g., T-cell-dependent immune responses)than Belatacept. It is assumed that the mutant molecule CTLA-4 according to this invention having one or more improved properties compared to Belatacept are stronger than Belatacept, and, thus, are more powerful and thus more effective, suitable and advantageous than Belatacept, in the treatment of diseases, disorders or conditions in which immunosuppression is desirable, including such diseases, disorders or conditions for which it was shown that the protein Belatacept has a clinical effect, such as the viability of renal allograft in primates that are not human.

Safety, tolerance, pharmacokinetic properties, immunogenicity and clinical efficacy of a molecule according to this invention, such as mutant molecule CTLA-4 of the invention (for example, a mutant polypeptide, CTLA-4 EVA soluble or is th mutant protein, CTLA-4-Ig, which are described in detail below), the subject having an immune disease or disorder (such as rheumatoid arthritis, multiple sclerosis, psoriasis and the like), which impose specific dose of specific molecules by introducing (for example, parenteral, intravenous, or subcutaneous administration), can be determined using methods comparable to those used in clinical trials for Orencia®, using similar subjects. See, for example, addresses the world websites bms.com and orencia.com. For example, the extent to which the mutant molecule CTLA-4 of the invention (e.g., soluble mutant CTLA-4-Ig) are effective in reducing in subjects with rheumatoid arthritis (RA) progression of joint damage, weakening of the signs and symptoms of RA, including reduction of pain, can be estimated using methods similar to those that apply for Orencia® clinical trials using RA patients.

Safety, tolerance, pharmacokinetic properties, immunogenicity and clinical efficacy of a molecule according to this invention (for example, a mutant polypeptide, CTLA-4 EVA or soluble mutant fused protein, CTLA-4-Ig, as described in detail below) of the entity for which the desirable immunosuppression (e.g., the entity in which the transplanted tissue, cell, organ or tissue or organ from a donor) and to the concerning specific dose being specific by introducing (for example, parenteral, intravenous, or subcutaneous administration), can be determined using methods comparable to those used in clinical trials for Belatacept using similar subjects. See, for example, address to the world web site bms.com. For example, the extent to which the mutant molecule CTLA-4 of the invention (e.g., soluble mutant CTLA-4-Ig) effective in reducing transplant rejection kidney or renal transplant patient-recipient, which produced kidney transplant or kidney transplant may be estimated using techniques similar to those that apply to clinical research Belatacept using patients undergoing a kidney transplant or kidney transplant.

Molecules and methods according to this invention and other aspects of the invention are discussed in more detail below.

The polypeptides according to this invention

This invention provides new polypeptides having the common name "polypeptides according to this invention. It is implied that the expression of the polypeptides according to this invention includes variants and/or derivatives of the amino acid sequences described in this description. The polypeptides according to this invention include recombinant, not naturally occurring or mutant polypeptides, CTLA-4, to the verge bind CD80 and/or CD86 and/or which inhibit or suppress immune responses. The polypeptides according to this invention include recombinant fused proteins, including mutant polypeptide CTLA-4 according to this invention, and include Monomeric and dimeric forms of such fused proteins. The polypeptides according to this invention include multimer comprising one or more mutant polypeptides, CTLA-4 according to this invention. The invention also includes conjugates comprising one or more mutant polypeptides, CTLA-4 according to this invention. Some polypeptides according to this invention are soluble polypeptides. For example, as described in more detail below, the invention includes a soluble, fused proteins, including mutant polypeptide CTLA-4 EVA associated with different polypeptide (such as, for example, immunoglobulin polypeptide, such as, for example, Ig Fc polypeptide), which increases the solubility of the mutant polypeptide, CTLA-4 EVA.

As discussed in more detail below, in one aspect of the invention a variety of strategies mutagenesis and screening are used for education and identify new proteins that bind CD80 and/or CD86. In particular, such strategies are used for education and identify new polypeptides having improved ability to bind CD80 and/or CD86, including new mutant polypeptides, CTLA-4 with improved binding Affi the activity or the avidity to CD80 and/or CD86. The polypeptides according to this invention, which bind CD80 and/or CD86 ligands expressed on antigen-presenting cells affect or block the interaction of these ligands with CD28 receptors expressed on T-cells. In the T-cell co-stimulatory signal transmission, which is provided by the interaction of the receptor T-cell surface CD28 with B7 molecules (namely, CD80 and CD86), inhibited or blocked. Assume that such polypeptides suitable for prophylactic and therapeutic treatment of diseases, disorders and conditions in which modulation of the immune system (for example, T-cell responses) is preferred.

Mutant polypeptides, CTLA-4

In one aspect, the invention provides isolated or recombinant polypeptides, each of which includes an amino acid sequence that has at least, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 90,5%, 91%, 91,5%, 92%, 92,5%, 93%, 93,5%, 94%, 94,5%, 95%, 95,5%, 96%, 96,5%, 97%, 97,5%, 98%, 98,5%, 99%, 99,5% or 100% sequence identity with at least one amino acid sequence selected from the group comprising SEQ ID NOS:1-73 (for example, SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, and SEQ ID NO:73), where the polypeptide binds CD80 and/or CD86 or polypeptide fragment of CD80 and/or CD86 (or EVA each or both), and/or modulates or regulates the immune response. Some such polypeptides, such as each of the following in SEQ ID NOS:1-73, described as secreted or Mature mutant polypeptides, CTLA-4 EVA. Mutant polypeptides, CTLA-4 EVA listed in each of SEQ ID nos:1-73, do not include the signal peptide; he otsaila during processing, thereby forming a Mature or secretory polypeptide. Polypeptide fragment CD80 may contain, for example, the polypeptide extracellular domain of CD80 polypeptide, such as, for example, human CD80 EVA polypeptide ("hCD80 EVA"). Polypeptide fragment CD86 may contain, for example, the polypeptide extracellular domain of CD86 polypeptide, such as, for example, human CD86 EVA polypeptide ("hCD86 EVA"). Some such polypeptides bind CD80 and/or CD86 mammals or polypeptide fragments, such as, for example, CD80 go CD86 EVA m is capitalship. Some such polypeptides bind the human CD80 DT ("hCD80 and/or human CD86 DT ("hCD86") or polypeptide fragment, such as, e.g., hCD80 EVA or hCD86 EVA. In some such methods, at least one immune response is suppressed or inhibited.

Some such polypeptides have a binding affinity of or avidity for hCD80 or its fragment (e.g., hCD80 EVA), which is at least approximately equal to or greater than the binding affinity of or avidity of the polypeptide hCTLA-4 EVA to hCD80 or its fragment, respectively. The estimated full-hCD80 amino acid sequence that includes a signal peptide, EVA, transmembrane domain and cytoplasmic domain that is covalently linked together in this order, is presented in SEQ ID NO:195. The signal peptide comprises amino acid residues 1-34, EVA includes amino acid residues 35-242, transmembrane domain comprises amino acid residues 243-263, and cytoplasmic domain includes amino acid residues 264-288 SEQ ID NO:195. Amino acid sequence hCD80 EVA shown in SEQ ID NO:174. The sequence of the nucleic acid represented in SEQ ID NO:173, encodes DT human CD80 signal peptide (N-end) and human CD80 EVA.

Some such polypeptides have a binding affinity of or avidity for hCD86 or its fragment is (for example, hCD86 EVA), which is at least approximately equal to or greater than the binding affinity of or avidity of the polypeptide hCTLA-4 EVA to hCD86 or its fragment (e.g., EVA), respectively. The estimated full amino acid sequence hCD86, which includes the signal peptide, EVA, transmembrane domain and cytoplasmic domain that is covalently linked together in the order presented in SEQ ID NO:175, and illustrative nucleic acid encoding the estimated full hCD86 amino acid sequence shown in SEQ ID NO:176. Amino acid sequence hCD86 EVA shown in SEQ ID NO:180. Some such polypeptides have a binding affinity of or avidity for hCD86, which is at least approximately equal to or greater than the binding affinity of or avidity of the polypeptide LEA29Y EVA, having the sequence represented in SEQ ID nos:168 to hCD86. Illustrative polypeptides according to this invention, which have a binding affinity or avidity of binding to hCD86 or hCD86 EVA, which is at least equal to or greater than those for hCTLA-4 EVA and LEA29Y EVA (also called "A29YL104E" or "L104EA29Y" KJV) to hCD86 or hCD86 EVA, respectively, include, for example, but not limited to the following, those that involve amino acid sequence having at least, 95%, 96%, 97%, 98%, 9% or 100% sequence identity with a sequence of any of SEQ ID NOS:4, 10-12, 15, 17, 24, 26, 28, 35 and 61. See, for example, table 5 in example 4. The data presented in table 5 reflect the Monomeric interaction between representative CTLA-4-Ig and Monomeric CD86 EVA. The expression of LEA29Y (or A29YL104E, or L104EA29Y), unless specified to the contrary, refers to LEA29Y EVA (or A29YL104E, or L104EA29Y KJV).

Some such polypeptides include an amino acid sequence having amino acid length, approximately equal to the amino acid long extracellular domain of human CTLA-4. Such polypeptides can be described as mutant polypeptides, CTLA-4 EVA. Some such mutant polypeptides, CTLA-4 EVA include amino acid sequence that is from about 110 to about 138 amino acid residues, from about 112 to about 136 amino acid residues, from about 114 to about 134 amino acid residues, from about 116 to about 132 amino acid residues, from about 118 to about 130 amino acid residues, from about 119 to about 129 amino acid residues, from about 120 to about 128 amino acid residues, from about 121 to about 127 amino acid residues, from about 122 to about 126 amino acid residues, from about 123 to about 125 amino acid residues in length. Some such mutant polypeptides, CTLA-4 EVA include a sequence of length 124 amino acid residues. Illustrative polypeptides include, for example, is not limited to the following polypeptide, comprising amino acid sequence having at least, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with at least one amino acid sequence selected from the group comprising SEQ ID NOS:1-73, where the polypeptide binds CD80 and/or CD86 (or EVA each or both).

Some such polypeptides described above, including, for example, the isolated or recombinant polypeptides, each of which includes an amino acid sequence having at least, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,5% or 100% sequence identity with at least one amino acid sequence selected from the group of SEQ ID NOS:1-73, and which binds CD80 and/or CD86 and/or EVA, have the ability to modulate or regulate the immune response. One or more of a variety of immune responses can be modulated or regulated such polypeptides according to this invention, including, but not limited to the following, for example, T-cell activation or proliferation, cytokine synthesis or production of cytokine (e.g., production of TNF-α (tumor necrosis factor alpha), IFN-γ (IFN-γ), IL-2 (interleukin-2), etc.), induction of various markers of antiquarii (e.g., CD25, IL-2 receptor and the like), the synthesis or production of inflammatory molecules, inflammation, swelling of the joint, painful joints, pain, stiffness, levels of serum C-reactive protein, products anticollagen antibodies and/or T-cell-dependent(s) response(s) of antibodies). In some cases, this polypeptide has a greater ability to suppress or inhibit at least one immune response than the hCTLA-4 or hCTLA-4 EVA.

For example, some such polypeptides are capable of inhibiting T-cell activation or T-cell proliferation in in vitro tests. Examples 4-9 below, for example, show the ability of representative fused protein according to this invention, including representative mutant amino acid sequence of CTLA-4 EVA, such as those described herein, to inhibit T-cell proliferation in vitro. Some such polypeptides can inhibit or suppress the immune response in the subject in vivo, for example, through the introduction of a therapeutically or prophylactically effective amount of at least one such polypeptide to a subject in need of immunosuppressive therapy. Assume that some such polypeptides suitable for various applications, including, for example, but not limited to the following: preventive and/or therapeutic methods for treating diseases, disorders and conditions of the immune system, in which immunomodulation is lateley, as described in more detail below. It is expected that such polypeptides useful in the prophylactic and/or therapeutic methods for inhibiting or suppressing an immune response in a subject (e.g., in vivo treatment of diseases or disorders of the immune system of mammals, such as humans, in which immunoinhibitory or immunosuppression is desirable), the methods for inhibiting rejection of a tissue or organ transplanted from a donor to a recipient (e.g., a mammal, such as, for example, human), and other methods described in this description.

Additionally or alternatively, some such polypeptides have the ability to suppress or inhibit an immune response, which is at least approximately equal to or greater than the capacity of the hCTLA-4 or hCTLA-4 EVA to suppress or inhibit one or more types of immune responses. For example, some such polypeptides have the ability to inhibit T-cell activation or proliferation in vitro and/or in vivo assays and/or applications, such as those described above and in more detail below, which is at least approximately equal to or greater than the capacity of the hCTLA-4 or hCTLA-4 EVA to inhibit T-cell activation or proliferation in such applications. Additionally, some such polypeptides have the ability inhib is to be encoded or to suppress the immune response (for example, T-cell activation or proliferation, cytokine production, T-cell-dependent antibody response), which is greater than the ability of the polypeptide LEA29Y - specific mutant CTLA-4 EVA, comprising the amino acid sequence represented in SEQ ID NO:168, to inhibit or suppress an immune response. Examples 4-9 below, for example, compare the ability of representative fused protein of the invention includes mutant amino acid sequence of CTLA-4 EVA in this invention, to inhibit T-cell proliferation in vitro concerning the ability of the fused protein comprising a polypeptide hCTLA-4 EVA or LEA29Y, to inhibit T-cell proliferation in vitro. Assume that such molecules are effective for use for various therapeutic purposes, including the treatment of autoimmune diseases and disorders, and prophylactic and therapeutic methods for inhibiting rejection of an organ transplant, the cells or tissue.

Some such polypeptides may differ from any other, for example, by amino acid deletion, addition and/or replacement. Amino acid substitutions may be conservative or non-conservative substitution. See, for example, the section called "the Variability of the sequence.

In another aspect, the invention also provides the t isolated or recombinant polypeptides, each of which includes an amino acid sequence having at least, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with at least one amino acid sequence selected from the group comprising SEQ ID NOS:1-73, where the polypeptide binds Monomeric hCD80 Monomeric or hCD86 or EVA of each or both. Some such polypeptides have (1) the affinity of binding to Monomeric hCD86, which is almost equal to or greater than the binding affinity of Monomeric polypeptide hCTLA-4 or LEA29Y to Monomeric hCD86 or EVA, and (2) the affinity of binding to Monomeric hCD80, which is almost equal to or greater than the binding affinity of Monomeric hCTLA-4 to Monomeric hCD80. Polypeptide LEA29Y includes the amino acid sequence of SEQ ID NO:168. Some such polypeptides have a greater ability to suppress one or more immune responses described here (for example, T-cell activation/proliferation, synthesis/cytokine production, induction of activation markers, production of inflammatory molecules, inflammation, products anticollagen AT, T-cell-dependent AT the answer)than Monomeric hCTLA-4, Monomeric hCTLA-4 EVA or LEA29Y polypeptide.

The invention also provides isolated or recombinant polypeptides, each of which includes an amino acid sequence having, hence, is her least 95%, 96%, 97%, 98%, 99% or 100% sequence identity with at least one amino acid sequence selected from the group comprising SEQ ID NOS:1-73, where the polypeptide has a binding affinity of for hCD86 EVA or hCD80 EVA, which is almost equal to or greater than the binding affinity of hCTLA-4 EVA to hCD86 EVA or hCD80 EVA respectively. Some such polypeptides have an affinity binding to hCD86 EVA, which is greater than the binding affinity of the polypeptide hCTLA - 4 EVA (SEQ ID NO:159) or LEA29Y (SEQ ID NO:168) to hCD86 EVA. Some such polypeptides have an affinity binding to hCD80 EVA, which is greater than the binding affinity of hCTLA-4 EVA to hCD80 EVA. Some such polypeptides have the ability to suppress the immune response, in some cases, a greater ability to suppress one or more immune responses, including those described above and elsewhere in the description, the hCTLA-4 EVA or LEA29Y polypeptide.

In another aspect, the invention provides a variant of an isolated or recombinant CTLA-4 polypeptide comprising the amino acid sequence which (a) differs from the amino acid sequence of human CTLA-4 EVA presented in SEQ ID NO:159 in no more than 15 amino acid residues, no more than 14 amino acid residues, no more than 13 amino acid residues, no more than 12 amino acid residues, non-b is more than 11 amino acid residues, no more than 10 amino acid residues, no more than 9 amino acid residues, no more than 8 amino acid residues, no more than 7 amino acid residues, no more than 6 amino acid residues, no more than 5 amino acid residues (for example, not more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 amino acid residues), and (b) where the amino acid residue in position 24, 30, 32, 39, 41, 50, 54, 55, 56, 64, 65, 70, 85, 104 or 106 hCTLA-4 EVA amino acid sequence (SEQ ID NO:159) is substituted by another amino acid residue in the sequence variants of CTLA-4 polypeptide, where the position of the amino acid residues variants of CTLA-4 polypeptide numbered according to SEQ ID NO:159, and where the option of CTLA-4 polypeptide has an ability to bind CD80 or CD86 or an extracellular domain or fragment of each and/or has the ability to suppress or inhibit an immune response. Some of these options include one or more (e.g. one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen) of amino acid substitutions selected from the group comprising AU, A24S, S25A, G27H, K28N, T30N, T30D, TA, V32I, Q39K, Q39E, D41G, D41N, D41S, AM, A50G, M54K, ME, M54V, G55E, G55K, N56D, D63K, S64P, I65S, I65F, I65T, S70F, MA, M85V, MA, L104E, L104D, and I106M, I106F and I106L.

In another aspect, the invention provides isolated or recombinant mutant CTLA-4 polypep the ID, comprising the amino acid sequence which (a) differs from the amino acid sequence of the extracellular domain of human CTLA-4, is presented in SEQ ID NO:159, no more than 12 amino acid residues (for example, not more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid residues), and (b) includes two, three, four, five, six, seven, eight, nine, ten, eleven, or 12 amino acid substitutions selected from the group comprising amino acid position corresponding to amino acid positions 24, 30, 32, 50, 54, 55, 56, 64, 65, 70 and 104 of SEQ ID NO:159, where the amino acid position of the mutant polypeptide, CTLA-4 are numbered in accordance with SEQ ID NO:159 and where the mutant polypeptide, CTLA-4 has the ability to bind CD80 or CD86 or an extracellular domain or fragment of each, and/or has the ability to suppress or inhibit an immune response. Illustrative amino acid substitutions at these positions include, but are not limited to the following, conservative amino acid substitutions of amino acid residues represented in DT hCTLA-4 EVA and/or A24S/E (that is, A24S or AE), T30N/D/A (that is, T30N, or T30D, or TA), V32I/L/M/V, A50M/G, M54E/V/K, G55E/K/R, N56D/A, S64P/M/C, I65S/T/F/V, S70F/Y/W, L104E/M or any combination of these substitutions.

In another aspect, the invention provides isolated or recombinant polypeptides (e.g., mutant polypeptides, CTLA-4 EVA), each of which including the AET amino acid sequence, (a)that differs from the amino acid sequence selected from the group comprising SEQ ID NOS:1-73 by not more than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residue(s), and (b) where the amino acid residue in the amino acid sequence in the amino acid residue position 41, 50, 54, 55, 56, 64, 65, 70 or 85 is identical to the amino acid residue in the corresponding position of the specified selected amino acid sequence (e.g.the polypeptide selected from SEQ ID NOS:1-73), where the polypeptide binds CD80 and/or CD86 and/or the extracellular domain of each or both, and/or inhibits an immune response(s). In some cases, the polypeptide differs from the selected polypeptide (e.g., selected from SEQ ID NOS:1-73) by not more than 10, 9, 8, 7, or 6 amino acid residues (for example, not more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues), but the amino acid occupying one or more of the provisions of the amino acid residues 41, 50, 54, 55, 56, 64, 65, 70, and 85, an identical amino acid residue included in this position in the selected amino acid sequence (e.g., selected from SEQ NOS:1-73), and is not deleted or replaced by another amino acid. Some such polypeptides include an amino acid sequence that differs from the selected amino acid sequence at no more than 10, 9, 8, 7, or 6 amino acid residues (the example not more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues that includes the amino acid residues at one or several positions of amino acid residues 24, 30, 32, 41, 50, 54, 55, 56, 64, 65, 70, 85, 104 and 106 that are identical with amino acid residues in the corresponding positions in the selected amino acid sequence. Such polypeptides may differ from the selected amino acid sequence at the amino acid deletion(s), addition(s) and/or amino acid substitution(s) in position(s)that are not characterized by the fact that have the amino acid that is identical to the one that is in the selected sequence. Such polypeptides having binding affinity of or avidity for hCD86 or its fragment (e.g., hCD86 EVA), which is at least approximately equal to or greater than the binding affinity of or avidity of the polypeptide hCTLA-4 EVA or LEA29Y to hCD86 or its fragment (e.g., EVA), respectively, are included. Some such polypeptides have a binding affinity of or avidity for hCDSO or its fragment (for example, hCDSO EVA), which is at least approximately equal to or greater than the binding affinity of or avidity of the polypeptide hCTLA-4 EVA to hCD80 or its fragment, respectively. Some such polypeptides include an amino acid sequence having a length approximately equal to aminokislot is th length of the hCTLA-4 EVA for example, 118 to 130, 119-129, 120-128, 121-127, 122-126, 123-125, or 124 the amino acid residue in length.

Some such polypeptides capable of inhibiting one or more of a variety of immune responses, including, for example, T-cell activation, T-cell proliferation, synthesis or production of cytokine (e.g., production of TNF-α, IFN-γ, IL-2), induction of activation markers (e.g., CD25, IL-2 receptor), inflammation, production of inflammatory molecules, products anticollagen AT, and/or T-cell-dependent AT the answer(s)). Some such polypeptides have a greater ability to inhibit one or more of these immune responses than the polypeptide hCTLA-4, hCTLA-4 EVA or polypeptide LEA29Y. For example, some such polypeptides are capable of inhibiting T-cell activation or T-cell proliferation in in vitro tests. Examples 4-9, for example, compare the ability of representative fused protein according to this invention includes mutant amino acid sequence of CTLA-4 EVA this invention to inhibit T-cell proliferation in vitro concerning the ability of the fused protein comprising a polypeptide hCTLA-4 EVA or LEA29Y, do it. Some such polypeptides can inhibit or suppress the immune response in the subject in vivo, namely through the introduction of a therapeutically or prophylactically effective amount of at least one that is on the polypeptide to the subject, in need of immunosuppressive therapy. Assume that such polypeptides suitable for various applications, including, for example, but not limited to, prophylactic and/or therapeutic treatment of diseases, disorders and conditions of the immune system, in which the suppression of immune response is desired, including, for example, a prophylactic and/or therapeutic treatment of autoimmune diseases and disorders, methods of inhibiting rejection of a tissue or organ transplanted from a donor to a recipient (e.g., a mammal, such as, for example, human), and other methods described in this description.

In another aspect, the invention provides isolated or recombinant polypeptides (e.g., mutant polypeptides, CTLA-4 EVA), each of which includes an amino acid sequence which (a) differs from the amino acid sequence of the extracellular domain of human CTLA-4, is presented in SEQ ID NO:159 in no more than 10 amino acid residues, no more than 9 amino acid residues, no more than 8 amino acid residues, no more than 7 amino acid residues, no more than 6 amino acid residues, no more than 5 amino acid residues, no more than 4 amino acid residues, no more than 3 amino acid residues, no more than 2 amino is slotnum balances or no more than 1 amino acid residue (e.g., not more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues), and (b) includes at least one amino acid substitution in position amino acid residue corresponding to position 50, 54, 55, 56, 64, 65, 70 or 85 relative to the amino acid sequence of SEQ ID NO:159, where the polypeptide binds hCD80 and/or hCD86 and/or EVA each or both, and/or inhibits an immune response (e.g., T-cell activation or proliferation, synthesis or production of cytokine (e.g., production of TNF-α, IFN-γ, IL-2), induction of activation markers (e.g., CD25, IL-2 receptor), inflammation, production of inflammatory molecules, products anticollagen AT and/or T-cell-dependent AT the answer), such as in vitro and/or in vivo methods and/or analyses, as described in more detail below. Some such polypeptides include an amino acid sequence with a length of 124 amino acid residues. Some such polypeptides include 2, 3, 4, 5 or 6 amino acid substitutions in positions relative to the amino acid sequence represented in SEQ ID NO:159, selected from the group including the position of the amino acid residues 50, 54, 55, 56, 64, 65, 70 and 85. Some such polypeptides include the amino acid substitution at position amino acid residue corresponding to position 104 and/or 30 relative to SEQ ID NO:159. Some such polypeptides include at least one of amino the PCI-e slot substitution relative to SEQ ID NO:159 in position 70 (optional S70F), position 64 (optional S64P), position 50 (optional A50M/G, for example, AM, A50G), position 54 (optional M54K/V, for example, MK), the position 65 (optional I65S), position 56 (optional N56D), position 55 (optional G55E/K, for example, G55E, G55K), position 85 (optional MA) and/or position 24 (optional A24E/S, for example, AE). Any such polypeptide can include amino acid substitution relative to SEQ ID NO:159 in position 104 (optional L104E/D, for example, L104E), position 30 (optional T30N/D/A, for example, T30N, T30D or TA) and/or position 32 (optional V32I). In some cases, the polypeptide comprises one or more substitutions in the amino acid position is relative to SEQ ID NO:159 selected from the group comprising AL, M54K, G55E, N56D, S64P, I65S and S70F. Some such polypeptides exhibit the affinity of binding to CD86 (e.g., hCD86) or CD86 EVA (e.g., hCD86 EVA), which is almost equal to or greater than the binding affinity of Monomeric hCTLA-4 EVA to or CD86 CD86 EVA respectively. Some such polypeptides exhibit the affinity of binding to CD80 (e.g., hCD80) or CD80 EVA (e.g., hCD80 EVA), which is greater than the binding affinity of Monomeric hCTLA-4 EVA to CD80 CD80 or EVA, respectively.

Some such polypeptides have the ability to suppress or inhibit one or more immune responses (e.g., T-cell activation or p is alifereti, production of the cytokine and the like), such as in vitro and/or in vivo. Some such polypeptides inhibit one or more of these immune responses to a greater extent than the polypeptide hCTLA-4, hCTLA-4 EVA or LEA29Y. It is assumed that such polypeptides have an effective application in various therapeutic applications, including preventive and/or therapeutic treatment of autoimmune diseases, disorders or preventive and/or therapeutic methods for inhibiting rejection of an organ transplant, the cells or tissue.

In another aspect, the invention provides isolated or recombinant polypeptides (e.g., mutant polypeptides, CTLA-4 EVA), each of which includes an amino acid sequence which (a) differs from the amino acid sequence of the extracellular domain of human CTLA-4, is presented in SEQ ID NO:159, no more than 6 amino acid residues (for example, not more than 1, 2, 3, 4, 5 or 6 amino acid residues), and (b) includes one or more substitutions at amino acid positions relative to SEQ ID NO:159, selected from groups, including AM, M54K, G55E, N56D, S64P, I65S and S70F, where the polypeptide binds hCD80 and/or hCD86 or an extracellular domain of each or both, and/or inhibits an immune response (e.g., T-cell activation or proliferation, synthesis or production of the cytokine, range of complete the Oia markers of activation, production of inflammatory molecules, inflammation, joint swelling or tenderness, pain, stiffness, levels of serum C-reactive protein, products anticollagen AT and/or T-cell-dependent AT the response and the like) in in vitro studies and/or in vivo methods. Assume that such polypeptides suitable for treatment of a subject suffering from diseases, disorders or conditions in which immunosuppressive therapy would be effective, including, for example, therapeutic and prophylactic methods of treatment of autoimmune diseases and disorders and prophylactic and therapeutic methods of inhibiting transplant rejection of organ, cell or tissue.

The invention also includes isolated or recombinant polypeptide that includes an amino acid sequence comprising (i)at least, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any amino acid sequence selected from the group comprising SEQ ID NOS:1-73, and (ii) the phenylalanine residue at amino acid position corresponding to position 70 of the specified amino acid sequence selected from the group comprising SEQ ID nos:1-73, where the polypeptide binds hCD80 and/or hCD86 or its extracellular domain and/or inhibits an immune response (e.g., T-cell activation or proliferation, synthesis or production of cyto is in, induction of activation markers, production of inflammatory molecules, inflammation, joint swelling or tenderness, pain, stiffness, levels of serum C-reactive protein, products anticollagen AT and/or T-cell-dependent AT the response and the like) in in vitro studies and/or in vivo methods. Some such polypeptides include one or more of the following with respect to the selected sequence: a glutamic acid residue at amino acid position corresponding to position 24; an asparagine residue at amino acid position corresponding to position 30; the isoleucine residue at amino acid position corresponding to position 32; methionine residue at the amino acid position corresponding to position 50; lysine residue in amino acid position corresponding to position 54; a glutamic acid residue at amino acid position corresponding to position 55; the aspartic acid residue at amino acid position corresponding to position 56; a Proline residue at amino acid position corresponding to position 64; a serine residue at amino acid position corresponding to position 65; and the glutamic acid residue at amino acid position corresponding to position 104. Assume that such polypeptides have an effective application in different fields of application, VK is UCA methods of treatment of autoimmune diseases and disorders, and methods of inhibiting rejection of an organ transplant, cells or tissue.

For example, in one non-limiting aspect, the invention includes isolated or recombinant polypeptide (e.g., mutant CTLA-4 EVA), which includes the amino acid sequence comprising (i)at least, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence SEQ ID NO:24, and (ii) the phenylalanine residue at amino acid position corresponding to position 70 of the amino acid sequence of SEQ ID NO:24, where the polypeptide binds hCD80 and/or hCD86 and/or EVA each or both, and/or inhibits an immune response in vitro and/or in vivo. The polypeptide can include at least one of the following relative to SEQ ID NO:24: glutamic acid residue at position 24; an asparagine residue at position 30; the isoleucine residue at position 32; methionine residue in position 50; the lysine residue in position 54; a glutamic acid residue in position 55; the aspartic acid residue in position 56; a Proline residue at position 64; a serine residue at position 65; and a glutamic acid residue in position 104.

The invention also includes isolated or recombinant polypeptide (e.g., a mutant polypeptide, CTLA-4 EVA), which binds hCD80 and/or hCD86 and/or EVA each or both) and/or inhibits an immune response (as described above), for example, in vitro and/or in vivo, where the polypeptide includes AMI is kislotno sequence, which (a) differs from the amino acid sequence of human CTLA-4 EVA polypeptide presented in SEQ ID NO:159 in no more than 6 amino acid residues, and (b) includes at least one amino acid substitution, where indicated, at least the amino acid substitution includes S70F, where the position of the amino acid residues are numbered according to SEQ ID NO:159. The polypeptide can also include at least one amino acid substitution selected from the group comprising AU, T30N, V32I, D41G, AM, M54K, G55E, N56D, S64P, I65S, M85A, L104E and I106F. Assume that such polypeptides have an efficient use in various applications, including the treatment of autoimmune diseases, and methods of inhibiting transplant rejection of organ, cell or tissue.

The invention also includes isolated or recombinant polypeptide comprising amino acid sequence which (a) differs from the amino acid sequence represented in SEQ ID NO:31 for no more than 6 amino acid residues, and (b) includes at least one of the following: a methionine residue at the position corresponding to position 50 of SEQ ID NO:31, a lysine residue at the position corresponding to position 54 of SEQ ID NO:31, a glutamic acid residue in the position corresponding to position 55 of SEQ ID NO:31, a Proline residue at the position equivalent is position 64 of SEQ ID NO:31, the serine residue at the position corresponding to position 65 of SEQ ID NO:31), the phenylalanine residue at the position corresponding to position 70 of SEQ ID NO:31, where the position of the amino acid residues are numbered according to SEQ ID NO:31, and the polypeptide binds CD80 and/or CD86 and/or EVA each or both, and/or inhibits an immune response, as described above in vitro and/or in vivo. The polypeptide can include a glutamic acid residue in the position corresponding to position 104, the aspartic acid residue in the position corresponding to position 30 and/or the isoleucine residue at the position corresponding to position 32 of SEQ ID NO:31. Assume that such polypeptides have an efficient use in various applications, including the treatment of rheumatism and disorders, and methods of inhibiting transplant rejection of organ, cell or tissue.

In another aspect, the invention provides isolated or recombinant polypeptide that includes an amino acid sequence comprising (i)at least, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence selected from the group comprising SEQ ID NOS:36-46 and 55, and (ii) a glutamic acid residue at amino acid position 55 of the specified selected amino acid sequence, where the polypeptide binds CD80 and/or CD86 or an extracellular house is each or both and/or suppresses an immune response. Immune responses, which can be suppressed include, for example, T-cell activation or proliferation, synthesis or production of cytokine (e.g., production of TNF-α, IFN-γ, IL-2), induction of activation markers (e.g., CD25, IL-2 receptor), inflammation, production of inflammatory molecules, products anticollagen AT and/or T-cell-dependent AT the answer). This amino acid sequence can also include a phenylalanine residue at amino acid position 70. This amino acid sequence can also include a Proline residue at position 64 and/or the asparagine residue in position 30. This amino acid sequence can also include a methionine residue in position 50 and/or the lysine residue in position 54.

In another aspect, the invention provides isolated or recombinant polypeptide that includes an amino acid sequence comprising (i)at least, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence selected from the group comprising SEQ ID NOS:28, 30, 36-46, 55-57 and 65-73, and (ii) a glutamic acid residue at amino acid position 55 of the specified selected amino acid sequence, where the polypeptide binds CD80 and/or CD86 or an extracellular domain of each or both and/or suppresses an immune response such as T-cell activate is or proliferation, the synthesis or production of cytokine (e.g., production of TNF-α, IFN-γ, IL-2), induction of activation markers (e.g., CD25, IL-2 receptor), inflammation, production of inflammatory molecules, products anticollagen AT and/or T-cell-dependent AT the answer. This amino acid sequence can also include a phenylalanine residue at amino acid position 70. This amino acid sequence can also include a Proline residue at position 64 and/or the asparagine residue in position 30. This amino acid sequence can also include a methionine residue in position 50 and/or the lysine residue in position 54.

Any polypeptide according to this invention described above can also include a peptide that facilitates secretion of the specified polypeptide. Thus, in one aspect, the invention provides isolated or recombinant polypeptide comprising (a) any polypeptide, as described above (e.g., mutant CTLA-4 EVA described above), and (b) a peptide that facilitates secretion of the expressed polypeptide from the host cell. The peptide is optionally a signal peptide. With the end of the signal peptide is typically covalently linked to the N-end of the polypeptide. The signal peptide can include the amino acid sequence having at least, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the IDA is lichnosti sequence with amino acid sequence SEQ ID NO:182 or SEQ ID NO:216. The signal peptide can include the amino acid sequence having at least, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence comprising amino acid residues 1-35, 1-37 1-36 or SEQ ID NO:160.

Any polypeptide according to this invention described above may also include a transmembrane domain and/or cytoplasmic domain. Thus, in one aspect, the invention provides isolated or recombinant polypeptide comprising (a) any polypeptide according to this invention described above (e.g., mutant CTLA-4 EVA described above), and (b) the transmembrane domain. Such protein may optionally also include the signal peptide, as described above, where the end of the signal peptide covalently linked to the N-end of the polypeptide according to this invention. With the end of the signal peptide is typically covalently linked to the N-end of the transmembrane domain. With the end of the transmembrane domain is typically covalently linked to the N-end of the cytoplasmic domain. In some cases, the transmembrane domain comprises amino acid sequence having at least, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence comprising amino acid residues 162-182 SEQ ID NO:160. In some cases, the cytoplasmic domain includes but is inoculate sequence, with at least, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence comprising amino acid residues 183-223 SEQ ID NO:160. Any of the above polypeptides may include one or more amino acid residues that are glycosylated or tahilramani.

The invention also includes multimer isolated or recombinant polypeptide comprising two or more polypeptides, where at least one of the polypeptides multimer is a mutant polypeptide, CTLA-4 according to this invention, as described herein (e.g., mutant CTLA-4 EVA or mutant CTLA-4-Ig). This multimer includes at least one polypeptide according to this invention and may also contain at least one additional polypeptide, which should not be a polypeptide according to this invention. For example, multimer may include at least one polypeptide according to this invention and at least one other polypeptide, which may be, for example, a wild-type polypeptide (e.g., hCTLA-4 EVA or hCTLA-4-Ig) and/or at least another mutant polypeptide (such as a mutant polypeptide that is not a polypeptide according to this invention). Some or all of the polypeptides in multimer (or the multimeric polypeptide) can be identical to each other, or in some the cases that all polypeptides in multimer may differ from each other. In some cases, a polypeptide multimer is a dimer comprising two polypeptide according to this invention, which may not necessarily be identical polypeptides (i.e glycosilated) or different polypeptides (i.e. heterodimer). In some cases, a polypeptide multimer is a tetramer comprising four polypeptide according to this invention. The tetramer can include four identical polypeptide (i.e., homotetramer) or any combination of the four polypeptides according to this invention, where at least one polypeptide is not identical to the other three polypeptides (i.e., heterotetramer). The invention also includes a tetramer with four identical polypeptide DT CTLA-4 EVA (e.g., hCTLA-4 KJV) or four identical DT CTLA-4-Ig (e.g., hCTLA-4-Ig). In some cases multimer able to wasuwat CD80 and/or CD86 (or EVA each or both) and/or to suppress or inhibit an immune response in vitro and/or in vivo methods (e.g., T-cell proliferation or activation, cytokine production, and so on). Some of these multimeric have a greater ability to suppress or inhibit an immune response in vitro and/or in vivo than hCTLA-4 or hCTLA-4-Ig (e.g., hCTLA-4-IgG2 or protein Orencia®). The polypeptides of multimers can be connected together, for example, covalent bridges, for example, through a disulfide bond between the od of them or more cysteine residues in one or more polypeptides.

Some such tetramer in this invention include the structure, schematically similar to that of antibodies, but in which each of the variable domains of the antibody substituted on any of the mutant polypeptide, CTLA-4 EVA in this invention, are described here. Heavy chain of the antibody comprises the variable domain (VHheavy chain fused with the immunoglobulin (Ig) CH1 domain (e.g., IgG2 SN), which merged with the hinge. The hinge merged with Ig CH2 domain (e.g., IgG2 CH2), which merged with Ig CH3 domain (e.g., IgG2 CH3). Light chain of the antibody comprises the variable domain (VL) light chain, merged With Ig Kappa (Ck) or lambda (Cλ) domain. Two heavy chains and two light chains covalently linked together by one or more disulfide bonds formed by cysteine residues in the heavy and light chains. The invention includes mutant CTLA-4 tetramer in which each of the variable domains of the heavy and light chains are replaced by the mutant polypeptide, CTLA-4 EVA on this invention. Thus, the tetramer consists of two light chains and two heavy chains. Each light chain comprises a mutant polypeptide, CTLA-4 EVA, merged with IgKorλdomain. Each heavy chain comprises a mutant CTLA-4 EVA, merged with Ig CH1 domain (e.g., IgG2 CH1), which merged with the hinge. The hinge merged with Ig CH2 domain (e.g., IG2 CH2), which merged with Ig CH3 domain (e.g., IgG2 CH3). Two heavy chains and two light chains covalently linked together by one or more disulfide bonds formed by cysteine residues in the heavy and light chains. This tetramer can be described as a tetramer, CTLA-4-Ig. Methods for making such tetramer CTLA-4-Ig are known and will be understood by specialists in this field. Tetramer CD4-Ig construct, which includes CD4 polypeptide and which will neutralize primary isolates of HIV type 1 described in Allaway, G. P. et al., AIDS Res. Hum. Retroviruses 11(5):533-9 (1995). The tetramer can include four identical four mutant polypeptides, CTLA-4 EVA or any combination of the four mutant polypeptides, CTLA-4 EVA in this invention so that at least one mutant CTLA-4 EVA is not identical to the other three mutant polypeptides, CTLA-4 EVA. Some such tetramer able to bind CD80 and/or CD86 with greater avidity of binding than hCTLA-4 (or hCTLA-4-Ig). Some such tetramer able to suppress or inhibit the immune response; in some cases, this tetramer has a greater ability to suppress or inhibit an immune response in in vitro studies or in vivo applications (e.g., T-cell proliferation or activation, cytokine production, and so on)than hCTLA-4 or hCTLA-4-Ig (e.g., hCTLA-4-IgG2 or Orencia®). Assume that multimer filed with the invention are effectively used in various applications, including methods of treatment of autoimmune diseases and disorders, and methods of inhibiting transplant rejection of organ, cell or tissue.

The invention also includes isolated or recombinant conjugate multimer composed of two or more conjugates, where at least one of the conjugates is the conjugate according to this invention, which includes a mutant polypeptide, CTLA-4 in this invention (e.g., mutant CTLA-4 EVA or mutant CTLA-4-Ig). Some or all of the conjugates in multimer can be identical to each other, or all of the conjugates in multimer may differ from each other. In some cases, the conjugate of multimer is a dimer, consisting of two conjugate or a tetramer comprising four conjugate according to this invention. Some of these conjugate multimer able to bind CD80 and/or CD86 (or EVA each or both) and/or to suppress or inhibit an immune response in vitro and/or in vivo. Conjugate molecules in multimeric can be connected together, for example, covalent bridges, for example, by disulfide bonds between one or more cysteine residues in one or more conjugates.

The invention includes isolated or recombinant dimer of a polypeptide comprising any two of the polypeptide according to this invention described above (for example, m is tanty CTLA-4 EVA described above), where the dimer has the avidity of binding to human CD86 or an extracellular domain, which is almost equal to or greater than the avidity of binding of the dimer, consisting of two extracellular domain of human CTLA-4 to human CD86 or an extracellular domain, respectively.

The invention includes isolated or recombinant polypeptide dimer comprising two polypeptide according to this invention described above (e.g., mutant CTLA-4 EVA described above), where the dimer has the avidity of binding to hCD80 or EVA, which is almost equal to or greater than the avidity of binding of a dimer comprising two polypeptide hCTLA-4 EVA (SEQ ID NO:159), hCD80 or EVA, respectively. For example, in a non-limiting aspect of the dimer may comprise two polypeptide, where each polypeptide consists of amino acid sequence having at least, 95%, 96%, 97%, 98%, 99% or 100% identity with a sequence selected from the group comprising SEQ ID NOS:1-73, and each polypeptide binds hCD80 and/or hCD86 and/or inhibits an immune response. In another non-limiting aspect of the dimer may comprise two polypeptide, where each polypeptide differs from the amino acid sequence of hCTLA-4 EVA (SEQ ID NO:159) at not more than 6 amino acid residues and comprises at least one substitution in the amino acid for which ogenyi relative to SEQ ID NO:159, selected from the group comprising AL, M54K, G55E, N56D, S64P, I65S and S70F; the polypeptide also includes optional replacement L104E, and each polypeptide binds hCD80 and/or hCD86 and/or inhibits an immune response.

In some cases, the dimer has the avidity of binding to hCD80 or EVA, which is almost equal to or greater than the avidity of binding of a dimer comprising two polypeptide hCTLA-4 EVA, hCD80 or EVA, respectively. In some cases, the dimer has the avidity of binding to hCD86 or EVA, which is greater than the avidity of binding of a dimer comprising two polypeptide LEA29Y, hCD86 or EVA, respectively, where each polypeptide LEA29Y includes the amino acid sequence represented in SEQ ID NO:168. In some cases, the dimer dissociates from the binding hCD86 or EVA with a speed that is less than the rate at which the dimer comprising two polypeptide hCTLA-4 EVA, dissociates from the binding hCD86 or EVA, respectively. In some cases, the dimer dissociates from the binding hCD86 or EVA with a speed that is less than the rate at which the dimer comprising two polypeptide LEA29Y, dissociates from the binding hCD86 or EVA, respectively, where each polypeptide LEA29Y includes the amino acid sequence represented in SEQ ID NO:168.

In some cases, the dimer associates with hCD86 or EVA with what corotu, which is greater than the rate at which the dimer comprising two hCTLA-4 EVA polypeptide that associates with hCD86 or EVA, respectively. In some cases, the dimer associates with hCD86 or EVA with a speed which is greater than the rate at which the dimer comprising two LEA29Y polypeptide that associates with hCD86 or EVA, respectively, where each LEA29Y polypeptide includes the amino acid sequence represented in SEQ ID NO:168.

In some cases, this dimer comprising a mutant CTLA-4 EVA, has a greater ability to suppress the immune response (for example, T-cell activation or proliferation, cytokine production, and so on)than the dimer, consisting of two extracellular domain of human CTLA-4, or two polypeptide LEA29Y.

Some of these dimers have equilibrium dissociation constant CD86 (KD), which is less than the equilibrium dissociation constant CD86 (KD) dimer comprising two polypeptide hCTLA-4 EVA or two polypeptide LEA29Y. Some of these dimers have equilibrium dissociation constant CD86 (KD), which is less than the equilibrium dissociation constant CD86 (KD) dimer comprising two polypeptide LEA29Y, each polypeptide LEA29Y includes the amino acid sequence represented in SEQ ID NO:168.

Some of these multimer comprising at least two polypeptide according to this image the structure (for example, dimer comprising two mutant polypeptide CTLA-4 EVA in this invention have an enhanced ability to suppress the immune response compared to multibeam full-hCTLA-4 of the same valence (i.e multimer, including the same number of full-CTLA-4 polypeptide). Some of these multimer comprising at least two polypeptide according to this invention have an enhanced ability to suppress the immune response compared to multibeam hCTLA-4 ECCD the same valence (i.e multimer, including the same number of polypeptides hCTLA-4 KJV).

Any polypeptide according to this invention, described above, may also include additional amino acid sequence, which enhances solubility, such as immunoglobulin (Ig) amino acid sequence, thereby forming, for example, soluble protein, as discussed in more detail below. Each polypeptide polypeptide multimer may also include additional amino acid sequence, which enhances solubility, such as Ig amino acid sequence, thereby forming, for example, soluble protein. Thus, for example, each polypeptide dimer comprising two or more polypeptides according to this invention, as described above, may also include additional aminoxy the pilot sequence, which enhances solubility, such as Ig amino acid sequence, thereby forming, for example, soluble protein.

Assume that such polypeptides and the dimers of this invention are effective for use in various applications, including the treatment of autoimmune diseases and disorders, and methods of inhibiting transplant rejection of organ, cell or tissue.

As previously discussed, the sequence of the Mature protein hCTLA-4 usually starts with a methionine residue at amino acid position 38 of the full sequence of the protein hCTLA-4, is presented in SEQ ID NO:160, and amino acid residues of the sequence of the Mature protein hCTLA-4 usually numbered starting with this residue of methionine as the first amino acids (i.e. amino acid occupying position 1).

Some of the mutant polypeptides, CTLA-4 according to this invention (including Monomeric and dimeric fused proteins and multimeric polypeptides) include at least one amino acid substitution in amino acid position corresponding to amino acid position in the sequence of the Mature protein hCTLA-4, which is outside the classical hCTLA-4/hB7-2 binding region (see, for example, Schwartz et al., Nature 410:604-608 (2001)), including, but not limited to the following, for example, any of the amino acids what's provisions 24, 41, 54, 55, 56, 64, 65, 70 and 85. In General, the person skilled in the art could not assume that amino acid substitution in any of the above provisions(24, 41, 54, 55, 56, 64, 65, 70 and/or 85), or any combination of one or more substitutions selected from the group provisions 24, 41, 54, 55, 56, 64, 65, 70 and 85 will have the ability to enhance the binding affinity of or avidity hCTLA-4 to hB7-2, will have an enhanced ability to inhibit the interaction SV positive with B7-2-positive cells or will provide a greater ability to suppress or inhibit an immune response than, e.g., hCTLA-4 EVA or hCTLA-4-Ig (e.g., T-cell activation or proliferation, synthesis or cytokine production, induction of activation markers, the synthesis or production of inflammatory molecules, products anticollagen antibodies, T-cell-dependent antibody response and the like). Also a specialist in this area could not assume that a particular amino acid replacement(s) or a combination of specific amino acid substitutions described herein, in any of the above provisions(24, 41, 54, 55, 56, 64, 65, 70 and/or 85), or any combination of such provisions will have the ability to enhance the binding affinity of or avidity hCTLA-4 to hB7-2, to have an enhanced ability to inhibit the interaction SV positive with B7-2-positive cells or to provide greater capacity is dawlati or to inhibit the immune response, than, e.g., hCTLA-4 EVA or hCTLA-4-Ig.

Mutant CTLA-4 fused proteins

The invention also provides new isolated and recombinant fused proteins, which include the first polypeptide, which, at least, is one of the polypeptides according to this invention described above and elsewhere in this description (such as mutant polypeptide CTLA-4 in this invention, such as, for example, a mutant polypeptide, CTLA-4 KJV) is linked or fused to the second polypeptide, thereby forming a fused protein. The second polypeptide facilitates the secretion or expression of the first polypeptide. Illustrative of the mutant polypeptides, CTLA-4 EVA include those that include the sequence identified as SEQ ID NOS:1-73. This invention includes a fused proteins, including immunoglobulin (Ig) domains, such as Ig Fc domain fused or attached biologically active part of this invention, such as mutant polypeptides, CTLA-4 according to this invention. Assume that the fused protein according to this invention is suitable as a preventive and/or therapeutic agents for prophylactic and/or therapeutic treatment of various diseases, disorders and conditions of the immune system, in which immunomodulation and/or immunosuppression is effective, diagnostic studies, and prepared for what I medicaments or agents, having immunomodulatory and/or immunosuppressive activity or property, as described in more detail in this description.

Slit proteins according to this invention, comprising a mutant polypeptide, CTLA-4 and Ig polypeptide (e.g., Ig Fc), typically referred to as mutant fused proteins, CTLA-4-Ig. Any fused protein according to this invention, including Monomeric and dimeric fused protein according to this invention, described in more detail below and in the examples, may include as an Ig polypeptide, such as, for example, the polypeptide Ig Fc, as described here and everywhere in this invention above and below. The second polypeptide can be connected directly with the first polypeptide. For example, the N-end of the second polypeptide (e.g., Ig polypeptide, such as an Ig Fc polypeptide may be covalently fused directly with the end of the first polypeptide according to this invention (for example, a mutant polypeptide, CTLA-4 KJV). Alternatively, the second polypeptide may be connected indirectly with the first polypeptide in which amino acid linker sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more amino acid residues, is included between the first and second polypeptides. In cases where the linker is enabled, the N-end amino acid linker sequence is typically covalently fused with the end of the first polypeptide (e.g., mutant CTLA-4 EVA), and N is the end of the second polyp is putida (for example, Ig polypeptide, such as Ig Fc) usually covalently fused with the C-end amino acid linker sequence.

In some cases, the second polypeptide includes at least part of the Ig polypeptide, such as, for example, one or more domains of the Ig constant region of the heavy chain. The second polypeptide can include a hinge, CH2 domain and CH3 domain polypeptide Ig. In some cases, the second polypeptide comprises the Fc domain of the polypeptide Ig DT (namely, a polypeptide Ig Fc DT), such as, for example, the Fc domain of human polypeptide Ig DT (namely, human polypeptide Ig Fc DT). As discussed herein, Ig polypeptide can be of different types, including, for example, a mammal, e.g. human, mouse, Primate, non-human (e.g., monkey, gorilla), cat, dog, horse, etc. and may be from different classes (e.g., IgG, IgM, IgE and the like) and subclasses (for example, IgG include IgG1, IgG2, IgG4, and the like), and may include an Fc domain or portion of any such Ig polypeptide. Amino acid sequence and nucleic acid sequence of Ig polypeptides such various types known in the art.

In one aspect, the invention provides a new isolated or recombinant fused proteins, each of which includes an isolated or recombinant mutant polypeptide CTLA-4 on nomu the invention, described above (e.g., mutant CTLA-4 EVA), covalently linked or fused directly or indirectly (via an amino acid linker sequence)at its C-end N-end polypeptide Ig Fc, namely, the Fc domain of an Ig polypeptide. Any of the fused protein according to this invention, including Monomeric and dimeric mutant fused proteins, CTLA-4-Ig in this invention, described in more detail below and in the examples, can include a polypeptide Ig Fc, as described here and everywhere, above and below in this description. Polypeptide Ig Fc usually includes the hinge region, CH2 domain and CH3 domain polypeptide Ig. Polypeptide Ig Fc may occur from various species including, for example, human, mouse, Primate, and the like, and may include Ig Fc polypeptide wild-type (e.g., IgG1, IgG2 or IgG4 DT). Illustrative of the human IgG Fc polypeptides include, for example, but not limited to the following, human IgG1, human IgG2, human IgG4, etc. Amino acid sequence of illustrative human IgG1 Fc presented in SEQ ID NO:185. Amino acid sequence of human illustrative polypeptides IgG2 Fc is presented in SEQ ID NOS:184 and 218, respectively. Alternatively, the polypeptide Ig Fc may include mutant polypeptide Ig. For example, a mutant IgG1 Fc, in which one or more cysteine residues substituted for another amino acid (e.g., OS is atok serine), thereby removing one or more disulfide bonds formed between two Ig chains or in which one or more residues of Proline is substituted by another amino acid (e.g., Proline) to reduce effector functions (reduced Fc receptor binding)may be included in a mutant protein, CTLA-4-Ig. Amino acid sequence illustrative mutant polypeptide IgG1 Fc shown in SEQ ID NO:186. The invention includes an isolated or recombinant protein, such as mutant dimer CTLA-4-Ig or mutant CTLA-4-Ig monomer that includes at least one recombinant mutant polypeptide, CTLA-4, described above, is connected at its C-end N-end of the recombinant polypeptide Ig Fc, including amino acid sequence having at least, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence selected from the group comprising SEQ ID NOS:184 (human polypeptide IgG2 Fc), 185 (human polypeptide IgG1 Fc), 186 (mutant polypeptide IgG1 Fc) and 218 (human polypeptide IgG2 Fc without C-terminal lysine residue (K)).

In one aspect of the putative amino acid sequence of mutant fused protein, CTLA-4-Ig in this invention includes the following segments: the sequence of the signal peptide that facilitates secretion of the fused protein (the example signal peptide hCTLA-4 (SEQ ID NO:182 or SEQ ID NO:216)); a mutant polypeptide, CTLA-4 EVA, and the mutant polypeptide, CTLA-4 EVA typically, but not necessarily, includes from about 118 to 130 amino acid residues, and usually about 124 amino acid residues in length; and Ig Fc polypeptide. Illustrative of the mutant polypeptides, CTLA-4 EVA include those described above and below. In some cases, not included in any one amino acid linker sequence between the end of the mutant polypeptide, CTLA-4 EVA and N-end of the human polypeptide Ig Fc; that is, From the end of the mutant polypeptide, CTLA-4 EVA covalently fused directly to the N-end of the polypeptide Ig Fc in mutant fused protein, CTLA - 4-Ig. If necessary, however, mutant CTLA-4-Ig can include a linker (e.g., one or more amino acid residues) between the end of the mutant polypeptide, CTLA-4 EVA and N-end of the human polypeptide Ig Fc. The signal peptide of the proposed Monomeric mutant fused protein, CTLA-4-Ig in this invention is generally cleaved from the N-terminal mutant fused protein, CTLA-4 Ig during processing and, thus, Mature or secretory mutant fused protein, CTLA-4-Ig in this invention typically does not include the sequence of the signal peptide. Dimer fused protein comprising two Monomeric mutant fused protein, CTLA-4-Ig, usually formed during CL is accurate processing through the formation of covalent disulfide bonds between (1) cysteine residues in the mutant CTLA-4 EVA and IgG2 Fc one such monomer fused protein and (2) cysteine residues in the mutant CTLA-4 EVA and IgG2 Fc of the second (usually, but not necessarily identical) monomer fused protein.

This invention includes a fused dimeric proteins (also called dimer fused protein), each of which includes two Monomeric fused protein according to this invention. The dimer may comprise two identical or different Monomeric fused protein. Dimeric protein formed by means of connection(s) between the two Monomeric fused proteins. Dimeric protein comprising two Monomeric fused protein, usually formed during cellular processing through the formation of covalent disulfide bonds between cysteine residues in one monomer fused protein and cysteine residues in the second monomer fused protein. Thus, in some cases, mutant protein, CTLA-4-Ig in this invention is expressed as a dimer comprising two Monomeric fused protein according to this invention.

In one aspect, the invention provides isolated or recombinant dimeric mutant protein, CTLA-4-Ig, comprising two Monomeric fused protein, where each Monomeric protein comprises a mutant polypeptide, CTLA-4 EVA in this invention, as described in detail above and below, fused at its C-end with Ig Fc polypeptide. The dimer is formed during cellular processing through the formation of covalent di is olefinic linkages between cysteine residues in the mutant CTLA-4 EVA and Ig Fc one monomer fused protein and cysteine residues in the mutant CTLA-4 EVA and the second monomer fused protein Ig Fc. Two Monomeric fused protein is typically, but not necessarily, include an identical sequence. Secretiruema or Mature form of mutant fused protein, CTLA-4-Ig does not include the signal peptide, as the signal peptide is typically cleaved from the N-Terminus of the protein during processing. Estimated mutant protein, CTLA-4-Ig includes the signal peptide, the C-end is typically covalently linked to the N-end of the mutant protein, CTLA-4-Ig. N-end of each monomer Mature mutant fused protein, CTLA-4-Ig typically includes a methionine (M).

As a non-limiting example, the invention provides fused dimeric proteins composed of two Monomeric fused protein, CTLA-4-Ig, where each Monomeric mutant protein, CTLA-4-Ig includes a mutant polypeptide, CTLA-4 EVA, linked at its C-end N-end polypeptide Ig Fc, where the mutant polypeptide, CTLA-4 EVA includes an amino acid sequence selected from any of SEQ ID NOS:1-73. In some such fused dimeric proteins of two Monomeric fused protein covalently linked together by covalent disulfide bond. Formed during cellular processing between the cysteine residue at position 120 in each amino acid sequence of CTLA-4 mutant EVA. Alternatively, or additionally, two Monomeric fused protein covalently linked together by covalent disulfide tie the Yu, formed between one or more cysteine residues in the Ig Fc polypeptide of the first monomer fused protein and one or more cysteine residues in the Ig Fc polypeptide of the second monomer fused protein. Monomeric fused proteins can be joined together many disulfide bonds (e.g., one, two, three, four or more disulfide bonds), formed during cellular processing between cysteine residues present in their respective Ig polypeptide Fcx. In some cases, each Monomeric protein composed of the same polypeptide Ig Fc (for example, human IgG2 Fc, as shown in, for example, SEQ ID NO:184 or 218), and covalent disulfide bond(s) may be formed during cellular processing between cysteine residues in equivalent positions in each Ig Fc polypeptide.

Illustrative of the mutant polypeptide, CTLA-4 EVA is D3-12 mutant polypeptide CTLA-4 EVA, comprising the amino acid sequence of SEQ ID NO:11. Illustrative mutant protein, CTLA-4-Ig in this invention is D3-12 mutant polypeptide CTLA-4 EVA, covalently linked or fused directly (without a linker) at its C-end N-end of the human IgG2 Fc polypeptide presented in SEQ ID NO:218, thereby forming a fused protein D3-12-IgG2 presented in SEQ ID NO:205, or covalently linked and the and fused directly (without a linker) at its C-end N-end of the human polypeptide IgG2 Fc, presented in SEQ ID NO:184, thereby forming D3-12-IgG2 protein represented in SEQ ID NO:74. Sequence of SEQ ID NO:74 differs from that SEQ ID NO:205 on one matter - namely additional lysine residue is present at the C-end of SEQ ID NO:74. We found experimentally by analysis using liquid chromatography / mass spectrometry (IHMS or similar that Mature CTLA-4-Ig protein formed in Cho (Chinese hamster ovary) cells by transfection of the expression vector, comprising a nucleotide sequence encoding a mutant CTLA-4 EVA, such as, for example, D3-12 EVA amino acid sequence represented in SEQ ID NO:11, and hIgG2 Fc polypeptide presented in SEQ ID NO:184, usually does not include the estimated C-terminal lysine residue (K), as expected for the basis of hIgG2 Fc sequence represented in SEQ ID NO:184.

For example, the nucleotide sequence of SEQ ID NO:153 encodes the signal peptide hCTLA-4 protein D3-12-IgG2 and includes the stop codon TAA at its C-end. The codon AAA, which encodes a lysine residue, directly preceding the stop codon TAA in the sequence SEQ ID NO:153. The putative amino acid sequence of Mature D3-12-IgG2 fused protein obtained by transfection of the expression vector comprising the nucleotide sequence of SEQ ID NO:153 in Cho cells is shown in SEQ ID NO74. The signal peptide is not present in the Mature form of a fused protein D3-12-IgG2, as it is cleaved during processing with the formation of a Mature fused protein. Nevertheless we found, based on MSIH analysis that in this case, Mature D3-12-IgG2 usually does not include the estimated C-terminal lysine residue, as expected on the basis of the nucleotide sequence of SEQ ID NO:153. Rather, the obtained amino acid sequence of Mature D3-12-IgG2 obtained in this way is that shown in SEQ ID NO:205. Assume that the C-terminal lysine polypeptide IgG2 Fc cleaved during processing or prior to secretion.

Assume that the products of protein D3-12-IgG2 using cell lines other mammal by transfection of this vector comprising the nucleotide sequence of SEQ ID NO:153 in such a cell of a mammal (e.g., COS cells, and the like) will produce similar protein D3-12-IgG2, which lacks the intended C-terminal lysine residue, through a similar mechanism of processing or secretion.

Dimeric protein D3-12-IgG2 includes two such monomer D3-12-IgG2, connected together by one or more disulfide bonds formed during cellular processing through the formation of covalent disulfide bonds between cysteine residues. D3-12-IgG2 and dragonlily proteins according to this invention can be formed, for example, using methods described in example 3. For example, the sequence of nucleic acids encoding a polypeptide D3-12 (e.g., SEQ ID NO:90) can be cloned into a vector merge IgG2 Fc, mammalian cells can be transfection with the vector, and the resulting protein can be expressed (typically in dimeric form), purified and analyzed as described in example 3.

Another illustrative mutant polypeptide CTLA-4 EVA is D3-54 mutant polypeptide CTLA-4 EVA, comprising the amino acid sequence of SEQ ID NO:36, and illustrative mutant protein, CTLA-4-Ig includes D3-54 mutant polypeptide CTLA-4 EVA, covalently linked or fused directly (without a linker) at its C-end N-end hIgG2 Fc polypeptide presented in SEQ ID NO:218 (without C-terminal lysine), thereby forming a fused protein D3-54-IgG2, presented in SEQ ID NO:211, or covalently linked or fused directly (without a linker) at its C-end N-end polypeptide hIgG2 Fc presented in SEQ ID NO:184 (with a C-terminal lysine), thereby forming D3-54-IgG2 protein represented in SEQ ID NO:197. As discussed above, experimental analysis determines that the Mature protein D3-54-IgG2, educated in Cho cells, usually does not include the estimated C-terminal lysine residue. Assume that the C-terminal lysine hIgG2 Fc cleaved in Ho is e processing or before secretion, resulting in a sequence of the fused protein D3-54-IgG2 presented in SEQ ID NO:211. As mentioned above, D3-29-IgG2 can be formed using the methods of example 3. Dimeric D3-54-IgG2 protein consists of two D3-54-IgG2 monomer, connected together by one or more disulfide bonds formed during cellular processing through the formation of covalent disulfide bonds between cysteine residues. The sequence of the nucleic acid represented in SEQ ID NO:201, encodes a fused protein represented in SEQ ID NOS:197 and 211.

Other fused protein according to this invention can similarly include a mutant polypeptide, CTLA-4 EVA, linked or merged with hIgG2 (SEQ ID NO:218, or 184). Illustrative Mature mutant fused proteins, CTLA-4-IgG2 in this invention include, for example, amino acid sequences of each of SEQ ID NOS:74-79, 197-200, 205-214, and 219-222. Each of the amino acid sequences of SEQ ID NOS:74-79, 197-200, 220 and 222 includes a C-terminal lysine residue; the C-terminal lysine residue is usually cleaved during processing or prior to secretion, resulting in amino acid sequence without C-terminal lysine presented in SEQ ID NOS:205-210, 211-214, 219 and 221, respectively.

Figure 10 is a schematic illustration showing an illustrative configuration is or structure illustrative mutant fused protein, CTLA-4-IgG2 according to this invention. Two identical Monomeric mutant fused protein, CTLA-4-IgG2 shown schematically, each includes Mature mutant polypeptide CTLA-4 EVA, covalently linked at its C-end N-end of the human IgG2 Fc polypeptide. Every human IgG2 polypeptide includes a human IgG2 hinge region, CH2 domain and CH3 domain. Also shows illustrative amino acid residues present in the joints between EVA and Ig polypeptide F. Amino acid residues at the joints between these components may vary depending on the amino acid sequence of a mutant CTLA-4 EVA and/or Ig amino acid sequence. This dimeric mutant protein, CTLA-4-IgG2 is the result of forming at least one disulfide bond between cysteine residues in the same positions in the two mutant monomers fused protein, CTLA-4-IgG2. Residues of cysteine (C), potentially involved in the formation of disulfide bonds between the two monomers, marked with asterisks. The signal peptide of each monomer fused protein is normally cleaved during processing, and thus, the indirect (Mature) protein usually does not include the sequence of the signal peptide. Amino acid sequence of the human polypeptide IgG2, which includes the hinge, CH2 domain and CH3 d is Myung-human IgG2, shown in SEQ ID NO:184. In an alternative aspect, the amino acid sequence of the human polypeptide IgG2, which includes the hinge region, CH2 domain and CH3 domain of human IgG2 shown in SEQ ID NO:218; in this case, the polypeptide IgG2 does not include the C-terminal lysine (K) residue in comparison with the sequence SEQ ID NO:184.

Properties of mutant fused proteins, CTLA-4-Ig in this invention, described in detail in this description can be compared with the properties of one or more reference Ig fused proteins, such as, e.g., hCTLA-4-IgG1, hCTLA-4-IgG2, protein Orencia®, LEA29Y-Ig. The properties that can be compared include, for example, the ability to bind CD80 and/or CD86 (and/or CD80-Ig and/or CD86-Ig) and/or the ability to inhibit or suppress an immune response (e.g., T-cell activation or proliferation, cytokine production, and so on). The Mature hCTLA-4-IgG1 protein normally exists in solution as a dimer fused protein hCTLA-4-IgG1 comprising two identical Monomeric protein hCTLA-4-IgG1, each Monomeric hCTLA-4-IgG1 protein includes a polypeptide hCTLA-4 EVA (SEQ ID NO:159), associated with the polypeptide IgG1 Fc. The Mature protein hCTLA-4-IgG2 usually exists in solution as a dimer fused protein hCTLA-4-IgG2, consisting of two identical Monomeric protein hCTLA-4-IgG2, each Monomeric protein hCTLA-4-IgG2 (SEQ ID NO:162) includes a polypeptide hCTLA-4 EVA (SEQ ID NO:159), SV is related to the IgG2 Fc polypeptide. The Mature protein Orencia® dimer fused protein consisting of two identical Monomeric Orencia® fused protein, each Monomeric protein (SEQ ID NO:164) includes hCTLA-4 EVA polypeptide (SEQ ID NO:159), associated with a specific mutant IgG1-polypeptide (SEQ ID NO:186). The Mature protein LEA29Y-Ig usually exists in solution as a dimer fused protein LEA29Y-Ig, consisting of two identical Monomeric fused protein LEA29Y-Ig, each Monomeric LEA29Y-Ig protein (SEQ ID NO:166) includes specific mutant polypeptide CTLA-4 EVA (SEQ ID NO:168), associated with a specific mutant IgG1-polypeptide (SEQ ID NO:186). Assume that two monomer fused protein Orencia® dimer covalently linked together one by a disulfide bond formed between cysteine residue at position 120 of each hCTLA-4-mutant IgG1 monomer, and that no disulfide bonds formed between two mutant polypeptides IgGI Fc.

Some mutant fused proteins, CTLA-4 bind CD80 (e.g., hCD80 and/or CD86 (e.g., hCD86). Some such mutant fused proteins, CTLA-4-Ig is associated protein CD80-Ig and/or CD86 protein-Ig. Illustrative fused proteins CD80-Ig include protein hCD80-mIg (SEQ ID NO:225), which includes human CD80 EVA associated with mouse Ig polypeptide Fc; and protein hCD80-hIgG1 (SEQ ID NO:171), which includes the sequence hCD80 EVA associated with human polypeptide the IgGI Fc. Illustrative fused proteins CD86-Ig include protein hCD86-mIg (SEQ ID NO:226), which includes hCD86 EVA (SEQ ID NO:180)associated with mouse Ig polypeptide Fc; and the Mature protein hCD86-hIgG1 (SEQ ID NO:178), which includes the sequence hCD86 EVA (SEQ ID NO:180)associated with human polypeptide IgG1 Fc (SEQ ID NO:185). Illustrative nucleic acid sequences encoding the fused protein hCD86-mIg and hCD80-mIg shown in SEQ ID NOS:227 and 228, respectively.

In one aspect, the invention provides isolated or recombinant protein, comprising (a) a polypeptide comprising amino acid sequence that has at least, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,5% or 100% sequence identity with at least one amino acid sequence selected from the group comprising SEQ ID NOS:1-73, and (b) Ig Fc polypeptide (for example, hIgG2 Fc), where the protein binds CD80 and/or CD86, and/or CD80-Ig and/or CD86-Ig protein, and/or showing the ability to inhibit or suppress an immune response. Polypeptide Ig Fc may include amino acid sequence having at least, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence selected from the group of SEQ ID NO:184, 185, 186 and 218. In some cases, With the end of the polypeptide (a) covalently linked to the N - end of the polypeptide Ig Fc (b). Some such mutant SL is made proteins, CTLA-4-Ig bind CD80 and/or CD86 mammal (for example, hCD80 and/or hCD86), and/or CD80-Ig and/or CD86-Ig protein. CD80-Ig may contain, for example, human CD80 EVA associated with Ig Fc (e.g., hCD80-Ig). In one embodiment of the invention hCD80-Ig - human CD80 EVA associated with human Ig Fc (hCD80-hIg); in another embodiment of the invention, hCD80-Ig - human CD80 EVA associated with mouse Ig Fc (hCD80-mIg). In one embodiment of the invention hCD86-Ig - human CD86 EVA associated with human Ig Fc (hCD86-hIg); in another embodiment of the invention, hCD86-Ig - human CD86 EVA associated with mouse Ig Fc (hCD86-mIg). Some of these fused proteins have the ability to inhibit or suppress one or more of various immune responses, such as, for example, T-cell activation, T-cell proliferation, synthesis or production of cytokine (e.g., production of TNF-α, IFN-γ, IL-2), induction of activation markers (e.g., CD25, IL-2 receptor) or inflammatory molecules, inflammation, products anticollagen AT and/or T-cell-dependent AT the answer(s) in in vitro and/or in vivo studies and/or methods. It is assumed that such fused proteins are efficiently used in various applications, including methods of treatment of diseases and disorders of the immune system (e.g., autoimmune diseases) and methods of inhibiting transplant rejection of organ, cell or tissue is, as is discussed below.

In another aspect, the invention provides isolated or recombinant mutant dimer fused protein, CTLA-4-Ig, comprising two Monomeric mutant fused protein, CTLA-4-Ig coupled via at least one disulfide bond formed between two cysteine residues present in each Monomeric mutant CTLA-4-Ig fused protein. Each monomer mutant fused protein, CTLA-4-Ig includes: (a) a mutant polypeptide, CTLA-4 EVA, comprising amino acid sequence having at least, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,5% or 100% sequence identity with at least one amino acid sequence selected from the group comprising SEQ ID NOS:1-73, and (b) Ig Fc polypeptide (e.g., MgG2 Fc), where the dimer fused protein binds CD80 and/or CD86, and/or CD80-Ig and/or CD86-Ig, and/or showing the ability to inhibit or suppress an immune response. In some cases, With the end of the polypeptide (a) covalently linked or fused to the N-end of the polypeptide Ig Fc (b). Polypeptide Ig Fc may include amino acid sequence having at least, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence selected from the group comprising SEQ ID NO:184-186 and 218. In some cases dimer fused protein formed covalent disulfide bond between residue qi is Thein at amino acid position 120 of each amino acid sequence of a mutant CTLA-4 EVA or amino acid position corresponding to the position 120 in each amino acid sequence is mutated relative to the amino acid sequence of hCTLA-4 EVA presented in SEQ ID NO:159. Some of these dimers fused protein have the ability to inhibit or suppress one or more of various immune responses, such as, for example, T-cell activation, T-cell proliferation, synthesis or production of cytokine (e.g., production of TNF-α, IFN-γ, IL-2), induction of activation markers (e.g., CD25, IL-2 receptor) or inflammatory molecules, inflammation, production anticollagen AT and/or T-cell-dependent AT the answer(s) in in vitro and/or in vivo studies and/or the ways. Assume that such dimers fused protein are efficiently used in various applications, including methods of treatment of diseases and disorders of the immune system (e.g., autoimmune diseases) and methods of inhibiting transplant rejection of organ, cell or tissue, as discussed below.

Some such mutant monomers fused protein, CTLA-4-Ig have binding affinity for hCD86 or hCD86 EVA, which is at least equal to or greater than those for hCTLA-4 EVA and LEA29 to hCD86 or hCD86 EVA respectively. See, for example, table 5 in example 4. Mutant polypeptide CTLA-4 EVA, present in some of these dimeric and Monomeric fused proteins, vkluchennosti sequence, having the amino acid length, approximately equal to the amino acid length of the hCTLA-4 EVA. For example, some such mutant polypeptides, CTLA-4 EVA include amino acid sequence that is about 110-138, 112-136, 114-134, 116-132, 118 to 130, 119-129, 120-128, 121-127, 122-126 or 123-125 amino acid residues in length. Some such mutant polypeptides, CTLA-4 EVA include a sequence of 124 amino acid residues. Illustrative of the mutant polypeptides, CTLA-4 EVA include, for example, but not limited to, those that include an amino acid sequence having at least, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with at least one amino acid sequence selected from the group comprising SEQ ID NOS:1-73, where such a mutant polypeptide, CTLA-4 EVA binds CD80 and/or CD86 (or EVA each or both) and/or has the ability to inhibit the immune response.

Some such mutant dimers fused protein, CTLA-4-Ig have the avidity of binding to hCD86 and/or hCD86-Ig, which is at least approximately equal to or greater than the avidity of binding of the dimer fused protein hCTLA-4-Ig (e.g., hCTLA-4-IgG2 or hCTLA-4-IgG1 dimer), Orencia® dimer and/or LEA29Y-Ig dimer to hCD86 and/or hCD86-Ig, respectively. Some of these dimers fused protein have the avidity of binding to hCD86 and/or hCD86-mIg, which is 2-10 times (2x-10x), 5-10 times (5x-10x), 10-20 RA is (10x-20x), 20-40 times (20x - 40x) or more than 40 times (>40) is greater than the avidity of binding of the dimer Orencia® for hCD86 and/or hCD86-mIg. See, for example, illustrative fused dimeric proteins according to this invention in table 3 below. Alternative or additionally, some such dimers fused protein have the avidity of binding to hCD80 and/or hCD80-Ig, which is at least approximately equal to or greater than the avidity of binding of the hCTLA-4-Ig dimer (e.g., dimer hCTLA-4-IgG2 or hCTLA-4-IgG1), dimer Orencia® and/or dimer LEA29Y-Ig to hCD80 and/or hCD80-Ig, respectively. Some of these dimers fused protein have the avidity of binding to hCD80 and/or hCD86-mIg, which is 0.5-2 times (0,5x-2), 2-4 times (2-4) or more than 2 times (>2) is greater than the avidity of binding of the dimer Orencia® for hCD86 and/or hCD86-mIg. See, for example, illustrative fused dimeric proteins according to this invention in table 4 below.

Some such mutant dimers fused protein, CTLA-4-Ig dissociate from connecting hCD86 and/or hCD86-Ig at a rate that is less than the rate at which hCTLA-4-Ig dimer (e.g., dimer hCTLA-4-IgG2 or hCTLA-4-IgG1), dimer Orencia® and/or dimer LEA29Y-Ig dissociates from the binding hCD86 and/or hCD86-Ig, respectively. Some of these fused proteins associated with or contact hCD86 and/or hCD86-Ig at a rate that is at least equal to or greater than the rate at which the dimer hCTLA-4-Ig (e.g., dimer hCTLA-4-IgG2 or hCTLA-4-IG1), Orencia® dimer and/or LEA29Y-Ig dimer associates with hCD86 and/or hCD86-Ig, respectively. For some of these dimers fused protein, the equilibrium dissociation constant (KDfor binding assays between CD86 (or CD86-Ig) and dimer fused protein according to this invention is less than the equilibrium dissociation constant (KDfor binding assays between CD86 (or CD86-Ig) and hCTLA-4-Ig dimer (for example, a dimer hCTLA-4-IgG2 or hCTLA-4-IgG1), dimer Orencia® and/or dimer LEA29Y-Ig. See, for example, illustrative dimers fused protein according to this invention in table 3. For some of these dimers fused protein, the equilibrium dissociation constant (KDfor binding assays between CD80 (or CD80-Ig) and dimer fused protein according to this invention is approximately equal to or less than the equilibrium dissociation constant (KDfor binding assays between CD80 (or CD80-Ig) and dimer hCTLA-4-Ig (e.g., dimer hCTLA-4-IgG2 or hCTLA-4-IgGl), dimer Orencia® dimer LEA29Y-Ig. See, for example, illustrative dimers fused protein according to this invention in table 4.

Some such mutant dimers fused protein, CTLA-4-Ig have the ability to inhibit or suppress an immune response (e.g., to inhibit T-cell activation or proliferation, inhibit the production of cytokines and the like), which is at least approximately equal to or greater than pursue the activity of the dimer hCTLA-4-Ig (e.g., dimer hCTLA-4-IgG2 or hCTLA-4-IgG1), dimer Orencia® and/or dimer LEA29Y-Ig to inhibit or suppress the specified immune response, respectively. For example, some of these dimers fused protein is able to inhibit T-cell activation or T-cell proliferation in in vitro studies. Examples 4-9 below, for example, demonstrate the ability of representative dimers fused protein according to this invention, comprising the amino acid sequence of a representative mutant CTLA-4 EVA, to inhibit T-cell proliferation in vitro. Some of these dimers are able to inhibit or suppress the immune response in the subject in vivo, for example, through the introduction of a therapeutically or prophylactically effective amount of at least one dimer to a subject in need of immunosuppressive therapy. It is assumed that some of these dimers fused protein usable in various fields, including, for example, but not limited to, prophylactic and/or therapeutic methods for inhibiting or suppressing an immune response in a subject suffering from immune diseases or disorders, in which immunosuppression is desirable (e.g., autoimmune diseases), and methods of inhibiting transplant rejection tissue, cell or organ from a donor to a recipient.

Some of that is their dimers have varying ability to modulate or suppress the transmission of the signal through CD28, because they have different consistent avidity binding to CD80 and CD86. Such dimers are suitable in applications in which differential control T-cell responses is desirable, including therapeutic and prophylactic methods of treatment of diseases and disorders of the immune system, such as, for example, immunodeficiency diseases and disorders (such as, PA, PC, psoriasis and the like). Illustrative dimer fused proteins comprising polypeptides according to this invention having the above-mentioned differential CD80/CD86 the avidity of binding and immunoinhibitory properties shown in example 4.

Some such mutant dimers CTLA-4-Ig have the ability to suppress or inhibit an immune response, which is at least approximately equal to or greater than the ability of the protein hCTLA-4 or dimer hCTLA-4-Ig to suppress or inhibit one or more types of immune responses. For example, some of these dimers have the ability to inhibit T-cell activation or proliferation in vitro and/or in vivo studies and/or applications, such as those described above and below, which is at least approximately equal to or greater than the capacity of the hCTLA-4 protein or hCTLA-4-Ig dimer (e.g., Orencia®, hCTLA-4-IgG2 dimer or dimer hCTLA-4-IgG1) to inhibit T-cell activation or proliferation in such applications is. Additionally, some of these dimers have the ability to inhibit or suppress an immune response (e.g., T-cell activation or proliferation, cytokine production, T-cell-dependent antibody response), which is greater than the ability of the dimer LEA29Y-Ig to inhibit or suppress an immune response. Examples 4-9, for example, compare the ability of a representative dimer fused protein according to this invention, comprising the amino acid sequence of a mutant CTLA-4 EVA in this invention, to inhibit T-cell proliferation in vitro with the ability of the dimeric hCTLA-4-IgG2, Orencia® and LEAY29-Ig to inhibit T-cell proliferation in vitro. See, for example, tables 6-9 below. Some of these dimers have the ability to bind hCD80 and/or hCD86 (or hCD80-Ig and/or hCD86-Ig), and the ability to inhibit or suppress the immune response in vitro and/or in vivo studies and/or applications, such as those described above and in more detail below (e.g., in vivo method, which is administered therapeutically or prophylactically effective amount of at least one such dimer). Some of these dimers have the avidity of binding to hCD80 and/or hCD86 (or hCD80-Ig and/or hCD86-Ig), which is at least approximately equal to or greater than the avidity of binding of the hCTLA-4 protein, a dimeric hCTLA-4-Ig (e.g., hCTLA-4-IgG2, Orencia®) or dimeric LEA29Y-Ig to hCD80 and/or hD86 (or hCD80-Ig and/or hCD86-Ig), respectively, and the ability to inhibit the immune response that is at least equal to or greater than the ability of the protein hCTLA-4, dimeric hCTLA-4-Ig (e.g., hCTLA-4-IgG2, Orencia®) or dimeric LEA29Y-Ig to inhibit the immune response. Assume that these mutant dimers fused protein, CTLA-4-Ig is suitable in various applications, including, for example, a prophylactic and/or therapeutic treatment of diseases, disorders and conditions of the immune system, as discussed in more detail below.

In another aspect, the invention provides isolated or recombinant dimer fused protein (e.g., mutant dimer fused protein, CTLA-4-Ig), consisting of two identical Monomeric fused protein (for example, two Monomeric mutant fused protein, CTLA-4-Ig), where each such Monomeric protein includes an amino acid sequence having at least, 90%, 90,5%, 91%, 91,5%, 92%, 92,5%, 93%, 93,5%, 94%, 94,5%, 95%, 95,5%, 96%, 96,5%, 97%, 97,5%, 98%, 98,5%, 99%, 99,5% or 100% identity with the amino acid sequence selected from the group comprising SEQ ID NOS:74-79, 197-200, 205-214, and 219-222, where the dimer binds CD80 and/or CD86 (and/or CD80-Ig and/or CD86-Ig, such as hCD80-mIg and/or hCD86-mIg, respectively), and/or has the ability to inhibit the immune response, including those described above, as well as below. Each of the amino acid sequences listed in SEQ ID NOS:74-79, 197-200, 205-214, and 219-222 p is ecstasy a Mature mutant CTLA-4 EVA fused at its C-end N-end IgG2 Fc polypeptide, and each such sequence can be called mutant fused protein, CTLA-4-Ig. Amino acid sequence of each of SEQ ID NOS:74-79, 197-200, 220 and 222 are identical to SEQ ID NOS:205-214, 219 and 221, except that SEQ ID NOS:74-79, 197-200, 220 and 222, which includes a lysine at the C-end.

In another aspect, the invention provides isolated or recombinant monomer fused protein comprising amino acid sequence having at least, 90%, 90,5%, 91%, 91,5%, 92%, 92,5%, 93%, 93,5%, 94%, 94,5%, 95%, 95,5%, 96%, 96,5%, 97%, 97,5%, 98%, 98,5%, 99%, 99,5% or 100% identity with the amino acid sequence comprising amino acid residues 1-351 any of SEQ ID NOS:74-79, 197-200, 205-214, and 219-222, where the protein binds CD80 and/or CD86 (and/or CD80-Ig and/or CD86-Ig, such as hCD80-mIg and/or hCD86-mIg, respectively), and/or has the ability to inhibit the immune response, including those described above and below.

In another aspect, the invention provides isolated or recombinant dimer fused protein consisting of two identical Monomeric fused protein, where each such Monomeric protein includes an amino acid sequence having at least, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence comprising amino acid residues 1-351 any of SEQ ID NOS:74-79, 197-200, 205-214, and 219-222, where b is Lok specified dimer fused protein binds CD80 and/or CD86 (and/or CD80-Ig and/or CD86-Ig, such as hCD80-mIg and/or hCD86-mIg, respectively), and/or has the ability to inhibit the immune response, including those described above and below.

The invention also provides mutant Monomeric protein, CTLA-4-Ig, including amino acid sequence having at least, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence selected from the group comprising SEQ ID NOS:74-79, 197-200, 205-214, and 219-222, where specified Monomeric protein binds CD80 and/or CD86 (and/or CD80-Ig and/or CD86-Ig, such as hCD80-mIg and/or hCD86-mIg, respectively), and/or has the ability to inhibit the immune response. Some of these monomers fused protein dimers have the ability to inhibit or suppress one or more immune responses, including, for example, T-cell activation or proliferation, synthesis or production of cytokine (e.g., production of TNF-α, IFN-γ, IL-2), induction of activation markers (e.g., CD25, IL-2 receptor), inflammation, production anticollagen AT and/or T-cell-dependent AT the answer(s)) in in vitro and/or in vivo studies and/or methods (e.g., in vivo in a subject suffering from diseases, disorders or conditions in which immunosuppressive therapy would be effective and which is administered a therapeutically effective amount of such fused dimeric protein, as discussed in more detail the who). Assume that such monomers fused protein and the dimers are suitable in various applications, including therapeutic and/or prophylactic treatment of diseases of the immune system, including those discussed below.

In another aspect, the invention provides isolated or recombinant dimer fused protein (e.g., mutant dimer fused protein, CTLA-4-Ig)comprising two Monomeric fused protein (e.g., Monomeric mutant protein, CTLA-4-Ig), where each such Monomeric protein comprises: (1) a polypeptide (for example, the mutant polypeptide extracellular domain of CTLA-4), which includes amino acid sequence that differs from amino acid sequence selected from the group comprising SEQ ID NOS:1-73 by no more than 6 amino acid residues (for example, not more than 1, 2, 3, 4, 5 or 6 amino acid residues), and where the amino acid residue in the amino acid sequence in position 41, 50, 54, 55, 56, 64, 65, 70 or 85 is identical to the amino acid residue in the corresponding position of the specified selected amino acid sequence (e.g., a polypeptide selected from SEQ ID NOS:1-73), and (2) a polypeptide Ig Fc (e.g., IgG2 Fc), where the dimer fused protein binds CD80 and/or CD86 (and/or CD80-Ig and/or CD86-Ig), and/or inhibits an immune response (e.g., T-cell activation or proliferation, etc the production of the cytokine, the induction of markers of activation or inflammatory molecules, products anticollagen AT, T-cell-dependent AT the answers and others) in in vitro and/or in vivo studies and/or methods, as discussed in more detail below. The invention also includes isolated or recombinant Monomeric protein, as described above, which binds CD80 and/or CD86 (and/or CD80-Ig and/or CD86-Ig) and/or induces an immune response in vitro or in vivo. In the dimer fused protein two Monomeric fused protein (e.g., mutant monomer CTLA-4-Ig) optional covalently joined together by one or more disulfide bonds through cysteine residues in each monomer, and two monomer is usually identical to each other. In some cases, the mutant polypeptide, CTLA-4 EVA in this dimer fused protein or monomer different from the selected polypeptide (e.g., selected from SEQ ID NOS:1-73) no more than 6 amino acid residues, but the amino acid occupying position 41, 50, 54, 55, 56, 64, 65, 70 or 85, identical amino acid residue included in these provisions in the selected amino acid sequence; that is, the amino acid residue in this position cannot be removed or replaced. Some such mutant polypeptides, CTLA-4 EVA in this fused protein include amino acid sequence that differs from amino acid selected placentas is in search of not more than 6 amino acid residues and includes the amino acid residues at positions 24, 30, 32, 41, 50, 54, 55, 56, 64, 65, 70, 85, 104 and 106 that are identical with amino acid residues in the corresponding positions in the selected amino acid sequence. Such a mutant polypeptide, CTLA-4 EVA may differ from the selected amino acid sequence at the amino acid deletion(s), addition(s) and/or amino acid replacement(s). Amino acid substitutions may be conservative or non-conservative substitution. See, for example, the "Variability of the sequence. Some of these fused dimeric proteins have the avidity of binding to hCD86 or hCD86-Ig, which is at least approximately equal to or greater than the avidity of binding of the hCTLA-4, dimeric hCTLA-4-Ig dimer LEA29Y-Ig or protein Orencia® for hCD86 or hCD86-Ig, respectively. Some of these Monomeric fused proteins have a binding affinity of or avidity for hCD86, hCD86-Ig or hCD86 EVA, which is at least approximately equal to or greater than the binding affinity of or avidity Monomeric hCTLA-4, Monomeric hCTLA-4-Ig or Monomeric LEA29Y-Ig to hCD86, hCD86-Ig or hCD86 EVA respectively. Alternative or additionally, some such fused dimeric proteins have the avidity of binding to hCD80 or hCD80-Ig, which is at least approximately equal to or greater than the avidity hCTLA-4 or hCTLA-4-Ig to hCD80 respectively. Alternative or additionally, some such Monomeric sliceable have a binding affinity of or avidity for hCD80, hCD80-Ig or hCD80 EVA, which is at least approximately equal to or greater than the binding affinity of or avidity Monomeric hCTLA-4 or Monomeric hCTLA-4-Ig to hCD80, hCD80-Ig or hCD80 EVA respectively. In some cases, the mutant polypeptide, CTLA-4 EVA in such a dimer or monomer fused protein comprises the amino acid sequence, having a length approximately equal to the amino acid length of the hCTLA-4 EVA, for example, about 118 to 130, 119-129, 120-128, 121-127, 122-126, 123-125 or 124 amino acid residues in length. N-terminal polypeptide Ig Fc (e.g., IgG2 Fc IgG1 Fc, IgG4 Fc or mutant IgG Fc, which reduces effector function or binding of the Fc receptor may be covalently linked or fused directly or indirectly (via a linker, including, for example, 1-10 amino acid residues) with the end of the mutant polypeptide, CTLA-4 EVA. Polypeptide Ig Fc may include amino acid sequence having at least, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence selected from the group comprising SEQ ID NOS:184-186 and 218, for example, any of SEQ ID NO:184, 185, 186 and 218.

Some such mutant dimers CTLA-4-Ig and monomers fused protein can inhibit one or more of various immune responses, including, for example, T-cell activation, T-cell proliferation, synthesis or production of cytokine (for example, about what uccio TNF-α, IFN-γ, IL-2), induction of activation markers (e.g., CD25, IL-2 receptor) or inflammatory molecules, inflammation, production anticollagen AT and/or T-cell-dependent AT the answer(s). Some such mutant dimers CTLA-4-Ig have a greater ability to inhibit one or more of these immune responses than hCTLA-4, dimeric hCTLA-4-Ig or dimeric LEA29Y-Ig. Examples 4-9, for example, present data that compares the ability of a representative dimer fused protein according to this invention, comprising a mutant polypeptide, CTLA-4 EVA this invention to inhibit T-cell proliferation in vitro with the ability of the dimeric hCTLA-4-Ig or dimeric LEA29Y-Ig to do it. Some such mutant monomers CTLA-4-Ig have a greater ability to inhibit one or more of these immune responses than Monomeric hCTLA-4, Monomeric hCTLA-4-Ig or Monomeric LEA29Y-Ig. Some of these monomers and dimers are able to inhibit or suppress the immune response in the subject in vivo, for example, by administration of a therapeutically or prophylactically effective amount of at least one such polypeptide to a subject in need of immunosuppressive therapy. Assume that such fused proteins are efficiently used in various applications, including methods of treating diseases, disorders or conditions in which immunosupressor the Naya therapy would be effective, such as preventive and/or therapeutic treatment of autoimmune diseases and disorders, and methods of inhibiting transplant rejection of organ, cell or tissue.

In another aspect, the invention provides dimer isolated or recombinant protein (e.g., mutant dimer fused protein, CTLA-4-Ig)comprising two Monomeric fused protein (for example, two Monomeric mutant fused protein, CTLA-4-Ig), where each such Monomeric protein comprises: (1) a mutant polypeptide extracellular domain of CTLA-4 (KJV), including amino acid sequence which (a) differs from the amino acid sequence selected from the group comprising SEQ ID NOS:1-73 by no more than 6 amino acid residues (for example, not more than 1, 2, 3, 4, 5 or 6 amino acid residues), and (b) includes at least one amino acid substitution in amino acid position corresponding to position 50, 54, 55, 56, 64, 65, 70 or 85 relative to the amino acid sequence of SEQ ID NO:159; and (2) a polypeptide Ig Fc, where dimer fused protein binds CD80 and/or CD86 (and/or CD80-Ig and/or CD86-Ig) and/or inhibits an immune response (for example, T-cell activation or proliferation, cytokine production, induction of activation markers, inflammation, production anticollagen antibodies, T-cell-dependent antibody response and the like) in in vitro and/or in vivo studies the studies and/or methods, as described in more detail below. The invention also includes isolated or recombinant Monomeric protein, as described above, which binds CD80 and/or CD86 (and/or CD80-Ig and/or CD86-Ig) and/or induces an immune response in vitro or in vivo. In some cases, CD80 is hCD80 and CD86 is hCD86. In the dimer fused protein two Monomeric fused protein (e.g., mutant monomer CTLA-4-Ig) optional covalently joined together by one or more disulfide bonds through cysteine residues in each monomer, and two monomer is usually identical to each other. N-terminal polypeptide Ig Fc (e.g., IgG2 Fc IgG1 Fc, IgG4 Fc or mutant IgG Fc, which reduces effector function or binding to Fc receptor), can be covalently linked or fused directly or indirectly (via a linker, including, for example, 1-10 amino acid residues) with the end of the mutant polypeptide, CTLA-4 EVA. Ig Fc polypeptide can include amino acid sequence having at least, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence selected from the group comprising SEQ ID NOS:184-186 and 218.

Some of these dimers or monomers fused protein include mutant polypeptide CTLA-4 EVA, which includes the amino acid sequence, having a length approximately equal to the amino acid length of the hCTLA-4 EVA, for example, 118 to 130, 119-19, 120-128, 121-127, 122-126 or 123-125 amino acid residues in length. Some such mutant polypeptides, CTLA-4 EVA in such a dimer or monomer fused protein include amino acid sequence with a length of 124 amino acid residue. Some such mutant polypeptides, CTLA-4-EVA include 2, 3, 4, 5 or 6 amino acid substitutions in positions relative to the sequence represented in SEQ ID NO:159 selected from the group comprising a position 50, 54, 55, 56, 64, 65, 70 and 85. Some such mutant polypeptides, CTLA-4 EVA also include amino acid replacement at a position corresponding to position 104 and/or 30 relative to SEQ ID NO:159. Some such mutant polypeptides, CTLA-4 EVA contain at least one amino acid substitution relative to SEQ ID NO:159 in position 70 (optional S70F), position 64 (optional S64P), position 50 (optional AM), position 54 (optional M54K/V, for example, M54K), the position 65 (optional I65S), position 56 (optional N56D), position 55 (optional G55E), position 85 (optional MA) and/or position 24 (optional A24E/S, for example, AE). Any such mutant polypeptide CTLA-4 EVA may also include amino acid substitution relative to SEQ ID NO:159 in position 104 (optional L104E/D, for example, L104E), position 30 (optional T30N/D/A, for example, T30N, T30D or TA) and/or position 32 (optional V32I). Some such m is tantie polypeptides CTLA-4 EVA contain, at least one substitution at amino acid position is relative to SEQ ID NO:159 selected from the group comprising AL, M54K, G55E, N56D, S64P, I65S and S70F. Some such mutant polypeptides, CTLA-4 EVA include 2, 3, 4, 5, or 6 amino acid substitutions in positions relative to SEQ ID NO:159 selected from the group comprising AL, MG, G55E. N56D, S64P, I65S and S70F.

Some such mutant dimers CTLA-4-Ig show avidity binding to CD86 (e.g., hCD86) or dimeric CD86-Ig (e.g., hCD86-Ig), which is almost equal to or greater than the avidity of binding of the hCTLA-4 protein, a dimeric hCTLA-4-Ig (e.g., CTLA-4-IgG1 or CTLA-4-IgG2), protein Orencia® dimer LEA29Y-Ig to CD86 or dimeric CD86-Ig, respectively. Some of these dimers have the avidity of binding to CD80 (e.g., hCD80) or dimeric CD80-Ig (e.g., hCD80-Ig), which is greater than the avidity of binding of the hCTLA-4, dimeric hCTLA-4-Ig, protein Orencia® and/or dimeric LEAY29-Ig to CD80 or dimeric CD80-Ig, respectively.

Some such mutant monomers CTLA-4-Ig show the binding affinity of or avidity for CD86 (e.g., hCD86) or CD86-Ig (e.g., hCD86-Ig), which is almost equal to or greater than the binding affinity of or avidity Monomeric hCTLA-4, Monomeric hCTLA-4-Ig or Monomeric LEA29Y-Ig to or CD86 CD86-Ig, respectively. Some of these monomers have the binding affinity of or avidity for CD80 (e.g., hCD80) or CD80-Ig (e.g., hCD80-Tg), which is more, che is the binding affinity of or avidity Monomeric hCTLA-4 or Monomeric hCTLA-4-Ig (e.g., Monomeric CTLA-4-IgG1 or CTLA-4-IgG2) to CD80 or dimeric CD80-Ig, respectively.

Some such mutant dimers CTLA-4-Ig and monomers have the ability to suppress or inhibit one or more immune responses, including those described above and elsewhere in this description (for example, T-cell activation or proliferation, cytokine production, induction of activation markers, inflammation, production anticollagen antibodies, T-cell-dependent antibody responses, in vitro and/or in vivo studies and/or methods (e.g., in vivo in a subject suffering from diseases, disorders or conditions in which immunosuppressive therapy would be effective and which is administered a therapeutically effective amount of at least one such mutant dimer CTLA-4-Ig). Some such mutant dimers CTLA-4-Ig inhibit one or more of these immune responses to a greater extent than hCTLA-4, dimeric hCTLA - 4-Ig (e.g., dimeric, CTLA-4-IgG1 or CTLA-4-IgG2), protein Orencia® and/or dimeric LEAY29-Ig. Some such mutant monomers CTLA-4-Ig inhibit one or more of these immune responses to a greater extent than Monomeric hCTLA-4, Monomeric hCTLA-4-Ig and/or Monomeric LEAY29-Ig. Assume that these mutant dimers CTLA-4-Ig and the monomers are effectively used in various applications, including the treatment of autoimmune diseases and the violations and methods of inhibiting rejection of an organ transplant, cells or tissue.

In another aspect, the invention provides isolated or recombinant dimer fused protein (e.g., mutant dimer fused protein, CTLA-4-Ig)comprising two Monomeric fused protein (e.g., Monomeric mutant protein, CTLA-4-Ig), where each such Monomeric protein comprises: (1) a polypeptide (e.g., mutant extracellular domain of CTLA-4), including amino acid sequence that (i) has at least, 95%, 96%, 97%, 98%, 99% or 100% identity sequence of any amino acid sequence selected from the group comprising SEQ ID NOS:1-73, and (ii) includes a phenylalanine residue at amino acid position corresponding to position 70 of the specified amino acid sequence selected from the group comprising SEQ ID NO:1-73; and (2) Ig Fc polypeptide (e.g., IgG2 Fc IgG1 Fc, IgG4 Fc or mutant IgG Fc, which reduces effector function or binding to Fc receptor), where the dimer fused protein binds CD80 (e.g., hCD80 and/or CD86 (e.g., hCD86) (and/or CD80-Ig, for example, hDC80-Ig and/or CD86-Ig, e.g., hCD86-Ig), and/or has the ability to inhibit the immune response in vitro or in vivo. The invention also includes isolated or recombinant Monomeric protein, as described above, which binds CD80 (e.g., hCD80 and/or CD86 (e.g., hCD86) (and/or CD80-Ig, for example, hDC80-Ig and/or CD86-Ig, for example, hCD86Ig) and/or induces an immune response in vitro or in vivo. In some cases, the Ig Fc polypeptide comprises a sequence having at least, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence selected from the group comprising SEQ ID NOS:184-186 and 218. N-terminal polypeptide Ig Fc can be covalently linked or fused directly or indirectly (via a linker, including, for example, 1-10 amino acids) with the end of the mutant polypeptide, CTLA-4 EVA.

In some such mutant dimers CTL-4-Ig or monomers mutant polypeptide CTLA-4 EVA includes one or more of the following relative to the specified amino acid sequence selected: the glutamic acid residue at amino acid position corresponding to position 24; an asparagine residue at amino acid position corresponding to position 30; the isoleucine residue at amino acid position corresponding to position 32; methionine residue at the amino acid position corresponding to position 50; lysine residue in amino acid position corresponding to position 54; a glutamic acid residue at amino acid position corresponding to position 55; the aspartic acid residue at amino acid position corresponding to position 56; a Proline residue at amino acid position corresponding to position 64; a serine residue at amino acid position, with testwuide position 65; and the glutamic acid residue at amino acid position corresponding to position 104. For example, some such mutant polypeptides, CTLA-4 EVA in these mutant dimers CTLA-4-Ig or monomers include amino acid sequence comprising (i)at least, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence SEQ ID NO:24, and (ii) the phenylalanine residue at amino acid position corresponding to position 70 of the amino acid sequence of SEQ ID NO:24, where the dimer fused protein binds hCD80 and/or hCD86 and/or hCD80-Ig and/or hCD86-Ig) and/or inhibits an immune response in vitro and/or in vivo studies and/or methods. Some such mutant polypeptides, CTLA-4 EVA in these mutant dimers CTLA-4-Ig or monomers include one or more of the following relative to SEQ ID NO:24: glutamic acid residue at position 24; an asparagine residue at position 30; the isoleucine residue at position 32; methionine residue in position 50; the lysine residue in position 54; a glutamic acid residue in position 55; the aspartic acid residue in position 56; a Proline residue at position 64; a serine residue at position 65; and a glutamic acid residue in position 104.

Some such mutant dimers CTLA-4-Ig show avidity binding to CD86 (e.g., hCD86) or dimeric CD86-Ig (e.g., hCD86-Ig), which PR is chicosci equal to or greater than the avidity of binding of the hCTLA-4, dimeric hCTLA-4-Ig, protein Orencia® dimer LEA29Y-Ig to CD86 or dimeric CD86-Ig, respectively. Some of these dimers have the avidity of binding to CD80 (e.g., hCD80) or dimeric CD80-Ig (e.g., hCD80-Ig), which is greater than the avidity of binding of the hCTLA-4, dimeric hCTLA-4-Ig or protein Orencia® to CD80 or dimeric CD80-Ig, respectively.

Some such mutant monomers CTLA-4-Ig show the binding affinity of or avidity for CD86 (e.g., hCD86) or CD86-Ig (e.g., hCD86-Ig), which is almost equal to or greater than the binding affinity of or avidity Monomeric hCTLA-4, Monomeric hCTLA-4-Ig and/or Monomeric LEA29Y-Ig to or CD86 CD86-Ig, respectively. Some of these monomers have the binding affinity of or avidity for CD80 (e.g., hCD80) or CD80-Ig (e.g., hCD80-Ig), which is greater than the binding affinity of or avidity Monomeric hCTLA-4 or Monomeric hCTLA-4-Ig to CD80 or dimeric CD80-Ig, respectively.

Some such mutant dimers CTLA-4-Ig and monomers have the ability to suppress or inhibit one or more immune responses (e.g., T-cell activation or proliferation, cytokine production, induction of activation markers, inflammation, production anticollagen AT, T-cell-dependent AT the answers), in in vitro and/or in vivo studies and/or methods (e.g., in vivo in a subject suffering from a disease, disrupting the ia or the state of the immune system, when immunosuppressive therapy would be effective and which is administered a therapeutically effective amount of at least one such mutant dimer CTLA-4-Ig). Some such mutant dimers CTLA-4-Ig have the ability to suppress or inhibit one or more of these immune responses to a greater extent than hCTLA-4, dimeric hCTLA-4-Ig (e.g., dimeric, CTLA - 4-IgG1 or CTLA-4-IgG2), protein Orencia® and/or dimeric LEAY29-Ig. Some such mutant monomers CTLA-4-Ig have the ability to suppress or inhibit one or more of these immune responses to a greater extent than Monomeric hCTLA-4, Monomeric hCTLA-4-Ig and/or Monomeric LEAY29-Ig. Assume that these mutant dimers CTLA-4-Ig and the monomers are effectively used in various therapeutic and/or prophylactic treatment of diseases or disorders in which immunosuppressive treatment would be effective, including, for example, methods of treatment of autoimmune diseases, and methods of inhibiting transplant rejection of organ, cell or tissue.

In another aspect, the invention provides isolated or recombinant dimer fused protein (e.g., mutant dimer fused protein, CTLA-4-Ig)comprising two Monomeric fused protein (e.g., Monomeric mutant fused proteins, CTLA-4-Ig), where each such Monomeric protein comprises: (1) p is dipeptide (for example, mutant extracellular domain of CTLA-4), comprising a amino acid sequence which (a) differs from the amino acid sequence of the polypeptide extracellular domain of human CTLA-4, is presented in SEQ ID NO:159, no more than 6 amino acid residues, and (b) includes at least one amino acid substitution, where indicated, at least the amino acid substitution includes S70F, where the position of the amino acid residues are numbered according to SEQ ID NO:159; and (2) IgG Fc polypeptide (e.g., IgG2 Fc IgG1 Fc, IgG4 Fc or mutant IgG Fc, which reduces effector function or binding to Fc receptor), where the dimer binds hCD80 and/or hCD86 and/or hCD86-Ig and/or hCD86-Ig), and/or inhibits an immune response (e.g., T-cell activation or proliferation, cytokine production, induction of activation markers, inflammation, production anticollagen antibodies, T-cell-dependent antibody responses, etc.) in in vitro and/or in vivo studies and/or methods, as discussed in detail below. The invention also includes isolated or recombinant Monomeric protein, as described above, which binds CD80 (e.g., hCD80 and/or CD86 (e.g., hCD86) (and/or CD80-Ig, for example, hDC80-Ig and/or CD86-Ig, e.g., hCD86-Ig) and/or induces an immune response in vitro or in vivo. Polypeptide Ig Fc may include a sequence having at least, 95%, 96%, 97%, 98%, 99 or 100% identity with the amino acid sequence, selected from the group comprising SEQ ID NOS:184-186 and 218. N-terminal polypeptide Ig Fc can be covalently linked or fused directly or indirectly (via a linker, including, for example, 1-10 amino acids) with the end of the mutant polypeptide, CTLA-4 EVA. In some such mutant dimers or monomers, CTLA-4-Ig mutant polypeptide CTLA-4 EVA also includes at least one amino acid substitution selected from the group comprising AU, T30N, V32I, D41G, AM, M54K, G55E, N56D, S64P, I65S, MA, L104E and I106F. In some such mutant dimers or monomers, CTLA-4-Ig, a mutant polypeptide, CTLA-4 EVA also includes replacement of L104E and/or two, three or four additional substitutions selected from the group of substitutions: T30N, V32I, AM, M54K, G55E, N56D, S64P and I65S.

Some of these dimers have the avidity of binding to CD86 (e.g., hCD86) or dimeric CD86-Ig (e.g., hCD86-Ig), which is almost equal to or greater than the avidity of binding of the hCTLA-4, dimeric hCTLA-4-Ig and/or protein Orencia® for CD86 or dimeric CD86-Ig, respectively. Some of these dimers have the avidity of binding to CD80 (e.g., hCD80) or dimeric CD80-Ig (e.g., hCD80-Ig), which is greater than the avidity of binding of the hCTLA-4, dimeric hCTLA-4-Ig and/or Orencia® to CD80 or dimeric CD80-Ig, respectively. Some of these monomers exhibit a binding affinity of or avidity for CD86 (e.g., hCD86) or CD86-Ig (e.g., hCD86-Ig), which is almost equal to or larger than affine shall be binding or avidity Monomeric hCTLA-4, Monomeric hCTLA-4-Ig or Monomeric LEA29Y-Ig to or CD86 CD86-Ig, respectively. Some of these monomers have the binding affinity of or avidity for CD80 (e.g., hCD80) or CD80-Ig (e.g., hCD80-Ig), which is greater than the binding affinity of or avidity Monomeric hCTLA-4 or Monomeric hCTLA-4-Ig to CD80 or dimeric CD80-Ig, respectively.

Some such mutant dimers CTLA-4-Ig and monomers have the ability to suppress or inhibit one or more immune responses (e.g., T-cell activation or proliferation, cytokine production, induction of activation markers, inflammation, production anticollagen antibodies, T-cell-dependent antibody responses, in vitro and/or in vivo studies and/or methods (e.g., in vivo in a subject suffering from diseases, disorders or conditions in which immunosuppressive therapy would be effective and which is administered a therapeutically effective amount of at least one such mutant dimer CTLA-4-Ig). Some of these dimers have the ability to suppress or inhibit one or more of these immune responses to a greater extent than hCTLA-4, dimeric hCTLA-4-Ig (e.g., dimeric, CTLA-4-IgG1 or CTLA-4-IgG2), Orencia® protein and/or dimeric LEAY29-Ig. Some of these monomers have the ability to suppress or inhibit one or more of these immune responses to a greater extent than the monomial is hydrated hCTLA-4, Monomeric hCTLA-4-Ig and/or Monomeric LEAY29-Ig. Assume that these mutant dimers CTLA-4-Ig and the monomers are effectively used in various therapeutic and/or prophylactic treatment of diseases or disorders in which immunosuppressive treatment would be effective, including, for example, methods of treatment of autoimmune diseases and disorders, and methods of inhibiting transplant rejection of organ, cell or tissue.

In another aspect, the invention provides isolated or recombinant dimer fused protein (e.g., mutant dimer fused protein, CTLA-4-Ig)comprising two Monomeric fused protein (for example, a mutant protein, CTLA-4-Ig), where each such Monomeric protein comprises: (1) a polypeptide (e.g., mutant extracellular domain of CTLA-4), comprising a amino acid sequence which (a) differs from the amino acid sequence of SEQ ID NO:31 for no more than 6 amino acid residues, and (b) includes, at least one of the following: a methionine residue at the position corresponding to position 50 of SEQ ID NO:31, a lysine residue at the position corresponding to position 54 of SEQ ID NO:31, a glutamic acid residue in the position corresponding to position 55 of SEQ ID NO:31, a Proline residue at a position corresponding to position 64 of SEQ ID NO:31), the serine residue at the position equivalent is the position 65 of SEQ ID NO:31, the phenylalanine residue at the position corresponding to position 70 of SEQ ID NO:31, where the position of the amino acid residues are numbered according to SEQ ID NO:31; and (2) a polypeptide Ig Fc, where specified dimer binds hCD80 and/or hCD86 and/or hCD86-Ig and/or hCD86-Ig) and/or inhibits an immune response. The invention also includes isolated or recombinant Monomeric protein, as described above, which binds CD80 (e.g., hCD80 and/or CD86 (e.g., hCD86) (and/or CD80-Ig, for example, hDC80-Ig and/or CD86-Ig, e.g., hCD86-Ig) and/or induces an immune response in vitro or in vivo. Ig Fc polypeptide may include IgG2 Fc IgG1 Fc, IgG4 Fc or mutant IgG Fc, which has reduced effector function, or Fc receptor binding. Ig Fc polypeptide may include a sequence having at least, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence selected from the group comprising SEQ ID NOS:184-186 and 218. N-terminal polypeptide Ig Fc can be covalently linked or fused directly or indirectly (via a linker, including, for example, 1-10 amino acids) with the end of the mutant polypeptide, CTLA-4 EVA. In some such dimers or monomers mutant polypeptide CTLA-4 EVA includes a glutamic acid residue in the position corresponding to position 104, the aspartic acid residue in the position corresponding to position 30, and/or the isoleucine residue at the position correspond to the eat to position 32 of SEQ ID NO:31.

Some of these dimers have the avidity of binding to CD86 (e.g., hCD86) or dimeric CD86-Ig (e.g., hCD86-Ig), which is almost equal to or greater than the avidity of binding of the hCTLA-4 protein, a dimeric hCTLA-4-Ig, protein Orencia® and/or dimeric LEAY29-Ig to CD86 or dimeric CD86-Ig, respectively. Some of these dimers have the avidity of binding to CD80 (e.g., hCD80) or dimeric CD80-Ig (e.g., hCD80-Ig), which is greater than the avidity of binding of the hCTLA-4, dimeric hCTLA-4-Ig and/or protein Orencia® to CD80 or dimeric CD80-Ig, respectively. Some of these monomers exhibit a binding affinity of or avidity for CD86 (e.g., hCD86) or CD86-Ig (e.g., hCD86-Ig), which is almost equal to or greater than the binding affinity of or avidity Monomeric hCTLA-4, Monomeric hCTLA-4-Ig or Monomeric LEA29Y-Ig to or CD86 CD86-Ig, respectively. Some of these monomers have the binding affinity of or avidity for CD80 (e.g., hCD80) or CD80-Ig (e.g., hCD80-Ig), which is greater than the binding affinity of or avidity Monomeric hCTLA-4 or Monomeric hCTLA-4-Ig to CD80 or dimeric CD80-Ig, respectively.

Some such mutant dimers CTLA-4-Ig and monomers have the ability to suppress or inhibit one or more immune responses (e.g., T-cell activation or proliferation, cytokine production, induction of activation markers, inflammation, production anticollagen antibodies,T-cell-dependent antibody responses in vitro and/or in vivo, as discussed in detail below. Some of these dimers have the ability to suppress one or more of these immune responses to a greater extent than hCTLA-4, dimeric hCTLA-4-Ig, protein Orencia® and/or dimeric LEAY29-Ig. Some of these monomers have the ability to suppress or inhibit one or more of these immune responses to a greater extent than Monomeric hCTLA-4 or Monomeric hCTLA-4-Ig. Assume that these mutant dimers CTLA-4-Ig and the monomers are effectively used in various therapeutic and/or prophylactic treatment of diseases or disorders of the immune system, in which immunosuppressive treatment would be effective (for example, autoimmune diseases and disorders, and methods of inhibiting transplant rejection of organ, cell or tissue).

Any such dimeric or Monomeric mutant dimer or monomer fused protein, CTLA-4-Ig, described above, can also include a peptide that facilitates secretion of the fused protein from the host cell. The peptide is an optional signal peptide. With the end of the signal peptide is typically covalently linked to the N-end of the fused protein. The signal peptide can include the amino acid sequence having at least, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence SEQ ID NO:182 or SEQ ID NO:216. The signal is eptid may include amino acid sequence, with at least, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence comprising amino acid residues 1-35, 1-37 1-36 or SEQ ID NO:160. Moreover, as discussed below, any such Monomeric or dimeric mutant protein, CTLA-4-Ig, described above, may include one or more amino acid residues that are glycosylated or pegylated.

The invention also provides the Mature/secretory mutant protein, CTLA-4-IgG2 length of 352 amino acids and includes a mutant polypeptide, CTLA-4 EVA, containing 124 amino acid residue and the human IgG2 Fc polypeptide, comprising 228 amino acid residue. Illustrative of the mutant polypeptides, CTLA-4 EVA include those polypeptides that include the sequence identified by any of SEQ ID NOS:1-73. Illustrative mutant fused proteins, CTLA-4-IgG2 include those that include the amino acid sequence identified by any of SEQ ID NOS:74-79, 197-200, 205-214, and 219-222. If necessary amino acids of the Mature mutant CTLA-4-IgG2 fused protein can be numbered, starting with the first amino acid residue of the mutant CTLA-4-IgG2 (namely the first residue mutant polypeptide CTLA-4 KJV). In some aspects, the first residue mutant fused protein, CTLA-4-IgG2 (or mutant CTLA-4 KJV) is Soboh is methionine, and thus, the numbering of the amino acids of mutant fused protein, CTLA-4-IgG2 (or mutant CTLA-4 EVA) will begin with a methionine (designated as amino acid residue 1).

The invention also includes isolated or recombinant multimeric fused proteins comprising two or more mutant fused proteins, CTLA-4-Ig, described above. In some cases multimer is a dimer fused protein comprising two mutant fused protein, CTLA-4-Ig, which may be identical fused proteins (i.e glycosilated) or different fused proteins (i.e. heterodimer). In some cases multimer is a tetramer protein, which includes four mutant polypeptide, CTLA-4-EVA on this invention. The tetramer can include four identical mutant polypeptide CTLA-4 EVA (that is, homotetramer) or any combination of the four mutant CTLA-4 EVA polypeptides according to this invention, so that all four mutant polypeptide CTLA-4 EVA are not identical (i.e., heterotetramer). Some of these multimeric bind CD80 and/or CD86 (and/or hCD80-Ig and/or hCD86-Ig) and/or suppress or inhibit an immune response.

This invention includes a soluble form of any of the polypeptides, fused protein and multimers described above. Also includes a soluble form of the conjugate according to this invention, is described below. The solution is that the molecules of this invention, for example, soluble polypeptides, fused dimeric proteins, Monomeric fused proteins, multimeric and the conjugates of this invention are not United or United or associated with the cell. Some of these soluble molecules are in solution or is able to circulate, for example, into fluid (e.g., in the body of the subject). The signal peptide may generally be used to facilitate secretion of this molecule, but the signal peptide is cleaved during the secretion of the molecule from the host cell. Thus, in most cases, soluble molecule, such as a soluble polypeptide, dimeric protein, Monomeric protein or multimer, does not include the signal peptide. As discussed above, the mutant polypeptide extracellular domain of CTLA-4 in this invention can be associated with Ig molecule, including, for example, part of the Ig polypeptide, such as, for example, the polypeptide Ig Fc, resulting in soluble protein. Thus, in one aspect, the invention includes a soluble mutant fused proteins, CTLA-4-Ig, which include any of the mutant polypeptide, CTLA-4 EVA in this invention, as described herein, fused or associated with at least part of an Ig polypeptide, such as, for example, wild-type Ig Fc (for example, human IgG2 Fc) or mutant Ig Fc polypeptide. Such rest the action mutant fused proteins, CTLA-4-Ig can be Monomeric or dimeric fused proteins and include those mutant monomers CTLA-4-Ig and dimers fused protein, described in detail above and elsewhere in this description, including the examples below. As described in detail above and elsewhere in this description, some of these soluble Monomeric and dimeric fused proteins may have the ability to bind CD80 and/or CD86 and/or an ability to suppress or inhibit an immune response (e.g., T-cell activation or proliferation in vitro and/or in vivo applications.

Assume that such soluble molecules according to this invention are particularly effective in various applications, including, for example, therapeutic and prophylactic methods of treatment of diseases and disorders of the immune system (e.g., autoimmune diseases), and prophylactic and therapeutic methods for inhibiting transplant rejection cells, organ or tissue. Soluble molecules according to this invention, for example, soluble recombinant mutant polypeptides, CTLA-4 EVA, Monomeric and dimeric mutant fused proteins, CTLA-4-Ig, mutant CTLA-4 EVA conjugates, mutant compared CTLA-4-Ig, multimer, including mutant polypeptides, CTLA-4 EVA or mutant CTLA-4-Ig, multimer, including mutant conjugates, CTLA-4, or a mutant CTLA-4-Ig conjugates of the invention that bind CD80 and/or CD86, when administered to a subject in a therapeutically or prophylactically effective amount to inhibit usaimage the due between endogenous CD80 and/or CD86 and endogenous CD28, thereby, inhibiting the subject's immune system response or an immune system attack on healthy tissue, organs and/or cells of the subject. In cases where the subject is the recipient of the healthy tissues, organs and/or cells of the donor (for example, when the subject is the recipient received donor tissue graft or cage or graft-body), such soluble molecules inhibit the interaction between endogenous CD80 and/or CD86 and endogenous CD28, thereby inhibiting harmful response or an immune system attack on the subject of healthy tissue, organs and/or cells obtained by the entity from a donor. By suppressing response or immune system attacks healthy tissues of the body side effects (eg, pain, joint inflammation and the like)associated with such response or immune system attack on healthy tissue, organs and/or cells of the subject can be reduced, and disorders arising from such a response or attack can be delayed or prevented.

Methods of measuring binding affinely and avednesday polypeptides according to this invention described above, including, for example, mutant polypeptides, CTLA-4 EVA, dimeric and Monomeric mutant fused proteins, CTLA-4-Ig and multimer according to this invention, well-known experts in this field and include, for example, but not limiting what is next, Biacore™ technology (GE Healthcare), isothermal titrimetric microcalorimetry (MicroCal LLC, Northampton, MA), ELISA, methods of phage display measure the affinity of binding and FACS methods. Biacore methods are described in more detail in example 4 below. FACS or other ways of sorting are described in more detail above and elsewhere in this description. Methods of measurement of avednesday binding polypeptides according to this invention to hCD80 and/or hCD86 using phage ELISA described in example 2 below.

Ways of defining and measuring T-cell responses induced by molecules of this invention (including, for example, mutant polypeptides, CTLA-4 EVA, dimeric and Monomeric mutant fused proteins, CTLA-4-Ig and multimer in this invention are well known to specialists in this field. T-cell activation is typically characterized physiological events, including, for example, T-cell-associated synthesis of cytokines (such as IFN-γ production) and the induction of activation markers (e.g., CD25, IL-2 receptor). CD4+ T cells recognize their immunogenic peptides in the context of MHC class II molecules, whereas CD8+ T cells recognize their immunogenic peptides in the context of MHC class I molecules. Illustrative methods of assessing and measuring the ability of the molecules according to this invention, described above, to inhibit or suppress T-cell activation and/or T-cell PR is literaly or block the transmission of the signal through CD86 and/or CD80 described in examples 5-8 and elsewhere in this specification.

Polypeptides, Monomeric and dimeric fused proteins and multimer according to this invention, including those discussed above, optionally also include additional amino acids, such as methionine added to the N-end and/or peptide marker for the purification or identification. The polypeptides according to this invention, including those discussed above, optionally also include an amino acid subsequence for purification, such as, for example, the subsequence selected from the epitope of the marker FLAG marker polyhistidine sequence, and a GST (glutathione S-transferase) fusion.

In addition, as discussed in more detail below, the invention includes isolated, recombinant or synthetic nucleic acid encoding all polypeptides, fused proteins and multimer according to this invention described above and in more detail below.

Identity sequence

As discussed above, in one aspect, the invention includes isolated or recombinant polypeptide that includes an amino acid sequence having at least, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,5% or 100% sequence identity with the amino acid sequence selected from the group comprising SEQ ID NOS:1-73, where the polypeptide binds CD80 or CD86 or wncl the exact each domain and/or has the ability to suppress or inhibit an immune response. In another aspect, as described in detail below, the invention provides isolated or recombinant nucleic acid comprising a polynucleotide sequence that encodes a polypeptide comprising amino acid sequence having at least, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,5% or 100% sequence identity with at least one amino acid sequence selected from the group comprising SEQ ID NOS:1-73, where the polypeptide has an ability to bind CD80 and/or CD86 and/or EVA and/or has the ability to suppress the immune response, or a complementary polynucleotide sequence.

The degree to which the sequence (polypeptide or nucleic acid) is similar to the other provides a definition similar structural and functional properties of two sequences. Accordingly, in the context of this invention, sequences that have a sequence similar to any given sequence, for example, are characteristic of the present invention. Sequences that have the percentage of sequence identities, as defined below, are characteristic of the present invention. Can be used various ways of determining the relationship of the sequences, including Virabyan the e manually and alignment and sequence analysis using the computer. Available a variety of computer programs for sequence alignment, or they can be created by an expert in the field.

As noted above, the nucleic acid sequences and polypeptides that are applied to the subject of this invention need not be identical, but can be essentially identical to the corresponding nucleic acid sequence according to this invention or of the polypeptide according to this invention, respectively. For example, the polypeptides according to this invention can be subject to various changes, such as one or more amino acid insertions, deletions and/or substitutions, or conservative or nonconservative, including those cases where such changes might provide for certain advantages in their use, such as when therapeutic or prophylactic use, or the introduction or diagnostic use. Nucleic acid according to this invention may also be subjected to various changes, such as one or more substitutions of one or more nucleic acids in one or more codons, so that a particular codon encodes the same or a different amino acid, leading to silent variations (for example, a mutation in the nucleotide sequence in the silent mutations in inoculates sequence, for example, when the encoded amino acid is not changed by primulaceae nucleic acid) or namoloasa variations, one or more divisions of one or more nucleic acids (or codons) in the sequence, one or more additions or insertions of one or more nucleic acids (or codons) in the sequence, the cleavage or one or more otsepleniya one or more nucleic acids (or codons) in the sequence. Nucleic acids can also be modified to include one or more codons that are optimal expression in an expression system (e.g., bacterial or mammalian), and, if necessary, the specified one or more codons still encode the same(s) amino acid(s). Such alteration of nucleic acids may provide certain advantages in their therapeutic or prophylactic use, or the introduction, or diagnostic use. Nucleic acids and polypeptides can be modified in various ways, provided that they include a sequence essentially identical (as defined below) of the sequence corresponding to the nucleic acid or polypeptide according to this invention.

The terms "identical" or "identity" in the context of the Vuh or more sequences of nucleic acids or polypeptides, refers to two or more sequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned with the maximum similarity as determined using the algorithm of comparison of the sequences described below, or by using visual analysis. "Percentage of sequence identity" ("% identity") of the analyzed sequence with a reference (namely, given a sequence means that the sequence is identical (namely on the basis of amino acid-to-amino acid to the amino acid sequence or on the basis of the nucleotide-to-nucleotide for polynucleotide sequences) by a certain percentage with a given sequence through the comparison length.

The percentage of sequence identity ("% sequence identity" or "% identity") of the analyzed sequences with a given sequence can be calculated as follows. First, the optimal alignment of two sequences is determined using the algorithm of comparison of the sequences with specific alignment options. This definition of optimal alignment is conducted using a computer or may be the calculated manually as is described below. Then two optimally aligned sequences are compared through a comparison of the length and determine the number of positions in the optimal alignment, in which identical residues are in both sequences, thereby ensuring that the number of matching positions. The number of matching positions is then divided by the total number of positions compared length (which, unless specified to the contrary, is the length of the sequence) and then multiply the result by 100 to obtain the percentage identity to the sequence of the studied sequence with a given sequence.

With respect to amino acid sequences, typically one sequence is considered as a "reference sequence" (e.g., amino acid sequence according to this invention), which compares one or more other sequences, namely "the analyzed sequence(s)" (e.g., sequence present in the database sequences). The comparison algorithm uses certain sequences alignment options to determine the optimal alignment between the reference sequence and the analyzed sequence(s). When comparing a given sequence database posledovatelno is her such as, for example, GENBANK® database (a database of Genetic Sequences; the Department of health and human services U.S.) or GENESEQ® database (Thomson Derwent; also available as DGENE® database on STN), usually only the specified sequence and alignment options enter in the computer; optimal alignment between the reference sequence and each of the investigated sequence returns back to a certain number of analyzed sequences.

1. Determination of the optimal alignment

Two amino acid sequences are optimally aligned"when they are aligned using certain parameters, namely the specific matrix of amino acid substitutions, the penalty for introducing a gap (also known as the penalty for opening a gap) and the penalty at the gap continued to achieve the highest weight similarities, possible for that pair of sequences. BLOSUM62 matrix (Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89(22): 10915-10919) is often used by default as a matrix replacement algorithms alignment of amino acid sequences (such as BLASTP, as described below). The penalty for introducing a gap impose for embedding single amino acid gap in one of the aligned sequences, and the penalty at the gap continued impose for each position OS the Atka in the gap. If not defined the opposite, the alignment parameters used here are the following: BLOSUM62 matrix replacement, the penalty for introducing a gap = 11 and the penalty for continued gap = 1. The weight of the alignment is determined by amino acid positions of each sequence, with which the alignment begins and ends (for example, land clearing), and optional insertion of a gap or multiple gaps in both sequences, so as to lead to the highest possible weight of similarity.

Because the optimal alignment between two or more sequences can be defined manually (as described below), a process facilitated by the application running on the computer alignment algorithm such as BLAST® (National Library of Medicine), for example, BLASTP for amino acid sequences and BLASTN for nucleic acids sequences, described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402 and available to the masses through resources such as the website of the National Center for Biotechnology Information (NCBI). When applied computerized BLAST interface, if there is an option to use "filter low complexity", then this option should be turned off (i.e. no filter).

The optimal alignment between two amino acid sequences can also be determined by counting manually BLASTP (i.e. without a computer) using the same alignment options, defined above (matrix = BLOSUM62, the penalty for opening a gap = 11 and the penalty for continued gap = 1). For a start, the two sequences of initial level visual inspection. The initial weight of the alignment then calculate as follows: for each individual alignment position (namely, for each pair of aligned residues), the numerical value is assessed according to the BLOSUM62 matrix (figure 13). The sum of the values set for each pair of residues in the alignment represents the weight of the alignment. If the two sequences to be aligned are similar to a high degree, this is often the initial alignment ensures the highest possible weight alignment. The alignment with the highest possible weight of the alignment is the optimal alignment based on the parameters used for the alignment.

Examples of the calculation manually indicators alignment of the two sequences shown in figures 14A-14D. Figure 14A shows the calculation of the weight equalization for arbitrary alignment (alignment 14A) "given" sequence defined here as the remains 39-53 sequence of human CTLA-4 EVA (SEQ ID NO:159), and "search" the sequence defined here as the remains 40-54 D3 (SEQ ID NO:61). The numerical value obtained from the BLOSUM62 matrix for each Viron is authorized pairs of amino acids, shown under each position in the alignment.

Figure 14C shows the weight equalization for optimal alignment of the two sequences. To render each identical pair of amino acids in the alignment shown in bold. The alignment in figure 14B (alignment 14B) below leads to the highest possible weight equalization (the sum of the amounts shown under each aligned position of the two sequences; any other alignment of these two sequences, with or without gaps, will result in a lower weight alignment.

In some cases, a higher weight alignment can be obtained by embedding one or more gaps in the alignment. Whatever gap was built in alignment, prescribe a penalty for opening a gap and in addition assign a penalty on the gap continued for each position of the residue in the gap. Thus, with the use of alignment options described above (including the penalty for opening a gap = 11 and the penalty for continued gap = 1), the gap of one residue in the alignment will correspond to the value- (11+(1×1))=-12, assigned to the gap; the gap of the two residues will correspond to the value- (11+(2×1))=-13, assigned to the gap, etc. This calculation is repeated for each new gap built into alignment.

The following is an example that demonstrates how strava is their a gap in the alignment can lead to a higher weight, alignment, despite the penalty for introducing a gap. Figure 14C shows the alignment (alignment 14C) "given" sequence defined here as the remains 39-53 sequence of human CTLA-4 EVA (SEQ ID NO:159), and "search" the sequence defined here as residues 41-55 D3 (SEQ ID NO:61), but in this case without amino acids 49-50, which was removed. Alignment 14C, which is the best possible alignment without embedding any gaps, leads to weight equalization 34.

The alignment in figure 14D (alignment 14D) shows the effect of embedding the gap of two residues in the lower sequence in weight alignment. Although the total penalty for introducing a gap is 13 (the penalty for opening a gap 11 and 2-fold penalty for gap continued 1), the total weight of the alignment of two sequences increases to 43. Alignment D below leads to the highest possible weight of the alignment and is, thus, an optimal alignment of the two sequences; any other alignment of these two sequences (with or without gaps) will lead to lower weight alignment.

It should be clear that the examples of calculations of alignment of the sequences described above, which use a relatively short sequence, are provided for illustrative purposes. In practice, using the appropriate alignment parameters (BLOSUM62 matrix, the penalty for opening a gap = 11 and the penalty for continued gap = 1) in General refers to amino acid sequences with a length of 85 amino acids and more. NCBI web site provides the following alignment options for sequences of other lengths that are acceptable for computer and count the alignment manually, using the same procedure as described above. For sequences 50-85 amino acids in length, the optimal parameters are BLOSUM80 matrix (Henikoff and Henikoff, see above), the penalty for opening a gap = 10, and the penalty for continued gap = 1. For sequences 35-50 amino acids in length, optimal parameters are RAM matrix (Dayhoff, M.O., Schwartz, R. & Orcutt, B.C. (1978) "A model of evolutionary change in proteins" in Atlas of Protein Sequence and Strucrute, vol. 5, suppl. 3, M.O. Dayhoff (ed.), pp.345-352, Natl. Biomed. Res. Found., Washington, DC), the penalty for opening a gap = 10, and the penalty for continued gap = 1. For sequences of length less than 35 amino acids optimal parameters are RASO matrix (Dayhoff, M.O., see above), the penalty for opening a gap = 9 and the penalty for continued gap = 1.

2. The calculation of the percent identity

As soon as the sequence is optimally aligned, calculate the percentage identity of the investigated sequence relative to the sequence by counting the number of provisions in the optimal alignment, which contain the antibodies of identical residue pairs, divide this value by the number of residues compared to the length (also called the plot of comparison), which, unless specified to the contrary, is the number of residues in defined sequence, and multiply the resulting number by 100. Coming back to the alignments above, in each example the sequence, denoted as the set (upper) sequence has a length of 15 amino acids. In the alignment In 12 pairs of aligned amino acid residues (shown in bold) are identical in the optimal alignment sequence (top) with the analyzed sequences (bottom). Thus, this particular study sequence has (12/15)×100=80% identity with a full-sized 15 residues of the sequence; in other words, the analyzed sequence in the alignment is at least 80% identity of amino acid sequence with a given sequence. In alignment D 11 pairs of amino acid residues (shown in bold) in the optimal alignment are identical; therefore, this particular study sequence has (11/15)×100=73,3% identity with the sequence with a total length of 15 amino acids; in other words, the analyzed sequence alignment D is at least 73% ID is tecnost amino acid sequence with the sequence.

As applied to polypeptides, the terms "substantial identity" (or "essentially identical") usually means that when two amino acid sequences (namely, a given sequence and the sequence under consideration) are optimally aligned using BLASTP algorithm (either manually or using a computer) using acceptable parameters described above, we analyzed the sequence has at least, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,5% or 100% amino acid sequence identity with the sequence. In some cases, substantial identity exists when comparing the lengths of at least 100 amino acid residues, such as, for example, at least, 110, 115, 118, 119, 120, 121, 122, 123, 124, 125, 130, 135, 140, 145, 150, 200, 250, 300, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 375, 400, 450 or 500 amino acid residues.

Similarly, as applied in the context of two nucleic acids sequences, the expression substantially identical (or substantially identical) means that when two nucleic acid sequences (namely the set and test sequence) are optimally aligned using BLASTN algorithm (either manually or using a computer) with the use of acceptable parameters, described below, studies the target sequence has at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,5% or 100% identity to the nucleic acid sequences with a given sequence. The parameters used for sequence alignment of nucleic acids, are as follows: remuneration for match 1, penalty for mismatch -3 penalty to make the gap 5, the penalty for a gap continued 2 (matrix substitutions not used in BLASTN algorithm). In some cases, substantial identity exists when comparing the lengths of at least 300 nucleotide residues, for example at least, 330, 345, 354, 357, 360, 363, 366, 369, 362, 365, 375, 390, 405, 420, 435, 450, 600, 750, 900, 1035, 1038, 1041, 1044, 1047, 1050, 1053, 1056, 1059, 1062, 1065, 1068, 1071, 1074, 1077, 1080, 1200, 1350 or 1500 nucleotide the remnants.

Can be used by other programs alignment of sequences known in the art. The ALIGN program gives the optimal global (total) alignment of two sequences of proteins or nucleic acids using a modification of the dynamic programming algorithm described in Myers and Miller, CABIOS 4: 11-17 (1988). The ALIGN program is usually, though not necessarily, used for the weighted end gaps. If there are penalties for opening or gap continued, they most often are in the range of from about -5 to -15 and 0 to -3, respectively, more mainly about -12 and the t 0.5 to -2, respectively, for alignments of amino acid sequences and -10 to -20 and from -3 to -5, respectively, more about -16 and -4, respectively, for alignments of sequences of nucleic acids. The ALIGN program is also described in Pearson et al, Proc. Natl. Acad. Sci. USA 85:2444-48 (1988), and Pearson et al, Meth. Enzymol. 18:63-98 (1990).

Alternatively, and particularly for multiple sequence analysis (i.e. comparison of more than three sequences) can be used by the program CLUSTALW (described in, for example, Thompson et al., Nucl. Acids Res. 22:4673-4680 (1994)). The program CLUSTALW is an algorithm that is acceptable for multiple alignments of DNA and amino acid sequences (Thompson et al., Nucl. Acids Res. 22:4673-4680 (1994)). CLUSTALW performs multiple pairwise comparisons between groups of sequences and combines them multiple alignment on the basis of homology. In one aspect, the penalties for opening a gap and the gap continued was 10 and 0.05, respectively. Alternative or additionally, the program CLUSTALW works with the use of "dynamic" (versus "fast") settings. Typically, the analysis of nucleotide sequences with CLUSTALW carried out using BESTFIT matrix, whereas amino acid sequences were analyzed with the use of variable settings BLOSUM matrices depending on the level of identity between sequences (e.g., as used is implemented in CLUSTALW version 1.6 program, available from the San Diego Supercomputer Center (SDSC) or version W 1,8, available from the European Bioinformatics Institute, Cambridge, UK). Mainly, CLUSTALW settings are set for SDSC CLUSTALW default settings (for example, with respect to special penalties for hydrophilic gap in the analysis of amino acid sequences). The program CLUSTALW also described in, for example, Higgins et al, CABIOS 8(2): 189-91 (1992), Thompson et al, Nucleic Acids Res. 22:4673-80 (1994), and Jeanmougin et al., Trends Biochem. Sci. 2:403-07 (1998).

In alternative format identity or percent identity between a particular pair of aligned amino acid sequences refers to the percentage of identical amino acid sequence, which is obtained by CLUSTALW analysis (for example, version W 1,8), counting the number of identical matches in the alignment and dividing this number of identical matches on more of (i) the length of the aligned sequences, and (and) 96, and with the application with the following default settings of ClustalW to get slow/accurate pairwise alignments is the Penalty for opening a gap: 10; Penalty for gap continued: 0,10; Matrix comparison of proteins: the series gonnet on; Matrix comparing DNA: IUB; slow/fast pairwise alignment Toggle = slow or full alignment.

Another useful algorithm for determining percent identity or percent similarity is the FASTA algorithm, which is th described in Pearson et al., Proc Natl. Acad. Sci. USA 85:2444 (1988), and Pearson, Methods Enzymol. 266:227-258 (1996). Typical parameters used in the FASTA alignment of DNA sequences to calculate percent identity are optimized, BL50 Matrix 15: -5, k-tuple (same plot) = 2; the penalty for linking = 40, optimize = 28; the penalty for introducing a gap = -12, the penalty for continuing gap = -2; width = 16.

Other acceptable algorithms include the BLAST and BLAST 2.0 algorithms, which facilitate the analysis of at least two amino acid or nucleotide sequences by alignment of selected sequences against multiple sequences in the database (e.g., GenSeq), or, if they are modified, with additional algorithm, such as BL2SEQ, between the two selected sequences. Software for conducting BLAST analyses is publicly available from the National Center for Biotechnology Information (NCBI) (address global web site ncbi.nlm.nih.gov). BLAST algorithm involves first defining pairs of sequences with high weight (HSP) by the definition of short words of length W in the specified sequence, which either match or satisfy some positive threshold value weight T when aligned with a word of the same length in a database sequence. T refers to the weight limit of neighboring words (Altschul t al., see above). These initial hit neighboring words are as the seed for the initial search to detect longer HSP containing them. Hit words are in both directions along each sequence, while aggregate alignment can be increased. The aggregate indicators are calculated using, for nucleotide sequences, the parameters M (reward score for each pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequence matrix replacement is used to calculate the total. The sequel to the hit words in each direction is stopped when: aggregate alignment falls on the value of X from its maximum achievable value; aggregate indicator goes to zero or below, due to the accumulation of one or more alignments of residues with negative result; or reaches the end of each sequence. The parameters of the BLAST algorithm W, T, and X determine the sensitivity and speed of the alignment. BLASTN program (for nucleotide sequences) can be used with the length of words (W) is equal to 11, an expectation (E) of 10, M=5, N=-4 and a comparison of the two circuits. For amino acid sequences, the BLASTP program (e.g., BLASTP 2,0,14; June 29, 2000) can be is used with the length of the words, equal to 3, and expectation (E)equal to 10. BLOSUM62 matrix substitutions (see Henikoff &Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:of 10,915) uses alignment (B)of 50, expectation (E)of 10, M=5, N=-4, and a comparison of both circuits. Again, as in other acceptable algorithms, the severity of the comparison may be increased until the program determines only the sequences that are more closely associated with those in the order listed here (for example, a polypeptide comprising amino acid sequence having at least, 85, 90, 91, 92, 93, 49, 95, 96, 97, 98, 99% or 100% identity with the amino acid sequence selected from SEQ ID NOS:1-79, 197-200, 205-214, and 219-222; or a nucleic acid comprising a nucleotide sequence having at least, 85, 90, 91, 92, 93, 49, 95, 96, 97, 98, 99% or 100% identity with a nucleotide sequence selected from any of SEQ ID NOS:80-158, 201-204, 223 and 224, or she complementary nucleotide sequence.

BLAST algorithm also performs a statistical analysis of similarity or identity between two sequences (see, e.g., Karlin &Altschul, (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of the similarity or identity, provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides a criterion for the probability with which a match between two nucleotide or aminoxy the pilot sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the analyzed nucleic acid to the reference nucleic acid is less than about 0.2, such as less than about 0.01, or less than about 0.001 in.

BLAST software analysis also or alternatively can be modified by software filtering of low complexity, such as DUST or SEG programs, which are mainly integrated into the BLAST operating systems (see, for example, Wootton et al., Comput. Chem. 17:149-63 (1993), Altschul et al., Nat. Genet. 6:119-29 (1991), Hancock et al., Comput. Appl. Biosci. 10:67-70 (1991), and Wootton et al, Meth. Enzymol. 266:554-71 (1996)). In these aspects if you use the equality lambda, suitable settings for equality between 0.75 and 0.95, and including between 0.8 and 0.9. If in such an aspect, using indicators of the existence of a gap (or weight gap), the indicator of the existence of the gap is typically between about -5 and -15, usually about 10, and most often the measure of the gap is typically between about 0 to -5, such as between 0 and -3 (for example, -0,5). Similar parameters gap can be used in other programs as appropriate. BLAST programs and principles associated with them are described in, for example, Altschul et al., J. Mol. Biol. 215:403-10 (1990), Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-68 (199) (modified Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-77 (193)), and Altschul et al., Nucl. Acids Res. 25:3389-3402 (1997).

Another example of a useful algorithm is a built-in a PILEUP software. A PILEUP program creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments in order to show the relationship and the percentage of sequence identity or percent similarity of sequences. PILEUP uses a simplification of the way of progressive alignment Feng &Doolittle (1987) J. Mol. Evol. 35:351-360, which is similar to the method described in Higgins & Sharp (1989) CABIOS 5:151-153. The program can align up to 300 sequences, each with a maximum length of 5000 nucleotides or amino acids. The multiple alignment procedure begins with a pairwise alignment of the two most similar sequences, forming a cluster of two aligned sequences. This cluster is then level with the next most similar sequence or cluster of aligned sequences. Two clusters of sequences aligned by a simple continuation of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive pairwise alignments. The program works by identifying specific sequences and their and inability or nucleotide coordinates for regions of sequence comparison and by using certain parameters of the program. Using a PILEUP reference sequence is compared with other analyzed sequences to determine the percent sequence identity or percent similarity of sequences) using the specified parameters. Illustrative parameters for a PILEUP program of the following: the mass gap default (3,00), the standard penalty for a gap continued (0,10), and weighted end gaps. PILEUP is a component of a software package GCG sequence analysis, for example, version 7.0 (Devereaux et al. (1984) Nucl. Acids Res. 12:387-395).

Other useful algorithms for the analysis of identity include an algorithm for local homology Smith and Waterman (1981) Adv. Appl. Math. 2:482, the algorithm homology alignment Needleman and Wunsch (1970) J. Mol. Biol. 48:443, and a way to search for similarities Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444. Computerized implementation of these algorithms (e.g., GAP, BESTFIT, FASTA and TFASTA) are provided in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, WI.

Variation sequences

As discussed above, in one aspect, the invention provides isolated or recombinant polypeptide mutant extracellular domain of CTLA-4, which includes the amino acid sequence which (a) differs from the amino acid sequence selected from the group comprising SEQ ID NOS1-73 no more than 6 amino acid residues (for example, not more than 1, 2, 3, 4, 5 or 6 amino acid residues), where the mutant polypeptide, CTLA-4 EVA binds CD80 and/or CD86 and/or the extracellular domain of each or both and/or inhibits an immune response. Such amino acid(s) substitution(s) include(s) conservative(s) of amino acid(s) substitution(s).

As a non-limiting example, a polypeptide according to this invention may have an amino acid sequence that differs from SEQ ID NO:1 in General for up to 6 amino acids (which can be a combination of amino acid substitutions, deletions and/or insertions, including those described above). In some cases none, some or all of the replacement is the replacement of the corresponding group of substitutions defined below.

Amino acid substitutions according to the invention may include, but are not limited to, one or more conservative amino acid substitutions. Conservative replacement amino acid residue usually includes replacement member of the same functional class of amino acid residue at the residue that belongs to the same functional class (identical amino acid residues are considered as functionally homologous or conservative in the calculation of functional homology). Table-conservative substitutions, providing functionally similar amino acids are well known in the art. One example represent the stations in table 1, which leads six illustrative groups containing amino acids, which can be considered "conservative substitutions" for each other.

Table 1
Group conservative substitutions of amino acid residues
1Alanine (A)Glycine (G)Serine (S)Threonine (T)
2Aspartic acid (D)Glutamic acid (E)
3Asparagine (N)Glutamine (Q)
4Arginine (R)Lysine (K)Histidine (H)
5Isoleucine (I)Leucine (L)Methionine (M)Valine (V)
6Phenylalanine (F) Tyrosine (Y)Tryptophan (W)

We can assume other groups of substitutions of amino acids. For example, amino acids can be grouped according to similar functions or chemical structure or composition (e.g., acidic, basic, aliphatic, aromatic, containing sulfur). For example, the aliphatic group may contain: Glycine (G), Alanine (A), Valine (V), Leucine (L), Isoleucine (I). Other groups containing amino acids, which are regarded as conservative substitutions for one another include: aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan (W); sulfur-containing: Methionine (M), Cysteine (C); basic: Arginine (R), Lysine (K), Histidine (H); acidic: Aspartic acid (D), Glutamic acid (E); non-polar uncharged residues: Cysteine (C), Methionine (M) and Proline (P); uncharged hydrophilic residues: Serine (S), Threonine (T), Asparagine (N) and Glutamine (Q). See also, Creighton (1984) Proteins, W.H. Freeman and Company, for additional groupings of amino acids. The enumeration of amino acid sequences here, together with the foregoing groups, replacement, provides a brief enumeration of all conservatively substituted amino acid sequence.

More conservative substitutions are in the classes of amino acid residues described above, which also or viola is rnative may be suitable. Conservative groups for replacements that are more conservative, include: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Thus, for example, in one particular aspect, the invention provides isolated or recombinant polypeptide comprising amino acid sequence that has at least, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with SEQ ID NO:1 (or SEQ ID NOS:1-79, 197-200, 205-214, and 219-222) and which differs from the sequence of SEQ ID NO:1, mainly (for example, at least, 50%, 60%, 70%, 75%, 80%, 90%), if not completely, on such a more conservative amino acid substitutions.

Additional groups of amino acid substitutions, which can also be used, can be determined using the principles described in, for example, Creighton (1984) PROTEINS: STRUCTURE AND MOLECULAR PROPERTIES (2d Ed. 1993), W.H. Freeman and Company. In some aspects, at least, 33%, 50%, 60%, 70% or more (e.g., at least, 75%, 80%, 90%, 95%, 96%, 97% or more substitutions in the variant amino acid sequences include the substitution of one or several amino acid residues in the amino acid sequence of this invention the residues that are in the same functional homology class (as determined using any suitable classification system, so the AK ones described above), and that amino acid residues amino acid sequence that is replaced.

Conservatively substituted variations of amino acid sequences according to this invention include substitutions of a small percentage, usually less than 10%, 9%, 8%, 7% or 6% of the amino acids amino acid sequence, or more typically less than 5%, 4%, 3%, 2% or 1% of amino acids amino acid sequences, conservatively selected amino acid of the same group of conservative substitutions.

The invention includes polypeptides that include amino acid variations in the amino acid sequence of this invention, are described here. As discussed above, in one aspect, the invention provides isolated or recombinant polypeptides (e.g., mutant polypeptides, CTLA-4, such as, for example, mutant polypeptides, CTLA-4 EVA), each of which includes an amino acid sequence having at least, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,5% or 100% sequence identity with at least one amino acid sequence selected from the group comprising SEQ ID NOS:1-73, where the polypeptide binds CD80 and/or CD86 or polypeptide fragment of CD80 and/or CD86 (or EVA each or both) and/or suppresses an immune response. Such polypeptides may vary on the well or several amino acid deletions, additions or substitutions including one or more conservative or nonconservative substitutions, providing, however, that polypeptides have described functional properties. In a particular aspect, the invention provides polypeptide variants that include conservatively modified variations of any such polypeptide described herein, such as, for example, one that includes an amino acid sequence selected from the group of SEQ ID NOS:1-73.

As also discussed above, in another aspect, the invention provides isolated or recombinant fused proteins (e.g., mutant fused proteins, CTLA-4-Ig), each of which includes an amino acid sequence having at least, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,5% or 100% sequence identity with at least one amino acid sequence selected from the group comprising SEQ ID NOS:74-79, 197-200, 205-214, and 219-222, where the protein binds CD80 and/or CD86 (and/or CD80-Ig and/or CD86-Ig) and/or suppresses an immune response. Such fused proteins can differ by one or more amino acid deletions, additions or substitutions, including one or more conservative or nonconservative substitutions, providing, however, that slit proteins have described functional properties. In particular and is the aspect the invention provides polypeptide variants which include conservatively modified variations of any such fused protein described herein, such as, for example, one that includes an amino acid sequence selected from the group of SEQ ID NOS:74-79, 197-200, 205-214, and 219-222.

It also provides polypeptide variants of any isolated or recombinant polypeptide according to this invention described above or elsewhere in this description, where the amino acid sequence of the polypeptide variant differs from the corresponding amino acid sequence of the reference polypeptide by one or more conservative substitutions of amino acid residues, although non-conservative substitution is sometimes possible or even preferred (examples of such non-conservative substitutions are also discussed in this description). For example, the polypeptide sequence variants may differ from the mutant amino acid sequence of CTLA-4 on one or more substitutions of amino acid residues in the mutant amino acid sequence of CTLA-4 EVA and one or more amino acid residues having similar tc (it is a balance that has homology weight with respect to the residue in the corresponding amino acid sequence, which is substituted). The weight (and hence the size) amino acid residues of the polypeptide may be significant is about to change the structure of the polypeptide. Conservatism, based on the weight, or homology is based on the fact whether non-identical corresponding amino acid with a positive indicator on one of the matrices, based on the weight described herein (e.g., BLOSUM50 matrix; RAM matrix).

Like the above-described functional amino acid classes of naturally occurring amino acid residues can be divided into groups of conservatism by weight (which is divided between "strong" and "weak" groups of conservatism). The eight most used strong groups of conservatism by weight are Ser Thr Ala, Asn Glu Gln Lys Asn His Gln Lys Asn Asp Glu Gln, Gln His Arg Lys Met lie Leu Val Met He Leu, Phe, His, Tyr and Phe Tyr Trp. Weak group of conservatism by weight include Cys Ser Ala Ala Thr Val Ser Ala Gly Ser Thr Asn Lys Ser Thr Pro Ala, Ser Gly Asn Asp Ser Asn Asp Glu Gln Lys Asn Asp Glu Gln His Lys Asn Glu Gln His Arg Lys Phe Val Leu He Met and His Phe Tyr. Some versions of the program sequence analysis CLUSTAL W provide an analysis of the strengths and weaknesses of conservative groups by weight at the outlet of alignment, thus offering a traditional technique for determination of conservatism by weight (e.g., CLUSTAL W, provided by SDSC, which is usually used with SDSC default settings). In some aspects, at least, 33%, 50%, 60%, 70%, 80% or 90% of substitutions in such polypeptide variant include replacement, where the remainder with conservatism by weight, Amin replaces the acidic amino acid residue sequence, which belongs to the same group of conservatism by weight. In other words, the percentage of substitutions is stored in the expressions of the characteristics of the weight of amino acid residue.

The polypeptide sequence variants may differ from the mutant polypeptide, CTLA-4 in this invention one or more amino acid replacements at one or more amino acid residue having similar hydrophilic profile (i.e. which are similar hydrophilicity) with replaced by (the original) the remnants of the mutant polypeptide CTLA-4. A hydrophilicity profile can be determined using the Kyte-Doolittle index values for each naturally occurring amino acids in the following index: I (+4,5), V (+4,2), L (+3,8), F (A+2.8) (+2,5), M(+1,9); (+1,8), G (-0,4), T (A-0.7), S (-0,8), W (-0,9), Y (-1,3). P (-1,6), N (-3,2); E (for 3,5), Q (for 3,5), D (for 3,5), N (for 3,5), K (-3,9) and R (-4,5) (see, for example, U.S. Patent No. 4554101 and Kyte &Doolittle, J. Molec. Biol. 157:105-32 (1982) for further discussion). At least, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or 100% amino acid residues in the variant amino acid sequences that are not identical to the corresponding residues in identical or functionally homologous to the mutant amino acid sequence of CTLA-4 as described in this description ("the closest homolog)and its homologue may be selected from any of SEQ ID NOS:1-73, show me what her than +/-2 change hydrophilicity, including less than +/-1 changes in hydrophilicity and less than +/-0,5 changes hydrophilicity relative to the non-identical amino acid residue in the corresponding position in the closest homologue. The variant polypeptide may be a complete change in hydrophilicity relative to its the closest homolog selected from the group of SEQ ID NOS:1-73, less than about 150, less than about 100 and/or less than about 50 (e.g., less than about 30, 20 or 10).

Examples of typical amino acid substitutions that have similar or identical hydrophilicity include replacement of arginine-lysine, replacement of the glutamate-aspartate, replacement of serine-threonine, replacement of the glutamine-asparagine and replacement of valine-leucine-isoleucine. Algorithms and software, such as GREASE program, available from SDSC, provide the traditional method for fast calculation of profile hydrophilicity amino acid sequence. As a substantial proportion (e.g., at least about 33%), if not more (at least 50%) or almost all (e.g., about 65, 80, 90, 95, 96, 97, 98, 99%) amino acid substitutions in the polypeptide sequence variants will often have a measure of hydrophilicity similar to amino acid residue, which they replace in (reference) amino acid sequence, assume that the sequence of polipeptidnaja shows the output of the program GREASE, similar to the amino acid sequence. For example, in a particular aspect suggest that the polypeptide variant of SEQ ID NO:61 is the output of the GREASE program (or similar program), which is more similar to the output of the GREASE obtained by introducing amino acid sequence of SEQ ID NO:61, than that obtained with the use of the polypeptide DT CTLA-4 (e.g., hCTLA-4), which can be determined by visual examination or by comparison with computer graphics (e.g., graphic overlapping/alignment) and/or numerical output, obtained by analysis of the variant sequence and SEQ ID NO:1 on the program.

Conservative amino acid residues in the expression of functional homology, homology by weight and hydrophilic characteristics also apply to other variants of amino acid sequences according to the invention, including, but not limited to the following, for example, amino acid sequence variants, where amino acid sequence selected from the group comprising SEQ ID NOS:1-79197-200, 205-214, and 219-222.

In a particular aspect, the invention includes at least one such polypeptide variant comprising the amino acid sequence that differs from recombinant amino acid sequence selected from the group of SEQ ID NOS:1-79, 197-200, 205-214, and 29-222, where the amino acid sequence of the variant has at least one substitution of amino acid residue selected according to the conservative by weight or homology by weight or similar hydrophilicity profile, as discussed above. Such polypeptide variants described above, typically have the ability to bind CD80 and/or CD86 and/or the ability to inhibit at least one type of immune response, as described above and in more detail in the examples.

The sequence of signal peptides

The polypeptides according to this invention may also optionally include any reasonable number and type of additional amino acid sequences, such as one or more peptide fragments. In one embodiment, such a polypeptide according to this invention also includes a signal peptide. In General, the signal peptide directs the recombinant polypeptide in the endoplasmic reticulum when the recombinant polypeptide is expressed in a cell of the animal. The signal sequence that directs the movement of organelles and/or secretion of at least part of the polypeptide upon expression in a cell may be included. Such sequences are usually present in immature (and it is not fully held processing) the form of the polypeptide and sequentially removed/evaluation of the proposed degradation of cellular proteasome to obtain the Mature form of the protein. For example, a mutant polypeptide, CTLA-4 or a protein according to this invention may include any suitable signal sequence or combination of signal sequence that directs the polypeptide to the extracellular compartments, such as a sequence that directs the polypeptide to the fact that it was transported (for example, translirovalsya) (for example, so that the protein is processed or released from) the endoplasmic reticulum or the secretory route of transmission (e.g., ER, the Golgi apparatus and other related secretion of organelles and cell compartments), core, and/or which directs the polypeptide to secretarials of cells, translirovalsya in the cell membrane and reached the second cell, which is separate from the cells from which it is secreted. In this respect, the polypeptide can include the extracellular guide sequence (or "sorting signal"), which directs the polypeptide to endosomal and/or the lysosomal compartment(s) or other compartment, reaching MHC II to stimulate CD4+ and/or CD8+ T-cell presentation and response, such as lysosomal/endosomal-guide sorting the output signal from protein 1 associated with a membrane (e.g., LAMP-1 - see, for example, Wu et al. Proc. Natl. Acad. Sci. USA 92:161-75 (1995) and Ravipraskash et al., Virology 290:74-82 (2001)), a part of him or homologue (see, for example, U.S. Patent No. 5633234) or other acceptable lysosomal, endosomal and/or ER guide sequence (see, for example, U.S. Patent No. 6248565). In some aspects to the extracellular guide sequence can be desirable, that it was located near or adjacent to confirmed /identified epitope sequence(s) in the polypeptide, which can be determined using techniques known in the technical field, thus increasing the probability of T-cell presentation of polypeptide fragments that include such epitope(s). Such polypeptides can be expressed from an isolated, recombinant or synthetic DNA or RNA, is delivered to the cell host one or more transfer vectors nucleotides, including, for example, one or more of the gene transfer vectors, which are described later in this description.

The polypeptide can include a signal sequence that directs the polypeptide to the endoplasmic reticulum (ER) (for example, facilitates ER translocation of the polypeptide when the polypeptide is expressed in a cell of a mammal. The polypeptide may include any reasonable ER-guide sequence. Many ER-rails sequences known in the sphere of the technology. Examples of such signal sequences are described in U.S. Patent No. 5846540. The most commonly used ER/secretory signal sequences include the signal sequence of the yeast factor alpha, and virus signal sequence of a mammal, such as a signal sequence of the herpes virus gD. Illustrative signal peptides for the production of E. coli include STII or Ipp signal sequence of E. coli. Examples of signal sequences are described in, for example, U.S. Patent No. 4690898, 5284768, 5580758, 5652139 and 5932445. Acceptable signal sequence can be identified by experts in the field. For example, SignalP program (described in, for example, Nielsen et al. (1997) Protein Engineering 10: 1-6), publicly available from Center for Biological Sequence Analysis at the world website cbs.dtu.dk/services/SignalP, or similar software for sequence analysis, able to identify the domains that are similar to the signal sequence, can be used. Related methods for determining suitable signal peptides described in Nielsen et al., Protein Eng. 10(1): 1-6 (1997). The sequence can be analyzed manually according to the characteristics usually associated with signal sequences, as described in, for example, the Application for the European patent No. 0621337, Zheng and Nicchitta (1999) J. Biol. Chem. 274(51): 36623-30, and Ng et al. (1996) J. Cll Biol. 134(2):269-78.

Additional aspects

Any polypeptide according to this invention (including any protein in this invention) may be present as part of a larger amino acid sequence that occurs when the addition of one or more domains or subsequences to stabilize, or definition, or purification of the polypeptide. Such domains or subsequences can be covalently attached to the polypeptide according to this invention, as is clear to a person skilled in the art, and can form the construct. Amino acid sequence for purification may include, for example, epitope marker, FLAG marker, polyhistidine sequence, a GST fusion, or any other subsequence detection/purification or "marker", known in the art. These additional domains or a subsequence or have little or no effect on the activity of the polypeptide according to this invention or can be removed using stages of processing after synthesis, such as treatment with protease, the inclusion intein or similar.

Any polypeptide according to this invention (including any fused protein according to this invention may also include one or more modified amino acids. The modified amino acid can be, e.g. the, glycosylated amino acid, a pegylated amino acid, farnesiani amino acid, acetylated amino acid, biotinylated amino acid, an amino acid conjugated to a lipid part, and/or an amino acid conjugated to an organic derivational agent. The presence of modified amino acids may be preferred in, for example, (a) extend the half-life of the polypeptide in the serum and/or functional time half-life in vivo, (b) the reduction of antigenicity or immunogenicity of the polypeptide, (C) increase the security of the polypeptide, (d) increasing the bioavailability, (e) decrease effector function and/or (f) the reduction or inhibition of the undesired self (for example, the formation of aggregates) between two or more molecules according to this invention (such as between two or more dimers fused protein according to this invention). The amino acid(s) are modified, for example, co-translational or post-translational during recombinant production (e.g., N-linked glycosylation sites N-X-S/T motifs during expression in mammalian cells) or modified by synthetic methods.

The polypeptides according to this invention (including fused protein according to this invention)described herein may also be modified by choosing the mi methods, for example, by post-translational modification and/or synthetic modifications or variations thereof. For example, polypeptides or fused proteins according to this invention can be acceptable glycosylated, usually by expression in a cell of a mammal. For example, in one aspect the invention provides glycosylated polypeptides that can bind to CD86 and/or CD80 and/or have the ability to suppress the immune response (for example, T-cell proliferation or activation, as described herein, where each specified glycosylated polypeptide includes an amino acid sequence having at least, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with a sequence selected from the group comprising SEQ ID NOS:1-79, 197-200, 205-214, and 219-222.

The polypeptides according to this invention can be the object for any number of additional forms suitable for post-translational and/or synthetic modifications or variations. For example, the invention provides a protein that copies the polypeptides according to this invention. Peptide mimetics described in, for example, U.S. Patent No. 5668110 and the references cited therein.

In another aspect, the polypeptide or protein according to this invention can be modified by the addition of protective groups for the side chains of one or more amino acids of the polypeptide Il the fused protein. Such protective groups can facilitate the transport of the polypeptide or fused protein through the membrane(s), if necessary, or through a specific tissue(s), for example, by reducing the hydrophilicity and increasing the lipophilicity of the polypeptide or fused protein. Examples of acceptable protective groups include ether protective groups, amine protective group, acyl protective group and the protective group of carboxylic acids, which are known in the art (see, for example, U.S. Patent No. 6121236). Synthetic fused protein according to this invention can take any suitable form. For example, protein can be structurally modified from its naturally occurring configuration for forming a cyclic peptide or other structurally modified peptide.

The polypeptides according to this invention can also be associated with one or more non-protein polymer, typically a hydrophilic synthetic polymer, such as polyethylene glycol (PEG), polypropyleneglycol or polyoxyalkylene, using techniques well known in the art such as described in, for example, U.S. Patents№4179337, 4301144, 4496689, 4640835, 4670417 and 4791192, or similar polymer, such as polyvinyl alcohol or polyvinylpyrrolidone (PVP).

The invention includes conjugates comprising at least one polyp is Ted on this invention (for example, mutant polypeptide CTLA-4 EVA, dimeric or Monomeric mutant CTLA-4-Ig, a multimeric mutant polypeptide CTLA-4 EVA, multimeric mutant CTLA-4-Ig) and polipeptidnoi part. It is implied that the term "conjugate" (or interchangeably "conjugated polypeptide") refers to a heterogeneous (in the sense of composition or chimeric) molecule formed by the covalent joining of one or more polypeptides to one or more polipeptides parts. The expression "covalent connection" means that the polypeptide and polipeptidna part either directly covalently bonded to each other, or indirectly covalently connected to each other via an intermediate part or parts, such as a bridge, spacer, or linkage sections or parts, using group attach, present in the polypeptide. Mainly, the conjugate is soluble in appropriate concentrations and conditions, namely, soluble in physiological fluids, such as blood. Examples of conjugated polypeptides according to this invention include glycosylated and/or pegylated polypeptides. The expression "unconjugated polypeptide" may be used to refer to the polypeptide part of the conjugate. This conjugate is usually binds CD80 (e.g., hCD80 and/or CD86 (e.g., hCD86 and/or the extracellular domain of each Il is both (including hCD80-Ig and/or hCD86-Ig) and/or has the ability to inhibit the immune response. Such an immune response may include, but is not limited to the following, for example, T-cell activation or proliferation, synthesis/cytokine production, induction of activation markers, production of inflammatory molecules, inflammation, production anticollagen AT and/or T-cell-dependent AT otvetin.ru polypeptides include those that have at least, 95%, 96%, 97%, 98%, 99% or 100% identity with a sequence selected from the group of SEQ ID NOS:1-79, 197-200, 205-214, and 219-222.

The expression "polipeptidna part" refers to a molecule that is capable of conjugality with a group of attachment of the polypeptide according to this invention. Preferred examples of such molecules include polymer molecules, parts of Sugars, lipophilic compounds or organic derivateservlet agents. When used in the context of the conjugate, as described here, you need to understand that polipeptidna relates to the polypeptide part of the conjugate through a group of attachment of the polypeptide.

The expression "polymer molecule" refers to a molecule formed by covalent linkage of two or more monomers, where none of the monomers is not an amino acid residue, except in those cases where the polymer is human albumin or other common protein of plasma. The expression "polymer" may be used interchangeable with the expression "polymer molecule".

Site N-glycosylase has the sequence N-X-S/T/C, where X is any amino acid residue except Proline, N is asparagine and S/T/C is either serine, threonine or cysteine, preferably serine or threonine, and most predominantly threonine.

"The site of O-glycosylation" HE includes a group of serine or threonine residue.

The expression "group attachment" refers to a group of amino acid residue polypeptide that is able to connect with a corresponding polipeptides part, such as a polymer molecule or the sugar. Non-limiting examples of acceptable groups of attachment and some appropriate polipeptide part are shown in table 2 below.

-CONH2
Table 2
Acceptable group attachment and examples of relevant polipeptidnyh parts
Group attached to
of
Amino acidExamples polipeptides partExamples of methods of conjugation/activated PEGLink
-NH2N-end, LysPolymer, such as PEG MPEG-SPA MPEG-NHS, MPEG-ALDNektar Inc. 2003 Catalog; see also Nektar Therapeutics, 2005-06 Catalog
-COOHWith the end, Asp, GluPolymer, such as PEG Part sugarMPEG-Hz In vitro bindingNektar, Inc. 2003 Catalog;see also NectarTherapeutics 2005-06 Catalog
-SHCysPolymer, such as PEG Part sugarMPEG-VS MPEG-MAL (MPEG-maleimide) In vitro bindingNektar Inc. 2003 Catalog; Nektar Therapeutics 2005-2006 Catalog; Delgado et al., Critical Reviews in Therapeutic Drug Carrier Systems 9(3,4):249-304 (1992)
-HESer, Thr, OH-Part sugarIn vitro O-linked glycosylation
-CONH2Asn as part of the site of N-glycosylationPart sugarIn vitro N-glycosylation
Aromati-
ical
balance
Phe, Tight, TGRPart sugarIn vitro binding
GinPart sugarIn vitro bindingYan and Wold, Biochemistry, 1984,Jul31; 23(16): 3759-65
Aldehyde KetoneOxygen-containing hydrocarbonPolymer, such as PEG, PEG-hydrazidePegylationAndresz et al., 1978, Makromol. Chem. 179:301; WO 92/16555, WO 00/23114
GuanidinoArgPart sugarIn vitro bindingLundblad and Noyes, Chemical Reagents for Protein Modification, CRC Press Inc. Boca Raton, FI
Imidazol-
Noah ring
HisPart sugarIn vitro bindingAs for guanidino

For in vivo N-glycosylation, the term "group attachment" is used in a different way, indicating the amino acid residues constituting an N-glycosylation (with the sequence N-X-S/T/C, where X is any amino acid residue except Proline, N is asparagine and S/T/C - or series, threonine or cysteine, preferably serine or threonine, and most predominantly threonine). Although sparganosis the rest of the site N-glycosylation is the to which is attached a piece of sugar in the process of glycosylation, such attachment can be achieved, while there are other amino acid residues of the N-glycosylation. Accordingly, when polipeptidna part is part of the sugar and the conjugation is carried out by N-glycosylation, the expression "amino acid residue including a group attached to polipeptides part", as used in connection with changes in the amino acid sequence of the polypeptide according to this invention should be understood that one, two or all of amino acid residues constituting an N-glycosylation, are subject to change in such a way that or functional N-glycosylation is embedded in the amino acid sequence, removing from the specified sequence, or a functional N-glycosylation remains in amino acid sequence (for example, by substitution of residue serine, which is already part of the site of N-glycosylation at threonine residue, and Vice versa).

The expression "embed" (namely "built-in" amino acid residue "embedding" amino acid residue) primarily refers to the replacement of an existing amino acid residue for another amino acid residue, but may also mean the insertion of extra aminokislotnogo the remainder.

The expression "delete" (namely the "remote" amino acid residue "delete" amino acid residue) primarily refers to the substitution of amino acid residue that should be removed to another amino acid residue, but may also mean a deletion (without replacement) amino acid residue that need to be removed.

The expression "amino acid residue including a group attached to polipeptides part" means that the amino acid residue is what binds polipeptidna part (in the case of built-amino acid residue) or with which it was associated (in the case of remote amino acid residue).

By removing and/or embedding amino acid residues, including a group attached to polipeptides side, it is possible to specifically adapt the polypeptide according to this invention, in order to create a molecule that is more suitable for conjugation with polipeptides part by choice, to optimize the conjugation pattern (for example, to ensure the optimal location polipeptidnyh parts on the surface of the polypeptide and, thus, for example, to effectively shield epitopes and other surface parts of the polypeptide, without significantly altering its function). For example, the embedding of the groups attached modifies the content of specific amino acid residues in the polypeptide is, with whom contact polipeptide part, which results in a more efficient, specific and/or extensive conjugation. By removing one or more groups attach it becomes possible to avoid conjugation with polipeptides part in parts of the polypeptide, in which the conjugation is undesirable, for example, from amino acid residue is localized at or near a functional site of the polypeptide (since conjugation at such a site may result in inactivation or reduced CD80 - or SV-binding or reduced immunosuppressive activity of the conjugate). Also it may be preferable to delete the group attach, localized near another group attachment.

Amino acid residue including a group attached to polipeptides part, or an existing balance, either the remote or built-in residue selected on the basis of nature polipeptides parts and in some cases on the basis of the used method of conjugation. For example, when polipeptidna part is a polymer molecule, such as a molecule, a derivative of polyethylene glycol (PEG) or polyalkylated (PJSC), the amino acid residues capable of functioning as a group, attach, can be selected from the group comprising cysteine, lysine or the N-terminal amino is the SCP polypeptide), aspartic acid, glutamic acid, histidine and arginine. When polipeptidna part is part of the sugar, the group attached is a site in vivo or in vitro N - or O-glycosylation, mainly the site of N-glycosylation.

In some cases, in part owned by a mutant polypeptide, CTLA-4, the conjugate according to this invention, group attachment, localized in or near the binding site of the receptor, is removed, for example, substitution of amino acid residue comprising such a group. In some cases, amino acid residues, including a group attached to polipeptides parts, such as cysteine or lysine, often not embed in or near the binding site of the receptor mutant polypeptide CTLA-4.

Mutant polypeptide CTLA-4 in this invention can be modified so as to escape and, thereby, modify or destroy or otherwise inactivate the epitope present in the mutant polypeptide, CTLA-4, by conjugation with polipeptides part. The epitope of mutant polypeptides, CTLA-4 can be determined using methods known in the field of technology, also known as the mapping of epitopes, see, for example, Romagnoli et al., J. Biol. Chem. 380(5):553-9 (1999), DeLisser HM, Methods Mol Biol, 1999, 96:11-20, Van de Water et al., Clin. Immunol. Immunopathol. 85(3):229-35 (1997), Saint-Remy JM, Toxicology 119(1):77-81 (1997).

The exact number of groups prikra is to be placed, available for conjugation and present in the mutant polypeptide, CTLA-4, depends on the effect that it is desirable to achieve with conjugation. The effect that is to be obtained, for example, depends on the nature and degree of conjugation (e.g., identity polipeptides part number polipeptidnyh parts, desirable or capable of conjugality with the polypeptide, when they should be conjugated or when pairing should be avoided and the like). For example, if necessary reduced immunogenicity, the number (and containment) attachment must be significant in order to escape most or all of the epitopes. This usually happens when a large part of the mutant polypeptide, CTLA-4 shielded. Effective shielding of epitopes are usually obtained when the total number of groups attach available for conjugation, is in the range 1-6 groups attached, for example, 1-5, for example, in the range of 1-3, e.g. 1, 2 or 3 groups of attachment.

The functional half-life in vivo may depend on the molecular weight of the conjugate and the number of groups attach necessary to ensure increased time half-life, thus, depends on the molecular weight polipeptides part of interest. Some such conjugates 1-6 include, for example, 1-5, for example, 1-3, e.g. 1, 2 or 3 Nepal the peptide part, each of which has a molecular weight of about 100-2000 daltons (Da), for example, about 200 Da, about 300 Da, 400 Da, about 600, about 900 Da, about 1000 Da or about 2-40 kDa, e.g., about 2 kDa, about 5 kDa, about 12 kDa, about 15 kDa, about 20 kDa, about 30 kDa, 40 kDa or 60 kDa.

In the conjugate according to this invention, some, most or essentially all capable of conjugation group attach busy corresponding polipeptides part.

The conjugate according to this invention can exhibit one or more of the following improved properties: (a) an increased half-life in serum and/or functional half-life in vivo, (b) reduced antigenicity or immunogenicity, (C) increased safety, (d) enhanced bioavailability, (e) reduced effector function, or (f) reduced or inhibited the self-Association (e.g., reduced formation unit) between two or more molecules according to this invention. For example, the conjugate may exhibit reduced immunogenicity compared to the hCTLA-4 or compared to the corresponding unconjugated polypeptide, for example, a reduction of at least 10%, such as a reduction of at least 25%, such as a reduction of at least 50%, for example, a reduction of at least 75% in comparison with unconjugated polypeptide or compared the Oia with hCTLA-4. The conjugate may exhibit increased functional in vivo half-life and/or increased half-life in serum compared to a reference molecule, such as hCTLA-4 or compared to the corresponding unconjugated polypeptide. Specific preferred conjugates are those conjugates, where the ratio between functional in vivo by time-life (or half-life in serum) specified conjugate and functional in vivo half-life or half-life in serum) of the specified reference molecule is at least 1.25 times, such as at least 1,50, such as at least about 1.75, such as at least 2, such as at least 3, such as at least 4, such as at least, 5, such as at least 6, such as at least 7, such as at least 8. The half-life is determined in the traditional way in an experimental animal such as a rat or monkey, and can be based on intravenous or subcutaneous injection. In an additional aspect, the conjugate may exhibit increased bioavailability compared to the reference molecule, such as hCTLA-4 or the corresponding non-conjugated polypeptide.

Polymer molecule, which must be attached to the polypeptide can be any acceptable polymer molecule such as a natural or synthetic Homo-polymer or heteropolymer, typically with a molecular weight in the range 300-100000 Yes, such as 300-20000 Yes, more predominantly in the range of 500-10000 Yes, even more predominantly in the range of 500-5000 Yes.

Examples of homopolymers include polyol (i.e. poly-OH), polyamine (namely poly-NH2) and polycarboxylic acid (i.e. poly-COOH). Heteropolymer is a polymer that includes one or more different linking groups such as, for example, hydroxyl group and amine group. Examples of acceptable polymer molecules include polymer molecules selected from the group comprising polyalkylene (PAO), including polyalkyleneglycol (PAG), such as polyethylene glycol (PEG) and polypropylenglycol (BCP), an extensive Page, polyvinyl alcohol (PVA), polycarboxylate, poly-(vinylpyrrolidone), polyethylene-co-anhydride malic acid, polystyrene-co-anhydride malic acid, dextran, including carboxymethylation, or any other biopolymer suitable for reducing immunogenicity and/or increased functional in vivo time-life and/or serum of time half-life. Another example of a polymer molecule is human albumin or other common protein of plasma. In General, polymers, derivatives polyalkyleneglycol, are biocompatible, non-toxic, panthenyl, non-immunogenic, have different t the WA water solubility and easy excretiruyutza of living organisms.

PEG is the preferred polymer molecule to use as it has only a few reactive groups capable of cross-linking compared to, for example, polysaccharides such as dextran and the like. In particular, monofunctional PEG, for example, monomethoxypolyethylene (MPEG), is the subject of interest due to the fact that his chemistry connections is relatively simple (only one reactive group available for conjugation with the groups attached to the polypeptide). Therefore, the risk of cross-linking is eliminated, causing polypeptide conjugates are more homogeneous and the reaction of the polymer molecules with the polypeptide easier to control. When the molecule is pegylated, it usually involves 1, 2, 3, 4 or 5 molecules of polyethylene glycol (PEG). Each molecule of PEG may have a molecular weight from about 5 kDa (kilo daltons) to 100 kDa, including, for example, about 10 kDa, about 12 kDa, about 20 kDa, about 40 kDa. Acceptable PEG molecules are available from Shearwater polymers, Inc. and Enzon, Inc. and can be selected from the SS-PEG, NPC-PEG, aldehyde-PEG, MPEG SPA, MPEG-SCM, MPEG-PTS, SC-PEG, trailerfunny MPEG (U.S. Patent No. 5880255) or oxycarbonyl-hydroxy-N-dicarboximide-PEG (U.S. Patent No. 5122614).

In one aspect, the invention provides isolated or synthetic conjugate, comprising: (a) the polypeptide is about this invention (for example, mutant polypeptide CTLA-4 EVA, dimeric or Monomeric mutant CTLA-4-Ig, a multimeric mutant polypeptide CTLA-4 EVA, multimeric mutant CTLA-4-Ig); and (b)at least one polipeptidnoi part, namely, for example, 1-10, 1-9, 1-8, 1-7, 1-7, 1-6, 1-5, 1-4, 1-3, 1, 2 or 3 polipeptide part attached to the polypeptide, where the conjugate binds CD80 (e.g., hCD80 and/or CD86 (e.g., hCD86) and/or the extracellular domain of each or both (including hCD80-Ig and/or hCD86-Ig), and/or has the ability to induce an immune response (e.g., T-cell-dependent immune response). Illustrative polypeptides include those that have at least, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence selected from the group of SEQ ID NOS:1-79, 197-200, 205-214, and 219-222. In some cases, the conjugate includes one polipeptidnoi part. In some cases, the conjugate comprises two, three, four or more polipeptidnyh parts. In some cases, the amino acid sequence of the polypeptide conjugate includes one or more substitutions, each of which embeds group attached to polipeptides part (for example, substitution of amino acid residue amino acid sequence of another residue, which includes a group of attachments for polipeptides part, or insertion in the amino acid sequence of additional and inoculating residue, which includes a group of attachments for polipeptides part).

The conjugate may comprise two or more polypeptides according to this invention. In some cases polipeptidna part covalently attached to each or both of such polypeptides. If the conjugate comprises two or more identical polypeptide according to this invention, the same type and number polipeptidnyh parts are normally attached to each such polypeptide, usually in the same way to the appropriate group(s) attached to each polypeptide. As noted above, polipeptidna part may contain, for example, a sugar molecule, which may not necessarily be attached to the site of N-glycosylation, or polymer, such as, for example, a portion of the glycol. Part of polyethylene glycol may be covalently attached to a cysteine residue or a lysine residue of the polypeptide according to this invention. In some cases, part of the polyethylene glycol is covalently attached to the N-terminal amino group of the polypeptide. Conjugate comprising a mutant CTLA-4-Ig in this invention, can be described as a mutant CTLA-4-Ig conjugate according to this invention. Multimer conjugates are also included. Multimeric conjugates include two or more conjugates, where at least one conjugate is a conjugate according to this izaberete the Oia, comprising at least one polypeptide according to this invention. Conjugates in the multimeric conjugate can be, but need not be identical to each other.

As discussed above, the polypeptides according to this invention, including fused protein according to this invention can generally be subject to glycosylation. Polypeptides and fused proteins according to this invention may also be (or be modified so as to become subject to other forms of post-translational modifications including, for example, hydroxylation, the attachment of lipid-derived lipids, methylation, miesterioasa, tahilramani, phosphorylation and sulfation. Other post-translational modification, which may also be subject to a polypeptide or protein according to this invention, include acetylation, acylation, ADP-ribosylating, amidation, covalent attachment of flavina, covalent attachment part heme, covalent attachment of a nucleotide or of a derived nucleotide, covalent attachment of phosphatidylinositol, cross-linking, cyclization, forming a disulfide bond, demethylation, formirovanie, the formation of anchor GPI (glycosyl phosphatidylinositol), salt, oxidation, proteolytic processing, prenisolone, racemization, got the plan, marginalrevenue and ubiquitinate. Other common protein modifications are described in, for example, Creighton, see above, Seifter et al., Meth. Enzymol. 18:626-646 (1990) and Rattan et al., Ann. NY Acad. Sci. 663:48-62 per (1992). Post-translational modification of polypeptides or fused protein expressed from the nucleic acids in the cells of the host, differ depending on the type of host or host cell, which is expressed peptide. For example, glycosylation often does not occur in bacterial hosts such as E. Coli, and differs significantly in baculovirus systems compared to cellular systems of the mammal. Accordingly, when glycosylation is desired (which is the usual case for most of the polypeptides of the present invention), the polypeptide or protein to be expressed (produced) in glycosylated host, generally a eukaryotic cell (e.g., the cell is mammalian or insect cell). Modifications of the polypeptide or fused protein expression and post-translational modification can be confirmed by any acceptable method, including, for example, x-ray diffraction, NMR (nuclear magnetic resonance) imaging, mass spectrometry and/or chromatography (e.g., reverse phase chromatography, affinity chromatography or gas-liquid chromatography).

On peptide or protein also or alternatively may include any reasonable number of non naturally occurring amino acid (for example, 3 amino acids) and/or alternative amino acids (e.g., selenocysteine), or amino acid analogs, such as those listed in the MANUAL OF PATENT EXAMINING PROCEDURE § 2422 (7th Revision - 2000), which can be embedded in protein synthesis, for example by solid-phase protein synthesis (as described in, for example, Merrifield, Adv. Enzymol. 32:221-296 (1969) and other references cited there). The polypeptide or protein according to this invention may also be modified by incorporating at least one modified amino acid. The inclusion of one or more modified amino acids may be predominant in, for example, (a) the increase in serum time half-life of the polypeptide or fused protein, (b) reducing the antigenicity of the polypeptide or fused protein, or (C) enhancing the security of the polypeptide or fused protein. The amino acid(s) modify, for example, co-translationally or excision during recombinant production (e.g., N-linked glycosylation site at the N-X-S/T motifs during expression in mammalian cells) or modify synthetic methods. Non-limiting examples of a modified amino acids include glycosylated amino acids, sulfated amino acids, prenisolone (for example, farnesylation, geranylgeranylation) amino acid, acetylated AMI is akisato, acylated amino acid, a pegylated amino acid, biotinylated amino acid, carboxylterminal amino acid, a phosphorylated amino acid, and the like. References acceptable to the guidance of the specialist in this field regarding the modification of amino acids, are very numerous in the literature. Example protocols can be found in Walker (1998) Protein Protocols on CD-ROM Humana Press, Towata, NJ. The modified amino acid may be selected from a glycosylated amino acid, a pegylated amino acid, farnesiani amino acids, acetylated amino acids, biotinylated amino acids, amino acids, conjugated with lipid part, and amino acids, conjugated with organic derivational agent.

The invention also provides polypeptides (including fused proteins)having the above described characteristics, which also include additional amino acid sequences that affect biological function (e.g., immunogenicity, direction, and/or half-life) of the polypeptide (or fused protein).

The polypeptide or protein according to this invention may also include a guide sequence that is different from or in addition to the signal sequence. For example, the polypeptide or protein can include a sequence that h is tasked with a specific receptor on the cell type (for example, the monocyte, dendritic cell, or a related cell) to provide targeted delivery of the polypeptide to such cells and/or associated tissues. Signal sequences are described above and include localized/anchored in the membrane of the sequence (e.g., a stop-transfer sequence, GPI anchor sequence) and the like.

Especially suitable merger partner for the polypeptide according to this invention (including fused protein according to this invention) is a peptide sequence that facilitates purification of the polypeptide, for example, the subsequence for purification of the polypeptide. Polynucleotide according to this invention may include the coding sequence, fused inside the reading frame with the marker amino acid sequence, e.g., facilitates purification of the encoded polypeptide. Such peptide domains, facilitates purification, or a subsequence for purification of the polypeptide include, but are not limited to the following, peptides, chelating a metal, such as histidine-tryptophan modules that provide cleansing on immobilizovannoi metal, such as hexa-his-tag peptide or other polyhistidine sequence, the sequence encoding such a marker embedded in a pQE vector, available from QIAGEN, Inc. (Chatsworth, California), after which outermost, which binds glutathione (for example, glutathione-8-transferase (GST)), hemaglutinin () marker (corresponding to an epitope derived hemagglutinin protein of influenza virus; Wilson et al., Cell 37:767 (1984)), the protein sequence that binds maltose, FLAG epitope used in the purification system FLAGS extension/affinity (Immunex Corp, Seattle, WA) (commercially available FLAG epitopes are also available from Kodak (New Haven, Connecticut)), E-epitope marker (E-token), thioredoxin (TRX), avidin, and the like. Epitope markers that facilitate the purification described in the art (see, for example, Whitehorn et al., Biotechnology 13:1215-19 (1995)). The polypeptide can include e-his (his-tag) token, which may include polyhistidine sequence and the sequence of the anti-e-epitope (Pharmacia Biotech Catalog); e-his markers can be obtained using standard techniques. The inclusion of the polypeptide linker sequence, tsepliaeva protease, between the domain of purification and the polypeptide is suitable to facilitate purification. His-tag residues facilitate purification on IMIAC (affinity chromatography using immobilized metal (IMAC)as described in Porath et al. Protein Expresion and Purifying 3:263-281 (1992)), whereas the site of cleavage of enterokinase provides a way of separating the polypeptide from the fused protein. pGEX vectors (Promega; Madison, WI) may also be used for expression of foreign the of polypeptides in the form of a fused protein with glutathione-S-reductase (GST). In General, such fused proteins are soluble and can easily be purified from lysed cells by adsorption to the ligand-agarose pellets (for example, glutathione-agarose in the case of GST-fusion) followed by elution in the presence of free ligand. Additional examples of subsequences that facilitate purification of the polypeptide, and their use for protein purification are described in, for example, in Published International Application WO 00/15823. After expression of the polypeptide of interest, and selecting such partners of merger or otherwise, as described above, can be used stages of the refolding of the protein, if necessary, in completing configuration of the Mature polypeptide.

Protein according to this invention may also include one or more additional peptide fragments or peptide parts, which provide the definition of the fused protein. For example, a known peptide fragment or a portion (e.g., green fluorescent protein (GFP), β-galactosidase or their designated domain) can be embedded in a protein. Additional marker molecules that can be conjugated to the polypeptide according to this invention, include radionuclides, enzymes, fluorophores, ligands, small molecules and the like. Such securing partners merge especially wagniv fused proteins, used in diagnostic methods discussed in this description.

In another aspect, the polypeptide according to this invention may include a merge partner, which ensures the stability of the polypeptide, the secretion of the polypeptide (different from the signal direction) or both of these characteristics. For example, the polypeptide may include immunoglobulin (Ig) domain, such as an IgG polypeptide comprising an Fc hinge region, CH2 domain and CH3 domain, which provides stability and/or secretion of the polypeptide.

Peptide fragments fused protein or peptide portions may be associated in any suitable manner. A variety of polypeptide fragments or parts of the fused protein can be covalently combined (e.g., via a peptide or disulfide bond). Polypeptide fragments or parts can be merged directly (for example, With the end of the antigenic or immunogenic sequence according to this invention can be fused to the N-end of the sequence for purification or heterologous immunogenic sequence). Protein may include any reasonable number of modified linkages, for example, ISOStAR, within or between peptide units. Alternative or additionally, the protein can include a peptide linker between one or more polypeptide fragments the AMI or parts which includes one or more amino acid sequences that do not form part of the biologically active peptide of the parts. Any acceptable peptide linker can be used. Such a linker may be of any appropriate size. Typically, the linker is less than about 30 amino acid residues, less than about 20 amino acid residues, and/or less than 10 amino acid residues. The linker mainly can include or consist of neutral amino acid residues. Acceptable linkers in General are described in, for example, U.S. Patent No. 5990275, 6010883, 6197946 and the Application for the European Patent 0035384. If the separation of peptide fragments or peptide of the parts is desired, it can be used by the linker, which facilitates the separation. An example of such a linker is described in U.S. Patent No. 4719326. Flexible linkers, which usually consist of combinations of glycine and/or serine residues, can be pre-emptive. Examples of such linkers are described in, for example, McCafferty et al., Nature 348:552-554 (1990), Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988), Glockshuber et al., Biochemistry 29:1362-1367 (1990), and Cheadle et al., Molecular Immunol. 29:21-30 (1992), Bird et al., Science 242:423-26 (1988), and U.S. Patent No. 5672683, 6165476 and 6132992.

The use of the linker may also reduce unwanted immune response to a protein formed by the merger of two peptide fragments or peptide parts, the cat is which can lead to unintended MHC I and/or MHC II epitope present in the fused protein. In addition to applying the linker identified unwanted epitope sequence and related sequences can be pegylated (e.g., by inserting the lysine residues to promote attachment of PEG), to screen the identified epitopes from symptoms. Other methods for reducing the immunogenicity of fused protein according to this invention can be used together with the introduction of the fused protein, include the techniques described in U.S. Patent No. 6093699.

Obtaining polypeptides

Recombinant methods of education polypeptides for the production and extraction of polypeptides according to this invention (including fused protein according to this invention) is described below. In addition to recombinant production, the polypeptides may be produced by direct peptide synthesis using solid-phase techniques (see, for example, Stewart et al. (1969) Solid-Phase Peptide Synthesis, W.H. Freeman Co, San Francisco; Merrifield (1963) J. Am. Chem. Soc 85:2149-2154). Synthesis of a peptide can be carried out using methods manually or automatically. Automated synthesis may be achieved, for example, using Applied Biosystemes 43 IA Peptide Synthesizer (Perkin Elmer, Foster City, Calif.) according to the manufacturer's instructions. For example, the subsequence may be chemically synthesized separately and compatible with the STN using chemical methods to ensure the mutant polypeptides, CTLA-4, or their functional fragments. Alternatively, such sequences may be supplied by many companies that specialize in the production of polypeptides. Most frequently, the polypeptides according to this invention produce expressive encoding nucleic acid and recovering the polypeptide, for example, as described below.

The invention provides methods of production of the polypeptides (including fused proteins) according to this invention. One such method includes embedding in the cell population of any nucleic acid described herein, which is functionally linked to a regulatory sequence effective to obtain the encoded polypeptide, culturing the cells in the cellular environment to obtain the polypeptide and the release of the polypeptide from the cells or the cell environment. Use the amount of nucleic acid that is sufficient to facilitate the capture of cells (transfection) and/or expression of the polypeptide. The cellular environment can be any described herein and in the examples. Additional environment known to specialists in this field. Nucleic acid embedded in these cells in any of these delivery methods, including, for example, an injection device, the injection needle, gene gun, electroporation (e.g., a device for electroporation of DNA, Inovio Biomedical Corp. (San Diego)), transdermal delivery, passive C the grip, etc. Nucleic acid according to this invention may be part of a vector such as a recombinant expression vector including a DNA plasmid vector, viral vector or any vector described herein. Nucleic acid or a vector comprising the nucleic acid according to this invention, can be prepared and formulated as described herein above and in the examples below. Such nucleic acid or expression vector can be integrated into a population of mammalian cells in vivo, or selected mammalian cells (e.g. tumor cells) can be removed from the mammal, and the expression vector nucleic acid is integrated ex vivo in a population of such cells in a quantity sufficient to cause the capture and expression of the encoded polypeptide. Or nucleic acid or a vector comprising the nucleic acid according to this invention, produced using cultured cells in vitro. In one aspect, a method of obtaining a polypeptide according to this invention includes embedding into a population of cells a recombinant expression vector comprising any nucleic acid described herein, the number and the formula, to ensure capture of the vector and expression of the polypeptide; introducing the expression vector into a mammal in any format embed/delivery described herein; and the allocation of polypeptid is from a mammal or a by-product of a mammal. Acceptable cell hosts, expression vectors, methods of transfection of host cells with the expression vector comprising the sequence of the nucleic acid encoding the polypeptide according to this invention, cell cultures and procedures for obtaining and recovery of such polypeptide from the cell culture are described in detail below in the section entitled "Nucleic acid according to this invention. Additional methods of production are discussed in the examples below.

As noted above, the polypeptides according to this invention (which include fused protein according to this invention can be subjected to various changes, such as one or more insertions of amino acids or nucleic acids, deletions and substitutions, either conservative or nonconservative, including, for example, such changes, which can provide certain advantages in their use, for example, in their therapeutic or prophylactic use, or the introduction or diagnostic use. Educational procedures variants of polypeptides with the use of amino acid substitutions, deletions, insertions and additions are routine in the art. Polypeptides, and their variants, with the necessary ability to bind CD80 and/or CD86 or their fragments (e.g., EVA) or the ability to suppress the immune response in vitro or in vivo, as opisanoj this description, to date identified in the research, well-known experts in this field, and in the research described here. See, for example, research presented in the examples below.

Nucleic acid according to this invention, discussed in more detail below, may also be subjected to various changes, such as one or more substitutions of one or more nucleic acids in one or more codons, so that a particular codon encodes the same or different amino acids, leading to a conservative or non-conservative substitution, or one or more deletions of one or more nucleic acids in the sequence. Nucleic acids can also be modified to include one or more codons that are optimal expression in an expression system (e.g., mammal cells or the expression system of a mammal), and, if necessary, these one or more codons continue to encode the same(s) amino acid(s). Educational procedures variants of nucleic acids using substitutions, deletions, insertions and additions and degenerate codons of the nucleic acid, are routine in the art, and variants of nucleic acids encoding polypeptides having the required properties, episondes (for example, the ability to bind CD80 and/or CD86 and/or suppress the immune response in vitro or in vivo), on the date determined by application of the research described here. Such alteration of nucleic acids may provide certain advantages in their therapeutic or prophylactic use, or the introduction or diagnostic use. In one aspect, the nucleic acids and polypeptides can be modified in many ways, provided that they include a sequence of nucleic acid or amino acid sequence essentially identical to the nucleic acid sequence corresponding to a nucleic acid that encodes a mutant polypeptide, CTLA-4, or a mutant polypeptide, CTLA-4 according to this invention, respectively.

NUCLEIC ACID ACCORDING to THIS INVENTION

The invention provides isolated or recombinant nucleic acids (also referred to in this description as polynucleotide), collectively referred to as "nucleic acids of this invention (or polynucleotide of the invention")that encode the polypeptides according to this invention. Nucleic acid according to this invention, including all described below, suitable for recombinant production (e.g., expression) of the polypeptides according to this invention, usually through the expression of plasma is odnogo of the expression vector, including the sequence encoding the polypeptide or fragment; therapeutic; prevention; as diagnostic tools; as diagnostic probes for the presence of complementary or partially complementary nucleic acids (including the application for the determination of nucleic acid wild-type CTLA-4). For example, a nucleic acid according to this invention, including all described below, is suitable, as they encode polypeptides that are useful for the suppression or inhibition of the immune response (for example, T-cell activation, T-cell proliferation, synthesis or production of cytokine (e.g., production of TNF-α, IFN-γ, IL-2), induction of activation markers (e.g., CD25, IL-2 receptor), inflammation, production anticollagen antibodies and/or T-cell-dependent antibody response in vitro and/or in vivo applications, including, for example, a prophylactic and/or therapeutic treatment of diseases, disorders and conditions of the immune system, in which the suppression of immune response is desired (for example, methods of treatment of autoimmune diseases and disorders, and methods of inhibiting rejection of the graft tissue, cell or organ from a donor to a recipient). Nucleic acid according to this invention can also be incorporated into expression vectors suitable for gene therapy is, vaccination with DNA and immunosuppressive therapy. Additional applications of nucleic acids and vectors of this invention comprising such a nucleic acid set forth in this description.

In one aspect the invention provides an isolated or recombinant nucleic acid comprising a nucleotide sequence encoding any polypeptide (including any protein and the like) according to this invention described above in the section entitled "Polypeptides according to this invention and throughout the present description. The invention also provides isolated or recombinant nucleic acid comprising a nucleotide sequence encoding a combination of two or more of any of the polypeptides (including any fused proteins) according to this invention described above and elsewhere in this description. Also included is a nucleic acid that encodes any polypeptide according to this invention, such as, for example, a mutant polypeptide, CTLA-4 EVA or mutant protein, CTLA-4-Ig, which includes a sequence of codons, significantly optimizing the expression in the host is a mammal, such as man.

For example, in one aspect the invention provides an isolated or recombinant nucleic acid comprising a polynucleotide sequence that encodes polypeptide, comprising the amino acid sequence that has at least, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,5% or 100% sequence identity with at least one amino acid sequence selected from the group comprising SEQ ID NOS:1 to 73, or a complementary polynucleotide sequence, where the polypeptide binds CD80 and/or CD86 or polypeptide fragment of CD80 and/or CD86 (e.g., the extracellular domain of CD80 and/or CD86 and/or suppresses an immune response in vitro and/or in vivo, or its complementary polynucleotide sequence. Additional details regarding the functional properties and characteristics of such polypeptides are discussed above in the section "Polypeptides according to this invention. Some such nucleic acids encode a polypeptide comprising amino acid sequence having amino acid length, approximately equal to the amino acid length of the hCTLA-4 EVA; as, for example, 110-138, 112-132, 118 to 130,119-129, 120-128, 121-127, 122-126, 123-125 or 124 amino acid residues. Illustrative nucleic acids that encode mutant polypeptides, CTLA-4 EVA, include the sequence shown in SEQ ID NOS:1-73, but not limited to the following, for example, a nucleic acid having the nucleotide sequence shown in SEQ ID NOS:80-152, respectively. For example, illustrative nuclei the OIC acid, encoding the polypeptide presented in SEQ ID NO:1 (clone D3-1), is a nucleic acid represented in SEQ ID:80. Also included fragments of any of these nucleic acids, where this fragment encodes a polypeptide that binds CD80 and/or CD86 and/or EVA each or both, and/or has the ability to suppress the immune response.

In another aspect, the invention provides isolated or recombinant nucleic acid comprising a polynucleotide sequence that encodes the polypeptide (for example, a mutant polypeptide, CTLA-4 EVA), which includes the amino acid sequence of (a), which differs from the amino acid sequence selected from the group comprising SEQ ID NOS:1-73 by no more than 6 amino acid residues (for example, not more than 1, 2, 3, 4, 5, or 6 amino acid residues), and (b) where the amino acid residue in the amino acid sequence in position 41, 50, 54, 55, 56, 64, 65, 70 or 85 is identical to the amino acid residue in the corresponding position of the specified amino acid sequence selected from the group comprising SEQ ID NOS:1-73, where the polypeptide binds CD80 and/or CD86 and/or the extracellular domain of each or both and/or inhibits an immune response in vitro and/or in vivo, or a complementary polynucleotide sequence. That is, the amino acid residue in this position 41, 50, 54, 55, 56, 64, 65, 70 and the and 85 in such a selected amino acid sequence is not deleted or replaced. Some such nucleic acids encode polypeptides comprising a sequence that differs from the selected amino acid sequence at no more than 6 amino acid residues and which includes amino acid residues in 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and/or 14 positions selected from amino acid positions 24, 30, 32, 41, 50, 54, 55, 56, 64, 65, 70, 85, 104 and 106 that are identical with amino acid residues in the corresponding positions in the selected amino acid sequence. Such polypeptides may differ from the selected amino acid sequence at the amino acid deletion(s), addition(s) and/or amino acid replacement(s). Amino acid substitutions may be conservative or non-conservative substitution. Illustrative conservative substitutions are discussed in the section entitled "Variation sequences". Some such polypeptides comprise a sequence having a length of about 118 to 130, 119-129, 120-128, 121 - 127, 122-126, 123-125 or 124 amino acid residues. Additional details of the functional properties and characteristics of such polypeptides discussed above. Illustrative nucleic acid include, but are not limited to the following, for example, those that include the nucleotide sequence shown in SEQ ID NOS:80-152.

In another aspect the invention provides an isolated or recombinant the Yu nucleic acid, comprising a polynucleotide sequence that encodes a polypeptide (e.g., mutant CTLA-4 EVA), including amino acid sequence which (a) differs from the amino acid sequence of the extracellular domain of human CTLA-4, is presented in SEQ ID NO:159 in no more than 6 amino acid residues, and (b) includes at least one amino acid substitution in amino acid position corresponding to position 50, 54, 55, 56, 64, 65, 70 or 85 relative to the amino acid sequence of SEQ ID NO:159, where the polypeptide binds hCD80 and/or hCD86 and/or EVA each or both and/or inhibits an immune response in vitro and/or in vivo, or its complementary polynucleotide sequence. Some such nucleic acids encode polypeptides that include 2, 3, 4, 5 or 6 amino acid substitutions in positions relative to the sequence shown in SEQ ID NO:159 selected from the group comprising a position 50, 54, 55, 56, 64, 65, 70 and 85. Some such nucleic acids encode polypeptides that include amino acid replacement at a position corresponding to position 104 and/or 30 relative to SEQ ID NO:159. Some such nucleic acids encode polypeptides comprising at least one amino acid substitution relative to SEQ ID NO:159 in position 70 (for example, S70F), position 64 (for example, S64P), position 50 (for example,AM), position 54 (for example, M54K/V), the position 65 (for example, I65S), position 56 (for example, N56D), position 55 (for example, G55E), position 85 (for example, MA) and/or position 24 (for example, AA). Any such polypeptide can include amino acid substitution relative to SEQ ID NO:159 in position 104 (optional L104E/D, for example, L104E), position 30 (for example, T30N/D/A) and/or position 32 (for example, V32I). Some such nucleic acids encode polypeptides comprising one or more substitutions at amino acid positions relative to SEQ ID NO:159 selected from the group comprising AL, M54K, G55E, N56D, S64P, I65S and S70F. Some of the encoded polypeptides include a sequence having the amino acid length of about 118 to 130, 119-129, 120-128, 121-127, 122-126, 123-125 or 124 amino acid residues. For more details about the functional properties and characteristics of such polypeptides discussed above.

The invention also provides isolated or recombinant nucleic acid comprising a polynucleotide sequence that encodes a polypeptide (e.g., mutant CTLA-4 EVA), including amino acid sequence, comprising (i)at least, 95%, 96%, 97%, 98%, 99%, or 100% identity with any amino acid sequence selected from the group comprising SEQ ID NOS:1-73 and (ii) the phenylalanine residue at amino acid position equivalent is the position 70 of the specified amino acid sequence, selected from the group comprising SEQ ID nos:1-73, where the polypeptide binds hCD80 and/or hCD86 or EVA and/or inhibits an immune response, or a complementary polynucleotide sequence. Some such nucleic acids encode polypeptides that include one or more of the following with respect to the selected sequence: a glutamic acid residue at amino acid position corresponding to position 24; an asparagine residue at amino acid position corresponding to position 30; the isoleucine residue at amino acid position corresponding to position 32; methionine residue at the amino acid position corresponding to position 50; lysine residue in amino acid position corresponding to position 54; a glutamic acid residue at amino acid position corresponding to position 55; the aspartic acid residue at amino acid position corresponding to position 56; a Proline residue at amino acid position corresponding to position 64; the serine residue at amino acid position corresponding to position 65; and the glutamic acid residue at amino acid position corresponding to position 104. Some such nucleic acids encode polypeptides comprising amino acid sequence having a length of about 118 to 130, 119-129, 120-128, 121-127, 122-126, 123-125 or 124 amino the PCI-e slot residues. Additional details regarding the functional properties and characteristics of such polypeptides discussed above.

In another aspect, the invention provides isolated or recombinant nucleic acid comprising a polynucleotide sequence that encodes a polypeptide (e.g., mutant CTLA-4 EVA), including amino acid sequence which (a) differs from the amino acid sequence of the polypeptide of the human CTLA-4 EVA presented in SEQ ID NO:159, no more than 6 amino acid residues, and (b) includes at least one amino acid substitution, where specified, at least one amino acid substitution is a S70F, where the position of the amino acid residues are numbered according to SEQ ID NO:159, where the polypeptide that binds hCD80 and/or hCD86 and/or EVA each or both) and/or inhibits an immune response, or she complementary polynucleotide sequence. Some such nucleic acids encode a polypeptide that includes at least one amino acid substitution selected from the group comprising AU, T30N, V32I, D41G, AM, M54K, G55E, N56D, S64P, I65S, MA, L104E and I106F. Some such nucleic acids encode polypeptides comprising amino acid sequence having a length of about 118 to 130, 119-129, 120-128, 121-127, 122-126, 123-125 or 124 amino acid the residue. Additional details regarding the functional properties and characteristics of such polypeptides discussed above.

In another aspect, the invention provides isolated or recombinant nucleic acid comprising a polynucleotide sequence that encodes a polypeptide (e.g., mutant CTLA-4 EVA), including amino acid sequence which (a) differs from the amino acid sequence represented in SEQ ID NO:31 no more than 6 amino acid residues, and (b) includes at least one of the following: a methionine residue at the position corresponding to position 50 of SEQ ID NO:31, a lysine residue at the position corresponding to position 54 of SEQ ID NO:31, balance glutamic acid at the position corresponding to position 55 of SEQ ID NO:31, a Proline residue at a position corresponding to position 64 of SEQ ID NO:31), the serine residue at the position corresponding to position 65 of SEQ ID NO:31), the phenylalanine residue at the position corresponding to position 70 of SEQ ID NO:31, where the position of the amino acid residues are numbered according to SEQ ID NO:31, and where the polypeptide binds CD80 and/or CD86 and/or EVA each or both, and/or inhibits an immune response, or a complementary polynucleotide sequence. Some of the encoded polypeptides include glutamic acid residue at the position equivalent is the position 104, the aspartic acid residue in the position corresponding to position 30, and/or the isoleucine residue at the position corresponding to position 32 of SEQ ID NO:31. Some such nucleic acids encode polypeptides comprising amino acid sequence having a length of about 118 to 130, 119-129, 120-128, 121-127, 122-126, 123-125 or 124 amino acid residues. Additional details regarding the functional properties and characteristics of such polypeptides discussed above.

In another aspect, the invention provides isolated or recombinant nucleic acid comprising a polynucleotide sequence having at least, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,5% or 100% identity with at least one polynucleotide sequence selected from the group comprising SEQ ID NOS:80 - 158, 201-204, 223 and 224, or a complementary polynucleotide sequence, where the nucleic acid encodes a polypeptide that binds hCD80 and/or hCD86 and/or EVA each or both, and/or suppresses an immune response, or a complementary polynucleotide sequence. Illustrative nucleic acid that includes a polynucleotide sequence defined by SEQ ID NOS:80-158, encode illustrative polypeptides comprising the amino acid sequence defined by SEQ ID NOS:1-79, respectively. illustrative nucleic acid, comprising the polynucleotide sequence defined in SEQ ID N08:201-204, encode illustrative polypeptides comprising the amino acid sequence defined by SEQ ID NOS:197-200, respectively.

In another aspect, the invention includes an isolated or recombinant nucleic acid comprising: (a) a polynucleotide sequence having at least, 95%, 96%, 97%, 98%, 99% or 100% identity with the RNA polynucleotide sequence where RNA polynucleotide sequence includes a DNA sequence selected from the group comprising SEQ ID NOS:80-158, 201-204, 223 and 224, in which all teminalia nucleotide residues in the DNA sequence is replaced by Wratislavia nucleotide residues; (b) a complementary polynucleotide sequence (a); or (C) a fragment of any of polynucleotide sequence (a) or (b)where the nucleic acid encodes a polypeptide which (i) binds CD80 and/or CD86 and/or EVA each or both, and/or (ii) has the ability to suppress the immune response in vitro or in vivo (e.g., T-cell activation or proliferation, synthesis or production of cytokine (e.g., production of TNF-α, IFN-γ, IL-2), induction of activation markers (e.g., CD25, IL-2 receptor), inflammation, production anticollagen antibodies and/or T-cell-dependent antibody response), or polynucleotide after outalmost.

The invention includes an isolated or recombinant nucleic acid encoding any of multimer any polypeptide according to this invention described above (e.g., dimer, tetramer, etc.). As discussed in more detail in this description, a dimer comprising two polypeptide according to this invention (including the two fused protein), usually formed during cellular processing by one or more covalent disulfide bonds between cysteine residue(s) in the same polypeptide and the cysteine residue(s) in the second polypeptide. Other multimer can be formed in a similar manner. For example, in a non-limiting aspect, the invention provides isolated or recombinant nucleic acid comprising a polynucleotide sequence that encodes a recombinant dimer of a polypeptide, comprising two polypeptide, where each polypeptide consists of amino acid sequence having at least, 95%, 96%, 97%, 98%, 99% or 100% identity with a sequence selected from the group comprising SEQ ID NOS:1-73, where the dimer binds hCD80 and/or hCD86 and/or inhibits an immune response, or a complementary polynucleotide sequence. Also includes an isolated or recombinant nucleic acid comprising a polynucleotide sequence of alnost, which encodes a dimer of a polypeptide, comprising two polypeptide, where each polypeptide differs from the amino acid sequence of hCTLA-4 EVA (SEQ ID NO:159) at not more than 6 amino acid residues and comprises at least one substitution at amino acid position is relative to SEQ ID NO:159 selected from the group comprising AL, M54K, G55E, N56D, S64P, I65S and S70F; and where the polypeptide is not necessary to also include replacement of L104E where specified dimer binds hCD80 and/or hCD86 and/or inhibits an immune response or its complementary polynucleotide sequence. Additional details regarding the functional properties of these dimers are discussed above.

The invention also provides isolated or recombinant nucleic acid encoding any protein in this invention, including any multimeric protein according to this invention (e.g., dimers, tetramer etc). In one aspect the invention provides an isolated or recombinant nucleic acid comprising a polynucleotide sequence that encodes a protein comprising (a) a polypeptide comprising amino acid sequence that has at least, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,5% or 100% identity with at least one amino acid sequence selected from the group, including the setup portion of SEQ ID NOS:1-73, and (b) Ig polypeptide, where the protein binds CD80 and/or CD86 (and/or CD80-Ig and/or CD86-Ig) and/or has the ability to suppress the immune response, or a complementary polynucleotide sequence. The Ig polypeptide can include a polypeptide Ig Fc, including, for example, the polypeptide Ig Fc, including amino acid sequence having at least, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence selected from the group comprising SEQ ID NOS:184-186 and 218. Dimeric protein comprising two Monomeric fused protein, usually formed during cellular processing through the formation of covalent disulfide bonds between cysteine residues in one monomer fused protein and cysteine residues in the second monomer fused protein. Other multimer can be formed in a similar manner. Additional details regarding the functional properties and characteristics of such fused proteins discussed above.

In another aspect, the invention provides isolated or recombinant nucleic acid comprising a polynucleotide sequence which encodes a protein dimer (e.g., mutant dimer CTLA-4-Ig)comprising two Monomeric fused protein (e.g., Monomeric mutant CTLA-4-Ig), where each Monomeric protein comprises: (a) polypeptide (e.g., mutant CTLA-4 EVA), including the Mering amino acid sequence, with at least, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,5% or 100% identity with at least one amino acid sequence selected from the group comprising SEQ ID NOS:1-73, and (b) Ig polypeptide, where the dimer fused protein binds CD80 and/or CD86 (and/or CD80-Ig and/or CD86-Ig), and/or has the ability to inhibit or suppress an immune response, or a complementary polynucleotide sequence. Two Monomeric fused protein with the expression connected together by at least one disulfide bond formed between two cysteine residues present in each Monomeric mutant CTLA-4-Ig fused protein. Ig polypeptide can include a polypeptide Ig Fc, including, for example, the polypeptide Ig Fc comprising a sequence having at least, 95%, 96%, 97%, 98%, 99% or 100% identity with a sequence selected from the group comprising SEQ ID NOS:184-186 and 218. In some cases, With the end of the polypeptide (a) covalently linked or fused to the N-end of the polypeptide Ig Fc (b). Additional details regarding the functional properties and characteristics of such dimers are discussed above.

A stop codon (e.g., tga) are usually included at the end of each nucleic acid sequence when the sequence is included in the expression vector for the expression of the protein of interest. For example, each of the nucleotide n is sledovatelnot according to this invention, encoding a mutant polypeptide, CTLA-4, or a mutant protein, CTLA-4, may optionally also include a stop codon at the C-end, such as TAA. Different stop codons can replace TAA, such as TGA stop codon. The sequence of nucleic acids encoding a protein of wild-type (e.g., hCTLA-4-Ig, hCD86-Ig and the like), may also include a stop codon at its C-end. Each of the nucleotide sequences may optionally also include at N-end of the nucleotide sequence encoding a signal peptide to facilitate secretion of the mutant polypeptide, CTLA-4 or fused protein.

Illustrative of the mutant dimers fused protein, CTLA-4-Ig include those that include the amino acid sequence shown in SEQ ID NOS:74-79, 197-200, 205-214, and 219-222. Illustrative nucleic acid encoding a mutant fused proteins, CTLA-4 Ig SEQ ID NOS:74-79, 197-200, 220 and 222 shown in SEQ ID NO:153-158, 201-204 and p.223-224, respectively. Sequence fused proteins of SEQ ID NOS:205-210, 211-214, 219 and 221 are identical protein sequences SEQ ID NOS:74-79, 197-200, 220 and 222, respectively, except those protein sequences SEQ ID NOS:205-210 that do not include a C-terminal lysine (K) residue; as explained above, suppose that the estimated C-terminal lysine residue that is encoded AAA by codon immediately precedes the TAA stop codon of each poly is ucleotide sequence, cleaved from the resulting fused protein during processing or secretion. The nucleic acid sequences SEQ ID NOS:153-158, 201-204 and p.223-224 encoding sequence fused proteins of SEQ ID NOS:74-79, 197-200, 220 and 222, respectively, each of which after removal/loss of C-terminal K of the residue gives the sequence of the fused protein represented in SEQ ID NOS:205-210, 211-214, 219 and 221, respectively.

Each of the polynucleotide sequences SEQ ID NOS:153-158, 201-204 and p.223-224 also includes at its N-end nucleotide sequence encoding a signal peptide presented in SEQ ID NO:181 or 215, and the signal peptide necessarily cleaved from the Mature fused protein. Nucleotide residues 1-111 each of the polynucleotide sequences SEQ ID NOS:153-158, 201-204 and p.223-224, counting from N-Terminus of each polynucleotide sequence (nucleotide residues 1-111 shown in SEQ ID NO:215), encode a 37-amino acid residue DT hCTLA-4 signal peptide presented in SEQ ID NO:216, with signal peptide necessarily cleaved during expression monomer or dimer Mature mutant fused protein, CTLA-4; thus, for each of the nucleic acids sequences SEQ ID NOS:153-158, 201-204 p.223-224 and the first codon encoding the first amino acid residue (methionine) Mature IgG2 fused protein, contains nucleotide residues 112-114 decree is Noah nucleotide sequence. As noted above, in some cases, the sequence of the signal peptide can contain only the amino acid residues 1-35, as shown in SEQ ID NO:182, and the nucleotide sequence encoding this signal peptide composed of 35 amino acid residues shown in SEQ ID NO:181. However, the encoded lysine (K) and alanine (A) residues at positions 36 and 37, respectively (encoded by two codons AAA-GCC), not present in the resulting Mature mutant fused protein, CTLA-4-Ig, and assume that they hatshepsuts from Mature mutant fused protein, CTLA-4-Ig during processing. The sequence of the Mature mutant protein, CTLA-4 usually starts with a methionine residue present at position amino acid residues 38 to encode mutant fused protein, CTLA-4-Ig.

In another aspect, the invention provides isolated or recombinant nucleic acid comprising a polynucleotide sequence that encodes dimer fused protein (e.g., mutant dimer CTLA-4-Ig), consisting of two identical Monomeric fused protein (e.g., Monomeric mutant CTLA-4-Ig), where each such Monomeric protein includes an amino acid sequence having at least, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence selected from the group comprising SEQ ID NOS:74-79, 197-200, 205-214, and 219-22, where dimer fused protein binds CD80 and/or CD86 (and/or CD80-Ig and/or CD86-Ig) and/or has the ability to inhibit an immune response, or a complementary polynucleotide sequence. It also provides isolated or recombinant nucleic acid encoding such a Monomeric protein that binds CD80 and/or CD86 (and/or CD80-Ig and/or CD86-Ig) and/or has the ability to inhibit the immune response. Illustrative of the mutant dimers fused protein, CTLA-4-Ig include those that include amino acid sequences shown in SEQ ID NOS:74-79, 197-200, 220 and 222; illustrative nucleic acid encoding such mutant fused proteins, CTLA-4 Ig, which is expressed as mutant dimers fused protein, CTLA-4-Ig, include those that include a polynucleotide sequence represented in SEQ ID NOS:153-158, 201-204 and p.223-224, respectively. Additional illustrative mutant dimers fused protein, CTLA-4-Ig include the amino acid sequence of SEQ ID NOS:205-210, 211-214, 219 and 222, which are expressed as dimers fused protein; in these fused proteins lacking the C-terminal lysine residue, because it is usually cleaved during processing or prior to secretion. Illustrative nucleic acids encoding these sequences fused protein with C-terminal lysine (lysine is cleaved sequentially) include polynucleotide sequences SEQ ID NOS:153-158, 201-204 and p.223-224, respectively.

In another aspect, the invention provides isolated or recombinant nucleic acid comprising a polynucleotide sequence that encodes a protein, where the specified protein includes an amino acid sequence having at least, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence selected from the group comprising SEQ ID NOS:74-79, 197-200, 205-214, and 219-222, where the protein binds CD80 and/or CD86 (and/or CD80-Ig and/or CD86-Ig) and/or has the ability to inhibit an immune response, or a complementary polynucleotide sequence.

In another aspect, the invention includes an isolated or recombinant nucleic acid comprising a nucleotide sequence having at least, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with a polynucleotide sequence selected from the group comprising SEQ ID NOS:153-158, 201-204 and p.223-224, where this nucleic acid encodes a mutant protein dimer, CTLA-4-Ig, which binds CD80 and/or CD86 (and/or CD80-Ig and/or CD86-Ig) and/or has the ability to inhibit an immune response, or a complementary polynucleotide sequence.

In another aspect, the invention provides isolated or recombinant nucleic acid comprising polynucleotide the sequence, which encodes dimer fused protein (e.g., mutant dimer CTLA-4-Ig)comprising two Monomeric fused protein (e.g., Monomeric mutant CTLA-4-Ig), where each such Monomeric protein comprises: (1) a polypeptide (e.g., mutant CTLA-4 EVA), which includes amino acid sequence that differs from amino acid sequence selected from the group comprising SEQ ID NOS:1-73 by no more than 6 amino acid residues, and where the amino acid residue in the amino acid sequence in position 41, 50, 54, 55, 56, 64, 65, 70 or 85 is identical to the amino acid residue in the corresponding position of the specified selected amino acid sequence (e.g., a polypeptide selected from SEQ ID NOS:1-73), and (2) a polypeptide Ig Fc, where the dimer binds CD80 and/or CD86 (and/or CD80-Ig and/or CD86-Ig) and/or inhibits an immune response, or a complementary polynucleotide sequence. Additional details regarding the functional properties and characteristics of such dimers are discussed above. It also provides a recombinant or isolated nucleic acid comprising a nucleotide sequence that encodes such a Monomeric protein that binds CD80 and/or CD86 (and/or CD80-Ig and/or CD86-Ig) and/or has the ability to inhibit the immune response.

In another aspect, the invention provides isolated is consistent or recombinant nucleic acid, comprising a polynucleotide sequence that encodes dimer fused protein (e.g., mutant dimer CTLA-4-Ig)comprising two Monomeric fused protein (for example, two Monomeric mutant CTLA-4-Ig), where each such Monomeric protein comprises: (1) a polypeptide mutant CTLA-4 extracellular domain comprising amino acid sequence which (a) differs from the amino acid sequence selected from the group comprising SEQ ID NOS:1-73 by no more than 6 amino acid residues, and (b) includes at least one amino acid substitution in amino acid position corresponding to position 50, 54, 55, 56, 64, 65, 70 or 85 relative to the amino acid sequence of SEQ ID NO:159; and (2) Ig polypeptide, where the dimer fused protein binds CD80 and/or CD86 (and/or CD80-Ig and/or CD86-Ig) and/or inhibits an immune response, or a complementary polynucleotide sequence. Additional details regarding the functional properties and characteristics of such dimers are discussed above. Ig polypeptide can include a polypeptide Ig Fc, including, for example, the polypeptide Ig Fc comprising a sequence having at least, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with a sequence selected from the group comprising SEQ ID NOS:184-186 and 218. Some such polypeptides (a) contain at least one substitution in the amino acid is coherent position relative to SEQ ID NO:159, selected from the group comprising AL, MG, G55E, N56D, S64P, I65S and S70F. Some such polypeptides also include amino acid substitution relative to SEQ ID NO:159 in position 104 (for example, L104E/D), position 30 (for example, T30N/D/A) and/or position 32 (for example, V32I). It also provides a recombinant or isolated nucleic acid encoding such a Monomeric protein that binds CD80 and/or CD86 (and/or CD80-Ig and/or CD86-Ig) and/or has the ability to inhibit the immune response.

In another aspect, the invention includes an isolated or recombinant nucleic acid encoding a dimer fused protein (e.g., mutant dimer CTLA-4-Ig)comprising two Monomeric fused protein (e.g., Monomeric mutant CTLA-4-Ig), where each such Monomeric protein comprises: (1) a polypeptide (e.g., mutant CTLA-4 EVA), including amino acid sequence that (i) has at least, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence selected from the group comprising SEQ ID NOS:1-73, and (ii) includes a phenylalanine residue at amino acid position corresponding to position 70 of the specified amino acid sequence selected from the group comprising SEQ ID NO:1-73; and (2) Ig polypeptide, where the dimer fused protein binds CD80 and/or CD86 (and/or CD80-Ig and/or CD86-Ig) and/or has the ability Engibarov the th immune response, or a complementary polynucleotide sequence. Encoded Ig polypeptide can include a polypeptide Ig Fc, including amino acid sequence having at least, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence selected from the group comprising SEQ ID NOS:184-186 and 218. Some of these encoded dimers include one or more of the following relative to the specified selected amino acid sequence (1)(i): Glu residue at the position corresponding to position 24; Asn at position corresponding to position 30; Ile residue at the position corresponding to position 32; Met residue at the position corresponding to position 50; Lys residue at the position corresponding to position 54; Glu residue at the position corresponding to position 55; Asp residue at the position corresponding to position 56; Pro residue at the position corresponding to position 64; Ser residue in amino acid position corresponding to position 65; and the Glu residue in the position corresponding to position 104. Additional details regarding the functional properties and characteristics of such dimers are discussed above. It also provides a recombinant or isolated nucleic acid comprising a nucleotide sequence that encodes such a Monomeric protein that binds CD80 and/or CD86 (and/or CD80-Ig and/or CD86-Ig) and/or has the ability to inhibit many response.

In another aspect, the invention includes an isolated or recombinant nucleic acid encoding a dimer fused protein (e.g., mutant dimer CTLA-4-Ig)comprising two Monomeric fused protein (e.g., Monomeric mutant CTLA-4-Ig), where each such Monomeric protein comprises: (1) a polypeptide (e.g., mutant CTLA-4 EVA), including amino acid sequence which (a) differs from the amino acid sequence of the polypeptide of the human CTLA-4 EVA presented in SEQ ID NO:159, no more than 6 amino acid residues, and (b) includes at least one amino acid substitution, where indicated, at least the amino acid substitution includes S70F, where the position of the amino acid residues are numbered according to SEQ ID NO:159; and (2) the IgG Fc polypeptide, where the dimer binds hCD80 and/or hCD86 and/or hCD86-Ig, and/or hCD86-Ig) and/or inhibits an immune response, or a complementary polynucleotide sequence. Ig Fc polypeptide may include a sequence having at least, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence selected from the group comprising SEQ ID NOS:184-186 and 218. The encoded polypeptide (1) can also contain at least one amino acid substitution selected from the group comprising AU, T30N, V32I, D41G, AM, M54K, G55E, N56D, S64P, I65S, MA, L104E and I106F. The stage is leitlinie details about the functional properties and characteristics of such dimers are discussed above. It also provides a recombinant or isolated nucleic acid comprising a nucleotide sequence that encodes such a Monomeric protein that binds CD80 and/or CD86 (and/or CD80-Ig and/or CD86-Ig) and/or has the ability to inhibit the immune response.

In another aspect, the invention provides isolated or recombinant nucleic acid encoding a dimer fused protein (e.g., mutant dimer CTLA-4-Ig)comprising two Monomeric fused protein (e.g., Monomeric mutant CTLA-4-Ig), where each such Monomeric protein comprises: (1) a polypeptide (e.g., mutant CTLA-4 EVA), including amino acid sequence which (a) differs from the amino acid sequence of SEQ ID NO:31 for no more than 6 amino acid residues, and (b) includes at least one of the following: a methionine residue at the position corresponding to position 50 of SEQ ID NO:31, a lysine residue at the position corresponding to position 54 of SEQ ID NO:31, a glutamic acid residue in the position corresponding to position 55 of SEQ ID NO:31, a Proline residue at a position corresponding to position 64 of SEQ ID NO:31), the serine residue at the position corresponding to position 65 of SEQ ID NO:31), the phenylalanine residue at the position corresponding to position 70 of SEQ ID NO:31, where the position of the amino acid residues are numbered according to SEQID NO:31; and (2) Ig polypeptide, where the dimer binds hCD80 and/or hCD86 and/or hCD86-Ig, and/or hCD86-Ig) and/or inhibits an immune response, or a complementary polynucleotide sequence. Ig polypeptide can include a polypeptide Ig Fc, including, for example, the polypeptide Ig Fc comprising a sequence having at least, 95%, 96%, 97%, 98%, 99% or 100% identity with a sequence selected from the group comprising SEQ ID NOS:184-186 and 218. In some such dimers or monomers polypeptide (1) comprises a glutamic acid residue in the position corresponding to position 104, the aspartic acid residue in the position corresponding to position 30, and/or the isoleucine residue at the position corresponding to position 32 of SEQ ID NO:31. Additional details regarding the functional properties and characteristics of such dimers are discussed above. It also provides a recombinant or isolated nucleic acid comprising a nucleotide sequence that encodes such a Monomeric protein that binds CD80 and/or CD86 (and/or CD80-Ig and/or CD86-Ig) and/or has the ability to inhibit the immune response.

Also included in the invention are fragments of any of these nucleic acids according to this invention described above, where these fragments encode a polypeptide that binds hCD80 and/or hCD86 and/or EVA each or both and/or have osobnosti to suppress or inhibit an immune response. Many fragments of these nucleic acids will be to Express the polypeptides that bind hCD80 and/or hCD86 or EVA, or to suppress the immune response, and properties can be identified by appropriate experiments. Nucleotide fragments usually contain at least, 250, 300, 400, 500, 600, 700, 800, 900, 950, 1000 or more nucleotide bases.

The invention includes an isolated or recombinant nucleic acid that encodes a protein comprising the signal peptide and the polypeptide according to this invention (which includes dimeric or Monomeric protein according to this invention), such as a mutant polypeptide, CTLA-4 EVA or mutant protein, CTLA-4-Ig in this invention that binds CD80 and/or CD86 and/or suppresses an immune response in vitro or in vivo studies and/or methods, as described in detail in this description. The encoded sequence of the signal peptide that directs secretion of the Mature polypeptide through the membrane prokaryotic or eukaryotic cells, usually covalently linked to aminocom.com the specified polypeptide. Can be used a variety of signal peptides, including, for example, the sequence of the signal peptide shown in SEQ ID NO:182, which is encoded, for example, the nucleotide sequence represented in SQ ID NO:181, or the sequence of the signal peptide shown in SEQ ID NO:216, which is encoded, for example, the nucleotide sequence presented in SEQ ID NO:215. The invention also includes an isolated or recombinant nucleic acid that encodes a protein including the signal peptide, mutant polypeptide CTLA-4 EVA, a transmembrane domain and/or cytoplasmic domain, as discussed in detail above.

The sequence of the signal peptide of the full sized human protein, CTLA-4 can be used to direct the expression or secretion of recombinant mutant polypeptide CTLA-4 EVA or mutant fused protein, CTLA-4-Ig in this invention. In one aspect, the signal peptide (SP) protein hCTLA-4 includes amino acid residues 1-37 hCTLA-4 protein; this sequence is the signal peptide shown in SEQ ID NO:216. In this case, the Mature hCTLA-4 protein usually starts with a methionine residue at position 38, and amino acid residues of the Mature protein hCTLA-4 are numbered accordingly, since this residue is methionine, which is designated as the first amino acid (namely, position 1). Thus, the signal peptide, the peptide comprising the sequence represented in SEQ ID NO:216, may be fused or linked to the amino (N) end of the mutant polypeptide, CTLA-4 EVA or mutant fused protein, CTLA-4-Ig is about this invention, for example, through a covalent bond so as to facilitate the expression or secretion of the mutant polypeptide, CTLA-4 EVA or mutant fused protein, CTLA-4-Ig, respectively. Illustrative nucleic acid comprising a nucleotide sequence that encodes hCTLA-4 sequence of the signal peptide SEQ ID NO:216, shown in SEQ ID NO:215.

When the sequence of the signal peptide SEQ ID NO:216 merged using the N-end of the mutant polypeptide, CTLA-4 EVA or mutant fused protein, CTLA-4-Ig, expression or secretion of the specified polypeptide or fused protein, the signal peptide is cleaved, and the resulting Mature mutant polypeptide CTLA-4 EVA or Mature mutant protein, CTLA-4-Ig usually starts with a methionine residue at position 38, and amino acid residues of the Mature mutant polypeptide CTLA-4 EVA or Mature mutant fused protein, CTLA-4-Ig numbered, respectively, since this residue is methionine, which is denoted as the first amino acid (namely, position 1).

The invention includes isolated or recombinant polypeptide comprising a signal peptide (e.g., SEQ ID NO:216) and the mutant polypeptide, CTLA-4 EVA (for example, a sequence selected from the group of SEQ ID NOS:1-73), where the signal peptide is covalently linked to the N-end of the mutant polypeptide, CTLA-4 EVA. Also included is Yong in the invention is an isolated or recombinant polypeptide, including the signal peptide (e.g., SEQ ID NO:216) and mutant CTLA-4-Ig (e.g., a sequence selected from the group of SEQ ID NOS:74-79, 197-200, 205-214, and 219-222), where the signal peptide is covalently linked to the N-end of the mutant CTLA-4-Ig. It also provides isolated or recombinant nucleic acid comprising the nucleotide sequence (e.g., SEQ ID NO:215), coding for a signal peptide (e.g., SEQ NO:216) and the nucleotide sequence encoding a mutant polypeptide, CTLA-4 EVA (for example, a sequence selected from the group of SEQ ID NOS:1-73) or mutant protein, CTLA-4-Ig (e.g., a sequence selected from the group of SEQ ID NOS:74-79, 197-200, 205-214, and 219-222).

In an alternative aspect, the signal peptide full-hCTLA-4 protein comprises residues 1-35 full-size protein hCTLA-4; this signal peptide includes a peptide sequence represented in SEQ ID NO:182. In this case, two amino acid residue lysine (K) and alanine (A) at positions 36 and 37, respectively, protein hCTLA-4, however, are usually absent in Mature secretroom protein hCTLA-4, as determined by protein sequencing. Thus, the resulting Mature protein hCTLA-4 also begins with a methionine residue at position 38, and amino acid residues of the Mature protein hCTLA-4 numbered respectively from methionine residue at position 38 protein hCTLA-4 to the th designated as the first amino acid of the Mature protein hCTLA-4. Because amino acid residues lysine (K) and alanine (A) at positions 36 and 37, respectively, full-hCTLA-4 protein is not present in the resulting Mature protein hCTLA-4, it is believed that they have tsebelis from the Mature protein hCTLA-4 during processing. Illustrative nucleic acid comprising a nucleotide sequence that encodes a signal peptide sequence hCTLA-4a (SEQ ID NO:182), shown in SEQ ID NO:181.

Signal peptide, including a peptide sequence represented in SEQ ID NO:182, may be fused or linked to the N-end of the mutant polypeptide, CTLA-4 EVA or mutant fused protein, CTLA-4-Ig in this invention, for example, the covalent bond so as to facilitate the expression or secretion of the mutant polypeptide, CTLA-4 EVA or mutant fused protein, CTLA-4-Ig, respectively. When the sequence of the signal peptide SEQ ID NO:182 fused or linked to the N-end of the mutant polypeptide, CTLA-4 EVA or mutant fused protein, CTLA-4-Ig, expression or secretion of the polypeptide or fused protein, the signal peptide is cleaved, and the resulting Mature mutant polypeptide CTLA-4 EVA or Mature mutant protein, CTLA-4-Ig, however, usually starts with a methionine residue at position 38, and amino acid residues of the Mature mutant polypeptide CTLA-4 EVA or Mature mutant fused protein, CTLA-4-Ig numbered with the NGOs, since this residue is methionine, which is designated as the first amino acid (namely, position 1). Because amino acid residues lysine (K) and alanine (A) at positions 36 and 37, respectively, a full-sized protein hCTLA-4 not present in the resulting Mature mutant polypeptide CTLA-4 EVA or Mature mutant fused protein, CTLA-4-Ig, then I think they have tsebelis from the specified mutant polypeptide CTLA-4 EVA or mutant fused protein, CTLA-4 - Ig during processing.

The invention includes isolated or recombinant polypeptide comprising a signal peptide (e.g., SEQ ID NO:182) and the mutant polypeptide, CTLA-4 EVA (for example, a sequence selected from the group of SEQ ID NOS:1-73), where the signal peptide is covalently linked to the N-end of the mutant polypeptide, CTLA-4 EVA. Also, the invention includes isolated or recombinant polypeptide comprising a signal peptide (e.g., SEQ ID NO:182) and mutant CTLA-4-Ig (e.g., a sequence selected from the group of SEQ ID NOS:74-79, 197-200, 205-214, and 219-222), where the signal peptide is covalently linked to the N-end of the mutant CTLA-4-Ig. It also provides isolated or recombinant nucleic acid comprising the nucleotide sequence (e.g., SEQ ID NO:181), coding for a signal peptide (e.g., SEQ NO:182), and the nucleotide sequence encoding Muta is fair polypeptide CTLA-4 EVA (for example, sequence selected from the group of SEQ ID NOS:1-73) or mutant protein, CTLA-4-Ig (e.g., a sequence selected from the group of SEQ ID NOS:74-79, 197-200, 205-214, and 219-222).

Nucleic acid according to this invention may also include one or more acceptable additional nucleotide sequences. For example, taking into account that the polypeptide according to this invention (which comprises the protein according to this invention may include one or more additional amino acid sequences, such as, for example, the subsequence for purification of the polypeptide (such as, for example, the subsequence selected from the epitope of the marker FLAG marker polyhistidine sequence and GST merger), the sequence of the signal peptide and the like, this invention includes nucleic acids that encode these polypeptides, including such additional sequences. Illustrative signal peptides, which when expression is typically covalently linked to the N-terminal polypeptide according to this invention, discussed above. For example, nucleic acid encoding the amino acid sequence of any of SEQ ID NOS:1-79, 197-200, 205-214, and 219-222, may also include a nucleic acid encoding a signal peptide, such as the sequence of signals the th peptide SEQ ID NO:182, such as, for example, the nucleotide sequence shown in SEQ ID NO:181, or the sequence of the signal peptide shown in SEQ ID NO:216, which is encoded, for example, the nucleotide sequence presented in SEQ ID NO:215. Such nucleotide sequences can be directly fused together in an acceptable reading frame, so that the resulting nucleic acid includes a nucleotide sequence encoding a signal peptide according to this invention, and the nucleotide sequence encoding the polypeptide according to this invention.

Nucleic acid according to this invention can be isolated by any acceptable technique, which is known to specialists in this field. An isolated nucleic acid according to this invention (e.g., nucleic acid, which is obtained in the cell-master and forth substantially clear any acceptable method of purifying nucleic acids) can be re-integrated into the cell host or re-built in the cellular or other biological environment or composition, where it will no longer be the dominant species of nucleic acid and is no longer separated from other nucleic acids.

Virtually any isolated or recombinant nucleic acid according to this invention can be inserted in and is and is merged with acceptable more nucleic acid molecule (including, for example, but not limited to the following, chromosome, plasmid, an expression vector or expression cassette, the viral genome, a gene sequence, a linear element expression, bacterial genome, vegetable or synthetic genome chromosome, such as a synthetic chromosome mammal (SHM), or yeast or bacterial counterparts (namely, storage, or SHB) to form a recombinant nucleic acid using conventional techniques. As another example, the isolated nucleic acid according to this invention may be merged with the smaller nucleotide sequences, such as promoter sequences, immunostimulatory sequences and/or sequences encoding other amino acids, such as other antigenic epitopes and/or linker sequences for the formation of recombinant nucleic acids.

In some cases, recombinant or synthetic nucleic acid may be formed using methods of chemical synthesis, which are not applied in relation to the cell-master (for example, nucleic acid obtained by polymerase chain reaction (PCR) or methods for chemical synthesis, examples of which are described in this description).

Nucleic acids encoding the polypeptides (including fused proteins) on this image is the shadow can be of any acceptable chemical composition, which allows the expression of a polypeptide of this invention or other relevant biological activity (e.g., hybridization to other nucleic acids). Polynucleotide according to this invention can be in the form of RNA or in the form DNA, and include mRNA, krnk, recombinant or synthetic DNA and RNA and cDNA. Nucleic acid according to this invention are typically DNA molecules and usually double-stranded DNA molecules. However, single-stranded DNA, single-stranded RNA, double-stranded RNA and hybrid DNA/RNA nucleic acid, or a combination comprising any of the nucleotide sequences according to this invention, are also provided in this invention. Nucleic acid according to this invention may include any reasonable nucleotide base similar to the base and/or frame (for example, the frame formed of or including phosphothioate communication more often than fosfodiesterazu communication, for example, DNA comprising phosphothioate or phosphorothioate frame). Nucleic acid according to this invention, if it is single-stranded may be the coding chain or non-coding (namely, antisense or complementary) strand. In addition to the nucleotide sequence that encodes a polypeptide according to this invention (for example, the nucleotide posledovatelno the ü, which includes the coding sequence of the mutant polypeptide, CTLA-4 EVA or mutant CTLA-4-Ig), polynucleotide according to this invention may include one or more additional encoding nucleotide sequences to encode, for example, fused protein, the guide sequence (different from the signal sequence) or similar (more specific examples of which are also discussed in this description), and/or may include non-coding nucleotide sequences, such as introns, the terminal sequence, or 5' and/or 3' netransliruemye area, and these areas may be effective for the expression of the coding sequence in an acceptable host, and/or control elements, such as promoter (e.g., naturally occurring or recombinant or piratesbay promoter).

Modification of nucleic acids is especially valid in the 3rd position of the mRNA radonaway sequence that encodes such a polypeptide. In particular aspects, at least a portion of the nucleic acid includes phosphorothioate frame, built in, at least one synthetic nucleotide analogue in place of or in addition to naturally occurring nucleotides in the sequence of nucleic acids. Also or alternatively, a nucleic acid which may include the addition of bases, other than guanine, adenine, uracil, thymine and cytosine. Such modifications can be associated with a longer half-life and, thus, may be desirable in the vector nucleic acid according to this invention. Thus, in one aspect the invention provides recombinant nucleic acids and nucleic acid vectors (also discussed below), and the nucleic acids or vectors contain at least one of the above modifications, or any acceptable combination thereof, where the nucleic acid persists longer in the host-mammal than a substantially identical nucleic acid without such modification or modifications. Examples of modified and/or non-casinovip, not-Denisovich, not-Gurinovich, not-siminovich nucleotides that can be incorporated into the nucleotide sequence of this invention shown in, for example, MANUAL OF PATENT EXAMINING PROCEDURE § 2422 (7th Revision - 2000).

It should be understood that nucleic acid encoding at least one polypeptide according to this invention (which comprises the protein according to this invention, including those described above and elsewhere in this description are not limited to the sequence, which directly encodes for the expression or production of the polypeptide according to this invention. For example, nekleenov the I acid can include a nucleotide sequence, which results in a polypeptide according to this invention by intein-like expression (as described in, for example, Colson and Davis (1994) Mol. Environ. 12(3):959-63, Duan et al. (1997) Cell 89(4):555-64, Perler (1998) Cell 92(1): 1-4, Evans et al. (1999) Biopolymers 51(5):333-42 and de Grey, Trends Biotechnol. 18(9):394-99 (2000)), or a nucleotide sequence that includes a self-playeraudio introns (or other self-playeraudio RNA transcripts), which forms an intermediate sequence encoding a recombinant polypeptide (as described, for example, in U.S. Patent No. 6010884). Nucleic acid also or alternatively can include sequences that lead to other modifications of splicing at the RNA level to obtain an mRNA transcript encoding the polypeptide and/or at the DNA level through mechanisms of TRANS-splicing in front of transcription (principles relating to the mechanisms described in, for example, Chabot, Trends Genet. (1996) 12(11):472-78, Cooper (1997) Am. J. Hum. Genet. 61(2):259-66 and Hertel et al. (1997) Curr. Opin. Cell. Biol. 9(3):350-57). Due to the inherent degeneracy of the genetic code, some nucleic acid can encode a polypeptide according to this invention. Thus, for example, any of the specific nucleic acids described herein can be modified by replacing one or more codons equivalent codon (relative to amino acids, the code is generated by the codon) on the basis of the degeneracy of the genetic code. Other nucleotide sequences that encode a polypeptide having the same or a functionally equivalent sequence with the amino acid sequence of this invention can also be used for synthesis, cloning and ekspressirovali such a polypeptide.

In General, any of the nucleic acids according to this invention can be modified to increase expression in a particular host, using the techniques presented in this description for example, in relation to the above-described nucleic acids coding for the polypeptide according to this invention (for example, a nucleic acid encoding a mutant CTLA-4 EVA or mutant CTLA-4-Ig). Any of the nucleic acids according to this invention, as described herein, may be codon-optimized for expression in a particular mammal (usually men). Various techniques for optimizing the codon known in the art. Codons that are used most often in a particular host, called optimal codons, and those that are not used very often, are classified as rare or slabosalenuyu codons (see, for example, Zhang, S. P. et al. (1991) Gene 105:61-72). Codons can be substituted to reflect the predominant use of codon host, the process is called "optimization Kodo is s or control over the body of statistical deviation from uniformity in the use of codons". Optimized coding sequence comprising codons that are preferred for a particular prokaryotic or eukaryotic host can be used to increase the speed broadcast or to obtain recombinant RNA transcripts having desirable properties, such as increased half-life compared to transcripts obtained from the non-optimized sequence. Methods to obtain sequences with optimized codons are known (see, for example, E. et al. (1989) Nuc. Acids Res. 17:477-508). Translational stop codons can also be modified to reflect the preference of the owner. For example, the preferred stop-codons for S. cerevisiae and mammals is UAA and UGA, respectively. The preferred stop codon for monocotyledonous plants is UGA, whereas insects and E. coli prefer the use of UAA as a stop-codon (see, for example, Dalphin, M.E. et al. (1996) Nuc. Acids Res. 24:216-218). The location of the codons in relation to other codons may also affect the biological properties of nucleic acids sequences and modification of nucleic acids to provide the location context of the codon-specific master, has also been considered by the inventors. Thus, the sequence of nucleic acids on this and the finding may include the nucleotide sequence with optimized codons, and it is often used with optimized code and/or codanova couple (namely, the context of the codon optimized for specific species (e.g., the polypeptide can be expressed from a polynucleotide sequence optimized for expression in humans, by replacing "rare" human codons, based on the frequency of codon usage or context of the codon, for example, using techniques such as those described in Buckingham et al. (1994) Biochimie 76(5):351-54 and U.S. Patent No. 5082767, 5786464 and 6114148).

Nucleic acid according to this invention can be modified by clipping or one or more residues from the C-terminal part of the sequence. Additional different stop or termination codons can be included at the end of the nucleotide sequence, which is also discussed below.

One or more nucleic acids according to this invention can be incorporated into a vector, cellular environment or the environment of the host, which encodes the nucleotide sequence according to this invention is a heterologous gene.

Polynucleotide in this invention include polynucleotide sequences that encode any polypeptide according to this invention (or a polypeptide fragment)that binds CD80 and/or CD86 and/or p is Dawley immune response, polynucleotide that hybridizing under at least stringent conditions with one or more such polynucleotide sequences described herein, polynucleotide sequences complementary to any such polynucleotide sequences, and variants, analogs and homologous derivatives of all those that described above. The coding sequence refers to a nucleotide sequence that encodes a particular polypeptide or domain subsequence, region or fragment of the specified polypeptide. The coding sequence can encode a mutant polypeptide, CTLA-4 or a fragment having a functional property, such as the ability to bind CD80 and/or CD86 and/or to inhibit or suppress an immune response. Nucleic acid according to this invention may include appropriate coding sequence of the mutant polypeptide, CTLA-4 on this invention and its variants, analogs and homologous derivatives.

Nucleic acid according to this invention can also be detected in combination with a typical composite structures of nucleic acids, including in the presence of media, buffers, excipients, fillers, solvents and the like, which are known to experts in this field.

Unless the decree is but otherwise, the specific sequence of nucleic acid described herein, also includes its conservatively modified variants (for example, replacement of the degenerate codon) and complementary sequences, as well as a precisely defined sequence. Specifically, substitution of degenerate codons can be achieved by the formation of sequences in which the third position of one or more selected (or all) codons is substituted by the remnants of mixed bases and/or deoxyinosine residues (Batzer et al. (1991) Nucl. Acid Res. 19:5081; Ohtsuka et al. (1985) J. Biol. Chem. 260:2605-2608; Cassol et al. (1992); Rossolini et al. (1994) Mol. Cell. Probes 8:91-98).

Hybridization of nucleic acids

As noted above, this invention includes nucleic acids that hybridize to the target nucleic acid according to this invention, such as, for example, polynucleotide selected from the group comprising SEQ ID NOS:80-158, 201-204, 223 and 224, or a complementary polynucleotide sequence, where the hybridization covers essentially the entire length of the target nucleic acid. Hybridized nucleic acid can hybridisierung with the nucleotide sequence according to this invention, such as, for example, SEQ ID NO:80, under at least stringent conditions or under at least high stringency conditions. Known medium strict, strict, and with the flax stringent hybridization conditions for the experiments on the hybridization of nucleic acids. Examples of factors that can be combined to achieve such levels of severity, are briefly discussed in this specification.

Nucleic acids "hybridize"when they contact you, usually in solution. Nucleic acids hybridize under various well-described physico-chemical forces, such as hydrogen bond, excluding solvent, the stacking interaction and the like. An extensive guide to the hybridization of nucleic acids can be found in P. Tijssen (1993) LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY-HYBRIDIZATION WITH NUCLEIC ACID PROBES, vol. 24, part I, chapter 2, "Overview of principles of hybridization and the strategy of nucleic acid probe assays," (Elsevier, NY) (hereinafter "Tijssen"), as well as in Ausubel, see above, Hames and Higgins (1995) GENE PROBES 1, IRL Press at Oxford University Press, Oxford, England (Hames and Higgins 1) and Hames and Higgins (1995) GENE PROBES 2, IRL Press at Oxford University Press, Oxford, England (Hames and Higgins 2) provide details of the synthesis, the formation of labels, definitions and quantitative analysis of DNA and RNA, including oligonucleotides.

Determining that two sequences of nucleic acids are essentially identical is that the two molecules hybridize to each other under at least stringent conditions. The phrase "hybridized specifically to" refers to the binding, the formation of duplex or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions, is when such a sequence is present in a complex mixture (e.g., total cellular) DNA or RNA. "Bind(et) essentially" refers to complementary hybridization between the nucleic acid probe and the target nucleic acid and embraces minor mismatches that can be adjusted by reducing the severity of environments hybridization to obtain the desired target polynucleotide sequence.

"Stringent hybridization conditions of washing" and "stringent hybridization conditions" in the context of experiments on hybridization of nucleic acids, such as southern and Northern hybridization, are dependent on the sequence and are different under different settings. An extensive guide to the hybridization of nucleic acids can be found in Tijssen (1993), see above, and in Hames and Higgins 1 and Hames and Higgins 2, see above.

In General, conditions of high stringency are chosen so that hybridization occurs at from about 5°C or less than the melting temperature (Tm) for the specific sequence at a defined ionic strength and pH. Tmis the temperature (under defined ionic strength and pH)at which 50% of the analyzed sequence's hybrid with a perfectly matched probe. In other words, Tmdefines the temperature at which the duplex nucleic acid is 50% denatured under these conditions and so dstanley direct measurement of the stability of the hybrid nucleic acid. Thus, Tmcorresponds to the temperature corresponding to the middle point in the transition from helix to random spiral; it depends on the length, nucleotide composition and ionic strength for nucleotides long. Typically, when "strict conditions" the probe will be hybridisierung with its target subsequence, but not with any other sequences. "Very strict conditions" is chosen to be equal to Tmfor a particular probe.

Tmduplex DNA-DNA can be calculated by applying the equality (I): Tm(°C)=81,5°C+16,6 (log10M)+0,41 (%G+C) is 0.72 (%f) - 500/n, where M is both molarity of the monovalent cations (usually Na+), (%G+C) is the percentage of nucleotides guanosine (G) and cytosine (C), (%f) is the percentage of formalin and n is the number of nucleotide bases (namely, length) of the hybrid. See Rapley, R. and Walker, J. M. eds., MOLECULAR BIOMETHODS HANDBOOK (1998), Humana Press, Inc. (hereinafter Rapley and Walker), Tijssen (1993), see above. Tmduplex RNA-DNA can be calculated using expression (2): Tm(°C)=79,8°C+18,5 (log10M)+of 0.58 (%G+C) and 11.8(%G+C)2- 0,56 (%f) - 820/n, where M is both molarity of the monovalent cations (usually Na+), (%G+C) is the percentage of nucleotides guanosine (G) and cytosine (C), (%f) is the percentage of formamide and n is the number of nucleotide bases (namely, length) of the hybrid. Id. Equations 1 and 2 above are usually only suitable for hybrid duplexes length of more than about 1 10-200 nucleotides. Id. Tmsequences of nucleic acids shorter than 50 nucleotides, can be calculated as follows: Tm(°C)=4(G+C)+2(A+T), where A (adenine), T (thymine), and G is the number of matching nucleotides.

In General, dehybridization material nucleic acids are removed by a series of cleaning, the severity of which can be adjusted depending on the desired results, for the analysis of hybridization. The washing conditions of low stringency (for example, using a higher salt concentration and lower temperature) increases the sensitivity, but may lead to non-specific hybridization signals and high background signals. Conditions of high stringency (for example, using a lower salt concentration and higher temperature, which is close to the temperature of hybridization) reduces the background signal, usually leaving only the specific signal transmission. Additional guidance regarding such hybridization techniques is provided in, for example, Rapley and Walker, see above (in particular, with respect to such hybridization experiments, part I, Chapter 2, "Overview of principles of hybridization and the strategy of nucleic acid probe assays"), Elsevier, New York, as well as in Ausubel, see above, Sambrook, see above, Watson, see above, Hames and Higgins (1995) GENE PROBES 1, IRL Press at Oxford Univ. Press, Oxford, England, and Hames and Higgins (1995) GENE PROBES 2, IRL Press, Oxford Univ. Press, Oxford, ngland.

Illustrative strict (or normal stringency) conditions for analysis of at least two nucleic acid, comprising at least 100 nucleotides include incubation in solution or on a filter in a southern or Northern blot band, which includes 50% formalin (or wavelengths) 1 milligram (mg) of heparin at 42°C, while hybridization over night. The hillshade normal severity can be carried out using, for example, solutions containing 0,2x SSC washing at about 65°C for about 15 minutes (see Sambrook, see above for a description of SSC buffer). Frequently washing the normal rigor about the post-cleaning low stringency to remove background probe signal. The hillshade low severity can be conducted, for example, a solution containing 2x SSC at 40°C for about 15 minutes. Washing high severity can be done with the use of solutions containing 0.15 M NaCl at 72°C for about 15 minutes. The example of washing medium (normal) severity, less stringent than the normal washing severity, described above, for duplex, for example, more than 100 nucleotides, can be carried out in a solution comprising 1x SSC at 45°C for 15 minutes. An example of cleaning a low severity for a duplex of, for example, more than 100 nucleotides, can be carried out in a solution of 4-6x SSC at 40°C for 15 minutes. For short IU is OK (for example, about 10 to 50 nucleotides), stringent conditions typically involve salt concentrations of less than about 1.0 M Na+ion, typically about 0.01 to 1.0 M Na+ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is typically at least about 30°C. Stringent conditions can also be achieved by the addition of destabilizing agents such as formamide.

Illustrative conditions of medium stringency include incubation over night at 37°C in a solution comprising 20% formalin (or wavelengths), 0,5x SSC, 50 mm sodium phosphate (pH of 7.6), 5x denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured hydrodynamically fragmentirovannoj DNA salmon sperm, followed by washing the filters for washing in 1x SSC at about 37-50°C, or essentially similar conditions, such as medium stringent conditions, as described in Sambrook, see above, and/or Ausubel, see above.

Conditions of high stringency conditions, which use, for example, (1) low ionic strength and high temperature for washing, for example, of 0.015 M sodium chloride/0,0015 M sodium citrate/0.1% sodium dodecyl sulfate (SDS) at 50°C, (2) use of denaturing agent in the process of hybridization, such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin (BSA)/a 0.1% Ficoll/0.1% polyvinylpyrrolidone (PVP)/50 mm sodium phosphate buffer at pH 6.5 with 750 mm sodium chloride, 75 mm sodium citrate at 42 is, or (3) employ 50% of formamide, 5x SSC (0.75 M NaCl, Of 0.075 M sodium citrate), 50 mm sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x denhardt's solution, DNA salmon sperm treated with ultrasound (50 µg/ml), 0.1% of SDS, and 10% dextran sulfate at 42°C, with cleaning at (i) 42°C in 0,2x SSC, (ii) at 55°C in 50% formamide and (iii) at 55°With 0,1x SSC (mostly in combination with EDTA (ethylenediaminetetraacetic acid)).

In General, the ratio of signal to noise ratio was 2 or 2.5-5 times more (or higher)than that observed for an unrelated probe in the particular hybridization analysis, which identifies the definition of a specific hybridization. Determining at least stringent hybridization between two sequences in the context of the present invention determines a strong structural similarity or homology with, for example, the nucleic acids according to this invention.

As noted, "highly stringent" conditions are selected to be about 5°C or lower than the melting temperature (Tm) for the specific sequence at a defined ionic strength and pH. Target sequences that are closely related to or identical with the nucleotide sequence of interest (e.g., "probe") can be identified under conditions of high stringency. Conditions of low stringency is suitable for sequences that are less complementary. motri, for example, Rapley and Walker; Sambrook, all see above.

Comparative hybridization can be used to identify nucleic acid according to this invention, and this method is comparative hybridization is the preferred method for distinguishing nucleic acids according to this invention. The definition of highly stringent hybridization between two nucleotide sequences in the context of the present invention defines a relatively strong structural similarity/homology with, for example, nucleic acids, presented in the order listed in this description. Highly stringent hybridization between two nucleotide sequences shows degree of similarity or homology of structure, composition of the nucleotide bases, locations, or order that is greater than the defined stringent conditions of hybridization. In particular, the definition of highly stringent hybridization in the context of the present invention determines a strong structural similarity or structural homology (for example, nucleotide structure of part of the grounds, location or order) with, for example, nucleic acids, presented in the order listed in this description. For example, it is desirable definition of the analyzed nucleic acids that hybridize with Fig the administrative nucleic acids in this description under strict conditions.

Thus, measurement of stringent hybridization is the ability to hybridisierung with nucleic acid according to this invention (for example, nucleic acid that includes a polynucleotide sequence selected from the group comprising SEQ ID NOS:80-158, 201-204, 223 and 224, or a complementary polynucleotide sequence) under conditions of high stringency, or very stringent conditions, or conditions of hybridization of ultra-high severity or conditions of hybridization super-super high severity). Stringent hybridization conditions (including, for example, highly stringent, super high severity or super-super high stringency conditions of hybridization and washing can be easily determined empirically for any of the analyzed nucleic acids.

For example, when determining highly stringent conditions of hybridization and washing conditions of hybridization and washing is gradually increased (e.g., increasing temperature, decreasing salt concentration, increasing the concentration of detergent and/or increasing the concentration of organic solvents such as formaldehyde, by hybridization or washing)until it matches the selected criteria. For example, the conditions of hybridization and washing is gradually increased until a probe comprising one or more nucleic acids sequences selected from the group comprising SEQ ID NOS:80-158, 201-204, 22 and 224 and the complementary polynucleotide sequence, do not link to the full match complementary target (again, a nucleic acid, comprising at least one sequence of a nucleic acid selected from the group comprising SEQ ID NOS:80-158, 201-204, 223 and 224 or a complementary polynucleotide sequence), with signal-to-noise ratio that is at least 2.5 times, and optional 5 times or more higher than that observed for hybridization of the probe with a different target. Mismatched target may include nucleic acid corresponding to, for example, the polypeptide-encoding nucleic acid sequence of CTLA-4.

Usually hybridization analysis is performed under conditions of hybridization, is selected so that the nucleic acid comprising a sequence that is completely complementary to the described reference (or known) nucleotide sequence (e.g., SEQ ID NO:80), was hybridisable with recombinant antigen-encoding sequence (e.g., a variant of the nucleotide sequence of the nucleic acid SEQ ID NO:80) with at least about 5, 7 or 10 the highest value of the ratio of signal to noise ratio than that observed for hybridization of the completely complementary nucleic acid with a nucleic acid that includes a nucleotide sequence that least about 80 or 90% identical to the reference nucleic acid. Such conditions may be considered as defining for specific hybridization.

The above-described hybridization conditions can be adjusted, or selected alternative conditions of hybridization to achieve any desired level of rigor to select the hybridized nucleic acid sequences. For example, the above-mentioned highly stringent conditions of hybridization and washing can be gradually increased (e.g., by increasing temperature, decreasing salt concentration, increasing the concentration of detergent and/or an increase in the concentration of organic solvents such as formaldehyde, by hybridization or washing)until it matches the selected set of criteria. For example, the conditions of hybridization and washing can be gradually increased until the desired probe does not contact with the same complementary target with a signal-to-noise ratio that is at least about 2.5 times, and optionally, at least about 5 times (for example, about 10 fold, about 20 fold, about 50 fold, about 100 fold or even about 500 times higher than the signal-to-noise ratio observed in the hybridization of the probe with the nucleic acid according to this invention, such as a nucleic acid encoding a polypeptide DT CTLA-4 EVA.

The formation and modification of nucleic acids

Nucleic acids according to izopet the tion can be obtained and/or formed using any of the acceptable methods of synthesis, manipulation and/or selection or combinations thereof. Illustrative procedures are described below. For example, polynucleotide according to this invention are usually obtained by standard methods of nucleic acid synthesis, such as solid-phase synthesis techniques known in the art. In such methods, the fragments of up to about 100 bases usually are synthesized separately and then combined (e.g., methods of enzymatic or chemical ligation or by recombinant methods, mediated polymerase) to generate essentially the nucleic acid sequence of any desired length. The synthesis of nucleic acids according to this invention can also be facilitated (or alternative implemented) by chemical synthesis using, for example, the classic phosphoramidite method, which is described in, for example, Beaucage et al. (1981) Tetrahedron Letters 22:1859-69, or the method described in Matthes et al. (1984) EMBO J. 3:801-05, for example, as it is typically practiced in automated synthesis methods. Nucleic acid according to this invention can also be obtained by using an automatic DNA synthesizer. Other methods for synthesis of nucleic acids and related principles are described in, e.g., Itakura et al., Annu. Rev. Biochem. 53:323 (1984), Itakura et al.. Science 198:1056 (1984), and Ike et al., Nucl. Acid Res. 11:477 (1983).

Traditionally performed in the order the nucleic acid can be obtained from various commercial sources, such as the Midland Certified Reagent Company (mcrc@olygos.com). Great American Gene Company (world wide web website address genco.com), ExpressGen Inc. (address to the world web site expressgen.com), Operon Technologies Inc. (Alameda, CA). Likewise custom-made peptides and antibodies can be obtained from any of numerous sources, for example, PeptidoGenic (pkim@ccnet.com), HTI Bio-products, Inc. (address to the world web site htibio.com), and BMA Biomedicals Ltd. (U.K.), Bio.Synthesys, Inc.

Certain nucleotides according to this invention can also be obtained by screening cDNA libraries using oligonucleotide tags that can be hybridisierung with or PCR to amplify polynucleotide that encode the polypeptides according to this invention. Procedures for screening and selection of cDNA clones and PCR amplification procedure is well known to specialists in this field; illustrative of the procedures described below (see, for example, the procedures described in the examples below). Such techniques are described in, for example, Berger and Kimmel, "Guide to Molecular Cloning Techniques," Methods in Enzymol. Vol.152, Acad. Press, Inc., San Diego, CA ("Berger"); Sambrook, see above; and Ausubel, see above. Some nucleic acid according to this invention can be obtained by changing the nature of the skeleton, for example, using mutagenesis, in vitro recombination (e.g., dicing), or oligonucleotide recombination. In other cases, such polynucleotide can be obtained i silico (computer simulation) or by means of oligonucleotide recombination, as described in the references cited in this specification.

Methods of recombinant DNA, suitable for modification of nucleic acids are well known in the art (for example, restriction endonuclease cleavage, ligation, reverse transcription, and cDNA products, and PCR). Suitable methods of recombinant DNA technology and principles related to them are described in, for example, Mulligan (1993) Science 260:926-932, Friedman (1991) THERAPY FOR GENETIC DISEASES, Oxford University Press, Ibanez et al. (1991) EMBO J. 10:2105-10, Ibanez et al. (1992) Cell 69:329-41 (1992), and U.S. Patent№4440859, 4530901, 4582800, 4677063, 4678751, 4704362, 4710463, 4757006, 4766075 and 4810648, and more specifically described in Sambrook et al. (1989) MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Press, and the third edition thereof (2001), Ausubel et al. (1994-1999), Current Protocols in Molecular Biology, Wiley Interscience Publishers (with Greene Publishing Associates for some editions), Berger, see above, and Watson, see above.

Modified coding sequences

When necessary, the nucleic acid according to this invention can be modified to increase or enhance expression in a particular host by modifying the sequence in the light of codon usage and/or context of the codon, this particular owner(s)in which the expression of the nucleic acid is desirable. Codons that are used most often in a particular host, called optimal codons, and those that are not used PTS is ery often, classified as rare or poorly-used codons (see, for example, Zhang, S. P. et al. (1991) Gene 105:61-72). Codons can be substituted to reflect the preferred codons used by the host, the process is called a "codon optimization" or "control characteristic of the organism statistical deviation from uniformity in the use of codons".

Optimized coding sequence comprising codons that are preferred for a particular prokaryotic or eukaryotic host can be used to increase the speed broadcast or for the production of recombinant RNA transcripts having desirable properties, such as increased half-life, than transcripts derived from the non-optimized sequence. Methods for obtaining the codon-optimized sequences are known (see, for example, Murray, E. et al. (1989) Nucl. Acids Res. 17:477-508). Translational stop codons can also be modified to reflect the preference of the owner. For example, the preferred stop-codons for S. cerevisiae and mammals is UAA and UGA, respectively. The preferred stop codon for monocotyledonous plants is UGA, whereas insects and E. coli prefer the use of UAA as a stop-codon (see, for example, Dalphin, M. E. et al. (1996) Nucl. Acids Res. 24:216-218, for discussion). RA is the position of the codons in relation to other codons can also affect the biological properties of nucleic acid sequences, and modification of nucleic acids to provide the location context of the codon-specific master also envisioned by the inventors. Thus, the sequence of nucleic acid according to this invention may include the nucleotide sequence with optimized codons, namely, frequently used codon optimized and/or radonaway pair (namely, the context of the codon optimized for specific species (for example, the polypeptide may be expressed from a polynucleotide sequence optimized for expression in humans by replacing "rare" human codons based on the frequency of codon usage or context of the codon, for example, using techniques such as those described in Buckingham et al. (1994) Biochimie 76(5):351-54 and U.S. Patent No. 5082767, 5786464 and 6114148). For example, the invention provides nucleic acid comprising a variant of the nucleotide sequence of SEQ ID NO:80, where a variant of the nucleotide sequence differs from the nucleotide sequence of SEQ ID NO:80 replacement "rare" codons for a particular host on codons, usually expressed in the host, where the codons encode the same amino acid residue that is substituted by "rare" codons in SEQ ID NO:80.

VECTORS, VECTOR COMPONENTS AND systems for the EXPRESSION

This image is giving also includes recombinant constructs comprising one or more nucleic acids according to this invention, as broadly described above. Such constructs can include a vector such as a plasmid, cosmid, phage, virus, viral particle, viral-like particle, a bacterial artificial chromosome (BSH), yeast artificial chromosome (USDA) or the like, or dereplication vector, such as a liposome, naked or conjugated DNA, DNA-microparticles, in which is embedded at least one sequence of nucleic acid according to this invention, in a direct or reverse orientation. In a particular aspect of this case for the construct also includes one or more regulatory sequences, including, for example, a promoter functionally linked to a nucleic acid sequence according to this invention (for example, nucleic acid encoding the isolated or recombinant mutant polypeptide CTLA-4 EVA or dimeric or Monomeric mutant CTLA-4-Ig). A large number of appropriate vectors and promoters are known to specialists in this field and is commercially available. In some cases, a vector such as a virus or virus-like particle, can also or alternatively include one or more polypeptides according to this invention, such as, for example, built in Obolo is the virus or virus-like particles. The vectors may be suitable as a delivery agent for delivery or administration to a subject of exogenous genes or proteins. The vectors of this invention, including those described herein, are suitable as delivery agents for delivery or administration of nucleic acids and/or polypeptides according to this invention.

General articles that describe molecular biological techniques useful in the present description, including the use of vectors, promoters and many other relevant topics, include Berger, see above, Sambrook (1989), see above and Ausubel, see above. Examples of techniques sufficient to guide the person skilled in the art on methods, in vitro amplification, including the following: polymerase chain reaction (PCR), ligase chain reaction (LCR), QΞ-replicata amplification and other RNA polymerase mediated techniques (e.g., first NASBA), for example, for the production of the homologous nucleic acids of this invention can be found in Berger, Sambrook, and Ausubel, all see above, as well as Mullis et al. (1987) U.S. Patent No. 4683202; PCR Protocols: a Guide to Methods and Applications (Innis et al., eds.) Academic Press Inc. San Diego, CA (1990) ("Innis"); Amheim & Levinson (October 1, 1990) C&EN 36-47; the Journal OfNIH Research (1991) 3:81-94; (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173 - 1177; Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878; Lomeli et al. (1989) J. Clin. Chem. 35:1826-1831; Landegren et al. (1988) Science 241:1077-1080; Van Brunt (1990) Biotechnology 8:291-294; Wu and Wallace (1989) Gene 4:560-569; Barringer et al. (1990) gene 89: 117-122, and Soknanan and Malek (1995) Biotechnology 13:563-564.

PCR in General refers to the procedure where the exact amount of a specific part of the nucleic acid (e.g., RNA or DNA) is amplified by methods well known in the art (see, for example. U.S. patent No. 4683195 and other references cited above). In General, the sequence information from the ends of the region of interest, or outside this field is used to design oligonucleotide primers. Such primers will be identical or similar to a sequence opposite chains of the matrix that you want to amplify. 5' terminal nucleotides opposite chains may coincide with the ends of the amplified material. PCR can be used to amplify specific RNA or specific DNA sequences, recombinant DNA or RNA sequences, DNA and RNA sequences from total genomic DNA, and cDNA transcribed from total cellular RNA, bacteriophobic or plasmid sequences, etc. PCR is one example, but it is not only an example of the method of polymerase reactions, nucleic acid amplificatoare the sample nucleic acid, comprising applying another (e.g., known) nucleic acid as a primer. Improved methods of cloning in vitro amplified nucleic keys is described in Wallace et al., U.S. patent No. 5426039. Improved methods of amplifying large nucleic acids by PCR are summarized in Cheng et al. (1994) Nature 369:684 685 and the references cited in them that generate PCR amplicons up to 40 thousand base pairs (kb). The person skilled in the art should be understood that essentially any RNA can be converted into double-stranded DNA suitable for processing by enzymes, continuing PCR and sequencing using reverse transcriptase polymerase. See Ausubel, Sambrook, and Berger, all of the above.

Nucleic acid according to this invention can be incorporated into any one of a variety of vectors, such as expression vectors, expressed polypeptides, including, for example, the polypeptide according to this invention. Can be used in the expression vectors compatible with cells prokaryotic host; prokaryotic expression vectors known in the field of technology and commercially available. Such vectors include, but are not limited to the following, for example, BLUESCRIPT vector (Stratagene), the T7 expression vector (Invitrogen), pET vectors (Novagen) and similar prokaryotic vectors.

An alternative can be used in the expression vectors compatible with eukaryotic host; such eukaryotic expression vectors are known in the field of technology and commercially available. Such, etc) the market include, but not limited to the following, for example, pCMV vectors (e.g., Invitrogen), pIRES vector (Clontech), pSG5 vector (Stratagene), pCDNA3,l (Invitrogen Life Technologies), pCDNA3 (Invitrogen Life Technologies), Ubiquitous Chromatin Opening Element (UCOE™expression vector (Millipore) and similar eukaryotic expression vectors. UCOE™ vector is typically used for production of the protein in mammalian cells (e.g., Cho cells). According Millipore, technology UCOE™ expression makes it difficult transgenic silencing and stable gene expression in a high level, regardless of the location of chromosomal integration. See Millipore website on the world address millipore.com. Illustrative UCOE the expression vector, in which, for example, a nucleic acid according to this invention can be integrated, represents the UCOE expression vector, named CET1019AS-puro-SceI, which is available under license from Millipore. Information about UCOE vectors expression CET1019AS-puro-SceI can be found in, for example, John Wynne, "UCOE™ Technology Maximizes Protein Expression", BioProcess Internatonal 4(7): 104-105 (July/August 2006) (RP1725ENOO) (available at world wide web address millipore.com/bibliography/tech1/rp1725en00); for more information about this vector and licensing of this vector from Millipore available on the web site Millipore, including, for example, the web address millipore.com/company/cp3/ucoe_licensing and millipore.com/techpublications/tech1/ps1013en00. Thus, for example, a DNA sequence encoding a mutant CTLA-4 is KD (for example, SEQ ID NO:36 or SEQ ID NO:50), merged with the DNA sequence coding IgG2 Fc polypeptide (e.g., SEQ ID NO:184), resulting in DNA sequence SEQ ID NO:201, embedded in UCOE CET1019AS vector (Millipore) and the resulting DNA plasmid can be used for transpency cells of the host.

The expression vectors include chromosomal, achromosome and synthetic DNA sequences, e.g., derivatives of SV40, bacterial vectors (e.g., S. typhimurium, S. typhi, S. flexneri, Listeria monocytogenes, B. anthracis); plasmids; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA; viral DNA or RNA vector, including, for example, the vaccine virus, adeno-associated virus (AAV), adenovirus, Semliki Forest virus (for example, Notka et al., Biol. Chem. 380:341-52 (1999), poxvirus (e.g., MVA), alphavirus (for example, virus Venezuelan encephalomyelitis of horses (VEE)virus, Western encephalomyelitis of horses (WEE)virus, Eastern encephalomyelitis of horses (EEE)), vesicular stomatitis virus (air force, or VSV), fowlpox virus, false rabies, herpes simplex viruses, retroviruses, and many others. Any vector that transfers genetic material into the cell and, if necessary replication, which is replicative and viable in the appropriate host, can be used. Viral and bacterial in story, serve as carriers for delivery, can be weakened; the attenuation must be sufficient to reduce, if not eliminate, the induction of unwanted symptoms. Figure 1 is a schematic illustration of an illustrative plasmid expression vector kDNK mutant CTLA-4-Ig, which encodes a mutant CTLA-4-Ig in this invention. Additional details about the appropriate expression vectors are provided below, including examples.

The vector according to this invention, comprising a sequence of nucleic acid according to this invention, as described herein, as well as acceptable promoter or control sequence, may be used to transform suitable host in order to permit the host to Express the protein. Examples of suitable expression hosts include: bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium; fungal cells, such as Saccharomyces cerevisiae, Pichiapastoris and Neurospora crassa; insect cells such as Drosophila and Spodoptera frugiperda; mammalian cells such as Chinese hamster ovary (Chinese Hamster Ovary or Cho (e.g., Cho-K1), COS (for example COS-I, COS-7), kidney baby hamster (we used the PDH, or KSS), and kidney cells of a human embryo (PCHE, or NECK) (for example, HEK 293)cells, Bowes melanoma cells and plants. It is clear that not all cells or cell whether the AI should be able to produce fully functional polypeptides according to this invention or their fragments. The invention is not limited to used cells masters. Additional details regarding acceptable host cells described below.

In bacterial systems, a variety of expression vectors may be selected depending on the implied application for the desired polypeptide or its fragment. For example, when large quantities of a particular polypeptide or its fragments is necessary for the induction of antibodies, can be desirable, vectors which direct high level expression of fused protein that moments cleaned. Such vectors include, but are not limited to the following, the multifunctional E. coli cloning vectors and expression vectors such as BLUESCRIPT (Stratagene), in which the sequence encoding a nucleotide of interest (for example, the nucleotide sequence encoding a recombinant mutant CTLA-4-Ig), can be Legerova in the vector inside the reading frame with sequences for the amino-terminal Met and the subsequent 7 residues of beta-galactosidase so as to obtain a hybrid protein; pIN vectors (Van Heeke &Schuster (1989) J. Biol. Chem. 264:5503-5509); pET vectors (Novagen, Madison WI); and the like.

Similarly, in the yeast Saccharomyces cerevisiae a number of vectors comprising constitutive or inducible promoters such as alpha factor, alcoholecstasy and PGH may b shall be used for the production of polypeptides according to this invention. For reviews, see Ausubel, see above, Berger, see above, and Grant et al. (1987) Meth. Enzymol. 153:516-544.

In cells masters mammal can be used in various expression systems, such as systems based viruses. In cases, when using adenovirus as an expression vector, the coding sequence of optional Legerova in complex adenovirusna transcription/translation, consisting of the late promoter and triple leader sequence. Insert into Nesmelova E1 or E3 region of the viral genome leads to a viable virus which is capable to Express the polypeptide of interest in infected cells-the masters (Logan and Shenk (1984) Proc. Natl. Acad. Sci. USA 81:3655-3659). In addition, transcription enhancers, such as enhancer sarcoma virus Rosa (HRV, or RSV), are used to increase expression in cells masters mammal.

The vector, for example an expression vector, or polynucleotide according to this invention may include one or more sequences control expression. The sequence of the control expression is usually associated with and/or functionally linked to the nucleic acid sequence according to this invention, such as a nucleic acid encoding a recombinant mutant polypeptide CTLA-4 EVA or recombinant mutant protein, CTLA-4-Ig. The follower is ity control expression this is usually a nucleotide sequence that induces, enhances or controls the expression (typically a transcription of another nucleotide sequence. Acceptable sequences control expression, which can be used include the promoter, including a constitutive promoter, inducible promoter and/or repressed promoter, enhancer for amplification of the expression of the initiating sequence, terminating the transmission sequence, the sequence control of splicing and the like.

When the nucleic acid according to this invention is included in the vector, the nucleic acid is usually functionally connected with the appropriate sequence of control transcription (promoter) to direct mRNA synthesis. Promoters are particularly important influence on the level of expression of the recombinant polypeptide. You can use any acceptable promoter. Examples of acceptable promoters include the promoter of cytomegalovirus (CMV) with or without the first intron (intron A), HIV long terminal repeated the promoter, the promoter phosphoglycerate kinase (PGA, or PGK), the promoters of the virus Sarcoma of Rosa (HRV, or RSV), such as the RSV long terminal repeated (LTR) promoters, SV40 promoters, the promoters of the virus tumors in the mammary gland of mice (WOMEN, or MMTV)promoters of HSV(the virus is Rastogi herpes), such as Lap2 promoter or promoter thymidine kinase of herpes simplex virus (as described in, for example, Wagner et al. (1981) Proc. Natl. Acad. Sci. 78:144-145), promoters derived SV40 virus and Epstein-Barr, the promoters of adeno-associated virus (AAV), such as the P5 promoter, metallothionein promoters (for example, metallothionine promoter sheep or metallothionine the murine promoter (see, e.g., Palmiter et al. (1983) Science 222:809-814), the promoter of the human ubiquitin C, the E. coli promoters such as the lac and trp promoters, the promoter of phage lambda PLand other promoters known to control expression of genes in prokaryotic or eukaryotic cells (or directly in the cell, or virus that infected cell). Promoters that exhibit strong constitutive background expression in mammals, especially in humans, such as CMV promoters such as the CMV immediate-early promoter (described in, for example, U.S. Patent No. 5168062, 5385839, 5688688 and 5658759), and promoters that have substantial sequence identity with CMV promoters, can be used. Recombinant promoters having reinforcing properties, such as the Publication of the International Application WO 02/00897 can also be used.

The promoter, which is functionally linked to the nucleic acid according to this invention for the expression of a nucleic acid may have any at lemy mechanism of action. Thus, the promoter can be, for example, the following: "inducible" promoter (for example, the promoter of the growth hormone, metallothionine the promoter, the promoter of heat shock proteins, EV the promoter, the promoter, causing hypoxia, a promoter induced by irradiation or adenoviral MLP promoter and triple leader), inducible-repressed promoter, growing gradually promoter (for example, the promoter globin gene, or tissue-specific promoter (e.g., the promoter of α-actin smooth muscle cells, the promoter IA light chain of myosin, or the promoter cadherin vascular endothelium). Acceptable inducible promoters include Edison and additonaly similar-inducible promoters. Additonaly similar-inducible promoters are commercially available, for example, from Stratagene (La Jolla, CA). If necessary, the nucleic acid according to this invention can be induced with the use of inducible expression systems with switching genes. Examples of such expression systems with switching genes include the Tet-On™ System Gene Expression and Tet-Off™ System Gene Expression, respectively (Clontech, Palo Alto, CA; see, for example, Clontech Catalog 2000, pg. 110-111 for a detailed description of each such system). The inducible promoter can be any promoter that is activated and/or deactivated in response to acceptable with the drove. Additional inducible promoters include arabinose-inducible promoters, steroid-inducible promoters (for example, the glucocorticoid-inducible promoters), as well as pH, stress, and heat-inducible promoters.

The promoter can be, and often is native to the host, or a promoter derived from a virus that infects a particular owner (for example, the promoter of the human beta-actin promoter, human or EF1α promoter derived from the human AAV, functionally linked to the nucleic acid of interest), especially when you simply prevent silencing of gene expression due to immunological responses of the host to sequence, which is not always present in the host. Can also be used bidirectional promoter system (as described in, for example. U.S. patent No. 5017478)associated with numerous nucleotide sequences of interest.

Other appropriate promoters and principles related to the selection, application and design acceptable promoters are presented in, for example, Wemer (1999) Mamm Genome 10(2): 168-75, Walther et al. (1996) J. Mol. Med. 74(7):379-92, Novina (1996) Trends Genet. 12(9):351-55, Hart (1996) Semin. Oncol. 23(1):154-58, Gralla (1996) Curr. Opin. Genet. Dev. 6(5):526-30, Fassler et al. (1996) Methods Enzymol 273:3-29, Ayoubi et al. (1996), 10(4) FASEB J 10(4):453-60, Goldsteine et al. (1995) Biotechnol. Annu. Rev. 1:105-28, Azizkhan et al. (199) Crit. Rev. Eukaryot. Gene Expr. 3(4):229-54, Dynan (1989) Cell 58(1): 1-4, Levine (1989) Cell 59(3):405-8, and Berk et al. (1986) Annu. Rev. Genet. 20:45-79, and U.S. Patent No. 6194191. Other useful promoters can be identified using a Database of Eukaryotic Promoters (publication 68) (available on the worldwide website epd.isb-sib.ch/) and other similar databases, such as Database Regulatory Sites of Transcription (TRRD) (version 4.1) (available on the world web site bionet.nsc.ru/trrd/) and the Database of Transcription Factors (TRANSFAC) (available on the world web site transfac.gbf.de/TRANSFAC/index.html).

Alternatively, the promoter, especially in RNA vectors and constructs a vector or nucleic acid according to this invention may include one or more internal sites planting ribosomes (IRES), the IRES-coding sequences or enhancers of RNA sequences (analogues consensus Kozak sequence), such as the leader sequence of the virus tobacco masiki.

Vector or polynucleotide according to this invention may include activating sequence (UAS), such as Gal4 activator sequence (see, for example, U.S. Patent 6133028) or other acceptable upper regulatory sequence (see, for example, U.S. Patent No. 6204060).

Vector or polynucleotide according to this invention may include a consensus of th is sequence Kozak, which is functional in a cell of a mammal. The Kozak sequence may be naturally occurring or modified sequence, such as a modified consensus Kozak sequence, described in U.S. Patent No. 6107477.

Specific triggering signals may contribute to the efficiency of translation of the coding sequence according to this invention, such as the nucleotide sequence encoding a mutant polypeptide, CTLA-4 EVA. Such signals may be included in the vector of this invention. These signals may include, for example, initiating ATG codon and adjacent sequences. In cases where the coding sequence, its initiator codon and the upper sequence is inserted at the appropriate expression vector, no additional transcriptional control signals. However, in cases where the inserted coding sequence (for example, the sequence encoding the Mature protein) or its part shall be given the exogenous signals control the transcription of nucleic acids, including initiating ATG codon. Moreover, the initiating codon must be in the correct reading frame to ensure the full transcription of the insert. Exogenous transcriptional elements and initiating to the dons can be of various origins : both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers that are acceptable to the cellular system, which is used (see, for example, Scharf et al., Results Probl. Cell. Differ. 20:125-62 (1994); and Bittner et al., Meth. Enzymol. 153:516-544 (1987)). Acceptable enhancers include enhancer sarcoma virus Rosa (HRV, or RSV) and RTE enhancers described in U.S. Patent No. 6225082.

The specialist in this area should be clear that embedding the start codon (ATG) at the 5' end of a specific nucleotide sequence of interest, usually leads to the addition of N-terminal methionine to the encoded amino acid sequence, when the sequence is expressed in the cell of a mammal (other modifications may occur in bacterial and/or other eukaryotic cells, such as embedding formyl-methionine residue to the start codon). For expression of the nucleic acid according to this invention in eukaryotic cells start codon and the nucleotide sequence encoding a signal peptide, typically include at the 5' end of the nucleic acid sequence according to this invention (for example, SEQ ID NO:80) and the termination codon usually include on-end of the nucleic acid (e.g., SEQ ID NO:80). Illustrative sequence of the signal peptide represents the selected signal peptide hCTLA-4 (SEQ ID NO:182); the sequence of nucleic acids encoding a signal peptide activator tissue plasminogen shown in SEQ ID NO:181. Another illustrative sequence of the signal peptide is a sequence of the signal peptide hCTLA-4 (SEQ ID NO:216), which is encoded by the sequence of the nucleic acid represented in SEQ ID NO:215.

The termination sequence is discussed in detail below.

Such elements can be included in a vector construct which choose to use. When the expression of the polypeptide variant encoded by the nucleic acid (e.g., SEQ ID NO:80), will initially include the N-terminal methionine residue and the sequence of the signal peptide. However, the N-terminal methionine and the sequence of the signal peptide will be chipped off during secretion, thereby forming the encoded polypeptide (e.g., SEQ ID NO:1).

The level of expression of a nucleic acid according to this invention (or the corresponding polypeptide according to this invention) can be estimated using any acceptable technique. Examples include Northern blot analysis (discussed in, e.g., McMaster et al., Proc. Natl. Acad. Sci. USA 74(11):4835-38 (1977) and Sambrook, see below), reverse transcriptase-polymerase chain reaction (RT-PCR) as described in, for example, U.S. Patent No. 5601820 and Zaheer et al., Neurochem. Res. 20:1457-63 (195)), and in situ hybridization techniques (as described in, for example. U.S. patents No. 5750340 and 5506098). Quantitative determination of proteins can also be carried out by Lowry analysis and other quantitative analyses of protein (see, for example, Bradford, Anal. Biochem. 72:248-254 (1976); Lowry et al., J. Biol. Chem. 193:265 (1951)). Western blot analysis of recombinant polypeptides according to this invention, obtained from a lysate of cells, transfection with polynucleotide encoding such recombinant polypeptides, is another acceptable method for assessing the levels of expression of the recombinant polypeptide.

The vector, for example an expression vector, or polynucleotide according to this invention may include a binding site with the ribosome for translation initiation and termination of transcription field. Acceptable transcription termination region is, for example, a polyadenylation sequence, which facilitates the cleavage and polyadenylation of the RNA transcript derived from the DNA sequence. Can be used any acceptable polyadenylation sequence, including synthetic optimized sequence, and a polyadenylation sequence BGR (bovine growth hormone), the gene for human growth hormone, virus polyoma, TK (thymidine kinase), EBV (Epstein-Barr), beta globin rabbit, and papillomavirus, including human papilloma viruses and BPV (bovine papilloma virus). Acceptable sequence for polyadenylation (poly) also include SV40 (human sarcoma virus-40) polyadenylation sequence and BGR the poly a sequence. Such the poly a sequence is described in, for example, Goodwin et al. (1998) Nucleic Acids Res. 26(12):2891-8, Schek et al. (1992) Mol. Cell. Biol. 12(12):5386-93, and van den Hoffet al. (1993) Nucleic Acids. 21(21):4987-8. Additional principles related to the selection of an appropriate polyadenylation sequences, are described in, for example, Levitt et al. (1989) Genes Dev. 1989 3(7): 1019-1025, Jacob et al. (1990) Crit. Rev. Eukaryot. Gene Expr. 1(1):49-59, Chen et al. (1995) Nucleic Acids Res. 23(14):2614-2620, Moreira et al. (1995) EMBO J. 14(15):3809-3819, Carswell et al. (1989) Mol. Cell. Biol. 9(10):4248-4258.

Vector or polynucleotide according to this invention may also include site-specific recombination sites, which can be used to modulate transcription of the nucleotide sequence of interest, as described in, for example, U.S. Patent No. 4959317, 5801030 and 6063627, the Application for the European patent No. 0987326 and publication of International Application no WO 97/09439.

Vector or polynucleotide according to this invention may also include a nucleic acid encoding the sequence secretion/localization direction polypeptide expression to a desired cellular compartment, membrane, or organelle, or direction for the polypeptide secretion in periplasmatic space or in the medium of cell cultures. Such sequences are known in the field of engineering and include a leader peptide secretion or signal peptides, guides the sequence of organelles (e.g., nuclear localization sequence, the signals hold ER (endoplasmic reticulum), mitochondrial transition sequences, chloroplast transition sequence), a membrane localization sequence/anchor sequence (e.g., a stop portable sequence, GPI anchor sequence) and the like. Polynucleotide according to this invention can be fused, for example, in reading frame with such a nucleic acid coding sequence for secretion and/or localization. Polypeptides expressed such polynucleotide according to this invention may include amino acid sequence corresponding to the sequence(s) secretion and/or localization.

In addition, the vector or polynucleotide according to this invention may include one or more breeding marker nucleotide sequences or genes to provide a phenotypic trait for selection of transformed host cells, such as resistance to dihydrofolate reductase, resistance to neomycin, resistance to G418, resistant to puromycin and/or resistance to b is actinidine culture of eukaryotic cells, or such as resistance to E. Coli to tetracycline or ampicillin.

Vector or polynucleotide according to this invention may also include a replication source, suitable for implementation in the microorganism. Used bacterial source replication (Ori) is mainly for those who do not have a retroactive effect on gene expression in mammalian cells. Examples of suitable sequences of the replication source include phage f1 ori, RK2 oriV, pUC ori and pSC1O1 ori. The sequence of the replication source include CoIEI ori and p15 (available from Plasmid pACYC1W, New England Biolab, Inc.), alternative another poorly copied ori sequence (similar to p15) may be desirable in some cases. Nucleic acid is in this case preferably acts as the shuffled vector capable of replicating and/or to be expressed in eukaryotic and prokaryotic hosts (for example, a vector comprising the sequence of the replication source, identified in eukaryotes and prokaryotes).

This invention includes a naked DNA or RNA vector, including, for example, a linear element expression (as described in, for example, Sykes and Johnston (1997) Nat Biotech 17:355-59), competitively vector nucleic acids (as described in, for example, U.S. Patent No. 6077835 and/or Publication of the International Application no WO 00/70087), plasmid vector, such as pCDNA3,1, BR322, Fig 19/18, or pUC 118/119, vector "Midge" nucleic acids of the minimum size (as described in, for example, Schakowski et al. (2001) Mol. Ther. 3:793-800) or as precipitiously construct the vector nucleic acid, such as Car precipitiously construct (as described in, for example. Publication of the International Application WO 00/46147, Benvenisty and Reshef (1986) Proc. Natl. Acad. Sci. USA 83:9551-55, Wigler et al. (1978), Cell 14:725, and Coraro and Pearson (1981) Somatic Cell Genetics 7:603), including nucleic acid according to this invention. For example, the invention provides a naked DNA plasmid comprising SEQ ID NO:80, functionally associated with the CMV promoter or a variant of the CMV promoter and acceptable polyadenylation sequence. Bare nucleotide vectors and their use are known in the art (see, for example, U.S. Patent No. 5589466 and 5973972).

The vector according to this invention is usually an expression vector, which is acceptable for expression in a bacterial system, a eukaryotic system, the system of a mammal or other system (which is the opposite of a vector designed for replicating nucleic acid sequence without expression, which can be called as a cloning vector). For example, in one aspect the invention provides a bacterial expression vector comprising the nucleic acid sequence Dunn is th invention (for example, the sequence of the nucleic acid encoding a recombinant mutant CTLA-4-Ig). Acceptable vectors include, for example, vectors that direct the expression of high levels fused proteins pre-treated (for example, the multifunctional E. coli cloning vectors and expression vectors such as BLUESCRIPT (Stratagene), pIN vectors (Van Heeke &Schuster, J. Biol. Chem. 264:5503-5509 (1989); pET vectors (Novagen, Madison WI); and the like). While such bacterial expression vectors can be used ekspressirovannoj specific polypeptides according to this invention picobello according to this invention mainly expressed in eukaryotic cells, and therefore, the invention also provides a eukaryotic expression vectors.

The expression vector may be a vector suitable for expression of the nucleic acid according to this invention in a yeast cell. Any vector suitable for expression in a yeast system can be used. Acceptable vectors for use in, for example, Saccharomyces cerevisiae include, for example, vectors comprising constitutive or inducible promoters such as alpha factor, alcoholised and PGH (described in reviews Ausubel, see above, Berger, see above, and Grant et al., Meth. Enzymol. 153:516-544 (1987)). Typically, the expression vector will be a vector suitable for expression of the nucleic acid in this and the finding in the cell of the animal, such as an insect cell (e.g., SF-9 cell) or a cell of a mammal (e.g., Cho cell, 293 cell, HeLa cell, human fibroblast cell or similar well-described cell). Acceptable expression vectors mammal known in the art (see, for example, Kaufman, Mol. Biotechnol. 16(2): 151-160 (2000), Van Craenenbroeck, Eur. J. Biochem. 267(18):5665-5678 (2000), Makrides, Protein Expr. Purif. 17(2):183-202 (1999), and Yarranton, Curr. Opin. Biotechnol. 3(5):506-511 (1992)). Acceptable plasmid expression vectors of the cells of the insect is also known (Braun, Biotechniques 26(6): 1038-1040: 1042 (1999)).

The expression vectors can usually be introduced into the cell host, which can be a eukaryotic cell (such as mammal cells, yeast cell or a plant cell or a prokaryotic cell such as a bacterial cell. On the embedding of the vector nucleic acid or expression vector into the cell host (e.g., transfection) may interfere with transfection-mediated FORATOM calcium (see, for example, the way co-deposition of calcium phosphate Graham et al., Virology 52:456-457 (1973)), DEAE-Dextran mediated transfection, electroporation, gene or vaccine gun, injection, lipofection and neoliticheskaya shipping or other traditional methods (see, for example, Kriegler, GENE TRANSFER AND EXPRESSION: A LABORATORY MANUAL, Stockton Press (1990); see Davis, L, Dibner, M, and Battey, L, BASIC METHODS IN MOLECULAR BIOLOGY (1986) to describe vivo i, ex vivo and in vitro methods). Cells, including these and other vectors of this invention, are an important part of the invention.

In one aspect the invention provides an expression vector comprising: (i) a first polynucleotide sequence that encodes a first polypeptide comprising amino acid sequence having at least, 95%, 96% 97%, 98%, 99% or 100% sequence identity with at least one amino acid sequence selected from the group comprising SEQ ID NOS:1-73, where the first polypeptide binds human CD86 and/or human CD80 and/or the extracellular domain of each or both and/or suppresses an immune response and (ii) a second polynucleotide sequence that encodes a second polypeptide comprising a hinge region, CH2 domain and CH3 domain of the immunoglobulin (Ig) polypeptide. Ig polypeptide is not necessarily a human Ig Fc polypeptide (e.g., IgG1, IgG2, IgG4, and the like) or mutant Ig Fc polypeptide (e.g. a polypeptide Ig Fc, in which one or more cysteine residues substituted by another amino acid (e.g., a serine residue), thereby removing one or more disulfide bonds formed between two Ig chains, or in which one or more prolinnova residue is substituted by another amino acid (e.g., Proline) to reduce factorey function (reduced binding to Fc receptor). In another aspect the invention provides an expression vector comprising a nucleotide sequence encoding a protein having at least, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with at least one amino acid sequence selected from the group comprising SEQ ID NOS:74-79, 197-200, 205-214, and 219-222.

Additional nucleic acid provided by the invention include Comedy. Any acceptable Kemeny vector can be used for replication, transfer and expression of nucleic acid sequence according to this invention. Typically, cosmid includes bacterial oriV, breeding marker antibiotic cloning site and one or two cos site, derived from bacteriophage lambda. Cosmid can be shuffled by kosmidou or kosmidou mammal, including SV40 oriV and, preferably, an acceptable marker(s) selection of a mammal. Cosignee vectors are also described in, for example, clear Hohn et al. (1988) Biotechnology 10: 113-27.

Nucleic acid according to this invention can be included in and/or introduced to the owner or cage-owner in a form acceptable carrier delivery (namely, vector). The vector may be any acceptable vector, including chromosomal, achromosome and vectors of synthetic nucleic acids or other vectors described above, and may include l is the buoy combination of the above elements of expression and/or other elements in the sequence, facilitate transfection and/or stimulating the expression. Examples of such vectors include viruses, bacterial plasmids, phages, Comedy, phagemid, derivatives of SV40, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and ragovoy DNA, and vectors of viral nucleic acids (RNA or DNA), polylysine and bacterial cells.

Delivery of recombinant DNA sequence according to this invention can be implemented naked DNA plasmid or a plasmid associated with one or more agents that facilitate transfection, which is also discussed in this description. Plasmid DNA vector may be any appropriate combination of characteristics. Plasmid DNA vectors can include a strong promoter/enhancer region (for example, human CMV, RSV, SV40, SL3-3, V or HIV LTR promoter), efficient poly(A) termination sequence, a source of replication for plasmid product in E. coli, a gene resistant to antibiotics as a breeding marker, and suitable cloning site (for example, polylinker). Specific plasmid vector for delivering the nucleic acids according to this invention in this regard are shown in figure 1; the design and specifications of this vector is described in the Examples below.

In another aspect the invention provides a vector Neocleous key is lots comprising at least one nucleic acid or polypeptide according to this invention. This vector Neocleous acid includes, for example, but not limited to: recombinant virus conjugate of viral nucleic acid and protein (which, together with the recombinant viral particles may sometimes be referred to as a viral vector), or cell, such as recombinant (usually weak) Salmonella, Shigella, Listeria, and Bacillus Calmette-Guerin (BCG) bacterial cells. Thus, for example, the invention provides a viral vector, the vector insect, bacterial vector or a vector of the plant, comprising a nucleic acid sequence according to this invention. Any acceptable viral, insect, plant or bacterial vector can be used in this regard and many well-known in the field of technology. The viral vector may include any number of viral polynucleotides, separate (vector of viral nucleic acid), or more frequently in combination with one or more (typically two, three or more viral proteins that facilitate the delivery, replication and/or expression of the nucleic acid according to this invention in a suitable cell host.

In one aspect of intracellular bacteria (such as Listeria monocytogenes) can be used to deliver nucleic acids this is obreteniyu. Illustrative bacterial vector for plasmid DNA delivery of one or more nucleic acids according to this invention is a Listeria monocytogenes (Lieberman et al., Vaccine 20:2007-2010 (2002)).

The invention includes recombinant or isolated viral vectors, which are modified so as to include one or more nucleic acids or polypeptides according to this invention. The viral vector may include polynucleotide, including all or part of the viral genome, the conjugate of viral protein and nucleic acid, virus-like particle (HPV), a vector similar to that described in U.S. Patent No. 5849586 and publication of International Application no WO 97/04748, or the intact viral particle comprising one or more viral nucleic acids and viral vector is usually manufactured so that it included at least one nucleic acid and/or polypeptide according to this invention. Viral vector (namely, the recombinant virus can include viral particle wild-type or modified viral particle, specific examples of which are discussed below. Different viruses are commonly used as vectors for delivery of exogenous nucleic acids, including at least one nucleic acid according to this invention, such as a nucleic acid encoding a mutant CTLA-4 In The D or mutant CTLA-4-Ig, as described here. Such vectors include recombinante modified coated or uncoated membrane DNA and RNA viruses, usually selected from baculoviridiae, parvoviridiae, picomoviridiae, herpesveridiae, from the poxviridae, adenoviridiae or picomnaviridiae. Viral vectors can be wild type or can be modified by techniques of recombinant nucleic acids to be insufficient replication competent or replication conditional replication.

The viral vector may be a vector, which require the presence of another vector or wild-type virus for replication and/or expression (namely, the helper-dependent virus, such as adenovirus vector amplicon. Typically, such viral vectors include viral particle wild-type or viral particle that is modified in its protein content and/or content of the nucleic acid to increase transgenic ability or ease of transfection and/or expression of nucleic acids (examples of such vectors include amplicon HSV/AAV). The viral genome can be modified to include inducible promoters that allow for replication or expression only under certain conditions.

The viral vector may be derived from or include a virus that usually infects animals, mostly vertebrates, such as mammalian is the following, including, for example, of people. Acceptable viral vector particles in this regard include, for example, an adenoviral vector particles (including any virus, or arising from virus adenoviridiae), adeno-associated viral vector particles (AAV vector particles) or other parvoviruses and parvovirus vector particles, papilloma virus vector particles, the viral vector Semliki-Forest, flavivirus vectors, picornavirus vectors, alphavirus vectors, viral vectors, herpes virus, poxvirus vectors, retroviral vectors, including lentiviral transfer vectors. Examples of such viruses and viral vectors are described in, for example. Fields Virology, see above. Fields et al., eds., VIROLOGY, Raven Press, Ltd., New York (3rd ed., 1996 and 4th ed., 2001), ENCYCLOPEDIA OF VIROLOGY, R.G. Webster et al., eds., Academic Press (2nd ed., 1999), FUNDAMENTAL VIROLOGY, Fields et al., eds., Lippincott-Raven (3rd ed., 1995), Levine, "Viruses," Scientific American Library, No. 37 (1992), MEDICAL VIROLOGY, D.O. White et al., eds., Academic Press (2nd ed. 1994), and INTRODUCTION TO MODERN VIROLOGY, Dimock, NJ. et al., eds., Blackwell Scientific Publications, Ltd. (1994).

Viral vectors that can be used with nucleic acids according to this invention and the methods described here include adeno-associated viral vectors, which are described in the reviews, for example, Carter (1992) Curr. Opinion Biotech. 3:533-539 (1992) and Muzcyzka (1992) Curr. Top.Microbiol. Immunol. 158:97-129 (1992). Additional types and aspects of AAV vectors are described in, for example, Buschacher et al., Blood 5(8):2499-504, Carter, Contrib. Environ. 4:85-86 (2000), mith-Arica, Curr. Cardiol. Rep.3(1):41-49 (2001), Taj, J. Biomed. Sci. 7(4):279-91 (2000), Vigna et al., J. Gene Med. 2(5):308-16 (2000), Klimatcheva et al., Front. Biosci. 4:D481-96 (1999), Lever et al., Biochem. Soc. Trans. 27(6):841-47 (1999), Snyder, J. Gene Med. 1(3): 166-75 (1999), Gerich et al., Knee Surg. Sports Traumatol. Arthrosc. 5(2): 118-23 (1998), and During, Adv. Drug Deliv. Review 27(1):83-94 (1997), and U.S. Patent№4797368, 5139941, 5173414, 5614404, 5658785, 5858775 and 5994136, and other references discussed in this description). Adeno-associated viral vectors can be constructed and/or purified using methods listed, for example, in U.S. Patent No. 4797368 and Laughlin et al., Gene 23:65-73 (1983).

Alphavirus vectors can be vectors for gene delivery in other contexts. Alphavirus vectors known in the technical field and are described in, for example, Carter (1992) Curr Opinion Biotech 3:533-539, Schlesinger Expert Opin. Biol. Ther. (2001) 1(2): 177-91, Polo et al., Dev. Biol. (Basel). 2000; 104:181-5, Wahlfors et al., Gene Ther. (2000) 7(6):472-80, Publications International Application no WO 01/81609, WO 00/39318, WO 01/81553, WO 95/07994, WO 92/10578.

Another preferred group of viral vectors are viral vectors, herpes. Examples are described in, for example, Lachmann et al., Curr. Opin. Mol. Ther. (1999) 1(5):622 - 32, Fraefel et al., Adv. Virus Res. (2000) 55:425-51, Huard et al., Neuromuscul. Disord. (1997) 7(5):299-313, Frenkel et al., Gene Ther. (1994) Suppl 1:S40-6, U.S. Patent No. 6261552 and 5599691.

Retroviral vectors, including lentiviral transfer vectors can also be the preferred carriers for gene delivery to specific contexts. There are various retroviral vectors, known is haunted in the art. Examples of retroviral vectors are described in, for example. Miller, Curr Top Environ. Immunol. (1992) 158:1-24, Weber et al, Curr. Opin. Mol. Ther. (2001) 3(5):439-53, Hu et al, Pharmacol. Rev. (2000) 52(4):493-511, Kirn et al., Adv. Virus Res. (2000) 55:545-63, Palu et al., Rev. Med. Virol. (2000) 10(3): 185-202, Takeuchi et al., Adv. Exp. Med. Biol. (2000) 465:23-35, U.S. Patent No. 6326195, 5888502, 5580766 and 5672510.

Baculovirus vectors are another preferred group of viral vectors, particularly for the production of polypeptides according to this invention. Production and use of baculovirus vectors are known (see, for example, Kost, Curr. Opin. Biotechnol. 10(5):428-433 (1999); Jones, Curr. Opin. Biotechnol. 7(5):512-516 (1996)). When the vector is used in therapeutic applications, the vector is chosen so that it was able to infect (or in the case of vector nucleic acid to transactivate or transform) target cells, which should be the desired therapeutic effect.

Adenoviral vectors can also be suitable viral vectors for gene transfer. Adenoviral vectors are well known in the technical field and are described in, for example, Graham et al. (1995) Mol. Biotechnol. 33(3):207-220, Stephenson (1998) Clin. Diagn. Virol. 10(2-3): 187-94, Jacobs (1993) Clin Sci (Lond). 85(2): 117-22, U.S. Patent No. 5922576, 5965358 and 6168941 and Publications of International Applications WO 98/22588, WO 98/56937, WO 99/15686, WO 99/54441 and WO 00/32754. Adenoviral vectors, viral vectors, herpes and Sindbis viral vectors suitable DL the practical application of the invention and acceptable to the organism in vivo transduction and expression of nucleic acids according to this invention, in General described in, for example. Jolly (1994) Cancer Gene Therapy 1:51-64, Latchman (1994) Molec. Biotechnol. 2:179-195, and Johanning et al. (1995) Nucl. Acids Res. 23:1495-1501.

The viral vector can be deficient in replication in the cell host. Adeno-associated viral (AAV) vectors, which are naturally deficient in replication in the absence of complementary adenoviruses or, at least, adenoviral gene products (which are provided, for example, helper virus, plasmid, or the cell of complementaria), included in this invention. Under "scarce replication realize that the viral vector includes a gene that lacks at least one is required for replication of gene function. Deficiency gene, gene function or gene or genomic region, as used in this description, referred to as a deletion of genetic material of the viral genome, sufficient to reduce or remove a function of a gene whose sequence of nucleic acid is removed in whole or in part. Gene functions required for replication are those gene functions that are required for replication (i.e. distribution) deficient in replication of the viral vector. Essential gene functions of viral vector particles differ depending on the type of viral vector particles of interest. Examples of scarce toreplicate viral vector particles described in, for example, Marconi et al., Proc. Natl. Acad. Sci. USA 93(21):11319-20 (1996), Johnson and Friedmann, Methods Cell Biol. 43 (pt. A):211-30 (1994), Timiryasova et al., J. Gene Med. 3(5):468-77 (2001), Burton et al., Stem Cells 19(5):358-77 (2001), Kim et al., Virology 282(1): 154-67 (2001), Jones et al., Virology 278(1): 137-50 (2000), Gill et al., J. Med. Virol. 62(2): 127-39 (2000). Other scarce replication vectors based on simple MLV vectors (Miller et al. (1990) Mol. Cell Biol. 10:4239; Kolberg (1992) J. NIH Res. 4:43, and Cometta et al. (1991) Hum. Gene. Ther. 2:215). Canary pox vectors are preferred in the infection of human cells, but being naturally incapable of replication in them (namely, without genetic modification).

The basic design of recombinant viral vectors is well understood in the art and include the use of standard molecular biological techniques such as those described in, for example, Sambrook et al., MOLECULAR CLONING: a LABORATORY MANUAL (Cold Spring Harbor Press 1989) and third edition (2001), Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Wiley Interscience Publishers, 1995), and Watson, see above, and some other references mentioned in this description. For example, adenoviral vectors can be constructed and/or purified using methods listed, for example, Graham et al., Mol. Biotechnol. 33(3):207-220 (1995), U.S. Patent No. 5965358, Donthine et al., Gene Ther. 7(20): 1707-14 (2000), and other references described here. Adeno-associated viral vectors can be constructed and/or purified using methods described, e.g. the, in U.S. Patent No. 4797368 and Laughlin et al., Gene 23:65-73 (1983). Similar techniques known in the field of technology in relation to other viral vectors, especially in relation to viral vectors, herpes (see, for example, Lachman et al., Curr. Opin. Mol. Ther. 1(5):622-32 (1999)), lentiviruses vectors and other retroviral vectors. In General, the viral vector comprises inserting nucleic acid (e.g., an adenoviral vector wild type may include the insertion of up to 3 KB without deletions), or, more typically, includes one or more deletions of the viral genome in order to accommodate the insertion of nucleic acid and complementary nucleic acids, as necessary, and to prevent replication in the cells of the masters.

Non-viral vectors, such as DNA plasmids, naked nucleic acids and nucleic acid in the complex in the carrier for delivery, such as liposome, can also be associated with molecules that direct the vector to a specific region in the host (for example, a particular organ, tissue and/or cell type). For example, the nucleotide can be conjugated with guides in protein, such as a viral protein that binds to a receptor or protein that binds to a receptor specific target (for example, by modification of methods in Wu et al., J. Biol. Chem. 263(29): 14621-24 (1988)). Known directed cationic lipid compositions (the show is, for example, U.S. Patent No. 6120799). Other techniques for directing the genetic constructs described in Published International Application no WO 99/41402.

The owners of the expression

This invention also provides engineered cells-owners, transduced, transfection or transformed with the vector according to this invention (for example, a cloning vector or expression vector or nucleic acid according to this invention. Designed cell host can be cultured in appropriate nutrient media modified acceptable for activating promoters, selecting transformants, or amplifying the nucleic acid of interest. Culturing conditions, such as temperature, pH and the like, are such as were previously used for the host cell selected for expression, and will be obvious to experts in the field and in the references cited herein, including, for example, Freshney (1994) Culture of Animal Cells, a Manual of Basic Technique, 3rd edition, Wiley - Liss, New York, and references cited therein. The polypeptides according to this invention, encoded by such vectors or nucleic acids according to this invention, expressed in such cells masters and can be isolated by standard techniques. For example, polypeptides that are released in CL the exact culture, can be isolated from the culture using ultracentrifugation or similar methods.

The polypeptides according to this invention can be obtained in various masters of expression, including, but not limited to the following: animal cells such as mammalian cells (e.g., Cho cells), including human and non-human cells primates, and non-animal cells such as plants, yeast, fungi, bacteria and the like. Examples of suitable expression hosts include bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium; fungal cells, such as Saccharomyces cerevisiae, Pichia pastoris, and Neurospora crassa; insect cells such as Drosophila and Spodoptera frugiperda; mammalian cells such as Cho (e.g., Cho-K1), COS (for example COS-1, COS-7), BHK and HEK (such as HEK 293) cells, melanoma cells Bowes, and plant cells. As noted above, the invention is not limited to used cells masters. In addition to Sambrook, Berger and Ausubel, all see above, the details of the cell culture can be found in, for example, Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley & Sons, Inc. New York, NY; Gamborg and Phillips (eds.) (1995) Plant Cell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual, Springer-Verlag (Berlin Heidelberg NY); Atlas & Parks (eds.) The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, FL. These cells are the masters can be adapted to growth in serum-free, protein-free medium, the medium without animal component is in, such as, for example, chemically defined (CD) medium (such as, for example, CD OptiCHO™ (Invitrogen, #12681) using procedures known in the field of engineering.

The invention provides a cell(s), including any one or more nucleic acids, vectors or other constructs according to this invention (for example, a construct expressing mutant CTLA-4 EVA or mutant CTLA-4-Ig)described herein or any combination of them. Also included cell that includes one or more of any of the polypeptides, antibodies, or fused proteins, or other constructs of this invention, described herein, or any combination of one or more of this. The cell of this invention is typically an isolated or recombinant cell, and may include cell owner. Such a cell, for example a recombinant cell may be modified by transformation, transfection and/or infection of at least one nucleic acid, vector or other construct of this invention. Such a cell can be a eukaryotic cell (e.g., mammal cells, yeast or plants) or prokaryotic cell (e.g., a bacterial cell) and can be transformed to any such construct according to this invention using various known methods, including, for example, Tr is specchio, mediated by calcium phosphate (see, for example, the way co-deposition of calcium phosphate, transfection mediated OEE(diethylaminoethyl)-dextran, electroporation (Irving et al., cell 64:891-901 (1991)), gene or vaccine gun, injection, lipofection and biolistics delivery or other traditional methods, as noted above. See also Inovio Biomedical Corp. methods and electroporation technology at the world site inovio.com.

Strain host cell does not necessarily selected for its ability to modulate the expression of the built-in sequences or to process the expressed protein in the desired manner. Such modifications of the protein include, but are not limited to the following: acetylation, carboxylation, glycosylation, phosphorylation, lipiduria and acylation. Different cells in a host, such as E. coli, Bacillus sp., the yeast cells or mammalian cells such as Cho, HeLa, BHK, MDCK, HEK 293, WI38, etc. have specific cellular apparatus and characteristic mechanisms for such post-translational action and can be chosen to ensure the correct modification and processing of the embedded foreign protein.

Nucleic acid according to this invention can be embedded in a suitable cell host (in culture or in an organism of the owner) in order to permit the host to Express the boe is OK of interest (e.g., mutant CTLA-4 EVA or mutant CTLA-4-Ig). Any acceptable a host cell may be transformed/transducible nucleic acids according to this invention. Examples of suitable expression hosts include: bacterial cells, such as E. coli, Streptomyces, Bacillus sp., and Salmonella typhimurium; fungal cells, such as Saccharomyces cerevisiae, Pichia pastoris, and Neurospora crassa; insect cells such as Drosophila and Spodoptera frugiperda; mammalian cells such as Vero cells, HeLa cells, Cho cells (such as Cho-K1), COS cells, WI38 cells, NIH-3T3 cells (and other fibroblast cells such as MRC-5 cells), MDCK cells, KB cells, SW-13 cells, MCF7 cells, BHK cells, HEK-293 cells, cells, Bowes melanoma cells and plants etc.

The invention also provides cell owners who transducible, transformed or transfection with at least one nucleic acid or vector of this invention. As discussed above, the vector of this invention generally includes a nucleic acid according to this invention. Cell owners are genetically engineered (e.g., translotsirovannoi, transformed, infected or transfectional) with the vectors of this invention, which may be, for example, a cloning vector or expression vector. The vector may be in formalised, a viral particle, a phage, weakened bacteria or any other suitable vector. Cells masters acceptable to the transduction and/or infection with viral vectors of this invention for the production of recombinant polypeptides according to this invention and/or for replication of the viral vector according to this invention include the above-described cells. Examples of cells that are shown as acceptable for packaging of viral vector particles described in, for example, Polo et al., Proc. Natl. Acad. Sci. 96(8):4598-603 (1999), Farson et al., J. Gene Med. 1(3): 195-209 (1999), Sheridan et al., Mol. Ther. 2(3):262-75 (2000), Chen et al., Gene Ther. 8(9):697-703 (2001), and Pizzaro et al., Gene Ther. 8(10):737-745 (2001). For scarce replication of viral vectors, such as AAV vectors, complementary cell lines, cell lines transformed with helper virus or cell line transformed with a plasmid encoding the essential genes necessary for replication of the viral vector.

Designed cell host can be cultured in an acceptable nutrient media modified acceptable for activating promoters, selecting transformants or amplifying the gene of interest. Cell host can be cultured in a medium containing serum or serum-free medium. Cell host can be cultured in serum-free, protein-free, environment, the e containing animal components, including, for example, chemically defined medium (for example, CD OptiCHO™ (Invitrogen, #12681)). The cellular environment can be supplemented, if necessary, additives, known to specialists in this field, such as, for example, one or more amino acids, such as L-glutamine (for example, 2% volume/volume of 200 mm L-glutamine (Invitrogen, #25031)). Culturing conditions, such as temperature, pH and the like, are such as previously used for the host cell selected for expression, and will be obvious to experts in the field and from the references cited herein, including, for example, ANIMAL CELL TECHNOLOGY, Rhiel et al., eds., (Kluwer Academic Publishers 1999), Chaubard et al., Genetic Erig. News 20(18) (2000), Hu et al., ASM News 59:65-68 (1993), No et al., Biotechnol. Prog. 1:209-215 (1985), Martin et al., Biotechnol. (1987), Freshney, CULTURE OF ANIMAL CELLS: A MANUAL OF BASIC TECHNIQUE, 4th ed. (Wiley, 2000), Mather, INTRODUCTION TO CELL AND TISSUE CULTURE: THEORY AND TECHNIQUE, (Plenum Press, 1998), Freshney, CULTURE OF IMMORTALIZED CELLS, 3rd ed. (John Wiley & Sons, 1996), CELL CULTURE: ESSENTIAL TECHNIQUES, Doyle et al, eds. (John Wiley & Sons 1998), and GENERAL TECHNIQUES OF CELL CULTURE, Harrison et al., eds. (Cambridge Univ. Press 1997).

Nucleic acid may also be stored, replicated and/or expressed in plant cells. Methods related to the culture of plant cells, are described in, for example, Raue et al. (1992) Raue et al. (1992) PLANT CELL AND TISSUE CULTURE IN LIQUID SYSTEMS John Wiley & Sons, Inc. New York, NY; Gamborg and Phillips (eds.) (1995) PLANT CELL, TISSUE AND ORGAN CULTURE: FUNDAMENTAL METHODS SPRINGER LAB MANUAL, Springer-Verlag (erlin Heidelberg New York) and Plant Molecular Biology (1993) R.R.D. Croy (ed.) Bios Scientific Publishers, Oxford, U.K. ISBN 012 198370 6. Environment cell cultures in General is given in Atlas and Parks (eds.) THE HANDBOOK OF MICROBIOLOGICAL MEDIA (1993) CRC Press, Boca Raton, FL.

For long products with a high yield of recombinant proteins can be used for stable expression systems. For example, cell lines which stably Express a polypeptide according to this invention, can be transducible with expression vectors, including viral sources of replication and/or endogenous elements of expression and breeding marker gene. After injection of the vector to the cells in the cell line can be allowed to grow for 1-2 days in an enriched media before they are transferred to selective medium. The purpose of breeding marker is confirmation of resistance to selection, and its presence allows growth and repair of cells which successfully Express the built-in sequence. For example, the steady accumulation of stably transformed cells can be proliferatory with using techniques of tissue culture, it is acceptable for the cell type. Serum-free medium to date are available (e.g., JRH Biosciences, SAFC Biosciences, Sigma-Aldrich Corporation, the world site sigmaaldrich.com). Serum-free medium or air-conditioned environment (e.g., growth medium pre-assembled from netr spectrophonic or native cell cultures) can be preferred for production of the protein or stored in cell banks in some cases.

This invention includes the immortal cells or cell lines comprising one or more polypeptides (including, for example, dimeric or Monomeric fused proteins and multimeric polypeptides, conjugates, nucleic acids or vectors of this invention.

Cell host transformed with the expression vector and/or polynucleotides, optionally cultured under conditions suitable for expression and recovery of the encoded protein from cell culture. The polypeptide or its fragment, obtained such a recombinant cell may be secretively, membrane-bound or contained intracellularly depending on the sequence and/or vector. The expression vectors comprising polynucleotide encoding a Mature polypeptide according to this invention, can be designed with signal sequences which direct secretion of the Mature polypeptide through the membrane prokaryotic or eukaryotic cells. Such signal sequences are usually built into the vector, so that the signal sequence is expressed at the N end of the polypeptide according to this invention. Principles related to such signal sequences are discussed in this description.

Production and recovery of polypeptide

After transduction pickup is acceptable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced by appropriate means (for example, temperature shift or chemical induction) and cells are cultured for an additional period. Cells are typically harvested by centrifugation, destroy physical or chemical methods, and the resulting crude extract is subjected to additional purification. Microbial cells used in the expression of proteins can be destroyed by any conventional method, including the cycle of freezing and thawing, sonication, mechanical disruption, or use of cell lytic agents, or other methods that are well known to specialists in this field.

As noted, many references are available for the culture and production of many cells, including cells of the bacterial, plant, animal (especially mammalian) and archaebacterial origin. See, for example, Sambrook, Ausubel, and Berger (all see above), and Freshney (1994) Culture of Animal Cells, a Manual of Basic Technique, Third edition Wiley-Liss, New York, and references cited there; Doyle and Griffiths (1997) Mammalian Cell Culture: Essential Techniques, John Wiley and Sons, NY; Humason (1979) Animal Tissue Techniques, fourth edition, W.H. Freeman and Company; and Ricciardelli, et al. (1989) In vitro Cell Dev. Biol. 25:1016 1024. For the culture of plant cells and regeneration of Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley & Sons, Inc. New York, N.Y.; Gamborg and Phillips (eds.) (1995) Plant Cell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual, Springer - Verlag (Berlin Heidelberg New York) and Plant Molecular Biology (1993) R. R. D. Croy, Ed. BiosScientific Publishers, Oxford, U.K. ISBN 0 12 198370 6. Environment cell cultures in General is given in Atlas and Parks (eds.) The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, FL. For more information about cell culture can be found in available commercial literature such as the Life Science Research Cell Culture Catalogue (1998) from Sigma-Aldrich, Inc. (St. Louis, Mo.) ("Sigma-LSRCCC") and, for example, the Plant Culture Catalogue and supplement (1997) also from Sigma-Aldrich, Inc (St. Louis, Mo.) ("Sigma-PCCS").

The polypeptides according to this invention can be recovered and purified from recombinant cell cultures by any of numerous methods well known in the field of engineering, including the deposition of ammonium sulfate or ethanol, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography (e.g., using any of the guides of the systems outlined in this description), hydroxylapatite chromatography and lecithin chromatography. The stages of the refolding of the protein can be used, if desirable, in completing configuration of the Mature protein. In the end, high-performance liquid chromatography (HPLC) can be used in the final stages of purification. In addition to the references noted above, various methods of purification are well known in the art including, for example, shown in Sandana (1997) Bioseparation of Proteins, Aademic Press, Inc.; and Bollag et al. (1996) Protein Methods, 2.sup.nd Edition, Wiley-Liss, NY; Walker (1996) The Protein Protocols Handbook Humana Press, NJ, Harris and Angal (1990) Protein Purification Applications: A Practical Approach, IRL Press at Oxford, Oxford, England; Harris and Angal Protein Purification Methods: A Practical Approach, IRL Press at Oxford, Oxford, England; Scopes (1993) Protein Purification: Principles and Practice 3.sup.rd Edition Springer Verlag, NY; Janson and Ryden (1998) Protein Purification: Principles, High Resolution Methods and Applications, Second Edition Wiley-VCH, NY; and Walker (1998) Protein Protocols on CD-ROM Humana Press, NJ.

The person skilled in the art it is clear that the fused protein according to this invention (for example, a mutant protein, CTLA-4-Ig) can be obtained in various ways described herein, including, for example, shown in example 1 for the formation of LEA29Y-Ig. For example, in the case EEA29Y-coding nucleic acid sequence of nucleic acids encoding a mutant polypeptide, CTLA-4 EVA on this invention (e.g., D3-54 polypeptide), can be cloned in IgG2 Fc vector merge to obtain a vector encoding a mutant protein, CTLA-4-Ig (e.g., D3-54-IgG2), stable Cho-K1 cells expressing this mutant protein, CTLA-4-Ig can be obtained by transfectional such cells with a vector encoding a mutant protein, CTLA-4-Ig, and the obtained mutant protein, CTLA-4-Ig (e.g., D3-54-IgG2) can be expressed (usually in dimeric form) and purified as described in example 1.

In vitro expression system

Cell-free system TRANS is ipli/broadcast can also be used to generate recombinant polypeptides according to this invention or their fragments using DNA and/or RNA according to this invention or their fragments. Some such systems are commercially available. General guidance on protocols for in vitro transcription and translation is Tymms (1995) IN VITRO AND REDUCED TRANSLATION PROTOCOLS: METHODS IN MOLECULAR BIOLOGY, Volume 37, Garland Publishing, New York.

The METHODS ACCORDING to the INVENTION

Polypeptides (including, for example, dimeric and Monomeric fused proteins and multimeric polypeptides, conjugates, compositions, nucleic acids, vectors and cells of this invention exhibit a variety of properties and characteristics, and it is assumed that they are suitable in various applications, including, but not limited to the following, for example, the prophylactic or therapeutic treatment of various diseases of the immune system, disorders and conditions of the immune system in which the modulation or regulation of the immune system and responses of the immune system can be effective. For example, suppose that the polypeptides, conjugates, compositions, nucleic acids, vectors and cells of this invention, which have the ability to bind CD80 and/or CD86 or EVA each or both and/or the ability to inhibit the immune response, suitable for prophylactic and/or therapeutic methods for inhibiting or suppressing an immune response in a subject, methods of inhibiting transplant rejection tissue, cell or organ from donor to recipient and the other is x, described in this description. Assume that some such polypeptides, conjugates, compositions, nucleic acids, vectors and cells of this invention are suitable in the methods of modulating or inhibiting the interaction of T cells expressing CD28 and/or CTLA-4 with B7-positive cells.

In one aspect therapeutic or prophylactic methods of this invention include an introduction to the subject an effective amount of at least one such polypeptide (including, for example, fused protein, multimer etc.), conjugates, compositions, nucleic acids, vectors and/or cells, to suppress or inhibit an immune response. In therapeutic context, the subject is usually a subject affected by a disease, disorder or condition of the immune system, and the introduction of conduct to prevent further development of the diseases, disorders or conditions. For example, the introduction of a molecule of this invention to a subject suffering from a disease of the immune system (e.g., autoimmune diseases), can lead to suppression or inhibition of such attacks the immune system or biological responses associated with it. Suppressing this immune system attacks healthy tissues obtained physical symptoms (e.g. pain, joint inflammation, joint swelling, or disease is ness), resulting from or associated with such an attack on healthy tissue can be reduced or eliminated, and biological and physical injury resulting from or associated with the attack of the immune system may be reduced, delayed or stopped.

In the preventive context, the subject may be infected, susceptible to or who believes he is a disease, disorder or condition of the immune system, and the introduction usually leads to preventing the development of diseases, disorders or conditions, inhibits or eliminates the symptoms, signs or biological responses associated with them, prevents damage to the body, which is their potential result and/or maintains or improves physical functioning entity.

In one aspect the invention provides a method of modulating the interaction of T cells expressing CD28 and/or CTLA-4 with B7-positive cells, where the method includes contact B7-positive cells with at least one of the following in an effective amount to modulate the interaction of B7-positive cells with SV-positive T-cells and/or CTLA-4-positive T-cells: (1) the polypeptide according to this invention (for example, a mutant polypeptide, CTLA-4-EVA or dimeric or Monomeric mutant protein CTLA-4-Ig); (2) multimer, including one Il is several polypeptides according to this invention (for example, dimer comprising any two of such polypeptide, or tetramer, including any four of such polypeptide); (3) a conjugate comprising at least one polypeptide according to this invention; (4) the nucleic acid according to this invention (for example, nucleic acid encoding a polypeptide according to this invention); (5) a vector comprising a nucleic acid according to this invention or encoding the polypeptide according to this invention; (6) the cell or population of cells comprising a polypeptide, nucleic acid, the conjugate and/or a vector according to this invention; or (7) the composition according to this invention, where the interaction of B7-positive cells with CD28-positive T-cells and/or CTLA-4-positive T-cells is modulated. Typically, the modulatory effect of this inhibitory effect, so that the interaction of B7-positive cells with CD28-positive T-cells and/or CTLA-4-positive T-cells is inhibited. In some cases, B7-positive cells are antigen-presenting cells (APC). In some such methods, the interaction of B7-2-positive cells (e.g., AIC, expressing B7-2 (CD86 with CD28-positive T-cells is inhibited. In some such methods, the interaction of B7-1-positive cells (e.g., AIC, expressing B7-1 (CD80)) SV-positive T-cells is inhibited.

In another aspect of the invention providing the em method of inhibiting the interaction of CD28-positive T cells and/or CTLA-4-positive T cells with B7-positive cells, where the method includes contact B7-positive cells (e.g., B7-1-positive cells and/or B7-2-positive cells) with at least one of the following molecules or components according to this invention in an effective amount to inhibit the interaction of CD28-positive T cells and/or CTLA-4-positive T cells with B7-positive cells: (1) the polypeptide according to this invention (for example, a mutant polypeptide, CTLA-4-EVA or dimeric or Monomeric mutant protein, CTLA-4-Ig); (2) multimer comprising one or more polypeptides according to this invention (e.g., dimer, comprising any two of such polypeptide, or tetramer, including any four of such polypeptide); (3) a conjugate comprising at least one polypeptide according to this invention; (4) the nucleic acid according to this invention (for example, nucleic acid encoding a polypeptide according to this invention); (5) a vector comprising a nucleic acid according to this invention or encoding the polypeptide according to this invention; (6) the cell or population of cells comprising a polypeptide, nucleic acid, the conjugate and/or a vector according to this invention; and/or (7) the composition according to this invention, where the interaction SV-positive T cells and/or CTLA-4-positive T cells with B7-positive cells is inhibited. In some cases, B7-positive cells E. what about the APK. In some cases, the interaction of CD28-positive T cells with hB7-1-positive cells and/or hB7-2-positive cells is inhibited. In some such methods, the inhibition of the interaction of CD28-positive T cells with hB7-1-positive cells and/or hB7-2-positive cells leads to suppression or inhibition of one or more of the following: T-cell activation or proliferation, the synthesis or production of cytokine (e.g., production of TNF-α, IFN-γ, IL-2), induction of various activation markers (e.g., CD25, IL-2 receptor), inflammation, joint swelling or pain, the level of serum C-reactive protein, production of antibodies anticollagen and/or T-cell-dependent response(s) of antibodies.

In some such methods, at least one such molecule or component according to this invention is administered to a subject in an effective amount to inhibit the interaction of endogenous D28-positive T cells with endogenous B7-1-positive cells and/or B7-2-positive cells in the subject. In some such methods, the interaction of endogenous SV-positive T cells with endogenous B7-positive cells expressing B7-2 (CD86) or B7-1 (CD80), inhibited. In some cases, B7-positive cells is APC that Express B7-2 or B7-1, and the interaction of B7-2 or B7-1 with CD28-positive T-cells is inhibited. In kotoryj cases, the interaction of both B7-2 and B7-1, expressed on APC with CD28-positive T-cells is inhibited.

In another aspect the invention provides a method of suppressing an immune response in vitro or in vivo. The method includes the contact B7-positive cells with at least one of the following molecules or components according to this invention in an effective amount to suppress the immune response: (1) the polypeptide according to this invention (for example, a mutant polypeptide, CTLA-4-EVA or dimeric or Monomeric mutant protein, CTLA-4-Ig); (2) multimer comprising one or more polypeptides according to this invention (e.g., dimer, comprising any two of such polypeptide, or tetramer, including any four of such polypeptide); (3) conjugate comprising at least one polypeptide according to this invention; (4) the nucleic acid according to this invention (for example, nucleic acid encoding a polypeptide according to this invention); (5) a vector comprising a nucleic acid according to this invention or encoding the polypeptide according to this invention; (6) the cell or population of cells comprising a polypeptide, nucleic acid, the conjugate and/or a vector according to this invention; and/or (7) the composition according to this invention, where the immune response is thus suppressed. One or more immune responses can be suppressed, including, for example, T-cell on the Board, T-cell proliferation or activation, synthesis or production of the cytokine, inflammation, joint swelling or pain, serum levels of C-reactive protein, products anticollagen antibodies and/or T-cell-dependent response(s) antibodies. In such methods, including contact B7-positive cells with a polypeptide according to this invention, the polypeptide binds to B7-1 (e.g., human B7-1), expressed on B7-positive cells, and/or binds B7-2 (for example, human B7-2), expressed on B7-positive cells. In some cases, B7-positive cells is agriculture. In some cases, the immune response is suppressed in vitro, such as, for example, in vitro study, including those described in detail herein (see, e.g., examples below). In some cases, the immune response is suppressed in vivo in a subject who is administered an effective amount to suppress the immune response, namely, for example, in therapeutic or prophylactic methods of treatment (for example, a method of treatment of rheumatoid arthritis, such as rheumatoid arthritis, or other autoimmune diseases), discussed in detail in this specification.

In another aspect the invention provides a method of suppressing an immune response in a subject (e.g. a mammal such as man). The method comprises the administration to a subject, nujdayas is this, at least one of the following molecules or components according to this invention in a therapeutically or prophylactically effective amount (e.g. a therapeutically or prophylactically effective dose), which suppresses the immune response in the subject: (1) the polypeptide according to this invention (e.g., mutant CTLA-4-EVA or dimeric or Monomeric mutant protein polypeptide CTLA-4-Ig); (2) multimer comprising one or more polypeptides according to this invention (e.g., dimer, comprising any two of such polypeptide, or tetramer, including any four of such polypeptide); (3) a conjugate comprising at least one polypeptide according to this invention; (4) the nucleic acid according to this invention (for example, nucleic acid encoding a polypeptide according to this invention); (5) a vector comprising a nucleic acid according to this invention or encoding the polypeptide according to this invention; (6) the cell or population of cells comprising a polypeptide, nucleic acid, the conjugate and/or a vector according to this invention; and/or (7) the composition according to this invention, where the immune response is thus suppressed in the subject.

In another aspect the invention provides a method of treatment of a subject having an immune disease or disorder modulated by interaction of endogenous T cells with endog nimi cells, expressing CD80 and/or CD86. The method comprises the administration to a subject requiring such treatment, a therapeutically effective amount of the following: (1) the polypeptide according to this invention (for example, a mutant polypeptide, CTLA-4-EVA or dimeric or Monomeric mutant protein, CTLA-4-Ig); (2) multimer comprising one or more polypeptides according to this invention (e.g., dimer, comprising any two of such polypeptide, or tetramer, including any four of such polypeptide); (3) a conjugate comprising at least one polypeptide according to this invention; (4) nucleic acid according to this invention (for example, nucleic acid encoding a polypeptide according to this invention); (5) a vector comprising a nucleic acid according to this invention or encoding the polypeptide according to this invention; (6) the cell or population of cells comprising a polypeptide, nucleic acid, the conjugate and/or a vector according to this invention; and/or (7) the composition according to this invention, thereby providing the treatment of immune diseases or disorder of a subject. If the subject is a person, CD80 - human CD80, CD86 - human CD86 and CD28 - human CD28. In some such methods, the interaction between endogenous T cells expressing CD28, and endogenous cells expressing CD86, and/or engage the cells, expressing CD80 inhibited.

I believe that various diseases or immune system disorders including rheumatoid or autoimmune disease or disorder can be effectively treated with the use of one or more molecules according to this invention described herein, such as, for example, a mutant polypeptide, CTLA-4 EVA (e.g., any of SEQ ID NOS:1-73, such as, for example, D3-54 (SEQ ID NO:36), D3-69 (SEQ ID NO:50), or D3-27 (SEQ ID NO:24) mutant CTLA-4 EVA)or protein (e.g., D3-54-IgG2 (SEQ ID NO:197 or 211), D3-69-IgG2 (SEQ ID NO:199 or 213), D3-29-IgG2 (SEQ ID NO:79 or 210)). Disease or disorder of the immune system may be or include, for example, but not limited to: Addison's disease, allergies, circular alopecia, Alzheimer's disease, vasculitis associated with antineutrophil cytoplasmic antibodies (LIKE), ankylosing spondylitis, antiphospholipid syndrome (Hughes Syndrome), arthritis, asthma, atherosclerosis, atherosclerotic plaque, autoimmune disease (e.g. lupus, PA, PC, diffuse toxic goiter, and the like), autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune disease of the inner ear, autoimmune lymphoproliferative syndrome, autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, azoospermia, Behcet's disease, a disease Berger, bullous pemphigoid, cardiomyopathy what I cardiovascular disease, celiac disease-sprue/gluten enteropathy, chronic fatigue syndrome and immune dysfunction (SHADES), chronic idiopathic polyneuritis, chronic inflammatory demyelination, polyradiculoneuropathy (CIPD), chronic relapsing polyneuropathy (Guillain-Barre syndrome), a syndrome Cerca-Strauss (ESS), zubtsovy pemphigoid syndrome cold agglutination (SHA), chronic obstructive pulmonary disease, CREST syndrome, Crohn's disease, dermatitis herpetiformis disease, dermatomyositis, diabetes, discoid lupus, eczema, acquired bullous epidermis, essential mixed cryoglobulinemia type, syndrome Evan, exophthalmos, fibromyalgia syndrome?, a disease or disorder associated with graft, diffuse toxic goiter, GVHD, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA nephropathy, immunoproliferative the disease or disorder (e.g., psoriasis), inflammatory bowel disease (IBD), insulin-dependent diabetes mellitus (IDDM), interstitial lung disease, juvenile diabetes, juvenile arthritis, juvenile idiopathic arthritis (Jia), Kawasaki syndrome, syndrome of Eaton-Lambert zoster Wilson, lupus, lupus nephritis, Lymphoscytic Lypophisitis, Meniere's disease, syndrome Miller-Fisher/acute diffuse encephalomyelopathy, mixed disorder of connective tissue, multiple sclerosis (PC), fibrositis, myalgic encephalomyelitis (me), myasthenia gravis, eye inflammation, leaf bladderwort common bladderwort, malignant anemia, Nowotny polyarteritis, polyandry, pluriglandular syndrome (Syndrome Whitaker), polymyalgia rheumatica, polymyositis, primary agammaglobulinemia, primary biliary cirrhosis/autoimmune cholangiopathy, psoriasis, psoriatic arthritis, Reynaud phenomenon, Reiter syndrome/reactive arthritis, restenosis, rheumatic fever, rheumatism, rheumatoid Arthritis, sarcoidosis syndrome Schmidt, scleroderma, Sjδrgen''s Syndrome, graft rejection, solid organ (kidney, heart, liver, lung and the like), a syndrome bound in human systemic lupus erythematosus (SLE), systemic scleroderma, Takayasu's arteritis, temporal arteritis diagnostics/giant cell arteritis diagnostics, thyroiditis, type 1 diabetes, type 2 diabetes, ulcerative colitis, uveitis, vasculitis, vitiligo, Wegener's granulomatosis, and the prevention or suppression of immune response that is associated with the rejection of the donor tissue, cells, tissue or organ transplanted subject to the recipient. Associated with graft diseases or disorders include disease graft-versus-host (GVDH), such as associated with bone marrow transplantation, and immune disorders, who lausiaca the result of or related to the rejection of an organ transplant, tissue or cells (e.g., tissue or cell allografts or xenografts), including, for example, skin grafts, muscle, neurons, Islands, bodies, parenchymal liver cells, etc. In relation to the transplant donor tissue, cells, tissue or whole body subject to the recipient, believe that such molecules according to this invention described herein (e.g., a mutant polypeptide, CTLA-4 EVA or mutant protein, CTLA-4-Ig) can be effective in preventing acute rejection of such a transplant to the recipient and/or for long-term maintenance therapy to prevent rejection of such a transplant to the recipient (for example, inhibition of transplant rejection insulin-producing islet cells from a donor to a subject, recipient suffering from diabetes).

The invention includes any such mutant polypeptide CTLA-4 EVA or mutant protein, CTLA-4-Ig on this invention for use in suppressing an immune response associated with at least one of the above diseases or disorders of the immune system. Also provided the use of any such mutant polypeptide CTLA-4 EVA or mutant fused protein, CTLA-4-Ig in this invention in the production of medication to suppress the immune response, at least, one of them of the above diseases or disorders of the immune system.

An effective amount of a molecule according to this invention, such as, e.g., mutant CTLA-4 EVA polypeptide (e.g., D3-54, D3-69, D3-29, D3-56, D3-75) or protein Ig, including mutant polypeptide CTLA-4 EVA on this invention (e.g., D3-54-IgG2, D3-69-IgG2, D3-29-IgG2, D3-56-IgG2, D3-75-IgG2, respectively), for suppressing an immune response in a subject or treating immune diseases or disorders modulated by interaction of endogenous T cells with endogenous cells expressing CD80 and/or CD86 with the subject in the methods described herein can include from about 0.0001 of milligrams per kilogram (mg/kg) weight of subject to about 200 milligrams per kilogram (mg/kg) of body weight of the subject, namely, for example, from about 0,001 milligrams per kilogram (mg/kg) of body weight of subject to about 100 milligrams per kilogram (mg/kg) weight of the subject or, for example, from about 0.001 mg/kg weight of the subject to at least about 0,005, 0,01, 0,025, 0,05, 0,1, 0,2, 0,3, 0,4, 0,5, 0,6, 0,7, 0,8, 0,9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 50 or 75 mg/kg body weight of the subject. One or more immune responses can be suppressed for a subject, including, for example, T-cell response, T-cell activation or proliferation, synthesis or production of cytokine (e.g., production of TNF-α, IFN-γ, IL-2, and so on), the induction of various activation markers (e.g., CD25, IL-2 receptor and the like), the synthesis or production of inflammatory molecules, inflammation, the hoots of the joint, painful joints, pain, stiffness, levels of serum C-reactive protein, products anticollagen antibodies and/or T-cell-dependent response(s) antibodies. An effective amount of a molecule or component of the present invention to suppress the immune response may be an amount that suppresses the immune response or symptom or sign determined or measured quantity. The immune response may be partially or completely suppressed. An effective amount for the treatment of immune diseases or disorders can be a number, which simplifies, reduces or eliminates at least one symptom or biological response or effect associated with the disease or disorder, prevent the development of diseases or disorders or improves physical functioning entity.

An effective amount of a molecule or component of the present invention for modulating or inhibiting the interaction of T cells expressing CD28 and/or CTLA-4 with B7-positive cells may be the amount that modulates or inhibits the binding between B7-positive cells and SV-positive and/or CTLA-4-positive T-cells, respectively. This binding interaction(I) may be partially or fully modulated or ingibirovalo.

In some such methods Muta is fair dimer fused protein, CTLA-4-Ig in this invention is administered to a subject in a therapeutically or prophylactically effective amount (or dose), sufficient to suppress the immune response, to treat immune disease or disorder modulated by interaction of T cells with B7-expressing cells, or to modulate or inhibit the interaction of T cells expressing CD28 and/or CTLA-4 with B7-positive cells. Dimer fused protein for injection is a usually soluble Ig dimer fused protein. In some such methods, an effective amount or dose of the dimer fused protein according to this invention includes from about 0,001 milligrams per kilogram (mg/kg) of body weight of subject to about 200 milligrams per kilogram (mg/kg) of body weight of the subject (such as, for example, human), or from about 0.001 mg/kg to about 300 mg/kg body weight of the subject. For example, the effective amount or dose of the dimer fused protein can include from about 0.001 mg/kg body weight of the subject to at least about 0,005, 0,01, 0,05, 0,1, 0,2, 0,3, 0,4, 0,5, 0,6, 0,7, 0,8, 0,9, 1, 2, 3, 4, 5, 10, 25, 30, 40, 50, 60, 75, 80, 90, 100, 125, 150, 175, 200, 225, 250 or 300 mg/kg body weight of the subject (such as, for example, a person, including an adult). In some cases, the effective amount or dose that ranges from about 0,001 milligrams (mg) to about 50 milligrams per kilogram (kg) of body weight of a subject, including, but not limited to the following, for example, from about 0.01 mg/kg to about 100 mg/kg body weight of the subject (e.g. human), from about 0.0 to mg/kg to about 50 mg/kg body weight of subject, or from about 0.01 mg/kg to about 25 mg/kg weight of a subject; for example, about 0.05 mg/kg, 0.075 mg/kg, 0.1 mg/kg, 0.15 mg/kg, 0.2 mg/kg, 0.25 mg/kg, 0.3 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg, 5 mg/kg, about 10 mg/kg, 20 mg/kg, 25 mg/kg 50 mg/kg, 75 mg/kg or 100 mg/kg body weight of the subject (for example, an adult human patient) to introduce the subject. In some cases, the effective amount or dose of the dimer fused protein is from about 2 to 10 mg/kg, about 3 to 10 mg/kg, about 3 to 5 mg/kg, about 5 to 10 mg/kg, 0.1 to 5 mg/kg, about 0.05 to 1.0 mg/kg, about 0.05 to 3 mg/kg, about 0.05 to 2.0 mg/kg, about 0.05 to 1.0 mg/kg, about 0.1 to 2.0 mg/kg, about 0.1 to 3.0 mg/kg, about 0.1 to 0.5 mg/kg, about 0.1 to 0.8 mg/kg, about 0.1 to 0.6 mg/kg, about 0.01 mg/kg to about 0.05 mg/kg, about 0.01 mg/kg to about 0.1 mg/kg, about 0.01 mg/kg to about 0.05 mg/kg, about 0.01 mg/kg to about 1 mg/kg, about 0.01 to about 5 mg/kg, about 0.01 mg/kg to about 3 mg/kg, about 0.05 mg/kg to about 2.5 mg/kg, about 0.1 mg/kg to about 1 mg/kg, about 0.1 mg/kg to about 5 mg/kg, about 0.2 to 1 mg/kg, about 0.2 to 0.6 mg/kg, about 0.2 to 0.5 mg/kg, about 0.3 to 1 mg/kg, about 0.3 to 0.6 mg/kg, about 0.3 to 0.5 mg/kg weight of a subject. In some cases, the effective amount or dose of less than about 500 mg for a subject weighing less than 60 kg (for example, less than about 100 mg, 75 mg, 50 mg, 25 mg, 12.5 mg or 10 mg), less than about 750 mg for a subject weighing 60-100 kg (e.g., less than about 150 mg, 100 mg, 75 mg, 37.5 mg, or 20 mg) or men who e than about 1000 mg for a subject, weighing more than 100 kg (for example, less than about 500 mg, 100 mg, 50 mg, 25 mg or 10 mg).

In another aspect, in some such methods, the invention of the mutant protein, CTLA-4-Ig in this invention is administered to a subject in a therapeutically or prophylactically effective amount or dose, which, for example, sufficient to suppress the immune response, to treat immune disease or disorder modulated by interaction of T cells with B7-expressing cells, or to modulate or inhibit the interaction of T cells expressing CD28 and/or CTLA-4 with B7-positive cells. The effective amount or dose of fused protein, which is usually soluble fused protein may include from about 0.001 mg/kg to about 300 mg/kg, about 0.001 mg/kg to about 200 mg/kg, or about 0.001 mg/kg to about 300 mg/kg body weight of the subject (e.g. human). In one aspect, the effective amount or dose of the fused protein comprises from about 0.001 mg/kg to at least about 0,005, 0,01, 0,05, 0,1, 0,2, 0,3, 0,4, 0,5, 0,6, 0,7, 0,8, 0,9, 1, 2, 3, 4, 5, 10, 25, 30, 40, 50, 60, 75, 80, 90, 100, 125, 150, 175, 200, 225, 250 or 300 mg/kg body weight of the subject. In another aspect, an effective amount or dose of from about 0.01 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 50 mg/kg or from about 0.01 mg/kg to about 25 mg/kg weight of a subject. Illustrative doses or amounts include about 0.05 mg/kg, 0.075 mg/kg, 0.1 mg/who g 0.15 mg/kg, 0.2 mg/kg, 0.25 mg/kg, 0.3 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg, 5 mg/kg, about 10 mg/kg, 20 mg/kg, 25 mg/kg 50 mg/kg, 75 mg/kg and 100 mg/kg body weight of the subject (e.g., an adult). In another aspect, an effective amount or dose of the slit protein is from about 2 to 10 mg/kg, about 3 to 10 mg/kg, about 3 to 5 mg/kg, about 5 to 10 mg/kg, 0.1 to 5 mg/kg, about 0.05 to 1.0 mg/kg, about 0.05 to 3 mg/kg, about 0.05 to 2.0 mg/kg, about 0.05 to 1.0 mg/kg, about 0.1 to 2.0 mg/kg, about 0.1 to 3.0 mg/kg, about 0.1 up to 0.5 mg/kg, about 0.1 to 0.8 mg/kg, about 0.1 to 0.6 mg/kg, about 0.01 mg/kg to about 0.05 mg/kg, about 0.01 mg/kg to about 0.1 mg/kg, about 0.01 mg/kg to about 0.05 mg/kg, about 0.01 mg/kg to about 1 mg/kg, about 0.01 to about 5 mg/kg, about 0.01 mg/kg to about 3 mg/kg, about 0.05 mg/kg to about 2.5 mg/kg, about 0.1 mg/kg to about 1 mg/kg, about 0.1 mg/kg to about 5 mg/kg, about 0.2 to 1 mg/kg, about 0.2 to 0.6 mg/kg, about 0.2 to 0.5 mg/kg, about 0.3 to 1 mg/kg, about 0.3 to 0.6 mg/kg, about 0.3 to 0.5 mg/kg weight of a subject. In some aspects, the effective amount or dose of less than about 500 mg for a subject weighing less than 60 kg (for example, less than about 100 mg, 75 mg, 50 mg, 25 mg, 12.5 mg or 10 mg, less than about 750 mg for a subject weighing between 60-100 kg (e.g., less than about 150 mg, 100 mg, 75 mg, 37.5 mg, or 20 mg), or less than about 1000 mg for a subject weighing more than 100 kg, (e.g. the, less than about 500 mg, 100 mg, 50 mg, 25 mg or 10 mg).

The effective amount or dose of the vector nucleic acid, vector, composition, and/or cells according to this invention sufficient to similarly suppress the immune response or modulate, treat immune disease or disorder modulated by interaction of T cells with B7-expressing cells, or to modulate or inhibit the interaction of T cells expressing CD28 and/or CTLA-4 with B7-positive cells can be determined. For example, if the vector encoding such a dimer fused protein according to this invention, it is necessary to introduce the subject, the person skilled in the art can determine the number of vectors for introduction, so that desired a therapeutically or prophylactically effective amount of dimer fused protein could be produced from the subject.

Illustrative dimers fused protein according to this invention include any of those described in detail above and herein, including, for example, dimer fused protein consisting of two identical monomer fused protein, where each monomer fused protein comprises a mutant polypeptide, CTLA-4 EVA in this invention, fused at its C-end N-end polypeptide Ig Fc (e.g., IgG2 Fc, IgG1, IgG4, or mutant polypeptide Ig Fc, which reduces effector function). Illustrative mutant polypep the d CTLA-4 EVA - is a peptide comprising the amino acid sequence selected from the group comprising SEQ ID NOS:1-73. Illustrative dimer fused protein is a dimer comprising two monomer fused protein, where each monomer fused protein comprises the amino acid sequence selected from the group consisting of SEQ ID NOS:74-79, 197-200, 205-214, and 219-222. Typically, two Monomeric fused protein fused dimeric protein covalently linked together via at least one disulfide bond formed between cysteine(s) residue(s)present in each monomer.

In any of the ways described above, a molecule or a component according to this invention (for example, a polypeptide (including, for example, dimeric or Monomeric protein or polypeptide multimer), conjugate, nucleic acid, vector, composition and/or cells of this invention) can be administered to the subject in the form of a composition. The composition typically comprises at least one molecule or component and a filler, carrier or solvent. The composition may include a pharmaceutical composition comprising at least one molecule or component and a pharmaceutically acceptable excipient, carrier or solvent (e.g., PBS). the pH of the compositions according to this invention is usually in the range from about pH 6.0 to about pH 9,0, including the example from about pH 6.5 to about pH 8.5, typically from about pH 7.0 to about pH 8.0. In one aspect, the pH of the compositions according to this invention is usually in the range from about pH 3 to about pH 10, about pH 4 to about pH 10, about pH 5 to about pH 9, from about pH 6 to about pH 9, from about pH 5.5 to about pH 8.5, from about pH 6.0 to about pH 6,7, from about pH 6.0 to about pH 6.5, about pH 6.2 to about pH 8,7, from about pH 6.5 to about pH 7.5, about pH 6.2 to about pH 7,0, from about pH 6.3 to about pH 6.8, from about pH 6.4 to about pH 6.8 and about pH 7.0 to about pH 7.4. In one aspect, the composition comprising at least one molecule or a component according to this invention, such as, for example, a mutant protein, CTLA-4-Ig has a pH value of pH 5.5, pH 6.0, pH of 6.1, pH of 6.2, pH 6.3, pH 6.4, pH 6.5, pH 6.6, pH of 6.7, pH 6.8, pH 6.9, pH 7.0, pH of 7.1, pH of 7.2, pH of 7.3, pH 7.4, pH 7.5, pH of 7.6, the pH of 7.7, pH of 7.8, pH 7,9, pH 8.0, pH 8.1, pH of 8.2, pH 8.3, pH of 8.4, pH 8.5, pH of 8.6, a pH of 8.7, pH 8.8, pH of 8.9, pH of 9.0, the pH of 9.1, pH of 9.2, the pH of 9.3, a pH of 9.4, a pH of 9.5, the pH of 9.6, a pH of 9.7, the pH of 9.8, pH, or pH of 9.9 and 10.0. Some of the compositions of this invention comprise one or more salts (e.g. sodium chloride, sodium phosphate, calcium chloride and the like), one or more buffers (such as HEPES (N-2-hydroxyethyl-piperazine-N-2-econsultancy acid), sodium citrate, sodium phosphate (e.g., Na2HPO4ZNa3PO4), succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate, Tris(hydroxym the Teal)aminomethan (Tris) and the like), one, two, three, four, five or more saccharides or sugars (e.g. sucrose, mannose, maltose, trehalose, dextrose and the like), and/or one, two, three, four or more polyalcohol or sugar alcohols (e.g. mannitol, sorbitol, glycol, glycerol, arabitol, Eritrea, xylitol, ribitol, lactitol and the like). One, two, three, four, five or more monosaccharides, disaccharides and/or polysaccharides can be included in the composition. The composition according to this invention may include any of the concentration of such molecules or component, effective to suppress the immune response when administered to a subject. For example, in some such manner (including, for example, the ways in which immunosuppression is desirable, such as, but not limited to the following, for example, the treatment of rheumatoid arthritis or similar immune disorders, or for inhibiting transplant rejection tissues, cells, tissue or organ from a donor to a subject, recipient), pharmaceutical composition, comprising a pharmaceutically acceptable carrier, excipient or diluent and dimer fused protein according to this invention, is administered to a subject (for example, parenterally, subcutaneously, intravenously, intramuscularly, etc), where the pharmaceutical composition comprises dimer fused protein this invention in a concentration of from about 0.001 mg/ml to OK the lo 200 mg/ml, about 0.001 mg/ml to about 300 mg/ml, about 0.01 mg/ml to about 200 mg/ml, about 0.01 mg/ml to about 250 mg/ml, about 0.1 mg/ml to about 200 mg/ml, about 0.001 mg/ml to about 100 mg/ml, about 0.001 mg/ml to about 90 mg/ml, about 0.01 mg/ml to about 90 mg/ml, about 0.01 mg/ml to about 75 mg/ml, about 0.1 to about 80 mg/ml, about 0.1 to about 75 mg/ml, about 0.1 to about 60 mg/ml, about 0.1 to about 50 mg/ml, about 0.1 to about 40 mg/ml, about 0.1 to about 30 mg/ml, about 1 to about 90 mg/ml, about 1 to about 80 mg/ml, about 1 to about 75 mg/ml, about 1 to about 60 mg/ml, about 1 to about 50 mg/ml, about 1 to about 40 mg/ml, about 1 to about 30 mg/ml, about 1 to about 20 mg/ml, about 1 to about 10 mg/ml, about 1 to about 5 mg/ml, about 5 to about 90 mg/ml, about 5 to about 80 mg/ml, about 5 to about 75 mg/ml, about 5 to about 60 mg/ml, about 5 to about 50 mg/ml, about 5 to about 40 mg/ml, about 5 to about 30 mg/ml, about 5 to about 20 mg/ml, about 5 to about 10 mg/ml, about 1 to about 5 mg/ml, about 10 to about 75 mg/ml, about 25 mg/ml to about 75 mg/ml, about 30 mg/ml to about 60 mg/ml, about 25 to about 50 mg/ml, about 50 mg/ml to about 100 mg/ml, including for example, about 1 mg/ml, 5 mg/ml, 10 mg/ml, about 15 mg/ml, about 25 mg/ml, about 30 mg/ml, about 40 mg/ml, about 50 mg/ml, about 60 mg/ml, about 70 mg/ml, about 80 mg/ml, about 90 mg/ml or 100 mg/ml Provides for other concentrations. In some methods of the invention described here, including some and therapeutic and prophylactic methods the amount of any such compositions (e.g., pharmaceutical compositions)comprising a protein according to this invention range from about 0.01 milliliters (ml) to about 10 ml, about 0.01 ml to about 5 ml, about 0.1 ml to about 5 ml, about 0.5 ml to about 2 ml, about 1 ml to about 2 ml, including, for example, the volume of 0.01 ml of 0.025 ml, 0.05 ml, 0.1 ml, 0.2 ml, 0.3 ml, 0.4 ml, 0.5 ml, 0.75 ml, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 10 ml, 20 ml, 25 ml, 50 ml, 75 ml, 100 ml, 200 ml, 250 ml, 300 ml, 500 ml, 1000 ml, etc. is administered to the subject through a separate I/p/,/m or/p injection. The details of the illustrative compositions of this invention are also discussed in this specification.

The effective amount or dose of a molecule according to this invention, which impose a particular subject may vary depending on, for example, diseases, disorders or condition to be treated, the strength of particular mutant CTLA-4 molecules of this invention (namely, its effectiveness) (e.g., mutant dimer CTLA-4-Ig fused protein according to this invention), which is subject to the introduction, the method of introduction of the molecules and the individual ability of the subject to carry a specific amount of a specific molecule. For example, in the method for suppressing an immune response in a subject having rheumatoid arthritis (RA) or a method of treatment of RA, the effective amount or dose of the mutant dimer CTLA-4-Ig in question is the invention (for example, D3-29-IGg2, D3-54-IgG2, D3-56-IgG2, D3-69-IgG2, D3-75-IgG2 and so on), which is subject to the introduction of the subject may be determined based on many factors, including the effect of mutant dimer CTLA-4-Ig, the method of introduction of the dimer and/or the severity of symptoms or signs of rheumatoid arthritis in the subject. In one aspect, the effective amount or dose of a specific mutant dimer CTLA-4-Ig in this invention can be determined by comparing the strength of such a mutant dimer CTLA-4-Ig with that of the dimer Orencia®. Dose dimer Orencia®, is effective for the treatment of rheumatoid arthritis and related disorders, known in the technical field. For example, dimer Orencia® is usually introduced intravenously to a person suffering from rheumatoid arthritis in a dose from about 10 mg Orencia® per kilogram (kg) of body weight. Mutant dimer CTLA-4-Ig on this invention, which is about X times stronger than Orencia®, can be entered (for example, intravenously, subcutaneously, or otherwise described herein) to a person suffering from rheumatoid arthritis, the dose, which is about "X" times less than the dose of the dimer Orencia® to achieve a therapeutic effect (e.g., suppression of the immune response), which is approximately equivalent to that observed for the dimer Orencia®. If you need a higher therapeutic effect, proportionally increased quantity or dose of the mutant dimer CTLA-4-Ig can is to be defined and introduced the man.

In any of the ways described herein, a molecule or a component according to this invention (for example, a polypeptide (including, for example, dimeric or Monomeric protein or polypeptide multimer), conjugate, nucleic acid, vector, composition and/or the cell according to this invention may be introduced parenterally, subcutaneously or intravenously or as described in this description. Molecule or a component according to this invention can be introduced in a therapeutically effective amount of one, two, three or four times a month, twice a week, once a fortnight (every two weeks or once in two months (every two months). The introduction may continue during the period 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months or longer (e.g., one, two, three, four or more years, including throughout the life of the subject).

Any of the methods described herein may also include an introduction to the subject an effective amount of at least one additional therapeutic or immunosuppressive agent, or connection. Thus, for example, the invention provides a method of suppressing an immune response comprising the administration to a subject in need this, (1) an effective amount of at least one of the first immunosuppressive agent, where each of the first immunosuppressive agent represents aliphatic, nucleic acid, vector, composition, and/or a cell according to this invention, and (2) an effective amount of at least one second immunosuppressive agent, where the immune response in the subject is inhibited.

Various additional therapeutic or immunosuppressive agents (which are not molecules of this invention can be used or put together with a molecule according to this invention (e.g., polypeptide, nucleic acid, vector, composition, and/or a cell according to this invention). Such agents include, for example, Antirheumatic drug, disease modifying (PPMS) (takoyaki, for example, methotrexate (MTX), a cytokine antagonist (e.g., IL-2 or IL-6 antagonist), steroid compound (e.g., corticosteroid, glucosteroid, such as prednisone or methylprednisone), a nonsteroidal compound, sodium or magnesium salicylate, ibuprofen, acetylsalicylic acid, acetaminophen, an antibody, a biological agent, which blocks the synthesis of products of anti-inflammatory cytokine, Raptiva® efalizumab, anti-inflammatory agent or compound and nonsteroidal anti-inflammatory drug (NSAID). Such additional therapeutic or immunosuppressive agent can be administered to the subject in a pharmaceutical composition that includes an additional is the agent and a pharmaceutically acceptable excipient or carrier. The effective amount or dose of the agent, be inserted will depend on the specific agent. Some of these agents are currently used in immunosuppressive therapies, and acceptable dosage can be determined on the basis of diseases, disorders or condition to be treated, and the ability of the entity to transfer a specific amount or dose and immunosuppressive efficiencies agent. Illustrative doses for immunosuppressive agents described above, which are not molecules according to this invention, are known. Additional immunosuppressive agent that is not a molecule according to this invention, may be introduced simultaneously with, or before or after the introduction of the molecules of this invention (e.g., mutant fused protein, CTLA-4-Ig).

The treatment regimen, including dose, schedule of administration, route of administration (for example, intravenous injection, subcutaneous injection and the like) and a pharmaceutical composition comprising at least one molecule or a component according to this invention, may vary depending on the diseases, disorders or condition to be treated. One or more such molecules or components according to this invention can be administered to the subject; each such molecule or component does not necessarily require the maintenance in the same pharmaceutical compositions, the same techniques, the same amount or with the same schedule of dosing frequency.

In some such methods, for example, about 1 ml of the pharmaceutical composition comprising a pharmaceutically acceptable excipient, carrier or solvent with the concentration of dimer fused protein according to this invention, amounting to about 50 mg/ml, injected subcutaneously to a subject (e.g., adult)in need of immunosuppression (for example, to a subject suffering from rheumatoid arthritis). This initial dose is 50 mg of dimer fused protein. For the subject with a body weight of about 100 kg, this initial dose with 0.5 mg of the dimer fused protein per kg of body weight of the subject. Second the same amount of injected subcutaneously after one or two weeks after the first dose. Also dose injected subcutaneously every week, every two weeks or once a month or more or less often if necessary. I believe that such compositions and formats introduction suitable for treatment of humans suffering from rheumatoid arthritis or other immune disorders in which immunosuppression is desirable, or for inhibiting transplant rejection tissues, cells, tissue or organ from a human donor to a human recipient.

Treatment of rheumatoid arthritis

Rheumatoid art is it is one of the most common systemic inflammatory autoimmune diseases and, as calculated, affects 1-2% of the adult population. See, for example, Dipiro, J. T., Rheumatoid arthritis, PHARMACOTHERAPY: A PATHOPHYSIOLOGIC APPROACH, 1671-1682 (Talbert, R.T. et al. eds., McGraw-Hill, New York, 6thed. 2005). The disease is characterized by hyperplasia of the synovial membrane and infiltration of inflammatory cells, including activated T-cells. Activated T-cells play a Central role in the development of rheumatoid arthritis by stimulating different cell types to obtain proinflammatory cytokines, such as IL-1, IL-6 and TNF-alpha antibodies, and matrix metalloproteinases (Hoffman, R.W., Front. Biosci. 6:1369-1378 (2001); Choy, E.K. et al., N. Engl. J. Med 344:907-916 (2001)). A significant contribution of T cells in the development of rheumatoid arthritis makes T-cell activation, which is a rational target for therapeutic intervention. I believe that these inflammatory molecules cause an inflammatory response, tissue damage (e.g., damage to the joint and the pain associated with rheumatoid arthritis.

Costimulate T cells, mediated by interactions between CD28 receptor and CD80 and/or CD86 ligand(s) is necessary for activation of T cells (Riley, J. L. et al., Blood 105:13-21 (2005)). It is shown that therapeutic or prophylactic agents that are antagonists CD80/CD86 - CD28 co-stimulatory pathways such as Orencia® (Abatacept) protein, which is what I soluble dimeric hCTLA-4-Ig fused protein, are clinically effective in the treatment of rheumatoid arthritis (Kremer, J. M. et al., Ann. Intern. Med. 144:865-876 (2006); Genovese, M.C. et al., N. Engl. J. Med. 353:1114-1123 (2005)). I believe that Abatacept demonstrates immunosuppressive function by binding to CD80 and/or CD86 ligands on antigen-presenting cells when administered to a subject (e.g., adult) in vivo in a therapeutically or prophylactically effective amount, thus preventing the interaction of each or both of these ligands with the CD28 receptor on T-cells.

Currently, the proven effectiveness of Abatacept in the treatment of patients who are older people with RA from moderate to severe, which showed inadequate response to one or more PPS, such as methotrexate or TNF antagonists. Abatacept is administered to an adult RA patient at a dose of 10 mg/kg of body weight of the subject by intravenous infusion. After the first dose, the second and third doses of 10 mg/kg fused protein is administered to the subject after two and four weeks respectively after the first dose. Subsequent doses are administered every four weeks (i.e. once a month). I believe that intravenous infusion of Abatacept required to deliver a high dose level required to obtain the desired efficiency in the treatment of rheumatoid arthritis.

Other modern treatment of rheumatoid ar is Rita include the introduction of non-specific immunosuppressive agents, such as methotrexate, steroid and non-steroid anti-inflammatory drugs. Additionally proved the effectiveness of biological agents that target specific proinflammatory cytokines, such as TNF-α (e.g., Remicade® infliximab, Enbrel® intercept, Humira® adalimumab) and IL-1 (e.g., Kineret®, anakinra). However, many of these therapies have significant side effects - some of which are toxic - especially with a long reception.

Despite the availability of various methods of treatment there is a significant unmet need for treatment of RA. For example, 60% of patients-people with RA who have had previous ineffective PMS treatment, and 80% of patients-people with RA who had ineffective previous anti-TNF therapy was not achieved ACR50 indicators after treatment with Orencia for 6 months (J.M. Kremer et al., Ann. Intern. Med. 144:865-876 (2006); Genovese, M.C. et al., N. Engl. J. Med. 353:1114-11 (2005)). Studies of response to dose using Abatacept and Belatacept (LEA29Y-Ig) fused protein in the treatment of RA in adults have shown that the efficacy was dose-dependent and was not saturated at the highest dose level, which was analysed (Kremer, J.M. et al., N. Engl. J. Med. 349:1907-1915(2003); Moreland, L.W. et al., Arthrit. Rheum. 46:1470-1479 (2002)).

Soluble dimeric mutant CTLA-4-Ig in this invention, having the higher is vidnosti binding to hCD80 and/or hCD86, than Abatacept, believed to be able to show a stronger immunosuppressive effects than Abatacept when administered to a subject with RA. Such mutant CTLA-4-Ig binds a similar amount of CD80 and/or CD86 ligands at lower concentrations than Abatacept.

Mutant CTLA-4-Ig with higher avidity binding to CD80 or CD86 and a slower dissociation rate from CD80 or CD86, respectively, is more time spent on this ligand. This increased the time spent, as I believe, is associated with greater efficiency in vivo. Assume that such mutant CTLA-4-Ig can be effective in therapeutic or prophylactic treatment of a subject with RA in a dose that is lower than the Abatacept. That is, suppose that such mutant CTLA-4-Ig can reach the degree of efficiency equivalent to that for Abatacept, when administered to a subject with RA in a dose that is less than the introduction of Abatacept at a dose of 10 mg/kg of body weight of the subject. This invention provides a soluble dimeric mutant fused proteins, CTLA-4-Ig different avednesday binding to hCD80 and/or hCD86. Soluble dimeric mutant fused proteins, CTLA-4-Ig, which have, in fact, more high-avidity binding to hCD86 than Abatacept may have a degree of efficiency equivalent to that of Abatacept when administered to a subject with RA in a dose that is essentially smaller than the W hat to Abatacept. The introduction of lower doses of such mutant CTLA-4-Ig can be provided by using a more acceptable route of administration (e.g. subcutaneous injection)than are used at present for the introduction of Abatacept (intravenous injection).

Also suggest that soluble mutant protein, CTLA-4-Ig on this invention with higher immunosuppressive force than Abatacept or Belatacept protein, will provide a higher level of effectiveness in the treatment of RA patients. Assume that more immunosuppressive mutant CTLA-4-Ig is able to eliminate the symptoms associated with RA, and to inhibit the development of harmful physical effects of RA is more effective than Abatacept. Such mutant CTLA-4-Ig can be formulated in a pharmaceutically acceptable solvent, excipient or carrier (e.g., PBS) at a concentration in the range from 0.1 to 200 mg/ml Treatment of a subject with RA can be achieved by introducing to the subject a therapeutically or prophylactically effective amount (dose) of mutant CTLA-4-Ig by subcutaneous injection or intravenous infusion at an acceptable specific frequency of dosing (eg, initial dose, followed by one dose 2-4 times per month, one dose per month or one dose every two months). The dose will depend on the severity of the disease or symptoms subjection, there may be a quantity or dose of the mutant CTLA-4-Ig no more than about 10 mg/kg (including, for example, about 1 mg/kg, 0.5 mg/kg, 0.25 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg or 9 mg/kg) of body weight of the subject. More immunosuppressive mutant CTLA-4-Ig may allow less frequent dosing schedule (e.g., once every two months)than the schedule of dosage, which is usually used with Abatacept. Alternatively, the amount or dose of a mutant CTLA-4-Ig is greater than about 10 mg/kg weight of a subject (for example, from about 10 mg/kg to about 100 mg/kg, from about 10 mg/kg to about 25 mg/kg, about 10 mg/kg to about 50 mg/kg, about 10 mg/kg to about 75 mg/kg and the like, including, for example, about 15 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 75 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg) can be administered to the subject with RA, if painful condition and/or symptoms of the subject require such amount or dose.

The effective amount or dose of the mutant dimer CTLA-4-Ig in this invention for the treatment of RA in humans who suffer from it, can be determined based on various factors such as the strength of the mutant dimer CTLA-4-Ig, the method of introduction of the dimer and/or the severity of the symptoms or signs of rheumatoid arthritis in the subject. For example, the effective amount or dose of the mutant dimer CTLA-4-Ig in this invention can be determined by cf is Vania strength of such a dimer with the same for Orencia® dimer and determine the amount or dose of the mutant dimer CTLA-4-Ig, which will provide the desired immunosuppressive effect compared to Orencia® (e.g., improved or approximately equivalent effect) based on the amount or dose of Orencia®, which is usually administered to a human subject exhibiting similar symptoms or signs of RA.

In one embodiment of the invention, for example, the invention provides a method of treating rheumatoid arthritis in a subject in need of such treatment, where the method includes introduction to the subject an effective amount of a soluble dimeric mutant fused protein, CTLA-4-Ig in this invention, for example, by intravenous or subcutaneous injection. The effective amount or dose, which may include from about 0,001 milligrams (mg) to about 10 milligrams per kilogram (kg) of body weight of a subject, including, but not limited to the following, for example, from about 0.01 mg/kg to about 10 mg/kg weight of a subject, from about 0.05 mg/kg 0.1 mg/kg 0.2 mg/kg, 0.25 mg/kg, 0.3 mg/kg, 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg or 10 mg/kg of body weight of an adult human patient is administered to the subject. In some cases, the effective amount or dose of from about 2 to 10 mg/kg, about 3 to 10 mg/kg, about 3 to 5 mg/kg, about 5 to 10 mg/kg, 0.1 to 5 mg/kg of body weight, about 0.05 to 1.0 mg/kg, about 0.05 to 3 mg/kg of body weight, about 0.05 to 2.0 mg/kg, about 0.05 to 1.0 mg/kg, about 0.05 is about 2.0 mg/kg, about 0.1 to 2.0 mg/kg, about 0.1 to 3.0 mg/kg, about 0.1 to 0.5 mg/kg, about 0.1 to 0.8 mg/kg, about 0.1 to 0.6 mg/kg, about 0.2 to 1 mg/kg, about 0.2 to 0.6 mg/kg, about 0.2 to 0.5 mg/kg, about 0.3 to 1 mg/kg, about 0.3 to 0.6 mg/kg, about 0.3 to 0.5 mg/kg weight of a subject. In some cases, the effective amount or dose of less than about 500 mg for a subject weighing less than 60 kg (for example, less than about 100 mg, 75 mg, 50 mg, 25 mg or 12.5 mg), less than about 750 mg for a subject weighing between 60-100 kg (e.g., less than about 150 mg, 100 mg, 75 mg, 37.5 mg, or 20 mg), or less than about 1000 mg for a subject weighing more than 100 kg (for example, less than about 500 mg, 100 mg, 50 mg, 25 mg or 10 mg). After the first dose, subsequent equivalent doses administered at intervals of 1, 2, 4, 8, 10, 12, 14 or 16 weeks. The frequency of subsequent dosage determines if necessary.

This mutant fusion of CTLA-4-Ig can be formulated with a pharmaceutically acceptable excipient, carrier or solvent to obtain a pharmaceutical composition that is acceptable for administration to a subject (e.g. a mammal, including humans). The concentration of the fused protein in the composition may be in the range from about 0.01 mg/ml to about 300 mg/ml, or from about 0.01 mg/ml to about 200 mg/ml, including, but not limited to the following, for example, from about 0.1 mg/ml to about 300 mg/ml, from about 0.1 mg/ml to about 200 is g/ml, from about 0.1 mg/ml to about 100 mg/ml, from about 0.5 mg/ml to about 100 mg/ml, from about 0.5 mg/ml to about 50 mg/ml, from about 1 to about 100 mg/ml, from about 1 to about 75 mg/ml, from about 5 to about 75 mg/ml, from about 10 to about 75 mg/ml, from about 10 to about 60 mg/ml, from about 25 to about 60 mg/ml, from about 30 to about 60 mg/ml, from about 25 to about 50 mg/ml, from about 40 to about 50 mg/ml, from about 25 mg/ml or about 50 mg/ml Other compositions, including those discussed above and below, also included in the scope of this invention.

Assume that such treatment reduces one or more signs and/or symptoms associated with rheumatoid arthritis, such as inflammation, pain, joint swelling, joint pain and stiffness, of the subject. Such treatment may reduce the development of disease in a patient. For example, such treatment may reduce the development of structural damage to the patient. Such treatment may improve physical functioning entity.

Methods of inhibiting transplant rejection tissues, cells, tissue or organ

In another aspect the invention provides a method of inhibiting rejection or suppression of the immune response associated with the transplantation of tissues, cells, skin or organ from a donor to a subject, recipient, where the method includes introducing the subject to the recipient a therapeutically effective the number of one or more of the following: (1) the polypeptide according to this invention (for example, mutant polypeptide, CTLA-4-EVA or dimeric or Monomeric mutant protein, CTLA-4-Ig); (2) multimer comprising one or more polypeptides according to this invention (e.g., dimer, comprising any two of such polypeptide, or tetramer, including any four of such polypeptide); (3) a conjugate comprising at least one polypeptide according to this invention; (4) the nucleic acid according to this invention (for example, nucleic acid encoding a polypeptide according to this invention); (5) a vector comprising nucleic acid according to this invention or encoding the polypeptide according to this invention; (6) the cell or population of cells comprising a polypeptide, nucleic acid, the conjugate and/or a vector according to this invention; and/or (7) the composition according to this invention, thereby inhibiting graft rejection tissues, cells, skin, or body subject to the recipient. The donor and recipient may be the same species or different species. The donor or the recipient may be a mammal, such as human, non-human Primate (e.g., monkey, gorilla), sheep, cat, dog, pig, cow, horse, etc.

In some such methods, the polypeptide, conjugate, vector and/or cell of this invention administered to a subject, the recipient prior to, simultaneously with or after the transplantation of tissues, cells, skin or body. The effect is positive number usually includes from about 0.001 mg/kg weight of subject to about 200 mg/kg body weight of the subject. In some such methods, for example, the effective amount includes from about 0,001 milligrams per kilogram (mg/kg) weight of the subject to at least about 0,005, 0,01, 0,05, 0,1, 0,2, 0,3, 0,4, 0,5, 0,6, 0,7, 0,8, 0,9, 1, 2, 3, 4, 5, 10, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250 or 300 milligrams per kilogram (mg/kg) of body weight of the subject. In some such methods, the effective amount includes from about 0,001 milligrams per kilogram (mg/kg) weight of the subject to at least about 0,005, 0,01, 0,05, 0,1, 0,2, 0,5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 50 or 75 milligrams per kilogram (mg/kg) of body weight of the subject. The polypeptide, conjugate, nucleic acid, vector and/or cell of this invention can be administered to the subject to the recipient in the process, before or immediately after transplantation. Alternative or additionally, such a molecule according to this invention can be introduced through one or more hours after transplantation, the next day after transplantation and/or daily thereafter or at least once a week, at least once in two weeks or at least once per month after transplantation, if necessary, up to 12, 24 or 36 or more months, or longer if necessary. An organ transplant may involve any organ, such as kidney, liver, heart or lung.

The effective amount or dose of the mutant molecules CTLA-4 and Yes the efforts of the invention (for example, mutant dimer fused protein, CTLA-4-Ig), subject to introduction to the subject is the recipient of an organ transplant, tissue or cells in order to inhibit graft rejection (or to suppress the immune response associated with such a transplant), usually determined on the basis of the strength of such a molecule, the method of administration, type of transplantation (e.g., cells, tissue, organ), history and/or the severity of symptoms or signs at the subject-transplant recipient's immune(s) answer(s)confirming the rejection of the graft. For example, the effective amount or dose of the mutant dimer CTLA-4-Ig on this invention (e.g., D3-29-IGg2, D3-54-IgG2, D3-56-IgG2, D3-69-IgG2, D3-75-IgG2 dimer and the like) can be determined by comparing the strength of such a dimer with that of the dimer Belatacept. Effective dose of Belatacept suitable to prevent or suppress the immune response associated with transplantation of kidney/renal transplantation, are known. For example, Belatacept administered by intravenous infusion to a person after kidney transplantation of kidney donors in the amount or dose of about 5 mg or 10 mg per kilogram of body weight per month. Mutant dimer CTLA-4-Ig on this invention, which is about "X" times more powerful than Belatacept can be entered (for example, intravenously, subcutaneously, or otherwise described herein) the forehead is ECU, which has a kidney transplant in the amount or dose, which is approximately X times less than the dose of Belatacept to achieve a therapeutic effect (e.g., suppression of the immune response), approximately equivalent to that for Belatacept. If you need a higher therapeutic effect can be determined and entered proportionally increased quantity or dose of the dimer fused protein, CTLA-4-Ig in this invention.

In another aspect the invention provides a method of treating transplant rejection tissue, cell or organ (e.g., graft rejection, solid organ (e.g. kidney, liver, lung, heart, etc.)) for a subject who has received such a tissue, cell or organ from a donor, where the method comprises the administration to the recipient a therapeutically effective amount of at least one polypeptide, conjugate, nucleic acids, vectors and/or cells according to this invention, thereby inhibiting rejection of the donor graft tissue, cells or body subject to the recipient. The polypeptide, conjugate, nucleic acid, vector and/or cells of this invention can be administered to the subject prior to, simultaneously with or after the transplantation of cells, tissue or organ.

In one aspect the invention provides a method of inhibiting rejection of transplantation of islet cell is from a donor to a subject, recipient, who needs it, where the method includes introduction to the subject an effective amount or dose of the mutant molecule CTLA-4 in this invention (e.g., mutant fused protein, CTLA-4-Ig) prior to, simultaneously with or after islet transplantation(s) cell(s) from the pancreas of a donor subject. The subject (i.e. the person) usually suffers from diabetes (e.g., IDDM), and this method is suitable for treatment of a subject who has been diagnosed with or suffering from diabetes. Transplantation of pancreatic islet known in the art. Typically, the pancreatic islets are removed from the pancreas of a deceased donor organ, cleaned and treated and implanted the principal recipient suffering from diabetes. After transplantation of beta cells in the pancreatic islets begin to produce and release insulin, thereby reducing the need to subject the recipient to insulin.

In such methods of inhibiting transplant rejection mutant molecule CTLA-4 in this invention (e.g., mutant CTLA-4-Ig) can be formulated with a pharmaceutically acceptable excipient, carrier or diluent to form a pharmaceutical composition that is acceptable for administration to a subject (e.g. a mammal, including humans). Some of these methods include the introduction of farmacevticheskoi composition, including pharmaceutically acceptable excipient, carrier or diluent and mutant dimer CTLA-4-Ig in this invention, having a concentration from about 0.01 mg/ml to about 300 mg/ml, or about 0.01 mg/ml to about 200 mg/ml, including, but not limited to the following, for example, from about 0.1 mg/ml to about 300 mg/ml, from about 0.1 mg/ml to about 200 mg/ml, about 0.1 mg/ml to about 100 mg/ml, about 0.5 mg/ml to about 100 mg/ml, about 0.5 mg/ml to about 50 mg/ml, about 1 to about 100 mg/ml, about 1 to about 75 mg/ml, about 5 to about 75 mg/ml, about 10 to about 75 mg/ml, about 10 to about 60 mg/ml, about 25 to about 60 mg/ml, about 30 to about 60 mg/ml, about 25 to about 50 mg/ml, about 40 to about 50 mg/ml, about 25 mg/ml or about 50 mg/ml Other compositions, including those discussed in detail above and below are also included in the scope of the invention.

Methods of inhibiting an immune response

In another aspect, the invention includes the use of the polypeptide (including, for example, dimeric or Monomeric protein or a multimeric polypeptide, conjugate, nucleic acid, vector or cell of this invention for the production of medicaments for the inhibition or suppression of the immune response in a mammal (e.g. human or non-human Primate). Immune responses, which can be suppressed include, for example, T-cell activation or prolifer the tion, the synthesis or production of cytokines, induction of activation markers, the synthesis or production of inflammatory molecules, inflammation, production anticollagen AT, T-cell-dependent AT the response.

The invention also includes the use of the polypeptide (including, for example, dimeric or Monomeric protein or a multimeric polypeptide, conjugate, nucleic acid, vector or cell of this invention for the production of medicaments for the treatment of immune diseases or disorders. Immune disease or disorder can be, which is mediated by the interaction of T cells with CDSO-positive cells and/or SW-positive cells in the mammal. Immune disease or disorder can be a disorder or disease of the immune system, such as rheumatism, or disorder, or an autoimmune disease or autoimmune disorder. Such immune disease or disorder may be or include, for example, but not limited to the following:

Addison's disease, allergies, circular alopecia, Alzheimer's disease, vasculitis associated with antineutrophil cytoplasmic antibodies (LIKE), ankylosing spondylitis, antiphospholipid syndrome (Hughes syndrome), arthritis, asthma, atherosclerosis, atherosclerotic plaque, autoimmune disease (e.g. lupus, PA, PC, diffuse toksicheskie and the like), autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune disease of the inner ear, autoimmune lymphoproliferative syndrome, autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, azoospermia, Behcet's disease, syndrome behceta, disease Berger, bullous pemphigoid, cardiomyopathy, cardiovascular disease, celiac disease-sprue/gluten enteropathy, chronic fatigue syndrome and immune dysfunction (SHADES), chronic idiopathic polyneuritis, chronic inflammatory demyelination, polyradiculoneuropathy (CIPD), chronic relapsing polyneuropathy (Guillain-Barre syndrome), a syndrome Cerca-Strauss (ESS), scar pemphigoid syndrome cold agglutination (The SHA), chronic obstructive pulmonary disease, CREST syndrome, Crohn's disease, dermatitis herpetiformis disease, dermatomyositis, diabetes, discoid lupus, eczema, acquired bullous epidermis, essential mixed cryoglobulinemia type, syndrome Evan, exophthalmos, fibromyalgia syndrome?, a disease or disorder associated with graft, diffuse toxic goiter, GVHD, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA nephropathy, immunoproliferative the disease or disorder (e.g., psoriasis), inflammatory bowel disease (IBD), Insa is insability diabetes mellitus (IDDM), interstitial lung disease, juvenile diabetes, juvenile arthritis, juvenile idiopathic arthritis (Jia), Kawasaki syndrome, myasthenic syndrome Lambert-Eaton zoster Wilson, lupus, lupus nephritis, Lymphoscytic Lypophisitis, Meniere's disease, syndrome, Miller-Fisher/acute diffuse encephalomyelopathy mixed disease of connective tissue, multiple sclerosis (PC), fibrositis, myalgic encephalomyelitis (me), myasthenia gravis, eye inflammation, leaf bladderwort common bladderwort, malignant anemia, Nowotny polyarteritis, polyandry, pluriglandular syndrome (syndrome Whitaker), polymyalgia rheumatica, polymyositis, the primary agammaglobulinemia, primary biliary cirrhosis/autoimmune cholangiopathy, psoriasis, psoriatic arthritis, Reynaud phenomenon, Reiter syndrome/reactive arthritis, restenosis, rheumatic fever, rheumatism, rheumatoid arthritis, sarcoidosis syndrome Schmidt, scleroderma, Sjogren Syndrome, graft rejection, solid organ (kidney, heart, liver, lung and the like), a syndrome bound in human systemic lupus erythematosus (SLE), systemic scleroderma, Takayasu's arteritis, temporal arteritis diagnostics/giant cell arteritis diagnostics, thyroiditis, type 1 diabetes, type 2 diabetes, ulcerative colitis, uveitis, vasculitis, vitiligo, Wegener's granulomatosis and warning lipocalin immune response, associated with the rejection of the donor tissue, cells, tissue or organ or organ transplanted subject to the recipient.

The invention also provides the use of the polypeptide, conjugate, nucleic acid, vector or cell of this invention for the production of medicaments for the inhibition of the interaction of CD80-positive cells and/or CD86-positive cells with CD28-positive and/or CTLA-4-positive T-cells. In another aspect, the invention includes the use of the polypeptide, conjugate, nucleic acid, vector or cell of this invention for the production of medicaments for the treatment of transplant rejection of a tissue or organ (e.g., graft rejection, solid organ (e.g. kidney, lung, liver, heart, etc.)) in a mammal.

Evaluation of immune responses

Immune responses that are suppressed by the polypeptide, nucleic acid, vector, virus, pseudovirus, HPV or composition according to this invention can be measured by any acceptable technique. Examples of suitable methodologies for assessing immune responses include the following: flow cytometry, research by Western blot turns, immunohistochemical studies, thus, radioimmunoassays (RIA) and enzyme immunoassays. Enzyme immunoassays include enzyme-linked immuno is ramochnie assays (ELIFA), and the enzyme-linked immunosorbent assays (ELISA), including sandwich ELISA and competitive ELISA studies. HPLC and capillary electrophoresis (CE) can also be used in immunoassays for the determination of complexes of antibodies and agents that target. Basic guide that provides such methods and related principles are described in, for example, Harlow and Lane (1988) ANTIBODIES, A LABORATORY MANUAL, Cold Spring Harbor Publications, New York, Hampton R et al. (1990) SEROLOGICAL METHODS A LABORATORY MANUAL, APS Press, St. Paul Minn., Stevens (1995) CLINICAL IMMUNOLOGY AND SEROLOGY: A LABORATORY PERSPECTIVE, CRC press, Bjerrum (1988) HANDBOOK OF IMMUNOBLOTTING OF PROTEINS, Vol.2, Zoa (1995) DIAGNOSTIC IMMUNOPATHOLOGY: LABORATORY PRACTICE AND CLINICAL APPLICATION, Cambridge University Press, Folds (1998) CLINICAL DIAGNOSTIC IMMUNOLOGY: PROTOCOLS IN QUALITY ASSURANCE AND STANDARDIZATION, Blackwell Science Inc., Bryant (1992) LABORATORY IMMUNOLOGY &SEROLOGY 3rd edition, W Saunders Co., and Maddox D E et al. (1983) J. Exp. Med. 158:1211. Guidance on ELISA techniques and related principles are described in, for example, Reen (1994) Methods Mol. Biol. 32:461-6, Goldberg et al. (1993) Curr. Opin. Immunol. 5(2):278-81, Voller et al. (1982) Lab. Res. Methods Biol. Med. 5:59-81, Yolken et al. (1983) Ann. NY Acad. Sci. 420:381-90, Vaughn et al. (1999) Am. J. Trop. Med. Hyg. 60(4):693-8, and Kuno et al. (1991) J. Virol. Methods 33(1-2): 101-13. Guidance on flow cytometry provided in, for example. Diamond (2000) IN LIVING COLOR: PROTOCOLS IN FLOW CYTOMETRY AND CELL SORTING, Springer Verlag, Jaroszeki (1998) FLOW CYTOMETRY PROTOCOLS, 1st Ed., Shapiro (1995) PRACTICAL FLOW CYTOMETRY, 3rd edition, Rieseberg et al. (2001) Appl. Environ. Biotechnol. 56(3-4):350-60, Scheffold and Kem (2000) J. Clin. Immunol. 20(6):400-7, and McSharry (1994) Clin. Environ. Rev. (4):576-604.

Cytotoxic and other T-cell immune responses can also be measured by any acceptable IU odili. Examples of such techniques include ELISpot analysis (in particular, IFN-gamma ELISpot), intracellular cytokine staining (GSC) (in particular in combination with FACS analysis), staining of CD8+ T cell tetramer/FACS, standard and modified tests T-cell proliferation, CTL (cytotoxic T lymphocytes) analysis of the release of chrome, the analysis is limited breeding (AOP, or LDA), and CTL killing research. Guidelines and principles related to research of T-cell proliferation, as described in, for example, Plebanski and Burtles (1994) J. Immunol. Meth. 170:15, Sprent et al. (2000) Philos. Trans. R. Soc. Lond. In Biol. Sci. 355(1395):317-22 and Messele et al. (2000) Clin. Diagn. Lab. Immunol. 7(4):687-92. LDA is described in, for example, Sharrock et al. (1990) Immunol. Today 11:281-286. ELISpot studies and related principles are described in, for example, Czerinsky et al. (1988) J. Immunol. Meth. 110:29-36, Olsson et al. (1990) J. Clin. Invest. 86:981-985, Schmittel et al. (2001) J. Immunol. Meth. 247(1-2): 17-24, Ogg and McMichael (1999) Immunol. Lett. 66(1-3):77-80, Schmittel et al. (2001) J. Immunol. Meth. 247(1-2): 17-24, Kurane et al. (1989) J. Exp. Med. 170(3):763-75, Chain et al. (1987) J. Immunol. Meth. 99(2):221-8, Czerkinsky et al. (1988) J. Immunol. Meth. 110:29-36, and U.S. Patent No. 5750356 and 6218132. Tetramer studies are discussed in, e.g., Skinner et al. (2000) J. Immunol. 165(2):613-7. Other T-cell analytical techniques described in Hartel et al. (1999) Scand. J. Immunol. 49(6):649-54 and Parish et al. (1983) J. Immunol. Meth. 58(1-2):225-37.

T-cell activation can also be analyzed by measuring CTL activity or expression of activation antigens, such as I-2 receptor, CD69 or HLA-DR molecules. Proliferation of purified T cells can be measured in the analysis of the reaction mixed culture of lymphocytes (SCR-reaction); such studies are well known in the art.

ELISpot studies measure the number of T cells secreting specific cytokines, such as IFN-γ or TNF-α, which is a marker of T-cell effectors. Cytokine-specific ELISA kits are commercially available (for example, IFN-γ-specific ELISPot available from R&D Systems, Minneapolis, MN).

Additional ways to evaluate and measure the ability of the molecules of this invention (e.g., polypeptides according to this invention, including, for example, soluble mutant fused proteins, CTLA-4-Ig in this invention) to suppress or inhibit T-cell activation and/or T-cell proliferation described in examples 5-8 in the Examples section below.

Methods introduction

In any of the ways described herein, the pharmaceutical composition for injection comprising the appropriate pharmaceutically acceptable excipient or carrier (e.g., PBS) and an effective amount of a molecule according to this invention, such as a polypeptide (e.g., mutant CTLA-4 EVA or Monomeric, dimeric or multimeric mutant CTLA-4-Ig), or a conjugate according to this invention may be introduced parenterally, intramuscularly, in traperitoneal, intravenous, subdermal, transdermal, subcutaneously or intradermally to the owner. Alternatively, it may be used neoliticheskie methods of protein delivery (delivery of vaccine cannon) (examples of which are discussed elsewhere in this description). Any other acceptable technique can also be used. The introduction of the polypeptide can be facilitated by using liposomes. Any such method of delivery can be used for delivery of the polypeptide or conjugate according to this invention together with any therapeutic or prophylactic method described here.

As the following discussion primarily relates to nucleic acids, it should be understood that it applies equally to the vector nucleic acid according to this invention. Nucleic acid according to this invention or the composition may be administered to the host in any acceptable way of introduction. In some aspects of the invention, the introduction of nucleic acid is parenteral (e.g. subcutaneous (s.c), intramuscular (Im.) or intradermal (Id.)), local or transdermal. Nucleic acid can be introduced directly into the tissue, such as muscle, by injection using a needle or similar device. See, for example, Nabel et al. (1990), above; Wolffet al. (1990) Science 247:1465-1468), Robbins (1996) Gene Therapy Protocols, Humana Press, NJ, and Joner (1993) Gene Targeting: A Practical Approach, IRL Press, Oxford, England, and U.S. Patent No. 5580859 and 5589466. Other methods, such as "neoliticheskaya or mediated by particle transformation (see, for example. U.S. patent No. 4945050 and 5036006, Sanford et al., J. Particulate Sci. Tech. 5:27-37 (1987), Yang et al., Proc. Natl. Acad. Sci. USA 87:9568-72 (1990), and Williams et al., Proc. Natl. Acad. Sci. USA 88:2726-30 (1991)). These methods are suitable not only for in vivo injection of DNA in a subject such as a mammal, but also for ex vivo modification of cells for re-injection into the mammal (which is also discussed in this description).

For a standard introduction of a gene gun, vector or nucleic acid of interest, precipitious on the surface of microscopic metal granules. Microprojector dispersed shock wave or an expanding gas of helium, they penetrate the tissue to a depth of several layers of cells. For example, Accel™ Appliance Delivery Gene produced Agacetus, Inc. Middleton WI, is acceptable for use in this embodiment. Nucleic acid or vector can be introduced using techniques such, for example, intramuscularly, intradermally, subdermally, subcutaneously and/or intraperitoneally. Additional devices and methods related to balistically delivery, can be found in the Publications of International Applications WO 99/2796, WO 99/08689, WO 99/04009 and WO 98/10750 and U.S. Patent No. 5525510, 5630796, 5865796 and 6010478.

Nucleic acid mo is et to be entered together with the agent, to facilitate transfection, examples of which were discussed above. Nucleic acid can be introduced locally and/or through the delivery of lipid particles (unlike balistically delivery of solid particles). Examples of such methods of delivery of nucleic acids, compositions and additional constructs that may be acceptable as carriers for delivery of nucleic acids according to this invention are provided in, for example, U.S. Patents№5591601, 5593972, 5679647, 5697901, 5698436, 5739118, 5770580, 5792751, 5804566, 5811406, 5817637, 5830876, 5830877, 5846949, 5849719, 5880103, 5922687, 5981505, 6087341, 6107095, 6110898 and Publications of International applications No. WO 98/06863, WO 98/55495 and WO 99/57275.

Alternatively, the nucleic acid can be administered to the host by way of gene delivery based on liposomes. Illustrative methods and principles related to gene delivery based on liposomes, are provided in, for example, Debs and Zhu (1993) WO 93/24640; Mannino and Gould-Fogerite (1988) Biotechniques 6(7):682-691; Rose U.S. Patent No. 5279833; Brigham (1991) Publication of the International Application WO 91/06309; Brigham et al. (1989) Am. J. Med. Sci. 298:278-281; Nabel et al. (1990) Science 249:1285-1288; Hazinski et al. (1991) Am. J. Resp. Cell Molec. Biol. 4:206-209; and Wang and Huang (1987) Proc. Natl. Acad. Sci. USA 84:7851-7855), and Feigner et al. (1987) Proc. Natl Acad. Sci. USA 84:7413-7414). Acceptable liposomal pharmaceutically acceptable compositions which can be used to deliver nucleic acids, are also described here.

Any number nekleenov the th acid according to this invention can be used in the methods according to this invention. For example, the necessary nucleic acid may be formulated in pharmaceutically acceptable excipient or carrier and administered to a subject, so that the encoded polypeptide or conjugate is produced in the subject in an amount that is considered effective to, for example, to suppress the immune response in the subject to inhibit an interaction between endogenous B7-positive cells and CD28-positive cells in the subject or to inhibit rejection of the graft tissue, cell, organ or tissue or organ. In one format, when the nucleic acid is injected through the injection is administered from about 50 micrograms (μg) to 100 mg of nucleic acid. In one illustrative application to suppress the immune response, the pharmaceutical composition comprising PBS and the amount of DNA vector that encodes an effective amount of a mutant polypeptide, CTLA-4, is administered by injection or electroporation, or other appropriate delivery method (e.g., gene gun, penetrating through the skin, and lipofectin) to a subject in need of treatment (e.g., a subject suffering from immune diseases or disorders, in which immunosuppressive treatment is desirable). Illustrative vector shown in figure 1.

The number of DNA plasmids for use in the methods according to this invention, to the Yes, the introduction is carried out by means of a gene gun, for example, is often from about 100 to about 1000 times less than the amount used for direct injection (for example, by standard injection needle). Despite this sensitivity, at least about 1 μg nucleic acid can be used in such methods balistically delivery.

RNA or DNA viral vector systems may be suitable for delivery of nucleic acids encoding the polypeptides according to this invention. Viral vectors can be introduced directly to a subject in vivo, or they can be used for treatment of cells in vitro, and the modified cells are administered to the subject in the format of ex vivo. Suitable viral vectors include those discussed above, such as adeno-associated, adenoviral, retroviral, lentivirinae and viral vectors, herpes simplex. With such a viral vector nucleic acid according to this invention can be easily transfection cells and the target tissue of the subject. Additionally retroviral, lentivirusnye and adeno-associated viral methods of gene transfer may be possible to integrate the nucleic acid according to this invention into the host genome, thus leading to prolonged expression built nucleic acids.

Delivery of viral vector according to this invention, in the with, at least one nucleic acid according to this invention to a subject, say, are able to suppress immune response in the subject, which introduce the vector. Not necessarily, some preventive and/or therapeutic methods according to this invention is practiced with a acceptable dosage of the viral vector, sufficient to inhibit detectable immune response. Any acceptable viral vector comprising a nucleic acid according to this invention, in any acceptable concentration, can be used to suppress the immune response. For example, entity-the owner may be introduced population of retroviral vectors (examples of which are described in, for example, Buchscher et al. (1992) J. Virol. 66(5) 2731-2739, Johann et al. (1992) J. Virol. 66 (5):1635-1640 (1992), Sommerfelt et al. (1990) Virol. 176:58-59, Wilson et al. (1989) J. Virol. 63:2374-2378, Miller et al., J. Virol. 65:2220-2224 (1991), Wong-Staal et al., PCT/US94/05700, and Rosenburg and RPPC i (1993) in FUNDAMENTAL IMMUNOLOGY, Third edition Paul (ed.) Raven Press, Ltd., New York and the references therein), AAV vector (as described in, for example. West et al. (1987) Virology 160:38-47, Kotin (1994) Human Gene Therapy 5:793-801, Muzyczka (1994) J. Clin. Invest. 94:1351, Tratschin et al. (1985) Mol. Cell. Biol. 5(11):3251-3260, U.S. Patent No. 4797368 and 5173414 and Publication of the International Application WO 93/24641), or an adenoviral vector (as described in, for example, Berns et al. (1995) Ann. NY Acad. Sci. 772:95-104; AIi et al. (1994) Gene Ther. 1:367-384; Haddada et al. (1995) Curr. Top. Environ. Immunol. 199 (Pt 3):297-306)to give immunosuppressive levels of expression of the NUS is einevoll acid, included in the vector, thereby causing the desired immunosuppressive response. Other acceptable types of viral vectors described in the present description (including alternative examples of acceptable retrovirus, AAV and adenoviral vectors).

Acceptable conditions of infection by these and other types of viral vector particles described in, for example, Bachrach et al., J. Virol, 74(18), 8480-6 (2000), Mackay et al., J. Virol, 19(2), 620-36 (1976), FIELDS VIROLOGY, see above. Additional procedures suitable for the production and use of viral vectors, are provided in, for example, "Practical Molecular Virology: Viral Vectors for Gene Expression" in METHODS IN MOLECULAR BIOLOGY, vol. 8, Collins, M. Ed., (Humana Press, 1991), VIRAL VECTORS: BASIC SCIENCE AND GENE THERAPY, 1st Ed. (Cid-Arregui et al., Eds.) (Eaton Publishing, 2000), "Viral Expression Vectors," in CURRENT TOPICS IN MICROBIOLOGY AND IMMUNOLOGY, Oldstone et al., Eds. (Springer-Verlag, NY, 1992), and "Viral Vectors" in CURRENT COMMUNICATIONS IN BIOTECHNOLOGY. Gluzman and Hughes, eds. (Cold Spring Harbor Laboratory Press, 1988).

Toxicity and therapeutic efficacy of vectors or viruses that include one or more molecules according to this invention, determined using standard pharmaceutical procedures in cell cultures or experimental animals. Can be defined BN50(the minimum lethal dose for 50% of the population) and/or the ED50(the dose therapeutically effective in 50% of the population) using procedures presented in this description and in other ways known in the Anna field of technology. See also S. Plotkin and W. Orenstein, VACCINES (W.. Saunders Co. 1999, 3rd ed.) to confirm doses for known viral vaccines. Nucleic acids, polypeptides, proteins, fused proteins, the transduced cells and other compositions according to the present invention may be imposed in the amount specified, for example, using BN50composition and side effects at various concentrations acceptable in relation to the mass and overall health of the patient. Thus, for example, the invention provides a method of inducing an immune response by introducing a dose equal to or greater than the ED50pharmaceutically acceptable compositions comprising a population of virus-like particles or viruses (e.g., weak or deficient in replication of the virus), which comprise the polypeptide or nucleic acid according to this invention. The introduction can be carried out through individual dose or divided doses (or joint introduction, serial introduction or their combinations. Methods of introduction and protocols described in, for example, Plotkin (VACCINES), see above, and other references cited in this description. In a related sense, the methods of assessing dose of the nucleic acid, polypeptide, vector and cellular compositions effective for the induction of immunity described in, for example, the Application for the European Patent No. 1156333 and what the media kit, quoted in it.

The viral vector may be directed to specific tissues, cells and/or organs of a subject such as a mammal. Examples of such vectors are described above. For example, the viral vector or the vector nucleic acid can be used for the selective delivery of a nucleic acid sequence according to this invention to monocytes, dendritic cells, and cells associated with dendritic cells (e.g. keratinocytes associated with Langerhans cells), T cells and/or b-cells. The viral vector can be deficient in replication of the viral vector. Particle viral vector can also be modified to reduce the immune response of the host to viral vector, thereby providing a persistent 919 gene expression. Such "hidden" vectors are described in, for example, Martin, Exp. Mol. Pathol. 66(1):3-7 (1999), Croyle et al., J. Virol. 75(10):4792-801 (2001), Rollins et al., Hum. Gene Ther. 7(5):619-26 (1996), Ikeda et al., J. Virol. 74(10):4765-75 (2000), Halbert et al., J. Virol. 74(3): 1524-32 (2000), and Published International Application no WO 98/40509. Alternative or additionally, the viral vector particles can be entered using the strategy chosen to reduce the immune response of the host to the vector particles. Strategies for reducing an immune response to the viral vector particle with the introduction of the owner provided in, for example, Maione et al., Proc. Natl. Acad. Sci. USA 98(11):5986-91 (2001), Morral t al., Proc. Natl. Acad. Sci. USA 96(22):2816-21 (1999), Pastore et al., Hum. Gene Ther. 10(11):1773-81 (1999), Morsy et al., Proc. Natl. Acad. Sci. USA 95(14):7866-71 (1998), Joos et al., Hum. Gene Ther. 7(13): 1555-66 (1996), Kass-Eisler et al., Gene Ther. 3(2):154-62 (1996), U.S. Patent No. 6093699, 6211160, 6225113, Application for U.S. patent No. 2001-A.

The skin and muscles in General are the preferred targets for the introduction of polypeptides, conjugates, nucleic acids and vectors of this invention, any appropriate method. Thus, delivery of the polypeptide, conjugate, nucleic acid or vector of this invention in or through the skin of a subject (e.g. a mammal) is a sign of the invention. Such molecules according to this invention can be introduced into a pharmaceutically acceptable solution for injection into or through the skin, such as intramuscularly or intraperitoneally. The introduction may also be transdermal devices or, more typically, balistically delivery of the polypeptide, conjugate, nucleic acid and/or vector to, in or through the skin of the subject or in the muscle of the subject. Transdermal device provided by the invention described herein, for example, can be applied on the skin to the owner within a reasonable period of time, so that there was sufficient transfer polynucleotide and/or vector to a subject, thereby suppressing immune response in a subject or inhibiting the rejection of transpl ntata tissue cells or tissue. Muscle introduction more often facilitated by the injection of a liquid solution comprising the polypeptide, polynucleotide or the vector according to this invention. Specific cells that can be target include dendritic cells, other agriculture, In cells, monocytes, T cells (including T-helper cells) and cells associated with the immune system cells (such as keratinocytes or other cells associated with Langerhans cells). The direction of the vectors and nucleic acids according to this invention described in this specification. This guided introduction can be nucleic acids or vectors comprising nucleic acid functionally associated with cell and/or tissue-specific promoters, examples of which are known in the technical field.

Polynucleotide according to this invention can be introduced any acceptable delivery system, so that was the expression of the recombinant polypeptide in the host, leading to suppression of the immune response, inhibition of an interaction between B7-positive cells and CD28-positive or inhibiting transplant rejection tissue, cell, organ or tissue or organ. For example, an effective amount of a population of bacterial cells, comprising a nucleic acid according to this invention can be introduced sub is KTU, leading to the expression of recombinant mutant polypeptide CTLA-4 according to this invention, and suppression of immune response in the subject. Bacterial cells developed for gene delivery mammal known in the field of engineering.

The introduction of polynucleotide or vector according to this invention, the subject is facilitated through the use of electroporation for the efficient number of cells or effective tissue orientation, so that the nucleic acid and/or vector of the captured cells and expressed in them, leading to the production of recombinant polypeptide according to this invention in these and subsequent suppression of the immune response in the subject.

METHODS PRODUCTION AND PURIFICATION

The invention also provides methods for creating and purification of polypeptides, nucleic acids, vectors and cells of this invention. In one aspect the invention provides a method of creating a recombinant polypeptide according to this invention by incorporating the nucleic acid according to this invention into a population of cells in the cellular environment, cultivation of cells in the environment (within the time and under conditions acceptable to the desired level of gene expression) to obtain the polypeptide and the selection of the polypeptide from the cells, the cellular environment, or both. Nucleic acid typically is functionally connected with the reg is Torno sequence, effective for expression of the polypeptide encoded by the nucleic acid.

The polypeptide can be isolated from cell lysates, cell supernatants and/or cellular environment is different acceptable methods known in the art, including, for example, various types of chromatography of cell lysates and/or cell supernatants. For example, the polypeptide can be isolated from cell lysates and/or cell environment by first concentrating the cellular environment using centrifugal filters (Amicon), alternatively, by precipitation of proteins with ammonium sulfate or polyethylene glycol, and then resuspended polypeptides in PBS or other acceptable buffers. The polypeptide can then be purified using either exclusion chromatography on Sephacryl S-400 column (Amersham Biosciences), as described in, for example, Hjorth, R. and J. Moreno-Lopez, J. Virol. Methods 5:151-158 (1982), or other affinity chromatography, or centrifugation in 20-60% sucrose gradients as described in, for example, Konish et al., Virology 188:714-720 (1992). Fractions containing the desired polypeptides can be identified using ELISA or SDS-PAGE with subsequent silver staining and Western blot turns. The required fractions are collected together and concentrated. Sucrose in gradient centrifugation fractions can be removed with the use of the gel is Oh filtration on PD-I0 column (Amersham Biosciences). Additional methods of purification include those described in the examples below and hydrophobic interaction chromatography (Diogo, M. M. et al., J. Gene Med. 3:577-584 (2001)), and any other acceptable methods known in the art.

Any acceptable method of purification, which is known in the art, may also be used. Methods of purification of the polypeptide, are known in the art include those described in, for example, Sandana (1997) BIOSEPARATION OF PROTEINS, Academic Press, Inc., Bollag et al. (1996) PROTEIN METHODS, 2ndEdition Wiley-Liss, NY, Walker (1996) THE PROTEIN PROTOCOLS HANDBOOK Humana Press, NJ, Harris and Angal (1990) PROTEIN PURIFICATION APPLICATIONS: A PRACTICAL APPROACH, IRL Press at Oxford, Oxford, England, Scopes (1993) PROTEIN PURIFICATION: PRINCIPLES AND PRACTICE 3rdEdition Springer Verlag, NY, Janson and Ryden (1998) PROTEIN PURIFICATION: PRINCIPLES, HIGH RESOLUTION METHODS AND APPLICATIONS, Second Edition Wiley-VCH, NY; and Walker (1998) PROTEIN PROTOCOLS ON CD-ROM Humana Press, NJ. Cells that are acceptable for production of the polypeptide, are known in the technical field and are discussed elsewhere in this description (e.g., Vero cells, 293 cells, BHK, Cho (e.g., Cho-K1), and COS cells can be acceptable). Cells can be lysed by any acceptable method, including, for example, sonication, microregiunea, physical separation, the French press lysis or lysis-based detergent.

In one aspect the invention provides a method for purification of the polypeptide according to this invention, which includes transforming the reception is emnd host cell with a nucleic acid according to this invention (for example, recombinant nucleic acid that encodes a recombinant polypeptide comprising amino acid sequence SEQ ID NO:1) cells of the host (for example, Cho cell or a 293 cell), lizirovania cells acceptable method of lysis (e.g., sonication, detergent lysis or other acceptable method) and exposure lysate affinity purification on a chromatographic column comprising a resin which contains at least one new antibody according to this invention (usually monoclonal antibody according to this invention) or antigennegative fragment, so the lysate becomes enriched with essential polypeptide (e.g. a polypeptide, comprising the amino acid sequence of SEQ ID NO:1).

In another aspect the invention provides a method for purification of such target polypeptide, where the method differs from the above-described method in that the nucleic acid comprising a nucleotide sequence encoding a protein that includes a polypeptide according to this invention (for example, SEQ ID NO:1) and an acceptable token (for example, e-epitope/his marker), and purification of the polypeptide improved techniques immunoaffinity, lentil-Lyudinovo affine core chromatography, affinity chromatography using immobilized metal (IMAC) or metal-chelating and what Finney chromatography (MSAS). Additional methods of purification described in this specification.

In another aspect the invention provides a method of producing the polypeptide according to this invention, where the method includes embedding into a population of cells a recombinant expression vector comprising a nucleic acid according to this invention, culturing the cells in the cellular environment at acceptable conditions sufficient for expression of the nucleic acid from the vector, and the production of the polypeptide encoded by the nucleic acid, and the secretion of the polypeptide from the cells, the cellular environment, or both. Selection of cells based on the desired processing of the polypeptide and is based on acceptable vector (for example, E. coli cells are preferred for bacterial plasmids, whereas 293 cells preferred for scrambled plasmid mammal and/or adenoviruses, especially E1-deficient adenovirus).

In another aspect, the invention includes a method of obtaining a polypeptide, where the method includes: (a) embedding in a population of cells a recombinant expression vector comprising at least one nucleic acid according to this invention, which encodes a polypeptide according to this invention; (b) introducing the expression vector into a mammal; and (C) isolation of the polypeptide from the mammal or from a by-product of a mammal.

polypeptide according to this invention can also be obtained by culturing cells or population of cells according to this invention (which, for example, have been transformed with a nucleic acid according to this invention, which encodes such a polypeptide) under conditions sufficient for expression of the polypeptide and recovering the polypeptide expressed in the cell or cell, using standard techniques known in the art.

In another aspect the invention provides a method of producing the polypeptide according to this invention, which includes (a) embedding in a population of cells a nucleic acid according to this invention, where the nucleic acid is functionally linked to a regulatory sequence effective to obtain the polypeptide encoded by the nucleic acid; (b) culturing the cells in the cellular environment to obtain the polypeptide; and (C) isolation of the polypeptide from the cells or the cell environment. Also included in this invention cultivated cell, which embed the vector according to this invention (for example, the expression vector according to this invention).

Also includes the method of producing the polypeptide according to this invention, which includes embedding a nucleic acid that encodes the specified polypeptide in the cell population in an environment where cells can be the expression of the nucleic acid content of cells under conditions in which the nucleic acid is expressed, and then the separation is of the polypeptide of the environment.

In another aspect the invention provides a method of creating a fused protein. The method includes: (1) culturing a host cell transformed with nucleic acid in the cell environment, where the nucleic acid comprises: (i) a first nucleotide sequence that encodes a polypeptide having at least 95% identity with the amino acid sequence selected from the group comprising SEQ ID NOS:1-73, where the polypeptide binds CD86 and/or CD80, and/or the extracellular domain of each CD86 or CD80, and (ii) a second nucleotide sequence encoding a polypeptide Ig Fc, including the hinge, CH2 domain and CH3 domain, the resulting nucleic acid is expressed and forms a fused protein; and (2) the restoration of the fused protein. Any Ig Fc polypeptide may be used, including for example, IgG1 Fc, IgG2 Fc, IgG4 Fc or mutant Ig Fc polypeptide. In some such methods, the nucleic acid also comprises a third nucleotide sequence that encodes or secretory signal peptide, the functionally associated with the merged protein, and protein is secreted from the host cells as a disulfide-linked dimer fused protein comprising identical first and second fused proteins, disulfide-linked dimer fused protein recovered from the cell environment. In some such methods disulfide tie the config dimer fused protein formed by covalent disulfide bond between the cysteine residue of the first fused protein and the cysteine residue of the second fused protein. In some such methods protein recovered from the cell environment, host cell or periplasm host cell.

In another aspect the invention provides a molecule isolated or recombinant nucleic acid comprising a nucleotide sequence that encodes (i) a first polypeptide containing an amino acid sequence having at least 95% sequence identity with at least one amino acid sequence selected from the group comprising SEQ ID NOS:1-73, where the first polypeptide binds CD80 and/or CD86 and/or the extracellular domain of each or both, and (ii) a second polypeptide comprising a hinge, CH2 domain and CH3 domain of an IgG polypeptide. The second polypeptide can include any acceptable Ig polypeptide discussed herein, including, for example, one that contains the amino acid sequence of SEQ ID NO:184, or SEQ ID NO:218.

In another aspect the invention provides a method of forming a soluble dimer fused protein. The method includes culturing the host cell transformed with an expression vector comprising a nucleotide sequence that encodes a soluble dimer fused protein according to this invention. Illustrative fused proteins include those that contain amino acid sequence of any of SEQ ID nos:74-79, 197-200, 205-214, and 219-222. The vector includes a nucleotide sequence that facilitates expression of the fused protein (for example, the nucleotide sequence encoding a signal peptide). Protein is secreted from the host cells as a disulfide-linked dimer fused protein consisting of two identical fused protein, and a disulfide-linked dimer fused protein recovered from the cell environment. In some such methods, a disulfide-linked dimer fused protein formed by covalent disulfide bond between a cysteine residue on each fused protein. Dimer fused protein usually recover from the cellular environment, host cell or periplasm host cell. Example 12 provides an illustrative procedure for the formation of stable transfectional cell lines expressing mutant CTLA4-Ig in this invention, the formation of mutant fused protein CTLA4-Ig fused protein and purification of the mutant fused protein from the culture.

In addition to recombinant production of the polypeptides according to this invention can be obtained by direct peptide synthesis using solid-phase techniques (see, for example, Stewart et al. (1969) SOLID-PHASE PEPTIDE SYNTHESIS, W.H. Freeman Co., San Francisco and Merrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154). Peptide synthesis can be carried out using techniques carried out manually or ed is automatically. Automated synthesis may be achieved, for example, using Applied Biosystems 43 IA peptide synthesizer (Perkin Elmer, Foster City, Calif.) in accordance with the manufacturer's instructions. For example, the subsequence may be chemically synthesized separately and combined using chemical methods to obtain the polypeptide of this invention or its fragments. Alternatively, synthesized polypeptides can be obtained from numerous companies specializing in the production of polypeptides. Most frequently, the polypeptides according to this invention is obtained by ekspressirovali encoding nucleic acid and recovering the polypeptide, for example, as described above.

The invention includes a method of producing the polypeptide according to this invention, including the incorporation of nucleic acid according to this invention, the vector according to this invention, or combinations thereof, in an animal such as a mammal (including, for example, rat, non-human Primate, bat, monkey, pig or chicken), so that the polypeptide according to this invention expressively in the animal, and the polypeptide isolated from the animal product or by-product of the animal. The secretion of the polypeptide from the animal product or by-product of the animal can be made by any acceptable technique dependent in the tee from the animal and the desired recovery strategy. For example, the polypeptide can be recovered from the sera of mice, monkeys or pigs expressing the polypeptide according to this invention. Transgenic animals (including the aforementioned mammals), including at least one nucleic acid according to this invention, are included in this invention. The transgenic animal may have a nucleic acid that is integrated into the genome of its host (for example, using the AAV vector, lentiviruses vector biolistics techniques, carried out with sequences that stimulate integration and the like) or may have a nucleic acid which contains epigramme (for example, in reintegravano plasmid vector or by insertion in at viral vector). Pyromania vectors can be designed for shorter gene expression than the integrated vectors. Vectors based on RNA often especially preferred in this regard.

COMPOSITION

The invention also provides new and useful compositions comprising at least one component according to this invention, such as, for example, at least one polypeptide (including, for example, fused proteins and multimeric polypeptide, conjugate, nucleic acid, vector, virus, virus-like particle (HPV) and/or cell of this invention or the any combination of the medium filler or solvent. Carrier, filler or solvent may be pharmaceutically acceptable carrier, excipient or solvent. Such a composition may include any reasonable amount of any reasonable number of polypeptides, conjugates, nucleic acids, vectors, viruses, HPV and/or cells according to this invention. Also provided pharmaceutical compositions comprising at least one polypeptide, conjugate, nucleic acid, vector, virus, HPV and/or cell, or any combination thereof, and a pharmaceutically acceptable carrier, excipient or solvent. Such compositions are suitable for the methods according to this invention described herein, including, for example, methods of suppressing immune responses.

For example, in one non-limiting embodiment, the invention provides a composition including a filler, a solvent or carrier and at least one polypeptide according to this invention (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more polypeptides), such as a mutant polypeptide, CTLA-4 EVA (e.g., any of SEQ ID NOS:1-73) or mutant protein, CTLA-4-Ig (e.g., any of SEQ ID NOS:74-79, 197-200, 205-214, and 219-222), where at least one polypeptide is present in the composition in amounts effective to suppress the immune response, including, for example, immune(s) answer(s), uvlechennyi(s) in graft rejection and/or autoimmunity, to inhibit rejection of the transplanted graft tissue, cell or organ or to inhibit the interaction of endogenous B7-positive cells with CD28-positive T-cells in a subject who is administered a composition.

Also included in this invention is a pharmaceutical composition comprising a pharmaceutically acceptable excipient, solvent or carrier and an effective amount of one or more of such components according to this invention. The effective amount may be therapeutically or prophylactically effective amount or dose for use in therapeutic or prophylactic method described here, such as the method of treating an autoimmune disease or a method of inhibiting transplant rejection tissues, cells, tissue or organ from a donor subject to the recipient.

The composition (or pharmaceutical composition) can be any non-toxic composition, which has no effect on the immunosuppressive properties of the polypeptide, conjugate, nucleic acid, vector, virus, HPV or cells of this invention that it contains. The composition may include one or more fillers, solvents or carriers, and pharmaceutical composition comprises one or more pharmaceutically acceptable fillers, solvents, Il the media. Wide range of available carriers, solvents and fillers known in the technical field and can be included in compositions and pharmaceutical compositions according to this invention. For example, various aqueous media can be used, for example, distilled or purified water, sterile saline, buffered saline, such as phosphate-buffered saline (PBS), and the like are preferred for compositions for injection of the polypeptide, fused protein, conjugate, nucleic acid, vector, virus, HPV and/or cells of this invention. Numerous acceptable excipients, carriers and solvents for injection of therapeutic proteins are known in the art. Such solutions are mostly sterile and generally free of undesirable matter. The composition can be sterilized by conventional, well known sterilization techniques. The compositions of this invention can include pharmaceutically acceptable excipients, if necessary, to maintain physiological conditions. Such substances include, for example, agents for regulating the pH, buferiruemoi agents and agents for regulating toychest, including, for example, sodium acetate, sodium ascorbate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. To the objection to the invention, including pharmaceutical compositions may also include one or more components, such as solvents, fillers, salts, buffers, surfactants, amplificatory, detergents (e.g., non-ionic detergent or emulsifier such as Tween®-20, Tween®-40, Tween®-60, Tween®-80, plutonomy F-68 and the like), stabilizers (for example, sugar or protein-free amino acids), preservatives, tissue fixatives, solvents and/or other materials acceptable for inclusion in a pharmaceutical composition.

Examples of acceptable components, which can be used in the pharmaceutical compositions described in, for example, Berge et al., J. Pharm. Sci. 66(1): 1-19 (1977), Wang and Hanson, J. Parentheral. Sci. Tech. 42:S4-S6 (1988), U.S. Patent No. 6165779 and 6225289 and in this description. Pharmaceutical compositions may also include preservatives (such as benzyl alcohol, sodium azide, t-cresol and the like), antioxidants, metal chelators (such as methionine, EDTA and the like) and/or other excipients known to specialists in this field. Examples of appropriate pharmaceutically acceptable carriers for use in the pharmaceutical compositions described in, for example, Urquhart et al., Lancet 16:367 (1980), Lieberman et al., PHARMACEUTICAL DOSAGE FORMS - DISPERSE SYSTEMS (2nd ed., Vol.3, 1998), Ansel et al., PHARMACEUTICAL DOSAGE FORMS &DRUG DELIVERY SYSTEMS (7th ed. 2000), Martindale, THE EXTRA PHARMACOPEIA (31st edition), Remington''s PHARMACEUTICAL SCIENCES (16th-20th editions), THE PHRMACOLOGICAL BASIS OF THERAPEUTICS, Goodman and Gilman, Eds. (9th ed. - 1996), WILSON AND GISVOLDS TEXTBOOK OF ORGANIC MEDICINAL AND PHARMACEUTICAL CHEMISTRY, Delgado and Remers, Eds. (10th ed. - 1998) and U.S. Patent No. 5708025 and 5994106. Principles of formulating pharmaceutically acceptable compositions are described in, for example, Platt, Clin. Lab Med. 7:289-99 (1987), Aulton, PHARMACEUTICS: the SCIENCE OF DOSAGE FORM DESIGN, Churchill Livingstone (New York) (1988), EXTEMPORANEOUS ORAL LIQUID DOSAGE PREPARATIONS, CSHP (1998), and "Drug Dosage," J. Kans. Med. Soc. 70(1):30-32 (1969). Additional pharmaceutically acceptable carriers, especially acceptable for the introduction of vectors, described in, for example, published international application WO 98/32859.

The compositions of this invention, including pharmaceutical compositions can include one or more aqueous carriers or excipients (including, for example, pharmaceutically acceptable carriers or excipients and one or more components, such as one or more buffers, one or more salts, one or more detergents or emulsification and/or one or more sugars. The buffer system is generally acceptable to maintain the pH of the composition in the range, which contributes to the stability of the molecule according to this invention, which is present in the composition (e.g., mutant CTLA-4-Ig). Illustrative buffers for use in the composition include, but are not limited to the following, for example, the buffer N-2-hydroxyethylpiperazine-N'-2-aminoethane sulfonic acid (HEPES), citrate buffer (n is an example, a mixture of potassium, sodium and trinitrate sodium, a mixture of sodium citrate and citric acid, a mixture of citric acid and trinitrate sodium, a mixture of Mononitrate sodium and potassium sodium, a mixture of citric acid and Mononitrate sodium), sodium phosphate buffer (for example, a mixture diphosphate and sodium triphosphate sodium (Na2HPO4/Na3PO4), a mixture of dibasic sodium phosphate and monobasic phosphate), acetate buffer (for example, a mixture of acetic acid and sodium acetate, the mixture of acetic acid and sodium hydroxide), his-tag buffer, Tris buffer, Tris-Malinovsky buffer, alternately buffer (for example, a mixture of succinic acid and sodium hydroxide, a mixture of succinic acid and monosaccate sodium, a mixture of succinic acid and disuccinate sodium, a mixture of mononucleate sodium and disuccinate sodium), Malinovsky buffer, imidazole buffer, tartrate buffer, fumaric buffer, gluconate buffer, oxalate buffer, lactate buffer, acetate buffer and the like, or a combination of any of them (for example, a cocktail of citrate and acetate buffers, and the like). The concentration of buffer in the composition may be any that is acceptable for molecules(molecules) according to this invention (e.g., mutant CTLA-4-Ig), is included in the solution composition, such as, but not limited to the following, for example, in the range from about 1 mm to about 00 mm about 1 mm to about 50 mm, about 5 mm to about 50 mm or about 5 mm to about 25 mm, including, for example, 1 mm, 5 mm, 10 mm, 15 mm, 20 mm, 25 mm, 30 mm, 40 mm, 50 mm, such as, for example, 20 mm HEPES buffer, 20 mm potassium sodium-trinary-citrate buffer, 20 mm alternately buffer and the like).

Illustrative salts for use in the composition include, but are not limited to the following, for example, water-soluble salts, including organic salt or inorganic salt (for example, water-soluble inorganic salt such as sodium chloride, magnesium chloride, sodium bicarbonate, potassium chloride, calcium chloride and ammonium chloride and the like, or any pharmaceutically acceptable or physiologically compatible salt. Illustrative of the salt concentration in the solution compositions include, but are not limited to the following, for example, in the range from about 1 mm to about 150 mm, about 10 mm to about 125 mm, or about 75 mm to about 125 mm, including, for example, 10 mm, 50 mm, 75 mm, 100 mm, 125 mm, 150 mm (such as, for example, 100 mm NaCl).

Illustrative of sugar or hydrocarbons for use in the composition include, but are not limited to the following, for example, sucrose, maltose, trehalose, dextrose, mannose, raffinose, lactose, maltodextrin, dextran, sucrose and the like, in a concentration range including, but not limited to the following, for example, from about 0.1% to about 10% by weight sugar, about 1% of d is about 5% by weight of sugar or about 1% to about 3% by weight of sugar, including for example, 0,1%, 0,5%, 1%, 1,5%, 2%, 2,5%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% by weight of sugar (for example, 2% by weight of sucrose, 2% by weight of trehalose or 2% by weight mannose) by weight of the composition. Illustrative sugar alcohols for use in the composition include, but are not limited to the following, for example, mannitol, sorbitol, glycol, glycerol, arabitol, Eritrea, xylitol, ribitol, lactitol and the like in a concentration range, including, but not limited to the following, for example, from about 0.1% to about 10% by weight of a sugar alcohol, about 1-5%, about 1-3%, including for example, 0,1%, 0,5%, 1%, 1,5%, 2%, 2,5%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% by weight of a sugar alcohol by weight of the composition.

The osmolarity of the compositions according to this invention, including pharmaceutical compositions, generally similar to the osmolarity of the blood serum, which is in the range from about 250 to about 350 milliosmoles per kilogram (mOsm/kg) of water. The salt concentration in the composition is typically less than 125 mm. The concentrations of salt and sugar can be adjusted or modified so that the osmolarity of the composition ranged from about 250-350 mOsm/kg of water.

Illustrative detergents or amplificatory for use in the composition include, but are not limited to the following, for example, Polysorbate, such as Tween®-20, Tween®-40, Tween®-60, Tween®-80, or plutonomy F-68 in the range, including but not limited to following the m, for example, from about 0,001% to about 0.2% by weight of the detergent or emulsifier by weight of the composition, including, for example, 0,001%, 0,002%, 0,003%, 0,004%, 0,005%, 0,006%, 0,007%, 0,008%, 0,009%, 0,01%, 0,02%, 0,03%, 0,04%, 0,05%, 0,075% and 0.1% by weight of the detergent or emulsifier (for example, Tween®-20, Tween®-40, Tween®-60, Tween®-80, or plutonomy F-68) weight of the composition.

The compositions of this invention, including pharmaceutical compositions, can include a polymer, such as PEG molecule in a concentration sufficient to reduce or inhibit unwanted Association between two or more molecules according to this invention, such as, for example, two or more mutant dimers fused protein, CTLA-4-Ig in this invention. The composition may include two or more different polymers (e.g., PEG). The polymer (for example, the PEG molecule) usually has a molecular weight from about 200 Da to about 8000 Da (for example, about 200, 300, 400, 600, 900, 1000, 1450, 3350, 4500 or 8000 Yes, available from Dow Chemical). I think that adding polymer (such as PEG molecules) to the composition reduces the formation of unwanted aggregates, especially unwanted aggregates two or more dimers fused protein according to this invention.

The compositions of this invention, including pharmaceutical compositions may include a cyclic oligosaccharide, such as a cyclodextrin (e.g., Captisol® (Cydex)). In od the second aspect, the composition includes two or more different cyclic oligosaccharides. Adding cyclic(them) of the oligosaccharide(s) to the composition improves the solubility, stability, bioavailability and/or the dosage of the active pharmaceutical ingredient(s) (e.g., mutant molecules CTLA-4).

the pH of the compositions of this invention, including pharmaceutical composition may be in the range of from about pH 3 to about pH 10, about pH 4 to about pH 10, about pH 5 to about pH 9, from about pH 6 to about pH 9, from about pH 5.5 to about pH 8.5, from about pH 6.0 to about pH 6,7, from about pH 6.0 to about pH 6.5, about pH 6.2 to about pH 8,7, from about pH 6.5 to about pH 8.5, from about pH 6.5 to about pH 7.5, about pH 6.2 to about pH 7.0, about pH 6.3 to about pH 6.8, from about pH 6.4 to about pH 6.8, from about pH 7.0 to about pH 8.0, about pH 7.0 to about pH 7.4. In one aspect a composition comprising a molecule according to this invention, such as, e.g., mutant CTLA-4-IgG2, have a pH level of pH 5.0, pH of 5.1, a pH of 5.2, pH of 5.3, the pH of 5.4, and 5.5, pH of 5.6, the pH of 5.7, a pH of 5.8, pH of 5.9, pH 6.0, pH of 6.1, pH of 6.2, pH 6.3, pH 6.4, pH 6.5, pH 6.6, pH of 6.7, pH 6.8, pH 6.9, pH 7.0, pH of 7.1, pH of 7.2, pH of 7.3, pH 7.4, pH 7.5, pH of 7.6, the pH of 7.7, pH of 7.8, pH 7,9, pH 8.0, pH 8.1, pH of 8.2, pH 8.3, pH of 8.4, pH 8.5, pH of 8.6, a pH of 8.7, pH 8.8, pH of 8.9, pH of 9.0, the pH of 9.1, pH of 9.2, the pH of 9.3, a pH of 9.4, a pH of 9.5, the pH of 9.6, a pH of 9.7, the pH of 9.8, pH, or pH of 9.9 and 10.0.

In one aspect the invention provides a composition according to this invention, comprising a filler or carrier (including, for example, the pharmaceutical composition, including the expansion of its pharmaceutically acceptable excipient or carrier and an effective amount of any of the polypeptide, CTLA-4, multimer, dimer, conjugate, fused protein or a dimer fused protein according to this invention, described elsewhere in this description, and also includes a buffer capable of maintaining the pH of the composition in the range from about pH 3 to about pH 10, water, optional non-ionic detergent, optional salt and optionally a sugar alcohol, a monosaccharide, disaccharide or polysaccharide. Some such compositions are at physiological pH. Some such compositions have a pH from about 4 to about 7.5, about 5.0 to about 7.5, or from about 6.4 to about 6.6, including, for example, about pH 6.5, about pH 7.4 or pH 7.5. Some such compositions include a buffer in a concentration of from about 1 mm to about 100 mm, about 1 mm to about 50 mm, about 5 mm to about 35 mm, about 10 mm to about 25 mm, including, for example, about 20 mm, 25 mm or 30 mm. Some such compositions include a buffer selected from the group comprising HEPES buffer, citrate buffer, alternately buffer, acetate buffer, citrate buffer, Malinovsky buffer, phosphate buffer and Tris buffer. Some such compositions include a buffer selected from the group comprising HEPES buffer, sodium-citrate buffer and sodium alternately buffer. Some such compositions, the pH is from about 6.0 to about 6.7 and the buffer is sodium succinate or sodium citrate. Some such compositions, the pH is about 7.0 to about 7.7 and the Ufer - it HEPES. Some such compositions also include a sugar alcohol or a saccharide, where the saccharide is a monosaccharide, a disaccharide (e.g. sucrose or trehalose), or polysaccharide. Some such compositions include salt present in a concentration from about 1 mm to about 50 mm, including, for example, about 20 mm, 25 mm or 30 mm. Some such compositions include non-ionic detergent, such as, for example, non-ionic detergent selected from the group consisting from the group consisting of Tween®-80, Tween®-60, Tween®-40, Tween®-20 or plurality F-68.

In some such compositions (including pharmaceutical compositions)described in the paragraph above, the polypeptide of multimer, dimer, the conjugate protein or a dimer fused protein is present in a concentration in the range of about 1 mg/ml (weight/volume) to about 200 mg/ml (weight/volume), about 25 mg/ml (weight/volume) to about 100 mg/ml (weight/volume), about 50 mg/ml to about 300 mg/ml, optionally in the range from about 50 mg/ml (weight/volume) to about 100 mg/ml (weight/volume). Some such compositions include an effective amount of the polypeptide, multimer, dimer, conjugate, fused protein or fused dimer of the protein from about 0.1 mg/kg to about 15 mg/kg, and the composition is administered to a mammal (e.g. human). Some such compositions include an effective amount of the polypeptide, multimer, dimer, conjugate, fused protein or a dimer SL is that protein from about 0.5 mg/kg to about 10 mg/kg, and the composition is administered parenterally. Some such compositions include an effective amount of the polypeptide, multimer, dimer, conjugate, fused protein or fused dimer of the protein from about 0.1 mg/kg to about 5 mg/kg, and optionally about 0.5 mg/kg, and the composition is administered subcutaneously. Some such compositions include an effective amount of the polypeptide, multimer, dimer, conjugate, fused protein or fused dimer of the protein from about 5 mg/kg to about 15 mg/kg (not necessarily about 10 mg/kg), and the composition is injected. Some such compositions polypeptide, multimer, dimer, the conjugate protein or a dimer fused protein includes an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the amino acid sequence SEQ ID NO:36. Some such compositions polypeptide, multimer, dimer, the conjugate protein or a dimer fused protein includes an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the amino acid sequence SEQ ID NO:50. Some such compositions are sterile and/or isotonic with blood. Some such compositions are liquids which their compositions. Some such compositions are in liquid or dried form, where the dried form selected from the group including lyophilized form, shape, air-dried, and the form spray dried.

Illustrative aspect of the invention provides a pharmaceutical composition comprising: (i) protein, CTLA-4-Ig in this invention, having a concentration from about 1 mg/ml to about 300 mg/ml (e.g., about 1 mg/ml to about 100 mg/ml, about 50 mg/ml or about 100 mg/ml etc) (optional dimeric protein); (ii) a buffer having a buffer capacity of about pH 5.0 to about pH 9.0 in a concentration from about 5 mm to about 50 mm; (iii) a pharmaceutically acceptable solvent to give a composition indicated volume; (iv) sugar in a concentration of from about 0.5% to about 10% by weight of sugar by weight of the composition; (v) salt concentration from about 1 mm to about 200 mm; (vi) optional non-ionic detergent (such as Tween®-20, Tween®-40, Tween®-60, Tween®-80, or plutonomy F-68) at a concentration of from about 0.01 mg/ml to about 0.5 mg/ml, for example, about 0.01 mg/ml up to about 0.1 mg/ml; and (vii) optionally cyclic oligosaccharide (e.g., cyclodextrin (Captisol®), where the pH of the composition is in the range from about pH 5.0 to about pH 8.0. Illustrative protein, CTLA-4-Ig in this invention include those that comprise amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with at least one amino acid sequence selected from the group comprising SEQ ID NOS:74-79, 197-200, 205-214, and 219-222 (optional, for example, selected from the group comprising SEQ ID NOS:197, 199, 211 and 213), where the protein binds CD80 and/or CD86 and/or its extracellular domain and/or suppresses the immune answer. Such fused proteins can be in Monomeric or dimeric form.

In another aspect the invention provides a pharmaceutical composition comprising: (i) a conjugate comprising a protein, CTLA-4-Ig on this invention (optional dimeric protein) and polipeptidnoi part, covalently attached to the fused protein, where specified, the conjugate has a concentration from about 1 mg/ml to about 300 mg/ml (e.g., about 1 mg/ml to about 100 mg/ml, about 50 mg/ml or about 100 mg/ml and the like); (ii) a buffer having a buffer capacity of from about pH 5.0 to about pH 8.0 at a concentration of from about 5 mm to about 50 mm; (iii) pharmaceutically acceptable solvent to give the composition a certain amount; (iv) sugar at a concentration of about 0.5% to about 10% by weight of sugar by weight of the composition; (v) salt concentration from about 1 mm to about 200 mm; and (vi) optional non-ionic detergent (such as Tween®- 20, Tween®-40, Tween®-60, Tween®-80, or plutonomy F-68) at a concentration of from about 0.01 mg/ml to about 0.5 mg/ml, e.g. the R is about 0.01 mg/ml to about 0.1 mg/ml, where the pH of the composition is in the range from about pH 5.0 to about pH 8.0. The conjugate may include one, two, three, four or more polipeptidnyh parts. Each polipeptidna part may include a polymer (e.g., PEG or RW) or of the sugar. In some cases polipeptidna part is a polymer molecule such as PEG molecule. Polymer molecule may have any desired molecular weight depending on the desired functional effect (e.g., increased half-life, reduced the Association between molecules fused protein and the like). In some cases, for example, the polymer is PEG having a molecular weight from about 1 kDa to about 100 kDa (e.g., 1, 2, 2,5, 3, 5, 8, 10, 12, 20, 25, 30, 40, 60 kDa and the like). Polipeptidna part (for example, sugar or polymer molecule) covalently attached to the group of attachment of amino acid residue fused protein using standard procedures, as described above. Illustrative fused proteins, CTLA-4-Ig include those that include an amino acid sequence having at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with at least one amino acid consequently the stew, selected from the group comprising SEQ ID NOS:74-79, 197-200, 205-214, and 219-222 (optional, for example, selected from the group comprising SEQ ID NOS:197, 199, 211 and 213), where the protein binds CD80 and/or CD86 and/or its extracellular domain and/or suppresses an immune response. Such fused proteins can be in Monomeric or dimeric form.

In another aspect the invention provides a pharmaceutical composition comprising: (a) a polypeptide comprising amino acid sequence having at least about 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence selected from the group of SEQ ID NOS:1-73, such as, for example, SEQ ID NOS:36 and 50, where the specified polypeptide is present in a concentration range from about 1 to about 200 mg/ml (weight/volume); (b) a buffer, having a buffer capacity of about pH 5.0 to about pH 8.0 to a concentration range from about 5 mm to about 50 mm; (C) pharmaceutically acceptable solvent to give the composition a certain amount; (d) a sugar at a concentration of from 0.5% to 10% by weight; (e) salt in a concentration from about 1 mm to about 200 mm; and (f) optional detergent, where the pH is in the range from about pH 5.0 to about pH 8.0. In certain of such pharmaceutical compositions (a) the polypeptide comprises the amino acid sequence of SEQ ID NO:36, present in a concentration range from about 50 mg/ml to about 100 mg/ml; (b) a buffer which is HEPES buffer, present in a concentration from about 20 mm; (C) pharmaceutically acceptable solvent is a water; (d) sugar is a sucrose or trehalose at a concentration of 2% by weight; (e) salt is sodium chloride at a concentration of about 100 mm; and (f) optionally a detergent selected from the group including Tween®-80, Tween®-60, Tween®-40, Tween®-20 or plurality F-68 concentration less than or equal to about 0.1 mg/ml, where the pH of the composition is about pH 7.4. Other such compositions (a) the polypeptide comprises the amino acid sequence of SEQ NO:50, present in a concentration range from about 50 mg/ml to about 100 mg/ml; (b) the buffer is a sodium citrate buffer present at a concentration of about 20 mm; (C) pharmaceutically acceptable solvent is a water; (d) sugar is a sucrose or trehalose at a concentration of 2% by weight; (e) the salt is sodium chloride at a concentration of about 100 mm; and (f) optionally a detergent selected from groups containing Tween®-80, Tween®-60, Tween®-40, Tween®-20 or plurality F-68 concentration less than or equal to about 0.1 mg/ml, where the pH is about pH 6.5.

In another aspect the invention provides a pharmaceutical composition comprising: (a) a polypeptide comprising amino acid sequence having at least 96%, at least 9%, at least 98% or at least 99% sequence identity with the amino acid sequence SEQ ID NO:36; and (b) HEPES or sodium citrate buffer (for example, a mixture of potassium, sodium and trinitrate sodium, a mixture of sodium citrate and citric acid, a mixture of citric acid and trinitrate sodium, a mixture of Mononitrate sodium and potassium, sodium or a mixture of citric acid and Mononitrate sodium).

Also provides a pharmaceutical composition comprising: (a) a polypeptide comprising amino acid sequence having at least 97%, at least 98% or at least 99% sequence identity with the amino acid sequence SEQ ID NO:36; and (b) a pharmaceutically acceptable excipient or pharmaceutically acceptable carrier, where the composition has a pH from about 7 to about 8. Some of these pharmaceutical compositions have a pH of about 7.4 or 7.5. Some of these pharmaceutical compositions include HEPES or sodium citrate buffer.

Also provides a pharmaceutical composition comprising: (a) a polypeptide comprising amino acid sequence having at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the amino acid sequence SEQ ID NO:50; and (b) sodium-citrate buffer (for example, a mixture of potassium is sodium and trinitrate sodium, a mixture of sodium citrate and citric acid, a mixture of citric acid and trinitrate sodium, a mixture of Mononitrate sodium and potassium, sodium or a mixture of citric acid and Mononitrate sodium).

Also provides a pharmaceutical composition comprising: (a) a polypeptide comprising amino acid sequence having at least 97%, at least 98% or at least 99% sequence identity with the amino acid sequence SEQ ID NO:50; and (b) a pharmaceutically acceptable excipient or pharmaceutically acceptable carrier, where the composition has a pH from about 6 to about 7. Some of these pharmaceutical compositions have a pH around 6.5. Some of these pharmaceutical compositions include sodium citrate buffer.

In one illustrative aspect of the invention provides a pharmaceutical composition comprising from about 1 mg/ml to about 300 mg/ml fused protein, CTLA-4-Ig on this invention (e.g., D3-54-IgG2) (for example, from about 1 mg/ml to about 100 mg/ml, for example about 50 mg/ml or about 100 mg/ml), which typically is expressed as a dimeric protein in 20 mm HEPES buffer in water, 100 mm NaCl, 2% by weight of sucrose by weight of the composition, pH 7.4, including optional non-ionic detergent (such as Tween®-20, Tween®-40, Tween®-60, Tween®-80, or plutonomy F-68) at a concentration of from about 0.01 mg/ml to about 0.5 mg/ml, for example the eye is about 0.01 mg/ml to about 0.1 mg/ml, and optionally including polyethylene glycol (PEG)such as PEG molecule having a molecular weight from about 200 daltons (Da) to about 8000 Da (for example, about 200, 300, 400, 600, 900, 1000, 1450, 3350, 4500 or 8000 Yes, available from Dow Chemical). In another illustrative aspect of the invention provides a pharmaceutical composition comprising from about 1 mg/ml to about 300 mg/ml fused protein, CTLA-4-Ig on this invention (e.g., D3-69-IgG2), which typically is expressed as a dimeric protein in 20 mm sodium citrate buffer in water, 100 mm NaCl, 2% by weight of sucrose by weight of the composition, pH 6.5, including optional non-ionic detergent (such as Tween®-20, Tween®-40, Tween®-60, Tween®-80, or plurality F-68) at a concentration of from about 0.01 mg/ml to about 0.5 mg/ml, for example about 0.01 mg/ml to about 0.1 mg/ml, and optionally including a PEG molecule, such as PEG molecule having a molecular weight from about 200 Da to about 8000 Da (for example, about 200, 300, 400, 600, 900, 1000, 1450, 3350, 4500 or 8000 Yes, available from Dow Chemical).

The invention includes capacity for compositions according to this invention, comprising a molecule according to this invention (e.g., mutant molecule, CTLA-4, such as a mutant CTLA-4-Ig) and a filler, a solvent or carrier. The composition may be a pharmaceutical composition comprising a molecule according to this invention and a pharmaceutically acceptable excipient, solvent is or the media. Containers include, but are not limited to the following, for example, ampules (for example, glass ampoule, such as a glass vial (type 1), autoinjector, syringes handle (fixed dose or variable dose) and pre-filled syringes or other acceptable containers. If necessary, the container may contain one or more pre-defined doses molecules according to this invention, which are effective for suppressing the immune response or treatment of immune diseases or disorders as described herein. Some such containers suitable for administration of the compositions contained therein, to a subject suffering from immune diseases or disorders (e.g., autoinjector, syringes, pens, pre-filled syringes and the like). Some of these containers allow you to make a self-introduction of the composition by the subject (for example, syringes, pens, autoinjector, pre-filled syringes and the like).

It also provides stable compositions or formulations of the molecule (e.g., mutant CTLA-4 EVA or mutant CTLA-4-Ig) according to this invention, including the pharmaceutically acceptable compositions of the molecules according to this invention with a pharmaceutically acceptable carrier. In another aspect, the invention includes dried by sublimation or freeze-dried com is osili or formulations. The expression "dried by sublimation" or "dried" in General refers to the state of matter, which was subjected to the procedure of drying, such as drying by sublimation or lyophilization, where at least 50% humidity is eliminated. Procedures reliabilitiy and freeze-drying are well known in the art (see, for example, LYOPHILIZATION OF BIOPHARMACEUTICALS, Vol.2 BIOTECHNOLOGY: PHARMACEUTICAL ASPECTS (Henry R. Costantino et al. eds., 2004), U.S. Patent No. 6436897, a Publication of the international application WO 06/104852) and will be understood by a person skilled in the art. Any acceptable procedure lyophilization can be used or modified to be acceptable, a specialist in the art in the preparation of freeze-dried compositions according to this invention. Dried by sublimation, air-dried, spray dried or freeze-dried composition is usually prepared from the liquid such as solution, suspension, emulsion or other Fluid that is subject to drying by sublimation, drying in air, spray drying or lyophilization, usually includes all the components (except liquid such as water)that will be present in the final recovered liquid composition. Thus, dried by sublimation, air-dried, spray dried or freeze-dried composition will have chelation the local composition of the liquid composition (e.g., pharmaceutical compositions) during recovery. Illustrative compositions according to this invention, including pharmaceutical compositions, comprising a molecule according to this invention (for example, a mutant protein, CTLA-4-Ig in this invention, such as, for example, SEQ ID NO:197, 199, 211 or 213), which are described in this application can be dried by sublimation, air-dried, spray dried or freeze-dried to obtain a stable dried by sublimation, air-dried, spray dried or freeze-dried compositions, respectively, using standard procedures known in the art. See, for example, the illustrative procedure described in LYOPHILIZATION OF BIOPHARMACEUTICALS, see above.

For example, the capacity (e.g., ampoule, such as a glass ampoule containing liquid composition of the molecules of this invention (e.g., mutant CTLA-4 - Ig protein according to this invention, such as, for example, SEQ ID NO:197, 199, 211 or 213), which is then subjected to freeze-drying, can be dried using standard procedures known to specialists in this field of technology. See, for example, LYOPHILIZATION OF BIOPHARMACEUTICALS, see above. Dried molecule according to this invention (e.g., mutant CTLA-4-Ig in this invention) can be consistently recovered liquid is awn to obtain the recovered liquid composition. Lyophilized formulations are usually recover by adding an aqueous solution to dissolve the lyophilized formulations. Any acceptable aqueous liquid or solution can be used for recovery of freeze-dried formulations. Lyophilized formulations are often restored with the use of sterile or distilled water, but solutions, including carriers, fillers, solvents, buffers and/or other components, including those described herein, can be used for recovery.

In one aspect the invention provides a pharmaceutical composition in lyophilized form, where the composition includes from about 1 mg/ml to about 300 mg/ml fused protein, CTLA-4-Ig on this invention, which typically is expressed as a dimeric protein, in an acceptable buffer (e.g., HEPES, potassium sodium-trinitrate sodium and the like) in water at a concentration that is acceptable to maintain the desired pH (e.g., about pH 6.0 to about pH 7.5), salt (for example, 50 mm NaCl), sugar (e.g., 4-6% by weight of sucrose based on the weight of the composition), and optionally including a non-ionic detergent (Tween®-20, Tween®-40, Tween®-60, Tween®-80, or plutonomy F-68) at a concentration of from about 0.01 mg/ml to about 0.5 mg/ml, for example about 0.01 mg/ml to about 0.1 mg/ml (e.g., Tween®-20, Tween®-60, Tween®-80, or Plutonian the th F-68). Freeze-dried form of pharmaceutical compositions typically include a lower salt concentration and higher concentration of sugar compared to deliverydiovan liquid composition.

In one particular aspect the invention provides a stable lyophilized composition for therapeutic introduction when restoring sterile water, which comprises a therapeutically effective amount of a molecule according to this invention, and optionally one or more of the following pharmaceutically acceptable components: (a) a sugar or saccharide such as sucrose, mannose, dextrose or trehalose in an amount of from about 1% by weight to about 10% by weight; (b) a detergent or emulsifier such as Tween®-20, Tween®-40, Tween®-60, Tween®-80, or plutonomy F-68; (C) isotonic agent or salt, such as an inorganic salt (e.g. sodium chloride)in a concentration from 0 mm to about 50 mm (including, for example, concentration, above); (d) acceptable buffer to maintain the pH of the composition in the range that correlates with the stability of the molecule; (e) dispersing agent (for example, in a quantity sufficient for long-term dispersion of the molecules according to this invention, such as, for example, from 0.001 weight/% vol. up to about 1.0 wt/vol%) (for example, Polysorbate, such as Tween®-20, Tween®-40, Tween®-60, or Tween®-80 or plurality F-68); and (f) Stabi who isator (for example, saccharide, dextrans, low molecular weight group of PEG, such as PEG, with MB (molecular weight from about 200 Da to about 8000 Da (for example, 200, 300, 400, 600, 900, 1000, 1450, 3350, 4500 or 8000 Yes) or preservative. In some such stable lyophilised compositions molecule according to this invention is a recombinant or isolated protein according to this invention, such as a fused protein comprising amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with at least one amino acid sequence selected from the group comprising SEQ ID NOS:74-79, 197-200, 205-214, and 219-222 (optional, for example, selected from the group comprising SEQ ID NOS:197, 199, 211 and 213), where the protein binds CD80 and/or CD86 and/or its extracellular domain and/or suppresses an immune response. Such fused proteins can be in Monomeric or dimeric form.

Illustrative amounts of each component in the freeze-dried compositions include those described above and here. In one aspect, the buffer is selected so as to maintain the pH of the composition in the range from about pH 3 to about pH 8, from about pH 4 to about pH 7.5. Freeze-dried composition according to this invention, including recombinant mutant protein, CTLA-4-g according to this invention, is usually stable at from -80° to +40°C and/or essentially retains its biological activity for at least one week, one or more months (e.g., six months, one year, two years, three years, four years or more when stored at room temperatures (for example, from about 22°to about 30°C). When restoring fluid (e.g., sterile water for injection (VDI)) lyophilized composition is acceptable for injection (for example, in/in (IV), p/(subcutaneously), parenteral,/m (im), and/d (intradermal), and/p (intraperitoneal) and the like) to a subject (e.g. human).

The invention also provides a kit comprising a lyophilized or dried by sublimation composition comprising freeze-dried or dried by sublimation molecule according to this invention (for example, a mutant protein, CTLA-4-Ig, such as, for example, SEQ ID NO:197, 199, 211 or 213) in the first vessel (e.g., ampoule, such as glass vials) and the instructions for restoring dried by sublimation or freeze-dried composition with a liquid (for example, sterile water, VDI or buffer). Optionally, the kit also includes a second capacitance (e.g., a vial, such as a glass ampoule)containing a sufficient amount of liquid (for example, sterile water, VDI of Albufera) for the recovery of freeze-dried or dried by sublimation of the composition into the liquid composition. In this case, recovery is achieved by use of a syringe to remove the required amount of water from the second vessel and the introduction of water in the first tank. The first container then shake gently for introducing molecules of this invention (e.g., fused protein) in solution. The kit may include a device(a) for the recovery of freeze-dried or dried by sublimation of the composition and/or the introduction of the recovered liquid composition. Illustrative devices include, but are not limited to the following, for example, two-component mixing syringe, dual-chamber syringe and double autoinjector. One component or chamber contains the freeze-dried composition, and the second component or chamber contains liquid for recovery. Using such devices, recovery is usually carried out just before the introduction and restored the composition is typically administered parenterally (for example, by injection (subcutaneously)/(IV)/m (im), and/d (intradermal)).

The composition or the pharmaceutical composition according to this invention may include or be in the form of liposomes. Acceptable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids and the like. The preparation of such a liposome is lnyh compounds described in, for example, U.S. Patent No. 4837028 and 4737323.

The form of the compositions or pharmaceutical compositions may be dictated, at least in part by the introduction of the polypeptide, conjugate, nucleic acid, vector, virus, HPV or cells of interest. Since there are many possible ways of introduction, the form of the pharmaceutical composition and its components may change. For example, when transmucosal or transdermal administration wetting agents acceptable to the barrier that must be overcome, can be included in the composition. Such wetting agents are in General known in the art and include, for example, for transmucosal administration, detergents, bile acids and derivatives of fuseboy acid. Otherwise transmucosally introduction can be facilitated by use of nasal sprays or suppositories.

The most common form for the compositions according to this invention, including pharmaceutical compositions, is an injection. Pharmaceutically acceptable compositions for administration by injection, usually include one or more acceptable liquid carriers such as water, petroleum jelly, physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ), PBS or oil. Liquid pharmaceutical compositions may also include physiological Sol is howling solution a solution of dextrose (or a solution of another saccharide), alcohols (e.g. ethanol), polyols (polyhydric alcohols, such as mannitol, sorbitol and the like) or a glycol such as ethylene glycol, propylene glycol, PEG molecules, covering agents, which provide the necessary fluidity, such as lecithin, isotonic agents, such as mannitol or sorbitol, organic esters, such as etiolate, and agents that prevent absorption such as aluminum monostearate and gelatin. Composition for injection may be in the form of a pyrogen free and no stable aqueous solution. An aqueous solution for injection may include isotonic media, such as sodium chloride, injection of ringer's solution, dextrose, saturated with lactate solution for injection, ringer's or equivalent media delivery (e.g., solution for injection sodium chloride/dextrose). Formulations acceptable for injection intra-articular, intravenous, intramuscular, intradermal, subdermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile solutions for injection, which may include solvents, joint solvents, antioxidants, reducing agents, chelating agents, buffers, bacteriostatic agents, antimicrobial preservatives and dissolved substances, which render the formulation and otoniel the recipient's blood, which it is administered (e.g., PBS and/or salt solutions such as 0.1 M NaCl), and aqueous and non-aqueous sterile suspensions that can include suspendresume agents, sigalda agents, thickeners, emulsification, stabilizers and preservatives.

The introduction of the polypeptide, conjugate, nucleic acid, vector, virus, pseudovirus, HPV or cells of this invention (or compositions, including any such component may be facilitated by a device for delivering formed from any acceptable material. Examples of acceptable matrix materials for the production of neuoranatomical devices for administration include hydroxyapatite, organic glass, aluminates, or other ceramic materials. In some applications, the insulating agent, such as carboxymethylcellulose (CMC), methylcellulose or hypromellose (HPMC), can be used to link to a specific component with a device for localized delivery.

Nucleic acid or vector of this invention can be formulated with one or more poloxamers, polyoxyethylene/polyoxypropylene block copolymers or other surface-active substances or mesopotamie lipophilic substances for delivery of nucleic acid or vector to a cell population or tissue or skin su is the target. See, for example, U.S. Patent No. 6149922, 6086899 and 5990241.

Nucleic acids and vectors of the invention can be associated with one or more agents that enhance the transfection. In some embodiments of the invention, nucleic acid and/or vector nucleic acid according to this invention are typically associated with one or more salts, providing stability, carriers (e.g., PEG) and/or formulations that promote transfection (for example, salts of sodium phosphate, dextranase carriers, carriers of iron oxide or media balistically delivery ("gene gun"), such as gold granule or powder carriers). See, for example, U.S. Patent No. 4945050. Additional agents that enhance the transfection include viral particles, which nucleic acid or the vector nucleic acid can be conjugated to the agent, precipitating calcium phosphate, protease, lipase, a solution of beauvechain, saponin, a lipid (e.g., charged lipid, liposome (e.g., cationic liposome), the peptide or protein complex that facilitates transfection (e.g., poly(ethylenimine), polylysine or complex of viral protein and nucleic acid), virosome or modified cell or lecocephala structure (e.g., merged cell).

Nucleic acids and vectors in this image is the shadow can also be delivered in vivo or ex vivo methods of electroporation, including, for example, those described in U.S. Patent No. 6110161 and 6261281 and Widera et al., J. of Immunol. 164:4635-4640 (2000).

Transdermal introduction of a component according to this invention (e.g., polypeptide, conjugate, nucleic acid, vector, virus, HPV and/or cells according to this invention may be facilitated by using a transdermal patch comprising such a component in any acceptable compositions in any acceptable form. Such devices - transdermal patches - are provided by the invention. For example, such a component may be contained in the liquid reservoir, the reservoir for a drug in the device-plaster, or, alternatively, the component may be dispersed through the material acceptable for inclusion in a device of simple monolithic transdermal patch. Typically, the patch includes immunosuppressive amount of at least one such component, such as the amount effective to suppress immune response in the subject in contact with the plaster. Examples of such devices-adhesive known in the art. The device is the patch may be a passive device, and a device capable of iontophoretically delivery of at least one such component to the skin or tissue of the subject.

Composition, especially a pharmaceutical composition, may include any of als is the dose at least one such component according to this invention (e.g., polypeptide, conjugate, nucleic acid, vector, virus, HPV and/or cells sufficient to achieve the desired immunosuppressive response in the subject after administration. The exact dosage can be determined using any reasonable methodology and instructions for determining that accurately known in the art. In simple analyze the scheme dosage low dose of the composition administered analyzed entity or system (for example, animal models, cell-free system or whole-cell system research). Dosage is often determined by the effectiveness of a particular component, subject to the introduction, the condition of the subject, the body weight of the subject and/or target region of the subject to be treated. The size of the dose is also determined by the existence, nature and breadth of the manifestation of any side effects that accompany the introduction of any such specific component specific subject. Principles related to the dosage of therapeutic and preventive agents are, for example, Platt, Clin. Lab Med. 7:289-99 (1987), "Drug Dosage," J. Kans. Med. Soc. 70(1):30-32 (1969), and other references described herein (e.g., Remington's, see above).

For example, a therapeutically effective amount of the polypeptide according to this invention for started the Noah dosage for the treatment of autoimmune diseases may include from about 0.001 mg/kg body weight of subject to about 100 mg/kg body weight of the subject, such as, for example, from about 0,001 milligrams per kilogram (mg/kg) of body weight of subject to about 100 milligrams per kilogram (mg/kg weight of a subject, or, for example, from about 0.001 mg/kg weight of a subject to at least about 0,005, 0,01, 0,025, 0,05, 0,1, 0,2, 0,25, 0,3, 0,4, 0,5, 0,6, 0,7, 0,8, 0,9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90 or 100 mg/kg body weight of the subject. This dosage may be any appropriate Protocol, such as the introduction of daily, weekly, or biweekly, or any combination thereof (e.g., about 0, 1, 2, 4, 5, 6 and 7 days every week after that, or about 0, 1, 2, 4 and 6 weeks), followed by 1-, 2-, 3-monthly intervals, and any acceptable method of delivery, such as, for example, by electroporation or subcutaneous (s/C, or s.c.), intramuscular (I/m, or i.m.), intravenous (IV or i.v.), or intraperitoneal (I/p, or I.P. Pavlova.), subdermal, transdermal, parenteral or intradermal (and/or i.d.) the injection. In some cases, the polypeptide according to this invention is usually administered in the form of a soluble polypeptide, such as, for example, a protein comprising a mutant polypeptide, CTLA-4 EVA in this invention, covalently associated with Ig Fc polypeptide. For example, the pharmaceutical composition comprising the mutant protein, CTLA-4-Ig on this invention in a pharmaceutically acceptable carrier, solvent or is omnitele, may be entered in any acceptable way (for example, intradermally, intravenously or subcutaneously) in the effective quantity depending on autoimmune diseases (e.g. rheumatoid arthritis) or condition to be treated (for example, inhibition of transplant rejection tissues, cells, tissue or organ or whole organ from a donor subject to the recipient).

In one illustrative aspect of the invention provides a method of suppressing an immune response in a subject in need thereof, comprising administration to the subject the pharmaceutical composition, including, for example, from about 1 mg/ml to about 300 mg/ml, including from about 25 mg/ml to about 150 mg/ml (e.g., 50 or 100 mg/ml) D3-54-IgG2 fused protein in 20 mm HEPES buffer in water, 100 mm NaCl, 2% by weight sucrose, pH 7.4, where the subject is suffering from autoimmune disorders (for example, rheumatoid arthritis). In another illustrative aspect of the invention provides a method of inhibiting transplant rejection tissues, cells, tissue or organ or whole organ from a donor subject to the recipient, including an introduction to the subject is the recipient of the pharmaceutical composition comprising from about 25 mg/ml to about 100 mg/ml (e.g., 50 or 100 mg/ml) D3-69-IgG2 fused protein in 20 mm sodium citrate buffer in water, 100 mm NaCl, 2% by weight sucrose, pH 6.5.

Also provided songs which I viral vector, which includes a carrier or filler and the viral vector according to this invention. Pharmaceutical compositions comprising a pharmaceutically acceptable carrier or excipient and the viral vector, are also provided by this invention. The quantity or dosage of the viral vector particles or nucleic acid that encodes a viral vector particles, depends on: (1) type of viral vector particles in the source vector, including, but not limited to the following, for example, whether the vector alphavirus vector, Semliki-Forest virus vector, adenoviral vector, adeno-associated (AAV) viral vector, flavivirus vector, papilloma virus vector and/or viral vector, herpes simplex (HSV), (2) whether the transgenic vector expressing or carrying recombinant peptide vector, (3) host and (4) other considerations discussed above. In General, in respect of the gene transfer vectors pharmaceutically acceptable composition comprises at least about 1×102viral vector particles in a volume of about 1 ml (e.g., at least about 1×102up to 1×108particles in about 1 ml). Higher doses may also be acceptable (e.g., at least about 1×106about 1×108about 1×109about 1×1010particles/ml).

The invention thus is e provides a composition including a pharmaceutical composition), including the Assembly of two or more polypeptides or conjugates according to this invention. Moreover, the invention provides a composition including a pharmaceutical composition comprising a population of one or more multimeric polypeptides or multimeric conjugates according to this invention. As noted above, the pharmaceutical compositions include a pharmaceutically acceptable excipient, solvent or carrier.

SETS

The invention also provides kits comprising one, two, three or more polypeptides (e.g., mutant polypeptides, CTLA-4 EVA mutant fused proteins, CTLA-4-Ig, including fused dimeric proteins, conjugates, nucleic acids, vectors, cells and/or compositions according to this invention. Kits according to this invention does not necessarily contain: (1) at least one polypeptide (e.g., mutant protein, CTLA-4-Ig), conjugate, nucleic acid, vector, HPV, cell and/or the composition according to this invention; (2) optionally, at least one second immunosuppressive agent (e.g., nonsteroidal anti-inflammatory agent, methotrexate, steroid, the TNFα antagonist and the like); (3) instructions for using any of the methods described herein, including a therapeutic or preventive method and instructions for using any component defined in (a) or (2); (4) capacity for content, at least one such component, defined in (1) or (2); and/or (5) packaging materials. One or more polypeptides (e.g., mutant protein, CTLA-4-Ig), conjugates, nucleic acids, vectors, HPV, cells and/or compositions according to this invention, optionally with one or more second immunosuppressive agents, can be Packed in packaging, dispensers and kits for administration to a subject such as a mammal, including humans. For a particular therapeutic or prophylactic method determines the effective amount of each of such polypeptide (e.g., mutant fused protein, CTLA-4-Ig), conjugate, nucleic acid, vector, HPV, cells and/or compositions of this invention or optional second immunosuppressive agent (e.g., dose) and is provided with one or several doses. One or more polypeptides (e.g., mutant protein, CTLA-4-Ig), conjugates, nucleic acids, vectors and/or cells of the composition and, if necessary, an optional second immunosuppressive agent can be provided in powder (e.g., lyophilized or liquid form and can be formulated with excipient or carrier (including, for example, pharmaceutically acceptable excipient or carrier), the fact itself is m forming composition (including, for example, a pharmaceutical composition). Provided packages or dispensers that include one or more uniform dosage forms. Typically, instructions for the introduction of such components are provided with the package, along with the corresponding marking on the label, which shows that the connection is acceptable for treatment of the indicated conditions.

Information confirming the possibility of carrying out the invention

The following examples also illustrate the invention but should not be construed as limiting its scope in any way.

Example 1

This example provides a description of how to create LEA29Y-Ig fused protein, which was used as a control and for comparison purposes in the study Biacore™ binding and activity-based cells.

The creation of a DNA plasmid vector encoding LEA29Y-Ig protein.

This example describes the creation of a DNA plasmid vector that encodes LEA29Y-Ig protein. LEA29Y-Ig includes specific known mutant polypeptide CTLA-4 EVA, named "LEA29Y" (or "LEA"or "L104EA29Y", or "A29YL104E"), which is covalently linked at its C-end N-end specific mutant human IgG1 Fc polypeptide. LEA29Y polypeptide is a mutant polypeptide, CTLA-4 EVA, comprising the amino acid sequence that differs from and is inoculates sequence of human CTLA-4 extracellular domain two mutations - A29Y replacement and L104E substitute - where the provisions of 29 and 104 are numbered by reference to the amino acid sequence of the human polypeptide CTLA-4 EVA, with the first amino acid residue of human CTLA-4, is designated as the position of amino acid residue 1. See U.S. Patent No. 7094874. The plasmid vector pCDNA3.1-LEA, which includes a nucleotide sequence that encodes LEA29Y-Ig, create to obtain this fused protein.

DNA encoding LEA29Y-Ig, is formed by PCR Assembly using overlapping oligonucleotides designed on the basis of sequence homology with the nucleotide sequence that encodes a LEA29Y-Ig, is presented in SEQ ID NO:167. Oligonucleotides design, create and assemble using standard procedures, well known to experts in this field, and they can include the stop and start codons and restriction sites if necessary. Procedures for PCR amplification, which are used here, also well known in the art. See, for example, Berger, Ausubel and Sambrook, all see above.

The oligonucleotides were harvested in 100 μl PCR reaction with 1 μm of oligonucleotides, 1x Taq buffer (Qiagen; #201225) and 200 μm dNTP (deoxynucleotide) for 30 cycles of amplification (94°C, 30 sec; 60°C, 30 sec; 72°C, 60 sec). Amplified DNA purified using the QiaQuick PCR spin columns (Qiagen, Cat.#28104) and treated with restriction enzymes NheI and SacII. The fragments separated by electrophoresis on agarose gel, purified using the kit for gel extraction Qiaquick (Qiagen, #28704) in accordance with the manufacturer's recommendations and are ligated into similarly treated with enzymes plasmid kDNK 3.1 (+) (Invitrogen, Cat. #V790-20). The ligation products transformed into cells lO E. coli (Qiagen, Cat. #S-10) in accordance with the manufacturer's recommendations. The obtained cells are incubated overnight at 37°C in LB medium containing 50 μg/ml of carbenicillin, with stirring at 250 rpm and then used to prepare maxiprep (Qiagen; #12362) flow plasmid DNA (hereinafter in this description referred to as plasmid vector pCDNA3.1-LEA).

The plasmid vector pCDNA3.1-LEA identical plasmid vector kDNK mutant vector, CTLA-4-IgG2, shown in figure 1, except that the sequence of nucleic acids encoding a mutant polypeptide, CTLA-4-IgG2, replaced by a sequence of nucleic acid that encodes a LEA29Y-Ig protein. Nucleic acid encoding a signal peptide of human CTLA-4, include the nucleotide sequence encoding a signal peptide.

The sequence of the nucleic acid encoding the estimated LEA29Y-Ig protein represented in SEQ ID NO:167. SEQ ID NO:167 includes a nucleotide sequence encoding a signal peptide (in the example, amino acid residues 1-37 SEQ ID NO:165). Amino acid sequence of the alleged LEA29Y-Ig and Mature LEA29Y-Ig fused proteins (without signal peptide) is shown in SEQ ID NOS:165 and 166, respectively. As noted in figure 2C, putative amino acid sequence of LEA29Y-Ig includes the following segments: a putative signal peptide (amino acid residues 1-37), LEA29Y EVA polypeptide (amino acid residues 38-161), linker (amino acid residue 162) and mutant (modified) Fc domain of human IgG1 polypeptide (amino acid residues 163-394). Amino acid residues at the joints between these different segments are also shown in figure 2C. Namely, it is shown the last four amino acid residue signal peptide, the first five and the last five amino acid residues LEA29Y EVA, one of the linker amino acid residue (Q) and the first five and the last five amino acid residues of mutant IgG1 Fc polypeptide.

The signal peptide is usually cleaved during processing, and thus, the secretory protein (namely, the Mature protein) LEA29Y-Ig does not contain the usual sequence of the signal peptide. Mature/secretiruema form LEA29Y-Ig, which has a total number of 357 amino acids include amino acid residues 38-394 (full sequence without signal peptide) predpoll the emnd sequence, presented in SEQ ID NO:165, and begins with the amino acid sequence: methionine-histidine-valine-alanine. SEQ ID NO:165 includes a signal peptide (e.g., residues 1-37) at its N end; the signal peptide is typically cleaved, forming the Mature protein represented in SEQ ID NO:166. If necessary, amino acids of the Mature form may be numbered starting with The sequence Met-His-VaI-Ala, Met labeling as the first residue (for example, the KJV includes amino acid residues are numbered 1-124), as in Mature LEA29Y-Ig fused protein having the sequence represented in SEQ ID NO:166. In one aspect, the sequence of SEQ ID NO:165 or 166 does not include the C-terminal lysine residue; the residue can be chipped off during processing or prior to secretion.

Protein sequence LEA29Y-Ig fused protein is described in U.S. Patent No. 7094874. Specifically SEQ ID NO:4 of U.S. Patent No. 7094874 shows the protein sequence, encoding an immature form Monomeric LEA29Y-Ig. In U.S. Patent No. 7094874 LEA29Y-Ig protein is called "L104EA29YIg." Mature LEA29Y-Ig protein comprising the sequence represented in SEQ ID NO:166 presented here differs from the sequence of the fused protein represented in SEQ ID NO:4 in U.S. Patent No. 7094874 as SEQ ID NO:4 of U.S. Patent No. 7094874 includes a signal peptide (namely, residues 1-26 SEQ ID NO:4). This signal peptidomics cleaved during processing, and thus, Mature (secretiruema) form LEA29Y-Ig fused protein usually does not include the sequence of the signal peptide. SEQ ID NO:3 of U.S. Patent No. 7094874 represents a sequence of nucleic acids that encodes L104EA29YIg protein (namely, LEA29Y-Ig).

LEA29Y-Ig usually exists in solution as a dimeric protein consisting of two identical Monomeric fused protein. In this case, each Monomeric Mature LEA29Y-Ig protein includes LEA29Y EVA (SEQ ID NO:168) polypeptide fused at its C-end N-end mutant IgG1 Fc (SEQ ID NO:186). Two LEA29Y-Ig monomer covalently linked together by disulfide bonds formed between cysteine residues in each monomer, thereby forming LEA29Y-Ig dimer fused protein. LEA29Y-Ig dimer is a form molecules fused protein used in the studies described in these examples, except where specified otherwise.

The creation of stable Cho-K1 cell line expressing the LEA29Y-Ig protein.

Stable cell line was created for education mnogomillionnyj quantities LEA29Y-Ig fused protein discussed above.

Transfection of Cho-K1 cells

Cho-K1 cells were seeded with a density of 1×106in T-175 flasks (BD Falcon, #353112)containing 40 ml of growth medium (DMEM/F12 medium (Invitrogen, #10565-018), supplemented with 10% fetal bovine serum (FBS) (Hyclone, #SV30014.03) and 1x PS (Penicillin + Article shall atomizing) (Invitrogen, #15140-122)). Cells were incubated for 24 hours (h) at 37°C and then was transfectional with 10 μg plasmid Maxiprep DNA (e.g. plasmid vector encoding LEA29Y-Ig, as described above), mixed with 60 μl of Fugene 6 (Roche, #11814443001) in accordance with the conditions recommended by the manufacturer. Cells were incubated for 2 days (days) at 37°C in growth medium and then 10 days. on Wednesday selection (growth medium containing 300 μg/ml of geneticin (Invitrogen, #10131-027)), replacing the medium every 2 days. The medium was removed and cells were dissipated by adding 3 ml of 0.05% trypsin (Invitrogen, Cat. #25300-054) and incubation at 37°C for 3 min Scattered cells were diluted in 10 ml growth medium and collected by centrifugation at 1000 rpm for 5 min at room temperature (RT) in GH-3,8 rotor (Beckman Coulter, #360581). After elimination of the supernatant, the cells suspended in 1 ml of growth medium, filtered through 40 µm membrane (BD Falcon, #352340) and brought to a density of 1×106cells/ml

Department of the unique clones.

Using the facility for cell sorting (Dako, MoFIo), living cells individually dissipated in 96-cellular tablets for culture (Sigma-Aldrich, #CLS-3596)containing 200 μl/cell growth medium containing 25% conditioned medium (growth medium previously gathered from retrospectively (or native) cell cultures). After incubation at 37°C for 10-14 d is her cell dissipated through hydrolysis by trypsin and transferred to new plates to culture, containing 200 μl/cell growth environment. Cells were cultured at 37°C in growth medium until cell density reached 70% of confluentes (approximately 14 days in the environment, which was replaced every 7 days).

Determination of the clones

Clones expressing high levels of recombinant LEA29Y-Ig fused protein was determined using the dot-blot analysis (analysis by hybridization of macromolecules by diffusion through point hole in the matrix) and Western analysis. For dot-blot analysis, 100 µl of medium was collected from each cell 96-cellular tablets for culture and transferred to nitrocellulose membranes (Whatman, #10439388) in accordance with the manufacturer's recommendations. The membrane was washed twice with 200 ml of PBST (PBST is a phosphate-sauverny saline (PBS)+0.05% of Tween®-20) for 10 min at room temperature (RT) and then incubated with PBST containing 5% non-fat dry milk (EMD, #1.15363.0500) for 1 hour (h) at RT. Membranes were washed as described above and incubated for 1 hour at RT in 20 ml of PBST containing antibody goat against human Ig conjugated to horseradish peroxidase (HRP) - (Vector Labs, #BA-3000)diluted to 1:4000. Membranes were washed as described above and incubated for 1 hour at RT with PBST containing streptavidin-HRP reagent (BD Biosciences, #554066)diluted 1:2000. Membranes were washed as described above, and signals the s was determined using ECL reagent (electrochemical lysis) to determine Western blot (Amersham, Cat. #RPN2132) in accordance with the conditions recommended by the manufacturer. Positive clones identified by high signal intensity (namely, expressing high levels fused protein), was dissipated trypsinogen hydrolysis and transferred to 6-cellular tablets for culture (BD Falcon, Cat. #353046)containing 2 ml/cell growth environment. After incubation at 37°C for 3-4 days, cells were dissipated trypsinogen hydrolysis and transferred to T-75 flasks (BD Falcon, Cat. #353136)containing 20 ml of growth medium. After incubation at 37°C for 2 days, 100 μl of the medium was collected and analyzed in relation to the levels of protein expression by Western analysis. For Western analysis equal amounts (15 μl) of medium from each cell culture were driven through 4-12% Bi-Tris NuPAGE gels (Invitrogen, #NP0322BOX) in MES (MES 2-(N-morpholino)econsultancy acid, pH 7,3) movable buffer (Invitrogen, #NP0002) in accordance with the conditions recommended by the manufacturer. Proteins transferred from gels to nitrocellulose membranes (Invitrogen, Cat. #LC2001) using elektroprenos in accordance with the conditions recommended by the manufacturer. Membranes were processed as described above for dot-blotting and positive clones (expressing the protein of interest) was measured by signal intensity and average molecular weight. Positive clones dissipated trypsin is the first hydrolysis, as described above, and placed in T-175 flasks containing 40 ml of the growth medium.

Obtaining and purification of LEA29Y-Ig fused protein

Breeding crops roller bottles

Stable Cho-K1 cell line, which was transfection, as described above, with a nucleic acid that encodes a protein of interest, grew to confluently in T-175 flasks containing 40 ml of growth medium (DMEM/F-12 medium (Invitrogen, #10565-018), supplemented with 10% FBS (Hyclone #SV30014,03) and 1x PS (Invitrogen, #15140-122)). The cells were collected by incubation in 3 ml of 0.05% trypsin (Invitrogen, Cat. #25300-054) for 3 min at 37°C, was dissolved in 12 ml of growth medium and then transferred in roller flasks (Coming, Cat. #431191)containing 250 ml of growth medium. After incubation of cultures in roller bottles at 37°C in humidified roller incubator for 2 days, the medium was removed and replaced by 250 ml of fresh growth medium. Cultures were incubated for 2 days at 37°C and the medium was replaced by 250 ml UltraCHO medium (UltraCHO medium (BioWhittaker, Cat. #12-724), supplemented with 1/1000 EX-CYTE (Serological Proteins, Cat. #81129N) and 1x PS)). After incubation for 2 days at 37°C. medium was replaced by 250 ml of fresh UltraCHO medium. Cultures were incubated for 2 days at 37°C and the medium was replaced by 250 ml of medium receipt (DMEM/F-12 medium, supplemented with 1/100x ITSA (Life Technologies #51300-044), 1/1000x EX-CYTE and 1x PS). In the process of obtaining the medium was collected and replaced with fresh medium obtained is I every day.

Purification of protein

The environment of the receiving culture roller bottles were cleared by centrifugation at 2500 x g for 30 min at RT followed by filtration through a 0.2 μm membrane (VWR, Cat. #73520-986). The medium was concentrated 10 times using filtration tangential flow using a 10 kDa MWCO membranes (Millipore, Cat. #RSS) and then used for affinity chromatography of protein And use of BioCad vision HPLC system. Ig protein was associated with Poros 20 Protein-A resin (Applied Biosystem, Cat. #1-5029-01) in PBS buffer, washed with the same buffer, was suirable with 80 mm citrate buffer (pH 4.0)containing 160 mm of sodium chloride, and then neutralize by addition of 2M Tris-base. The protein solution was subjected to the final dialysis for 6 liters (l) PBS using a 10 kDa MWCO membranes (Pierce, Cat. #R).

Example 2

This example describes illustrative methods for creating and screening libraries of mutant CTLA-4 to change activities linking human CD80 and/or human CD86 rahovym display.

Protein human CD80-Ig

Fused proteins of human CD80-Ig ("hCD80-Ig") and human CD86-Ig ("hCD86-Ig") was used as ligands in the experiments of phage panning and phage ELISA for determination of mutant molecules CTLA-4, which bind the human CD80 ("hCD80 and/or human CD86 ("hCD86 and/or the extracellular domain of each or about them. Fused proteins of human CD80-Ig (also called "hB7.1-Ig or hB7-1-Ig") and human CD86-Ig (also called "hB2.1-Ig" or "hB2-1-Ig") available from R&D Systems (Minneapolis, MN).

A representative nucleic acid sequence encoding the presumed protein of human CD80-IgG1 DT, which includes the signal peptide of human CD80, human CD80 EVA and human IgG1 Fc, shown in SEQ ID NO:172. Prospective and Mature amino acid sequence hCD80-IgG1 fused protein shown in SEQ ID NO:170, and SEQ ID NO:171, respectively. Estimated protein represented in SEQ ID NO:170, includes a human CD80 EVA DT, covalently fused at its C-end N-end of the human IgG1 Fc polypeptide, and includes a signal peptide at its N end. The signal peptide is typically cleaved, forming the Mature protein CD80-Ig, is presented in SEQ ID NO:171.

Human CD80-IgG1 usually in this description to reduce as hCD80-Ig. As shown in figure 2A, the putative amino acid sequence of the fused protein hCD80-Ig (also designated as CD80-IgG1") includes the following segments: a putative signal peptide (amino acid residues 1-34), human CD80 EVA (amino acid residues 35-242), linker (amino acid residues 243-245) and the polypeptide of the human IgGI Fc (amino acid residues 246-476). Amino acid residues in the joints is between these different segments are shown in figure 2A. In particular, it is shown the last four amino acid residue signal peptide, the first five and the last five amino acid residues of human CD80 EVA amino acid residues of the linker (GVT) and the first five and the last five amino acid residues of the human IgG1 Fc polypeptide. In fused protein CD80-Ig three residue GVT present as a cloning artifact (or linker) between the end of CD80 EVA (which ends with amino acid residues FPDN) and N-end polypeptide IgG1 Fc (which begins with amino acid residues PK.SC). This GVT cloning artifact or a linker shown in the proposed and Mature CD80-Ig amino acid sequences presented in SEQ ID NO:170 and 171, respectively.

The signal peptide is usually cleaved during processing, and thus, the secretory protein (namely, the Mature protein) hCD80-Ig usually does not contain a signal peptide. Mature/secretiruema form hCD80-Ig, which has a total number of 442 amino acids include amino acid residues 35-476 (full sequence without signal peptide) SEQ ID NO:170 and begins with a sequence of amino acids: valine-isoleucine-histidine-valine. If necessary, amino acids of the Mature form may be numbered, starting with valine (Val) sequence Val-Ile-His-Val, indicating Val as the first residue (for example,the KJV includes amino acid residues, numbered 1-208), as in the Mature form hCD80-Ig, including the amino acid sequence represented in SEQ ID NO:171.

Protein hCD80-Ig usually exists in solution as a dimeric protein consisting of two identical Monomeric Mature fused protein hCD80-Ig. In this case, each Monomeric Mature protein hCD80-Ig (SEQ ID NO:171) includes a human CD80 EVA (SEQ ID NO:174), fused at its C-end N-end of the human IgG1 Fc (SEQ ID NO:185). Two hCD80-Ig monomer covalently linked together by disulfide bonds formed between cysteine residues in each monomer, thereby forming a dimer fused protein hCD80-Ig. Dimer fused protein hCD80-Ig is a form molecules fused protein used in the studies described in these examples, unless precisely defined callback.

Representative nucleic acid encoding a prospective full-sized polypeptide of human CD80 shown in SEQ ID NO:196. The sequence of the nucleic acid represented in SEQ ID NO:196, encodes the signal peptide of human CD80, EVA, transmembrane domain and cytoplasmic domain, and includes a TAA stop codon at the C-end.

Protein human CD86-Ig

A representative sequence of nucleic acids encoding the presumed amino acid sequence of human CD86-human IgG1 (typical is about having in the present description, the abbreviation "hCD86-Ig") fused protein, shown in SEQ ID NO:179. This sequence is a nucleic acid includes a nucleotide sequence encoding a signal peptide Mature human CD86-human IgG1 fused protein. The putative amino acid sequence of the fused protein hCD86-Ig shown in SEQ ID NO:177, and illustrative nucleic acid encoding the presumed protein hCD86-Ig shown in SEQ ID NO:179.

As shown in figure 2B, the putative amino acid sequence of the fused protein hCD86-Ig includes the following segments: a putative signal peptide (amino acid residues 1-23), the extracellular domain of human CD86 (amino acid residues 24-243), the linker sequence (amino acid residues 244-246), and the polypeptide of the human IgG1 Fc (amino acid residues 247-477). Amino acid residues at the joints between these different segments are also shown in figure 2B. In particular, it is shown the last four amino acid residue signal peptide, the first five and the last seven amino acid residues of human CD86 EVA amino acid residues of the linker (GVT) and the first five and the last five amino acid residues of the polypeptide of the human IgG1 Fc.

Signal peptide CD86 usually cleaved from the intended polypeptide hCD86-Ig during processing, and thus, the secretory white fused the human CD86-Ig (namely, the Mature protein) usually does not include the signal peptide. Mature/secretiruema form hCD86-Ig, which has a total of 454 amino acids include amino acid residues 24-477 (full sequence without signal peptide) SEQ ID NO:177 and starts with the following sequence of amino acid residues: alanine-Proline-leucine. If necessary, amino acids of the Mature fused protein can be numbered, starting with residue alanine (Ala) sequence Ala-Pro-Leu, indicating Ala as the first residue (for example, the KJV includes amino acid residues are numbered 1-218), as in the Mature form hCD86-Ig, including the amino acid sequence represented in SEQ ID NO:178. The Mature protein (SEQ ID NO:178) includes protein DT HCD86 EVA, covalently fused at its C-end N-end polypeptide hIgG1 Fc.

Protein hCD86-Ig usually exists in solution as a dimeric protein consisting of two identical Monomeric Mature fused protein hCD86-Ig. In this case, each Monomeric Mature protein CD86-Ig (SEQ ID NO:178) includes a human CD86 EVA (SEQ ID NO:180), fused at its C-end N-end of the human IgG1 Fc (SEQ ID NO:185). Two monomer hCD86-Ig covalently linked together by disulfide bonds formed between cysteine residues in each monomer, thereby forming a dimer fused protein hCD86-Ig. Dimer fused protein hCD86-Ig is a form melekalikimaka protein, used in the studies described in these examples, if not precisely defined callback.

In fused protein CD86-Ig (e.g., proposed and Mature forms), three residue GVT present as a cloning artifact (or linker) between the end of CD86 EVA and N-end polypeptide IgG1 Fc. In another aspect of human CD86 EVA protein DT includes an amino acid sequence comprising amino acid residues 1-218 SEQ ID NO:180 (that is, excluding the last two C-terminal amino acid residue (RR)).

Protein Orencia®

As an additional control and for comparison purposes, were purchased commercially available protein, known as protein Orencia® (Bristol-Myers Squibb Co., Princeton, NJ). Protein Orencia® consists of human CTLA-4 extracellular domain DT, covalently fused at its C-end N-end specific mutant IgG1 Fc polypeptide. Protein Orencia® is a dimeric protein consisting of two identical Monomeric fused protein, covalently bonded together by disulfide bonds formed between cysteine residues present in each Monomeric fused protein. Amino acid sequence of each Mature Orencia® monomer fused protein shown in SEQ ID NO:164 and consists of the following segments: extracellular domain DT of human CTLA-4 (amino acid residues 1-124), linker follower of the awn (amino acid residue 125) and the mutant polypeptide IgG1 Fc (amino acid residues 126-357). Each monomer fused protein Orencia® has a structure similar to that of the LEA29Y-Ig monomer fused protein is shown schematically in figure 2C, except that LEA29Y EVA substituted on the human CTLA-4 EVA DT, and not one signal peptide is not present in both monomers fused protein Orencia®, as each monomer is indirect or Mature protein. Amino acid sequence of the immature forms of the monomer Orencia® (which includes the signal peptide and the sequence of nucleic acids encoding an immature form Orencia® monomer fused protein shown in SEQ ID NO:8 and SEQ ID NO:7, respectively, of U.S. patent No. 7094874. How to create and use fused protein Orencia® is also described in U.S. patent No. 7094874.

The creation of DNA sequences encoding mutant polypeptides, CTLA-4

Methods directed development was used to create libraries of recombinant not naturally occurring polynucleotide encoding the recombinant polypeptide mutant extracellular domain of CTLA-4. Protein and nucleotide sequence of numerous naturally occurring homologues of mammalian CTLA-4 are known. See, for example, the national Center for Biotechnology Information (NCBI). The diversity of sequences defined by the different nature of the homologues of the extracellular domain of the mammalian CTLA-4, used in ways directed development to create libraries of recombinant polynucleotides encoding the mutant polypeptide domain of CTLA-4 EVA. Treatments directed development include, for example, in vitro procedures recombination and mutagenesis, as essentially described in Stemmer, Proc. Natl. Acad. Sci. USA 91:10747-10751 (1994); Chang et al. Nature Biotech 17:793-797 (1999); publication of the International application no WO 98/27230; and U.S. patent No. 6117679 and 6537776.

Education libraries of DNA sequences encoding mutant polypeptides, CTLA-4

Mutant DNA sequence encoding the recombinant mutant polypeptides, CTLA-4 EVA amplified from reactions Assembly by PCR using forward and reverse primers, designed on the basis of sequence homology. The primers were designed, created and assembled using standard procedures, well known to experts in the art and they include the stop and start codons and restriction sites if necessary. Procedures for PCR amplification, which were used, are also well known in the art. See, for example, Berger, Ausubel and Sambrook, all see above. Illustrative direct and reverse primers include, but are not limited to the following: forward primer (5'-CTATTGCTACGGCCGCTATGGCCMTKCACGTCGCTCAACCAGCCGTCGTACTCGCGTCC-3') (SEQ ID NO:191) and reverse primer (5'-GTGATGGTGATGGTGTGCGGCCGCATCAGA-3') (SEQ ID NO:192). 5 the CL reaction Assembly was used as a template in a 100 μl PCR reaction with 1 μm of forward and reverse primers, Taq buffer (Qiagen; #201225) and 200 μm dNTP for 15 cycles of amplification (94°C 30 sec; 50°C 30 sec; 72°C 40 sec). Amplified DNA encoding the mutant polypeptide, CTLA-4 EVA, were treated with enzymes (SfiI and NotI) and fragments were separated by electrophoresis on agarose gel, purified using Qiaquick kit gel extraction (Qiagen, #28704) according to the manufacturer's instructions and ligated into similarly treated with enzymes vector phage display pSB0124 (a procedure similar to that described in Chang et al., Nature Biotech 17:793-797 (1999)). The resulting library ligation was transformed via electroporation into TOP10 E. coli cells (Invitrogen, Inc; #S-50) according to the conditions recommended by the manufacturer. Transformed cells were incubated in LB (broth, Luria)containing 50 μg/ml carbenicillin at 250 rpm overnight at 37°C and then used for cooking maxiprep (Qiagen; #12362) runoff library DNA in accordance with the conditions recommended by the manufacturer.

Screening of phage libraries, demonstrating mutant polypeptides, CTLA-4, which has improved the avidity of binding to human CD80 and/or human CD86

The formation of phage display libraries of mutant polypeptides, CTLA-4

The library DNA (for example, a library of DNA sequences encoding mutant CTLA-4 KJV) transformed electropores is she in TG-1 E. coli cells (Stratagene; #200123) in accordance with the conditions recommended by the manufacturer. The culture was grown under conditions fiamengo selection (LB medium containing carbenicillin 50 μg/ml) for 1-2 formations, infected helper phage M13KO7 (at a multiplicity of infection of 5-10) and incubated with shaking at 250 rpm overnight at 37°C, With double selection for phagemid (carbenicillin 50 μg/ml) and helper phage (kanamycin 70 µg/ml). The culture was purified by centrifugation (6000 rpm, 15 min, 4°C in a Sorvall TC the rotor) and phage particles were besieged by incubation 32 ml of culture supernatant with 8 ml of PEG/NaCl solution (20% PEG-8000; 2.5 M NaCl) on ice for 30 min followed by centrifugation (9500 rpm, 40 min, 4°C in a Sorvall 600TC the rotor). Phage precipitate suspended in 1 ml PBS containing 1% BSA (bovine serum albumin, Sigma; #A7906), was transferred to a microcentrifuge tube and purified by centrifugation (maximum speed, 5 min, CT in Eppendorf table centrifuge).

Panning mutant phage laboratories, CTLA-4

Phage libraries were separated using panning in five rounds against hCD80-Ig or fused proteins hCD86-Ig using standard conditions. See, e.g., Lowman et al., Biochemistry 12;30(45): 10832-10838 (1991); Smith, G.P. et al., Chem. Rev. 97:391-410 (1997). Each round of panning includes: (a) binding of phage showing m tantie polypeptides CTLA-4 EVA with ligands hCD80-Ig or hCD86-Ig; (b) removing unbound phage; (C) elution of bound peroxidase phage; and (d) amplification lirovannomu phage for the next round of panning. An aliquot of the phage from each round were transducible in E. coli cells to obtain individual colonies of transduct.

The definition of a mutant CTLA-4 with enhanced avidity binding to human CD80 and/or human CD86 using phage ELISA.

Individual colonies obtained from each round of panning, inoculable in 96-cellular tablets for culture (NUNC; #243656)containing 150 μl/cell 2 x DT (yeast-tripton) medium containing 50 μg/ml of carbenicillin, and incubated at 250 rpm overnight at 37°C. the overnight culture was used to inoculate Globaloney blocks (Scienceware; #378600001)containing 600 μl/cell in the same environment. Cultures were incubated at 250 rpm for 2 hours at 37°C., infected with M13KO7 helper phage (multiplicity of infection (CI) 5-10) and then incubated at 250 rpm overnight at 37°C, With double selection markers phagemid and helper phage (carbenicillin 50 μg/ml and kanamycin 70 µg/ml, respectively). The culture was purified by centrifugation at 4000 rpm for 20 min at 4°C in Beckman GH 3.8 the rotor. ELISA plates (NUNC; #449824) were coated by adding 50 μl/cell PBS containing 10 µg/ml hCD80-Ig or hCD86-Ig, Inc. and who has bated overnight at 4°C. The tablets were washed three times with 200 μl/cell PBST and blocked by adding 200 μl/cell PBS containing 3% nonfat dry milk, and incubated at RT for 1 hour (HR). 25 μl/cell phage supernatants of globalunlock unit was transferred to the ELISA tablets containing 25 μl/cell 6% nonfat dry milk, and the plates were incubated for 1 hour at room temperature (RT). The tablets were washed three times with 200 μl/cell PBST and incubated with 50 μl/cell HRP-conjugated anti-M13 monoclonal antibody (GE Healthcare, #27-9421-01)diluted 1:5000 in PBST containing 3% nonfat dry milk for 1 hour at RT. The tablets were washed three times with 200 μl/cell PBST and the signal was determined using TMB substrate kit (Pierce; #34021) under the conditions recommended by the manufacturer. Mutant polypeptides, CTLA-4 EVA, showing increased avidity of binding to human CD80 and/or human CD86 (which was measured with increased avidity to hCD80-Ig and/or hCD86-Ig), compared to the avidity of binding of human CTLA-4 to human CD80 and/or human CD86 (which was measured using the avidity of binding of human CTLA-4 EVA to hCD80-Ig and/or hCD86-Ig), was chosen for further analysis.

Example 3

Creating vector IgG2 Fc fused protein

Plasmid expression vector IgG2-Fc fused protein was created to obtain the fused protein, vkluchaya what about the mutant polypeptide, CTLA-4 EVA according to the invention and of the human IgG2 Fc polypeptide. DNA encoding human IgG2 Fc polypeptide, was created by PCR amplification of human leukocyte cDNA (BD Biosciences, Cat. #HL4050AH) using direct primer (5'-AAGCTGTC ACCGGTGGATCGATCCCGAACCCTGCCCTGATTCTGATGAGCGCAAATGTTGTGTCGAGTGCCCACCGT-3') (SEQ ID NO:189) and reverse primer (5'-CAGAATTCATTATTTACCCGGAGACAGGGAGAGGCTCTTCTG-3') (SEQ ID NO:190). The primers were designed, created and assembled using standard techniques, well known to experts in the art and they include the stop and start codons and restriction sites if necessary. The PCR amplification procedure used here, are also well known in the art. See, for example, Berger, Ausubel and Sambrook, all see above. 50-100 ng of cDNA was used as a template in a 100 μl PCR reaction with 1 μm of forward and reverse primers, Taq buffer (Stratagene; #200435) and 200 μm dNTP for 25 cycles of amplification (94°C, 30 sec; 55°C, 30 sec; 72°C, 60 sec). PCR product was purified using QiaQuick PCR spin columns (Qiagen #28106) in accordance with the conditions recommended by the manufacturer, and was treated with the restriction enzymes AgeI and EcoRI. PCR-treated enzyme fragment was separated by electrophoresis on agarose gel and purified using Qiaquick kit gel extraction (Qiagen, #28704) in accordance with the conditions recommended by the manufacturer. A modified version of pCDNA3.1-LEA vector (described above), which contains the AgeI restriction site in galinou sequence of CTLA-4 (built-in as a silent mutation), can be processed with AgeI and EcoRI and Legerova fragment mentioned above. The product of ligation transform in disposable lO E. coli cells (Invitrogen Cat. #S-03) in accordance with the conditions recommended by the manufacturer. Transformed cells are incubated in LB (broth, Luria)containing 50 μg/ml of carbenicillin, at 250 rpm overnight at 37°C and then used to create maxiprep (Qiagen; #12362) flow of plasmid DNA in accordance with the conditions recommended by the manufacturer.

The obtained plasmid expression vector is denoted as a vector expression kDNK IgG2 Fc fused protein. This vector is identical to plasmid vector kDNK mutant CTLA-4 - IgG2, shown in figure 1, except that the sequence of nucleic acids encoding a mutant polypeptide, CTLA-4 EVA removed. The sequence of nucleic acids encoding a signal peptide in rdnk IgG2 Fc vector merge (which in the figure 1 can be any acceptable encodes a signal peptide nucleotide sequence)is a nucleic acid encoding a signal peptide of human CTLA-4 (SEQ ID NO:181 or SEQ ID NO:215). This vector merge IgG2 Fc does not include any nucleic acid encoding a mutant polypeptide, CTLA-4 EVA according to the invention, or any other polypeptide, CTLA-4 EVA.

Cloning nucleotide consistent is telestai, encoding the mutant polypeptide, CTLA-4 in the vector merge IgG2 Fc

To obtain mutant polypeptides, CTLA-4 in the form of soluble Fc-fused proteins each of the DNA sequences encoding mutant polypeptides, CTLA-4 EVA, identified as those that have improved the avidity of binding to human CD80 and/or human CD86 (compared to the avidity of binding of human CTLA-4-EVA to human CD80 and/or human CD86) from screening ragovoy library, clone in the vector IgG2 Fc fused protein described above, using, for example, the following procedure.

DNA sequences encoding mutant polypeptides, CTLA-4 EVA, showing a higher avidity binding to hCD80-Ig and/or hCD86-Ig, first restored from the vector phage display PCR amplification using forward and reverse primers, designed on the basis of the homology of the sequences according to the number of nucleotide residues (for example, 30-60 nucleotides) at the N - and C-ends of the mutant polypeptides, CTLA-4 EVA and application of known engineering standard procedures. See, for example, the procedures described in, for example, Berger, Ausubel and Sambrook, all see above. For example, in one illustrative aspect, the DNA sequence encoding the mutant polypeptide, CTLA-4 EVA, restored from the vector phage display PCR amplification with p the direct physical alteration primer (5'-GGAATACCGGTTTTTTGTAAAGCCATGCACGTCGCTCAACCAGCCGTCGTACTC-3') (SEQ ID NO:191) and reverse primer (5'-GGCACTCAGATCTACGTCATCGATCCCGAA-3') (SEQ ID NO:192). 10 nanograms (ng) of plasmid DNA (vector phage display containing the nucleotide sequence encoding a mutant CTLA-4 EVA) was used as a template in a 100 μl PCR reaction with 1 μm of forward and reverse primers described above, Taq buffer (Stratagene; #200435) and 200 μm dNTP for 25 cycles of amplification (94°C, 30 sec; 55°C, 30 sec; 72°C, 60 sec). PCR product was purified using QiaQuick PCR Spin columns (Qiagen #28106) in accordance with the conditions recommended by the manufacturer, and was treated with the restriction enzymes AgeI and ClaI. The fragments were separated by electrophoresis on agarose gel, purified using Qiaquick kit gel extraction (Qiagen, #28704) in accordance with the conditions recommended by the manufacturer, and ligated into similarly treated with enzymes plasmid IgG2 Fc vector merge. The ligation product was transformed into disposable lO E. coli cells (Invitrogen Cat. #S-03) in accordance with the conditions recommended by the manufacturer. Transformed cells were incubated in LB (broth, Luria)containing 50 μg/ml of carbenicillin, at 250 rpm overnight at 37°C and then used to create maxiprep (Qiagen; #12362) flow of plasmid DNA in accordance with the conditions recommended by the manufacturer.

The obtained plasmid expression vector that includes a nucleic acid encoding a mutant protein, CTLA-4-IgG2 to izopet the tion, identified as plasmid expression vector kDNK mutant CTLA-4 EVA IgG2 Fc. A schematic representation of this vector is shown in figure 1. This vector includes the Bla promoter; gene resistant to ampicillin; pUC source; the signal sequence of the SV40 polyadenylation (poly); f1 source; the SV40 promoter; gene resistant to neomycin; CMV promoter to facilitate expression of mutant fused protein, CTLA-4-Ig of the invention (including, for example, the signal peptide of human CTLA-4, mutant polypeptide CTLA-4 EVA and the polypeptide of the human IgG2 Fc); the sequence of nucleic acids encoding a signal peptide of human CTLA-4 (SEQ ID NO:181 or SEQ ID NO:215); the sequence of the nucleic acid encoding a mutant polypeptide, CTLA-4 EVA according to the invention (including but not limited to the following, for example, the nucleotide sequence encoding any one of SEQ ID NOS:80-152); illustrative sequence of nucleic acids encoding a human IgG2 Fc polypeptide shown in SEQ ID NO:183 or SEQ ID NO:217; and polymermineral signal sequence of bovine growth hormone (BGR). The nucleic acid sequence SEQ ID NO:183 encodes a polypeptide hIgG2 Fc with C-terminal lysine residue (K) (SEQ ID NO:184); the nucleic acid sequence SEQ ID NO:217 encodes hIgG2 Fc polypeptide without C-terminal lysine residue (SEQ ID NO:218).

Plasmid vector IgG2 Fc fused protein may also be used to obtain a fused protein of human CTLA-4-IgG2 ("hCTLA-4-Ig"). In this case, the sequence of nucleic acids encoding human CTLA-4 EVA (for example, SEQ ID NO:193), clone in plasmid vector IgG2 Fc fused protein using standard cloning procedures similar to those described above in place of the nucleotide sequence that encodes a mutant CTLA-4 EVA. Protein hCTLA-4-Ig usually exists in solution as a dimeric protein consisting of two identical Monomeric Mature fused protein hCTLA-4-Ig. In this case, each Monomeric Mature protein hCTLA-4-Ig includes a human CTLA-4 EVA (SEQ ID NO:159) fused at its C-end N-end of the human IgG2 Fc (SEQ ID NO:218, or SEQ ID NO:184). Two monomer hCTLA-4-Ig covalently linked together by disulfide bonds formed between cysteine residues in each monomer, thereby forming a dimer fused protein hCTLA-4-Ig. Mature dimer fused protein hCTLA-4-Ig is a form fused protein used in the studies described in these examples, except when precisely defined callback.

We found experimentally that the fused protein of human CTLA-4-Ig or mutant protein, CTLA-4-Ig, educated in Cho cells by transfection of the expression vector, comprising a nucleotide sequence encoding a hCTLA-4-Ig or mutant protein, CTLA-4-Ig and polypeptide hIgG2 Fc presented in SEQ ID NO:184, usually does not include one expects the range of C-terminal lysine residue (K), as determined by MSGH analysis; thus, hIgG2 Fc amino acid sequence of hCTLA-4-IgG2 or mutant CTLA-4-IgG2 is that shown in SEQ ID NO:218, where the amino acid sequence of hIgG2 Fc usually does not include the C-terminal lysine residue compared to the amino acid sequence represented in SEQ ID NO:184.

Transient transfection of COS cells

COS-7 cells were grown to 80-90% of confluently in T-175 flasks containing 40 ml of growth medium (DMEM/F12 medium (Invitrogen, Cat. #10565-018), supplemented with 10% FBS (Hyclone Cat. #SV30014,03) and 1 x PSG (penicillin, streptomycin and glutamine) (Invitrogen, Cat. #10378-016)). Immediately prior to transfection of cells with plasmid expression vector, the medium was removed and replaced with 35 ml of medium expression (OptiMem media (Gibco #51985)containing 1x PSG). Plasmid DNA (10 µg) (for example, the expression vector kDNK mutant CTLA-4 EVA IgG2 Fc encoding the mutant protein, CTLA-4-IgG2 according to the invention) was mixed with FuGENEβ transfection reagent (Roche #11815075001) in a volume ratio of 1:3 and added to 1 ml of growth medium. This mixture was then added slowly in a T-175 flask and was told gently to mix. After incubation at 37°C for 3 days, the medium was collected, added with fresh medium of expression and the culture incubated for an additional 3 days.

A similar procedure can be used to generate a COS - cells, transfection similar plasmid vector encoding plasmid vector human CTLA-4-IgG2 or pCDNA3.1-LEA coding LEA29Y-Ig.

Purification of proteins

Supernatant from transfection cultures (for example, including cells, transfection with kDNK mutant CTLA-4 EVA IgG2 Fc vector encoding the mutant protein, CTLA-4-Ig) was purified by centrifugation at 1000x g for 10 min at RT and filtered through a 0.2 μm membrane (Nalgene, VWR #73520-982). Proteins were purified by affinity chromatography protein And using INSTRUMENT Explorer HPLC system (GE Healthcare). The mutant protein, CTLA-4-Ig was associated with Hitrap Protein a FF columns (GE Healthcare, #17-5079-01) in PBS buffer, washed with the same buffer, was suirable with 100 mm citrate buffer (pH 4.0) and then neutralize by adding 1/10 volume of 2M Tris-base. The buffer in the protein solution in the end was replaced with PBS using a dialysis using a 10 kDa MWCO membranes (Pierce, Cat. #R).

A similar procedure can be used for purification of the fused protein of human CTLA-4-IgG2 or LEA29Y-Ig.

Calculation of quality protein. Analysis of SDS/PAGE (polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate)

The apparent molecular weight (MB) of purified mutant fused protein, CTLA-4-Ig of the invention was measured using SDS/PAGE analysis under non conditions. When non conditions of the mutant protein, CTLA-4-Ig is about the invention typically exists as a dimeric protein, comprising two Monomeric mutant fused protein, CTLA-4-Ig. In one aspect of the dimer is a glycosilated, consisting of two identical mutant monomer fused protein, CTLA-4-Ig. In one aspect, each monomer fused protein, CTLA-4-Ig includes Mature/secretory mutant CTLA-4 EVA, fused at its C-end N-end of the human polypeptide IgG2 Fc. Two mutant CTLA-4-Ig monomer covalently linked together by disulfide bonds formed between cysteine residues in each monomer. Glycosilated is a form of mutant molecules fused protein according to the invention, generally described in these examples, if only not precisely defined inverse. The data presented in these examples are homodimers mutant fused protein, CTLA-4-Ig, if only not precisely defined callback.

Analysis of SDS/PAGE was carried out as follows. 2 µg of purified protein was added to 20 μl LDS sample buffer (Invitrogen #NP0007) and dispersed on NuPAGE 4-12% bis-Tris gels (Invitrogen #NP0321BOX) in 1x MES sodium dodecyl sulfate (SDS) /PAGE mobile buffer (Invitrogen #NP0002), in accordance with the conditions recommended by the manufacturer. Gels were stained by incubation in 50 ml SimplyBlue SafeStain (Invitrogen #LC6060) for 1 hour with gentle agitation at RT. The gels were subjected to removal of dye by two incubation with 200 ml of water for 1 hour with gentle shaking at RT and processed in the buffer wysu the air traffic management (Bio RAD 161-0752) in accordance with the terms and conditions recommended by the manufacturer.

Representative SDS/PAGE gel of two illustrative of homodimeric mutant fused proteins, CTLA-4-Ig of the invention (designated as clones D3 and D4) and the fused protein Orencia®, which served as a control comparison, shown in figure 3. D3-Ig dimer fused protein comprises two identical D3-Ig monomer fused protein, which is covalently linked by disulfide bonds formed between cysteine residues in each D3-Ig monomer. Each D3-Ig monomer includes a mutant polypeptide, CTLA-4 EVA (called "D3"), comprising the amino acid sequence represented in SEQ ID NO:61, merged directly (i.e. without the "linker" amino acid residue(s)) at its C-end N-end hIgG2 Fc polypeptide presented in SEQ ID NO:218. D4-Ig dimer fused protein similarly includes two identical D4-Ig monomer, which are covalently linked by disulfide bonds formed between cysteine residues in each monomer D3-Ig. Each Monomeric D4-IgG2 protein comprises a mutant polypeptide, CTLA-4 EVA (called "D4"), comprising the amino acid sequence represented in SEQ ID NO:62, merged directly (i.e. without the "linker" amino acid residue(s)) at its C-end N-end hIgG2 Fc amino acid sequence represented in SEQ ID NO:218.

As explained, discovered experimentally that mutant the th protein, CTLA-4-Ig, produced in Cho cells using a vector comprising the nucleotide sequence encoding the estimated hIgG2 Fc polypeptide presented in SEQ ID NO:184, usually does not include the estimated C-terminal lysine residue (K), as determined MSIH analysis; thus, hIgG2 Fc amino acid sequence of a mutant CTLA-4-IgG2, described here, is that shown in SEQ ID NO:218, where MgG2 Fc amino acid sequence usually does not include the C-terminal lysine residue compared to the amino acid sequence represented in SEQ ID NO:184.

Analysis of SDS/PAGE was carried out on all drugs protein to confirm the quality of the protein in terms of the apparent molecular weight, protein concentration and purity. The results of SDS/PAGE analyses were the same for all protein drugs. From the results illustrative of the gel shown in figure 3, peeled D3-IgG2 and D4-IgG2 dimers fused protein had seemingly MB approximately 80 kDa, which consisted of the proposed MB illustrative patterns homodimeric mutant CTLA-4-IgG2 fused protein represented in the figure 10 (estimated MB=78-79 kDa). (Purified monomers mutant protein, CTLA-4-IgG2 usually have visible MB 39-40 kDa.) Protein bands on the gel shown in figure 3, were stained with the respective intensities; this confirms the accuracy of measuring the value of protein concentration for different samples. For the purity of the protein band with lower MB can be seen in figure 3 the seeming MB approximately 44 kDa, which consists of the proposed MB Monomeric IgG2 fused protein. The relative intensity of this band with lower MB consequently is low and amounts to less than 5% of the total protein.

Similar analyses SDS/PAGE can be used to assess the purity of the fused proteins of human CTLA-4-IgG2 or LEA29Y-Ig obtained using methods similar to those described above.

Endotoxin Analysis

The levels of endotoxin mutant fused proteins, CTLA-4 was measured using QCL-1000 kit for research use amebocyte lysate "limulus" (LAL test) (Cambrex #50-648U), following the manufacturer's recommendations on the conditions. The maximum level of endotoxin proteins used in cell research, is 10 endotoxin units (in this case)/mg protein.

Similar analyses can be used to measure levels of endotoxin protein preparations of human CTLA - 4-IgG2 or LEA29Y-Ig.

Analysis of gel-chromatography (SEC)

The aggregation levels of the protein (including the levels of aggregation of mutant polypeptides, CTLA-4 and control polypeptides) were measured by gel-chromatography using Dionex BioLC system (Dionex). Protein (5 µg) were dispersed through the column Tosoh G3000Wx1 (Tosoh Bioscience) in PBS concentration in the second buffer using 20 min isocratic elution and determine the absorbance (a) at 214 nanometers (nm). The maximum level of aggregation for the protein used in the further analyses, was 10%.

SEC analysis was performed on all protein preparations to ensure the quality of protein in the characteristics of the aggregation levels of the protein. The results were the same for all protein drugs and representative profile of elution from the SEC analysis of mutant dimer fused protein, CTLA-4-Ig (D3-Ig) is shown in figure 4. y-axis shows units of malabsorbtion (may); x-axis shows the elution time in minutes. Structure D3-Ig dimer above in the section "Analysis of SDS/PAGE. This purified mutant dimer CTLA-4-Ig in more homogeneous in size does not contain high levels of aggregated species. Other mutant fused proteins, CTLA-4-Ig of the invention were analyzed similarly and showed similar results (data not shown). It was found that the purified mutant fused proteins, CTLA-4-Ig of the invention are homogeneous in size and do not contain high levels (>10%) of the aggregated proteins. It is important to confirm the aggregate state of the purified mutant fused proteins, CTLA-4-Ig of the invention, as strongly aggregated mutant fused proteins, CTLA-4-Ig can bind with high avidity of the molecule of human CD80 and/or human CD86 and, thus, may exhibit greater biological activity.

Example 4

smirenje the avidity of binding of the mutant fused proteins, CTLA-4-Ig to slit proteins human CDSO-mouse Ig (hCD80-mIg) and human CD86-mouse Ig (hCD86-mIg) using surface plasma resonance (SPR) (Biacore™ Analysis).

This example describes the procedure for screening mutant CTLA-4-Ig fused proteins for improved avidity binding to hCD80-mIg and/or hCD86-mIg ligands using Biacore interaction analysis. In the nomenclature used to describe this type of analysis, immobilizovannyi binding partner is called a "ligand", as a binding partner in the mobile phase is called the "analyte". Fused proteins containing Ig domain, usually form a dimeric structure in solution using a strong Association between the two Ig domains. Except where specified to the contrary, believe that such dimeric conformations exist for slit proteins described in this example (namely, mutant CTLA-4-Ig, fused protein Orencia®, LEA29Y-Ig, hCD80-mIg and hCD86-mIg). The term "avidity" usually refer to the strength of binding between a dimeric analytes (e.g., mutant fused proteins, CTLA-4-Ig) and dimeric ligands (e.g., fused proteins hCD80-mIg or hCD86-mIg). Increase the avidity of binding described for mutant fused proteins, CTLA-4-Ig, is the result of an increase in the affinity of binding between each domain of CTLA-4 EVA and its corresponding ligand. The power of the avidity of binding is usually described in terms of the equilibrium dissociation constants (KD), which describes the molar concentration of analyte at which 50% of the available ligand binds in equilibrium./p>

In this method of screening Biacore touch the chips were derivateservlet with ligands hCD80-mIg or hCD86-mIg, and mutant fused proteins, CTLA-4-Ig in the buffer was poured on the sensor chips coated with ligands. We measured the ability of the mutant molecules fused protein, CTLA-4-Ig to bind to specific binding partner (namely, hCD80-mIg or hCD86-mIg). Control fused proteins (namely, human CTLA-4-IgG2 protein, protein Orencia® and mutant protein LEA29Y-Ig) was also poured on the sensor chips coated with ligands, and similarly evaluated the ability of these molecules to contact hCD80-mIg or hCD86-mIg for comparison. Using Biacore system is the rate constant of Association (kon) and dissociation (koffprotein of interest bound to ligands hCD80-mIg or hCD86-mIg can be estimated and used to calculate the equilibrium dissociation constants, KD. Human CTLA-4-IgG2 protein that includes a polypeptide DT human CTLA-4 EVA, merged with the polypeptide of human IgG2, can serve as wild-type "control" human CTLA-4-Ig. In addition or alternatively, as a protein Orencia® consists of the polypeptide DT human CTLA-4 EVA, merged with the modified polypeptide IgGI Fc, it also effectively serves as a control wild-type human CTLA-4-Ig for comparison purposes. Defined mutant SL is made proteins, CTLA-4-Ig, with increased avidity of binding to hCD80-mIg and/or hCD86-mIg, compared to human CTLA-4-IgG2, fused protein Orencia® and/or LEA29Y-Ig. As discussed in more detail below, the increased avidity of binding of the mutant fused protein, CTLA-4-Ig of the invention to hCD80-mIg and/or hCD86-mIg, compared to the avidity of binding of the fused protein Orencia® (namely, hCTLA-4-IgG1) and/or LEA29Y-Ig to hCD80-mIg and/or hCD86-mIg, is not the result of differences between IgG2, present in the mutant molecules, CTLA-4-Ig and mutant IgG1 present in molecules Orencia® or LEA29Y-Ig.

All analyses Biacore™ was performed on a Biacore™ 2000 system (GE Healthcare) at room temperature (RT, 25°C). HBS-EP buffer (10 mm HEPES (pH 7.4), 150 mm NaCl, 3 mm EDTA, 0.005% of the surfactant P20) was used as a line buffer for all experiments.

Standard kinetic study

Standard kinetic study measures the kinetic parameters of the binding of dimeric ligand (e.g., fused protein hCD80-mIg or fused protein hCD86-mIg), covering touch the chips, and a dimer of an analyte (e.g., mutant fused proteins, CTLA-4-Ig of the invention) in the mobile phase. Antibody rabbit against mouse IgG (GE Healthcare, #BR-1005-14) immobilizerpower on SM sensor chips (GE Healthcare, #BR-1000-14) according to the manufacturer's Protocol. The antibody was diluted to 30 μg/ml in buffer for immobilization (10 mm sodium acetate, pH 5.0 (BR-1003-51). At a flow rate of 5 ál/min a sensor chip CM activated 35 ál injection of a mixture of 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) (formed by mixing equal volumes of 11.5 mg/ml EDC and 75 mg/ml NHS) (GE Healthcare, #BR-1000-50), with a further 35 ál injection of undiluted antibody. Unreacted sites were redeemed 35 μl of 1 M ethanolamine-HCL, pH 8.5 (GE Healthcare, #BR-1000-50). This procedure usually gives at the output of 15000 units response (SW) associated antibodies. The ligands of the human CTLA-4 to CD80-mouse Ig (hCD80-mIg) (Ancell, #510-820) or human CD86-mouse Ig (hCD86-mIg) (Ancell, #509-820) was associated with sensor chips coated with antibody by injection of 10 µl or 16 μl, respectively ligand solution (2 mg/ml protein in HBS-EP buffer [10 mm HEPES pH 7.4, 150 mm NaCl, 3 mm EDTA, 0.005% of (volume/volume) surfactant P-20, GE Healthcare, #BR-1001-88]) at a flow rate of 10 ál/min Levels capture ligand was usually 135-170 SW. Mutant proteins, CTLA-4-Ig or diluted in HBS-EP buffer and cast on the sensor chips coated with a ligand for 2 min at 30 μl/min, followed 5 min incubation with HBS-EP buffer containing no protein at the same flow rate. For mutant fused proteins, CTLA-4-Ig, which has a very low rate of dissociation from the hCD86-mIg, kinetic studies were performed with the use of a longer time of dissociation (in the example, 20 min). Rmaxthe signal levels for the mutant proteins, CTLA-4-Ig was lying in the range from about 70-100 SW. Recovery between cycles was carried out 3-minute incubation with 10 mm glycine buffer (pH 1,7) at 50 μl/min New chips were subjected 4-5 cycles capture/binding/repair before use in the actual experiments. Data from the reference cell containing only the captured antibody rabbit against mouse IgG, subtracted from the data obtained from the current cell containing captured hCD80-mIg or hCD86-mIg. Typically, 8 dilutions of mutant proteins, CTLA-4-Ig in the range from 500 nm to 0.2 nm were analyzed against a blank sample (only HBS-EP buffer).

Write sensogram from the typical Biacore analysis are shown in figure 5. This figure shows the response (SW) time in seconds (sec)), which is formed by linking the following three fused proteins with fused protein hCD86-mIg: (1) protein Orencia® (serving as a control and for comparison) (Bristol-Myers Squibb Co.; see, for example, Larson CF. et al, Am. J. Trans. 5:443-453 (2005)); (2) LEA29Y-Ig protein (for comparison); and (3) illustrative mutant protein, CTLA-4-Ig of the invention, designated "clone D3 (also called D3-IgG2). Protein D3-IgG2 includes two identical Monomeric fused protein that covalently joined together by one or more disulfide bonds formed between cysteine residues in which each monomer. See discussion in the section "Analysis of SDS/PAGE" above. Each Monomeric protein includes Mature mutant polypeptide CTLA-4 EVA presented in SEQ ID NO:159, covalently fused at its C-end N-end of the human IgG2 polypeptide presented in EQ ID NO:184 or 218. Other fused dimeric proteins of the invention may include structures similar to those observed in fused dimeric protein D3-IgG2 - except that D3 mutant polypeptide CTLA-4 EVA each monomer fused protein is replaced with another mutant polypeptide CTLA-4 EVA.

Phase Association reflects the binding between the analyte of interest and the ligand of interest. In figure 5 the phase of the Association for each analyte is represented by the curve in time to point in time indicated by the arrow, and is characterized by the binding of the analyte (D3-IgG2, Orencia® protein or ligand LEA29Y-Ig) with hCD86-mIg. The rate at which the analyte associates with the ligand hCD86-mIg, reflected on the curve - see, for example, a sharp increase in the units of the answer, starting from about 510 seconds.

Phase dissociation analysis starts from the time indicated by the arrow in figure 5. During phase dissociation of the analyte and the ligand dissociate from their binding conformation. In figure 5 the rate at which the analyte dissociates from the ligand hCD86-mIg, presents reduction units response speed reduction unit is response time). Based on these data, can be defined relative rate constants of dissociation (off rate, koffor kd) and the rate constants of dissociation ("on" speed, konor ka). The overall avidity of the interaction can be described as KD, (koff)/ (kon). The increased avidity of binding is often manifested in lower speeds dissociation. If the reduced rate of dissociation is accompanied by an equal, higher or slightly lower speed Association, so that the calculated equilibrium dissociation constant KDbude below, the avidity will be more. In this case, the mutant protein, CTLA-4-Ig, which has the avidity of binding to the hCD86-mIg ligand, which is greater than the avidity of binding of the fused protein Orencia® to the same ligand, will also have a lower rate of dissociation from the ligand than Orencia® protein. The mutant protein, CTLA-4-Ig, which has the avidity of binding to the hCD86-mIg ligand, which is greater than the avidity of binding of the LEA29Y-Ig to the same ligand, will also have a lower rate of dissociation from the ligand than LEA29Y-Ig.

Figure 5 shows that the rate of dissociation of the LEA29Y-Ig from the hCD86-mIg ligand is significantly less than the rate of dissociation of the protein Orencia® from the same ligand. LEA29Y-Ig also has a speed Association to associate with hCD86-mIg SAS who put the which is observed for the fused protein Orencia®. Thus, LEA29Y-Ig has a higher avidity for hCD86-mIg than protein Orencia®. This discovery is consistent with previous studies describing the LEA29Y-Ig as having a higher avidity of binding to the ligand hCD86-mIg than protein Orencia® (Larson CF. et al., Am. J. Trans. 5:443-453 (2005)). Figure 5 also shows that the mutant D3-IgG2 protein according to the invention has a lower dissociation rate of ligand hCD86-mIg than or Orencia®, or LEA29Y-Ig protein. Protein D3-IgG2 also has a similar (but somewhat faster) speed Association to associate with hCD86-mIg ligand compared with the speeds of the Association each Orencia® or fused proteins LEA29Y-Ig to the ligand hCD86-mIg. Thus, D3-IgG2 protein shows a higher avidity binding to the hCD86-mIg ligand than as Orencia®protein LEA29Y-Ig. Therefore, I believe the protein D3-IgG2 binds native CD86, including, for example, CD86, expressed in vivo on agriculture in mammals, such as humans, with higher avidity binding. This consideration is also confirmed by functional cell research, discussed in the examples below.

Biacore analysis was carried out using the other mutant CTLA-4-Ig fused protein according to the invention. In addition, Biacore analysis was performed using hCD80-mIg-covered touch the chip is in and mutant fused proteins, CTLA-4-Ig of the invention. The rate of dissociation and the avidity of binding of these mutant molecules were determined and compared with rates of dissociation and the avidity of binding of Orencia® and fused proteins LEA29Y-Ig. Representative results are shown in tables 3 and 4 and are discussed in more detail below.

Standard Biacore analysis of the data

After exclusion of parts recovery and capture sensorama, curves were zeroed to 5 seconds on average for all curves in approximately 10 s before the injection of the sample. No-load curve subtracted from each of the analyzed curve. Data were analyzed using the software BIAevaluation version 4.1, available from GE Healthcare) using the function "Under kinetic characteristics, synchronous ka/kd". The start time of the injection was defined as the time until the phase of the Association, where the curves were close to zero. The selection of data for phase Association was started approximately 10 seconds after the start time of the injection and was completed in approximately 10 seconds before the end time of injection. The end time of the injection was defined as the time until the occurrence of any signal peaks associated with the phase dissociation. Phase dissociation chose from 10 seconds after the end time of injection and inclusive 280-295 sec 5 minute phase dissociation. 1:1 Langmuir model describes the reaction a + b <=> AV. This model represents the binding of the individual ligand with a separate protein, of interest (e.g., receptor). 1:1 Langmuir model of the program BIAevaluation used to determine the rate constants of Association (ka) and the rate constants of dissociation (kdand to calculate the equilibrium dissociation constants KD. KD=kd/ka. KD=([A][V])/[AB]. The equilibrium dissociation constant KDinversely proportional to the equilibrium constant of Association KA. KD=1/KA. Equation rate for reaction (analyte plus the ligand In giving the complex AB), where a = input analyte, In = free ligand and t = time, represent: d[B]/dt=-(ka[A][V]-kd[AB]) and d[AB]/dt=ka[A][B]-kd[AB]. Replacing R, Biacore response unit (SW) at this time, [AV], Rmax-R [V], and the concentration of the analyte) for [A], the net rate, expressed in Biacore units, is a dR/dt=kaC(Rmax-R)-kdR R with t0=0, In[0]=Rmaxand AB[0]=0 SW, with a total response = [AB]+RI. Shift (RI) was close to zero, and Rmax, kaand kdmeet globally for all curves.

Standard kinetic studies and data analyses were carried out on protein drugs mutant fused protein, CTLA-4-Ig of the invention, and LEA29Y-Ig, and fused proteins Orencia®. Tables 3 and 4 summarize the data binding for the representative mutant fused proteins, CTLA-4-Ig of the invention.

Table 3 pre who is the avidity of binding of representative mutant CTLA-4-Ig fused proteins to hCD86-mIg, what was measured in the standard Biacore study described above. Specifically, the table 3 shows the name of each mutant fused protein, CTLA-4Ig; identification number sequence (SEQ ID NO), the corresponding amino acid sequence of Monomeric mutant CTLA-4 EVA fused protein; equilibrium dissociation constant (KD (molar value (M)), defined on the basis of the avidity of binding of the mutant protein to the hCD86-mIg; and the avidity of binding of each mutant CTLA-4-Ig to hCD86-mIg relative to the avidity of binding of the fused protein Orencia® for hCD86-mIg. This relative avidity of binding (shown in the far right column) are shown as the fold increase in the avidity of binding of the mutant fused protein to the hCD86-mIg compared to the avidity of binding of the fused protein Orencia® for hCD86-mIg. Each mutant protein, CTLA-4-Ig of the invention in table 3 usually exists in solution as a dimeric protein consisting of two identical Monomeric fused protein, where each Monomeric protein comprises a mutant polypeptide, CTLA-4 EVA (for example, named D1, D1T, D2, D3, D4 and so on), merged directly on it-end N-end IgG2 Fc polypeptide comprising the sequence of SEQ ID NO:184 or 218. Each dimeric mutant CTLA-4-Ig can be created using standard techniques known in the art. Alternatively, each of the first such dimeric mutant dimer fused protein, CTLA-4-Ig can be created using methods shown in example 3 above. Briefly, the sequence of nucleic acids encoding a specific mutant CTLA-4 EVA, defined in table 3 (for example, the sequence of nucleic acids encoding a mutant amino acid sequence of CTLA-4 EVA, defined in table 3), can be cloned into a vector merge IgG2 Fc, mammalian cells can be transfection with the vector, and the resulting protein can be expressed, purified and analyzed as described in example 3. Illustrative nucleic acid sequence for each mutant CTLA-4 EVA shown in the list of sequences that are included in this description. Orencia® protein, which consists of two Monomeric fused protein, where each Monomeric protein comprises wild-type human CTLA-4 EVA, merged with a modified IgG1 Fc serves as a reference point, namely with avidity binding to the hCD86-mIg equal to 1. KDvalues for LEA29 Y-Ig fused protein and human CTLA-4-Ig fused protein is also presented. In addition, it also presents several-fold improvement in the hCD86-mIg the avidity of binding of the LEA29Y-Ig compared to the hCD86-mIg avidity binding protein Orencia®. The avidity of binding of CTLA-4-IgG2 and Orencia® fused proteins to hCD86-mIg approximately equal, confirming that the differences between human IgG2 with the polypeptide present in human CTLA-4IgG2, and modified IgG1, present in the Orencia® protein, contribute little, if any, contribute, contribution towards hCD86-mIg affinely binding of these molecules. As discussed in more detail in example 5 below, we confirmed that the differences in immunosuppressive functional activities between mutant CTLA-4-Ig polypeptides according to the invention and white Orencia® (or LEA29Y-Ig) can not be assigned to their respective areas of Ig Fc, including different IgG isotypes.

As shown in table 3, representative mutant fused proteins, CTLA-4-Ig of the invention have hCD86-mIg the avidity of binding that: (1)at least approximately equal to or greater than the avidity of binding of human CTLA-4-IgG2 ("hCTLA-4-IgG2") (which consists of two covalently linked monomer fused protein, where each such Monomeric protein comprises a polypeptide of the human CTLA-4 EVA, merged with IgG2 Fc polypeptide); (2)at least approximately equal to or greater than the avidity of binding protein Orencia® hCD86-mIg ligand; and/or (3)at least approximately equal to or greater than the avidity of binding of the LEA29Y-Ig ligand hCD86-mIg. Significantly improve the avidity of binding to the ligand hCD86-mIg relative to the avidity of binding protein Orencia® hCD86-mIg determined for each CTLA-4-Ig mutant (4th column in table 3).

Found that most of the mutant fused protein CTLA4-Ig are the dissociation rate from the fused protein hCD86-mIg, which is less than the rate of dissociation of the fused protein Orencia® hCD86-mIg (data not shown). Found that many mutant fused proteins, CTLA-4-Ig have the speed Association with hCD86-mIg greater than the velocity of the Association fused protein Orencia® with the same ligand (data not shown).

All mutant fused proteins, CTLA-4-Ig, are presented in table 3, had hCD86-mIg equilibrium dissociation constants (Ko), which were lower than the hCD86-mIg equilibrium dissociation constant of human CTLA-4-IgG2 or fused protein Orencia®. Moreover, most mutant fused proteins, CTLA-4-Ig, are presented in table 3, had hCD86-mIg equilibrium dissociation constants were lower than hCD86-mIg equilibrium constant LEA29Y-Ig.

All mutant fused proteins, CTLA-4-Ig, are presented in table 3, had the avidity of binding to the hCD86-mIg, which is larger than the avidity of binding of human CTLA-4-IgG2 or fused protein Orencia® for hCD86-mIg (calculated times improvements for hCD86-mIg the avidity of the binding relative to the fused protein Orencia® shown in the 4th column of table 3). Moreover, many mutant fused proteins, CTLA-4-Ig, are presented in table 3, had the avidity of binding to the hCD86-mIg, which is larger than the avidity of binding of the LEA29Y-Ig to hCD86-mIg (4th column of table 3).

Mutant CTLA-4-Ig of the invention, which has a higher avidity binding hCD86-mIg ligand than Orencia® LEA29Y-Ig protein, is likely to have an increased in vivo immunosuppressive effect in comparison with the hCTLA-4-IgG2, Orencia®, or fused protein LEA29Y-Ig, respectively, such as, for example, therapeutic and/or prophylactic methods for suppressing an immune response in a subject (e.g., in vivo treatment of diseases or disorders of the immune system of mammals, such as humans, in which immunoinhibitory or immunosuppression are desirable), methods for inhibiting transplant rejection of a tissue or organ from donor to recipient (e.g., a mammal, such as, for example, people), and other methods described in this description.

Table 3
Protein (dimer)SEQ ID NO* (namely, mutant CTLA-4 VDK)KD(M) hCD86 EVAThe avidity of binding Monomeric hCD86 EVA (compared to the dimer fused protein Orencia®)
Human CTLA-4-IgG2162of 3.95×10-101,32
dimer fused protein Orencia®1645,23×10-101
LEA29Y-Ig166of 1.05×10-10-5,23×10-105x-10x
D1-IgG258to 2.65×10-10-5,23×10-102x-10x
D1T-IgG2595,23×10-10-2,62×10-1010x-20x
D2-IgG2602,62×10-10-1,31×10-1020x-40x
D3-IgG261<is 1.31×10-10>40
D4-IgG262to 2.65×10-10-5,23×10-102x-10x
D5-IgG263to 2.65×10-10-5,23×10-102x-10x
D6-IgG264to 2.65×10-10-5,23×10-102x-10x
D20-IgG2652,62×10-10-1,31×10-10 20x-40x
D21-IgG2662,62×10-10-1,31×10-1020x-40x
D23-IgG267<is 1.31×10-10>40
D24-IgG268<is 1.31×10-10>40
D26-IgG269<is 1.31×10-10>40
D27-IgG270<is 1.31×10-10>40
D28-IgG271<is 1.31×10-10>40

35
Protein (dimer)SEQ ID NO* (namely, mutant CTLA-4 KJV)KD(M) hCD86 EVAThe avidity of binding Monomeric hCD86 EVA (compared to the dimer fused protein Orencia®)
D29-IgG272<is 1.31×10 -10>40
D31-IgG2732,62×10-10-1,31×10-1020x-40x
D3-1-IgG21<is 1.31×10-10>40
D3-2-IgG222,62×10-10-1,31×10-1020x-40x
D3-3-IgG232,62×10-10-1,31×10-1020x-40x
D3-4-IgG24<is 1.31×10-10>40
D3-5-IgG252,62×10-10-1,31×10-1020x-40x
D3-6-IgG262,62×10-10-1,31×10-1020x-40x
D3-7-IgG27<is 1.31×10-10>40
D3-8-IgG28 2,62×10-10-1,31×10-1020x-40x
D3-9-IgG29<is 1.31×10-10>40
D3-11-IgG210<l,31×10-10>40
D3-12-IgG211<is 1.31×10-10>40
D3-14-IgG212<is 1.31×10-10>40
D3-15-IgG213<is 1.31×10-10>40
D3-16-IgG214<is 1.31×10-10>40
D3-17-IgG215<is 1.31×10-10>40
D3-19-IgG2162,62×10-10-1,31×10-1020x-40x
D3-20-IgG2172,62×10-10-1,1×10 -1020x-40x
D3-21-IgG2182,62×10-10-1,31×10-1020x-40x
D3-22-IgG2192,62×10-10-1,31×10-1020x-40x
D3-23-IgG2202,62×10-10-1,31×10-1020x-40x
D3-24-IgG2212,62×10-10-1,31×10-1020x-40x
D3-25-IgG2222,62×10-10-1,31×10-1020x-40x
D3-26-IgG2232,62×10-10-1,31×10-1020x-40x
D3-27-IgG2242,62×10-10-1,31×10-1020x-40x
D3-28-IgG2252,62×10-10-1,31×10-1020x-40x
D3-29-IgG2 26<is 1.31×10-10>40
D3-30-IgG2272,62×10-10-1,31×10-1020x-40x
D3-31-IgG228<is 1.31×10-10>40
D3-32-IgG229<is 1.31×10-10>40
D3-33-IgG230<is 1.31×10-10>40
D3-34-IgG2312,62×10-10-1,31×10-1020x-40x
D3-39-IgG2325,23×10-10-2,62×10-1010x-20x
D3-50-IgG2332,62×10-10-1,31×10-1020x-40x
D3-52-IgG2345,23×10-10-2,62×10-1010x-20x
D3-53-IgG2<is 1.31×10-10>40
D3-54-IgG236<is 1.31×10-10>40
D3-56-IgG238<is 1.31×10-10>40
D3-62-IgG244<is 1.31×10-10>40
D3-65-IgG247<is 1.31×10-10>40
D3-66-IgG248<is 1.31×10-10>40
D3-69-IgG250<is 1.31×10-10>40
D3-70-IgG251<is 1.31×10-10>40
D3-71-IgG252<is 1.31×10-10>40
D3-72-IgG253<is 1.31×10-10 >40
D3-73-IgG254<is 1.31×10-10>40
D3-74-IgG255<is 1.31×10-10>40
D3-75-IgG256<is 1.31×10-10>40
D3-76-IgG257<is 1.31×10-10>40

* Note: Identification numbers of the sequences (SEQ ID NOS), are presented in table 3 for human CTLA-4-IgG2, fused proteins Orencia®, LEA29Y-Ig, represent the amino acid sequences of these Ig fused proteins, respectively. SEQ ID nos shown in table 3 for each mutant fused protein, CTLA-4-Ig, refers to the amino acid sequence of the mutant CTLA-4 EVA certain mutant fused protein, CTLA-4-Ig. Data refer to a mutant CTLA-4-Ig, including mutant CTLA-4 EVA (e.g., any of SEQ ID NOS:1-48 and 58-73), fused at its N-end C-end IgG2 Fc polypeptide presented in SEQ ID NO:184 or 218. Methods for the image is of such fused proteins known in the technical field and are described here. However, as noted above, found experimentally that the mutant CTLA-4-Ig produced in Cho cells using a vector comprising the nucleotide sequence encoding the estimated hIgG2 Fc polypeptide presented in SEQ ID NO:184, usually does not include the estimated C-terminal lysine residue (K); thus, hgG2 Fc amino acid sequence of a mutant CTLA-4-IgG2, is that shown in SEQ ID NO:218, where hIgG2 Fc amino acid sequence does not include the C-terminal lysine residue in comparison with the sequence presented in SEQ ID NO:184. Illustrative sequence fused protein, which does not include the C-terminal lysine residue shown in SEQ ID NOS:205-214, 219 and 221.

Table 4 represents the avidity of binding of the mutant CTLA-4-Ig fused proteins with hCD80-mIg, which was measured by a standard Biacore analysis. Specifically table 4 shows the name of the clone mutant fused protein, CTLA-4-Ig; identification number sequence (SEQ ID NO), the corresponding amino acid sequence of Monomeric mutant CTLA-4 EVA fused protein; equilibrium dissociation constant (KD(Both molarity (M)) to analyze the avidity of binding of the ligand hCD80-mIg; and the avidity of binding of the mutant fused protein, CTLA-4-Ig to hCD80-mIg ligand relative to the avidity of St. the statements fused protein Orencia® to the same ligand. For each mutant protein shown to significantly improve the avidity of binding to hCD80-mIg ligand compared to the avidity of binding of the fused protein Orencia® hCD80-mIg ligand (4th column in table 4). Orencia® protein serves as a benchmark, namely with avidity binding hCD80-mIg, adopted 1. Mutant fused proteins, CTLA-4-Ig usually exist as dimeric fused proteins in solution. As shown in table 4, dimeric mutant fused proteins, CTLA-4-Ig of the invention have the avidity of binding to the ligand hCD80-mIg that: (1)at least approximately equal to or greater than the avidity of binding of human CTLA-4-IgG2; (2)at least approximately equal to or greater than the avidity of binding of the fused protein Orencia® to hCD80-mIg ligand; and/or (3)at least approximately equal to or greater than the avidity of binding of the LEA29Y-Ig to hCD80-mIg ligand. As discussed above, each dimeric mutant dimer CTLA-4-Ig can be created using the methods described in example 3 above.

Many mutant fused proteins, CTLA-4-Ig was found to have a dissociation rate from hCD80-mIg fused protein, which is almost equal to or greater than the rate of dissociation of the fused protein Orencia® from the same ligand (data not shown). Some mutant fused proteins, CTLA-4-Ig was found to have a rate of Association with hCD80-mIg, approximately equal to or greater than the soon the be Association fused protein Orencia® with the same ligand (data not shown).

Many mutant fused proteins, CTLA-4-Ig, are presented in table 4, have hCD80-mIg equilibrium dissociation constants (KD), which is lower than hCD80-mIg equilibrium dissociation constant of human CTLA-4-IgG2 or fused protein Orencia®. Moreover, some mutant fused proteins, CTLA-4-Ig, are presented in table 4, have hCD80-mIg equilibrium dissociation constants, which are at least approximately equal hCD80-mIg equilibrium constant LEA29Y-Ig.

Many mutant fused proteins, CTLA-4-Ig, are presented in table 4, have the avidity of binding to hCD80-mIg, which is larger than the avidity of binding of human CTLA-4-IgG2 or fused protein Orencia® to the same ligand (significantly improve hCD80-mIg the avidity of the binding relative to the fused protein Orencia® shown in the 4th column). Additionally, some mutant fused proteins, CTLA-4-Ig, are presented in table 4, have the avidity of binding to hCD80-mIg, which at least approximately equal to the avidity of binding of the LEA29Y-Ig to the same ligand.

Mutant CTLA-4-Ig of the invention, which has a higher avidity binding to hCD80-mIg, than has hCTLA-4-IgG1, Orencia®, or LEA29Y-Ig protein is likely to have an increased in vivo immunosuppressive effect in comparison with the hCTLA-4-IgG2, Orencia® or fused protein LEA29Y-Ig, respectively, such as, for example, therapeutic and/or prophylactic methods for podavini the immune response in a subject (for example, in vivo treatment of diseases or disorders of the immune system of mammals, such as humans, in which immunoinhibitory or immunosuppression are desirable), methods for inhibiting transplant rejection of a tissue or organ from a donor to a recipient (e.g., a mammal, such as, for example, a person) and other methods described in this description.

Table 4
Protein (dimer)SEQ ID NO* (namely, mutant CTLA-4 VDK)KD(M) hCD80 EVAThe avidity of binding Monomeric hCD80 EVA (compared to the dimer fused protein Orencia®)
hCTLA-4-IgG21626,55×10-101,h
dimer fused protein Orencia®1648,77×10-101
LEA29Y-Ig1664,39×10-10-2,19×10-102-4
D1-IgG258<4,39×10-10 >2
D1T-IgG259<4,39×10-10>2
D2-IgG260<4,39×10-10>2
D3-IgG261of 1.75×10-9-4,39×10-100,5x-2x
D4-IgG262of 1.75×10-9-4,39×10-100,5x-2x
D5-IgG263of 1.75×10-9-4,39×10-100,5x-2x
D6-IgG264of 1.75×10-9-4,39×10-100,5x-2x
D20-IgG265of 1.75×10-9-4,39×10-100,5x-2x
D21-IgG266of 1.75×10-9-4,39×10-100,5x-2x
D23-IgG267<4,39×10-10 >2
D24-IgG268of 1.75×10-9-4,39×10-100,5x-2x
D26-IgG269of 1.75×10-9-4,39×10-100,5x-2x
D27-IgG270of 1.75×10-9-4,39×10-100,5x-2x
D28-IgG271of 1.75×10-9-4,39×10-100,5x-2x
D29-IgG272of 1.75×10-9-4,39×10-100,5x-2x
D31-IgG273of 1.75×10-9-4,39×10-100,5x-2x
D3-1-IgG21of 1.75×10-9-4,39×10-100,5x-2x
D3-2-IgG22of 1.75×10-9-4,39×10-100,5x-2x
D3-3-IgG23 -9-4,39×10-100,5x-2x
D3-4-IgG24of 1.75×10-9-4,39×10-100,5x-2x
D3-5-IgG25of 1.75×10-9-4,39×10-100,5x-2x
D3-6-IgG26of 1.75×10-9-4,39×10-100,5x-2x
D3-7-IgG27of 1.75×10-9-4,39×10-100,5x-2x
D3-8-IgG28of 1.75×10-9-4,39×10-100,5x-2x
D3-9-IgG29of 1.75×10-9-4,39×10-100,5x-2x
D3-11-IgG210of 1.75×10-9-4,39×10-100,5x-2x
D3-12-IgG211<4,39×10-10>2
D3-14-IgG2 12of 1.75×10-9-4,39×10-100,5x-2x
D3-15-IgG213of 1.75×10-9-4,39×10-100,5x-2x
D3-16-IgG214of 1.75×10-9-4,39×10-100,5x-2x
D3-17-IgG215of 1.75×10-9-4,39×10-100,5x-2x
D3-19-IgG216of 1.75×10-9-4,39×10-100,5x-2x
D3-20-IgG217of 1.75×10-9-4,39×10-100,5x-2x
D3-21-IgG218of 1.75×10-9-4,39×10-100,5x-2x
D3-22-IgG219of 1.75×10-9-4,39×10-100,5x-2x
D3-23-IgG220of 1.75×10-9-4,39×10-100,5x-2x
D3-24-IgG221of 1.75×10-9-4,39×10-100,5x-2x
D3-25-IgG222of 1.75×10-9-4,39×10-100,5x-2x
D3-27-IgG223of 1.75×10-9-4,39×10-100,5x-2x
D3-28-IgG224of 1.75×10-9-4,39×10-100,5x-2x
D3-29-IgG225of 1.75×10-9-4,39×10-100,5x-2x
D3-30-IgG226of 1.75×10-9-4,39×10-100,5x-2x
D3-31-IgG227of 1.75×10-9-4,39×10-100,5x-2x

Protein (dimer)SEQ ID NO* (namely, mutant CTLA-4 VDK)KD(M) hCD80 EVAAVI is of binding Monomeric hCD80 EVA (compared to the dimer fused protein Orencia®)
D3-32-IgG228<4,39×10-10>2
D3-33-IgG229of 1.75×10-9-4,39×10-100,5x-2x
D3-34-IgG230of 1.75×10-9-4,39×10-100,5x-2x
D3-39-IgG231of 1.75×10-9-4,39×10-100,5x-2x
D3-50-IgG232of 1.75×10-9×4,39×10-100,5x-2x
D3-52-IgG233of 1.75×10-9-4,39×10-100,5x-2x
D3-53-IgG235<4,39×10-10>2
D3-54-IgG236of 1.75×10-9-4,39×10-100,5x-2x
D3-56-IgG238of 1.75×10-9-4,39×10 -100,5x-2x
D3-62-IgG244<4,39×10-10>2
D3-65-IgG247<4,39×10-10>2
D3-66-IgG248<4,39×10-10>2
D3-69-IgG250<4,39×10-10>2
D3-70-IgG251<4,39×10-10>2
D3-71-IgG252<4,39×10-10>2
D3-72-IgG253<4,39×10-10>2
D3-73-IgG254<4,39×10-10>2
D3-74-IgG255<4,39×10-10>2
D3-75-IgG256<4,39×10-10>2
D3-76-IgG257of 1.75×10-9-4,39×10-100,5x-2x
* Note: SEQ ID NOS shown in table 4 for human CTLA-4-IgG2, fused proteins Orencia®, LEA29Y-Ig, represent the amino acid sequences of these fused proteins, respectively. SEQ ID nos shown in table 4 for each mutant fused protein, CTLA-4-Ig, determines the amino acid sequence of a mutant CTLA-4 EVA certain mutant fused protein, CTLA-4-Ig. Data refer to a mutant CTLA-4-Ig, including mutant CTLA-4 EVA (e.g., any of SEQ ID NOS:1-48 and 58-73), fused at its N-end C-end IgG2 Fc polypeptide presented in SEQ ID NO:184 or 218. However, we found experimentally using MSIH analysis of mutant CTLA-4-Ig produced in Cho cells using a vector comprising the nucleotide sequence encoding the estimated MgG2 Fc polypeptide presented in SEQ ID NO:184, usually does not include the estimated C-terminal lysine residue (K); thus, hIgG2 Fc amino acid sequence of a mutant CTLA-4-IgG2 is that p is the cauldron in SEQ ID NO:218, where hIgG2 Fc amino acid sequence usually does not include the C-terminal lysine residue compared to the sequence represented in SEQ ID NO:184. Illustrative sequence fused protein, which does not include the C-terminal lysine residue shown in SEQ ID NOS:205-214, 219 and 221.

Monomeric kinetic studies

To further confirm the binding properties of the mutant CTLA-4-Ig fused proteins were obtained using monovalent kinetic binding analysis. These studies measure the kinetics of binding of divalent analyzed protein (e.g., dimeric mutant fused protein, CTLA-4-Ig), which covered the touch chips, and monovalent ligand (treated with papain hCD86-mIg) in the mobile phase. Antibody goat against human IgG (Jackson ImmunoResearch, #109-005-098) was associated with SM sensor chips according to the manufacturer's protocols, typically with the output of 15000 units response (SW). The ligand was captured by incubation of sensor chips, antibody-coated with 10 μl of 2 μg/ml solution of dimeric mutant CTLA-4-Ig fused proteins in HBS-EP buffer at a flow rate of 10 ál/min Levels capture ligand was typically 25-80 SW. Monomeric ligands hCD86-mIg (generated by treatment with papain and protein-And sepharose adsorption hCD86-mIg, as described in Hermanson, G.T. BIOCONJUGATE TECHNIQUES, Academic Press, 1996) is asfodeli in HBS-EP buffer and poured through touch chips, covered the analyzed protein for 2 min at 30 μl/min, 2 min, followed by incubation in HBS-EP buffer that does not contain protein, at the same flow rate. Recovery between cycles was performed by 3-minute incubation with 10 mm glycine buffer (pH 1,7) at 30 μl/min Typical 8 dilutions Monomeric proteins analyzed in the range from 3000 nm to 0.2 nm were analyzed against a blank sample (only HBS-EP buffer) in two Parallels. Rmax signal levels to bind to the mutant CTLA-4-Ig proteins were in the range of 10-60 SW.

For kinetic analysis of data from Monomeric binding studies were selected, as described above, except that the selection of data Association was started and finished within 5 seconds from the start time and the end of injection, and the data selection dissociation was started after 5 seconds after the end time of injection, and usually included 1-60 sec period dissociation. These data are further analyzed in relation to the equations of dynamic equilibrium of affinely using BIAevaluation software. Levels of dynamic equilibrium binding for each concentration (Answerequally("Req") values) were averaged in the range of 5-20 sec about the completion of injection of the sample using BIAevaluation "main alignment:average" function. The steady-state affinity was determined from the graph Req vs concentration with the use of the group BIAevaluation program according to the formula Req=K A×With×Rmax/(KA×S×n+1), where C is the concentration of the analyzed agent, and n, the factor steric interference is 1, and KD=1/KA. In some cases, observed the actual nonspecific binding represented residual flat projection of the R-values after dissociation. These data were corrected by subtraction of the residual R-values from the values of Req. KDthen was calculated in GraphPad Prism program (GraphPad Software, Inc.) using the model of "one site-specific binding".

It is a Monomeric binding study was performed on a subset of representative mutant proteins, CTLA-4-Ig and the results are summarized in table 5. In General, the degree of improvement hCD86 EVA binding mutant fused proteins, CTLA-4-Ig relatively LEA29Y-Ig observed in Monomeric binding studies was similar to that observed for hCD86-mIg-binding mutant fused proteins, CTLA-4-Ig relatively LEA29Y-Ig in the standard kinetic studies. These results confirm the assumption that the observed improvements in the kinetic characteristics of binding to mutant proteins are the result of actual improvements affinely binding mutant proteins (e.g., compared to Orencia® and/or LEA29Y-Ig fused proteins) and not due to possible artifacts caused by high valence gregorova the different protein preparations.

Table 5
Protein (dimer)SEQ ID NO* (a that is, mutant CTLA-4 VDK)KD(M) Monomeric hCD86 EVA.The binding affinity of Monomeric hCD86 EVA (compared with LEA29Y-Ig)
LEA29Y-Ig166by 1.68×10-61
D3-IgG261<to 3.36×10-7>5x
D3-04-IgG24<to 3.36×10-7>5x
D3-11-IgG210<to 3.36×10-7>5x
D3-12-IgG211<to 3.36×10-7>5x
D3-14-IgG212<to 3.36×10-7>5x
D3-17-IgG215<to 3.36×10-7 >5x
D3-20-IgG2178,4×10-7-3,36×10-72x-5x
D3-27-IgG2248,4×10-7-3,36×10-72x-5x
D3-29-IgG226<to 3.36×10-7>5x
D3-31-IgG228<to 3.36×10-7>5x
D3-53-IgG235<to 3.36×10-7>5x
* Note: the SEQ ID NO shown in table 5 for LEA29Y-Ig, is the number of the amino acid sequence of LEA29Y-Ig fused protein. SEQ ID nos shown in table 5 for each mutant fused protein, CTLA-4-Ig, determines the amino acid sequence of a mutant CTLA-4 EVA that is present in a mutant CTLA-4-Ig fused protein. Data refer to a mutant CTLA-4-Ig, including mutant CTLA-4 EVA (e.g., any of SEQ ID NOS:4, 10-12, 15, 17, 24, 26, 28, 35 and 61), fused at its N-end C-end IgG2 Fc polypeptide presented in SEQ ID NO:184 or 218. However, the it was found experimentally using MSIH analysis, that mutant CTLA-4-Ig produced in Cho cells using a vector comprising the nucleotide sequence encoding the estimated hIgG2 Fc polypeptide presented in SEQ ID NO:184, usually does not include the estimated C-terminal lysine residue (K); thus, hIgG2 Fc amino acid sequence of a mutant CTLA-4-IgG2 is that shown in SEQ ID NO:208, and hIgG2 Fc amino acid sequence usually does not include the C-terminal lysine residue compared to the sequence represented in SEQ ID NO:184. Illustrative sequence fused protein, which does not include the C-terminal lysine residue shown in SEQ ID NOS:205-214, 219 and 221. Illustrative sequence fused protein, which include C-terminal lysine residue shown in SEQ ID NOS:74-79, 197-200, 220 and 222.

Example 5

Measurement of biological activity of CTLA-4 mutants using studies of proliferation of human IPC (peripheral blood mononuclear cells) (stimulation of anti-CDS antibodies)

It is shown that CTLA-4-Ig and its specific variants are strong inhibitors of T-cell proliferation in vitro (see, e.g., Larson et al., Am. J. Transplantat, 443). To measure improved activity of mutant proteins, CTLA-4-Ig in these studies produced an analysis of the proliferation of peripheral blood mononuclear cells (IPC).

Human blood (freshly harvested donor program) was diluted with an equal volume of PBS and was fractionally to allocate IPC using Histopaque (Sigma, #10771) Ficoll gradient in accordance with the conditions recommended by the manufacturer. The IPC were diluted in growth medium (DMEM/F12 medium (Invitrogen, #10565-018), supplemented with 10% FBS (Hyclone #SV30014,03) and 1x PSG (Invitrogen, #10378-016)), and added to 96-cellular tablets for culture (BD Biosciences, #353077) at a density of 1×10 cells/cell. The analyzed compounds were serially diluted in growth medium and added to cells in three Parallels. Cell proliferation was initiated by addition of mouse antibodies against human CD3 (BD Pharmingen: 555329) to a final concentration of 5 μg/ml After incubation at 37°C for 2 days (days)3H thymidine (GE Healthcare, #TRK758-5MCI) was added at a concentration of 1 MX/cell and the plates were incubated at 37°C for an additional 16 hours. Cells were collected cell collector (FilterMate Omnifilter-96 Harvester) using conditions recommended by the manufacturer and measured embedding3H thymidine using a scintillation counter (Wallac Trilux, #1450-421). The degree of embedding3H thymidine (capture3H thymidine) is the exponent of the T-cell proliferation. Embedding3H-thymidine was measured using standard techniques. The proliferation of T-cells is expressed as the average of the number per minute (CME) from three parallel cells.

Data cell proliferation was analyzed by the program GraphPad Prism 5 using the model curve for non-linear regression (sigmoidal response to dose, variable slope) and the selection of the method of least squares. Shows the results of the parameters IC50 (also shown as IC50) and their associated 95% confidence intervals (table 6). Figure 6 shows curves of cell proliferation from a representative analysis of the IPC proliferation (using stimulation with anti-CD3 antibodies), including a list of illustrative mutant CTLA-4-Ig fused protein according to the invention - namely, D3-04-IgG2, D3-11-IgG2, D3-12-IgG2 and D3-14-IgG2 fused proteins. The graph depicts the dependency injection3H thymidine (quantities per minute (KVM)) of protein concentration (nanomoles (nm)). Embedding3H thymidine (capture3H thymidine), which is expressed in the degree of cell proliferation, was measured by standard methods.

Orencia®, LEA29Y-Ig fused proteins were included in the study as controls for comparison. These results indicate that the mutant CTLA-4-Ig Ig fused proteins according to the invention have significantly higher strength or more ability than Orencia® and/or LEA29Y-Ig protein(proteins) in the inhibition or suppression of polyclonal T cell activation or T-cell proliferation in vitro.

Analysis of the IPC proliferation was carried out using the other mu is based CTLA-4-Ig fused protein according to the invention. Table 6 provides summary data for a representative list of mutant CTLA-4-Ig fused protein according to the invention. Table 6 presents a comparison of the average values of IC50 (nanomoles (nm)) for illustrative mutant CTLA-4-Ig fused proteins against controls (Orencia®, hCTLA-4-IgG2 and LEA29Y-Ig fused proteins) in the analysis of the IPC proliferation (stimulation of anti-CD3 antibodies). The IC50 value represents the concentration of compounds (e.g., mutant fused protein, CTLA-4-Ig, hCTLA-4-IgG2, Orencia® or LEA29Y-Ig), which is required for 50% inhibition of T-cell proliferation in vitro. IC50 values from separate experiments were averaged to obtain mean values of IC50 were used for statistical analyses. Univariate analysis ANOVA with a posteriori test Dannetta or Bonferroni was used to compare mutant CLTA-4-Ig fused proteins and hCTLA-4-IgG2 with Orencia® or LEA29Y-Ig fused protein, respectively (CW. Dunnett, New Tables for Multiple Comparisons with Control, Biometrics 20(3):482-491 (Sept. 1964); Abdi, Herve, The Bonferroni and Sidak corrections for multiple comparisons", in ENCYCLOPEDIA OF MEASUREMENT AND STATISTICS (NJ. Salkind, ed., Thousand Oaks, CA 2007); also available at world wide web site utdallas.edu/~herve/Abdi-Bonferroni2007-pretty.pdf.). Statistical analysis of Ig fused protein consisting of one of the following mutant CTLA-4 EVA polypeptides clone D24, D3-07, D3-15 and D3-16 - were not, as n=1. The term "(mean log IC50)" represents the standard deviation from the average C is achene log IC50.

Table 6
Compilation of data from illustrative studies IPC proliferation using stimulation with anti-CD3 antibody
Protein (dimer)The average IC50 (nm)The mean log IC50 (nm)WITH (average log IC50) (nm)
dimer fused protein Orencia®5,160,710,51
hCTLA-4-Ig8,460,930,43
LEA29Y-Ig0,481-0,320,54
D3-04-IgG20,051,2-1,270,45
D3-07-IgG20,12-0,93BUT
D3-ll-IgG20,081,2-1,070,40
D3-12-IgG2 1,2-1,270,17
D3-14-IgG20,071,2-1,170,15
D3-15-IgG20,21-0,69BUT
D3-17-IgG20,041,2-1,360,37
D3-20-IgG20,071,2-1,190,37
D3-26-IgG20,151-0,830,49
D3-27-IgG20,101,2-1,000,41
D3-29-IgG20,071,2of 1.130,53
D3-30-IgG20,181-0,740,35
D3-31-IgG20,041,2-1,420,25
D3-32-IgG20,101-1,000,47
D3-33-IgG20,121-0,940,69
D3-34-IgG20,4110.39 per0,18
D3-39-IgG20,471-0,330,21
D3-50-IgG20,351-0,460,23
D3-52-IgG20,4910.31 supranational0,20
D3-53-IgG20,021,2-1,690,24
D3-54-IgG20,171-0,770,22
D3-56-IgG20,081-,080,20
D3-62-IgG20,051,2-,29 0,23
D3-69-IgG20,051,2-,340,19
D3-70-IgG20,081-,100,10
D3-71-IgG20,05-,32BUT
D3-72-IgG20,031,2-,540,19
D3-73-IgG20,231-0,640,25
D3-75-IgG20,081-1,110,33
D3-76-IgG20,131-0,880,36
D3-IgG20,141-0,850,13
D24-IgG20,12-0,93BUT

Superscripts, before the purposes of table 6, are as follows:1Statistically different from Orencia with p<0.05 as determined using one way ANOVA with a posteriori test Dannetta.2Statistically different from the LEA with p<0.05 as determined using one way ANOVA with a posteriori test Bonferroni. Slit proteins, including D3-7, D3-15, D3-71 and D24 mutant CTLA-4 EVA analyzed once, and therefore were not conducted any statistical comparison. Note: the Expression "BUT" in table 6 means "undefined".

Statistical analysis shows that all mutant fused proteins, CTLA-4-Ig, which were analyzed in at least two separate studies and denoted by an upper index (1) (see table 6), were statistically higher in strength compared to Orencia® and hCTLA-4-IgG2 fused protein (p<0,05) (namely, have a greater ability to suppress or inhibit T-cell proliferation in in vitro assays IPC than Orencia® and hCTLA-4-IgG2 fused proteins). Those marked with an upper index (2) (see table 6), also statistically superior in strength LEA29Y-Ig protein (p<0,05) (namely, have a greater ability to suppress or inhibit T-cell proliferation in in vitro assays IPC than LEA29Y-Ig protein), which was determined using univariate analysis ANOVA with a posteriori tests Dunn the TTA and Bonferroni, discussed above. Not any statistical difference between the human CTLA-4 fused proteins comprising either a modified IgG1 Fc (as in Orencia®), or wild-type IgG2 Fc (as in hCTLA4-IgG2). This study implies that differences in functional activities, are presented in table 6 between the mutant CTLA-4-Ig fused protein according to the invention (each of which includes a human IgG2 Fc) and either Orencia®, or hCTLA4-IgG2 were a direct consequence of amino acid changes (namely, amino acid substitutions), produced in CTLA-4 EVA region. Differences in functional activity between the mutant CTLA-4-Ig fused protein according to the invention and, for example, Orencia® (which includes a modified IgG1 Fc), were not a consequence of differences in their respective Ig Fc amino acid sequences.

Assume that, having increased the ability of the mutant CTLA-4-Ig fused protein according to the invention to suppress or inhibit T-cell proliferation in in vitro studies compared to Orencia®, LEA29Y-Ig, and/or hCTLA-4-IgG2 fused proteins, such mutant proteins will also be increased immunosuppressive capacity in in vivo therapeutic and/or prophylactic methods compared to Orencia®, LEA29Y-[g and/or hCTLA-4-IgG2 fused proteins. Each mutant protein, CTLA-4-Ig of the invention, it is believed, has the most ways is the ability to suppress or inhibit T-cell proliferation in vivo methods or applications regarding Orencia®, LEA29Y-Ig, and/or hCTLA-4-IgG2 fused proteins, such as therapeutic and/or prophylactic methods for suppressing or inhibiting an immune response in a subject (e.g., in vivo treatment of diseases or disorders of the immune system in a mammal, such as, for example, people), methods for suppressing or inhibiting transplant rejection of a tissue or organ from a donor to a recipient (e.g., a mammal, such as for example, a person) and/or other treatment or diagnostic methods described in this specification.

Applying the same statistical analyses, we found no statistical differences between Orencia® fused protein containing the human CTLA-4 EVA-mutant IgG1) and human CTLA-4-IgG2. Thus, it is assumed that differences in Ig domains of these molecules (namely, a mutant IgG1 fused protein Orencia® and human IgG2 hCTLA-4-IgG2) do not affect the functionality of these molecules. See figure 11. The inhibition of proliferation observed with higher doses, fused protein Orencia®, was not significantly different from that observed with CTLA-4-IgG2, indicating that their respective immunosuppressive activity are not displaced in accordance with their different IgG isotypes, but are rather a result of their hCTLA-4 EVA polypeptides. Thus, differences in immunosuppressive activities m the waiting mutant CTLA-4-Ig polypeptides according to the invention and Orencia® fused protein (or LEA29Y-Ig, because it contains the same Ig, as Orencia® protein) cannot be attributed to their respective Fc regions comprising different IgG isotypes.

The invention includes a Monomeric mutant CTLA-4 EVA proteins that have the ability and in some cases a greater ability to suppress or inhibit T-cell activation or proliferation than Monomeric protein of human CTLA-4 or its extracellular domain. Also provided Monomeric mutant fused proteins, CTLA-4 EVA, who have the ability and in some cases a greater ability to suppress or inhibit T-cell activation or proliferation than Monomeric protein hCTLA-4-Ig or its extracellular domain. Also included in the scope of the invention mutant protein dimers CTLA-4 EVA, who have the ability and in some cases a greater ability to suppress or inhibit T-cell activation or proliferation than dimer comprising two human extracellular domain of CTLA-4. Some mutant dimers fused protein, CTLA-4 EVA according to the invention (for example, mutant dimers fused protein, CTLA-4-EVA-Ig) have the ability and in some cases a greater ability to suppress or inhibit T-cell activation or proliferation than hCTLA-4-IgG2 dimer fused protein, Orencia® dimer fused protein and/or LEA29YIg dimer fused protein.

Example 6

Measurement of biological the practical activity of mutant CTLA-4-Ig molecules using studies of human CD4 +T-cell proliferation.

It is shown that human CTLA-4-Ig and its specific variants inhibit T-cell proliferation by blocking the transmission signal CD80 and CD86 through CD28 (Linsley P.S., Immunity 1:793-801 (1994); S.R. Larson et al., Am. J. Transplant. 5:443-453 (2005)). Because mutant CTLA-4-Ig proteins according to the invention have improved avidity binding to CD86-Ig ligand, doing research CD4+T-cell proliferation to measure the activity of mutant proteins, CTLA-4-Ig in blocking the transmission of the signal through CD86.

Creating a DNA sequence that encodes a full-sized human CD86 protein

Created plasmid pCDNA3,1 hB7,2 FL, to encode a full-sized human CD86 protein for expression on the surface transfection cells. DNA encoding human CD86, was obtained by PCR amplification of cDNA derived from human leukocytes (BD Biosciences, Cat# HL4050AH) using forward and reverse oligonucleotide primers, designed on the basis of sequence homology with the nucleotide sequence encoding CD86 shown in SEQ ID NO:176. The primers were designed, created and assembled using standard techniques, well known to experts in the art and they include the stop and start codons and restriction sites if necessary. The procedure used PCR amplification so the e is well known in the art. Such techniques are described in, for example, Berger, Ausubel and Sambrook, all see above. 50 nanograms (ng) of cDNA used as a template in a 100 μl PCR reaction with 1 μm forward and reverse primers, Herculase Polymerase buffer (Stratagene; #600260) and 200 μm dNTP for 30 cycles of amplification (94°C, 30 sec; 50°C, 30 sec; 72°C, 60 sec). PCR product was purified using QiaQuick PCR spin columns (Qiagen #28106) and was treated with restriction enzymes Kpnl and Notl. The fragments were separated by electrophoresis on agarose gel, purified using Qiaquick kit gel extraction (Qiagen, #28704) according to the manufacturer's instructions and ligated into similarly treated with enzymes plasmid kDNK 3.1(+) (Invitrogen, Cat. #V790-20). The ligation products were transformed into TOP10 E. coli cells (Qiagen, Cat. #S-10) in accordance with the manufacturer's recommendations. Transformed cells were incubated in LB (broth, Luria)containing 50 μg/ml carbenicillin at 250 rpm overnight at 37°C and then used to create maxiprep (Qiagen; #12362) flow of plasmid DNA in accordance with the conditions recommended by the manufacturer.

The putative amino acid sequence of a full-sized human CD86 protein shown in SEQ ID NO:175. In this sequence of amino acid residues 1-23 include a projected signal sequence, amino acid residues 24-241 include human CD86, unclutch the first domain, amino acid residues 242-270 include a transmembrane domain, and amino acid residues 271-329 include cytoplasmic domain.

The creation of stable cell lines expressing human CD86 on the cell surface

HEK293 cells were grown to 80-90% of confluently in T-75 flasks containing 20 ml of growth medium (DMEM/F12 medium (Invitrogen, Cat. #10565-018), supplemented with 10% FBS (Hyclone Cat. #SV30014,03) and 1x PSG (Invitrogen, Cat. #10378-016)). The cells were transfectional with 10 μg of plasmid DNA (pCDNA3,1 hB7,2 FL)mixed with 60 μl of Fugene 6 (ROCHE, #11814443001) in accordance with the conditions recommended by the manufacturer. Cells were incubated for 2 days (days) at 37°C in growth medium and then incubated for 10 days. At 37°C in the environment of selection (growth medium containing 300 μg/ml of geneticin (Invitrogen, #10131-027)), replacing the medium every 2 days. To ensure FACS sorting transfection cells were stained with FITC-labeled anti-CD86 antibody (BD Biosciences, #555) in accordance with the conditions recommended by the manufacturer. Using cell sorting device (Dako, MoFlo)skipping FITC signal, CD86-positive cells individually sorted into 96-cellular tablets for culture (Sigma-Aldrich, #CLS-3596)containing 200 μl/cell growth medium containing 25% conditioned medium (growth medium, previously collected from retrospectively (or native) cell cultures). the donkey incubation at 37°C for 13-19 days, cells were dissipated trypsinogen hydrolysis and transferred into 24-cellular tablets for culture containing 0.5 ml/cell growth environment. After incubation at 37°C for 7 days, cells were dissipated trypsinogen hydrolysis and transferred to T-75 flasks containing 20 ml of growth medium. Finite cell lines were chosen on the basis of high levels of CD86 expression on the cell surface, which was measured by FACS analysis of cells stained with FITC-labeled anti-CD86 antibody using FACS Caliber (BD Biosciences), in accordance with the conditions recommended by the manufacturer.

Research CD4+T-cell proliferation

CD4+T cells were enriched to >96% of the human leukocyte preparations films (Stanford University Blood Center, Stanford, CA) using EasySep set of positive selection of human CD4 (StemCell Technologies, #18052R) with a magnetic cell separator (RoboSep, StemCell Technologies, #20000), following the manufacturer's recommendations. Enriched CD4+T cells were brought to a density of 1×106cells/ml in Yssel medium (Gemini Bio-Products, #400-102), supplemented with 10% FBS (Hyclone SV30014,03), and added to 96-cellular tablets for tissue culture at a concentration of 50 μl/cell. NECK cells expressing membranectomy human CD86, were irradiated at 6000 rad (Stanford Research Institute, Menio Park, CA), brought up to 1×106cells/ml in the same medium and added to the plates for culture in Konz is Tracii 50 μl/cell. The analyzed compounds were serially diluted in the same medium and added to cells in three Parallels. Cell proliferation was initiated by addition of mouse antibodies against human CD3 (BD Pharmingen: 555329) to a final concentration of 5 μg/ml After incubation at 37°C for 3 days.3H thymidine (GE Healthcare, #TRK75S-5MCI) was added at a concentration of 1 MX/cell and the plates were incubated at 37°C. the next 18 hours. Cells were harvested using cell collector (Perkin Elmer Filter Harvester D961962) and3H thymidine was measured using a liquid scintillation counter (Wallac Trilux 1450) in accordance with the conditions recommended by the manufacturer. Data cell proliferation was analyzed by the program GraphPad Prism 5 using equation variable slope (Y=bottom+(top-bottom)/(1+10^((LogIC50-X)(slope))) to obtain IC50 for each of the analyzed compounds. The term "(LogIC50-X)(slope)is the exponent in the equation.

Figure 7 shows curves of cell proliferation from representative studies of CD4+T-cell proliferation, including an illustrative list of mutant CTLA-4-Ig fused protein according to the invention - namely, D3-04-IgG2, D3-11-IgG2, D3-12-IgG2 and D3-14-IgG2. Orencia®, LEA29Y-Ig fused proteins included in the analysis as controls for comparison. Shows a graph of the dependency injection3H thymidine (CME) concentration (nm) of the protein. Embedding3H time the on - it is a measure of the degree of cell proliferation, it was measured by standard methods. These results indicate that mutant fused proteins, CTLA-4-Ig of the invention have significantly higher strength or more ability than Orencia® and/or LEA29Y-Ig fused proteins in the inhibition or suppression of CD86 costimulation in vitro.

This analysis of CD4+T-cell proliferation was carried out on some other mutant fused protein, CTLA-4-Igx according to the invention. Table 7 presents summary data for illustrative list of mutant CTLA-4-Ig fused protein according to the invention. Table 7 presents a comparison of average values of IC50 (nanomoles (nm)) for illustrative mutant CTLA-4-Ig fused proteins against reference controls (Orencia®, LEA29Y-Ig, and fused proteins of human CTLA-4-IgG2) using analysis of CD4+T-cell proliferation. IC50 values from separate experiments were averaged to obtain mean values of IC50, which was used in the statistical analyses. The term "(mean log IC50)" represents the standard deviation from the average log IC50.

Table 7
Summary of illustrative studies of CD4+T-cell proliferation
Protein (dimer) The average IC50 (nm)The mean log IC50 (nm)WITH (average log IC50) (nm)
dimer fused protein Orencia®1,560,190,22
hCTLA-4-Ig2,240,350,28
LEA29Y-Ig0,21-0,670,25
D3-02-IgG20,03-1,56BUT
D3-03-IgG20,041,2-1,400,11
D3-04-IgG20,031,2-1,470,02
D3-06-IgG20,051-1,320,03
D3-11-IgG20,031,2-1,520,07
D3-12-IgG20,041,2of-1.45 0,09
D3-14-IgG20,041,2-1,370,11
D3-15-IgG20,021,2-1,620,08
D3-17-IgG20,031,2-1,470,11
D3-20-IgG20,041,2-1,400,11
D3-27-IgG20,041,2-1,370,10
D3-29-IgG20,041,2-1,360,13
D3-31-IgG20,031,2-1,580,21
D3-34-IgG20,051,2-1,280,13
D3-39-IgG20,051,2-1,350,10
D3-50-IgG20,061,2 -1,230,24
D3-52-IgG20,071,2-1,140,24
D3-53-IgG20,021,2-1,700,25
D3-54-IgG20,041,2-1,400,12
D3-56-IgG20,041,2-1,410,07
D3-62-IgG20,041,2-1,440,09
D3-65-IgG20,061,2-1,260,14
D3-69-IgG20,031,2-1,590,14
D3-70-IgG20,051,2-1,300,06
D3-71-IgG20,041,2-1,430,09
D3-72-IgG2 0,031,2-1,480,05
D3-73-IgG20,061,2-1,260,18
D3-75-IgG20,031,2-1,150,12

D2-IgG20,031,2-1,520,18
D3-IgG20,041,2of-1.450,17
The upper indices are presented in table 7 are as follows:1Statistically different from Orencia with p<0.05, which was determined using single-way ANOVA with a posteriori test Dannetta.2Statistically different from the LEA with p<0.05, which was determined using single-way ANOVA with a posteriori test Bonferroni. Protein, including D3-02 mutant CTLA-4 EVA analyzed once, so could not provide a statistical comparison.

Statistical analysis shows that all mutant fused proteins, CTLA-4-Ig, which were analyzed in at least the Vuh separate research and denoted by an upper index (1) (see table 7), statistically superior in strength Orencia® and hCTLA-4-IgG2 fused proteins (p<0,05) (namely, have a greater ability to suppress or inhibit T-cell proliferation in vitro CD44+T-cell research than Orencia® and hCTLA-4-IgG2 fused proteins). Those that were indicated by the upper index (2) (see table 7), also statistically superior in strength LEA29Y-Ig protein (p<0,05) (namely, have a greater ability to suppress or inhibit T-cell proliferation in vitro CD4+T-cell research than LEA29Y-Ig protein), which was determined using univariate analysis ANOVA with a posteriori tests Dannetta and Bonferroni discussed above. Not any statistical difference between the fused proteins of human CTLA-4, including any modified IgGI Fc (as in Orencia®), or wild-type IgG2 Fc (as in hCTLA4-IgG2). This study implies that differences in functional activities, are presented in table 7, between mutant fused proteins, CTLA-4-Ig of the invention (each of which include human IgG2 Fc) and either Orencia®, or hCTLA4-IgG2 were a direct consequence of amino acid changes (namely, amino acid substitutions made in the field of CTLA-4 EVA. Differences in functional activity between the mutant CTLA-4-Ig fused protein according to the invention and, for example, Orencia® (which includes modificar the bath IgGI Fc) were not a consequence of differences in their respective Ig Fc amino acid sequences.

Assume that, having increased the ability of the mutant CTLA-4-Ig proteins according to the invention to suppress or inhibit C-mediated costimulation T cells (e.g., human T cells) in vitro studies compared to Orencia®, LEA29Y-Ig, and/or hCTLA-4-IgG2 fused proteins, such mutant proteins will also be increased immunosupressor power in in vivo therapeutic and/or prophylactic methods compared to Orencia®, LEA29Y-Ig, and/or human fused proteins, CTLA-4-Ig (e.g., human CTLA-4-IgG2 ("hCTLA-4-IgG2")) respectively. In one aspect mutant fused proteins, CTLA-4-Ig of the invention are believed to have a greater ability to suppress or inhibit SW-mediated costimulation T cells (e.g., human T cells) in vivo methods or applications compared to Orencia®, LEA29Y-Ig, and/or fused proteins of human CTLA-4-Ig (e.g., hCTLA-4-IgG2), respectively, such as, for example, therapeutic and/or prophylactic methods for suppressing or inhibiting an immune response in a subject (for example, in vivo treatment of diseases or disorders of the immune system in a mammal, such as, for example, people), methods for suppressing or inhibiting transplant rejection of a tissue or organ from donorrecipient (for example, a mammal, such as, for example, a person) and/or other treatment or diagnostic methods described in this specification.

Example 7

Measurement of the biological activity of mutant molecules CTLA-4-Ig using research on the proliferation of human IPC (antigenic stimulation of memory).

Activation of T-cell memory is an important aspect of autoimmunity (Rogers NJ., et al. Eur. J. Immunol 35:2909-2919 (2005)). To measure the immunosuppressive activity of mutant proteins, CTLA-4-Ig in this respect, the study of proliferation of peripheral blood mononuclear cells (IPC) was carried out using the PPD antigenic stimulation.

Human blood (svejesobranna on donor program) was diluted with an equal volume of PBS and was fractionally to allocate IPC using Histopaque (Sigma, #10771) Ficoll gradient in accordance with the conditions recommended by the manufacturer. IPC was diluted in RPMI medium (Sigma, #R8758), supplemented with 10% FBS (Hyclone # SV30014.03) and 1x PSG (penicillin, streptomycin and glutamine) (Invitrogen, #10378-016) and added to 96-cellular tablets for culture (BD Biosciences, #353077) at a density of 1×105cells/cell. The analyzed compounds were serially diluted in the same medium and added to cells in four Parallels. Cell proliferation was initiated by addition of PPD antigen (purified protein derivative from Mycobacterium tuberculosis, Mycos, #P-1000-001) to of course the concentration of 5 µg/ml After incubation at 37°C for 5 days.3H thymidine (GE Healthcare, #TRK758-5MCs) was added at a concentration of 1 MX/cell and the plates were incubated at 37°C. the next 18 hours. Cells were collected cell collector (FilterMate Omnifilter-96 Harvester, Perkin Elmer) using conditions recommended by the manufacturer, and was measured by embedding3H thymidine using a scintillation counter (Wallac Trilux, #1450-421). Data cell proliferation was analyzed using GraphPad Prism 5 software using model-curve non-linear regression (sigmoidal response to dose, variable slope) and the selection of the method of least squares. Recorded IC50(or "IC50") parameters and their associated 95% confidence intervals.

Figure 8 shows curves of cell proliferation from a representative analyses of the IPC proliferation, including a list of illustrative mutant CTLA-4-Ig fused protein according to the invention - namely, D3-IgG2, D3-12-IgG2, D3-17-IgG2, D3-29-IgG2 fused proteins. Orencia®, LEA29Y-Ig fused proteins were included in the analyses as controls for comparison. Shows a graph of the dependency injection3H thymidine (CME) of protein concentration (nm). Embedding3H thymidine, which indicates the degree of cell proliferation, was measured by standard methods. These results show that in one aspect mutant fused proteins, CTLA-4-Ig on izaberete the Oia have significantly more power than Orencia® and/or LEA29Y-Ig proteins in the inhibition or suppression of cell proliferation T-cell memory in vitro (e.g., mutant proteins, CTLA-4-Ig have more power than Orencia® and/or LEA29Y-Ig proteins in the inhibition or suppression of cell proliferation T-cell memory in vitro studies of cell proliferation of human IPC (antigenic stimulation of memory).

This analysis of the IPC proliferation to PPD antigen stimulation was carried out on some other mutant fused protein, CTLA-4-Igx according to the invention. Table 8 presents summary data of the illustrative list of mutant fused proteins, CTLA-4-Ig of the invention. Table 8 presents a comparison of average values of IC50 (nanomoles (PM)) for illustrative mutant fused proteins, CTLA-4-Ig against reference controls (Orencia®, LEA29Y-Ig fused proteins) using the analysis of the IPC proliferation with antigenic stimulation of memory. IC50 values from separate experiments were averaged to obtain mean values of IC50, which was used in the statistical analyses. The term "(mean log IC50)" represents the standard deviation from the average log IC50.

Table 8
Summary of illustrative studies IPC proliferation using PPD antigenic stimulation
Protein (dimer)The average IC50 (nm)The mean log IC50 (nm)WITH (average log IC50) (nm)
Dimer fused protein Orencia®of 5.920,670,37

LEA29Y-Ig0,31-0,710,53
D3-IgG20,03-1,49BUT
D3-12-IgG20,06-1,270,17
D3-14-IgG20,07-1,170,15
D3-17-IgG20,07-1,240,25
D3-20-IgG20,13-0,910,18
D3-27-IgG20,17-0,840,30
D3-29-IgG2on 07 -1,160,18
D3-34-IgG20,51-0,29BUT
D3-50-IgG20,28be 0,55BUT
D3-53-IgG20,05-1,29BUT
D3-54-IgG20,01-2,00BUT
D3-56-IgG20,01-1,92BUT
D3-69-IgG20,01-2,00BUT
D3-71-IgG20,01-1,87BUT
D3-75-IgG20,01-2,02BUT
D3-76-IgG20,01-1,89BUT
Note: the Expression "BUT" in table 8 indicates "not determined the flax".

Statistical analyses show that all analyzed mutant fused proteins, CTLA-4-Ig statistically superior in strength as Orencia®and fused proteins LEA29Y-Ig p<0.05, which was determined by phase analysis ANOVA with a posteriori tests Dannetta and Bonferroni, respectively, which were discussed above (e.g., mutant fused proteins, CTLA-4-Ig have more power than Orencia® and/or LEA29Y-Ig fused proteins in the inhibition or suppression of the proliferation of T-memory cells in vitro analyses of cell proliferation of human IPC (antigenic stimulation of memory)).

Assume that, having increased the ability of the mutant fused proteins, CTLA-4-Ig of the invention to suppress or inhibit the proliferation of T-cell memory (e.g., human T-cell memory) in in vitro studies compared to Orencia® and/or LEA29Y-Ig fused proteins, such mutant proteins will also be increased immunosuppressive capacity in in vivo therapeutic and/or prophylactic methods or applications compared to Orencia®, LEA29Y-Ig, and/or fused proteins of human CTLA-4-Ig (e.g., human CTLA-4-IgG2). In one aspect, the mutant proteins, CTLA-4-Ig of the invention are believed to have a greater ability to suppress or inhibit the proliferation of T-cell memory (e.g., human T-cell memory in vivo method or application than the Orencia®, LEA29Y-Ig, and/or fused proteins of human CTLA-4-Ig (e.g., human CTLA-4-IgG2), such as, for example, therapeutic and/or prophylactic methods for suppressing or inhibiting an immune response in a subject (e.g., in vivo treatment of diseases or disorders of the immune system in a mammal, such as, for example, people), methods for suppressing or inhibiting transplant rejection of a tissue or organ from a donor to a recipient (e.g., a mammal, such as, for example, a person) and/or other methods of treatment or the diagnostic methods described in this specification.

Example 8

Measurement of the biological activity of mutant molecules CTLA-4-Ig using research SCR reaction (reaction of the mixed culture of lymphocytes).

CTLA-4-Ig and his ways are powerful inhibitors of the primary allotees in vitro (Vaughan, A. N. et al., J. Immunol. 165:3175-3181 (2000); Wallace, P. M., et al. Transplantation 58:602-610 (1994)). To measure improved activity of mutant CTLA-4-Ig proteins in such studies conducted analysis of cell proliferation of human reactions mixed culture of lymphocytes (SCR reaction).

Human blood (freshly program donors-people) was diluted with an equal volume of PBS and was fractionally to allocate IPC using Histopaque (Sigma, #10771) Ficoll gradient in accordance with the conditions recommended PR is the producer. The IPC from a single donor were dissolved in RMPI medium (Sigma, #R8758) supplemented with 10% FBS (Hyclone # SV30014,03) and 1x PSG (Invitrogen, #10378-016) and added to 96-cellular tablets for culture (BD Biosciences, #353077) at a density of 1×105cells/cell. The IPC from another donor were irradiated at 2500 glad bred in the same environment and were added in the same tablet with a density of 1×105cells/cell. The analyzed compounds were serially diluted in the same medium and added to cells in four Parallels. After incubation at 37°C for 5 days3H thymidine (GE Healthcare, #TRK758-5MCi) was added at a concentration of 1 MX/cell and the plates were incubated at 37°C. the next 18 hours. Cells were collected cell collector (FilterMate Omnifilter-96 Harvester, Perkin Elmer) using conditions recommended by the manufacturer, and was measured by embedding3H thymidine using a scintillation counter (Wallac Trilux, #1450-421). Data cell proliferation were analyzed by GraphPad Prism 5 software using model-curve non-linear regression (sigmoidal response to dose, variable slope) and the selection of the method of least squares. Recorded IC50 parameters and their associated 95% confidence intervals.

Figure 9 shows curves of cell proliferation from a representative analyses of proliferation SCR reaction, including illustrative mutant protein, CTLA-4-Ig of the invention: D3 - IgG2. Orencia®, LEA29Y-Ig merged be the key were included as controls for comparison. The graph shows the dependency injection3H thymidine (CME) of protein concentration (nm). Embedding3H thymidine, which indicates the degree of cell proliferation, was measured by standard methods. These results show that D3-IgG2 has a significantly greater effect than Orencia® and/or LEA29Y-Ig fused proteins in the inhibition or suppression of the primary allostimulatory T cells in vitro (e.g., D3-IgG2 has a greater ability to suppress or inhibit primary allostimulatory T-cell proliferation in vitro analysis of the SCR reaction than Orencia® or LEA29Y-Ig fused proteins).

This study SCR reaction was carried out on some other mutant fused proteins, CTLA-4-Igx according to the invention. Table 9 provides summary data for illustrative list of mutant CTLA-4-Ig fused protein according to the invention. Table 9 presents the comparison of mean values of IC50 (nanomoles (nm)) for illustrative mutant fused proteins, CTLA-4-Ig against reference controls (Orencia®, LEA29Y-Ig fused proteins) in the study SCR reaction. IC50 values from two separate experiments were averaged to obtain the mean IC50. The term "(mean log IC50)" represents the standard deviation from the average log IC50. The mean IC50 values from mutant CTLA-4-Ig fused proteins, are presented in table 9, were lower than the corresponding average values of IC50 Orencia®, LEA29Y-Ig fused proteins.

In one aspect, the invention provides mutant fused proteins, CTLA-4-Ig, which is considered superior in strength Orencia® and/or LEA29Y-Ig fused proteins (e.g., mutant fused proteins, CTLA-4-Ig is considered to have a greater ability to suppress or inhibit primary allostimulatory T-cell proliferation in vitro study SCR reaction than Orencia® and/or LEA29Y-Ig fused proteins).

Assume that on the basis of the predicted increased abilities mutant fused proteins, CTLA-4-Ig of the invention to suppress or inhibit primary allostimulatory T cells (e.g., human T cells) in vitro studies compared to Orencia® and/or LEA29Y-Ig fused proteins, such mutant proteins should also be increased immunosuppressive capacity in in vivo therapeutic and/or prophylactic methods or applications compared to Orencia® and/or LEA29Y-Ig fused proteins.

In one aspect mutant fused proteins, CTLA-4-Ig of the invention have a much greater impact than Orencia®, LEA29Y-Ig, and/or human CTLA-4-Ig (e.g., human CTLA-4-IgG2) fused proteins in the inhibition or suppression of the primary allostimulatory T cells in vitro (e.g., mutant fused proteins, CTLA-4 - Ig have a greater ability to suppress or inhibit primary allostimulatory T-cell proliferation in vitro study SCR reaction than Oencia®, LEA29Y-Ig, and/or human CTLA-4-Ig) fused proteins. In one aspect, the mutant CTLA-4-Ig of the invention, it is believed, has a greater ability to suppress or inhibit primary allostimulatory T cells (e.g., human T cells) in vivo method or application compared to Orencia®, LEA29Y-Ig, and/or fused proteins of human CTLA-4-Ig, such as, for example, therapeutic and/or prophylactic methods for suppressing or inhibiting an immune response in a subject (e.g., in vivo treatment of diseases or disorders of the immune system in a mammal, such as, for example man), methods for suppressing or inhibiting transplant rejection of a tissue or organ from a donor to a recipient (e.g., a mammal, such as, for example, a person) and/or other treatment or diagnostic methods described in this specification.

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Table 9
Summary of illustrative studies SCR-reaction
Protein (dimer)The average IC50 (nm)The mean log IC50 (nm)WITH (average log IC50) (nm)
Dimer fused protein Orencia®12,53 1,050,23
LEA29Y-Ig0,92-0,190,48
D3-IgG20,06-1,26BUT
D3-12-IgG20,09-1,110,34
D3-14-IgG20,08-1,200,43
D3-17-IgG20,09-1,150,43
D3-20-IgG20,14-1,110,64
D3-27-IgG20,13-1,290,86
D3-29-IgG20,11-1,140,54
D3-34-IgG20,10-1,090,39
D3-53-IgG20,02-1,640,11
D3-54-IgG20,07of 1.13BUT
D3-56-IgG20,06-1,23BUT
D3-69-IgG20,04-1,41BUT
D3-71-IgG20,05-1,32BUT
D3-75-IgG20,08-1,11BUT
D3-76-IgG20,08-1,11BUT
Note: the Expression "BUT" in table 9 means "undefined".

Example 9

The patient is an adult, suffering from rheumatoid arthritis, you may get treatment of soluble mutant fused protein, CTLA-4-Ig in the following way. Preparing a pharmaceutical composition comprising a soluble mutant protein, CTLA-4-Ig of the invention and a pharmaceutically acceptable excipient or carrier (e.g., PBS). Illustrative of soluble mutant protein, CTLA-4-Ig includes two identical monomial what situations mutant fused protein, CTLA-4-Ig, connected together by one or more disulfide bonds, where each such Monomeric protein comprises a mutant polypeptide, CTLA-4 EVA according to the invention, comprising the amino acid sequence selected from any of SEQ ID NOS:1-73, fused at its C-end N-end of the human IgG2 Fc polypeptide. Illustrative fused proteins include those that include the amino acid sequence shown in any of SEQ ID NOS:74-79, 197-200, 205-214, and 219-222. Such fused proteins are typically expressed in dimeric form. The concentration of the fused protein in the pharmaceutical composition may be in the range from about 0.05 mg/ml to about 200 mg/ml, from about 1 mg/ml to about 150 mg/ml, from about 25 mg/ml to about 120 mg/ml, from about 1 mg/ml to about 100 mg/ml, from about 25 mg/ml to about 100 mg/ml, from about 50 mg/ml to about 100 mg/ml, from about 50 mg/ml to about 75 mg/ml from about 100 mg/ml to about 150 mg/ml and the like. For example, the concentration of the fused protein in the pharmaceutical composition may be about 1 mg/ml, 5 mg/ml, 10 mg/ml, 15 mg/ml, 25 mg/ml, 30 mg/ml, 40 mg/ml, 50 mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml, 100 mg/ml, 150 mg/ml or 200 mg/ml. pH of such a pharmaceutical composition is about pH 4 to about pH 10, including about pH 5 to about pH 9 to about pH 6.5 to about pH 8.5, mainly about pH 6.0 to pH 8.0, about 6.5 to pH 7.5, or about pH 7.0 to about pH 8.0.

Treatment of rheumatoid arthritis, patsie is that carried out by the introduction of a therapeutically effective amount of a mutant CTLA-4-Ig to the patient (for example, effective dose) intravenous or subcutaneous injection. The injection can be, for example, the patient's arm, torso or leg. Enter the effective dose of mutant fused protein, CTLA-4-Ig is typically, but not limited to the following, is, for example, from about 0.01 mg/kg to about 100 mg/kg of body weight of an adult human patient, such as, for example, from about 0.01-5.0 mg/kg, about 0.01-3.0 mg/kg, about 0.05-2.5 mg/kg, about 0.1-2.0 mg/kg, about 0.1-1.0 mg/kg, about 0.01-0.05 mg/kg, about 0.5-1.5 mg/kg, about 1.0-4.0 mg/kg, about 1.0-3.0 mg/kg, about 1.0-2.0 mg/kg, including but not limited to, about 0.01 mg/kg, 0.05 mg/kg, 0.075 mg/kg, 0.1 mg/kg, 0.15 mg/kg, 0.2 mg/kg, 0.25 mg/kg, 0.3 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 1.25 mg/ml, 1.5 mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, 25 mg/kg 50 mg/kg, 75 mg/kg or 100 mg/kg body weight of the patient lead to the patient. Alternatively, it may be used in an effective amount, or dose, or the dose range described in the Methods according to the invention" above. Dose fused protein, subject to the introduction, is determined based on the strength of the fused protein and/or the severity of symptoms or signs of rheumatoid arthritis in the patient. The total number of mutant fused protein, CTLA-4-Ig, enter the patient may be, for example, from about 0.01 mg to about 100 mg, typically from about 1 mg to 100 mg, from about 10 mg to 100 mg, from about 10 mg to about 75 mg, or from about 10 to about 50 mg. is the volume of the pharmaceutical composition, enter the patient is determined based on the concentration of the fused protein in the composition and doses of fused protein to be introduction. For subcutaneous injection one to two ml of the pharmaceutical composition comprising a protein, usually injected per injection. For intravenous injections can be introduced acceptable amount of a pharmaceutical composition comprising a protein.

After injection of the initial dose to the patient can be entered a second identical dose fused protein subcutaneously (e.g., p/injection) or intravenously (for example,/injection) through, for example, 1, 2, 3, or 4 weeks after the initial dose. Schedule of dosage may be as follows: one dose every two weeks, one dose per month, one dose every two months, etc. depending on, for example, the patient's condition. Subsequent doses can be introduced every four weeks or more or less frequently, if necessary. The frequency of dosage may vary depending on the condition of the patient and may depend on the severity of symptoms or signs of rheumatoid arthritis patient.

In an illustrative aspect, the amount of pharmaceutical composition comprising the mutant dimer fused protein, CTLA-4-Ig of the invention (such as D3-29-IgG2, D3-54-IgG2, D3-56-IgG2, D3-69-IgG2, etc.) and pharmaceutically acceptable filler, sufficient to provide a dose of dimer fused belcolle 0.5 mg/kg body weight, injected with subcutaneous injection of the person suffering from rheumatoid arthritis once a week or once a month, if necessary, depending on the patient's condition and response to the medication.

In another illustrative aspect, the amount of pharmaceutical composition comprising a dimer fused protein, CTLA-4-Ig of the invention (such as D3-29-IgG2, D3-54-IgG2, D3-56-IgG2, D3-69-IgG2, etc.) and pharmaceutically acceptable filler, sufficient to provide a dose of dimer fused protein of about 10 mg/kg, administered intravenously to a person suffering from rheumatoid arthritis, once a week or once a month, if necessary, depending on the patient's condition and response to drug. Standard/procedure can be used for such an introduction. For example, the pharmaceutical composition may be infusion in the human body with another liquid, such as sterile saline solution, dextrose or other isotonic solution, using standard continuous intravenous drip infusions through a standard intravenous access devices.

It is assumed that each of the above-described method of treatment by p/or/injection reduces or alleviates one or more signs, symptoms or biological responses associated with rheumatoid arthritis, such as, n is the sample, inflammation, tenderness, joint swelling, joint pain, tissue atrophy and stiffness in a patient. Such treatment may reduce the development of disease in a patient, in particular, connective, muscle, and bone tissues. For example, such treatment may reduce the development of damage or degradation of the connective tissue, atrophy of muscle tissue, bones, joints, cartilage and/or the vertebral column and the like of the patient. Additional clinical symptoms, including skin damage, Central nervous system or organs may also be reduced or relieved. Such treatment may also improve physical functioning of the patient.

Example 10

The patient is an adult receiving maintenance therapy to prevent rejection of an organ transplant, may receive treatment soluble mutant fused protein, CTLA-4-Ig in the following way. Preparing a pharmaceutical composition comprising a soluble mutant protein, CTLA-4-Ig of the invention and a pharmaceutically acceptable excipient or carrier (e.g., PBS or the like). Illustrative of soluble mutant protein, CTLA-4-Ig includes two identical Monomeric mutant fused protein, CTLA-4-Ig, connected together by one or more disulfide bonds, where each such Monomeric protein comprises a mutant is the first polypeptide CTLA-4 EVA according to the invention, comprising the amino acid sequence selected from any of SEQ ID NOS:1-73, fused at its C-end N-end of the human IgG2 Fc polypeptide. Illustrative fused proteins include those that include the amino acid sequence shown in any of SEQ ID NOS:74-79, 197-200, 205-214, and 219-222. The concentration of the fused protein in the pharmaceutical composition may be in the range from about 0.05 mg/ml to about 200 mg/ml, from about 1 mg/ml to about 150 mg/ml, from about 25 mg/ml to about 120 mg/ml, from about 1 mg/ml to about 100 mg/ml, from about 25 mg/ml to about 100 mg/ml, from about 50 mg/ml to about 100 mg/ml, from about 50 mg/ml to about 75 mg/ml from about 100 mg/ml to about 150 mg/ml and the like. For example, the concentration of the fused protein in the pharmaceutical composition may be about 1 mg/ml, 5 mg/ml, 10 mg/ml, 15 mg/ml, 25 mg/ml, 30 mg/ml, 40 mg/ml, 50 mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml, 100 mg/ml, 150 mg/ml or 200 mg/ml. pH of such a pharmaceutical composition is about pH 4 to about pH 10, including about pH 5 to about pH 9 to about pH 6.5 to about pH 8.5, about pH 6.0 to pH 8.0, about 6.5 to pH 7.5, or about pH 7.0 to about pH 8.0

Maintenance therapy to prevent or suppress the rejection of an organ transplant is carried out by introducing a therapeutically effective amount of a mutant CTLA-4-Ig to the patient (e.g., effective dose), who received an organ transplant (e.g., transplant the kidney) by intravenous or subcutaneous injection. The injection can be, for example, the patient's arm, torso or leg. Effective dose of mutant fused protein, CTLA-4-Ig for the introduction, which is usually, but not limited to the following, for example, from about 0.01 mg/kg to about 100 mg/kg of body weight of an adult human patient, such as, for example, from about 0.01-5.0 mg/kg, about 0.01-3.0 mg/kg, about 0.05-2.5 mg/kg, about 0.1-2.0 mg/kg, about 0.1-1.0 mg/kg, about 0.01-0.05 mg/kg, about 0.5 to 1.5 mg/kg, about 1.0-4.0 mg/kg, about 1.0-3.0 mg/kg, about 1.0-2.0 mg/kg, including but not limited to, about 0.01 mg/kg, 0.05 mg/kg, 0.075 mg/kg, 0.1 mg/kg, 0.15 mg/kg, 0.2 mg/kg, 0.25 mg/kg, 0.3 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 1.25 mg/ml, 1.5 mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, 25 mg/kg 50 mg/kg, 75 mg/kg, or 100 mg/kg body weight of the patient, enter the patient. Alternatively, the effective amount or dose, or the dose range described above in the section "Methods according to the invention can be used. Dose fused protein, subject to the introduction, is determined based on the strength of the fused protein and/or the severity of symptoms or signs of rejection of an organ transplant patient. The total number of mutant fused protein, CTLA-4-Ig, enter the patient may be, for example, from about 0.01 mg to about 100 mg, typically from about 1 mg to 100 mg, from about 10 mg to 100 mg, from about 10 mg to about 75 mg, or from about 10 to about 50 mg. Amount of the pharmaceutical compositions is AI, enter the patient is determined depending on the concentration of the fused protein in the composition and doses of fused protein, be inserted. Protein can be entered on any day after the transplantation, for example 1, 4, 7, 14, 28, 56, 84 day etc. after transplantation. For subcutaneous injection is usually administered one to two ml of the pharmaceutical composition. For intravenous injections can be introduced acceptable amount of a pharmaceutical composition comprising a protein.

After injection of the initial dose to the patient can be entered a second identical dose fused protein subcutaneously or intravenously through 1, 2, 3, or 4 weeks after the initial dose. Schedule of dosage may be as follows: one dose every two weeks, one dose every month, one dose every two months, etc. depending on, for example, the patient's condition. Subsequent doses can be introduced every four weeks or more or less frequently, if necessary, and continue, if necessary, on a monthly basis. The frequency of dosing may vary depending on the condition of the patient and may depend on the severity of symptoms or signs of rejection of an organ transplant patient.

Assume that such treatment reduces or alleviates one or more signs, symptoms or biological responses associated with rejection of an organ transplant, that is them, for example, acute rejection of the transplanted organ, chronic rejection of the transplanted organ function decline transplanted organ, increased levels of creatinine in the patient's serum and/or increased infiltration of T cells in transplantirovannam body. Such treatment may reduce the likelihood of rejection of the transplanted organ by the immune system of the patient.

Example 11

Pharmacokinetic study of mutant fused proteins, CTLA-4-IgG2 in rats

The concentration in serum therapeutic agent after external introduction of a living organism strongly affects therapeutic efficacy and determined the pharmacokinetic (PK) evaluation. In the following procedure, FC profiles were evaluated in rats for representative mutant CTLA-4-IgG2 analyzed products D3-29-IgG2, D3-54-IgG2, D3-56-IgG2, D3-69-IgG2 and D3-75-IgG2 in comparison with both the analyzed products: hCTLA-4-IgG2 and Orencia® protein. Scheme of the experiment and the data and their interpretation are described below.

Scheme of the experiment on live animals

Verified by weight of male rats Hans Wistar used after acclimatization period, which amounted to at least 5 days. The dosage amounts of the analyzed product was calculated for individual animals based on their weight, so that all received 1 mg/kg analyzer is imago product. A typical 150-gram (g) rat, thus, received 150 μl metered volume, while each of the analyzed product was prepared at a concentration of 1 mg/ml in PBS. Separate introduction of mutant tested product CTLA-4-IgG2, described above, hCTLA-4IgG2 or fused protein Orencia® delivered either by intravenous (IV or IV) bolus or subcutaneous (abbreviated as "p/C" or "SC") way. The scope of the study was sufficient for a minimum of four fences blood samples of 300 μl in each moment of time, thus limiting the amount of blood withdrawn from any given rat, was not more than 10% of the total blood volume. Dynamics of blood sampling was like before the introduction of the dose), 5 minutes (min), 30 min, 2 hours (h), 4 hour, 8 hour, 1 day (days), 2 days, 3 days, 4 days, 6 days, 8D, 10 DN., 12 Nam. and 14 days. before the introduction, and 5 min, 30 min, 2 hours, 4 hours, 8 hours, 1 days, 2 days, 3 days, 4 days, 6 days, 8D, 10 DN., 11 days, 12 days, 13 days, 14 days. and 15 days. after a dose for a/V and s/C injections, respectively. Serum was prepared by their individual blood samples and analyzed using ELISA to calculate the percentage entered the analyzed product.

Method FC ELISA

The mutant protein, CTLA-4-IgG2, hCTLA-4IgG2 or Orencia®, present in the serum samples, was associated with human CDSO-mouse IgG (described above)which had previously covered microturbulence tablets. The definition of Khujand who have been adding antibodies goat against human IgG, conjugated to horseradish peroxidase (HRP) (Jackson ImmunoResearch #109-035-098). Quantification was performed by use of the chromogenic HRP substrate 3,3',5,5'-tetramethylbenzidine (TMB), with the addition of hydrogen peroxide (Kem-En-TEC #A), causing the reaction was stopped by adding 0,2N sulfuric acid (H2SO4) and was measured by optical absorbance at 450 nm on a spectrophotometer. Serum samples pre-diluted 1/20 to add on ELISA tablet, so that the matrix was normalized to 5% rat serum, and further diluted in 5% rat serum to eight dilutions in General. The concentration of each diluted serum sample was quantitatively determined against a standard curve obtained with the use of titrations of the same analyzed product introduced by syringe in 5% rat serum. A standard curve ranging from 10 to 0.078 ng/ml and the exact range was determined so that it was twice the background value to 5 ng/ml in 5% rat serum. The standard curve was assessed using quality control (QC) analyzed the same product, prepared in 5% rat serum at high, medium and low concentrations of the exact range of the standard curve. Criterion validity QC was observed for QC concentrations within 20% of the estimated QC is concentratie. The concentration of individual serum sample was obtained by averaging the concentrations indicated for breeding with optical densities (OD) within the accurate range of the standard curve. At least, used 4 individual serum samples for calculation of average concentration in the serum in each nominal point in time.

FC Parameters

Figures 15A and 15C show FC profiles for fused protein Orencia®, hCTLA-4-IgG2 and mutant fused proteins, CTLA-4-IgG2, introduced in quantities of 1 mg/kg as single (A) intravenous (IV) bolus or subcutaneous (SC) injection to rats. Trims errors represent the standard deviation from the mean (). Dashed line shows the lower limit of quantitation for ELISA (~3 ng/ml for the analyzed product in 100% serum). The average concentration of serum in each nominal point in time after the introduction of the analyzed product at time 0 h includes data on semi-log profile concentration-time in figure 15.

The average concentrations in the serum used for PK parameters using WinNonLin model 201 (in/in - bolus) or model 200 (extravascular introduction)/(BB) or s.c. (PC) ways of dosing, respectively. Table 10 results summary of key PK parameters for p/and/in routes of administration, respectively./p>

Table 10
Summary table of PK parameters for fused protein Orencia®, hCTLA-4 - IgG2 and mutant fused proteins, CTLA-4-IgG2, introduced in quantities of 1 mg/kg as single BB or s/C bolus to rats.
ConnectionPathKmax(ug/l)T1/2 (hour)AUC (hour*ng/l)CL (ml/HR/kg)Vz (ml/kg).
OrenciaPC3,370,03912,6258,13
CTLA-4lgG2PC5,945,08381,277,49
D3-29PC4,611,26501,5of 24.90
D3-54PC 4,523,65281,964,52
D3-56PC7,423,710491,032,67
D3-69PC6,028,19671,041,97
D3-75PC9,0of 56.412040,867,59
OrenciaBB22,342,37281,3783,9
CTLA-4lgG2BB66,249,824830,4029,0
D3-29BB43,033,0 9121,1052,2
D3-54BB20,639,410340,9755,0
D3-56BB34,215,318220,5512,1
D3-69BB81,983,021330,4756,1
D3-75BB30,165,423260,4340,5

Kmaxrefers to the maximum serum concentration of the analyzed product. Is the terminal half-life (T 1/2) is the time taken in hours for which the concentration of the analyzed product in the serum is reduced by half during the terminal phase of the concentration profile. The area under the curve concentration in the serum of the time (the PC) count from zero to infinity using the trapezoid rule. Clearance (CL) calculated using equation dose/CPD, whereas the volume of distribution (Vz) of the terminal phase calculated using equation CL/k.

Factor bioavailability (F) for each compound are presented in table 11 and was determined by calculating CPD through subcutaneous path/ CPD through intravenous route.

Table 11
Comparison of the bioavailability of the fused protein Orencia®, hCTLA-4-Ig2, D3-29-IgG2, D3-54-IgG2, D3-56-IgG2, D3-69-IgG2 and D3-75-IgG2 fused proteins
ConnectionBioavailability
Orencia0,54
CTLA-4-Ig20,34
D3-290,71
D3-540,51
D3-560,58
D3-690,45
D3-750,52

Protein human CTLA-4IgG2 and Orencia® showed similar PK profiles, despite their differences in the structure of IgG, assuming that human IgG2 and mutated human IgGI Fc portion of these COO is responsible analyzed products have comparable FC activity if the functional domain is a constant. We can conclude that any changes observed in FC profile for the mutant fused proteins, CTLA-4-IgG2 compared with the fused protein Orencia®, will thus contribute to differences in the functional domain and shall not affect the Fc part.

For each mutant fused protein, CTLA-4-IgG2, elimination was slow, as evidenced by long-time values of half-life and a large area under the curves. Half-life ranged from 11,2 to 56.4 hours for PC dosing, ranging from 15.3 to 83.0 hours (h) to IV dosing compared to Orencia® fused protein which had a half-life of 70,0 or 42.3 hours with the introduction of PC or BB paths, respectively. The value of CPD for mutant fused proteins, CTLA-4-IgG2 were on average higher than those for fused protein Orencia®. After PC dosing was 879,5+/-281,6 hour·kg·ng/l/mg compared with 391 h·kg·ng/l/mg for PC fused protein Orencia®, whereas BB introduction gave the average CPD 1645,5+/-641,1 hour·kg·ng/l/mg compared to 728 hour·kg·ng/l/mg for CENTURIES fused protein Orencia®.

The volume of distribution was similar for both routes of administration, which mutant fused proteins, CTLA-4-IgG2 were distributed outside of the serum, but within extravascular fluid, which was confirmed by the average value 44,8 ml/kg, which is higher than the reference plasma volume 30 ml/kg, and within R is ferentinou border extracellular fluid 300 ml/kg for the standard rats (Davies, C. et al., Pharm. Res. 10(7): 1093-95 (1993)).

Bioavailability was also similar for most mutant fused proteins, CTLA-4-IgG2, with an average value of 0.6+/-0,1, which compared favorably with 0,53 bioavailability fused protein Orencia®.

In the end, Kmaxwas in General higher for the mutant CTLA-4-IgG2 fused proteins compared with the fused protein Orencia®. Values of Kmaxwas in the range of from 4.5 to 9.3 ág/l for PC dosing and from 20,6 to 81.9 µg/l for IV dosing in comparison with fused protein Orencia®, which had Kmax3,3 22,3 or µg/l with the introduction of PC or BB paths, respectively.

In General, FC data showed that all analyzed mutant CTLA-4-IgG2 fused proteins usually had FC profile is not lower than protein Orencia® when administered to rats in amounts of 1 mg/kg, PC or BB ways, and that IgG1 or IgG2 Fc portions were comparable when the functional domain was constant.

Example 12

This example describes a method of creating a stable transfectional cell lines for ekspressirovali mutant fused protein CTLA4-Ig of the invention with the use of cell lines for routine laboratory scale production of mutant fused protein CTLA4-Ig and purification of the mutant CTLA-4-Ig fused proteins from cellular environments expression. Although this example specifically describes a method of creating a stable transfectional cell line Cho-K1 DL is ekspressirovali D3-54-IgG2 fused protein, the use of such cells for laboratory scale production and purification of the protein from the media, the methods described herein may be used with any mutant fused protein, CTLA-4-Ig of the invention and/or any acceptable cell line described above.

The creation of stable transfectional cell line

Materials

Cho-K1 native (retrospektywnie) cell: Cho-K1 cells adapted to serum-free suspension grown in a chemically defined medium (Cell ID: M4-PeM-0436-l 12-01), maintained in the vapor phase of liquid nitrogen (Dewar MVE1536P). One vessel M4-PeM-0436-112-01 championed and cultivated with CD OptiCHO™ medium (Invitrogen, #12681) in shake flasks. Culture provided cells for transfection and air-conditioned environment for the growth and cloning. All cultures were grown at 37°C, 5% CO2.

Plasmid: DNA encoding a D3-54 mutant CTLA-4 EVA, fused with the Fc region of human IgG2a, was built in CET1019AS UCOE vector (Millipore) and the resulting plasmid CET1019AS-D3-54-IgG2 was used for all transpency.

The cell culture medium: CD Opti CHO (Invitrogen #12681) chemically defined medium devoid of animal component, supplemented with 2% volume/volume of 200 mm L-glutamine (Invitrogen #25031), was used for all cultures.

Air-conditioned environment: air-Conditioned environment was obtained by culturing the parental Cho-K1 cell lines is in CD Opti CHO environment. When the number of cells >5×105cells/ml of cell culture was centrifuged and the supernatant conditioned medium was filtered under sterile conditions.

The conditioned medium were prepared fresh each day of use or kept at 2-8°C for up to 7 days. 50% conditioned medium: air-Conditioned environment (see above) combined with an equal volume of fresh cell environment; prepared fresh every day use.

Analytical methods

Count cells and determine viability was carried out on Cedex Cedex or HiRes cell counter (Innovatis).

Initial screening for clones expressing the mutant protein, CTLA-4-Ig (D3-54-IgG2) for products was performed using ELISA. ELISA tablets covered hCD80-mouse Ig fused protein throughout the night. The next day, the samples to be analyzed, was transferred to the ELISA tablets in 50 - and 200-fold dilution in two Parallels. After two hours of incubation was added anti-human IgG-HRP antibody and incubated for 30 minutes. The tablets were treated with TMB and analyzed at 450 nm. Recorded raw data of optical densities (OD).

Quantitative determination of the concentration of the fused protein D3-54-IgG2 was conducted by way of protein HPLC using Poros And/20 column (ABI # 1-5024-12). Used two buffers: Buffer A: 50 mm phosphoric acid, 150 mm potassium chloride, pH 7,6±0, and Buffer B: 50 mm phosphoric acid, 150 mm potassium chloride, pH 2,5±0,1. Both buffer additionally contained an added 5% isopropanol. Equilibration and washing after injection of the sample was conducted with 42% Buffer a and 58% Buffer (pH 6.5). Elution was with a linear gradient of 12% Buffer a and 88% Buffer B for 1 minute.

Procedure

Native attached Cho-K1 cells used to create stable cell lines used in GMP production D3-54-IgG2 fused protein, adapted to growth in suspension in a chemically specific CD OptiCHO™ medium. The vessel with these native Cho-K1 cells defended and were grown in 125 ml shake flasks containing CD OptiCHO™ medium to a density of 5×105viable cells/ml 2×106viable cells resuspendable in 400 μl of 50% conditioned medium and combined with 20 μg of plasmid DNA (D3-54-IgG2 in CET1019AS UCEO vector) in a ditch. Electroporation was performed with a gene pulse generator Xcell (BioRad) at 320 Volts (V) with the length of the rectangular pulse 15 milliseconds (PC). Spent duplicate transfection and then merged cells. Merged cells was transferred into a T-25 flask containing 5 ml of 50% conditioned medium and incubated for two days.

Transfection cells either directly distributed in 96-cellular tablets for cloning or cultivated with the selection of antibiotics until I got a stable pool and then distributed in 96-cellular tablets. Two days after electroporation, the culture was diluted to 1250 cells/ml in conditioned medium containing 8 μg/ml puromycin for selection pressure. The cells were distributed in 96-cellular tablets with 200 μl of the cell (250 cells/cell). The plates were incubated for about 10 days to kill retrospektywnie and temporarily-transfection cells. After 10-12 days each cell on each tablet visually examined to identify cells from individual colonies. These cells later re-examined to confirm the presence of a healthy colonies that are acceptable for transfer to 24-cellular tablets.

Two days after electroporation, the culture was centrifuged and resuspendable 50% conditioned medium containing 7 μg/ml puromycin for selection pressure. A control flask was inoculable retrospektivnyi cells in the same environment. Based on continuous research optimization, the concentration of puromycin was increased to 8 mcg/ml after 3 days. Stable pool was formed in 10-12 days after selection, when all cells in the control flask were killed. The expression product in a stable pool was confirmed using protein HPLC and confirmed that the viability of the culture was >95%. Cells were serially diluted in air-conditioned environment without PUR is mizina to a final concentration of 3.8 cells/ml Cells were seeded in 96-cellular tablets in the amount of 200 μl per-cell (75 cells/tablet or 0.8 cells/cell).

After one day each cell in each plate was visually examined to identify cells with individual cells or colonies. The next day, cells from individual colonies of 2-4 cells were selected. Any cell with more than two colonies were removed. The second statement was confirmed selections. Cells were then re-examined to confirm that they contain a healthy colony acceptable for transfer to 24-cellular tablets.

Clones from 96-cellular tablets were transferred to 24-cellular tablets containing 1 ml of conditioned medium on cell with 8 μg/ml puromycin. The internal contents of the selected cells from 96-cellular tablets with a single colony was transferred into individual cells in 24-cellular tablets. 200 μl were taken from each of the new cells in 24-cellular tablet for washing the corresponding cells in 96-cellular tablet and moved back. For backup, 200 μl of conditioned medium containing 8 μg/ml puromycin was added back to each well in 96-cellular tablets.

After 1-3 days selected sample from each cell in a 24-cellular tablets and analyzed for the D3-54-IgG2 expression using ELISA. Clones for further use were selected on the base of original values OP ELISA, demonstrate acceptable growth.

Top 35-40 clones, based on the ELISA results and the observed growth was transferred into a T-25 flask containing 5 ml of the conditioned medium with 8 μg/ml puromycin. Internally the contents of each of the selected cells from 24-cellular tablets carried in the individual T-25 flask. The remaining cells in the cells were washed with the same medium and added to the appropriate T-25 flask. For backup 1 ml of conditioned medium containing 8 μg/ml puromycin was added back to each well in a 24-cellular tablets.

The number of clones was further reduced by selecting clones with the highest productivity. Cells resuspendable in fresh medium at a concentration of 1-2×105viable cells/ml and seeded in KZT25 flasks (5 ml culture) or 125 ml shake flasks (12 ml culture). Cultures were incubated for 22-24 hours and then determined the final cell density and viability and taking samples to determine the concentration of the protein product by HPLC. Productivity was calculated by dividing the total protein produced by the total number of viable cells in the flask, the term culture. Units were transferred to the pictograms on the cage for a day or "PKD". Clones were sequenced according to their values PKD, but the clones that did not show significant growth, have been omitted Upper clones were transferred into 125 ml shake flasks for cryopreservation and further studies of growth and productivity.

Clones, subject to further analysis, were sown in 250 ml shake flasks (1×105viable cells/ml in 50 ml of fresh medium. When cell density reached 1×106viable cells/ml, the culture was transferred into a new shake flask, again with 1×105viable cells/ml in 50 ml of fresh medium. The transfer was repeated one more time. During this third transfer every day taking samples of the culture to determine the number of cells, viability and concentration of the product using protein HPLC.

Top clones expressing D3-54-IgG2 protein, based on the speed of growth and specific products were selected for sublimirovanny. Subclavian conducted a limited breeding, as described in the section above, for stable pools, except that puromycin not used at any time. Distribution, screening and evaluation of subclones was performed as described above.

Selected subclones re-transferred in shake flasks at approximately 90 days to assess the stability of the products. At each passage, cells were seeded in 125 ml shake flasks (1×105viable cells/ml in 25 ml of fresh medium. Culture endured every 3-4 days. Before each passage was measured by cell density and viability and is birali samples to determine the concentration of the product protein HPLC.

Selected clones and subclones were kryokonservierung from time to organize the PKD values to different points in the dimension stability. Prepared fresh environment with freezing 90% of the growth medium and 10% DMSO (dimethyl sulfoxide) (Sigma). Cells from cultures in shake flask was centrifuged and resuspendable environment freezing at densities in the range of 2-10×106cells/ml Cell suspension in 1 ml aliquot was transferred into a cryogenic vessels. Cryogenic vessels were placed in isopropanolamine containers for frozen and kept at -80°C over night. Frozen vessels transferred into the vapor phase of liquid nitrogen, kept for the next day.

Obtaining mutant fused protein CTLA4-IgG2.

Creosoted containing 1 ml volume of Cho-K1 cells expressing D3-54-IgG2 protein was thawed and grown in 125 ml shake flasks containing CD OptiCHO™ medium, at 37°C and 5% CO2to a density of 5×105viable cells/ml Some of the flasks were combined with oscillatory capacity for inoculation in the amount of 1-2×105 cells/ml in 5 or 10 l volume CD OptiCHO™ medium with 4 mm glutamine. Culture of oscillatory containers were kept in the incubator at 37°C with 5% CO2on the device swinging platforms with 18-22 rpm and an angle of 8 degrees for equipment purchased from Sartorius Stedim Biotech. Every day OTB the morals of the culture samples for analysis of cell number, viability, levels of nutrients, metabolic profile and level of expression of the mutant CTLA4-IgG2 (namely, D3-54-IgG2) using protein HPLC. The culture was harvested, when the viability was reduced to ~50%, usually within 9-11 days after inoculation. The material of the cell culture was purified by filtration using a combination of deep filtration and sterile filtration and either used immediately for further processing, or kept at 2-8°C.

Purification of mutant fused proteins, CTLA-4-IgG2

Mutant fused protein CTLA4-IgG2 (D3-54-IgG2) was purified protein by affinity chromatography using INSTRUMENT Explorer HPLC system (GE Healthcare). The mutant protein, CTLA-4-Ig (D3-54-IgG2) was associated with MabSelect Protein FF column (GE Healthcare, #17-5079-01) in PBS buffer (Invitrogen)were loaded at a concentration of ~10 mg/ml of medium chromatography, washed with the same buffer, was suirable 100 mm citrate buffer (pH 4.0) and then neutralized by adding 1/10 volume of 2M Tris-base. Protein a purified sample was further treated with diafiltrate using system filtration tangential flow (TFF) buffer is replaced with 20 mm Tris-Cl, pH 7.5.

The protein sample is replaced with the buffer was further purified by anion exchange chromatography on Q-Sepharose column loaded at a concentration of ~10 mg/ml density loading. Bound protein was suirable using 20 volume of the column (the K) linear NaCl gradient of 0-500 mm NaCl in 20 mm Tris-Cl, pH 7.5. The main peak fractions were collected and the concentration was determined by measuring the absorbance at 280 nm.

The purity of the protein was confirmed by SDS-PAGE analysis and the content of the monomer was determined using gel-HPLC method. The samples were stored in aliquot at 2-8°C or at -20°C for long periods of time before use.

As the foregoing invention has been described in some detail with the purpose of explanation and understanding, the expert in this area should be clear from the reading of this description that various changes in form and detail may be made without limitation of the valid scope of the invention. It is clear that the materials, examples, and embodiments of the invention described here is given only for illustrative purposes and do not imply limitation and that various modifications or changes in this world should be implied by the specialists in the art and are included within the meaning and scope of this application and scope of the invention claimed in the claims. All publications, patent applications, patents or other document referred to here are included with references in their entirety for all purposes to the same extent as if each individual publication, patent, patent application or other document separately has been specifically defined the n by including links in this description in its entirety for all purposes and was shown in its entirety in the present description. In case of conflict, this description of the invention, including definitions, will be monitored.

1. Recombinant dimer fused protein comprising two Monomeric fused protein coupled via at least one disulfide bond formed between two cysteine residues present in each Monomeric mutant fused protein, each Monomeric protein comprises (a) a polypeptide having a polypeptide sequence presented in SEQ ID NO: 36, and (b) Ig Fc polypeptide, where C is the end of the polypeptide is covalently bonded directly or indirectly via a linker to the N-end of the polypeptide Ig Fc and where dimer fused protein binds to human CD80 or human CD86 or an extracellular domain any of them and has great ability to suppress immune response than the dimer fused protein LEA29Y-Ig, where dimer fused protein LEA29Y-Ig includes two Monomeric fused protein LEA29Y-Ig, each of which includes the amino acid sequence of SEQ ID NO: 166, where recombinant dimer fused protein is to inhibit or suppress an immune response in a mammal.

2. Dimer fused protein according to claim 1, in which the IgFc polypeptide is a polypeptide IgG2 Fc.

3. Dimer fused protein according to claim 1, in which each Monomeric protein contains the polypeptide sequence shown in SEQ ID NO: 197 or 211.

4. Re ominantly dimer fused protein, comprising two Monomeric fused protein coupled via at least one disulfide bond formed between two cysteine residues present in each Monomeric mutant fused protein, each Monomeric protein comprises (a) a polypeptide having a polypeptide sequence presented in SEQ ID NO: 50, and (b) Ig Fc polypeptide, where C is the end of the polypeptide is covalently bonded directly or indirectly via a linker to the N-end of the polypeptide Ig Fc and where dimer fused protein binds to human CD80 or human CD86 or an extracellular domain of any of them and has greater ability to suppress immune response than the dimer fused protein LEA29Y-Ig, where dimer fused protein LEA29Y-Ig includes two Monomeric fused protein LEA29Y-Ig, each of which includes the amino acid sequence of SEQ ID NO: 166, where recombinant dimer fused protein is to inhibit or suppress an immune response in a mammal.

5. Dimer fused protein according to claim 4, in which the IgFc polypeptide is a polypeptide IgG2 Fc.

6. Dimer fused protein according to claim 4, in which each Monomeric protein contains a polypeptide sequence presented in SEQ ID NO: 199 or 213.

7. Dimer fused protein according to claim 1, which has an equilibrium dissociation constant CD86 (KD), which is less than the equilibrium constant dis is ociali CD86 (K D) dimer fused protein LEA29Y-Ig, and dimer fused protein LEA29Y-Ig includes two Monomeric fused protein LEA29Y-Ig, each of which includes the amino acid sequence of SEQ ID NO: 166.

8. Recombinant nucleic acid which encodes a Monomeric protein dimer fused protein according to any one of claims 1 to 3.

9. Recombinant nucleic acid which encodes a Monomeric protein dimer fused protein according to any one of claims 4 to 6.

10. Expressing a vector comprising the nucleic acid of claim 8.

11. Expressing a vector comprising a nucleic acid according to claim 9.

12. Recombinant a host cell to obtain a dimer fused protein according to any one of paragraphs. 1-3, including:
(a) the nucleic acid of claim 8 and/or
(b) the vector of claim 10.

13. Recombinant a host cell to obtain a dimer fused protein according to any one of claims 4 to 6, including:
(a) nucleic acid according to claim 9 and/or
(b) vector under item 11.

14. Pharmaceutical composition comprising a pharmaceutically acceptable excipient or pharmaceutically acceptable carrier and dimer fused protein according to any one of claims 1 to 3, where the pharmaceutical composition is for inhibiting or suppressing an immune response in a mammal.

15. Pharmaceutical composition comprising a pharmaceutically acceptable excipient or pharmaceutically acceptable carrier and dim the R fused protein according to any one of claims 4 to 6, where the pharmaceutical composition is for inhibiting or suppressing an immune response in a mammal.

16. The use of dimer fused protein according to any one of claims 1 to 3 in the manufacture of a medicine for:
(i) inhibiting or suppressing an immune response in a mammal;
(ii) treatment of diseases or disorders of the immune system or
(iii) treatment of transplant rejection of an organ or tissue from a mammal.

17. The use of dimer fused protein according to any one of claims 4 to 6 in the manufacture of a medicine for:
(i) inhibiting or suppressing an immune response in a mammal;
(ii) treatment of diseases or disorders of the immune system or
(iii) treatment of transplant rejection of an organ or tissue from a mammal.

18. A method of obtaining a dimer fused protein according to any one of claims 1 to 3, comprising culturing the host cell under item 12 in the culture medium and the allocation of the dimer fused protein from the host cell environment of the host cell or periplasm host cell.

19. A method of obtaining a dimer fused protein according to any one of claims 4 to 6, comprising culturing the host cell according to item 13 in the culture medium and the allocation of the dimer fused protein from the host cell environment of the host cell or periplasm host cell.



 

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FIELD: biotechnologies.

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FIELD: biotechnology.

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10 cl, 6 dwg, 1 tbl, 8 ex

FIELD: biotechnologies.

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3 cl, 1 dwg, 1 tbl

FIELD: biotechnology.

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4 ex

FIELD: biotechnology.

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5 cl, 10 dwg, 1 tbl, 4 ex

FIELD: biotechnologies.

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EFFECT: improvement of the method.

5 cl, 1 dwg, 3 tbl, 4 ex

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