Antibodies of interferon alfa receptor 1 and their application

FIELD: medicine.

SUBSTANCE: claimed are versions of separated monoclonal antibody, specific to INNAR-1. Described are: bispecific molecule, immunoconjugate and compositions for treatment of IFNAR-1-mediated diseases and disorders based on monoclonal antibody. Also described are methods of inhibiting biological activity of type I interferons, method of treating diseases and disorders, mediated by type I interferon with application of antibody. Claimed are nucleic acid, which codes antibody, vector for antibody expression, cell, transformed by vector, as well as method of obtaining antibodies and antibody-producing hybridoma.

EFFECT: application of invention provides novel IFNAR-1 inhibiting antibodies, which block IFNAR-1 and bind its other epitope, in comparison with known antibody 64G12.

29 cl, 15 dwg, 6 tbl, 9 ex

 

This application claims the priority of provisional application U.S. No. 60/581747, filed June 21, 2004; the contents of which are incorporated into the present application by reference in full.

The LEVEL of TECHNOLOGY

Interferons (IFN) type I (IFN-α, IFN-β, IFN-ω, IFN-τ) represent a family of structurally related cytokines with antiviral, antitumor and immunomodulatory effects (Hardyet al. (2001)Blood,97: 473; Cutrone and Langer (2001)J.Biol.Chem.276: 17140). Locus IFNα person includes two subfamilies. The first subfamily consists of 14 nearely genes and 4 pseudogenes having a homology of not less than 80%. The second subfamily αII or omega (ω), contains 5 pseudogenes and 1 functional gene, which is 70% homologous genes IFNα (Weissman and Weber (1986)Prog.Nucl.Acid Res. Mol. Biol.,33: 251-300). The IFNα subtypes have different kinds of specific activity, but have the same biological spectrum (Streuliet al. (1981)Proc.Natl.Acad.SciUSA78:2848) and the same cell receptor (Agnet M.et. al.in “Interferon 5” Ed.I. Gresser p. 1-22, Academic Press, London 1983).

Interferon β (IFNβ) is encoded by a single gene, which has approximately 50% homology with the genes of IFNα.

IFN-γ secreted by activated lymphocytes, it does not have any homology with interferon alpha/beta and doesn't interact with their receptor.

Useinterface human type I associated with a receptor on the cell surface (IFN alpha receptor, IFNAR), consisting of two transmembrane proteins, i.e. IFNAR-1 and IFNAR-2 (Uzeet. al.(1990)Cell,60:225; Novicket al. (1994)Cell,77:391). IFNAR-1 is important for high-affinity binding and differential specificity of the IFNAR complex (Cutrone, 2001, see above). Although the functional differences of each of the subtypes of IFN type I were not set, assume that each of them can perform various interactions with components of the receptor IFNAR, leading to the emergence of potentially different signals (Cooket al. (1996)J.Biol.Chem.271: 13448). In particular, studies using mutant forms of IFNAR-1 and IFNAR-2 suggests that the signals of alpha - and beta-interferons different transmitted receptor due to different interactions with the respective circuits (Lewerenz et al. (1998)J.Mol.Biol.282: 585).

Originally functional studies of type I interferons were focused on natural protection against viral infections (Halleret al. (1981)J.Exp.Med.,154:199; Lindenmannet al. (1981)Methods Enzymol.78: 181). However, in more recent studies, interferons type I is considered as a powerful immunoregulatory cytokines in adaptive immune response. Specifically, it was shown that type I interferons facilitate the differentiation of "untrained" T-cells in the direction of Th1 (Brinkmannet al. (1993)J.Exp.Med,178: 1655), increased production is at antibodies (Finkelman et al. (1991)J.Exp.Med.174:1179) and maintain the functional activity and the persistence of memory T-cells (Santiniet al.(2000)J.Exp.Med.191:1777; Toughet al.(1996)Science,272:1947).

In modern works of numerous research groups it is assumed that IFN-α can enhance the maturation or activation of dendritic cells (DC) (Santini,et al. (2000)J.Exp.Med.191:1777; Luftet al. (1998)J.Immunol.161:1947; Luftet al. (2002)Int.Immunol.14:367; Radvanyiet al.(1999)Scand.J.Immunol.50:499). In addition, the increased expression of type I interferons have been described in numerous autoimmune diseases (Fouliset al. (1987)Lancet2: 1423; Hookset al. (1982)Arthritis Rheum.25:396; Hertzoget al. (1988)Clin. Immunol.Immunopathol.48:192; Hopkins and Meager (1988)Clin.Exp.Immunol.73:88; Arvin and Miller (1984)Arthritis Rheum.27:582). The most studied examples of these diseases are insulin-dependent diabetes mellitus (IDDM) (Foulis (1987), see above) and systemic lupus erythematosus (SLE) (Hooks (1982), see above)that are associated with elevated levels of IFN-α, as well as rheumatoid arthritis (RA) (Hertzog (1988), Hopkins and Meager (1988), Arvin and Miller (1984), see above), in which a more important role could be played by IFN-β.

In addition, it was reported that interferon α exacerbates the underlying disease in patients with psoriasis and multiple sclerosis and cause SLE-like syndrome in patients with RAS who were absent autoimmune diseases. It was also shown that interferon causes glomerulonephritis in normal mice and accelerates the beginning of a spontaneous autoimmune disease in NZB mice/W. in Addition, it was shown that in some cases, IFN-α therapy leads to undesirable side effects, including fever and neurological disorders. Therefore, there is a pathological condition in which inhibition of the activity of type I interferons may have a beneficial effect in a patient, and there is a need for tools that are effective for inhibiting the activity of interferon type I.

The INVENTION

The present invention relates to the selected monoclonal human antibodies that bind to IFNAR-1 and inhibit the biological activity of interferon type I, preferably several interferon type I. in Addition, these antibodies do not bind to the same epitope as anti-IFNAR-1 antibody mouse, i.e. 64G12.

In one aspect the present invention relates to the selected human antibody or its antigen-binding site, and the antibody specific binds to IFNAR-1 and shows one or more of the following properties:

a) binds to IFNAR-1 with KDequal to 1·10-7M, or with a higher affinity;

b) inhibits the biological activity of short interest is Perunov type I;

c) inhibits the activity of IFN-α 2b in the analysis of Daudi cell proliferation;

d) inhibits the activity of IFN omega in the analysis of Daudi cell proliferation;

e) inhibits the secretion of IP-10 managername the peripheral blood cells induced IFN-α 2b;

f) inhibits the secretion of IP-10 managername the peripheral blood cells induced IFN omega;

g) inhibits the development of dendritic cell-mediated plasma systemic lupus erythematosus; and

h) binds to a different epitope than the monoclonal antibody 64G12 mouse (ECACC, Depository No. 92022605).

Preferred antibodies of the present invention is specific in contact with the receptor 1 interferon alpha man, and the binding is carried out with KD1·10-8M or greater affinity, or with KD1·10-9M or greater affinity, or with KD5·10-10M or greater affinity, or with KD2·10-10M or greater affinity.

In one aspect the present invention relates to the selected monoclonal antibody or its antigen-binding site comprising the variable region of the heavy chain, which is the product of or derived genes VH 4-34 or 5-51, with the indicated antibody specific binds to a receptor 1 interferon alpha man. In another aspect the invention relates to the selected monoclonal and what the antibodies or antigen-binding portion, including the variable region of light chain, which is the product of or derived genes VK L18 or A27, with the indicated antibody specific binds to a receptor 1 interferon alpha man. In another aspect the invention relates to the selection of a monoclonal antibody to human anatomy or the antigen-binding site, including:

(a) the variable region of the heavy chain, which is the product of or derived genes VH 4-34 or 5-51 person; and

(b) variable region light chain, which is the product of or derived genes VK L18 or A27 person;

where the antibody is specific binds to a receptor 1 interferon alpha man.

In preferred embodiments, the implementation of the antibody comprises the variable region of the heavy chain gene VH 4-34 man and a variable region light chain gene VK L18 person or antibody comprises the variable region of the heavy chain gene VH 5-51 man and a variable region light chain gene VK A27 person.

In another aspect the invention relates to the selection of a monoclonal antibody to human anatomy or the antigen-binding site, including:

variable region of the heavy chain of a human, comprising the sequence of CDR1, CDR2 and CDR3; and a variable region light chain of a human, comprising the sequence of CDR1, CDR2 and CDR3, and:

(a) the pic shall egovernance CDR3 variable region of the heavy chain of a human being includes the amino acid sequence selected from the group consisting of amino acid sequence SEQ ID NO: 9, 10, 11 and 12 and their conservative modifications;

(b) sequence of CDR3 of the variable region of the light chain of a human being includes the amino acid sequence selected from the group consisting of amino acid sequence SEQ ID NO: 21, 22, 23 and 24 and their conservative modifications;

(c) antibody-specific binds to a receptor 1 interferon alpha man with an affinity for binding to at least 1·10-8M or greater; and

(d) the antibody inhibits the biological activity of at least one interferon type I.

Preferably, the CDR2 sequence of the variable region of the heavy chain of a human being includes the amino acid sequence selected from the group consisting of amino acid sequence SEQ ID NO: 5, 6, 7 and 8 and their conservative modifications; and the CDR2 sequence of the variable region of the light chain of a human being includes the amino acid sequence selected from the group consisting of amino acid sequence SEQ ID NO: 17, 18, 19 and 20 and their conservative modifications. Preferably the CDR1 sequence of the variable region of the heavy chain of a human being includes the amino acid sequence selected from the group consisting of amino acid sequence SEQ ID NO: 1, 2, 3 and 4 and their conservative mod is ficacy; and the CDR1 sequence of the variable region of the light chain of a human being includes the amino acid sequence selected from the group consisting of amino acid sequence SEQ ID NO: 13, 14, 15 and 16 and their conservative modifications.

In another aspect the invention relates to the selection of a monoclonal antibody to human anatomy or the antigen-binding site comprising the variable region of the heavy chain of human rights and the variable region of the light chain, with:

(a) the variable region of the heavy chain of a human being includes the amino acid sequence that is at least 80% homologous to the amino acid sequence selected from the group consisting of SEQ ID NO: 25, 26, 27 and 28;

(b) the variable region of the light chain of a human being includes the amino acid sequence that is at least 80% homologous to the amino acid sequence selected from the group consisting of SEQ ID NO: 29, 30, 31 and 32;

(c) antibody-specific binds to a receptor 1 interferon alpha man with an affinity for binding to at least 1·10-8M or greater; and

(d) the antibody inhibits the biological activity of at least one interferon type I.

Preferred antibodies of the present invention include the selected monoclonal human antibodies or their antigen-binding sites, including:

p> (a) the variable region of the heavy chain CDR1 of a person, comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 1, 2, 3, and 4;

(b) the variable region of the heavy chain CDR2 of a person, comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 5, 6, 7 and 8;

(c) variable region of the heavy chain CDR3 of a person, comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 9, 10, 11 and 12;

(d) variable region light chain CDR1 of a person, comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 13, 14, 15 and 16;

(e) variable region light chain CDR2 of a person, comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 17, 18, 19 and 20; and

(f) variable region light chain CDR3 of a person, comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 21, 22, 23 and 24;

moreover, the antibody is specific binds to a receptor 1 interferon alpha man with an affinity for binding to at least 1·10-8M or greater.

Preferred combinations of CDR regions include the following:

(a) the variable region of the heavy chain CDR1 of a person, comprising SEQ ID NO:1;

(b) the variable region of the heavy chain CDR2 of a person, comprising SEQ ID NO:5;

(c) variable region of the heavy chain CDR3 of a person comprising SEQ ID NO:9;

(d) variable region light chain CDR1 of a person, comprising SEQ ID NO:13;

(e) variable region light chain CDR2 of a person, comprising SEQ ID NO:17; and

(f) variable region light chain CDR3 of a person, comprising SEQ ID NO:21.

(a) the variable region of the heavy chain CDR1 of a person, comprising SEQ ID NO:2;

(b) the variable region of the heavy chain CDR2 of a person, comprising SEQ ID NO:6;

(c) variable region of the heavy chain CDR3 of a person, comprising SEQ ID NO:10;

(d) variable region light chain CDR1 of a person, comprising SEQ ID NO:14;

(e) variable region light chain CDR2 of a person, comprising SEQ ID NO:18; and

(f) variable region light chain CDR3 of a person, comprising SEQ ID NO:22.

(a) the variable region of the heavy chain CDR1 of a person, comprising SEQ ID NO:3;

(b) the variable region of the heavy chain CDR2 of a person, comprising SEQ ID NO:7;

(c) variable region of the heavy chain CDR3 of a person, comprising SEQ ID NO:11;

(d) variable region light chain CDR1 of a person, comprising SEQ ID NO:15;

(e) variable region light chain CDR2 of a person, comprising SEQ ID NO:19; and

(f) variable region light chain CDR3 of a person, comprising SEQ ID NO:23.

(a) the variable region of the heavy chain CDR1 of a person, comprising SEQ ID NO:4;

(b) the variable region of the heavy chain CDR2 of a person, comprising SEQ ID NO:8;

(c) variable region of the heavy chain CDR3 of a person, comprising SEQ ID NO:12;

(d) variable region light chain CDR1 of a person, comprising SEQ ID NO:16;

(e) variable region light chain CDR2 of a person, comprising SEQ ID NO:20; and

(f) variable region light chain CDR3 of a person, comprising SEQ ID NO:24.

Other preferred antibodies of the present invention include the selected monoclonal human antibodies or their antigen-binding sites, including:

(a) the variable region of the heavy chain of a human, comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 25, 26, 27 and 28; and

(b) the variable region of the light chain of a human, comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 29, 30, 31 and 32;

moreover, the antibody is specific binds to a receptor 1 interferon alpha man with an affinity for binding to at least 1·10-8M or greater.

Preferred combinations of the heavy and light chains include the following:

(a) the variable region of the heavy chain of a human, comprising the amino acid sequence of SEQ ID NO:25; and

(b) the variable region of the light chain of a human, comprising the amino acid sequence of SEQ ID NO:29.

(a) the variable region of the heavy chain of a human, comprising the amino acid sequence of SEQ ID NO:26; and

(b) the variable region of the light chain of a human, comprising the amino acid after outermost SEQ ID NO:30.

(a) the variable region of the heavy chain of a human, comprising the amino acid sequence of SEQ ID NO:27; and

(b) the variable region of the light chain of a human, comprising the amino acid sequence of SEQ ID NO:31.

(a) the variable region of the heavy chain of a human, comprising the amino acid sequence of SEQ ID NO:28; and

(b) the variable region of the light chain of a human, comprising the amino acid sequence of SEQ ID NO:32.

Another aspect of the invention relates to antibodies that compete for binding to IFNAR-1 with the control antibody, is developed in the present invention. Accordingly, in another embodiment, the invention relates to:

the selected monoclonal antibody or its antigen-binding site, and the antibody is involved in cross-competition with a control antibody for binding to the receptor 1 interferon alpha man, where the reference antibody selected from the group consisting of:

(a) an antibody comprising the variable region of the heavy chain containing the amino acid sequence of SEQ ID NO:25; and the variable region of the light chain containing the amino acid sequence of SEQ ID NO:29;

(b) an antibody comprising the variable region of the heavy chain containing the amino acid sequence of SEQ ID NO:26; and the variable region of the light chain is, containing the amino acid sequence of SEQ ID NO:30;

(c) an antibody comprising the variable region of the heavy chain containing the amino acid sequence of SEQ ID NO: 27; and the variable region of the light chain containing the amino acid sequence of SEQ ID NO:31; and

(d) an antibody comprising the variable region of the heavy chain containing the amino acid sequence of SEQ ID NO:28; and the variable region of the light chain containing the amino acid sequence of SEQ ID NO:32.

In some embodiments implementing the invention relates to a human antibody or its antigen-binding site, and the antibody does not bind to the same epitope as monoclonal antibody 64G12 mouse (ECACC, Depository No. 92022605) (i.e. not involved in cross-competition with him).

Antibodies of the present invention can be of any isotype. Preferred antibodies are isotypes IgG1, IgG3 or IgG4. Antibodies of the present invention can be full-size antibody comprising the variable and constant region, or they can be their antigen-binding fragments, such as antibodies, single chain or Fab fragments or Fab'2.

In addition, the invention relates to immunoconjugate comprising the antibody of the present invention or an antigen-binding site, linked to the s with a therapeutic tool such as a cytotoxin or a radioactive isotope. The invention also relates to bispecific molecule comprising the antibody or the antigen-binding site according to the present invention, associated with the second functional fragment having binding specificity different from that described antibody or its antigen-binding site.

The compositions containing the antibody or its antigen-binding site, or immunoconjugate, or bispecific molecule of the present invention and, in addition, pharmaceutically acceptable carrier, also belong to the present invention.

Molecules of nucleic acids encoding antibodies or their antigen-binding sites of the present invention, also included in the scope of the invention, as well as expression vectors comprising such nucleic acids, and cells of the host containing such expression vectors. In addition, the invention relates to transgenic mice with transgenes heavy and light chains of human immunoglobulin, and mouse Express the antibodies of the present invention, as well as hybridomas derived from such mice, and hybridoma produce antibodies of the present invention.

In addition, the invention relates to methods for "second generation" of antibodies anti-IFNAR-1, based on the sequences of an antibody and-IFNAR-1, developed in the present invention. For example, the invention relates to a method for producing antibodies anti-IFNAR-1, including:

(a) obtaining: (i) sequence variable regions of the heavy chain of the antibody comprising the CDR1 sequence that is selected from the group consisting of SEQ ID NO: 1, 2, 3 and 4, a CDR2 sequence that is selected from the group consisting of SEQ ID NO: 5, 6, 7 and 8; and a CDR3 sequence that is selected from the group consisting of SEQ ID NO: 9, 10, 11 and 12; or (ii) the sequence of the variable region of the light chain of the antibody comprising the CDR1 sequence that is selected from the group consisting of SEQ ID NO: 13, 14, 15 and 16, a CDR2 sequence that is selected from the group consisting of SEQ ID NO: 17, 18, 19 and 20, and a CDR3 sequence that is selected from the group consisting of SEQ ID NO: 21, 22, 23 and 24;

(b) modifying at least one amino acid residue in at least one variable region sequence of the antibody, and the above sequence selected from the sequences of variable regions of heavy chain antibodies and sequence of the variable region of the light chain of the antibody, to create at least one altered sequences of the antibodies; and

(c) the expression of the modified sequence of the antibody in the form of protein.

In addition, the present invention relates to a method of inhibiting biologists the definition of interferon type I expressed on cells receptor 1 interferon alpha, including the interaction of cells with the antibody of the present invention, in order to inhibit the biological activity of interferon type I. the Invention also relates to a method of treating diseases or disorders in an individual, mediated by interferon type I, in case of need of such treatment, comprising the administration to an individual antibody or its antigen-binding site according to the present invention, in order to treat a disease mediated by interferon type I. a Disease mediated by interferon type I, may, for example, be a disease mediated by interferon-alpha.

Examples of diseases or disorders that can be effectively applied methods of the present invention include systemic lupus erythematosus, insulin-dependent diabetes mellitus, inflammatory bowel disease, multiple sclerosis, psoriasis, autoimmune thyroiditis, rheumatoid arthritis, glomerulonephritis, HIV infection, AIDS, transplant rejection and graft-versus-host.

Other distinctive features and advantages of the present invention will become apparent from the following detailed description and examples, which should not be construed as limiting. The contents of all references, positions in those in GenBank, patents, and published the data of patent applications cited in the text of this application, directly incorporated into the present application by reference.

BRIEF DESCRIPTION of DRAWINGS

On figa shows the nucleotide sequence (SEQ ID NO:33) and amino acid sequence (SEQ ID NO:25) variable regions of the heavy chain of the monoclonal antibody 3F11 person. Selection CDR1(SEQ ID NO:1), CDR2(SEQ ID NO:5) and CDR3(SEQ ID NO:9).

On FIGU shows the nucleotide sequence (SEQ ID NO:37) and amino acid sequence (SEQ ID NO:29) variable region of the light chain of the monoclonal antibody 3F11 person. Selection CDR1(SEQ ID NO:13), CDR2(SEQ ID NO:17), and CDR3(SEQ ID NO:21).

On figa shows the nucleotide sequence (SEQ ID NO:34) and amino acid sequence (SEQ ID NO:26) variable regions of the heavy chain of the monoclonal antibody 4G5 person. Selection CDR1(SEQ ID NO:2), CDR2(SEQ ID NO:6) and CDR3(SEQ ID NO:10).

On FIGU shows the nucleotide sequence (SEQ ID NO:38) and amino acid sequence (SEQ ID NO:30) variable region light chain monoclonal antibodies 4G5 person. Selection CDR1(SEQ ID NO:14), CDR2(SEQ ID NO:18) and CDR3(SEQ ID NO:22).

On figa shows the nucleotide sequence (SEQ ID NO:35) and amino acid sequence (SEQ ID NO:27) variable regions of the heavy chain of the monoclonal antibody 11E2 person. Selection CDR1(SEQ ID NO:3), CDR2(SEQ ID NO:7) and CDR3(SEQ ID NO:11).

Nafig shows the nucleotide sequence (SEQ ID NO:39) and amino acid sequence (SEQ ID NO:31) variable region light chain monoclonal antibodies 11E2 person. Selection CDR1(SEQ ID NO:15), CDR2(SEQ ID NO:19) and CDR3(SEQ ID NO:23).

On figa shows the nucleotide sequence (SEQ ID NO:36) and amino acid sequence (SEQ ID NO:28) variable regions of the heavy chain of the monoclonal antibody 9D4 person. Selection CDR1(SEQ ID NO:4), CDR2(SEQ ID NO:8), and CDR3(SEQ ID NO:12).

On FIGU shows the nucleotide sequence (SEQ ID NO:40) and amino acid sequence (SEQ ID NO:32) variable region light chain monoclonal antibodies 9D4 person. Selection CDR1(SEQ ID NO:16), CDR2(SEQ ID NO:20), and CDR3(SEQ ID NO:24).

Figure 5 shows the alignment of amino acid sequences of variable regions of the heavy chain 3F11 with the amino acid sequence of germline VH 4-34 human (SEQ ID NO:41).

Figure 6 shows the alignment of amino acid sequences of variable regions of the heavy chain 4G5 with the amino acid sequence of germline VH 4-34 human (SEQ ID NO:41).

7 shows the alignment of amino acid sequences of variable regions of the heavy chain 11E2, and 9D4 with the amino acid sequence of germline VH 5-51 human (SEQ ID NO:42).

On Fig shows the alignment of amino acid sequences of variable region of the light chain 3F11 with the amino acid sequence of germline VK L18 human (SEQ ID NO:43).

Figure 9 shows the combination of aminokislot the th sequence of the variable region of the light chain 4G5 with the amino acid sequence of germline VK L18 human (SEQ ID NO:43).

Figure 10 shows the alignment of amino acid sequences of variable region of the light chain 11E2, and 9D4 with the amino acid sequence of germline VK A27 human (SEQ ID NO:44).

11 is a graph showing the results of experiments showing that a monoclonal antibody human, namely 3F11 directed against IFNAR-1 person, not competes with a monoclonal antibody 64G12 mouse for binding to IFNAR-1.

DETAILED description of the INVENTION

The present invention relates to the selection of monoclonal antibodies that bind to the receptor 1 interferon alpha (IFNAR-1) and which is able to block the action of interferon type I. In the present invention developed a dedicated antibodies, methods of obtaining these antibodies, immunoconjugates and bispecific molecules comprising such antibodies and pharmaceutical compositions containing these antibodies, immunoconjugates or bispecific molecules of the present invention. In addition, the present invention relates to methods of using these antibodies to inhibit the binding of interferon type I with IFNAR-1 cells expressing IFNAR-1, for example, in the treatment of immune-mediated disorders in an individual, including autoimmune disorders, transplant rejection and reaction transplantatio host (GVHD).

For easier understanding of the present invention at the beginning define some terms. Additional definitions are provided in the course of the detailed description.

The terms "receptor 1 interferon alpha, IFNAR-1 and antigen IFNAR-1" are used interchangeably, and include variants, isoforms, variants, homologues of IFNAR-1 person, and its analogs having at least one common epitope with IFNAR-1. Accordingly, human antibodies of the present invention may, in some cases to engage in cross-interaction with IFNAR-1 other species besides humans, or other proteins that are structurally related with IFNAR-1 person (for example, homologues of IFNAR-1 person). In other cases, the antibodies may be completely specific for IFNAR-1, and may not manifest signs of the ability to cross-interactions of other types.

The complete cDNA sequence of IFNAR-1 person has inventory number of those in GenBank NM_000629.

The term "type I interferon" in the present description is designed to indicate the family members of molecules of type I interferons, which are ligands for IFNAR-1 (i.e. family members of molecules of type I interferons, which are able to communicate with IFNAR-1). Examples of ligands related to type I interferons are interferon alpha 1, 2a, 2b, 4, 5, 6, 7, 8, 10, 14, 16, 17, 21, interfere the beta and interferon omega.

The term "immune response" refers to the action of, for example, lymphocytes, antigen presenting cells, phagocytes, granulocytes, and soluble macromolecules produced by these cells or the liver (including antibodies, cytokines, and complement), which results in selective damage to, or destruction, or removal from the human body penetrated him pathogens, cells or tissues infected with pathogens, cancerous cells, or, in cases of autoimmunity or pathological inflammation, normal cells or tissues.

The term "path of signal transduction" refers to the biochemical links between different molecules, which converts the signal, which play a role in the transmission of signal from one part of cell to another part. In the present description, the phrase "cell surface receptor" includes, for example, molecules and complexes of molecules able to receive the signal and send this signal through the cytoplasmic membrane of the cells. An example of a "cell surface receptor" of the present invention is the receptor IFNAR-1.

The term "antibody" in the framework of the present description includes whole antibodies and any antigen-binding fragments (i.e., "antigen-binding sites") or single chains. The term "antibody" refers to glycoproteins containing at least two heavy chains (H) and two light the chain (L), connected by disulfide bonds, or an antigen-binding sites. Each heavy chain is included variable region heavy chain (abbreviated as indicated in the text VH) and constant region of the heavy chain. The constant region of the heavy chain consists of three domains, CH1, CH2 and CH3. In each of the light chains included variable region light chain (abbreviated as indicated in the text VL) and a constant region light chain. The constant region of the light chain consists of a single domain, denoted by CL. Region VH and VL can be further subdivided into the field of hypervariability, called scopes, complementarity determining (CDR), which are interleaved with the more conservative fragments, referred to as frame regions (FR). Each of the chains VH and VL is composed of three CDRs and four FR located from aminobenzo to carboxilic in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (for example, cell effectors) and the first component (Clq) of the classical complement system.

The term "antigen-binding site" of an antibody (or simply "plot antibodies") this is the Scripture refers to one or more fragments of antibodies, which retain the ability-specific contact with the antigen (for example, IFNAR-1). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-sized antibodies. Examples of binding fragments encompassed by the term "antigen-binding site" of an antibody include (i) a Fab fragment, i.e. a monovalent fragment consisting of the VL domains, VH, CL and CH1; (ii) the fragment F(ab')2i.e. bivalent fragment comprising two Fab fragment linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH domains and CH1; (iv) a Fv fragment consisting of domains VL and VH one shoulder antibodies; (v) a dAb fragment (Wardet al. (1989)Nature,341:544-546), which consists of a VH domain; and (vi) the allocated region complementarity determining (CDR). In addition, although the two domains of the Fv fragment, i.e. VL and VH, encoded by separate genes, they can be joined by a synthetic linker using recombinant methods, the linker gives an opportunity to get them in the form of a single protein chain in which a pair of areas VL and VH form monovalent molecules (known as single-chain Fv (scFv); see, for example, Birdet al. (1988)Science,242: 423-426; and Hustonet al. (1988)Proc.Natl.Acad.Sci.USA85: 5879-5883). Have in mind that such single-chain antibodies are also covered by the term "antigen-binding site antibodies. These fragments of antibodies obtained using standard techniques known to experts in the art, and the fragments are subjected to selection based on their usefulness in the same manner as are intact antibodies.

The term "isolated antibody" in the present invention is designed to indicate the antibody, which is essentially free of other antibodies having other antigenic specificnosti (for example, the selected antibody that is specific binds to IFNAR-1, largely free from antibodies that have been specifically bind to other antigens that differ from IFNAR-1). However, the selected antibody that is specific binds to IFNAR-1, may have cross-reactivity to other antigens, such as molecules IFNAR-1 other species. In addition, the selected antibody can be substantially free of other cellular substances and/or chemical compounds.

The terms "monoclonal antibody" or "composition based on monoclonal antibody" in the present description relate to the drug based on the antibody molecules of the same molecular composition. Composition based on monoclonal antibodies reveals a single binding specificity and affinity for a particular epitope.

It is assumed that in the present description, the term "human antibody" includes antibodies available is variable regions, in which as a framework and CDR region derived from immunoglobulin sequences of the germline of human cells. Furthermore, if the antibody contains a constant region, the constant region also is derived from immunoglobulin sequences of the germline of human cells. Human antibodies of the present invention may include amino acid residues not encoded immunoglobulin sequences of the germ line of human cells (e.g., mutations introduced by random or site-specific mutagenesisin vitroor somatic mutationin vivo). However, in the present description is not intended that the term "human antibody" includes antibodies by CDR sequences derived from the germ line cells of other mammalian species, such as mice, are grafted into frame sequences of a person.

The term "monoclonal human antibody" refers to antibodies, demonstrating a single binding specificity which have variable regions in which both the frame and the CDR region derived from immunoglobulin sequences of the germline of human cells. In one embodiment, the implementation of a monoclonal human antibodies produced by hybridomas, which includes B-cells derived from Tran the gene animals not related to the human race, such as a transgenic mouse, having a genome that comprises the heavy chain transgene person and a light chain transgene person, merged with immortalizing cell.

The term "recombinant human antibody" in the present description includes all human antibodies that are formed, expressed, created or isolated by means of recombinant technology, such as (a) antibodies isolated from an animal (e.g., mice)that are transgenic or transpromotional, to obtain from them the genes of the human immunoglobulin or hybridoma (described below), (b) antibodies isolated from host cells transformed for expression of human antibodies, for example, from transfection, (c) antibodies isolated from a recombinant, combinatorial libraries of antibodies person, and (d) antibodies obtained, expressed, created or selected by any other means that involves splicing of immunoglobulin gene sequences person with other DNA sequences. Such recombinant human antibodies have variable regions in which the frame and the CDR region derived from immunoglobulin sequences of the germline of human cells. However, in some embodiments, the implementation of such recombinant antibodies man can be subjected to in vitromutagenesis (or, if you use an animal that is transgenic due to the presence of Ig sequences person,in vivosomatic mutagenesis) and thus the amino acid sequences of regions VH and VL of the recombinant antibodies are sequences that, being derived from and being related to the sequences of the VH and VL germline human cell lines may not exist in nature in the range of antibody germline manin vivo.

In the present description, the term "isotype" refers to the class of antibodies (e.g., IgM or IgGI), which encoded the genes for the constant region of the heavy chain.

The term "specific binding" refers in this description to the binding of an antibody to a given antigen. Typically, the antibody binds with a dissociation constant (KD)equal to 10-7M or less, and the binding with the given antigen occurs with KDthat is at least two times less than the KDbinding of this antibody with non-specific antigen (e.g., BSA, casein), which differs from the set or a closely-related antigen. The phrases "an antibody recognizing an antigen" and "an antibody specific for the antigen" in the present description are interchangeable with the term "antibody, which is specific linked with antig the nom.

The term "KACC" or "Ka" in the present invention is designed to indicate the speed of the Association upon the specific interaction of antibody-antigen, whereas the term "Kdis" or "Kd" in the present description is intended to indicate that the dissociation rate at the specific interaction of antibody-antigen. The term "KD" in the present description is intended to indicate the dissociation constant, which is obtained from the relation KdKa(i.e. Kd/Ka) and is expressed in units of molar concentration (M). Values of KDantibodies can be determined using methods well-established in the art. The preferred method of determining the KDantibodies is the use of surface plasmon resonance, preferably using a biosensor system such as the Biacore system®.

In the present description, the term "high affinity" for IgG antibody refers to an antibody having KD10-8M or less, more preferably 10-9M or less and even more preferably 10-10M or less. However, linking a "high affinity" can be changed to antibodies of other isotypes. For example, tying with "high affinity" for an IgM isotype refers to an antibody having KD10-7M or less, more preferably 10-8M or less.

In the present description, the term "subject" includes humans and animals, non-human kind. The term "animal not belonging to the human race" includes all vertebrates, such as mammals and not mammals, such as primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc.

In the following sections, various aspects of the present invention is described with further details.

Antibodies anti-IFNAR-1

Antibodies of the present invention are characterized by certain functional features or properties of the antibodies. For example, these antibodies specific contact IFNAR-1, preferably IFNAR-1 person. In addition, antibodies can engage in cross-interaction with IFNAR-1 one or more human primates, such as cynomolgus monkeys and/or rhesus monkeys. Preferably the antibody of the present invention binds to IFNAR-1 with high affinity, for example, with KDequal to 10-7M or less, more preferably KDequal to 10-8M or less, or 10-9M or less, or even 5·10-10M or less, or 2·10-10M or less.

In addition, antibodies of the present invention is able to inhibit the biological activity of interferon type I. These antibodies inhibit the biological activity of the hile least one interferon type I and preferably inhibit the biological activity of a large number of type I interferons (i.e. at least two, more preferably at least three, or at least four or at least five or at least six or at least seven or at least eight or at least nine, or at least ten, or at least 11 or at least 12 or at least 13 or at least 14 or at least 15 different subtypes of type I interferons). In a preferred embodiment, the antibody inhibits the biological activity following interferon type I: α1, α2a, α2b, α4, α5, α6, α7, α8, α10, α14, α16, α17, and α21, beta and omega. In other preferred embodiments, the implementation of the antibody inhibits the activity of lymphoblastoid interferon and/or leukocyte interferon.

The ability of antibodies to inhibit the biological activity of type I interferons can be tested in one or more assays known in the art. Non-limiting examples include inhibition of Daudi cell proliferation mediated inhibition of interferon type I inhibition caused by interferon type I expression of IP-10 managername cells perifericheskoi blood (PBMC), inhibition of the development of dendritic cell-mediated plasma systemic lupus erythematosus (SLE), as well as the inhibition of the antiviral activity of interferon type I. Antibody "inhibits the biological activity of interferon type I"if the but this inhibits the activity of at least 20%, more preferably at least 30%, even more preferably at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90%, compared to a nonspecific control antibody.

In preferred embodiments, the implementation of the antibody inhibits the activity of IFN-α 2b in the analysis, based on the proliferation of Daudi cells, inhibits the activity of IFN omega in the analysis, based on the proliferation of Daudi cells, inhibits the secretion of IP-10 by PBMC cells induced IFN-α 2b or IFN omega, and/or inhibits the development of dendritic cell-mediated plasma of SLE.

In another preferred embodiment, the antibody is not involved in cross-competition (i.e. binds to a different epitope) with anti-IFNAR-1 antibody 64G12 mouse (deposited as ECACC, Depository No. 92022605).

In the examples described in more detail analyses to assess the functional activity of anti-IFNAR antibodies. Preferred antibodies of the present invention exhibit at least one, more preferably two, three, four, five or more of the following properties:

a) specific contact IFNAR-1 (preferably IFNAR-1 person);

b) contact with IFNAR-1 with high affinity, so that KDis 1·10-8M or greater affinity;

c) inhibit the biological activity of multiple interferon type I;

p> d) inhibit the activity of IFN-α 2b in the analysis of Daudi cell proliferation;

(e) inhibit the activity of IFN omega in the analysis of Daudi cell proliferation;

f) inhibit the secretion of IP-10 managername the peripheral blood cells induced IFN-α 2b;

g) inhibit the secretion of IP-10 managername the peripheral blood cells induced IFN omega;

h) inhibit the development of dendritic cell-mediated plasma systemic lupus erythematosus; and

i) contact with another epitope (e.g., does not participate in cross-competition)than monoclonal antibody 64G12 mouse (ECACC, Depository No. 92022605).

The antibody of the present invention may be any combination of the above functional features and/or functionalities described in the examples.

Monoclonal antibody 3F11, 4G5, 11E2, and 9D4

Preferred antibodies of the present invention are monoclonal antibodies person 3F11, 4G5, 11E2, and 9D4, isolated and structurally characterized as described in the examples. Amino acid sequence of VH 3F11, 4G5, 11E2, and 9D4 shown in SEQ ID NO: 25, 26, 27 and 28, respectively. Amino acid sequence of VL 3F11, 4G5, 11E2, and 9D4 shown in SEQ ID NO: 29, 30, 31 and 32, respectively.

Considering the fact that each of these antibodies can bind to IFNAR-1, sequences of VH and VL can be "mixed and matched in pairs for the create other anti-IFNAR-1 binding molecules of the present invention. The binding of such "mixed and matched in pairs of antibodies with IFNAR-1 can be studied with the use described in this application analyses of binding (e.g., ELISA) and/or with the use of functional assays of inhibition of interferon type I described in the examples. Preferably, if the chain VH and VL mixed and matched in pairs, a VH sequence from a particular pair of VH/VL is replaced by a similar structure by a sequence of VH. Similarly, preferably, a VL sequence of specific pairs of VH/VL is replaced by a similar structure by a sequence of VL. For example, sequences of VH and VL of the antibody 3F11 and 4G5 particularly suitable for mixing and matching pair, since these antibodies use sequences of VH and VL derived from the same germline sequences of lines (VH 4-34 and VK L18), and thus, they exhibit structural similarity. In addition, sequences of VH and VL 11E2, and 9D4 particularly suitable for mixing and matching pair, since these antibodies use sequences of VH and VL derived from the same germline sequences of lines (VH 5-51 and VK A27), and thus, they exhibit structural similarity.

Accordingly, in one aspect the present invention relates to the selected monoclonal antibody or its antigen-binding site, including:

(a) the var is abelow region of the heavy chain, comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 25, 26, 27 and 28; and

(b) variable region light chain comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 29, 30, 31 and 32;

where the antibody is specific bound IFNAR-1.

Preferred combinations of the heavy and light chains include:

(a) a variable region of a heavy chain including amino acid sequence SEQ ID NO: 25; and (b) variable region light chain comprising the amino acid sequence of SEQ ID NO: 29; or

(a) a variable region of a heavy chain including amino acid sequence SEQ ID NO: 26; and (b) variable region light chain comprising the amino acid sequence of SEQ ID NO: 30; or

(a) a variable region of a heavy chain including amino acid sequence SEQ ID NO: 27; and (b) variable region light chain comprising the amino acid sequence of SEQ ID NO: 31; or

(a) a variable region of a heavy chain including amino acid sequence SEQ ID NO: 28; and (b) variable region light chain comprising the amino acid sequence of SEQ ID NO: 32.

In another aspect, the present invention relates to antibodies which comprise heavy and light chain CDR1, CDR2 and CDR3 of an antibody 3F11, 4G5, 11E2, and 9D4 or combinations thereof. Amino acid placentas the activity VH CDR1 3F11, 4G5, 11E2, and 9D4 shown in SEQ ID NO: 1, 2, 3 and 4. Amino acid sequence of VH CDR2 3F11, 4G5, 11E2, and 9D4 shown in SEQ ID NO: 5, 6, 7 and 8. Amino acid sequence of the VH CDR3 3F11, 4G5, 11E2, and 9D4 shown in SEQ ID NO: 9, 10, 11 and 12. Amino acid sequences of the VK CDR1 3F11, 4G5, 11E2, and 9D4 shown in SEQ ID NO: 13, 14, 15 and 16. Amino acid sequences of the VK CDR2 3F11, 4G5, 11E2, and 9D4 shown in SEQ ID NO: 17, 18, 19 and 20. Amino acid sequences of the VK CDR3 3F11, 4G5, 11E2, and 9D4 shown in SEQ ID NO: 21, 22, 23 and 24. Region CDR selected using the Kabat system (Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242).

Considering the fact that each of these antibodies can bind to IFNAR-1, and that antigen-binding specificity is provided primarily by the regions CDR1, 2 and 3, the sequence of the VH CDR1, 2 and 3 and VK CDR1, 2 and 3 can be "mixed and matched in pairs" (i.e., CDRs from different antibodies can be mixed and matched in pairs, although each antibody must contain a VH CDR1, 2 and 3 and VK CDR1, 2 and 3) to create other anti-IFNAR-1 binding molecules according to the present invention. The binding of such "mixed and matched in pairs of antibodies with IFNAR-1 can be investigated using analyses of binding described above and in the examples (e.g., ELISA). Preferably, if the sequence VH CDR mixed and matched in pairs, the sequence of CDR1, CDR2 and/or CDR3 of a particular pic is egovernance VH replaced by a similar structure sequence (sequences), CDR. Similarly, if the sequence VK CDR mixed and matched in pairs, the sequence of CDR1, CDR2 and/or CDR3 of a specific sequence VK is preferably replaced by a similar structure sequence (sequences), CDR. For example, the sequence of the VH CDR1 of the antibody 3F11 and 4G5 have some structural similarity and, therefore, suitable for mixing and matching pair. As another example, the sequence of the VH CDR1 of the antibody 11E2, and 9D4 have some structural similarity and, therefore, suitable for mixing and matching pair. As another example, the sequence VK CDR1 of the antibody 3F11 and 4G5 have some structural similarity. As another example, the sequence of the VH CDR1 of the antibody 11E2, and 9D4 have some structural similarity. An ordinary specialist in the art will easily understand that the new sequences of VH and VL can be created by substituting one or more sequences of the CDR regions of VH and/or VL, structurally similar sequences from the CDR sequences disclosed in the present description for monoclonal antibody 3F11, 4G5, 11E2, and 9D4.

Accordingly, in another aspect, the invention relates to the selected monoclonal antibody or its antigen-binding site, including:

(a) the variable region of the heavy chain CDR1, including the surrounding amino acid sequence, selected from the group consisting of SEQ ID NO: 1, 2, 3, and 4;

(b) variable region heavy chain CDR2 comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 5, 6, 7 and 8;

(c) variable region heavy chain CDR3 comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 9, 10, 11 and 12;

(d) variable region light chain CDR1 comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 13, 14, 15 and 16;

(e) variable region light chain CDR2 comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 17, 18, 19 and 20; and

(f) variable region light chain CDR3 comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 21, 22, 23 and 24;

moreover, the antibody is specific binds to IFNAR-1.

In preferred embodiments, the implementation of the antibody includes:

(a) the variable region of the heavy chain CDR1 comprising SEQ ID NO:1;

(b) variable region heavy chain CDR2 comprising SEQ ID NO:5;

(c) variable region heavy chain CDR3 comprising SEQ ID NO:9;

(d) variable region light chain CDR1 comprising SEQ ID NO:13;

(e) variable region light chain CDR2 comprising SEQ ID NO:17; and

(f) variable region light chain CDR3 comprising SEQ ID NO:21.

In another preferred embodiment, titulo includes:

(a) the variable region of the heavy chain CDR1 comprising SEQ ID NO:2;

(b) variable region heavy chain CDR2 comprising SEQ ID NO:6;

(c) variable region heavy chain CDR3 comprising SEQ ID NO:10;

(d) variable region light chain CDR1 comprising SEQ ID NO:14;

(e) variable region light chain CDR2 comprising SEQ ID NO:18; and

(f) variable region light chain CDR3 comprising SEQ ID NO:22.

In another preferred embodiment, the antibody includes:

(a) the variable region of the heavy chain CDR1 comprising SEQ ID NO:3;

(b) variable region heavy chain CDR2 comprising SEQ ID NO:7;

(c) variable region heavy chain CDR3 comprising SEQ ID NO:11;

(d) variable region light chain CDR1 comprising SEQ ID NO:15;

(e) variable region light chain CDR2 comprising SEQ ID NO:19; and

(f) variable region light chain CDR3 comprising SEQ ID NO:23.

In another preferred embodiment, the antibody includes:

(a) the variable region of the heavy chain CDR1 comprising SEQ ID NO:4;

(b) variable region heavy chain CDR2 comprising SEQ ID NO:8;

(c) variable region heavy chain CDR3 comprising SEQ ID NO:12;

(d) variable region light chain CDR1 comprising SEQ ID NO:16;

(e) variable region light chain CDR2 comprising SEQ ID NO:20; and

(f) variable region light chain CDR3 comprising SEQ ID NO:24.

Antibodies to the verge associated with the same epitope as 3F11, 4G5, 11E2, and 9D4

In another embodiment, the invention relates to antibodies that bind to the same epitope on IFNAR-1 person, and monoclonal antibody 3F11, 4G5, 11E2, or 9D4 (having the sequence VH,shown in SEQ ID NO: 25, 26, 27 and 28, respectively, and VL sequence shown in SEQ ID NO: 29, 30, 31 and 32, respectively). Such antibodies can be identified based on their ability to engage in cross-competition with 3F11, 4G5, 11E2, or 9D4 in standard assays of binding to IFNAR-1. The capacity of the investigated antibodies to inhibit the binding of 3F11, 4G5, 11E2, or 9D4 with IFNAR-1 person demonstrates that the test antibody can compete with 3F11, 4G5, 11E2, or 9D4 for binding to IFNAR-1 of human and, thus, to contact the same epitope IFNAR-1 person, and antibody 3F11, 4G5, 11E2, or 9D4. In a preferred embodiment, the antibody that binds to the same epitope IFNAR-1 person, and 3F11, 4G5, 11E2, or 9D4, is a monoclonal antibody man. Such monoclonal antibodies can be obtained and allocated as described in the examples.

In another preferred embodiment, the antibody binds to a different epitope (i.e., not involved in cross-competition)than monoclonal antibody 64G12 mouse (ECACC, Depository No. 92022605).

Antibodies, is within a specific sequence germ lines

In some embodiments, the implementation of the antibody of the present invention includes the variable region of the heavy chain of the gene heavy chain immunoglobulin specific germ line and/or the variable region of the light chain of the gene light chain immunoglobulin specific germ line.

For example, in a preferred embodiment, the invention relates to the selection of anti-IFNAR-1 monoclonal antibody or its antigen-binding site, and the antibody:

(a) includes the variable region of the heavy chain of the genes VH 4-34 or 5-51 person;

(b) includes the variable region of the light chain of the genes VK L18 or A27 person; and

(c) antibody-specific binds to IFNAR-1.

Examples of antibodies having VH and VK of VH 4-34 and VK L18, respectively, include 3F11 and 4G5. Examples of antibodies having VH and VK of VH 5-51 and VK A27, respectively, include 11E2, and 9D4.

In the present description, the human antibody comprises variable regions of the heavy or light chains "of"or "derived from"or "which is the product of certain sequences germ lines if the variable regions of antibodies derived from a system that uses the genes of the immunoglobulin germ-line person. Such systems include immunosorbant transgenic mice with genes of human immunoglobulin of interest, antigen is or screening of libraries of genes of human immunoglobulin in the phage-display system of interest antigen. The antibody of the person against whom the terms "of"or "derived from"or "being a product" immunoglobulin sequence to the germline of the person can be identified by itself, by comparing the amino acid sequences of human antibodies with amino acid sequences of immunoglobulin germline of man and selection of the immunoglobulin sequences of the germline of the person that most closely matches (i.e. the highest % compliance) sequences of human antibodies. The antibody of the person against whom the terms "of"or "derived from"or "being a product" immunoglobulin sequence specific germline person, may contain differences in amino acids compared to the germline sequence, due to, for example, natural somatic mutations or intentional introduction of site-directed mutations. However, the selected human antibody amino acid sequence, typically at least 90% identical to the amino acid sequence encoded by a gene of the immunoglobulin germline person, and contains amino acid residues that define human nature human antibodies when compared with immunoglob Lisovyi amino acid sequences germ lines of other species (e.g., sequences germ line of the mouse). In some cases, the antibody may be at least 95% or even at least 96%, 97%, 98% or 99% identical in amino acid sequence, amino acid sequence, encoded by a gene of the immunoglobulin germ line. As a rule, the human antibody derived from the specific sequences of the germline of the person, not be more than 10 differences in amino acid residues compared to the amino acid sequence encoded by a gene of the immunoglobulin germline person. In some cases, the antibody can be detected not more than 5, or even no more than 4, 3, 2 or 1 differences in amino acid residues compared to the amino acid sequence encoded by a gene of the immunoglobulin germ line.

Homologous antibodies

In yet another embodiment, the antibody of the present invention includes the variable regions of the heavy and light chains containing amino acid sequences that are homologous amino acid sequences described in the present application preferred antibodies, and the antibodies retain the desired functional properties of the anti-IFNAR-1 antibodies of the present invention. For example, the invention relates to the selected mo is acanalonia the antibody or its antigen-binding site, including the variable region of the heavy chain and the variable region of light chain, in which:

(a) the variable region of the heavy chain comprises the amino acid sequence that is at least 80% homologous to the amino acid sequence selected from the group consisting of SEQ ID NO: 25, 26, 27 and 28;

(b) the variable region of the light chain comprises the amino acid sequence that is at least 80% homologous to the amino acid sequence selected from the group consisting of SEQ ID NO: 29, 30, 31 and 32;

(c) antibody-specific binds to IFNAR-1 and exhibits at least one of the functional properties described in this application, preferably more of the described in the application properties.

In other embodiments, implementation of the amino acid sequences of VH and/or VL can be 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to the above sequences. Antibody, wherein the VH region and VL are high (i.e. 80% or more) degree of homology regions VH and VL of the above sequences can be obtained by mutagenesis (e.g., site-directed or PCR-mediated mutagenesis) of nucleic acid molecules encoding SEQ ID NO: 33, 34, 35, 36, 37, 38, 39 or 40 and then to test whether changing the encoded antibodies to the preservation of its functions (i.e. the functions set out above in paragraphs. (c), (d) the (e)) with the use described in this application functional tests.

In the present description, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. The percent identity between two sequences is a function of the number of identical positions, which are present in the two sequences at the same time (i.e. the % homology = number of identical positions/total number of positions ×100), taking into account the number of gaps and the length of each gap, which should be introduced for optimal alignment of the two sequences. Comparison of sequences and determination of percent identity between two sequences can be performed using a mathematical algorithm, as described in the following non-limiting example.

The percent identity between two amino acid sequences can be determined using algorithm E.Meyers and W.Miller (Comput. Appl.Biosci.,4:11-17 (1988)), which was incorporated into the ALIGN program (version 2.0) using table weight residues PAM120, setting gap length penalty”equal to 12, and the parameter “gap penalty”equal to 4. In addition, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J.Mol.Biol.48:444-453 (1970)), which was included in the program GAP GCG software package available on the website http://www.gcg.com), using either matrix Blossum 62, or a PAM250 matrix, and the parameter “gap weight” 16, 14, 12, 10, 8, 6 or 4 and a length weight” 1, 2, 3, 4, 5 or 6.

In addition to or as an alternative, the protein sequence of the present invention can additionally be used as "item database" to search public databases to, for example, identify related sequences. Such searches can be performed with the XBLAST program (version 2.0) of Altschul,et al. (1990)J.Mol.Biol.215:403-10. To obtain amino acid sequences homologous to the antibody molecules of the present invention, it is possible to perform a search of the protein BLAST using the XBLAST program, under parameters “score”=50, “wordlength”=3. To obtain missed alignments with the purposes of comparison, may be used by the program Gapped BLAST, as described in Altschul,et al. (1997)Nucleic Acid Res.25(17):3389-3402. When using the programs BLAST and Gapped BLAST can be used the parameters of the respective programs by default (e.g., XBLAST and NBLAST). Cm. http://www.ncbi.nlm.nih.gov.

Antibodies with conservative modifications

In some embodiments, the implementation of the antibody of the present invention includes a variable region heavy chain comprising sequences of CDR1, CDR2 and CDR3, and a variable region light chain, which contains asuu sequences CDR1, CDR2 and CDR3, where one or more of these CDR sequences include certain amino acid sequences based on the preferred antibodies described in this application (for example, 3F11, 4G5, 11E2, and 9D4), or conservative modifications, and where the antibodies retain the desired functional properties of the anti-IFNAR-1 antibodies of the present invention. Accordingly, the invention relates to the selected monoclonal antibody or its antigen-binding site comprising the variable region of the heavy chain comprising sequences of CDR1, CDR2 and CDR3, and a variable region light chain comprising sequences of CDR1, CDR2 and CDR3, and:

(a) the sequence of CDR3 of the variable region of the heavy chain contains an amino acid sequence selected from the group consisting of amino acid sequence SEQ ID NO: 9, 10, 11 and 12 and their conservative modifications;

(b) sequence of CDR3 of the variable region of the light chain contains an amino acid sequence selected from the group consisting of amino acid sequence SEQ ID NO: 21, 22, 23 and 24 and their conservative modifications; and

(c) antibody-specific binds to IFNAR-1 and exhibits at least one of the functional properties described in this application, preferably more of the described in the application properties.

In others the GOM embodiment, the CDR2 sequence of the variable region of the heavy chain comprises the amino acid sequence, selected from the group consisting of amino acid sequence SEQ ID NO: 5, 6, 7 and 8 and their conservative modifications; and the CDR2 sequence of the variable region of the light chain comprises the amino acid sequence selected from the group consisting of amino acid sequence SEQ ID NO: 17, 18, 19 and 20 and their conservative modifications. In yet another embodiment, the CDR1 sequence of the variable region of the heavy chain comprises the amino acid sequence selected from the group consisting of amino acid sequence SEQ ID NO: 1, 2, 3 and 4 and their conservative modifications; and the CDR1 sequence of the variable region of the light chain comprises the amino acid sequence selected from the group consisting of amino acid sequence SEQ ID NO: 13, 14, 15 and 16 and their conservative modifications.

In this application, the term "conservative modifications sequences" is intended to indicate modification of amino acid sequences that do not have a significant impact or not significantly alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include substitutions, additions or deletions of amino acids. Modifications to the antibody of the present invention can be made using the conventional methods, known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative substitutions of amino acids include substitutions, where amino acid residue is substituted with amino acid residue having a similar side chain. In the technique were identified family of amino acid residues having similar side chains. These families include amino acids with basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, Proline, phenylalanine, methionine), the side chains with beta-branching (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in the CDR regions of the antibodies of the present invention can be replaced by other amino acid residues with side chain from the same family and the altered antibody can be tested from the point of view of preserving functions (i.e. the functions set out above in paragraphs. (c), (d) and (e)) with the use described in this application, the functionality is selected tests.

Designed and modified antibodies

In addition to the above, the antibody of the present invention can be obtained using an antibody having one or more sequences of VH and/or VL disclosed in this application, as the starting material for constructing a modified antibody, which may have properties different from the original antibody. The antibody can be engineered by modifying one or more residues within one or both variable regions (i.e. VH and/or VL), for example in one or more CDR regions and/or in one or more frame regions. In addition to or as an alternative, the antibody can be constructed by modifying residues in a constant region (regions), for example, to change the effector functions (functions) antibodies.

One of the possible types of design variable region is the transplantation of CDR. Antibodies predominantly interact with target antigens through amino acid residues that are located in the light and heavy chains in six complementarity determining regions (CDR). Therefore, different antibody amino acid sequences in the CDRs differ to a greater extent than outside the CDR. Because the sequences of CDRs are responsible for most of the interactions, antibody-antigen, there is a possibility of the expression of recombinant antibodies that mimic the properties of natural antibodies by constructing expression vectors that include CDR sequence of specific natural antibodies, transplanted to a frame sequence of a different antibody with different properties (see, e.g., Riechmann, L.et al. (1998)Nature,332:323-327; Jones, P.et al.(1986)Nature,321: 522-525; Oueen, C.et al(1989)Proc.Natl.Acad.See.USA86:10029-10033; U.S. Patent No. 5 225 539 issued Winter, and U.S. patent No. 5530101; 5585089; 5693762 and 6180370 issued by the Queen and co-authors).

Accordingly, another variant of implementation of the present invention is related to a selected monoclonal antibody or its antigen-binding site comprising: a variable region heavy chain comprising sequences of CDR1, CDR2 and CDR3 comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 1, 2, 3 and 4, SEQ ID NO: 5, 6, 7 and 8, and SEQ ID NO: 9, 10, 11 and 12, respectively, and a variable region light chain comprising sequences of CDR1, CDR2 and CDR3 comprising amino acid sequences selected from the group consisting of SEQ ID NO: 13, 14, 15 and 16, SEQ ID NO: 17, 18, 19 and 20 and SEQ ID NO: 21, 22, 23 and 24, respectively. Thus, these antibodies contain sequences of CDRs of VH and VL of a monoclonal antibody 3F11, 4G5, 11E2, or 9D4, and, in addition to the, they may contain different framework sequences from these antibodies.

These frame sequences can be obtained from public DNA databases or published reference information, which includes sequences of antibody genes germ lines. For example, the DNA sequence of a germ-line genes for the variable regions of the heavy and light chains can be found in the database “VBase” sequences germ line person (available at the Internet address www.mrc-cpe.cam.ac.uk/vbase), as well as in Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Tomlinson, I.M., et al. (1992) “The Repertoire of Human Germline VH Sequences Reveals about Fifty Groups of VH Segments with Different Hypervariable Loops”. J. Mol. Biol., 227:776-798; and Cox, J. P.L. et al. (1994) “A Directory of Human Germ-line VH Segments Reveals a Strong Bias in their Usage”. Eur. J. Immunol., 24:827-836; and the content of each of these sources directly incorporated into the present application by reference.

The preferred frame sequences for use in the antibodies of the present invention are those sequences that are structurally similar to frame sequences available in the selected antibodies of the present invention, for example close to the frame sequences VH 4-34 and VL L18 available monoclonal antibody 3F11 and 4G5, Il is to frame sequences of VH 5-51 and VK A27, available in monoclonal antibodies 11E2, and 9D4. The sequence of the VH CDR1, 2 and 3, SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12, and the sequence of VL CDR1, 2 and 3, SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 and 24 can be transplanted in a frame region, which have the same sequences that were found in the gene for the immunoglobulin germ line, which is formed of a frame sequence, or the CDR sequence can be transplanted in a frame region that contain one or more mutations compared to the germline sequences of lines. For example, it was found that in some cases it is advantageous to expose the mutation of residues in the framework regions to maintain or improve the antigen-binding ability of the antibodies (see, for example, U.S. patent No. 5530101; 5585089; 5693762 and 6180370 issued by the Queen and co-authors).

Another type of modification of the variable region is the mutation of amino acid residues in regions CDR1, CDR2 and/or CDR3 of VH and/or VL, in order thus to improve one or more binding properties (e.g., affinity) of interest antibodies. To introduce mutations (mutations) can be carried out site-directed mutagenesis or PCR-mediated mutagenesis, and the effect on the binding of an antibody or other interesting functional properties may be evaluated in the analysis ofin vitroili> in vivoas described in this application and shown in the examples. Preferably (as discussed above) are conservative modifications. The mutation can be a substitution, addition and deletion of amino acids, but preferably they are substitutions. In addition, as a rule, in the field of the CDR change not more than five residues.

Accordingly, in another embodiment, the invention developed a dedicated anti-IFNAR-1 monoclonal antibodies or their antigen-binding sites comprising variable regions of the heavy chains containing: (a) a VH region CDR1 comprising amino acid sequence selected from the group consisting of SEQ ID NO: 1, 2, 3 and 4, or the amino acid sequence in which there are one, two, three, four or five substitutions, deletions or additions of amino acids compared to the amino acid sequence selected from the group consisting of SEQ ID NO: 1, 2, 3 and 4; (b) a VH region CDR2 comprising amino acid sequence selected from the group consisting of SEQ ID NO: 5, 6, 7 and 8, or the amino acid sequence in which there are one, two, three, four or five substitutions, deletions or additions of amino acids compared to the amino acid sequence selected from the group consisting of SEQ ID NO: 5, 6, 7 and 8; (c) a VH region CDR3 comprising the amino acid sequence is selected from the group consisting of SEQ ID NO: 9, 10, 11 and 12, or the amino acid sequence in which there are one, two, three, four or five substitutions, deletions or additions of amino acids compared to the amino acid sequence selected from the group consisting of SEQ ID NO: 9, 10, 11 and 12; (d) a VL region CDR1 comprising amino acid sequence selected from the group consisting of SEQ ID NO: 13, 14, 15 and 16, or the amino acid sequence in which there are one, two, three, four or five substitutions, deletions or additions of amino acids compared to the amino acid sequence selected from the group consisting of SEQ ID NO: 13, 14, 15 and 16; (e) a VL CDR2 comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 17, 18, 19 and 20, or the amino acid sequence in which there are one, two, three, four or five substitutions, deletions or additions of amino acids compared to the amino acid sequence selected from the group consisting of SEQ ID NO: 17, 18, 19 and 20; and (f) a VL region CDR3 comprising amino acid sequence selected from the group consisting of SEQ ID NO: 21, 22, 23 and 24, or an amino acid sequence in which there are one, two, three, four or five substitutions, deletions or additions of amino acids compared to the amino acid sequence selected from the group consisting of SEQ ID NO: 17,18, 19 and 20.

Engineered antibodies of the present invention include antibodies that were made modifications to the frame residues in the VH and/or VL, for example, to improve properties of antibodies. Typically, these frame modifications are made to reduce the immunogenicity of the antibody. For example, one approach is to "reverse mutation" of one or more residues frame to the corresponding germline sequence. More specifically, the antibody that has undergone somatic mutation, may contain in frame region residues that differ from the germline sequence from which the obtained antibody. These residues can be identified by comparing the frame sequences of the antibodies with sequences germ lines, of which the obtained antibody. For example, for 3F11 amino acid residue No. 43 (FR2) VH represents a threonine, whereas this residue in the corresponding sequence of germline VH 4-34 represents alanine (see figure 5). To return the sequence of this frame region to its germline configuration, the somatic mutations can be "reverse mutation" to the germline sequence, using, for example, site-directed mutagenesis or PCR-mediated m is Magenta (for example, the residue 43 VH 3F11 can be turned from threonine to alanine by "reverse mutation"). As another example, amino acid residue No. 81 VH antibodies 4G5 (FR3) represents asparagine, whereas this residue in the corresponding sequence of germline VH 4-34 is a lysine (see Fig.6). To return these sequences frame region to their germline configuration, the somatic mutations can be "reverse mutation of asparagine to lysine. As another example, for antibodies 11E2, and 9D4 amino acid residue No. 28 (FR1) of VH is an isoleucine whereas this residue in the corresponding sequence of germline VH 5-51 represents serine (see Fig.7). To return these sequences frame region to their germline configuration, the somatic mutations can be "reverse mutation from isoleucine to serine. It is assumed that these victims of "reverse mutation" antibodies are also included in the scope of the present invention.

Another type of frame modifications includes one or more mutating residues in the framework region, or even within one or more CDR regions, to remove T-cell epitopes, in order to reduce potential immunogenicity of the antibody. This approach is also called "demonizaci the St and it is described in more detail in patent publication U.S. No. 20030153043 Carr and co.

In addition or alternative to modifications made in the framework or CDR regions, antibodies of the present invention can be engineered to include modifications within the Fc region, typically to modify one or more functional properties of antibodies, such as time-life in serum, complement fixation, binding of the Fc receptor and/or antigen-dependent cellular cytotoxicity. In addition, the antibody of the present invention may be chemically modified (for example, the antibody can be attached to one or more chemical fragments) or be subject to modification to alter its glycosylation, i.e. again to change one or more functional properties of the antibody. Each of these embodiments are described in more detail below. The numbering of the residues in the Fc region corresponds to the EU index of Kabat.

In one of the embodiments of the hinge region of CH1 change so that in this area changes the number of cysteine residues, such as increases or decreases. This approach is additionally described in U.S. patent No. 5677425 Bodmer and co-authors. The number of cysteine residues in the hinge region, CH1 change, for example, to facilitate Assembly of the light and heavy chains or to increase or decrease the stability is nitela.

In another embodiment, the hinge region of the Fc antibody is subjected to mutation to reduce biological time half-life of the antibody. More specifically, one or more mutations of amino acids injected into the border region domains CH2-CH3 hinge Fc fragment so that antibody becomes degraded binding protein A Staphylococcyl (SpA), compared with the binding of natural Fc-hinge domain SpA. This approach is described in more detail in U.S. patent No. 6165745 Ward and co.

In another embodiment, the antibody is modified to increase its biological half-life. There are different approaches. For example, can be entered one or more of the following mutations: T252L, T254S, T256F, as described in U.S. patent No. 6277375 issued by the Ward. On the other hand, for longer biological half-life of the antibody may be subjected to changes in the areas CH1 or CL for inclusion epitope binding regenerating receptor, taken from two loops of a CH2 domain of the Fc region of IgG, as described in U.S. patent No. 5869046 and 6121022 Presta and co-authors.

In yet another embodiment, the Fc region change by replacing at least one amino acid residue with other amino acid residue to change effector functions (functions) antibodies. For example, one or more amino acids selected from the amino acid OST Dow 234, 235, 236, 237, 297, 318, 320 and 322 can be replaced with other amino acid residues, such that the antibody acquires an altered affinity for the effector ligand, but retains the antigen-binding capacity of the original antibody. Effector ligand, which changes the affinity may be, for example, Fc receptor, a component of complement C1. This approach is described in more detail in U.S. patent No. 5624821 and 5648260 Winter and co-authors.

In another example, one or more amino acids selected from amino acid residues 329, 331 and 322 can be replaced with another amino acid residue, so that changes the binding of an antibody to C1q and/or reduced or eliminated in the presence of complement-dependent cytotoxicity (CDC). This approach is described in more detail in U.S. patent No. 6194551 Idusogie and co-authors.

In another example, alter one or more amino acid residues at positions 231 and 239, thereby altering the ability of antibodies to stop the complement. This approach is additionally described in PCT publication WO 94/29351 Bodmer and co-authors.

In another example, the Fc region modified to increase the ability of the antibody to mediate dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody to the Fcγ receptor by modifying one or more amino acid residues at the following positions: 238, 239, 248, 249, 252, 254, 255, 56, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439. This approach is additionally described in PCT publication WO 00/42072 Presta. In addition, it was carried out the mapping of binding sites IgG1 person to FcγR1, FcγRII, FcγRIII and FcRn, and were described variants with improved binding (see. Shields, R.L.et al.(2001)J.Biol.Chem.276:6591-6604). It has been shown that certain mutations at positions 256, 290, 298, 333, 334 and 339 improve binding to FcγRIII. In addition, it was shown that these combined products mutations improve binding to FcγRIII: T256A/S298A, S298A/E333A, S298A/K224A and S298A/E333A/K334A.

In yet another embodiment, the modification is subjected to glycosylation of the antibody. For example, can be obtained deglycosylated antibody (i.e., the antibody does not have glycosylation). Glycosylation may be subject to changes, for example, to increase the affinity of the antibody to the antigen. Such modifications of the carbohydrate composition can be, for example, by changing one or more glycosylation sites in the sequence of the antibody. For example, can be conducted by replacing one or more amino acid residues, which leads to the destruction of one or more sites of glycosylation frame variabililty, in order to eliminate glycosylation at this site. This deglycosylation may increase the affinity of the antibody to the antigen. A similar approach is described with more detail in U.S. patent No. 5714350 and 6350861 Co and co-authors.

In addition to or as an alternative, can be obtained such an antibody that has an altered type of glycosylation, such as hepatocanalicular antibody having reduced the number fucosamine residues or an antibody having increased the number of bisecting GlcNac structures. It is shown that such modified model glycosylation increase ADCC ability of antibodies. These carbohydrate modifications can be effected, for example, by expression of antibodies in the cell host with a modified complex mechanisms of glycosylation. Cells with a modified complex mechanisms of glycosylation are described in the art and can be used as host cells in which expression of recombinant antibodies of the present invention, to obtain thereby the antibody with altered glycosylation. For example, in EP 1176195 Hanai and co-authors described cell line with a functionally disrupted FUT8 gene, which encodes fucosyltransferase, so that antibodies expressed in this cell line, identify gipoholesterinova. In the publication the purpose PCT WO 03/035835 Presta describes a variant of the cell lines CHO, namely cells Lec 13, with reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hepatocanalicular antibodies expressed in this cell the owner (see also Shields, R.L.et al. (2002)J.Biol.Chem.277:26733-26740). In PCT publication WO 99/54342 Umana and co-authors describe cell lines created for the expression of the glycoprotein-modifying glycosyltransferases (e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)), so that antibodies expressed in the developed cell lines revealed an increased amount bisecting GlcNac structures, resulting in increased ADCC activity of the antibodies (see also Umanaet al. (1999)Nat. Biotech. 17:176-180).

Another modification of the antibodies according to the present application, which is discussed in the present invention, is pegylation. The antibody can be paglinawan, for example, to increase its biological (e.g., in serum) time-life. For the implementation of paglierani antibody is the antibody or its fragment, as a rule, enter into interaction with polyethylene glycol (PEG), such as reactive complex ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups attached to the antibody or its fragment. Preferably, the pegylation is carried out by means of acylation reaction or reactions the alkylation with a reactive PEG molecule (or an analogous reactive water-soluble polymer). In the present description, the term "polyethylene glycol" is used to denote all forms of PEG that have been used to obtain the derivatives of other proteins, such as mono(C1-C10)alkoxy or aryloxy of glycols or polietilenglikolya. In some embodiments, the implementation of the antibody that is supposed to PageLayout is deglycosylated antibody. Methods paglierani proteins known in the art, and they can be applied to the antibodies of the present invention. See, for example, EP 0154316 Nishimuraet al. and EP 0401384 Ishikawaet al.

The methods of constructing antibodies

Thus, in another aspect of the present invention, the structural features of anti-IFNAR-1 antibodies of the present invention, for example 3F11, 4G5, 11E2, and 9D4, are used to create structurally related anti-IFNAR-1 antibodies that retain at least one functional property of the antibodies of the present invention, such as binding to IFNAR-1. For example, one or more CDR regions 3F11, 4G5, 11E2, or 9D4 or products of their mutations can be recombinante combined with known frame areas and/or other CDRs to create additional recombinante-designed anti-IFNAR-1 antibodies of the present invention, as discussed above. Other types of modifications include modifications described in the previous RA matter. The source material for fashion design are one or several sequences of numbers VH and/or VL, developed in the present invention, or one or more of the CDR regions of these sequences. To create engineered antibodies, there is no need really to obtain, for example, to Express in the form of protein) antibody having one or more of the number of sequences of the VH and/or VL, developed in the present invention, or one or more of their areas CDR. Rather, the information contained in the sequence (the sequence)is used as source material to create a "second generation" sequence (sequences)obtained from the initial sequence (sequence), and then receive a "second generation" sequence (sequences) and Express in the form of protein.

Accordingly, in another embodiment, the invention relates to a method for producing an anti-IFNAR-1 antibodies, including:

(a) obtaining: (i) sequence variable regions of the heavy chain of the antibody containing a CDR1 sequence selected from the group consisting of SEQ ID NO: 1, 2, 3 and 4, a CDR2 sequence selected from the group consisting of SEQ ID NO: 5, 6, 7 and 8, and/or a CDR3 sequence selected from the group consisting SEQ ID NO: 9, 10, 11 and 12; and (ii) the sequence of the variable region of the light chain of the antibody containing a CDR1 sequence selected from the group consisting of SEQ ID NO: 13, 14, 15 and 16, a CDR2 sequence selected from the group consisting of SEQ ID NO: 17, 18, 19 and 20, and/or a CDR3 sequence selected from the group consisting of SEQ ID NO: 21, 22, 23 and 24;

(b) modifying at least one amino acid residue in the first sequence of the antibody and/or the second sequence antibodies to create at least one altered sequences of the antibodies; and

(c) obtaining a modified sequence of the antibody; and

(d) the expression of the modified sequence of the antibody in the form of protein.

To obtain and expression of the modified sequence of the antibody can be used standard molecular biology techniques.

Preferably, the antibody encoded by the altered sequence (sequences), this is the antibody that retains one, some or all of the functional properties described in this application anti-IFNAR-1 antibodies, and these functional properties include, but are not limited to:

(i) binding to IFNAR-1;

(ii) inhibition of binding of interferon type I with IFNAR-1;

(iii) binding to live cells expressing IFNAR-1 person;

(iv) binding to IFNAR-1 person Dequal to 10-8M or less (for example, 10-9M or 10-10M or less);

(v) binding to a unique epitope on IFNAR-1 (to eliminate the possibility that monoclonal antibodies with complimentary activities when used in combination would take part in the competition for binding to the same epitope).

Functional properties of the modified antibodies can be assessed using standard assays known in the art and/or described in this application. For example, the ability of antibodies to contact IFNAR-1 can be determined using standard assays of binding, such as those described in the examples (e.g., ELISA).

In some embodiments, the implementation of the methods of constructing antibodies according to the present invention, mutations can be introduced randomly or selectively in the whole sequence encoding an anti-IFNAR-1 antibody, or part thereof (for example, the sequence encoding 3F11, 4G5, 11E2, or 9D4), and the resulting modified anti-IFNAR-1 antibodies can be screened on the basis of the binding activity and/or other functional properties as described in this application. Mutation techniques have been described in the art. For example, in published under PCT WO 02/092780 Short describes how to create and screening of mutations antibodies using saturating Mut is Genesis, Assembly of synthetic staple or a combination thereof. On the other hand, in PCT publication WO 03/074679 Lazar and co-authors described methods using computational screening methods to optimize physiochemical properties of antibodies.

Molecules of nucleic acids encoding antibodies of the present invention

Another aspect of the present invention relates to nucleic acid molecules that encode the antibodies of the present invention. The nucleic acid may be present in whole cells or cell lysate, or in a partially purified or substantially pure form. Nucleic acid is "vydelennoi" or "given in almost pure state", when it is purified from other cellular components or other contaminants, such as other cellular nucleic acids or proteins, by standard techniques, including processing alkaline/SDS stratification in CsCl, column chromatography, agarose gel electrophoresis and others well known in the art. Cm. F.Ausubel,et al., ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. Nucleic acids of the present invention may constitute, for example, DNA or RNA, and may contain or not contain intron sequences. In a preferred embodiment, nucleic acid isone cDNA molecule.

Nucleic acids of the present invention can be obtained by using standard techniques of molecular biology. For antibodies expressed by hybridomas (e.g., hybridomas derived from transgenic mice carrying the genes of human immunoglobulin, as described hereinafter)generated by hybridoma cDNA encoding the light and heavy chain antibodies, can be obtained by standard methods of PCR amplification or cDNA cloning. For antibodies, obtained from the library of immunoglobulin genes (e.g., using the technique of phage display), nucleic acid encoding the antibody can be recovered from the library.

Preferred nucleic acid molecules of the present invention are molecules that encode sequences of VH and VL of a monoclonal antibody 3F11, 4G5, 11E2, and 9D4. DNA sequences, coding sequences of VH and VL 3F11 shown in SEQ ID NO: 33 and 37 respectively. DNA sequences, coding sequences of VH and VL 4G5 shown in SEQ ID NO: 34 and 38, respectively. DNA sequences, coding sequences of VH and VL 11E2 shown in SEQ ID NO: 35 and 39, respectively. DNA sequences, coding sequences of VH and VL 9D4 shown in SEQ ID NO: 36 and 40, respectively.

If the DNA fragments encoding the segments VH and VL, already have ever been is Holocene, these DNA fragments can be further processed using standard techniques for working with recombinant DNA, for example to turn the genes for the variable region genes to full-chain antibody, the genes of the Fab fragment or a scFv gene. During these transformations the DNA fragment that encodes a VL or VH, is operationally associated with another DNA fragment coding for a different protein sequence, such as a constant region or a flexible linker to the antibody. The term "operationally linked" in this context is used to denote that two fragments of DNA are combined in such a way that the amino acid sequences encoded by the two DNA fragments are stored in a frame are read.

The selected DNA encoding the VH region can be converted to a full-size heavy chain gene using operational binding encodes a VH DNA to another DNA molecule that encodes a constant region of the heavy chain (CH1, CH2 and CH3). Gene-sequences of the constant regions of the heavy chains of a man known in the art (see, for example, Kabat, E.A., et al. (1991) Sequenced of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242)and DNA fragments corresponding to these areas can be obtained by standard PCR amplification. The constant region of the heavy chain can be constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or Ig, but most preferably are constant regions of IgG1 or IgG4. For gene fragment Fab heavy chain encoding a VH DNA can be operationally linked to another DNA molecule encoding only the constant region CH1 of the heavy chain.

The selected DNA encoding the VL region can be turned into a full-sized light chain gene (as well as in gene Fab light chain) using operational binding VL-encoding DNA to another DNA molecule that encodes a constant region of light chain, i.e. CL. Gene-sequences of constant regions of light chains of a man known in the art (see, for example, Kabat, E.A., et al. (1991) Sequenced of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242)and DNA fragments corresponding to these areas can be obtained by standard PCR amplification. Constant region light chain can be a constant regions of Kappa or lambda, but most preferably constant region Kappa.

To create a scFv gene, the VH-and VL-encoding DNA fragments are operationally linked to another fragment encoding a flexible linker, such as encoding the amino acid sequence (Gly4-Ser)3so that sequences of VH and VL can be expressed as a contiguous single-chain protein, with the regions VH and VL linked by a flexible linker (see, for example, Birdet al., (1988)Science,242:423-426; Hustonet al. (1988)Proc.Natl.Acad.Sci USA85:5879-5883; McCaffertyet al., (1990)Nature,348:552-554).

Obtaining monoclonal antibodies of the present invention

Monoclonal antibodies (mAb) of the present invention can be obtained in a variety of ways, including the traditional methodology of work with monoclonal antibodies, such as standard methods of hybridization of somatic cells developed by Kohler and Milstein (1975)Nature,256:495. Although preferred are methods of hybridization of somatic cells, in principle, can be applied to other methods of obtaining monoclonal antibodies, such as viral or oncogenic transformation of B lymphocytes.

Preferred animal system for obtaining the hybrid is the murine system. Method for producing a hybrid in mice is very well developed. In the technique known protocols of immunization and methods of selection of immunized splenocytes for fusion. Also known cells of the partners in the merger (for example, myeloma cells mouse) and methods of the merge.

Chimeric or humanized antibodies of the present invention can be derived from the sequences of monoclonal mouse antibodies, obtained as described above. DNA encoding the heavy and light chains of immunoglobulins, can the be obtained using standard techniques of molecular biology from the appropriate hybridoma mouse and designed it contains not a mouse (e.g., human) immunoglobulin sequences. For example, to create a chimeric antibody variable regions of the mouse may be linked to constant regions of a human using known in the art methods (see, for example, U.S. patent No. 4816567 issued by Cabilly and co-authors). To create gumanitarnogo antibodies region CDR mouse can be embedded in the frame of the person using methods known in the art (see, for example, U.S. patent No. 5225539 issued Winter, and U.S. patent No. 5530101; 5585089; 5693762 and 6180370 issued by the Queen and co-authors).

In a preferred embodiment, antibodies of the present invention are monoclonal antibodies person. Such monoclonal human antibodies directed against IFNAR-1, can be obtained with the use of transgenic or transamazonic mice with the human, not the immune system of the mouse. These transgenic and transamazonia mice include mice, which in this application are referred to as HuMAb mice and KM mice, respectively, and together are referred to as "mice with human Ig".

The HuMAb Mouse® (Medarex, Inc.) contains minilogue immunoglobulin gene in humans that encode neeregirovannye sequence heavy (μ and γ) and κ light chains of human immunoglobulin, together with targeted mutations is, which inactivate endogenous loci µ and κ chains (see, e.g., Lonberg,et al. (1994)Nature,368(6474):856-859). Accordingly, the mouse shows reduced expression of mouse IgM or κ, and in response to immunization, the introduced transgenes heavy and light chains of human experience switching classes and somatic mutation, generating IgGκ monoclonal person with high affinity (Lonberg, N.et al. (1994),see above; overview in Lonberg, N. (1994)Handbook of Experimental Pharmacology,113: 49-101; Lonberg, N. and Huszar, D. (1995)Intern.Rev. Immunol.13:65-93, and Harding, F. and Lonberg, N. (1995)Ann.N.Y.Acad.Sci.764:536-546). The collection and use of HuMAb mice and modifications of the genome, carried out by such mice, is further described in Taylor, L.et al.(1992) Nucleic Acids Research,20:6287-6295; Chen, J.et al. (1993)International Immunology,5:647-656; Tuaillonet al. (1993) Proc. Natl. Acad. Sci. USA90:3720-3724: Choiet al. (1993) Nature Genetics 4:117-123; Chen, J.et al. (1993) EMBO J.12:821-830; Tuaillon et al. (1994)J. Immunol,152: 2912-2920; Taylor, L. et al. (1994)International Immunology,6:579-591; and Fishwild, D. et al. (1996)Nature Biotecnology,14:845-851, the contents of all of the listed works directly incorporated into the present application in its entirety by reference. Additionally, see U.S. patents№№ 5545806; 5569825; 5625126; 5633425; 5789650; 5877397; 5661016; 5814318; 5874299 and 5770429; all issued to Lonberg and Kay; U.S. patent No. 5545807 issued Surani and collaborators; PCT publication number WO92/03918, WO93/12227, WO94/25585, WO97/13852, WO8/24884 and WO99/45962 all Lonberg and Kay; as well as PCT publication no WO 01/14424 Korman and co-authors.

In another embodiment, human antibodies of the present invention can be obtained by using mice, which are carriers of the immunoglobulin sequences of human transgenes or transpromotional, such as mice carrying the transgene heavy chain of human and transhumanity light chain of a human. Such mice, referred to in the present description "mouse KM", is described in detail in PCT publication no WO 02/43478 Ishida and co-authors.

In the technique known alternative transgenic animal systems expressing the genes of human immunoglobulin, and these systems can be used to generate anti-IFNAR-1 antibodies of the present invention. For example, can be used an alternative transgenic system, called Xenomouse (Abgenix, Inc.); such mice are described, for example, in U.S. patent No. 5939598; 6075181; 6114598; 6150584 and 6162963 issued by Kucherlapati and co-authors.

In addition, in the technique there are alternative transamazonia animal systems expressing immunoglobulin genes of the person, and they can be used to generate anti-IFNAR-1 antibodies of the present invention. For example, can be applied to the mouse being carriers as transhumanity heavy chain human and transhumanity light chain of a man called "TC mice is"; such mice are described in Tomizukaet al. (2000)Proc.Natl.Acad.Sci. USA97:722-727. In addition, in the technique have been described in cows carrying transfromation heavy and light chains of the person (Kuroiwaet al. (2002)Nature Biotechnology,20:889-894)and can be used to generate anti-IFNAR-1 antibodies of the present invention.

Monoclonal human antibodies of the present invention can also be obtained using techniques of phage display technique for screening libraries of human immunoglobulin genes. In the technique developed such techniques of phage display for the selected antibodies person. See, for example, U.S. patent No. 5223409; 5403484; and 5571698 issued by Ladner and collaborators; U.S. patent No. 5427908 and 5580717 issued Dower and collaborators; U.S. patent No. 5969108 and 6172197 issued by McCafferty and collaborators; and U.S. patent№№ 5885793; 6521404; 6544731; 6555313; 6582915 and 6593081 issued by Griffiths and co.

Monoclonal human antibodies of the present invention can also be obtained with the use of SCID mice, which are reproduced in immune cells, so that when immunization is generated immune response with production of antibodies person. Such mice are described, for example, in U.S. patent No. 5476996 and 5698767 issued by Wilson and co-authors.

Immunization of mice with human Ig

If for the production of human antibodies according to the present invention using mice with Ig che is oweka, such mice can be immunized with purified or enriched preparations of antigen IFNAR-1 and/or cells expressing IFNAR-1, as described in Lonberg, N.et al. (1994)Nature,368(6474):856-859; Fishwild, D.et al(1996)Nature Biotechnology,14: 845-851; and PCT publications WO 98/24884 and WO 01/14424. Preferably, when the first infusion mouse must be aged 6-16 weeks. For example, immunization of mice with human Ig can be applied intraperitoneal purified or enriched preparation (5-50 μg) of antigen IFNAR-1. If immunizations using a purified or enriched preparation of antigen IFNAR-1 does not result in antibodies, mice can also be immunized with cells expressing IFNAR-1, for example a line of T-cells, to stimulate immune responses.

In example 1 hereinafter described method of generating a fully monoclonal antibodies human IFNAR-1. Lessons learned related to the various antigens has shown that transgenic mice provide an immune response, when the initial intraperitoneally (IP) immunization with antigen in complete Freund's adjuvant, with subsequent weekly immunizations (up to 6) with antigen in incomplete Freund's adjuvant. However, it was found that other adjuvants, in addition to adjuvant-blockers are also effective. In addition, there was n Geno, that whole cells in the absence of adjuvant have high immunogenicity. For the immune response can be observed in the course of the immunization Protocol using plasma samples obtained at retroorbital bleeding. This plasma may be subjected to screening using ELISA (as described below), and mice with sufficient titers of anti-IFNAR-1 of human immunoglobulin can be used for fusions. The effect on the mouse can be amplified by the intravenous injection of antigen for 3 days before the killing and removal of the spleen. It is expected that for each immunization may be necessary to perform 2-3 merge. For each antigen, as a rule, subjected to immunization from 6 to 24 mice. Usually use both lines HCo7 and HCo12. In addition, both the transgene HCo7 and HCo12 and can be played in single mouse having two different heavy chain transgene person (HCo7/HCo12).

Generation of hybridomas that produce monoclonal human antibodies of the present invention

To generate hybridomas producing monoclonal human antibodies of the present invention, splenocytes or cells of lymph nodes of immunized mice can be isolated and fused with suitable cells immortalizing lines, such as lines of murine myeloma. Received hybridoma can be subjected to screening criterion is robotki antigen-specific antibodies. For example, a suspension of single cells of spleen lymphocytes obtained from immunized mice can be fused in a quantitative ratio of one to six P3X63-Ag8.653 cells " non-secretory mouse myeloma (ATCC, CRL 1580) with 50% PEG. Cells are placed on the tablet in an amount of about 2·105in a flat-bottomed microtiter plate, followed by a two-week incubation in selective medium containing 20% fetal serum, 18% air-conditioned environment "653", 5% Origen (IGEN), 4 mm L-glutamine, 1 mm sodium pyruvate, 5 mm HEPES, 0.055 mm 2-mercaptoethanol, 50 u/ml penicillin, 50 mg/ml streptomycin, 50 mg/ml gentamicin and 1X HAT (Sigma; the HAT is added 24 hours after the fusion). After about two weeks, cells can be cultured in an environment in which the HAT is replaced by HT. Then the individual wells can be explored by way of ELISA for monoclonal antibodies IgM and IgG person. If there is extensive growth of hybridoma, the environment is usually possible to explore 10-14 days. Hybridoma, secreting antibodies, can be re-placed on the tablet, again subjected to screening, and in case of repeated positive result for IgG human monoclonal antibodies, can be subcloned at least twice by using serial dilutions. Then the stable subclones can be cultivatedin vitrosince C is poured generation of small amounts of antibody in tissue culture medium for identification.

For purification of monoclonal antibodies person selected hybridoma can be grown in two-liter roller flasks for the purification of monoclonal antibodies. Before carrying out affinity chromatography with protein a-separate (Pharmacia, Piscataway, N.J.) supernatant can be subjected to filtration and concentration. To guarantee purity suirvey IgG can be checked by gel-electrophoresis and high performance liquid chromatography. The buffer solution can be replaced with PBS and the concentration can be determined by OD280 (optical density) using the extinction coefficient of 1.43. The obtained monoclonal antibodies can be divided into aliquots and stored at -80°C.

Generating transfection that produce monoclonal human antibodies of the present invention

Human antibodies of the present invention can also be obtained in the cell-the owner called transfective, using, for example, is well known in the art methods based on combinatii or recombinant DNA, and methods transfection of genes (e.g., Morrison, S. (1985) Science, 229:1202).

For example, in order to Express the antibody or its fragment, using standard techniques of molecular biology (e.g., PCR amplification or cDNA cloning using hybridoma, which expresses-the interest antibody) can be obtained from DNA molecules, encoding a full size light and heavy chain or part thereof, and these DNA molecules can be incorporated into expression vectors so that the genes were operationally linked to transcriptional and translational control sequences. In this context, the term "operationally linked" is intended to indicate that the antibody gene is integrated into a vector such that transcriptional and translational control sequences within the vector perform the intended function control transcription and translation of genes antibodies. The expression vector and control the expression of the sequence are chosen so that they were compatible with the used for the expression of the host-cell. Gene light chain antibody gene and the heavy chain antibodies can be included in a separate vectors, or more frequently both genes include in the same expression vector. Genes antibodies include in the expression vector by standard methods (for example, the binding of complementary restriction sites on the fragment of the antibody gene and the vector or stapling "blunt ends", if restriction sites are absent). Variable regions of the heavy and light chains of the antibodies described in this invention can be used to create full-size genes of any isotypes of antibodies by incorporating them into expression vectors already code is their constant region of the heavy chain and light chain of the desired isotype, so that the VH segment is operationally linked in the vector to the segment (segments) CH and the VL segment is operationally linked in the vector to the segment of the CL. As a Supplement or alternative to recombinant expression vector can encode a signal peptide that facilitates secretion of the chain of the antibody from the host cell. Gene chain antibodies can be cloned into a vector so that the signal peptide was linked in frame with aminocom.com gene chain antibodies. The signal peptide may be a signal peptide immunoglobulin or a heterologous signal peptide (i.e. the signal peptide, non-immunoglobulin protein).

In addition to genes chain antibodies, recombinant expression vectors of the present invention carry regulatory sequences that control the expression of genes chain antibodies in the cell host. It is assumed that the term "regulatory sequence" includes a promoter, enhancer, and other controls expression (e.g., polyadenylation signals)that control the transcription or translation of genes chain antibodies. Such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, CA (1990)). Specialist in the art will take into account that the creation of expression vector, including the selection of regulatory consistently the TEI, may depend on such factors as the choice of host cell to be transformed, the desired level of expression of the protein, etc. of the Preferred regulatory sequences for expression in the cells of the host mammal include viral elements that lead to high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV), monkey virus 40 (SV40), adenovirus (such as a large late promoter of adenovirus (AdMLP)) and polyoma. On the other hand, can be used regulatory sequences of non-viral nature, such as the promoter of ubiquitin or the promoter of β-globin. You can also apply regulatory elements, created on the basis of sequences from other sources, such as the system of SRα promoter, which contains sequences from the early promoter of SV40, the long terminal repeat of the virus T-cell leukemia human type I (Takebe, Y.et al.(1988)Mol.Cell. Biol.8:466-472).

In addition to genes chain antibodies and regulatory sequences, the recombinant expression vectors of the present invention may include additional sequences, such as sequences that regulate replication of the vector in the cells of the host (for example, sites of replication initiation) and the gene of the breeding marker. Gene breeding marker facilitates selection of host cells, in which was introduced the vector (see, for example, U.S. patent No. 4399216, 4634665 and 5179017, all Axel and co-authors). For example, as a rule, breeding gene marker attaches to the cells of the host, which was introduced in the vector, resistance to drugs, such as G418, hygromycin or methotrexate. Preferred genes of breeding token include gene digidrofolatreduktazy (DHFR) (for use in dhfr-cells-owners with the selection/amplification methotrexate) and Neogene (for G418 selection).

For the expression of light and heavy chain vector (vectors) expression encoding the light and heavy chains, transferout in cell host using standard techniques. It is assumed that various forms of the term "transfection" cover a wide range of techniques commonly used for introducing exogenous DNA into a prokaryotic or eukaryotic cell host, such as electroporation, precipitation with calcium phosphate, transfection with DEAE-dextran and the like, Although it is theoretically possible to Express the antibodies of the present invention in either prokaryotic or eukaryotic cells-hosts, expression of antibodies in eukaryotic cells, and preferably in cells of the host mammal is the most preferred because such eukaryotic cells, and in particular glue the Ki mammals, more likely than prokaryotic cells to assemble and secrete antibodies with appropriate spatial structure and immunological activity. Expression of antibody genes in prokaryotic cells, as reported, is ineffective to obtain a high yield of active antibody (Boss, M.A. and Wood, C.R. (1985)Immunology Today,6:12-13).

Preferred cells of mammalian hosts for expression of recombinant antibodies of the present invention include cells of the Chinese hamster ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub and Chasin, (1980)Proc.Natl.Acad.Sci. USA77:4216-4220, used with a DHFR breeding marker, for example, as described in R.J.Kaufman and P.A.Sharp (1982)Mol.Biol.159:601-621), NSO myeloma cells, COS cells and SP2 cells. In particular, another preferred expression system for use with NSO myeloma cells system is the GS gene expression disclosed in WO 87/04462, WO 89/01036 and EP 338841. When recombinant expression vectors encoding antibody genes, introduced into cells of mammalian hosts, antibody occurs when the cultivation of the host cells for a period of time sufficient to allow expression of the antibody in the cells of the host, more preferably for the secretion of antibodies into the culture medium which produces the cells of the host. Antibodies which may be isolated from the culture medium using standard methods of protein purification.

Determination of the binding characteristics of an antibody to an antigen

Antibodies of the present invention can be tested for binding to IFNAR-1, for example, using a standard ELISA. Briefly, microtiter tablets cover purified IFNAR-1 at a concentration of 0.25 μg/ml in PBS and then blocked with 5% bovine serum albumin in PBS. Antibody at various dilutions (e.g., dilutions of plasma IFNAR-1-immunized mice) are added to each well and incubated for 1-2 hours at 37°C. the Tablets are washed with a mixture of PBS/Tween and then incubated with secondary reagent (e.g., in the case of human antibodies, goat anti-human IgG Fc-specific polyclonal reagent), conjugated with alkaline phosphatase for 1 hour at 37°C. After washing tablets treated with pNPP substrate (1 mg/ml) and analyzed OD when 405-650. It is preferable to merge will use the mouse showing the highest titers.

As mentioned above, the ELISA can also be used for selection of hybridomas that show positive reactivity against the immunogen IFNAR-1. Hybridoma that bind to IFNAR-1 with high avidity, are subclavian and further define the characteristics. One clone from each hybridoma, which retains the reactivity of the original cells (according ELISA), can be selected for the create 5-10 vials with banks of cells, when stored at -140°C, and for the purification of antibodies.

To purify anti-IFNAR-1 antibodies, selected hybridoma can be grown in two-liter roller flasks for the purification of monoclonal antibodies. Supernatant can be subjected to filtration and concentrated before affinity chromatography with protein a-separate (Pharmacia, Piscataway, NJ). Suirvey immunoglobulin to guarantee purity can be checked by gel electrophoresis and high performance liquid chromatography. The buffer solution can be replaced with PBS, and the concentration can be determined by OD280 using the extinction coefficient of 1.43. Monoclonal antibodies can be divided into aliquots and stored at -80°C.

In order to determine contacted if selected anti-IFNAR-1 monoclonal antibodies with a single epitopes, each antibody can be biotinylation using commercially available reagents (Pierce, Rockford, IL). Study competitive binding using unlabeled monoclonal antibodies and biotinylated monoclonal antibodies can be performed using tablets ELISA coated with IFNAR-1, as described above. Binding of biotinylated mAb can be detected using the probe strep-avidin-alkaline phosphatase.

To determine the isotype of purified antibodies m which may be performed ELISA analysis of isotype using reagents specific to antibodies specific isotype. For example, to determine the isotype of monoclonal human antibodies, the wells of microtiter plates may be coated over night at 4°C with 1 µg/ml anti-human immunoglobulin. After blocking with 1% BSA contents of the tablets enter into interaction with 1 μg/ml or less a test of monoclonal antibodies or purified isotype control samples at room temperature within one or two hours. Then the contents of the wells can be entered into cooperation with either human IgG1 or conjugated probes IgM-specific alkaline phosphatase person. The tablets are treated and analyzed as described above.

To demonstrate binding of monoclonal antibodies to live cells expressing IFNAR-1, can be used in flow cytometry. Briefly, cell lines expressing IFNAR-1 (grown under standard growth conditions)are mixed with monoclonal antibodies at various concentrations in PBS containing 0.1% BSA and 10% fetal serum of calves, and then incubated at 37°C for 1 hour. After washing, the cells enter into interaction with labeled anti-human IgG fluorescein antibody under the same conditions in which it was implemented primary antibody staining. Samples can be analyzed on the FACScan instrument using with the STS light and side scattering at excitation fluorescence of individual cells. An alternative analysis using fluorescence microscopy can be performed either in addition to flow cytometry, or instead of it. Cells can be stained in accordance with the description above and examined by fluorescence microscopy. This method makes possible the visualization of individual cells, but may have reduced sensitivity depending on the density of the antigen.

Further, anti-IFNAR-1 human IgG can be tested for reactivity against the antigen IFNAR-1 by Western blot. Briefly, cell extracts obtained from cells expressing IFNAR-1, can be obtained and subjected to electrophoresis in a gel sodium dodecyl sulphate - polyacrylamide. After electrophoresis the separated antigens are transferred to a membrane of nitrocellulose, blocked with 10% fetal calf serum and conduct the test with the test monoclonal antibodies. The binding of human IgG can be detected using anti-human IgG - alkaline phosphatase and developed with substrate tablets BCIP/NBT (Sigma Chem.Co., St. Louis, MO).

Immunoconjugate

In another aspect of the present invention includes anti-IFNAR-1 antibody or fragment is conjugated to a therapeutic fragment, such as a cytotoxin, a drug (e.g., immuno shall epressant) or radiotoxins. Such conjugates in the present application is referred to as the "immunoconjugate". Immunoconjugate that include one or more cytotoxins called "immunotoxins". A cytotoxin or cytotoxic agent includes any agent that is harmful to cells (e.g., kills). Examples include Taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracene, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as their analogues and homologues. Therapeutic agents also include, for example, antimetabolites (e.g. methotrexate, 6-mercaptopurine, 6-tioguanin, cytarabine, 5-fluorouracil, dacarbazine), alkylating means (for example, mechlorethamine, tiapa chlorambucil, melphalan, carmustine (BSNU) and lomustin (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and CIS-dichlorodiammine platinum (II) (DDP) cisplatin), anthracyclines (e.g. daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin and astromicin (AMC)), and antimitoticescoy tools (e.g., vincristine and vinblastine).

Other preferred examples of terapeuticas the x cytotoxins, which can be conjugated with antibodies of the present invention, include duocarmycin, calicheamicin, maytansine and auristatin, as well as their derivatives. An example of a conjugate antibodies with calicheamicin is a commercially available product (MylotargTM; Wyeth-Ayerst).

Cytotoxins can be conjugated with antibodies of the present invention using existing technology linker technology. Examples of the types of linkers, which were used for conjugation of the cytotoxin to the antibody include, but are not limited to the above, hydrazones, thioethers, esters, disulfides and peptideatlas linkers. Can be selected by the linker, which, for example, is susceptible to cleavage in the lysosomal compartment at low pH values or sensitive to cleavage by the action of proteases, such as proteases, mainly expressed in tumor tissue, such as cathepsins (such as cathepsins B, C, D).

For a more detailed discussion of the types of cytotoxins, linkers and methods of conjugation of therapeutic agents and antibodies, see also Saito, G.et al.(2003)Adv. Drug Deliv. Rev.55:199-215; Trail, P.A. et al. (2003)Cancer Immunol. Immunother.52:328-337; Payne, G. (2003) Cancer Cell.3:207-212; Allen, T.M. (2002)Nat. Rev. Cancer2:750-763; Pastan, I. and Kreitman, R.J. (2002)Curr. Opin. Investig. Drugs.3:1089-1091; Senter, P.D. and Springer, C.J. (2001)Adv. Drug. Deliv. Rev.53

Antibodies of the present invention can also be conjugated with radioactive isotopes for a cytotoxic radioactive pharmaceuticals, also referred to as radioimmunoconjugates. Examples of radioactive isotopes that can be conjugated with antibodies for diagnostic and therapeutic applications, include, but are not limited to the above, the iodine131, indium111, yttrium90and Lu177. Methods of obtaining radioimmunoconjugates technique. Examples of commercially available radioimmunoconjugates include ZevalinTM(IDEC Pharmaceuticals) and BexxarTM(Corixa Pharmaceuticals), and similar techniques can be applied to obtain radioimmunoconjugates, including antibodies of the present invention.

Conjugates of the antibody of the present invention can be used for modifying a given biological response, and we should not assume that the fragments of the medicinal product limited range of classical chemical medicines. For example, drug fragment may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, enzyme-active toxins, or their active fragments, such as abrin, ricin A, pseudomonades exotoxin or diphtheria Tox is n; a protein such as tumor necrosis factor or interferon-γ; or biological reaction modifiers, such as, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte-macrophage colony-stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.

Well-known techniques of conjugation of such therapeutic fragments of antibodies, see, e.g., Amonet al.,"Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeldet al.(eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstromet al.,"Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinsonet al.(eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe. "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pincherael al.(eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy. Baldwinet al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpeet al."The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev., 62:119-58(1982).

Bispecific molecules

In another aspect, the present invention relates to bispecific molecules, including anti-IFNAR-1 antibody of the present invention or its fragment. The antibody of the present invention or an antigen-binding site can be transformed into derived or linked to another functional molecule, such as other pepti the om or protein (e.g., another antibody or ligand for a receptor), to obtain bispecific molecule, which is associated with at least two different binding sites or by target molecules. In fact, the antibody of the present invention can be converted into derived or associated with more than one other functional molecule to obtain multispecific molecules that bind to more than two different binding sites and/or by target molecules; and we mean that in the present description such multispecific molecules are also covered by the term "bispecific molecule". To create bispecific molecules of the present invention, the antibody of the present invention can be functionally linked (e.g., by chemical combination, genetic fusion, noncovalent Association or otherwise) with one or more other binding molecules, such as another antibody, antibody fragment, peptide or binding mimetic, so formed bispecific molecule.

Accordingly, the present invention includes bispecific molecules containing at least one first specific binding fragment for IFNAR-1 and the second specific binding fragment to another target epitope. In a specific embodiment, the present image is the shadow of the second target epitope is an Fc receptor, for example, human FcγRI (CD64) or Fcα receptor human (CD89). Therefore, the invention includes bispecific molecules able to bind to effector cells expressing FcγR, FcαR or FcεR (e.g., monocytes, macrophages or polymorphisim cells (PMN)), and to target cells expressing IFNAR-1. These bispecific molecules target IFNAR-1 expressing cells to effector cell and trigger different kinds of activity of effector cells, mediated by Fc receptor, such as phagocytosis IFNAR-1 expressing cell-dependent antibodies cell-mediated cytotoxicity (ADCC), cytokine release, or generation of superoxide anion.

In the embodiment of the present invention, in which bispecific molecule is multispecific, this molecule can further include the third specific binding fragment, in addition to the specific binding of anti-Fc fragment and specific binding of anti-IFNAR-1 fragment. In one embodiment, the implementation of the third specific binding fragment represents a portion of antifactor gain (EF), such as a molecule that binds to a surface protein involved in cytotoxic activity, and thereby enhances the immune response against target cells. "The plot of antifactor Wuxi is possible" can be an antibody, functional fragment of the antibody or ligand that binds to the molecule, for example an antigen or a receptor, and thereby leads to increased exposure of the binding determinants on the Fc receptor or antigen target cells. "The plot of antifactor gain" can bind the Fc receptor or antigen target cells. On the other hand, the plot of antifactor gain can contact the object, which is different from the object, which contact the first and second specific binding fragments. For example, the plot of antifactor gain can bind cytotoxic T-cells (e.g., via CD2, CD3, CD8, CD28, CD4, CD40, ICAM-1 or other immune cells, which leads to increased immune response against target cells).

In one of the embodiments bispecific molecules of the present invention include as a specific binding fragment of at least one antibody or its fragment, such as Fab, Fab', F(ab')2, Fv or single-chain Fv. The antibody may also be a dimmer light chain or heavy chain, or any minimal fragment such as an Fv fragment or single-chain design, as described Ladner and co-authors in U.S. patent No. 4946778, the content of which is directly incorporated into the present application by reference.

In one embodiment, the implementation of the specificity of St. the statements with the Fcγ receptor is provided a monoclonal antibody the binding of which is not blocked by human immunoglobulin G (IgG). In the present description, the term "IgG receptor" refers to any of the eight genes γ-chain, located on chromosome 1. These genes encode a total of twelve isoforms of the transmembrane or soluble receptors, which are grouped into three classes of Fcγ receptors: FcγRI (CD64), FcγRII(CD32) and FcγRIII(CD16). In one of the preferred embodiments the Fcγ receptor is Vysocany human FcγRI. FcγRI is a molecule mass of 72 kDa, which shows high affinity for Monomeric IgG (108-109M-1).

Preparation and properties of some preferred anti-Fcγ monoclonal antibodies are described by Fanger and co-authors in PCT publication WO 88/00052 and U.S. patent No. 4954617, the contents of which are fully incorporated into the present application by reference. These antibodies bind to an epitope FcγRI, FcγRII or FcγRIII on the site, which is different from the binding site Fcγ receptor and, thus, their binding is not blocked substantially by physiological levels of IgG. Specific anti-FcγRI antibodies that are applicable in the present invention are mAb 22, mAb 32, mAb 44, mAb 62 and mAb197. Hybridoma producing mAb 32, available from the American type culture collection (ATCC), inventory No. HB9469. In other embodiments, the implementation of anti-Fcγ receptor Academy of Sciences of the United Italo is humanitarian form of monoclonal antibody 22 (H22). Preparation and properties of antibody H22 described in Graziano, R.F.et al. (1995)J.Immunol,155(10):4996-5002 and PCT publication WO 94/10332. The cell line producing the antibody H22, was deposited in the American type culture collection with the designation HA022CL1 and has inventory No. CRL 11177.

In other preferred embodiments, the implementation of the specificity of binding to the Fc receptor is provided an antibody that binds to a receptor IgA person, for example Fc-alpha receptor (FcαRI(CD89)), the binding of which is preferably not blocked immunoglobulin A person (IgA). Have in mind that the term "IgA receptor" includes the product α-gene (FcαRI), located on chromosome 19. It is known that this gene encodes several alternative playerowner transmembrane isoforms, weighing from 55 to 110 kDa. FcαRI(CD89) is constitutively expressed on monocytes/macrophages, eosinophilic and neutrophilic granulocytes, but not on the populations effektornyh cells. FcαRI has medium affinity (≈5·107M-1) to both isoforms IgA1 and IgA2, which is increased under the action of cytokines such as G-CSF or GM-CSF (Morton, H.C.et al. (1996)Critical Reviews in Immunology,16:423-440). Were described four FcαRI-specific monoclonal antibodies, identified as A3, A59, A62 and A77, which bind with FcαRI outside of the domain, a ligand-binding IgA (Monteiro, R.C.et al. (1992)J.Immunol .148:1764).

FcαRI and FcγRI are preferred trigger receptors for use in bispecific molecules of the present invention, because they (1) are mainly expressed on immune effector cells, such as monocytes, PMN, macrophages and dendritic cells; (2) expressed in large quantities (for example, 5000-100000 per cell); (3) are mediators of the activity of cytokines (e.g., ADCC, phagocytosis); (4) mediate the increased presentation of antigens, including those aimed at their own antigens.

Although preferred are monoclonal human antibodies other antibodies that can be used in bispecific molecules of the present invention are murine, chimeric and humanized monoclonal antibodies.

Bispecific molecules of the present invention can be obtained by conjugation of the components of the specific binding fragments, such as anti-FcR and anti-IFNAR-1 fragments, using known in the art methods. For example, each of the specific binding fragments bispecific molecules can be obtained separately and then anywhereman with another fragment. If specific binding fragments are proteins or peptides may be administered to a large number of linking or cross-linking reagents to Cove the Lenten conjugation. Examples of cross-linking reagents include protein A, carbodiimide, N-Succinimidyl-S-acetylthiourea (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-Succinimidyl-3-(2-pyridylthio)propionate (SPDP) and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) (see, e.g., Karpovskyet al. (1984)J.Exp.Med.160:1686; Liu, MAet al. (1985)Proc. Natl. Acad. Sci. USA82:8648). Other methods include the methods described in the following publications Paulus (1985) Behring Ins.Mitt. No.78, 118-132; Brennanet al. (1985)Science,229:81-83 Glennie andet al.(1987)J.Immunol.139:2367-2375. Preferred reagents for conjugation are SATA, sulfo-SMCC, both of these reagents can be purchased from Pierce Chemical Co. (Rockford, IL).

If specific binding fragments are antibodies, they can be conjugated via sulfhydryl binding of the C-ends of the hinge regions of the two heavy chains. In a particularly preferred embodiment, the hinge region is altered so that it is up to conjugation contained an odd number of sulfhydryl residues, preferably one.

On the other hand, both specific binding fragment can be encoded in the same vector and expressed and collected in the same cell as the host. This method is particularly applicable if bispecific molecule represented yet a mAb · mAb, mAb · Fab, Fab · F(ab')2or ligand · Fab protein. Bispecific molecule of the present invention may be single-stranded molecule comprising one single-chain antibody and the determinants of binding or single-stranded bispecific molecule comprising two determinants of binding. Bispecific molecules can include at least two single-stranded molecules. Methods of obtaining bispecific molecules described, for example, in U.S. patent No. 5260203; U.S. patent No. 5455030; U.S. patent No. 4881175; U.S. patent No. 5132405; U.S. patent No. 5091513; U.S. patent No. 5476786; U.S. patent No. 5013653; U.S. patent No. 5258498; and U.S. patent No. 5482858.

Linking bispecific molecules to their specific targets can be confirmed by, for example, enzyme-linked immunosorbent assay (ELISA), radioimmune assay (RIA), FACS analysis, biological analysis (e.g., growth inhibition), or Western blotting. Each of these methods of analysis typically finds interesting complexes protein-antibody through the use of labeled reagent (e.g., antibodies)specific interest complex. For example, complexes FcR-antibody can be detected using, for example, the antibody or its fragment associated with an enzyme that recognizes and is specific linked with complexes of antibody-FcR. With others the hand, these complexes can be detected using any of the many other methods of immunological analysis. For example, the antibody can be radioactively labeled and used in a radioimmunoassay analysis (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, and this publication is incorporated into the present application by reference). The radioactive isotope can be detected by such means as the use of γ-counter or a scintillation counter or autoradiography.

The pharmaceutical composition

In another aspect, the present invention relates to compositions, such as pharmaceutical compositions, containing one monoclonal antibody or its antigen-binding site (sites), or a combination of such bodies or sections of the present invention, in combination with a pharmaceutically acceptable carrier. Such compositions can include one antibody, or a combination (for example, two or more different antibodies), or immunoconjugate, or bispecific molecules of the present invention. For example, the pharmaceutical composition of the present invention can include a combination of antibodies (or immunoconjugates, or bispecific antibodies)that bind to different epitopes on the target antigen or that have additional active the property.

The pharmaceutical compositions of the present invention can also be administered in combination therapy, i.e. in conjunction with other tools. For example, the combination therapy can include an anti-IFNAR-1 antibody of the present invention in combination with at least one immunosuppressive agent.

In the present description, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and fungicide, isotonic and slows the absorption means and the like that are physiologically compatible. Preferably the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e. the antibody immunoconjugate or bispecific molecule, may be coated with an appropriate substance to secure the connection from the action of acids and other natural conditions that may inactivate the current connection.

Pharmaceutical compounds of the present invention can include one or more pharmaceutically acceptable salts. The term "pharmaceutically acceptable salt" refers to a salt that retains the desired biological act shall want to make the parent compound and does not give any undesired Toxicological effects (see, for example, Berge, S.M.,et al. (1977)J.Pharm. Sci.66:1-19). Examples of such salts include acid-additive and primary additive salt. Acid additive salts include salts derived from nontoxic inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, Hydrobromic, uudistoodetena, phosphoric and the like, as well as from nontoxic organic acids, such as aliphatic mono - and dicarboxylic acids, phenylsilane alcamovia acid, hydroxyalkanoate acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc. Basically additive salts include salts derived from alkali and alkaline earth metals such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N,N'-dibenziletilendiaminom, N-methylglucamine, chloroprocaine, choline, diethanolamine, Ethylenediamine, procaine and the like

In addition, the pharmaceutical composition of the present invention may also include pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, bottled hydrox the anisole (BHA), bottled hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol and the like; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTU), sorbitol, tartaric acid, phosphoric acid, etc.

Examples of suitable aqueous and nonaqueous carriers which may be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), and suitable mixtures of vegetable oils, such as olive oil, and suitable for injectable organic esters, such as etiloleat. Proper mobility can be supported, for example, the use of covering materials, such as lecithin, in the case of dispersions maintaining the desired particle size and application of surfactants.

The described compositions may also contain auxiliary substances such as preservatives, wetting means, emulsifiers and dispersing funds. Protection from the presence of microorganisms may be ensured both by sterilization procedures, see above, and the inclusion of various antibacterial and antifungal materials such as paraben, chlorobutanol, phenol, sorbic acid, etc. may Also be desirable to include in the compositions isotonic means, such as sa is Ara, sodium chloride, etc. in Addition, prolonged absorption of injectable dosage forms may be achieved by the introduction of agents which delay absorption such as aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for extemporanea the sterile preparation suitable for injection solutions or suspensions. The use of such media and agents for pharmaceutically active substances is known in the art. In the pharmaceutical compositions of the present invention assumes the use of any conventional medium or agent, if they are not incompatible with the current connection. In addition, the composition may include additional active ingredients.

Therapeutic compositions typically must be sterile and stable under conditions of manufacture and storage. The composition may take the form of a solution, microemulsion, liposome, or other specified structure suitable for high concentration of the drug. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g. glycerol, propylene glycol, liquid polyethylene glycol and the like), and suitable mixtures. Proper mobility can be supported, for example, the R, application of covering materials, such as lecithin, in the case of dispersions maintaining the desired particle size and application of surfactants. In many cases it will be preferable to include in the composition isotonic means, such as sugar, polyalcohol, such as lures and sorbitol, or sodium chloride. Prolonged absorption of injectable compositions can be achieved by introducing in the compositions of agents that delay absorption, for example monostearate salt and gelatin.

Sterile suitable for injection solutions can be obtained by introducing the active compound in the required amount in an appropriate solvent together with one of the above ingredients, or a combination, if required, followed by sterilization using microfiltration. Basically dispersion obtained by introduction of the active compound into a sterile vehicle, which contains the basic dispersion medium and the other required ingredients listed above. In the case of sterile powders for the preparation of suitable sterile injectable solutions, the preferred methods of obtaining are drying in the vacuum drying and freezing (freeze-drying), which allow to obtain a powder of the active ingredient and any additional desired ingredient from the th received previously sterile-filtered solution.

The amount of active ingredient that may be combined with the substance of the media to produce a dosage form of a single dosage will vary depending on the exposed treatment of the subject and the particular route of administration. The amount of active ingredient that may be combined with the substance of the media to produce a dosage form of a single dosage will generally be that amount of the composition, which leads to a therapeutic effect. Mainly based on a percentage scale, this number will be in the range from about 0.01 percent to about 99 percent of active ingredient, preferably from about 0.1 percent to about 70 percent, most preferably from about 1 percent to about 30 percent of active ingredient in combination with pharmaceutically acceptable carrier.

Requirements for the dosage adjusted to provide the optimum desired response (e.g., therapeutic response). For example, can be made of a single bolus may be entered several individual doses in a certain period of time or the dose may be proportionally reduced or increased depending on the requirements of the current situation in the treatment process. Especially, it is preferable to get a pair of Teraline composition in the form of dosage forms of a single dosage to ease of administration and uniformity of dosage. In the present description, the term "dosage form of a single dosage" refers to physically discrete units suitable as a dose for a single injection of subjects treated; each unit contains a predetermined quantity of active compound calculated to obtain the desired therapeutic effect, in combination with the required pharmaceutical carrier. Specification of dosage forms of a single dosage of the present invention is determined and directly dependent on (a) specific characteristics of the active compound and the particular therapeutic result to be achieved, and (b) limitations of sensitivity in individual patients, existing in the technique of blending, similar to that used for the treatment of the current connection.

With the introduction of the antibody, the dosage ranges from about 0.0001 to 100 mg/kg and more from 0.01 to 5 mg/kg of body weight of the patient taking the drug. For example, the dosage can be 0.3 mg/kg of body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight or in the range of 1-10 mg/kg typical treatment regimen determines the introduction once a week, once every two weeks, once every three weeks, once a month, once every three months, once every 3-6 IU azev. Preferred dosing regimens for anti-IFNAR-1 antibodies of the present invention include 1 mg/kg body weight or 3 mg/kg of body weight when administered intravenously, and the antibody is administered using one of the following schedules: (i) every four weeks for six dosages, then every three months; (ii) every three weeks; (iii) a single injection of 3 mg/kg of body weight, then 1 mg/kg of body weight every three weeks.

In some methods, two or more monoclonal antibodies with different specificnosti linking entered at the same time, and in this case the dosage of each of the injected antibody is in the specified ranges. Antibodies usually do for many reasons. The intervals between individual introductions may be, for example, weekly, monthly, three-month or one-year. Intervals can also be unequal and determined in accordance with measuring levels of antibodies to the target antigen in the patient's blood. In some ways regulate the dosage to achieve the concentration of antibodies in the plasma of about 1-1000 μg/ml and in some methods about 25-300 μg/ml

On the other hand, the antibodies can be administered in the form of compositions with prolonged release, and it requires less frequent administration. Dosage and frequency of introduction of change in a time-dependent half-life ant the body in the body of the patient. As a rule, most prolonged time of life demonstrate human antibodies, followed by humanized antibodies, chimeric antibodies and antibodies, non-human antibodies. Dosage and frequency of injection can vary depending on whether the treatment is prophylactic or therapeutic. For prophylactic use relatively low doses over a relatively long time intervals over a long period of time. Some patients continue to receive treatment throughout life. Therapeutic application sometimes requires relatively high doses in relatively short periods of time up until the development of the disease will not slow down or terminated, and preferably until then, until the patient demonstrates partial or complete relief of symptoms. After this drug can be administered to a patient in a preventive mode.

Actual dosage levels of active ingredients in the pharmaceutical compositions of the present invention can be modified in order to obtain quantities of the active ingredient which is effective to achieve the desired therapeutic response in a particular patient, for a given composition and the method of administration, without p is oyavleniy toxicity to the patient. The selected dosage level will depend upon a wide range of pharmacokinetic factors including the activity of a specific applied composition of the present invention or a complex ester, salt or amide, route of administration, time of administration, rate of excretion of the specific applicable connection, duration of the treatment, other drugs, compounds and/or substances used in combination with the particular compositions used, age, sex, weight, painful condition, General health and prior medical history of the patient being treated, and like factors well known in medicine.

"Therapeutically effective dosage" of an anti-IFNAR-1 antibodies of the present invention preferably reduces the severity of disease symptoms, an increase in the frequency and duration of symptom-free periods, or a prevention of ill health or disability due to disease progression. If, for example, systemic lupus erythematosus (SLE), a therapeutically effective dose preferably prevents further deterioration of the physical symptoms associated with SLE, such as, for example, pain, fatigue and weakness. A therapeutically effective dose also preferably prevents or slows the onset of SLE, for example, that may be VC is positive, if you have initial or preliminary signs of the disease. It also includes slow chronic development associated with SLE. Laboratory tests used in the diagnosis of SLE include chemical analysis, Hematology, serology and radiology. Accordingly, any clinical or biochemical analysis, which controls any of the foregoing, can be used to determine whether this dose is therapeutically effective for the treatment of SLE. Ordinary specialist in the art would be capable of setting the number of, on the basis of such factors as the size of the subject, the severity of symptoms in the subject, as well as the specific composition or the selected route of administration.

The compositions of the present invention can be one or more ways using one or more of the variety of ways known in the art. As understood by a person skilled in the technical field, the path and/or the form of the introduction will vary depending on the desired results. The preferred route of administration of antibodies of the present invention include intravenous, intramuscular, intradermal, intraperitoneally, subcutaneous, spinal or other parenteral routes of administration, e.g. by injection or infusion. The phrase "parenteral centuries the Denia" in the present description means the forms of introduction, non-intestinal and local administration, usually by injection or infusion, i.e. not limited to, intravenous, intramuscular, intraarterial, intrathecal, intra-articular, intraorbital, intracardiac, intradermal, intraperitoneally, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and vnutrigrudne injections and infusions.

On the other hand, antibodies of the present invention can be aparentally ways, such as local and epidermal route of administration or through the mucous membranes, for example, intranasally, orally, vaginally, rectally, sublingually or tapicerki.

Preparations of active compounds can be obtained by the use of carriers that will protect the compound against rapid release, i.e. can be applied compositions with controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Can be used biorstwami, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyarteritis and polylactic acid. A large number of ways of making such formulations are patented or known to all specialists in this the field of technology. See, for example,Sustained and Controlled Release Drug Delivery Systems, J.R.Robinson, ed., Marcel Dekker, Inc., New York, 1978.

Therapeutic compositions can be administered using known in the art medical devices. For example, in a preferred embodiment, a therapeutic composition of the present invention can be entered using a needleless hypodermic injector, such as the device disclosed in U.S. patents№№ 5399163; 5383851; 5312335; 5064413; 4941880; 4790824; or 4596556. Examples of well-known implants and modules that are applicable in the present invention include: U.S. patent No. 4487603 in which the disclosed implantable microinfusion pump for dispensing medicines with adjustable speed; U.S. patent No. 4486194, which revealed a therapeutic device for administering drugs through the skin; U.S. patent No. 4447233, in which is disclosed a pump for infusion of drugs designed to deliver drugs at the exact speed of infusion; U.S. patent No. 4447224, in which is disclosed an implantable infusion device with a variable flow for continuous drug delivery; U.S. patent No. 4439196, which is disclosed the osmotic delivery system of drugs that have multi-chamber compartments; and U.S. patent No. 4475196, which disclosed the osmotic delivery system of drugs. These patents included in this is avko by reference. Many other such implants, delivery systems, and modules are well-known specialists in this field of technology.

In some embodiments, the implementation of a monoclonal human antibodies of the present invention may be incorporated in such compositions, to ensure the proper distribution ofin vivo. For example, the barrier the blood-brain (BBB) prevents the penetration of many high hydrophilic compounds. To ensure penetration of therapeutic compounds of the present invention through the BBB (if desired), they can be included in the composition, for example, on the basis of liposomes. To become familiar with methods of making liposomes, see, for example, U.S. patents 4522811; 5374548; and 5399331. Liposomes can include one or more fragments, which are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see, for example, V.V.Ranade (1989)J.Clin.Pharmacol.29:685). Typical target fragments include folate or Biotin (see, for example, U.S. patent 5416016 issued and Low co-authors); mannoside (Umezawa et al. (1988)Biochem.Biophys.Res.Commun.153:1038); antibodies (P.G.Bloeman et al. (1995)FEBS Lett.357:140; M.Owaiset al.(1995)Antimicrob.Agents Chemother.39:180); surface-active receptor protein A (Briscoeet al. (1995)Am.J.Physiol.1233:134); p120 (Schreieret al. (1994)J.Biol.Chem.269:9090); see also K.Keinanen; M.L.Laukkanen (1994)FEBS Ltt. 346:123; J.J.Killion:I.J.Fidler (1994)Immunomethods.4:273.

Applications and methods of the present invention

Antibodies (also immunoconjugate and bispecific molecules) of the present invention have diagnostic and therapeutic applicationsin vitroandin vivo. For example, these molecules can be introduced into cells in culture, for example,in vitroorex vivoor entity, for example,in vivofor treatment, prevention or diagnosis of a large number of disorders. It is assumed that the term "entity" in the present description includes humans and animals that do not belong to the human race. Animals include all vertebrates, e.g., mammals and insects memleketim, such as not belonging to the human race primates, sheep, dogs, cats, cows, horses, chickens, amphibians and reptiles. The methods developed in the present invention are particularly suitable for the treatment of patients among people with a disorder associated with aberrant or inappropriate expression of interferon type I (e.g., over-expression).

If antibodies to IFNAR-1 is administered together with another agent, both of these drugs can be administered in any order or simultaneously. For example, anti-IFNAR-1 antibody of the present invention can be used in combination with one or more of the following environments is TV: anti-IFNα antibody antibody against IFNγ receptor, soluble IFNγ receptor, anti-TNF antibody, antibody against TNF receptor and/or soluble TNF receptor (see, for example, U.S. patent No. 5888511). In addition, anti-IFNAR-1 antibody of the present invention can be used in combination with an antagonist of Flt3 ligand (see, for example, an application for U.S. patent No. 2002/0160974).

In one of the embodiments of the antibodies (and immunoconjugates and bispecific molecules) of the present invention can be used to determine levels of IFNAR-1, or levels of cells which Express IFNAR-1. This can be achieved, for example, the introduction of the interaction of the sample (e.g. samplein vitro) and the control sample with the anti-IFNAR-1 antibody under conditions that allow formation of a complex between the antibody and IFNAR-1. Any complexes formed by the antibody and IFNAR-1, find and compare in study and control sample. When using the compositions of the present invention may be implemented, for example, standard detection methods well known in the art, such as ELISA and flow cytometry.

Accordingly, in one aspect the invention additionally relates to methods of detecting the presence of IFNAR-1 (e.g., antigen IFNAR-1 person) in a sample or measuring the amount of IFNAR-1, including introduction to interoperability is brazza and the control sample with the antibody of the present invention or an antigen-binding site, which specific contact IFNAR-1, under conditions that allow formation of a complex between the antibody or its fragment and IFNAR-1. Then install the formation of the complex, and the difference in complex formation between the sample and the control sample is indicative the presence of IFNAR-1 in the sample.

Also in the scope of the present invention are kits comprising the compositions of the present invention (e.g., antibodies, human antibodies, immunoconjugates and bispecific molecules) and instructions for use. Such a kit may further comprise at least one additional reagent, or one or more additional antibodies of the present invention (e.g., an antibody having a complementary activity which binds to an epitope on the target antigen, different from the first antibody). The kits typically include a label with instructions on the intended application of the contents of the kit. The term "label" includes any written or recorded material deposited on the kit or supplied to him or any other image accompanying the kit.

IFNAR-1 is one of the cellular receptors of type I interferons and interferon type I, as you know, are immunoregulatory cytokines that are involved including differential is iruku T-cells, the production and activity of antibodies and memory T-cells. In addition, increased expression of interferon type I was observed in many autoimmune diseases, HIV-infection, when transplant rejection and graft-versus-host (GVHD). Accordingly, anti-IFNAR-1 antibodies (as well as immunoconjugate and bispecific molecules) of the present invention, which inhibit the functional activity of type I interferons, may be used in a variety of clinical indications involving aberrant or unwanted activity of interferon type I. Therefore, the present invention relates to a method of inhibiting diseases and disorders mediated by interferon type I, and the method includes the introduction of the antibody or its antigen-binding site according to the present invention (or immunoconjugate, or bispecific molecules of the present invention), in order to treat a disease or disorder mediated by interferon type I.

Specific examples of autoimmune conditions that can be applied to the antibodies of the present invention, include, but are not limited to, the following: systemic lupus erythematosus (SLE), insulin-dependent diabetes mellitus (IDDM), inflammatory bowel disease (IBD) (including Crohn's disease, I have public colitis and coeliac disease), multiple sclerosis (MS), psoriasis, autoimmune thyroiditis, rheumatoid arthritis (RA) and glomerulonephritis. In addition, the compositions of the antibodies of the present invention can be used for inhibiting or preventing transplant rejection or for the treatment of graft-versus-host (GVHD) or in the treatment of HIV/AIDS.

High levels of IFNα were observed in the serum of patients with systemic lupus erythematosus (SLE) (see, for example, Kim et al. (1987)Clin.Exp.Immunol.70:562-569). In addition, it was shown that the introduction of IFNα, for example, in the treatment of cancer or viral infections causes SLE (Garcia-Porrua et al. (1998)Clin.Exp.Rheumatol.16:107-108). Accordingly, in another embodiment, the anti-IFNAR-1 antibodies of the present invention can be used in the treatment of SLE by introducing antibodies to a subject in need of treatment. The antibody can be used alone or in combination with other agents against SLE, such as non-steroidal anti-inflammatory drugs (NSAIDs), analgesics, corticosteroids (eg, prednisone, hydrocortisone), immunosuppressants (such as cyclophosphamide, azathioprine and methotrexate, antimalarial drugs such as hydroxychloroquine) and biological drugs, which inhibit the production of antibodies against ds (double-stranded DNA) (e.g., LJP 394).

IFNα is also involved in pathological the Oia diabetes type I. For example, it was reported about the presence of immunoreactive IFNα in the beta cells of the pancreas in patients with diabetes type I (Fouliset al. (1987)Lancet.2:1423-1427). In addition, it was shown that prolonged use of IFNα in antiviral therapy causes diabetes type I (Waguriet al. (1994)Diabetes Res.Clin.Pract.23:33-36). Accordingly, in another embodiment, the anti-IFNAR-1 antibodies of the present invention can be used in the treatment of type I diabetic by injecting antibodies to a subject, if necessary, treatment. The antibody can be used alone or in combination with other antidiabetic agents such as insulin.

It was shown that antibodies to IFNAR are effective in animal models of inflammatory bowel diseases (see patent application U.S. 60/465155). Thus, anti-IFNAR-1 antibodies of the present invention can be used in the treatment of inflammatory bowel disease (IBD), including ulcerative colitis and Crohn's disease, by introducing antibodies to a subject, if necessary, treatment. This antibody can be used alone or in combination with other anti-IBD means, such as drugs, containing mesalamine (including sulfasalazin and other products containing 5-aminosalicylic acid (5-ASA), such as olsalazine and balsalazide), non-steroidal protivoop the extreme drugs (NSAID), analgesics, corticosteroids (eg, prednisone, hydrocortisone), TNF inhibitors (including adalimumab (Humira®), etanercept (Enbrel®) and infliximab (Remicade®)), immunosuppressants (such as 6-mercaptopurine, azathioprine and cyclosporine A), as well as antibiotics.

We also observed that treatment with IFNα induces autoimmune thyroiditis (Monzaniet al. (2004)Clin.Exp.Med.3:199-210; Prummel and Laurberg (2003)Thyroid.13:547-551). Accordingly, in another embodiment, the anti-IFNAR antibodies of the present invention can be used in the treatment of autoimmune thyroid diseases, including autoimmune primary hypothyroidism, graves ' disease, Hashimoto's thyroiditis and destructive thyroiditis with hypothyroidism, by introducing antibodies to a subject in need of treatment. This antibody can be used by itself or in combination with other agents or treatments, such as antithyroid drugs, radioactive iodine and partial thyroidectomy.

Elevated levels of type I interferons, in particular IFNβ was observed in the serum of patients with RA (see, e.g., Hertzoget al. (1988)Clin.Immunol.Immunopath.48:192). Thus, in one embodiment, the implementation of anti-IFNAR-1 antibodies of the present invention can be used in the treatment of RA by introducing these antibodies to a subject in case the need of such treatment. Antibodies can be used by themselves or in combination with one or more other anti-RA means, such as non-steroidal anti-inflammatory drugs (NSAIDs), COX-2 inhibitors, analgesics, corticosteroids (eg, prednisone, hydrocortisone), gold, immunosuppressants (e.g. methotrexate), means for depleting populations of B-cells (e.g., RituxanTM), agonists B-cells (e.g., LymphoStat-BTM) and anti-TNF-α tools (for example, EMBRELTM, HUMIRA® and REMICADETM).

It was reported that the introduction of IFNα exacerbate psoriasis. Accordingly, in another embodiment, the anti-IFNAR-1 antibody of the present invention can be used in the treatment of psoriasis and psoriatic arthritis by injecting antibodies to a subject in need of such treatment. The antibody can be used by itself or in combination with one or more other therapies for psoriasis, such as therapy, local therapy (e.g., local injection of corticosteroids) or systemic therapy (e.g. methotrexate, a synthetic retinoid, cyclosporine), anti-TNFα drug (e.g., EMBRELTM, HUMIRA® and REMICADETM) and the inhibitor of T-cells (for example, RaptivaTM).

In addition, high levels of IFNα were observed in the blood of patients with HIV infection, and their presence is predictive sign of the development of AIDS (DeStefanoet al/i> . (1982)J.Infec.Disease.146:451; Vadhan-Rajet al. (1986)Cancer Res.46:417). Thus, in another embodiment, the anti-IFNAR-1 antibody of the present invention is used in the treatment of HIV infection or AIDS by introducing antibodies to a subject, if necessary, treatment. The antibody can be used alone or in combination with other agents against HIV, such as nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, protease inhibitors and fusion inhibitors.

It was demonstrated that antibodies to IFNAR-1 are effective in the inhibition of allograft rejection and prolong the longevity of the allograft (see, for example, Toveyet al. (1996)J.Leukoc.Biol.59:512-517; Benizriet al. (1998)J.Interferon Cytokine Res.18:273-284). Accordingly, anti-IFNAR-1 antibodies of the present invention can also be used in recipients of grafts for inhibiting rejection of allografts and/or the extension of the durability of allografts. The present invention relates to a method of inhibiting transplant rejection by the introduction of anti-IFNAR-1 antibodies of the present invention to a recipient of a transplant, if necessary, treatment. Examples of tissue grafts that can be treated include, but are not limited to,liver, lungs, kidneys, heart, small intestine and insular cells of the pancreas, it also includes the treatment of graft-versus-host (GVHD). The antibody can be used by itself or in combination with other agents for inhibiting transplant rejection, such as immunosuppressive tools (e.g., cyclosporine, azathioprine, methylprednisolone, prednisolone, prednisone, mycophenolate mofetil, sirolimus, rapamycin, tacrolimus), anti-infective tools (e.g., acyclovir, clotrimazole, ganciclovir, nystatin, trimethoprimsulfamethoxazole), diuretics (for example, bumetanide, furosemide, metolazone) and antiulcer drugs (e.g. cimetidine, famotidine, lansoprazole, omeprazole, ranitidine, sukralfat).

Further, the present invention is illustrated by the following examples, which should not be construed as additional constraints. The contents of all illustrative material and all references, patents and published patent applications cited throughout this specification, is expressly incorporated in this application by reference.

Example 1:Obtaining monoclonal antibodies against IFNAR-1

Antigen

Soluble IFNAR-1, containing the extracellular domain of IFNAR-1, obtained by recombinant methods and used as an anti-Christ. Jena for immunization.

Transgenic HuMab mouse

Fully monoclonal antibodies person to IFNAR-1 was obtained with the use of lines of HuMab transgenic mice of the HCo7, HCo12 and HCo7·HCo12, each of which expresses genes of human antibodies. In each of these lines of mice, the endogenous gene Kappa light chain mouse was subjected to a homozygous disruption, as described in Chenet al. (1993)EMBO J. 12:811-820 and endogenous gene of the heavy chain of the mouse was subjected to a homozygous disruption, as described in example 1 was published under PCT WO 01/09187. Each of these lines of mice carries the transgene Kappa light chain KCo5 human, as described in Fishwildet al. (1996)Nature Biotechnology.14:845-851. Line HCo7 is the heavy chain transgene HCo7 human, as described in U.S. patent No. 5545806; 5625825 and 5545807. Line HCo12 is the heavy chain transgene HCo12 human, as described in example 2 was published under PCT WO 01/09187. Line HCo7·HCo12 bears as a transgene HCo7 and the transgene, HCo12, and it was received by the joint breeding individuals of the previous two lines.

Immunization of mice HuMab

To obtain a fully monoclonal antibodies person to IFNAR-1, HuMab mice were immunized with purified recombinant IFNAR-1 as antigen. The General scheme of immunization of HuMab mice is described in Lonberg, N.et al(1994)Nature.368 (6474): 856-859; Fishwild, D.et al. (1996)Nature Biotechnology.14: 845-851 and published under PCT WO 98/24884. If the first infusion Academy of Sciences of the Egan age mice were 6 to 16 weeks. A purified recombinant preparation (5-50 μg) of soluble antigen IFNAR-1 was administered to immunize the HuMab mice administered intraperitoneally, subcutaneously (Sc) or by injection into the paw pads.

Transgenic mice were immunized twice with antigen in complete Freund's adjuvant or Freund Ribi, administered intraperitoneally (IP), subcutaneously (Sc) or via pads (FP), then through 3-21 days followed IP, Sc or FP immunization (in total up to 11 immunizations) with the antigen in incomplete Freund's adjuvant or Ribi. For the immune reaction was observed using retroorbital bleeding. Was carried out by screening plasma using ELISA (as described below), and mice with sufficient titers of anti-IFNAR-1 of human immunoglobulin were used for fusions. For 2 or 3 days before the killing and removal of the spleen immune response of mice was supported by the intravenous injection of antigen. Typically, for each antigen was performed 10-35 m. For each antigen were immunized several dozen mice.

Selection of HuMab mice producing anti-IFNAR-1 antibodies

To select HuMab mice producing antibodies that bind to IFNAR-1, serum from immunized mice were examined by the ELISA method, as described D.Fishwild and co-authors (1996). Briefly, microtiter tablets were coated with purified recombinant IFNAR-1 fromE. coliat a concentration of 1-2 µg/ml in PBS, 50 µl/well, which was inkubirovali at 4°C overnight, then blocked with 200 µl/well of 5% serum of chickens in PBS/Tween (0.05 per cent). Plasma from IFNAR-1 immunized mice at various dilutions was added to each well and incubated for 1-2 hours at room temperature. The tablets were washed in PBS/Tween and then incubated with goat Fc polyclonal antibody against human IgG, conjugated with horseradish peroxidase (HRP)for 1 hour at room temperature. After washing tablets were treated with ABTS substrate (Sigma, A-1888, 0.22 mg/ml) and analyzed by spectrophotometer, defining OD 415-495. Mice that demonstrated the highest titers of anti-IFNAR-1 antibodies were used for fusions. The merge was performed, as described below, and supernatant hybridomas were tested for anti-IFNAR-1 activity by ELISA method.

Development of hybridomas producing monoclonal antibodies person to IFNAR-1

The fusion of mouse splenocytes, isolated from the HuMab mice with cell line murine myeloma in the presence of PEG was carried out using standard protocols. Then there was the screening of the obtained hybridomas according to the criterion production of antigen-specific antibodies. Carried out the merger of the suspension of individual cells of the splenic lymphocytes from immunized mice with one-fourth part by the number of SP2/0 " non-secretory of murine myeloma cells (ATCC, CRL 1581) with 50% PEG (Sigma). The cells were distributed in flat-bottomed microtic the oval tablets in the amount of about 1·10 5/well, followed about two week incubation in selective medium containing 10% fetal calf serum, 10% conditioned medium P388D1 (ATCC, CRL TIB-63), 3-5% Origen (IGEN) in DMEM (Mediatech, CRL 10013, with high glucose, L-glutamine and sodium pyruvate) c supplements 5 mm HEPES, 0.055 mm 2-mercaptoethanol, 50 mg/ml gentamycin and 1·HAT (Sigma, SRL P-7185). After 1-2 weeks the cells were cultured in the environment in which the HAT was replaced with HT. Then the wells are individually subjected to the screening method is ELISA (described above) on anti-IFNAR-1 human monoclonal IgG antibodies. If there was extensive growth of hybridoma, the environment is typically investigated in 10-14 days. Hybridoma, Sekretareva antibodies, re-distributed on the plates were again subjected to screening and in case of repeated positive result for IgG human anti-IFNAR-1 monoclonal antibodies were subclinically at least twice using serial dilutions. Then the stable subclones were culturedin vitroto generate small amounts of antibody in tissue culture medium for later retrieval performance.

The hybrid clones 3F11, 4G5, 11E2, and 9D4 were selected for further analysis.

Example 2:Obtaining structural characteristics of monoclonal antibodies person 3F11, 4G5, 11E2, and 9D4

Sequence of cDNA encoding the variable regions of heavy and light chain is th monoclonal antibody 3F11, 4G5, 11E2, and 9D4, was obtained from hybrid 3F11, 4G5, 11E2, and 9D4, respectively, using standard PCR techniques and sequenced using standard methods of DNA sequencing.

Nucleotide and amino acid sequences of variable regions of the heavy chain 3F11 shown in figa and SEQ ID NO: 33 and 25, respectively.

Nucleotide and amino acid sequences of variable region of the light chain 3F11 shown in figv and SEQ ID NO: 37 and 29, respectively.

Comparison of immunoglobulin sequences of the heavy chain 3F11 with known immunoglobulin sequences of the heavy chain germ-line human showed that the heavy chain 3F11 used VH segment embryonic cell line of human VH 4-34, unspecified D segment, and a JH segment germline JH 6b person. The combination of a VH sequence 3F11 with a sequence of germline VH 4-34 shown in figure 5. Further analysis of the VH sequence 3F11 using the CDR define areas in Kabat led to the identification of CDR1, CDR2 and CDR3 regions of the heavy chain as shown in figa and 5 and SEQ ID NO: 1, 5, and 9, respectively.

Comparison of immunoglobulin sequence to the light chain 3F11 with known immunoglobulin sequences of the light chain germ-line person demonstrated that in the light chain 3F11 VL segment germline cells VK L18 man and a JK segment germline JK 5 man. The combination of VL sequence 3F11 sequence germline VK L18 shown in Fig. Further analysis of the VL sequence 3F11 using the CDR define areas in Kabat led to the identification of CDR1, CDR2 and CDR3 regions of the light chain, as shown in figv and 8, and in SEQ ID NO: 13, 17 and 21, respectively.

Nucleotide and amino acid sequences of variable regions of the heavy chain 4G5 shown in figa and SEQ ID NO:34 and 26, respectively.

Nucleotide and amino acid sequences of variable region of the light chain 4G5 shown in figv and SEQ ID NO:38 and 30, respectively.

Comparison of immunoglobulin sequences of the heavy chain 4G5 with known immunoglobulin sequences of the heavy chain germ-line human showed that the heavy chain 4G5 used VH segment embryonic cell line VH 4-34 man, unidentified D segment, and a JH segment germline JH 4b person. The combination of a VH sequence 4G5 with a sequence of germline VH 4-34 shown in Fig.6. Further analysis of the VH sequence 4G5 using the CDR define areas in Kabat led to the identification of CDR1, CDR2 and CDR3 regions of the heavy chain as shown in figa and 6 and SEQ ID NO: 2, 6 and 10, respectively.

Comparison of immunoglobulin sequence to the light chain 4G5 known immunoglobulin sequences of the light chain germ-line human demonstrated in the light chain 4G5 used VL segment germline cells VK L18 man and a JK segment germline JK 2 people. The combination of VL sequence 4G5 sequence germline VK L18 shown in Fig.9. Further analysis of the VL sequence 4G5 using the CDR define areas in Kabat led to the identification of CDR1, CDR2 and CDR3 regions of the light chain, as shown in figv and 9 and SEQ ID NO: 14, 18 and 22, respectively.

Nucleotide and amino acid sequences of variable regions of the heavy chain 11E2 shown in figa and SEQ ID NO: 35 and 27, respectively.

Nucleotide and amino acid sequences of variable region of the light chain 11E2 shown in figv and SEQ ID NO: 39 and 31, respectively.

Comparison of immunoglobulin sequences of the heavy chain 11E2 with known immunoglobulin sequences of the heavy chain germ-line human showed that the heavy chain 11E2 derives or has a high degree of similarity VH segment embryonic cell line VH 5-51 man, unidentified D segment, and a JH segment germline JH 4b person. The combination of a VH sequence 11E2 with a sequence of germline VH 5-51 shown in Fig.7. Further analysis of the VH sequence 11E2 using the system definition CDR regions according to Kabat is rival to identify CDR1, CDR2 and CDR3 regions of the heavy chain as shown in figa and 7 and SEQ ID NO: 3, 7 and 11, respectively.

Comparison of immunoglobulin sequence to the light chain 11E2 with known immunoglobulin sequences of the light chain germ-line person demonstrated that in the light chain 11E2 used VL segment germline cells VK A27 man and a JK segment germline JK 5 man. The combination of VL sequence 11E2 sequence germline VK A27 shown in figure 10. Further analysis of the VL sequence 11E2 using the CDR define areas in Kabat led to the identification of CDR1, CDR2 and CDR3 regions of the light chain, as shown in figv and 10 and SEQ ID NO:15, 19 and 23, respectively.

Nucleotide and amino acid sequences of variable regions of the heavy chain 9D4 shown in figa and SEQ ID NO:36 and 28, respectively.

Nucleotide and amino acid sequences of variable region of the light chain 9D4 shown in figv and SEQ ID NO:40 and 32, respectively.

Comparison of immunoglobulin sequences of the heavy chain 9D4 with known immunoglobulin sequences of the heavy chain germ-line human showed that the heavy chain 9D4 derives or has a high degree of similarity VH segment embryonic cell line VH 5-51 person, not the plant D segment, and a JH segment germline JH 4b person. The combination of a VH sequence 9D4 with a sequence of germline VH 5-51 shown in Fig.7. Further analysis of the VH sequence 9D4 using the CDR define areas in Kabat led to the identification of CDR1, CDR2 and CDR3 regions of the heavy chain as shown in figa and 7 and SEQ ID NO:4, 8, and 12, respectively.

Comparison of immunoglobulin sequence to the light chain of 9D4 with known immunoglobulin sequences of the light chain germ-line person demonstrated that in the light chain 9D4 used VL segment germline cells VK A27 man and a JK segment germline JK 5 man. The combination of VL sequence 9D4 sequence germline VK A27 shown in figure 10. Further analysis of the VL sequence 9D4 using the CDR define areas in Kabat led to the identification of CDR1, CDR2 and CDR3 regions of the light chain, as shown in figv and 10 and SEQ ID NO:16, 20 and 24, respectively.

Example 3:Monoclonal anti-IFNAR-1 antibodies inhibit human biological activity of interferon α2b

Cell line Daudi, derived from B-lymphoblastic lymphoma Bernita person, expresses high levels of IFNAR-1, and growth of these cells is inhibited by interferon type I. For measuring functional blocking the ability of anti-IFNAR-1 antibodies were performed the VA various analysis tools namely, the analysis of cell proliferation and reporter analysis.

In the first analysis of Daudi cells were cultured with interferon α2b in the presence or absence of antibody and proliferation was measured by uptake of3[H]-thymidine. Cells Daudi (ATCC CCL-213) were grown in RPMI containing 10% FCS and 2 mm beta-mercaptoethanol (Wednesday). Cells were centrifuged and resuspendable at a concentration of 1·106cells/ml in medium with the addition of 1% albumin human serum (medium and HS). To each well of 96-hole tablet was added 100 μl of 200 U/ml interferon α2b (Schering Corporation), containing the appropriate concentration of antibody. To the wells were added with 100 μl of Daudi cells in the environment and HS and then the plates were incubated for 48 hours at 37°C. the Tablets were labeled 1 mccoury3[H]-thymidine and incubated for another 24 hours. Delete the contents of the wells, gathering his 96-hole fiber filter plate, and made calculations of radioactivity using a scintillation counter TopCount (Packard). Build a graph of counts per minute as a function of the concentration of antibodies and analyzed the data using nonlinear regression, i.e. sigmoidal dependence of the dose-response (variable slope)using the software Prism (San Diego, CA).

In the second analysis U937 cells were transfusional construct, in which the interferon-stimulated feeling is sustained fashion item is associated with a reporter gene (ISRE-RG), and he measured the ability of humanized anti-IFNAR-1 antibodies block induced by interferon expression of reporter gene. Cells were grown in RPMI containing 10% FCS and 2 mm betareceptor (Wednesday). Cells (1·106cells/ml) resuspendable in the environment with the addition of 2% human serum. In a 96-well plate was added 100 μl of cells. Antibody serially diluted in medium containing 200 U/ml interferon α2b (Schering corporation), and to each well was added 100 μl. The plates were incubated over night at 37°C. After this incubation were measured reporter gene expression using flow cytometry. Build a graph of the average geometric fluorescence intensity as a function of the concentration of antibodies and analyzed the data using nonlinear regression, i.e. sigmoidal dependence of the dose-response (variable slope)using the software Prism (San Diego, CA).

Using two methods of analysis described above, compared the efficacy of monoclonal antibody 3F11, mouse anti-IFNAR-1 antibody 64G12 (ECACC, Depository No. 92022605) and gumanitarnogo anti-IFNAR-1 antibodies D1 H3K1 (additionally described in the application U.S. serial No. 60/465058). Antibody 3F11 demonstrated in 5-10 times more effective than antibody mouse, and 6-30 times greater efficiency than humanitariannet antibody. The result is summarized below in table 1.

Table 1
Blocking the ability of anti-IFNAR-1 antibodies person in relation to IFN alpha 2b
The isotypeCell proliferation (Daudi) EC50(nm)The ISRE reporter-RG (U937) EC50(nm)
64G12m IgG13,16,0
D1 H3K1h IgG19,38,0
3F11h IgG10,31,2

Example 4:Monoclonal anti-IFNAR-1 antibodies inhibit human biological activity of IFN omega

Investigated the ability of anti-IFNAR-1 antibodies person to inhibit the reaction IFN omega using analysis of Daudi cell proliferation, as described above in example 3. In each well of 96-hole tablet was added 100 μl of 200 U/ml interferon omega (PBL), containing antibody at appropriate concentrations. Human antibodies 3F11, 4G5, 11E2, and 9D4 was 4-18 times more effective (as measured by EC50)than the antibody 64G12 mouse. Results summer the Vanir below in table 2.

Table 2
Blocking the ability of monoclonal anti-IFNAR-1 antibodies person in relation to IFN omega
The isotypeCell proliferation (Daudi) EC50(nm)
64G12m IgG15,5
D1 H3K1h IgG130,7
3F11h IgG10,6
4G5h IgG11,4
11E2h IgG10,3
9D4h IgG10,3

Example 5:Monoclonal anti-IFNAR-1 antibodies inhibit human biological activity of IFN different types of I

As indicated in example 3, interferon alpha inhibits proliferation of Daudi cells (lymphoma Berkata, ATCC #CCL-213) dose-dependent way. Neutralizing antibody that blocks the binding of interferon to its receptor, can restore proliferation. Applying the analysis about what operacii cells, investigated the specificity of the purified anti-IFN alpha antibodies man, testing the blocking natural lymphoblastoid IFNα, natural leukocyte interferon, 13 subtypes of recombinant IFN alpha, IFN beta or IFN omega.

The Daudi cells were grown in culture medium (RPMI 1640, with the addition of 10% FCS, 1x2-ME, L-glutamine, penicillin and streptomycin) with additive and without the addition of IFNα in 96-well flat-bottomed tablet for culturing cells. Each of the tested interferon type I researched when EC50and mixed with 2 multiples of serial dilutions of anti-IFNAR-1 antibody 3F11, usually from 50 µg/ml (312 nm) to 381 PG/ml (2,4 PM). The mixture of antibody/IFN was added to Daudi cells in 96-well-plate with a bottom to a final density of 1·104of Daudi cells/100 μl/well, and incubated at 37°C in atmosphere of 5% CO2within 72 hours. Proliferation was analyzed by adding MTS (Promega), 20 μl/well, and was measured by OD at 490 nm with a further 3-hour incubation. The number of viable cells was proportional to the registered value of OD. Expected percentage of blocking interferon in relation to the proliferation of Daudi cells in the absence of IFN (=100% block) and in the presence of only IFN (=0% blocking). Results for antibody 3F11 was calculated in accordance with the degree of blocking, receiving the specificity profile of IFNα subtype. the results showed the antibody 3F11 person against receptor 1 interferon alpha inhibits the action of numerous subtypes of interferon alpha, including IFNα 6, 2b, 2a, 1, 16, 10, 8, 5, 14, 17, 7, 4 and 21, as well as lymphoblastoid IFN, leukocyte IFN and IFN omega. 3F11 is an inhibitor of IFN beta lower level, although there was a greater than 50% inhibition. The percentage of blocking and standard deviation for various interferons shown below in table 3.

Table 3
Inhibition of various interferon type I antibody 3F11
3F11 IFN blocking (%) at 1000·Ab
IFNAverageStandard deviation
Lymphoblastoid IFN94,92,9
IFNα 6107,16,6
IFNα 2b101,90,4
IFNα 2a103,13,0
IFNα 1111,61,9
Leukocyte IFN109,41,4
IFNα 16105,71,4
IFNα 1096,75,5
IFNα 887,52,6
IFNα 5105,1a 3.9
IFNα 14100,31,4
IFNα 1799,82,4
IFNα 7102,83,2
IFNα 4100,52,5
IFNα 21104,42,3
IFN-beta53,01,7
IFN-omega107,11,3

Example 6:The inhibition induced IFN secretion of IP-10 anti-IFNAR-1 antibodies

It was shown that addition of IFN alpha 2b to cell cultures is an integral part of the environment induces the secretion of IP-10 normal managername the peripheral blood cells (PBMNC). Investigated the activity of anti-IFNAR-1 antibody 3F11 person in the inhibition induced by interferon secretion of IP-10 normal PBMNC cultures by analysis of binding by ELISA method.

Cells PBMNC were incubated in culture medium (RPMI 1640+10% FBS+1% human serum) with leukocyte IFN, IFN alpha 2b or IFNω within 24-48 hours.

Supernatant collected and analyzed for the concentration of IP-10/CXCL10, using the kit for the quantitative sandwich ELISA (Quantikine®, R&D Systems) in dilution 1:30, according to the manufacturer's recommendations. The results of the analysis demonstrated that monoclonal antibodies person 3F11 inhibit induced leukocyte IFN, recombinant IFNα 2b and recombinant IFNω the secretion of IP-10 normal PBMNC culture. These results are shown in table 4.

Table 4
Inhibition of IFN-induced expression of IP-10 on normal PBMNC antibodies
Used
antibody
The absence of IFN
IP-10 (PG/ml)
Leukocyte
IFN
IP-10 (PG/ml)
IFN alpha 2b
IP-10 (PG/ml)
IFN omega
IP-10 (PG/ml)
The absence of antibodies907 266527392904
3F11 (5 µg/ml)387854745674
Control Ig (5 μg/ml)838351231173960
* Cultures were added 100 U/ml each subtype IFN

Example 7:Analysis of cross-competition monoclonal anti-IFNAR-1 antibodies man

To identify, contact Lee monoclonal human antibodies to the same epitope as monoclonal antibody 64G12 mouse, conducted analysis of cross-competition ELISA to determine, arbitrate whether these antibodies for one binding epitope.

96-well tablets covered soluble IFNAR-1 person, derived from CHO cells, at a concentration of 1 μg/ml in freshly prepared DPBS in the amount of 100 μl/well (Mediatech). Monoclonal antibodies person 3F11, 4G5, 11E2, and 9D4 at a concentration of 20 μg/ml was added to the number of holes 1 and serially diluted in the ratio 1:2 in the hole relating to the series from 1 to 12, followed by incubation for 45 minutes. Added monoclonal the antibody 64G12 mouse at a concentration of EC 75equal to 0.3 ág/ml, 50 μl per well and incubated the plates for 30 minutes. Tablets 3 times washed with automatic device for washing tablets Elx 405 (BIO-TEK Instruments). Since affinity purification of F(ab')2 goat antimurine IgG (Fcγ specific) antibody with peroxidase diluted at a ratio of 1:3000 in PBS and added as a conjugate for detection (Jackson ImmunoResearch Laboratories, cat. 115-036-0710). After incubation for 1 hour tablets 3 times washed with automatic device for washing tablets Elx 405. The ABTS solution (800 ál of ABTS solution, 8 μl of 30% H2O2and 100 ml of citrate-phosphate buffer) at a concentration of 27.8 mg/ml was added to each well and incubated for 20 minutes. The tablets were read at 415 nm using 490 nm as a reference wavelength. The results are shown figure 11. The results show that the monoclonal anti-IFNAR-1 antibodies person, namely 3F11, 4G5, 11E2, and 9D4, not compete with 64G12 for binding to IFNAR-1 and, thus, are associated with a different epitope on IFNAR-1 than 64G12.

Example 8:Inhibition antibodies development of dendritic cell-mediated SLE plasma

SLE plasma induces the development of dendritic cells from normal human monocytes. In this example, purified monoclonal anti-IFNAR-1 antibody man 3F11 had inhibit the study of the development of dendritic cells, which was assessed by the ability of antibodies to inhibit the induction of cell surface markers CD38, MHC class I and CD123 under the action of SLE plasma.

25 ml of leukocyte film was diluted in PBS 4 times. The sample was divided by 4·50-ml conical tubes and was introduced in the form of a layer of 15 ml of medium for fractionation of lymphocytes (ICN Biomedicals). After a 30-min rotation at 500g leukocyte layer containing PBMC were removed and washed with PBS. Cells resuspendable in the cultural environment 4·106cells/ml Monocytes were isolated by incubation of PBMC (2,0·107cells/5 ml/25 cm3the flask) for 1.5 hours at 37°C in culture medium with two subsequent leaching of the non-adhesive cells. After the second wash, the cells were cultured in medium containing 1% human serum, inactivated by heating. In flask cultures were added dvadtsatipyatiletnei the plasma of SLE patients plus/minus neutralizing antibodies and isotype control (30 μg/ml); the mixture of IFN Alfa-2b (100 and 10 IU/ml) and 25% normal human plasma was used as a positive control for the induction of markers. Flasks were incubated at 37°C in an atmosphere of 5% CO2from three to seven days. From each flask was collected, air-conditioned environment, and provided the suspended cells by centrifugation at 1000 rpm on the rotor Sorvall RTH-750. Precipitated cells were retained on ldwi layer of fluid was frozen at -80°C for ELISA.

Adhesive cells were isolated from the flask by washing PBS (2 ml) followed by 15-minute incubation in versene (EDTU) (3 ml) if necessary. At the end of incubation with versinon flask was otkarmlivali and finally washed with PBS (2 ml). PBS, verse together with washed cells were combined with cells isolated from collected kondicionirovanie environment. The combined cell suspension was centrifuged at 1000 rpm on the rotor Sorvall RTH-750, the precipitate resuspendable up in 300 µl of staining buffer (PBS + 0,1M EDTU + 2% FBS +1% HS) and distributed 100 μl/well in 96-well plate with a V-shaped bottom. The tablet was subjected to pulse centrifugation at 2800 rpm on the rotor Sorvall RTH-750 and precipitated cells resuspendable 25 ál/well labeled fluorochrome antibodies as follows: (1) mouse anti-MHC class I-FITC + mouse anti-CD38-PE, and (2) the control isotype mouse IgG-FITC+mouse IgG-PE. Protected from light, the plates were incubated on ice for 45 minutes. Cells were washed three times by adding 200 µl of staining buffer followed by pulse centrifugation and then resuspendable in 200 μl of 2% paraformaldehyde in PBS. Staining of dendritic cells were analyzed by flow cytometry using a Becton Dickinson FACScaliburTM. The graph of a forward scattering, side scattering were drawn open to remove pollutants glue the OK from the analysis. Monoclonal anti-IFNAR-1 antibody inhibits human IFN alpha-dependent development of dendritic cells, as demonstrated using the normalized expression of markers of cell surface MHC class I, CD38 and CD123 in the presence of 3F11. The results are shown below in table 5, where part of the table (A) and (B) summarize the results for the sample of two representatives of donors SLE.

Table 5
Inhibition of maturation of dendritic cells
(A)
Plasma donor No. 40* (13,3 IU/ml**)
Conditions of cultivationMHC class ICD123CD38
MFIMFIMFI
0 IFN/ml1481440
10 IFN/ml2001944
100 IFN/ml2292663
02062247
3F111151332
HuIgG1 (isotype control)1942262
(B)
Plasma donor No. 59* (75,3 IU/ml**)
Conditions of cultivationMHC class ICD123CD38
0 IFN/ml2291158
10 IFN/ml2711286
100 IFN/ml29413112
0202 1562
3F11112822
HuIgG1 (isotype control)2661455

Example 9: Analysis of the binding of anti-IFNAR-1 antibodies person with Daudi cells or human managername the peripheral blood cells by Scatchard

Menagerie cells of peripheral blood were obtained from fresh blood according to standard protocols using the separation step Ficol gradient. The Daudi cells were obtained from ATCC and were grown in RPMI containing 10% fetal calf serum (FBS). Cells were twice washed in RPMI containing 10% FBS at 4 degrees, and the number of cells brought up to 4·107cells/ml in RPMI medium containing 10% fetal calf serum (binding buffer). Tablets Millipore (MAFB NOB) were covered with 1% fat-free dried milk in water and kept at 4°C over night. The tablets were washed in binding buffer and added 25 μl of unlabeled antibody (1000-fold excess) in binding buffer in the control wells of 96-well filter plate fiberglass Millipore (nonspecific binding, NSB). Twenty-five microlitres pure buffer was added to the completed well with the maximum binding (total binding). Added twenty-five microlitres125I-anti-IFNAR-1 antibodies at varying concentrations and 25 μl of Daudi cells or managernew cells in human peripheral blood (4·107cells/ml) in binding buffer. The plates were incubated for 2 hours on a shaker at 200 rpm at 4°C. after the incubation tablets Millipore washed twice with 0.2 ml of cold binding buffer. The filters were removed and counted in a gamma counter. Determination of equilibrium binding was performed using parameters of a single binding site using the software Prism (San Diego, CA).

According to the results of the above analysis of binding by Scatchard, values of KDantibodies to Daudi cells and for managernew cells in human peripheral blood was approximately 0.2 nm and 0.5 nm, respectively.

Sequence listing

VK CDR2 amino acid 4G5
SEQ ID NO:Sequence
1VH CDR1 amino acid 3F11
2VH CDR1 amino acid 4G5
3VH CDR1 amino acid 11E2
4VH CDR1 amino acid 9D4
5VH CDR2 amino acid 3F11
6VH CDR2 amino acid 4G5
7VH CDR2 amino acid 11E2
8VH CDR2 amino acid 9D4
9VH CDR3 amino acid 3F11
10VH CDR3 amino acid 4G5
11VH CDR3 amino acid 11E2
12VH CDR3 amino acid 9D4
13VK CDR1 amino acid 3F11
14VK CDR1 amino acid 4G5
15VK CDR1 amino acid 11E2
16VK CDR1 amino acid 9D4
17VK CDR2 amino acid 3F11
18
19VK CDR2 amino acid 11E2
20VK CDR2 amino acid 9D4
21VK CDR3 amino acid 3F11
22VK CDR3 amino acid 4G5
23VK CDR3 amino acid 11E2
24VK CDR3 amino acid 9D4
25VH amino acid 3F11
26VH amino acid 4G5
27VH amino acid 11E2
28VH amino acid 9D4
29VK amino acid 3F11
30VK amino acid 4G5
31VK amino acid 11E2
32 VK amino acid 9D4
33VH nucleotide 3F11
34VH nucleotide 4G5
35VH nucleotide 11E2
36VH nucleotide 9D4
37V nucleotide 3F11
38V nucleotide 4G5
39V nucleotide 11E2
40V nucleotide 9D4
41A.K. germline VH 4-34
42A.K. germline VH 5-51
43A.K. germline VK L-18
44A.K. germline VK A27
is the

1. The selected monoclonal antibody human, which is associated with specific receptor 1 interferon alpha man (IFNAR-1), or antigennegative plot containing:
(a) the variable region of the heavy chain CDR1 of man, containing SEQ ID NO: 4;
(b) the variable region of the heavy chain CDR2 of man, containing SEQ ID NO: 8;
(c) variable region of the heavy chain CDR3 of man, containing SEQ ID NO: 12;
(d) variable region light chain CDR1 of man, containing SEQ ID NO: 16;
(e) variable region light chain CDR2 of man, containing SEQ ID NO: 20; and
(f) variable region light chain CDR3 of man, containing SEQ ID NO: 24.

2. The selected monoclonal antibody human, which is associated with specific receptor 1 interferon alpha man (IFNAR-1), or antigennegative plot containing:
(a) the variable region of the heavy chain of man, containing the amino acid sequence of SEQ ID NO: 28; and
(b) the variable region of the light chain of man, containing the amino acid sequence of SEQ ID NO: 32.

3. The human antibody according to any one of claims 1 and 2 or 26-32, which does not bind to the same epitope as monoclonal antibody 64G12 mouse (Depository number ESAS No. 92022605).

4. Composition for treating diseases and disorders mediated by interferon type I, containing a therapeutically effective the active amount of the antibody or its antigennegative plot according to any one of claims 1 and 2 or 26-32, and pharmaceutically acceptable carrier.

5. Immunoconjugate for the treatment of diseases and disorders mediated by interferon type I, containing the antibody or antigennegative plot according to any one of claims 1 and 2 or 26-32 associated with therapeutic tool.

6. Immunoconjugate according to claim 5, further containing a pharmaceutically acceptable carrier.

7. Immunoconjugate according to claim 5, in which therapeutic agent is a cytotoxin.

8. Immunoconjugate according to claim 7, further containing a pharmaceutically acceptable carrier.

9. Immunoconjugate according to claim 5, in which therapeutic agent is a radioactive isotope.

10. Immunoconjugate according to claim 9, further containing a pharmaceutically acceptable carrier.

11. Bispecific molecule for the treatment of diseases and disorders mediated by interferon type I, containing the antibody or antigennegative plot according to any one of claims 1 and 2 or 26-32 associated with the second functional fragment having a different binding specificity compared with the indicated antibody or antigennegative plot.

12. A composition comprising a therapeutically effective amount bispecific molecule according to claim 11 and a pharmaceutically acceptable carrier, for treating diseases and disorders mediated by interferon type I.

13. The allotment is fair molecule of nucleic acid, encoding the antibody or antigennegative plot according to any one of claims 1 and 2 or 26-32.

14. The expression vector comprising the nucleic acid molecule according to item 13.

15. A host cell containing the expression vector by 14.

16. The antibody according to any one of claims 1, 2, 3 or 27-33 obtained by immunization with the recombinant protein IFNAR-1 transgenic mouse NSO, NSO and NSO×NCO carrying the transgenes of the heavy and light chains of human immunoglobulin.

17. Hybridoma producing the antibody according to any one of claims 1, 2, 3, 26-32, and hybridoma obtained from transgenic mouse NSO, NSO and NSO×Na.

18. The method of obtaining anti-IFNAR-1 antibody according to any one of claims 1, 2 or 26-32, including:
(a) receive:
(i) sequence variable regions of heavy chain antibodies containing the sequence of CDR1 SEQ ID NO: 4, a CDR2 sequence SEQ ID NO: 8; and the CDR3 sequence SEQ ID NO: 12; or (ii) the sequence of the variable region of the light chain antibodies containing the sequence of CDR1 SEQ ID NO: 16, a CDR2 sequence SEQ ID NO: 20, and the CDR3 sequence SEQ ID NO: 24;
(b) modifying at least one amino acid residue of at least one variable region sequence of the antibody, and the above sequence selected from the sequences of variable regions of heavy chain antibodies and sequence of the variable region Le is coy chain antibodies, to create at least one altered sequences of the antibodies; and
(C) the expression of the modified sequence of the antibody in the form of a protein, where the antibody inhibits the biological activity of IFN-β; inhibits the activity of IFN-α2b in the analysis of Daudi cell proliferation; inhibits the activity of IFN omega in the analysis of Daudi cell proliferation; inhibits secretion of IP-10 by mononuclear cells in peripheral blood and induced IFN-α2b; inhibits secretion of IP-10 by mononuclear cells in peripheral blood and induced IFN omega; inhibits the development of dendritic cells, mediated by plasma of a patient with systemic lupus erythematosus; and binds to the epitope other than the epitope binding monoclonal antibody 64G12 mouse (Depository number ESAS No. 92022605).

19. A method of inhibiting the biological activity of interferon type I on the cell expressing the receptor 1 interferon alpha, including the interaction of cells with the antibody according to any one of claims 1, 2 or 26-32, in order to inhibit the biological activity of interferon type I.

20. The method of treatment of diseases or disorders mediated by interferon type I, the individual, including the introduction of individual antibodies or antigennegative plot, according to any one of claims 1, 2 or 26-32, for the treatment of diseases mediated by interferon type I, the individual.

21. The way what about the claim 20, where the disease is mediated by interferon type I, is a disorder mediated by interferon-alpha.

22. The method according to claim 20, where the disease or disorder is a systemic lupus erythematosus.

23. The method according to claim 20, where the disease or disorder selected from the group consisting of insulin-dependent diabetes mellitus, inflammatory bowel disease, multiple sclerosis, psoriasis, autoimmune thyroiditis, rheumatoid arthritis and glomerulonephritis.

24. The method according to claim 20, where the disease or disorder is an HIV infection or AIDS.

25. The method according to claim 20, where the disease or disorder is a graft rejection or graft-versus-host.

26. The selected monoclonal human antibody, which binds to the receptor 1 interferon alpha man (IFNAR-1), or antigennegative plot containing the variable region of the heavy chain comprising sequences of CDR1, CDR2 and CDR3, and a variable region light chain comprising sequences of CDR1, CDR2 and CDR3, where:
(a) the sequence of CDR3 of the variable region of the heavy chain contains the amino acid sequence of SEQ ID NO: 12, and conservative modifications specified sequence; and
(b) sequence of CDR3 of the variable region of the light chain contains aminokislotnoi sequence of SEQ ID NO: 24 and conservative modifications specified sequence.

27. The antibody according p, where the CDR2 sequence of the variable region of the heavy chain contains the amino acid sequence of SEQ ID NO: 8 and conservative modifications specified sequence;
the CDR2 sequence of the variable region of the light chain contains the amino acid sequence of SEQ ID NO: 20, and conservative modifications specified sequence.

28. The antibody according to item 27, where the CDR1 sequence of the variable region of the heavy chain contains the amino acid sequence of SEQ ID NO: 4, and conservative modifications specified sequence; the sequence of the variable regions CDR1 light chain contains the amino acid sequence of SEQ ID NO: 16, and conservative modifications specified sequence.

29. The selected monoclonal human antibody, which binds to the receptor 1 interferon alpha man (IFNAR-1), or antigennegative plot, in which:
(a) the variable region of the heavy chain contains an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 28; and
(b) variable region light chain contains an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 32.



 

Same patents:

FIELD: medicine.

SUBSTANCE: there are offered versions of a monoclonal antibody specific to GPVI polypeptide, peptide or its naturally occurred version. A based antithrombotic composition and a method for preparing thereof are described. The versions of methods for inhibition and treatment of various thrombocyte aggregation mediated diseases are disclosed. An antithrombotic set and hybridoma for producing the monoclonal antibody are described.

EFFECT: use of the invention provides the antibodies inhibiting thrombocyte aggregation that can find the further application in medicine for treating various thromboses.

13 cl, 16 dwg, 9 tbl, 10 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to immunology and biotechnology. Claimed are versions of antibodies or their functional fragments, which are bound with receptor of human insulin-like growth factor I IGF-IR, and/or natural binding of its ligands IGF1 and/or IGF2 and/or are capable of specific inhibition of tyrosine kinase activity of said IGF-IR. Antibodies contain respective CDR sections of light and heavy chains. Described is mouse hybridoma I-3193 for production of antibodies. Composition for prevention or treatment of cancer, based on antibody application. Described is application of antibodies and/or composition for obtaining respective medication. Claimed is method of diagnostics in vitro of diseases, caused by over-expression or insufficient expression of receptor IGF-I based on antibodies..

EFFECT: invention application ensures antibodies able to bind with isophorms A and B insulin, insulin/ IGF-1 of hybrid receptors which can be applied in medicine for tumour treatment.

15 cl, 8 dwg, 2 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: there are offered specific antibodies linked at least with KIR2DL1, KIR2DL2, KIR2DL3 human receptor, neutralise KIR-mediated NK cytolergy inhibition in relation to Cw3+ or Cw4+ target-cells. There are described: B-lymphocyte hybrid cell for producing the antibodies, versions of the method for producing the antibody, as well as a method for detecting a NK-cell, a method for purifying the NK-cells with the use of the antibody and versions of the pharmaceutical antibody composition. Using the antibody for preparing a medicinal agent is offered.

EFFECT: use of the invention provides producing the antibody which controls NK cytolergy of various types, intensifies cytotoxicity, increases NK cytolergy or cytotoxicity in individuals.

63 cl, 13 dwg, 3 tbl, 8 ex

FIELD: medicine; pharmacology.

SUBSTANCE: allocated human monoclonal antibodies which specifically bind a receptor of the epidermal growth factor (EGFR), and also corresponding compositions on the basis of antibodies and a biospecific molecule are described. Human antibodies can be received with use of the transgenic mouse capable to formation of set of isotypes of human monoclonal antibodies by recombination V-D-J and switching of isotypes. The pharmaceutical compositions containing human antibodies for treatment or prevention of diseases, mediated by expression EGFR, the transgenic animals distinct from a human, the specified expressing antibodies, hybridomes and transfectomes which produce human antibodies are also presented. Ways of therapy and diagnostics of the diseases mediated by expression EGFR, with use of human antibodies or their antigen-binding of fragments, and also methods of growth suppression of the cells expressing EGFR, and an induction of cytolysis of the specified cells are described.

EFFECT: invention allows obtaining therapeutic and diagnostic preparations of antibodies with improved properties.

53 cl, 22 dwg, 4 tbl, 11 ex

FIELD: biotechnology, immunology, medicine, oncology.

SUBSTANCE: strain of hybrid cultured mammalian cells Mus musculus VKPM H-98 is prepared by the hybridoma technology method. This strain is a producer of monoclonal antibodies possessing individual specificity to hypoglycosidated and deglycosidated isoforms of tumor-associated human antigen Muc I. Productivity of the strain and specificity of produced antibodies is estimated based on the immunoenzyme assay using some markers of specificity: natural purified antigen Muc I isolated from human milk; VNTR22-polypeptide; synthetic monomeric polypeptide (TR1); deglycosidated antigen Muc I (de-Muc I) prepared by chemical oxidation of natural Muc I; hypoglycosidated antigen Muc I (o-Muc I) prepared by periodate oxidation of natural Muc I. Monoclonal antibodies produced by the claimed strain recognize clinically significant isoforms of antigen Muc I and allows assaying its concentrations in human serum blood in carrying out the early diagnosis of tumors. Invention can be used in preparing monoclonal antibodies to tumor-associated human antigen Muc I.

EFFECT: valuable properties of strain.

2 dwg, 3 ex

FIELD: biotechnology, immunology, medicine, oncology.

SUBSTANCE: strain of hybrid cultured mammalian cells Mus musculus VKPM H-97 is prepared by the hybridoma technology method. This strain represents a producer of monoclonal antibodies possessing specificity to conformation-dependent of tumor-associated human antigen Muc I. Productivity of the strain and specificity of produced antibodies is estimated based on immunoenzyme assay using some markers of specificity: natural purified antigen Muc I isolated from human milk; VNTR22-polypeptide; synthetic monomeric polypeptide (TR1); deglycosidated antigen Muc I (de-Muc I) prepared by chemical oxidation of natural Muc I; hypoglycosidated antigen Muc I (o-Muc I) prepared by periodate oxidation of natural Muc I. Monoclonal antibodies produced by the claimed strain recognize clinically significant isoforms of Muc I antigen and allows assaying its concentration in human serum blood in carrying out early diagnosis. Invention can be used for preparing monoclonal antibodies to tumor-associated human antigen Muc I.

EFFECT: valuable properties of strain.

2 dwg, 3 ex

FIELD: medicine, biotechnology.

SUBSTANCE: invention proposes variants of antibodies showing specificity to peptide domain located by both side of hinged site R76S77 in pro-BNP(1-108). Indicated antibodies recognize specifically also circulating pro-BNP(1-108) in human serum or plasma samples but they don't recognize practically peptides BNP(1-76) or BNP(77-108). Also, invention describes variants of peptides used in preparing antibodies. Amino acid sequence is given in the invention description. Also, invention discloses methods for preparing indicated antibodies and among of them by using indicated peptides. Also, invention describes methods for preparing antibody-secreting hybridoma, and hybridoma is disclosed prepared by indicated method. Also, invention describes a monoclonal antibody secreted by hybridoma 3D4 and deposited at number CNCM I-3073. Also, invention discloses variants for diagnosis of cardiac insufficiency in vitro and by using antibodies proposed by the invention. Also, invention describes a set used for detecting pro-BNP(1-108) in a biological sample. Using this invention simplifies detection of pro-BNP(1-108) circulating in human serum or plasma samples and provides specific detection of pro-BNP(1-108) that can be used in early diagnosis of human cardiac insufficiency.

EFFECT: valuable medicinal properties of antibodies.

24 cl, 16 dwg, 5 tbl, 20 ex

FIELD: immunology.

SUBSTANCE: invention relates to immunoenzyme analysis and can be used for assay of von Willebrand factor. Method involves immunoenzyme analysis wherein monoclonal antibody 5C3 is used as an immobilizing antibody, and a mixture of biotin-labeled monoclonal antibodies 2H2 and 7D12 is used as a detecting antibody. Also, invention relates to monoclonal antibodies produced by the strain of hybridoma cultured cells Mus musculus L. and directed against von Willebrand factor, and to strains of hybrid cultured cells Mus musculus L. producing indicated monoclonal antibodies. Invention provides the development of highly sensitive method for assay of von Willebrand factor.

EFFECT: improved method for analysis.

9 cl, 1 tbl, 2 dwg, 3 ex

New virus of plants // 2411290

FIELD: agriculture.

SUBSTANCE: first plant or its part is exposed to infection dose of ToTV. Then plants with no or slight symptoms of disease are identified.

EFFECT: plants identified in such a manner as resistant to virus are used as donor ones to cross with recipient plants, and plants resistant to ToTV are chosen from descendant plants.

8 cl, 7 dwg, 5 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: invention refers to oligonucleotide with the sequence SEQ ID NO: 1 or to its functional homolog (nucleotide sequence is disclosed in the description). Oligonucleotide is used to induce apoptosis of neoplastic B-cells and to treat B-lymphoid neoplasm in a subject mammal. According to the invention, oligonucleotide is used as an ingredient of a pharmaceutical composition for treating B-lymphoid neoplasm in the subject mammal in a combination with a B-lymphoid neoplasm drug specified among chemotherapeutic drugs, immunotherapeutic drugs, particularly CD20 antibody, or drugs used in radiation therapy.

EFFECT: oligonucleotide under the invention induces apoptosis of neoplastic B-cells, increases CD40 expression on neoplastic B-cells and stimulates IL-10 formation.

17 cl, 7 dwg, 2 tbl, 9 ex

FIELD: medicine.

SUBSTANCE: invention refers to producing versions of group I Poaceae (holy grass) allergen, also can be used either for specific immunotherapy (hyposensitisation) of patients with grass pollen allergy, or for preventive immunotherapy of grass pollen allergies. The produced versions are characterised by Cys41 Ser, Cys57Ser, Cys69Ser, Cys72Ser, Cys77Ser, Cys83Ser and Cysl39Ser substitutes in a Phi p1 mature protein sequence. Also, a structure of the allergen versions can be presented with no fragments relevant to amino acid residues 1-6, 1-30, 92-104, 115-119, 175-185 and 213-220 or 1-6, 115-119 and 213-220 as a part of a primary sequence of Phi p1 mature protein.

EFFECT: invention allows producing a version of group I Poaceae allergen characterised lower IgE responsiveness as compared with common wild allergen and substantially maintained responsiveness to T-lymphocytes.

8 cl, 9 dwg, 2 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: recombinant plasmid pFastBac-B17R DNA carries a cowpox virus genome fragment. BvB 17RG recombinant baculovirus strain is produced with the use of recombinant plasmid pFastBac-B 17R DNA and deposited in the Microorganism Cultures Collection of the Federal State Research Institution 'State Research Centre for Virology and Biotechnology 'Vector' of Federal Service for Supervision of Consumer Rights Protection and Human Welfare' (FGUN GNC VB 'Vector' of Rospotrebnadzor) under No. V-388, characterised as a producer of a soluble analogue protein of cowpox interferons type 1 cell receptor.

EFFECT: extended spectrum of preparations of new generation.

2 cl, 5 dwg, 8 ex

FIELD: medicine.

SUBSTANCE: there are offered versions of an angiopoietin-2 (Ang-2) specific antibody and a pharmaceutical antibody composition for treatment of various diseases associated with angiopoietin-2 overexpression. Also there are described methods of inhibition, modulation and treatment of various diseases mediated by angiopoietin-2 activity. There are offered: coding nucleic acid, an expression vector and a vector-transformed cell, as well as a method for producing antibodies.

EFFECT: use of the invention ensures new high-cytotoxicity antibodies (according to ELISA analysis IC50=0,35 nM) comparable with a common antibody Ab536 that further can find application in medicine.

22 cl, 2 dwg, 11 tbl, 6 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology and specifically to obtaining versions of glycoprotein IV alpha polypeptide of human thrombocytes (GPIbalpha) and can be used in medicine to treat vascular disorders. Using a recombinant technique, a polypeptide is obtained, which contains substitutes in SEQ ID NO:2 selected from: Y276F K237V C65S; K237V C65S; Y276F C65S; or Y276F Y278F Y279F K237V C65S. The obtained polypeptide is used to inhibit bonding of leucocytes to biological tissue or for treating disorders associated with activation of thrombocytes.

EFFECT: invention enables to obtain GPIbalpha polypeptide which bonds with von Willebrand factor with affinity which is at least 10 times higher than in natural GPIbα polypeptide, and also has low affinity for bonding with alpha-thrombin, lower aggregation and/or high resistance to proteolysis relative the polypeptide with SEQ ID NO:2.

41 cl, 3 dwg, 8 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: to obtain microbe-resistant plants plant cells are transformed with vector, which contains polynucleotide structure, which codes defensin polypeptide, which has amino-acid sequence C-x (3)-C-x (7, 9)-C-C, C-C-(8)-C-x-C and C-x-C-x (8, 11)- C.

EFFECT: obtaining polypeptide, which can be also applied as drug or veterinary medication, as well as in forage for animals.

23 cl, 4 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: there is claimed isolated human antibody or its fragment, which binds to human EGFR. Antibody contains corresponding CDR areas of light and heavy chain. Its conjugate with anti-neoplastic means or marker is described. Also described are: coding nucleic acid, expression vector, recombinant cell-host for obtaining antibodies and method of inhibiting growth of tumor, expressing EGFR on the basis of antibody.

EFFECT: application of invention provides antibodies with affinity comparable or higher, than in IMC-C225, which neutralises EGFR activation, what can be applied in medicine for treatment of tumours.

36 cl, 14 dwg, 6 tbl, 13 ex

FIELD: medicine.

SUBSTANCE: invention represents mutant photoprotein for determining intracellular calcium simultaneously in different cell organelles, which possesses light0emitting function and is characterised by amino acid sequence of wild type calcium-regulated photoprotein, in which natural residues of phenylalanine, related to conservative regions of said sequence and corresponding to residue 119 of photoprotein obelin Obelia longissima, as well as to residue 88 of obelin Obelia longissima, are substituted with other residues, different from phenylalanine, substitution of which results in the reduction of sensitivity of calcium-regulated photoprotein to calcium ions in comparison with wild type photoprotein and change of emitted light wavelength in comparison with wild type photoprotein. Invention also relates to one more mutant protein, which has substitution in position 144 of photoprotein obelin Obelia longissima. Invention also relates to DNA, which codes said proteins, as well as to vectors for expression of proteins.

EFFECT: invention allows to obtain novel forms of photoprotein with changed sensitivity to calcium and changes wavelength of emitted light.

13 cl, 5 dwg, 4 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: present invention relates to immunology and biotechnology. The invention discloses versions of a cytotoxically active CD3-specific binding structure. The structure comprises a first domain specifically binding to human CD3 and an Ig-derived second binding domain which is specific to molecules on the cell surface. The invention describes a coding nucleic acid, a vector for expressing the structure and an eukaryotic cell transformed by the vector. The invention discloses versions of compositions based on the structure for treating, preventing or alleviating various diseases and corresponding methods of treating the diseases. A method of obtaining the structure is disclosed.

EFFECT: use of the invention provides a structure with low immunological potency, which has cytotoxicity comparable to the initial structure, which may find further use in medicine.

60 cl, 18 dwg, 15 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: binding molecule represents a CD45RO and CD45RB chimeric antibody. The molecule contains two domains with consistent hypervariable sites CDR1, CDR2 and CDR3, and CDR1', CDR2' and CDR3', CDR1 has amino acid sequence NYIIH, CDR2 has amino acid sequence YFNPYNHGTKYNEKFKG, and CDR3 has amino acid sequence SGPYAWFDT. CDR1' has amino acid sequence RASQNIGTSIQ, CDR2' has amino acid sequence SSSESIS, and CDR3' has amino acid sequence QQSNTWPFT. Related coding polynucleotide is described.

EFFECT: use of the invention allows to induce immunosuppression, to inhibit T-cell response and primary lymphocyte reaction in the mixed culture, to prolong survival time in mice with severe combined immunodeficiency SCID.

6 cl, 5 dwg, 2 tbl, 8 ex

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