Antibodies of high affinity to il-6-receptor of humans

FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed invention relates to field of immunology and biotechnology. Claimed are: versions of antibody and antigen-binding fragments of antibody to receptor Il-6 of humans. Considered are: isolated molecule of nucleic acid and vector which contains it. Described are: system "host-vector" and method of obtaining antibody or its antigen-binding fragment, as well as application of antibody or its antigen-binding fragment for obtaining medication.

EFFECT: invention application provides novel antibodies to receptor IL-6 of humans, which can be applied in therapy of IL-6- mediated diseases.

11 cl, 5 tbl, 7 ex

 

The technical field to which the invention relates.

Interleukin-6 (IL-6) is a pleiotropic cytokine produced by immune and non-immune cells, which plays a crucial role in the regulation of immune responses, acute phase reactions and hematopoiesis. It binds to soluble and associated with the cell membrane IL-6R (α-chain) with the formation of the binary complex, and this complex is able to interact with the associated with the cellular membrane gp130 (β-chain), induces the formation of the complex signal containing two of each of IL-6, IL-6R and gp130.

Antibodies to hIL-6R described in patents US 5670373, 5795965, 5817790, 6410691 and EP 409607B1. therapeutic methods described in patents US 5888510 and 6723319.

The invention

In the first aspect, the invention provides human antibodies, preferably recombinant human antibodies, that specifically bind the receptor of interleukin-6 human (hIL-6R). These antibodies are characterized by binding to hIL-6R with high affinity and slow dissociation kinetics and the ability to neutralize the activity of IL-6. These antibodies can be full-sized (e.g., antibody IgG1 or IgG2) or may contain only antigennegative part (e.g., Fab fragment, F(ab')2or scFv) and can be modified to influence the functionality of, for example, for the elimination statocniritiery functions (Reddy et al. (2000) J. Immunol. 164:1925-1933). In the preferred embodiment, this invention provides an antibody or antigennegative fragment that binds IL-6-receptor human (SEQ ID NO: 1) with KDabout 500 PM or less, as measured by surface plasmon resonance. In a more specific embodiment, this antibody or antigennegative fragment has KDless than 300 PM or less, 200 PM or even less than 100 PM. In a different implementation, this antibody or antigennegative fragment blocks the activity of hIL-6 with IC50250 PM or less, as measured by the bioanalysis using luciferase. In more specific embodiments, the implementation, the antibody or antigennegative fragment shows IC50150 PM or less.

In related aspects, this antibody or antigennegative fragment according to this invention binds to hIL-6R with an affinity at least 2 times higher than the affinity with which it binds IL-6R monkeys. In more preferred embodiments, implementation, this antibody or antigennegative fragment protein binds hIL-6R (SEQ ID NO: 1) with an affinity that is approximately 3 times higher relative to its affinity binding to IL-6R monkeys (extracellular domain Macaca fascicularis shown in SEQ ID NO: 251).

In one embodiment, the antibody or ntaganzwa part of the antibodies according to this invention contains the variable region of the heavy chain (HCVR), selected from the group consisting of SEQ ID NO: 3, 227, 19, 231, 35, 51, 67, 83, 99, 115, 131, 147, 239, 241, 163, 179, 235, 195 and 211 or essentially similar sequence. In a more specific embodiment, this antibody or antigennegative fragment further comprises a variable region light chain (LCVR)selected from the group consisting of SEQ ID NO: 11, 229, 27, 233, 43, 59, 75, 91, 107, 123, 139, 155, 171, 187, 237, 203 and 219 or essentially similar sequence. In specific embodiments, implementation, this antibody or antigennegative fragment contains a pair HCVR/LCVR selected from the group consisting of SEQ ID NO: 3/11; 227/229; 19/27; 231/233; 35/43; 51/59; 67/75; 83/91; 99/107; 115/123; 131/139; 147/155; 239/155; 241/155; 163/171; 179/187; 235/237; 195/203 and 211/219 or essentially similar sequence.

In a second aspect, this invention provides the selected molecules are nucleic acids that encode the antibody or antigennegative fragment of the antibody of this invention. In one embodiment, the nucleic acid molecule according to this invention encodes an antibody or its fragment containing HCVR described above. In specific embodiments, the implementation, the nucleic acid molecule encoding a HCVR selected from the group consisting of SEQ ID NO: 2, 226, 18, 230, 34, 50, 66, 82, 98, 114, 130, 146, 238, 240, 162, 178, 234, 194 and 210 or essentially identical sequences. In a related aspect, this invention provide the supports selected nucleic acid molecule, LCVR encoding described above. In specific embodiments, implementation, this molecule is a nucleic acid encoding a LCVR, is a nucleotide sequence selected from the group consisting of SEQ ID NO: 10, 228, 26, 232, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 236, 202 and 218 or essentially identical to the sequence.

In a third aspect, this invention describes an antibody or antigennegative fragment containing the domain complementarity determining section 3 (CDR3) of the heavy chain domain and light chain CDR3, where

domain CDR3 of the heavy chain contains the amino acid sequence of the formula X1- X2- X3- X4- X5- X6- X7- X8- X9- X10- X11- X12- X13- X14- X15- X16- X17- X18- X19(SEQ ID NO: 247), where X1=Ala, X2=Lys, X3=Gly, X4=Arg X5=Asp, X6=Ser or Ala, X7=Phe, X8=Asp; X9=Ile, X10=Pro or is absent, X11=Phe or is absent, X12=Val or absent, X13=Tyr or absent, X14=Tyr or absent, X15=Tyr or absent, X16=Gly or absent, X17=Met or absent, X18=Asp or absent, and X19=Val or absent; and

domain CDR3 light chain contains the amino acid sequence of the formula X1- X2- X3- X4- X5X 6- X7- X8- X9(SEQ ID NO: 250), where X1=Gln, X2=Gln or His, X3=Ala, X4=Asn or Tyr, X5=Ser, X6=Phe, X7=Pro, X8=Pro and X9=Thr.

In a more specific embodiment, this antibody or antigennegative fragment further comprises

domain CDR1 heavy chain containing the amino acid sequence of the formula X1- X2- X3- X4- X5- X6- X7- X8(SEQ ID NO: 245), where X1=Gly or Arg, X2=Phe, X3=Thr, X4=Phe, X5=Asp, X6=Asp, X7=Tyr and X8=Ala;

domain CDR2 of the heavy chain containing the amino acid sequence of the formula X1- X2- X3- X4- X5- X6- X7- X8(SEQ ID NO: 246), where X1=Ile or Val, X2=Ser, X3=Trp, X4=Asn, X5=Ser, X6=Gly, X7=Ser and X8=Ile;

domain CDR1 light chain containing the amino acid sequence of the formula X1- X2- X3- X4- X5- X6(SEQ ID NO: 248), where X1=Gln, X2=Gly, X3=Ile, X4=Ser, X5=Ser and X6=Trp; and

domain CDR2 light chain containing the amino acid sequence of the formula X1- X2- X3(SEQ ID NO: 249), where X1=Gly or Ala, X2=Ala and X3=Ser.

In a fourth aspect, this invention describes an antibody or antigennegative fragment containing:

domain CDR3 of the heavy chain selected from the group consisting of SEQ ID NO: 25, 153, 9, 185, 41, 57, 73, 89, 105, 121, 137, 169, 201 and 217; and

domain light chain CDR3 selected from the group consisting of SEQ ID NO: 33, 161, 17, 193, 49, 65, 81, 97, 113, 129, 145, 177, 209 and 225.

In a more specific embodiment, the antibody or antigennegative fragment further comprises:

domain heavy chain CDR1 selected from the group consisting of SEQ ID NO: 21, 149, 5, 181, 37, 53, 69, 85, 101, 117, 133, 165, 197 and 213;

domain heavy chain CDR2 selected from the group consisting of SEQ ID NO: 23, 151, 7, 183, 39, 55, 71, 87, 103, 119, 135, 167, 199 and 215;

domain light chain CDR1 selected from the group consisting of SEQ ID NO: 29, 157, 13, 189, 45, 61, 77, 93, 109, 125, 141, 173, 205 and 221; and

domain light chain CDR2 selected from the group consisting of SEQ ID NO: 31, 159, 15, 191, 47, 63, 79, 95, 111, 127, 143, 175, 207 and 223.**

In specific embodiments, implementation, this antibody or antigennegative fragment contains a sequence of CDR of the heavy chain SEQ ID NO: 21, 23, 25 and sequence of the CDR of the light chain of SEQ ID NO: 29, 31, 33; sequence of CDR of the heavy chain SEQ ID NO: 149, 151, 153 and sequence of the CDR of the light chain of SEQ ID NO: 157, 159, 161; sequence of CDR of the heavy chain SEQ ID NO: 5, 7, 9 and sequence of the CDR of the light chain of SEQ ID NO: 13, 15, 17; and sequence of the heavy chain CDR SEQ ID NO: 181, 183, 185 and sequence of the CDR of the light chain of SEQ ID NO: 189, 191, 193.

In the fifth aspect, this invention describes a selected nucleic acid molecule, the code is the dominant antibody or antihistamie fragments of this invention, where the antibody or its fragment contains

domain CDR3 of the heavy chain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 24, 152, 8, 184, 40, 56, 72, 88, 104, 120, 136, 168, 200 and 216; and

domain CDR3 light chain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 32, 160, 16, 192, 48, 64, 80, 96, 112, 128, 144, 176, 208 and 224; and their essentially identical nucleic acid sequences.

In a more specific embodiment, provided with a selected nucleic acid molecule encoding the antibody or antigennegative fragment according to this invention, where the antibody or its fragment contains

domain CDR1 of the heavy chain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 20, 148, 4, 180, 36, 52, 68, 84, 100, 116, 132, 164, 196 and 212;

domain CDR2 of the heavy chain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 22, 150, 6, 182, 38, 54, 70, 86, 102, 118, 134, 166, 198 and 214;

domain CDR1 light chain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 28, 156, 12, 188, 44, 60, 76, 92, 108, 124, 140, 172, 204 and 220; and

domain CDR2 light chain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 30, 158, 14, 190, 30, 46, 62, 78, 94, 110, 126, 142, 174, 206 and 222; and their essentially identical to the sequence of nucleic sour is.

The invention includes anti-hIL-6R antibody or antihistamie fragments with modified pattern (distribution) of glycosylation. In some applications, it may be applicable modification to remove unwanted sites of glycosylation or antibody devoid fukanou part of the molecule on the oligosaccharide chain, for example, to increase antibody-dependent cellular cytotoxicity (ADCC) (see Shield et al. (2002) JBC 277:26733). In other applications, can be produced by modification of galactosylceramide for modification complementability cytotoxicity (CDC).

In other aspects, the invention provides recombinant expressing vectors carrying the nucleic acid molecule of this invention, and cells of the hosts, which were introduced such vectors, and methods for producing antibodies or antigenspecific fragments of this invention is obtained by culturing host cells of this invention. This is a host cell can be prokaryotic or eukaryotic cell, it is preferable that the host-cell is an E. coli cell or mammal cells, such as cell SNO.

In the following aspect, this invention describes a pharmaceutical composition comprising the human antibody or antigennegative fragment of the antibody, which specifically with azeveda with hIL-6R, and pharmaceutically acceptable carrier.

The following aspects, this invention describes methods of inhibiting the activity of IL-6 person using antibodies or antigennegative part of this invention. In one embodiment, this antibody includes a therapeutic method comprising the introduction of an antibody of this invention or its fragment to a subject, a person suffering from disorders that can be treated or relieved by inhibition of the activity of IL-6. This disorder can be, for example, arthritis, including chronic rheumatoid arthritis; inflammatory bowel disease, including Crohn's disease and ulcerative colitis; systemic lupus erythematosus and inflammatory diseases.

In other aspects, the invention provides the use of antibodies or antigennegative fragment of the antibody, as defined above, in the preparation of medicines for use in the reduction or inhibition of IL-6-mediated diseases or disorders in humans. In a related aspect, this invention provides an antibody or antigennegative fragment of the antibody, as defined above, for use in the reduction or inhibition of IL-6-mediated diseases or disorders in humans.

Other objectives and advantages will become apparent when considering the following detailed description.

DETAILED description of the INVENTION

Before describing the methods of the present invention should be understood that this invention is not limited to the described specific ways and experimental conditions, as such methods and conditions may vary. It should also be understood that as used in this description, the terminology is intended solely for the purpose of describing particular embodiments and is not intended to be limiting, as the scope of the present invention will be limited only by the attached claims.

If nothing else is specified, all technical and scientific terms used in the present invention, have the same meaning as is understood by a person of ordinary skill in the field that belongs to this invention. Although in the practice or testing of the present invention can be used any methods and materials similar or equivalent to the methods and materials described herein, will now be described the preferred methods and materials.

The term "human IL6R" (hIL-6R) refers in this context to the receptor of the cytokine person that specifically binds interleukin-6 (IL-6). The extracellular domain of hIL-6R shown in SEQ ID NO: 1.

The term “antibody” refers, in this context, the molecules of the immunoglobulin is, contains four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain contains a variable region heavy chain (abbreviated herein as HCVR or VH) and a constant region of the heavy chain. The constant region of the heavy chain contains three domains, CN, CH2 and CH3. Each light chain contains a variable region light chain (abbreviated as LCVR or VL) and a constant region light chain. The constant region of the light chain consists of a single domain (CL1). VH - and VL-region can be further subdivided into hypervariable region, called complementarity determining regions (oblasts) (CDR), with the inserted regions that are more conservative, called frame regions (FR). Each 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.

The term "antigennegative part of the antibody (or simply "antibody" or "antibody fragment"refers, in this context, to one or more fragments of an antibody that retain the ability to specifically bind with the antibody (e.g., hIL-6R). It was shown that antigennegative function of antibodies can be performed by fragments of a full-sized antibodies. Examples of binding fragments is s, covered by the term "antigennegative part of the antibody include (i) a Fab fragment, a monovalent antibody comprising the VL domains, VH, CH1 CL1 and; (ii) F(ab')2-fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment (Ward et al. (1989) Nature 241 :544-546), which consists of a VH domain; and (vi) selected complementarity determining region (CDR). In addition, although the two domains of Fv fragment, VL and VH, are coded by different genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to form a single contiguous chain in which the VL - and VH-region mate with the formation of monovalent molecules (known as single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). It is assumed that such single-chain antibodies are also included in the term "antigennegative part of the antibody. Other forms of single-chain antibodies, such as diamela, also included in this term (see, e.g., Holliger et al. (1993) Proc. Natl. Acad Sci. USA 90:6444-6448).

“Neutralizing" or “blocking" antibody, in this context, refers to the antibody, the binding of which to hIL-6R leads to inhibition of the biological activity of the hIL-6. This inhibition of the biological activity of hIL-6 can be assessed by measuring one or more indicators of biological activity of hIL-6, known in this field, such as hIL-6-induced cellular activity and the binding of hIL-6 hIL-6R (see examples below).

“CDR" or complementarity determining plot is a plot of hypervariability inserted at intervals in areas that are more conservative, called “framework regions” (FR). In different variants of implementation of the anti-hIL-6R antibody or fragment according to this invention, these FR can be identical to the sequences of the germline of the person or may be natural or artificially modified. The group of CDR can be defined as amino acid consensus sequence, for example, in one embodiment, the anti-hIL-6R-antibody or antigennegative fragment of this invention can be described as containing the domain CDR3 of the heavy chain containing the amino acid sequence of the formula X1- X2- X3- X4- X5- X6- X7- X8- X9- X10- X11- X12- X13- X14- X15- X16- X17- X18- X19(SEQ ID NO: 247), where X1=Ala, X2=Lys, X3=Gly, X4=Arg X5=Asp, X6=Ser or Ala, X7=Phe, X =Asp; X9=He, X10=Pro or is absent, X11=Phe or is absent, X12=Val or absent, X13=Tyr or absent, X14=Tyr or absent, X15=Tyr or absent, X16=Gly or absent, X17=Met or absent, X18=Asp or absent, and X19=Val or absent; and CDR3 domain light chain containing the amino acid sequence of the formula X1- X2- X3- X4- X5- X6- X7- X8- X9(SEQ ID NO: 250), where X1=Gln, X2=Gln or His, X3=Ala, X4=Asn or Tyr, X5=Ser, X6=Phe, X7=Pro, X8=Pro and X9=Thr.

The term "surface plasmon resonance", in this context, refers to an optical phenomenon that makes possible the analysis of interactions real-time detection of changes in protein concentrations within a biosensor matrix, for example using the BIAcore™ system (Pharmacia Biosensor AB).

The term "epitope" means an antigenic determinant, which interacts with specific antigennegative site in the variable region of the antibody molecules, known as Pratap. Individual antigen can have more than one epitope. Epitopes can be either conformational or linear. Conformational epitope formed spatially arranged side by side amino acids from different segments of the line is Oh polypeptide chain. Linear epitope formed adjacent amino acid residues in the polypeptide chain. In some circumstances, the epitope may include sacharine part, a phosphoryl group or sulfonylurea group antigen.

The term "substantial identity" or "substantially identical", when it refers to nucleic acid or its fragment, indicates that under optimal mapping with appropriate nucleotide insertions or deletions with another nucleic acid molecule (or its complementary chain) has the nucleotide sequence identity in at least about 95%, or more preferably at least approximately 96%, 97%, 98% or 99% nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed below.

As applied to polypeptides, the term "substantial identity" or "essentially identical” means that two peptide sequences, when optimally mapping, such as the programs GAP or BESTFIT using mass gaps by default, have at least 95% sequence identity, even more preferably at least 98%, or 99% sequence identity. Preferably, the position of the residues that are not identical, according to the evidence of conservative amino acid substitutions. “Conservative amino acid substitution" is a substitution in which the amino acid residue is replaced with another amino acid residue having a side chain (R group) with similar chemical properties (e.g. charge or hydrophobicity). Usually conservative amino acid substitution does not significantly alter the functional properties of the protein. In cases where two or more amino acid sequences differ from each other conservative substitutions, the percent identity or similarity of the sequence may be adjusted in the direction of correction of the conservative nature of the substitution. See, for example, Pearson (1994) Methods Mol. Biol. 24: 307-331. Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amesterdam side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine and tryptophan; 5) basic side chains: lysine, arginine and histidine; 6) acidic side chains: aspartate and glutamate and 7) sulfur-containing side chains: cysteine and methionine. Preferred groups of conservative substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-TSA is tat and asparagine-glutamine. Alternatively, a conservative replacement is any change having a positive value in the matrix log-likelihood PAM250 described in gonnet on et al. (1992) Science 256: 1443-45. A "moderately conservative" replacement is any change having a non-negative value in the matrix log-likelihood PAM250.

The similarity of the sequences for polypeptides, which is also referred to as sequence identity, usually measured using the software sequence analysis. Software analysis of protein matches similar sequences using similarity measurements assigned to various substitutions, deletions and other modifications including amino acid substitutions. For example, software GCG contains programs such as Gap and Bestfit, which can be used with default parameters to determine the homology of sequences or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild-type protein and its muteena. See, for example, the GCG Version 6.1. Polypeptide sequences can also be compared using FASTA using the default settings or recommended settings, programs in GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides is sopostavlenija and the percentage sequence identity of the regions best match between the requested sequences and sequences of search (Pearson (2000) supra). Other preferred algorithm when comparing sequences of this invention with a database containing a large number of sequences from different organisms is the computer program BLAST, especially blastp or tblastn, using the default settings. See, for example, Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389 402.

Antibodies man

Methods of generating human antibodies include, for example, technology Velocimmune™ (Regeneron Pharmaceuticals), XenoMouse™ (Green et al. (1994) Nature Genetics 7:13-21; Abgenix), the approach minilogues" and phage display (see, for example, patent US 5545807, patent US 6787637). Velocimmune technology™ (patent US 6596541) includes a method of generating highly specific full-length human antibodies to the selected antigen. This technology involves the generation of a transgenic mouse, having a genome containing the variable regions of heavy and light chains of a human, functionally associated with the endogenous loci of constant region of the mouse so that the mouse produces the antibody containing the variable region of a human and a constant region of the mouse in response to antigenic stimulation. DNA encoding the variable regions of the heavy and light chains of the antibody is isolated and functionally linked to DNA that encodes a constant region of the heavy and light chains of human rights. For the eat this DNA Express in the cell, able to Express fully human antibody. In a specific embodiment, this cell is the cell SNO.

Antibodies may be therapeutically applicable in blocking the interaction of ligand-receptor or inhibition of the interaction of the receptor component, and not by killing cells through the fixation of complement (complement-dependent cytotoxicity) (CDC) and through participation in antibody-dependent cell-mediated cytotoxicity (ADCC). The constant region of the antibody is important in the ability of the antibody to fix complement and mediate cleocinonline cytotoxicity Thus, the isotype of the antibody can be selected on the basis of whether it is desirable that this antibody was posredovano cytotoxicity.

The human immunoglobulins may exist in two forms that are associated with the heterogeneity of hinge region. In one form, the immunoglobulin molecule contains stable chutyrehzveznuyu design approximately 150-160 kDa, in which the dimers are held together by a disulfide bond interaction of the heavy chain. In the second form, these dimers are not connected together megamachine disulfide bonds, and formed molecule is approximately 75-80 kDa, composed of covalently linked to the light chain and the heavy chain (poloitical). Section is whether these forms was extremely difficult, even after affinity purification. The frequency of occurrence of the second form in different intact IgG isotypes due to, but not limited to, structural differences associated with the isotype hinge region of the antibody. Indeed, the replacement of single amino acids in the hinge region of IgG4 hinge can significantly reduce the occurrence of the second form (Angal et al. (1993) Molecular Immunology 30:105) to levels normally observed using IgG1 hinge person. The invention includes antibodies having one or more mutations in the hinge region, in the region of CH2 or CH3, which may be desirable, for example, in production, to improve the yield of the desired form antibodies.

Antibodies according to this invention preferably receive using Velocimmune technology™. Transgenic mouse in which the endogenous variable regions of the heavy and light chains of immunoglobulin are replaced by corresponding variable regions of a human, stimulate interest antigen and remove lymphatic cells (such as b-cells from these mice that Express antibodies. These lymph cells can be fused with a myeloma cell line to obtain immortalized hybridoma cell lines, and such hybridoma cell lines are screened and selected for Eden is eficacia hybridoma cell lines, which produce antibodies specific against interest antigen. DNA encoding a variable region of the heavy chain and light chain can be selected and associated with the desired izotopicheskii constant regions of the heavy chain and light chain. Such protein antibodies can be obtained in the cell, such as cell SNO. Alternatively, DNA encoding the antigen-specific chimeric antibody or the variable domains of the light and heavy chains, can be isolated directly from antigen-specific lymphocytes.

In one embodiment, this transgenic mouse contains up to 18 functional gene variable regions of the heavy chain of human and 12 functional gene variable regions of the light chain of the Kappa man. In another embodiment, this transgenic mouse contains up to 39 gene variable regions of the heavy chain of human and 30 genes for the variable regions of the light chain of the Kappa man. In another embodiment, this transgenic mouse contains up to 80 gene variable regions of the heavy chain of human and 40 genes for the variable regions of the light chain of the Kappa man.

Usually antibodies according to this invention have a very high affiniscape, usually with KDapproximately 10-9approximately 10-12M when measured by binding to the antigen Lieb is in the form immobilized on a solid phase, either solution phase antibody.

First, select the chimeric antibodies of high affinity, having a variable region of a human and a constant region of a mouse. As described below, these antibodies are characterized and selected for desirable characteristics, including binding affinity against hIL-6R, the ability to block hIL-6 and/or selectivity for protein man. The constant region of the mouse replaces the desired constant region of a person to generate fully human antibodies of this invention, for example, IgG4 or IgG1 wild-type or modified (for example, SEQ ID NO: 242, 243, 244). Even though the constant region may vary in accordance with the specific application, the characteristics of the binding of the antigen with high affinity and specificity against the target is localized in the variable regions.

Mapping of epitopes and related technologies

For screening for antibodies that bind to a specific epitope can be a routine cross-blocking analysis, such as analysis, described in Antibodies: A Laboratory Manual, 1988, Cold Spring Harbor Laboratory, Harlow and Lane, eds. Other ways include Leninskaya mutants, peptide blots (Reineke (2004) Methods Mol Biol 248:443-63) or analyses of peptide cleavage, as described in the examples below. In addition, can be used as the e ways as cut epitopes, extraction epitopes and chemical modification of antigens (Tomer (2000) Protein Science: 9: 487-496).

Analysis using modifications (MAP), also known as analysis of antibody structure-based antigen (ASAP)is a method that classifies a large number of monoclonal antibodies (mAbs)directed against the same antigen, in accordance with similarities profile of the binding of each antibody with surfaces chemically or enzymatically modified antigens (Publication of patent US 2004/0101920). Each category may reflect a unique epitope, or clearly different from the epitope presented another category or overlapping epitope, presents a different category. This technology makes possible quick filtering genetically identical antibodies, so that the characteristic can be focused on genetically different antibodies. When using hybrid screening MAR may facilitate identification of rare hybridoma clones with the desired characteristics. MAR can be used for sorting hIL-6R antibodies of this invention on a group of antibodies that bind different epitopes.

Agents applicable for changes in the structure of the immobilized antigen are enzymes, such as, for example, proteolytic enzymes, and chemical the agents. Antigenic protein can be immobilized either on the surfaces of the biosensor chip, or polystyrene granules. The latter can be processed using, for example, such analysis as multiplex analysis detection using Luminex™ (Luminex Corp., TX). Because the ability Luminex™ to perform multiplex analysis with as many as 100 different types of pellets, Luminex™ provides almost unlimited antigenic surface with various modifications, which leads to improved resolution analysis of the epitopes of antibodies in comparison with biosensor analysis.

Therapeutic introduction and final form

The introduction of therapeutic substances in accordance with this invention will be carried out with suitable carriers, excipients, and other agents, which include in the finished shape to provide improved transfer, delivery, tolerance, etc. Many suitable ready-made forms can be found in pharmaceutical formulary known to all pharmaceutical chemists: Remington''s Pharmaceutical science (15th ed, Mack Publishing Company, Easton, Pa., 1975), in particular, part 87, written Blaug, Seymour, in this Handbook. These forms include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)containing vesicles (such as Lipofectin™), DNA-conjug is you, anhydrous absorption pastes, emulsions of the type oil-in-water and water-in-oil emulsion of carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. Any of the previous mixes may be appropriate in treatments and therapies in accordance with this invention, provided that the active ingredient in this final form is not inactivated in this ready-made form, and this finished form is physiologically compatible and tolerable for the particular route of administration. Cm. also Powell et al. PDA (1998) J Pharm Sci Technol. 52:238-311 and references cited in this work, for further information related to the excipients and carriers are well known to pharmaceutical chemists.

EXAMPLES

The following examples are offered to meet specialists of ordinary skill in this field a complete disclosure and description of how to make and use the methods and compositions of the present invention, and are not intended to limit the scope of the material considered by the authors as their invention. Attempts have been made to ensure accuracy in terms of numbers (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. If not indicated otherwise, parts are mass h is s, molecular weight is average molecular weight, temperature is the temperature in degrees centigrade Celsius and the pressure is atmospheric or nearly atmospheric pressure.

Example 1. Generation of human antibodies to IL-6-receptor human.

Immunization of rodents can be performed by any means known in the art (see, for example, Harlow and Lane (1988) supra; Malik and Lillehoj, Antibody techniques: Academic Press, 1994, CA). In one preferred embodiment, the antigen hIL-6R injected directly mice that contain loci DNA encoding as the variable region of the heavy chain Ig, and the variable region of light chain Kappa person (Velocimmune™, Regeneron Pharmaceuticals, Inc.; US 6596541), with an adjuvant to stimulate the immune response. Such adjuvant includes complete and incomplete beta-blockers, system adjuvants MPL+TDM (Sigma) or RIBI (muramyldipeptide) (see O Hagan, Vaccine Adjuvant, by Human Press, 2000, NJ). This adjuvant can prevent the rapid dissipation of polypeptides sequestration of antigen in the local depot and may contain factors that can stimulate an immune response of the host. In one embodiment, hIL-6R injected directly in the form of a DNA plasmid that contains the gene hIL-6R and expresses hIL-6R using the apparatus of the expression of host cellular proteins with the formation of the antigen for which peptide in vivo. In both approaches, the immunization scheme requires multiple injections at intervals of the order of several weeks. The immune response antibodies are monitored using standard antigen-specific immunoassay. Upon reaching their animals maximum immune response by expressing the antibody In cells collected and merged with mouse myeloma cells to maintain their viability, obtaining hybridoma cells. For selection of the desired functional monoclonal antibodies air-conditioned environment of these hybridoma cells or transfected cells were subjected to screening for specificity, antigennegative the affinity and efficacy of blocking the binding of hIL-6 hIL-6R (as described below).

Example 2. Anti-hIL-6R antibody, generated through the direct allocation of splenocytes

DNA encoding the domains VH and VL can be selected directly from the positive single antigen b cells. Briefly, immunized hIL-6Rα transgenic mouse autonational and collected splenocytes. Erythrocytes were removed by lysis with subsequent deposition of the collected splenocytes. Resuspendable splenocytes are first incubated with a cocktail (mixture) human IgG, FITC-anti-mFc and Biotin-IL6Ra for 1 hour. Stained cells were washed twice SFR, then stained with a cocktail IG human and mouse, APC-anti-mIgM and SA-PE for one hour. These stained cells were washed once SFR and were analyzed by flow cytometry on a MoFlo (Cytomation). Each IgG positive, IgM-negative and antigen-positive B-cells was subjected to sorting and were sown in individual wells of 96-hole tablet. RT-PCR of antibody genes from these cells was performed according to the method described by Wang et al. (2000) J Immunol Methods 244:217-225). Briefly, cDNA for each individual b-cells synthesized using RT-PCR. Then, each obtained FROM the product tsalala and transferred in two corresponding holes on two 96-well plates. One set received FROM products initially amplified using PCR using the 5'degenerate primer specific in relation to the leader sequence of the variable region of the heavy chain of IgG, and the 3'primer-specific in relation to the constant region of the heavy chain of the mouse, for the formation of the amplicon. Then this amplicon amplified again by means of PCR using a set of 5'-degenerate primers specific in relation to the frame 1 variable regions of the heavy chain of human IgG, and built the 3'primer-specific in relation to the constant region of the heavy chain of the mouse. Another set obtained FROM the products initially amplified using PCR using 5'-degenerate is Reimer, specific in relation to the leader sequence of the variable region of the light chain Kappa man, and the 3'primer-specific in relation to the constant region of the light chain Kappa mouse, with the formation of the amplicon. Then this amplicon amplified again by means of PCR using a set of 5'-degenerate primers specific in relation to the frame 1 variable region light chain Kappa man, and built the 3'primer-specific in relation to the constant region of the light chain Kappa mouse. These PCR products of the heavy chain and light chain were cloned in Sap I-linearized vectors of antibodies containing a constant region IgGI heavy chain and the variable region of light chain Kappa, respectively. Plasmid heavy chain has a website lox2272 and lox511 site flanking the expression cassette of the heavy chain. In addition, directly to the right of lox2272 in this plasmid the heavy chain has the gene for resistance to hygromycin who is devoid of promoter and initiating ATG. The gene of resistance to hygromycin also associated transcription with the right gene eGFP via an IRES sequence. Plasmid light chain has a website loxP and lox2272 site flanking the expression cassette light chain. In addition, this plasmid light chain has the SV40 promoter directly in front of the ATG in lox2272 site, so that after integration into a suitable the tapped-host this lox2272-proximal promoter of SV40 and the initiating ATG of the light chain plasmids become contiguous with the gene of resistance to hygromycin plasmid heavy chain in the correct reading frame, allowing transcription and translation of genes of resistance to hygromycin and eGFP. Then purified recombinant plasmid having the sequence of the variable region of the heavy chain, and the plasmid having the sequence of the variable region of the light chain from the same b cells were combined and were transicional, together with a plasmid that expresses the Cre recombinases, in a modified cell line host CHO. This modified cell line host CHO contains, from 5' to 3'loxP site, eCFP, lox2272 site, DsRed and lox511 site in transcriptionally active locus. Then this a host cell CHO can be selected flow cytometry in the form of positive blue staining, positive red staining and negative in relation to the green staining of the cells. When transfection of recombinant plasmids expressing the genes for the heavy chain and light chain, together with a plasmid expressing the Cre recombinases, site-specific recombination-mediated recombinase Cre leads to the integration of these plasmids antibodies in the chromosomal locus containing the lox sites, and replacement of genes eCFP and DsRed. Then the recombinants can be selected as a negative against blue staining, negative in relation to red staining and positive green staining of cells flowing qi is ometries. Thus, cells SNO, transfetsirovannyh recombinant plasmid having the sequence of the variable region of the heavy chain, and the plasmid having the sequence of the variable region of the light chain from the same cells were subjected to sorting flow cytometry and correct recombinants, which find a negative against blue staining, negative in relation to red staining and positive green staining phenotype were isolated and established stable expressing recombinant antibody cell line Cho of the selected clones.

Example 3. Definition antigennegative affinity

KDbinding of the antigen with selected antibodies, described above, was determined by analysis of surface kinetics on the biosensor real-time surface plasmon resonance (BIAcore™). More specifically, the affinity of these antibodies against IL-6R person was measured using a BIAcore® 2000 or BIAcore® 3000. This antibody was caught on the surface of the anti-mouse IgG-antibodies and subjected to the action of different concentrations of recombinant protein hIL-6R in Monomeric or dimeric form. Performed kinetic analysis using the software BIAevaluation™ to obtain the rate constants of Association and dissociation.

The affinity of the binding is of these antibodies with hIL-6R also measured either against hybridoma-air-conditioned environments or in respect of purified proteins competitive immunoassay-based tablets. Proteins antibody was purified using Protein G affinity chromatography from the conditioning hybridoma cell environment, which was depleted in relation to bovine IgG (Invitrogen). For competitive ELISA, briefly, a constant amount of antibodies at different levels of pre-mixed with serial dilutions of the antigenic protein, hIL-6R-hFc, in the range of 0-10 μg/ml and incubated for two hours at room temperature to reach equilibrium pseudocatalase between this antibody and the antigen. Then these solutions was transferred into a 96-well pre-coated hIL-6R-hFc tablets to allow the free antibody in these mixtures to contact deposited on the tablet hIL-6R-hFc. These tablets generally covered 1-2 µg/ml protein hIL-6R-hFc in solution SFR over night at 4°C with subsequent nonspecific BSA-blocking. After removing excess antibody in the solution associated with the tablet antibodies were detected by reagent HRP-conjugated goat polyclonal antibody against mouse IgG or IgA, and showed using either colorimetric or chemiluminescent substrates. The dependence of the signals from the concentration of antigen in solution was analyzed using a 4-parametric analysis of graphic represent the tion using the software Prism™ software (Graph Pad) and reported as IC 50. Conducted competitive immunoassay with the use of device Kinexa™ solution phase as the stationary phase (Sapidyne Inc.).

The results are shown in table 1 (control: humanitariannet monoclonal antibody to IL-6R person (Patent US 5817790 SEQ ID NO: 69 and 71). Antibody (amino acid sequence HCVR and LCVR): VQ8A9-6 (3, 11); VQ8F11-21 (19, 27); VV7G4-1 (35, 43); VV7G4-10 (51, 59) VV6C10-1 (67, 75); VV6C10-3 (83, 91); VV6C10-4 (99, 107); VV6F12-11 (115, 123); VVA6-11 (131, 139); VV6A9-5 (147, 155), VV3D8-4 (163, 171); VV1G4-7 (179, 187); 248982-13-1-E5 (195, 203); 248982-13-2-A9 (211, 219). KDmonomer and dimer were determined using a BIAcore™; KDin solution using Kinexa™; IC50using ELISA assays (n.d.=not defined).

Table 1
Antigennegative affinity
AntibodyKDThe monomer (nm)KDDimer (nm)KDin solution, the Monomer (nm)IC50ELISA, Dimer (nm)
VQ8A9-60,2220,1010,1200,004
VQ8F11-210,0670,023 0,0090,008
VV3D8-42,4100,1721,9100,013
VV6A9-50,0970,146to 0.0320,005
VV1G4-70,2250,0700,1970,041
VV6C10-10,267to 0.0322,0500,010
VV6F12-11n.d.n.d.n.d.0,033
VV7G4-10n.d.n.d.n.d.1,980
VV9A6-11n.d.n.d.n.d.0,347
VV6C10-3n.d.n.d.n.d.0,009
248982-13-1-E50,9870,785n.d.0,360
248982-13-2-A92,870n.d.n.d.0,054
Control1,790n.d.1,960n.d.

Example 4. Neutralization activity of hIL-6

Blocking hIL-6 activity anti-hIL-6R antibodies of this invention were subjected to screening using immunoassays block hIL-6, in vitro bioassays hIL-6-dependent growth of cells and surface plasmon resonance (BIAcore™). The immunoassay was used for screening the ability of test antibodies to block the binding of hIL-6 hIL-6R, and the in vitro bioanalysis used to determine the effectiveness of these antibodies to neutralize hIL-6R-mediated transmission of cellular signals.

For immunoassay recombinant protein hIL-6 was applied to a 96-well plate in SFR-buffer over night at 4°C. This tablet is used to capture the free hIL-6R-hFc from solutions of samples of antibodies and the number of captured hIL-6R-hFc was determined in accordance with the standard curve. Solutions of the samples consisted of postoyanno the amount of recombinant protein hIL-6R-hFc (100 PM) and varying amounts of antibodies, either untreated hybridoma-air-conditioned environment, either in the form of purified protein antibodies, in the range 0 to approximately 50 nm in the form of serial dilutions. This mixture of antibody-antigen were incubated at room temperature for ~2 hours to allow binding of the antibody-antigen to reach equilibrium. Then these steady solutions of the samples was transferred into a covered hIL-6 tablets for the measurement of free hIL-6R-hFc. After 1-hour linking the tablet was washed and bound hIL-6R-hFc were detected using HRP-conjugated goat polyclonal anti-hFc antibody (Jackson lmmuno Research) and showed using TMB substrate (BD Pharmigen). IC50defined as the amount of antibody required to reduce by 50% hIL-6R-hFc, informative in regard to tablet ligand hIL-6. The results are shown in the first column of table 2.

Additionally, the ability of a test antibody to block the binding of hIL-6 receptor hIL-6R was determined using surface plasmon resonance. Molecules purified antigen hIL-6R-hFc was captured goat polyclonal antibody against human IgG immobilized on the surface of a CM-5 through aminosilane, to a density of 250 GB. A solution of hIL-6 (0.25 ml, 50 nm) was injectively on the surface of the receptor and associated hIL-6 was detected (the first injection of IL-6). Then bound hIL-6 is alali pulse 3 M MgCl 2with the subsequent addition of the conditioning buffer. Anti-hIL-6R-antibody in hybridoma-air-conditioned environment was injectively captured on the surface of the receptor, followed by the second injection of hIL-6. Percentage reduction in the binding of hIL-6 originating from a pre-formed complex of the antibody-receptor, was used as an evaluation to determine hIL-6-blockers among newlocation (second column, table 2).

Table 2
Neutralization of binding of hIL-6
AntibodyInhibition of binding of hIL-6R/hIL-6, IC50(nm)Inhibition IC50(nM)Inhibition of binding of hIL-6R/hIL-6, (%)Inhibition of cell proliferation XG-1, IC50(nm)The luciferase activity HepG2/Stat3, IC50(nm)
VQ8A9-60,39680,400,097
VQ8F11-210,12980,62is 0.135
W3D8-4 0,6193>100n.d.
W6A9-50,351001,100,188
W1G4-71,10341,800,578
W6C10-14,6061>6,90n.d.
W6F12-112,20n.d.n.d.n.d.
W7G4-1013,00n.d.n.d.n.d.
W9A6-110,50n.d.n.d.n.d.
W6C10-30,06n.d.n.d.n.d.
Control2,20911,5 0,854

The ability of hIL-6R antibodies to block the activity of hIL-6 in vitro was measured in hIL-6-dependent myeloma line XG-1. Cells XG-1, supported in hIL-6-containing medium, washed twice, not containing hIL-6 medium and were cultured for ~24 hours in not containing hIL-6 environment for the depletion of the remaining hIL-6. Then starving the cells were besieged by centrifugation and resuspendable in medium at 4×105cells per ml and were sown 20,000 cells per well in 96-well tablet for tissue culture. Purified proteins antibodies were serially diluted in medium and added to the seeded cells at concentrations in the range from 0 to 50 nm. Then the wells were added recombinant hIL-6 to a final concentration of 8 PM. The cells were allowed to grow for ~72 hours at 37°C in humidified 5% CO2thermostat. At the end of the period growth of living cells was measured using a set of CCK-8 (Dojindo, Japan). IC50was determined as described above and are listed in the third column of table 2.

The ability of antibodies hIL-6R to block the activity of hIL-6 was measured in vitro in hIL-6-sensitive cell line human hepatoma, HepG2. The HepG2 cells were transfusional reporter plasmid containing sensitive to STAT3 (carrier signal and activator of transcription 3) the element associated with the gene luciferase. Transfetsirovannyh cells were trypsinization, adrucil is whether and resuspendable in the medium at approximately 2.5×10 5cells per ml and were sown 20,000 cells per well in 96-well tablet for tissue culture. Purified proteins antibodies were serially diluted in medium and added to the seeded cells at concentrations in the range from 0 to 100 nm. Then the wells were added recombinant hIL-6 to a final concentration of 50 PM. The reaction was measured after incubation of these cells for 6 hours at 37°C in humidified 5% CO2thermostat. Luciferase activity was measured by the system for analysis of luciferase Steady-Glo™ (Promega). IC50was determined as described above and are listed in the fourth column of table 2.

Example 5. A variety of epitopes binding

Competitive immunoassay of antibody binding was performed using as a control gumanitarnogo antibodies to IL-6R person. Briefly, 96-well ELISA (solid) plate was covered with 20 ng per well of recombinant protein hIL-6R over night at 4°C. After blocking of nonspecific binding of BSA, the binding sites hIL-6R on one half of the tablet saturated binding of the control antibody by adding 500 ng of control on the hole, and the other half of the tablet was added only buffer for binding. After three hours of binding at room temperature purified antibodies were added at a final concentration of 50 ng/ml with preexisting control is the major antibody in the hole or without preexisting control in the hole. After one hour of additional binding free antibody was washed and associated with the tablet antibody was detected with HRP-conjugated polyclonal goat antibody against mouse IgG or IgA and the tablet showed using chromatic HRP substrates and record the optical density at 450 nm. Derived cent of the binding of anti-hIL-6R antibodies in the presence of the control antibody is shown in the list in table 3 below. Conducted a similar experiment using the technology of surface plasmon resonance (table 3). Both methods gave consistent results. Antibodies VQ8F11, VV3D8, VV6A9, VV6C10-1 was associated epitopes, coincides with a control antibody; whereas antibodies VQ8A9, VV1G4, VV6F12, VV7G4, VV9A6 and VV6C10-3, apparently, are associated with different epitopes, as binding to the antigen is not blocked by this control antigen. Partial competition may result from steric obstacles from the side of the first related antibodies, even in the case where the epitopes may not be coincident.

Table 3
Competition for binding to the antigen with a control antibody
AntibodyBIAcore™ (%decrease the) Immunoassay (%decrease)
VQ8A9-6263
VQ8F11-219679
W3D8-49784
W6A9-59684
W1G4-7123
W6C10-19080
W6F12-11n.d.3
W7G4-10n.d.26
W9A6-11n.d.18
W6C10-3n.d.1

Example 6. Cross-binding ability between species

Four antibodies were tested for cross reactivity with recombinant protein hIL-6R monkeys using technology BIAcore™. In short, the biosensor chip, which was immobilized goat polyclonal anti-mouse Fc-anti the ate, used for the presentation of monoclonal anti-hIL-6R antibodies to a density of approximately 75 EN. On the surface this antibody was injectively recombinant Monomeric protein IL-6R (extracellular domain, SEQ ID NO: 251, Macaca fasciculahs) in the concentration range of 1.25-40 nm. The binding of this receptor with this antibody and the dissociation of the bound complex was subjected to real-time monitoring. Received as the rate constant of Association (ka)and the rate constant of dissociation (kd) and expected KD(table 4).

Table 4
Comparison of the affinity of binding to IL-6R humans and monkeys
AntibodyAntigenka (M-1with-1)kd (-1)KD(nm)
ControlIL6R man1,74E+051,67E-040,963
IL6R monkeys1,44E+051,68E-041,170
VQ8F11-21IL6R people the century 8,51 E+054,38E-050,051
IL6R monkeys3,39E+054,86E-050,143
VV1G4-7IL6R man2,57E+056,18E-050,240
IL6R monkeysno binding
VV6A9-5IL6R man5,18E+05to 8.41 E-05rate £ 0.162
IL6R monkeys5,00E+057,70E-050,154
VQ8A9-6IL6R man7,32E+052,76E-040,377
IL6R monkeys7,31E+054,16E-040,569

Among the four tested anti the ate, VQ8F11, VV6A9 and VQ8A9 strongly reacted with the receptor monkeys with values of KDwhich differ approximately 1.5 to approximately 3 times greater binding with the receptor, respectively. VV1G4, which is not blocked by the control antibody (table 3), did not bind to the receptor monkeys, despite the strong binding with the receptor of a person with KD241 PM.

Example 7. The action of the constant region on the binding affinity of

The affinity of binding to Monomeric hIL-6R four antibodies with mouse IgG, IgG1 or IgG4 human (wild-type or modified), was determined using BIAcore™, as described above, except when used surface goat polyclonal anti-Fc-human antibodies for capture hIgG antibodies. Monomeric hIL-6R was injectively at concentrations of 12.5, 6,25, 3,12 and 1.56 nm. The ability of these antibodies to neutralize hIL-6-dependent signal transduction HepG2/STAT3 was also identified in the luciferase assay (IC50). IC50for different IgG isotypes were similar, which suggests a lack of action isotype on the affinity of antibodies against the antigen.

Table 5
Comparison of IgG isotypes
Antibody IgGka (M-1with-1)kd (-1)KD(nm)-IC50(nm)
hlgG16,22 E+054,54E-050,0730,150
hlgG47,17E+055,22E-050,0730,228
VQ8F11-21mlgG2a7,86E+055,27E-050,067is 0.135
modhlgG48,81E+054,705-050,0530,249
hlgG11,09E+062,60E-040,2380,130
VQ8A9-6hlgG41 17E+062,35E-040,2010,185
mlgG19,95E+052,21E-040,2220,097
hlgG17,12E+058,87E-050,1250,204
VV6A9-5hlgG45,67E+057,64X-05is 0.1350,343
mlgG2a7,72E+057,52E-050,0970,188
hlgG13,34E+057,92E-050,2370,767
VQ1G4-21hlgG42,73E+059,18E-050,3360,528
mlgG2a3,41E+057,66E-050,225 0,578

1. The antibody or antigennegative fragment that specifically bind the receptor of interleukin-6 human (hIL-6R), where the indicated antibody or antigennegative fragment selected from the group consisting of:
(a) antibodies or antigennegative fragment containing the domain CDR1 of the heavy chain, containing SEQ ID NO:21, CDR2 domain of the heavy chain, containing SEQ ID NO:23, domain CDR3 of the heavy chain, containing SEQ ID NO:25, and the domain of the light chain CDR1, containing SEQ ID NO:29, a CDR2 domain light chain containing SEQ ID NO:31, the domain CDR3 light chain containing SEQ ID NO:33;
(b) antibodies or antigennegative fragment containing the domain CDR1 of the heavy chain, containing SEQ ID NO:149, CDR2 domain of the heavy chain, containing SEQ ID NO:151, the domain CDR3 of the heavy chain, containing SEQ ID NO:153, and the domain of the light chain CDR1, containing SEQ ID NO:157, domain CDR2 light chain containing SEQ ID NO:159, domain CDR3 light chain containing SEQ ID NO:161;
(c) antibodies or antigennegative fragment containing the domain CDR1 of the heavy chain, containing SEQ ID NO:5, domain CDR2 of the heavy chain, containing SEQ ID NO:7, domain CDR3 of the heavy chain, containing SEQ ID NO:9, and the domain of the light chain CDR1, containing SEQ ID NO:13, a CDR2 domain light chain containing SEQ ID NO:15, domain CDR3 light chain containing SEQ ID NO:17, and
(d) antibodies or antigennegative fragment containing the domain CDR1 of the heavy chain, containing SEQ ID NO:181, a CDR2 domain of the heavy chain, the content is the seer SEQ ID NO:183, domain CDR3 of the heavy chain, containing SEQ ID NO:185, domain and light chain CDR1, containing SEQ ID NO:189, domain CDR2 light chain containing SEQ ID NO:191, domain CDR3 light chain containing SEQ ID NO:193.

2. The antibody or antigennegative fragment according to claim 1, containing the variable region of the heavy chain having SEQ ID NO:19, and a variable region light chain having SEQ ID NO:27.

3. The antibody or antigennegative fragment according to claim 1, containing the variable region of the heavy chain having SEQ ID NO:147, and a variable region light chain having SEQ ID NO:155.

4. The antibody or antigennegative fragment according to claim 1, containing the variable region of the heavy chain having SEQ ID NO:3, and the variable region light chain having SEQ ID NO: 11.

5. The antibody or antigennegative fragment according to claim 1, containing the variable region of the heavy chain having SEQ ID NO:179, and a variable region light chain having SEQ ID NO:187.

6. The selected nucleic acid molecule encoding the antibody or antigennegative fragment according to any one of the preceding paragraphs.

7. Vector for ekspressirovali antibodies or antigennegative fragment that specifically bind IL-6R person containing the nucleic acid molecule of claim 6.

8. System the host-vector to obtain antibodies or antigennegative fragment that specifically bind IL-6R person, containing ve the tor according to claim 7 in cage-master, selected from the E. coli cells and cells SNO.

9. A method of obtaining antibodies to IL-6R or antigennegative fragment comprising growing cells of the host-vector of claim 8 and removing the antibody or antibody fragment.

10. The use of antibodies or antigennegative fragment according to any one of claims 1 to 5 in getting medicines for use in the reduction or inhibition of IL-6-mediated diseases or disorders in humans.

11. The method according to claim 10, where IL-6-mediated disease or disorder selected from the group consisting of rheumatoid arthritis, inflammatory bowel disease and systemic lupus erythematosus.



 

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8 cl, 22 dwg, 8 tbl, 9 ex

FIELD: medicine.

SUBSTANCE: substance of the invention involves a humanised human osteopontin antibody containing a variable region of a heavy chain consisting of the amino acid sequence SEQ ID NO:1 and a variable region of a light chain consisting of the amino acid sequence SEQ ID NO:3. Furthermore, the invention involves a polynucleotide containing a sequence coding the variable region of the respective light and heavy chains of the humanised antibody, an expression vector containing polynucleotide, a host cell, a medicine, a method of producing the humanised antibody, a medicine for treating an autoimmune disease, a method of treating, and application of the humanised antibody for producing a pharmaceutical agent.

EFFECT: advantage of the invention consists in creation of the humanised antibody exhibiting improved activity or stability, than activity and stability of standard human osteopontin antibodies.

13 cl, 14 ex, 1 tbl, 16 dwg

FIELD: medicine.

SUBSTANCE: there are offered versions of antibodies and their antigen-binding IL-13, particularly human IL-13 specific fragments. There are described: a pharmaceutical composition, a pharmaceutical compound of the antibody, versions of coding and hybridising nucleic acids and expression vectors. There are offered versions of: cells and methods of producing the antibody, methods of treating IL-13 associated disorders. A method of IL-13 detection in a sample is described.

EFFECT: use of the invention provides new IL-13 antibodies with KD about 10-10 M which can be used for diagnosing, preventing or treating one or more IL-13 associated diseases.

87 cl, 37 dwg, 5 tbl, 6 ex

Glypican-3 antibody // 2427588

FIELD: medicine.

SUBSTANCE: versions of antibodies bound with glypican-3 in a site with amino acid residues 1-563 are offered. Each version is characterised by the fact that it contains three CDRs of a light chain and three CDRs of a heavy chain. There are described: coding polynucleotide, and also a based expression and a host cell on the basis of the vector. There are disclosed: a method of producing the antibody with using a host cell, a cell growth inhibitor on the basis of the antibody, versions of application of the antibody for treating cancer or hepatoma. There is described peptide for producing glypican-3 antibodies containing residues 546-551 of glypican-3. The offered new antibodies exhibit higher cytotoxicity as compared with known glypican-3 antibodies and are specific to a certain site of glypican-3.

EFFECT: invention use can find further application in cancer therapy.

16 cl, 20 dwg, 2 tbl, 27 ex

FIELD: medicine.

SUBSTANCE: what is offered is a human OX40L antibody containing a light chain and a heavy chain each of which contains respectively three CDRs of the light chain and three CDRs of the heavy chain. There are described: a coding polynucleotide, and also an expression vector and a host cell including coding polynucleotide. There are disclosed: a method of producing and a method of treating with using the antibody.

EFFECT: use of the invention can find further application in therapy of the OX40L mediated immune disorders.

28 cl, 8 dwg, 1 tbl, 7 ex

FIELD: medicine.

SUBSTANCE: versions of the CD38 specific antibodies and their functional fragments are offered. Each version is characterised by the fact that it contains three CDRs of a light chain and three CDRs of a heavy chain. There are described: a coding polynucleotide, and also an expression vector and a host cell including coding polynucleotide. There are disclosed: a pharmaceutical and diagnostic compositions, a method of treating, a method of detecting CD38 in erythrocyte, a method of inducing specific CD38 expressing tumour cell killing with using the antibody. The offered new antibodies exhibit the unexpected properties: to bind minipig's CD38 and to cause cross-linked specific CD38 expressing cell killing.

EFFECT: use of the invention can find further application in therapy of the CD38 mediated disorders.

87 cl, 37 dwg, 4 tbl, 6 ex

Il2 antibodies // 2425054

FIELD: medicine, pharmaceutics.

SUBSTANCE: humanised monoclonal antibody and its active fragment under the invention neutralises human IL2 activity by binding with said human IL2 prior to, during and/or after binding of said human IL2 with human IL2 receptor. A variable light chain region of said antibody contains an adherent amino acid KAPKA sequence in its second frame region, and in addition, in the CDR1-CDR3 regions, contains the amino acid sequences presented in SEQ ID NO 1-3 disclosed in the description, while a variable heavy chain region contains in the CDR1 - CDR3 regions, amino acid sequences presented in SEQ ID NO 4-6 disclosed in the description. The invention describes a polynecleotide molecule coding the antibody under the invention or its active fragment, a pharmaceutical composition based on said antibody or its active fragment exhibiting human IL2 neutralising action, and also an application of said antibody or its active fragment or the polynecleotide molecule coding it for preparing a drug which optionally contains an auxiliary anti-inflammatory or anticancer agent for treating inflammatory diseases or tumours, respectively.

EFFECT: production of the alternative specific IL2 activity inhibitors which directly bind with human IL2.

24 cl, 18 dwg, 4 tbl, 9 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and immunology. Versions of a human CD37 specific antibody are presented, each containing a variable site of a light and heavy chain. A coding nucleic acid and a based expression vector are described. There are disclosed: a host cell containing a vector, a method of producing the antibody with using the cell, and also a composition for decreasing the B-cell count and a method of treating diseases associated with aberrant B-cell activity, with using the CD37-specific antibodies.

EFFECT: use of the invention provides specific loss of 80% BJAB cells at the antibody concentration 10 mcg/ml, and also increases the survival rate of Daudi mice as compared with Rhithuximab therapy that can find application for treating various tumours.

39 cl, 39 dwg, 7 tbl, 18 ex

FIELD: medicine.

SUBSTANCE: offered are versions of antibodies each of which is specifically bound with IGF-IR, inhibits its activity and is its antagonist, not exhibiting substantially IGF-IR agonist activity. Each of the antibodies is characterised at least by the presence of a variable area of a heavy and easy chain. There are described: antibody conjugates with cytotoxic agents, and also versions of a pharmaceutical composition for cancer diagnosing and therapy, methods of cancer treatment and diagnosing, a cancer diagnosing reagent - on the basis of the antibodies. There are disclosed: a method of producing the antibodies; nucleic acid (NA) coding the antibody; an expression vector containing NA. Offered is a hybridoma EM 164 producing the antibody under the invention, deposited in ATCC, No. PTA-4457, and also the use of the antibody for IGF-IR linkage. The use of the invention presents the antibodies which allow inhibiting MCF cell growth approximately in 12 times that is higher approximately in 5 times than hen using the antibody IR3, and can be used for cancer diagnosing and treatment expressing higher levels of receptor IGF-I, such as breast cancer, colon cancer, lung cancer, prostate cancer, ovarian cancer, synovial carcinoma and pancreatic cancer.

EFFECT: more efficient diagnosing and treatment of said cancers.

58 cl, 28 dwg, 10 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: by means of expression vector into plant cell introduced are nucleotide sequences, coding light and heavy chains of antibody, binding human VEGF. Antibody against human VEGF can be used, in particular, for reduction of microvascular permeability of human tumours and treatment of diabetic and age-related neovascular retinopathy.

EFFECT: antibody production in plant cells provides possibility of its obtaining in industrial scale, at a significantly lower cost than in obtaining in expression system on mammalian cell culture base, presence in obtained preparation of antibody of causative agents of prion, mycoplasmal and viral diseases of mammals is excluded.

39 cl, 11 dwg, 2 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology, and represents a polyclonal Nogo antibody. Also, there are presented a pharmaceutical composition and application of the antibody for preparing a drug.

EFFECT: invention can effectively used for treating central nervous system disorders.

6 cl, 79 dwg, 2 tbl

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