High-affinity human il-4 receptor antibodies

FIELD: medicine.

SUBSTANCE: what is offered is an antibody or its antigen-binding fragment which specifically coupling hlL-4R with KD less than 200-pM measured with using surface plasmon resonance. What is described is a recovered nucleic acid molecule coding the antibody, and a based vector for producing the antibody. There are disclosed a host-vector system for producing the antibody or its antigen-binding fragment, and a method for producing the substances stated above with using such system. What is disclosed is using the antibody or antigen-binding fragment for preparing a drug for relieving (inhibiting) hlL-4R mediated diseases. What is disclosed is a composition on the basis of the antibody or antigen-binding fragment to be used in a method for treating a hlL-4R mediated disease or disorder in humans.

EFFECT: inventions can find application in therapy of the hlL-4R mediated diseases.

15 cl, 3 dwg, 5 tbl, 6 ex

 

The LEVEL of TECHNOLOGY

Interleukin-4 (IL-4, also known as a factor that stimulates B-cells, or BSF-1) was initially characterized with respect to its ability to stimulate the proliferation of B-cells in response to low concentrations of antibodies directed to surface immunoglobulin. It is shown that IL-4 has a wide range of biological activities, including stimulation of growth of T-cells, mast cells, granulocytes, megakaryocytes and erythrocytes. IL-4 induces the expression of molecules of the major histocompatibility complex class II in resting B-cells and increases the secretion of IgE isotypes IgG1 and stimulated B-cells.

Biological activity of IL-4 mediated by specific receptors of IL-4 on the cell surface. The IL-4 receptor alpha human (hIL-4R) (SEQ ID NO: 1) is described, for example, in U.S. patent No. 5599905, 5767065 and 5840869. Antibodies to hIL-4R described in U.S. patent No. 5717072.

Methods for producing antibodies suitable as therapeutic agents for humans, include the creation of chimeric antibodies and humanized antibodies (see, for example, US 6949245). See, e.g., WO 94/02602 (Abgenix) and US 6596541 (Regeneron Pharmaceuticals) (both publications are included in this description by reference), describes methods of obtaining transgenic mice capable of producing human antibodies.

The methods of using antibodies to hIL-4R described in U.S. patent No. 574146, 5985280 and 6716587.

The INVENTION

In the first aspect of the invention relates to human antibodies, preferably recombinant human antibodies human-specific bind the receptor for interleukin-4 (hIL-4R). Human antibodies are binding to hIL-4R with high affinity and ability to neutralize the activity of hIL-4. In specific embodiments, human antibodies are capable of blocking the binding of the complex hIL-13/hIL-13R1 c hIL-4R and thus to inhibit the signal transmission by means of hIL-13. Antibodies can be full-sized (for example, the antibody is IgG1 or IgG4), or may contain only antigennegative part (e.g., Fab fragment, F(ab')2or scFv) and can be modified to affect functionality, for example to eliminate residual effector functions (Reddy et al. (2000) J. Immunol. 164: 1925-1933).

In one embodiment, the invention relates to an antibody or antigennegative fragment which is specific binds hIL-4R (SEQ ID NO: 1) with KDapproximately 200 PM or less, which is measured using surface plasmon resonance. In a more specific embodiment, the antibody or antigennegative part have KDless than about 150 PM, or less than about 50 PM, or less than about 20 PM. In various embodiments, the antibody or antigennegative fragment b is ciruit activity of hIL-4 with IC 50about 200 PM or less, as measured in bioanalysis luciferase STAT6. In more specific embodiments, the antibody or antigennegative fragment have the IC50approximately 150 PM or less, or about 100 PM or less, or even about 50 PM or less, as measured in bioanalysis luciferase. In various embodiments, the antibody or antigennegative fragment blocks the activity of hIL-13 with IC50approximately 100 PM or less, as measured in bioanalysis luciferase STAT6. In more specific embodiments, the antibody or antigennegative fragment have the IC50approximately 75 PM or less, or about 50 PM or less, or even about 20 PM or less.

In the second aspect, the antibody according to the invention contains a variable region heavy chain (HCVR)selected from the group consisting of sequences

or essentially similar sequence.

In the third aspect, the antibody according to the invention contains a variable region light chain (LCVR)selected from the group consisting of sequences

or essentially similar sequence.

In one embodiment, the antibody or antibody fragment according to the invention contains HCVR and LCVR (HCVR/LCVR)selected from the group consisting of sequences

In a preferred embodiment, the antibody or antibody fragment contain a HCVR/LCVR selected from the sequences SEQ ID NO: 51/59, 259/267, 275/283, 291/299, 579/59 or 581/59.

In the fourth aspect of the invention relates to nucleic acid molecules coding for HCVR, the nucleic acid molecule is a nucleotide sequence selected from the group consisting of sequences

or substantially identical sequence having with them, at least 95% homology.

In the fifth aspect of the invention relates to nucleic acid molecules coding for LCVR, the nucleic acid molecule is a sequence selected from the group consisting of sequences

or substantially identical sequence having with them, at least 95% homology.

In one embodiment, the antibody according to the invention contains a HCVR and a LCVR encoded by a pair of nucleotide sequences selected from the group consisting of sequences

In a preferred embodiment, the antibody or antibody fragment contain a HCVR/LCVR encoded by a nucleotide sequence selected from the sequences SEQ ID NO: 50/58, 258/266, 274/282, 290/298, 578/58 or 80/58.

In the sixth aspect, the hallmark of the invention is an antibody or antigennegative fragment antibodies that are specific bind hIL-4R comprising three complementarity determining region heavy chain and three complementarity determining region light chain (CDR), where

CDR1 of the heavy chain contains the amino acid sequence of the formula X1-X2-X3-X4-X5-X6-X7-X8(SEQ ID NO: 582), where X1= Gly; X2= Tyr or Phe; X3= Thr or I; X4= Phe; X5= Asn or Arg; X6= Ser; X7= Tyr and X8= Gly;

CDR2 of the heavy chain contains the amino acid sequence of the formula X1-X2-X3-X4-X5-X6-X7-X8(SEQ ID NO: 583), where X1= Ile, X2= Ser or Arg, X3= Thr or Tyr, X4= Tyr or Asp, X5= Asn or Gly, X6= Gly or Ser, X7= Lys or Asn and X8= Thr;

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-X20-X21(SEQ ID NO: 584), where X1= Ala or Val, X2= Arg or Lys, X3= Asp or Glu, X4= Gly or Glu, X5= Ala or Arg, X6= Arg or Ser, X7= Ile or Gly, X8= Val or Ser, X9= Val or Trp, X10 = Ala or Phe, X11= Gly or Asp, X12= Thr or Pro, X13= Thr or absent, X14= Pro or is absent, X15= Tyr or absent, X16= Tyr or absent, X17= Tyr or absent, X18= Gly or absent, X19= Met or absent, X20= Asp or absent, and X21= Val or absent;

CDR1 light chain contains the amino acid sequence of the formula X1-X2-X3-X4-X5-X6(SEQ ID NO: 585), where X1= Gln, X2= Asp or Ala, X3= Ile, X4= Ser or Asn, X5= Asn or Ile and X6= Trp or Phe;

CDR2 light chain contains the amino acid sequence of the formula X1-X2-X3(SEQ ID NO: 586), where X1= Ala or Val, X2= Ala or Thr and X3= Ser; and

CDR3 light chain contains the amino acid sequence of the formula X1-X2-X3-X4-X5-X6-X7-X8-X9(SEQ ID NO: 587), where X1= Gln, X2= Gln, X3= Ala or Tyr, X4= Asn, X5= Ser, X6= Phe or His, X7= Pro, X8= Ile or Trp and X9= Thr.

In the seventh aspect of the invention relates to an antibody or antigennegative fragment containing the CDR3 of the heavy chain and light chain CDR3, where domain CDR3 of the heavy chain selected from the group consisting of sequences SEQ ID NO: 9, 25, 41, 57, 73, 89, 105, 121, 137, 153, 169, 185, 201, 217, 233, 249, 265, 281, 297, 313, 329, 345, 361 377; and CDR3 domain is egcoa chain selected from the group consisting of SEQ ID NO: 17, 33, 49, 65, 81, 97, 113, 129, 145, 161, 177, 193, 209, 225, 241, 257, 273, 289, 305, 321, 337, 353, 369 and 385. In a preferred embodiment, the CDR3 region of the heavy chain and light chain are selected from pairs of sequences SEQ ID NO: 57 and 65; 265 and 273; 281 and 289; and 297 and 305.

In the following embodiment, the hallmark of the invention is a human antibody or antibody fragment, containing the domain heavy chain CDR1 selected from the group consisting of sequences SEQ ID NO: 5, 21, 37, 53, 69, 85, 101, 117, 133, 149, 165, 181, 197, 213, 229, 245, 261, 277, 293, 309, 325, 341, 357 and 373, or essentially similar sequence; domain heavy chain CDR2 selected from the group consisting of sequences SEQ ID NO: 7, 23, 39, 55, 71, 87, 103, 119, 135, 151, 167, 183, 199, 215, 231, 247, 263, 279, 295, 311, 327, 343, 359 and 375, or essentially similar sequence; domain CDR3 of the heavy chain selected from the group consisting of sequences SEQ ID NO: 9, 25, 41, 57, 73, 89, 105, 121, 137, 153, 169, 185, 201, 217, 233, 249, 265, 281, 297, 313, 329, 345, 361 and 377, or essentially similar sequence; domain light chain CDR1 selected from the group consisting of sequences SEQ ID NO: 13, 29, 45, 61, 77, 93, 109, 125, 141, 157, 173, 189, 205, 221, 237, 253, 269, 285, 301, 317, 333, 349, 365 and 381 or essentially similar sequence; the domain of the light chain CDR2 selected from the group consisting of sequences SEQ ID NO: 15, 31, 47, 63, 79, 95, 111, 127, 143, 159, 175, 191, 207, 223, 239, 255, 271, 287, 303, 319, 335, 351, 367 and 383, or substantially similar item is coherence; domain and light chain CDR3 selected from the group consisting of sequences SEQ ID NO: 17, 33, 49, 65, 81, 97, 113, 129, 145, 161, 177, 193, 209, 225, 241, 257, 273, 289, 305, 321, 337, 353, 369 and 385 or substantially similar sequences. In a preferred embodiment, the antibody or antigennegative fragment contain a region CDR heavy chain SEQ ID NO: 53, 55, 57 and the field of the CDR of the light chain of SEQ ID NO: 61, 63, 65; region CDR heavy chain SEQ ID NO: 261, 263, 265 and region CDR light chain SEQ ID NO: 269, 271, 273; region CDR heavy chain SEQ ID NO: 277, 279, 281 and region CDR light chain SEQ ID NO: 285, 287, 289; and the region CDR heavy chain SEQ ID NO: 293, 295, 297 and region CDR light chain SEQ ID NO: 301, 303, 305.

In the eighth aspect of the invention relates to an antibody or antigennegative fragment containing the three CDRs of a HCVR and three CDRs from the LCVR, while HCVR/LCVR selected from the group consisting of 51/59, 579/59, 581/59, 259/267, 275/283 and 291/299.

In one embodiment, the distinguishing feature of the invention is a human antibody or antibody fragment, containing the CDR3 of the heavy chain and light chain, where the CDR3 of the heavy chain is encoded by a nucleotide sequence selected from the group consisting of sequences SEQ ID NO: 8, 24, 40, 56, 72, 88, 104, 120, 136, 152, 168, 184, 200, 216, 232, 248, 264, 280, 296, 312, 328, 344, 360 and 376; and CDR3 light chain is encoded by a nucleotide sequence selected from the group consisting of sequences SEQ ID NO: 16, 32, 48, 64, 80, 96, 112, 128, 144, 160, 176, 192, 208, 224, 240, 256, 272, 288, 304, 320, 336, 352, 368 and 84.

In the following embodiment, the hallmark of the invention is a human antibody or antibody fragment, containing the domain CDR1 of the heavy chain encoded by a nucleotide sequence selected from the group consisting of sequences SEQ ID NO: 4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164, 180, 196, 212, 228, 244, 260, 276, 292, 308, 324, 340, 356 and 372 or substantially identical sequence having with them, at least 95% homology; domain CDR2 of the heavy chain encoded by a nucleotide sequence selected from the group consisting of sequences SEQ ID NO: 6, 22, 38, 54, 70, 86, 102, 118, 134, 150, 166, 182, 198, 214, 230, 246, 262, 278, 294, 310, 326, 342, 358 and 374 or substantially identical sequence having with them, at least 95% homology; domain CDR3 of the heavy chain encoded by a nucleotide sequence selected from the group consisting of sequences SEQ ID NO: 8, 24, 40, 56, 72, 88, 104, 120, 136, 152, 168, 184, 200, 216, 232, 248, 264, 280, 296, 312, 328, 344, 360 and 376 or substantially similar sequence having with them, at least 95% homology; domain CDR1 light chain encoded by a nucleotide sequence selected from the group consisting of sequences SEQ ID NO: 12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172, 188, 204, 220, 236, 252, 268, 284, 300, 316, 332, 348, 364 and 380 or substantially similar sequence having with them, at least 95% homology; domain CDR2 light chain encoded by a nucleotide PEFC is a sequence, selected from the group consisting of sequences SEQ ID NO: 14, 30, 46, 62, 78, 94, 110, 126, 142, 158, 174, 190, 206, 222, 238, 254, 270, 286, 302, 318, 334, 350, 366 and 382 or substantially similar sequence having with them, at least 95% homology; and CDR3 domain light chain encoded by a nucleotide sequence selected from the group consisting of sequences SEQ ID NO: 16, 32, 48, 64, 80, 96, 112, 128, 144, 160, 176, 192, 208, 224, 240, 256, 272, 288, 304, 320, 336, 352, 368 and 384, or substantially similar sequence having with them, at least 95% homology. In a preferred embodiment, the antibody or antigennegative fragment contains the region of heavy chain CDR encoded by nucleotide sequences SEQ ID NO: 52, 54, 56, and area light chain CDR encoded by nucleotide sequences SEQ ID NO: 60, 62, 64; field heavy chain CDR encoded by nucleotide sequences SEQ ID NO: 260, 262, 264, and area light chain CDR encoded by nucleotide sequences SEQ ID NO: 268, 270, 272; region heavy chain CDR encoded by nucleotide sequences SEQ ID NO: 276, 278, 280, and area light chain CDR encoded by nucleotide sequences SEQ ID NO: 284, 286, 288; and the field of the CDR of the heavy chain encoded by the nucleotide sequences SEQ ID NO: 292, 294, 296, and area light chain CDR encoded by nucleotide sequences SEQ ID NO: 300, 302, 304.

The invention encompasses anti-hIL-4R-is nitela, with a modified pattern of glycosylation. In some applications it may be useful modification to remove unwanted sites of glycosylation or antibody that lacks fucose residue present in the oligosaccharide chain, for example, to enhance the function of antibody-dependent cellular cytotoxicity (ADCC) (see Shield et al. (2002) JBC 277: 26733). In other applications can be carried out modification of galactosylceramide to modify the complement-dependent cytotoxicity (CDC).

In the ninth aspect of the invention relates to recombinant expressing vectors carrying the nucleic acid molecule according to the invention, and to cells-owners in which put such vectors, and methods of producing antibodies or antigenspecific fragments according to the invention, obtained by culturing host cells according to the invention. The host-cell can be prokaryotic or eukaryotic cell, preferably a cell-host cell is E. coli or mammal cells, such as CHO cell.

In the tenth aspect, the hallmark of the invention is a composition comprising a recombinant human antibody, which is specific binds hIL-4R, and an acceptable carrier.

In the eleventh aspect, the hallmark of izopet the deposits are methods of inhibiting the activity of hIL-4 with the use of antibodies or antigennegative part according to the invention. In specific embodiments, the antibodies according to the invention also block the binding of the complex hIL-13/hIL-13R1 with hIL-4R. In one embodiment, the method comprises contacting hIL-4R with the antibody according to the invention or its antigennegative part, so that inhibited the activity of hIL-4 and hIL-4 and hIL-13. In another embodiment, the method includes the introduction of antibodies according to the invention or its antigennegative part of the person suffering from a disorder that weakens when the inhibition activity of hIL-4 and hIL-4 and hIL-13. Disorder that can be treated is any disease or condition which is improved, reduced, inhibited or prevented by removal, inhibition or reduction of the activity of hIL-4 and hIL-4 and hIL-13.

In addition, the scope of the invention includes the use of antibodies or antigennegative fragment of the antibody according to any one of claims 1-9 in the production of medicines for use to decrease or inhibition of IL-4-mediated diseases or disorders in humans.

Mediated IL-4 violation or related disorders that can be treated with antibodies or fragments of antibodies according to the invention include, for example, arthritis (including septic arthritis), herpetiformis disease, chronic idiopathic urticaria, scleroderma, hypertrophic the prioritization of scarring, the Whipple's disease, benign prostatic hyperplasia, pulmonary disorders such as asthma in mild, moderate or severe form, inflammatory disorders such as inflammatory bowel disease, allergic reactions, Kawasaki disease, sickle cell disease, syndrome Cerca-Strauss, diffuse toxic goiter, pre-eclampsia, Sjogren syndrome, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, tuberculosis, and nephrosis.

Other objects and advantages will be apparent upon further consideration of the following detailed description.

BRIEF DESCRIPTION of FIGURES

Fig. 1(A-C). Evaluation of the profile of antibody binding obtained in the analysis of sequential binding on the basis of the OCTETTM. Fig. 1(A) is a histogram showing the results obtained in the case where the first antibody, which are associated antigen, is a control antibody. Fig. 1(B): the first antibody, which are associated antigen, is VAB16F3-1. Fig. 1(C): the first antibody, which are associated antigen, is VAK5H4-4. Second antibody: 1 = control; 2 = VX4E7-9; 3 = VX3F7-6; 4 = VAB16G-1; 5 = VAB16F3-1; 6 = VAB15C8-17; 7 = VAB11G8-1; 8 = VAB10C1-5; 9 = VAB10G8-19; 10 = VAB8G10-1; 11 = VAB7B9-3; 12 = VAB6C10-14; 13 = VAB5C5-11; 14 = VAB3B4-10; 15 = VAB4D5-3; 16 = VAB1H1-2; 17 = VAK5H4-4; 18 = VAK7G8-5; 19 = VAK8G11-13; 20 = VAK9C6-11; 21 = VK10G6-7; 22 = VAK11D4-1; 23 = VAK12B11-9; and 24 = VAK10G12-5. Where there's no shading bars show the level of binding of the first antibody, the shaded bars show the additional binding of the second antibody.

DETAILED DESCRIPTION

Before describing the methods, it should be stated that this invention is not limited to specific methods and described the conditions of the experiments, as such methods and conditions may vary. Also it should be understood that the terminology used in the present description, is used only to describe specific options and is not intended to be limiting, as the scope of the present invention will be limited only by the attached claims.

Unless otherwise noted, all technical and scientific terms used in this description, have the same meaning, which is usually understood by the specialist in the area that includes the present invention. Although the practical implementation or testing of the present invention can use any methods and materials similar or equivalent to the methods and materials described in this publication, the following describes the preferred methods and materials.

Definitions

The term "IL4R man" (hIL-4R) is used in this sense refers to a receptor of a cytokine of the person who specification who but binds interleukin-4 (IL-4), IL-4Rα (SEQ ID NO: 1). The term "interleukin-13 man" (hIL-13) refers to a cytokine that binds to a receptor specific IL-13, and the term "complex hIL-13/hIL-13R1" refers to the complex formed upon binding of hIL-13 with complex hIL-13R1, and this complex binds to a receptor hIL-4, initiating biological activity.

It is implied that the term "antibody" is used in this sense refers to immunoglobulin molecules, containing the four polypeptide chains, two heavy (H) chains and two light (L) chains connected by disulfide bonds. Each heavy chain contains a variable region heavy chain (commonly referred to in this description HCVR or VH) and a constant region of the heavy chain. The constant region of the heavy chain contains three domains, CH1, CH2 and CH3. Each light chain contains a variable region light chain (commonly referred to in this description LCVR or VL) and a constant region light chain. The constant region of the light chain contains a single domain (CL1). Region VH and VL can be further subdivided into the field of hypervariability called complementarity determining regions (CDR), between which are more conservative area called frame regions (FR). Each VH and VL is composed of three CDRs and four FR, which are distributed from the amino end carboxyl end shadowmania: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

The term "antigennegative part of the antibody (or simply "antibody" or "antibody fragment") is used in this sense refers to one or more fragments of an antibody that retain the ability-specific contact with the antigen (e.g., hIL-4R). It was shown that antigennegative function of antibodies can be performed by fragments of a full-sized antibodies. Examples of binding fragments encompassed by the term "antigennegative part of the antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL domains, VH, CH1 CL1 and; (ii) F(ab')2-fragment, a bivalent fragment comprising two F(ab)'fragments linked by a disulfide bond in the hinge region; (iii) a Fd fragment consisting of the VH domains and CH1; (iv) an Fv fragment consisting of domains VL and VH one shoulder antibody, (v) a dAb fragment (Ward et al. (1989) Nature 241: 544-546), which consists of a VH domain; and (vi) an isolated region, complementarity determining (CDR). In addition, although the two domains of Fv fragment, VL and VH, are encoded by separate genes, they can be linked using recombination synthetic linker that enables you to obtain one continuous chain in which the VL region and a VH constitute a pair with the formation of monovalent molecules (known as single-chain Fv (scFv); see e.g ird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883. This also implies that such single-chain antibodies covered by the term "antigennegative part of the antibody. Other forms of single-chain antibodies, such as dimeric antibodies, also fall under this term (see, e.g., Holliger et al. (1993) Proc. Natl. Acad Sci. USA 90: 6444-6448).

"Neutralizing" or "blocking" antibody used in this sense refers to the antibody, the binding of which hIL-4R leads to inhibition of the biological activity of hIL-4 and/or hIL-13. This inhibition of the biological activity of hIL-4 and/or IL-13 can be assessed by measuring one or more indicators of biological activity of hIL-4 and/or hIL-13, known in this field, such as induced hIL-4 and/or IL-13 activation of cells and the binding of hIL-4, hIL-4R (see examples below).

"CDR" or complementarity determining region is a region of hypervariability, with scattered inside areas that are more conservative, called "framework regions" (FR). In other embodiments, the anti-hIL-4R antibody or fragment according to the invention FR can be identical to the sequences of the germline of the person or may be natural or artificially modified.

The term "surface plasmon resonance" is used in esteem description sense refers to an optical phenomenon, which allows the analysis of the interactions in real time by recording the changes in the concentrations of proteins on a biosensor matrix, for example using the BIAcore systemTM(Pharmacia Biosensor AB).

The term "epitope" means an antigenic determinant, which interacts with specific antigennegative plot variable regions of antibody molecules, known as Pratap. One antigen may have more than one epitope. Epitopes can be either conformational or linear. Conformational epitope formed spatially close amino acids from different parts of the linear polypeptide chain. Linear epitope formed adjacent amino acid residues in the polypeptide chain. In some cases, the epitope may contain some residual sugars, groups of phosphoryla or sulfonylurea group antigen.

The term "substantial identity" or "essentially identical" in that case when he refers to nucleic acid or its fragment, indicates that under optimal alignment with appropriate insertions or deletions of nucleotides with another nucleic acid (or complementary to her thread) is the identity of the nucleotide sequences constituting at least about 95% and more preferably at least about 96%, 97%, 98% or 99% n is cleotide grounds measured using any well-known algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed below.

With respect to polypeptides, the term "substantial similarity" or "essentially similar" means that two peptide sequences, when optimally aligned, for example, using the programs GAP or BESTFIT using the weight estimates for a space 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, differ in the result of conservative amino acid substitutions. "Conservative amino acid substitution" is a substitution in which the amino acid residue substituted with other amino acid residue having a side chain (R group) with similar chemical properties (e.g. charge or hydrophobicity). In General, a conservative amino acid substitution does not significantly alter the functional properties of the protein. In cases when two or more amino acid sequences differ from each other conservative substitutions, sequence identity in percent or degree of similarity can be improved by adjusting the conservative nature C the exchange. The ways of making this adjustment are well known to specialists in this field. 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) containing amide 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. A preferred group of conservative substitutions of amino acids: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate and asparagine-glutamine. Alternative conservative replacement is any change having a positive value when evaluated using matrix logarithmic likelihood function PAM250 described in gonnet on et al. (1992) Science 256: 1443-45. "Moderately conservative" replacement is any change having a positive value when evaluated using matrix logarithmic likelihood function PAM250.

The similarity of the sequences in the case of polypeptides, which is also called identity follower of the awn, usually measured using a computer program sequence analysis. Computer analysis program of protein matches similar sequences using the parameters of similarity with regard to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For example, the computer program 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 mutant variant. See, for example, the GCG Version 6.1. Polypeptide sequences can also be compared using FASTA with its default settings or recommended parameters, the program in GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignment and gives the percent identity of the sequences in the areas of best alignment between the requested and the analyzed sequences (Pearson (2000), supra). Other preferred algorithm when comparing a sequence according to the invention with a database containing a large number of sequences from different organisms is the computer program BLAST, especially BLASTP or TBLASTN the application of 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 person

Methods for producing human antibodies include, for example, VeloclmmuneTM(Regeneron Pharmaceuticals), the method XenoMouseTM(Green et al. (1994) Nature Genetics 7: 13-21; Abgenix), the way "minilogue" and phage display (see, for example, US 5545807, US 6787637). Methodology VeloclmmuneTM(US 6596541) includes a method of obtaining a high-affinity fully human antibodies to the selected antigen.

Rodents can immunize any method known in the art (see, for example, Harlow and Lane (1988), supra; Malik and Lillehoj (1994) Antibody techniques, Academic Press, CA). In a preferred embodiment, the antigen hIL-4R injected directly mice, which are the loci of the DNA coding and the variable region of the heavy chain and the variable region of the light chain Ig Kappa person (VeloclmmuneTM, Regeneron Pharmaceuticals, Inc.; US 6596541), with an adjuvant to stimulate the immune response, for example, complete or incomplete adjuvant's adjuvant, the adjuvant MPL+TDM (Sigma) or RIBI (muramyl-dipeptides) (see O Hagan (2000) Vaccine Adjuvant, Human Press, NJ). Adjuvant can prevent the rapid dissipation of the polypeptide in the sequestration of antigen in the local depot and may contain factors that can stimulate the immune response of the host. Methodology VeloclmmuneTMis to create a transgenic mouse, have her genome, containing the variable regions of heavy and light chains of a human, functionally associated with the endogenous loci of constant regions 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 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. Then DNA Express in the cell, is able to Express fully human antibody. In a specific embodiment, the cell is a CHO cell.

Antibodies may be therapeutically applicable in blocking the interaction of ligand-receptor or inhibition of the interaction between the components of the receptor, and not to cause cell death in the fixation of complement (complementability cytotoxicity) (CDC) and participation-dependent antibody-mediated cell cytotoxicity (ADCC). The constant region of the antibody is important to ensure the ability of the antibody to fix complement and mediate dependent cell cytotoxicity. Thus, the isotype of the antibody can be selected on the basis of whether mediating the cytotoxicity of the antibody.

The human immunoglobulins may exist in two forms, the quiet associated with the heterogeneity of hinge. In one form, the immunoglobulin molecule contains a stable structure of the four chains with a molecular mass of approximately 150-160 KD, in which the dimers are held together miaocheng by a disulfide bond between the heavy chains. In the second form dimers are not bound by miaocheng disulfide bonds and the molecule with a molecular mass of approximately 75-80 KD formed covalently linked light and heavy chains (poloitical). These forms are very difficult to separate even after affinity purification. The frequency of the second form in a variety of intact IgG isotypes is a consequence of, but not limited to structural differences associated with the hinge region isotype antibodies. Really a single amino acid substitution 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 present invention encompasses antibodies having one or more mutations in the hinge region, in the region of CH2 or CH3, which may be desirable, for example, upon receipt, to increase the yield of the desired form antibodies.

Antibodies according to the invention preferably are obtained from applying the methodology VeloclmmuneTM. Transgenic mouse in which the endogenous variable regions of heavy and light is EPA immunoglobulin are replaced by corresponding variable regions of a human, stimulate interest antigen and lymphatic cells (such as B-cells) are recovered from the mice that Express antibodies. Lymph cells can be merged with the line of myeloma cells to obtain an immortalized cell line of hybridoma, and such cell line of hybridoma subjected to screening and selection in order to identify the cell line of hybridoma that produce antibodies that are specific against interest antigen. DNA encoding a variable region of the heavy chain and light chain can be selected and associated with the required izotopicheskii constant regions of the heavy chain and light chain. Such protein antibodies can be produced in a cell such as a CHO cell. Alternative DNA encoding the antigen-specific chimeric antibody or variable region light and heavy chains, can be isolated directly from antigen-specific lymphocytes.

In one embodiment, the 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, a 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 yet another variant transcinnamic contains up to 80 gene variable regions of the heavy chain of human and 40 gene variable region light chain Kappa man.

In General, the antibodies according to the present invention have a very high affinity, typically have KD's about 10-9up to 10-12M, which is measured by binding to the antigen, or immobilized on a solid phase or in solution.

First, select high-affinity chimeric antibodies having a variable region of a human and a constant region of a mouse. As described below, characterize antibodies and subjected to selection in relation to the desired properties, including affinity binding to hIL-4R, the ability to block the binding of hIL-4, hIL-4R and/or selectivity with respect to the squirrel man. The constant region of the mouse replace the required constant regions of a human to create a fully human antibodies according to the invention, for example wild-type or modified IgG4 or IgG1 (e.g., SEQ ID NO: 588, 589, 590). While choosing the constant region may vary depending on the particular application, such properties as high-affinity antigen binding and specificity towards the target, inherent in the variable regions.

Mapping of epitopes and related methods

For the implementation of screening for antibodies that bind to a specific epitope, it is possible to carry out routine analysis of cross-blocking, such as the analysis described in Antibodies: A aboratory Manual 1988 Cold Spring Harbor Laboratory, Harlow and Lane, eds. Other methods include the analysis of mutants obtained by alanine scanning peptide blots (Reineke (2004) Methods Mol. Biol, 248: 443-63) or analysis of the cleavage peptides. In addition, you can use techniques such as cut epitope, the destruction of the epitope and chemical modification of antigens (Tomer (2000) Protein Science 9: 487-496).

The profile analysis based on modification (MAP), also known as profile analysis based on the structure of the antigen (ASAP)is a method that allows to classify large amounts of monoclonal antibodies (MAB)directed against the same antigen on the basis of similarity of the profile of the binding of each antibody with a chemically or enzymatically modified surface antigen (publication of the application for the grant of U.S. patent No. 2004/0101920). Each category may reflect a unique epitope, either explicitly different or partially overlapping with the epitope presented in the other category. This methodology allows us to quickly filter genetically identical antibodies, so that the characteristic can be focused on genetically different antibodies. In the case of use in screening a hybrid MAP can facilitate the identification of rare clones of hybridoma with the required properties. MAP can be used to sort hIL-4R antibodies according to the invention on groups of the antibody, binding different epitopes.

Agents applicable for changes in the structure of the immobilized antigen are enzymes, such as proteolytic enzymes and chemical agents. Protein antigen can be immobilized either on the surfaces of biosensor chips or beads of polystyrene. The latter can be subjected to analysis, such as multiplex analysis for registration LuminexTM(Luminex Corp., TX). Because LuminexTMable to perform multiplex analysis of up to 100 different types of beads, LuminexTMprovides almost unlimited surface antigens with different modifications, which leads to an increased resolution in the profile analysis of the epitopes of antibodies using biosensor analysis.

Therapeutic introduction and drugs

The introduction of therapeutic agents according to the invention can be implemented together with suitable carriers, excipients, and other agents, including drugs, to provide improved transfer, delivery, tolerance, and the like. Many candidate drugs can be found in reference books, the well-known specialists in the field of pharmaceutical chemistry: Remington: The Science and Practice of Pharmacy (2003, 20thed, Lippincott Williams and Wilkins). These drugs include, for example, powders, pastes, ointments, jellies, waxes, is of asle, lipids containing lipid (cationic or anionic) vesicles (such as LIPOFECTINTM), DNA conjugates, anhydrous intake pastes, emulsions of the type oil-in-water" and "water in oil", carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. Any of the above mixtures may be suitable for treatment and therapy according to the present invention, provided that the active ingredient in the drug is not inactivated by other components of the drug, and the drug is physiologically compatible and tolerable if used route of administration. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J. Pharm. Sci. Technol. 52: 238-311 and cited in this work publications for additional information related to the excipients and carriers, well known to experts in the field of pharmaceutical chemistry.

Therapeutic molecules according to the invention it is possible to enter the patient in a way that is suitable for this testimony, for example, parenterally, topically or by inhalation. In the case of the injection of the antagonist can be entered, for example, intra-articular, intravenous, intramuscular route, in the lesion, intraperitoneal or subcutaneous routes by bolus injection or continuous infusion. It is assumed, and the locale is the first introduction to the place of illness or injury, as, for example, transdermal delivery and slow release from implants. Delivery by inhalation include, for example, nasal or oral inhalation, application of the spray, inhalation antagonist in aerosol form and the like. Other alternatives include eye drops, oral medications, including pills, syrups, cakes, or chewing gum; and local products, such as lotions, gels, sprays and ointments.

Specific dosage and frequency of injection can vary depending on such factors as the route of administration, nature and severity of the disease being treated, whether the condition is acute or chronic, and the weight and General state of health of the patient. The appropriate dose can be determined by methods known in the art, for example in clinical trials, which may include studies using increasing doses. Therapeutic molecules according to the invention it is possible to introduce one or more times. In specific embodiments, the antibody or antibody fragment is administered over a period of time comprising at least a month or more, for example one, two or three months, or even indefinitely. For the treatment of chronic conditions is usually the most effective long-term treatment. However, to ensure the Stryj States may be sufficient introduction for more than short periods, for example from one to six weeks. In General, therapeutic agent is administered until symptoms in a patient with significant medical point of view, the degree of improvement compared to the initial level on the chosen indicator or indicators. The level of IL-4 can be controlled during and/or after treatment with a therapeutic molecule according to the invention. Ways to measure levels of IL-4 in serum-known in this field, for example using ELISA, etc.

Therapeutic use and combination therapy

Antibodies and antibody fragments according to the invention is applicable for the treatment of diseases and disorders, a condition in which you can improve, which can inhibit or weaken by reducing the activity of IL-4. Such disorders include disorders characterized by abnormal or excessive expression of IL-4 or abnormal response of the host to the production of IL-4. Associated with IL-4 disorders that can be treated with antibodies or fragments of antibodies include, for example, arthritis (including septic arthritis), herpetiformis disease, chronic idiopathic urticaria, scleroderma, hypertrophic scarring, Whipple's disease, benign prostatic hyperplasia, pulmonary disorders such as asthma (mild, moderate or severe), inflammatory disorders such as vos is liteline bowel disease, allergic reactions, Kawasaki disease, sickle cell disease, syndrome Cerca-Strauss, diffuse toxic goiter, pre-eclampsia, Sjogren syndrome, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, tuberculosis, atopic dermatitis, ulcerative colitis, fibrosis and nephrosis (see U.S. 7186809).

The invention relates to a combination therapy, in which the anti-IL-4R-antibody or antibody fragment is administered in combination with a second therapeutic agent. Introduction of joint and combined therapy is not limited to the simultaneous introduction, and include treatment regimens in which the anti-IL-4R-antibody or antibody fragment is administered at least once during treatment, which involves injecting the patient with at least one other therapeutic agent. The second therapeutic agent may be another antagonist of IL-4, such as another antibody/fragment antibodies or soluble cytokine receptor, IgE antagonist, a drug for asthma (corticosteroids, non-steroidal drugs, beta-agonists, leukotriene antagonists, xantina, fluticasone, salmeterol, albuterol), which can be delivered by inhalation, or other suitable means. In a specific embodiment, anti-IL-4R-antibody or antibody fragment with the according to the invention can be introduced with an antagonist of IL-1, such as rilonacept, or an antagonist of IL-13. The second means may include one or more antagonists of leukotriene receptor for the treatment of such disorders, such as allergic inflammatory diseases such as asthma and allergies. Examples of antagonists of leukotriene receptor include, without limitation montelukast, pranlukast and zafirlukast. The second tool may contain an inhibitor of a cytokine, such as one or more TNF (etanercept, ENBRELTM), the antagonist of IL-9, IL-5 or IL-17.

EXAMPLES

The following examples are offered in order to provide the experts in this field a complete disclosure and description of how to obtain and apply the compositions and methods according to the invention, and the examples are not intended to limit the scope, which the inventors regard as their invention. Attempts to ensure the accuracy in terms of quantitative indicators (e.g., amounts, temperature, etc.), but keep in mind some errors and deviations. Unless otherwise stated, parts are parts by weight, molecular weight is average molecular weight, temperatures are in degrees centigrade, and pressure is atmospheric or close to atmospheric.

Example 1. Obtaining human antibodies to the IL-4 receptor of the person.

Mice with DNA loci encoding and variable region of the heavy chain and the variable region of the light chain Ig Kappa person (VeloclmmuneTM, Regeneron Pharmaceuticals, Inc.; U.S. patent No. 6596541), were immunized IL-4R human (hIL-4R, SEQ ID NO: 1). hIL-4R has introduced direct injection of purified antigen in Freund or indirectly by introduction of a DNA sequence hIL-4R in a DNA plasmid that contains the gene hIL-4R and expresses hIL-4R, using the apparatus of expression of proteins of the host cell, giving antigenic polypeptide in vivo. To obtain an optimal immune response was performed subsequent booster immunization of animals every 3-4 weeks and fences blood was performed after 10 after each booster immunization to assess the development of the response against the antigen.

When the mice reached a maximum immune response, expressing antibodies B cells are collected and merged with myeloma cells of mice with the formation of the hybrid. Necessary from a functional point of view monoclonal antibodies were collected by screening, air-conditioned environment hybrid or transfected cells in relation to specificity, affinity of binding of the antigen and the effectiveness of blocking the binding of hIL-4, hIL-4R (described below).

Selected antibodies, which have the desired binding affinity of antigen, efficiency and/or the ability to block the binding of hIL-4 with hIL4R, included (HCVR/LCVR):

Example 2. Determination of the affinity of binding the antigen.

The binding affinity of (KDselected antibody against hIL-4R were determined using an analysis that uses biosensors based on surface plasmon resonance in real time (BIAcoreTM2000). Briefly, the antibody can be immobilized on the surface of polyclonal antibodies against GAM IgG mouse, created by direct chemical binding of IgG GAM with BIAcore chipTMwith the formation of an immobilized on the surface of the antibody. Various concentrations (ranging from 12.5 nm to of 0.625 nm) monomer hIL-4R (R&D Systems) or dimeric hIL-4R-hFc was applied by injection to the surface with immobilized antibodies. Binding of the antigen with the antibody and the dissociation of the bound complex was observed in real time. Equilibrium dissociation constants (KD) and the rate constants of dissociation was determined through kinetic analysis using computer programs for data evaluation BIA. A computer program for data evaluation BIA also used to calculate the half-life of dissociable complex antigen/antibody (T1/2). The results are shown in table 1. Control: a fully human anti-IL-4R-antibody (U.S. patent No. 7186809; SEQ ID NO: 0 and 12). The results of kinetic analyses revealed that the selected antibodies contained high-affinity antibodies that are able to communicate with dimeric receptor with KDless than 1 nm. Also tested the binding affinity of anti-hIL-4R antibodies to IL-4R or mice, or monkeys (Macaca fascicularis). Selected antibodies (1) not cross-react with IL-4R mouse; and (2) either could not bind IL-4R monkeys, or bound IL-4R monkeys with very low affinity.

The binding affinity of the antibody-antigen was also evaluated using the analysis of competitive binding in solution-based ELISA. Briefly, antibodies (purified protein at a concentration of 1 or 3.3 ng/ml) was pre-mixed with serial dilutions of the antigenic protein (Monomeric or dimeric) in the range from 0 to 10 μg/ml Solutions of mixtures of antibody and antigen is then incubated for two to four hours at room temperature to reach equilibrium binding. Then measured the free antibody in the composition using a quantitative ELISA "sandwich". Briefly, 96-well tablets MaxisorpTM(VWR, West Chester, PA) were coated with 2 μg/ml protein hIL-4R-hFc in PBS overnight at 4ºC, followed by blocking of non-specific binding protein BSA. Then the solutions of mixtures of antibody-antigen was transferred into a covered PLA is SETI Maxisorb TMwith the subsequent one-hour incubation. Then the tablets were washed with buffer for washing and associated with the tablet antibodies were detected using a reagent: conjugated with HRP polyclonal antibody goat against mouse IgG (Jackson ImmunoResearch) or conjugated with HRP polyclonal antibodies goats against human IgG (Jackson ImmunoResearch) in the case of the control antibody and processed using colorimetric substrates such as BD OptEIATM(BD Biosciences Pharmingen, San Diego, CA). After stopping the reaction with 1 M phosphoric or sulphuric acid was detected optical density at 450 nm and the data were analysed using the computer program GraphPadTMThe Prism. The dependence of the signals from the concentration of antigen in solution was determined using an analysis based on the adjustment of four parameters, and were expressed as IC50the concentration of antigen required to achieve a 50% reduction of signal samples from antibodies in the absence of antigen in solution. IC50was determined as described above and summarized in table 2. In a preferred embodiment, the antibody or antibody fragment according to the invention has the IC50for dimeric hIL-4R, which is about 20 PM or less, and the IC50for Monomeric hIL-4R approximately 150 PM or less, or about 100 PM or less, which is measured in the analysis of competition in the solution-based ELISA.

Example 3. Inhibition of the interaction between hIL-4 and hIL-4R

Organized the screening ability of the selected antibodies to block the binding of hIL-4, hIL-4R in the analysis block hIL-4 BIAcoreTMand efficiency was measured in a quantitative immunoassay blocking hIL-4, which is described below. The results are summarized in table 3.

The ability of antibodies to block the binding of hIL-4 receptor hIL-4R was determined using surface plasmon resonance. Purified molecules hIL-4R-hFc caught polyclonal antibody goat IgG against human immobilized on a CM-5 with density EN 260 to obtain a surface receptor. Then IL-4 (0.25 ml, 50 nm) through injections were applied to the coated receptor surface and recorded the amount of bound hIL-4 (first injection of hIL-4). Associated hIL-4 was then removed pulsed exposure to 3 M MgCl2with subsequent exposure to the cleaning buffer. Then were injected with purified anti-hIL4R-antibody coated receptor surface with a subsequent second injection of hIL-4 at the same concentration (second injection of hIL-4). The reduction of binding of hIL-4 percent in the prior formation of a complex of the antibody to the receptor are shown in table 3.

In order to further assess the ability to block the binding of hIL-4, hIL-4 was performed quantitative immunoassay. Briefly, solutions 25 PM hIL-4R-Fc was pre-mixed with protein antibodies in the range from ~50 nm to 0 nm in serial dilutions with subsequent one-hour incubation at room temperature. The concentration of free hIL-4R-Fc (not associated with the antibody) was determined using specific for hIL-4R ELISA "sandwich". Tablets for registration hIL-4R was prepared by linking biotinylated hIL-4 (0.5 μg/ml) streptavidin coated 96-well plates. Pre-linked samples, the antibody-antigen was transferred into a covered hIL-4 tablet for registration. After one-hour incubation at room temperature the tablet to register washed and associated with the tablet hIL-4R-hFc were identified using conjugated with HRP polyclonal antibody goat against hFc, and processed using colorimetric substrates such as BD OptEIATM(BD Biosciences Pharmingen, San Diego, CA). After stopping the reaction with 1 M phosphoric or sulphuric acid was detected optical density at 450 nm and the data were analysed using the computer program GraphPadTMThe Prism. IC50defined as the amount of antibody required to reduce 50% of the detected IL-4R-hFc, which is associated with hIL-4, associated with the tablet hIL-4. In a preferred embodiment, the antibody or antibody fragment according to the invention has the IC50block 25 PM hIL-4R or less comprimere 50 PM or less than about 40 um, or less than about 30 PM, or less than about 20 PM measurements in ELISA.

Example 4. Neutralization of the biological action of hIL-4 in vitro

The path mediated by IL-4 signal transduction is described in detail in the literature (for example, see the review Hebenstreit et al. 2006 Cytokine Growth Factor Rev.17(3): 173-88, 2006). IL-4 can stimulate two receptor complex, type I and type II. The receptor complexes of type I are formed as a result of binding of IL-4 to IL-4R and subsequent heterodimerization with the common gamma-chain. Alternative complex IL4/IL4R can heterodimerization with 1 receptor IL-13 with the formation of receptor complexes of type II. Complexes of type I and type II transmit a signal mainly through STAT6. Therefore evaluated the ability of the selected antibodies to block the transmission of the signal through STAT6, as described below.

Created a line of cells with high sensitivity to hIL-4 and hIL-13. The HEK293 cells stably was transfusional STAT6 man and a plasmid with luciferase reporter for STAT6 and maintained in growth medium (DMEM, 10% FBS, L-glutamine, penicillin, streptomycin). Strong receptor-mediated IL-4 response was reached, when added 10 PM hIL-4 in the growth medium STAT6-transfected HEK293 cells. For biological response analysis hIL-4 cells were washed once in slice d is I analysis (Optimem I (Gibco) plus of 0.1% FBS) and were sown on 1×10 4cells/well (96-well plate) in 80 μl of medium for analysis. Purified antibodies were serially diluted in medium for analysis (final concentration in the range from 20 nm to 0) and 10 μl each of the test antibodies were added to cells together with 10 μl of hIL-4 (fixed final concentration of 10 PM). The cells are then incubated at 37 degrees C, 5% CO2within 6 hours. The degree of response of the cells was measured in the analysis of luciferase (Promega Biotech). The results are shown in table 4.

The inhibition is dependent on IL-4 biological activity in vitro was also confirmed using either cell line erythroblasts person, TF1, or a modified cell line TF1, sverkhekspressiya IL-13Rα (TF1/A12). In this bioanalysis 20000 cells were sown in each well of 96-well plate in a medium RPMI 1640 containing 10% FBS, 2 mm L-glutamine and penicillin and streptomycin. Twenty-five microlitres purified antibodies in the range from 0 to 50 nm (final concentration) was added together with 25 μl of recombinant protein hIL-4 to a final concentration of either 50 PM in case of cell line TF1, either 20 PM in the case of cell line TF1/A12. Then the cells were given the opportunity to grow for 3 days at 37 degrees C and a finite number of cells was measured using CCK8 kit (Dojindo, Japan). The ability of antibodies to block the growth of cells TF-1 are shown in table 4 as the antibody concentration required to achieve 50% is Nigeria cell proliferation.

Example 5. Neutralization of the biological action of hIL-13 in vitro

As it was shown that IL-4R is a modulator of the activity of IL-13 through its binding to a complex of IL-13/IL-13R, the selected antibodies were tested for their ability to block the activity of IL-13 using a modified analysis of STAT6-luciferase HEK293 described above, with the modification consisted in the replacement of 10 PM IL-4 on 40 PM hIL-13. Antibodies were also evaluated in relation to the effectiveness of blocking the activity of hIL-13 in the analysis of the cell line TF-1, described above, using hIL-13 at a concentration of 150 PM in the presence of 0-50 nm antibody. The results are shown in table 5.

Example 6. Evaluation of the profile of binding antibodies

Profile of binding antibodies can be set, defining the impact that the antibody (associated with its antigen) may have on the ability of a panel of different antibodies subsequently bind the same antigen. For example, the antigen can be immobilized on the substrate with the formation of antigen coated surface antigen coated surface can be saturated with antibody and then antigen coated and saturated with antibody surface may be subjected to a panel of other antibodies. Article the stump binding panel of other antibodies with antigen coated and saturated with antibody surface gives the profile of antibody binding sites. Based on OCTETTManalyses of serial link used to obtain a profile of antibody binding. Briefly, a group of 24 FA biosensors containing streptavidin high binding efficiency (ForteBio, Inc., Menlo Park, CA) is first incubated with the antigen, Biotin-hIL-4R-hFc, at a concentration of 2 µg/ml for 10 min at 30ºC in order to reach saturation, and the amount of bound antigen was measured by changing the thickness (nm) of the biological layer due to the associated protein, which is directly measured by the shift of the wavelength. Associated with Biotin-hIL-4R-hFc biosensors then incubated with the first antibody (control antibody) at a concentration of 50 μg/ml for 15 min at 30ºC in order to reach saturation, and the amount of bound control antibodies were measured by changing the thickness (nm) of the biological layer. Each biosensor associated with the control antibody, then incubated with one of the panels from 24 different anti-hIL-4R-antibody (second antibody) at a concentration of 50 μg/ml for 15 min at 30ºC, and the amount of bound second antibody was measured by changing the thickness of the biological layer. The same analysis was repeated using as the first antibody anti-hIL-4R-antibody or VAB16F3-1 or VAK5H4-4. The results are shown in Fig. 1A-C.

Selected anti-hIL-4R antibodies was also evaluated using Western blot (Dan is haunted not shown). Briefly, the monomer hIL-4R (200 ng per lane) and His-tagged monomer mfIL-4R (200 ng per lane) were subjected to electrophoresis in SDS-page-gels, using as a reducing and non buffer for samples. Each of the four separate gels were transferred to PVDF-membrane and each membrane was subjected to one of the first four antibodies: anti-His-Ab (Qiagen), VAB 16F3-1, VAK 5H4-4 or control anti-hIL-4R antibodies, using as the second antibody or conjugated with HRP antibody goat against mIgG, or antibody against hIgG (Pierce). All three anti-hIL-4R antibodies learned unrestored form of hIL-4R. Only VAB 16F3-1 and VAK 5H4-4 revealed a restored form of hIL-4R. None of the anti-hIL-4R antibodies did not detect any IL-4R monkeys.

1. The antibody or antigennegative fragment that is specific bind hIL-4R (SEQ ID N0:1) with KDapproximately 200 PM or less, which is measured using surface plasma resonance, containing sequence HCVR/LCVR selected from 579/59 and 581/59.

2. The antibody or antigennegative fragment according to claim 1, which Express the KDagainst hIL-4R (SEQ ID N0:1), constituting less than 150 PM.

3. The antibody or antigennegative fragment according to claim 1, which Express the KDagainst hIL-4R (SEQ ID N0:1), constituting less than 50 PM.

4. An isolated nucleic acid molecule encoding the antibody or antigenspecific fragment according to any one of the preceding paragraphs.

5. A vector containing the nucleic acid sequence according to claim 4, for obtaining antibodies or antigennegative fragment antibodies that are specific bind the IL-4 receptor.

6. System host - vector to obtain antibodies or antigennegative fragment antibodies that are specific bind the IL-4 receptor, containing the vector according to claim 5 in a suitable cell host.

7. System host - vector according to claim 6, in which a host cell is prokaryotic or eukaryotic cell selected from the E. coli cell or SNO.

8. The method of obtaining anti-IL-4R antibodies or antigennegative fragment, which includes the cultivation of the cells of the host system is the vector of claim 6 or 7 in conditions that ensure the production of the antibody or its fragment, and the allocation expressed thus antibody or fragment.

9. The use of antibodies or antigennegative fragment of the antibody according to any one of claims 1 to 3 in the manufacture of a medicinal product for the purpose of weakening or mediated inhibition of IL-4 diseases or disorders in humans.

10. The antibody or antigennegative fragment according to any one of claims 1 to 3 for use in the method of treating diseases or disorders in human, which disease or disorder is improving, attenuated or inhibited by removal, inhibition or reduced the I activity of interleukin-4 (hIL-4).

11. The use according to claim 9, in which the disease or disorder is selected from arthritis, herpetiformis disease, chronic idiopathic urticaria, scleroderma, hypertrophic scarring, Whipple's disease, benign prostate hyperplasia, pulmonary disorders, inflammatory disorders, allergic reactions, Kawasaki disease, sickle cell disease, syndrome Cerca-Strauss, diffuse toxic goiter, pre-eclampsia, Sjogren syndrome, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, tuberculosis, atopic dermatitis, ulcerative colitis, fibrosis and nephrosis.

12. The application of claim 11, in which the specified pulmonary infringement is asthma specified inflammatory violation is an inflammatory bowel disease or specified arthritis is a septic arthritis.

13. The antibody or antigennegative fragment of claim 10, where the disease or disorder is selected from arthritis, herpetiformis disease, chronic idiopathic urticaria, scleroderma, hypertrophic scarring, Whipple's disease, benign prostate hyperplasia, pulmonary disorders, inflammatory disorders, allergic reactions, Kawasaki disease, sickle cell disease, is indrema Cerca-Strauss, diffuse toxic goiter, pre-eclampsia, Sjogren syndrome, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, tuberculosis, atopic dermatitis, ulcerative colitis, fibrosis and nephrosis.

14. The antibody or antigennegative fragment according to item 13, where the specified pulmonary infringement is asthma specified inflammatory violation is an inflammatory bowel disease or specified arthritis is a septic arthritis.

15. A composition comprising an effective amount of the antibody or antigennegative fragment according to any one of claims 1 to 3, and an acceptable carrier for use in the method of treating diseases or disorders in human, which disease or disorder is improving, attenuated or inhibited by removal, inhibition or reduction of the activity of interleukin-4 (hIL-4).



 

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FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, virology and medicine. What is disclosed is a recombinant protein containing one or more polypeptides carrying one or more epitopes of one or more human papilloma virus HPV antigens. Said polypeptides are embedded in one or various permissive sites of adenylatecyclase (CyaA) or its fragments. What is also disclosed is the polypeptide coding such protein and their use in expression systems for producing immunogenic compositions and drugs. The invention can be used in medicine.

EFFECT: CyaA fragment possesses the property of said adenylatecyclase protein for targeted interaction with antigen-presenting cells.

60 cl, 21 dwg, 1 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: invention describes novel polynucleotide and amino acid sequences of Brachyspira hyodysenteriae, which can be used to diagnose diseases in animals, caused by B. hyodysenteriae, to treat or prevent diseases associated with infection with B. hyodysenteriae. The invention describes a cell containing a plasmid containing a polynucleotide, for treating and preventing a disease associated with infection of an animal with B. Hyodysenteriae. The invention describes immunogenic and vaccine compositions for generating immune response in an animal, which contain a polypeptide, a polynucleotide, a cell or a plasmid for treating or preventing infection of animals by B. hyodysenteriae, as well as sets of instruments for diagnosis which contain a monoclonal antibody, capable of biding the disclosed polypeptide or a polypeptide or polynucleotide. The invention enables to successfully diagnose diseases caused by B. hyodysenteriae, prevent or treat animals infected with B. hyodysenteriae. The sequences described herein can be used for diagnosis and therapeutic and/or preventive treatment of animals from diseases caused by other types of Brachyspira, including B. intermedia, B. suantatina, B. alvinipulli, B. aalborgi, B. innocens, B. murdochii and B. pilosicoli.

EFFECT: high efficiency of using the composition.

39 cl, 4 tbl

FIELD: medicine.

SUBSTANCE: invention relates to versions of glucoamylase with altered properties and to methods of glucoamylase versions application.

EFFECT: reduced formation of condensation products and increase of glucose output during starch conversion.

22 cl, 2 tbl, 1 ex

FIELD: chemistry.

SUBSTANCE: recombinant technique is used to obtain a fused polypeptide with activity of interleukin-7, containing a modified human IL-7 molecule in which the T-cell epitope is modified to reduce T-cell response against IL-7, and the Fc part of an immunoglobulin molecule which is fused through its C-end with the N-end of said modified IL-7 molecule. The obtained polypeptide is used in a pharmaceutical composition to stimulate immune response in a patient.

EFFECT: invention enables to obtain a polypeptide with interleukin-7 activity, having low immunising capacity.

8 cl, 43 dwg, 14 tbl, 12 ex

FIELD: medicine.

SUBSTANCE: there are offered versions of antibodies specific to CD22 epitope located from amino acid 22 to amino acid 240 CD22. There are disclosed: a coding polynucleotide, an expression vector, a based host cell and a method of producing an antibody with the use of the cell. There are described versions of a method of CD22 detection on the basis of the antibodies. There are disclosed versions of the CD22 immunoconjugate and based pharmaceutical compositions for treating disturbed B-cell proliferation, and also versions of a method of treating with the use of the pharmaceutical composition. There is disclosed a method of B-cell proliferation inhibition on a basis the immunoconjugate. There are described versions of an engineered cystein-substituted antibody specific to CD22 with one or more free cysteines of thiol reactance within the range 0.6 to 1.0. There are disclosed versions of the "antibody-drug" conjugate, the immunoconjugate and pharmaceutical formulaitons for treating disturbed B-cell proliferation. There are also described a method for protein CD22 detection in a sample on the basis of the immunoconjugate, a method for B-cell detection and a method of treating a malignant tumour on the basis of the "antibody-drug" conjugate. There are disclosed: a product for treating disturbed B-cell proliferation on the basis of the pharmaceutical formulation and a method of producing the "antibody-drug" conjugate.

EFFECT: use of the invention provides new specific CD22 antibodies and the based drugs of acceptable therapeutic efficacy with lower toxicity that can find application in therapy of tumours.

227 cl, 25 dwg, 16 tbl, 14 ex

FIELD: immunology and bioengineering.

SUBSTANCE: present invention refers to immunology and bioengineering. The variants of an antisubstance that is specific in relation to at least one globulomer Aβ(20-42) have been suggested. Each of the variants is characterized by the fact that it includes VH and VL parts; each of these parts contains three corresponding CDR. The antisubstance antigen-binding section has been revealed. They described: a coding nucleic acid and the vector that contains it, and a host cell that bears the vector that are used for the antisubstance production. The way of antisubstance production with the use of a cell has been discovered. The suggested inventions can find their application in therapy and diagnostics of Alzheimer's disease and other amyloid diseases.

EFFECT: antosubstances that can be used in therapy and diagnostics of Alzheimer's disease and other amyloid diseases.

10 cl, 28 dwg, 9 tbl, 12 ex

FIELD: medicine.

SUBSTANCE: there are offered versions of antibodies specific to CD22 epitope located from amino acid 22 to amino acid 240 CD22. There are disclosed: a coding polynucleotide, an expression vector, a based host cell and a method of producing an antibody with the use of the cell. There are described versions of a method of CD22 detection on the basis of the antibodies. There are disclosed versions of the CD22 immunoconjugate and based pharmaceutical compositions for treating disturbed B-cell proliferation, and also versions of a method of treating with the use of the pharmaceutical composition. There is disclosed a method of B-cell proliferation inhibition on a basis the immunoconjugate. There are described versions of an engineered cystein-substituted antibody specific to CD22 with one or more free cysteines of thiol reactance within the range 0.6 to 1.0. There are disclosed versions of the "antibody-drug" conjugate, the immunoconjugate and pharmaceutical formulaitons for treating disturbed B-cell proliferation. There are also described a method for protein CD22 detection in a sample on the basis of the immunoconjugate, a method for B-cell detection and a method of treating a malignant tumour on the basis of the "antibody-drug" conjugate. There are disclosed: a product for treating disturbed B-cell proliferation on the basis of the pharmaceutical formulation and a method of producing the "antibody-drug" conjugate.

EFFECT: use of the invention provides new specific CD22 antibodies and the based drugs of acceptable therapeutic efficacy with lower toxicity that can find application in therapy of tumours.

227 cl, 25 dwg, 16 tbl, 14 ex

FIELD: medicine.

SUBSTANCE: by recombinant method obtained is fused protein, which contains natural molecule of human erythropoetine with cysteine residue near its C-end and Fc fragment of humal IgG, containing hinge region, N-end of said Fc fragment is connected to said C-end of said erythropoetine molecule, and said Fc fragment is natural, excluding mutation, consisting in substitution of cysteine residue in said hinge region, located the nearest of all to said erythropoetine molecule, with non-cysteine residue, which resulted in the fact that first cysteine residue of said hinge region, located the nearest of all to said N-end, is separated, by, at least, 12 or 17 amino acids from said cysteine residue of said erythropoetine molecule. Obtained peptide is used for stimulation of erythropoesis in mammal.

EFFECT: invention makes it possible to obtain fused protein, which possesses erythropoetine activity, has prolonged time of half-life in vivo in comparison with native human erythropoetine.

43 cl, 20 dwg, 10 ex

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

FIELD: medicine.

SUBSTANCE: there are offered: IL-6 receptor antibody, a coding gene, a vector and a host cell for producing the antibody, a method for producing the antibodies, and a pharmaceutical composition for treating IL-6-related diseases containing the antibody.

EFFECT: use of the invention provides new humanised IL-6 receptor antibodies that can find further application in therapy of the IL-6-mediated diseases.

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

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to a drug for multiple sclerosis which is presented in the form of a pharmaceutical composition and contains an activated potentiating form of human gamma-interferon (IFN-γ) antibodies and an activated potentiating form of brain-specific protein S-100 antibodies as an additive intensifying component. Using the drug in a method for treating multiple sclerosis involves in introduction of the activated potentiating form of human gamma-interferon (IFN-γ) in combination with the intensifying component in the form of the activated potentiating form of very-low-dose affinity purified brain-specific protein S-100 antibodies.

EFFECT: use of the inventions allows higher clinical effectiveness in multiple sclerosis.

9 cl, 1 ex

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