Bifunctional antagonists of tumor necrosis factor-alpha and transforming growth factor-beta and their applications

Abstract

FIELD: biotechnology.SUBSTANCE: bifunctional antagonist molecule that specifically binds to TNF-α and TGF-β ligand. The said molecule comprises any of the amino acid sequences of SEQ ID NO: 30-31 and 42 in combination with any of the amino acid sequences of SEQ ID NO: 11, 15, 19, 23, 27 and 43. The invention also provides a pharmaceutical composition containing such a molecule, as well as a method for treating or preventing conditions such as pulmonary arterial hypertension (PAH) and pulmonary fibrosis.EFFECT: molecule that simultaneously and effectively neutralizes TNF-α -mediated signaling and TGF-β -mediated signal transmission.4 cl, 10 dwg, 8 tbl, 5 ex

Description

Related patent applications

[0001] This application claims priority to U.S. Provisional Application No. 63 / 104,850, filed October 23, 2020, which is incorporated herein by reference in its entirety. Prior Art

[0002] Tumor necrosis factor-α (TNF-α) mediates NF-κB (nuclear factor kappa B)-associated signaling and plays an important role in various physiological and pathological processes, including cell proliferation, differentiation, apoptosis, modulation of immune responses, and the induction of inflammation. TNF acts through two receptors, TNFR1 (TNF receptor-1) and TNFR2 (TNF receptor-2). TNF-α plays a key role in inflammatory responses, programmed cell death, and tissue necrosis. Enhanced TNF-α-mediated signaling is associated with a number of inflammatory diseases, including rheumatoid arthritis, ankylosing spondylitis, Crohn's disease, and psoriasis, and anti-TNF therapeutics such as adalimumab, infliximab, and etanercept have been shown to be highly effective in the treatment of such inflammatory diseases.Elevated TNF-α levels and enhanced TNF-α-mediated signaling are also implicated in the pathogenesis and progression of many other disease states, including anemia, leukemia, multiple myeloma, fibrosis, hypertension, muscle wasting, bone loss, neurodegeneration, sepsis, fibrosis, pain, chronic kidney disease, liver disease, and heart failure.

[0003] Transforming growth factor-beta (TGF-β), including TGF-β1, TGF-β2, and TGF-β3, mediates Smad2 / 3-associated signaling by binding to and activating its high-affinity cell surface receptors TGFβRII and TGFβRIIB. TGF-β plays a critical role in regulating a wide range of biological activities, including immune function, cell proliferation and differentiation, epithelial-mesenchymal transition, fibrogenesis, hematopoiesis, myogenesis, and bone remodeling. Elevated TGF-β levels and subsequent upregulation of Smad2 / 3-mediated signaling are implicated in the pathogenesis and progression of many disease states, including cancer, anemia, bone metastasis, bone loss, fibrosis, pain, muscle loss, insulin resistance, chronic kidney disease, liver disease, and cardiovascular disease.

[0004] Increasing evidence indicates that many complex disorders involve the simultaneous activation of the TNF-α-mediated NF-κB signaling pathway and the TGF-β-mediated Smad2 / 3 signaling pathway, the activities of which contribute to pathogenesis and progression. Examples of such complex disorders include certain hematopoietic disorders such as intractable anemia and myelodysplastic syndromes, cardiovascular diseases such as pulmonary hypertension and congestive heart failure, bone lesions such as bone metastasis and bone fracture, organ failure such as renal failure, liver failure, or myelodystrophy, and fibrosing diseases such as nonalcoholic steatohepatitis, cirrhosis, and pulmonary fibrosis, and pain such as nociceptive or neuropathic pain.

[0005] Current treatment options for these complex disorders are limited. Due to the involvement of more than one disease-associated signaling mechanism in these disorders, currently available therapies, which were designed to target a single disease mechanism, typically have poor efficacy and low response rates. Since both the TNF-α-NF-kB signaling pathway and the TGF-β-Smad2 / 3 signaling pathway are fundamentally involved in the pathogenesis and progression of the disease, it is crucial to develop new bifunctional antagonists that can simultaneously inhibit these two disease-related signaling pathways. Description of the invention

[0006] According to one aspect of the present invention, novel bifunctional antagonist molecules based on polypeptides are provided, specifically designed to simultaneously and effectively neutralize TNF-α-mediated signaling and TGF-β-mediated signaling. In some embodiments, the bifunctional antagonist molecule is designed as shown in FIG. 1. In some embodiments, the bifunctional antagonist molecule is designed as shown in FIG. 2. In some embodiments, the bifunctional antagonist molecule is designed as shown in FIG. 3.

[0007] In some embodiments, the bifunctional antagonist molecule is a bifunctional molecule comprising a first antigen-binding molecule that specifically binds to a TNF-α ligand (“TNF-α-binding polypeptide”) and a second antigen-binding molecule that specifically binds to TGF-β (“TGF-β-binding polypeptide”).In some embodiments, the "TNF-α-binding polypeptide" is selected from the group consisting of an antibody to TNF, a fragment of an antibody to TNF, extracellular domains (ECDs) of wild-type TNFR1 and TNFR2, modified extracellular domains of TNFR1 and TNFR2, and a phage-displayed polypeptide that targets a TNF-α ligand, and the "TGF-β-binding polypeptide" is selected from the group consisting of an antibody to TGF-β, a fragment of an antibody to TGF-β, extracellular domains (ECDs) of type 2 TGF-β receptors (including TGFpRIIA and TGFβRIIB), wild-type extracellular domains of modified TGFβRIIA and TGFβRIIB, and a phage-displayed antagonist polypeptide that targets a TGF-β ligand.

[0008] In some embodiments, the bifunctional molecule comprises an isolated antibody or antigen-binding fragment thereof that specifically binds to TNF-α, and an isolated antibody or antigen-binding fragment thereof that specifically binds to a TGF-β ligand. In some embodiments, the isolated antibody or antigen-binding fragment thereof is selected from the group consisting of monoclonal antibodies (mAbs), polyclonal Abs, Ab fragments (e.g., Fab, Fab', F(ab')2, Fv, Fc, etc.), chimeric Abs, mini-Abs or domain Abs (dAbs), dual-specificity Abs, bispecific Abs, heteroconjugate Abs, single-chain Abs (SCAs), single-chain variable fragments (scFv), humanized Abs, fully human Abs, and any other modified configuration of an immunoglobulin (Ig) molecule that contains an antigen-recognizing site with the desired specificity.In some embodiments, the bifunctional molecule comprises an isolated antibody or antigen-binding fragment thereof selected from the group consisting of a fully human, a humanized, and a chimeric antibody.

[0009] In some embodiments, the first antigen-binding molecule specifically binds to a TNF ligand comprising the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the first antigen-binding molecule specifically binds to a TNF ligand comprising the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the first antigen-binding molecule specifically binds to a TNF ligand comprising the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the first antigen-binding molecule specifically binds to a TNF ligand comprising the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the first antigen-binding molecule specifically binds to a TNF ligand comprising the amino acid sequence set forth in SEQ ID NO: 5.

[0010] In some embodiments, the second antigen-binding molecule specifically binds to a TGF-β ligand comprising the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the second antigen-binding molecule specifically binds to a TGF-β ligand comprising the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the second antigen-binding molecule specifically binds to a TGF-β ligand comprising the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the second antigen-binding molecule specifically binds to a TGF-β ligand comprising the amino acid sequence set forth in SEQ ID NO: 9.

[0011] In some embodiments, the first antigen-binding molecule that specifically binds to a TNF-α ligand is an isolated antibody selected from the group consisting of an antibody comprising the heavy chain amino acid sequence set forth in SEQ ID NO: 10; an antibody comprising the light chain amino acid sequence set forth in SEQ ID NO: 11; an antibody comprising the heavy chain amino acid sequence set forth in SEQ ID NO: 10 and the light chain amino acid sequence set forth in SEQ ID NO: 11; an antibody comprising the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 12; an antibody comprising the light chain variable region amino acid sequence set forth in SEQ ID NO: 13;and an antibody comprising the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO: 12 and the amino acid sequence of the light chain variable region set forth in SEQ ID NO: 13.;

[0012] In some embodiments, the first antigen-binding molecule that specifically binds to a TNF-α ligand is an isolated antibody selected from the group consisting of an antibody comprising the heavy chain amino acid sequence set forth in SEQ ID NO: 14; an antibody comprising the light chain amino acid sequence set forth in SEQ ID NO: 15; an antibody comprising the heavy chain amino acid sequence set forth in SEQ ID NO: 14 and the light chain amino acid sequence set forth in SEQ ID NO: 15; an antibody comprising the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 16; an antibody comprising the light chain variable region amino acid sequence set forth in SEQ ID NO: 17;and an antibody comprising the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO: 16 and the amino acid sequence of the light chain variable region set forth in SEQ ID NO: 17.;

[0013] In some embodiments, the first antigen-binding molecule that specifically binds to a TNF-α ligand is an isolated antibody selected from the group consisting of an antibody comprising the heavy chain amino acid sequence set forth in SEQ ID NO: 18; an antibody comprising the light chain amino acid sequence set forth in SEQ ID NO: 19; an antibody comprising the heavy chain amino acid sequence set forth in SEQ ID NO: 18 and the light chain amino acid sequence set forth in SEQ ID NO: 19; an antibody comprising the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 20; an antibody comprising the light chain variable region amino acid sequence set forth in SEQ ID NO: 21;and an antibody comprising the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO: 20 and the amino acid sequence of the light chain variable region set forth in SEQ ID NO: 21.;

[0014] In some embodiments, the first antigen-binding molecule that specifically binds to a TNF-α ligand is an isolated antibody selected from the group consisting of an antibody comprising the heavy chain amino acid sequence set forth in SEQ ID NO: 22; an antibody comprising the light chain amino acid sequence set forth in SEQ ID NO: 23; an antibody comprising the heavy chain amino acid sequence set forth in SEQ ID NO: 22 and the light chain amino acid sequence set forth in SEQ ID NO: 23; an antibody comprising the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 24; an antibody comprising the light chain variable region amino acid sequence set forth in SEQ ID NO: 25;and an antibody comprising the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO: 24 and the amino acid sequence of the light chain variable region set forth in SEQ ID NO: 25.

[0015] In some embodiments, the second antigen-binding molecule that specifically binds to a TGF-β ligand is an isolated antibody selected from the group consisting of an antibody comprising the heavy chain amino acid sequence set forth in SEQ ID NO: 26; an antibody comprising the light chain amino acid sequence set forth in SEQ ID NO: 27; an antibody comprising the heavy chain amino acid sequence set forth in SEQ ID NO: 26 and the light chain amino acid sequence set forth in SEQ ID NO: 27; an antibody comprising the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 28; an antibody comprising the light chain variable region amino acid sequence set forth in SEQ ID NO: 29;and an antibody comprising the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO: 28 and the amino acid sequence of the light chain variable region set forth in SEQ ID NO: 29.;

[0016] In some embodiments, the bifunctional antagonist molecule is a bifunctional molecule comprising a first antigen-binding molecule that specifically binds to a TNF-α ligand and a second antigen-binding molecule that specifically binds to a TGF-β ligand, wherein the TNF-α ligand-binding molecule is selected from the group of polypeptides comprising the amino acid sequence set forth in SEQ ID NOs: 1-5 and 10-25, and the TGF-β ligand-binding molecule is selected from the group of polypeptides comprising the amino acid sequence set forth in SEQ ID NOs: 6-9 and 26-29.

[0017] In some embodiments, the bifunctional antagonist molecule is a bifunctional molecule comprising a first antigen-binding molecule that specifically binds to a TNF-α ligand and a second antigen-binding molecule that specifically binds to a TGF-β ligand, wherein the bifunctional molecule is selected from the group consisting of: a bifunctional molecule comprising the amino acid sequence of a heavy chain set forth in SEQ ID NO: 30 and the amino acid sequence of a light chain set forth in SEQ ID NO: 11; a bifunctional molecule comprising the amino acid sequence of a heavy chain set forth in SEQ ID NO: 31 and the amino acid sequence of a light chain set forth in SEQ ID NO: 11; and a bifunctional molecule comprising the heavy chain amino acid sequence set forth in SEQ ID NO: 10 and the light chain amino acid sequence set forth in SEQ ID NO: 11.

[0018] In some embodiments, the bifunctional antagonist molecule is a bifunctional molecule comprising a first antigen-binding molecule that specifically binds to a TNF-α ligand and a second antigen-binding molecule that specifically binds to a TGF-β ligand, wherein the bifunctional molecule is selected from the group consisting of: a bifunctional molecule comprising the amino acid sequence set forth in SEQ ID NO: 32; a bifunctional molecule comprising the amino acid sequence set forth in SEQ ID NO: 33; a bifunctional molecule comprising the amino acid sequence set forth in SEQ ID NO: 34; a bifunctional molecule comprising the amino acid sequence set forth in SEQ ID NO: 35; a bifunctional molecule comprising the amino acid sequence set forth in SEQ ID NO: 36; a bifunctional molecule comprising the amino acid sequence set forth in SEQ ID NO: 37;a bifunctional molecule comprising the amino acid sequence set forth in SEQ ID NO: 38; a bifunctional molecule comprising the amino acid sequence set forth in SEQ ID NO: 39; a bifunctional molecule comprising the amino acid sequence set forth in SEQ ID NO: 40; and a bifunctional molecule comprising the amino acid sequence set forth in SEQ ID NO: 41.

[0019] According to another aspect of the present invention, there are provided isolated nucleic acid molecules comprising a polynucleotide encoding a bifunctional antagonist molecule of the present invention. In some embodiments, the isolated nucleic acid molecules comprise the polynucleotides described herein and further comprise a polynucleotide encoding at least one heterologous protein described herein. In some embodiments, the nucleic acid molecules further comprise polynucleotides encoding linkers or hinge linkers described herein.

[0020] According to another aspect of the present invention, there are provided vectors comprising the nucleic acids described herein. In some embodiments, the vector is an expression vector. According to another aspect of the present invention, there are provided isolated cells comprising the nucleic acids of the invention. In some embodiments, the cell is a host cell comprising an expression vector of the invention. According to another aspect, there are provided methods for producing bifunctional antagonist molecules by culturing host cells under conditions conducive to the expression of proteins or polypeptides.

[0021] According to another aspect, a method for producing a bifunctional antagonist molecule is provided, comprising a first antigen-binding molecule that specifically binds to TNF-α and a second antigen-binding molecule that specifically binds to TGF-β, as described in this application, comprising the steps of a) transforming a host cell with vectors containing polynucleotides encoding said bifunctional antagonist molecule, b) culturing the host cell under conditions suitable for expressing the bifunctional antagonist molecule, and c) recovering the bifunctional antagonist molecule from the culture. The invention also encompasses a bifunctional antagonist molecule obtained by the method of the invention.

[0022] According to another aspect of the present invention, there is provided a pharmaceutical composition comprising isolated bifunctional antagonist molecules in admixture with a pharmaceutically acceptable carrier.

[0023] According to another aspect of the present invention, a method for treating or preventing various complex disease states in the pathogenesis of which activation of both the TNF-α-mediated NF-κB signaling pathway and the TGF-β-mediated Smad2 / 3 signaling pathway is involved.

[0024] In some embodiments, the novel bifunctional antagonist molecules of the present invention may find broad application in the treatment of various disorders, including, but not limited to, the following conditions: blood diseases: ineffective erythropoiesis, anemia, pancytopenia, myelodysplastic syndromes; fibrosing diseases: NASH (non-alcoholic steatohepatitis), liver fibrosis, pulmonary fibrosis, renal fibrosis, polycystic kidney disease, myocardial fibrosis, muscle fibrosis, myelofibrosis, skin fibrosis, hand fibrosis, and ocular fibrosis; cancer: multiple myeloma, acute myeloid leukemia, melanoma; cancer treatment in combination with checkpoint inhibitors such as antibodies to programmed death protein 1 (PD1), programmed death protein ligand 1 (PDL1), and cytotoxic T-lymphocyte antigen 4 (CTL4), or using chimeric antigen receptor (CAR) T cells;Neuromuscular disorders: muscular dystrophy, spinal muscular atrophy, spinal cord injury, stroke; Pain: nociceptive pain, neuropathic pain; Wasting disorders: sarcopenia, cancer cachexia, anorexia nervosa; Bone disorders: bone metastasis, bone fragility, bone fracture, osteopenia, osteoporosis; Cardiovascular disorders: pulmonary hypertension, pulmonary arterial hypertension, myocardial infarction, heart failure; Metabolic disorders: insulin resistance, diabetic nephropathy, chronic kidney disease; Inflammatory diseases: rheumatoid arthritis, inflammatory bowel disease; Infections: severe acute respiratory syndrome coronavirus (SARS-CoV), cytokine storm syndrome, sepsis; and Trauma: burn injury.

[0025] According to another aspect of the invention, there are provided uses of bifunctional antagonist molecules for the preparation of a medicament for the treatment of any disorder or condition described in this application.Brief Description of the Figures

[0026] FIG. 1 shows a representative bifunctional antagonist molecule of the present invention. The “TNF-binding polypeptide” illustrated in this diagram refers to any polypeptide having the ability to bind to TNF-α, which includes, but is not limited to, an anti-TNF antibody, an anti-TNF antibody fragment, the extracellular domains (ECDs) of wild-type TNFR1 and TNFR2, the extracellular domains of modified TNFR1 and TNFR2, and a phage-displayed polypeptide that targets TNF-α. The “TGF-β-binding polypeptide” illustrated in this diagram refers to any polypeptide having the ability to bind to TGF-β (i.e.,TGF-β1, TGF-β2, or TGF-β3), which includes, but is not limited to, an anti-TGF-β antibody, an anti-TGF-β antibody fragment, the extracellular domains (ECDs) of wild-type TGF-β receptors type 2 (including TGF-βRIIA and TGF-βRIIB), the extracellular domains of modified TGF-βRIIA and TGF-βRIIB, and phage-displayed antagonist polypeptides that are capable of binding and neutralizing TGF-β. The term "linker" shown in this diagram refers to various methods of fusing different fusion partners to the polypeptide to create bispecific and multispecific molecules, which includes, but is not limited to, the use of any peptide linker or chemical linker.

[0027] FIG. 2 shows two representative bifunctional antagonist molecules of the present invention, wherein: (A) the TNF-α-binding polypeptide is an antibody to TNF-α, and the TGF-β-binding polypeptide is the ECD of the TGF-β receptor attached via a linker to the constant region 3 of the heavy chain (CH3) of the antibody to TNF-α; or (B) the TGF-β-binding polypeptide is an antibody to TGF-β, and the TNF-α-binding polypeptide is the ECD of the TNF receptor attached via a linker to the CH3 of the heavy chain of the antibody to TGF-β. In alternative embodiments, the TGF-β receptor ECD (or TNF receptor ECD) is linked to an anti-TNF-α antibody (or anti-TGF-β antibody) via a linker through the heavy chain variable region (VH) of the antibody. In alternative embodiments, the TGF-β receptor ECD (or TNF receptor ECD) is linked to an anti-TNF-α antibody (or anti-TGF-β antibody) via a linker through the light chain variable region (VL) of the antibody.In alternative embodiments, the TGF-β receptor ECD (or TNF receptor ECD) is linked via a linker to an anti-TNF-α antibody (or anti-TGF-β antibody) at an internal site, rather than at the CH3 sites of the heavy chain, VL, or VH of the antibody.

[0028] FIG. 3 shows a representative bifunctional antagonist molecule of the present invention in the form of a bispecific antibody that comprises (A) variable regions (VH and VL) derived from an anti-TGF-β antibody and (B) variable regions (VH and VL) from an anti-TNF antibody. It should be noted that although the bispecific antibody illustrated in FIG. 3 is shown in one specific configuration, bispecific antibodies comprising variable regions from both an anti-TGF-β antibody and an anti-TNF antibody can be designed by those skilled in the art in a wide variety of configurations.

[0029] FIG. 4 shows line graphs showing that the bifunctional antagonist molecule A119 effectively neutralizes TNF-α, TGF-β1, and TGF-β3 in cell-based assays. The 50% inhibition concentration (IC) values ​​were calculated. 50 ), expressing TNF-α-neutralizing and TGF-β-neutralizing activities at the cellular level, and plotted using Prism software (from GraphPad Software); RLU - relative light units.

[0030] FIG. 5 shows line graphs showing that the bifunctional antagonist molecule A120 effectively neutralizes TNF-α, TGF-β1, and TGF-β3 in cell-based assays. IC values ​​were calculated 50 , expressing TNF-α-neutralizing and TGF-β-neutralizing activities at the cellular level, and plotted using Prism software (from GraphPad Software).

[0031] FIG. 6 shows the changes in proliferation and morphology of primary human pulmonary arterial smooth muscle cells (PASMCs) under different treatment conditions, showing that the bifunctional antagonist A120 was highly effective in preventing TNFα- and TGF-β1-induced hyperplasia and hypertrophy of PASMCs.

[0032] FIG. 7 shows histograms showing that under conditions of elevated TNF-α and TGF-β1 levels, A120 was more effective than anti-TNF antibody or TGFRTI-Fc in preventing both PASMC hyperplasia and hypertrophy. Morphometric analysis of PASMC was performed using ImageJ software.

[0033] FIG. 8 shows histological images of hematoxylin and eosin (H&E)-stained lung sections and a histogram of Ashcroft scores, showing that A120 was more effective than anti-TNF antibody or TGFRII-Fc in attenuating lung tissue injury and fibrosis in mice with bleomycin-induced pulmonary fibrosis.

[0034] FIG. 9 shows histological images of lung sections with Masson trichrome staining and a histogram for quantitative analysis of the collagen deposition area, showing that A120 attenuated pulmonary fibrosis more effectively than the anti-TNF antibody or TGFRII-Fc in bleomycin-treated mice.

[0035] Fig. 10 shows histological images after staining for αSMA (alpha-smooth muscle actin) in lung sections, showing that A120 was more effective than anti-TNF antibody or TGFRII-Fc in preventing the induction of αSMA in lung tissues of bleomycin-treated mice. The lung sections were immunochemically stained with anti-αSMA antibody and horseradish peroxidase (HRP)-labeled secondary antibody. Method(s) for carrying out the invention Definitions

[0036] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer composed of amino acid residues. In some embodiments, "peptides," "polypeptides," and "proteins" are chains of amino acids whose alpha-carbon atoms are linked to each other via peptide bonds. Consequently, the terminal amino acid at one end of the chain (the amino terminus) has a free amino group, while the terminal amino acid at the other end of the chain (the carboxy terminus) has a free carboxyl group. As used herein, the term "amino terminus" (abbreviated N-terminus) refers to the free α-amino group of the amino acid located at the amino terminus of the peptide, or to the α-amino group (imino group, when participating in the formation of the peptide bond) of the amino acid at any other position in the peptide.Similarly, the term "carboxy-terminus" refers to a free carboxyl group at the carboxy-terminus of a peptide or a carboxyl group of an amino acid at any other position in the peptide. Peptides also include essentially any polyamino acid, including, but not limited to, peptide mimetics, such as amino acids linked by an ether linkage as opposed to an amide linkage.

[0037] The polypeptides of the invention include polypeptides modified in any way and for any reason, such as to: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) change binding affinity for forming protein complexes, (4) change binding affinity, and (5) impart other or modify existing physicochemical or functional properties.

[0038] The term "amino acid substitution" as used herein refers to the replacement of one amino acid in a polypeptide at a specific position in the original polypeptide sequence with another amino acid. Amino acid substitutions can be made using genetic or chemical methods well known in the art. For example, single or multiple amino acid substitutions (e.g., conservative amino acid substitutions) can be made in a natural sequence (e.g., at a position in a polypeptide outside the domain(s) forming(s) intermolecular contacts). A "conservative amino acid substitution" refers to the replacement of an amino acid in a polypeptide with a functionally similar amino acid.Each of the six groups below contains amino acids whose substitutions for each other are conservative: 1) alanine (A), serine (S), and threonine (T); 2) aspartic acid (D) and glutamic acid (E); 3) asparagine (N) and glutamine (Q); 4) arginine (R) and lysine (K); 5) isoleucine (I), leucine (L), methionine (M), and valine (V); 6) phenylalanine (F), tyrosine (Y), and tryptophan (W).

[0039] The term "non-conservative amino acid substitution" refers to the replacement of a member of one of these classes with a member of another class. When making such changes according to the various embodiments, the hydrophobicity index of amino acids should be taken into account. Each amino acid has been assigned a hydrophobicity index based on its hydrophobicity and charge characteristics. The indices are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9) and arginine (-4.5).

[0040] The importance of the hydrophobicity index of amino acids in imparting interactive biological function to a protein is known in the art (see, for example, Kyte et al., 1982, J. Mol. Biol., 157: 105-131). It is known that some amino acids can be replaced with other amino acids having a similar hydrophobicity index or value and still maintain similar biological activity. When making changes based on the hydrophobicity index, some embodiments include substitution of amino acids whose hydrophobicity indices are within ±2. Some embodiments include those that are within ±1, and some embodiments include those that are within ±0.5.

[0041] It is also known in the art that substitution of similar amino acids can be effectively carried out taking into account hydrophilicity, particularly if it is intended to create a biologically functional protein or peptide for use in the immunological embodiments described in this application. In some embodiments, the highest local average hydrophilicity of a protein, determined by the hydrophilicity of amino acids located nearby one another, correlates with its immunogenicity and antigenicity, i.e., with the biological property of the protein.

[0042] The following hydrophilicity values ​​were assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±0.1); glutamate (+3.0±0.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±0.1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5) and tryptophan (-3.4). When making changes taking into account similar hydrophilicity values, in some embodiments, the replacement of amino acids whose hydrophilicity values ​​are within ±2 is included, in some embodiments, those that are within ±1 are included, and in some embodiments, those that are within ±0.5 are included.

[0043] Typical amino acid substitutions are shown in Table 1.

[0044] A skilled artisan can identify suitable variants of the polypeptides described herein using well-known techniques. In some embodiments, a skilled artisan can identify suitable regions of the molecule that can be altered without disrupting activity by targeting regions that are not believed to be important for activity. In other embodiments, a skilled artisan can identify residues and portions of the molecule that are conserved among similar polypeptides. In further embodiments, even regions that may be important for biological activity or structure can be subjected to conservative amino acid substitutions without disrupting biological activity or without adversely affecting the structure of the polypeptide.

[0045] Furthermore, a person skilled in the art can review structure-function studies that identify residues in similar polypeptides that are important for activity or structure. Based on such comparisons, the skilled person can predict the importance of amino acid residues in a polypeptide that correspond to amino acid residues important for activity or structure in similar polypeptides. The skilled person can select chemically similar amino acid substitutions for such predicted important amino acid residues.

[0046] A person skilled in the art can also analyze the three-dimensional structure and amino acid sequence relative to that of similar polypeptides. Based on this information, a person skilled in the art can predict the arrangement of amino acid residues in the polypeptide based on its three-dimensional structure. In some embodiments, a person skilled in the art may prefer not to make radical changes to amino acid residues predicted to be on the surface of the polypeptide, as such residues may be involved in important interactions with other molecules. Furthermore, a person skilled in the art can create test variants containing a single amino acid substitution at each desired amino acid residue. These variants can then be screened using activity assays known to those skilled in the art.Such variants can be used to gather information about suitable variants. For example, if a change to a specific amino acid residue was found to result in a disruption, an undesirable reduction, or unacceptable activity, then variants with that change could be prevented from being generated. In other words, based on the information gained from such routine experiments, one skilled in the art can easily identify amino acids for which further substitutions should be avoided, either alone or in combination with other mutations.

[0047] The terms "polypeptide fragment" and "truncated polypeptide" as used herein refer to a polypeptide that has a deletion at the amino terminus and / or carboxy terminus compared to the corresponding full-length protein. In some embodiments, the length of such fragments can be, for example, at least 5, at least 10, at least 25, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1000 amino acids.In some embodiments, the length of the fragments may also be, for example, at most 1000, at most 900, at most 800, at most 700, at most 600, at most 500, at most 450, at most 400, at most 350, at most 300, at most 250, at most 200, at most 150, at most 100, at most 50, at most 25, at most 10, or at most 5 amino acids. The fragment may further comprise, either at one of its two or both ends, one or more additional amino acids, such as an amino acid sequence from another naturally occurring protein (e.g., an Fc or leucine zipper domain) or an artificial amino acid sequence (e.g., an artificial linker sequence).

[0048] The terms "polypeptide variant," "hybrid polypeptide," and "mutant polypeptide" as used herein refer to a polypeptide that comprises an amino acid sequence wherein one or more amino acid residues are inserted into, deleted from, and / or substituted in the amino acid sequence compared to another polypeptide sequence.In some embodiments, the number of amino acid residues to be inserted, deleted, or replaced can be, for example, at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 25, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 350, at least 400, at least 450, or at least 500 amino acids in length. Hybrids of the present invention include fusion proteins.

[0049] A “derivative” of a polypeptide is a polypeptide that has been chemically modified, such as by conjugation with another chemical moiety such as, for example, polyethylene glycol, albumin (e.g., human serum albumin), phosphorylation, and glycosylation.

[0050] The term "% sequence identity" is used interchangeably with the term "% identity" in this specification and refers to the level of amino acid sequence identity between two or more peptide sequences or the level of nucleotide sequence identity between two or more nucleotide sequences when aligned using a sequence alignment program. For example, as used in this specification, 80% identity means the same as 80% sequence identity determined by a specific algorithm and means that a given sequence is at least 80% identical to another sequence of a different length.In some embodiments, the % identity is selected from, for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or higher sequence identity with a given sequence. In some embodiments, the % identity is in the range of, for example, from about 60% to about 70%, from about 70% to about 80%, from about 80% to about 85%, from about 85% to about 90%, from about 90% to about 95%, or from about 95% to about 99%.

[0051] The term "% sequence homology" is used interchangeably with the term "% homology" in this specification and refers to the level of amino acid sequence homology between two or more peptide sequences or the level of nucleotide sequence homology between two or more nucleotide sequences when aligned using a sequence alignment program. For example, as used in this specification, 80% homology means the same as 80% sequence homology determined using a specific algorithm, and accordingly, a homologue of a given sequence has more than 80% sequence homology over the entire length of that sequence.In some embodiments, the % homology is selected from, for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or higher sequence homology with a given sequence. In some embodiments, the % homology is in the range of, for example, from about 60% to about 70%, from about 70% to about 80%, from about 80% to about 85%, from about 85% to about 90%, from about 90% to about 95%, or from about 95% to about 99%.

[0052] Typical computer programs that can be used to detect identity between two sequences include, but are not limited to, the BLAST (Baseline Local Alignment Search Tool) package of programs such as BLASTN (BLAST for nucleotides), BLASTX (a version of BLAST that translates the target nucleotide sequence into the encoded amino acids and then compares it with an existing database of protein amino acid sequences) and TBLASTX (a version of BLAST that translates the target nucleotide sequence into amino acid sequences and then compares it with the translated sequences of a database of sequenced nucleic acids), BLASTP (BLAST for proteins), and TBLASTN (a program for comparing a target amino acid sequence with the translated sequences of a database of sequenced nucleic acids), publicly available online at the National Center for Biotechnology Information (NCBI) website. See alsoAltschul et al., J. Mol. Biol., 215: 403–10, 1990 (with special reference to the published default settings, i.e., w=4, t=17) and Altschul et al., Nucleic Acids Res., 25: 3389–3402, 1997. Sequence searches are typically performed using the BLASTP program by evaluating a given amino acid sequence against amino acid sequences in the GenBank protein sequence database and other published databases. BLASTX is preferred for searching nucleic acid sequences that have been translated in all reading frames by comparison with amino acid sequences in the GenBank protein sequence database and other published databases. Both BLASTP and BLASTX are run using default parameters of a gap-introduction penalty of 11.0 and a gap-extension penalty of 1.0, and using the BLOSUM-62 scoring matrix.

[0053] In addition to calculating the percentage of sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity of two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA, 90: 5873–5787, 1993). One measure of similarity that the BLAST algorithm can produce is the smallest sum probability (P(N)), which estimates the probability with which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid sequence is considered to be similar to a comparison sequence if the smallest sum probability when comparing the test nucleic acid sequence with the comparison nucleic acid sequence is, for example, less than about 0.1, less than about 0.01, or less than about 0.001.

[0054] The term “modification” as used herein refers to any manipulation of the peptide backbone (e.g., amino acid sequence) or any post-translational modifications (e.g., glycosylation) of a polypeptide.

[0055] The term "antigen-binding molecule" as used herein refers in its broadest sense to a molecule that specifically binds to an antigenic determinant. Examples of antigen-binding molecules include antibodies, antibody fragments, and antigen-binding scaffold proteins. "An antigen-binding molecule that binds to the same epitope" as a reference molecule refers to an antigen-binding molecule that blocks the binding of the reference molecule to its antigen in a competitive assay by 50% or more, and conversely, the reference molecule blocks the binding of the antigen-binding molecule to its antigen in a competitive assay by 50% or more.

[0056] As used herein, the term "antigen-binding site" refers to the portion of an antigen-binding molecule that specifically binds to an antigenic determinant. More specifically, the term "antigen-binding site" refers to a portion of an antibody that contains a region that specifically binds to part or all of an antigen and is complementary to part or all of the antigen. If the antigen is large, the antigen-binding molecule can bind only to a specific portion of the antigen, which is called an epitope. The antigen-binding site can be represented, for example, by one or more variable domains (also called variable regions). Preferably, the antigen-binding site comprises the variable region of the light chain (VL) of the antibody and the variable region of the heavy chain (VH) of the antibody.

[0057] As used herein, the term "antigenic determinant" is synonymous with the terms "antigen" and "epitope" and refers to a site (e.g., a continuous stretch of amino acids or a conformational configuration consisting of different stretches of non-contiguous amino acids) on a polypeptide macromolecule to which an antigen-binding moiety binds, forming an antigen-binding moiety-antigen complex. Useful antigenic determinants can be found, for example, on the surfaces of tumor cells, on the surfaces of virally infected cells, on the surfaces of other diseased cells, on the surface of immune cells, in free form in blood serum and / or in the extracellular matrix (ECM). The proteins used in this application as antigens may be any proteins in native form derived from any vertebrate, including mammals, such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified.In some embodiments, the antigen is a human protein.

[0058] The term "antibody" is used herein in the broadest sense and encompasses various antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided that they exhibit the desired antigen-binding activity.

[0059] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a specific source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0060] The term "humanized" antibody refers to a chimeric antibody comprising amino acid residues from the hypervariable regions (HVRs) of a non-human immunoglobulin and amino acid residues from the framework regions (FRs) of a human immunoglobulin. In some embodiments, a humanized antibody will comprise substantially all variable domains, at least one variable domain and typically two variable domains, wherein all or substantially all HVRs (e.g., complementarity determining regions (CDRs)) correspond to those of a non-human antibody, and all or substantially all FRs correspond to those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. The term "humanized form" of an antibody, for example, a non-human antibody, refers to an antibody that has been humanized.Other forms of "humanized antibodies" encompassed by the present invention are those in which the constant region is further modified or altered compared to that of the parent antibody to achieve the properties of the invention, particularly with respect to binding to the first component of the complement system (Clq) and / or binding to the Fc receptor (FcR).

[0061] A "human" antibody is an antibody that has an amino acid sequence corresponding to that of an antibody produced by a human being or a human cell, or that is derived from a non-human source and utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody containing antigen-binding residues from a non-human antibody.

[0062] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope, excluding possible antibody variants, such as those containing naturally occurring mutations or arising during the preparation of the monoclonal antibody preparation, which variants are typically present in small amounts. Unlike polyclonal antibody preparations, which typically include different antibodies that target different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation targets a single determinant of an antigen.

[0063] The term "monospecific" antibody, as used herein, means an antibody that has one or more binding sites, each of which binds to the same epitope on the same antigen. The term "bispecific" means that the antibody has the ability to specifically bind to at least two different antigenic determinants, for example, there are two binding sites, each formed by a pair consisting of the variable domain of the antibody heavy chain (VH) and the variable domain of the antibody light chain (VL), binding to different antigens or to different epitopes on the same antigen. Such a bispecific antibody represents a 1+1 format.Other bispecific antibody formats include 2+1 formats (containing two binding sites for the first antigen or epitope and one binding site for the second antigen or epitope) or 2+2 formats (containing two binding sites for the first antigen or epitope and two binding sites for the second antigen or epitope). Typically, a bispecific antibody contains two antigen-binding sites, each with specificity for a different antigenic determinant.

[0064] The term "valent" as used throughout this application means the presence of a certain number of binding sites in an antigen-binding molecule. In essence, the terms "bivalent," "tetravalent," and "hexavalent" mean the presence of two binding sites, four binding sites, and six binding sites, respectively, in an antigen-binding molecule. The bispecific antibodies of the invention are at least "bivalent" and may be "trivalent" or "multivalent" (e.g., "tetravalent" or "hexavalent"). In some embodiments, the antibodies of the present invention have two or more binding sites and are bispecific. That is, antibodies can be bispecific even in cases where there are more than two binding sites (i.e., when the antibody is trivalent or multivalent).In particular, the invention relates to bispecific divalent antibodies having one binding site for each antigen to which they specifically bind.

[0065] The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to that of a native antibody. "Native antibodies" refer to naturally occurring immunoglobulin molecules with varying structures. For example, native IgG antibodies are heterotetrameric glycoproteins with a molecular weight of approximately 150,000 daltons, composed of two light chains and two heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called the variable heavy domain or heavy chain variable domain, followed by three constant domains (CHI, CH2, and CH3), also called the heavy chain constant regions.Similarly, starting from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called the variable light domain or light chain variable domain, followed by a light chain constant domain (CL), also called the light chain constant region. The heavy chain of an antibody can be classified into one of five types, called alpha (IgA), delta (IgD), epsilon (IgE), gamma (IgG), or mu (IgM), with some further divided into subtypes, such as gamma 1 (IgG1), gamma 2 (IgG2), gamma 3 (IgG3), gamma 4 (IgG4), alpha 1 (IgA1), and alpha 2 (IgA2). The light chain of an antibody can be classified into one of two types, called kappa and lambda, based on the amino acid sequence of its constant domain.

[0066] The term "antibody fragment" refers to a molecule, other than an intact antibody, that contains a portion of an intact antibody that binds to an antigen to which an intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies, triabodies, tetrabodies, cross-linked Fab fragments (crossFab); linear antibodies; single-chain antibody molecules (e.g., scFv); multispecific antibodies derived from antibody fragments; and single-domain antibodies. For a review of some antibody fragments, see Hudson et al., Nat. Med., 9, 129–134 (2003). For a review of scFv fragments, see, e.g., Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp.269-315 (1994); see also WO 93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458.For a discussion of Fab and F(ab')2 fragments containing salvage receptor binding epitope residues and having an increased half-life in vivo, see U.S. Patent No. 5,869,046. Diabodies are antibody fragments with two antigen-binding sites, which may be bivalent or bispecific, see, e.g., EP 404097; WO 1993 / 01161; Hudson et al., Nat. Med., 9, 129–134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90, 6444–6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med., 9, 129–134 (2003). Single-domain antibodies are antibody fragments comprising all or part of the variable domain of an antibody's heavy chain or all or part of the variable domain of an antibody's light chain. In some embodiments, the single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Patent No. 6,248,516 B1).Additionally, antibody fragments comprise single-chain polypeptides that exhibit characteristics of a VH domain, namely, the ability to form complexes with a VL domain, or a VL domain, namely, the ability to form complexes with a VH domain to form a functional antigen-binding site and thereby provide the antigen-binding properties of full-length antibodies. Antibody fragments can be produced by various methods, including, but not limited to, proteolytic cleavage of an intact antibody, as well as production by recombinant host cells (e.g., E. coli or phage), as described in this application.

[0067] Papain digestion of intact antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each containing the variable domains of the heavy and light chains, as well as the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Thus, as used herein, the term "Fab fragment" refers to an antibody fragment containing a light chain fragment containing the VL domain and the constant domain of the light chain (CL), and the VH domain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of several residues at the carboxy terminus of the CH1 domain of the heavy chain, including one or more cysteine ​​residues from the hinge region of the antibody. Fab'-SH are Fab' fragments in which the cysteine ​​residue(s) of the constant domains bear(s) a free thiol group.After treatment with pepsin, a P(ab')2 fragment is formed, which has two antigen-binding sites (two Fab fragments) and part of the Fc region.

[0068] "Single-chain Fab fragment" or "scFab" is a polypeptide consisting of a heavy chain variable domain (VH) of an antibody, a constant domain 1 (CH1) of an antibody, a light chain variable domain (VL) of an antibody, a light chain constant domain (CL) of an antibody and a linker, wherein said antibody domains and said linker are connected in the direction from the N-terminus to the C-terminus according to one of the following orders: a) VH-CH1-linker-VL-CL, b) VL-C1,-linker-VH-CH1, c) VH-linker-VL-CH1 or d) VL-CH1-linker-VH-CL; and wherein said linker is a polypeptide consisting of at least 30 amino acids, preferably 32-50 amino acids. These single-chain Fab fragments are stabilized by a natural disulfide bond between the CL domain and the CH1 domain.In addition, these single-chain Fab molecules can be further stabilized by the formation of interchain disulfide bonds through the insertion of cysteine ​​residues (e.g., at position 44 in the variable region of the heavy chain and at position 100 in the variable region of the light chain according to Kabat numbering).

[0069] A "single-chain variable fragment (scFv)" is a fusion protein based on the variable regions of the heavy (VH) and light (VL) chains of an antibody, connected by a short linker peptide containing ten to about 25 amino acids. Typically, the linker is enriched with glycine residues to impart flexibility and serine or threonine residues to improve solubility and can connect either the N-terminus of VH to the C-terminus of VL or vice versa. Such a protein retains the specificity of the original antibody despite the removal of the constant regions and the introduction of a linker. Antibodies in the form of scFvs are described, for example, in Houston, JS, Methods in Enzymol., 203 (1991) 46-96.In addition, antibody fragments contain single-chain polypeptides that have the characteristics of a VH domain, namely, the ability to form complexes with a VL domain, or a VL domain, namely, the ability to form complexes with a VH domain to form a functional antigen-binding molecule and thereby to provide the antigen-binding properties of full-length antibodies.

[0070] The term "Fc domain" or "Fc region" is used herein to define the C-terminal region of an antibody heavy chain, which contains at least a portion of the constant region. The term includes a native sequence Fc region and a variant Fc region. In particular, the Fc region of the human IgG heavy chain extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. In the amino acid sequences of heavy chains, a C-terminal lysine is always present; however, variants without a C-terminal lysine are also included in the present invention.

[0071] The Fc region of IgG comprises the CH2 domain of IgG and the CH3 domain of IgG. The "CH2 domain" of the Fc region of human IgG typically extends from the amino acid residue at approximately position 231 to the amino acid residue at approximately position 340. In one embodiment, a carbohydrate chain is attached to the CH2 domain. The CH2 domain referred to herein may be a native sequence CH2 domain or a variant CH2 domain. The "CH3 domain" comprises a region of residues from the C-terminus to the CH2 domain in the Fc region (i.e., from the amino acid residue at approximately position 341 to the amino acid residue at approximately position 447 in IgG). The CH3 region referred to herein may be a native sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain with an introduced "protrusion" ("knob") in one chain thereof and with a corresponding introduced "cavity" ("hole") in the other chain thereof; see U.S. Patent No. 5,821,333, expressly incorporated herein by reference).Such CH3 domain variants can be used to promote heterodimerization of two non-identical heavy chains of the antibodies described in this application. Unless otherwise indicated herein, the assignment of amino acid residue numbers in the Fc region or constant region is performed according to the European Union (EU) numbering system, also referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991.

[0072] The knob-into-hole technology is described, for example, in U.S. Patent Nos. 5,731,168, 7,695,936; Ridgway et al., Prot. Eng., 9, 617-621 (1996) and Carter, J. Immunol. Meth., 248, 7-15 (2001). Typically, this method involves introducing a protrusion ("knob") on the interface of a first polypeptide and a corresponding cavity ("cavity") on the interface of a second polypeptide such that the protrusion can be positioned in the cavity to promote heterodimer formation and inhibit homodimer formation. The protrusions are constructed by replacing amino acids with smaller side chains from the interface of the first polypeptide with amino acids with larger side chains (e.g., tyrosine or tryptophan).Compensatory cavities of identical or similar size to the protrusions are created at the interface of the second polypeptide by replacing amino acids with larger side chains with smaller side chains (e.g., alanine or threonine). The protrusion and cavity can be created by altering the nucleic acid encoding the polypeptides, for example, by site-directed mutagenesis or peptide synthesis. In a specific embodiment, the modification resulting in the formation of the protrusion comprises the amino acid substitution T366W in one of the two Fc domain subunits, and the modification resulting in the formation of the cavity comprises the amino acid substitutions T366S, L368A, and Y407V in the second of the two Fc domain subunits.In a further specific embodiment, the Fc domain subunit containing the knob-forming modification further comprises the amino acid substitution S354C, and the Fc domain subunit containing the hole-forming modification further comprises the amino acid substitution Y349C. The introduction of these two cysteine ​​residues results in the formation of a disulfide bridge between the two Fc region subunits, thereby further stabilizing the dimer (Carter, J. Immunol. Methods, 248, 7-15 (2001)).

[0073] The "region equivalent to the immunoglobulin Fc region" is intended to include naturally occurring allelic variants of the immunoglobulin Fc region, as well as variants having changes that result in substitutions, additions, or deletions, but which do not substantially reduce the ability of the immunoglobulin to mediate effector functions (such as antibody-dependent cellular cytotoxicity). For example, one or more amino acids may be deleted from the N-terminus or C-terminus of the immunoglobulin Fc region without significant loss of biological function. Such variants may be selected in accordance with general rules known in the art to have minimal effect on activity (see, e.g., Bowie, JU et al., Science, 247: 1306-10 (1990)).

[0074] The term “effector functions” refers to biological activities that are intrinsic to the Fc region of an antibody and that vary depending on the antibody isotype. Examples of antibody effector functions include: Clq binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (e.g., B cell receptor), and B cell activation.

[0075] An “activating Fc receptor” is an Fc receptor that, upon interaction with the Fc region of an antibody, triggers signaling events that stimulate the cell bearing the receptor to perform effector functions. Activating Fc receptors include Fc gamma receptors (FcγR)IIIa (cluster of differentiation (CD) 16a), FcγRI (CD64), FcγRIIa (CD32), and FcaRI (CD89). A particular activating Fc receptor is human FcγRIIIa (see UniProt, accession no. P08637, version 141).

[0076] A "blocking" antibody or "antagonist" antibody is an antibody that inhibits or reduces the biological activity of the antigen to which it binds. In some embodiments, blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen. For example, the bispecific antibodies of the invention block signaling through TNF-α and TGF-β to inhibit the TNF-α-NF-kB signaling pathway and the TGF-β-Smad2 / 3 signaling pathway.

[0077] As used herein, the term "specific binding" means that such binding to the antigen is selective and can be distinguished from unwanted or non-specific interactions. The ability of an antigen-binding molecule to bind to a specific antigen can be measured either by an enzyme-linked immunosorbent assay (ELISA) or by other methods known to those skilled in the art, such as by surface plasmon resonance (SPR) (as analyzed on a BIAcore instrument) (Liljeblad et al., Glyco. J., 17, 323-329 (2000)) and using conventional binding assays (Heeley, Endocr. Res., 28, 217-229 (2002)).

[0078] The term "affinity" or "binding affinity" as used herein refers to the strength of the total sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). The affinity of a molecule X for its partner Y can typically be represented by the dissociation constant (K D ), which is the ratio of the rate constants of dissociation and association (k off and k on , respectively). A specific method for measuring affinity is surface plasmon resonance (SPR). As used herein, the term "high affinity" antibody refers to an antibody having a K D 10 -9 M or lower and more specifically 10 -10 M or lower for the target antigen. The term "low affinity" antibody refers to an antibody having a K D 10 -8M or higher. The term "weakened binding" as used herein refers to a decrease in the affinity of the respective interaction as measured, for example, by SPR. Conversely, "enhanced binding" refers to an increase in binding affinity for the respective interaction.

[0079] The terms "a bispecific antibody comprising a first antigen-binding molecule that specifically binds to TNF-α and a second antigen-binding molecule that specifically binds to TGF-β", "a bispecific antibody that specifically binds to TNF-α and TGF-β", "a bispecific antigen-binding molecule having specificity for TNF-α and TGF-β" are used interchangeably herein and refer to a bispecific antibody that is capable of binding to TNF-α and TGF-β with sufficient affinity, whereby the antibody is useful as a diagnostic and / or therapeutic agent in the case of targeting TNF-α and TGF-β.

[0080] The terms “anti-TNF-α antibody” and “antibody comprising an antigen-binding site that binds to TNF-α” refer to an antibody capable of binding to TNF-α, especially to a TNF-α polypeptide expressed on the surface of a cell, with sufficient affinity, whereby the antibody is useful as a diagnostic and / or therapeutic agent in the case of targeting TNF-α. In one embodiment, the extent of binding of an antibody to a non-TNF-α protein is less than about 10% of the extent of binding of the antibody to TNF-α as measured, for example, by radioimmunoassay (RIA) or flow cytometry (FACS (fluorescence-activated cell sorting)) or in a surface plasmon resonance assay using a biosensor system such as the Biacore® system.In some embodiments, the antigen-binding molecule that binds to human TNF-α has a K value corresponding to the binding affinity. D for binding to human TNF-α, amounting to, for example, 10 -8 M to 10 -13 M. In one preferred embodiment, the K value corresponding to the binding affinities D determined in a surface plasmon resonance assay using the extracellular domain (ECD) of human TNF-α (TNF-α ECD) for binding affinity to TNF-α. The term "anti-TNF-α antibody" also encompasses bispecific antibodies that are capable of binding to TNF-α and a second antigen.

[0081] The terms "anti-TGF-β antibody" and "antibody comprising an antigen-binding site that binds to TGF-β" refer to an antibody capable of binding to TGF-β, especially to a TGF-β polypeptide expressed on the surface of a cell, with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent for targeting TGF-β. In one embodiment, the extent of binding of the anti-TGF-β antibody to a non-TGF-β protein other than TGF-β is less than about 10% of the extent of binding of the antibody to TGF-β, as measured, for example, by radioimmunoassay (RIA) or flow cytometry (FACS), or in a surface plasmon resonance assay using a biosensor system such as the Biacore® system. In some embodiments, the antigen-binding molecule that binds to human TGF-β has a K value corresponding to the binding affinity Dfor binding to human TGF-β, amounting to, for example, 10 -8 M to 10 -13 M. In one preferred embodiment, the K value corresponding to the binding affinities D determined by surface plasmon resonance using the extracellular domain (ECD) of human TGF-β (TGF-β ECD) for binding affinity to TGF-β. The term "anti-TGF-β antibody" also encompasses bispecific antibodies that are capable of binding to TGF-β and a second antigen.

[0082] The term "fusion protein" as used herein refers to a fusion polypeptide molecule comprising the products of two or more genes that originally encode different proteins, wherein the components of the fusion protein are joined to each other by peptide bonds either directly or through peptide linkers. The term "fused" as used herein refers to components that are joined by peptide bonds either directly or through one or more peptide linkers.

[0083] "Linker" refers to a molecule that connects two other molecules either covalently or through ionic, van der Waals, or hydrogen bonds, such as a nucleic acid molecule that hybridizes to one complementary sequence at the 5' end and to another complementary sequence at the 3' end, thereby joining two non-complementary sequences. "Cleavable linker" refers to a linker that can be cleaved or otherwise disrupted to separate the two components joined by the cleavable linker. Typically, cleavable linkers are cleaved by enzymes, typically peptidases, proteases, nucleases, lipases, and the like. Cleavable linkers can also be cleaved by environmental factors such as changes in temperature, pH, salt concentration, etc.

[0084] The term "peptide linker" as used herein refers to a peptide comprising one or more amino acids, typically about 2-20 amino acids. Peptide linkers are known in the art or described in this application. Suitable non-immunogenic linker peptides include, for example, peptide linkers (G4S) n , (SG4) n or G4(SG4) n Typically, "n" is a number between 1 and 10, typically between 2 and 4.

[0085] "Pharmaceutical composition" refers to a composition suitable for pharmaceutical use in an animal. The pharmaceutical composition contains a pharmacologically effective amount of an active agent and a pharmaceutically acceptable carrier. "Pharmacologically effective amount" refers to that amount of the agent that is effective to achieve the intended pharmacological result. The term "pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers, fillers, buffers and excipients, such as phosphate-buffered saline, 5% aqueous dextrose solution and emulsions such as oil-in-water or water-in-oil emulsions, and wetting agents and / or adjuvants of various types. Suitable pharmaceutical carriers and compositions are described in Remington's Pharmaceutical Sciences, 21st Ed., 2005, Mack Publishing Co, Easton.A "pharmaceutically acceptable salt" is a salt in the form of which a compound can be prepared for pharmaceutical use, including, for example, metal salts (sodium, potassium, magnesium, calcium, etc.) and salts of ammonia or organic amines.

[0086] As used herein, the term "treatment" (and its grammatical variations such as "treat" or "treating") refers to a clinical intervention in an attempt to modify the natural course of a disease in the individual being treated and may be administered either prophylactically or as part of a clinical laboratory diagnosis. Desirable effects of treatment include, but are not limited to, preventing the onset or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, alleviating the intensity of symptoms or temporarily alleviating the disease state, and inducing remission or improving prognosis. As used herein, the term "alleviate" a disease, disorder, or condition means reducing the severity and / or decreasing the frequency of symptoms of the disease, disorder, or condition.In addition, references in this description to "treatment" include references to curative, palliative, and prophylactic treatment.

[0087] The term "effective amount" or "therapeutically effective amount" as used herein refers to an amount of a compound or composition sufficient to treat a particular disorder, condition, or disease, such as to ameliorate, alleviate, reduce, and / or delay one or more of its symptoms. With respect to cancers or other unwanted cell proliferation, an effective amount includes an amount sufficient to: (1) reduce the number of cancer cells; (2) reduce the size of the tumor; (3) suppress, delay, slow to some extent, and preferably stop the infiltration of cancer cells into peripheral organs; (4) suppress (i.e.(5) inhibiting tumor growth; (6) preventing or delaying tumor onset and / or recurrence; and / or (7) alleviating to some extent one or more symptoms associated with cancer. The effective amount may be administered in one or more administration sessions.

[0088] The phrase "administration" or "procurement of administration" refers to the actions taken by a healthcare professional (e.g., a physician) or a person in control of a patient's healthcare who supervises and / or authorizes the administration of the agent(s) / compound(s) dispensed to the patient. Providing administration may include diagnosing and / or determining an appropriate treatment regimen and / or prescribing a specific agent(s) / compound(s) to the patient. Such prescribing may include, for example, filling out a prescription form, noting it in a medical record, and the like. If this application describes administration, the phrase "procurement of administration" is also intended to be used.

[0089] The terms "patient," "individual," and "subject" may be used interchangeably and refer to a mammal, preferably a human or non-human primate, but also to domesticated mammals (e.g., canine or feline), laboratory mammals (e.g., mouse, rat, rabbit, hamster, guinea pig), and farm mammals (e.g., horse, cattle, pig, sheep). In some embodiments, the patient may be a human (e.g., adult male, adult female, adolescent male, adolescent female, child male, child female) under the care of a physician or other healthcare professional) in a hospital, mental health facility, outpatient setting, or other clinical context.In some embodiments, the patient may be an immunocompromised patient or a patient with a weakened immune system, including, but not limited to, patients with primary immunodeficiency, acquired immunodeficiency syndrome (AIDS); cancer patients and transplant patients receiving certain immunosuppressive drugs; and patients with inherited diseases affecting the immune system (e.g., congenital agammaglobulinemia, congenital IgA deficiency). In some embodiments, the patient has an immunogenic cancer, including, but not limited to, bladder cancer, lung cancer, melanoma, and other cancers that have been reported to have a high mutation rate (Lawrence et al., Nature, 499(7457):214-218, 2013).

[0090] The term "immunotherapy" refers to cancer treatment methods that include, but are not limited to, treatment using depleting antibodies to specific tumor antigens; treatment using antibody-drug conjugates; treatment with agonist, antagonist, or blocking antibodies to costimulatory or coinhibitory molecules (immune checkpoints) such as CTLA-4, PD-1, OX-40, CD 137, GITR (glucocorticoid-induced tumor necrosis factor receptor), LAG3 (leukocyte activation gene 3), TIM-3 (T-cell immunoglobulin and mucin domain-containing molecule 3), SIRP (signal regulatory protein), CD40, CD47, Siglec (sialic acid-binding immunoglobulin-like lectin) 8, Siglec 9, Siglec 15, TIGIT (T-cell immunoreceptor with Ig and ITIM (immunoreceptor tyrosine-linked inhibitory motif) domains), and VISTA (suppressor of T-cell activation containing immunoglobulin V region);Treatment with bispecific T cell recruiter antibodies (BiTE®), such as blinatumomab Treatment including administration of biological response modifiers, such as IL-2 (interleukin-2), IL-12, IL-15, IL-21, GM-CSF (granulocyte macrophage colony-stimulating factor), IFN-α (interferon-alpha), IFN-β and IFN-γ Treatment with therapeutic vaccines, such as sipuleucel-T Treatment with bacillus Calmette-Guérin (BCG) Treatment with dendritic cell vaccines or peptide vaccines based on tumor antigens Treatment with chimeric antigen receptor (CAR) T cells Treatment with CAR-expressing natural killer (CAR-NK) cells Treatment with tumor infiltrating lymphocytes (TILs) treatment using adoptive transfer of antitumor T cells (ex vivo expanded and / or transgenic for TCR (T cell receptor)); treatment using TALL-104 cells;and treatment with immunostimulatory agents such as toll-like receptor (TLR) CpG agonists and imiquimod.

[0091] The term "resistant or poorly responsive cancer" refers to tumor cells or cancer that are not responsive to previous anticancer therapy, including, for example, chemotherapy, surgery, radiation therapy, stem cell transplantation, and immunotherapy. Tumor cells may be resistant or poorly responsive at the beginning of treatment, or they may become resistant or poorly responsive as treatment progresses. Poorly responsive tumor cells include tumor cells that do not respond at the beginning of treatment or respond initially for a short period of time but are not responsive to treatment. Poorly responsive tumor cells also include tumor cells that are responsive to anticancer therapy but do not respond to subsequent therapies.For the purposes of this invention, difficult-to-treat tumor cells also encompass tumor cells that appear to be suppressed by anticancer therapy but recur within five years, sometimes up to ten years or longer, after treatment is stopped. Anticancer therapy may employ chemotherapy alone, radiation alone, targeted therapy alone, immunotherapy alone, surgery alone, or combinations thereof. For ease of description and without limitation, the terms "difficult-to-treat tumor cells" and "resistant tumor" are used interchangeably.

[0092] The term "polymer" as used in this description generally includes, but is not limited to, homopolymers; copolymers, such as, for example, block copolymers, graft, random and alternating copolymers; and terpolymers; and blends and variants thereof. Furthermore, unless otherwise specifically limited, the term "polymer" will include all possible geometric configurations of the substance. Such configurations include, but are not limited to, isotactic, syndiotactic and random configurations.

[0093] The term "polynucleotide" refers to a polymer composed of nucleotide units. Polynucleotides include naturally occurring nucleic acids such as deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA"), as well as nucleic acid analogs. Nucleic acid analogs include those that contain non-naturally occurring bases, nucleotides that form linkages with other nucleotides other than the natural phosphodiester bond, or that contain bases attached via linkages other than phosphodiester bonds. Thus, nucleotide analogs include, for example and without limitation, phosphorothioates, phosphorodithioates, phosphorotriesters, phosphoramidates, boranophosphates, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, peptide nucleic acids (PNA), and the like. Such polynucleotides can be synthesized, for example, using an automated DNA synthesizer.Typically, the term "nucleic acid" refers to larger polynucleotides. The term "oligonucleotide" typically refers to shorter polynucleotides, typically consisting of no more than approximately 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C), with "U" replacing "T."

[0094] In this application, conventional notations are used to describe polynucleotide sequences: the left end of a single-stranded polynucleotide sequence is the 5' end; the left direction of a double-stranded polynucleotide sequence is called the 5' direction. The direction from 5' to 3', in which nucleotides are added to nascent RNA transcripts, is called the direction of transcription. The strand of DNA having the same sequence as the mRNA is called the "coding strand"; sequences on the DNA strand having the same sequence as the mRNA transcribed from it and which are located in the 5' direction relative to the 5' end of the RNA transcript are called "upstream sequences"; Sequences on the DNA strand that have the same sequence as the RNA and that are located in the 3' direction relative to the 3' end of the encoded RNA transcript are called "downstream sequences."

[0095] The term "complementary" refers to the topological compatibility or match between the interacting surfaces of two polynucleotides. As a result, these two molecules can be described as complementary, and furthermore, the characteristics of the contact surface are complementary. A first polynucleotide is complementary to a second polynucleotide if the nucleotide sequence of the first polynucleotide is substantially identical to the nucleotide sequence of the polynucleotide-binding partner of the second polynucleotide, or if the first polynucleotide can hybridize with the second polynucleotide under stringent hybridization conditions.

[0096] The term "hybridizes specifically to" or "specific hybridization" or "hybridizes selectively to" refers to the binding, duplex formation, or hybridization of a nucleic acid molecule preferentially to a specific nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., in total cellular DNA or RNA). The term "stringent conditions" refers to conditions under which each probe will hybridize preferentially to its target subsequence and to a lesser extent to other sequences, or will not hybridize to them at all. "Stringent hybridization" and "stringent hybridization wash conditions" in the context of nucleic acid hybridization experiments such as Southern hybridization and Northern hybridization are sequence-dependent and vary under different environmental conditions.A detailed guide to nucleic acid hybridization can be found in Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, part I, chapter 2, "Overview of principles of hybridization and the strategy of nucleic acid probe assays," Elsevier, NY; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 3. rd ed., NY; and Ausubel et al., eds., Current Edition, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, NY.

[0097] Generally, the high stringency hybridization and washing conditions are selected so that the temperature is approximately 5°C lower than the melting temperature (T пл ) for a specific sequence at specific values ​​of ionic strength and pH. T плis the temperature (at specific ionic strength and pH values) at which the target sequence hybridizes 50% with a perfectly matched probe. Very stringent conditions are chosen such that the temperature is equal to T плfor a particular probe. An example of stringent hybridization conditions for hybridization of complementary nucleic acids that have more than about 100 complementary residues on a Southern or Northern blot filter is 50% formalin with 1 mg heparin at 42°C, with hybridization performed overnight. An example of high-stringency wash conditions is a wash with 0.15 M NaCl at 72°C for about 15 minutes. An example of stringent wash conditions is a wash with 0.2 x SSC at 65°C for 15 minutes. See Sambrook et al. for a description of an SSC-based buffer. A high-stringency wash may be preceded by a low-stringency wash to remove background signal from the probe. A typical medium stringency wash for a duplex containing, for example, more than about 100 nucleotides, is a wash with 1 x SSC at 45°C for 15 minutes.A typical low-stringency wash for a duplex containing, for example, more than approximately 100 nucleotides, is 4-6 x SSC at 40°C for 15 minutes. Generally, a signal-to-noise ratio (SNR) that is 2-fold (or greater) greater than that of an unrelated probe in a given hybridization assay indicates detection of specific hybridization.

[0098] The term "primer" refers to a polynucleotide that has the ability to specifically hybridize to a specified polynucleotide template and provide an initiation point for the synthesis of a complementary polynucleotide. Such synthesis occurs when the polynucleotide primer is placed under conditions that allow synthesis, i.e., the presence of nucleotides, a complementary polynucleotide template, and a polymerization agent such as DNA polymerase. A primer is typically single-stranded, but may be double-stranded. Primers are typically deoxyribonucleic acids, but a wide variety of synthetic and natural primers are useful for many applications. A primer is complementary to the template to which it is designed to hybridize and serves as a site for the initiation of synthesis, but does not necessarily reflect the exact sequence of the template. In such a case, specific hybridization of the primer to the template depends on the stringency of the hybridization conditions.Primers can be labeled with, for example, chromogenic, radioactive or fluorescent moieties and can be used as detectable moieties.

[0099] The term "probe," when used in reference to a polynucleotide, refers to a polynucleotide capable of specifically hybridizing to a specified sequence of another polynucleotide. A probe specifically hybridizes to a target complementary polynucleotide but does not necessarily reflect the exact complementary sequence of the template. In such cases, specific hybridization of the probe to the target depends on the stringency of the hybridization conditions. Probes may be labeled with labels, such as chromogenic, radioactive, or fluorescent moieties, and these can be used as detectable moieties. In cases where a probe provides an initiation point for the synthesis of a complementary polynucleotide, this probe can also serve as a primer.

[0100] A "vector" is a polynucleotide that can be used to introduce another nucleic acid linked to it into a cell. One type of vector is a "plasmid," which refers to a linear or circular double-stranded DNA molecule into which additional nucleic acid segments can be ligated. Another type of vector is a viral vector (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), in which additional DNA segments can be introduced into the viral genome. Some vectors are capable of autonomous replication within the host cell into which they are introduced (e.g., bacterial vectors containing a bacterial origin of replication and mammalian episomal vectors). Other vectors (e.g., non-episomal mammalian vectors) integrate into the host cell's genome after introduction into the host cell and are subsequently replicated along with the host genome.An "expression vector" is a type of vector that can direct the expression of a selected polynucleotide.

[0101] A "regulatory sequence" is a nucleic acid that affects the expression (e.g., the level, duration, or location of expression) of a nucleic acid to which it is operably linked. For example, a regulatory sequence may exert its effect on the regulated nucleic acid directly or through the action of one or more other molecules (e.g., polypeptides that bind the regulatory sequence and / or the nucleic acid). Examples of regulatory sequences include promoters, enhancers, and other expression-regulating elements (e.g., polyadenylation signals). Other examples of regulatory sequences are described, for example, in Goeddel, 1990, Gene Expression Technology: Methods in Enzymology, 185, Academic Press, San Diego, Calif, and Baron et al., 1995, Nucleic Acids Res., 23: 3605-06.A nucleotide sequence is "operably linked" to a regulatory sequence if the regulatory sequence affects the expression (e.g., the level, duration, or location of expression) of the nucleotide sequence.

[0102] A "host cell" is a cell that can be used to express a polynucleotide of the invention. The host cell can be a prokaryotic cell, such as E. coli, or it can be a eukaryotic cell, such as a unicellular eukaryotic cell (e.g., a yeast or other fungus), a plant cell (e.g., a cell of a plant such as tobacco or tomato), an animal cell (e.g., a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or an insect cell), or a hybridoma. Typically, the host cell is a cultured cell that can be transformed or transfected with a nucleic acid encoding a polypeptide, which can then be expressed in the host cell. The phrase "recombinant host cell" can be used to refer to a host cell that has been transformed or transfected with a nucleic acid to be expressed.A host cell may also be a cell that contains a nucleic acid but does not express it at the desired level, unless a regulatory sequence is introduced into the host cell that becomes operably linked to the nucleic acid. Clearly, the term "host cell" refers not only to the specific cell being claimed but also to the progeny or potential progeny of such a cell. Since certain modifications may occur in subsequent generations due to, for example, mutation or environmental influences, such progeny may not actually be identical to the parent cell, but they are still included within the scope of the term as used herein.

[0103] The term "isolated molecule" (where the molecule is, for example, a polypeptide or a polynucleotide) is a molecule that, by virtue of its origin or source, (1) is not associated with naturally associated components that accompany it in its native state, (2) is substantially free of other molecules of the same species, (3) is expressed by a cell of a different species, or (4) is not found in nature. Thus, a molecule that is chemically synthesized or expressed in a cellular system different from the cell from which it naturally originates will be "isolated" from the components with which it is naturally associated. Isolation using purification methods well known in the art can also produce a molecule that is substantially free of naturally associated components.The purity or homogeneity of molecules can be analyzed using a variety of techniques well known in the art. For example, the purity of a polypeptide sample can be analyzed using polyacrylamide gel electrophoresis and staining the gel to visualize the polypeptide using methods well known in the art. For certain purposes, higher resolution can be achieved using high-performance liquid chromatography (HPLC) or other purification techniques well known in the art.

[0104] A protein or polypeptide is considered "substantially pure," "substantially homogeneous," or "substantially purified" if at least about 60% to 75% of the sample contains one type of polypeptide. The polypeptide or protein may be monomeric or multimeric. A substantially pure polypeptide or protein will typically contain about 50%, 60%, 70%, 80%, or 90% (w / w) of the protein in the sample, most commonly about 95%, and will preferably be greater than 99% pure. The purity or homogeneity of a protein can be determined by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein sample followed by visualization of a single polypeptide band by staining the gel with a dye well known in the art. For certain purposes, higher resolution can be achieved using HPLC or other means well known in the purification art.

[0105] The term "label" or "labeled" as used herein refers to the incorporation of another molecule into an antibody. In one embodiment, the label is a detectable marker, such as the incorporation of a radiolabeled amino acid or the attachment of biotinylated moieties to a polypeptide that can be detected using labeled avidin (e.g., streptavidin containing a fluorescent marker), or enzymatic activity that can be detected using optical or calorimetric methods). In another embodiment, the label or marker may be a therapeutic agent, such as a drug- or toxin-based conjugate. Various methods for labeling polypeptides and glycoproteins are known in the art and can be used. Examples of labels for polypeptides include, but are not limited to, the following: radioactive isotopes or radionuclides (e.g., 3 H, 14 WITH,15 N, 35 S, 90 Y, 99 Тс, 111 In, 125 I, 131I), fluorescent labels (e.g., FITC (fluorescein isothiocyanate), rhodamine, lanthanide-phosphorus complexes), enzymatic labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinylated groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., paired leucine zipper sequences, binding sites for secondary antibodies, metal-binding domains, epitope labels), magnetic agents such as gadolinium chelates, toxins such as pertussis toxin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoside, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracinatedione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin and their analogs or homologues.In some embodiments, the labels are attached via spacer arms of varying lengths to reduce potential steric hindrance.

[0106] The term "heterologous" as used herein refers to a composition or state that is not native or does not occur in nature, such as one that can be achieved by replacing a naturally occurring composition or state with one derived from another source. Similarly, expression of a protein in an organism different from the organism in which the protein is naturally expressed constitutes a heterologous expression system, and the protein is a heterologous protein.

[0107] It is understood that the aspects and embodiments of the invention described in this application include “consisting of” and / or “consisting essentially of” aspects and embodiments.

[0108] The use of the term "about" in relation to a value or parameter given in this description includes (and describes) variations that relate to this value or parameter as such. For example, a description referring to the term "about X" includes a description of "X" itself.

[0109] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It is understood that aspects and variants of the invention described in this application include "consisting of" and / or "consisting essentially of" aspects and variants. Tumor necrosis factor ligands

[0110] TNF is an immune-modulating cytokine essential for immune processes. Unregulated TNF activity can lead to the development of inflammatory diseases. Excessive amounts of TNF expressed in cells are associated with the development of immune diseases, including rheumatoid arthritis, Crohn's disease, psoriatic arthritis, and inflammatory bowel disease. TNF function requires binding to its two receptors, TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2). Blocking the interaction of TNF with TNFR has been successfully used in the development of a therapeutic method for the treatment of inflammatory or autoimmune diseases.

[0111] In some embodiments, the bifunctional antagonist of the present invention is a bifunctional molecule comprising a first antigen-binding molecule that specifically binds to a TNF-α ligand and a second antigen-binding molecule that specifically binds to a TGF-β ligand. In some embodiments, the bifunctional molecule has the ability to bind to a TNF-α ligand having an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NO: 1-5:

[0112] In some embodiments, the bifunctional molecule has the ability to bind to a TNF-α ligand having an amino acid sequence selected from the group consisting of the amino acid sequences shown in Table 2. TGF-β ligands

[0113] Transforming growth factor-beta (TGF-β), including TGF-β1, TGF-β2, and TGF-β3, mediates Smad2 / 3 signaling by binding to and activating their high-affinity cell surface receptors TGFβRII and TGFβRIIB. TGF-β plays a critical role in regulating a wide range of biological activities, including immune function, cell proliferation and differentiation, epithelial-mesenchymal transition, fibrogenesis, hematopoiesis, myogenesis, bone remodeling, and cancer progression and metastasis. Elevated TGF-β levels and subsequent upregulation of Smad2 / 3 signaling are implicated in the pathogenesis and progression of many disease states, including cancer, anemia, bone metastasis, bone loss, fibrosis, pain, muscle loss, insulin resistance, chronic kidney disease, liver disease, and cardiovascular disease.

[0114] In some embodiments, the bifunctional molecule of the present invention has the ability to bind to a TGF-β ligand having an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NO: 6-9: human TGF-β receptor II, isoform 1 Human TGF-β receptor II ECD, isoform 1 (TGF-β RIIB-ECD) human TGF-β receptor II isoform 2 Human TGF-β receptor II ECD, isoform 2 (TGF-β RIIA-ECD)

[0115] In some embodiments, the bifunctional molecule has the ability to bind to a TGF-β ligand having an amino acid sequence selected from the group consisting of the amino acid sequences shown in Table 3. Antibodies to TNF-α and / or TGF-β and antibody fragments

[0116] Methods for creating new antibodies that bind to ligands and / or receptors of TNF-α and / or TGF-β are known to those skilled in the art. For example, a method for creating a monoclonal antibody that specifically binds to a ligand of TNF-α and / or TGF-β may include administering to a mouse an amount of an immunogenic composition containing a ligand of TNF-α and / or TGF-β, effective to stimulate a detectable immune response, obtaining antibody-producing cells (e.g., spleen cells) from the mouse and fusing these antibody-producing cells with myeloma cells to obtain antibody-producing hybridomas and testing the antibody-producing hybridomas to identify a hybridoma producing a monoclonal antibody that specifically binds to a ligand of TNF-α and / or TGF-β.Once produced, the hybridoma can be expanded in cell culture, possibly under conditions whereby the hybridoma-derived cells produce a monoclonal antibody that specifically binds to the TNF-α and / or TGF-β ligand. The monoclonal antibody can be purified from the cell culture. A variety of techniques are then used to test the antigen / antibody interaction to identify particularly desirable antibodies.

[0117] Other suitable methods can be used to produce or isolate antibodies with the appropriate specificity, including, for example, methods that involve selecting a recombinant antibody from a library or that rely on immunization of transgenic animals (e.g., mice) capable of producing a complete repertoire of human antibodies. See, for example, Jakobovits et al., Proc. Natl. Acad. Sci. (USA), 90: 2551-2555, 1993; Jakobovits et al., Nature, 362: 255-258, 1993; Lonberg et al., U.S. Patent No. 5,545,806; and Surani et al., U.S. Patent No. 5,545,807.

[0118] Antibodies can be engineered in a variety of ways. They can be produced as single-chain antibodies (including small modular immunopharmaceuticals or SMIP™), Fab and F(ab')2 fragments, and so on. Antibodies can be humanized, chimerized, deimmunized, or fully human. Numerous publications describe many types of antibodies and methods for engineering such antibodies. For example, see U.S. Patent Nos. 6,355,245, 6,180,370, 5,693,762, 6,407,213, 6,548,640, 5,565,332, 5,225,539, 6,103,889, and 5,260,203.

[0119] Chimeric antibodies can be produced by recombinant DNA methods known in the art. For example, a gene encoding the Fc constant region of a mouse (or other species) monoclonal antibody molecule is digested with restriction enzymes to remove the portion encoding the mouse Fc region and replaced with the equivalent portion of a gene encoding the Fc constant region of a human antibody (see Robinson et al., International Patent Application Publication PCT / US86 / 02269; Akira, et al., European Patent Application 184187; Taniguchi, M., European Patent Application 171496; Morrison et al., European Patent Application 173494; Neuberger et al., International Patent Application WO 86 / 01533; Cabilly et al., U.S. Patent No. 4,816,567; Cabilly et al., European Patent Application 125023; Better et al., Science, 240:1041-1043, 1988; Liu et al., Proc. Natl. Acad. Sci. (USA), 84: 3439-3443, 1987; Liu et al., J. Immunol., 139: 3521-3526, 1987; Sun et al., Proc. Natl. Acad. Sci.(USA), 84: 214-218, 1987; Nishimura et al., Cane. Res., 47: 999-1005, 1987; Wood et al., Nature, 314: 446-449, 1985; Shaw et al., J. Natl. Cancer Inst, 80: 1553-1559, 1988).

[0120] Methods for humanizing antibodies are described in the art. In some embodiments, a humanized antibody has one or more amino acid residues introduced from a non-human source in addition to CDRs from a non-human source. Humanization can be carried out essentially according to the method of Winter and co-workers (Jones et al., Nature, 321: 522-525, 1986; Riechmann et al., Nature, 332: 323-327, 1988; Verhoeyen et al., Science, 239: 1534-1536, 1988), by replacing hypervariable region sequences with the corresponding sequences from a human antibody. Accordingly, such “humanized” antibodies are chimeric antibodies (US Patent No. 4,816,567), in which a substantially smaller region than the variable region of an intact human antibody is replaced with the corresponding sequence from a non-human species.In practice, humanized antibodies are usually human antibodies in which some hypervariable region residues and possibly some framework residues have been replaced by residues from analogous sites in rodent antibodies.

[0121] U.S. Patent No. 5,693,761 to Queen et al. describes an improvement on Winter et al.'s method for humanizing antibodies and is based on the premise that loss of avidity is attributed to problems associated with structural motifs in the humanized framework that, due to steric or other chemical incompatibility, interfere with the folding of the CDRs into the binding conformation found in the murine antibody. To address this issue, Queen reports using human antibody framework sequences that have close linear peptide sequence homology to the framework sequences of the murine antibody to be humanized. Accordingly, Queen's methods focus on cross-species comparison of framework sequences.Typically, all available human antibody variable region sequences are compared to a specific mouse antibody sequence, and the percent identity for the corresponding framework residues is calculated. To obtain framework sequences for humanization work, the human antibody variable region with the highest percent identity is selected. Queen also reports the importance of preserving certain amino acid residues from the mouse antibody framework in the humanized framework, which are essential for maintaining the CDR in a binding-competent conformation. Potential criticality is assessed using molecular models. Typically, candidate residues for preservation are those located in a linear sequence near the CDR region or physically within 6 Å (0.6 nm) of any CDR residue.

[0122] Another method for humanizing antibodies, called "framework shuffling," involves creating a combinatorial library with the variable CDR regions from a non-human antibody fused in frame to a pool of individual human germline antibody frameworks (Dall'Acqua et al., Methods, 36:43, 2005). These libraries are then screened to identify clones encoding humanized antibodies that retain good binding.

[0123] Methods for creating fully human antibodies are described in the art. For example, a method for producing an antibody to TNF-α or an antigen-binding fragment thereof includes the steps of synthesizing a library of human antibodies using phage, screening this library using a TNF-α polypeptide or an antibody-binding portion thereof, isolating phage that has bound to the TNF-α polypeptide, and producing an antibody from this phage.As another example, one method for producing an antibody library for use in phage display methods comprises the steps of immunizing a non-human animal containing human immunoglobulin loci with a TNF-α polypeptide or an antigenic portion thereof to generate an immune response, recovering antibody-producing cells from the immunized animal, isolating RNA encoding the heavy and light chains of the antibodies of the invention from the recovered cells, reverse-transcriptizing the RNA to produce complementary DNA (cDNA), amplifying this cDNA using primers, and inserting this cDNA into a phage display vector so that the antibodies are expressed on the surface of the phage. Recombinant anti-TNF-α antibodies of the invention can be produced in this manner.

[0124] The recombinant human antibodies to TNF-α and / or TGF-β of the invention can also be isolated by screening a combinatorial library of recombinant antibodies. Preferably, the library is a phage display library for scFv generated using cDNA for V L and V Hhuman phage display libraries derived from mRNA isolated from B cells. Methods for generating and screening such libraries are known in the art. Kits for generating phage display libraries are commercially available (e.g., Pharmacia Recombinant Phage Antibody System, catalog #27-9400-01; and Stratagene SurfZAP™ Phage Display Kit, catalog #240612). There are also other methods and reagents that can be used to generate and screen antibody libraries (see, e.g., U.S. Patent No. 5,223,409; PCT Application Publication Nos. WO 92 / 18619, WO 91 / 17271, WO 92 / 20791, WO 92 / 15679, WO 93 / 01288, WO 92 / 01047, WO 92 / 09690; Fuchs et al., Bio / Technology, 9: 1370–1372 (1991); Hay et al., Hum. Antibod. Hybridomas, 3: 81–85, 1992; Huse et al., Science, 246: 1275–1281, 1989; McCafferty et al., Nature, 348: 552-554, 1990; Griffiths et al., EMBO J., 12: 725-734, 1993; Hawkins et al., J. Mol. Biol., 226: 889-896, 1992; Clackson et al., Nature, 352: 624-628, 1991; Gram et al., Proc. Natl. Acad.Sci. (USA), 89: 3576-3580, 1992; Garrad et al., Bio / Technology, 9: 1373-1377, 1991; Hoogenboom et al., Nuc. Acid Res., 19: 4133-4137, 1991; and Barbas et al., Proc. Natl. Acad. Sci. (USA), 88: 7978-7982, 1991), all of which are incorporated herein by reference.

[0125] Human antibodies are also produced by immunizing a non-human transgenic animal that contains several or all of the human heavy chain and light chain loci with a human IgE antigen, such as a XenoMouse™ mouse (Abgenix, Inc. / Amgen, Inc.-Fremont, Calif.). XenoMouse™ mice are engineered mouse strains that contain large fragments of the human heavy chain and light chain loci and lack the ability to produce mouse antibodies. See, e.g., Green et al., Nature Genetics, 7: 13-21, 1994 and U.S. Patent Nos. 5916771, 5939598, 5985615, 5998209, 6075181, 6091001, 6114598, 6130364, 6162963, and 6150584. XenoMouse™ mice produce a fully human antibody repertoire similar to an adult human and generate antigen-specific human antibodies.In some embodiments, XenoMouse™ mice comprise approximately 80% of the human antibody V gene repertoire by introducing fragments, in a human germline configuration, of the heavy chain loci and the kappa light chain loci of several meganucleotides into a yeast artificial chromosome (YAC). In other embodiments, XenoMouse™ mice further comprise approximately the entire human lambda light chain locus. See Mendez et al., Nature Genetics, 15: 146-156, 1997; Green and Jakobovits, J. Exp. Med., 188: 483-495, 1998; and WO 98 / 24893. According to one aspect of the present invention, a method is provided for producing antibodies to TNF-α and / or TGF-β from non-human, non-mice animals by immunizing non-human transgenic animals that contain human immunoglobulin loci with a TNF-α and / or TGF-β polypeptide. Such animals can be created using the methods described in the aforementioned documents. Antibodies to TNF-α.

[0126] The anti-TNF-α antibody, adalimumab (HUMIRA® by Abbvie; DrugBank DB00051), approved for use by the U.S. Food and Drug Administration (FDA), has been used to treat humans. In some embodiments of the present invention, the anti-TNF-α antibody is a human antibody or antigen-binding fragment comprising the heavy chain amino acid sequence set forth in SEQ ID NO: 10: a human antibody or antigen-binding fragment comprising the light chain amino acid sequence set forth in SEQ ID NO: 11: or a human antibody or antigen-binding fragment comprising the heavy chain amino acid sequence set forth in SEQ ID NO: 10 and the light chain amino acid sequence set forth in SEQ ID NO: 11; a human antibody or antigen-binding fragment comprising the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 12: , a human antibody or antigen-binding fragment comprising the amino acid sequence of the light chain variable region set forth in SEQ ID NO: 13: or a human antibody or antigen-binding fragment comprising the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO: 12 and the amino acid sequence of the light chain variable region set forth in SEQ ID NO: 13.

[0127] In some embodiments, the invention provides antibodies comprising a heavy chain, a light chain, or both a heavy chain and a light chain; a heavy chain variable region, a light chain variable region, or both a heavy chain variable region and a light chain variable region; wherein the heavy chain, light chain, heavy chain variable region, or light chain variable region comprises a sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% identity to the amino acid sequences as set forth in SEQ ID NO: 10, 11, 12, or 13; Moreover, this antibody specifically binds to human TNF-α.

[0128] The FDA-approved anti-TNF-α antibody, infliximab (REMICADE® by Centocor; DrugBank DB00065), has been used to treat humans. In some embodiments of the present invention, the anti-TNF-α antibody is a human antibody or antigen-binding fragment comprising the heavy chain amino acid sequence set forth in SEQ ID NO: 14: a human antibody or antigen-binding fragment comprising the light chain amino acid sequence set forth in SEQ ID NO: 15: or a human antibody or antigen-binding fragment comprising the heavy chain amino acid sequence set forth in SEQ ID NO: 14 and the light chain amino acid sequence set forth in SEQ ID NO: 15; a human antibody or antigen-binding fragment comprising the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 16: a human antibody or antigen-binding fragment comprising the amino acid sequence of a light chain variable region as set forth in SEQ ID NO: 17: or a human antibody or antigen-binding fragment comprising the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO: 16 and the amino acid sequence of the light chain variable region set forth in SEQ ID NO: 17.

[0129] In some embodiments, the invention provides antibodies comprising a heavy chain, a light chain, or both a heavy chain and a light chain; a heavy chain variable region, a light chain variable region, or both a heavy chain variable region and a light chain variable region; wherein the heavy chain, light chain, heavy chain variable region, or light chain variable region comprises a sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% identity to the amino acid sequences as set forth in SEQ ID NO: 14, 15, 16, or 17; Moreover, this antibody specifically binds to human TNF-α.

[0130] The FDA-approved anti-TNF-α antibody, certolizumab pegol (CIMZIA® from UCB; DrugBank DB08904), has been used to treat humans. In some embodiments of the present invention, the anti-TNF-α antibody is a human antibody or antigen-binding fragment comprising the heavy chain amino acid sequence set forth in SEQ ID NO: 18: a human antibody or antigen-binding fragment comprising the light chain amino acid sequence set forth in SEQ ID NO: 19: or a human antibody or antigen-binding fragment comprising the heavy chain amino acid sequence set forth in SEQ ID NO: 18 and the light chain amino acid sequence set forth in SEQ ID NO: 19; a human antibody or antigen-binding fragment comprising the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 20: a human antibody or antigen-binding fragment comprising the amino acid sequence of a light chain variable region as set forth in SEQ ID NO: 21: or a human antibody or antigen-binding fragment comprising the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO: 20 and the amino acid sequence of the light chain variable region set forth in SEQ ID NO: 21.

[0131] In some embodiments, the invention provides antibodies comprising a heavy chain, a light chain, or both a heavy chain and a light chain; a heavy chain variable region, a light chain variable region, or both a heavy chain variable region and a light chain variable region; wherein the heavy chain, light chain, heavy chain variable region, or light chain variable region comprises a sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% identity to the amino acid sequences as set forth in SEQ ID NO: 18, 19, 20, or 21; Moreover, this antibody specifically binds to human TNF-α.

[0132] The FDA-approved anti-TNF-α antibody, golimumab (SIMPONI® by Janssen Biotech; DrugBank DB06674), has been used to treat humans. In some embodiments of the present invention, the anti-TNF-α antibody is a human antibody or antigen-binding fragment comprising the heavy chain amino acid sequence set forth in SEQ ID NO: 22: a human antibody or antigen-binding fragment comprising the light chain amino acid sequence set forth in SEQ ID NO: 23: or a human antibody or antigen-binding fragment comprising the heavy chain amino acid sequence set forth in SEQ ID NO: 22 and the light chain amino acid sequence set forth in SEQ ID NO: 23; a human antibody or antigen-binding fragment comprising the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 24: a human antibody or antigen-binding fragment comprising the amino acid sequence of a light chain variable region as set forth in SEQ ID NO: 25: or a human antibody or antigen-binding fragment comprising the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO: 24 and the amino acid sequence of the light chain variable region set forth in SEQ ID NO: 25.

[0133] In some embodiments, the invention provides antibodies comprising a heavy chain, a light chain, or both a heavy chain and a light chain; a heavy chain variable region, a light chain variable region, or both a heavy chain variable region and a light chain variable region; wherein the heavy chain, light chain, heavy chain variable region, or light chain variable region comprises a sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% identity to the amino acid sequences as set forth in SEQ ID NO: 22, 23, 24, or 25; Moreover, this antibody specifically binds to human TNF-α. Antibodies to TGF-I3

[0134] In some embodiments of the present invention, the anti-TGF-β antibody is an anti-TGF-β antibody that is a human antibody or antigen-binding fragment comprising the heavy chain amino acid sequence set forth in SEQ ID NO: 26: a human antibody or antigen-binding fragment comprising the light chain amino acid sequence set forth in SEQ ID NO: 27: or a human antibody or antigen-binding fragment comprising the heavy chain amino acid sequence set forth in SEQ ID NO: 26 and the light chain amino acid sequence set forth in SEQ ID NO: 27; a human antibody or antigen-binding fragment comprising the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 28: a human antibody or antigen-binding fragment comprising the amino acid sequence of a light chain variable region as set forth in SEQ ID NO: 29: or a human antibody or antigen-binding fragment comprising the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO: 28 and the amino acid sequence of the light chain variable region set forth in SEQ ID NO: 29.

[0135] In some embodiments, the invention provides antibodies comprising a heavy chain, a light chain, or both a heavy chain and a light chain; a heavy chain variable region, a light chain variable region, or both a heavy chain variable region and a light chain variable region; wherein the heavy chain, light chain, heavy chain variable region, or light chain variable region comprises a sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% identity to the amino acid sequences as set forth in SEQ ID NOs: 26, 27, 28, and 29; Moreover, this antibody specifically binds to human TGF-β. Linkers

[0136] In some embodiments, a first antigen-binding molecule that specifically binds to a TNF-α ligand is connected to a second antigen-binding molecule that specifically binds to a TGF-β ligand via a linker and / or a hinge linker peptide. The linker or hinge linker may be an artificial sequence of 5, 10, 15, 20, 30, 40 or more amino acids that are somewhat free of secondary structure or exhibit an α-helix conformation.

[0137] A peptide linker provides the formation of a covalent bond and additional structural and / or spatial flexibility between protein domains. As is known in the art, peptide linkers contain amino acid residues that impart flexibility, such as glycine and serine. In some embodiments, a peptide linker may include 1-100 amino acids. In some embodiments, the spacer may comprise a GGGSGGGS motif (SEQ ID NO: 51). In other embodiments, the linker may comprise a GGGGS motif (SEQ ID NO: 54)n, where n is an integer from 1 to 10. In other embodiments, the linker may also contain amino acids other than glycine and serine. In another embodiment, the linker may comprise other protein motifs, including, but not limited to, alpha-helix conformation sequences such as AEAAAKEAAAAKEAAAKA (SEQ ID NO: 49).In some embodiments, the length and composition of the linker can be adjusted to optimize activity or redesignability, including, but not limited to, expression level and aggregation susceptibility. In another embodiment, the peptide linker can be a simple chemical bond, such as an amide bond (e.g., by chemical conjugation with polyethylene glycol (PEG)).

[0138] Typical peptide linkers are shown in Table 4. Bifunctional antagonist molecules

[0139] According to the present invention, there are provided novel bifunctional antagonist molecules based on polypeptides, specifically designed to simultaneously and effectively neutralize TNF-α-mediated signaling and TGF-β-mediated signaling and comprising a first antigen-binding molecule that specifically binds to a TNF-α ligand, and a second antigen-binding molecule that specifically binds to a TGF-β ligand. In some embodiments, the bifunctional molecule comprises an isolated antibody or an antigen-binding fragment thereof that specifically binds to TNF-α, and an isolated antibody or an antigen-binding fragment thereof that specifically binds to a TGF-β ligand.It is important to note that these bifunctional antagonists also have advantageous properties such as manufacturability, stability, binding affinity, biological activity, cell-targeting specificity, targeting efficiency, and reduced toxicity.Typical bifunctional antagonist molecules.

[0140] In some embodiments, the bifunctional antagonist molecules of the present invention are selected from the group of molecules designed and comprising fusion partners described in Table 5.

[0141] In some embodiments, the bifunctional antagonist molecule of the present invention is selected from the group of molecules described in Table 6. Polynucleotides

[0142] According to another aspect of the present invention, isolated nucleic acid molecules are provided, comprising a polynucleotide encoding a bifunctional antagonist molecule of the present invention. The claimed nucleic acids can be single-stranded or double-stranded. Such nucleic acids can be DNA or RNA molecules. DNA includes, for example, cDNA, genomic DNA, synthetic DNA, DNA amplified using the polymerase chain reaction (PCR), and combinations thereof. Genomic DNA encoding bifunctional antagonist molecules is obtained from genomic libraries, which are available for a number of species. Synthetic DNA is obtained by chemical synthesis of overlapping oligonucleotide fragments, followed by assembly of the fragments to recreate part or all of the coding regions and flanking sequences.RNA can be obtained using prokaryotic expression vectors that perform high-level mRNA synthesis, such as vectors utilizing the T7 phage promoter and RNA polymerase. cDNA is obtained from libraries prepared from mRNA isolated from various tissues expressing the bifunctional antagonist molecule. The DNA molecules of the invention include full-length genes, as well as polynucleotides and fragments thereof. A full-length gene may also include sequences encoding an N-terminal signal sequence.

[0143] In some embodiments, the isolated nucleic acid molecules comprise the polynucleotides described in this application and further comprise a polynucleotide encoding at least one heterologous protein described in this application. In some embodiments, the nucleic acid molecules further comprise polynucleotides encoding linkers or hinge linkers described in this application.

[0144] In some embodiments, the recombinant nucleic acids of the present invention may be operably linked to one or more regulatory nucleotide sequences in the expression construct. Regulatory sequences are known in the art and are selected to achieve targeted expression of the bifunctional antagonist molecule. Accordingly, the term "regulatory sequence" includes promoters, enhancers, and other expression regulatory elements. Exemplary regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, Calif. (1990).Typically, said one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosome binding sites, transcription start and stop sequences, translation start and stop sequences, and enhancer or activator sequences. Constitutive or inducible promoters known in the art are encompassed by the present invention. Promoters may be either natural promoters or hybrid promoters that combine elements of more than one promoter. The expression construct may be present in the cell as an episome, such as a plasmid, or the expression construct may be integrated into the chromosome. In some embodiments, the expression vector contains a selectable marker gene that allows for the selection of transfected host cells.Selectable marker genes are well known in the art and will vary depending on the host cell used.

[0145] According to another aspect of the present invention, the claimed nucleic acid is provided as part of an expression vector comprising a nucleotide sequence encoding a bifunctional antagonist molecule and operably linked to at least one regulatory sequence. The term "expression vector" refers to a plasmid, phage, virus, or vector for expressing a polypeptide from a polynucleotide sequence. Vectors suitable for expression in host cells are readily available, and nucleic acid molecules are inserted into these vectors using standard recombinant DNA techniques. Such vectors may include a wide variety of expression control sequences that control the expression of the DNA sequence when operably linked thereto and that can be used in these vectors to express DNA sequences encoding a bifunctional antagonist molecule.Such useful expression control sequences include, for example, the early and late promoters of simian virus 40 (SV40), the tet promoter, the immediate-early promoter of adenovirus or cytomegalovirus, the promoters of respiratory syncytial virus (RSV), the lac system, the trp system, the TAC or TRC system, the T7 promoter, which directs the expression of phage T7 RNA polymerase, the major operator and promoter regions of phage lambda, the control regions of the phage fd coat protein, the promoter of 3-phosphoglycerate kinase or other glycolytic enzymes, acid phosphatase promoters such as PhoS, promoters of yeast a-mating factors, the polyhedrin gene promoter of the baculovirus system, and other sequences that are known to regulate the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.It should be understood that the design of an expression vector may depend on factors such as the choice of host cell to be transfected and / or the type of protein desired to be expressed. Furthermore, the vector copy number, the ability to regulate that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered.

[0146] The recombinant nucleic acid of the present invention can be obtained by ligating the cloned gene or a portion thereof into a vector suitable for expression in either prokaryotic cells or eukaryotic cells (yeast, bird, insect or mammalian cells), or both. Expression means for producing the recombinant bifunctional antagonist molecule include plasmids and other vectors. For example, suitable vectors include the following types of plasmids: pBR322-based plasmids, pEMBL-based plasmids, pEX-based plasmids, pBTac-based plasmids and pUC-based plasmids, intended for expression in prokaryotic cells such as E. coli.

[0147] Some mammalian expression vectors contain both prokaryotic sequences to facilitate vector propagation in bacteria and one or more eukaryotic transcription units that are expressed in eukaryotic cells. Vectors based on pcDNAI / amp, pcDNAI / neo, pRc / CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo, and pHyg are examples of mammalian expression vectors suitable for transfection of eukaryotic cells. Some of these vectors are modified with sequences from bacterial plasmids, such as pBR322, to facilitate replication and selection for drug resistance in both prokaryotic and eukaryotic cells. Alternatively, derivatives of viruses such as bovine papillomavirus (BPV-1) or Epstein-Barr virus (pHEV, based on pREP and p205) can be used for transient expression of proteins in eukaryotic cells.Examples of other viral (including retroviral) expression systems can be found below under the description of delivery systems for gene therapy. The various methods used to produce plasmids and to transform host microorganisms are well known in the art. For other expression systems suitable for both prokaryotic and eukaryotic cells, as well as general recombination techniques, see Molecular Cloning: A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch, and Maniatis (Cold Spring Harbor Laboratory Press, 1989), Chapters 16 and 17. In some cases, expression of recombinant polypeptides using a baculovirus expression system may be desirable. Examples of such baculovirus expression systems include pVL-based vectors (such as pVL1392, pVL1393, and pVL941), pAcUW-based vectors (such as pAcUW1), and pBlueBac-based vectors (such as B-gal containing pBlueBac III).

[0148] In some embodiments, the vector will be designed to produce the claimed bifunctional antagonist molecule in Chinese hamster ovary (CHO) cells, such as the Pcmv-Script vector (Stratagene, La Jolla, Calif.), pcDNA4 vectors (Invitrogen, Carlsbad, Calif.), and pCI-neo vectors (Promega, Madison, Wis.). It is understood that the claimed gene constructs can be used to cause expression of the claimed bifunctional antagonist molecule in cells propagated in culture, for example, to obtain proteins, including fusion proteins or protein variants, for purification.

[0149] Accordingly, the present invention also relates to methods for producing the claimed bifunctional antagonist molecules. For example, a host cell transfected with an expression vector encoding a bifunctional antagonist molecule can be cultured under appropriate conditions allowing expression of the bifunctional antagonist molecule. The bifunctional antagonist molecule can be secreted and isolated from a mixture of cells and a medium containing the bifunctional antagonist molecule. Alternatively, the bifunctional antagonist molecule can remain in the cytoplasmic or membrane fraction, in which case the cells are collected, lysed, and the protein is isolated. Cell culture includes host cells, media, and other byproducts. Media suitable for cell culture are well known in the art.

[0150] The polypeptides and proteins of the present invention can be purified using protein purification methods well known to those skilled in the art. These methods include, at a first level, crude fractionation of protein and non-protein fractions. After separating the peptides or polypeptides from other proteins, the peptide or polypeptide of interest can be further purified using chromatographic and electrophoretic methods in order to achieve partial or complete purification (or purification to a state of homogeneity). The term "isolated polypeptide" or "purified polypeptide" used herein is intended to refer to a composition that can be separated from other components, wherein the polypeptide is purified to any degree of purity compared to its natural state. Thus, the term "purified polypeptide" also refers to a polypeptide that does not contain environmental components with which it may occur in nature.Typically, "purified" will refer to a polypeptide composition that has been fractionated to remove various other components, and that composition substantially retains its expressed biological activity. When the term "substantially purified" is used, the designation will refer to a peptide or polypeptide composition wherein the polypeptide or peptide forms a major component of the composition, for example, comprising about 50%, about 60%, about 70%, about 80%, about 85%, or about 90% or more of the proteins in the composition.

[0151] Those skilled in the art will be well aware of various methods suitable for use in purification. Such methods include, for example, precipitation using ammonium sulfate, PEG, antibodies (immunoprecipitation), and the like, or by heat denaturation followed by centrifugation; chromatography such as affinity chromatography (on protein A columns), ion exchange, gel filtration, reversed phase, hydroxyapatite chromatography, hydrophobic chromatography; isoelectric focusing; gel electrophoresis; and a combination of these methods. It is generally known and believed in the art that the order of the various purification steps can be changed or that some steps can be omitted and still result in a suitable method for producing a substantially purified polypeptide. Pharmaceutical compositions

[0152] According to another aspect of the present invention, there is provided a pharmaceutical composition comprising the isolated bifunctional antagonist molecules in admixture with a pharmaceutically acceptable carrier. Such pharmaceutically acceptable carriers are well known and understood by those skilled in the art and have been described in detail (see, for example, Remington's Pharmaceutical Sciences, 18th Edition, A.R. Gennaro, ed., Mack Publishing Company, 1990). Pharmaceutically acceptable carriers can be included for the purpose of modifying, maintaining, or preserving, for example, pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or permeability of the composition. Such pharmaceutical compositions can affect the physical state, stability, rate of release of the polypeptide in vivo, and rate of elimination of the polypeptide in vivo. Suitable pharmaceutically acceptable carriers include,but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite, or sodium hydrosulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrate, phosphate, other organic acids); bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides and other carbohydrates (such as glucose, mannose, or dextrins); proteins (such as serum albumin, gelatin, or immunoglobulins); coloring agents; flavoring and diluents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium ion); preservatives (such as benzalkonium chloride,benzoic acid, salicylic acid, thiomersal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (such as glycerol, propylene glycol, or polyethyleneglycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEGs, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, chlesterol, tyloxapal); stability-enhancing agents (sucrose or sorbitol); tonicity-enhancing agents (such as alkali metal halides (preferably sodium or potassium chloride), mannitol, sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants,

[0153] The main excipient or carrier in the pharmaceutical composition may be either aqueous or non-aqueous in nature. For example, a suitable excipient or carrier may be water for injection, saline, or artificial cerebrospinal fluid, possibly supplemented with other substances commonly used in parenteral compositions. Physiologically buffered saline or saline mixed with serum albumin are other typical diluents. Other typical pharmaceutical compositions comprise a Tris buffer of approximately pH 7.0-8.5 or an acetate buffer of approximately pH 4.0-5.5, which may further include sorbitol or a suitable substitute.In one embodiment of the present invention, the compositions can be prepared for storage by mixing the selected composition, having the desired degree of purity, with optional formulation agents (Remington's Pharmaceutical Sciences, supra) in the form of a lyophilized tablet or aqueous solution. Furthermore, the therapeutic composition can be prepared in the form of a lyophilisate using appropriate excipients, such as sucrose. The optimal pharmaceutical composition will be determined by one of ordinary skill in the art depending on, for example, the intended route of administration, delivery format, and desired dosage.

[0154] When parenteral administration is intended, therapeutic pharmaceutical compositions can be in the form of a pyrogen-free parenterally acceptable aqueous solution containing the desired bifunctional antagonist molecule in a pharmaceutically acceptable diluent. In particular, a suitable diluent for parenteral injection is sterile distilled water, in which the polypeptide-based composition is prepared as a sterile isotonic solution with a suitable preservative. In some embodiments, pharmaceutical compositions suitable for injection administration can be prepared as aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or buffered saline. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran.In addition, suspensions of the active compounds can be prepared as appropriate oil-based injection suspensions. The suspension may also contain suitable stabilizers or agents to enhance the solubility of the compounds and allow for the preparation of highly concentrated solutions.

[0155] In some embodiments, therapeutic pharmaceutical compositions can be prepared for targeted delivery using a colloidal dispersion system. Colloidal dispersion systems include macromolecular complexes, nanocapsules, microspheres, granules, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Examples of lipids suitable for preparing liposomes include phosphatidyl-containing compounds such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Illustrative phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine, and distearoylphosphatidylcholine. Targeting of liposomes is also possible based on, for example, organ specificity, cell specificity and organelle specificity, and this is known in the art.

[0156] In some embodiments, oral administration of the pharmaceutical compositions is contemplated. Pharmaceutical compositions that are administered in this manner may be prepared without carriers or with such carriers that are commonly used in the preparation of solid dosage forms such as tablets and capsules. In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), one or more therapeutic compounds of the present invention may be mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dibasic calcium phosphate, and / or with any of the following: (1) fillers or dry diluents, such as starches, lactose, sucrose, glucose, mannitol and / or silicic acid; (2) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and / or acacia; (3) wetting agents,such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain types of silicates and sodium carbonate; (5) dissolution retarders, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also contain buffering agents. Solid compositions of this type may also be used as fillers in soft and hard gelatin capsules using excipients such as lactose or milk sugar,and high molecular weight polyethylene glycols and the like. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, peanut, corn, germ, olive, castor and sesame oils), glycerin, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan and mixtures thereof. In addition to inert diluents, oral compositions may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents,flavoring agents, dyes, fragrances and preservatives,

[0157] In some embodiments, topical administration of the pharmaceutical compositions to either the skin or mucous membranes is contemplated. Compositions for topical use may further include one or more agents from a wide range of those known to be effective as skin or stratum corneum permeability enhancers. Examples include 2-pyrrolidone, N-methyl-2-pyrrolidone, dimethylacetamide, dimethylformamide, propylene glycol, methyl or isopropyl alcohol, dimethyl sulfoxide, and azone. Additional agents may also be included to prepare a cosmetically acceptable composition. Examples include fats, waxes, oils, dyes, fragrances, preservatives, stabilizers, and surface-active agents. Keratolytic agents, such as those known in the art, may also be included. Examples include salicylic acid and sulfur.Dosage forms for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active compound can be mixed under sterile conditions with a pharmaceutically acceptable carrier and any necessary preservatives, buffers, or propellants. Ointments, pastes, creams, and gels may contain, in addition to the claimed compound of this invention (e.g., a bifunctional antagonist molecule), excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc, and zinc oxide, or mixtures thereof.

[0158] Additional pharmaceutical compositions contemplated for use in this application include formulations containing polypeptides in sustained- or controlled-delivery formulations. Those skilled in the art are also familiar with methods for preparing a variety of other sustained- or controlled-delivery agents, such as liposomal carriers, biodegradable microparticles or porous granules, and injectable depot preparations.

[0159] An effective amount of a pharmaceutical composition for therapeutic use will depend, for example, on the therapeutic characteristics and goals. Thus, one skilled in the art will understand that appropriate dosage levels for treatment will vary, in part, depending on the molecule being delivered, the indication for which the polypeptide is used to treat, the route of administration, and the size (body weight, body surface area, or organ size) and condition (age and general health) of the patient. Accordingly, the attending physician can titrate the dosage and change the route of administration to obtain the optimal therapeutic effect. A typical dosage can range from about 0.1 mg / kg up to about 100 mg / kg or more, depending on the factors mentioned above. Compositions based on the polypeptide can preferably be administered by injection or administered intravenously.Long-acting pharmaceutical compositions can be administered every three to four days, once weekly, or twice weekly, depending on the half-life and elimination rate of the specific composition. The frequency of administration will depend on the pharmacokinetic parameters of the polypeptide used in the composition. Typically, the composition is administered until a dosage that provides the desired effect is achieved. Thus, the composition can be administered as a single dose or as multiple doses (at the same or different concentrations / dosages) over a period of time, or as a continuous infusion. Subsequent adjustments to the appropriate dosage are typically made. Appropriate dosages can be established using relevant dose-response data.

[0160] The route of administration of the pharmaceutical composition corresponds to known methods, for example, orally, by injection by intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, intraportal, intralesional routes, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous or intraperitoneal routes; as well as intranasal, enteral, topical, sublingual, urethral, ​​vaginal or rectal routes, via sustained-release systems or via implantation devices. If desired, the compositions can be administered by bolus injection or continuously by infusion or using an implantation device. Alternatively or in addition, the composition can be administered locally by implanting a membrane, sponge or other appropriate material onto which the desired molecule is absorbed or encapsulated.If an implantable device is used, such a device can be implanted into any suitable tissue or any suitable organ, and delivery of the desired molecule can be accomplished by diffusion, sustained-release bolus, or continuous administration.Therapeutic applications.

[0161] According to another aspect of the present invention, a method for treating or preventing various complex disease states in the pathogenesis of which activation of both the TNF-α-mediated NF-κB signaling pathway and the TGF-β-mediated Smad2 / 3 signaling pathway is involved.

[0162] In some embodiments, the novel bifunctional antagonist molecules of the present invention may find broad application in the treatment of various disorders including, but not limited to, anemia, inflammation, pulmonary hypertension, heart failure, renal failure, muscular dystrophy, arthritis, organ fibrosis, and cancer in a subject comprising administering to said subject a therapeutically effective amount (either as monotherapy or in a combination therapy regimen) of the bifunctional antagonist molecule of the present invention in a pharmaceutically acceptable carrier, wherein such administration reduces muscle loss and / or loss of muscle function. In particular, the bifunctional antagonist molecule of the present invention is useful for the treatment of various complex diseases including, but not limited to, blood diseases (such as ineffective erythropoiesis, pancytopenia,myelodysplastic syndromes, bone marrow failure, leukemia, beta-thalassemia, and sickle cell anemia), fibrosing diseases (such as nonalcoholic steatohepatitis or NASH, cirrhosis, pulmonary fibrosis, renal fibrosis, polycystic kidney disease, myocardial fibrosis, muscle fibrosis, myelofibrosis, cutaneous fibrosis, and ocular fibrosis), muscular dystrophies (MDs) (such as Duchenne (muscular) dystrophy (DMD), Becker MD, limb-girdle MD, myotonic MD, and facioscapulohumeral dystrophy (FSHD)), myositis (such as polymyositis and dermatomyositis), myopathies (including hereditary myopathy and acquired myopathy), motor neuron diseases (such as mitochondrial dystrophy Lou Gehrig's disease or amyotrophic lateral sclerosis (ALS)), neurodegenerative diseases (such as Parkinson's disease, Huntington's disease, and Alzheimer's disease), cancer cachexia, sarcopenia, bone fragility disorders (such as bone fracture and cancer metastasis),chronic heart failure, chronic kidney disease (CKD), diabetes, chronic obstructive pulmonary disease (COPD), cytokine storm events due to infections (such as AIDS, tuberculosis, SARS-CoV, and sepsis), arthritis, including rheumatoid arthritis (RA) and osteoarthritis (OA), trauma (such as burns or motorcycle accidents), including critical illness treatment in the intensive care unit (ICU), denervation (such as stroke or spinal cord injury), prolonged bed rest, sarcopenic obesity and age-related muscle and bone loss (including postmenopausal osteoporosis and age-related sarcopenia), organ or tissue transplantation (such as heart transplantation, kidney transplantation, and liver transplantation), and various malignancies (such as leukemia, melanoma, breast cancer, multiple myeloma, prostate cancer glands, lung cancer, pancreatic cancer, stomach cancer, ovarian cancer,colorectal cancer, brain cancer, bladder cancer, and head and neck cancer), as monotherapy or in combination with an immune checkpoint inhibitor such as anti-PD1, anti-PDL1, and anti-CTL4 antibodies.

[0163] According to the present invention, there is provided a method for treating a cardiovascular disease in a subject, comprising administering to said subject a therapeutically effective amount (either as monotherapy or in a combination therapy regimen) of a bifunctional antagonist molecule of the present invention in a pharmaceutically acceptable carrier, wherein such administration reduces inflammation and fibrosis of the vascular systems and muscles, including smooth muscle, cardiac muscle and skeletal muscle. In particular, the bifunctional antagonist molecule of the present invention is useful for the treatment of heart failure, pulmonary hypertension, including pulmonary arterial hypertension, myocarditis, coronary heart disease, myocardial infarction, cardiac arrhythmias, heart valve dysfunction, cardiomyopathy, pericardial disease, aortic dysfunction, Marfan syndrome and cardiac muscle atrophy.

[0164] According to the present invention, there is provided a method for treating cardiac dysfunction or heart failure in a subject, comprising administering an effective amount of a bifunctional antagonist molecule to the subject. The modulation can improve the cardiac function in said subject by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%. Improvement in cardiac function can be assessed by echocardiography to measure 1) cardiac pumping function with emphasis on the volume of blood ejected and the efficiency of ejection and 2) myocardial function with emphasis on the force of myocardial contraction.

[0165] According to the present invention, methods for treating metabolic disorders in a subject are provided, comprising administering to said subject a therapeutically effective amount (either as monotherapy or in a combination therapy regimen) of a bifunctional antagonist molecule of the present invention in a pharmaceutically acceptable carrier. In particular, the bifunctional antagonist molecule of the present invention is useful for treating a metabolic disease selected from obesity, dyslipidemia, diabetes, insulin resistance, sarcopenic obesity, steatosis and metabolic syndrome, as well as diabetic myopathy, nephropathy, neuropathy, retinopathy, bone loss, impaired glucose tolerance, hyperglycemia and androgen deprivation.

[0166] According to the present invention, there is provided a method of treating cancer cells in a subject, comprising administering to said subject a therapeutically effective amount (either as monotherapy or in a combination therapy regimen) of a bifunctional antagonist molecule of the present invention in a pharmaceutically acceptable carrier, wherein such administration inhibits the growth and / or proliferation of a cancer cell. In particular, the bifunctional antagonist molecule of the present invention is useful for the treatment of a disorder characterized as cancer. Such disorders include, but are not limited to, solid tumors, such as cancers of the breast, respiratory tract, brain, reproductive organs, gastrointestinal tract, urinary tract, eye, liver, skin, head and neck, thyroid, parathyroid gland and their distant metastases, lymphomas, sarcomas, multiple myeloma and leukemia.Examples of breast cancer include, but are not limited to, invasive ductal carcinoma, invasive lobular carcinoma, ductal carcinoma in situ, and lobular carcinoma in situ. Examples of respiratory tract cancers include, but are not limited to, small cell and non-small cell lung cancer, bronchial adenoma, and pleuropulmonary blastoma. Examples of brain cancers include, but are not limited to, brainstem and hypothalamic glioma, cerebellar and cerebral astrocytoma, medulloblastoma, ependymoma, and neuroectodermal and pineal tumors. Tumors of the male reproductive organs include, but are not limited to, prostate and testicular cancer. Tumors of the female reproductive organs include, but are not limited to, endometrial, cervical, ovarian, vaginal, and vulvar cancers, as well as uterine sarcoma.Tumors of the digestive tract include, but are not limited to, anal cancer, colon cancer, colorectal cancer, esophageal cancer, gallbladder cancer, stomach cancer, pancreas cancer, rectum cancer, small intestine cancer, and salivary gland cancer. Tumors of the urinary tract include, but are not limited to, cancers of the bladder, penis, kidney, renal pelvis, ureter, and urethra. Eye cancers include, but are not limited to, intraocular melanoma and retinoblastomas. Examples of liver cancers include, but are not limited to, hepatocellular carcinoma (hepatocellular carcinomas with or without the fibrolamellar variant), cholangiocarcinoma (carcinoma of the intrahepatic bile ducts), and mixed-lineage hepatocellular cholangiocarcinoma. Skin cancers include, but are not limited to, squamous cell carcinoma, Kaposi's sarcoma, malignant melanoma, Merkel cell skin cancer, and non-melanoma skin cancer.Head and neck cancers include, but are not limited to, nasopharyngeal cancer and cancer of the lip and oral cavity. Lymphomas include, but are not limited to, AIDS-associated lymphoma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, Hodgkin's disease, and central nervous system lymphoma. Sarcomas include, but are not limited to, soft tissue sarcoma, osteosarcoma, malignant fibrous histiocytoma, lymphosarcoma, and rhabdomyosarcoma. Leukemias include, but are not limited to, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, and hairy cell leukemia. In some embodiments, the cancer will be a cancer characterized by high levels of TNF-α and TGF-β expression, such as pancreatic cancer, gastric cancer, ovarian cancer, colorectal cancer, melanoma, leukemia, lung cancer, prostate cancer, brain cancer, bladder cancer, and head and neck cancer.

[0167] According to the present invention, there is provided a method for treating chronic kidney disease (CKD) in a subject, comprising administering to said subject a therapeutically effective amount (either as monotherapy or in a combination therapy regimen) of a bifunctional antagonist molecule of the present invention in a pharmaceutically acceptable carrier, wherein such administration attenuates the loss of renal function and prevents the loss of muscle mass or inhibits renal fibrosis. In particular, the bifunctional antagonist molecule of the present invention is useful for the treatment of CKD, including renal failure, interstitial fibrosis and renal dialysis, as well as protein-energy malnutrition (PEW) associated with CKD.The modulation can improve CKD or PEW in said subject by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. Improvement in renal function can be assessed by measuring the urine protein / creatinine ratio (PCR) and glomerular filtration rate (GFR). Improvement in PEW can be assessed by measuring serum albumin and inflammatory cytokine levels, protein synthesis and degradation rates, body weight, muscle mass, physical activity, and diet-related effects.

[0168] According to the present invention, methods for treating an autoimmune disease in a subject are provided, comprising administering to said subject a therapeutically effective amount (either as monotherapy or in a combination therapy regimen) of a bifunctional antagonist molecule of the present invention in a pharmaceutically acceptable carrier. In particular, the bifunctional antagonist molecule of the present invention is useful for the treatment of an autoimmune disorder selected from multiple sclerosis, diabetes (type 1), glomerulonephritis, severe myasthenia, psoriasis, systemic sclerosis and systemic lupus erythematosus, polymyositis and primary biliary cirrhosis.

[0169] The present invention provides methods for treating arthritis in a subject, comprising administering to said subject a therapeutically effective amount (either as monotherapy or in a combination therapy regimen) of a bifunctional antagonist molecule of the present invention in a pharmaceutically acceptable carrier. In particular, the bifunctional antagonist molecule of the present invention is useful for the treatment of arthritis selected from rheumatoid arthritis and osteoarthritis.

[0170] According to the present invention, methods for treating anorexia in a subject are provided, comprising administering to said subject a therapeutically effective amount (either as monotherapy or in a combination therapy regimen) of a bifunctional antagonist molecule of the present invention in a pharmaceutically acceptable carrier. In particular, the bifunctional antagonist molecule of the present invention is useful for the treatment of anorexia selected from anorexia nervosa and anorexia-cachexia syndrome.

[0171] According to the present invention, methods for treating a liver disease in a subject are provided, comprising administering to said subject a therapeutically effective amount (either as monotherapy or in a combination therapy regimen) of a bifunctional antagonist molecule of the present invention in a pharmaceutically acceptable carrier. In particular, the bifunctional antagonist molecule of the present invention is useful for treating a liver disease selected from non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, alcoholic fatty liver disease, liver cirrhosis, liver failure, autoimmune hepatitis, and hepatocellular carcinoma.

[0172] According to the present invention, methods of transplanting organs or tissues in a subject are provided, comprising administering to said subject a therapeutically effective amount (either as monotherapy or in a combination therapy regimen) of a bifunctional antagonist molecule of the present invention in a pharmaceutically acceptable carrier. In particular, the bifunctional antagonist molecule of the present invention is useful in the treatment of the consequences of transplantation selected from transplantations of such organs as the heart, kidney, liver, lung, pancreas, intestine and thymus, or from transplantations of such tissues as bone, tendon, cornea, skin, heart valve, nerves and veins.

[0173] According to the present invention, methods of treating anemia in a subject are provided, comprising administering to said subject a therapeutically effective amount (either as monotherapy or in a combination therapy regimen) of a bifunctional antagonist molecule of the present invention in a pharmaceutically acceptable carrier. In some embodiments, the anemia is selected from various anemia-related disorders, including cancer-associated anemia, chemotherapy-induced anemia, chronic kidney disease-associated anemia, iron deficiency anemia, iron overload and hemochromatosis, thalassemia, sickle cell anemia, aplastic anemia, myelodysplastic syndromes, pancytopenia and bone marrow failure.

[0174] The present invention provides methods for treating fibrosis in a subject, comprising administering a therapeutically effective amount of the pharmaceutical compositions of the invention to the subject in need thereof. In one embodiment, the subject is a human.In some embodiments, the fibrosis is selected from pulmonary fibrosis (such as idiopathic pulmonary fibrosis and cystic fibrosis), liver fibrosis (such as non-alcoholic steatohepatitis and cirrhosis), airway fibrosis (such as asthma), myocardial fibrosis (such as myocardial infarction, diastolic dysfunction, or valvular heart disease), renal fibrosis (such as interstitial fibrosis), myelofibrosis, idiopathic retroperitoneal fibrosis, nephrogenic fibrosing dermatopathy, intestinal fibrosis in inflammatory bowel disease (including Crohn's disease), keloid, scleroderma, systemic sclerosis, fibrosis of the hand (e.g., Dupuytren's contracture), fibrosis of the eye, and arthrofibrosis.

[0175] The present invention provides methods for treating pain in a subject, comprising administering a therapeutically effective amount of the pharmaceutical compositions of the invention to the subject in need thereof. In one embodiment, the subject is a human. In some embodiments, the pain is selected from neuropathic pain, inflammatory pain, or cancer pain.

[0176] According to the present invention, methods for treating a bone disease in a subject are provided, comprising administering a therapeutically effective amount of the pharmaceutical compositions of the invention to the subject in need thereof. In one embodiment, the subject is a human. In some embodiments, the bone disease is selected from osteomalacia, osteoporosis, osteogenesis imperfecta, progressive fibrous osteodysplasia, corticosteroid-induced bone loss, bone fracture, and bone metastasis.

[0177] According to the present invention, there is provided a method for inhibiting the loss of muscle mass and / or muscle function in a subject, comprising administering an effective amount of a bifunctional antagonist molecule to the subject. The modulation can attenuate the loss of muscle mass and / or function in said subject by at least 5%, 10%, at least 25%, at least 50%, at least 75%, or at least 90%. Inhibition of muscle mass and muscle function loss can be assessed by using imaging methods and physical strength tests. Examples of imaging methods for assessing muscle mass include dual-energy X-ray absorptiometry (DEXA), magnetic resonance imaging (MRI), and computed tomography (CT).Examples of tests to assess muscle function include the grip strength test, the stair-climbing test, the Short Physical Performance Battery (SPPB), and the 6-minute walk test, as well as the determination of the maximum inspiratory pressure (MIP) and maximum expiratory pressure (MEP), which are used to measure the strength of the respiratory muscles.

[0178] The term "therapeutically effective amount" or "therapeutically effective dose" refers to that amount of a therapeutic agent administered that will alleviate to some extent one or more symptoms of the disorder being treated.

[0179] The therapeutically effective dose can first be estimated based on the results of cell culture assays by determining the IC 50 The dose can then be tested in animal models to obtain a range of circulating plasma concentrations that includes the IC 50, determined in cell culture. This information can be used to more accurately determine the doses beneficial for humans. Plasma levels can be measured, for example, by HPLC. The exact composition, route of administration, and dosage can be individually selected by a physician, depending on the patient's condition.

[0180] Administration regimens can be adjusted to achieve the optimal desired response (e.g., therapeutic or prophylactic). For example, administration can be performed as a single bolus, or several divided doses (multiple, repeat, or maintenance) can be administered over a period of time, and the dose can be proportionally tapered or decreased depending on the severity of the therapeutic situation. It is particularly advantageous to prepare parenteral compositions in unit dosage form for ease of administration and dosage uniformity.The term "unit dosage form" as used herein refers to physically discrete units suitable as unitary dosages for the mammals to be treated, each unit containing the active compound in a predetermined quantity calculated to produce the desired therapeutic effect, together with the required pharmaceutical carrier. The definition of unit dosage forms in accordance with the present invention will be dictated primarily by the unique characteristics of the antibody and the specific therapeutic or prophylactic effect intended to be achieved.

[0181] Thus, it will be apparent to one skilled in the art, given the invention disclosed herein, that the dose and administration regimen are adjusted in accordance with methods well known in the therapeutic field. That is, the maximum tolerated dose can be readily established, and an effective amount providing a detectable therapeutic benefit to a subject can also be determined, as well as the timing requirements for administering each agent in order to provide a detectable therapeutic benefit to the subject can be formulated. Accordingly, although certain doses and administration regimens are provided herein by way of example, these examples in no way limit the dose and administration regimen that may be proposed to a subject in the case of practicing the present invention.

[0182] It should be noted that dosage amounts may vary depending on the type and severity of the condition being alleviated and may include single or multiple doses. It should also be understood that for any particular subject, specific administration regimens should be adjusted over time based on the individual needs and professional judgment of the person administering or supervising the administration of the compositions, and that the dosage ranges provided herein are only typical and are not intended to limit the scope of the claimed composition or its practical use. In addition, the basis for choosing an administration regimen using the compositions of the invention may be a number of factors, including the type of disease, age, weight, gender, health status of the subject, severity of the condition, route of administration, and the specific antibody used.Thus, the administration regimen may vary widely but can be determined according to routine practice using standard methods. For example, doses can be adjusted based on pharmacokinetic or pharmacodynamic parameters, which may include clinical effects, such as toxic effects, and / or laboratory parameters. Thus, the present invention encompasses intra-individual dose escalation, as determined by one skilled in the art. Determining appropriate dosages and regimens is well known in the relevant art, and one skilled in the art will understand how to accomplish this upon familiarity with the teachings described herein.

[0183] A typical non-limiting range of once-daily administration for a therapeutically or prophylactically effective amount of the bifunctional antagonist molecule of the invention may be from 0.001 to 100 mg / kg, from 0.001 to 90 mg / kg, from 0.001 to 80 mg / kg, from 0.001 to 70 mg / kg, from 0.001 to 60 mg / kg, from 0.001 to 50 mg / kg, from 0.001 to 40 mg / kg, from 0.001 to 30 mg / kg, from 0.001 to 20 mg / kg, from 0.001 to 10 mg / kg, from 0.001 to 5 mg / kg, from 0.001 to 4 mg / kg, from 0.001 to 3 mg / kg, from 0.001 to 2 mg / kg, from 0.001 to 1 mg / kg, from 0.010 to 50 mg / kg, from 0.010 to 40 mg / kg, from 0.010 to 30 mg / kg, from 0.010 to 20 mg / kg, from 0.010 to 10 mg / kg, from 0.010 to 5 mg / kg, from 0.010 to 4 mg / kg, from 0.010 to 3 mg / kg, from 0.010 to 2 mg / kg, from 0.010 to 1 mg / kg, from 0.1 to 50 mg / kg, from 0.1 to 40 mg / kg, from 0.1 to 30 mg / kg, from 0.1 to 20 mg / kg, from 0.1 to 10 mg / kg, from 0.1 to 5 mg / kg, from 0.1 to 4 mg / kg, from 0.1 to 3 mg / kg, from 0.1 to 2 mg / kg, from 0.1 to 1 mg / kg, from 1 to 50 mg / kg, from 1 to 40 mg / kg,from 1 to 30 mg / kg, from 1 to 20 mg / kg, from 1 to 10 mg / kg, from 1 to 5 mg / kg, from 1 to 4 mg / kg, from 1 to 3 mg / kg, from 1 to 2 mg / kg, or from 1 to 1 mg / kg of body weight. It should be noted that dosage amounts may vary depending on the type and severity of the condition being alleviated. It should also be understood that for any particular subject, specific administration regimens will need to be adjusted over time based on the individual needs and the professional judgment of the person administering or supervising the administration of the compositions, and that the dosage ranges given herein are typical only and are not intended to limit the scope of the claimed composition or its practical application.

[0184] In some embodiments, the total administered dose will achieve an antibody concentration in the blood plasma in the range of, for example, from about 1 to 1000 μg / ml, from about 1 to 750 μg / ml, from about 1 to 500 μg / ml, from about 1 to 250 μg / ml, from about 10 to 1000 μg / ml, from about 10 to 750 μg / ml, from about 10 to 500 μg / ml, from about 10 to 250 μg / ml, from about 20 to 1000 μg / ml, from about 20 to 750 μg / ml, from about 20 to 500 μg / ml, from about 20 to 250 μg / ml, from about 30 up to 1000 mcg / ml, from about 30 to 750 mcg / ml, from about 30 to 500 mcg / ml, from about 30 to 250 mcg / ml.

[0185] The toxicity and therapeutic index of the pharmaceutical compositions of the invention can be determined using standard pharmaceutical techniques on cell cultures or experimental animals, for example, to determine the LD 50(lethal dose for 50% of the population) and ED 50 (the dose therapeutically effective for 50% of the population). The ratio of doses corresponding to toxic and therapeutic effects is the therapeutic index and can be expressed as the LD ratio. 50 / ED 50 Compositions that exhibit high therapeutic indices are generally preferred.

[0186] The dosing frequency for administering the pharmaceutical composition based on the bifunctional antagonist molecule depends on the nature of the therapy and the specific disease being treated. The subject may receive treatment at regular intervals, such as once a week or once a month, until the desired therapeutic result is achieved. Typical dosing frequencies include, but are not limited to: once a week without a break; once a week, once every two weeks; once every two weeks; once every three weeks; once a week without a break for two weeks, then once a month; once a week without a break for three weeks, then once a month; once a month; once every two months; once every three months; once every four months; once every five months; or once every six months or once a year. Combination therapy

[0187] As used herein, the terms "co-administration," "co-administered," and "in combination with" when referring to a bifunctional antagonist molecule of the invention and one or more other therapeutic agents are intended to mean, refer to, and include the following: the simultaneous administration of such a combination of a bifunctional antagonist molecule of the invention and a therapeutic agent(s) to a subject in need of treatment, in the case of preparing a single dosage form based on such components, from which said components are released in the body of said subject at substantially the same time;substantially simultaneous administration of such a combination of a bifunctional antagonist molecule according to the invention and a therapeutic agent(s) to a subject in need of treatment, in the case of preparing, on the basis of such components, separately from each other, separate dosage forms that are taken by said subject at substantially the same time, whereupon the release of said components in the body of said subject is carried out at substantially the same time;the sequential administration of such a combination of a bifunctional antagonist molecule according to the invention and a therapeutic agent(s) to a subject in need of treatment, in the case of preparing, on the basis of such components, separate from each other, separate dosage forms that are taken by said subject one after the other with a significant time interval in each case between doses, after which the release of said components in the body of said subject is carried out essentially at different points in time;and sequential administration of such a combination of a bifunctional antagonist molecule according to the invention and a therapeutic agent(s) to a subject in need of treatment, in the case of preparing a joint single dosage form based on such components, from which said components are released in a controlled manner, after which their release in the body of said subject is carried out simultaneously, sequentially and / or with overlapping at the same time and / or at different times, wherein each part can be administered either by the same route or by different routes.;

[0188] According to another aspect, the present invention relates to methods for treating diseases associated with muscle tissue wasting in a subject, comprising administering a combination of a) a therapeutically effective amount of a bifunctional antagonist molecule of the present invention; and b) a second agent. Such a combination therapy can be particularly effective against a disease associated with muscle tissue wasting that is resistant to treatment or poorly responsive to treatment using only the second agent. In some embodiments, the second agent is selected from growth hormone, ghrelin, insulin-like growth factor type 1 (IGF1), antagonists of inflammatory cytokines such as TNF-alpha and TNF-alpha, IL-6, IL-1, and their receptors, and other antagonists of myostatin and activin A and their receptors.

[0189] In some embodiments, combination therapy comprises administering a composition based on a bifunctional antagonist molecule and a second agent simultaneously, either as part of the same pharmaceutical composition or in separate pharmaceutical compositions. In some embodiments, the composition based on a bifunctional antagonist molecule and the composition based on the second agent are administered sequentially, i.e., the composition based on the bifunctional antagonist molecule is administered either before or after the composition based on the second agent.

[0190] In some embodiments, the administration of the composition based on the bifunctional antagonist molecule and the composition based on the second agent is performed simultaneously, i.e., the periods of administration of the composition based on the bifunctional antagonist molecule and the composition based on the second agent overlap with each other.

[0191] In some embodiments, the administration of the composition based on the bifunctional antagonist molecule and the composition based on the second agent is not performed simultaneously. For example, in some embodiments, the administration of the composition based on the bifunctional antagonist molecule is stopped before the administration of the composition based on the second agent. In some embodiments, the administration of the composition based on the second agent is stopped before the administration of the composition based on the bifunctional antagonist molecule.

[0192] The following examples are given to more fully illustrate the invention, but are not construed as limiting its scope. Example 1

[0193] The bifunctional antagonist molecules of the present invention can be produced according to recombinant DNA techniques well known to those skilled in the art. This example generally describes the production of bifunctional antagonist molecules.

[0194] cDNAs encoding various novel bifunctional antagonist polypeptides were generated using gene synthesis and subcloned into mammalian expression plasmids. CHO cells were transiently or stably transfected with mammalian expression plasmids encoding individual bifunctional antagonist polypeptides. Transiently transfected CHO cells or pools of stably transfected CHO cells were grown in high-density suspension cultures in a shaking CO2 incubator at 32°C for 6–8 days. Culture media were collected after passing through a 0.22-μm filter cell (Millipore Corporation, MA). Recombinantly expressed bifunctional polypeptides were purified from culture media using protein A affinity chromatography using an AKTA rapid-performance liquid chromatography (FPLC) system (GE Healthcare). Example 2

[0195] The activities of individual bifunctional antagonists toward binding to human ligands were measured by biolayer interferometry (BI) using the Octet RED96 system (ForteBIO, Pall Corporation, USA). The binding assay was first performed by capturing the bifunctional polypeptide antagonists onto biosensors. To measure the association and dissociation rates, the biosensors with captured bifunctional antagonists were placed in wells containing ligands (such as TNF-α, TGF-β1, and TGF-β1) at different concentrations diluted in 1× kinetics measurement buffer for 10 min, then incubated for 10-20 min in 1× kinetics measurement buffer. It should be noted that all tested ligands were produced recombinantly, taking into account their corresponding human sequences.Sensors with captured bifunctional polypeptide antagonists were also placed in wells containing 1x kinetics measurement buffer to ensure uniform subtraction of the reference value to compensate for the natural dissociation of the captured bifunctional antagonists. Binding sensorgrams were collected using the 8-channel detection mode on the biosensor. Data were collected and analyzed using FortéBIO data acquisition software, version 11.1 (FortéBIO, Pall Corporation, USA).

[0196] As shown in Table 7, A119 and A120 bind to TNF-α, TGF-β1 and TGF-β3 with high affinity values.

[0197] The binding affinity of A131, a representative bifunctional antagonist of the present invention developed in the form of a bispecific antibody, to ligands was examined in a BLI assay. As shown in Table 8, A131 is capable of binding with high affinity to both TNF-α and TGF-β. Table 8 TNF-α binding affinity, KD (M) Binding affinity to TGF-β1, KD (M) Binding affinity to TGF-β2, KD (M) Binding affinity to TGF-β3, KD (M) A131 ~1,0E-12 ~1,0E-12 ~1,0E-8 ~5,0E-11 Example 3

[0198] In this example, the neutralizing activities of bifunctional antagonists were investigated using cell-based NF-κB and Smad2 / 3 reporter assays with the ability to detect TNF-α-mediated signaling and TGF-β-mediated signaling.

[0199] Smad2 / 3 signaling assay. To measure the activities of TGF-β-mediated signaling pathways in cell cultures, a luciferase reporter gene-engineered cell line, C2C12-CAGA-luc, with the ability to detect Smad2 / 3 signaling was used. To measure the neutralizing activities of bifunctional antagonists, 1 nM human TGF-β1, TGF-β2, or TGF-β3 ligand solution was pre-incubated with each bifunctional antagonist at increasing concentrations of 0.00004 nM, 0.0004 nM, 0.004 nM, 0.04 nM, 0.4 nM, 4 nM, 40 nM, and 400 nM for 1 hour at room temperature. The reaction mixtures were then added to cultures of C2C12-CAGA-luc-bearing cells. After incubation for 5 hours in a CO2 incubator at 37°C, luciferase activity in cultures containing the C2C12-CAGA-luc reporter gene was measured using a LuminoSkan Ascent instrument (Thermo Scientific).IC50 values ​​were analyzed and plotted using Prism software (from GraphPad Software).

[0200] NF-κB signaling assay. To determine the TNF-α-neutralizing activity, a stably transfected luciferase reporter gene cell line, K536-NF-κB-luc, with the ability to detect TNF-α-mediated NF-κB signaling was used to quantify the IC50 value of each bifunctional antagonist in blocking TNF-α-mediated NF-κB signaling. Specifically, to measure TNF-α-neutralizing activity, human TNF-α at a final concentration of 0.02 nM was pre-incubated with individual bifunctional antagonists at increasing concentrations of 0.00001 nM, 0.0001 nM, 0.001 nM, 0.01 nM, 0.1 nM, 1.0 nM, 10 nM, and 100 nM for 1 hour at room temperature. The reaction mixtures were then added to cultures of cells carrying the K536-NF-κB-luc reporter gene.After incubation for 5 hours in a CO2 incubator at 37°C, luciferase activity was measured in cell cultures with the K536-NF-kB-luc reporter gene using a LuminoSkan Ascent instrument (Thermo Scientific). IC values. 50 Calculated and plotted graphs using Prism software (from GraphPad Software).

[0201] As shown in FIG. 4 and FIG. 5, A119 and A120 had a strong neutralizing effect on TNF-α, TGF-β1 and TGF-β3 in the cell-based assay. Example 4

[0202] Pulmonary arterial hypertension (PAH) is characterized by thickening of the pulmonary artery walls due to proliferation and hypertrophy of pulmonary arterial smooth muscle cells, as well as increased deposition of extracellular matrix. TNF-α and TGF-β levels have been shown to be significantly elevated in lung tissue from patients with PAH and in animal models of PAH. Inhibition of TNF-α or TGF-β has been shown to attenuate pulmonary hypertension. Therefore, TNF-α and TGF-β are both involved in the pathogenesis of PAH.

[0203] In this example, the ability of A120, a bifunctional TNF-α and TGF-β antagonist, to affect pulmonary arterial smooth muscle cell remodeling was investigated compared with that of an anti-TNF antibody and TGFRII-Fc by using a culture of primary human pulmonary arterial smooth muscle cells in the presence of TNF-α and TGF-β.

[0204] Primary human pulmonary arterial smooth muscle cells (from the American Type Culture Collection (ATCC)) were grown in vascular smooth muscle medium supplemented with 10% fetal bovine serum (FBS) in a CO2 incubator at 37°C. After reaching 80% confluency, the cells were passaged into the medium supplemented with 0.2% FBS and incubated under different conditions with or without the addition of different agents as follows: 1) no addition, 2) with TNF-α, 3) with TGF-β1, 4) with TNF-α + TGF-β1, 5) with TNF-α + TGF-β1 + anti-TNF antibody, 6) with TNF-α + TGF-β1 + TGFRII-Fc and 7) with TNF-α + TGF-β1 + A120. After 72 hours of incubation, photographs of the cell cultures were taken using an inverted microscope coupled with a digital camera, and morphometric analysis was performed using ImageJ software.

[0205] FIG. 6 and FIG. 7 show the effects of TNF-α, TGF-β1, and a combination of TNF-α and TGF-β1 on the proliferation and size of primary human pulmonary arterial smooth muscle cells (PASMCs). In addition, these figures also show the effects of an anti-TNF antibody, a TGF-β-neutralizing protein TGFRII-Fc, and a bifunctional antagonist A120, respectively, on the proliferation and size of PASMCs in cell cultures under the conditions of exogenous addition of TNF-α and TGF-β1. The data demonstrate that addition of TNF-α to the culture resulted in PASMC hyperplasia due to enhanced cell proliferation (FIG. 6, panel B), addition of TGF-β1 to the culture resulted in PASMC hypertrophy due to increased cell size (FIG. 6, panel C), and addition of TNF-α and TGF-β1 in combination to the culture resulted in both PASMC hyperplasia and hypertrophy due to simultaneous enhancement of cell proliferation and increase in cell size (FIG. 6, panel D).Thus, following exposure to elevated levels of TNF-α and TGF-β1, PASMCs underwent highly significant pathological remodeling characterized by both hyperplasia and hypertrophy. Furthermore, under conditions of elevated TNF-α and TGF-β1, inhibition of TNF-α with anti-TNF antibody prevented PASMC hyperplasia but not hypertrophy (FIG. 6, panel E), inhibition of TGF-β1 by TGFRII-Fc attenuated PASMC hypertrophy but not hyperplasia (FIG. 6, panel F), and simultaneous inhibition of TNF-α and TGF-β1 by A120 prevented both PASMC hyperplasia and hypertrophy (FIG. 6, panel G). Morphometric analysis of cell number (FIG. 7, panel A) and cell size (FIG. 7, panel B) indicated that, compared with anti-TNF or TGFRII-Fc antibody, A120 was more effective in suppressing pathological remodeling in PASMCs.The excellent ability of A120 to inhibit both PASMC hyperplasia and hypertrophy suggests that the use of the novel bifunctional TNF-α and TGF-β antagonists described in the present invention represents a promising new therapeutic approach for the treatment of RAS. Example 5.

[0206] Fibrosis, a pathological process characterized by the replacement of functional tissue with fibrous tissue, can occur in virtually any tissue or organ, leading to a variety of fibrosing diseases. As a primary fibrosis-associated condition, idiopathic pulmonary fibrosis (IPF) involves extensive lung tissue damage due to persistent inflammation, excessive collagen fibril formation, and their deposition in the extracellular matrix. TNF-α and TGF-β expression levels have been shown to be elevated in lung tissue from patients with IPF, as well as in animal models of pulmonary fibrosis. Elevated TNF-α levels mediate pulmonary inflammation and also play a role in the transition from pulmonary inflammation to fibrosis, while elevated TGF-β levels may directly contribute to pulmonary fibrosis by stimulating excessive collagen fibril formation.

[0207] In this example, A120, a novel bifunctional TNF-α and TGF-β antagonist, was evaluated against TNF antibody and TGFRII-Fc, respectively, for its ability to counteract fibrosis in a mouse model of bleomycin-induced pulmonary fibrosis.

[0208] Study of a mouse model of bleomycin-induced pulmonary fibrosis. All procedures involving animals were approved by the Institutional Animal Care Committee. Eight-week-old male C57BL / 6 mice were purchased from Jackson Laboratories. Mice were maintained on a 12-hour light / dark cycle and had ad libitum access to water and laboratory rodent chow. Mice were acclimatized for 1 week before treatment. Bleomycin (Sigma) was dissolved in sterile 0.9% saline and administered at a single dose of 0.5 mg / kg per animal. Control animals received saline only. All animals received either bleomycin or saline instillation via intratracheal injection (IT) on day 0.Mice were randomly assigned to the following groups: (1) IT saline (control); (2) IT bleomycin (bleomycin); (3) IT bleomycin plus TGFRII-Fc (bleomycin+TGFRII-Fc); (4) IT bleomycin plus TNF antibody (bleomycin+TNF Ab); (4) IT bleomycin plus A120 (bleomycin+A120). Starting from day -2, groups of mice separately received TGFRII-Fc, TNF antibody, and A120, respectively, once a week by subcutaneous (s.c.) administration at a dose of 5-10 mg / kg, normalized for the molecular weight of each agent. After two weeks of treatment, the animals were sacrificed, right lung tissue was collected into cassettes, and fixed in neutral-buffered formalin. For histological evaluation, the samples were dehydrated in a series of gradually varying ethanol concentrations, clarified in xylene, and embedded in paraffin.Sections were cut at a thickness of 4–6 μm and stained with hematoxylin and eosin (H&E), Masson's trichrome (MT), and an antibody to alpha-SMA conjugated with HRP. Fibrosing lung lesions were assessed histologically using the Ashcroft scale (Hübner et al. 2008. PMID: 18476815; DOI: 10.2144 / 000112729). Ashcroft scores were assigned at 10x magnification.

[0209] FIG. 8 shows the histological images of H&E-stained lung sections and the Ashcroft scores for lung sections from control mice and mice with bleomycin-induced pulmonary fibrosis. The data indicate that the simultaneous inhibition of TNF-α and TGF-β by A120 significantly suppressed bleomycin-induced pulmonary fibrosis, while the anti-TNF antibody or TGFRII-Fc showed a moderate suppression of bleomycin-induced pulmonary fibrosis. Analysis of the Ashcroft scores revealed that treatment with A120 resulted in a greater attenuation of pulmonary fibrosis compared with treatment with the anti-TNF antibody or TGFRII-Fc.

[0210] FIG. 9 shows histological images of lung sections stained with Masson's trichrome and the results of quantitative analysis of the collagen deposition area in lung sections from control and bleomycin-treated mice. The data showed that A120 was able to more effectively reduce bleomycin-induced collagen deposition compared with the TNF antibody or TGFRII-Fc.

[0211] FIG. 10 shows the results of immunochemical staining for alpha-smooth muscle actin (αSMA), a fibrosis marker, in lung sections from control and bleomycin-treated mice. The data indicate that, compared with the TNF antibody or TGFRII-Fc, A120 more effectively attenuated the induction of αSMA immunoreactivity in bleomycin-treated mice.

[0212] Taken together, the data obtained in mice with bleomycin-induced pulmonary fibrosis demonstrate that A120, through simultaneous neutralization of TNF-α and TGF-β, is more effective in preventing fibrosis than an anti-TNF antibody or TGFRII-Fc. The enhanced anti-fibrotic effect of A120 suggests that the use of the novel bifunctional TNF and TGF-β antagonists described in this invention represents a promising new approach for the treatment of various fibrosing diseases.

[0213] All articles and methods disclosed and claimed in this specification can be made and implemented without undue experimentation in light of the present invention. Although the articles and methods of this invention have been described in terms of preferred embodiments, it will be obvious to those skilled in the art that variations of these articles and methods can be used without departing from the spirit and scope of the invention. All such variations and equivalents obvious to those skilled in the art, both now existing and those later developed, are considered to be within the spirit and scope of the invention, as defined by the appended claims. All patents, patent applications, and publications mentioned in this specification are indicative of the levels of ordinary skill in the art to which this invention pertains.All patents, patent applications, and publications are herein incorporated by reference in their entirety for all purposes and to the same extent as if each individual publication were specifically and individually indicated to be incorporated by reference in its entirety for all purposes. The invention illustratively described in this application may suitably be practiced in the absence of any element(s) not specifically described in this application. It should therefore be understood that although the present invention has been particularly described by means of preferred embodiments and optional features, those skilled in the art may modify and vary the concepts disclosed herein, and such modifications and variations are considered to be included within the scope of the invention as defined by the appended claims. Sequence Listings

[0214] The nucleic acid and amino acid sequences provided in the accompanying sequence listing are shown using standard one-letter abbreviations for nucleotide bases and three-letter codes for amino acids as defined in 37 CFR 1.822.SEQ ID NOs: 1-5 delineate the amino acid sequences of various TNF ligands.SEQ ID NOs: 6-9 delineate the amino acid sequences of various TGF-β ligands.SEQ ID NOs: 10, 14, 18, and 22 delineate the amino acid sequences of the heavy chain of various antibodies that specifically bind to TNF-α ligand.SEQ ID NOs: 11, 15, 19, and 23 delineate the amino acid sequences of the light chain of various antibodies that specifically bind to TNF-α ligand.SEQ ID NOs: 12, 16, 20 and 24 set forth the amino acid sequences of the variable region of the heavy chain of various antibodies that specifically bind to TNF-α ligand. SEQ ID NOs: 13, 17, 21 and 25 set forth the amino acid sequences of the variable region of the light chain of various antibodies that specifically bind to TNF-α ligand. SEQ ID NO: 26 sets forth the amino acid sequence of the heavy chain of an antibody that specifically binds to TGF-β ligand. SEQ ID NO: 27 sets forth the amino acid sequence of the light chain of an antibody that specifically binds to TGF-β ligand. SEQ ID NO: 28 sets forth the amino acid sequence of the variable region of the heavy chain of an antibody that specifically binds to TGF-β ligand. SEQ ID NO: 29 sets forth the amino acid sequence of the variable region of the light chain of an antibody that specifically binds with TGF-β ligand.SEQ ID NOs: 30-31 set forth the amino acid sequences of the heavy chain of a bifunctional antagonist molecule that specifically binds to a TNF-α ligand and to a TGF-β ligand. SEQ ID NOs: 32-41 set forth the amino acid sequences of various bifunctional antagonist molecules that specifically bind to a TNF-α ligand and that specifically bind to a TGF-β ligand. SEQ ID NO: 42 set forth the amino acid sequence of the heavy chain of a bifunctional antagonist molecule that specifically binds to a TNF-α ligand and to a TGF-β ligand. SEQ ID NO: 43 set forth the amino acid sequence of the light chain of a bifunctional antagonist molecule that specifically binds to a TNF-α ligand and to a TGF-β ligand. SEQ ID NOs: 44-63 set forth the amino acid sequences of various peptide linkers. SEQUENCE LISTINGS. Human TGF-β receptor II isoform 1 Human TGF-β receptor II ECD, isoform 1 (TGF-β RIIB-ECD) Human TGF-β receptor II, isoform 2 Human TGF-β receptor II ECD, isoform 2 (TGF-β RIIA-ECD) Amino acid sequence of the heavy chain of the antibody to TNF-α Amino acid sequence of the light chain of the antibody to TNF-α Amino acid sequence of the variable region of the heavy chain of the antibody to TNF-α Amino acid sequence of the light chain variable region of the antibody to TNF-α Amino acid sequence of the heavy chain of the antibody to TNF-α Amino acid sequence of the light chain of the antibody to TNF-α Amino acid sequence of the variable region of the heavy chain of the antibody to TNF-α Amino acid sequence of the light chain variable region of the antibody to TNF-α Amino acid sequence of the heavy chain of the antibody to TNF-α Amino acid sequence of the light chain of the antibody to TNF-α Amino acid sequence of the variable region of the heavy chain of the antibody to TNF-α Amino acid sequence of the light chain variable region of the antibody to TNF-α Amino acid sequence of the heavy chain of the antibody to TNF-α Amino acid sequence of the light chain of the antibody to TNF-α Amino acid sequence of the variable region of the heavy chain of the antibody to TNF-α Amino acid sequence of the light chain variable region of the antibody to TNF-α Amino acid sequence of the heavy chain of the antibody to TGF-β Amino acid sequence of the light chain of the antibody to TGF-β Amino acid sequence of the variable region of the heavy chain of the antibody to TGF-β Amino acid sequence of the light chain variable region of an antibody to TGF-β Amino acid sequence of the heavy chain of A119 Amino acid sequence of the heavy chain of A12Q Amino acid sequence of A121 Amino acid sequence of A122 Amino acid sequence of A123 Amino acid sequence of A124 Amino acid sequence of A125 Amino acid sequence of A126 Amino acid sequence of A127 Amino acid sequence of A128 Amino acid sequence of A129 Amino acid sequence of A130 Amino acid sequence of the heavy chain of A131 Amino acid sequence of the light chain of A131 Peptide linker sequence GGGSGGGSGGGS (SEQ ID NO: 44). Peptide linker sequence GGGS (SEQ ID NO: 45). Peptide linker sequence GSSGGSGGSGGSG (SEQ ID NO: 46). Peptide linker sequence GSSGT (SEQ ID NO: 47). Peptide linker sequence GGGGSGGGGSGGGS (SEQ ID NO: 48). Peptide linker sequence AEAAAKEAAAKEAAAKA (SEQ ID NO: 49). Peptide linker sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 50). Peptide linker sequence GGGSGGGS (SEQ ID NO: 51). Peptide linker sequence GS (SEQ ID NO: 52). Peptide linker sequence GGS (SEQ ID NO: 53). Peptide linker sequence GGGGS (SEQ ID NO: 54). Peptide linker sequence GGSG (SEQ ID NO: 55). Peptide linker sequence SGGG (SEQ ID NO: 56). Peptide linker sequence GSGS (SEQ ID NO: 57). Peptide linker sequence GSGSGS (SEQ ID NO: 58). Peptide linker sequence GSGSGSGS (SEQ IDNO: 59). Peptide linker sequence GSGSGSGSGS (SEQ ID NO: 60). Peptide linker sequence GSGSGSGSGSGS (SEQ ID NO: 61). Peptide linker sequence GGGGSGGGGS (SEQ ID NO: 62). Peptide linker sequence GGGGSGGGGSGGGGS (SEQ ID NO: 63). ---> SEQUENCE LISTING <110> HAN, HQZHOU, XIAOLAN <120> Bifunctional Tumour Necrosis Factor-Alpha and Transforming Growth Factor-Beta Antagonists and Their Applications <130> CACAG1.0008WO <160> 63 <170> PatentIn version 3.5 <210> 1 <211> 190 <212> PRT <213> Homo sapiens <400> 1Ile Tyr Pro Ser Gly Val Ile Gly Leu Val Pro His Leu Gly Asp Arg1 5 10 15Glu Lys Arg Asp Ser Val Cys Pro Gln Gly Lys Tyr Ile His Pro Gln20 25 30Asn Asn Ser Ile Cys Cys Thr Lys Cys His Lys Gly Thr Tyr Leu Tyr35 40 45Asn Asp Cys Pro Gly Pro Gly Gln Asp Thr Asp Cys Arg Glu Cys Glu50 55 60Ser Gly Ser Phe Thr Ala Ser Glu Asn His Leu Arg His Cys Leu Ser65 70 75 80Cys Ser Lys Cys Arg Lys Glu Met Gly Gln Val Glu Ile Ser Ser Cys85 90 95Thr Val AspArg Asp Thr Val Cys Gly Cys Arg Lys Asn Gln Tyr Arg100 105 110His Tyr Trp Ser Glu Asn Leu Phe Gln Cys Phe Asn Cys Ser Leu Cys115 120 125Leu Asn Gly Thr Val His Leu Ser Cys Gln Glu Lys Gln Asn Thr Val130 135 140Cys Thr Cys His Ala Gly Phe Phe Leu Arg Glu Asn Glu Cys Val Ser145 150 155 160Cys Ser Asn Cys Lys Lys Ser Leu Glu Cys Thr Lys Leu Cys Leu Pro165 170 175Gln Ile Glu Asn Val Lys Gly Thr Glu Asp Ser Gly Thr Thr180 185 190<210> 2<211> 235<212> PRT<213> Homo sapiens<400> 2Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser1 5 10 15Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met Cys Cys20 25 30Ser Lys Cys Ser Pro Gly Gln His Ala Lys Val Phe Cys Thr Lys Thr35 40 45Ser Asp Thr Val Cys Asp Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu50 55 60Trp Asn Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser65 70 75 80Asp Gln Val Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys85 90 95Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys100 105 110Arg Leu Cys AlaPro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala115 120 125Arg Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro Cys Ala Pro130 135 140Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His145 150 155 160Gln Ile Cys Asn Val Val Ala Ile Pro Gly Asn Ala Ser Met Asp Ala165 170 175Val Cys Thr Ser Thr Ser Pro Thr Arg Ser Met Ala Pro Gly Ala Val180 185 190His Leu Pro Gln Pro Val Ser Thr Arg Ser Gln His Thr Gln Pro Thr195 200 205Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser Phe Leu Leu Pro Met Gly210 215 220Pro Ser Pro Pro Ala Glu Gly Ser Thr Gly Asp225 230 235<210> 3<211> 242<212> PRT<213> Homo sapiens<400> 3Ile Tyr Pro Ser Gly Val Ile Gly Leu Val Pro His Leu Gly Asp Arg1 5 10 15Glu Lys Arg Asp Ser Val Cys Pro Gln Gly Lys Tyr Ile His Pro Gln20 25 30Asn Asn Ser Ile Cys Cys Thr Lys Cys His Lys Gly Thr Tyr Leu Tyr35 40 45Asn Asp Cys Pro Gly Pro Gly Gln Asp Thr Asp Cys Arg Ser Cys Glu50 55 60Asp Ser Thr Tyr Thr Gln Leu Trp Asn Trp Val Pro Glu Cys Leu Ser65 70 75 80Cys Gly Ser Arg Cys Ser Ser AspGln Val Glu Thr Gln Ala Cys Thr85 90 95Arg Glu Gln Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp Tyr Cys Ala100 105 110Leu Ser Lys Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys Cys115 120 125Arg Pro Gly Phe Gly Val Ala Arg Pro Gly Thr Glu Thr Ser Asp Val130 135 140Val Cys Lys Pro Cys Ala Pro Gly Thr Phe Ser Asn Thr Thr Ser Ser145 150 155 160Thr Asp Ile Cys Arg Pro His Gln Ile Cys Asn Val Val Ala Ile Pro165 170 175Gly Asn Ala Ser Met Asp Ala Val Cys Thr Ser Thr Ser Pro Thr Arg180 185 190Ser Met Ala Pro Gly Ala Val His Leu Pro Gln Pro Val Ser Thr Arg195 200 205Ser Gln His Thr Gln Pro Thr Pro Glu Pro Ser Thr Ala Pro Ser Thr210 215 220Ser Phe Leu Leu Pro Met Gly Pro Ser Pro Pro Ala Glu Gly Ser Thr225 230 235 240Gly Asp<210> 4<211> 240<212> PRT<213> Homo sapiens<400> 4Ile Tyr Pro Ser Gly Val Ile Gly Leu Val Pro His Leu Gly Asp Arg1 5 10 15Glu Lys Arg Asp Ser Val Cys Pro Gln Gly Lys Tyr Ile His Pro Gln20 25 30Asn Asn Ser Ile Cys Cys Thr Lys Cys His Lys Gly Thr Tyr Leu Tyr35 40 45Asn Asp Cys ProGly Pro Gly Gln Asp Thr Asp Cys Arg Glu Cys Glu50 55 60Ser Gly Ser Phe Thr Ala Ser Glu Asn His Leu Arg His Cys Leu Ser65 70 75 80Cys Ser Lys Cys Arg Lys Glu Met Gly Gln Val Glu Ile Ser Ser Cys85 90 95Thr Val Asp Arg Asp Thr Val Cys Gly Cys Arg Lys Asn Gln Tyr Arg100 105 110His Tyr Trp Ser Glu Asn Leu Phe Gln Cys Phe Asn Cys Ser Leu Cys115 120 125Leu Asn Gly Thr Val His Leu Ser Cys Gln Glu Lys Gln Asn Thr Val130 135 140Cys Pro Cys Ala Pro Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp145 150 155 160Ile Cys Arg Pro His Gln Ile Cys Asn Val Val Ala Ile Pro Gly Asn165 170 175Ala Ser Met Asp Ala Val Cys Thr Ser Thr Ser Pro Thr Arg Ser Met180 185 190Ala Pro Gly Ala Val His Leu Pro Gln Pro Val Ser Thr Arg Ser Gln195 200 205His Thr Gln Pro Thr Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser Phe210 215 220Leu Leu Pro Met Gly Pro Ser Pro Pro Ala Glu Gly Ser Thr Gly Asp225 230 235 240<210> 5<211> 145<212> PRT<213> Homo sapiens<400> 5Ile Tyr Pro Ser Gly Val Ile Gly Leu Val Pro His Leu Gly Asp Arg1 5 10 15GluLys Arg Asp Ser Val Cys Pro Gln Gly Lys Tyr Ile His Pro Gln20 25 30Asn Asn Ser Ile Cys Cys Thr Lys Cys His Lys Gly Thr Tyr Leu Tyr35 40 45Asn Asp Cys Pro Gly Pro Gly Gln Asp Thr Asp Cys Arg Glu Cys Glu50 55 60Ser Gly Ser Phe Thr Ala Ser Glu Asn His Leu Arg His Cys Leu Ser65 70 75 80Cys Ser Lys Cys Arg Lys Glu Met Gly Gln Val Glu Ile Ser Ser Cys85 90 95Thr Val Asp Arg Asp Thr Val Cys Gly Cys Arg Lys Asn Gln Tyr Arg100 105 110His Tyr Trp Ser Glu Asn Leu Phe Gln Cys Phe Asn Cys Ser Leu Cys115 120 125Leu Asn Gly Thr Val His Leu Ser Cys Gln Glu Lys Gln Asn Thr Val130 135 140Cys145<210> 6<211> 567<212> PRT<213> Homo sapiens<400> 6Met Gly Arg Gly Leu Leu Arg Gly Leu Trp Pro Leu His Ile Val Leu1 5 10 15Trp Thr Arg Ile Ala Ser Thr Ile Pro Pro His Val Gln Lys Ser Val20 25 30Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro35 40 45Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln50 55 60Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro65 70 75 80Gln GluVal Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr85 90 95Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile100 105 110Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys115 120 125Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn130 135 140Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Leu145 150 155 160Leu Leu Val Ile Phe Gln Val Thr Gly Ile Ser Leu Leu Pro Pro Leu165 170 175Gly Val Ala Ile Ser Val Ile Ile Ile Phe Tyr Cys Tyr Arg Val Asn180 185 190Arg Gln Gln Lys Leu Ser Ser Thr Trp Glu Thr Gly Lys Thr Arg Lys195 200 205Leu Met Glu Phe Ser Glu His Cys Ala Ile Ile Leu Glu Asp Asp Arg210 215 220Ser Asp Ile Ser Ser Thr Cys Ala Asn Asn Ile Asn His Asn Thr Glu225 230 235 240Leu Leu Pro Ile Glu Leu Asp Thr Leu Val Gly Lys Gly Arg Phe Ala245 250 255Glu Val Tyr Lys Ala Lys Leu Lys Gln Asn Thr Ser Glu Gln Phe Glu260 265 270Thr Val Ala Val Lys Ile Phe Pro Tyr Glu Glu Tyr Ala Ser Trp Lys275 280 285Thr Glu Lys Asp Ile Phe Ser Asp Ile AsnLeu Lys His Glu Asn Ile290 295 300Leu Gln Phe Leu Thr Ala Glu Glu Arg Lys Thr Glu Leu Gly Lys Gln305 310 315 320Tyr Trp Leu Ile Thr Ala Phe His Ala Lys Gly Asn Leu Gln Glu Tyr325 330 335Leu Thr Arg His Val Ile Ser Trp Glu Asp Leu Arg Lys Leu Gly Ser340 345 350Ser Leu Ala Arg Gly Ile Ala His Leu His Ser Asp His Thr Pro Cys355 360 365Gly Arg Pro Lys Met Pro Ile Val His Arg Asp Leu Lys Ser Ser Asn370 375 380Ile Leu Val Lys Asn Asp Leu Thr Cys Cys Leu Cys Asp Phe Gly Leu385 390 395 400Ser Leu Arg Leu Asp Pro Thr Leu Ser Val Asp Asp Leu Ala Asn Ser405 410 415Gly Gln Val Gly Thr Ala Arg Tyr Met Ala Pro Glu Val Leu Glu Ser420 425 430Arg Met Asn Leu Glu Asn Val Glu Ser Phe Lys Gln Thr Asp Val Tyr435 440 445Ser Met Ala Leu Val Leu Trp Glu Met Thr Ser Arg Cys Asn Ala Val450 455 460Gly Glu Val Lys Asp Tyr Glu Pro Pro Phe Gly Ser Lys Val Arg Glu465 470 475 480His Pro Cys Val Glu Ser Met Lys Asp Asn Val Leu Arg Asp Arg Gly485 490 495Arg Pro Glu Ile Pro Ser Phe Trp Leu Asn His Gln Gly Ile Gln Met500505 510Val Cys Glu Thr Leu Thr Glu Cys Trp Asp His Asp Pro Glu Ala Arg515 520 525Leu Thr Ala Gln Cys Val Ala Glu Arg Phe Ser Glu Leu Glu His Leu530 535 540Asp Arg Leu Ser Gly Arg Ser Cys Ser Glu Glu Lys Ile Pro Glu Asp545 550 555 560Gly Ser Leu Asn Thr Thr Lys565<210> 7<211> 137<212> PRT<213> Homo sapiens<400> 7Thr Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val1 5 10 15Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys20 25 30Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn35 40 45Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala50 55 60Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His65 70 75 80Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser85 90 95Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe100 105 110Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser115 120 125Glu Glu Tyr Asn Thr Ser Asn Pro Asp130 135<210> 8<211> 592<212> PRT<213> Homo sapiens<400> 8Met Gly Arg GlyLeu Leu Arg Gly Leu Trp Pro Leu His Ile Val Leu1 5 10 15Trp Thr Arg Ile Ala Ser Thr Ile Pro Pro His Val Gln Lys Ser Asp20 25 30Val Glu Met Glu Ala Gln Lys Asp Glu Ile Ile Cys Pro Ser Cys Asn35 40 45Arg Thr Ala His Pro Leu Arg His Ile Asn Asn Asp Met Ile Val Thr50 55 60Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp65 70 75 80Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys85 90 95Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val100 105 110Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp115 120 125Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro130 135 140Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met145 150 155 160Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu165 170 175Glu Tyr Asn Thr Ser Asn Pro Asp Leu Leu Leu Val Ile Phe Gln Val180 185 190Thr Gly Ile Ser Leu Leu Pro Pro Leu Gly Val Ala Ile Ser Val Ile195 200 205Ile Ile Phe Tyr Cys Tyr Arg Val Asn Arg Gln Gln Lys Leu SerSer210 215 220Thr Trp Glu Thr Gly Lys Thr Arg Lys Leu Met Glu Phe Ser Glu His225 230 235 240Cys Ala Ile Ile Leu Glu Asp Asp Arg Ser Asp Ile Ser Ser Thr Cys245 250 255Ala Asn Asn Ile Asn His Asn Thr Glu Leu Leu Pro Ile Glu Leu Asp260 265 270Thr Leu Val Gly Lys Gly Arg Phe Ala Glu Val Tyr Lys Ala Lys Leu275 280 285Lys Gln Asn Thr Ser Glu Gln Phe Glu Thr Val Ala Val Lys Ile Phe290 295 300Pro Tyr Glu Glu Tyr Ala Ser Trp Lys Thr Glu Lys Asp Ile Phe Ser305 310 315 320Asp Ile Asn Leu Lys His Glu Asn Ile Leu Gln Phe Leu Thr Ala Glu325 330 335Glu Arg Lys Thr Glu Leu Gly Lys Gln Tyr Trp Leu Ile Thr Ala Phe340 345 350His Ala Lys Gly Asn Leu Gln Glu Tyr Leu Thr Arg His Val Ile Ser355 360 365Trp Glu Asp Leu Arg Lys Leu Gly Ser Ser Leu Ala Arg Gly Ile Ala370 375 380His Leu His Ser Asp His Thr Pro Cys Gly Arg Pro Lys Met Pro Ile385 390 395 400Val His Arg Asp Leu Lys Ser Ser Asn Ile Leu Val Lys Asn Asp Leu405 410 415Thr Cys Cys Leu Cys Asp Phe Gly Leu Ser Leu Arg Leu Asp Pro Thr420 425 430Leu Ser ValAsp Asp Leu Ala Asn Ser Gly Gln Val Gly Thr Ala Arg435 440 445Tyr Met Ala Pro Glu Val Leu Glu Ser Arg Met Asn Leu Glu Asn Val450 455 460Glu Ser Phe Lys Gln Thr Asp Val Tyr Ser Met Ala Leu Val Leu Trp465 470 475 480Glu Met Thr Ser Arg Cys Asn Ala Val Gly Glu Val Lys Asp Tyr Glu485 490 495Pro Pro Phe Gly Ser Lys Val Arg Glu His Pro Cys Val Glu Ser Met500 505 510Lys Asp Asn Val Leu Arg Asp Arg Gly Arg Pro Glu Ile Pro Ser Phe515 520 525Trp Leu Asn His Gln Gly Ile Gln Met Val Cys Glu Thr Leu Thr Glu530 535 540Cys Trp Asp His Asp Pro Glu Ala Arg Leu Thr Ala Gln Cys Val Ala545 550 555 560Glu Arg Phe Ser Glu Leu Glu His Leu Asp Arg Leu Ser Gly Arg Ser565 570 575Cys Ser Glu Glu Lys Ile Pro Glu Asp Gly Ser Leu Asn Thr Thr Lys580 585 590<210> 9<211> 162<212> PRT<213> Homo sapiens<400> 9Thr Ile Pro Pro His Val Gln Lys Ser Asp Val Glu Met Glu Ala Gln1 5 10 15Lys Asp Glu Ile Ile Cys Pro Ser Cys Asn Arg Thr Ala His Pro Leu20 25 30Arg His Ile Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val35 4045Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys50 55 60Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys65 70 75 80Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu85 90 95Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His100 105 110Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu115 120 125Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp130 135 140Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn145 150 155 160Pro Asp<210> 10<211> 451<212> PRT<213> Homo sapiens<400> 10Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr20 25 30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val35 40 45Ser Ala Ile Thr Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val50 55 60Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr TyrCys85 90 95Ala Lys Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Tyr Trp Gly100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser115 120 125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys210 215 220Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly225 230 235 240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His260 265 270Glu Asp Pro Glu Val Lys Phe Asn Val Tyr Val Asp Gly Val Glu Val275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr290 295 300Arg Val Val Ser ValLeu Thr Val Leu His Gln Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val340 345 350Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser355 360 365Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu370 375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met420 425 430His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser435 440 445Pro Gly Lys450<210> 11<211> 214<212> PRT<213> Homo sapiens<400> 11Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Tyr20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile35 40 45Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly5055 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Val Ala Thr Tyr Tyr Cys Gln Arg Tyr Asn Arg Ala Pro Tyr85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser195 200 205Phe Asn Arg Gly Glu Cys210<210> 12<211> 121<212> PRT<213> Homo sapiens<400> 12Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr20 25 30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val35 40 45Ser Ala Ile Thr Trp Asn Ser Gly HisIle Asp Tyr Ala Asp Ser Val50 55 60Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys85 90 95Ala Lys Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Tyr Trp Gly100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser115 120 <210> 13 <211> 107 <212> PRT <213> Homo sapiens <400> 13Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Tyr20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile35 40 45Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Val Ala Thr Tyr Tyr Cys Gln Arg Tyr Asn Arg Ala Pro Tyr85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys100 105 <210> 14 <211> 450 <212> PRT <213> Artificial <220> <223> Amino acid sequence of the heavy chain of a chimeric antibody to TNF-alpha <400> 14Glu Val Lys Leu Glu Glu Ser Gly GlyGly Leu Val Gln Pro Gly Gly1 5 10 15Ser Met Lys Leu Ser Cys Val Ala Ser Gly Phe Ile Phe Ser Asn His20 25 30Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val35 40 45Ala Glu Ile Arg Ser Lys Ser Ile Asn Ser Ala Thr His Tyr Ala Glu50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ala65 70 75 80Val Tyr Leu Gln Met Thr Asp Leu Arg Thr Glu Asp Thr Gly Val Tyr85 90 95Tyr Cys Ser Arg Asn Tyr Tyr Gly Ser Thr Tyr Asp Tyr Trp Gly Gln100 105 110Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val115 120 125Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala130 135 140Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser145 150 155 160Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val165 170 175Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro180 185 190Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys195 200 205Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp210 215 220Lys ThrHis Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly225 230 235 240Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile245 250 255Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu260 265 270Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His275 280 285Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg290 295 300Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys305 310 315 320Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu325 330 335Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr340 345 350Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu355 360 365Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp370 375 380Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val385 390 395 400Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp405 410 415Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His420 425 430Glu Ala Leu His Asn His Tyr ThrGln Lys Ser Leu Ser Leu Ser Pro435 440 445Gly Lys450 <210> 15 <211> 214 <212> PRT <213> Artificial <220> <223> The amino acid sequence of the light chain of the chimeric antibody to TNF-alpha <400> 15Asp Ile Leu Leu Thr Gln Ser Pro Ala Ile Leu Ser Val Ser Pro Gly1 5 10 15Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Phe Val Gly Ser Ser20 25 30Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Tyr 45 Lys 45Lys Ser Glu Ser Met Ser Gly Ile Pro Ser Arg Phe Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Thr Val Glu Ser65 70 75 80Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Ser His Ser Trp Pher 95 95 Ser Gly Pher Asn Leu Glu Val Lys Arg Thr Val Ala Ala100 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Phe Tyr Pro Arg Glu Ala130 130 135 135 135 135 GLys Val Gl A Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser165 170 175 Ser ThrLeu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser195 200 205Phe Asn Arg Gly Glu Cys210 <210> 16 <211> 120 <212> PRT <213> Artificial <220> <223> Amino acid sequence of the heavy chain variable region of a chimeric antibody to TNF-alpha <400> 16Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Met Lys Leu Ser Cys Val Ala Ser Gly Phe Ile Phe Ser Asn His20 25 30Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val35 40 45Ala Glu Ile Arg Ser Lys Ser Ile Asn Ser Ala Thr His Tyr Ala Glu50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ala65 70 75 80Val Tyr Leu Gln Met Thr Asp Leu Arg Thr Glu Asp Thr Gly Val Tyr85 90 95Tyr Cys Ser Arg Asn Tyr Tyr Gly Ser Thr Tyr Asp Tyr Trp Gly Gln100 105 110Gly Thr Thr Leu Thr Val Ser Ser115 120 <210> 17 <211> 107 <212> PRT <213> Artificial <220> <223> Amino acid sequence of the light chain variable region of the chimericantibodies to TNF-alpha <400> 17Asp Ile Leu Leu Thr Gln Ser Pro Ala Ile Leu Ser Val Ser Pro Gly1 5 10 15Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Phe Val Gly Ser Ser20 25 30Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile35 40 45Lys Tyr Ala Ser Glu Ser Met Ser Gly Ile Pro Ser Arg Phe Ser Gly50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Thr Val Glu Ser65 70 75 80Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Ser His Ser Trp Pro Phe85 90 95Thr Phe Gly Ser Gly Thr Asn Leu Glu Val Lys100 105 <210> 18 <211> 229 <212> PRT <213> Artificial <220> <223> Amino acid sequence of the heavy chain of a humanized antibody to TNF-alpha <400> 18Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Val Phe Thr Asp Tyr20 25 30Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met35 40 45Gly Trp Ile Asn Thr Tyr Ile Gly Glu Pro Ile Tyr Ala Asp Ser Val50 55 60Lys Gly Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr AlaTyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys85 90 95Ala Arg Gly Tyr Arg Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr100 105 110Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro115 120 125Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly130 135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln165 170 175Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser180 185 190Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser195 200 205Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr210 215 220His Thr Cys Ala Ala225 <210> 19 <211> 214 <212> PRT <213> Artificial <220> <223> Amino acid sequence of the light chain of a humanized antibody to TNF-alpha <400> 19Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn20 25 30Val AlaTrp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Ala Leu Ile35 40 45Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Tyr Arg Phe Ser Gly50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ile Tyr Pro Leu85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser195 200 205Phe Asn Arg Gly Glu Cys210 <210> 20 <211> 118 <212> PRT <213> Artificial <220> <223> Amino acid sequence of the heavy chain variable region of a humanized antibody to TNF-alpha <400> 20Glu ValGln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Val Phe Thr Asp Tyr20 25 30Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met35 40 45Gly Trp Ile Asn Thr Tyr Ile Gly Glu Pro Ile Tyr Ala Asp Ser Val50 55 60Lys Gly Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys85 90 95Ala Arg Gly Tyr Arg Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr100 105 110Leu Val Thr Val Ser Ser115 <210> 21 <211> 107 <212> PRT <213> Artificial <220> <223> Amino acid sequence of the light chain variable region of a humanized antibody to TNF-alpha <400> 21Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Ala Leu Ile35 40 45Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Tyr Arg Phe Ser Gly50 55 60Ser Gly Ser Gly Thr Asp PheThr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ile Tyr Pro Leu85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys100 105<210> 22<211> 456<212> PRT<213> Homo sapiens<400> 22Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser Ser Tyr20 25 30Ala Met His Trp Val Arg Gln Ala Pro Gly Asn Gly Leu Glu Trp Val35 40 45Ala Phe Met Ser Tyr Asp Gly Ser Asn Lys Lys Tyr Ala Asp Ser Val50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys85 90 95Ala Arg Asp Arg Gly Ile Ala Ala Gly Gly Asn Tyr Tyr Tyr Tyr Gly100 105 110Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser115 120 125Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr130 135 140Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro145 150 155 160Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu ThrSer Gly Val165 170 175His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser180 185 190Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile195 200 205Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val210 215 220Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala225 230 235 240Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro245 250 255Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val260 265 270Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val275 280 285Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln290 295 300Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln305 310 315 320Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala325 330 335Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro340 345 350Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr355 360 365Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser370 375 380Asp IleAla Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr385 390 395 400Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr405 410 415Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe420 425 430Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys435 440 445Ser Leu Ser Leu Ser Pro Gly Lys450 455<210> 23<211> 215<212> PRT<213> Homo sapiens<400> 23Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Tyr Ser Tyr20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile35 40 45Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro85 90 95Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala100 105 110Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser115 120 125Gly Thr Ala Ser Val Val Cys Leu Leu AsnAsn Phe Tyr Pro Arg Glu130 135 140Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser145 150 155 160Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu165 170 175Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val180 185 190Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys195 200 205Ser Phe Asn Arg Gly Glu Cys210 215<210> 24<211> 126<212> PRT<213> Homo sapiens<400> 24Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser Ser Tyr20 25 30Ala Met His Trp Val Arg Gln Ala Pro Gly Asn Gly Leu Glu Trp Val35 40 45Ala Phe Met Ser Tyr Asp Gly Ser Asn Lys Lys Tyr Ala Asp Ser Val50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys85 90 95Ala Arg Asp Arg Gly Ile Ala Ala Gly Gly Asn Tyr Tyr Tyr Tyr Gly100 105 110Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser115 120 125<210>25 <211> 108 <212> PRT <213> Homo sapiens <400> 25Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Tyr Ser Tyr20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile35 40 45Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro Pro85 90 95Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys100 105 <210> 26 <211> 447 <212> PRT <213> Artificial <220> <223> Amino acid sequence of the heavy chain of TGF-beta antibody <400> 26Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Asn20 25 30Val Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met35 40 45Gly Gly Val Ile Pro Ile Val Asp Ile Ala Asn Tyr Ala Gln Arg Phe50 55 60Lys Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr ThrTyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys85 90 95Ala Ser Thr Leu Gly Leu Val Leu Asp Ala Met Asp Tyr Trp Gly Gln100 105 110Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val115 120 125Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala130 135 140Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser145 150 155 160Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val165 170 175Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro180 185 190Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys195 200 205Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro210 215 220Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val225 230 235 240Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr245 250 255Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu260 265 270Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys275 280 285Thr Lys Pro Arg GluGlu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser290 295 300Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys 303333 335Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro340 345 350Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu355 360 365Val Lys Gly Phe Tyr Pro Ser Asp Val Glu Tr30 Ser 375 380Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser385 390 395 400Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg405 410 415Trp Gln Gly P Gly P He Ser Met Ser His Ser Leu420 425 430His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys435 440 445 <210> 27 <211> 215 <212> PRT <213> Artificial <220> <223> The amino acid sequence of the mild receptor antibody to TGF-beta <400> 27Glu Thr Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Leu Gly Ser Ser20 25 30Tyr LeuAla Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Pro Gly Ile Pro Asp Arg Phe Ser50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Ala Asp Ser Pro85 90 95Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala100 105 110Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser115 120 125Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu130 135 140Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser145 150 155 160Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu165 170 175Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val180 185 190Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys195 200 205Ser Phe Asn Arg Gly Glu Cys210 215 <210> 28 <211> 120 <212> PRT <213> Artificial <220> <223> Amino acid sequence of the heavy chain variable region of the antibody to TGF-beta <400> 28Gln Val Gln LeuVal Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Asn20 25 30Val Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met35 40 45Gly Gly Val Ile Pro Ile Val Asp Ile Ala Asn Tyr Ala Gln Arg Phe50 55 60Lys Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Thr Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys85 90 95Ala Ser Thr Leu Gly Leu Val Leu Asp Ala Met Asp Tyr Trp Gly Gln100 105 110Gly Thr Leu Val Thr Val Ser Ser115 120 <210> 29 <211> 108 <212> PRT <213> Artificial <220> <223> Amino acid sequence of the light chain variable region of an antibody to TGF-beta <400> 29Glu Thr Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Leu Gly Ser Ser20 25 30Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Pro Gly Ile Pro Asp Arg Phe Ser50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu ThrIle Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Ala Asp Ser Pro85 90 95Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys100 105<210> 30<211> 603<212> PRT<213> Artificial<220><223> Аминокислотная последовательность тяжелой цепи A119<400> 30Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr20 25 30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val35 40 45Ser Ala Ile Thr Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val50 55 60Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys85 90 95Ala Lys Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Tyr Trp Gly100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser115 120 125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155 160SerTrp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys210 215 220Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly225 230 235 240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr290 295 300Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val340 345 350Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser355 360 365Leu Thr Cys Leu Val Lys Gly PheTyr Pro Ser Asp Ile Ala Val Glu370 375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met420 425 430His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser435 440 445Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly450 455 460Gly Ser Thr Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met465 470 475 480Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys485 490 495Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met500 505 510Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys515 520 525Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val530 535 540Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala545 550 555 560Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr565 570 575Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp AsnIle Ile580 585 590Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp595 600<210> 31<211> 628<212> PRT<213> Artificial<220><223> Аминокислотная последовательность тяжелой цепи A120<400> 31Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr20 25 30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val35 40 45Ser Ala Ile Thr Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val50 55 60Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys85 90 95Ala Lys Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Tyr Trp Gly100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser115 120 125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala165 170 175Val Leu Gln SerSer Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys210 215 220Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly225 230 235 240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr290 295 300Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val340 345 350Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser355 360 365Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu370 375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn TyrLys Thr Thr Pro Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met420 425 430His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser435 440 445Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly450 455 460Gly Ser Thr Ile Pro Pro His Val Gln Lys Ser Asp Val Glu Met Glu465 470 475 480Ala Gln Lys Asp Glu Ile Ile Cys Pro Ser Cys Asn Arg Thr Ala His485 490 495Pro Leu Arg His Ile Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly500 505 510Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser515 520 525Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser530 535 540Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn545 550 555 560Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro565 570 575Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met580 585 590Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser595 600605Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr610 615 620Ser Asn Pro Asp625<210> 32<211> 580<212> PRT<213> Artificial<220><223> Аминокислотная последовательность A121<400> 32Leu Val Pro His Leu Gly Asp Arg Glu Lys Arg Asp Ser Val Cys Pro1 5 10 15Gln Gly Lys Tyr Ile His Pro Gln Asn Asn Ser Ile Cys Cys Thr Lys20 25 30Cys His Lys Gly Thr Tyr Leu Tyr Asn Asp Cys Pro Gly Pro Gly Gln35 40 45Asp Thr Asp Cys Arg Glu Cys Glu Ser Gly Ser Phe Thr Ala Ser Glu50 55 60Asn His Leu Arg His Cys Leu Ser Cys Ser Lys Cys Arg Lys Glu Met65 70 75 80Gly Gln Val Glu Ile Ser Ser Cys Thr Val Asp Arg Asp Thr Val Cys85 90 95Gly Cys Arg Lys Asn Gln Tyr Arg His Tyr Trp Ser Glu Asn Leu Phe100 105 110Gln Cys Phe Asn Cys Ser Leu Cys Leu Asn Gly Thr Val His Leu Ser115 120 125Cys Gln Glu Lys Gln Asn Thr Val Cys Thr Cys His Ala Phe Phe Leu130 135 140Arg Glu Asn Glu Cys Val Ser Cys Ser Asn Cys Lys Lys Ser Leu Glu145 150 155 160Cys Thr Lys Leu Cys Leu Pro Gln Ile Glu Asn Val Lys Gly Thr Glu165 170175Asp Ser Gly Thr Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly180 185 190Gly Gly Gly Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro195 200 205Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe210 215 220Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val225 230 235 240Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe245 250 255Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro260 265 270Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr275 280 285Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val290 295 300Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala305 310 315 320Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg325 330 335Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly340 345 350Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro355 360 365Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser370 375 380Phe Phe Leu Tyr Ser Lys LeuThr Val Asp Lys Ser Arg Trp Gln Gln385 390 395 400Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His405 410 415Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly420 425 430Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ile Pro Pro His435 440 445Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly450 455 460Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser465 470 475 480Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser485 490 495Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn500 505 510Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro515 520 525Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met530 535 540Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser545 550 555 560Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr565 570 575Ser Asn Pro Asp580<210> 33<211> 605<212> PRT<213> Artificial<220><223> Аминокислотная последовательность A122<400> 33Leu Val ProHis Leu Gly Asp Arg Glu Lys Arg Asp Ser Val Cys Pro1 5 10 15Gln Gly Lys Tyr Ile His Pro Gln Asn Asn Ser Ile Cys Cys Thr Lys20 25 30Cys His Lys Gly Thr Tyr Leu Tyr Asn Asp Cys Pro Gly Pro Gly Gln35 40 45Asp Thr Asp Cys Arg Glu Cys Glu Ser Gly Ser Phe Thr Ala Ser Glu50 55 60Asn His Leu Arg His Cys Leu Ser Cys Ser Lys Cys Arg Lys Glu Met65 70 75 80Gly Gln Val Glu Ile Ser Ser Cys Thr Val Asp Arg Asp Thr Val Cys85 90 95Gly Cys Arg Lys Asn Gln Tyr Arg His Tyr Trp Ser Glu Asn Leu Phe100 105 110Gln Cys Phe Asn Cys Ser Leu Cys Leu Asn Gly Thr Val His Leu Ser115 120 125Cys Gln Glu Lys Gln Asn Thr Val Cys Thr Cys His Ala Phe Phe Leu130 135 140Arg Glu Asn Glu Cys Val Ser Cys Ser Asn Cys Lys Lys Ser Leu Glu145 150 155 160Cys Thr Lys Leu Cys Leu Pro Gln Ile Glu Asn Val Lys Gly Thr Glu165 170 175Asp Ser Gly Thr Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly180 185 190Gly Gly Gly Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro195 200 205Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe LeuPhe210 215 220Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val225 230 235 240Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe245 250 255Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro260 265 270Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr275 280 285Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val290 295 300Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala305 310 315 320Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg325 330 335Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly340 345 350Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro355 360 365Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser370 375 380Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln385 390 395 400Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His405 410 415Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly420 425 430Ser Gly GlyGly Gly Ser Gly Gly Gly Gly Ser Thr Ile Pro Pro His435 440 445Val Gln Lys Ser Asp Val Glu Met Glu Ala Gln Lys Asp Glu Ile Ile450 455 460Cys Pro Ser Cys Asn Arg Thr Ala His Pro Leu Arg His Ile Asn Asn465 470 475 480Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu485 490 495Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser500 505 510Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu515 520 525Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu530 535 540Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu545 550 555 560Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly565 570 575Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn580 585 590Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp595 600 605<210> 34<211> 634<212> PRT<213> Artificial<220><223> Аминокислотная последовательность A123<400> 34Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser1 5 10 15Thr Cys Arg Leu Arg Glu Tyr TyrAsp Gln Thr Ala Gln Met Cys Cys20 25 30Ser Lys Cys Ser Pro Gly Gln His Ala Lys Val Phe Cys Thr Lys Thr35 40 45Ser Asp Thr Val Cys Asp Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu50 55 60Trp Asn Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser65 70 75 80Asp Gln Val Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys85 90 95Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys100 105 110Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala115 120 125Arg Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro Cys Ala Pro130 135 140Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His145 150 155 160Gln Ile Cys Asn Val Val Ala Ile Pro Gly Asn Ala Ser Met Asp Ala165 170 175Val Cys Thr Ser Thr Ser Pro Thr Arg Ser Met Ala Pro Gly Ala Val180 185 190His Leu Pro Gln Pro Val Ser Thr Arg Ser Gln His Thr Gln Pro Thr195 200 205Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser Phe Leu Leu Pro Met Gly210 215 220Pro Ser Pro Pro Ala Glu Gly Ser Thr Gly Asp Gly Gly Gly Gly Ser225 230 235240Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Pro Lys Ser Cys Asp245 250 255Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly260 265 270Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile275 280 285Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu290 295 300Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His305 310 315 320Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg325 330 335Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys340 345 350Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu355 360 365Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr370 375 380Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu385 390 395 400Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp405 410 415Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val420 425 430Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp435 440 445Lys Ser Arg Trp Gln Gln GlyAsn Val Phe Ser Cys Ser Val Met His450 455 460Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro465 470 475 480Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly485 490 495Ser Thr Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile500 505 510Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe515 520 525Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser530 535 540Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val545 550 555 560Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys565 570 575His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala580 585 590Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe595 600 605Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe610 615 620Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp625 630<210> 35<211> 659<212> PRT<213> Artificial<220><223> Аминокислотная последовательность A124<400> 35Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro Glu Pro GlySer1 5 10 15Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met Cys Cys20 25 30Ser Lys Cys Ser Pro Gly Gln His Ala Lys Val Phe Cys Thr Lys Thr35 40 45Ser Asp Thr Val Cys Asp Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu50 55 60Trp Asn Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser65 70 75 80Asp Gln Val Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys85 90 95Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys100 105 110Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala115 120 125Arg Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro Cys Ala Pro130 135 140Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His145 150 155 160Gln Ile Cys Asn Val Val Ala Ile Pro Gly Asn Ala Ser Met Asp Ala165 170 175Val Cys Thr Ser Thr Ser Pro Thr Arg Ser Met Ala Pro Gly Ala Val180 185 190His Leu Pro Gln Pro Val Ser Thr Arg Ser Gln His Thr Gln Pro Thr195 200 205Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser Phe Leu Leu Pro Met Gly210 215 220Pro Ser Pro Pro Ala Glu Gly SerThr Gly Asp Gly Gly Gly Gly Ser225 230 235 240Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Pro Lys Ser Cys Asp245 250 255Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly260 265 270Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile275 280 285Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu290 295 300Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His305 310 315 320Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg325 330 335Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys340 345 350Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu355 360 365Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr370 375 380Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu385 390 395 400Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp405 410 415Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val420 425 430Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu ThrVal Asp435 440 445Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His450 455 460Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro465 470 475 480Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly485 490 495Ser Thr Ile Pro Pro His Val Gln Lys Ser Asp Val Glu Met Glu Ala500 505 510Gln Lys Asp Glu Ile Ile Cys Pro Ser Cys Asn Arg Thr Ala His Pro515 520 525Leu Arg His Ile Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala530 535 540Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr545 550 555 560Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile565 570 575Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp580 585 590Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr595 600 605His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys610 615 620Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser625 630 635 640Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser645 650 655Asn ProAsp<210> 36<211> 633<212> PRT<213> Artificial<220><223> Аминокислотная последовательность A125<400> 36Leu Val Pro His Leu Gly Asp Arg Glu Lys Arg Asp Ser Val Cys Pro1 5 10 15Gln Gly Lys Tyr Ile His Pro Gln Asn Asn Ser Ile Cys Cys Thr Lys20 25 30Cys His Lys Gly Thr Tyr Leu Tyr Asn Asp Cys Pro Gly Pro Gly Gln35 40 45Asp Thr Asp Cys Arg Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu Trp50 55 60Asn Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser Asp65 70 75 80Gln Val Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys Thr85 90 95Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys Arg100 105 110Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala Arg115 120 125Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro Cys Ala Pro Gly130 135 140Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His Gln145 150 155 160Ile Cys Asn Val Val Ala Ile Pro Gly Asn Ala Ser Met Asp Ala Val165 170 175Cys Thr Ser Thr Ser Pro Thr Arg Ser Met Ala Pro Gly Ala Val His180 185 190Leu Pro Gln Pro ValSer Thr Arg Ser Gln His Thr Gln Pro Thr Pro195 200 205Glu Pro Ser Thr Ala Pro Ser Thr Ser Phe Leu Leu Pro Met Gly Pro210 215 220Ser Pro Pro Ala Glu Gly Ser Thr Gly Asp Gly Gly Gly Gly Ser Gly225 230 235 240Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Pro Lys Ser Cys Asp Lys245 250 255Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro260 265 270Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser275 280 285Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp290 295 300Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn305 310 315 320Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val325 330 335Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu340 345 350Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys355 360 365Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr370 375 380Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr385 390 395 400Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp IleAla Val Glu Trp Glu405 410 415Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu420 425 430Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys435 440 445Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu450 455 460Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly465 470 475 480Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser485 490 495Thr Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val500 505 510Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys515 520 525Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn530 535 540Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala545 550 555 560Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His565 570 575Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser580 585 590Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe595 600 605Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser610 615620Glu Glu Tyr Asn Thr Ser Asn Pro Asp625 630<210> 37<211> 658<212> PRT<213> Artificial<220><223> Аминокислотная последовательность A126<400> 37Leu Val Pro His Leu Gly Asp Arg Glu Lys Arg Asp Ser Val Cys Pro1 5 10 15Gln Gly Lys Tyr Ile His Pro Gln Asn Asn Ser Ile Cys Cys Thr Lys20 25 30Cys His Lys Gly Thr Tyr Leu Tyr Asn Asp Cys Pro Gly Pro Gly Gln35 40 45Asp Thr Asp Cys Arg Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu Trp50 55 60Asn Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser Asp65 70 75 80Gln Val Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys Thr85 90 95Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys Arg100 105 110Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala Arg115 120 125Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro Cys Ala Pro Gly130 135 140Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His Gln145 150 155 160Ile Cys Asn Val Val Ala Ile Pro Gly Asn Ala Ser Met Asp Ala Val165 170 175Cys Thr Ser Thr Ser Pro Thr Arg Ser Met Ala Pro GlyAla Val His180 185 190Leu Pro Gln Pro Val Ser Thr Arg Ser Gln His Thr Gln Pro Thr Pro195 200 205Glu Pro Ser Thr Ala Pro Ser Thr Ser Phe Leu Leu Pro Met Gly Pro210 215 220Ser Pro Pro Ala Glu Gly Ser Thr Gly Asp Gly Gly Gly Gly Ser Gly225 230 235 240Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Pro Lys Ser Cys Asp Lys245 250 255Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro260 265 270Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser275 280 285Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp290 295 300Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn305 310 315 320Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val325 330 335Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu340 345 350Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys355 360 365Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr370 375 380Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr385 390 395 400CysLeu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu405 410 415Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu420 425 430Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys435 440 445Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu450 455 460Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly465 470 475 480Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser485 490 495Thr Ile Pro Pro His Val Gln Lys Ser Asp Val Glu Met Glu Ala Gln500 505 510Lys Asp Glu Ile Ile Cys Pro Ser Cys Asn Arg Thr Ala His Pro Leu515 520 525Arg His Ile Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val530 535 540Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys545 550 555 560Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys565 570 575Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu580 585 590Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His595 600 605Asp Phe Ile Leu Glu Asp Ala AlaSer Pro Lys Cys Ile Met Lys Glu610 615 620Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp625 630 635 640Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn645 650 655Pro Asp<210> 38<211> 630<212> PRT<213> Artificial<220><223> Аминокислотная последовательность A127<400> 38Leu Val Pro His Leu Gly Asp Arg Glu Lys Arg Asp Ser Val Cys Pro1 5 10 15Gln Gly Lys Tyr Ile His Pro Gln Asn Asn Ser Ile Cys Cys Thr Lys20 25 30Cys His Lys Gly Thr Tyr Leu Tyr Asn Asp Cys Pro Gly Pro Gly Gln35 40 45Asp Thr Asp Cys Arg Glu Cys Glu Ser Gly Ser Phe Thr Ala Ser Glu50 55 60Asn His Leu Arg His Cys Leu Ser Cys Ser Lys Cys Arg Lys Glu Met65 70 75 80Gly Gln Val Glu Ile Ser Ser Cys Thr Val Asp Arg Asp Thr Val Cys85 90 95Gly Cys Arg Lys Asn Gln Tyr Arg His Tyr Trp Ser Glu Asn Leu Phe100 105 110Gln Cys Phe Asn Cys Ser Leu Cys Leu Asn Gly Thr Val His Leu Ser115 120 125Cys Gln Glu Lys Gln Asn Thr Val Pro Cys Ala Pro Gly Thr Phe Ser130 135 140Asn Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro HisGln Ile Cys Asn145 150 155 160Val Val Ala Ile Pro Gly Asn Ala Ser Met Asp Ala Val Cys Thr Ser165 170 175Thr Ser Pro Thr Arg Ser Met Ala Pro Gly Ala Val His Leu Pro Gln180 185 190Pro Val Ser Thr Arg Ser Gln His Thr Gln Pro Thr Pro Glu Pro Ser195 200 205Thr Ala Pro Ser Thr Ser Phe Leu Leu Pro Met Gly Pro Ser Pro Pro210 215 220Ala Glu Gly Ser Thr Gly Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly225 230 235 240Ser Gly Gly Gly Gly Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr245 250 255Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe260 265 270Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro275 280 285Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val290 295 300Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr305 310 315 320Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val325 330 335Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys340 345 350Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser355 360365Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro370 375 380Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val385 390 395 400Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly405 410 415Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp420 425 430Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp435 440 445Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His450 455 460Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly465 470 475 480Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ile Pro485 490 495Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr Asp Asn500 505 510Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg515 520 525Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile530 535 540Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg545 550 555 560Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys565 570 575Leu Pro Tyr His Asp PheIle Leu Glu Asp Ala Ala Ser Pro Lys Cys580 585 590Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser595 600 605Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr610 615 620Asn Thr Ser Asn Pro Asp625 630<210> 39<211> 655<212> PRT<213> Artificial<220><223> Аминокислотная последовательность A128<400> 39Leu Val Pro His Leu Gly Asp Arg Glu Lys Arg Asp Ser Val Cys Pro1 5 10 15Gln Gly Lys Tyr Ile His Pro Gln Asn Asn Ser Ile Cys Cys Thr Lys20 25 30Cys His Lys Gly Thr Tyr Leu Tyr Asn Asp Cys Pro Gly Pro Gly Gln35 40 45Asp Thr Asp Cys Arg Glu Cys Glu Ser Gly Ser Phe Thr Ala Ser Glu50 55 60Asn His Leu Arg His Cys Leu Ser Cys Ser Lys Cys Arg Lys Glu Met65 70 75 80Gly Gln Val Glu Ile Ser Ser Cys Thr Val Asp Arg Asp Thr Val Cys85 90 95Gly Cys Arg Lys Asn Gln Tyr Arg His Tyr Trp Ser Glu Asn Leu Phe100 105 110Gln Cys Phe Asn Cys Ser Leu Cys Leu Asn Gly Thr Val His Leu Ser115 120 125Cys Gln Glu Lys Gln Asn Thr Val Pro Cys Ala Pro Gly Thr Phe Ser130 135 140Asn Thr Thr Ser SerThr Asp Ile Cys Arg Pro His Gln Ile Cys Asn145 150 155 160Val Val Ala Ile Pro Gly Asn Ala Ser Met Asp Ala Val Cys Thr Ser165 170 175Thr Ser Pro Thr Arg Ser Met Ala Pro Gly Ala Val His Leu Pro Gln180 185 190Pro Val Ser Thr Arg Ser Gln His Thr Gln Pro Thr Pro Glu Pro Ser195 200 205Thr Ala Pro Ser Thr Ser Phe Leu Leu Pro Met Gly Pro Ser Pro Pro210 215 220Ala Glu Gly Ser Thr Gly Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly225 230 235 240Ser Gly Gly Gly Gly Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr245 250 255Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe260 265 270Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro275 280 285Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val290 295 300Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr305 310 315 320Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val325 330 335Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys340 345 350Lys Val Ser Asn Lys Ala Leu Pro Ala Pro IleGlu Lys Thr Ile Ser355 360 365Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro370 375 380Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val385 390 395 400Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly405 410 415Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp420 425 430Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp435 440 445Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His450 455 460Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly465 470 475 480Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Ile Pro485 490 495Pro His Val Gln Lys Ser Asp Val Glu Met Glu Ala Gln Lys Asp Glu500 505 510Ile Ile Cys Pro Ser Cys Asn Arg Thr Ala His Pro Leu Arg His Ile515 520 525Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro530 535 540Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln545 550 555 560Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro565 570575Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr580 585 590Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile595 600 605Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys610 615 620Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn625 630 635 640Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp645 650 655<210> 40<211> 536<212> PRT<213> Artificial<220><223> Аминокислотная последовательность A129<400> 40Leu Val Pro His Leu Gly Asp Arg Glu Lys Arg Asp Ser Val Cys Pro1 5 10 15Gln Gly Lys Tyr Ile His Pro Gln Asn Asn Ser Ile Cys Cys Thr Lys20 25 30Cys His Lys Gly Thr Tyr Leu Tyr Asn Asp Cys Pro Gly Pro Gly Gln35 40 45Asp Thr Asp Cys Arg Glu Cys Glu Ser Gly Ser Phe Thr Ala Ser Glu50 55 60Asn His Leu Arg His Cys Leu Ser Cys Ser Lys Cys Arg Lys Glu Met65 70 75 80Gly Gln Val Glu Ile Ser Ser Cys Thr Val Asp Arg Asp Thr Val Cys85 90 95Gly Cys Arg Lys Asn Gln Tyr Arg His Tyr Trp Ser Glu Asn Leu Phe100 105 110Gln Cys Phe Asn Cys SerLeu Cys Leu Asn Gly Thr Val His Leu Ser115 120 125Cys Gln Glu Lys Gln Asn Thr Val Cys Gly Gly Gly Gly Ser Gly Gly130 135 140Gly Gly Ser Gly Gly Gly Gly Ser Glu Pro Lys Ser Cys Asp Lys Thr145 150 155 160His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser165 170 175Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg180 185 190Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro195 200 205Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala210 215 220Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val225 230 235 240Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr245 250 255Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr260 265 270Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu275 280 285Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys290 295 300Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser305 310 315 320Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr ProPro Val Leu Asp325 330 335Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser340 345 350Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala355 360 365Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys370 375 380Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr385 390 395 400Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr405 410 415Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp420 425 430Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys435 440 445Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val450 455 460Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp465 470 475 480Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro485 490 495Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met500 505 510Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu515 520 525Glu Tyr Asn Thr Ser Asn Pro Asp530 535<210> 41<211> 561<212> PRT<213>Artificial<220><223> Аминокислотная последовательность A130<400> 41Leu Val Pro His Leu Gly Asp Arg Glu Lys Arg Asp Ser Val Cys Pro1 5 10 15Gln Gly Lys Tyr Ile His Pro Gln Asn Asn Ser Ile Cys Cys Thr Lys20 25 30Cys His Lys Gly Thr Tyr Leu Tyr Asn Asp Cys Pro Gly Pro Gly Gln35 40 45Asp Thr Asp Cys Arg Glu Cys Glu Ser Gly Ser Phe Thr Ala Ser Glu50 55 60Asn His Leu Arg His Cys Leu Ser Cys Ser Lys Cys Arg Lys Glu Met65 70 75 80Gly Gln Val Glu Ile Ser Ser Cys Thr Val Asp Arg Asp Thr Val Cys85 90 95Gly Cys Arg Lys Asn Gln Tyr Arg His Tyr Trp Ser Glu Asn Leu Phe100 105 110Gln Cys Phe Asn Cys Ser Leu Cys Leu Asn Gly Thr Val His Leu Ser115 120 125Cys Gln Glu Lys Gln Asn Thr Val Cys Gly Gly Gly Gly Ser Gly Gly130 135 140Gly Gly Ser Gly Gly Gly Gly Ser Glu Pro Lys Ser Cys Asp Lys Thr145 150 155 160His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser165 170 175Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg180 185 190Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His GluAsp Pro195 200 205Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala210 215 220Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val225 230 235 240Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr245 250 255Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr260 265 270Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu275 280 285Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys290 295 300Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser305 310 315 320Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp325 330 335Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser340 345 350Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala355 360 365Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys370 375 380Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr385 390 395 400Ile Pro Pro His Val Gln Lys Ser Asp Val Glu Met Glu Ala Gln Lys405 410 415Asp GluIle Ile Cys Pro Ser Cys Asn Arg Thr Ala His Pro Leu Arg420 425 430His Ile Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys435 440 445Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp450 455 460Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu465 470 475 480Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn485 490 495Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp500 505 510Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys515 520 525Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu530 535 540Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro545 550 555 560Asp<210> 42<211> 586<212> PRT<213> Artificial<220><223> Аминокислотная последовательность тяжелой цепи A131<400> 42Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Asn20 25 30Val Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met35 40 45GlyGly Val Ile Pro Ile Val Asp Ile Ala Asn Tyr Ala Gln Arg Phe50 55 60Lys Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Thr Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys85 90 95Ala Ser Thr Leu Gly Leu Val Leu Asp Ala Met Asp Tyr Trp Gly Gln100 105 110Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly115 120 125Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly130 135 140Gly Leu Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser145 150 155 160Gly Phe Thr Phe Asp Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro165 170 175Gly Lys Gly Leu Glu Trp Val Ser Ala Ile Thr Trp Asn Ser Gly His180 185 190Ile Asp Tyr Ala Asp Ser Val Glu Gly Arg Phe Thr Ile Ser Arg Asp195 200 205Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu210 215 220Asp Thr Ala Val Tyr Tyr Cys Ala Lys Val Ser Tyr Leu Ser Thr Ala225 230 235 240Ser Ser Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser245 250 255Ala Ser Thr Lys Gly Pro Ser Val Phe ProLeu Ala Pro Ser Ser Lys260 265 270Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr275 280 285Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser290 295 300Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser305 310 315 320Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr325 330 335Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys340 345 350Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys355 360 365Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro370 375 380Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys385 390 395 400Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp405 410 415Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu420 425 430Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu435 440 445His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn450 455 460Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly465 470475 480Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu485 490 495 495Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr500 505 510Pro Ser Asp Ile Ala Val Glu Tr Glu Gl Pro Glu Sern Glu485 490 490 495 Leu Thr Lys Asn515 520 525Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe530 535 540Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn545 550 555 Phe 56 Phe 56 Met Ala His Val His Ser Glu Ser Asn His Tyr Thr565 570 575Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys580 585 <210> 43 <211> 337 <212> PRT <213> Artificial <220> <223> The amino acid sequence of the light chain of A131 <400> 43Glu Thr Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Leu Gly Ser Ser20 25 30Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Leu Leu 45 Leu 45 Leu Leu Gly Ala Ser Ser Arg Ala Pro Gly Ile Pro Asp Arg Phe Ser50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val TyrTyr Cys Gln Gln Tyr Ala Asp Ser Pro85 90 95Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Gly Gly Gly Gly100 105 110Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr115 120 125Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile130 135 140Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Tyr Leu Ala Trp Tyr Gln145 150 155 160Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Thr165 170 175Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr180 185 190Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr195 200 205Tyr Tyr Cys Gln Arg Tyr Asn Arg Ala Pro Tyr Thr Phe Gly Gln Gly210 215 220Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile225 230 235 240Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val245 250 255Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys260 265 270Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu275 280 285Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu ThrLeu290 295 300Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr305 310 315 320His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu325 330 335Cys <210> 44 <211> 12 <212> PRT <213> Artificial <220> <223> Peptide linker sequence <400> 44Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser1 5 10 <210> 45 <211> 4 <212> PRT <213> Artificial <220> <223> Peptide linker sequence <400> 45Gly Gly Gly Ser1 <210> 46 <211> 13 <212> PRT <213> Artificial <220> <223> Peptide linker sequence <400> 46Gly Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly1 5 10 <210> 47 <211> 5 <212> PRT <213> Artificial <220> <223> Peptide linker sequence <400> 47Gly Ser Ser Gly Thr1 5 <210> 48 <211> 14 <212> PRT <213> Artificial <220> <223> Peptide linker sequence <400> 48Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser1 5 10 <210> 49 <211> 17 <212> PRT <213> Artificial <220> <223> Peptide linker sequence <400> 49Ala Glu Ala Ala Ala Lys Glu Ala Ala AlaLys Glu Ala Ala Ala Lys1 5 10 15Ala <210> 50 <211> 20 <212> PRT <213> Artificial <220> <223> Peptide linker sequence <400> 50Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly1 5 10 15Gly Gly Gly Ser20 <210> 51 <211> 8 <212> PRT <213> Artificial <220> <223> Peptide linker sequence <400> 51Gly Gly Gly Ser Gly Gly Gly Ser1 5 <210> 52 <211> 5 <212> PRT <213> Artificial <220> <223> Peptide linker sequence <400> 52Gly Ser Gly Ser Ser1 5 <210> 53 <211> 4 <212> PRT <213> Artificial <220> <223> Peptide linker sequence <400> 53Gly Gly Ser Ser1 <210> 54 <211> 5 <212> PRT <213> Artificial <220> <223> Peptide linker sequence <400> 54Gly Gly Gly Gly Ser1 5 <210> 55 <211> 4 <212> PRT <213> Artificial <220> <223> Peptide linker sequence <400> 55Gly Gly Ser Gly1 <210> 56 <211> 4 <212> PRT <213> Artificial <220> <223> Peptide linker sequence <400> 56Ser Gly Gly Gly1 <210> 57 <211> 4 <212> PRT <213> Artificial <220> <223> Subsequencepeptide linker <400> 57Gly Ser Gly Ser1 <210> 58 <211> 6 <212> PRT <213> Artificial <220> <223> Peptide linker sequence <400> 58Gly Ser Gly Ser Gly Ser1 5 <210> 59 <211> 8 <212> PRT <213> Artificial <220> <223> Peptide linker sequence <400> 59Gly Ser Gly Ser Gly Ser Gly Ser1 5 <210> 60 <211> 10 <212> PRT <213> Artificial <220> <223> Peptide linker sequence <400> 60Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser1 5 10 <210> 61 <211> 12 <212> PRT <213> Artificial <220> <223> Peptide linker sequence <400> 61Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser1 5 10 <210> 62 <211> 10 <212> PRT <213> Artificial <220> <223> Peptide linker sequence <400> 62Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser1 5 10 <210> 63 <211> 15 <212> PRT <213> Artificial <220> <223> Peptide linker sequence <400> 63Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser1 5 10 15<---

Claims

1. An isolated bifunctional antagonist molecule comprising a first antigen-binding molecule that specifically binds to a TNF-α (tumor necrosis factor-alpha) ligand and a second antigen-binding molecule that specifically binds to a TGF-β (transforming growth factor-beta) ligand, wherein the bifunctional antagonist molecule comprises any of the amino acid sequences of SEQ ID NOs: 30-31 and 42 in combination with any of the amino acid sequences of SEQ ID NOs: 11, 15, 19, 23, 27 and 43 and simultaneously and effectively neutralizes TNF-α-mediated signaling and TGF-β-mediated signaling.

2. An isolated bifunctional antagonist molecule according to claim 1, selected from the group consisting of: a bifunctional molecule comprising the amino acid sequence of a heavy chain as set forth in SEQ ID NO: 30 and the amino acid sequence of a light chain as set forth in SEQ ID NO: 11; a bifunctional molecule comprising the amino acid sequence of a heavy chain as set forth in SEQ ID NO: 31 and the amino acid sequence of a light chain as set forth in SEQ ID NO:

11.

3. A pharmaceutical composition for neutralizing TNF-α-mediated signaling and TGF-β-mediated signaling, containing a therapeutically effective amount of a bifunctional antagonist molecule according to any one of claims 1, 2 in a mixture with a pharmaceutically acceptable carrier.

4. A method for treating or preventing a disease state, the pathogenesis of which involves activation of both the TNF-α-mediated NF-κB (nuclear factor kappa B)-associated signaling pathway and the TGF-β-mediated Smad2 / 3-associated signaling pathway, selected from pulmonary arterial hypertension (PAH) and pulmonary fibrosis, comprising administering to said subject a therapeutically effective amount of the composition of claim 3.

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