Activin and tumor necrosis factor-alpha bifunctional antagonist and its use

Abstract

The present disclosure provides novel bifunctional antagonist polypeptides comprising at least one TNF-cs binding domain and at least one activin binding domain, which have a high ability to sequester TNF-α and activin or activin-related ligands in parallel. Pharmaceutical compositions of such bifunctional polypeptide antagonists and their use for treating various complex disease states whose pathogenesis involves the activation of both the TNF-α-mediated NF-KB signaling pathway and the activin-mediated Smad2 / 3 signaling pathway are also provided.

Description

[Technical Field]

[0001] Related patent applications This application claims the interests of U.S. Provisional Application No. 63 / 113,918, filed on 15 November 2020, and U.S. Provisional Application No. 63 / 104,765, filed on 23 October 2020, each of which is incorporated herein by reference in whole. [Background technology]

[0002] Background technology Tumor necrosis factor-α (TNF-α) mediates NF-κB signaling and plays a crucial role in various physiological and pathological processes, including cell proliferation, differentiation, apoptosis, and modulation of immune responses and induction of inflammation. TNF acts through two receptors, TNFR1 (TNF receptor-1) and TNFR2 (TNF receptor-2). TNF-α plays a vital role in inflammatory responses, programmed cell death, and tissue necrosis. Increased TNF-α signaling has been linked to numerous inflammatory diseases, including rheumatoid arthritis, ankylosing spondylitis, Crohn's disease, and psoriasis, and anti-TNF therapies, such as adalimumab, infliximab, and etanercept, have been shown to be highly effective in treating such inflammatory diseases. Elevated TNF-α levels and increased TNF-α signaling have also been linked to the pathogenesis and progression of many other disease conditions, including anemia, leukemia, multiple myeloma, fibrosis, hypertension, muscle wasting, osteopenia, neurodegeneration, sepsis, pain, chronic kidney disease, liver disease, and heart failure.

[0003] Activins, including activin A, activin B, and activin AB as a subset of the TGF-β superfamily, and activin-related proteins, including myostatin (GDF-8) and GDF-11, mediate Smad2 / 3 signaling through the binding and activation of their high-affinity receptors, ActRIIA and ActRIIB, on the cell surface. Activins and related proteins play essential roles in regulating a wide range of biological activities, including mesoderm induction, cell differentiation, myogenesis, bone remodeling, hematopoiesis, fibrosis, and reproductive physiological functions. The secreted glycoprotein, follistatin (FST), binds to activins and activin-related ligands, negatively regulating their signaling activity. Overexpression of activin and related ligands, as well as increased Smad2 / 3 signaling, has been linked to the pathogenesis and progression of many distressing conditions, such as cancer, anemia, bone metastases, bone fragility, fractures (factures), muscle wasting disorders, cachexia, pulmonary hypertension, fibrosis, pain, insulin resistance, chronic kidney disease, liver disease, myocardial infarction, and heart failure.

[0004] Growing evidence indicates that many complex disorders involve parallel activation of the TNF-α-mediated NF-κB signaling pathway and the activin-mediated Smad2 / 3 signaling pathway, whose activity promotes pathogenesis and progression. Examples of such complex disorders include certain hematological disorders, e.g., refractory anemia and myelodysplastic syndromes; cardiovascular diseases, e.g., pulmonary hypertension and congestive heart failure; bone disorders, e.g., bone metastases and fractures; organ failure, e.g., renal failure, hepatic failure or bone marrow failure; fibrous diseases, e.g., non-alcoholic steatohepatitis, cirrhosis and pulmonary fibrosis; and pain, e.g., nociceptive or neuropathic pain.

[0005] Current treatment options for these complex disorders are limited. Due to the involvement of one or more disease signaling mechanisms in these disorders, currently available therapies designed to target a single disease mechanism typically exhibit poor efficacy and low response rates. Since both the TNF-α-NF-κB signaling pathway and the activin-Smad2 / 3 signaling pathway are essentially involved in disease pathogenesis and progression, developing novel bifunctional antagonists with the ability to inhibit both disease signaling pathways in parallel is clearly important. [Overview of the project]

[0006] Disclosure of the invention In one embodiment, the present invention provides a novel polypeptide-based bifunctional antagonist molecule specifically designed to simultaneously neutralize TNF-α signaling and activin signaling in a potent manner. In various embodiments, the bifunctional antagonist molecule is designed as depicted in Figure 1. In various embodiments, the bifunctional antagonist molecule is designed as depicted in Figure 2. In various embodiments, the bifunctional antagonist molecule is designed as depicted in Figure 3.

[0007] In various embodiments, a bifunctional antagonist molecule is a bifunctional molecule comprising a first antigen-binding molecule ("TNF-binding polypeptide") that specifically binds to a TNF-α ligand and a second antigen-binding molecule ("activin-binding polypeptide") that specifically binds to activin or an activin-related ligand. In various embodiments, the "TNF-binding polypeptide" is selected from the group consisting of anti-TNF antibodies, fragments of anti-TNF antibodies, wild-type TNFR1 and TNFR2 extracellular domains (ECDs), modified TNFR1 and TNFR2 extracellular domains, and phage display-derived polypeptides that target TNF-α, while the "activin-binding polypeptide" is selected from the group consisting of anti-activin antibodies (including anti-activin A antibodies and anti-activin B antibodies), fragments of anti-activin antibodies, and wild-type activin 2A receptor (ActRIIA) or activin 2B receptor (ActRIIB) extracellular domains. The selection includes, but is not limited to, activin (ECD), modified ActRIIA and ActRIIB extracellular domains, wild-type and modified native activin-binding proteins such as follistatin, follistatin-like proteins and propeptides, and phage display-derived polypeptides that target activin or activin-related ligands, and any polypeptides capable of binding to activin (i.e., activin A, activin B, or activin AB) and / or activin-related ligands (i.e., GDF8 or GDF11).

[0008] In various embodiments, the bifunctional molecule comprises an isolated antibody or its antigen-binding fragment that specifically binds to TNF-α, and an isolated antibody or its antigen-binding fragment that specifically binds to activin or an activin-related ligand. In various embodiments, the isolated antibody or its antigen-binding fragment is selected from the group consisting of monoclonal Ab (mAb), polyclonal Ab, Ab fragment (e.g., Fab, Fab', F(ab')2, Fv, Fc, etc.), chimeric Ab, mini-Ab or domain Ab (dAb), bispecific Ab, bispecific Ab, heteroconjugate Ab, single-chain Ab (SCA), single-chain variable region fragment (ScFv), humanized Ab, fully human Ab, and any other modified configuration of an immunoglobulin (Ig) molecule containing an antigen recognition site of the required specificity. In various embodiments, the bifunctional molecule comprises an isolated antibody or its antigen-binding fragment selected from the group consisting of fully human, humanized, and chimeric antibodies.

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

[0010] In various embodiments, the second antigen-binding molecule specifically binds to activin or an activin-related ligand containing the amino acid sequence described in SEQ ID NO: 6. In various embodiments, the second antigen-binding molecule specifically binds to activin or an activin-related ligand containing the amino acid sequence described in SEQ ID NO: 7. In various embodiments, the second antigen-binding molecule specifically binds to activin or an activin-related ligand containing the amino acid sequence described in SEQ ID NO: 8. In various embodiments, the second antigen-binding molecule specifically binds to activin or an activin-related ligand containing the amino acid sequence described in SEQ ID NO: 9. In various embodiments, the second antigen-binding molecule specifically binds to activin or an activin-related ligand containing the amino acid sequence described in SEQ ID NO: 10. In various embodiments, the second antigen-binding molecule specifically binds to activin or an activin-related ligand containing the amino acid sequence described in SEQ ID NO: 11. In various embodiments, the second antigen-binding molecule specifically binds to activin or an activin-related ligand containing the amino acid sequence described in SEQ ID NO: 12. In various embodiments, the second antigen-binding molecule specifically binds to an activin or activin-related ligand containing the amino acid sequence described in SEQ ID NO: 13. In various embodiments, the second antigen-binding molecule specifically binds to an activin or activin-related ligand containing the amino acid sequence described in SEQ ID NO: 14.

[0011] In various embodiments, the first antigen-binding molecule that specifically binds to the TNF-α ligand is an isolated antibody selected from the group consisting of: an antibody containing the heavy chain amino acid sequence described in SEQ ID NO: 15; an antibody containing the light chain amino acid sequence described in SEQ ID NO: 17; an antibody containing the heavy chain amino acid sequence described in SEQ ID NO: 15 and the light chain amino acid sequence described in SEQ ID NO: 17; an antibody containing the heavy chain variable region amino acid sequence described in SEQ ID NO: 16; an antibody containing the light chain variable region amino acid sequence described in SEQ ID NO: 18; and an antibody containing the heavy chain variable region amino acid sequence described in SEQ ID NO: 16 and the light chain variable region amino acid sequence described in SEQ ID NO: 18.

[0012] In various embodiments, the first antigen-binding molecule that specifically binds to the TNF-α ligand is an isolated antibody selected from the group consisting of: an antibody containing the heavy chain amino acid sequence described in SEQ ID NO: 19; an antibody containing the light chain amino acid sequence described in SEQ ID NO: 21; an antibody containing the heavy chain amino acid sequence described in SEQ ID NO: 19 and the light chain amino acid sequence described in SEQ ID NO: 21; an antibody containing the heavy chain variable region amino acid sequence described in SEQ ID NO: 20; an antibody containing the light chain variable region amino acid sequence described in SEQ ID NO: 22; and an antibody containing the heavy chain variable region amino acid sequence described in SEQ ID NO: 20 and the light chain variable region amino acid sequence described in SEQ ID NO: 22.

[0013] In various embodiments, the first antigen-binding molecule that specifically binds to the TNF-α ligand is an isolated antibody selected from the group consisting of: an antibody containing the heavy chain amino acid sequence described in SEQ ID NO: 23; an antibody containing the light chain amino acid sequence described in SEQ ID NO: 25; an antibody containing the heavy chain amino acid sequence described in SEQ ID NO: 23 and the light chain amino acid sequence described in SEQ ID NO: 25; an antibody containing the heavy chain variable region amino acid sequence described in SEQ ID NO: 24; an antibody containing the light chain variable region amino acid sequence described in SEQ ID NO: 26; and an antibody containing the heavy chain variable region amino acid sequence described in SEQ ID NO: 24 and the light chain variable region amino acid sequence described in SEQ ID NO: 26.

[0014] In various embodiments, the first antigen-binding molecule that specifically binds to the TNF-α ligand is an isolated antibody selected from the group consisting of: an antibody containing the heavy chain amino acid sequence described in SEQ ID NO: 27; an antibody containing the light chain amino acid sequence described in SEQ ID NO: 29; an antibody containing the heavy chain amino acid sequence described in SEQ ID NO: 27 and the light chain amino acid sequence described in SEQ ID NO: 29; an antibody containing the heavy chain variable region amino acid sequence described in SEQ ID NO: 28; an antibody containing the light chain variable region amino acid sequence described in SEQ ID NO: 30; and an antibody containing the heavy chain variable region amino acid sequence described in SEQ ID NO: 28 and the light chain variable region amino acid sequence described in SEQ ID NO: 30.

[0015] In various embodiments, the second antigen-binding molecule that specifically binds to activin or an activin-related 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: 31; an antibody comprising the light-chain amino acid sequence set forth in SEQ ID NO: 33; an antibody comprising the heavy-chain amino acid sequence set forth in SEQ ID NO: 31 and the light-chain amino acid sequence set forth in SEQ ID NO: 33; an antibody comprising the heavy-chain variable region amino acid sequence set forth in SEQ ID NO: 32; an antibody comprising the light-chain variable region amino acid sequence set forth in SEQ ID NO: 34; and an antibody comprising the heavy-chain variable region amino acid sequence set forth in SEQ ID NO: 32 and the light-chain variable region amino acid sequence set forth in SEQ ID NO: 34.

[0016] In various embodiments, the second antigen-binding molecule that specifically binds to activin or an activin-related 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: 35; an antibody comprising the light-chain amino acid sequence set forth in SEQ ID NO: 37; an antibody comprising the heavy-chain amino acid sequence set forth in SEQ ID NO: 35 and the light-chain amino acid sequence set forth in SEQ ID NO: 37; an antibody comprising the heavy-chain variable region amino acid sequence set forth in SEQ ID NO: 36; an antibody comprising the light-chain variable region amino acid sequence set forth in SEQ ID NO: 38; and an antibody comprising the heavy-chain variable region amino acid sequence set forth in SEQ ID NO: 36 and the light-chain variable region amino acid sequence set forth in SEQ ID NO: 38.

[0017] In various 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 activin or an activin-related ligand, wherein the TNF-α ligand-binding molecule is selected from the group of polypeptides comprising the amino acid sequences set forth in SEQ ID NOs: 1-5 and 15-30, and wherein the activin or activin-related ligand-binding molecule is selected from the group of polypeptides comprising the amino acid sequences set forth in SEQ ID NOs: 6-14 and 31-38.

[0018] In various 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 activin or an activin-related ligand, wherein the bifunctional molecule comprises a heavy-chain amino acid sequence described in SEQ ID NO: 39 and a light-chain amino acid sequence described in SEQ ID NO: 17; a heavy-chain amino acid sequence described in SEQ ID NO: 40 and a light-chain amino acid sequence described in SEQ ID NO: 17; a heavy-chain amino acid sequence described in SEQ ID NO: 41 and a light-chain amino acid sequence described in SEQ ID NO: 42 and a light-chain amino acid sequence described in SEQ ID NO: 17; a heavy-chain amino acid sequence described in SEQ ID NO: 43 and a light-chain amino acid sequence described in SEQ ID NO: 17; a heavy-chain amino acid sequence described in SEQ ID NO: 44 and a light-chain amino acid sequence described in SEQ ID NO: 17; a heavy-chain amino acid sequence described in SEQ ID NO: 45 and a light-chain amino acid sequence described in SEQ ID NO: 17 Functional molecules; bifunctional molecules containing the heavy chain amino acid sequence described in SEQ ID NO: 46 and the light chain amino acid sequence described in SEQ ID NO: 17; bifunctional molecules containing the heavy chain amino acid sequence described in SEQ ID NO: 47 and the light chain amino acid sequence described in SEQ ID NO: 17; bifunctional molecules containing the heavy chain amino acid sequence described in SEQ ID NO: 31 and the light chain amino acid sequence described in SEQ ID NO: 33; bifunctional molecules containing the heavy chain amino acid sequence described in SEQ ID NO: 35 and the light chain amino acid sequence described in SEQ ID NO: 37; bifunctional molecules containing the heavy chain amino acid sequence described in SEQ ID NO: 57 and the light chain amino acid sequence described in SEQ ID NO: 33; bifunctional molecules containing the heavy chain amino acid sequence described in SEQ ID NO: 58 and the light chain amino acid sequence described in SEQ ID NO: 37; bifunctional molecules containing the heavy chain amino acid sequence described in SEQ ID NO: 68 and the light chain amino acid sequence described in SEQ ID NO: 33; bifunctional molecules containing the heavy chain amino acid sequence described in SEQ ID NO: 69 and the light chain amino acid sequence described in SEQ ID NO: 37; bifunctional molecules containing the heavy chain amino acid sequence described in SEQ ID NO: 70 and the light chain amino acid sequence described in SEQ ID NO: 71;Furthermore, it is selected from the group consisting of bifunctional molecules containing the heavy chain amino acid sequence described in SEQ ID NO: 72 and the light chain amino acid sequence described in SEQ ID NO: 73.

[0019] In various 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 activin or an activin-related ligand, wherein the bifunctional molecule comprises the amino acid sequence described in SEQ ID NO: 48; the amino acid sequence described in SEQ ID NO: 49; the amino acid sequence described in SEQ ID NO: 50; the amino acid sequence described in SEQ ID NO: 51; the amino acid sequence described in SEQ ID NO: 52; the amino acid sequence described in SEQ ID NO: 53; the amino acid sequence described in SEQ ID NO: 54; and SEQ ID NO: 5 The group is selected from the following: a bifunctional molecule containing the amino acid sequence described in 5; a bifunctional molecule containing the amino acid sequence described in SEQ ID NO: 56; a bifunctional molecule containing the amino acid sequence described in SEQ ID NO: 59; a bifunctional molecule containing the amino acid sequence described in SEQ ID NO: 60; a bifunctional molecule containing the amino acid sequence described in SEQ ID NO: 61; a bifunctional molecule containing the amino acid sequence described in SEQ ID NO: 62; a bifunctional molecule containing the amino acid sequence described in SEQ ID NO: 63; a bifunctional molecule containing the amino acid sequence described in SEQ ID NO: 64; a bifunctional molecule containing the amino acid sequence described in SEQ ID NO: 65; a bifunctional molecule containing the amino acid sequence described in SEQ ID NO: 66; and a bifunctional molecule containing the amino acid sequence described in SEQ ID NO: 67.

[0020] In another aspect, the present disclosure provides an isolated nucleic acid molecule comprising a polynucleotide encoding a bifunctional antagonist molecule of the present disclosure. In various embodiments, the isolated nucleic acid molecule comprises the polynucleotides described herein and further comprises a polynucleotide encoding at least one heterologous protein described herein. In various embodiments, the nucleic acid molecule further comprises a polynucleotide encoding a linker or hinge linker described herein.

[0021] In another aspect, the present disclosure provides a vector comprising the nucleic acids described herein. In various embodiments, the vector is an expression vector. In another aspect, the present disclosure provides an isolated cell comprising the nucleic acids of the present disclosure. In various embodiments, the cell is a host cell comprising the expression vector of the present disclosure. In another aspect, provided is a method of manufacturing a bifunctional antagonist molecule, by culturing a host cell under conditions that promote the expression of the protein or polypeptide.

[0022] In another aspect, provided is a method of manufacturing a bifunctional antagonist molecule comprising a first antigen-binding molecule that specifically binds to TNF-α described herein and a second antigen-binding molecule that specifically binds to activin, the method comprising: a) transforming a host cell with a vector comprising a polynucleotide encoding the bifunctional antagonist molecule; b) culturing the host cell under conditions suitable for the expression of the bifunctional antagonist molecule; and c) recovering the bifunctional antagonist molecule from the culture. The present invention also encompasses bifunctional antagonist molecules produced by the method of the present invention.

[0023] In another aspect, the present disclosure provides a pharmaceutical composition comprising an isolated bifunctional antagonist molecule mixed with a pharmaceutically acceptable carrier.

[0024] In another embodiment, the disclosure provides methods for treating or preventing various complex disease conditions, such as inflammatory anemia, pulmonary hypertension, muscular dystrophy, and organofibrosis, whose pathogenesis involves activation of both the TNF-α-mediated NF-κB signaling pathway and the activin-mediated Smad2 / 3 signaling pathway.

[0025] In various embodiments, the novel bifunctional antagonist molecule of the present invention is used to treat the following conditions: blood disorders: ineffective erythropoiesis, anemia, pancytopenia, myelodysplastic syndrome; fibrotic diseases: NASH, hepatic fibrosis, pulmonary fibrosis, renal fibrosis, polycystic kidney disease, cardiac fibrosis, muscular fibrosis, myelofibrosis, dermatofibrosis, tendon fibrosis, hand fibrosis, and ocular fibrosis; cancers: multiple myeloma, acute myeloid leukemia, melanoma, sarcoma, lung cancer, esophageal cancer, pancreatic cancer, colorectal cancer, liver cancer, head and neck cancer, endometrial cancer, ovarian cancer, and breast cancer; and checkpoint inhibitors, such as anti-PD1, anti-PDL1, and anti-CTL4 antibodies. It may have broad applications for the treatment of various disorders, including but not limited to: combined cancer treatment; neuromuscular diseases: muscular dystrophy, spinal muscular atrophy, spinal cord injury, stroke; pain: nociceptive pain, neuropathic pain; wasting disorders: sarcopenia, cancer cachexia, anorexia nervosa; bone disorders: bone metastases, bone fragility, fractures, osteopenia, osteoporosis; cardiovascular diseases: pulmonary hypertension, myocardial infarction, heart failure; metabolic disorders: insulin resistance, diabetic nephropathy, chronic kidney disease; inflammatory diseases: rheumatoid arthritis, inflammatory bowel disease; infections: SARS-CoV, cytokine storm syndrome, sepsis; and trauma: burns.

[0026] In another embodiment, the present disclosure provides the use of a bifunctional antagonist molecule for manufacturing a pharmacopoeia for the treatment of any disorder or condition described herein. [Brief explanation of the drawing]

[0027] [Figure 1]Figure 1 illustrates a representative bifunctional antagonist molecule of the present invention. The “TNF-binding polypeptide” shown in this diagram refers to any polypeptide with the ability to bind to TNF-α, including, but not limited to, anti-TNF antibodies, fragments of anti-TNF antibodies, wild-type TNFR1 and TNFR2 extracellular domains (ECDs), modified TNFR1 and TNFR2 extracellular domains, and phage display-derived polypeptides that target TNF-α. The “activin-binding polypeptide” shown in this diagram refers to any polypeptide capable of binding to activin (i.e., activin A, activin B, or activin AB) and / or activin-related ligands (i.e., GDF8 or GDF11), including but not limited to anti-activin antibodies (including anti-activin A and anti-activin B antibodies), fragments of anti-activin antibodies, the extracellular domain (ECD) of wild-type activin 2A receptor (ActRIIA) or activin 2B receptor (ActRIIB), modified ActRIIA and ActRIIB extracellular domains, wild-type and modified native activin-binding proteins, such as follistatin, follistatin-like proteins and propeptides, and phage display-derived polypeptides that target activin or activin-related ligands. The “linker” shown in this diagram refers to various methods of fusing different polypeptide fusion partners to produce bispecific and polyspecific molecules, including but not limited to the use of any peptide linker or chemical linker. [Figure 2]Figure 2 depicts two representative bifunctional antagonist molecules of the present invention, wherein (A) the TNF-α-binding polypeptide is an anti-TNF-α antibody and the activin-binding polypeptide is an activin receptor ECD attached via a linker to the heavy chain CH3 of the anti-TNF-α antibody; or (B) the activin-binding polypeptide is an anti-activin antibody and the TNF-α-binding polypeptide is a TNF receptor ECD attached via a linker to the heavy chain CH3 of the anti-activin antibody. In alternative embodiments, the activin receptor ECD (or TNF receptor ECD) is attached to the anti-TNF-α antibody (or anti-activin antibody) via a linker in the heavy chain variable region (VH) of the antibody. In alternative embodiments, the activin receptor ECD (or TNF receptor ECD) is attached to the anti-TNF-α antibody (or anti-activin antibody) via a linker in the light chain variable region (VL) of the antibody. In an alternative embodiment, the activin receptor ECD (or TNF receptor ECD) is attached to the anti-TNF-α antibody (or anti-activin antibody) via a linker at an internal site rather than at the CH3, VL, or VH site of the antibody's heavy chain. [Figure 3] Figure 3 illustrates a representative bifunctional antagonist molecule of the present invention in the form of a bispecific antibody, comprising (A) variable regions (VH and VL) derived from an anti-activin A antibody and (B) variable regions (VH and VL) from an anti-TNF antibody. While the bispecific antibody illustrated in Figure 3 is shown in one specific configuration, it should be noted that bispecific antibodies comprising variable regions from both anti-activin A and anti-TNF antibodies can be constructed in a variety of configurations by those skilled in the art. [Figure 4] Figure 4 depicts line graphs showing that A109, A110, A305, and A711 potently neutralize TNF-α and activin A in cell-based assays. Cell-based TNF-α and activin neutralization IC50 values ​​were calculated and plotted using Prism software (GraphPad Software). [Figure 5]Figure 5 illustrates line graphs showing the neutralization of activin A-related ligands activin B, GDF-8, and GDF-11 by A109 and A110. Cell-based activin neutralization IC50 values ​​were calculated and plotted using Prism software (GraphPad Software). [Figure 6]Figure 6A depicts changes in cell proliferation and morphology of human primary pulmonary artery smooth muscle cells (PASMCs) under different treatment conditions, showing that the bifunctional antagonist A109 was highly effective in preventing TNFα and activin A-induced hyperplasia and hypertrophy of PASMCs. Figure 6B depicts changes in cell proliferation and morphology of human primary pulmonary artery smooth muscle cells (PASMCs) under different treatment conditions, showing that the bifunctional antagonist A109 was highly effective in preventing TNFα and activin A-induced hyperplasia and hypertrophy of PASMCs. Figure 6C depicts changes in cell proliferation and morphology of human primary pulmonary artery smooth muscle cells (PASMCs) under different treatment conditions, showing that the bifunctional antagonist A109 was highly effective in preventing TNFα and activin A-induced hyperplasia and hypertrophy of PASMCs. Figure 6D depicts changes in cell proliferation and morphology of human primary pulmonary artery smooth muscle cells (PASMCs) under different treatment conditions, showing that the bifunctional antagonist A109 was highly effective in preventing TNFα and activin A-induced hyperplasia and hypertrophy of PASMCs. Figure 6E depicts changes in cell proliferation and morphology of human primary pulmonary artery smooth muscle cells (PASMCs) under different treatment conditions, showing that the bifunctional antagonist A109 was highly effective in preventing TNFα and activin A-induced hyperplasia and hypertrophy of PASMCs. Figure 6F depicts changes in cell proliferation and morphology of human primary pulmonary artery smooth muscle cells (PASMCs) under different treatment conditions, showing that the bifunctional antagonist A109 was highly effective in preventing TNFα and activin A-induced hyperplasia and hypertrophy of PASMCs. Figure 6G depicts changes in cell proliferation and morphology of human primary pulmonary artery smooth muscle cells (PASMCs) under different treatment conditions, showing that the bifunctional antagonist A109 was highly effective in preventing TNFα and activin A-induced hyperplasia and hypertrophy of PASMCs. [Figure 7]Figure 7 depicts a bar graph showing that A109 was more effective than anti-TNF antibodies or ActRIIA-Fc in preventing both hyperplasia and hypertrophy of PASMCs in the context of elevated TNF-α and activin A. Morphometric analysis of PASMCs was performed using ImageJ software. [Figure 8] Figure 8 depicts histological images of H&E-stained lung sections and a bar graph of the Ashcraft score, showing that A109 was more effective than anti-TNF antibodies or ActRIIA-Fc in reducing lung tissue damage and fibrosis in bleomycin-induced pulmonary fibrosis mice. [Figure 9] Figure 9 depicts histological images of Masson's trichrome-stained lung sections and bar graphs representing quantitative analysis of collagen deposition area, showing that A109 was more effective than anti-TNF antibody or ActRIIA-Fc in attenuating pulmonary fibrosis in bleomycin-induced pulmonary fibrosis mice. [Figure 10] Figure 10 depicts a histological image of αSMA staining in lung sections, showing that A109 was more effective than anti-TNF antibody or ActRIIA-Fc in preventing the induction of αSMA in lung tissue of bleomycin-induced pulmonary fibrosis mice. Lung sections were immunostained using anti-αSMA antibody and HRP-labeled secondary antibody. [Figure 11] Figure 11 depicts a representative microCT image of trabecular bone volume, showing that A109 administration increased bone mass in normal mice. [Figure 12] Figure 12 depicts the results of microCT imaging regarding distal femoral parameters, showing that A109 administration led to a significant increase in bone volume and density in CD1 mice. [Modes for carrying out the invention]

[0028] Forms for implementing this disclosure definition The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. In various embodiments, “peptide,” “polypeptide,” and “protein” are chains of amino acids whose alpha carbons are linked through peptide bonds. The terminal amino acid at one end of the chain (amino-terminus) therefore has a free amino group, and the terminal amino acid at the other end of the chain (carboxy-terminus) has a free carboxyl group. Where used herein, the term “amino-terminus” (abbreviated as N-terminus) refers to the free α-amino group on the amino acid at the amino-terminus of a peptide or the α-amino group (imino group if participating in a peptide bond) of an amino acid at any other position within the peptide. Similarly, the term “carboxy-terminus” refers to the free carboxyl group at the carboxy-terminus of a peptide or the carboxyl group of an amino acid at any other position within the peptide. Peptides also include, but are not limited to, peptide mimes, such as amino acids linked by ethers as opposed to amide bonds, essentially any polyamino acid.

[0029] The polypeptides of this disclosure include polypeptides that have been modified in any way and for any reason to (1) reduce susceptibility to protein hydrolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinity, and (5) confer or alter other physicochemical or functional properties.

[0030] When used herein, amino acid "substitution" refers to the substitution of one amino acid at a specific position in the parent polypeptide sequence with a different amino acid within the polypeptide. Amino acid substitutions can be produced using genetic or chemical methods well known in the art. For example, one or more amino acid substitutions (e.g., conserved amino acid substitutions) may occur in naturally occurring sequences (e.g., in portions of the polypeptide outside domains that form intermolecular contacts). A "conserved amino acid substitution" refers to the substitution of an amino acid in a polypeptide with a functionally similar amino acid. Each of the following six groups contains amino acids that are conserved substitutions with respect to each other. 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)

[0031] A "non-conservative amino acid substitution" refers to the substitution of a member of one of these classes with a member from another class. In making such a change, the hydroxyl index of the amino acids may be considered according to various embodiments. Each amino acid is assigned a hydroxyl index based on its hydrophobic and electrostatic characteristics. These 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); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0032] The importance of hydroxyl amino acid indices in conferring interactive biological functions to proteins is well understood in the art (see, for example, Kyte et al., 1982, J.Mol.Biol.157:105-131). It is known that certain amino acids can be substituted with other amino acids having similar hydroxyl indices or scores, while still retaining similar biological activity. When making changes based on hydroxyl indices, various embodiments include substitutions of amino acids whose hydroxyl indices are within ±2; various embodiments include substitutions within ±1; and various embodiments include substitutions within ±0.5.

[0033] Substitutions of similar amino acids can be effectively carried out based on hydrophilicity, and this is also understood in the Art, particularly when the resulting biologically functional protein or peptide is intended for use in immunological embodiments, as disclosed herein. In various embodiments, the maximum local mean hydrophilicity of a protein, such that it is governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., the biological properties of the protein.

[0034] The following hydrophilic values ​​are assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0 ± 1); glutamic acid (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 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 based on similar hydrophilicity values, various embodiments include substitutions of amino acids whose hydrophilicity value is within ±2, within ±1, and within ±0.5.

[0035] Examples of amino acid substitutions are listed in Table 1. TIFF0007857306000001.tif183170

[0036] Those skilled in the art can determine preferred variants of the polypeptides described herein using well-known techniques. In various embodiments, those skilled in the art may identify preferred segments of the molecule that can be altered without disrupting activity by targeting regions considered insignificant for activity. In other embodiments, those skilled in the art can identify conserved molecular residues and segments among similar polypeptides. In further embodiments, segments that may be important for biological activity or structure may be subjected to conserved amino acid substitution without disrupting biological activity or adversely affecting the polypeptide structure.

[0037] Additionally, those skilled in the art may consider structure-function studies to identify residues in similar polypeptides that are important for activity or structure. Taking such comparisons into account, those skilled in the art can predict the importance of amino acid residues in a polypeptide corresponding to amino acid residues that are important for activity or structure in similar polypeptides. Those skilled in the art may select chemically similar amino acid substitutions for such predicted important amino acid residues.

[0038] Those skilled in the art can also analyze the three-dimensional structure of similar polypeptides and the amino acid sequences associated with that structure. Considering such information, those skilled in the art may predict the alignment of amino acid residues of the polypeptide with respect to its three-dimensional structure. In various embodiments, those skilled in the art may choose not to make radical changes to amino acid residues that are expected to be on the surface of the polypeptide, because such residues may be involved in important interactions with other molecules. Furthermore, those skilled in the art may generate 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 preferred variants. For example, if a change to a particular amino acid residue is found to result in disruption, undesirable reduction, or unsuitable activity, variants with such changes can be avoided. In other words, based on the information gathered from such routine experiments, those skilled in the art can easily determine which amino acids should be avoided, either alone or in combination with other mutations.

[0039] The terms “polypeptide fragment” and “cleaved polypeptide,” as used herein, refer to polypeptides having amino-terminal and / or carboxy-terminal deletions compared to the corresponding full-length protein. In various embodiments, the fragments may 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 length. In various embodiments, the fragment can also be, for example, up to 1000, up to 900, up to 800, up to 700, up to 600, up to 500, up to 450, up to 400, up to 350, up to 300, up to 250, up to 200, up to 150, up to 100, up to 50, up to 25, up to 10, or up to 5 amino acids in length. The fragment may further include one or more additional amino acids at either or both of its ends, for example, a sequence of amino acids from different naturally occurring proteins (e.g., Fc or leucine zipper domain) or an artificial amino acid sequence (e.g., an artificial linker sequence).

[0040] The terms “polypeptide variant,” “hybrid polypeptide,” and “polypeptide mutant,” as used herein, refer to a polypeptide comprising an amino acid sequence in which one or more amino acid residues are inserted, deleted, and / or substituted compared to another polypeptide sequence. In various embodiments, the number of amino acid residues inserted, deleted, or substituted 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 acid lengths. The hybrids of this disclosure include fusion proteins.

[0041] A polypeptide "derivative" is a polypeptide that has been chemically modified, for example, by conjugation to another chemical moiety, such as polyethylene glycol, albumin (e.g., human serum albumin), phosphorylation, and glycosylation.

[0042] The term “% sequence identity” (sequence identity %) is used herein interchangeably with the term “% identity” (identity %) 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 herein, 80% identity means the same as 80% sequence identity determined by a defined algorithm, meaning that a given sequence is at least 80% identical to another sequence. In various embodiments, 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 to a given sequence. In various embodiments, the identity percentage is, for example, within the range of about 60% to about 70%, about 70% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 99%.

[0043] The term “% sequence homology” (sequence homology%) is used herein interchangeably with the term “% homology” (homology%) 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 herein, 80% homology means the same as 80% sequence homology determined by a defined algorithm, so that homologs of a given sequence have a sequence homology higher than 80% over the length of a given sequence. In various embodiments, 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 to a given sequence. In various embodiments, the homology percentage is, for example, in the range of about 60% to about 70%, about 70% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 99%.

[0044] Exemplary computer programs that can be used to determine identity between two sequences include, but are not limited to, a set of BLAST programs publicly available on the NCBI website, such as BLASTN, BLASTX, and TBLASTX, BLASTP, and TBLASTN. See also Altschul et al., J.Mol.Biol.215:403-10, 1990 (the published default settings, i.e., parameters w=4, t=17, are particularly referenced) and Altschul et al., Nucleic Acids Res., 25:3389-3402, 1997. Sequence searches are typically performed using the BLASTP program when evaluating a given amino acid sequence against amino acid sequences in GenBank Protein Sequences and other public databases. The BLASTX program is preferred for searching for nucleic acid sequences translated in all reading frames against amino acid sequences in GenBank Protein Sequences and other public databases. Both BLASTP and BLASTX are run using the default parameters of an open gap penalty of 11.0 and an extended gap penalty of 1.0, and utilize the BLOSUM-62 matrix.

[0045] In addition to calculating the sequence identity percentage, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA, 90:5873-5787, 1993). One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indicator of the probability that a match between two nucleotide or amino acid sequences will occur by chance. For example, if the minimum sum probability in the comparison of the test nucleic acid with the reference nucleic acid is, for example, less than approximately 0.1, less than approximately 0.01, or less than approximately 0.001, the nucleic acid is considered similar to the reference sequence.

[0046] The terms “modification” or “alteration,” as used herein, refer to any manipulation of the peptide backbone (e.g., amino acid sequence) or post-translational modifications (e.g., glycosylation) of a polypeptide.

[0047] The term "antigen-binding molecule," as used herein, refers in its broadest sense to a molecule that specifically binds to an antigen-determining factor. Examples of antigen-binding molecules are antibodies, antibody fragments, and scaffold antigen-binding 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 by 50% or more in a competitive assay, and conversely, a reference molecule that blocks the binding of the antigen-binding molecule to its antigen by 50% or more in a competitive assay.

[0048] As used herein, the term “antigen-binding site” refers to a portion of an antigen-binding molecule that specifically binds to an antigen-determining factor. More specifically, the term “antigen-binding site” refers to a portion of an antibody that specifically binds to a portion or all of an antigen and includes a compartment complementary to that portion or all of the antigen. If the antigen is large, the antigen-binding molecule may bind to only a specific portion of the antigen, which is referred to as an epitope. The antigen-binding site may be provided, for example, by one or more variable domains (also called variable regions). Preferably, the antigen-binding site includes an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

[0049] As used herein, the term “antigen-determinant” is synonymous with “antigen” and “epitope” and refers to a site on a polypeptide macromolecule (e.g., a conformational arrangement made up of a continuous stretch of amino acids or different regions of discontinuous amino acids) to which an antigen-binding moiety binds to form an antigen-binding moiety-antigen complex. Useful antigen-determinants may be found, for example, on the surface of tumor cells, on the surface of virus-infected cells, on the surface of other diseased cells, on the surface of immune cells, free in serum, and / or in the extracellular matrix (ECM). Proteins useful as antigens herein, unless otherwise indicated, may be proteins in any native form from any vertebrate source, including mammals, e.g., primates (e.g., humans) and rodents (e.g., mice and rats). In various embodiments, the antigen is a human protein.

[0050] In this specification, the term "antibody" is used in its broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific and polyspecific antibodies (e.g., bispecific antibodies), and antibody fragments insofar as they exhibit desired antigen-binding activity.

[0051] The term "chimeric" antibody refers to an antibody in which the heavy chain and / or light chain portion originates from a specific source or species, while the rest of the heavy chain and / or light chain originates from a different source or species.

[0052] A “humanized” antibody refers to a chimeric antibody containing amino acid residues from a non-human HVR and amino acid residues from a human FR. In certain embodiments, a humanized antibody contains substantially all of at least one, and typically two, variable domains, where all or substantially all of the HVR (e.g., CDR) corresponds to that of a non-human antibody, and all or substantially all of the FR corresponds to that of a human antibody. A humanized antibody may optionally contain at least a portion of the antibody constant region derived from a human antibody. A “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization. Other forms of “humanized antibodies” encompassed by the present invention are antibodies in which the constant region has been additionally modified or altered from that of the original antibody to produce the properties according to the present invention, particularly with respect to C1q binding and / or Fc receptor (FcR) binding.

[0053] A "human" antibody is an antibody that is produced by a human or human cell, or has an amino acid sequence that corresponds to the amino acid sequence of an antibody derived from a non-human source that utilizes the human antibody repertoire or other human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0054] When used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies constituting the population are identical and / or bind to the same epitope, except for possible variant antibodies, such as naturally occurring mutations or those arising during the manufacture of the monoclonal antibody preparation, and such variants are generally present in trace amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies directed to different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed to a single determinant on an antigen.

[0055] The term "monospecific" antibody, as used herein, refers to an antibody having one or more binding sites, each binding to the same epitope of the same antigen. The term "bispecific" means that an antibody can specifically bind to at least two distinct antigen-determinants, for example, each of the two binding sites being formed by a pair of antibody heavy chain variable domains (VH) and antibody light chain variable domains (VL) that bind to different antigens or different epitopes on the same antigen. Such a bispecific antibody is in a 1+1 format. Other bispecific antibody formats are a 2+1 format (containing two binding sites for a first antigen or epitope and one binding site for a second antigen or epitope) or a 2+2 format (containing two binding sites for a first antigen or epitope and two binding sites for a second antigen or epitope). Typically, a bispecific antibody contains two antigen-binding sites, each specific to a different antigen-determinant.

[0056] The term "valent," as used within this application, refers to the presence of a specified number of binding sites in an antigen-binding molecule. Therefore, the terms "bivalent," "tetravalent," and "hexavalent" refer to the presence of two, four, and six binding sites, respectively, in the antigen-binding molecule. The bispecific antibodies according to the present invention are at least "bivalent," and may be "trivalent" or "polyvalent" (e.g., "tetravalent" or "hexavalent"). In various embodiments, the antibodies of the present invention are bispecific, having two or more binding sites. That is, an antibody can be bispecific even if it has more than two binding sites (i.e., the antibody is trivalent or polyvalent). In particular, the present invention relates to a bispecific bivalent antibody having one binding site for each antigen to which it specifically binds.

[0057] The terms “full-length antibody,” “intact antibody,” and “whole antibody” are used interchangeably herein to refer to antibodies having a structure substantially similar to that of a native antibody. “Native antibody” refers to naturally occurring immunoglobulin molecules with a variety of structures. For example, a native IgG class antibody is a heterotetrameric glycoprotein with approximately 150,000 daltons, composed of two disulfide-linked light chains and two heavy chains. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy chain domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3), also called heavy chain constant regions. Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light chain domain or light chain variable domain, followed by a light chain constant domain (CL), also called a light chain constant region. The heavy chain of an antibody can be assigned to one of five types called alpha (IgA), delta (IgD), epsilon (IgE), gamma (IgG), or mu (IgM), some of which can be further divided into subtypes, e.g., 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 assigned to one of two types called kappa and lambda, based on the amino acid sequence of its constant domain.

[0058] An "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies, triabodies, tetrabodies, cross-Fab fragments; linear antibodies; single-chain antibody molecules (e.g., scFv); and polyspecific antibodies and single-domain antibodies formed from antibody fragments, including but not limited to these. For a review of a particular antibody fragment, see Hudson et al., Nat Med 9, 129-134 (2003). For a review of the scFv fragment, see, for example, Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); and also International Publication No. 93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For a discussion of the Fab and F(ab')2 fragments containing salvage receptor-binding epitope residues and having increased in vivo half-lives, see U.S. Patent No. 5,869,046. A diabody is an antibody fragment having two antigen-binding sites, which may be bivalent or bispecific; see, for example, European Patent No. 404,097; International Publication No. 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). A single-domain antibody is an antibody fragment containing all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (see, for example, Domantis, Inc., Waltham, Mass.; U.S. Patent No. 6,248,516 B1).Additionally, the antibody fragment comprises a single-chain polypeptide having a VH domain that can be assembled with a VL domain into a functional antigen-binding site, or a VL domain that can be assembled with a VH domain into a functional antigen-binding site, thereby providing the antigen-binding properties of a full-length antibody. The antibody fragment may be produced by a variety of techniques, including but not limited to production by recombinant host cells (e.g., Escherichia coli or phages), as described herein, in addition to the proteolytic degradation of intact antibodies.

[0059] Papain digestion of an intact antibody produces two identical antigen-binding fragments, also known as “Fab” fragments, each containing heavy and light chain variable domains, as well as the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. As used herein, the term “Fab fragment” therefore refers to the light chain fragment containing the VL domain and the constant domain (CL) of the light chain, and the antibody fragment containing the VH domain and the first constant domain (CH1) of the heavy chain. The Fab' fragment is distinguished from the Fab fragment by the addition of several residues at the carboxyl terminus of the heavy chain CH1 domain, which contains one or more cysteines from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine ​​residue of the constant domain has a free thiol group. Pepsin treatment yields an F(ab')2 fragment having two antigen-binding sites (two Fab fragments) and a portion of the Fc region.

[0060] A "single-chain Fab fragment" or "scFab" is a polypeptide comprising an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL), and a linker, wherein the antibody domain and the linker have one of the following sequences in the direction from the N-terminus to the C-terminus: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1, or d) VL-CH1-linker-VH-CL; and the linker is a polypeptide of at least 30 amino acids, preferably 32 to 50 amino acids. The single-chain Fab fragment is stabilized via a native disulfide bond between the CL domain and the CH1 domain. Additionally, these single-chain Fab molecules can be further stabilized by the formation of interchain disulfide bonds via the insertion of cysteine ​​residues (e.g., at position 44 in the variable heavy chain and position 100 in the variable light chain, according to Kabat numbering).

[0061] A "single-chain variable fragment (scFv)" is a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of an antibody, linked by a short linker peptide of 10 to approximately 25 amino acids. The linker is usually rich in serine or threonine for solubility, in addition to glycine for flexibility, and can connect the N-terminus of the VH to the C-terminus of the VL, or vice versa. This protein retains the specificity of the original antibody despite the removal of the constant region and the introduction of the linker. scFv antibodies are described, for example, in Houston, JS, Methods in Enzymol. 203 (1991) 46-96. Additionally, the antibody fragment contains a single-chain polypeptide that possesses the characteristics of a VH domain that allows it to assemble into a functional antigen-binding molecule together with the VL domain, or vice versa, thereby providing the antigen-binding properties of a full-length antibody.

[0062] In this specification, the terms “Fc domain” or “Fc region” are used to define the C-terminal region of an antibody heavy chain that includes at least a portion of the constant region. The terms include native sequence Fc regions and variant Fc regions. In particular, the human IgG heavy chain Fc region extends from Cys226 or Pro230 of the heavy chain to the carboxyl terminus. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. The amino acid sequence of the heavy chain is always shown with the C-terminal lysine, but variants without the C-terminal lysine are included in the present invention.

[0063] The IgG Fc region contains IgG CH2 and IgG CH3 domains. The “CH2 domain” of the human IgG Fc region typically extends from approximately position 231 to approximately position 340. In one embodiment, a carbohydrate chain is attached to the CH2 domain. The CH2 domain as used herein may be a native sequence CH2 domain or a variant CH2 domain. The “CH3 domain” includes a stretch of residues at the C-terminus relative to the CH2 domain in the Fc region (i.e., from approximately position 341 to approximately position 447 of IgG). The CH3 region as used herein may be a native sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain having an introduced “nob” on one chain and a corresponding introduced “hole” on the other chain; see U.S. Patent No. 5,821,333 expressly incorporated herein by reference). Such a variant CH3 domain may be used to facilitate heterodimerization of two non-identical antibody heavy chains as described herein. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system, also known 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.

[0064] The “knob-into-hole” technique is described, for example, in U.S. Patent No. 5,731,168; No. 7,695,936; Ridgway et al., Prot Eng 9,617-621 (1996); and Carter, J Immunol Meth 248,7-15 (2001). Generally, the method involves introducing a ridge ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") at the interface of a second polypeptide, so that the ridge can be positioned within the cavity to promote heterodimerization and inhibit homodimerization. The ridge is constructed by replacing a smaller amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). Compensatory cavities of the same or similar size as the bulges are created at the interface of the second polypeptide by replacing a larger amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine). The bulges and cavities can be created, for example, by modifying the nucleic acid encoding the polypeptide by site-directed mutagenesis, or by peptide synthesis. In a specific embodiment, the knob modification includes the amino acid substitution T366W in one of the two subunits of the Fc domain, and the hole modification includes the amino acid substitutions T366S, L368A, and Y407V in the other of the two subunits of the Fc domain. In a further specific embodiment, the subunit of the Fc domain containing the knob modification additionally includes the amino acid substitution S354C, and the subunit of the Fc domain containing the hole modification additionally includes the amino acid substitution Y349C. The introduction of these two cysteine ​​residues results in the formation of a disulfide bridge between the two subunits of the Fc region, thereby further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0065] The “region equivalent to the Fc region of immunoglobulins” is intended to include variants that, in addition to naturally occurring allele variants of the Fc region of immunoglobulins, have modifications that produce substitutions, additions, or deletions but do not substantially reduce the immunoglobulin’s ability to mediate effector functions (e.g., antibody-dependent cytotoxicity). For example, one or more amino acids may be deleted from the N-terminus or C-terminus of the Fc region of immunoglobulins without substantial loss of biological function. Such variants may be selected according to general rules known in the art to have the least effect on activity (see, for example, Bowie, JU et al., Science 247:1306-10 (1990)).

[0066] The term "effector function" refers to the biological activity attributed to the Fc region of an antibody, which varies with antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0067] An "activated Fc receptor" is an Fc receptor that, after engagement by the Fc region of an antibody, triggers a signaling event that stimulates receptor-hosting cells to perform effector functions. Activated Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89). A specific activated Fc receptor is human FcγRIIIa (see UniProt accession number P08637, version 141).

[0068] 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, a blocking antibody or antagonist antibody substantially or completely inhibits the biological activity of the antigen. For example, the bispecific antibodies of the present invention block signaling through TNF-α and activin so as to inhibit the TNF-α-NF-κB signaling pathway and the activin-Smad2 / 3 signaling pathway.

[0069] As used herein, “specific binding” means that binding is selective for an antigen and can be distinguished from undesirable or nonspecific interactions. The ability of an antigen-binding molecule to bind to a specific antigen can be measured through either enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) techniques (analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)), and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)).

[0070] The terms "affinity" or "binding affinity," as used herein, refer to the total strength 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 molecule X for its partner Y can generally be expressed by the dissociation constant (KD), which is the ratio of the dissociation and association rate constants (koff and kon, respectively). A specific method for measuring affinity is surface plasmon resonance (SPR). As used herein, the term "high affinity" of an antibody means that the antibody has a 10% affinity for its target antigen. -9 M or lower, especially 10 -10 This refers to having an M or lower Kd. The term "low affinity" for an antibody means that the antibody has 10 -8This refers to having M or a higher Kd. The term "reduced binding," as used herein, refers to a decrease in affinity for each interaction, for example, as measured by SPR. Conversely, "increased binding" refers to an increase in binding affinity for each interaction.

[0071] The terms “bispecific antibody comprising a first antigen-binding molecule that specifically binds to TNF-α and a second antigen-binding molecule that specifically binds to activin,” “bispecific antibody that specifically binds to TNF-α and activin,” and “bispecific antigen-binding molecule specific to TNF-α and activin” are used interchangeably herein and refer to a bispecific antibody having the ability to bind to TNF-α and activin with sufficient affinity to be useful in targeting TNF-α and activin as a diagnostic and / or therapeutic agent.

[0072] The terms “anti-TNF-α antibody” and “antibody containing an antigen-binding site that binds to TNF-α” refer to an antibody that has the ability to bind to TNF-α, particularly TNF-α polypeptides expressed on the cell surface, with sufficient affinity to be useful in targeting TNF-α as a diagnostic and / or therapeutic agent. In one embodiment, the degree of binding of an anti-TNF-α antibody to an unrelated non-TNF-α protein is lower than about 10% of the binding of the antibody to TNF-α, when measured, for example, by radioimmunoassay (RIA) or flow cytometry (FACS) or by surface plasmon resonance assay using a biosensor system, such as the Biacore® system. In a particular embodiment, the antigen-binding molecule that binds to human TNF-α is, for example, 10 -8 M~10 -13M has a KD value of binding affinity for binding to human TNF-α. In one preferred embodiment, the KD value of each binding affinity is determined in a surface plasmon resonance assay using the extracellular domain (ECD) of human TNF-α (TNF-α-ECD) for TNF-α binding affinity. The term “anti-TNF-α antibody” also encompasses bispecific antibodies that have the ability to bind to TNF-α and a second antigen.

[0073] The terms “anti-activin antibody” and “antibody containing an antigen-binding site that binds to activin” refer to an antibody that has the ability to bind to activin, particularly activin polypeptides expressed on the cell surface, with sufficient affinity to be useful in targeting activin as a diagnostic and / or therapeutic agent. In one embodiment, the degree of binding of an anti-activin antibody to unrelated non-activin proteins is lower than about 10% of the binding of the antibody to activin, as measured, for example, by radioimmunoassay (RIA) or flow cytometry (FACS) or by surface plasmon resonance assay using a biosensor system, such as the Biacore® system. In a particular embodiment, the antigen-binding molecule that binds to human activin is, for example, 10 -8 M~10 -13 M has a KD value of binding affinity for binding to human activin. In one preferred embodiment, the KD value of each binding affinity is determined in a surface plasmon resonance assay using the extracellular domain (ECD) of human activin (activin-ECD) for activin binding affinity. The term “anti-activin antibody” also includes bispecific antibodies that have the ability to bind to activin and a second antigen.

[0074] As used herein, the term "fusion protein" refers to a fusion polypeptide molecule comprising two or more genes that originally encoded separate proteins, wherein the components of the fusion protein are linked to each other by peptide bonds, either directly or through a peptide linker. As used herein, the term "fused" refers to components linked by peptide bonds, either directly or through one or more peptide linkers.

[0075] A "linker" is a molecule that joins two other molecules, either covalently or through ionic, van der Waals or hydrogen bonds, for example, 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. A "cleavable linker" is a linker that can be cleaved or otherwise severed to separate two components connected by the cleavable linker. Cleavable linkers are generally cleaved by enzymes, typically peptidases, proteases, nucleases, and lipases, etc. Cleavable linkers may also be cleaved by environmental cues, such as changes in temperature, pH, salt concentration, etc.

[0076] As used herein, the term "peptide linker" refers to a peptide comprising one or more amino acids, typically about 2 to 20 amino acids. Peptide linkers are known in the art or described herein. Suitable, non-immunogenic linker peptides include, for example, (G4S) n , (SG4) n or G4(SG4)n peptide linkers. "n" is generally a number from 1 to 10, typically 2 to 4.

[0077] "Pharmaceutical composition" refers to a composition suitable for medicinal use in animals. A pharmaceutical composition contains a pharmacologically effective amount of an activator and a pharmaceutically acceptable carrier. "Pharmacologically effective amount" refers to the amount of agent effective to produce the intended pharmacological result. "pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers, media, buffers, and excipients, e.g., phosphate-buffered saline solution, 5% dextrose aqueous solution, and emulsions, e.g., oil / water or water / oil emulsion, and various types of wetting agents and / or adjuvants. Suitable pharmaceutical carriers and formulations are listed in Remington's Pharmaceutical Sciences, 21st Ed. 2005, Mack Publishing Co, Easton. "pharmaceutically acceptable salt" refers to a salt that can be incorporated into a compound for medicinal use, e.g., metal salts (such as sodium, potassium, magnesium, calcium, etc.) and salts of ammonia or organic amines.

[0078] As used herein, “treatment” (and its grammatical variations, e.g., “treat” or “treating”) refers to a clinical intervention in an attempt to alter the natural course of a disease in the treated individual, which may be performed either preventively or during the course of a clinical pathological condition. The desired effects of treatment include, but are not limited to, prevention of the onset or recurrence of the disease, reduction of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction of the rate of disease progression, remission or temporary relief of the disease state, and improved or better prognosis. As used herein, “reducing” a disease, disorder, or condition means reducing the severity and / or frequency of the symptoms of the disease, disorder, or condition. Furthermore, references to “treatment” herein include references to curative, palliative, and preventive treatments.

[0079] The terms “effective dose” or “therapeutic effective dose,” as used herein, refer to an amount of a compound or composition sufficient to treat a specified disorder, condition or disease, for example, to alleviate, reduce, and / or delay one or more of its symptoms. With respect to cancer or other undesirable cell proliferation, an effective dose includes an amount sufficient to (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, delay, slow to some extent, preferably halt, cancer cell infiltration into peripheral organs; (iv) inhibit tumor metastasis (i.e., slow to some extent, preferably halt); (v) inhibit tumor growth; (vi) prevent or delay tumor development and / or recurrence; and / or (vii) alleviate to some extent one or more of the symptoms associated with cancer. An effective dose may be administered in one or more doses.

[0080] The phrases “administer” or “cause to be administered” refer to the act of controlling and / or permitting the administration of the agent / compound in question to a patient, performed by a healthcare professional (e.g., a physician) or the person responsible for the patient’s medical care. Causing to be administered may involve a diagnosis and / or determination of an appropriate treatment regimen, as well as / or the prescription of a specific agent / compound for the patient. Such a prescription may include, for example, writing a prescription form and annotating a medical record. Where administration is described herein, “causing to be administered” is also assumed.

[0081] The terms “patient,” “individual,” and “subject” are interchangeable and may refer to mammals, preferably humans or non-human primates, but may also refer to domesticated mammals (e.g., dogs or cats), laboratory mammals (e.g., mice, rats, rabbits, hamsters, guinea pigs), and agricultural mammals (e.g., horses, cattle, pigs, sheep). In various embodiments, the patient may be a human being (e.g., adult males, adult females, adolescent males, adolescent females, boys, girls) under the care of a physician or other healthcare professional in an outpatient setting or other clinical setting, such as a hospital or psychiatric nursing facility. In various embodiments, the patient may include, but is not limited to, immunocompromised patients or patients with a weakened immune system, including patients with primary immunodeficiency, AIDS; patients with cancer; and transplant patients taking certain immunosuppressants; and patients with genetic disorders affecting the immune system (e.g., congenital agammaglobulinemia, congenital IgA deficiency). In various embodiments, patients have immunogenic cancers, including but not limited to bladder cancer, lung cancer, melanoma, and other cancers that have been reported to have a high rate of mutation (Lawrence et al., Nature, 499(7457):214-218, 2013).

[0082] The term "immunotherapy" includes treatments using depletion antibodies against specific tumor antigens; treatments using antibody-drug conjugates; treatments using agonists, antagonists, or blocking antibodies against costimulatory or coinhibitory molecules (immune checkpoints), e.g., CTLA-4, PD-1, OX-40, CD137, GITR, LAG3, TIM-3, SIRP, CD40, CD47, Siglec 8, Siglec 9, Siglec 15, TIGIT, and VISTA; treatments using bispecific T-cell engaging antibodies (BiTE®), e.g., blinatumomab; and biological response modifiers, e.g., IL-2, IL-12, IL-15, IL-21, GM-CSF, IFN-α, IFN-α. This refers to cancer treatments that include, but are not limited to, treatments involving the administration of N-β and IFN-γ; treatments using therapeutic vaccines, such as cypluce-T; treatments using Bacillus calmette-Guérin (BCG); treatments using dendritic cell vaccines or tumor antigen peptide vaccines; treatments using chimeric antigen receptor (CAR)-T cells; treatments using CAR-NK cells; treatments using tumor-infiltrating lymphocytes (TILs); treatments using adoptively transferred antitumor T cells (ex vivo expanding and / or TCR transgenic); treatments using TALL-104 cells; and treatments using immunostimulants, such as the Toll-like receptor (TLR) agonists CpG and imiquimod.

[0083] "Resistant or refractory cancer" refers to tumor cells or cancer that do not respond to previous anticancer therapies, including, for example, chemotherapy, surgery, radiotherapy, stem cell transplantation, and immunotherapy. Tumor cells may be resistant or refractory at the beginning of treatment, or may become resistant or refractory during treatment. Refractory tumor cells include tumors that do not respond at the start of treatment, or that initially respond for a short period but do not respond to treatment. Refractory tumor cells also include tumors that respond to treatment with anticancer therapy but do not respond to subsequent rounds of therapy. For the purposes of this invention, refractory tumor cells also include tumors that appear to be inhibited by treatment with anticancer therapy but recur within 5 years, possibly 10 years, or longer after treatment is discontinued. Anticancer therapy may be a chemotherapeutic agent alone, radiotherapy alone, targeted therapy alone, immunotherapy alone, surgery alone, or a combination thereof. For ease of description and not limited thereto, it is understood that refractory tumor cells are interchangeable with resistant tumors.

[0084] The term "polymer," as used herein, generally includes, but is not limited to, homopolymers; copolymers, such as block, graft, random, and alternating copolymers; and ternary copolymers; as well as blends and modifications thereof. Furthermore, unless otherwise specifically limited, the term "polymer" includes all possible geometric configurations of a material. These configurations include, but are not limited to, isotactic, syndiotactic, and random symmetries.

[0085] "Polynucleotides" refer to polymers 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 containing bases not naturally occurring, nucleotides involved in linking with other nucleotides besides naturally occurring phosphodiester bonds, or bases attached through linkages other than phosphodiester bonds. Therefore, nucleotide analogs include, for example and without limitation, phosphorothioates, phosphorodithioates, phosphorotryesters, phosphoramidates, boranophosphates, methylphosphonates, chiral-methylphosphonates, 2-O-methylribonucleotides, and peptide-nucleic acid (PNA). Such polynucleotides can be synthesized, for example, using automated DNA synthesizers. The term "nucleic acid" typically refers to large polynucleotides. The term "oligonucleotide" typically refers to short polynucleotides, generally about 50 nucleotides or fewer. When a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), it is understood that this also includes an RNA sequence in which "U" replaces "T" (i.e., A, U, G, C).

[0086] Conventional notation is used herein to describe polynucleotide sequences: the left-hand end of a single-stranded polynucleotide sequence is the 5' end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5'-direction. The direction of nucleotide addition from 5' to 3' into a nascent RNA transcript is referred to as the transcription direction. The DNA strand having the same sequence as the mRNA is referred to as the "coding strand"; the sequence on the DNA strand having the same sequence as the mRNA transcribed from that DNA and located 5' relative to the 5' end of the RNA transcript is referred to as the "upstream sequence"; and the sequence on the DNA strand having the same sequence as the RNA and located 3' relative to the 3' end of the coding RNA transcript is referred to as the "downstream sequence".

[0087] "Complementary" refers to the topological compatibility or matching of the interacting surfaces of two polynucleotides. Therefore, two molecules can be described as complementary, and furthermore, the features of their contact surfaces are complementary to each other. 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 to the second polynucleotide under stringent hybridization conditions.

[0088] "Specific hybridization" or "selective hybridization" refers to the preferential binding, duplexing, or hybridization of a nucleic acid molecule to a particular nucleotide sequence under stringent conditions, given its presence in a complex mixture of DNA or RNA (e.g., a whole cell). The term "stringent conditions" refers to conditions under which a probe preferentially hybridizes to its target subsequence and to a lower degree, or not hybridizes at all, to other sequences. "Stringent hybridization" and "stringent hybridization conditions" in the context of nucleic acid hybridization experiments, such as Southern and Northern hybridization, are sequence-dependent and vary under different environmental parameters. Comprehensive guides on 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, 3rd sup.rd ed., NY; and Ausubel et al., ed., Current Edition, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, NY.

[0089] Generally, highly stringent hybridization and washing conditions are selected to be approximately 5°C lower than the thermal melting point (Tm) for a particular sequence at a defined ionic strength and pH. Tm is the temperature at which 50% of the target sequence hybridizes to a perfectly matched probe (under defined ionic strength and pH). Very stringent conditions are selected to be equal to the Tm for a particular probe. An example of stringent hybridization conditions for hybridization of complementary nucleic acids with approximately 100 or more complementary residues on a filter in Southern or Northern blots is 50% formalin with 1 mg of heparin at 42°C, with hybridization performed overnight. An example of highly stringent washing conditions is 0.15 M NaCl, 72°C, for approximately 15 minutes. An example of stringent washing conditions is washing with 0.2 × SSC, 65°C, for 15 minutes. See Sambrook et al. for a description of SSC buffers. High-stringency washes may be preceded by low-stringency washes to remove background probe signals. For example, an exemplary medium-stringency wash for double helices with approximately 100 or more nucleotides is 1 × SSC, 45°C, 15 minutes. For example, an exemplary low-stringency wash for double helices with approximately 100 or more nucleotides is 4–6 × SSC, 40°C, 15 minutes. In general, a signal-to-noise ratio twice (or higher) than that observed for unrelated probes in a particular hybridization assay indicates the detection of specific hybridization.

[0090] A "primer" refers to a polynucleotide that has the ability to specifically hybridize to a designated polynucleotide template and provide a starting point for the synthesis of a complementary polynucleotide. Such synthesis occurs when the polynucleotide primer is placed under conditions that induce synthesis, i.e., in the presence of a nucleotide, a complementary polynucleotide template, and a polymerization agent, such as DNA polymerase. Primers are typically single-stranded, but may also be double-stranded. Primers are typically deoxyribonucleic acid, but a variety of synthetic and naturally occurring primers are useful for many applications. A primer is complementary to the template to which it is designed to hybridize in order to serve as a site for the initiation of synthesis, but does not need to reflect the exact sequence of the template. In such cases, the specific hybridization of the primer to the template depends on the stringency of the hybridization conditions. Primers can be labeled, for example, with a chromogenic, radioactive, or fluorescent moiety and used as a detectable moiety.

[0091] When used in relation to polynucleotides, "probe" refers to a polynucleotide that has the ability to specifically hybridize to a specified sequence of another polynucleotide. The probe specifically hybridizes to the target complementary polynucleotide, but does not need to reflect the exact complementary sequence of the template. In such cases, the specific hybridization of the probe to the target depends on the stringency of the hybridization conditions. The probe may be labeled with, for example, a pigment-generating, radioactive, or fluorescent moiety and used as a detectable moiety. In cases where the probe provides a starting point for the synthesis of the complementary polynucleotide, the probe can also be a primer.

[0092] A "vector" is a polynucleotide that can be used to introduce another nucleic acid, to which it is ligated, into a cell. One type of vector is a "plasmid," which refers to a linear or circular double-stranded DNA molecule to which an additional nucleic acid segment can be ligated. Another type of vector is a viral vector (e.g., replication-deficient retroviruses, adenoviruses, and adeno-associated viruses), to which an additional DNA segment can be introduced into the viral genome. Certain vectors have the ability to autonomously replicate in the host cell into which they are introduced (e.g., bacterial vectors containing bacterial origins of replication and episomatic mammalian vectors). Other vectors (e.g., non-episomatic mammalian vectors) are integrated into the host cell's genome upon introduction into the host cell and thereby replicate together with the host genome. An "expression vector" is a type of vector that can direct the expression of a selected polynucleotide.

[0093] A “regulatory sequence” is a nucleic acid that influences the expression (e.g., level, timing, or location) of the nucleic acid to which it is operably ligated. A regulatory sequence can exert its effect, for example, directly on the nucleic acid being regulated, or through the action of one or more other molecules (e.g., polypeptides that bind to the regulatory sequence and / or nucleic acid). Examples of regulatory sequences include promoters, enhancers, and other expression regulatory elements (e.g., polyadenylation signals). Further 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 ligated” to a regulatory sequence if the regulatory sequence influences the expression (e.g., level, timing, or location) of the nucleotide sequence.

[0094] "Host cell" refers to a cell that may be used to express the polynucleotides of this disclosure. A host cell may be a prokaryote, e.g., Escherichia coli, or a eukaryote, e.g., a single-celled eukaryote (e.g., yeast or other fungi), a plant cell (e.g., tobacco or tomato plant cell), an animal cell (e.g., human cell, monkey cell, hamster cell, rat cell, mouse cell, or insect cell), or a hybridoma. Typically, a 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 term "recombinant host cell" may be used to describe a host cell that has been transformed or transfected with the nucleic acid to be expressed. A host cell may also be a cell that contains a nucleic acid but does not express the nucleic acid at the desired level until a regulatory sequence is introduced into the host cell so that it is operably linked to the nucleic acid. It is understood that the term host cell refers not only to a specific target cell but also to the offspring or potential offspring of such a cell. Because certain modifications may occur in subsequent generations, for example, due to mutation or environmental influences, such offspring may not actually be identical to the parent cells, but such offspring still fall within the scope of the term as used herein.

[0095] The term “isolated molecule” (where the molecule is, for example, a polypeptide or polynucleotide) is defined as a molecule that, for reasons of its origin or source of origin, (1) is not associated with naturally occurring components that are present with it in its native state, (2) substantially does not contain other molecules from the same species, (3) is expressed by cells from a different species, or (4) does not exist in nature. Therefore, a molecule that is chemically synthesized or expressed in a cellular system different from the cells from which the molecule originates naturally is “isolated” from its naturally occurring components. A molecule may also be made substantially free of naturally occurring components by isolation using purification techniques well known in the art. The purity or homogeneity of a molecule may be assayed by a number of means well known in the art. For example, the purity of a polypeptide sample may be assayed using polyacrylamide gel electrophoresis and gel staining for visualization of polypeptides using techniques well known in the art. For certain purposes, higher resolution may be provided by using HPLC or other means well known in the art for purification.

[0096] A protein or polypeptide is considered "substantially pure," "substantially homogeneous," or "substantially purified" if at least about 60% to 75% of the sample exhibits a single species of polypeptide. Polypeptides or proteins may be monomers or polymers. Substantially pure polypeptides or proteins typically constitute about 50%, 60%, 70%, 80%, or 90% w / w of a protein sample, more commonly about 95% w / w, and preferably more than 99% pure. The purity or homogeneity of a protein may be indicated by numerous means known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by staining the gel with stains known in the art to visualize a single polypeptide band. For certain purposes, higher resolution may be provided by using HPLC or other means known in the art for purification.

[0097] The terms “labeling” or “labeled,” as used herein, refer to the incorporation of another molecule into an antibody. In one embodiment, labeling is the incorporation of a detectable marker, e.g., a radiolabeled amino acid, or the attachment of a biotinyl moiety to a polypeptide that can be detected by a marked avidin (e.g., a streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or calorimetry). In another embodiment, the label or marker may be a therapeutic agent, e.g., a drug conjugate or a toxin. Various methods for labeling polypeptides and glycoproteins are known in the art and can be used. Examples of labeling for polypeptides are: radioisotopes or radionuclides (e.g., 3 H, 14 C, 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131I) Fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzyme labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal-binding domains, epitope tags), magnetic agents (e.g., gadolinium chelate), toxins (e.g., pertussis toxin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracendione This includes, but is not limited to, dione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as their analogs or homologs. In various embodiments, the labels are attached by spacer arms of varying lengths to reduce potential steric hindrance.

[0098] When used herein, the term "heterogeneous" refers to a composition or state that is neither native nor naturally occurring, for example, one that can be achieved by replacing an existing natural composition or state with one derived from another source. Similarly, the expression of a protein in an organism other than the organism in which it is naturally expressed constitutes a heterogeneous expression system and a heterogeneous protein.

[0099] It is understood that the aspects and embodiments of the disclosure described herein include aspects and embodiments that "consist of" and / or "essentially consist of".

[0100] References to values ​​or parameters “about” in this specification include (and describe) variations directed toward the value or parameter itself. For example, a statement referring to “about X” includes a statement of “X.”

[0101] As used herein and in the appended claims, the singular “a,” “or,” and “the” refer to multiple subjects unless the context otherwise explicitly states. The aspects and variations of the disclosure described herein are understood to include aspects and variations consisting of and / or essentially consisting of.

[0102] Tumor necrosis factor ligand TNF is an immunomodulatory cytokine required for immune processes. Unregulated activity of TNF can lead to the development of inflammatory diseases. Excessive expression of TNF in cells is associated with the development of immune diseases, including rheumatoid arthritis, Crohn's disease, psoriatic arthritis, and inflammatory bowel disease. The function of TNF requires binding to its two receptors, TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2). Blocking the interaction between TNF and TNFR has been successfully developed as a therapy in the treatment of inflammatory or autoimmune diseases.

[0103] In various 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 activin or an activin-related ligand. In various 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 amino acid sequences described in SEQ ID NOs: 1 to 5. Human TNFR1 ECD IYPSGVIGLVPHLGDREKRDSVCPQGKYIHPQNNSICCTKCHKGTYLYNDCPGPGQDTDCRECESGSFTASENHLRHCLSCSKCRKEMGQVEISSCTVDRDTVCGCRKNQYRHYWSENLFQCFNCSLCLNGTVHLSCQEKQNTVCTCHAGFFLRENECVSCSNCKKSLECTKLCLPQIENVKGTEDSGTT(Sequence No. 1) Human TNFR2 ECD LPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDTVCDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCALSKQEGCRLCAPLRKCRPGFGVARPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAIPGNASMDAVCTSTSPTRSMAPGAVHLPQPVSTRSQHTQPTPEPSTAPSTSFLLPMGPSPPAEGSTGD (Sequence ID 2) Human TNFR1 / CRD1-TNFR2 / CRD2 / 3 / 4 IYPSGVIGLVPHLGDREKRDSVCPQGKYIHPQNNSICCTKCHKGTYLYNDCPGPGQDTDCRSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCALSKQEGCRL CAPLRKCRPGFGVARPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAIPGNASMDAVCTSTSPTRSMAPGAVHLPQPVSTRSQHTQPTPEPSTAPSTSFLLPMGPSPPAEGSTGD (Sequence ID 3) Human TNFR1 / CRD1 / 2 / 3-TNFR2 / CRD4 IYPSGVIGLVPHLGDREKRDSVCPQGKYIHPQNNSICCTKCHKGTYLYNDCPGPGQDTDCRECESGSFTASENHLRHCLSCSKCRKEMGQVEISSCTVDRDTVCGCRKNQYRHYWSENLFQCFNCSLCLNGTVHLSCQEKQNTVCPCAPGTFSNTTSSTDICRPHQICNVVAIPGNASMDAVCTSTSPTRSMAPGAVHLPQPVSTRSQHTQPTPEPSTAPSTSFLLPMGPSPPAEGSTGD (Sequence ID 4) TNFR1 / ΔCRD4 IYPSGVIGLVPHLGDREKRDSVCPQGKYIHPQNNSICCTKCHKGTYLYNDCPGPGQDTDCRECESGSFTASENHLRHCLSCSKCRKEMGQVEISSCTVDRDTVCGCRKNQYRHYWSENLFQCFNCSLCLNGTVHLSCQEKQNTVC(Sequence ID 5)

[0104] In various 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 listed in Table 2: TIFF0007857306000002.tif40170

[0105] Activin and activin-related ligands Activins, including activin A, activin B, and activin AB, as well as activin-related proteins, including myostatin (GDF-8) and GDF-11, mediate Smad2 / 3 signaling through the binding and activation of their high-affinity receptors, ActRIIA and ActRIIB, on the cell surface. Activins and related proteins play essential roles in regulating a wide range of biological activities, including mesoderm induction, cell differentiation, myogenesis, bone remodeling, hematopoiesis, fibrosis, and reproductive physiological functions. The secreted glycoprotein, follistatin (FST), binds to activins and activin-related ligands, negatively regulating their signaling activity.

[0106] In various embodiments, the bifunctional molecules of the present invention have the ability to bind to activin or activin-related ligands having an amino acid sequence selected from the group consisting of the amino acid sequences described in SEQ ID NOs: 6-14. Human ActRIIA-ECD ETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNPVTPKPP(Sequence ID 6) Human ActRIIB-ECD ETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEV TYEPPPTAPT(Sequence ID 7) Human follistatin 315 GNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEAACSSGVLLEVKHSGSCNSISEDTEEEEEDEDQDYSFPISSILEW (Sequence ID 8) Human follistatin ΔHBS (modified follistatin) GNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGQSCVVDQTGSPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEAACSSGVLLEVKHSGSCNSISEDTEEEEEDEDQDYSFPISSILEW (Sequence ID 9) Human follistatin 288 GNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEAACSSGVLLEVKHSGSCN (Sequence ID 10) Modified Human ActRIIB ECD ETRECIYYNANWELERTNQSGLERCEGDQDKRLHCYASWRNSSGTIELVKKGCWLDDINCYDRQECVATKENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPT(Sequence ID 11) Modified Human ActRIIB ECD ETRECIYYNANWELERTNQSGLERCYGDKDKRRHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPT(Sequence ID 12) Modified Human ActRIIB ECD ETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWDDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPT(Sequence ID 13) Modified Human ActRIIA ECD GAILGRSETQECLFYNANWELERTNQTGVEPCEGEKDKRLHCYATWRNISGSIEIVKKGCWLDDFNCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS(Sequence ID 14)

[0107] In various embodiments, the bifunctional molecule has the ability to bind to an activin or activin-related ligand having an amino acid sequence selected from the group consisting of the amino acid sequences listed in Table 3: TIFF0007857306000003.tif70170

[0108] TNF-α and / or activin antibodies and antibody fragments Methods for generating novel antibodies that bind to TNF-α and / or activin ligands and / or receptors are known to those skilled in the art. For example, a method for generating monoclonal antibodies that specifically bind to TNF-α and / or activin ligands may include administering an immunogenic composition containing TNF-α and / or activin ligands in an amount effective enough to stimulate a detectable immune response to a mouse, obtaining antibody-producing cells from the mouse (e.g., cells from the spleen), fusing these antibody-producing cells with myeloma cells to obtain antibody-producing hybridomas, and testing the antibody-producing hybridomas to identify hybridomas that produce monoclonal antibodies that specifically bind to TNF-α and / or activin ligands. Once hybridomas are obtained, they can optionally be propagated in cell culture under conditions that allow hybridoma-derived cells to produce monoclonal antibodies that specifically bind to TNF-α and / or activin ligands. The monoclonal antibodies may be purified from the cell culture. Various different techniques are available to test antigen / antibody interactions and identify particularly desirable antibodies.

[0109] Other suitable methods are available for producing or isolating antibodies of the required specificity, including, for example, selecting recombinant antibodies from a library or relying 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.

[0110] Antibodies can be manipulated in numerous ways. Antibodies can be produced as single-chain antibodies (including small modular immunopharmaceuticals or SMIPs®), Fab and F(ab')2 fragments, etc. Antibodies can be humanized, chimeric, deimmunized, or fully human antibodies. Numerous publications describe many types of antibodies and methods for manipulating such antibodies. See, for example, U.S. Patents 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.

[0111] Chimeric antibodies can be produced by recombinant DNA techniques known in the art. For example, the gene encoding the Fc constant region of a mouse (or other species) monoclonal antibody molecule is digested with restriction enzymes to remove the mouse Fc encoding region and replaced with an equivalent portion of the gene encoding the human Fc constant region (Robinson et al., International Publication PCT / US86 / 02269; Akira, et al., European Patent Application No. 184,187; Taniguchi, M., European Patent Application No. 171,496; Morrison et al., European Patent Application No. 173,494; Neuberger et al., International Publication 86 / 01533; Cabilly et al., U.S. Patent No. 4,816,567; Cabilly et al., European Patent Application No. 125,023; 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. al., Canc. Res., 47:999-1005, 1987; Wood et al., Nature, 314:446-449, 1985; and Shaw et al., J. Natl Cancer Inst., 80:1553-1559, 1988).

[0112] Methods for humanizing antibodies have been described in the Art. In some embodiments, the humanized antibody has one or more amino acid residues introduced from a non-human source in addition to the non-human CDR. Humanization can essentially be carried out by substituting the hypervariable region sequence with the corresponding sequence of the human antibody, according to the method of Winter and collaborators (Jones et al., Nature, 321:522-525, 1986; Riechmann et al., Nature, 332:323-327, 1988; Verhoeyen et al., Science, 239:1534-1536, 1988). Thus, such a “humanized” antibody is a chimeric antibody (U.S. Patent No. 4,816,567) in which substantially less of the intact human variable region is replaced by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and, in some cases, some framework region residues are replaced with residues from similar sites in rodent antibodies.

[0113] Queen et al.'s U.S. Patent No. 5,693,761 discloses a refinement in Winter et al. for humanizing antibodies, based on the premise that avidity loss can be attributed to problems in structural motifs in the humanized framework that interfere with the folding of the CDR into a binding-enabled conformation found in the mouse antibody due to steric or other chemical incompatibility. To address this problem, Queen instructs the use of a human framework sequence in a linear peptide sequence that is closely homologous to the framework sequence of the mouse antibody to be humanized. Thus, Queen's method focuses on comparing framework sequences across species. Typically, all available human variable region sequences are compared to a specific mouse sequence, and the percentage of identity between the corresponding framework residues is calculated. The human variable region with the highest percentage is selected to provide a framework sequence for the humanization project. Queen also instructs that it is important to retain certain amino acid residues from the mouse framework that are essential to supporting the CDR in the binding-enabled conformation in the humanized framework. Potential essentiality is assessed from molecular models. Candidate residues for retention are typically those adjacent to a CDR in a linear sequence, or physically within 6 Å of any CDR residue.

[0114] Another method for humanizing antibodies, referred to as "framework shuffling," relies on generating a combinatorial library containing non-human CDR variable regions fused in-frame to a pool of individual human germline frameworks (Dall'Acqua et al., Methods, 36:43, 2005). The library is then screened to identify clones encoding humanized antibodies that retain good binding.

[0115] Methods for producing fully human antibodies have been described in the Art. For example, a method for producing an anti-TNF-α antibody or its antigen-binding fragment comprises the steps of synthesizing a library of human antibodies on phages, screening the library using the TNF-α polypeptide or its antibody-binding moiety, isolating phages that bind to the TNF-α polypeptide, and obtaining antibodies from the phages. Another example is a method for preparing a library of antibodies for use in phage display technology, comprising the steps of immunizing a non-human animal containing a human immunoglobulin locus with the TNF-α polypeptide or its antigenic moiety to induce an immune response, extracting antibody-producing cells from the immunized animal; isolating RNA encoding the heavy and light chains of the antibody of the present invention from the extracted cells, reverse transcribing the RNA to produce cDNA, amplifying the cDNA using primers, and inserting the cDNA into a phage display vector to express the antibody on phages. The recombinant anti-TNF-α antibody of the present invention may be obtained in this manner.

[0116] The recombinant human anti-TNF-α and / or activin antibody of the present invention may also be isolated by screening a recombinant combinatorial antibody library. Preferably, the library is prepared from mRNA isolated from B cells. L and V HThis is an scFv phage display library generated using cDNA. Methods for preparing 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 no. 27-9400-01; and Stratagene SurfZAP® phage display kit, catalog no. 240612). Other methods and reagents may be used in the generation and screening of antibody display libraries (e.g., U.S. Publication No. 5,223,409; PCT International Publication No. 92 / 18619, International Publication No. 91 / 17271, International Publication No. 92 / 20791, International Publication No. 92 / 15679, International Publication No. 93 / 01288, International Publication No. 92 / 01047, International Publication No. 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. 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. 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. See al., Proc. Natl. Acad. Sci. (USA), 88:7978-7982, 1991; all are incorporated herein by reference).

[0117] Human antibodies are also produced by immunizing non-human transgenic animals, such as XenoMouse® animals (Abgenix, Inc. / Amgen, Inc.--Fremont, Calif.), which contain some or all of the human immunoglobulin heavy and light chain loci in their genome, with human IgE antigens. XenoMouse® mice are engineered mouse strains that contain large fragments of the human immunoglobulin heavy and light chain loci and are deficient in mouse antibody production. See, for example, Green et al., Nature Genetics, 7:13-21, 1994 and U.S. Patents 5,916,771, 5,939,598, 5,985,615, 5,998,209, 6,075,181, 6,091,001, 6,114,598, 6,130,364, 6,162,963 and 6,150,584. XenoMouse® mice produce an adult-like human repertoire of fully human antibodies and generate antigen-specific human antibodies. In some embodiments, XenoMouse® mice contain approximately 80% of the human antibody V gene repertoire through the introduction of megabase-sized germline fragments of human heavy chain and kappa light chain loci in yeast artificial chromosomes (YACs). In other embodiments, XenoMouse® mice further contain substantially all human lambda light chain loci. See Mendez et al., Nature Genetics, 15:146-156, 1997; Green and Jakobovits, J.Exp.Med., 188:483-495, 1998; and International Publication No. 98 / 24893. In one embodiment, the present invention provides a method for producing anti-TNF-α and / or activin antibodies from non-human, non-mouse animals by immunizing non-human transgenic animals containing human immunoglobulin loci with TNF-α and / or activin polypeptides. Such animals can be produced using the methods described in the literature referenced above.

[0118] Anti-TNF-α antibody The FDA-approved anti-TNF-α antibody, adalimumab (Abbvie HUMIRA®; DrugBank DB00051), is used to treat humans. In various 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 described in SEQ ID NO: 15: EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNVYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 15) Human antibodies or antigen-binding fragments containing the light chain amino acid sequence described in Sequence ID No. 17: DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 17) Alternatively, a human antibody or antigen-binding fragment containing the heavy chain amino acid sequence described in SEQ ID NO: 15 and the light chain amino acid sequence described in SEQ ID NO: 17; Human antibodies or antigen-binding fragments containing the heavy chain variable region amino acid sequence described in SEQ ID NO: 16: EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSS(Sequence ID 16) Human antibodies or antigen-binding fragments containing the light chain variable region amino acid sequence described in Sequence ID No. 18: DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIK(Sequence ID 18) Alternatively, it is a human antibody or antigen-binding fragment containing the heavy chain variable region amino acid sequence described in SEQ ID NO: 16 and the light chain variable region amino acid sequence described in SEQ ID NO: 18.

[0119] In various embodiments, the present invention provides an antibody 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; the heavy chain, light chain, heavy chain variable region, or light chain variable region comprising 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 with the amino acid sequence described in SEQ ID NOs. 15, 16, 17, or 18; and the antibody specifically binds to human TNF-α.

[0120] The FDA-approved anti-TNF-α antibody, infliximab (Centocor REMICADE®; DrugBank DB00065), is used to treat humans. In various embodiments of the present invention, the anti-TNF-α antibody is a human antibody or antigen-binding fragment containing the heavy chain amino acid sequence described in SEQ ID NO: 19: EVKLEESGGGLVQPGGSMKLSCVASGFIFSNHWMNWVRQSPEKGLEWVAEIRSKSINSATHYAESVKGRFTISRDDSKSAVYLQMTDLRTEDTGVYYCSRNYYGSTYDYWGQGT TLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence ID 19) Human antibodies or antigen-binding fragments containing the light chain amino acid sequence described in Sequence ID No. 21: DILLTQSPAILSVSPGERVSFSCRASQFVGSSIHWYQQRTNGSPRLLIKYASESMSGIPSRFSGSGSGTDFTLSINTVESEDIADYYCQQSHSWPFTFGSGTNLEVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 21) Alternatively, a human antibody or antigen-binding fragment containing the heavy chain amino acid sequence described in SEQ ID NO: 19 and the light chain amino acid sequence described in SEQ ID NO: 21; Human antibodies or antigen-binding fragments containing the heavy chain variable region amino acid sequence described in Sequence ID No. 20: EVKLEESGGGLVQPGGSMKLSCVASGFIFSNHWMNWVRQSPEKGLEWVAEIRSKSINSATHYAESVKGRFTISRDDSKSAVYLQMTDLRTEDTGVYYCSRNYYGSTYDYWGQGTTLTVSS (Sequence ID 20) Human antibodies or antigen-binding fragments containing the light chain variable region amino acid sequence described in Sequence ID No. 22: DILLTQSPAILSVSPGERVSFSCRASQFVGSSIHWYQQRTNGSPRLLIKYASESMSGIPSRFSGSGSGTDFTLSINTVESEDIADYYCQQSHSWPFTFGSGTNLEVK(Sequence ID 22) Alternatively, it is a human antibody or antigen-binding fragment containing the heavy chain variable region amino acid sequence described in SEQ ID NO: 20 and the light chain variable region amino acid sequence described in SEQ ID NO: 22.

[0121] In various embodiments, the present invention provides an antibody 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; 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 with the amino acid sequence described in SEQ ID NOs: 19, 20, 21, or 22; and the antibody specifically binds to human TNF-α.

[0122] The FDA-approved anti-TNF-α antibody, certolizumab-pegol (UCB CIMZIA®; DrugBank DB08904), is used to treat humans. In various 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 described in SEQ ID NO: 23: EVQLVESGGGLVQPGGSLRLSCAASGYVFTDYGMNWVRQAPGKGLEWMGWINTYIGEPIYADSVKGRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCARGYRSYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCAA (Sequence ID 23) Human antibodies or antigen-binding fragments containing the light chain amino acid sequence described in Sequence ID No. 25: DIQMTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKALIYSASFLYSGVPYRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNIYPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 25) Alternatively, a human antibody or antigen-binding fragment containing the heavy chain amino acid sequence described in SEQ ID NO: 23 and the light chain amino acid sequence described in SEQ ID NO: 25; Human antibodies or antigen-binding fragments containing the heavy chain variable region amino acid sequence described in Sequence ID No. 24: EVQLVESGGGLVQPGGSLRLSCAASGYVFTDYGMNWVRQAPGKGLEWMGWINTYIGEPIYADSVKGRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCARGYRSYAMDYWGQGTLVTVSS (Sequence ID 24) Human antibodies or antigen-binding fragments containing the light chain variable region amino acid sequence described in Sequence ID No. 26: DIQMTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKALIYSASFLYSGVPYRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNIYPLTFGQGTKVEIK (Sequence ID 26) Alternatively, it is a human antibody or antigen-binding fragment containing the heavy chain variable region amino acid sequence described in SEQ ID NO: 24 and the light chain variable region amino acid sequence described in SEQ ID NO: 26.

[0123] In various embodiments, the present invention provides an antibody 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; the heavy chain, light chain, heavy chain variable region, or light chain variable region comprising 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 with the amino acid sequence described in SEQ ID NOs: 23, 24, 25, or 26; and the antibody specifically binds to human TNF-α.

[0124] The FDA-approved anti-TNF-α antibody, golimumab (Janssen Biotech SIMPONI®; DrugBank DB06674), is used to treat humans. In various embodiments of the present invention, the anti-TNF-α antibody is a human antibody or antigen-binding fragment containing the heavy chain amino acid sequence described in SEQ ID NO: 27: QVQLVESGGGVVQPGRSLRLSCAASGFIFSSYAMHWVRQAPGNGLEWVAFMSYDGSNKKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRGIAAGGNYYYYGMDVW GQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 27) Human antibodies or antigen-binding fragments containing the light chain amino acid sequence described in Sequence ID No. 29: EIVLTQSPATLSLSPGERATLSCRASQSVYSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 29) Alternatively, a human antibody or antigen-binding fragment containing the heavy chain amino acid sequence described in SEQ ID NO: 27 and the light chain amino acid sequence described in SEQ ID NO: 29; Human antibodies or antigen-binding fragments containing the heavy chain variable region amino acid sequence described in Sequence ID No. 28: QVQLVESGGGVVQPGRSLRLSCAASGFIFSSYAMHWVRQAPGNGLEWVAFMSYDGSNKKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRGIAAGGNYYYYGMDVWGQGTTVTVSS(Sequence ID 28) Human antibodies or antigen-binding fragments containing the light chain variable region amino acid sequence described in SEQ ID NO: 30: EIVLTQSPATLSLSPGERATLSCRASQSVYSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPFTFGPGTKVDIK(Sequence ID 30) Alternatively, it is a human antibody or antigen-binding fragment containing the heavy chain variable region amino acid sequence described in SEQ ID NO: 28 and the light chain variable region amino acid sequence described in SEQ ID NO: 30.

[0125] In various embodiments, the present invention provides an antibody 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; the heavy chain, light chain, heavy chain variable region, or light chain variable region comprising 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 with the amino acid sequence described in SEQ ID NOs: 27, 28, 29, or 30; and the antibody specifically binds to human TNF-α.

[0126] Anti-activin antibodies In various embodiments of the present invention, the anti-activin antibody is a human antibody or antigen-binding fragment containing the heavy chain amino acid sequence described in SEQ ID NO: 31: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGLSWVRQAPGQGLEWMGWIIPYNGNTNSAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYFCARDRDYGVNYDAFDIWGQG TMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNVYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 31) Human antibodies or antigen-binding fragments containing the light chain amino acid sequence described in Sequence ID No. 33: SYEVTQAPSVSVSPGQTASITCSGDKLGDKYACWYQQKPGQSPVLVIYQDSKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQAWDSSTAVFGGGTKLTVLRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 33) Alternatively, a human antibody or antigen-binding fragment containing the heavy chain amino acid sequence described in SEQ ID NO: 31 and the light chain amino acid sequence described in SEQ ID NO: 33; Human antibodies or antigen-binding fragments containing the heavy chain variable region amino acid sequence described in Sequence ID No. 32: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGLSWVRQAPGQGLEWMGWIIPYNGNTNSAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYFCARDRDYGVNYDAFDIWGQGTMVTVSS(Sequence ID 32) Human antibodies or antigen-binding fragments containing the light chain variable region amino acid sequence described in SEQ ID NO: 34: SYEVTQAPSVSVSPGQTASITCSGDKLGDKYACWYQQKPGQSPVLVIYQDSKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQAWDSSTAVFGGGTKLTVL(Sequence ID 34) Alternatively, a human antibody or antigen-binding fragment containing the heavy chain variable region amino acid sequence described in SEQ ID NO: 32 and the light chain variable region amino acid sequence described in SEQ ID NO: 34. It is an anti-activin A antibody.

[0127] In various embodiments, the present invention provides an antibody 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; the heavy chain, light chain, heavy chain variable region, or light chain variable region comprising 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 with the amino acid sequence described in SEQ ID NOs: 31, 32, 33, or 34; and the antibody specifically binds to human activin A.

[0128] In various embodiments of the present invention, the anti-activin antibody is a human antibody or antigen-binding fragment containing the heavy chain amino acid sequence described in SEQ ID NO: 35: QVQLQESGPGLVKPSETLSLTTCTVSGGSFSSHFWSWIRQPPGKGLEWIGYILYTGGTSFNPSLKSRVSMSVGTSKNQFSLKLSSVTAADTAVYYCARARSGITFTGIIVPGSFDI WGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGP PCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (Sequence ID 35) Human antibodies or antigen-binding fragments containing the light chain amino acid sequence described in Sequence ID No. 37: EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 37) Alternatively, a human antibody or antigen-binding fragment containing the heavy chain amino acid sequence described in SEQ ID NO: 35 and the light chain amino acid sequence described in SEQ ID NO: 37; Human antibodies or antigen-binding fragments containing the heavy chain variable region amino acid sequence described in SEQ ID NO: 36: QVQLQESGPGLVKPSETLSLTCTVSGGSFSSHFWSWIRQPPGKGLEWIGYILYTGGTSFNPSLKSRVSMSVGTSKNQFSLKLSSVTAADTAVYYCARARSGITFTGIIVPGSFDIWGQGTMVTVSS (Sequence ID 36) Human antibodies or antigen-binding fragments containing the light chain variable region amino acid sequence described in SEQ ID NO: 38: EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK (Sequence ID 38) Alternatively, a human antibody or antigen-binding fragment containing the heavy chain variable region amino acid sequence described in SEQ ID NO: 36 and the light chain variable region amino acid sequence described in SEQ ID NO: 38. It is an anti-activin A antibody.

[0129] In various embodiments, the present invention provides an antibody 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; the heavy chain, light chain, heavy chain variable region, or light chain variable region comprising 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 with the amino acid sequence described in SEQ ID NOs. 35, 36, 37, or 38; and the antibody specifically binds to human activin A.

[0130] Linker In various embodiments, a first antigen-binding molecule that specifically binds to a TNF-α ligand is attached to a second antigen-binding molecule that specifically binds to activin or an activin-related ligand by a linker and / or hinge-linker peptide. The linker or hinge-linker may be an artificial sequence of amino acids between 5, 10, 15, 20, 30, 40 or more that exhibits an α-helix conformation, or it may be relatively free of secondary structures.

[0131] Peptide linkers provide covalent linkage between protein domains as well as additional structural and / or spatial flexibility. As is known in the art, peptide linkers contain flexible amino acid residues, such as glycine and serine. In various embodiments, peptide linkers may contain 1 to 100 amino acids. In various embodiments, the spacer may contain the motif GGGSGGGS (SEQ ID NO: 81). In other embodiments, the linker may contain the motif GGGGS (SEQ ID NO: 84)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 contain other protein motifs, including but not limited to α-helix conformation sequences, such as AEAAAKEAAAKEAAAKA (SEQ ID NO: 79). In various embodiments, the length and composition of the linker may be tuned to optimize developability, including but not limited to activity, expression level, and aggregation tendency. In another embodiment, the peptide linker may be a simple chemical bond, such as an amide bond (e.g., by chemical conjugation of PEG).

[0132] Exemplary peptide linkers are provided in Table 4: TIFF0007857306000004.tif119170

[0133] Dual-function antagonist molecule The present invention provides novel polypeptide-based bifunctional antagonist molecules specifically designed to simultaneously neutralize TNF-α signaling and activin signaling in a potent manner, 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 activin or an activin-related ligand. In various embodiments, the bifunctional molecule comprises an isolated antibody or its antigen-binding fragment that specifically binds to TNF-α, and an isolated antibody or its antigen-binding fragment that specifically binds to activin or an activin-related ligand. Importantly, these bifunctional antagonists also offer advantageous properties, such as productivity, stability, binding affinity, biological activity, specific targeting of certain cells, targeting efficiency, and reduced toxicity.

[0134] Exemplary bifunctional antagonist molecules In various embodiments, the bifunctional antagonist molecule of the present invention is selected from the group of molecules including the designed and fusion partners listed in Table 5: TIFF0007857306000005.tif236170TIFF0007857306000006.tif234170TIFF0007857306000007.tif131170

[0135] In various embodiments, the bifunctional antagonist molecule of the present invention is selected from the group of molecules listed in Table 6. TIFF0007857306000008.tif150170

[0136] Polynucleotides In another embodiment, the Disclosure provides isolated nucleic acid molecules comprising polynucleotides encoding the bifunctional antagonist molecules of the Disclosure. The nucleic acid of interest may be single-stranded or double-stranded. Such nucleic acids may be DNA or RNA molecules. DNA includes, for example, cDNA, genomic DNA, synthetic DNA, PCR-amplified DNA, and combinations thereof. Genomic DNA encoding the bifunctional antagonist molecules can be obtained from genomic libraries available for a number of species. Synthetic DNA can be obtained by the chemical synthesis of overlapping oligonucleotide fragments, followed by the assembly of fragments to reconstruct parts or all of the coding region and adjacent sequences. RNA can be obtained from prokaryotic expression vectors that direct high levels of mRNA synthesis, for example, vectors using the T7 promoter and RNA polymerase. cDNA can be obtained from libraries prepared from mRNA isolated from various tissues expressing the bifunctional antagonist molecules. The DNA molecules of the Disclosure include, in addition to the full-length gene, its polynucleotides and fragments. The full-length gene may also include a sequence encoding an N-terminal signal sequence.

[0137] In various embodiments, the isolated nucleic acid molecule comprises a polynucleotide described herein and further comprises a polynucleotide encoding at least one heterologous protein described herein. In various embodiments, the nucleic acid molecule further comprises a polynucleotide encoding a linker or hinge linker described herein.

[0138] In various embodiments, the recombinant nucleic acids of this disclosure may be operably ligated to one or more regulatory nucleotide sequences in an expression construct. Regulatory sequences are recognized in the art and are selected to direct the expression of a bifunctional antagonist molecule. Thus, 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, the 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 termination sequences, translation start and termination sequences, and enhancer or activator sequences. Constitutive or inducible promoters, as known in the art, are assumed by this disclosure. Promoters may be either naturally occurring promoters or hybrid promoters combining elements of one or more promoters. Expression constructs may reside in cells on episomes, e.g., plasmids, or expression constructs may be inserted into chromosomes. In various embodiments, the expression vector contains a selection marker gene to enable the selection of transformed host cells. The selection marker gene is well known in the art and varies with the host cell used.

[0139] In another aspect of this disclosure, the nucleic acid of interest is provided in an expression vector comprising a nucleotide sequence encoding a bifunctional antagonist molecule and operably ligated 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. Suitable vectors for expression in host cells are readily available, and the nucleic acid molecule is inserted into the vector using standard recombinant DNA techniques. Such vectors may include a variety of expression regulatory sequences that control the expression of the DNA sequence which, when operably ligated to the DNA sequence, can be used in these vectors to express the DNA sequence encoding the bifunctional antagonist molecule. Such useful expression regulatory sequences include, for example, the early and late promoters of SV40, the tet promoter, the earliest promoters of adenovirus or cytomegalovirus, the RSV promoter, the lac system, the trp system, the TAC or TRC system, the T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter regions of phage lambda, regulatory regions for fd coat proteins, promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, acid phosphatases, such as the promoter of PhoS, the promoter of the yeast α junction factor, the polyhedron promoter of the baculovirus system, and other sequences known to regulate the expression of genes in prokaryotic or eukaryotic cells or their viruses, as well as various combinations thereof. It should be understood that the design of expression vectors may depend on factors such as the selection of the host cell to be transformed and / or the type of protein whose expression is desired. Furthermore, the copy number of the vector, the ability to control the copy number and expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered.

[0140] The recombinant nucleic acids of this disclosure may be prepared by ligating a cloned gene or portion thereof into a vector suitable for expression in prokaryotic cells, eukaryotic cells (yeast, birds, insects, or mammals), or both. Expression media for the preparation of recombinant bifunctional antagonist molecules include plasmids and other vectors. For example, suitable vectors include plasmids of the type for expression in prokaryotic cells, e.g., Escherichia coli: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids, and pUC-derived plasmids.

[0141] Some mammalian expression vectors contain both a prokaryotic sequence to promote vector replication in bacteria and one or more eukaryotic transcription units expressed in eukaryotic cells. pcDNAI / amp, pcDNAI / neo, pRc / CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo, and pHyg-derived vectors are examples of mammalian expression vectors suitable for eukaryotic cell transfection. Some of these vectors have been modified with sequences from bacterial plasmids, e.g., pBR322, to promote replication and drug resistance selection in both prokaryotic and eukaryotic cells. Alternatively, derivatives of viruses, e.g., bovine papillomavirus (BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived, and p205) may be used for transient protein expression in eukaryotic cells. Examples of other viral (including retrovirus) expression systems can be found in the following description of gene therapy delivery systems. Various methods used in plasmid preparation and host organism transformation are well known in the art. For general recombination procedures, as well as other suitable expression systems for both prokaryotic and eukaryotic cells, see Chapters 16 and 17 of Molecular Cloning: A Laboratory Manual, 2nd Ed., Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 1989). In some cases, it may be desirable to express recombinant polypeptides using baculovirus expression systems. Examples of such baculovirus expression systems include pVL-derived vectors (e.g., pVL1392, pVL1393 and pVL941), pAcUW-derived vectors (e.g., pAcUW1), and pBlueBac-derived vectors (e.g., B-gal-containing pBlueBac III).

[0142] In various embodiments, vectors are designed for the production of a target bifunctional antagonist molecule in CHO cells, such as the Pcmv-Script vector (Stratagene, La Jolla, Calif.), the pcDNA4 vector (Invitrogen, Carlsbad, Calif.), and the pCI-neo vector (Promega, Madison, Wis.). As is evident, the target gene construct can be used to induce the expression of a target bifunctional antagonist molecule in cells propagated in culture, for example, to produce a protein, including a fusion protein or variant protein, for purification.

[0143] Therefore, this disclosure further relates to methods for producing the bifunctional antagonist molecule of interest. For example, host cells transfected with an expression vector encoding the bifunctional antagonist molecule can be cultured under appropriate conditions to allow expression of the bifunctional antagonist molecule to occur. The bifunctional antagonist molecule may be secreted and isolated from a mixture of cells and a culture medium containing the bifunctional antagonist molecule. Alternatively, the bifunctional antagonist molecule may be retained in the cytoplasm or in a membrane fraction, the cells may be harvested and lysed, and the protein isolated. The cell culture comprises host cells, culture medium, and other by-products. Suitable culture media for cell culture are well known in the art.

[0144] The polypeptides and proteins of this disclosure may be purified according to protein purification techniques well known to those skilled in the art. These techniques involve, at one level, coarse fractionation of proteinaceous and nonproteinaceous fractions. Once the peptides or polypeptides have been isolated from other proteins, the peptides or polypeptides of interest may be further purified using chromatography and electrophoresis techniques to achieve partial or complete purification (or purification to homogeneity). The terms “isolated polypeptide” or “purified polypeptide,” as used herein, are intended to mean a composition in which the polypeptide has been purified to any degree compared to its naturally occurring state and is isolated from other components. A purified polypeptide therefore also means the polypeptide that has been freed from the environment in which it may naturally exist. Generally, “purified” refers to a polypeptide composition that has been subjected to fractionation to remove various other components and substantially retains its expressed biological activity. When the term “substantially purified” is used, this designation refers to a peptide or polypeptide composition in which the polypeptide or peptide forms the main component of the composition, for example, about 50%, about 60%, about 70%, about 80%, about 85%, or about 90% or more of the proteins in the composition.

[0145] Various techniques suitable for use in purification are well known to those skilled in the art. These include, for example, precipitation using ammonium sulfate, PEG, and antibodies (immunoprecipitation) or by thermal denaturation followed by centrifugation; chromatography, such as affinity chromatography (protein-A column), ion exchange, gel filtration, reversed phase, hydroxyl apatite, hydrophobic interaction chromatography; isoelectric focusing; gel electrophoresis; and combinations of these techniques. As is generally known in the art, the order in which various purification steps are performed may be changed or certain steps may be omitted, but this is still considered to result in a suitable method for preparing substantially purified polypeptides.

[0146] Pharmaceutical composition In another embodiment, the disclosure provides a pharmaceutical composition comprising an isolated bifunctional antagonist molecule mixed with a pharmaceutically acceptable carrier. Such pharmaceutically acceptable carriers are well known, understood and extensively described to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Edition, edited by ArGennaro, Mack Publishing Company, 1990). The pharmaceutically acceptable carrier may be included, for example, for the purpose of modifying, maintaining, or preserving the pH, volumetric osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or permeation of the composition. Such a pharmaceutical composition may affect the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the polypeptide.Suitable pharmaceutically acceptable carriers include amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (e.g., boric acid, bicarbonate, Tris-HCl, citric acid, phosphoric acid, or other organic acids); fillers (e.g., mannitol or glycine); chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); complexing agents (e.g., caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides and other carbohydrates (e.g., glucose, mannose, or dextrin); proteins (e.g., serum albumin, gelatin, or immunoglobulin); colorants; flavoring agents and diluents; emulsifiers; hydrophilic polymers (e.g., polyvinylpyrrolidone) This includes, but is not limited to, low molecular weight polypeptides; salt-forming counterions (e.g., sodium); preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (e.g., glycerin, propylene glycol, or polyethylene glycol); sugar alcohols (e.g., mannitol or sorbitol); suspending agents; surfactants or wetting agents (e.g., Pluronic acid, PEG, sorbitan esters, polysorbates, e.g., polysorbate 20, polysorbate 80, Triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancers (sucrose or sorbitol); tonicity enhancers (e.g., alkali metal halides (preferably sodium or potassium chloride, mannitol, sorbitol); delivery media; diluents; excipients and / or pharmaceutical aids).

[0147] The primary medium or carrier in a pharmaceutical composition may be either aqueous or non-aqueous. For example, suitable mediums or carriers may be water for injection, physiological saline solution, or artificial cerebrospinal fluid, which may optionally be supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary mediums. Other exemplary pharmaceutical compositions include Tris buffer at approximately pH 7.0–8.5, or acetate buffer at approximately pH 4.0–5.5, which may further contain sorbitol or a suitable substitute thereof. In one embodiment of this disclosure, a composition may be prepared for storage by mixing a selected composition having a desired degree of purity in the form of a lyophilized cake or aqueous solution with an optional formulation agent (Remington's Pharmaceutical Sciences, cited above). Furthermore, therapeutic compositions may be formulated as lyophilized products using appropriate excipients, such as sucrose. The optimal pharmaceutical composition will be determined by those skilled in the art, for example, depending on the intended route of administration, the delivery format, and the desired dosage.

[0148] When parenteral administration is intended, the therapeutic pharmaceutical composition may be in the form of a pyrogen-free, parenterally acceptable aqueous solution containing the desired bifunctional antagonist molecule in a pharmaceutically acceptable medium. A particularly preferred medium for parenteral injection is sterile distilled water, in which the polypeptide is formulated as a sterile isotonic solution that can be properly stored. In various embodiments, a pharmaceutical formulation suitable for injectable administration may be formulated in an aqueous solution, preferably in a physiologically compatible buffer, such as Hanks' solution, Ringer's solution, or physiologically buffered saline. The aqueous injection suspension may contain a substance that increases the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Additionally, the suspension of the active compound may be prepared as a suitable oily injection suspension. Optionally, the suspension may also contain a suitable stabilizer or agent to increase the solubility of the compound, which may allow for the preparation of a highly concentrated solution.

[0149] In various embodiments, therapeutic pharmaceutical compositions may be formulated for targeted delivery using colloidal dispersion systems. Colloidal dispersion systems include polymer complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Examples of lipids useful in liposome production include phosphatidyl compounds, e.g., phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Exemplary phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine, and distearoylphosphatidylcholine. Liposome targeting is also possible, for example, based on organ specificity, cell specificity, and organelle specificity, which is known in the art.

[0150] In various embodiments, oral administration of pharmaceutical compositions is envisioned. Pharmaceutical compositions administered in this manner may be formulated with or without carriers conventionally used in the formulation of solid dosage forms, such as tablets and capsules. In solid dosage forms for oral administration (e.g., capsules, tablets, pills, sugar-coated tablets, powders, and granules), one or more therapeutic compounds of the present disclosure may be one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or the following: (1) fillers or bulking agents, such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia; (3) wetting agents, such as glycerol; (4) disintegrants, such as (5) Dissolution retarders, e.g., paraffin; (6) Absorption enhancers, e.g., quaternary ammonium compounds; (7) Wetting agents, e.g., cetyl alcohol and glycerol monostearate; (8) Absorbents, e.g., kaolin and bentonite clay; (9) Lubricants, e.g., talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof; and (10) Colorants may be mixed with any of these. In the case of capsules, tablets and pills, the pharmaceutical composition may also contain buffering agents. Similar types of solid compositions may also be used as fillers in soft and hard-filled gelatin capsules using high molecular weight polyethylene glycol, in addition to excipients such as lactose or milk sugar. Liquid dosing forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs.In addition to the active ingredient, the liquid dosage form may also contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (especially cottonseed, peanut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, as well as mixtures thereof. Besides inert diluents, the oral composition may also contain adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, colorants, fragrances, and preservatives.

[0151] In various embodiments, topical administration of the pharmaceutical composition to either the skin or mucous membranes is envisioned. Topical formulations may further include one or more of a variety of agents known to be effective as skin or stratum corneum permeability enhancers. Examples of these include 2-pyrrolidone, N-methyl-2-pyrrolidone, dimethylacetamide, dimethylformamide, propylene glycol, methyl or isopropyl alcohol, dimethyl sulfoxide, and azon. Additional agents may be included to make the formulation cosmetically acceptable. Examples of these include fats, waxes, oils, pigments, fragrances, preservatives, stabilizers, and surfactants. Keratinolants, such as those known to those skilled 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 may be mixed under sterile conditions with a pharmaceutically acceptable carrier and any required preservatives, buffers, or propellants. The ointments, pastes, creams, and gels may contain, in addition to the compounds of this disclosure (e.g., bifunctional antagonist molecules), excipients such as animal and vegetable fats, oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silicic acid, talc, and zinc oxide, or mixtures thereof.

[0152] Additional pharmaceutical compositions envisioned for use herein include formulations involving polypeptides in sustained or controlled delivery formulations. Various other sustained or controlled delivery means, such as liposome carriers, biodegradable microparticles or porous beads, and techniques for formulating depot injections are also known to those skilled in the art.

[0153] The effective dose of a therapeutically used pharmaceutical composition depends, for example, on the therapeutic context and purpose. Those skilled in the art will understand that the appropriate dosage level for a treatment will therefore vary in part depending on the molecule being delivered, the indication for which the polypeptide is used, the route of administration, and the patient's size (body weight, body surface or organ size) and condition (age and overall health). Thus, clinicians can titrate the dosage and modify the route of administration to obtain the optimal therapeutic effect. Typical dosages may range from about 0.1 mg / kg to about 100 mg / kg or more, depending on the factors mentioned above. Polypeptide compositions may preferably be administered by intravenous injection or administration. Long-acting pharmaceutical compositions may be administered every 3-4 days, weekly, or bi-weekly, depending on the half-life and clearance rate of the particular formulation. The frequency of administration depends on the pharmacokinetic parameters of the polypeptide in the formulation used. Typically, the composition is administered until a dosage is reached that achieves the desired effect. The composition may therefore be administered as a single dose, or as multiple doses (of the same or different concentrations / doses) over time, or as a series of infusions. Further refinement of the appropriate dosage is carried out as usual. The appropriate dosage may be confirmed through the use of appropriate dose-response data.

[0154] The route of administration of the pharmaceutical composition follows known methods, for example, by oral, intravenous, intraperitoneal, intracerebral (intraparum), intraventricular, intramuscular, intraocular, intraarterial, intraportal, intrafocal, intramedullary, intrathecal, intraventricular, percutaneous, subcutaneous, or intraperitoneal injection; otherwise, by intranasal, intraintestinal, topical, sublingual, urethral, ​​transvaginal, or rectal means, by a continuous-release system, or by an implantable device. If desired, the composition may be administered continuously by bolus injection or infusion, or by an implantable device. Alternatively or additionally, the composition may be administered topically via implantation of a membrane, sponge, or other suitable material in which the desired molecule is absorbed or encapsulated. When an implantable device is used, the device may be implanted in any suitable tissue or organ, and the delivery of the desired molecule may be done by diffusion, time-release bolus, or continuous administration.

[0155] therapeutic use In another embodiment, the present disclosure provides methods for treating or preventing various complex disease conditions whose pathogenesis involves activation of both the TNF-α-mediated NF-κB signaling pathway and the activin-mediated Smad2 / 3 signaling pathway.

[0156] In various embodiments, the novel bifunctional antagonist molecules of the present invention may have a broad application for the treatment of a variety of disorders in subjects, including but not limited to the following conditions: anemia, inflammation, pulmonary hypertension, heart failure, renal failure, muscular dystrophy, arthritis, organofibrosis, and cancer, the treatment comprising administering to the subject a therapeutically effective dose (as either monotherapy or in a combination therapy regimen) of the bifunctional antagonist molecule of the present disclosure in a pharmaceutically acceptable carrier, such administration attenuating loss of muscle mass and / or loss of muscle function.In particular, the bifunctional antagonist molecules of this disclosure are intended for use as monotherapy or in combination with immune checkpoint inhibitors, such as anti-PD1, anti-PDL1, and anti-CTL4 antibodies, or chimeric antigen receptor (CAR) T-cell therapy, for hematological disorders (e.g., ineffective erythropoiesis, pancytopenia, myelodysplastic syndrome, bone marrow failure, leukemia, beta-thalassemia, and sickle cell disease), fibrotic diseases (e.g., non-alcoholic steatohepatitis or NASH, cirrhosis, pulmonary fibrosis, renal fibrosis, polycystic kidney disease, cardiac Visceral fibrosis, myofibrosis, myelofibrosis, dermatofibrosis, hand fibrosis, and ocular fibrosis), muscular dystrophy (e.g., DMD, Becker MD, limb-girdle muscular dystrophy, myotonic MD, and FSHD), myositis (e.g., polymyositis and dermatomyositis), myopathy (including hereditary myopathy and acquired myopathy), motor neuron disease (e.g., Lou Gehrig's disease or ALS), neurodegenerative diseases (e.g., Parkinson's disease, Huntington's disease, and Alzheimer's disease), cancer cachexia, sarcopenia, bone Fragility disorders (e.g., fractures and metastases in cancer), cardiovascular diseases (e.g., pulmonary hypertension, Marfan syndrome, myocardial infarction, and chronic heart failure), chronic kidney disease (CKD), diabetes, chronic obstructive pulmonary disease (COPD), cytokine storms resulting from infections (e.g., AIDS, tuberculosis, SARS-CoV, and sepsis), arthritis including rheumatoid arthritis (RA) and osteoarthritis (OA), trauma (e.g., burns or motorcycle accidents), ICU life support, denervation (e.g., stroke or It is useful in the treatment of various complex diseases, including, but not limited to, 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 (e.g., heart, kidney, and liver transplants), and various malignancies (e.g., leukemia, melanoma, breast cancer, multiple myeloma, prostate cancer, lung cancer, pancreatic cancer, stomach cancer, ovarian cancer, colorectal cancer, brain cancer, bladder cancer, and head and neck cancer).

[0157] This disclosure provides a method for treating cardiovascular disease in a subject, comprising administering to the subject a therapeutically effective dose (either as monotherapy or in a combination therapy regimen) of the bifunctional antagonist molecule of this disclosure in a pharmaceutically acceptable carrier, wherein such administration reduces inflammation and fibrosis of the vascular system, as well as of muscle, including smooth muscle, cardiac muscle, and skeletal muscle. In particular, the bifunctional antagonist molecule of this disclosure is useful in treating heart failure, pulmonary hypertension (including pulmonary arterial hypertension), myocarditis, coronary artery disease, myocardial infarction, cardiac arrhythmias, cardiac valve disease, cardiomyopathy, pericardial disease, aortic disease, Marfan syndrome, and cardiac atrophy.

[0158] This disclosure provides 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. Modulation may improve the cardiac function of the 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 may be assessed by echocardiography to measure cardiac pump function, focusing on 1) the volume of blood pumped and the efficiency of pumping, and 2) myocardial function, focusing on the strength of myocardial contraction.

[0159] This disclosure provides a method for treating a metabolic disorder in a subject, comprising administering to the subject a therapeutically effective dose (either as monotherapy or in a combination therapy regimen) of the bifunctional antagonist molecule of this disclosure in a pharmaceutically acceptable carrier. In particular, the bifunctional antagonist molecule of this disclosure is useful in treating metabolic disorders selected from obesity, dyslipidemia, diabetes mellitus, insulin resistance, sarcopenic obesity, steatosis, and metabolic syndrome, as well as diabetic myopathy, nephropathy, neuropathy, retinopathy, osteopenia, impaired glucose tolerance, hyperglycemia, and androgen depletion.

[0160] This disclosure provides a method for treating cancer cells in a subject, comprising administering to the subject a therapeutically effective dose (either as monotherapy or in a combination therapy regimen) of a bifunctional antagonist molecule of this disclosure in a pharmaceutically acceptable carrier, wherein such administration inhibits the growth and / or proliferation of cancer cells. In particular, the bifunctional antagonist molecule of this disclosure is useful in treating disorders characterized as cancer. Such disorders include, but are not limited to, solid tumors, such as cancers of the breast, airway, brain, genitals, gastrointestinal tract, urinary tract, eye, liver, skin, head and neck, thyroid, parathyroid and their distant metastases, lymphoma, sarcoma, 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 airway cancer include, but are not limited to, bronchial adenoma and pleuriblastoma, in addition to small cell and non-small cell lung cancer. Examples of brain cancers include, but are not limited to, brainstem and hypothalamic (hypophthalmic) gliomas, cerebellar and cerebral astrocytomas, medulloblastomas, ependymomas, as well as neuroectoderm and pineal gland tumors. Tumors of the male reproductive organs include, but are not limited to, prostate and testicular cancers. Tumors of the female reproductive organs include, but are not limited to, endometrial, cervical, ovarian, vaginal, and vulvar cancers, as well as uterine sarcomas. Tumors of the gastrointestinal tract include, but are not limited to, anal, colon, colorectal, esophageal, gallbladder, stomach, pancreatic, rectum, small intestine, and salivary gland cancers. Tumors of the urinary tract include, but are not limited to, bladder, penile, kidney, renal pelvis, ureter, and urethral cancers. Eye cancers include, but are not limited to, intraocular melanoma and retinoblastoma. Liver cancers include, but are not limited to, hepatocellular carcinoma (hepatocellular carcinoma with or without lamellar variants), cholangiocarcinoma (intrahepatic cholangiocarcinoma), and mixed hepatocellular carcinoma. Skin cancers include, but are not limited to, squamous cell carcinoma, Kaposi's sarcoma, malignant melanoma, Merkel cell carcinoma, and non-melanoma skin cancer. Head and neck cancers include, but are not limited to, nasopharyngeal cancer and lip cancer. Lymphomas include, but are not limited to, AIDS-associated lymphoma, non-Hodgkin lymphoma, cutaneous T-cell lymphoma, Hodgkin's disease, and central nervous system lymphomas.Sarcomas include, but are not limited to, soft tissue sarcomas, osteosarcomas, malignant fibrous histiocytoma, lymphosarcoma, and rhabdomyosarcoma. Leukemias include, but are not limited to, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, and hairy cell leukemia. In certain embodiments, cancers are cancers with high expression of TNF-α and activin (e.g., activin A, activin B, activin AB), such as pancreatic cancer, gastric cancer, ovarian cancer, colorectal cancer, melanoma, leukemia, lung cancer, prostate cancer, brain cancer, bladder cancer, and head and neck cancers.

[0161] This disclosure provides a method for treating chronic kidney disease (CKD) in a subject, comprising administering to the subject a therapeutically effective dose (as either monotherapy or a combination therapy regimen) of the bifunctional antagonist molecule of this disclosure in a pharmaceutically acceptable carrier, wherein such administration attenuates loss of renal function and prevents muscle loss or inhibits renal fibrosis. In particular, the bifunctional antagonist molecule of this disclosure is useful in treating CKD, including renal failure, interstitial fibrosis, and hemodialysis, as well as in treating protein-energy-wasting (PEW) associated with CKD. Modulation may improve the subject's CKD or PEW 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 protein / creatinine ratio (PCR) and glomerular filtration rate (GFR) in urine. Improvement in PEW can be assessed by measuring serum levels of albumin and inflammatory cytokines, rates of protein synthesis and degradation, body weight, muscle mass, physical activity, and nutritional outcomes.

[0162] This disclosure provides a method for treating an autoimmune disease in a subject, comprising administering to the subject a therapeutically effective dose (either as monotherapy or in a combination therapy regimen) of the bifunctional antagonist molecule of this disclosure in a pharmaceutically acceptable carrier. In particular, the bifunctional antagonist molecule of this disclosure is useful in treating autoimmune disorders selected from multiple sclerosis, diabetes mellitus (type 1), glomerulonephritis, myasthenia gravis, psoriasis, systemic sclerosis and systemic lupus erythematosus, polymyositis, and primary biliary cirrhosis.

[0163] This disclosure provides a method for treating arthritis in a subject, comprising administering to the subject a therapeutically effective dose (either as monotherapy or in a combination therapy regimen) of the bifunctional antagonist molecule of this disclosure in a pharmaceutically acceptable carrier. In particular, the bifunctional antagonist molecule of this disclosure is useful in treating arthritis selected from rheumatoid arthritis and osteoarthritis.

[0164] This disclosure provides a method for treating anorexia in a subject, comprising administering to the subject a therapeutically effective dose (either as monotherapy or as part of a combination therapy regimen) of the bifunctional antagonist molecule of this disclosure in a pharmaceutically acceptable carrier. In particular, the bifunctional antagonist molecule of this disclosure is useful in treating anorexia selected from anorexia nervosa and anorexia-cachexia syndrome.

[0165] This disclosure provides a method for treating liver disease in a subject, comprising administering to the subject a therapeutically effective dose (either as monotherapy or as part of a combination therapy regimen) of the bifunctional antagonist molecule of this disclosure in a pharmaceutically acceptable carrier. In particular, the bifunctional antagonist molecule of this disclosure is useful in treating liver diseases selected from non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, alcoholic fatty liver disease, cirrhosis, hepatic failure, autoimmune hepatitis, and hepatocellular carcinoma.

[0166] This disclosure provides a method for performing organ or tissue transplantation in a subject, comprising administering to the subject a therapeutically effective dose (as either monotherapy or in a combination therapy regimen) of the bifunctional antagonist molecule of this disclosure in a pharmaceutically acceptable carrier. In particular, the bifunctional antagonist molecule of this disclosure is useful in transplantation procedures selected from organ transplantation of the heart, kidney, liver, lung, pancreas, intestine, and thymus, or tissue transplantation of bone, tendon, cornea, skin, heart valve, nerve, and vein.

[0167] This disclosure provides a method for treating anemia in a subject, comprising administering to the subject a therapeutically effective dose (either as monotherapy or in a combination therapy regimen) of the bifunctional antagonist molecule of this disclosure in a pharmaceutically acceptable carrier. In various embodiments, the anemia is selected from a variety of anemia disorders, including cancer-related anemia, chemotherapy-induced anemia, chronic kidney disease-related anemia, iron deficiency anemia, thalassemia, iron and hemochromatosis, sickle cell disease, aplastic anemia, myelodysplastic syndrome, pancytopenia, and bone marrow failure.

[0168] This disclosure provides a method for treating fibrosis in a subject, comprising administering a therapeutically effective amount of the pharmaceutical composition of the present invention to a subject in need thereof. In one embodiment, the subject is a human subject. In various embodiments, the fibrosis is selected from pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis and cystic fibrosis), hepatic fibrosis (e.g., non-alcoholic steatohepatitis and cirrhosis), airway fibrosis (e.g., asthma), cardiac fibrosis (e.g., myocardial infarction, diastolic dysfunction or heart valve disease), renal fibrosis (e.g., interstitial fibrosis), myelofibrosis, idiopathic retroperitoneal fibrosis, nephrogenic fibrous skin disorders, intestinal fibrosis in inflammatory bowel disease (inducing Crohn's disease), keloids, scleroderma, systemic sclerosis, hand fibrosis (e.g., Dupuytren's contracture), ocular fibrosis, and arthral fibrosis.

[0169] This disclosure provides a method for treating pain in a subject, comprising administering a therapeutically effective amount of the pharmaceutical composition of the present invention to a subject in need thereof. In one embodiment, the subject is a human subject. In various embodiments, the pain is selected from neuropathic pain, inflammatory pain, or cancer pain.

[0170] This disclosure provides a method for treating a bone disease in a subject, comprising administering a therapeutically effective amount of the pharmaceutical composition of the present invention to a subject in need thereof. In one embodiment, the subject is a human subject. In various embodiments, the bone disease is selected from osteomalacia, osteoporosis, osteogenesis imperfecta, fibrodysplasia ossificans progressive, corticosteroid-induced osteopenia, fractures, and bone metastases.

[0171] This disclosure provides 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 may attenuate the loss of muscle mass and / or function in the subject by at least 5%, 10%, at least 25%, at least 50%, at least 75%, or at least 90%. The inhibition of muscle mass and function loss may be evaluated using imaging techniques and physical strength tests. Examples of imaging techniques for muscle mass evaluation include dual-energy X-ray absorptiometry (DEXA), magnetic resonance imaging (MRI), and computed tomography (CT). Examples of muscle function tests include grip strength tests, stair climbing tests, short physical performance batteries (SPPB), and 6-minute walk tests, as well as maximum inspiratory pressure (MIP) and maximum expiratory pressure (MEP) used to measure respiratory muscle strength.

[0172] The "therapeutic dose" refers to the amount of a therapeutic agent administered that alleviates one or more symptoms of the disorder being treated to some degree.

[0173] The effective therapeutic dose is IC 50The dose can first be estimated from the cell culture assay by determining the IC. 50 Formulas can be formulated in animal models to achieve the circulating plasma concentration range, including [specific parameters]. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by HPLC. The exact composition, route of administration, and dosage can be selected by individual physicians, taking into account the conditions of the subject.

[0174] Dosage regimens can be adjusted to provide the optimal desired response (e.g., therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses (multiple, repeated, or maintenance) may be administered over time, and the dose may be proportionally reduced or increased as indicated by the urgency of the therapeutic situation. Formulating parenteral compositions in dosing unit forms is particularly advantageous for ease of administration and uniformity of dosage. When used herein, dosing unit forms refer to physically separate units suitable as unit doses for the mammalian subject being treated; each unit contains a predetermined amount of the active compound calculated to produce the desired therapeutic effect in relation to the required pharmaceutical carrier. The specifications for dosing unit forms in this disclosure are primarily governed by the unique characteristics of the antibody and the specific therapeutic or prophylactic effect to be achieved.

[0175] Therefore, those skilled in the art will understand, based on the disclosures provided herein, that doses and administration regimens are adjusted according to methods well known in the therapeutic field. That is, the maximum acceptable dose can be readily established, the effective dose that provides a detectable therapeutic benefit to the subject can also be determined, and the time requirements for administering each agent to provide a detectable therapeutic benefit to the subject can be similarly determined. Thus, while certain doses and administration regimens are illustrated herein, these examples are by no means limited to the doses and administration regimens that may be provided to the subject in practice of this disclosure.

[0176] It should be noted that the dosage values ​​may vary with the type and severity of the condition to be alleviated, and may include one or more doses. It should be further understood that for any particular subject, a specific dosage regimen should be adjusted over time according to the individual needs and the professional judgment of the person administering or supervising the administration of the composition, and that the dosage ranges described herein are illustrative and not intended to limit the scope or implementation of the claimed composition. Furthermore, dosage regimens using the compositions of this disclosure may be based on a variety of factors, including the type of disease, the subject's age, weight, sex, medical condition, severity of the condition, route of administration, and the specific antibody used. Therefore, dosage regimens can vary widely but can be routinely determined using standard methods. For example, doses may be adjusted based on pharmacokinetic or pharmacodynamic parameters, which may include clinical effects, e.g., toxic effects and / or experimental values. Therefore, this disclosure includes dose escalation within a subject as determined by those skilled in the art. The determination of appropriate dosages and regimens is well known in the relevant art and will be understood to be attainable by those skilled in the art once the teachings disclosed herein are provided.

[0177] An exemplary, non-limiting daily dosing range for therapeutic or prophylactic effective doses of the bifunctional antagonist molecules of this disclosure is 0.001–100 mg / kg, 0.001–90 mg / kg, 0.001–80 mg / kg, 0.001–70 mg / kg, 0.001–60 mg / kg, 0.001–50 mg / kg, 0.001–40 mg / kg, 0 .001~30mg / kg, 0.001~20mg / kg, 0.001~10mg / kg, 0.001~5mg / kg, 0.001~4mg / kg, 0.001~3mg / kg, 0.001~2mg / kg, 0.001~1mg / kg, 0.010~50mg / kg, 0.010~40mg / kg, 0.010~30mg / kg, 0.010~2 0mg / kg, 0.010~10mg / kg, 0.010~5mg / kg, 0.010~4mg / kg, 0.010~3mg / kg, 0.010~2mg / kg, 0.01 0~1mg / kg, 0.1~50mg / kg, 0.1~40mg / kg, 0.1~30mg / kg, 0.1~20mg / kg, 0.1~10mg / kg, 0.1~5mg / k The dosage may be g, 0.1-4 mg / kg, 0.1-3 mg / kg, 0.1-2 mg / kg, 0.1-1 mg / kg, 1-50 mg / kg, 1-40 mg / kg, 1-30 mg / kg, 1-20 mg / kg, 1-10 mg / kg, 1-5 mg / kg, 1-4 mg / kg, 1-3 mg / kg, 1-2 mg / kg, or 1-1 mg / kg body weight. It should be noted that the dosage values ​​may vary with the type and severity of the condition to be alleviated. It should be further understood that for any particular subject, a specific dosage regimen should be adjusted over time according to the individual needs and the professional judgment of the person administering or supervising the administration of the composition, and that the dosage ranges described herein are illustrative only and are not intended to limit the scope or implementation of the claimed composition.

[0178] In various embodiments, the total dose administered achieves plasma antibody concentrations within the following ranges, for example: approximately 1-1000 μg / ml, approximately 1-750 μg / ml, approximately 1-500 μg / ml, approximately 1-250 μg / ml, approximately 10-1000 μg / ml, approximately 10-750 μg / ml, approximately 10-500 μg / ml, approximately 10-250 μg / ml, approximately 20-1000 μg / ml, approximately 20-750 μg / ml, approximately 20-500 μg / ml, approximately 20-250 μg / ml, approximately 30-1000 μg / ml, approximately 30-750 μg / ml, approximately 30-500 μg / ml, and approximately 30-250 μg / ml.

[0179] The toxicity and therapeutic index of the pharmaceutical compositions disclosed herein are, for example, LD50. 50 (A lethal dose for 50% of the population) and ED 50 The therapeutically effective dose (the dose that is therapeutically effective in 50% of the population) can be determined by standard medical procedures in cell cultures or experimental animals. The dose ratio between the toxic dose and the therapeutically effective dose is the therapeutic index, and the therapeutic index is the ratio LD50. 50 / ED 50 It can be expressed as follows. Compositions exhibiting a large therapeutic index are generally preferred.

[0180] The frequency of administration of a pharmaceutical composition of a bifunctional antagonist molecule depends on the nature of the therapy and the specific disease being treated. The subject may be treated at regular intervals, for example, weekly or monthly, until the desired therapeutic outcome is achieved. Exemplary dosing frequencies include, but are not limited to, once a week without breaks; once a week every other week; once every two weeks; once every three weeks; once a week for two weeks without breaks, then monthly; once a week for three weeks without breaks, then monthly; monthly; once every two months; once every three months; once every four months; once every five months; or once every six months; or annually.

[0181] Combination therapy When used herein, the terms “combined administration,” “administered in combination,” and “in combination with” referring to the bifunctional antagonist molecules and one or more other therapeutic agents of the Disclosure are intended to mean, refer to, and include: simultaneous administration of such combination of the bifunctional antagonist molecules and therapeutic agents of the Disclosure to a subject in need of treatment, wherein the components are formulated together to constitute a single dosage form that releases the components to the subject at substantially the same time; or substantially simultaneous administration of such combination of the bifunctional antagonist molecules and therapeutic agents of the Disclosure to a subject in need of treatment, wherein the components are formulated separately from each other to constitute separate dosage forms that are ingested by the subject at substantially the same time, and the ingested components release the components to the subject at substantially the same time. In cases where; a sequential administration of such combination of the bifunctional antagonist molecule and therapeutic agent of the present disclosure to a subject requiring treatment, wherein such components are formulated separately from each other to constitute separate dosing forms ingested by the subject over a continuous period of time with a significant time interval between each administration, and the components are released to the subject at substantially different times in the ingestion; and a sequential administration of such combination of the bifunctional antagonist molecule and therapeutic agent of the present disclosure to a subject requiring treatment, wherein such components are formulated together to constitute a single dosing form that releases the components in a controlled manner, and in the release they are released to the subject in parallel, sequentially, and / or overlapping at the same and / or different times, and each part may be administered by the same or different routes.

[0182] In another embodiment, the present disclosure relates to a method for treating a muscle-wasting disease in a subject, comprising the administration of a therapeutically effective amount of a bifunctional antagonist molecule of the present disclosure; and b) a combination of the second agent. This combination therapy may be particularly effective for muscle-wasting diseases that are resistant or refractory to treatment using the second agent alone. In various embodiments, the second agent is selected from growth hormone, ghrelin, IGF1, inflammatory cytokines such as TNF-alpha and TNF-alpha, IL-6, IL-1 and antagonists to their receptors, and myostatin and activin A and other antagonists to their receptors.

[0183] In various embodiments, combination therapy involves simultaneously administering a composition of a bifunctional antagonist molecule and a composition of a second agent, either in the same pharmaceutical composition or in separate pharmaceutical compositions. In various embodiments, the composition of the bifunctional antagonist molecule and the composition of the second agent are administered sequentially, i.e., the composition of the bifunctional antagonist molecule is administered either before or after the administration of the composition of the second agent.

[0184] In various embodiments, the administration of the bifunctional antagonist molecule composition and the second agent composition occurs simultaneously; that is, the administration periods of the bifunctional antagonist molecule composition and the second agent composition overlap with each other.

[0185] In various embodiments, the administration of the composition of the bifunctional antagonist molecule and the composition of the second agent are asynchronous. For example, in various embodiments, the administration of the composition of the bifunctional antagonist molecule is completed before the administration of the composition of the second agent.

[0186] The following embodiments are provided for the purpose of better illustrating the present disclosure, but are not intended to limit its scope. [Examples]

[0187] Example 1 The bifunctional antagonist molecules of this disclosure can be prepared according to recombinant DNA techniques well known to those skilled in the art. The preparation of the bifunctional antagonist molecules is generally described in these examples.

[0188] cDNAs encoding various novel bifunctional antagonist polypeptides were generated via gene synthesis and subcloned into mammalian expression plasmids. CHO cells were transiently or stably transfected with mammalian expression plasmids encoding individual bifunctional polypeptide antagonists. Transiently transfected CHO cells or stably transfected CHO pools were grown for 6–8 days in high-density suspension cultures in a 32°C CO2 shaking incubator. The culture medium was collected after passing through a 0.22 μm filter unit (Millipore Corporation, MA). Recombinantly expressed bifunctional polypeptides were purified from the culture medium via protein A affinity chromatography using an AKTA PFLC system (GE Healthcare).

[0189] Example 2 In this example, the human ligand-binding activity of several bifunctional antagonist molecules was measured by biolayer interferometry (BLI) using Octet RED96 (ForteBIO, Pall Corporation, USA). Binding analysis was performed by capturing the bifunctional polypeptide antagonists on a biosensor. To measure the rates of association and dissociation, the biosensor containing the captured bifunctional antagonists was immersed for 10 minutes in wells containing different concentrations of ligands (e.g., TNF-α, activin A, activin B, activin AB, myostatin, and GDF-11) diluted in 1x kinetic buffer, followed by 10–20 minutes in 1x kinetic buffer. The sensor containing the captured bifunctional polypeptide antagonists was also immersed in a well containing 1x kinetic buffer to allow for single reference subtraction to compensate for the innate dissociation of the captured bifunctional antagonists. Binding sensorgrams were collected for the biosensor using an 8-channel detection mode. Data was acquired and analyzed using ForteBIO data acquisition software v11.1 (ForteBIO, Pall Corporation, USA).

[0190] As shown in Table 7, binding data indicate that A101, A102, A103, A105, A109, A110, A305, and A711 have the ability to bind with high affinity to both TNF-α and activin A. TIFF0007857306000009.tif123170

[0191] The ligand-binding affinity of A785, a representative bifunctional antagonist of the present invention designed in the form of a bispecific antibody, was investigated using BLI analysis. As shown in Table 8, A785 can bind to both TNF-α and activin A with high affinity. TIFF0007857306000010.tif34170

[0192] Example 3 In this example, the neutralizing activity of the bifunctional antagonist was investigated using cell-based NF-κB and Smad2 / 3 reporter assays capable of sensing TNF-α and activin signaling.

[0193] Smad2 / 3 signaling assay. Using a modified luciferase reporter cell line, C2C12-CAGA-luc, capable of sensing Smad2 / 3 signaling, activin A, activin B, activin AB, myostatin, and GDF-11 signaling activity was measured in cell cultures. To measure the neutralizing activity of bifunctional antagonists, 2 nM human ligands (activin A, activin B, activin AB, myostatin, or GDF-11) were pre-incubated at room temperature for 1 hour with increasing concentrations of each bifunctional antagonist (0.00004 nM, 0.0004 nM, 0.004 nM, 0.04 nM, 0.4 nM, 4 nM, 40 nM, and 400 nM). The reaction mixture was then added to C2C12-CAGA-luc cell cultures. Luciferase activity of C2C12-CAGA-luc reporter cultures was measured using LuminoSkan Ascent (Thermo Scientific) after 5 hours of incubation in a 37°C CO2 incubator. IC was measured using Prism software (GraphPad Software). 50 The values ​​were analyzed and plotted.

[0194] NF-κB signaling assay. To measure TNF-α neutralizing activity, the IC50 values ​​of each bifunctional antagonist blocking TNF-α-mediated NF-κB signaling were quantified using the K536-NF-κB-luc stable, transfected luciferase reporter cell line capable of sensing 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 at room temperature for 1 hour with increasing concentrations of individual bifunctional antagonists at 0.00001 nM, 0.0001 nM, 0.001 nM, 0.01 nM, 0.1 nM, 1.0 nM, 10 nM, and 100 nM. The reaction mixture was then added to the K536-NF-κB-luc reporter cell culture. Luciferase activity of K536-NF-κB-luc reporter cultures was measured using Luminoskan Ascent (Thermo Scientific) after 5 hours of incubation at 37°C in a CO2 incubator. Cell-based TNF-α neutralization IC50 50 The values ​​were calculated and plotted using Prism software (GraphPad Software).

[0195] Exemplary data on the cell-based neutralizing activity of novel bifunctional antagonist molecules A109, A110, A305, and A711 against activin A and activin A-related ligands are shown in Figures 4 and 5. A109, A110, A305, and A711 potently inhibited TNF-α-mediated NF-κB signaling at a concentration of 2 nM with IC50 values ​​ranging from 0.1 nM to 0.36 nM against TNF-α (see Figure 4, upper panel). In parallel, A109, A110, A305, and A711 also potently inhibited activin A-mediated Smad2 / 3 signaling at a concentration of 2 nM with IC50 values ​​ranging from 2.0 to 4.5 nM against activin A (see Figure 4, lower panel). As shown in Figure 4, A109 and A110 also strongly neutralized ligands functionally related to activin A, activin B, GDF-8, and GDF11.

[0196] Example 4 Pulmonary artery hypertension (PAH) is characterized by increased extracellular matrix deposition, in addition to thickening of the pulmonary artery wall due to proliferation and hypertrophy of pulmonary artery smooth muscle cells. TNF-α and activin A have been shown to be significantly elevated in the lung tissue of patients with PAH and in animal models of PAH. Inhibition of either TNF-α or activin A has been shown to attenuate pulmonary hypertension. Therefore, both TNF-α and activin A are linked to the pathogenesis of PAH.

[0197] In this example, the ability of A109, a bifunctional antagonist of TNF-α and activin A, to affect pulmonary artery smooth muscle cell remodeling was investigated using human primary pulmonary artery smooth muscle cell cultures in the presence of TNF-α and activin A, compared with that of anti-TNF antibody and ActRIIA-Fc.

[0198] Human primary pulmonary artery smooth muscle cells (ATCCs) were grown in vascular smooth muscle medium supplemented with 10% FBS in a 37°C CO2 incubator. Once 80% confluence was reached, the cells were replated into medium supplemented with 0.2% FBS and incubated under different conditions with or without the addition of various agents, as follows: 1) none, 2) TNF-α, 3) activin A, 4) TNF-α + activin A, 5) TNF-α + activin A + anti-TNF antibody, 6) TNF-α + activin A + ActRIIA-Fc, and 7) TNF-α + activin A + A119. After 72 hours of incubation, cell cultures were photographed using an inverted microscope coupled with a digital camera, and morphometric analysis was performed using ImageJ software.

[0199] Figures 6 and 7 show the effects of TNF-α, activin A, and combinations of TNF-α and activin A on cell proliferation and cell size in human primary pulmonary artery smooth muscle cells (PASMCs). Furthermore, the figures also show the effects of anti-TNF antibodies, ActRIIA-Fc and A109, respectively, on PASMC proliferation and size in the context of exogenously added TNF-α and activin A. The data demonstrate that the addition of TNF-α to the culture resulted in PASMC hyperplasia due to accelerated cell proliferation (Figure 6, Panel B), the addition of activin A to the culture led to PASMC hypertrophy due to an increase in cell size (Figure 6, Panel C), and the addition of TNF-α and activin A in combination to the culture resulted in both PASMC hyperplasia and hypertrophy due to parallel increases in cell proliferation and cell size (Figure 6, Panel D). Therefore, combined exposure to elevated TNF-α and activin A led to more extensive pathological remodeling of PASMCs characterized by both hyperplasia and hypertrophy. Furthermore, in the context of elevated TNF-α and activin A, inhibition of TNF-α with anti-TNF antibodies prevented hyperplasia but not hypertrophy in PASMCs (Figure 6, Panel E), inhibition of activin A with ActRIIA-Fc attenuated hypertrophy but not hyperplasia in PASMCs (Figure 6, Panel F), and parallel inhibition of TNF-α and activin A with A109 prevented both hyperplasia and hypertrophy in PASMCs (Figure 6, Panel G). Morphometric analyses of cell number (Figure 7, Panel A) and cell size (Figure 7, Panel B) demonstrated that only A109 could completely prevent both hyperplasia and hypertrophy in PASMC cultures. Therefore, A109 was more effective than anti-TNF antibodies or ActRIIA-Fc in preventing pathological remodeling of PASMCs. The superior ability of A109 to prevent both hyperplasia and hypertrophy of PASMCs suggests that the novel bifunctional antagonists of TNF-α and activin disclosed herein represent a promising new therapeutic approach for treating PAH.

[0200] Example 5 Fibrosis, a pathological process characterized by the functional replacement of fibrous tissue, can occur in virtually any type of tissue or organ and lead to a variety of fibrous diseases. As a major fibrous state, idiopathic pulmonary fibrosis (IPF) involves extensive damage to lung tissue due to persistent inflammation and excessive collagen fibril production and deposition in the extracellular matrix. Expression levels of TNF-α, as well as activins including activin A and activin B, have been shown to be upregulated in the lung tissue of patients with IPF and in animal models of pulmonary fibrosis. Elevated TNF-α mediates pneumonia and also plays a role in the transition from pneumonia to fibrosis, while elevated activin A and activin B can directly promote pulmonary fibrosis by stimulating the overproduction of collagen fibrils.

[0201] In this example, the ability to combat fibrosis in a mouse model of bleomycin-induced pulmonary fibrosis was evaluated by comparing A109, a bifunctional antagonist of TNF-α and activin, with that of an anti-TNF antibody and ActRIIA-Fc, respectively.

[0202] A mouse model study of bleomycin-induced pulmonary fibrosis. All animal-involved procedures were approved by the Animal Experimentation Committee. Eight-week-old male C57BL / 6 mice were purchased from Jackson Laboratories. Mice were maintained in a 12-hour light / dark cycle and given free access to water and laboratory rodent feed. Mice were acclimatized for one week before treatment. Bleomycin (Sigma) was dissolved in sterile 0.9% saline and administered as a single dose of 0.5 mg / kg per animal. Control animals were given saline alone. All animals received intratracheal injection (IT) of either bleomycin or saline on day 0. Mice were randomly assigned to one of the following groups: (1) IT saline (control); (2) IT bleomycin (bleomycin); (3) IT bleomycin with ActRIIA-Fc treatment (bleomycin + ActRIIA-Fc); (4) IT bleomycin with TNF antibody treatment (bleomycin + TNF Ab); (5) IT bleomycin with A109 treatment (bleomycin + A109). Starting on day 2, ActRIIA-Fc, anti-TNF, and A-109 were administered to each mouse group once a week via SC administration at doses of 5-10 mg / kg normalized by the molecular weight of each agent. After 2 weeks of treatment, animals were dissected, right lung tissue was collected in cassettes and fixed in neutral buffered formalin. For histological evaluation, samples were dehydrated in graded ethanol series, clarified in xylene, and embedded in paraffin. Sections 4–6 μm thick were cut and stained with hematoxylin and eosin (H&E), Masson's trichrome (TM), and HRP-linked anti-alpha-SMA antibodies. Fibrous lung injury was histologically evaluated using the Ashcroft scoring system (Hubner et al. 2008. PMID:18476815; DOI:10.2144 / 000112729). Ashcroft scores were analyzed at 10X magnification.

[0203] Figure 8 shows histological images of H&E-stained lung sections and Ashcraft scores for lung sections from control and bleomycin-inducible animals with and without treatment. Data indicate that treatment with A109 significantly reduced bleomycin-induced pulmonary fibrosis, while treatment with TNF antibody or ActRIIA-Fc moderately inhibited bleomycin-induced pulmonary fibrosis. Analysis of Ashcraft scores revealed that A109 treatment resulted in a greater reduction in pulmonary fibrosis compared to TNF antibody treatment or ActRIIA-Fc treatment.

[0204] Figure 9 depicts histological images and quantitative analysis of collagen deposition area of ​​Masson trichrome-stained lung sections from control and bleomycin-treated mice. The data indicate that A109 reduced bleomycin-induced collagen deposition more effectively than anti-TNF antibodies or ActRIIA-Fc.

[0205] Figure 10 shows immunochemical staining of alpha-smooth muscle actin (αSMA), a marker of fibrosis, in lung sections from control and bleomycin-treated mice. The data indicate that the bifunctional molecule A109 more effectively attenuated the induction of αSMA immunoreactivity in bleomycin-treated mice compared to anti-TNF antibody or ActRIIA-Fc.

[0206] In summary, data from bleomycin-induced pulmonary fibrosis mice demonstrate that A109, a bifunctional antagonist capable of simultaneously neutralizing TNF-α and activin, is more effective than anti-TNF antibodies or the activin-neutralizing protein, ActRIIA-Fc, in combating fibrosis. The enhanced anti-fibrotic effect of A109 suggests that the novel bifunctional antagonist of TNF and activin presented in this invention represents a promising new approach for treating fibrotic diseases.

[0207] Example 6 TNF-α and activin A have been shown to play important roles in regulating bone remodeling. In addition to TNF-α, activin A expression levels are upregulated in bone damage, and elevated levels of both TNF-α and activin A are linked to the pathogenesis of osteopenia. In this example, the effects of A109, a novel bifunctional antagonist of activin A and TNF-α, on bone were investigated in mice.

[0208] Experimental Methods. Ten-week-old female CD1 mice were purchased from Envigo. The mice were randomly assigned to two groups (n=6). The treatment group received subcutaneous A109 at 30 mg / kg once a week, while the control group received subcutaneous PBS. After 4 weeks of treatment, the animals were euthanized by carbon dioxide inhalation, and the right femur was dissected for microCT imaging. Skyscan's Skyscan 1172 microCT Instrument and CTrecon and CTan software were used for bone imaging analysis. Briefly, the femur was placed in a styrofoam mold mounted on the machine's rotating stage. The bone was scanned, and the image acquisitions were reconstructed for analysis. 3D and 2D microCT imaging analyses were performed on the trabecular and cortical bones, respectively, to obtain parameters related to bone volume and density.

[0209] Figure 11 shows representative microCT images of trabecular bone volume. The data suggest that A109 administration resulted in increased bone mass in normal mice. Quantitative analysis of microCT data (Figure 12) further revealed that A109 administration significantly increased bone volume and density in CD1 mice.

[0210] These results demonstrate that A109, a novel bifunctional antagonist of activin A and TNF-α, has the ability to enhance bone mass in mice.

[0211] All articles and methods disclosed and claimed in this application can be manufactured and performed without excessive experimentation in light of the present invention. Although the articles and methods of the present invention are described in terms of preferred embodiments, it will be apparent to those skilled in the art that variations can be applied to the articles and methods without deviating from the spirit and scope of the present invention. All such variations and equivalents, whether existing or subsequently developed, which are apparent to those skilled in the art, are considered to be within the spirit and scope of the present invention as defined by the appended claims. All patents, patent applications, and publications referenced herein indicate the level of those skilled in the art in the field relating to the present invention. All patents, patent applications, and publications are incorporated herein by reference in whole for all purposes, and to the same extent that each individual publication is specifically and individually indicated to be incorporated by reference in whole for any and all purposes. The present invention, preferably described exemplary herein, may be carried out in the absence of any elements not specifically disclosed herein. Therefore, although the present invention is particularly disclosed by preferred embodiments and optional features, it should be understood that modifications and variations of the concepts disclosed herein may be taken by those skilled in the art, and that such modifications and variations are considered to fall within the scope of the present invention as defined by the appended claims.

[0212] Sequence List The nucleic acid and amino acid sequences listed in the attached sequence listing are shown using standard abbreviations for nucleotide bases and three-letter codes for amino acids, as defined in 37 CFR 1.822. Sequence IDs 1-5 are amino acid sequences of various TNF ligands. Sequence IDs 6-14 are amino acid sequences of various activin-related ligands. Sequence IDs 15, 19, 23, and 27 are the amino acid sequences of the heavy chains of various antibodies that specifically bind to TNF-α ligands. Sequence IDs 16, 20, 24, and 28 are amino acid sequences of the heavy chain variable regions of various antibodies that specifically bind to TNF-α ligands. Sequence IDs 17, 21, 25, and 29 are the amino acid sequences of the light chains of various antibodies that specifically bind to TNF-α ligands. Sequence IDs 18, 22, 26, and 30 are amino acid sequences of the light chain variable regions of various antibodies that specifically bind to TNF-α ligands. Sequence IDs 31 and 35 are amino acid sequences of the heavy chain of antibodies that specifically bind to activin-related ligands. Sequence IDs 32 and 36 are amino acid sequences of the heavy chain variable region of antibodies that specifically bind to activin-related ligands. Sequence IDs 33 and 37 are amino acid sequences of the light chains of antibodies that specifically bind to activin-related ligands. Sequence IDs 34 and 38 are amino acid sequences of the light chain variable region of antibodies that specifically bind to activin-related ligands. Sequence IDs 39-47 are the amino acid sequences of the heavy chains of bifunctional antagonist molecules that specifically bind to TNF-α ligands and activin or activin-related ligands. Sequence IDs 48-56 are amino acid sequences of various bifunctional antagonist molecules that specifically bind to TNF-α ligands and specifically bind to activin or activin-related ligands. Sequence IDs 57 and 58 are the amino acid sequences of the heavy chain variable region of an antibody that specifically binds to activin A ligand. Sequence IDs 59-67 are amino acid sequences of various bifunctional antagonist molecules that specifically bind to TNF-α ligands and specifically bind to activin or activin-related ligands. Sequence IDs 68 and 69 are amino acid sequences of the heavy chain variable region of antibodies that specifically bind to activin A ligand. Sequence IDs 70 and 72 are the amino acid sequences of the heavy chains of bifunctional antagonist molecules that specifically bind to TNF-α ligand and activin or activin-related ligands. Sequence IDs 71 and 73 are the amino acid sequences of the heavy chains of bifunctional antagonist molecules that specifically bind to TNF-α ligand and activin or activin-related ligands. Sequence IDs 74-93 are amino acid sequences of various peptide linker sequences. Sequence List Human TNFR1 ECD IYPSGVIGLVPHLGDREKRDSVCPQGKYIHPQNNSICCTKCHKGTYLYNDCPGPGQDTDCRECESGSFTASENHLRHCLSCSKCRKEMGQVEISSCTVDRDTVCGCRKNQYRHYWSENLFQCFNCSLCLNGTVHLSCQEKQNTVCTCHAGFFLRENECVSCSNCKKSLECTKLCLPQIENVKGTEDSGTT(Sequence No. 1) Human TNFR2 ECD LPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDTVCDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCALSKQEGCRLCAPLRKCRPGFGVARPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAIPGNASMDAVCTSTSPTRSMAPGAVHLPQPVSTRSQHTQPTPEPSTAPSTSFLLPMGPSPPAEGSTGD (Sequence ID 2) Human TNFR1 / CRD1-TNFR2 / CRD2 / 3 / 4 IYPSGVIGLVPHLGDREKRDSVCPQGKYIHPQNNSICCTKCHKGTYLYNDCPGPGQDTDCRSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCALSKQEGCRLCAPLRKCRPGFGVARPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAIPGNASMDAVCTSTSPTRSMAPGAVHLPQPVSTRSQHTQPTPEPSTAPSTSFLLPMGPSPPAEGSTGD (Sequence ID 3) Human TNFR1 / CRD1 / 2 / 3-TNFR2 / CRD4 IYPSGVIGLVPHLGDREKRDSVCPQGKYIHPQNNSICCTKCHKGTYLYNDCPGPGQDTDCRECESGSFTASENHLRHCLSCSKCRKEMGQVEISSCTVDRDTVCGCRKNQYRHYWSENLFQCFNCSLCLNGTVHLSCQEKQNTVCPCAPGTFSNTTSSTDICRPHQICNVVAIPGNASMDAVCTSTSPTRSMAPGAVHLPQPVSTRSQHTQPTPEPSTAPSTSFLLPMGPSPPAEGSTGD (SEQ ID NO: 4) TNFR1 / ΔCRD4 IYPSGVIGLVPHLGDREKRDSVCPQGKYIHPQNNSICCTKCHKGTYLYNDCPGPGQDTDCRECESGSFTASENHLRHCLSCSKCRKEMGQVEISSCTVDRDTVCGCRKNQYRHYWSENLFQCFNCSLCLNGTVHLSCQEKQNTVC (SEQ ID NO: 5) Human ActRIIA-ECD ETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNPVTPKPP (SEQ ID NO: 6) Human ActRIIB-ECD ETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPT (SEQ ID NO: 7) Human follistatin 315 GNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEAACSSGVLLEVKHSGSCNSISEDTEEEEEDEDQDYSFPISSILEW (Sequence ID 8) Human follistatin ΔHBS (modified follistatin) GNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGQSCVVDQTGSPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEAACSSGVLLEVKHSGSCNSISEDTEEEEEDEDQDYSFPISSILEW (Sequence ID 9) Human follistatin 288 GNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEAACSSGVLLEVKHSGSCN (Sequence ID 10) Modified Human ActRIIB ECD ETRECIYYNANWELERTNQSGLERCEGDQDKRLHCYASWRNSSGTIELVKKGCWLDDINCYDRQECVATKENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPT(Sequence ID 11) Modified Human ActRIIB ECD ETRECIYYNANWELERTNQSGLERCYGDKDKRRHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPT(Sequence ID 12) Modified Human ActRIIB ECD ETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWDDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPT(Sequence ID 13) Modified Human ActRIIA ECD GAILGRSETQECLFYNANWELERTNQTGVEPCEGEKDKRLHCYATWRNISGSIEIVKKGCWLDDFNCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS(Sequence ID 14) Anti-TNF-α antibody heavy chain amino acid sequence EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNVYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 15) Amino acid sequence of the variable region of the heavy chain of anti-TNF-α antibody EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSS(Sequence ID 16) Light chain amino acid sequence of anti-TNF-α antibody DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 17) Amino acid sequence of the variable region of the light chain of anti-TNF-α antibody DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIK(Sequence ID 18) Anti-TNF-α antibody heavy chain amino acid sequence EVKLEESGGGLVQPGGSMKLSCVASGFIFSNHWMNWVRQSPEKGLEWVAEIRSKSINSATHYAESVKGRFTISRDDSKSAVYLQMTDLRTEDTGVYYCSRNYYGSTYDYWGQGT TLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence ID 19) Amino acid sequence of the variable region of the heavy chain of anti-TNF-α antibody EVKLEESGGGLVQPGGSMKLSCVASGFIFSNHWMNWVRQSPEKGLEWVAEIRSKSINSATHYAESVKGRFTISRDDSKSAVYLQMTDLRTEDTGVYYCSRNYYGSTYDYWGQGTTLTVSS(Sequence ID 20) Light chain amino acid sequence of anti-TNF-α antibody DILLTQSPAILSVSPGERVSFSCRASQFVGSSIHWYQQRTNGSPRLLIKYASESMSGIPSRFSGSGSGTDFTLSINTVESEDIADYYCQQSHSWPFTFGSGTNLEVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 21) Amino acid sequence of the variable region of the light chain of anti-TNF-α antibody DILLTQSPAILSVSPGERVSFSCRASQFVGSSIHWYQQRTNGSPRLLIKYASESMSGIPSRFSGSGSGTDFTLSINTVESEDIADYYCQQSHSWPFTFGSGTNLEVK (SEQ ID NO: 22) Anti-TNF-α antibody heavy chain amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGYVFTDYGMNWVRQAPGKGLEWMGWINTYIGEPIYADSVKGRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCARGYRSYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCAA (SEQ ID NO: 23) Amino acid sequence of the variable region of the heavy chain of anti-TNF-α antibody EVQLVESGGGLVQPGGSLRLSCAASGYVFTDYGMNWVRQAPGKGLEWMGWINTYIGEPIYADSVKGRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCARGYRSYAMDYWGQGTLVTVSS (SEQ ID NO: 24) Light chain amino acid sequence of anti-TNF-α antibody DIQMTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKALIYSASFLYSGVPYRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNIYPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 25) Amino acid sequence of the variable region of the light chain of anti-TNF-α antibody DIQMTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKALIYSASFLYSGVPYRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNIYPLTFGQGTKVEIK (Sequence ID 26) Anti-TNF-α antibody heavy chain amino acid sequence QVQLVESGGGVVQPGRSLRLSCAASGFIFSSYAMHWVRQAPGNGLEWVAFMSYDGSNKKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRGIAAGGNYYYYGMDVW GQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 27) Amino acid sequence of the variable region of the heavy chain of anti-TNF-α antibody QVQLVESGGGVVQPGRSLRLSCAASGFIFSSYAMHWVRQAPGNGLEWVAFMSYDGSNKKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRGIAAGGNYYYYGMDVWGQGTTVTVSS(Sequence ID 28) Light chain amino acid sequence of anti-TNF-α antibody EIVLTQSPATLSLSPGERATLSCRASQSVYSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 29) Amino acid sequence of the variable region of the light chain of anti-TNF-α antibody EIVLTQSPATLSLSPGERATLSCRASQSVYSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPFTFGPGTKVDIK(Sequence ID 30) Anti-activin antibody heavy chain amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGLSWVRQAPGQGLEWMGWIIPYNGNTNSAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYFCARDRDYGVNYDAFDIWGQG TMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNVYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 31) Amino acid sequence of the variable region of the heavy chain of anti-activin A antibody QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGLSWVRQAPGQGLEWMGWIIPYNGNTNSAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYFCARDRDYGVNYDAFDIWGQGTMVTVSS(Sequence ID 32) Light chain amino acid sequence of anti-activin A antibody SYEVTQAPSVSVSPGQTASITCSGDKLGDKYACWYQQKPGQSPVLVIYQDSKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQAWDSSTAVFGGGTKLTVLRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 33) Amino acid sequence of the variable region of the light chain of anti-activin A antibody SYEVTQAPSVSVSPGQTASITCSGDKLGDKYACWYQQKPGQSPVLVIYQDSKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQAWDSSTAVFGGGTKLTVL(Sequence ID 34) Anti-activin A antibody heavy chain amino acid sequence QVQLQESGPGLVKPSETLSLTTCTVSGGSFSSHFWSWIRQPPGKGLEWIGYILYTGGTSFNPSLKSRVSMSVGTSKNQFSLKLSSVTAADTAVYYCARARSGITFTGIIVPGSFDI WGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGP PCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (Sequence ID 35) Amino acid sequence of the variable region of the heavy chain of anti-activin A antibody QVQLQESGPGLVKPSETLSLTCTVSGGSFSSHFWSWIRQPPGKGLEWIGYILYTGGTSFNPSLKSRVSMSVGTSKNQFSLKLSSVTAADTAVYYCARARSGITFTGIIVPGSFDIWGQGTMVTVSS (Sequence ID 36) Light chain amino acid sequence of anti-activin A antibody EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 37) Amino acid sequence of the variable region of the light chain of anti-activin A antibody EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK (Sequence ID 38) A109 heavy chain amino acid sequence (Sequence ID 39) A201 heavy chain amino acid sequence (Sequence No. 40) A110 heavy chain amino acid sequence (Sequence ID 41) A301 heavy chain amino acid sequence (Sequence ID 42) A304 heavy chain amino acid sequence (Sequence No. 43) A410 heavy chain amino acid sequence (Sequence ID 44) A401 heavy chain amino acid sequence (Sequence No. 45) A404 heavy chain amino acid sequence (Sequence No. 46) A407 heavy chain amino acid sequence (Sequence ID 47) A710 amino acid sequence (Sequence No. 48) A711 amino acid sequence IYPSGVIGLVPHLGDREKRDSVCPQGKYIHPQNNSICCTKCHKGTYLYNDCPGPGQDTDCRECESGSFTASENHLRHCLSCSKCRKEMGQVEISSCTVDRDTVCGCRKNQYRHYWSENLFQCFNCSLCLNGTVHLSCQ EKQNTVCTCHAGFFLRENECVSCSNCKKSLECTKLCLPQIENVKGTEDSGTTGGGGSGGGGSGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEV TYEPPPTAPT (Sequence ID 49) A713 amino acid sequence (Sequence ID 50) A714 amino acid sequence (Sequence ID 51) A715 amino acid sequence (Sequence ID 52) A716 amino acid sequence (Sequence No. 53) A712 amino acid sequence (Sequence No. 54) A717 amino acid sequence (Sequence ID 55) A718 amino acid sequence (Sequence ID 56) A719 heavy chain amino acid sequence (Sequence ID 57) A720 heavy chain amino acid sequence (Sequence ID 58) A211 amino acid sequence (Sequence ID 59) A305 amino acid sequence LPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDTVCDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCALSKQEGCRLCAPLRKCRPGFGVARPGTETSDVVCKPCAPGTFSN TTSSTDICRPHQICNVVAIPGNASMDAVCTSTSPTRSMAPGAVHLPQPVSTRSQHTQPTPEPSTAPSTSFLLPMGPSPPAEGSTGDGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEV TYEPPPTAPT (Sequence ID 60) A213 amino acid sequence (Sequence ID 61) A214 amino acid sequence (Sequence ID 62) A215 amino acid sequence (Sequence No. 63) A216 amino acid sequence (Sequence ID 64) A105 amino acid sequence (Sequence ID 65) A217 amino acid sequence (Sequence ID 66) A218 amino acid sequence (Sequence ID 67) A219 heavy chain amino acid sequence (Sequence No. 68) A220 heavy chain amino acid sequence (Sequence ID 69) A785 heavy chain amino acid sequence (Sequence ID 70) A785 light chain amino acid sequence SYEVTQAPSVSVSPGQTASITCSGDKLGDKYACWYQQKPGQSPVLVIYQDSKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQAWDSSTAVFGGGTKLTVLGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 71) A786 heavy chain amino acid sequence (Sequence ID 72) A786 light chain amino acid sequenceEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 73) Peptide linker sequence GGGSGGGSGGGS (SEQ ID NO: 74) Peptide linker sequence GGGS (SEQ ID NO: 75) Peptide linker sequence GSSGGSGGSGGSG (SEQ ID NO: 76) Peptide linker sequence GSSGT (SEQ ID NO: 77) Peptide linker sequence GGGGSGGGGSGGGS (Sequence ID 78) Peptide linker sequence AEAAAKEAAAKEAAAKA (SEQ ID NO: 79) Peptide linker sequence GGGGSGGGGSGGGGSGGGGS (Sequence 80) Peptide linker sequence GGGSGGGS (SEQ ID NO: 81) Peptide linker sequence GS (SEQ ID NO: 82) Peptide linker sequence GGS (SEQ ID NO: 83) Peptide linker sequence GGGGS (SEQ ID NO: 84) Peptide linker sequence GGSG (SEQ ID NO: 85) Peptide linker sequence SGGG (SEQ ID NO: 86) Peptide linker sequence GSGS (SEQ ID NO: 87) Peptide linker sequence GSGSGS (Sequence ID 88) Peptide linker sequence GSGSGSGS (SEQ ID NO: 89) Peptide linker sequence GSGSGSGSGS (SEQ ID NO: 90) Peptide linker sequence GSGSGSGSGSGS (SEQ ID NO: 91) Peptide linker sequence GGGGSGGGGS (SEQ ID NO: 92) Peptide linker sequence GGGGSGGGGSGGGGS (SEQ ID NO: 93)

Claims

1. An isolated bifunctional antagonist molecule comprising a first antigen-binding molecule that specifically binds to a TNF ligand and a second antigen-binding molecule that specifically binds to activin or an activin-related ligand, The isolated bifunctional antagonist molecule simultaneously neutralizes TNF signaling and activin signaling. The isolated bifunctional antagonist molecule is selected from the group consisting of: a bifunctional molecule having the heavy chain amino acid sequence described in SEQ ID NO: 39 and the light chain amino acid sequence described in SEQ ID NO: 17; a bifunctional molecule having the heavy chain amino acid sequence described in SEQ ID NO: 41 and the light chain amino acid sequence described in SEQ ID NO: 17; a bifunctional molecule having the amino acid sequence described in SEQ ID NO: 60; and a bifunctional molecule having the amino acid sequence described in SEQ ID NO:

49. An isolated, bifunctional antagonist molecule characterized by the following features.

2. A pharmaceutical composition comprising a therapeutically effective amount of the bifunctional antagonist molecule according to claim 1, mixed with a pharmaceutically acceptable carrier.

3. An isolated nucleic acid molecule comprising a polynucleotide encoding the bifunctional antagonist molecule described in claim 1.

4. A recombinant vector comprising the nucleic acid molecule described in claim 3.

5. A host cell comprising the recombinant vector described in claim 4.

6. A method for producing a bifunctional antagonist molecule according to claim 1, comprising: a) transforming host cells with a vector comprising a polynucleotide encoding the bifunctional antagonist molecule; b) culturing the host cells under conditions suitable for the expression of the bifunctional antagonist molecule; and c) recovering the bifunctional antagonist molecule from the culture.

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