Bifunctional antagonists of activin and tumor necrosis factor-alpha and their applications

Claims

1. An isolated bifunctional antagonist molecule comprising a first antigen-binding molecule that specifically binds to a tumor necrosis factor (TNF) ligand and a second antigen-binding molecule that specifically binds to activin or an activin-related ligand, wherein the bifunctional antagonist molecule simultaneously and effectively neutralizes TNF-α-mediated signaling and activin-mediated signaling.

2. The isolated bifunctional antagonist molecule of claim 1, wherein the first antigen-binding molecule specifically binds to a TNF ligand selected from the group consisting of TNF ligands comprising the amino acid sequence set forth in SEQ ID NOs: 1-5, and wherein the second antigen-binding molecule specifically binds to activin or an activin-related ligand selected from the group consisting of activin or activin-related ligands comprising the amino acid sequence set forth in SEQ ID NOs: 6-14.

3. An isolated bifunctional antagonist molecule according to any one of claims 1-2, wherein the first antigen-binding molecule that specifically binds to a TNF ligand ("TNF-binding polypeptide") is selected from the group consisting of an antibody to TNF, a fragment of an antibody to TNF, extracellular domains (ECDs) of wild-type TNFR1 (TNF receptor 1) and TNFR2 (TNF receptor 2), modified extracellular domains of TNFR1 and TNFR2, and a phage-displayed polypeptide that targets a TNF-α ligand;and wherein the second antigen-binding molecule that specifically binds to activin or an activin-related ligand ("activin-binding polypeptide") is selected from the group consisting of an antibody to activin (including an antibody to activin A and an antibody to activin B), a fragment of an antibody to activin, extracellular domains (ECDs) of the wild-type activin receptor type 2A (ActRIIA) or the wild-type activin receptor type 2B (ActRIIB), extracellular domains of modified ActRIIA and ActRIIB, native activin-binding proteins, wild-type and modified, such as follistatin, follistatin-like protein and propeptide, and a phage-displayed polypeptide that targets activin or an activin-related ligand.

4. An isolated bifunctional antagonist molecule according to any one of claims 1-3, wherein the TNF-binding polypeptide is selected from the group of polypeptides comprising the amino acid sequence set forth in SEQ ID NO: 1-5, and the activin-binding polypeptide is selected from the group of polypeptides comprising the amino acid sequence set forth in SEQ ID NO: 6-14.

5. An isolated bifunctional antagonist molecule according to any one of claims 1-3, wherein the TNF-binding polypeptide comprises an isolated TNF antibody or antigen-binding fragment thereof, and wherein the activin-binding polypeptide comprises an isolated activin antibody or antigen-binding fragment thereof.

6. The isolated bifunctional antagonist molecule of claim 5, wherein the isolated anti-TNF antibody or antigen-binding fragment thereof and the isolated anti-activin antibody or antigen-binding fragment thereof are selected from the group consisting of monoclonal Abs (mAbs), polyclonal Abs, Ab fragments (e.g., Fab, Fab', F(ab')2, Fv, Fc, etc.), chimeric Abs, mini-Abs or domain Abs (dAbs), dual-specificity Abs, bispecific Abs, heteroconjugate Abs, single-chain Abs (SCAs), single-chain variable fragments (scFvs), humanized Abs, fully human Abs, and any other modified configuration of an immunoglobulin (Ig) molecule that contains an antigen-recognizing site with the desired specificity.

7. An isolated bifunctional antagonist molecule according to any one of claims 5-6, wherein the isolated antibody or antigen-binding fragment thereof is selected from the group consisting of a fully human, a humanized, and a chimeric antibody.

8. The isolated bifunctional antagonist molecule of any one of claims 5-7, wherein the TNF-binding polypeptide 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: 15; an antibody comprising the light chain amino acid sequence set forth in SEQ ID NO: 17; an antibody comprising the heavy chain amino acid sequence set forth in SEQ ID NO: 15 and the light chain amino acid sequence set forth in SEQ ID NO: 17; an antibody comprising the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 16; an antibody comprising the light chain variable region amino acid sequence set forth in SEQ ID NO: 18;an antibody comprising the amino acid sequence of a heavy chain variable region as set forth in SEQ ID NO: 16 and the amino acid sequence of a light chain variable region as set forth in SEQ ID NO: 18; an antibody comprising the amino acid sequence of a heavy chain as set forth in SEQ ID NO: 19; an antibody comprising the amino acid sequence of a light chain as set forth in SEQ ID NO: 21; an antibody comprising the amino acid sequence of a heavy chain as set forth in SEQ ID NO: 19 and the amino acid sequence of a light chain as set forth in SEQ ID NO: 21; an antibody comprising the amino acid sequence of a heavy chain variable region as set forth in SEQ ID NO: 20; an antibody comprising the amino acid sequence of a light chain variable region as set forth in SEQ ID NO: 22;an antibody comprising the amino acid sequence of a heavy chain variable region as set forth in SEQ ID NO: 20 and the amino acid sequence of a light chain variable region as set forth in SEQ ID NO: 22; an antibody comprising the amino acid sequence of a heavy chain as set forth in SEQ ID NO: 23; an antibody comprising the amino acid sequence of a light chain as set forth in SEQ ID NO: 25; an antibody comprising the amino acid sequence of a heavy chain variable region as set forth in SEQ ID NO: 23 and the amino acid sequence of a light chain as set forth in SEQ ID NO: 25; an antibody comprising the amino acid sequence of a heavy chain variable region as set forth in SEQ ID NO: 24; an antibody comprising the amino acid sequence of a light chain variable region as set forth in SEQ ID NO: 26;an antibody comprising the amino acid sequence of a heavy chain variable region as set forth in SEQ ID NO: 24 and the amino acid sequence of a light chain variable region as set forth in SEQ ID NO: 26; an antibody comprising the amino acid sequence of a heavy chain as set forth in SEQ ID NO: 27; an antibody comprising the amino acid sequence of a light chain as set forth in SEQ ID NO: 29; an antibody comprising the amino acid sequence of a heavy chain as set forth in SEQ ID NO: 27 and the amino acid sequence of a light chain as set forth in SEQ ID NO: 29; an antibody comprising the amino acid sequence of a heavy chain variable region as set forth in SEQ ID NO: 28; an antibody comprising the amino acid sequence of a light chain variable region as set forth in SEQ ID NO: 30;and an antibody comprising the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO: 28 and the amino acid sequence of the light chain variable region set forth in SEQ ID NO: 30.; 9. The isolated bifunctional antagonist molecule of any one of claims 5-8, wherein the activin-binding polypeptide 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;an antibody comprising the amino acid sequence of a heavy chain variable region as set forth in SEQ ID NO: 32 and the amino acid sequence of a light chain variable region as set forth in SEQ ID NO: 34; an antibody comprising the amino acid sequence of a heavy chain as set forth in SEQ ID NO: 35; an antibody comprising the amino acid sequence of a light chain as set forth in SEQ ID NO: 37; an antibody comprising the amino acid sequence of a heavy chain as set forth in SEQ ID NO: 35 and the amino acid sequence of a light chain as set forth in SEQ ID NO: 37; an antibody comprising the amino acid sequence of a heavy chain variable region as set forth in SEQ ID NO: 36; an antibody comprising the amino acid sequence of a light chain variable region as set forth in SEQ ID NO: 38;and an antibody comprising the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO: 36 and the amino acid sequence of the light chain variable region set forth in SEQ ID NO: 38.; 10. The isolated bifunctional antagonist molecule of any one of claims 1-2, wherein the bifunctional molecule is selected from the group consisting of: a bifunctional molecule comprising the heavy chain amino acid sequence set forth in SEQ ID NO: 39 and the light chain amino acid sequence set forth in SEQ ID NO: 17; a bifunctional molecule comprising the heavy chain amino acid sequence set forth in SEQ ID NO: 40 and the light chain amino acid sequence set forth in SEQ ID NO: 17; a bifunctional molecule comprising the heavy chain amino acid sequence set forth in SEQ ID NO: 41 and the light chain amino acid sequence set forth in SEQ ID NO: 17; a bifunctional molecule comprising the heavy chain amino acid sequence set forth in SEQ ID NO: 42 and the light chain amino acid sequence set forth in SEQ ID NO: 17;a bifunctional molecule comprising the heavy chain amino acid sequence set forth in SEQ ID NO: 43 and the light chain amino acid sequence set forth in SEQ ID NO: 17; a bifunctional molecule comprising the heavy chain amino acid sequence set forth in SEQ ID NO: 44 and the light chain amino acid sequence set forth in SEQ ID NO: 17; a bifunctional molecule comprising the heavy chain amino acid sequence set forth in SEQ ID NO: 45 and the light chain amino acid sequence set forth in SEQ ID NO: 17; a bifunctional molecule comprising the heavy chain amino acid sequence set forth in SEQ ID NO: 46 and the light chain amino acid sequence set forth in SEQ ID NO: 17; a bifunctional molecule comprising the heavy chain amino acid sequence set forth in SEQ ID NO: 47 and the light chain amino acid sequence set forth in SEQ ID NO: 17;a bifunctional molecule 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; a bifunctional molecule 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; a bifunctional molecule comprising the heavy chain amino acid sequence set forth in SEQ ID NO: 57 and the light chain amino acid sequence set forth in SEQ ID NO: 33; a bifunctional molecule comprising the heavy chain amino acid sequence set forth in SEQ ID NO: 58 and the light chain amino acid sequence set forth in SEQ ID NO: 37; a bifunctional molecule comprising the heavy chain amino acid sequence set forth in SEQ ID NO: 68 and the light chain amino acid sequence set forth in SEQ ID NO: 33;and a bifunctional molecule comprising the heavy chain amino acid sequence set forth in SEQ ID NO: 69 and the light chain amino acid sequence set forth in SEQ ID NO: 37.; 11. The isolated bifunctional antagonist molecule of any one of claims 1-2, wherein the bifunctional molecule is selected from the group consisting of: a bifunctional molecule comprising the amino acid sequence set forth in SEQ ID NO: 48; a bifunctional molecule comprising the amino acid sequence set forth in SEQ ID NO: 49; a bifunctional molecule comprising the amino acid sequence set forth in SEQ ID NO: 50; a bifunctional molecule comprising the amino acid sequence set forth in SEQ ID NO: 51; a bifunctional molecule comprising the amino acid sequence set forth in SEQ ID NO: 52; a bifunctional molecule comprising the amino acid sequence set forth in SEQ ID NO: 53; a bifunctional molecule comprising the amino acid sequence set forth in SEQ ID NO: 54; a bifunctional molecule comprising the amino acid sequence set forth in SEQ ID NO: 55;a bifunctional molecule comprising the amino acid sequence set forth in SEQ ID NO: 56; a bifunctional molecule comprising the amino acid sequence set forth in SEQ ID NO: 59; a bifunctional molecule comprising the amino acid sequence set forth in SEQ ID NO: 60; a bifunctional molecule comprising the amino acid sequence set forth in SEQ ID NO: 61; a bifunctional molecule comprising the amino acid sequence set forth in SEQ ID NO: 62; a bifunctional molecule comprising the amino acid sequence set forth in SEQ ID NO: 63; a bifunctional molecule comprising the amino acid sequence set forth in SEQ ID NO: 64; a bifunctional molecule comprising the amino acid sequence set forth in SEQ ID NO: 65; a bifunctional molecule comprising the amino acid sequence set forth in SEQ ID NO: 66;and a bifunctional molecule comprising the amino acid sequence set forth in SEQ ID NO: 67.; 12. A pharmaceutical composition comprising a therapeutically effective amount of a bifunctional antagonist molecule according to any one of claims 1-11 in a mixture with a pharmaceutically acceptable carrier.

13. A method for treating or preventing a disease state in the pathogenesis of which activation of both the TNF-α-mediated NF-κB (nuclear factor kappa B)-associated signaling pathway and the activin-mediated Smad2 / 3-associated signaling pathway is involved, comprising administering to said subject a therapeutically effective amount of the composition of claim 12 to the subject.

14. The method according to claim 13, wherein the disease state is selected from the group consisting of: blood diseases, ineffective erythropoiesis, anemia, pancytopenia, myelodysplastic syndromes;Fibrosing diseases: NASH (non-alcoholic steatohepatitis), liver fibrosis, pulmonary fibrosis, renal fibrosis, polycystic kidney disease, myocardial fibrosis, muscle fibrosis, myelofibrosis, cutaneous fibrosis, ocular fibrosis, multiple myeloma, acute myeloid leukemia, melanoma, muscular dystrophy, spinal muscular atrophy, spinal cord injury, stroke, nociceptive pain, neuropathic pain, sarcopenia, cancer cachexia, anorexia nervosa, bone metastasis, bone fragility, bone fracture, osteopenia, osteoporosis, pulmonary hypertension, myocardial infarction, heart failure, insulin resistance, diabetic nephropathy, chronic kidney disease, rheumatoid arthritis, coronavirus-induced inflammatory bowel disease severe acute respiratory syndrome (SARS-CoV), cytokine storm syndrome, sepsis and burn injury.; 15. An isolated nucleic acid molecule comprising a polynucleotide encoding a bifunctional antagonist molecule according to any one of claims 1-11.

16. A recombinant vector containing a nucleic acid molecule according to claim 15.

17. A host cell containing the recombinant vector according to claim 16.

18. A method for producing a bifunctional antagonist molecule according to any one of claims 1-11, comprising the steps of a) transforming a host cell with vectors containing polynucleotides encoding said bifunctional antagonist molecule, b) culturing the host cell under conditions suitable for expressing the bifunctional antagonist molecule, and c) recovering the bifunctional antagonist molecule from the culture.