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

Claims

1. An isolated bifunctional antagonist molecule comprising a first antigen-binding molecule that specifically binds to a TNF (tumor necrosis factor) ligand and a second antigen-binding molecule that specifically binds to a TGF-β (transforming growth factor) ligand, wherein the bifunctional antagonist molecule simultaneously and effectively neutralizes TNF-α-mediated signaling and TGF-β-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 a TGF-β ligand selected from the group consisting of TGF-β ligands comprising the amino acid sequence set forth in SEQ ID NOs: 6-9.

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-α ligand ("TNF-α-binding polypeptide") is selected from the group consisting of an antibody to TNF-α, a fragment of an antibody to TNF-α, extracellular domains (ECDs) of wild-type TNFR1 (TNF-α receptor 1) and TNFR2 (TNF-α 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 a TGF-β ligand ("TGF-β-binding polypeptide") is selected from the group consisting of an antibody to TGF-β, a fragment of an antibody to TGF-β, extracellular domains (ECDs) of the wild-type TGF-β receptor type 2 (including TGFRIIA and TGFRIIB), modified extracellular domains of TGFRIIA and TGFRIIB, and a phage-displayed antagonist polypeptide that targets a TGF-β 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 TGF-β-binding polypeptide is selected from the group of polypeptides comprising the amino acid sequence set forth in SEQ ID NO: 6-9.

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 TGF-β-binding polypeptide comprises an isolated TGF-β 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-TGF-β 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 required 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: 10; an antibody comprising the light chain amino acid sequence set forth in SEQ ID NO: 11; an antibody comprising the heavy chain amino acid sequence set forth in SEQ ID NO: 10 and the light chain amino acid sequence set forth in SEQ ID NO: 11; an antibody comprising the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 14; an antibody comprising the light chain variable region amino acid sequence set forth in SEQ ID NO: 15;an antibody comprising the amino acid sequence of a heavy chain variable region as set forth in SEQ ID NO: 14 and the amino acid sequence of a light chain variable region as set forth in SEQ ID NO: 15; an antibody comprising the amino acid sequence of a heavy chain as set forth in SEQ ID NO: 18; an antibody comprising the amino acid sequence of a light chain as set forth in SEQ ID NO: 19; an antibody comprising the amino acid sequence of a heavy chain variable region as set forth in SEQ ID NO: 18 and the amino acid sequence of a light chain as set forth in SEQ ID NO: 19; an antibody comprising the amino acid sequence of a heavy chain variable region as set forth in SEQ ID NO: 22; an antibody comprising the amino acid sequence of a light chain variable region as set forth in SEQ ID NO: 23;and an antibody comprising the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO: 22 and the amino acid sequence of the light chain variable region set forth in SEQ ID NO: 23.; 9. An isolated bifunctional antagonist molecule according to any one of claims 5-8, wherein the TGF-β 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: 26; an antibody comprising the light chain amino acid sequence set forth in SEQ ID NO: 27; an antibody comprising the heavy chain amino acid sequence set forth in SEQ ID NO: 26 and the light chain amino acid sequence set forth in SEQ ID NO:

27.

10. An isolated bifunctional antagonist molecule according to any one of claims 1, 2, selected from the group consisting of: a bifunctional molecule comprising the amino acid sequence of a heavy chain as set forth in SEQ ID NO: 30 and the amino acid sequence of a light chain as set forth in SEQ ID NO: 11; a bifunctional molecule comprising the amino acid sequence of a heavy chain as set forth in SEQ ID NO: 31 and the amino acid sequence of a light chain as set forth in SEQ ID NO: 11; and a bifunctional molecule comprising the amino acid sequence of a heavy chain as set forth in SEQ ID NO: 10 and the amino acid sequence of a light chain as set forth in SEQ ID NO:

11.

11. An isolated bifunctional antagonist molecule according to any one of claims 1, 2, selected from the group consisting of: a bifunctional molecule comprising the amino acid sequence set forth in SEQ ID NO: 32; a bifunctional molecule comprising the amino acid sequence set forth in SEQ ID NO: 33; a bifunctional molecule comprising the amino acid sequence set forth in SEQ ID NO: 34; a bifunctional molecule comprising the amino acid sequence set forth in SEQ ID NO: 35; a bifunctional molecule comprising the amino acid sequence set forth in SEQ ID NO: 36; a bifunctional molecule comprising the amino acid sequence set forth in SEQ ID NO: 37; a bifunctional molecule comprising the amino acid sequence set forth in SEQ ID NO: 38; a bifunctional molecule comprising the amino acid sequence set forth in SEQ ID NO: 39; a bifunctional molecule comprising the amino acid sequence set forth in SEQ ID NO: 40; and a bifunctional molecule comprising the amino acid sequence set forth in SEQ ID NO:

41.

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 TGF-β-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, skin fibrosis, ocular fibrosis, hand 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, pulmonary arterial hypertension, myocardial infarction, heart failure, insulin resistance, diabetic nephropathy, chronic kidney disease, rheumatoid arthritis, inflammatory bowel disease caused by severe acute respiratory syndrome coronavirus (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.