Pharmaceutical compositions comprising protein complexes

Abstract

Provided are pharmaceutical compositions comprising alpha-2-macroglobulin (A2M) and a serine protease protein such as porcine pancreatic elastase (PPE), which are bound together in a protein complex that retains the CD95 protease cleavage and cancer-cell killing activities of the serine protease, but sterically hinders binding of the serine protease to fibrinogen and serine protease inhibitors; and related methods of use and manufacture for treating diseases such as cancers.

Description

The abstract provides pharmaceutical compositions comprising α-2-macroglobulin (A2M) and serine protease proteins such as porcine pancreatic elastase (PPE), wherein the A2M and the serine protease proteins are bound together in a protein complex that retains the CD95 protease cleavage and cancer cell killing activity of the serine protease, but spatially hinders the binding of the serine protease to fibrinogen and serine protease inhibitors; and related uses and methods of preparation for treating diseases such as cancer.

Claims

Attorney Docket No: OPNI-009 / 02WO 332575-2061 Claims 1. A pharmaceutical composition, comprising a protein complex of: (a) alpha-2-macroglobulin (A2M) proteins; and (b) serine protease proteins, wherein (a) and (b) are present in the composition at a molar ratio [(a):(b)] of about 1:3 to about 1:

1.

2. The pharmaceutical composition of claim 1, wherein the A2M proteins of (a) and the serine protease proteins of (b) are bound together in the protein complex, and optionally wherein the protein complex: (i) retains CD95 (Fas Receptor) protease cleavage activity and cancer cell-killing activity of (b); (ii) sterically hinders binding of (b) to fibrinogen and reduces or inhibits fibrinogen cleavage activity of (b); and (iii) sterically hinders binding of (b) to serine protease inhibitors (including alpha-1 antitrypsin (A1AT)).

3. The pharmaceutical composition of claim 1 or 2, wherein (a) comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to a sequence selected from Table A1, or a functional fragment thereof.

4. The pharmaceutical composition of claim 3, wherein the functional fragment thereof comprises, consists, or consists essentially of about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1300, or 1400 contiguous amino acids of a sequence selected from Table A1.

5. The pharmaceutical composition of claim 4, wherein the functional fragment thereof is composed of approximately residues 1-1400, 1-1300, 1-1200, 1-1100, 1-1000, 1-900, 1-800, 1-700, 1-600, 1-500, 1-400, 1-300, 1-200, 100-1400, 100-1300, 100-1200, 100-1100, 100-1000, 100-900, 100-800, 100-700, 100-600, 100-500, 100-400, 100-300, 100-200, 200-1400, 200-1300, 200-1200, 200-1100, 200-1000, 200-900, 200-800, 200-700, 200-600, 200-500, 200-400, 200-300, 300-1400, 300-1300, 300-1200, 300-1100, 300-1000, 300-900, 300-800, 300-700, 300-600, 300-500, 300-400, 400-1400, 400-1300, 400-1200, 400-1100, 400-1000, 400-900, 400-800, 400-700, 400-600, 400-500, 500-1400, 500-1300, 500-1200, 500-1100, 500-1000, 500-900, 500-800, 500-700, 500-600, 600- 1400, 600-1300, 600-1200, 600-1100, 600-1000, 600-900, 600-800, 600-700, 700-1400, 700-1300, 700-1200, 700-1100, 700-1000, 700-900, 700-800, 800-1400, 800-1300, 800-1200, 800-1100, 800- 1000, 800-900, 900-1400, 900-1300, 900-1200, 900-1100, 900-1000, 1000-1400, 1000-1300, 1000-Attorney Docket No: OPNI-009 / 02WO 332575-2061 1200, 1000-1100, 1100-1400, 1100-1300, 1100-1200, 1200-1400, or 1200-1300 of a sequence selected from Table A1. The pharmaceutical composition of any one of claims 1-5, wherein (a) is conjugated or fused to an antibody, or an antigen binding fragment thereof.

7. The pharmaceutical composition of claim 6, wherein the antibody, or antigen binding fragment thereof, specifically binds to a tumor-associated antigen (TAA) or tumor-specific antigen (TSA).

8. The pharmaceutical composition of any one of claims 1-7, wherein (b) is selected from a porcine pancreatic elastase (PPE) protein, a human neutrophil elastase (ELANE) protein, a human cathepsin G (CTSG) protein, a human proteinase 3 (PR3) protein, and a granzyme B protein.

9. The pharmaceutical composition of claim 8, wherein: the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 5, and which retains the Q211F amino acid substitution; the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 6, and which retains the T55A amino acid substitution; the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 7, and which retains the Q211F and T55A amino acid substitutions; the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 8, and which retains the N241A amino acid substitution; the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 9, and which retains the N241Y amino acid substitution; the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 10, and which retains the R75A amino acid substitution; the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 11, and which retains the R75E amino acid substitution;Attorney Docket No: OPNI-009 / 02WO 332575-2061 the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 12, and which retains the Q211A amino acid substitution; the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 13, and which retains the R237A amino acid substitution; the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 14, and which retains the S214A amino acid substitution; the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 15, and which retains the D74A amino acid substitution; and the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO:

16.

10. The pharmaceutical composition of claim 8, wherein: the human ELANE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 17; the human CTSG protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 18; the human PR3 protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 19; or the human granzyme B protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO:

20.

11. The pharmaceutical composition of any one of claims 1-10, wherein (a) and (b) are present in the composition at a molar ratio of about 1:3, 1:2.9, 1: 2.8, 1:2.7, 1:2.6, 1:2.5, 1:2.4, 1:2.3, 1: 2.1, 1:2, 1:1.9, 1:1.8, 1:1.7, 1:1.6, 1:1.5, 1:1.4, 1:1.3, 1:1.2, 1:1.1, or 1:

1.

12. The pharmaceutical composition of claim 11, wherein (a) and (b) are present in the composition at a molar ratio of about 1:

2.

13. A method of treating, ameliorating the symptoms of, and / or reducing the progression of, a cancer in a subject in need thereof, comprising administering to the subject a pharmaceutical composition of any one of claims 1-12.Attorney Docket No: OPNI-009 / 02WO 332575-2061 14. The method of claim 13, wherein the cancer is a primary cancer or a metastatic cancer, and is selected from one or more of melanoma (optionally metastatic melanoma), breast cancer (optionally triple-negative breast cancer, TNBC), kidney cancer (optionally renal cell carcinoma), pancreatic cancer, bone cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer (NSCLC), mesothelioma, leukemia (optionally lymphocytic leukemia, chronic myelogenous leukemia, acute myeloid leukemia, or relapsed acute myeloid leukemia), multiple myeloma, lymphoma, hepatoma (hepatocellular carcinoma), sarcoma, B-cell malignancy, ovarian cancer, colorectal cancer, glioma, glioblastoma multiforme, meningioma, pituitary adenoma, vestibular schwannoma, primary CNS lymphoma, primitive neuroectodermal tumor (medulloblastoma), bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancers, cervical cancer, testicular cancer, thyroid cancer, and stomach cancer.

15. The method of claim 13 or 14, wherein administration (optionally intravenous administration) of the pharmaceutical composition does not substantially increase prothrombin time or partial thromboplastin time in the subject.

16. The method of any one of claims 13-15, wherein administration of the pharmaceutical composition increases cancer cell-killing in the subject by about or at least about 2-fold, 5-fold, 10- fold, 50-fold, 100-fold, 500-fold, or 1000-fold or more relative to a control or reference.

17. The method of any one of claims 13-16, comprising administering the pharmaceutical composition to the subject by parenteral administration.

18. The method of claim 17, wherein the parenteral administration is intravenous administration.

19. A method of manufacturing a pharmaceutical composition comprising a protein complex, by combining: (a) alpha-2-macroglobulin (A2M) proteins; and (b) serine protease proteins, into a composition at a molar ratio [(a):(b)] of about 1:3 to about 1:1, thereby manufacturing the pharmaceutical composition comprising the protein complex.

20. The method of claim 19, comprising recombinantly producing (a) prior to combining with (b).Attorney Docket No: OPNI-009 / 02WO 332575-2061 21. The method of claim 19, comprising purifying (a) from plasma of a human subject prior to combining with (b).

22. The method of any one of claims 19-21, comprising recombinantly producing (b) prior to combining with (a).

23. The method of any one of claims 19-22, which comprises combining (a) and (b) at a molar ratio [(a):(b)] of about 1:3, 1:2.9, 1: 2.8, 1:2.7, 1:2.6, 1:2.5, 1:2.4, 1:2.3, 1: 2.1, 1:2, 1:1.9, 1:1.8, 1:1.7, 1:1.6, 1:1.5, 1:1.4, 1:1.3, 1:1.2, 1:1.1, or 1:1 24. The method of claim 23, which comprises combining (a) and (b) at a molar ratio [(a):(b)] of about 1:

2.

25. The method of any one of claims 19-24, wherein the A2M proteins of (a) and the serine protease proteins of (b) are bound together in the protein complex, and optionally wherein the protein complex: (i) retains CD95 (Fas Receptor) protease cleavage activity and cancer cell-killing activity of (b); (ii) sterically hinders binding of (b) to fibrinogen and reduces or inhibits fibrinogen cleavage activity of (b); and (iii) sterically hinders binding of (b) to serine protease inhibitors (including alpha-1 antitrypsin (A1AT)).

26. The method of any one of claims 19-25, wherein (a) comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to a sequence selected from Table A1, or a functional fragment thereof.

27. The method of claim 26, wherein the functional fragment thereof comprises, consists, or consists essentially of about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1300, or 1400 contiguous amino acids of a sequence selected from Table A1.

28. The method of claim 27, wherein the functional fragment thereof is composed of approximately residues 1-1400, 1-1300, 1-1200, 1-1100, 1-1000, 1-900, 1-800, 1-700, 1-600, 1-500, 1-400, 1-300, 1-200, 100-1400, 100-1300, 100-1200, 100-1100, 100-1000, 100-900, 100-800, 100- 700, 100-600, 100-500, 100-400, 100-300, 100-200, 200-1400, 200-1300, 200-1200, 200-1100, 200- 1000, 200-900, 200-800, 200-700, 200-600, 200-500, 200-400, 200-300, 300-1400, 300-1300, 300- 1200, 300-1100, 300-1000, 300-900, 300-800, 300-700, 300-600, 300-500, 300-400, 400-1400, 400-Attorney Docket No: OPNI-009 / 02WO 332575-2061 1300, 400-1200, 400-1100, 400-1000, 400-900, 400-800, 400-700, 400-600, 400-500, 500-1400, 500- 1300, 500-1200, 500-1100, 500-1000, 500-900, 500-800, 500-700, 500-600, 600-1400, 600-1300, 600-1200, 600-1100, 600-1000, 600-900, 600-800, 600-700, 700-1400, 700-1300, 700-1200, 700- 1100, 700-1000, 700-900, 700-800, 800-1400, 800-1300, 800-1200, 800-1100, 800-1000, 800-900, 900-1400, 900-1300, 900-1200, 900-1100, 900-1000, 1000-1400, 1000-1300, 1000-1200, 1000-1100, 1100-1400, 1100-1300, 1100-1200, 1200-1400, or 1200-1300 of a sequence selected from Table A1.

29. The method of any one of claims 19-28, wherein (a) is conjugated or fused to an antibody, or an antigen binding fragment thereof.

30. The method of claim 29, wherein the antibody, or antigen binding fragment thereof, specifically binds to a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA).

31. The method of any one of claims 19-30, wherein (b) is selected from a porcine pancreatic elastase (PPE) protein, a human neutrophil elastase (ELANE) protein, a human cathepsin G (CTSG) protein, a human proteinase 3 (PR3) protein, and a human granzyme B protein.

32. The method of claim 31, wherein: the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 5, and which retains the Q211F amino acid substitution; the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 6, and which retains the T55A amino acid substitution; the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 7, and which retains the Q211F and T55A amino acid substitutions; the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 8, and which retains the N241A amino acid substitution; the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 9, and which retains the N241Y amino acid substitution; the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 10, and which retains the R75A amino acid substitution;Attorney Docket No: OPNI-009 / 02WO 332575-2061 the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 11, and which retains the R75E amino acid substitution; the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 12, and which retains the Q211A amino acid substitution; the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 13, and which retains the R237A amino acid substitution; the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 14, and which retains the S214A amino acid substitution; the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 15, and which retains the D74A amino acid substitution; and the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO:

16.

33. The method of claim 31, wherein: the human ELANE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 17; the human CTSG protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 18; the human PR3 protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 19; or the human granzyme B protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO:

20.

34. The method of any one of claims 19-33, further comprising the step of testing the pharmaceutical composition in one or more activity assays selected from one or more of a CD95 cleavage assay (optionally in the presence of a serine protease inhibitor such as A1AT), a fibrinogen cleavage assay, and a cancer cell-killing assay.

35. The method of claim 32, wherein the pharmaceutical composition cleaves CD95 (optionally in the presence of the serine protease inhibitor such as A1AT), does not substantially cleave fibrinogen, and / or has cancer cell-killing activity.

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