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 activity of the serine protease, but spatially hinders binding of the serine protease to fibrinogen and a serine protease inhibitor; and related use and preparation methods for treating diseases such as cancer.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202480029289.1 (22) Application Date 2024.04.04 (30) Priority Data 63 / 456,916 2023.04.04 US 63 / 623,030 2024.01.19 US (85) PCT International Application Entering National Phase Date 2025.10.30 (86) PCT International Application Application Data PCT / US2024 / 022956 2024.04.04 (87) PCT International Application Publication Data WO2024 / 211493 EN 2024.10.10 (71) Applicant Onsilex Pharmaceuticals, Inc. Address: California, USA (72) Inventors: Court R. Turnalev Becker, Cui Chang (74) Patent Agency: Beijing Ying Sai Jia Hua Intellectual Property Agency Co., Ltd. 11204 Patent Attorneys: Zu Luxia, Hong Xin (51) Int.Cl. C12N 9 / 64 (2006.01) A61K 38 / 48 (2006.01) (54) Invention Title: Pharmaceutical Composition Containing Protein Complex (57) Abstract: Provides a pharmaceutical composition comprising α-2-macroglobulin (A2M) and a serine protease protein such as porcine pancreatic elastase (PPE), wherein the A2M and the serine protease protein are bound together in a protein complex, wherein the protein complex retains the CD95 protease cleavage and cancer cell killing activity of the serine protease, but spatially impedes the binding of the serine protease to fibrinogen and serine protease inhibitors; and related use and preparation methods for treating diseases such as cancer. Claims 5 pages, Description 47 pages, Sequence Listing (Electronic Publication), Drawings 50 pages, CN 121263518 A 2026.01.02 CN 1 21 26 35 18 A 1. A pharmaceutical composition comprising a protein complex of: (a) α-2-macroglobulin (A2M) protein; and (b) a serine protease protein, wherein (a) and (b) are present in the composition in a molar ratio of about 1:3 to about 1:1 [(a):(b)]. 2. The pharmaceutical composition according to claim 1, wherein the A2M protein of (a) and the serine protease protein of (b) are bound together in the protein complex, and optionally wherein the protein complex: (i) retains the CD95 (Fas receptor) protease cleavage activity and cancer cell killing activity of (b); (ii) spatially hinders the binding of (b) to fibrinogen and reduces or inhibits the fibrinogen cleavage activity of (b); and(iii) Spatially hinders the binding of (b) to serine protease inhibitors, including α-1 antitrypsin (A1AT). 3. The pharmaceutical composition of claim 1 or 2, wherein (a) comprises, is composed of, or is substantially composed of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% of an amino acid sequence selected from Table A1 or a functional fragment thereof. 4. The pharmaceutical composition of claim 3, wherein the functional fragment comprises, is composed of, or is substantially composed of about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1300, or 1400 consecutive amino acids selected from Table A1. 5. The pharmaceutical composition according to 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, 900-1300, 1000-1400, 1000-1300, 1000-1200, 1000-1100, 1100-1400, 1100- 6. The pharmaceutical composition according to any one of claims 1 to 5, wherein (a) is conjugated or fused with an antibody or an antigen-binding fragment thereof. 7. The pharmaceutical composition according to claim 6, wherein the antibody or an antigen-binding fragment thereof specifically binds to a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA). 8. The pharmaceutical composition according to any one of claims 1 to 7, wherein (b) is selected from porcine pancreatic elastase (PPE) protein, human neutrophil elastase (ELANE) protein, human tissue proteinase G (CTSG) protein, human proteinase 3 (PR3) protein, and granzyme B protein. 9. The pharmaceutical composition according to claim 8, wherein: the PPE protein comprises, is composed of, or is substantially composed of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the Q211F amino acid substitution; the PPE protein comprises, is composed of, or is substantially composed of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the T55A amino acid substitution; the PPE protein comprises, is composed of, or is substantially composed of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the Q211F and T55A amino acid substitutions; the PPE protein comprises, is ... 8. An amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains, the amino acid substitution of N241A, or is composed of, or is substantially composed of, the amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains, the amino acid substitution of N241Y, or is composed of, or is substantially composed of, the amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains, the amino acid substitution of N241Y;The PPE protein comprises, is composed of, or is substantially composed of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the R75A amino acid substitution, of SEQ ID NO: 10; the PPE protein comprises, is composed of, or is substantially composed of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the R75E amino acid substitution, of SEQ ID NO: 11; the PPE protein comprises, is composed of, or is substantially composed of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the Q211A amino acid substitution, of SEQ ID NO: 12; the PPE protein comprises, is composed of, or is substantially composed of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the R237A amino acid substitution, of SEQ ID NO: 13; the PPE protein comprises, is composed of, or is substantially composed of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the R237A amino acid substitution, of SEQ ID NO: 10; the PPE protein comprises, is composed of, and ... NO: 14 is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, or constitutes, an amino acid sequence that retains the S214A amino acid substitution; the PPE protein comprises, is composed of, or is substantially composed of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, or retains the D74A amino acid substitution; and the PPE protein comprises, is composed of, or is substantially composed of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to ...5%, 98%, 99%, or 100% identical to, or is substantially composed of, an amino acid sequence that is at least 80%, 85%, 95%, 98%, 99%, or 100% identical to, or is substantially composed of, an amino acid sequence that is at least 80%, 85%, 95%, 98%, 99%, or 100% identical to, or is substantially composed of 10. The pharmaceutical composition of claim 8, wherein: the human ELANE protein comprises, is composed of, or is substantially composed of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO: 17; the human CTSG protein comprises, is composed of, or is substantially composed of, at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO: 18; the human PR3 protein comprises, is composed of, or is substantially composed of, at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO: 19; or the human granzyme B protein comprises, is composed of, or is substantially composed of, at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO: 20.11. The pharmaceutical composition according to any one of claims 1 to 10, wherein (a) and (b) are present in the composition in 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 according to claim 11, wherein (a) and (b) are present in the composition in a molar ratio of about 1:2. 13. A method for treating a subject with cancer, improving symptoms of said cancer, and / or reducing the progression of said cancer, the method comprising administering to the subject the pharmaceutical composition according to any one of claims 1 to 12. 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 the following: melanoma (optionally metastatic melanoma), breast cancer (optionally triple-negative breast cancer), ... (Claims 2 / 5, page 3, CN 121263518 A) TNBC, renal cell carcinoma (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 myeloid leukemia, acute myeloid leukemia, or relapsed acute myeloid leukemia), multiple myeloma, lymphoma, liver cancer (hepatocellular carcinoma), sarcoma, B-cell malignancy, ovarian cancer, colorectal cancer, glioma, glioblastoma multiforme, meningioma, pituitary adenoma, vestibular schwannoma, primary CNS lymphoma, primitive neuroectodermal tumor (neuroblastoma), bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancer, cervical cancer, testicular cancer, thyroid cancer, and gastric cancer. 15. The method according to claim 13 or 14, wherein the administration of said pharmaceutical composition (optionally intravenous) does not significantly increase prothrombin time or partial prothrombin kinase time in said subject. 16. The method of any one of claims 13 to 15, wherein administration of the pharmaceutical composition increases cancer cell killing in the subject by about or at least about 2, 5, 10, 50, 100, 500, or 1000 times or more relative to a control or reference. 17. The method of any one of claims 13 to 16, comprising administering the pharmaceutical composition to the subject via parenteral administration. 18. The method of claim 17, wherein the parenteral administration is intravenous administration. 19. A method for preparing a pharmaceutical composition comprising a protein complex, the method being carried out by combining the following in a molar ratio of about 1:3 to about 1:1 [(a):(b)]:(a) α-2-macroglobulin (A2M) protein; and (b) serine protease protein, thereby preparing the pharmaceutical composition comprising the protein complex. 20. The method of claim 19, wherein (a) is recombinantly generated prior to combination with (b). 21. The method of claim 19, wherein (a) is purified from plasma of a human subject prior to combination with (b). 22. The method of any one of claims 19 to 21, wherein (b) is recombinantly generated prior to combination with (a). 23. The method according to any one of claims 19 to 22, comprising combining (a) and (b) in 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 [(a):(b)]. 24. The method according to claim 23, comprising combining (a) and (b) in a molar ratio of about 1:2 [(a):(b)]. 25. The pharmaceutical composition according to any one of claims 19 to 24, wherein the A2M protein of (a) and the serine protease protein of (b) are bound together in the protein complex, and optionally wherein the protein complex: (i) retains the CD95 (Fas receptor) protease cleavage activity and cancer cell killing activity of (b); (ii) spatially inhibits the binding of (b) to fibrinogen and reduces or inhibits the fibrinogen cleavage activity of (b); and (iii) spatially inhibits the binding of (b) to serine protease inhibitors (including α-1 antitrypsin (A1AT)). 26. The method according to any one of claims 19 to 25, wherein (a) comprises, is composed of, or is substantially composed of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, constitutes, or is substantially composed of a sequence selected from Table A1 or a functional fragment thereof. 27. The method of claim 26, wherein the functional fragment comprises, is composed of, or is substantially composed of about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1300, or 1400 consecutive amino acids selected from the sequence in Table A1. 28. The method of claim 27, wherein the functional fragment consists of approximately [number missing] residues selected from the sequence in Table A1.1-1400, 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- Composed of 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. 29. The method according to any one of claims 19 to 28, wherein (a) is conjugated or fused with an antibody or an antigen-binding fragment thereof. 30. The method of claim 29, wherein the antibody or its antigen-binding fragment specifically binds to a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA).31. The method according to any one of claims 19 to 30, wherein (b) is selected from porcine pancreatic elastase (PPE) protein, human neutrophil elastase (ELANE) protein, human tissue protease G (CTSG) protein, human protease 3 (PR3) protein and human granzyme B protein. 32. The method of claim 31, wherein: the PPE protein comprises, is composed of, or is substantially composed of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, SEQ ID NO: 5, and retains the Q211F amino acid substitution; the PPE protein comprises, is composed of, or is substantially composed of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, SEQ ID NO: 6, and retains the T55A amino acid substitution; the PPE protein comprises, is composed of, or is substantially composed of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, SEQ ID NO: 7, and retains the Q211F and T55A amino acid substitutions; the PPE protein comprises, is composed of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, SEQ ID NO: 5 ... The PPE protein comprises, consists of, or is substantially composed of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the N241A amino acid substitution, of the same amino acid sequence as SEQ ID NO: 9; the PPE protein comprises, consists of, or is substantially composed of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the N241Y amino acid substitution, of the same amino acid sequence as SEQ ID NO: 10; the PPE protein comprises, consists of, or is substantially composed of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the R75A amino acid substitution, of the same amino acid sequence as SEQ ID NO: 11; the PPE protein comprises, consists of, or is substantially composed of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the R75E amino acid substitution, of the same amino acid sequence as SEQ ID NO: 9; the PPE protein comprises, consists of, and ... N241A amino acid substitution, of the same amino acid sequence as SEQ ID NO: 9; the PPE protein comprises, consists of, 12. At least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retaining the amino acid sequence substituted with Q211A, constitute, or substantially constitute the amino acid sequence of SEQ ID NO: 13. At least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retaining the amino acid sequence substituted with R237A, constitute, or substantially constitute the amino acid sequence of SEQ ID NO: 13.14. The PPE protein comprises, is composed of, or is substantially composed of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the amino acid substitution of S214A; and the PPE protein comprises, is composed of, or is substantially composed of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the amino acid substitution of D74A; and the PPE protein comprises, is composed of, or is substantially composed of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and ...5%, 98%, 99%, or 100% identical to, and is substantially composed of, an amino acid sequence that is at least 80%, 85%, 95%, 98%, 99%, or 100% identical to, and is substantially composed of, an amino acid sequence that is at least 80%, 85%, 95%, 98%, 99%, or 100% identical to, and is substantially composed of, an amino acid sequence that is at least 80%, 85%, 95%, 98%, 99%, or 10 33. The method of claim 31, wherein: the human ELANE protein comprises, is composed of, or is substantially composed of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO: 17; the human CTSG protein comprises, is composed of, or is substantially composed of, at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO: 18; the human PR3 protein comprises, is composed of, or is substantially composed of, at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO: 19; or the human granzyme B protein comprises, is composed of, or is substantially composed of, at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO: 20. 34. The method according to any one of claims 19 to 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 AlAT), a fibrinogen cleavage assay, and a cancer cell killing assay. 35. The method according to claim 32, wherein the pharmaceutical composition cleaves CD95 (optionally in the presence of the serine protease inhibitor such as AlAT), substantially does not cleave fibrinogen, and / or has cancer cell killing activity. Claims 5 / 5 Page 6 CN 121263518 A Pharmaceutical composition comprising a protein complex

[0001] Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 623,030, filed January 19, 2024, and U.S. Provisional Application No. 63 / 456,916, filed April 4, 2023, pursuant to 35 USC § 119(e), each of which is incorporated herein by reference in its entirety.

[0003] Statement Regarding the Sequence Listing

[0004] The sequence listing XML associated with this application is provided in XML file format and is hereby incorporated by reference in this specification. The name of the XML file containing the sequence listing XML is OPNI_009_02WO_ST26.xml. The XML file is approximately 28,746 bytes long, was created on March 28, 2024, and was filed electronically through the USPTO Patent Centre. Technical Field

[0005] This disclosure relates to 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 impedes 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. Background Art

[0006] Precision medicine, which aims to optimize the efficiency or therapeutic benefits of specific patient populations through the use of genetic or molecular analysis, has gained tremendous appeal in the treatment of cancer. Identifying specific genomic abnormalities that (i) confer the risk of developing cancer; (ii) affect tumor growth; and (iii) regulate metastasis has defined how cancer is diagnosed, determined how targeted therapies are developed and implemented, and shaped cancer prevention strategies.

[0007] The need for precision medicine in cancer is largely based on the failure to identify targetable properties in tumor cells that distinguish them from healthy, non-cancerous cells. Indeed, while radiation and / or chemotherapy have the ability to effectively kill many (if not most) cancer cells, their efficacy is severely limited by cytotoxic effects on non-cancerous cells. These findings confirm that rapid cell division, i.e., the properties targeted by radiation and chemotherapy, are not unique enough to cancer cells to achieve the specificity required to limit a wide range of side effects.

[0008] It has been shown that certain serine proteases or elastases are selectively toxic to cancer cells but relatively non-toxic to normal cells or other healthy cells (see, for example, WO 2018 / 232273). However, there is a need in the art to identify optimal enzyme compositions that can possess such selective cancer cell toxicity but also have reduced side effects, and thus improve the pharmacokinetics and overall clinical usability of such compositions. Summary of the Invention

[0009] Embodiments of this disclosure include pharmaceutical compositions comprising a protein complex of: (a) an α-2-macroglobulin (A2M) protein; and (b) a serine protease protein, wherein (a) and (b) are present in the composition in a molar ratio of about 1:3 to about 1:1 [(a):(b)]. In some specifications, page 1 / 47, CN 121263518 AIn the embodiments, the A2M protein of (a) and the serine protease protein of (b) are bound together in the protein complex, for example, wherein the protein complex: (i) retains the CD95 (Fas receptor) protease cleavage activity and cancer cell killing activity of (b); (ii) spatially inhibits the binding of (b) to fibrinogen and reduces or inhibits the fibrinogen cleavage activity of (b); and (iii) spatially inhibits the binding of (b) to serine protease inhibitors (including plasma serine protease inhibitors, such as α-1 antitrypsin (A1AT)).

[0010] In some embodiments, (a) comprises, is composed of, or is substantially composed of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, constitutes, or is substantially composed of a sequence selected from Table A1 or a functional fragment thereof. In some embodiments, the functional fragment comprises, consists of, or is substantially composed of about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1300, or 1400 consecutive amino acids selected from the sequences in Table A1. In some embodiments, the functional fragment 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- The values ​​are 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.

[0011] In some embodiments, (a) is conjugated or fused with an antibody or its antigen-binding fragment. In some embodiments, the antibody or its antigen-binding fragment specifically binds to a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA).

[0012] In some embodiments, (b) is selected from porcine pancreatic elastase (PPE) protein, human neutrophil elastase (ELANE) protein, human tissue protease G (CTSG) protein, human protease 3 (PR3) protein and granzyme B protein. In specific embodiments: the PPE protein comprises, is composed of, or is substantially composed of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, SEQ ID NO: 5, and retains the Q211F amino acid substitution; the PPE protein comprises, is composed of, or is substantially composed of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, SEQ ID NO: 6, and retains the T55A amino acid substitution; the PPE protein comprises, is composed of, or is substantially composed of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, SEQ ID NO: 7, and retains the Q211F and T55A amino acid substitutions; the PPE protein comprises, is composed of, or is substantially composed of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, SEQ ID NO: 8, and retains the N241A amino acid substitution; The PPE protein contains the protein associated with SEQ ID NO:9. At least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, or consisting of, an amino acid sequence with the N241Y amino acid substitution, and retained; Specification 2 / 47 page 8 CN 121263518 A The PPE protein comprises, consists of, or consists of, an amino acid sequence with the R75A amino acid substitution, and at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, or consisting of, an amino acid sequence with the R75E ...A amino acid substitution, and at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, or consisting of, an amino acid sequence with the R75A amino acid substitution, and at least 80%, 85%, 90%, 95%, 98%, 12. The PPE protein comprises, is composed of, or is substantially composed of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the amino acid substitution of Q211A; 12. The PPE protein comprises, is composed of, or is substantially composed of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the amino acid substitution of R237A; 13. The PPE protein comprises, is composed of, or is substantially composed of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the amino acid substitution of S214A; 14. The PPE protein comprises, is composed of, or is substantially composed of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the amino acid substitution of D74A; and 15 ... an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the amino acid substitution of D74A; and 15. The PPE protein comprises, is composed of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the amino acid substitution of Q211A 16. At least 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO: 17, or consisting of or substantially consisting of the same amino acid sequence as SEQ ID NO: 17; the human ELANE protein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO: 18, or consisting of or substantially consisting of the same amino acid sequence as SEQ ID NO: 18; the human PR3 protein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO: 19, or consisting of or substantially consisting of the same amino acid sequence as SEQ ID NO: 19; or the human granzyme B protein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO: 19.20 has at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the same amino acid sequence, is composed of, or is substantially composed of.

[0014] In some embodiments, (a) and (b) are present in the composition in 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. In a specific embodiment, (a) and (b) are present in the composition in a molar ratio of about 1:2.

[0015] It also includes methods for treating cancer in a subject in need, improving symptoms of said cancer and / or reducing the progression of said cancer, said methods comprising administering the pharmaceutical composition described herein to said subject.

[0016] In some embodiments, the cancer is a primary cancer or a metastatic cancer, and is selected from one or more of the following: 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 myeloid leukemia, acute myeloid leukemia or relapsed acute myeloid leukemia), multiple myeloma, lymphoma, liver cancer (hepatocellular carcinoma), sarcoma, B-cell malignancy, ovarian cancer, colorectal cancer, glioma, glioblastoma multiforme, meningioma, pituitary adenoma, vestibular schwannoma, primary CNS lymphoma, primitive neuroectodermal tumor (neuroblastoma), bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancer, cervical cancer, testicular cancer, thyroid cancer, and gastric cancer.

[0017] In some embodiments, administration of the pharmaceutical composition (optionally intravenous) does not significantly increase prothrombin time or partial prothrombin kinase time in the subject. In some embodiments, administration of the pharmaceutical composition increases cancer cell killing in the subject by about or at least about 2, 5, 10, 50, 100, 500, or 1000 times or more compared to a control or reference to the specification page 3 / 47 9 CN 121263518 A.

[0018] Some embodiments include administration of the pharmaceutical composition to the subject via parenteral administration. In some embodiments, the parenteral administration is intravenous administration.

[0019] A method for preparing a pharmaceutical composition comprising a protein complex is also included, the method being carried out by combining the following in a molar ratio of about 1:3 to about 1:1 [(a):(b)]: (a) α-2-macroglobulin (A2M) protein; and (b) serine protease protein,The pharmaceutical composition comprising the protein complex is thus prepared.

[0020] Some embodiments include recombinantly generating (a) prior to combination with (b). Some embodiments include purification (a) from plasma of a human subject prior to combination with (b). Specific embodiments include recombinantly generating (b) prior to combination with (a).

[0021] In some embodiments, the preparation method comprises combining (a) and (b) in 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 [(a):(b)]. Specific embodiments comprise combining (a) and (b) in a molar ratio of about 1:2 [(a):(b)].

[0022] In some embodiments, the A2M protein of (a) and the serine protease protein of (b) are bound together in the protein complex, comprising the protein complex that: (i) retains the CD95 (Fas receptor) protease cleavage activity and cancer cell killing activity of (b); (ii) spatially inhibits the binding of (b) to fibrinogen and reduces or inhibits the fibrinogen cleavage activity of (b); and (iii) spatially inhibits the binding of (b) to serine protease inhibitors (including α-1 antitrypsin (A1AT)).

[0023] In some embodiments, (a) comprises, is composed of, or is substantially composed of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, constitutes, or is substantially composed of a sequence selected from Table A1 or a functional fragment thereof. In some embodiments, the functional fragment comprises, consists of, or is substantially composed of about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1300, or 1400 consecutive amino acids selected from the sequences in Table A1. In some embodiments, the functional fragment 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 Composed of 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. Instructions for Use, Page 4 / 47, 10 CN 121263518 A

[0024] In some embodiments, (a) is conjugated or fused with an antibody or its antigen-binding fragment. In some embodiments, the antibody or its antigen-binding fragment specifically binds to a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA).

[0025] In some embodiments, (b) is selected from porcine pancreatic elastase (PPE) protein, human neutrophil elastase (ELANE) protein, human tissue protein G (CTSG) protein, human proteinase 3 (PR3) protein, and human granzyme B protein.

[0026] In some embodiments: the PPE protein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 5 andThe PPE protein contains at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the amino acid sequence identical to SEQ ID NO: 6 and retains the amino acid sequence substituted with T55A, or consists of or is substantially composed of it; the PPE protein contains at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the amino acid sequence identical to SEQ ID NO: 7 and retains the amino acid sequences substituted with Q211F and T55A, or consists of or is substantially composed of it; the PPE protein contains at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the amino acid sequence identical to SEQ ID NO: 8 and retains the amino acid sequence substituted with N241A, or consists of or is substantially composed of it; the PPE protein contains the amino acid sequence identical to SEQ ID NO: 6 and retains the amino acid sequence substituted with T5 ... The PPE protein comprises, consists of, or is substantially composed of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the N241Y amino acid substitution, of the same amino acid sequence as SEQ ID NO: 10, and retains the R75A amino acid substitution, of the same amino acid sequence as SEQ ID NO: 11, and retains the R75E amino acid substitution, of the same amino acid sequence as SEQ ID NO: 12, and retains the Q211A amino acid substitution, of the same amino acid sequence as SEQ ID NO: 12, and is substantially composed of, the same amino acid sequence as SEQ ID NO: 10, and retains the R75A amino acid substitution, of the same amino acid sequence as SEQ ID NO: 10, and is substantially composed of, the same amino acid sequence as SEQ ID NO: 10, and is substantially composed of, the same amino acid sequence as SEQ ID NO: 10, and is substantially composed of, the same amino acid sequence as SEQ ID NO: 11, and is substantially composed of, the same amino acid sequence as SEQ ID NO: 12, and retains the Q211A amino acid substitution, of the same amino acid sequence as SEQ ID NO: 10, and is substantially composed of, ... 13. The PPE protein comprises, is composed of, or is substantially composed of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the amino acid substitution of R237A; the PPE protein comprises, is composed of, or is substantially composed of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the amino acid substitution of S214A; the PPE protein comprises, is composed of, or is substantially composed of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the amino acid substitution of D74A; and the PPE protein comprises, is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, the amino acid sequence that ...5%, 98%, 99%, or 100% identical to, the amino acid sequence that is at least 80%, 85%, 95%, 98%, 99%, or 100% identical to, the amino acid sequence that is at least 80%, 85Amino acid sequence, constituted therewith, or substantially constituted therewith.

[0027] In some embodiments: the human ELANE protein comprises, constituted therewith, or substantially constituted therewith, at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO: 17; the human CTSG protein comprises, constituted therewith, or substantially constituted therewith, at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO: 18; the human PR3 protein comprises, constituted therewith, or substantially constituted therewith, at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO: 19; or the human granzyme B protein comprises, constituted therewith, or substantially constituted therewith, at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO: 20.

[0028] Certain methods further include testing one or more steps of the pharmaceutical composition in one or more activity assays selected from one or more of the following: CD95 cleavage assay (optionally in the presence of a serine protease inhibitor such as A1AT), fibrinogen cleavage assay, and cancer cell killing assay. In some embodiments, the pharmaceutical composition cleaves CD95 (optionally in the presence of the serine protease inhibitor such as A1AT), substantially does not cleave fibrinogen, and / or has cancer cell killing activity.

[0029] Figure 1 illustrates the formation and structure of a protein complex comprising human A2M protein and a serine protease protein. The protease binds to and cleaves the decoy region of the A2M homotetramer, thereby inducing a conformational change that incorporates the serine protease protein into the protein complex.

[0030] Figures 2A-2B show the activity of mutant F (MutF) alone on tumor growth (2A) and lung metastasis number (2B) after IV injection (day 0 and day 1) in the 4T1 tumor model. Figure 2C shows the effect of MutF on prothrombin time 5 minutes after IV injection.

[0031] Figure 3A shows the MutF activity with different ratios of A2M:MutF in the presence of its inhibitor A1AT. Figure 3B shows the MutF activity at an A2M:MutF ratio of 1:2 in the presence of different concentrations of the inhibitor A1AT. Figure 3C shows the MutF activity at an A2M:MutF ratio of 1:2 in plasma.

[0032] Figure 4A shows the A2M:MutF product after cation exchange column separation in the presence or absence of A1AT.MutF activity and concentration in different fractions. Figure 4B shows the MutF activity and concentration in different fractions of the A2M:MutF product after size exclusion column separation with or without A1AT. Figures 4C-4D show that the A2M:MutF complex is stable over a wide pH range, as measured by enzyme activity (4C) and A1AT protection (4D). Figure 4E shows that the A2M:MutF complex is stable in multiple freeze-thaw cycles, as measured by enzyme activity.

[0033] Figures 5A-5D show the MutF activity in various cell lysates after treatment in serum-free medium (SFM) for 30 minutes with or without A1AT.

[0034] Figure 6A shows CD95-cut Coomassie blue staining after incubation with MutF or A2M:MutF at different ratios for 30 minutes. Figure 6B shows Western blots of fibrinogen after incubation with MutF or A2M:MutF at different ratios for 1 hour. A2M:MutF cleaves CD95 at a 1:2 ratio with the same efficiency as MutF alone (shown by double bands), but does not cleave fibrinogen as MutF alone (shown by bottom band). Figure 6C shows the fluorescence signal of cleaved elastin after incubation with PBS, MutF alone, or the A2M:MutF protein complex.

[0035] Figure 7A shows the prothrombin time in mouse plasma 5 minutes after IV injection of 480 μg MutF or A2M:MutF (1:2) protein complex. Figure 7B shows the partial prothrombin kinase time in mouse plasma 5 minutes after IV injection of 480 μg MutF or A2M:MutF (1:2) protein complex. Figure 7C shows the concentration of fibrinogen in mouse plasma 5 minutes after IV injection of 480 μg MutF or A2M:MutF (1:2) protein complex.

[0036] Figure 8A shows the cytotoxicity assays of various mouse cancer cell lines by the MutF or A2M:MutF (1:2) protein complex at 400 nM. Figure 8B shows the broad cytotoxicity of the A2M:MutF protein complex against cancer cells of different anatomical origins, and Figure 8C shows that the complex does not kill non-cancer cells. Figure 8D shows the antitumor effects of MutF and the A2M:MutF (1:2) protein complex in the CT26 model after a 100 μg IT injection on day 0. Figure 8E shows that the A2M:MutF protein complex has an improved functional PK profile (enzymatic activity in plasma) compared to MutF alone after intravenous administration. Figure 8F shows the favorable immune profile induced by the A2M:MutF protein complex in the CT26 model (from left to right in each figure).From left to right: PBS, MutF, A2M:MutF). Figure 8G shows that the A2M:MutF protein complex induces a tumor antigen-specific CD8+ T cell response in the CT26 model (PBS, MutF, A2M:MutF from left to right in each figure). Specification 6 / 47 pages 12 CN 121263518 A

[0037] Figure 9A shows that the A2M:MutF protein complex has a wide therapeutic window compared to doxorubicin and oxaliplatin, as shown by killing human ovarian cancer cells without killing non-cancerous cells in patients. Figure 9B shows that A2M:MutF kills cancer cells equally from both chemotherapy-naïve and chemotherapy-treated patients compared to doxorubicin and oxaliplatin, which shows reduced killing of cancer cells from chemotherapy-treated patients relative to chemotherapy-naïve patients.

[0038] Figure 10A shows that the A2M:MutF protein complex induces ICD markers in CT26 and A549 cells. Figure 10B shows that the A2M:MutF protein complex induces ICD markers in tumor cells derived from human ovarian patients (CTRL, A2M:MutF, and oxaliplatin from left to right in each figure).

[0039] Figures 11A-11B show tumor growth after treatment in a mouse CT26 tumor model. Figure 11C shows tumor weight at 15 days after treatment (11C from left to right: mediator every other day, A2M:MutF 100 μg daily, A2M:MutF 200 μg every other day, A2M:MutF 400 μg every 4 days).

[0040] Figures 12A-12B show that the A2M:MutF protein complex effectively attenuates tumor growth in the Jh-BALB / c CT26 colorectal cancer model. Figures 12C-12D show the A2M:MutF protein complex treatment of primary and metastatic tumors in the Jh-C57BL / 6 B16F10 melanoma model. Figure 12E shows the efficacy of the A2M:MutF protein complex across a range of tumors with variable immune status.

[0041] Figures 13A-13C show that the A2M:MutF protein complex has improved antitumor efficacy relative to SoC chemotherapy (oxaliplatin) without observed toxicity.

[0042] Figure 14A shows the efficacy of the A2M:MutF protein complex in a human xenograft model of lung cancer. Figure 14B summarizes the efficacy of the A2M:MutF protein complex across various prostate cancer, colon cancer, and lung cancer models. Figure 14C shows that the A2M:MutF protein complex effectively kills tumor cells derived from human ovarian patients (from patient CDX_O02) in a xenograft mouse model, and Figure 14D summarizes the efficacy of the A2M:MutF protein complex in this model in three ovarian cancer patients.Efficacy. Figures 14E-14F show that the A2M:MutF protein complex effectively kills patient-derived breast cancer cells in vitro and in vivo. Figure 14G summarizes the in vivo efficacy of the A2M:MutF protein complex across a variety of human tumors and shows that the efficacy is independent of tumor genetic or immune status.

[0043] Figure 15 shows that mice treated with the A2M:MutF protein complex were tumor-free (5 / 11) after initial stimulation with CT26 colorectal cancer cells, and all of these mice (5 / 5) remained tumor-free after re-stimulation with CD26 cells. Detailed Description

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Although similar or equivalent methods, materials, compositions, reagents, cells to any of the methods, materials, compositions, reagents, cells described herein may be used in practice or testing of the subject matter of this disclosure, preferred methods and materials are described. All publications and references cited in this specification, including but not limited to patents and patent applications, are incorporated herein by reference in their entirety, as if each individual publication or reference were expressly and individually indicated to be incorporated herein by reference as fully illustrated. Any patent application claiming priority in this application is also incorporated herein by reference in its entirety in the manner described above with respect to the publications and references.

[0045] Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, liposome transfection). Enzymatic reactions and purification techniques may be performed according to the manufacturer's instructions or as commonly practiced in the art or as described herein. These and related techniques and procedures may generally be performed according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout this specification. Unless specifically defined herein, the nomenclature used in conjunction with the molecular biology, analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein, as well as the laboratory procedures and techniques of said molecular biology, analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry, are well-known and commonly used nomenclature and laboratory procedures and techniques in the art. Standard techniques can be used in recombinant technologies, molecular biology, microbiology, chemical synthesis, chemical analysis, drug preparation, formulation and delivery, and patient treatment.

[0046] For the purposes of this disclosure, the following terms are defined as follows.

[0047] The article “a / an” is used herein to refer to one or more (i.e., at least one) grammatical object of said article. For example, “element” includes “an element,” “one or more elements,” and / or “at least one element.”

[0048] “About” means a quantity, level, value, number, frequency, percentage, size, size, quantity, weight, or length that varies by as much as 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% relative to a reference quantity, level, value, number, frequency, percentage, size, quantity, weight, or length.

[0049] “Antagonist” means a biological or chemical agent that interferes with or otherwise reduces the physiological effects of another agent or molecule. In some cases, antagonists bind specifically to another agent or molecule. This includes full antagonists and partial antagonists.

[0050] “Agonist” means a biological or chemical agent that increases or enhances the physiological effects of another agent or molecule. In some cases, agonists bind specifically to another agent or molecule. This includes full agonists and partial agonists.

[0051] As used herein, the term “amino acid” is intended to refer to naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and imitators. For example, naturally occurring amino acids include the 20 (L)-amino acids used in protein biosynthesis, as well as other amino acids such as 4-hydroxyproline, hydroxylysine, desmodium, isodesmodium, homocysteine, citrulline, and ornithine. Non-naturally occurring amino acids include those known to those skilled in the art, such as (D)-amino acids, leucine, valine, p-fluorophenylalanine, ethionine, etc. Amino acid analogs include modified forms of naturally occurring and non-naturally occurring amino acids. Such modifications may include, for example, substitution or replacement of chemical groups and portions on the amino acid, or derivatization of the amino acid. Amino acid mimics include organic structures that exhibit functionally similar properties, such as charge and charge spacing characteristics, to a reference amino acid. For example, an organic structure mimicking arginine (Arg or R) would have a positively charged portion located in a similar molecular space and having the same mobility as the e-amino group of the side chain of a naturally occurring Arg amino acid. Mimics also include constrained structures to maintain optimal spacing and charge interactions between the amino acid or amino acid functional groups. Those skilled in the art know or can determine which structures constitute functionally equivalent amino acid analogs and amino acid mimics.

[0052] As used herein, a subject who is “at risk” of developing a disease or experiencing an adverse reaction may or may not have a detectable disease or disease symptoms, and may or may not have exhibited a detectable disease or disease symptoms prior to the treatments described herein. “At risk” means that the subject has one or more risk factors, which are measurable parameters associated with the development of a disease as described herein and known in the art. A subject with one or more of these risk factors is more likely to develop a disease or experience an adverse reaction than a subject without one or more of these risk factors.

[0053] "Biocompatible" means a material or compound that generally does not impair the biological function of cells or subjects and does not cause any degree of unacceptable toxicity (including allergies and disease states).

[0054] The term "binding" means a direct association between two molecules resulting from interactions such as covalent, electrostatic, hydrophobic, and ionic and / or hydrogen bonding (including interactions such as salt bridges and water bridges).

[0055] "Coding sequence" means any nucleic acid sequence that contributes to the encoding of a polypeptide product of a gene. Conversely, the term "non-coding sequence" means any nucleic acid sequence that does not directly contribute to the encoding of a polypeptide product of a gene.

[0056] Throughout this disclosure, unless the context otherwise requires, the words "comprise," "comprises," and "comprising" are to be understood as implying inclusion of the stated steps or elements or groups of steps or elements, but not excluding any other steps or elements or groups of steps or elements.

[0057] "Comprising..." means including and limited to anything following the phrase "comprising...". Thus, the phrase "comprising..." indicates that the listed element is necessary or mandatory, and other elements may be absent. "Substantially comprising..." means including any element listed following the phrase, and limited to other elements that do not interfere with or facilitate the activity or action specified for the listed element in this disclosure. Thus, the phrase "substantially comprising..." indicates that the listed element is necessary or mandatory, but other elements are optional and may be present or absent depending on whether they substantially affect the activity or action of the listed element.

[0058] The terms "endotoxin-free" or "substantially endotoxin-free" generally mean that the composition, solvent, and / or blood vessels contain at most trace amounts (e.g., amounts that have no clinically adverse physiological effects on the subject) of endotoxin and preferably contain undetectable amounts of endotoxin. Endotoxins are toxins associated with certain microorganisms, such as bacteria (usually Gram-negative bacteria), but can also be found in Gram-positive bacteria such as Listeria monocytogenes. The most common endotoxins are lipopolysaccharides (LPS) or lipooliposomes (LOS) found in the outer membranes of various Gram-negative bacteria and representing the central pathogenic characteristic of these bacteria in their ability to cause disease. Small amounts of endotoxins in the human body can produce fever, decreased blood pressure, inflammation, and activation of coagulation, as well as other adverse physiological effects.

[0059] Therefore, in pharmaceutical manufacturing, it is generally desirable to remove most or all of the endotoxins from the pharmaceutical product and / or pharmaceutical container.All trace amounts of endotoxins are considered harmful because even small amounts can have adverse effects on humans. A depyrogenation oven can be used for this purpose, as the decomposition of most endotoxins typically requires temperatures exceeding 300°C. For example, based on primary packaging materials such as syringes or vials, a combination of a glass temperature of 250°C and a holding time of 30 minutes is usually sufficient to achieve a 3-log reduction in endotoxin levels. Other methods for removing endotoxins are envisioned, including, for example, chromatography and filtration as described herein and known in the art.

[0060] Endotoxins can be detected using conventional techniques known in the art. For example, the horseshoe crab blood lysate assay is a very sensitive assay for detecting the presence of endotoxins. In this test, very low levels of LPS can cause detectable coagulation in the horseshoe crab lysate due to the strong enzyme cascade amplifying the reaction. Endotoxins can also be quantified by enzyme-linked immunosorbent assay (ELISA). To be substantially free of endotoxins, endotoxin levels may be below about 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.08, 0.09, 0.1, 0.5, 1.0, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, or 10 EU / mg of active compound. Typically, 1 ng of lipopolysaccharide (LPS) corresponds to about 1–10 EU.

[0061] The term “half-maximum effective concentration” or “EC50” refers to the concentration of the agent (e.g., a protein complex) as described herein that induces a response between baseline and maximum after some specific exposure time; thus, the EC50 of a graded dose-response curve represents the concentration at which the compound is observed to have 50% of its maximum effect. EC50 also represents the plasma concentration required to obtain 50% of the maximum effect in vivo. Similarly, "EC90" refers to the concentration of a pharmaceutical agent or composition at which the maximum effect is observed. "EC90" can be calculated based on "EC50" and the Hill slope, or determined directly from data using conventional knowledge in the art. In some embodiments, the EC50 of the pharmaceutical agent is less than about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, or 500 nM. In some embodiments, the EC50 value of the pharmaceutical agent is about 1 nM or less.

[0062] The "half-life" of a drug can refer to the drug's activity relative to its activity when applied to the serum or tissues of an organism.Half-life refers to the time taken for a drug to lose half of its pharmacological, physiological, or other activity relative to any other defined point in time. Half-life can also refer to the time taken for the amount or concentration of a drug to decrease by half relative to such amount or concentration when applied to the serum or tissues of an organism, or relative to any other defined point in time, from the initial amount applied to the serum or tissues of an organism. Half-life can be measured in serum and / or any one or more selected tissues.

[0063] The term "heterogeneous" refers to a feature or element in a polypeptide or encoding polynucleotide that originates from a source different from the wild-type polypeptide or encoding polynucleotide, such as a feature from a species different from the wild-type, or a non-natural engineered feature.

[0064] The terms "modulation" and "alteration" include "increase," "enhancement," or "stimulation," and "decrease" or "reduction" in a quantity or degree generally statistically or physiologically significant relative to a control. The “increased,” “stimulating,” or “enhanced” amount is generally a “statistically significant” amount and may include an increase of about 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, or 1000 times compared to the amount produced by the no composition (e.g., in the absence of the drug) or the control composition. The “reduced” or “lower” amount is generally a “statistically significant” amount and may include a reduction of about or at least about 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, or 1000 times less than that produced by the no composition (e.g., the absence of the drug) or the control composition. Examples of comparative and “statistically significant” amounts are described herein.

[0065] The terms “polypeptide,” “protein,” and “peptide” are used interchangeably and refer to polymers of amino acids not limited to any particular length. The term “enzyme” includes polypeptide or protein catalysts. As used herein, “preprotein,” “preenzyme,” or “zymogen” refers to an inactive (or substantially inactive) protein or enzyme that is typically activated by protease cleavage of an activating peptide to produce an active protein or enzyme. The terminology includes modifications such as myristylation, sulfation, glycosylation, phosphorylation, and the addition or deletion of a signal sequence. The term “polypeptide” or “protein” means one or more chains of amino acids, each chain containing amino acids covalently linked by peptide bonds, and said polypeptide or protein may comprise amino acids non-covalently and / or covalently linked by peptide bonds, having been derived from natural proteins (i.e., from naturally occurring cells, specifically non-recombinant cells, or genetically engineered).A polypeptide is a multiple strand of a protein (produced by a cell or recombinant cell) and comprises a molecule having the amino acid sequence of a natural protein or a molecule having one or more amino acids of a natural sequence with deletions, additions, and / or substitutions. In some embodiments, the polypeptide is a “recombinant” polypeptide produced by a recombinant cell containing one or more recombinant DNA molecules, which are typically made from heterologous polynucleotide sequences or combinations of polynucleotide sequences that cannot be found otherwise in the cell.

[0066] The terms “polynucleotide” and “nucleic acid” include mRNA, RNA, cRNA, cDNA, and DNA. The terms generally refer to a polymeric form of nucleotides with a length of at least 10 bases, either ribonucleotides, deoxynucleotides, or modified forms of any type of nucleotide. The terms include single-stranded and double-stranded forms of DNA. The terms “isolated DNA”, “isolated polynucleotide”, and “isolated nucleic acid” refer to molecules that have been isolated from total genomic DNA of a specific species. Therefore, the isolated DNA fragment encoding the polypeptide refers to a DNA fragment containing one or more coding sequences but which is essentially isolated from or purified from the total genomic DNA of the species from which the DNA fragment was obtained. This also includes non-coding polynucleotides that do not encode the polypeptide (e.g., primers, probes, oligonucleotides). It also includes recombinant vectors, including, for example, expression vectors, viral vectors, plasmids, phage particles, bacteriophages, viruses, etc.

[0067] Additional coding or non-coding sequences may be present within the polynucleotides described herein, but are not required to be present, and the polynucleotide may be, but is not required to be linked to other molecules and / or supporting materials. Therefore, polynucleotides or expressible polynucleotides, regardless of the length of the coding sequence itself, can be combined with other sequences, such as expression control sequences.

[0068] “Expression control sequences” include regulatory sequences of nucleic acids or corresponding amino acids, such as promoters, precursors, enhancers, introns, recognition motifs of RNA or DNA-binding proteins, polyadenylation signals, terminators, internal ribosome entry sites (IRES), secretion signals, subcellular localization signals, etc., which are capable of influencing the transcription or translation or subcellular or cellular localization of coding sequences in host cells. An exemplary expression control sequence is described in the following literature: Goeddel; *Gene Expression Technology: Methods in Enzymology*, 185, Academic Press, San Diego, California (1990).

[0069] A “promoter” is a DNA regulatory region that binds to RNA polymerase in a cell and initiates transcription of a downstream (3' direction) coding sequence. As used herein, the promoter sequence is defined at its 3' end by a transcription start site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at a level detectable above background. The transcription start site can be found within the promoter sequence and the protein-binding domain (common sequence) responsible for RNA polymerase binding (conveniently defined by mapping with nuclease S1). Eukaryotic promoters typically may, but do not always, contain a “TATA” box and a “CAT” box. Prokaryotic promoters contain a Shine-Dalgarno sequence in addition to the -10 and -35 common sequences.

[0070] A large number of promoters from a variety of different sources, including constitutive, inducible, and repressive promoters, are well known in the art. Representative sources include, for example, viral, mammalian, insect, plant, yeast, and bacterial cell types, and suitable promoters from these sources are readily available or can be synthesized based on sequences publicly available online or from depositories such as ATCC and other commercial or private sources. Promoters can be unidirectional (i.e., initiating transcription in one direction) or bidirectional (i.e., initiating transcription in the 3' or 5' direction). Non-limiting examples of promoters include, for example, the T7 bacterial expression system, the pBAD(araA) bacterial expression system, the cytomegalovirus (CMV) promoter, the SV40 promoter, and the RSV promoter. Inducible promoters include the Tet system (US Patents 5,464,758 and 5,814,618), the ecdysone-inducible system (No. 7,112,715, Indra et al., Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci.) (1996) 93(8): 3346-3351; T-REx™ system (Invitrogen Carlsbad, CA)), LacSwitch® (Stratagene, San Diego, CA)), and the Cre-ERT tamoxifen-inducible recombinase system (Indra et al., Nucleic Acid Research (Nuc. Acid. Res.) (1999) 27(22): 4324-4327; Nucleic Acid Research (2000) 28(23): e99; US Patent No. 7,112,715). Number; and Kramer and Fussenegger, *Methods in Molecular Biology* (2005) 308:123-144) or any promoter known in the art suitable for expression in desired cells.

[0071] "Expressible polynucleotide" includes cDNA, RNA, mRNA or other polynucleotides that contain at least one coding sequence and optionally at least one expression control sequence (e.g., transcription and / or translation regulatory elements) and can express the encoded polypeptide when introduced into cells.

[0072] The term “isolated” polypeptide or protein as used herein means that the test protein: (1) is free from at least some other proteins that are normally found with the test protein in nature; (2) is substantially free from other proteins from the same source, for example, from the same species; (3) is expressed by cells from different species; (4) has been isolated from the test protein in at least about 50% of a polynucleotide, lipid, carbohydrate or other material associated with it in natural inoculation; (5) is not associated with the protein portion of the “isolated protein” associated with it in nature (through covalent or non-covalent interactions); (6) is operatively associated with polypeptides that are not associated with the test protein in nature (through covalent or non-covalent interactions); or (7) is not present in nature. Such isolated proteins may be encoded by genomic DNA, cDNA, mRNA or other RNA, or may have a synthetic source, or any combination thereof. In some embodiments, the isolated protein is substantially free of proteins or peptides or other contaminants found in its natural environment that would interfere with its use (therapeutic, diagnostic, preventative, research, or otherwise).

[0073] In some embodiments, the “purity” of any given agent in the composition can be defined. For example, some compositions may include agents such as peptide agents, measured by protein or weight-to-weight ratio, such as, but not limited to, by high-performance liquid chromatography (HPLC), with a purity of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, encompassing all decimals and ranges therebetween, said HPLC being a well-known form of column chromatography commonly used in biochemistry and analytical chemistry for the separation, identification, and quantification of compounds.

[0074] The term “reference sequence” generally refers to a nucleic acid coding sequence or amino acid sequence to which another sequence is compared. All peptide and polynucleotide sequences described herein are included as reference sequences, including sequences described by name and sequences described in tables and sequence listings.

[0075] Some embodiments include bioactive “variants” and “fragments” of the proteins / peptides described herein, as well as polynucleotides encoding them. The “variants” contain one of the following relative to a reference polypeptide or polynucleotide (see, for example, the tables and sequence listings).Variant polypeptides or polynucleotides may have multiple substitutions, additions, deletions, and / or insertions. The variant polypeptides or polynucleotides contain an amino acid or nucleotide sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity, similarity, or homology to the reference sequence as described herein, and substantially retain the activity of the reference sequence. This also includes sequences consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 or more amino acids or nucleotides, or sequences that differ from a reference sequence by the addition, deletion, insertion or substitution of said amino acids or nucleotides and substantially retain at least one active element of said reference sequence. In some embodiments, additions or deletions include C-terminal and / or N-terminal additions and / or deletions.

[0076] As used herein, the term “sequence identity” or, for example, “a sequence that is 50% identical to…” refers to the degree to which sequences are identical on a nucleotide-by-nucleotide or amino acid-by-amino acid basis within a comparison window. Therefore, the "sequence identity percentage" can be calculated as follows: compare two optimally aligned sequences within a comparison window, determine the number of positions in which the same nucleic acid bases (e.g., A, T, C, G, I) or the same amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) appear in both sequences to produce the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiply the result by 100 to produce the sequence identity percentage. The optimal alignment of sequences for the comparison window can be achieved through a computerized implementation of the algorithm (GAP, BESTFIT, FASTA, and TFASTA in version 7.0 of the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive Madison, Wisconsin, USA), or by examining and determining the best alignment produced by any of the chosen methods (i.e., producing the highest percentage of homology within the comparison window). Reference can also be made to, for example, the BLAST family of programs disclosed by Altschul et al., Nucleic Acid Research 25:3389, 1997.

[0077] The term “solubility” refers to the property of a pharmaceutical agent described herein to dissolve in a liquid solvent and form a homogeneous solution. Solubility is generally expressed as concentration and is the mass of solute per unit volume of solvent (g of solute per kg of solvent, g / dL (100 mL), mg / ml, etc.), molar concentration, mass molar concentration, mole fraction, or other similar concentration descriptions. The maximum equilibrium amount of solute that can dissolve each unit volume of solvent is the solubility of the solute in the solvent under specific conditions including temperature, pressure, pH, and the properties of the solvent. In some embodiments, solubility is measured at physiological pH or other pH values, such as pH 5.0, pH 6.0, pH 7.0, pH 7.4, pH 7.6, pH 7.8, or pH 8.0 (e.g., about pH 5–8). In some embodiments, solubility is measured in water or physiological buffers such as PBS or NaCl (with or without NaPO4). In specific embodiments, solubility is measured at relatively low pH (e.g., pH 6.0) and relatively high salt concentrations (e.g., 500 mM NaCl and 10 mM NaPO4). In some embodiments, solubility is measured in biological fluids (solvents) such as blood or serum. In some embodiments, the temperature may be approximately room temperature (e.g., approximately 20°C, 21°C, 22°C, 23°C, 24°C, 25°C) or approximately body temperature (37°C). In some embodiments, the agent has a solubility of at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 mg / ml at room temperature or 37°C.

[0078] “Subject” or “subject in need” or “patient” or “patient in need” includes mammalian subjects such as human subjects.

[0079] “Substantially” or “truly” means almost completely or thoroughly, for example, 95%, 96%, 97%, 98%, 99%, or higher for some given amount.

[0080] “Statistically significant” means that the result could not have been accidental. Statistical significance can be determined by any method known in the art. Commonly used measures of significance include the p-value, which is the frequency or probability of the observed event occurring if the null hypothesis is true. If the obtained p-value is less than the significance level, the null hypothesis is rejected. In simple cases, the significance level is defined as a p-value of 0.05 or less.

[0081] “Treatment response” refers to symptom improvement (whether or not it is sustained) based on the administration of one or more therapeutic agents.

[0082] As used herein, the terms “therapeutic effective amount,” “therapeutic dose,” “preventive effective amount,” or “diagnostic effective amount” are the amounts of a pharmaceutical agent required to elicit a desired biological response after administration.

[0083] As used herein, “treatment” of a subject (e.g., a mammal, such as a human) or cell is any type of intervention intended to alter the natural course of an individual or cell. Treatment includes, but is not limited to, administration of a pharmaceutical composition and may be preventative or administered after the onset of a pathological event or after exposure to a pathogen. “Preventative” treatment is also included, which may be designed to reduce the rate of progression of the treated disease or symptom, delay the onset of said disease or symptom, or reduce the severity of its onset. “Treatment” or “prevention” does not necessarily indicate the complete eradication, cure, or prevention of a disease or symptom or its associated symptoms.

[0084] The term “wildtype” refers to the gene or gene product (e.g., a polypeptide) most commonly observed in a population and is therefore arbitrarily designed as a “normal” or “wildtype” form of the gene.

[0085] Unless otherwise explicitly stated, each embodiment in this specification applies to every other embodiment.

[0086] Protein Complexes

[0087] The embodiments of this disclosure generally relate to pharmaceutical compositions comprising protein complexes of α-2-macroglobulin (A2M) protein and serine protease proteins such as PPE. Here, certain serine proteases are capable of killing cancer cells upon direct contact with or administration to a tumor (e.g., intratumoral administration), regardless of their genetic abnormalities, and are relatively harmless to non-cancer cells or healthy cells (see, for example, WO 2018 / 232273; WO / 2020 / 132465; PCT / US2021 / 046453; and PCT / 2021 / 046467). However, in some cases, systemic administration of independent serine proteases such as PPE can adversely affect coagulation, for example, by inducing fibrinogen cleavage. This disclosure relates in part to the discovery that a protein complex of human A2M and a serine protease, such as PPE, not only spatially protects the serine protease from the effects of serine protease inhibitors in plasma while retaining CD95 protease cleavage and cancer cell killing activity, but also reduces the negative effects of serine protease on coagulation, for example, by spatially inhibiting or otherwise reducing the ability of the complex serine protease to cleave fibrinogen. This disclosure further relates to the discovery of a series of optimal molar ratios between [A2M protein] and [serine protease protein] in the protein complex, which provide a balance between retaining the CD95 cleavage and cancer cell killing activity of the serine protease and reducing its negative effects on coagulation, as measured, for example, by reducing prothrombin time or reducing fibrinogen cleavage relative to the serine protease alone.

[0088] Therefore, some embodiments include pharmaceutical compositions comprising a protein complex of: (a) an α-2-macroglobulin (A2M) protein; and (b) a serine protease protein, wherein (a) and (b) are present in the composition in a molar ratio of about 1:3 to about 1:1 [(a):(b)], including the A2M protein of (a) and the serine protease protein of (b) bound together in the protein complex. In some embodiments, the protein complex: (i) retains the CD95 (Fas receptor) protease cleavage activity and cancer cell killing activity of (b); (ii) spatially inhibits the binding of (b) to fibrinogen, thereby reducing or inhibiting the fibrinogen cleavage activity of (b); and (iii) spatially inhibits the binding of (b) to a serine protease inhibitor such as α-1 antitrypsin (A1AT), thereby protecting (b) from inhibition by the serine protease inhibitor.

[0089] In some embodiments, the A2M proteins are bound together in the protein complex as A2M monomers or A2M polymers, for example as A2M dimers, such as A2M homodimers, as A2M trimers, such as A2M homotrimers, or as A2M tetramers, such as A2M homotetramers. In specific embodiments, the A2M proteins of (a) are bound together as A2M homotetramers, and the serine protease protein of (b) is bound to the protein complex by the A2M homotetramer. In specific embodiments, each protein complex consists of a set of A2M homotetramers (i.e., four A2M proteins, as (i) four whole A2M proteins or as (ii) up to eight A2M fragments generated by the serine protease cleaving the decoy region of the whole A2M protein, since the serine protease is added to the A2M homotetramer complex) and two serine protease proteins (see, for example, Figure 1).

[0090] In some embodiments, the protein complex spatially prevents (b) from binding to larger molecules but not to smaller molecules. That is, as described above, the protein complex spatially prevents (b) from binding to larger molecules such as serine protease proteins (e.g., ALAT), fibrinogen, and / or plasma antibodies. Therefore, in some embodiments, the protein complex protects (b) from inhibition by serine proteases and also reduces / inhibits (b)'s ability to cleave fibrinogen. In some cases, for example, where the serine protease of (b) is a non-human protein such as PPE, the protein complex protects (b) from the effects of anti-PPE plasma antibodies or the generation of anti-PPE plasma antibodies. In some cases, this provides clinical advantage when administering non-human protein drugs such as PPE to humans, which can...Anti-drug antibodies are generated in other ways. In contrast, the protein complex does not spatially impede the binding of (b) to smaller molecules such as CD95. Therefore, in a specific embodiment, (b) in the protein complex cleaves CD95 and kills cancer cells, but does not substantially cleave fibrinogen.

[0091] The pharmaceutical compositions and protein complexes described herein contain α-2-macroglobulin (A2M) protein, such as human A2M protein. A2M is a highly conserved protease inhibitor present in plasma at relatively high concentrations (0.1–6 mg / ml) (Bhattacharjee et al., Journal of Biol. Chem. 275: 26806–11, 2000). It typically exists as a tetramer of four identical ~180 kDa subunits forming a hollow cylindrical structure. It can present multiple target peptide bonds to proteases that attack its central “decoy” domain. Human A2M “captures” serine proteases such as PPE: Here, after the serine protease binds to and cleaves the decoy region, a conformational change is induced in A2M, which captures the serine protease in such a way that the protease retains activity against low molecular weight substrates but exhibits significantly reduced activity against high molecular weight substrates (see, for example, Vandooren and Itoh, Frontiers in Immunology, 12, 2021; and Harwood et al., Molecular & Cellular Proteomics, 20, 2021). The amino acid sequences of full-length and mature (signal peptide-free) human A2M are provided in Table A1 below. Specification page 14 / 47 20 CN 121263518 A

[0092] Specification page 15 / 47 21 CN 121263518 A

[0093] Therefore, in some embodiments, the A2M protein portion of the protein complex comprises, is composed of, or is substantially composed of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the same amino acid sequence as, selected from the sequences in Table A1 or their functional fragments. In some embodiments, the functional fragment comprises, is composed of, or is substantially composed of about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1300, or 1400 consecutive amino acids selected from the sequences in Table A1.

[0094] For example, in a specific embodiment, the functional fragment consists of approximately residues 1-1400 selected from the sequence in Table A1.1-1300, 1-1200, 1-1100, 1-1000, 1-900, 1-800, 1-700, 1-600, 1-500, 1-400, 1-300, 1-200, Instruction manual 16 / 47 pages 22 CN 121263518 A 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, 1000-1200, 1000-1100, 1100-1400, 1100-1300, 1100- It is composed of 1200, 1200-1400, or 1200-1300. In some embodiments, its functional fragment can form an A2M homotetramer and spatially inhibit the binding of serine proteases to serine protease inhibitors (such as A1AT), thus preserving the serine protein.The conformation of an enzyme with CD95 protease cleavage activity and cancer cell killing activity, and / or an inhibitory or otherwise reduced ability of serine proteases to cleave fibrinogen, captures or otherwise binds serine proteases (such as PPE) to the protein complex.

[0095] In some embodiments, the A2M portion of the protein complex improves uptake of cancer cells relative to serine proteases alone. For example, A2M binds to LPR1 and GRP78 receptors expressed on normal cells and cancer cells. Indeed, elevated GRP78 levels are generally associated with higher pathological grades, recurrence, and poor patient survival in breast cancer, liver cancer, prostate cancer, colon cancer, and gastric cancer (see, for example, Lee, Cancer Res. 67:3496–3499, 2007), and in some cases, the ability of A2M to bind GRP78 improves selective targeting of cancer cells expressing GRP78.

[0096] In some embodiments, the A2M portion of the protein complex is fused to or otherwise conjugated to an antibody or its antigen-binding fragment. In some embodiments, the antibody or its antigen-binding fragment specifically binds to a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA). Exemplary TAAs and TSAs include, but are not limited to: alpha-fetoprotein (AFP), epithelial tumor antigen (ETA), tyrosinase, human Her2 / neu, Her1 / EGF receptor (EGFR), Her3, A33 antigen, B7H3, CD5, CD19, CD20, CD22, CD23 (IgE receptor), melanoma-associated antigen (MAGE), C242 antigen, 5T4, IL-6, IL-13, vascular endothelial growth factor VEGF (e.g., VEGF-A), VEGFR-1, VEGFR-2, VEGR-3, NRP2, CD30, CD33, CD37, CD40, etc. CD44, CD51, CD52, CD56, CD74, CD80, CD152, CD200, CD221, CCR4, HLA-DR, CTLA-4, NPC-1C, tendinin, vimentin, insulin-like growth factor 1 receptor (IGF-1R), alpha-fetoprotein, insulin-like growth factor 1 (IGF-1), carbonic anhydrase 9 (CA-IX), carcinoembryonic antigen (CEA), guanylate cyclase C, NY-ESO-1, p53, survivin, integrin αvβ3, integrin α5β1, folate receptor 1, transmembrane glycoprotein NMB, fibroblast activating protein α (FAP), glycoprotein 75, TAG-72, MUC1, MUC16 (or CA-125), phosphatidylserine, prostate-specific membrane antigen (PSMA), NR-LU-13 antigen.TRAIL-R1, tumor necrosis factor receptor superfamily member 10b (TNFRSF10B or TRAIL-R2), SLAM family member 7 (SLAMF7), EGP40 pan-cancer antigen, B cell activating factor (BAFF), platelet-derived growth factor receptor, glycoprotein EpCAM (17-1A), programmed death-1, protein disulfide isomerase (PDI), regenerating liver phosphatase 3 (PRL-3), prostatic acid phosphatase, Lewis-Y antigen, GD2 (disialyl ganglioside expressed on neuroectodermal-derived tumors), phosphatidylinositol proteoglycan-3 (GPC3), and mesothelin.

[0097] The pharmaceutical compositions and protein complexes described herein comprise serine proteases. Examples of serine proteases include porcine pancreatic elastase (PPE), human neutrophil elastase (ELANE), human tissue protease G (CTSG), human protease 3 (PR3), and human granzyme B (GZMB). The amino acid sequence of an exemplary full-length wild-type serine protease preprotein is provided in Table S1 below.

[0098]

[0099] Therefore, in some embodiments, the serine protease protein comprises, is composed of, or is substantially composed of a full-length serine protease preprotein selected from Table S1, said full-length serine protease preprotein including biologically active variants and fragments thereof. In specific embodiments, the serine protease comprises, is composed of, or is substantially composed of at least 80%, 85%, 90%, 95%, 98%, or 100% of the same amino acid sequence as the sequence selected from Table S1.

[0100] In some embodiments, the serine protease protein is composed of the active peptidase domain of a serine protease. Exemplary peptidase domain sequences of PPE (including its exemplary mutants), human ELANE, human CTSG, and human PR3 are provided in Table S2 below. Specification 18 / 47 pages 24 CN 121263518 A

[0101] Specification 19 / 47 pages 25 CN 121263518 A Specification 20 / 47 pages 26 CN 121263518 A

[0102] Therefore, in some embodiments, the serine protease protein comprises, is composed of, or is substantially composed of a serine protease peptidase domain sequence selected from Table S2, said serine protease peptidase domain sequence including biologically active variants and fragments thereof. In specific embodiments, the serine protease protein comprises, is composed of, or is substantially composed of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 100% identical to a sequence selected from Table S2.

[0103] In some embodiments, the serine protease protein is a PPE protein, for example, wherein:The PPE protein comprises, is composed of, or is substantially composed of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, SEQ ID NO: 5, and retains the Q211F amino acid substitution; the PPE protein comprises, is composed of, or is substantially composed of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, SEQ ID NO: 6, and retains the T55A amino acid substitution; the PPE protein comprises, is composed of, or is substantially composed of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, SEQ ID NO: 7, and retains the Q211F and T55A amino acid substitutions; the PPE protein comprises, is composed of, or is substantially composed of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, SEQ ID NO: 8, and retains the N241A amino acid substitution; The PPE protein comprises, is composed of, or is substantially composed of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the N241Y amino acid substitution, of SEQ ID NO: 9; the PPE protein comprises, is composed of, or is substantially composed of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the R75A amino acid substitution, of SEQ ID NO: 10; the PPE protein comprises, is composed of, or is substantially composed of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the R75E amino acid substitution, of SEQ ID NO: 11; the PPE protein comprises, is composed of, or is substantially composed of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the Q211A amino acid substitution, of SEQ ID NO: 12; the PPE protein comprises, is composed of, or is substantially composed of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the Q211A amino acid substitution, of SEQ ID NO: 9; the PPE protein comprises, is composed of, and ... NO: 13 The PPE protein comprises, is composed of, or is substantially composed of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the amino acid substitution of R237A; The PPE protein comprises, is composed of, or is substantially composed of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the amino acid substitution of S214A; The PPE protein comprises, is composed of, or is substantially composed of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the amino acid substitution of D74A; andThe PPE protein comprises, is composed of, or is substantially composed of the same amino acid sequence as SEQ ID NO: 16, at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% as specified on page 21 / 47 of the specification, CN 121263518 A.

[0104] In some embodiments, the serine protease protein is a human ELANE protein, for example, wherein the human ELANE protein comprises, is composed of, or is substantially composed of the same amino acid sequence as SEQ ID NO: 17, at least 80%, 85%, 90%, 95%, 98%, 99%, or 100%. In some embodiments, the serine protease protein is a human CTSG protein, for example, wherein the human CTSG protein comprises, is composed of, or is substantially composed of the same amino acid sequence as SEQ ID NO: 18, at least 80%, 85%, 90%, 95%, 98%, 99%, or 100%. In some embodiments, the serine protease protein is a human PR3 protein, for example, wherein the human PR3 protein comprises, is composed of, or is substantially composed of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO: 19. In some embodiments, the serine protease protein is a human granzyme B protein, for example, wherein the human granzyme B protein comprises, is composed of, or is substantially composed of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO: 20.

[0105] As described above, in some embodiments, (a) and (b) are present in the composition in a molar ratio of [(a):(b)] in the range of about 1:3 to about 1:1, for example, 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. In a specific aspect, the molar ratio defined herein retains the CD95 protease cleavage and cancer cell killing activity of the serine protease while spatially hindering its binding to serine protease inhibitors (e.g., ALAT), fibrinogen, and / or plasma antibodies, thereby protecting it from inhibition by the serine protease inhibitors or plasma antibodies, and inhibiting or otherwise reducing its ability to cleave fibrinogen.

[0106] Thus, in some embodiments, the protein complex retains the ability to cleave CD95 (Fas receptor) and substantially does not cleave fibrinogen. In some embodiments, the protein complex described herein has about or at least about 50%, 60%,70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% or more of the corresponding serine protease protein's own CD95 protease cleavage and / or cancer cell killing activity (e.g., in the presence of serine protease inhibitors in plasma such as A1AT). In some embodiments, the protein complexes described herein have about or less than about 50%, 40%, 30%, 20%, 10%, 5%, or less of the corresponding serine protease protein's own fibrinogen protease cleavage activity. CD95 cleavage activity, cancer cell killing activity, fibrinogen cleavage activity, and coagulation properties can be measured according to conventional techniques in the art (see Examples). For example, more generally, serine protease activity can be monitored using a colorimetric substrate activity assay (N-methoxysuccinoyl-Ala-Ala-Pro-Val p-nitroaniline), and CD95 and / or fibrinogen cleavage can be measured directly (e.g., Western blotting). As needed, protease cleavage activity can be measured in the presence of serine protease inhibitors such as A1AT. Cancer cell killing activity can be measured in vitro or in vivo, and the effect on in vivo coagulation can be measured, for example, by routine assays such as prothrombin (PT) time and partial thromboplastin (PTT) time (see Examples).

[0107] In some embodiments, the protein complex described herein is generated in vivo or in vitro, for example, in cells by contacting cells or a subject with one or more expressible polynucleotides encoding (a) α-2-macroglobulin (A2M) protein and (b) serine protease protein. The protein complex is then formed in the cell. “Expressible polynucleotide” includes DNA, cDNA, RNA, mRNA or other polynucleotides comprising at least one coding sequence for (a) and / or (b) and optionally at least one expression control sequence (e.g., transcriptional and / or translational regulatory elements) and capable of expressing the encoded protein when introduced into cells (e.g., the subject’s cells). Some embodiments involve contacting in vitro cells with one or more expressible polynucleotides encoding (a) and (b) and administering the cells to a subject.

[0108] Exemplary viral vectors that can be used to deliver expressible polynucleotides include adenovirus vectors, herpesvirus vectors, vaccinia virus vectors, adeno-associated virus (AAV) vectors, and retroviral vectors such as lentivirus vectors. Examples of retroviral vectors include, but are not limited to, vectors based on Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), SIV, BIV, HIV, and Rous sarcoma virus (RSV). In certain embodiments, expressible polynucleotides can be delivered...The polynucleotide is a modified RNA or a modified mRNA polynucleotide, for example, a non-naturally occurring RNA analog. In some embodiments, the modified RNA or mRNA polypeptide contains one or more modified or non-natural bases. In some embodiments, the modified mRNA contains one or more modified or non-natural nucleotide links. Expressible RNA polynucleotides for delivering encoded proteins are described, for example, Kormann et al., Nature Biotechnology 29:154-7, 2011; and U.S. Applications 2015 / 0111248; 2014 / 0243399; 2014 / 0147454; and 2013 / 0245104, all of which are incorporated herein by reference in their entirety.

[0109] In some embodiments, the protein complexes described herein have one or more improved biological, physical, and / or pharmacokinetic properties relative to the corresponding serine protease protein itself. The protein complexes described herein can be used in any of the compositions, methods, and / or kits described herein.

[0110] Methods of Use and Pharmaceutical Compositions

[0111] Some embodiments include methods for treating a disease or condition in a subject of need, improving symptoms of said disease or condition, and / or reducing the progression of said disease or condition, said methods comprising administering to said subject a composition comprising the protein complexes as described herein. In a particular embodiment, said disease is cancer, i.e., the subject of need has, is suspected of having, or is at risk of developing cancer.

[0112] In a particular embodiment, said cancer is primary cancer or metastatic cancer. In a specific embodiment, the cancer is selected from one or more of the following: melanoma (optionally metastatic melanoma), breast cancer (optionally triple-negative breast cancer, TNBC), kidney cancer (optionally renal cell carcinoma), pancreatic cancer, bone cancer, prostate cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer (NSCLC), squamous cell lung cancer), mesothelioma, leukemia (optionally lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, or relapsed acute myeloid leukemia), multiple myeloma, lymphoma, liver cancer (hepatocellular carcinoma), sarcoma, B-cell malignancy, ovarian cancer, colorectal cancer, glioma, glioblastoma multiforme, meningioma, pituitary adenoma, vestibular schwannoma, primary CNS lymphoma, primitive neuroectodermal tumor (neuroblastoma), bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancer, cervical cancer, testicular cancer, thyroid cancer, and gastric cancer.

[0113] In some embodiments, as described above, the cancer is a metastatic cancer. Further, for the above-described cancers, exemplary metastatic cancers include, but are not limited to: bladder cancer that has metastasized to the bones, liver, and / or lungs; cancer that has metastasized to the bones,Breast cancer of the brain, liver, and / or lungs; colorectal cancer that has metastasized to the liver, lungs, and / or peritoneum; kidney cancer that has metastasized to the adrenal glands, bones, brain, liver, and / or lungs; lung cancer that has metastasized to the adrenal glands, bones, brain, liver, and / or other lung sites; melanoma that has metastasized to the bones, brain, liver, lungs, and / or skin / muscles; ovarian cancer that has metastasized to the liver, lungs, and / or peritoneum; pancreatic cancer that has metastasized to the liver, lungs, and / or peritoneum; prostate cancer that has metastasized to the adrenal glands, bones, liver, and / or lungs; gastric cancer that has metastasized to the liver, lungs, and / or peritoneum; thyroid cancer that has metastasized to the bones, liver, and / or lungs; and uterine cancer that has metastasized to the bones, liver, lungs, peritoneum, and / or vagina, etc.

[0114] In some embodiments, administration (e.g., intravenous administration) of a protein complex or composition containing a serine protease substantially does not affect the subject's coagulation, for example, it substantially does not increase the prothrombin time or partial prothrombin kinase time in the subject (e.g., relative to the administration of the corresponding serine protease alone). In some embodiments, administration of the protein complex containing the serine protease described herein has a significantly reduced effect on coagulation in the subject compared to administration of the corresponding serine protease alone.

[0115] Methods for treating cancer can be combined with other treatment modalities. For example, the combination therapies described herein can be administered to the subject before, during, or after other therapeutic interventions, including symptomatic care, radiotherapy, surgery, transplantation, hormone therapy, photodynamic therapy, antibiotic therapy, or any combination thereof. Symptomatic care includes administration of corticosteroids to reduce cerebral edema, headache, cognitive impairment, and vomiting, and administration of anticonvulsants to reduce seizures. Radiotherapy includes whole-brain irradiation, fractionated radiotherapy, and radiosurgery such as stereotactic radiosurgery, which can be further combined with conventional surgery.

[0116] Therefore, some embodiments include combination therapies for treating cancer, the combination therapy comprising methods of treating and improving symptoms of cancer or inhibiting cancer progression in a subject in need, the methods comprising administering to the subject a composition of a protein complex described herein in combination with at least one additional agent, such as an immunotherapeutic agent, a chemotherapy agent, a hormone therapy agent, and / or a kinase inhibitor. In some embodiments, administration of the composition, only relative to the additional agent, increases susceptibility of cancer to the additional agent (e.g., an immunotherapeutic agent, a chemotherapy agent, a hormone therapy agent, and / or a kinase inhibitor) by about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, or 90%.100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2000%, or more.

[0117] Certain combination therapies employ one or more cancer immunotherapeutic agents or "immunotherapy agents." In some cases, immunotherapy agents modulate the immune response of a subject, for example, to increase or maintain a cancer-related or cancer-specific immune response, and thereby result in increased suppression of immune cells or reduction of cancer cells. Exemplary immunotherapy agents include peptides, such as antibodies and their antigen-binding fragments, ligands, and small peptides, and mixtures thereof. Immunotherapy agents also include small molecules, cells (e.g., immune cells such as T cells), various cancer vaccines, gene therapy agents, or other polynucleotide-based agents, including viral agents such as oncolytic viruses, and other agents known in the art. Thus, in some embodiments, cancer immunotherapy agents are selected from one or more of immune checkpoint modulators, cancer vaccines, oncolytic viruses, cytokines, and cell-based immunotherapies.

[0118] In some embodiments, the cancer immunotherapy agent is an immune checkpoint modulator. Specific examples include “antagonists” of one or more inhibitory immune checkpoint molecules and “agonists” of one or more stimulating immune checkpoint molecules. Typically, immune checkpoint molecules are components of enhancing (co-stimulatory) or de-stimulating signals of the immune system, and targeting these components has therapeutic potential for cancer because cancer cells may disrupt the natural function of immune checkpoint molecules (see, for example, Sharma and Allison, Science 348:56-61, 2015; Topalian et al., Cancer Cell 27:450-461, 2015; Pardoll, Nature Reviews Cancer 12:252-264, 2012). In some embodiments, immune checkpoint modulators (e.g., antagonists, agonists) “bind” or “specifically bind” to one or more immune checkpoint molecules as described herein.

[0119] In some embodiments, immune checkpoint modulators are antagonists or inhibitors of one or more inhibitory immune checkpoint molecules. Exemplary inhibitory immune checkpoint molecules include: programmed death-ligand 1 (PD-L1), programmed death-ligand 2 (PD-L2), programmed death 1 (PD-1), T cell activation V domain Ig inhibitor (VISTA), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), indoleamine 2,3-dioxygenase (IDO), tryptophan 2,3-dioxygenase (TDO), T cell immunoglobulin domain and mucin domain 3 (TIM-3), lymphocyte activation gene-3 (LAG-3), and B and T lymphocyte...The agents include the cytoatogenic molecule BTLA, CD160, and the T-cell immune receptor (TIGIT) with Ig and ITIM domains.

[0120] In some embodiments, the agents are PD-1 (receptor) antagonists or inhibitors that have been shown to target the restoration of immune function in the tumor setting (see, for example, Phillips et al., International Journal of Immunology 27:39-46, 2015). PD-1 is a cell surface receptor belonging to the immunoglobulin superfamily and expressed on T cells and progenitor B cells. PD-1 interacts with two ligands, PD-L1 and PD-L2. PD-1 acts as a suppressive immune checkpoint molecule, for example, by reducing or preventing T cell activation, which in turn reduces autoimmunity and promotes self-tolerance. The inhibitory effect of PD-1 is accomplished at least in part through a dual mechanism of promoting apoptosis of antigen-specific T cells in lymph nodes while also reducing apoptosis of regulatory T cells (suppressive T cells). Some examples of PD-1 antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to PD-1 and reduce one or more of its immunosuppressive activities, such as its downstream signaling pathways or its interaction with PD-L1. Specific examples of PD-1 antagonists or inhibitors include the antibodies nivolumab, pembrolizumab, PDR001, MK-3475, AMP-224, AMP-514, and pidilizumab and their antigen-binding fragments (see, for example, U.S. Patents 8,008,449, 8,993,731, 9,073,994, 9,084,776, 9,102,727, and 9,102,728). Applications Nos. 9,181,342, 9,217,034, 9,387,247, 9,492,539, 9,492,540, and U.S. Applications Nos. 2012 / 0039906 and 2015 / 0203579.

[0121] In some embodiments, the agent is a PD-L1 antagonist or inhibitor. As described above, PD-L1 is one of the natural ligands of the PD-1 receptor. General examples of PD-L1 antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to PD-L1 and reduce one or more of its immunosuppressive activities (e.g., its binding to the PD-1 receptor). Specific examples of PD-L1 antagonists include the antibody atezolizumab (MPDL3280A), averulin, etc.Monoclonal antibodies (avelumab) (MSB0010718C) and durvalumab (MEDI4736) and their antigen-binding fragments (see, for example, U.S. Patent Nos. 9,102,725; 9,393,301; 9,402,899; 9,439,962).

[0122] In some embodiments, the agent is a PD-L2 antagonist or inhibitor. As described above, PD-L2 is one of the natural ligands of the PD-1 receptor. General examples of PD-L2 antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to PD-L2 and reduce one or more immunosuppressive activities of its immunosuppressive activity (e.g., its binding to the PD-1 receptor).

[0123] In some embodiments, the agent is a VISTA antagonist or inhibitor. VISTA is approximately 50 kDa in size and belongs to the immunoglobulin superfamily (which has an IgV domain) and the B7 family. It is primarily expressed in leukocytes, and its transcription is partially controlled by p53. There is evidence that VISTA can act as both a ligand and receptor on T cells to suppress T cell effector function and maintain peripheral tolerance. VISTA is produced at high levels in tumor-infiltrating lymphocytes such as myeloid-derived suppressor cells and regulatory T cells, and its blockade with antibodies leads to delayed tumor growth in mouse models of melanoma and squamous cell carcinoma. Exemplary anti-VISTA antagonist antibodies include, for example, the antibodies described in WO 2018 / 237287, which is incorporated herein by reference in its entirety.

[0124] In some embodiments, the agent is a CTLA-4 antagonist or inhibitor. CTLA4 or CTLA-4 (cytotoxic T-lymphocyte-associated protein 4), also known as CD152 (differentiation cluster 152), is a protein receptor that acts as a suppressive immune checkpoint molecule, for example, by transmitting an inhibitory signal to the T cell when the T cell binds to CD80 or CD86 on the surface of the antigen-presenting cell. General examples of CTLA-4 antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to CTLA-4. Specific examples include the antibodies ipilimumab and tremelimumab, and their antigen-binding fragments. It is believed that at least some of the activity of ipilimumab is mediated by antibody-dependent cell-mediated cytotoxicity (ADCC) killing by inhibiting the CTLA-4 inhibitor Treg.

[0125] In some embodiments, the agent is an IDO antagonist or inhibitor or a TDO antagonist or inhibitor. IDO and TDO are tryptophan-degrading enzymes with immunosuppressive properties. For example, IDO is known to inhibit T cells and NK cells, producing and activatingTregs and myeloid-derived suppressor cells, and promote tumor angiogenesis. General examples of IDO and TDO antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to IDO or TDO (see, for example, Platten et al., Frontiers in Immunol. 5: 673, 2014) and reduce or inhibit one or more immunosuppressive activities. Specific examples of IDO antagonists or inhibitors include indoximod (NLG-8189), 1-methyl-tryptophan (1MT), β-carboline (norharmane, 9H-pyrido[3,4-b]indole), rosmarinic acid, and epacadostat (see, for example, Sheridan, Nature Biotechnology 33:321-322, 2015). Specific examples of agents or inhibitors include 680C91 and LM10 (see, for example, Pilote et al., Proceedings of the National Academy of Sciences of the United States of America (PNAS USA.) 109:2497-2502, 2012).

[0126] In some embodiments, the agent is a TIM-3 antagonist or inhibitor. T cell immunoglobulin domain and mucin domain 3 (TIM-3) is expressed on activated human CD4+ T cells and regulates Th1 and Th17 cytokines. TIM-3 also acts as a negative regulator of Th1 / Tc1 function by triggering cell death upon interaction with its ligand, galactagogue-9. TIM-3 contributes to a suppressive tumor microenvironment, and its overexpression is associated with poor prognosis in various cancers (see, for example, Li et al., Acta Oncol. 54:1706-13, 2015). General examples of TIM-3 antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to TIM-3 and reduce or inhibit one or more of its immunosuppressive activities.

[0127] In some embodiments, the agent is a LAG-3 antagonist or inhibitor. Lymphocyte activation gene-3 (LAG-3) is expressed on activated T cells, natural killer cells, B cells, and plasmacytoid dendritic cells. It negatively regulates T cell proliferation, activation, and homeostasis in a manner similar to that of CTLA-4 and PD-1 (see, for example, Workman and Vignali, *European Journal of Cancer* 33: 970-9, 2003; and Workman et al., *Journal of Immunology* 172: 5450-5).LAG3 (2004) has been reported to play a role in Treg suppression (see, for example, Huang et al., Immunology 21: 503-13, 2004). LAG3 also keeps CD8+ T cells in a tolerant state and, in combination with PD-1, maintains CD8 T cell exhaustion. General examples of LAG-3 antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to LAG-3 and inhibit one or more of its immunosuppressive activities. Specific examples include the antibody BMS-986016 and its antigen-binding fragment.

[0128] In some embodiments, the agent is a BTLA antagonist or inhibitor. B and T lymphocyte attenuator (BTLA; CD272) expression is induced during T cell activation and it inhibits T cells by interacting with the tumor necrosis family receptor (TNF-R) and the B7 cell surface receptor family. BTLA is a ligand for tumor necrosis factor (receptor) superfamily member 14 (TNFRSF14), also known as a herpesvirus entry mediator (HVEM). The BTLA-HVEM complex negatively regulates T cell immune responses, for example, by inhibiting the function of human CD8+ cancer-specific T cells (see, for example, Derré et al., J Clin Invest 120:157-67, 2009). General examples of BTLA antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to BTLA-4 and reduce one or more of its immunosuppressive activities.

[0129] In some embodiments, the agent is an HVEM antagonist or inhibitor, for example, an antagonist or inhibitor that specifically binds to HVEM and interferes with its interaction with BTLA or CD160. General examples of HVEM antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to HVEM, optionally reduce HVEM / BTLA and / or HVEM / CD160 interactions, and thereby reduce one or more immunosuppressive activities of HVEM's immunosuppressive activity.

[0130] In some embodiments, the agent is a CD160 antagonist or inhibitor, for example, an antagonist or inhibitor that specifically binds to CD160 and interferes with its interaction with HVEM. General examples of CD160 antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to CD160, optionally reduce CD160 / HVEM interactions, and thereby reduce or inhibit one or more immunosuppressive activities of its immunosuppressive activity.

[0131] In some embodiments, the agent is a TIGIT antagonist or inhibitor. T cell Ig and ITIM domains (TIGIT) are co-inhibitory receptors found on the surface of various lymphocytes and inhibit antitumor immunity, for example, through Tregs.(Kurtulus et al., Journal of Clinical Investigation 125:4053-4062, 2015). General examples of TIGIT antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to TIGIT and reduce one or more of its immunosuppressive activities (see, for example, Johnston et al., Cancer Cell 26:923-37, 2014).

[0132] In some embodiments, the immune checkpoint modulator is an agonist of one or more stimulating immune checkpoint molecules. Exemplary stimulating immune checkpoint molecules include CD40, OX40, glucocorticoid-induced TNFR family-associated gene (GITR), CD137 (4-1BB), CD27, CD28, CD226, and herpesvirus entry mediator (HVEM).

[0133] In some embodiments, the agent is a CD40 agonist. CD40 is expressed on antigen-presenting cells (APCs) and some malignancies. Its ligand is CD40L (CD154). On APCs, this linkage leads to the upregulation of co-stimulatory molecules, potentially bypassing the need for T-cell help in antitumor immune responses. CD40 agonist therapy plays an important role in APC maturation and its migration from tumors to lymph nodes, thereby enhancing antigen presentation and T-cell activation. Anti-CD40 agonist antibodies produce substantial responses and durable anticancer immunity in animal models, effects at least in part mediated by cytotoxic T cells (see, for example, Johnson et al., Clin Cancer Research 21: 1321-1328, 2015; and Vonderheide and Glennie, Clin Cancer Research 19:1035-43, 2013). General examples of CD40 agonists include antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to CD40 and increase one or more of its immunostimulatory activities. Specific examples include CP-870, 893, dacetuzumab, Chi Lob 7 / 4, ADC-1013, CD40L, rhCD40L, and their antigen-binding fragments. Specific examples of CD40 agonists include, but are not limited to, APX005 (see, for example, US 2012 / 0301488) and APX005M (see, for example, US 2014 / 0120103).

[0134] In some embodiments, the agent is an OX40 agonist. OX40 (CD134) promotes the expansion of effector T cells and memory T cells and inhibits the differentiation and activation of regulatory T cells (see, for example, Croft et al., Immunol Rev.).229:173-91, 2009). Its ligand is OX40L (CD252). Since OX40 signaling affects both T cell activation and survival, it plays an important role in initiating antitumor immune responses in lymph nodes and maintaining antitumor immune responses in the tumor microenvironment. General examples of OX40 agonists include one or more antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to OX40 and increase its immunostimulatory activity. Specific examples include OX86, OX-40L, Fc-OX40L, GSK3174998, MEDI0562 (humanized OX40 agonist), MEDI6469 (mouse OX4 agonist), and MEDI6383 (OX40 agonist) and their antigen-binding fragments.

[0135] In some embodiments, the agent is a GITR agonist. Glucocorticoid-induced TNFR family-associated genes (GITR) increase T cell proliferation, inhibit the inhibitory activity of Tregs, and prolong the survival of T effector daughter cells. It has been shown that GITR agonists promote antitumor responses through loss of Treg lineage stability (see, for example, Schaer et al., Cancer Immunol Res. 1:320-31, 2013). These different mechanisms demonstrate that GITR plays an important role in initiating immune responses in lymph nodes and in maintaining immune responses in tumor tissues. Its ligand is GITRL. General examples of GITR agonists include antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to GITR and increase one or more of its immunostimulatory activities. Specific examples include GITRL, INCAGN01876, DTA-1, MEDI1873, and their antigen-binding fragments.

[0136] In some embodiments, the agent is a CD137 agonist. CD137 (4-1BB) is a member of the tumor necrosis factor (TNF) receptor family, and cross-linking of CD137 enhances T cell proliferation, IL-2 secretion, survival, and cytolytic activity. CD137-mediated signaling also protects T cells, such as CD8+ T cells, from activation-induced cell death. General examples of CD137 agonists include antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to CD137 and increase one or more of its immunostimulatory activities. Specific examples include CD137 (or 4-1BB) ligands (see, for example, Shao and Schwarz, *Journal of Leukoc Biol.* 89:21-9, 2011) and the antibody utomilumab, which includes its antigen-binding fragment.

[0137] In some embodiments, the agent is a CD27 agonist. Stimulation of CD27 increases the antigen specificity of untreated T cells.It expands and contributes to the long-term maintenance of T-cell memory and T-cell immunity. Its ligand is CD70. Targeting stimulation of human CD27 with agonist antibodies activates T cells and anti-tumor immunity (see, for example, Thomas et al., Tumor Immunology Manual 27 / 47 pp. 33 CN 121263518 A (Oncoimmunology.) 2014;3:e27255. doi:10.4161 / onci.27255; and He et al., Journal of Immunology 191:4174-83, 2013). General examples of CD27 agonists include antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to CD27 and increase one or more of its immunostimulatory activities. Specific examples include CD70 and the antibodies varlilumab and CDX-1127 (1F5), including their antigen-binding fragments.

[0138] In some embodiments, the agent is a CD28 agonist. CD28 is constitutively expressed on CD4+ T cells and some CD8+ T cells. Its ligands include CD80 and CD86, and its stimulation increases T cell proliferation. General examples of CD28 agonists include antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to CD28 and increase one or more of their immunostimulatory activities. Specific examples include CD80, CD86, antibody TAB08, and its antigen-binding fragment.

[0139] In some embodiments, the agent is a CD226 agonist. CD226 is a stimulatory receptor that shares a ligand with TIGIT, and unlike TIGIT, binding to CD226 enhances T cell activation (see, for example, Kurtulus et al., *Journal of Clinical Investigation* 125:4053-4062, 2015; Bottino et al., *Journal of Experimental Medicine* 1984:557-567, 2003; and Tahara-Hanaoka et al., *International Journal of Immunology* 16:533-538, 2004). General examples of CD226 agonists include antibodies or antigen-binding fragments or small molecules or ligands (e.g., CD112, CD155) that specifically bind to CD226 and increase one or more of its immunostimulatory activities.

[0140] In some embodiments, the agent is an HVEM agonist. Herpesvirus entry mediator (HVEM), also known as tumor necrosis factor receptor superfamily member 14 (TNFRSF14), is a human cell surface receptor of the TNF receptor superfamily. HVEM is found on a variety of cells, including T cells, APCs, and other immune cells. Unlike other receptors, HVEM is present at high levels on resting T cells.HVEM signaling is expressed and downregulated upon activation. It has been shown that HVEM signaling plays an important role in the early stages of T cell activation and during the expansion of tumor-specific lymphocyte populations in lymph nodes. General examples of HVEM agonists include antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to HVEM and increase one or more of their immunostimulatory activities.

[0141] In some embodiments, the immunotherapeutic agent is a bispecific or multispecific antibody. For example, certain bispecific or multispecific antibodies are capable of (i) binding to and inhibiting one or more inhibitory immune checkpoint molecules, and also (ii) binding to and activating one or more stimulatory immune checkpoint molecules. In some embodiments, the bispecific or multispecific antibody (i) binds to and inhibits one or more of PD-L1, PD-L2, PD-1, CTLA-4, IDO, TDO, TIM-3, LAG-3, BTLA, CD160, and / or TIGIT, and also (ii) binds to and activates one or more of CD40, OX40, glucocorticoid-induced TNFR family-associated gene (GITR), CD137 (4-1BB), CD27, CD28, CD226, and / or herpesvirus entry mediator (HVEM).

[0142] In some embodiments, the immunotherapeutic agent is a cancer vaccine. In some embodiments, the cancer vaccine is selected from one or more of Oncophage, optionally Gardasil or Cervarix human papillomavirus HPV vaccines, optionally Engerix-B, Recombivax HB or Twinrix hepatitis B vaccines, and sipuleucel-T (Provenge). In some embodiments, the cancer vaccine comprises or expresses TAA or TSA as described herein.

[0143] In some embodiments, the immunotherapeutic agent is an oncolytic virus. In some embodiments, the oncolytic virus is selected from one or more of the following: talimogene laherparepvec (T-VEC), Coxsackievirus A21 (CAVATAK™), Oncorine (H101), Pelareorep (REOLYSIN®), Seneca Valley virus (NTX-010), Senecavirus SVV-001, ColoAd1, SEPREHVIR (HSV-1716), CGTG-102 (Ad5 / 3-D24-GMCSF), GL-ONC1, MV-NIS, and DNX-2401.

[0144] In some embodiments, the cancer immunotherapeutic agent is a cytokine. Exemplary cytokines include interferon.(IFN)-α, IL-2, IL-12, IL-7, IL-21, and granulocyte-macrophage colony-stimulating factor (GM-CSF). Specification 28 / 47 pages 34 CN 121263518 A

[0145] In some embodiments, the cancer immunotherapy agent is a cell-based immunotherapy, such as a therapy utilizing immune cells, said immune cells including ex vivo immune cells, such as lymphocytes, natural killer (NK) cells, macrophages, and / or dendritic cells (DCs). In some embodiments, the lymphocytes comprise T cells, such as cytotoxic T lymphocytes (CTLs). See, for example, June, *Journal of Clinical Investigation* 117: 1466–1476, 2007; Rosenberg and Restifo, *Science* 348: 62–68, 2015; Cooley et al., *Biol Blood Marrow Transplant.* 13: 33–42, 2007; and Li and Sun, *Chin J Cancer Res.* 30: 173–196, 2018, for descriptions of adoptive T-cell and NK-cell immunotherapy. In some embodiments, the T cells comprise cancer antigen-specific T cells targeting at least one cancer antigen. In some embodiments, the cancer antigen-specific T cells are selected from one or more of the following: chimeric antigen receptor (CAR) modified T cells, T cell receptor (TCR) modified T cells, tumor-infiltrating lymphocytes (TILs), and peptide-induced T cells. In specific embodiments, CAR-modified T cells target CD-19 (see, for example, Maude et al., Blood 125:4017-4023, 2015). In some cases, ex vivo immune cells are autologous cells obtained from the patient to be treated.

[0146] Some combination therapies employ one or more chemotherapeutic agents, such as small molecule chemotherapeutic agents. Non-limiting examples of chemotherapeutic agents include alkylating agents, antimetabolites, cytotoxic antibiotics, topoisomerase inhibitors (type I or II), and antimicrotubule agents, etc.

[0147] Examples of alkylating agents include: nitrogen mustard (e.g., dichloromethyldiethylamine, cyclophosphamide, nitrogen mustard, melphalan, chlorambucil, ifosfamide, and busulfan), nitrosourea (e.g., N-nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), formustine, and streptozotocin), tetrazine, etc.(e.g., dacarbazine, mitozolomide, and temozolomide), aziridine (e.g., thiotepa, mytomycin, and diaziquone (AZQ)), cisplatin and its derivatives (e.g., carboplatin and oxaliplatin), and atypical alkylating agents (optionally procarbazine and hexamethylmelamine).

[0148] Examples of antimetabolites include: antifolate agents (e.g., methotrexate and pemetrexed), fluoropyrimidines (e.g., 5-fluorouracil and capecitabine), deoxynucleoside analogs (e.g., ancitabine, enocitabine, cytarabine, gemcitabine, decitabine, azacitidine, fludarabine, nelarabine, cladribine, clofarabine, fludarabine, and pentostatin) and thiopurines (e.g., thioguanine and mecaptopurine)).

[0149] Examples of cytotoxic antibiotics include anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, aclarubicin, and mitoxantrone), bleomycins, mitomycin C, mitoxantrone, and actinomycin. Examples of topoisomerase inhibitors include camptothecin, irinotecan, topotecan, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, merbarone, and aclarubicin.

[0150] Examples of antimicrotubule agents include taxanes (e.g., paclitaxel and docetaxel) and vinca alkaloids (e.g., vinblastine, vincristine, vindesine, vinorelbine).

[0151] The various chemotherapeutic agents described herein may be combined with any one or more of the protein complexes described herein and used according to any one or more of the methods or compositions described herein.

[0152] Some combination therapies employ at least one hormonal agent. General examples of hormonal agents include hormone agonists and hormone antagonists. Specific examples of hormone agonists include: progestins (progesterone), corticosteroids (e.g., prednisolone, methylprednisolone, dexamethasone), insulin-like growth factor, VEGF-derived angiogenesis and lymphangiogenesis factors (e.g., VEGF-A, VEGF-A145, VEGF-A165, VEGF-C, VEGF-D, PIGF-2), fibroblast growth factor (FGF), galactagogue, hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF)-β, androgens, estrogens, and somatostatin analogs. Examples of hormone antagonists include hormone synthesis inhibitors, such as aromatase inhibitors and gonadotropin-releasing hormone (GnRH) agonists (e.g., leuprolide, goserelin, triptorelin, histrelin), including analogues thereof. Hormone receptor antagonists are also included, such as selective estrogen receptor modulators (SERMs, e.g., tamoxifen, raloxifene, toremifene) and antiandrogens (e.g., flutamide, bicalutamide, nilutamide).

[0153] Hormone pathway inhibitors are also included, such as antibodies against hormone receptors. Examples include inhibitors of IGF receptors (e.g., IGF-IR1), such as cixutumab, dalotuzumab, and fentuzumab.Inhibitors of vascular endothelial growth factor receptors 1, 2, or 3 (VEGFR1, VEGFR2, or VEGFR3), such as alacizumab pegol, bevacizumab, icrucumab, and ramucirumab; inhibitors of TGF-β receptors R1, R2, and R3, such as fresolimumab and metelimumab; inhibitors of c-Met, such as naxitamab; and inhibitors of EGF receptors, such as cetuximab and depatuxizumab. Inhibitors of the following receptors: mafodotin, futuximab, imgatuzumab, lapituximab emtansine, matuzumab, modotuximab, necitumumab, nimotuzumab, panitumumab, tomuzotuximab, and zalutumumab; inhibitors of the FGF receptor, such as aprotumab ixadotin and bemarituzumab; and inhibitors of the PDGF receptor, such as olaratumab or tovetumab.

[0154] The various hormone therapeutic agents described herein may be combined with any one or more of the protein complexes described herein and used according to any one or more of the methods or compositions described herein.

[0155] Certain combination therapies employ at least one kinase inhibitor, including tyrosine kinase inhibitors. Examples of kinase inhibitors include, but are not limited to: adavosertib, afanitib, aflibercept, axitinib, bevacizumab, bosutinib, cabozantinib, cetuximab, cobimetinib, crizotinib, dasatinib.(dasatinib), entrectinib, erdafitinib, erlotinib, fostamitinib, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, mubritinib, nilotinib, panitumab, pazopanib, pegaptanib, panatinib package insert 30 / 47 pages 36 CN 121263518 A (ponatinib), ranibizumab b), regorafenib, ruxolitinib, sorafenib, sunitinib, SU6656, tofacitinib, trastuzumab, vandetanib, and vemurafenib.

[0156] The various kinase inhibitors described herein may be combined with any one or more of the protein complexes described herein and used according to any one or more of the methods or compositions described herein.

[0157] In some embodiments, the methods and compositions described herein increase cancer cell killing in a subject by about or at least about 2, 5, 10, 50, 100, 500, or 1000 times or more relative to a control or reference. In some embodiments, relative to a control or reference (e.g., relative to the corresponding serine protease itself), the methods and compositions described herein increase the immune response of the subject by about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2000% or more, or about 2, 5, 10, 50, 100, 500, or 1000 or more times, including where the immune response is an anticancer immune response.

[0158] In some embodiments, the methods and compositions described herein increase the median survival time of the subject by 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 15 weeks, 20 weeks, 25 weeks, 30 weeks, 40 weeks or more. In some embodiments, the methods or compositions described herein increase the median survival of subjects by 1 year, 2 years, 3 years, or more.In the embodiments, the methods and pharmaceutical compositions increase progression-free survival by 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, or longer. In some embodiments, the methods and pharmaceutical compositions described herein increase progression-free survival by 1 year, 2 years, 3 years, or longer.

[0159] In some embodiments, the methods and compositions described herein are sufficient to cause tumor regression, for example, as indicated by a statistically significant reduction in tumor survival, such as a reduction in tumor mass of at least 10%, 20%, 30%, 40%, 50%, or more, or as indicated by a change in scan size (e.g., a statistically significant reduction). In some embodiments, the methods and compositions described herein are sufficient to result in stable disease. In some embodiments, the methods and compositions described herein are sufficient to reduce the clinical relevance of symptoms of a specific disease indication known to a skilled clinician.

[0160] As described above, for in vivo use, for the treatment of human or non-human mammalian diseases or for testing, the protein complexes described herein are generally incorporated into one or more therapeutic or pharmaceutical compositions, including veterinary therapeutic compositions, prior to administration.

[0161] Therefore, certain embodiments relate to pharmaceutical or therapeutic compositions comprising protein complexes as described herein. In some cases, the pharmaceutical or therapeutic composition comprises one or more of the protein complexes described herein in combination with a pharmaceutically or physiologically acceptable carrier or excipient. Some pharmaceutical or therapeutic compositions further comprise at least one additional agent, such as an immunotherapeutic agent, chemotherapeutic agent, hormone therapy agent, and / or kinase inhibitor as described herein.

[0162] In certain embodiments, the pharmaceutical or therapeutic composition comprising the protein complex is substantially pure on a protein-based or weight-by-weight basis, for example, the purity of the composition is at least about 80%, 85%, 90%, 95%, 98%, or 99% on a protein-based or weight-by-weight basis.

[0163] In some embodiments, as known in the art, the protein complexes described herein do not form aggregates, have desired solubility, and / or have an immunogenic profile suitable for humans. Therefore, in some embodiments, the pharmaceutical or therapeutic composition comprising the protein complex is substantially non-aggregating. For example, certain compositions contain less than about 10% (on a protein basis) of high molecular weight aggregates, or less than about 5% of high molecular weight aggregates, or less than about 4% of high molecular weight aggregates, or less than about 3% of high molecular weight aggregates, or less than about 2% of high molecular weight aggregates, or less than about 1% of high molecular weight aggregates as specified on page 31 / 47 of the specification, CN 121263518 A.

[0164] In some embodiments, the protein complex is concentrated to about or at least about 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, etc.mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6, 0.7, 0.8, 0.9, 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, 10 mg / ml, 11, 12, 13, 14, or 15 mg / ml, and formulated for biotherapeutic use.

[0165] To prepare a therapeutic or pharmaceutical composition, an effective or desired amount of one or more protein complexes is mixed with any pharmaceutical carrier or excipient known to those skilled in the art to suit a particular dosage and / or administration mode. The pharmaceutical carrier may be liquid, semi-liquid, or solid. Solutions or suspensions for parenteral, intradermal, subcutaneous, or topical application may include, for example, sterile diluents (such as water), saline solutions (e.g., phosphate-buffered saline; PBS), fixative oils, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; antimicrobial agents (such as benzyl alcohol and methylparaben), antioxidants (such as ascorbic acid and sodium bisulfite), and chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); buffers (such as acetates, citrates, and phosphates). If administered intravenously (e.g., via IV infusion), suitable carriers include physiological saline or phosphate-buffered saline (PBS), and solutions containing thickeners and solubilizers such as glucose, polyethylene glycol, polypropylene glycol, and mixtures thereof.

[0166] The administration of the protein complexes described herein, either in their pure form or in a suitable therapeutic or pharmaceutical composition, may be carried out by any acceptable method of pharmaceutical administration for similar uses. Therapeutic or pharmaceutical compositions can be prepared by combining a composition containing a protein complex with a suitable physiologically acceptable carrier, diluent, or excipient, and can be formulated into a solid, semi-solid, liquid, or gaseous form, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalers, gels, microspheres, and aerosols. Furthermore, other pharmaceutically active ingredients (including other small molecules as described elsewhere herein) and / or suitable excipients, such as salts, buffers, and stabilizers, may, but need not, be present in the composition.

[0167] Administration can be achieved through a variety of different routes, including oral, parenteral, nasal, intravenous, intradermal, intramuscular, subcutaneous, or topical administration. Preferred administration methods depend on the nature of the condition to be treated or prevented. Specific embodiments include administration via IV infusion.

[0168] The carrier may include, for example, a pharmaceutically or physiologically acceptable carrier, excipient, or stabilizer that is non-toxic to cells or mammals exposed to it at the doses and concentrations employed. Physiologically acceptable carriers are typically aqueous pH buffer solutions. Examples of physiologically acceptable carriers include buffer solutions such as phosphoric acid, citric acid, and other organic acids.Antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) peptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming ions such as sodium; and / or nonionic surfactants such as polysorbate 20 (TWEEN™), polyethylene glycol (PEG), and poloxamers (PLURONICS™).

[0169] In some embodiments, one or more agents may be encapsulated, for example, by agglomeration techniques or by interfacial polymerization into prepared microcapsules (e.g., hydroxymethyl cellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in crude emulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, edited by Oslo A., (1980). One or more particles or liposomes may further contain other therapeutic or diagnostic agents.

[0170] The precise dosage and duration of treatment are functions of the disease being treated and can be determined empirically using known testing protocols or by testing the composition in model systems known in the art and inferring from thereto. See also specification 32 / 47 pages 38 CN 121263518 A Controlled clinical trials are conducted. Dosage may also vary depending on the severity of the symptom to be alleviated. Pharmaceutical compositions are typically formulated and administered to achieve a therapeutically useful effect while minimizing undesirable side effects. The composition may be administered once or may be divided into many smaller doses administered at intervals. For any particular subject, a specific dosage regimen may be adjusted over time according to individual needs.

[0171] Thus, typical routes of administration of these and related therapeutic compositions or pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, oral, rectal, vaginal, and intranasal administration. The term parenteral, as used herein, includes subcutaneous injection, intravenous, intramuscular, intrasternal injection, or infusion techniques. The therapeutic compositions or pharmaceutical compositions according to certain embodiments of this disclosure are formulated to allow the active ingredient contained therein to be bioavailable when the composition is administered to a subject or patient. The composition to be administered to a subject or patient may be in the form of one or more dose units, wherein, for example, tablets may be single dose units, and containers of pharmaceutical agents in the form of aerosols described herein may contain multiple dose units.The actual methods for preparing such dosage forms are known or obvious to those skilled in the art; see, for example, Remington: The Science and Practice of Pharmacy, 20th edition (Philadelphia College of Pharmacy and Science, 2000). The composition to be administered will typically contain a therapeutically effective amount of the medicament described herein for the treatment of the disease or condition of interest.

[0172] The therapeutic or pharmaceutical composition may be in solid or liquid form. In one embodiment, the carrier is granular, and thus the composition is, for example, in tablet or powder form. The carrier may be liquid, while the composition is, for example, an oral oil, an injectable liquid, or an aerosol suitable for, for example, inhalation. When intended for oral administration, the pharmaceutical composition is preferably in solid or liquid form, wherein semi-solid, semi-liquid, suspension, and gel forms are included within the forms considered solid or liquid herein. Some embodiments include sterile injectable solutions.

[0173] As a solid composition for oral administration, the pharmaceutical composition can be formulated into powders, granules, compressed tablets, pills, capsules, chewing gum, sheets, etc. Such solid compositions typically contain one or more inert diluents or edible carriers. In addition, one or more of the following may be present: binders such as carboxymethyl cellulose, ethyl cellulose, microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch, lactose, or dextrin; disintegrants such as alginate, sodium alginate, Primogel, or corn starch; lubricants such as magnesium stearate or Sterotex; flow aids such as colloidal silica; sweeteners such as sucrose or saccharin; flavoring agents such as peppermint, methyl salicylate, or orange flavoring; and coloring agents. When the pharmaceutical composition is in capsule form, for example, gelatin capsules, in addition to the materials of the types described above, the pharmaceutical composition may also contain liquid carriers such as polyethylene glycol or oil.

[0174] The therapeutic or pharmaceutical composition may be in liquid form, such as an elixir, syrup, solution, emulsion, or suspension. As two examples, the liquid may be for oral administration or for delivery by injection. When intended for oral administration, the preferred composition, in addition to the compounds of the present invention, contains one or more of a sweetener, preservative, dye / coloring agent, and flavor enhancer. In compositions intended for injection administration, one or more of a surfactant, preservative, wetting agent, dispersant, suspending agent, buffer, stabilizer, and isotonic agent may be included.

[0175] Liquid therapeutic or pharmaceutical compositions, whether or not they are solutions, suspensions, or other similar forms, may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, etc.The solution, isotonic sodium chloride, fixed oil such as synthetic monoglycerides or diglycerides that can be used as a solvent or suspension medium, polyethylene glycol, glycerol, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetate, citrate or phosphate and agents for tension regulation such as sodium chloride or dextran. Parenteral preparations may be packaged in ampoules made of glass or plastic, single-use syringes or multi-dose vials. Physiological saline is a preferred adjuvant. Injectable pharmaceutical compositions are preferably sterile.

[0176] Liquid therapeutic compositions or pharmaceutical compositions intended for parenteral or oral administration should contain an amount of the pharmaceutical agent to obtain an appropriate dosage. Typically, this amount is at least 0.01% of the pharmaceutical agent of interest in the composition. When intended for oral administration, this amount can vary between 0.1% and 70% by weight of the composition. Some oral therapeutic or pharmaceutical compositions contain between about 4% and about 75% of the agent of interest. In some embodiments, therapeutic or pharmaceutical compositions and formulations are prepared such that parenteral dose units contain between 0.01% and 10% by weight of the agent of interest before dilution.

[0177] The therapeutic or pharmaceutical composition may be intended for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment, or gel matrix. For example, the matrix may comprise one or more of the following: petrolatum, lanolin, polyethylene glycol, beeswax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. Thickeners may be present in the therapeutic or pharmaceutical composition intended for topical administration. If intended for transdermal administration, the composition may comprise a transdermal patch or an iontophoresis device.

[0178] The therapeutic or pharmaceutical composition may be intended for rectal administration, for example, in the form of a suppository, which will melt in the rectum and release the drug. Compositions for rectal administration may contain an oily matrix as a suitable non-irritating excipient. Such matrices include, but are not limited to, lanolin, cocoa butter, and polyethylene glycol.

[0179] Therapeutic or pharmaceutical compositions may include various materials that alter the physical form of a solid or liquid dosage unit. For example, a composition may include a material that forms a coating around the active ingredient. The material forming the coating is generally inert and may be selected from, for example, sugars, shellac, and other enteric coating agents. Alternatively, the active ingredient may be encapsulated in a gelatin capsule. Therapeutic or pharmaceutical compositions in solid or liquid form may include components that bind to the pharmaceutical agent and thereby facilitate the delivery of the compound. Suitable components that can function in this way include monoclonal or polyclonal antibodies, one or more proteins, or liposomes.

[0180] Therapeutic or pharmaceutical compositions may consist essentially of dose units that can be administered as aerosols. The term aerosol is used to refer to a variety of systems, from colloidal systems to systems consisting of pressurized packaging. Delivery can be carried out by liquefying or compressing gas or by a suitable pump system dispensing the active ingredient. Aerosols can be delivered in single-phase, biphase, or triphase systems to deliver the active ingredient. Aerosol delivery includes necessary containers, activators, valves, sub-containers, etc., which may collectively form a kit. Preferred aerosols can be determined by those skilled in the art without extensive experimentation.

[0181] The compositions described herein can be prepared using a carrier that protects the pharmaceutical agent from rapid elimination from the body, such as timed-release formulations or coatings. Such carriers include controlled-release formulations, such as, but not limited to, implants and microencapsulated delivery systems, and biodegradable biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid, and other substances known to those skilled in the art.

[0182] Therapeutic or pharmaceutical compositions can be prepared using methods well known in the pharmaceutical industry. For example, a therapeutic or pharmaceutical composition intended for administration by injection may comprise one or more salts, buffers, and / or stabilizers, and form a solution together with sterile distilled water. Surfactants may be added to promote the formation of a homogeneous solution or suspension. Surfactants are compounds that non-covalently interact with a pharmaceutical agent to promote its dissolution or homogeneous suspension in an aqueous delivery system.

[0183] The therapeutic or pharmaceutical composition may be administered in a therapeutically effective amount, which will vary depending on a variety of factors, including the activity of the specific compound used; the metabolic stability and duration of action of the compound; the subject's age, weight, general health condition, sex, and diet; the administration pattern and time; the excretion rate; the combination of drugs; the severity of the specific condition or symptom; and the subject undergoing the therapy. In some cases, the therapeutically effective daily dose (for a 70 kg mammal) is from about 0.001 mg / kg (i.e., ~0.07 mg) to about 100 mg / kg (i.e., ~7.0 g) as per the specification (pages 34 / 47, CN 121263518 A); preferably, the therapeutically effective dose (for a 70 kg mammal) is from about 0.01 mg / kg (i.e., ~0.7 mg) to about 50 mg / kg (i.e., ~3.5 g); more preferably, the therapeutically effective dose (for a 70 kg mammal) is from about 1 mg / kg (i.e., ~70 mg) to about 25 mg / kg (i.e., ~1.75 g). In some embodiments, the therapeutically effective dose is administered on a weekly, bi-weekly, or monthly basis. In specific embodiments, the therapeutically effective dose is, for example, weekly or bi-weekly at doses of about 1-10 or 1-5 mg / kg or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg.Or administered monthly.

[0184] The combination therapies described herein may comprise administration of a single drug dose formulation containing a protein complex and an additional therapeutic agent (e.g., an immunotherapy agent, a chemotherapy agent, a hormone therapy agent, a kinase inhibitor), and administration of a composition comprising a protein complex and an additional therapeutic agent in its own separate drug dose formulation. For example, the protein complex and the additional therapeutic agent may be administered to the subject together as a single parenteral dose composition (e.g., in a saline solution or other physiologically acceptable solution), or each agent may be administered in a separate parenteral dose formulation. When using separate dose formulations, the compositions may be administered substantially simultaneously, i.e., concurrently, or separately and alternately, i.e., sequentially and in any order; combination therapies should be understood to include all of these regimens.

[0185] A patient care kit is also included, comprising (a) a protein complex as described herein; and optionally (b) at least one additional therapeutic agent (e.g., an immunotherapy agent, a chemotherapy agent, a hormone therapy agent, a kinase inhibitor). In some kits, (a) and (b) are in separate therapeutic compositions. In some kits, (a) and (b) are in the same therapeutic composition.

[0186] Kits described herein may also include one or more additional therapeutic agents or other components suitable for or desired for the therapeutic indication or for a desired diagnostic application. Kits described herein may also include one or more syringes or other components required or desired to facilitate the intended delivery mode (e.g., stents, implantable receptacles, etc.).

[0187] In some embodiments, the patient care kit contains separate containers, dividers, or compartments for the composition and informational material. For example, the composition may be contained in a bottle, vial, or syringe, and the informational material may be contained in association with the container. In some embodiments, individual elements of the kit are contained in a single, undivided container. For example, the composition is contained in a bottle, vial, or syringe with an informational material labeled thereon. In some embodiments, the kit includes a plurality of (e.g., a package) separate containers, each containing one or more unit dosage forms of a protein complex (e.g., dosage forms described herein), and optionally at least one additional therapeutic agent. For example, the kit includes multiple syringes, ampoules, foil pouches, or blister packs, each containing a single unit dose of the protein complex, and optionally at least one additional therapeutic agent. The container of the kit may be airtight, waterproof (e.g., impermeable to changes in moisture or evaporation), and / or light-proof.

[0188] The patient care kit optionally includes a device suitable for administering the composition, such as a syringe, inhaler, dropper (e.g., eye dropper), swab (e.g., cotton swab or wooden swab), or any such delivery device. In some embodiments, the device is...An implantable device for dispensing a metered dose of a pharmaceutical agent. Also includes, for example, a method for providing a kit by combining the components described herein.

[0189] Preparation and Purification Systems

[0190] Some embodiments include methods and related compositions for preparing, expressing, and purifying the protein complexes or protein components described herein. For example, some embodiments relate to methods for preparing or otherwise preparing pharmaceutical compositions comprising protein complexes, the methods being carried out by combining the following in a molar ratio of about 1:3 to about 1:1 [(a):(b)]: (a) α-2-macroglobulin (A2M) protein; and (b) serine protease protein, thereby preparing the pharmaceutical composition comprising the protein complex. Specific embodiments include combining (a) and (b) into the composition at molar ratios of approximately 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 [(a):(b)].

[0191] A2M and / or serine protease proteins can be prepared or obtained by purification from biological samples or by recombinant technology. For example, in some embodiments, A2M protein and / or serine protease protein are obtained by purifying proteins from the blood or plasma of mammalian subjects, such as human subjects. Specific embodiments include obtaining and purifying A2M protein from the plasma of a human subject prior to combination with a serine protease protein. Methods for purifying A2M from human plasma (plasma-rich A2M; or A2M-PPP) are known in the art (see, for example, Jordan et al., Pain Physician. 23(2):229-23, 2020; U.S. Patent No. 9,352,021).

[0192] Recombinant proteins can be conveniently prepared using standard protocols, such as those described, for example, in Sambrook et al., (1989, ibid.), particularly Sections 16 and 17; Ausubel et al., (1994, ibid.), particularly Chapters 10 and 16; and Coligan et al., Current Protocols in Protein Science (John Wiley & Sons, Inc., 1995–1997), Chapters 1, 5, and 6. As a general example, recombinant proteins can be prepared by including one or more of the following steps.The procedure for the steps is as follows: (a) preparing a vector or construct comprising a polynucleotide sequence encoding the protein described herein, said vector or construct being operatively linked to one or more regulatory elements; (b) introducing said vector or construct into a host cell; (c) culturing said host cell to express said protein; and (d) isolating said protein from said host cell.

[0193] To express a desired polypeptide, the nucleotide sequence of a protein may be inserted into a suitable expression vector, i.e., a vector containing the necessary elements of the coding sequence for transcription and translation insertion. Methods well known to those skilled in the art can be used to construct expression vectors containing sequences encoding the polypeptide of interest and suitable transcription and translation control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo gene recombination. Such techniques are described in the following literature: Sambrook et al., Molecular Cloning, A Laboratory Manual (1989), and Ausubel et al., Current Protocols in Molecular Biology (1989).

[0194] Various expression vector / host systems are known and can be utilized to contain and express polynucleotide sequences. These include, but are not limited to: microorganisms, such as bacteria transformed with recombinant phage, plasmid, or copious DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculoviruses); plant cell systems transformed with viral expression vectors (e.g., cauliflower mosaic virus CaMV; tobacco mosaic virus TMV) or bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems including mammalian cells and more specifically human cell systems.

[0195] The “control elements” or “regulatory sequences” present in the expression vector are those untranslated regions of the vector—enhancers, promoters, 5' and 3' untranslated regions—that interact with host cell proteins to perform transcription and translation. The strength and specificity of such elements can vary. Depending on the vector system and host used, any number of suitable transcription and translation elements, including constitutive and inducible promoters, can be used. For example, when cloning in bacterial systems, inducible promoters such as the heterozygous lacZ promoter of the PBLUESCRIPT phage particle (Stratagene, La Jolla, California) or the PSPORT1 plasmid (Gibco BRL, Gaithersburg, Maryland) can be used. In mammalian cell systems, promoters derived from mammalian genes or mammalian viruses are generally preferred.The promoter. If it is necessary to generate cell lines containing multiple copies of the sequence encoding a polypeptide, then SV40 or EBV-based vectors can be advantageously used with appropriate optional markers.

[0196] In bacterial systems, multiple expression vectors can be selected depending on the intended use of the polypeptide to be expressed. For example, when a large quantity is required, a vector that guides high-level expression of an easily purified protein can be used. Such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors, such as BLUESCRIPT (Stratagene), in which the sequence encoding the polypeptide of interest can be linked using the same reading frame to a vector having an amino-terminal Met sequence and the following 7 residues of β-galactosidase to produce a mixed protein; pIN vectors (Van Heeke and Schuster, Journal of Biochemistry 264:5503 5509 (1989)), etc. The pGEX vector (Promega, Madison, Wisconsin) can also be used to express recombinant proteins as fusion proteins containing glutathione S-transferase (GST). Generally, such fusion proteins are soluble and can be readily purified from lysed cells by adsorption to glutathione-glucose beads followed by elution in the absence of free glutathione. Proteins prepared in such systems can be engineered to include heparin, thrombin, or factor XA protease cleavage sites, allowing arbitrary release of the clonal polypeptide of interest from the GST moiety.

[0197] Some embodiments employ E. coli-based expression systems (see, for example, Structural Genomics Consortium et al., Nature Methods 5:135-146, 2008). These and related embodiments may rely partially or entirely on ligation-independent cloning (LIC) to produce suitable expression vectors. In specific embodiments, protein expression may be controlled by a T7 RNA polymerase (e.g., a pET vector sequence). These and related embodiments can utilize expression host strain BL21 (DE3), a DE3 lysinogen of BL21 that supports T7-mediated expression and lacks the lon and ompT proteases used to improve target protein stability. Expression host strains carrying plasmids encoding tRNAs rarely used in *E. coli*, such as ROSETTA™ (DE3) and Rosetta 2 (DE3) strains, are also included. Reagents sold under the trademarks BENZONASE® nucleases and BUGBUSTER® protein extraction reagents can also be used to improve cell lysis and sample handling. For cell culture, self-inducing media can be used to improve...This type of expression system is more efficient than many others, including high-throughput systems. Culture media (e.g., the OVERNIGHT EXPRESS™ self-inducible system) gradually induce protein expression via metabolic transfer without the need for artificial inducers such as IPTG. Certain embodiments employ hexahistine tags (such as those sold under the trademark HIS TAG® fusions) followed by immobilized metal affinity chromatography (IMAC) purification or related techniques. However, in some respects, clinical-grade proteins can be isolated from *E. coli* inclusion bodies without or without affinity tags (see, for example, Shimp et al., *Protein Expression and Purification* 50:58-67, 2006). As another example, some embodiments can employ cold-shock-induced high-yield production systems in *E. coli*, as overexpression of proteins in *E. coli* at low temperatures improves the solubility and stability of *E. coli* (see, for example, Qing et al., *Nature Biotechnology* 22:877-882, 2004).

[0198] High-density bacterial fermentation systems are also included. For example, high-density culture of Ralstonia eutropha allows for protein production at cell densities exceeding 150 g / L and expression of recombinant proteins at titers exceeding 10 g / L.

[0199] In yeast Saccharomyces cerevisiae, many vectors containing constitutive or inducible promoters such as α-factor, alcohol oxidase, and PGH can be used. For reviews, see Ausubel et al., (ibid.) and Grant et al., Methods Enzymol. 153:516-544 (1987). Pichia pandoris expression systems are also included (see, for example, Li et al., Nature Biotechnology 24, 210-215, 2006; and Hamilton et al., Science, 301:1244, 2003). Some embodiments include yeast systems engineered to selectively glycosylate proteins, including yeasts with humanized N-glycosylation pathways, etc. (see, for example, Hamilton et al., Science 313:1441-1443, 2006; Wildt et al., Nature Reviews Microbiol. 3:119-28, 2005; and Gerngross et al., Nature Biotechnology 22:1409-1414, 2004; U.S. Patents 7,629,163; 7,326,681 and 7,029,872). Examples only.It is stated that recombinant yeast cultures can be grown in Fernbach Flasks or fermenters of 15L, 50L, 100L and 200L, etc., as per the instructions on pages 37 / 47, CN 121263518 A.

[0200] In the case of using plant expression vectors, the expression of the sequence encoding the polypeptide can be driven by any of a number of promoters. For example, viral promoters such as the 35S and 19S promoters of CaMV can be used alone or in combination with the ω leader sequence from TMV (Takamatsu, Journal of the European Society for Molecular Biology (EMBO J.) 6:307-311 (1987)). Alternatively, plant promoters such as the small subunit of RUBISCO or the heat shock promoter can be used (Coruzzi et al., Journal of the European Society for Molecular Biology 3:1671-1680 (1984); Broglie et al., Science 224:838-843 (1984); and Winter et al., Results Probl. Cell Differ. 17:85-105 (1991)). These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. Such techniques are described in many generally available reviews (see, for example, McGraw Hill’s Hobbs, Yearbook of Science and Technology, pp.191-196, (1992)).

[0201] Insect systems can also be used to express peptides of interest. For example, in one such system, the alfalfa silver-striped armyworm nucleopolyhedrovirus (AcNPV) is used as a vector to express exogenous genes in fall armyworm or white armyworm cells. The sequence encoding the polypeptide can be cloned into a non-essential region of the virus, such as the polyhedral protein gene, and placed under the control of the polyhedral protein promoter. Successful insertion of the polypeptide-coding sequence will inactivate the polyhedral protein gene and produce a recombinant virus lacking the coat protein. This recombinant virus can then be used to infect, for example, fall armyworm or white armyworm cells that can express the polypeptide of interest (Engelhard et al., Proceedings of the National Academy of Sciences 91:3224-3227 (1994)). Baculovirus expression systems are also included, including those utilizing SF9, SF21, and T. ni cells (see, for example, Murphy and Piwnica-Worms, Current Protocol for Protein Science, Chapter 5: Unit 5.4).2001). Insect systems can provide post-translational modifications similar to those of mammalian systems.

[0202] In mammalian host cells, many virus-based expression systems are generally available. For example, when using adenovirus as an expression vector, the sequence encoding the polypeptide of interest can be linked to an adenoviral transcription / translation complex consisting of a late promoter and a triple leader sequence. Insertion into the non-essential E1 or E3 region of the viral genome can be used to obtain a live virus capable of expressing the polypeptide in infected host cells (Logan and Shenk, Proceedings of the National Academy of Sciences 81:3655-3659 (1984)). In addition, transcriptional enhancers such as the Raoult sarcoma virus (RSV) enhancer can be used to increase expression in mammalian host cells.

[0203] Examples of useful mammalian host cell lines include the monkey kidney CV1 cell line transformed from SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell lines (293 or 293 cells for growth subclones in suspension culture, Graham et al., J. Gen. Virol. 36:59 (1977)); juvenile hamster kidney cells (BHK, ATCC CCL 10); mouse supporting cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical tumor cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat hepatocytes (BRL). 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); murine mammary tumors (MMT 060562, ATCC CCL51); TR1 cells (Mather et al., Annals of the New York Academy of Sciences 383:44-68 (1982)); MRC 5 cells; FS4 cells; and human hepatocellular carcinoma cell line (Hep G2). Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proceedings of the National Academy of Sciences 77:4216 (1980)); and myeloma cell lines such as NSO and Sp2 / 0. For a review of certain mammalian host cell lines suitable for protein production, see, for example, Yazaki and Wu, *Methods in Molecular Biology*, Vol. 248 (edited by BKC Lo).Humana Press, Totowa, NJ (2003), pp. 255-268. (Instruction manual 38 / 47 pages, 44 CN 121263518 A Totowa, NJ.). Certain preferred mammalian cell expression systems include expression systems based on CHO and HEK293 cells. Mammalian expression systems can utilize attached cell lines, for example, in T-flasks, roller flasks, or cell factories, or in suspension cultures, for example, in 1 L and 5 L rotary flasks, 5 L, 14 L, 40 L, 100 L, and 200 L stirred tank bioreactors, or 20 / 50 L and 100 / 200 L WAVE bioreactors, etc., as known in the art.

[0204] Cell-free expression of proteins is also included. These and related embodiments typically utilize purified RNA polymerases, ribosomes, tRNA, and ribonucleotides; these reagents can be produced from cells or from cell-based expression systems.

[0205] Specific initiation signals can also be used to achieve more efficient translation of sequences encoding the polypeptide of interest. Such signals include the ATG start codon and adjacent sequences. When the sequence encoding the polypeptide, its start codon, and the upstream sequence are inserted into a suitable expression vector, additional transcription or translation control signals may not be required. However, when only the coding sequence or a portion thereof is inserted, an exogenous translation control signal including the ATG start codon should be provided. Furthermore, the start codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and start codons can be of various origins, both natural and synthetic. The efficiency of expression can be enhanced by including enhancers suitable for the specific cell system used, such as enhancers described in the literature (Scharf et al., Results Probl. Cell Differ. 20:125-162 (1994)).

[0206] Additionally, a host cell line can be selected because it is capable of modulating the expression of the inserted sequence or processing the expressed protein in the desired manner. Such modifications to peptides include, but are not limited to, post-translational modifications such as acetylation, carboxylation, glycosylation, phosphorylation, esterification, and acylation. Post-translational processing of protein “precursor” forms can also be used to facilitate proper insertion, folding, and / or function. In addition to bacterial cells, different host cells with specific cellular mechanics and characteristic mechanisms, such as yeast, CHO, HeLa, MDCK, HEK293, and W138, with or even lacking such post-translational activity, can be selected to ensure proper modification and processing of foreign proteins.

[0207] Stable expression is generally preferred for long-term, high-yield production of recombinant proteins. For example, expression can be used...A vector is used to transform cell lines stably expressing the polynucleotide of interest. The expression vector may contain a replicating viral-originating and / or endogenous expression element, and an optional marker gene on the same vector or on a separate vector. After vector introduction, cells may be allowed to grow in an enriched medium for approximately 1–2 days before being transferred to a selective medium. The purpose of the optional marker is to confer resistance to the selection, and its presence allows for the growth and recovery of cells successfully expressing the introduced sequence. Resistant clones of stably transformed cells can be proliferated using tissue culture techniques suitable for the cell type. Transient production, such as by transient transfection or infection, is also possible. Exemplary mammalian expression systems suitable for transient production include HEK293 and CHO-based systems.

[0208] Any number of selection systems can be used to recover transformed or transduced cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase gene (Wigler et al., Cell 11:223-232 (1977)) and the adenine phosphoribosyltransferase gene (Lowy et al., Cell 22:817-823 (1990)), which can be used in tk-cells or aprt-cells, respectively. Similarly, resistance to antimetabolites, antibiotics, or herbicides can be used as a basis for selection; for example, dhfr (Wigler et al., Proceedings of the National Academy of Sciences 77:3567-70 (1980)) confers resistance to methotrexate; npt (Colbere-Garapin et al., Journal of Molecular Biology 150:1-14 (1981)) confers resistance to aminoglycosides, neomycin, and G-418; and als or pat (Murry, ibid.) confers resistance to chlorfluazuron and phosphinotricin acetyltransferase, respectively. Other alternative genes have been described, such as trpB, which allows cells to use indole instead of tryptophan, or hisD, which allows cells to use histidine instead of histidine (Hartman and Mulligan, Proceedings of the National Academy of Sciences 85: 8047-51 (1988)). The use of visible markers has become increasingly popular for such markers as green fluorescent protein (GFP) and other fluorescent proteins (e.g., RFP, YFP), anthocyanins, β-glucuronidase and its substrate GUS, luciferase and its substrate luciferin. These visible markers are widely used not only for identifying transformants but also for quantifying the amount of transient or stable protein expression attributable to a specific vector system (see, for example, Rhodes et al.).

[0209] High-throughput protein production systems or microproduction systems are also included. In some aspects, for example, hexahistine fusion tags can be used for protein expression and purification on metal chelate-modified slide surfaces or MagneHis Ni particles (see, for example, Kwon et al., BMC Biotechnol. 9:72, 2009; and Lin et al., Molecular Biology Methods 498:129-41, 2009). High-throughput cell-free protein expression systems are also included (see, for example, Sitaraman et al., Molecular Biology Methods 498:229-44, 2009).

[0210] Various protocols for detecting and measuring the expression of polynucleotide-encoded products using binders or antibodies (such as product-specific polyclonal or monoclonal antibodies) are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), Western blotting, radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS). These and other assays are described in Hampton et al., *Serological Methods*, *Laboratory Manual* (1990) and Maddox et al., *Journal of Experimental Medicine* 158:1211-1216 (1983).

[0211] A wide variety of labeling and conjugation techniques are known to those skilled in the art and can be used in a variety of nucleic acid and amino acid assays. Methods for generating labeled hybridization or PCR probes for detecting sequences associated with polynucleotides include oligolabeling, nick translation, end labeling, or PCR amplification using labeled nucleotides. Alternatively, the sequence or any portion thereof can be cloned into a vector for the production of mRNA probes. Such vectors are commercially available and known in the art and can be used to synthesize RNA probes in vitro by adding appropriate RNA polymerases such as T7, T3, or SP6 and labeled nucleotides. These procedures can be performed using a variety of commercially available kits. Suitable reporter molecules or markers that can be used include radionuclides, enzymes, fluorescence, chemiluminescence or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, etc.

[0212] Host cells transformed with one or more polynucleotide sequences of interest can be cultured under conditions suitable for protein expression and recovery from cell cultures. Some specific embodiments utilize serum-free cell expression systems. Examples include HEK293 cells and CHO cells that can be grown in serum-free media (see, for example, Rosser et al., Protein Expression and Purification 40:237-43,2005; and U.S. Patent No. 6,210,922).

[0213] Proteins produced by recombinant cells can be secreted or contained within cells depending on the sequence and / or vector used. As those skilled in the art will understand, expression vectors containing polynucleotides can be designed to contain a signal sequence that guides the secretion of the encoded polypeptide through the prokaryotic or eukaryotic cell membrane. Other recombinant constructs can be used to link a sequence encoding a polypeptide of interest with a nucleotide sequence encoding a polypeptide domain, which will facilitate the purification and / or detection of soluble proteins. Examples of such domains include cleavable and cleavable affinity purification and epitope tags, such as avidin, FLAG tags, polyhistidine tags (e.g., 6xHis), cMyc tags, V5 tags, glutathione S-transferase (GST) tags, etc.

[0214] Proteins produced by recombinant cells can be purified and characterized according to a variety of techniques known in the art. Exemplary systems for protein purification and analysis of protein purity include rapid protein liquid chromatography (FPLC) (e.g., AKTA and Bio-Rad FPLC systems) and high-performance liquid chromatography (HPLC) (e.g., Beckman and Waters HPLC). Exemplary chemical reactions for purification include ion exchange chromatography (e.g., Q, S), size exclusion chromatography, salt gradients, affinity purification (e.g., Ni, Co, FLAG, maltose, glutathione, protein A / G), gel filtration, reversed phase, ceramic HYPERD® ion exchange chromatography, and hydrophobic interaction columns (HIC). Analytical methods such as SDS-PAGE (e.g., Coomassie brilliant blue staining, silver staining), immunoblotting, Bradford, and ELISA are also included, which can be used during any step of the production or purification process and are typically used to measure the purity of protein compositions.

[0215] The method also includes methods for concentrating the proteins or protein complexes described herein, and compositions comprising concentrated, soluble proteins or protein complexes. In some aspects, such concentrated solutions of proteins or protein complexes contain proteins at concentrations of about or at least about 5 mg / mL, 8 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, or more.

[0216] In some aspects, such compositions may be substantially monodisperse, meaning that when evaluated, for example, by size exclusion chromatography, dynamic light scattering, or analytical ultracentrifugation, the protein or protein complex is primarily (i.e., at least about 90% or more) present in an apparent molecular weight form.

[0217] In some aspects, such compositions have a purity of at least about 90% (based on protein), or in some aspects...The purity is less than about 95%, or in some embodiments, at least 98%. Purity can be determined by any conventional analytical method known in the art.

[0218] In some aspects, such compositions have a high molecular weight aggregate content of less than about 10% compared to the total amount of protein present, or in some embodiments, such compositions have a high molecular weight aggregate content of less than about 5%, or in some aspects, such compositions have a high molecular weight aggregate content of less than about 3%, or in some embodiments, a high molecular weight aggregate content of less than about 1%. The high molecular weight aggregate content can be determined by a variety of analytical techniques, including, for example, size exclusion chromatography, dynamic light scattering, or analytical ultracentrifugation.

[0219] Examples of concentration methods considered herein include lyophilization, which is typically used when the solution contains a small number of soluble components other than the protein of interest. Lyophilization is typically performed after HPLC and can remove most or all volatile components from the mixture. Ultrafiltration techniques are also included, which typically employ one or more selectively permeable membranes to concentrate the protein solution. The membrane allows water and small molecules to pass through the protein and retains the protein; the solution can be pressed against the membrane by other techniques such as mechanical pumps, air pressure, or centrifugation.

[0220] In some embodiments, the protein or protein complex in the composition has a purity of at least about 90%, as measured according to conventional techniques in the art. In some embodiments, such as diagnostic compositions or certain pharmaceutical or therapeutic compositions, the protein or protein complex in the composition has a purity of at least about 95%, or at least about 97%, 98%, or 99%. In some embodiments, such as when used as a reference or research reagent, the protein or protein complex may be of lower purity and may have a purity of at least about 50%, 60%, 70%, or 80%. Purity can be measured overall or can be measured with respect to selected components such as other proteins, for example, based on protein purity.

[0221] The purified protein or protein complex can also be characterized according to its biological characteristics. Binding affinity and binding kinetics can be measured using a variety of techniques known in the art, such as Biacore® and related techniques utilizing surface plasmon resonance (SPR), an optical phenomenon that enables real-time detection of unlabeled interactants. SPR-based biosensors can be used to determine activity concentrations, screen, and characterize affinity and kinetics. The presence or level of one or more biological activities can be measured by in vitro or cell-based assays, optionally functionally coupled to readouts or indicators such as fluorescent or luminescent indicators of the biological activities described herein.

[0222] In some embodiments, as described above, the composition is substantially free of endotoxins, including, for example, about 95% endotoxin-free, preferably about 99% endotoxin-free, and more preferably about 99.99% endotoxin-free. As described herein, it can be based on…The presence of endotoxins is detected using conventional techniques in the art. In specific embodiments, the protein or protein complex is prepared from eukaryotic cells, such as mammalian or human cells in a substantially serum-free culture medium. In some embodiments, as described herein, the composition has an endotoxin content of less than about 10 EU / mg protein, or less than about 5 EU / mg protein, less than about 3 EU / mg protein, or less than about 1 EU / mg protein. Specification 41 / 47 pages 47 CN 121263518 A

[0223] In some embodiments, the composition comprises less than about 10% wt / wt high molecular weight aggregates, or less than about 5% wt / wt high molecular weight aggregates, or less than about 2% wt / wt high molecular weight aggregates, or less than or less than about 1% wt / wt high molecular weight aggregates.

[0224] Protein-based analytical assays and methods are also included, which can be used to assess characteristics such as protein purity, size, solubility, and degree of aggregation. Protein purity can be assessed in a variety of ways. For example, purity can be assessed based on primary structure, higher-order structure, size, charge, hydrophobicity, and glycosylation. Examples of methods for evaluating primary structure include N-terminal and C-terminal sequencing and peptide mapping (see, for example, Allen et al., *Biologicals* 24:255–275, 1996). Examples of methods for evaluating higher-order structure include circular dichroism (see, for example, Kelly et al., *Biochim Biophys Acta.* 1751:119–139, 2005), fluorescence spectroscopy (see, for example, Meagher et al., *Journal of Biochemistry* 273:23283–89, 1998), FT-IR, amide hydrogen-deuterium exchange kinetics, differential scanning calorimetry, NMR spectroscopy, and immunoreactivity with conformationally sensitive antibodies. Higher-order structure can also be assessed based on various parameters, such as pH, temperature, or added salts. Examples of methods for evaluating protein characteristics, such as size, include analytical ultracentrifugation and size exclusion HPLC (SEC-HPLC), and exemplary methods for measuring charge include ion exchange chromatography and isoelectric focusing. Hydrophobicity can be evaluated, for example, by reversed-phase HPLC and hydrophobic interaction chromatography HPLC. Glycosylation can affect pharmacokinetics (e.g., clearance), conformation or stability, receptor binding, and protein function, and can be evaluated, for example, by mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy.

[0225] As described above, some embodiments include using SEC-HPLC to evaluate protein characteristics, such as purity, size (e.g., size uniformity), or degree of aggregation, and / or to purify proteins, as well as other uses. Gel filtration chromatography (GFC) is also included.The term SEC in gel permeation chromatography (GPC) refers to a chromatographic method in which molecules in solution are separated in a porous material based on their size, or more specifically based on their hydrodynamic volume, diffusion coefficient, and / or surface properties. This method is commonly used to separate biomolecules and to determine the molecular weight and molecular weight distribution of polymers. Typically, biological or protein samples (e.g., protein extracts produced according to the protein expression methods provided herein and protein extracts known in the art) are loaded into an exclusion column of a selected size having a defined stationary phase (porous material), preferably a phase that does not interact with the proteins in the sample. In some respects, the stationary phase consists of inert particles packed into a dense three-dimensional matrix within a glass or steel column. The mobile phase can be pure water, an aqueous buffer, an organic solvent, or a mixture thereof. The stationary phase particles typically have pores and / or channels that allow only molecules smaller than a certain size to enter. Therefore, large particles are excluded from these pores and channels, and their limited interaction with the stationary phase results in them eluting as “completely excluded” peaks at the start of the experiment. Smaller molecules that can embed into the pores are removed from the flowing mobile phase, and the time it takes for them to become fixed in the stationary phase pores depends in part on the distance they penetrate into the pores. Their removal from the mobile phase results in a longer elution time from the column, and separation between particles occurs based on the difference in particle size. An exclusion column of a given size has a range of separable molecular weights. Generally, molecules larger than the upper limit will not be captured by the stationary phase, molecules smaller than the lower limit will enter the solid phase completely and elute as a single band, and molecules within this range will elute at different rates, defined by their characteristics such as hydrodynamic volume. For examples of the application of these methods in pharmaceutical proteins, see Bruner et al., *Journal of Pharmaceutical and Biomedical Analysis* 15: 1929–1935, 1997.

[0226] Protein purity for clinical applications is also discussed, for example, by Anicetti et al. (Trends in Biotechnology. 7:342-349, 1989). Recent techniques for analyzing protein purity include, but are not limited to, LabChip GXII (an automated platform for rapid analysis of proteins and nucleic acids), which provides high-throughput analysis of protein titers, sizes, and purity. In some non-limiting embodiments, this can be achieved by utilizing a combination of chromatographic materials in at least two orthogonal steps (see, for example, Therapeutic Proteins: Methods and Protocols).Clinical-grade proteins or protein complexes are obtained from *Protocols.*, Volume 308, edited by Smales and James, Humana Press Inc., 2005. Typically, the protein preparations are substantially free of endotoxins, as measured according to techniques known in the art and described herein.

[0227] Protein solubility assays are also included. Such assays can be used, for example, to determine optimal growth and purification conditions for recombinant production, to optimize the selection of one or more buffer solutions, and to optimize the selection of proteins or protein complexes and their variants. Solubility or aggregation can be evaluated based on a variety of parameters including temperature, pH, salt, and the presence or absence of other additives. Examples of solubility screening assays include, but are not limited to, microplate-based methods for measuring protein solubility using turbidity or other measures as endpoints; high-throughput assays for analyzing the solubility of purified recombinant proteins (see, for example, Stenvall et al., Acta Biochimica et Biophysica Sinica 1752:6-10, 2005); assays for monitoring and measuring protein folding and in vivo solubility using structural complementarity of gene marker proteins (see, for example, Wigley et al., Nature Biotechnology 19:131-136, 2001); and electrochemical screening of recombinant protein solubility in Escherichia coli using scanning electrochemical microscopy (SECM) (see, for example, Nagamine et al., Biotechnology and Bioengineering 96:1008-1013, 2006), etc. Proteins or protein complexes exhibiting increased solubility (or reduced aggregation) can be identified or selected using conventional techniques in the art, including simple in vivo assays of protein solubility (see, for example, Maxwell et al., Protein Science 8:1908-11, 1999).

[0228] Protein solubility and aggregation can also be measured using dynamic light scattering techniques. Aggregation is a general term encompassing several types of interactions or characteristics, including soluble / insoluble, covalent / non-covalent, reversible / irreversible, and native / denatured interactions and characteristics. For protein therapeutics, the presence of aggregates is generally considered undesirable due to concerns that they may cause immunogenic reactions (e.g., small aggregates) or adverse events upon administration (e.g., microparticles). Dynamic light scattering refers to a technique that can be used to determine the size distribution curves of small particles in a suspension or polymer such as a protein in solution. This technique is also known as photon correlation spectroscopy (PCS) or quasi-elastic light scattering (QELS), which uses dispersionLight scattering is used to measure the diffusion rate of protein particles. Fluctuations in scattering intensity can be observed due to the Brownian motion of molecules and particles in solution. This motion data can be routinely processed to derive the size distribution of the sample, where the size is given by the Stokes radius or hydrodynamic radius of the protein particles. The hydrodynamic size depends on both mass and shape (consistent). Dynamic scattering can detect the presence of very small amounts of aggregated proteins (< 0.01% by weight), even in samples containing large masses. It can also be used to compare the stability of different formulations, including applications that rely on real-time monitoring of changes at high temperatures. Therefore, some embodiments include the use of dynamic light scattering to analyze the solubility and / or presence of aggregates in samples containing proteins or protein complexes of the present disclosure.

[0229] Although some detailed description of the foregoing embodiments has been given by way of illustration and example for purposes of clarity, it should be readily understood by those skilled in the art, based on the teachings of this disclosure, that certain changes and modifications may be made therein without departing from the spirit or scope of the appended claims. The following examples are provided by way of illustration only and not by way of limitation. Those skilled in the art will readily recognize the various non-critical parameters that can be altered or modified to produce substantially similar results.

[0230] Examples

[0231] Example 1

[0232] Specification for the characterization of A2M:PPE protein complex, pages 43 / 47, 49 CN 121263518 A

[0233] Experiments were conducted to test the characterization of protein complexes composed of A2M and PPE protein 'mutant F' (MutF, SEQ ID NO: 5) in different molar ratios.

[0234] A1AT protection assay. MutF (400 nM) was mixed with different concentrations of A2M to achieve various molar ratios, followed by incubation at 37°C for 30 minutes. Protection was performed by adding 10 μL of sample to 384 black well plates coated with 10 μL of PBS or 10 μL of A1AT 2 μM, or in duplicate coated with different concentrations. 20 μL of substrate AAPV-AMC at a concentration of 100 μM was added. Kinetics were measured on a Varioskan LUX for 16 reads: 380 nm ex / 460 nm em (5 nm bandwidth), 37°C, top, 100 ms, 2 min. Vo was calculated as the activity measurement.

[0235] As shown in Figures 3A-3B, the 1:2 molar ratio of the A2M:MutF protein complex provided optimal protection against A1AT (3A), and gradually increasing A1AT concentrations did not interfere with the protection of A2M against MutF (3B).

[0236] Plasma protection assay.MutF (400 nM) was mixed with 200 nM A2M to achieve a 1:2 molar ratio (A2M: MutF), and then incubated in PBS at 37°C for 30 min. Plasma protection assays were performed in 384 black-well coated plates by adding 10 μL of sample to 10 μL of PBS or 10 μL of mouse plasma sample in duplicate. 20 μL of substrate AAPV-AMC at a concentration of 100 µM was added. Kinetics were measured on a Varioskan LUX for 16 reads: 380 nm ex / 460 nm (5 nm bandwidth), 37°C, top, 100 ms, 2 min. Vo was calculated as the activity measurement.

[0237] As shown in Figure 3C, when combined with A2M:MutF at a 1:2 molar ratio, plasma itself inhibits MutF activity, but does not inhibit its activity.

[0238] N17350-A2M was purified by column chromatography. MutF was mixed with A2M at a 1:2 molar ratio (A2M:MutF) and then incubated at 37°C for 30 min. Protein purification was performed using an AKTA pure chromatography system. For cation exchange columns, the protein mixture was buffer-exchanged to 50 mM sodium acetate (pH 5.0) solution using a PD10 desalting column. The sample was loaded onto a pre-washed / equilibrated HiTrap SP column with 0.5 M NaCl and 50 mM sodium acetate (pH 5.0) and eluted in 20 fractions (1 mL / fraction). For size exclusion columns, the protein mixture was buffer-exchanged to 50 mM sodium phosphate and 150 mM NaCl (pH 7) using a PD10 desalting column. The sample was loaded onto a pre-washed Superose 6 10 / 300 increase. The flow rate was set to 0.5 mL / min to collect 1 mL / fraction. Each fraction was diluted 1:250 in PBS for A2M:MutF A1AT protection assay. Protein concentrations were measured using the Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific, see manufacturer’s instructions). For the A1AT protection assay, 10 μL of each fraction dilution was coated in duplicate with 10 μL of PBS or 10 μL of 2 μM A1AT, or at different concentrations, in 384 black plates. 20 μL of substrate AAPV-AMC at a concentration of 100 µM was added. Kinetics were measured on a Varioskan LUX for 16 reads: 380 nm ex / 460 nm em (5 nm bandwidth), 37 °C, top, 100 ms, 2 min. Vo was calculated as the activity measurement.

[0239] The results in Figures 4A-4E show that the A2M:MutF protein complex is stable. Figure 4A shows the results in the presence or absence of...MutF activity and concentration of different fractions of the A2M:MutF complex after cation exchange column separation in the presence of A1AT. Figure 4B shows the MutF activity and concentration of different fractions of the A2M:MutF complex after size exclusion column separation in the presence or absence of A1AT. Figures 4C-4D show that the A2M:MutF complex is stable over a wide pH range, as measured by enzyme activity (4C) and A1AT protection (4D). Figure 4E shows that the A2M:MutF complex is stable in multiple freeze-thaw cycles, as measured by enzyme activity.

[0240] Activity and A1AT protection in cell lysates. Tumor cells were trypsinized, washed with PBS, counted, and diluted in serum-free medium. Add 40k tumor cells to a V-type plate, followed by 40 μL of different conditions (SFM, A2M, 400 nM N17350, or premixed A2M:MutF (800 nM–400 nM)). Incubate the plate at 37°C for 30 min. After incubation, centrifuge the plate at 300 x g for 5 min and wash each well with an additional 200 μL of PBS. Add 25 μL of cytoplasmic lysis buffer (10 mM HEPES pH 8, 10 mM KCl, 0.1 mM EDTA, 0.3% NP-40) to each well and vortex for 10 seconds every 5 min for 20 min on ice (page 44 / 47, CN 121263518 A). Centrifuge the plate at 300 x g for 5 min. Carefully transfer the supernatant to a new 96-well plate for A2M:MutF activity and protection assays. Here, 10 μL of supernatant was coated in duplicate with 10 μL of PBS or 10 μL of 2 μM A1AT, or at different concentrations, into 384 black well plates. 20 μL of substrate AAPV-AMC at a concentration of 100 µM was added. Kinetics were measured on a Varioskan LUX for 16 reads: 380 nm ex / 460 nm em (5 nm bandwidth), 37°C, top, 100 ms, 2 min. Vo was calculated as the activity measurement.

[0241] The results in Figures 5A-5D show that the A2M:MutF protein complex can enter cells and remain in the intracellular protein complex form because A1AT cannot inhibit its activity.

[0242] CD95-C cleavage assay. MutF was mixed with different concentrations of A2M to achieve various molar ratios, followed by incubation at 37°C. Incubate in PBS for 30 minutes. Incubate recombinant CD95-C terminal protein (Wuxi) with premixed A2M:MutF protein complex at 37°C for 1 hour at different molar ratios. Load the sample containing 1 μg CD95-C into 17.5% of the solution.SDS-PAGE gels were prepared and stained with One-step Blue (Biotium). Gels were imaged using an iBright 1500.

[0243] As shown in Figure 6A, the A2M:MutF protein complex cleaved CD95 as efficiently as MutF alone in a molar ratio range of 1:2 to 1:16.

[0244] Fibrinogen cleavage assay. MutF was mixed with different concentrations of A2M to achieve various molar ratios and then incubated in PBS at 37°C for 30 minutes. Human fibrinogen (Sigma) was incubated with premixed A2M:MutF protein complex at different molar ratios for 1 hour at 37°C. Samples containing 5 μg of fibrinogen were loaded onto 4–15% gels and fibrinogen was detected by Western blotting using anti-fibrinogen (Cell Signaling Technology). Gels were imaged using an iBright 1500.

[0245] As shown in Figure 6B, the A2M:MutF protein complex does not cleave fibrinogen compared to MutF alone.

[0246] Elastin cleavage assay. MutF was mixed with different concentrations of A2M to achieve various molar ratios, and then incubated in PBS at 37 °C for 30 min. Elastin-F (1:100) was incubated with the premixed A2M:MutF protein complex overnight at 37 °C, and the fluorescence signal of degradation was measured in the supernatant.

[0247] As shown in Figure 6C, the A2M:MutF protein complex does not cleave elastin compared to MutF alone.

[0248] Coagulation assay. Mice were injected with 200 μL of 480 μg MutF or a 1:2 molar ratio of purified A2M:MutF protein complex. Blood was collected in sodium citrate tubes and centrifuged at 1500 x g for 15 min. 300 μl of plasma was frozen and transported to IDEXX for analysis of prothrombin time, partial prothrombin kinase time, and fibrinogen concentration.

[0249] The results in Figures 7A-7C show that the A2M:MutF protein complex did not induce a coagulation effect compared to MutF itself, while the former increased PT and PTT times and resulted in a decrease in fibrinogen levels.

[0250] Cell killing assay. Approximately 20K-40K cancer cells were plated in black-well-coated 96-well plates and incubated overnight for sedimentation. On the second day, each well was washed once with 200 μL of serum-free medium, followed by three separate treatments. 24 hours after treatment, the cells were treated with pre-diluted calcein-AM solution (Thermo Fisher Scientific, 4 μM).Incubate together with HBSS containing Ca2+Mg2+ for 45 minutes. Pour calcein AM and add 100 μL of HBSS containing Ca2+Mg2+ to each well, and measure fluorescence at 485 / 520 nm (20 bandwidth) using Varioskan LUX.

[0251] As shown in Figure 8A, the A2M:MutF protein complex with a molar ratio of 1:2 induced the killing of various cancer cells in vitro, and in most cases the activity was comparable to that of MutF itself. Figure 8B shows the broad cytotoxicity of the A2M:MutF (1:2) protein complex against cancer cells of different anatomical origins, and Figure 8C shows that the complex does not kill non-cancer cells. Specification 45 / 47 pages 51 CN 121263518 A

[0252] In vivo efficacy. Approximately one million CT26 cells were implanted into the flanks of 7-8 week old BALB / c mice. Once the tumor reached approximately 80 mm3, it was treated with 100 μg MutF, 100 μg A2M:MutF (1:2), or 1.4 mg A2M (corresponding to the amount that produces a 1:2 A2M:MutF molar ratio). The tumor was monitored every two days.

[0253] As shown in Figure 8D, the A2M:MutF protein complex at a molar ratio of 1:2 induced the killing of cancer cells in vivo, comparable to the cytotoxic activity of MutF itself. Figure 8E shows that, after intravenous administration, the A2M:MutF protein complex had an improved functional PK profile (enzymatic activity in plasma) compared to MutF alone. Figure 8F shows that the A2M:MutF protein complex induced a favorable immune profile in the CT26 model (PBS, MutF, A2M:MutF from left to right in each figure). Figure 8G shows that the A2M:MutF protein complex induced a tumor antigen-specific CD8+ T cell response in the CT26 model (PBS, MutF, and A2M:MutF are shown from left to right in each figure). The induction of effectors and memory T cells indicates a functional adaptive immune response.

[0254] Evaluation of selective cancer cell killing in ovarian cancer patient samples. Cancer cells and non-cancer cells were isolated from primary tumors, intraperitoneal (IP) fluid, omental adipose tissue (a common metastatic site), and blood from six ovarian cancer patients. The following cells were isolated and tested: cancer cells (fibroblasts, CD45+ cells, and EpCAM+ / highly cell-depleted digested tumors), non-cancer immune cells (neutrophils or CD45+ depleted digested omental cells in tumor tissue), peripheral blood mononuclear cells (PBMCs; B cells, T cells, monocytes, and NK cells), fibroblasts (isolated from tumor samples using a fibroblast isolation kit), and IP cells (composed of >90% CD45+ immune cells, including B cells, T cells, myeloid cells, etc.).Cells (NK cells). The isolated cells were plated and exposed to A2M:MutF, doxorubicin (standard of care for chemotherapy), or oxaliplatin (standard of care for chemotherapy) for 24 hours. Cell viability was assessed by calcein-AM.

[0255] Figure 9A shows that the A2M:MutF protein complex has a wide therapeutic window compared to doxorubicin and oxaliplatin, as demonstrated by killing human ovarian cancer cells without killing non-cancer cells in patients. Figure 9B shows that A2M:MutF kills cancer cells equally from both chemotherapy-naïve and chemotherapy-treated patients compared to doxorubicin and oxaliplatin, demonstrating reduced killing of cancer cells from chemotherapy-treated patients relative to chemotherapy-naïve patients.

[0256] Immunogenic cell death (ICD) was assessed. CT26 (mouse colon), A549 (human lung), and ovarian cancer patient cells were treated for 24 hours with A2M:MutF and oxaliplatin (high concentrations of ICD inducers known in certain cell types). Immunogenic cell markers, including HSP70, ATP release, HMGB1, and CALR, were evaluated (see, for example, Fucikova et al., Cell Death and Disease. 11(11): 1013, 2020).

[0257] Figure 10A shows that the A2M:MutF protein complex induces ICD markers in CT26 and A549 cells. Figure 10B shows that the A2M:MutF protein complex induces ICD markers in tumor cells derived from human ovarian patients (CTRL, A2M:MutF, and oxaliplatin from left to right in each figure).

[0258] The dosage and schedule of A2M:MutF were evaluated. CT26 colon cancer cells were injected into the flanks of mice and allowed to grow until they reached approximately 80 mm3. Multiple intravenous doses of the A2M:MutF protein complex or mediator were administered as follows: ● Mediator: Days 0, 2, 4, and 6, and Days 8, 10, 12, and 14; ● A2M:MutF 100 μg: every day for 2 weeks; ● A2M:MutF 200 μg: Days 0, 2, 4, 6, and Days 8, 10, 12, and 14 (every other day); and ● A2M:MutF 400 μg: Days 0, 4, 8, and 12 (every 4 days).

[0259] Tumor growth and weight were assessed. Figures 11A-11B show tumor growth after treatment, and Figure 11C shows tumor weight 15 days after treatment (11C from left to right: mediator every other day, A2M:MutF 100 μg daily, A2M:MutF [Instructions for Use] 46 / 47 pages 52 CN 121263518 A 200)

[0260] Antitumor efficacy was evaluated in syngeneic mouse tumor models. CT26 (a highly immunogenic model of the colon), MC38 (a warm immunogenic model of the colon), and B16F10 (a cold immunogenic model of skin cancer and lung metastases) cancer cells were injected into the flanks of syngeneic B-cell-deficient Jh-BALB / c or C57BL / 6 mice and allowed to grow until they reached approximately 80–100 mm3. The A2M:MutF protein complex (400 μg) or a mediator was injected intravenously every other day for 2–3 weeks. Tumor growth was monitored.

[0261] Figures 12A–12B show that the A2M:MutF protein complex effectively attenuated tumor growth in the Jh-BALB / c CT26 colorectal cancer model. Figures 12C-12D show the A2M:MutF protein complex treatment of primary and metastatic tumors in a Jh-C57BL / 6 B16F10 melanoma model. Figure 12E shows the efficacy of the A2M:MutF protein complex across a range of tumors with variable immune status.

[0262] Evaluation of antitumor efficacy relative to SoC chemotherapy: CT26 colon cancer cells were injected into the flanks of syngeneic B-cell-deficient Jh-BALB / c mice and allowed to grow until they reached approximately 80-100 mm3. The A2M:MutF protein complex (400 μg, every other day for 3 weeks) or oxaliplatin (6 mg / kg on days 0 and 2) was administered intravenously. Tumor growth and overall survival were monitored.

[0263] Figures 13A-13C show that the A2M:MutF protein complex had improved antitumor efficacy relative to SoC chemotherapy (oxaliplatin) without the observation of toxicity.

[0264] The antitumor efficacy against human cancer cells was evaluated in xenograft models (NU / NU and NCG female mice). HCT116 (human colorectal cancer), HT29 (human colorectal cancer), PC3 (human prostate cancer), NCI-H358 (human lung cancer), A549 (human lung cancer), ovarian patient-derived (CDX) model, and breast cancer (PDX) model cancer cells were injected into the flanks of mice and allowed to grow until they reached approximately 80–100 mm3. The A2M:MutF protein complex (400 μg) or a mediator was administered intravenously every other day for 2–3 weeks. Tumor growth and overall survival were monitored.

[0265] Figure 14A shows the efficacy of the A2M:MutF protein complex in a human xenograft model of lung cancer. Figure 14B summarizes the efficacy of the A2M:MutF protein complex across various prostate cancer, colon cancer, and lung cancer models. Figure 14C shows that A2M:The A2M:MutF protein complex effectively killed human ovarian patient-derived tumor cells (from patient CDX_O02) in a xenograft mouse model, and Figure 14D summarizes the efficacy of the A2M:MutF protein complex across three ovarian cancer patients in this model (patient history: diagnosis, grade 3; treatment, CDX_O01 and CDX_O03 were chemotherapy-naïve and CDX_O02 was treated with 3 cycles of carboplatin + paclitaxel + Keytruda). Figures 14E-14F show that the A2M:MutF protein complex effectively killed patient-derived breast cancer cells in vitro and in vivo (patient history: diagnosis, breast cancer HER2- and ER+; treatment, chemotherapy-naïve). Figure 14G summarizes the in vivo efficacy of the A2M:MutF protein complex across various human tumors and shows that the efficacy is independent of tumor genetic or immune status.

[0266] Figure 15 shows that mice treated with the A2M:MutF protein complex were tumor-free after initial challenge with CT26 colorectal cancer cells (5 / 11), and all of these mice (5 / 5) remained tumor-free after rechallenge with CD26 cells. This study demonstrates that treatment with the A2M:MutF protein complex induces a tumor-specific immune memory response. Instruction manual page 47 / 47, page 53, CN 121263518 A, Figure 1; Instruction manual figure 1 / 50, page 54, CN 121263518 A, Figure 2A; Instruction manual figure 2 / 50, page 55, CN 121263518 A, Figure 2B; Instruction manual figure 3 / 50, page 56, CN 121263518 A, Figure 2C; Instruction manual figure 4 / 50, page 57, CN 121263518 A, Figure 3A; Instruction manual figure 5 / 50, page 58, CN 121263518 A, Figure 3B; Instruction manual figure 6 / 50, page 59, CN 121263518 A, Figure 3C; Instruction manual figure 7 / 50, page 60, CN 121263518 A, Figure 4A; Instruction manual figure 8 / 50, page 61, CN 121263518 A, Figure 4B; Instruction manual figure 9 / 50, page 62, CN 121263518 A Figure 4C: Instruction manual drawing, page 10 / 50, 63 CN 121263518 A Figure 4D: Instruction manual drawing, page 11 / 50, 64 CN 121263518 A Figure 4E: Instruction manual drawing, page 12 / 50, 65 CN 121263518 A Figure 5A: Instruction manual drawing, page 13 / 50, 66 CN 121263518 A Figure 5B: Instruction manual drawingPage 14 / 50, 67 CN 121263518 A, Figure 5C, Instruction Manual Drawing; Page 15 / 50, 68 CN 121263518 A, Figure 5D, Figure 6A, Instruction Manual Drawing; Page 16 / 50, 69 CN 121263518 A, Figure 6B, Figure 6C, Instruction Manual Drawing; Page 17 / 50, 70 CN 121263518 A, Figure 7A, Instruction Manual Drawing; Page 18 / 50, 71 CN 121263518 A, Figure 7B, Instruction Manual Drawing; Page 19 / 50, 72 CN 121263518 A, Figure 7C, Instruction Manual Drawing; Page 20 / 50, 73 CN 121263518 A, Figure 8A, Instruction Manual Drawing; Page 21 / 50, 74 CN 121263518 A, Figure 8B, Instruction Manual Drawing; Page 22 / 50, 75 CN 121263518 A, Figure 8C, Instruction Manual Drawing; Page 23 / 50 Page 76 CN 121263518 A Figure 8D Instruction Manual Drawing 24 / 50 Page 77 CN 121263518 A Figure 8E Instruction Manual Drawing 25 / 50 Page 78 CN 121263518 A Figure 8F Instruction Manual Drawing 26 / 50 Page 79 CN 121263518 A Figure 8G Instruction Manual Drawing 27 / 50 Page 80 CN 121263518 A Figure 9A Instruction Manual Drawing 28 / 50 Page 81 CN 121263518 A Figure 9B Instruction Manual Drawing 29 / 50 Page 82 CN 121263518 A Figure 10A Instruction Manual Drawing 30 / 50 Page 83 CN 121263518 A Figure 10B Instruction Manual Drawing 31 / 50 Page 84 CN 121263518 A Figure 11A Instruction Manual Drawing 32 / 50 Page 85 CN 121263518 A Figure 11B Instruction Manual Appendix 33 / 50 Page 86 CN 121263518 A Figure 11C Instruction Manual Appendix 34 / 50 Page 87 CN 121263518 A Figure 12A Instruction Manual Appendix 35 / 50 Page 88 CN 121263518 A Figure 12B Instruction Manual Appendix 36 / 50 Page 89 CN 121263518 A Figure 12C Instruction Manual Appendix 37 / 50 Page 90 CN 121263518 A Figure 12D Instruction Manual AppendixFigure 38 / 50, page 91, CN 121263518 A; Figure 12E, Instruction Manual Drawing; Figure 39 / 50, page 92, CN 121263518 A; Figure 13A, Instruction Manual Drawing; Figure 40 / 50, page 93, CN 121263518 A; Figure 13B, Instruction Manual Drawing; Figure 41 / 50, page 94, CN 121263518 A; Figure 13C, Instruction Manual Drawing; Figure 42 / 50, page 95, CN 121263518 A; Figure 14A, Instruction Manual Drawing; Figure 43 / 50, page 96, CN 121263518 A; Figure 14B, Instruction Manual Drawing; Figure 44 / 50, page 97, CN 121263518 A; Figure 14C, Instruction Manual Drawing; Figure 45 / 50, page 98, CN 121263518 A; Figure 14D, Instruction Manual Drawing; Figure 46 / 50, page 99, CN 121263518 A; Figure 14E Instruction manual figures 47 / 50, page 100, CN 121263518 A, Figure 14F; Instruction manual figures 48 / 50, page 101, CN 121263518 A, Figure 14G; Instruction manual figures 49 / 50, page 102, CN 121263518 A, Figure 15; Instruction manual figures 50 / 50, page 103, CN 121263518 A

Claims

1. A pharmaceutical composition comprising the following protein complex: (a) α-2-macroglobulin (A2M) protein; and (b) Serine protease protein, (a) and (b) are present in the composition in a molar ratio of about 1:3 to about 1:1 [(a):(b)].

2. The pharmaceutical composition according to claim 1, wherein the A2M protein of (a) and the serine protease protein of (b) are bound together in the protein complex, and optionally wherein the protein complex: (i) Retains the CD95 (Fas receptor) protease cleavage activity and cancer cell killing activity of (b); (ii) Spatially hinders the binding of (b) to fibrinogen and reduces or inhibits the fibrinogen cleavage activity of (b); and (iii) Spatially hinders the binding of (b) to serine protease inhibitors, including α-1 antitrypsin (A1AT) 3. The pharmaceutical composition according to claim 1 or 2, wherein (a) comprises, is composed of, or is substantially composed of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, constitutes, or is substantially composed of a sequence selected from Table A1 or a functional fragment thereof.

4. The pharmaceutical composition according to claim 3, wherein the functional fragment comprises, is composed of, or is substantially composed of about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1300, or 1400 consecutive amino acids selected from the sequence in Table A1.

5. The pharmaceutical composition according to claim 4, wherein the functional fragment thereof comprises 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, 10 0-200, 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, 700-1 Composed of 200, 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.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein (a) is conjugated or fused with an antibody or an antigen-binding fragment thereof.

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

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

9. The pharmaceutical composition according to claim 8, wherein: The PPE protein comprises, consists of, or is substantially composed of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the Q211F amino acid substitution, of SEQ ID NO:

5. The PPE protein comprises, consists of, or is substantially composed of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the T55A amino acid substitution, of SEQ ID NO:

6. The PPE protein comprises, consists of, or is substantially composed of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the Q211F and T55A amino acid substitutions of SEQ ID NO:

7. The PPE protein comprises, consists of, or is substantially composed of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the N241A amino acid substitution. The PPE protein comprises, consists of, or is substantially composed of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the N241Y amino acid substitutions of SEQ ID NO:

9. The PPE protein comprises, consists of, or is substantially composed of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the R75A amino acid substitution. The PPE protein comprises, is composed of, or is substantially composed of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the R75E amino acid substitutions of SEQ ID NO:

11. The PPE protein comprises, consists of, or is substantially composed of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the Q211A amino acid substitution of, SEQ ID NO:

12. The PPE protein comprises, consists of, or is substantially composed of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the amino acid substitution of R237A. The PPE protein comprises, consists of, or is substantially composed of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the amino acid substitution of S214A of SEQ ID NO:

14. The PPE protein comprises, is composed of, or is substantially composed of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the D74A amino acid substitution, of SEQ ID NO: 15; and The PPE protein contains, is composed of, or is substantially composed of the same amino acid sequence as SEQ ID NO: 16, at least 80%, 85%, 90%, 95%, 98%, 99%, or 100%.

10. The pharmaceutical composition according to claim 8, wherein: The human ELANE protein comprises, is composed of, or is substantially composed of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO:

17. The human CTSG protein comprises, is composed of, or is substantially composed of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO:

18. The human PR3 protein comprises, is composed of, or is substantially composed of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO: 19; or The human granzyme B protein comprises, consists of, or is substantially composed of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO:

20.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein (a) and (b) are present in the composition in 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 according to claim 11, wherein (a) and (b) are present in the composition in a molar ratio of about 1:

2.

13. A method for treating a subject with cancer, improving symptoms of said cancer, and / or reducing the progression of said cancer, the method comprising administering to said subject a pharmaceutical composition according to any one of claims 1 to 12.

14. The method of claim 13, wherein the cancer is a primary or metastatic cancer, and is selected from one or more of the following: melanoma (optionally metastatic melanoma), breast cancer (optionally triple-negative breast cancer, TNBC), renal cell carcinoma (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 myeloid leukemia, acute myeloid leukemia, or relapsed acute myeloid leukemia), multiple myeloma, lymphoma, hepatocellular carcinoma, sarcoma, B-cell malignancy, ovarian cancer, colorectal cancer, glioma, glioblastoma multiforme, meningioma, pituitary adenoma, vestibular schwannoma, primary CNS lymphoma, primitive neuroectodermal tumor (neuroblastoma), bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancer, cervical cancer, testicular cancer, thyroid cancer, and gastric cancer.

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

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

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

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

19. A method for preparing a pharmaceutical composition comprising a protein complex, the method being carried out by combining the following items in a molar ratio of about 1:3 to about 1:1 [(a):(b)]: (a) α-2-macroglobulin (A2M) protein; and (b) Serine protease protein, The pharmaceutical composition comprising the protein complex is thus prepared.

20. The method of claim 19, wherein it comprises recombining (a) prior to combination with (b).

21. The method of claim 19, comprising purifying (a) from the plasma of a human subject prior to combination with (b).

22. The method according to any one of claims 19 to 21, wherein it comprises recombining (b) prior to combination with (a).

23. The method according to any one of claims 19 to 22, comprising a combination of (a): (b) in molar ratios 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, comprising combining (a) and (b) in a molar ratio of about 1:2 [(a):(b)].

25. The pharmaceutical composition according to any one of claims 19 to 24, wherein the A2M protein of (a) and the serine protease protein of (b) are bound together in the protein complex, and optionally wherein the protein complex: (i) Retains the CD95 (Fas receptor) protease cleavage activity and cancer cell killing activity of (b); (ii) Spatially hinders the binding of (b) to fibrinogen and reduces or inhibits the fibrinogen cleavage activity of (b); and (iii) Spatially hinders the binding of (b) to serine protease inhibitors, including α-1 antitrypsin (A1AT) 26. The method according to any one of claims 19 to 25, wherein (a) comprises, is composed of, or is substantially composed of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, constitutes, or is substantially composed of a sequence selected from Table A1 or a functional fragment thereof.

27. The method of claim 26, wherein the functional fragment comprises, is composed of, or is substantially composed of, about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1300, or 1400 consecutive amino acids selected from the sequences in Table A1.

28. The method of claim 27, wherein the functional fragment comprises 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, 4 00-1100, 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, 700-12 Composed of 00, 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.

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

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

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

32. The method according to claim 31, wherein: The PPE protein comprises, consists of, or is substantially composed of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the Q211F amino acid substitution, of SEQ ID NO:

5. The PPE protein comprises, consists of, or is substantially composed of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the T55A amino acid substitution, of SEQ ID NO:

6. The PPE protein comprises, consists of, or is substantially composed of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the Q211F and T55A amino acid substitutions of SEQ ID NO:

7. The PPE protein comprises, consists of, or is substantially composed of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the N241A amino acid substitution. The PPE protein comprises, consists of, or is substantially composed of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the N241Y amino acid substitutions of SEQ ID NO:

9. The PPE protein comprises, consists of, or is substantially composed of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the R75A amino acid substitution. The PPE protein comprises, is composed of, or is substantially composed of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the R75E amino acid substitutions of SEQ ID NO:

11. The PPE protein comprises, consists of, or is substantially composed of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the Q211A amino acid substitution of, SEQ ID NO:

12. The PPE protein comprises, consists of, or is substantially composed of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the amino acid substitution of R237A. The PPE protein comprises, consists of, or is substantially composed of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the amino acid substitution of S214A of SEQ ID NO:

14. The PPE protein comprises, is composed of, or is substantially composed of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to, and retains the D74A amino acid substitution, of SEQ ID NO: 15; and The PPE protein contains, is composed of, or is substantially composed of the same amino acid sequence as SEQ ID NO: 16, at least 80%, 85%, 90%, 95%, 98%, 99%, or 100%.

33. The method according to claim 31, wherein: The human ELANE protein comprises, is composed of, or is substantially composed of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO:

17. The human CTSG protein comprises, is composed of, or is substantially composed of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO:

18. The human PR3 protein comprises, is composed of, or is substantially composed of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO: 19; or The human granzyme B protein comprises, consists of, or is substantially composed of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO:

20.

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

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

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