Pharmaceutical composition comprising protein complex

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

[0001] Cross Reference to Related Applications

[0002] This application claims the benefit under 35 U.S.C. § 119(e) 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, each of which is incorporated by reference in its entirety.

[0003] Statement as to Federally Sponsored Research

[0004] The Sequence Listing associated with this application is provided in XML format and is hereby incorporated by reference into the specification. The name of the XML file containing the Sequence Listing is OPNI_009_02WO_ST26.xml. The XML file is about 28,746 bytes, was created on March 28, 2024, and is being submitted electronically via the USPTO Patent Center. TECHNICAL FIELD

[0005] The present disclosure relates to pharmaceutical compositions comprising alpha-2-macroglobulin (A2M) and a serine protease protein such as porcine pancreatic elastase (PPE) bound together in a protein complex that retains the CD95 protease cleavage and cancer cell killing activity of the serine protease but sterically hinders the binding of the serine protease to fibrinogen and serine protease inhibitors; and related uses and methods of manufacture for treating diseases such as cancer. BACKGROUND

[0006] Precision medicine, which aims to optimize the efficacy or therapeutic benefit for specific patient populations through the use of genetic or molecular analysis, has gained tremendous appeal in the context of cancer treatment. The identification of (i) specific genomic abnormalities that confer risk of developing cancer; (ii) influence tumor growth; and (iii) modulate 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 a targetable property in tumor cells that distinguishes them from healthy, non-cancerous cells. In fact, despite the ability of radiation and / or chemotherapy to effectively kill many, if not most, cancer cells, its efficacy is severely limited by cytotoxic effects on non-cancerous cells. These findings confirm that the property of rapid cell division, which radiotherapy and chemotherapy target, is not sufficiently unique to cancer cells to enable the specificity required to limit widespread side effects.

[0008] Certain serine proteases or elastases have been shown to have selective toxicity to cancer cells, but relative non-toxicity to normal or otherwise healthy cells (see, e.g., WO 2018 / 232273). However, there is a need in the art to identify optimal enzyme compositions that can have such selective cancer cell toxicity but also have reduced side effects, and thus improve the pharmacokinetics and overall clinical utility of such compositions. SUMMARY

[0009] Embodiments of the present disclosure include pharmaceutical compositions comprising a protein complex of: (a) an alpha-2-macroglobulin (A2M) protein; and (b) a serine protease protein, wherein (a) and (b) are present in the composition in a molar ratio [(a):(b)] of about 1:3 to about 1:1. In certain embodiments, the A2M protein of (a) and the serine protease protein of (b) are bound together in the protein complex, e.g., wherein the protein complex: (i) retains the CD95 (Fas receptor) protease cleavage activity and cancer cell killing activity of (b); (ii) sterically hinders the binding of (b) to fibrinogen and reduces or inhibits the fibrinogen cleavage activity of (b); and (iii) sterically hinders the binding of (b) to serine protease inhibitors, including plasma serine protease inhibitors, such as alpha-1 antitrypsin (A1AT).

[0010] In some embodiments, (a) comprises, consists of, or is substantially composed of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% the same amino acid sequence as, selected from the sequence in 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 sequence 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 from the sequence selected from Table A1. -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, 400-11 00, 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 Composed of 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.

[0011] In some embodiments, (a) it 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 protein G (CTSG) protein, human protease 3 (PR3) protein, and granzyme B protein. In specific embodiments: 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 amino acid substitution of Q211A. 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%.

[0013] In some embodiments: 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.

[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 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), 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 intravenously) 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.

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

[0019] The method also includes 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.

[0020] Some embodiments involve recombining (a) prior to combination with (b). Some embodiments involve purifying (a) from the plasma of a human subject prior to combination with (b). Specific embodiments involve recombining (b) prior to combination with (a).

[0021] In some embodiments, the preparation method comprises combinations (a) and (b) of 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)]]. Specific embodiments include combinations (a) and (b) of molar ratios of approximately 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, including 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)

[0023] In some embodiments, (a) comprises, consists of, or is substantially composed of at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% the same amino acid sequence as, selected from the sequence in 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 sequence 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 from the sequence selected from Table A1. -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, 400-11 00, 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 Composed of 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.

[0024] In some embodiments, (a) it 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 protease G (CTSG) protein, human protease 3 (PR3) protein and human granzyme B protein.

[0026] In some embodiments: 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 amino acid substitution of Q211A. 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%.

[0027] In some embodiments: 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.

[0028] Some methods further include 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. In some embodiments, 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. Attached Figure Description

[0029] Figure 1 The formation and structure of a protein complex containing human A2M protein and a serine protease protein are shown. 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 The activity of mutant F (MutF) alone on tumor growth (2A) and the number of lung metastases (2B) after IV injection (day 0 and day 1) in the 4T1 tumor model is shown. Figure 2C The effect of MutF on prothrombin time was shown 5 minutes after IV injection.

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

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

[0033] Figures 5A-5DThe activity of MutF in various cell lysates after treatment in serum-free medium (SFM) for 30 minutes in the presence or absence of A1AT is shown.

[0034] Figure 6A The images show Coomassie blue staining of CD95 cuts after incubation with MutF or A2M:MutF for 30 minutes at different ratios. Figure 6B The images show the protein blots of fibrinogen after incubation with MutF or A2M:MutF for 1 hour at different ratios. A2M:MutF cleaved CD95 as efficiently as MutF alone at a 1:2 ratio (shown by the double bands), but did not cleave fibrinogen as MutF alone (shown by the bottom band). Figure 6C Fluorescent signals of cleaved elastin after incubation with PBS, MutF alone, or the A2M:MutF protein complex are shown.

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

[0036] Figure 8A The cell killing assays of various mouse cancer cell lines by the MutF or A2M:MutF (1:2) protein complex at 400 nM are shown. Figure 8B The A2M:MutF protein complex was shown to have broad cytotoxicity against cancer cells of different anatomical origins, and Figure 8C The study showed that the complex did not kill non-cancer cells. Figure 8D The antitumor effect of the MutF and A2M:MutF (1:2) protein complex in the CT26 model was demonstrated after a 100 μg IT injection on day 0. Figure 8E The results show that, after intravenous administration, the A2M:MutF protein complex has an improved functional PK profile (enzymatic activity in plasma) compared to MutF alone. Figure 8F The A2M:MutF protein complex induces a favorable immune profile in the CT26 model (PBS, MutF, and A2M:MutF are shown from left to right in each figure). Figure 8GAs shown, the A2M:MutF protein complex induced tumor antigen-specific CD8+ T cell responses in the CT26 model (PBS, MutF, and A2M:MutF are shown from left to right in each figure).

[0037] Figure 9A The results show that, compared to doxorubicin and oxaliplatin, the A2M:MutF protein complex has a broad therapeutic window, as demonstrated by killing human ovarian cancer cells without killing non-cancerous cells in patients. Figure 9B The results showed that A2M:MutF killed cancer cells isolated from both chemotherapy-naïve and chemotherapy-treated patients equivalently to doxorubicin and oxaliplatin, indicating reduced killing of cancer cells isolated from chemotherapy-treated patients compared to chemotherapy-naïve patients.

[0038] Figure 10A The results show that the A2M:MutF protein complex induces ICD markers in CT26 and A549 cells. Figure 10B As shown, 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 The growth of the treated tumor in the mouse CT26 tumor model is shown. Figure 11C Tumor weights at 15 days post-treatment are shown (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 The results show that the A2M:MutF protein complex effectively attenuates tumor growth in the Jh-BALB / c CT26 colorectal cancer model. Figures 12C-12D The results show that the A2M:MutF protein complex was used to treat primary and metastatic tumors in the Jh-C57BL / 6 B16F10 melanoma model. Figure 12E The A2M:MutF protein complex demonstrates efficacy across a range of tumors with variable immune status.

[0041] Figures 13A-13C The results showed that the A2M:MutF protein complex had improved antitumor efficacy compared to SoC chemotherapy (oxaliplatin), without any observed toxicity.

[0042] Figure 14A The efficacy of the A2M:MutF protein complex in a human xenograft model of lung cancer was demonstrated. Figure 14BThe efficacy of the A2M:MutF protein complex across various prostate cancer, colon cancer, and lung cancer models was summarized. Figure 14C The A2M:MutF protein complex was shown to effectively kill tumor cells derived from human ovarian patients (from patient CDX_O02) in a xenograft mouse model, and Figure 14D The efficacy of the A2M:MutF protein complex in this model in three ovarian cancer patients was summarized. Figures 14E-14F The results show that the A2M:MutF protein complex effectively kills patient-derived breast cancer cells both in vitro and in vivo. Figure 14G The in vivo efficacy of the A2M:MutF protein complex across a variety of human tumors was summarized, and the efficacy was shown to be independent of tumor genetic or immune status.

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

[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. While methods, materials, compositions, reagents, and cells similar to or equivalent to those 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 elucidated. Any patent application claiming priority to this application is also incorporated herein by reference in its entirety as described above with respect to the publications and references.

[0045] Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, liposome transfection). Enzymatic reactions and purification techniques can 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 can 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, 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 nomenclature and laboratory procedures and techniques well known and commonly used in the art. Standard techniques can be used for 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] This article uses the article “a / an” to refer to one or more (i.e., at least one) grammatical objects of the article. For example, “element” includes “a element”, “one or more elements” and / or “at least one element”.

[0048] "Approximately" means a quantity, level, value, number, frequency, percentage, size, size, quantity, weight, or length that varies by up to 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] An "antagonist" is a biological or chemical agent that interferes with or otherwise reduces the physiological effects of another drug or molecule. In some cases, antagonists bind specifically to another drug or molecule. They include complete antagonists and partial antagonists.

[0050] An "agonist" is a biological or chemical agent that increases or enhances the physiological effects of another drug or molecule. In some cases, agonists bind specifically to another drug or molecule. They include 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 mimics. 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, for example, (D)-amino acids, leucine, valine, p-fluorophenylalanine, ethionine, etc., known to those skilled in the art. 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, for example, organic structures exhibiting functionally similar properties to a reference amino acid, such as charge and charge spacing characteristics. 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 amino acids or amino acid functional groups. Those skilled in the art know or can identify which structurally and functionally equivalent amino acid analogs and amino acid mimics exist.

[0052] As used herein, a subject at "risk" for 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 that a material or compound will not normally impair the biological function of cells or subjects and will not cause any degree of unacceptable toxicity (including allergies and disease states).

[0054] The term "association" refers to the 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] A “coding sequence” refers to any nucleic acid sequence that contributes to the encoding of the polypeptide product of a gene. Conversely, the term “non-coding sequence” refers to any nucleic acid sequence that does not directly contribute to the encoding of the polypeptide product of a gene.

[0056] Throughout this disclosure, unless the context otherwise requires, the words “comprise,” “comprises,” and “comprising” will be understood to imply inclusion of the stated steps or elements or groups of steps or elements, but do not exclude any other steps or elements or groups of steps or elements.

[0057] The phrase “consisting of…” means including and limited to anything that follows the phrase “consisting of…”. Therefore, the phrase “consisting of…” indicates that the listed element is necessary or mandatory, and other elements may be absent. The phrase “substantially constitutes…” means including any element listed after the phrase, and is limited to other elements that do not interfere with or facilitate the activities or actions specified for the listed elements in this disclosure. Therefore, the phrase “substantially constitutes…” 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 activities or actions of the listed elements.

[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., ， Endotoxins (in amounts that have no adverse clinical physiological effects on the subject) and preferably contain undetectable amounts of endotoxins. Endotoxins are toxins associated with certain microorganisms, such as bacteria (usually Gram-negative bacteria), but can also be found in organisms such as Listeria monocytogenes (…). Listeria monocytogenes Endotoxins are found in Gram-positive bacteria, such as lipopolysaccharides (LPS) and lipoolipoproteins (LOS). The most common endotoxins are found in the outer membranes of various Gram-negative bacteria and are central pathogenic features representing these bacteria's ability to cause disease. Small amounts of endotoxins in the human body can produce fever, decreased blood pressure, inflammation, and activated coagulation, as well as other adverse physiological effects.

[0059] Therefore, in pharmaceutical manufacturing, it is generally desirable to remove most or all trace amounts of endotoxins from pharmaceutical products and / or pharmaceutical containers, as even small amounts can have adverse effects on humans. Depyrogen ovens 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 often 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-based assay using horseshoe crab amoeboid cell lysate is a highly sensitive method for detecting the presence of endotoxins. In this test, very low levels of LPS can induce detectable coagulation in the horseshoe crab lysate due to the strong amplification of the reaction by the enzyme cascade. Endotoxins can also be quantified by enzyme-linked immunosorbent assay (ELISA). To be substantially endotoxin-free, endotoxin levels can be below approximately 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 approximately 1–10 EU.

[0061] The term "half-maximum effective concentration" or "EC" 50 "" refers to the concentration of the agent (e.g., a protein complex) that, after some specific exposure time, induces a response between baseline and maximum; therefore, the EC50 of the graded dose-response curve... 50 EC50 indicates the concentration at which a compound is observed to have 50% of its maximum effect. EC50 also indicates the plasma concentration required to achieve 50% of the maximum effect in vivo. Similarly, “EC50”... 90 "This refers to the concentration of a drug or composition at which the maximum effect is observed." (EC) 90 "It can be calculated based on 'EC50' and the Hill slope, or it can be determined directly from the data using conventional knowledge in the art. In some embodiments, the EC50 of the agent..." 50 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 EC of the agent... 50 The value is approximately 1 nM or less.

[0062] The "half-life" of a drug can refer to the time it takes for the drug to lose half of its pharmacological, physiological, or other activity relative to its initial activity in the serum or tissues of a living organism, or relative to any other defined time point. The "half-life" can also refer to the time it takes for the amount or concentration of a drug to decrease by half relative to its initial amount in the serum or tissues of a living organism, or relative to any other defined time point. 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 that of 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 “modification” and “alteration” include an increase, enhancement, or stimulation, as well as a decrease or reduction, relative to a control, generally in a statistically or physiologically significant amount or degree. The “increased,” “stimulated,” 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., 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 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., no drug) or the control composition. Examples of comparisons 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 terms include modifications such as myristylation, sulfation, glycosylation, phosphorylation, and the addition or deletion of a signal sequence. The terms “polypeptide” or “protein” mean one or more amino acid chains, each chain containing amino acids covalently linked by peptide bonds, and said polypeptide or protein may comprise multiple chains having the sequence of a native protein (i.e., a protein produced by naturally occurring cells, specifically non-recombinant cells, or genetically engineered or recombinant cells) linked together non-covalently and / or covalently by peptide bonds, and comprise molecules having the amino acid sequence of a native protein or molecules having one or more amino acids of the native sequence with deletions, additions, and / or substitutions. In some embodiments, the polypeptide is a “recombinant” polypeptide produced by recombinant cells containing one or more recombinant DNA molecules, which are typically made up of 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. These 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 the total genomic DNA of a specific species. Therefore, an isolated DNA fragment encoding a polypeptide refers to a DNA fragment containing one or more coding sequences but which is substantially 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 polypeptides (e.g., non-coding polynucleotides that do not encode polypeptides). ， Primers, probes, and oligonucleotides. It also includes recombinant vectors, such as expression vectors, viral vectors, plasmids, phage particles, bacteriophages, and viruses.

[0067] The polynucleotides described herein may, but need not, contain additional coding or non-coding sequences, and the polynucleotides may, but need not, 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 their 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 can affect 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 contain, 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 wide variety of promoters, 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 personal 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. […], Proceedings of the National Academy of Sciences (PNAS) (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 (1999) 27(22): 4324-4327; Nucleic Acid Research (2000) 28(23): e99; US Patent No. 7,112,715; and Kramer and Fussenegger, […] ... Methods in Molecular Biology (2005) 308:123-144 or any promoter known in the art that is suitable for expression in the desired cells.

[0071] "Expressible polynucleotides" include 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 a cell.

[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, e.g., from the same species; (3) is expressed by cells from a 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 from proteins or polypeptides 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 peptides, measured by protein or weight-to-weight ratio, as measured by, but not limited to, 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 polypeptide and polynucleotide sequences described herein are included as reference sequences, including those described by name and those described in the tables and sequence listings.

[0075] Some embodiments include bioactive “variants” and “fragments” of the proteins / peptides described herein, as well as polynucleotides encoding them. A “variant” contains one or more substitutions, additions, deletions, and / or insertions relative to a reference polypeptide or polynucleotide (see, for example, tables and sequence listings). The variant polypeptide or polynucleotide contains 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 retains the activity of the reference sequence. It 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 in that the amino acids or nucleotides are added, deleted, inserted or substituted, and substantially retain at least one active element of the 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, containing “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 by comparing two optimally aligned sequences within a comparison window, determining the number of positions in which identical nucleic acid bases (e.g., A, T, C, G, I) or identical 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, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying 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 typically 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 said solute in said 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 "essentially" means almost entirely or completely, for example, 95%, 96%, 97%, 98%, 99% or higher for some given quantity.

[0080] "Statistically significant" means that the result could not have occurred by chance. 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] "Therapeutic 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” refer to the amount of a drug required to elicit the desired biological response after administration.

[0083] As used herein, “treatment” for 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, the administration of a pharmaceutical composition and may be preventative or administered after the onset of a pathological event or after exposure to a pathogen. It also includes “preventative” treatment, 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 "wild type" refers to the gene or gene product (e.g., polypeptide) that is most commonly observed in a population and is therefore arbitrarily engineered to be in the "normal" or "wild type" form of the gene.

[0085] Unless otherwise expressly stated, each embodiment in this specification is applicable to every other embodiment.

[0086] protein complex

[0087] The embodiments of this disclosure generally relate to pharmaceutical compositions comprising a protein complex of an α-2-macroglobulin (A2M) protein and a serine protease protein 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 protein complexes of human A2M and serine proteases, such as PPE, not only sterically protect serine proteases from serine protease inhibitors in plasma while retaining CD95 protease cleavage and cancer cell killing activity, but also reduce the negative effects of serine proteases on coagulation, for example, by sterically inhibiting or otherwise reducing the ability of the complex serine proteases 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 proteases and reducing their negative effects on coagulation, as measured, for example, by reducing prothrombin time or reducing fibrinogen cleavage relative to the serine proteases 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 protein is bound in the protein complex as an A2M monomer or an A2M polymer, for example as an A2M dimer, such as an A2M homodimer, as an A2M trimer, such as an A2M homotrimer, or as an A2M tetramer, such as an A2M homotetramer. In specific embodiments, the A2M protein in (a) is bound as an A2M homotetramer, and the serine protease protein in (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 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 may otherwise generate anti-drug antibodies. In contrast, the protein complex does not spatially prevent (b) from binding to smaller molecules such as CD95. Therefore, in specific embodiments, (b) in the protein complex cleaves CD95 and kills cancer cells but substantially does not 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.

[0092]

[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% 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 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 from the sequence selected from Table A1. 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, 700-120 Composed of 0, 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.In some embodiments, the functional fragment is capable of forming an A2M homotetramer and capturing or otherwise binding a serine protease (such as PPE) to a protein complex in a conformation that spatially hinders the binding of a serine protease to a serine protease inhibitor (such as A1AT), retains the CD95 protease cleavage activity and cancer cell killing activity of the serine protease, and / or inhibits or otherwise reduces the ability of the serine protease to cleave fibrinogen.

[0095] In some embodiments, the A2M moiety 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 both normal and cancer cells. Indeed, elevated GRP78 levels are generally associated with higher pathological grades, recurrence, and poor patient survival in breast, liver, prostate, colon, and gastric cancers (see, for example, Lee, Cancer Res. 67:3496–3499, 2007), and in some cases, the ability of A2M to bind to GRP78 improves selective targeting of GRP78-expressing cancer cells.

[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, 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 TRAI) L-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 tumors), phosphatidylinositol proteoglycan-3 (GPC3), and mesothelin.

[0097] The pharmaceutical compositions and protein complexes described herein contain serine protease proteins. Examples of serine proteases include porcine pancreatic elastase (PPE), human neutrophil elastase (ELANE), human cathepsin G (CTSG), human protease 3 (PR3), and human granzyme B (GZMB). The amino acid sequences of exemplary full-length wild-type serine protease proproteins are 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 an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 100% identical to a 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.

[0101]

[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, 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 amino acid substitution of Q211A. 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%.

[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 at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO: 17. 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, at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO: 18. 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)] ranging from 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 preserves the CD95 protease cleavage and cancer cell killing activity of the serine protease while spatially hindering its binding to serine protease inhibitors (e.g., A1AT), 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] Therefore, in some embodiments, the protein complex retains the ability to cleave CD95 (Fas receptor) and substantially 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 CD95 protease cleavage and / or cancer cell killing activity of the corresponding serine protease protein itself (e.g., in the presence of serine protease inhibitors in plasma such as A1AT). In some embodiments, the protein complex described herein has about or less than about 50%, 40%, 30%, 20%, 10%, 5%, or less of the fibrinogen protease cleavage activity of the corresponding serine protease protein itself. 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-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroaniline), and CD95 and / or fibrinogen cleavage can be measured directly (e.g., Western blotting). If necessary, 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 its effect on in vivo coagulation can be measured, for example, by routine assays such as prothrombin (PT) time and activated partial thromboplastin (PTT) time (see examples).

[0107] In some embodiments, the protein complexes described herein are generated in vivo or in vitro, for example, in cells by contacting cells or a subject with one or more expressible polynucleotides encoding (a) an α-2-macroglobulin (A2M) protein and (b) a serine protease protein. The protein complexes are then formed within the cells. “Expressible polynucleotides” include 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., a transcriptional and / or translational regulatory element) and capable of expressing the encoded protein when introduced into cells (e.g., a 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, the expressible polynucleotide is modified RNA or 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 linkages. 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 possess 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] Method of use and pharmaceutical composition

[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 the subject a composition comprising a protein complex 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, the cancer is either 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-mentioned cancers, exemplary metastatic cancers include, but are not limited to: bladder cancer that has metastasized to the bones, liver, and / or lungs; breast cancer that has metastasized to the bones, 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; stomach 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) of a protein complex or composition containing a serine protease has substantially no effect on the subject's coagulation; for example, it substantially does not increase the subject's prothrombin time or partial prothrombin kinase time (e.g., relative to 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 the subject's coagulation compared to administration of the corresponding serine protease alone.

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

[0116] Therefore, some embodiments include combination therapies for treating cancer, the combination therapies comprising methods for 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%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2000%, or more.

[0117] Some combination therapies employ one or more cancer immunotherapeutic agents, or "immunotherapy agents." In some cases, immunotherapy agents modulate a subject's immune response, for example, to increase or maintain a cancer-related or cancer-specific immune response, thereby leading to increased suppression of immune cells or a reduction in cancer cells. Exemplary immunotherapy agents include peptides, such as antibodies and their antigen-binding fragments, ligands, and small peptides, as well as 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, cancer immunotherapeutic agents are immune checkpoint modulators. 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, an immune checkpoint modulator is an antagonist or inhibitor 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), B and T lymphocyte attenuator (BTLA), CD160, and T cell immune receptor (TIGIT) having Ig and ITIM domains.

[0120] In some embodiments, the agent is a PD-1 (receptor) antagonist or inhibitor, which has 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 its immunosuppressive activity, such as its downstream signaling pathways or their 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. Patent Nos. 8,008,449 and 8,993,730). Applications No. 1, 9,073,994, 9,084,776, 9,102,727, 9,102,728, 9,181,342, 9,217,034, 9,387,247, 9,492,539, 9,492,540, and U.S. Application 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 immunosuppressive activities, such as their binding to the PD-1 receptor. Specific examples of PD-L1 antagonists include the antibodies atezolizumab (MPDL3280A), 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; and 9,439,962).

[0122] In some embodiments, the agent is a PD-L2 antagonist or inhibitor. As mentioned 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, such as those associated with 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, those 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 a T cell when the T cell binds to CD80 or CD86 on the surface of an 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 the inhibitory factor Treg that inhibits CTLA-4.

[0125] In some embodiments, the agent is an IDO antagonist or inhibitor or a TDO antagonist or inhibitor. IDOs and TDOs are tryptophan-catabolizing enzymes with immunosuppressive properties. For example, IDOs are known to inhibit T cells and NK cells, generate and activate Tregs 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 IDOs or TDOs (see, for example, Platten et al., Frontiers in Immunology 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 TDO antagonists or inhibitors include 680C91 and LM10 (see, for example, Pilotte 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, 2004), and has been reported to play a role in Treg suppression (see, for example, Huang et al., *Immunity* 21: 503-13, 2004). LAG3 also maintains CD8+ T cells in a tolerogenic 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 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 tumor necrosis family receptors (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., *Journal of Clinical Investigation (J ClinInvest)* 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, such as 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, such as 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 interaction, and thereby reduce or inhibit one or more of their immunosuppressive activities.

[0131] In some embodiments, the agent is a TIGIT antagonist or inhibitor. The T-cell Ig and ITIM domains (TIGIT) are co-inhibitory receptors found on the surface of various lymphocytes and, for example, inhibit antitumor immunity via 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 Cells* 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 their 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, an effect at least partially mediated by cytotoxic T cells (see, for example, Johnson et al., Clin Cancer Res. 21: 1321-1328, 2015; and Vonderheide and Glennie, Clin Cancer Res. 19:1035-43, 2013). General examples of CD40 agonists include antibodies, antigen-binding fragments, 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, ChiLob 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., Immunology Review 229:173-91, 2009). Its ligand is OX40L (CD252). Because 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 (GITRs) increase T cell proliferation, inhibit the suppressive activity of Tregs, and prolong the survival of T effector daughter cells. GITR agonists have been shown to 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 the important role of GITRs 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 GITRs and increase one or more of their 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, *J 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 antigen-specific amplification of untreated T cells and contributes to the long-term maintenance of T cell memory and T cell immunity. Its ligand is CD70. Targeting of human CD27 with agonist antibodies stimulates T cell activation and anti-tumor immunity (see, for example, Thomas et al., *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), which include their antigen-binding fragments.

[0138] In some embodiments, the agent is a CD28 agonist. CD28 is constitutively expressed 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 expressed at high levels on resting T cells and is downregulated upon activation. HVEM signaling has been shown to play 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 its 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's human papillomavirus (HPV) vaccine, optionally Engerix-B, Recombivax HB or Twinrix's hepatitis B vaccine, 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), Pelareoreep (REOLYSIN®), Seneca Valley virus (NTX-010), Seneca virus (… 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 immunotherapy 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).

[0145] In some embodiments, cancer immunotherapies are cell-based immunotherapies, such as therapies utilizing immune cells, including ex vivo immune cells such as lymphocytes, natural killer (NK) cells, macrophages, and / or dendritic cells (DCs). In some embodiments, 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 immunotherapies. In some embodiments, 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 a specific embodiment, CAR-modified T cells target CD19 (see, for example, Maude et al., Blood 125:4017-4023, 2015). In some cases, the ex vivo immune cells are autologous cells obtained from the patient to be treated.

[0146] Some combination therapies use 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.

[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), fotemustine, and streptozotocin), and tetrazine (e.g., dacarbazine). arbazine, 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 hormone therapy agent. General examples of hormone therapy 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 their analogues. Also included are hormone receptor antagonists, such as selective estrogen receptor modulators (SERMs, e.g., tamoxifen, raloxifene, toremifene) and antiandrogens (e.g., flutamide, bicalutamide, nilutamide).

[0153] This also includes inhibitors of hormonal pathways, such as antibodies targeting hormone receptors. Examples include inhibitors of IGF receptors (e.g., IGF-IR1), such as cixutumumab, dalotuzumab, figitumumab, ganytumab, isitiratumab, and robatumumab; and inhibitors of vascular endothelial growth factor receptors 1, 2, or 3 (VEGFR1, VEGFR2, or VEGFR3), such as alicizumab. pegol, bevacizumab, icrucumab, ramucirumab; TGF-β receptor inhibitors R1, R2, and R3, such as fresolimumab and metelimumab; c-Met inhibitors, such as naxitamab; EGF receptor inhibitors, such as cetuximab and depatuxizumab. Inhibitors of the following receptors: mafodotin, futuximab, imgatuzumab, lapituximabemtansine, 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] Some 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, entrectinib, erdafitinib, erlotinib, fostamitinib, gefitinib, ibrutinib, and imatinib. The following are listed: tinib, lapatinib, lenvatinib, mubritinib, nilotinib, panitumab, pazopanib, pegaptanib, ponatinib, ranibizumab, regorafenib, ruxolitinib, sorafenib, sunitinib, SU6656, tofacitinib, trastuzumab, vandetanib, and vemuafenib.

[0156] The various kinase inhibitors described herein can 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, the methods and compositions described herein increase the immune response in a 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 times or more, including where the immune response is an anticancer immune response, relative to a control or reference (e.g., relative to the corresponding serine protease itself).

[0158] In some embodiments, the methods and compositions described herein increase median survival in subjects by 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40 weeks, or longer. In some embodiments, the methods or compositions described herein increase median survival in subjects by 1 year, 2 years, 3 years, or longer. In some embodiments, the methods and pharmaceutical compositions increase progression-free survival by 2, 3, 4, 5, 6, 7, 8, 9, 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 induce 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 alleviate clinically relevant symptoms of a specific disease indication known to a skilled clinician.

[0160] As described above, for in vivo use, for the treatment of diseases in humans or non-human mammals, 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, some 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 pharmaceutical 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 is known in the art, the protein complexes described herein do not form aggregates, have the desired solubility, and / or have an immunogenic profile suitable for humans. Therefore, in some embodiments, pharmaceutical or therapeutic compositions comprising protein complexes are substantially non-aggregating. For example, some 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.

[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, 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 biological therapeutic 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 formulation 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 protein complexes described herein, either in their pure form or in a suitable therapeutic or pharmaceutical composition, may be administered via any acceptable method of pharmaceutical administration for similar purposes. Therapeutic or pharmaceutical compositions may be prepared by combining a composition containing the protein complex with a suitable physiologically acceptable carrier, diluent, or excipient, and may be formulated into formulations in solid, semi-solid, liquid, or gaseous forms, 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 various routes, including oral, parenteral, nasal, intravenous, intradermal, intramuscular, subcutaneous, or local administration. The preferred mode of administration depends on the nature of the condition to be treated or prevented. Specific embodiments include administration via intravenous infusion.

[0168] Carriers may include, for example, pharmaceutically or physiologically acceptable carriers, excipients, or stabilizers that are non-toxic to cells or mammals exposed to them at the doses and concentrations used. Physiologically acceptable carriers are typically aqueous pH buffer solutions. Examples of physiologically acceptable carriers include buffers 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 pharmaceutical 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 inferred by testing the composition in model systems known in the art. Controlled clinical trials can also be conducted. The dosage may also vary depending on the severity of the symptom to be alleviated. Drug compositions are typically formulated and administered to achieve a therapeutically useful effect while minimizing undesirable side effects. The composition can be administered once or divided into many smaller doses administered at intervals. For any given subject, the specific dosage regimen can be adjusted over time according to individual needs.

[0171] Therefore, typical routes of administration of these and related therapeutic or pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, oral, rectal, vaginal, and intranasal administration. As used herein, the term parenteral includes subcutaneous injection, intravenous, intramuscular, intrasternal injection, or infusion techniques. The therapeutic 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 for aerosol forms of pharmaceutical preparations described herein may contain multiple dose units. Practical methods for preparing such dosage forms are known or obvious to those skilled in the art; see, for example, Remington: Pharmaceutical Science and Practice (…). Remington: The Science and Practice of Pharmacy (Philadelphia College of Pharmacy and Science, 2000). The composition to be administered will typically contain a therapeutically effective amount of the medicine described herein for the treatment of the disease or condition of interest.

[0172] Therapeutic or pharmaceutical compositions may be in solid or liquid form. In one embodiment, the carrier is granular, so 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 intended 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 flavorings; and colorants. When the pharmaceutical composition is in capsule form, such as 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 oils.

[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 in solution, suspension, or other similar form, may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixative oils such as synthetic monoglycerides or diglycerides that can be used as solvents or suspension media, 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 acetates, citrates, or phosphates; and agents for tension regulation such as sodium chloride or dextran. Parenteral preparations may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic. Physiological saline is a preferred adjuvant. Injectable pharmaceutical compositions are preferably sterile.

[0176] Liquid therapeutic or pharmaceutical compositions intended for parenteral or oral administration should contain an amount of the pharmaceutical agent to obtain a suitable 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 about 70% by weight of the composition. Some oral therapeutic or pharmaceutical compositions contain between about 4% and about 75% of the pharmaceutical agent of interest. In some embodiments, therapeutic or pharmaceutical compositions and formulations are prepared such that a parenteral dose unit contains between 0.01% and 10% by weight of the pharmaceutical agent of interest before dilution.

[0177] Therapeutic or pharmaceutical compositions may be intended for topical application, 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 application. If intended for transdermal application, the composition may comprise a transdermal patch or an iontophoresis device.

[0178] Therapeutic or pharmaceutical compositions may be intended for rectal administration, for example, in the form of suppositories, which dissolve 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 solid or liquid dosage units. For example, a composition may include a material that forms a coating around an active ingredient. The coating material is typically 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 dosage units that can be administered as aerosols. The term aerosol is used to refer to a variety of systems, ranging from colloidal systems to systems consisting of pressurized packaging. Delivery can be carried out by liquefying or compressing gases or by a suitable pump system dispensing the active ingredient. Aerosols can be delivered in single-phase, two-phase, or three-phase systems to deliver the active ingredient. Aerosol delivery includes the necessary containers, activators, valves, sub-containers, etc., which can 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 a timed-release formulation or a coating. Such carriers include controlled-release formulations, such as, but not limited to, implants and microencapsulated delivery systems, as well as 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 pharmaceutical agents to promote the dissolution or homogeneous suspension of the agent 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 timing; 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); 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 administered, for example, at a dose of about 1-10 or 1-5 mg / kg or about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg / kg weekly, bi-weekly or monthly.

[0184] The combination therapies described herein may comprise the administration of a single drug dose formulation containing a protein complex and an additional therapeutic agent (e.g., an immunotherapeutic agent, a chemotherapeutic agent, a hormonal therapy, a kinase inhibitor), as well as the 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 as 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 therapy should be understood to include all of these regimens.

[0185] The kit also includes a patient care kit comprising (a) a protein complex as described herein; and optionally (b) at least one additional therapeutic agent (e.g., an immunotherapeutic 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] The kit described herein may also include one or more additional therapeutic agents or other components suitable for or intended for use in the therapeutic indication or for intended diagnostic applications. The kit described herein may also include one or more syringes or other components required or desired to facilitate the intended delivery modality (e.g., stents, implantable reservoirs, 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 may be 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 pack) separate containers, each containing one or more unit dosage forms of the protein complex (e.g., dosage forms described herein), and optionally at least one additional therapeutic agent. For example, the kit includes a plurality of 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 opaque.

[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 measured dose of the medication. Methods for providing the kit, for example, by combining the components described herein, are also included.

[0189] Preparation and purification system

[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 a composition 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 [(a):(b)].

[0191] A2M and / or serine protease proteins can be prepared or obtained by purification from biological samples or by recombinant techniques. For example, in some embodiments, A2M proteins and / or serine protease proteins are obtained by purifying proteins from the blood or plasma of mammalian subjects, such as human subjects. Specific embodiments include obtaining and purifying A2M proteins from the plasma of human subjects prior to combination with serine protease proteins. 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 readily 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 a procedure comprising one or more of the following steps: (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 the protein can be inserted into a suitable expression vector, i.e., a vector containing the necessary elements of the coding sequence inserted for transcription and translation. Methods well known to those skilled in the art can be used to construct expression vectors containing sequences encoding the polypeptide of interest and appropriate transcriptional and translational 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 used to contain and express polynucleotide sequences. These include, but are not limited to: microorganisms, such as bacteria transformed with recombinant bacteriophage, 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 expression vectors are those untranslated regions of the vector—enhancers, promoters, 5' and 3' untranslated regions—that interact with host cell proteins to enable transcription and translation. The strength and specificity of these elements can vary. Depending on the vector system and host used, any number of suitable transcriptional and translational 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 phagemid (Stratagene, La Jolla, Calif.) or the PSPORT1 plasmid (Gibco BRL, Gaithersburg, Md.) can be used. In mammalian cell systems, promoters derived from mammalian genes or mammalian viruses are generally preferred. If it is necessary to generate cell lines containing multiple copies of a sequence encoding a polypeptide, SV40 or EBV-based vectors can be advantageously used with appropriate selectable markers.

[0196] In bacterial systems, multiple expression vectors can be selected based on the intended use of the polypeptide to be expressed. For example, when large quantities are required, vectors that guide high-level expression of easily purified proteins 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 ligated into a vector with an amino-terminal Met sequence and the following 7 residues of β-galactosidase using the same reading frame to produce a hybrid protein; pIN vectors (Van Heeke and Schuster, J. Biol. Chem. 264:5503 5509 (1989)); and pGEX vectors (Promega, Madison, Wisconsin) can also be used to express recombinant proteins as fusion proteins with glutathione S-transferase (GST). Generally, such fusion proteins are soluble and can be readily purified from lysed cells by adsorption onto 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 desired clonal polypeptide from the GST moiety.

[0197] Some embodiments employ *E. coli*-based expression systems (see, for example, Structural Genomics Consortium, *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 may utilize expression host strain BL21 (DE3), a DE3 lysinogen of BL21 that supports T7-mediated expression and lacks the lon and ompT proteases for improving target protein stability. Expression host strains carrying plasmids encoding tRNAs rarely used in *E. coli*, such as *ROSETTA*, are also included. ™ (DE3) and Rosetta 2 (DE3) strains. 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-inducible media can improve the efficiency of many expression systems, including high-throughput expression systems. This type of medium (e.g., the OVERNIGHTEXPRESS™ self-inducible system) gradually induces protein expression through metabolic transfer without the need for artificial inducers such as IPTG. Specific embodiments utilize hexahistine tags (such as those under the trademark HIS). The TAG® fusion is labeled for sale, 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 of *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] This also includes high-density bacterial fermentation systems. For example, high-density culture of Ralstonia eutropha allows for protein production at cell densities exceeding 150 g / L and recombinant protein expression at titers exceeding 10 g / L.

[0199] In *Saccharomyces cerevisiae*, numerous vectors containing constitutive or inducible promoters such as α-factors, alcohol oxidases, and PGH can be used. For reviews, see Ausubel et al. (ibid.) and Grant et al., *Methods Enzymol.* 153:516-544 (1987). Also included are *Pichia pandoris* expression systems (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 (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). By way of example only, recombinant yeast cultures can be grown in Fernbach flasks or fermenters of 15L, 50L, 100L, and 200L.

[0200] When using plant expression vectors, the expression of the sequence encoding the polypeptide can be driven by any of many 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 (EMBO J.)* 3:1671-1680 (1984); Broglie et al., *Science* 224:838-843 (1984); and Winter et al., *Results Probl. CellDiffer.* 17:85-105 (1991)). These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. Such technologies are described in many commonly 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 peptide can be cloned into a non-essential region of the virus, such as the polyhedromic protein gene, and placed under the control of the polyhedromic protein promoter. Successful insertion of the peptide-coding sequence will inactivate the polyhedromic protein gene and produce a recombinant virus lacking the coat protein. The recombinant virus can then be used to infect, for example, fall armyworm or white armyworm cells that can express the peptide of interest (Engelhard et al., Proceedings of the National Academy of Sciences 91:3224-3227 (1994)). Baculovirus expression systems are also included, including baculovirus expression systems 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 regions of the viral genome can be used to obtain live viruses capable of expressing the polypeptide in infected host cells (Logan and Shenk, Proceedings of the National Academy of Sciences 81:3655-3659 (1984)). Additionally, 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, ATCCCCL 10); mouse Support 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); and Buffalo rat hepatocytes (BRL3A, 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. Some 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] This also includes cell-free protein expression. These and related examples typically utilize purified RNA polymerase, 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 transcriptional or translational control signals may not be necessary. However, when only the coding sequence or a portion thereof is inserted, an exogenous translational 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. Expression efficiency can be enhanced by including enhancers suitable for the specific cell system used, such as those described in the literature (Scharf et al., ResultsProbl. Cell Differ. 20:125-162 (1994)).

[0206] Additionally, host cell lines can be selected because they can regulate the expression of the inserted sequence or process 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, lipidation, and acylation. Post-translational processing, including cleavage of the protein's "precursor" form, can also be used to facilitate proper insertion, folding, and / or function. Besides bacterial cells, different host cells with specific cellular mechanics and characteristic mechanisms, such as yeast, CHO, HeLa, MDCK, HEK293, and W138, with or without such post-translational activity, can be selected to ensure proper modification and processing of foreign proteins.

[0207] For long-term, high-yield production of recombinant proteins, stable expression is generally preferred. For example, expression vectors can be used to transform cell lines stably expressing the polynucleotide of interest. These vectors may contain replicating viral-origin and / or endogenous expression elements, as well as selectable marker genes on the same vector or on separate vectors. After vector introduction, cells can be allowed to grow in enriched media for approximately 1–2 days before being transferred to selective media. The purpose of the selectable marker is to confer resistance to the selected gene, and its presence allows for the growth and recovery of cells that successfully express the introduced sequence. Resistant clones of stably transformed cells can be proliferated using tissue culture techniques suitable for the cell type. Transient production can also be employed, such as through transient transfection or infection. Exemplary mammalian expression systems suitable for transient production include HEK293 and CHO-based systems.

[0208] Any number of selection systems can be used to restore 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 confers resistance to methotrexate (Wigler et al., Proceedings of the National Academy of Sciences 77:3567-70 (1980)); npt confers resistance to aminoglycosides, neomycin, and G-418 (Colbere-Garapin et al., Journal of Molecular Biology 150:1-14 (1981)); and als or pat confers resistance to chlorfluazuron and phosphinotricin acetyltransferase, respectively (Murry, ibid.). Other selectable genes have been described, such as trpB, which allows cells to utilize indole instead of tryptophan, or hisD, which allows cells to utilize 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 in 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 not only widely used to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system (see, for example, Rhodes et al., Methods in Molecular Biology 55:121-131 (1995)).

[0209] This also includes high-throughput protein production systems or microproduction systems. 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 various 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 generation 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 labels 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 engineered to contain a signal sequence that guides the secretion of the encoded polypeptide across the prokaryotic or eukaryotic cell membrane. Other recombinant constructs can be used to link sequences encoding polypeptides of interest with nucleotide sequences encoding polypeptide domains, 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 generated from recombinant cells can be purified and characterized using 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), etc. 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 generation or purification process and are typically used to measure the purity of protein compositions.

[0215] It 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 respects, such compositions can 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 respects, such compositions have a purity of at least about 90% (based on protein), or in some respects at least about 95% purity, or in some embodiments at least 98% purity. Purity can be determined by any conventional analytical method known in the art.

[0218] In some respects, 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 respects, 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 in this article include lyophilization, which is typically used when the solution contains a few soluble components other than the protein of interest. Lyophilization is usually performed after HPLC and can remove most or all volatile components from the mixture. Ultrafiltration is also included, which typically uses one or more selectively permeable membranes to concentrate protein solutions. The membrane allows water and small molecules to pass through the protein while retaining the protein; the solution can be pressed against the membrane using other techniques such as mechanical pumps, pneumatic 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 have a lower purity and may have a purity of at least about 50%, 60%, 70%, or 80%. Purity can be measured holistically or with respect to selected components such as other proteins, for example, based on protein purity.

[0221] Purified proteins or protein complexes can also be characterized based on their biological properties. 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. The presence of endotoxins can be detected using conventional techniques in the art, as described herein. 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.

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

[0224] This also includes protein-based analytical assays and methods that can be used to assess characteristics such as protein purity, size, solubility, and 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 conformation-sensitive antibodies. Higher-order structures can also be assessed based on various parameters, such as pH, temperature, or added salts. Examples of methods for assessing 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 assessed, 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 assessed, for example, by mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy.

[0225] As described above, some embodiments include the use of 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. SEC, which also includes gel filtration chromatography (GFC) and 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. These methods are typically 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 size-selective size size exclusion column having a defined stationary phase (porous material), preferably a phase that does not interact with the proteins in the sample. In some aspects, 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 a “completely excluded” peak 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 flow 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. In general, molecules larger than the upper limit will not be captured by the stationary phase, molecules smaller than the lower limit will completely enter the solid phase and elute as a single band, and molecules within these ranges will elute at different rates, defined by their properties 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. (Trendsin 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, clinical-grade proteins or protein complexes can be obtained by utilizing a combination of chromatographic materials in at least two orthogonal steps, as well as other methods (see, for example, Therapeutic Proteins: Methods and Protocols. Vol. 308, edited by Smales and James, Humana Press Inc., 2005). Typically, protein preparations are substantially free of endotoxins, as measured according to techniques known in the art and described herein.

[0227] This also includes protein solubility assays. 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 assessed 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, Stevanall et al., Acta Biochimica et Biophysica Sinica 1752:6-10, 2005); assays for monitoring and measuring protein folding and in vivo solubility using genetic marker protein structural complementarity (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 with 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 Sci. 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 induce immunogenic reactions (e.g., small aggregates) or cause 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 suspensions or polymers such as proteins in solution. This technique, also known as photon correlation spectroscopy (PCS) or quasi-elastic light scattering (QELS), uses scattered light 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 particle. The hydrodynamic size depends on both mass and shape (consistent). Dynamic light 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 using 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 the foregoing embodiments have been described in detail by way of illustration and example for the purpose of clarity, it will be readily apparent to those skilled in the art, based on the teachings of this disclosure, that certain changes and modifications can 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 various non-critical parameters that can be changed or modified to produce substantially similar results.

[0230] Example

[0231] Example 1

[0232] Characteristics of the A2M:PPE protein complex

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

[0234] A1AT protection assayMutF (400 nM) was mixed with different concentrations of A2M to achieve various molar ratios, followed by incubation at 37 °C for 30 min. Protective assays were performed by adding 10 μL of sample to 384-well black plates coated with 10 μL of PBS or 10 μL of A1AT 2 μM, or in duplicate coated at different concentrations. 20 μL of the 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] like Figures 3A-3B As shown, the 1:2 molar ratio of the A2M:MutF protein complex provides optimal protection against A1AT (3A), and gradually increasing A1AT concentration does 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), followed by incubation 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] like Figure 3C As shown, when combined with A2M:MutF at a molar ratio of 1:2, plasma itself inhibits MutF activity, but does not inhibit its activity.

[0238] Purification of N17350-A2M by column chromatographyMutF and A2M were mixed to a 1:2 molar ratio (A2M:MutF) and 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) using a PD10 desalting column. The sample was loaded onto a pre-washed / equilibrated HiTrap SP column containing 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. Using Pierce TM The BCA Protein Assay Kit (Thermo Fisher Scientific, see manufacturer's instructions) was used to measure protein concentration. For the A1AT protection assay, 10 μL of each fractional 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 100 µM AAPV-AMC substrate 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] Figures 4A-4E The results show that the A2M:MutF protein complex is stable. Figure 4A The activity and concentration of MutF in different fractions of the A2M:MutF complex after cation exchange column separation are shown, with or without A1AT. Figure 4B The activity and concentration of MutF in different fractions of the A2M:MutF complex after size exclusion column separation are shown, with or without A1AT. Figures 4C-4D As shown, the A2M:MutF complex is stable over a wide pH range, as measured by enzyme activity (4C) and A1AT protection (4D). Figure 4E As shown, as measured by enzyme activity, the A2M:MutF complex is stable in multiple freeze-thaw cycles.

[0240] Activity in cell lysates and A1AT protectionTumor cells were trypsinized, washed with PBS, counted, and diluted in serum-free medium. 40k tumor cells were added 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)). The plate was incubated at 37°C for 30 min. After incubation, the plate was centrifuged at 300 x g for 5 min and each well was washed with an additional 200 μL of PBS. 25 μL of cytoplasmic lysis buffer (10 mM HEPES pH 8, 10 mM KCl, 0.1 mM EDTA, 0.3% NP-40) was added to each well and the plate was vortexed for 10 seconds every 5 min for 20 min on ice. The plate was centrifuged at 300 x g for 5 min. The supernatant was carefully transferred to a new 96-well plate for A2M:MutF activity and protection assays. Here, 10 μL of supernatant was washed with 10 μL of PBS or 10 μL of LA1AT 2. μM or two copies of different concentrations were coated into 384 black plates. 20 μL of substrate AAPV-AMC at a concentration of 100 µM were 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] Figures 5A-5D The results showed that the A2M:MutF protein complex can enter the cell 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, and then incubated in PBS at 37°C for 30 minutes. Recombinant CD95-C terminal protein (Wuxi) was incubated with premixed A2M:MutF protein complexes at different molar ratios for 1 hour at 37°C. Samples containing 1 μg of CD95-C were loaded onto 17.5% SDS-PAGE gels and stained with One-step Blue (Biotium). The gels were imaged using an iBright 1500.

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

[0244] Fibrinogen cleavage assayMutF 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 complexes 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 (Cellsignaling Technology). Gel imaging was performed using an iBright 1500.

[0245] like Figure 6B As shown, the A2M:MutF protein complex does not cleave fibrinogen, compared to MutF which cleaves fibrinogen 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 minutes. 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] like Figure 6C As shown, the A2M:MutF protein complex does not cleave elastin, compared to MutF which cleaves elastin alone.

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

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

[0250] Cell killing assayApproximately 20K–40K cancer cells were seeded in black-coated 96-well plates and incubated overnight for sedimentation. The following day, each well was washed once with 200 μL of serum-free medium, followed by three separate treatments. Twenty-four hours after treatment, the cells were incubated for 45 minutes with pre-diluted calcein-AM solution (Thermo Fisher Scientific, 4 μM, HBSS containing Ca²⁺ and Mg²⁺). Calcein AM was poured off, and 100 μL of HBSS containing Ca²⁺ and Mg²⁺ was added to each well. Fluorescence was measured at 485 / 520 nm (20-bandwidth) using Varioskan LUX.

[0251] like Figure 8A As shown, 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, its activity was comparable to that of MutF itself. Figure 8B The A2M:MutF (1:2) protein complex was shown to have broad cytotoxicity against cancer cells of different anatomical origins, and Figure 8C The study showed that the complex did not kill non-cancer cells.

[0252] In vivo efficacy Approximately one million CT26 cells were implanted into the flank and abdomen of 7-8 week old BALB / c mice. Once the tumor reached approximately 80 mm... 3 The tumor was treated with 100 ug MutF, 100 ug A2M:MutF (1:2), or 1.4 mg A2M (the amount corresponding to the production of a 1:2 A2M:MutF molar ratio). The tumor was monitored every two days.

[0253] like Figure 8D As shown, the A2M:MutF protein complex with a molar ratio of 1:2 induces the killing of cancer cells in vivo, with cytotoxic activity comparable to that of MutF itself. Figure 8E The results show that, after intravenous administration, the A2M:MutF protein complex has an improved functional PK profile (enzymatic activity in plasma) compared to MutF alone. Figure 8F The A2M:MutF protein complex induces a favorable immune profile in the CT26 model (PBS, MutF, and A2M:MutF are shown from left to right in each figure). Figure 8G As shown, the A2M:MutF protein complex induced tumor antigen-specific CD8+ T cell responses 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 samplesCancer 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 in tumor tissue or CD45+ depleted digested omental cells), 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, and 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 using calcein-AM.

[0255] Figure 9A The results show that, compared to doxorubicin and oxaliplatin, the A2M:MutF protein complex has a broad therapeutic window, as demonstrated by killing human ovarian cancer cells without killing non-cancerous cells in patients. Figure 9B The results showed that A2M:MutF killed cancer cells isolated from both chemotherapy-naïve and chemotherapy-treated patients equivalently to doxorubicin and oxaliplatin, indicating reduced killing of cancer cells isolated from chemotherapy-treated patients compared to chemotherapy-naïve patients.

[0256] Evaluation of immunogenic cell death (ICD) CT26 (mouse colon), A549 (human lung), and ovarian cancer patient cells were treated for 24 hours with A2M:MutF and oxaliplatin (high concentrations of an ICD inducer 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 The results show that the A2M:MutF protein complex induces ICD markers in CT26 and A549 cells. Figure 10B As shown, 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] Evaluation of dose and schedule of A2M:MutF CT26 colon cancer cells were injected into the flanks of mice and allowed to grow until they reached approximately 80 mm. 3Until then. Administer multiple intravenous doses of the A2M:MutF protein complex or mediator as follows: ● Mediator: Day 0, Day 2, Day 4 and Day 6, and Day 8, Day 10, Day 12 and Day 14; ● A2M:MutF 100 μg: Every day for 2 weeks (daily); ● A2M:MutF 200 μg: Day 0, Day 2, Day 4, Day 6, and Day 8, Day 10, Day 12, and Day 14 (every other day); and ● A2M:MutF 400 μg: Day 0, Day 4, Day 8 and Day 12 (every 4 days).

[0259] Assess tumor growth and weight. Figures 11A-11B The growth of the treated tumor is shown, and Figure 11C Tumor weights at 15 days post-treatment are shown (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).

[0260] Evaluation of anti-tumor efficacy 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 mm. 3 Continue intravenous injection of A2M:MutF protein complex (400 μg) or a carrier every other day for 2-3 weeks. Monitor tumor growth.

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

[0262] Evaluation of anti-tumor 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 mm. 3Until then. Administer intravenous A2M:MutF protein complex (400 μg, every other day for 3 weeks) or oxaliplatin (6 mg / kg on days 0 and 2). Monitor tumor growth and overall survival.

[0263] Figures 13A-13C The results showed that the A2M:MutF protein complex had improved antitumor efficacy compared to SoC chemotherapy (oxaliplatin), without any observed toxicity.

[0264] Evaluation of anti-tumor efficacy against human cancer cells 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 mm. 3 Continue intravenous injection of A2M:MutF protein complex (400 μg) or a carrier every other day for 2–3 weeks. Monitor tumor growth and overall survival.

[0265] Figure 14A The efficacy of the A2M:MutF protein complex in a human xenograft model of lung cancer was demonstrated. Figure 14B The efficacy of the A2M:MutF protein complex across various prostate cancer, colon cancer, and lung cancer models was summarized. Figure 14C The results show 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 The efficacy of the A2M:MutF protein complex in this model across three ovarian cancer patients (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 The results show that the A2M:MutF protein complex effectively kills patient-derived breast cancer cells in vitro and in vivo (patient history: diagnosis, breast cancer HER2- and ER+; treatment, no chemotherapy). Figure 14G The in vivo efficacy of the A2M:MutF protein complex across a variety of human tumors was summarized, and the efficacy was shown to be independent of tumor genetic or immune status.

[0266] Figure 15The results showed 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) maintained their tumor-free status after rechallenge with CD26 cells. This study demonstrates that treatment with the A2M:MutF protein complex induces a tumor-specific immune memory response.

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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