Pharmaceutical composition containing a protein complex

Abstract

A pharmaceutical composition comprising α2-macroglobulin (A2M) and a serine protease protein such as porcine pancreatic elastase (PPE) is provided, wherein A2M and the serine protease protein such as PPE are bound as a protein complex, and this protein complex retains the CD95 protease cleavage activity and cancer cell killing activity of the serine protease, while sterically inhibiting the binding of the serine protease to fibrinogen and a serine protease inhibitor. Related methods of use and manufacturing for treating diseases such as cancer are also provided.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims an interest under Section 119(e) of the United States Patent Act with respect to U.S. Provisional Application No. 63 / 623,030 filed on 19 January 2024 and U.S. Provisional Application No. 63 / 456,916 filed on 4 April 2023, both of which are incorporated herein by reference in their entirety.

[0002] Description regarding sequence listings The sequence listing XML related to this application is provided in XML file format and is incorporated herein by reference. The name of the XML file containing the sequence listing XML is OPNI_009_02WO_ST26.xml. The XML file is approximately 28,746 bytes in size, was created on March 28, 2024, and is filed electronically through the USPTO Patent Centre.

[0003] This disclosure relates to a pharmaceutical composition comprising α2-macroglobulin (A2M) and a serine protease protein such as porcine pancreatic elastase (PPE), wherein A2M and the serine protease protein such as PPE are bound as a protein complex, and this protein complex retains the CD95 protease cleavage activity and cancer cell killing activity of the serine protease, while sterically inhibiting the binding of the serine protease to fibrinogen and a serine protease inhibitor, as well as related methods of use and manufacturing for treating diseases such as cancer. [Background technology]

[0004] Explanation of related technologies Precision medicine, designed to optimize efficiency or therapeutic benefit for specific patient groups by using genetic or molecular profiling, has achieved great success in treating cancer. Identifying specific genomic abnormalities that (i) contribute to the risk of developing cancer, (ii) affect tumor growth, and (iii) modulate metastasis defines how cancer is diagnosed, determines how targeted therapies are developed and implemented, and shapes cancer prevention strategies.

[0005] The need for precision medicine in cancer is primarily based on the inability to identify targetable characteristics of tumor cells that distinguish them from healthy, non-cancerous cells. Indeed, while radiation and / or chemotherapy have the ability to effectively kill a large number of cancer cells, if not the majority, their effectiveness is significantly limited by their cytotoxic effects on non-cancerous cells. These findings suggest that rapid cell division, a targetable characteristic of radiation and chemotherapy, is not sufficiently specific to cancer cells to achieve the specificity required to limit widespread side effects.

[0006] Certain serine proteases or elastase enzymes have been shown to be selectively toxic to cancer cells but relatively non-toxic to normal or other healthy cells (see, for example, WO2018 / 232273). However, in the art, there is a need to identify optimal enzyme compositions that possess such selective cancer cytotoxicity while simultaneously reducing side effects, thereby improving the pharmacokinetics and overall clinical utility of such compositions. [Overview of the project]

[0007] The embodiments of this disclosure are, (a) α2 macroglobulin (A2M) protein, and (b) A pharmaceutical composition comprising a protein complex consisting of serine protease proteins, (a) and (b) are present in the composition in a molar ratio of approximately 1:3 to approximately 1:1 [(a):(b)]. In certain embodiments, the A2M protein of (a) and the serine protease protein of (b) are bound in a protein complex, for example, the protein complex is (i)(b) retains CD95 (Fas receptor) protease cleavage activity and cancer cell killing activity, (ii) Sterically inhibits the binding of (b) to fibrinogen, thereby reducing or inhibiting the fibrinogen cleavage activity of (b), (iii)(b) sterically inhibits the binding of (iii)(b) to serine protease inhibitors (plasma serine protease inhibitors, including α1-antitrypsin (A1AT)).

[0008] In some embodiments, (a) consists of, or essentially consists of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to a sequence or functional fragment thereof selected from Table A1. In some embodiments, the functional fragment consists of, or essentially consists of, a sequence containing about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1300, or 1400 consecutive amino acids of a sequence selected from Table A1.In some embodiments, the functional fragment is approximately 1-1400, 1-1300, 1-1200, 1-1100, 1-1000, 1-900, 1-800, 1-700, 1-600, 1-500, 1-400, 1-300, 1-200, 100-1400, 100-1300, 100-1200, 100-1100, 100-1000, 100-900, 100-800, 100-700, 100-600, 100-500, 100-400, 100-300, 100-200, 200-1400, 200-1300, 200-1200, 200-1100, 200-1000, 200-900, 200-800, 200-700, 200-600, 200-500, 200-400, 200-300, 300-1400, 300-1300, 300-1200, 300-1100, 300-1000, 300-900, 300-800, 300-700, 300-600, 300-500, 300-400, 400-1400, 400-1300, 400-1200, 400-110 0, 400~1000, 400~900, 400~800, 400~700, 400~600, 400~500, 500~1400, 500~1300, 500~1200, 500~1100, 500~1000, 500~900, 500~800, 500~700, 500~600, 600~1400, 600~1300, 600~1200, 600~1100, 600~1000, 600~900, 600~800, 600~700, 700~1400, 700~1300, 700~1200, 700~ It consists of residues of 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.

[0009] In certain embodiments, (a) is conjugated to or fused 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 tumor-specific antigen (TSA).

[0010] In some embodiments, (b) is selected from porcine pancreatic elastase (PPE) protein, human neutrophil elastase (ELANE) protein, human cathepsin G (CTSG) protein, human proteinase 3 (PR3) protein, and granzyme B protein. In certain embodiments, The PPE protein consists of, or essentially consists of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 5, and that sequence retains the Q211F amino acid substitution. The PPE protein consists of, or essentially consists of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 6, and that sequence retains the T55A amino acid substitution. The PPE protein consists of, or essentially consists of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 7, and that sequence retains the Q211F and T55A amino acid substitutions. The PPE protein consists of, or essentially consists of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 8, and that sequence retains the N241A amino acid substitution. The PPE protein consists of, or essentially consists of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 9, and that sequence retains the N241Y amino acid substitution. The PPE protein consists of, or essentially consists of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 10, and that sequence retains the R75A amino acid substitution. The PPE protein consists of, or essentially consists of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 11, and that sequence retains the R75E amino acid substitution. The PPE protein consists of, or essentially consists of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 12, and that sequence retains the Q211A amino acid substitution. The PPE protein consists of, or is essentially composed of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 13, and that sequence retains the R237A amino acid substitution. The PPE protein consists of, or is essentially composed of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 14, and that sequence retains the S214A amino acid substitution. The PPE protein consists of, or is essentially composed of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 15, and that such sequence retains the D74A amino acid substitution, and The PPE protein consists of, or essentially consists of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 16.

[0011] In some embodiments, Human ELANE protein consists of, or essentially consists of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 17. Human CTSG proteins consist of, or essentially consist of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 18. Human PR3 protein contains, or essentially consists of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 19, or Human granzyme B protein consists of, or essentially consists of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 20.

[0012] In some embodiments, (a) and (b) are present in the 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. In certain embodiments, (a) and (b) are present in the composition in molar ratios of about 1:2.

[0013] The disclosure also includes methods for treating cancer in a subject, improving its symptoms, and / or slowing its progression, which include administering the pharmaceutical compositions described herein to a subject in need thereof.

[0014] In some embodiments, the cancer is a primary or metastatic cancer and is selected from one or more of melanoma (optionally metastatic melanoma), breast cancer (optionally triple-negative breast cancer, TNBC), kidney cancer (optionally renal cell carcinoma), pancreatic cancer, bone cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer (NSCLC), mesothelioma, leukemia (optionally lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, or relapsed acute myeloid leukemia), multiple myeloma, lymphoma, hepatocellular carcinoma (hepatocarcinoma), sarcoma, B-cell malignancy, ovarian cancer, colorectal cancer, glioma, glioblastoma multiforme, meningioma, pituitary adenoma, vestibular schwannoma, primary CNS lymphoma, primitive neuroectodermal tumor (medulloblastoma), bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancer, cervical cancer, testicular cancer, thyroid cancer, and gastric cancer.

[0015] In some embodiments, administration of the pharmaceutical composition (optionally intravenous administration) does not substantially increase the prothrombin time or partial thromboplastin 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-fold, 5-fold, 10-fold, 50-fold, 100-fold, 500-fold, or 1000-fold or more compared to a control or reference.

[0016] Certain embodiments include administering the pharmaceutical composition to a subject by parenteral administration. In some embodiments, the parenteral administration is intravenous administration.

[0017] Also included is a method of manufacturing a pharmaceutical composition comprising a protein complex, (a) an α2-macroglobulin (A2M) protein, and (b) a serine protease protein, are combined in the composition at a molar ratio [(a):(b)] of about 1:3 to about 1:1, thereby manufacturing a pharmaceutical composition comprising a protein complex.

[0018] Certain embodiments include recombinant production of (a) before combining with (b). Some embodiments include purification of (a) derived from human subject plasma before combining with (b). Certain embodiments include recombinant production of (b) before combining with (a).

[0019] In some embodiments, the manufacturing method involves combining (a) and (b) in a molar ratio 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)]. Certain embodiments involve combining (a) and (b) in a molar ratio of approximately 1:2 [(a):(b)].

[0020] In some embodiments, the A2M protein (a) and the serine protease protein (b) are bound in a protein complex, and the protein complex is (i)(b) retains CD95 (Fas receptor) protease cleavage activity and cancer cell killing activity, (ii) Sterically inhibits the binding of (b) to fibrinogen, thereby reducing or inhibiting the fibrinogen cleavage activity of (b), (iii)(b) sterically inhibits the binding of serine protease inhibitors (including α1-antitrypsin (A1AT)).

[0021] In some embodiments, (a) consists of, or essentially consists of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to a sequence or functional fragment thereof selected from Table A1. In some embodiments, the functional fragment consists of, or essentially consists of, a sequence containing about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1300, or 1400 consecutive amino acids of a sequence selected from Table A1.In some embodiments, the functional fragment is approximately 1-1400, 1-1300, 1-1200, 1-1100, 1-1000, 1-900, 1-800, 1-700, 1-600, 1-500, 1-400, 1-300, 1-200, 100-1400, 100-1300, 100-1200, 100-1100, 100-1000, 100-900, 100-800, 100-700, 100-600, 100-500, 100-400, 100-300, 100-200, 200-1400, 200-1300, 200-1200, 200-1100, 200-1000, 200-900, 200-800, 200-700, 200-600, 200-500, 200-400, 200-300, 300-1400, 300-1300, 300-1200, 300-1100, 300-1000, 300-900, 300-800, 300-700, 300-600, 300-500, 300-400, 400-1400, 400-1300, 400-1200, 400-110 0, 400~1000, 400~900, 400~800, 400~700, 400~600, 400~500, 500~1400, 500~1300, 500~1200, 500~1100, 500~1000, 500~900, 500~800, 500~700, 500~600, 600~1400, 600~1300, 600~1200, 600~1100, 600~1000, 600~900, 600~800, 600~700, 700~1400, 700~1300, 700~1200, 700~ It consists of residues of 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.

[0022] In some embodiments, (a) is conjugated to or fused 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 tumor-specific antigen (TSA).

[0023] In certain embodiments, (b) is selected from porcine pancreatic elastase (PPE) protein, human neutrophil elastase (ELANE) protein, human cathepsin G (CTSG) protein, human proteinase 3 (PR3) protein, and human granzyme B protein.

[0024] In some embodiments, The PPE protein consists of, or essentially consists of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 5, and that sequence retains the Q211F amino acid substitution. The PPE protein consists of, or essentially consists of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 6, and that sequence retains the T55A amino acid substitution. The PPE protein consists of, or essentially consists of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 7, and that sequence retains the Q211F and T55A amino acid substitutions. The PPE protein consists of, or essentially consists of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 8, and that sequence retains the N241A amino acid substitution. The PPE protein consists of, or essentially consists of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 9, and that sequence retains the N241Y amino acid substitution. The PPE protein consists of, or essentially consists of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 10, and that sequence retains the R75A amino acid substitution. The PPE protein consists of, or essentially consists of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 11, and that sequence retains the R75E amino acid substitution. The PPE protein consists of, or essentially consists of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 12, and that sequence retains the Q211A amino acid substitution. The PPE protein consists of, or is essentially composed of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 13, and that sequence retains the R237A amino acid substitution. The PPE protein consists of, or is essentially composed of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 14, and that sequence retains the S214A amino acid substitution. The PPE protein consists of, or is essentially composed of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 15, and that such sequence retains the D74A amino acid substitution, and The PPE protein consists of, or essentially consists of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 16.

[0025] In some embodiments, Human ELANE protein consists of, or essentially consists of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 17. Human CTSG proteins consist of, or essentially consist of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 18. Human PR3 protein contains, or essentially consists of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 19, or Human granzyme B protein consists of, or essentially consists of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 20.

[0026] Certain methods further include one or more steps of testing the pharmaceutical composition in one or more activity assays selected from one or more of the following: a CD95 cleavage assay (optionally, in the presence of a serine protease inhibitor such as A1AT), a fibrinogen cleavage assay, and a cancer cell killing assay. In some embodiments, the pharmaceutical composition cleaves CD95 (optionally, in the presence of a serine protease inhibitor such as A1AT), substantially does not cleave fibrinogen, and / or has cancer cell killing activity. [Brief explanation of the drawing]

[0027] [Figure 1] Figure 1 shows the formation and structure of a protein complex containing human A2M and serine protease proteins. The protease binds to and cleaves the bait region of the A2M homotetramer, inducing a structural change that incorporates the serine protease protein into the protein complex. [Figure 2A]Figures 2A and 2B show the effects of intravenous injection of mutant F (MutF) alone on tumor growth (2A) and the number of lung metastases (2B) in a 4T1 tumor model (days 0 and 1). [Figure 2B] Figures 2A and 2B show the effects of intravenous injection of mutant F (MutF) alone on tumor growth (2A) and the number of lung metastases (2B) in a 4T1 tumor model (days 0 and 1). [Figure 2C] Figure 2C shows the effect of MutF on prothrombin time 5 minutes after intravenous injection. [Figure 3A] Figure 3A shows MutF activity at different A2M:MutF ratios in the presence of the inhibitor A1AT. [Figure 3B] Figure 3B shows MutF activity when the A2M:MutF ratio is 1:2 in the presence of different concentrations of the inhibitor A1AT. [Figure 3C] Figure 3C shows MutF activity when the A2M:MutF ratio in plasma is 1:2. [Figure 4A] Figure 4A shows the MutF activity and concentration in different fractions of the A2M:MutF product after cation exchange column separation, with and without A1AT. [Figure 4B] Figure 4B shows the MutF activity and concentration in different fractions of the A2M:MutF product after size exclusion column separation, with and without A1AT. [Figure 4C] Figures 4C–4D show that the A2M:MutF complex is stable over a wide pH range, as measured by enzyme activity (4C) and A1AT protection (4D). [Figure 4D] Figures 4C–4D show that the A2M:MutF complex is stable over a wide pH range, as measured by enzyme activity (4C) and A1AT protection (4D). [Figure 4E] Figure 4E shows that the A2M:MutF complex is stable across multiple freeze / thaw cycles, as measured by enzyme activity. [Figure 5A]Figures 5A–5D show MutF activity in various cell lysates after 30 minutes of treatment in serum-free medium (SFM) with and without A1AT. [Figure 5B] Figures 5A–5D show MutF activity in various cell lysates after 30 minutes of treatment in serum-free medium (SFM) with and without A1AT. [Figure 5C] Figures 5A–5D show MutF activity in various cell lysates after 30 minutes of treatment in serum-free medium (SFM) with and without A1AT. [Figure 5D] Figures 5A–5D show MutF activity in various cell lysates after 30 minutes of treatment in serum-free medium (SFM) with and without A1AT. [Figure 6A] Figure 6A shows Coomassie blue staining of CD95 sections after incubation with MutF or A2M:MutF in different ratios for 30 minutes. [Figure 6B] Figure 6B shows Western blots of fibrinogen after incubation for 1 hour with MutF or A2M:MutF at different ratios. A2M:MutF at a ratio of 1:2 cleaves CD95 as efficiently as MutF alone (indicated by the double band), but does not cleave fibrinogen as well as MutF alone (indicated by the lower band). [Figure 6C] Figure 6C shows the fluorescence signals of cleaved elastin after incubation with PBS, MutF alone, or with the A2M:MutF protein complex. [Figure 7A] Figure 7A shows the prothrombin time in mouse plasma 5 minutes after intravenous injection of 480 ug of MutF or A2M:MutF(1:2) protein complex. [Figure 7B] Figure 7B shows the partial thromboplastin time in mouse plasma 5 minutes after intravenous injection of 480 ug of MutF or A2M:MutF(1:2) protein complex into mice. [Figure 7C]Figure 7C shows the concentration of fibrinogen in mouse plasma 5 minutes after intravenous injection of 480 ug of MutF or A2M:MutF(1:2) protein complex. [Figure 8A] Figure 8A shows cell-killing assays against various mouse cancer cell lines using MutF or A2M:MutF(1:2) protein complexes at 400 nM. [Figure 8B] Figure 8B shows the broad cytotoxicity of the A2M:MutF protein complex against cancer cells of different anatomical origins. [Figure 8C] Figure 8C shows that the complex does not kill non-cancer cells. [Figure 8D] Figure 8D shows the antitumor effects of MutF and the A2M:MutF(1:2) protein complex after intravenous injection of 100 ug on day 0 in the CT26 model. [Figure 8E] Figure 8E shows that the A2M:MutF protein complex has an improved functional PK profile (enzyme activity in plasma) after intravenous administration compared to MutF alone. [Figure 8F] Figure 8F shows that the A2M:MutF protein complex induces a favorable immune profile in the CT26 model (from left to right in each graph: PBS, MutF, and A2M:MutF). [Figure 8G] Figure 8G shows that the A2M:MutF protein complex induces a tumor antigen-specific CD8+ T cell response in the CT26 model (from left to right in each graph: PBS, MutF, and A2M:MutF). [Figure 9A] Figure 9A demonstrates that the A2M:MutF protein complex has a broad therapeutic range, as shown by its ability to kill human ovarian cancer cells without killing non-cancerous cells from the patient, in contrast to doxorubicin and oxaliplatin. [Figure 9B]Figure 9B shows that A2M:MutF demonstrated comparable killing power against cancer cells isolated from chemotherapy-naive and chemotherapy-treated patients, while doxorubicin and oxaliplatin, in contrast, showed reduced killing power against cancer cells isolated from chemotherapy-treated patients compared to chemotherapy-naive patients. [Figure 10A] Figure 10A shows that the A2M:MutF protein complex induces ICD markers in CT26 and A549 cells. [Figure 10B] Figure 10B shows that the A2M:MutF protein complex induces ICD markers in tumor cells derived from human ovarian patients (from left to right in each graph: CTRL, A2M:MutF, and oxaliplatin). [Figure 11A] Figures 11A-11B show tumor growth after treatment in a mouse CT26 tumor model. [Figure 11B] Figures 11A-11B show tumor growth after treatment in a mouse CT26 tumor model. [Figure 11C] Figure 11C shows the tumor weight 15 days after treatment (Figure 11 shows, from left to right, vehicle every other day, A2M:MutF 100 μg daily, A2M:MutF 200 μg every other day, and A2M:MutF 400 μg every four days). [Figure 12A] Figures 12A-12B show that the A2M:MutF protein complex effectively suppresses tumor growth in the Jh-BALB / c CT26 colorectal cancer model. [Figure 12B] Figures 12A-12B show that the A2M:MutF protein complex effectively suppresses tumor growth in the Jh-BALB / c CT26 colorectal cancer model. [Figure 12C] Figures 12C–12D show that the A2M:MutF protein complex treats primary tumors and metastases in the Jh-C57BL / 6 B16F10 melanoma model. [Figure 12D] Figures 12C–12D show that the A2M:MutF protein complex treats primary tumors and metastases in the Jh-C57BL / 6 B16F10 melanoma model. [Figure 12E] Figure 12E shows that the A2M:MutF protein complex is effective in various tumors with different immunological states. [Figure 13A] Figures 13A-13C show that the A2M:MutF protein complex exhibits improved antitumor efficacy compared to SoC chemotherapy (oxaliplatin) without the observation of toxicity. [Figure 13B] Figures 13A-13C show that the A2M:MutF protein complex exhibits improved antitumor efficacy compared to SoC chemotherapy (oxaliplatin) without the observation of toxicity. [Figure 13C] Figures 13A-13C show that the A2M:MutF protein complex exhibits improved antitumor efficacy compared to SoC chemotherapy (oxaliplatin) without the observation of toxicity. [Figure 14A] Figure 14A shows the efficacy of the A2M:MutF protein complex in a human xenograft model of lung cancer. [Figure 14B] Figure 14B summarizes the efficacy of the A2M:MutF protein complex across various models of prostate cancer, colon cancer, and lung cancer. [Figure 14C] Figure 14C shows that the A2M:MutF protein complex effectively kills tumor cells (derived from patient CDX_O02) from human ovarian patients in a xenograft mouse model. [Figure 14D] Figure 14D summarizes the efficacy of the A2M:MutF protein complex across three ovarian cancer patients in this model. [Figure 14E] Figures 14E–14F demonstrate that the A2M:MutF protein complex effectively kills patient-derived breast cancer cells in vitro and in vivo. [Figure 14F] Figures 14E–14F demonstrate that the A2M:MutF protein complex effectively kills patient-derived breast cancer cells in vitro and in vivo. [Figure 14G]Figure 14G summarizes the in vivo efficacy of the A2M:MutF protein complex in various human tumors, demonstrating that its efficacy is independent of tumor genetics or immune status. [Figure 15] Figure 15 shows that mice treated with the A2M:MutF protein complex were tumor-free after the initial trial using CT26 colorectal cancer cells (5 / 11), and all of these mice (5 / 5) remained tumor-free during the subsequent trial using CD26 cells. [Modes for carrying out the invention]

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this disclosure belongs. Any methods, materials, compositions, reagents, and cells similar or equivalent to those described herein may be used in carrying out or testing the subject matter of this disclosure, but preferred methods and materials are described. All publications and references, including but not limited to patents and patent applications cited herein, are incorporated herein by reference in their entirety, as if each individual publication or reference were to be specifically and individually incorporated herein as being fully incorporated by reference. Any patent application for which this application claims priority is also incorporated herein by reference in its entirety, in the manner described above for publications and references.

[0029] Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzyme reactions and purification techniques may be performed according to the manufacturer's specifications, or as commonly achieved in the art, or as described herein. These and related techniques and procedures may generally be performed according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout this specification. Unless otherwise specified, the nomenclature used in connection with molecular biology, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry, as well as their laboratory procedures and techniques, are well known and commonly used in the art. Standard techniques may be used for recombinant techniques, molecular biological, microbiological, chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, as well as for patient treatment.

[0030] For the purposes of this disclosure, the following terms are defined below.

[0031] The articles "a" and "an" are used herein to refer to one or more (i.e., at least one) grammatical objects of an article. For example, "element" includes "one element," "one or more elements," and / or "at least one element."

[0032] "Approximately" means a quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length that varies by approximately 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% relative to the reference quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length.

[0033] An "antagonist" refers to a biological or chemical substance that inhibits or reduces the physiological effects of another drug or molecule. In some cases, antagonists specifically bind to other drugs or molecules. This includes complete and partial antagonists.

[0034] An "agonist" refers to a biological or chemical substance that increases or enhances the physiological effects of another drug or molecule. In some cases, agonists specifically bind to other drugs or molecules. This includes complete and partial agonists.

[0035] As used herein, the term “amino acid” is intended to mean both native and unnatural amino acids, as well as both amino acid analogs and mimetic compounds. Native amino acids include the 20(L) amino acids utilized in protein biosynthesis, as well as others such as 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine, homocysteine, citrulline, and ornithine. Unnatural amino acids include, for example, (D)-amino acids, norleucine, norvaline, p-fluorophenylalanine, and ethionine, which are known to those skilled in the art. Amino acid analogs include modified forms of native and unnatural amino acids. Such modifications may include, for example, substitution or exchange of chemical groups and moieties to or by derivatization of amino acids. Amino acid mimetic compounds include organic structures that exhibit functionally similar properties, such as the charge and charge spacing characteristics of a reference amino acid. For example, an organic structure mimicking arginine (Arg or R) would have a positively charged moiety located in a similar molecular space and possessing the same degree of mobility as the e-amino group of the side chain of the naturally occurring Arg amino acid. The mimetic also involves a constrained structure to maintain optimal spacing and charge interactions between amino acids or amino acid functional groups. Those skilled in the art know or can determine which structures construct functionally equivalent amino acid analogs and amino acid mimetic compounds.

[0036] As used herein, subjects “at risk” of developing a disease or adverse reaction may or may not have a detectable disease or symptoms of a disease, and may or may not exhibit a detectable disease or symptoms of a disease prior to the treatment methods described herein. “At risk” means that a subject has one or more risk factors, which are measurable parameters that correlate with the development of diseases described herein and known in the art. Subjects having one or more of these risk factors have a higher probability of developing the disease or will have an adverse reaction than subjects not having one or more of these risk factors.

[0037] "Biocompatibility" refers to a material or compound that is generally not harmful to the biological function of cells or objects and does not result in any unacceptable degree of toxicity, including allergenicity and pathological conditions.

[0038] The term "bond" refers to a direct relationship between two molecules resulting from covalent, electrostatic, hydrophobic, and ionic and / or hydrogen bonding interactions, including, for example, salt and water bridges.

[0039] A "coding sequence" refers to any nucleic acid sequence that contributes to coding the polypeptide product of a gene. In contrast, the term "non-coding sequence" refers to any nucleic acid sequence that does not directly contribute to coding the polypeptide product of a gene.

[0040] Throughout this disclosure, unless otherwise required by context, the words “comprise,” “comprises,” and “comprising” are understood to imply the inclusion of the described step or element or group of steps or elements, but not the exclusion of other steps or elements or groups of steps or elements.

[0041] "Consists of" means including and being limited to what follows the phrase "consists of." Thus, the phrase "consists of" indicates that the enumerated elements are necessary or essential, and the other elements are not required. "Essentially consists of" means including any elements enumerated after the phrase, and being limited to other elements that do not interfere with or contribute to the activity or action of the enumerated elements as expressed in this disclosure. Thus, the phrase "essentially consists of" indicates that the enumerated elements are necessary or essential, but the other elements are optional and may or may not be present, depending on whether they substantially affect the activity or action of the enumerated elements.

[0042] The terms “endotoxin-free” or “substantially endotoxin-free” generally refer to compositions, solvents, and / or blood vessels containing the maximum trace amount of endotoxin (e.g., an amount that does not have clinically harmful physiological effects on the subject), and preferably an undetectable amount of endotoxin. Endotoxins are toxins associated with certain microorganisms, such as bacteria, typically Gram-negative bacteria, although endotoxins can be found in Gram-positive bacteria such as Listeria monocytogenes. The most commonly found endotoxins are lipopolysaccharides (LPS) or lipooligosaccharides (LOS) found on the outer membranes of various Gram-negative bacteria, representing a central pathogenic feature in the disease-causing ability of these bacteria. Small amounts of endotoxin in humans can produce harmful physiological effects, including fever, decreased blood pressure, and activation of inflammation and coagulation.

[0043] Therefore, in pharmaceutical production, it is often desirable to remove most or all traces of endotoxins from drug products and / or drug containers, as even small amounts can cause adverse effects on humans. Since temperatures above 300°C are typically required to decompose most endotoxins, depyrogenation ovens can be used for this purpose. For example, based on the primary packaging material, such as a syringe or vial, 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 being considered, for example, including chromatography and filtration methods described herein and known in the art.

[0044] Endotoxins can be detected using conventional techniques known in the art. For example, the Limulus Amoebocyte Lysate assay, which utilizes horseshoe crab blood, is a highly sensitive assay for detecting the presence of endotoxins. In this test, very low levels of LPS can cause detectable coagulation of Limulus lysate due to a potent enzyme cascade that amplifies the reaction. Endotoxins can also be quantified by enzyme-linked immunosorbent assay (ELISA). Since they are substantially endotoxin-free, endotoxin levels may be 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 less than 10 EU / mg of active compounds. Typically, 1 ng of lipopolysaccharide (LPS) is equivalent to approximately 1 to 10 EU.

[0045] "Semi-maximum effective concentration" or "EC 50 The term "EC" refers to the concentration of the drug (e.g., protein complex) described herein, which induces a response between baseline and maximum after several specified exposure times, and thus the EC of the graded dose-response curve. 50EC50 represents the concentration of the compound at which 50% of its maximum effect is observed. EC50 also represents the plasma concentration required to obtain 50% of the maximum effect in vivo. Similarly, EC50 represents the concentration at which 50% of the maximum effect is observed. 90 "EC" refers to the concentration of a drug or composition at which 90% of its maximum effect is observed. 90 The EC50 can be calculated from the Hill gradient or determined directly from the data using conventional knowledge in the art. In some embodiments, the EC50 of the drug 50 These values ​​are approximately 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 less than 500 nM. In some embodiments, the drug will have an EC50 value of approximately 1 nM or less.

[0046] The “half-life” of a drug may refer to the time it takes for the drug to lose half of its pharmacological, physiological, or other activity compared to such activity at the time of administration to the serum or tissue of an organism, or at any other defined point in time. “Half-life” may also refer to the time it takes for the amount or concentration of a drug to be reduced by half of the initial dose administered to the serum or tissue of an organism, compared to such amount or concentration at the time of administration to the serum or tissue of an organism, or at any other defined point in time. Half-life may be measured in serum and / or any one or more selected tissues.

[0047] The term "heterogeneous" refers to features or elements of a polypeptide or polynucleotide that originate from a source different from that of the wild-type polypeptide or the polynucleotide it encodes, for example, features from a species different from the wild type, or manipulated features of non-natural origin.

[0048] The terms “modulate” and “alter” typically include “increase,” “enhance,” or “stimulate,” as well as “decrease,” or “reduce,” by a statistically significant or physiologically significant amount or degree compared to a control. The amount “increased,” “stimulated,” or “enhanced” is typically a “statistically significant” amount and may include an increase of about or at least about 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, or 1000 times greater than the amount produced by the control composition or in the absence of the composition (e.g., the absence of the drug). The amount “reduced” or “reduced” is typically a “statistically significant” amount and may include reductions of about or at least less than 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 the amount produced without the composition (e.g., in the absence of the drug) or by the control composition. Examples of comparisons and “statistically significant” amounts are described herein.

[0049] 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, “proprotein,” “proenzyme,” or “enzyme precursor” refers to an inactive (or substantially inactive) protein or enzyme that is typically activated by protease cleavage of an activated peptide to produce an active protein or enzyme. The terms include modifications such as myristoylation, sulfation, glycosylation, phosphorylation, and addition or deletion of signal sequences. The terms “polypeptide” or “protein” mean one or more chains of amino acids, each chain containing amino acids covalently linked by peptide bonds, and such polypeptide or protein may contain multiple chains non-covalently and / or covalently linked together by peptide bonds, including native proteins, i.e., proteins of natural origin, particularly those produced by non-recombinant cells, or molecules having sequences of genetically engineered or recombinant cells and possessing the amino acid sequence of a native protein, or molecules having deletions, additions to, and / or substitutions of one or more amino acids from the native sequence. In certain embodiments, the polypeptide is a “recombinant” polypeptide produced by a recombinant cell containing one or more recombinant DNA molecules, and is typically made from heterogeneous polynucleotide sequences or combinations of polynucleotide sequences not otherwise found in the cell.

[0050] The terms “polynucleotide” and “nucleic acid” include mRNA, RNA, cRNA, cDNA, and DNA. The terms typically refer to macromolecular forms of nucleotides, either ribonucleotides or deoxynucleotides, or modified forms of either type of nucleotide, with a length of at least 10 bases. The terms include single-stranded and double-stranded forms of DNA. The terms “isolated DNA,” “isolated polynucleotide,” and “isolated nucleic acid” refer to molecules isolated without containing the total genomic DNA of a particular species. Therefore, an isolated DNA segment encoding a polypeptide refers to a DNA segment containing one or more coding sequences, and further, being isolated substantially apart from or freely purified from the total genomic DNA of the species from which the DNA segment is obtained. It also includes non-coding polynucleotides that do not encode polypeptides (e.g., primers, probes, oligonucleotides). Furthermore, it includes recombinant vectors, such as expression vectors, viral vectors, plasmids, cosmids, phagemids, phages, and viruses.

[0051] Additional coding or non-coding sequences may be present within the polynucleotides described herein, although this is not essential, and polynucleotides may be bound to other molecules and / or supporting materials, although this is not essential. Therefore, polynucleotides or expressible polynucleotides can be combined with other sequences, such as expression regulatory sequences, regardless of the length of the coding sequence itself.

[0052] “Regulatory sequences” include corresponding amino acid regulatory sequences for nucleic acids, or for promoters, leaders, enhancers, introns, recognition motifs for RNA or DNA-binding proteins, polyadenylation signals, terminators, intrasequence ribosome entry sites (IRESs), secretory signals, and intracellular localization signals, which have the ability to influence the transcription or translation of coding sequences within host cells, or their location within or between cells. Exemplary regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).

[0053] A "promoter" is a DNA regulatory region capable of binding to an intracellular RNA polymerase and initiating the transcription of a downstream (3' direction) coding sequence. As used herein, a promoter sequence is bound at its 3' end by a transcription start site, extends upstream (5' direction), and contains the minimum number of bases or elements necessary to initiate transcription at a detectable level above the background. The transcription start site (conveniently defined by mapping to nuclease S1) may be found within the promoter sequence and the protein-binding domain (consensus sequence) involved in RNA polymerase binding. Eukaryotic promoters may, often but not always, contain "TATA" and "CAT" boxes. Prokaryotic promoters contain the Shine-Dalgano sequence in addition to the -10 and -35 consensus sequences.

[0054] Numerous promoters, including constitutive, inducible, and repressive promoters from various different sources, are well known in the art. Typical sources include, for example, viruses, mammals, insects, plants, yeasts, and bacterial cell types, and suitable promoters from these sources can be readily available or constructed based on sequences published online, or synthetically from deposits such as ATCC, and other commercial or individual sources. Promoters may be unidirectional (i.e., initiating transcription in one direction) or bidirectional (i.e., initiating transcription in either 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. Examples of inducible promoters include the Tet system (US Patent Nos. 5,464,758 and 5,814,618), the Ecdysone-inducible system (No et al., Proc. Natl. Acad. Sci. (1996) 93(8):3346-3351), the T-REx™ system (Invitrogen Carlsbad, CA), LacSwitch® (Stratagene, San Diego, CA), and the Cre-ERT tamoxifen-inducible recombinase system (Indra et al. Nuc. Acid. Res. (1999) 27(22):4324-4327; Nuc. Acid. Res. (2000) 28(23):e99, US Patent No. 7,112,715, and Kramer & Fussenegger, Methods) Examples include Mol. Biol. (2005) 308:123-144), or any promoter known in the art that is suitable for expression in the desired cells.

[0055] "Expressable polynucleotides" include cDNA, RNA, mRNA or other polynucleotides containing at least one coding sequence and optionally at least one expression regulatory sequence, such as transcriptional and / or translational regulatory elements, which can express cells or encoded polypeptides upon introduction into cells.

[0056] The term “isolated” polypeptide or protein as used herein means that the protein in question (1) does not contain at least some other proteins that would typically be found in nature, (2) does not contain essentially other proteins from the same source, e.g., from the same species, (3) is expressed by cells of a different species, (4) is isolated from at least about 50% polynucleotides, lipids, carbohydrates, or other substances associated in nature, (5) the “isolated protein” is not associated (by covalent or non-covalent interactions) with any part of a protein associated in nature, (6) is manipulably associated (by covalent or non-covalent interactions) with a polypeptide not associated in nature, or (7) is not of natural origin. Such isolated proteins may be encoded by genomic DNA, cDNA, mRNA, or other RNA, or any combination thereof, which may be of synthetic origin. In certain 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, prophylactic, research, or otherwise).

[0057] In certain embodiments, the "purity" of any given agent in a composition may be defined. For example, a particular composition may include, but is not limited to, agents such as polypeptide agents that, when measured by high-performance liquid chromatography (HPLC), a well-known form of column chromatography frequently used in biochemistry and analytical chemistry for separating, identifying, and quantifying compounds, have a purity of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% on a protein-based or weight-based basis, and includes all minorities and ranges in between.

[0058] The term "reference sequence" generally refers to a nucleic acid coding sequence or amino acid sequence that another sequence is being compared to. All polypeptide and polynucleotide sequences described herein are included as reference sequences, including those described by name, as well as those described in the tables and sequence lists.

[0059] Certain embodiments include biologically active “variants” and “fragments” of the proteins / polypeptides described herein, as well as polynucleotides encoding them. A “variant” contains one or more substitutions, additions, deletions, and / or insertions to a reference polypeptide or polynucleotide (see, for example, the Table and Sequence List). A variant polypeptide or polynucleotide contains an amino acid or nucleotide sequence that has at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more of sequence identity, similarity, or homology with a reference sequence, as described herein, and substantially retains the activity of the reference sequence. Furthermore, the sequence may consist of a reference sequence formed by the addition, deletion, insertion, or substitution 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, or 150 or more amino acids or nucleotides, or may differ from such a sequence but substantially retains the activity of at least one of the reference sequences. In certain embodiments, the addition or deletion may include C-terminal and / or N-terminal additions and / or deletions.

[0060] As used herein, the term “sequence identity,” or for example, “50% identical sequences,” refers to the extent to which sequences are identical at the nucleotide or amino acid level across a comparison window. Thus, the “percentage of sequence identity” can be calculated by comparing two optimally aligned sequences across 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) occur in both sequences to obtain 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 obtain the percentage of sequence identity. The optimal alignment of sequences for aligning the comparison window can be achieved by a computerized implementation of the algorithm (GAP, BESTFIT, FASTA, and TFASTA from Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, Wis., USA) or by one of a variety of selected methods, through inspection and best alignment (i.e., resulting in the highest percentage of homology across the comparison window). For example, the BLAST family of programs may also be referenced, as disclosed by Altschul et al., Nucl. Acids Res. 25:3389, 1997.

[0061] The term "solubility" refers to the property of the agent described herein to dissolve in a liquid solvent and form a homogeneous solution. Solubility is typically expressed as concentration, by any of the following: mass of solute per unit volume of solvent (e.g., g, g / dL (100 mL), mg / mL of solute per 1 kg of solvent), molar concentration, molar concentration fraction, or any other similar description of concentration. The maximum equilibrium amount of solute that can dissolve per unit volume of solvent is the solubility of that solute in that solvent under specified conditions, including temperature, pressure, pH, and the properties of the solvent. In certain embodiments, solubility is measured at physiological pH or other pH, e.g., pH 5.0, pH 6.0, pH 7.0, pH 7.4, pH 7.6, pH 7.8, or pH 8.0 (e.g., approximately pH 5–8). In certain embodiments, solubility is measured in water or a physiological buffer such as PBS or NaCl (with or without NaPO4). In certain embodiments, solubility is measured at relatively low pH (e.g., pH 6.0) and relatively high salt (e.g., 500 mM NaCl and 10 mM NaPO4). In certain embodiments, solubility is measured in a biological fluid (solvent) such as blood or serum. In certain embodiments, the temperature can be about room temperature (e.g., about 20, 21, 22, 23, 24, 25°C) or about body temperature (37°C). In certain embodiments, the drug 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.

[0062] "Subject" or "Subject requiring it" or "Patient" or "Patient requiring it" includes mammalian subjects such as human subjects.

[0063] "Substantially" or "essentially" means, for example, nearly whole or complete of a given quantity, such as 95%, 96%, 97%, 98%, 99%, or more.

[0064] "Statistically significant" means that the result was unlikely to have occurred by chance. Statistical significance can be determined by any method known in the art. A commonly used measure of significance is the p-value, which is the frequency or probability that the observed event would occur if the null hypothesis were true. If the resulting p-value is less than the significance level, the null hypothesis is rejected. In simple terms, the significance level is defined as a p-value of 0.05 or less.

[0065] "Therapeutic response" refers to the improvement (whether or not it is sustained) of symptoms based on the administration of one or more medications.

[0066] As used herein, “therapeutic dose,” “therapeutic dose,” “preventive dose,” and “diagnostic dose” refer to the amount of drug required to elicit a desired biological response after administration.

[0067] As used herein, “treatment” of an object (e.g., a mammal such as a human) or cells is any type of intervention used in an attempt 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 carried out either prophylactically or after the onset of a pathological event or contact with a pathogen. It also includes “prophylactic” treatment, which may be directed to reduce the rate of progression of the disease or condition being treated, delay the onset of the disease or condition, or reduce the severity of its onset. “Treatment” or “prevention” does not necessarily imply the complete eradication, cure, or prevention of the disease or condition or its associated symptoms.

[0068] The term "wild-type" refers to a gene or gene product (e.g., polypeptide) that is most frequently observed in a population and is therefore arbitrarily designed to be a "normal" or "wild-type" form of the gene.

[0069] Each embodiment described herein applies to all other embodiments unless otherwise expressly stated.

[0070] protein complex Embodiments of this disclosure generally relate to pharmaceutical compositions comprising a protein complex of an α2-macroglobulin (A2M) protein and a serine protease protein such as PPE. Here, certain serine proteases can kill cancer cells, regardless of their genetic abnormalities, upon direct contact with or administration to a tumor (e.g., intratumoral administration), and are relatively harmless to non-cancerous or healthy cells (see, e.g., WO2018 / 232273, WO2020 / 132465, PCT / US2021 / 046453, and PCT / 2021 / 046467). However, in some cases, systemic administration of independent serine proteases such as PPE may adversely affect coagulation, for example, by inducing fibrinogen cleavage. This disclosure is partly related to the discovery that a protein complex of human A2M with a serine protease such as PPE not only sterically protects the serine protease from serine protease inhibitors in plasma while retaining CD95 protease cleavage activity and cancer cell killing activity, but also mitigates the adverse effects of serine proteases on coagulation, for example, by sterically inhibiting or otherwise reducing the ability of the complexed serine protease to cleave fibrinogen. This disclosure further relates to the discovery of an optimal molar ratio range between [A2M protein]:[serine protease protein] in the protein complex, which provides a balance between retaining the CD95 cleavage and cancer cell killing activity of the serine protease and reducing its negative effects on coagulation, as measured, for example, by reduced prothrombin time or reduced fibrinogen cleavage compared to serine protease alone.

[0071] Accordingly, certain embodiments include a pharmaceutical composition comprising a protein complex of (a) α2-macroglobulin (A2M) protein and (b) 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)], and the A2M protein of (a) and the serine protease protein of (b) are bound to each other in a 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) reduces or inhibits the fibrinogen cleavage activity of (b) by sterically inhibiting its binding to fibrinogen, and (iii) protects (b) from inhibition by serine protease inhibitors such as α1 antitrypsin (A1AT).

[0072] In certain embodiments, A2M proteins bind to each other in protein complexes as A2M monomers or A2M polymers, for example, as A2M dimers such as A2M homodimers, as A2M trimers such as A2M homotrimers, or as A2M tetramers such as A2M homotetramers. In certain embodiments, the A2M proteins in (a) bind to each other as A2M homotetramers, and the serine protease protein in (b) is bound to the protein complex by the A2M homotetramers. In certain embodiments, each protein complex consists of one set of A2M homotetramers (i.e., four A2M proteins as (i) four complete A2M proteins, or (ii) up to eight A2M fragments resulting from the cleavage of the bait region of the complete A2M proteins by the serine protease when the serine protease binds to the A2M homotetramer complex) and two serine protease proteins (see, for example, Figure 1).

[0073] In certain embodiments, the protein complex sterically inhibits the binding of (b) to larger molecules but not to smaller molecules. That is, as described above, the protein complex sterically inhibits the binding of (b) to larger molecules such as serine protease proteins (e.g., A1AT), fibrinogen, and / or plasma antibodies. Therefore, in some embodiments, the protein complex protects (b) from inhibition by serine proteases and reduces / inhibits (b)'s ability to cleave fibrinogen. In some cases, for example, if 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 or anti-PPE plasma antibodies. In some cases, this provides a clinical benefit, as administration of non-human protein drugs such as PPE to humans can normally induce anti-drug antibodies. In contrast, the protein complex does not sterically inhibit the binding of (b) to small molecules such as CD95. Therefore, in certain embodiments, (b) in the protein complex cleaves CD95 and kills cancer cells, but does not substantially cleave fibrinogen.

[0074] The pharmaceutical compositions and protein complexes described herein include α2-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., J. Biol. Chem. 275:26806-11, 2000). It often exists as a tetramer consisting of four identical subunits of approximately 180 kDa, forming a hollow cylindrical structure. In its central "bait domain," it can present multiple target peptide bonds to the protease it attacks. Human A2M "captures" serine proteases such as PPE, where the serine protease binds to and cleaves the bait region of A2M. This cleavage induces a conformational change in A2M, which significantly reduces the protease's activity toward high molecular weight substrates while retaining its activity toward low 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 (without signal peptide) human A2M are provided in Table A1 below. [Table 1]

[0075] In some embodiments, the A2M protein of the protein complex consists of, or essentially consists of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to a sequence selected from Table A1, or a functional fragment thereof. In some embodiments, the functional fragment consists of, or essentially consists of, a sequence containing about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1300, or 1400 consecutive amino acids of a sequence selected from Table A1.

[0076] For example, in a particular embodiment, the functional fragment is approximately 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, 100-2 00, 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, 400~1 100, 400~1000, 400~900, 400~800, 400~700, 400~600, 400~500, 500~1400, 500~1300, 500~1200, 500~1100, 500~1000, 500~900, 500~800, 500~700, 500~600, 600~1400, 600~1300, 600~1200, 600~1100, 600~1000, 600~900, 600~800, 600~700, 700~1400, 700~1300, 700~1200, 700 It consists of residues of ~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 can capture or otherwise bind a serine protease (such as PPE) to a protein complex in a configuration that forms an A2M homotetramer, sterically inhibiting the binding of the serine protease to a serine protease inhibitor such as A1AT, preserving the CD95 protease cleavage activity and cancer cell killing activity of the serine protease, and / or inhibiting or otherwise reducing the serine protease's ability to cleave fibrinogen.

[0077] In certain embodiments, the A2M portion of the protein complex improves uptake into cancer cells compared to serine proteases alone. For example, A2M binds to LPR1 receptors and GRP78 receptors expressed in normal and cancer cells. Indeed, elevated GRP78 levels commonly correlate with increased pathological malignancy, recurrence, and decreased patient survival in breast, liver, prostate, colon, and gastric cancers (see, e.g., Lee, Cancer Res. 67:3496-3499, 2007), and in some cases, A2M's ability to bind to GRP78 improves selective targeting of cancer cells expressing GRP78.

[0078] In some embodiments, the A2M portion of the protein complex is fused to or 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 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, Tenacin, 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), Guanylyl cyclase C, N Y-ESO-1, p53, Survivin, Integrin αvβ3, Integrin α5β1, Folic Acid Receptor 1, Transmembrane Glycoprotein NMB, Fibroblast-Activating Protein Alpha (FAP), Glycoprotein 75, TAG-72, MUC1, MUC16 (or CA-125), Phosphatidylserine, Prostate-Specific Membrane Antigen (PSMA), NR-LU-13 Antigen, TRAIL-R1, Tumor Necrosis Factor Receptor Superfamily Member 10b (TNFRSF10B or TRAIL- This includes R2), SLAM family member 7 (SLAMF7), EGP40 pan-cancer antigen, B-cell activator (BAFF), platelet-derived growth factor receptor, glycoprotein EpCAM (17-1A), programmed death 1, protein disulfide isomerase (PDI), regenerative liver phosphatase 3 (PRL-3), prostatic acid phosphatase, Lewis-Y antigen, GD2 (a disialoganglioside expressed in neuroectoderm-derived tumors), glypican-3 (GPC3), and mesothelin.

[0079] 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 proteinase 3 (PR3), and human granzyme B (GZMB). Exemplary full-length, wild-type serine protease proprotein amino acid sequences are provided in Table S1 below. [Table 2]

[0080] Therefore, in certain embodiments, the serine protease protein comprises, or essentially comprises, a full-length serine protease proprotein selected from Table S1, which includes a biologically active variant and its fragments. In certain embodiments, the serine protease comprises, or essentially comprises, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 100% identical to a sequence selected from Table S1.

[0081] In certain embodiments, the serine protease protein consists of the active peptidase domain of the serine protease. Exemplary peptidase domain sequences of PPE (including its exemplary variants), human ELANE, human CTSG, and human PR3 are provided in Table S2 below. [Table 3-1] [Table 3-2]

[0082] Therefore, in some embodiments, the serine protease protein comprises, or essentially comprises, a serine protease peptidase domain sequence selected from Table S2, which includes a biologically active variant and fragment thereof. In certain embodiments, the serine protease protein comprises, or essentially comprises, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 100% identical to the sequence selected from Table S2.

[0083] In some embodiments, the serine protease protein is a PPE protein, for example, The PPE protein consists of, or essentially consists of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 5, and that sequence retains the Q211F amino acid substitution. The PPE protein consists of, or essentially consists of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 6, and that sequence retains the T55A amino acid substitution. The PPE protein consists of, or essentially consists of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 7, and that sequence retains the Q211F and T55A amino acid substitutions. The PPE protein consists of, or essentially consists of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 8, and that sequence retains the N241A amino acid substitution. The PPE protein consists of, or essentially consists of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 9, and that sequence retains the N241Y amino acid substitution. The PPE protein consists of, or essentially consists of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 10, and that sequence retains the R75A amino acid substitution. The PPE protein consists of, or essentially consists of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 11, and that sequence retains the R75E amino acid substitution. The PPE protein consists of, or essentially consists of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 12, and that sequence retains the Q211A amino acid substitution. The PPE protein consists of, or is essentially composed of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 13, and that sequence retains the R237A amino acid substitution. The PPE protein consists of, or is essentially composed of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 14, and that sequence retains the S214A amino acid substitution. The PPE protein consists of, or is essentially composed of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 15, and that such sequence retains the D74A amino acid substitution, and The PPE protein consists of, or essentially consists of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 16.

[0084] In some embodiments, the serine protease protein is the human ELANE protein, for example, the human ELANE protein consists of, or essentially consists of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 17. In some embodiments, the serine protease protein is the human CTSG protein, for example, the human CTSG protein consists of, or essentially consists of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 18. In some embodiments, the serine protease protein is the human PR3 protein, for example, the human PR3 protein consists of, or essentially consists of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 19. In some embodiments, the serine protease protein is a human granzyme B protein, for example, the human granzyme B protein comprises, or essentially comprises, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 20.

[0085] As described above, in certain embodiments, (a) and (b) are present in the composition in molar ratios ranging from approximately 1:3 to approximately 1:1 [(a):(b)], such as 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. In certain embodiments, the molar ratios defined herein protect serine proteases from inhibition by serine protease inhibitors (e.g., A1AT), fibrinogen, and / or plasma antibodies, while preserving their CD95 protease cleavage and cancer cell-killing activity, thereby inhibiting or reducing their ability to cleave fibrinogen.

[0086] Therefore, in certain embodiments, the protein complex retains the ability to cleave CD95 (Fas receptor) but substantially does not cleave fibrinogen. In some embodiments, the protein complexes described herein possess 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 (e.g., in the presence of a plasma serine protease inhibitor such as A1AT). In certain embodiments, the protein complexes described herein possess about 50, 40, 30, 20, 10, or 5% or less of the fibrinogen protease cleavage activity of the corresponding serine protease protein. CD95 cleavage activity, cancer cell killing activity, fibrinogen cleavage activity, and coagulation properties can be measured according to the usual art (see Examples). For example, serine protease activity can more commonly be monitored using a chromogenic substrate activity assay (N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide), and CD95 and / or fibrinogen cleavage can be directly measured (e.g., by Western blotting). If necessary, protease cleavage activity can be measured in the presence of a serine protease inhibitor such as A1AT. Cancer cell killing activity can be measured in vitro or in vivo, and the effect on coagulation in vivo can be measured by common assays such as prothrombin (PT) time and partial thromboplastin (PTT) time (see Examples).

[0087] In some embodiments, the protein complexes described herein are generated in vivo or ex vivo, for example, in cells, by contacting cells or a subject with one or more expressible polynucleotides encoding (a) α2-macroglobulin (A2M) protein and (b) serine protease protein. The protein complexes are then formed within the cells. The “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 regulatory sequence, such as transcriptional and / or translational regulatory elements, which can express the encoded proteins upon introduction into cells, for example, cells of a subject. Certain embodiments include contacting ex vivo cells with one or more expressible polynucleotides encoding (a) and (b), and administering the cells to a subject.

[0088] Exemplary viral vectors that can be used to deliver expressible polynucleotides include retroviral vectors such as adenovirus vectors, herpesvirus vectors, vaccinia virus vectors, adeno-associated virus (AAV) vectors, and lentivirus vectors. Examples of retroviral vectors include, but are not limited to, Moloney's mouse leukemia virus (MoMuLV), Harvey's mouse sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), SIV, BIV, HIV, and Rous sarcoma virus (RSV) based vectors. In certain embodiments, the expressible polynucleotide is a modified RNA or modified mRNA polynucleotide, for example, a non-natural RNA analog. In certain 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 interbondings. Expressible RNA polynucleotides for delivering encoded proteins are described, for example, in Kormann et al., Nat Biotechnol. 29:154-7, 2011, and in U.S. Patent Applications 2015 / 0111248, 2014 / 0243399, 2014 / 0147454, and 2013 / 0245104, which are incorporated in their entirety by reference.

[0089] In some embodiments, the protein complexes described herein have one or more improved biological, physical, and / or pharmacokinetic properties compared to the corresponding serine protease protein alone. The protein complexes described herein can be used in any of the compositions, methods, and / or kits described herein.

[0090] Method of use and pharmaceutical composition Certain embodiments include methods for treating, improving the symptoms of, and / or reducing the progression of a disease or condition in a subject that requires such treatment, improvement, and / or reduction of its progression, the method comprising administering to the subject a composition comprising a protein complex as described herein. In certain embodiments, the disease is cancer, i.e., the subject requiring it has cancer, is suspected of having cancer, or is at risk of having cancer.

[0091] In certain embodiments, cancer is either primary or metastatic cancer. In certain embodiments, cancer is 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 carcinoma of the lung), mesothelioma, leukemia (optionally lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, or relapsed leukemia). One or more of the following are selected: 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 (medulloblastoma), bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancer, cervical cancer, testicular cancer, thyroid cancer, and gastric cancer.

[0092] In some embodiments, as described above, the cancer is metastatic cancer. In addition to the cancers described above, exemplary metastatic cancers include, but are not limited to, bladder cancer with metastasis to the bone, liver, and / or lungs; breast cancer with metastasis to the bone, brain, liver, and / or lungs; colorectal cancer with metastasis to the liver, lungs, and / or peritoneum; kidney cancer with metastasis to the adrenal glands, bone, brain, liver, and / or lungs; lung cancer with metastasis to the adrenal glands, bone, brain, liver, and / or other lung sites; melanoma with metastasis to the bone, brain, liver, lungs, and / or skin / muscles; ovarian cancer with metastasis to the liver, lungs, and / or peritoneum; pancreatic cancer with metastasis to the liver, lungs, and / or peritoneum; prostate cancer with metastasis to the adrenal glands, bone, liver, and / or lungs; gastric cancer with metastasis to the liver, lungs, and / or peritoneum; thyroid cancer with metastasis to the bone, liver, and / or lungs; and uterine cancer with metastasis to the bone, liver, lungs, peritoneum, and / or vagina.

[0093] In certain embodiments, administration (e.g., intravenous administration) of a serine protease-containing protein complex or composition substantially affects coagulation in the subject, for example, not substantially increasing prothrombin time or partial thromboplastin time in the subject (e.g., compared to administration of the corresponding serine protease alone). In certain embodiments, administration of the serine protease-containing protein complex described herein significantly reduces the effect on coagulation in the subject compared to administration of the corresponding serine protease alone.

[0094] Methods for treating cancer may be combined with other treatments. For example, the combination therapies described herein may be administered to a subject before, during, or after other therapeutic interventions, including symptomatic treatment, radiotherapy, surgery, transplantation, hormone therapy, photodynamic therapy, antibiotic therapy, or any combination thereof. Symptomatic treatments include the administration of corticosteroids to reduce cerebral edema, headache, cognitive impairment, and vomiting, as well as the administration of anticonvulsants to reduce seizures. Radiotherapy includes radiosurgery such as whole-brain irradiation, fractionated radiotherapy, and stereotactic radiosurgery, which may be further combined with conventional surgery.

[0095] Certain embodiments include combination therapies for treating cancer, which include methods for treating cancer, improving its symptoms, or inhibiting its progression in subjects that require treatment of cancer, improvement of its symptoms, or inhibition of its progression, and which include administering to a subject in combination with at least one additional agent, e.g., an immunotherapy agent, a chemotherapeutic agent, a hormonal therapy agent, and / or a kinase inhibitor. In some embodiments, the administration of the composition enhances the sensitivity of cancer to the additional agent (e.g., an immunotherapy agent, a chemotherapeutic agent, a hormonal 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, 2000, 300, 400, 500, 600, 700, 800, 900, 1000, 2000% or more compared to the additional agent alone.

[0096] Certain combination therapies employ one or more cancer immunotherapeutic agents, or “immunotherapeutic agents.” In certain cases, the immunotherapeutic agent modulates the target immune response, for example, to increase or maintain a cancer-related or cancer-specific immune response, thereby resulting in increased immune cell inhibition or reduction of cancer cells. Exemplary immunotherapeutic agents include polypeptides, e.g., antibodies and their antigen-binding fragments, ligands, and small peptides, as well as mixtures thereof. Immunotherapeutic agents also include small molecule compounds, cells (e.g., immune cells such as T cells), various cancer vaccines, gene therapies or other polynucleotide agents (e.g., viral agents such as oncolytic viruses), and others known in the art. Thus, in certain embodiments, the cancer immunotherapeutic agent is selected from one or more of immune checkpoint modulators, cancer vaccines, oncolytic viruses, cytokines, and cell-system immunotherapies.

[0097] In certain embodiments, cancer immunotherapy 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. Generally, immune checkpoint molecules are components of the immune system that either signal upward (co-stimulatory molecules) or signal downward, and their targeting has therapeutic potential in cancer because cancer cells can interfere with the innate function of immune checkpoint molecules (see, e.g., 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" to or "specifically bind" to one or more immune checkpoint molecules as described herein.

[0098] 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 cell death ligand 1 (PD-L1), programmed cell death ligand 2 (PD-L2), programmed cell death 1 (PD-1), V-domain Ig inhibitor of T cell activation (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 attenuation factor (BTLA), CD160, and T cell immune receptor having Ig and ITIM domains (TIGIT).

[0099] In certain embodiments, the drug is a PD-1 (receptor) antagonist or inhibitor, and its targeting has been shown to reclaim immune function in the tumor environment (see, e.g., Phillips et al., Int Immunol. 27:39-46, 2015). PD-1 is a cell surface receptor belonging to the immunoglobulin superfamily and expressed on T cells and pro-B cells. PD-1 interacts with two ligands, PD-L1 and PD-L2. PD-1 functions as an inhibitory immune checkpoint molecule, for example, by reducing or preventing T cell activation, thereby reducing autoimmunity and promoting autoimmune tolerance. The inhibitory effect of PD-1 is achieved, at least in part, through a dual mechanism that promotes apoptosis of antigen-specific T cells in lymph nodes, while also reducing apoptosis in regulatory T cells (suppressor T cells). Some examples of PD-1 antagonists or inhibitors include antibodies, antigen-binding fragments, or small molecules that specifically bind to PD-1 and reduce one or more of its immunosuppressive activities, such as its downstream signaling or its interaction with PD-L1. Specific examples of PD-1 antagonists or inhibitors include the antibodies nivolumab, pembrolizumab, PDR001, MK-3475, AMP-224, AMP-514, and pizilizumab, as well as their antigen-binding fragments (e.g., U.S. Patent Nos. 8,008,449, 8,993,731, and 9,073,994). See also U.S. Patent Applications Nos. 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. Patent Applications Nos. 2012 / 0039906 and 2015 / 0203579).

[0100] In some embodiments, the drug is a PD-L1 antagonist or inhibitor. As described above, PD-L1 is one of the natural ligands for the PD-1 receptor. Common examples of PD-L1 antagonists or inhibitors include antibodies, antigen-binding fragments, or small molecules that specifically bind to PD-L1 and reduce one or more of its immunosuppressive activities, such as its binding to the PD-1 receptor. Specific examples of PD-L1 antagonists include the antibodies atezolizumab (MPDL3280A), avelumab (MSB0010718C), and durvalumab (MEDI4736), as well as their antigen-binding fragments (see, for example, U.S. Patents 9,102,725, 9,393,301, 9,402,899, and 9,439,962).

[0101] In some embodiments, the drug is a PD-L2 antagonist or inhibitor. As described above, PD-L2 is one of the natural ligands for the PD-1 receptor. Common examples of PD-L2 antagonists or inhibitors include antibodies, antigen-binding fragments, or small molecules that specifically bind to PD-L2 and reduce one or more of its immunosuppressive activities, such as its binding to the PD-1 receptor.

[0102] In certain embodiments, the drug is a VISTA antagonist or inhibitor. VISTA is approximately 50 kDa in size and belongs to the immunoglobulin superfamily (possessing one IgV domain) and the B7 family. It is primarily expressed on leukocytes, and its transcription is partially regulated by p53. There is evidence that VISTA may act as both a ligand and receptor on T cells to inhibit T cell effector function and maintain peripheral immune tolerance. VISTA is produced at high levels in tumor-infiltrating lymphocytes such as myeloid suppressor cells and regulatory T cells, and antibody inhibition results in delayed tumor growth in mouse models of melanoma and squamous cell carcinoma. An exemplary anti-VISTA antagonist antibody is, for example, the antibody described in WO2018 / 237287, which is incorporated in its entirety by reference.

[0103] In some embodiments, the drug is a CTLA-4 antagonist or inhibitor. CTLA4 or CTLA-4 (cytotoxic T lymphocyte-associated protein 4), also known as CD152 (cluster of differentiated 152), is a protein receptor that functions as an inhibitory immune checkpoint molecule by transmitting inhibitory signals to T cells when it binds to CD80 or CD86 on the surface of antigen-presenting cells, for example. Common 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, as well as their antigen-binding fragments. At least part of the activity of ipilimumab is thought to be mediated by killing CTLA-4-expressing suppressor Tregs via antibody-dependent cell-mediated cytotoxicity (ADCC).

[0104] In some embodiments, the agent is an IDO antagonist or inhibitor, or a TDO antagonist or inhibitor. IDO and TDO are tryptophan-degrading enzymes with immunosuppressive properties. For example, IDO is known to suppress T cells and NK cells, generate and activate Treg and myeloid suppressor cells, and promote tumor angiogenesis. Common examples of IDO and TDO antagonists or inhibitors include antibodies, antigen-binding fragments, or small molecules that specifically bind to IDO or TDO (see, e.g., Platten et al., Front Immunol. 5:673, 2014) and reduce or inhibit one or more immunosuppressive activities. Specific examples of IDO antagonists or inhibitors include indoximod (NLG-8189), 1-methyltryptophan (1MT), β-carboline (norharmane, 9H-pyrido[3,4-b]indole), rosmarinic acid, and epacadostat (see, e.g., Sheridan, Nature Biotechnology. 33:321-322, 2015). Specific examples of TDO antagonists or inhibitors include 680C91 and LM10 (see, e.g., Pilotte et al., PNAS USA. 109:2497-2502, 2012).

[0105] In some embodiments, the agent is a TIM-3 antagonist or inhibitor. T cell immunoglobulin domain and mucin domain 3 (TIM-3) are expressed on activated human CD4+ T cells and regulate Th1 and Th17 cytokines. TIM-3 also acts as a negative regulator of Th1 / Tc1 function by inducing cell death through interaction with its ligand, galectin-9. TIM-3 contributes to the suppressive tumor microenvironment, and its overexpression is associated with poor prognosis in various cancers (see, e.g., Li et al., Acta Oncol. 54:1706-13, 2015). Common examples of TIM-3 antagonists or inhibitors include antibodies, antigen-binding fragments, or small molecules that specifically bind to TIM-3 and reduce or inhibit one or more of its immunosuppressive activities.

[0106] 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 has been reported to negatively regulate T cell proliferation, activation, and homeostasis in a manner similar to CTLA-4 and PD-1 (see, e.g., Workman and Vignali, European Journal of Immun. 33:970-9, 2003, and Workman et al., Journal of Immun. 172:5450-5, 2004), and to play a role in Treg suppression (see, e.g., Huang et al., Immunity. 21:503-13, 2004). LAG3 also maintains CD8+ T cells in an immunotolerative state and, in combination with PD-1, maintains CD8 T cell exhaustion. Common examples of LAG-3 antagonists or inhibitors include antibodies, antigen-binding fragments, or small molecules that specifically bind to LAG-3 and inhibit one or more of its immunosuppressive activities. Specific examples include the antibody BMS-986016 and its antigen-binding fragment.

[0107] In some embodiments, the agent is a BTLA antagonist or inhibitor. Expression of B- and T-lymphocyte attenuation factor (BTLA, CD272) is induced during T cell activation and inhibits T cells through interactions with tumor necrosis family receptors (TNF-R) and B7 family cell surface receptors. BTLA is a ligand for tumor necrosis factor (receptor) superfamily member 14 (TNFRSF14), also known as herpesvirus entry mediator (HVEM). The BTLA-HVEM complex negatively modulates the T cell immune response, for example, by inhibiting the function of human CD8+ cancer-specific T cells (see, e.g., Derre et al., J Clin Invest 120:157-67, 2009). Common 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.

[0108] In some embodiments, the agent is an HVEM antagonist or inhibitor, for example, an antagonist or inhibitor that specifically binds to HVEM and interferes with its interaction with BTLA or CD160. Common examples of HVEM antagonists or inhibitors include antibodies, antigen-binding fragments, or small molecules that specifically bind to HVEM and, optionally, reduce the interaction between HVEM / BTLA and / or HVEM / CD160, thereby reducing one or more of the immunosuppressive activities of HVEM.

[0109] In some embodiments, the agent is a CD160 antagonist or inhibitor, for example, an antagonist or inhibitor that specifically binds to CD160 and interferes with its interaction with HVEM. Common examples of CD160 antagonists or inhibitors include antibodies, antigen-binding fragments, or small molecules that specifically bind to CD160 and selectively reduce the CD160 / HVEM interaction, thereby reducing or inhibiting one or more of its immunosuppressive activities.

[0110] In some embodiments, the drug is a TIGIT antagonist or inhibitor. T cell Ig and ITIM domain (TIGIT) are co-inhibitory receptors found on the surface of various lymphocytes, suppressing antitumor immunity, for example, via Tregs (Kurtulus et al., J Clin Invest. 125:4053-4062, 2015). Common examples of TIGIT antagonists or inhibitors include antibodies, antigen-binding fragments, or small molecules that specifically bind to TIGIT and reduce one or more of its immunosuppressive activities (see, for example, Johnston et al., Cancer Cell. 26:923-37, 2014).

[0111] In certain embodiments, an immune checkpoint modulator is an agonist of one or more stimulating immune checkpoint molecules. Exemplary stimulating immune checkpoint molecules include CD40, OX40, glucocorticoid-inducible TNFR family-related genes (GITR), CD137(4-1BB), CD27, CD28, CD226, and herpesvirus entry mediator (HVEM).

[0112] In some embodiments, the drug is a CD40 agonist. CD40 is expressed in antigen-presenting cells (APCs) and some malignancies. Its ligand is CD40L (CD154). In APCs, ligation results in upward regulation of costimulatory molecules, potentially bypassing the need for T cell support in the antitumor immune response. CD40 agonist therapy plays a crucial role in APC maturation and their migration from tumors to lymph nodes, resulting in high antigen presentation and T cell activation. Anti-CD40 agonist antibodies produce substantial responses and sustained anti-cancer immunity in animal models, at least partially mediated by cytotoxic T cells (see, e.g., Johnson et al. Clin Cancer Res. 21:1321-1328, 2015, and Vonderheide and Glennie, Clin Cancer Res. 19:1035-43, 2013). Common examples of CD40 agonists include antibodies or 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, dasetuzumab, Chi Lob7 / 4, ADC-1013, CD40L, rhCD40L, and their antigen-binding fragments. Specific examples of CD40 agonists include, but are not limited to, APX005 (see, e.g., US2012 / 0301488) and APX005M (see, e.g., US2014 / 0120103).

[0113] In some embodiments, the agent is an OX40 agonist. OX40 (CD134) promotes the proliferation of effector and memory T cells and suppresses the differentiation and activity of regulatory T cells (see, e.g., Croft et al., Immunol Rev. 229:173-91, 2009). Its ligand is OX40L (CD252). Because OX40 signaling affects both T cell activation and survival, it plays a crucial role in the initiation of antitumor immune responses in lymph nodes and the maintenance of antitumor immune responses in the tumor microenvironment. Common examples of OX40 agonists include antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to OX40 and increase one or more of its immunostimulatory activities. Specific examples include OX86, OX-40L, Fc-OX40L, GSK3174998, MEDI0562 (humanized OX40 agonist), MEDI6469 (mouse OX4 agonist), and MEDI6383 (OX40 agonist), as well as their antigen-binding fragments.

[0114] In some embodiments, the drug is a GITR agonist. Glucocorticoid-inducible TNFR family-related genes (GITRs) increase T cell proliferation, inhibit Treg suppressive activity, and prolong T effector cell survival. GITR agonists have been shown to promote antitumor responses through loss of Treg lineage stability (see, e.g., Schaer et al., Cancer Immunol Res. 1:320-31, 2013). These diverse mechanisms indicate that GITR plays a crucial role in initiating immune responses in lymph nodes and maintaining immune responses in tumor tissue. Its ligand is GITRL. Common examples of GITR agonists include antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to GITR and increase one or more of its immunostimulatory activities. Specific examples include GITRL, INCAGN01876, DTA-1, MEDI1873, and their antigen-binding fragments.

[0115] In some embodiments, the agent is a CD137 agonist. CD137(4-1BB) is a member of the tumor necrosis factor (TNF) receptor family, and crosslinking 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. Common 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, e.g., Shao and Schwarz, J Leukoc Biol. 89:21-9, 2011) and the antibody utomilumab, including its antigen-binding fragments.

[0116] In some embodiments, the agent is a CD27 agonist. Stimulation of CD27 increases antigen-specific proliferation of naive T cells, contributing to T cell memory and the long-term maintenance of T cell immunity. Its ligand is CD70. Targeting human CD27 with agonist antibodies stimulates T cell activation and anti-tumor immunity (see, e.g., Thomas et al., Oncoimmunology. 2014;3:e27255.doi:10.4161 / onci.27255, and He et al., J Immunol. 191:4174-83, 2013). Common 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, as well as the antibodies varlilumab and CDX-1127(1F5), and their antigen-binding fragments.

[0117] In some embodiments, the drug is a CD28 agonist. CD28 is constitutively expressed in CD4+ T cells and some CD8+ T cells. Its ligands include CD80 and CD86, and its stimulation increases T cell proliferation. Common 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 its immunostimulatory activities. Specific examples include CD80, CD86, the antibody TAB08, and its antigen-binding fragments.

[0118] In some embodiments, the drug is a CD226 agonist. CD226 is a stimulatory receptor that shares a ligand with TIGIT, and, contrary to TIGIT, the involvement of CD226 enhances T cell activation (see, e.g., Kurtulus et al., J Clin Invest. 125:4053-4062, 2015, Bottino et al., J Exp Med. 1984:557-567, 2003, and Tahara-Hanaoka et al., Int Immunol. 16, 533-538, 2004). Common 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.

[0119] In some embodiments, the drug 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 highly expressed on resting T cells and is downwardly regulated upon activation. HVEM signaling has been shown to play a crucial role in the early stages of T cell activation and during the proliferation of tumor-specific lymphocyte populations in lymph nodes. Common 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.

[0120] In certain embodiments, the immunotherapy agent is a bispecific or multispecific antibody. For example, a particular bispecific or multispecific antibody may (i) bind to one or more inhibitory immune checkpoint molecules to inhibit them, and (ii) bind to one or more stimulating immune checkpoint molecules to cause them to act. In certain embodiments, the bispecific or multispecific antibody may (i) bind to one or more of PD-L1, PD-L2, PD-1, CTLA-4, IDO, TDO, TIM-3, LAG-3, BTLA, CD160, and / or TIGIT to inhibit them, and (ii) bind to one or more of CD40, OX40 glucocorticoid-inducible TNFR family-related genes (GITR), CD137 (4-1BB), CD27, CD28, CD226, and / or herpesvirus entry mediators (HVEMs) to cause them to act.

[0121] In some embodiments, the immunotherapy agent is a cancer vaccine. In certain embodiments, the cancer vaccine is selected from one or more of the following: Oncophage, optionally Gardasil or Celvarix (human papillomavirus HPV vaccines), optionally Engelix-Hepatitis B vaccine, Recomvivax-Hepatitis HB vaccine, or Matrix, and SippleCel-T (Provenge). In some embodiments, the cancer vaccine contains or expresses TAA or TSA as described herein.

[0122] In some embodiments, the immunotherapy agent is an oncolytic virus. In some embodiments, the oncolytic virus was selected from one or more of the following: talimogene laherparepvec (T-VEC), coxsackievirus A21 (CAVATAK®), Oncorine (H101), pelareorep (REOLYSIN®), Seneca Valley virus (NTX-010), Senecavirus SVV-001, ColoAd1, SEPREHVIR (HSV-1716), CGTG-102 (Ad5 / 3-D24-GMCSF), GL-ONC1, MV-NIS, and DNX-2401.

[0123] In certain 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).

[0124] In certain embodiments, cancer immunotherapy agents are immune cell-based therapies, including cell-system immunotherapy, such as ex vivo-derived immune cells, including lymphocytes, natural killer (NK) cells, macrophages, and / or dendritic cells (DCs). In some embodiments, lymphocytes include T cells, such as cytotoxic T lymphocytes (CTLs). For example, see June, J Clin Invest. 117:1466-1476, 2007, Rosenberg and Restifo, Science. 348:62-68, 2015, Cooley et al., Biol. of Blood and Marrow Transplant. 13:33-42, 2007, and Li and Sun, Chin J Cancer Res. 30:173-196, 2018. In some embodiments, T cells include cancer antigen-specific T cells directed towards at least one cancer antigen. In some embodiments, 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 certain embodiments, CAR modified T cells are targeted toward CD-19 (see, e.g., Maude et al., Blood. 125:4017-4023, 2015). In some cases, ex vivo-derived immune cells are autologous cells obtained from the patient being treated.

[0125] Certain combination therapies involve the use of one or more chemotherapeutic agents, such as small molecule chemotherapeutic agents. Non-exclusive examples of chemotherapeutic agents include alkylating agents, antimetabolites, cytotoxic antibiotics, topoisomerase inhibitors (type I or type II), and antimicrotubule agents.

[0126] Examples of alkylating agents include nitrogen mustards (e.g., mechloretamine, cyclophosphamide, mustine, melphalan, chlorambucil, ifosfamide, and busulfan), nitrosoureas (e.g., N-nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine, and streptozotocin), tetrazines (e.g., dacarbazine, mitozolomide, and temozolomide), aziridines (e.g., thiotepa, mitomycin, diazicone (AZQ)), cisplatins and their derivatives (e.g., carboplatin and oxaliplatin), and non-classical alkylating agents (optionally, procarbazine and hexamethylmelamine).

[0127] Examples of antimetabolites include antifolic acid agents (e.g., methotrexate and pemetrexed), fluoropyrimidines (e.g., 5-fluorouracil and capecitabine), deoxynucleoside analogs (e.g., ancitabine, enokitabine, cytarabine, gemcitabine, decitabine, azacitidine, fludarabine, nerarabine, cladribine, clopharabine, fludarabine, and pentostatin), and thiopurines (e.g., thioguanine and mercaptopurine).

[0128] Examples of cytotoxic antibiotics include anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, acralubicin, and mitoxantrone), bleomycin, mitomycin C, mitoxantrone, and actinomycin. Examples of topoisomerase inhibitors include camptothecin, irinotecan, topotecan, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, melbaron, and acralubicin.

[0129] Examples of antimicrotubule agents include taxanes (e.g., paclitaxel and docetaxel) and vinca alkaloids (e.g., vinblastine, vincristine, vindesine, and vinorelbine).

[0130] The various chemotherapeutic agents described herein may be combined with one or more of the protein complexes described herein and may be used according to one or more of the methods or compositions described herein.

[0131] Certain combination therapies involve the use of at least one hormone therapy agent. Common examples of hormone therapy agents include hormone agonists and hormone antagonists. Specific examples of hormone agonists include progestins, corticosteroids (e.g., prednisolone, methylprednisolone, dexamethasone), insulin-like growth factor, VEGF-derived angiogenic and lymphangiogenic factors (e.g., VEGF-A, VEGF-A145, VEGF-A165, VEGF-C, VEGF-D, PIGF-2), fibroblast growth factor (FGF), galectin, hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF)-beta, androgens, estrogens, and somatostatin analogs. Examples of hormone antagonists include hormone synthesis inhibitors such as aromatase inhibitors, gonadotropin-releasing hormone (GnRH) agonists (e.g., leuprolide, goserelin, triptorelin, histrelin), and 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).

[0132] This also includes hormone pathway inhibitors such as antibodies directed at hormone receptors. Examples include IGF receptor inhibitors (e.g., IGF-IR1) such as cictumumab, darotuzumab, figtumumab, ganitumumab, istiratumumab, and lobatumumab; vascular endothelial growth factor receptor 1, 2, or 3 (VEGFR1, VEGFR2, or VEGFR3) inhibitors such as aracizumab pegol, bevacizumab, icluscumab, and ramucirumab; TGF-beta receptor R1, R2, and R3 inhibitors such as fresolimmumab and meterimumab; and naxi. Examples include c-Met inhibitors such as tamab, EGF receptor inhibitors such as cetuximab, depatuxizumab mafodotin, futuximab, imugatuzumab, laprituximab emtansine, matuzumab, modotuximab, nesitumumab, nimotuzumab, panitumumab, tomzotuximab, and zaltumumab, FGF receptor inhibitors such as aplutamab ixadotin and bemarituzumab, and PDGF receptor inhibitors such as olaratumab and tobetumab.

[0133] The various hormonal therapeutic agents described herein may be combined with one or more of the protein complexes described herein and may be used according to one or more of the methods or compositions described herein.

[0134] Certain combination therapies involve the use of at least one kinase inhibitor, including tyrosine kinase inhibitors. Examples of kinase inhibitors include, but are not limited to, adavocertib, afanitib, aflibercept, axitinib, bevacizumab, bosutinib, cabozantinib, cetuximab, cobimetinib, crizotinib, dasatinib, antrecutinib, erdafitinib, erlotinib, fostamitinib, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, mbritinib, nilotinib, panitumumab, pazopanib, pegaptanib, ponatinib, ranibisumab, regorafenib, ruxolitinib, sorafenib, sunitinib, SU6656, tofatinib, trastezumab, vandetanib, and bemafenib.

[0135] The various kinase inhibitors described herein may be combined with one or more of the protein complexes described herein and may be used according to one or more of the methods or compositions described herein.

[0136] In some embodiments, the methods and compositions described herein increase cancer cell killing in a subject by about or at least about 2x, 5x, 10x, 50x, 100x, 500x, or 1000x or more compared to a control or reference. In some embodiments, the methods and compositions described herein include cases where the immune response is an anti-cancer immune response, and increase the immune response in a subject by about or at least about 5x, 10x, 15x, 20x, 25x, 30x, 35x, 40x, 45x, 50x, 60x, 70x, 80x, 90x, 100x, 200x, 300x, 400x, 500x, 600x, 700x, 800x, 90x, 100x, 2000x or more compared to a control or reference (e.g., compared to the corresponding serine protease alone).

[0137] In some embodiments, the methods and compositions described herein increase the median survival time of subjects by 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 15 weeks, 20 weeks, 25 weeks, 30 weeks, or 40 weeks or more. In certain embodiments, the methods and compositions described herein increase the median survival time of subjects by 1 year, 2 years, or 3 years or more. In some embodiments, the methods and pharmaceutical compositions increase progression-free survival by 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks or more. In certain embodiments, the methods and pharmaceutical compositions described herein increase progression-free survival by 1 year, 2 years, or 3 years or more.

[0138] In certain embodiments, the methods and compositions described herein are sufficient to result in tumor regression, which is indicated, for example, by a statistically significant reduction in viable tumor volume, and the reduction is indicated, for example, by a reduction of at least 10%, 20%, 30%, 40%, 50% or more in tumor mass, or by a change in scan dimensions (e.g., a statistically significant reduction). In certain embodiments, the methods and compositions described herein are sufficient to result in stabilization. In certain embodiments, the methods and compositions described herein are sufficient to result in a clinically relevant reduction in the symptoms of certain disease indications known to clinicians skilled in the art.

[0139] As described above, for in vivo use for the treatment or testing of diseases in humans or non-human mammals, the protein complexes described herein are generally incorporated into one or more therapeutic or pharmaceutical compositions, including a veterinary therapeutic composition, prior to administration.

[0140] Accordingly, certain embodiments relate to pharmaceutical or therapeutic compositions comprising protein complexes described herein. In some cases, the pharmaceutical or therapeutic composition comprises one or more of the protein complexes described herein, combined with a pharmaceutically or physiologically acceptable carrier or excipient. Certain pharmaceutical or therapeutic compositions further comprise at least one additional agent described herein, such as an immunotherapeutic agent, a chemotherapeutic agent, a hormonal therapy agent, and / or a kinase inhibitor.

[0141] In certain embodiments, the pharmaceutical or therapeutic composition comprising the protein complex is substantially pure on a protein-based or weight-based basis, for example, the composition having a purity of at least about 80%, 85%, 90%, 95%, 98%, or 99% on a protein-based or weight-based basis.

[0142] In some embodiments, the protein complexes described herein have the desired solubility and / or immunogenicity profile that is suitable for use in humans, as is known in the art, and does not form aggregates. Accordingly, in some embodiments, the pharmaceutical or therapeutic composition comprises a protein complex that is substantially aggregate-free. For example, certain compositions contain less than about 10% (protein-based) of high molecular weight aggregated protein, or less than about 5% of high molecular weight aggregated protein, or less than about 4% of high molecular weight aggregated protein, or less than about 3% of high molecular weight aggregated protein, or less than about 2% of high molecular weight aggregated protein, or less than 1% of high molecular weight aggregated protein.

[0143] 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 use.

[0144] To prepare therapeutic or pharmaceutical compositions, one or more effective or desired amounts of protein complexes are mixed with any pharmaceutical carrier or excipient known to those skilled in the art to be suitable for a particular drug and / or mode of administration. The pharmaceutical carrier may be liquid, semi-liquid, or solid. Solutions or suspensions used 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, glycerin, propylene glycol, or other synthetic solvents, antimicrobial agents (e.g., benzyl alcohol, methylparaben), antioxidants (e.g., ascorbic acid and sodium bisulfite), and chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)), and buffers (e.g., acetates, citrates, and phosphates). For intravenous administration (e.g., IV infusion), suitable carriers include saline or phosphate-buffered saline (PBS), as well as solutions containing thickeners and solubilizers such as glucose, polyethylene glycol, polypropylene glycol, and mixtures thereof.

[0145] Administration of the protein complexes described herein, either in pure form or in appropriate therapeutic or pharmaceutical compositions, may be carried out via any acceptable mode of administration of the agent for delivering similar utility. Therapeutic or pharmaceutical compositions may be prepared by combining the protein complexes with appropriate physiologically acceptable carriers, diluents, or excipients, and may be formulated into preparations in solid, semi-solid, liquid, or gaseous forms such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microparticles, and aerosols. In addition, other pharmaceutically active ingredients (including other low molecular weights as described elsewhere herein), as well as / or suitable excipients such as salts, buffers, and stabilizers, may be present in the compositions, although these are not essential.

[0146] Administration can be achieved by various routes, including oral, parenteral, nasal, intravenous, intradermal, intramuscular, subcutaneous, or topical. The preferred mode of administration depends on the nature of the condition being treated or prevented. Specific embodiments include administration by IV infusion.

[0147] The carrier may include, for example, a pharmaceutically or physiologically acceptable carrier, excipient, or stabilizer that is nontoxic to the cells or mammals to which it is exposed at the dose and concentration used. In many cases, the physiologically acceptable carrier is an aqueous pH buffer solution. Examples of physiologically acceptable carriers include buffers such as phosphates, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; 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, or other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as polysorbate 20 (TWEEN®), polyethylene glycol (PEG), and polyoxamers (PLURONICS®).

[0148] In some embodiments, one or more agents may be encapsulated in microcapsules (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmetassylate) microcapsules, respectively) prepared by coacervation techniques or interfacial polymerization, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed., (1980). The particles or liposomes may further comprise other therapeutic or diagnostic agents.

[0149] The precise dose and duration of treatment are functional to the disease being treated and can be determined empirically using known test protocols or by testing the composition in a model system known in the art and extrapolating therefrom. Controlled clinical trials may also be conducted. The dose may also vary depending on the severity of the condition being alleviated. Pharmaceutical compositions are generally formulated and administered to produce therapeutically beneficial effects while minimizing undesirable side effects. The composition may be administered as a single dose or divided into several smaller doses administered at time intervals. For any particular subject, a specific dosing regimen may be adjusted over time according to individual needs.

[0150] Accordingly, typical routes for administering these and related therapeutic or pharmaceutical compositions include, without limitation, oral, topical, transdermal, inhalation, parenteral, sublingual, oral cavity, rectal, vaginal, and nasal. As used herein, the term parenteral includes subcutaneous, intravenous, intramuscular, intrasternal injection, or infusion techniques. The therapeutic or pharmaceutical compositions according to specific embodiments of this disclosure are formulated such that the active ingredients contained therein become bioavailable at the time of administration of the composition to a subject or patient. The composition to be administered to a subject or patient may take the form of one or more dosage units; for example, a tablet may be a single dosage unit, and a container of a drug described herein in aerosol form may hold multiple dosage units. Practical methods for preparing such dosage forms are known or obvious to those skilled in the art; see, for example, Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000). The administered composition will typically contain a therapeutically effective amount of the agent described herein for the treatment of the disease or condition of interest.

[0151] Therapeutic or pharmaceutical compositions may be in solid or liquid form. In one embodiment, the carrier is fine particles, and as a result, the composition is, for example, in tablet or powder form. The carrier may also be liquid, and the composition is, for example, an oral oil, an injectable liquid, or an aerosol, which is useful, for example, for inhalation administration. When intended for oral administration, the pharmaceutical composition is preferably either in solid or liquid form, and semi-solid, semi-liquid, suspension, and gel forms are included within the forms considered as either solid or liquid form herein. Certain embodiments include sterile injectable solutions.

[0152] As a solid composition for oral administration, the pharmaceutical composition may be formulated in the form of a powder, granules, compressed tablets, pills, capsules, chewing gum, wafers, etc. Such solid compositions typically contain one or more inert diluents or food carriers. In addition, one or more of the following may be present: binders such as carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch, lactose, or dextrin; disintegrants such as alginic acid, sodium alginate, Primogel, or corn starch; lubricants such as magnesium stearate or Sterotex; lubricants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; flavorings such as peppermint, methyl salicylate, or orange flavoring; and colorants. When the pharmaceutical composition is in the form of a capsule, for example, a gelatin capsule, it may contain a liquid carrier such as polyethylene glycol or oil in addition to the above types of materials.

[0153] Therapeutic or pharmaceutical compositions may be in the form of liquids, such as elixirs, syrups, solutions, emulsions, or suspensions. Liquids may, as two examples, be intended for oral administration or delivery by injection. When intended for oral administration, preferred compositions contain, in addition to the compound, one or more of the following: sweeteners, preservatives, dyes / colorants, and flavorings. Compositions intended for administration by injection may contain one or more of the following: surfactants, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, and isotonic agents.

[0154] Liquid therapeutic or pharmaceutical compositions, whether in solution, suspension or other similar forms, may contain one or more of the following adjuvants: water for injection, saline, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixing oils such as synthetic mono or diglycerides that can function as a solvent or suspension medium, sterile diluents such as polyethylene glycol, glycerin, propylene glycol, or other solvents, antimicrobial 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 isotonic agents such as sodium chloride or glucose. Parenteral preparations may be sealed in ampoules made of glass or plastic, disposable syringes, or multi-dose vials. Physiological saline is a preferred adjuvant. Injectable pharmaceutical compositions are preferably sterile.

[0155] Liquid therapeutic or pharmaceutical compositions intended for either parenteral or oral administration should contain a certain amount of the drug so that a suitable dose is obtained. Typically, this amount is at least 0.01% of the drug of interest in the composition. When intended for oral administration, this amount can vary to 0.1% to about 70% of the weight of the composition. Certain oral therapeutic or pharmaceutical compositions contain about 4% to about 75% of the drug of interest. In certain embodiments, therapeutic or pharmaceutical compositions and preparations are prepared so that parenteral dose units contain 0.01% to 10% by weight of the drug of interest before dilution.

[0156] Therapeutic or pharmaceutical compositions may be intended for topical administration, in which case the carrier may preferably comprise a solution, emulsion, ointment, or gel base. The base may comprise one or more of the following: diluents such as petrolatum, lanolin, polyethylene glycol, beeswax, mineral oil, water, and alcohol, as well as emulsifiers and stabilizers. Thickeners may be present in the therapeutic or pharmaceutical composition for topical administration. When intended for transdermal administration, the composition may comprise a transdermal patch or an iontophoresis device.

[0157] Therapeutic or pharmaceutical compositions may be intended for rectal administration, for example, in the form of suppositories that dissolve in the rectum and release the drug. Compositions for rectal administration may contain oily bases as suitable non-irritating excipients. Such bases include, without limitation, lanolin, cocoa butter, and polyethylene glycol.

[0158] Therapeutic or pharmaceutical compositions may contain various materials that modify the physical form of solid or liquid dose units. For example, a composition may contain a material that forms a coating shell around an active ingredient. The material forming the coating shell is typically inert and can 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 contain components that bind to a drug and thereby assist in the delivery of the compound. Suitable components that can act with this ability include monoclonal or polyclonal antibodies, one or more proteins, or liposomes.

[0159] Therapeutic or pharmaceutical compositions may essentially consist of dosing units that can be administered as aerosols. The term aerosol is used to describe a variety of systems, ranging from colloidal in nature to systems consisting of pressurized packages. Delivery may be by liquefaction or compressed gas, or by a suitable pump system for dispensing the active ingredient. Aerosols may be delivered in single-phase, two-phase, or three-phase systems to deliver the active ingredient. Aerosol delivery may involve necessary containers, activators, valves, sub-containers, etc., which together may form a kit. Without excessive experimentation, those skilled in the art can determine a preferred aerosol.

[0160] The compositions described herein may be prepared using carriers that protect the drug from rapid removal from the body, such as time-release formulations or coatings. Such carriers include, but are not limited to, controlled-release formulations such as implants and microcapsule delivery systems, as well as biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, polyorthoester, polylactic acid, and others known to those skilled in the art.

[0161] Therapeutic or pharmaceutical compositions may be prepared by methodologies well known in the pharmaceutical art. For example, a therapeutic or pharmaceutical composition intended to be administered by injection may contain one or more salts, buffers, and / or stabilizers, with sterile distilled water to form a solution. Surfactants may be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that interact non-covalently with a drug to facilitate its dissolution or homogeneous suspension in an aqueous delivery system.

[0162] Therapeutic or pharmaceutical compositions may be administered in therapeutically effective doses and will vary depending on a variety of factors, including the activity of the specific compound used, the metabolic stability and duration of the compound's action, age, weight, overall health, sex, and the subject's diet, mode and timing of administration, excretion rate, drug combinations, the severity of a particular disorder or condition, and the subject receiving treatment. In some cases, the therapeutically effective daily dose is about 0.001 mg / kg (i.e., about 0.07 mg) to about 100 mg / kg (i.e., about 7.0 g) (for a 70 kg mammal), preferably about 0.01 mg / kg (i.e., about 0.7 mg) to about 50 mg / kg (i.e., about 3.5 g) (for a 70 kg mammal), and more preferably about 1 mg / kg (i.e., about 70 mg) to about 25 mg / kg (i.e., about 1.75 g) (for a 70 kg mammal). In some embodiments, the therapeutically effective dose is administered weekly, bi-weekly, or monthly. In certain embodiments, the therapeutically effective dose is administered weekly, bi-weekly, or monthly, for example, at doses of approximately 1 to 10 or 1 to 5 mg / kg, or approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg.

[0163] The combination therapies described herein may include the administration of a single pharmaceutical dosage form containing a protein complex and an additional therapeutic agent (e.g., an immunotherapy agent, a chemotherapeutic agent, a hormone therapy agent, a kinase inhibitor), as well as the administration of a composition containing the protein complex and the additional therapeutic agent in a separate pharmaceutical dosage form of its own. For example, the protein complex and the additional therapeutic agent may be administered together to a subject in a single parenteral dose composition, such as a saline solution or other physiologically acceptable solution, or each agent may be administered in a separate parenteral dosage form. When separate dosage forms are used, the compositions may be administered essentially simultaneously, i.e., at the same time, or separately at time-staggered intervals, i.e., sequentially and in any order, and the combination therapy is understood to include all of these regimens.

[0164] Also included are patient care kits comprising (a) the protein complex described herein, and optionally, (b) at least one additional therapeutic agent (e.g., an immunotherapy agent, a chemotherapeutic agent, a hormone therapy agent, a kinase inhibitor). In certain kits, (a) and (b) are in separate therapeutic compositions. In some kits, (a) and (b) are in the same therapeutic composition.

[0165] The kits described herein may also include one or more additional therapeutic agents or other components that are suitable or desired for the indication being treated or for the desired diagnostic use. The kits described herein may also include one or more syringes or other components that are necessary or desired to facilitate the intended mode of delivery (e.g., stent, implantable depot).

[0166] In some embodiments, the patient care kit includes separate containers, dividers, or compartments for the composition and informational materials. For example, the composition may be contained in a bottle, vial, or syringe, and the informational materials may be contained in association with the container. In some embodiments, the separate elements of the kit are contained in a single, unseparated container. For example, the composition is contained in a bottle, vial, or syringe, to which the informational materials are attached in the form of labels. In some embodiments, the kit comprises a plurality of individual containers (e.g., packs), 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 comprises a plurality of syringes, ampoules, foil packets, or blister packs, each containing a single unit dose of the protein complex and optionally, at least one additional therapeutic agent. The containers of the kit may be airtight, waterproof (e.g., impermeable to changes or evaporation of moisture), and / or lightfast.

[0167] The patient care kit optionally includes a device suitable for administering the composition, such as a syringe, inhalant, infusion device (e.g., eye dropper), swab (e.g., cotton swab or wooden swab), or any such delivery device. In some embodiments, the device is a portable device for dispensing a quantitative dose of the drug. Methods of providing the kit also include, for example, combining the components described herein.

[0168] Manufacturing and refining systems Certain embodiments include methods and related compositions for producing, expressing, and purifying protein complexes or protein components described herein. For example, a particular embodiment relates to a method for producing or preparing a pharmaceutical composition comprising a protein complex, the method comprising mixing (a) α2-macroglobulin (A2M) protein and (b) serine protease protein in a composition in a molar ratio of about 1:3 to about 1:1 [(a):(b)]. Certain embodiments involve combining (a) and (b) in a composition in a molar ratio 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)].

[0169] A2M and / or serine protease proteins can be prepared or obtained by purification from biological samples or by recombinant technology. For example, in some embodiments, A2M and / or serine protease proteins are obtained by purifying proteins from the blood or plasma of a mammalian subject, e.g., a human subject. Certain embodiments involve obtaining and purifying the A2M protein from the plasma of a human subject before combining it with a serine protease protein. Methods for purifying A2M from human plasma (plasma-enriched 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).

[0170] Recombinant proteins can be prepared using standard protocols such as those described in Sambrook, et al., (1989, above), particularly sections 16 and 17; Ausubel et al., (1994, above), particularly chapters 10 and 16; and Coligan et al., Current Protocols in Protein Science (John Wiley & Sons, Inc. 1995–1997), particularly 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 containing a polynucleotide sequence encoding a protein described herein, which is operably linked to one or more regulatory elements; (b) introducing the vector or construct into host cells; (c) culturing the host cells to express the protein; and (d) isolating the protein from the host cells.

[0171] To express a desired polypeptide, the protein-coding nucleotide sequence can be inserted into a suitable expression vector, i.e., a vector containing the elements necessary for the transcription and translation of the inserted coding sequence. Methods well known to those skilled in the art can be used to construct an expression vector containing the polypeptide of interest and the sequences encoding the appropriate transcription and translation regulatory elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in Sambrook et al., Molecular Cloning, A Laboratory Manual (1989), and Ausubel et al., Current Protocols in Molecular Biology (1989).

[0172] Various expression vectors / 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 bacteriophages, plasmids, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus); 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, more specifically human cell systems.

[0173] The “regulatory elements” or “regulatory sequences” present in an expression vector are their untranslated regions—enhancers, promoters, and 5' and 3' untranslated regions—that interact with host cell proteins to carry out transcription and translation. Such elements can vary in their intensity and specificity. Depending on the vector system and host used, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. For example, when cloning in a bacterial system, inducible promoters such as the hybrid lacZ promoter of the PBLUESCRIPT phagemide (Stratagene, La Jolla, Calif.) or PSPORT1 plasmid (Gibco BRL, Gaithersburg, Md.) may be used. In mammalian cell systems, promoters from mammalian genes or mammalian viruses are generally preferred. When it is necessary to generate cell lines containing multiple copies of the polypeptide-encoding sequence, SV40 or EBV-based vectors may be advantageously used with appropriate selectable markers.

[0174] In bacterial systems, several expression vectors may be selected depending on the intended use of the expressed polypeptide. For example, when large quantities are needed, vectors that direct high levels of easily purified protein expression may be used. Such vectors, though not limited to them, include polyfunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene), such as the pIN vector (Van Heeke & Schuster, J. Biol. Chem. 264:5503 5509 (1989)), in which the sequence encoding the polypeptide of interest can be ligated into the vector in a frame with an amino-terminal Met sequence and seven subsequent residues of β-galactosidase so that a hybrid protein is produced. The pGEX vector (Promega, Madison, Wis.) can also be used to express recombinant proteins as fusion proteins using glutathione S-transferase (GST). Generally, such fusion proteins are soluble and can be readily purified from lysed cells by adsorption to glutathione-agarose beads, followed by elution in the presence of free glutathione. Proteins produced in such systems may be designed to include heparin, thrombin, or factor XA protease cleavage sites so that the cloned polypeptide of interest can be freely released from the GST moiety.

[0175] Certain embodiments utilize an E. coli-based expression system (see, e.g., Structural Genomics Consortium et al., Nature Methods. 5:135-146, 2008). These and related embodiments may rely on partially or entirely ligation-independent cloning (LIC) to generate suitable expression vectors. In certain embodiments, protein expression may be controlled by T7 RNA polymerase (e.g., the pET vector series). These and related embodiments may utilize the expression host strain BL21(DE3), a λDE3 lysogen of BL21 that supports T7-mediated expression and lacks lon and ompT proteases for improved target protein stability. Also included are expression host strains carrying plasmids encoding tRNAs rarely used in E. coli, such as the ROSETTA®(DE3) strain and the Rosetta 2(DE3) strain. Cell lysis and sample processing can also be improved using reagents marketed under the trademarks BENZONASE® nuclease and BUGBUSTER® Protein Extraction Reagent. For cell culture, automated induction media can improve the efficiency of many expression systems, including high-throughput expression systems. This type of medium (e.g., OVERNIGHT EXPRESS® Autoinduction System) gradually induces protein expression via metabolic shifts without the addition of artificial inducers such as IPTG. Certain embodiments utilize hexahistidine tags (e.g., those marketed under the trademark HIS·TAG® fusion), followed by immobilized metal affinity chromatography (IMAC) purification, or related techniques. However, in certain embodiments, clinical-grade proteins can be isolated from E. coli-containing organisms without or without the use of affinity tags (see, e.g., Shimp et al., Protein Expr Purif. 50:58-67, 2006).As a further example, certain embodiments may employ a cold shock-induced high-yield production system for Escherichia coli, where protein overexpression in Escherichia coli at low temperatures improves their solubility and stability (see, for example, Qing et al., Nature Biotechnology. 22:877-882, 2004).

[0176] This also includes high-density bacterial fermentation systems. For example, culturing Ralstonia eutropha at high cell density enables protein production at cell densities exceeding 150 g / L and recombinant protein expression at titers exceeding 10 g / L.

[0177] In the yeast *Saccharomyces cerevisiae*, several vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH may be used. For reviews, see Ausubel et al. (above) and Grant et al., *Methods Enzymol.* 153:516-544 (1987). This also includes the Pichia pandoris expression system (see, for example, Li et al., *Nature Biotechnology.* 24, 210-215, 2006, and Hamilton et al., *Science* 301:1244, 2003). Specific embodiments include, among other things, yeast systems manipulated to selectively glycosylate proteins, including yeast having a humanized N-glycosylation pathway (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). Simply as an example, recombinant yeast cultures can be grown in Fernbach Flasks or 15 L, 50 L, 100 L, and 200 L fermentation apparatuses, among other things.

[0178] When plant expression vectors are used, the expression of the polypeptide-encoding sequence can be driven by one of several promoters. For example, viral promoters such as the 35S and 19S promoters of CaMV can be used alone or in combination with the omega-leader sequence from TMV (Takamatsu, EMBO J.6:307-311 (1987)). Alternatively, plant promoters or heat shock promoters such as the small subunit of RUBISCO can be used (Coruzzi et al., EMBO J.3:1671-1680 (1984), Broglie et al., Science 224:838-843 (1984), and Winter et al., Results Probl. Cell Differ. 17:85-105 (1991)). These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. Such techniques are described in several publicly available reviews (see, for example, Hobbs in McGraw Hill, Yearbook of Science and Technology, pp. 191–196 (1992)).

[0179] Insect systems can also be used to express polypeptides of interest. For example, in one such system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector for expressing exogenous genes in Spodoptera frugiperda or Trichoplusia cells. The polypeptide-coding sequence can be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under the control of the polyhedrin promoter. Successful insertion of the polypeptide-coding sequence inactivates the polyhedrin gene and produces a recombinant virus lacking the coat protein. The recombinant virus can then be used to infect S. frugiperda or Trichoplusia cells, for example, in which the polypeptide of interest can be expressed (Engelhard et al., Proc. Natl. Acad. Sci. USA 91:3224-3227 (1994)). This also includes baculovirus expression systems, including those utilizing SF9, SF21, and T.ni cells (see, for example, Murphy and Piwnica-Worms, Curr Protoc Protein Sci. Chapter 5: Unit 5.4, 2001). Insect systems may offer post-translational modifications similar to those found in mammalian systems.

[0180] Several virus-based expression systems are commonly available in mammalian host cells. For example, when adenovirus is used as an expression vector, the sequence encoding the polypeptide of interest can be ligated to an adenovirus transcription / translation complex consisting of a late promoter and a three-part reader sequence. Insertions into non-essential E1 or E3 regions of the viral genome can be used to obtain viable viruses capable of expressing polypeptides in infected host cells (Logan & Shenk, Proc. Natl. Acad. Sci. USA 81:3655-3659 (1984)). In addition, transcriptional enhancers, such as Roussarcoma virus (RSV) enhancers, can be used to increase expression in mammalian host cells.

[0181] Examples of useful mammalian host cell lines include the CV1 monkey kidney cell line transformed with SV40 (COS-7, ATCC CRL 1651), human embryonic kidney cell line (293 or 293 cells sub-cloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)), baby hamster kidney cells (BHK, ATCC CCL 10), mouse Sertoli 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 cancer cells (HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), and buffalo rat liver cells (BRL 3A, ATCC CRL Examples of useful mammalian host cell lines include human lung cells (W138, ATCC CCL 75), human liver cells (Hep G2, HB 8065), mouse mammary tumor cells (MMT 060562, ATCC CCL51), TR1 cells (Mather et al., Annals NYAcad.Sci.383:44-68(1982)), MRC 5 cells, FS4 cells, and human hepatocellular carcinoma cell lines (Hep G2). Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., PNAS USA 77:4216(1980)), as well as myeloma cell lines such as NSO and Sp2 / 0. For a review of specific mammalian host cell lines suitable for protein production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 255-268. Specific preferred mammalian cell expression systems include CHO and HEK293 cell-based expression systems.Mammalian expression systems can utilize, among others known in the art, for example, T-flasks, roller bottles, or attached cell lines in cell factories, or suspension cultures in, for example, 1 L and 5 L spinners, 5 L, 14 L, 40 L, 100 L and 200 L stirred tank bioreactors, or 20 / 50 L and 100 / 200 L WAVE bioreactors.

[0182] This also includes cell-free expression of proteins. These and related embodiments typically utilize purified RNA polymerase, ribosomes, tRNA, and ribonucleotides, which can be generated from cells or by extraction from cell-based expression systems.

[0183] Specific start signals can also be used to achieve more efficient translation of the sequence encoding the polypeptide of interest. Such signals include the ATG start codon and its adjacent sequences. If the polypeptide, its start codon, and the sequence encoding the upstream sequence are inserted into a suitable expression vector, additional transcriptional or translational regulatory signals may not be required. However, if only the coding sequence, or a portion thereof, is inserted, an exogenous translational regulatory signal, including the ATG start codon, should be provided. Furthermore, the start codon should be within the correct reading frame to ensure translation of the entire insertion. Exogenous translational elements and start codons can be of various origins, both natural and synthetic. Expression efficiency can be enhanced by the inclusion of enhancers appropriate for the specific cell line being used, such as those described in the literature (Scharf et al., Results Probl. Cell Differ. 20:125-162 (1994)).

[0184] In addition, host cell lines may be selected for their ability to regulate the expression of the inserted sequence or to process the expressed protein in a desired manner. Such modifications of polypeptides include, but are not limited to, post-translational modifications such as acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing that cleaves the "prepro" form of the protein may also be used to facilitate correct insertion, folding, and / or function. In addition to bacterial cells, different host cells such as yeast, CHO, HeLa, MDCK, HEK293, and W138, which may or may not have specific cellular and characteristic mechanisms for such translational activity, may be selected to ensure the correct modification and processing of foreign proteins.

[0185] For long-term, high-yield recombinant protein production, stable expression is generally preferred. For example, cell lines that stably express a polynucleotide of interest can be transformed using expression vectors that may contain a viral replication origin and / or endogenous expression elements, as well as a selectable marker gene, on the same or separate vector. After vector introduction, cells may be allowed to grow in concentrated medium for about 1-2 days before being switched to selective medium. The purpose of the selectable marker is to confer resistance to selection, and its presence allows for the growth and harvesting of cells that successfully express the introduced sequence. Resistant clones of stably transformed cells can be grown using tissue culture techniques appropriate to the cell type. Transient production, such as transient transfection or infection, can also be used. Exemplary mammalian expression systems suitable for transient production include HEK293 and CHO-based systems.

[0186] Any number of selection systems can be used to recover transformed or transduced cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223-232 (1977)) and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817-823 (1990)) genes, which can be recruited into tk- or aprt- cells, respectively. Furthermore, resistance to antimetabolites, antibiotics, or herbicides can be used as a basis for selection; for example, dhfr (Wigler et al., Proc. Natl. Acad. Sci. USA 77:3567-70 (1980)) conferring resistance to methotrexate, npt (Colbere-Garapin et al., J. Mol. Biol. 150:1-14 (1981)) conferring resistance to aminoglycosides, neomycin, and G-418, and als or pat (Murry, above) conferring resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively. Additional selectable genes, such as trpB, which allows cells to utilize indole instead of tryptophan, or hisD, which allows cells to utilize histidine instead of histinol, have been described (Hartman & Mulligan, Proc. Natl. Acad. Sci. USA 85:8047-51 (1988)). The use of visible markers, such as green fluorescent protein (GFP) and other fluorescent proteins (e.g., RFP, YFP), anthocyanins, β-glucuronidase and its substrate GUS, and luciferase and its substrate luciferin, is widely used not only to identify transformants but also to quantify the amount of transient or stable protein expression resulting from specific vector systems (e.g., Rhodes et al., Methods Mol. Biol. 55:121-131 (1995)).

[0187] This also includes high-throughput protein production systems or micro-production systems. Specific embodiments may utilize, for example, hexahistidine fusion tags for protein expression and purification on metal-chelated sliding surfaces or MagneHis Ni-Particles (see, e.g., Kwon et al., BMC Biotechnol. 9:72, 2009, and Lin et al., Methods Mol Biol. 498:129-41, 2009). High-throughput cell-free protein expression systems are also included (see, e.g., Sitaraman et al., Methods Mol Biol. 498:229-44, 2009).

[0188] Various protocols for detecting and measuring the expression of polynucleotide-encoded products using product-specific binders or antibodies such as polyclonal or monoclonal antibodies are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), Western immunoblotting, radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS). These and other assays are described, among others, in Hampton et al., Serological Methods, a Laboratory Manual (1990), and Maddox et al., J. Exp. Med. 158:1211-1216 (1983).

[0189] A wide range 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. Means for generating labeled hybridization or PCR probes for detecting sequences associated with polynucleotides include oligo-labeling, nick translation, end-labeling, or PCR amplification using labeled nucleotides. Alternatively, a sequence, or any part thereof, can be cloned into a vector for the production of mRNA probes. Such vectors are known and commercially available in the art and can be used to synthesize RNA probes in vitro by adding a suitable RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures can be carried out using various commercially available kits. Suitable reporter molecules or labels that can be used include radionuclides, enzymes, fluorescent agents, chemiluminescent agents, or colorants, and substrates, cofactors, inhibitors, magnetic particles, etc.

[0190] 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. Certain embodiments utilize serum-free cell expression systems. Examples include HEK293 cells and CHO cells that can be grown in serum-free medium (see, for example, Rosser et al., Protein Expr. Purif. 40:237-43, 2005, and U.S. Patent No. 6,210,922).

[0191] Proteins produced by recombinant cells may be secreted or contained within the cell, depending on the sequence and / or vector used. As will be understood by those skilled in the art, polynucleotide-containing expression vectors may be designed to contain a signal sequence that directs the secretion of the encoded polypeptide across the prokaryotic or eukaryotic cell membrane. Other recombinant constructs may be used to conjugate a sequence encoding a polypeptide of interest to a nucleotide sequence encoding a polypeptide domain, which will facilitate the purification and / or detection of the soluble protein. Examples of such domains include cleavable and non-cleavable affinity purifiers, as well as epitope tags such as avidin, FLAG tags, polyhistidine tags (e.g., 6xHis), cMyc tags, V5- tags, glutathione S-transferase (GST) tags, and others.

[0192] Proteins produced by recombinant cells can be purified and characterized according to various techniques known in the art. Exemplary systems for performing protein purification and analyzing protein purity include high-performance 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 chemicals for purification, among those known in the art, 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). Also included are analytical methods such as SDS-PAGE (e.g., Coomassie, silver staining), immunoblotting, Bradford, and ELISA, which can typically be used during any step of the production or purification process to measure the purity of the protein composition.

[0193] Also included herein are methods for concentrating the proteins or protein complexes described herein, and compositions comprising the concentrated soluble proteins or protein complexes. In some embodiments, 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 higher.

[0194] In some embodiments, such compositions may be substantially monodisperse, meaning that the protein or protein complex is predominantly (i.e., at least about 90%) in a single apparent molecular weight form when evaluated, for example, by size exclusion chromatography, dynamic light scattering, or analytical ultracentrifugation.

[0195] In some embodiments, such compositions have a purity of at least about 90%, or in some embodiments, at least about 95%, or in some embodiments, at least 98%. Purity can be determined by any conventional analytical method known in the art.

[0196] In some embodiments, such compositions have a high molecular weight aggregate content of less than about 10% relative 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 embodiments, such compositions have a high molecular weight aggregate content of less than about 3%, or in some embodiments, such compositions have a high molecular weight aggregate content of less than about 1%. The high molecular weight aggregate content can be determined by various analytical techniques, including, for example, size exclusion chromatography, dynamic light scattering, or analytical ultracentrifugation.

[0197] An example of the concentration approach intended herein is lyophilization, which is typically used when the solution contains little to no soluble components other than the protein of interest. Lyophilization is often performed after HPLC and can remove most or all volatile components from the mixture. Ultrafiltration techniques are also included, which typically use one or more selectively permeable membranes to concentrate the protein solution. The membranes allow water and low molecular weights to pass through while retaining the protein, and the solution can be pressed against the membrane by mechanical pumps, gas pressure, or centrifugation, among other techniques.

[0198] In certain embodiments, the protein or protein complex in the composition has a purity of at least about 90% when measured according to the conventional art of the art. In certain embodiments, such as diagnostic compositions or certain pharmaceutical or therapeutic compositions, the protein or protein complex in the composition has a purity of at least about 95%, or at least about 97%, 98%, or 99%. In some embodiments, such as when used as a reference or research reagent, the protein or protein complex may be of lower purity, having a purity of at least about 50%, 60%, 70%, or 80%. Purity may be measured as a whole or against selected components such as other proteins, and may be, for example, protein-based purity.

[0199] Purified proteins or protein complexes can also be characterized according to their biological characteristics. Binding affinity and binding kinetics can be measured according to various techniques known in the art, such as Biacore® and related technologies, which utilize surface plasmon resonance (SPR), an optical phenomenon that enables real-time detection of unlabeled interacting substances. SPR-based biosensors can be used to determine activity concentrations, screening, and characterization in terms of both affinity and kinetics. The presence or level of one or more biological activities can be measured according to in vitro or cell-based assays as described herein, and they are functionally and optionally bound to readouts or indicators, such as fluorescence or luminescence indicators of biological activity.

[0200] In certain embodiments, as described above, the composition is substantially endotoxin-free, for example, containing about 95% endotoxin-free, preferably about 99% endotoxin-free, and more preferably about 99.99% endotoxin-free. The presence of endotoxins can be detected according to the conventional techniques of the art described herein. In certain embodiments, the protein or protein complex is prepared from eukaryotic cells, such as mammalian or human cells, in substantially serum-free medium. In certain embodiments, as described herein, the composition has an endotoxin content of less than about 10 EU / mg of protein, or less than about 5 EU / mg of protein, less than about 3 EU / mg of protein, or less than about 1 EU / mg of protein.

[0201] In certain embodiments, the composition comprises high molecular weight aggregates of less than about 10% by weight, or less than about 5% by weight, or less than about 2% by weight, or less than about 1% by weight.

[0202] For example, this includes protein-based analytical assays and methods that can be used to evaluate, among other properties, protein purity, size, solubility, and degree of aggregation. Protein purity can be assessed in several 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- and C-terminal sequencing, as well as peptide mapping (see, e.g., Allen et al., Biologicals. 24:255-275, 1996). Examples of methods for evaluating higher-order structure include circular dichroism (see, e.g., Kelly et al., Biochim Biophys Acta. 1751:119-139, 2005), fluorescence spectroscopy (see, e.g., Meagher et al., J. Biol. Chem. 273:23283-89, 1998), FT-IR, amide hydrogen-deuterium exchange dynamics, differential scanning calorimetry, NMR spectroscopy, and immunoassay using structure-sensitive antibodies. Higher-order structure can also be evaluated as a function of various parameters such as pH, temperature, or added salts. Examples of methods for evaluating protein properties such as size include analytical ultracentrifugation and size exclusion HPLC (SEC-HPLC), and exemplary methods for measuring charge include ion exchange chromatography and isoelectric focusing. Hydrophobicity can be evaluated, for example, by reversed-phase HPLC and hydrophobic interaction chromatography-HPLC. Glycosylation can affect pharmacokinetics (e.g., clearance), conformation or stability, receptor binding, and protein function, and can be evaluated, for example, by mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy.

[0203] As described above, certain embodiments include the use of SEC-HPLC for evaluating protein properties such as purity, size (e.g., size homogeneity), or degree of aggregation, and / or for purifying proteins, among other applications. SEC, also including gel filtration chromatography (GFC) and gel permeation chromatography (GPC), refers to a chromatographic method in which molecules in solution are separated by a porous material based on their size, or more specifically, on their hydrodynamic volume, diffusion coefficient, and / or surface properties. The process is commonly used to separate biomolecules and determine the molecular weight and molecular weight distribution of polymers. Typically, a biological or protein sample (such as a protein extract produced according to a protein expression method provided herein and known in the art) is loaded into a selected size exclusion column having a defined stationary phase (porous material), preferably a phase that does not interact with the proteins in the sample. In certain embodiments, the stationary phase consists of inert particles packed in a high-density three-dimensional matrix within a glass or steel column. The mobile phase may be pure water, aqueous buffer, organic solvent, or a mixture thereof. Stationary phase particles typically have small pores and / or channels that allow only molecules below a certain size to enter. Therefore, larger particles are excluded from these pores and channels, and their limited interaction with the stationary phase causes them to elute as "completely excluded" peaks at the start of the experiment. Smaller molecules that can fit within the pores are removed from the flowing mobile phase, and the time they spend immobilizing in the stationary phase pores depends, in part, on how far they penetrate into the pores. Their removal from the mobile phase flow results in separation between particles based on their size differences, causing them to take longer to elute from the column. A given size exclusion column has a range of molecular weights that can be separated. Overall, molecules larger than the upper limit will not be captured by the stationary phase, molecules smaller than the lower limit will enter the solid phase completely and elute as a single band, and molecules within the range will elute at different rates defined by their properties, such as hydrodynamic volume.For practical examples of these methods using pharmaceutical proteins, see Bruner et al., Journal of Pharmaceutical and Biomedical Analysis. 15:1929-1935, 1997.

[0204] Protein purity for clinical applications has also been discussed, for example, by Anicetti et al. (Trends in Biotechnology. 7:342-349, 1989). More recent techniques for analyzing protein purity include, without limitation, the LabChip GXII, an automated platform for rapid analysis of proteins and nucleic acids, which provides high-throughput analysis of protein titer, sizing, and purity. In certain 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, among other methods (see, for example, Therapeutic Proteins: Methods and Protocols. Vol.308, Eds., Smales and James, Humana Press Inc., 2005). Typically, protein agents are substantially endotoxin-free when measured according to techniques known in the art and techniques described herein.

[0205] Protein solubility assays are also included. Such assays may be used, for example, to determine optimal growth and purification conditions for recombinant production, to optimize the selection of buffers, and to optimize the selection of proteins or protein complexes and their variants. Solubility or aggregation may be evaluated according to 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 that measure protein solubility using turbidity or other measurements as endpoints; high-throughput assays for the analysis of the solubility of purified recombinant proteins (see, e.g., Stenvall et al., Biochim Biophys Acta. 1752:6-10, 2005); assays that use structural complementation of genetic marker proteins to monitor and measure protein folding and solubility in vivo (see, e.g., 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, e.g., Nagamine et al., Biotechnology and Bioengineering. 96:1008-1013, 2006). Proteins or protein complexes exhibiting increased solubility (or reduced aggregation) may be identified or selected according to conventional techniques in the art, including simple in vivo assays for protein solubility (see, for example, Maxwell et al., Protein Sci. 8:1908-11, 1999).

[0206] Protein solubility and aggregation can also be measured by dynamic light scattering techniques. Aggregation is a general term encompassing several types of interactions or features, including soluble / insoluble, covalent / non-covalent, reversible / irreversible, and intrinsic / denatured interactions and features. For protein therapeutics, the presence of aggregates is typically considered undesirable due to concerns that the aggregates may cause immunogenic reactions (e.g., small aggregates) or adverse events at administration (e.g., microparticles). Dynamic light scattering refers to a technique that can be used to determine the size distribution profile of small particles in suspension 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. Variations in scattering intensity can be observed due to Brownian motion of molecules and particles in solution. This motion data can conventionally be processed to derive the size distribution for a sample, where size is given by the Stokes radius or hydrodynamic radius of the protein particles. Hydrodynamic size depends on both mass and shape (morphology). Dynamic scattering can detect the presence of very small amounts of aggregated proteins (<0.01% by weight) even in samples containing a wide range of masses. It can also be used to compare the stability of different formulations, for example, in applications that rely on real-time monitoring of changes at high temperatures. Accordingly, certain embodiments include the use of dynamic light scattering to analyze the solubility and / or presence of aggregates in samples containing the proteins or protein complexes of this disclosure.

[0207] While the embodiments described above have been explained in some detail by examples and embodiments for the purpose of clarity of understanding, it will be readily apparent to those skilled in the art that certain changes and modifications can be made in light of the teachings of this disclosure without departing from the spirit or scope of the appended claims. The following embodiments are provided by illustration only and not by limitation. Those skilled in the art will readily recognize various non-essential parameters that can be changed or modified to produce essentially similar results. [Examples]

[0208] Example 1 Characteristics of the A2M:PPE protein complex We conducted experiments to test the properties of protein complexes consisting of A2M and the PPE protein "Mutant F" (MutF, SEQ ID NO: 5) in different molar ratios.

[0209] A1AT protection assay. MutF (400 nM) was mixed with A2M at different molar ratios and incubated at 37°C for 30 minutes. The protection assay was performed on a 384-well black coated plate. 10 μL of sample and either 10 μL of PBS, 10 μL of 2 μM A1AT, or different concentrations of A1AT were added to each well, and duplicate measurements were performed. 20 μL of 100 μM substrate AAPV-AMC was added. The reaction rate was measured using Varioskan LUX: excitation wavelength 380 nm / fluorescence wavelength 460 nm (bandwidth 5 nm), 37°C, top reading, measurement time 100 ms, 16 readings over 2 minutes. Vo was calculated as the activity value.

[0210] As shown in Figures 3A-3B, the A2M:MutF protein complex in a molar ratio of 1:2 provides the best protection against A1AT (3A), and increasing the concentration of A1AT does not impair the protection of MutF by A2M (3B).

[0211] Plasma protection assay. MutF (400 nM) and 200 nM A2M were mixed in a 1:2 molar ratio (A2M:MutF) and incubated in PBS at 37°C for 30 minutes. The plasma protection assay was performed on a 384-well black coated plate. 10 μL of sample and either 10 μL of PBS or 10 μL of mouse plasma sample were added to each well, and duplicate measurements were performed. 20 μL of 100 μM substrate AAPV-AMC was added. The reaction rate was measured using Varioskan LUX: excitation wavelength 380 nm / fluorescence wavelength 460 nm (bandwidth 5 nm), 37°C, top reading, measurement time 100 ms, 16 readings over 2 minutes. Vo was calculated as the activity value.

[0212] As shown in Figure 3C, plasma inhibits the activity of MutF alone, but does not inhibit the activity of the A2M:MutF complex at a molar ratio of 1:2.

[0213] Purification of N17350-A2M by column chromatography. MutF was mixed with A2M in a 1:2 molar ratio (A2M:MutF) and incubated at 37°C for 30 minutes. An AKTA Pure chromatography system was used for protein purification. For cation exchange columns, the protein mixture was buffer-exchanged to a 50 mM sodium acetate pH 5.0 solution using a PD10 desalting column. The sample was packed into a HiTrap SP column equilibrated with 0.5 M NaCl and 50 mM sodium acetate pH 5.0, and eluted in 20 fractions of 1 mL each. For samples used in size exclusion columns, the protein mixture buffer was replaced with a 50 mM sodium phosphate, 150 mM sodium chloride pH 7 solution using a PD10 desalting column. The sample was packed into a pre-washed and equilibrated Superose 6 10 / 300 Increase column. The flow rate was set to 0.5 mL / min, and 1 mL was taken per fraction. Each fraction was diluted 1:250 with PBS and subjected to the A2M:MutF A1AT protection assay. Protein concentrations were measured using the Pierce® BCA protein assay kit (ThermoFisher Scientific, see manufacturer's instructions). In the A1AT protection assay, 10 μL of each fraction dilution was added to a 384-well black plate, and 10 μL of PBS, or 10 μL of 2 μM A1AT, or different concentrations of A1AT were added in a dual measurement. 20 μL of 100 μM substrate AAPV-AMC was added. The reaction rate was measured using Varioskan LUX: excitation wavelength 380 nm / fluorescence wavelength 460 nm (bandwidth 5 nm), 37°C, top reading, measurement time 100 ms, 16 readings over 2 minutes. Vo was calculated as the activity value.

[0214] The results in Figures 4A–4E demonstrate the stability of the A2M:MutF protein complex. Figure 4A shows MutF activity and concentration in different fractions of the A2M:MutF complex after cation exchange column separation, in and out of the presence of A1AT. Figure 4B shows MutF activity and concentration in different fractions of the A2M:MutF complex after size exclusion column separation, in and out of the presence of A1AT. Figures 4C–4D demonstrate that the A2M:MutF complex is stable over a wide pH range, as measured by enzyme activity (4C) and A1AT protection (4D). Figure 4E demonstrates that the A2M:MutF complex is stable over multiple freeze / thaw cycles, as measured by enzyme activity.

[0215] Activity and A1AT protection in cell lysates. Tumor cells were trypsinized, washed with PBS, counted, and diluted in serum-free medium. 40,000 tumor cells were added to a V-bottom plate, followed by 40 μL of various conditions (SFM, A2M, 400 nM N17350, or pre-mixed A2M:MutF (800 nM-400 nM)). The plate was incubated at 37°C for 30 minutes. After incubation, the plate was centrifuged at 300 x g for 5 minutes and then washed with 200 μL of PBS in each well. 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 incubated on ice for 20 minutes, vortexed for 10 seconds every 5 minutes. The plate was centrifuged at 300 x g for 5 minutes. The supernatant was carefully transferred to a new 96-well plate and used for the A2M:MutF activity and protection assay. Here, 10 μL of the supernatant was added to a 384-well black plate, and 10 μL of PBS, or 10 μL of 2 μM A1AT, or different concentrations of A1AT were added in a dual measurement. 20 μL of 100 μM substrate AAPV-AMC was added. The reaction rate was measured using Varioskan LUX: excitation wavelength 380 nm / fluorescence wavelength 460 nm (bandwidth 5 nm), 37°C, top reading, measurement time 100 ms, 16 readings over 2 minutes. Vo was calculated as the activity value.

[0216] The results in Figures 5A-5D show that the A2M:MutF protein complex can enter cells and remain in the protein complex form within the cell because A1AT cannot inhibit its activity.

[0217] CD95-C cleavage assay. MutF was mixed with A2M at different concentrations in various molar ratios and incubated in PBS at 37°C for 30 minutes. Recombinant CD95-C terminal protein (Wuxi) was incubated with pre-mixed A2M:MutF protein complexes at different molar ratios at 37°C for 1 hour. Samples containing 1 ug of CD95-C were applied to a 17.5% SDS-PAGE gel and stained with One-step Blue (Biotium). The gel was imaged using iBright 1500.

[0218] As shown in Figure 6A, the A2M:MutF protein complex exists in a molar ratio range of 1:2 to 1:16 and cleaves CD95 as efficiently as MutF alone.

[0219] Fibrinogen cleavage assay. MutF was mixed with A2M at different concentrations in various molar ratios and incubated in PBS at 37°C for 30 minutes. Human fibrinogen (Sigma) was incubated with pre-mixed A2M:MutF protein complexes at different molar ratios at 37°C for 1 hour. Samples containing 5 ug of fibrinogen were loaded onto 4-15% gels, and fibrinogen was detected by Western blotting using anti-fibrinogen (cell signaling technology). The gels were imaged using iBright 1500.

[0220] As shown in Figure 6B, the A2M:MutF protein complex does not cleave fibrinogen, but MutF alone does.

[0221] Elastin cleavage assay. MutF was mixed with A2M at different concentrations in various molar ratios and incubated in PBS at 37°C for 30 minutes. Elastin-F (1:100) was incubated overnight at 37°C with a pre-mixed A2M:MutF protein complex, and the degradation fluorescence signal was measured in the supernatant.

[0222] As shown in Figure 6C, the A2M:MutF protein complex does not cleave elastin, but MutF alone does cleave elastin.

[0223] Coagulation assay. Mice were injected with 200 μL of 480 μg of MutF or purified A2M:MutF protein complex in a 1:2 molar ratio. Blood was collected in a sodium citrate tube and centrifuged at 1500 x g for 15 minutes. 300 μl of plasma was frozen and sent to IDEXX for analysis of prothrombin time, partial thromboplastin time, and fibrinogen concentration.

[0224] The results in Figures 7A-7C show that the A2M:MutF protein complex does not induce coagulation effects compared to MutF itself, which in contrast increases PT and PTT counts and leads to a decrease in fibrinogen levels.

[0225] Cell killing assay. Approximately 20,000–40,000 cancer cells were seeded in a 96-well plate coated with black wells, and the cells were cultured statically overnight for adhesion stabilization. On day 2, each well was washed once with 200 μL of serum-free medium, followed by three different treatments. 24 hours after treatment, the cells were incubated for 45 minutes in a pre-diluted Calcein-AM solution (ThermoFisher, 4 μM in Ca2+Mg2+-containing HBSS). The Calcein AM was dumped, and 100 μL of Ca2+Mg2+-containing HBSS was added to each well. Fluorescence was measured at 485 / 520 nm (20 bandwidth) using Varioskan LUX.

[0226] As shown in Figure 8A, the A2M:MutF protein complex in a 1:2 molar ratio induces the killing of various cancer cells in vitro, and its activity is in most cases comparable to that of MutF alone. Figure 8B shows the broad cytotoxicity of the A2M:MutF(1:2) protein complex against cancer cells of different anatomical origins, and Figure 8C shows that the complex does not kill non-cancer cells.

[0227] In vivo efficacy. Approximately 1 million CT26 cells were transplanted into the flank of 7-8 week old BALB / c mice. The tumor was approximately 80 mm. 3 Upon reaching a certain level, the tumor was treated with 100 ug of MutF, 100 ug of A2M:MutF (1:2), or 1.4 mg of A2M (corresponding to an amount that results in an A2M:MutF molar ratio of 1:2). The tumor was monitored every two days.

[0228] As shown in Figure 8D, the A2M:MutF protein complex in a 1:2 molar ratio induces cancer cell killing in vivo, and its activity is comparable to that of MutF alone. Figure 8E shows that the A2M:MutF protein complex has an improved functional PK profile (enzyme activity in plasma) compared to MutF alone after intravenous administration. Figure 8F shows that the A2M:MutF protein complex induces a favorable immune profile in the CT26 model (from left to right in each graph: PBS, MutF, A2M:MutF). Figure 8G shows that the A2M:MutF protein complex induces a tumor antigen-specific CD8+ T cell response in the CT26 model (from left to right in each graph: PBS, MutF, A2M:MutF). The induction of effector and memory T cells indicates a functionally adaptive immune response.

[0229] Evaluation of selective cancer cell killing in ovarian cancer patient samples. Cancer and non-cancer cells were isolated from the primary tumors, intraperitoneal fluid (IP), ovarian adipose tissue (common metastatic site), and blood of six ovarian cancer patients. The following cells were isolated and tested: cancer cells (digested tumors with fibroblasts, CD45+ cells, and EpCAM+ / high cells removed), non-cancerous immune cells (digested ovarian cells with neutrophils or CD45+ cells removed from tumor tissue), peripheral blood mononuclear cells (PBMCs; B cells, T cells, monocytes, and NK cells), fibroblasts (isolated from tumor samples using a fibroblast isolation kit), and IP cells (>90% CD45+ immune cells, including B cells, T cells, myeloid cells, and NK cells). The isolated cells were seeded and exposed to A2M:MutF, doxorubicin (standard therapeutic chemotherapy), or oxaliplatin (standard therapeutic chemotherapy) for 24 hours. Cell viability was evaluated by Calcein-AM.

[0230] Figure 9A shows that the A2M:MutF protein complex has a broad therapeutic window, as shown by killing human ovarian cancer cells without killing non-cancer cells from patients, in contrast to doxorubicin and oxaliplatin. Figure 9B shows that A2M:MutF shows equivalent killing against cancer cells isolated from chemotherapy-naive and chemotherapy-treated patients, while doxorubicin and oxaliplatin, in contrast, show a decrease in killing against cancer cells isolated from chemotherapy-treated patients compared to chemotherapy-naive patients.

[0231] Evaluation of immunogenic cell death (ICD). CT26 (mouse colon), A549 (human lung), and ovarian patient cancer cells were treated with A2M:MutF and oxaliplatin (known to induce ICD in certain cell types at high concentrations) for 24 hours. Immunogenic cell markers including HSP70, ATP release, HMGB1, and CALR were evaluated (see, for example, Fucikova et al., Cell death Dis. 11(11):1013, 2020).

[0232] Figure 10A shows that the A2M:MutF protein complex induces ICD markers in CT26 cells and A549 cells. Figure 10B shows that the A2M:MutF protein complex induces ICD markers in tumor cells derived from human ovarian cancer patients (from left to right in each graph: CTRL, A2M:MutF, oxaliplatin).

[0233] Evaluation of the 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 3 The A2M:MutF protein complex or vehicle was administered intravenously multiple times as follows: · Vehicle: on days 0, 2, 4, and 6, and on days 8, 10, 12, and 14, · A2M:MutF 100 μg: daily for 2 weeks (every day), · A2M:MutF 200 μg: on days 0, 2, 4, 6 and 8, 10, 12 and 14 (every other day), and · A2M:MutF 400 μg: on days 0, 4, 8, and 12 (every 4 days).

[0234] Tumor growth and weight were evaluated. Figures 11A - 11B show tumor growth after treatment, and Figure 11C shows tumor weight on day 15 after treatment (Figure 11 shows, from left to right, vehicle every other day, A2M:MutF 100 μg daily, A2M:MutF 200 μg every other day, A2M:MutF 400 μg every 4 days).

[0235] Evaluation of the antitumor effect in syngeneic mouse tumor models. Each cancer cell of CT26 (highly immunogenic colon cancer model), MC38 (moderately immunogenic colon cancer model), B16F10 (cold immunogenic skin cancer and lung metastasis model) was injected into the flanks of syngeneic B cell - deficient Jh - BALB / c mice or C57BL / 6 mice and allowed to grow until they reached approximately 80 - 100 mm 3 The A2M:MutF protein complex (400 μg) or vehicle was injected intravenously every other day for 2 - 3 weeks. Tumor growth was monitored.

[0236] Figures 12A - 12B show that the A2M:MutF protein complex effectively suppresses tumor growth in the Jh - BALB / c CT26 colorectal cancer model. Figures 12C - 12D show that the A2M:MutF protein complex treats primary tumors and metastases in the Jh - C57BL / 6 B16F10 melanoma model. Figure 12E shows that the A2M:MutF protein complex shows efficacy in various tumors with different immunological states.

[0237] Evaluation of antitumor effect compared with 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 3 in size. The A2M:MutF protein complex (400 μg every other day for 3 weeks) or oxaliplatin (6 mg / kg on days 0 and 2) was injected intravenously. Tumor growth and overall survival were monitored.

[0238] Figures 13A - 13C show that the A2M:MutF protein complex has an improved antitumor effect compared to SoC chemotherapy (oxaliplatin) with no toxicity observed.

[0239] Evaluation of antitumor effect against human cancer cells in xenograft models (NU / NU and NCG female mice). Cancer cells of 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 were injected into the flanks of mice and allowed to grow until they reached approximately 80 - 100 mm 3 in size. The A2M:MutF protein complex (400 μg) or vehicle was injected intravenously every other day for 2 - 3 weeks. Tumor growth and overall survival were monitored.

[0240] Figure 14A shows the efficacy of the A2M:MutF protein complex in a human xenograft model of lung cancer. Figure 14B summarizes the efficacy of the A2M:MutF protein complex across various models of prostate cancer, colon cancer, and lung cancer. Figure 14C shows that the A2M:MutF protein complex effectively kills tumor cells derived from human ovarian patients (from patient CDX_O02) in a xenograft mouse model, and Figure 14D summarizes the efficacy of the A2M:MutF protein complex across three ovarian cancer patients in this model (all patient history: diagnosis was grade 3; treatment status: CDX_O01 and CDX_O03 were chemotherapy-naive; CDX_O02 had received 3 cycles of treatment with carboplatin + paclitaxel + keytruda). Figures 14E–14F demonstrate that the A2M:MutF protein complex effectively kills patient-derived breast cancer cells in vitro and in vivo (patient history: diagnosis of HER2-negative and ER-positive breast cancer, treatment status: chemotherapy-naive). Figure 14G summarizes the in vivo efficacy of the A2M:MutF protein complex in various human tumors and shows that its efficacy is independent of tumor genetics or immune status.

[0241] Figure 15 shows that mice treated with the A2M:MutF protein complex were tumor-free after the initial trial using CT26 colorectal cancer cells (5 / 11), and all of these mice (5 / 5) remained tumor-free upon a second trial using CD26 cells. This study suggests that treatment with the A2M:MutF protein complex induces a tumor-specific immunomemory response.

Claims

1. A pharmaceutical composition, (a) α2-macroglobulin (A2M) protein, and (b) Contains a protein complex consisting of serine protease proteins, (a) and (b) are pharmaceutical compositions present in the composition in a molar ratio of approximately 1:3 to approximately 1:1 [(a):(b)].

2. The A2M protein in (a) and the serine protease protein in (b) are bound to each other as a protein complex, and optionally the protein complex is formed (i)(b) retains the CD95 (Fas receptor) protease cleavage activity and cancer cell killing activity, (ii) Sterically inhibit the binding of (b) to fibrinogen, thereby reducing or inhibiting the fibrinogen cleavage activity of (b), The pharmaceutical composition according to claim 1, wherein (iii)(b) sterically inhibits the binding of serine protease inhibitors (including α1-antitrypsin (A1AT)).

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

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

5. The aforementioned functional Y fragments are selected from Table A1 in the sequence approximately 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, 20 0-1300, 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, 4 00-900, 400-800, 400-700, 400-600, 400-500, 500-1400, 500-1300, 500-1200, 500-1100, 500-1000, 500-900, 500-800, 500-700, 500-600, 600-1400, 600-1300, 600-1200, 600-1100, 600-1000, 600-900, 600-800, 600-700, 700-1400, 700-1300, 700-1200, 700-1100, 700-1000 The pharmaceutical composition according to claim 4, comprising 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 residues.

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

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

8. (b) is a 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 cathepsin G (CTSG) protein, human proteinase 3 (PR3) protein, and granzyme B protein.

9. The PPE protein comprises, or essentially comprises, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 5, wherein the sequence retains the Q211F amino acid substitution. The PPE protein comprises, or essentially comprises, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 6, wherein the sequence retains the T55A amino acid substitution. The PPE protein comprises, or essentially comprises, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 7, wherein the sequence retains the Q211F and T55A amino acid substitutions. The PPE protein comprises, or essentially comprises, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 8, wherein the sequence retains the N241A amino acid substitution. The PPE protein comprises, or essentially comprises, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 9, wherein the sequence retains the N241Y amino acid substitution. The PPE protein comprises, or essentially comprises, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 10, wherein the sequence retains the R75A amino acid substitution. The PPE protein comprises, or essentially comprises, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 11, wherein the sequence retains the R75E amino acid substitution. The PPE protein comprises, or essentially comprises, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 12, wherein the sequence retains the Q211A amino acid substitution. The PPE protein comprises, or essentially comprises, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 13, wherein the sequence retains the R237A amino acid substitution. The PPE protein comprises, or essentially comprises, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 14, wherein the sequence retains the S214A amino acid substitution. The PPE protein comprises, or essentially comprises, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 15, and the sequence retains the D74A amino acid substitution, and The pharmaceutical composition according to claim 8, wherein the PPE protein comprises, or essentially comprises, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:

16.

10. The aforementioned human ELANE protein comprises, or essentially comprises, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:

17. The human CTSG protein comprises, or essentially comprises, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:

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

20.

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

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, improving the symptoms of, and / or delaying the progression of cancer in a subject requiring the use of the pharmaceutical composition described in any one of claims 1 to 12.

14. The method according to claim 13, wherein the cancer is primary cancer 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, hepatocellular carcinoma, sarcoma, B-cell malignancy, ovarian cancer, colorectal cancer, glioma, glioblastoma multiforme, meningioma, pituitary adenoma, vestibular schwannoma, primary CNS lymphoma, primitive neuroectodermal tumor (medulloblastoma), bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancer, cervical cancer, testicular cancer, thyroid cancer, and gastric cancer.

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

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

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

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

19. A method for producing a pharmaceutical composition containing a protein complex, (a) α2-macroglobulin (A2M) protein, and (b) Serine protease protein, A method for producing a pharmaceutical composition containing the protein complex by combining it in a composition in a molar ratio of approximately 1:3 to approximately 1:1 [(a):(b)].

20. The method according to claim 19, comprising recombinantly producing (a) before combining it with (b).

21. The method according to claim 19, comprising purifying (a) from the plasma of a human subject before combining it with (b).

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

23. The method according to any one of claims 19 to 22, comprising combining (a) and (b) in a molar ratio 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)].

24. The method according to claim 23, comprising combining (a) and (b) in a molar ratio of approximately 1:2 [(a):(b)].

25. The A2M protein in (a) and the serine protease protein in (b) are bound to each other as a protein complex, and optionally the protein complex is formed (i)(b) retains the CD95 (Fas receptor) protease cleavage activity and cancer cell killing activity, (ii) Sterically inhibit the binding of (b) to fibrinogen, thereby reducing or inhibiting the fibrinogen cleavage activity of (b), The method according to any one of claims 19 to 24, wherein the binding of (iii)(b) to a serine protease inhibitor (including α1-antitrypsin (A1AT)) is sterically inhibited.

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

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

28. The aforementioned functional Y fragments are selected from Table A1 in the sequence approximately 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, 20 0-1300, 200-1200, 200-1100, 200-1000, 200-900, 200-800, 200-700, 200-600, 200-500, 200-400, 200-300, 300-1400, 300-1300, 300-1200, 300-1100, 300-1000, 300-900, 300-800, 300-700, 300-600, 300-500, 300-400, 400-1400, 400-1300, 400-1200, 400-1100, 400-1000, 400-900, 400-800, 400-700, 400-600, 400-500, 500-1400, 500-1300, 500-1200, 500-1100, 500-1000, 500-900, 500-800, 500-700, 500-600, 600-1400, 600-1300, 600-1200, 600-1100, 600-1000, 600-900, 600-800, 600-700, 700-1400, 700-1300, 700-1200, 700-1100, 700-10 The method according to claim 27, comprising the terms 00, 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. (a) is conjugated or fused to an antibody or an antigen-binding fragment, the method according to any one of claims 19 to 28.

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

31. (b) 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 cathepsin G (CTSG) protein, human proteinase 3 (PR3) protein, and human granzyme B protein.

32. The PPE protein comprises, or essentially comprises, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 5, wherein the sequence retains the Q211F amino acid substitution. The PPE protein comprises, or essentially comprises, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 6, wherein the sequence retains the T55A amino acid substitution. The PPE protein comprises, or essentially comprises, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 7, wherein the sequence retains the Q211F and T55A amino acid substitutions. The PPE protein comprises, or essentially comprises, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 8, wherein the sequence retains the N241A amino acid substitution. The PPE protein comprises, or essentially comprises, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 9, wherein the sequence retains the N241Y amino acid substitution. The PPE protein comprises, or essentially comprises, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 10, wherein the sequence retains the R75A amino acid substitution. The PPE protein comprises, or essentially comprises, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 11, wherein the sequence retains the R75E amino acid substitution. The PPE protein comprises, or essentially comprises, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 12, wherein the sequence retains the Q211A amino acid substitution. The PPE protein comprises, or essentially comprises, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 13, wherein the sequence retains the R237A amino acid substitution. The PPE protein comprises, or essentially comprises, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 14, wherein the sequence retains the S214A amino acid substitution. The PPE protein comprises, or essentially comprises, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 15, and the sequence retains the D74A amino acid substitution, and The method according to claim 31, wherein the PPE protein comprises, or essentially comprises, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:

16.

33. The aforementioned human ELANE protein comprises, or essentially comprises, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:

17. The human CTSG protein comprises, or essentially comprises, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:

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

20.

34. The method 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 the following: a CD95 cleavage assay (optionally in the presence of a serine protease inhibitor such as A1AT), a fibrinogen cleavage assay, and a cancer cell killing assay.

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

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