Pharmaceutical compositions comprising protein complexes

Description

"Pharmaceutical compositions comprising protein complexes" CROSS-REFERENCE TO RELATED REQUESTS

[001] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 623,030, filed January 19, 2024; and U.S. Provisional Application No. 63 / 456,916, filed April 4, 2023, each of which is incorporated by reference in its entirety. STATEMENT REGARDING SEQUENCE LISTING

[002] The XML Sequence Listing associated with this application is provided in XML file format and is incorporated herein by reference in the descriptive report. The name of the XML file containing the XML Sequence Listing is OPNI_009_02WO_ST26.xml. The XML file is approximately 28,746 bytes in size, was created on March 28, 2024, and is being submitted electronically via the USPTO Patent Center. FUNDAMENTALS Technical Field

[003] This disclosure relates to pharmaceutical compositions comprising alpha-2-macroglobulin (A2M) and a serine protease protein, such as porcine pancreatic elastase (PPE), which combine into a protein complex that retains the CD95 protease cleavage and cancer cell-killing activities of serine protease, but sterically prevents the binding of serine protease to fibrinogen and serine protease inhibitors; and related methods of use and manufacture for treating diseases, such as cancers. Description of the Related Technique

[004] Precision medicine, designed to optimize therapeutic efficiency or benefit for specific patient groups using genetic or molecular profiles, has gained enormous traction in cancer treatment. Identifying specific genomic abnormalities that (i) confer risk of developing Petition 870250109095, dated 11 / 27 / 2025, page 6 / 150 2 / 102 cancer, (ii) influence tumor growth and (iii) regulate metastasis defined how cancer is diagnosed, determined how targeted therapies are developed and implemented, and shaped cancer prevention strategies.

[005] The need for precision medicine in cancer is largely based on the failure to identify targetable properties in tumor cells that distinguish them from healthy, non-cancerous cells. In fact, while radiation and / or chemotherapy have the ability to effectively kill many, if not most, cancer cells, their effectiveness is severely limited by cytotoxic effects on non-cancerous cells. These findings demonstrate that rapid cell division, a property targeted by radiotherapy and chemotherapy, is not unique enough for cancer cells to achieve the specificity needed to limit extensive side effects.

[006] Certain serine protease and elastase enzymes have been shown to be selectively toxic to cancer cells but relatively non-toxic to normal or healthy cells (see, for example, WO 2018 / 232273). However, there is a need in the art to identify ideal enzyme compositions that are capable of such selective toxicity to cancer cells but also have reduced side effects and thus refine the pharmacokinetics and overall clinical utility of such compositions. BRIEF SUMMARY

[007] Embodiments of the present disclosure include a pharmaceutical composition comprising a protein complex of: (a) alpha-2-macroglobulin (A2M) proteins; and (b) serine protease proteins, wherein (a) and (b) are present in the composition in a molar ratio [(a):(b)] of about 1:3 to about 1:1. In certain embodiments, the A2M proteins of (a) and the serine protease proteins of (b) are linked together in the protein complex, for example, wherein the protein complex: Petition 870250109095, dated 11 / 27 / 2025, page 7 / 150 3 / 102 (i) retains the CD95 protease cleavage activity (Fas receptor) and the cancer cell killing activity of (b); (ii) sterically prevents the binding of (b) to fibrinogen and reduces or inhibits the fibrinogen cleavage activity of (b); and (iii) sterically prevents the binding of (b) to serine protease inhibitors (including serine protease inhibitors in plasma, for example, alpha-1 antitrypsin (A1AT)).

[008] In some embodiments, (a) comprises, 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 thereof comprises, consists of, or essentially consists of about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1300, or 1400 contiguous amino acids from a sequence selected from Table A1. In some modalities, the functional fragment thereof is composed of 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, 400-1100, 400-1000, 400-900, 400-800, 400-700, 400-600, 400-500, 500-1400, 500-1300, 500-1200, 500-1100, 500-1000, 500-900, 500-800, 500-700, 500-600, 600-1400, 600-1300, 600-1200, 600-1100, 600-1000 600-900, 600-800, 600-700, 700-1400, 700-1300, 700-1200, 700-1100, 700-1000, 700-900, 700-800, 800-1400, 800-1300, 800-1200, 800-1100, 800-1000, 800-900, 900-1400, 900-1300, 900-1200, 900-1100, 900-1000, 1000-1400, 1000-1300, 1000-1200, 1000-1100 1100-1400, 1100-1300, 1100-1200, 1200-1400 or 1200-1300 residues of a selected sequence of, Petition 870250109095, dated 11 / 27 / 2025, page 8 / 150 4 / 102 Table A1.

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

[010] In some embodiments, (b) is selected from a porcine pancreatic elastase (PPE), a human neutrophil elastase (ELANE), a human cathepsin G protein (CTSG), a human proteinase 3 (PR3), and a human granzyme B protein. In particular embodiments: a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 5, and retaining the Q211F amino acid substitution; a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 6, and retaining the T55A amino acid substitution; a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 7, and retaining the amino acid substitutions Q211F and T55A; a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 8, and retaining the N241A amino acid substitution; a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 9, and retaining the substitution of Petition 870250109095, dated 11 / 27 / 2025, p. 9 / 150 5 / 102 amino acid N241Y; a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 10, and retaining the R75A amino acid substitution; a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 11, and retaining the R75E amino acid substitution; a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 12, and retaining the Q211A amino acid substitution; a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 13, and retaining the R237A amino acid substitution; a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 14, and retaining the S214A amino acid substitution; a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 15, and retaining the D74A amino acid substitution; and a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 16. Petition 870250109095, dated 11 / 27 / 2025, p. 10 / 150 6 / 102

[011] In some modalities: a modified human ELANE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 17; a modified human CTSG protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 18; The human PR3 protein comprises, consists 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; or the human granzyme B protein comprises, 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.

[012] In some embodiments, (a) and (b) are present in the composition in a molar ratio of about 1:3, 1:2.9, 1:2.8, 1:2.7, 1:2.6, 1:2.5, 1:2.4, 1:2.3, 1:2.1, 1:2, 1:1.9, 1:1.8, 1:1.7, 1:1.6, 1:1.5, 1:1.4, 1:1.3, 1:1.2, 1:1.1 or 1:1. In specific embodiments, (a) and (b) are present in the composition in a molar ratio of about 1:2.

[013] Also included are methods of treatment, improvement of symptoms and / or reduction of cancer progression in a subject in need, comprising the administration of the pharmaceutical composition to the subject described in this document.

[014] In some modalities, the cancer is either a primary cancer or a metastatic cancer and is selected from one or more melanomas (optionally metastatic melanoma), breast cancer (optionally triple-negative breast cancer, TNBC), renal cancer (optionally renal cell carcinoma), pancreatic cancer, bone cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer (NSCLC), Petition 870250109095, dated 11 / 27 / 2025, page 11 / 150 7 / 102 mesothelioma, leukemia (optionally lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, or relapsed acute myeloid leukemia), multiple myeloma, lymphoma, hepatoma (hepatocellular carcinoma), sarcoma, B-cell malignancy, ovarian cancer, colorectal cancer, glioma, glioblastoma multiforme, meningioma, pituitary adenoma, vestibular schwannoma, primary CNS lymphoma, primitive neuroectodermal tumor (medulloblastoma), bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancer, cervical cancer, testicular cancer, thyroid cancer, and stomach cancer.

[015] In some embodiments, administration (optionally intravenous administration) of the pharmaceutical composition does not substantially increase prothrombin time or partial thromboplastin time in the subject. In some embodiments, administration of the pharmaceutical composition increases cancer cell death in the subject by approximately or at least approximately 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 500-fold, or 1000-fold or more relative to a control or reference.

[016] Certain modalities involve administering the pharmaceutical composition to the subject by parenteral administration. In some modalities, parenteral administration is intravenous administration.

[017] Also included are methods for manufacturing a pharmaceutical composition comprising a protein complex, combining: (a) alpha-2-macroglobulin (A2M) proteins; and (b) serine protease proteins, in a composition in a molar ratio [(a):(b)] of about 1:3 to about 1:1, thus manufacturing the pharmaceutical composition comprising the protein complex.

[018] Certain embodiments comprise recombinant production (a) before combining with (b). Some embodiments comprise purifying (a) a Petition 870250109095, dated 11 / 27 / 2025, page 12 / 150 8 / 102 from the plasma of a human subject before combining with (b). Particular embodiments comprise recombinantly producing (b) before combining with (a).

[019] In some embodiments, the manufacturing methods comprise combining (a) and (b) in a molar ratio [(a):(b)] of about 1:3, 1:2.9, 1:2.8, 1:2.7, 1:2.6, 1:2.5, 1:2.4, 1:2.3, 1:2.1, 1:2, 1:1.9, 1:1.8, 1:1.7, 1:1.6, 1:1.5, 1:1.4, 1:1.3, 1:1.2, 1:1.1 or 1:1. Specific embodiments comprise combining (a) and (b) in a molar ratio [(a):(b)] of about 1:2.

[020] In some embodiments, the A2M proteins of (a) and the serine protease proteins of (b) join together in the protein complex, including wherein the protein complex: (i) retains the CD95 protease cleavage activity (Fas receptor) and the cancer cell killing activity of (b); (ii) sterically prevents the binding of (b) to fibrinogen and reduces or inhibits the fibrinogen cleavage activity of (b); and (iii) sterically prevents the binding of (b) to serine protease inhibitors (including alpha-1 antitrypsin (A1AT)).

[021] In some embodiments, (a) comprises, 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 thereof comprises, consists of, or essentially consists of about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1300, or 1400 contiguous amino acids from a sequence selected from Table A1. In some modalities, the functional fragment thereof is composed of 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, Petition 870250109095, dated 11 / 27 / 2025, p. 13 / 150 9 / 102 200-1100, 200-1000, 200-900, 200-800, 200-700, 200-600, 200-500, 200-400, 200-300, 300-1400, 300-1300, 300-1200, 300-1100, 300-1000, 300-900, 300-800, 300-700, 300-600, 300-500, 300-400, 400-1400, 400-1300, 400-1200, 400-1100, 400-1000, 400-900, 400-800, 400-700, 400-600, 400-500, 500-1400, 500-1300, 500-1200, 500-1100, 500-1000, 500-900, 500-800, 500-700, 500-600, 600-1400, 600-1300, 600-1200, 600-1100, 600-1000, 600-900, 600-800, 600-700, 700-1400, 700-1300, 700-1200, 700-1100, 700-1000 700-900, 700-800, 800-1400, 800-1300, 800-1200, 800-1100, 800-1000, 800-900, 900-1400, 900-1300, 900-1200, 900-1100, 900-1000, 1000-1400, 1000-1300, 1000-1200, 1000-1100, 1100-1400, 1100-1300, 1100-1200, 1200-1400 or 1200-1300 residues from a sequence selected from Table A1.

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

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

[024] In some modalities: a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 5, and retaining the Q211F amino acid substitution; a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 6, and retaining the substitution of Petition 870250109095, dated 11 / 27 / 2025, p. 14 / 150 10 / 102 amino acid T55A; a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 7, and retaining the amino acid substitutions Q211F and T55A; a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 8, and retaining the N241A amino acid substitution; a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 9, and retaining the N241Y amino acid substitution; a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 10, and retaining the R75A amino acid substitution; a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 11, and retaining the R75E amino acid substitution; a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 12, and retaining the Q211A amino acid substitution; a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 13, and retaining the substitution of Petition 870250109095, dated 11 / 27 / 2025, p. 15 / 150 11 / 102 amino acid R237A; a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 14, and retaining the S214A amino acid substitution; a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 15, and retaining the D74A amino acid substitution; and a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 16.

[025] In some modalities: a modified human ELANE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 17; a modified human CTSG protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 18; The human PR3 protein comprises, consists 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; or the human granzyme B protein comprises, 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.

[026] Certain methods further comprise the step of testing the pharmaceutical composition in one or more activity assays selected from one or more CD95 cleavage assays (optionally in the presence of an inhibitor of Petition 870250109095, dated 11 / 27 / 2025, page 16 / 150 12 / 102 serine protease inhibitor, such as A1AT), a fibrinogen cleavage assay, and a cancer cell death assay. In some embodiments, the pharmaceutical composition cleaves CD95 (optionally in the presence of a serine protease inhibitor, such as A1AT), does not substantially cleave fibrinogen, and / or has cancer cell death activity. BRIEF DESCRIPTION OF THE FIGURES

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

[028] Figures 2A-2B show the activity of Mutant F (MutF) alone in the 4T1 tumor model after IV injection (day 0 and 1) on tumor growth (2A) and the number of pulmonary metastases (2B). Figure 2C shows the effects of MutF on prothrombin time 5 min after IV injection.

[029] Figure 3A shows the activity of MutF at a different A2M:MutF ratio in the presence of its inhibitor A1AT. Figure 3B shows the activity of MutF at a 1:2 A2M:MutF ratio in the presence of its inhibitor A1AT at a different concentration. Figure 3C shows the activity of MutF at a 1:2 A2M:MutF ratio in plasma.

[030] Figure 4A shows the activity and concentration of MutF in the different fractions of the A2M:MutF product after separation from the cation exchange column in the presence or absence of A1AT. Figure 4B shows the activity and concentration of MutF in the different fractions of the A2M:MutF product after separation from the size exclusion column in the presence or absence of A1AT. Figures 4C-4D show that the A2M:MutF complexes are stable over a wide pH range, as measured by enzymatic activity (4C) and A1AT protection (4D). Figure 4E shows that the A2M:MutF complexes are stable throughout Petition 870250109095, dated 11 / 27 / 2025, page 17 / 150 13 / 102 of multiple freeze / thaw cycles, as measured by enzymatic activity.

[031] Figures 5A-5D show MutF activity in various cell lysates after 30 min of treatment in serum-free medium (SFM) in the presence or absence of A1AT.

[032] Figure 6A shows the Coomassie Blue staining of CD95 cleavage after 30 min of incubation with MutF or A2M:MutF at different ratios. Figure 6B shows a Western blot for fibrinogen after 1 hour of incubation with MutF or A2M:MutF at different ratios. A2M:MutF at a 1:2 ratio cleaves CD95 as efficiently as MutF alone (shown by the double band), but does not cleave fibrinogen relative to MutF alone (shown by the lower band). Figure 6C shows the fluorescent signal of cleaved elastin after incubation with PBS, MutF alone, or A2M:MutF protein complexes.

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

[034] Figure 8A shows a cell death assay in various mouse cancer cell lines by MutF or the A2M:MutF (1:2) protein complex at 400 nM. Figure 8B shows broad cytotoxicity of the A2M:MutF protein complex against cancer cells of different anatomical origin, and Figure 8C shows that the complex does not kill non-cancerous cells. Figure 8D shows the antitumor effects of MutF and the A2M:MutF (1:2) protein complex in a CT26 model after IT injection of 100 µg on day 0. Figure 8E shows that the A2M:MutF protein complex has a functional PK profile. Petition 870250109095, dated 11 / 27 / 2025, page 18 / 150 14 / 102 improved (enzymatic 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 is PBS, MutF, A2M:MutF). Figure 8G shows that the A2M:MutF protein complex elicits a tumor antigen-specific CD8+ T cell response in the CT26 model (from left to right in each graph is PBS, MutF, A2M:MutF).

[035] Figure 9A shows that the A2M:MutF protein complex, in contrast to doxorubicin and oxaliplatin, has a wide therapeutic window, as shown by the elimination of human ovarian cancer cells without killing non-cancerous cells from patients. Figure 9B shows that A2M:MutF kills cancerous cells comparatively from patients without chemotherapy and those treated with chemotherapy, in contrast to doxorubicin and oxaliplatin, which show reduced elimination of cancerous cells isolated from patients treated with chemotherapy compared to patients without chemotherapy.

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

[037] Figures 11A-11B show tumor growth after treatment in the CT26 mouse tumor model. Figure 11C shows tumor weight 15 days after treatment (11C from left to right: vehicle on alternate days, A2M:MutF 100 μg daily, A2M:MutF 200 μg on alternate days, A2M:MutF 400 μg every 4 days).

[038] Figures 12A-12B show that the A2M:MutF protein complex effectively attenuates tumor growth in the colorectal cancer model. Petition 870250109095, dated 11 / 27 / 2025, page 19 / 150 15 / 102 BALB / c CT26. Figures 12C-12D show that the A2M:MutF protein complex treats primary tumor and metastasis in a Jh-C57BL / 6 B16F10 melanoma model. Figure 12E shows that the A2M:MutF protein complex exhibits efficacy in a variety of tumors with varying immune status.

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

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

[041] Figure 15 shows that mice treated with A2M:MutF protein complexes are tumor-free (5 / 11) after initial challenge with CT26 colorectal cancer cells and that all of these mice (5 / 5) retain their tumor-free status after further challenge with CD26 cells. DETAILED DESCRIPTION

[042] Unless otherwise defined, all technical and scientific terms used in this document have the same meaning commonly understood by those skilled in the art to which the disclosure pertains. Although Petition 870250109095, dated 11 / 27 / 2025, page 20 / 150 16 / 102 Any methods, materials, compositions, reagents, cells, similar or equivalent to those described in this document that may be used in the practice or testing of the subject matter of this disclosure, preferred methods and materials are described. All publications and references, including, but not limited to, patents and patent applications, cited in this descriptive report are incorporated herein by reference in their entirety, as if each individual publication or reference were specifically and individually indicated for incorporation by reference herein as being fully established. Any patent application to which this application claims priority is also incorporated by reference herein in its entirety in the manner described above for publications and references.

[043] Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to the manufacturer's specifications or as commonly performed in the art or as described in this document. These techniques and related procedures may generally be performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout this descriptive report. Unless specific definitions are provided, the nomenclature used in connection with, and the laboratory procedures and techniques of molecular biology, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described in this document are those well known and commonly used in the art.Standard techniques can be used for recombinant technology, molecular biology, microbiology, chemical syntheses, chemical analyses, pharmaceutical preparation, formulation and distribution, and patient treatment.

[044] For the purposes of this disclosure, the following terms are Petition 870250109095, dated 11 / 27 / 2025, p. 21 / 150 17 / 102 defined below.

[045] The articles “o / a” and “um / uma” are used in this document to refer to one or more of (i.e., at least one) of the article’s grammatical object. For example, “um elemento” includes “um elemento”, “um ou mais elementos” and / or “pelo menos um elemento”.

[046] By approximately means a quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight or length that varies by up to 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% for a reference quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight or length.

[047] An antagonist refers to a biological or chemical agent that interferes with or reduces the physiological action of another agent or molecule. In some cases, the antagonist binds specifically to the other agent or molecule. Full and partial antagonists are included.

[048] An agonist refers to the biological or chemical agent that increases or potentiates the physiological action of another agent or molecule. In some cases, the agonist binds specifically to the other agent or molecule. Full and partial agonists are included.

[049] As used in this document, the term amino acid is intended to mean naturally occurring and unnaturally occurring amino acids, as well as amino acid analogs and mimetics. Naturally occurring amino acids include the 20 (L)-amino acids used during protein biosynthesis, as well as others such as 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine, homocysteine, citrulline, and ornithine, for example. Unnaturally occurring amino acids include, for example, (D)-amino acids, norleucine, norvaline, p-fluorophenylalanine, ethionine, and the like, which are known to a person skilled in the art. Amino acid analogs include modified forms of naturally occurring and unnaturally occurring amino acids. Such modifications may include, for example, substitution or Petition 870250109095, dated 11 / 27 / 2025, page 22 / 150 18 / 102 Substitution of chemical groups and moieties in the amino acid or by derivatization of the amino acid. Amino acid mimetics include, for example, organic structures that exhibit functionally similar properties, such as charge characteristics and charge spacing of the reference amino acid. For example, an organic structure mimicking arginine (Arg or R) would have a positive charge fraction located in similar molecular space and having the same degree of mobility as the ε-amino group of the naturally occurring amino acid Arg side chain. Mimics also include structures constrained to maintain optimal spacing and charge interactions of the amino acid or amino acid functional groups. Those skilled in the art know or can determine which structures constitute functionally equivalent amino acid analogs and amino acid mimetics.

[050] As used in this document, a subject at risk of developing a disease or adverse reaction may or may not have detectable disease or symptoms of the disease, and may or may not have exhibited detectable disease or symptoms of the disease prior to the treatment methods described in this document. “At risk” denotes that a subject has one or more risk factors, which are measurable parameters that correlate with the development of a disease, as described in this document and known in the art. A subject with one or more of these risk factors has a greater probability of developing disease or an adverse reaction than a subject without one or more of these risk factors.

[051] Biocompatible refers to materials or compounds that are generally not harmful to the biological functions of a cell or subject and that will not result in any degree of unacceptable toxicity, including allergenic and disease states.

[052] The term bond refers to a direct association between two molecules, due, for example, to covalent, electrostatic, hydrophobic and ionic interactions and / or hydrogen bonds, including interactions such as Petition 870250109095, dated 11 / 27 / 2025, p. 23 / 150 19 / 102 salt bridges and water bridges.

[053] By coding sequence we mean any nucleic acid sequence that contributes to the polypeptide product code of a gene. In contrast, the term non-coding sequence refers to any nucleic acid sequence that does not directly contribute to the polypeptide product code of a gene.

[054] Throughout this disclosure, unless the context requires otherwise, the words includes, include and including will be understood as implying the inclusion of a stated step or element or group of steps or elements, but not the exclusion of any other step or element or group of steps or elements.

[055] By "consisting of" is meant including and limited to everything that follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the listed elements are mandatory or required, and that no other element may be present. By "essentially consisting of" is meant including any elements listed after the phrase and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure of the listed elements. Thus, the phrase "essentially consisting of" indicates that the listed elements are mandatory or required, but that other elements are optional and may or may not be present, depending on whether or not they materially affect the activity or action of the listed elements.

[056] The term endotoxin-free or substantially endotoxin-free generally refers to compositions, solvents and / or containers that contain at most trace amounts (e.g., amounts without clinically adverse physiological effects for a subject) of endotoxin and preferably undetectable amounts of endotoxin. Endotoxins are toxins associated with certain microorganisms, such as bacteria, usually gram-negative bacteria, although endotoxins can be found in gram-positive bacteria, such as Petition 870250109095, dated 11 / 27 / 2025, page 24 / 150 20 / 102 Listeria monocytogenes. The most prevalent endotoxins are lipopolysaccharides (LPS) or lipo-oligosaccharides (LOS) found in the outer membrane of various Gram-negative bacteria and represent a central pathogenic feature in the ability of these bacteria to cause disease. Small amounts of endotoxin in humans can produce fever, decreased blood pressure, and activation of inflammation and coagulation, among other adverse physiological effects.

[057] Therefore, in pharmaceutical production, it is often desirable to remove most or all traces of endotoxin from pharmaceutical products and / or drug containers, because even small amounts can cause adverse effects in humans. A depyrogenation oven can be used for this purpose, as temperatures above 300°C are typically required to break down most endotoxins. For example, based on the primary packaging material, such as syringes or vials, the 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 of endotoxin removal are contemplated, including, for example, chromatography and filtration methods, as described in this document and known in the art.

[058] Endotoxins can be detected using routine techniques known in the art. For example, the Limulus Amoebocyte Lysate assay, which uses horseshoe crab blood, is a very sensitive assay for detecting the presence of endotoxin. In this test, very low levels of LPS can cause detectable coagulation of the limulus lysate due to a powerful enzymatic cascade that amplifies this reaction. Endotoxins can also be quantified by enzyme-linked immunosorbent assay (ELISA). To be substantially free of endotoxins, endotoxin levels may be less than approximately 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.08, 0.09, 0.1, 0.5, 1.0, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, or 10 EU / mg of active compound. Typically, 1 ng of lipopolysaccharide (LPS) Petition 870250109095, dated 11 / 27 / 2025, p. 25 / 150 21 / 102 corresponds to approximately 1-10 EU.

[059] The term “half-maximum effective concentration” or “EC50” refers to the concentration of an agent (e.g., a protein complex) as described in this document at which it induces an intermediate response between the baseline and the maximum after a specified exposure time; the EC50 of a graded dose-response curve therefore represents the concentration of a compound at which 50% of its maximum effect is observed. EC50 also represents the plasma concentration required to obtain 50% of a maximum effect in vivo. Similarly, “EC90” refers to the concentration of an agent or composition at which 90% of its maximum effect is observed. The “EC90” can be calculated from the “EC50” and Hill slope, or it can be determined directly from the data using routine knowledge of the art.In some embodiments, the EC50 of an agent is less than 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 500 nM. In some embodiments, an agent will have an EC50 of approximately 1 nM or less.

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

[061] The term heterologous refers to a feature or element in a coding polypeptide or polynucleotide that is derived from a source Petition 870250109095, dated 11 / 27 / 2025, page 26 / 150 22 / 102 different from the wild-type polypeptide or coding polynucleotide, for example, a resource from a species different from the wild type, or an unnatural engineered resource.

[062] The terms modulate and alter include increasing, potentiating, or stimulating, as well as decreasing or reducing, typically by a statistically significant or physiologically significant amount or degree relative to a control. An increased, stimulated, or augmented amount 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 more than the amount produced by no composition (e.g., the absence of agent) or a control composition.A decreased or reduced quantity is typically a statistically significant quantity and may include a decrease of approximately, or at least approximately, 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, or 1000 times less than the quantity produced by no composition (e.g., the absence of an agent) or a control composition. Examples of comparisons and “statistically significant” values ​​are described in this document.

[063] The terms polypeptide, protein, and peptide are used interchangeably and refer to a polymer of amino acids not limited to any specific length. The term enzyme includes polypeptides or protein catalysts. As used in this document, a proprotein, proenzyme, or zymogen refers to an inactive (or substantially inactive) protein or enzyme that is normally activated by protease cleavage of an activating peptide to generate an active protein or enzyme. Terms include modifications such as myristoylation, sulfation, glycosylation, phosphorylation, and the addition or deletion of signal sequences. The terms polypeptide or protein mean one or more amino acid chains, wherein each chain comprises Petition 870250109095, dated 11 / 27 / 2025, page 27 / 150 23 / 102 amino acids covalently linked by peptide bonds, wherein said polypeptide or protein may comprise a plurality of non-covalent and / or covalently linked chains joined together by peptide bonds, possessing the sequence of native proteins, i.e., proteins produced by naturally occurring and specifically non-recombinant cells, or genetically modified or recombinant cells, and comprise molecules possessing the amino acid sequence of the native protein, or molecules with deletions, additions 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 comprising one or more recombinant DNA molecules, which are typically made of heterologous polynucleotide sequences or combinations of polynucleotide sequences that would not otherwise be found in the cell.

[064] The term polynucleotide and nucleic acid includes mRNA, RNA, cRNA, cDNA, and DNA. The term typically refers to the polymeric form of nucleotides of at least 10 bases in length, ribonucleotides or deoxynucleotides, or a modified form of any type of nucleotide. The term includes single-stranded and double-stranded forms of DNA. The terms isolated DNA and isolated polynucleotide and isolated nucleic acid refer to a molecule that has been isolated free from total genomic DNA of a particular species. Therefore, an isolated DNA segment encoding a polypeptide refers to a segment of DNA that contains one or more coding sequences but is substantially isolated or purified from the total genomic DNA of the species from which the DNA segment is obtained. Also included are non-coding polynucleotides (e.g., primers, probes, oligonucleotides), which do not encode a polypeptide.Also included are recombinant vectors, including, for example, expression vectors, viral vectors, plasmids, cosmids, phagemids, phages, viruses and the like. Petition 870250109095, dated 11 / 27 / 2025, page 28 / 150 24 / 102

[065] Additional coding or non-coding sequences may, but do not necessarily, be present within a polynucleotide described in this document, and a polynucleotide may, but not necessarily, be linked to other molecules and / or support materials. Therefore, a polynucleotide or expressible polynucleotide, regardless of the length of the coding sequence itself, may be combined with other sequences, for example, expression control sequences.

[066] Expression control sequences include regulatory sequences of nucleic acids or the corresponding amino acids, such as promoters, leaders, enhancers, introns, recognition motifs for RNA or DNA-binding proteins, polyadenylation signals, terminators, internal ribosome entry sites (IRES), secretion signals, subcellular localization signals, and the like, which have the ability to affect the transcription or translation, or subcellular or cellular localization of a coding sequence in a host cell. Exemplary expression control sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).

[067] A “promoter” is a regulatory region of DNA capable of binding to RNA polymerase in a cell and initiating transcription of a downstream coding sequence (3' direction). As used in this document, the promoter sequence is bounded at its 3' end by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at detectable levels above the background. A transcription initiation site (conveniently defined by mapping with S1 nuclease) can be found within a promoter sequence, as well as protein-binding domains (consensus sequences) responsible for RNA polymerase binding. Eukaryotic promoters may often, but not always, contain “TATA” boxes and “CAT” boxes. Promoters Petition 870250109095, dated 11 / 27 / 2025, page 29 / 150 25 / 102 prokaryotes contain Shine-Dalgarno sequences in addition to the consensus sequences -10 and -35.

[068] A large number of promoters, including constitutive, inducible, and repressible promoters, from a variety of different sources are well known in the art. Representative sources include, for example, viral, mammalian, insect, plant, yeast, and bacterial cell types, and suitable promoters from these sources are readily available or can be made synthetically, based on publicly available sequences online or, for example, from repositories such as ATCC, as well as other commercial or individual sources. Promoters can be unidirectional (i.e., initiate transcription in one direction) or bidirectional (i.e., initiate transcription in the 3' or 5' direction). Non-limiting examples of promoters include, for example, the bacterial T7 expression system, the bacterial pBAD (araA) expression system, the cytomegalovirus (CMV) promoter, the SV40 promoter, and the RSV promoter.Inducible promoters include the Tet system (U.S. Patents 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 tamoxifen-inducible recombinase system Cre-ERT (Indra et al. Nuc. Acid. Res. (1999) 27 (22): 4324-4327; Nuc. Acid. Res. (2000) 28 (23): e99; U.S. Patent No. 7,112,715; and Kramer & Fussenegger, Methods Mol. Biol. (2005) 308: 123-144) or any promoter known in the art suitable for expression in the desired cells.

[069] An expressible polynucleotide includes a cDNA, RNA, mRNA or other polynucleotide comprising at least one coding sequence and, optionally, at least one expression control sequence, for example, a transcription and / or translation regulatory element, and which can express an encoded polypeptide after introduction into a cell.

[070] The term polypeptide or isolated protein referred to in this document Petition 870250109095, dated 11 / 27 / 2025, p. 30 / 150 26 / 102 means that a protein in question (1) is free from at least some other proteins with which it would normally be found in nature, (2) is essentially free from other proteins of the same source, for example, of the same species, (3) is expressed by a cell of a different species, (4) has been separated from at least about 50 percent of the polynucleotides, lipids, carbohydrates or other materials with which it is associated in nature, (5) is not associated (by covalent or non-covalent interaction) with portions of a protein with which the isolated protein is associated in nature, (6) is operationally associated (by covalent or non-covalent interaction) with a polypeptide with which it is not associated in nature, or (7) does not occur in nature. Such an isolated protein may be encoded by genomic DNA, cDNA, mRNA or other RNA, may be of synthetic origin or any combination thereof.In certain embodiments, the isolated protein is substantially free of proteins or polypeptides or other contaminants found in its natural environment that could interfere with its use (therapeutic, diagnostic, prophylactic, research or other).

[071] In certain embodiments, the purity of any given agent in a composition may be defined. For example, certain compositions may comprise an agent such as a polypeptide agent that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% pure on a protein or weight-weight basis, including all decimal places and intermediate ranges, as measured, for example, and in no way limitingly, by high-performance liquid chromatography (HPLC), a well-known method of column chromatography frequently used in biochemistry and analytical chemistry to separate, identify and quantify compounds.

[072] The term reference sequence generally refers to a nucleic acid coding sequence, or amino acid sequence, to which another sequence is being compared. All polypeptide sequences and Petition 870250109095, dated 11 / 27 / 2025, p. 31 / 150 27 / 102 polynucleotides described in this document are included as reference sequences, including those described by name and those described in the Tables and Sequence Listing.

[073] Certain embodiments include variants and biologically active fragments of the proteins / polypeptides described in this document and the polynucleotides that encode them. Variants contain one or more substitutions, additions, deletions and / or insertions relative to a reference polypeptide or polynucleotide (see, for example, the Tables and the Sequence Listing). A variant polypeptide or polynucleotide comprises a nucleotide or amino acid sequence with at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity or similarity or homology with a reference sequence, as described in this document, and retains substantially the activity of that reference sequence.Also included are sequences that consist of or differ from reference sequences 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, 150 or more amino acids or nucleotides and that retain substantially less of the activity of that reference sequence. In certain embodiments, the additions or deletions include C-terminal and / or N-terminal additions and / or deletions.

[074] The terms sequence identity or, for example, comprising a sequence 50% identical to, as used in this document, refer to the extent to which sequences are identical on a nucleotide-by-nucleotide base or amino acid-by-amino acid base within a comparison window. Thus, a percentage of sequence identity can be calculated by comparing two perfectly aligned sequences within the comparison window, determining the number of positions where the identical nucleic acid base (e.g., A, T, C, G, I) or amino acid residue is found. Petition 870250109095, dated 11 / 27 / 2025, p. 32 / 150 28 / 102 identical (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) occurs in both sequences to yield 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 sequence identity percentage. Optimal sequence alignment to align a comparison window can be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, Wis., USA) or by inspection, and the best alignment (i.e., resulting in the highest percentage of homology in the comparison window) generated by any of several selected methods.Reference can also be made to the BLAST family of programs, as, for example, reported by Altschul et al., Nucl. Acids Res. 25:3389, 1997.

[075] The term solubility refers to the property of an agent described in this document to dissolve in a liquid solvent and form a homogeneous solution. Solubility is typically expressed as a concentration, either by mass of solute per unit volume of solvent (g of solute per kg of solvent, g per dL (100 mL), mg / mL, etc.), molarity, molality, mole fraction, or other similar descriptions of concentration. The maximum equilibrium amount of solute that can be dissolved per amount of solvent is the solubility of that solute in that solvent under the specified conditions, including temperature, pressure, pH, and the nature of the solvent. In certain embodiments, solubility is measured at physiological pH or another pH, for example, at pH 5.0, pH 6.0, pH 7.0, pH 7.4, pH 7.6, pH 7.8, or pH 8.0 (e.g., about pH 5-8). In certain methods, solubility is measured in water or a physiological buffer, such as PBS or NaCl (with or without NaPO4).In specific applications, solubility is measured at a relatively lower pH (e.g., Petition 870250109095, dated 11 / 27 / 2025, page 33 / 150 29 / 102 pH 6.0) and a relatively higher salt concentration (e.g., 500mM NaCl and 10mM NaPO4). In certain embodiments, solubility is measured in a biological fluid (solvent), such as blood or serum. In certain embodiments, the temperature may be close to room temperature (e.g., around 20, 21, 22, 23, 24, 25°C) or close to body temperature (37°C). In certain embodiments, an agent has a solubility of at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90 or 100 mg / ml at room temperature or at 37°C.

[076] A subject or a subject in need of it or a patient or a patient in need of it includes a mammalian subject, such as a human subject.

[077] “Substantially” or “essentially” means almost entirely or completely, for example, 95%, 96%, 97%, 98%, 99% or more of a given quantity.

[078] “Statistically significant” means that it was unlikely that the result occurred by chance. Statistical significance can be determined by any method known in the art. Commonly used significance measures include the p-value, which is the frequency or probability with which the observed event would occur if the null hypothesis were true. If the p-value obtained is less than the significance level, then the null hypothesis is rejected. In simple cases, the significance level is set at a p-value of 0.05 or less.

[079] Therapeutic response refers to the improvement of symptoms (sustained or not) based on the administration of one or more therapeutic agents.

[080] As used in this document, the terms therapeutically effective amount, therapeutic dose, prophylactically effective amount, or diagnostically effective amount refers to the quantity of an agent needed to elicit the desired biological response after administration. Petition 870250109095, dated 11 / 27 / 2025, page 34 / 150 30 / 102

[081] As used in this document, treatment of a subject (e.g., a mammal, such as a human) or a cell is any type of intervention used in an attempt to alter the natural course of the subject or cell. Treatment includes, but is not limited to, administration of a pharmaceutical composition and may be performed prophylactically or subsequent to the onset of a pathological event or contact with an etiological agent. Also included are “prophylactic” treatments, which may be aimed at reducing the rate of progression of the disease or condition being treated, delaying the onset of that disease or condition, or reducing the severity of its onset. “Treatment” or “prophylaxis” does not necessarily indicate complete eradication, cure, or prevention of the disease or condition, or symptoms associated with it.

[082] The term “wild type” refers to a gene or gene product (e.g., a polypeptide) that is observed most frequently in a population and is therefore arbitrarily designated as the “normal” or “wild type” form of the gene.

[083] Each embodiment in this specification shall apply to all other embodiments, unless expressly stated otherwise. Protein Complexes

[084] The embodiments of this disclosure generally relate to pharmaceutical compositions comprising protein complexes of alpha-2-macroglobulin (A2M) proteins and serine protease proteins, such as PPE. Herein, certain serine proteases are capable of killing cancer cells by direct contact or administration to tumors (e.g., intratumoral administration), regardless of their genetic abnormalities, and are relatively harmless to non-cancerous or healthy cells (see, for example, WO 2018 / 232273; WO / 2020 / 132465; PCT / US2021 / 046453; and PCT / 2021 / 046467). However, in some cases, systemic administration of independent serine proteases, such as PPE, may negatively affect the Petition 870250109095, dated 11 / 27 / 2025, page 35 / 150 31 / 102 coagulation, for example, by inducing fibrinogen cleavage. This disclosure relates in part to the discovery that protein complexes of human A2M and human serine proteases, such as PPE, not only sterically protect serine protease from serine protease inhibitors in plasma, maintaining CD95 protease cleavage and cancer cell killing activity, but also reduce the negative impact of serine protease on coagulation, for example, by sterically inhibiting or reducing the ability of the complexed serine protease to cleave fibrinogen.This disclosure also refers to the discovery of an ideal molar ratio range between [A2M proteins]:[serine protease proteins] in the protein complex that provides a balance between retaining CD95 cleavage and the cancer cell-killing activity of serine protease and reducing its negative impact on coagulation, as measured, for example, by reduced prothrombin time or decreased fibrinogen cleavage compared to serine protease alone.

[085] Certain embodiments therefore include pharmaceutical compositions comprising a protein complex of: (a) alpha-2-macroglobulin (A2M) proteins; and (b) serine protease proteins, wherein (a) and (b) are present in the composition in a molar ratio [(a):(b)] of about 1:3 to about 1:1, including wherein the A2M proteins of (a) and the serine protease proteins of (b) are linked together in the protein complex. In some embodiments, the protein complex: (i) retains the CD95 protease (Fas receptor) cleavage activity and cancer cell killing activity of (b); (ii) sterically prevents the binding of (b) to fibrinogen, thereby reducing or inhibiting the fibrinogen cleavage activity of (b); and (iii) sterically prevents (b) from binding to serine protease inhibitors, such as alpha-1 antitrypsin (A1AT), thereby protecting (b) from inhibition by serine protease inhibitors.

[086] In certain embodiments, A2M proteins join in the protein complex as A2M monomers or A2M multimers, for example, as Petition 870250109095, dated 11 / 27 / 2025, page 36 / 150 32 / 102 A2M dimers, such as A2M homodimers, such as A2M trimers, such as A2M homotrimers or such as A2M tetramers, such as A2M homotetramers. In specific embodiments, the A2M proteins of (a) join as A2M homotetramers, and the serine protease proteins of (b) are linked by the A2M homotetramers in the protein complex. In specific embodiments, each protein complex is composed of a set of A2M homotetramers (i.e., four A2M proteins, either as (i) four whole A2M proteins or as (ii) up to eight A2M fragments resulting from the cleavage of the bait region of the whole A2M proteins by serine proteases as they bind to the A2M homotetramer complex) and two serine protease proteins (see, for example, Figure 1).

[087] In certain embodiments, the protein complex sterically prevents the binding of (b) to larger molecules, but does not sterically prevent the binding of (b) to smaller molecules. That is, as noted above, the protein complex sterically prevents the binding of (b) to larger molecules, such as serine protease proteins (e.g., A1AT), fibrinogen, and / or plasma antibodies. Thus, in some embodiments, the protein complex protects (b) from inhibition by serine proteases and also reduces / inhibits the ability of (b) 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 will protect (b) from anti-PPE plasma antibodies or from the generation of anti-PPE plasma antibodies. In some cases, this provides clinical advantages in administering non-human protein drugs, such as PPE, to humans, who may generate anti-drug antibodies.In contrast, the protein complex does not sterically prevent the binding of (b) to smaller molecules, such as CD95. Thus, in specific embodiments, (b) in the protein complex cleaves CD95 and kills cancer cells, but does not substantially cleave fibrinogen.

[088] The pharmaceutical composition and protein complexes described in this Petition 870250109095, dated 11 / 27 / 2025, page 37 / 150 33 / 102 documents comprise an alpha-2-macroglobulin (A2M) protein, for example, a 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 of four identical ~180 kDa subunits forming a hollow, cylinder-like structure. It may present multiple target peptide bonds to attack proteases in its central "bait" domain.Human A2M “captures” serine proteases, such as PPE: here, after the serine protease binds to and cleaves the bait region, a conformational change is induced in A2M that captures the serine protease in such a way that the protease remains active towards low molecular weight substrates but has significantly reduced activity towards high molecular weight substrates (see, for example, Van Gateen and Itoh, Frontiers in Immunology, 12, 2021; and Harwood et al., Molecular & Cellular Proteomics, 20, 2021). The amino acid sequence of complete and mature human A2M (without signal peptide) is provided in Table A1 below. Table A1. A2M Proteins Protein Sequence SEQ ID NO: Complete human A2M signal peptide underlined decoy region in bold and underlined MGKNKLLHPSLVLLLLVLLPTDASVSGKPQYMVLVPSLLHTETTEKGCVLL SYLNETVTVSASLESVRGNRSLFTDLEAENDVLHCVAFAVPKSSSNEEVM FLTVQVKGPTQEFKKRTTVMVKNEDSLVFVQTDKSIYKPGQTVKFRVVS MDENFHPLNELIPLVYIQDPKGNRIAQWQSFQLEGGLKQFSFPLSSEPFQ GSYKVVVQKKSGGRTEHPFTVEEFVLPKFEVQVTVPKIITILEEEMNVSVC GLYTYGKPVPGHVTVSICRKYSDASDCHGEDSQAFCEKFSGQLNSHGCF YQQVKTKVFQLKRKEYEMKLHTEAQIQEEGTVVELTGRQSSEITRTITKLS FVKVDSHFRQGIPFFGQVRLVDGKGVPIPNKVIFIRGNEANYYSNATTDE HGLVQFSINTTNVMGTSLTVRVNYKDRSPCYGYQWVSEEHEEAHHTAYL VFSPSKSFVHLEPMSHELPCGHTQTVQAHYILNGGTLLGLKKLSFYYLIMA KGGIVRTGTHGLLVKQEDMKGHFSISIPVKSDIAPVARLLIYAVLPTGDVIG DSAKYDVENCLANKVDLSFSPSQSLPASHAHLRVTAAPQSVCALRAVDQ SVLLMKPDAELSASSVYNLLPEKDLTGFPGPLNDQDNEDCINRHNVYINGI TYTPVSSTNEKDMYSFLEDMGLKAFTNSKIRKPKMCPQLQQYEMHGPE GLRVGFYESDVMGRGHARLVHVEEPHTETVRKYFPETWIWDLVVVNSA GVAEVGVTVPDTITEWKAGAFCLSEDAGLGISSTASLRAFQPFFVELTMPYSVIRGEAFTLKATVLNYLPKCIRVSVQLEASPAFLAVPVEKEQAPHCICA NGRQTVSWAVTPKSLGNVNFTVSAEALESQELCGTEVPSVPEHGRKDT VIKPLLVEPEGLEKETTFNSLLCPSGGEVSEELSLKLPPNVVEESARASVS VLGDILGSAMQNTQNLLQMPYGCGEQNMVLFAPNIYVLDYLNETQQLTP EIKSKAIGYLNTGYQRQLNYKHYDGSYSTFGERYGRNQGNTWLTAFVLK TFAQARAYIFIDEAHITQALIWLSQRQKDNGCFRSSGSLLNNAIKGGVEDE VTLSAYITIALLEIPLTVTHPVVRNALFCLESAWKTAQEGDHGSHVYTKALL 22 Petition 870250109095, dated 11 / 27 / 2025, page 38 / 150 34 / 102 AYAFALAGNQDKRKEVLKSLNEEAVKKDNSVHWERPQKPKAPVGHFYE PQAPSAEVEMTSYVLLAYLTAQPAPTSEDLTSATNIVKWITKQQNAQGGF SSTQDT WALH ALSKYG AATFTRTGKAAQVTIQSSGTFSSKFQVD NNN RL LLQQVSLPELPGEYSMKVTGEGCVYLQTSLKYNILPEKEEFPFALGVQTL PQTCDEPKAHTSFQISLSVSYTGSRSASNMAIVDVKMVSGFIPLKPTVKM LERSNHVSRTEVSSNHVLIYLDKVSNQTLSLFFTVLQDVPVRDLKPAIVKV YDYYETDEFAIAEYNAPCSKDLGNA A2M humana madura região isca em negrito e sublinhada SVSGKPQYMVLVPSLTHETTETKGCVLLSYLNETVTVSASLESVRGNRSL FTDLEAENDVLHCVAFAVPKSSSNEEVMFLTVQVKGPTQEFKKRTTVMV KNEDSLVFVQTDKSIYKPGQTVKFRVVSMDENFHPLNELIPLVYIQDPKG NRIAQWQSFQLEGGLKQFSFPLSSEPFQGSYKVVVQKKSGGRTEHPFTV EEFVLPKFEVQVTVPKIITILEEEMNVSVCGLYTYGKPVPGHVSICRKYS DASDCHGEDSQAFCEKFSGQLNSHGCFYQQVKTKVFQLKRKEYEMKLH TEAQIQEEGTVVELTGRQSSEITRTITKLSFVKVDSHFRQGIPFFGQVRLV DGKGVPIPNKVIFIRGNEANYYSNATTDEHGLVQFSINTTNVMGTSLTVRV NYKDRSPCYGYQWVSEEHEAHHTAYLVFSPSKSFVHLEPMSHELPCG HTQTVQAHYILNGGTLLGLKKLSFYYLIMAKGGIVRTGTHGLLVKQEDMK GHFSISIPVKSDIAPVARLLIYAVLPTGDVIGDSAKYDVENCLANKVDLSFSPSQSLPASHAHLRVTAAPQSVCALRAVDQSVLLMKPDAELSASSVYNLL PEKDLTGFPGPLNDQDNEDCINRHNVYINGITYTPVSSTNEKDMYSFLED MGLKAFTNSKIRKPKMCPQLQQYEMHGPEGLRVGFYESDVMGRGHAR LVHVEEPHTETVRKYFPETWIWDLVVVNSAGVAEVGVTVPDTITEWKAG AFCLSEDAGLGISSTASLRAFQPFFVELTMPYSVIRGEAFTLKATVLNYLP KCIRVSVQLEASPAFLAVPVEKEQAPHCICANGRQTVSWAVTPKSLGNV NFTVSAEALESQELCGTEVPSVPEHGRKDTVIKPLLVEPEGLEKETTFNSL LCPSGGEVSEELSLKLPPNVVEESARASVSVLGDILGSAMQNTQNLLQM PYGCGEQNMVLFAPNIYVLDYLNETQQLTPEIKSKAIGYLNTGYQRQLNY KHYDGSYSTFGERYGRNQGNTWLTAFVLKTFAQARAYIFIDEAHITQALI WLSQRQKDNGCFRSSGSLLNNAIKGGVEDEVTLSAYITIALLEIPLTVTHP VVRNALFCLESAWKTAQEGDHGSHVYTKALLAYAFALAGNQDKRKEVLK SLNEEAVKKDNSVHWERPQKPKAPVGHFYEPQAPSAEVEMTSYVLLAYL TAQPAPTSEDLTSATNIVKWITKQQNAQGGFSSTQDTVVALHALSKYGAA TFTRTGKAAQVTIQSSGTFSSKFQVDNNNRLLLQQVSLPELPGEYSMKVT GEGCVYLQTSLKYNILPEKEEFPFALGVQTLPQTCDEPKAHTSFQISLSVS YTGSRSASNMAIVDVKMVSGFIPLKPTVKMLERSNHVSRTEVSSNHVLIY LDKVSNQTLSLFFTVLQDVPVRDLKPAIVKVYDYYETDEFAIAEYNAPCSK DLGNA 23

[089] Thus, in some embodiments, the A2M protein portion of the protein complex comprises, 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 thereof comprises, consists of, or essentially consists of about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1300, or 1400 contiguous amino acids from a sequence selected from Table A1.

[090] For example, in specific modalities, the functional fragment of the same is composed of approximately 1-1400, 1-1300, 1-1200, 1-1100, 1-1000, Petition 870250109095, dated 11 / 27 / 2025, page 39 / 150 35 / 102 1-900, 1-800, 1-700, 1-600, 1-500, 1-400, 1-300, 1-200, 100-1400, 100-1300, 100-1200, 100-1100, 100-1000, 100-900, 100-800, 100-700, 100-600, 100-500, 100-400, 100-300, 100-200, 200-1400, 200-1300, 200-1200, 200-1100, 200-1000, 200-900, 200-800, 200-700, 200-600, 200-500, 200-400, 200-300, 300-1400, 300-1300, 300-1200, 300-1100, 300-1000, 300-900, 300-800, 300-700, 300-600, 300-500, 300-400, 400-1400, 400-1300, 400-1200, 400-1100, 400-1000, 400-900, 400-800, 400-700, 400-600, 400-500 500-1400, 500-1300, 500-1200, 500-1100, 500-1000, 500-900, 500-800, 500-700, 500-600, 600-1400, 600-1300, 600-1200, 600-1100, 600-1000, 600-900, 600-800, 600-700, 700-1400, 700-1300, 700-1200, 700-1100, 700-1000, 700-900, 700-800, 800-1400, 800-1300, 800-1200, 800-1100, 800-1000, 800-900, 900-1400, 900-1300, 900-1200, 900-1100, 900-1000, 1000-1400, 1000-1300, 1000-1200, 1000-1100, 1100-1400, 1100-1300, 1100-1200, 1200-1400 or 1200-1300 residues from a sequence selected from Table A1.In some embodiments, the functional fragment thereof is able to form an A2M homotetramer and capture or otherwise bind serine protease (such as PPE) to the protein complex in a configuration that sterically prevents the binding of serine protease to serine protease inhibitors such as A1AT, retains the CD95 protease cleavage activity and the serine protease cancer cell killing activity, and / or inhibits or reduces the ability of serine protease to cleave fibrinogen.

[091] In certain modalities, the A2M portion of the protein complex enhances uptake in cancer cells compared to serine protease alone. For example, A2M binds to LPR1 and GRP78 receptors, which are expressed in both normal and cancerous cells. In fact, elevated GRP78 levels generally correlate with higher pathological grade, recurrence, and lower patient survival in breast, liver, prostate, colon, and gastric cancers (see, for example, Lee, Cancer Res. 67:3496-3499, 2007), and in some cases, A2M's ability to bind to GRP78 enhances selective targeting to Petition 870250109095, dated 11 / 27 / 2025, page 40 / 150 36 / 102 cancer cells expressing GRP78.

[092] In some embodiments, the A2M portion of the protein complex is fused or otherwise conjugated to an antibody, or an antigen-binding fragment thereof. In some embodiments, the antibody, or antigen-binding fragment thereof, binds specifically to a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA). Exemplary TAAX and TSAs include, among others, alpha-fetoprotein (AFP), epithelial tumor antigen (ETA), tyrosinase, Her2 / human neutrophil, 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, VEGFR2, VEGR-3, NRP2, CD30, CD33, CD37, CD40, CD44, CD51, CD52, CD56, CD74, CD80, CD152, CD200, CD221, CCR4, HLA-DR, CTLA-4, NPC-1C, tenascin, vimentin, receptor of insulin-like growth factor 1 (IGF-1R), alpha-fetoprotein,Insulin-like growth factor 1 (IGF-1), carbonic anhydrase 9 (CA-IX), carcinoembryonic antigen (CEA), guanylyl cyclase C, NYESO-1, p53, survivin, integrin αvβ3, integrin α5β1, folate receptor 1, transmembrane glycoprotein NMB, fibroblast-activating protein alpha (FAP), glycoprotein 75, TAG-72, MUC1, MUC16 (or CA-125), phosphatidylserine, prostate-specific membrane antigen (PMSA), NR-LU-13 antigen, TRAIL-R1, tumor necrosis factor receptor 10b superfamily member (TNFRSF10B or TRAIL-R2), SLAM family member 7 (SLAMF7), pancarcinoma antigen EGP40, B-cell activating factor (BAFF), receptor-derived growth factor platelets, EpCAM glycoprotein (17-1A), programmed cell death-1, protein disulfide isomerase (PDI), Regenerative Liver Phosphatase 3 (PRL-3), prostatic acid phosphatase, Lewis-Y antigen, GD2 (a disialoganglioside expressed in tumors of neuroectodermal origin), glypican-3 (GPC3), and mesothelin. Petition 870250109095, dated 11 / 27 / 2025, page 41 / 150 37 / 102

[093] The pharmaceutical composition and protein complexes described in this document comprise a serine protease protein. 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 complete wild-type serine protease proprotein amino acid sequences are provided in Table S1 below. Table S1. Complete serine protease sequences Name Sequence SEQ ID NO: Proprotein PPE WT, signal peptide underlined MLRLLVVASLVLYGHSTQDFPETNARWGGTEAQRNSWPS QISLQYRSGSSWAHTCGGTLIRQNWVMTAAHCVDRELTFR WVGEHNLNQNDGTEQYVGVQKIVVHPYWNTDDVAAGYDI ALLRLAQSVTLNSYVQLGVLPRAGTILANNSPCYITGWGLTR TNGQLAQTLQQAYLPTVDYAICSSSSYWGSTVKNSMVCAG GDGVRSGCQGDSGGPLHCLVNGQYAVHGVTSFVSRLGCN VTRKPTVFTRVSAYISWINNVIASN 1 ELANE human WT, peptidase domain residues 30-247 or residues 30-267, signal peptide underlined MTLGRRLACLFLACVLPALLLGGTALASEIVGGRRARPHAW PFMVSLQLRGGHFCGATLIAPNFVMSAAHCVANVNVRAVR WLGAHNLSRREPTRQVFAVQRIFENGYDPVNLLNDIVILQL NGSATINANVQVAQLPAQGRRLGNGVQCLAMGWGLLGRN RGIASVLQELNVTVVTSLCRRSNVCTLVRGRQAGVCFGDSG SPLVCNGLIHGIASFVRGGCASGLYPDAFAPVAQFVNWIDSII QRSEDNPCPHPRDPDPASRTH 2 CTSG humana WT, peptidase domain residues 21-243 or residues 21-255,underlined signal peptide MQPLLLLLAFLLPTGAEAGEIIGGRESRPHSRPYMAYLQIQS PAGQSRCGGFLVREDFVLTAAHCWGSNINVTLGAHNIQRR ENTQQHITARRAIRHPQYNQRTIQNDIMLLQLSRRVRRNRN VNPVALPRAQEGLRPGTLCTVAGWGRVSMRRGTDTLREV QLRVQRDRQCLRIFGSYDPRRQICVGDRRERKAAFKGDSG GPLLCNNVAHGIVSYGKSSGVPPEVFTRVSSFLPWIRTTMR SFKLLDQMETPL 3 PR3 human WT, peptidase domain residues 28-248, underlined signal peptide MAHRPPSPALASVLLALLLSGAARAAEIVGGHEAQPHSRPY MASLQMRGNPGSHFCGGTLIHPSFVLTAAHCLRDIPQRLVN WLGAHNVRTQEPTQQHFSVAQVFLNNYDAENKLNDVLLIQ LSSPANLSASVATVQLPQQDQPVPHGTQCLAMGWGRVGA HDPPAQVLQELNVTVVTFFCRPHNICTFVPRRKAGICFGDS GGPLICDGIIQGIDSFVIWGCATRLFPDFFTRVALYVDWIRST LRRVEAKGRP 4 Human granzyme B WT, peptidase domain residues 21-245, signal peptide underlined MQPILLLLAFLLLPRADAGEIIGGHEAKPHSRPYMAYLMIWD QKSLKRCGGFLIRDDFVLTAAHCWGSSINVTLGAHNIKEQEP TQQFIPVKRPIPHPAYNPKNFSNDIMLLQLERKAKRTRAVQP LRLPSNKAQVKPGQTCSVAGWGQTAPLGKHSHTLQEVKMT VQEDRKCESDLRHYYDSTIELCVGDPEIKKTSFKGDSGGPL VC N KVAQGIVSYGRNNGMPPRACTKVSSFVHWIKKTM KRY 21,

[094] Thus, in certain embodiments, the serine protease protein comprises, consists of, or essentially consists of a complete serine protease proprotein selected from Table S1, including biologically different variants. Petition 870250109095, dated 11 / 27 / 2025, page 42 / 150 38 / 102 active and fragments thereof. In specific embodiments, the serine protease comprises, consists of, or essentially consists of an amino acid sequence that is at least 80, 85, 90, 95, 98, or 100% identical to a sequence selected from Table S1, or a functional fragment thereof.

[095] In certain embodiments, the serine protease protein is composed of the active peptidase domain of a serine protease. Exemplary peptidase domain sequences of PPE (including exemplified mutants thereof), human ELANE, human CTSG, and a human PR3 are provided in Table S2 below. Table S2. Peptidase Dominant Sequences Exemplificativas Nome Sequência SEQ ID NO: PPE F mutante Q211F WGGTEAQRNSWPSQISLQYRSGSSWAHTCGGTLIRQNWVMTAAHCV DRELTFRVWGEHNLNQNDGTEQYVGVQKIVVHPYWNTDDVAAGYDIA LLRLAQSVTLNSYVQLGVLPRAGTILANNSPCYITGWGLTRTNGQLAQT LQQAYLPTVDYAICSSSSYWGSTVKNSMVCAGGDGVRSGCFGDSGGP LHCLVNGQYAVHGVTSFVSRLGCNVTRKPTVFTRVSAYISWINNVIASN 5 PPE H mutante T55A WGGTEAQRNSWPSQISLQYRSGSSWAHACGGTLIRQNWVMTAAHCV DRELTFRVWGEHNLNQNDGTEQYVGVQKIVVHPYWNTDDVAAGYDIA LLRLAQSVTLNSYVQLGVLPRAGTILANNSPCYITGWGLTRTNGQLAQT HCV DRELTFRVWGEHNLNQNDGTEQYVGVQKIVVHPYWNTDDVAAGYDIA LLRLAQSVTLNSYVQLGVLPRAGTILANNSPCYITGWGLTRTNGQLAQT LQQAYLPTVDYAICSSSSYWGSTVKNSMVCAGGDGVRSGCFGDSGGP LHCLVNGQYAVHGVTSFVSRLGCNVTRKPTVFTRVSAYISWINNVIASN 7 PPE A mutant N241A WGGTEAQRNSWPSQISLQYRSGSSWAHTCGGTLIRQNWVMTAAHCVDRELTFRVWGEHNLNQNDGTEQYVGVQKIVVHPYWNTDDVAAGYDIA LLRLAQSVTLNSYVQLGVLPRAGTILANNSPCYITGWGLTRTNGQLAQT LQQAYLPTVDYAICSSSSYWGSTVKNSMVCAGGDGVRSGCQGDSGGP LHCLVNGQYAVHGVTSFVSRLGCAVTRKPTVFTRVSAYISWINNVIASN 8 PPE B mutante N241Y WGGTEAQRNSWPSQISLQYRSGSSWAHTCGGTLIRQNWVMTAAHCV DRELTFRVWGEHNLNQNDGTEQYVGVQKIVVHPYWNTDDVAAGYDIA LLRLAQSVTLNSYVQLGVLPRAGTILANNSPCYITGWGLTRTNGQLAQT LQQAYLPTVDYAICSSSSYWGSTVKNSMVCAGGDGVRSGCQGDSGGP LHCLVNGQYAVHGVTSFVSRLGCYVTRKPTVFTRVSAYISWINNVIASN 9 PPE C mutante R75A WGGTEAQRNSWPSQISLQYRSGSSWAHTCGGTLIRQNWVMTAAHCV DAELTFRVWGEHNLNQNDGTEQYVGVQKIVVHPYWNTDDVAAGYDIA LLRLAQSVTLNSYVQLGVLPRAGTILANNSPCYITGWGLTRTNGQLAQT LQQAYLPTVDYAICSSSSYWGSTVKNSMVCAGGDGVRSGCQGDSGGP LHCLVNGQYAVHGVTSFVSRLGCNVTRKPTVFTRVSAYISWINNVIASN 10 PPE D mutante R75E WGGTEAQRNSWPSQISLQYRSGSSWAHTCGGTLIRQNWVMTAAHCV DEELTFRWVGEHNLNQNDGTEQYVGVQKIVVHPYWNTDDVAAGYDIA LLRLAQSVTLNSYVQLGVLPRAGTILANNSPCYITGWGLTRTNGQLAQT LQQAYLPTVDYAICSSSSYWGSTVKNSMVCAGGDGVRSGCQGDSGGP LHCLVNGQYAVHGVTSFVSRLGCNVTRKPTVFTRVSAYISWINNVIASN 11 PPE Emutant VVGGTEAQRNSWPSQISLQYRSGSSWAHTCGGTLIRQNWVMTAAHCV DRELTFRVVVGEHNLNQNDGTEQYVGVQKIVVHPYWNTDDVAAGYDIA 12 Petition 870250109095, dated 11 / 27 / 2025, page 43 / 150 39 / 102 Q211A LLRLAQSVTLNSYVQLGVLPRAGTILANNSPCYITGWGLTRTNGQLAQT LQQAYLPTVDYAICSSSSYWGSTVKNSMVCAGGDGVRSGCAGDSGGP LHCLVNGQYAVHGVTSFVSRLGCNVTRKPTVFTRVSAYISWINNVIASN PPE G mutante R237A WGGTEAQRNSWPSQISLQYRSGSSWAHTCGGTLIRQNWVMTAAHCV DRELTFRVWGEHNLNQNDGTEQYVGVQKIVVHPYWNTDDVAAGYDIA LLRLAQSVTLNSYVQLGVLPRAGTILANNSPCYITGWGLTRTNGQLAQT LQQAYLPTVDYAICSSSSYWGSTVKNSMVCAGGDGVRSGCQGDSGGP LHCLVNGQYAVHGVTSFVSALGCNVTRKPTVFTRVSAYISWINNVIASN 13 PPE I mutante S214A WGGTEAQRNSWPSQISLQYRSGSSWAHTCGGTLIRQNWVMTAAHCV DRELTFRVWGEHNLNQNDGTEQYVGVQKIVVHPYWNTDDVAAGYDIA LLRLAQSVTLNSYVQLGVLPRAGTILANNSPCYITGWGLTRTNGQLAQT LQQAYLPTVDYAICSSSSYWGSTVKNSMVCAGGDGVRSGCQGDAGGP LHCLVNGQYAVHGVTSFVSRLGCNVTRKPTVFTRVSAYISWINNVIASN 14 PPE J mutante D74A WGGTEAQRNSWPSQISLQYRSGSSWAHTCGGTLIRQNWVMTAAHCV ARELTFRVWGEHNLNQNDGTEQYVGVQKIVVHPYWNTDDVAAGYDIA LLRLAQSVTLNSYVQLGVLPRAGTILANNSPCYITGWGLTRTNGQLAQT LQQAYLPTVDYAICSSSSYWGSTVKNSMVCAGGDGVRSGCQGDSGGP LHCLVNGQYAVHGVTSFVSRLGCNVTRKPTVFTRVSAYISWINNVIASN 15 PPE WTWGGTEAQRNSWPSQISLQYRSGSSWAHTCGGTLIRQNWVMTAAHCV DRELTFRVWGEHNLNQNDGTEQYVGVQKIVVHPYWNTDDVAAGYDIA LLRLAQSVTLNSYVQLGVLPRAGTILANNSPCYITGWGLTRTNGQLAQT LQQAYLPTVDYAICSSSSYWGSTVKNSMVCAGGDGVRSGCQGDSGGP LHCLVNGQYAVHGVTSFVSRLGCNVTRKPTVFTRVSAYISWINNVIASN 16 human ELANE IVGGRRARPHAWPFMVSLQLRGGHFCGATLIAPNFVMSAAHCVANVNV RAVRWLGAHNLSREPTRQVFAVQRIFENGYDPVNLLNDIVILQLNGSA TINANVQVAQLPAQGRRLGNGVQCLAMGWGLLGRNRGIASVLQELNVT WTSLCRRSNVCTLVRGRQAGVCFGDSGSPLVCNGLIHGIASFVRGGC ASGLYPDAFAPVAQFVNWIDSIIQRSEDNPCPHPRDPDPASRTH 17 human CTSG IIGGRESRPHSRPYMAYLQIQSPAGQSRCGGFLVREDFVLTAAHCWGS NINVTLGAHNIQRRENTQQHITARRAIRHPQYNQRTIQNDIMLLQLSRRV RRNRNVNPVALPRAQEGLRPGTLCTVAGWGRVSMRRGTDTLREVQLR VQRDRQCLRIFGSYDPRRQICVGDRRERKAAFKGDSGGPLLCNNVAHG IVSYGKSSGVPPEVFTRVSSFLPWIRTTMRSFKLLDQMETPL 18 human PR3 IVGGHEAQPHSRPYMASLQMRGNPGSHFCGGTLIHPSFVLTAAHCLRDI PQRLVNVVLGAHNVRTQEPTQQHFSVAQVFLNNYDAENKLNDVLLIQLS SPANLSASVATVQLPQQDQPVPHGTQCLAMGWGRVGAHDPPAQVLQ ELNVTVVTFFCRPHNICTFVPRRKAGICFGDSGGPLICDGIIQGIDSFVIWGCATRLFPDFFTRVALYVDWIRSTLRRVEAKGRP 19 Human Granzyme B IIGGHEAKPHSRPYMAYLMIWDQKSLKRCGGFLIRDDFVLTAAHCWGSS INVTLGAHNIKEQEPTQQFIPVKRPIPHPAYNPKNFSNDIMLLQLERKAKR TRAVQPLRLPSNKAQVKPGQTCSVAGWGQTAPLGKHSHTLQEVKMTV QEDRKCESDLRHYYDSTIELCVGDPEIKKTSFKGDSGGPLVCNKVAQGI VSYGRNNGMPPRACTKVSSFVHWIKKTMKRY 20

[096] Thus, in some embodiments, the serine protease protein comprises, consists of, or essentially consists of a serine protease peptidase domain sequence selected from Table S2, including biologically active variants and fragments thereof. In specific embodiments, the serine protease protein comprises, consists of, or essentially consists of an amino acid sequence that is at least 80, 85, 90, 95, 98, or 100% identical to a sequence selected from Table S2, or a functional fragment of Petition 870250109095, dated 11 / 27 / 2025, page 44 / 150 40 / 102 same.

[097] In some embodiments, the serine protease protein is a PPE protein, for example, where: a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 5, and retaining the Q211F amino acid substitution; a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 6, and retaining the T55A amino acid substitution; a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 7, and retaining the amino acid substitutions Q211F and T55A; a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 8, and retaining the N241A amino acid substitution; a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 9, and retaining the N241Y amino acid substitution; a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 10, and retaining the R75A amino acid substitution; a modified PPE protein comprising, consisting of, or consisting of Petition 870250109095, dated 11 / 27 / 2025, p. 45 / 150 41 / 102 essentially in an amino acid sequence that is at least 80, 85, 90, 95, 98, 99 or 100% identical to SEQ ID NO: 11, and that retains the R75E amino acid substitution; a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 12, and retaining the Q211A amino acid substitution; a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 13, and retaining the R237A amino acid substitution; a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 14, and retaining the S214A amino acid substitution; a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 15, and retaining the D74A amino acid substitution; and a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 16. In some embodiments, the serine protease protein is a human ELANE protein, for example, wherein the human ELANE protein comprises, 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 a human CTSG protein, for example, wherein the human CTSG protein comprises, consists of, or consists of Petition 870250109095, dated 11 / 27 / 2025, p. 46 / 150 42 / 102 essentially in 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 a human PR3 protein, for example, wherein the human PR3 protein comprises, 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, wherein the human granzyme B protein comprises, 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.

[098] As noted above, in certain embodiments, (a) and (b) are present in the composition in a molar ratio of [(a):(b)] ranging from about 1:3 to about 1:1, for example, a molar ratio of about 1:3, 1:2.9, 1:2.8, 1:2.7, 1:2.6, 1:2.5, 1:2.4, 1:2.3, 1:2.1, 1:2, 1:1.9, 1:1.8, 1:1.7, 1:1.6, 1:1.5, 1:1.4, 1:1.3, 1:1.2, 1:1.1 or 1:1. In specific aspects, the molar ratio defined in this document retains the CD95 protease cleavage and serine protease cancer cell killing activity, sterically preventing binding to serine protease inhibitors (e.g., A1AT), fibrinogen, and / or plasma antibodies, thus protecting it from inhibition by serine protease inhibitors or plasma antibodies, and inhibiting or reducing its ability to cleave fibrinogen.

[099] Thus, in certain embodiments, the protein complex retains the ability to cleave CD95 (Fas receptor) and does not substantially cleave fibrinogen. In some embodiments, a protein complex described in this document has approximately or at least approximately 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 on its own (e.g., in the presence of plasma serine protease inhibitors such as A1AT). In certain embodiments, a protein complex Petition 870250109095, dated 11 / 27 / 2025, page 47 / 150 The 43 / 102 described in this document has approximately or less than approximately 50, 40, 30, 20, 10, 5% or less of the fibrinogen protease cleavage activity of the corresponding serine protease protein alone. CD95 cleavage activity, cancer cell killing activity, fibrinogen cleavage activity, and coagulation properties can be measured according to routine techniques in the art (see Examples). For example, serine protease activity can most commonly be monitored using a colorimetric substrate activity assay (pN-Methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide) and CD95 and / or fibrinogen cleavage can be measured directly (e.g., Western blot). As desired, protease cleavage activity can be measured in the presence of serine protease inhibitors such as A1AT.The activity of cancer cell killing can be measured in vitro or in vivo, and the effects on coagulation in vivo can be measured, for example, by routine assays such as prothrombin time (PT) and partial thromboplastin time (PTT) (see Examples).

[0100] In some embodiments, a protein complex described in this document is generated in vivo or ex vivo, for example, in a cell by contacting a cell or subject with one or more expressible polynucleotides encoding (a) alpha-2-macroglobulin (A2M) proteins and (b) serine protease proteins. The protein complexes then form within the cell. An expressible polynucleotide includes a DNA, cDNA, RNA, mRNA, or other polynucleotide comprising at least one coding sequence for (a) and / or (b) and, optionally, at least one expression control sequence, for example, a transcription and / or translation regulatory element, and which can express an encoded polypeptide (e.g., a modified PPE proprotein) after introduction into a cell, for example, a cell in the subject. Certain embodiments include placing a cell ex vivo in contact with one or more expressible polynucleotides encoding (a) and (b) and administering the cell to a Petition 870250109095, dated 11 / 27 / 2025, page 48 / 150 44 / 102 subject.

[0101] Exemplary viral vectors that can be used to deliver an expressible polynucleotide include adenoviral vectors, herpesvirus vectors, vacciniavirus vectors, adeno-associated virus (AAV) vectors, and retroviral vectors such as lentiviral vectors. Examples of retroviral vectors include, but are not limited to, Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), SIV-based vectors, BIV, HIV, and sarcoma virus (RSV). In particular embodiments, the expressible polynucleotide is a modified RNA or modified mRNA polynucleotide, for example, an unnatural RNA analog. In certain embodiments, the modified RNA or mRNA polypeptide comprises one or more modified or unnatural bases. In some embodiments, the modified mRNA comprises one or more modified or unnatural internucleotide bonds.RNA polynucleotides expressible for delivering an encoded protein are described, for example, in Kormann et al., Nat Biotechnol. 29:154-7, 2011; and U.S. Application Nos. 2015 / 0111248; 2014 / 0243399; 2014 / 0147454; and 2013 / 0245104, which are incorporated by reference in their entirety.

[0102] In some embodiments, a protein complex described in this document has one or more enhanced biological, physical and / or pharmacokinetic properties, relative to the corresponding serine protease protein alone. The protein complexes described in this document may be used in any of the compositions, methods and / or kits described in this document. Methods of Use and Pharmaceutical Compositions

[0103] Certain modalities include methods of treatment, symptom improvement and / or reduction of the progression of a disease or condition in a subject in need, comprising administering to the subject a composition comprising a protein complex, as described in this document. In Petition 870250109095, dated 11 / 27 / 2025, page 49 / 150 45 / 102 particular modalities, the disease is cancer, that is, the subject in need has, is suspected of having, or is at risk of having cancer.

[0104] In particular modalities, cancer is either primary cancer or metastatic cancer. In specific modalities, the cancer is selected from one or more of the following: melanoma (optionally metastatic melanoma), breast cancer (optionally triple-negative breast cancer, TNBC), renal 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, hepatoma (hepatocellular carcinoma), sarcoma, B-cell malignancy, ovarian cancer, colorectal cancer, glioma, glioblastoma multiforme, meningioma, pituitary adenoma, vestibular schwannoma, primary CNS lymphoma, primitive neuroectodermal tumor (medulloblastoma), bladder cancer, uterine cancer, cancer of esophagus,Brain cancer, head and neck cancer, cervical cancer, testicular cancer, thyroid cancer, and stomach cancer.

[0105] In some modalities, as noted above, the cancer is a metastatic cancer. In addition to the cancers listed above, exemplary metastatic cancers include, without limitation, bladder cancers that have metastasized to the bone, liver, and / or lungs; breast cancers that have metastasized to the bones, brain, liver, and / or lungs; colorectal cancers that have metastasized to the liver, lungs, and / or peritoneum; kidney cancers that have metastasized to the adrenal glands, bones, brain, liver, and / or lungs; lung cancers that have metastasized to the adrenal glands, bones, brain, liver, and / or other lung sites; melanomas that have metastasized to the bone, brain, liver, lung, and / or skin / muscle; ovarian cancers that have metastasized to the liver, lung, and / or peritoneum; pancreatic cancers that have metastasized to the Petition 870250109095, dated 11 / 27 / 2025, page 50 / 150 46 / 102 liver, lung and / or peritoneum; prostate cancers that have metastasized to the adrenal glands, bones, liver and / or lungs; stomach cancers that have metastasized to the liver, lung and / or peritoneum; thyroid cancers that have metastasized to the bones, liver and / or lungs; and uterine cancers that have metastasized to the bones, liver, lung, peritoneum and / or vagina; among others.

[0106] In certain embodiments, the administration (e.g., intravenous administration) of the serine protease-containing protein composition or complex does not substantially affect coagulation in the subject, for example, it does not substantially increase prothrombin time or partial thromboplastin time in the subject (e.g., in relation to the administration of the corresponding serine protease alone). In certain embodiments, the administration of a serine protease-containing protein complex described in this document has a significantly reduced impact on coagulation in the subject compared to the administration of the corresponding serine protease alone.

[0107] Cancer treatment methods can be combined with other therapeutic modalities. For example, a combination therapy described in this document may be administered to a subject before, during, or after other therapeutic interventions, including symptomatic care, radiotherapy, surgery, transplantation, hormone therapy, photodynamic therapy, antibiotic therapy, or any combination thereof. Symptomatic care includes the administration of corticosteroids to reduce cerebral edema, headaches, cognitive dysfunction, and vomiting, and the administration of anticonvulsants to reduce seizures. Radiotherapy includes whole-brain irradiation, fractionated radiotherapy, and radiosurgery, such as stereotactic radiosurgery, which may be combined with traditional surgery.

[0108] Certain modalities thus include combination therapies to treat cancers, including methods of treating and improving symptoms, or inhibiting the progression of a cancer in a subject in need, Petition 870250109095, dated 11 / 27 / 2025, page 51 / 150 47 / 102 comprising administering to the subject a composition of a protein complex described in this document in combination with at least one additional agent, for example, an immunotherapy agent, a chemotherapeutic agent, a hormonal therapeutic agent and / or a kinase inhibitor. In some embodiments, administration of the composition potentiates the susceptibility of the cancer to the additional agent (e.g., immunotherapy agent, chemotherapeutic agent, hormonal therapeutic agent, and / or kinase inhibitor) by approximately or at least approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000% or more compared to the additional agent alone.

[0109] Certain combination therapies employ one or more cancer immunotherapy agents, or immunotherapy agents. In certain cases, an immunotherapy agent modulates a subject's immune response, for example, to increase or maintain a cancer-related or cancer-specific immune response, and thus results in increased inhibition of immune cells or reduction of cancer cells. Examples of immunotherapy agents include polypeptides, for example, antibodies and their antigen-binding fragments, ligands, and small peptides and mixtures thereof. Also included as immunotherapy agents are small molecules, cells (e.g., immune cells such as T cells), various cancer vaccines, gene therapy, or other polynucleotide-based agents, including viral agents such as oncolytic viruses and others known in the art.Thus, in certain modalities, the cancer immunotherapy agent is selected from one or more immune checkpoint modulators, cancer vaccines, oncolytic viruses, cytokines, and cell-based immunotherapies.

[0110] In certain modalities, the cancer immunotherapy agent is an immune checkpoint modulator. Particular examples include antagonists of one or more immune checkpoint inhibitory molecules and agonists of one or more immune checkpoint stimulatory molecules. Petition 870250109095, dated 11 / 27 / 2025, page 52 / 150 48 / 102 Generally, immune checkpoint molecules are components of the immune system that activate a signal (co-stimulatory molecules) or decrease a signal, the targeting of which has therapeutic potential in cancer because cancer cells can disrupt the natural function of immune checkpoint molecules (see, for example, Sharma and Allison, Science. 348:56-61, 2015; Topalian et al., Cancer Cell. 27:450-461, 2015; Pardoll, Nature Reviews Cancer. 12:252-264, 2012). In some modalities, the immune checkpoint modulating agent (e.g., antagonist, agonist) binds to or specifically targets one or more immune checkpoint molecules, as described in this document.

[0111] In some embodiments, the immune checkpoint modulating agent is an antagonist or inhibitor of one or more inhibitory immune checkpoint molecules. Examples of inhibitory immune checkpoint molecules include Programmed Death Ligand 1 (PD-L1), Programmed Death Ligand 2 (PD-L2), Programmed Death 1 (PD-1), V-domain Ig T cell activation suppressor (VISTA), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), indoleamine 2,3-dioxygenase (IDO), tryptophan 2,3-dioxygenase (TDO), T cell immunoglobulin domain and mucin domain 3 (TIM-3), lymphocyte activation gene-3 (LAG-3), B and T lymphocyte attenuator (BTLA), CD160, and T cell immunoreceptor with Ig and ITIM domains (TIGIT).

[0112] In certain modalities, the agent is a PD-1 (receptor) antagonist or inhibitor, targeting which has been shown to restore immune function in the tumor environment (see, for example, Phillips et al., Int Immunol. 27:39-46, 2015). PD-1 is a cell surface receptor belonging to the immunoglobulin superfamily and is 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 molecule of the immune checkpoint, for example, by reducing or preventing T cell activation, which in turn reduces autoimmunity and promotes self-tolerance. Petition 870250109095, dated 11 / 27 / 2025, page 53 / 150 49 / 102 The inhibitory effect of PD-1 is achieved, at least in part, through a dual mechanism of promoting apoptosis in antigen-specific T cells in lymph nodes while simultaneously reducing apoptosis in regulatory T cells (suppressor T cells). Some examples of PD-1 antagonists or inhibitors include an antibody or antigen-binding fragment or small molecule that specifically binds to PD-1 and reduces one or more of its immunosuppressive activities, for example, its downstream signaling or its interaction with PD-L1. Specific examples of PD1 antagonists or inhibitors include nivolumab, pembrolizumab, PDR001, MK-3475, AMP-224, AMP-514, and pidilizumab antibodies and antigen-binding fragments thereof (see, for example, U.S. Patents Nos. 8,008,449; 8,993,731; 9,073,994; 9,084,776; 9,102,727; 9,102,728; 9,181,342; 9,217,034; 9,387,247; 9,492,539; 9,492,540; and U.S. Application Nos. 2012 / 0039906; 2015 / 0203579).

[0113] In some embodiments, the agent is a PD-L1 antagonist or inhibitor. As noted above, PD-L1 is one of the natural ligands for the PD-1 receptor. General examples of PD-L1 antagonists or inhibitors include an antibody or antigen-binding fragment or small molecule that specifically binds to PD-L1 and reduces one or more of its immunosuppressive activities, for example, its binding to the PD-1 receptor. Specific examples of PD-L1 antagonists include the antibodies atezolizumab (MPDL3280A), avelumab (MSB0010718C), and durvalumab (MEDI4736) and their antigen-binding fragments (see, for example, U.S. Patents Nos. 9,102,725; 9,393,301; 9,402,899; 9,439,962).

[0114] In some embodiments, the agent is a PD-L2 antagonist or inhibitor. As noted above, PD-L2 is one of the natural ligands for the PD-1 receptor. General examples of PD-L2 antagonists or inhibitors include an antibody or antigen-binding fragment or small molecule that specifically binds to PD-L2 and reduces one or more of its activities. Petition 870250109095, dated 11 / 27 / 2025, p. 54 / 150 50 / 102 immunosuppressants, for example, their binding to the PD-1 receptor.

[0115] In certain embodiments, the agent is an antagonist or inhibitor of VISTA. VISTA is approximately 50 kDa in size and belongs to the immunoglobulin superfamily (possesses an IgV domain) and the B7 family. It is primarily expressed on white blood cells and its transcription is partially controlled by p53. There is evidence that VISTA can act as both a ligand and a receptor on T cells to inhibit T cell effector function and maintain peripheral tolerance. VISTA is produced at high levels in tumor-infiltrating lymphocytes, such as myeloid-derived suppressor cells and regulatory T cells, and its blockade with an antibody results in tumor growth retardation in mouse models of melanoma and squamous cell carcinoma. Examples of anti-VISTA antagonist antibodies include, for example, the antibodies described in WO 2018 / 237287, which is incorporated by reference in its entirety.

[0116] In some embodiments, the agent is a CTLA-4 antagonist or inhibitor. CTLA4 or CTLA-4 (cytotoxic T-cell-associated protein 4), also known as CD152 (cluster of differentiation 152), is a protein receptor that functions as an inhibitory immune checkpoint molecule, for example, transmitting inhibitory signals to T cells when bound to CD80 or CD86 on the surface of antigen-presenting cells. General examples of CTLA-4 antagonists or inhibitors include an antibody or antigen-binding fragment or small molecule that specifically binds to CTLA-4. Particular examples include the antibodies ipilimumab and tremelimumab and their antigen-binding fragments. At least part of the activity of ipilimumab is believed to be mediated by antibody-dependent cell-mediated cytotoxicity (ADCC) killing of suppressor Tregs expressing CTLA-4.

[0117] In some embodiments, the agent is an IDO antagonist or inhibitor, or a TDO antagonist or inhibitor. IDO and TDO are catabolic enzymes. Petition 870250109095, dated 11 / 27 / 2025, p. 55 / 150 51 / 102 of tryptophan with immunoinhibitory properties. For example, IDO is known to suppress T cells and NK cells, generate and activate Tregs and myeloid-derived suppressor cells, and promote tumor angiogenesis. General examples of IDO and TDO antagonists or inhibitors include an antibody or antigen-binding fragment or small molecule that specifically binds to IDO or TDO (see, for example, Platten et al., Front Immunol. 5: 673, 2014) and reduces or inhibits 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, for example, Sheridan, Nature Biotechnology, 33:321-322, 2015). Specific examples of TDO antagonists or inhibitors include 680C91 and LM10 (see, for example, Pilotte et al., PNAS USA. 109:2497-2502, 2012).

[0118] In some embodiments, the agent is a TIM-3 antagonist or inhibitor. The T-cell immunoglobulin domain and mucin domain 3 (TIM-3) is expressed on activated human CD4+ T cells and regulates Th1 and Th17 cytokines. TIM-3 also acts as a negative regulator of Th1 / Tc1 function, triggering cell death by interacting with its ligand, galectin-9. TIM-3 contributes to the suppressive tumor microenvironment, and its overexpression is associated with poor prognosis in a variety of cancers (see, for example, Li et al., Acta Oncol. 54:1706-13, 2015). General examples of TIM-3 antagonists or inhibitors include an antibody or antigen-binding fragment or small molecule that specifically binds to TIM-3 and reduces or inhibits one or more of its immunosuppressive activities.

[0119] In some embodiments, the agent is an antagonist or inhibitor of LAG-3. Lymphocyte activating gene 3 (LAG-3) is expressed on activated T cells, natural killer cells, B cells, and plasmacytoid dendritic cells. It negatively regulates cell proliferation, activation, and T cell homeostasis, similarly to CTLA-4 and PD-1 (see, for example, Workman and Vignali). Petition 870250109095, dated 11 / 27 / 2025, page 56 / 150 52 / 102 LAG3 has been reported to play a role in the suppressive function of Treg cells (see, for example, Huang et al., Immunity. 21: 503-13, 2004). LAG3 also maintains CD8+ T cells in a tolerogenic state and combines with PD-1 to maintain CD8+ T cell exhaustion. General examples of LAG-3 antagonists or inhibitors include an antibody or antigen-binding fragment or small molecule that specifically binds to LAG-3 and inhibits one or more of its immunosuppressive activities. Specific examples include the antibody BMS-986016 and its antigen-binding fragments.

[0120] In some embodiments, the agent is a BTLA antagonist or inhibitor. B and T lymphocyte attenuator (BTLA; CD272) expression is induced during T cell activation and inhibits T cells through interaction with tumor necrosis factor family (TNF-R) receptors and the B7 family of cell surface receptors. BTLA is a ligand for the tumor necrosis factor (receptor) superfamily, member 14 (TNFRSF14), also known as herpes virus entry mediator (HVEM). BTLA-HVEM complexes negatively regulate T cell immune responses, for example, by inhibiting the function of human cancer-specific CD8+ T cells (see, for example, Derré et al., J Clin Invest 120:157-67, 2009). General examples of BTLA antagonists or inhibitors include an antibody or antigen-binding fragment or small molecule that specifically binds to BTLA-4 and reduces one or more of its immunosuppressive activities.

[0121] In some embodiments, the agent is an HVEM antagonist or inhibitor, for example, an antagonist or inhibitor that specifically binds to HVEM and interferes with its interaction with BTLA or CD160. General examples of HVEM antagonists or inhibitors include an antibody or antigen-binding fragment or small molecule that specifically binds to HVEM, optionally reduces the HVEM / BTLA and / or HVEM / CD160 interaction and thus reduces Petition 870250109095, dated 11 / 27 / 2025, page 57 / 150 53 / 102 one or more of the immunosuppressive activities of HVEM.

[0122] In some embodiments, the agent is a CD160 antagonist or inhibitor, for example, an antagonist or inhibitor that specifically binds to CD160 and interferes with its interaction with HVEM. General examples of CD160 antagonists or inhibitors include an antibody or antigen-binding fragment or small molecule that specifically binds to CD160, optionally reduces the CD160 / HVEM interaction, and thus reduces or inhibits one or more of its immunosuppressive activities.

[0123] In some modalities, the agent is a TIGIT antagonist or inhibitor. T cell Ig and ITIM domain (TIGIT) is a co-inhibitory receptor that is found on the surface of a variety of lymphoid cells and suppresses antitumor immunity, for example, via Tregs (Kurtulus et al., J Clin Invest. 125: 4053-4062, 2015). General examples of TIGIT antagonists or inhibitors include an antibody or antigen-binding fragment or small molecule that specifically binds to TIGIT and reduces one or more of its immunosuppressive activities (see, for example, Johnston et al., Cancer Cell. 26:923-37, 2014).

[0124] In certain modalities, the immune checkpoint modulating agent is an agonist of one or more immune checkpoint stimulatory molecules. Exemplary immune checkpoint stimulatory molecules include CD40, OX40, glucocorticoid-induced TNFR family-related gene (GITR), CD137 (4-1BB), CD27, CD28, CD226, and herpes virus entry mediator (HVEM).

[0125] In some modalities, the agent is a CD40 agonist. CD40 is expressed on antigen-presenting cells (APCs) and in some malignancies. Its ligand is CD40L (CD154). On APCs, binding results in the upregulation of co-stimulatory molecules, potentially bypassing the need for T cell assistance in an antitumor immune response. A Petition 870250109095, dated 11 / 27 / 2025, p. 58 / 150 54 / 102 CD40 agonist therapy plays an important role in APC maturation and its migration from the tumor to the lymph nodes, resulting in elevated antigen presentation and T cell activation. Anti-CD40 agonist antibodies produce substantial responses and durable anticancer immunity in animal models, an effect mediated at least in part by cytotoxic T cells (see, for example, Johnson et al. Clin Cancer Res. 21: 1321-1328, 2015; and Vonderheide and Glennie, Clin Cancer Res. 19:1035-43, 2013). General examples of CD40 agonists include an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to CD40 and enhances one or more of its immunostimulatory activities. Specific examples include CP870,893, dacetuzumab, Chi Lob 7 / 4, ADC-1013, CD40L, rhCD40L and their antigen-binding fragments.Specific examples of CD40 agonists include, but are not limited to, APX005 (see, for example, US 2012 / 0301488) and APX005M (see, for example, US 2014 / 0120103).

[0126] In some modalities, the agent is an OX40 agonist. OX40 (CD134) promotes the expansion of effector and memory T cells and suppresses the differentiation and activity of regulatory T cells (see, for example, Croft et al., Immunol Rev. 229:173-91, 2009). Its ligand is OX40L (CD252). Since OX40 signaling influences both T cell activation and survival, it plays a key role in initiating an antitumor immune response in the lymph node and in maintaining the antitumor immune response in the tumor microenvironment. General examples of OX40 agonists include an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to OX40 and enhances one or more of its immunostimulatory activities. Specific examples include OX86, OX-40L, Fc-OX40L, GSK3174998, MEDI0562 (a humanized OX40 agonist), MEDI6469 (a murine OX4 agonist), and MEDI6383 (an OX40 agonist) and their antigen-binding fragments. Petition 870250109095, dated 11 / 27 / 2025, p. 59 / 150 55 / 102

[0127] In some modalities, the agent is a GITR agonist. The gene-related TNFR family-induced glucocorticoid (GITR) enhances T cell expansion, inhibits the suppressive activity of Tregs, and extends the survival of effector T cells. GITR agonists have been shown to promote an antitumor response through loss of Treg lineage stability (see, for example, Schaer et al., Cancer Immunol Res. 1:320-31, 2013). These diverse mechanisms show that GITR plays an important role in initiating the immune response in lymph nodes and maintaining the immune response in tumor tissue. Its ligand is GITRL. General examples of GITR agonists include an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to GITR and enhances one or more of its immunostimulatory activities. Specific examples include GITRL, INCAGN01876, DTA-1, MEDI1873 and their antigen-binding fragments.

[0128] In some embodiments, the agent is a CD137 agonist. CD137 (4-1BB) is a member of the tumor necrosis factor (TNF) receptor family, and cross-linking of CD137 increases T cell proliferation, IL-2 secretion, survival, and cytolytic activity. CD137-mediated signaling also protects T cells, such as CD8+ T cells, from activation-induced cell death. General examples of CD137 agonists include an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to CD137 and enhances one or more of its immunostimulatory activities. Specific examples include the CD137 (or 4-1BB) ligand (see, for example, Shao and Schwarz, J Leukoc Biol. 89:21-9, 2011) and the antibody utomilumab, including its antigen-binding fragments.

[0129] In some modalities, the agent is a CD27 agonist. Stimulation of CD27 increases antigen-specific expansion of naive T cells and contributes to T cell memory and long-term maintenance of Petition 870250109095, dated 11 / 27 / 2025, page 60 / 150 56 / 102 T-cell immunity. Its ligand is CD70. Targeting human CD27 with an agonist antibody stimulates T-cell activation and antitumor immunity (see, for example, Thomas et al., Oncoimmunology. 2014;3:e27255. doi:10.4161 / onci.27255; and He et al., J Immunol. 191:4174-83, 2013). General examples of CD27 agonists include an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to CD27 and enhances one or more of its immunostimulatory activities. Specific examples include CD70 and the antibodies varlilumab and CDX-1127 (1F5), including their antigen-binding fragments.

[0130] In some embodiments, the agent is a CD28 agonist. CD28 are constitutively expressed CD4+ T cells, some CD8+ T cells. Their ligands include CD80 and CD86, and their stimulation increases T cell expansion. General examples of CD28 agonists include an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to CD28 and increases one or more of its immunostimulatory activities. Specific examples include CD80, CD86, the TAB08 antibody and their antigen-binding fragments.

[0131] In some modalities, the agent is a CD226 agonist. CD226 is a stimulatory receptor that shares ligands with TIGIT and, unlike TIGIT, CD226 involvement increases T cell activation (see, for example, 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.). General examples of CD226 agonists include an antibody or antigen-binding fragment or small molecule or ligand (e.g., CD112, CD155) that specifically binds to CD226 and increases one or more of its immunostimulatory activities.

[0132] In some forms, the agent is an HVEM agonist. The herpesvirus entry mediator (HVEM), also known as a member of Petition 870250109095, dated 11 / 27 / 2025, p. 61 / 150 The 57 / 102 tumor necrosis factor receptor 14 (TNFRSF14) superfamily is a human cell surface receptor of the TNF receptor superfamily. HVEM is found on a variety of cells, including T cells, APCs, and other immune cells. Unlike other receptors, HVEM is expressed at high levels on resting T cells and downregulated upon activation. HVEM signaling has been shown to play a crucial role in the early stages of T cell activation and during the expansion of tumor-specific lymphocyte populations in lymph nodes. General examples of HVEM agonists include an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to HVEM and enhances one or more of its immunostimulatory activities.

[0133] In certain modalities, the immunotherapy agent is a bispecific or multispecific antibody. For example, certain bispecific or multispecific antibodies are capable of (i) binding to and inhibiting one or more inhibitory molecules of the immune checkpoint and also (ii) binding to and agonizing one or more stimulatory molecules of the immune checkpoint. In certain embodiments, a bispecific or multispecific antibody (i) binds to and inhibits one or more of PD-L1, PD-L2, PD-1, CTLA-4, IDO, TDO, TIM-3, LAG-3, BTLA, CD160 and / or TIGIT, and also (ii) binds to and agonizes one or more of CD40, Glucocorticoid-Induced TNFR Family Related Gene (GITR), CD137 (4-1 BB), CD27, CD28, CD226, and / or Herpes Virus Entry Mediator (HVEM).

[0134] In some embodiments, the immunotherapy agent is a cancer vaccine. In certain embodiments, the cancer vaccine is selected from one or more of Oncophage, a human papillomavirus HPV vaccine optionally Gardasil or Cervarix, a hepatitis B vaccine optionally Engerix-B, Recombivax HB or Cellrix, and sipuleucel-T (Provenge). In some embodiments, the cancer vaccine comprises or expresses a TAA or TSA, as described in this document. Petition 870250109095, dated 11 / 27 / 2025, page 62 / 150 58 / 102

[0135] In some embodiments, the immunotherapy agent is an oncolytic virus. In some embodiments, the oncolytic virus is selected from one or more of the following: Talimogene laherparepvec (T-VEC), coxsackievirus A21 (CAVATAK™), Oncorine (H101), pelareorep (REOLYSIN®), Seneca Valley virus (NTX-010), Senecavirus SVV-001, ColoAd1, SEPREHVIR (HSV-1716), CGTG-102 (Ad5 / 3D24-GMCSF), GL-ONC1, MV-NIS, and DNX-2401.

[0136] In certain modalities, the immunotherapy agent against cancer is a cytokine. Examples of cytokines include interferon (IFN)^, IL-2, IL-12, IL-7, IL-21, and granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0137] In certain modalities, the immunotherapy agent against cancer is cell-based immunotherapy, for example, a therapy that utilizes immune cells, including ex vivo derived immune cells such as lymphocytes, natural killer (NK) cells, macrophages, and / or dendritic cells (DCs). In some modalities, the lymphocytes comprise T cells, for example, cytotoxic T lymphocytes (CTLs). See, for example, 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, for descriptions of adoptive T-cell and NK-cell immunotherapies. In some modalities, the T cells comprise cancer antigen-specific T cells, which are directed against at least one cancer antigen.In some modalities, cancer antigen-specific T cells are selected from one or more chimeric antigen receptor-modified (CAR) T cells, T cell receptor-modified (TCR) T cells, tumor infiltrating lymphocytes (TILs), and peptide-induced T cells. In specific modalities, the CAR-modified T cell is targeted against CD-19 (see, for example, Maude et al., Blood. 125:4017-4023, 2015). In some cases, the ex vivo derived immune cells are autologous cells, which are obtained from the patient to be treated.

[0138] Certain combination therapies employ one or more agents Petition 870250109095, dated 11 / 27 / 2025, page 63 / 150 59 / 102 chemotherapeutic agents, for example, small molecule chemotherapeutic agents. Non-limiting examples of chemotherapeutic agents include alkylating agents, antimetabolites, cytotoxic antibiotics, topoisomerase inhibitors (type 1 or type II) and antimicrotubule agents, among others.

[0139] Examples of alkylating agents include nitrogen mustards (e.g., mechlorethamine, 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, and diaziquone (AZQ)), cisplatins and their derivatives (e.g., carboplatin and oxaliplatin), and non-classical alkylating agents (optionally procarbazine and hexamethylmelamine).

[0140] Examples of antimetabolites include antifolates (e.g., methotrexate and pemetrexed), fluoropyrimidines (e.g., 5-fluorouracil and capecitabine), deoxynucleoside analogs (e.g., ancitabine, enocitabine, cytarabine, gemcitabine, decitabine, azacitidine, fludarabine, nelarabine, cladribine, clofarabine, fludarabine, and pentostatin), and thiopurines (e.g., thioguanine and mercaptopurine).

[0141] Examples of cytotoxic antibiotics include anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, aclarrubicin, and mitoxantrone), bleomycins, mitomycin C, mitoxantrone, and actinomycin. Examples of topoisomerase inhibitors include camptothecin, irinotecan, topotecan, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, merbarone, and aclarrubicin.

[0142] Examples of antimicrotubule agents include taxanes (e.g., paclitaxel and docetaxel) and vinca alkaloids (e.g., vinblastine, vincristine, vindesine, vinorelbine).

[0143] The various chemotherapeutic agents described in this document Petition 870250109095, dated 11 / 27 / 2025, p. 64 / 150 60 / 102 can be combined with any one or more of the protein complexes described in this document and used in accordance with any one or more of the methods or compositions described in this document.

[0144] Certain combination therapies employ at least one hormonal therapeutic agent. General examples of hormonal therapeutic agents include hormonal agonists and hormonal antagonists. Specific examples of hormonal agonists include progestogen (progestin), corticosteroids (e.g., prednisolone, methylprednisolone, dexamethasone), insulin-like growth factors, 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 analogues.Examples of hormonal antagonists include inhibitors of hormone synthesis, such as aromatase inhibitors and gonadotropin-releasing hormone (GnRH) agonists (e.g., leuprolide, goserelin, triptorelin, histrelin), including their analogues. Also included are hormone receptor antagonists, such as selective estrogen receptor modulators (SERMs; e.g., tamoxifen, raloxifene, toremifene) and antiandrogens (e.g., flutamide, bicalutamide, nilutamide).

[0145] Also included are inhibitors of the hormonal pathway, such as antibodies directed against hormone receptors. Examples include IGF receptor inhibitors (e.g., IGF-IR1), such as cixutumumab, dalotuzumab, figitumumab, ganitumab, istiratumab, and robatumumab; vascular endothelial growth factor receptor 1, 2, or 3 (VEGFR1, VEGFR2, or VEGFR3) inhibitors such as alacizumab pegol, bevacizumab, icrucumab, ramucirumab; TGF-beta R1, R2, and R3 receptor inhibitors, such as fresolimumab and metelimumab; c-Met inhibitors, such as naxitamab; receptor inhibitors Petition 870250109095, dated 11 / 27 / 2025, page 65 / 150 61 / 102 EGF inhibitors, such as cetuximab, depatuxizumab mafodotin, futuximab, imgatuzumab, laprituximab emtansine, matuzumab, modotuximab, necitumumab, nimotuzumab, panitumumab, tomuzotuximab, and zalutumumab; FGF receptor inhibitors such as aprutumab ixadotin and bemituzumab; and PDGF receptor inhibitors such as olaratumab and tovetumab.

[0146] The various hormonal therapeutic agents described in this document may be combined with any one or more of the protein complexes described in this document and used in accordance with any one or more of the methods or compositions described in this document.

[0147] Certain combination therapies employ at least one kinase inhibitor, including tyrosine kinase inhibitors. Examples of kinase inhibitors include, without limitation, adavosertib, afanitib, aflibercept, axitinib, bevacizumab, bosutinib, cabozantinib, cetuximab, cobimetinib, crizotinib, dasatinib, entrectinib, erdafitinib, erlotinib, fostamitib, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, mubritinib, nilotinib, panitumumab, pazopanib, pegaptanib, ponatinib, ranibizumab, regorafenib, ruxolitinib, sorafenib, sunitinib, SU6656, tofacitinib, trastuzumab, vandetanib, and vemuafenib.

[0148] The various kinase inhibitors described in this document can be combined with any one or more of the protein complexes described in this document and used according to any one or more of the methods or compositions described in this document.

[0149] In some embodiments, the methods and compositions described in this document increase cancer cell death in the subject by approximately or at least approximately 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 500-fold, or 1000-fold or more relative to a control or reference. In some embodiments, the methods and compositions described in this document increase an immune response in the subject by approximately or at least approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, Petition 870250109095, dated 11 / 27 / 2025, p. 66 / 150 62 / 102 1000, 2000% or more, or by about 2 times, 5 times, 10 times, 50 times, 100 times, 500 times, or 1000 times or more, relative to a control or reference (e.g., relative to a corresponding serine protease alone), including where the immune response is an anticancer immune response.

[0150] In some embodiments, the methods and compositions described in this document increase a subject's median survival time by 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 15 weeks, 20 weeks, 25 weeks, 30 weeks, 40 weeks or more. In certain embodiments, the methods and compositions described in this document increase a subject's median survival time by 1 year, 2 years, 3 years or more. In some embodiments, the pharmaceutical methods and compositions increase progression-free survival by 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks or more. In certain embodiments, the pharmaceutical methods and compositions described in this document increase progression-free survival by 1 year, 2 years, 3 years or more.

[0151] In certain modalities, the methods and compositions described in this document are sufficient to result in tumor regression, for example, as indicated by a statistically significant decrease in the amount of viable tumor, for example, at least 10%, 20%, 30%, 40%, 50% or more decrease in tumor mass, or by altered scan dimensions (e.g., statistically significant decreases). In certain modalities, the methods and compositions described in this document are sufficient to result in stable disease. In certain modalities, the methods and compositions described in this document are sufficient to result in a clinically relevant reduction in the symptoms of a specific disease indication known to the experienced clinician.

[0152] For in vivo use, as noted above, for the treatment of Petition 870250109095, dated 11 / 27 / 2025, page 67 / 150 63 / 102 disease or test of human or non-human mammal, the protein complexes described in this document are generally incorporated into one or more therapeutic or pharmaceutical compositions prior to administration, including veterinary therapeutic compositions.

[0153] Thus, certain embodiments refer to pharmaceutical or therapeutic compositions comprising a protein complex, as described in this document. In some cases, a pharmaceutical or therapeutic composition comprises one or more of the protein complexes described in this document in combination with a pharmaceutically or physiologically acceptable carrier or excipient. Certain pharmaceutical or therapeutic compositions further comprise at least one additional agent, for example, an immunotherapy agent, a chemotherapeutic agent, a hormonal therapeutic agent and / or a kinase inhibitor as described in this document.

[0154] In particular embodiments, pharmaceutical or therapeutic compositions comprising a protein complex are substantially pure on a protein or weight-weight basis, for example, the composition has a purity of at least about 80%, 85%, 90%, 95%, 98% or 99% on a protein basis or on a weight-weight basis.

[0155] In some embodiments, the protein complexes described in this document do not form aggregates, have a desired solubility, and / or have an immunogenicity profile suitable for use in humans, as known in the art. Thus, in some embodiments, the pharmaceutical and therapeutic composition comprising a protein complex is substantially free of aggregates. For example, certain compositions comprise less than about 10% (on a protein basis) of aggregated high molecular weight proteins, or less than about 5% of aggregated high molecular weight proteins, or less than about 4% of aggregated high molecular weight proteins, or less than about 3% of aggregated high molecular weight proteins, or less than about 2% Petition 870250109095, dated 11 / 27 / 2025, page 68 / 150 64 / 102 high molecular weight aggregated proteins, or less than about 1% high molecular weight aggregated proteins.

[0156] In some embodiments, the protein complexes are 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 are formulated for biotherapeutic uses.

[0157] To prepare a therapeutic or pharmaceutical composition, an effective or desired amount of one or more protein complexes is mixed with any pharmaceutical carriers or excipients known to those skilled in the art to be suitable for the agent and / or particular route of administration. A pharmaceutical carrier may be liquid, semi-liquid or solid. Solutions or suspensions used for parenteral, intradermal, subcutaneous or topical application may include, for example, a sterile diluent (such as water), saline solution (e.g., phosphate-buffered saline; PBS), fixed oil, polyethylene glycol, glycerin, propylene glycol or other synthetic solvent; antimicrobial agents (such as benzyl alcohol and methylparabens); antioxidants (such as ascorbic acid and sodium bisulfite) and chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); buffers (such as acetates, citrates and phosphates).If administered intravenously (e.g., by IV infusion), suitable carriers include physiological saline or phosphate-buffered saline (PBS) and solutions containing thickening and solubilizing agents such as glucose, polyethylene glycol, polypropylene glycol, and mixtures thereof.

[0158] The administration of protein complexes described in this document, in pure form or in an appropriate therapeutic or pharmaceutical composition, may be carried out by any of the accepted modes of administration of agents to serve similar purposes. Therapeutic or pharmaceutical compositions may be prepared by combining a composition Petition 870250109095, dated 11 / 27 / 2025, page 69 / 150 65 / 102 containing a protein complex with a suitable physiologically acceptable carrier, diluent or excipient, and may be formulated in solid, semi-solid, liquid or gaseous preparations, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres and aerosols. In addition, other pharmaceutically active ingredients (including other small molecules as described elsewhere in this document) and / or suitable excipients, such as salts, buffers and stabilizers, may, but do not necessarily, be present in the composition.

[0159] Administration can be performed via a variety of different routes, including oral, parenteral, nasal, intravenous, intradermal, intramuscular, subcutaneous, or topical. Preferred modes of administration depend on the nature of the condition being treated or prevented. Particular modalities include administration by IV infusion.

[0160] Carriers may include, for example, pharmaceutically or physiologically acceptable transporters, excipients, or stabilizers that are not toxic to the cell or mammal to be exposed at the dosages and concentrations employed. Often, the physiologically acceptable vehicle is a pH-buffered aqueous solution.Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight polypeptides (less than about 10 residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counter-ions such as sodium; and / or nonionic surfactants such as polysorbate 20 (TWEEN™), polyethylene glycol (PEG), and poloxamers (PLURONICS™) and the like. Petition 870250109095, dated 11 / 27 / 2025, page 70 / 150 66 / 102

[0161] In some embodiments, one or more agents may be trapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin microcapsules and poly-(methylmethacrylate) microcapsules, respectively), 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 also comprise other therapeutic or diagnostic agents.

[0162] The precise dosage and duration of treatment are a function of the disease being treated and can be determined empirically using known testing protocols or by testing the compositions in model systems known in the art and extrapolating from them. Controlled clinical trials may also be conducted. Dosages may also vary with the severity of the condition to be alleviated. A pharmaceutical composition is generally formulated and administered to exert a therapeutically useful effect while minimizing undesirable side effects. The composition may be administered once or may be divided into several smaller doses to be administered at time intervals. For any specific subject, specific dosage regimens may be adjusted over time according to individual need.

[0163] Typical routes of administration of these and related therapeutic or pharmaceutical compositions therefore include, without limitation, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal and intranasal. The term parenteral as used in this document includes subcutaneous, intravenous, intramuscular, intrasternal injections or infusion techniques. The therapeutic or pharmaceutical compositions according to certain embodiments of this disclosure are formulated so as to allow the Petition 870250109095, dated 11 / 27 / 2025, p. 71 / 150 67 / 102 The active ingredients contained therein are bioavailable after administration of the composition to a subject or patient. Compositions to be administered to a subject or patient may take the form of one or more dosage units, where, for example, a tablet may be a single dosage unit and a container of an agent described herein in aerosol form may contain a plurality of dosage units. The actual methods of preparation of such dosage forms are known, or will be apparent, to those skilled in the art; for example, see Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000). The composition to be administered will typically contain a therapeutically effective amount of an agent described herein for the treatment of a disease or condition of interest.

[0164] A therapeutic or pharmaceutical composition may be in the form of a solid or liquid. In some embodiments, the carriers are particulate, so that the compositions are, for example, in the form of a tablet or powder. The carriers may be liquid, the compositions being, for example, an oral oil, injectable liquid or an aerosol, which is useful, for example, in inhalation administration. When intended for oral administration, the pharmaceutical composition is preferably in solid or liquid form, where semi-solid, semi-liquid forms, suspensions and gels are included in the forms considered in this document as solid or liquid. Certain embodiments include sterile injectable solutions.

[0165] As a solid composition for oral administration, the pharmaceutical composition may be formulated as a powder, granule, tablet, pill, capsule, chewing gum, biscuit, or similar. Such a solid composition will typically contain one or more inert diluents or edible vehicles. In addition, one or more of the following may be present: binders such as carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, Petition 870250109095, dated 11 / 27 / 2025, page 72 / 150 68 / 102 Tragacanth gum or gelatin; excipients such as starch, lactose or dextrins; disintegrating agents such as alginic acid, sodium alginate, Primogel, corn starch and the like; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate or orange flavor; and a coloring agent. When the pharmaceutical composition is in the form of a capsule, for example, a gelatin capsule, it may contain, in addition to the above-mentioned materials, a liquid carrier such as polyethylene glycol or oil.

[0166] The therapeutic or pharmaceutical composition may be in the form of a liquid, for example, an elixir, syrup, solution, emulsion, or suspension. The liquid may be for oral administration or for distribution by injection, as two examples. When intended for oral administration, the preferred composition contains, in addition to the present compounds, one or more sweetening agents, preservatives, colorants / coloring agents, and flavor enhancers. In a composition intended for administration by injection, one or more surfactants, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers, and isotonic agents may be included.

[0167] Liquid therapeutic or pharmaceutical compositions, whether solutions, suspensions or other similar forms, may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono- or diglycerides that may serve as a solvent or suspension medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for adjusting tonicity such as sodium chloride or Petition 870250109095, dated 11 / 27 / 2025, page 73 / 150 69 / 102 dextrose. The parenteral preparation may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic. Physiological saline solution is a preferred adjuvant. An injectable pharmaceutical composition is preferably sterile.

[0168] A therapeutic or pharmaceutical liquid composition intended for parenteral or oral administration must contain an amount of an agent such that an adequate dosage is obtained. Normally, this amount is at least 0.01% of the agent of interest in the composition. When intended for oral administration, this amount may vary between 0.1% and about 70% of the weight of the composition. Certain oral therapeutic or pharmaceutical compositions contain between about 4% and about 75% of the agent of interest. In certain embodiments, therapeutic or pharmaceutical compositions and preparations are prepared so that a parenteral dosage unit contains between 0.01% and 10% by weight of the agent of interest before dilution.

[0169] Therapeutic or pharmaceutical compositions may be intended for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel base. The base, for example, may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, beeswax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. Thickening agents may be present in a therapeutic or pharmaceutical composition for topical administration. If intended for transdermal administration, the composition may include a transdermal patch or iontophoresis device.

[0170] Therapeutic or pharmaceutical compositions may be intended for rectal administration, for example in the form of a suppository, which will melt in the rectum and release the drug. The composition for rectal administration may contain an oily base as a suitable non-irritating excipient. Such bases include, without limitation, lanolin, cocoa butter and polyethylene glycol. Petition 870250109095, dated 11 / 27 / 2025, page 74 / 150 70 / 102

[0171] The therapeutic or pharmaceutical composition may include various materials that modify the physical form of a solid or liquid dosage unit. For example, the composition may include materials that form a coating shell around the active ingredients. The materials that form the coating shell are typically inert and may be selected from, for example, sugar, shellac, and other enteric coating agents. Alternatively, the active ingredients may be enclosed in a gelatin capsule. Therapeutic or pharmaceutical compositions in solid or liquid form may include a component that binds to the agent and thus assists in the distribution of the compound. Suitable components that may act in this capacity include monoclonal or polyclonal antibodies, one or more proteins, or a liposome.

[0172] The therapeutic or pharmaceutical composition may consist essentially of dosage units that can be administered as an aerosol. The term aerosol is used to designate a variety of systems ranging from those of a colloidal nature to systems consisting of pressurized packets. Delivery may be by a liquefied or compressed gas or by a suitable pump system dispensing the active ingredients. Aerosols may be supplied in single-phase, two-phase, or three-phase systems to deliver the active ingredients. The aerosol delivery includes the necessary container, activators, valves, sub-containers, and the like, which together may form a kit. One skilled in the art, without undue experimentation, may determine the preferred aerosols.

[0173] The compositions described in this document may be prepared with carriers that protect the agents against rapid elimination from the body, such as sustained-release formulations or coatings. Such carriers include controlled-release formulations, such as, but not limited to, microencapsulated implants and delivery systems, and biodegradable, Petition 870250109095, dated 11 / 27 / 2025, page 75 / 150 71 / 102 biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid and others known to those skilled in the art.

[0174] Therapeutic or pharmaceutical compositions may be prepared by a methodology well known in the pharmaceutical art. For example, a therapeutic or pharmaceutical composition intended to be administered by injection may comprise one or more salts, buffers and / or stabilizers, with sterile distilled water to form a solution. A surfactant may be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that interact non-covalently with the agent in order to facilitate the homogeneous dissolution or suspension of the agent in the aqueous administration system.

[0175] Therapeutic or pharmaceutical compositions may be administered in a therapeutically effective amount, which will vary depending on a variety of factors including the activity of the specific compound employed; the metabolic stability and duration of action of the compound; the age, body weight, general health, sex and diet of the subject; the mode and time of administration; the rate of excretion; the combination of drugs; the severity of the particular disorder or condition; and the subject in therapy. In some cases, a therapeutically effective daily dose is (for a 70 kg mammal) from about 0.001 mg / kg (i.e. ~0.07 mg) to about 100 mg / kg (i.e. ~7.0 g); preferably a therapeutically effective dose is (for a 70 kg mammal) from about 0.01 mg / kg (i.e. ~0.7 mg) to about 50 mg / kg (i.e. ~3.5 g); More preferably, a therapeutically effective dose is (for a 70 kg mammal) from about 1 mg / kg (i.e., ~70 mg) to about 25 mg / kg (i.e., ~1.75 g).In some modalities, the therapeutically effective dose is administered weekly, bi-weekly, or monthly. In specific modalities, the therapeutically effective dose is administered weekly, bi-weekly, or monthly. Petition 870250109095, dated 11 / 27 / 2025, p. 76 / 150 72 / 102 monthly, for example, at a dose of about 1-10 or 1-5 mg / kg, or about 1, 2, 3, 4.5, 6, 7, 8, 9 or 10 mg / kg.

[0176] The combination therapies described in this document may include administration of a single pharmaceutical dosage formulation containing a protein complex and an additional therapeutic agent (e.g., immunotherapy agent, chemotherapeutic agent, hormonal therapeutic agent, kinase inhibitor), as well as the administration of compositions comprising a modified PPE protein or proprotein and an additional therapeutic agent in their own separate pharmaceutical dosage formulation. For example, a protein complex and an additional therapeutic agent may be administered to the subject together in a single parenteral dosage composition, such as in a saline solution or other physiologically acceptable solution, or each agent may be administered in separate parenteral dosage formulations.When separate dosage formulations are used, the compositions may be administered essentially at the same time, i.e., concomitantly, or at staggered times separately, i.e., sequentially and in any order; combination therapy is understood to include all these regimens.

[0177] Also included are patient care kits comprising (a) a protein complex, as described in this document; and optionally (b) at least one additional therapeutic agent (e.g., immunotherapy agent, chemotherapeutic agent, hormonal therapeutic agent, 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.

[0178] The kits in this document may also include one or more additional therapeutic agents or other components suitable or desired for the indication to be treated or for the intended diagnostic application. The kits in this document may also include one or more syringes or other components necessary or desired to facilitate an intended method of distribution (by Petition 870250109095, dated 11 / 27 / 2025, p. 77 / 150 73 / 102 example, stents, implantable depots, etc.).

[0179] In some embodiments, a patient care kit contains separate containers, dividers, or compartments for the compositions and informational materials. For example, the composition(s) may be contained in a bottle, vial, or syringe, and the informational material(s) may be contained in association with the container. In some embodiments, the separate elements of the kit are contained in a single indivisible container. For example, the composition is contained in a bottle, vial, or syringe that has the informational material attached in the form of a label. In some embodiments, the kit includes a plurality (e.g., a package) of individual containers, each containing one or more unit dosage forms (e.g., a dosage form described in this document) of a protein complex and, optionally, at least one additional therapeutic agent.For example, the kit includes a plurality of syringes, ampoules, aluminum packages, or blister packs, each containing a single unit dose of a protein complex and, optionally, at least one additional therapeutic agent. The kit containers may be airtight, waterproof (e.g., impervious to changes in humidity or evaporation), and / or lightproof.

[0180] The patient treatment kit optionally includes a suitable device for administering the composition, for example, a syringe, inhalant, dropper (e.g., dropper), swab (e.g., a cotton swab or wood swab), or any other administration device. In some embodiments, the device is an implantable device that dispenses measured doses of the agents. Methods for providing a kit are also included, for example, by combining the components described in this document. Manufacturing and purification systems

[0181] Certain embodiments include methods and related compositions for manufacturing, expressing, and purifying a protein complex or protein component. Petition 870250109095, dated 11 / 27 / 2025, page 78 / 150 74 / 102 described in this document. For example, certain embodiments relate to methods of manufacturing or preparing a pharmaceutical composition comprising a protein complex, combining: (a) alpha-2-macroglobulin (A2M) proteins; and (b) serine protease proteins in a composition at a molar ratio [(a):(b)] of about 1:3 to about 1:1, thereby manufacturing the pharmaceutical composition comprising the protein complex. Some embodiments include combining (a) and (b) in a composition at a molar ratio [(a):(b)] of about 1:3, 1:2.9, 1:2.8, 1:2.7, 1:2.6, 1:2.5, 1:2.4, 1:2.3, 1:2.1, 1:2, 1:1.9, 1:1.8, 1:1.7, 1:1.6, 1:1.5, 1:1.4, 1:1.3, 1:1.2, 1:1.1 or 1:1.

[0182] A2M and / or serine protease proteins can be prepared or obtained by purification from a biological sample or by recombinant techniques. For example, in some embodiments, A2M and / or serine protease proteins are obtained by purifying the protein from the blood or plasma of a mammalian subject, for example, a human subject. Specific embodiments include obtaining and purifying A2M proteins from the plasma of a human subject prior to combination with serine protease proteins. Methods for purifying A2M from human plasma (plasma-enriched A2M; or A2MPPP) are known in the art (see, for example, et al., Pain Physician. 23(2):229-23, 2020; U.S. Patent No. 9,352,021).

[0183] Recombinant proteins can be conveniently prepared using standard protocols as described, for example, in Sambrook, et al., (1989, supra), in particular Sections 16 and 17; Ausubel et al., (1994, supra), in particular Chapters 10 and 16; and Coligan et al., Current Protocols in Protein Science (John Wiley & Sons, Inc. 1995-1997), in particular Chapters 1, 5 and 6. As a general example, a protein can be prepared by a procedure that includes one or more of the following steps: (a) preparing a vector or construct comprising a polynucleotide sequence encoding a protein described in this document, which is operationally linked to one or more regulatory elements; Petition 870250109095, dated 11 / 27 / 2025, page 79 / 150 75 / 102 (b) introduce the vector or construct into a host cell; (c) culture the host cell to express the protein; and (d) isolate the protein from the host cell.

[0184] To express a desired polypeptide, a nucleotide sequence encoding a protein can be inserted into appropriate expression vectors, that is, vectors containing the elements necessary for the transcription and translation of the inserted coding sequence. Methods that are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding a polypeptide of interest and appropriate transcription and translation control 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).

[0185] A variety of vector / host expression systems are known and can be used to contain and express polynucleotide sequences. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems, including mammalian cells and, more specifically, human cell systems.

[0186] The control elements or regulatory sequences present in an expression vector are those untranslated regions of the vector enhancers, promoters, 5' and 3' untranslated regions - which Petition 870250109095, dated 11 / 27 / 2025, p. 80 / 150 76 / 102 interact with host cellular proteins to perform transcription and translation. These elements can vary in their strength and specificity. Depending on the vector system and host used, any number of suitable transcription and translation elements can be used, including constitutive and inducible promoters. For example, when cloning in bacterial systems, inducible promoters such as the lacZ hybrid promoter of the PBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or the PSPORT1 plasmid (Gibco BRL, Gaithersburg, Md.) and the like can be used. In mammalian cell systems, promoters from mammalian genes or mammalian viruses are generally preferred. If it is necessary to generate a cell line containing multiple copies of the sequence encoding a polypeptide, SV40- or EBV-based vectors can be advantageously used with an appropriate selectable marker.

[0187] In bacterial systems, several expression vectors can be selected depending on the intended use for the expressed polypeptide. For example, when large quantities are needed, vectors that direct high-level expression of proteins that are easily purified can be used. Such vectors include, among others, multifunctional E. coli cloning and expression vectors, such as BLUESCRIPT (Stratagene), in which the sequence encoding the polypeptide of interest can be ligated into the vector structure with sequences for the Met amino terminus and the subsequent 7 β-galactosidase residues to produce a hybrid protein; pIN vectors (Van Heeke & Schuster, J. Biol. Chem. 264:5503-5509 (1989)); and similar vectors. pGEX vectors (Promega, Madison, Wis.) can also be used to express foreign polypeptides such as fusion proteins with glutathione S-transferase (GST).In general, these fusion proteins are soluble and can be easily purified from lysed cells by adsorption onto glutathione agarose beads followed by elution in the presence of free glutathione. Proteins produced in such systems can be engineered to include cleavage sites. Petition 870250109095, dated 11 / 27 / 2025, page 81 / 150 77 / 102 of heparin, thrombin, or factor XA protease so that the cloned polypeptide of interest can be released from the GST portion at will.

[0188] Certain modalities employ E. coli-based expression systems (see, for example, Structural Genomics Consortium et al., Nature Methods. 5:135-146, 2008). These and related modalities may depend partially or entirely on ligation-independent cloning (LIC) to produce a suitable expression vector. In specific modalities, protein expression may be controlled by a T7 RNA polymerase (e.g., pET vector series). These and other related modalities may utilize the BL21(DE3) expression host strain, a BL21 DE3 λ lysogen that supports T7-mediated expression and is deficient in lon and ompT proteases to improve target protein stability. Also included are expression host strains carrying plasmids encoding tRNAs rarely used in E. coli, such as ROSETTA™ (DE3) and Rosetta 2 (DE3) strains.Cell lysis and sample handling can also be improved using reagents sold under the trademarks BENZONASE® nuclease and BUGBUSTER® Protein Extraction Reagent. For cell culture, autoinducing media can improve the efficiency of many expression systems, including high-throughput expression systems. Media of this type (e.g., OVERNIGHT EXPRESS™ Autoinduction System) gradually induce protein expression through metabolic bypass without the addition of artificial inducing agents such as IPTG. Particular modalities employ hexahistidine markers (such as those sold under the trademark HIS*TAG® fusions), followed by purification by immobilized metal affinity chromatography (IMAC) or related techniques. In certain respects, however, clinical-grade proteins can be isolated from E. coli inclusion bodies with or without the use of affinity markers (see, for example, Shimp et al., Protein Expr Purif.).50:58-67, 2006). As another example, certain modalities may employ a production system of. Petition 870250109095, dated 11 / 27 / 2025, page 82 / 150 78 / 102 high yield of E. coli induced by cold shock, because the overexpression of proteins in Escherichia coli at low temperature improves their solubility and stability (see, for example, Qing et al., Nature Biotechnology. 22:877-882, 2004).

[0189] High-density bacterial fermentation systems are also included. For example, high-density cell culture of Ralstonia eutropha allows protein production at cell densities greater than 150 g / L and the expression of recombinant proteins at titers greater than 10 g / L.

[0190] In the yeast Saccharomyces cerevisiae, several vectors containing constitutive or inducible promoters, such as factor alpha, alcohol oxidase, and PGH, can be used. For reviews, see Ausubel et al. (supra) and Grant et al., Methods Enzymol. 153:516-544 (1987). Also included are the expression systems of Pichia pandoris (see, for example, Li et al., Nature Biotechnology. 24, 210-215, 2006; and Hamilton et al., Science, 301:1244, 2003). Certain embodiments include yeast systems that are engineered to selectively glycosylate proteins, including yeasts possessing humanized N-glycosylation pathways, among others (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., NatureBiotechnology. 22:1409-1414, 2004; U.S. Patents Nos. 7,629,163; 7,326,681; and 7,029,872).By way of example only, recombinant yeast cultures can be grown in Fernbach flasks or fermenters of 15L, 50L, 100L and 200L, among others.

[0191] In cases where plant expression vectors are used, the expression of polypeptide-coding sequences can be driven by any 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 of TMV (Takamatsu, EMBO J. 6:307-311 (1987)). Alternatively, plant promoters such as the subunit can be used. Petition 870250109095, dated 11 / 27 / 2025, page 83 / 150 79 / 102 small RUBISCO or heat shock promoters (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 generally available reviews (see, for example, Hobbs in McGraw Hill, Yearbook of Science and Technology, pp. 191-196 (1992)).

[0192] An insect system can also be used to express a polypeptide of interest. For example, in one such system, the nuclear polyhedrosis virus Autographa californica (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia cells. The sequences encoding the polypeptide 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-encoding sequence will render the polyhedrin gene inactive and produce the recombinant virus without a coat protein. The recombinant viruses can then be used to infect, for example, S. frugiperda cells or Trichoplusia cells in which the polypeptide of interest can be expressed (Engelhard et al., Proc. Natl. Acad. Sci. USA 91:3224-3227 (1994)).Also included are baculovirus expression systems, including those using 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 can provide post-translational modifications similar to mammalian systems.

[0193] In mammalian host cells, several virus-based expression systems are generally available. For example, in cases where an adenovirus is used as an expression vector, sequences encoding a polypeptide of interest can be linked to an adenovirus transcription / translation complex consisting of the late promoter and the sequence Petition 870250109095, dated 11 / 27 / 2025, page 84 / 150 80 / 102 tripartite leader. Insertion into a non-essential E1 or E3 region of the viral genome can be used to obtain a viable virus capable of expressing the polypeptide in infected host cells (Logan & Shenk, Proc. National Acad. Sci. USA 81:3655-3659 (1984)). Furthermore, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, can be used to increase expression in mammalian host cells.

[0194] Examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 or 293 subcloned cells 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 carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TR1 cells (Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma cell line (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)); and myeloma cell lines, such as NSO and Sp2 / 0. For a review of certain mammalian host cell lines suitable for protein production, see, for example, Yazaki and Wu, Methods in Molecular Biology, vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 255-268. Certain preferred mammalian cell expression systems include CHO and HEK293 cell-based expression systems. Mammalian expression systems may utilize attached cell lines, for example, Petition 870250109095, dated 11 / 27 / 2025, page 85 / 150 81 / 102 in T-flasks, rotary bottles or cell factories, or suspension cultures, for example, in 1L and 5L centrifuges, 5L, 14L, 40L, 100L and 200L shake tank bioreactors, or 20 / 50L and 100 / 200L WAVE bioreactors, among others known in the art.

[0195] Also included is cell-free expression of proteins. These and related modalities typically utilize purified RNA polymerase, ribosomes, tRNA, and ribonucleotides; these reagents can be produced by cell extraction or from a cell-based expression system.

[0196] Specific initiation signals can also be used to achieve more efficient translation of sequences encoding a polypeptide of interest. Such signals include the ATG initiation codon and adjacent sequences. In cases where the sequences encoding the polypeptide, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be necessary. However, in cases where only the coding sequence, or a portion thereof, is inserted, exogenous translational control signals, including the ATG initiation codon, must be provided. Furthermore, the initiation codon must be in the correct reading frame to ensure translation of the entire insertion. Exogenous translational elements and initiation codons can be of various origins, both natural and synthetic.The efficiency of expression can be increased by including enhancers that are appropriate for the particular cellular system that is used, such as those described in the literature (Scharf. et al., Results Probl. Cell Differ. 20:125-162 (1994)).

[0197] In addition, a host cell strain can be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired manner. Such polypeptide modifications include, but are not limited to, post-translational modifications such as Petition 870250109095, dated 11 / 27 / 2025, page 86 / 150 82 / 102 acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing that cleaves a “prepro” form of the protein can also be used to facilitate correct insertion, folding, and / or function. Different host cells, such as yeast, CHO, HeLa, MDCK, HEK293, and W138, as well as bacterial cells, which possess or even lack specific cellular machinery and characteristic mechanisms for such post-translational activities, can be chosen to ensure the correct modification and processing of the foreign protein.

[0198] For long-term, high-yield production of recombinant proteins, 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 viral origins of replication and / or endogenous expression elements and a selectable marker gene in the same or a separate vector. After vector introduction, cells can grow for about 1-2 days in an enriched medium before being switched to a selective medium. The purpose of the selectable marker is to confer resistance to selection, and its presence allows for the growth and recovery of cells that successfully express the introduced sequences. Resistant clones of stably transformed cells can be proliferated using tissue culture techniques appropriate for the cell type.Transient production, such as through transfection or transient infection, can also be employed. Exemplary mammalian expression systems that are suitable for transient production include systems based on HEK293 and CHO.

[0199] Any number of selection systems can be used to recover transformed or transduced cell lines. These include, but are not limited to, herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223-232 (1977)) and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817-823). Petition 870250109095, dated 11 / 27 / 2025, page 87 / 150 83 / 102 (1990)) genes that can be employed in tk- or aprt- cells, respectively. In addition, resistance to antimetabolites, antibiotics, or herbicides can be used as a basis for selection; for example, dhfr conferring resistance to methotrexate (Wigler et al., Proc. Natl. Acad. Sci. USA 77:3567-70 (1980)); npt conferring resistance to aminoglycosides, neomycin, and G-418 (Colbere-Garapin et al., J. Mol. Biol. 150:1-14 (1981)); and ais or pat conferring resistance to chlorsulfuron and phosphinothricin acetyltransferase, respectively (Murry, supra). Additional selectable genes have been described, for example, trpB, which allows cells to use indole instead of tryptophan, or hisD, which allows cells to use histinol instead of histidine (Hartman & Mulligan, Proc. National Acad. Science USA 85:8047-51 (1988)).The use of visible markers gained popularity with 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, being widely used not only to identify transformants but also to quantify the amount of transient or stable protein expression attributable to a specific vector system (see, for example, Rhodes et al., Methods Mol. Biol. 55:121-131 (1995)).

[0200] High-throughput protein production systems or microproduction systems are also included. Certain aspects may utilize, for example, hexahistidine fusion markers for protein expression and purification on metal chelate-modified slide surfaces or Ni MagneHis particles (see, for example, 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, for example, Sitaraman et al., Methods Mol Biol. 498:229-44, 2009).

[0201] A variety of protocols for detecting and measuring the expression of polynucleotide-encoded products, using linking agents or Petition 870250109095, dated 11 / 27 / 2025, page 88 / 150 84 / 102 antibodies, such as polyclonal or monoclonal antibodies specific for the product, are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), western immunoblots, radioimmunoassays (RIA), and fluorescence-activated cell screening (FACS). These and other assays are described, among other places, in Hampton et al., Serological Methods, a Laboratory Manual (1990) and Maddox et al., J. Exp. Med. 158:1211-1216 (1983).

[0202] A wide variety of markers and conjugation techniques are known to those skilled in the art and can be used in various nucleic acid and amino acid assays. Means of producing labeled hybridization or PCR probes to detect polynucleotide-related sequences include oligolabeling, translation cleavage, end-labeling, or PCR amplification using a labeled nucleotide. Alternatively, sequences, or any portions thereof, can be cloned into a vector for the production of an mRNA probe. These vectors are known in the art, are commercially available, and can be used to synthesize RNA probes in vitro by adding an appropriate RNA polymerase, such as T7, T3, or SP6, and labeled nucleotides. These procedures can be performed using a variety of commercially available kits.Suitable reporter molecules or markers that can be used include radionuclides, enzymes, fluorescent, chemiluminescent or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles and the like.

[0203] Host cells transformed with one or more polynucleotide sequences of interest can be cultured under conditions suitable for the expression and recovery of the cell culture protein. Certain specific modalities utilize serum-free cell expression systems. Examples include HEK293 cells and CHO cells that can be cultured 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). Petition 870250109095, dated 11 / 27 / 2025, page 89 / 150 85 / 102

[0204] A protein produced by a recombinant cell can be secreted or contained intracellularly depending on the sequence and / or vector used. As will be understood by those skilled in the art, expression vectors containing polynucleotides can be designed to contain signal sequences that direct the secretion of the encoded polypeptide across a prokaryotic or eukaryotic cell membrane. Other recombinant constructs can be used to link sequences encoding a polypeptide of interest to the nucleotide sequence encoding a polypeptide domain that will facilitate the purification and / or detection of soluble proteins. Examples of such domains include cleavable and non-cleavable affinity purification and epitope markers such as avidin, FLAG markers, polyhistidine markers (e.g., 6xHis), cMyc markers, V5 markers, glutathione S-transferase (GST) markers, and others.

[0205] The protein produced by a recombinant cell can be purified and characterized according to a variety of techniques known in the art. Exemplary systems for performing protein purification and analyzing protein purity include fast protein liquid chromatography (FPLC) (e.g., AKTA and Bio-Rad FPLC systems), high-performance liquid chromatography (HPLC) (e.g., Beckman and Waters HPLC). Exemplary chemistries for purification include ion-exchange chromatography (e.g., Q, S), size-exclusion chromatography, salt gradients, affinity purification (e.g., Ni, Co, FLAG, maltose, glutathione, protein A / G), gel filtration, reversed-phase, HYPERD® ceramic ion-exchange chromatography, and hydrophobic interaction (HIC) columns, among others known in the art.Also included are analytical methods such as SDS-PAGE (e.g., Coomassie, silver staining), immunoblotting, Bradford, and ELISA, which can be used during any stage of the production or purification process, typically to measure the purity of the protein composition. Petition 870250109095, dated 11 / 27 / 2025, page 90 / 150 86 / 102

[0206] Also included are methods of concentrating a protein or protein complex described in this document and compositions comprising the concentrated soluble protein or protein complex. In some respects, such concentrated solutions of a protein or protein complex comprise proteins at a concentration of about or at least about 5 mg / mL, 8 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, or more.

[0207] In some respects, such compositions may be substantially monodisperse, meaning that a protein or protein complex exists primarily (i.e., at least about 90% or more) in an apparent molecular weight form when assessed, for example, by size exclusion chromatography, dynamic light scattering, or analytical ultracentrifugation.

[0208] In some respects, such compositions have a purity (based on protein) of at least about 90%, or in some respects at least about 95% purity, or in some embodiments at least 98% purity. Purity can be determined by any routine analytical method known in the art.

[0209] In some aspects, such compositions have a high molecular weight aggregate content of less than about 10% compared to the total amount of protein present, or in some embodiments, such compositions have a high molecular weight aggregate content of less than about 5%, or in some aspects, such compositions have a high molecular weight aggregate content of less than about 3%, or in some embodiments, a high molecular weight aggregate content of less than about 1%. The high molecular weight aggregate content can be determined by a variety of analytical techniques, including, for example, size exclusion chromatography, dynamic light scattering, or analytical ultracentrifugation.

[0210] Examples of concentration approaches covered in this document include lyophilization, which is typically employed when the solution Petition 870250109095, dated 11 / 27 / 2025, page 91 / 150 87 / 102 contains few soluble components besides 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 employ one or more selectively permeable membranes to concentrate a protein solution. The membrane allows water and small molecules to pass through while retaining the protein; the solution can be forced against the membrane by mechanical pump, gas pressure, or centrifugation, among other techniques.

[0211] In certain embodiments, a protein or protein complex in a composition has a purity of at least about 90%, as measured according to routine techniques in the art. In certain embodiments, such as diagnostic compositions or certain pharmaceutical or therapeutic compositions, a protein or protein complex in a composition has a purity of at least about 95%, or at least about 97% or 98% or 99%. In some embodiments, such as when used as a reference or research reagent, a protein or protein complex may be of lower purity and may have a purity of at least about 50%, 60%, 70% or 80%. Purity may be measured globally or in relation to selected components, such as other proteins, for example, protein-based purity.

[0212] Purified proteins or protein complexes can also be characterized according to their biological characteristics. Binding affinity and binding kinetics can be measured according to a variety of techniques known in the art, such as Biacore® and related technologies that utilize surface plasmon resonance (SPR), an optical phenomenon that allows the detection of unlabeled interactants in real time. SPR-based biosensors can be used in the determination of active concentration, screening, and characterization in terms of affinity and kinetics. The presence or levels of one or more biological activities can be measured according to assays. Petition 870250109095, dated 11 / 27 / 2025, page 92 / 150 88 / 102 in vitro or cell-based, which are optionally functionally coupled to a readout or indicator, such as a fluorescent or luminescent indicator of biological activity, as described in this document.

[0213] In certain embodiments, as noted above, a composition is substantially free of endotoxin, including, for example, about 95% endotoxin-free, preferably about 99% endotoxin-free, and more preferably about 99.99% endotoxin-free. The presence of endotoxins can be detected according to routine techniques in the art, as described in this document. In specific embodiments, a protein or protein complex is produced from a eukaryotic cell, such as a mammalian or human cell, in a substantially serum-free medium. In certain embodiments, as noted in this document, a 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.

[0214] In certain embodiments, a composition comprises less than about 10% by weight / weight of high molecular weight aggregates, or less than about 5% by weight / weight of high molecular weight aggregates, or less than about 2% by weight / weight of high molecular weight aggregates, or less than about 1% w / w of high molecular weight aggregates.

[0215] Also included are protein-based assays and analytical methods, which can be used to assess, for example, purity, size, solubility, and degree of protein aggregation, among other characteristics. 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 assessing primary structure include N- and C-terminal sequencing and peptide mapping (see, for example, Allen et al., Biologicals. 24:255-275, 1996). Examples of methods Petition 870250109095, dated 11 / 27 / 2025, p. 93 / 150 Methods to assess higher-order structure include circular dichroism (see, for example, Kelly et al., Biochim Biophys Acta. 1751:119-139, 2005), fluorescence spectroscopy (see, for example, Meagher et al., J. Biol. Chem. 273:23283-89, 1998), FT-IR, hydrogen-deuterium amide exchange kinetics, differential scanning calorimetry, NMR spectroscopy, and immunoreactivity with conformation-sensitive antibodies. Higher-order structure can also be assessed as a function of a variety of parameters, such as pH, temperature, or added salts. Examples of methods for assessing protein characteristics, such as size, include analytical ultracentrifugation and size-exclusion HPLC (SEC-HPLC), and exemplary methods for measuring charge include ion-exchange chromatography and isoelectric focusing. Hydrophobicity can be assessed, for example, by reverse-phase HPLC and hydrophobic interaction chromatography HPLC.Glycosylation can affect pharmacokinetics (e.g., clearance), conformation or stability, receptor binding, and protein function, and can be assessed, for example, by mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy.

[0216] As noted above, certain modalities include the use of SEC-HPLC to evaluate protein characteristics such as purity, size (e.g., size homogeneity) or degree of aggregation and / or to purify proteins, among other uses. SEC, also including gel filtration chromatography (GFC) and gel permeation chromatography (GPC), refers to a chromatographic method in which molecules in solution are separated in a porous material based on their size, or more specifically on their hydrodynamic volume, diffusion coefficient, and / or surface properties. The process is generally used to separate biological molecules and to determine molecular weights and molecular weight distributions of polymers. Typically, a biological or protein sample (such as a protein extract produced according to the protein expression methods provided in this document and Petition 870250109095, dated 11 / 27 / 2025, page 94 / 150 (90 / 102 known in the art) is loaded onto a selected size-exclusion column with a defined stationary phase (the porous material), preferably a phase that does not interact with the sample proteins. In certain respects, the stationary phase is composed of inert particles packed in a dense three-dimensional array within a glass or steel column. The mobile phase can be pure water, an aqueous buffer, an organic solvent, or a mixture thereof. The stationary phase particles typically have small pores and / or channels that allow only the entry of molecules below a certain size. Large particles are therefore excluded from these pores and channels, and their limited interaction with the stationary phase causes them to elute as a “totally excluded” peak early in the experiment.Smaller molecules, which can fit within the pores, are removed from the mobile phase flow, and the time they spend immobilized within the stationary phase pores depends, in part, on how far into the pores they penetrate. Their removal from the mobile phase flow causes them to take longer to elute from the column and results in a separation between particles based on size differences. A given size-exclusion column has a range of molecular weights that can be separated. In general, molecules larger than the upper limit will not be trapped 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 examples of these methods in practice with pharmaceutical proteins, see Bruner et al., Journal of Pharmaceutical and Biomedical Analysis. 15: 1929-1935, 1997.

[0217] Protein purity for clinical applications is also discussed, for example, by Anicetti et al. (Trends in Biotechnology. 7:342-349, 1989). More recent techniques for analyzing protein purity include, but are not limited to, the LabChip GXII, an automated platform for rapid analysis of proteins and nucleic acids, which provides high-throughput titration analysis, Petition 870250109095, dated 11 / 27 / 2025, page 95 / 150 91 / 102 Protein sizing and purity analysis. In certain non-limiting embodiments, clinical-grade proteins or protein complexes can be obtained using 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, the protein agents are substantially free of endotoxins, as measured according to techniques known in the art and described in this document.

[0218] Protein solubility assays are also included. Such assays can be used, for example, to determine the ideal growth and purification conditions for recombinant production, to optimize the choice of buffers, and to optimize the choice of a protein or protein complex and its variants. Solubility or aggregation can be evaluated according to a variety of parameters, including temperature, pH, salts, and the presence or absence of other additives. Examples of solubility screening assays include, without limitation, microplate-based methods for measuring protein solubility using turbidity or another measure as an endpoint, high-throughput assays for analyzing the solubility of purified recombinant proteins (see, for example, Stenvall et al., Biochim Biophys Acta.1752:6-10, 2005), assays that use the structural complementation of a genetic marker protein to monitor and measure protein folding and solubility in vivo (see, for example, Wigley et al., Nature Biotechnology. 19:131-136, 2001) and electrochemical screening of recombinant protein solubility in Escherichia coli using scanning electrochemical microscopy (SECM) (see, for example, Nagamine et al., Biotechnology and Bioengineering. 96:1008-1013, 2006), among others. A protein or protein complex with increased solubility (or reduced aggregation) can be identified or selected according to routine techniques in the art, including simple in vivo assays for protein solubility (see, for example, Maxwell et al., Protein. Petition 870250109095, dated 11 / 27 / 2025, page 96 / 150 92 / 102 Sci. 8:1908-11, 1999).

[0219] Protein solubility and aggregation can also be measured by dynamic light scattering techniques. Aggregation is a general term encompassing various types of interactions or characteristics, including soluble / insoluble, covalent / non-covalent, reversible / irreversible, and native / denatured interactions and characteristics. For protein therapeutics, the presence of aggregates is usually considered undesirable due to concerns that aggregates may cause an immunogenic reaction (e.g., small aggregates) or adverse events on administration (e.g., particulate matter). 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 scattering rate of protein particles. Fluctuations in scattering intensity can be observed due to the Brownian motion of molecules and particles in solution. This motion data can be conventionally processed to derive a size distribution for the sample, where the size is given by the Stokes radius or hydrodynamic radius of the protein particle. The hydrodynamic size depends on both mass and shape (conformation). Dynamic scattering can detect the presence of very small amounts of aggregated protein (<0.01 wt%), even in samples containing a wide range of masses. It can also be used to compare the stability of different formulations, including, for example, applications that rely on real-time monitoring of changes at elevated temperatures.Consequently, certain methods include the use of dynamic light scattering to analyze the solubility and / or presence of aggregates in a sample containing a protein or protein complex. Petition 870250109095, dated 11 / 27 / 2025, page 97 / 150 93 / 102 disclosure.

[0220] Although the foregoing embodiments have been described in some detail by way of illustration and example for the sake of clarity of understanding, it will be readily apparent to one skilled in the art in the light of the teachings of this disclosure that certain changes and modifications may be made without departing from the spirit or scope of the appended claims. The following examples are provided by way of illustration only and not by way of limitation. Those skilled in the art will readily recognize a variety of non-critical parameters that may be altered or modified to produce essentially similar results. EXAMPLES Example 1 Characteristics of A2M:PPE Protein Complexes

[0221] Experiments were performed to test the characteristics of protein complexes composed of different molar ratios of A2M and 'mutant F' of the PPE protein (MutF, SEQ ID NO: 5).

[0222] A1AT Protection Assay. MutF (400nM) was mixed with different concentrations of A2M to achieve various molar ratios, followed by 30 minutes of incubation at 37°C. A protection assay was performed on a 384-well-coated black plate by adding 10uL of the sample with 10uL of PBS or 10uL of 2uM A1AT or at different concentrations in duplicate. 20μL of AAPV-AMC substrate at a concentration of 100μM were added. Kinetics were measured on Varioskan LUX: 380 nm ex / 460 nm em (5nm bandwidth), 37°C, top, 100 ms, 2 min for 16 readings. Vo was calculated as an activity measurement.

[0223] As shown in Figures 3A-3B, A2M:MutF protein complexes in a 1:2 molar ratio provide the best protection against A1AT (3A), and increasing the concentration of A1AT does not impair MutF protection by A2M. Petition 870250109095, dated 11 / 27 / 2025, page 98 / 150 94 / 102 (3B).

[0224] Plasma protection assay. MutF (400nM) was mixed with 200nM A2M to achieve a molar ratio of 1:2 (A2M:MutF), followed by 30 minutes of incubation at 37°C in PBS. A plasma protection assay was performed on a well-coated 384 black plate by adding 10uL of sample with 10uL of PBS or 10uL of mouse plasma samples in duplicate. 20μL of AAPV-AMC substrate at a concentration of 100μM were added. Kinetics were measured on Varioskan LUX: 380 nm ex / 460 nm (5nm bandwidth), 37°C, top, 100 ms, 2 min for 16 readings. Vo was calculated as a measure of activity.

[0225] As shown in Figure 3C, plasma inhibits MutF activity on its own, but does not inhibit its activity when complexed with A2M:MutF in a molar ratio of 1:2.

[0226] Purification of N17350-A2M by column chromatography. MutF was mixed with A2M to achieve a molar ratio of 1:2 (A2M:MutF), followed by a 30-minute incubation at 37°C. The AKTA pure chromatography system was used for protein purification. For the cation exchange column, the PD10 desalting column was used to buffer the protein mixture in 50 mM sodium acetate solution, pH 5.0. Samples were loaded onto the pre-washed / equilibrated HiTrap SP column with 0.5 M NaCl, 50 mM sodium acetate, pH 5.0, eluting in 20 fractions (1 mL / fraction). For the size exclusion column, the PD10 desalting column was used to buffer the protein mixture in 50 mM sodium phosphate, 150 mM NaCl, pH 7. Samples were loaded into pre-washed Superose 6 magnification 10 / 300. The flow rate was set to 0.5 mL / min to collect 1 mL per fraction. Each fraction was diluted 1:250 in PBS for A1AT protection assays of A2M:MutF.Protein concentration was measured using the Pierce™ MBC protein assay kit (ThermoFisher Scientific, see manufacturer's instructions). For the assay of... Petition 870250109095, dated 11 / 27 / 2025, page 99 / 150 95 / 102 A1AT protection, 10 μL of each dilution fraction were coated onto a 384 black well plate with 10 μL of PBS or 10 μL of 2 μM A1AT or at different concentrations in duplicate. 20 μL of the AAPV-AMC substrate at a concentration of 100 μM were added. Kinetics were measured on Varioskan LUX: 380 nm ex / 460 nm em (5 nm bandwidth), 37°C, top, 100 ms, 2 min for 16 readings. Vo was calculated as a measure of activity.

[0227] The results in Figures 4A-4E show that the A2M:MutF protein complex is stable. Figure 4A shows the activity and concentration of MutF in the different fractions of the A2M:MutF complex after separation from the cation exchange column in the presence or absence of A1AT. Figure 4B shows the activity and concentration of MutF in the different fractions of the A2M:MutF complex after separation from the size exclusion column in the presence or absence of A1AT. Figures 4C-4D show that the A2M:MutF complexes are stable over a wide pH range, as measured by enzymatic activity (4C) and A1AT protection (4D). Figure 4E shows that the A2M:MutF complexes are stable across multiple freeze / thaw cycles, as measured by enzymatic activity.

[0228] A1AT activity and 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-shaped bottom plate, followed by the addition of 40 µL of different conditions (SFM, A2M, 400 nm N17350, or premixed A2M:MutF (800 nm-400 nm)). The plate was incubated at 37°C for 30 minutes. After incubation, the plate was centrifuged at 300 x g for 5 minutes and washed with an additional 200 µL of PBS per well. 25 µL of cytoplasmic lysis buffer were added to each well (10 mM HEPES pH 8, 10 mM KCl, 0.1 mM EDTA, 0.3% NP-40) and incubated on ice for 20 minutes with 10-second vortex cycles 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 Petition 870250109095, dated 11 / 27 / 2025, pp. 100 / 150 96 / 102 for measuring the activity and protection assay of A2M:MutF. Here, 10 μL of the supernatant were coated onto 384 black well plates with 10 μL of PBS or 10 μL of 2 μM A1AT, or at different concentrations in duplicate. 20 μL of the AAPV-AMC substrate at a concentration of 100 μM were added. Kinetics were measured on Varioskan LUX: 380 nm ex / 460 nm em (5 nm bandwidth), 37°C, top, 100 ms, 2 min for 16 readings. Vo was calculated as an activity measurement.

[0229] The results in Figures 5A-5D show that A2M:MutF protein complexes can enter cells and remain in the form of an intracellular protein complex, since A1AT is not able to inhibit their activity.

[0230] CD95-C cleavage assay. MutF was mixed with different concentrations of A2M to achieve various molar ratios, followed by a 30-minute incubation at 37°C in PBS. Recombinant terminal CD95-C protein (Wuxi) was incubated with premixed A2M:MutF protein complexes at different molar ratios for 1 h at 37°C. Samples containing 1 µg of CD95-C were loaded onto 17.5% SDS-PAGE gels and stained with One-step Blue (Biotium). The gels were photographed using iBright 1500.

[0231] As shown in Figure 6A, A2M:MutF protein complexes ranging from a molar ratio of 1:2 to 1:16 cleave CD95 as efficiently as MutF alone.

[0232] Fibrinogen cleavage assay. MutF was mixed with different concentrations of A2M to achieve various molar ratios, followed by a 30-minute incubation at 37°C in PBS. Human fibrinogen (Sigma) was incubated with premixed A2M:MutF protein complexes at different molar ratios for 1 hour at 37°C. Samples containing 5µg of fibrinogen were loaded onto 4-15% gels, and fibrinogen was detected by Western blot with an anti-fibrinogen (Cell Signaling Technology). The gels were photographed using an iBright 1500. Petition 870250109095, dated 11 / 27 / 2025, pp. 101 / 150 97 / 102

[0233] As shown in Figure 6B, the A2M:MutF protein complexes do not cleave fibrinogen, in comparison with MutF alone, which does cleave fibrinogen.

[0234] Elastin cleavage assay. MutF was mixed with different concentrations of A2M to achieve various molar ratios, followed by a 30-minute incubation at 37°C in PBS. Elastin-F (1:100) was incubated with premixed A2M:MutF protein complexes overnight at 37°C, and the degraded fluorescent signal was measured in the supernatant.

[0235] As shown in Figure 6C, the A2M:MutF protein complexes do not cleave elastin, in comparison with MutF alone, which does cleave elastin.

[0236] Coagulation assay. Mice were injected with 200 μL of 480 μg MutF or purified A2M:MutF protein complexes at a molar ratio of 1:2. Blood was collected in sodium citrate tubes and centrifuged at 1500 x g for 15 minutes. 300 μL of plasma were frozen and sent to IDEXX for analysis of prothrombin time, partial thromboplastin time, and fibrinogen concentration.

[0237] The results in Figures 7A-7C show that the A2M:MutF protein complexes do not induce coagulation effects, compared to MutF alone, which, in contrast, increases PT and PTT times and causes reductions in fibrinogen levels.

[0238] Cell death assay. Approximately 20K-40K cancer cells were plated onto 96-well plates coated with black wells overnight to settle. On the second day, each well was washed with 200µL of serum-free medium once, followed by different treatments in triplicate. 24h after treatment, the cells were incubated with pre-diluted Calcein-AM solution (ThermoFisher, 4µM in HBSS with Ca2+Mg2+) for 45 minutes. Calcein-AM was poured off and 100µL of HBSS with Ca2+Mg2+ was added to each well and the Petition 870250109095, dated 11 / 27 / 2025, pp. 102 / 150 98 / 102 fluorescence was measured by Varioskan LUX at 485 / 520nm (20 bandwidth).

[0239] As shown in Figure 8A, A2M:MutF protein complexes in a 1:2 molar ratio induce the death of a variety of cancer cells in vitro, comparable in most cases to the activity of MutF alone. Figure 8B shows broad cytotoxicity of the A2M:MutF (1:2) protein complex against cancer cells of different anatomical origin, and Figure 8C shows that the complex does not kill non-cancerous cells.

[0240] In vivo efficacy. Approximately one million CT26 cells were implanted into the flank of 7-8 week-old BALB / c mice. Once the tumors reached ~80 mm3, the tumors were treated with 100µg of MutF, 100µg of A2M:MutF (1:2) or 1.4mg of A2M (corresponding to the amount to generate the A2M:MutF molar ratio in 1:2). The tumors were monitored every two days.

[0241] As shown in Figure 8D, A2M:MutF protein complexes at a molar ratio of 1:2 induce cancer cell death in vivo, comparable to the cell death activity of MutF alone. Figure 8E shows that the A2M:MutF protein complex has an improved functional PK profile (enzymatic activity in plasma) compared to MutF alone after intravenous administration. Figure 8F shows that the A2M:MutF protein complex induces a favorable immune profile in the CT26 model (from left to right in each graph is PBS, MutF, A2M:MutF). Figure 8G shows that the A2M:MutF protein complex elicits a tumor antigen-specific CD8+ T cell response in the CT26 model (from left to right in each graph is PBS, MutF, A2M:MutF). The induction of effector and memory T cells is indicative of a functional adaptive immune response.

[0242] Evaluation of selective cancer cell death in samples from ovarian cancer patients. Cancerous and non-cancerous cells were isolated from primary tumors, intraperitoneal fluid (IP), and adipose tissue. Petition 870250109095, dated 11 / 27 / 2025, pp. 103 / 150 99 / 102 omental (common metastatic site) and blood from 6 patients with ovarian cancer. The following cells were isolated and tested: cancerous cells (tumor digested with fibroblast depletion, CD45+ cells, and EpCAM+ / high cells), non-cancerous immune cells (omental digested cells with neutrophil depletion or CD45+ cells 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 (composed of >90% CD45+ immune cells, including B, T, myeloid, and NK cells). Isolated cells were plated and exposed to A2M:MutF, doxorubicin (standard-of-care chemotherapy), or oxaliplatin (standard-of-care chemotherapy) for 24 hours. Cell viability was assessed by calcein-AM.

[0243] Figure 9A shows that the A2M:MutF protein complex, in contrast to doxorubicin and oxaliplatin, has a wide therapeutic window, as shown by the elimination of human ovarian cancer cells without killing non-cancerous cells from patients. Figure 9B shows that A2M:MutF kills cancerous cells comparatively from patients without chemotherapy and those treated with chemotherapy, in contrast to doxorubicin and oxaliplatin, which show reduced elimination of cancerous cells isolated from patients treated with chemotherapy compared to patients without chemotherapy.

[0244] Evaluation of immunogenic cell death (ICD). CT26 (murine colon), A549 (human lung) and ovarian cancer patient cells were treated with A2M:MutF and oxaliplatin (a known ICD inducer in certain cell types at high concentrations) for 24 hours. Immunogenic cell markers were evaluated, including HSP70, ATP release, HMGB1 and CALR (see, for example, Fucikova et al., Cell Death Dis. 11(11): 1013, 2020).

[0245] Figure 10A shows that the A2M:MutF protein complex induces ICD markers in CT26 and A549 cells. Figure 10B shows that the complex Petition 870250109095, dated 11 / 27 / 2025, pp. 104 / 150 100 / 102 protein A2M:MutF induces ICD markers in tumor cells derived from human ovarian patients (from left to right in each graph are CTRL, A2M:MutF, oxaliplatin).

[0246] Dose and schedule assessment of A2M:MutF. CT26 colon cancer cells were injected into the flanks of mice and cultured to approximately 80 mm3. Multiple intravenous doses of A2M:MutF protein complex or vehicle were administered as follows: • Vehicle: Days 0, 2, 4 and 6 and Days 8, 10, 12 and 14; • A2M:MutF 100 μg: Every day for 2 weeks (daily); • A2M:MutF 200 μg: Days 0, 2, 4, 6 and Days 8, 10, 12 and 14 (on alternate days); and • A2M:MutF 400 μg: Days 0, 4, 8 and 12 (every 4 days).

[0247] Tumor growth and weight were evaluated. Figures 11A and 11B show tumor growth after treatment and Figure 11C shows tumor weight 15 days after treatment (11C from left to right: vehicle on alternate days, A2M:MutF 100 μg daily, A2M:MutF 200 μg on alternate days, A2M:MutF 400 μg every 4 days).

[0248] Evaluation of antitumor efficacy in syngeneic mouse tumor models. CT26 (highly immunogenic colon model), MC38 (hot immunogenic colon model) and B16F10 (cold immunogenic model of skin cancer and lung metastasis) cancer cells were injected into the flanks of syngeneic B-cell deficient Jh-BALB / c or C57BL / 6 mice and cultured to approximately 80-100 mm3. A2M:MutF protein complex (400 μg) or vehicle was injected intravenously on alternate days for 2-3 weeks. Tumor growth was monitored.

[0249] Figures 12A-12B show that the A2M:MutF protein complex effectively attenuates tumor growth in the JhBALB / c CT26 colorectal cancer model. Figures 12C-12D show that the A2M:MutF protein complex Petition 870250109095, dated 11 / 27 / 2025, pp. 105 / 150 101 / 102 treats primary tumor and metastasis in a Jh-C57BL / 6 B16F10 melanoma model. Figure 12E shows that the A2M:MutF protein complex exhibits efficacy in a variety of tumors with varying immune status.

[0250] Evaluation of antitumor efficacy compared to SoC chemotherapy. CT26 colon cancer cells were injected into the flanks of syngeneic B-cell deficient JhBALB / c mice and cultured to approximately 80-100 mm3. A2M:MutF protein complex (400 μg on alternate days for 3 weeks) or oxaliplatin (6 mg / kg on days 0 and 2) were injected intravenously. Tumor growth and overall survival were monitored.

[0251] Figures 13A-13C show that the A2M:MutF protein complex improved antitumor efficacy compared to chemotherapy with SoC (oxaliplatin) without observed toxicity.

[0252] Evaluation of antitumor efficacy against human cancer cells in xenograft models (NU / NU and NCG female mice). HCT116 (human colorectal cancer), HT29 (human colorectal cancer), PC3 (human prostate cancer), NCI-H358 (human lung cancer), A549 (human lung cancer), ovarian patient-derived model (CDX), and breast cancer model (PDX) cells were injected into the flanks of mice and cultured to approximately 80-100 mm3. A2M:MutF protein complex (400 μg) or vehicle was injected intravenously on alternate days for 2-3 weeks. Tumor growth and overall survival were monitored.

[0253] 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 in a variety of prostate cancer, colon cancer, and lung cancer models. Figure 14C shows that the A2M:MutF protein complex effectively kills human tumor cells derived from ovarian patients (from patient CDX_O02) in a model of Petition 870250109095, dated 11 / 27 / 2025, pp. 106 / 150 Figure 14D summarizes the efficacy of the A2M:MutF protein complex in this model in three ovarian cancer patients (patient histories: diagnosis, Grade 3; treatment, CDX_O01 and CDX_O03 without chemotherapy and CDX_O02 treated with 3 cycles of carboplatin+paclitaxel+keytruda). Figures 14E-14F show that the A2M:MutF protein complex effectively kills patient-derived breast cancer cells in vitro and in vivo (patient history: diagnosis, HER2- and ER+ breast cancer; treatment, no prior chemotherapy). Figure 14G summarizes the in vivo efficacy of the A2M:MutF protein complex in a variety of human tumors and shows that efficacy is independent of tumor genetics or immune status.

[0254] Figure 15 shows that mice treated with A2M:MutF protein complexes are tumor-free (5 / 11) after initial challenge with CT26 colorectal cancer cells and that all of these mice (5 / 5) retain their tumor-free status after further challenge with CD26 cells. This study suggests that treatment with A2M:MutF protein complexes induces a tumor-specific immune memory response.

Claims

1. Pharmaceutical composition CHARACTERIZED in that it comprises a protein complex of: (a) alpha-2-macroglobulin (A2M) proteins; and (b) serine protease proteins, wherein (a) and (b) are present in the composition in a molar ratio [(a):(b)] of about 1:3 to about 1:

1.

2. Pharmaceutical composition according to claim 1, CHARACTERIZED in that the A2M proteins of (a) and the serine protease proteins of (b) are joined in the protein complex and, optionally, in that the protein complex: (i) retains the CD95 protease cleavage activity (Fas receptor) and the cancer cell killing activity of (b); (ii) sterically prevents the binding of (b) to fibrinogen and reduces or inhibits the fibrinogen cleavage activity of (b); and (iii) sterically prevents the binding of (b) to serine protease inhibitors (including alpha-1 antitrypsin (A1AT)).

3. Pharmaceutical composition, according to claim 1 or 2, CHARACTERIZED in that (a) it comprises, 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.

4. Pharmaceutical composition, according to claim 3, CHARACTERIZED in that the functional fragment thereof comprises, consists of, or essentially consists of about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1300, or 1400 contiguous amino acids of a sequence selected from Table A1. Petition 870250088726, dated 09 / 30 / 2025, page 9 / 20 2 / 11 5. Pharmaceutical composition, according to claim 4, CHARACTERIZED in that the functional fragment thereof is composed of 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, 400-1100, 400-1000, 400-900, 400-800, 400-700, 400-600, 400-500, 500-1400, 500-1300, 500-1200, 500-1100, 500-1000, 500-900, 500-800, 500-700, 500-600, 600-1400, 600-1300, 600-1200, 600-1100, 600-1000, 600-900, 600-800, 600-700, 700-1400 700-1300, 700-1200, 700-1100, 700-1000, 700-900, 700-800, 800-1400, 800-1300, 800-1200, 800-1100, 800-1000, 800-900, 900-1400, 900-1300, 900-1200,9001100, 900-1000, 1000-1400, 1000-1300, 1000-1200, 1000-1100, 1100-1400, 11001300, 1100-1200, 1200-1400 or 1200-1300 residues of a sequence selected from Table A1.

6. Pharmaceutical composition, according to any one of claims 1-5, CHARACTERIZED in that (a) it is conjugated or fused to an antibody, or an antigen-binding fragment thereof.

7. Pharmaceutical composition, according to claim 6, CHARACTERIZED in that the antibody, or antigen-binding fragment thereof, binds specifically to a tumor-associated antigen (TAA) or tumor-specific antigen (TSA).

8. Pharmaceutical composition, according to any one of claims 1-7, CHARACTERIZED in that (b) is selected from porcine pancreatic elastase protein (PPE), human neutrophil elastase protein Petition 870250088726, dated 09 / 30 / 2025, page 10 / 20 3 / 11 (ELANE), human cathepsin G protein (CTSG), human proteinase 3 (PR3) and granzyme B protein.

9. Pharmaceutical composition, according to claim 8, CHARACTERIZED in that: a modified PPE protein comprising, consisting of or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99 or 100% identical to SEQ ID NO: 5, and retaining the amino acid substitution Q211F; a modified PPE protein comprising, consisting of or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99 or 100% identical to SEQ ID NO: 6, and retaining the amino acid substitution T55A; a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 7, and retaining the amino acid substitutions Q211F and T55A;a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 8, and retaining the N241A amino acid substitution; a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 9, and retaining the N241Y amino acid substitution; a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 10, and retaining the R75A amino acid substitution; a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 11, and retaining the R75E amino acid substitution;Petition 870250088726, dated 09 / 30 / 2025, p. 11 / 20 4 / 11 a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 12, and retaining the Q211A amino acid substitution; a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 13, and retaining the R237A amino acid substitution; a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 14, and retaining the S214A amino acid substitution; a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 15, and retaining the D74A amino acid substitution;and a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 16.; 10. Pharmaceutical composition, according to claim 8, CHARACTERIZED in that: a modified human ELANE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99 or 100% identical to SEQ ID NO: 17; a modified human CTSG protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99 or 100% identical to SEQ ID NO: 18; a human PR3 protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99 or 100% identical to SEQ ID NO: 19; or Petition 870250088726, dated 09 / 30 / 2025, p. 12 / 20 5 / 11 The human granzyme B protein comprises, 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.

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

1.

12. Pharmaceutical composition according to claim 11, CHARACTERIZED in that (a) and (b) are present in the composition in a molar ratio of about 1:

2.

13. A method for treating, improving symptoms and / or reducing the progression of cancer in a subject in need, CHARACTERIZED in that it comprises administering a pharmaceutical composition, as defined in any one of claims 1-12.

14. Method according to claim 13, CHARACTERIZED in that the cancer is a primary cancer or a metastatic cancer and is selected from one or more melanomas (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, hepatoma (hepatocellular carcinoma), sarcoma, B-cell malignancy, ovarian cancer, colorectal cancer, glioma, glioblastoma multiforme, meningioma, pituitary adenoma, vestibular schwannoma, primary CNS lymphoma, tumor primitive neuroectodermal (medulloblastoma), bladder cancer, uterine cancer, esophageal cancer,brain cancer, head and neck cancer, cervical cancer, testicular cancer, thyroid cancer, and stomach cancer.

15. Method, according to claim 13 or 14, CHARACTERIZED in that the administration (optionally intravenous administration) of the pharmaceutical composition does not substantially increase the prothrombin time or the partial thromboplastin time in the subject.

16. Method, according to any one of claims 13-15, CHARACTERIZED in that administration of the pharmaceutical composition increases the death of cancer cells 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 in relation to a control or reference.

17. Method, according to any one of claims 13-16, CHARACTERIZED in that it comprises administering the pharmaceutical composition to the subject by parenteral administration.

18. Method according to claim 17, CHARACTERIZED in that parenteral administration is intravenous administration.

19. Method of manufacturing a pharmaceutical composition CHARACTERIZED in that it comprises a protein complex, combining: (a) alpha-2-macroglobulin (A2M) proteins; and (b) serine protease proteins, in a composition in a molar ratio [(a):(b)] of about 1:3 to about 1:1, thereby manufacturing the pharmaceutical composition comprising the protein complex.

20. Method according to claim 19, CHARACTERIZED in that it comprises recombinantly producing (a) before combining with (b).

21. Method according to claim 19, CHARACTERIZED in that it comprises purifying (a) from the plasma of a human subject before combining with (b). Petition 870250088726, dated 09 / 30 / 2025, p. 14 / 20 7 / 11 22. Method, according to any one of claims 19-21, CHARACTERIZED in that it comprises recombinantly producing (b) before combining with (a).

23. Method, according to any one of claims 19-22, CHARACTERIZED in that it comprises combining (a) and (b) in a molar ratio [(a):(b)] of about 1:3, 1:2.9, 1:2.8, 1:2.7, 1:2.6, 1:2.5, 1:2.4, 1:2.3, 1:2.1, 1:2, 1:1.9, 1:1.8, 1:1.7, 1:1.6, 1:1.5, 1:1.4, 1:1.3, 1:1.2, 1:1.1 or 1:

1.

24. Method according to claim 23, CHARACTERIZED in that it comprises combining (a) and (b) in a molar ratio [(a):(b)] of about 1:

2.

25. Method according to any one of claims 19-24, CHARACTERIZED in that the A2M proteins of (a) and the serine protease proteins of (b) join in the protein complex and, optionally, in that the protein complex: (i) retains the CD95 protease cleavage activity (Fas receptor) and cancer cell killing activity of (b); (ii) sterically prevents the binding of (b) to fibrinogen and reduces or inhibits the fibrinogen cleavage activity of (b); and (iii) sterically prevents the binding of (b) to serine protease inhibitors (including alpha-1 antitrypsin (A1AT)).

26. Method according to any one of claims 19-25, CHARACTERIZED in that (a) it comprises, 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.

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

28. Method according to claim 27, characterized in that the functional fragment thereof is composed of approximately 1-1400, 11300, 1-1200, 1-1100, 1-1000, 1-900, 1-800, 1-700, 1-600, 1-500, 1-400, 1-300, 1-200, 100-1400, 100-1300, 100-1200, 100-1100, 100-1000, 100-900, 100-800, 100-700, 100-600, 100-500, 100-400, 100-300, 100-200, 200-1400, 200-1300, 200-1200, 200-1100, 200-1000, 200-900, 200-800, 200-700, 200-600, 200-500, 200-400, 200-300, 300-1400, 300-1300, 300-1200, 300-1100, 300-1000, 300-900, 300-800, 300-700, 300-600, 300-500, 300-400, 400-1400, 400-1300 400-1200, 400-1100, 400-1000, 400-900, 400-800, 400-700, 400-600, 400-500, 500-1400, 500-1300, 500-1200, 500-1100, 500-1000, 500-900, 500-800, 500-700, 500-600, 600-1400, 600-1300, 600-1200, 600-1100, 600-1000, 600-900, 600-800, 600-700, 700-1400 700-1300, 700-1200, 700-1100, 700-1000, 700-900, 700-800, 800-1400, 800-1300, 800-1200, 800-1100, 800-1000, 800-900, 900-1400, 900-1300, 900-1200, 900-1100, 900-1000,1000-1400, 1000-1300, 1000-1200, 1000-1100, 1100-1400, 1100-1300, 1100-1200, 1200-1400 or 1200-1300 residues of a sequence selected from Table A1.

29. Method according to any one of claims 19-28, CHARACTERIZED in that (a) it is conjugated or fused to an antibody, or an antigen-binding fragment thereof.

30. Method according to claim 29, CHARACTERIZED in that the antibody, or antigen-binding fragment thereof, binds specifically to a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA).

31. Method, according to any one of claims 19-30, CHARACTERIZED in that (b) is selected from a porcine pancreatic elastase protein (PPE), a human neutrophil elastase protein (ELANE), a human cathepsin G protein (CTSG), a human proteinase 3 (PR3) and a human granzyme B protein.

32. Method according to claim 31, CHARACTERIZED in that: a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99 or 100% identical to SEQ ID NO: 5, and retaining the Q211F amino acid substitution; a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99 or 100% identical to SEQ ID NO: 6, and retaining the T55A amino acid substitution; a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 7, and retaining the amino acid substitutions Q211F and T55A;a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 8, and retaining the N241A amino acid substitution; a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 9, and retaining the N241Y amino acid substitution; a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 10, and retaining the R75A amino acid substitution; a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 11, and retaining the R75E amino acid substitution;a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99 or 100% identical to SEQ ID NO: 12, and retaining the Q211A amino acid substitution; a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99 or 100% identical to SEQ ID NO: 13, and retaining the R237A amino acid substitution; a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 14, and retaining the S214A amino acid substitution; a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 15, and retaining the D74A amino acid substitution;and a modified PPE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 16.; 33. Method according to claim 31, CHARACTERIZED in that: a modified human ELANE protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 17; a modified human CTSG protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 18; a human PR3 protein comprising, consisting of, or essentially consisting of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 19; or the human granzyme B protein comprises, consists 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.

34. Method, according to any one of claims 19-33, CHARACTERIZED in that it further comprises the step of testing the pharmaceutical composition in one or more activity assays selected from one or more of a CD95 cleavage assay (optionally in the presence of a serine protease inhibitor, such as A1AT), a fibrinogen cleavage assay and a cancer cell death assay.

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

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