Serine proteases for treating therapy-resistant cancers

Abstract

Provided are methods and compositions related to the use of serine proteases such as porcine pancreatic elastase (PPE) for treating cancers that are resistant or cross-resistant to one or more drug therapies.

Description

[0001] Attorney Docket: OPN 1-014 / 01 WO 332575-2132

[0002] SERINE PROTEASES FOR TREATING THERAPY-RESISTANT CANCERS

[0003] Cross-Reference to Related Applications

[0004] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 769,367, filed March 10, 2025, which is incorporated by reference in its entirety.

[0005] Statement Regarding Sequence Listing

[0006] The Sequence Listing XML associated with this application is provided in XML file format and is hereby incorporated by reference into the specification. The name of the XML file containing the Sequence Listing XML is OPNI_014_01 WO_ST26.xmL The XML file is about 28,747 bytes, was created on March 9, 2026, and is being submitted electronically via USPTO Patent Center.

[0007] BACKGROUND

[0008] Technical Field

[0009] The present disclosure relates to the use of serine proteases such as porcine pancreatic elastase (PPE) for treating cancers that are resistant or cross-resistant to one or more drug therapies.

[0010] Description of the Related Art

[0011] Drug resistance continues to represent a major limiting factor to effectively treating cancers (see, for example, Vasan et aL, Nature. 575: 299-309, 2019). Moreover, patients who develop resistance to one drug can exhibit cross-resistance to others, both within and across drug classes, narrowing treatment options and limiting therapeutic efficacy (see, for example, Loria, et al., Front. Oncol. 12: 877380, 2022). Potential solutions include the identification of novel drugs and improved pharmacological principles that result in deeper responses.

[0012] Serine protease or elastase enzymes that target the ELANE (neutrophil elastase) pathway are selectively toxic to cancer cells but relatively non-toxic to normal or otherwise healthy cells (see, for example WO 2018 / 232273). However, there is a need in the art to determine if such agents retain broad efficacy against cancers that are resistant or crossresistant to standard of care drug therapies, including immunotherapies, chemotherapies, and targeted therapies.

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[0015] BRIEF SUMMARY

[0016] Embodiments of the present disclosure include methods of treating, ameliorating the symptoms of, and / or reducing the progression of, a cancer in a subject in need thereof, wherein the cancer is resistant (or refractory) to a drug therapy, comprising administering to the subject a serine protease protein or an expressible polynucleotide encoding the serine protease protein. In some embodiments, the cancer is resistant (for example, cross-resistant) to a drug therapy selected from an immunotherapy, a chemotherapy, and a targeted therapy.

[0017] In certain embodiments, the immunotherapy is a checkpoint inhibitor (CPi) selected from a Programmed Death 1 (PD-1) inhibitor, a Programmed Death-Ligand 1 (PD-L1) inhibitor, a Cytotoxic T-Lymphocyte-Associated protein 4 (CTLA-4) inhibitor, a Programmed Death-Ligand 2 (PD-L2) inhibitor, a V-domain Ig suppressor of T cell activation (VISTA) inhibitor, an Indoleamine 2,3-dioxygenase (IDO) inhibitor, a tryptophan 2,3-dioxygenase (TDO) inhibitor, a T-cell Immunoglobulin domain and Mucin domain 3 (TIM-3) inhibitor, a Lymphocyte Activation Gene-3 (LAG-3) inhibitor, a B and T Lymphocyte Attenuator (BTLA) inhibitor, a CD160 inhibitor, and a T-cell immunoreceptorwith Ig and ITIM domains (TIGIT) inhibitor. In some embodiments, the PD-1 inhibitor is selected from cemiplimab, dostarlimab, nivolumab, pembrolizumab, and pidilizumab; the PD-L1 inhibitor is selected from atezolizumab, avelumab, and durvalumab; the CTLA-4 inhibitor is selected from ipilimumab and tremelimumab; and / or the LAG3 inhibitor is relatlimab (BMS-986016).

[0018] In some embodiments, the immunotherapy is a co-stimulatory agonist selected from a CD40 agonist, an 0X40 agonist, a Glucocorticoid-Induced TNFR Family Related Gene (GITR) agonist, a CD137 (4-1 BB) agonist, a CD27 agonist, a CD28 agonist, a CD226 agonist, an ICOS agonist, and a Herpes Virus Entry Mediator (HVEM) agonist.

[0019] In some embodiments, the chemotherapy is selected from one or more of paclitaxel, doxorubicin, oxaliplatin, and carboplatin. In particular embodiments, the chemotherapy is paclitaxel, which is cross-resistant to one or more of doxorubicin, oxaliplatin, and carboplatin. In certain embodiments, the chemotherapy is doxorubicin, which is cross-resistant to one or more of paclitaxel, oxaliplatin, and carboplatin. In some embodiments, the chemotherapy is oxaliplatin, which is cross-resistant to one or more of paclitaxel, doxorubicin, and carboplatin. In some embodiments, the chemotherapy is carboplatin, which is cross-resistant to one or more of paclitaxel, doxorubicin, and oxaliplatin.

[0020] In some embodiments, the chemotherapy is selected from an alkylating agent, an antimetabolite, a cytotoxic antibiotic, a topoisomerase inhibitor (type 1 or type II), and an anti-

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[0023] microtubule agent, optionally wherein the chemotherapy is part of a conjugate, optionally an antibody-drug conjugate (ADC) or peptide drug conjugate (PDC). In specific embodiments, the alkylating agent is selected from nitrogen mustards (optionally mechlorethamine, cyclophosphamide, mustine, melphalan, chlorambucil, ifosfamide, or busulfan), nitrosoureas (optionally N-Nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine, or streptozotocin), tetrazines (optionally dacarbazine, mitozolomide, or temozolomide), aziridines (optionally thiotepa, mytomycin, ordiaziquone (AZQ)), cisplatins and derivatives thereof (optionally carboplatin or oxaliplatin), and non-classical alkylating agents (optionally procarbazine and hexamethylmelamine); the anti-metabolite is selected from anti-folates (optionally methotrexate or pemetrexed), fluoropyrimidines (optionally 5-fluorouracil or capecitabine), deoxynucleoside analogues (optionally ancitabine, enocitabine, cytarabine, gemcitabine, decitabine, azacitidine, fludarabine, nelarabine, cladribine, clofarabine, fludarabine, or pentostatin), and thiopurines (optionally thioguanine or mercaptopurine); the cytotoxic antibiotic is selected from anthracyclines (optionally doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, aclarubicin, or mitoxantrone), bleomycins, mitomycin C, mitoxantrone, and actinomycin; the topoisomerase inhibitor is selected from camptothecin, irinotecan, topotecan, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, merbarone, and aclarubicin; or the anti-microtubule agent is selected from taxanes (optionally paclitaxel or docetaxel) and vinca alkaloids (optionally vinblastine, vincristine, vindesine, or vinorelbine).

[0024] In some embodiments, the targeted therapy is a KRAS inhibitor, a HER-targeted therapy (optionally a HER1 , HER2, HER3, or HER4-targeted therapy), a BRAF inhibitor, a poly (ADP-ribose) polymerase (PARP) inhibitor, a MEK1 / MEK2 kinase inhibitor, a phosphatidylinositol 3-kinase (PI3K) signaling pathway-targeted therapy optionally a PI3K inhibitor, an AKT inhibitor, an EGFR-targeted therapy, a cyclin dependent kinase (CDK) 4 / 6 inhibitor, an androgen receptor inhibitor, or a NOTCH inhibitor. In certain embodiments, the KRAS inhibitor is a multi-KRAS inhibitor, a KRAS G12C inhibitor, a KRAS G12D inhibitor, or a KRAS G12V inhibitor, optionally wherein the subject has a KRAS G12 substitution, optionally G12C, G12D, G12R, or G12V. In specific embodiments, the KRAS inhibitor is selected from sotorasib (AMG-510), MRTX-1257, adagrasib, JDQ443, divarasib / GDC-6036, LY353798, MRTX-1133, RMC-9805, HRS-4642, ASP3082, TD010, VRTX153, QTX3046, QTX3544, VRTX144, ERAS-4, NT-0300, PP-008, JAB-22000, AFNT-212, and M DG2021.

[0025] In some embodiments, the resistance is an acquired resistance or acquired crossresistance. In some embodiments, the subject is currently undergoing or has previously

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[0028] undergone the drug therapy, including wherein the subject has previously undergone more than one drug therapy and is cross-resistant to the more than one drug therapy. Particular methods comprise selecting the subject for treatment with the serine protease based on the cancer in the subject being resistant (or refractory) to the drug therapy, or cross-resistant to more than one drug therapy. Specific embodiments include selecting the subject for treatment based on the cancer having an acquired resistance to the drug therapy or acquired cross-resistance to more than one drug therapy.

[0029] In some embodiments, the serine protease protein is selected from a porcine pancreatic elastase (PPE) protein (optionally SEQ ID NO: 5), a human neutrophil elastase (ELANE) protein, a human cathepsin G (CTSG) protein, a human proteinase 3 (PR3) protein, and a human granzyme B protein, which optionally targets the ELANE pathway by proteolytically activating in a cancer cell the CD95 death domain and causing histone H1 translocation, which leads to DNA damage, mitochondrial dysfunction, and activation of cell death effectors in the cancer cell.

[0030] In some embodiments, the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 5, and which retains the Q211 F amino acid substitution;

[0031] the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 6, and which retains the T55A amino acid substitution;

[0032] the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 7, and which retains the Q211 F and T55A amino acid substitutions;

[0033] the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 8, and which retains the N241Aamino acid substitution;

[0034] the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 9, and which retains the N241Y amino acid substitution;

[0035] the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 10, and which retains the R75A amino acid substitution;

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[0038] the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 11 , and which retains the R75E amino acid substitution;

[0039] the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 12, and which retains the Q211 A amino acid substitution;

[0040] the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 13, and which retains the R237A amino acid substitution;

[0041] the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 14, and which retains the S214A amino acid substitution;

[0042] the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 15, and which retains the D74A amino acid substitution;

[0043] the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ I D NO: 16;

[0044] the human ELANE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 17;

[0045] the human CTSG protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 18;

[0046] the human PR3 protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 19; or

[0047] the human granzyme B protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ I D NO: 20.

[0048] Some embodiments include administering the serine protease protein as part of a protein complex of:

[0049] (a) alpha-2-macroglobulin (A2M) proteins; and

[0050] (b) the serine protease proteins,

[0051] wherein (a) and (b) are present in the composition at a molar ratio [(a):(b)] of about 1 :3 to about 1:1. In some embodiments, the A2M proteins of (a) and the serine protease proteins of (b) are bound together in the protein complex, and optionally wherein the protein complex: (i) retains CD95 (Fas Receptor) protease cleavage activity and cancer cell-killing activity of (b); (ii) sterically hinders binding of (b) to fibrinogen and reduces or inhibits fibrinogen cleavage activity

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[0054] of (b); and (iii sterically hinders binding of (b) to serine protease inhibitors (including alpha-1 antitrypsin (A1AT)).

[0055] In some embodiments, (a) comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to a sequence selected from Table A1, or a functional fragment thereof. In some embodiments, (a) and (b) are present in the composition at a molar ratio of about 1 :3, 1 :2.9, 1 : 2.8, 1 :2.7, 1 :2.6, 1 :2.5, 1 :2.4, 1:2.3, 1 : 2.1 , 1:2, 1:1.9, 1 :1.8, 1 :1.7, 1 :1.6, 1 :1.5, 1 :1.4, 1 :1.3, 1:1.2, 1 :1.1 , or 1 :1. In specific embodiments, (a) and (b) are present in the composition at a molar ratio of about 1 :2.

[0056] In some embodiments, the cancer is a primary cancer or a metastatic cancer, and is selected from one or more of melanoma (optionally metastatic melanoma), breast cancer (optionally triple-negative breast cancer, TNBC), kidney cancer (optionally renal cell carcinoma), pancreatic cancer, bone cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer (NSCLC), mesothelioma, leukemia (optionally lymphocytic leukemia, chronic myelogenous leukemia, acute myeloid leukemia, or relapsed acute myeloid leukemia), multiple myeloma, lymphoma, hepatoma (hepatocellular carcinoma), sarcoma, B-cell malignancy, ovarian cancer, colorectal cancer, glioma, glioblastoma multiforme, meningioma, pituitary adenoma, vestibular schwannoma, primary CNS lymphoma, primitive neuroectodermal tumor (medulloblastoma), bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancers, cervical cancer, testicular cancer, thyroid cancer, and stomach cancer.

[0057] In some embodiments, administering the serine protease protein (or expressible polynucleotide encoding the serine protease protein) increases cell-killing of the resistant cancer cell in the subject by about or at least about 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 500-fold, or 1000-fold or more relative to a control or reference. In some embodiments, administering the serine protease protein (or expressible polynucleotide encoding the serine protease protein) results in tumor regression in the subject, optionally as indicated by a statistically significant decrease in the amount of viable tumor or tumor mass, optionally at least about a 10%, 20%, 30%, 40%, 50% or more decrease in tumor mass.

[0058] Certain embodiments comprise administering the serine protease protein (or expressible polynucleotide encoding the serine protease protein) to the subject by parenteral administration or by intra-tumoral administration. In some embodiments, the parenteral administration is intravenous administration.

[0059] BRIEF DESCRIPTION OFTHE DRAWINGS

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[0062] Figures 1 A-1 B show the effects of MutF (400|jg / 100mm3, intra-tumoral), anti-PD-1 , or IgG control antibodies (10mg / kg; i.p.; days 0, 3, 6, 9) on tumor growth in PD-1 -sensitive (Fig. 1A) and PD-1 resistant (Fig.1 B) CT26 models; n =7 mice / group. * p<0.05, two-way ANOVA. Results are mean ± SEM. Arrows indicate MutF treatment.

[0063] Figures 2A-2B shows the efficacy of MutF and A2M:MutF treatment on cancer cells that are cross-resistant to chemotherapeutic agents, including cancer cells resistant to paclitaxel (Fig. 2A), carboplatin (Fig.2B), oxaliplatin (Fig. 2C), and doxorubicin (Fig.2D).

[0064] Figure 3 shows the efficacy of MutF and A2M:MutF treatment on cancer cells that are cross-resistant to KRAS inhibitors.

[0065] DETAILED DESCRIPTION

[0066] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the disclosure belongs. Although any methods, materials, compositions, reagents, cells, similar or equivalent similar or equivalent to those described herein can be used in the practice or testing of the subject matter of the present disclosure, preferred methods and materials are described. All publications and references, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference in their entirety as if each individual publication or reference were specifically and individually indicated to be incorporated by reference herein as being fully set forth. 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.

[0067] 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 manufacturer’s specifications or as commonly accomplished in the art or as described herein. These and related techniques and procedures may be generally 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 the present specification. Unless specific definitions are provided, the nomenclature utilized in connection with, and the laboratory procedures and techniques of, molecular biology, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques may be used for recombinant technology, molecular biological,

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[0070] microbiological, chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0071] For the purposes of the present disclosure, the following terms are defined below. The articles “a” and “an” are used herein to refer to one or to more than one (i.e„ to at least one) of the grammatical object of the article. Byway of example, “an element” includes “one element”, “one or more elements” and / or “at least one element”.

[0072] By “about” is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 % to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0073] An “antagonist” or “inhibitor” refers to a biological or chemical agent that interferes with or otherwise reduces the physiological action of another agent or molecule. In some instances, the antagonist specifically binds to the other agent or molecule. Included are full and partial antagonists.

[0074] An “agonist” refers to a biological or chemical agent that increases or enhances the physiological action of another agent or molecule. In some instances, the agonist specifically binds to the other agent or molecule. Included are full and partial agonists.

[0075] As used herein, the term “amino acid” is intended to mean both naturally occurring and non-naturally occurring amino acids as well as amino acid analogs and mimetics. Naturally-occurring amino acids include the 20 (L)-amino acids utilized during protein biosynthesis as well as others such as 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine, homocysteine, citrulline and ornithine, for example. Non-naturally 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 and non-naturally occurring amino acids. Such modifications can include, for example, substitution or replacement of chemical groups and moieties on the amino acid or by derivatization of the amino acid. Amino acid mimetics include, for example, organic structures which exhibit functionally similar properties such as charge and charge spacing characteristic of the reference amino acid. For example, an organic structure which mimics arginine (Arg or R) would have a positive charge moiety located in similar molecular space and having the same degree of mobility as the e-amino group of the side chain of the naturally occurring Arg amino acid. Mimetics also include constrained structures so as to maintain optimal spacing and charge interactions of the amino acid or of the amino acid functional groups. Those skilled in

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[0078] the art know or can determine what structures constitute functionally equivalent amino acid analogs and amino acid mimetics.

[0079] As used herein, a subject “at risk” of developing a disease, or adverse reaction may or may not have detectable disease, or symptoms of disease, and may or may not have displayed detectable disease or symptoms of disease prior to the treatment methods described herein. “At risk” denotes that a subject has one or more risk factors, which are measurable parameters that correlate with development of a disease, as described herein and known in the art. A subject having one or more of these risk factors has a higher probability of developing disease, or an adverse reaction than a subject without one or more of these riskfactor(s).

[0080] “Biocompatible” refers to materials or compounds which are generally not injurious to biological functions of a cell or subject and which will not result in any degree of unacceptable toxicity, including allergenic and disease states.

[0081] The term “binding” refers to a direct association between two molecules, due to, for example, covalent, electrostatic, hydrophobic, and ionic and / or hydrogen-bond interactions, including interactions such as salt bridges and water bridges.

[0082] By “coding sequence” is meant any nucleic acid sequence that contributes to the code for the polypeptide product of a gene. By contrast, the term “non-coding sequence” refers to any nucleic acid sequence that does not directly contribute to the code for the polypeptide product of a gene.

[0083] Throughout this disclosure, unless the context requires otherwise, the words “comprise,” “comprises,” and “comprising” will be understood to imply 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.

[0084] By “consisting of” is meant including, and limited to, whatever follows the phrase “consisting of.”Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially 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 for the listed elements. Thus, the phrase “consisting essentially of” indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they materially affect the activity or action of the listed elements.

[0085] The term “endotoxin free” or “substantially endotoxin free” relates generally to compositions, solvents, and / orvessels that contain at most trace amounts (e.g., amounts

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[0088] having no clinically adverse physiological effects to a subject) of endotoxin, and preferably undetectable amounts of endotoxin. Endotoxins are toxins associated with certain microorganisms, such as bacteria, typically gram-negative bacteria, although endotoxins may be found in gram-positive bacteria, such as Listeria monocytogenes. The most prevalent endotoxins are lipopolysaccharides (LPS) or lipo-oligo-saccharides (LOS) found in the outer membrane of various Gram-negative bacteria, and which represent a central pathogenic feature in the ability of these bacteria to cause disease. Small amounts of endotoxin in humans may produce fever, a lowering of the blood pressure, and activation of inflammation and coagulation, among other adverse physiological effects.

[0089] Therefore, in pharmaceutical production, it is often desirable to remove most or all traces of endotoxin from drug products and / or drug containers, because even small amounts may cause adverse effects in humans. A depyrogenation oven may be used for this purpose, as temperatures in excess of 300°C are typically required to break down most endotoxins. For instance, based on 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 removing endotoxins are contemplated, including, for example, chromatography and filtration methods, as described herein and known in the art.

[0090] Endotoxins can be detected using routine techniques known in the art. For example, the Limulus Amoebocyte Lysate assay, which utilizes blood from the horseshoe crab, is a very sensitive assay for detecting presence of endotoxin. In this test, very low levels of LPS can cause detectable coagulation of the limulus lysate due a powerful enzymatic cascade that amplifies this reaction. Endotoxins can also be quantitated by enzyme-linked immunosorbent assay (ELISA). To be substantially endotoxin free, endotoxin levels may be less than about 0.001 , 0.005, 0.01 , 0.02, 0.03, 0.04, 0.05, 0.06, 0.08, 0.09, 0.1 , 0.5, 1.0, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, or 10 EU / mg of active compound. Typically, 1 ng lipopolysaccharide (LPS) corresponds to about 1-10 EU.

[0091] The term “half maximal effective concentration” or “EC5o” refers to the concentration of an agent (for example, serine protease, protein complex) as described herein at which it induces a response halfway between the baseline and maximum after some specified exposure time; the EC50 of a graded dose response curve therefore represents the concentration of a compound at which 50% of its maximal effect is observed. EC50 also represents the plasma concentration required for obtaining 50% of a maximum effect in vivo. Similarly, the “EC90” refers to the concentration of an agent or composition at which 90% of its maximal effect is

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[0094] observed. The “EC9o” can be calculated from the “EC5o” and the Hill slope, or it can be determined from the data directly, using routine knowledge in the art. In some embodiments, the EC5O of an agent is less than about 0.01 , 0.05, 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200or 500 nM. In some embodiments, an agent will have an EC50value of about 1 nM or less.

[0095] The “half-life” of an agent can refer to the time it takes for the agent to lose half of its pharmacologic, physiologic, or other activity, relative to such activity at the time of administration into the serum or tissue of an organism, or relative to any other defined timepoint. “Half-life” can also refer to the time it takes for the amount or concentration of an agent to be reduced by half of a starting amount administered into the serum or tissue of an organism, relative to such amount or concentration at the time of administration into the serum or tissue of an organism, or relative to any other defined time-point. The half-life can be measured in serum and / or any one or more selected tissues.

[0096] The term “heterologous” refers to a feature or element in a polypeptide or encoding polynucleotide that is derived from a different source than the wild-type polypeptide or encoding polynucleotide, for example, a feature from a different species than the wild-type, or a non-natural, engineered feature.

[0097] The terms “modulating” and “altering” include “increasing,” “enhancing” or “stimulating,” as well as “decreasing” or “reducing,” typically in a statistically significant or a physiologically significant amount or degree relative to a control. An “increased,” “stimulated” or “enhanced” amount is typically a “statistically significant” amount, and may include an increase that is 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-fold more than the amount produced by no composition (e.g., the absence of agent) or a control composition. A “decreased” or “reduced” amount is typically a “statistically significant” amount, and may include a decrease that 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-fold less than the amount produced by no composition (e.g., the absence of an agent) or a control composition. Examples of comparisons and “statistically significant” amounts are described herein.

[0098] The terms “polypeptide,” “protein”, and “peptide” are used interchangeably and refer to a polymer of amino acids not limited to any particular length. The term “enzyme” includes polypeptide or protein catalysts. As used herein a “proprotein”, “proenzyme”, or “zymogen” refers to an inactive (or substantially inactive) protein or enzyme, which typically is activated by protease cleavage of an activation peptide to generate an active protein or enzyme. The terms

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[0101] include modifications such as myristoylation, sulfation, glycosylation, phosphorylation and addition or deletion of signal sequences. The terms “polypeptide” or “protein” means one or more chains of amino acids, wherein each chain comprises amino acids covalently linked by peptide bonds, and wherein said polypeptide or protein can comprise a plurality of chains non-covalently and / or covalently linked together by peptide bonds, having the sequence of native proteins, that is, proteins produced by naturally-occurring and specifically non-recombinant cells, or genetically-engineered or recombinant cells, and comprise molecules having the amino acid sequence of the native protein, or molecules having deletions from, additions to, and / or substitutions of one or more amino acids of the native sequence. In certain embodiments, the polypeptide is a “recombinant” polypeptide, produced by recombinant cell that comprises 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.

[0102] The term “polynucleotide” and “nucleic acid” includes mRNA, RNA, cRNA, cDNA, and DNA. The term typically refers to polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide. The term includes single 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 of total genomic DNA of a particular species. Therefore, an isolated DNA segment encoding a polypeptide refers to a DNA segment that contains one or more coding sequences yet is substantially isolated away from, or purified free from, 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, phage, viruses, and the like.

[0103] Additional coding or non-coding sequences may, but need not, be present within a polynucleotide described herein, and a polynucleotide may, but need not, be linked to other molecules and / or support materials. Hence, 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.

[0104] “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

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[0107] ability to affect the transcription or translation, or subcellular, or cellular location 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).

[0108] A “promoter” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3’ direction) coding sequence. As used herein, the promoter sequence is bounded at its 3’ terminus by the transcription initiation site and extends upstream (5’ direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. A transcription initiation site (conveniently defined by mapping with nuclease S1) can be found within a promoter sequence, as well as protein binding domains (consensus sequences) responsible forthe binding of RNA polymerase. Eukaryotic promoters can often, but not always, contain “TATA” boxes and “CAT” boxes. Prokaryotic promoters contain Shine- Dalgarno sequences in addition to the -10 and -35 consensus sequences.

[0109] 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 sequences publicly available on line or, for example, from depositories such as the ATCC as well as other commercial or individual sources. Promoters can be unidirectional (i.e., initiate transcription in one direction) or bi-directional (i.e., initiate transcription in either a 3’ or 5’ direction). Non-limiting examples of promoters include, for example, the T7 bacterial expression system, pBAD (araA) bacterial expression system, the cytomegalovirus (CMV) promoter, the SV40 promoter, the RSV promoter. Inducible promoters include the Tet system, (US 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-RExTM system (Invitrogen Carlsbad, CA), LacSwitch® (Stratagene, (San Diego, CA) and the Cre-ERT tamoxifen inducible recombinase system (Indra et al. Nuc. Acid. Res. (1999) 27 (22): 4324-4327; Nuc. Acid. Res. (2000) 28 (23): e99; US Patent No.

[0110] 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.

[0111] An “expressible polynucleotide” includes a cDNA, RNA, mRNA or other polynucleotide that comprises at least one coding sequence and optionally at least one expression control sequence, for example, a transcriptional and / or translational regulatory element, and which can express an encoded polypeptide upon introduction into a cell.

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[0114] The term “isolated” polypeptide or protein referred to herein means that a subject protein (1 ) is free of at least some other proteins with which it would typically be found in nature, (2) is essentially free of other proteins from the same source, e.g., from the same species, (3) is expressed by a cell from a different species, (4) has been separated from at least about 50 percent of 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 operably 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 can be encoded by genomic DNA, cDNA, mRNA or other RNA, of may be of synthetic origin, or any combination thereof. In certain embodiments, the isolated protein is substantially free from proteins or polypeptides or other contaminants that are found in its natural environment that would interfere with its use (therapeutic, diagnostic, prophylactic, research or otherwise).

[0115] In certain embodiments, the “purity” of any given agent in a composition may be defined. For instance, 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 basis or a weight-weight basis, including all decimals and ranges in between, as measured, for example and by no means limiting, by high performance liquid chromatography (HPLC), a well-known form of column chromatography used frequently in biochemistry and analytical chemistry to separate, identify, and quantify compounds.

[0116] The term “reference sequence” refers generally to a nucleic acid coding sequence, or amino acid sequence, to which another sequence is being compared. All polypeptide and polynucleotide sequences described herein are included as references sequences, including those described by name and those described in the Tables and the Sequence Listing.

[0117] Certain embodiments include biologically active “variants” and “fragments” of the proteins / polypeptides described herein, and the polynucleotides that encode the same.

[0118] “Variants” contain one or more substitutions, additions, deletions, and / or insertions relative to a reference polypeptide or polynucleotide (see, e.g., the Tables and the Sequence Listing). A variant polypeptide or polynucleotide comprises an amino acid or nucleotide 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 to a reference sequence, as described herein, and substantially retains the activity of that reference sequence. Also included are sequences that consist of or differ from a 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,

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[0121] 20, 30, 40, 50, 60,70, 80, 90, 100, 110, 120, 130, 140, 150 or more amino acids or nucleotides and which substantially retain at least one activity of that reference sequence. In certain embodiments, the additions or deletions include C-terminal and / or N-terminal additions and / or deletions.

[0122] The terms “sequence identity” or, for example, comprising a “sequence 50% identical to,” as used herein, refer to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a “percentage of sequence identity” may be calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) orthe identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Vai, Leu, lie, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the 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 homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al., Nucl. Acids Res. 25:3389, 1997.

[0123] The term “solubility” refers to the property of an agent described herein 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 dissolve 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 other 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 embodiments, solubility is measured in water or a physiological buffer such as PBS or NaCl (with or without NaPO4). In specific embodiments, solubility is measured at relatively lower pH (e.g., pH 6.0) and relatively higher salt (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 can

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[0126] be about room temperature (e.g., about 20, 21 , 22, 23, 24, 25°C) or about 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.

[0127] A “subject” or a “subject in need thereof” or a “patient” or a “patient in need thereof” includes a mammalian subject such as a human subject.

[0128] “Substantially” or “essentially” means nearly totally or completely, for instance, 95%, 96%, 97%, 98%, 99% or greater of some given quantity.

[0129] By “statistically significant,” it is meant that the result was unlikely to have occurred by chance. Statistical significance can be determined by any method known in the art. Commonly used measures of significance include the p-value, which is the frequency or probability with which the observed event would occur, if the null hypothesis were true. If the obtained p-value is smaller than the significance level, then the null hypothesis is rejected. In simple cases, the significance level is defined at a p-value of 0.05 or less.

[0130] “Therapeutic response” refers to improvement of symptoms (whether or not sustained) based on administration of one or more therapeutic agents.

[0131] As used herein, the terms “therapeutically effective amount”, “therapeutic dose,” “prophylactically effective amount,” or “diagnostically effective amount” is the amount of an agent needed to elicit the desired biological response following administration.

[0132] As used herein, “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 individual or cell. Treatment includes, but is not limited to, administration of a pharmaceutical composition, and may be performed either prophylactically or subsequent to the initiation of a pathologic event or contact with an etiologic agent. Also included are “prophylactic” treatments, which can be directed to 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 associated symptoms thereof.

[0133] The term “wild-type” refers to a gene or gene product (e.g., a polypeptide) that is most frequently observed in a population and is thus arbitrarily designed the “normal” or “wild-type” form of the gene.

[0134] Each embodiment in this specification is to be applied to every other embodiment unless expressly stated otherwise.

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[0137] Drug Resistant Cancers

[0138] Embodiments of the present disclosure relate generally to the discovery that serine protease therapy, for instance, targeting the “ELANE pathway,” retains anti-cancer efficacy across a broad range of drug resistant cancers, including cancers that have a primary or secondary (acquired) resistance to one or more immunotherapies, chemotherapies, and / or targeted therapies, including combinations thereof. The ELANE pathway refers to the proteolytic activation of the CD95 death domain and histone H1 translocation, which leads to DNA damage, mitochondrial dysfunction, and activation of cell death effectors (see, for example, Cui et al., Cell. 184(12):3163-3177, 2021 ). Certain embodiments thus relate to methods of treating, ameliorating the symptoms of, and / or reducing the progression of, a cancer in a subject in need thereof, wherein the cancer is resistant (or refractory) to a drug therapy, comprising administering to the subject a serine protease protein such as PPE (or an expressible polynucleotide encoding the serine protease protein).

[0139] The terms “resistant” or “refractory” are used interchangeably herein to refer to a cancer that does not significantly respond to, has previously failed to respond to, or has become non-responsive (e.g., via selection) to, at least one drug therapy. Resistance can be “primary,” that is, the cancer at the outset is intrinsically resistant to the therapy, or “acquired,” that is, the cancer became resistant during therapy, e.g., via selection. Thus, in some instances, the resistant to the drug therapy is acquired resistance, including wherein the subject is currently undergoing or has previously undergone the drug therapy. In some instances, the subject is undergoing or has previously undergone more than one drug therapy and is cross-resistant to at least one or all of the drug therapies.

[0140] Certain embodiments include the step of selecting the subject for treatment with the serine protease therapy (ELANE-targeted therapy) based on the cancer in the subject being resistant (or refractory) to the drug therapy, or cross-resistant to more than one drug therapy. Some embodiments thus include the steps of (a) identifying a subject that is resistant or refractory to at least one drug therapy (e.g., the subject has an acquired resistance), and (b) administering the serine protease therapy to the subject based on said identification. In particular embodiments, the subject’s cancer is resistant (or cross-resistant) to a drug therapy selected from: an immunotherapy, a chemotherapy, and / or a targeted therapy such as a KRAS (Kirsten rat sarcoma) inhibitor therapy. In some embodiments, the subject’s cancer is crossresistant to an immunotherapy and a chemotherapy. In particular embodiments, the subject’s cancer is cross-resistant to an immunotherapy and a targeted therapy. In certain embodiments, the subject’s cancer is cross-resistant to a chemotherapy and a targeted therapy. In specific

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[0143] embodiments, the subject’s cancer is cross-resistant to an immunotherapy, a chemotherapy, and a targeted therapy such as a KRAS inhibitor therapy.

[0144] In some embodiments, the cancer is resistant to an immunotherapy. The term “immunotherapy” refers to a therapy (therapeutic agent) that modulates the immune response of a subject, for example, to increase or maintain a cancer-related or cancer-specific immune response, and thereby results in increased immune cell activity against cancer cells. General examples of immunotherapies include immune checkpoint modulatory agents, including checkpoint inhibitors (CPIs; see, for example, J Clin Med. 12(13) :4301 , 2023) and co-stimulatory agonists (see, for example, Pourakbari etal., EXCLI J. 20:1055-1085, 2021; and Sanmamed et al., Seminars in Oncology. 42: 640-655, 2015).

[0145] In certain embodiments, the cancer is resistant or refractory to a checkpoint inhibitor therapy. General examples of checkpoint inhibitors include Programmed Death 1 (PD-1 ) inhibitors, Programmed Death-Ligand 1 (PD-L1) inhibitors, Cytotoxic T-Lymphocyte-Associated protein 4 (CTLA-4) inhibitors, Programmed Death-Ligand 2 (PD-L2) inhibitors, V-domain Ig suppressorof T cell activation (VISTA) inhibitors, Indoleamine 2,3-dioxygenase (IDO) inhibitors, tryptophan 2,3-dioxygenase (TDO) inhibitors, T-cell Immunoglobulin domain and Mucin domain 3 (TIM-3) inhibitors, Lymphocyte Activation Gene-3 (LAG-3) inhibitors, B and T Lymphocyte Attenuator (BTLA) inhibitors, CD160 inhibitors, and T-cell immunoreceptor with Ig and ITIM domains (TIGIT) inhibitors, including cancers that are resistant or cross-resistant to any combination of the foregoing.

[0146] Specific embodiments include methods of treating cancers that are resistant to PD-1 and / or PD-L1 inhibitors, including cancers with primary or acquired resistance to PD-1 / PD-L1 blockade (see, for example, Nowicki et aL, Cancer J. 24(1):47-53, 2018). PD-1 is a cell surface receptor that belongs 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 immune checkpoint molecule, for example, by reducing or preventing the activation of T-cells, which in turn reduces autoimmunity and promotes self-tolerance. The inhibitory effect of PD-1 is accomplished at least in part through a dual mechanism of promoting apoptosis in antigen specific T-cells in lymph nodes while also reducing apoptosis in regulatory T cells (suppressor T cells). Particular examples of PD-1 inhibitors include cemiplimab, dostarlimab, nivolumab, pembrolizumab, pidilizumab, PDR001 , MK-3475, AMP-224, and AMP-514, including antigenbinding fragments thereof (see, e.g., U.S. Patent Nos. 8,008,449; 8,993,731 ; 9,073,994;

[0147] 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). As noted above, PD-L1 is one of the

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[0150] natural ligands for the PD-1 receptor. Certain examples of PD-L1 inhibitors include atezolizumab, avelumab, and durvalumab, including antigen-binding fragments thereof (see, e.g., U.S. Patent Nos. 9,102,725; 9,393,301 ; 9,402,899; 9,439,962).

[0151] Certain embodiments include treating cancers that are resistant to CTLA-4 inhibitors. CTLA4 or CTLA-4 (cytotoxic T-lymphocyte-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, by transmitting inhibitory signals to T-cells when it is bound to CD80 or CD86 on the surface of antigen-presenting cells. Examples of CTLA-4 inhibitors include ipilimumab and tremelimumab, including antigen-binding fragments thereof.

[0152] Also included are methods of treating cancers that are resistant to LAG3 inhibitors. Lymphocyte Activation Gene-3 (LAG-3) is expressed on activated T-cells, natural killer cells, B-cells and plasmacytoid dendritic cells. It negatively regulates cellular proliferation, activation, and homeostasis of T-cells, in a similar fashion to CTLA-4 and PD-1 (see, e.g., Workman and Vignali. European Journal of Immun. 33: 970-9, 2003; and Workman et al., Journal of Immun.

[0153] 172: 5450-5, 2004), and has been reported to play a role in Treg suppressive function (see, e.g., 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. Exemplary LAG3 inhibitors include relatlimab (BMS-986016) and antigen-binding fragments thereof.

[0154] Some embodiments include treating cancers that are resistant or cross-resistant to more than one checkpoint inhibitor, for example, cancers that are resistant or cross-resistant to any combination of PD-1 / PD-L1 inhibitor therapy, CTLA-4 inhibitor therapy, and / or LAG3 inhibitor therapy.

[0155] In some embodiments, the cancer is resistant or refractory to a co -stimulatory agonist therapy. General examples of co-stimulatory agonists include CD40 agonists, 0X40 agonists, Glucocorticoid-Induced TNFR Family Related Gene (GITR) agonists, CD137 (4-1 BB) agonists, CD27 agonists, CD28 agonists, CD226 agonists, ICOS agonists, and Herpes Virus Entry Mediator (HVEM) agonists, including cancers that are resistant or cross-resistant to any combination of the foregoing.

[0156] In certain embodiments, the cancer is resistant or refractory to a chemotherapy.

[0157] General classes of chemotherapies include alkylating agents, anti-metabolites, cytotoxic antibiotics, topoisomerase inhibitors (type 1 ortype II), and anti-microtubule agents. Certain embodiments include cancers that are resistant or cross-resistant to any combination of the foregoing general classes of chemotherapies.

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[0160] 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, mytomycin, and diaziquone (AZQ)), cisplatins and derivatives thereof (e.g., carboplatin and oxaliplatin), and non-classical alkylating agents (optionally procarbazine and hexamethylmelamine).

[0161] Examples of anti-metabolites include anti-folates (e.g., methotrexate and pemetrexed), fluoropyrimidines (e.g., 5-fluorouracil and capecitabine), deoxynucleoside analogues (e.g., ancitabine, enocitabine, cytarabine, gemcitabine, decitabine, azacitidine, fludarabine, nelarabine, cladribine, clofarabine, fludarabine, and pentostatin), and thiopurines (e.g., thioguanine and mercaptopurine).

[0162] Examples of cytotoxic antibiotics include anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, aclarubicin, and mitoxantrone), bleomycins, mitomycin C, mitoxantrone, and actinomycin. Examples of topoisomerase inhibitors include camptothecin, irinotecan, topotecan, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, merbarone, and aclarubicin.

[0163] Examples of anti-microtubule agents include taxanes (e.g., paclitaxel and docetaxel) and vinca alkaloids (e.g., vinblastine, vincristine, vindesine, vinorelbine).

[0164] In specific embodiments, the cancer is resistant or cross-resistant to one or more of chemotherapies selected from paclitaxel, doxorubicin, oxaliplatin, and carboplatin, including combinations thereof. For instance, in some embodiments, the cancer resistant to paclitaxel (e.g., by acquired resistance during prior paclitaxel therapy), and in some instances is also cross-resistant to one or more of doxorubicin, oxaliplatin, and carboplatin. In some embodiments, the cancer is resistant to doxorubicin (e.g., by acquired resistance during prior doxorubicin therapy), and in certain instances is also cross-resistant to one or more of paclitaxel, oxaliplatin, and carboplatin. In particular embodiments, the cancer is resistant to oxaliplatin (e.g., by acquired resistance during prior oxaliplatin therapy), and in some instances is also cross-resistant to one or more of paclitaxel, doxorubicin, and carboplatin. In certain embodiments, the cancer is resistant to carboplatin (e.g., by acquired resistance during prior carboplatin therapy), and in certain instances is also cross-resistant to one or more of paclitaxel, doxorubicin, and oxaliplatin.

[0165] In some embodiments, the cancer is resistant to a targeted therapy, or therapies that target the genetic changes or mutations that turn healthy cells into cancer cells (see, for

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[0168] example, Tsimeridou, Cancer Chemother. Pharmacol. 76:1113-1132, 2015; Zhou and Li, BMC Med 20: 90, 2022). Non-limiting examples of targeted therapies include KRAS inhibitors, HER-targeted therapies (e.g., HER1 , HER2, HER3, HER4-targeted therapies), BRAF inhibitors, poly (ADP-ribose) polymerase (PARP) inhibitors, MEK1 / MEK2 kinase inhibitors, phosphatidylinositol 3-kinase (PI3K) signaling pathway-targeted therapies including PI3K inhibitors, AKT inhibitors, EGFR-targeted therapies, cyclin dependent kinase (CDK) 4 / 6 inhibitors, androgen receptor inhibitors, NOTCH inhibitors, and others. Also included are cancers that are resistant or crossresistant to any combination of the foregoing.

[0169] Specific embodiments include methods of treating a cancer that is resistant or crossresistant to one or more KRAS inhibitors (see, for example, Isermann et al., Trends in Cancer. 11 : 91-116, 2025; Singhal et al., Nature Med. 30: 969-983, 2024; Akhave et aL, Mol Cancer Ther. 21 :1645-1651 , 2022). General examples of KRAS inhibitors include multi-KRAS inhibitors or pan-KRAS inhibitors (see, for example, Kim et al., Nature. 619: 160-166, 2023) and relatively specific KRAS inhibitors such as KRAS G12C inhibitors, KRAS G12D inhibitors, and KRAS G12V inhibitors. Particular examples of KRAS inhibitors include sotorasib (AMG-510), MRTX-1257, adagrasib, JDQ443, divarasib / GDC-6036, LY353798, MRTX-1133, RMC-9805, RMC-6291 , RMC-8839, HRS-4642, ASP3082, TD010, VRTX153, QTX3046, QTX3544, VRTX144, ERAS-4, NT-0300, PP-008, JAB-22000, AFNT-212, and MDG2021. KRAS inhibitors are generally used in patients with a corresponding KRAS mutation. Thus, in certain embodiments, a subject that is resistant or cross-resistant to one or more KRAS inhibitors (e.g., from prior or current KRAS inhibitor therapy) has a genetic KRAS mutation, for example, a KRAS G12 substitution such as a G12C, G12D, G12R, or G12V substitution; a KRAS G13 substitution; a KRAS Q61 substitution; ora KRAS A146 substitution (see, for example, Isermann et aL, 2025, supra). In particular embodiments, the KRAS-resistant cancer is a lung cancer (e.g., NSCLC), a pancreatic ductal adenocarcinoma (PDAC), or a colorectal cancer.

[0170] In some instances, the cancer is resistant to a drug therapy as part of a drug delivery system or formulation such as a liposome, polymeric nanoparticle, gold nanoparticle, dendrimer, exosome, magnetic nanoparticle, biomimetic nanocarrier, carbon-based nanomaterial, nanozyme, or other (see, for example, Liu et aL, Front OncoL 14: 1507958, 2024). In some embodiments, the cancer is resistant to a conjugate drug therapy. Examples of conjugates include antibody-drug conjugates (ADCs) and peptide-drug conjugates (PDCs), for instance, comprising a chemotherapy.

[0171] The methods and compositions described herein comprise administering a serine protease protein or an expressible polynucleotide that encodes the serine protease protein. In

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[0174] particular embodiments, the serine protease (protein or polynucleotide) targets the ELANE pathway by proteolytically-activating in a cancer cell the CD95 death domain and causing histone H1 translocation - a mechanism that leads to DNA damage, mitochondrial dysfunction, and / or activation of cell death effectors in the cancer cell. Thus, in certain embodiments, the serine protease cleaves CD95 (optionally in the presence of the serine protease inhibitor such as A1 AT) and has cancer cell-killing activity. CD95 cleavage activity, cancer cell-killing activity, and pharmacokinetic characteristics (e.g., half-life, tumor penetration) can be measured according to routine techniques in the art (see, the Examples). For instance, serine protease activity generally can be monitored using a colorimetric substrate activity assay (N-Methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide), and CD95 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. Cancer cell-killing activity can be measured in vitro or in vivo.

[0175] 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). The amino acid sequences of exemplary full-length, wild-type serine protease proproteins are provided in Table S1 below.

[0176]

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[0178]

[0179] Thus, in certain embodiments, the serine protease protein comprises, consists, or consists essentially of a full-length, serine protease proprotein selected from Table S1 , including biologically active variants and fragments thereof. In specific embodiments, the serine protease comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, or 100% identical to a sequence selected from Table S1.

[0180] 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 exemplary mutants thereof), human ELANE, human CTSG, and a human PR3 are provided in Table S2 below.

[0181]

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[0184]

[0185] Thus, in some embodiments, the serine protease protein comprises, consists, or consists essentially 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, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, or 100% identical to a sequence selected from Table S2.

[0186] In some embodiments, the serine protease protein is a PPE protein, for example, wherein:

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[0189] the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 5, and which retains the Q211 F amino acid substitution;

[0190] the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 6, and which retains the T55A amino acid substitution;

[0191] the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 7, and which retains the Q211 F and T55A amino acid substitutions;

[0192] the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 8, and which retains the N241Aamino acid substitution;

[0193] the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 9, and which retains the N241Y amino acid substitution;

[0194] the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 10, and which retains the R75A amino acid substitution;

[0195] the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 11 , and which retains the R75E amino acid substitution;

[0196] the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 12, and which retains the Q211 A amino acid substitution;

[0197] the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 13, and which retains the R237A amino acid substitution;

[0198] the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 14, and which retains the S214A amino acid substitution;

[0199] the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 15, and which retains the D74A amino acid substitution; and

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[0202] the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ I D NO: 16.

[0203] In some embodiments, the serine protease protein is a human ELANE protein, for example, wherein the human ELANE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 17. In some embodiments, the serine protease protein is a human CTSG protein, for instance, wherein the human CTSG protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 18. In some embodiments, the serine protease protein is a human PR3 protein, for example, wherein the human PR3 protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 19. In some embodiments, the serine protease protein is a human granzyme B protein, for example, wherein the human granzyme B protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 20.

[0204] Some embodiments include administering the serine protease protein, or pharmaceutical composition comprising the same, as part of a protein complex with alpha-2-macroglobulin (A2M) proteins (see, for example, PCT / US2024 / 022956, incorporated by reference in its entirety). For instance, in certain embodiments, the protein complex or pharmaceutical composition is composed of: (a) alpha-2-macroglobulin (A2M) proteins; and (b) the serine protease proteins, wherein (a) and (b) are present in the composition at a molar ratio [(a):(b)] of about 1 :3 to about 1 :1 , including wherein the A2M proteins of (a) and the serine protease proteins of (b) are bound together in the protein complex. Thus, certain of the pharmaceutical composition and protein complexes described herein comprise an alpha-2-macroglobulin (A2M) protein, for example, a human A2M protein. The amino acid sequence of full-length and mature (w / o signal peptide) human A2M is provided in Table A1 below.

[0205]

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[0208]

[0209] Thus, in some embodiments, the A2M protein portion of the protein complex comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to a sequence selected from Table A1 , or a functional fragment thereof. In some embodiments, the functional fragment thereof comprises, consists, or consists essentially of about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1300, or 1400 contiguous amino acids of a sequence selected from Table A1. In certain embodiments, (a) and (b) are present in the composition at a molar ratio of [(a) : (b)] that ranges from about 1 :3 to about

[0210] 333102695Attorney Docket: OPNI-014 / 00US 332575-2088

[0211] 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.

[0212] 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, specifically binds to a tumor-associated antigen (TAA) or tumor-specific antigen (TSA). Exemplary TAAs and TSAs include, without limitation, alphafetoprotein (AFP), epithelial tumor antigen (ETA), tyrosinase, human Her2 / neu, Her1 / EGF receptor (EGFR), Her3, A33 antigen, B7H3, CD5, CD19, CD20, CD22, CD23 (IgE Receptor), melanoma associated antigen (MAGE), C242 antigen, 5T4, IL-6, IL-13, vascular endothelial growth factor VEGF (e.g., VEGF-A) VEGFR-1 , VEGFR-2, VEGR-3, NRP2, CD30, CD33, CD37, CD40, CD44, CD51 , CD52, CD56, CD74, CD80, CD152, CD200, CD221 , CCR4, HLA-DR, CTLA-4, NPC-1 C, tenascin, vimentin, insulin-like growth factor 1 receptor (IGF-1 R), alpha-fetoprotein, insulin-like growth factor 1 (IGF-1 ), carbonic anhydrase 9 (CA-IX), carcinoembryonic antigen (CEA), guanylyl cyclase C, NY-ESO-1 , p53, survivin, integrin avp3, integrin a5pi , folate receptor 1, transmembrane glycoprotein NMB, fibroblast activation protein alpha (FAP), glycoprotein 75, TAG-72, MUC1, MLJC16 (orCA-125), phosphatidylserine, prostate-specific membrane antigen (PSMA), NR-LU-13 antigen, TRAIL-R1 , tumor necrosis factor receptor superfamily member 10b (TNFRSF10B orTRAIL-R2), SLAM family member 7 (SLAMF7), EGP40 pancarcinoma antigen, B-cell activating factor (BAFF), platelet-derived growth factor receptor, glycoprotein EpCAM (17-1A), Programmed Death-1 , protein disulfide isomerase (PDI), Phosphatase of Regenerating Liver 3 (PRL-3), prostatic acid phosphatase, Lewis-Y antigen, GD2 (a disialoganglioside expressed on tumors of neuroectodermal origin), glypican-3 (GPC3), and mesothelin.

[0213] In some embodiments, a serine protease protein or protein complex described herein is generated in vivo or ex vivo, for example, in a cell by contacting a cell or subject with one or more expressible polynucleotides that encode the serine protein (optionally in combination with A2M proteins if the method relates to administering a protein complex). An “expressible polynucleotide” includes a DNA, cDNA, RNA, mRNA or other polynucleotide that comprises at least one coding sequence for (a) and / or (b) and optionally at least one expression control sequence, for example, a transcriptional and / or translational regulatory element, and which can express the encoded protein(s) upon introduction into the cell, for example, a cell in the subject. Certain embodiments include contacting an ex vivo cell with the one or more expressible polynucleotides that encode the serine protease protein (optionally A2M) and administering the cell to a subject.

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[0216] Exemplary viral vectors that can be utilized to deliver an expressible polynucleotide include adenoviral vectors, herpes virus vectors, vaccinia virus 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, BIV, HIV and Rous Sarcoma Virus (RSV)-based vectors. In particular embodiments, the expressible polynucleotide is a modified RNA or modified mRNA polynucleotide, for example, a non-naturally occurring RNA analog. In certain embodiments, the modified RNA or mRNA polypeptide comprises one or more modified or non-natural bases. In some embodiments, the modified mRNA comprises one or more modified or non-natural internucleotide linkages. Expressible RNA polynucleotides 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 entireties.

[0217] As noted above, the methods and compositions described herein relate to treating a cancer in a subject in need thereof. Thus, in particular embodiments, a subject in need thereof has, is suspected of having, or is at risk for having, a cancer. In particular embodiments, the cancer is a primary cancer or a metastatic cancer. In specific embodiments, the cancer is selected from one or more of melanoma (optionally metastatic melanoma), breast cancer (optionally triple-negative breast cancer, TNBC), kidney cancer (optionally renal cell carcinoma), pancreatic cancer, bone cancer, prostate cancer, lung cancer (for example, small cell lung cancer, non-small cell lung cancer (NSCLC), squamous cell lung carcinoma), mesothelioma, leukemia (optionally lymphocytic leukemia, chronic myelogenous leukemia, acute myeloid leukemia, or relapsed acute myeloid leukemia), multiple myeloma, lymphoma, hepatoma (hepatocellular carcinoma), sarcoma, B-cell malignancy, ovarian cancer, colorectal cancer, glioma, glioblastoma multiforme, meningioma, pituitary adenoma, vestibular schwannoma, primary CNS lymphoma, primitive neuroectodermal tumor (medulloblastoma), bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancers, cervical cancer, testicular cancer, thyroid cancer, and stomach cancer.

[0218] In some embodiments, as noted above, the cancer is a metastatic cancer. Further to the above cancers, exemplary metastatic cancers include, without limitation, bladder cancers which have metastasized to the bone, liver, and / or lungs; breast cancers which have metastasized to the bone, brain, liver, and / or lungs; colorectal cancers which have metastasized to the liver, lungs, and / or peritoneum; kidney cancers which have metastasized to the adrenal glands, bone, brain, liver, and / or lungs; lung cancers which have metastasized to

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[0221] the adrenal glands, bone, brain, liver, and / or other lung sites; melanomas which have metastasized to the bone, brain, liver, lung, and / or skin / muscle; ovarian cancers which have metastasized to the liver, lung, and / or peritoneum; pancreatic cancers which have metastasized to the liver, lung, and / or peritoneum; prostate cancers which have metastasized to the adrenal glands, bone, liver, and / or lungs; stomach cancers which have metastasized to the liver, lung, and / or peritoneum; thyroid cancers which have metastasized to the bone, liver, and / or lungs; and uterine cancers which have metastasized to the bone, liver, lung, peritoneum, and / or vagina; among others.

[0222] The methods for treating cancers can be combined with other therapeutic modalities. For example, a combination therapy described herein can 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 administration of corticosteroids, to reduce cerebral edema, headaches, cognitive dysfunction, and emesis, and administration of anticonvulsants, to reduce seizures. Radiotherapy includes whole-brain irradiation, fractionated radiotherapy, and radiosurgery, such as stereotactic radiosurgery, which can be further combined with traditional surgery.

[0223] Certain embodiments thus include combination therapies for treating cancers, including methods of treating ameliorating the symptoms of, or inhibiting the progression of, a cancer in a subject in need thereof, comprising administering to the subject a serine protease protein or protein complex described herein 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.

[0224] In some embodiments, the methods and compositions described herein increase cancer cell-killing in the subject by about or at least about 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 500-fold, or 1000-fold or more relative to a control or reference. In some embodiments, the methods and compositions described herein increase an immune response in the subject by about or at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000% or more, or by about 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 500-fold, or 1000-fold or more, relative to a control or reference (e.g., relative to a corresponding serine protease on its own), including wherein the immune response is an anticancer immune response.

[0225] In some embodiments, the methods and compositions described herein increase median survival time of a subject by 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10

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[0228] weeks, 15 weeks, 20 weeks, 25 weeks, 30 weeks, 40 weeks, or longer. In certain embodiments, the methods and compositions described herein increase median survival time of a subject by 1 year, 2 years, 3 years, or longer. In some embodiments, the methods and pharmaceutical compositions increase progression-free survival by 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks or longer. In certain embodiments, the methods and pharmaceutical compositions described herein increase progression-free survival by 1 year, 2 years, 3 years, or longer.

[0229] In certain embodiments, the methods and compositions described herein 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 a 10%, 20%, 30%, 40%, 50% or greater decrease in tumor mass, or by altered (e.g., decreased with statistical significance) scan dimensions. In certain embodiments, the methods and compositions described herein are sufficient to result in stable disease. In certain embodiments, the methods and compositions described herein are sufficient to result in clinically relevant reduction in symptoms of a particular disease indication known to the skilled clinician.

[0230] For in vivo use, as noted above, for the treatment of human or non-human mammalian disease or testing, the serine protease proteins (or expressible polynucleotides encoding the same) or protein complexes described herein are generally incorporated into one or more therapeutic or pharmaceutical compositions prior to administration, including veterinary therapeutic compositions.

[0231] Thus, certain embodiments relate to pharmaceutical or therapeutic compositions that comprise a serine protease protein (or expressible polynucleotides encoding the same) or protein complex, as described herein. In some instances, a pharmaceutical or therapeutic composition comprises one or more of the serine proteinase proteins or protein complexes described herein 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 herein.

[0232] In particular embodiments, the pharmaceutical or therapeutic compositions comprising a serine protease protein or protein complex is substantially pure on a protein basis or a 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 a weight-weight basis.

[0233] In some embodiments, the serine protease proteins or protein complexes described herein do not form aggregates, have a desired solubility, and / or have an immunogenicity profile

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[0236] that is suitable for use in humans, as known in the art. Thus, in some embodiments, a pharmaceutical or therapeutic composition comprising a serine protease protein or protein complex is substantially aggregate-free. For example, certain compositions comprise less than about 10% (on a protein basis) high molecular weight aggregated proteins, or less than about 5% high molecular weight aggregated proteins, or less than about 4% high molecularweight aggregated proteins, or less than about 3% high molecular weight aggregated proteins, or less than about 2 % high molecular weight aggregated proteins, or less than about 1 % high molecular weight aggregated proteins.

[0237] In some embodiments, the serine protease proteins or 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.

[0238] To prepare a therapeutic or pharmaceutical composition, an effective or desired amount of one or more serine protease proteins (or expressible polynucleotide) or protein complexes is mixed with any pharmaceutical carrier(s) or excipient known to those skilled in the art to be suitable for the particular agent and / or mode 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 methyl parabens); 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.

[0239] Administration of serine protease proteins or protein complexes described herein, in pure form or in an appropriate therapeutic or pharmaceutical composition, can be carried out via any of the accepted modes of administration of agents for serving similar utilities. The therapeutic or pharmaceutical compositions can be prepared by combining a serine protease protein- or protein complex-containing composition with an appropriate physiologically acceptable carrier, diluent or excipient, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. In addition,

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[0242] other pharmaceutically active ingredients (including other small molecules as described elsewhere herein) and / or suitable excipients such as salts, buffers and stabilizers may, but need not, be present within the composition.

[0243] Administration may be achieved by a variety of different routes, including oral, parenteral, nasal, intravenous, intradermal, intramuscular, subcutaneous, or topical. Preferred modes of administration depend upon the nature of the condition to be treated or prevented. Particular embodiments include administration by IV infusion or by intratumoral injection.

[0244] Carriers can include, for example, pharmaceutically- or physiologically-acceptable carriers, excipients, or stabilizers that are non-toxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically-acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; 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; saltforming counterions such as sodium; and / or nonionic surfactants such as polysorbate 20 (TWEEN™) polyethylene glycol (PEG), and poloxamers (PLURONICS™), and the like.

[0245] In some embodiments, one or more agents can be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate)microcapsules, respectively), in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington’s Pharmaceutical Sciences, 16th edition, Oslo, A., Ed., (1980). The particle(s) or liposomes may further comprise other therapeutic or diagnostic agents.

[0246] The precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by testing the compositions in model systems known in the art and extrapolating therefrom. Controlled clinical trials may also be performed. 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 one time, or may be divided into a number of smaller doses to be

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[0249] administered at intervals of time. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need.

[0250] Typical routes of administering these and related therapeutic or pharmaceutical compositions thus include, without limitation, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques. Therapeutic or pharmaceutical compositions according to certain embodiments of the present disclosure are formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a subject or patient. Compositions that will 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 a herein described agent in aerosol form may hold a plurality of dosage units. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this 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 treatment of a disease or condition of interest.

[0251] Atherapeutic or pharmaceutical composition may be in the form of a solid or liquid. In one embodiment, the carrier(s) are particulate, so that the compositions are, for example, in tablet or powder form. The carrier(s) may be liquid, with the compositions being, for example, an oral oil, injectable liquid or an aerosol, which is useful in, for example, inhalatory administration. When intended for oral administration, the pharmaceutical composition is preferably in either solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid. Certain embodiments include sterile, injectable solutions.

[0252] As a solid composition for oral administration, the pharmaceutical composition may be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like. Such a solid composition will typically contain one or more inert diluents or edible carriers. In addition, one or more of the following may be present: binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, gum tragacanth 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 flavoring; and a

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[0255] coloring agent. When the pharmaceutical composition is in the form of a capsule, for example, a gelatin capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or oil.

[0256] 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 delivery by injection, as two examples. When intended for oral administration, preferred composition contain, in addition to the present compounds, one or more of a sweetening agent, preservatives, dye / colorant and flavor enhancer. In a composition intended to be administered by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent may be included.

[0257] The liquid therapeutic or pharmaceutical compositions, whetherthey be solutions, suspensions or other like form, may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer’s solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Physiological saline is a preferred adjuvant. An injectable pharmaceutical composition is preferably sterile.

[0258] A liquid therapeutic or pharmaceutical composition intended for either parenteral or oral administration should contain an amount of an agent such that a suitable dosage will be obtained. Typically, this amount is at least 0.01 % of the agent of interest in the composition. When intended for oral administration, this amount may be varied to be 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 to 10% by weight of the agent of interest prior to dilution.

[0259] The 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, bee wax, mineral oil, diluents such as water and alcohol, and

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[0262] 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.

[0263] The therapeutic or pharmaceutical compositions may be intended for rectal administration, in the form, for example, of a suppository, which will melt in the rectum and release the drug. The composition for rectal administration may contain an oleaginous base as a suitable nonirritating excipient. Such bases include, without limitation, lanolin, cocoa butter, and polyethylene glycol.

[0264] The therapeutic or pharmaceutical composition may include various materials, which 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 encased in a gelatin capsule. The therapeutic or pharmaceutical compositions in solid or liquid form may include a component that binds to agent and thereby assists in the delivery of the compound. Suitable components that may act in this capacity include monoclonal or polyclonal antibodies, one or more proteins or a liposome.

[0265] The therapeutic or pharmaceutical composition may consist essentially of dosage units that can be administered as an aerosol. The term aerosol is used to denote a variety of systems ranging from those of colloidal nature to systems consisting of pressurized packages. Delivery may be by a liquefied or compressed gas or by a suitable pump system that dispenses the active ingredients. Aerosols may be delivered in single phase, bi-phasic, or tri-phasic systems in order to deliver the active ingredient(s). Delivery of the aerosol includes the necessary container, activators, valves, subcontainers, and the like, which together may form a kit. One of ordinary skill in the art, without undue experimentation may determine preferred aerosols.

[0266] The compositions described herein may be prepared with carriers that protect the agents against rapid elimination from the body, such as time release formulations or coatings. Such carriers include controlled release formulations, such as, but not limited to, implants and microencapsulated delivery systems, and biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid and others known to those of ordinary skill in the art.

[0267] The therapeutic or pharmaceutical compositions may be prepared by 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 of salts, buffers and / or

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[0270] stabilizers, with sterile, distilled water so as to form a solution. A surfactant may be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that non-covalently interact with the agent so as to facilitate dissolution or homogeneous suspension of the agent in the aqueous delivery system.

[0271] The therapeutic or pharmaceutical compositions may be administered in a therapeutically effective amount, which will vary depending upon a variety of factors including the activity of the specific compound employed; the metabolic stability and length 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 drug combination; the severity of the particular disorder or condition; and the subject undergoing therapy. In some instances, 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 embodiments, the therapeutically effective dose is administered on a weekly, bi-weekly, or monthly basis. In specific embodiments, the therapeutically effective dose is administered on a weekly, bi-weekly, or monthly basis, 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.

[0272] The combination therapies described herein may include administration of a single pharmaceutical dosage formulation, which contains a serine protease protein or protein complex and an additional therapeutic agent (e.g., immunotherapy agent, chemotherapeutic agent, hormonal therapeutic agent, kinase inhibitor), as well as administration of compositions comprising a serine protease protein or protein complex and an additional therapeutic agent in its own separate pharmaceutical dosage formulation. For example, a serine protease protein or protein complex and an additional therapeutic agent can 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 administered in separate parenteral dosage formulations. Where separate dosage formulations are used, the compositions can be administered at essentially the same time, i.e., concurrently, or at separately staggered times, i.e., sequentially and in any order; combination therapy is understood to include all these regimens.

[0273] Also included are patient care kits, comprising (a) serine protease protein or a protein complex, as described herein; and optionally (b) at least one additional therapeutic agent (e.g., immunotherapy agent, chemotherapeutic agent, hormonal therapeutic agent, kinase inhibitor).

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[0276] In certain kits, (a) and (b) are in separate therapeutic compositions. In some kits, (a) and (b) are in the same therapeutic composition.

[0277] The kits herein may also include a one or more additional therapeutic agents or other components suitable or desired for the indication being treated, or for the desired diagnostic application. The kits herein can also include one or more syringes or other components necessary or desired to facilitate an intended mode of delivery (e.g., stents, implantable depots, etc.).

[0278] In some embodiments, a patient care kit contains separate containers, dividers, or compartments for the composition(s) and informational material(s). For example, the composition(s) can be contained in a bottle, vial, or syringe, and the informational material(s) can be contained in association with the container. In some embodiments, the separate elements of the kit are contained within a single, undivided container. For example, the composition is contained in a bottle, vial or syringe that has attached thereto the informational material in the form of a label. In some embodiments, the kit includes a plurality (e.g., a pack) of individual containers, each containing one or more unit dosage forms (e.g., a dosage form described herein) of a protein complex and optionally at least one additional therapeutic agent. For example, the kit includes a plurality of syringes, ampules, foil packets, or blister packs, each containing a single unit dose of a protein complex and optionally at least one additional therapeutic agent. The containers of the kits can be airtight, waterproof (e.g., impermeable to changes in moisture or evaporation), and / or light-tight.

[0279] The patient care kit optionally includes a device suitable for administration of the composition, e.g., a syringe, inhalant, dropper (e.g., eye dropper), swab (e.g., a cotton swab or wooden swab), or any such delivery device. In some embodiments, the device is an implantable device that dispenses metered doses of the agent(s). Also included are methods of providing a kit, e.g., by combining the components described herein.

[0280] EXAMPLES

[0281] Example 1

[0282] PPE Avoids Cross-Resistance to Anti-Cancer Drug Therapies Patients who develop resistance to one drug can exhibit cross-resistance to others, both within and across drug classes, complicating treatment options and limiting therapeutic efficacy (see, for example, Loria et al., Front. Oncol. 12: 877380, 2022). Given the prevalence of chemotherapies, immunotherapies, and targeted therapies in the clinic, experiments were

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[0285] performed to investigate whether resistance or cross-resistance to various anti-cancer therapies affects the efficacy of PPE (MutF) therapy.

[0286] Materials & Methods

[0287] Cell killing assays - Cancer cells or non-cancer cells were plated in complete growth media and grown to 80-90% confluence. For MutF or A2M:MutF protein complexes (see PCT / US2024 / 022956 for preparation of protein complexes of serine proteases with A2M), cells were washed with serum-free media (SFM), treated with various doses of MutF or A2M:MutF, and incubated at 37°C for 24h. For chemotherapies or KRAS inhibitors, cells were treated with various doses of drugs in complete media and incubated at 37°C for 72h. All treated cells were washed with HBSS, incubated with Calcein AM solution (C1430, Invitrogen, 4pg / mL) for 40min, washed with HBSS, and fluorescence was measured at 485 / 520nm (Varioskan LUX).

[0288] Cellular resistance and cross-resistance assays - Cancer cells were initially treated with MutF (100nM), A2M:MutF, oxaliplatin (100uM), DOX (10uM), paclitaxel (20uM), carboplatin (100uM) and AMG-510 (1 uM) and MRTX1133 (1 uM) for 24-72h to produce -90% killing. The cells were regrown to confluence and re-treated with those cytotoxic agents for five cycles. For chemotherapy and KRAS inhibitors, higher doses were required in successive cycles to achieve 90% killing while MutF concentrations did not require adjustment. Serially passaged nontreated cells (R0) and cells killed five successive times (R5) were treated with its corresponding cytotoxic agents to assess drug resistance. R0 and R5 cells were also treated with other cytotoxic agents in the same class to assess cross-resistance.

[0289] Generation ofanti-PD-1 resistant CT26 tumors - Balb / c female mice were inoculated with 1x10sCT26 cells on the rear right flank and treated with anti-PD-1 (10mg / kg, i. p. , days 0,3,6). Tumors were excised from mice that did not respond to anti-PD-1 therapy 10 to 14 days after the first treatment. Excised tumors were dissociated with collagenase type IV (Gibco), washed with PBS, and plated in RPMI culture media supplemented with 10% FBS (Gibco) and 1 % Antibiotic-Antimycotic (Gibco). Cells were passaged at least two times, inoculated into new recipient mice, and the anti-PD-1 treatment protocol was repeated. This cycle was performed a total of six times to develop CT26 tumor-bearing mice resistant to anti-PD-1 therapy.

[0290] Tumor treatments - All tumors were grown to ~100-500mm3in size prior to initiating treatments. Tumors were measured three times per week with calipers and tumor volume was calculated as width x width x height x 0.52. Mice were euthanized using CO2and underwent subsequent cervical dislocation in accordance with IACUC protocols. Single agent studies: Tumor-bearing mice were treated with either MutF (400pg / 100mm3, intra-tumoral, days 0, variable), or MRTX-1257 inhibitor (100mg / kg, oral, daily for 2 weeks), carboplatin (100mg / kg, i.p.,

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[0293] days 0,7), oxaliplatin (6mg / kg, i.p., days 0,2), cyclophosphamide (1 OOmg / kg, i.p., days 0,2), anti-CTLA-4 (5mg / kg, i.p., days 0,3,6), or anti-PD-1 (1 Omg / kg, i.p., days 0,3,6).

[0294] Results

[0295] Immunotherapy Resistance -CT26 tumors, initially sensitive to checkpoint inhibitors (CPIs), were serially-passaged in mice treated with an anti-PD-1 antibody to create a PD-1-resistant model (see, for example, Memon et al., Cancer Cell. 42, 209-224. e9, 2024). As shown in Figures 1A-1 B, PD-1 -resistant tumors showed resistance to anti-PD-1 treatment; in contrast, PPE (MutF) effectively killed the PD-1 resistant tumors.

[0296] Chemotherapy Resistance - Previously established control (R0) and chemotherapyresistant (R5) cells were treated with their corresponding drug to confirm resistance. Control (R0) and resistant cells (R5) were also treated with MutF (500nM, 24h), paclitaxel (PTX, 0.6pM, 72h), doxorubicin (DOX, 1.9pM, 72h), oxaliplatin (OXP, 33.3pM, 72h), orcarboplatin (CAB, 66.7pM, 72h) and cell viability was assessed; n=3 / group. * p<0.05, two-way ANOVA. Results are presented as mean ± SEM. As shown in Figures 2A-2B, MutF and A2M:MutF effectively killed cancer cells that show cross-resistance to chemotherapeutic agents, including cancer cells resistant to paclitaxel (Fig. 2A), carboplatin (Fig. 2B), oxiplatin (Fig.2C), and doxorubicin (Fig.

[0297] 2D).

[0298] Targeted Therapy Resistance - Previously established control (R0) and KRAS inhibitorresistant (R5) cells were treated with their corresponding drug to confirm resistance. Control (R0) and resistant cells (R5) were also treated with MutF (500nM, 24h) and KRAS inhibitors AMG-510 (82pM, 72h) and MRTX-1257 (206pM, 72h), and cell viability was assessed; n=3 / group. * p<0.05, two-way ANOVA. Results are presented as mean ± SEM. As shown in Figure 3, MutF and A2M:MutF effectively killed cancer cells that show cross-resistance to KRAS inhibitors.

[0299] The foregoing results evidence that targeting the ELANE pathway elicits robust antitumor effects across a wide range of patient treatment histories, as commonly encountered in early-phase clinical trials and clinical practice. The results also evidence the clinical utility of targeting the ELANE pathway in the select population of difficult-to-treat cancer patients that show resistance or cross-resistance to drug therapies, including acquired resistance or acquired cross-resistance to prior drug therapies, examples of which include any combination of immunotherapies, chemotherapies, and / or targeted therapies such as KRAS inhibitor therapies.

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[0301] 333102695

Claims

Attorney Docket: OPNI-014 / 00US 332575-2088CLAIMS1. A method of treating, ameliorating the symptoms of, and / or reducing the progression of, a cancer in a subject in need thereof, wherein the cancer is resistant (or refractory) to a drug therapy, comprising administering to the subject a serine protease protein or an expressible polynucleotide encoding the serine protease protein.

2. The method of claim 1 , wherein the cancer is resistant (optionally crossresistant) to a drug therapy selected from an immunotherapy, a chemotherapy, and a targeted therapy.

3. The method of claim 2, wherein the immunotherapy is a checkpoint inhibitor (CPi) selected from a Programmed Death 1 (PD-1) inhibitor, a Programmed Death-Ligand 1 (PD-L1) inhibitor, a Cytotoxic T-Lymphocyte-Associated protein 4 (CTLA-4) inhibitor, a Programmed Death-Ligand 2 (PD-L2) inhibitor, a V-domain Ig suppressor of T cell activation (VISTA) inhibitor, an Indoleamine 2,3-dioxygenase (IDO) inhibitor, a tryptophan 2,3-dioxygenase (TDO) inhibitor, a T-cell Immunoglobulin domain and Mucin domain 3 (TIM-3) inhibitor, a Lymphocyte Activation Gene-3 (LAG-3) inhibitor, a B and T Lymphocyte Attenuator (BTLA) inhibitor, a CD160 inhibitor, and a T-cell immunoreceptor with Ig and ITIM domains (TIGIT) inhibitor.

4. The method of claim 3, wherein the PD-1 inhibitor is selected from cemiplimab, dostarlimab, nivolumab, pembrolizumab, and pidilizumab; the PD-L1 inhibitor is selected from atezolizumab, avelumab, and durvalumab; the CTLA-4 inhibitor is selected from ipilimumab and tremelimumab; and / or the LAG3 inhibitor is relatlimab (BMS-986016).

5. The method of claim 2, wherein the immunotherapy is a co-stimulatory agonist selected from a CD40 agonist, an 0X40 agonist, a Glucocorticoid-Induced TNFR Family Related Gene (GITR) agonist, a CD137 (4-1 BB) agonist, a CD27 agonist, a CD28 agonist, a CD226 agonist, an ICOS agonist, and a Herpes Virus Entry Mediator (HVEM) agonist.

6. The method of claim 2, wherein the chemotherapy is selected from one or more of paclitaxel, doxorubicin, oxaliplatin, and carboplatin.

7. The method of claim 6, wherein the chemotherapy is paclitaxel, which is crossresistant to one or more of doxorubicin, oxaliplatin, and carboplatin.41333102695Attorney Docket: OPNI-014 / 00US 332575-20888. The method of claim 6, wherein the chemotherapy is doxorubicin, which is cross-resistant to one or more of paclitaxel, oxaliplatin, and carboplatin.

9. The method of claim 6, wherein the chemotherapy is oxaliplatin, which is crossresistant to one or more of paclitaxel, doxorubicin, and carboplatin.

10. The method of claim 6, wherein the chemotherapy is carboplatin, which is cross-resistant to one or more of paclitaxel, doxorubicin, and oxaliplatin.

11. The method of claim 2, wherein the chemotherapy is selected from an alkylating agent, an anti-metabolite, a cytotoxic antibiotic, a topoisomerase inhibitor (type 1 or type II), and an anti-microtubule agent, optionally wherein the chemotherapy is part of a conjugate, optionally an antibody-drug conjugate (ADC) or peptide drug conjugate (PDC).

12. The method of claim 11 , whereinthe alkylating agent is selected from nitrogen mustards (optionally mechlorethamine, cyclophosphamide, mustine, melphalan, chlorambucil, ifosfamide, or busulfan), nitrosoureas (optionally N-Nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine, or streptozotocin), tetrazines (optionally dacarbazine, mitozolomide, or temozolomide), aziridines (optionally thiotepa, mytomycin, ordiaziquone (AZQ)), cisplatins and derivatives thereof (optionally carboplatin or oxaliplatin), and non-classical alkylating agents (optionally procarbazine and hexamethylmelamine);the anti-metabolite is selected from anti-folates (optionally methotrexate or pemetrexed), fluoropyrimidines (optionally 5-fluorouracil or capecitabine), deoxynucleoside analogues (optionally ancitabine, enocitabine, cytarabine, gemcitabine, decitabine, azacitidine, fludarabine, nelarabine, cladribine, clofarabine, fludarabine, or pentostatin), and thiopurines (optionally thioguanine or mercaptopurine);the cytotoxic antibiotic is selected from anthracyclines (optionally doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, aclarubicin, or mitoxantrone), bleomycins, mitomycin C, mitoxantrone, and actinomycin;the topoisomerase inhibitor is selected from camptothecin, irinotecan, topotecan, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, merbarone, and aclarubicin; or42333102695Attorney Docket: OPNI-014 / 00US 332575-2088the anti-microtubule agent is selected from taxanes (optionally paclitaxel or docetaxel) and vinca alkaloids (optionally vinblastine, vincristine, vindesine, or vinorelbine).

13. The method of claim 2, wherein the targeted therapy is a KRAS inhibitor, a HER-targeted therapy (optionally a HER1, HER2, HER3, or HER4-targeted therapy), a BRAF inhibitor, a poly (ADP-ribose) polymerase (PARP) inhibitor, a MEK1 / MEK2 kinase inhibitor, a phosphatidylinositol 3-kinase (PI3K) signaling pathway-targeted therapy optionally a PI3K inhibitor, an AKT inhibitor, an EGFR-targeted therapy, a cyclin dependent kinase (CDK) 4 / 6 inhibitor, an androgen receptor inhibitor, or a NOTCH inhibitor.

14. The method of claim 13, wherein the KRAS inhibitor is a multi-KRAS inhibitor, a KRAS G12C inhibitor, a KRAS G12D inhibitor, or a KRAS G12V inhibitor, optionally wherein the subject has a KRAS G12 substitution, optionally G12C, G12D, G12R, or G12V.

15. The method of claim 14, wherein the KRAS inhibitor is selected from sotorasib (AMG-510), MRTX-1257, adagrasib, JDQ443, divarasib / GDC-6036, LY353798, MRTX-1133, RMC-9805, HRS-4642, ASP3082, TD010, VRTX153, QTX3046, QTX3544, VRTX144, ERAS-4, NT-0300, PP-008, JAB-22000, AFNT-212, and MDG2021.

16. The method of any one of claims 1-15, wherein the resistance is an acquired resistance or acquired cross-resistance.

17. The method of any one of claims 1 -16, wherein the subject is currently undergoing or has previously undergone the drug therapy, optionally wherein the subject has previously undergone more than one drug therapy and is cross-resistant to the more than one drug therapy.

18. The method of any one of claims 1 -17, comprising selecting the subject for treatment with the serine protease based on the cancer in the subject being resistant (or refractory) to the drug therapy, or cross-resistant to more than one drug therapy.

19. The method of any one of claims 1 -18, wherein the serine protease protein is selected from a porcine pancreatic elastase (PPE) protein (optionally SEQ ID NO: 5), a human neutrophil elastase (ELANE) protein, a human cathepsin G (CTSG) protein, a human proteinase43333102695Attorney Docket: OPNI-014 / 00US 332575-20883 (PR3) protein, and a human granzyme B protein, which optionally targets the ELANE pathway by proteolytically activating in a cancer cell the CD95 death domain and causing histone H1 translocation, which leads to DNA damage, mitochondrial dysfunction, and activation of cell death effectors in the cancer cell.

20. The method of claim 19, wherein:the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 5, and which retains the Q211 F amino acid substitution;the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 6, and which retains the T55A amino acid substitution;the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 7, and which retains the Q211 F and T55A amino acid substitutions;the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 8, and which retains the N241Aamino acid substitution;the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 9, and which retains the N241Y amino acid substitution;the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 10, and which retains the R75A amino acid substitution;the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 11 , and which retains the R75E amino acid substitution;the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 12, and which retains the Q211 A amino acid substitution;the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 13, and which retains the R237A amino acid substitution;44333102695Attorney Docket: OPNI-014 / 00US 332575-2088the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 14, and which retains the S214A amino acid substitution;the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 15, and which retains the D74A amino acid substitution;the PPE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ I D NO: 16;the human ELANE protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 17;the human CTSG protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 18;the human PR3 protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ ID NO: 19; orthe human granzyme B protein comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to SEQ I D NO: 20.

21. The method of any one of claims 1 -20, comprising administering the serine protease protein as part of a protein complex of:(a) alpha-2-macroglobulin (A2M) proteins; and(b) the serine protease proteins,wherein (a) and (b) are present in the composition at a molar ratio [(a):(b)] of about 1 :3 to about 1:1.

22. The method of claim 21 , wherein the A2M proteins of (a) and the serine protease proteins of (b) are bound together in the protein complex, and optionally wherein the protein complex:(i) retains CD95 (Fas Receptor) protease cleavage activity and cancer cell-killing activity of (b);(ii) sterically hinders binding of (b) to fibrinogen and reduces or inhibits fibrinogen cleavage activity of (b); and(iii) sterically hinders binding of (b) to serine protease inhibitors (including alpha-1 antitrypsin (A1AT)).45333102695Attorney Docket: OPNI-014 / 00US 332575-208823. The method of claim 21 or 22, wherein (a) comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 98, 99, or 100% identical to a sequence selected from Table A1 , or a functional fragment thereof.

24. The method of any one of claims 21 -23, wherein (a) and (b) are present in the composition at a molar ratio of about 1 :3, 1 :2.9, 1 : 2.8, 1 :2.7, 1 :2.6, 1 :2.5, 1 :2.4, 1:2.3, 1 : 2.1 , 1:2, 1:1.9, 1 :1.8, 1 :1.7, 1 :1.6, 1 :1.5, 1 :1.4, 1 :1.3, 1:1.2, 1:1.1, or 1:1.

25. The method of claim 24, wherein (a) and (b) are present in the composition at a molar ratio of about 1 :2.

26. The method of any one of claims 1 -25, wherein the cancer is a primary cancer or a metastatic cancer, and is selected from one or more of melanoma (optionally metastatic melanoma), breast cancer (optionally triple-negative breast cancer, TNBC), kidney cancer (optionally renal cell carcinoma), pancreatic cancer, bone cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer (NSCLC), mesothelioma, leukemia (optionally lymphocytic leukemia, chronic myelogenous leukemia, acute myeloid leukemia, or relapsed acute myeloid leukemia), multiple myeloma, lymphoma, hepatoma (hepatocellular carcinoma), sarcoma, B-cell malignancy, ovarian cancer, colorectal cancer, glioma, glioblastoma multiforme, meningioma, pituitary adenoma, vestibular schwannoma, primary CNS lymphoma, primitive neuroectodermal tumor (medulloblastoma), bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancers, cervical cancer, testicular cancer, thyroid cancer, and stomach cancer.

27. The method of any one of claims 1 -26, wherein administering the serine protease protein (or expressible polynucleotide encoding the serine protease protein) increases cell-killing of the resistant cancer cell in the subject by about or at least about 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 500-fold, or 1000-fold or more relative to a control or reference.

28. The method of any one of claims 1 -27, wherein administering the serine protease protein (or expressible polynucleotide encoding the serine protease protein) results in tumor regression in the subject, optionally as indicated by a statistically significant decrease in the amount of viable tumor or tumor mass, optionally at least about a 10%, 20%, 30%, 40%, 50% or more decrease in tumor mass.46333102695Attorney Docket: OPNI-014 / 00US 332575-208829. The method of any one of claims 1 -28, comprising administering the serine protease protein (or expressible polynucleotide encoding the serine protease protein) to the subject by parenteral administration or by intra-tumoral administration.

30. The method of claim 29, wherein the parenteral administration is intravenous administration.47333102695

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