WO2025094148A1

WO2025094148A1 - Histone h1 levels as biomarkers for cancer therapy

Info

Publication number: WO2025094148A1
Application number: PCT/IB2024/060843
Authority: WO
Inventors: Chang CUI; Court Turner
Original assignee: Onchilles Pharma Inc
Current assignee: Onchilles Pharma Inc
Priority date: 2023-11-03
Filing date: 2024-11-02
Publication date: 2025-05-08
Anticipated expiration: 2026-05-03

Abstract

Provided is the use of histone H1 levels as a biomarker for ELANE pathway-related cancer therapies.

Description

HISTONE H1 LEVELS AS BIOMARKERS FOR CANCER THERAPY

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and the benefit of U.S. Provisional Application No. 63/547,267, filed November 3, 2023, which is incorporated by reference in its entirety.

STATEMENT REGARDING SEQUENCE LISTING

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_011 _01 WO_ST26.xml. The XML file is about 42,942 bytes, was created on October 31 , 2024, and is being submitted electronically via USPTO Patent Center.

BACKGROUND

Technical Field

The present disclosure relates to the use of histone H1 levels as a biomarker for ELANE pathway- related cancer therapies.

Description of the Related Art

Precision medicine, which is designed to optimize efficiency or therapeutic benefit for particular groups of patients by using genetic or molecular profiling, has gained tremendous traction for treating cancer. Identifying the specific genomic abnormalities that (i) confer risk of developing cancer, (ii) influence tumor growth, and (iii) regulate metastasis have defined how cancer is diagnosed, determined how targeted therapies are developed and implemented, and shaped cancer prevention strategies.

The need for precision medicine in cancer is largely based on the failure to identify targetable properties in cancerous tumor cells that distinguish them from healthy, non- cancerous cells. Indeed, although radiation and/or chemotherapies have the capacity to effectively kill many if not most cancer cells, their efficacy is severely limited by cytotoxic effects on non-cancer cells. These findings demonstrate that rapid cell division, a property targeted by radiation therapy and chemotherapy, is not unique enough to cancer cells to achieve the specificity required to limit extensive side effects.

It has been shown that certain serine protease enzymes and their proteolytic substrates, for example, CD95 DD polypeptides, are selectively toxic to cancer cells but relatively non-toxic to normal or otherwise healthy cells. However, there is a need in the art to characterize the molecular bases for such selective toxicity, and thus better identify cancers that will respond optimally to ELANE pathway- related therapies comprising serine proteases or DD polypeptides.

BRIEF SUMMARY

Embodiments of the present disclosure relate to methods for treating a cancer in a subject, comprising (a) determining histone H1 levels in a sample of cancer tissue from the subject; and (b) administering a pharmaceutical composition to the subject if histone H1 levels in the cancer tissue are increased relative to a control or reference, wherein the pharmaceutical composition comprises, (i) a serine protease selected from a porcine pancreatic elastase (PPE) polypeptide (optionally SEQ ID NO: 27) and a human neutrophil elastase (ELANE) polypeptide, or an expressible polynucleotide that encodes the serine protease; or (ii) a death domain (DD) polypeptide, wherein the DD polypeptide is a C-terminal fragment of human CD95 (SEQ ID NO: 1) that induces apoptosis of cancer cells, or an expressible polynucleotide that encodes the DD polypeptide.

In certain embodiments, the histone H1 levels are selected from one or more of histone H1 .0, H1 .1 , H1 .2, H1 .3, H1 .4, H1 .5, H1 .6, H1 .7, H1 .8, H1 .9, and H1 .10 levels. In specific embodiments, the histone H1 levels are selected from one or more of histone H1 .0, H1 .2, H1 .4, and H1 .5 levels. Some embodiments include determining histone H1 levels in the sample of cancer tissue by immunohistochemistry (IHC) optionally chromogenic or fluorescent IHC, enzyme linked immunosorbent assay (ELISA), flow cytometry, quantitative mass spectrometry, qPCR, or Western blot on a human histone H1 protein. Certain embodiments comprise administering the pharmaceutical composition to the subject if histone H1 levels in the cancer tissue are increased by about or at least about 1 .2, 1 .3, 1 .4, 1 .5, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 50, or 100-fold or more relative to histone H1 levels of the control or reference, optionally wherein the control is a healthy or non-cancerous tissue.

In certain embodiments: the PPE polypeptide comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 6 (WT pro-PPE), or the ELANE polypeptide comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 7 (WT pro-ELANE).

In certain embodiments, the PPE polypeptide or ELANE polypeptide comprises, in an N- terminal to C-terminal orientation, a signal peptide, a modified activation peptide, and a peptidase domain, wherein the modified activation peptide comprises a heterologous protease cleavage site that is cleavable by a protease selected from a metalloprotease, an aspartyl protease, and a cysteine protease. In certain embodiments, the metalloprotease, aspartyl protease, or cysteine protease is selected from matrix metalloproteinase-12 (MMP12), cathepsin D (CTSD), cathepsin C (CTSD), and cathepsin L (CTSL), and optionally wherein the heterologous protease cleavage site is selected from Table S3, including the MMP12 cleavage site of SEQ ID NOs: 8-10, the CTSD cleavage site of SEQ ID NOs: 11-12, the CTSC cleavage site of SEQ ID NO: 13, or the CTSL cleavage site of SEQ ID NOs: 14-16. In some embodiments, the PPE polypeptide comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to a sequence selected from SEQ ID NOs: 17-25 (Table S4), which retains the heterologous protease cleavage site.

In certain embodiments, the PPE polypeptide comprises at least one amino acid alteration in the peptidase domain (SEQ ID NO: 26), wherein the at least one alteration is at a residue selected from one or more of Q211 , T55, D74, R75, S214, R237, and N241 , the residue numbering being defined by SEQ ID NO: 6 (WT pro-PPE). In some embodiments, the at least one amino acid alteration is selected from one or more of Q211 F, T55A, D74A, R75A, R75E, Q211 A, S214A, R237A, N241A, and N241Y, the residue numbering being defined by SEQ ID NO: 6 (WT pro-PPE).

In certain embodiments, the PPE polypeptide comprises, consists, or consists essentially of a peptidase domain selected from: an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 27, and which retains the Q211 F amino acid substitution; an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 28, and which retains the T55A amino acid substitution; an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 29, and which retains the N241A amino acid substitution; an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 30, and which retains the N241 Yamino acid substitution; an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 31 , and which retains the R75A amino acid substitution; an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 32, and which retains the R75E amino acid substitution; an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 33, and which retains the Q211 A amino acid substitution; an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 34, and which retains the R237A amino acid substitution; an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 35, and which retains the S214A amino acid substitution; and an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 36, and which retains the D74A amino acid substitution.

In certain embodiments, the pharmaceutical composition comprises a protein complex of:

(A) alpha-2-macroglobulin (A2M) proteins; and

(B) the serine protease proteins, optionally the ELANE or PPE polypeptides as defined herein, 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 of (B); and

(iii) sterically hinders binding of (B) to serine protease inhibitors (including alpha-1 antitrypsin (A1 AT)).

In certain 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 particular 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 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.

In certain embodiments, the DD polypeptide comprises, consists, or consists essentially of: an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to a selected from SEQ ID NOs: 2-5 and which induces apoptosis of cancer cells; an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 2 and which induces apoptosis of cancer cells; an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 3 and which induces apoptosis of cancer cells; an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 4 and which induces apoptosis of cancer cells; or an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 5 and which induces apoptosis of cancer cells.

In certain embodiments, the expressible polynucleotide is a virus or viral vector optionally selected from adenoviral vectors, herpes virus vectors, vaccinia virus vectors, adeno-associated virus (AAV) vectors, myxoma virus vectors, and retroviral vectors (optionally lentiviral vectors), or a modified mRNA polynucleotide.

Some embodiments include obtaining the sample of cancer tissue from the subject. In certain embodiments, the sample of cancer tissue is a blood sample, a surgical sample, a biopsy sample, a pleural effusion sample, or an ascetic fluid sample obtained from the subject, optionally selected from one or more of a white blood cell, breast, lung, gastrointestinal (stomach, colon, rectal), ovarian, pancreatic, liver, bladder, cervical, neuronal, uterine, salivary gland, kidney, prostate, thyroid, skin, head and neck, or muscle tissue sample.

In certain embodiments, the subject is a human subject.

In some embodiments, the cancer is a solid tumor. In certain embodiments, the cancer is a hematological malignancy. In particular embodiments, the cancer is selected from one or more of bladder cancer, blood cancer, bone cancer, bone marrow cancer, brain/nervous system cancer (optionally glioblastoma), breast cancer (optionally triple negative breast cancer), colon or colorectal cancer, esophageal cancer, gastrointestinal cancer, head cancer, kidney cancer, liver cancer, lung cancer (optionally small cell lung cancer (SCLC)), nasopharynx cancer, neck cancer (optionally head and neck cancer), ovarian cancer, pancreatic cancer, gallbladder cancer, prostate cancer, skin cancer (optionally melanoma, cutaneous squamous cell carcinoma, Merkel cell carcinoma), stomach cancer, testicular cancer, tongue cancer, uterine cancer, multiple myeloma, and embryonal rhabdomyosarcoma.

Some embodiments include administering the pharmaceutical composition to the subject by systemic administration or intratumoral injection. Certain embodiments include administering the pharmaceutical composition to the subject by parenteral administration, optionally intravenous or subcutaneous administration. Some embodiments comprise administering the pharmaceutical composition to the subject in combination with an immune checkpoint modulatory agent, a chemotherapeutic agent, a hormonal therapeutic agent, and/or a kinase inhibitor. In certain embodiments, the immune checkpoint modulatory agent is a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor.

Also included are patient care kits, comprising: (a) reagents for determining histone H1 levels in a sample of cancer tissue from a subject, including cancer tissue and non-cancerous tissue; and (b) pharmaceutical composition, comprising, (i) a serine protease selected from a porcine pancreatic elastase (PPE) polypeptide and a human neutrophil elastase (ELANE) polypeptide, or an expressible polynucleotide that encodes the serine protease; or (ii) a death domain (DD) polypeptide, wherein the DD polypeptide is a C-terminal fragment of human CD95 (SEQ ID NO: 1 ) that induces apoptosis of cancer cells, or an expressible polynucleotide that encodes the DD polypeptide.

In certain embodiments, (a) comprises reagents for performing a diagnostic assay selected from one or more of immunohistochemistry (IHC) optionally chromogenic or fluorescent IHC, enzyme linked immunosorbent assay (ELISA), flow cytometry, quantitative mass spectrometry, qPCR, or Western blot on a human histone H1 protein. In certain embodiments, the human histone H1 protein is selected from histone H1 .0, H1 .1 , H1 .2, H1 .3, H1 .4, H1 .5, H1 .6, H1 .7, H1 .8, H1 .9, and H1 .10. In specific embodiments, the human histone H1 protein is selected from histone H1 .0, H1 .2, H1 .4, and H1 .5.

In certain embodiments, the PPE polypeptide or ELANE polypeptide comprises, in an N- terminal to C-terminal orientation, a signal peptide, a modified activation peptide, and a peptidase domain, wherein the modified activation peptide comprises a heterologous protease cleavage site that is cleavable by a protease selected from a metalloprotease, an aspartyl protease, and a cysteine protease. In certain embodiments, the metalloprotease, aspartyl protease, or cysteine protease is selected from matrix metalloproteinase-12 (MMP12), cathepsin D (CTSD), cathepsin C (CTSD), and cathepsin L (CTSL), and optionally wherein the heterologous protease cleavage site is selected from Table S3, including the MMP12 cleavage site of SEQ ID NOs: 8-10, the CTSD cleavage site of SEQ ID NOs: 11-12, the CTSC cleavage site of SEQ ID NO: 13, or the CTSL cleavage site of SEQ ID NOs: 14-16. In certain embodiments, the PPE polypeptide comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to a sequence selected from SEQ ID NOs: 17-25 (Table S4), which retains the heterologous protease cleavage site.

In some embodiments, the PPE polypeptide comprises at least one amino acid alteration in the peptidase domain (SEQ ID NO: 26), wherein the at least one alteration is at a residue selected from one or more of Q211 , T55, D74, R75, S214, R237, and N241 , the residue numbering being defined by SEQ ID NO: 6 (WT pro-PPE). In certain embodiments, the at least one amino acid alteration is selected from one or more of Q211 F, T55A, D74A, R75A, R75E, Q211A, S214A, R237A, N241A, and N241Y, the residue numbering being defined by SEQ ID NO: 6 (WT pro-PPE).

(A) alpha-2-macroglobulin (A2M) proteins; and (B) the serine protease proteins, optionally the ELANE or PPE polypeptides defined herein, wherein (A) and (B) are present in the composition at a molar ratio [(A):(B)] of about 1 :3 to about 1 :1 .

In certain embodiments, the A2M proteins of (A) and the serine protease proteins of (B) are bound together in the protein complex, and optionally wherein the protein complex:

In certain 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 certain 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 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.

In certain embodiments, the DD polypeptide comprises, consists, or consists essentially of: an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to a selected from SEQ ID NOs: 2-5 and which induces apoptosis of cancer cells; an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 2 and which induces apoptosis of cancer cells; an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 3 and which induces apoptosis of cancer cells; an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 4 and which induces apoptosis of cancer cells; or an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 5 and which induces apoptosis of cancer cells. In certain embodiments, the expressible polynucleotide is a virus or viral vector optionally selected from adenoviral vectors, herpes virus vectors, vaccinia virus vectors, adeno-associated virus (AAV) vectors, myxoma virus vectors, and retroviral vectors (optionally lentiviral vectors), or a modified mRNA polynucleotide.

Certain embodiments include a patient care kit described herein for use in diagnosing and/or treating a cancer in a subject. Also included is the use of a patient care kit described herein in the manufacture of a kit or medicament for diagnosing and/or treating a cancer in a subject.

In certain embodiments, the cancer is a solid tumor. In certain embodiments, the cancer is a hematological malignancy. In specific embodiments, the cancer is selected from one or more of bladder cancer, blood cancer, bone cancer, bone marrow cancer, brain/nervous system cancer (optionally glioblastoma), breast cancer (optionally triple negative breast cancer), colon or colorectal cancer, esophageal cancer, gastrointestinal cancer, head cancer, kidney cancer, liver cancer, lung cancer (optionally small cell lung cancer (SCLC)), nasopharynx cancer, neck cancer (optionally head and neck cancer), ovarian cancer, pancreatic cancer, gallbladder cancer, prostate cancer, skin cancer (optionally melanoma, cutaneous squamous cell carcinoma, Merkel cell carcinoma), stomach cancer, testicular cancer, tongue cancer, uterine cancer, multiple myeloma, and embryonal rhabdomyosarcoma.

In certain embodiments, the pharmaceutical composition is administered to the subject by systemic administration or intratumoral injection. In certain embodiments, the pharmaceutical composition is administered to the subject by parenteral administration, optionally intravenous or subcutaneous administration.

Some patient care kits are for use in combination with (or further comprise) an immune checkpoint modulatory agent, a chemotherapeutic agent, a hormonal therapeutic agent, and/or a kinase inhibitor. In certain embodiments, the immune checkpoint modulatory agent is a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A-1C show that Histone H1 levels are elevated in cancer cells and correlated with killing by PPE Mutant F (MutF). Figure 1A shows H1 .0 and H1 .2 mRNA levels and EC50 values of human cancer and non-cancer cell lines (RNA data was obtained from TCGA database. * TCGA data was unavailable; values were measured by qRT-PCR, normalized to A549 cells, and converted to nTPM). Figure 1B shows the correlation of H1 .0 and H1 .2 protein levels (quantified by flow cytometry) and MutF EC50 values across multiple cancer cell lines (1 B). Figure 1C shows cytosolic and total H1 .0 and H1 .2 levels in cancer cells pre- and posttreatment with serum-free media (SFM, control) or MutF (200nM, 4h) as measured by flow cytometry. Results are expressed as percent cytosolic.

Figures 2A-2C show validation in ovarian cancer (OvCa) patient samples. Figure 2A shows the experimental design. Figure 2B shows representative MutF killing curve (left) and H1 .0, H1 .2 mRNA levels in purified cancer cells (CCs) and non-cancer cells (NCs) from tumors. Figure 2C shows the effect of MutF (500nM), doxorubicin (10uM), or oxaliplatin (100uM) on cancer and non-cancer cells isolated from OvCa patients. IP = intraperitoneal cells; PBMCs = peripheral blood mononuclear cells.

Figures 3A-3E show that MutF effectively regresses tumors in multiple pre-clinical models. Figures 3A-3C show the effects of a single intratumoral dose of MutF in syngeneic mouse models (3A; n=7/group), xenograft mouse models (3B; n= 5/group), and ovarian patient cell-derived xenograft models (3C, n=5/group). Figure 3D shows the experimental design and Figure 3E shows the effects of a single intratumoral dose of MutF on larger CT26 tumors (3E, n=5/group). *, p<0.05, two-way ANOVA. Results are mean ± SEM.

Figures 4A-4C show that repeated intratumoral dosing with MutF eliminates tumors in multiple pre-clinical models. Figure 4A shows tumor growth in the 4T1 model (injected on Days 0, 8); tumor growth (left) and lung metastases (right). Figure 4B shows tumor growth in the HCT116 model (injected on Days 0, 10, 22, 44) and the HT29 model (injected on Days 0, 8, 16, 26). Figure 4C shows tumor growth in the NCI-H358 model (injected on Days 0, 8, 16) and the PC3 model (injected on Days 0, 14, 23). Red arrows indicates treatment days. n=5/group. *, p<0.05, Student’s t-test. Results are mean ± SEM.

Figures 5A-5F show that histone H1 levels are elevated in human tumors and correlated with disease progression/malignancy. Figures 5A-5C show histone H1 .0 levels and Figures 5D- 5F show histone H1 .2 levels in breast cancer, melanoma, and head and neck cancer tissues.

Figure 6 shows that histone H1 levels are elevated in cancer cells relative to non-cancer cells isolated from ovarian cancer patients.

Figures 7A-7B show that PPE (MutF) induces H1 translocation to the cytosol in cancer cells (Fig. 7A) but not in non-cancer cells (Fig. 7B) from ovarian cancer patients.

Figures 8A-8C show that elevated histone H1 levels are associated with selective PPE (MutF) killing in vitro and in vivo (CDX model) of cells isolated from ovarian cancer patients. Figure 8A shows H1 protein and RNA levels, Figure 8B shows primary cell killing in vitro, and Figure 8C shows the results from an in vivo CDX model using an ovarian cancer patient-derived cell line. Detailed Description

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.

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

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. By way of example, “an element” includes “one element”, “one or more elements” and/or “at least one element”.

By “about” is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by up to 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. 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.

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.

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

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 risk factor(s). “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.

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.

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.

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.

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.

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

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 agent or compound. Typically, 1 ng lipopolysaccharide (LPS) corresponds to about 1 -10 EU.

The term “half maximal effective concentration” or “EC₅o” refers to the concentration of an agent (for example, a protein complex) as described herein at which it induces a response halfway between the baseline and maximum after some specified exposure time; the EC₅o of a graded dose response curve therefore represents the concentration of an agent or compound at which 50% of its maximal effect is observed. EC₅o 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 observed. The “EC₉₀” can be calculated from the “EC50” and the Hill slope, or it can be determined from the data directly, using routine knowledge in the art. In some embodiments, the EC₅₀ 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, 200 or 500 nM. In some embodiments, an agent will have an EC₅0 value of about 1 nM or less.

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.

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.

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.

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 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.

An “expressible polynucleotide” includes an mRNA, RNA, cRNA, cDNA, and DNA 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, for example, a cell in a subject.

In some 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 nonnatural bases, for example, a nucleotide base other than adenine (A), guanine (G), cytosine (C), thymine (T), and/or uracil (U). In some embodiments, the modified mRNA comprises one or more modified or non-natural internucleotide linkages. Expressible RNA polynucleotides for delivering an encoded therapeutic polypeptide 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.

In some embodiments, various viruses or 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, myxoma virus vectors, and retroviral vectors. In some instances, the retroviral vector is a derivative of a murine or avian retrovirus, or is a lentiviral vector. Examples of retroviral vectors in which a single foreign gene can be inserted 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). A number of additional retroviral vectors can incorporate multiple genes. All of these vectors can transfer or incorporate a gene for a selectable marker so that transduced cells can be identified and generated. By inserting a polypeptide sequence of interest into the viral vector, along with another gene that encodes the ligand for a receptor on a specific target cell, for example, the vector may be made target specific. Retroviral vectors can be made target specific by inserting, for example, a polynucleotide encoding a protein. Illustrative targeting may be accomplished by using an antibody to target the retroviral vector. Those of skill in the art will know of, or can readily ascertain without undue experimentation, specific polynucleotide sequences which can be inserted into the retroviral genome to allow target specific delivery of the retroviral vector.

In certain instances, the expressible polynucleotides described herein are engineered for localization within a cell, potentially within a specific compartment such as the nucleus, or are engineered for secretion from the cell or translocation to the plasma membrane of the cell. In exemplary embodiments, the expressible polynucleotides are engineered for nuclear localization.

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, phages, viruses, and the like.

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.

“Expression control sequences” include regulatory sequences of nucleic acids, or the corresponding amino acids, such as promoters, leaders, enhancers, introns, recognition motifs for RNA, or DNA binding proteins, polyadenylation signals, terminators, internal ribosome entry sites (IRES), secretion signals, subcellular localization signals, and the like, which have the ability to affect the transcription or translation, or subcellular, or cellular 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).

A “promoter” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiatingtranscription 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 for the 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.

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. 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.

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).

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 agents.

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.

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

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) or the 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.

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 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.

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.

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

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.

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

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.

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.

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.

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

Embodiments of the present disclosure relate generally to the discovery that increased histone H1 levels can serve as a biomarker or companion diagnostic for identifying cancers that will respond optimally to ELANE pathway- related therapies. Certain embodiments thus relate to methods for treating a cancer in a subject, comprising (a) determining histone H1 levels in a sample of cancer tissue from the subject (for example, intracellular histone H1 levels); and (b) administering a pharmaceutical composition to the subject if histone H1 levels in the cancer tissue are increased relative to a control or reference, wherein the pharmaceutical composition comprises, (i) a serine protease or an expressible polynucleotide that encodes the serine protease; or (ii) a death domain (DD) polypeptide, or an expressible polynucleotide that encodes the DD polypeptide. In some embodiments, the serine protease is a porcine pancreatic elastase (PPE) polypeptide or a human neutrophil elastase (ELANE) polypeptide. In some embodiments, the DD polypeptide a C-terminal fragment of human CD95 (SEQ ID NO: 1) that induces apoptosis of cancer cells.

Histone H1 plays a dominant role in establishing the compaction state of an array of nucleosomes as well as influencing the conformation. H1 has three domains: a central globular domain that binds near the entry/exit site of linker DNA on the nucleosome, an extended N- terminus, and an extended C-terminus. The C-terminus is particularly rich in the basic amino acids lysine and arginine and, thus, has a strong propensity to bind DNA. Human histone H1 has 11 subtypes or isoforms (see, for example, Hergeth and Schneider, EMBO Reports. 16:1439-1453, 2015), and embodiments of the present disclosure can utilize any one or more of the H1 subtypes. Thus, in certain embodiments, the histone H1 levels are selected from one or more of histone H1 .0, H1.1 , H1.2, H1.3, H1.4, H1.5, H1.6, H1.7, H1.8, H1.9, and H1.10 levels. In preferred embodiments, the histone H1 levels are selected from one or more of histone H1 .0, H1 .2, H1 .4, and H1 .5 levels. Certain embodiments comprise administeringthe pharmaceutical composition to the subject if histone H1 levels in the cancer tissue are increased by about or at least about 1 .2, 1 .3, 1 .4, 1 .5, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 50, or 100-fold or more relative to histone H1 levels of the control or reference, including wherein the control is a healthy or non- cancerous tissue.

Histone H1 levels in a sample of cancer or non-cancer tissue can be determined by any variety of methods. For example, histone H1 levels can be determined by immunohistochemistry (IHC) optionally chromogenic or fluorescent IHC, enzyme linked immunosorbent assay (ELISA), flow cytometry, quantitative mass spectrometry (see, for example, Rozanova et al., Methods Mol Biol. 2228:85-116, 2021 ), qPCR (RT-qPCR, or quantitative reverse transcription polymerase chain reaction), or Western blot on a human histone H1 protein, among othertechniques. Certain embodiments thus include the step of determining or detecting or measuring histone H1 levels in a sample of cancer tissue from a subject in need thereof. Also included is the step of comparing the histone H1 levels in a sample of cancer tissue relative to that of a control or reference.

Examples of a “reference” include a value, amount, sequence, or other characteristic obtained from a database or a non-cancerous tissue from one or more controls, for example, one or more healthy or non-cancerous control subjects (e.g., a population of healthy or non- cancerous control subjects), or one or more corresponding non-cancerous control tissues from the subject being tested. Typically, a “corresponding” non-cancerous control tissue is obtained from the same type of tissue as the cancer tissue being tested. As with the cancer tissue, the histone H1 levels from a non-cancerous control can be determined by any variety of methods, including, for example, by IHC, for example, chromogenic or fluorescent IHC, ELISA, quantitative mass spectrometry, qPCR, or Western blot on a human histone H1 protein.

In some embodiments, the sample of cancer tissue (or non-cancerous control tissue) is a blood sample, a surgical sample, a biopsy sample, a pleural effusion sample, or an ascetic fluid sample from the subject. Particular examples of samples of cancer tissues (or non- cancerous control tissues) include white blood cell, lung, breast, gastrointestinal (stomach, colon, rectal), ovarian, pancreatic, liver, bladder, cervical, neuronal, uterine, salivary gland, kidney, prostate, thyroid, skin, head and neck, or muscle tissues. Certain embodiments include the step of obtaining the sample of cancer tissue (or non-cancerous control tissue) from the subject, for example, prior to determining histone H1 levels. In some embodiments, the subject is a human subject.

In certain embodiments, a pharmaceutical composition comprises a serine protease or an expressible polynucleotide that encodes the serine protease. Generally, serine proteases in nature are produced as an inactive “proprotein” (or “proenzyme”, “zymogen”) composed of a signal peptide, an activation peptide comprising a native or wild-type protease cleavage site, and an active peptidase domain. The proprotein is activated by protease cleavage of the activation peptide to release the enzymatically-active, peptidase domain. In some embodiments, the serine protease is PPE or human ELANE, including proproteins (e.g., pro- PPE, pro-ELANE) and fragments and variants thereof. Exemplary pro-PPE and pro-ELANE polypeptide sequences are provided in Table S1.

Therefore, in some embodiments, the serine protease is a PPE polypeptide that comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 6 (WT pro-PPE). In some instances the serine protease is an ELANE polypeptide that comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 7 (WT pro-ELANE). Also included are variants and/or fragments thereof, some of which include “activatable” proproteins (i.e., activated by proteolytic cleavage at the activation peptide domain to release the proteolytically-active peptidase domain), and some of which include activated polypeptide variants and fragments thereof (e.g., peptidase domain). In some embodiments, administration of the pro-PPE or pro-ELANE and protease cleavage of the activation peptide, for example, at a cancer or tumor site in vivo, generates an active PPE or ELANE peptidase domain, which has increased cancer cell-killing activity relative to the proprotein form. In certain embodiments, the cancer cell-killing activity of the active peptidase domain is increased 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 that of the proprotein.

Certain examples of variants include PPE and ELANE polypeptides (e.g., proproteins of SEQ ID NOs: 6 or 7) that comprise a modified “activation peptide”, which allows them to be selectively activated by proteases associated with the tumor microenvironment (see, for example, WO 2022/040288, incorporated by reference). That is, the activation peptide containing the native protease cleavage site is replaced or otherwise modified so that it is not cleavable (or not substantially cleavable) by its native protease under suitable conditions (e.g., in vivo, in vitro, for example, using a colorimetric substrate activity assay), but is instead cleavable by a protease that is present at relatively higher levels in cancer tissues or at tumor sites.

For instance, in some embodiments, a variant or modified PPE or ELANE polypeptide comprises, in an N-terminal to C-terminal orientation, a signal peptide, a modified activation peptide, and a peptidase domain, wherein the modified activation peptide comprises a heterologous protease cleavage site that is cleavable by a protease selected from a metalloprotease, an aspartyl protease, and a cysteine protease. In particular embodiments, the heterologous protease cleavage site is cleavable by matrix metalloproteinase-12 (MMP12), cathepsin D (CTSD), cathepsin C (CTSC), or cathepsin L (CTSL). The amino acid sequences of exemplary protease cleavage sites are provided in Table S3.

In certain embodiments, a modified activation peptide has a heterologous protease cleavage site that comprises, consists, or consists essentially of an amino acid sequence selected from Table S3. For example, in some embodiments, the modified activation peptide comprises a protease cleavage site selected from SEQ ID NOs: 8-10, and is cleavable by MMP12; or the modified activation peptide comprises a protease cleavage site selected from SEQ ID NOs: 11 -12, and is cleavable by CTSD; or the modified activation peptide comprises a protease cleavage site of SEQ ID NO: 13, and is cleavable by CTSC; or the modified activation peptide comprises a protease cleavage site selected from SEQ ID NOs: 14-16, and is cleavable byCTSL.

Examples of modified PPE proproteins having a heterologous protease cleavage site, as described herein, are provided in Table S4 below.

Thus, in certain embodiments, the PPE polypeptide comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to a sequence selected from Table S4 (e.g., SEQ ID NOs: 17-25), which retains the heterologous protease cleavage site.

In some embodiments, the PPE polypeptide (e.g., pro-PPE) comprises the wild-type PPE peptidase domain (SEQ ID NO: 26), and in some embodiments, the PPE peptidase domain is a variant or fragment thereof, for example, which comprises an amino acid sequence that is at least 80, 85, 90, 95, 98, or 99% identical to SEQ ID NO: 7, and which has serine protease activity and/or cancer cell-killing activity. Thus, additional examples of variants include PPE polypeptides (e.g., pro-PPE) that comprise at least one amino acid alteration in the peptidase domain (SEQ ID NO: 26), some of which have increased cancer cell-killing activity, and/or reduced binding to or interaction with a human alpha-1 antitrypsin (A1AT) protein, relative to that of the wild-type PPE peptidase domain. Particular examples of variants include a PPE peptidase domain that has at least one alteration at a residue selected from one or more of Q211 , T55, D74, R75, S214, R237, and N241 , the residue numbering being defined by SEQ ID NO: 6 (WT pro-PPE). In specific embodiments, the at least one amino acid alteration is selected from one or more of Q211 F, T55A, D74A, R75A, R75E, Q211 A, S214A, R237A, N241 A, and N241Y, the residue numbering being defined by SEQ ID NO: 6 (WT pro-PPE) (see, for example, WO 2022/040281 , incorporated by reference).

The wild-type PPE peptidase domain and exemplary PPE peptidase variants are provided in Table S5.

Thus, in certain embodiments, the PPE polypeptide comprises a peptidase domain selected from: an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 27, and which retains the Q211 F amino acid substitution; an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 28, and which retains the T55A amino acid substitution; an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 29, and which retains the N241 A amino acid substitution; an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 30, and which retains the N241 Y amino acid substitution; an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 31 , and which retains the R75A amino acid substitution; an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 32, and which retains the R75E amino acid substitution; an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 33, and which retains the Q211 A amino acid substitution; an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 34, and which retains the R237A amino acid substitution; an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 35, and which retains the S214A amino acid substitution; and an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 36, and which retains the D74A amino acid substitution.

In some embodiments, the pharmaceutical composition comprises a protein complex of: (A) alpha-2-macroglobulin (A2M) proteins; and (B) serine protease proteins, as described herein (for example, ELANE or PPE proteins such as SEQ ID NO: 27), 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. In some embodiments, the protein complex: (i) retains the CD95 (Fas Receptor) protease cleavage activity and cancer cell-killing activity of (B); (ii) sterically hinders binding of (B) to fibrinogen, thereby reducing or inhibiting the fibrinogen cleavage activity of (B); and (iii) sterically hinders binding of (B) to serine protease inhibitors such as alpha-1 antitrypsin (A1 AT), thereby protecting (B) from inhibition by the serine protease inhibitors (see, for example, U.S. Provisional Application No. 63/456,916, incorporated by reference).

In certain embodiments, the A2M proteins are bound together in the protein complex as A2M monomers or A2M multimers, for example, as A2M dimers such as A2M homodimers, as A2M trimers such as A2M homotrimers, or as A2M tetramers such as A2M homotetramers. In specific embodiments, the A2M proteins of (A) are bound together as A2M homotetramers, and the serine protease proteins of (B) are bound by the A2M homotetramers into the protein complex. In specific embodiments, each protein complex is composed of one set of A2M homotetramers (that is, four A2M proteins, either as (i) four whole A2M proteins or as (ii) up to eight A2M fragments which result from cleavage of the bait region of the whole A2M proteins by the serine protease(s) as the latter join the A2M homotetramer complex) and two serine protease proteins.

In certain embodiments, the protein complex sterically hinders binding of (B) to larger molecules but does not sterically hinder binding of (B) to smaller molecules. That is, as noted above, the protein complex sterically hinders binding of (B) to larger molecules such as serine protease proteins (e.g., A1 AT), fibrinogen, and/or plasma antibodies. Thus, in some embodiments, the protein complex protects (B) from inhibition by serine proteases and also reduces/inhibits the ability of (B) to cleave fibrinogen. In some instances, for example, where the serine protease of (B) is a non-human protein such as PPE, the protein complex protects (B) from anti-PPE plasma antibodies orthe generation of anti-PPE plasma antibodies. In some instances, such provide clinical advantages in the administration of non-human protein drugs such as PPE to humans, which can otherwise generate anti-drug antibodies. In contrast, the protein complex does not sterically hinder binding of (B) to smaller molecules such as CD95. Thus, in specific embodiments, (B) in the protein complex cleaves CD95 and kills cancer cells but does not substantially cleave fibrinogen.

The pharmaceutical composition and protein complexes described herein comprise an alpha-2-macroglobulin (A2M) protein, for example, a human A2M protein. A2M is a highly conserved protease inhibitor present in plasma at relatively high concentrations (0.1-6 mg/ml) (Bhattacharjee et al., J. Biol. Chem. 275: 26806-11 , 2000). It often exists as a tetramer of four identical ~180 kDa subunits that forms a hollow cylinder-like structure. It can present multiple target peptide bonds to attacking proteases in its central “bait” domain. Human A2M “traps” serine proteases such as PPE: here, after the serine protease binds to and cleaves the bait region, a conformational change is induced in A2M which traps the serine protease in a way that the protease remains active against low molecular weight substrates but has significantly reduced activity against high molecular weight substrates (see, for example, Vandooren and Itoh, Frontiers in Immunology, 12, 2021 ; and Harwood et al., Molecular & Cellular Proteomics, 20, 2021 ). The amino acid sequence of full-length and mature (w/o signal peptide) human A2M is provided in Table A1 below.

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.

For instance, in specific embodiments, the functional fragment thereof is composed of approximately residues 1 -1400, 1 -1300, 1-1200, 1 -1100, 1 -1000, 1 -900, 1 -800, 1-700, 1-600, 1 - 500, 1-400, 1-300, 1 -200, 100-1400, 100-1300, 100-1200, 100-1100, 100-1000, 100-900, 100- 800, 100-700, 100-600, 100-500, 100-400, 100-300, 100-200, 200-1400, 200-1300, 200-1200, 200-1100, 200-1000, 200-900, 200-800, 200-700, 200-600, 200-500, 200-400, 200-300, 300- 1400, 300-1300, 300-1200, 300-1100, 300-1000, 300-900, 300-800, 300-700, 300-600, 300-500, 300-400, 400-1400, 400-1300, 400-1200, 400-1100, 400-1000, 400-900, 400-800, 400-700, 400- 600, 400-500, 500-1400, 500-1300, 500-1200, 500-1100, 500-1000, 500-900, 500-800, 500-700, 500-600, 600-1400, 600-1300, 600-1200, 600-1100, 600-1000, 600-900, 600-800, 600-700, 700- 1400, 700-1300, 700-1200, 700-1100, 700-1000, 700-900, 700-800, 800-1400, 800-1300, 800- 1200, 800-1100, 800-1000, 800-900, 900-1400, 900-1300, 900-1200, 900-1100, 900-1000, 1000-1400, 1000-1300, 1000-1200, 1000-1100, 1100-1400, 1100-1300, 1100-1200, 1200-1400, or 1200-1300 of a sequence selected from Table A1. In some embodiments, the functional fragment thereof is capable of forming an A2M homotetramer and trapping or otherwise binding the serine protease (such as PPE) into the protein complex in a configuration which sterically hinders binding of the serine protease to serine protease inhibitors such as A1 AT, retains CD95 protease cleavage activity and cancer-cell killing activity of the serine protease, and/or inhibits or otherwise reduces the ability of the serine protease to cleave fibrinogen.

In certain embodiments, the A2M portion of the protein complex improves uptake into cancer cells relative to the serine protease alone. For instance, A2M binds to the LPR1 and GRP78 receptors, which are expressed on normal and cancer cells. Indeed, elevated GRP78 levels generally correlate with higher pathologic grade, recurrence, and poor patient survival in breast, liver, prostate, colon, and gastric cancers (see, for example, Lee, Cancer Res. 67:3496- 3499, 2007), and in some instances the ability of A2M to bind GRP78 improves selective targeting to cancer cells that express GRP78.

As noted above, 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 1 :1 , for example, a molar ratio of about 1 :3, 1 :2.9, 1 : 2.8, 1 :2.7, 1 :2.6, 1 :2.5, 1 :2.4, 1 :2.3, 1 : 2.1 , 1 :2, 1 :1.9, 1 :1.8, 1 :1.7, 1 :1.6, 1 :1.5, 1 :1.4, 1 :1.3, 1 :1.2, 1 :1 .1 , or 1 :1 . In specific aspects, the molar ratio defined herein retains the CD95 protease cleavage and cancer cell-killing activity of the serine protease while sterically hindering it from binding to serine protease inhibitors (e.g., A1AT), fibrinogen and/or plasma antibodies, thereby protecting it from inhibition by serine protease inhibitors or plasma antibodies, and inhibiting or otherwise reducing its ability to cleave fibrinogen.

Thus, in certain embodiments, the protein complex retains the ability to cleave CD95 (Fas Receptor) and does not substantially cleave fibrinogen. In some embodiments, a protein complex described herein has about or at least about 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000% or more of the CD95 protease cleavage and/or cancer cellkilling activity of the corresponding serine protease protein on its own (for example, in the presence of serine protease inhibitors in plasma such as A1AT). In certain embodiments, a protein complex described herein has about or less than about 50, 40, 30, 20, 10, 5% or less of the fibrinogen protease cleavage activity of the corresponding serine protease protein on its own.

In certain embodiments, a pharmaceutical composition comprises a death domain (DD) polypeptide, wherein the DD polypeptide is a C-terminal fragment of human CD95 (SEQ ID NO: 1) that induces apoptosis of cancer cells, or an expressible polynucleotide that encodes the DD polypeptide. Exemplary CD95 and DD sequences are provided in Table D1.

Thus, in some embodiments, the DD polypeptide comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to a selected from Table D1 (for example, SEQ ID NOs: 2-5) and which induces apoptosis of cancer cells. Induction of apoptosis in cancer cells can be measured according to routine techniques in the art (see, for example, WO 2020/132465; and WO 2018/232273, incorporated by reference). Serine protease activity, CD95 cleavage activity, cancer cell-killing activity, fibrinogen cleavage activity, and coagulation properties can be measured according to routine techniques in the art. For example, serine protease activity can be monitored using a colorimetric substrate activity assay (N-Methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide), and cancer cell-killing activity can be measured in vitro or in vivo. CD95 and/or fibrinogen cleavage can be measured directly (e.g., Western blot). As desired, protease cleavage activity can be measured in the presence of serine protease inhibitors such as A1 AT. Cancer cell-killing activity can be measured in vitro or in vivo, and effects on coagulation in vivo can be measured, for example, by routine assays such as prothrombin (PT) time and partial thromboplastin (PTT) time.

As noted above, embodiments of the present disclosure relate generally to methods of treating, ameliorating the symptoms of, or inhibiting the progression of, a cancer in a subject in need thereof. In some embodiments, the cancer is a solid tumor. In certain embodiments, the cancer is a hematological malignancy. In particular embodiments, the cancer is selected from one or more of bladder cancer, blood cancer, bone cancer, bone marrow cancer, brain/nervous system cancer (optionally glioblastoma), breast cancer (optionally triple negative breast cancer), colon or colorectal cancer, esophageal cancer, gastrointestinal cancer, head cancer, kidney cancer, liver cancer, lung cancer (optionally small cell lung cancer (SCLC)), nasopharynx cancer, neck cancer (optionally head and neck cancer), ovarian cancer, pancreatic cancer, gallbladder cancer, prostate cancer, skin cancer (optionally melanoma, cutaneous squamous cell carcinoma, Merkel cell carcinoma), stomach cancer, testicular cancer, tongue cancer, uterine cancer, multiple myeloma, and embryonal rhabdomyosarcoma.

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.

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 pharmaceutical composition 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. In some embodiments, administeringthe pharmaceutical composition enhances the susceptibility of the cancer to the additional agent (for example, immunotherapy agent, chemotherapeutic agent, hormonal therapeutic agent, and or kinase inhibitor) by about or at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000% or more relative to the additional agent alone.

Certain combination therapies employ one or more cancer immunotherapy agents, or “immunotherapy agents”. In certain instances, an immunotherapy agent modulates the immune response of a subject, for example, to increase or maintain a cancer-related or cancerspecific immune response, and thereby results in increased immune cell inhibition or reduction of cancer cells. Exemplary immunotherapy agents include polypeptides, for example, antibodies and antigen-binding fragments thereof, ligands, and small peptides, and mixtures thereof. Also include as immunotherapy agents are small molecules, cells (e.g., immune cells such as T-cells), various cancer vaccines, gene therapy or other polynucleotide-based agents, including viral agents, and others known in the art. Thus, in certain embodiments, the cancer immunotherapy agent is selected from one or more of immune checkpoint modulatory agents, cancer vaccines, cytokines, and cell-based immunotherapies.

In certain embodiments, the cancer immunotherapy agent is an immune checkpoint modulatory agent. Particular examples include “antagonists” or “inhibitors: of one or more inhibitory immune checkpoint molecules, and “agonists” of one or more stimulatory immune checkpoint molecules. Generally, immune checkpoint molecules are components of the immune system that either turn up a signal (co-stimulatory molecules) or turn down a signal, the targeting of which has therapeutic potential in cancer because cancer cells can perturb the natural function of immune checkpoint molecules (see, e.g., Sharma and Allison, Science. 348:56-61 , 2015; Topalian et al., Cancer Cell. 27:450-461 , 2015; Pardoll, Nature Reviews Cancer. 12:252-264, 2012). In some embodiments, the immune checkpoint modulatory agent (e.g., antagonist, agonist) “binds” or “specifically binds” to the one or more immune checkpoint molecules, as described herein.

In some embodiments, the immune checkpoint modulatory agent is an antagonist or inhibitor of one or more inhibitory immune checkpoint molecules. Exemplary inhibitory immune checkpoint molecules include Programmed Death-Ligand 1 (PD-L1 ), Programmed Death- Ligand 2 (PD-L2), Programmed Death 1 (PD-1 ), V-domain Ig suppressor of T cell activation (VISTA), Cytotoxic T-Lymphocyte-Associated protein 4 (CTLA-4), Indoleamine 2,3- dioxygenase (IDO), tryptophan 2,3-dioxygenase (TDO), T-cell Immunoglobulin domain and Mucin domain 3 (TIM-3), Lymphocyte Activation Gene-3 (LAG-3), B and T Lymphocyte Attenuator (BTLA), CD160, and T-cell immunoreceptor with Ig and ITIM domains (TIGIT). In specific embodiments, the immune checkpoint modulatory agent is a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor. Specific examples of PD-1 antagonists or inhibitors include the antibodies nivolumab, pembrolizumab, PDR001 , MK-3475, AMP-224, AMP-514, and pidilizumab, and antigen-binding fragments thereof (see, e.g., U.S. Patent Nos. 8,008,449; 8,993,731 ; 9,073,994; 9,084,776; 9,102,727; 9,102,728; 9,181 ,342; 9,217,034; 9,387,247; 9,492,539; 9,492,540; and U.S. Application Nos. 2012/0039906; 2015/0203579). Specific examples of PD-L1 antagonists include the antibodies atezolizumab (MPDL3280A), avelumab (MSB0010718C), and durvalumab (MEDI4736), and antigen-bindingfragments thereof (see, e.g., U.S. Patent Nos. 9,102,725; 9,393,301 ; 9,402,899; 9,439,962). Particular examples of CTLA-4 inhibitors include the antibodies ipilimumab and tremelimumab, and antigen-binding fragments thereof. At least some of the activity of ipilimumab is believed to be mediated by antibody-dependent cell-mediated cytotoxicity (ADCC) killing of suppressor Tregs that express CTLA-4.

In certain embodiments, the immune checkpoint modulatory agent is an agonist of one or more stimulatory immune checkpoint molecules. Exemplary stimulatory immune checkpoint molecules include CD40, 0X40, Glucocorticoid-Induced TNFR Family Related Gene (GITR), CD137 (4-1 BB), CD27, CD28, CD226, and Herpes Virus Entry Mediator (HVEM).

Also included are patient care kits, comprising (a) reagents for determining histone H1 levels in a sample of cancer tissue from a subject, including cancer tissue and non-cancerous tissue; and (b) pharmaceutical composition, comprising, (i) a serine protease, or an expressible polynucleotide that encodes the serine protease; or (ii) a DD polypeptide, or an expressible polynucleotide that encodes the DD polypeptide. In some embodiments, a) comprises reagents for performing a diagnostic assay selected from one or more of immunohistochemistry (IHC) optionally chromogenic or fluorescent IHC, enzyme linked immunosorbent assay (ELISA), flow cytometry, quantitative mass spectrometry, qPCR, or Western blot on a human histone H1 protein. Reagents for performing any one or more of the diagnostic assays described herein are known in the art. In some embodiments, the serine protease is a porcine pancreatic elastase (PPE) polypeptide or a human neutrophil elastase (ELANE) polypeptide, as described herein. In some embodiments, the DD polypeptide a C-terminal fragment of human CD95 (SEQ ID NO: 1) that induces apoptosis of cancer cells, as described herein.

Certain embodiments include a patient care kit described herein for use in diagnosing and/or treating a cancer in a subject, for example, a human subject. Also included is the use of a patient care kit described herein for diagnosing and/or treating a cancer in a subject, for example, a human subject.

For in vivo use, as noted herein, the serine proteases, DD polypeptides, or expressible polynucleotides described herein are generally incorporated into one or more pharmaceutical compositions prior to administration. Thus, certain embodiments relate to pharmaceutical compositions, comprising a pharmaceutically acceptable carrier and a serine protease, DD polypeptides, or expressible polynucleotide described herein.

To prepare a pharmaceutical composition, an effective or desired amount of one or more therapeutic agents (e.g., proteins/polypeptides, polynucleotides, viruses) 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, semiliquid or solid. Solutions or suspensions used for administration may include, for example, any one or more of a sterile diluent (such as water), saline solution (e.g., NaCl, phosphate buffered saline (PBS)), fixed oil(s), polyethylene glycol, glycerin, propylene glycol or other synthetic solvent, antimicrobial agents (e.g., benzyl alcohol, methyl parabens), antioxidants (e.g., ascorbic acid, sodium bisulfite), chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)), and/or buffers (e.g., acetates, citrates, phosphates). If administered intravenously (e.g., by IV infusion), exemplary suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, or polypropylene glycol, including mixtures thereof.

The compositions described herein may be prepared with carriers that protect the therapeutic agents against rapid elimination from the body, such as time release formulations or coatings. Such carriers include controlled release formulations, inclulding 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.

The compositions may be administered in a therapeutically effective amount, which will vary depending upon a variety of factors including the activity of the specific agent employed; the metabolic stability and length of action of the agent; 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 daily, weekly, bi-weekly, or monthly basis.

Administration may be achieved by a variety of different routes, and in some methods include systemic administration and intratumoral injection. Particular examples include administering an agent or composition to a subject by parenteral, oral, topical, pulmonary, or rectal administration. In some embodiments, the parenteral administration is intravenous, subcutaneous, intraperitoneal, intrathecal, intracerebral, epidural, intramuscular, intradermal, vaginal, or intracarotid administration.

Preferred modes of administration will depend upon the nature of the condition to be treated or prevented. Particular embodiments include administration by IV infusion.

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 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 administered at intervals of time. For any particular subject, specific dosage regimens may be adjusted over time accordingto the individual need.

Pharmaceutical compositions that will be administered to a subject or patient may take the form of one or more dosage units. 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.

Although the foregoing embodiments have been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this disclosure that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. The following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of noncritical parameters that could be changed or modified to yield essentially similar results. EXAMPLES

Example 1

Serine Protease-Mediated Pathway Selectively Kills Cancer cells and Activates Adaptive Immunity Without Inducing Resistance Following Repeated Dosing

Experiments were performed to evaluate histone H1 as a potential biomarker for serine protease therapy with MutF, and test the effects of MutF as a monotherapy.

In vitro studies. Human and murine cancer and non-cancer cells (cell lines, primary cells from healthy donors and ovarian cancer patients) were treated with MutF and cell viability was quantified by calcein-AM and immunogenic cell death (ICD) markers. To evaluate histone H1 as a potential biomarker, the correlation between H1 levels in cancer cells and MutF killing was evaluated and compared to H1 levels in normal and tumor tissues in patients.

In vivo studies. A single dose of MutF was delivered intratu morally into multiple tumor models (syngeneic, xenograft, and CDX). Effects on primary and metastatic (4T1 ) tumor growth, immunology, and survival were assessed as a monotherapy. CT26 tumor-free mice were rechallenged to examine immune memory.

To evaluate histone H1 levels in vivo, histone H1 .0 and H1 .2 levels were quantified by immunohistochemistry on tumor microarrays containing normal, adjacent normal, and various tumor grades, sourced from Biocore USA. Slides were stained with antibodies specific to histones H1 .0 and H1 .2. Tumor CD45- and CD45+ cells, as well as PBMCs and intraperitoneal cells, were also isolated from ovarian cancer patients, and H1 .0 and H1 .2 level were quantified by ELISA and normalized to total cell protein levels. H1 .0 and H1 .2 levels in cancer and noncancer cells isolated from ovarian patient sample were also quantified by flow cytometry. Cytoplasm fixation and permeabilization were applied to obtain cytosolic histone levels. Nuclear fixation and permeabilization was applied to obtain total histone levels. Primary cancer cells were defined as CD45-FAP- cells, while non-cancer cells include FAP+ fibroblasts and CD45+ immune cells.

Primary cancer cells (CD45-FAP- population) and non-cancer cells (FAP+ and CD45+ population) were isolated from ovarian cancer patients. H1 RNA and protein levels (ELISA & western blot) were quantified. Primary cancer cell killing was quantified by Calcein AM. Cancer cells were subsequently cultured through multiple passages to establish a cell line, which was implanted into mice to generate a CDX model. The efficacy of MutF was then evaluated in this CDX model. Results and Conclusions

Broad, direct, and selective cancer cell killing by MutF. MutF selectively killed primary cancer cells from ovarian cancer (OvCa) patients and over 50 different cancer cell lines. The efficacy of MutF killing was correlated with histone H1 isoform levels, which were upregulated in cancer cells versus non-cancer cells. A single intratumoral dose of MutF regressed tumors in syngeneic, xenograft, and OvCa CDX models, spanning multiple cancer cell genetics, anatomical origin, and size. Figures 1 A-1C show that histone H1 levels are elevated in cancer cells and correlated with killing by MutF. Figures 2A-2C show validation of anti-tumor activity in ovarian cancer (OvCa) patient samples (experimental design shown in Fig. 2A). Figures 3A-3E show that MutF effectively regresses tumors in multiple pre-clinical models.

Figures 5A-5F show that histone H1 levels are elevated in human tumors and correlated with disease progression/malignancy. Figures 5A-5C show histone H1 .0 levels and Figures 5D- 5F show histone H1 .2 levels in breast cancer, melanoma, and head and neck cancer tissues. Figure 6 shows that histone H1 levels are elevated in cancer cells relative to non-cancer cells isolated from ovarian cancer patients. Figures 7A-7B show that PPE (MutF) induces H1 translocation to the cytosol in cancer cells (Fig. 7A) but not in non-cancer cells (Fig. 7B) from ovarian cancer patients. Figures 8A-8C show that elevated histone H1 levels are associated with selective PPE (MutF) killing in vitro and in vivo (CDX model) of cells isolated from ovarian cancer patients. Figure 8A shows H1 protein and RNA levels, Figure 8B shows primary cell killing in vitro, and Figure 8C shows the results from an in vivo CDX model using an ovarian cancer patient-derived cell line. Here, PPE (MutF) shows significantly improved and selective cancer cell-killing relative to the carboplatin standard of care.

MutF is unable to induce tumor resistance. Figures 4A-4 show that repeated intratumoral dosing with MutF eliminates tumors in multiple pre-clinical models, and that cancer cells are not able to generate resistance to MutF following repeated treatment.

Taken together, the foregoing data not only demonstrate that MutF selectively kills cancer cells and produces complete responses in mice, but also identifies histone H1 as an unexpected indicator of efficacy for ELANE pathway-related cancer therapies in humans, such as therapies with serine proteases or DD polypeptides or expressible polynucleotides that encode the same.

Claims

1 . A method for treating a cancer in a subject, comprising

(a) determining histone H1 levels in a sample of cancer tissue from the subject; and

(b) administering a pharmaceutical composition to the subject if histone H1 levels in the cancer tissue are increased relative to a control or reference, wherein the pharmaceutical composition comprises,

(i) a serine protease selected from a porcine pancreatic elastase (PPE) polypeptide (optionally SEQ ID NO: 27) and a human neutrophil elastase (ELANE) polypeptide, or an expressible polynucleotide that encodes the serine protease; or

(ii) a death domain (DD) polypeptide, wherein the DD polypeptide is a C- terminal fragment of human CD95 (SEQ ID NO: 1) that induces apoptosis of cancer cells, or an expressible polynucleotide that encodes the DD polypeptide.

2. The method of claim 1 , wherein the histone H1 levels are selected from one or more of histone H1 .0, H1.1 , H1.2, H1.3, H1.4, H1.5, H1.6, H1.7, H1.8, H1.9, and H1.10 levels.

3. The method of claim 2, wherein the histone H1 levels are selected from one or more of histone H1 .0, H1 .2, H1 .4, and H1 .5 levels.

4. The method of any one of claims 1-3, comprising determining histone H1 levels in the sample of cancer tissue by immunohistochemistry (IHC) optionally chromogenic or fluorescent IHC, enzyme linked immunosorbent assay (ELISA), flow cytometry, quantitative mass spectrometry, qPCR, or Western blot on a human histone H1 protein.

5. The method of any one of claims 1-4, comprising administering the pharmaceutical composition to the subject if histone H1 levels in the cancer tissue are increased by about or at least about 1 .2, 1 .3, 1 .4, 1 .5, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 50, or 100-fold or more relative to histone H1 levels of the control or reference, optionally wherein the control is a healthy or non-cancerous tissue.

6. The method of any one of claims 1 -5, wherein: the PPE polypeptide comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 27 or SEQ ID NO: 6 (WT pro-PPE), or the ELANE polypeptide comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 7 (WT pro-ELANE).

7. The method of anyone of claims 1-6, wherein the PPE polypeptide or ELANE polypeptide comprises, in an N-terminal to C-terminal orientation, a signal peptide, a modified activation peptide, and a peptidase domain, wherein the modified activation peptide comprises a heterologous protease cleavage site that is cleavable by a protease selected from a metalloprotease, an aspartyl protease, and a cysteine protease.

8. The method of claim 7, wherein the metalloprotease, aspartyl protease, or cysteine protease is selected from matrix metalloproteinase-12 (MMP12), cathepsin D (CTSD), cathepsin C (CTSD), and cathepsin L (CTSL), and optionally wherein the heterologous protease cleavage site is selected from Table S3, includingthe MMP12 cleavage site of SEQ ID NOs: 8- 10, the CTSD cleavage site of SEQ ID NOs: 11 -12, the CTSC cleavage site of SEQ ID NO: 13, or the CTSL cleavage site of SEQ ID NOs: 14-16.

9. The method of claim 8, wherein the PPE polypeptide comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to a sequence selected from SEQ ID NOs: 17-25 (Table S4), which retains the heterologous protease cleavage site.

10. The method of any one of 1-9, wherein the PPE polypeptide comprises at least one amino acid alteration in the peptidase domain (SEQ ID NO: 26), wherein the at least one alteration is at a residue selected from one or more of Q211 , T55, D74, R75, S214, R237, and N241 , the residue numbering being defined by SEQ ID NO: 6 (WT pro-PPE).

11 . The method of claim 10, wherein the at least one amino acid alteration is selected from one or more of Q211 F, T55A, D74A, R75A, R75E, Q211 A, S214A, R237A, N241 A, and N241 Y, the residue numbering being defined by SEQ ID NO: 6 (WT pro-PPE).

12. The method of claim 11 , wherein the PPE polypeptide comprises, consists, or consists essentially of a peptidase domain selected from: an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 27, and which retains the Q211 F amino acid substitution; an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 28, and which retains the T55A amino acid substitution; an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 29, and which retains the N241A amino acid substitution; an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 30, and which retains the N241 Yamino acid substitution; an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 31 , and which retains the R75A amino acid substitution; an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 32, and which retains the R75E amino acid substitution; an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 33, and which retains the Q211 A amino acid substitution; an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 34, and which retains the R237A amino acid substitution; an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 35, and which retains the S214A amino acid substitution; and an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 36, and which retains the D74A amino acid substitution.

13. The method of any one of claims 1-12, wherein the pharmaceutical composition comprises a protein complex of:

(A) alpha-2-macroglobulin (A2M) proteins; and

(B) the serine protease proteins, optionally the ELANE or PPE polypeptides defined in any one of claims 6-12, wherein (A) and (B) are present in the composition at a molar ratio [(A):(B)] of about 1 :3 to about 1 :1 .

14. The method of claim 13, 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

15. The method of claim 13 or 14, 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.

16. The method of claim 15, wherein 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.

17. The method of any one of claims 13-16, 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.

18. The method of claim 17, wherein (A) and (B) are present in the composition at a molar ratio of about 1 :2.

19. The method of anyone of claims 1-4, wherein the DD polypeptide comprises, consists, or consists essentially of: an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to a selected from SEQ ID NOs: 2-5 and which induces apoptosis of cancer cells; an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 2 and which induces apoptosis of cancer cells; an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 3 and which induces apoptosis of cancer cells; an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 4 and which induces apoptosis of cancer cells; or an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 5 and which induces apoptosis of cancer cells.

20. The method of any one of claims 1-12 or 19, wherein the expressible polynucleotide is a virus or viral vector optionally selected from adenoviral vectors, herpes virus vectors, vaccinia virus vectors, adeno-associated virus (AAV) vectors, myxoma virus vectors, and retroviral vectors (optionally lentiviral vectors), or a modified mRNA polynucleotide.

21 . The method of any one of claims 1-20, comprising obtaining the sample of cancer tissue from the subject.

22. The method of any one of claims 1-21 , wherein the sample of cancer tissue is a blood sample, a surgical sample, a biopsy sample, a pleural effusion sample, or an ascetic fluid sample obtained from the subject, optionally selected from one or more of a white blood cell, breast, lung, gastrointestinal (stomach, colon, rectal), ovarian, pancreatic, liver, bladder, cervical, neuronal, uterine, salivary gland, kidney, prostate, thyroid, skin, head and neck, or muscle tissue sample.

23. The method of any one of claims 1-22, wherein the subject is a human subject.

24. The method of any one of claims 1-23, wherein the cancer is a solid tumor.

25. The method of any one of claims 1-23, wherein the cancer is a hematological malignancy.

26. The method of any one of claims 1-25, wherein the cancer is selected from one or more of bladder cancer, blood cancer, bone cancer, bone marrow cancer, brain/nervous system cancer (optionally glioblastoma), breast cancer (optionally triple negative breast cancer), colon or colorectal cancer, esophageal cancer, gastrointestinal cancer, head cancer, kidney cancer, liver cancer, lung cancer (optionally small cell lung cancer (SCLC)), nasopharynx cancer, neck cancer (optionally head and neck cancer), ovarian cancer, pancreatic cancer, gallbladder cancer, prostate cancer, skin cancer (optionally melanoma, cutaneous squamous cell carcinoma, Merkel cell carcinoma), stomach cancer, testicular cancer, tongue cancer, uterine cancer, multiple myeloma, and embryonal rhabdomyosarcoma.

27. The method of anyone of claims 1-26, comprising administerin the pharmaceutical composition to the subject by systemic administration or intratumoral injection.

28. The method of claim 27, comprising administering the pharmaceutical composition to the subject by parenteral administration, optionally intravenous or subcutaneous administration.

29. The method of any one of claims 1-28, comprising administering the pharmaceutical composition to the subject in combination with an immune checkpoint modulatory agent, a chemotherapeutic agent, a hormonal therapeutic agent, and/or a kinase inhibitor.

30. The method of claim 29, wherein the immune checkpoint modulatory agent is a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor.

31. A patient care kit, comprising:

(a) reagents for determining histone H1 levels in a sample of cancer tissue from a subject, including cancertissue and non-cancerous tissue; and

(b) pharmaceutical composition, comprising,

(i) a serine protease selected from a porcine pancreatic elastase (PPE) polypeptide and a human neutrophil elastase (ELANE) polypeptide, or an expressible polynucleotide that encodes the serine protease; or

32. The patient care kit of claim 31 , wherein (a) comprises reagents for performing a diagnostic assay selected from one or more of immunohistochemistry (IHC) optionally chromogenic or fluorescent IHC, enzyme linked immunosorbent assay (ELISA), flow cytometry, quantitative mass spectrometry, qPCR, or Western blot on a human histone H1 protein.

33. The patient care kit of claim 32, wherein the human histone H1 protein is selected from histone H1 .0, H1 .1 , H1 .2, H1 .3, H1 .4, H1 .5, H1 .6, H1 .7, H1 .8, H1 .9, and H1 .10.

34. The patient care kit of claim 33, wherein the human histone H1 protein is selected from histone H1 .0, H1 .2, H1 .4, and H1 .5.

35. The patient care kit of any one of claims 31 -34, wherein: the PPE polypeptide comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 6 (WT pro-PPE), or the ELANE polypeptide comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 7 (WT pro-ELANE).

36. The patient care kit of any one of claims 31 -35, wherein the PPE polypeptide or ELANE polypeptide comprises, in an N-terminal to C-terminal orientation, a signal peptide, a modified activation peptide, and a peptidase domain, wherein the modified activation peptide comprises a heterologous protease cleavage site that is cleavable by a protease selected from a metalloprotease, an aspartyl protease, and a cysteine protease.

37. The patient care kit of claim 36, wherein the metalloprotease, aspartyl protease, or cysteine protease is selected from matrix metalloproteinase-12 (MMP12), cathepsin D (CTSD), cathepsin C (CTSD), and cathepsin L (CTSL), and optionally wherein the heterologous protease cleavage site is selected from Table S3, including the MMP12 cleavage site of SEQ ID NOs: 8-10, the CTSD cleavage site of SEQ ID NOs: 11 -12, the CTSC cleavage site of SEQ ID NO: 13, or the CTSL cleavage site of SEQ ID NOs: 14-16.

38. The patient care kit of claim 37, wherein the PPE polypeptide comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to a sequence selected from SEQ ID NOs: 17-25 (Table S4), which retains the heterologous protease cleavage site.

39. The patient care kit of any one of 31 -38, wherein the PPE polypeptide comprises at least one amino acid alteration in the peptidase domain (SEQ ID NO: 26), wherein the at least one alteration is at a residue selected from one or more of Q211 , T55, D74, R75, S214, R237, and N241 , the residue numbering being defined by SEQ ID NO: 6 (WT pro-PPE).

40. The patient care kit of claim 39, wherein the at least one amino acid alteration is selected from one or more of Q211 F, T55A, D74A, R75A, R75E, Q211 A, S214A, R237A, N241 A, and N241 Y, the residue numbering being defined by SEQ ID NO: 6 (WT pro-PPE).

41 . The patient care kit of claim 40, wherein the PPE polypeptide comprises, consists, or consists essentially of a peptidase domain selected from: an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 27, and which retains the Q211 F amino acid substitution; an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 28, and which retains the T55A amino acid substitution; an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 29, and which retains the N241A amino acid substitution; an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 30, and which retains the N241 Yamino acid substitution; an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 31 , and which retains the R75A amino acid substitution; an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 32, and which retains the R75E amino acid substitution; an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 33, and which retains the Q211 A amino acid substitution; an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 34, and which retains the R237A amino acid substitution; an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 35, and which retains the S214A amino acid substitution; and an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 36, and which retains the D74A amino acid substitution.

42. The patient care kit of any one of claims 31 -41 , wherein the pharmaceutical composition comprises a protein complex of:

(A) alpha-2-macroglobulin (A2M) proteins; and

(B) the serine protease proteins, optionally the ELANE or PPE polypeptides defined in any one of claims 35-41 , wherein (A) and (B) are present in the composition at a molar ratio [(A):(B)] of about 1 :3 to about 1 :1 .

43. The patient care kit of claim 42, 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:

44. The patient care kit of claim 42 or 43, 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.

45. The patient care kit of claim 44, wherein 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.

46. The patient care kit of any one of claims 42-45, 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.

47. The patient care kit of claim 46, wherein (A) and (B) are present in the composition at a molar ratio of about 1 :2.

48. The patient care kit of any one of claims 31 -34, wherein the DD polypeptide comprises, consists, or consists essentially of: an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to a selected from SEQ ID NOs: 2-5 and which induces apoptosis of cancer cells; an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 2 and which induces apoptosis of cancer cells; an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 3 and which induces apoptosis of cancer cells; an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 4 and which induces apoptosis of cancer cells; or an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 5 and which induces apoptosis of cancer cells.

49. The patient care kit of any one of claims 31-41 or 48, wherein the expressible polynucleotide is a virus or viral vector optionally selected from adenoviral vectors, herpes virus vectors, vaccinia virus vectors, adeno-associated virus (AAV) vectors, myxoma virus vectors, and retroviral vectors (optionally lentiviral vectors), or a modified mRNA polynucleotide.

50. The patient care kit of any one of claims 31-49 for use in diagnosing and/or treating a cancer in a subject.

51 . Use of the patient care kit of any one of claims 31 -49 in the manufacture of a kit or medicament for diagnosing and/or treating a cancer in a subject.

52. The patient care kit of claim 50 or the use of claim 51 , wherein the cancer is a solid tumor.

53. The patient care kit of claim 50 or the use of claim 51 , wherein the cancer is a hematological malignancy.

54. The patient care kit or use of any one of claims 50-53, wherein the cancer is selected from one or more of bladder cancer, blood cancer, bone cancer, bone marrow cancer, brain/nervous system cancer (optionally glioblastoma), breast cancer (optionally triple negative breast cancer), colon or colorectal cancer, esophageal cancer, gastrointestinal cancer, head cancer, kidney cancer, liver cancer, lung cancer (optionally small cell lung cancer (SCLC)), nasopharynx cancer, neck cancer (optionally head and neck cancer), ovarian cancer, pancreatic cancer, gallbladder cancer, prostate cancer, skin cancer (optionally melanoma, cutaneous squamous cell carcinoma, Merkel cell carcinoma), stomach cancer, testicular cancer, tongue cancer, uterine cancer, multiple myeloma, and embryonal rhabdomyosarcoma.

55. The patient care kit or use of any one of claims 50-54, wherein the pharmaceutical composition is administered to the subject by systemic administration or intratumoral injection.

56. The patient care kit or use of claim 55, wherein the pharmaceutical composition is administered to the subject by parenteral administration, optionally intravenous or subcutaneous administration.

57. The patient care kit or use of any one of claims 50-56, for use in combination with (or further comprising) an immune checkpoint modulatory agent, a chemotherapeutic agent, a hormonal therapeutic agent, and/or a kinase inhibitor.

58. The patient care kit or use of claim 57, wherein the immune checkpoint modulatory agent is a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor.