TRPV6 binding peptide-drug conjugates

Abstract

Provided are TRPV6 binding peptide-drug conjugates (PDCs), methods of synthesizing the PDCs, and related compositions and methods for treating diseases such as cancers.

Description

[0001] TRPV6 BINDING PEPTIDE-DRUG CONJUGATES

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit under 35 U. S. C. § 119(e) of U. S. Provisional Application No. 63 / 715,439, filed November 1, 2024, which is incorporated by reference in its entirety.

[0004] STATEMENT REGARDING SEQUENCE LISTING

[0005] 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 SORI_003_01WO_ST26.xml. The XMLfile is about 2,799 bytes, was created on October 28, 2025, and is being submitted electronically via USPTO Patent Center.

[0006] BACKGROUND

[0007] Technical Field

[0008] Embodiments of the present disclosure relate to TRPV6 binding peptide-drug conjugates (PDCs), methods of synthesizing the PDCs, and related compositions and methods for treating diseases such as cancers.

[0009] Description of the Related Art

[0010] Protein- or peptide-drug conjugates (PDCs) offer a useful modality for targeted drug delivery in the treatment of cancer, showing improved efficacy and reduced side effects (see, for example, J. Med. Chem. 64(1): 216-23, 2021). Transient Receptor Potential (TRP) channels, subfamily vanilloid (TRPV), member 6 (TRPV6) is expressed on certain cancers, and TRPV6 binding peptides can target such cancers (see, for example, U. S. Patent No. 10,064,964).

[0011] However, it has proven difficult to synthesize TRPV6-inhibitor PDCs (see, for example, WO 2017 / 136769). Thus, there is a need in the art for optimal TRPV6 binding PDCs and improved methods of synthesizing the same.

[0012] BRIEF SUMMARY

[0013] Embodiments of the present disclosure include a peptide-drug conjugate (PDC), selected from a PCD of:

[0014] Formula (I)

[0015]

[0016] including pharmaceutically-acceptable salts thereof.

[0017] Also included are pharmaceutical compositions, comprising a pharmaceutically-acceptable carrier and a PDC as described herein. Some embodiments include methods of treating a cancer in a subject in need thereof, comprising administering to the subject in need thereof a pharmaceutical composition or PDC described herein. In certain embodiments, the cancer expresses or over-expresses Transient Receptor Potential (TRP) channels, subfamily vanilloid (TRPV), members (TRPV6).

[0018] Some embodiments comprise:

[0019] (a) determining TRPV6 levels in a subject, optionally in a sample of cancer tissue or in a cancer cell from the subject; and

[0020] (b) administering the pharmaceutical composition to the subject if TRPV6 levels in the subject are increased relative to a control.

[0021] In certain embodiments, the cancer is selected from one or more of prostate cancer (optionally hormone-resistant prostate cancer), breast cancer, thyroid cancer, colon or colorectal cancer, ovarian cancer (optionally ovarian epithelial tumor), melanoma (e.g., metastatic melanoma, ocular melanoma), pancreatic cancer, bone cancer, small cell lung cancer, non-small cell lung cancer (NSCLC), mesothelioma, leukemia (e.g., lymphocytic leukemia, chronic myelogenous leukemia, acute myeloid leukemia, relapsed acute myeloid leukemia), lymphoma, hepatoma (hepatocellular carcinoma), hepatobiliary cancer, sarcoma, B-cell malignancy (optionally mature B-cell neoplasm), neuroepithelial tumor, glioma, glioblastoma multiforme, meningioma, pituitary adenoma, vestibular schwannoma, primary CNS lymphoma, primitive neuroectodermal tumor (medulloblastoma), kidney cancer (e.g., renal cell carcinoma optionally renal clear cell carcinoma, renal non-clear cell carcinoma), bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancers, cervical cancer, testicular cancer, adrenocortical carcinoma, seminoma, thymic epithelial tumor, plural mesothelioma, non-seminomatous germ cell tumor, and stomach cancer.

[0022] Particular embodiments comprise administering to the subject at least one additional agent selected from one or more of a cancer immunotherapy agent, a chemotherapeutic agent, a hormonal therapeutic agent, and a kinase inhibitor. In certain embodiments, the PDC and the at least one additional agent are administered separately, as separate compositions. In some embodiments, the PDC and the at least one additional agent are administered together as part of the same therapeutic composition.

[0023] In certain embodiments, the cancer immunotherapy agent is selected from one or more of an immune checkpoint modulatory agent, a cancer vaccine, an oncolytic virus, a cytokine, and a cell-based immunotherapies. In certain embodiments, the immune checkpoint modulatory agent is an immune checkpoint antagonist / inhibitor of Programmed Death-Ligand 1 (PD-L1), Programmed Death 1 (PD-1), Programmed Death-Ligand 2 (PD-L2), 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), V-domain Ig suppressor of T cell activation (VISTA), B and T Lymphocyte Attenuator (BTLA), CD160, Herpes Virus Entry Mediator (HVEM), and T-cell immunoreceptor with Ig and ITIM domains (TIGIT).

[0024] In some embodiments:

[0025] the antagonist is a PD-L1 and / or PD-L2 antagonist optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds thereto, atezolizumab (MPDL3280A), avelumab (MSB0010718C), and durvalumab (MEDI4736), optionally wherein the cancer is selected from one or more of colorectal cancer, melanoma, breast cancer, non-small-cell lung carcinoma, bladder cancer, and renal cell carcinoma;

[0026] the antagonist is a PD-1 antagonist optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds thereto, nivolumab, pembrolizumab, MK-3475, AMP-224, AMP-514PDR001, and pidilizumab, optionally wherein the PD-1 antagonist is nivolumab and the cancer is optionally selected from one or more of Hodgkin’s lymphoma, melanoma, non-small cell lung cancer, hepatocellular carcinoma, renal cell carcinoma, and ovarian cancer;

[0027] the PD-1 antagonist is pembrolizumab and the cancer is optionally selected from one or more of melanoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, and urothelial cancer;

[0028] the antagonist is a CTLA-4 antagonist optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds thereto, ipilimumab, tremelimumab, optionally wherein the cancer is selected from one or more of melanoma, prostate cancer, lung cancer, and bladder cancer;

[0029] the antagonist is an IDO antagonist optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds thereto, indoximod (NLG-8189), 1-methyl-tryptophan (1 MT), p-Carboline (norharmane; 9H-pyrido[3,4-b]indole), rosmarinic acid, and epacadostat, and wherein the cancer is optionally selected from one or more of metastatic breast cancer and brain cancer optionally glioblastoma multiforme, glioma, gliosarcoma or malignant brain tumor;

[0030] the antagonist is a TDO antagonist optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds thereto, 680C91, and LM10; the antagonist is a TIM-3 antagonist optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds thereto;

[0031] the antagonist is a LAG-3 antagonist optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds thereto, and BMS-986016;

[0032] the antagonist is a VISTA antagonist optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds thereto;

[0033] the antagonist is a BTLA, CD160, and / or HVEM antagonist optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds thereto;

[0034] the antagonist is a TIGIT antagonist optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds thereto.

[0035] In certain embodiments, the immune checkpoint modulatory agent is an immune checkpoint agonist of OX40, CD40, Glucocorticoid-Induced TNFR Family Related Gene (GITR), CD137 (4-1 BB), CD27, CD28, CD226, and Herpes Virus Entry Mediator (HVEM).

[0036] In specific embodiments:

[0037] the agonist is an OX40 agonist optionally selected from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds thereto, OX86, Fc-OX40L, and GSK3174998;

[0038] the agonist is a CD40 agonist optionally selected from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds thereto, CP-870,893, dacetuzumab, Chi Lob 7 / 4, ADC-1013, and rhCD40L, and wherein the cancer is optionally selected from one or more of melanoma, pancreatic carcinoma, mesothelioma, and hematological cancers optionally lymphoma such as Non-Hodgkin’s lymphoma;

[0039] the agonist is a GITR agonist optionally selected from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds thereto, INCAGN01876, DTA-1, and MEDI1873;

[0040] the agonist is a CD137 agonist optionally selected from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds thereto, utomilumab, and 4-1 BB ligand;

[0041] the agonist is a CD27 agonist optionally selected from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds thereto, varlilumab, and CDX-1127 (1 F5); the agonist is a CD28 agonist optionally selected from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds thereto, and TAB08; and / or

[0042] the agonist is an HVEM agonist optionally selected from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds thereto.

[0043] In certain embodiments, the cancer vaccine is selected from one or more of Oncophage, a human papillomavirus HPV vaccine optionally Gardasil or Cervarix, a hepatitis B vaccine optionally Engerix-B, Recombivax HB, or Twinrix, and sipuleucel-T (Provenge), or comprises a cancer antigen selected from one or more of human Her2 / neu, Her1 / EGF receptor (EGFR), Her3, A33 antigen, B7H3, CD5, CD19, CD20, CD22, CD23 (IgE Receptor), MAGE-3, C242 antigen, 5T4, IL-6, IL-13, vascular endothelial growth factor VEGF (e.g., VEGF-A) VEGFR-1, VEGFR-2, CD30, CD33, CD37, CD40, CD44, CD51, CD52, CD56, CD74, CD80, CD152, CD200, CD221, CCR4, HLA-DR, CTLA-4, NPC-1C, tenascin, vimentin, insulin-like growth factor 1 receptor (IGF-1 R), alpha-fetoprotein, insulin-like growth factor 1 (IGF-1 ), carbonic anhydrase 9 (CA-IX), carcinoembryonic antigen (CEA), guanylyl cyclase C, NY-ESO-1, p53, survivin, integrin av|33, integrin a5|31, folate receptor 1, transmembrane glycoprotein NMB, fibroblast activation protein alpha (FAP), glycoprotein 75, TAG-72, MUC1, MUC16 (or CA-125), phosphatidylserine, prostate-specific membrane antigen (PMSA), NR-LU-13 antigen, TRAIL-R1, tumor necrosis factor receptor superfamily member 10b (TNFRSF10B orTRAIL-R2), SLAM family member 7 (SLAMF7), EGP40 pancarcinoma antigen, B-cell activating factor (BAFF), platelet-derived growth factor receptor, glycoprotein EpCAM (17-1 A), Programmed Death-1, protein disulfide isomerase (PDI), Phosphatase of Regenerating Liver 3 (PRL-3), prostatic acid phosphatase, Lewis-Y antigen, GD2 (a disialoganglioside expressed on tumors of neuroectodermal origin), glypican-3 (GPC3), and mesothelin, optionally wherein the subject has or is at risk for having a cancerthat comprises the corresponding cancer antigen.

[0044] In certain embodiments, the oncolytic virus selected from one or more of talimogene laherparepvec (T-VEC), coxsackievirus A21 (CAVATAK™), Oncorine (H101), pelareorep (REOLYSIN®), Seneca Valley virus (NTX-010), Senecavirus SW-001, ColoAdl, SEPREHVIR (HSV-1716), CGTG-102 (Ad5 / 3-D24-GMCSF), GL-ONC1, MV-NIS, and DNX-2401.

[0045] In some embodiments, the cytokine selected from one or more of interferon (IFN)-a, IL-2, IL-12, IL-7, IL-21, and Granulocyte-macrophage colony-stimulating factor (GM-CSF). In certain embodiments, the cell-based immunotherapy agent comprises cancer antigen-specific T-cells, optionally ex v / o-derived T-cells. In certain embodiments, the cancer antigen-specific T-cells are selected from one or more of chimeric antigen receptor (CAR)-modified T-cells, and T-cell Receptor (TCR)-modified T-cells, tumor infiltrating lymphocytes (TILs), and peptide-induced T-cells.

[0046] In particular embodiments, the at least one chemotherapeutic agent is selected from one or more of an alkylating agent, an anti-metabolite, a cytotoxic antibiotic, a topoisomerase inhibitor (type 1 ortype II), and an anti-microtubule agent.

[0047] In certain embodiments:

[0048] the alkylating agent is selected from one or more of nitrogen mustards (optionally mechlorethamine, cyclophosphamide, mustine, melphalan, chlorambucil, ifosfamide, and busulfan), nitrosoureas (optionally N-Nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine, and streptozotocin), tetrazines (optionally dacarbazine, mitozolomide, and temozolomide), aziridines (optionally thiotepa, mytomycin, and diaziquone (AZQ)), cisplatins and derivatives thereof (optionally carboplatin and oxaliplatin), and non-classical alkylating agents (optionally procarbazine and hexamethylmelamine);

[0049] the anti-metabolite is selected from one or more of anti-folates (optionally methotrexate and pemetrexed), fluoropyrimidines (optionally 5-fluorouracil and capecitabine), deoxynucleoside analogues (optionally ancitabine, enocitabine, cytarabine, gemcitabine, decitabine, azacitidine, fludarabine, nelarabine, cladribine, clofarabine, fludarabine, and pentostatin), and thiopurines (optionally thioguanine and mercaptopurine);

[0050] the cytotoxic antibiotic is selected from one or more of anthracyclines (optionally doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, aclarubicin, and mitoxantrone), bleomycins, mitomycin C, mitoxantrone, and actinomycin;

[0051] the topoisomerase inhibitor is selected from one or more of camptothecin, irinotecan, topotecan, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, merbarone, and aclarubicin; and / or

[0052] the anti-microtubule agent is selected from one or more of taxanes (optionally paclitaxel and docetaxel) and vinca alkaloids (optionally vinblastine, vincristine, vindesine, vinorelbine).

[0053] In certain embodiments, the at least one hormonal therapeutic agent is a hormonal agonist or a hormonal antagonist. In some embodiments, the hormonal agonist is selected from one or more of a progestogen (progestin), a corticosteroid (optionally prednisolone, methylprednisolone, or dexamethasone), insulin like growth factors, VEGF derived angiogenic and lymphangiogenic factors (optionally VEGF-A, VEGF-A145, VEGF-A165, VEGF-C, VEGF-D, PIGF-2), fibroblast growth factor (FGF), galectin, hepatocyte growth factor (HGF), platelet derived growth factor (PDGF), transforming growth factor (TGF)-beta, an androgen, an estrogen, and a somatostatin analog.

[0054] In certain embodiments, the hormonal antagonist is selected from one or more of a hormone synthesis inhibitor, optionally an aromatase inhibitor or a gonadotropin-releasing hormone (GnRH) or an analog thereof, and a hormone receptor antagonist, optionally a selective estrogen receptor modulator (SERM) or an anti-androgen, or an antibody directed against a hormonal receptor, optionally cixutumumab, dalotuzumab, figitumumab, ganitumab, istiratumab, robatumumab, alacizumab pegol, bevacizumab, icrucumab, ramucirumab, fresolimumab, metelimumab, naxitamab, cetuximab, depatuxizumab mafodotin, futuximab, imgatuzumab, laprituximab emtansine, matuzumab, modotuximab, necitumumab, nimotuzumab, panitumumab, tomuzotuximab, zalutumumab, aprutumab ixadotin, bemarituzumab, olaratumab, ortovetumab.

[0055] In some embodiments, the kinase inhibitor is selected from one or more of adavosertib, afanitib, aflibercept, axitinib, bevacizumab, bosutinib, cabozantinib, cetuximab, cobimetinib, crizotinib, dasatinib, entrectinib, erdafitinib, erlotinib, fostamitinib, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, mubritinib, nilotinib, panitumumab, pazopanib, pegaptanib, ponatinib, ranibizumab, regorafenib, ruxolitinib, sorafenib, sunitinib, SU6656, tofacitinib, trastuzumab, vandetanib, and vemuafenib.

[0056] Some embodiments include a process of manufacturing a PDC of Formula (I)

[0057]

[0058] comprising:

[0059] i) reacting a compound of Formula 1-1 with a compound of Formula I-2 in the presence of a coupling reagent, thereby producing a compound of Formula I-3

[0060]

[0061] ii) reacting a compound of Formula I-3 in the presence of a deprotecting reagent, thereby producing a compound of Formula 1-4

[0062]

[0063] wherein:

[0064] PG1is an oxygen protecting group; and

[0065] PG2is a nitrogen protecting group.

[0066] In certain embodiments, wherein the coupling reagent is trichlorobenzoyl chloride. In certain embodiments, the deprotecting reagent is an organic base. In some embodiments, the deprotecting reagent is diethylamine. In certain embodiments, PG1is para-methoxybenzyl (PMB). In certain embodiments, PG2is fluorenylmethyloxycarbonyl (Fmoc).

[0067] Some embodiments include a process of manufacturing a PDC of Formula (II)

[0068]

[0069] comprising:

[0070] i) reacting a compound of Formula 11-1 with a compound that provides an oxygen protecting group, thereby producing a compound of Formula 11-2

[0071]

[0072] II-1 II-2, and

[0073] ii) reacting a compound of Formula 11-2 with a compound of Formula 11-3 in the presence of a coupling reagent, thereby producing a compound of Formula 11-4

[0074]

[0075] wherein:

[0076] PG3is an oxygen protecting group; and

[0077] PG4is a nitrogen protecting group.

[0078] In certain embodiments, the compound that provides an oxygen protecting group is benzyl bromide. In certain embodiments, the coupling reagent is 1 -ethyl-3-(3-dimethylaminopropyl)carbodiimide. In certain embodiments, PG3is benzyl. In certain embodiments, PG4is tert-butyloxycarbonyl.

[0079] Also included are methods of manufacturing a PDC of Formula (III), or a pharmaceutically-acceptable salt thereof, comprising:

[0080] i) reacting a compound of Formula 111-1 with a compound of Formula HI-2, thereby producing a compound of Formula HI-3

[0081]

[0082] ii) reacting the compound of Formula HI-3 with a peptide of Formula HI-4, thereby producing a conjugate of Formula III, wherein the peptide is H-Cys-Lys-Glu-Phe-Leu-His-Pro-Ser-Lys- Val-Asp- Leu-Pro-Arg-OH (SEQ ID NO: 2)

[0083]

[0084] Certain embodiments include a PDC manufactured according to a process or method described herein.

[0085] Also included are pharmaceutical compositions, comprising a pharmaceutically-acceptable carrier and a PDC manufactured according to a process or method described herein. Also included are methods of treating a cancer in a subject in need thereof, comprising administering to the subject a PDC manufactured according to a process or method described herein, including pharmaceutical compositions comprising the PDC.

[0086] BRIEF DESCRIPTION OF THE DRAWINGS

[0087] Figures 1A-1B show the stability of the “S73” lactam in human serum (Fig.1A) and mouse serum (Fig. 1 B) serum. Figures 1C-1D show the stability of the “S74” lactone in human serum (Fig.1C) and mouse serum (Fig.1D). Figure 1E shows the half-life (2.8 hrs) of SBI-1401 in human serum.

[0088] Figures 2A-2C show the effects of the “S73” lactam (Fig.2A), the “S74” lactone (Fig. 2B), and SBI-1401 (Fig.2C) on PC3 tumor growth in nude mice xenograft studies.

[0089] Figures 3A-3B show the effects of S73 lactam (Fig.3A) and S74 lactone (Fig.3B) on mice body weight in a PC3 nude mice xenograft study. Arrows indicate treatment days.

[0090] Figure 4A shows percent body weight change during the S73 treatment period.

[0091] Treatment days are indicated with an arrow. Treatment discontinuation for the 5.0 mg / kg treatment is indicated by a dotted line. Figure 4B shows percent body weight change during the S74 treatment period. Treatment days are indicated with an arrow. Treatment discontinuation for the 2.4, 3.5 and 5.0 mg / kg treatments is indicated by a dotted line.

[0092] Figure 5 shows RNA sequencing (RNA-Seq) expression data from the TCGA Pan-Cancer cohort, which was analyzed via the cBioPortal platform to evaluate TRPV6 mRNA expression across multiple tumor types. Cancer types were ranked based on the median fold-change in TRPV6 expression relative to matched normal tissues. Transcript quantification was performed using next-generation RNA sequencing data. Expression values for individual tumor samples, represented as blue dots, are displayed on a log2scale, where a value of +1 corresponds to a two-fold upregulation compared with normal tissues, and values below 0 indicate downregulation.

[0093] DETAILED DESCRIPTION

[0094] Unless defined otherwise, all technical and scientific terms used herein have the same meaningas 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.

[0095] 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.

[0096] 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”.

[0097] By “about” is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0098] 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 or inhibitor specifically binds to the other agent or molecule. Included are full and partial antagonists.

[0099] 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.

[0100] 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.

[0101] 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).

[0102] “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.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] 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.

[0108] 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. Adepyrogenation 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.

[0109] Endotoxins can be detected using routine techniques known in the art. For example, the Limulus Amoebocyte Lysate assay, which utilizes blood from the horseshoe crab, is a very sensitive assay for detecting presence of endotoxin. In this test, very low levels of LPS can cause detectable coagulation of the limulus lysate due a powerful enzymatic cascade that amplifies this reaction. Endotoxins can also be quantitated by enzyme-linked immunosorbent assay (ELISA). To be substantially endotoxin free, endotoxin levels may be less than about 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.08, 0.09, 0.1, 0.5, 1.0, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, or 10 EU / mg of active compound. Typically, 1 ng lipopolysaccharide (LPS) corresponds to about 1-10 EU.

[0110] The term “half maximal effective concentration” or “EC50” refers to the concentration of an agent (for example, a PDC) 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 a 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 “EC5o” and the Hill slope, or it can be determined from the data directly, using routine knowledge in the art. In some embodiments, the EC5o of an agent is less than about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200 or500 nM. In some embodiments, an agent will have an EC5o value of about 10 nM or less.

[0111] The term “half maximal inhibitory concentration” or “IC5o” refers to the concentration of an agent (for example, a PDC) as described herein at which it inhibits a response halfway between the baseline and maximum after some specified exposure time; the IC5o of a graded dose response curve therefore represents the concentration of a compound at which 50% of its maximal inhibitory effect of a specific biological or biochemical function is observed. IC5o also represents the plasma concentration required for obtaining 50% of a maximum inhibitory effect in vivo. Similarly, the “IC90” refers to the concentration of an agent or composition at which 90% of its maximal inhibitory effect is observed. The “IC90” can be calculated from the “IC50” and the Hill slope, or it can be determined from the data directly, using routine knowledge in the art. In some embodiments, the IC50of 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 IC5o value of about 10 nM or less.

[0112] 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.

[0113] 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.

[0114] 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.

[0115] 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.

[0116] 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. 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).

[0117] “Pharmaceutically acceptable salt” includes both acid and base addition salts.

[0118] “Pharmaceutically acceptable acid addition salt” refers to those salts which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and which are formed with inorganic acids such as, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1 -hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, and the like.

[0119] “Pharmaceutically acceptable base addition salt” refers to those salts which retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Preferred inorganic salts are the ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0120] In certain embodiments, the “purity” of any given agent in a composition may be defined. For instance, certain compositions may comprise an agent such as a polypeptide agent that is at least 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% pure on a protein basis or a weight-weight basis, including all decimals and ranges in between, as measured, for example and by no means limiting, by high performance liquid chromatography (HPLC), a well-known form of column chromatography used frequently in biochemistry and analytical chemistry to separate, identify, and quantify compounds.

[0121] 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.

[0122] 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.

[0123] “Substantially” or “essentially” means nearly totally or completely, for instance, 95%, 96%, 97%, 98%, 99% or greater of some given quantity.

[0124] 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.

[0125] “Therapeutic response” refers to improvement of symptoms (whether or not sustained) based on administration of one or more therapeutic agents.

[0126] 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.

[0127] 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.

[0128] 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.

[0129] Each embodiment in this specification is to be applied to every other embodiment unless expressly stated otherwise.

[0130] Peptide-drug Conjugates (PDCs) Embodiments of the present disclosure relate, in pertinent part, to protein- or peptide-drug conjugates (PDCs). The terms “protein” and “peptide” are used interchangeably herein. The PDCs comprise a TRPV6 binding peptide conjugated via a linker to a payload such as cryptophycin or monomethyl auristatin E (MMAE). In specific embodiments, the PDC is selected from a PDC of:

[0131] Formula (I)

[0132]

[0133] including pharmaceutically-acceptable salts thereof.

[0134] Formula (I) is an amide PDC composed of a TRPV6 binding peptide (KEFLHPSKVDLPR; SEQ ID NO: 1 ) conjugated via a linker to a cryptophycin 52 payload (S73 lactam). Formula (II) is a lactone PDC composed of a TRPV6 binding peptide (KEFLHPSKVDLPR; SEQ ID NO: 1) conjugated via a linker to a cryptophycin 52 payload (S74 lactone). Formula (III) is a PDC composed of a TRPV6 binding peptide (CKEFLHPSKVDLPR; SEQ ID NO: 2) conjugated via a linkerto a monomethyl auristatin E (MMAE) payload (SBI-1401).

[0135] The synthesis schemes for the PDCs of Formula (I), Formula (II), and / or Formula (III) are described herein (see Examples 1 -3). The PDCs can be used in any of the methods and compositions described herein.

[0136] Methods of Use and Therapeutic Compositions

[0137] Certain embodiments include methods of treating, ameliorating the symptoms of, and / or inhibitingthe progression of, a cancer in a subject in need thereof, comprising administeringto the subject a PDC of Formula (I), Formula (II), or Formula (III), as described herein, for example, as part of a pharmaceutical composition.

[0138] In certain embodiments, the cancer over-expresses TRPV6, for example, relative to non-cancerous cells from the corresponding tissue type. Here, TRPV6 is a member of the super family of Transient Receptor Potential (TRP) channels, subfamily vanilloid (TRPV), member 6, and is selective for Ca2+ ions. It is also highly expressed in a variety of cancer tissues including prostate, colon, breast, thyroid, and ovarian carcinomas, among others. Its expression coincides with cancer progression, suggesting that it can drive cancer cell growth.

[0139] The methods and therapeutic compositions described herein can be used in the treatment of any variety of cancers. In some embodiments, the subject or patient has a cancer selected from one or more of pancreatic cancer, bone cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer (NSCLC), mesothelioma, leukemia (e.g., lymphocytic leukemia, chronic myelogenous leukemia, acute myeloid leukemia, relapsed acute myeloid leukemia), lymphoma, hepatoma (hepatocellular carcinoma or HCC), sarcoma, B-cell malignancy, melanoma (e.g., metastatic melanoma), breast cancer (for example, estrogen receptor positive (ER+), estrogen receptor negative (ER-), Her2 positive (Her2+), Her2 negative (Her2-), or a combination thereof, e.g., ER+ / Her2+, ER+ / Her2-, ER- / Her2+, or ER- / Her2-; or “triple negative” breast cancer which is estrogen receptor-negative, progesterone receptornegative, and HER2-negative), ovarian cancer, colorectal cancer, glioma (e.g., astrocytoma, oligodendroglioma, ependymoma, or a choroid plexus papilloma), glioblastoma multiforme (e.g., giant cell gliobastoma or a gliosarcoma), meningioma, pituitary adenoma, vestibular schwannoma, primary CNS lymphoma, primitive neuroectodermal tumor (medulloblastoma), kidney cancer (e.g., renal cell carcinoma), bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancers, cervical cancer, testicular cancer, stomach cancer, virus-induced tumors such as, for example, papilloma virus-induced carcinomas (e.g., cervical carcinoma, cervical cancer), adenocarcinomas, herpes virus-induced tumors (e.g. Burkitt’s lymphoma, EBV-induced B-cell lymphoma), hepatitis B-induced tumors (hepatocellular carcinomas), HTLV-1 -indued and HTLV-2-induced lymphomas, acoustic neuroma, lung cancers (e.g., lung carcinoma, bronchial carcinoma), small-cell lung carcinomas, pharyngeal cancer, anal carcinoma, glioblastoma, rectal carcinoma, astrocytoma, brain tumors, retinoblastoma, basalioma, brain metastases, medulloblastomas, vaginal cancer, pancreatic cancer, testicular cancer, Hodgkin’s syndrome, meningiomas, Schneeberger disease, hypophysis tumor, Mycosis fungoides, carcinoids, neurinoma, spinalioma, Burkitt’s lymphoma, laryngeal cancer, renal cancer, thymoma, corpus carcinoma, bone cancer, non-Hodgkin’s lymphomas, urethral cancer, CUP syndrome, head / neck tumors, oligodendroglioma, vulval cancer, intestinal cancer, colon carcinoma, oesophageal cancer (e.g., oesophageal carcinoma), wart involvement, tumors of the small intestine, craniopharyngeomas, ovarian carcinoma, genital tumors, ovarian cancer (e.g., ovarian carcinoma), pancreatic cancer (e.g., pancreatic carcinoma), endometrial carcinoma, liver metastases, penile cancer, tongue cancer, gall bladder cancer, leukemia, plasmocytoma, and lid tumor, among others described herein and known in the art.

[0140] In some embodiments, as noted above, the cancer is a metastatic cancer. Further to the above cancers, exemplary metastatic cancers include, without limitation, bladder cancers which have metastasized to the bone, liver, and / or lungs; breast cancers which have metastasized to the bone, brain, liver, and / or lungs; colorectal cancers which have metastasized to the liver, lungs, and / or peritoneum; kidney cancers which have metastasized to the adrenal glands, bone, brain, liver, and / or lungs; lung cancers which have metastasized to the adrenal glands, bone, brain, liver, and / or other lung sites; melanomas which have metastasized to the bone, brain, liver, lung, and / or skin / muscle; ovarian cancers which have metastasized to the liver, lung, and / or peritoneum; pancreatic cancers which have metastasized to the liver, lung, and / or peritoneum; prostate cancers which have metastasized to the adrenal glands, bone, liver, and / or lungs; stomach cancers which have metastasized to the liver, lung, and / or peritoneum; thyroid cancers which have metastasized to the bone, liver, and / or lungs; and uterine cancers which have metastasized to the bone, liver, lung, peritoneum, and / or vagina; among others.

[0141] Some embodiments include using TRPV6 levels as a companion diagnostic for treatment with a PDC described herein (see, for example, Stewart, J Cancer. 11(2): 374-387, 2020). For instance, certain embodiments include (a) measuring or otherwise determining TRPV6 levels in a subject, for example, in a sample of cancer tissue or in a cancer cell from the subject; and (b) administering a PDC-containing pharmaceutical composition to the subject if TRPV6 levels are increased relative to a control.

[0142] Certain embodiments include determiningTRPV6 protein levels in the subject. The presence, absence, amount, levels, or cellular localization (e.g., surface, subcellular) of TRPV6 protein in a biological sample can be measured according to any variety of techniques in the art. For instance, certain embodiments may employ standard protein-based methodologies and detectors. Examples include immunohistochemistry (IHC), immunofluorescence (IF), Western blotting, immunoprecipitation, enzyme-linked immunosorbent assays (ELISA), slot blotting, and peptide mass fingerprinting. Certain embodiments may employ cell-sorting or cell visualization or imaging devices / techniques to visualize, detect, and / or quantitate the amount or levels of TRPV6 protein. Examples include flow cytometry (or FACS), immunofluorescence analysis (IFA), and in situ hybridization techniques, such as fluorescent in situ hybridization (FISH).

[0143] Specific embodiments include the use of IHC or IF analysis. Examples include chromogenic immunohistochemistry (CIH), for example, wherein an anti-TRPV6 antibody is conjugated to an enzyme, such as peroxidase (immunoperoxidase), which catalyzes a colorproducing reaction (see, for example, Ramos-Vara, “Technical Aspects of Immunohistochemistry”. Veterinary Pathol. 42: 405-426, 2005). In IF analysis, an anti-TRPV6 antibody is labeled directly or indirectly with a fluorophore, such as fluorescein or rhodamine, which allows visualization by light microscopy with a fluorescent microscope. Certain embodiments employ primary (or direct) IF analysis, wherein a primary anti-TRPV6 antibody is chemically linked to a fluorophore. Certain embodiments employ secondary (or indirect) IF analysis, which requires at least two antibodies; an unlabeled primary anti-TRPV6 antibody that specifically binds the target molecule, and one or more secondary antibodies, which carries the fluorophore(s) and binds to the primary antibody.

[0144] In specific embodiments, the IHC or IF assay comprises a multiplex IHC or IF assay (see, for example, Tan, Wei Chang Colin et al. “Overview of multiplex immunohistochemistry / immunofluorescence techniques in the era of cancer immunotherapy.” Cancer communications (London, England) vol. 40,4 (2020): 135-153). Multiplex IHC or IF includes contacting the biological sample with at least one additional antibody that specifically binds to an additional marker of interest, which has or utilizes a different (direct or indirect) detectable label. In particular embodiments, the additional marker of interest is selected from one or more of signal transduction pathway molecules (for example, VEGF-C, VEGF-A, EGF, IGF, FGF, TGF- beta, VEGFR1, VEGFR2, VEGFR3, CCR7, EGFR1, EGFR2, PDGFR, TGFR1, TGFR2, TGFR3, c-MET); EMT markers such as mesenchymal markers (for example, N-cadherin, E-cadherin, OB-cadherin, ZO-1, a5|31 integrin 1, aV|36 integrin, Syndecan-1, FSP1, Cytokeratin, a-SMA, Vimentin 1, p-Catenin), epithelial markers (for example, CDH1, EPCAM, claudins, and cytokeratins), and related transcription factors (for example, Snail, Slug, ZEB1, ZEB2, and Twist); lymphangiogenesis markers (for example, lymphatic vessel endothelial hyaluronan receptor-1, or LYVE-1); fibrosis markers (for example, collagen fibers, matricellular proteins such as tenascin-C, a-SMA); and immune activation / exhaustion markers and immune modulators (for example, CD45RA, CD45RO, CD27, CD62L, CD95, PD-1, PD-L1, CD80, CD86, CXCR4, BLC, SCD30, MCP-2, IP-10, APRIL, SIL-2R, IL7, MIF, MIP-1 b, SCF, SDF-1a, sTNF-RI).

[0145] Some embodiments include determiningTRPV6 mRNA levels in the subject. The presence, absence, amount, or levels of TRPV6 RNAor mRNA in a biological sample can be measured according to any variety of techniques in the art. For instance, mRNA levels can be measured according to standard RNA-based methodologies and detectors, including RT-PCR (see, for example, Moul et al., Clin Prostate Cancer. 1 (1 ):42-50, 2002) such as quantitative competitive (QC) RT-PCR, RNA sequencing (RNA-seq, for example Nie et al., Dose Response.

[0146] 17(1): 1559325819833474, 2019), RNA in situ hybridization (for example, branched DNA assays), and other techniques known in the art.

[0147] Also included are combination therapies, comprising administering the PDC to the subject in combination with at least one additional anti-cancer agent. In some instances, the PDC and the anti-cancer agent are administered separately, for example, in separate pharmaceutical compositions and at the same or different times. In some embodiments, the PDC and the anti-cancer agent are administered as part of the same therapeutic composition, at the same time. Examples of anti-cancer agents include cancer immunotherapy agents, chemotherapeutic agents, hormonal therapeutic agents, and kinase inhibitors.

[0148] 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-bindingfragments 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 such as oncolytic viruses, 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, oncolytic viruses, cytokines, and cell-based immunotherapies.

[0149] In certain embodiments, the cancer immunotherapy agent is an immune checkpoint modulatory agent. Particular examples include “antagonists” 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.

[0150] 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 Igand ITIM domains (TIGIT).

[0151] In certain embodiments, the agent is a PD-1 (receptor) antagonist or inhibitor, the targeting of which has been shown to restore immune function in the tumor environment (see, e.g., Phillips et al., Int Immunol. 27:39-46, 2015). PD-1 is a cell surface receptorthat belongs to the immunoglobulin superfamily and is expressed on T cells and pro-B cells. PD-1 interacts with two ligands, PD-L1 and PD-L2. PD-1 functions as an inhibitory immune checkpoint molecule, forexample, by reducingor preventingthe activation of T-cells, which in turn reduces autoimmunity and promotes self-tolerance. The inhibitory effect of PD-1 is accomplished at least in part through a dual mechanism of promoting apoptosis in antigen specific T-cells in lymph nodes while also reducing apoptosis in regulatoryT cells (suppressorT cells). Some examples of PD-1 antagonists or inhibitors include an antibody or antigen-binding fragment or small molecule that specifically binds to PD-1 and reduces one or more of its immune-suppressive activities, for example, its downstream signaling or its interaction with PD-L1. Specific examples of PD-1 antagonists or inhibitors include the antibodies nivolumab, pembrolizumab, PDR001, MK-3475, AMP-224, AMP-514, and 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.

[0152] Application Nos. 2012 / 0039906; 2015 / 0203579).

[0153] In some embodiments, the agent is a PD-L1 antagonist or inhibitor. As noted above, PD-L1 is one of the natural ligands for the PD-1 receptor. General examples of PD-L1 antagonists or inhibitors include an antibody or antigen-binding fragment or small molecule that specifically binds to PD-L1 and reduces one or more of its immune-suppressive activities, for example, its bindingto the PD-1 receptor. Specific examples of PD-L1 antagonists include the antibodies atezolizumab (MPDL3280A), avelumab (MSB0010718C), and durvalumab (MEDI4736), and antigen-binding fragments thereof (see, e.g., U. S. Patent Nos. 9,102,725; 9,393,301; 9,402,899; 9,439,962).

[0154] In some embodiments, the agent is a PD-L2 antagonist or inhibitor. As noted above, PD-L2 is one of the natural ligands for the PD-1 receptor. General examples of PD-L2 antagonists or inhibitors include an antibody or antigen-binding fragment or small molecule that specifically binds to PD-L2 and reduces one or more of its immune-suppressive activities, for example, its bindingto the PD-1 receptor.

[0155] In certain embodiments, the agent is a VISTA antagonist or inhibitor. VISTA is approximately 50 kDa in size and belongs to the immunoglobulin superfamily (it has one IgV domain) and the B7 family. It is primarily expressed in white blood cells, and its transcription is partially controlled by p53. There is evidence that VISTA can act as both a ligand and a receptor on T cells to inhibit T cell effector function and maintain peripheral tolerance. VISTA is produced at high levels in tumor-infiltrating lymphocytes, such as myeloid -de rived suppressor cells and regulatory T cells, and its blockade with an antibody results in delayed tumor growth in mouse models of melanoma and squamous cell carcinoma. Exemplary anti-VISTA antagonist antibodies include, for example, the antibodies described in WO 2018 / 237287, which is incorporated by reference in its entirety.

[0156] In some embodiments, the agent is a CTLA-4 antagonist or inhibitor. CTLA4 or CTLA-4 (cytotoxic T-lymphocyte-associated protein 4), also known as CD152 (cluster of differentiation 152), is a protein receptor that functions as an inhibitory immune checkpoint molecule, for example, by transmitting inhibitory signals to T-cells when it is bound to CD80 or CD86 on the surface of antigen-presenting cells. General examples CTLA-4 antagonists or inhibitors include an antibody or antigen-binding fragment or small molecule that specifically binds to CTLA-4. Particular examples include the antibodies ipilimumab and tremelimumab, and antigenbinding 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.

[0157] In some embodiments, the agent is an IDO antagonist or inhibitor, or a TDO antagonist or inhibitor. IDO and TDO are tryptophan catabolic enzymes with immune-inhibitory properties. For example, IDO is known to suppress T-cells and NK cells, generate and activate Tregs and myeloid-derived suppressor cells, and promote tumor angiogenesis. General examples of IDO and TDO antagonists or inhibitors include an antibody or antigen-binding fragment or small molecule that specifically binds to IDO orTDO (see, e.g., Flatten et al., Front Immunol. 5: 673, 2014) and reduces or inhibits one or more immune-suppressive activities. Specific examples of IDO antagonists or inhibitors include indoximod (NLG-8189), 1-methyl-tryptophan (1 MT), 0-Carboline (norharmane; 9H-pyrido[3,4-b]indole), rosmarinic acid, and epacadostat (see, e.g., Sheridan, Nature Biotechnology. 33:321-322, 2015). Specific examples of TDO antagonists or inhibitors include 680C91 and LM10 (see, e.g., Pilotte et al., PNAS USA. 109:2497-2502, 2012).

[0158] In some embodiments, the agent is a TIM-3 antagonist or inhibitor. T-cell Immunoglobulin domain and Mucin domain 3 (TIM-3) is expressed on activated human CD4+ T-cells and regulates Th1 and Th17 cytokines. TIM-3 also acts as a negative regulator of Th1 / Tc1 function by triggering cell death upon interaction with its ligand, galectin-9. TIM-3 contributes to the suppressive tumor microenvironment and its overexpression is associated with poor prognosis in a variety of cancers (see, e.g., Li et al., Acta Oncol. 54:1706-13, 2015). General examples of TIM-3 antagonists or inhibitors include an antibody or antigen-binding fragment or small molecule that specifically binds to TIM-3 and reduces or inhibits one or more of its immune-suppressive activities.

[0159] In some embodiments, the agent is a LAG-3 antagonist or inhibitor. Lymphocyte Activation Gene-3 (LAG-3) is expressed on activated T-cells, natural killer cells, B-cells and plasmacytoid dendritic cells. It negatively regulates cellular proliferation, activation, and homeostasis of T-cells, in a similar fashion to CTLA-4 and PD-1 (see, e.g., Workman and Vignali. European Journal of Immun. 33: 970-9, 2003; and Workman et al., Journal of Immun. 172: 5450-5, 2004), and has been reported to playa role in Treg suppressive function (see, e.g., Huang et al., Immunity. 21: 503-13, 2004). LAG3 also maintains CD8+ T-cells in a tolerogenic state and combines with PD-1 to maintain CD8 T-cell exhaustion. General examples of LAG-3 antagonists or inhibitors include an antibody or antigen-binding fragment or small molecule that specifically binds to LAG-3 and inhibits one or more of its immune-suppressive activities. Specific examples include the antibody BMS-986016, and antigen-binding fragments thereof.

[0160] In some embodiments, the agent is a BTLA antagonist or inhibitor. B- and T-lymphocyte attenuator (BTLA; CD272) expression is induced during activation of T-cells, and it inhibits T-cells via interaction with tumor necrosis family receptors (TNF-R) and B7 family of cell surface receptors. BTLA is a ligand for tumor necrosis factor (receptor) superfamily, member 14 (TNFRSF14), also known as herpesvirus entry mediator (HVEM). BTLA-HVEM complexes negatively regulate T-cell immune responses, for example, by inhibiting the function of human CD8+ cancer-specific T-cells (see, e.g., Derre et al., J Clin Invest 120:157-67, 2009). General examples of BTLA antagonists or inhibitors include an antibody or antigen-binding fragment or small molecule that specifically binds to BTLA-4 and reduce one or more of its immune-suppressive activities.

[0161] In some embodiments, the agent is an HVEM antagonist or inhibitor, for example, an antagonist or inhibitor that specifically binds to HVEM and interferes with its interaction with BTLAor CD160. General examples of HVEM antagonists or inhibitors include an antibody or antigen-binding fragment or small molecule that specifically binds to HVEM, optionally reduces the HVEM / BTLA and / or HVEM / CD160 interaction, and thereby reduces one or more of the immune-suppressive activities of HVEM.

[0162] In some embodiments, the agent is a CD160 antagonist or inhibitor, for example, an antagonist or inhibitor that specifically binds to CD160 and interferes with its interaction with HVEM. General examples of CD160 antagonists or inhibitors include an antibody or antigenbindingfragment or small molecule that specifically binds to CD160, optionally reduces the CD160 / HVEM interaction, and thereby reduces or inhibits one or more of its immune-suppressive activities.

[0163] In some embodiments, the agent is a TIGIT antagonist or inhibitor. T cell Ig and ITIM domain (TIGIT) is a co-inhibitory receptor that is found on the surface of a variety of lymphoid cells, and suppresses antitumor immunity, for example, via Tregs (Kurtulus et al., J Clin Invest.

[0164] 125:4053-4062, 2015). General examples of TIGIT antagonists or inhibitors include an antibody or antigen-binding fragment or small molecule that specifically binds to TIGIT and reduce one or more of its immune-suppressive activities (see, e.g., Johnston et al., Cancer Cell. 26:923-37, 2014).

[0165] 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).

[0166] In some embodiments, the agent is a CD40 agonist. CD40 is expressed on antigen-presenting cells (APC) and some malignancies. Its ligand is CD40L (CD154). On APC, ligation results in upregulation of costimulatory molecules, potentially bypassing the need forT-cell assistance in an antitumor immune response. CD40 agonist therapy plays an important role in APC maturation and their migration from the tumor to the lymph nodes, resulting in elevated antigen presentation and T cell activation. Anti-CD40 agonist antibodies produce substantial responses and durable anticancer immunity in animal models, an effect mediated at least in part by cytotoxic T-cells (see, e.g., Johnson et al. Clin Cancer Res. 21: 1321-1328, 2015; and Vonderheide and Glennie, Clin Cancer Res. 19:1035-43, 2013). General examples of CD40 agonists include an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to CD40 and increases one or more of its immunostimulatory activities. Specific examples include CP-870,893, dacetuzumab, Chi Lob 7 / 4, ADC-1013, CD40L, rhCD40L, and antigen-binding fragments thereof. Specific examples of CD40 agonists include, but are not limited to, APX005 (see, e.g., US 2012 / 0301488) and APX005M (see, e.g., US 2014 / 0120103).

[0167] In some embodiments, the agent is an 0X40 agonist. 0X40 (CD134) promotes the expansion of effector and memory T cells, and suppresses the differentiation and activity of T-regulatory cells (see, e.g., Croft et al., Immunol Rev. 229:173-91, 2009). Its ligand is OX40L ( CD252). Since 0X40 signaling influences both T-cell activation and survival, it plays a key role in the initiation of an anti-tumor immune response in the lymph node and in the maintenance of the anti-tumor immune response in the tumor microenvironment. General examples of 0X40 agonists include an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to 0X40 and increases one or more of its immunostimulatory activities. Specific examples include 0X86, OX-40L, FC-OX40L, GSK3174998, MEDI0562 (a humanized 0X40 agonist), MEDI6469 (murine 0X4 agonist), and MEDI6383 (an 0X40 agonist), and antigenbindingfragments thereof.

[0168] In some embodiments, the agent is a GITR agonist. Glucocorticoid-Induced TNFR family Related gene (GITR) increasesT cell expansion, inhibits the suppressive activity of Tregs, and extends the survival of T-effector cells. GITR agonists have been shown to promote an antitumor response through loss of Treg lineage stability (see, e.g., Schaer et al., Cancer Immunol Res. 1:320-31, 2013). These diverse mechanisms show that GITR plays an important role in initiating the immune response in the lymph nodes and in maintaining the immune response in the tumor tissue. Its ligand is GITRL. General examples of GITR agonists include an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to GITR and increases one or more of its immunostimulatory activities. Specific examples include GITRL, INCAGN01876, DTA-1, MEDI1873, and antigen-binding fragments thereof.

[0169] In some embodiments, the agent is a CD137 agonist. CD137 (4-1 BB) is a member of the tumor necrosis factor (TNF) receptor family, and crosslinking of CD137 enhances T-cell proliferation, IL-2 secretion, survival, and cytolytic activity. CD137-mediated signaling also protects T-cells such as CD8+ T-cells from activation-induced cell death. General examples of CD137 agonists include an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to CD137 and increases one or more of its immunostimulatory activities. Specific examples include the CD137 (or 4-1 BB) ligand (see, e.g., Shao and Schwarz, J Leukoc Biol. 89:21-9, 2011) and the antibody utomilumab, including antigen-binding fragments thereof.

[0170] In some embodiments, the agent is a CD27 agonist. Stimulation of CD27 increases antigen-specific expansion of naive T cells and contributes to T-cell memory and long-term maintenance of T-cell immunity. Its ligand is CD70. The targeting of human CD27 with an agonist antibody stimulates T-cell activation and antitumor immunity (see, e.g., Thomas et al., Oncoimmunology. 2014;3:e27255. doi:10.4161 / onci.27255; and He et al., J Immunol.

[0171] 191:4174-83, 2013). General examples of CD27 agonists include an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to CD27 and increases one or more of its immunostimulatory activities. Specific examples include CD70 and the antibodies varlilumab and CDX-1127 (1 F5), including antigen-binding fragments thereof.

[0172] In some embodiments, the agent is a CD28 agonist. CD28 is constitutively expressed CD4+ T cells some CD8+ T cells. Its ligands include CD80 and CD86, and its stimulation increases T-cell expansion. General examples of CD28 agonists include an antibody or antigenbinding fragment or small molecule or ligand that specifically binds to CD28 and increases one or more of its immunostimulatory activities. Specific examples include CD80, CD86, the antibody TAB08, and antigen-binding fragments thereof.

[0173] In some embodiments, the agent is CD226 agonist. CD226 is a stimulating receptor that shares ligands with TIGIT, and opposite to TIGIT, engagement of CD226 enhances T-cell activation (see, e.g., Kurtulus et al., J Clin Invest. 125:4053-4062, 2015; Bottino et al., J Exp Med.

[0174] 1984:557-567, 2003; and Tahara-Hanaoka et al., Int Immunol. 16:533-538, 2004). General examples of CD226 agonists include an antibody or antigen-binding fragment or small molecule or ligand (e.g., CD112, CD155) that specifically binds to CD226 and increases one or more of its immunostimulatory activities. In some embodiments, the agent is an HVEM agonist. Herpesvirus entry mediator (HVEM), also known as tumor necrosis factor receptor superfamily member 14 (TNFRSF14), is a human cell surface receptor of the TNF-receptor superfamily. HVEM is found on a variety of cells including T-cells, APCs, and other immune cells. Unlike other receptors, HVEM is expressed at high levels on resting T-cells and down-regulated upon activation. It has been shown that HVEM signaling plays a crucial role in the early phases of T-cell activation and during the expansion of tumor-specific lymphocyte populations in the lymph nodes. General examples of HVEM agonists include an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to HVEM and increases one or more of its immunostimulatory activities.

[0175] In certain embodiments, the immunotherapy agent is a bi-specific or multi-specific antibody. For instance, certain bi-specific or multi-specific antibodies are able to (i) bind to and inhibit one or more inhibitory immune checkpoint molecules, and also (ii) bind to and agonize one or more stimulatory immune checkpoint molecules. In certain embodiments, a bi-specific or multi-specific antibody (i) binds to and inhibits one or more of PD-L1, PD-L2, PD-1, CTLA-4, IDO, TDO, TIM-3, LAG-3, BTLA, CD160, and / or TIGIT, and also (ii) binds to and agonizes one or more of CD40, 0X40 Glucocorticoid-Induced TNFR Family Related Gene (GITR), CD137 (4-1 BB), CD27, CD28, CD226, and / or Herpes Virus Entry Mediator (HVEM).

[0176] In some embodiments, the immunotherapy agent is a cancer vaccine. In certain embodiments, the cancer vaccine is selected from one or more of Oncophage, a human papillomavirus HPV vaccine optionally Gardasil or Cervarix, a hepatitis B vaccine optionally Engerix-B, Recombivax HB, or Twinrix, and sipuleucel-T (Provenge). In some embodiments, the cancer vaccine comprises or expresses a TAAorTSAas described herein.

[0177] In some embodiments, the immunotherapy agent is an oncolytic viruses. In some embodiments, the oncolytic virus selected from one or more of talimogene laherparepvec (T-VEC), coxsackievirus A21 (CAVATAK™), Oncorine (H101), pelareorep (REOLYSIN®), Seneca Valley virus (NTX-010), Senecavirus SW-001, ColoAdl, SEPREHVIR (HSV-1716), CGTG-102 (Ad5 / 3-D24-GMCSF), GL-ONC1, MV-NIS, and DNX-2401.

[0178] In certain embodiments, the cancer immunotherapy agent is a cytokine. Exemplary cytokines include interferon (IFN)-a, IL-2, IL-12, IL-7, IL-21, and Granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0179] In certain embodiments, the cancer immunotherapy agent is cell-based immunotherapy, for example, a therapy that utilizes immune cells, including ex vivo-derived immune cells, such as lymphocytes, natural killer (NK) cells, macrophages, and / or dendritic cells (DCs). In some embodiments, the lymphocytes comprise T-cells, for example, cytotoxic T-lymphocytes (CTLs). See, for example, June, J Clin Invest. 117: 1466-1476, 2007; Rosenberg and Restifo, Science. 348:62-68, 2015; Cooley et al., Biol, of Blood and MarrowTransplant. 13:33-42, 2007; and Li and Sun, Chin J Cancer Res. 30:173-196, 2018, for descriptions of adoptive T-cell and NK cell immunotherapies. In some embodiments, the T-cells comprise cancer antigenspecific T-cells, which are directed against at least one cancer antigen. In some embodiments, the cancer antigen-specific T-cells are selected from one or more of chimeric antigen receptor (CAR)-modified T-cells, T-cell Receptor (TCR)-modified T-cells, tumor infiltrating lymphocytes (TILs), and peptide-induced T-cells. In specific embodiments, the CAR-modified T-cell is targeted against CD-19 (see, e.g., Maude et al., Blood. 125:4017-4023, 2015). In some instances, the ex vivo-derived immune cells are autologous cells, which are obtained from the patient to be treated.

[0180] Certain combination therapies employ one or more chemotherapeutic agents, for example, small molecule chemotherapeutic agents. Non-limiting examples of chemotherapeutic agents include alkylating agents, anti-metabolites, cytotoxic antibiotics, topoisomerase inhibitors (type 1 or type II), and anti-microtubule agents, among others.

[0181] Examples of alkylating agents include nitrogen mustards (e.g., mechlorethamine, cyclophosphamide, mustine, melphalan, chlorambucil, ifosfamide, and busulfan), nitrosoureas (e.g., N-Nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine, and streptozotocin), tetrazines (e.g., dacarbazine, mitozolomide, and temozolomide), aziridines (e.g., thiotepa, mytomycin, and diaziquone (AZQ)), cisplatins and derivatives thereof (e.g., carboplatin and oxaliplatin), and non-classical alkylating agents (optionally procarbazine and hexamethylmelamine).

[0182] Examples of anti-metabolites include anti-folates (e.g., methotrexate and pemetrexed), fluoropyrimidines (e.g., 5-fluorouracil and capecitabine), deoxynucleoside analogues (e.g., ancitabine, enocitabine, cytarabine, gemcitabine, decitabine, azacitidine, fludarabine, nelarabine, cladribine, clofarabine, fludarabine, and pentostatin), and thiopurines (e.g., thioguanine and mercaptopurine).

[0183] Examples of cytotoxic antibiotics include anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, aclarubicin, and mitoxantrone), bleomycins, mitomycin C, mitoxantrone, and actinomycin. Examples of topoisomerase inhibitors include camptothecin, irinotecan, topotecan, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, merbarone, and aclarubicin. Examples of anti-microtubule agents include taxanes (e.g., paclitaxel and docetaxel) and vinca alkaloids (e.g., vinblastine, vincristine, vindesine, vinorelbine).

[0184] The various chemotherapeutic agents described herein can be combined with any one or more of the PDCs described herein, and used according to any one or more of the methods or compositions described herein.

[0185] Certain combination therapies employ at least one hormonal therapeutic agent.

[0186] General examples of hormonal therapeutic agents include hormonal agonists and hormonal antagonists. Particular examples of hormonal agonists include progestogen (progestin), corticosteroids (e.g., prednisolone, methylprednisolone, dexamethasone), insulin like growth factors, VEGF derived angiogenic and lymphangiogenic factors (e.g., VEGF-A, VEGF-A145, VEGF-A165, VEGF-C, VEGF-D, PIGF-2), fibroblast growth factor (FGF), galectin, hepatocyte growth factor (HGF), platelet derived growth factor (PDGF), transforming growth factor (TGF)-beta, androgens, estrogens, and somatostatin analogs. Examples of hormonal antagonists include hormone synthesis inhibitors such as aromatase inhibitors and gonadotropin-releasing hormone (GnRH)s agonists (e.g., leuprolide, goserelin, triptorelin, histrelin) including analogs thereof. Also included are hormone receptor antagonist such as selective estrogen receptor modulators (SERMs; e.g., tamoxifen, raloxifene, toremifene) and anti-androgens (e.g., flutamide, bicalutamide, nilutamide).

[0187] Also included are hormonal pathway inhibitors such as antibodies directed against hormonal receptors. Examples include inhibitors of the IGF receptor (e.g., IGF-IR1 ) such as cixutumumab, dalotuzumab, figitumumab, ganitumab, istiratumab, and robatumumab; inhibitors of the vascular endothelial growth factor receptors 1, 2 or 3 (VEGFR1, VEGFR2 or VEGFR3) such as alacizumab pegol, bevacizumab, icrucumab, ramucirumab; inhibitors of the TGF-beta receptors R1, R2, and R3 such as fresolimumab and metelimumab; inhibitors of c-Met such as naxitamab; inhibitors of the EGF receptor such as cetuximab, depatuxizumab mafodotin, futuximab, imgatuzumab, laprituximab emtansine, matuzumab, modotuximab, necitumumab, nimotuzumab, panitumumab, tomuzotuximab, and zalutumumab; inhibitors of the FGF receptor such as aprutumab ixadotin and bemarituzumab; and inhibitors of the PDGF receptor such as olaratumab and tovetumab.

[0188] The various hormonal therapeutic agents described herein can be combined with any one or more of the PDCs described herein, and used according to any one or more of the methods or compositions described herein.

[0189] Certain combination therapies employ at least one kinase inhibitor, including tyrosine kinase inhibitors. Examples of kinase inhibitors include, without limitation, adavosertib, afanitib, aflibercept, axitinib, bevacizumab, bosutinib, cabozantinib, cetuximab, cobimetinib, crizotinib, dasatinib, entrectinib, erdafitinib, erlotinib, fostamitinib, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, mubritinib, nilotinib, panitumumab, pazopanib, pegaptanib, ponatinib, ranibizumab, regorafenib, ruxolitinib, sorafenib, sunitinib, SU6656, tofacitinib, trastuzumab, vandetanib, and vemuafenib.

[0190] The various kinase inhibitors described herein can be combined with any one or more of the PDCs described herein, and used according to any one or more of the methods or compositions described herein.

[0191] In some embodiments, the methods and compositions described herein increase cancer cell-killing in the subject by about or at least about 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 500-fold, or 1000-fold or more relative to a control or reference. In some embodiments, the methods and compositions described herein increase an immune response in the subject by about or at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000% or more, or by about 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 500-fold, or 1000-fold or more, relative to a control or reference (e.g., relative to a corresponding serine protease on its own), including wherein the immune response is an anticancer immune response.

[0192] In some embodiments, the methods and compositions described herein increase median survival time of a subject by 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 15 weeks, 20 weeks, 25 weeks, 30 weeks, 40 weeks, or longer. In certain embodiments, the methods and compositions described herein increase median survival time of a subject by 1 year, 2 years, 3 years, or longer. In some embodiments, the methods and pharmaceutical compositions increase progression-free survival by 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks or longer. In certain embodiments, the methods and pharmaceutical compositions described herein increase progression-free survival by 1 year, 2 years, 3 years, or longer.

[0193] In certain embodiments, the methods and compositions described herein are sufficient to result in tumor regression, for example, as indicated by a statistically significant decrease in the amount of viable tumor, for example, at least a 10%, 20%, 30%, 40%, 50% or greater decrease in tumor mass, or by altered (e.g., decreased with statistical significance) scan dimensions. In certain embodiments, the methods and compositions described herein are sufficient to result in stable disease. In certain embodiments, the methods and compositions described herein are sufficient to result in clinically relevant reduction in symptoms of a particular disease indication known to the skilled clinician. For in vivo use, as noted above, for the treatment of human or non-human mammalian disease or testing, the PDCs described herein are generally incorporated into one or more therapeutic or pharmaceutical compositions prior to administration, including veterinary therapeutic compositions.

[0194] Thus, certain embodiments relate to pharmaceutical or therapeutic compositions that comprise a PDC, as described herein. In some instances, a pharmaceutical or therapeutic composition comprises one or more of the PDCs described herein in combination with a pharmaceutically- or physiologically-acceptable carrier or excipient. Certain pharmaceutical or therapeutic compositions further comprise at least one additional agent, for example, an immunotherapy agent, a chemotherapeutic agent, a hormonal therapeutic agent, and / or a kinase inhibitor as described herein.

[0195] In particular embodiments, the pharmaceutical or therapeutic compositions comprising a PDC is substantially pure on a protein basis or a weight-weight basis, for example, the composition has a purity of at least about 80%, 85%, 90%, 95%, 98%, or 99% on a protein basis or a weight-weight basis.

[0196] In some embodiments, the PDCs described herein do not form aggregates, have a desired solubility, and / or have an immunogenicity profile that is suitable for use in humans, as known in the art. Thus, in some embodiments, a pharmaceutical or therapeutic composition comprising a PDC is substantially aggregate-free. For example, certain compositions comprise less than about 10% (on a protein basis) high molecular weight aggregated proteins, or less than about 5% high molecular weight aggregated proteins, or less than about 4% high molecular weight aggregated proteins, or less than about 3% high molecularweight aggregated proteins, or less than about 2 % high molecularweight aggregated proteins, or less than about 1% high molecularweight aggregated proteins.

[0197] In some embodiments, the PDCs are concentrated to about or at least about 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6, 0.7, 0.8, 0.9, 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, 10 mg / ml, 11, 12, 13, 14 or 15 mg / ml and are formulated for biotherapeutic uses.

[0198] To prepare a therapeutic or pharmaceutical composition, an effective or desired amount of one or more PDCs is mixed with any pharmaceutical carrier(s) or excipient known to those skilled in the art to be suitable for the particular agent and / or mode of administration. A pharmaceutical carrier may be liquid, semi-liquid or solid. Solutions or suspensions used for parenteral, intradermal, subcutaneous or topical application may include, for example, a sterile diluent (such as water), saline solution (e.g., phosphate buffered saline; PBS), fixed oil, polyethylene glycol, glycerin, propylene glycol or other synthetic solvent; antimicrobial agents (such as benzyl alcohol and methyl parabens); antioxidants (such as ascorbic acid and sodium bisulfite) and chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); buffers (such as acetates, citrates and phosphates). If administered intravenously (e.g., by IV infusion), suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, polypropylene glycol and mixtures thereof.

[0199] Administration of PDCs described herein, in pure form or in an appropriate therapeutic or pharmaceutical composition, can be carried out via any of the accepted modes of administration of agents for serving similar utilities. The therapeutic or pharmaceutical compositions can be prepared by combining a PDC-containing composition with an appropriate physiologically acceptable carrier, diluent or excipient, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. In addition, other pharmaceutically active ingredients (including other small molecules as described elsewhere herein) and / or suitable excipients such as salts, buffers and stabilizers may, but need not, be present within the composition.

[0200] Administration may be achieved by a variety of different routes, including oral, parenteral, nasal, intravenous, intradermal, intramuscular, subcutaneous, or topical. Preferred modes of administration depend upon the nature of the condition to be treated or prevented. Particular embodiments include administration by IV infusion.

[0201] Carriers can include, for example, pharmaceutically- or physiologically-acceptable carriers, excipients, or stabilizers that are non-toxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically-acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecularweight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; saltforming counterions such as sodium; and / or nonionic surfactants such as polysorbate 20 (TWEEN™) polyethylene glycol (PEG), and poloxamers (PLURONICS™), and the like. In some embodiments, one or more agents can be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate)microcapsules, respectively), in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington’s Pharmaceutical Sciences, 16th edition, Oslo, A., Ed., (1980). The particle(s) or liposomes may further comprise other therapeutic or diagnostic agents.

[0202] The precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by testing the compositions in model systems known in the art and extrapolating therefrom. Controlled clinical trials may also be performed. Dosages may also vary with the severity of the condition to be alleviated. A pharmaceutical composition is generally formulated and administered to exert a therapeutically useful effect while minimizing undesirable side effects. The composition may be administered one time, or may be divided into a number of smaller doses to be administered at intervals of time. For any particular subject, specific dosage regimens may be adjusted overtime according to the individual need.

[0203] Typical routes of administering these and related therapeutic or pharmaceutical compositions thus include, without limitation, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques. Therapeutic or pharmaceutical compositions according to certain embodiments of the present disclosure are formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a subject or patient. Compositions that will be administered to a subject or patient may take the form of one or more dosage units, where for example, a tablet may be a single dosage unit, and a container of a herein described agent in aerosol form may hold a plurality of dosage units. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000). The composition to be administered will typically contain a therapeutically effective amount of an agent described herein, for treatment of a disease or condition of interest.

[0204] Atherapeutic or pharmaceutical composition may be in the form of a solid or liquid. In one embodiment, the carrier(s) are particulate, so that the compositions are, for example, in tablet or powder form. The carrier(s) may be liquid, with the compositions being, for example, an oral oil, injectable liquid or an aerosol, which is useful in, for example, inhalatory administration. When intended for oral administration, the pharmaceutical composition is preferably in either solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid. Certain embodiments include sterile, injectable solutions.

[0205] As a solid composition for oral administration, the pharmaceutical composition may be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like. Such a solid composition will typically contain one or more inert diluents or edible carriers. In addition, one or more of the following may be present: binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, Primogel, corn starch and the like; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate or orange flavoring; and a coloring agent. When the pharmaceutical composition is in the form of a capsule, for example, a gelatin capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or oil.

[0206] The therapeutic or pharmaceutical composition may be in the form of a liquid, for example, an elixir, syrup, solution, emulsion or suspension. The liquid may be for oral administration or for delivery by injection, as two examples. When intended for oral administration, preferred composition contain, in addition to the present compounds, one or more of a sweetening agent, preservatives, dye / colorant and flavor enhancer. In a composition intended to be administered by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent may be included.

[0207] The liquid therapeutic or pharmaceutical compositions, whether they be solutions, suspensions or other like form, may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer’s solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Physiological saline is a preferred adjuvant. An injectable pharmaceutical composition is preferably sterile.

[0208] A liquid therapeutic or pharmaceutical composition intended for either parenteral or oral administration should contain an amount of an agent such that a suitable dosage will be obtained. Typically, this amount is at least 0.01 % of the agent of interest in the composition. When intended for oral administration, this amount may be varied to be between 0.1 and about 70% of the weight of the composition. Certain oral therapeutic or pharmaceutical compositions contain between about 4% and about 75% of the agent of interest. In certain embodiments, therapeutic or pharmaceutical compositions and preparations are prepared so that a parenteral dosage unit contains between 0.01 to 10% by weight of the agent of interest prior to dilution.

[0209] The therapeutic or pharmaceutical compositions may be intended for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel base. The base, for example, may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, bee wax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. Thickening agents may be present in a therapeutic or pharmaceutical composition for topical administration. If intended for transdermal administration, the composition may include a transdermal patch or iontophoresis device.

[0210] The therapeutic or pharmaceutical compositions may be intended for rectal administration, in the form, for example, of a suppository, which will melt in the rectum and release the drug. The composition for rectal administration may contain an oleaginous base as a suitable nonirritating excipient. Such bases include, without limitation, lanolin, cocoa butter, and polyethylene glycol.

[0211] The therapeutic or pharmaceutical composition may include various materials, which modify the physical form of a solid or liquid dosage unit. For example, the composition may include materials that form a coating shell around the active ingredients. The materials that form the coating shell are typically inert, and may be selected from, for example, sugar, shellac, and other enteric coating agents. Alternatively, the active ingredients may be encased in a gelatin capsule. The therapeutic or pharmaceutical compositions in solid or liquid form may include a component that binds to agent and thereby assists in the delivery of the compound. Suitable components that may act in this capacity include monoclonal or polyclonal antibodies, one or more proteins or a liposome. The therapeutic or pharmaceutical composition may consist essentially of dosage units that can be administered as an aerosol. The term aerosol is used to denote a variety of systems ranging from those of colloidal nature to systems consisting of pressurized packages. Delivery may be by a liquefied or compressed gas or by a suitable pump system that dispenses the active ingredients. Aerosols may be delivered in single phase, bi-phasic, ortri-phasic systems in order to deliver the active ingredient(s). Delivery of the aerosol includes the necessary container, activators, valves, sub-containers, and the like, which together may form a kit. One of ordinary skill in the art, without undue experimentation may determine preferred aerosols.

[0212] The compositions described herein may be prepared with carriers that protect the agents against rapid elimination from the body, such as time release formulations or coatings. Such carriers include controlled release formulations, such as, but not limited to, implants and microencapsulated delivery systems, and biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid and others known to those of ordinary skill in the art.

[0213] The therapeutic or pharmaceutical compositions may be prepared by methodology well known in the pharmaceutical art. For example, a therapeutic or pharmaceutical composition intended to be administered by injection may comprise one or more of salts, buffers and / or stabilizers, with sterile, distilled water so as to form a solution. A surfactant may be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that non-covalently interact with the agent so as to facilitate dissolution or homogeneous suspension of the agent in the aqueous delivery system.

[0214] The therapeutic or pharmaceutical compositions may be administered in a therapeutically effective amount, which will vary depending upon a variety of factors including the activity of the specific compound employed; the metabolic stability and length of action of the compound; the age, body weight, general health, sex, and diet of the subject; the mode and time of administration; the rate of excretion; the drug combination; the severity of the particular disorder or condition; and the subject undergoing therapy. In some instances, a therapeutically effective daily dose is (for a 70 kg mammal) from about 0.001 mg / kg (i.e., ~ 0.07 mg) to about 100 mg / kg (i.e., ~ 7.0 g); preferably a therapeutically effective dose is (for a 70 kg mammal) from about 0.01 mg / kg (i.e., ~ 0.7 mg) to about 50 mg / kg (i.e., ~ 3.5 g); more preferably a therapeutically effective dose is (for a 70 kg mammal) from about 1 mg / kg (i.e., ~ 70 mg) to about 25 mg / kg (i.e., ~ 1.75 g). In some embodiments, the therapeutically effective dose is administered on a weekly, bi-weekly, or monthly basis. In specific embodiments, the therapeutically effective dose is administered on a weekly, bi-weekly, or monthly basis, for example, at a dose of about 1 -10 or 1 -5 mg / kg, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg.

[0215] The combination therapies described herein may include administration of a single pharmaceutical dosage formulation, which contains a PDC and an additional therapeutic (e.g., anti-cancer agent such as an immunotherapy agent, chemotherapeutic agent, hormonal therapeutic agent, kinase inhibitor), as well as administration of compositions comprising a PDC and an additional therapeutic agent in its own separate pharmaceutical dosage formulation. For example, a PDC and an additional therapeutic agent can be administered to the subject together in a single parenteral dosage composition such as in a saline solution or other physiologically acceptable solution, or each agent administered in separate parenteral dosage formulations. Where separate dosage formulations are used, the compositions can be administered at essentially the same time, i.e., concurrently, or at separately staggered times, i.e., sequentially and in any order; combination therapy is understood to include all these regimens.

[0216] Also included are patient care kits, comprising (a) a PDC, as described herein; and optionally (b) at least one additional therapeutic agent (e.g., anti-cancer agent such as an immunotherapy agent, chemotherapeutic agent, hormonal therapeutic agent, kinase inhibitor). In certain kits, (a) and (b) are in separate therapeutic compositions. In some kits, (a) and (b) are in the same therapeutic composition.

[0217] The kits herein may also include a one or more additional therapeutic agents or other components suitable ordesired forthe indication beingtreated, orforthe desired diagnostic application. The kits herein can also include one or more syringes or other components necessary or desired to facilitate an intended mode of delivery (e.g., stents, implantable depots, etc.).

[0218] In some embodiments, a patient care kit contains separate containers, dividers, or compartments for the composition(s) and informational material(s). For example, the composition(s) can be contained in a bottle, vial, or syringe, and the informational material(s) can be contained in association with the container. In some embodiments, the separate elements of the kit are contained within a single, undivided container. For example, the composition is contained in a bottle, vial or syringe that has attached thereto the informational material in the form of a label. In some embodiments, the kit includes a plurality (e.g., a pack) of individual containers, each containing one or more unit dosage forms (e.g., a dosage form described herein) of a PDC and optionally at least one additional therapeutic agent. For example, the kit includes a plurality of syringes, ampules, foil packets, or blister packs, each containing a single unit dose of a PDC and optionally at least one additional therapeutic agent. The containers of the kits can be airtight, waterproof (e.g., impermeable to changes in moisture or evaporation), and / or light-tight.

[0219] The patient care kit optionally includes a device suitable for administration of the composition, e.g., a syringe, inhalant, dropper (e.g., eye dropper), swab (e.g., a cotton swab or wooden swab), or any such delivery device. In some embodiments, the device is an implantable device that dispenses metered doses of the agent(s). Also included are methods of providing a kit, e.g., by combiningthe components described herein.

[0220] All publications, patent applications, and issued patents cited in this specification are herein incorporated by reference as if each individual publication, patent application, or issued patent were specifically and individually indicated to be incorporated by reference.

[0221] Although the foregoing invention has 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 certain changes and modifications may be made thereto without departing from the spirit or scope of the description or appended claims. The following examples are provided by way of illustration only and not byway 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.

[0222] EXAMPLES

[0223] Example 1

[0224] Synthesis Scheme for PDC of Formula (I)

[0225] An improved scheme was designed and implemented to synthesize an amide PDC (S73 lactam) composed of a TRPV6 binding peptide (KEFLHPSKVDLPR; SEQ ID NO: 1) conjugated via a linker to a cryptophycin 52 payload.

[0226] Step 1.

[0227]

[0228] 68 mg {confirmed by HNMR.i used in next step without purification.96 g (confirmed by HNMR) used in next step without purification.20 g (confirmed by HNMR) used in n ext step without purification

[0229] Step 3.

[0230]

[0231] Step 4.

[0232]

[0233] Step 6.

[0234]

[0235] Step 7.

[0236]

[0237] Step 9.

[0238]

[0239] Step 10.

[0240]

[0241] Step 12.

[0242]

[0243] Example 2

[0244] Synthesis Scheme for PDC of Formula (II)

[0245] An improved scheme was designed to synthesize a lactone PDC (S74 lactone) composed of a TRPV6 binding peptide (KEFLHPSKVDLPR; SEQ ID NO: 1) conjugated via a linker to a cryptophycin 52 payload. Step 1.

[0246]

[0247] Step 2.

[0248]

[0249] Step 3.

[0250]

[0251] 1.5 g (89.9% purity) 1 1 g 810 mg (crude, confirmed by HNMR) 16 g (91.1% purity)

[0252] 12.2 g (crude, confirmed by HNMR) 3.3 g (96.4% purity) '

[0253] 26 g (96.2% purity). 24.8 g (crude, confirmed by HNMR)

[0254] Step 4.

[0255]

[0256] 810 mg (crude, confirmed by HNMR) 510 mg (crude, confirmed by HNMR) 12.2 g (crude, confirmed by HNMR) 8.5 g (crude, confirmed by HNMR) 24.8 g (crude, confirmed by HNMR) 16.5 g (crude, confirmed by HNMR)

[0257] Step 5.

[0258]

[0259] 510 mg (crude, confirmed by HNMR) purified and afford 730 mg (98% purity, confirmed by HNMR) 8.5 g (crude, confirmed by HNMR) purified and afford 10.9 g (99% purity, confirmed by HNMR) 16.5 g (crude, confirmed by HNMR) purified and afford 27 g (97.4% purity, confirmed by HNMR)

[0260] Step 6.

[0261]

[0262] Step 7.

[0263]

[0264] Step 8.

[0265]

[0266] Step 9.

[0267]

[0268] Step 10.

[0269]

[0270] Step 11.

[0271]

[0272] Step 12.

[0273]

[0274] Step 13.

[0275]

[0276] Step 14.

[0277]

[0278] Step 15.

[0279]

[0280] Step 16.

[0281]

[0282] Example 3

[0283] Synthesis Scheme for PDC of Formula (III)

[0284] An improved scheme was designed to synthesize a PDC composed of a TRPV6 binding peptide (CKEFLHPSKVDLPR; SEQ ID NO: 2) conjugated via a linker to a monomethyl auristatin E (MMAE) payload (SBI-1401).

[0285] Step 1.

[0286]

[0287] Example 4

[0288] In Vitro Anti-Cancer Activity Experiments were performed to asses the in vitro anticancer activity of the S73 lactam (Formula I), the S74 lactone (Formula II), and SBI-1401 (Formula III) against a panel of human cancer cell lines.

[0289] Tumor cell plating: The tumor cells were seeded at the appropriate density cells / well in 96-well flat-bottom microtitration plates (5000 cells / well). Cells were seeded in 90 pL of drug free adequate medium (see § 3.1.3.) and incubated at 37°C under 5% CO2.

[0290] Treatments: The day after seeding, cells were treated with the 3 compounds (S73 lactam, S74 lactone, and the positive control paclitaxel) at 9 doses in duplicate wells.

[0291] For Paclitaxel, the top dose tested was 1 pM (followed by 500, 100, 50, 10, 5, 1, 0.5, 0.1 nM). It was prepared as a stock solution of 1 mM in DMSO. Paclitaxel was diluted from stock solution in culture medium and 10 pL was added to the cells (as a 10x concentration). Ten pL of DMSO 0.1 % was used as negative control.

[0292] For the 2 test substances S73 lactam and S74 lactone, the top dose was 500 nM (followed by 100, 50, 10, 5, 1, 0.5, 0.1, 0.05 nM) final concentration on cells. For SBI-1401, the top dose was 12,000 followed by 2400, 480, 96, 19.2, 3.84, 0.77, 0.15, 0.03 and 0.01 nM. Test articles were prepared as a stock solution of 2 mM in Dulbecco’s Phosphate Buffered Saline (DPBS) or Phosphate Buffered Saline (PBS) and then diluted to 5 pM working solution with (DPBS or PBS) and then diluted from the working solution in culture medium, with 10 pL added to the cells (as a 10x concentration). Ten pL of DPBS or PBS was used as negative control.

[0293] Cells were incubated with treatment for 72 hours at 37°C under 5% CO2.

[0294] Viability read-out: Compound toxicity after 3 days incubation was monitored by using the CellTiter-Glo Luminescent Cell Viability Assay (Promega, Ref: G7572). 100 pLof the CellTiter-Glo reagent was added to the wells. Plates were placed on an orbital shaker for 2 minutes to induce cell lysis and incubated for 10 minutes at room temperature to stabilize luminescent signal. Then, plates were read using Envision Multilabel Reader (PerkinElmer).

[0295] The IC5o on cell viability was calculated with XLfit Software usingthe fitting model - 205 non-linear regression. The data is shown in Table E1 below.

[0296]

[0297]

[0298] Overall, the results showed that that both S73 lactam and the S74 lactone have similar potent activity with IC50S in the low single-digit nM range. Moreover, the IC5o values are better than paclitaxel, another class of tubulin inhibitor that represents a standard of care for treating ovarian cancer, esophageal cancer, breast cancer, lung cancer, Kaposi’s sarcoma, melanoma, prostate cancer, cervical cancer, bladder cancer, and pancreatic cancer, among other solid tumor cancers. SBI-1401 is less efficacious than paclitaxel and the other PDCs against PC3 with an IC5o of 32.1 nM, compared to less than 10 nM for paclitaxel and the other PDCs.

[0299] Example 5

[0300] In Vitro Stability in Serum

[0301] Experiments were performed to evaluate the in vitro stability of the S73 lactam (Formula I), the S74 lactone (Formula II), and SBI-1401 (Formula III) in human and / or mouse serum.

[0302] PDC and IS quench preparation: Stock solutions of the PDCs (S73, S74, and SBI-1401) and SOR-C27 internal standard (IS) were prepared by dissolving lyophilized PDCs or SOR-C27 in filter sterilized DPBS solution to give a 2.0 mM solution. The PDC stock solutions were aliquoted into 100 pL single use aliquots and stored at -20 °C until use.

[0303] The quench solution was prepared by adding 480 pL of 2 mM SOR-C27 (in DPBS) and 480 pL of 8.5% H3PO4 (in water) to 23,040 pL of methanol. The quench solution (40 pM SOR-C27, 0.17% H3PO4in methanol) was aliquoted into eighty 300 pLaliquots in 1.5 mL protein LoBind tubes and stored at -80 °C until day of analysis when it was moved to -20 °C.

[0304] PDC preparation and stability incubation: The human or mouse serum and 2 mM stock solutions of PDC were thawed at room temperature prior to preparing PDC serum solutions. To assess stability of either S73 orS74 in human or mouse serum, orSBI-1401 in human serum, a 150 pL of 2 mM PDC was added to 1850 pL of serum (pre-warmed to 37 °C in 2 mL protein LoBind tube) at to for a final concentration of 150 pM PDC in serum. The human serum was from a single female donor, while the mouse serum was pooled from male CD-1 mice. The serum tube(s) containing the PDC were incubated up to 3 days in a 37°C, with continuous slow agitation (150 rpm; Stuart Orbital incubator).

[0305] Sampling and Processing: AttO, 60, 120, 360, 1440, 2880 and 4320 minutes (0, 1, 2, 6, 24, 48 and 72 hours) the tube(s) was mixed for 5 sec and briefly, spun at 1000 x gto collect serum to bottom of the tube. Then 100 pL sample (n = 2) was removed and added to a tube containing 300 pL of internal control quench solution (40 pM SOR-C27 internal standard and 0.17% H3PO4 in methanol). The quenched sample was then centrifuged for 5 min at 5000 x gat room temperature. The supernatant loaded onto Nanosep 10 kDa filter tube (cat# OD010C34) and centrifuged at 14,000 xg for 20 min at room temperature. Removed 60 pL of the flow through and added to HPLC vial and immediately analyzed by high-performance liquid chromatography (HPLC). The remaining flow-through was stored at -80 °C as back-up samples for analysis.

[0306] HPLC analysis: The flow-through was analyzed using a PDC HPLC method. The PDC peak area (mAU / min) for each time-point was normalized against the internal control and analyzed in Excel as percentage of TO remaining.

[0307] As shown in Figures 1A-1 D, the S73 lactam and S74 lactone have a long half-life in human serum of 20.2 and 30.3 hours, respectively. Both PDCs also have a long half-life in CD1 mouse serum of 18.6 and 21.5 hours, respectively. Figure 1E shows the half-life (2.8 hrs) of SBI-1401 in human serum.

[0308] Example 6

[0309] In Vivo Efficacy in Xenograft Studies

[0310] Studies were performed to assess the in vivo efficacy of the S73 lactam (Formula I), the S74 lactone (Formula II), and SBI-1401 (Formula III) in male BALB / c nude mice bearing PC-3 tumors. Doses of S73 and S74 for in vivo efficacy determination were 0.5, 1.0, 1.5 and 2.0 mg / kg administered by i.v. injection. Doses of SBI-1401 at 6 and 9 mg / kg were also administered by i.v. injection.

[0311] Cancer cell line: The PC-3 cell line is a bone metastasis of a grade IV prostatic adenocarcinoma isolated from a 62-year-old male Caucasian (3).

[0312] Cell culture method: PC-3 tumor cells were grown as monolayer at 37°C in a humidified atmosphere (5% CO2, 95% air). The culture medium was RPM11640 supplemented with 1% Glutamax and 10% fetal bovine serum.

[0313] Tumor cells are adherent to plastic flasks. For passages and experimental use, tumor cells will be detached from the culture flask by a 5-minute treatment with Accutase (Gibco, A1110501) and neutralized by the addition of culture medium containing 10% FBS.

[0314] Cells were counted and viability assessed using a 0.25% trypan blue exclusion assay.

[0315] Animals: Seventy-five (75) healthy male BALB / c Nude (CAnN. Cg-Foxnl nu / Crl) mice, 5 - 6 weeks old at reception, were obtained from Charles River. Tumor induction: Tumors were induced by subcutaneous injection of 10x106PC-3 cells in 200 L of RPM11640 without phenol red into the right flank of 75 male animals. PC-3 tumor cell implantation was performed 24 to 72 hours after a whole-body irradiation with a gammasource (1.2 Gy).

[0316] Randomization: Animals were randomized based on their individual tumor volume. Randomization was performed when values reach a mean of 200 mm3with a 50 mm3std. Animals 54 / 75 were randomized into nine groups of six animals each. Homogeneity between groups was evaluated by an analysis of variance (ANOVA).

[0317] Treatment: The treatments were administered by intravenous injection (IV) into the caudal vein. The recommended pH formulation for IV administration is pH 5.0 - 8.0 (min pH 3.0). The administration volume was 10 mL / kg. Treatment started on the day of randomization (DR). There were nine (9) treatment groups:

[0318] 1- Control (4 treatments, Day 0, 4, 8 and 14 post randomization)*

[0319] 2- S73 lactam 0.5 mg / kg (4 treatments, Day 0, 4, 8 and 14 post randomization)* 3- S73 lactam 1.0 mg / kg (4 treatments, Day 0, 4, 8 and 14 post randomization)* 4- S73 lactam 1.5 mg / kg (4 treatments, Day 0, 4, 8 and 14 post randomization)* 5- S73 lactam 2.0 mg / kg (4 treatments, Day 0, 4, 8 and 14 post randomization)* 6- S74 lactone 0.5 mg / kg (4 treatments, Days 0, 7, 14 and 21 post randomization)** 7- S74 lactone 1.0 mg / kg (4 treatments, Days 0, 7, 14 and 21 post randomization)** 8- S74 lactone 1.5 mg / kg (4 treatments, Days 0, 7, 14 and 21 post randomization)** 9- S74 lactone 2.0 mg / kg (4 treatments, Days 0, 7, 14 and 21 post randomization)** * The original treatment schedule was Day 0, 4 and 8. Because the tumour growth in the control group a 4thdose was added after 6 days.

[0320] ** The original treatment schedule was Day 0, 4 and 8. Because of the toxicity observed in groups 1.5 and 2.0 mg / kg groups the dosing was changed for a weekly dosing and a 4thdose was added.

[0321] There were three (3) treatment groups for SBI-1401:

[0322] 1 - Control (3 treatments, Day 1, 5, and 9 post randomization).

[0323] 2- SBI-1401 6.0 mg / kg (3 treatments, Day 1, 5, and 9 and 14 post randomization).

[0324] 3- SBI-1401 9.0 mg / kg (3 treatments, Day 1, 5, and 9 post randomization).

[0325] Clinical monitoring: All study data was scheduled and recorded. Animal viability and behavior was observed daily. Aclinical follow-up will be performed if deemed necessary. Body weights were measured a minimum of twice a week. The length and width of the tumor was measured a minimum of twice a week with calipers. Animals were terminated 6 weeks post treatment initiation S73 and S74, 30 days post treatment initiation forSBI-1401, orfor humane reasons as per the associated animal care protocol.

[0326] Results: A summary of the treatment groups is shown below:

[0327] 1 - Control (4 treatments, Day 27, 31, 35 and 41 )

[0328] 2- S73 lactam 0.5 mg / kg (4 treatments, Day 27, 31, 35 and 41 )

[0329] 3- S73 lactam 1.0 mg / kg (4 treatments, Day 27, 31, 35 and 41 )

[0330] 4- S73 lactam 1.5 mg / kg (4 treatments, Day 27, 31, 35 and 41 )

[0331] 5- S73 lactam 2.0 mg / kg (4 treatments, Day 27, 31, 35 and 41 )

[0332] 6- S74 lactone 0.5 mg / kg (4 treatments, Day 27, 34, 41 and 48)

[0333] 7- S74 lactone 1.0 mg / kg (4 treatments, Day 27, 34, 41 and 48)

[0334] 8- S74 lactone 1.5 mg / kg (Group dead after 1 dose)

[0335] 9- S74 lactone 2.0 mg / kg (Group dead after 1 dose)

[0336] The S74 lactone was more toxic in the BALB / c nude mice compared to the CD1 mice from the maximum tolerated dose (MTD) study (not shown). All mice from the 1.5 and 2.0 mg / kg group died after only 1 dose. The 0.5 and 1.0 mg / kg treatment groups are fine. No significant toxicity signs were observed in mice treated with S73. One mouse died at day 41 in the 0.5 mg / kg group and the study coordinator mentioned that it is likely due to anesthesia side effects because no abnormal finding was seen during autopsy and was not drug-related.

[0337] Figures 2A-2B show the results for S73 and S74 from Day 69 of the study. There is a clear dose dependent response to treatment for both the S73 lactam (Fig.2A) and S74 lactone (Fig.2B). The S731.0, 1.5, and 2.0 mg / kg groups showed significant dose-dependent tumour growth inhibition and tumour growth reduction compared to tumour size at the start of treatment. For example, in the control group at day 69 the tumor size average was 1960 mm3and after treatment with S73 at 2 mg / kg the tumor size average was 65 mm3.

[0338] The S741.0 mg / kg group also showed significant tumour growth inhibition and tumour growth reduction compared to tumour size at the start of treatment. For instance, the 1.0 mg / kg group showed a significant reduction in tumor size at day 69 compared to control (142 mm3versus 1657 mm3respectively). Interestingly, the tumour growth inhibition results of the S73 and S741.0 mg / kg group are very similar until day 46. Body weight of the surviving mice from the S73 and S74 treatment groups were similar (Figures 3A-3C).

[0339] Figure 2C shows the treatment for SBI-1401 at days 1, 5, and 9 of the study. Some tumor regression was observed at 6.0 and 9.0 mg / kg. Statistical tumor reduction compared to control was observed at day 15 for 6.0 and 9.0 mg / kg. Example 7

[0340] In Vivo Maximal Tolerated Dose(MTD)

[0341] Experiments were performed to evaluate the MTD of the S73 lactam (Formula I), the S74 lactone (Formula II), and SBI-1401 (Formula III) in mice.

[0342] Animals: Fifty-five (55) healthy female CD1 (Crl: CD1 (ICR)) mice, 5 - 6 weeks old at reception, were used for the MTD Study.

[0343] Randomization: Animals were randomized based on their individual body weight.

[0344] Animals (55 / 55) were randomized into eleven groups of five animals each. Homogeneity between groups was tested by an analysis of variance (ANOVA).

[0345] Treatments: The treatments were administered by intravenous injection (IV) into the caudal vein. The recommended pH formulation for IV administration was pH 5.0 - 8.0 (min pH 3.0). The administration volume was 10 mL / kg. Treatment started on DO (day of randomization). The treatment schedule is shown in Table E2 below.

[0346]

[0347] Daily monitoring of mice for body weight.

[0348] Daily monitoring of mice for behavior and survival at pre-dose and 2-6 hours post dose. Daily scoring of animals to evaluate apparition of signs of potential peripheral neuropathy as secondary side effect (e.g., reduced exploratory mobility, abnormal posture).

[0349] Daily measurement of food intake from DO to D12 (estimation for each cage of 5 mice). Terminal necropsy and macroscopic examination on D12.

[0350] Collection from all terminated mice of organs with gross lesions. They will be placed in formalin and kept for further analysis if required. At the time of termination, collection of blood from 3 mice per group for blood biochemistry (liver and kidney enzymes including ALP, ALT and AST) as well as blood hematology (RBC, WBC, reticulocyte, neutrophil and platelet).

[0351] Results: Maximal tolerated doses (MTD) in CD1 mice injected IV Q4Dx3 with the peptide drug conjugates (PDC) S73 lactam or S74 lactone were determined based on clinical observations, weight loss, food consumption, and survival. Weight loss was consistent with the severity of the clinical symptoms for both PDCs more particularly for S74 (Figures 4A-4B). No (or minimal) weight loss was observed for most mice treated with S73 at all doses (Figure 4A).

[0352] Significant toxicity was observed at higher doses for S74 (Figure 4B).

[0353] For S74 no weight loss was observed in the 0.6 and 1.2 mg / kg groups (Figure 4B).

[0354] However, evident weight loss was observed after only one treatment for the 2.4, 3.5, and 5.0 mg / kg groups. Treatment was interrupted forthe 3.5 and 5.0 mg / kg groups afteronly one dose. Weight loss worsened after the second treatment for the 2.4 mg / kg group and the third treatment was interrupted. Upon treatment interruption, all the surviving mice immediately started to gain back weight. The average weight recovery time is not linear and was 1 day forthe 2.4 mg / kg group, 4 days forthe 3.5 mg / kg group and >7 days forthe 5 mg / kg group (Figure 4B).

[0355] The MTD for S73 is likely between 3.5 and 5 mg / kg, while the MTD for S74 is likely between 1.2 and 2.4 mg / kg. Although S73 and S74 showed very similar in vitro IC50s, serum stability, and blood biochemistry, the S74 lactone is significantly and unexpectedly more toxic than the S73 lactam.

Claims

CLAIMS1. A peptide-drug conjugate (PDC), selected from a PDC of:Formula (I)Formula (III)including pharmaceutically-acceptable salts thereof.

2. A pharmaceutical composition, comprising a pharmaceutically-acceptable carrier and a PDC of claim 1.

3. A method of treating a cancer in a subject in need thereof, comprising administeringto the subject in need thereof a pharmaceutical composition of claim 2.

4. The method of claim 3, wherein the cancer expresses or over-expresses Transient Receptor Potential (TRP) channels, subfamily vanilloid (TRPV), member 6 (TRPV6).

5. The method of claim 3, comprising:(a) determining TRPV6 levels in a subject, optionally in a sample of cancer tissue or in a cancer cell from the subject; and(b) administering the pharmaceutical composition to the subject if TRPV6 levels in the subject are increased relative to a control.

6. The method of any one of claims 3-5, wherein the cancer is selected from one or more of prostate cancer (optionally hormone-resistant prostate cancer), breast cancer, thyroid cancer, colon or colorectal cancer, ovarian cancer (optionally ovarian epithelial tumor), melanoma (e.g., metastatic melanoma, ocular melanoma), pancreatic cancer, bone cancer, small cell lung cancer, non-small cell lung cancer (NSCLC), mesothelioma, leukemia (e.g., lymphocytic leukemia, chronic myelogenous leukemia, acute myeloid leukemia, relapsed acute myeloid leukemia), lymphoma, hepatoma (hepatocellular carcinoma), hepatobiliary cancer, sarcoma, B-cell malignancy (optionally mature B-cell neoplasm), neuroepithelial tumor, glioma, glioblastoma multiforme, meningioma, pituitary adenoma, vestibular schwannoma, primary CNS lymphoma, primitive neuroectodermal tumor (medulloblastoma), kidney cancer (e.g., renal cell carcinoma optionally renal clear cell carcinoma, renal non-clear cell carcinoma), bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancers, cervical cancer, testicular cancer, adrenocortical carcinoma, seminoma, thymic epithelial tumor, plural mesothelioma, non-seminomatous germ cell tumor, and stomach cancer.

7. The method of any one of claims 3-6, comprising administering to the subject at least one additional agent selected from one or more of a cancer immunotherapy agent, a chemotherapeutic agent, a hormonal therapeutic agent, and a kinase inhibitor.

8. The method of claim 7, wherein the PDC and the at least one additional agent are administered separately, as separate compositions.

9. The method of claim 7, wherein the PDC and the at least one additional agent are administered together as part of the same therapeutic composition.

10. The method of any one of claims 7-9, wherein the cancer immunotherapy agent is selected from one or more of an immune checkpoint modulatory agent, a cancer vaccine, an oncolytic virus, a cytokine, and a cell-based immunotherapies.

11. The method of claim 10, wherein the immune checkpoint modulatory agent is an immune checkpoint antagonist / inhibitor of Programmed Death-Ligand 1 (PD-L1), Programmed Death 1 (PD-1), Programmed Death-Ligand 2 (PD-L2), 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), V-domain Ig suppressor of T cell activation (VISTA), B and T Lymphocyte Attenuator (BTLA), CD160, Herpesvirus Entry Mediator (HVEM), and T-cell immunoreceptor with Igand ITIM domains (TIGIT).

12. The method of claim 11, wherein:the antagonist is a PD-L1 and / or PD-L2 antagonist optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds thereto, atezolizumab (MPDL3280A), avelumab (MSB0010718C), and durvalumab (MEDI4736), optionally wherein the cancer is selected from one or more of colorectal cancer, melanoma, breast cancer, non-small-cell lung carcinoma, bladder cancer, and renal cell carcinoma;the antagonist is a PD-1 antagonist optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds thereto, nivolumab, pembrolizumab, MK-3475, AMP-224, AMP-514PDR001, and pidilizumab, optionally wherein the PD-1 antagonist is nivolumab and the cancer is optionally selected from one or more of Hodgkin’s lymphoma, melanoma, non-small cell lung cancer, hepatocellular carcinoma, renal cell carcinoma, and ovarian cancer;the PD-1 antagonist is pembrolizumab and the cancer is optionally selected from one or more of melanoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, and urothelial cancer;the antagonist is a CTLA-4 antagonist optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds thereto,ipilimumab, tremelimumab, optionally wherein the cancer is selected from one or more of melanoma, prostate cancer, lung cancer, and bladder cancer;the antagonist is an IDO antagonist optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds thereto, indoximod (NLG-8189), 1-methyl-tryptophan (1 MT), 0-Carboline (norharmane; 9H-pyrido[3,4-b]indole), rosmarinic acid, and epacadostat, and wherein the cancer is optionally selected from one or more of metastatic breast cancer and brain cancer optionally glioblastoma multiforme, glioma, gliosarcoma or malignant brain tumor;the antagonist is a TDO antagonist optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds thereto, 680C91, and LM10;the antagonist is a TIM-3 antagonist optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds thereto;the antagonist is a LAG-3 antagonist optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds thereto, and BMS-986016;the antagonist is a VISTA antagonist optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds thereto;the antagonist is a BTLA, CD160, and / or HVEM antagonist optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds thereto;the antagonist is a TIGIT antagonist optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds thereto.

13. The method of claim 10, wherein the immune checkpoint modulatory agent is an immune checkpoint agonist of OX40, CD40, Glucocorticoid-Induced TNFR Family Related Gene (GITR), CD137 (4-1 BB), CD27, CD28, CD226, and Herpes Virus Entry Mediator (HVEM).

14. The method of claim 15, wherein:the agonist is an OX40 agonist optionally selected from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds thereto, OX86, Fc-OX40L, and GSK3174998;the agonist is a CD40 agonist optionally selected from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds thereto, CP-870,893, dacetuzumab, Chi Lob 7 / 4, ADC-1013, and rhCD40L, and wherein the cancer is optionally selected from one or more of melanoma, pancreatic carcinoma, mesothelioma, and hematological cancers optionally lymphoma such as Non-Hodgkin’s lymphoma;the agonist is a GITR agonist optionally selected from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds thereto, INCAGN01876, DTA-1, and MEDI1873;the agonist is a CD137 agonist optionally selected from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds thereto, utomilumab, and 4-1 BB ligand;the agonist is a CD27 agonist optionally selected from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds thereto, varlilumab, and CDX-1127 (1 F5);the agonist is a CD28 agonist optionally selected from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds thereto, and TAB08; and / orthe agonist is an HVEM agonist optionally selected from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds thereto.

15. The method of claim 10, wherein the cancer vaccine is selected from one or more of Oncophage, a human papillomavirus HPV vaccine optionally Gardasil or Cervarix, a hepatitis B vaccine optionally Engerix-B, Recombivax HB, or Twinrix, and sipuleucel-T (Provenge), or comprises a cancer antigen selected from one or more of human Her2 / neu, Her1 / EGF receptor (EGFR), Her3, A33 antigen, B7H3, CD5, CD19, CD20, CD22, CD23 (IgE Receptor), MAGE-3, C242 antigen, 5T4, IL-6, IL-13, vascular endothelial growth factor VEGF (e.g., VEGF-A) VEGFR-1, VEGFR-2, CD30, CD33, CD37, CD40, CD44, CD51, CD52, CD56, CD74, CD80, CD152, CD200, CD221, CCR4, HLA-DR, CTLA-4, NPC-1 C, tenascin, vimentin, insulin-like growth factor 1 receptor (IGF-1 R), alpha-fetoprotein, insulin-like growth factor 1 (IGF-1 ), carbonic anhydrase 9 (CA-IX), carcinoembryonic antigen (CEA), guanylyl cyclase C, NY-ESO-1, p53, survivin, integrin av|33, integrin a5|31, folate receptor 1, transmembrane glycoprotein NMB, fibroblast activation protein alpha (FAP), glycoprotein 75, TAG-72, MUC1, MUC16 (or CA-125), phosphatidylserine, prostate-specific membrane antigen (PMSA), NR-LU-13 antigen, TRAIL-R1, tumor necrosis factor receptor superfamily member 10b (TNFRSF10B or TRAIL-R2), SLAM family member 7 (SLAMF7), EGP40 pancarcinoma antigen, B-cell activating factor (BAFF), platelet-derived growth factor receptor, glycoprotein EpCAM (17-1 A),Programmed Death-1, protein disulfide isomerase (PDI), Phosphatase of Regenerating Liver 3 (PRL-3), prostatic acid phosphatase, Lewis-Y antigen, GD2 (a disialoganglioside expressed on tumors of neuroectodermal origin), glypican-3 (GPC3), and mesothelin, optionally wherein the subject has or is at risk for having a cancerthat comprises the corresponding cancer antigen.

16. The method of claim 10, wherein the oncolytic virus selected from one or more of talimogene laherparepvec (T-VEC), coxsackievirus A21 (CAVATAK™), Oncorine (HI 01 ), pelareorep (REOLYSIN®), Seneca Valley virus (NTX-010), Senecavirus SW-001, ColoAdl, SEPREHVIR (HSV-1716), CGTG-102 (Ad5 / 3-D24-GMCSF), GL-ONC1, MV-NIS, and DNX-2401.

17. The method of claim 10, wherein the cytokine selected from one or more of interferon (IFN)-a, IL-2, IL-12, IL-7, IL-21, and Granulocyte-macrophage colony-stimulating factor (GM-CSF).

18. The method of claim 17, wherein the cell-based immunotherapy agent comprises cancer antigen-specific T-cells, optionally ex v / o-derived T-cells.

19. The method of claim 18, wherein the cancer antigen-specific T-cells are selected from one or more of chimeric antigen receptor (CAR)-modified T-cells, and T-cell Receptor (TCR)-modified T-cells, tumor infiltrating lymphocytes (TILs), and peptide-induced T-cells.

20. The method of any one of claims 7-9, wherein the at least one chemotherapeutic agent is selected from one or more of an alkylating agent, an anti-metabolite, a cytotoxic antibiotic, a topoisomerase inhibitor (type 1 or type II), and an anti-microtubule agent.

21. The method of claim 20, wherein:the alkylating agent is selected from one or more of nitrogen mustards (optionally mechlorethamine, cyclophosphamide, mustine, melphalan, chlorambucil, ifosfamide, and busulfan), nitrosoureas (optionally N-Nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine, and streptozotocin), tetrazines (optionally dacarbazine, mitozolomide, and temozolomide), aziridines (optionally thiotepa, mytomycin, and diaziquone (AZQ)), cisplatins and derivatives thereof (optionally carboplatinand oxaliplatin), and non-classical alkylating agents (optionally procarbazine and hexamethylmelamine);the anti-metabolite is selected from one or more of anti-folates (optionally methotrexate and pemetrexed), fluoropyrimidines (optionally 5-fluorouracil and capecitabine), deoxynucleoside analogues (optionally ancitabine, enocitabine, cytarabine, gemcitabine, decitabine, azacitidine, fludarabine, nelarabine, cladribine, clofarabine, fludarabine, and pentostatin), and thiopurines (optionally thioguanine and mercaptopurine);the cytotoxic antibiotic is selected from one or more of anthracyclines (optionally doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, aclarubicin, and mitoxantrone), bleomycins, mitomycin C, mitoxantrone, and actinomycin;the topoisomerase inhibitor is selected from one or more of camptothecin, irinotecan, topotecan, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, merbarone, and aclarubicin; and / orthe anti-microtubule agent is selected from one or more of taxanes (optionally paclitaxel and docetaxel) and vinca alkaloids (optionally vinblastine, vincristine, vindesine, vinorelbine).

22. The method of any one of claims 7-9, wherein the at least one hormonal therapeutic agent is a hormonal agonist or a hormonal antagonist.

23. The method of claim 22, wherein the hormonal agonist is selected from one or more of a progestogen (progestin), a corticosteroid (optionally prednisolone, methylprednisolone, or dexamethasone), insulin like growth factors, VEGF derived angiogenic and lymphangiogenic factors (optionally VEGF-A, VEGF-A145, VEGF-A165, VEGF-C, VEGF-D, PIGF-2), fibroblast growth factor (FGF), galectin, hepatocyte growth factor (HGF), platelet derived growth factor (PDGF), transforming growth factor (TGF)-beta, an androgen, an estrogen, and a somatostatin analog.

24. The method of claim 22, wherein the hormonal antagonist is selected from one or more of a hormone synthesis inhibitor, optionally an aromatase inhibitor or a gonadotropinreleasing hormone (GnRH) or an analog thereof, and a hormone receptor antagonist, optionally a selective estrogen receptor modulator (SERM) or an anti-androgen, or an antibody directed against a hormonal receptor, optionally cixutumumab, dalotuzumab, figitumumab, ganitumab, istiratumab, robatumumab, alacizumab pegol, bevacizumab, icrucumab, ramucirumab,fresolimumab, metelimumab, naxitamab, cetuximab, depatuxizumab mafodotin, futuximab, imgatuzumab, laprituximab emtansine, matuzumab, modotuximab, necitumumab, nimotuzumab, panitumumab, tomuzotuximab, zalutumumab, aprutumab ixadotin, bemarituzumab, olaratumab, ortovetumab.

25. The method of any one of claims 7-9, wherein the kinase inhibitor is selected from one or more of adavosertib, afanitib, aflibercept, axitinib, bevacizumab, bosutinib, cabozantinib, cetuximab, cobimetinib, crizotinib, dasatinib, entrectinib, erdafitinib, erlotinib, fostamitinib, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, mubritinib, nilotinib, panitumumab, pazopanib, pegaptanib, ponatinib, ranibizumab, regorafenib, ruxolitinib, sorafenib, sunitinib, SU6656, tofacitinib, trastuzumab, vandetanib, and vemuafenib.

26. A process of manufacturing a PDC of Formula (I)comprising:i) reacting a compound of Formula 1-1 with a compound of Formula I-2 in the presence of a coupling reagent, thereby producing a compound of Formula I-3ii) reacting a compound of Formula I-3 in the presence of a deprotecting reagent, thereby producing a compound of Formula I-4wherein:PG1is an oxygen protecting group; andPG2is a nitrogen protecting group.

27. The process of claim 26, wherein the coupling reagent is trichlorobenzoyl chloride.

28. The process of claim 26, wherein the deprotecting reagent is an organic base.

29. The process of claim 26, wherein the deprotecting reagent is diethylamine.

30. The process of claim 26, wherein PG1is para-methoxybenzyl (PMB).

31. The process of claim 26, wherein PG2is fluorenylmethyloxycarbonyl (Fmoc).

32. A process of manufacturing a PDC of Formula (II)comprising:i) reacting a compound of Formula 11-1 with a compound that provides an oxygen protecting group, thereby producing a compound of Formula 11-2, andii) reacting a compound of Formula 11-2 with a compound of Formula 11-3 in the presence of a coupling reagent, thereby producing a compound of Formula 11-4wherein:PG3is an oxygen protecting group; andPG4is a nitrogen protecting group.

33. The process of claim 26, wherein the compound that provides an oxygen protecting group is benzyl bromide.

34. The process of claim 26, wherein the coupling reagent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

35. The process of claim 26, wherein PG3is benzyl.

36. The process of claim 26, wherein PG4is tert-butyloxycarbonyl.

37. A process of manufacturing a PDC of Formula (III), or a pharmaceutically-acceptable salt thereof, comprising:i) reacting a compound of Formula 111-1 with a compound of Formula HI-2, thereby producing a compound of Formula HI-3ii) reacting the compound of Formula HI-3 with a peptide of Formula HI-4, thereby producing a conjugate of Formula III, wherein the peptide is H-Cys-Lys-Glu-Phe-Leu-His-Pro- Ser-Lys- Val-Asp- Leu-Pro-Arg-OH (SEQ ID NO: 2)38. A PDC manufactured according to the method of any one of claims 26-37.

39. A pharmaceutical composition, comprising a pharmaceutically-acceptable carrier and a PDC manufactured according to anyone of claims 26-37.

40. A method of treating a cancer in a subject in need thereof, comprising administeringto the subject a pharmaceutical composition of claim 39.

41. The method of claim 40, wherein the cancer expresses or over-expresses Transient Receptor Potential (TRP) channels, subfamily vanilloid (TRPV), member 6 (TRPV6).

42. The method of claim 41, comprising:(a) determining TRPV6 levels in a subject, optionally in a sample of cancer tissue or in a cancer cell from the subject; and(b) administering the pharmaceutical composition to the subject if TRPV6 levels in the subject are increased relative to a control.

43. The method of any one of claims 40-42, wherein the cancer is selected from one or more of prostate cancer (optionally hormone-resistant prostate cancer), breast cancer, thyroid cancer, colon or colorectal cancer, ovarian cancer (optionally ovarian epithelial tumor),melanoma (e.g., metastatic melanoma, ocular melanoma), pancreatic cancer, bone cancer, small cell lung cancer, non-small cell lung cancer (NSCLC), mesothelioma, leukemia (e.g., lymphocytic leukemia, chronic myelogenous leukemia, acute myeloid leukemia, relapsed acute myeloid leukemia), lymphoma, hepatoma (hepatocellular carcinoma), hepatobiliary cancer, sarcoma, B-cell malignancy (optionally mature B-cell neoplasm), neuroepithelial tumor, glioma, glioblastoma multiforme, meningioma, pituitary adenoma, vestibular schwannoma, primary CNS lymphoma, primitive neuroectodermal tumor (medulloblastoma), kidney cancer (e.g., renal cell carcinoma optionally renal clear cell carcinoma, renal non-clear cell carcinoma), bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancers, cervical cancer, testicular cancer, adrenocortical carcinoma, seminoma, thymic epithelial tumor, plural mesothelioma, non-seminomatous germ cell tumor, and stomach cancer.

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