Methods of treating, ameliorating or preventing cancer

Abstract

Described herein is a method of treating, ameliorating and / or preventing a cancer in a subject in need thereof. The method comprises administering to the subject an effective amount of a compound that down regulates amphiregulin (AREG); a platinum-based chemotherapy agent; and a microtubule-stabilizing chemotherapy agent. Also described herein is a kit for performing the method.

Description

[0001] METHODS OF TREATING, AMELIORATING OR PREVENTING CANCER

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] The present application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 647,883, filed July 24, 2024, which is incorporated herein by reference in its entirety.

[0004] SEQUENCE LISTING

[0005] The XML file named "371288-3009W01(00094)_Seq Listing.xml" created on June 23, 2025, comprising 2,895 bytes, is hereby incorporated by reference in its entirety.

[0006] BACKGROUND

[0007] High-grade serous ovarian cancer (HGSOC) is a lethal gynecologic malignancy in which chemoresistant recurrence rates remain high. Patients with HGSOC often respond well to chemotherapy. However, cancer recurrence after remission is high in HGSOC patients. HGSOC patients have demonstrated overall low response rates to clinically available immunotherapies

[0008] Therefore, there is a need for effective treatment for HGSOC. The present study addresses this need.

[0009] SUMMARY

[0010] In some aspects, the present invention is directed to the following non-limiting embodiments:

[0011] Method of treating, ameliorating and / or preventing cancer

[0012] In some aspects, the present invention is directed to a method of treating, ameliorating and / or preventing a cancer in a subject in need thereof.

[0013] In some embodiments, the method comprising administering to the subject an effective amount of: a compound that down regulates amphiregulin (AREG); a platinum-based chemotherapy agent; and a microtubule-stabilizing chemotherapy agent.

[0014] In some embodiments, the compound that down regulates AREG downregulates the expression level or the activity of AREG. In some embodiments, the compound that down regulates AREG is a small molecule inhibitor of AREG.

[0015] In some embodiments, the compound that down regulates AREG is a protein inhibitor of AREG.

[0016] In some embodiments, the compound that down regulates AREG is a nucleic acid that downregulates the expression level and / or activity of AREG by RNA interference, and / or an expression vector expressing the nucleic acid that downregulates the expression level and / or activity of AREG by RNA interference.

[0017] In some embodiments, the compound that down regulates AREG is a ribozyme that downregulates the expression level and / or activity of AREG, and / or an expression vector expressing the ribozyme.

[0018] In some embodiments, the compound that down regulates AREG is an expression vector comprising an expression cassette, and the expression cassette expresses CRISPR components that downregulate the expression level and / or activity of AREG by CRISPR knockout or CRISPR knockdown.

[0019] In some embodiments, the compound that down regulates AREG is a trans-dominant negative mutant protein of AREG, and / or an expression vector that expresses the trans-dominant negative mutant protein of AREG.

[0020] In some embodiments, the compound that down regulates AREG is a neutralizing antibody against AREG.

[0021] In some embodiments, the platinum-based chemotherapy agent is at least one selected from the group consisting of: cisplatin, carboplatin, heptaplatin, iproplatin, lobaplatin, miriplatin, nedaplatin, oxaliplatin, picoplatin, phenanthriplatin, PN149, pyriplatin, satraplatin, tetraplatin, and triplatin tetranitrate.

[0022] In some embodiments, the microtubule-stabilizing chemotherapy agent is at least one selected from the group consisting of an epothilone chemotherapy agent, a laulimalide chemotherapy agent, peloruside A, and a taxane chemotherapy agent.

[0023] In some embodiments, the microtubule-stabilizing chemotherapy agent is BMS-310705, cabazitaxel, docetaxel, epothilone A, epothilone B, epothilone C, epothilone D, epothilone E, epothilone F, fludelone (KOS-1584), ixabepilone (BMS-247550), paclitaxel, an antibody drug conjugate (ADC) comprising ravtansine (DM4), or an ADC comprising monomethyl auristatin. In some embodiments, the compound that down regulates AREG is a neutralizing antibody against AREG; the platinum-based chemotherapy agent is carboplatin; and the microtubule-stabilizing chemotherapy agent is paclitaxel.

[0024] In some embodiments, apart from the compound that down regulates amphiregulin, the platinum-based chemotherapy agent, and the microtubule-stabilizing chemotherapy agent, the subject is not administered any additional active compounds for treating, ameliorating, and / or preventing the cancer.

[0025] In some embodiments, the administration comprises a first phase administration and a second phase administration after the first phase administration.

[0026] In some embodiments, the subject is administered the compound that down regulates AREG, the platinum-based chemotherapy agent, and the microtubule-stabilizing chemotherapy agent in the first administration phase.

[0027] In some embodiments, the subject is administered the compound that down regulates AREG but not the platinum-based chemotherapy agent, or the microtubule-stabilizing chemotherapy agent in the second administration phase.

[0028] In some embodiments, the cancer is an ovarian cancer, optionally a serous ovarian cancer; optionally a high-grade serous ovarian cancer (HGSOC).

[0029] In some embodiments, the cancer is a breast a cancer, optionally a triple negative breast cancer (TNBC).

[0030] In some embodiments, the cancer is an endometrial cancer, optionally a serous endometrial cancer, optionally a high-grade serous endometrial cancer.

[0031] In some embodiments, the cancer is a carcinosarcoma.

[0032] Kit

[0033] In some aspects, the present invention is directed to kit.

[0034] In some embodiments, the kit comprises: a compound that down regulates amphiregulin (AREG); a platinum-based chemotherapy agent; a microtubule-stabilizing chemotherapy agent; and an instructional manual.

[0035] In some embodiments, the instruction manual instructs that the compound that down regulates AREG, the platinum-based chemotherapy agent and the microtubule-stabilizing chemotherapy agent are to be administered to a subject in need thereof to treat, ameliorate and / or prevent a cancer.

[0036] In some embodiments, the compound that down regulates AREG downregulates the expression level or the activity of AREG.

[0037] In some embodiments, the compound that down regulates AREG is a small molecule inhibitor of AREG.

[0038] In some embodiments, the compound that down regulates AREG is a protein inhibitor of AREG.

[0039] In some embodiments, the compound that down regulates AREG is a nucleic acid that downregulates the expression level and / or activity of AREG by RNA interference, and / or an expression vector expressing the nucleic acid that downregulates the expression level and / or activity of AREG by RNA interference.

[0040] In some embodiments, the compound that down regulates AREG is a ribozyme that downregulates the expression level and / or activity of AREG, and / or an expression vector expressing the ribozyme.

[0041] In some embodiments, the compound that down regulates AREG is an expression vector comprising an expression cassette, and the expression cassette expresses CRISPR components that downregulate the expression level and / or activity of AREG by CRISPR knockout or CRISPR knockdown.

[0042] In some embodiments, the compound that down regulates AREG is a trans-dominant negative mutant protein of AREG, and / or an expression vector that expresses the trans-dominant negative mutant protein of AREG.

[0043] In some embodiments, the compound that down regulates AREG is a neutralizing antibody against AREG.

[0044] In some embodiments, the platinum-based chemotherapy agent is at least one selected from the group consisting of: cisplatin, carboplatin, heptaplatin, iproplatin, lobaplatin, miriplatin, nedaplatin, oxaliplatin, picoplatin, phenanthriplatin, PN149, pyriplatin, satraplatin, tetraplatin, and triplatin tetranitrate.

[0045] In some embodiments, the microtubule-stabilizing chemotherapy agent is at least one selected from the group consisting of an epothilone chemotherapy agent, a laulimalide chemotherapy agent, peloruside A, and a taxane chemotherapy agent. In some embodiments, the microtubule-stabilizing chemotherapy agent is BMS-310705, cabazitaxel, docetaxel, epothilone A, epothilone B, epothilone C, epothilone D, epothilone E, epothilone F, fludelone (KOS-1584), ixabepilone (BMS-247550), paclitaxel, an antibody drug conjugate (ADC) comprising ravtansine (DM4), or an ADC comprising monomethyl auristatin.

[0046] In some embodiments, the compound that down regulates AREG is a neutralizing antibody against AREG; the platinum-based chemotherapy agent is carboplatin; and the microtubule-stabilizing chemotherapy agent is paclitaxel.

[0047] In some embodiments, apart from the compound that down regulates amphiregulin, the platinum-based chemotherapy agent, and the microtubule-stabilizing chemotherapy agent, the kit does not comprise another therapeutic agent for treating, ameliorating, and / or preventing the cancer.

[0048] In some embodiments, the instructional manual instructs that the administration comprises a first phase administration and a second phase administration after the first phase administration.

[0049] In some embodiments, the instructional manual instructs that the subject is to be administered the compound that down regulates AREG, the platinum-based chemotherapy agent, and the microtubule-stabilizing chemotherapy agent in the first administration phase.

[0050] In some embodiments, the instructional manual instructs that the subject is to be administered the compound that down regulates AREG but not the platinum-based chemotherapy agent, or the microtubule-stabilizing chemotherapy agent in the second administration phase.

[0051] In some embodiments, the cancer is an ovarian cancer, optionally a serous ovarian cancer; optionally a high-grade serous ovarian cancer (HGSOC).

[0052] In some embodiments, the cancer is a breast a cancer, optionally a triple negative breast cancer (TNBC).

[0053] In some embodiments, the cancer is an endometrial cancer, optionally a serous endometrial cancer, optionally a high-grade serous endometrial cancer.

[0054] In some embodiments, the cancer is a carcinosarcoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] The following detailed description of exemplary embodiments will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating, nonlimiting embodiments are shown in the drawings. It should be understood, however, that the instant specification is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0056] Figs. 1A-1D illustrate certain aspects of the bioinformatic analysis of AREG in ovarian cancer, in accordance with some embodiments. TCGA Ovarian Cancer Nature 2011 cohort was employed to compare (Fig. 1A) mRNA expression of AREG in patients with platinum sensitive (?? = 197) or resistant (?? = 90) disease. The TCGA Ovarian Cancer Firehose Legacy was used to examine AREG mRNA expression stratified by upper and lower quartile (Fig. IB) mutation count ( i = 41, upper quartile, n = 52, lower quartile) and (Fig. 1C) tumor mutational burden (n = 79, for both upper and lower quartile). Fig. ID: TIDE cox proportional hazards model analysis of OS using TCGA ovarian cohort data demonstrating z-score of interaction effect between AREG and CTLs. *p < 0.05, **p < 0.005, as indicated. TCGA, The Cancer Genome Atlas; TIDE, Tumor Immune Dysfunction Exclusion; CTLs, Cytotoxic T lymphocytes; OS, overall survival.

[0057] Figs. 2A-2C demonstrate that rAREG drives tumor intrinsic immune changes that promote immune evasion and ovarian pathogenesis. Fig. 2A: Volcano plot demonstrating differential gene expression in OVCAR8 cells stimulated with 200 ng / mL of rAREG relative to BSA control, measured by NanoString Human PanCancer 10360. Fig. 2B: Top differentially expressed genes in OVCAR8 rAREG treated cells relative to BSA control, with Benjamini- Hochberg adjusted ^-values listed. Fig. 2C: Gene set analysis with top pathway changes and significance scores listed in rAREG treated AREG cells relative to BSA control.

[0058] Figs. 3A-3H illustrate certain aspects of the qPCR analysis of rAREG stimulated PEA1 cells, in accordance with some embodiments. Fig. 3A: CXCL1, Fig. 3B: DUSP5, Fig. 3C: IL-11 Fig. 3D: CXCL2, and Fig. 3E: IL-6 mRNA levels in PEA1 cells stimulated with 200 ng / mL rAREG for 1 h and analyzed via qPCR. Spearman Rank Correlation analysis of mRNA expression (RNA Seq V2 RSEM) of AREG with (Fig. 3F) DUSP5, (Fig. 3GG) CXCL2, and (Fig. 3H) IL-6 using TCGA-OV Firehose Legacy cohort (n = 307). Error bars represent standard deviation of > 3 biological replicates. *p < 0.05 as indicated. TCGA, The Cancer Genome Atlas. Figs. 4A-4E demonstrate that AREG exposure leads to prominent upregulation of STAT3 signaling in HGSOC cells, in accordance with some embodiments. Fig. 4A: Phospho-proteome profder analysis of fold-change expression of phosphor-proteins in rAREG (200 ng / mL) treated OVCAR8 and PEA1 cells relative to BSA control. Fig. 4B: Western blot analysis of rAREG (200 ng / mL) exposed 0VCAR8WT and PEA1 cells of STAT3, pSTAT3, PD-L1, with respective GADPH loading controls at various indicated timepoints. ELISA levels of IL-6 in conditioned media of (Fig. 4C) OVCAR8 and (Fig. 4D) PEA1 cells following 200 ng / mL of rAREG exposure at 2 and 4 h or BSA control. Fig. 4E: Western blot analysis of downstream EGFR cell growth pathways p-ERK / ERK and pAKT / AKT with respective GAPDH loading controls in OVCAR8 and PEA1 cells following rAREG treatment at various indicated timepoints. *p < 0.05, as indicated. ELISA, enzyme-linked immunosorbent assay.

[0059] Figs. 5A-5D demonstrate that AREG compromises PBMC cytotoxicity, in accordance with some embodiments. Cell viability analysis (Fig. 5AA) OVCAR8 and (Fig. 5B) PEA1 cells following 24-h co-cultured with PBMCs+ BSA control or PBMCs+ 200 ng / mL of rAREG. qPCR analysis of (Fig. 5C) IFNy and (Fig. 5D) IL-2 in PBMCs treated with BSA control, or rAREG for 2 h. Error bars represent standard deviation of > 3 biological replicates. *p < 0.05, as indicated, PBMCs, peripheral blood mononuclear cells.

[0060] Figs. 6A-6G illustrate certain aspects of the Multiplex cytokine and chemokine analysis of C57BL / 6 ID8p53 mouse ascites and serum following in vivo rAREG exposure, in accordance with some embodiments. Concentrations of IL-2 in (Fig. 6A) serum and (Fig. 6B) ascites of mice treated with saline control (n = 5) or 400 ug / kg of rAREG (n = 5). IL-5 levels in the ascites (Figs. 6C-6D) serum in mice exposed to saline control or rAREG. Fig. 6E: Fractalkine ascites, Fig. 6F: IL-11 ascites, and Fig. 6G: IL-20 serum levels in saline and rAREG treated mice. *p < 0.05 as indicated.

[0061] Figs. 7A-7B demonstrate that AREG exposure in vivo leads to a reduction in intratumoral cytotoxic CD8+T cells, in accordance with some embodiments. Fig. 7A: Representative images and associated (Fig. 7B) Fluorescent Immunohistochemistry analysis of intratumoral CD8+T cells levels denoted by the number of positive cells per field in saline control (n = 3) and 400 pg / kg rAREG exposed (n = 4) mice. *p < 0.05, as indicated.

[0062] Figs. 8A-8B illustrate certain aspects of the intratumoral analysis of CD4+T cells following in vivo rAREG exposure, in accordance with some embodiments. Fig. 8A: Representative images and associated (Fig. 8B) Fluorescent Immunohistochemistry analysis of intratumoral CD4+T cells (number of positive cells per field) in saline control (n = 3) and 400 pg / kg rAREG exposed (n = 4) mice, ns, non-significant.

[0063] Figs. 9A-9B demonstrate that AREG exposure leads to an upregulation of intratumoral PD-L1 expression, in accordance with some embodiments. Fluorescent Immunohistochemistry analysis of (Fig. 9A) of PD-L1 in saline control (n = 3) and 400 pg / kg rAREG exposed (n = 3) mice, demonstrated by mean intensity (pixels) with (Fig. 9B) representative images. *p < 0.05, as indicated.

[0064] Figs. 10A-10B demonstrate that targeting AREG in combination with carboplatin synergistically reduces HGSOC cell viability, in accordance with some embodiments. Cell viability analysis of (Fig. 10A) OVCAR8 and (Fig. 10B) PEA2 cells pre-treated with carboplatin (300 pM for OVCAR8 cells, 400 pm for PEA2 cells) for 24 h and then stimulated with 30 pM of AREG neutralizing antibody or corresponding IgG control for an additional 48 h. Error bars represent standard deviation of > 3 biological replicates. **p < 0.005, ***p < 0.0005, ****p < 0.00005, as indicated. AREG nab: AREG neutralizing antibody.

[0065] Figs. 11A-1 ID demonstrate that AREG is not associated with HGSOC survival outcomes, in accordance with some embodiments. Kaplan-Meier Plotter analysis using TCGA analysis and Gene Expression Omnibus Series data in patients with stage 3 and 4 ovarian cancer, demonstrating AREG’s association with PFS stratified by (Fig. 11 A) lower quartile and (Fig. 1 IB) upper quartile AREG levels. AREG’s association with OS stratified by (Fig. 11C) lower quartile and (Fig. 1 ID) upper quartile AREG levels. TCGA, The Cancer Genome Atlas; PFS, progression-free survival; OS, overall -survival.

[0066] Figs. 12A-12E illustrate certain aspects of the PEA1 qPCR analysis at 2 h rAREG exposure timepoint, in accordance with some embodiments. qPCR analysis CXCL1 (Fig. 12B) DUSP5 (Fig. 12C) IL-11 (Fig. 12D) CXCL2 (Fig. 12E) IL-6 of PEA1 cells stimulated with 200 ng / ml of rAREG or BSA control for 1 h. ns, non-significant.

[0067] Fig. 13 demonstrate that small molecule JAK / STAT inhibition does not affect AREG levels in HGSOC cells, in accordance with some embodiments. Western blot analysis of AREG levels and corresponding GAPDH loading control in PEA1 and OVCAR8 cells following 10 pm of ruxolitinib treatment for 48 h. Fig. 14 illustrate certain aspects of the qPCR analysis of GRZB in PBMCs. ns, nonsignificant; PBMC, peripheral blood mononuclear cell.

[0068] Figs. 15A-15B demonstrate the synergistic effects of combining AREG inhibition with microtubule stabilization agent and platinum-based chemotherapy agent in treating ovarian cancer in a mouse model, in accordance with some embodiments. C57 / BL6 mice were administered intraperitoneally with ID8p53’ ‘ cells to form tumor. 20 days post inoculation, the animals received mock treatment, carboplatin / paclitaxel treatment, AREG neutralizing antibody treatment, or carboplatin / paclitaxel / AREG neutralizing antibody combination treatment. Fig. 15 A: tumor weight as percentages of total body weight 7-days post treatment. Fig. 15B: tumor weight 7-days post treatment.

[0069] Figs. 16A-16C illustrate certain aspects of the combinatorial AREG and chemotherapy survival study, in accordance with some embodiments. Fig. 16A: Kaplan-Meier survival analysis of mice across all four treatment groups revealed mice treated with a dual AREG and chemotherapy blockade experienced an improved survival. Fig. 16B: Ascites volume was significantly lower in mice treated with a dual AREG and chemotherapy blockade compared to IgG control and IgG+carbo / pax. Fig. 16C: No significant differences in tumor weights were identified, however median tumor weights in the dual AREG and chemotherapy weights were the lowest. Ordinary one-way ANOVA with Tukey’s multiple comparisons test were performed for these analyses. *p<0.05; ns, non-significant.

[0070] Figs. 17A-17D illustrate certain aspects of the circulating immune changes resulting from dual AREG and chemotherapy blockade, in accordance with some embodiments. Fig. 17A: Serum eotaxin levels across all treatment groups. Spearman’s rank correlation analysis between tumor size and (Fig. 17B) IL-4, (Fig. 17C) IL-12p40, and (Fig. 17D) Eotaxin. *p<0.05.

[0071] Figs. 18A-18B illustrate certain aspects of the fluorescent IHC analysis of tryptase and CP A3 in AREG nab treated tumors, in accordance with some embodiments. Fig. 18A: Tryptase+CPA3+ mast cells counts in combinatorial AREG nab and chemotherapy treated tumors. Fig. 18B: Representative images for treatment group. (n=3, mice per group)*p<0.05, *ns, non-significant.

[0072] Figs. 19A-19D illustrate certain aspects of the expanded 10 day on-treatment in vivo AREG nab study in a ID8p53' ' HGSOC model, in accordance with some embodiments. Fig. 19A: Percent (%) tumor weight to body weight of mice treated with carboplatin / paclitaxel (c / p) , AREG nab, or IgG control. Organ weights (g) of (Fig. 19B) livers, (Fig. 19C) kidneys, and (Fig. 19D) spleens were all non-significantly different across the four treatment groups.

[0073] Fig. 20 illustrates certain aspects of the cell viability analysis example of HGSOC patient spheroid, in accordance with some embodiments. HGSOC patient spheroid treated alone pretreated with 300pM of carboplatin and corresponding DMSO control, followed by a 48-hour treatment of 30pM of AREG nab. or corresponding IgG control. p<0.05.

[0074] Fig. 21 demonstrates that dual AREG and chemotherapy blockade is superior compared to dual EGFR and chemotherapy blockade, in accordance with some embodiments. HGSOC chemoresistant PEA2 cells treated in combination with an AREG nab (30pM) and carboplatin (300pM) demonstrate a significant reduction in cell viability, compared to carboplatin and IgG (30 pM). EGFR inhibition (gefitinib, 5pM) alone did not produce a significant difference in tumor cell death compared to EGFR inhibition in combination with carboplatin. *p<0.05, *ns, non-significant.

[0075] Figs. 22A-22B illustrate certain aspects of the AREG expression in endometrial cancer, in accordance with some embodiments. qPCR analysis of human endometrial cell lines (Figs. 22A) RL95 and (Figs. 22B) KLE stimulated with DMSO control or chemotherapy for 48 hours.

[0076] Figs. 23A-23B illustrate certain aspects of the AREG expression in metastatic TNBC, in accordance with some embodiments. Figs. 23A-23B: Representative images (Fig. 23 A) and mean intensity analysis (Fig. 23B) of AREG expression in early (E0771) and metastatic (4T1) mouse tumors. Fig. 23C: qPCR analysis of AREG expression in mTNBC 4T1 cells treated with chemotherapy for 48 hours.

[0077] Figs. 24A-24B illustrate certain aspects of the AREG expression in early TNBC, in accordance with some embodiments. Figs. 24A-24B: Representative images (Fig. 24A) and integrated optical density analysis (Fig. 24B) of AREG expression in early E0771 mouse tumors treated with carboplatin and paclitaxel once weekly for two weeks.

[0078] DETAILED DESCRIPTION

[0079] The following disclosure provides many different embodiments, or examples, for implementing different features of the provided subject matter. Specific examples of components and arrangements are described below to simplify the present disclosure. These are, of course, merely examples and are not intended to be limiting. For example, the formation of a first feature over or on a second feature in the description that follows may include embodiments in which the first and second features are formed in direct contact, and may also include embodiments in which additional features may be formed between the first and second features, such that the first and second features may not be in direct contact. In addition, the present disclosure may repeat reference numerals and / or letters in the various examples. This repetition is for the purpose of simplicity and clarity and does not in itself dictate a relationship between the various embodiments and / or configurations discussed.

[0080] In the study described herein (“the present study”), it was discovered that, although the inhibition of amphiregulin (AREG) in a mouse model of high grade serous ovarian cancer (HGSOC) did not achieve desirable anticancer efficacy, the combination of the AREG inhibition with an exemplary platinum-based chemotherapy agent (carboplatin) and an exemplary microtubule stabilizing chemotherapy agent (paclitaxel) achieved significant reduction of tumor growth (see e.g., Figs. 15A-15B), and increased survival (see e.g., Fig. 16A), without significantly affecting the organs in the treated animal subjects (see e.g., Figs. 19B-19D). Furthermore, this combination appears to be more effective than the dual EGFR and chemotherapy blockade (e.g., gefitinib and carboplatin) (see e.g., Fig. 21).

[0081] Accordingly, in some aspects, the present invention is directed to a method and a kit for treating, ameliorating, and / or preventing cancer in a subject in need thereof.

[0082] Definitions

[0083] As used herein, each of the following terms has the meaning associated with it in this section. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Generally, the nomenclature used herein and the laboratory procedures in animal pharmacology, pharmaceutical science, peptide chemistry, and organic chemistry are those well-known and commonly employed in the art. It should be understood that the order of steps or order for performing certain actions is immaterial, so long as the present teachings remain operable. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting; information that is relevant to a section heading may occur within or outside of that particular section. All publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference.

[0084] In the application, where an element or component is said to be included in and / or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components and can be selected from a group consisting of two or more of the recited elements or components.

[0085] In the methods described herein, the acts can be carried out in any order, except when a temporal or operational sequence is explicitly recited. Furthermore, specified acts can be carried out concurrently unless explicit claim language recites that they be carried out separately. For example, a claimed act of doing X and a claimed act of doing Y can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process.

[0086] In this document, the terms "a," "an," or "the" are used to include one or more than one unless the context clearly dictates otherwise. The term "or" is used to refer to a nonexclusive "or" unless otherwise indicated. The statement "at least one of A and B" or "at least one of A or B" has the same meaning as "A, B, or A and B."

[0087] "About" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, in certain embodiments ±5%, in certain embodiments ±1%, in certain embodiments ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0088] A "disease" is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate.

[0089] A "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.

[0090] A disease or disorder is "alleviated" if the severity of a symptom of the disease or disorder, the frequency with which such a symptom is experienced by a patient, or both, is reduced. In one aspect, the terms "co-administered" and "co-admini strati on" as relating to a subject refer to administering to the subject a compound and / or composition of the disclosure along with a compound and / or composition that may also treat or prevent a disease or disorder contemplated herein. In certain embodiments, the co-administered compounds and / or compositions are administered separately, or in any kind of combination as part of a single therapeutic approach. The co-administered compound and / or composition may be formulated in any kind of combinations as mixtures of solids and liquids under a variety of solid, gel, and liquid formulations, and as a solution.

[0091] As used herein, the term "pharmaceutical composition" or "composition" refers to a mixture of at least one compound useful within the disclosure with a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates administration of the compound to a patient. Multiple techniques of administering a compound exist in the art including, but not limited to, subcutaneous, intravenous, oral, aerosol, inhalational, rectal, vaginal, transdermal, intranasal, buccal, sublingual, parenteral, intrathecal, intragastrical, ophthalmic, pulmonary, and topical administration.

[0092] As used herein, the term "pharmaceutically acceptable" refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively non-toxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0093] As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the disclosure within or to the patient such that it may perform its intended function. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation, including the compound useful within the disclosure, and not injurious to the patient. Some examples of materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives. As used herein, "pharmaceutically acceptable carrier" also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound useful within the disclosure, and are physiologically acceptable to the patient. The "pharmaceutically acceptable carrier" may further include a pharmaceutically acceptable salt of the compound useful within the disclosure. Other additional ingredients that may be included in the pharmaceutical compositions used in the practice of the disclosure are known in the art and described, for example in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.

[0094] As used herein, the language "pharmaceutically acceptable salt" refers to a salt of the administered compound prepared from pharmaceutically acceptable non-toxic acids and bases, including inorganic acids, inorganic bases, organic acids, inorganic bases, solvates, hydrates, and clathrates thereof.

[0095] As used herein, a "pharmaceutically effective amount," "therapeutically effective amount," or "effective amount" of a compound is that amount of compound that is sufficient to provide a beneficial effect to the subject to which the compound is administered.

[0096] As used herein, the term "prevent" or "prevention" means no disorder or disease development if none had occurred, or no further disorder or disease development if there had already been development of the disorder or disease. Also considered is the ability of one to prevent some or all of the symptoms associated with the disorder or disease.

[0097] As used herein, the terms "subject" and "individual" and "patient" can be used interchangeably and may refer to a human or non-human mammal or a bird. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals. In certain embodiments, the subject is human.

[0098] As used herein, the term "treatment" or "treating" is defined as the application or administration of a therapeutic agent, i.e., a compound useful within the disclosure (alone or in combination with another pharmaceutical agent), to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient (e. , for diagnosis or ex vivo applications), who has a disease or disorder and / or a symptom of a disease or disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease or disorder and / or the symptoms of the disease or disorder. Such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics.

[0099] Method of Treating, Ameliorating and / or Preventing Cancer In some aspects, the instant specification is directed to a method of treating, ameliorating and / or preventing cancer.

[0100] In some embodiments, the method comprises administering to the subject an effective amount of: a compound that down regulates amphiregulin (AREG); a platinum-based chemotherapy agent; and a microtubule-stabilizing chemotherapy agent.

[0101] In some embodiments, the platinum-based chemotherapy agent is an alkylating agent. In some embodiments, the platinum-based chemotherapy agent covalently binds to nucleic acid molecules in a cancer cell, such as cross-link DNA molecules, thereby damaging or killing the cancer cell.

[0102] In some embodiments, the platinum-based chemotherapy agent is at least one selected from the group consisting of: cisplatin, carboplatin, heptaplatin, iproplatin, lobaplatin, miriplatin, nedaplatin, oxaliplatin, picoplatin, phenanthriplatin, PN149, pyriplatin, satraplatin, tetraplatin, and triplatin tetranitrate.

[0103] In some embodiments, the microtubule-stabilizing chemotherapy agent stabilizes microtubules in cancer cells by the prevention of depolymerization of the tubulin molecules forming the microtubules, leading to cell death.

[0104] In some embodiments, the microtubule-stabilizing chemotherapy agent is at least one selected from the group consisting of an epothilone chemotherapy agent, a laulimalide chemotherapy agent, peloruside A, and a taxane chemotherapy agent, or an antibody-drug conjugate (ADC) comprising the same.

[0105] In some embodiments, the microtubule-stabilizing chemotherapy agent is BMS-310705, cabazitaxel, docetaxel, epothilone A, epothilone B, epothilone C, epothilone D, epothilone E, epothilone F, fludelone (KOS-1584), ixabepilone (BMS-247550), paclitaxel, or an antibody-drug conjugate (ADC) comprising the same.

[0106] In some embodiments, the microtubule-stabilizing chemotherapy agent is an antibody drug conjugate (ADC) that include a microtubule stabilizing agent in the payload thereof. Nonlimiting examples of the ADSs include mirvetuximab soravtansine (which includes ravtansine (DM4)), ADCs including monomethyl auristatin (MMAE), and the like.

[0107] In some embodiments, the compound that down regulates AREG is a neutralizing antibody against AREG; the platinum-based chemotherapy agent is carboplatin; and the microtubule-stabilizing chemotherapy agent is paclitaxel. In some embodiments, apart from the compound that down regulates amphiregulin, the platinum-based chemotherapy agent, and the microtubule-stabilizing chemotherapy agent, the subject is not administered any additional active compounds for treating, ameliorating, and / or preventing the cancer.

[0108] In some embodiments, the administration comprises a first phase administration and a second phase administration after the first phase administration.

[0109] In some embodiments, the subject is administered the compound that down regulates AREG, the platinum-based chemotherapy agent, and the microtubule-stabilizing chemotherapy agent in the first administration phase.

[0110] In some embodiments, the subject is administered the compound that down regulates AREG but not the platinum-based chemotherapy agent, or the microtubule-stabilizing chemotherapy agent in the second administration phase.

[0111] In some embodiments, a length of the first administration phase is about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 10 days, about 15 days, about 20 days, about 30 days, or any ranges therebetween.

[0112] In some embodiments, a length of the second administration phase is about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 10 days, about 15 days, about 20 days, about 30 days, or any ranges therebetween.

[0113] In some embodiments, the cancer is an ovarian cancer, optionally a serous ovarian cancer; optionally a high-grade serous ovarian cancer (HGSOC). In some embodiments, the cancer is an ovarian , primary peritoneal, or fallopian tube cancer, such as an ovarian , primary peritoneal, or fallopian tube cancer with high grade serous histopathology.

[0114] Biologically, cancers such as triple negative breast cancer (TNBC), endometrial cancer and carcinosarcoma and serous ovarian cancer are similar and / or have overlap in some tumor genomic mutations. For example, AREG has comparable mRNA expressions in breast cancer, endometrial cancer, and endometrial carcinoma. Furthermore, these cancers are also treated with the same frontline chemotherapy as HGSOC (e.g., carboplatin and paclitaxel).

[0115] In some embodiments, the cancer is a breast a cancer, optionally a triple negative breast cancer (TNBC).

[0116] In some embodiments, the cancer is an endometrial cancer, optionally a serous endometrial cancer, optionally a high-grade serous endometrial cancer. In some embodiments, the cancer is a carcinosarcoma.

[0117] In some embodiments, the subject is a mammal.

[0118] In some embodiments, the subject is a human.

[0119] Downregulating AREG

[0120] In some embodiments, the compound that down regulates AREG downregulates the expression level or the activity of AREG.

[0121] In some embodiments, the compound that downregulates the expression level or the activity of AREG acts at the genomic level. For example, the expression level of AREG can be down-regulated by gene knockout, such as CRISPR knockout and other knockout techniques.

[0122] In some embodiments, the compound that downregulates the expression level or the activity of AREG acts at the transcriptional level or the translational level. For example, the expression level of AREG can be down-regulated by gene knockdown, such as by RNA interference technique, ribozyme knockdown, or CRISPR knockdown.

[0123] In some embodiments, the compound that downregulates the expression level or the activity of AREG acts at the post-translational level. For example, the expression level of AREG can be down-regulated by targeted protein degradation, such as proteolysis-targeting chimera (PROTAC) and other protein degradation strategies. For example, the activity of AREG can be down-regulated by small molecules inhibitors of AREG, antibodies that neutralizes AREG, and trans-dominant negative mutant of AREG.

[0124] In some embodiments, the compound that downregulates the expression level or the activity of AREG includes a small molecule inhibitor of AREG, a protein inhibitor of AREG, or a compound that downregulates the expression level and / or activity of AREG by RNA interference, by ribozyme, by CRISPR knockout / knockdown, or by producing a trans-dominant negative mutant, and so forth.

[0125] In some embodiments, the compound contemplated herein can be delivered by a vector, such as a plasmid or a viral vector. One of ordinary skill in the art would understand that such vectors can be used to deliver compounds in the form of nucleic acids, such as RNA or DNA. Such vectors are described herein below.

[0126] In certain embodiments, the compound contemplated herein (including but not limited to nucleic acids) can be more efficiently delivered to the cell nucleus by coupling the compound with the monoclonal anti-DNA antibody 3E10, which penetrates living cells and localizes in the nucleus without causing any apparent harm to the cell (Hansen JE, et al., Intranuclear protein transduction through a nucleoside salvage pathway. J Biol Chem 2007;282:20790-3; see also WO 2020 / 047353 and WO 2021 / 042060, all of which are incorporated herein in their entireties by reference). 3E10 and its single-chain variable fragment (3E10 scFv) have been developed as an intracellular delivery system for macromolecules. After localizing in the cell nucleus, 3E10 scFv is largely degraded within 4 hours, thus further minimizing any potential toxicity.

[0127] In certain embodiments, the compounds contemplated herein can be more efficiently delivered to tissue by coupling with certain protein fragments, called “pHLIP” (pH (Low) Insertion Peptide), which allow for the cargo to accumulate in acidic environments within the body. In certain embodiments, a polypeptide with a predominantly hydrophobic sequence long enough to span a membrane lipid bilayer as a transmembrane helix (TM) and comprising one or more dissociable groups inserts across a membrane spontaneously in a pH-dependent fashion placing one terminus inside cell. The polypeptide conjugated with various functional moieties delivers and accumulates them at cell membrane with low extracellular pH. The functional moiety conjugated with polypeptide terminus placed inside cell are translocated through the cell membrane in cytosol. The peptide and its variants or non-peptide analogs can be used to deliver therapeutic, prophylactic, diagnostic, imaging, gene regulation, cell regulation, or immunologic agents to or inside of cells in vitro or in vivo in tissue at low extracellular pH. See also US20080233107, WO2012 / 021790, US20120039990, US20120142042, US20150051153, US20150086617, and US20150191508, all of which are incorporated herein in their entireties by reference.

[0128] Downregulating AREG by small molecule inhibitors

[0129] In some embodiments, the compound that downregulates the expression level or the activity of AREG includes a small molecule that inhibits the activity of AREG. As used herein, the term “small molecule” refers to a molecule having a size of less than 2000, 1800, 1600, 1400, 1200, 1000, 800, or 600 Daltons.

[0130] In some embodiments, the small molecule inhibitor comprises a PROTAC or a Proteolysis Targeting Chimeric Molecule. PROTACs are heterobifunctional nanomolecules that can target any protein for ubiquitination and degradation. In certain embodiments, the PROTAC contemplated in the present invention comprises a group that is recognized by the E3 ubiquitin ligase and a group that is recognized by AREG. The PROTAC is able to simultaneously bind to the AREG and the E3 ligase. Formation of such trimeric complex formation leads to the transfer of ubiquitins to the AREG, marking it for degradation. PROTAC molecules possess good tissue distribution and the ability to target intracellular proteins, thus can be directly applied to cells or injected into animals without the use of vectors. PROTACS useful within the invention can be prepared using any known compound that binds to and / or recognizes and / or inhibits AREG, which is linked through a linker to an E3 ubiquitin ligase, such as but not limited to those described in WO 2013 / 106643, WO 2013 / 106646, and WO 2019 / 148055.

[0131] Downregulating AREG by protein inhibitors of AREG

[0132] In some embodiments, the compound that downregulates the expression level or the activity of AREG includes a protein that downregulates the expression level or the activity of AREG.

[0133] Since AREG is a transmembrane glycoprotein with an extracellular portion, one of ordinary skill in the art would understand that antibodies, such as neutralizing antibodies, are effective in down regulating AREG.

[0134] Examples of monoclonal and / or polyclonal antibodies that target AREG include 66433-1- IG (clone 1A1G9) available from Proteintech (Rosemont, IL, USA), 17-5370-42 (clone AREG559) and PA5-102501 available from Invitrogen (Waltham, MA, USA), BS-3847R available from Bioss (Woburn, MA), MAB262-100 available from R&D Systems (Minneapolis, MN, USA), and any humanized derivatives thereof.

[0135] In some embodiments, the protein that downregulates the expression level and / or activity of AREG is administered in form of a protein. In some embodiments, the protein that downregulates the expression level and / or activity of AREG is administered in form of a nucleic acid that expresses the protein, such as an expression vector. The expression vector is described in the “Vector” section elsewhere in the instant specification.

[0136] Downregulating AREG by RNA Interference In some embodiments, the compound that downregulates the activity or expression level of AREG includes a nucleic acid that downregulates the activity and / or expression level of AREG by the means of RNA interreference.

[0137] In some embodiments, the nucleic acid that downregulates the expression level of AREG by the means of RNA interreference includes an isolated nucleic acid. In other embodiments, the modulator is an RNAi molecule (such as but not limited to siRNA and / or shRNA and / or miRNAs) or antisense molecule, which inhibits AREG expression and / or activity. In yet other embodiments, the nucleic acid comprises a promoter / regulatory sequence, such that the nucleic acid is preferably capable of directing expression of the nucleic acid. Thus, the instant specification provides expression vectors and methods for the introduction of exogenous DNA into cells with concomitant expression of the exogenous DNA in the cells such as those described, for example, in Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York) and as described elsewhere herein.

[0138] In certain embodiments, siRNA is used to decrease the level of AREG. RNA interference (RNAi) is a phenomenon in which the introduction of double-stranded RNA (dsRNA) into a diverse range of organisms and cell types causes degradation of the complementary mRNA. In the cell, long dsRNAs are cleaved into short 21-25 nucleotide small interfering RNAs, or siRNAs, by a ribonuclease known as Dicer. The siRNAs subsequently assemble with protein components into an RNA-induced silencing complex (RISC), unwinding in the process. Activated RISC then binds to complementary transcript by base pairing interactions between the siRNA antisense strand and the mRNA. The bound mRNA is cleaved and sequence specific degradation of mRNA results in gene silencing. See, for example, U.S. Patent No. 6,506,559; Fire et al., 1998, Nature 391 (19):306-311; Timmons et al., 1998, Nature 395:854; Montgomery et al., 1998, TIG 14 (7):255-258; Engelke, Ed., RNA Interference (RNAi) Nuts & Bolts of RNAi Technology, DNA Press, Eagleville, PA (2003); and Hannon, Ed., RNAi A Guide to Gene Silencing, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2003). Soutschek et al. (2004, Nature 432: 173-178) describes a chemical modification to siRNAs that aids in intravenous systemic delivery. Optimizing siRNAs involves consideration of overall G / C content, C / T content at the termini, Tm and the nucleotide content of the 3' overhang. See, for instance, Schwartz et al., 2003, Cell, 115:199-208 and Khvorova et al. , 2003, Cell 115:209-216. Therefore, the instant specification also includes methods of decreasing levels of AREG using RNAi technology.

[0139] In certain embodiments, the instant specification provides a vector comprising an siRNA or antisense polynucleotide. In other embodiments, the siRNA or antisense polynucleotide inhibits the expression of AREG. The incorporation of a desired polynucleotide into a vector and the choice of vectors is well-known in the art.

[0140] In certain embodiments, the expression vectors described herein encode a short hairpin RNA (shRNA) inhibitor. shRNA inhibitors are well known in the art and are directed against the mRNA of a target, thereby decreasing the expression of the target. In certain embodiments, the encoded shRNA is expressed by a cell, and is then processed into siRNA. For example, in certain instances, the cell possesses native enzymes (e.g., dicer) that cleaves the shRNA to form siRNA.

[0141] The siRNA, shRNA, or antisense polynucleotide can be cloned into a number of types of vectors as described elsewhere herein. For expression of the siRNA or antisense polynucleotide, at least one module in each promoter functions to position the start site for RNA synthesis.

[0142] In order to assess the expression of the siRNA, shRNA, or antisense polynucleotide, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected using a viral vector. In certain embodiments, the selectable marker may be carried on a separate piece of DNA and used in a cotransfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers are known in the art and include, for example, antibiotic-resistance genes, such as neomycin resistance and the like.

[0143] Following the generation of the siRNA polynucleotide, a skilled artisan will understand that the siRNA polynucleotide has certain characteristics that can be modified to improve the siRNA as a therapeutic compound. Therefore, in some embodiments, the siRNA polynucleotide is further designed to resist degradation by modifying it to include phosphorothioate, or other linkages, methylphosphonate, sulfone, sulfate, ketyl, phosphorodithioate, phosphoramidate, phosphate esters, and the like (see, e.g., Agrwal et al., 1987, Tetrahedron Lett. 28:3539-3542; Stec et al, 1985 Tetrahedron Lett. 26:2191-2194; Moody et al., 1989 Nucleic Acids Res. 12:4769-4782; Eckstein, 1989 Trends Biol. Sci. 14:97-100; Stein, In: Oligodeoxynucleotides. Antisense Inhibitors of Gene Expression, Cohen, ed., Macmillan Press, London, pp. 97-117 (1989)).

[0144] Any polynucleotide may be further modified to increase its stability in vivo. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and / or 3' ends; the use of phosphorothioate or 2' O-methyl rather than phosphodi ester linkages in the backbone; and / or the inclusion of nontraditional bases such as inosine, queosine, and wybutosine and the like, as well as acetyl- methyl-, thio- and other modified forms of adenine, cytidine, guanine, thymine, and uridine.

[0145] In certain embodiments, an antisense nucleic acid sequence expressed by a plasmid vector is used to inhibit AREG protein expression. The antisense expressing vector is used to transfect a mammalian cell or the mammal itself, thereby causing reduced endogenous expression of AREG.

[0146] Antisense molecules and their use for inhibiting gene expression are well known in the art (see, e.g., Cohen, 1989, In: Oligodeoxyribonucleotides, Antisense Inhibitors of Gene Expression, CRC Press). Antisense nucleic acids are DNA or RNA molecules that are complementary, as that term is defined elsewhere herein, to at least a portion of a specific mRNA molecule (Weintraub, 1990, Scientific American 262:40). In the cell, antisense nucleic acids hybridize to the corresponding mRNA, forming a double-stranded molecule thereby inhibiting the translation of genes.

[0147] The use of antisense methods to inhibit the translation of genes is known in the art, and is described, for example, in Marcus-Sakura (1988, Anal. Biochem. 172:289). Such antisense molecules may be provided to the cell via genetic expression using DNA encoding the antisense molecule as taught by Inoue, 1993, U.S. Patent No. 5,190,931.

[0148] Alternatively, antisense molecules of the instant specification may be made synthetically and then provided to the cell. Antisense oligomers of between about 10 to about 30, and more preferably about 15 nucleotides, are preferred, since they are easily synthesized and introduced into a target cell. Synthetic antisense molecules contemplated by the instant specification include oligonucleotide derivatives known in the art which have improved biological activity compared to unmodified oligonucleotides (see U.S. Patent No. 5,023,243).

[0149] Downregulating AREG by ribozyme In some embodiments, the compound that down regulates the activity or expression level of AREG includes a ribosome that inhibits AREG protein expression.

[0150] A ribozyme is used to inhibit AREG protein expression. Ribozymes useful for inhibiting the expression of a target molecule may be designed by incorporating target sequences into the basic ribozyme structure which are complementary, for example, to the mRNA sequence encoding AREG. Ribozymes are antisense RNAs which have a catalytic site capable of specifically cleaving complementary RNAs. Therefore, ribozymes having sequence complementary to AREG mRNA sequences are capable of downregulating the expression of AREG by reduces the level of AREG mRNA. Ribozymes targeting AREG, may be synthesized using commercially available reagents (Applied Biosystems, Inc., Foster City, CA) or they may be genetically expressed from DNA encoding them. In some embodiments, the DNA encoding the ribozymes are incorporated in a vector, which is described in the “Vector” section elsewhere in the instant specification.

[0151] Downregulating AREG by CRISPR knockout / knockdown and other knockouts / knockdown techniques

[0152] In some embodiments, the compound that down regulates the activity or expression level of AREG comprises a nucleic acid that down regulates the expression level of AREG by the means of CRISPR knockout.

[0153] In some embodiments, the compound down regulates the activity or expression level of AREG comprises a CRISPR / Cas9 system for knocking out AREG.

[0154] The CRISPR / Cas9 system is a facile and efficient system for inducing targeted genetic alterations. Target recognition by the Cas9 protein requires a “seed” sequence within the guide RNA (gRNA) and a conserved di-nucleotide containing protospacer adjacent motif (PAM) sequence upstream of the gRNA-binding region. The CRISPR / Cas9 system can thereby be engineered to cleave virtually any DNA sequence by redesigning the gRNA in cell lines (such as 293T cells), primary cells, and CAR T cells. The CRISPR / Cas9 system can simultaneously target multiple genomic loci by co-expressing a single Cas9 protein with two or more gRNAs, making this system uniquely suited for multiple gene editing or synergistic activation of target genes.

[0155] The Cas9 protein and guide RNA form a complex that identifies and cleaves target sequences. Cas9 is comprised of six domains: REC I, REC II, Bridge Helix, PAM interacting, HNH, and RuvC. The Red domain binds the guide RNA, while the Bridge helix binds to target DNA. The HNH and RuvC domains are nuclease domains. Guide RNA is engineered to have a 5' end that is complementary to the target DNA sequence. Upon binding of the guide RNA to the Cas9 protein, a conformational change occurs activating the protein. Once activated, Cas9 searches for target DNA by binding to sequences that match its protospacer adjacent motif (PAM) sequence. A PAM is a two or three nucleotide base sequence within one nucleotide downstream of the region complementary to the guide RNA. In one non-limiting example, the PAM sequence is 5'-NGG-3'. When the Cas9 protein finds its target sequence with the appropriate PAM, it melts the bases upstream of the PAM and pairs them with the complementary region on the guide RNA. Then the RuvC and HNH nuclease domains cut the target DNA after the third nucleotide base upstream of the PAM.

[0156] One non-limiting example of a CRISPR / Cas system used to inhibit gene expression, CRISPRi, is described in U.S. Patent Appl. Publ. No. US2014 / 0068797. CRISPRi induces permanent gene disruption that utilizes the RNA-guided Cas9 endonuclease to introduce DNA double stranded breaks which trigger error-prone repair pathways to result in frame shift mutations. A catalytically dead Cas9 lacks endonuclease activity. When coexpressed with a guide RNA, a DNA recognition complex is generated that specifically interferes with transcriptional elongation, RNA polymerase binding, or transcription factor binding. This CRISPRi system efficiently represses expression of targeted genes.

[0157] CRISPR / Cas gene disruption occurs when a guide nucleic acid sequence specific for a target gene and a Cas endonuclease are introduced into a cell and form a complex that enables the Cas endonuclease to introduce a double strand break at the target gene. In certain embodiments, the CRISPR / Cas system comprises an expression vector, such as, but not limited to, an pAd5F35-CRISPR vector. In other embodiments, the Cas expression vector induces expression of Cas9 endonuclease. Other endonucleases may also be used, including but not limited to, T7, Cas3, Cas8a, Cas8b, CaslOd, Csel, Csyl, Csn2, Cas4, CaslO, Csm2, Cmr5, Fokl, other nucleases known in the art, and any combinations thereof.

[0158] In certain embodiments, inducing the Cas expression vector comprises exposing the cell to an agent that activates an inducible promoter in the Cas expression vector. In such embodiments, the Cas expression vector includes an inducible promoter, such as one that is inducible by exposure to an antibiotic (e g., by tetracycline or a derivative of tetracycline, for example doxycycline). However, it should be appreciated that other inducible promoters can be used. The inducing agent can be a selective condition (e.g., exposure to an agent, for example an antibiotic) that results in induction of the inducible promoter. This results in expression of the Cas expression vector.

[0159] In certain embodiments, guide RNA(s) and Cas9 can be delivered to a cell as a ribonucleoprotein (RNP) complex. RNPs are comprised of purified Cas9 protein complexed with gRNA and are well known in the art to be efficiently delivered to multiple types of cells, including but not limited to cancer cells, neurons, stem cells and immune cells (Addgene, Cambridge, MA, Mirus Bio LLC, Madison, WI).

[0160] The guide RNA is specific for a genomic region of interest and targets that region for Cas endonuclease-induced double strand breaks. The target sequence of the guide RNA sequence may be within a loci of a gene or within a non-coding region of the genome. In certain embodiments, the guide nucleic acid sequence is at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more nucleotides in length.

[0161] Guide RNA (gRNA), also referred to as "short guide RNA" or "sgRNA", provides both targeting specificity and scaffolding / binding ability for the Cas9 nuclease. The gRNA can be a synthetic RNA composed of a targeting sequence and scaffold sequence derived from endogenous bacterial crRNA and tracrRNA. gRNA is used to target Cas9 to a specific genomic locus in genome engineering experiments. Guide RNAs can be designed using standard tools well known in the art.

[0162] In the context of formation of a CRISPR complex, "target sequence" refers to a sequence to which a guide sequence is designed to have some complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex. A target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides. In certain embodiments, a target sequence is located in the nucleus or cytoplasm of a cell. In other embodiments, the target sequence may be within an organelle of a eukaryotic cell, for example, mitochondrion or nucleus. Typically, in the context of an endogenous CRISPR system, formation of a CRISPR complex (comprising a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands in or near (e.g., within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs) the target sequence. As with the target sequence, it is believed that complete complementarity is not needed, provided this is sufficient to be functional.

[0163] In certain embodiments, one or more vectors driving expression of one or more elements of a CRISPR system are introduced into a host cell, such that expression of the elements of the CRISPR system direct formation of a CRISPR complex at one or more target sites. For example, a Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence could each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more of the elements expressed from the same or different regulatory elements may be combined in a single vector, with one or more additional vectors providing any components of the CRISPR system not included in the first vector. CRISPR system elements that are combined in a single vector may be arranged in any suitable orientation, such as one element located 5' with respect to ("upstream" of) or 3' with respect to ("downstream" of) a second element. The coding sequence of one element may be located on the same or opposite strand of the coding sequence of a second element, and oriented in the same or opposite direction. In certain embodiments, a single promoter drives expression of a transcript encoding a CRISPR enzyme and one or more of the guide sequence, tracr mate sequence (optionally operably linked to the guide sequence), and a tracr sequence embedded within one or more intron sequences (e.g., each in a different intron, two or more in at least one intron, or all in a single intron).

[0164] In certain embodiments, the CRISPR enzyme is part of a fusion protein comprising one or more heterologous protein domains (e.g. about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains in addition to the CRISPR enzyme). A CRISPR enzyme fusion protein may comprise any additional protein sequence, and optionally a linker sequence between any two domains. Examples of protein domains that may be fused to a CRISPR enzyme include, without limitation, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity. Additional domains that may form part of a fusion protein comprising a CRISPR enzyme are described in U.S. Patent Appl. Publ. No. US20110059502, incorporated herein by reference. In certain embodiments, a tagged CRISPR enzyme is used to identify the location of a target sequence.

[0165] Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids in mammalian and non-mammalian cells or target tissues. Such methods can be used to administer nucleic acids encoding components of a CRISPR system to cells in culture, or in a host organism. Non-viral vector delivery systems include DNA plasmids, RNA (e.g., a transcript of a vector described herein), naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell (Anderson, 1992, Science 256:808-813; and Yu, etal., 1994, Gene Therapy 1: 13-26).

[0166] In certain embodiments, the CRISPR / Cas is derived from a type II CRISPR / Cas system. In other embodiments, the CRISPR / Cas system is derived from a Cas9 protein. The Cas9 protein can be from Streptococcus pyogenes, Streptococcus thermophilus, or other species.

[0167] In general, Cas proteins comprise at least one RNA recognition and / or RNA binding domain. RNA recognition and / or RNA binding domains interact with the guiding RNA. Cas proteins can also comprise nuclease domains (i.e., DNase or RNase domains), DNA binding domains, helicase domains, RNAse domains, protein-protein interaction domains, dimerization domains, as well as other domains. The Cas proteins can be modified to increase nucleic acid binding affinity and / or specificity, alter an enzymatic activity, and / or change another property of the protein. In certain embodiments, the Cas-like protein of the fusion protein can be derived from a wild type Cas9 protein or fragment thereof. In other embodiments, the Cas can be derived from modified Cas9 protein. For example, the amino acid sequence of the Cas9 protein can be modified to alter one or more properties (e.g., nuclease activity, affinity, stability, and so forth) of the protein. Alternatively, domains of the Cas9 protein not involved in RNA-guided cleavage can be eliminated from the protein such that the modified Cas9 protein is smaller than the wild type Cas9 protein. In general, a Cas9 protein comprises at least two nuclease (i.e., DNase) domains. For example, a Cas9 protein can comprise a RuvC-like nuclease domain and a HNH- like nuclease domain. The RuvC and HNH domains work together to cut single strands to make a double-stranded break in DNA. (Jinek, et al., 2012, Science, 337:816-821). In certain embodiments, the Cas9-derived protein can be modified to contain only one functional nuclease domain (either a RuvC-like or a HNH-like nuclease domain). For example, the Cas9-derived protein can be modified such that one of the nuclease domains is deleted or mutated such that it is no longer functional (i.e., the nuclease activity is absent). In some embodiments in which one of the nuclease domains is inactive, the Cas9-derived protein is able to introduce a nick into a double-stranded nucleic acid (such protein is termed a "nickase"), but not cleave the doublestranded DNA. In any of the above-described embodiments, any or all of the nuclease domains can be inactivated by one or more deletion mutations, insertion mutations, and / or substitution mutations using well-known methods, such as site-directed mutagenesis, PCR-mediated mutagenesis, and total gene synthesis, as well as other methods known in the art.

[0168] In one non-limiting embodiment, a vector drives the expression of the CRISPR system. The art is replete with suitable vectors that are useful in the instant specification. The vectors to be used are suitable for replication and, optionally, integration in eukaryotic cells. Typical vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence. The vectors of the instant specification may also be used for nucleic acid standard gene delivery protocols. Methods for gene delivery are known in the art (U.S. Patent Nos. 5,399,346, 5,580,859 & 5,589,466, incorporated by reference herein in their entireties).

[0169] Further, the vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (4thEdition, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 2012), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, Sindbis virus, gammaretrovirus and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0170] In some embodiments, the compound that down regulates the activity or expression level of AREG comprises a nucleic acid that down regulates the expression level of AREG by the means of CRISPR knockdown. CRISPR knockdown includes, but not limited to, CRISPRCasl3 knockdown. (See e.g., Mendez-Mancilla et al., Cell Chemical Biology 29, 1-7, 2021 Jul 27, and Kushawah et al., Dev Cell. 2020 Sep 28;54(6):805-817. The entireties of which are incorporated herein by reference). In some embodiments, the present invention includes any other methods for effecting gene knockdown and / editing, which allow for deletion and / or inactivation of AREG, such as but not limited to those described in WO 2018 / 236840 (which is incorporated herein in its entirety by reference).

[0171] Downregulating AREG by inactivating and / or sequestering

[0172] In some embodiments, the compound that downregulates the activity or expression level of AREG includes a protein that downregulates the activity of AREG by inactivating and / or sequestering AREG. In some embodiment, the compound includes a nucleic acid that express the protein that downregulates the activity of AREG by inactivating and / or sequestering AREG. In some embodiments, the compound includes an expression vector that express the protein that downregulates the activity of AREG by inactivating and / or sequestering AREG (see “Vector” section for descriptions on vectors).

[0173] In some embodiments, the compound that downregulates the expression level of AREG is a trans-dominant negative mutant of AREG, and / or a nucleic acid or a vector expressing the trans-dominant negative mutant of AREG.

[0174] Kit

[0175] In some aspects, the instant specification is directed to a kit, such as a kit for carrying out the methods described herein.

[0176] In some embodiments, the kit comprises a compound that down regulates amphiregulin (AREG); a platinum-based chemotherapy agent; a microtubule-stabilizing chemotherapy agent; and an instructional manual.

[0177] In some embodiments, the compound that down regulates AREG, the platinum-based chemotherapy agent, and the microtubule-stabilizing chemotherapy agent are the same as or similar to those described elsewhere herein, such as in the “Method of Treating, Ameliorating and / or Preventing Cancer” section.

[0178] In some embodiments, the instruction manual instructs that the compound that down regulates AREG, the platinum-based chemotherapy agent and the microtubule-stabilizing chemotherapy agent are to be administered to a subject in need thereof to treat, ameliorate and / or prevent a cancer, such as according to the method described herein. Vectors

[0179] Vectors can increase the stability of the nucleic acids, make the delivery easier, or allow the expression of the nucleic acids or protein products thereof in the cells.

[0180] Therefore, in some embodiments, the protein inhibitors or the nucleic acids that that down regulates the activity or expression level of AREG is incorporated into a vector.

[0181] In some embodiments, the instant specification relates to a vector, including the nucleic acid sequence of the instant specification or the construct of the instant specification. The choice of the vector will depend on the host cell in which it is to be subsequently introduced. In certain embodiments, the vector of the instant specification is an expression vector. Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells. In certain embodiments, the expression vector is selected from the group consisting of a viral vector, a bacterial vector and a mammalian cell vector. Prokaryote- and / or eukaryote-vector based systems can be employed for use with the instant specification to produce polynucleotide, or their cognate polypeptides. Many such systems are commercially and widely available.

[0182] In some embodiments, the vector is a viral vector. Viral vector technology is well known in the art and is described, for example, in virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno- associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers. (See, e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Pat. No. 6,326,193.

[0183] In some embodiments, the viral vector is a suitable adeno-associated virus (AAV), such as the AAV1-AAV8 family of adeno-associated viruses. In some embodiments, the viral vector is a viral vector that can infect a human. The desired nucleic acid sequence, such as the nucleic acids that downregulates AREG described above, can be inserted between the inverted terminal repeats (ITRs) in the AAV. In various embodiments, the viral vector is an AAV2 or an AAV8. The promoter can be a thyroxine binding globulin (TBG) promoter. In various embodiments, the promoter is a human promoter sequence that enables the desired nucleic acid expression in the brain. The AAV can be a recombinant AAV, in which the capsid comes from one AAV serotype and the ITRs come from another AAV serotype. In various embodiments, the AAV capsid is selected from the group consisting of a AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, and a AAV8 capsid. In various embodiments, the ITR in the AAV is at least one ITR selected from the group consisting of a AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, and an AAV8 ITR. In various embodiments, the instant specification contemplates an AAV8 viral vector (recombinant or non-recombinant) containing a desired nucleic acid expression sequence and at least one promoter sequence that, when administered to a subject, causes elevated systemic expression of the desired nucleic acid. In some embodiments, the viral vector is a recombinant or non-recombinant AAV2 or AAV5 containing any of the desired nucleic acid expression sequences described herein. In some embodiments, the AAV is an engineered AAVs for delivering nucleic acid across the blood brain barrier to the central and peripheral nervous systems, such as those as described by Chan et al., Nat Neurosci. 2017 Aug; 20(8): 1172-1179. The entirety of this reference is incorporated herein by reference.

[0184] In some embodiments, the vector in which the nucleic acid sequence is introduced is a plasmid that is or is not integrated in the genome of a host cell when it is introduced in the cell. Illustrative, non-limiting examples of vectors in which the nucleotide sequence of the instant specification or the gene construct of the instant specification can be inserted include a tet-on inducible vector for expression in eukaryote cells.

[0185] The vector may be obtained by conventional methods known by persons skilled in the art (Sambrook et al., 2012). In certain embodiments, the vector is a vector useful for transforming animal cells.

[0186] In certain embodiments, the recombinant expression vectors may also contain nucleic acid molecules which encode a peptide or peptidomimetic inhibitor of the instant specification, described elsewhere herein.

[0187] A promoter may be one naturally associated with a gene or polynucleotide sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment and / or exon. Such a promoter can be referred to as "endogenous." Similarly, an enhancer may be one naturally associated with a polynucleotide sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages will be gained by positioning the coding polynucleotide segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a polynucleotide sequence in its natural environment. A recombinant or heterologous enhancer refers also to an enhancer not normally associated with a polynucleotide sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers not "naturally occurring," i.e., containing different elements of different transcriptional regulatory regions, and / or mutations that alter expression. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and / or nucleic acid amplification technology, including PCR™, in connection with the compositions disclosed herein (U.S. Patent 4,683,202, U.S. Patent 5,928,906). Furthermore, it is contemplated the control sequences that direct transcription and / or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well.

[0188] It will be important to employ a promoter and / or enhancer that effectively directs the expression of the DNA segment in the cell type, organelle, and organism chosen for expression. Those of skill in the art of molecular biology generally know how to use promoters, enhancers, and cell type combinations for protein expression. The promoters employed may be constitutive, tissue-specific, inducible, and / or useful under the appropriate conditions to direct high-level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins and / or peptides. The promoter may be heterologous or endogenous.

[0189] The recombinant expression vectors may also contain a selectable marker gene which facilitates the selection of transformed or transfected host cells. Suitable selectable marker genes are genes encoding proteins such as G418 and hygromycin which confer resistance to certain drugs, P-galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or an immunoglobulin or portion thereof such as the Fc portion of an immunoglobulin preferably IgG. The selectable markers may be introduced on a separate vector from the nucleic acid of interest.

[0190] Combination Therapies

[0191] In some embodiments, apart from the compounds herein, the subject is further administered at least one additional agent that treats, ameliorates, and / or prevents a disease and / or disorder contemplated herein. In other embodiments, the compound and the at least one additional agent are co-administered to the subject. In yet other embodiments, the compound and the at least one additional agent are co-formulated.

[0192] The compounds contemplated within the disclosure are intended to be useful when combined, and / or in combination with one or more additional compounds. These additional compounds may comprise compounds of the present disclosure and / or at least one additional agent for treating cancers, and / or at least one additional agent that treats one or more diseases or disorders contemplated herein.

[0193] A synergistic effect may be calculated, for example, using suitable methods such as, for example, the Sigmoid-Emax equation (Holford & Scheiner, 1981, Clin. Pharmacokinet. 6:429- 453), the equation of Loewe additivity (Loewe & Muischnek, 1926, Arch. Exp. Pathol Pharmacol. 114:313-326) and the median-effect equation (Chou & Talalay, 1984, Adv. Enzyme Regul. 22:27-55). Each equation referred to above may be applied to experimental data to generate a corresponding graph to aid in assessing the effects of the drug combination. The corresponding graphs associated with the equations referred to above are the concentration-effect curve, isobologram curve and combination index curve, respectively.

[0194] Administration / Dosage / Formulations

[0195] The regimen of administration may affect what constitutes an effective amount. The therapeutic formulations contemplated within the disclosure may be administered to the subject either prior to or after the onset of a disease and / or disorder contemplated herein. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations contemplated within the disclosure may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.

[0196] Administration of the compositions contemplated within the disclosure to a patient, preferably a mammal, more preferably a human, may be carried out using known procedures, at dosages and for periods of time effective to treat a disease and / or disorder contemplated herein in the patient. An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the state of the disease or disorder in the patient; the age, sex, and weight of the patient; and the ability of the therapeutic compound contemplated within the disclosure to treat a disease and / or disorder contemplated herein in the patient. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A non-limiting example of an effective dose range for a therapeutic compound contemplated within the disclosure is from about 1 and 5,000 mg / kg of body weight / per day. One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.

[0197] Actual dosage levels of the active ingredients in the pharmaceutical compositions contemplated within the disclosure may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0198] In particular, the selected dosage level depends upon a variety of factors including the activity of the particular compound employed, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds or materials used in combination with the compound, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well, known in the medical arts.

[0199] A medical doctor, e.g, physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds contemplated within the disclosure employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0200] In particular embodiments, it is especially advantageous to formulate the compound in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the patients to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The dosage unit forms contemplated within the disclosure are dictated by and directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding / formulating such a therapeutic compound for the treatment of a disease and / or disorder contemplated herein. In certain embodiments, the compositions of the disclosure are formulated using one or more pharmaceutically acceptable excipients or carriers. In certain embodiments, the pharmaceutical compositions of the disclosure comprise a therapeutically effective amount of a compound of the disclosure and a pharmaceutically acceptable carrier.

[0201] The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.

[0202] In certain embodiments, the compositions of the disclosure are administered to the patient in dosages that range from one to five times per day or more. In another embodiment, the compositions of the disclosure are administered to the patient in range of dosages that include, but are not limited to, once every day, every two, days, every three days to once a week, and once every two weeks. It is readily apparent to one skilled in the art that the frequency of administration of the various combination compositions of the disclosure varies from individual to individual depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors. Thus, the disclosure should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any patient is determined by the attending physical taking all other factors about the patient into account.

[0203] Compounds of the disclosure for administration may be in the range of from about 1 pg to about 10,000 mg, about 20 pg to about 9,500 mg, about 40 pg to about 9,000 mg, about 75 pg to about 8,500 mg, about 150 pg to about 7,500 mg, about 200 pg to about 7,000 mg, about 3050 pg to about 6,000 mg, about 500 pg to about 5,000 mg, about 750 pg to about 4,000 mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about 20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 30 mg to about 1,000 mg, about 40 mg to about 900 mg, about 50 mg to about 800 mg, about 60 mg to about 750 mg, about 70 mg to about 600 mg, about 80 mg to about 500 mg, and any and all whole or partial increments therebetween.

[0204] In some embodiments, the dose of a compound of the disclosure is from about 1 mg and about 2,500 mg. In some embodiments, a dose of a compound of the disclosure used in compositions described herein is less than about 10,000 mg, or less than about 8,000 mg, or less than about 6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in some embodiments, a dose of a second compound as described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof.

[0205] In certain embodiments, the present disclosure is directed to a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound of the disclosure, alone or in combination with a second pharmaceutical agent; and instructions for using the compound to treat, prevent, or reduce one or more symptoms of a cancer in a patient.

[0206] Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for intracranially, intrathecal , oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration, known to the art. The pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and / or aromatic substances and the like. They may also be combined where desired with other active agents, e.g., other analgesic agents.

[0207] Routes of administration of any of the compositions of the disclosure include oral, nasal, rectal, intravaginal, parenteral, buccal, sublingual or topical. The compounds for use in the disclosure may be formulated for administration by any suitable route, such as for oral or parenteral, for example, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.

[0208] Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions that would be useful in the present disclosure are not limited to the particular formulations and compositions that are described herein.

[0209] Oral Administration

[0210] For oral application, particularly suitable are tablets, dragees, liquids, drops, suppositories, or capsules, caplets and gelcaps. The compositions intended for oral use may be prepared according to any method known in the art and such compositions may contain one or more agents selected from the group consisting of inert, non-toxic pharmaceutically excipients that are suitable for the manufacture of tablets. Such excipients include, for example an inert diluent such as lactose; granulating and disintegrating agents such as cornstarch; binding agents such as starch; and lubricating agents such as magnesium stearate. The tablets may be uncoated or they may be coated by known techniques for elegance or to delay the release of the active ingredients. Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert diluent.

[0211] For oral administration, the compounds of the disclosure may be in the form of tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., polyvinylpyrrolidone, hydroxypropylcellulose or hydroxypropylmethylcellulose); fillers (e.g., cornstarch, lactose, microcrystalline cellulose or calcium phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrates (e.g., sodium starch gly collate); or wetting agents (e.g., sodium lauryl sulphate). If desired, the tablets may be coated using suitable methods and coating materials such as OPADRY™ film coating systems available from Colorcon, West Point, Pa. (e.g., OP ADR Y™ OY Type, OYC Type, Organic Enteric OY-P Type, Aqueous Enteric OY-A Type, OY-PM Type and OP ADR Y™ White, 32K 18400). Liquid preparation for oral administration may be in the form of solutions, syrups or suspensions. The liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats); emulsifying agent (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters or ethyl alcohol); and preservatives (e.g., methyl or propyl p-hydroxy benzoates or sorbic acid).

[0212] The present disclosure also includes a multi-layer tablet comprising a layer providing for the delayed release of one or more compounds of the disclosure, and a further layer providing for the immediate release of another medication. Using a wax / pH-sensitive polymer mix, a gastric insoluble composition may be obtained in which the active ingredient is entrapped, ensuring its delayed release.

[0213] Parenteral Administration

[0214] For parenteral administration, the compounds of the disclosure may be formulated for injection or infusion, for example, intravenous, intramuscular or subcutaneous injection or infusion, or for administration in a bolus dose and / or continuous infusion. Suspensions, solutions or emulsions in an oily or aqueous vehicle, optionally containing other formulatory agents such as suspending, stabilizing and / or dispersing agents may be used.

[0215] Additional Administration Forms

[0216] Additional dosage forms of this disclosure include dosage forms as described in U.S.

[0217] Patents Nos. 6,340,475; 6,488,962; 6,451,808; 5,972,389; 5,582,837; and 5,007,790. Additional dosage forms of this disclosure also include dosage forms as described in U.S. Patent Applications Nos. 20030147952; 20030104062; 20030104053; 20030044466; 20030039688; and 20020051820. Additional dosage forms of this disclosure also include dosage forms as described in PCT Applications Nos. WO 03 / 35041; WO 03 / 35040; WO 03 / 35029; WO 03 / 35177; WO 03 / 35039; WO 02 / 96404; WO 02 / 32416; WO 01 / 97783; WO 01 / 56544; WO 01 / 32217; WO 98 / 55107; WO 98 / 11879; WO 97 / 47285; WO 93 / 18755; and WO 90 / 11757. Controlled Release Formulations and Drug Delivery Systems

[0218] In certain embodiments, the formulations of the present disclosure may be, but are not limited to, short-term, rapid-offset, as well as controlled, for example, sustained release, delayed release and pulsatile release formulations.

[0219] The term sustained release is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that may, although not necessarily, result in substantially constant blood levels of a drug over an extended time period. The period of time may be as long as a month or more and should be a release which is longer that the same amount of agent administered in bolus form.

[0220] For sustained release, the compounds may be formulated with a suitable polymer or hydrophobic material which provides sustained release properties to the compounds. As such, the compounds for use the method of the disclosure may be administered in the form of microparticles, for example, by injection or in the form of wafers or discs by implantation.

[0221] In certain embodiments of the disclosure, the compounds of the disclosure are administered to a patient, alone or in combination with another pharmaceutical agent, using a sustained release formulation.

[0222] The term delayed release is used herein in its conventional sense to refer to a drug formulation that provides for an initial release of the drug after some delay following drug administration and that mat, although not necessarily, includes a delay of from about 10 minutes up to about 12 hours.

[0223] The term pulsatile release is used herein in its conventional sense to refer to a drug formulation that provides release of the drug in such a way as to produce pulsed plasma profiles of the drug after drug administration.

[0224] The term immediate release is used in its conventional sense to refer to a drug formulation that provides for release of the drug immediately after drug administration.

[0225] As used herein, short-term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes and any or all whole or partial increments thereof after drug administration after drug administration.

[0226] As used herein, rapid-offset refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and any and all whole or partial increments thereof after drug administration.

[0227] Dosing

[0228] The therapeutically effective amount or dose of a compound of the present disclosure depends on the age, sex and weight of the patient, the current medical condition of the patient and the progression of the cancer in the patient being treated. The skilled artisan is able to determine appropriate dosages depending on these and other factors.

[0229] A suitable dose of a compound of the present disclosure may be in the range of from about 0.01 mg to about 5,000 mg per day, such as from about 0.1 mg to about 1,000 mg, for example, from about 1 mg to about 500 mg, such as about 5 mg to about 250 mg per day. The dose may be administered in a single dosage or in multiple dosages, for example from 1 to 4 or more times per day. When multiple dosages are used, the amount of each dosage may be the same or different. For example, a dose of 1 mg per day may be administered as two 0.5 mg doses, with about a 12-hour interval between doses.

[0230] It is understood that the amount of compound dosed per day may be administered, in nonlimiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days. For example, with every other day administration, a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on.

[0231] In the case wherein the patient's status does improve, upon the doctor's discretion the administration of the modulator of the disclosure is optionally given continuously; alternatively, the dose of drug being administered is temporarily reduced or temporarily suspended for a certain length of time (i.e., a "drug holiday"). The length of the drug holiday optionally varies between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. The dose reduction during a drug holiday includes from 10%-100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. Once improvement of the patient's conditions has occurred, a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, is reduced, as a function of the patient's condition, to a level at which the improved disease is retained. In certain embodiments, patients require intermittent treatment on a long-term basis upon any recurrence of symptoms and / or infection.

[0232] The compounds for use in the method of the disclosure may be formulated in unit dosage form. The term "unit dosage form" refers to physically discrete units suitable as unitary dosage for patients undergoing treatment, with each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, optionally in association with a suitable pharmaceutical carrier. The unit dosage form may be for a single daily dose or one of multiple daily doses (e. ., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.

[0233] Toxicity and therapeutic efficacy of such therapeutic regimens are optionally determined in cell cultures or experimental animals, including, but not limited to, the determination of the LDso (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between the toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio between LD50 and ED50. Capsid assembly modulators exhibiting high therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are optionally used in formulating a range of dosage for use in human. The dosage of such capsid assembly modulators lies preferably within a range of circulating concentrations that include the ED50 with minimal toxicity. The dosage optionally varies within this range depending upon the dosage form employed and the route of administration utilized.

[0234] Those skilled in the art recognizes, or is able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents were considered to be within the scope of this disclosure and covered by the claims appended hereto. For example, it should be understood, that modifications in assay and / or reaction conditions, with art-recognized alternatives and using no more than routine experimentation, are within the scope of the present application.

[0235] It is to be understood that wherever values and ranges are provided herein, all values and ranges encompassed by these values and ranges, are meant to be encompassed within the scope of the present disclosure. Moreover, all values that fall within these ranges, as well as the upper or lower limits of a range of values, are also contemplated by the present application.

[0236] Examples

[0237] The instant specification further describes in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless so specified. Thus, the instant specification should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0238] Example 1: Enhanced amphiregulin exposure promotes modulation of the high grade serous ovarian cancer tumor immune microenvironment

[0239] High grade serous ovarian cancer (HGSOC) is a lethal gynecologic malignancy in which chemoresistant recurrence rates remain high. Furthermore, HGSOC patients have demonstrated overall low response rates to clinically available immunotherapies. Amphiregulin (AREG), a low affinity epidermal growth factor receptor ligand is known to be significantly upregulated in HGSOC patient tumors following neoadjuvant chemotherapy exposure. While much is known about AREG’s role in oncogenesis and classical immunity, it is function in tumor immunology has been comparatively understudied. Therefore, the objective of this present study was to elucidate how increased AREG exposure impacts the ovarian tumor immune microenvironment (OTIME). Using NanoString IO 360 and protein analysis, it was revealed that treatment with recombinant AREG led to prominent upregulation of genes associated with ovarian pathogenesis and immune evasion (CXCL8, CXCL1, CXCL ) along with increased STAT3 activation in HGSOC cells. In vitro co-culture assays consisting of HGSOC cells and peripheral blood mononuclear cells (PBMCs) stimulated with recombinant AREG (rAREG) led to significantly enhanced tumor cell viability. Moreover, PBMCs stimulated with rAREG exhibited significantly lower levels of l Ny and IL-2. In vivo rAREG treatment promoted significant reductions in circulating levels of IL -2 and IL-5. Intratumoral analysis of rAREG treated mice revealed a significant reduction in CD8+T cells coupled with an upregulation of PD-L1. Finally, combinatorial treatment with an AREG neutralizing antibody and carboplatin led to a synergistic reduction of cell viability in HGSOC cell lines 0VCAR8 and PEA2. Overall, this study demonstrates AREG’s ability to modulate cytotoxic responses within the OTIME and highlights its role as a novel HGSOC immune target.

[0240] Example 1-1:

[0241] High grade serous ovarian cancer (HGSOC) is the most lethal of all gynecologic malignancies with a 5-year survival rate just below 50%, due to the fact that patients are frequently diagnosed at an advanced stage and possess high recurrence rates 12-18 months after initially achieving remission. Furthermore, recurrent HGSOC tumors are heavily chemoresistant and therefore do not always respond to traditional platinum-taxane based chemotherapies that are utilized in the frontline setting. In recent years, targeted approaches such as the anti-angiogenic therapy bevacizumab and the poly (ADP-ribose) polymerase (PARP) inhibitor olaparib have been implemented in the maintenance setting as standard of care for HGSOC patients. However, with the exception of BRCA1 / 2 mutated patients who significantly benefit from olaparib treatment, these targeted therapies have not had profound effects on overall HGSOC survival rates. In addition, the majority of HGSOC patients derive no significant benefit from clinically available immunotherapies, as numerous clinical trials have demonstrated low response rates to programmed cell death protein 1 (PD-1) based therapies, despite the fact that intratumoral T cells are known to be highly prognostic in ovarian cancer. Hence, it has been theorized that the muted response to clinically available immunotherapies can be attributed to the uniquely immunosuppressive ovarian tumor immune microenvironment (OTIME), which is composed of high levels of T regulatory cells (Tregs), adipose tissue, and cancer associated fibroblasts (CAFs) that collectively contribute to tumor immune evasion and further drive ovarian pathogenesis.

[0242] In an effort to identify novel immune targets that are more representative of the unique OTIME, a genomic analysis was performed in matched diagnostic biopsy and interval debulking HGSOC patient tissue, obtained both prior to and following neoadjuvant chemotherapy (NACT) exposure to characterize OTIME adaptations. This analysis revealed that the gene amphiregulin (AREG) exhibited the highest fold-upregulation of out a panel of 770 of the most commonly studied immune oncology genes. AREG is a secreted glycoprotein and low-affinity epidermal growth factor receptor (EGFR) ligand and has an established role in promoting ovarian cell proliferation, metastasis, cancer sternness, and therapy resistance in ovarian cancer. Furthermore, in classical immunity, AREG is thought to function as a Th2 cytokine that controls inflammation and downregulates adaptive immune responses. However, there are limited studies evaluating AREG’s role in tumor immunology. Therefore, the present study sought to begin to elucidate the impact of AREG on multiple aspects of the OTIME.

[0243] Example 1-2: Materials and methods

[0244] Cell culture

[0245] HGSOC cell lines PEA1 / PEA2 cells were obtained from Millipore Sigma and cultured in RPMI 1640 supplemented with 2 mM Glutamine, 2 mM Sodium Pyruvate, and 10% Fetal Bovine Serum (FBS) and 1% penicillin / streptomycin. OVCAR8 HGSOCs were obtained from American Type Culture Collection (ATCC) and ID8 p53 cells were generously gifted by the Freiman lab at Brown University that were originally generated by the McNeish lab at the University of Glasgow. Both OVCAR8 and ID8 p53 cells were cultured in Dulbecco Modified Eagle Medium (DMEM) supplemented with 10% FBS and 1% penicillin / streptomycin. All cells were kept in a 37°C / 5% CO2 humidified chamber. Cells were treated with 200 ng / mL human recombinant AREG (rAREG; R&D Systems, 262-AR-100) or with BSA control at various timepoints (15 min- 4 h). HGSOC cells were treated with 10 pM of ruxolitinib (Selleckchem, S1378) or DMSO control (Sigma Aldrich, D54879) for 48 h.

[0246] RNA isolation and NanoString nCounter® PanCancer 10360

[0247] OVCAR8 and PEA1 cells were stimulated with 200 ng / mL of rAREG or BSA control for 2 h. RNA isolation was performed using the Trizol extraction / LiCl high salt precipitation and NanoString nCounter® PanCancer 10360 was performed as described in James et al. (Front. Immunol. 13, 965331). A total of three biological replicates per treatment in each cell line were submitted for analysis.

[0248] NanoString nCounter® PanCancer 10360 analysis

[0249] Data was analyzed in nSolver Advanced Analysis software and ROSALIND® (https: / / rosalind.bio / ), with a HyperScale architecture developed by ROSALIND, Inc. (San Diego, CA). The QC step generated read distribution percentages, violin plots identify heatmaps, and sample MDS plots. Normalization, fold changes and / .--values were calculated using criteria provided by NanoString® (https: / / nanostring.com). Control and rAREG samples were used to construct groups, respective to each cell line. ROSALIND® follows the nCounter® Advanced Analysis protocol of dividing counts within a lane by the geometric mean of the normalizer probes from the same lane. Housekeeping probes to be used for normalization are selected based on the geNorm algorithm as implemented in the NormqPCR R library described in Perkins et al. (BMC Genomics 13, 296). Fold changes andp-values are calculated using the fast method as described in the nCounter® Advanced Analysis 2.0 User

[0250] Manual (nanostring.com). The Benjamini -Hochberg method of estimating false discovery rates (FDR) was used to adjust / -values. The clustering of genes for the final heatmap of differentially expressed genes was performed using the Partitioning Around Medoids (PAM) method using the fpc R library that takes into the account the direction and type of all signals on the pathway, the position, role and type of every gene, etc. Hypergeometric distribution was employed to analyze the enrichment of pathways, gene ontology domain structure, and other ontologies. The topGO R library was employed to determine local similarities and dependencies between GO terms in order to perform Elim pruning correction. Interpro, NCB, MSigDB, REACT0ME, and WikiPathways databases were referenced for enrichment analysis. Enrichment was calculated relative to a set of background genes relevant to this experiment. RCC files were deposited in NCBFs Gene Expression Omnibus (GEO) and are accessible through GEO series accession number GSE252495.

[0251] RNA isolation and quantitative PCR

[0252] RNA isolation and quantitative PCR was performed as described in James et al. (Front. Immunol. 13, 965331). Validated human primers were purchased from Bio-Rad (CXCL1, DUSP5, IL-11, CXCL2, IL6, IFNy, IL-2, GZME). Custom primer sequences (Invitrogen) are as follows:

[0253] 18s rRNA-F-CCGCGGTTCTATTTTGTTGG (SEQ ID NO:1) 18s rRNA-R-GGCGCTCCCTCTTAATCATG (SEQ ID N0:2)

[0254] Phosphoproteomics

[0255] 0VCAR8 and PEA1 cells were treated with 200 ng / mL of rAREG or BSA control for 15 min, and then protein was collected in lysis buffer supplied by the Proteome Profiler Human Phospho-Kinase Array Kit (R&D Systems, ARY003C). Manufacturer’s instructions were followed and membranes were developed using the Bio-Rad ChemiDoc Imaging System. ImageJ was employed to perform background subtraction and measure spot density.

[0256] Western blot

[0257] Protein was extracted from cell pellets using Cell Lysis Buffer (Cell Signaling 9803) with 1 mM of a protease inhibitor cocktail (AbCam, ab65621). Concentrations for all extracted proteins were determined by the DC Protein Assay (Bio-Rad Laboratories, 5000116). Equal amounts of proteins were boiled at 70°C with Novex Sample Reducing Agent (Life Technologies, NP009) and NuPAGE LDS sample buffer (ThermoFisher Scientific, NP0007) into a 4%-12% gradient SurPAGE™ Bis-Tris Gel (GeneScript, M00652). The gel was transferred using a semi-dry method to methanol activated PVDF membrane using the Trans-Blot Turbo RTA Transfer Kit PVDF (Bio-Rad, 1704273), Trans-Blot Turbo 5x Transfer Buffer (Bio-Rad, 10026938), and the Bio-Rad Trans-Blot Turbo Transferring System (1.3A-25V) for 10 min. Membranes were then blocked in 5% milk in phosphate-buffered saline with 0.05% Tween 20 (PBS-T) for 30 min at room temperature, and primary antibodies were incubated overnight at 4°C diluted in 5% milk in PBS-T. Secondary antibodies were then diluted in 5% milk in PBS-T for 1 h at room temperature. Membranes were washed with PBS-T in between primary and secondary incubations and following the secondary incubation. Clarity™ Western ECL substrate (Biorad, 102030779 [peroxide solution], 102030787 [luminol / enhancer solution]) was used to detect HRP-tagged secondary antibodies. The Bio-Rad ChemiDoc Imaging System was used to image all blots and GAPDH was employed as a loading control. Antibodies and dilutions were as follows:

[0258] STAT3 (Cell Signaling, 4904S, 1 :500) or (Proteintech, 60199-1-lg, 1:500) Phospho-STAT3 (Cell Signaling, 9145S, 1 :500) PD-L1 (Proteintech, 66248-1-lg, 1 :500)

[0259] GAPDH (Santa Cruz Biotechnology, 47724, 1 : 1,000)

[0260] ERK (Cell Signaling, 9102S, 1 :500) or (Proteintech, 11257-1-AP, 1 :500)

[0261] Phospho-ERK (Cell Signaling, 4376SS, 1 :500) AKT (Proteintech, 60203 -2- 1g, 1 :500) Phospho-AKT (Proteintech, 28731-1-AP, 1 :500) AREG (Protientech, 16036-1-AP, 1 :500)

[0262] Anti -Rabbit (Cell Signaling, 7074S, 1 :1,000)

[0263] Anti-Mouse (Cell Signaling, 7076S, 1 : 1,000)

[0264] Enzyme-linked immunosorbent assay (ELISA)

[0265] OVCAR8 and PEA1 cells were treated with 200 ng / mL of rAREG or BSA control for 2 and 4 h. Their respective media was collected and secreted levels of IL-6 were examined using a commercially available IL-6 ELISA kit (ab 178013). Media was diluted 4-fold using the kit provided Sample Diluent NS solution. Manufacturer’s instructions were followed with the endpoint reading at 450 nm. All samples were run in duplicate, with three biological replicates of each sample.

[0266] Cell viability assays

[0267] HGSOC and peripheral blood mononuclear cell (PBMC) co-cultures

[0268] HGSOC cells were seeded in a 96-well plate (20,000 cells / well) and allowed to grow for 24-h. PBMCs (HumanCells Biosciences, PBMC-C10M) were co-cultured with HGSOC cells in a 5: 1 ratio and stimulated with 200 ng / mL of rAREG or BSA control. After 24 h, 10 pL / well of CellTiter 96® Aqueous One Solution Cell proliferation MTS Assay (Promega, G3580), incubated for 1 h at 37°C / 5% CO2, and finally read at 492 nm to assess cell viability.

[0269] AREG neutralizing antibody and chemotherapy treatments

[0270] PEA2 and OVCAR8 cells were seeded in a 96-well plate (20,000 cells / well) and allowed to grow for 24-h. Cells were pre-treated with carboplatin (400 pM for PEA2, 300 pM for OVCAR8; Santa Cruz Biotechnology, CAS 4157.5-94-4) or DMSO control (Sigma Aldrich, D54879) for 24-h, and with 30 pM of AREG neutralizing antibody (R&D Systems, MAB262- 100) or corresponding IgG control (MAB002) for 48-h prior to cell viability assessment as described above.

[0271] Animals

[0272] C57BL / 6 mice were purchased from Jackson Laboratories (strain#000664) All animal protocols were approved by the Brown University Animal Care and Use Committee (#22-09- 0002) and performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. This protocol was reviewed and acknowledged by the Lifespan University Institutional Animal Care and Use Committee (#505422).

[0273] In vivo treatment and tissue collection

[0274] 7-week-old C57BL / 6 mice were inoculated with five million ID8p53 cells intraperitoneally (IP). 28-day post tumor inoculation, mice were treated with either rAREG (400 pg / kg; R&D Systems, 989-AR-CF) or saline, daily for a maximum of 6 days until large ascites formation, at which point mice were euthanized by carbon dioxide inhalation. Tissue was harvested and immediately fixed in a 1 : 10 formalin solution overnight and then placed in 30%, 50%, and 70% ethanol for 30 min each. Previously fixed tumors were then submitted to the Brown University Molecular Pathology Core for standard paraffin embedding and 5 pM serial sectioning.

[0275] Mouse ascites and serum multiplex assays

[0276] Ascites was collected from mice post-mortem and then spun at 5,000 g for 10 min at 4°C. Whole blood was collected via cardiac puncture post-mortem into serum separator tubes, allowed to clot for 30 min and then spun at 3,000 g for 15 min at 4°C. Both ascites supernatant and serum was collected and stored at -80°C. Ascites and serum from mice treated with rAREG (n = 5) and saline ( = 5) were analyzed using a Mouse Cytokine / Chemokine 44-Plex Discovery Assay® Array (MD44) by Eve Technologies (Calgary, Canada), to simultaneously determine the levels of the of the following immune factors: Eotaxin, Erythropoietin, 6Ckine, Fractalkine, G- CSF, GM-CSF, IFNB1, IFNy, IL-la, IL-ip, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL- 11, IL-12p40, IL-12p70, IL-13, IL-15, IL-16, IL-17, IL-20, IP-10, KC, LIF, LIX, MCP-1, M- CSF, MDC, MIG, MIP-la, MIP-ip, MIP-3a, MIP-3B, RANTES, TARC, TNFa, VEGF-A. Each analyte was bound to a differently colored / fluorescent bead to allow for simultaneous detection of all of the aforementioned immune factors in a single assay. A bead analyzer (Bio-Plex 200) first activates the fluorescent dye via laser, then excites the streptavidin-phycoerythrin fluorescent conjugate with a second laser, allowing for measurement of each specific analyte. Each sample was performed in duplicate. Fluorescent immunohistochemistry

[0277] FFPE mouse tumors were baked for 2 h at 65°C and then washed in SafeClear xylene substitute, 100% ethanol, 95% ethanol, 70% ethanol, deoxygenated water, and FTA Hemagglutination buffer for 10 min at each wash on a shaker. Antigen retrieval was then performed via Antigen retrieval solution (IX; Vector Laboratories, H-3300) and heated at 95°C for 20 min. Slides were blocked in 5% horse serum diluted in FTA Hemagglutination buffer and incubated overnight in primary antibody at 4°C. Secondary antibody was then added for 1 h in the dark at room temperature. Between each step slides were washed with FTA Hemagglutination buffer. Lastly, slides were cover-slipped with D API containing mounting medium (Vector Laboratories, H-1200). Primary and secondary antibodies and respective dilutions were as follows:

[0278] CD8 (Proteintech, 29896-1-AP, 1 :50)

[0279] PD-L1 (Proteintech, 66248-1-lg, 1:50)

[0280] CD4 (Proteintech, 677886-1-lg, 1 :50)

[0281] CD45 (Proteintech, 20103-1-AP, 1 :50)

[0282] CD45 (Proteintech, 67786-1-lg, 1 :50)

[0283] Anti-Rabbit DyLight™488 (Vector Laboratories, DL1488, 1 : 1,000) Anti -Mouse DyLight™594 (Vector Laboratories, DL2594, 1 : 1,000)

[0284] Image analysis

[0285] For PD-L1 intensity and CD8+ / CD4+ T cell counts, three and five randomly selected fields per case were selected based on DAPI staining, respectively. Images were acquired via a spinning disk confocal Nikon Eclipse Ti microscope at ax20 objective. Image processing and analysis was performed utilizing ImageJ. For PD-L1 staining analysis, images were thresholded for specific staining and mean intensity was calculated. For CD8+and CD4+T cells, the total number of positive cells co-stained with CD45 and DAPI were counted. Representative images were taken atx20 or x40.

[0286] Statistical analysis Statistical analyses were performed in GraphPad Prism. Student t-tests were performed to determine differences in control and rAREG treated cell lines and mice. All , ’-values reported with the exception of ROSALIND NanoString Analysis were 2-tailed and unadjusted. cBioPortal cBioPortal was used to analyze TCGA ovarian serous cystadenocarcinoma cohorts from the Firehose Legacy ( / / = 617) or Nature 2011 (n = 489) studies. AREG’s association with platinum status (Nature 2011), tumor mutational burden (TMB), mutation count, and Spearman’s rank correlation analysis with genes of interest (Firehose Legacy) were determined.

[0287] Kaplan-Meier plotter analysis

[0288] The Kaplan-Meier Plotter ovarian cancer analysis was used to examine the association of AREG with progression-free survival (PFS) and overall survival (OS) in stage III-IV, grade 3 serous ovarian cancer using either the lower or upper quartile as a cutoff.

[0289] Tumor immune dysfunction and exclusion

[0290] Tumor Immune Dysfunction and Exclusion (TIDE) query gene analysis was employed to examine AREG and cytotoxic T lymphocyte levels and T-cell dysfunction score / z-score of interaction between AREG and cytotoxic lymphocytes (CTLs) in a Cox proportional hazard model. TCGA ovarian cancer cohort was used by TIDE for these analyzes. Briefly, an interaction test within the multivariate Cox-PH regression was applied to identify AREG genomic levels in association with the T cell dysfunction phenotype. Then the Cox-PH survival regression was employed to test how CTL levels interact with AREG in the tumor to affect overall survival outcomes. The linear model Hazard was solved (=aXCTL+&XV+ 7XCTLxV+C) using the Cox-PH regression, where the CTL level is estimated from the bulk-tumor expression average of cytotoxicity T cell markers (CD8A, CD8B, GZMA, G7MB, PRF1). The death hazard within the Cox-PH model was estimated via patient survival clinical outcome, the variable V is the expression level of the candidate gene in the test (in this case AREG). The T cell dysfunction score listed is defined as the Wald test z score, which represents the coefficient <7, dived by its standard error. The -value listed was adjusted using the Benjamini -Hochberg method. Example 1-3: Bioinformatic analysis of AREG in ovarian cancer

[0291] AREG is significantly upregulated in HGSOC patient tumors following NACT exposure compared to matched pre-treatment diagnostic biopsy specimens. Therefore, using publicly available datasets the present study first sought to uncover AREG’s relationship to clinical outcomes in HGSOC. TCGA ovarian cancer cohort analysis revealed that AREG mRNA levels were significantly (p = 0.007) upregulated in patients defined as having a chemoresistant versus sensitive platinum status (Fig. 1A). As approximately 80% of patients are defined as platinum sensitive, this small population of patients defined as chemoresistant exhibits an exceptionally poor survival of 6 months or less Luvero et al. (Crit. Rev. Oncol. Hematol. 140, 28-38). Interestingly, Kaplan Meier curve analysis of publicly available GSE and TCGA databases found no significant association between AREG expression and progression-free survival (PFS) or overall survival (OS; Figs. 11A-11D).

[0292] Further bioinformatic analysis revealed that despite being associated with chemoresistant disease, AREG mRNA levels were significantly (p < 0.05) higher in patients with a higher mutation count and tumor mutational burden (TMB), when stratified by quartile (Figs. 1B-1C). Moreover, Tumor Immune Dysfunction and Exclusion (TIDE) analysis revealed that higher levels ofz .(.7 were significantly (continuous z-score, 3.15, p = 0.00136) associated with a cytotoxic lymphocyte (CTL) dysfunction phenotype (Fig. ID). Overall, these results demonstrate that despite higher AREG levels detected in patient tumors with a higher TMB count, AREG was also associated with chemoresistant disease and T cell dysfunction.

[0293] Example 1-4: AREG exposure leads to tumor intrinsic immune changes that drive ovarian pathogenesis and immune evasion

[0294] Next, in order to recapitulate the high levels of AREG that are seen in post-NACT treated HGSOC tumors, the present study stimulated the HGSOC cell lines OVCAR8 and PEA1 with 200 ng / mL recombinant AREG (rAREG) and respective controls for 2 h. Extracted RNA was subjected to NanoString IO 360 analysis with the goal of broadly capturing tumor intrinsic changes resulting from increased AREG exposure. Unexpectedly, the present study found no significant differences in PEA1 treated cells. However, in OVCAR8 cells, several genes were significantly upregulated relative to control, including CXCL8 (3.49-fold, p = 1.59e- 06), EGR1 (2.47-fold, / ? = 2.87e-06), CXCLJ (2.43-fold, p = 2.63e-05), DUSP5 (2.05-fold, p = 5.08e-09), LIE (2.03-fold, p = 1.41 e-07), CXCL2 (1 .66-fold, p = 2.98e-08), and IL-1 1 (1.44- fold, p = 1.86e-06; Figs. 2A-2B). Furthermore, gene set analysis revealed prominent changes in Wnt, MAPK, Notch, TGF-beta, JAK-STAT, and cytokine and chemokine signaling, as well as changes in cytotoxicity, metabolic stress and myeloid and lymphoid compartment (Fig. 2C), showcasing that increased AREG leads to significant tumor intrinsic immune changes that can contribute to cell proliferation, migration, and angiogenesis, while simultaneously promoting tumor immune suppression.

[0295] Following the NanoString analysis, the present study re-treated PEA1 cells with 200 ng / mL of rAREG and collected RNA at an earlier Ih timepoint. The present study performed quantitative PCR (qPCR) with the goal of examining levels of differentially expressed genes (DEGs) identified in OVCAR8 cells. The present study found that mRNA levels of C CL 1 (2.53-fold, p = 0.036), DUSP5 (2.82-fold, p = 0.008), IL-1 J (4.01-fold, p = 0.012), CXCL2 (3.36-fold, p = 0.029), and IL6 (2.84-fold, p = 0.013), were all significantly increased following Ih rAREG exposure (Figs. 3A-3E), with no significant changes at 2 h stimulation (Figs. 12A-12E), confirming the NanoString analysis observations. The discrepancies in OVCAR8 and PEA1 could potentially be explained by the fact that it is known that OVCAR8 cells harbor Erbb2 and 764 mutations, which could lead to differential rAREG effects. Finally, the present study observed that the DEGs DUSP5 (r = 0.487, p < 0.0001), CXCL2 (r = 0.355, p < 0.0001), and IL-6 (r = 0.401, p < 0.0001) were amongst some of the top correlative genes with AREG in the TCGA ovarian cancer cohort (Figs. 3F-3H), adding a further degree of clinical relevance to the NanoString analysis.

[0296] Examples 1-5: AREG promotes upregulation of downstream EGFR cell growth pathways

[0297] As the EGFR pathway is upstream of numerous cancer cell growth, the present study employed a commercially available proteome profiler array to unbiasedly uncover notable signaling changes in HGSOC cells following rAREG exposure. Interestingly, the present study found that STAT3 expression was upregulated 2.93-fold in OVCAR8 and 1.63-fold in PEA1 cells after only 15 min of exposure (Fig. 4A). Western blot analysis was employed to validate findings and compare phospho-STAT3 (p-STAT3) levels at multiple timepoints, which revealed the highest upregulation of p-STAT3 at 1 h and 4 h in OVCAR8 and PEA1 cells, respectively (Fig. 4B). Moreover, the present study found that programmed-death ligand 1 (PD-L1), a major immune target downstream of the STAT3 pathway, was also increased strikingly starting at 30 min following rAREG treatment in both OVCAR8 and PEA1 cells (Fig. 4B).

[0298] To further examine AREG’s influence on the STAT3 pathway, the present study evaluated secreted levels of the pro-inflammatory and major STAT3 -associated cytokine, IL-6, in media from rAREG stimulated HGSOC cells. At both 2 h and 4 h time points following rAREG exposure, IL-6 levels in conditioned media were 21.1-fold and 49.6-fold higher in OVCAR8 cells, respectively and 12.2-fold higher at both 2 h and 4 h post-rAREG exposure timepoints in PEA1 cells (IL-6 levels reached or exceeded the upper limit of detection at 500 pg / mL; Figs. 4C-4D). As IL-6 possesses the unique ability to induce STAT3 target genes, which in turn produce multifaceted downstream effects that drive tumor cell growth, angiogenesis, invasion, metastasis, and immunosuppression, the results highlight AREG’s indirect pro-tumorigenic effects through IL-6 stimulation. In addition, the present study treated OVCAR8 and PEA1 cells with ruxolitinib, a small molecule JAK / STAT3 inhibitor, which resulted in unaltered AREG levels (Fig. 13), suggesting that STAT3 does not have a bidirectional influence on AREG in HGSOC.

[0299] Finally, Western blot analysis revealed that rAREG exposure led to activation of additional tumor cell growth pathways downstream of EGFR, illustrated by increased p-ERK and p-AKT levels starting at 15 min of exposure in both OVCAR8 and PEA1 cells (Fig. 4E). Taken together, these results showcase that AREG greatly contributes to the activation of numerous cell growth pathways in HGSOC, with predominant effects on STAT3 and its associated targets.

[0300] Example 1-6: AREG reduces cytotoxic immune response in vitro

[0301] AREG leads to tumor intrinsic immune changes that drive ovarian pathogenesis and promote immune evasion. The present study sought to evaluate if increased AREG exposure affects cytotoxic immune responses. To investigate this phenomenon, the present study cocultured OVCAR8 and PEA1 cells with peripheral blood mononuclear cells (PBMCs) that were stimulated with or without rAREG for 24-h. The present study observed significantly (p = 0.001) reduced viability of 38.8% in OVCAR8 cells stimulated with PBMCs + BSA control compared to a 24.2% reduction in viability in cells stimulated with PBMCs + rAREG (Fig. 5A). Similarly, PEA1 cells co-cultured with PBMCs + BSA demonstrated a 22.6% reduction in viability, compared to 14.4% with PBMCs + rAREG (p = 0.007; Fig. 5B). Furthermore, the present study stimulated PBMCs alone with rAREG and performed qPCR analysis, which revealed a significant (p < 0.05) decrease in both IL-2 and IFNy (Figs. 5C-5D), in addition to a trend toward reduced GZMB levels (Fig. 14). Collectively, these studies show that increased AREG dampens PBMCs’ ability to promote tumor cell death potentially through the reduction of cytokines crucially responsible for carrying out cytotoxic immune responses.

[0302] Example 1-7: In vivo AREG exposure predominantly drives immunosuppresive adaptations within the OTIME

[0303] In order to characterize the effect of AREG on the ovarian tumor immune microenvironment, the present study carried out an immunocompetent in vivo study using an ID8p53 C57BL / 6 model in which mice were treated with 400 pg / kg of rAREG or a saline control. Ascites and serum obtained post-euthanasia were submitted for multiplex cytokine and chemokine analysis which revealed a significant (p = 0.026) reduction of IL-2 levels in ascites of mice treated with rAREG compared to saline control mice (Fig. 6A). A similar reduction in IL-2 levels was seen in rAREG treated mouse serum, however this did not reach significance (p = 0.097) (Fig. 6B). This result corroborates the in vitro findings that rAREG exposure leads to reduced IL-2 mRNA levels in PBMCs. Interestingly, the present study also observed that mice treated with rAREG had significantly (p < 0.05) reduced ascites and serum levels of IL-5, a pro- inflammatory cytokine that is primarily responsible for eosinophil production (Figs. 6C-6D). Furthermore, a significant (p = 0.034) reduction of Fractalkine, also known as CX3CL1 was observed in rAREG treated mouse ascites, which has been found to be a key mediator in of cytotoxic T cell immunity and associated with improved prognosis in numerous cancer subtypes (Fig. 6E). In addition, a significant (p = 0.004) reduction in IL-11, an IL-6 associated cytokine was also was also observed (Fig. 6F), which was observed to be upregulated in a tumor-intrinsic setting (Figs. 2A and 3C). Finally, there was a significant (p = 0.028) increase in IL-20 (Fig. 6G), a potent inflammatory cytokine that is classically associated with psoriasis and rheumatoid arthritis but has also been shown to promote tumorigenesis through promoting cellular proliferation and migration.

[0304] In addition to evaluating circulating changes within the OTIME, the present study further observed a significant (p = 0.0212) reduction in the average number of intratumoral CD8+T cells, with an average of five positive CD8+T cells per field in saline tumors compared to one positive CD8+T cell per field in mice exposed to rAREG (Figs. 7A-7B). Conversely, the present study observed no significant changes in CD4+T cell populations (Figs. 8A-8B). Finally, these tumors were also stained for PD-L1, which revealed significantly (f = 0.009) higher mean intensity levels of PD-L1 in tumors treated with rAREG compared to saline control (Figs. 9A- 9B), recapitulating the results in HGSOC cell lines.

[0305] Example 1-8: Combinatorial AREG inhibition and carboplatin promotes synergistic HGSOC cell death

[0306] Finally, the present study have targeted AREG in vitro using an AREG neutralizing antibody (nab) in combination with carboplatin. HGSOC cell lines OVCAR8 and PEA2 (the chemoresistant counterpart to PEA1), were employed for this experiment. Both cell lines were pre-treated with carboplatin for 24 h and then treated with either an IgG control or AREG nab for 48 h. In both cell lines, it was observed that co-treatment with carboplatin and an AREG nab led to a significant (p < 0.005) reduction in cell viability compared to either carboplatin or AREG nab treatment alone, with the most striking reduction in chemoresistant PEA2 cells where combinatorial treatment produced a 73% reduction in viability compared to DMSO control (Figs. 10A-10B). While these cell viability assays were performed in an immune devoid context, it will be pertinent to validate these findings using an immunocompetent in vivo model.

[0307] Example 1-9:

[0308] The present study revealed that enhanced AREG exposure produced multifaceted effects within the OTIME that collectively drive tumor immune evasion. Using bioinformatic analyses the present study failed to observe an association between AREG expression and patient survival. The analysis revealed AREG’s upregulation in chemoresistant ovarian cancer patients, further strengthening the observation that AREG’s significant upregulation in HGSOC patient tumors following exposure to frontline carboplatin and paclitaxel.

[0309] In this present investigation the present study first sought to specifically uncover how elevated AREG expression impacts tumor intrinsic immune changes. Interestingly, the present study found through the NanoString analysis that exposure of HGSOC cells to AREG led to an upregulation in genes related tumor cell growth, angiogenesis, and immune evasion. Most notably, the present study saw significant changes in angiogenic factors CXC 8 and VEGFA, as well as prominent changes in CXCLl and CXCL2, two chemokines known to contribute to chemoresistance via the recruitment of myeloid derived suppressor cells (MDSCs) and known to be associated with ovarian tumorigenesis. Finally, pathway analysis revealed substantial upregulation of genes associated with STAT3 and MAPK / ERK signaling in HGSOC cells. Increased STAT3 and MAPK / ERK activation was confirmed via Western blot, while simultaneously detecting increased AKT pathway activation following rAREG exposure.

[0310] Using in vitro and in vivo models, this investigation has established that AREG compromises cytotoxic immune responses in HGSOC. AREG’s specific function in the context of tumor immunology has been comparatively understudied. The present study have shown for the first time in HGSOC that elevated AREG exposure in vivo leads to a reduction in intratumoral CD8+T cells.

[0311] Out of an extensive panel of chemokines and cytokines, IL-5 was found to be significantly downregulated by in vivo rAREG exposure in both ascites and serum.

[0312] The present study also saw a marked reduction of IL-2 in ascites from mice exposed to rAREG. Overall, the data here shows that AREG treatment leads to the pronounced downregulation of a vital pro-inflammatory, clinically relevant HGSOC cytokine IL-2.

[0313] The present study has shown that even in an immune devoid context, targeting AREG in combination with HGSOC standard of care chemotherapy synergistically promotes HGSOC cell death.

[0314] In conclusion, this study demonstrates that AREG promotes immunomodulation within the OTIME and leads to the reduction of cytotoxic responses, indicating its putative role as a novel HGSOC immune target. In addition, AREG’s function in promoting chemoresistance and PD-L1 immune dysfunction provides strong rationale for combinatorial approaches with HGSOC standard of care chemotherapy and PD-1 based immunotherapy.

[0315] Example 2-1: Combinatorial standard of care chemotherapy and amphiregulin inhibition synergizes to reduce tumor burden in an immunocompetent high grade serous ovarian cancer in vivo model In the study described in Example 2-1, it was discovered that Amphiregulin inhibition synergizes with standard of care chemotherapy to reduce tumor burden in a high grade serous ovarian cancer in vivo model.

[0316] The purpose of this current study was to evaluate the efficacy of targeting AREG alone and in combination with HGSOC standard of care chemotherapy in an HGSOC immunocompetent in vivo model.

[0317] In the present study, ID8p53' ' cells were administered intraperitoneally in C57 / BL6 mice. 20 days post-inoculation, mice were either treated with saline control or carboplatin (30 mg / kg) and paclitaxel (15 mg / kg) once and then treated three times with either a commercially available AREG neutralizing antibody (25ug) or respective IgG control. The four treatment groups were as follows: Saline + IgG, Carboplatin / Paclitaxel + IgG, AREG neutralizing antibody + Saline, and Carboplatin / Paclitaxel + AREG neutralizing antibody. 7-days post treatment all mice were euthanized and tumors, serum, and ascites if applicable were collected post-mortem. Tumors were weighted and the volume of ascites from each mouse was collected. Serum was submitted for multiplex cytokine / chemokine analysis to Eve Technologies and levels of each analyte were correlated with tumor burden.

[0318] While all mice were euthanized 7-days post-treatment, 60% of mice in both the Saline + IgG and Carboplatin / Paclitaxel + IgG groups had met a humane survival endpoint, while only 20% of mice in the AREG neutralizing antibody + Saline and Carboplatin / Paclitaxel + AREG neutralizing antibody groups had. Referring to Figs. 15A-15B, one way ANOVA with Tukey’s multiple comparison test revealed that mean tumor weights were significantly (p<0.05) lower in the combinatorial group, Carboplatin / Paclitaxel + AREG neutralizing antibody (0.47g), compared to Saline+ IgG (0.886g) and Carboplatin / Paclitaxel+ IgG (1.07g). No significant differences were detected between AREG neutralizing antibody + Saline (0.836g) and Carboplatin / Paclitaxel + AREG neutralizing antibody. It was found that serum levels of Eotaxin, a chemokine involved in promoting anti-tumor immune response significantly inversely correlated with tumor burden (r=-0.5661, p=0.009) while tumor burden positively significantly correlated with cytokines and chemokines associated with promoting tumor progression IL-4 (r=0.5226, 0.0187), and IL-12p40 (r=0.4925, p=0.0274).

[0319] This study strongly suggests that AREG represents a novel immunomodulatory therapy in HGSOC. Example 2-2: Combinatorial AREG and Chemotherapy Improved Survival

[0320] Referring to Figs. 16A-16C, the combinatorial AREG and chemotherapy improved survival, and reduced ascites volume in the immunocompetent HGSOC mice model (C57 / BL6 mice intraperitoneally administered with ID8p53’ ' cells).

[0321] Referring to Fig. 16A, Kaplan-Meier survival analysis of mice across all four treatment groups revealed mice treated with a dual AREG and chemotherapy blockade experienced an improved survival.

[0322] Referring to Fig. 16B, ascites volume was significantly lower in mice treated with a dual AREG and chemotherapy blockade compared to IgG control and IgG + carbo / pax.

[0323] Referring to Fig. 16C, no significant differences in tumor weights were identified, however median tumor weights in the dual AREG and chemotherapy weights were the lowest. Ordinary one-way ANOVA with Tukey’s multiple comparisons test were performed for these analyses. *p<0.05; ns, non-significant.

[0324] Example 2-3: Combinatorial AREG and Chemotherapy Resulted in Circulating Immune Changes

[0325] Referring to Figs. 17A-17D, the circulating immune changes resulted from dual AREG and chemotherapy blockade.

[0326] Fig. 17A shows serum eotaxin levels across all treatment groups. Combinatorial AREG and chemotherapy treatment resulted in significantly elevated serum eotaxin levels.

[0327] Fig. 17B shows Spearman’s rank correlation analysis between tumor size (g) and serum IL-4 levels

[0328] Fig. 17C shows Spearman’s rank correlation analysis between tumor size (g) and serum IL-12p40 levels

[0329] Fig. 17D shows Spearman’s rank correlation analysis between tumor size (g) and serum eotaxin levels.

[0330] Example 2-4: Fluorescent IHC analysis of Mast Cells in AREG nab Treated Tumors

[0331] Referring to Figs. 18A-18B, the present study performed a fluorescent IHC analysis of tryptase and CP A3 in AREG nab treated tumors. While chemotherapy with Carbo / pax alone caused a significant increase in Tryptase+ and CPA3+ mast cell in the tumor tissues, the combinatorial AREG and chemotherapy treatment significantly combated the increase in Tryptase+ and CPA3+ mast cells observed from chemotherapy treatment alone.

[0332] Example 2-5: An Expanded 10 day On-Treatment in vivo AREG nab Study in a ID8p53 / _immunocompetent HGSOC model

[0333] Referring to Figs. 19A-19D, the present study performed an expanded 10 day on- treatment in vivo AREG nab study in an immunocompetent ID8p53 HGSOC model.

[0334] Referring to Fig. 19A, the combination of AREG nab and carboplatin / paclitaxel significantly reduced the tumor weight to body weight ratio in the HGSOC mice model as compared to animals treated with AREG nab alone or mock treatment.

[0335] Referring to Figs. 19B-19D, the organ weights of livers (Fig. 19B), kidneys (Fig. 19C), and spleens (Fig. 19D) were similar among the four treatment groups.

[0336] Example 2-6: Combination of AREG Inhibition and Chemotherapy Reduced Cell Viability in HGSOC Patient-Derived Spheroids

[0337] Referring to Fig. 20, the present study performed a cell viability analysis with HGSOC patient spheroids. HGSOC patient spheroid treated alone pre-treated with 300pM of carboplatin and corresponding DMSO control, followed by a 48-hour treatment of 30pM of AREG nab. or corresponding IgG control. The results show that the combination of AREG nab and carboplatin significantly reduced cell viability in the spheroids, as compared to the control and the treatment with carboplatin alone.

[0338] Example 2-7: Dual AREG and Chemotherapy Blockade Is Superior Compared to Dual EGFR and Chemotherapy blockade

[0339] Fig. 21 demonstrates that dual AREG and chemotherapy blockade is superior compared to dual EGFR and chemotherapy blockade, in accordance with some embodiments. HGSOC chemoresistant PEA2 cells treated in combination with an AREG nab (30pM) and carboplatin (300pM) demonstrate a significant reduction in cell viability, compared to carboplatin and IgG (30 pM). EGFR inhibition (gefitinib, 5pM) alone did not produce a significant difference in tumor cell death compared to EGFR inhibition in combination with carboplatin. *p<0.05, *ns, non-significant.

[0340] Example 2-8: Chemotherapy Compounds Increase AREG Levels in Endometrial Cancer Cell Lines

[0341] Referring to Figs. 22A-22B, the present study analyzed the AREG expression in endometrial cancer by performing qPCR analysis of human endometrial cell lines (Figs. 22A) RL95 and (Figs. 22B) KLE stimulated with DMSO control or chemotherapy for 48 hours.

[0342] The chemotherapy compounds (carboplatin / paclitaxel) significantly increased the AREG mRNA levels in both RL95 and KLE cell lines (both are human endometrial carcinoma cell lines).

[0343] Example 2-9: AREG Expression in Metastatic TNBC

[0344] Referring to Figs. 23A-23C and 24A-24B, the present study analyzed the AREG expression in TNBC tissues.

[0345] Referring to Figs. 23A-23B, AREG expression levels are significantly higher in metastatic TNBC tumor tissues (induced by 4T1 cells in mice), as compared to earlier stage TNBC tumor tissues (induced by E0771 cells in mice).

[0346] Referring to Figs. 23C and 24A-24B, treatments with carboplatin / paclitaxel increased AREG levels in both the metastatic 4T1 TNBC cells and the early stage E0771 TNBC tumor tissues.

[0347] ENUMERATED EMBODIMENTS

[0348] In some aspects, the present invention is directed to the following non-limiting embodiments:

[0349] Embodiment 1 : A method of treating, ameliorating and / or preventing a cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of: a compound that down regulates amphiregulin (AREG); a platinum-based chemotherapy agent; and a microtubule-stabilizing chemotherapy agent. Embodiment 2: The method of Embodiment 1, wherein the compound that down regulates AREG downregulates the expression level or the activity of AREG.

[0350] Embodiment 3: The method of Embodiment 1 or 2, wherein the compound that down regulates AREG is at least one selected from the group consisting of: a small molecule inhibitor of AREG; a protein inhibitor of AREG; a nucleic acid that downregulates the expression level and / or activity of AREG by RNA interference, and / or an expression vector expressing the nucleic acid that downregulates the expression level and / or activity of AREG by RNA interference; a ribozyme that downregulates the expression level and / or activity of AREG, and / or an expression vector expressing the ribozyme; an expression vector comprising an expression cassette, wherein the expression cassette expresses CRISPR components that downregulate the expression level and / or activity of AREG by CRISPR knockout or CRISPR knockdown; and a trans-dominant negative mutant protein of AREG, and / or an expression vector that expresses the trans-dominant negative mutant protein of AREG.

[0351] Embodiment 4: The method of Embodiment 3, wherein the compound that down regulates AREG is a neutralizing antibody against AREG.

[0352] Embodiment 5: The method of any one of Embodiments 1-4, wherein the at least one of the following applies:

[0353] (a) the platinum-based chemotherapy agent is at least one selected from the group consisting of: cisplatin, carboplatin, heptaplatin, iproplatin, lobaplatin, miriplatin, nedaplatin, oxaliplatin, picoplatin, phenanthriplatin, PN149, pyriplatin, satraplatin, tetraplatin, and triplatin tetranitrate; or

[0354] (b) the microtubule-stabilizing chemotherapy agent is at least one selected from the group consisting of an epothilone chemotherapy agent, a laulimalide chemotherapy agent, peloruside

[0355] A, and a taxane chemotherapy agent.

[0356] Embodiment 6: The method of any one of Embodiments 1-5, wherein the microtubulestabilizing chemotherapy agent is BMS-310705, cabazitaxel, docetaxel, epothilone A, epothilone

[0357] B, epothilone C, epothilone D, epothilone E, epothilone F, fludelone (KOS-1584), ixabepilone (BMS-247550), paclitaxel, an antibody drug conjugate (ADC) comprising ravtansine (DM4), or an ADC comprising monomethyl auristatin.

[0358] Embodiment 7: The method of any one of Embodiments 1-6, wherein: the compound that down regulates AREG is a neutralizing antibody against AREG; the platinum-based chemotherapy agent is carboplatin; and the microtubule-stabilizing chemotherapy agent is paclitaxel.

[0359] Embodiment 8: The method of any one of Embodiments 1-7, wherein, apart from the compound that down regulates amphiregulin, the platinum-based chemotherapy agent, and the microtubule-stabilizing chemotherapy agent, the subject is not administered any additional active compounds for treating, ameliorating, and / or preventing the cancer.

[0360] Embodiment 9: The method of any one of Embodiments 1-8, wherein the administration comprises a first phase administration and a second phase administration after the first phase administration, and wherein

[0361] (a) the subject is administered the compound that down regulates AREG, the platinumbased chemotherapy agent, and the microtubule-stabilizing chemotherapy agent in the first administration phase; and

[0362] (b) the subject is administered the compound that down regulates AREG but not the platinum-based chemotherapy agent, or the microtubule-stabilizing chemotherapy agent in the second administration phase.

[0363] Embodiment 10: The method of any one of Embodiments 1-9, wherein at least one of the following applies:

[0364] (a) the cancer is an ovarian cancer, optionally a serous ovarian cancer; optionally a highgrade serous ovarian cancer (HGSOC);

[0365] (b) the cancer is a breast a cancer, optionally a triple negative breast cancer (TNBC);

[0366] (c) the cancer is a endometrial cancer, optionally a serous endometrial cancer, optionally a high-grade serous endometrial cancer; or

[0367] (d) the cancer is a carcinosarcoma.

[0368] Embodiment 11 : A kit comprising: a compound that down regulates amphiregulin (AREG); a platinum-based chemotherapy agent; a microtubule-stabilizing chemotherapy agent; and an instructional manual instructing that the compound that down regulates AREG, the platinum-based chemotherapy agent and the microtubule-stabilizing chemotherapy agent are to be administered to a subject in need thereof to treat, ameliorate and / or prevent a cancer.

[0369] Embodiment 12: The kit of Embodiment 11, wherein the compound that down regulates AREG downregulates the expression level or the activity of AREG.

[0370] Embodiment 13: The kit of Embodiment 11 or 12, wherein the compound that down regulates AREG is at least one selected from the group consisting of: a small molecule inhibitor of AREG; a protein inhibitor of AREG; a nucleic acid that downregulates the expression level and / or activity of AREG by RNA interference, and / or an expression vector expressing the nucleic acid that downregulates the expression level and / or activity of AREG by RNA interference; a ribozyme that downregulates the expression level and / or activity of AREG, and / or an expression vector expressing the ribozyme; an expression vector comprising an expression cassette, wherein the expression cassette expresses CRISPR components that downregulate the expression level and / or activity of AREG by CRISPR knockout or CRISPR knockdown; and a trans-dominant negative mutant protein of AREG, and / or an expression vector that expresses the trans-dominant negative mutant protein of AREG.

[0371] Embodiment 14: The kit of Embodiment 13, wherein the compound that down regulates AREG is a neutralizing antibody against AREG.

[0372] Embodiment 15: The kit of any one of Embodiments 11-14, wherein the at least one of the following applies:

[0373] (a) the platinum-based chemotherapy agent is at least one selected from the group consisting of: cisplatin, carboplatin, heptaplatin, iproplatin, lobaplatin, miriplatin, nedaplatin, oxaliplatin, picoplatin, phenanthriplatin, PN149, pyriplatin, satraplatin, tetraplatin, and triplatin tetranitrate; or

[0374] (b) the microtubule-stabilizing chemotherapy agent is at least one selected from the group consisting of an epothilone chemotherapy agent, a laulimalide chemotherapy agent, peloruside A, and a taxane chemotherapy agent. Embodiment 16: The kit of any one of Embodiments 1 1-15, wherein the microtubulestabilizing chemotherapy agent is BMS-310705, cabazitaxel, docetaxel, epothilone A, epothilone B, epothilone C, epothilone D, epothilone E, epothilone F, fludelone (KOS-1584), ixabepilone (BMS-247550), paclitaxel, an antibody drug conjugate (ADC) comprising ravtansine (DM4), or an ADC comprising monomethyl auristatin.

[0375] Embodiment 17: The kit of any one of Embodiments 11-16, wherein: the compound that down regulates AREG is a neutralizing antibody against AREG; the platinum-based chemotherapy agent is carboplatin; and the microtubule-stabilizing chemotherapy agent is paclitaxel.

[0376] Embodiment 18: The kit of any one of Embodiments 11-17, wherein, apart from the compound that down regulates amphiregulin, the platinum-based chemotherapy agent, and the microtubule-stabilizing chemotherapy agent, the kit does not comprise another therapeutic agent for treating, ameliorating, and / or preventing the cancer.

[0377] Embodiment 19: The kit of any one of Embodiments 11-18, wherein the instructional manual instructs that the administration comprises a first phase administration and a second phase administration after the first phase administration, and wherein

[0378] (a) the subject is to be administered the compound that down regulates AREG, the platinum-based chemotherapy agent, and the microtubule-stabilizing chemotherapy agent in the first administration phase; and

[0379] (b) the subject is to be administered the compound that down regulates AREG but not the platinum-based chemotherapy agent, or the microtubule-stabilizing chemotherapy agent in the second administration phase.

[0380] Embodiment 20: The kit of any one of Embodiments 11-19, wherein at least one of the following applies:

[0381] (a) the cancer is an ovarian cancer, optionally a serous ovarian cancer; optionally a highgrade serous ovarian cancer (HGSOC);

[0382] (b) the cancer is a breast a cancer, optionally a triple negative breast cancer (TNBC);

[0383] (c) the cancer is a endometrial cancer, optionally a serous endometrial cancer, optionally a high-grade serous endometrial cancer; or

[0384] (d) the cancer is a carcinosarcoma. The foregoing outlines features of several embodiments so that those skilled in the art may better understand the aspects of the present disclosure. Those skilled in the art should appreciate that they may readily use the present disclosure as a basis for designing or modifying other processes and structures for carrying out the same purposes and / or achieving the same advantages of the embodiments introduced herein. Those skilled in the art should also realize that such equivalent constructions do not depart from the spirit and scope of the present disclosure, and that they may make various changes, substitutions, and alterations herein without departing from the spirit and scope of the present disclosure.

Claims

CLAIMSWhat is claimed is:

1. A method of treating, ameliorating and / or preventing a cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of: a compound that down regulates amphiregulin (AREG); a platinum-based chemotherapy agent; and a microtubule-stabilizing chemotherapy agent.

2. The method of claim 1, wherein the compound that down regulates AREG downregulates the expression level or the activity of AREG.

3. The method of claim 1 or 2, wherein the compound that down regulates AREG is at least one selected from the group consisting of: a small molecule inhibitor of AREG; a protein inhibitor of AREG; a nucleic acid that downregulates the expression level and / or activity of AREG by RNA interference, and / or an expression vector expressing the nucleic acid that downregulates the expression level and / or activity of AREG by RNA interference; a ribozyme that downregulates the expression level and / or activity of AREG, and / or an expression vector expressing the ribozyme; an expression vector comprising an expression cassette, wherein the expression cassette expresses CRISPR components that downregulate the expression level and / or activity of AREG by CRISPR knockout or CRISPR knockdown; and a trans-dominant negative mutant protein of AREG, and / or an expression vector that expresses the trans-dominant negative mutant protein of AREG.

4. The method of claim 3, wherein the compound that down regulates AREG is a neutralizing antibody against AREG.

5. The method of any one of claims 1-4, wherein the at least one of the following applies:(a) the platinum -based chemotherapy agent is at least one selected from the group consisting of: cisplatin, carboplatin, heptaplatin, iproplatin, lobaplatin, miriplatin, nedaplatin, oxaliplatin, picoplatin, phenanthriplatin, PN149, pyriplatin, satraplatin, tetraplatin, and triplatin tetranitrate; or(b) the microtubule-stabilizing chemotherapy agent is at least one selected from the group consisting of an epothilone chemotherapy agent, a laulimalide chemotherapy agent, peloruside A, and a taxane chemotherapy agent.

6. The method of any one of claims 1-5, wherein the microtubule-stabilizing chemotherapy agent is BMS-310705, cabazitaxel, docetaxel, epothilone A, epothilone B, epothilone C, epothilone D, epothilone E, epothilone F, fludelone (KOS-1584), ixabepilone (BMS-247550), paclitaxel, an antibody drug conjugate (ADC) comprising ravtansine (DM4), or an ADC comprising monomethyl auristatin.

7. The method of any one of claims 1-6, wherein: the compound that down regulates AREG is a neutralizing antibody against AREG; the platinum-based chemotherapy agent is carboplatin; and the microtubule-stabilizing chemotherapy agent is paclitaxel.

8. The method of any one of claims 1-7, wherein, apart from the compound that down regulates amphiregulin, the platinum-based chemotherapy agent, and the microtubule-stabilizing chemotherapy agent, the subject is not administered any additional active compounds for treating, ameliorating, and / or preventing the cancer.

9. The method of any one of claims 1-8, wherein the administration comprises a first phase administration and a second phase administration after the first phase administration, and wherein(a) the subject is administered the compound that down regulates AREG, the platinumbased chemotherapy agent, and the microtubule-stabilizing chemotherapy agent in the first administration phase; and(b) the subject is administered the compound that down regulates AREG but not the platinum-based chemotherapy agent, or the microtubule-stabilizing chemotherapy agent in the second administration phase.

10. The method of any one of claims 1-9, wherein at least one of the following applies:(a) the cancer is an ovarian cancer, optionally a serous ovarian cancer; optionally a highgrade serous ovarian cancer (HGSOC);(b) the cancer is a breast a cancer, optionally a triple negative breast cancer (TNBC);(c) the cancer is a endometrial cancer, optionally a serous endometrial cancer, optionally a high-grade serous endometrial cancer; or(d) the cancer is a carcinosarcoma.

11. A kit compri sing : a compound that down regulates amphiregulin (AREG); a platinum-based chemotherapy agent; a microtubule-stabilizing chemotherapy agent; and an instructional manual instructing that the compound that down regulates AREG, the platinum-based chemotherapy agent and the microtubule-stabilizing chemotherapy agent are to be administered to a subject in need thereof to treat, ameliorate and / or prevent a cancer.

12. The kit of claim 11, wherein the compound that down regulates AREG downregulates the expression level or the activity of AREG.

13. The kit of claim 11 or 12, wherein the compound that down regulates AREG is at least one selected from the group consisting of: a small molecule inhibitor of AREG; a protein inhibitor of AREG; a nucleic acid that downregulates the expression level and / or activity of AREG by RNA interference, and / or an expression vector expressing the nucleic acid that downregulates the expression level and / or activity of AREG by RNA interference;a ribozyme that downregulates the expression level and / or activity of AREG, and / or an expression vector expressing the ribozyme; an expression vector comprising an expression cassette, wherein the expression cassette expresses CRISPR components that downregulate the expression level and / or activity of AREG by CRISPR knockout or CRISPR knockdown; and a trans-dominant negative mutant protein of AREG, and / or an expression vector that expresses the trans-dominant negative mutant protein of AREG.

14. The kit of claim 13, wherein the compound that down regulates AREG is a neutralizing antibody against AREG.

15. The kit of any one of claims 11-14, wherein the at least one of the following applies:(a) the platinum-based chemotherapy agent is at least one selected from the group consisting of: cisplatin, carboplatin, heptaplatin, iproplatin, lobaplatin, miriplatin, nedaplatin, oxaliplatin, picoplatin, phenanthriplatin, PN149, pyriplatin, satraplatin, tetraplatin, and triplatin tetranitrate; or(b) the microtubule-stabilizing chemotherapy agent is at least one selected from the group consisting of an epothilone chemotherapy agent, a laulimalide chemotherapy agent, peloruside A, and a taxane chemotherapy agent.

16. The kit of any one of claims 11-15, wherein the microtubule-stabilizing chemotherapy agent is BMS-310705, cabazitaxel, docetaxel, epothilone A, epothilone B, epothilone C, epothilone D, epothilone E, epothilone F, fludelone (KOS-1584), ixabepilone (BMS-247550), paclitaxel, an antibody drug conjugate (ADC) comprising ravtansine (DM4), or an ADC comprising monomethyl auristatin.

17. The kit of any one of claims 11-16, wherein: the compound that down regulates AREG is a neutralizing antibody against AREG; the platinum-based chemotherapy agent is carboplatin; and the microtubule-stabilizing chemotherapy agent is paclitaxel.

18. The kit of any one of claims 11-17, wherein, apart from the compound that down regulates amphiregulin, the platinum-based chemotherapy agent, and the microtubule-stabilizing chemotherapy agent, the kit does not comprise another therapeutic agent for treating, ameliorating, and / or preventing the cancer.

19. The kit of any one of claims 11-18, wherein the instructional manual instructs that the administration comprises a first phase administration and a second phase administration after the first phase administration, and wherein(a) the subject is to be administered the compound that down regulates AREG, the platinum-based chemotherapy agent, and the microtubule-stabilizing chemotherapy agent in the first administration phase; and(b) the subject is to be administered the compound that down regulates AREG but not the platinum-based chemotherapy agent, or the microtubule-stabilizing chemotherapy agent in the second administration phase.

20. The kit of any one of claims 11-19, wherein at least one of the following applies:(a) the cancer is an ovarian cancer, optionally a serous ovarian cancer; optionally a highgrade serous ovarian cancer (HGSOC);(b) the cancer is a breast a cancer, optionally a triple negative breast cancer (TNBC);(c) the cancer is a endometrial cancer, optionally a serous endometrial cancer, optionally a high-grade serous endometrial cancer; or(d) the cancer is a carcinosarcoma.

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