Methods and compositions for the treatment of breast cancer

Abstract

The present disclosure provides compositions and methods for the treatment of cancer. The present disclosure further provides therapeutic and pharmaceutical compositions comprising an immune checkpoint inhibitor and a CD38 inhibitor. Aspects of the disclosure further relate to methods for treating breast cancer, including triple-negative breast cancer.

Description

TITLE OF THE INVENTIONMETHODS AND COMPOSITIONS FOR THE TREATMENT OF BREAST CANCERCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority of U.S. Provisional Appl. Ser. No 63 / 725,849, filed November 27, 2024, the entire disclosure of which is incorporated herein by reference.FIELD OF THE INVENTION

[0002] The present disclosure relates to the field of cancer therapeutics, and more specifically to compositions and methods for targeting the immune and metabolic dysregulation of cancer.BACKGROUND OF THE INVENTION

[0003] Triple -negative breast cancer (TNBC) is a subtype of breast cancer that is characterized by the lack of estrogen receptors (ERs), progesterone receptors (PRs) and human epidermal growth factor receptor 2 (HER2) expression. Such tumors, which are diagnosed in 10-15% of breast cancer patients, are typically aggressive and particularly challenging to treat due to the absence of aforementioned receptors, fewer clinical targets, and fewer treatment options. Women with TNBC have high relapse and low survival rates. Over 75% of women with TNBC succumb to metastases within 5 years after diagnosis. Management of TNBC is a substantial challenge due to its heterogeneity and absence of well-defined molecular targets. TNBCs have high cellular plasticity - a characteristic associated with flexible phenotypic states adopted by cells which fuels intratumoral heterogeneity and contributes to tumor progression, metastasis, and therapy resistance. There is lack of targeted treatments to address tumor cell plasticity. Hence a critical need to elucidate these regulatory networks and develop targeted therapies for TNBC which can be extended to other carcinomas with plasticity.

[0004] Cells can display plasticity through epithelial-to-mesenchymal transition (EMT), a process that alters migratory and invasive capabilities of cells allowing dissemination and metastases and facilitates the generation of cancer stem- like cells (CSCs) that further accelerate tumor initiation, drug resistance and metastasis. During EMT, cells typically transition from expressing epithelial markers like E-cadherin and cytokeratin 8 to mesenchymal markers such as vimentin and fibronectin, and specific EMT-inducing transcription factors (EMT-TF)1US_ACTIVE\131595479W-1including Zebl, Twist, Snail, and Slug. However, recent studies report that tumor cells do not transition completely to the mesenchymal state but rather maintain a “hybrid EM’’ state with epithelial and mesenchymal properties. The epithelial- mesenchymal plasticity (EMP) reflects the capacity of cells to interconvert between phenotypic states and enhances fitness to overcome selective pressures. Acquisition of the hybrid EM state has been linked to tumor heterogeneity and therapy resistance. Hybrid EM cells migrate collectively as clusters playing a pivotal role in seeding metastases. Hybrid EM state also plays a role in modulating the tumor immune microenvironment (TIME). However, critical questions such as the key drivers within hybrid EM TNBC tumors that promote metastasis and immune evasion remain unanswered.

[0005] In recent years, the US Food and Drug Administration (FDA) has granted approval to a few innovative targeted therapies for the treatment of TNBCs. Notably, a recent study indicated growing promise of immune checkpoint blockade (ICB), in conjunction with chemotherapy for prolonged survival in metastatic TNBC patients. This subsequently led to approval of a combined treatment of atezolizumab-mediated PD-L1 blockade with nab- paclitaxel. However, not every tumor expressing PD-L1 responds to anti PD-L1 therapy, and the overall response rates and clinical advantages of ICB in TNBC are limited, which might be due to interference from immune-suppressive cells within the TIME. In addition, there is a need for more in-depth investigation into how these phenotypic states within tumors influence immunotherapy response since cancer cells can toggle between epithelial and mesenchymal states in response to microenvironmental cues. This area of research has been relatively unexplored and deserves greater attention.SUMMARY OF THE INVENTION

[0006] In one aspect, the present disclosure provides methods of treating a subject afflicted with or at risk of developing cancer, the methods comprising administering to the subject an effective amount of an immune checkpoint inhibitor and an effective amount of a CD38 inhibitor. In certain embodiments, the immune checkpoint inhibitor inhibits activity of an immune checkpoint molecule selected from the group consisting of programmed death- 1 (PD- 1), programmed death ligand- 1 (PD-L1), cytotoxic T lymphocyte associated antigen 4 (CTLA- 4), T cell immunoglobulin and mucin protein-3 (TIM-3), lymphocyte activation gene-3 (LAG- 3), programmed death ligand-2 (PD-L2), B and T lymphocyte attenuator (BTLA), T cell immunoreceptor with immunoglobulin and ITIAM domains (TIGIT), PVRIG (CD112R), VISTA (B7-H5), B7 homolog 4 (B7-H4), CD200, CD328, and CD329. In some embodiments,2US_ACTIVE\131595479W-1the immune checkpoint inhibitor is selected from the group consisting of atezolizumab, ipilimumab, nivolumab, pembrolizumab, avelumab, durvalumab, cemiplimab, tislelizumab, toripalimab, dostarlimab, retifanlimab, and relatlimab. In further embodiments, the immune checkpoint inhibitor is atezolizumab. In further embodiments, the CD38 inhibitor is selected from the group consisting of 78c, daratumumab, isatuximab, CD38-dircctcd CAR-T cells, GBR 1342, TAK-079, TAK-169, and MK-0159. In yet further embodiments, the CD38 inhibitor is 78c.

[0007] In certain embodiments, the immune checkpoint inhibitor is atezolizumab and the CD38 inhibitor is 78c. In certain embodiments, the effective amount of the immune checkpoint inhibitor is about 1 mg / kg to about 2500 mg / kg body weight, about 10 mg / kg to about 2000 mg / kg body weight, about 50 mg / kg to about 1750 mg / kg body weight, about 100 mg / kg to about 1500 mg / kg body weight, about 200 mg / kg to about 1200 mg / kg body weight, about 100 mg / kg to about 800 mg / kg body weight, about 100 mg / kg to about 600 mg / kg body weight, about 200 mg / kg body weight to about 500 mg / kg body weight, or about 200 mg / kg to about 400 mg / kg body weight. In some embodiments, the effective amount of the CD38 inhibitor is about 1 mg / kg to about 2500 mg / kg body weight per day, about 10 mg / kg to about 2000 mg / kg body weight per day, about 50 mg / kg to about 1750 mg / kg body weight per day, about 100 mg / kg to about 1500 mg / kg body weight per day, about 200 mg / kg to about 1200 mg / kg body weight per day, about 100 mg / kg to about 800 mg / kg body weight per day, about 100 mg / kg to about 600 mg / kg body weight per day, about 200 mg / kg body weight to about 500 mg / kg body weight per day, or about 200 mg / kg to about 400 mg / kg body weight per day.

[0008] In further embodiments, the cancer is selected from the group consisting of breast cancer, lung cancer, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), brain cancer, glioblastoma, medulloblastoma, skin cancer, melanoma, pancreatic cancer, colorectal cancer, appendiceal cancer, hematopoietic cancer, B-cell lymphoma, leukemia, myeloma, head and neck cancer, prostate cancer, kidney cancer, bladder cancer, liver cancer, esophageal cancer, stomach cancer, thyroid cancer, small bowel adenocarcinoma, hepatobiliary cancer, gynecological cancer, cervical cancer, uterine cancer, and ovarian cancer. In yet further embodiments, the cancer is breast cancer, for example triple-negative breast cancer (TNBC). In certain embodiments, the cancer is resistant to chemotherapy, immunotherapy, or a small molecule inhibitor.

[0009] In certain embodiments, the subject is a mammalian subject, for example a human subject. In further embodiments, administering comprises oral administration, buccal3US_ACTIVE\131595479W-1administration, injection, microneedle administration, vaginal administration, inhalation, intraosseous administration, transnasal application, topical administration, transdermal application, or rectal administration. In certain embodiments, the disclosed methods further comprise administering a second therapy to said subject, and said second therapy may comprise chemotherapy, radiation therapy, immunotherapy, or surgery. In yet further embodiments, the disclosed methods comprise administering a pharmaceutical composition comprising the effective amount of the immune checkpoint inhibitor or the effective amount of the CD38 inhibitor to said subject. For example, administering a first pharmaceutical composition comprising the effective amount of the immune checkpoint inhibitor and a second pharmaceutical composition comprising the effective amount of the CD38 inhibitor to said subject. In another example, the pharmaceutical composition may comprise the effective amount of the immune checkpoint inhibitor and the effective amount of the CD38 inhibitor.

[0010] The instant disclosure further provides a pharmaceutical composition comprising an effective amount of an immune checkpoint inhibitor and an effective amount of a CD38 inhibitor. In certain embodiments, the immune checkpoint inhibitor inhibits activity of an immune checkpoint molecule selected from the group consisting of programmed death- 1 (PD- I), programmed death ligand- 1 (PD-L1), cytotoxic T lymphocyte associated antigen 4 (CTLA- 4), T cell immunoglobulin and mucin protein-3 (TIM-3), lymphocyte activation gene-3 (LAG- 3), programmed death ligand-2 (PD-L2), B and T lymphocyte attenuator (BTLA), T cell immunoreceptor with immunoglobulin and ITIAM domains (TIGIT), PVRIG (CD112R), VISTA (B7-H5), B7 homolog 4 (B7-H4), CD200, CD328, and CD329. In further embodiments, the immune checkpoint inhibitor is selected from the group consisting of atezolizumab, ipilimumab, nivolumab, pembrolizumab, avelumab, durvalumab. cemiplimab, tislelizumab, toripalimab, dostarlimab, retifanlimab, and relatlimab. For example, the immune checkpoint inhibitor may be atezolizumab. In yet further embodiments, the CD38 inhibitor is selected from the group consisting of 78c, daratumumab, isatuximab, CD38- CAR-1' cells, GBR 1342, TAK-079, TAK-169, and MK-0159. For example, the CD38 inhibitor may be 78c. In certain embodiments of the pharmaceutical composition, the immune checkpoint inhibitor is atezolizumab and the CD38 inhibitor is 78c. In some embodiments, the disclosed pharmaceutical composition is formulated for oral administration, buccal administration, injection, microneedle administration, vaginal administration, inhalation, intraosseous administration, transnasal application, topical administration, transdermal application, or rectal4US_ACTIVE\131595479W-1administration. In further embodiments, the pharmaceutical composition is serum-free, endotoxin-free, or sterile.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0012] FIG. 1 demonstrates that CD38 is a key protein associated with hybrid EM breast carcinomas. FIG. 1, Panel A - Immunofluorescence images of syngeneic murine mammary cancer cell lines for the indicated EMT markers. E-cadherin, vimentin, and DAPI (scale bar: 100pm). FIG. 1, Panel B - Immunofluorescence images of syngeneic murine mammary tumor sections corresponding to cell lines in FIG. 1, Panel A, for the indicated EMT markers. E- cadherin, vimentin, and DAPI (scale bar: 50pm). FIG. 1, Panel C - Data from Reverse Phase Protein Array (RPPA). Heatmap showing differentially expressed proteins in Epithelial (E), Mesenchymal (M), and hybrid (EM) murine mammary carcinoma tumors. FDR-adjusted P< 0.05 and N=3 per tumor. FIG. 1, Panel D - Quantification of CD38 expression in EM, E, M tumors. FIG. 1, Panel E - Immunofluorescence images of EM tumor sections (4T1, EMT6) showing CD38 expression on panCK+) tumor cells. (Scale bar: 20pm). FIG. 1, Panel F - Pathways predicted by Ingenuity Pathway Analysis to be activated in hybrid EM tumors, with z score>2 and p<0.05 based on the differential protein expression in FIG. 1, Panel C. Data are represented as mean ± SEM. Significance by one-way ANOVA. *, P<0.05; **, P<0.01; ***, P<0.001; ns, not significant.

[0013] FIG. 2 demonstrates that CD38 is a key protein associated with hybrid EM breast carcinomas. FIG. 2, Panel A - Immunofluorescence images of syngeneic murine mammary cancer cell lines for the indicated EMT markers. Cytokeratin 8, vimentin, and DAPI (nuclear stain in blue) (scale bar: 100pm). FIG. 2, Panel B - Immunofluorescence analysis of syngeneic murine mammary tumors for the indicated EMT markers. Cytokeratin 8, vimentin, and DAPI (nuclear stain) (scale bar: 50pm). FIG. 2, Panel C, Western blot analysis of lysates from syngeneic murine mammary tumors for the indicated EMT markers. FIG. 2, Panel D - Western blot analysis showing CD38 expression in EM, E, and M tumors.

[0014] FIG. 3 demonstrates that CD38 expression is necessary to drive breast cancer invasion and metastasis. FIG. 3, Panel A-C - RT-qPCR (A), western blot analysis (B), and flow5US_ACTIVE\131595479W-1cytometry analysis (C) for CD38 expression in EMT6 cells with stable knockdown by short hairpin RNA (shRNA) compared to scramble control. FIG. 3, Panel D - Representative images from migration assay (left) (scale bar: 200 pm) and bar graph showing number of cells migrated in CD38 KD group compared to SCR control group (right). FIG. 3, Panel E - Representative images from invasion assay (left) (scale bar: 200 pm) and bar graph showing number of cells invaded in CD38 KD group compared to SCR control group (right) FIG. 3, Panel F - Primary tumor volume of tumors arising from EM 1'6 CD38 KD cells compared to SCR control tumors. N= 10 mice per group. FIG. 3, Panel G, H - Analysis to confirm knockdown of CD38 in tumors by western blot (Panel G) and immunohistochemical staining images (left) (scale bar: 20 pm) and quantification showing % positive CD38 cells (right) (Panel H). FIG. 3, Panel I, J - CD38 knockdown cells form a significantly lower number of circulating tumor cell (CTC) colonies compared to control. Representative image (Panel I) and quantification showing number of CTC colonies (Panel J). FIG. 3, Panel K, L - CD38 knockdown cells display significantly reduced potential to form lung metastases compared to control. Representative H&E images with black arrows showing metastatic nodules (scale bar: 5 mm) (Panel K) and quantification showing area of metastases per mouse (Panel L). Data are represented as mean ± SEM. Significance by one-way ANOVA. *, P<0.05; **, P<0.01; ***, P<0.001; ns, not significant.

[0015] FIG. 4 demonstrates that CD38 overexpression promotes breast cancer invasion and metastasis. FIG. 4, Panel A-C - RT-qPCR (A), western blot analysis (B), and flow cytometry analysis (C) for CD38 expression in EMT6 cells with stable overexpression compared to vector control. FIG. 4, Panel D - Representative images from migration assay (left) (scale bar: 200 pm) and bar graph showing number of cells migrated in CD38 KD group compared to SCR control group (right) FIG. 4, Panel E - Representative images from invasion assay (left) (scale bar: 200 pm) and bar graph showing number of cells invaded in CD38 KD group compared to SCR control group ( right) FIG. 4, Panel F - Primary tumor volume of tumors arising from EMT6 CD38 OE cells compared to vector control tumors. N=10 mice per group FIG. 4, Panel G, H - Analysis to confirm overexpression of CD38 in tumors by western blot analysis (Panel G) and immunohistochemical staining images (left) (scale bar: 20 pm) and quantification showing % positive CD38 cells (right) (Panel H). FIG. 4, Panel I, J - CD38 overexpressing cells form a significantly higher number of circulating tumor cell (CTC) colonies compared to control. Representative image (Panel I) and bar graph showing quantification of CTC colonies (Panel J) FIG. 4, Panel K, L - CD38 overexpressing cells display more potential to form lung metastases compared to control as shown by6US_ACTIVE\131595479W-1representative H&E images with black arrows showing metastatic nodules (Panel K) (scale bar: 5 mm) and quantification showing area of metastases (Panel L). Data are represented as mean ± SEM. Significance by the two-tailed unpaired Student’s t-test. *, P<0.05; **, PcO.Ol; ***, P<0.001; ns, not significant.

[0016] FIG. 5 demonstrates that CD38 expression correlates with EMT phenotype in EMT6 model and is necessary to drive breast cancer invasion and metastasis in 4T1 murine models. EIG. 5, Panel A and B - Western blot analysis (Panel A) and flow cytometry analysis (Panel B) for CD38 expression in 4T1 cells with stable knockdown by shRNA compared to scramble control. FIG. 5, Panel C - Representative images from migration assay (left) (scale bar: 200 pm) and bar graph showing number of 4T1 cells migrated in CD38 KD group compared to SCR control group ( right). FIG. 5, Panel D - Representative images from invasion assay (left) (scale bar: 200 pm) and bar graph showing number of 4T1 cells migrated in CD38 KD group compared to SCR control group (right). FIG. 5, Panel E - Primary tumor volume of tumors arising from 4T1 CD38 KD cells compared to SCR control tumors. N=7 mice per group. FIG. 5, Panel F - Bar graph showing quantification of CTC colonies in 4T1 tumor-bearing mice with CD38 knockdown cells. N=5 mice per group. FIG. 5, Panel G and H - 4T1 tumor-bearing mice with CD38 knockdown cells display significantly reduced potential to form lung metastases compared to control. Representative H&E images with black arrows showing metastatic nodules (scale bar: 5 mm) (Panel G) Bar graph quantification showing area of metastases per mouse (Panel H). FIG. 5, Panel I - Representative images from acini formation assay (left) (scale bar: 200 pm) and bar graph showing number of acini formed in CD38 KD group compared to SCR control group (right). FIG. 5, Panel J, K - qRT-PCR (Panel J) and western blot analysis (Panel K) for indicated EMT markers in EMT6 cells with CD38 knockdown Data are represented as mean ± SEM. Significance by one-way ANOVA. *, P<0.05; **, PcO.Ol; ***, P<0.001; ns, not significant.

[0017] FIG. 6 demonstrates that CD38 overexpression promotes breast cancer invasion and metastasis in D2A1 murine cells. FIG. 6, Panel A, B - Western blot analysis (A) and Flow cytometry (B) for CD38 expression in D2A1 cells with stable overexpression compared to vector control. FIG. 6, Panel C - Representative images from migration assay (left) (scale bar: 200 pm) and bar graph showing number of cells migrated in D2A1 CD38 OE group compared to Vector control group (right). FIG. 6, Panel D - Representative images from invasion assay (left) (scale bar: 200 pm) and bar graph showing number of cells invaded in D2A1 CD38 OE group compared to Vector control group (right). Representative images from acini formation assay (left) (scale bar: 200 pm) and bar graph showing number of acini formed in EMT6 CD387US_ACTIVE\131595479W-1OE group compared to vector control group (.right). FIG. 6, Panel G, qRT-PCR (F) and western blot analysis (Panel G) for indicated EMT markers in EMT6 cells with CD38 OE. FIG. 6, Panel H - Flow cytometry for CD38 expression in F3II cells with stable overexpression compared to vector control. FIG. 6, Panel I - Western blot analysis in F3II cells with CD38 OE for the indicated EMT markers. FIG. 6, Panel J - Representative images from acini formation assay (left) (scale bar: 200 pm) and bar graph showing number of acini formed in F31I CD38 OE group compared to vector control group (right); FIG. 6, Panel K - Primary tumor volume of tumors arising from F3II CD38 OE cells compared to vector control tumors. N=10 mice per group. FIG. 6, Panel L - Bar graph showing area of lung metastases per mouse in F3II tumor-bearing mice with CD38 OE. Data are represented as mean ± SEM. Significance by the two-tailed unpaired Student’s t-test. *, P<0.05; **, P<0.01; ***, PcO.OOl; ns, not significant.

[0018] FIG. 7 demonstrates the ability of tumor derived CD38 to regulate T cells in the TME. FIG. 7, Panel A - Bar graph representing the percentage of CD8+T cells after gating on the CD45+population in the indicated tumors. Each data point represents a tumor from one mouse, N=3-6 mice / group and represent pooled values from two independent experiments. FIG. 7, Panel B - Heatmap indicating the percentage of CD8+T cells expressing the indicated cytokines as determined by flow cytometry. FIG. 7, Panel C - Flow cytometry quantification of T cells as percentage of CD8+T cells co-expressing TIM3, PD-1. FIG. 7, Panel D - Flow cytometry quantification of T cells as percentage of CD4+T cells co-expressing CD25, FOXP3. FIG. 7 , Panel E - Bar graph representing the percentage of CD8+T cells after gating on the CD45+population in the indicated tumors. Each data point represents a tumor from one mouse, N=5 mice / group and represent pooled values from two independent experiments. FIG. 7, Panel F - Heatmap indicating the percentage of CD8+T cells expressing the indicated cytokines as determined by flow cytometry. FIG. 7, Panel G - Flow cytometry quantification of T cells as percentage of CD62Lhl8hCD44LowCD4+T cells. FIG. 7, Panel H - Flow cytometry quantification of T cells as percentage of CD4+T cells co-expressing CD25 and FOXP3. FIG. 7 , Panel I - Immunohistochemistry (IHC) staining images of EMT6 tumor sections with CD38 knockdown and overexpression from (left to right) for CD8a (scale bar: 50 pm). FIG. 7, Panel J - Bar graph showing quantification of % positive area of CD8a cells at the tumor core with Image! using the mean of up to 3 representative images from each tumor section, with n = 3-4 tumor sections for each experimental group. FIG. 7, Panel K - Bar graph showing quantification of % positive area of CD8a cells at the tumor periphery with Image! using the mean of up to 3 representative images from each tumor section, with n = 3-8US_ACTIVE\131595479W-14 tumor sections for each experimental group. FIG. 7, Panel L - Immunohistochemical staining of EMT6 tumor sections with CD38 knockdown and overexpression from (left to right) for FOXP3 (scale bar: 50 pm). FIG. 7, Panel M - Bar graph showing quantification of % positive area for FOXP3 cells with ImageJ using the mean of up to 3 representative images from each tumor section, with n = 3-4 tumor sections for each experimental group. Data arc represented as mean ± SEM. Significance by the two-tailed unpaired Student’s t-test and / or one-way ANOVA *, Pc0.05; **, PcO.Ol; PcO.OOl; ns, not significant.

[0019] FIG. 8 demonstrates the ability of tumor derived CD38 to regulate TAMs. FIG. 8, Panel A - Bar graph representing the flow cytometry quantification as percentage of GDI 1B+F480+total macrophages expressing iNOS and MHC II. Each data point represents a tumor from one mouse N=3-6 mice / group and represent pooled values from two independent experiments. FIG. 8, Panel B-D - Tumors lacking CD38 downregulate immunosuppressive molecules as represented by percentage of CD11B+F480+total macrophages expressing PD- L1 (Panel B), CD206 (Panel C) and CSF1R (Panel D). FIG. 8, Panels E-II - Tumors overexpressing CD38 upregulate immunosuppressive molecules as represented by percentage of CD11B+F480+total macrophages expressing A2AR (Panel E), PD-L1 (Panel F), CD206 (Panel G) and CSF1R (Panel H). FIG. 8, Panel I - Immunohistochemical staining images (left) and quantification of tumor sections showing CD206 expression in vector control vs CD38 overexpression tumors (scale bar; 50 pm). FIG. 8 Panel J - Bar graph showing quantification of % positive CD206 cells with ImageJ using the mean of up to 3 representative images from each tumor section, with n = 3-4 tumor sections for each experimental group. Data are represented as mean ± SEM. Significance by the two-tailed unpaired Student’s t-test and / or one-way ANOVA *, P<0.05; **, PcO.Ol; ***, PcO.OOl; ****, PcO.OOOl; ns, not significant.

[0020] FIG. 9 demonstrates tumor immune microenvironment (TIME) changes in 4T1 murine models with CD38 knockdown. FIG. 9, Panel A - Representative flow cytometry plots for CD4+T cells in 4T1 tumor model for CD38 knockdown (left) and bar graph representing CD4+T cell numbers in the indicated tumors (right). N=4 mice per group. FIG. 9, Panel B, Representative flow cytometry plots for CD4+T cells expressing CD25 and FOXP3 in 4T1 tumor model for CD38 knockdown (left) and bar graph representing cell numbers in the indicated tumors (right). N=4 mice per group. FIG. 9, Panel C - Representative flow cytometry plots for GDI lb+Gr-l+myeloid derived suppressor cells (MDSCs) in the 4T1 tumor model for CD38 knockdown (left) and bar graph representing MDSC numbers in the indicated tumors (right). N=4 mice per group. FIG 9, Panel D - Representative flow cytometry plots for percentage of GDI lb+F480+total macrophages expressing MHC II and Arginase-1 in the 4T1 tumor model9US_ACTIVE\131595479W-1for CD38 knockdown (left) and bar graph representing TAM numbers in the indicated tumors ( right). N=4 mice per group. Panel E - Representative flow cytometry plots for percentage of CDl lb+F480+total macrophages expressing iNOS in the 4T1 tumor model for CD38 knockdown (left) and bar graph representing TAM numbers in the indicated tumors (right). N=4 mice per group. Data arc represented as mean ± SEM. Significance by one-way ANOVA. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001; ns, not significant.

[0021] FIG. 10 demonstrates that CD38 expression correlates with suppressive immune cells subsets in breast cancer patients. FIG. 10, Panel A - Immunohistochemical staining for CD38 in tumor microarrays from breast cancer patients (N=123). Representative images of cell staining intensity are shown (scale bar; 50 pm). FIG. 10, Panel B-D - Spearman correlation plots showing positive correlation between tumoral CD38 expression and (B) PD-L1 on CD3+T cells (C) PD-L1 on CD68+macrophages and (D) CD45RO+FOXP3+T cells. FIG. 10, Panel E - Survival graph from Kaplan Meier plotter database representing recurrence -free survival with high and low CD38 expression in patients with EMT-enriched TNBC. FIG. 10, Panel F - Survival graph from Kaplan Meier plotter database representing distant metastases-free survival with high and low CD38 expression in patients with EMT-enriched TNBC.

[0022] FIG. 11 demonstrates that CD38 expression correlates with immune checkpoints and exhibits lower survival trend in breast cancer patients. FIG. 11, Panel A-E - Spearman correlation plots comparing CD38 mRNA expression with indicated markers. The gene expression data, obtained from the UCSC Xena database (http: / / xena.ucsc.edu / ), was derived from 1,092 TCGA-BRCA samples and is presented in log2(TPM+0.001) format. FIG. 11, Panel F - Overall survival in TNBC patients with tumoral CD38 expression. Data from 1 : 1 matched cases by stage, race, subtype, and age + / - 5 years.

[0023] FIG. 12 demonstrates that targeting CD38 in combination with Anti-PD-Ll ICB delays primary tumor growth. FIG. 12, Panel A - Schematic representation of experimental outline for EMT6- tumor bearing mice receiving indicated treatments (N=7 mice / group). FIG. 12, Panel B - Kinetics for primary tumor growth of EMT6 tumor-bearing mice receiving indicated treatments. FIG. 12, Panel C - graph showing comparison between primary tumor weight from EMT6 tumor-bearing mice receiving indicated treatments. FIG. 12, Panel D - Visual representation of tumors at the end of experiment (Day 22). FIG. 12, Panel E - Bar graph showing comparison between CTC colony numbers present in the blood of EMT6 tumorbearing mice receiving indicated treatments. FIG. 12, Panel F - Representative image for CTC colony numbers. FIG. 12, Panel G - Bar graph showing comparison between lung metastases from EMT6 tumor-bearing mice receiving indicated treatments. FIG. 12, Panel H -10US_ACTIVE\131595479W-1Immunohistochemical images for Ki67 in tumor sections from EMT-6 tumor bearing mice. Representative images of cell staining intensity are shown (scale bar; 50 pm) (left) Bar graph showing quantification of % positive area for Ki67 cells with Image.! using the mean of up to3 representative images from each tumor section, with n = 3-4 tumor sections for each experimental group, (right). FIG. 12, Panel I - Immunohistochemical images for cleaved caspase 3 in tumor sections from EMT-6 tumor bearing mice. Representative images of cell staining intensity are shown (scale bar; 50 pm) (left) Bar graph showing quantification of % positive area for cleaved caspase 3 cells with Imagel using the mean of up to 3 representative images from each tumor section, with n = 3-4 tumor sections for each experimental group. ( right).

[0024] FIG. 13 demonstrates that combination treatment induces metabolic and immunological changes in EMT6 tumor- bearing murine models. FIG. 13, Panel A - Bar graph showing relative abundance of NAD+ in vehicle and treatment groups. FIG. 13, Panel B - Bar graph showing quantification of % positive area for CD8a cells within the tumor core quantified with Imagel using the mean of up to 3 representative images from each tumor section, with n = 3-4 tumor sections for each experimental group. FIG. 13, Panel C - Immunohistochemical images for CD69 in tumor sections from EMT6 tumor bearing mice. Representative images of cell staining intensity arc shown (scale bar; 50 pm) (left). Bar graph showing quantification of % positive area for CD69 cells quantified with Imagel using the mean of up to 3 representative images from each tumor section, with n = 3-4 tumor sections for each experimental group. ( right).

[0025] FIG. 14 demonstrates that targeting CD38 in combination with Anti-PD-Ll ICB evokes anti-tumor immune response. FIG. 14, Panel A - Immunohistochemical images for CD8ot in tumor sections from EMT-6 tumor bearing mice. Representative images of cell staining intensity are shown (scale bar; 50 pm) (left) Bar graph showing quantification of % positive area for CD8a cells with ImageJ using the mean of up to 3 representative images from each tumor section, with n - 3-4 tumor sections for each experimental group ( right). FIG. 14, Panel B - Immunohistochemical images for Granzyme B in tumor sections from EMT-6 tumor bearing mice. Representative images of cell staining intensity are shown (scale bar; 50 pm) (left) Bar graph showing quantification of % positive area for Granzyme B cells with ImageJ using the mean of up to 3 representative images from each tumor section, with n = 3-4 tumor sections for each experimental group (right). FIG. 14, Panel C - Immunohistochemical images for FOXP3 in tumor sections from EMT-6 tumor bearing mice. Representative images of cell staining intensity are shown (scale bar; 50 pm) (left) Bar graph showing quantification11US_ACTIVE\131595479W-1of % positive area for F0XP3 cells with ImageJ using the mean of up to 3 representative images from each tumor section, with n = 3-4 tumor sections for each experimental group (right). FIG. 14, Panel C - The working model comparing the effects of CD38 expression versus CD38 targeting on the TIME and metastasis of hybrid EM tumors. Data arc represented as mean ± SEM. Significance by the two-tailed unpaired Student t test. *, P<0.05; **, P<0.01; ***, P<0.001; ns, not significant.DETAILED DESCRIPTION OF THE INVENTION

[0026] Highly metastatic cancers such as triple negative breast cancer (TNBC) are often characterized by tumor cells with both epithelial and mesenchymal traits known as hybrid EM carcinoma cells. Recent studies highlight the enhanced metastatic potential of the hybrid EM phenotype. However, molecular insights and targetable vulnerabilities within cancer subtypes characterized by hybrid EM cells remain elusive.

[0027] The present disclosure demonstrates that EM tumors are enriched in CD38, an immunosuppressive molecule associated with worse clinical outcomes in liquid malignancies. It is further demonstrated that altering tumor cell CD38 expression impacts migratory, invasive, and metastatic capabilities of hybrid EM cells, while abrogation of CD38 expression triggers an immuncstimulatory response thereby preventing the assembly of an immunosuppressive microenvironment in hybrid EM tumors. The present disclosure further demonstrates a positive correlation between CD38 and PD-L1 in TNBC patient samples.

[0028] In view of this, the present disclosure provides compositions and methods for treatment of subjects afflicted with or at risk of developing a cancer, particularly a cancer subtype characterized by EM hybrid tumor cells. In certain embodiments, the present disclosure provides methods of treating a subject afflicted with or at risk of developing cancer comprising administering to the subject an effective amount of a CD38 inhibitor together with an effective amount of an immune checkpoint inhibitor.A. CD38 Inhibitors

[0029] CD38 is an ectoenzyme belonging to the ribosyl cyclase family that is found on the surface on many immune cells including T cells, B cells, natural killer cells. CD38 plays pivotal roles in regulation of extracellular metabolites, intracellular Ca2+, and signaling pathways, however its specific role in tumor cell migration or invasion is not well understood.12US_ACTIVE\131595479W-1

[0030] Inhibitors of CD38 may act through various mechanisms to reduce the activity or function of CD38. In some embodiments, a CD38 inhibitor of the present disclosure may result in decreased cell migration, motility, and / or invasion. As used herein the term “CD38 inhibitor” refers to a compound that may bind to, decrease, prevent, delay activation, inactivate, desensitize, or downrcgulatc the activity or expression of CD38. Inhibitors of CD38 may also include, in certain embodiments, genetically modified versions of CD38, e.g., versions with altered activity, as well as naturally occurring and synthetic antagonists, antibodies, peptides, cyclic peptides, nucleic acids, antisense molecules, ribozymes, small chemical molecules, and the like.

[0031] Assays for inhibitors include, e.g., expressing CD38 in vitro, in cells, or in cell membranes, applying putative inhibitor compounds, and then determining the functional effects on activity, as described herein. Test samples or assays comprising CD38 that are treated with a potential inhibitor may be compared to a control sample lacking the inhibitor in order to determine the extent of inhibition. Control samples to which a test sample or assay is compared may be assigned a relative protein activity value of 100%. Inhibition of CD38 is achieved when the activity value of the test sample relative to the control sample is less than about 80%, including less than about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, and about 1%, including all ranges and values derivable therebetween.

[0032] Any CD38 inhibitor may be used according to the embodiments of the present disclosure. Non- limiting examples of such therapeutic molecules include a protein, a peptide, a polypeptide, an RNA molecule, a peptidornimetic, an siRNA molecule, a gRNA molecule, or a small molecule. Exemplary CD38 inhibitors include, but are not limited to 78c, daratumumab, isatuximab, CD38-directed CAR-T cells, GBR 1342, TAK-079, TAK-169, and MK-0159.B. Immune Checkpoint Inhibitors

[0033] As used herein the terms “immune checkpoint inhibitor,” “ICT,” “immune checkpoint blockade,” and “ICB” refer to a composition that blocks an immune checkpoint. Immune checkpoints are a normal part of the immune system and prevent an overly robust immune response. When an immune checkpoint is blocked by an immune checkpoint inhibitor, immune cells are able to mount a more robust immune response. Such a robust immune response may be beneficial, for example, for killing cancer cells. In some embodiments, an13US_ACTIVE\131595479W-1immune checkpoint inhibitor may promote an increased T cell response. Non-limiting examples of immune checkpoint inhibitors include inhibitors of programmed death- 1 (PD-1), programmed death ligand- 1 (PD-L1), cytotoxic T lymphocyte associated antigen 4 (CTLA-4), T cell immunoglobulin and mucin protein-3 (TIM-3), lymphocyte activation gene-3 (LAG-3), programmed death ligand-2 (PD-L2), B and T lymphocyte attenuator (BTLA), T cell immunoreceptor with immunoglobulin and ITIAM domains (TIGIT), PVRIG (CD112R), VISTA (B7-H5), B7 homolog 4 (B7-H4), CD200, CD328, and CD329.

[0034] In some embodiments, an immune checkpoint inhibitor may be a small molecule inhibitor, an antibody, an antibody fragment, an antigen binding protein, or an antigen binding fragment. The term “antibody” as used herein refers to an intact immunoglobulin of any isotype or an antibody fragment that can compete with an intact antibody for specific binding to the target antigen. An “antigen binding fragment” as used herein refers to a portion of a protein which is capable of binding specifically to an antigen. The term “antigen binding protein” as used herein refers to any protein that binds a specified target antigen. An antigen binding protein includes but is not limited to antibodies and antigen binding fragments. Antibodies of the present disclosure, may include but are not limited to chimeric, humanized, fully human, and bispecific antibodies. An intact antibody may comprise, in certain embodiments, two full-length heavy chains and two full-length light chains. Tn other embodiments, however, an antibody may include fewer chains. For example, antibodies naturally occurring in camelids can comprise only heavy chains. Antibodies can be derived from a single source or may be chimeric. As used herein the term “chimeric antibody” refers to an antibody that comprises portions that are derived from two different antibodies or an antibody variable region derived from one species paired with a constant region from a different species. The antigen binding proteins, antibodies, and binding fragments of the present disclosure may be produced using any technique known in the art. Non-limiting examples of such techniques include production in hybridomas, production by recombinant DNA techniques, and production by enzymatic or chemical cleavage of intact antibodies. An antibody or antigen binding fragment may include, in many embodiments, two full-length heavy chains and two full-length light chains. In some embodiments, an antibody, antigen binding fragment, or an antigen binding protein may include an antibody derivative, an antibody variant, an antibody fragment, or an antibody mutant. Non-limiting examples of antibodies, antigen binding fragments, and antigen binding proteins include monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies,14US_ACTIVE\131595479W-1antibody mimetics, chimeric antibodies, humanized antibodies, human antibodies, antibody fusions, antibody conjugates, peptibodies, and fragments thereof.

[0035] Test samples or assays that are treated with a potential immune checkpoint inhibitor may be compared to a control sample lacking the inhibitor in order to determine the extent of inhibition. Control samples to which a test sample or assay is compared may be assigned a relative protein activity value of 100%. Immune checkpoint inhibition is achieved when the activity value of the test sample relative to the control sample is less than about 80%, including less than about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, and about 1%, including all ranges and values derivable therebetween.

[0036] Any immune checkpoint inhibitor may be used according to the embodiments of the present disclosure. According to the present disclosure, any therapeutic molecule may be used as an immune checkpoint inhibitor. Non-limiting examples of such therapeutic molecules include a protein, a peptide, a polypeptide, an RNA molecule, a peptidomimetic, an siRNA molecule, a gRNA molecule, or a small molecule.

[0037] Exemplary immune checkpoint inhibitors include, but are not limited to, atezolizumab, ipilimumab, nivolumab, pembrolizumab, avelumab, durvalumab, cemiplimab, tislelizumab, toripalimab, dostarlimab, retifanlimab, and relatlimab.C. Therapeutic and Pharmaceutical Compositions

[0038] In certain aspects, the present disclosure provides pharmaceutical and therapeutic compositions comprising an immune checkpoint inhibitor and a CD38 inhibitor. In some embodiments, the immune checkpoint inhibitor and / or CD38 inhibitors of the present disclosure may be combined with a pharmaceutically acceptable carrier. As used herein, a “pharmaceutically acceptable carrier,” “pharmaceutically acceptable adjuvant,” or “adjuvant” refers to reagents, cells, compounds, materials, compositions, and / or dosage forms that are not only compatible with the immune checkpoint inhibitors, CD38 inhibitors, or other agents to be administered therapeutically, but also are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other complication commensurate with a reasonable benefit / risk ratio. Also included may be an agent that modifies the effect of other agents and is useful in preparing a therapeutic compound or pharmaceutical compound or composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable. Such an15US_ACTIVE\131595479W-1agent may be added to a therapeutic composition or pharmaceutical composition to modify for example the cellular target, cellular localization, or cellular uptake of an immune checkpoint inhibitor or a CD38 inhibitor as described herein. Such an agent may include any excipient, diluent, earner, or adjuvant that is acceptable for pharmaceutical use. Such an agent may be non-naturally occurring, or may be naturally occurring, but not naturally found in combination with other agents in the therapeutic or pharmaceutical composition.

[0039] As used herein, a “therapeutic compound” or “therapeutic composition” refers to a composition comprising an immune checkpoint inhibitor and / or a CD38 inhibitor of the present disclosure. In some embodiments, a therapeutic composition has the activity of reducing the activity, function, or expression of immune checkpoint molecules. In certain embodiments, a therapeutic composition has the activity of reducing the activity, function, or expression of CD38 in a subject as described herein. In one embodiment, the composition is capable of reducing, stabilizing, or eliminating tumor growth or tumor progression in a subject. In another embodiment, the composition is capable of reducing, stabilizing, or eliminating tumor size in a subject. In another embodiment, the composition is capable of reducing, stabilizing, or eliminating tumor cell migration, mobility, invasion, or metastasis in a subject. In particular embodiments, a therapeutic composition of the present disclosure is capable of reducing or increasing the expression of genes associated with the oxidationreduction process, G protein-coupled receptor signaling, Rho GTPase signaling, oxidative phosphorylation, the electron transport chain, nucleotide metabolic processes, lipid metabolic processes, carboxylic acid metabolic processes, regulation of the ERK1 / 2 cascade, regulation of the mitotic cell cycle, NADH dehydrogenase complex assembly, cAMP-mediated signaling, or the tricarboxylic acid cycle.

[0040] A compound or composition of the present disclosure is meant to encompass a composition suitable for administration to a subject, such as a mammal, particularly a human subject. In general, a therapeutic composition is sterile, and preferably free of contaminants that are capable of eliciting an undesirable response within the subject (e.g., the compound(s) in the composition are pharmaceutical grade). Therapeutic compositions may be designed for administration to subjects in need thereof via a number of different routes of administration including oral, intravenous, intraarticular, intraarterial, buccal, rectal, parenteral, intraperitoneal, intradermal, intratracheal, intramuscular, subcutaneous, inhalation, vaginal, intraosseous, transnasal, injection, microneedle, topical, and transdermal. The appropriate dosage of a composition, as described herein, may be determined based on the type of disease16US_ACTIVE\131595479W-1to be treated, the severity and course of the disease, the clinical condition of the individual, clinical history, response to the treatment, and the discretion of the attending physician. In some embodiments, therapeutic compositions provided by the present disclosure may include various "unit doses." A unit dose is defined as containing a predetermined quantity of the therapeutic composition. The quantity to be administered, and the particular route and formulation, is within the skill of determination of those in the clinical arts. A unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time. In some aspects, a unit dose comprises a single administrable dose.

[0041] The phrase “effective amount” as used herein refers to a concentration or amount of a therapeutic compound or composition as described herein, a reagent, or another agent, which is effective for producing an intended result, including treatment of cancer as described herein. With respect to the administration of a therapeutic compound as disclosed herein, an effective amount may be any effective range or concentration. The exact dose will depend on the purpose of the treatment, and one of skill in the art will be able to determine such a dose using techniques known in the art.

[0042] Therapeutic compounds or compositions may be provided to a subject in a single dose or multiple doses and as such provided in single-dose or multi-dose containers, such as sealed ampules or vials. Such containers may be sealed to preserve sterility of the composition until use. In general, compositions as described herein may be stored as suspensions, solutions, or emulsions in oily or aqueous vehicles. Alternatively, such a composition may be stored in a freeze-dried condition requiring only the addition of a sterile liquid carrier immediately prior to use. In some embodiments, the therapeutic compounds or compositions may be stored in tablet form.

[0043] In some embodiments, an inhibitor, pharmaceutical composition, or therapeutic composition of the present disclosure may administered at an effective amount of about 1 mg / kg to about 2500 mg / kg body weight, about 10 mg / kg to about 2000 mg / kg body weight, about 50 mg / kg to about 1750 mg / kg body weight, about 100 mg / kg to about 1500 mg / kg body weight, about 200 mg / kg to about 1200 mg / kg body weight, about 100 mg / kg to about 800 mg / kg body weight, about 100 mg / kg to about 600 mg / kg body weight, about 200 mg / kg body weight to about 500 mg / kg body weight, about 200 mg / kg to about 400 mg / kg body weight, about 0.5 mg / kg to about 2500 mg / kg body weight, about 10 mg / kg to about 2000 mg / kg body weight, about 50 mg / kg to about 1750 mg / kg body weight, about 200 mg / kg to about 1000 mg / kg body weight, about 200 mg / kg to about 800 mg / kg body weight, about 40017US_ACTIVE\131595479W-1mg / kg to about 600 mg / kg body weight, about 0.5 mg / kg to about 20 mg / kg body weight, about 0.5 mg / kg to about 15 mg / kg body weight, about 0.5 mg / kg to about 10 mg / kg body weight, about 0.5 mg / kg to about 9 mg / kg body weight, about 0.5 mg / kg to about 8 mg / kg body weight, about 0.5 mg / kg to about 7 mg / kg body weight, about 0.5 mg / kg to about 6 mg / kg body weight, about 0.5 mg / kg to about 5 mg / kg body weight, about 0.5 mg / kg to about 4 mg / kg body weight, about 0.5 mg / kg to about 3 mg / kg body weight, about 0.5 mg / kg to about 2 mg / kg body weight, or about 0.5 mg / kg to about 1.5 mg / kg body weight, including all ranges and values derivable therebetween. In one embodiment, an effective amount of an inhibitor as described herein may be represented as a daily effective amount, a twice daily effective amount, a bi-daily effective amount, a weekly effective amount, or a monthly effective amount.

[0044] Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the patient, the route of administration, the intended goal of treatment (alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance or other therapies a subject may be undergoing.

[0045] As used herein, “subject” or “patient” refers to animals, including humans, who are treated with the inhibitors, therapeutic compounds, or compositions or in accordance with the methods described herein. For diagnostic or research applications, a wide variety of mammals may be suitable subjects, including rodents (e.g., mice, rats, hamsters), rabbits, primates, and swine, such as inbred pigs. In particular embodiments, a subject in need of therapy may be any subject who comprises a cell that exhibits a mutation in a subunit of a SWI / SNF chromatin remodeling complex as described herein. Non-limiting examples of diseases or conditions that a subject of the present disclosure may be afflicted with or at risk of developing include breast cancer (including triple negative breast cancer), lung cancer, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), brain cancer, glioblastoma, medulloblastoma, skin cancer, melanoma, pancreatic cancer, colorectal cancer, appendiceal cancer, hematopoietic cancer, B-cell lymphoma, leukemia, myeloma, head and neck cancer, prostate cancer, kidney cancer, bladder cancer, liver cancer, esophageal cancer, stomach cancer, thyroid cancer, small bowel adenocarcinoma, hepatobiliary cancer, gynecological cancer, cervical cancer, uterine cancer, and ovarian cancer.

[0046] In certain embodiments, a subject of the present disclosure may comprise a cancer that is resistant to chemotherapy, immunotherapy, or a small molecule inhibitor. Chemotherapy,18US_ACTIVE\131595479W-1immunotherapy, and small molecule inhibitors for the treatment of cancer are known in the art and any such agent may be used according to the embodiments of the present disclosure. Non-limiting examples of such agents include alkylating agents, antimetabolites, anti-tumor antibiotics, topoisomerase inhibitors, mitotic inhibitors, immune checkpoint inhibitors, immunomodulators, cytokines, monoclonal antibodies, CAR T therapy, oncolytic virus therapy, cancer vaccines, adoptive cell therapy, targeted antibodies, kinase inhibitors, epigenetic modulators, enzyme inhibitors, and chemokine receptors antagonists.

[0047] A composition, as described herein, may include, in particular embodiments, a combination of therapeutic agents. In some embodiments, a composition as described here may be administered as a single composition or as more than one composition. Different compositions as provided herein, in certain embodiments, may be administered by the same route of administration or by different routes of administration.

[0048] A pharmaceutical composition of the present disclosure may comprise, in certain embodiments, in immune checkpoint inhibitor and / or a CD38 inhibitor. Any immune inhibitor and any CD38 inhibitor known in the art may be used in a pharmaceutical composition of the present disclosure. Non-limiting examples of such CD38 inhibitors include, but are not limited to 78c, daratumumab, isatuximab, CD38-directed CAR-T cells, GBR 1342, TAK-079, TAK-169, and MK-0159. Non-limiting examples of such immune checkpoint inhibitors include inhibitors of programmed death- 1 (PD-1), programmed death ligand- 1 (PD-L1), cytotoxic T lymphocyte associated antigen 4 (CTLA-4), T cell immunoglobulin and mucin protein-3 (TIM-3), lymphocyte activation gene-3 (LAG-3), programmed death ligand-2 (PD-L2), B and T lymphocyte attenuator (BTLA), T cell immunoreceptor with immunoglobulin and ITIAM domains (TIGIT), PVRIG (CD112R), VISTA (B7-H5), B7 homolog 4 (B7-H4), CD200, CD328, and CD329. In certain examples, immune checkpoint inhibitors include, but are not limited to, atezolizumab, ipilimumab, nivolumab, pembrolizumab, avelumab, durvalumab, cemiplimab, tislelizumab, toripalimab, dostarlimab, retifanlimab, and relatlimab.

[0049] In certain embodiments, an immune checkpoint inhibitor or CD38 inhibitor of the present disclosure may include a therapeutic peptide or nucleic acid. Any therapeutic peptide or nucleic acid known in the art to reduce the activity, function, or expression of an immune checkpoint molecule or CD38 may be used according to the embodiments of the present disclosure. In particular embodiments, the therapeutic peptide or therapeutic nucleic acid may function in or encode a protein or small RNA molecule associated with reduction of an19US_ACTIVE\131595479W-1immune checkpoint molecule or CD38 activity, expression, or function. A pharmaceutical composition of the present disclosure may comprise, in some embodiments, a targeting molecule or a nanoparticle for delivery of a therapeutic peptide or nucleic acid. In one embodiment, the targeting molecule or nanoparticle may be cell-specific or tissue-specific. Numerous such targeting molecules and nanoparticlcs arc known in the art and any such targeting molecule may be used according to certain embodiments of the present disclosure. In certain embodiments, a composition of the present disclosure may be modified with or conjugated to a peptide, a protein, a colloidal molecule, or a polymer to facilitate delivery or adsorption. The pharmaceutical composition of the present disclosure, in some embodiments, may be serum-free, endotoxin-free, or sterile.

[0050] A peptide or polynucleotide molecule for use according to the compositions of the present disclosure may, in some embodiments, be a recombinant peptide or nucleic acid. As used herein, the term “recombinant” refers to a polynucleotide molecule, protein, or cell that is not naturally present, or is not naturally present in the same form or structure and was created by human intervention. In one embodiment, a recombinant polynucleotide may be a DNA molecule or may be an RNA molecule. A recombinant polynucleotide molecule or a recombinant polypeptide molecule or protein may comprise, in certain embodiments, a combination of two or more polynucleotide or polypeptide sequences that do not naturally occur together in the same manner, such as a polynucleotide molecule or protein that comprises at least two polynucleotide or protein sequences that are operably linked but heterologous with respect to each other. As used herein the term “heterologous” refers to a polynucleotide molecule or protein that is not naturally present or is not naturally present in the same form or structure and was created by human intervention. For example, a heterologous polynucleotide molecule or protein may not naturally occur in the cell being transformed or may be expressed in a manner or genomic context that differs from the natural expression pattern or genomic context found in the cell being transformed. The heterologous polynucleotide molecule or protein, in some embodiments, may be overexpressed in the cell being transformed. In certain embodiments, a recombinant polynucleotide molecule, protein, construct, or vector may comprise any combination of two or more polynucleotide or protein sequences in the same molecule which are heterologous to one another, such that the combination is man-made and not normally found in nature. As used herein, the phrase “not normally found in nature” means not found in nature without human intervention. A recombinant polynucleotide or protein molecule, may comprise, for example, polynucleotide or protein sequences that are separated20US_ACTIVE\131595479W-1from other polynucleotide or protein sequences that exist in proximity to each other in nature. A recombinant polynucleotide or protein molecule may also comprise, for example, polynucleotide or protein sequences that are adjacent to or contiguous with other polynucleotide or protein sequences that are not naturally in proximity with each other. Such a recombinant polynucleotide molecule, protein, or expression construct may also refer to a polynucleotide or protein molecule or sequence that has been genetically engineered or constructed outside of a cell. For example, a recombinant polynucleotide molecule may comprise any engineered or man-made plasmid, vector, or expression construct, and may include a linear or circular DNA molecule. Such plasmids, vectors, and expression constructs may comprise, for example, various maintenance elements including, but not limited to, a heterologous promoter sequence, a prokaryotic origin of replication, or a selectable marker.

[0051] In certain aspects, a therapeutic composition of the present disclosure may comprise an immune checkpoint inhibitor or a CD38 inhibitor of the present disclosure and a second therapeutic agent or a detectable label. Non-limiting example of therapeutic agents or detectable labels that may be used according to the present disclosure include a chemotherapeutic agent, an immunotherapeutic agent, a mitochondrial therapeutic agent, a neurotherapeutic agent, a metabolic therapeutic agent, or a radiotherapeutic agent. Nonlimiting examples of detectable labels that may be used according to embodiments of the present disclosure include a paramagnetic ion, a radioactive isotope, a fluorochrome, an NMR- detectable agent, or an X-ray imaging agent. As used herein, the term "label" refers to a directly or indirectly detectable compound or composition that is conjugated directly or indirectly to the composition to be detected. In certain embodiments, an inhibitor, a polynucleotide molecule, protein, or cell may be labeled to generate a labeled composition. In particular embodiments, labeled compositions also include sequences which are conjugated a polynucleotide molecule that will provide a signal upon expression of the inserted sequences, such as green fluorescent protein (GFP) and the like. The label may be detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition that is detectable. Labels may be suitable for small scale detection or for high-throughput screening. As such, suitable labels include, but are not limited to radioisotopes, fluorochromes, chemiluminescent compounds, dyes, and proteins, including enzymes. Labels may be simply detected or may be quantified. In certain embodiments, labels that may be quantified provide numerically reportable value. In luminescence or fluorescence assays, the detectable response may be21US_ACTIVE\131595479W-1generated directly using a luminophore or fluorophore associated with an assay component involved in binding, or indirectly using a luminophore or fluorophore associated with another (e.g., reporter or indicator) component.

[0052] In certain embodiments, the compositions and methods for treating an individual described herein may be combined with any other composition or method of treatment known in the art. The compositions and methods may be administered in any suitable manner known in the art. For example, a first and a second therapeutic agent or inhibitor may be administered sequentially (at different times) or concurrently (at the same time). In some aspects, a first and a therapeutic agent or inhibitor may be administered in separate compositions. In certain embodiments, a first and a second cancer treatment or inhibitor may be administered in the same composition.

[0053] Non-limiting examples of additional treatment modalities that may be included in combination with the compositions and methods provided herein include a therapeutic agent or surgery. In specific embodiments, the methods and compositions of the present disclosure may be combined with other therapies directed towards the treatment of cancer as described herein.

[0054] The term "about" is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. The use of the term "or" in the claims is used to mean "and / or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive. When used in conjunction with the word "comprising" or other open language in the claims, the words "a" and "an" denote "one or more," unless specifically noted otherwise. The terms "comprise," "have," and "include" are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as "comprises," "comprising," "has," "having," "includes," and "including," are also open-ended. For example, any method that "comprises," "has," or "includes" one or more steps is not limited to possessing only those one or more steps and also covers other unlisted steps. Similarly, any system or method that "comprises," "has," or "includes" one or more components is not limited to possessing only those components and covers other unlisted components.

[0055] Other objects, features, and advantages of the present disclosure are apparent from detailed description provided herein. It should be understood, however, that the detailed description and any specific examples provided, while indicating specific embodiments of the22US_ACTIVE\131595479W-1disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description. Any embodiment of the present disclosure may be used in combination with any other embodiment described herein.

[0056] All references herein are incorporated herein by reference in their entirety.EXAMPLES

[0057] The following examples are included to illustrate embodiments of the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of the invention. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.Example 1: TNBC Cell Line- Derived Hybrid EM tumors Show a Significant Enrichment of CD38.

[0058] The expression of epithelial and mesenchymal markers was evaluated in a panel of syngeneic murine breast cancer cell lines (FIG. 1, Panel A, FIG. 2, Panel A) as well as histopathological sections of corresponding tumor sections (FIG. 1, Panel B, FIG. 2, Panel B). These experiments revealed that cell lines and the derived tumors can be classified into three phenotypes: epithelial-like (E), mesenchymal-like (M), and hybrid epithelial / mesenchymal-like (EM). E cell lines and derived tumors were characterized by strong enrichment of E-cadhcrin and cytokcratin 8 (CK8) with little or no expression of vimentin, M cell lines and derived tumors did not express epithelial markers and expressed a high level of vimentin. The hybrid EM cell lines and derived tumors co-expressed E and M markers as shown by two different combinations; E-cadherin and vimentin (FIG. 1 Panel A and B), CK8 and vimentin, (FIG. 2, Panel A and B). These findings were corroborated by western blot analyses of E, EM, and M tumors (FIG. 2, Panel C).23US_ACTIVE\131595479W-1

[0059] To identify novel proteins enriched in the hybrid EM state, a reverse phase protein array (RPPA) was performed on whole tumor protein lysates from E, EM, and M murine models (FIG. 1, Panel C). Of the 480 proteins detected, CD38 was found to be significantly enriched in the lysates of hybrid EM tumors relative to E and M tumor lysates (FIG. 1, Panel D). This was further validated by western blot (FIG. 2, Panel D) and immunofluorescence staining (FIG. 1, Panel E) that revealed CD38 expression on tumor cells specifically. Upon detailed analysis under the expertise of a pathologist, tumor specific CD38 expression was identified and quantified among E, EM an M murine tumor sections (Table 1). It was found that the hybrid EM tumors showed an overall higher expression of tumoral CD38 compared to E and M groups, which complements the observation from the RPPA dataset. Furthermore, the Ingenuity Pathway Analysis on the entire set of differentially expressed proteins predicted the upregulation / downregulation of several pathways, out of which the top 5 relevant pathways were identified (FIG. 1, Panel F) namely, p53 signaling, VEGF signaling, IL- 15 production, IL-8 signaling and NAD signaling pathway. Interestingly, CD38 is a critical component of the NAD signaling pathway, which was the most upregulated amongst the pathways. This complements the observation from RPPA data showing enrichment of CD38 preferentially in hybrid EM tumors. While the role of CD38 has been extensively studied in hematological malignancies, consequences of CD38 expression within a solid tumor microenvironment remain elusive, more specifically, within a TNBC setting. The primary focus became to assess the impact of this multifaceted enzyme on the immune remodeling and metastasis of hybrid EM tumors and ultimately scrutinize the molecular, clinical, and therapeutic relevance of targeting this protein in TNBCs, an uncharted realm.24US_ACTIVE\131595479W-1Table 1. Analysis of Tumoral CD38 expression in murine samples.Example 2: CD38 Expression on Tumor Cells is Necessary to Promote Invasion and Metastasis.

[0060] To determine whether CD38 is necessary for invasion and metastasis, two important features of cells enriched in EMT, shRNA-mediated knockdown of CD38 was performed in hybrid EM cell line EMT6 (FIG. 3, Panel A-C). EMT6 is a murine mammary carcinoma cell line that has intermediate levels of CD38 expression (FIG. 3, Panel C, FIG. 4, Panel C) which makes it an excellent model system to characterize knockdown and overexpression. Additionally, in immunocompetent syngeneic mice, EMT6 cells form tumors and spontaneous metastases, primarily to lungs, which makes it a relevant model to study invasion, metastases as well as the immune response to tumor formation. Hence, CD38 was independently inhibited by using two different shRNAs in EMT6 and 4T1 murine cells and their knockdown was confirmed by RT-qPCR (FIG. 3, Panel A), western blot (FIG. 3, Panel B, FIG. 5, Panel A), and flow cytometry (FIG. 3, Panel C, FIG. 4, Panel B). CD38 knockdown25US_ACTIVE\131595479W-1caused a significant decrease in migration (FIG. 3, Panel D, FIG. 5, Panel C) and invasion (FIG. 3, Panel E, FIG. 5, Panel D). To assess cell behavior upon CD38 knockdown in 3D, which is more physiologically relevant, matrigel assays were utilized and formation of acini was quantified. It was observed that CD38 knockdown cells formed significantly fewer acini compared to scramble control (FIG. 5, Panel I). To study the effect of CD38 knockdown on metastasis, the EMT6 scramble control and CD38 knockdown cells were implanted orthotopic ally in syngeneic WT Balb / cJ mice (n=10 mice / group), and tumor growth was monitored for 4 weeks. There was no significant difference in tumor growth between the scramble control and the CD38 knockdown cohorts (Fig. 3, Panel F). The maintenance of CD38 knockdown in murine tumors was confirmed by western blotting and immunohistochemistry (Fig. 3, Panel G and H). Despite no differences in primary tumor volume, CD38 knockdown significantly hampered the invasive capacity of primary tumor cells as confirmed by the reduced number of circulating tumor cell (CTC) colonies in blood isolated from the scramble and knockdown murine models (Fig. 3, Panel I and J). Concomitantly, decreased metastatic ability was observed by quantifying the area of lung metastatic nodules and by hematoxylin and eosin (H&E) stained images (Fig. 3, Panel K and L). Similarly, 4T1 murine models (n=8 mice / group) implanted with CD38 knockdown cells showed a non-significant difference in primary tumor growth (Fig. 5, Panel E) but significantly decreased number of CTC colonies (Fig. 5, Panel F) and area of lung metastatic nodules (Fig. 5, Panel G and H). This data strongly suggests that CD38 expression is required for breast cancer metastasis. To elucidate whether CD38 regulates the hybrid epithelial / mesenchymal (E / M) state, the expression levels of key EMT markers (E-cadherin, CK8, Zebl, Vimentin) were assessed upon CD38 knockdown in EMT6 cells. CD38 knockdown significantly decreased CD38 expression, which was accompanied by an increase in mRNA expression of E-cadherin and CK8 and a decrease in Zebl and Vimentin expression (Fig. 5, Panel J). This indicates a shift towards an epithelial phenotype. Western blot analysis confirmed the mRNA expression data, showing increased E-cadherin and CK8 levels and decreased Zebl and Vimentin levels upon CD38 knockdown (Fig. 5, Panel K).Example 3: CD38 expression on tumor cells is sufficient to promote invasion and metastasis.

[0061] Since CD38 expression on tumor cells is necessary to drive invasion and metastasis, it was next ascertained whether CD38 is also sufficient to drive invasion and metastasis. For this, CD38 was constitutively overexpressed by lentiviral transduction of CD38 cDNA in26US_ACTIVE\131595479W-1EMT6 and D2A1 murine cells and overexpression was confirmed by RT-qPCR (Fig. 4, Panel A), western blot (Fig. 4, Panel B, Fig. 6, Panel A), and flow cytometry (Fig. 4, Panel C, Fig. 6, Panel B). Increased CD38 expression enhanced the migratory potential of EMT6 cells (Fig. 4, Panel D) and also increased their invasiveness (Fig. 4, Panel E). This effect was analogous to the phenotype observed in another hybrid EM murine mammary carcinoma cell line- D2A1 , where CD38 overexpression significantly increased cell migration and invasion (Fig. 6, Panel C and D), respectively.

[0062] To assess cell behavior upon CD38 overexpression in 3D, matrigel assays were utilized and the formation of acini was quantified. It was observed that cells overexpressing CD38 formed significantly higher number of acini compared to vector control (Fig. 6, Panel E). CD38 has been reported to interact with CD31 (PECAM-1), a cell adhesion molecule involved in endothelial cell migration and angiogenesis. The CD38- CD31 interaction could enhance cell-cell adhesion and signaling pathways that promote cell migration and invasion. Given the specific enrichment of CD38 in the hybrid EM dataset (Fig. 1, Panel D), the direct correlation between CD38 and the hybrid EM state was investigated. To elucidate this relationship, EMT markers in EMT6 cells were evaluated, and it was observed that CD38 overexpression maintained the mRNA (Fig. 6, Panel F) and protein (Fig. 6, Panel G) expression of both epithelial markers (E-cadherin and CK8) and mesenchymal markers (Vimentin, Zebl, and Snail). These findings suggest that CD38 plays a role in maintaining the hybrid EM phenotype. This phenotype is associated with enhanced migratory and invasive properties of tumor cells, indicating that CD38 may regulate the EMT process by preserving the hybrid EM state. To investigate the metastatic ability of CD38 in vivo, CD38 constitutively overexpressing cells were implanted orthotopically in WT Balb / cJ mice and allowed tumor cells to spontaneously metastasize. The CD38 overexpression cohort (n=10 mice / group) showed no significant difference compared to control tumors, however an increased trend in primary tumor volume at the end of experiment was noted (Fig. 4, Panel F). It was confirmed that the primary tumors maintained CD38 protein overexpression (Fig. 4, Panel G and H). Although CD38 overexpression had no significant effect on primary tumor growth, it did have a significant impact on entry of primary tumor cells into the circulation as confirmed by quantifying the number of CTC colonies in blood isolated from control and CD38 overexpression murine models (Fig. 4, Panel I and J). Remarkably, CD38 overexpression showed an increased number of lung metastatic nodules as determined by quantifying the lung metastatic nodules and H&E-stained images (Fig. 4,27US_ACTIVE\131595479W-1Panel K and L). Constitutive overexpression of CD38 was achieved in F3II murine cells, which naturally lack CD38 expression but have abundant E-cadherin and cytokeratin 8 (previously classified as an E cell line), via lentiviral transduction of CD38 cDNA. This overexpression was confirmed through flow cytometry (Fig. 6, Panel H) and western blot (Fig. 6, Panel I). Detailed western blot analysis for EMT markers revealed that F3II cells overexpressing CD38 maintained a hybrid EM phenotype, co-expressing both epithelial (E- cadherin, cytokeratin 8, EpCAM) and mesenchymal (Vimentin, Zebl) markers (Fig. 6, Panel I). Similar to the EMT6 model, F3II cells with CD38 overexpression formed significantly more acini in a 3D Matrigel setting compared to vector controls (Fig. 5, Panel J). In vivo studies demonstrated that the CD38 overexpression cohort (n=10 mice / group) exhibited an increased trend in primary tumor volume (Fig. 6, Panel K) as well as the area of lung metastatic nodules (Fig. 6, Panel L). In summary, these findings indicate the sufficiency of CD38 to promote invasion and metastasis in EMT6, D2A1 and F3II murine models.Example 4: CD38 expression on tumor cells alters the localization and function of T cells in the TIME to drive metastasis.

[0063] Residence of cancer cells in alternating epithelial and mesenchymal states alters their immunomodulatory properties and drives their resistance to immunotherapy by inducing immune cell exhaustion. Thus, it was next investigated whether CD38 knockdown induces changes in TIME to a less immunosuppressive microenvironment and whether overexpression of CD38 induces a more immunosuppressive microenvironment in hybrid EM murine models. In addition to reduction of metastases, the immune-competent syngeneic hosts bearing CD38 knockdown tumors showed a significant increase in the numbers of CD8+T cells relative to the SCR control tumors (Fig. 7, Panel A).

[0064] Furthermore, the CD8+T cells present in each of these knockdown tumors expressed higher levels of CD8+T-cell effector markers granzyme B, IFNy and TNFa (Fig. 7, Panel B) and significantly decreased exhaustion markers PD-1 and TIM-3 (Fig. 7, Panel C) relative to the CD8+T cells present in control tumors. Strikingly, increase in the numbers of CD8+T cells was accompanied by a decrease in numbers of immunosuppressive Tregs (CD4+CD25+FOXP3+) (Fig. 7, Panel D). In contrast, overexpression tumors had significantly lower CD8+T cell number as (Fig. 7, Panel E). Additionally, CD8+T cells in the overexpression tumors showed a dampened effector response compared to control, as evidenced by lower expression of CD8+ T-cell effector markers (Fig. 7, Panel F) and fewer effector memory CD4+T cells (Fig. 7, Panel G) with significant induction of Tregs (Fig. 7, Panel H).28US_ACTIVE\131595479W-1Mobilization of T cells to the tumor core is crucial for a favorable anti-tumor immune response. Accordingly, the localization of CD8+T cells was investigated and it was found that CD38 knockdown tumors recruited CD8+T cells into the tumor core in comparison to overexpression tumors which restricted most CD8+T cells to periphery as confirmed by immunohistochemical staining of tumor sections (Fig. 7, Panel I) and analysis of % positive CD8+T cells in tumor core (Fig. 7, Panel J) versus tumor periphery (Fig. 7, Panel K). It was confirmed that the presence of Tregs in CD38 knockdown and overexpression tumor sections via immunohistochemical staining for FOXP3 (Fig. 7, Panel L and M). Taken together, these observations indicate that knockdown of CD38 in hybrid EM tumors induces an immunostimulatory response with heightened numbers and infiltration of cytotoxic CD8+T cells which are crucial to mount an anti-tumor immune response.Example 5: TAMs in the TIME display phenotypic changes in a CD38-dependent manner.

[0065] To further characterize the TME, the numbers and localization of tumor associated macrophages (TAMs) were examined, which play pivotal roles in influencing various immune cells in the TME including CD8+T cells. TAMs exist along a spectrum of Ml (anti-tumor) to M2 (pro-tumor) phenotypic states exhibiting plasticity. It was found that tumors with CD38 knockdown showed increased expression of Ml -like macrophage markers such as iNOS and MHC-II relative to SCR control tumors (Fig. 8, Panel A). Strikingly, increased expression levels of the aforementioned Ml -like markers were accompanied by significant decreases in TAMs expressing immunosuppressive markers such as Programmed Death-Ligand 1 (PD-L1) (Fig. 8, Panel B), CD206 (Fig. 8, Panel C), and Colony Stimulating Factor Receptor 1 (CSF1R) (Fig. 8, Panel D). Increased PD-L1 expression on TAMs has been shown to affect CD8+T cell metabolism and induce direct CD8+T cell apoptosis via PD-1 / PD-L1 axis while the CSF1 / CSF-1R axis is vital to recruitment and accumulation of immunosuppressive TAMs that are adept at polarizing from Ml -like / anti -tumor to the M2-likc / pro-tumor phenotype. Hence, the presence of such surface molecules can be an important contributor to immunesuppression and further accelerate metastasis. As mentioned earlier, adenosine is generated in CD38’s enzymatic reaction cascade via CD203 A and CD73 enzymes. The adenosine receptor A2 (A2AR) binds to adenosine in the TME and further accelerates tumor development, anti-inflammatory response, and immune tolerance. Most notably, tumors with CD38 overexpression significantly upregulated A2AR expression on surface of TAMs (Fig. 8, Panel E). This was complemented by subsequent29US_ACTIVE\131595479W-1increases in the aforementioned immunosuppressive markers on TAMs such as PD-L1, CD206, CSF1R (Fig. 8, Panel F-H).

[0066] Immunohistochemical staining for CD206 expression confirmed the observation from flow cytometry analyses and underscores the higher infiltration of CD206+TAMs in ovcrcxprcssion tumors compared to knockdown tumors (Fig. 8, Panel I and J). Tumors arising from hybrid EM 4T1 CD38 knockdown cells were also analyzed using flow cytometry and similar changes in the TIME were found; an increase in CD4+T cells (Fig. 9, Panel A) and decrease in Tregs (Fig. 9, Panel B). Lack of CD38 expression in 4T1 cancer cells led to a decrease in myeloid-derived suppressor cells (MDSCs) (Fig. 9, Panel C). Interestingly, 4T1 murine model displayed reduced M2-like TAMs characterized by MHC-IIloArg-lhlexpression (Fig. 9, Panel D) and increased Ml-like TAMs characterized by iNOS expression (Fig. 9, Panel E). These analyses expose the pivotal contribution of CD38 tumoral expression to inducing suppressive TIME in hybrid EM tumors.Example 6: CD38 expression correlates with the presence of PD-L1+immune cells in breast cancer patients

[0067] To determine the clinical implications of tumoral CD38, human breast carcinoma specimens were analyzed for CD38 expression by immunohistochemical staining (Table 2). Among the 123 breast cancer tissue microarrays, it was found that 13% (n=16 positive cases, n=107 negative cases) of cases exhibited positive staining for tumoral CD38 expression (Fig. 10, Panel A, Table 3). Despite the less frequent tumoral occurrence, a positive correlation was observed between tumoral CD38 expression and the presence of PD-E1+immune cells in the TIME of patient samples (Table 4), specifically, PD-L1+CD3+immune cells (Fig. 10, Panel B), PD-L1+CD68+macrophages (Fig. 10, Panel C), andCD3+CD45RO+FOXP3+T cells (Fig. 10, Panel D). This is consistent with experimental data regarding the tumor immune microenvironment (TIME), specifically, increased PD-L1 expression on macrophages (Fig. 8, Panel B). PD-L1+immune cell subsets are implicated in immunosuppression in cancer patients. Additionally, it was determined from publicly available breast cancer datasets that CD38 expression positively correlates with immune checkpoints such as PD-L1 (Fig. 11, Panel A), TIM3 (Fig. 11, Panel B), LAG3 (Fig. 11, Panel C), ICOS (Fig. 11, Panel D) and CTLA-4 (Fig. 11 , Panel E) which suggests a possible link between CD38 and CD8+T cell exhaustion as well as active suppressive immune response. Moreover, a trend toward reduced overall survival was observed in patients with high tumoral CD38 expression (Fig. 11, Panel F). A negative correlation was also observed between CD3830US_ACTIVE\131595479W-1expression and recurrence-free and metastases -free survival specifically in patient datasets with EMT-enriched mesenchymal subtype of TNBC (Fig. 10, Panel E and F). Taken together, these results strongly suggest an important role of tumoral CD38 in altering the TIME and TNBC patient survival.31US_ACTIVE\131595479W-1Table 2. TMA patient characteristics.32US_ACTIVE\131595479W-1Table 3. CD38 Tumoral expression descriptive statistics.33US_ACTIVE\131595479W-1Table 4. Mean, standard deviation and Spearman correlation matrix with tumoral CD38 H score.34US_ACTIVE\131595479W-1Example 7: Targeting CD38 in combination with anti-PD-Ll ICB reduces primary tumor growth and potentiates an anti-tumor immune response.

[0068] Given the statistically strong positive correlation between CD38 and PD-L1 expression in TNBC patient samples, it was hypothesized that coinhibition of CD38 and PD-L1 in breast cancer would impact growth and the TIME of hybrid EM tumors. To test this, EMT6 breast carcinoma cells were orthotopically injected into immunocompetent, syngeneic hosts and the mice were treated with the anti PD-L1 mAB, Atezolimumab, 5mg / kg and / or with the CD38 inhibitor 78c, lOmg / kg (Fig. 12, Panel A). 78c is a small molecule known to act as “NAD booster” with an improved pharmacokinetic profile compared to other molecules. EMT6 tumor-bearing mice that received either anti-PD-Ll treatment or 78c treatment showed a reduction in tumor volume and weight compared to control mice. Strikingly, mice that received the combination treatments (i.e., anti-PD-Ll + 78c) had the lowest tumor volume (Fig. 12, Panel B) and weight (Fig. 12, Panel C and D). Taken together, these results indicate that CD38 inhibition has the potential to enhance immune checkpoint inhibitor efficacy and underscores the importance of CD38 inhibition in sensitizing heterogenous tumors frequently observed in TNBC patients.

[0069] Metastasis is the major cause of breast cancer-related deaths. Hence, the functional role of CD38 was evaluated in regulating the presence of CTCs in blood circulation and metastatic colonization of hybrid EM cells to lungs. EMT6-tumor bearing mice treated with 78c, atezolizumab (Atz), and a combination had a significant reduction in the number of CTC colonies in blood (Fig. 12, Panel E and F) and fewer lung metastatic nodules relative to the lungs of mice that received either control or single-agent treatments (Fig. 12, Panel G). Markers for tumor proliferation (Ki67) and apoptosis (cleaved Caspase 3) were also assessed by immunohistochemical staining of tumor sections. Compared to controls and single drugtreatment groups, the combination treatment resulted in the lowest levels of proliferation (Fig. 12, Panel H) and the highest numbers of apoptotic tumor cells (Fig. 12, Panel 1). Given that 78c is recognized as an NAD+ booster, the relative abundance of NAD was evaluated in vehicle and treatment groups. The results indicated that tumors treated with 78c and the combination therapy exhibited significantly higher NAD levels compared to the vehicle groups and even Atz (Fig. 13, Panel A). This elevation in NAD levels suggests a potential therapeutic advantage of 78c in enhancing antitumor responses. NAD and its metabolites are critical for various cellular processes, including energy metabolism, DNA repair, and gene expression regulation. Increasing NAD availability in tumor cells can disrupt metabolic homeostasis,35US_ACTIVE\131595479W-1rendering them more susceptible to therapeutic interventions. This highlights the uniqueness of CD38 therapeutics and provides a rationale for combining CD38 inhibitors with immunotherapies .

[0070] Furthermore, the localization of CD8+ T cells within the tumor sections was assessed and increases in single-drug treatment groups were found compared to controls (Fig. 14, Panel A), but only combination treated group had CD8+T cells infiltrated into the tumor core (Fig. 13, Panel B). Such localization of CD8+T cells is crucial for a favorable anti-tumor immune response. Complementing this was a sharp increase in the expression of granzyme B+ T cells infiltrated into the combination treated tumors (Fig. 14, Panel B). Moreover, CD8+T cells from the combination-treated tumors expressed significantly higher levels of CD69, a marker associated with early activation of T cells (Fig. 13, Panel C). Lastly, there was a steep decline in FOXP3+Tregs within the combination-treated group (Fig. 14, Panel C). Overall, these data indicate an anti-tumor immune response in both single-drug and combination treatment with a much-enhanced effect within the combination group with CD8+T cells that are not only active but also cytotoxic. This illustrates the therapeutic benefit of co-targeting CD38 with PD-L1.

[0071] These results indicate that a hybrid EM phenotypic state harbors enrichment of the immunosuppressive molecule, CD38, influencing the assembly of an immunosuppressive microenvironment thereby accelerating the steps (migration, invasion, CTCs) in the metastatic cascade. Additionally, targeting CD38 is critical for regulating the susceptibility of these tumors to antitumor immune attack and elimination by anti-PD-Ll ICB therapy (Fig. 14, Panel C).* * *

[0072] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments or aspects, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.36US_ACTIVE\131595479W-1

Claims

CLAIMS1. A method of treating a subject afflicted with or at risk of developing cancer, the method comprising administering to the subject an effective amount of an immune checkpoint inhibitor and an effective amount of a CD38 inhibitor.

2. The method of claim 1 , wherein the immune checkpoint inhibitor inhibits activity of an immune checkpoint molecule selected from the group consisting of programmed death- 1 (PD- 1), programmed death ligand- 1 (PD-L1), cytotoxic T lymphocyte associated antigen 4 (CTLA- 4), T cell immunoglobulin and mucin protein-3 (TIM-3), lymphocyte activation gene-3 (LAG- 3), programmed death ligand-2 (PD-L2), B and T lymphocyte attenuator (BTLA), T cell immunoreceptor with immunoglobulin and ITIAM domains (TIGIT), PVRIG (CD112R), VISTA (B7-H5), B7 homolog 4 (B7-H4), CD200, CD328, and CD329.

3. The method of claim 1, wherein the immune checkpoint inhibitor is selected from the group consisting of atezolizumab, ipilimumab, nivolumab, pembrolizumab, avelumab, durvalumab, cemiplimab, tislelizumab, toripalimab, dostarlimab, retifanlimab, and relatlimab.

4. The method of claim 3, wherein the immune checkpoint inhibitor is atezolizumab.

5. The method of claim 1 , wherein the CD38 inhibitor is selected from the group consisting of 78c, daratumumab, isatuximab, CD38-directed CAR-T cells, GBR 1342, TAK- 079, TAK-169, and MK-0159.

6. The method of claim 5, wherein the CD38 inhibitor is 78c.

7. The method of claim 1, wherein the immune checkpoint inhibitor is atezolizumab and the CD38 inhibitor is 78c.

8. The method of claim 1, wherein the effective amount of the immune checkpoint inhibitor is about 1 mg / kg to about 2500 mg / kg body weight, about 10 mg / kg to about 2000 mg / kg body weight, about 50 mg / kg to about 1750 mg / kg body weight, about 100 mg / kg to about 1500 mg / kg body weight, about 200 mg / kg to about 1200 mg / kg body weight, about 100 mg / kg to about 800 mg / kg body weight, about 100 mg / kg to about 600 mg / kg body weight, about 200 mg / kg body weight to about 500 mg / kg body weight, or about 200 mg / kg to about 400 mg / kg body weight.

9. The method of claim 1, wherein the effective amount of the CD38 inhibitor is about 1 mg / kg to about 2500 mg / kg body weight per day, about 10 mg / kg to about 2000 mg / kg body37US_ACTIVE\131595479W-1weight per day, about 50 mg / kg to about 1750 mg / kg body weight per day, about 100 mg / kg to about 1500 mg / kg body weight per day, about 200 mg / kg to about 1200 mg / kg body weight per day, about 100 mg / kg to about 800 mg / kg body weight per day, about 100 mg / kg to about 600 mg / kg body weight per day, about 200 mg / kg body weight to about 500 mg / kg body weight per day, or about 200 mg / kg to about 400 mg / kg body weight per day.

10. The method of claim 1, wherein the cancer is selected from the group consisting of breast cancer, lung cancer, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), brain cancer, glioblastoma, medulloblastoma, skin cancer, melanoma, pancreatic cancer, colorectal cancer, appendiceal cancer, hematopoietic cancer, B-cell lymphoma, leukemia, myeloma, head and neck cancer, prostate cancer, kidney cancer, bladder cancer, liver cancer, esophageal cancer, stomach cancer, thyroid cancer, small bowel adenocarcinoma, hepatobiliary cancer, gynecological cancer, cervical cancer, uterine cancer, and ovarian cancer.

11. The method of claim 10, wherein the cancer is breast cancer.

12. The method of claim 11, wherein the cancer is triple-negative breast cancer (TNBC).

13. The method of claim 1, wherein the cancer is resistant to chemotherapy, immunotherapy, or a small molecule inhibitor.

14. The method of claim 1, wherein the subject is a mammalian subject.

15. The method of claim 14, wherein the subject is a human subject.

16. The method of claim 1, wherein said administering comprises oral administration, buccal administration, injection, microneedle administration, vaginal administration, inhalation, intraosseous administration, transnasal application, topical administration, transdermal application, or rectal administration.

17. The method of claim 1, further comprising administering a second therapy to said subject.

18. The method of claim 17, wherein said second therapy is selected from the group consisting of chemotherapy, radiation therapy, immunotherapy, and surgery.

19. The method of claim 1 , further comprising administering a pharmaceutical composition comprising the effective amount of the immune checkpoint inhibitor or the effective amount of the CD38 inhibitor to said subject.38US_ACTIVE\131595479W-120. The method of claim 1, further comprising administering a first pharmaceutical composition comprising the effective amount of the immune checkpoint inhibitor and a second pharmaceutical composition comprising the effective amount of the CD38 inhibitor to said subject.

21. The method of claim 19, wherein the pharmaceutical composition comprises the effective amount of the immune checkpoint inhibitor and the effective amount of the CD38 inhibitor.

22. A pharmaceutical composition comprising an effective amount of an immune checkpoint inhibitor and an effective amount of a CD38 inhibitor.

23. The pharmaceutical composition of claim 22, wherein the immune checkpoint inhibitor inhibits activity of an immune checkpoint molecule selected from the group consisting of programmed death-1 (PD- 1), programmed death ligand- 1 (PD-L1), cytotoxic T lymphocyte associated antigen 4 (CTLA-4), T cell immunoglobulin and mucin protein-3 (TIM-3), lymphocyte activation gene-3 (LAG-3), programmed death ligand-2 (PD-L2), B and T lymphocyte attenuator (BTLA), T cell immunoreceptor with immunoglobulin and ITIAM domains (T1GIT), PVRIG (CD112R), VISTA (B7-H5), B7 homolog 4 (B7-H4), CD200, CD328, and CD329.

24. The pharmaceutical composition of claim 22, wherein the immune checkpoint inhibitor is selected from the group consisting of atezolizumab, ipilimumab, nivolumab, pembrolizumab, avelumab, durvalumab, cemiplimab, tislelizumab, toripalimab, dostarlimab, retifanlimab, and relatlimab.

25. The pharmaceutical composition of claim 24, wherein the immune checkpoint inhibitor is atezolizumab.

26. The pharmaceutical composition of claim 22, wherein the CD38 inhibitor is selected from the group consisting of 78c, daratumumab, isatuximab, CD38-directed CAR-1' cells, GBR 1342, TAK-079, TAK-169, and MK-0159.

27. The pharmaceutical composition of claim 26, wherein the CD38 inhibitor is 78c.

28. The pharmaceutical composition of claim 22, wherein the immune checkpoint inhibitor is atezolizumab and the CD38 inhibitor is 78c.

29. The pharmaceutical composition of claim 22, wherein said pharmaceutical composition is formulated for oral administration, buccal administration, injection, microneedle39US_ACTIVE\131595479W-1administration, vaginal administration, inhalation, intraosseous administration, transnasal application, topical administration, transdermal application, or rectal administration.

30. The pharmaceutical composition of claim 22, wherein the pharmaceutical composition is serum-free, endotoxin-free, or sterile.40US_ACTIVE\131595479W-1

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