WO2022109227A1

WO2022109227A1 - Combined cancer therapy of b7-h3 and cd47 immune checkpoint inhbitior and methods of use

Info

Publication number: WO2022109227A1
Application number: PCT/US2021/060025
Authority: WO
Inventors: Julide Tok Celebi; Min Hsu; C. Martin TIPHAINE; E. Ramon PARSONS
Original assignee: Icahn School Of Medicine At Mount Sinai
Priority date: 2020-11-19
Filing date: 2021-11-19
Publication date: 2022-05-27

Abstract

The present disclosure generally relates to compositions and methods for treating a solid tumor in a subject, wherein administering compositions having a combination of an inhibitor of B7-H3 (CD276) and an inhibitor of CD47-SIRPα can inflame a cold tumor microenvironment.

Description

COMBINED CANCER THERAPY OF B7-H3 AND CD47 IMMUNE CHECKPOINT INHBITIOR AND METHODS OF USE

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application 63/115,699 filed November 19, 2020, the disclosure of which is hereby incorporated by reference in its entirety.

GOVERNMENTAL RIGHTS

[0002] This invention was made with government support under CAI 77940 awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD

[0003] The present disclosure generally relates to compositions and methods for treating a solid tumor in a subject, wherein administering compositions having a combination of an inhibitor of B7-H3 (CD276) and an inhibitor of CD47-SIRPa can inflame a cold tumor microenvironment.

BACKGROUND

[0004] Immunotherapies have shown to be effective for treating a variety of immune-related diseases, including cancers. However, many cancers appear to be resistant to such therapies due to a number of immunological obstacles. These can include the ability of tumors to foster a tolerant microenvironment and the activation of a plethora of immunosuppressive mechanisms, which may act in concert to counteract effective immune responses. For instance, tumor-associated macrophages, marrow-derived suppressor cells, tumor-associated neutrophils, cancer-associated fibroblasts, and regulatory T cell interactions actively promote tumorigenesis.

[0005] Significant advances in checkpoint inhibitor development and use have indicated that such immunotherapies could be a hopeful therapeutic option in for treating solid cancerous tumors. Whereas some subjects positively respond to such therapies, a large percentage of cancer patients never demonstrate a clinical response or stabilized disease after treatment with one or more checkpoint inhibitors. As such, reversing the immunosuppressed microenvironment and switching to an inflamed and immune infiltrated phenotype, identifying immune signaling axes that mechanistically function via divergent pathways, and ultimately finding effective therapeutic combinations are of interest in the cancer field. Accordingly, there is a need in the field to develop methods of treating solid tumors, such as melanomas, that are resistant to currently available immunotherapies.

SUMMARY

[0006] An aspect of the present disclosure provides methods for treating a solid tumor in a subject. In some embodiments, methods can include administering to a subject in need thereof an effective amount a composition, the composition having an inhibitor of B7-H3 (CD276) and an inhibitor of CD47-SIRPa. In accordance with these embodiments, compositions herein may be pharmaceutical compositions comprising an inhibitor of B7-H3 (CD276), an inhibitor of CD47- SIRPa, and/or at least one pharmaceutically acceptable excipient. In some embodiments, a subject in need thereof can be non-responsive to anti-programmed cell death 1/programmed cell death ligand 1 (PD1/PDL1) therapy. In some embodiments, a subject can be a human patient having a solid tumor selected from the group of pancreatic ductal adenocarcinoma (PDA), colorectal cancer (CRC), melanoma, breast cancer, lung cancer, upper and lower gastrointestinal malignancies, squamous cell head and neck cancer, genitourinary cancer, ovarian cancer, sarcomas, or any combination thereof. In some embodiments, a subject can be a human patient having a melanoma. [0007] In certain embodiments, methods disclosed herein can include administration of a composition and/or pharmaceutical composition herein wherein the effective amount of the composition and/or pharmaceutical composition herein may be sufficient to activate the innate immune system. In some embodiments, methods disclosed herein can include administration of a composition and/or pharmaceutical composition herein wherein the effective amount of the composition and/or pharmaceutical composition herein may be sufficient to activate at least one population of innate immune system cells. In some embodiments, populations of innate immune system cells activated by the methods herein can be macrophages, dendritic cells (DCs) and natural killer (NK) cells. In some embodiments, methods disclosed herein can include administration of a composition and/or pharmaceutical composition herein wherein the effective amount of the composition and/or pharmaceutical composition herein may be sufficient to reduce tumor volume. [0008] In certain embodiments, methods herein can include administration of an inhibitor of B7-H3. In some embodiments, methods herein can include administration of an inhibitor of B7- H3 wherein the inhibitor of B7-H3 can be a small molecule, a peptide, a polynucleotide, a genetically modified cell or an antibody, an antibody fragment, or any combination thereof. In some embodiments, an inhibitor of B7-H3 can be an antibody. In some embodiments, an inhibitor of B7-H3 can be a full-length antibody or an antigen-binding fragment thereof. In some embodiments, an inhibitor of B7-H3 can be a human antibody or a humanized antibody. In some embodiments, an inhibitor of B7-H3 can be an isolated nucleic acid or set of nucleic acids, which collectively encodes an antibody. In some embodiments, an inhibitor of B7-H3 can be enoblituzumab, omburtamab, orlotamab, BP-102, DS-7300, 8H-9, TCB-005, YBL-015, or any combination thereof. In some embodiments, an inhibitor of B7-H3 can be a B7-H3-specific chimeric antigen receptor T (CAR-T) cell.

[0009] In certain embodiments, methods herein can include administration of an inhibitor of CD47-SIRPa. In some embodiments, an inhibitor of CD47-SIRPa can be a CD47-ligand inhibitor, a SIRPa-receptor inhibitor, or any combination thereof. In some embodiments, methods herein can include administration of a CD47-ligand inhibitor. In some embodiments, methods herein can include administration of a CD47-ligand inhibitor wherein the CD47-ligand inhibitor can be a small molecule, a peptide, a polynucleotide, a genetically modified cell or an antibody, an antibody fragment or a combination thereof. In some embodiments, a CD47-ligand inhibitor can be an antibody. In some embodiments, a CD47-ligand inhibitor can be a full-length antibody or an antigen-binding fragment thereof. In some embodiments, a CD47-ligand inhibitor can be a human antibody or a humanized antibody. In some embodiments, a CD47-ligand inhibitor can be an isolated nucleic acid or set of nucleic acids, which collectively encodes an antibody. In some embodiments, a CD47-ligand inhibitor can be a CD47-specific chimeric antigen receptor T (CAR- T) cell. In some embodiments, methods herein can include administration of a SIRPa-receptor inhibitor. In some embodiments, methods herein can include administration of a SIRPa-receptor inhibitor wherein the SIRPa-receptor inhibitor can be a small molecule, a peptide, a polynucleotide, a genetically modified cell or an antibody, an antibody fragment or a combination thereof. In some embodiments, a SIRPa-receptor inhibitor can be an antibody. In some embodiments, a SIRPa-receptor inhibitor can be a full-length antibody or an antigen-binding fragment thereof. In some embodiments, a SIRPa-receptor inhibitor can be a human antibody or a humanized antibody. In some embodiments, a SIRPa-receptor inhibitor can be a SIRPa -specific chimeric antigen receptor T (CAR-T) cell. In some embodiments, an inhibitor of CD47-SIRPa can be RRx-001, a dinitroazetidine derivative, Hu5F9-G4, CC-90002, TTI-621, ALX148, SRF231, SHR-1603, IBI188, ST-1901, SGNCD-47M, or any combination thereof.

[0010] Another aspect of the present disclosure provides methods for inflaming a cold tumor microenvironment. In certain embodiments, a method of inflaming a cold tumor microenvironment as disclosed herein can include administering to the cold tumor microenvironment a composition having an inhibitor of B7-H3 (CD276) and an inhibitor of CD47-SIRPa. In some embodiments, a tumor having a cold tumor microenvironment can be a solid tumor. In some embodiments, a tumor having a cold tumor microenvironment can be pancreatic ductal adenocarcinoma (PDA), colorectal cancer (CRC), melanoma, breast cancer, lung cancer, upper and lower gastrointestinal malignancies, squamous cell head and neck cancer, genitourinary cancer, ovarian cancer, and/or sarcoma. In some embodiments, a tumor having a cold tumor microenvironment can be melanoma. In some embodiments, a tumor having a cold tumor microenvironment can have at least one of B7-H3 overexpression, overactive P-catenin signaling, or any combination thereof. In some embodiments, a tumor having a cold tumor microenvironment can have at least one genetic mutation comprising BRAF^V600E, loss of PTEN, or a combination thereof.

[0011] In certain embodiments, methods of inflaming a cold tumor microenvironment disclosed herein can increase of at least one interferon, increase of CD8+ T cells, increase of CD44 expression in CD4+ cells, in CD8+ cells, or both, increased TNFalpha expression in T cells, or any combination thereof.

[0012] Another aspect of the present disclosure provides pharmaceutical compositions for treating a solid tumor in a subject. In some embodiments, pharmaceutical compositions for treating a solid tumor in a subject as disclosed herein can include an inhibitor of B7-H3 (CD276) and an inhibitor of CD47-SIRPa. In some embodiments, pharmaceutical compositions disclosed herein can further include at least one pharmaceutically acceptable excipient. In some embodiments, pharmaceutical compositions disclosed herein can include an inhibitor of B7-H3 (CD276) and an inhibitor of CD47-SIRPa in a dosage amount that may restore CD8+ T cell effector function in a cold tumor microenvironment. [0013] In certain embodiments, kits are provided for transport, storage and use in treating or reducing the size of, reducing expansion of a tumor, and/or inflaming a cold tumor microenvironment as disclosed herein.

BRIEF DESCRIPTION OF THE FIGURES

[0014] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to the drawing in combination with the detailed description of specific embodiments presented herein.

[0015] Figs. 1A-1K are images depicting that B7-H3 was overexpressed in melanomas and regulated that tumor cell-mediated signals that influenced the tumor microenvironment. Fig. 1A shows a representative image of an immunoblot detection of B7-H3 protein expression in a panel of human primary (n = 12) and metastatic (n = 15) melanoma cell lines, and human melanocytes (n = 1). Fig. IB shows a graph that quantifies B7-H3 mRNA expression in a panel of human primary (n = 12) and metastatic (n = 15) melanoma cell lines, and human melanocytes (n = 1) as assessed by quantitative real-time PCR (n = 28). Fig. 1C shows a graph of B7-H3 protein expression in a melanoma tissue arrays consisting of metastatic melanomas (n = 46) examined for B7-H3 protein expression by immunohistochemistry. Fig. ID shows a representative image of positive immunostaining of B7-H3 protein expression in a tissue within the melanoma tissue array; Scale bar: 60 pm. Fig. IE is a graph showing that siRNA knockdown was confirmed in two human metastatic melanoma cell lines, Hs. 688(A)T and Hs. 839T, that were transfected with scrambled or B7-H3-specific siRNAs in triplicates using quantitative real-time PCR to assay for two isoforms of B7-H3. Fig. IF is an image of a heatmap of gene expression in Hs. 688(A)T and Hs. 839T cells that were transfected with scrambled or B7-H3-specific siRNAs, where RNA sequencing identified differentially expressing genes between the control and the silenced groups that were consistently up- or down- regulated in both cell lines. Fig. 1G is a representative immunoblot for B7-H3 showing B7-H3 protein expression in a panel of murine melanoma cell lines and immortalized murine melanocytes (melan-a). Fig. 1H is a graph showing mRNA levels of B7-H3 as analyzed in a panel of murine melanoma cell lines and immortalized murine melanocytes (melan-a) by quantitative real-time PCR. Figs. II and 1J show that B7-H3 or P-catenin was silenced by gene-specific siRNAs in YUMM2.1 cells, silencing was confirmed by immunoblot (Fig. 1H), and mRNAs obtained from these cells were processed for B7-H3 downstream targets (shown in Fig. IF) in triplicates using quantitative real-time PCR (Fig. 1J). Fig. II is an image showing Reverse Phase Protein Array (RPPA) data for 899 cancer cell lines from 24 organs, including 50 melanoma cell lines, downloaded from Cancer Cell Line Encyclopedia (CCLE) where each row indicates a differentially expressed gene and each column represents a tumor sample and is color-coded on the basis of median-centered log2 gene expression levels. Fig. IK is an image showing a heatmap depicting a high-level correlation between WNT-signaling genes and B7-H3, and anti-correlation with VAV1.

[0016] Figs. 2A-2E are images depicting that B7-H3 inhibition led to an inflamed tumor microenvironment in P-catenin activated tumors. FIG. 2A is a graph showing tumor growth curves from tumor-bearing C57BL/6J mice that were subcutaneously injected with YUMM2.1 cells (1 x 10⁶), harboring the Braf^V600E/wt/Pten'^/7Cdkn2^+/'/Bcat^loxex3/wt genotype, to generate tumors where the mice were challenged with isotype control, anti-B7-H3 (600 pg, three times per week), anti-PD-1 (300 pg, every five days), or both (anti-B7-H3 and anti-PD-1) when tumor size reached 20-50 mm³ (day 9). Treatments were continued until the end of the experiment. Tumor growth curves were determined by measuring tumor volume three times a week. The arrow indicates the first day when the drug or control was injected into mice. Fig. 2B is an image showing immune cell subgroups across treatment groups in RNA from the tumor (bulk) in triplicates after RNA was subjected to RNA sequencing. Enriched immune cell subgroups across treatment groups are shown. Fig. 2C is an image of a heatmap depicting selected genes and innate immune signals differentially expressed between the control and anti-B7-H3 treatment group. Fig. 2D is an image showing innate immune signals identified as enriched by RNA sequencing in the anti-B7-H3 treatment group where the tumors were harvested from tumor-bearing C57BL/6J mice that were subcutaneously injected with either YLTMM2.1 cells or YUMMERl .7 cells to generate tumors. Fig. 2E is an image of a heatmap depicting selected genes assessed in CD45+ immune cells that were sorted and subjected to ultra low RNA sequencing in triplicates in YLTMM2.1 tumors treated with anti-B7-H3, anti-PD-1, or control.

[0017] Figs. 3A-3I depict images showing that B7-H3 inhibition resulted in macrophage infiltration and activation. Figs. 3A-3C are representative images showing IBA-1 (Allograft Inflammatory Factor 1) immunohistochemistry of tumors in treatment groups with IgG control (Fig. 3A), anti-B7-H3 (Fig. 3B), or anti-PD-1 (Fig. 3C) showing activated macrophages within the tissue. Figs. 3D-3F are representative images showing CD47 expression of the tumor cells by immunohistochemistry in treatment groups with IgG control (Fig. 3D), anti-B7-H3 (Fig. 3E), or anti-PD-1 (Fig. 3F). Figs. 3G-3I are representative images showing SIRPa expression, a receptor for CD47 in immune subpopulations such as macrophages and DCs, in treatment groups with IgG control (Fig. 3G), anti-B7-H3 (Fig. 3H), or anti-PD-1 (Fig. 31).

[0018] Figs. 4A-4F depict images showing that therapeutic synergy was achieved when anti-

B7-H3 (or anti-PD-1) was combined with anti-CD47. Fig. 4A is a graph showing tumor growth curves of tumors that were generated with YUMM2.1 cells (IxlO⁶) from mice treated with isotype control, anti-B7-H3 (600 pg, three times per week), anti-CD47 (200 pg, three times per week), or both (anti-B7-H3 and anti-CD47) (n = 6 mice per group). Fig. 4B is a graph showing tumor growth curves of tumors that were generated with YUMM2.1 cells from mice treated with isotype control, anti-PD-1 (300 pg, every five days), anti-CD47 (200 pg, three times per week), or both (anti-PD- 1 and anti-CD47). Fig. 4C is a graph showing tumor growth curves of tumors that were generated with YUMM2.1 cells from NK cell depleted-mice, where the mice were pretreated with anti NK1.1 (200 pg, days 1 and 3) followed by 150 pg twice a week (n = 6 mice per group). Fig. 4D is a graph showing tumor growth curves of tumors that were generated with YUMM2.1 cells from CD8+ T cell depleted-mice, where the mice were pretreated with anti-CD8a 250 pg three times per week was used (n = 6 mice per group). Fig. 4E is a graph showing tumor growth curves of tumors that were generated with YUMMER1.7 cells (5 x 10⁵) harboring the Braf^V600E/wt/Pten'^/7Cdkn2'^/' genotype, from mice treated with anti-B7-H3, anti-PD-1, or both (anti-B7-H3 and anti-PD-1) once the tumor size reached 20-50 mm³ (n ^{= r}l mice per group). Fig. 4F is a graph showing tumor growth curves of tumors that were generated with YUMMER1.7 cells from mice treated with IgG, anti- B7-H3, anti-PD-1, anti-CD47, or combination of anti-B7-H3 and anti-CD47, or anti-PD-1 and anti-CD47.

[0019] Figs. 5A-5E depict images showing that therapeutic synergy was achieved when anti-

B7-H3 (or anti-PD-1) was combined with anti-CD47 in P-catenin activated tumors. Fig. 5A is a graph showing tumor growth curves for tumors generated by subcutaneous injection with YUMM2.1 cells (IxlO⁶) and treated with isotype control, anti-B7-H3 (600 pg, three times per week), anti-CD47 (200 pg, three times per week), or both (anti-B7-H3 and anti-CD47) (n = 6 mice per group). Quantitative results were analyzed by one-way ANOVA. Fig. 5B is a graph showing UMAP of immune cell distribution for CD45+ sorted cells subjected to single cell RNA sequencing (scRNA-sequencing) in the tumors described in Fig. 5A. Fig. 5C is a graph showing proportions of major immune cell subsets in each sample as analyzed by scRNA-sequencing. Fig. 5D is a heatmap depicting immune cell subtypes for each treatment group. The key for the x-axis is; I . DC (DC.103+1 IB-), 2. T cells (T.4); 3. Stromal cells (ST); 4. ILC (LIV.NK.DX5+); 5. B cells (B.Fo); 6. Macrophages (MFI05.II-); 7. ILC (ILC3); 8. NKT (NKT.4-); 9. Macrophages (MF.F480HI); 10. DC (DC.8-4-1 IB-); 11. NKT (NKT.44+); 12. B cells (B.CD19CONTROL); 13. T cells (T.8MEM), 14. T cells (T.8SP); 15. T cells (T.4SP), 16. Macrophages (MFI05.II+); 17 Stem cells (SC); 18. NKT (NKT.44-); 19. ILC (ILC1.CD127+); 20. Macrophages (MF.II+480LO); 21 . T cells (T.8NVE); 22. T cells (T.4.Pa); 23. Macrophages (MF. l 1C-11B+); 24. DC (DC.PDC.8- ); 25. B cells (B.GC); 26. Macrophages (MF.103-11B+); 27. Epithelial cells (Ep); 28. Macrophages (MF.II-480HI); 29. Fibroblasts (FI), 30. DC (DC.8+); 31. NKT (NKT.4+); 32. NK cells (NK.49CI+); 33. Macrophages (MF.103CLOSER); 34. T cells (T.4FP3-); 35. NK cells (NK.49C1-); 36 T cells (T.Tregs); 37. DC (DC.11B-); 38. Basophils (BA); 39. Monocytes (MO); 40. DC (DC.8-); 41. ILC (ILC2); 42. NK cells (NK.B2M-); 43. Macrophages (MF); 44 B cells (B.FrF); 45. T cells (T.4MEM); 46. Macrophages (MF.480INT.NAIVE); 47. T cells (T.4FP3+25+); 48. Endothelial cells (EC); 49. Tgd (Tgd); 50. Eosinophils (EO); 51. B cells (B.MEM); 52. T cells (T.4EFF); 53. DC (DC.PDC.8+); 54. T cells (T.ISP); 55. T cells (T.CD8); 56. T cells (T.8EFF); 57. Neutrophils (GN); 58. Macrophages (MF.l 1 CLOSER), 59. T cells (T.4.PLN); 60. T cells (T.DN); 61. B cells (preB); 62. T cells (T.CD4); 63. Macrophages (MF.169+11 CHI), 64. DC (DC.103-11B+). Fig. 5E is a beatmap showing differentially expressed genes (x-axis) between the treatment groups (y-axis).

[0020] Figs. 6A-6T depict images showing UMAP of differentially expressed genes GZMA (Figs. 6A-6D), GZMB (Figs. 6F-6I), HSPA1A (Figs. 6K-6N), HSPA1B (Figs. 6P-6S), and expression levels of GZMA (Fig. 6E), GZMB (Fig. 6J), HSPA1A (Fig. 60), an . HSPA 1B (Fig. 6T) in immune cell subsets. Significant P values are indicated.

[0021] Figs. 7A-7O depict images showing UMAP of differentially expressed genes CCL5 (Figs. 7A-7D), CXCR4 (Figs. 7F-7I), NKG7 (Figs. 7K-7N) and expression levels of CCL5 (Fig. 7E), CXCR4 (Fig. 7J), and NKG7 (Fig. 70) in immune cell subsets. Significant P values are indicated.

[0022] Figs. 8A-8E depict images showing enriched chemokine signaling upon combining anti-B7-H3 and anti-CD47. Fig. 8A shows a graph of tumor volume over time for tumors generated by subcutaneous injection of YUMM2.1 cells (IxlO⁶) into C57BL/6J mice and challenged with isotype control, anti-B7-H3, anti-PD-1, anti-CD47, or combination of anti-B7-H3 and anti-CD47, or anti-PD-1 and anti-CD47 (n = 6 mice per group). Quantitative results were analyzed by oneway ANOVA. Fig. 8B shows a graph of tumor volume over time for tumors generated by subcutaneous injection of YUMM2.1 cells (IxlO⁶) into C57BL/6 CCR5-/- mice and challenged with isotype control, anti-B7-H3, anti-PD-1, anti-CD47, or combination of anti-B7-H3 and anti- CD47, or anti-PD-1 and anti-CD47 (n = 6 mice per group). Quantitative results were analyzed by one-way ANOVA. Fig. 8C shows a graph of tumor volume over time for tumors generated by subcutaneous injection of YUMM2.1 cells (IxlO⁶) into C57BL/6 IL4R-/- mice and challenged with isotype control, anti-B7-H3, anti-PD-1, anti-CD47, or combination of anti-B7-H3 and anti- CD47, or anti-PD-1 and anti-CD47 (n = 5 mice per group). Quantitative results were analyzed by one-way ANOVA. Figs. 8D and 8E are RNA sequencing files of metastatic melanomas of the TCGA (n = 320) where signatures for T cell exclusion and dysfunction (M2 macrophages - M2, myeloid-derived suppressive cells -MDSC, cancer-associated fibroblasts - CAF, T cell exclusion, and T cell dysfunction) (Fig. 8D) and their correlations (Fig. 8E) with B7-H3, PD-1, PDL-1 and others were examined.

DETAILED DESCRIPTION

[0023] Immunotherapy using immune checkpoint inhibitors can be effective at treating cancer in a subset of patients having solid tumors, such as melanoma. While durable and complete responses are observed in patients, many do not respond or develop resistance. Objective response rate using immune checkpoint inhibitors, such as anti-PD-1 (B7-H1) and/or anti-CTLA-4, can range from 10% to 40%. The present disclosure provides, in part, a unique combination therapy of immune checkpoint inhibitors with higher response rates that likely act by exerting distinct immune regulatory functions to restore the innate immune system surrounding the tumor. [0024] B7-H3 (CD276) is an immune regulatory molecule that belongs to the B7 superfamily. It is expressed on the membrane of the cancer cell, but its receptor is unknown. Aberrant protein expression of B7-H3 is associated with poor prognosis in many cancers including melanoma. Monoclonal antibodies against B7-H3 are tested in preclinical and phase I clinical studies. Despite the importance of immune checkpoint molecules in the cancer field, B7-H3 is less well studied than other checkpoints and its functions remain less well delineated.

[0025] CD47 functions through a mechanism by which the cancer cell evades innate immune surveillance (macrophage immune checkpoint). CD47 is expressed on the membrane of the cancer cell. CD47 binds and activates SIRPa, an inhibitory protein expressed on the surface of myeloid cells - macrophages -, and functions as an anti-phagocytic or “don’t eat me signal”. Activation of SIRPa inhibits the phagocytic activity of macrophages. Blocking the CD47-SIRPa interaction allows macrophages to engulf cancer cells. Monoclonal antibodies inhibiting CD47 are tested in pre-clinical models, and phase I clinical trials are either completed or ongoing.

[0026] Provided herein are compositions that include an inhibitor of B7-H3 (CD276) and an inhibitor of CD47-SIRPa which are capable of treating a solid tumor in a subject, and uses thereof for activating at least one population of innate immune system cells in a cold tumor microenvironment.

I. Definitions

[0027] For the purposes of promoting an understanding of the principles of the present disclosure, reference will now be made to preferred embodiments and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the disclosure is thereby intended, such alteration and further modifications of the disclosure as illustrated herein, being contemplated as would normally occur to one skilled in the art to which the disclosure relates.

[0028] As used in the specification, articles “a” and “an” are used herein to refer to one or to more than one (i.e., at least one) of the grammatical object of the article. By way of example, “an element” means at least one element and can include more than one element.

[0029] “About” is used to provide flexibility to a numerical range endpoint by providing that a given value may be “slightly above” or “slightly below” the endpoint without affecting the desired result. The term “about” in association with a numerical value means that the numerical value can vary plus or minus by 5% or less of the numerical value.

[0030] Throughout this specification, unless the context requires otherwise, the word “comprise” and “include” and variations (e.g., “comprises,” “comprising,” “includes,” “including”) will be understood to imply the inclusion of a stated component, feature, element, or step or group of components, features, elements or steps but not the exclusion of any other integer or step or group of integers or steps.

[0031] As used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations where interpreted in the alternative (“or”).

[0032] As used herein, the transitional phrase “consisting essentially of’ (and grammatical variants) is to be interpreted as encompassing the recited materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention. Thus, the term “consisting essentially of’ as used herein should not be interpreted as equivalent to “comprising.” [0033] Moreover, the present disclosure also contemplates that in some embodiments, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0034] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise- indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure.

[0035] As used herein, "treatment," "therapy" and/or "therapy regimen" refer to the clinical intervention made in response to a disease, disorder or physiological condition manifested by a patient or to which a patient may be susceptible. The aim of treatment includes the alleviation or prevention of symptoms, slowing or stopping the progression or worsening of a disease, disorder, or condition and/or the remission of the disease, disorder or condition.

[0036] As used herein, “prevent” or “prevention” refers to eliminating or delaying the onset of a particular disease, disorder or physiological condition, or to the reduction of the degree of severity of a particular disease, disorder or physiological condition, relative to the time and/or degree of onset or severity in the absence of intervention.

[0037] The term “effective amount" or "therapeutically effective amount" refers to an amount sufficient to effect beneficial or desirable biological and/or clinical results.

[0038] As used herein, “individual”, “subject”, “host”, and “patient” can be used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, prophylaxis or therapy is desired, for example, humans, pets, livestock, horses or other animals. As used herein, the term "subject" and "patient" are used interchangeably herein and refer to both human and nonhuman animals. The term "nonhuman animals" of the disclosure includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dog, cat, horse, cow, chickens, amphibians, reptiles, and the like. In some embodiments, the subject can be a human. In other embodiments, the subject can be a human in need of treating a solid tumor (e.g., melanoma).

[0039] As used herein, “polynucleotide,” “nucleic acid” or “nucleic acid molecule” include deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), mRNA, oligonucleotides, and the like.

[0040] As used herein, “vector”, “expression vector” or “construct” refers to a nucleic acid used to introduce polynucleotides into a cell that has regulatory elements to provide expression of the heterologous nucleic acids in the cell. Vectors include but are not limited to plasmid, minicircles, yeast, and/or viral genomes. In some alternatives, the vectors are plasmid, minicircles, or viral genomes. In some alternatives, the vector is a viral vector. In some embodiments, the viral vector is a lentivirus. In some alternatives, the vector is a lentiviral vector. In some embodiments, the vector is a foamy viral vector, adenoviral vectors, retroviral vectors or lentiviral vectors.

[0041] As used herein, “chimeric antigen receptor” or “CAR” or “chimeric T cell receptor” refers herein to a synthetically designed receptor having a ligand binding domain of an antibody or another peptide sequence that binds to a molecule associated with the disease or disorder and is linked via a spacer domain to one or more intracellular signaling domains of a T cell or other receptors, such as a costimulatory domain. Chimeric receptor can also be referred to as artificial T cell receptors, chimeric T cell receptors, chimeric immunoreceptors, and chimeric antigen receptors (CARs).

IL Compositions

[0042] Aspects of the present disclosure include compositions encompassing an inhibitor of B7-H3 (CD276) and an inhibitor of CD47-SIRPa. Compositions herein can also be pharmaceutical compositions. a. B7-H3 Inhibitors

[0043] In certain embodiments, compositions herein can include at least one inhibitor of B7- H3 (CD276). In some embodiments, B7-H3 activity can be reduced through the use of an antagonist of the receptor, a partial antagonist or an antibody that either competes with the natural agonist, blocks the activity or encourages uptake of the B7-H3 molecule. In accordance with these embodiments, a B7-H3 inhibitor can be a small molecule, a peptide, a polynucleotide, a genetically modified cell or an antibody, an antibody fragment or a combination thereof. In certain embodiments, a B7-H3 inhibitor can be an anti- B7-H3 antibody or fragment thereof capable of binding to B7-H3 and inhibiting or reducing B7-H3 activity. In some embodiments, other B7-H3 inhibitors can include, but are not limited to, a B7-H3-specific siRNA, RNAi, microRNA or ribozyme. In some embodiments, an inhibitor of B7-H3 can be an isolated nucleic acid or set of nucleic acids, which collectively encodes an antibody. Other B7-H3 inhibitors and/or antagonists can include anti-B7-H3 specific antibody fragments (F(ab')2 or Fab'), single chain Fv, and Fc- fusion protein of B7-H3 ligands, e.g. Fc fusion proteins of 2B4 or CD2. In some embodiments, an anti-B7-H3 antibody used herein can be a polyclonal or monoclonal antibody. A “humanized” anti-B7-H3 antibody can be used if needed in order to avoid any potential use incompatibilities (e.g. adverse reactions when introducing the ILC2 cells exposed to such an antibody, if needed). In some embodiments, an inhibitor of B7-H3 can be a B7-H3-specific chimeric antigen receptor T (CAR-T) cell. In some embodiments, an inhibitor of B7-H3 suitable for use herein can be, but is not limited to, enoblituzumab, omburtamab, orlotamab, BP-102, DS-7300, 8H-9, TCB-005, YBL- 015, or a combination thereof. h. CD47-SIRPa Inhibitors [0044] In certain embodiments, compositions herein can include at least one inhibitor CD47- SIRPa. In some embodiments, CD47-activity can be reduced through the use of an antagonist of the receptor (SIRPa), a partial antagonist or an antibody that either competes with the natural agonist, blocks the activity or encourages uptake of the CD47 molecule. In some embodiments, a CD47-SIRPa inhibitor can be a CD47-ligand inhibitor. In some embodiments, a CD47-SIRPa inhibitor can be a SIRPa-receptor inhibitor.

[0045] In accordance with these embodiments, a CD47 inhibitor can be a small molecule, a peptide, a polynucleotide, a genetically modified cell or an antibody, an antibody fragment or a combination thereof. In some embodiments, a CD47 inhibitor can be an anti-CD47 antibody or fragment thereof capable of binding to CD47 and inhibiting or reducing CD47 activity. In other embodiments, other CD47 inhibitors can include, but are not limited to, a CD47-specific siRNA, RNAi, microRNA or ribozyme. In some embodiments, an inhibitor of CD47 can be an isolated nucleic acid or set of nucleic acids, which collectively encodes an antibody. Other CD47 inhibitors and/or antagonists can include anti-CD47 specific antibody fragments (F(ab')2 or Fab'), single chain Fv, and Fc-fusion protein of B7-H3 ligands, e.g. Fc fusion proteins of 2B4 or CD2. In some embodiments, an anti- CD47 antibody used herein can be a polyclonal or monoclonal antibody. A “humanized” anti-CD47 antibody can be used if needed in order to avoid any potential use incompatibilities. In some embodiments, an inhibitor of CD47 can be a CD47-specific chimeric antigen receptor T (CAR-T) cell.

[0046] In accordance with these embodiments, a SIRPa inhibitor can be a small molecule, a peptide, a polynucleotide, a genetically modified cell or an antibody, an antibody fragment or a combination thereof. In some embodiments, a SIRPa inhibitor can be an anti-SIRPa antibody or fragment thereof capable of binding to SIRPa and inhibiting or reducing SIRPa activity. In some embodiments, other SIRPa inhibitors can include, but are not limited to, a SIRPa-specific siRNA, RNAi, microRNA or ribozyme. In some embodiments, an inhibitor of SIRPa can be an isolated nucleic acid or set of nucleic acids, which collectively encodes an antibody. Other SIRPa inhibitors and/or antagonists can include anti- SIRPa specific antibody fragments (F(ab')2 or Fab'), single chain Fv, and Fc-fusion protein of SIRPa, e.g. Fc fusion proteins of 2B4 or CD2. In some embodiments, an anti-SIRPa antibody used herein can be a polyclonal or monoclonal antibody. A “humanized” anti-SIRPa antibody can be used if needed in order to avoid any potential use incompatibilities (e.g. adverse reactions when introducing the ILC2 cells exposed to such an antibody, if needed). In some embodiments, an inhibitor of SIRPa can be a SIRPa -specific chimeric antigen receptor T (CAR-T) cell.

[0047] In some embodiments, an inhibitor of CD47-SIRPa suitable for use herein can be, but is not limited to, RRx-001, a dinitroazetidine derivative, Hu5F9-G4, CC-90002, TTI-621, ALX148, SRF231, SHR-1603, IBI188, ST-1901, SGNCD-47M, or a combination thereof.

[0048] In certain embodiments, compositions herein may encompass an inhibitor of B7-H3 (CD276) and an inhibitor of CD47-SIRPa. In some embodiments, compositions herein may encompass an inhibitor of B7-H3 (CD276) and an inhibitor of CD47-SIRPa, wherein a B7-H3 inhibitor can be a small molecule, a peptide, a polynucleotide, a genetically modified cell or an antibody, an antibody fragment or a combination thereof and a CD47-SIRPa, can be a small molecule, a peptide, a polynucleotide, a genetically modified cell or an antibody, an antibody fragment or a combination thereof. In some embodiments, compositions herein may encompass a B7-H3-specific siRNA, RNAi, microRNA or ribozyme and a CD47-specific siRNA, RNAi, microRNA or ribozyme and/or a SIRPa-specific siRNA, RNAi, microRNA or ribozyme. In some embodiments, compositions herein may encompass an anti-B7-H3 antibody and an anti-CD47 antibody and/or an anti-SIRPa antibody. In some embodiments, compositions herein may encompass: enoblituzumab, omburtamab, orlotamab, BP-102, DS-7300, 8H-9, TCB-005, YBL- 015, or any combination thereof; and RRx-001, a dinitroazetidine derivative, Hu5F9-G4, CC- 90002, TTI-621, ALX148, SRF231, SHR-1603, IBI188, ST-1901, SGNCD-47M, or any combination thereof. c. Pharmaceutical Compositions

[0049] The B7-H3 inhibitors and/or CD47-SIRPa inhibitors, as described herein can be mixed with a pharmaceutically acceptable carrier (excipient) to form a pharmaceutical composition for use in treating a target disease. “Acceptable” means that the carrier must be compatible with the active ingredient of the composition (and preferably, capable of stabilizing the active ingredient) and not deleterious to the subject to be treated. Pharmaceutically acceptable excipients (carriers) including buffers, which are well known in the art. See, e.g., Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Areiams and Wilkins, Ed. K. E. Hoover. [0050] The pharmaceutical compositions to be used in the present methods can comprise pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions. (Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Areiams and Wilkins, Ed. K. E. Hoover). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations used, and comprise buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrans; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; saltforming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or nonionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). In some examples, the pharmaceutical composition described herein comprises liposomes containing the antibodies (or the encoding nucleic acids) which can be prepared by methods known in the art, such as described in Epstein, et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang, et al., Proc. Natl. Acad. Sci. USA 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556. Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.

[0051] In some embodiments, one or more of the B7-H3 inhibitors and/or CD47-SIRPa inhibitors, can be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are known in the art, see, e.g., Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing (2000).

[0052] In some embodiments, pharmaceutical compositions described herein can be formulated in sustained-release format. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the inhibitors, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained- release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers ofL-glutamic acid and 7 ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid- glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3- hydroxybutyric acid.

[0053] The pharmaceutical compositions to be used for in vivo administration must be sterile. This may be readily accomplished by, for example, filtration through sterile filtration membranes. Therapeutic compositions disclosed herein can be placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[0054] The pharmaceutical compositions described herein can be in unit dosage forms such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories, for oral, parenteral or rectal administration, or administration by inhalation or insufflation.

[0055] For preparing solid compositions such as tablets, the principal active ingredient can be mixed with a pharmaceutical carrier, e.g., conventional tableting ingredients such as com starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, e.g., water, to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention, or a non-toxic pharmaceutically acceptable salt thereof. When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. This solid preformulation composition is then subdivided into unit dosage forms of the type described above containing from 0.1 to about 900 mg of the active ingredient of the present invention. The tablets or pills of the novel composition can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer that serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate. Suitable surface-active agents (surfactant) include, in particular, non-ionic agents, such as poly oxy ethylenesorbitans (e.g., Tween™ 20, 40, 60, 80 or 85) and other sorbitans (e.g., Span™ 20, 40, 60, 80 or 85). Compositions with a surface-active agent are conveniently comprise between 0.05 and 5% surfaceactive agent, and can be between 0.1 and 2.5%. It are be appreciated that other ingredients may be added, for example mannitol or other pharmaceutically acceptable vehicles, if necessary.

[0056] Suitable emulsions may be prepared using commercially available fat emulsions, such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™ and Lipiphysan™. The active ingredient may be either dissolved in a pre-mixed emulsion composition or alternatively it may be dissolved in an oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, com oil or almond oil) and an emulsion formed upon mixing with a phospholipid (e.g. egg phospholipids, soybean phospholipids or soybean lecithin) and water. It are be appreciated that other ingredients may be added, for example glycerol or glucose, to adjust the tonicity of the emulsion. Suitable emulsions are typically contain up to 20% oil, for example, between 5 and 20%. The fat emulsion can comprise fat droplets between 0.1 and 1.0 .im, particularly 0.1 and 0.5 .im, and have a pH in the range of 5.5 to 8.0. The emulsion compositions can be those prepared by mixing an inhibitor with Intralipid™ or the components thereof (soybean oil, egg phospholipids, glycerol and water).

[0057] Pharmaceutical compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as set out above. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic effect. [0058] Compositions in preferably sterile pharmaceutically acceptable solvents may be nebulized by use of gases. Nebulized solutions may be breathed directly from the nebulizing device or the nebulizing device may be attached to a face mask, tent or intermittent positive pressure breathing machine. Solution, suspension or powder compositions may be administered, preferably orally or nasally, from devices which deliver the formulation in an appropriate manner. [0059] In some embodiments, pharmaceutical formulations herein may comprise an anti-B7- H3 antibody at a suitable concentration, for example, about 200 pg to about 1000 pg (e.g., about 200 pg, about 250 pg, about 300 pg, about 350 pg, about 400 pg, about 450 pg, about 500 pg, about 550 pg, about 600 pg, about 650 pg, about 700 pg, about 750 pg, about 800 pg, about 850 pg, about 900 pg, about 950 pg, about 1000 pg). In some embodiments, pharmaceutical formulations herein may comprise an anti-CD47 antibody at a suitable concentration, for example, about 50 pg to about 800 pg (e.g., about 50 pg, about 100 pg, about 150 pg, about 200 pg, about 250 pg, about 300 pg, about 350 pg, about 400 pg, about 450 pg, about 500 pg, about 550 pg, about 600 pg, about 650 pg, about 700 pg, about 750 pg, about 800 pg). In some embodiments, pharmaceutical formulations herein may compromise both an anti-B7-H3 antibody and an anti- CD47 antibody. In some embodiments, pharmaceutical formulations herein may comprise an anti- B7-H3 antibody at about 200 pg to about 1000 pg (e.g., about 200 pg, about 250 pg, about 300 pg, about 350 pg, about 400 pg, about 450 pg, about 500 pg, about 550 pg, about 600 pg, about 650 pg, about 700 pg, about 750 pg, about 800 pg, about 850 pg, about 900 pg, about 950 pg, about 1000 pg) and an anti-CD47 antibody at about 50 pg to about 800 pg (e.g., about 50 pg, about 100 pg, about 150 pg, about 200 pg, about 250 pg, about 300 pg, about 350 pg, about 400 pg, about 450 pg, about 500 pg, about 550 pg, about 600 pg, about 650 pg, about 700 pg, about 750 pg, about 800 pg). In some embodiments, pharmaceutical formulations herein may compromise an anti-B7-H3 antibody and an anti-CD47 antibody in two separate units. d. Packaging

[0060] In some embodiments, compositions disclosed herein may be packaged. In some aspects, packaging of a composition may be for storage, shipment, display for sale, or a combination thereof. In some embodiments, compositions may be packaged using one or more suitable materials known in the art. In some embodiments, compositions may be packaged using one or more suitable methods known in the art. In s some embodiments, the choice of packaging material and/or packaging method is dependent on the dosage form of a composition disclosed herein to be packaged.

[0061] In some embodiments, compositions disclosed herein may be packaged wherein packaging increases the length of time a composition can be stored. As used herein, the “shelflife” of a composition is the length of time after formulation that a composition can maintain one or more physiological effects following administration to a subject as detailed herein. In some embodiments, compositions disclosed herein may be packaged wherein packaging increases the shelf-life of a composition by about 1 week, about 1 month, or about 6 months. In some embodiments, compositions disclosed herein may be packaged wherein packaging increases the length of time a composition can be stored at about -85°C to about -75°C. In some embodiments, compositions disclosed herein may be packaged wherein packaging of at least one composition component increases the length of time that composition component can be stored at room temperature by about 1 week, about 1 month, or about 6 months. In some embodiments, compositions disclosed herein may be packaged wherein packaging of at least one composition component increases the length of time that composition component can be stored at about -85°C to about -75°C by about 1 week, about 1 month, or about 6 months.

III. Methods

[0062] The present disclosure provides methods for treating solid tumors. In some embodiments, the present disclosure provides methods for treating solid tumors (e.g., melanoma) that are not responsive to one or more anti-cancer therapies. In some embodiments, the present disclosure provides methods for treating solid tumors (e.g., melanoma) that are not responsive to anti-programmed cell death 1/programmed cell death ligand 1 (PD1/PDL1) therapy using any of the compositions having an inhibitor of B7-H3 (CD276) and an inhibitor of CD47-SIRPa as disclosed herein. Also provided in the present disclosure are methods of inflaming a cold tumor microenvironment using any of the compositions having an inhibitor of B7-H3 (CD276) and an inhibitor of CD47-SIRPa as disclosed herein.

[0063] In some embodiments, the present disclosure provides methods of treating a solid tumor. In some embodiments, a solid tumor can be pancreatic ductal adenocarcinoma (PDA), colorectal cancer (CRC), melanoma, breast cancer, lung cancer, upper and lower gastrointestinal malignancies, squamous cell head and neck cancer, genitourinary cancer, ovarian cancer, and sarcomas. In an exemplary example, a solid tumor is a melanoma.

[0064] In some embodiments, a solid tumor to be treated by methods disclosed herein can have B7-H3 overexpression, overactive P-catenin signaling, or a combination thereof. In some aspects, a solid tumor to be treated by methods disclosed herein can have at least one genetic mutation. In some examples, a solid tumor can have a genetic mutation of BRAF^V600E. In some examples, a solid tumor can have a genetic mutation resulting in the loss of PTEN.

[0065] Certain embodiments herein can provide methods for treating solid tumors (e.g., melanoma) in a subject in need thereof. The subject to be treated by the methods described herein can be a mammal, more preferably a human. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice and rats. A human subject who needs the treatment may be a human patient having, at risk for, or suspected of having a target disease/disorder, such as a solid tumor, including a melanoma.

[0066] In some embodiments, a subject to any of the methods herein can be any subject for whom treatment or therapy is desired. In some embodiments, a subject can have or can be suspected of having cancer, a tumor, or any combination thereof. In some embodiments, a subject can have or can be suspected of having one or more primary tumors, one or more metastatic tumors such as solid tumors or any combination thereof. In some embodiments, a subject can be a mammal. In some embodiments, a subject can be a human patient. In some embodiments, a human patient such as an adult, child, adolescent, toddler, young adult or infant or fetus who is in need of the methods herein can be identified by routine medical examination, e.g., laboratory tests, biopsy, magnetic resonance imaging (MRI) scans, ultrasound exams, and the like.

[0067] In some embodiments, a subject to be treated by the methods described herein can be a human patient having, suspected of having, or a risk for developing a solid tumor. In some embodiments, a subject to be treated by the methods described herein can have a tumor wherein the tumor has at least one B7H3⁺ cell. In some embodiments, a subject to be treated by the methods described herein can have a tumor wherein the tumor has at least one CD47⁺ cell. In some embodiments, a subject to be treated by the methods described herein can have a tumor wherein the tumor has at least one CD47⁺ cell/B7H3⁺ cell. [0068] In some examples, a human subject who needs the treatment may be a human patient having, at risk for, or suspected of having melanoma. Methods herein can be used to treat a human patient having a Stage 0, Stage I, Stage II, Stage III, or Stage IV melanoma. As used herein, a Stage 0 melanoma (melanoma in situ) has not grown deeper than the top layer of the skin (the epidermis). As used herein, a melanoma can be staged according to the Ameri can Joint Committee on Cancer (AJCC) TNM staging system. Using the AJCC TNM staging system for Melanomas: Stage 0 - The cancer is confined to the epidermis, the outermost skin layer and has not spread to nearby lymph nodes or to distant parts of the body; Stage I - The tumor is no more than 2 mm (2/25 of an inch) thick and might or might not be ulcerated and has not spread to nearby lymph nodes or to distant parts of the body; Stage II - The tumor is more than 1 mm thick and may be thicker than 4 mm, it might or might not be ulcerated, and the cancer has not spread to nearby lymph nodes or to distant parts of the body; Stage III - The tumor is no more than 4 mm thick and might or might not be ulcerated, the cancer has spread to 1, 2, 3, 4 or more nearby lymph nodes, and it has not spread to distant parts of the body, and Stage IV - The tumor can be any thickness and might or might not be ulcerated, the cancer might or might not have spread to nearby lymph nodes, and it has spread to distant lymph nodes or to organs such as the lungs, liver or brain.

[0069] In some embodiments, the subject is a human patient who is in need of enhancing immunity. For example, the human patient may have a solid tumor. Examples of solid tumor cancers include melanoma, pancreatic duct adenocarcinoma (PDA), colorectal cancer (CRC), melanoma, cholangiocarcinoma, breast cancer, lung cancer (for example, non-small cell lung cancer, NSCLC, and small cell lung cancer, SCLC), upper and lower gastrointestinal malignancies (including, but not limited to, esophageal, gastric, and hepatobiliary cancer), squamous cell head and neck cancer, genitourinary cancers, ovarian cancer, and sarcomas. A subject having a solid tumor can be identified by routine medical examination, e.g., laboratory tests, organ functional tests, CT scans, or ultrasounds. In some embodiments, the subject to be treated by the method described herein may be a human cancer patient who has undergone or is subjecting to an anticancer therapy, for example, chemotherapy, radiotherapy, immunotherapy, or surgery.

[0070] As used herein, the term “treating” refers to the application or administration of a composition including one or more active agents to a subject, who has a target disease or disorder, a symptom of the disease/disorder, or a predisposition toward the disease/disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disorder, a symptom of the disease or disorder, or the predisposition toward the disease or disorder.

[0071] Alleviating a target disease/disorder includes delaying the development or progression of the disease, or reducing disease severity or prolonging survival. Alleviating the disease or prolonging survival does not necessarily require curative results. As used therein, "delaying" the development of a target disease or disorder means to defer, hinder, slow, retard, stabilize, and/or postpone progression of the disease. This delay can be of varying lengths of time, depending on the history of the disease and/or individuals being treated. A method that “delays” or alleviates the development of a disease, or delays the onset of the disease, is a method that reduces probability of developing one or more symptoms of the disease in a given time frame and/or reduces extent of the symptoms in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a number of subjects sufficient to give a statistically significant result.

[0072] “Development” or “progression” of a disease means initial manifestations and/or ensuing progression of the disease. Development of the disease can be detectable and assessed using standard clinical techniques as well known in the art. However, development also refers to progression that may be undetectable. For purpose of this disclosure, development or progression refers to the biological course of the symptoms. “Development” includes occurrence, recurrence, and onset. As used herein “onset” or “occurrence” of a target disease or disorder includes initial onset and/or recurrence.

[0073] In some embodiments, the present disclosure provides methods for reducing, ameliorating, or eliminating one or more symptom(s) associated with a solid tumor. The treatment methods disclosed herein involve the combined therapy of an inhibitor of B7-H3 and an inhibitor of CD47-SIRPa. In some embodiments, the present disclosure provides methods of administering a combined therapy of an inhibitor of B7-H3 and an inhibitor of CD47-SIRPa for reducing, ameliorating, or eliminating one or more symptom(s) associated with a solid tumor. In some embodiments, the present disclosure provides methods of administering a combined therapy of an inhibitor of B7-H3 and an inhibitor of CD47-SIRPa for reducing, ameliorating, or eliminating one or more symptom(s) associated with a solid tumor wherein administering a combined therapy of an inhibitor of B7-H3 and an inhibitor of CD47-SIRPa as disclosed herein synergistically reduces, ameliorates, and/or eliminates one or more symptom(s) associated with a solid tumor. In some embodiments, methods of administering a combined therapy of an inhibitor of B7-H3 and an inhibitor of CD47-SIRPa herein reduces, ameliorates, and/or eliminates one or more symptom(s) associated with a solid tumor at a faster rate compared to the rate after administering a single therapy of an inhibitor of B7-H3 or a single therapy of an inhibitor of CD47-SIRPa to a subject. In some embodiments, methods of administering a combined therapy of an inhibitor of B7-H3 and an inhibitor of CD47-SIRPa herein reduces, ameliorates, and/or eliminates one or more symptom(s) associated with a solid tumor at a rate that can be at least about 10% faster, at least about 20% faster, at least about 30% faster, at least about 40% faster, or at least about 50% faster compared to the rate after administering a single therapy of an inhibitor of B7-H3 or a single therapy of an inhibitor of CD47-SIRPa to a subject. In some embodiments, methods of administering a combined therapy of an inhibitor of B7-H3 and an inhibitor of CD47-SIRPa herein reduces, ameliorates, and/or eliminates one or more symptom(s) associated with a solid tumor at a rate that can be about 10% to about 99% faster, about 15% to about 95% faster, or about 20% to about 90% faster compared to the rate after administering a single therapy of an inhibitor of B7- H3 or a single therapy of an inhibitor of CD47-SIRPa to a subject.

[0074] In some embodiments, an effective amount of the combined therapy of an inhibitor of B7-H3 and an inhibitor of CD47-SIRPa can be given to a subject having a solid tumor e.g., melanoma), wherein the subject is on a treatment involving the one or more chemotherapeutics. In some embodiments, an effective amount of the one or more chemotherapeutics are given to a subject having a solid tumor (e.g., melanoma), wherein the subject is on a treatment involving the combined therapy of an inhibitor of B7-H3 and an inhibitor of CD47-SIRPa. In some embodiments, an effective amount of the combined therapy of an inhibitor of B7-H3 and an inhibitor of CD47-SIRPa and an effective amount of the one or more chemotherapeutics are given to the subject, concurrently or sequentially.

[0075] In some embodiments, the methods of the present disclosure increase anti-tumor activity (e.g., reduce cell proliferation, tumor growth, tumor volume, tumor burden, tumor load, and/or number of metastatic lesions over time) as compared to levels prior to treatment or in a control subject. In some embodiments, the methods of the present disclosure increase anti-tumor activity (e.g., reduce cell proliferation, tumor growth, tumor volume, tumor burden, tumor load, and/or number of metastatic lesions over time) by at least about 10%, least about 20%, least about 25%, least about 30%, least about 40%, least about 50%, least about 60%, least about 70%, least about 75%, least about 80%, least about 85%, least about 90%, least about 95%, or more as compared to levels prior to treatment or in a control subject. In some embodiments, the methods of the present disclosure increase anti-tumor activity (e.g., reduce cell proliferation, tumor growth, tumor volume, tumor burden, tumor load, and/or number of metastatic lesions over time) by at about 5% to about 99%, about 10% to about 95%, or about 15% to about 90% as compared to levels prior to treatment or in a control subject. In some embodiments, the methods of the present disclosure synergistically increase anti-tumor activity (e.g., reduce cell proliferation, tumor growth, tumor volume, tumor burden, tumor load, and/or number of metastatic lesions over time) as compared to levels after treatment of a subject with a single therapy of an inhibitor of B7-H3 or a single therapy of an inhibitor of CD47-SIRPa.

[0076] In some embodiments, reduction may be measured by comparing cell proliferation, tumor growth, and/or tumor volume in a subject before and after administration of composition disclosed herein. In some embodiments, a method of treating or ameliorating a cancer in a subject herein can ablate, reverse, and/or attenuate one or more symptoms of the cancer. In some embodiments, a method of treating or ameliorating a cancer in a subject herein can allow one or more symptoms of the cancer to improve. In some embodiments, a method of treating or ameliorating a cancer in a subject herein can allow one or more symptoms of the cancer to improve by at least about 10%, least about 20%, least about 30%, least about 40%, least about 50%, least about 60%, least about 70%, least about 80%, least about 90%, least about 95%, or more as compared to levels prior to treatment. In some embodiments, a method of treating or ameliorating a cancer in a subject herein can allow one or more symptoms of the cancer to improve by at about 5% to about 99%, about 10% to about 95%, or about 15% to about 90% as compared to levels prior to treatment.

[0077] In some embodiments, before, during, and after the administration of a composition disclosed herein, cancerous cells and/or biomarkers in a subject can be measured in a biological sample, such as blood, serum, plasma, urine, peritoneal fluid, and/or a biopsy from a tissue or organ. [0078] In some embodiments, methods herein can include administration of a composition herein to reduce tumor volume, size, load, and/or burden in a subject to an undetectable size, or less than the subject's tumor volume, size, load, and/or burden prior to treatment. In some embodiments, methods herein can include administration of a composition herein to reduce tumor volume, size, load, and/or burden in a subject to an undetectable size, or to less than about 1%, less than about 2%, less than about 5%, less than about 10%, less than about 20%, less than about 25%, less than about 30%, less than about 40%, less than about 50%, less than about 60%, less than about 70%, less than about 75%, less than about 80%, or less than about 90% of the subject's tumor volume, size, load, and/or burden prior to treatment.

[0079] In some embodiments, methods herein can include administration of a composition herein to reduce the cell proliferation rate and/or tumor growth rate in a subject to an undetectable rate. In some embodiments, methods herein can include administration of a composition herein to reduce the cell proliferation rate and/or tumor growth rate in a subject to a rate less than the rate prior to treatment. In some embodiments, methods herein can include administration of a composition herein to reduce the cell proliferation rate and/or tumor growth rate in a subject to a rate less than about 1%, less than about 2%, less than about 5%, less than about 10%, less than about 20%, less than about 25%, less than about 30%, less than about 40%, less than about 50%, less than about 60%, less than about 70%, less than about 75%, less than about 80%, or less than about 90% of the rate prior to treatment.

[0080] In some embodiments, methods herein can include administration of a composition herein to reduce the development of and/or the number or size of metastatic lesions in a subject to an undetectable rate. In some embodiments, methods herein can include administration of a composition herein to reduce the development of and/or the number or size of metastatic lesions in a subject to a rate less than the rate prior to treatment. In some embodiments, methods herein can include administration of a composition herein to reduce the development of and/or the number or size of metastatic lesions in a subject to a rate less than about 1%, less than about 2%, less than about 5%, less than about 10%, less than about 20%, less than about 25%, less than about 30%, less than about 40%, less than about 50%, less than about 60%, less than about 70%, less than about 75%, less than about 80%, or less than about 90% of the rate prior to treatment. [0081] In some embodiments, the combined therapy of an inhibitor of B7-H3 and an inhibitor of CD47-SIRPa as described herein may be administered to a subject in need of the treatment at an amount sufficient to activate the innate immune system. In some embodiments, the combined therapy of an inhibitor of B7-H3 and an inhibitor of CD47-SIRPa as described herein may be administered to a subject in need of the treatment at an amount sufficient to activate the innate immune system in a tumor microenvironment by at least about 20% (e.g., least about 20%, least about 30%, least about 40%, least about 50%, least about 60%, least about 70%, least about 80%, least about 90% or greater). In some embodiments, the combined therapy of an inhibitor of B7-H3 and an inhibitor of CD47-SIRPa as described herein may be administered to a subject in need of the treatment at an amount sufficient to activate the innate immune system in a tumor microenvironment by about 20% to about 99%, by about 25% to about 95%, or by about 30% to about 90%. In some embodiments, the methods of the present disclosure synergistically activate the innate immune system in a tumor microenvironment compared to activation after treatment of a subject with a single therapy of an inhibitor of B7-H3 or a single therapy of an inhibitor of CD47- SIRPa.

[0082] In some embodiments, the combined therapy of an inhibitor of B7-H3 and an inhibitor of CD47-SIRPa as described herein may be administered to a subject in need of the treatment at an amount sufficient to activate at least one population of innate immune system cells in a tumor microenvironment by at least about 20% (e.g., at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or greater). In some embodiments, the combined therapy of an inhibitor of B7- H3 and an inhibitor of CD47-SIRPa as described herein may be administered to a subject in need of the treatment at an amount sufficient to activate at least one population of innate immune system cells in a tumor microenvironment by about 20% to about 99%, about 25% to about 95%, or about 30% to about 90%. In some embodiments, the combined therapy of an inhibitor of B7-H3 and an inhibitor of CD47-SIRPa as described herein may activate macrophages, dendritic cells (DCs), natural killer (NK) cells, or any combination thereof. In some embodiments, the combined therapy of an inhibitor of B7-H3 and an inhibitor of CD47-SIRPa as described herein may activate macrophages, dendritic cells (DCs), natural killer (NK) cells, or any combination thereof by at least about 20% (e.g., at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or greater). In some embodiments, the combined therapy of an inhibitor of B7-H3 and an inhibitor of CD47- SIRPa as described herein may activate macrophages, dendritic cells (DCs), natural killer (NK) cells, or any combination thereof. In some embodiments, the combined therapy of an inhibitor of B7-H3 and an inhibitor of CD47-SIRPa as described herein may activate macrophages, dendritic cells (DCs), natural killer (NK) cells, or any combination thereof by about 20% to about 99%, about 25% to about 95%, or about 30% to about 90%.

[0083] In some embodiments, the combined therapy of an inhibitor of B7-H3 and an inhibitor of CD47-SIRPa as described herein may be administered to a subject in need of the treatment at an amount sufficient to inflame a cold tumor microenvironment. As used herein, the terms “hot” and “cold” are routinely used to refer to T cell-infiltrated, inflamed but non-infiltrated, and non-inflamed tumors. Characteristics of hot tumors can include, but are not limited to the presence of tumor-infiltrating lymphocytes (TILs), expression of anti-programmed death-ligand 1 (PD-L1) on tumor-associated immune cells, possible genomic instability and the presence of a preexisting anti-tumor immune response. Characteristics of cold tumors can include, but are not limited to poorly infiltrated with T cells, immunologically ignorant (scarcely expressing PD-L1), high proliferation with low mutational burden (low expression of neoantigens), and low expression of antigen presentation machinery markers such as major histocompatibility complex class I (MHC I). In some embodiments, the combined therapy of an inhibitor of B7-H3 and an inhibitor of CD47-SIRPa as described herein may be administered to a subject in need of the treatment at an amount sufficient to inflame a cold tumor microenvironment by at least about 20% (e.g., at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or greater) in vivo. In some embodiments, the combined therapy of an inhibitor of B7-H3 and an inhibitor of CD47-SIRPa as described herein may be administered to a subject in need of the treatment at an amount sufficient to inflame a cold tumor microenvironment by about 20% to about 99%, about 25% to about 95%, or about 30% to about 90%. In some embodiments, inflaming a cold tumor microenvironment using the methods disclosed herein can increase of at least one interferon, increase of CD8+ T cells, increase of CD44 expression in CD4+ cells, in CD8+ cells, or both, increased TNFalpha expression in T cells, or a combination thereof. [0084] In some embodiments, the combined therapy of an inhibitor of B7-H3 and an inhibitor of CD47-SIRPa as described herein may be administered to a subject in need of the treatment at an amount sufficient to restore CD8+ T cell effector function in a cold tumor microenvironment.

[0085] In some embodiments, the combined therapy of an inhibitor of B7-H3 and an inhibitor of CD47-SIRPa as described herein may be administered to initiate immune-cell mediated cytotoxicity. In some embodiments, the combined therapy of an inhibitor of B7-H3 and an inhibitor of CD47-SIRPa as described herein may be administered to initiate CTL-cell mediated cytotoxicity. In some embodiments, the combined therapy of an inhibitor of B7-H3 and an inhibitor of CD47-SIRPa as described herein may be administered to initiate NK-cell mediated cytotoxicity. In some embodiments, the combined therapy of an inhibitor of B7-H3 and an inhibitor of CD47-SIRPa as described herein may be administered to modulate gene expression of GZMA, GZMB, NKG7, IL2R, HSPA1A, HSPA1, RANTES, CCL5, CCR5, CXCR4, CXCL12, or any combination thereof. In some embodiments, the combined therapy of an inhibitor of B7-H3 and an inhibitor of CD47-SIRPa as described herein may be administered to increase gene expression of GZMA, GZMB, NKG7, IL2R, HSPA1A, HSPAJ, RANTES, CCL5, CCR5, CXCR4, CXCL12, or any combination thereof In some embodiments, the combined therapy of an inhibitor of B7-H3 and an inhibitor of CD47-SIRPa as described herein may be administered to increase cytokine signaling in a subject. In some embodiments, the combined therapy of an inhibitor of B7- H3 and an inhibitor of CD47-SIRPa as described herein may be administered to increase cytokine signaling CCL5 (RANTES, CCL5/CCR5) signaling in a subject. In some embodiments, the combined therapy of an inhibitor of B7-H3 and an inhibitor of CD47-SIRPa as described herein may be administered to increase chemokine signaling in a subject. In some embodiments, the combined therapy of an inhibitor of B7-H3 and an inhibitor of CD47-SIRPa as described herein may be administered to increase CXCR4 (CXCR4/CXCL12) signaling in a subject.

[0086] Conventional methods, known to those of ordinary skill in the art of medicine, can be used to administer the compositions herein to a subject, depending upon the type of disease to be treated or the site of the disease. In some embodiments, the combined therapy of B7-H3 inhibitors and CD47-SIRPa inhibitors can be administered to a subject by intravenous infusion. Injectable compositions may contain various carriers such as vegetable oils, dimethylactamide, dimethyformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, and the like). For intravenous injection, water soluble antibodies can be administered by the drip method, whereby a pharmaceutical formulation containing the B7-H3 inhibitor and/or CD47-SIRPa inhibitor and a physiologically acceptable excipient can be infused. Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer’s solution or other suitable excipients. Intramuscular preparations, e.g., a sterile formulation of a suitable soluble salt form of the antibody, can be dissolved and administered in a pharmaceutical excipient such as Water-for-Inj ection, 0.9% saline, or 5% glucose solution.

[0087] In some embodiments, the combined B7-H3 inhibitor and CD47-SIRPa inhibitor therapy herein can be administered concurrently with the one or more chemotherapeutics. In some embodiments, the combined B7-H3 inhibitor and CD47-SIRPa inhibitor therapy can be administered before or after the one or more chemotherapeutics. In some embodiments, the B7- H3 inhibitor and CD47-SIRPa inhibitor therapy can be administered after administration of a combined therapy of the B7-H3 inhibitor and CD47-SIRPa inhibitor therapy and one or more chemotherapeutics. In some embodiments, the B7-H3 inhibitor therapy can be administered after administration of a combined therapy of the B7-H3 inhibitor and CD47-SIRPa inhibitor therapy. In some embodiments, the CD47-SIRPa inhibitor therapy can be administered after administration of a combined therapy of the B7-H3 inhibitor and CD47-SIRPa inhibitor therapy. In some embodiments, the one or more chemotherapeutics can be administered systemically. In some embodiments, the one or more chemotherapeutics is administered locally. In some embodiments, the one or more chemotherapeutics can be administered by intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-arterial, intra-articular, intrasynovial, intrathecal, intratumoral, oral, inhalation or topical routes. In one embodiment, the one or more chemotherapeutics is administered to the subject by intravenous infusion. The one or more chemotherapeutics may include an antimetabolite, a microtubule inhibitor, or a combination thereof. Antimetabolites include, for example, folic acid antagonist (e.g., methotrexate) and nucleotide analogs such as pyrimidine antagonist (e.g., 5-fluorouracil, foxuridine, cytarabine, capecitabine, and gemcitabine), purine antagonist (e.g., 6-mercaptopurine and 6-thioguanine), and adenosine deaminase inhibitor (e.g., cladribine, fludarabine and pentostatin).

[0088] In some embodiments, the methods are provided, the combined B7-H3 inhibitor and CD47-SIRPa inhibitor therapy can be administered prior to, concurrently, or subsequent administration of a checkpoint inhibitor that is not a B7-H3 inhibitor or a CD47-SIRPa inhibitor. In some embodiments, checkpoint inhibitors that are not a B7-H3 inhibitor or a CD47-SIRPa inhibitor can include a small molecule, a peptide, a polynucleotide, a genetically modified cell or an antibody, an antibody fragment or a combination thereof capable of inhibiting one or more checkpoint proteins. Non-limiting examples of checkpoint proteins that can be the target of a checkpoint inhibitor that is not a B7-H3 inhibitor or a CD47-SIRPa inhibitor can include CTLA- 4, PDL1, PDL2, PD1, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4 (belongs to the CD2 family of molecules and is expressed on all NK, y5, and memory CD8⁺ (aP) T cells), CD160 (also referred to as BY55), CGEN-15049, CHK 1 and CHK2 kinases, A2aR and various B-7 family ligands.

[0089] An effective amount of the pharmaceutical composition described herein can be administered to a subject (e.g., a human) in need of the treatment via a suitable route, systemically or locally. In some embodiments, a B7-H3 inhibitor and CD47-SIRPa inhibitor therapy may be administered by intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-arterial, intraarticular, intrasynovial, intrathecal, intratumoral, oral, inhalation or topical routes.

[0090] Determination of whether an amount of the combined B7-H3 inhibitor and CD47- SIRPa inhibitor therapy achieved a therapeutic effect would be evident to one of skill in the art. Effective amounts vary, as recognized by those skilled in the art, depending on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment. [0091] Empirical considerations, such as the half-life, generally contribute to the determination of the dosage. For example, where the combined B7-H3 inhibitor and CD47-SIRPa inhibitor therapy includes anti-B7-H3 and anti-CD47/SIRPa antibodies that are compatible with the human immune system, such as humanized antibodies or fully human antibodies, the half-life of the antibody may be prolonged. Frequency of administration may be determined and adjusted over the course of therapy, and is generally, but not necessarily, based on treatment and/or suppression and/or amelioration and/or delay of a target disease/disorder. Alternatively, sustained continuous release formulations of an antibody may be appropriate. Various formulations and devices for achieving sustained release are known in the art.

[0092] A subject having a target solid tumor as disclosed herein, for example, melanoma, can be identified by routine medical examination, e.g., laboratory tests, organ functional tests, genetic tests, interventional procedure (biopsy, surgery) any and all relevant imaging modalities,. In some embodiments, the subject to be treated by the method described herein is a human cancer patient who has undergone or is subjecting to an anti-cancer therapy, for example, chemotherapy, radiotherapy, immunotherapy, or surgery. In some embodiments, subjects may have received prior immuno-modulatory anti-tumor agents. Non-limiting examples of such immuno-modulatory agents include, but are not limited to as anti-PDl, anti-PD-Ll, anti-CTLA-4, anti-OX40, anti- CD137, and the like. In some embodiments, a subject shows disease progression through the treatment. In other embodiments, a subject is resistant to the treatment (either de novo or acquired). In some embodiments, such a subject is demonstrated as having advanced malignancies (e.g., inoperable or metastatic). Alternatively or in addition, in some embodiments, the subject has no standard therapeutic options available or ineligible for standard treatment options, which refer to therapies commonly used in clinical settings for treating the corresponding solid tumor.

[0093] In some embodiments, the subject herein may be a human patient having a refractory disease, for example, a refractory melanoma. As used herein, “refractory” refers to the tumor that does not respond to or becomes resistant to a treatment. In some embodiments, the subject herein may have a tumor that is resistant to at least one immune-modulatory anti-tumor agents. In some embodiments, the subject herein may have a tumor that is resistant to anti-PDl, anti-PD-Ll, or a combination thereof. In some embodiments, the subject may be a human patient having a relapsed disease, for example, a relapsed melanoma. As used herein, “relapsed” or “relapses” refers to a tumor that returns or progresses following a period of improvement (e.g., a partial or complete response) with treatment.

IV. Kits for Use in Therapy of Solid Tumors

[0094] The present disclosure also provides kits for use in treating or alleviating a solid tumor, for example, melanoma, PDA, CRC, HCC, or cholangiocarcinoma, and others described herein. Such kits can include one or more containers having an inhibitor of B7-H3 and an inhibitor of CD47-SIRPa. A kit provided herein can optionally have one or more chemotherapeutics.

[0095] In some embodiments, the kit can have instructions for use in accordance with any of the methods described herein. The included instructions can include a description of administration of the inhibitor of B7-H3 and/or inhibitor of CD47-SIRPa to treat, delay the onset, or alleviate a target disease as those described herein. In some embodiments, the kit can further comprise a description of selecting an individual suitable for treatment based on identifying whether that individual has the target disease, e.g., applying the diagnostic method as described herein. In still other embodiments, the instructions comprise a description of administering an antibody to an individual at risk of the target disease.

[0096] The instructions relating to the use of an inhibitor of B7-H3 and/or inhibitor of CD47- SIRPa can generally include information as to dosage, dosing schedule, and route of administration for the intended treatment. The containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Instructions supplied in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0097] The label or package insert indicates that the composition is used for treating, delaying the onset and/or alleviating the solid tumor. In some embodiments, instructions are provided for practicing any of the methods described herein.

[0098] The kits of this invention are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Also contemplated are packages for use in combination with a specific device, such as an inhaler, nasal administration device (e.g., an atomizer) or an infusion device such as a minipump. In some embodiments, a kit has a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). In some embodiments, the container also has a sterile access port (for example the container is an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an inhibitor of B7-H3 and/or inhibitor of CD47- SIRPa such as those described herein.

[0099] Kits may optionally provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container. In some embodiments, the invention provides articles of manufacture comprising contents of the kits described above.

General Techniques

[00100] The practice of the present invention are employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel, et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds., 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995). [00101] Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.

EXAMPLES

[00102] While the present disclosure has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the disclosure. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit, and scope of the present disclosure. All such modifications are intended to be within the scope of the disclosure.

Example 1. B7-H3 was overexpressed in melanoma and regulated tumor cell intrinsic signals that affected the tumor microenvironment

[00103] To determine the expression pattern of B7-H3 in melanoma, protein (n = 13 cell lines) and mRNA (n = 28 cell lines) levels were examined in a panel of primary and metastatic human melanoma cells and human melanocytes. Human primary cell lines tested included: WM853, WM902B, WM983A, WD1341D, WM1366, and WM1862. Human metastatic melanoma cell lines tested included: MeWo, SkMel28, Htl44, MM485, and 501Mel. All cell lines were grown and maintained in 2% Melanoma Medium (4:1 MCDB153 with 1.5 g/L sodium bicarbonate and Leibovitz’s L-15 medium with 2 mM L-glutamine, 0.005 mg/ml bovine insulin, 1.68 mM CaCh, 2% fetal bovine serum). All cells were maintained at 37°C in a 5% CO2 environment.

[00104] To determine the expression pattern of B7-H3 protein in the cell lines, cultured cells were harvested and resulting cell lysates subjected to immunoblotting. In brief, cells were solubilized in 200 pl of sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample buffer (4% sodium dodecyl sulfate, 60 mmol/L Tris, pH 6.8, 5% glycerol, 0.01% bromophenol blue, and 50 mmol/L mercaptoethanol), and heated to 95°C for 5 minutes. Whole cell protein extracts (20 to 40 pg) were separated on 6 to 15% sodium dodecyl sulfate-polyacrylamide gels. Proteins were electrophoretically transferred onto polyvinylidene difluoride membranes for 1 hour, washed in Tris-buffered saline (TBS; 100 mmol/L Tris-HCl, pH 7.5, and 150 mmol/L NaCl), before being blocked for 1 hour in TBS containing 0.1% Tween-20 and 5% milk (TBST-milk). Primary antibody (anti-human B7-H3) incubations were performed overnight at 4°C in TBST-milk followed by washing and then a 1-hour incubation with either anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibody diluted in TBST-milk. Immunocomplexes were visualized using a chemiluminescence system and detected on photographic film. After analysis, Western blots were stripped once and reprobed (anti-P-actin) to demonstrate even protein loading. [00105] To determine the expression pattern of B7-H3 mRNA in the cell lines, cultured cells were harvested and resulting cell lysates subjected to quantitative real-time PCR (qRT-PCR). In brief, total RNA was extracted from samples using the RNeasy Protect Mini Kit (Qiagen). The RNA Integrity Number of each sample was determined using the 2100 Bioanalyzer instrument (Agilent Technologies). Samples were selected with stringent criteria based on RNA Integrity Number of 7.0 or greater, and a minimum of 500 ng of total RNA. For validating the transcript signatures in melanoma cell lines, 1 pg total RNA was reverse transcribed to cDNA using RNA to cDNA EcoDry™ Premix (Random Hexamers) (Takara Bio). The cDNA was next assayed in quantitative real-time PCR (qPCR) using the QuantiNova™ SYBR® Green PCR Kit (Qiagen) on the CFX384™ Real-Time System (Bio-Rad). GAPDH was used as a housekeeping gene for normalization.

[00106] Metastatic cell lines showed significantly higher levels of B7-H3 expression both at the protein (Fig. 1A) and mRNA level (Fig. IB) as compared to primary melanoma cells, suggesting a role for B7-H3 during progression from primary disease to metastasis.

[00107] To assess the frequency of aberrant expression in metastatic disease, we next stained a tissue microarray consisting of metastatic human melanoma samples (n = 46) with anti-B7-H3 using immunohistochemistry. Briefly, 46 approximately 2 mm cores were retrieved from human melanoma samples and embedded into a single tissue microarray paraffin block. For immunohistochemistry, 5-pm sections of the tissue microarray paraffin block were subjected to antigen retrieval and blocking using Target Retrieval Solution and Serum-Free Protein Block (Dako), sequentially. Sections were further stained with diluted antibody with Background Reducing Components and Streptavidin/HRP (Dako). Signals were developed via the SignalStain® DAB Substrate Kit (Cell Signaling). Figs. 1C and ID show that high levels of membranous and cytoplasmic expression of B7-H3 were found in a significant portion of the cases - 61%.

[00108] To gain insight into B7-H3 functions, the downstream effectors of B7-H3 were examined. Two human metastatic melanoma cell lines were selected for the study, Hs. 688(A)T and Hs. 839T, with the highest levels of B7-H3 expression. Cells were transfected with either scrambled or B7-H3 specific siRNAs in triplicates and silencing was confirmed using quantitative RT-PCR according to the methods described in this example. The transcriptomes were also analyzed via RNA-sequencing. In brief, the total RNA extracted from the samples was subjected to ribosomal RNA depletion and further used as input for library construction by the Illumina TruSeq Standard Total RNA library Pre Gold kit (Illumina). The libraries were then sequenced on the Illumina HiSeq 2500 system (Illumina) using 2 x 100 bp paired-end protocol to a minimum mean coverage of 50x. Raw reads were quality filtered by trimming the read ends; bases with quality <20 were removed. Next, the quality at the 20th percentile was computed. The entire read was discarded if the quality at the 20th percentile was less than 15. Reads shorter than 40 bases after trimming were discarded as well. If one or more reads in the pair failed the quality check both reads were discarded. Paired-end RNA sequencing reads were mapped to the human reference genome (Ensembl annotation, build 37) and to sequences from the Repbase database of human repetitive elements (release 19) using the STAR aligner in a manner similar to that described in Dobin et al., Bioinformatics. 2013;29(l): 15-21, the disclosure of which is incorporated herein in its entirety. Aligned reads were assigned to genes using the featureCounts function of the Rsubread package via the external Ensembl annotation. This procedure generated the raw read counts for each gene. Gene expression in the form of log2 counts per million reads was computed and normalized across the samples using the TMM method as implemented in the cal cNormF actors function of edgeR package in a manner similar to that described in Robinson et al., Bioinformatics. 2010;26(l): 139-140 and Robinson et al., Genome Biol. 2010;l 1(3):R25, the disclosures of which are incorporated herein in their entirety. Differential expression analysis was performed using the limma software package. Expression data were used in conjunction with the weights computed by the voom transformation based on the mean-variance relationship of log read counts to calculate moderated t-statistics using empirical Bayes in a manner similar to that described in Law et al., Genome Biol. 2014;15(2):R29,the disclosure of which is incorporated herein in its entirety. For protein-coding RNA (mRNA), the gene signature differentiating each pair of subtypes was identified. For this analysis, only genes with counts per million reads greater than 10 in at least 2 samples were considered. To create a comprehensive global heatmap depicting the gene signature of different subtypes, genes to be differentially expressed with a Benjamini- Hochberg-corrected P value of P < 0.005 and a fold change of >1.5 were considered. Principal component analysis of the resulting sets of differentially expressed genes confirmed effective grouping of subtypes. To determine the biological modules that are affected in our differentially expressed gene set, the Database for Annotation, Visualization and Integrated Discovery (DAVID), Ingenuity Pathway Analysis, and the Gene Ontology database were used. Pathways were examoined with a Benjamini -Hochberg-adjusted P value of P < 0.05. The analysis revealed eleven up- and six down-regulated genes upon silencing of B7-H3 that were consistent across both cell lines (Figs. 1E-1F). Surprisingly, it was found that majority of B7-H3 downstream effectors are modulators of the tumor microenvironment -MMP3 (matrix metalloproteinase family), P3H1 and FBLN5 (collagen family), ITGA2 (integrin family), CXCL8 (IL8, inflammation), and interferon stimulating genes IFIT2 (antiviral responses), CMPK2 (antiviral responses), OASL (antiviral responses), and RSAD2 (antiviral responses). These data suggested that B7-H3, when overexpressed, had key functions in shaping the tumor microenvironment by suppressing antiviral and inflammatory responses, and by regulating matricellular proteins.

[00109] To study the effects of B7-H3 on the microenvironment, a suitable model system was searched for next. Prior studies on B7-H3 utilized the B 16 spontaneous mouse melanoma model that has several limitations, as it does not reflect the genetic alterations, ultraviolet-driven Protein and mRNA expression levels of B7-H3 in a panel of murine cells was examined by immunoblotting (Fig. 1G) blotting and quantitative RT-PCR (Fig. 1H), respectively using methods described earlier in this Example. While melan-a cells - an immortalized murine melanocyte line with loss of Cdkn2a - and B16 melanoma lines showed significantly low levels of B7-H3 expression, cells obtained from genetically engineered murine models (GEMM), in particular YUMM2.1 cells with activated P-catenin signaling (Braf^V600E/wt/Pten'^/7Cdkn2a^+/‘ /Bcat^loxex3/wt) revealed significantly high levels (Figs. 1G and 1H). Expression of B7-H3 was also notable in YUMM1.7/YUMMER1.7 cells (Braf^V600E/wt/Pten'^/7Cdkn2a'^/‘), but cells with activated P-catenin, YUMM2.1, had the highest level. [00110] Next, B7-H3 or P-catenin was silenced using gene-specific siRNAs. Fig. II shows that silencing of B7-H3 had no effect on P-catenin, but knocking down P-catenin lowered B7-H3 protein levels, suggesting that B7-H3 was in part modulated by P-catenin in YUMM2.1 cells. The downstream effectors of B7-H3 in YUMM2.1 murine cells were compared to those found in human melanomas by quantitative RT-PCR (Fig. 1J). Majority such s RAB27B and GPRC5A, as well as interferon stimulating genes - IFIT2, CMPK2, OASL, and RSAD2 - were validated. In aggregate, it was concluded that YUMM2.1 cells represented a suitable model and offer a unique advantage to study B7-H3 and its effects on the local microenvironment.

[00111] To address the relationship between B7-H3 and Wnt/p-catenin signaling in human cells, their correlation was examined by mining the Reverse Phase Protein Array dataset from the Cancer Cell Line Encyclopedia (CCLE, n = 899). A co-occurrence of B7-H3 and Wnt signaling pathway proteins was found including P-catenin, whereas anti-correlation was noted between B7- H3 and VAV1 (Fig. IK). In sum, B7-H3 overexpression co-occurred with activated P-catenin signaling in YUMM2.1 cells and a subset of human cancers. YUMM2.1 (as well as YLnMM1.7/YUMMER1.7) preclinical model represents a suitable system to study the local microenvironment regulated by B7-H3.

Example 2. Blockade of B7-H3, but not PD-1, resulted in an inflamed tumor microenvironment in a preclinical model of activated P-catenin signaling

[00112] Clinical and preclinical studies demonstrated that melanomas driven by P-catenin, in particular those co-occurring with BRAF^V600E and loss of PTEN as in YUMM2.1 model are highly metastatic and unresponsive to anti-PD-l/anti-CTLA-4, due to lack of immune cell infiltration and T-cell exclusion. This example aimed to investigate the tumor microenvironment changes driven by B7-H3 in a syngeneic tumor system (YUMM2.1) and explore therapeutic strategies by targeting B7-H3.

[00113] Immunocompetent C57BL/6J mice were injected with 1 x 10⁶ YUMM2.1 murine melanoma cells subcutaneously into the flanks of each mouse. Nine days after the first injection, 600 pg of IgG control, 600 pg of anti-B7-H3 (clone MJ18, BioXCell), 300 pg of anti-PDl (clone RMP1-14, BioXCell), or 600 pg of anti-B7-H3 and 300 pg of anti-PDl were injected intraperitoneally (i.p.) where IgG or anti-B7-H3 was injected three times a week and anti-PDl was injected every five days. Injections were continued until the resulting tumors reached 20-50 mm³. Antitumor activities and changes within the local milieu were then examined by RNA-sequencing. In brief, total RNA isolated from murine tumor bulk was subjected to RNA sequencing similar to methods described in example 1. Using the resulting sequencing data, first assessed was the quality of paired-end reads with FASTQC (vO.l 1.8). Next, reads were filtered with BBDUK from BBTOOLS (v37.53) to remove adapters, known artefacts, and quality trimmed (PHRED quality score < 10). Reads that became too short after trimming (N<60 bp) were discarded. Singleton reads (i.e. reads whose mate has been discarded) were not retained. The transcript-level quantification of cleaned data was estimated using SALMON (vl.0.0) by a quasi -mapping on Mus musculus GRCm38. The gene-level quantification was estimated using tximport library and converted 22,231 mouse gene expression to 15,091 ortholog human gene expression using biomaRt library. Differential expression was assessed on tumor samples under IgG treatment (n = 3), B7-H3 inhibitor treatment (n = 3), PD-1 inhibitor treatment (n = 3), and combination treatment of B7-H3 inhibitor with PD-1 inhibitor (n = 3) using R DESeq2 library (vl.81.1) similar to the methods described in Luo et al., BMC Bioinformatics. 2009; 10: 161, the disclsure of which is incoprotaed herein in its entirety. The differential expression between two conditions was called when the adjusted /^J- value was at 5% and log2 fold change was more than 1. Next, gene set enrichment analysis was performed that captured pathways perturbed towards both directions simultaneously using the 15,091 ranked genes identified in our dataset and annotated in ENSEMBL (v94) against the KEGG pathways using R GAGE (v2.28.2) and Pathview (vl.18.2) libraries. Heatmap was drawn using R ComplexHeatmap library (v2.0.0). Fig. 2A shows that tumor reduction was observed with either anti-B7-H3 or anti -PD-1 therapy, but no synergy was observed when combined. Even though the anti -PD-1 versus control appeared to be responsive to this treatment, the interpretation of the data on the murine system was regarded as unresponsive or resistant based on clinical trials and preclinical work, and that any effective treatment should flatten the growth curve significantly as compared to anti-PD-1 alone group.

[00114] To identify the composition of the immune cell infiltrate within the tumor milieu, next analyzed was the major immune cell subsets and functional categories (i.e. CD4+ T cells, CD8+ T cells, NK cells, dendritic cells, B cells, and macrophages) from the dissected tumors using bulk RNA sequencing as well as CD45+ sorted cells (ultra low RNA sequencing, each treatment group in triplicates) using methods described in the examples herein. As shown in Fig. 2B, immune cell lineage-specific transcript signatures, in particular innate (macrophages, DCs, and NK cells) populations of the immune system, were significantly upregulated in mice treated with anti-B7-H3 antibody as compared to the isotype control, or anti-PD-1, and to a lesser extent with the combination of both antibodies. It was noted that there was an upregulation of immune cell recruitment (CCR5), inflammatory (IL4R, JAK1/3, and STAT6), and macrophage inhibitory signals (CD47, SIRPa), as well as Interferon pathways (type I IFN (IFNAR2/JAK1/STAT1) and type II IFN (IFNGR1/2, JAK1/2)) upon B7-H3 inhibition in the P-catenin activated YUMM2.1 tumors (Fig. 2C).

[00115] It was next determined whether similar results were observed when YUMMER1.7 tumor model was used in an identical experiment. To generate the YUMMER1.7 tumor models, immunocompetent C57BL/6J mice were injected with 5 x IO⁵ YUMMER1.7 murine melanoma cells subcutaneously into the flanks of each mouse. Following injection of the YUMMER1.7 cells, mice were administered the same treatments in the same manner as that described above for the YUMM2.1 tumor model mice. While B7-H3 inhibition resulted in more immune cell infiltration in YUMMER1.7 tumors as compared to control or PD-1 inhibition, the degree of infiltration and inflammation was significantly higher in the P-catenin activated YUMM2.1 tumors in mice treated with B7-H3 inhibitor as compared to YUMMER1.7 tumors (Fig. 2D). CCR5 and IL4R (JAK1/3 and STAT6) signaling axes were elevated when B7-H3 is inhibited in both models, whereas there was increased type I IFN (IFNAR2/JAK1/STAT1) and type II IFN (IFNGR1/2, JAK1/2), CCR5/CCR1, and TLR4/RF5 signaling in the p-catenin activated YUMM2. 1 tumors. Macrophage signature, in particul r m crophage inhibitory signals (CD47, SIRPa) was confirmed using CD45+ RNA sequencing (Fig. 2E). Macrophage signature was also confirmed in harvested tumor tissue by immunohistochemistry using methods described in the examples herein. Figs. 3A-3C show IBA-1 (Allograft Inflammatory Factor 1) immunohistochemistry of tumors in treatment groups with IgG control, anti-B7-H3, or anti-PD-1 showing activated macrophages within the tissue whereas Figs. 3D-3F show CD47 expression of the tumor cells and Figs. 3G-3I show that SIRPa expression, a receptor for CD47 in immune subpopulations such as macrophages and DCs, was also indicated. An intense macrophage signal depicted by IBA-1 expression (activated macrophages) in the anti-B7-H3 group as compared to the others was noted (Figs. 3A-3C). All together, these data suggest that elevated levels of B7-H3 confer immunosuppressive signals, and its inhibition leads to reprogramming of the tumor microenvironment in favor of macrophage infiltration and potential macrophage checkpoint activation (CD47/Sirpa), enrichment of myeloid recruitment signals (CCR5), and pro-inflammatory signals (IL4R).

Example 3. Blockade ofB7-H3 together with CD47, as well as inhibition of PD-1 and CD47, were synergistic in reducing tumor growth in a preclinical model of activated f- catenin signaling

[00116] Since CD47 and its interacting partner SIRPa were significantly upregulated in tumors upon anti-B7-H3 treatment, it was next explored whether combining and co-targeting molecules signaling distinct pathways, B7-H3 and CD47, had a synergistic therapeutic effect. In addition to treating YUMM2.1 tumor models with IgG, anti-B7-H3, mice were also injected i.p. with 200 pg of anti-CD47 (clone MIAP301, BioXCell) three times per week. Growth patterns in YUMM2.1 tumors upon treatment with IgG, anti-B7-H3, anti-CD47, or combination of the two showed similar antitumor activities with anti-B7-H3 or anti-CD47 alone, but importantly significant tumor reduction and additive benefit was noted when B7-H3 and CD47 inhibitors were combined (Fig. 4A, Fig. 5A). It was then explored whether inhibiting PD-1 was synergistic with CD47 in this model, and it was found that such additive therapeutic benefit does exist, and this combination may be a candidate in the clinical setting as well (Fig. 4B, Fig. 8A).

[00117] Next, the YUMM2.1 tumor models were subjected to NK cell and CD8+ T cell depletion experiments. In brief, for NK cell depletion experiments, mice were pretreated with 200 pg of anti-NKl .1 (clone PK136, BioXCell) via i.p. on days 1 and 3 prior to the murine melanoma cell challenge, and then received treatment of 150 pg twice a week. For the CD8+ T cell depletion experiment, anti-CD8a (clone YTS169.4 BioXcell) 250 pg via i.p. three times per week was used. Tumor volumes were calculated using the following equation: 0.5 x 1 x w². NK cell and CD8+ T cell depletion experiments showed dependency to both antitumor effector mechanisms upon anti- B7-H3 treatment (Figs. 4C and 4D). It was next sought to determine therapeutic benefit of blocking B7-H3 and PD-1, B7-H3 and CD47, or PD-1 and CD47 in a different preclinical melanoma model, YUMMER1.7, driven by Braf ^600E/wt/Pten’^/7Cdkn2a'^/' and high levels of B7- H3. Experiments did not demonstrate any additive benefit of CD47 combination, nor by combining B7-H3 and PD-1 inhibitors together (Figs. 4E and 4F).

[00118] To determine the mechanism of tumor cytotoxicity, single cell RNA sequencing (scRNA sequencing) was performed on CD45+ sorted cells. In brief, CD45+ cells were sorted from murine tumor samples undergoing IgG (n = 3), B7-H3 inhibition (n = 3), CD47 inhibition ( = 3), and combination treatment with B7-H3 and CD47 inhibition (n = 3). Single cell suspensions were stained with diluted hashtag antibodies according to the New York Genome Center hashing protocol, similar to the methods described in Stoeckius et al., Genome Biol 19, 224 (2018), the disclosure of which is incorporated herein in its entirety. Hashed samples were pooled in equal amounts of live cells to achieve a target of 2 million cells/ml. The hashed sample pool was then loaded onto two lanes of the lOx Genomics NextGen 5’vl.l assay according to the manufacturer’s instructions with a targeted cell recovery of 20,000 cells. Gene expression libraries were made as per the lOx Genomics protocol. During cDNA amplification, hashtag oligonucleotides (HTO) were enriched with the addition of 3 pmol of a HTO Additive primer. The PCR product was isolated from the mRNA-derived cDNA via SPRI Select size selection and used for making HTO libraries according to the New York Genome Center hashing protocol. All libraries were quantified via Agilent 2100 hsDNA Bioanalyzer and KAPA library quantification kit (Roche). Gene expression libraries were sequenced at a targeted depth of 25,000 reads per cells, and HTO libraries were sequenced at a targeted read depth of 1,000 reads per cell. All libraries were sequenced on the Illumina NovaSeq S2 100 cycle kit using run parameters set to 28x8x0x60 (Rlxi7xi5xR2). For the scRNA sequencing data analysis, BCL files were first base-called and demultiplexed using cellranger mkfastq v5.0.1. Alignment to mouse reference mm 10 and feature counting were performed using cellranger count v5.0.1. Then, HTO barcodes were mapped to samples of origin. Briefly, a “key” matrix with biological samples as rows and HTO features as columns was created. A cell was populated with a value of “ 1 ” if the sample was supposed to be positive for the hashtag, “0” otherwise. For each barcode, pairwise distances with a cosine similarity metric were computed from the key matrix to generate a cosine similarity matrix. Thus, a barcode was initially assigned to a sample based on the largest cosine similarity to a sample. Additionally, for each barcode, a signal to noise ratio (sn) was calculated by subtracting its highest distance metric from its second highest distance metric. The sn ratio for each initially assigned sample usually followed a bi-modal distribution. In order to identify the local minimum of this distribution, four standard deviations were calculated from the mode of the right most identified local maximum. Only barcodes superior or equal to local minimum were kept as the final barcodes belonging to that sample. Data were then read and analyzed using Bioconductor libraries including DropletUtils (vl .12.3) and scater (v.1.20.1). The cells with a high percentage of mitochondria were filtered out and library size normalization was then performed. All data was merged using R BiocSingular library (vl.8.1), renormalized to adjust for differences in depth using R batchelor library (vl.8.1), dimensionality reduced using UMAP method, and then subjected to Louvain clustering. Using R SingleR (v.1.6.1) and celldex (vl.2.0) libraries, the clusters were annotated and each cell individually using the Wilcoxon method with the fine pruned labels from the Immunological Genome Project (See Heng et al., Nat Immunol 9, 1091-1094 (2008)) which consisted of microarray profiles of pure mouse immune cells. To identify differentially expressed genes between treatments, FindMarker of R Seurat library (v4.0.2) was used. Heatmaps were drawn using the R Compl exHeatmap library (v2.0.0).

[00119] An average of 838 cells per sample were sequenced with a total of 10,054 cells after quality control. Overall, major immune subsets were represented within the groups with similar distributions across treatment categories (Figs. 5B-5C). The immune cells were then subtyped based on known gene expression signatures. In the CD47 treated group, a predominance of NK cells (NK.49CI+), NKT (NKT.4+), and DCs ((DC.8-, DC.11B-, DC.8+, DC.PCD.8-) was observed together with macrophages, Tregs, and eosinophils. In the B7-H3 treated group, there was abundance of T cells (T.CD8, T.CD4, T.4EFF, T.4.Pa, T.4SP, T.DN, T.4PLN, T4. FP3+25, and T. ISP) along with macrophages, B cells, DCs and eosinophils (Fig. 5D). These data suggested recruitment of specific immune cell subsets and utilization of different mechanisms during tumor cell killing upon CD47 and B7-H3 inhibition.

[00120] Differentially expressed genes between the treatment groups were assessed to evaluate gene expression specific signals during cytolytic activity (Fig. 5E). CTL- and NK-cell mediated cytotoxicity (IgG vs. B7-H3/CD47) such as GZMA, GZMB, NKG7, IL2R as well as heat shock genes HSPA1A and HSPA1B (B7-H3 vs. B7-H3/CD47, CD47 vs. B7-H3/CD47) were enriched. Enrichment of cytokine signaling CCL5 (RANTES, CCL5/CCR5) upon B7-H3 inhibition, and CXCR4 (CXCR4/CXCL12) upon CD47 inhibition — tboth of which are critical for hematopoietic cell trafficking — was observed. In the data, CCL5 was unique to B7-H3 inhibition, whereas CXCR4 was observed with CD47 treatment.

[00121] Leveraging scRNA-sequencing, data were dissected further and immune cells that expressed these molecules were mapped out using UMAP as described herein (Figs. 6A-6T and Figs. 7A-7O) HSPA1A and HSPA1B genes not only expressed at high levels during tumor cytotoxicity (IgG vs. B7-H3/CD47, Figs. 6K-6T), but they expressed across most of the immune cell types ubiquitously, whereas GZMA and GZMB was mostly expressed in macrophages, NK cells, and T cells (Figs. 6A-6J). Expression details for CCL5, CXCR4, and NKG7 were also mapped out (Figs. 7A-7O).

[00122] In aggregate, these data showed that inhibition of B7-H3/CD47 in combination was effective in a well-characterized beta-catenin driven murine model. Both inhibitors utilized distinct immune cell populations during cytotoxicity. They employed known cytolytic mechanisms such as GZMA, GZMB, NKG7, HSPA1, and HSPAIB, and their expression patterns on immune cells was dissected herein.

Example 4. Therapeutic synergy was dependent on CCR5 and IL4R signaling [00123] Since TAM-associated inflammatory signals were consistently upregulated upon B7- H3 inhibition, the antitumor effects of co-targeting B7-H3 and CD47 were examined to determine if there was dependency on CCR5 or IL4R signaling. C57BL/6 CCR5-/- or IL4R-/- mice were used in the exemplary methods. Tumors were generated as described herein and the mice were treated with IgG control, B7-H3 inhibitor, and CD47 inhibitor alone or in combination according to methods described herein. As shown in Fig. 8A, the therapeutic synergy was robust but dissipated when these signaling axes were absent (Figs. 8B-8C). Without being bound by theory, the observed dissipation was likely due to a lack of tumor-associated macrophage and DC recruitment and antigen presentation. Similar results were obtained with the combination of PD-1 and CD47 inhibition (Figs. 8A-8C).

[00124] In order to determine whether B7-H3 conferred a suppressive microenvironment in human melanomas, the transcriptome data of the TCGA’s metastatic melanomas was next examined (n = 320). In brief) total RNA was isolated from human tumor tissues and subjected to RNA-sequencing. TCGA’s RNA-sequencing data (illuminahiseq_maseqv2-RSEM_genes, version 01/28/2016) and clinical phenotype (merge clinical, version, version 01/28/2016) were downloaded from firebrowse of Broad Institute. Analysis showed that B7-H3 correlated significantly with suppressive signals (M2 macrophage, myeloid derived suppressive cell (MDSC), T cell exclusion, and cancer associated fibroblast (CAF) signature), whereas it significantly anti-correlated with PD-1, and moderately with PD-L1, and IL-15 (Figs. 8D and 8E). [00125] In aggregate, these data provided an additional layer of evidence that B7-H3 conferred immunosuppressive signals and when inhibited, modulated the microenvironment. The data suggested cooperative and non-redundant functions of B7-H3- and CD47-driven signaling and provided evidence that their blockade was efficacious therapeutically in preclinical murine models resistant to PD-1 inhibition.

[00126] In sum, the data provided in the examples herein showed that B7-H3 and CD47 drove distinct signaling pathways in shaping the tumor microenvironment and their combined blockade resulted in cooperative cytotoxicity. The data also delineated independent pathways affecting the tumor microenvironment, as combining distinct immune cell targeting mechanism (i.e. CD8+ T cell effector function, NK cell cytotoxicity, macrophage-mediated phagocytosis) was a viable therapeutic strategy.

NUMBERED EMBODIMENTS

[00127] Notwithstanding the appended claims, the following numbered embodiments are also contemplated herein and form part of the instant disclosure:

[00128] 1. A method for treating a solid tumor in a subject, the method comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition, the pharmaceutical composition comprising an inhibitor of B7-H3 (CD276) and an inhibitor of CD47-SIRPa, wherein the subject is not responsive to anti-programmed cell death 1/programmed cell death ligand 1 (PD1/PDL1) therapy.

[00129] 2. The method of embodiment 1, wherein the subject is a human patient having a solid tumor selected from the group consisting of pancreatic ductal adenocarcinoma (PDA), colorectal cancer (CRC), melanoma, breast cancer, lung cancer, upper and lower gastrointestinal malignancies, squamous cell head and neck cancer, genitourinary cancer, ovarian cancer, and sarcomas.

[00130] 3. The method of embodiment 2, wherein the subject is a human patient having melanoma.

[00131] 4. The method of any one of embodiments 1-3, wherein the effective amount of the pharmaceutical composition is sufficient to activate the innate immune system. [00132] 5. The method of any one of embodiments 1-4, wherein the effective amount of the pharmaceutical composition is sufficient to activate at least one population of innate immune system cells.

[00133] 6. The method of embodiment 5, wherein the at least one population of innate immune system cells is selected from the group consisting of macrophages, dendritic cells (DCs) and natural killer (NK) cells.

[00134] 7. The method of any one of embodiments 1-6, wherein the effective amount of the pharmaceutical composition is sufficient to reduce tumor volume.

[00135] 8. The method of any one of embodiments 1-7, wherein the inhibitor of B7-H3 comprises at least one of a small molecule, a peptide, a polynucleotide, a genetically modified cell or an antibody, an antibody fragment or a combination thereof.

[00136] 9. The method of embodiment 8, wherein the inhibitor of B7-H3 is an antibody.

[00137] 10. The method of embodiment 9, wherein the antibody is a full-length antibody or an antigen-binding fragment thereof.

[00138] 11. The method of embodiment 9, wherein the antibody is a human antibody or a humanized antibody.

[00139] 12. The method of embodiment 8, wherein the inhibitor of B7-H3 is an isolated nucleic acid or set of nucleic acids, which collectively encodes an antibody.

[00140] 13. The method of embodiment 8, wherein the inhibitor of B7-H3 comprises at least one of enoblituzumab, omburtamab, orlotamab, BP-102, DS-7300, 8H-9, TCB-005, YBL-015, or a combination thereof.

[00141] 14. The method of any one of embodiments 1-7, wherein the inhibitor of B7-H3 comprises a B7-H3-specific chimeric antigen receptor T (CAR-T) cell.

[00142] 15. The method of any one of embodiments 1-7, wherein the inhibitor of CD47-

SIRPa is a CD47-ligand inhibitor, a SIRPa-receptor inhibitor, or a combination thereof.

[00143] 16. The method of embodiment 15, wherein the CD47-ligand inhibitor comprises at least one of a small molecule, a peptide, a polynucleotide, a genetically modified cell or an antibody, an antibody fragment or a combination thereof.

[00144] 17. The method of embodiment 16, wherein the CD47-ligand inhibitor is an antibody. [00145] 18. The method of embodiment 17, wherein the antibody is a full-length antibody or an antigen-binding fragment thereof.

[00146] 19. The method of embodiment 17, wherein the antibody is a human antibody or a humanized antibody.

[00147] 20. The method of embodiment 16, wherein the CD47-ligand inhibitor is an isolated nucleic acid or set of nucleic acids, which collectively encodes an antibody.

[00148] 21. The method of embodiment 15, wherein the CD47-ligand inhibitor is a CD47- specific chimeric antigen receptor T (CAR-T) cell.

[00149] 22. The method of embodiment 15, wherein the SIRPa-receptor inhibitor comprises at least one of a small molecule, a peptide, a polynucleotide, a genetically modified cell or an antibody, an antibody fragment or a combination thereof.

[00150] 23. The method of embodiment 22, wherein the SIRPa-receptor inhibitor is an antibody.

[00151] 24. The method of embodiment 23, wherein the antibody is a full-length antibody or an antigen-binding fragment thereof.

[00152] 25. The method of embodiment 23, wherein the antibody is a human antibody or a humanized antibody.

[00153] 26. The method of embodiment 22, wherein the SIRPa-receptor inhibitor is an isolated nucleic acid or set of nucleic acids, which collectively encodes an antibody.

[00154] 27. The method of embodiment 15, wherein the inhibitor of CD47-SIRPa comprises at least one of RRx-001, a dinitroazetidine derivative, Hu5F9-G4, CC-90002, TTI-621, ALX148, SRF231, SHR-1603, IBI188, ST-1901, SGNCD-47M, or a combination thereof.

[00155] 28. A method of inflaming a cold tumor microenvironment, the method comprising administering to the cold tumor microenvironment a composition comprising an inhibitor of B7- H3 (CD276) and an inhibitor of CD47-SIRPa.

[00156] 29. The method of embodiment 28, wherein a tumor having the cold tumor microenvironment is a solid tumor.

[00157] 30. The method of embodiment 29, the solid tumor is selected from the group consisting of pancreatic ductal adenocarcinoma (PDA), colorectal cancer (CRC), melanoma, breast cancer, lung cancer, upper and lower gastrointestinal malignancies, squamous cell head and neck cancer, genitourinary cancer, ovarian cancer, and sarcomas.

[00158] 31. The method of embodiment 30, wherein the solid tumor is melanoma.

[00159] 32. The method of any one of embodiments 29-31, wherein the solid tumor comprises at least one of B7-H3 overexpression, overactive P-catenin signaling, or a combination thereof.

[00160] 33. The method of any one of embodiments 29-33, wherein the solid tumor comprises at least one genetic mutation comprising BRAF^V600E, loss of PTEN, or a combination thereof.

[00161] 34. The method of any one of embodiments 28-33, wherein inflaming a cold tumor microenvironment comprises increase of at least one interferon, increase of CD8+ T cells, increase of CD44 expression in CD4+ cells, in CD8+ cells, or both, increased TNF alpha expression in T cells, or a combination thereof.

[00162] 35. The method of any one of embodiments 28-34, wherein the inhibitor of B7-H3 comprises at least one of a small molecule, a peptide, a polynucleotide, a genetically modified cell or an antibody, an antibody fragment or a combination thereof.

[00163] 36. The method of embodiment 35, wherein the inhibitor of B7-H3 is an antibody.

[00164] 37. The method of embodiment 36, wherein the antibody is a full-length antibody or an antigen-binding fragment thereof.

[00165] 38. The method of embodiment 36, wherein the antibody is a human antibody or a humanized antibody.

[00166] 39. The method of embodiment 35, wherein the inhibitor of B7-H3 is an isolated nucleic acid or set of nucleic acids, which collectively encodes an antibody.

[00167] 40. The method of embodiment 35, wherein the inhibitor of B7-H3 comprises at least one of enoblituzumab, omburtamab, orlotamab, BP-102, DS-7300, 8H-9, TCB-005, YBL-015, or a combination thereof.

[00168] 41. The method of any one of embodiments 28-34, wherein the inhibitor of B7-H3 comprises a B7-H3-specific chimeric antigen receptor T (CAR-T) cell.

[00169] 42. The method of any one of embodiments 28-34, wherein the inhibitor of CD47-

SIRPa is a CD47-ligand inhibitor, a SIRPa-receptor inhibitor, or a combination thereof. [00170] 43. The method of embodiment 42, wherein the CD47-ligand inhibitor comprises at least one of a small molecule, a peptide, a polynucleotide, a genetically modified cell or an antibody, an antibody fragment or a combination thereof.

[00171] 44. The method of embodiment 43, wherein the CD47-ligand inhibitor is an antibody.

[00172] 45. The method of embodiment 44, wherein the antibody is a full-length antibody or an antigen-binding fragment thereof.

[00173] 46. The method of embodiment 44, wherein the antibody is a human antibody or a humanized antibody.

[00174] 47. The method of embodiment 43, wherein the CD47-ligand inhibitor is an isolated nucleic acid or set of nucleic acids, which collectively encodes an antibody.

[00175] 48. The method of embodiment 42, wherein the CD47-ligand inhibitor is a CD47- specific chimeric antigen receptor T (CAR-T) cell.

[00176] 49. The method of embodiment 42, wherein the SIRPa-receptor inhibitor comprises at least one of a small molecule, a peptide, a polynucleotide, a genetically modified cell or an antibody, an antibody fragment or a combination thereof.

[00177] 50. The method of embodiment 49, wherein the SIRPa-receptor inhibitor is an antibody.

[00178] 51. The method of embodiment 50, wherein the antibody is a full-length antibody or an antigen-binding fragment thereof.

[00179] 52. The method of embodiment 50, wherein the antibody is a human antibody or a humanized antibody.

[00180] 53. The method of embodiment 49, wherein the SIRPa-receptor inhibitor is an isolated nucleic acid or set of nucleic acids, which collectively encodes an antibody.

[00181] 54. The method of embodiment 42, wherein the inhibitor of CD47-SIRPa comprises at least one of RRx-001, a dinitroazetidine derivative, Hu5F9-G4, CC-90002, TTI-621, ALX148, SRF231, SHR-1603, IBI188, ST-1901, SGNCD-47M, or a combination thereof.

[00182] 55. A pharmaceutical composition for treating a solid tumor in a subject, the pharmaceutical composition comprising an inhibitor of B7-H3 (CD276) and an inhibitor of CD47- SIRPa. [00183] 56. The pharmaceutical composition of embodiment 55 further comprising at least one pharmaceutically acceptable excipient.

[00184] 57. The pharmaceutical composition of embodiment 55 or embodiment 56, wherein the inhibitor of B7-H3 (CD276) and the inhibitor of CD47-SIRPa.comprises at least one of a small molecule, a peptide, a polynucleotide, a genetically modified cell or an antibody, an antibody fragment or a combination thereof.

[00185] 58. The pharmaceutical composition of any one of embodiments 55-57, wherein the inhibitor of B7-H3 comprises at least one of enoblituzumab, omburtamab, orlotamab, BP- 102, DS- 7300, 8H-9, TCB-005, YBL-015, or a combination thereof.

[00186] 59. The pharmaceutical composition of any one of embodiments 55-58, wherein the inhibitor of CD47-SIRPa comprises at least one of RRx-001, a dinitroazetidine derivative, Hu5F9- G4, CC-90002, TTI-621, ALX148, SRF231, SHR-1603, IBI188, ST-1901, SGNCD-47M, or a combination thereof.

[00187] 60. The pharmaceutical composition of any one of embodiments 55-59, wherein the inhibitor of B7-H3 (CD276) and the inhibitor of CD47-SIRPa are administered in a dosage amount that restores CD8+ T cell effector function in a cold tumor microenvironment.

[00188] 61. A composition for treating a solid tumor in a subject, the composition comprising at least one inhibitor of B7-H3 (CD276) and at least one inhibitor of CD47-SIRPa

[00189] 62. The composition of embodiment 61 further comprising at least one pharmaceutically acceptable excipient.

[00190] 63. The composition of either embodiment 61 or embodiment 62, wherein the at least one inhibitor of B7-H3 comprises at least one of a small molecule, a peptide, a polynucleotide, a genetically modified cell or an antibody, an antibody fragment, or any combination thereof.

[00191] 64. The composition of embodiment 63, wherein the at least one inhibitor of B7-H3 comprises an antibody.

[00192] 65. The composition of embodiment 63, wherein the at least one inhibitor of B7-H3 comprises an isolated nucleic acid or set of nucleic acids, which collectively encodes an antibody. [00193] 66. The composition of embodiment 63, wherein the at least one inhibitor of B7-H3 comprises a B7-H3-specific chimeric antigen receptor T (CAR-T) cell. [00194] 67. The composition of embodiment 63, wherein the at least one inhibitor of B7-H3 comprises enoblituzumab, omburtamab, orlotamab, BP-102, DS-7300, 8H-9, TCB-005, YBL-015, or any combination thereof.

[00195] 68. The composition of either embodiment 61 or embodiment 62, wherein the at least one inhibitor of CD47-SIRPa comprises at least one CD47-ligand inhibitor, at least one SIRPa- receptor inhibitor, or any combination thereof.

[00196] 69. The composition of embodiment 68, wherein the at least one inhibitor of CD47-

SIRPa comprises at least one of a small molecule, a peptide, a polynucleotide, a genetically modified cell or an antibody, an antibody fragment, or any combination thereof.

[00197] 70. The composition of embodiment 68, wherein the at least one CD47-ligand inhibitor comprises an antibody.

[00198] 71. The composition of embodiment 68, wherein the at least one CD47-ligand inhibitor comprises an isolated nucleic acid or set of nucleic acids, which collectively encodes an antibody.

[00199] 72. The composition of embodiment 68, wherein the at least one CD47-ligand inhibitor comprises a CD47-specific chimeric antigen receptor T (CAR-T) cell.

[00200] 73. The composition of embodiment 68, wherein the at least one SIRPa-receptor inhibitor comprises an antibody.

[00201] 74. The composition of embodiment 68, wherein the at least one SIRPa-receptor inhibitor comprises an isolated nucleic acid or set of nucleic acids, which collectively encodes an antibody.

[00202] 75. The composition of embodiment 68, wherein the at least one inhibitor of CD47-

SIRPa comprises RRx-001, a dinitroazetidine derivative, Hu5F9-G4, CC-90002, TTI-621, ALX148, SRF231, SHR-1603, IBI188, ST-1901, SGNCD-47M, or any combination thereof.

[00203] 76. A method of preventing, treating, or ameliorating a soil tumor in a subject in need thereof, the method comprising administering an effective amount of a composition of any one of embodiments 61-75, wherein the subject in need thereof has or is suspected of having a solid tumor that is not responsive to at least one anti-programmed cell death 1/programmed cell death ligand 1 (PD1/PDL1) therapy. [00204] 77. The method of embodiment 76, wherein the subject in need thereof is a human patient having or suspected of having a solid tumor selected from the group consisting of pancreatic ductal adenocarcinoma (PDA), colorectal cancer (CRC), melanoma, breast cancer, lung cancer, upper and lower gastrointestinal malignancies, squamous cell head and neck cancer, genitourinary cancer, ovarian cancer, and sarcomas.

[00205] 78. The method of embodiment 77, wherein the subject in need thereof is a human patient having or suspected of having melanoma.

[00206] 79. The method of any one of embodiments 76-78, wherein the solid tumor comprises at least one of B7-H3 overexpression, overactive P-catenin signaling, or any combination thereof. [00207] 80. The method of any one of embodiments 76-79, wherein the solid tumor comprises at least one genetic mutation comprising BRAF^V600E, loss of PTEN, or a combination thereof.

[00208] 81. The method of any one of embodiments 76-80, wherein administering an effective amount of a composition of any one of embodiments 61-75 activates the innate immune system of the subject in need thereof.

[00209] 82. The method of any one of embodiments 76-81, wherein administering an effective amount of a composition of any one of embodiments 61-75 to the subject in need thereof inflames a cold tumor microenvironment, wherein inflaming a cold tumor microenvironment comprises an increase of at least one interferon, an increase of CD8+ T cells, an increase of CD44 expression in CD4+ cells, an increase of CD44 expression in CD8+ cells, an increase of TNF alpha expression in T cells, or any combination thereof.

[00210] 83. A kit comprising a pharmaceutical composition according to any one of embodiments 55-75 and at least one container.

[00211] 84. The kit of embodiment 83 further comprising one or more chemotherapeutics.

Claims

CLAIMS What is claimed is:

1. A composition for treating a solid tumor in a subject, the composition comprising at least one inhibitor of B7-H3 (CD276) and at least one inhibitor of CD47-SIRPa

2. The composition of claim 1 further comprising at least one pharmaceutically acceptable excipient.

3. The composition of either claim 1 or claim 2, wherein the at least one inhibitor of B7-H3 comprises at least one of a small molecule, a peptide, a polynucleotide, a genetically modified cell or an antibody, an antibody fragment, or any combination thereof.

4. The composition of claim 3, wherein the at least one inhibitor of B7-H3 comprises an antibody.

5. The composition of claim 3, wherein the at least one inhibitor of B7-H3 comprises an isolated nucleic acid or set of nucleic acids, which collectively encodes an antibody.

6. The composition of claim 3, wherein the at least one inhibitor of B7-H3 comprises a B7- H3 -specific chimeric antigen receptor T (CAR-T) cell.

7. The composition of claim 3, wherein the at least one inhibitor of B7-H3 comprises enoblituzumab, omburtamab, orlotamab, BP-102, DS-7300, 8H-9, TCB-005, YBL-015, or any combination thereof.

8. The composition of either claim 1 or claim 2, wherein the at least one inhibitor of CD47- SIRPa comprises at least one CD47-ligand inhibitor, at least one SIRPa-receptor inhibitor, or any combination thereof.

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9. The composition of claim 8, wherein the at least one inhibitor of CD47-SIRPa comprises at least one of a small molecule, a peptide, a polynucleotide, a genetically modified cell or an antibody, an antibody fragment, or any combination thereof.

10. The composition of claim 8, wherein the at least one CD47-ligand inhibitor comprises an antibody.

11. The composition of claim 8, wherein the at least one CD47-ligand inhibitor comprises an isolated nucleic acid or set of nucleic acids, which collectively encodes an antibody.

12. The composition of claim 8, wherein the at least one CD47-ligand inhibitor comprises a CD47-specific chimeric antigen receptor T (CAR-T) cell.

13. The composition of claim 8, wherein the at least one SIRPa-receptor inhibitor comprises an antibody.

14. The composition of claim 8, wherein the at least one SIRPa-receptor inhibitor comprises an isolated nucleic acid or set of nucleic acids, which collectively encodes an antibody.

15. The composition of claim 8, wherein the at least one inhibitor of CD47-SIRPa comprises RRx-001, a dinitroazetidine derivative, Hu5F9-G4, CC-90002, TTI-621, ALX148, SRF231, SHR- 1603, IBI188, ST-1901, SGNCD-47M, or any combination thereof.

16. A method of preventing, treating, or ameliorating a soil tumor in a subject in need thereof, the method comprising administering an effective amount of a composition of any one of claims 1-15, wherein the subject in need thereof has or is suspected of having a solid tumor that is not responsive to at least one anti-programmed cell death 1/programmed cell death ligand 1 (PD1/PDL1) therapy.

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17. The method of claim 16, wherein the subject in need thereof is a human patient having or suspected of having a solid tumor selected from the group consisting of pancreatic ductal adenocarcinoma (PDA), colorectal cancer (CRC), melanoma, breast cancer, lung cancer, upper and lower gastrointestinal malignancies, squamous cell head and neck cancer, genitourinary cancer, ovarian cancer, and sarcomas.

18. The method of claim 17, wherein the subject in need thereof is a human patient having or suspected of having melanoma.

19. The method of any one of claims 16-18, wherein the solid tumor comprises at least one of B7-H3 overexpression, overactive P-catenin signaling, or any combination thereof.

20. The method of any one of claims 16-19, wherein the solid tumor comprises at least one genetic mutation comprising BRAF^V600E, loss of PTEN, or a combination thereof.

21. The method of any one of claims 16-20, wherein administering an effective amount of a composition of any one of claims 1-15 activates the innate immune system of the subject in need thereof.

22. The method of any one of claims 16-21, wherein administering an effective amount of a composition of any one of claims 1-15 to the subject in need thereof inflames a cold tumor microenvironment, wherein inflaming a cold tumor microenvironment comprises an increase of at least one interferon, an increase of CD8+ T cells, an increase of CD44 expression in CD4+ cells, an increase of CD44 expression in CD8+ cells, an increase of TNFalpha expression in T cells, or any combination thereof.

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