SIRPα-deficient macrophages for treating cancer

Abstract

As disclosed herein, SIRPα is essential for immune evasion by many different cancer types and cancer resistance to therapy, and reducing SIRPα levels on the TME can enhance antigen acquisition, processing, and presentation and reduce TME immunosuppression, thereby promoting tumor-specific T cell activation to eliminate tumors and generate adaptive immune responses consisting of memory T cells, circulating antibodies, and plasma cells, all of which may be specific for neo-antigens in the original cancer. Thus, activated SIRPαlow macrophages useful for the treatment of cancer are disclosed.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 271,930, filed October 26, 2021, which is incorporated by reference in its entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under Grant Nos. AI106839 and CA241271 awarded by the National Institutes of Health. The Government has certain rights in this invention. [Background technology]

[0003] Cancer remains a major threat to human health worldwide, despite various therapeutic efforts. Given that immune evasion is a hallmark of cancer, new immunotherapies, such as immune checkpoint blockade (ICB), chimeric antigen receptor (CAR)-T, cancer vaccination, and immunomodulatory radiotherapy (RT), have been developed to combat cancer, but these attempts have fallen short of fully meeting clinical needs due to low response rates and limited cancer types for which these treatments are effective. Thus, there is a pressing need for additional approaches and therapeutic innovations to circumvent immune elimination and improve the treatment of cancers resistant to current therapies. Summary of the Invention [Problem to be solved by the invention]

[0004] As disclosed herein, SIRPα is essential for immune evasion by many different cancer types and cancer resistance to RT, ICB and other immunomodulatory therapies. Reducing SIRPα expression or reducing SIRPα-mediated regulation can enhance antigen acquisition, processing, and presentation and reduce tumor microenvironment (TME) immunosuppression, thereby promoting the activation of tumor-specific T cells to eliminate tumors and generate an adaptive immune response consisting of T cells, circulating antibodies, and plasma cells, all of which can be specific for neo-antigens in the original cancer. [Means for solving the problem]

[0005] Thus, provided herein are activated SIRPα inhibitors for use in the treatment of cancer. low Disclosed are macrophages. In some embodiments, these activated SIRPα low The macrophages can be produced by obtaining a biological sample containing peripheral blood mononuclear cells (PBMCs) from a subject, isolating monocytes from the PBMCs, differentiating the monocytes in vitro to produce macrophages, contacting the macrophages with a SIRPα inhibitor, and contacting the macrophages with a macrophage activator, whereby SIRPα cell surface expression is significantly reduced (SIRPα activator) compared to untreated macrophages. low ), generating a population of macrophages with increased capacity for phagocytosis of cancer cells, proinflammatory responses, and immunogenic antigen presentation to activate tumor-specific T cells, thereby activating SIRPα low and producing a medicament for treating a cancer involving macrophages.

[0006] In some embodiments, the SIRPα inhibitor and the macrophage activator are administered sequentially, which may be in any order and may be separated by minutes, hours, or days, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours. In other embodiments, the SIRPα inhibitor and the macrophage activator are administered simultaneously or in parallel.

[0007] In some embodiments, the SIRPα inhibitor and the macrophage activator are present in the same composition. Thus, in some embodiments, the method includes isolating monocytes from peripheral blood mononuclear cells (PBMCs) in a biological sample, differentiating the monocytes in vitro to produce macrophages, and contacting the macrophages with a SIRPα expression inhibitor and a macrophage activator to produce activated macrophages (activated SIRPα) that have reduced SIRPα cell surface expression and increased phagocytic, proinflammatory and antigen presentation activity compared to untreated macrophages. low and generating a population of cultured cells (macrophages).

[0008] In some embodiments, the disclosed compositions and methods are used with any professional antigen-presenting cell. Professional antigen-presenting cells (APCs) are immune cells specialized to present antigens to T cells. The main types of professional APCs are dendritic cells (DCs), macrophages, and B cells, but can also include endothelial cells and, in some embodiments, granulocytes.

[0009] Accordingly, a method for treating cancer in a subject includes administering a therapeutically effective amount of activated SIRPα low Also disclosed are methods that include administering to a subject a therapeutically effective amount of activated SIRPα. low Macrophages are administered directly into the tumor (intratumoral administration) followed by tumor-directed in situ radiation therapy (FIG. 13A). In some embodiments, a therapeutically effective amount of activated SIRPα lowDirect administration of the macrophages to the tumor is preceded by tumor-directed in situ radiotherapy (FIG. 13B). In some embodiments, a therapeutically effective amount of activated SIRPα low Macrophages are administered directly to the tumor without tumor-directed in situ radiotherapy (FIG. 13C).

[0010] In some embodiments, a therapeutically effective amount of activated SIRPα low Macrophages are administered directly to the tumor, followed by tumor-directed in situ radiation therapy and intravenous (IV) administration of ICB therapy (FIG. 13D). In some embodiments, a therapeutically effective amount of activated SIRPα low Direct administration of macrophages to the tumor is preceded by tumor-directed in situ radiotherapy, followed by IV administration of ICB (FIG. 13E). In some embodiments, a therapeutically effective amount of activated SIRPα low Macrophages are administered directly into the tumor, followed by IV administration of ICB without tumor-directed in situ radiotherapy (Figure 13F).

[0011] In some embodiments, a therapeutically effective amount of activated SIRPα low Macrophages are administered IV, followed by tumor-directed in situ radiation therapy (FIG. 13G). In some embodiments, a therapeutically effective amount of activated SIRPα low Macrophages are administered IV, followed by tumor-directed in situ radiotherapy and IV administration of ICB (Figure 13H).

[0012] In some embodiments, a therapeutically effective amount of SIRPα that is not activated in in vitro culture. low Macrophages are administered IV, followed by tumor-directed in situ radiotherapy (FIG. 13I). In some embodiments, a therapeutically effective amount of SIRPα that is not activated in in vitro culture. low Macrophages are administered IV, followed by tumor-directed in situ radiotherapy and IV administration of ICB (Figure 13J).

[0013] Also provided herein is an in vitro expanded tumor-specific peripheral blood T (PBT) cell for use in the treatment of cancer, comprising obtaining a biological sample from a subject comprising peripheral blood mononuclear cells (PBMCs), isolating monocytes from the PBMCs, isolating peripheral blood T cells from the blood or the PBMCs, differentiating the monocytes in vitro to produce macrophages, contacting the macrophages with an inhibitor of SIRPα expression, and contacting the macrophages with an activator, whereby SIRPα cell surface expression is significantly reduced (SIRPα 1 + 1) compared to untreated macrophages. low ), generating a population of macrophages with increased capacity for phagocytosis, proinflammatory responses, and immunogenic antigen presentation against cancer cells; obtaining a biological sample from a subject, including a tumor biopsy or surgical tumor resection; and inducing activation of SIRPα. low Macrophages were co-cultured in vitro with cells from tumors to express tumor antigens (tumor-supplied SIRPα low Macrophages) and tumor-supplying SIRPα low Also disclosed are cells produced by the method comprising co-culturing macrophages in vitro with isolated PBT cells to expand the number of tumor-specific T cells, thereby producing a medicament for treating cancer comprising the in vitro grown tumor-specific PBT cells.

[0014] Thus, also disclosed are methods for treating cancer in a subject, comprising administering a therapeutically effective amount of in vitro grown tumor-specific PBT cells to the subject. In some embodiments, the in vitro grown PBT cells are administered to the subject by IV administration (FIG. 13K). In some embodiments, the in vitro grown PBT cells are administered to the subject by IV administration followed by tumor-directed in situ radiotherapy (FIG. 13L). In some embodiments, the in vitro grown PBT cells are administered to the subject by IV administration followed by IV administration of ICB (FIG. 13N). In some embodiments, the in vitro grown PBT cells are administered to the subject by IV administration followed by tumor-directed in situ radiotherapy and IV administration of ICB (FIG. 13M). In some embodiments, the in vitro grown PBT cells are administered to the subject by IV administration followed by tumor-directed in situ radiotherapy. In some embodiments, the in vitro grown PBT cells are administered to the subject by IV administration followed by tumor-directed in situ radiotherapy followed by IV administration of ICB.

[0015] Also provided herein is an in vitro tumor-specific T cell from a TIL cell, comprising obtaining a biological sample from a subject comprising peripheral blood mononuclear cells (PBMCs), isolating monocytes from the PBMCs, differentiating the monocytes in vitro to produce macrophages, contacting the macrophages with an inhibitor of SIRPα expression, and contacting the macrophages with an activator, whereby SIRPα cell surface expression is significantly reduced (SIRPα 1 + SIRPα 2 + SIRPα 3 + SIRPα 4 + SIRPα 5 + SIRPα 6 + SIRPα 7 + SIRPα 8 + SIRPα 9 + SIRPα 10 + SIRPα 11 + SIRPα 12 + SIRPα 13 + SIRPα 14 + SIRPα 15 + SIRPα 16 + SIRPα 17 + SIRPα 18 + SIRPα 19 + SIRPα 20 + SIRPα 21 + SIRPα 22 + SIRPα 23 + SIRPα 24 + SIRPα 25 + SIRPα 26 + SIRPα 27 + SIRPα 28 + SIRPα 29 + SIRPα 30 + SIRPα 31 + SIRPα 32 + SIRPα 33 + SIRPα 34 + SIRPα 35 + SIRPα 36 + SIRPα 37 + SIRPα 38 ​​+ SIRPα 29 + SIRPα 39 + SIRPα 39 + SIRPα 38 ​​+ SIRPα 39 + SIRPα 40 + SIRPα 41 + SIRPα 42 + SIRPα 43 + SIRPα 44 + SIRPα 45 + SIRPα 46 + SIRPα 47 + SI low ), generating a population of macrophages with increased capacity for phagocytosis, proinflammatory responses, and immunogenic antigen presentation against cancer cells; collecting a biological sample from a subject, including a tumor biopsy or surgical tumor resection; isolating tumor-infiltrating T lymphocyte (TIL) cells from the tumor biopsy; and administering activated SIRPα to the subject. low Macrophages were co-cultured in vitro with tumor cells from tumor samples to allow for phagocytosis and delivery of tumor antigens (tumor-supplied SIRPα low Obtaining tumor-delivering SIRPαlow Also disclosed are cells produced by the method comprising co-culturing macrophages with isolated TIL cells in vitro to expand the number of tumor-specific T cells, thereby producing a medicament for treating cancer comprising in vitro expanded tumor-specific T cells from the TILs.

[0016] Also disclosed herein is a method for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of in vitro grown tumor-specific T cells from TILs. In some embodiments, the in vitro grown tumor-specific T cells from TILs are administered to the subject by IV administration (Figure 13O). In some embodiments, the in vitro grown tumor-specific T cells from TILs are administered to the subject by IV administration, followed by tumor-directed in situ radiotherapy (Figure 13P). In some embodiments, the in vitro grown tumor-specific T cells from TILs are administered to the subject by IV administration, followed by IV administration of ICB (Figure 13R). In some embodiments, the in vitro grown tumor-specific T cells from TILs are administered to the subject by IV administration, followed by tumor-directed in situ radiotherapy and IV administration of ICB (Figure 13Q). In some embodiments, the in vitro grown tumor-specific T cells from TILs are administered to the subject by IV administration, followed by tumor-directed in situ radiotherapy. In some embodiments, in vitro expanded tumor-specific T cells from TILs are administered to a subject by IV administration prior to tumor-directed in situ radiotherapy, followed by IV administration of ICB.

[0017] In some embodiments, a "SIRPα inhibitor" suppresses the expression of SIRPα, inhibits the activity of SIRPα, reduces the presence of SIRPα on the surface of a cell, interferes with the interaction between SIRPα and CD47, activates phagocytosis, promotes antigen processing and presentation to T cells, promotes T cell activation, or a combination thereof.

[0018] In some embodiments, macrophage activators increase phagocytosis by macrophages, increase antigen processing and presentation activity and function of macrophages, increase the immune stimulatory capacity of macrophages, improve the T cell stimulatory function of macrophages, promote a pro-inflammatory (so-called M1) phenotype of macrophages, or enable macrophages to alter the TME to promote an immune response against cancer cells.

[0019] Also disclosed herein is a method for treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of an SHP-1 inhibitor in combination with RT, ICB, an oncolytic virus, or any combination thereof.

[0020] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. [Brief description of the drawings]

[0021] [Figure 1] FIG. 1 is a schematic diagram showing the activation and inhibition mechanisms controlling phagocytosis towards self / tumor cells, with particular reference to the role played by the SIRPα-CD47 signaling axis. [Figure 2A] Figure 1 shows the effect of intratumoral anti-PD-L1 on sc MC38 tumors in WT and SIRPα- / - mice. Two doses of anti-PD-L1 Ab (50 μg, BioXcell, clone10F.9G2) were given when tumors were freshly formed (≦50 mm3, day 8 / 11). [Figure 2B] Shows the effect of intratumoral anti-PD-L1 on sc MC38 tumors in WT and SIRPα- / - mice. Two doses of anti-PD-L1 Ab (50 μg, BioXcell, clone10F.9G2) were given when tumors grew larger (>200 mm3, 12 / 15 days) ± IFNγ and CpG (100 ng and 20 μg, respectively). [Diagram 3]Shown is the effect of intratumoral anti-PD-1 / L1 on subcutaneous PDA tumors Panc02 and KPC. Panc02 and KPC tumors of approximately 100 mm3 were given two doses of anti-PD-L1 Ab (50 μg each) via it. [Figure 4] Effect of αPD-L1 in combination with tumor radiation. 150-400 mm3 sc MC38, Pan02 and KPC tumors were irradiated with 8 Gy of X-rays followed by αPD-L1 Ab (50 μg, it) given once in SIRPα- / - mice or twice (3 days apart) in WT mice. [Figure 5A] We show that Sirpα− / − mice following eradication of MC38 tumors by treatment with αPD-L1+IFNγ / CpG(2x), or αPD-L1+8Gy radiation developed long-lasting immunity that prevented tumor reimplantation despite expansion of MC38 cells. [Figure 5B] Transfer of serum from tumor-eradicated Sirpα − / − mice into WT recipients conferred resistance to MC38 tumors: serum samples positively stained the MC38 cell surface. [Figure 5C] Transfer of splenic T cells conferred MC38 tumor resistance, and serum samples positively stained the MC38 cell surface. [Figure 6A] Figure 2 shows that Sirpα- / - mice demonstrate enhanced anti-tumor CD8 Tc in the TME upon treatment with αPD-L1±IFNγ / CpG or 8 Gy radiation (all data 5 days after treatment). [Figure 6B] p15E specificity and GranzB expression, as well as detection of Tem are shown. [Figure 6C] FIG. 1 shows an ex vivo cytotoxicity assay by co-incubating Tc isolated from tumors with MC38 o / n. [Figure 6D] Statistics for total Tc, GranzB+, and P15E+ subpopulations are shown. [Figure 7A] Figure 2 shows that SIRPα- / - mice upon treatment with αPD-L1+IFNγ / CpG or 8 Gy RT / IR (radiation treatment / irradiation) exhibited a decrease in CD4+Foxp3+Tregs in the TME. [Figure 7B] Figure 2 shows that SIRPα- / - mice upon treatment with αPD-L1+IFNγ / CpG or 8 Gy RT / IR (radiation treatment / irradiation) exhibited a decrease in CD4+Foxp3+Tregs in the TME. [Figure 7C] We show prominent Ly6C+ monocyte / MDSC infiltration in tumors following αPD-L1+8Gy RT in WT mice but absent in SIRPα− / − mice. [Figure 7D] Figure 2 shows tumor-associated leukocytes before and after αPD-L1 + 8Gy RT treatment. Data were collected 3 days after treatment. [Figure 8A] We show that ex vivo IFNγ / CpG-activated Sirpα− / − Mφ (BMDM, 0.5x106) were injected it into MC38 tumors together with αPD-L1 Ab (2x) and successfully induced tumor elimination. [Figure 8B] It shows an increase in tumor-specific Tc in the TME after αPD-L1+Sirpα− / − Mφ or various amounts of Sirpα− / − Mφ. [Figure 8C] Shown is an it injection of Sirpα − / − Mφ(2x) into MC38 tumors in WT mice. [Figure 9A] We show that CD47-induced SIRPα signaling inhibits Mφ antigen presentation and proinflammatory cytokine production.WT and Sirpα- / - BMDMs were stimulated with IFNγ / CpG in the presence or absence of CD47 (mCD47.ex) for 12 hours, followed by FACS and ELISA detection of cell surface protein expression and cytokines secreted into the medium. [Figure 9B] We show that CD47-induced SIRPα signaling inhibits Mφ antigen presentation and proinflammatory cytokine production.WT and Sirpα- / - BMDMs were stimulated with IFNγ / CpG in the presence or absence of CD47 (mCD47.ex) for 12 hours, followed by FACS and ELISA detection of cell surface protein expression and cytokines secreted into the medium. [Figure 10]Schematic diagram showing two-step inhibition by SIRPα- / -: 1) tumor CD47 phagocyte SIRPα via SHP-1 suppresses antigen presentation machinery; 2) APC SIRPα on T→CD47 inhibits T cell activation. [Figure 11] We show that the disclosed macrophage therapy treatment dramatically reduces SIRPα in human PBMC-derived Mφ (FIG. 11A), and phagocytic activation of treated SIRPαlow Mφ induces uptake of autologous RBCs (FIG. 11B) and human intestinal cancer cells HT29, T84, and Caco2, as well as THP1 leukemia cells (FIG. 11C). [Figure 12] FIG. 1 is a schematic diagram illustrating an embodiment of the disclosed macrophage therapy treatment. [Figure 13A] 13A-13R are schematic diagrams depicting steps of various embodiments of the disclosed method. As used in FIGS. 13A-13R, the term "Reagent A" refers to a SIRPα inhibitor and the term "Reagent B" refers to a macrophage activator. [Figure 13B] 13A-13R are schematic diagrams depicting steps of various embodiments of the disclosed method. As used in FIGS. 13A-13R, the term "Reagent A" refers to a SIRPα inhibitor and the term "Reagent B" refers to a macrophage activator. [Figure 13C] 13A-13R are schematic diagrams depicting steps of various embodiments of the disclosed method. As used in FIGS. 13A-13R, the term "Reagent A" refers to a SIRPα inhibitor and the term "Reagent B" refers to a macrophage activator. [Figure 13D] 13A-13R are schematic diagrams depicting steps of various embodiments of the disclosed method. As used in FIGS. 13A-13R, the term "Reagent A" refers to a SIRPα inhibitor and the term "Reagent B" refers to a macrophage activator. [Figure 13E] 13A-13R are schematic diagrams depicting steps of various embodiments of the disclosed method. As used in FIGS. 13A-13R, the term "Reagent A" refers to a SIRPα inhibitor and the term "Reagent B" refers to a macrophage activator. [Figure 13F] 13A-13R are schematic diagrams depicting steps of various embodiments of the disclosed method. As used in FIGS. 13A-13R, the term "Reagent A" refers to a SIRPα inhibitor and the term "Reagent B" refers to a macrophage activator. [Figure 13G] 13A-13R are schematic diagrams depicting steps of various embodiments of the disclosed method. As used in FIGS. 13A-13R, the term "Reagent A" refers to a SIRPα inhibitor and the term "Reagent B" refers to a macrophage activator. [Figure 13H] 13A-13R are schematic diagrams depicting steps of various embodiments of the disclosed method. As used in FIGS. 13A-13R, the term "Reagent A" refers to a SIRPα inhibitor and the term "Reagent B" refers to a macrophage activator. [Figure 13I] 13A-13R are schematic diagrams depicting steps of various embodiments of the disclosed method. As used in FIGS. 13A-13R, the term "Reagent A" refers to a SIRPα inhibitor and the term "Reagent B" refers to a macrophage activator. [Figure 13J] 13A-13R are schematic diagrams depicting steps of various embodiments of the disclosed method. As used in FIGS. 13A-13R, the term "Reagent A" refers to a SIRPα inhibitor and the term "Reagent B" refers to a macrophage activator. [Figure 13K] 13A-13R are schematic diagrams depicting steps of various embodiments of the disclosed method. As used in FIGS. 13A-13R, the term "Reagent A" refers to a SIRPα inhibitor and the term "Reagent B" refers to a macrophage activator. [Figure 13L] 13A-13R are schematic diagrams depicting steps of various embodiments of the disclosed method. As used in FIGS. 13A-13R, the term "Reagent A" refers to a SIRPα inhibitor and the term "Reagent B" refers to a macrophage activator. [Figure 13M]13A-13R are schematic diagrams depicting steps of various embodiments of the disclosed method. As used in FIGS. 13A-13R, the term "Reagent A" refers to a SIRPα inhibitor and the term "Reagent B" refers to a macrophage activator. [Figure 13N] 13A-13R are schematic diagrams depicting steps of various embodiments of the disclosed method. As used in FIGS. 13A-13R, the term "Reagent A" refers to a SIRPα inhibitor and the term "Reagent B" refers to a macrophage activator. [Figure 13O] 13A-13R are schematic diagrams depicting steps of various embodiments of the disclosed method. As used in FIGS. 13A-13R, the term "Reagent A" refers to a SIRPα inhibitor and the term "Reagent B" refers to a macrophage activator. [Figure 13P] 13A-13R are schematic diagrams depicting steps of various embodiments of the disclosed method. As used in FIGS. 13A-13R, the term "Reagent A" refers to a SIRPα inhibitor and the term "Reagent B" refers to a macrophage activator. [Figure 13Q] 13A-13R are schematic diagrams depicting steps of various embodiments of the disclosed method. As used in FIGS. 13A-13R, the term "Reagent A" refers to a SIRPα inhibitor and the term "Reagent B" refers to a macrophage activator. [Figure 13R] 13A-13R are schematic diagrams depicting steps of various embodiments of the disclosed method. As used in FIGS. 13A-13R, the term "Reagent A" refers to a SIRPα inhibitor and the term "Reagent B" refers to a macrophage activator. [Figure 14A] Local RT eliminates MC38 and PDA tumors in Sirpα- / - mice, but not in WT mice. RT scheme shown. MC38, Pan02 or KPC cells were implanted (5x105, sc) into the right flank of WT or Sirpα- / - mice, and various doses of X-ray irradiation (IR) were given when tumors reached >150 mm3. [Figure 14B]Local RT eliminates MC38 and PDA tumors Sirpα- / - mice, but not WT mice. Changes in tumor volume and survival are shown. Either a single fraction of IR (Fig. 14B and Fig. 14C) or three fractions (Fig. 14D) were given when tumors were 150-400mm3 >500mm3, respectively, except for some Sirpα- / - mice treated with two fractions (purple line, Fig. 14D). Blue lines indicate WT mice treated with 2x 8Gy (Fig. 4C) or 8Gy-4Gy-4Gy (Fig. 14D) and with anti-PD-L1 Ab (50μg, intratumor) given after each fraction. Data are representative of at least three independent experiments with different tumors (n=3-12 / group). Survival data were recorded up to 1.5 years after IR. [Figure 14C] Local RT eliminates MC38 and PDA tumors Sirpα- / - mice, but not WT mice. Changes in tumor volume and survival are shown. Either a single fraction of IR (Fig. 14B and Fig. 14C) or three fractions (Fig. 14D) were given when tumors were 150-400mm3 >500mm3, respectively, except for some Sirpα- / - mice treated with two fractions (purple line, Fig. 14D). Blue lines indicate WT mice treated with 2x 8Gy (Fig. 4C) or 8Gy-4Gy-4Gy (Fig. 14D) and with anti-PD-L1 Ab (50μg, intratumor) given after each fraction. Data are representative of at least three independent experiments with different tumors (n=3-12 / group). Survival data were recorded up to 1.5 years after IR. [Figure 14D]Local RT eliminates MC38 and PDA tumors Sirpα- / - mice, but not WT mice. Changes in tumor volume and survival are shown. Either a single fraction of IR (Fig. 14B and Fig. 14C) or three fractions (Fig. 14D) were given when tumors were 150-400mm3 >500mm3, respectively, except for some Sirpα- / - mice treated with two fractions (purple line, Fig. 14D). Blue lines indicate WT mice treated with 2x 8Gy (Fig. 4C) or 8Gy-4Gy-4Gy (Fig. 14D) and with anti-PD-L1 Ab (50μg, intratumoral) given after each fraction. Data are representative of at least three independent experiments with different tumors (n=3-12 / group). Survival data were recorded up to 1.5 years after IR. Serum cytokines assessed at the indicated times after IR are shown (n=5 / group). [Figure 14E] Figure 1 shows that focal RT eliminates MC38 and PDA tumors Sirpα- / - mice, but not WT mice. Representative images of MC38 and luciferase-expressing KPC tumors in WT and Sirpα- / - mice before and after a single 8 Gy IR are included. [Figure 14F]Local RT eliminates MC38 and PDA tumors in Sirpα− / − mice, but not in WT mice. Figure 14A shows the RT scheme. MC38, Pan02 or KPC cells were implanted (5x105, sc) into the right flank of WT or Sirpα− / − mice, and various doses of X-ray irradiation (IR) were given when tumors reached >150 mm3. Figures 14B-14D show the changes in tumor volume and survival. Except for some Sirpα− / − mice treated with two fractions (purple line, Figure 14D), either a single fraction of IR (Figures 14B and 14C) or three fractions (Figure 14D) were given when tumors were 150-400 mm3 >500 mm3, respectively. Blue lines indicate WT mice treated with 2x 8Gy (Fig. 4C) or 8Gy-4Gy-4Gy (Fig. 14D) and with anti-PD-L1 Ab (50 μg, intratumor) given after each fraction. Data are representative of at least three independent experiments of different tumors (n=3-12 / group). Survival data were recorded up to 1.5 years post-IR. Figure 14E contains representative images of MC38 and luciferase-expressing KPC tumors in WT and Sirpα- / - mice before and after a single 8Gy IR. Figure 14D shows serum cytokines assessed at the indicated times post-IR (n=5 / group). [Figure 15A] Sirpα- / - mice display RT-induced abscopal effect and long-lasting antitumor immunity. Abscopal effect in MC38 tumor-bearing mice. Primary tumors (>150 mm3) were irradiated (8 Gy) and 8-10 days later, a subset of mice with residual abscopal tumors received anti-PD-L1 Ab (αPD-L1; 100 μg, ip 2 x, 3 day intervals). Tumor volume and survival were recorded. Data are representative of 5 independent experiments (n=3-8 / group). [Figure 15B]Figure 1 shows that Sirpα- / - mice display RT-induced abscopal effect and long-lasting antitumor immunity. Figure 2 shows abscopal effect in mice bearing KPC tumors. Primary tumors (>150 mm3) were irradiated (8 Gy) and 8-10 days later, a subset of mice with residual abscopal tumors received anti-PD-L1 Ab (αPD-L1; 100 μg, ip 2 x, 3 day intervals). Tumor volume and survival were recorded. Representative images show WT and Sirpα- / - mice bearing luciferase-expressing KPC tumors implanted in both flanks, dorsal regions or peritoneal cavity before and after RT ± αPD-L1. Data are representative of 5 independent experiments (n=3-8 / group). [Figure 15C] Sirpα- / - mice display RT-induced abscopal effects and long-lasting antitumor immunity. Showing long-lasting antitumor immunity. After eradication of MC38 or PDA tumors (3 weeks post-RT), Sirpα- / - mice were challenged with three rounds of escalating doses of the same tumors. Data are representative of four independent experiments (n=3-8 / group). [Figure 15D] Sirpα- / - mice display RT-induced abscopal effects and long-lasting antitumor immunity. After eradication of MC38 or PDA tumors (3 weeks post-RT), Sirpα- / - mice were scored for tumor volume and survival with three rounds of dose-escalating inoculations of the same tumors. Data are representative of four independent experiments (n=3-8 / group). [Figure 15E] Sirpα- / - mice display RT-induced abscopal effects and long-lasting antitumor immunity. Ten days after the last inoculation, sera from MC38-resistant (containing anti-MC38 IgG) or tumor-naive Sirpα- / - mice were tested for antitumor IgG by cell surface immunostaining of the respective tumor cells. Splenic T cells from the same MC38-resistant or tumor-naive Sirpα- / - mice were transferred into WT recipients prior to MC38 engraftment and tumor growth was recorded. [Figure 15F]Sirpα- / - mice display RT-induced abscopal effects and long-lasting antitumor immunity. Ten days after the last inoculation, sera from MC38-resistant (containing anti-MC38 IgG) or tumor-naive Sirpα- / - mice were assessed for complement-dependent cytotoxicity (CDC) and macrophage phagocytosis for antitumor IgG by cell surface immunostaining of the respective tumor cells. Splenic T cells from the same MC38-resistant or tumor-naive Sirpα- / - mice were transferred into WT recipients prior to MC38 engraftment and tumor growth was recorded. [Figure 15G] Sirpα- / - mice display RT-induced abscopal effects and long-lasting antitumor immunity. Data are representative of three independent experiments (n=3 / group) and presented as the mean ± SD of triplicates. [Figure 16A] We show that Sirpα- / - macrophages, but not CD47 blockade, confer a complete response after IR. We show that depletion of intratumoral macrophages reduced RT efficacy in Sirpα- / - mice. MC38 or PDA tumors (>200 mm3) in Sirpα- / - mice were administered Cl2MDA liposomes or anti-CSF receptor antibody (αCSF1R) to deplete macrophages 2 days before and immediately after tumor 8 Gy IR. Data are representative of two independent experiments (n=3-4 / group). [Figure 16B] We show that Sirpα- / - macrophages, but not CD47 blockade, confer a complete response after IR. We show that depletion of intratumoral macrophages reduced RT efficacy in Sirpα- / - mice. MC38 or PDA tumors (>200 mm3) in Sirpα- / - mice were administered Cl2MDA liposomes or anti-CSF receptor antibody (αCSF1R) to deplete macrophages 2 days before and immediately after tumor 8 Gy IR. Data are representative of two independent experiments (n=3-4 / group). [Figure 16C]We show that Sirpα− / − macrophages, but not CD47 blockade, confer a complete response after IR. We show that combining RT with adoptive Sirpα− / − BMDM injection conferred tumor clearance in WT mice. MC38 tumors in WT mice were treated once (1x) or twice (2x, 3 days apart) with intratumoral (it) injections of Sirpα− / − BMDM (1x104 per mm3 tumor mass), anti-mouse CD47 blocking antibodies (αCD47, miap301, 100μg), soluble mouse SIRPα extracellular domain (mSIRPα, ex, 100μg), anti-MC38 serum (100μl, undiluted), or αCD47 plus anti-MC38 serum, with an additional 8Gy IR given 3 hours later. The same treatments were repeated 3 days later. Tumor volumes and animal survival were recorded. Data are representative of two independent experiments (n=3-5 / group). [Figure 16D] We show that Sirpα− / − macrophages, but not CD47 blockade, confer a complete response after IR. We show that combining RT with adoptive Sirpα− / − BMDM injection conferred tumor clearance in WT mice. MC38 tumors in WT mice were treated once (1x) or twice (2x, 3 days apart) with intratumoral (it) injections of Sirpα− / − BMDM (1x104 per mm3 tumor mass), anti-mouse CD47 blocking antibodies (αCD47, miap301, 100μg), soluble mouse SIRPα extracellular domain (mSIRPα, ex, 100μg), anti-MC38 serum (100μl, undiluted), or αCD47 plus anti-MC38 serum, with an additional 8Gy IR given 3 hours later. The same treatments were repeated 3 days later. Tumor volumes and animal survival were recorded. Data are representative of two independent experiments (n=3-5 / group). [Figure 16E]We show that Sirpα− / − macrophages, but not CD47 blockade, confer a complete response after IR. We show that combining RT with adoptive Sirpα− / − BMDM injection conferred tumor clearance in WT mice. MC38 tumors in WT mice were treated once (1x) or twice (2x, 3 days apart) with intratumoral (it) injections of Sirpα− / − BMDM (1x104 per mm3 tumor mass), anti-mouse CD47 blocking antibodies (αCD47, miap301, 100μg), soluble mouse SIRPα extracellular domain (mSIRPα, ex, 100μg), anti-MC38 serum (100μl, undiluted), or αCD47 plus anti-MC38 serum, with an additional 8Gy IR given 3 hours later. The same treatments were repeated 3 days later. Tumor volumes and animal survival were recorded. Data are representative of two independent experiments (n=3-5 / group). [Figure 16F] We show that Sirpα− / − macrophages, but not CD47 blockade, confer a complete response after IR. We show that combining RT with adoptive Sirpα− / − BMDM injection conferred tumor clearance in WT mice. MC38 tumors in WT mice were treated once (1x) or twice (2x, 3 days apart) with intratumoral (it) injections of Sirpα− / − BMDM (1x104 per mm3 tumor mass), anti-mouse CD47 blocking antibodies (αCD47, miap301, 100μg), soluble mouse SIRPα extracellular domain (mSIRPα, ex, 100μg), anti-MC38 serum (100μl, undiluted), or αCD47 plus anti-MC38 serum, with an additional 8Gy IR given 3 hours later. The same treatments were repeated 3 days later. Tumor volumes and animal survival were recorded. Data are representative of two independent experiments (n=3-5 / group). [Figure 16G]We show that Sirpα− / − macrophages, but not CD47 blockade, confer a complete response after IR. We show that combining RT with adoptive Sirpα− / − BMDM injection conferred tumor clearance in WT mice. MC38 tumors in WT mice were treated once (1x) or twice (2x, 3 days apart) with intratumoral (it) injections of Sirpα− / − BMDM (1x104 per mm3 tumor mass), anti-mouse CD47 blocking antibodies (αCD47, miap301, 100μg), soluble mouse SIRPα extracellular domain (mSIRPα, ex, 100μg), anti-MC38 serum (100μl, undiluted), or αCD47 plus anti-MC38 serum, with an additional 8Gy IR given 3 hours later. The same treatments were repeated 3 days later. Tumor volumes and animal survival were recorded. Data are representative of two independent experiments (n=3-5 / group). [Figure 16H] We show that Sirpα- / - macrophages, but not CD47 blockade, confer a complete response after IR. We show that combining RT with adoptive Sirpα- / - BMDM injection conferred tumor clearance in WT mice. MC38 tumors in WT mice were treated once (1x) or twice (2x, 3 days apart) with intratumoral (it) injections of Sirpα- / - BMDM (1x104 per mm3 tumor mass), anti-mouse CD47 blocking antibody (αCD47, miap301, 100 μg), soluble mouse SIRPα extracellular domain (mSIRPα, ex, 100 μg), anti-MC38 serum (100 μl, undiluted), or αCD47 plus anti-MC38 serum without further 8 Gy IR given 3 hours later. The same treatments were repeated 3 days later. Tumor volumes and animal survival were recorded. Data are representative of two independent experiments (n=3-5 / group). [Figure 16I]We show that Sirpα− / − macrophages, but not CD47 blockade, confer a complete response after IR. We show that combining RT with adoptive Sirpα− / − BMDM injection conferred tumor clearance in WT mice. MC38 tumors in WT mice were treated once (1x) or twice (2x, 3 days apart) with intratumoral (it) injections of Sirpα− / − BMDM (1x104 per mm3 tumor mass), anti-mouse CD47 blocking antibodies (αCD47, miap301, 100μg), soluble mouse SIRPα extracellular domain (mSIRPα, ex, 100μg), anti-MC38 serum (100μl, undiluted), or αCD47 plus anti-MC38 serum, with an additional 8Gy IR given 3 hours later. The same treatments were repeated 3 days later. Tumor volumes and animal survival were recorded. Data are representative of two independent experiments (n=3-5 / group). [Figure 17A] Figure 17 shows that radiation-activated Sirpα- / - macrophages drive a proinflammatory TME. Figure 17 shows that MC38 tumors in WT and Sirpα- / - mice before and after a single 8 Gy IR were analyzed for CD45+ tumor-infiltrating leukocyte populations and CD45- non-leukocytes by flow cytometry. The frequency of intratumoral F4 / 80high macrophages (Mφ) before and after IR was visualized by t-SNE (Figure 17B) and calculated per mg of tumor mass (Figure 17D). Data are representative of at least six independent experiments (Figures 17A-B) or pooled from three experiments (Figures 17C-D, n-12-16 / group). Figure 17 shows mRNA profiling of bulk tumors before and 12 hours after IR by Nanostring (nCounter Mouse Immunology Panel). [Figure 17B]Figure 17 shows that radiation-activated Sirpα- / - macrophages drive the proinflammatory TME. MC38 tumors in WT and Sirpα- / - mice before and after a single 8 Gy IR were analyzed for CD45+ tumor-infiltrating leukocyte populations and CD45- non-leukocytes by flow cytometry. The frequency of intratumoral F4 / 80high macrophages (Mφ) before and after IR was visualized by t-SNE (Figure 17B) and calculated per mg of tumor mass (Figure 17D). Data are representative of at least six independent experiments (Figures 17A-B) or pooled from three experiments (Figures 17C-D, n-12-16 / group). [Figure 17C] Figure 17 shows that radiation-activated Sirpα- / - macrophages drive the proinflammatory TME. MC38 tumors in WT and Sirpα- / - mice before and after a single 8 Gy IR were analyzed for CD45+ tumor-infiltrating leukocyte populations and CD45- non-leukocytes by flow cytometry. The frequency of intratumoral F4 / 80high macrophages (Mφ) before and after IR was visualized by t-SNE (Figure 17B) and calculated per mg of tumor mass (Figure 17D). Data are representative of at least six independent experiments (Figures 17A-B) or pooled from three experiments (Figures 17C-D, n-12-16 / group). [Figure 17D] Figure 17 shows that radiation-activated Sirpα- / - macrophages drive the proinflammatory TME. MC38 tumors in WT and Sirpα- / - mice before and after a single 8 Gy IR were analyzed for CD45+ tumor-infiltrating leukocyte populations and CD45- non-leukocytes by flow cytometry. The frequency of intratumoral F4 / 80high macrophages (Mφ) before and after IR was visualized by t-SNE (Figure 17B) and calculated per mg of tumor mass (Figure 17D). Data are representative of at least six independent experiments (Figures 17A-B) or pooled from three experiments (Figures 17C-D, n-12-16 / group). [Figure 17E]Figure 1 shows that radiation-activated Sirpα- / - macrophages drive a proinflammatory TME. GFP-positive Sirpα- / - BMDMs (GFP-Sirpα- / - M) injected intratumorally (it, 1x104 / mm3) into WT recipients were analyzed pre-IR and at different time points post-IR. Data are pooled from two independent experiments and presented as mean ± SD (n=3 / group). [Figure 17F] Radiation-activated Sirpα- / - macrophages drive a proinflammatory TME. MC38 intratumoral F4 / 80high macrophages in WT and Sirpα- / - mice (FIG. 17F) or in GFP-Sirpα- / - BMDM-injected WT recipients (FIG. 17G) were analyzed for antigen presentation and inflammatory phenotype before (-IR) and 12 hours after IR (8 Gy). Cell surface staining: MHC I / II, CD80 / 86, and PX40L; intracellular staining: IL-12, IFNα, and IL-10. Data are representative of three independent experiments (n=3-4 / group). [Figure 17G] Radiation-activated Sirpα- / - macrophages drive a proinflammatory TME. MC38 intratumoral F4 / 80high macrophages in WT and Sirpα- / - mice (FIG. 17F) or in GFP-Sirpα- / - BMDM-injected WT recipients (FIG. 17G) were analyzed for antigen presentation and inflammatory phenotype before (-IR) and 12 hours after IR (8 Gy). Cell surface staining: MHC I / II, CD80 / 86, and PX40L; intracellular staining: IL-12, IFNα, and IL-10. Data are representative of three independent experiments (n=3-4 / group). [Figure 17H] Radiation-activated Sirpα- / - macrophages promote proinflammatory TME. Heatmap (FIG. 17H) and scatter plot (FIG. 17I) show differential expression of antigen presentation, proinflammatory, and antiinflammatory related genes (n=3 mice / group). [Figure 17I]Figure 17 shows that radiation-activated Sirpα- / - macrophages promote a pro-inflammatory TME. mRNA profiling of bulk tumors pre-IR and 12 hours post-IR by Nanostring (nCounter Mouse Immunology Panel). Heatmap (Figure 17H) and scatter plot (Figure 17I) show differential expression of antigen presentation, pro-inflammatory and anti-inflammatory related genes (n=3 mice / group). [Figure 18A] Sirpα- / - macrophages drive robust tumor-specific Tc expansion after RT. TME analysis of CD8+ Tc and CD4+ Th among CD45+ tumor-infiltrating leukocytes in MC38, Pan02 or KPC tumors before and after single fraction 8 Gy IR. Representative of at least 6 independent experiments (n=4-6 / group). [Figure 18B] Sirpα- / - macrophages drive robust tumor-specific Tc proliferation after RT. IHC and IF staining of CD8+ Tc in MC38 tumors 3 days after IR is shown. Representative of at least 6 independent experiments (n=4-6 / group). [Figure 18C] Sirpα- / - macrophages drive robust tumor-specific Tc expansion after RT. Frequencies of Granzyme Bhigh (GranzB) and p15E+ Tc in the MC38 TME are shown. The frequency of CD44+CD62L- effector memory T cells (TEM) among p15E+ Tc was also determined. Representative of at least six independent experiments (n=4-6 / group). [Figure 18D] Sirpα- / - macrophages drive robust tumor-specific Tc proliferation after RT. Summary of intratumoral GranzBhigh and p15E+ Tc pre- and post-IR. Representative of at least six independent experiments (n=4-6 / group). [Figure 18E]Sirpα- / - macrophages drive robust tumor-specific Tc expansion after RT. Frequencies of P15E+ Tc and p15E+CD44+CD62L- TEM in peripheral blood and spleen of MC38-eradicated Sirpα- / - mice. Data are representative of three independent experiments (n=5 / group). [Figure 18F] We show that Sirpα- / - macrophages drive robust tumor-specific Tc proliferation after RT. WT mice bearing MC38 tumors were injected intratumorally with Sirpα- / - BMDMs via it (total of 2x106, tumor size approximately 200mm3) and iv routes (1x107 per mouse), followed by two rounds (3 days apart) of IR (8Gy). The frequency of p15+ and GranzB+ Tc was determined 3 days after the second round. Data are representative of three independent experiments (n=2-4 / group). [Figure 18G] Sirpα- / - macrophages drive robust tumor-specific Tc proliferation after RT. Tc from irradiated tumors were isolated (3 days after IR) and co-cultured with MC38 cells at various effector:target ratios as indicated for 6 or 24 hours. MC38 cell death was identified by propidium iodide (PI) staining. Data are presented as mean ± SD and are representative of three independent experiments (n=3 / group). [Figure 18H] Sirpα- / - macrophages drive robust tumor-specific Tc expansion after RT. Depletion of CD8 Tc (αCD8) or CD4 Th (αCD4) in MC38 tumors of Sirpα- / - mice before IR. Data are representative of two independent experiments (n=4 / group). [Figure 19A] We show that Sirpα- / - macrophages reduce tumor immune suppression after RT. MC38 tumors before and 3 days after IR were excised and analyzed for their cell counts for intratumoral immune populations. Note: Data for WT mice were without Sirpα- / - macrophage injection. Data are pooled from 4 independent experiments and presented as mean ± SD (n=12 / group). [Figure 19B] We show that Sirpα- / - macrophages reduce tumor immune suppression after RT. MC38 tumors before and 3 days after IR were excised and analyzed for intratumoral immune populations for their percentage. Note: Data for WT mice were without Sirpα- / - macrophage injection. Data are pooled from 4 independent experiments and presented as mean ± SD (n=12 / group). [Figure 19C] Sirpα- / - macrophages reduce tumor immune suppression after RT. Foxp3+ Tregs and IFNγ-producing Th1 among intratumoral CD4 T cells. Data are representative of three independent experiments (n=10-12 / group). [Figure 19D] Sirpα- / - macrophages reduce tumor immune suppression after RT. Foxp3+ Tregs and IFNγ-producing Th1 among intratumoral CD4 T cells. Data are representative of three independent experiments (n=10-12 / group). [Figure 19E] Sirpα- / - macrophages reduce tumor immunosuppression after RT. GranzB expression in NK cells is shown with mean fluorescence intensity (MFI) shown as mean ± SD. Data are representative of two independent experiments (n=5 / group). [Figure 19F] Sirpα- / - macrophages reduce tumor immune suppression after RT. Differential intratumoral infiltration of monocytes and PMNs in WT and Sirpα- / - mice after IR. Gating strategy (Fig. 19F, Fig. 19I) identifies monocytes (Ly6C+) and PMNs (Ly6G+) among CD11b+ myeloid cells and their numbers (Fig. 19G). Inhibition of T cell proliferation (Fig. 19H) was assayed in the presence of intratumoral myeloid cells. ROS production by PMNs (Fig. 19J) was assayed in the presence of DCFDA and 1 μM PMA. Data are representative of three independent experiments and presented as mean ± SD (n = 3-5 / group). [Figure 19G]Sirpα- / - macrophages reduce tumor immune suppression after RT. Differential intratumoral infiltration of monocytes and PMNs in WT and Sirpα- / - mice after IR. Gating strategy (Fig. 19F, Fig. 19I) identifies monocytes (Ly6C+) and PMNs (Ly6G+) among CD11b+ myeloid cells and their numbers (Fig. 19G). Inhibition of T cell proliferation (Fig. 19H) was assayed in the presence of intratumoral myeloid cells. ROS production by PMNs (Fig. 19J) was assayed in the presence of DCFDA and 1 μM PMA. Data are representative of three independent experiments and presented as mean ± SD (n = 3-5 / group). [Figure 19H] Sirpα- / - macrophages reduce tumor immune suppression after RT. Differential intratumoral infiltration of monocytes and PMNs in WT and Sirpα- / - mice after IR. Gating strategy (Fig. 19F, Fig. 19I) identifies monocytes (Ly6C+) and PMNs (Ly6G+) among CD11b+ myeloid cells and their numbers (Fig. 19G). Inhibition of T cell proliferation (Fig. 19H) was assayed in the presence of intratumoral myeloid cells. ROS production by PMNs (Fig. 19J) was assayed in the presence of DCFDA and 1 μM PMA. Data are representative of three independent experiments and presented as mean ± SD (n = 3-5 / group). [Figure 19I] Sirpα- / - macrophages reduce tumor immune suppression after RT. Differential intratumoral infiltration of monocytes and PMNs in WT and Sirpα- / - mice after IR. Gating strategy (Fig. 19F, Fig. 19I) identifies monocytes (Ly6C+) and PMNs (Ly6G+) among CD11b+ myeloid cells and their numbers (Fig. 19G). Inhibition of T cell proliferation (Fig. 19H) was assayed in the presence of intratumoral myeloid cells. ROS production by PMNs (Fig. 19J) was assayed in the presence of DCFDA and 1 μM PMA. Data are representative of three independent experiments and presented as mean ± SD (n = 3-5 / group). [Figure 19J]Sirpα- / - macrophages reduce tumor immune suppression after RT. Differential intratumoral infiltration of monocytes and PMNs in WT and Sirpα- / - mice after IR. Gating strategy (Fig. 19F, Fig. 19I) identifies monocytes (Ly6C+) and PMNs (Ly6G+) among CD11b+ myeloid cells and their numbers (Fig. 19G). Inhibition of T cell proliferation (Fig. 19H) was assayed in the presence of intratumoral myeloid cells. ROS production by PMNs (Fig. 19J) was assayed in the presence of DCFDA and 1 μM PMA. Data are representative of three independent experiments and presented as mean ± SD (n = 3-5 / group). [Figure 19K] Figure 1 shows that Sirpα- / - macrophages reduce tumor immune suppression after RT. Figure 2 shows that PMN infiltration promotes tumor regression. Intratumoral PMNs and other leukocytes (K) in 15 MC38 tumors from Sirpα- / - mice 3 days after IR, and regression analysis of intratumoral PMNs and percentage of tumor regression (L). Data are representative of three independent experiments and presented as mean ± SD of triplicate assays (n=15 / group). [Figure 19L] Figure 1 shows that Sirpα- / - macrophages reduce tumor immune suppression after RT. Figure 2 shows that PMN infiltration promotes tumor regression. Intratumoral PMNs and other leukocytes (K) in 15 MC38 tumors from Sirpα- / - mice 3 days after IR, and regression analysis of intratumoral PMNs and percentage of tumor regression (L). Data are representative of three independent experiments and presented as mean ± SD of triplicate assays (n=15 / group). [Figure 20A] We show that phagocytic Sirpα- / - macrophages function as APCs and activate tumor-specific Tc. We show that MC38 tumors (~300 mm3) were resected, dissected, and then cultured ex vivo immediately after IR (8 Gy), and some WT MC38 tumors were injected it with Sirpα- / - BMDMs (1x106 / mm3) immediately after resection prior to culture. Four days later, single cell suspensions were analyzed for Tc and Th in the CD45+ population. Data are representative of two independent experiments (n=3-5 / group). [Figure 20B] Figure 1 shows that phagocytic Sirpα- / - macrophages function as APCs and activate tumor-specific Tc. Figure 2 shows in vitro expansion of tumor-specific Tc from TILs by tumor-phagocytosing Sirpα- / - BMDM. Figure 3 shows the experimental scheme. [Figure 20C] We show that phagocytic Sirpα- / - macrophages function as APCs and activate tumor-specific Tc. We show in vitro expansion of tumor-specific Tc from TILs by tumor-phagocytosing Sirpα- / - BMDMs. Images of Tc (red, CD8 staining) conjugated with tumor antigen-loaded Sirpα- / - BMDMs (grey) are shown. Activation of Tc after 2 days of TIL-Sirpα- / - BMDM coculture was evident by robust Tc (but not Th) proliferation summarized as an increase in cell size), SSC and FSC. Data are representative of at least 5 independent experiments with TILs pooled from 3 tumors and 3 cocultures each. [Figure 20D] We show that phagocytic Sirpα- / - macrophages function as APCs and activate tumor-specific Tc. We show in vitro expansion of tumor-specific Tc from TILs by tumor-phagocytosing Sirpα- / - BMDMs. Activation of Tc after 2 days of TIL-Sirpα- / - BMDM coculture was evident by robust Tc (but not Th) proliferation summarized as increased cell size), increases in SSC and FSC, and GranzB expression. Data are representative of at least 5 independent experiments with TILs pooled from 3 tumors each and 3 cocultures. [Figure 20E]We show that phagocytic Sirpα- / - macrophages function as APCs and activate tumor-specific Tc. We show in vitro expansion of tumor-specific Tc from TILs by tumor-phagocytosing Sirpα- / - BMDMs. Activation of Tc after 2 days of TIL-Sirpα- / - BMDM coculture was evident by robust Tc (but not Th) proliferation, as indicated by enlarged cell size), increases in SSC and FSC, and CSFE dilution. Data are representative of at least 5 independent experiments with 3 cocultures and TILs pooled from 3 tumors each. [Figure 20F] We show that phagocytic Sirpα- / - macrophages function as APCs and activate tumor-specific Tc. We show in vitro expansion of tumor-specific Tc from TILs by tumor-phagocytosing Sirpα- / - BMDMs. Activation of Tc after 2 days of TIL-Sirpα- / - BMDM coculture was evident by robust Tc (but not Th) expansion summarized as enlarged cell size), increases in SSC and FSC, and increased frequency. Data are representative of at least 5 independent experiments with TILs pooled from 3 tumors each and 3 cocultures. [Figure 20G] We show that phagocytic Sirpα- / - macrophages function as APCs and activate tumor-specific Tc. We show in vitro expansion of tumor-specific Tc from TILs by tumor-phagocytosing Sirpα- / - BMDMs. Activation of Tc after 2 days of TIL-Sirpα- / - BMDM coculture was evident by robust Tc (but not Th) proliferation summarized as enlarged cell size), increases in SSC and FSC, and increased numbers. Data are representative of at least 5 independent experiments with TILs pooled from 3 tumors each and 3 cocultures. [Figure 20H]Figure 1 shows that phagocytic Sirpα- / - macrophages function as APCs and activate tumor-specific Tc. Cytotoxicity of Tc expanded by MC38 or KPC-loaded Sirpα- / - BMDMs was assessed by co-culture with MC38 or KPC cells, respectively, for 24 h at the indicated effector:target ratios. TILs with αCD3 / CD28 expanded Tc of approximately 70% were used as comparison. Data are representative of three independent experiments and presented as mean ± SD (n=3 / group). [Figure 20I] We show that phagocytic Sirpα- / - macrophages function as APCs and activate tumor-specific Tc. We show the efficacy of Tc-MC38 and Tc-KPC in vivo. WT mice bearing MC38 (Figure 20I) or KPC (Figure 20J) tumors were treated with Tc-MC38 or Tc-KPC (iv 5x106), ± total body radiation (WBI; 5Gy), and recombinant human IL-2 (ip 25,000IU, 2x daily for 5 days), or with the same number of TILs activated by αCD3 / CD28. MC38-Tc showed activated / migratory morphology compared to αCD3 / CD28-TIL. Data are representative of 5 independent experiments (n=3-4 mice / group). [Figure 20J] We show that phagocytic Sirpα- / - macrophages function as APCs and activate tumor-specific Tc. We show the efficacy of Tc-MC38 and Tc-KPC in vivo. WT mice bearing MC38 (Figure 20I) or KPC (Figure 20J) tumors were treated with Tc-MC38 or Tc-KPC (iv 5x106), ± total body radiation (WBI; 5Gy), and recombinant human IL-2 (ip 25,000IU, 2x daily for 5 days), or with the same number of TILs activated by αCD3 / CD28. MC38-Tc showed activated / migratory morphology compared to αCD3 / CD28-TIL. Data are representative of 5 independent experiments (n=3-4 mice / group). [Figure 21A]Scheme for controlling macrophage phagocytosis of cancer cells. Tumor-associated macrophages are predominantly inhibited by immunosuppressive cytokines / factors in TEM where the CD47-SIRPα axis is not essential, thereby indicating that CD47 blockade alone (FIG. 21B) does not induce phagocytosis. [Figure 21B] Scheme for controlling macrophage phagocytosis of cancer cells. Tumor-associated macrophages are predominantly inhibited by immunosuppressive cytokines / factors in TEM where the CD47-SIRPα axis is not essential, thereby indicating that CD47 blockade alone (FIG. 21B) does not induce phagocytosis. [Figure 21C] We show that SIRPANT's proprietary reagent, Phago-Act™, simultaneously downregulates SIRPα expression and activates macrophage phagocytosis to produce SIRPANT-M, which has the ability to potently phagocytose tumor cells, mediate antigen presentation, and activate tumor-specific T cell cytotoxicity and long-lasting adaptive immunity. [Figure 22A] We show that tumors upregulate SIRPα expression. We show that tumor-associated macrophages (TAMs), tumor-infiltrating dendritic cells (DCs), and myeloid-derived suppressor cells (MDSCs) express SIRPα expression that increases with tumor size, as detected by flow cytometry. MC38: mouse colorectal carcinoma, KPC: mouse pancreatic ductal adenocarcinoma, EL4: mouse T-cell lymphoma. [Figure 22B] We show that tumors upregulate SIRPα expression. We show that tumor-associated macrophages (TAMs), tumor-infiltrating dendritic cells (DCs), and myeloid-derived suppressor cells (MDSCs) express SIRPα expression that increases with tumor size, as detected by flow cytometry. MC38: mouse colorectal carcinoma, KPC: mouse pancreatic ductal adenocarcinoma, EL4: mouse T-cell lymphoma. [Figure 22C]Tumors upregulate SIRPα expression. IF staining of MC38 tumor sections is shown. Note: CD47 (also PD-L1, FIG. 22A) shows an increase in tumor cells along tumor growth, indicating stronger CD47-SIRPα regulation and greatly enhanced immune suppression in large tumors. [Figure 22D] We show that tumors upregulate SIRPα expression. Human PBMC-derived macrophages (human M) were treated with various cancer cell conditioned media and show increased SIRPa expression. HT29, Caco2 and T84: human colorectal cancer cells; MDA231, MDA-435, BT549 and T47D: human breast cancer cells, etc. [Figure 23A] We show that high SIRPα expression (SIRPαhigh) confers a strong immunosuppressive phenotype to macrophages and tumor resistance to therapy. We show that tumor-regulated SIRPαhigh-M and SIRPα- / --M are compared in producing pro- and anti-inflammatory cytokines induced by the presence of IFNy / LPS±tumor medium (TME) and / or CD47 ligation (CD47.ex). [Figure 23B] We show that high SIRPα expression (SIRPαhigh) confers a strong immunosuppressive phenotype and tumor resistance to therapy in macrophages. We show that SIRPαhigh-M increased IL-4-induced arginase-1 expression and reduced iNOS by IFNy / LPS, whereas SIRPα- / --M displayed the opposite expression. [Figure 23C]We show that high SIRPα expression (SIRPαhigh) confers a strong immunosuppressive phenotype to macrophages and tumor resistance to treatment. We show transcriptional analysis of SIRPαhigh and SIRPα− / − tumors in response to radiotherapy (RT). SIRPαhigh tumors induced little antigen presentation or proinflammatory response but enhanced immunosuppression indicated by increased TGFB and chemokines that attract MDSCs for wound healing and T cell inhibition, whereas SIRPα− / − tumors showed the opposite response, their immune landscape exhibiting a strong inflammatory response and immunogenic antigen presentation that activated tumoricidal activity of T cells. MC38: colorectal cancer, KPC and Pan02: pancreatic ductal adenocarcinoma. [Figure 23D] Figure 1 shows that high SIRPα expression (SIRPαhigh) confers a strong immunosuppressive phenotype in macrophages and tumor resistance to therapy. A comparison of the expression of antigen-presenting machinery on the cell surface between tumor-regulated SIRPαhigh-M and Phago-Act™-produced SIRPαLow / SIRPANT-M is shown. [Figure 24A] Figure 2 shows the mechanism of SIRPα regulation: Tumor immunosuppressive signals upregulate SIRPα, whose cytoplasmic ITIM is phosphorylated by Btk, leading to recruitment of SHP-2 and enhanced TME immunosuppression. [Figure 24B] We show the mechanism of SIRPα regulation. Under treatment, SIRPα via SFK-mediated ITIM phosphorylation recruits / activates SHP-1, which inhibits multipathway proinflammatory signals and confers treatment resistance. [Figure 24C] Figure 2 shows the SIRPα regulatory mechanism: Under pro- or anti-inflammatory stimulation, phosphorylated SIRPα ITIM in macrophages mediates individual binding to either SHP-1 or SHP-2, respectively. [Figure 24D] 1 shows the mechanism of SIRPα regulation, showing that SIRPα regulation is independent of, but enhanced by, extracellular ligation of CD47. [Figure 25A]Activation of Sirpα-deficient macrophages to phagocytose cancer cells. IL-17, LPS and IL-6 (10 ng / ml each) activate SIRPα- / --M to phagocytose B16 melanoma cells in co-culture. The figure also shows that SIRPα- / --M did not phagocytose in the absence of activation, and WT-M did not phagocytose with or without activation. B) SIRPα- / --M treated with a cocktail containing IL-6 (10 ng / ml), CpG and Polyl:C (100 ng / ml each) actively phagocytose LLC lung cancer cells, MC38 colorectal adenocarcinoma, EL4 lymphoma and Pan02 pancreatic cancer cells. [Figure 25B] Activation of Sirpα-deficient macrophages to phagocytose cancer cells. IL-17, LPS and IL-6 (10 ng / ml each) activate SIRPα- / --M to phagocytose B16 melanoma cells in co-culture. The figure also shows that SIRPα- / --M did not phagocytose in the absence of activation, and WT-M did not phagocytose with or without activation. B) SIRPα- / --M treated with a cocktail containing IL-6 (10 ng / ml), CpG and Polyl:C (100 ng / ml each) actively phagocytose LLC lung cancer cells, MC38 colorectal adenocarcinoma, EL4 lymphoma and Pan02 pancreatic cancer cells. [Figure 26A] 13 shows that IL-17A-treated SIRPα− / − mice eliminated B16 melanoma. [Figure 26B] Figure 1 shows that melanoma-eradicated SIRPα- / - mice developed anti-cancer immunity by anti-B16 Ab and the ability to resist reimplantation. WB: Detection of B16 membrane protein by ctl serum or anti-B16 serum from melanoma-eradicated SIRPα- / - mice. [Figure 26C] 1 shows that WT mice that received anti-B16 serum exhibited resistance to melanoma transplantation. [Figure 27A]Tumor elimination by RT in SIRPα- / - mice. MC38, Pan02 or KPC were implanted sc into WT or SIRPα- / - mice. After tumors were well formed (≥200 mm3), fractions of X-ray RT (4-15 Gy) were administered, followed by recording of tumor volume change and animal survival. Grey line: WT mice resisted 8 Gy RT plus two applications of anti-PD-L1 (100 μg, ip) 3 days apart. [Figure 27B] Tumor elimination by RT in SIRPα- / - mice. MC38, Pan02 or KPC were implanted sc into WT or SIRPα- / - mice. After tumors were well formed (≥200 mm3), fractions of X-ray RT (4-15 Gy) were administered, followed by recording of tumor volume change and animal survival. Grey line: WT mice resisted 8 Gy RT plus two applications of anti-PD-L1 (100 μg, ip) 3 days apart. [Figure 27C] Figure 1 shows tumor clearance by RT in SIRPα- / - mice.Intratumoral depletion of SIRPα- / -M abolished RT efficacy in SIRPα- / - mice. [Figure 27D] Figure 1 shows tumor elimination by RT in SIRPα- / - mice. Figure 2 shows that adoptive transfer of bone marrow-derived SIRPα- / --M to tumors in WT mice conferred tumor regression by PT. [Figure 28A] We show that tumor elimination in SIRPα- / - mice by IR was associated with the expansion of anti-tumor Tc expressed near GranzB (Figure 28A) and that this fraction was tumor antigen (p15E) specific, differentiated into TEM (CD44+CD62L-) (Figure 28B). [Figure 28B] We show that tumor elimination in SIRPα- / - mice by IR was associated with the expansion of anti-tumor Tc expressed near GranzB (Figure 28A) and that this fraction was tumor antigen (p15E) specific, differentiated into TEM (CD44+CD62L-) (Figure 28B). [Figure 28C]SIRPα− / − tumors also reduced Foxp3 Tregs (FIG. 28C) and decreased Ly6C+ MDSC infiltration but increased NKs after IR (FIG. 28D). [Figure 28D] SIRPα− / − tumors also reduced Foxp3 Tregs (FIG. 28C) and decreased Ly6C+ MDSC infiltration but increased NKs after IR (FIG. 28D). [Figure 29A] 1 shows up- and down-regulation of SIRPα expression in macrophages by cytokines, TLR agonists, steroids, and tumor-conditioned medium. 2 shows mouse bone marrow-derived macrophages. [Figure 29B] 1 shows up- and down-regulation of SIRPα expression in macrophages by cytokines, TLR agonists, steroids, and tumor-conditioned medium. 2 shows mouse bone marrow-derived macrophages. [Figure 29C] Figure 1 shows up- and down-regulation of SIRPα expression in macrophages by cytokines, TLR agonists, steroids, and tumor conditioned medium.Human PBMC-derived macrophages are shown. [Figure 29D] 1 shows up- and down-regulation of SIRPα expression in macrophages by cytokines, TLR agonists, steroids, and tumor conditioned media. FIG. 1 is a scheme of ex vivo production of SIRPαlow activated macrophages, SIRPANT-M, by Phago-Act™. [Figure 29E] Figure 1 shows up- and down-regulation of SIRPα expression in macrophages by cytokines, TLR agonists, steroids, and tumor-conditioned medium. Human SIRPANT-M resists phenotypic changes (re-expression of SIRPa) in the tumor state and maintains longevity. [Figure 29F] Figure 1 shows up- and down-regulation of SIRPα expression in macrophages by cytokines, TLR agonists, steroids, and tumor-conditioned medium. Figure 2 shows that human SIRPANT-M directly phagocytose human cancer cells. [Figure 30A]This shows that mouse SIRPANT-M directly phagocytose syngeneic cancer cells. The experimental scheme is shown. [Figure 30B] Figure 1 shows that mouse SIRPANT-M directly phagocytose syngeneic cancer cells. Sample microscopy results of SIRPANT-M phagocytosing EL4 lymphoma and MC38 colorectal adenocarcinoma cells are shown. [Figure 30C] Figure 1 shows that mouse SIRPANT-M directly phagocytose syngeneic cancer cells. Sample flow cytometry showing SIRPANT-M phagocytosis of MC38 cells. BMDM or SIRPANT-M were gated by CD11b+. [Figure 30D] This shows that mouse SIRPANT-M directly phagocytoses syngeneic cancer cells. Phagocytosis of syngeneic cancer cells at 4 hours is shown. ****p<0.0001. [Figure 31A] Human PBMC-derived macrophages (SIRPa*-M) were treated with TNFα and IL-17, or INFy, or Phag-Act (SIRPANT-M) for 2 days and then tested for phagocytosis against various human cancer cells. Only SIRPANT-M showed positive phagocytosis. [Figure 31B] Time course of SIRPANT-M phagocytosis is shown. [Figure 31C] Shows SIRPANT-M phagocytosis of NCI-60 human cancer panel at 4 hours. [Figure 31D] Microscopic images showing SIRPANT-M phagocytosis by HT29, T84, Caco2 and THP-1 are shown. [Figure 31E] We show that SIRPANT-M mediates phagocytosis independent of CD47 expression on cancer cells. [Figure 32A]Human SIRPANT-M shows enhanced phagocytosis towards X-ray irradiated human cancer cells. Human PBMC-derived SIRPANT-M (Fig. 32A) or SIRPα+-M (Fig. 32B) were incubated with various non-irradiated (-IR) or irradiated (8Gy) human cancer cells for 4 hours, followed by phagocytosis assessment. Sample fluorescence microscopy images (CD11b staining) showing that SIRPANT-M, but not SIRPα+-M, actively phagocytose irradiated OVCAR3 ovarian cancer cells and UACC-62 melanoma cells (CFSE). [Figure 32B] Human SIRPANT-M shows enhanced phagocytosis towards X-ray irradiated human cancer cells. Human PBMC-derived SIRPANT-M (Fig. 32A) or SIRPα+-M (Fig. 32B) were incubated with various non-irradiated (-IR) or irradiated (8Gy) human cancer cells for 4 hours, followed by phagocytosis assessment. Sample fluorescence microscopy images (CD11b staining) showing that SIRPANT-M, but not SIRPα+-M, actively phagocytose irradiated OVCAR3 ovarian cancer cells and UACC-62 melanoma cells (CFSE). [Figure 33A] Figure 1 shows that murine SIRPANT-M enhanced phagocytosis of irradiated cancer cells. Comparison of BMDM (SIRPα+) and SIRPANT-M for phagocytosis of non-irradiated (-IR) and irradiated (8 Gy) syngeneic tumor cells. [Figure 33B] Showing that murine SIRPANT-M enhanced phagocytosis of irradiated cancer cells. Microscopy and flow cytometry showing that SIRPANT-M, but not BMDM, actively phagocytose irradiated MC-38 cells. [Figure 33C] Figure 1 shows that mouse SIRPANT-M enhanced phagocytosis of radiation-treated cancer cells.Figure 2 shows a time course assay showing that SIRPANT-M enhanced phagocytosis of EL4 irradiated at various doses. [Figure 33D]We show that mouse SIRPANT-M enhanced phagocytosis of irradiated cancer cells. We show that non-ablative radiation did not induce apoptosis (PI / YO-PRO-1) or changes in cell surface CD47, but did increase calreticulin (CRT). [Figure 33E] We show that mouse SIRPANT-M enhanced phagocytosis of irradiated cancer cells. We show that non-ablative radiation did not induce apoptosis (PI / YO-PRO-1) or changes in cell surface CD47, but did increase calreticulin (CRT). [Figure 34A] Shown is SIRPANT-M activation phenotype and antigen-presenting capacity. Freshly obtained mouse BMDM (SIRPα+-M) was further treated with Phago-Act™ for 48 hours to induce SIRPANT-M. Shown is SIRPα expression in SIRPα+-M and SIRPANT-M before and after Phago-Act™ treatment. [Figure 34B] SIRPANT-M activation phenotype and antigen-presenting capacity. Freshly obtained mouse BMDMs (SIRPα+-M) were further treated with Phago-Act™ for 48 hours to induce SIRPANT-M. The capacity of SIRPANT-M vs. SIRPα+-M as antigen-presenting cells (APCs) assessed by their expression of MHC-I, MHC-Il, and the costimulatory molecules CD80 and CD86. [Figure 34C] SIRPANT-M activation phenotype and antigen-presenting capacity are shown. Freshly obtained mouse BMDMs (SIRPα+-M) were further treated with Phago-Act™ for 48 hours to induce SIRPANT-M. Inflammatory characteristics of SIRPANT-M vs. SIRPα+-M as assessed by their production of pro-inflammatory and anti-inflammatory cytokines are shown. [Figure 35A] Figure 1 shows mapping mRNA transcripts of SIRPANT-M from seven human PBMCs compared to donor-matched SIRPα+-M. Heatmap transcript analysis of genes involved in antigen presentation and pro- and anti-inflammatory responses. [Figure 35B]Figure 1 shows mapping mRNA transcripts of 7 human PBMC-derived SIRPANT-M compared to donor-matched SIRPα+-M. Figure 2 shows gene expression programs induced in SIRPANT-M by Phago-Act™. Display shows differentially regulated genes (total 2029, 1093 upregulated and 936 downregulated) categorized according to known or predicted function(s), literature and sequence similarity. [Figure 35C] Figure 1 shows mapping mRNA transcripts of 7 human PBMC-derived SIRPANT-M compared to donor-matched SIRPα+-M. Figure 2 shows scatter plots showing differences in gene expression in SIRPANT-M compared to SIRPα+-M. [Figure 36A] 1 shows an exemplary scheme showing that in vitro SIRPANT-M activates MC38-specific T cells and KPC-specific T cells from intratumoral TILs. [Figure 36B] Figure 36 shows that in vitro SIRPANT-M activates MC38- and KPC-specific T cells from intratumoral TILs. Figure 36B shows that SIRPANT-M (Figure 36C), but not SIRPα+-M, delivered tumor antigens induced CD8+ T cell proliferation from TILs. Minimal CD4+ T cell proliferation was detected (Figure 36D). [Figure 36C] Figure 36 shows that in vitro SIRPANT-M activates MC38- and KPC-specific T cells from intratumoral TILs. Figure 36B shows that SIRPANT-M (Figure 36C), but not SIRPα+-M, delivered tumor antigens induced CD8+ T cell proliferation from TILs. Minimal CD4+ T cell proliferation was detected (Figure 36D). [Figure 36D] Figure 36 shows that in vitro SIRPANT-M activates MC38- and KPC-specific T cells from intratumoral TILs. Figure 36B shows that SIRPANT-M (Figure 36C), but not SIRPα+-M, delivered tumor antigens induced CD8+ T cell proliferation from TILs. Minimal CD4+ T cell proliferation was detected (Figure 36D). [Figure 36E]In vitro SIRPANT-M activates MC38-specific and KPC-specific T cells from intratumoral TILs. SIRPANT-M after tumor antigen-mediated engagement with CD8 T cells (CD8 staining) for antigen presentation induces phagocytosis (FIG. 36E), CD8 T cell expansion (increasing SSC and FSC on day 2 (D2)) and proliferation (FIG. 36G). [Figure 36F] In vitro SIRPANT-M activates MC38-specific and KPC-specific T cells from intratumoral TILs. SIRPANT-M after tumor antigen-mediated engagement with CD8 T cells (CD8 staining) for antigen presentation induces phagocytosis (FIG. 36E), CD8 T cell expansion (increasing SSC and FSC on day 2 (D2)) and proliferation (FIG. 36G). [Figure 36G] In vitro SIRPANT-M activates MC38-specific and KPC-specific T cells from intratumoral TILs. SIRPANT-M after tumor antigen-mediated engagement with CD8 T cells (CD8 staining) for antigen presentation induces phagocytosis (FIG. 36E), CD8 T cell expansion (increasing SSC and FSC on day 2 (D2)) and proliferation (FIG. 36G). [Fig. 36H] Figure 2 shows that in vitro SIRPANT-M activates MC38-specific and KPC-specific T cells from intratumoral TILs. Figure 2 shows that SIRPANT-M-activated CD8 T cells against MC38 displayed increased reactivity with MC38-specific p15E and ADPGK epitopes, as well as highly expressed granzyme B. [Figure 36I] Figure 2 shows that in vitro SIRPANT-M activates MC38-specific and KPC-specific T cells from intratumoral TILs. Figure 2 shows that SIRPANT-M-activated CD8 T cells against MC38 displayed increased reactivity with MC38-specific p15E and ADPGK epitopes, as well as highly expressed granzyme B. [Figure 36J]Figure 1 shows that in vitro SIRPANT-M activates MC38- and KPC-specific T cells from intratumoral TILs. Figure 2 shows the cytotoxicity of in vitro SIRPANT-M-activated CD8 T cells against cancer. CD8 T cells expanded from MC38 TILs and KPC TILs (designated TMC38 and TKPC) were co-incubated (12 h) with healthy cultured MC38 and KPC cells at a T:cancer cell ratio of 1:1 or 1:3, respectively, followed by analysis of cancer cell death (J) compared to MC38 and KPC cells without T cell co-incubation (Ctl.). [Figure 36K] Figure 2 shows that in vitro SIRPANT-M activates MC38- and KPC-specific T cells from intratumoral TILs. Real-time imaging snapshots of TMC38 (arrow) killing MC38 cells are shown. [Figure 37] SIRPANT-M induces B16-gp33 antigen-specific CD8 T cell activation in vitro. Left: Experimental scheme. Right: Only SIRPANT-M supplied with B16gp33 robustly induced antigen (gp33)-specific T cell activation. [Figure 38A] Figure 1 shows SIRPANT-M intratumoral monotherapy treating early stage (small tumors) and late stage (large tumors) colorectal cancer MC38 and pancreatic ductal adenocarcinoma KPC (both sc). Dose-response study. Intratumoral injection (it) dosing strategy is shown. [Figure 38B] Shown is SIRPANT-M intratumoral monotherapy treating early stage (small tumors) and late stage (large tumors) colorectal cancer MC38 and pancreatic ductal adenocarcinoma KPC (both sc). Dose-response study. Shown is tracing of SIRPANT-M in MC38 after it injection, kinetics indicate that SIRPANT-M is present in the tumor for approximately 2 days. [Figure 38C]Figure 1 shows SIRPANT-M intratumoral monotherapy treating early stage (small tumors) and late stage (large tumors) colorectal cancer MC38 and pancreatic ductal adenocarcinoma KPC (both sc). Dose-response study. MC38 of various sizes (dashed lines) are shown treated with SIRPANT-M by it. Data show one of two to three cohorts of each size of MC38 tumors treated with D1 / 2 and D1 doses every 3 days for three doses starting on days 10, 12, 14, and 16 after MC38 implantation. [Figure 38D] Shown is SIRPANT-M intratumoral monotherapy treating early stage (small tumors) and late stage (large tumors) colorectal cancer MC38 and pancreatic ductal adenocarcinoma KPC (both sc). Dose-response study. Shown is overall survival of MC38-implanted mice treated with vehicle (PBS) control or 3x SIRPANT-M it at D1 / 2 and D1 doses. Data are pooled for 2-3 cohorts in each treatment group (n=10-22). [Figure 38E] Figure 1 shows SIRPANT-M intratumoral monotherapy treating early (small tumors) and late stage (large tumors) colorectal cancer MC38 and pancreatic ductal adenocarcinoma KPC (both sc). Dose-dependency study. KPCs of various sizes (dashed lines) are shown treated with SIRPANT-M by it. Data show one of two to three cohorts of each KPC tumor size treated with the D1 dose every 3 days for three doses starting on days 14, 16, and 18 after KPC implantation. Note: MC38 tumors generally grow faster than KPC tumors by it, and treatment doses were calculated according to tumor size. [Figure 38F] Shown is SIRPANT-M intratumoral monotherapy treating early stage (small tumors) and late stage (large tumors) colorectal cancer MC38 and pancreatic ductal adenocarcinoma KPC (both sc). Dose-response study. Shown is overall survival of KPC-implanted mice treated with vehicle (PBS) control or 3x SIRPANT-M it. at D1 dose. Data are pooled from 2-3 cohorts with a total of n=15-20 in each treatment group. [Figure 39A]Showing that SIRPANT-M therapy is tumor cross-sectional. Colorectal (MC38), pancreatic (Pan02), lung (LLC) or lymphoma (EL4) tumors (size 150-400 mm3) were treated with SIRPANT-M at D2 dose (it, 3x, every 3 days). One of 2-3 cohorts of each cancer type is shown. [Figure 39B] Shows that SIRPANT-M therapy is tumor-crossing. Shows overall survival of tumor-implanted mice treated with vehicle control (PBS) or D2 dose of SIRPANT-M by it. Data summarizes multiple cohorts of each type of cancer with treatment applied at different stages (tumor size). [Figure 39C] SIRPANT-M therapy is shown to be tumor cross-sectional. SIRPANT-M treating spontaneous triple-negative mammary adenocarcinoma in MMTV-PyMT mice (n=20) is shown. D1 dose of SIRPANT-M was injected intratumorally into the first tumor to develop on days 62 and 66, and into the largest tumor that developed subsequently on days 70, 74, 76, and 82, and 80. Only one tumor was treated at a time. Overall survival is shown as the number of surviving mice as a fraction. Median overall survival and Kaplan-Meier analysis are shown. [Figure 40A] Figure 1 shows that the combination of SIRPANT-M it and RT eliminates RT-refractory MC38 colorectal cancer and KPC as well as Pan02 pancreatic cancer. Figure 2 shows that mice bearing MC38, KPC and Pan02 cancers of different sizes were treated with two rounds of RT or RT plus SIRPANT-M it at the D2 dose. The treatment scheme for relatively small tumors was either 4Gy and 4Gy (tumors ≦200mm3, 3 days apart) or 8Gy and 8Gy (tumors 200-400mm3, 3 days apart) with or without immediate SIRPANT-M it after each RT fraction. For large tumors, 15Gy was used for the first treatment followed by 8Gy for the second treatment. A group of tumor-bearing mice was set as a control without treatment. [Figure 40B]Figure 40B shows that the combination of SIRPANT-M it and RT eliminates RT-refractory MC38 colorectal cancer and KPC, as well as Pan02 pancreatic cancer. Figure 40C shows the progression or regression of MC38 colorectal cancer in tumor-bearing mice after treatment for tumors of different sizes. [Figure 40C] Figure 40B shows that the combination of SIRPANT-M it and RT eliminates RT-refractory MC38 colorectal cancer and KPC, as well as Pan02 pancreatic cancer. Figure 40C shows the progression or regression of MC38 colorectal cancer in tumor-bearing mice after treatment for tumors of different sizes. [Figure 40D] We show that the combination of SIRPANT-M it and RT eliminates RT-refractory MC38 colorectal cancer and KPC and Pan02 pancreatic cancer. We show the progression or regression of KPC pancreatic cancer after treatment for their tumors of different sizes (Figure 40D) and overall survival of mice (Figure 40E). [Figure 40E] We show that the combination of SIRPANT-M it and RT eliminates RT-refractory MC38 colorectal cancer and KPC and Pan02 pancreatic cancer. We show the progression or regression of KPC pancreatic cancer after treatment for their tumors of different sizes (Figure 40D) and overall survival of mice (Figure 40E). [Diagram 40F] We show that the combination of SIRPANT-M it and RT eliminates RT-refractory MC38 colorectal cancer and KPC as well as Pan02 pancreatic cancer. We show the progression or regression of Pan02 pancreatic cancer after treatment for their tumors of different sizes (Figure 40F) and overall survival of mice (Figure 40G). [Figure 40G] We show that the combination of SIRPANT-M it and RT eliminates RT-refractory MC38 colorectal cancer and KPC as well as Pan02 pancreatic cancer. We show the progression or regression of Pan02 pancreatic cancer after treatment for their tumors of different sizes (Figure 40F) and overall survival of mice (Figure 40G). [Figure 41A]Shows dose-dependent efficacy of SIRPANT-M in combination with RT to treat MC38 colorectal cancer and KPC, and Pan02 pancreatic cancer. Well-established MC38, KPC, and Pan02 tumors with sizes <250 mm3 (blue line) or less (>300 mm3, red line) were treated with fractions of 8 Gy of X-ray radiation, followed by immediate (<30 min) it administration of SIRPANT-M at D1 / 2 (open circle) or D2 dose (closed square). The same treatment was repeated 3 days later (total of 2 times). Recording of tumor volume changes. [Figure 41B] Shows the dose-dependent efficacy of SIRPANT-M in combination with RT to treat MC38 colorectal cancer and KPC and Pan02 pancreatic cancer. Shows survival records of mice with no treatment, 8Gy RT only, or 8Gy RT plus various doses of SIRPANT-M it. Data includes mice given SIRPANT-M it at D1 / 2, D1, and D2 doses. [Figure 42A] We show that the combination of SIRPANT-Mi.t and RT induces a strong abscopal effect and eliminates KPC cancer lesions systemically. Mice were implanted with KPC / Luc pancreatic adenocarcinoma at multiple locations (Figure 40A). After tumor formation, one or two of the largest palpable tumors (red circles, all >200 mm3) were treated with a first round of SIRPANT-Mi.t at 8 Gy RT and a D1 dose, followed by two rounds of SIRPANT-Mi.t at 4 Gy RT and a D1 dose (each round was given 3 days apart). The control group (left) received three rounds of 8 Gy RT without SIRPANT-M. Whole-body luminescence imaging was performed before and after each treatment to document tumor growth or regression. [Figure 42B]We show that the combination of SIRPANT-Mi.t and RT induces a strong abscopal effect and eliminates KPC cancer lesions systemically. Mice were implanted with KPC / Luc pancreatic adenocarcinoma at multiple locations (Figure 40A). After tumor formation, one or two of the largest palpable tumors (red circles, all >200 mm3) were treated with the first round of SIRPANT-Mi.t at 8 Gy RT and D1 dose, followed by two rounds of SIRPANT-Mi.t at 4 Gy RT and D1 dose. (Each round was given at 3-day intervals). The control group (left) received three rounds of 8 Gy RT without SIRPANT-M. Whole-body luminescence imaging was performed before and after each treatment to record tumor growth or regression. Total tumor volume (Figure 42B) was calculated by the in vivo luminescence intensity of KPC / Luc cells, and animal survival (Figure 42C) was recorded. [Figure 42C] We show that the combination of SIRPANT-Mi.t and RT induces a strong abscopal effect and eliminates KPC cancer lesions systemically. Mice were implanted with KPC / Luc pancreatic adenocarcinoma at multiple locations (Figure 40A). After tumor formation, one or two of the largest palpable tumors (red circles, all >200 mm3) were treated with the first round of SIRPANT-Mi.t at 8 Gy RT and D1 dose, followed by two rounds of SIRPANT-Mi.t at 4 Gy RT and D1 dose. (Each round was given at 3-day intervals). The control group (left) received three rounds of 8 Gy RT without SIRPANT-M. Whole-body luminescence imaging was performed before and after each treatment to record tumor growth or regression. Total tumor volume (Figure 42B) was calculated by the in vivo luminescence intensity of KPC / Luc cells, and animal survival (Figure 42C) was recorded. [Figure 43A] We show that SIRPANT-M plus RT induces a potent abscopal effect that systemically clears MC38 colorectal cancer lesions. Mice were implanted with MC38 tumors on both flanks with the right primary site and received SIRPANT-M it plus RT treatment. The experimental scheme is shown. [Figure 43B]We show that SIRPANT-M plus RT induces a potent abscopal effect that systemically clears MC38 colorectal cancer lesions. Mice were implanted with MC38 tumors in both flanks, with the right primary tumor, and received SIRPANT-M it plus RT treatment. Tumor volume changes in both flanks are shown when the right primary tumor received treatment. [Figure 43C] We show that SIRPANT-M plus RT induces a potent abscopal effect that systemically clears MC38 colorectal cancer lesions. Mice were implanted with MC38 tumors in both flanks, with the right primary tumor, and received SIRPANT-M it plus RT treatment. Tumor volume changes in both flanks are shown when the right primary tumor received treatment. [Fig. 43D] Figure 43 shows that SIRPANT-M plus RT induces a strong abscopal effect that systemically clears MC38 colorectal cancer lesions. Mice were implanted with MC38 tumors on both flanks, with the primary tumor on the right side, and received SIRPANT-M it plus RT treatment. Survival records of mice with small and large primary and abscopal tumors are shown associated with Figure 43B and Figure 43C, respectively. Note: A single dose (20 μg, ip) of anti-PD-L1 was given to mice that initially had large abscopal tumors in Figure 43C to promote abscopal clearance. [Figure 43E] Figure 43 shows that SIRPANT-M plus RT induces a strong abscopal effect that systemically clears MC38 colorectal cancer lesions. Mice were implanted with MC38 tumors on both flanks, with the primary tumor on the right side, and received SIRPANT-M it plus RT treatment. Survival records of mice with small and large primary and abscopal tumors are shown associated with Figure 43B and Figure 43C, respectively. Note: A single dose (20 μg, ip) of anti-PD-L1 was given to mice that initially had large abscopal tumors in Figure 43C to promote abscopal clearance. [Figure 44A]Figure 1 shows the efficacy of SIRPANT-M it administration before or after RT. MC38 colorectal cancer and EL4 lymphoma established in C57BL6 mice were treated with SIRPANT-M it (D1 dose) either immediately (<3 hours), or 24 hours, or 48 hours before a fraction of 8Gy RT, or for the same length of time after RT. Tumor volume changes in response to the different treatments were recorded and compared to untreated controls and tumors treated with RT only. [Figure 44B] Figure 1 shows the efficacy of SIRPANT-M it administration before or after RT. Figure 2 shows the survival records of mice treated with different sequences of SIRPANT-M it and RT. [Figure 45A] We show the dose-dependent efficacy of SIRPANT-M when combined with RT to treat lung cancer (LLC), lymphoma (EL4), and two forms of triple-negative breast cancer (4T1 and PyMT). LLC lung cancer and EL4 lymphoma were implanted sc in C57BL6 mice. 4T1 breast cancer was implanted orthotopically in the mammary gland of Balb C mice. Female MMTV-PyMT mice spontaneously developed breast cancer at approximately 50 days of age. After palpable tumor formation, tumors were treated with their syngeneic SIRPANT-M at D1 / 2, D1, and D2 doses by it immediately after a fraction of 8 Gy RT. Treatments were repeated 3 days later (2 times in total). For PyMT mice, SIRPANT-M it and 8 Gy RT treatments were applied to the first palpable tumor, followed by additional treatments for other tumors that subsequently appeared, but only the largest tumor was treated each time. A total of 6x SIRPANT-M it and RT combination treatments were applied. [Figure 45B]We show the dose-dependent efficacy of SIRPANT-M when combined with RT to treat lung cancer (LLC), lymphoma (EL4), and two forms of triple-negative breast cancer (4T1 and PyMT). LLC lung cancer and EL4 lymphoma were implanted sc in C57BL6 mice. 4T1 breast cancer was implanted orthotopically in the mammary gland of Balb C mice. Female MMTV-PyMT mice spontaneously developed breast cancer at approximately 50 days of age. After palpable tumor formation, tumors were treated with their syngeneic SIRPANT-M at D1 / 2, D1, and D2 doses by it immediately after a fraction of 8 Gy RT. Treatments were repeated 3 days later (2 times in total). For PyMT mice, SIRPANT-M it and 8 Gy RT treatments were applied to the first palpable tumor, followed by additional treatments for other tumors that subsequently appeared, but only the largest tumor was treated each time. A total of 6x SIRPANT-M it and RT combination treatments were applied. [Figure 45C] We show the dose-dependent efficacy of SIRPANT-M when combined with RT to treat lung cancer (LLC), lymphoma (EL4), and two forms of triple-negative breast cancer (4T1 and PyMT). LLC lung cancer and EL4 lymphoma were implanted sc in C57BL6 mice. 4T1 breast cancer was implanted orthotopically in the mammary gland of Balb C mice. Female MMTV-PyMT mice spontaneously developed breast cancer at approximately 50 days of age. After palpable tumor formation, tumors were treated with their syngeneic SIRPANT-M at D1 / 2, D1, and D2 doses by it immediately after a fraction of 8 Gy RT. Treatments were repeated 3 days later (2 times in total). For PyMT mice, SIRPANT-M it and 8 Gy RT treatments were applied to the first palpable tumor, followed by additional treatments for other tumors that subsequently appeared, but only the largest tumor was treated each time. A total of 6x SIRPANT-M it and RT combination treatments were applied. [Figure 45D]We show the dose-dependent efficacy of SIRPANT-M when combined with RT to treat lung cancer (LLC), lymphoma (EL4), and two forms of triple-negative breast cancer (4T1 and PyMT). LLC lung cancer and EL4 lymphoma were implanted sc in C57BL6 mice. 4T1 breast cancer was implanted orthotopically in the mammary gland of Balb C mice. Female MMTV-PyMT mice spontaneously developed breast cancer at approximately 50 days of age. After palpable tumor formation, tumors were treated with their syngeneic SIRPANT-M at D1 / 2, D1, and D2 doses by it immediately after a fraction of 8 Gy RT. Treatments were repeated 3 days later (2 times in total). For PyMT mice, SIRPANT-M it and 8 Gy RT treatments were applied to the first palpable tumor, followed by additional treatments for other tumors that subsequently appeared, but only the largest tumor was treated each time. A total of 6x SIRPANT-M it and RT combination treatments were applied. [Figure 46] The timing and order of generating human SIRPαlow macrophages from PBMCs are shown. [Figure 47A] Treatment of KPC cancer with TPI-1 or TPI-1+RT is shown. [Figure 47B] Treatment of MC38 cancer with TPI-1 or TPI-1+RT is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] Before describing the present disclosure in more detail, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0023] Where a range of values ​​is provided, it is understood that, unless the context clearly dictates otherwise, each intervening value between the upper and lower limits of that range, to the tenth of the unit of the lower limit, and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any expressly excluded limitations in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this disclosure, the preferred methods and materials are now described herein.

[0025] All publications and patents cited herein are incorporated by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference herein, and are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0026] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has distinct components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the disclosure. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0027] The embodiments of the present disclosure employ, unless otherwise indicated, techniques of chemistry, biology, medicine, and the like, which are within the skill of the art.

[0028] Descriptions of the methods of the invention may include routine steps, such as collecting or obtaining a biological sample from a subject, or delivering or administering a composition to a subject, with processing steps of the invention. In such cases, it is understood that the methods of the invention may exclude any or all of the steps of collecting or obtaining a biological sample, or administering or delivering a composition to a subject.

[0029] The following examples are presented to provide those of ordinary skill in the art with a complete disclosure and description of how to practice the methods and use the therapeutic methods disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperatures, etc.), but some errors and deviations should be accounted for. Unless otherwise indicated, parts are parts by weight, temperature is in °C, and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20 °C and 1 atmosphere.

[0030] Before embodiments of the present disclosure are described in detail, it will be understood that, unless otherwise indicated, the disclosure is not limited to particular materials, reagents, reactants, manufacturing processes, etc., as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. The present disclosure also allows for steps to be carried out in different order, where this is logically possible.

[0031] definition It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. The term "about," when immediately preceding a number or numerical value, means that the number or numerical value is within a range of plus or minus 10%.

[0032] The term "subject" refers to any individual who is the target of administration or treatment. A subject can be a vertebrate, e.g., a mammal. Thus, a subject can be a human or veterinary patient. The term "patient" refers to a subject under the care of a clinician, e.g., a physician.

[0033] The term "therapeutically effective" refers to an amount of a composition used that is sufficient to achieve an outcome, which is a beneficial or desired result, such as, for example, amelioration of one or more causes or symptoms of a disease or disorder. Such amelioration need only involve reduction or alteration, not elimination. The therapeutically effective amount may vary depending on one or more of the subject and disease state being treated, the weight and age of the subject, the severity of the disease state, the method of administration, and the like.

[0034] The term "pharmaceutical acceptable" refers to compounds, substances, compositions, and / or dosage forms that are suitable, within the scope of sound medical judgment, for use in contact with human and animal tissues. Generally, a pharmaceutical acceptable moiety has one or more advantages that outweigh any adverse effects that the moiety may have. Adverse effects may include, for example, toxicity, irritation, allergic reaction, or other problems or complications that are commensurate with a reasonable benefit / risk ratio.

[0035] The term "carrier" means a compound, composition, substance, or structure that, when combined with a compound or composition, aids or facilitates the preparation, storage, administration, delivery, efficacy, selectivity, or any other characteristic of the compound or composition for its intended use or purpose. For example, a carrier can be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject.

[0036] The term "treatment" refers to the medical management of a patient with the intent of curing, alleviating, stabilizing, or preventing a disease, pathological condition, or disorder. The term includes active treatment, i.e., treatment specifically directed to ameliorating a disease, condition, or disorder, and also includes causal treatment, i.e., treatment directed to eliminating the cause of the associated disease, condition, or disorder. In addition, the term includes palliative treatment, i.e., treatment designed to relieve symptoms but not cure the disease, condition, or disorder; preventive treatment, i.e., treatment directed to minimize or partially or completely inhibit the onset of the associated disease, condition, or disorder; and supportive treatment, i.e., treatment used to complement another specific treatment directed to ameliorating the associated disease, condition, or disorder.

[0037] The term "agent" or "compound" as used herein refers to one or more chemicals or biological products (e.g., proteins, peptides, nucleic acids, polynucleotides, carbohydrate moieties), or a combination of chemicals and / or biological products. Depending on the identity of the "agent," it may be contacted with a cell in vitro or administered to a subject (e.g., to treat or prevent or control a disease or condition). In some embodiments, the agent is a protein, such as a cytokine, or an antibody. In some embodiments, the agent is a carbohydrate moiety, such as lipopolysaccharide (LPS). In some embodiments, the agent is a chemical, such as polyinosinic:polycytidylic acid (poly I:C). In some embodiments, the agent is a nucleic acid, such as a CpG oligonucleotide (ODN). The chemical or biological product may be, preferably but not necessarily a low molecular weight compound, a larger compound, or any organic or inorganic molecule, including modified and unmodified nucleic acids, such as antisense nucleic acids, RNAi, such as siRNA or shRNA, peptides, peptidomimetics, receptors, ligands, and antibodies, aptamers, polypeptides, nucleic acid analogs, or variants thereof. For example, the agent may be an oligomer of nucleic acid, amino acid, or carbohydrate, including, but not limited to, a protein, peptide, oligonucleotide, ribozyme, DNAzyme, glycoprotein, RNAi agent (e.g., siRNA), lipoprotein, aptamer, and modifications and combinations thereof. The agent may also be a naturally occurring or modified cell. In some embodiments, the active agent is a nucleic acid, such as an miRNA or a derivative or variant thereof.

[0038] As used herein, a "SIRPα inhibitor" is an agent that can promote a decrease in the expression level (e.g., protein, mRNA), decrease in function (e.g., signaling function), and / or decrease in interaction ability (e.g., interaction with CD47) of SIRPα. In some embodiments, a SIRPα inhibitor physically associates with SIRPα. In some embodiments, upon contact with a SIRPα-expressing cell, a SIRPα inhibitor can decrease the expression of SIRPα (e.g., cell surface expression of SIRPα), inhibit the activity of SIRPα, disrupt the interaction between SIRPα and CD47, or any combination thereof.

[0039] The term "inhibit" refers to a decrease in an activity, response, condition, disease, or other biological parameter. This can include, but is not limited to, the complete elimination of an activity, response, condition, or disease. It can also include, for example, a 10% reduction in an activity, response, condition, or disease compared to native or control levels. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100% reduction, or any amount in between, compared to native or control levels.

[0040] The term "radiation" refers to ionizing radiation consisting of fast moving energetic elementary particles, ions, or atoms or high energy electromagnetic waves. As used herein, the term "radiation" is used in a medical context and is used synonymously with "ionizing radiation," "irradiation," "radiotherapy," and "radiotherapy treatment." The term "tumor-directed radiation" refers to the medical use of beams of radiation that are aimed directly at a patient's tumor.

[0041] Compositions and methods Provided herein is a method for treating cancer in a subject, comprising administering a therapeutically effective amount of activated SIRPα. low Methods are disclosed that include administering to a subject activated SIRPα. lowMacrophages, in some embodiments, are produced by collecting a biological sample comprising peripheral blood mononuclear cells (PBMCs) from a subject, isolating monocytes from the PBMCs, culturing the monocytes in vitro to produce macrophages, and contacting the macrophages with a SIRPα inhibitor to produce macrophages with reduced SIRPα cell surface expression or activity compared to untreated macrophages (SIRPα inhibitors). low Macrophage) population and SIRPα low The macrophages are contacted with a macrophage activator to induce SIRPα low Activates macrophages, thereby activating SIRPα low producing macrophages.

[0042] In some embodiments, the SIRPα inhibitor and macrophage activator are contacted with the macrophages sequentially, which may be in any order and may be at intervals of minutes, hours, or days, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours apart. In other embodiments, the SIRPα inhibitor and macrophage activator are contacted with the macrophages simultaneously or in parallel.

[0043] In some embodiments, the SIRPα inhibitor and the macrophage activator are present in the same composition. Thus, in some embodiments, the method includes isolating monocytes from peripheral blood mononuclear cells (PBMCs) in a biological sample, differentiating the monocytes in vitro to produce macrophages, and contacting the macrophages with a composition comprising a SIRPα inhibitor and a macrophage activator to produce activated SIRPα. low and generating a population of macrophages. In some embodiments, the activated SIRPα low The macrophages exhibit reduced cell surface expression of SIRPα compared to control untreated macrophages. In some embodiments, activated SIRPα lowThe macrophages exhibit increased phagocytosis, pro-inflammatory activity, antigen presentation, or any combination thereof, compared to untreated macrophages.

[0044] In some embodiments, SIRPα low The macrophages have about a 90% decrease in SIRPα cell surface expression or activity compared to untreated macrophages, including about an 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% decrease compared to untreated macrophages. In some embodiments, activated SIRPα low Expression of SIRPα in macrophages is lower than expression of SIRPα in control untreated macrophages. In some embodiments, expression is cell surface expression of SIRPα. In some embodiments, activated SIRPα low Expression of SIRPα in the macrophages is at least about 50% (e.g., about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, or 100% (including all values ​​and subranges therebetween)) lower than expression of SIRPα in control untreated macrophages.

[0045] In some embodiments, activated SIRPα low The activity of SIRPα in macrophages is lower than the activity of SIRPα in control untreated macrophages. low The activity of SIRPα in the macrophages is at least about 50% (e.g., about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, or 100% (including all values ​​and subranges therebetween)) less than the activity of SIRPα in control untreated macrophages.

[0046] In some embodiments, activated SIRPα lowThe phagocytic activity, pro-inflammatory activity, and / or antigen-presenting activity of the macrophages are higher than the phagocytic activity, pro-inflammatory activity, and / or antigen-presenting activity of control untreated macrophages, respectively. low The phagocytic activity, proinflammatory activity, and / or antigen presenting activity of the macrophages is at least about 2% (e.g., about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 7 ... 5%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, about 1000%, about 10,000%, about 100,000%, about 1,000,000%, or about 10,000,000% (including all values ​​and sub-ranges therebetween) higher.

[0047] In some embodiments, one or more activated SIRPα low The phagocytic activity of the macrophages is greater than the phagocytic activity of control untreated macrophages. low The phagocytic activity of the macrophages is at least about 2% (e.g., about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%, about 10,000%, about 100,000%, about 1,000,000%, or about 10,000,000% (including all values ​​and subranges therebetween)) greater than the phagocytic activity of control untreated macrophages.

[0048] In some embodiments, one or more activated SIRPα lowThe proinflammatory activity of the macrophages is greater than the proinflammatory activity of control untreated macrophages. In some embodiments, one or more activated SIRPα low The proinflammatory activity of the macrophages is at least about 2% (e.g., about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%, about 10,000%, about 100,000%, about 1,000,000%, or about 10,000,000% (including all values ​​and subranges therebetween)) greater than the proinflammatory activity of control untreated macrophages.

[0049] In some embodiments, one or more activated SIRPα low The antigen-presenting activity of the macrophages is higher than that of control untreated macrophages. low The antigen-presenting activity of the macrophages is at least about 2% (e.g., about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%, about 10,000%, about 100,000%, about 1,000,000%, or about 10,000,000% (including all values ​​and subranges therebetween)) greater than the antigen-presenting activity of untreated control macrophages.

[0050] Various embodiments of the disclosed methods are illustrated in Figures 13A-13R. For example, in some embodiments, a therapeutically effective amount of activated SIRPα low Macrophages are administered directly to the tumor, followed by tumor-directed in situ radiation therapy (FIG. 13A). In some embodiments, a therapeutically effective amount of activated SIRPα lowThe macrophages are administered directly to the tumor, and are preceded by tumor-directed in situ radiation therapy (FIG. 13B). In some embodiments, a therapeutically effective amount of activated SIRPα low Macrophages are administered directly to the tumor without tumor-directed in situ radiotherapy (FIG. 13C).

[0051] In some embodiments, a therapeutically effective amount of activated SIRPα low Macrophages are administered directly to the tumor, followed by tumor-directed in situ radiotherapy and intravenous (IV) administration of ICB (FIG. 13D). In some embodiments, a therapeutically effective amount of activated SIRPα low Macrophages are administered directly to the tumor, preceded by tumor-directed in situ radiotherapy, followed by IV administration of ICB (FIG. 13E). low Macrophages are administered directly into the tumor, followed by IV administration of ICB without tumor-directed in situ radiotherapy (FIG. 13F).

[0052] In some embodiments, a therapeutically effective amount of SIRPα that is not activated in in vitro culture. low Macrophages are administered IV, followed by tumor-directed in situ radiation therapy (FIG. 13G). In some embodiments, a therapeutically effective amount of SIRPα that is not activated in in vitro culture. low Macrophages are administered IV, followed by tumor-directed in situ radiotherapy and IV administration of ICB (FIG. 13H).

[0053] In some embodiments, a therapeutically effective amount of activated SIRPα low Macrophages are administered IV, followed by tumor-directed in situ radiation therapy (FIG. 13I). In some embodiments, a therapeutically effective amount of activated SIRPα low Macrophages are administered IV, followed by tumor-directed in situ radiotherapy and IV administration of ICB (Figure 13J).

[0054] As shown in Figures 13K to 13R, activated SIRPα low Macrophages can also be co-cultured with cells from tumor biopsies to generate tumor-specific peripheral blood T (PBT) cells (Figures 13K-N) or tumor-infiltrating T lymphocyte (TIL) cells (Figures 13O-R).

[0055] In some embodiments, as an alternative to collecting a biological sample comprising PBMCs from a subject, the method includes collecting a biological sample comprising blood from a subject, or collecting a biological sample comprising peripheral blood leukocytes from a subject, or collecting a biological sample comprising an apheresis product from a subject, or collecting a biological sample comprising bone marrow from a subject, or collecting a biological sample comprising resected healthy tissue from a subject. Such biological samples may be used for isolation of monocytes, isolation of macrophages, isolation of T cells, or isolation of other cells.

[0056] Methods for isolating monocytes from a biological sample are well known in the art. Methods for isolating macrophages from a biological sample are well known in the art. Methods for culturing monocytes in vitro to produce macrophages are well known in the art.

[0057] Disclosed herein are agents that inhibit the activity of SIRPα or disrupt its interaction with CD47. Without being bound by theory, it is believed that inhibiting the activity or expression of SIRPα or disrupting its interaction with CD47 enhances the phagocytic activity of SIRPα-expressing cells and enhances the production of T cell-mediated adaptive immune responses.

[0058] The agent (SIRPα inhibitor) can be a chemical compound or an antibody (e.g., an anti-SIRPα monoclonal antibody) or other protein that inhibits the activity of SIRPα or interferes with its interaction with CD47. For example, the antibody or other protein can specifically bind to a target, such as SIRPα or a downstream component in a SIRPα-mediated pathway, without activating the bound target. The agent can be, for example, a soluble CD47 extracellular domain or a fragment thereof engineered by molecular techniques to be identical or different to the naturally occurring CD47 extracellular domain. Such an agent can bind to SIRPα but cannot activate SIRPα, thereby interfering with the interaction of SIRPα with CD47. The agent can be, for example, a soluble SIRPα extracellular domain or a fragment thereof engineered by molecular techniques to be identical or different to the naturally occurring SIRPα extracellular domain. Such an agent can bind to CD47 but cannot activate CD47, thereby interfering with the interaction of SIRPα with CD47. The agent may be a chemical compound or an antibody or other protein that causes a decrease in the amount of SIRPα present on the surface of the cell. The agent may be a chemical compound or an antibody or other protein that causes a decrease in the amount of SIRPα present on the surface of the cell by promoting endocytosis of surface-expressed SIRPα. The agent may be a chemical compound or an antibody or other protein that causes a decrease in the amount of SIRPα present on the surface of the cell by decreasing the expression level of the gene encoding SIRPα. The agent may be a cytokine, a growth factor, or a chemokine.

[0059] SIRPα can also be inhibited by inhibiting the SIRPα signaling pathway. Some tyrosine kinase inhibitors (e.g., those that target Src family tyrosine kinases and / or Btk) inhibit phosphorylation of the SIRPα cytoplasmic domain and recruitment of SHP-1 / 2. Therefore, these agents are useful in the present method. SIRPα can also be inhibited by inhibiting the SIRPα signaling pathway or its components further downstream than SHP-1 / 2.

[0060] Non-limiting examples of SHP-1 inhibitors that can be used in the disclosed methods include TPI-1 (0.1-5 mg / kg, 2-(2,5-dichlorophenyl)-1,4-benzoquinone), TPI-1a1 (0.1-5 mg / kg, 2-(2,5-dichlorophenyl)-2,4-benzoquinone), TPI-1a2 (0.1-5 mg / kg, 2-(3-chlorophenyl)-1,4-benzoquinone), TPI-1a3 (0.1-5 mg / kg, 2-phenylnaphthoquinone), TPI-1a4 (0.1-5 mg / kg, 2-(4-ethoxyphenyl)-1,4-benzoquinone), TPI-1a5 (0.1-5 mg / kg, 2-(4-methoxyphenyl)-1,4-benzoquinone), TPI-1a6 (0.1-5 mg / kg, 2-(4-methoxyphenyl)-1,4-benzoquinone), TPI-1a7 (0.1-5 mg / kg, 2-(4-methoxyphenyl)-1,4-benzoquinone), TPI-1a8 (0.1-5 mg / kg, 2-(4-methoxyphenyl)-1,4-benzoquinone), TPI-1a9 (0.1-5 mg / kg, 2-(4-methoxyphenyl)-1,4-benzoquinone), TPI-1a10 (0.1-5 mg / kg, 2-(4-methoxyphenyl)-1,4-benzoquinone), TPI-1a11 (0.1-5 mg / kg, 2-(4-methoxyphenyl)-1,4-benzoquinone), TPI-1a12 (0.1-5 mg / kg, 2-(4-methoxyphenyl)-1,4-benzoquinone), TPI-1a13 (0.1-5 mg / kg, 2-(4-methoxyphenyl)-1,4-benzoquinone), TPI-1a14 (0.1- (0.5-10 mg / kg, sodium stibogluconate), PTP inhibitor I (0.5-10 mg / kg, 2-bromo-1-(4-hydroxyphenyl)-ethanone), PTP inhibitor II (0.5-10 mg / kg, 2-bromo-1-(4-methoxyphenyl)-ethanone), PTP inhibitor III (0.5-10 mg / kg, 2-[4-(2-bromoacetyl)phenoxy]-acetic acid), PTP inhibitor IV (0.5-10 mg / kg, N,N'-[1,4-phenylenebis[(1-methylethylidene)-4,1-phenylene]]bis[1,1,1-trifluoro-methanesulfonamide), NSC 23922 (0.5-10 mg / kg, 3-aminocholestane), and NSC 87877 (0.5-10 mg / kg, 8-hydroxy-7-[2-(6-sulfo-2-naphthalenyl)diazenyl]-5-quinolinesulfonic acid).

[0061] In some embodiments, the SIRPα inhibitor suppresses the expression of SIRPα, inhibits the activity of SIRPα, reduces the abundance of SIRPα on the surface of cells, disrupts the interaction between SIRPα and CD47, activates phagocytosis, or any combination thereof. Methods for knocking down the expression of SIRPα in macrophages include in vitro treatment of macrophages with a cytokine or a cocktail of cytokines, with a chemokine or a cocktail of chemokines, with a growth factor or a cocktail of growth factors, with a cocktail of cytokines, chemokines, and / or growth factors, with an immune stimulatory molecule, with a cell signaling protein or other cell signaling molecule, or with any combination of the above. Knocking down the expression of SIRPα in macrophages can also be achieved by stimulating cell surface receptors or other cell receptors. Such stimulation can be by cross-linking the receptor. Receptor cross-linking can be mediated by an antibody or a cocktail of antibodies. Stimulation of cell receptors can also occur by treatment with small molecules or drugs.

[0062] Non-limiting examples of SIRPα inhibitors include IFNγ, IL-6, IL-1 family cytokines (e.g., IL-1α, IL-1β, IL-18, IL-33, IL-36α, IL-36β, IL-36γ, IL-36Ra, IL-37, IL-38), IL-12, IFNα, IFNβ, tumor necrosis factor alpha (TNFα), Toll-like receptor (TLR) agonists, or other molecules containing pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) (e.g., LPS, CpG, Poly I:C, LTA, PGN, flagellin, HMGB1, etc.), Pam3CSK4, zymosan, cytokines, chemokines, growth factors, and glucocorticoids such as methylprednisolone and dexamethasone. SIRPα inhibition can also be achieved by stimulating cell surface receptors or other cellular receptors. Such stimulation may be by cross-linking the receptors. Receptor cross-linking may be mediated by an antibody or a cocktail of antibodies. In some embodiments, the SIRPα inhibitor may be a combination of any of the SIRPα inhibitors listed.

[0063] In some embodiments, the SIRPα inhibitor is a mixture of 100 ng / mL IFNγ, 100 ng / mL IL-6, and 1 μg / mL CpG. In other embodiments, the SIRPα inhibitor is a mixture of IFNγ, IL-6, and CpG, where the concentration of IFNγ is 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, or 1000 ng / mL and the concentration of IL-6 is 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, or 1000 ng / mL. and the concentration of CpG is 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, or 500 nm / mL, or 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 μg / mL.

[0064] In some embodiments, macrophage activators increase phagocytosis by macrophages, increase antigen processing and presentation activity and function of macrophages, increase the immune stimulatory capacity of macrophages, improve the T cell stimulatory function of macrophages, promote a pro-inflammatory (so-called M1) phenotype of macrophages, enable macrophages to alter the TME to promote an immune response against cancer cells, or any combination thereof.

[0065] Non-limiting examples of macrophage activators include IL-1 family cytokines (e.g., IL-1α, IL-1β, IL-18, IL-33, IL-36α, IL-36β, IL-36γ, IL-36Ra, IL-37, IL-38, or others that may be identified in the future), IL-12, IFNα, IFNβ, tumor necrosis factor alpha (TNFα), Toll-like receptor (TLR) agonists (e.g., LPS, CpG, Poly I:C, LTA, PGN, flagellin, Pam3CSK4, zymosan, HMGB1, etc.) or other molecules containing pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs), cytokines, chemokines, growth factors, or glucocorticoids such as methylprednisolone and dexamethasone. Macrophage activation can also be achieved by stimulating cell surface receptors or other cellular receptors. Such stimulation may be by cross-linking the receptor. Receptor cross-linking may be mediated by an antibody or a cocktail of antibodies. Stimulation of cell receptors may also occur by treatment with small molecules or drugs, such as the PKC activator phorbol 12-myristate 13-acetate (PMA) and protein tyrosine phosphatase inhibitors such as pervanadate. Macrophages may also be activated by PMA, an agent that is a PKC stimulator and thus activates macrophages by stimulating the PKC-Syk pathway. Biologically active variants of these activators may be used as well. The macrophage activator may also be a ligand for a TLR (e.g., lipopolysaccharide (LPS), polyinosinic:polycytidylic acid (poly I:C), lipoteichoic acid (LTA), flagellin, GARDIQUIMOD™ (an imidazoquinoline compound currently manufactured by InvivoGen, CAS number 1020412-43-4), IMIQUIMOD™ (1-isobutyl-1H-imidazo[4,5-c]quinolin-4-amine, CAS number 99011-02-6), peptidoglycan (PDG), or CpG oligonucleotides).In some embodiments, the CpG oligonucleotide is a class A oligonucleotide (ODN), a class B ODN, a class C ODN, or any combination thereof. In some embodiments, the CpG oligonucleotide is a class B ODN. In some embodiments, the CpG oligonucleotide is ODN1826. In some embodiments, the CpG oligonucleotide is ODN BW006 (also known as ODN 684). Because both macrophages and some cancer cells (e.g., breast cancer cells) express TLRs, ligands for TLRs or agents that activate TLRs can be used as either SIRPα inhibitors or macrophage activators in compositions and methods for activating macrophages and subsequently treating cancer. In some embodiments, agents that activate macrophages, possibly by disrupting the interaction between SIRPα and CD47, can be surfactant proteins (e.g., surfactant proteins A, B, or D). Macrophages can also be activated by ionizing radiation.

[0066] In some embodiments, the macrophage activator is phorbol 12-myristate 13-acetate (PMA) at 20 nM, in other embodiments, the macrophage activator is PMA at a concentration of 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 25, 30, 40, 50, 60, 70, 80, 90, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, or 1000 nM.

[0067] The agent that activates macrophage phagocytosis of cancer cells can be a small molecule, an amino acid, a peptide, a nucleic acid (e.g., RNA or DNA), a protein (e.g., an antibody), or one or more combinations thereof. The agent can be naturally occurring, derived from a naturally occurring agent, or synthetic. In some embodiments, the agent activates the PKC-Syk pathway in the subject. For example, the agent can be a cytokine (e.g., IL-17, IL-1β, IFNγ, IL-6, or a biologically active variant thereof). The agent can also be lipopolysaccharide (LPS) or a biologically active variant thereof. In some embodiments, the agent can be IL-1, TNFα, PMA (phorbol 12-myristate 13-acetate), or a biologically active variant thereof. In certain embodiments, the disclosed method can include identifying an agent that activates macrophage phagocytosis of cancer cells.

[0068] When the agent is a nucleic acid, it may be a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA), or a DNA or RNA sequence containing one or more up to all artificial nucleic acid analogs. An agent containing a DNA sequence may contain multiple nucleobases including cytosine, guanine, adenine, and thymine, as well as other natural or synthetic nucleobases, or combinations thereof. Nucleobases may also include derivatives of C, G, A, or T, or synthetic nucleobases. In certain embodiments, the DNA sequence may be in one or more conformations including A-DNA, B-DNA, and Z-DNA. The DNA sequence may also be linear or branched. In certain embodiments, the DNA sequence may be single-stranded, double-stranded, or multi-stranded.

[0069] In some embodiments, the RNA can be messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), transfer messenger RNA (tmRNA), microRNA (miRNA), small interfering RNA (siRNA), CRISPR RNA, antisense RNA, pre-mRNA, or small nuclear RNA (snRNA). The RNA can also include multiple nucleobases including adenine, cytosine, guanine, or uracil, other natural nucleobases, or combinations thereof. In certain embodiments, the nucleobases can include derivatives of A, C, G, U, or synthetic nucleobases. The RNA can also be linear or branched. In certain embodiments, the RNA can be single-stranded, double-stranded, or multi-stranded.

[0070] In some embodiments, artificial nucleic acid analogs can include backbone analogs (e.g., hydrolysis-resistant RNA analogs, precursors of the RNA world (e.g., TNA, GNA, PNA)) or base analogs (e.g., nucleobase structural analogs, fluorophores, fluorescent base analogs, natural non-standard bases, base pairs, metallobase pairs).

[0071] In some embodiments, the protein may be an antibody, including but not limited to an IgG class antibody, a monoclonal antibody, an antibody fragment, a single chain antibody, or a single chain variable fragment. The antibody may be naturally occurring or non-naturally occurring.

[0072] In some embodiments, CD47, SIRPα, or the interaction between them can inhibit or inactivate one or more receptors. Thus, by inhibiting the expression or activity of SIRPα or suppressing the interaction between CD47 and SIRPα, the agent can activate one or more receptors. In certain embodiments, one or more receptors may also be activated by a macrophage activator. Thus, by inhibiting the expression or activity of SIRPα or suppressing the interaction between CD47 and SIRPα, the agent can enhance the activity of one or more receptors.

[0073] The present disclosure provides a method for administering an activated SIRPα antibody to a subject, comprising: (a) providing a macrophage; and (b) contacting the macrophage with a composition. low A method of producing macrophages is provided, the composition comprising a SIRPα inhibitor and an agent that enhances the phagocytic activity of macrophages. In some embodiments, the SIRPα inhibitor is an agent that suppresses expression of SIRPα. In some embodiments, step (a) comprises one or more of the following steps: (i) collecting a biological sample comprising peripheral blood mononuclear cells (PBMCs) from a subject; (ii) isolating monocytes from the PBMCs; and (iii) culturing the monocytes in vitro to produce macrophages.

[0074] In some embodiments, the method includes (i) collecting a biological sample comprising peripheral blood mononuclear cells (PBMCs) from a subject; (ii) isolating monocytes from the PBMCs; (iii) culturing the monocytes in vitro to produce macrophages; and (iv) contacting the macrophages with a composition comprising an agent that inhibits expression of SIRP-alpha and an agent that enhances phagocytic activity of the macrophages.

[0075] The present disclosure also relates to an activated SIRPα produced by any one of the methods disclosed herein. low Provides macrophages.

[0076] The present disclosure further provides compositions comprising an agent that inhibits expression of SIRP-alpha, an agent that enhances phagocytic activity of macrophages, or a combination thereof, hi some embodiments, the compositions comprise an agent that inhibits expression of SIRP-alpha and an agent that enhances phagocytic activity of macrophages.

[0077] In some embodiments, the agent that inhibits expression of SIRP-alpha is a cytokine. In some embodiments, the cytokine is a proinflammatory cytokine, such as an interferon. In some embodiments, the proinflammatory cytokine is IFNγ, IFNα, IL-1, IL-6, or any combination thereof.

[0078] In some embodiments, the agent that enhances the phagocytic activity of macrophages is a ligand for a toll-like receptor, an interleukin, tumor necrosis factor alpha (TNFα), or phorbol 12-myristate 13-acetate (PMA). In some embodiments, the ligand for a toll-like receptor is lipopolysaccharide (LPS), polyinosinic:polycytidylic acid (poly I:C), lipoteichoic acid (LTA), flagellin, imidazoquinoline, l-isobutyl-lH-imidazo[4,5-c]quinolin-4-amine, or a CpG oligonucleotide. In some embodiments, the ligand for a toll-like receptor is polyinosinic:polycytidylic acid (poly I:C), a CpG oligonucleotide, or a combination thereof. In some embodiments, the interleukin is IL-1, IL-1α, IL-1β, IL-6, or IL-17.

[0079] In some embodiments, the composition comprises IFNγ, IFNα, CpG, poly I:C, or any combination thereof. In some embodiments, the composition comprises IFNγ, IFNα, CpG oligonucleotides (e.g., ODN1826 and ODN BW006), and poly I:C. In some embodiments, the concentration of IFNγ in the composition is in the range of about 40 ng / ml to about 200 ng / ml, e.g., about 50 ng / ml, about 60 ng / ml, about 70 ng / ml, about 80 ng / ml, about 90 ng / ml, about 100 ng / ml, about 110 ng / ml, about 120 ng / ml, about 130 ng / ml, about 140 ng / ml, about 150 ng / ml, about 160 ng / ml, about 170 ng / ml, about 180 ng / ml, about 190 ng / ml, or about 200 ng / ml (including all values ​​and subranges therebetween).

[0080] In some embodiments, the concentration of IFNα in the composition is in the range of about 40 ng / ml to about 200 ng / ml, e.g., about 50 ng / ml, about 60 ng / ml, about 70 ng / ml, about 80 ng / ml, about 90 ng / ml, about 100 ng / ml, about 110 ng / ml, about 120 ng / ml, about 130 ng / ml, about 140 ng / ml, about 150 ng / ml, about 160 ng / ml, about 170 ng / ml, about 180 ng / ml, about 190 ng / ml, or about 200 ng / ml (including all values ​​and subranges therebetween).

[0081] In some embodiments, the concentration of the CpG oligodeoxynucleotide in the composition is in the range of about 1 μg / ml and about 10 μg / ml, e.g., about 1.5 μg / ml, about 2 μg / ml, about 2.5 μg / ml, about 3 μg / ml, about 3.5 μg / ml, about 4 μg / ml, about 4.5 μg / ml, about 5 μg / ml, about 5.5 μg / ml, about 6 μg / ml, about 6.5 μg / ml, about 7 μg / ml, about 7.5 μg / ml, about 8 μg / ml, about 8.5 μg / ml, about 9 μg / ml, about 9.5 μg / ml, or about 10 μg / ml (including all values ​​and sub-ranges therebetween).

[0082] In some embodiments, the concentration of Poly I:C in the composition is in the range of about 1 μg / ml and about 10 μg / ml, e.g., about 1.5 μg / ml, about 2 μg / ml, about 2.5 μg / ml, about 3 μg / ml, about 3.5 μg / ml, about 4 μg / ml, about 4.5 μg / ml, about 5 μg / ml, about 5.5 μg / ml, about 6 μg / ml, about 6.5 μg / ml, about 7 μg / ml, about 7.5 μg / ml, about 8 μg / ml, about 8.5 μg / ml, about 9 μg / ml, about 9.5 μg / ml, or about 10 μg / ml (including all values ​​and sub-ranges therebetween).

[0083] In some embodiments, the composition comprises about 40 ng / ml to about 200 ng / ml IFNγ, about 40 ng / ml to about 200 ng / ml IFNα, about 1 μg / ml and about 10 μg / ml CpG oligodeoxynucleotide, and about 1 μg / ml and about 10 μg / ml Poly I:C. In some embodiments, the composition comprises about 100 ng / ml IFNγ, about 100 ng / ml IFNα, about 2 μg / ml CpG oligodeoxynucleotide, and about 2 μg / ml Poly I:C.

[0084] In some embodiments, a therapeutically effective amount of macrophages is 50 million macrophages, 150 million macrophages, or 450 million macrophages. In some embodiments, a therapeutically effective amount of macrophages is in the range of about 1 million to about 1 billion macrophages (e.g., about 1 million, about 5 million, about 10 million, about 20 million, about 30 million, about 40 million, about 60 million, about 70 million, about 80 million, about 90 million, about 100 million, about 125 million, about 175 million, about 200 million, about 250 million, about 300 million, about 350 million, about 400 million, about 500 million, about 600 million, about 750 million, or about 1 billion (including all values ​​and sub-ranges therebetween)). In some embodiments, the therapeutically effective amount of macrophages is a function of tumor mass size. In some embodiments, the therapeutically effective amount of macrophages is a function of patient weight. In some embodiments, the therapeutically effective amount of macrophages is a function of patient age. In some embodiments, the therapeutically effective amount of macrophages is a function of a combination of tumor mass size, patient weight, and patient age.

[0085] In some embodiments, the method for treating cancer in a subject in need thereof includes administering a therapeutically effective amount of activated SIRPα in combination with a second-line therapy (or second-line therapeutic agent) that targets the cancer. low The method includes administering macrophages to a subject. In some embodiments, the cancer-targeting secondary therapeutic agent promotes inflammation. As used herein, the term "concomitantly" administered is understood to mean that two (or more) different therapies are delivered to a subject during the course of the subject's affliction with a disorder (such as cancer) such that the effects of the therapies on the patient overlap at some point. In certain embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so there is an overlap in terms of administration. This is sometimes referred to herein as "simultaneous" or "parallel" delivery. In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins, which may be referred to as "sequential" delivery.

[0086] In some embodiments, the treatment is more effective due to the combined administration.For example, the second treatment is more effective, for example, the second treatment is administered less than the second treatment is administered in the absence of the first treatment, or the second treatment reduces symptoms to a greater extent than the first treatment, or a similar situation is observed.The effect of the two treatments may be partially additive, completely additive, or more than additive (synergistic).

[0087] In some embodiments, the inflammation-promoting secondary therapeutic agent is one or more damage-associated molecular patterns (DAMPs). Non-limiting examples of DAMPs include high mobility group box 1 protein (HMGB1), heat shock proteins (HSPs), SNAP-associated proteins (SNAPINs), versican, biglycan, decorin, eosinophil-derived neurotoxin, surfactant protein A / D, beta-defensin 3, histones, serum amyloid A (SAA), beta amyloid (Aβ), beta 2-glycoprotein I, mRNA, tenascin-C, S100 proteins, high mobility group box 1 protein (HMGN1), biglycan, decorin, heparin sulfate, hyaluronic acid, fibrinogen, fibronectin, beta-diff. Ensin 2, surfactant protein A / D, lactoferrin, neutrophil elastase, peroxiredoxin, histones, serum amyloid A (SAA), ox-LDL, IgG-ribonucleoprotein complex, microRNA, mtDNA, F-actin, Sin3A-related protein 130, β-glucosylceramide, N-glycans, monosodium urate (MSU), glucose, cholesterol crystals, ATP, oxidized 1-palmitoyl-2-arachidonyl sn-glycero-3-phosphocholine (ox-PAPC), RNA transcribed from Alu elements (Alu-RNA), endogenous 5'pppRNA, unedited long self-dsRNA, endogenous retroviral RNA, cytoplasmic DNA, damaged nuclear DNA, advanced glycation end products (AGE), DNA, HSP70, peptidoglycan recognition protein 1 (PGLYRP1), actin, phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), cardiolipin, sulfatides, sphingomyelin, apolipoprotein A1 (APOA1), apolipoprotein DAMPs include protein A2 (APOA2), apolipoprotein B (APOB), apolipoprotein E (APOE), apolipoprotein J (APOJ), low density lipoprotein (LDL), high density lipoprotein (HDL), very low density lipoprotein (VLDL), Lp(a), HSP60, N-formyl peptides, cathepsin G, FAM19A4, annexin 1, Aβ42, serum amyloid A (SAA), low density lipoprotein (LL-37) and other peptides, ATP, UTP, UDP, ADP, cyclic-GMP-AMP (cGAMP), calcium ions, and ROS. In some embodiments, the DAMPs include any DAMPs described in Gong et al., Nature Reviews Immunology, Volume 20, February 2020, which is incorporated by reference in its entirety for all purposes.

[0088] In some embodiments, the inflammation-promoting secondary therapeutic agent is one or more ligands or other activators of DAMP-sensing receptors. Non-limiting examples of DAMP-sensing receptors include Toll-like receptors (TLRs) (e.g., TLR2, TLR3, TLR4, TLR7, TLR9), C-type lectin receptors (CLRs) (e.g., DNGR1, MINCLE, Dectin-1), NOD-like receptors (NLRs) (e.g., NLR-family pyrin domain-containing 3), RIG-1-like receptors (RLRs) (e.g., RIG-1, MDA5), cytoplasmic DNA sensors (CDSs) (e.g., These include cyclic-GMP-AMP synthase (cGAS), AIM2), RAGE receptors, TREMs (e.g., TREM1, TREM2), GPCRs (e.g., FPR1, FPR2, P2Y2R, P2Y6R, P2Y12R, CaSR, GPRC6A), stimulator of interferon genes (STING or transmembrane protein 173 (TMEM173)), and ion channels (e.g., TRPM2, other TRPs, P2X7R).

[0089] The present disclosure further provides: (a) an activated SIRPα as disclosed herein; low Compositions are provided that include a macrophage and (b) any one of the DAMPs disclosed herein, a ligand of any one of the DAMP-sensing receptors disclosed herein, or a combination thereof.

[0090] In some embodiments, the second therapeutic agent targets and / or inhibits the function of one or more T cell inhibitory receptors (IR). In some embodiments, the second therapeutic agent comprises one or more immunotherapeutic agents that target one or more T cell inhibitory receptors (IR). Further details regarding T cell inhibitory receptors (IR) can be found in Chauvin JM, Zarour HM. TIGIT in cancer immunotherapy. Journal for ImmunoTherapy of Cancer 2020;8:e000957, which is incorporated by reference in its entirety for all purposes. In some embodiments, the second therapeutic agent interferes with or inhibits the interaction between a T cell inhibitory receptor (IR) and its ligand. In some embodiments, the second therapeutic agent can bind to a T cell inhibitory receptor (IR). In some embodiments, the second therapeutic agent can bind to a ligand of a T cell inhibitory receptor (IR). In some embodiments, the second therapeutic agent that targets one or more T cell inhibitory receptors (IR) is a small molecule.

[0091] In some embodiments, the second therapeutic agent is an antibody, or an antigen-binding fragment thereof (e.g., a monoclonal antibody) capable of binding to a T cell inhibitory receptor (IR). Non-limiting examples of T cell inhibitory receptors (IR) are programmed death receptor 1 (PD-1), programmed death-ligand 1 (PD-L1), anti-cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), CD96 / TACTILE, CD112R / PVRIG, DNAM-1 / CD226, T cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT), T cell immunoglobulin and mucin domain-containing molecule-3 (TIM-3), and lymphocyte activation gene 3 (LAG-3).

[0092] In some embodiments, the second therapeutic agent is an anti-PD-1 antibody, an anti-CTLA-4 antibody, an anti-TIGIT antibody, an anti-CD96 antibody, an anti-CD112R antibody, an anti-DNAM-1 antibody, an anti-TIM-3 antibody, an anti-PD-L1 antibody, an anti-LAG3 antibody, an anti-OX40 antibody, and an anti-OX40L antibody, an antibody targeting a member of the tumor necrosis factor (TNF) receptor family, or any combination thereof. In some embodiments, the anti-TIGIT antibody can bind to the immunoglobulin tail tyrosine (ITT)-like motif of TIGIT. In some embodiments, the anti-TIGIT antibody can bind to the immunoreceptor tyrosine-based inhibitory motif (ITIM) of TIGIT.

[0093] In some embodiments, the secondary therapeutic agent is an antibody, or an antigen-binding fragment thereof (e.g., a monoclonal antibody), capable of binding to a ligand of a T cell inhibitory receptor (IR). In some embodiments, the secondary therapeutic agent is an antibody, or an antigen-binding fragment thereof (e.g., a monoclonal antibody), capable of binding to a ligand of TIGIT, such as CD155 (PVR / NECL-5), CD112 (PVRL2 / Nectin-2), or the Fap2 protein from the anaerobic gram-symbiotic bacterium Fusobacterium nucleatum. Thus, in some embodiments, the secondary therapeutic agent is an anti-CD155 antibody, an anti-CD112 antibody, or an anti-Fap2 antibody.

[0094] In some embodiments, the second line therapy comprises one or more immunotherapeutic agents targeting one or more different T cell inhibitory receptors (IR) and / or one or more ligands of a T cell inhibitory receptor (IR). Thus, in some embodiments, the second line therapy comprises an anti-CD155 antibody, an anti-CD112 antibody, an anti-Fap2 antibody, an anti-PD-1 antibody, an anti-CTLA-4 antibody, an anti-TIGIT antibody, an anti-CD96 antibody, an anti-CD112R antibody, an anti-DNAM-1 antibody, an anti-TIM-3 antibody, an anti-PD-L1 antibody, an anti-LAG3 antibody, or any combination thereof.

[0095] In some embodiments, the anti-PD-1 antibody is pembrolizumab, nivolumab, or cemiplimab-rwlc. In some embodiments, the anti-PD-L1 antibody is atezolizumab, avelumab, or durvalumab. In some embodiments, the anti-CTLA-4 antibody ipilimumab. In some embodiments, the anti-TIGIT antibody is tiragolumab, BMS-986207 (Bristol Myers Squibb), BGB-A1217 (BeiGene), OP-313M32 (Oncomed), AB154 (Arcus Biosciences), ASP8374 (Astella Pharma Global Development), MK-7684 (Merck Sharp & Dohme), or any combination thereof. In some embodiments, the anti-CD112R antibody is COM701 (Compugen). In some embodiments, the second-line therapeutic agent that targets DNAM-1 is LY3435151 (Eli Lilly and Company). In some embodiments, the second-line therapeutic agent that targets OX40 is GSK998 (GSK).

[0096] In some embodiments, the secondary therapeutic agent is an antibody that can act as an agonist. In some embodiments, the secondary therapeutic agent is an antibody that can act as an antagonist.

[0097] The present disclosure further provides: (a) an activated SIRPα as disclosed herein; low Compositions are provided that include macrophages and (b) any one or more of the immunotherapeutic agents that target one or more T cell inhibitory receptors (IR) and / or one or more ligands of a T cell inhibitory receptor (IR) disclosed herein. In some embodiments, the composition comprises (a) an activated SIRPα as disclosed herein. lowmacrophages; and (b) any one or more of an anti-CD155 antibody, an anti-CD112 antibody, an anti-Fap2 antibody, an anti-PD-1 antibody, an anti-CTLA-4 antibody, an anti-TIGIT antibody, an anti-CD96 antibody, an anti-CD112R antibody, an anti-DNAM-1 antibody, an anti-TIM-3 antibody, a PD-L1 antibody, an anti-LAG3 antibody, an anti-OX40 antibody, an anti-OX40L antibody, or any combination thereof.

[0098] In some embodiments, the secondary therapy is radiation. In some embodiments, the method further comprises treating the subject with an effective amount of tumor-directed in situ radiation therapy. For example, the tumor-directed radiation may be administered in an amount of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, or 25 Gray. The tumor-directed radiation may be administered in a single dose or in multiple doses. As disclosed herein, irradiation is administered immediately before, immediately after, or simultaneously with administration of macrophages. For example, irradiation can be administered 0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours prior to or after administration of the macrophages. As another example, irradiation can be administered 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days prior to or after administration of the macrophages.

[0099] In some embodiments, the radiation therapy is any form of energy or particle radiation commonly used in cancer treatment. In some embodiments, the radiation therapy is ionizing radiation. In some embodiments, the radiation is non-ionizing radiation. Non-ionizing radiation includes visible light, heat, radar, microwaves, and radio waves. Ionizing radiation includes X-rays, which are more energetic than non-ionizing radiation. Particle radiation includes alpha particles, beta particles, gamma rays, photons, carbon ions, heavy ions, muons, protons, electrons, and neutrons.

[0100] In some embodiments, the secondary therapy is an immune checkpoint inhibitor. In some embodiments, the method further comprises treating the subject with an immune checkpoint inhibitor, also known as immune checkpoint blockade. Treating the subject with an immune checkpoint inhibitor is also known as "immune checkpoint inhibitor therapy" or "immune checkpoint blockade therapy". In any of the methods, the macrophages and the immune checkpoint inhibitor can be administered simultaneously by the same or different routes of administration, or can be administered sequentially by the same or different routes of administration. For example, the immune checkpoint inhibitor can be administered 0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours before or after administration of the macrophages. As other examples, the immune checkpoint inhibitor can be administered 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days prior to or after administration of the macrophages.

[0101] When the agents are administered simultaneously by the same route of administration, they may be contained within a single formulation. Examples of immune checkpoint inhibitors include monoclonal antibodies targeting PD-1 (e.g., KEYTRUDA® (pembrolizumab), OPDIVO® (nivolumab), or LIBTAYO® (cemiplimab-rwlc)), PD-L1 (e.g., TECENTRIQ® (atezolizumab), Bavencio® (avelumab), or IMFINZI® (durvalumab)), CTLA-4 (e.g., YERVOY® (ipilimumab)), or other immune checkpoint proteins that may be identified or approved for use in humans in the future.

[0102] In some embodiments, the second line therapy is a chemotherapeutic agent. In some embodiments, the method further comprises treating the subject with a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent increases tumor damage signals. Non-limiting examples of known anti-cancer agents include abemaciclib, abiraterone acetate, Abraxane (paclitaxel albumin stabilized nanoparticle formulation), ABVD, ABVE, ABVE-PC, AC, acalabrutinib, AC-T, Actemra (tocilizumab), Adcetris (brentuximab vedotin), ADE, Ado-trastuzumab emtansine, Adriamycin (doxol), and rituximab. Bicine hydrochloride), afatinib maleate, Afinitor (everolimus), Aquinzeo (netupitant and palonosetron hydrochloride), Aldara (imiquimod), aldesleukin, Alecensa (alectinib), alectinib, alemtuzumab, Alimta (pemetrexed sodium), Alicopa (copanlisib hydrochloride), Alkeran for injection (melphalan hydrochloride), Alkeran tablets (melphalan), Aloxi (palonosetron hydrochloride), alpelisib, Alumbrig (brigutinib), Amers (aminolevulinic acid hydrochloride), amifostine, aminolevulinic acid hydrochloride, anastrozole, apalutamide, aprepitant, Aranesp (darbepoetin alfa), Aredia (pamidronate disodium), Arimidex (anastrozole), Aromasin (exemestane) arsenic trioxide, Arsera (ofatumumab), asparaginase erwinia chrysanthemumii, Asparas (calaspargase pegol-mknl), atezolizumab, avapritinib, Avastin (bevacizumab), avelumab, axicabtagene silolelucel, axitinib, Ivakit (avapritinib), azacitidine, azedra (iobenguane I 131), Barbara (erdafitinib), Bavencio (avelumab), BEACOPP, belantamab mafodotin-blmf, Beleodac (belinostat), belinostat, bendamustine hydrochloride, Bendeca (bendamustine hydrochloride), BEP, Besponsa (inotuzumab ozogamicin), bevacizumab, bexarotene, bicalutamide, BiCNU (carmustine), binimetinib,Blenrep (belantamab mafodotin-blmf), bleomycin sulfate, blinatumomab, Blincyto (blinatumomab), bortezomib, Bosulif (bosutinib), bosutinib, Birafutovi (encorafenib), brentuximab vedotin, brexcabtagene autolucel, Breyanzi (lysocabtagene maralelucel), brigitinib, Brukinsa (zanubrutinib), Bumel, busulfan, Busulfex (busulfan), cabazitaxel, Cabryvi (caplacizumab-yhdp), Cabometyx (cabozantinib) nib-S-malate), cabozantinib-S-malate, CAF, calaspargase pegol-mknl, calkens (acalabrutinib), campath (alemtuzumab), camptosar (irinotecan hydrochloride), capecitabine, caplacizumab-yhdp, capmatinib hydrochloride, CAPOX, carlac (fluorouracil-topical), carboplatin, CARBOPLATIN-TAXOL, carfilzomib, carmustine, carmustine implant, casodex (bicalutamide), CEM, cemiplimab-rwlc, ceritinib, ceritinib Rubizin (daunorubicin hydrochloride), Cervarix (recombinant HPV bivalent vaccine), cetuximab, CEV, chlorambucil, CHLORAMBUCIL-PREDNISONE, CHOP, cisplatin, cladribine, clofarabine, chloral (clofarabine), CMF, cobimetinib fumarate, Cometrik (cabozantinib-S-malate), copanlisib hydrochloride, COPDAC, Copictra (duvelisib), COPP, COPP-ABV, Cosmegen (dactinomycin), Cotellic (cobimetinib fumarate), Cryptomegaly (cryptomegaly), Zotinib, CVP, cyclophosphamide, Cyramza (ramucirumab), cytarabine, dabrafenib mesylate, dacarbazine, Dacogen (decitabine), dacomitinib, dactinomycin, Danielza (naxitamab-gqgk), daratumumab, daratumumab and hyaluronidase-fihj, darbepoetin alfa, darolutamide, Darazalex (daratumumab), Darazalex Faspro (daratumumab and hyaluronidase-fihj), dasatinib, daunorubicin hydrochloride, daunorubicin hydrochloride and cytarabine liposomal,Daurismo (glasdegib maleate), decitabine, decitabine and cedazuridine, defibrotide sodium, Defitelio (defibrotide sodium), degarelix, denileukin diftitox, denosumab, dexamethasone, dexrazoxane hydrochloride, dinutuximab, docetaxel, Doxil (doxorubicin hydrochloride liposomal), doxorubicin hydrochloride, doxorubicin hydrochloride liposomal, durvalumab, duvelisib, Efudex (fluorouracil--topical), Eligard (leuprorelin acetate), ERITECH (rasbri) case), Elence (epirubicin hydrochloride), elotuzumab, Eloxatin (oxaliplatin), eltrombopag olamine, Erzonris (taglaxofusp-erzs), emapalumab-lzsg, Emend (aprepitant), Empliti (elotuzumab), enasidenib mesylate, encorafenib, enfortumab vedotin-ejfv, Enhartz (Fam-trastuzumab deruxtecan-nxki), entrectinib, enzalutamide, epirubicin hydrochloride, EPOCH, epoetin alfa, Epogen (epoetin alfa), Erbitux (cetuximab), erdafitinib, eribulin mesylate, Erivage (vismodegib), Erleada (apalutamide), erlotinib hydrochloride, Erwinase (asparaginase erwinia chrysanthemum), Ethiol (amifostine), Etopofos (etoposide phosphate), etoposide, etoposide phosphate, everolimus, Evista (raloxifene hydrochloride), Evomela (melphalan hydrochloride), exemestane, 5-FU (fluorouracil injection), 5-FU (fluorouracil - topical), Fam-trastuzumab derc Stecan-nxki, Fairston (toremifene), Faridak (panobinostat lactate), Faslodex (fulvestrant), FEC, fedratinib hydrochloride, Femara (letrozole), filgrastim, Filmagon (degarelix), fludarabine phosphate, Fluoroplex (fluorouracil-topical), fluorouracil injection, fluorouracil-topical, flutamide, Folfiri, FOLFIRI-BEVACIZUMAB, FOLFIRI-CETUXIMAB, FOLFIRINOX, FOLFOX,FOLOTIN (pralatrexate), fostamatinib disodium, FULFILLA (pegfilgrastim), FU-LV, fulvestrant, GAMIFANT (emapalumab-lzsg), ​​GARDASIL (recombinant HPV quadrivalent vaccine), GARDASIL 9 (recombinant HPV nonavalent vaccine), GABRETO (pralsetinib), GAZYVA (obinutuzumab), gefitinib, gemcitabine hydrochloride, GEMCITABINE-CISPLATIN, GEMCITABINE-OXALIPLATIN, gemtuzumab ozogamicin, GEMZAR (gemcitabine Tabine hydrochloride), Gilotrif (afatinib dimaleate), gilteritinib fumarate, glasdegib maleate, Glivec (imatinib mesylate), Gliadel wafer (carmustine implant), glucarpidase, goserelin acetate, granisetron, granisetron hydrochloride, Granix (filgrastim), Halaven (eribulin mesylate), Hemangeol (propranolol hydrochloride), Herceptin Hylecta (trastuzumab and hyaluronidase-oysk), Herceptin (trastuzumab) , HPV bivalent vaccine, recombinant, HPV 9-valent vaccine, recombinant, HPV quadrivalent vaccine, recombinant, Hycamtin (topotecan hydrochloride), Hydrea (hydroxyurea), hydroxyurea, HyperCVAD, Ibrance (palbociclib), ibritumomab tiuxetan, ibrutinib, ICE, Iclusig (ponatinib hydrochloride), idamycin PFS (idarubicin hydrochloride), idarubicin hydrochloride, idelalisib, Idifa (enasidenib mesylate), Ifex (ifosfamide), ifosfamide, IL-2 (aldosterone) Suleukin), Imatinib mesylate, Imbruvica (ibrutinib), Imfinzi (durvalumab), Imiquimod, Imlijik (talimogene laherparepvec), Infugem (gemcitabine hydrochloride), Inlyta (axitinib), inotuzumab ozogamicin, Incovi (decitabine and cedazuridine), Inrevik (fedratinib hydrochloride), interferon alfa-2b, recombinant, interleukin-2 (aldesleukin), Intron A (recombinant interferon alfa-2b), iobenguane I 131, ipilimumab, Iressa (gefitinib), irinotecan hydrochloride,Irinotecan hydrochloride liposomal, Isatuximab-irfc, Istodax (romidepsin), Ivosidenib, Ixabepilone, Ixazomib citrate, Ixempra (ixabepilone), Jakafi (ruxolitinib phosphate), JEB, Jelmit (mitomycin), Jevtana (cabazitaxel), Kadcyla (Ado-trastuzumab emtansine), Kepivans (palifermin), Keytruda (pembrolizumab), Kisqali (ribociclib), Cosergo (selumetinib sulfate), Kymriah (tisagenlecleucel), Cyprolis (carfilzomib), Lanreotide acetate, Lapatinib tosylate, Larotrectinib sulfate, lenalidomide, lenvatinib mesylate, Lenvima (lenvatinib mesylate), letrozole, leucovorin calcium, Leukeran (chlorambucil), leuprorelin acetate, Levrankerastic (aminolevulinic acid hydrochloride), Libtayo (cemiplimab-rwlc), lysocabtagene malareucel, lomustine, Lonsurf (trifluridine and tipiracil hydrochloride), Lorbrena (lorlatinib), lorlatinib, Lumoxity (moxetumomab pasudotox-tdfk), Luprondepot (leuprorelin acetate), lurbinectedin, luspatercept-aamt, Lutathera (lutetium Lu 177-dotatate), Lutetium (Lu 177-dotatate), Lynparza (olaparib), Margenza (margetuximab-cmkb), margetuximab-cmkb, Marquibo (vincristine sulfate liposomal), Matulan (procarbazine hydrochloride), mechlorethamine hydrochloride, megestrol acetate, Mekinist (trametinib dimethyl sulfoxide), Mektobi (binimetinib), melphalan, melphalan hydrochloride, mercaptopurine, meth Nex (mesna), methotrexate sodium, methylnaltrexone bromide, midostaurin, mitomycin, mitoxantrone hydrochloride, mogamulizumab-kpkc, Monjuvi (tafasitamab-cxix), moxetumomab-passudotox-tdfk, Mozovir (plelixafor), MVAC, Muvasi (bevacizumab), Myleran (busulfan), Mylotarg (gemtuzumab ozogamicin),Nanoparticle paclitaxel (paclitaxel albumin-stabilized nanoparticle formulation), naxitamab-gqgk, necitumumab, nelarabine, neratinib maleate, Nerlinx (neratinib maleate), netupitant and palonosetron hydrochloride, Neulasta (pegfilgrastim), Neupogen (filgrastim), Nexavar (sorafenib tosylate), Nilandrone (nilutamide), nilotinib, Nilutamide, Ninlaro (ixazomib citrate), niraparibut tosylate hydrate, nivolumab, Nplate (romiplostim), Nuveka (darolutamide), Nivepria (pegfilgrastim), obinutuzumab, Odomzo (sonidegib), OEPA, ofatumumab, OFF, olaparib, omacetaxine mepesuccinate, Oncaspar (pegaspargase), ondansetron hydrochloride, Onivyde (irinotecan hydrochloride liposomal), Ontac (denileukin diftitox), Onureg (azacitidine), Opdivo (nivolumab) ), OPPA, Orgovix (relugolix), osimertinib mesylate, oxaliplatin, paclitaxel, paclitaxel albumin-stabilized nanoparticle formulation, PAD, Padoseb (enfortumab vedotin-ejfv), palbociclib, palifermin, palonosetron hydrochloride, palonosetron hydrochloride and netupitant, pamidronate disodium, panitumumab, panobinostat lactate, pazopanib hydrochloride, PCV, PEB, pegaspargase, pegfilgrastim, peginterferon alfa-2b, PEG-Intron (PEG Interferon alfa-2b), Pemazil (pemigatinib), pembrolizumab, pemetrexed sodium, pemigatinib, Perjeta (pertuzumab), pertuzumab, pertuzumab, trastuzumab, and hyaluronidase-zzxf, pexidartinib hydrochloride, Phesgo (pertuzumab, trastuzumab, and hyaluronidase-zzxf), Piqray (alpelisib), plerixafor, polatuzumab vedotin-piiq, Porivee (polatuzumab vedotin-piiq), pomalidomide, Pomalyst (pomalidomide), ponatinib hydrochloride, Portrazza (necitumumab), Potelizio (mogamulizumab-kpkc), pralatrexate, pralsetinib, prednisone, procarbazine hydrochloride, Procrit (epoetin alfa), Proleukin (aldesleukin), Prolia (denosumab), Promacta (eltrombopag olamine), propranolol hydrochloride, Provenzi (sipuleucel-T), Prinetol (mercaptopurine), Prixan (mercaptopurine), Quinloc (ripretinib), radium-223 dichloride, raloxifene hydrochloride, ramucirumab,Rasburicase, ravulizumab-cwvz, Revlozil (luspatercept-aamt), R-CHOP, R-CVP, recombinant human papillomavirus (HPV) bivalent vaccine, recombinant human papillomavirus (HPV) nonavalent vaccine, recombinant human papillomavirus (HPV) quadrivalent vaccine, recombinant interferon alfa-2b, regorafenib, liristor (methylnaltrexone bromide), relugolix, R-EPOCH, Retacrit (epoetin alfa), LetVimo (selpercatinib), Revlimid (lenalidomide), ribociclib, R-ICE, ripretinib, Rituxan (rituximab), Rituxan Hysera (rituximab and hyaluronidase human), rituximab, rituximab and hyaluronidase Human, Rolapitant hydrochloride, Romidepsin, Romiplostim, Rozlytrek (entrectinib), Rubidomycin (daunorubicin hydrochloride), Rubraca (rucaparib camsylate), Rucaparib camsylate, Ruxolitinib phosphate, Ridapt (midostaurin), Sacituzumab govitecan-hziy, Thankuso (granisetron), Sarcrisa (isatuximab-irfc), Sclerosol intrapleural aerosol (talc), Selinexol, Selpercatinib, Selumetinib sulfate, Siltuximab, Sipuleucel-T, Soltamox (tamoxifen citrate), Somatuline Depot (lanreotide acetate), Sonidegib, Sorafenib tosylate, Sprycel (dasatinib), STANFORD V, Sterile Talc Powder (Talc), Steritalc (Talc), Stivarga (Regorafenib), Sunitinib Malate, Sustole (Granisetron), Sutent (Sunitinib Malate), Silatron (Peginterferon alfa-2b), Silvant (Siltuximab), Synribo (Omacetaxine Mepesuccinate), Tabloid (Thioguanine), Tabrecta (Capsule) tafasitamab-cxix, tafinlar (dabrafenib mesylate), tagraxofusp-erzs, tagrisso (osimertinib mesylate), talazopaributosylate, talc, talimogene laherparepvec, tarzenna (talazopaributosylate), tamoxifen citrate, tarceva (erlotinib hydrochloride), targretin (bexarotene),Tasigna (nilotinib), Tavarisse (fostamatinib disodium), Taxotere (docetaxel), tazemetostat hydrobromide, Tazberik (tazemetostat hydrobromide), Tecartas (brexcavtagene autorucel), Tecentriq (atezolizumab), Temodar (temozolomide), temozolomide, temsirolimus, Tepadina (thiotepa), thalidomide, Thalomid (thalidomide), thioguanine, thiotepa, Tibsovo (ivosidenib), tisagenlecleucel, tocilizumab, Trac (fluorouracil-topical) ), topotecan hydrochloride, toremifene, Torisel (temsirolimus), Totecto (dexrazoxane hydrochloride), TPF, trabectedin, trametinib dimethyl sulfoxide, trastuzumab, trastuzumab and hyaluronidase-oysk, Treanda (bendamustine hydrochloride), Trexol (methotrexate sodium), trifluridine and tipiracil hydrochloride, Trisenox (arsenic trioxide), Trodelvy (sacituzumab govitecan-hziy), Truxima (rituximab), tucatinib, Tukysa (tucatinib), Tur alio (pexidartinib hydrochloride), Tykerb (lapatinib ditosylate), Uconix (umbralisib tosylate), Ultomiris (ravulizumab-cwvz), umbralisib tosylate, Undencyca (pegfilgrastim), Unituxin (dinutuximab), uridine triacetate, VAC, valrubicin, Valstar (valrubicin), vandetanib, VAMP, Varubi (rolapitant hydrochloride), Vectibix (panitumumab), VeIP, Velcade (bortezomib), vemurafenib, Venclexta (bene Toclax), venetoclax, Verzenio (abemaciclib), Vidaza (azacitidine), vinblastine sulfate, vincristine sulfate, vincristine sulfate liposomal, vinorelbine tartrate, VIP, vismodegib, Bistogard (uridine triacetate), Vitrakvi (larotrectinib sulfate), Vizinpro (dacomitinib), Voraxaze (glucarpidase), vorinostat, Votrient (pazopanib hydrochloride), Vixeos (daunorubicin hydrochloride and cytarabine liposomal), Xalkori (crizotinib),Zatomep (methotrexate sodium), Xeloda (capecitabine), XELIRI, XELOX, Xgeva (denosumab), Xofigo (radium-223 dichloride), Xospata (gilteritinib fumarate), Xpovio (selinexol), Xtandi (enzalutamide), Yervoy (ipilimumab), Yescarta (axicabtagene silol-eucel), Yondelis (trabectedin), Yonsa (abiraterone acetate), Zaltrap (Ziv-aflibercept), zanubrutinib, Zalxio (filgrastim), Zejula (niraparibut tosilate monohydrate) , Zelboraf (vemurafenib), Zepzelca (lurbinectedin), Zevalin (ibritumomab tiuxetan), Ziextenzo (pegfilgrastim), Zinecard (dexrazoxane hydrochloride), Zirabev (bevcizumab), Ziv-aflibercept, Zofran (ondansetron hydrochloride), Zoladex (goserelin acetate), zoledronic acid, Zolinza (vorinostat), Zometa (zoledronic acid), Zyclara (imiquimod), Zydelig (idelalisib), Zykadia (ceritinib), and Zytiga (abiraterone acetate).

[0103] In any of the methods, the macrophages and the chemotherapeutic agent can be administered simultaneously by the same or different routes of administration, or can be administered sequentially by the same or different routes of administration. For example, the chemotherapeutic agent can be administered 0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours before or after administration of the macrophages. As another example, the chemotherapeutic agent can be administered 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days before or after administration of the macrophages.

[0104] In some embodiments, the second line therapy is oncolytic virus therapy. In some embodiments, the method further comprises treating the subject with oncolytic virus therapy. Oncolytic viruses are viruses that preferentially infect and kill cancer cells. When infected cancer cells are destroyed by oncolysis, they release new infectious virus particles or virions to help destroy the remaining tumor. Oncolytic viruses are thought to stimulate the host's anti-tumor immune system response in addition to causing direct destruction of tumor cells. Adenoviruses, herpes viruses, measles viruses, coxsackie viruses, polio viruses, reo viruses, pox viruses, filo viruses, coronaviruses, equine encephalitis viruses, flaviviruses, arena viruses, influenza viruses, and Newcastle disease viruses, among others, are some of the oncolytic viruses in preclinical and clinical development for cancer therapy. In some embodiments, the oncovirus is vaccinia virus (VACV) or vesicular stomatitis virus (VSV).

[0105] In any of the methods, the macrophages and oncolytic virus therapy can be administered simultaneously by the same or different routes of administration, or can be administered sequentially by the same or different routes of administration. For example, the oncolytic virus therapy can be administered 0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours before or after administration of the macrophages. As another example, the oncolytic virus therapy can be administered 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days before or after administration of the macrophages.

[0106] Also provided herein is a method for treating cancer in a subject, comprising collecting a biological sample from the subject comprising peripheral blood mononuclear cells (PBMCs), isolating monocytes from the PBMCs, isolating peripheral blood T (PBT) cells from the PBMCs, culturing the monocytes in vitro to produce macrophages, and contacting the macrophages with a SIRPα inhibitor to produce macrophages having reduced SIRPα cell surface expression or activity compared to untreated macrophages (SIRPα inhibitors). low Macrophage) population and SIRPα low The macrophages are contacted with a macrophage activator to induce SIRPα low activating macrophages, collecting a biological sample from a subject, including a tumor biopsy, and activating SIRPα low Macrophages were co-cultured in vitro with cells from tumor biopsies (tumor-fed SIRPα low Macrophages) and tumor-supplying SIRPα low Also disclosed is a method comprising co-culturing macrophages with isolated PBT cells in vitro to expand the number of tumor-specific T cells and administering a therapeutically effective amount of the in vitro expanded PBT cells to a subject.

[0107] In some embodiments, the in vitro expanded PBT cells are administered to the subject by IV administration. In some embodiments, the in vitro expanded PBT cells are administered to the subject by IV administration, followed by tumor-directed in situ radiation therapy. In some embodiments, the in vitro expanded PBT cells are administered to the subject by IV administration, followed by IV administration of ICB. In some embodiments, the in vitro expanded PBT cells are administered to the subject by IV administration, followed by tumor-directed in situ radiation therapy and IV administration of ICB. In some embodiments, the in vitro expanded PBT cells are administered to the subject by IV administration, followed by tumor-directed in situ radiation therapy. In some embodiments, the in vitro expanded PBT cells are administered to the subject by IV administration, followed by tumor-directed in situ radiation therapy, followed by IV administration of ICB.

[0108] In some embodiments, the in vitro expanded PBT cells are administered to the subject by IV administration. In other embodiments, the in vitro expanded PBT cells are administered to the subject by intratumoral injection. In other embodiments, the in vitro expanded PBT cells are administered to the subject by injection into the tissue surrounding the tumor.

[0109] Also provided herein is a method for treating cancer in a subject, comprising collecting a biological sample from the subject comprising peripheral blood mononuclear cells (PBMCs), isolating monocytes from the PBMCs, culturing the monocytes in vitro to produce macrophages, and contacting the macrophages with a SIRPα inhibitor to produce macrophages having reduced SIRPα cell surface expression or activity compared to untreated macrophages (SIRPα inhibitors). low Macrophage) population and SIRPα low The macrophages are contacted with a macrophage activator to induce SIRPα low activating macrophages, collecting a biological sample from a subject including a tumor biopsy, isolating tumor infiltrating lymphocytes (TILs) from the tumor biopsy, and isolating activated SIRPα. low Macrophages were co-cultured in vitro with tumor cells from tumor biopsies (tumor-fed SIRPα low Macrophages) and tumor-supplying SIRPα low Also disclosed is a method comprising co-culturing macrophages with isolated TIL cells in vitro to expand the number of tumor-specific T cells, and administering a therapeutically effective amount of the in vitro tumor-specific T cells from the TIL cells to a subject.

[0110] In some embodiments, the in vitro tumor-specific T cells from TIL cells are administered to a subject by IV administration. In some embodiments, the in vitro tumor-specific T cells from TIL cells are administered to a subject by IV administration, followed by tumor-directed in situ radiation therapy. In some embodiments, the in vitro tumor-specific T cells from TIL cells are administered to a subject by IV administration, followed by IV administration of ICB. In some embodiments, the in vitro tumor-specific T cells from TIL cells are administered to a subject by IV administration, followed by tumor-directed in situ radiation therapy and IV administration of ICB. In some embodiments, the in vitro tumor-specific T cells from TIL cells are administered to a subject by IV administration, followed by tumor-directed in situ radiation therapy. In some embodiments, the in vitro tumor-specific T cells from TIL cells are administered to a subject by IV administration, followed by tumor-directed in situ radiation therapy, followed by IV administration of ICB.

[0111] In some embodiments, the TIL cells are tumor-infiltrating T lymphocytes. In some embodiments, the in vitro tumor-specific T cells from the TIL cells are administered to the subject by IV administration. In other embodiments, the in vitro tumor-specific T cells from the TIL cells are administered to the subject by intratumoral injection. In other embodiments, the in vitro tumor-specific T cells from the TIL cells are administered to the subject by injection into the tissue surrounding the tumor.

[0112] Various types of cancers and their metastases can be treated by the methods described herein. For example, cancers include adrenal gland cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, triple negative breast cancer, carcinoma, Castleman's disease, cervical cancer, colon / rectum (colorectal) cancer, endometrial cancer, esophageal cancer, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (gist), gestational trophoblastic disease, Hodgkin's disease, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, and the like. The tumor may be a cystic cyst, non-Hodgkin's lymphoma, oral and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, small intestine cancer, gastric cancer, testicular cancer, thymic cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, Wilms' tumor, melanoma, adenoma, carcinoma of solid tissue, carcinoma in situ, adenocarcinoma, hypoxic tumor, genitourinary cancer, head and neck cancer, nervous system cancer, benign lesion, or any combination thereof.

[0113] In some embodiments, the cancer is refractory to one or more of radiation therapy, chemotherapy, or immunotherapy (e.g., checkpoint blockade). In some embodiments, the cancer is colorectal cancer, pancreatic cancer, ovarian, metastatic triple-negative breast cancer, lung, or brain cancer.

[0114] The disclosed macrophage and / or immune checkpoint inhibitors ("agents") can be administered orally or parenterally. When administration is parenteral, the agent can be administered intravenously, intratumorally, intramuscularly, subcutaneously, intraperitoneally, intrapleurally, intrabronchially, intravaginally, topically, via the ear, eye or nose, sublingually, intrathecally, intrarectally, intracranially, or into the cerebrospinal fluid.

[0115] In some embodiments of the methods of treatment disclosed herein, activated SIRPα low Macrophages, or activated SIRPα as disclosed herein lowThe composition containing the macrophages is administered by injection, for example, by injection at the site of a tumor.

[0116] The mode of administration used for the second line therapeutic (e.g., any one of the DAMPs disclosed herein, any one of the ligands of a DAMP-sensing receptor disclosed herein, any one or more of the immunotherapeutics targeting a T cell inhibitory receptor (IR) and / or a ligand of a T cell inhibitory receptor (IR) disclosed herein) can depend on the nature (e.g., site of cancer) and severity of the condition being treated and can be determined by a physician.

[0117] In some embodiments, any one of the second line therapeutic agents disclosed herein, activated SIRPα low Macrophages or activated SIRPα low Routes of administration of compositions comprising macrophages include oral, enteral, transmucosal, rectal, intranasal, buccal (e.g., sublingual), vaginal, intrathecal, intraocular, transdermal, intrauterine (or intraovo), parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular (including administration to the skeleton, diaphragm and / or myocardium), intradermal, intrapleural, intracerebral, intraarticular, intravascular, or by injection), topical (e.g., both cutaneous and mucosal surfaces, including airway surfaces, and transdermal administration), intralymphatic, etc., as well as direct tissue or organ injection (e.g., liver, skeletal muscle, myocardium, diaphragm muscle, or brain).

[0118] In some embodiments, any one of the second line therapeutic agents disclosed herein, activated SIRPα low Macrophages or activated SIRPα low Administration of the composition comprising the macrophages and one or more secondary therapeutic agents is by injection, for example, by injection into the tumor site.

[0119] In various embodiments, the compositions disclosed herein (e.g., activated SIRPα lowThe macrophage-containing composition may be formulated in the form of a pill, capsule, granule, tablet, pallet, suspension, injection, infusion, suppository, continuous delivery system, syrup, tincture, ointment, cream, eye drop, ear drop, flush, wash, slow absorption depot, bandage, lozenge, or any pharma- ceutically acceptable form for use, or as a nutritional supplement.

[0120] The agents as disclosed herein may be formulated with conventional carriers and excipients, which may be selected according to normal practice. Tablets may typically contain excipients, glidants, fillers, binders, etc. Aqueous formulations may be prepared in sterile form and generally be isotonic if intended for delivery other than oral administration. The formulations may contain excipients (e.g., excipients described in Handbook of Pharmaceutical Excipients, 5th Ed.; Rowe, Sheskey, and Owen, Eds.; American Pharmacists Association; Pharmaceutical Press: Washington, DC, 2006). Excipients may include ascorbic acid or other antioxidants, chelating agents such as EDTA, carbohydrates such as dextrin, hydroxyalkylcellulose, hydroxyalkylmethylcellulose, stearic acid, etc.

[0121] For oral use, tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups or elixirs may be prepared. Compounds intended for oral use may be prepared according to any method known in the art for the manufacture of pharmaceutical compositions, and such compositions may contain one or more agents, including sweeteners, flavoring agents, coloring agents and preservatives, to provide a palatable preparation.

[0122] In some embodiments, activated SIRPα low Macrophages, or activated SIRPα as disclosed herein lowThe composition comprising macrophages is in the form of a sterile injectable preparation (e.g., a sterile injectable aqueous or oleaginous suspension). The suspension may be formulated according to methods known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent (e.g., prepared as a solution in 1,3-butanediol or as a lyophilized powder). Among the acceptable vehicles and solvents that may be used are water, Ringer's solution, and isotonic sodium chloride solution. In some embodiments, the vehicle is a buffer solution, such as phosphate buffered saline (PBS). In some embodiments, PBS contains at least about 0.1% sodium chloride (e.g., about 0.3%, about 0.5%, about 0.7%, about 0.9%, about 1.1%, or about 1.3%, including all values ​​and subranges therebetween). In some embodiments, PBS has a pH in the range of about 7 to 8, for example, about 7.2, about 7.4, about 7.6, or about 7.8 (including all values ​​and subranges therebetween). In some embodiments, PBS has about 0.9% sodium chloride and a pH of about 7.4. Additionally, sterile fixed oils can be conventionally used as a solvent or suspending medium. For this purpose, any bland fixed oil can be used (e.g., synthetic mono- or diglycerides). Fatty acids (e.g., oleic acid) can also be used in the preparation of injectables. In some embodiments, any of the disclosed compositions can further include serum, such as human serum. In some embodiments, the serum is GMP manufactured human AB serum.

[0123] The formulations can be presented in unit-dose or multi-dose containers (e.g., sealed ampoules and vials) and can be stored in a freeze-dried (lyophilized) condition requiring the addition of a sterile liquid carrier for injection (e.g., water) immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets of the kind previously described. Preferred unit-dose formulations can be those containing a daily dose or daily sub-dose of the active ingredient, as set forth hereinabove, or an appropriate fraction thereof.

[0124] The formulations can be presented in unit-dose or multi-dose containers (e.g., sealed ampoules and vials) and can be stored in a frozen (wet) state, requiring thawing of the formulation for injection immediately before use. Extemporaneous injection solutions and suspensions can be prepared by thawing the frozen formulations. The formulated suspensions can be presented in unit-dose or multi-dose containers (e.g., sealed ampoules and vials) and can be stored in a frozen (wet) state, requiring thawing of the formulated suspension, followed by centrifugation of the suspension and resuspension of the centrifuged pellet in fresh sterile injection vehicle, diluent or solvent. A preferred unit-dose formulation can be a formulation that contains a daily dose or daily sub-dose of active ingredient, or a suitable fraction thereof, as listed herein above.

[0125] If necessary, the compound of the subject of the present disclosure can be applied in conjunction with one or more inactive or inactive ingredients. The first agent and / or the second agent disclosed herein can be administered by any route appropriate for the condition to be treated. Suitable routes can include oral, rectal, nasal, topical (including buccal and sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural).

[0126] In some embodiments, the disclosed SIRPα inhibitors, macrophage activators, and radiation may also be combined with other active ingredients. The combination may be selected based on the condition being treated, the cross-reactivity of the ingredients, and the pharmacological properties of the combination. The agent may also be combined with one or more other active ingredients in a single dosage form for simultaneous or sequential administration to the patient. The combination therapy may be administered as a simultaneous or sequential regimen. When administered sequentially, the combination may be administered in two or more doses.

[0127] Generally, during alternation therapy, the effective dose of each active ingredient can be administered sequentially (i.e., sequentially), whereas in combination therapy, effective doses of two or more active ingredients can be administered together. Combination therapy can provide "synergy" or "synergistic effect" (i.e., the effect achieved when active ingredients are used together is greater than the sum of the effects produced by using the compounds separately). In certain embodiments, synergistic effect can be achieved when active ingredients are (1) co-formulated and administered or delivered simultaneously in a combined formulation, (2) delivered alternately or in parallel as separate formulations, or (3) by some other regimen. In alternation therapy, synergistic effect can also be achieved when compounds are administered or delivered sequentially (e.g., in separate tablets, pills, or capsules, or by different injections in separate syringes).

[0128] Aspects of the Disclosure Aspect 1. Activated SIRPα low 1. A method for producing macrophages, comprising: (a) isolating monocytes from peripheral blood mononuclear cells (PBMCs) in a biological sample; (b) differentiating monocytes in vitro to produce macrophages; (c) contacting the macrophage with a SIRPα inhibitor; (d) Macrophages were contacted with a macrophage activator, which significantly reduced SIRPα cell surface expression compared to untreated macrophages (SIRPα low) generating a population of macrophages; SIRPα low The method, wherein the macrophages have activated phagocytosis against cancer cells, an increased proinflammatory response, and increased immunogenic antigen presentation.

[0129] Embodiment 2. The method of embodiment 1, wherein the SIRPα inhibitor suppresses expression of SIRPα, reduces the abundance of SIRPα on the surface of the cell, inhibits the activity of SIRPα, disrupts the interaction between SIRPα and CD47, or a combination thereof.

[0130] Embodiment 3 The method of embodiment 1 or 2, wherein the SIRPα inhibitor comprises a cytokine, a TLR ligand, a glucocorticoid, or a combination thereof.

[0131] Aspect 4. The method of aspect 3, wherein the SIRPα inhibitor is selected from the group consisting of IFNα, IFNβ, IFNγ, IL-1, IL-6, IL-12, IL-18, LPS, CpG, Poly I:C, LTA, PGN, flagellin, Pam3CSK4, zymosan, and HMGB1.

[0132] Embodiment 5. The method of any one of embodiments 1-4, wherein the macrophage activator comprises a cytokine, a phorbol ester, a TLR ligand, or a combination thereof.

[0133] Aspect 6. The method of aspect 5, wherein the cytokine is selected from the group consisting of IFNα, IFNβ, IL-6, IL-1, IL-17, IL-18, TNFα, and IL-12.

[0134] Embodiment 7 The method of embodiment 5 or 6, wherein the phorbol ester comprises phorbol 12-myristate 13-acetate (PMA).

[0135] Aspect 8. The method of any one of aspects 5 to 7, wherein the TLR ligand is selected from the group consisting of LPS, CpG, Poly I:C, LTA, PGN, flagellin, Pam3CSK4, zymosan, and HMGB1.

[0136] Aspect 9. The method of any one of aspects 8 to 11, wherein the glucocorticoid comprises methylprednisolone or dexamethasone.

[0137] Aspect 10. The method of any one of aspects 1 to 10, wherein the SIRPα inhibitor and the macrophage activator are contacted with the macrophage sequentially.

[0138] Aspect 11. The method of any one of aspects 1 to 10, wherein the SIRPα inhibitor and the macrophage activator are contacted with the macrophages simultaneously or in parallel.

[0139] Embodiment 12. The method according to any one of embodiments 1 to 10, wherein the SIRPα inhibitor and the macrophage activator are present in the same composition.

[0140] Aspect 13. The method of aspect 12, wherein the composition comprises recombinant human interferon gamma (IFNγ), recombinant human interferon alpha A2 (IFNα), CpG oligodeoxynucleotides, and polyinosinic:polycytidylic acid (Poly I:C).

[0141] Embodiment 14 The method of any one of embodiments 1 to 13, wherein the SIRPα inhibitor comprises an SHP-1 inhibitor.

[0142] Aspect 15. The SHP-1 inhibitor is selected from the group consisting of TPI-1 (2-(2,5-dichlorophenyl)-1,4-benzoquinone), TPI-1a1 (2-(2,5-dichlorophenyl)-2,4-benzoquinone), TPI-1a2 (2-(3-chlorophenyl)-1,4-benzoquinone), TPI-1a3 (2-phenylnaphthoquinone), TPI-1a4 (2-(4-ethoxyphenyl)-1,4-benzoquinone), TPI-1a5 (2-(4-methoxyphenyl)-1,4-benzoquinone), ), SSG (sodium stibogluconate), PTP inhibitor I (2-bromo-1-(4-hydroxyphenyl)-ethanone), PTP inhibitor II (2-bromo-1-(4-methoxyphenyl)-ethanone), PTP inhibitor III (2-[4-(2-bromoacetyl)phenoxy]-acetic acid), PTP inhibitor IV (N,N'-[1,4-phenylenebis[(1-methylethylidene)-4,1-phenylene]]bis[1,1,1-trifluoro-methanesulfonamide), NSC 23922 (3-aminocholestane), and NSC 87877 (8-hydroxy-7-[2-(6-sulfo-2-naphthalenyl)diazenyl]-5-quinolinesulfonic acid).

[0143] Embodiment 16 The method of any one of embodiments 1 to 13, further comprising contacting the macrophage with a SHP-1 inhibitor.

[0144] Embodiment 17 The method of embodiment 16, wherein the SHP-1 inhibitor is an irreversible SHP-1 inhibitor.

[0145] Aspect 18: Activated SIRPα produced by the method according to any one of aspects 1 to 12 low A composition comprising a macrophage.

[0146] Embodiment 19. A method for producing in vitro grown tumor-specific peripheral blood T (PBT) cells, comprising: (a) isolating peripheral blood T (PBT) cells from a biological sample; (b) Activated SIRPα produced by the method of claim 1 lowMacrophages were co-cultured in vitro with cells from tumor biopsies to express tumor-supplied SIRPα low Producing macrophages; (c) Tumor-supplying SIRPα low co-culturing macrophages with the isolated PBT cells in vitro to expand the number of tumor-specific T cells, thereby producing in vitro expanded tumor-specific PBT cells.

[0147] A composition comprising in vitro grown tumor-specific PBT cells produced by the method of embodiment 19.

[0148] 21. A method for producing in vitro grown tumor infiltrating T-lymphocyte (TIL) cells, comprising: (a) isolating tumor-infiltrating T lymphocyte (TIL) cells from a tumor biopsy; (b) Activated SIRPα produced by the method of claim 1 low Macrophages were co-cultured in vitro with tumor cells from tumor biopsies to detect tumor-supplied SIRPα low Producing macrophages; (c) Tumor-supplying SIRPα low co-culturing macrophages with the isolated TIL cells in vitro to expand the number of tumor-specific T cells, thereby producing in vitro expanded tumor-specific T cells from the TILs.

[0149] Embodiment 22. A composition comprising in vitro tumor-specific T cells from TIL cells produced by the method according to embodiment 21.

[0150] Aspect 23. A method for treating a tumor in a subject, comprising administering to the subject a therapeutically effective amount of activated macrophages, in vitro grown tumor-specific PBT cells as described in aspect 18, in vitro grown TIL cells as described in aspect 20, or in vitro tumor-specific T cells from TIL cells as described in aspect 22, or any combination thereof.

[0151] Aspect 24. The method of aspect 23, further comprising treating the subject with tumor-directed radiation.

[0152] Embodiment 25 The method of embodiment 23 or 24, further comprising administering to the subject a therapeutically effective amount of an immune checkpoint inhibitor.

[0153] Embodiment 26 The method of embodiment 25, wherein the immune checkpoint inhibitor comprises an anti-PD1, anti-PD-L1, anti-CTLA4 antibody, or a combination thereof.

[0154] Embodiment 27 The method of any one of embodiments 23 to 26, wherein the subject is refractory to PD-1 blockade.

[0155] Embodiment 28 The method of any one of embodiments 23 to 27, further comprising treating the subject with an oncolytic virus.

[0156] Embodiment 29 The method of embodiment 23, wherein the oncolytic virus is a vesicular stomatitis virus.

[0157] Aspect 30. A composition comprising recombinant human interferon gamma (IFNγ), recombinant human interferon alpha A2 (IFNα), a CpG oligodeoxynucleotide, and polyinosinic:polycytidylic acid (Poly I:C).

[0158] Embodiment 31. The composition according to embodiment 30, wherein IFNγ is present at a concentration of 40 to 200 ng / ml.

[0159] Aspect 32. The composition according to aspect 30 or 31, wherein IFNα is present at a concentration of 40 to 200 ng / ml.

[0160] Embodiment 33. The composition according to any one of embodiments 25 to 27, wherein the CpG oligodeoxynucleotide is present in a concentration of 1 to 5 μg / ml.

[0161] Embodiment 34. The composition according to any one of embodiments 30 to 33, wherein Poly I:C is present in a concentration of 1 to 5 μg / ml.

[0162] Aspect 35. Activated SIRPα produced by a method comprising contacting macrophages from a subject with an effective amount of the composition according to any one of aspects 30 to 34. low A composition comprising a macrophage.

[0163] Aspect 36 The method of aspect 35, wherein the macrophage is a bone marrow-derived macrophage or a monocyte-derived macrophage.

[0164] Aspect 37. A method for treating a tumor in a subject, comprising administering to the subject a therapeutically effective amount of an SH domain-containing tyrosine phosphatase-1 (SHP-1) inhibitor and a therapeutically effective amount of radiation therapy, an immune checkpoint inhibitor, an oncolytic virus, or a combination thereof.

[0165] Aspect 38. The method of aspect 37, wherein the immune checkpoint inhibitor comprises an anti-PD1, anti-PD-L1, anti-CTLA4 antibody, or a combination thereof.

[0166] Aspect 39. The SHP-1 inhibitor is selected from the group consisting of TPI-1 (2-(2,5-dichlorophenyl)-1,4-benzoquinone), TPI-1a1 (2-(2,5-dichlorophenyl)-2,4-benzoquinone), TPI-1a2 (2-(3-chlorophenyl)-1,4-benzoquinone), TPI-1a3 (2-phenylnaphthoquinone), TPI-1a4 (2-(4-ethoxyphenyl)-1,4-benzoquinone), TPI-1a5 (2-(4-methoxyphenyl)-1,4-benzoquinone), 38. The method of embodiment 37, wherein the inhibitor is selected from the group consisting of N,N'-[1,4-phenylenebis[(1-methylethylidene)-4,1-phenylene]]bis[1,1,1-trifluoro-methanesulfonamide), NSC 23922 (3-aminocholestane), and NSC 87877 (8-hydroxy-7-[2-(6-sulfo-2-naphthalenyl)diazenyl]-5-quinolinesulfonic acid).

[0167] Embodiment 40. The method of any one of embodiments 23 to 29, further comprising administering to the subject one or more damage-associated molecular patterns (DAMPs).

[0168] Embodiment 41. The one or more DAMPs are selected from the group consisting of HMGB1, heat shock proteins (HSPs), SNAP-associated proteins (SNAPINs), versican, biglycan, decorin, eosinophil-derived neurotoxin, surfactant protein A / D, beta-defensin 3, histones, serum amyloid A (SAA), beta amyloid (Aβ), beta 2-glycoprotein I, mRNA, tenascin-C, S100 proteins, high mobility group box 1 protein (HMGN1), biglycan, decorin, heparin sulfate, hyaluronic acid, fibrinogen, fibronectin, beta-defensin 2, surfactant, and the like. Factant protein A / D, lactoferrin, neutrophil elastase, peroxiredoxin, histones, serum amyloid A (SAA), ox-LDL, IgG-ribonucleoprotein complex, microRNA, mtDNA, F-actin, Sin3A-related protein 130, β-glucosylceramide, N-glycans, monosodium urate (MSU), glucose, cholesterol crystals, ATP, oxidized 1-palmitoyl-2-arachidonyl sn-glycero-3-phosphocholine (ox-PAPC), RNA transcribed from Alu elements (Alu-RNA), endogenous 5′pppRNA, unedited long self-dsRNA, endogenous retroviral RNA, cytoplasmic DNA, damaged nuclear DNA, advanced glycation end products (AGE), DNA, HSP70, peptidoglycan recognition protein 1 (PGLYRP1), actin, phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), cardiolipin, sulfatide, sphingomyelin, apolipoprotein A1 (APOA1), apolipoprotein A2 (APOA2) , apolipoprotein B (APOB), apolipoprotein E (APOE), apolipoprotein J (APOJ), low density lipoprotein (LDL), high density lipoprotein (HDL), very low density lipoprotein (VLDL), Lp(a), HSP60, N-formylated peptides, cathepsin G, FAM19A4, annexin 1, Aβ42, serum amyloid A (SAA), low density lipoprotein (LL-37) and other peptides, ATP, UTP, UDP, ADP, cyclic-GMP-AMP (cGAMP), calcium ions, ROS, or any combination thereof.

[0169] 42. The method of any one of aspects 23 to 29, 40, and 41, further comprising administering to the subject an anti-CD155 antibody, an anti-CD112 antibody, an anti-Fap2 antibody, an anti-TIGIT antibody, an anti-CD96 antibody, an anti-CD112R antibody, an anti-DNAM-1 antibody, an anti-TIM-3 antibody, an anti-LAG3 antibody, or any combination thereof.

[0170] 43. The method of aspect 42, wherein the anti-TIGIT antibody is tiragolumab, BMS-986207, BGB-A1217, OP-313M32, AB154, ASP8374, MK-7684, or any combination thereof.

[0171] Although several embodiments of the invention have been described, it should be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims. EXAMPLES

[0172] Example 1: Immune checkpoint blockade (ICB) has been well-established for its exceptional efficacy in several types of cancer (Wei, SC, et al. Cancer Discov., 2018.8(9):1069-1086). Unfortunately, many cancer patients do not respond or become refractory to ICB, which is believed to be due to tumors and tumor microenvironment (TME) incorporating mechanisms that subvert T cell immunity (Jenkins, RW, et al. British Journal Of Cancer, 2018.118:9). In particular, colorectal cancer (CRC) and pancreatic cancer, especially pancreatic ductal adenocarcinoma (PDA), are well-known to show limited and poor response to ICB (<11% for CRC and <4% for PDA) (Brahmer, JR, et al. N Engl J Med., 2012.366(26):2455-2465). Although CRC and PDA are associated with a high mutational burden and therefore should be immunogenic, both CRC and PDA are associated with a deficiency of cytotoxic CD8 T cells (Tc) and T REGS and myeloid-derived suppressor cells (MDSCs), thereby compromising the efficacy of ICB (Kabacaoglu, D., et al. Frontiers in Immunology, 2018.9(1878); Emambux, S., et al. Expert Opin Biol Ther, 2018.18(5):561-573). Thus, there is an urgent need for therapeutic innovations to improve the efficacy of ICB in ICB-resistant cancers such as CRC and PDA.

[0173] SIRPα-deficient mice (Sirpα - / -Anti-PD-L1 antibody (αPD-L1 Ab) administration in WT mice resulted in potent anti-tumor immunity, achieving complete elimination of CRC and PDA in situ with a robustness not observed in WT mice and rarely reported elsewhere. SIRPα is an immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing signaling receptor whose canonical function is to inhibit professional phagocytes (e.g., macrophages (Mφ) dendritic cells (DCs)) from phagocytosing self / tumor cells through interaction with the self marker CD47 (Figure 1) (Veillette, A., et al. Trends Immunol, 2018.39(3):173-184). Despite the fact that many cancers exploit this mechanism by increasing CD47 expression (Willingham, SB, et al. Proc Natl Acad Sci USA, 2012.109(17):6662-7), simple depletion of the CD47-SIRPα axis or SIRPα signaling does not result in phagocytosis (as shown in Figure 1), an activity that cannot occur unless phagocytes are simultaneously stimulated by activation mechanisms such as those mediated by TLR agonists or proinflammatory cytokines (Bian, Z., et al. Proc Natl Acad Sci USA, 2016.113(37):E5434-43). Indeed, Sirpα - / - Mice showed minimal immune control in the absence of ICB against syngeneic non-immunogenic MC38 (CRC) and Panc02 as well as KPC (PDA) tumors. All of these tumors were tolerated and grew to form palpable primary tumors after subcutaneous (sc) implantation similar to WT mice (Figures 2 and 3). However, tumor-implanted Sirpα - / -Mice showed higher basal numbers of tumor-infiltrating T cells than WT mice. Considering that phagocytes, especially Mφ, are found to be abundant in these tumors (Cassetta, L., et al. Nature Reviews Drug Discovery, 2018.17:887), it is reasonable to hypothesize that ICB, which further activates Tc to locally increase cytotoxic cell damage, DAMP release, and proinflammatory cytokines (e.g., IFN-γ, TNFα, and IL-17), may be involved in the upregulation of Sirpα. - / - The question was whether SIRPα would activate tumor-associated phagocytes in mice to eliminate those tumors in the absence of SIRPα.

[0174] Two doses of αPD-L1 (50 μg each, BioXcell clone 10F.9G2) given to MC38 tumors (sc) downregulated Sirpα - / - Induced a robust antitumor immune response in mice (Figure 2) and 3 Direct removal of tumors or tumors that are relatively large (>200 mm 3 ) caused strong growth inhibition. - / - Although increasing the number of doses of αPD-L1 to eliminate larger tumors in mice may have yielded similar results, we used IFN-γ plus CpG together with αPD-L1 for two reasons: 1) the addition of proinflammatory cytokines / TLR agonists increases the expression of tumor-associated Sirpα - / - 2) by activating intratumoral phagocytes and focusing their activity, we are able to selectively target Sirpα rather than further reactivating exhausted T cells. - / - These results suggest that phagocytes play a key role in tumor elimination. Indeed, combining IFN-γ and CpG with αPD-L1 enhanced immunogenicity and upregulated Sirpα. - / -This resulted in complete elimination of larger MC38 tumors in WT mice (Figure 2B). The same αPD-L1 treatment(s) in WT mice produced only partial effects when tumors were small, or else provided little beneficial effect when tumors were larger, even in combination with CpG. (Note: these preliminary studies were performed giving αPD-L1 Ab via intratumoral (it) injection instead of intraperitoneal (ip) to conserve reagents).

[0175] αPD-L1 treatment also upregulates Sirpα - / - It was also tested against the PDA tumors Panc02 and KPC implanted in mice (sc), and again, complete responses were observed (Figure 3). In these experiments, tumors grew to approximately 100 mm when the first of two doses of αPD-L1 ± IFNγ / CpG was administered. 3 and the second dose was given 3 days later. - / - In mice, αPD-L1 alone strongly suppressed the growth of Panc02 and KPC tumors, and in some cases was sufficient for complete remission, whereas the combination with IFNγ / CpG consistently and completely eliminated these tumors. However, the same treatment had a minor effect in WT mice, where tumors continued to grow and quickly reached humane endpoints. As clinical trials are evaluating the combination of tumor radiation and checkpoint blockade (Gong, J., et al. J Immunother Cancer, 2018 6(1):46), we tested this method to enhance the efficacy of αPD-L1. Notably, Sirpα - / - Mice were treated with a single X-ray fraction of 8 Gy (RT) followed immediately by administration of αPD-L1, resulting in tumors with a size of >250 mm 3Radiation plus αPD-L1 resulted in complete elimination of even the most aggressive MC38, Panc02, and KPC tumors (Figure 4). In stark contrast, WT mice treated with two rounds of radiation plus αPD-L1 achieved only modest control of tumor progression. These tumor models, especially MC38 tumors, have been extensively studied in preclinical immunotherapy and radiation therapy (Deng, L., et al. J Clin Invest, 2014. 124(2):687-695; Vatner, RE, et al. Semin Radiat Oncol., 2015. 25(1):18-27; Ahn, G.-O., et al. Proc Natl Acad Sci US A., 2010. 107(18):8363-8368), in part due to their notoriously poor immunogenicity, thereby facilitating the identification of interventions that may be useful in difficult-to-treat cancers in the clinical setting. With the exception of efficacy shown only against relatively small tumors (Goding, SR, et al. J Immunol., 2018. 200(9): 3304-3311; Smilowitz, HM, et al. Cancer Immunol Immunother., 2016. 65(2): 127-139; Filatenkov, A., et al. Clin Cancer Res., 2015. 21(16): 3727-3739; Twyman-Saint Victor, C., et al. Nature, 2015. 520(7547): 373-7; Azad, A., et al. EMBO Mol Med., 2017. 9(2): 167-180), Sirpα - / - Such dramatic tumor regression and remission of the larger MC38 and PDA tumors in mice has rarely been seen or found in the literature.

[0176] In one study (Deng, L., et al. J Clin Invest, 2014.124(2):687-695), the mean age was about 50 mm 3 Treatment of small MC38 tumors with a combination of 20 Gy radiation and four doses of αPD-L1 induced sustained tumor regression, but recurrence occurred 10 days after treatment cessation. 3Another study treating Panc02 and KPC tumors with 12 Gy of radiation and four doses of αPD-L1 achieved only tumor growth delay (Azad, A., et al. EMBO Mol Med., 2017.9(2):167-180). Notably, αPD-L1 alone (small tumors, 50 mm 3 ), or all Sirpα treated with either αPD-L1 plus IFNγ / CpG or 8 Gy radiation (larger tumors, 100–250 mm3) - / - Mice survived (100%), remained tumor-free, and acquired strong, long-lasting adaptive immunity against their cancer. Two direct effects were observed: first, these mice were resistant to multiple attempts at tumor reimplantation (Figure 5A); second, tumor-resistant Sirpα - / - Adoptive transfer of serum or splenic T cells from donor mice to WT mice conferred tumor resistance in WT recipients (Figures 5B, 5C). Evaluation of serum samples from donor mice revealed the presence of IgG that directly labeled MC38 cells (Figure 5B).

[0177] Considering that Tc is important for mediating the anticancer effects of PD-L1 blockade (Wei, SC, et al. Cancer Discov., 2018.8(9):1069-1086), WT and Sirpα - / - We analyzed Tc infiltration in MC38 tumors before and after administration of αPD-L1 to mice. As shown in Figure 6A, Sirpα - / - Mice were able to grow much higher numbers of Tc in their tumors after αPD-L1 administration than WT mice, and even before treatment, Sirpα - / - Tumors showed higher basal levels of Tc (Figure 6A). Therefore, these findings suggest that αPD-L1 alone upregulates Sirpα compared with WT mice. - / - This explains why Sirpα exerted better efficacy in mice. - / - Treatment of the mice further increased tumor-infiltrating T cells to significant numbers, and total infiltrating leukocytes (CD45 +) even reaching >50% of the Tc in WT tumors. In contrast, Tc in WT tumors was only modestly increased after two doses of αPD-L1±IFNγ / CpG or 8 Gy RT.

[0178] Tc is Sirpα - / - Not only did they grow rapidly and infiltrate abundantly in tumors, they also displayed high levels of Granzyme B (GranzB) expression, suggesting their robust activation and potent cytotoxicity, and demonstrated remarkable specificity for tumor cells (Figure 6B), the latter assessed by reactivity to p15E-MHCI tetramers. MuLV p15E is an epitope that is absent in the host animal but specifically expressed on MC38 tumor cells (Kershaw, MH, et al. Cancer Research, 2001. 61(21):7920-7924; Bronte, V., et al. J Immunol., 2003. 171(12):6396-6405; Kim, E.-K., et al. Cancer Research, 2014. 74(22):6705-6716), and therefore represents a useful tumor-specific antigen to assess Tc tumor specificity. αPD-L1-treated Sirpα - / - Thirty to 40% of Tc in tumors were p15E-reactive, and this number was further increased to an astounding >70% when IFNγ / CpG or RT were combined. Among these p15E-reactive Tc, a significant fraction were CD44 + CD62L - It was found that effector memory cells (T EM ) differentiation into p15E-reactive Tc and T EM αPD-L1-treated Sirpα - / - Conversely, αPD-L1-treated WT mice harbored significantly fewer p15E-reactive Tc / T cells from an already smaller total Tc population. EM , which also produces mostly GranzB low , indicating poor Tc activation and cytotoxicity. Ex vivo cytotoxicity assays (Figure 6C) confirmed this fact, showing that treated Sirpα - / -It was shown that Tc isolated from tumors had strong killing ability against MC38, whereas those from WT tumors showed weak activity. Collectively, these findings suggest that SIRPα expression negatively affects both the quantity and quality of antitumor Tc, especially in response to αPD-L1.

[0179] Further characterization of the MC38 TME demonstrated that Sirpα expression following αPD-L1 treatment was increased in the MC38 TME, particularly when combined with IFNγ / CpG or RT. - / - Other differences between CD4 and WT mice were evident. These included: 1) CD4 FoxP3 + T REGS had more Sirpα than WT mice - / - 2) there was a much greater decrease in Ly6C in WT mice after αPD-L1+RT treatment. high There were monocyte / MDSC-infiltrated tumors, which were associated with Sirpα - / - As shown in Figure 7A, Sirpα - / - Tumors have undergone a transition from constituting >50% of the total CD4 T helper cell (Th) population to representing a minor fraction (<10%) of FoxP3 + T REGS This dramatic decrease in T REG The decrease was due to irradiated Sirpα - / - These results suggest that tumors have altered their immunogenicity and possibly removed many of the immunosuppressive barriers. However, WT mice were not able to tolerate T REGS reduces only moderately

[0180] After tumors were treated with αPD-L1 plus RT, significant numbers of Ly6C +Monocytes were found to infiltrate tumors in WT mice (Figure 7C), suggesting that WT tumors after radiation / Tc-mediated damage generated potent wound-healing signaling that recruited monocytes to function as MDSCs (Bian, Z., et al. Eur J Immunol., 2018. 48(3):532-542) and suppress Tc cytotoxicity while promoting tumor growth (Gabrilovich, DI, et al. Cancer Immunol Res., 2017. 5(1):3-8). Interestingly, this important tumor-supporting mechanism was also observed in Sirpα after the same treatment. - / - Again, WT mice and Sirpα mice were clearly lacking this gene. - / - These differences between mice and humans are surprising and will be investigated further.

[0181] Considering that Sirpα is expressed in myeloid phagocytes, these data therefore support the notion that intratumoral Sirpα - / - These results suggest that phagocytes played a central role in conferring αPD-L1 sensitivity and reprogramming the tumor immune landscape. Loss of SIRPα depletes the ITIMs-SHP1 / 2-mediated inhibitory pathway (Weiskopf, K., Eur J Cancer, 2017.76:100-109), a manipulation that may promote phagocytes, and the entire TME, toward inflammation induction and antigen presentation. In contrast, SIRPα signaling induced by increased CD47 on surrounding tumor cells strongly suppresses this activation. Sirpα - / - To examine whether phagocytes could produce similar immunogenic changes and enhance the efficacy of αPD-L1, Sirpα was expressed when transferred into WT tumors. - / - Mφ (bone marrow derived, BMDM, 5x10 5 Or 2x10 6) were treated ex vivo with IFNγ / CpG (6–12 h) to activate their phagocytic capacity and enhance antigen presentation (Kranzer, K., et al. Immunology, 2000.99(2):170-8), and then transformed large αPD-L1-refractory MC38 tumors (≥200 mm) in WT mice. 3 ) was intratumorally injected. Two hours later, one dose of αPD-L1 was given. As shown in Figure 8A, Sirpα - / - Mφ injection dramatically reversed the resistance of WT mice to αPD-L1, and two doses of this combination completely eliminated MC38 tumors. Again, this potent anticancer effect was associated with tumor-specific Tc proliferation (Figure 8B). These results support the conclusion that Sirpα - / - We support the hypothesis that phagocytes are essential for sensitization to ICB and also suggest the use of Sirpα as a therapeutic modality to reverse ICB-refractory states, particularly those present in currently incurable cancers with poor immunogenicity. - / - The use of phagocytes shows promise.

[0182] Interestingly, activated Sirpα - / - Mφ(2~5x10 6 Sirpα transfer alone into WT tumors also induced significant Tc proliferation and tumor regression (Figure 8C), suggesting that these Mφs performed immunogenic antigen presentation following phagocytosis of cancer cells / antigens (predicted function). - / -To test whether Mφs have an enhanced capacity for antigen presentation and whether SIRPα signaling negatively regulates it, we examined Mφ expression of MHC-I and MHC-II as well as the costimulatory molecules CD80 and CD86 upon IFNγ / CpG stimulation. As shown in Figure 9, IFNγ / CpG induced the expression of MHC-I / II and CD80 / CD86 on both Mφs, whereas ligation of SIRPα with CD47 (mCD47.ex) on WT Mφs strongly inhibited their expression. Furthermore, CD47-SIRPα interaction also markedly inhibited the production of inflammatory cytokines essential for immunogenic antigen presentation, such as IL-12, TNFα, and IL-6, in WT Mφs, whereas depletion of SIRPα increased their release.

[0183] These studies have demonstrated that Sirpα - / - Compelling results have been obtained that reveal a new anti-cancer mechanism mediated by phagocytes. These findings point to a central role for phagocytes / APCs in inducing anti-cancer immunity, whereas SIRPα functions as a key "brake" / barrier directing intrinsic phagocytosis of tumor cells, phagocytic APC antigen presentation, Tc activation, and TME immunosuppression. Notably, depleting SIRPα releases the full capacity of phagocytes / APCs to activate Tc and even remodel the TME to favor immunogenicity, together empowering ICBs to eliminate cancer. Indeed, the effect of Sirpα injected with αPD-L1 on eliminating poorly immunogenic MC38 tumors in WT mice was not significant. - / - The effectiveness of Mφ is quite astounding.

[0184] Example 2: result Focal RT is associated with Sirpα - / - Achieving a curative response against poorly immunogenic tumors in mice Subcutaneously implanted MC38 or PDA (Pan02 or KPC) cells express WT and Sirpα - / - The tumors grew similarly in mice. Once the tumors were well established (>150 mm 3), single or multiple fractions of X-ray radiation were given to treat the tumors. As shown (Figures 14A-14D), these RT regimens, even high-dose hypofractionated RT with PD-1 blockade (2x[8Gy+αPD-L1], Figure 14C), failed to control tumor burden in WT mice, where tumors rapidly progressed beyond the humane endpoint. These results are consistent with studies by others, indicating that these poorly immunogenic tumors were highly resistant to available therapies. However, single fractions of X-ray radiation (1 x 4, 8 or 20 Gy) significantly inhibited Sirpα - / - It produced a complete response in mice with MC38, Pan02 or KPC tumors (small tumors (<200 mm 3 ), but also larger ones (>300mm 3 ) also induced the elimination of all traces of Sirpα. - / - Cessation of tumor growth in mice occurred immediately after irradiation (IR), followed by sustained regression and complete clearance over several days (5–12 days). 3 Even in the case of , multiple fractionation regimens (8 Gy-4 Gy or 8 Gy-4 Gy-4 Gy, 3-day intervals) were used to suppress Sirpα - / - While we achieved complete ablation in these mice, the same strategy using αPD-L1 only provided a slight benefit to WT mice (Figure 14E).

[0185] Sirpα tumors treated with 4 or 8 Gy - / - All of the mice survived (100%) without any apparent side effects and remained tumor-free for the remainder of the study (>1.5 years) (Figure 14). Sirpα given 20 Gy - / - Although the mice showed rapid tumor regression, they developed a severe adverse response similar to the systemic inflammatory response syndrome (SIRS) with high release of proinflammatory cytokines, resulting in a mortality rate of 33%, although surviving mice (67%) recovered and remained tumor-free (>1.5 years). All irradiated Sirpα mice, not just those treated with 20 Gy, were negatively affected. - / -Mice showed elevated levels of TNFα, IL-6, IL-12, and IL-2 in their serum (FIG. 14F), whereas WT mice after RT did not show similar increases in cytokines. These differences were again consistent with the effect of Sirpα on RT. - / - Mice responded very well to the treatment, resulting in a significantly enhanced proinflammatory antitumor response.

[0186] Irradiated Sirpα - / - The abscopal effect and long-term immunity in mice. Further studies have shown that Sirpα eradicated tumors - / - We demonstrated that mice acquired robust and long-lasting antitumor cellular and humoral immunity. Two direct effects were observed: first, the RT-treated Sirpα - / - Mice showed effective abscopal tumor suppression; second, tumor eradication by Sirpα - / - The mice were resistant to relapse (FIGS. 15D-15F).

[0187] In rare cases, RT promotes an endogenous immune response that is robust enough to control tumor burden outside the irradiated area, i.e., the abscopal effect. To assess whether irradiation of the primary lesion can induce control of non-irradiated tumors, MC38 or PDA were implanted into both flanks of mice (some also in the dorsal region) and mice were randomly assigned to receive RT until the primary tumor (right flank) reached >150 mm. 3 As shown (Figures 15A-15C), Sirpα - / - In mice, RT treatment not only eliminated the primary tumor but also significantly inhibited the growth or induced regression of non-irradiated tumors in other regions. In a subset of experiments, abscopal KPC tumors were orthotopically implanted in the upper peritoneal / liver region, and these also showed regression with RT-induced elimination of the primary tumor (Figure 15D). The abscopal response was mediated by Sirpα - / -Although abscopal tumor regression was evident in irradiated mice, the regression of abscopal tumors was usually slower or incomplete compared to that of irradiated tumors. Given that the abscopal response is highly dependent on the tumoricidal effect mediated by cytotoxic CD8 T cells (Tc), we administered anti-PD-L1 to enhance Tc function, and this regimen significantly accelerated abscopal tumor elimination, achieving complete clearance in a few days (Figures 15B-15D). In contrast, WT mice did not show an abscopal response in all experiments (Figure 5).

[0188] After tumor clearance, a strong antitumor immune memory was induced by Sirpα - / - Despite being challenged with three rounds of inoculations of increasing MC38 or Pan02 reimplantation, each attempt was accompanied by Sirpα immunization with the same tumors that had previously been ablated. - / - The tumor-resistant mice failed to establish tumors in these mice (Figures 15E-15F). Serum samples from these tumor-resistant mice revealed antitumor immunoglobulins (polyclonal IgG) that directly labeled tumor cells (Figure 15G) and mediated tumor cell killing via complement-dependent cytotoxicity (CDC) and Fc-mediated phagocytosis (Figure 15H). The same tumor-resistant Sirpα - / - Adoptive transfer of splenic T cells from mice to WT mice also conferred immunological protection to the latter and eliminated tumor formation in recipients following attempted transplantation (Figure 15I).

[0189] Intratumoral Sirpα - / - Macrophages account for the complete response to local irradiation Sirpα - / - Macrophages express Sirpα - / - To determine whether RT efficacy in WT mice was underpinned by depletion of intratumoral macrophages with Cl2MDA liposomes or an antibody against the CSF receptor (αCSF1R) in these mice, we found that both strategies abolished the efficacy of RT (Figures 16A-16B). Furthermore, in WT mice, bone marrow-derived Sirpα - / - Macrophages (Sirpα - / -BMDM) were injected into MC38 or PDA tumors to test the RT response (Figure 16C). - / - BMDMs were injected directly into tumors (i.t. multipoint approach) or intravenously (i.v.), the latter approach based on the fact that Sirpα deficiency alone does not drive macrophages to phagocytose autologous cells. Both approaches demonstrated intratumoral Sirpα expression in WT mice. - / - Macrophage infusion was achieved, and neither administration route caused adverse reactions such as anemia. In particular, i.v. administration (1–2x10 in 200 μl PBS) 7 ) Pharmacokinetic analysis revealed that Sirpα - / - It was found that the majority of BMDMs extravasated within 12 h, with approximately 20–30% infiltrating into the tumor tissue, a phenomenon shown to be associated with the tumor-expressed monocyte / macrophage chemoattractant CCL2.

[0190] Intratumoral Sirpα - / - Macrophage injection radically enhanced the efficacy of RT in recipient WT mice in a dose-dependent manner (Figures 16D-16F). Endogenous intratumoral WT (Sirpα + ) Macrophages (mm 3 Approximately 1x10 per tumor mass 4 , Fig. 16E, inset) - / - Number of BMDM after two rounds of 8 Gy plus Sirpα - / - BMDM administration was performed in MC38, Pan02, and KPC tumors (all >200 mm 3 ) resulting in rapid regression and complete elimination of IR and 100% survival of treated WT mice (Figures 16D-16F). These experiments also demonstrated that Sirpα administered 1-3 hours before or immediately after IR - / - These results suggest that Sirpα expression in BMDMs was associated with similar antitumor efficacy. - / - We confirmed that macrophages account for the tumor response to RT. Similar tumor clearance efficacy was observed in Sirpα-positive mice (otherwise RT-refractory) with WT mice. - / -The fact that this was achieved by intratumoral injection of macrophages lends credibility to future RT treatment regimens combining engineered SIRPα-deficient macrophages to improve efficacy and abscopal effects.

[0191] CD47 blockade does not recapitulate Sirpα deficiency in RT Although both modalities disrupt the CD47-SIRPα axis, Sirpα - / - The compelling antitumor efficacy after RT conferred by macrophages could not be reproduced by CD47 blockade. Two CD47 blocking reagents, an antagonistic CD47 antibody (αCD47; miap301) and a soluble mouse SIRPα extracellular domain (mSIRPα.ex), as well as a rabbit Fc fusion protein, in combination with RT, inhibited the proliferation of MC38 and PDA tumors (all >200 mm) in WT mice. 3 To assess their effects, these agents were administered either before or immediately after IR, followed by Sirpα - / - As shown (Figures 16G-16I), neither αCD47 nor mSIRPα.ex, even at high doses, inhibited Sirpα - / - Combining IR with these CD47 blockers only slightly delayed tumor progression, not as effectively as BMDM. Furthermore, IR and αCD47 were not associated with tumor-eliminating Sirpα after three rounds of reimplantation challenge. - / - The triple combination regimen was further combined with high titer antisera against MC38 or PDA tumors obtained from mice (Figure 2). Despite significantly slowing tumor progression, an effect largely attributable to the antisera in combination with RT (Figure 16I), this triple combination regimen still failed to induce a complete response. It was also noted that neither the CD47 blocking reagent nor the antitumor serum, alone or in combination, inhibited tumor growth in the absence of IR. These results suggest that mere blockade of CD47 binding does not completely abrogate Sirpα signaling, and even partial activity appears to significantly impede RT-induced antitumor responses.

[0192] Radiation-activated Sirpα- / - Macrophages reshape the tumor microenvironment Sirpα after 8 Gy treatment - / - Analysis of the tumor microenvironment (TME) in the presence or absence of macrophages revealed that changes in the underlying immune landscape correlated with their differential response to IR. As shown (Figures 17A-17C), Sirpα - / - Mouse or Sirpα - / - Sirpα in either macrophage-injected tumor-bearing WT recipients - / - MC38 and PDA tumors, which contain macrophages, express Sirpα - / - Compared to tumors lacking macrophages (WT mice), tumors were rapidly infiltrated by large numbers of leukocytes (CD45+) after IR. By day 6 after IR, the majority of these were lymphocytes expressing Sirpα - / - Leukocytes infiltrating the TME frequently outnumbered tumor cells, which decreased as the tumor volume rapidly contracted. Interestingly, Sirpα expression before IR - / - >35% of all tumor leukocytes in mice express Sirpα - / - Macrophages, but this population was rapidly depleted after IR before detectable tumor regression (Figures 17B and 17D). Similarly, intratumorally injected Sirpα in WT recipients - / - Macrophages express endogenous WT (Sirpα) in the same tumors. + ) were not detected the day after IR, even though macrophages were not reduced in number (Figure ​(Figure17E). 17E). Instead, Sirpα + Intratumoral macrophage populations were not reduced when macrophages were injected or when IR was withheld. Further time course analysis during the 24-h window after IR demonstrated that intratumoral Sirpα - / - We revealed that the macrophage population remained unchanged up to 12 h after IR but rapidly declined thereafter (Figure ​(Figure17D). 17D). These findings support the role of Sirpα in RT-induced tumor clearance. - / - This is surprising given the essential role of macrophages (Figure 3) and the activation of Sirpα in tumor cells. - / -This suggested that mechanisms other than macrophage phagocytosis underlie the sustained tumor regression.

[0193] Sirpα - / - Despite the loss of macrophages, their initial response to IR-induced tumor damage triggered a cascade of events that led to immunogenic repolarization of the TME and eventual tumor elimination. As shown (Figures 17F-17G), immediately after IR (<12 h), Sirpα - / - Intratumoral Sirpα in both mice and tumor-bearing WT recipients - / - Macrophages displayed a robust proinflammatory signature and immunogenic antigen-presenting machinery with increased cell surface MHC-I, MHC-II, CD80, CD86 and OX40L, as well as expression of IL-12 and IFNα. Profiling of the inflammatory signature of bulk MC38, Pan02 and KPC tumors by Nanostring transcriptional analysis (Figures 17H-17I) revealed a similar, significantly altered TME after IR with widespread increases in the transcription of proinflammatory cytokines (IFNα / β / γ, IL-1α / β, IL-12, IL-18 and IL-33), immunogenic antigen-presenting costimulatory molecules (CD80, CD86, OX40L, IcosL, GITRL and CD40), T cell and neutrophil chemokines (CXCL1 / 2, CXCL8 etc.), and other notable molecules crucial for tumor resistance (CX3CR1, CCR7, IRF3, IRF7 etc.). Meanwhile, immunosuppressive cytokines such as TGFβ1 / 2 / 3 were significantly downregulated and Sirpα was upregulated after irradiation. - / - This indicates a shift in the TME phenotype towards a proinflammatory one and away from wound healing. - / - Irradiated tumors in WT mice without macrophage injection showed only weak proinflammatory transcription but a marked induction of TGFβ and their associated Sirpα + Macrophages showed a limited capacity for immunogenic antigen presentation but increased expression of IL-10, both of which suggest an increasingly immunosuppressive TME. These studies also demonstrated that Sirpα - / -or revealed subtle differences between the transcriptional profiles of non-irradiated MC38, Pan02, and KPC tumors in WT mice (Figure 17H).

[0194] SIRPα deficiency robustly induces tumor-specific cytotoxic CD8 T cells Sirpα - / - Among the many striking differences between the tumor milieu with and without macrophages, the population of tumor-infiltrating CD8 T cells (Tc) was significantly larger in the former. As shown (Figure 18A), Sirpα - / - Irradiation of MC38 or PDA tumors in mice resulted in a rapid expansion of intratumoral Tc, which represented nearly 40% of all tumor-infiltrating leukocytes within 24 h after IR and further increased to 50–70% by 3–6 days. - / - These large numbers of Tc in the TME were distributed throughout the tumor core and along the invasive edge (Figure 18B) and displayed high cytotoxicity and tumor specificity, as indicated by their high GranzB expression and reactivity with MuLV p15E-H2Kb tetramers (Figures 18C and 18D), respectively. MuLV p15E is an antigen expressed in MC38, Pan02, and KPC tumor cells, but not present in the host animal. Sirpaα - / - Approximately 30–50% of proliferating Tc in the TME are tumor-specific, and among them, a significant fraction expresses CD44 + CD62L - and effector memory T cells (T EM ) differentiation into tumor-specific (p15E + )Tc and T EM Sirpα - / - The increase in Tc against another MC38-specific tumor antigen, ADPGK, was also observed in MC38 tumor-irradiated Sirpα mice, and was readily detectable in the peripheral blood and spleen 2 weeks after tumor eradication (Figure 18E). - / - Sirpα in WT recipients was detected in mice. - / - Irradiation of macrophage-injected tumors similarly reduced the GranzB high p15E +In stark contrast, Sirpα induced robust proliferation of Tc after IR (Figure 18E). - / - WT mice lacking macrophages only generated a small population of intratumoral Tc, which is consistent with tumor-specific (p15E + ), and is largely non-cytotoxic (GranzB low Ex vivo cytotoxicity assays revealed that irradiated Sirpα - / - We confirmed that Tc isolated from macrophage-containing tumors were highly cytotoxic and capable of rapid (<3 h) elimination of cancer cells at low effector:target cell ratios (Figure ​(Figure18G),18), whereas Tc isolated from macrophage-containing tumors were not significantly different from Tc isolated from non-IR or non-Sirpα tumors in WT mice. - / - Tc from macrophage-injected tumors were inactive against tumor cells. Apparently, the massive expansion of tumor-specific Tc was important for IR-induced sustained tumor regression, but depletion of intratumoral Tc (αCD8), but not Th cells (αCD4), was associated with Sirpα - / - reduced the efficacy of RT in mice (Figure 18H).

[0195] activated SIRPα - / - Macrophages prevent compensatory immune suppression Further analysis revealed that Sirpα - / - We uncovered other distinct immune signatures that synergistically augmented tumoricidal activity in the irradiated TME, including macrophages. These included: 1) CD4 FoxP3 + T REGS Decrease in IFNγ + 1) Th1 proliferation; 2) a significant increase in NK cells; 3) a significant infiltration of proinflammatory PMN (polymorphonuclear leukocytes, neutrophils) and Ly6C high A striking lack of monocytes / MDSCs. Figures 19A-19B).

[0196] Despite maintaining a similar total population of intratumoral CD4 T cells (Th) (Fig. S19B), Sirpα - / - Tumors in mice express FoxP3 after IR + T REGSThere was a marked decrease in Th, from constituting >50% of total Th to only a small number (<10%), whereas IFNγ + The Th1 population expanded (Figures 19C-19D). This Th phenotype switch (T REG → Th1), suggesting an immunogenic shift within the TME that favors tumor elimination. - / - These antitumor TMEs in mice showed a four-fold increase in NK cells that also had high GranzB expression (FIG. 19E).

[0197] Consistent with reports showing that tumor damage caused by IR promotes a robust wound-healing response characterized by the recruitment of monocytes that function as MDSCs to suppress Tc immunity and promote tumor repair and growth, Sirpα - / - All irradiated MC38 and PDA tumors in WT mice without macrophage injections expressed Ly6C, which strongly inhibited Tc proliferation. high The tumor-promoting cells were highly infiltrated by monocytes (Figures 19F-19H). Surprisingly, this compensatory tumor-promoting mechanism was mediated by Sirpα - / - In mice, Sirpα expression was clearly absent, and instead tumor-infiltrating monocytes / MDSCs were reduced after IR. - / -- Mice developed a characteristic proinflammatory response with the release of proinflammatory cytokines (see Nanostring profiling and FIG. 14) and PMN (Ly6G high ), which produced high levels of reactive oxygen species (ROS), but there was no inhibition on Tc proliferation (Figures 19H-19J). - / - Approximately 20% of irradiated tumors in mice were extensively infiltrated by PMNs, a phenomenon that correlated with much faster tumor regression (Figures 19K-19L). Chemokine profiling supported these results, showing that Sirpα - / - We showed that tumors from mice secreted high levels of neutrophil-attracting CXCL8 after IR, whereas tumors from WT mice highly produced CCL2 to attract monocytes (see FIG. 17G). Ex vivo chemotaxis assays showed that tumors from mice secreted high levels of neutrophil-attracting CXCL8 after IR, whereas tumors from WT mice highly produced CCL2 to attract monocytes (see FIG. 17G).- / - Differential neutrophil and monocyte chemotaxis towards IL-1 and WT tumors was further confirmed.

[0198] Phagocytic SIRPα - / - Macrophages activate tumor-specific Tc in situ Intratumoral Sirpα after IR - / - The high expression of immunogenic antigen-presenting machinery, including MHC I / II and costimulatory molecules, in macrophages (Figure 17) suggested that these macrophages, following phagocytosis of tumor cells, functioned as antigen-presenting cells (APCs) and activated tumor-specific Tc by presenting tumor antigens. Sirpα - / - Given the magnitude and kinetics of Tc expansion in irradiated tumors, including macrophages, this antigen presentation event occurs primarily in situ and is driven by tissue-resident tumor-specific memory T cells (i.e., T EM and T RM ) was hypothesized to lead to anamnestic responses.

[0199] To test this hypothesis, we performed two experiments. First, we investigated the effect of Sirpα immediately (<30 min) after IR. - / - Tumor explants from mice without tumor-draining lymph nodes (TDLN) were cultured ex vivo (Figure 20A). Despite the absence of TDLN, these cultured tumor explants showed Tc proliferation similar to that seen in vivo. Sirpα was expressed in tumor explants from WT mice. - / - Injecting macrophages also induced intratumoral Tc proliferation. Second, an in vitro macrophage-TIL (tumor infiltrating T cell) coculture was established to examine Sirpα expression in tumor antigen presentation and activation of tumor-specific Tc. - / - In these experiments (shown in FIG. 20B), Sirpα - / - BMDMs were first incubated with irradiated MC38 or PDA tumor dissociates, which contained tumor cells and ICD debris, for phagocytosis of tumor antigens. After overnight incubation (16–18 h) for antigen processing, Sirpα - / -BMDMs express proinflammatory properties, increase immunogenic antigen-presenting machinery, and express Sirpα loaded with tumor antigens. - / - BMDMs were then co-cultured with TILs isolated from non-irradiated tumors of the same type. As shown (Figure 20C), Sirpα - / - Clear engagement between BMDMs and Tc was seen within 1 h of their coculture in a manner reminiscent of APC antigen presentation. These engaged Tc were less than 5% of the total Tc applied, suggesting that most other cells were likely "bystanders" and remained detached or nonadherent. After 1–3 days of coculture, Tc activation was evident by cell expansion (i.e., blasting), robust Tc proliferation, and expression of GranzB (Figures 20D–20E). Interestingly, Sirpα - / - APCs exclusively induced the proliferation of Tc, but not Th, in the TIL population, which was associated with Sirpα - / - This phenomenon reflects the proliferation of Tc in the irradiated TME by macrophages. After 8–10 days of culture in the presence of IL-2, a large number of Tc produced after more than 10 cycles of proliferation were detected by Sirpα. - / - were harvested from BMDM / APC-TIL cocultures (Figures 20F-20G).

[0200] Tumor cell killing assays confirmed the tumor specificity and potent cytotoxicity of these in vitro expanded Tc, which rapidly induced MC38 or PDA cell death at low effector:target ratios (1-3:1) (Figure 20H). Interestingly, despite their exceptional cytotoxicity, only a fraction (<5%) of these Tc inhibited p15E + , suggesting that the Tc population was polyclonal and that the majority recognized tumor cells through other tumor-associated antigens. The ability of Tc to eliminate established tumors in vivo was further evaluated. In these experiments, in vitro propagated Tc against MC38 or KPC (referred to as Tc-MC38 and Tc-KPC, respectively) was administered i.v. (5x10 6, 2x, 3 days apart). Prior to Tc injection, a subset of mice was preconditioned with total body irradiation (WBI; 5 Gy) and then subjected to iv injections of Tc with IL-2 (ip, 50,000 IU per day for 5 consecutive days). As shown (Figures 20I-20J), two rounds of Tc injections in WBI-conditioned mice plus IL-2 were administered at 400 mm 3 This resulted in complete clearance of over 100% of MC38 and KPC tumors and 100% survival. Similar Tc infusions without WBI and IL-2 achieved partial responses that significantly delayed tumor progression. For comparison, infusion of Tc / TILs expanded by antibody ligation of CD3 and CD28, a non-tumor specific method, was largely ineffective against established tumors or when cultured with tumor cells in vitro (Figures 20H-20I).

[0201] Consideration Although WT TME itself was unable to robustly activate Tc after IR, Sirpα - / - Injection of macrophages resulted in a robust response to IR and GranzB in tumor-bearing WT recipients high p15E + This resulted in rapid proliferation of Tc (Figure 18F).

[0202] Example 2: overview SIRPANT technology targets autologous SIRPα to drive potent anti-cancer innate and adaptive immunity to eliminate cancer. low This involves an innovative approach to engineer activated macrophages (SIRPANT-M): Patient monocytes (peripheral blood mononuclear cells [PBMCs]) obtained from peripheral blood apheresis are engineered ex vivo with SIRPANT's proprietary reagent, Phago-Act™, to produce significantly reduced signal regulatory protein alpha (SIRPα) expression (i.e., SIRPα low), generating macrophages with enhanced intrinsic capabilities for phagocytosis, proinflammatory activity, and immunogenic antigen presentation. When administered to the tumor mass, SIRPANT-M exerts potent anticancer activity, including ingesting tumor cells, reprogramming the tumor microenvironment (TME) to be proinflammatory, thereby reducing immunosuppression, and presenting tumor-associated neo-antigens that activate T cells in an immunogenic manner. As a result, large numbers of tumor-specific polyclonal cytotoxic T cells are activated to eliminate tumors and distant metastases, but this response also results in long-lasting cellular and humoral immunity that prevents cancer recurrence.

[0203] Since its development, SIRPANT-M as a cancer therapeutic approach has been thoroughly scrutinized in mouse cancer models of various solid tumors, including lymphoma, colorectal adenocarcinoma, pancreatic ductal adenocarcinoma, melanoma, lung cancer, and metastatic breast cancer. Among these tested cancers, several were late-stage and had large tumors with multiple distant lesions (metastases) that resisted combination therapy with immune checkpoint inhibitors (ICIs), radiation therapy (RT), CD47 blockade, tumor vaccines, and antitumor antibodies. However, treatment of these tumors with SIRPANT-M was highly effective in all cases, resulting in systemic elimination of tumor lesions and survival rates of up to 100%. Treated animals also exhibited features of long-lasting immune memory that effectively prevented cancer recurrence.

[0204] In addition to the in vivo proof-of-principle and efficacy studies completed in mouse cancer models, ex vivo human studies were performed using human SIRPANT-M to evaluate their phagocytosis against a panel of National Cancer Institute (NCI)-60 human tumor cell lines and activation of tumor-killing T cells from tumor-infiltrating lymphocytes (TILs) obtained from patient samples. Results confirmed that human SIRPANT-M has the potential for rapid elimination of cancer cells through both phagocytosis and potent induction of tumor-specific cytotoxic T cells.

[0205] The goal of SIRPANT is to translate the findings of these studies into clinical trials as an effective cellular immunotherapy to treat cancer. This cell therapy approach was selected based on extensive preclinical studies demonstrating that the efficacy of SIRPANT-M specifically for treating solid tumors cannot be replicated or even approached using ICIs, RT, chemotherapy, CD47 blocking reagents, or other therapies.

[0206] Research Overview In vivo proof-of-principle and efficacy studies were completed in mouse cancer models of lymphoma and various solid tumors, including syngeneic colorectal adenocarcinoma (MC38 cell line), pancreatic ductal adenocarcinoma (KPC and Pan02 cell lines), Lewis lung carcinoma (LLC), melanoma (B16 cell line), breast cancer 4T1 cell line (orthotopic implant), and metastatic breast cancer (mouse mammary tumor virus-polyoma intermediate tumor-antigen [MMTV-PyMT]). In all cases, SIRPANT-M treatment, especially when combined with local tumor radiation (TR), resulted in durable complete responses with abscopal effects, eliminating late-stage primary tumors with distant disease. All treated mice survived treatment without obvious adverse effects and achieved long-term post-treatment survival rates comparable to healthy mice housed in the same facility (>90%, >1 year). Data from these studies are summarized in Table 1.

[0207] Ex vivo human studies were performed to evaluate Phago-Act™-produced human SIRPANT-M for: a) phagocytosis against a global NCI-60 panel of human tumor cell lines and other human cancer cells, b) the ability to produce inflammatory cytokines and thereby drive a proinflammatory response, and c) expression of immunogenic antigen presentation machinery and activation of tumor-killing T cells from tumor-infiltrating lymphocytes (TILs) obtained from patient specimens. Results show that SIRPANT-M actively phagocytoses both healthy and irradiated cancer cells, whereas regulatory PBMC-derived macrophages fail to phagocytose. These studies also confirmed that human SIRPANT-M has the ability to drive a strong proinflammatory response and immunogenic antigen presentation that activates tumor-killing cytotoxic T cells. Further transcriptional profiling of SIRPANT-M prepared from six healthy volunteers of different genders and races / ethnicities showed biased proinflammatory expression and enhanced immunogenic antigen presentation machinery.

[0208] In summary, both preclinical in vitro and in vivo studies point to the potentially high efficacy of SIRPANT-M as a cross-cancer immunotherapy, empowering both innate and adaptive immunity to eliminate cancer.

[0209] Background and Mechanism of Action Macrophages are the most abundant leukocytes in the tumor microenvironment (TME) and play a central role in the immune system's ability to eliminate or tolerate cancer cells. One of the key mechanisms regulating macrophage activity is governed by SIRPα-mediated signaling, which in one aspect executes through activation of SHP-1, inhibiting: i) phagocytosis of cancer cells; ii) proinflammatory activation by toll-like receptor (TLR) agonists, interferons (IFNs), and other proinflammatory cytokines and cancer therapy inducers; and iii) expression of immunogenic mechanisms for antigen presentation to induce anti-cancer adaptive immunity. Conversely, by withholding cytokine receptor inhibitory SHP-2, SIRPα promotes signaling induced by immunosuppressive IL-4 / 13, IL-10, and TGFβ, thereby enhancing immune suppression within the TME and resistance to cancer. Details of these mechanisms are described in the following sections.

[0210] Regulation of macrophage phagocytosis of cancer cells CD47 is a ubiquitous marker of autologous cells and a cellular ligand for SIRPα. Cancer cells escape phagocytic elimination by inducing a strong SIRPα-mediated inhibition when their CD47 extracellularly ligates SIRPα on macrophages. However, despite some cancers showing high CD47 expression, many more cases (>50%), broadly representative of different cancer types, express little or no CD47 (The Human Pathology Atlas:CD47), and these cancers avoid immune elimination in vivo, even though their TME contains macrophages in abundance. Indeed, the mere depletion of CD47 or cognate SIRPα signaling does not result in phagocytosis, instead, additional phagocytic activation mechanism(s) endowed to macrophages are required to direct their phagocytic activity. These studies were initially directed at the CD47 (Cd47 - / - ) or SIRPα (Sirpα - / -), which are generally healthy but exhibit anemia due to aggressive hemophagocytosis when exposed to viral infection or under inflammatory conditions. - / -In ex vivo studies using macrophages, these macrophages are found to be quiescent unless treated with specific proinflammatory cytokines or TLR agonists, which subsequently render them phagocytic towards autologous and cancer cells, despite the lack of SIRPα-mediated inhibition. Along these lines, inflammatory cytokines including the IL-1 family (e.g., IL-1β and IL-18), IL-6, IL-17, TNFα, and type I IFNs (IFNα and IFNβ), but not IFNγ, are found, and all TLR agonists (LPS, CpG, LTA, Poly I:C, flagellin, etc.) activate macrophage phagocytosis, whereas the immunosuppressive cytokines IL-10 and TGFβ, as well as the steroid glucocorticoids, counter these proinflammatory factors by inhibiting macrophage phagocytic activation. Although the detailed underlying mechanisms remain undefined, this process likely involves specific phagocytic receptors that require proinflammatory cytokine / TLR-induced signaling for inside-out activation, which then mediates "universal" macrophage phagocytosis of self / cancer cells in the absence of CD47-SIRPα inhibition (Figure 21C). These studies collectively suggest that macrophage phagocytosis is controlled by multiple layers of activation and inhibition mechanisms, with front-line controls that determine whether macrophages are quiescent or activated for phagocytosis, and subsequent controls via the CD47-SIRPa axis that determine targets for phagocytosis. These knowledges partially explain why tumor-associated macrophages (TAMs) are generally non-phagocytic, even in the surroundings of CD47-poor / negative cancer cells. In parallel, this also explains why blocking CD47 (e.g., anti-CD47 antibodies) alone is insufficient to treat cancer (Figure 11B).Indeed, this therapeutic strategy requires the combination with modalities that activate phagocytosis, such as cancer-specific antibodies that activate phagocytosis via Fc receptors (rituximab for B-cell lymphomas) or chemotherapy reagents that increase cellular expression of calreticulin, which ligates macrophage-expressed LRP1, triggering phagocytosis (e.g., azacytidine for myelodysplastic syndromes [MDS] or acute myeloid leukemia [AML]).

[0211] Example 3: SIRPα contributes to TME immunosuppression By studying different solid tumors in mice, we found that SIRPα controls TME immunogenicity by enhancing the immunosuppressive phenotype of TAMs. Expression of SIRPα in TAMs, dendritic cells (DCs), and myeloid-derived suppressor cells (MDSCs) gradually increases as tumors grow (Figure 22), and the dynamic nature of SIRPα and its effects due to both cancer cells and TME produce factors that upregulate SIRPα expression, such as IL-10, IL-4, TGFβ, IL-17, etc. (see Figure 29). SIRPα expression in macrophages profoundly influences their response to pro- and anti-inflammatory stimuli, thus determining their subsequent effector functions. Macrophages with high levels of SIRPα (SIRPα high -M) and macrophages without itSirpα - / - -M and SIRPα low -M, the latter also called SIRPant-M), high We showed that M1-M preferentially adopt a hyperimmune suppressive phenotype characterized by elevated expression of IL-10, TGFβ, and arginase-1, a general resistance to proinflammatory activation, and reduced expression of antigen-presenting machinery (Figure 23). Even when exposed to strong proinflammatory stimuli such as LPS plus IFNγ, M1-M preferentially adopt a hyperimmune suppressive phenotype characterized by elevated expression of IL-10, TGFβ, and arginase-1, a general resistance to proinflammatory activation, and reduced expression of antigen-presenting machinery (Figure 23). high-M induced only weak expression of proinflammatory molecules, but highly expressed IL-10, with amounts equal to or exceeding the sum of their proinflammatory cytokine production. These dramatic results suggest that high SIRPα expression in the TME is essentially self-reinforcing, whereby the immunosuppressive TME upregulates SIRPα, which in turn further drives TAMs to enhance tumor immunosuppression. Furthermore, the ability of SIRPα to strongly inhibit proinflammatory signals suggests its role in TAM resistance, which undergoes a proinflammatory phenotype switch in response to therapeutic treatment. Indeed, high SIRPα expression promotes immunosuppression (IL-10) and drives TAM activation toward a wound-healing response under cancer therapy, promoting tumor recovery and progression. In support of this concept, LPS / IFNγ-treated SIRPα high -M was found to have increased production of the chemoattractant CCL2, which recruits monocytes / MDSCs to promote wound healing, but minimal secretion of CXCL1 / 2, which attracts proinflammatory neutrophils that promote tumor tissue damage.

[0212] SIRPα high In contrast to -M, Phago-Act™-treated SIRPα low Macrophages (also called SIRPANT-M) displayed the opposite, predominantly proinflammatory polarization and a less immunosuppressive phenotype in response to the same stimuli. - / - Similar to -M, SIRPANT-M had elevated levels of IL-12, IL-1β, IL-6, TNFα, and CXCL1 / 2, but not CCL2, but showed higher expression of antigen-presenting machinery, including MHC-I, MHC-II, and costimulatory molecules CD80, CD86, OX40L, CD40, etc. (Figure 23).

[0213] SIRPα regulates macrophage polarization signaling Mechanistic studies (Figure 24) revealed that macrophage immunophenotype and function are regulated by SIRPα through its cytoplasmic ITIM, which undergoes tyrosine phosphorylation upon macrophage stimulation and provides distinct docking sites for SHP-1 or SHP-2, the major cellular tyrosine phosphatases that regulate downstream signaling events. Phosphorylation of SIRPα ITIM requires tyrosine kinase activity induced by cytokines, TLR agonists, or other stimuli. Under tumor homeostasis, TAMs are constantly exposed to immunosuppressive cytokines (e.g., IL-4 / 13, IL-10) that activate Bruton's tyrosine kinase (Btk), which phosphorylates SIRPα ITIM in a manner that causes exclusive docking of SHP-2 but not SHP-1. This sequence of events, in which immunosuppressive cytokines activate Btk to drive SIRPα-SHP-2 binding, prevents SHP-2 from inhibiting IL-4 / 13R or IL-10R, thereby enhancing immunosuppressive signaling in macrophages (FIG. 24A). As a result of the addition of this pathway, SIRPα expression is further increased in TAMs, thus favorably controlling their phenotype.

[0214] Under proinflammatory conditions led by cancer therapy, immunomodulatory therapy, cytokines, TLR agonists or other stimuli, Src family tyrosine kinases (SFKs) are induced and phosphorylate the SIRPα ITIM (Figure 24B). Unlike Btk, SFKs phosphorylate the ITIM in a pattern that leads to the docking and activation of SHP-1. By dephosphorylating multiple proteins, SHP-1 reduces the IFNα / β / γ-mediated JAK-STAT and PI3k-Akt pathways that induce the antigen presentation machinery and the expression of costimulatory molecules (Kalbasi 2020). Similarly, SHP-1 inhibits the proinflammatory cytokine / TLR-mediated MAPK and NFκB pathways that exaggerate other proinflammatory signals, including those that activate phagocytosis, drive inflammation, and / or downregulate SIRPα expression (see Figure 29). In established tumors, SIRPα-SHP-1-mediated inhibition of inflammation, together with SIRPα-SHP-2-mediated enhancement of immune suppression, remain largely intact during cancer treatment, thus greatly suppressing or almost completely abolishing the efficacy of most therapeutic modalities. For example, high SIRPα expression of TLR-mediated processing-induced cell damage signals (DAMPs) in TAMs (SIRPα high ) skews the response to potent wound healing amplifying the production of IL-10, TGFβ and CCL2, the latter attracting MDSCs and inhibiting T cell-mediated anti-cancer immunity. Figure 24 shows the dichotomous regulation of SIRPα mediated by SHP-2 or SHP-1, either promoting an immunosuppressive macrophage phenotype (via SHP-2) or inhibiting proinflammatory macrophage activation and antigen presentation (via SHP-1). CD47 ligation is not required for SIRPα regulation (Figure 24D), but it induces structural change(s) in the cytoplasmic domain of SIRPα that promotes SIRPα ITIM phosphorylation by kinases, thereby enhancing the strength of SHP-1 / 2 docking and subsequent downstream regulation.

[0215] Based on these mechanistic understandings, the SIRPANT strategy is to therapeutically induce SIRPα via an ex vivo process. lowThe goal of this study is to produce SIRPANT-M macrophages, thereby avoiding the immunosuppressive TME and the strong SIRPα-mediated regulation therein that deactivates the effects of Phago-Act™ (see FIG. 29). Our previous studies have shown that, although Phago-Act™ has the ability to downregulate SIRPα and activate phagocytosis, injection of Phago-Act™ or other pro-inflammatory reagents into established tumors suppresses the response and minimizes SIRPα expression on TAMs or controls the tumor. Even multiple injections of Phago-Act™ in combination with tumor-directed radiation failed to reduce tumor burden and only moderately suppressed tumor growth. These results are particularly typical when challenging difficult-to-treat cancers such as MC38 colorectal cancer and pancreatic ductal adenocarcinomas KPC and Pan02, all of which contain a highly immunosuppressive TME and are therefore resistant to treatments such as tumor radiation (RT), anti-PD-1 / L1 checkpoint blockade, and combinations thereof.

[0216] Depleting SIRPα reprograms the TME, allowing for the elimination of cancer cells Although SIRPα depletion alone does not result in macrophages phagocytosing cancer cells, combining SIRPα depletion with cytokine / TLR agonist-mediated activation transforms macrophages into potent cancer-clearing phagocytes (Figure 25). - / - Studies using mice have found that these mice do not express the innate immunity that prevents tumor formation, but often show complete responses (CR) to immunomodulatory therapies and systemically eliminate even end-stage cancer with distant disease (metastasis). - / - Several syngeneic cancers have been tested in mice, including melanoma (B16), lymphoma (EL4), Lewis lung carcinoma (LLC), colorectal cancer (MC38), pancreatic ductal adenocarcinoma (Pan02, KPC), and DSS-AOM-induced spontaneous colorectal cancer. - / -Treating established tumors in mice with a simple cytokine plus TLR agonist scheme (Figure 26) or fractions of non-ablative X-ray RT (4-15 Gy) induced a dramatic anti-cancer response that led to rapid regression and eventual elimination (abscopal effect) of large tumors along with untreated distant lesions (Figures 27 and 28). Moreover, this strong anti-cancer response conferred long-lasting anti-cancer immunity that prevented recurrence. These solid tumors, especially MC38, KPC, Pan02 and LLC, are known to be difficult to treat in preclinical studies, and it is noteworthy that established tumors >200 mm3 have been shown to resist other effective therapies, including immune checkpoint inhibitors, RT and other combinations. In our experiments, administration of high doses of RT in combination with anti-PD-1 failed to control these tumors in WT mice. Indeed, we found that immune-modulatory treatments upregulate Sirpα - / - The degree of efficacy against these tumors in mice has not been seen in the literature or anywhere else.

[0217] During these studies, intratumoral Sirpα - / - -M shows that depletion of this population occurs after treatment with Sirpα - / - Sirpα suppressed the healing response in WT mice and thus played an important role in tumor elimination (Figure 27C). - / - Adoptive transfer of Sirpα-M dramatically reversed their resistance to treatment and conferred tumor regression (Figure 27D). - / - -M predicted anticancer responses, whereas treatment efficacy leading to tumor elimination was related to the expression of Sirpα in cancer cells. - / - -M is not solely due to enhanced phagocytosis, but rather due to activated Sirpα - / -The results of the study showed that CD8+ T cells in the TME were the result of tumor-specific induction by endothelial growth factor receptor 1 (EGFR)-M, which were found to acquire cancer antigens following phagocytosis and subsequent immunogenic antigen presentation that activated large numbers of tumoricidal T cells. The proliferation of cytotoxic T cells (CD8+) in the TME occurred rapidly after tumor treatment (24 hours) and coincided with the TME changing from "very cold" to "very hot" with respect to CD8 T cell infiltration (Figure 28A). These CD8 T cells displayed a combination of cancer specificity (p15E), potent tumoricidal capacity (granzyme B), and hallmarks of immune memory (CD44 + CD62L - , T EM ), and these attributes contributed to T cell-mediated abscopal inhibition and clearance of cancerous lesions (Figure 28B). Mechanistic studies revealed that Sirpα - / - -M is a tumor-specific memory T cell (i.e., T EM / T RM ) in situ was found to induce T cell activation, a response that is faster and much more robust than DC-mediated activation of naive T cells in lymphoid organs. In addition, activated Sirpα - / - The robust proinflammatory profile of Sirpα-M promoted antitumor responses that attracted antitumor neutrophils and cytotoxic NK cells while downregulating immunosuppressive Tregs and MDSCs, thereby forming a tumoricidal tissue niche that favored tumor elimination (Figures 28C-D). In contrast, Sirpα - / - Tumors lacking Sirpα-M responded to RT by directing the TME towards wound healing and enhanced immunosuppression by increasing TGFβ and MDSC infiltration. - / - Transcriptome profiling of various tumors (e.g., MC38, KPC, and Pan02) that contained or did not contain Sirpα - / - We confirmed that -M initiates a remarkable antitumor response and remodels the immune landscape to promote tumor elimination.

[0218] Discovery of a non-genetic approach to downregulate SIRPα in macrophages The SIRPANT strategy targets activated Sirpα as a central therapeutic weapon against cancer. - / - -phagocytosis-activated SIRPα, which displays similar characteristics to M low We employed a novel IFNγ-dependent phagocytosis inhibitor, SIRPANT-M, in macrophages. The development of SIRPANT-M was based on the discovery that IFNγ has no ability to activate phagocytosis, but dramatically reduces SIRPα protein expression in macrophages from mice and humans (Figures 29A-29C). Screening of other factors found that the cytokines IL-1β, IL-18, IL-6, IFNα, and IFNβ, as well as all TLR agonists tested so far (LPS, CpG, LTA, flagellin, Poly I:C, PGN, etc.), downregulate SIRPα while simultaneously activating phagocytosis. Unlike their ability to rapidly activate phagocytosis (1-6 hours), these factors require approximately 2 days to downregulate SIRPα (>90%), the mechanism of which involves cytokine- and TLR-mediated signaling, which in turn leads to the induction of three microRNAs (mir-17 / 20a / 106a) that inhibit the translation of SIRPα mRNA. Conversely, we found that the immunosuppressive cytokines IL-10, TGFβ, IL-4, and IL-13, as well as the proinflammatory cytokines IL-17 and TNFα, upregulate SIRPα expression, with the latter two also activating phagocytosis. In addition, we found that dexamethasone (DEX) and methylprednisolone (MP) downregulate SIRPα expression, however these glucocorticoids potently inhibit macrophage phagocytosis. These comprehensive studies investigating the dynamics of SIRPα expression and macrophage phagocytic activation informed the selection of reagents to generate therapeutically applicable SIRPANT-M. A cocktail of cytokines and TLR agonists formed the core of a proprietary reagent, Phago-Act™, which was thoroughly explored, retested, and optimized in countless iterations under various experimental conditions. In a single step of processing, Phago-Act™ potently downregulates SIRPα (SIRPα low), activates macrophage phagocytosis against cancer cells and endows macrophages with the expanded proinflammatory phenotype and immunogenic antigen-presenting capacity.

[0219] Phago-Act(trademark) The proprietary reagent Phago-Act™ contains four components: recombinant human interferon gamma (IFNγ), recombinant human interferon alpha A2 (IFNα), CpG oligodeoxynucleotides, and polyinosinic:polycytidylic acid (Poly I:C) and is used for ex vivo treatment of macrophages of both human and murine origin. In Phago-Act™, IFNγ can be present in the range of 40ng / ml to 200ng / ml, IFNα can be present in the range of 40ng / ml to 200ng / ml, CpG oligodeoxynucleotides can be present in the range of 1μg / ml and 5μg / ml, and Poly I:C can be present in the range of 1μg / ml and 5μg / ml. In certain embodiments of Phago-Act™, (IFNγ is present at a concentration of 100 ng / ml, IFNα is present at a concentration of 100 ng / ml, CpG oligodeoxynucleotide is present at a concentration of 2 μg / ml, and Poly I:C is present at a concentration of 2 μg / ml.

[0220] The combination of these reagents is the key intellectual property of the SIRPANT technology and is specially prepared under quality control to ensure efficacy and consistency. low To prepare activated macrophages, SIRPα derived from PBMCs prepared from cancer patients with M-CSF was used. + Treatment of 16-M mice with Phago-Act™ for 48 hours (2 days) (Figure 29D shows the workflow) significantly reduced SIRPα expression, resulting in phenotypically and functionally similar SIRPα expression to that seen when SIRPα is genetically knocked out. lowSIRPANT-M produces a population of macrophages that are highly resistant to phagocytosis. In addition to downregulating SIRPα, the Phago-Act™ formulation also simultaneously endows macrophages with a strong phagocytic capacity, a hyper-inflammatory phenotype, and increased expression of immunogenic antigen-presenting machinery. Ex vivo phenotypic analysis showed that SIRPANT-M maintained phenotypic stability and viability for at least 3 days after completion of Phago-Act™ treatment (Figure 29E), a period during which clinical practice could treat patients. Assays of SIRPANT-M phagocytosis confirmed their ability to engulf a range of cancer cells (Figure 29F; additional data in next section Pharmacology, Figures 30-31). Using a similar experimental setup, phagocytic evaluation of human SIRPANT-M was extended to the entire NCI-60 panel of human cancer cells (Figure 31). As expected, SIRPANT-M exhibited phagocytosis against all healthy cancer cells tested, and this phagocytic capacity was not incidental upon cancer cell expression of CD47 (R 2 = 0.0191). Additional testing of SIRPANT-M phagocytosis of non-apoptotic cancer cells treated with X-ray radiation showed further enhancement of phagocytosis as the irradiated cancer cells expressed DAMPs that promote phagocytosis. In contrast, macrophages prepared from the same donors without Phago-Act™ treatment (SIRPα + SIRPANT-M) was unable to phagocytose either healthy or irradiated cancer cells. Using the same method, we were also able to produce SIRPANT-M from mice, and mouse bone marrow-derived SIRPANT-M from different genetic backgrounds phagocytosed their syngeneic cancer cells, such as C57BL6 / J SIRPANT-M → B16, MC38, KPC, BALB / c SIRPANT-M → 4T1, and breast cancer cells isolated from palpable tumors in FVB / NJ SIRPANT-M → MMTV-PyMT mice (see Figures 30-31).

[0221] SIRPANT-M as an effective immunotherapy for cancer SIRPANT-M produced with Phago-Act™ is a marker for activated Sirpα - / -SIRPANT-M is functionally similar to Rag-1 and has an enhanced ability to activate both innate and adaptive immunity against cancer. SIRPANT-M has been extensively probed in vitro in a number of macrophage phenotypic and functional assays that assessed phagocytosis, pro- and anti-inflammatory responses, and antigen presentation to activate antigen-specific T cells (Figures 34-37). These in vitro studies have demonstrated that SIRPANT-M inhibits adaptive immunity (WT, Rag-1) and inhibits inflammatory responses (WT, Rag-1). - / - The results are complemented by a comprehensive in vivo evaluation of SIRPANT-M in various mouse cancer models across different genetic backgrounds (C57BL6 / J, BALB / C, FVB / NJ) with or without CD47 (nude), as well as in cancers of murine and human origin (Figures 38-45). All these studies demonstrate that SIRPANT-M holds promise as a highly effective immunotherapy for cancer patients by driving tumor neo-antigen-specific, polyclonal and long-lasting T cell and humoral immunity. This therapy does not recapitulate or overlap with any other therapy in practice or development, but is suitable to synergize with immune checkpoint blockade, RT, tumor vaccines and other immunomodulatory regimens. Unlike CD47 blockade, SIRPANT-M does not require cancer-specific antibodies or other methods to direct phagocytosis, thereby making it broadly suitable for many cancers. Indeed, preclinical studies support that SIRPANT-M is a unique tumor-crossing therapy applicable to most, if not all, types of cancer without prior identification of cancer-specific markers. In addition, with the exception of a transient increased inflammatory response associated with tumor clearance, no or minimal side effects were observed in SIRPANT-M treated mice, and animals in which tumors were cleared generally achieved long-term survival (>1 year post-treatment) without recurrence.

[0222] Example 4: Pharmacology of SIRPANT-M SIRPANT-M is a marker for autologous SIRPα produced by Phago-Act™ treatment. LowSIRPANT-M is an activated macrophage. The therapeutic efficacy of SIRPANT-M depends on three factors: i) its ability to phagocytose cancer cells, ii) its ability to drive a robust proinflammatory response in the tumor microenvironment, and iii) its ability to activate tumor-specific T cells that present tumor antigens and exert tumoricidal activity. The in vitro studies presented below focused on the evaluation of these SIRPANT-M properties.

[0223] Cancer cell phagocytosis Both mouse and human SIRPANT-M were produced according to standard operating procedures outlined in Figure 29D and then tested for phagocytosis against cancer cells of mouse or human origin, respectively.

[0224] Methods: Total bone marrow cells from mice of different genetic backgrounds (C57BL6 / J, BALB / C, or FVB / NJ) were cultured (RPMI 1640, 10% fetal bovine serum [FBS], 37°C, 5% CO) in the presence of macrophage colony-stimulating factor (M-CSF, 10 ng / ml) for 5 consecutive days. 2) to differentiate into macrophages (BMDM) in vitro. Differentiated macrophages were then treated with Phago-Act™ (mouse version) for 2 days to produce SIRPANT-M (Figure 31A). Phagocytosis assays were performed by co-culturing SIRPANT-M or control BMDM with healthy syngeneic cancer cells (CFSE-labeled) at a ratio of 1:2 (BMDM:cancer cells) for 4 hours (37°C), followed by evaluation and quantification of phagocytosis by fluorescence microscopy and / or flow cytometry (Figures 31B and 31C). The genetic backgrounds of the cancer cells were as follows: C57BL6 / J-B16F10, MC38, KPC, Pan02, LLC, and EL4; BALB / C-4T1; and FVB / NJ-PyMT breast cancer cells isolated from tumor-bearing MMTV-PyMT mice. SIRPANT-M and control BMDMs were tested against genetically matched syngeneic cancer cells. For fluorescence microscopy, phagocytosis was calculated as follows: (number of BMDMs that engulfed at least one cancer cell / 100 BMDMs in a field of view) x 100. For flow cytometry, phagocytosis was calculated as the number of BMDMs that engulfed at least one cancer cell / 100 BMDMs in a field of view x 100. + BMDM frequency was quantified and statistical significance was determined by Student's t-test.

[0225] Methods: Human PBMC-derived macrophages (SIRPα + -M) were treated with Phago-Act™ for 2 days to generate SIRPANT-M. An additional control was SIRPα + Phagocytosis assays were performed using adherent SIRPANT-M, control SIRPα, and IFNγ-treated SIRPANT-M. + -M, or other processed SIRPα +Phagocytosis was performed by co-incubating CFSE-M with healthy human cancer cells (obtained from the NCI-60 cell line repository) for various periods (37°C), followed by evaluation and quantification of phagocytosis by fluorescence microscopy and / or flow cytometry. Human cancer cells were labeled with CFSE and examined for their CD47 expression by flow cytometry to determine whether their CD47 expression influenced the magnitude of phagocytosis. Statistical significance was determined by one-way ANOVA and post-hoc Dunn's test. Correlation assessment between CD47 expression and phagocytosis was determined by linear regression analysis, and Pearson's coefficients are presented.

[0226] Conclusions: Both mouse bone marrow-derived SIRPANT-M and human PBMC-derived SIRPANT-M demonstrate the ability to directly phagocytose cancer cells in vitro. Furthermore, the ability of SIRPANT-M to phagocytose cancer cells occurs regardless of CD47 expression on the cancer cells. These studies confirm that Phago-Act™ treatment removes CD47-SIRPα-mediated inhibition and provides the activation that allows SIRPANT-M to robustly phagocytose cancer cells.

[0227] Methods: Healthy mice or human cancer cells were treated with non-ablative X-ray radiation (4 Gy, 8 Gy, or 15 Gy) followed by administration of mouse or human SIRPANT-M or control SIRPα. + Phagocytosis was then quantified by fluorescence microscopy and / or flow cytometry. Statistical significance was determined by either Student's t test or one-way ANOVA with post hoc Tukey's test.

[0228] Conclusion: Irradiation of cancer cells significantly increased their susceptibility to phagocytosis by SIRPANT-M. The data indicate that non-ablative radiation induces damage-associated molecules (such as calreticulin) on cancer cells that enhance SIRPANT-M phagocytosis, despite maintaining cancer cell viability and CD47 expression. In contrast, SIRPα +SIRPANT-M does not show a significant improvement in phagocytosis of irradiated cancer cells, partly due to the presence of CD47-SIRPa inhibition. However, blockade of CD47 by anti-CD47 Ab or CD47 deficiency in cancer cells did not improve phagocytosis of irradiated cancer cells by SIRPα, although the extent to which irradiated cancer cells are phagocytosed by SIRPANT-M was unparalleled. + -M improves phagocytosis only partially.

[0229] Inflammatory phenotype and antigen presentation mechanisms Methods: Freshly prepared mouse bone marrow-derived macrophages (BMDMs, SIRPα + Human PBMC-derived SIRPANT-M (+Phago-Act™) and control SIRPα were further treated with Phago-Act™ for 48 hours to induce SIRPANT-M. + Cell culture media of -M (-Phago-Act™) was collected and assayed for pro-inflammatory and anti-inflammatory cytokines by ELISA. Flow cytometry was performed to analyze cell surface expression of antigen-presenting machinery including MHC-I and -II, and costimulatory molecules CD80 and CD86. Total RNA was prepared for mRNA transcription analysis by Nanostring.

[0230] Conclusion: SIRPα + Compared to -M, SIRPANT-M displays an expanded proinflammatory phenotype characterized by increased expression of proinflammatory cytokines, reduced production of immunosuppressive IL-10, and increased expression of immunogenic antigen-presenting machinery including MHC-I / II and costimulatory molecules.

[0231] Methods: Human PBMC-derived SIRPANT-M and donor-matched SIRPα + Total RNA was isolated from seven samples (#1-7) of 14-M. The donors were healthy volunteers, including four men and three women, two of whom were white, two black, two Asian, and one mixed. These RNA samples were subjected to comprehensive sequencing to analyze the expression of over 10,000 genes.

[0232] Conclusion: Donor-matched SIRPα + Compared to SIRPANT-M, SIRPANT-M shows increased expression of genes related to immunogenic antigen presentation mechanisms, including MHC-I, MHC-II, CIITA, and costimulatory molecules (e.g., CD80 / 86 / 40 / 70, OX40L, 4-1BBL, ICAM-1), but decreased expression of nonclassical immune tolerance-associated HLA-G. SIRPANT-M also increases the expression of proinflammatory cytokines and chemokines (e.g., IL-1 / 6 / 12 / 18 / 23 / 27, IFNα / β / γ, TNFα, CXCL1 / 2 / 9 / 10 / 11), while decreasing the expression of anti-inflammatory IL-10, TGFα / β, TGFβR, and CCL2 / 18.

[0233] SIRPANT-M mediates antigen presentation and activates tumor antigen-specific T cells Methods: The experimental scheme is shown in Figure 36A. Mouse bone marrow-derived SIRPANT-M or control BMDM / SIRPα + -M were incubated overnight (approximately 18 hours at 37°C) with irradiated MC38 or KPC tumor cells to allow macrophages to phagocytose cancer cells and process tumor antigens. Tumor infiltrating lymphocytes (TILs) were obtained from resected MC38 or KPC tumors after collagenase digestion of tumor tissue, culture of dissociated cells, and collection of the non-adherent cell population, which was mostly T lymphocytes. Enriched TILs were then incubated at a TIL:macrophage ratio of 5:1 (1 × 10 per well in a 24-well plate) for 1 h at 4°C. 6 TILs and 2 × 10 5 SIRPANT-M or SIRPα + SIRPANT-M-TIL cocultures were then maintained in RPMI-1640 medium containing 10% FBS, 2 mM L-glutamine, and 50 μM β-mercaptoethanol for 8-10 days (37°C, 5% CO 2 ), and 50 IU / ml of recombinant IL-2 was added on day 2. The IL-2-containing medium was replenished every 3 days, and the cell density was maintained at 1 × 10 6The levels of CD8 T cells were kept below 1000 cells / ml. To examine T cell activation and proliferation, fluorescent microscopy and flow cytometry were performed 24 hours after co-culture (d2) to assess TIL-macrophage engagement and T cell expansion (Figures 36E-36F). T cell proliferation was assessed by CFSE dilution at various time points using flow cytometry (Figure 36G). (TILs were pre-labeled with CFSE prior to co-culture for Figures 36E-36G). The amount of CD8 T cells and CD4 T cells was also significantly correlated with SIRPα. + The number of CD8 T cells was determined after co-incubation with tumor-specific MHC tetramers p15E and ADPGK, both indicative of T cell specificity for cancer, in co-cultures containing tumor antigen-loaded SIRPANT-M and TILs (Figure 36B), and after co-incubation with SIRPANT-M that had phagocytosed and processed antigen (+ antigen) or when cancer cells were withheld (- antigen) (Figure 36C). Additional flow cytometry analysis was performed to determine the frequency and quantity of CD8 T cells that expressed granzyme B and were reactive to tumor-specific MHC tetramers p15E and ADPGK, both indicative of T cell specificity for cancer (Figures 36H-36I). In co-cultures containing tumor antigen-loaded SIRPANT-M and TILs, the total T cell number was 1×10 6 Approximately 2 x 10 cells 7 The number of T cells expanded by a typical 20-fold until 100,000 T cells were identified, of which >95% were CD8 T cells after 8–10 days of coculture. Depending on the tumor type, the expanded T cells were classified as T MC38 Or T KPC These were termed as serogroups and were tested for their tumoricidal toxicity against MC38 or KPC cancer cells, respectively, in vitro (Figures 36J-36K) and in vivo by adoptive T cell therapy.

[0234] Conclusion: These studies demonstrated that: i) tumor-engulfed SIRPANT-M are excellent antigen-presenting cells (APCs), which mediate immunogenic antigen presentation and robustly activate tumor-specific CD8+ cytotoxic T cells (CTLs) from TILs; ii) SIRPANT-M can mediate the induction of memory tumor-specific T cells (i.e., T EM / T RM) activates CD8 T cells through in situ calling of SIRPANT-M; iii) SIRPANT-M-mediated antigen presentation preferentially activates tumor-specific CD8+ cytotoxic T cells, but not CD4+ T helper cells (Th); iv) SIRPANT-M-activated CD8 T cells highly express granzyme B and exhibit polyclonal cancer specificity; v) SIRPANT-M-activated CD8 T cells are highly cytotoxic to cancer and rapidly eliminate cancer cells at relatively low effector:target ratios. These conclusions are consistent with in vivo experiments in mouse tumor models.

[0235] Methods: A similar experimental scheme was followed as detailed in Figure 36A, except that B16 melanoma-specific naive CD8 T cells were induced with SIRPANT-M or control BMDM / SIRPα. + BMDM / SIRPα-M were co-cultured with parental B16F10 melanoma cells or gp33-expressing B16F10 melanoma cells and subjected to multiple freeze-thaw cycles to induce immunogenic cell death and provide B16 antigen. Then, B16 antigen-loaded SIRPANT-M or control BMDM / SIRPα-M were co-cultured with parental B16F10 melanoma cells or gp33-expressing B16F10 melanoma cells and subjected to multiple freeze-thaw cycles to induce immunogenic cell death and provide B16 antigen. + -M, H-2D b Naive splenic CD8 from P14 transgenic mice expressing TCR specific for a restricted gp33 epitope + were co-cultured with T cells.

[0236] Conclusion: These experiments confirmed that SIRPANT-M is an excellent APC that can phagocytose tumor antigens, followed by antigen presentation and activation of antigen-specific naive CD8+ T cells.

[0237] Example 5: In vivo pharmacology studies The ability of SIRPANT-M to drive anti-cancer responses in vivo has been extensively tested in various preclinical cancer models in mice (C57BL6, BalbC, FVB / NJ) across various genetic backgrounds. These cancers include lymphoma, colorectal adenocarcinoma, melanoma, lung cancer, pancreatic ductal adenocarcinoma, metastatic breast cancer, carcinogen- and inflammation-induced colon cancer, and others. Among these tested cancers, some were late-stage cancers with large tumors with distant lesions (metastases). In all cases, SIRPANT-M exerts potent anti-cancer activity when administered to the tumor mass, exhibits direct phagocytosis of cancer cells, drives proinflammatory responses and downstream presentation of tumor-associated neo-antigens, and activates tumoricidal T cells in an immunogenic manner. As a result, large numbers of tumor-specific polyclonal cytotoxic T cells proliferate to combat the tumor and distant lesions (metastases), achieving (i) rapid and systemic elimination of solid tumors, and (ii) induction of long-lasting anti-cancer immune T cells and antibodies that prevent cancer recurrence.

[0238] The following section describes preclinical cancer therapy studies conducted in mice.

[0239] SIRPANT-M monotherapy Treatment: SIRPANT-M intratumoral injection (it) Dosage: D1 / 2=0.5x10 4 / mm 3 Tumor mass D1=1x10 4 / mm 3 Tumor mass D2=2x10 4 / mm 3 Tumor mass

[0240] Cancer types: i. colorectal adenocarcinoma MC38-C57BL6 syngraft, ii. pancreatic ductal adenocarcinoma (PDA) KPC-C57BL6 syngraft, iii. pancreatic ductal adenocarcinoma (PDA) Pan02 - - C57BL6 syngraft, iv. lung cancer LLC-C57BL6 syngraft, v. lymphoma EL4-C57BL6 syngraft, and vi. MMTV-PyMT triple negative metastatic breast cancer - FVB / NJ spontaneous.

[0241] Testing Procedure: Tumor model: In the syngeneic transplant model, healthy cultured EL4, MC38, LLC, KPC, Pan02 cancer cells (5x10 5 ) were suspended in 50 μl of PBS and implanted subcutaneously into WT C57BL6 mice (6-8 weeks, male or female). Palpable tumors generally formed after 10-18 days, with a growth rate dependent on the cancer type. Measurements of tumor length and width were taken using calipers, and tumor volume (V) was then calculated according to the formula: Volume = (Length × Width). 2 ) / 2. MMTV-PyMT mice were obtained from The Jackson Laboratory (002374 FVB / N-Tg(MMTV-PyVT) 634Mul / J). Female PyVT transgene carriers spontaneously develop palpable mammary tumors at approximately 2 months of age (mean latency 53 days).

[0242] Preparation of SIRPANT-M: Femurs were obtained from WT C57BL6 mice or male MMTV-PyVT mice. Bone marrow derived macrophages (BMDM) were generated by M-CSF, followed by treatment of BMDM with Phago-Act™ (37°C, 48 hours) to generate SIRPANT-M. Prior to use, SIRPANT-M was trypsinized from the culture dish and, after washing, the cells were cultured at 1x10 8 The cells were resuspended in PBS at 100 / ml and used for 0.5-3 h (kept on ice before use). Flow cytometry analysis confirmed that SIRPANT-M was SIRPa Low and increased expression of MHC-I, MHC-II, CD80, and CD86 was confirmed. Only genetically matched SIRPANT-M was used to treat tumors in mice of different backgrounds, such as treating EL4, MC38, LLC, KPC, and Pan02 tumors in C57BL6 mice with SIRPANT-M prepared from C57BL6 mice, and treating PyMT breast cancer in mice of the same background with SIRPANT-M prepared from FVB / NJ mice.

[0243] Tumor treatment: The dose of SIRPANT-M was calculated according to the tumor size. SIRPANT-M in PBS was injected it into the tumor following a multi-point injection mode, e.g., 2-4 injections from different directions or angles of the tumor, using an Exel-Comfort Point insulin injection needle (29G1 / 2), a procedure to improve SIRPANT-M diffusion in the tumor tissue. Treatment was repeated every 3 days, giving a total of 2-3 treatments.

[0244] Conclusions of the SIRPANT-M monotherapy study: SIRPANT-M potently inhibits tumor growth or induces tumor regression in a dose-dependent manner when administered it.

[0245] SIRPANT-M monotherapy significantly increased animal survival and, in the case of small tumors, produced complete responses with long-term survival.

[0246] The antitumor effect of SIRPANT-M was cross-sectional across tumor types, showing potent inhibition against all tumors tested.

[0247] Combination of SIRPANT-M and radiation therapy (RT) Treatment modality: 1-SIRPANT-M intratumoral injection (it) 2- Non-ablative tumor-focused x-ray radiation (RT) SIRPANT-M dose: D1 / 2=0.5x10 4 / mm 3 Tumor mass D1=1x10 4 / mm 3 Tumor mass D2=2x10 4 / mm 3 Tumor mass RT dose: X-ray 4Gy X-ray 8Gy X-ray 15Gy

[0248] Cancer types: i. colorectal adenocarcinoma MC38-C57BL6 syngeneic graft; ii. pancreatic ductal adenocarcinoma (PDA) KPC-C57BL6 syngeneic graft; iii. pancreatic ductal adenocarcinoma (PDA) Pan02-C57BL6 syngeneic graft; iv. lung cancer LLC-C57BL6 syngeneic graft; v. lymphoma EL4-C57BL6 syngeneic graft; vi. triple negative breast cancer (TNBC) 4T1-Balb C orthotopic graft; and vii. MMTV-PyMT triple negative breast cancer (TNBC)-FVB / NJ spontaneous.

[0249] Testing Procedure: Tumor models: Using the same procedures as in the last section (monotherapy), syngeneic transplant models of EL4, MC38, LLC, KPC and Pan02 tumors in WT C57BL6 mice were established. To establish distal lesions, 5x10 cells were used for the formation of primary tumors. 5 One location (e.g., the right flank) was implanted with 0.5–2 x 10 tumor cells, and other locations, such as the left flank, right and / or left armpit, and peritoneal cavity, were implanted with 0.5–2 x 10 tumor cells. 5 Tumor cells were implanted to form smaller "distal" lesions. In some experiments, two primary tumors were implanted along with multiple distal lesions. 4T1 orthotopic breast cancer was established in Balb C mice. In this model, 3x10 cells suspended in 50-μl PBS were implanted. 4 4T1 cells were injected into the mammary fat pad of 6-8 week-old female Balb C mice, typically resulting in the formation of palpable tumors 2 weeks after implantation. Establishment of MMTV-PyMT triple-negative metastatic breast cancers is described in the last section.

[0250] Preparation of SIRPANT-M: The same procedure (Figure 29D) was followed to prepare bone marrow-derived SIRPANT-M from C57BL6, MMTV-PyVT, or Balb C mice. Only genetically matched SIRPANT-M was used to treat tumors from mice with the same background to ensure homogeneity, such as using SIRPANT-M prepared from C57BL6 mice to treat EL4, MC38, LLC, KPC, and Pan02 tumors in C57BL6 mice, and using SIRPANT-M prepared from Balb C mice to treat 4T1 breast cancer transplanted into Balb C mice.

[0251] Tumor treatment: i) SIRPANT-M it- Freshly prepared SIRPANT-M, calculated according to the tumor size suspended in PBS, was injected into the tumor mass following a multi-point injection mode of 2-4 injections from different directions or angles of the tumor, for example using an Exel-Comfort Point insulin injection needle (29G1 / 2).

[0252] ii) Tumor RT: Tumor-bearing mice, anesthetized with ketamine (17.5 mg / ml, Henry Schein) and xylazine (2.5 mg / ml, Henry Schein), were placed in a customized jig with a lead holder so that only the primary tumor was exposed, and then irradiated with an RS-2000 Biological X-ray Irradiator (Rad Source Technology) at a dose rate of 1.2 Gy / min (160 kV, 25 mA) until 4 Gy, 8 Gy, 10 Gy, or 15 Gy was reached.

[0253] iii) Combination: SIRPANT-M it was administered either before or after a fraction of radiation given to the same tumor. We tested SIRPANT-M it given 0.5-48 hours before or at the same time after tumor-confined RT.

[0254] Study-1: Trial of SIRPANT-M it in combination with RT at different doses (4Gy, 8Gy or 15Gy) to treat RT-refractory colorectal adenocarcinoma MC38 and pancreatic ductal adenocarcinoma KPC and Pan02 at different stages (different tumor sizes). Partial data is shown in Figure 40.

[0255] Study-2: A trial of 8 Gy RT in combination with SIRPANT-M at various doses to treat RT-refractory colorectal adenocarcinoma MC38 and pancreatic cancers KPC and Pan02. Figure 41 shows partial data from the study.

[0256] Study-3: Testing the abscopal effect. Given that SIRPANT-M mediates its anticancer effects primarily through their immunogenic antigen presentation and activation of tumor-specific T cells, potent abscopal tumoricidal activity is expected thereby. In this study, SIRPANT-M was tested for its ability to induce the abscopal effect, resulting in the inhibition and / or clearance of distant cancer lesions (mimicking metastases).

[0257] Study-3-1: Testing the combination of SIRPANT-M and RT for abscopal effect to systemically eliminate KPC pancreatic cancer with distant lesions. KPC / Luc pancreatic adenocarcinoma tumors were implanted simultaneously in multiple locations, with one or two implants forming primary tumors. After tumor formation, the primary tumor(s) were treated with SIRPANT-M it plus RT for 2 or 3 cycles (3-day intervals) according to the 8Gy (1st)-4Gy-4Gy RT scheme, followed by immediate SIRPANT-M it at D2 dose, respectively. Other cancer lesions were untreated. Whole-body imaging was performed to monitor the primary and systemic KPC tumors for progression, regression, or clearance. Partial data is shown in Figure 42.

[0258] Study-3-2: Testing the combination of SIRPANT-M and RT for abscopal effect to eliminate MC38 colorectal cancer with distal disease. In this study, MC38 adenocarcinoma was implanted in both flanks. After tumor formation, the right-sided tumor (primary) was treated with SIRPANT-M it plus RT for two cycles (8 Gy in the first cycle, 4 Gy in the second cycle, 3 days apart), while the left-sided tumor was left untreated. One additional SIRPANT-M and 4 Gy RT treatment (3rd cycle) was performed in patients with primary tumors with a volume of ≥ 100 mm after two cycles of treatment. 3 Tumor volumes were measured on both flanks throughout treatment to monitor the abscopal effect and systemic MC38 tumor clearance. Partial data are shown in FIG.

[0259] Study-4: Testing the timing and sequence of administration of two modalities, SIRPANT-M it and RT. Studies were conducted to compare the efficacy of SIRPANT-M it given before and after tumor RT. These studies conclude that the two treatment modalities should be administered within a short time interval (3 hours) and that SIRPANT-M it given before or after tumor RT achieves similar efficacy. A longer time interval between the two modalities results in a decrease in treatment efficacy. Figure 44 shows data on MC38 colorectal cancer and EL4 lymphoma treated with two modalities in different sequences.

[0260] Study-5: Testing the combination of SIRPANT-M and RT to treat other RT-refractory cancers. In these studies, SIRPANT-M it was tested in combination with 8Gy RT to treat additional cancers including LLC lung cancer (sc), EL4 lymphoma (sc), 4T1 orthotopically implanted triple-negative breast cancer, and PyMT spontaneous triple-negative breast cancer in MMTV-PyMT mice. The efficacy of the combination of SIRPANT and RT was compared to treatment with the same dose of RT alone. Partial data is shown in Figure 45.

[0261] summary: Both in vitro and in vivo studies have demonstrated that Phago-Act™-produced SIRPANT-M is a potent anti-cancer immune initiator and that SIRPANT-M (SIRPa low Strategies using activated macrophages have been confirmed to be effective in eliminating cancer and metastases. The table below summarizes our in vivo studies using a D2 dose of SIRPANT-M administered by intratumoral injection (it). [Table 1]

[0262] Example 6: Considering that the mechanism by which SIRPANT-M achieves cancer elimination depends on the tumoricidal activity of activated tumor-specific T cells, combining SIRPANT-M+RT with checkpoint inhibitors that enhance T cell activity will expand the ability to eliminate tumors and clear distant lesions (metastases). In this example, we test these possibilities and use the generated data to determine clinical treatment schemes and modalities within IND protocols. Two lines of experiments test either SIRPANT-M+RT±anti-PD1 / L1 or anti-CTLA4 to treat pancreatic adenocarcinoma KPC or colorectal cancer MC38 in subcutaneous tumor models (IIB-1 and IIB-2). To closely mimic the treatment of cancers formed in humans, two additional lines of experiments test SIRPANT-M+RT±anti-PD1 / L1 or anti-CTLA4 against inflammation (DSS-colitis) or carcinogen (AOM)-induced colorectal neoplasms / cancers (IIB-3 and IIB-4). In contrast to syngeneic transplants such as subcutaneous models, which predispose to immune responses and do not form tumors in their natural location, DSS-AOM-induced colorectal cancer occurs at sites of inflammation, is associated with exacerbated colitis, and is induced by the presence of carcinogens that cause mutations in oncogenes and tumor suppressor genes. This cancer model therefore closely resembles how cancers form "naturally" in humans. Examples of such cancers include those that form in the lung, colon, ovary, breast, prostate, etc. The testing of SIRPANT-M treatment against this naturally occurring cancer supports its application to a wider variety of cancer patients.

[0263] In addition to optimizing cancer treatment strategies, human SIRPα low Design and test quality control (QC) assays required for CMC production of macrophages and human SIRPα from peripheral blood monocytes (PBMCs). low The current production of macrophages involves treatment with M-CSF for 5 days to differentiate macrophages and downregulating SIRPα. lowAccording to the diagram in Figure 46, which involves 48 hours of treatment with a proprietary drug "Phago-Act™" to generate macrophages, two QC assays, QC1 and QC2, are designed. QC1 is performed after 48 hours of Phago-Act™ treatment to confirm that the macrophages have achieved the desired phenotype and function. QC2 is performed after 48 hours of Phago-Act™ treatment to confirm that the macrophages have achieved the desired phenotype and function. low These assays are performed prior to administration of macrophages to patients to ensure sterility, cell viability, and other clinically relevant parameters. The design of QC1 / 2 is shown in Tables 2 and 3 and will test these assays. [Table 2] [Table 3]

[0264] Example 7: Inhibition of SHP-1 downstream of SIRPα as a potential therapy for cancer SIRPα mediates inhibitory regulation of macrophages through activation of the SH domain containing tyrosine phosphatase SHP-1, which subsequently mediates extensive protein dephosphorylation and terminates multiple cytokine and TLR-mediated activation pathways. In addition to downregulating SIRPα, SHP-1 inhibition was also tested as an alternative approach to deplete SIRPα-SHP-1-mediated inhibition.

[0265] The SHP-1 inhibitor TPI-1 (Kundu et al., J Immunol 2010 184:6529-6536) was purchased from Cayman Chemical (also available from Selleck Chemicals). TPI-1 was used as a single agent or in combination with RT to treat subcutaneously established colorectal cancer (CRC) MC38 and pancreatic ductal adenocarcinoma (PDA) KPC.

[0266] Testing the SHP-1 inhibitor TPI-1 for treating CRC and PDA tumors in vivo MC38 or KPC tumors are approximately 200 mm 3 Once the tumor volume reached 100 μg, tumors were intratumorally injected with 20 μg TPI-1 in 50 μl PBS (dosage was calculated according to 1 mg / kg body weight). Treatment was repeated 2 days later. For combination treatment, mice intratumorally injected with TPI were given 30 min to allow TPI to diffuse into the tumor tissue, followed by local 8 Gy X-ray irradiation. This TPI+8 Gy RT treatment was repeated 2 days later. Controls were untreated (no treatment) tumors or tumors treated with 8 Gy RT (RT only). Tumor volumes were measured every other day and calculated using the spheroid formula (V=a2b / 2), where a and b are the width and length of the tumor, respectively, in mm. The induction of tumor treatments on immune landscape changes in the TME was examined 48 hours after treatment. KPC tumors were also imaged with a bioluminescence imager. Figure 47A shows the results of KPC treatment, and Figure 47B shows the results of MC38.

[0267] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed invention belongs. Publications cited herein and the material for which they are cited are specifically incorporated herein by reference.

[0268] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims.

[0269] Numbered Embodiments The following list of embodiments is included herein for illustrative purposes only and is not intended to be exhaustive or limiting. Claimed subject matter is expressly not limited to the following embodiments.

[0270] Embodiment 1. Activated SIRPα low 1. A method for producing macrophages, comprising: (a) isolating monocytes from peripheral blood mononuclear cells (PBMCs) in a biological sample; (b) differentiating monocytes in vitro to produce macrophages; (c) contacting the macrophage with a SIRPα inhibitor; (d) Macrophages were contacted with a macrophage activator, which significantly reduced SIRPα cell surface expression compared to untreated macrophages (SIRPα low ) generating a population of macrophages; SIRPα low The method, wherein the macrophages have activated phagocytosis against cancer cells, an increased proinflammatory response, and increased immunogenic antigen presentation.

[0271] Embodiment 2. The method of claim 1, wherein the SIRPα inhibitor suppresses expression of SIRPα, reduces the abundance of SIRPα on the surface of a cell, inhibits the activity of SIRPα, disrupts the interaction between SIRPα and CD47, or a combination thereof.

[0272] Embodiment 3. The method of claim 1 or 2, wherein the SIRPα inhibitor comprises a cytokine, a TLR ligand, a glucocorticoid, or a combination thereof.

[0273] Embodiment 4. The method of any one of claims 1 to 3, wherein the SIRPα inhibitor is selected from the group consisting of IFNα, IFNβ, IFNγ, IL-1, IL-6, IL-12, IL-18, LPS, CpG, Poly I:C, LTA, PGN, flagellin, Pam3CSK4, zymosan, and HMGB1.

[0274] Embodiment 5. The method of any one of claims 1 to 4, wherein the macrophage activator comprises a cytokine, a phorbol ester, a TLR ligand, or a combination thereof.

[0275] Embodiment 6. The method of claim 5, wherein the cytokine is selected from the group consisting of IFNα, IFNβ, IL-6, IL-1, IL-17, IL-18, TNFα, and IL-12.

[0276] Embodiment 7. The method of claim 5, wherein the phorbol ester comprises phorbol 12-myristate 13-acetate (PMA).

[0277] Embodiment 8. The method of claim 5, wherein the TLR ligand is selected from the group consisting of LPS, CpG, Poly I:C, LTA, PGN, flagellin, Pam3CSK4, zymosan, and HMGB1.

[0278] Embodiment 9. The method of claim 3, wherein the glucocorticoid comprises methylprednisolone or dexamethasone.

[0279] Embodiment 10. The method of any one of claims 1 to 9, wherein the SIRPα inhibitor and the macrophage activator are contacted with the macrophage sequentially.

[0280] Embodiment 11. The method of any one of claims 1 to 9, wherein the SIRPα inhibitor and the macrophage activator are contacted with the macrophages simultaneously or in parallel.

[0281] Embodiment 12. The method of any one of claims 1 to 9 and 11, wherein the SIRPα inhibitor and the macrophage activator are present in the same composition.

[0282] Embodiment 13. The method of any one of claims 1 to 12, wherein the composition comprises recombinant human interferon gamma (IFNγ), recombinant human interferon alpha A2 (IFNα), CpG oligodeoxynucleotides, and polyinosinic:polycytidylic acid (Poly I:C).

[0283] Embodiment 14. The method of any one of claims 1 to 13, wherein the SIRPα inhibitor comprises an SHP-1 inhibitor.

[0284] Embodiment 15. The SHP-1 inhibitor is selected from the group consisting of TPI-1 (2-(2,5-dichlorophenyl)-1,4-benzoquinone), TPI-1a1 (2-(2,5-dichlorophenyl)-2,4-benzoquinone), TPI-1a2 (2-(3-chlorophenyl)-1,4-benzoquinone), TPI-1a3 (2-phenylnaphthoquinone), TPI-1a4 (2-(4-ethoxyphenyl)-1,4-benzoquinone), TPI-1a5 (2-(4-methoxyphenyl)-1,4-benzoquinone), TPI-1a6 (2-(4-methoxyphenyl)-1,4-benzoquinone), TPI-1a7 (2-(4-methoxyphenyl)-1,4-benzoquinone), TPI-1a8 (2-(4-methoxyphenyl)-1,4-benzoquinone), TPI-1a9 (2-(4-methoxyphenyl)-1,4-benzoquinone), TPI-1b (2-(4-methoxyphenyl)-1,4-benzoquinone), TPI-1b1 (2-(4-methoxyphenyl)-1,4-benzoquinone), TPI-1b2 (2-(4-methoxyphenyl)-1,4-benzoquinone), TPI-1b3 (2-(4-methoxyphenyl)-1,4-benzoquinone), TPI-1b4 (2-(4-methoxyphenyl)-1,4-benzoquinone), TPI-1b5 (2-(4-methoxyphenyl)-1,4-benzoquinone), TPI-1b6 (2-(4-methoxyphenyl)-1,4-benzoquinone), TPI-1b7 (2-(4-methoxyphenyl)-1,4-benzoquinone), TPI-1b8 (2-(4-methoxyphenyl)-1,4-benzoquinone), TPI-1b9 (2-(4-methoxyphenyl)-1,4-benzoquinone), TPI-1b10 (2-(4-methoxyphenyl) 15. The method of claim 14, wherein the inhibitor is selected from the group consisting of N,N'-[1,4-phenylenebis[(1-methylethylidene)-4,1-phenylene]]bis[1,1,1-trifluoro-methanesulfonamide), NSC 23922 (3-aminocholestane), and NSC 87877 (8-hydroxy-7-[2-(6-sulfo-2-naphthalenyl)diazenyl]-5-quinolinesulfonic acid).

[0285] Embodiment 16. The method of any one of claims 1 to 15, further comprising contacting the macrophage with a SHP-1 inhibitor.

[0286] Embodiment 17. The method of claim 16, wherein the SHP-1 inhibitor is an irreversible SHP-1 inhibitor.

[0287] Embodiment 18: Activated SIRPα produced by the method according to any one of claims 1 to 17. low A composition comprising a macrophage.

[0288] Embodiment 19. A method for producing in vitro grown tumor-specific peripheral blood T (PBT) cells, comprising: (a) isolating peripheral blood T (PBT) cells from a biological sample; (b) Activated SIRPα produced by the method of embodiment 1. low Macrophages were co-cultured in vitro with cells from tumor biopsies to express tumor-supplied SIRPα low Producing macrophages; (c) Tumor-supplying SIRPα low co-culturing macrophages with the isolated PBT cells in vitro to expand the number of tumor-specific T cells, thereby producing in vitro expanded tumor-specific PBT cells.

[0289] Embodiment 20. A composition comprising in vitro grown tumor-specific PBT cells produced by the method of claim 19.

[0290] Embodiment 21. A method for generating in vitro expanded tumor-specific T cells from tumor-infiltrating T lymphocytes (TILs), comprising: (a) isolating tumor-infiltrating T lymphocyte (TIL) cells from a tumor biopsy; (b) Activated SIRPα produced by the method of embodiment 1. low Macrophages were co-cultured in vitro with tumor cells from tumor biopsies to detect tumor-supplied SIRPα low Producing macrophages; (c) Tumor-supplying SIRPα low co-culturing macrophages with the isolated TIL cells in vitro to expand the number of tumor-specific T cells, thereby producing in vitro expanded tumor-specific T cells from the TILs.

[0291] Embodiment 22. A composition comprising in vitro expanded tumor-specific T cells from TILs produced by the method of claim 21.

[0292] Embodiment 23. A method for treating a tumor in a subject, comprising administering to the subject a therapeutically effective amount of the composition described in embodiment 18, the in vitro expanded tumor-specific PBT cells described in embodiment 20, the in vitro expanded tumor-specific T cells from TILs described in embodiment 22, or any combination thereof.

[0293] Embodiment 24. The method of claim 23, further comprising treating the subject with tumor-directed radiation.

[0294] Embodiment 25. The method of claim 23 or 24, further comprising administering to the subject a therapeutically effective amount of an immune checkpoint inhibitor.

[0295] Embodiment 26. The method of claim 25, wherein the immune checkpoint inhibitor comprises an anti-PD1, anti-PD-L1, anti-CTLA4 antibody, or a combination thereof.

[0296] Embodiment 27. The method of any one of claims 23 to 26, wherein the subject is refractory to PD-1 blockade.

[0297] Embodiment 28. The method of any one of claims 23 to 27, further comprising treating the subject with an oncolytic virus.

[0298] Embodiment 29. The method of claim 28, wherein the oncolytic virus is a vesicular stomatitis virus.

[0299] Embodiment 30. A composition comprising recombinant human interferon gamma (IFNγ), recombinant human interferon alpha A2 (IFNα), CpG oligodeoxynucleotides, and polyinosinic:polycytidylic acid (Poly I:C).

[0300] Embodiment 31. The composition of claim 30, wherein IFNγ is present at a concentration ranging from about 40 ng / ml to about 200 ng / ml.

[0301] Embodiment 32. The composition of claim 30 or 31, wherein IFNγ is present at a concentration of about 100 ng / mL.

[0302] Embodiment 33. The composition of any one of claims 30 to 32, wherein IFNα is present at a concentration ranging from about 40 ng / ml to about 200 ng / ml.

[0303] Embodiment 34. The composition of any one of claims 30 to 33, wherein IFNα is present at a concentration of about 100 ng / mL.

[0304] Embodiment 35. The composition of any one of claims 30 to 34, wherein the CpG oligodeoxynucleotide is present at a concentration ranging from about 1 μg / ml to about 5 μg / ml.

[0305] Embodiment 36. The composition according to any one of claims 30 to 35, wherein the CpG oligodeoxynucleotide is present in a concentration of 2 μg / ml.

[0306] Embodiment 37. The composition of any one of claims 30 to 36, wherein Poly I:C is present at a concentration ranging from about 1 μg / ml to about 5 μg / ml.

[0307] 38. The composition of any one of claims 30 to 37, wherein Poly I:C is present at a concentration of about 2 μg / ml.

[0308] Embodiment 39. The composition of any one of claims 30 to 38, wherein the composition comprises about 100 ng / ml IFNγ, about 100 ng / ml IFNα, about 2 μg / ml CpG oligodeoxynucleotide, and about 2 μg / ml Poly I:C.

[0309] Embodiment 40. Activated SIRPα produced by a method comprising contacting macrophages with an effective amount of the composition of any one of embodiments 30 to 39. low A composition comprising a macrophage.

[0310] Embodiment 41. One or more activated SIRPα low 1. A method for producing macrophages, comprising: (a) providing one or more macrophages; (b) contacting one or more macrophages with a composition according to any one of embodiments 30 to 40, thereby activating one or more SIRPα lowand producing macrophages.

[0311] Embodiment 42. The method of claim 41, wherein step (a) comprises: (i) collecting from the subject a biological sample comprising one or more peripheral blood mononuclear cells (PBMCs); (ii) isolating one or more monocytes from the PBMCs; and (iii) culturing the one or more monocytes in vitro to produce one or more macrophages.

[0312] Embodiment 43. The method of claim 42, wherein step (iii) comprises culturing one or more monocytes in the presence of a macrophage differentiation factor.

[0313] Embodiment 44. The method of claim 43, wherein the macrophage differentiation factor comprises macrophage colony stimulating factor (M-CSF), GM-CSF, IL-6, human serum, IL-4, IL-10, IFN-α, IL-1, TGF-β, or any combination thereof.

[0314] Embodiment 45. The method of any one of claims 42 to 44, wherein the biological sample is blood or serum.

[0315] Embodiment 46. The method of any one of claims 41 to 45, wherein the macrophage is a bone marrow-derived macrophage or a monocyte-derived macrophage.

[0316] Embodiment 47: Activated SIRPα produced by the method according to any one of claims 41 to 46. low A composition comprising a macrophage.

[0317] Embodiment 48. A method for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of a composition described in claim 40 or 47.

[0318] Embodiment 49. The method of any one of claims 23 to 29 and 48, further comprising administering to the subject one or more damage-associated molecular patterns (DAMPs).

[0319]

[0036] In one embodiment, the one or more DAMPs are selected from the group consisting of high mobility group box 1 protein (HMGB1), heat shock protein (HSP), SNAP-related protein (SNAPIN), versican, biglycan, decorin, eosinophil-derived neurotoxin, surfactant protein A / D, beta-defensin 3, histones, serum amyloid A (SAA), beta amyloid (Aβ), beta 2-glycoprotein I, mRNA, tenascin-C, S100 proteins, high mobility group box 1 protein (HMGN1), biglycan, decorin, heparin sulfate, hyaluronic acid, fibrinogen, fibronectin, beta-dipeptide, β ... Fensin 2, surfactant protein A / D, lactoferrin, neutrophil elastase, peroxiredoxin, histones, serum amyloid A (SAA), ox-LDL, IgG-ribonucleoprotein complex, microRNA, mtDNA, F-actin, Sin3A-related protein 130, β-glucosylceramide, N-glycans, monosodium urate (MSU), glucose, cholesterol crystals, ATP, oxidized 1-palmitoyl-2-arachidonyl sn-glycero-3-phosphocholine (ox-PAPC), RNA transcribed from Alu elements (Alu-RNA), endogenous 5′pppRNA, unedited long self-dsRNA, endogenous retroviral RNA, cytoplasmic DNA, damaged nuclear DNA, advanced glycation end products (AGE), DNA, HSP70, peptidoglycan recognition protein 1 (PGLYRP1), actin, phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), cardiolipin, sulfatides, sphingomyelin, apolipoprotein A1 (APOA1), apolipoprotein A2 (APOA2), 50. The method of claim 49, comprising apolipoprotein B (APOB), apolipoprotein E (APOE), apolipoprotein J (APOJ), low density lipoprotein (LDL), high density lipoprotein (HDL), very low density lipoprotein (VLDL), Lp(a), HSP60, N-for...

Claims

1. 1. A method for producing activated macrophages, comprising: (a) isolating monocytes; (b)i contacting the monocytes with a composition comprising macrophage colony-stimulating factor (M-CSF), GM-CSF, IL-6, human serum, IL-4, IL-10, IFN-α, IL-1, TGF-β, or any combination thereof to produce macrophages; ii. contacting the macrophage with a SIRPα inhibitor; and iii. contacting the macrophages with a macrophage activator, thereby generating a population of activated macrophages; differentiating in vitro monocytes isolated from peripheral blood mononuclear cells (PBMCs) in a biological sample obtained from a subject to produce macrophages by: A method comprising:

2. 10. The method of claim 1, wherein the SIRPα inhibitor comprises a cytokine, a TLR ligand, a glucocorticoid, or a combination thereof.

3. 2. The method of claim 1, wherein the SIRPα inhibitor is selected from the group consisting of IFNα, IFNβ, IFNγ, IL-1, IL-6, IL-12, IL-18, LPS, CpG, Poly I:C, LTA, PGN, flagellin, Pam3CSK4, zymosan, and HMGB1.

4. 10. The method of claim 1, wherein the macrophage activator comprises a cytokine, a phorbol ester, a TLR ligand, or a combination thereof.

5. 5. The method of claim 4, wherein the cytokine is selected from the group consisting of IFNα, IFNβ, IL-6, IL-1, IL-17, IL-18, TNFα, and IL-12.

6. 5. The method of claim 4, wherein the TLR ligand is selected from the group consisting of GARDIQUIMOD™ (CAS No. 1020412-43-4), IMIQUIMOD™ (CAS No. 99011-02-6), LPS, CpG, Poly I:C, LTA, PGN, flagellin, Pam3CSK4, zymosan, and HMGB1.

7. 10. The method of claim 1, wherein the SIRPα inhibitor and macrophage activator are present in the same composition.

8. 8. The method of claim 7, wherein the composition comprises recombinant human interferon gamma (IFNγ), recombinant human interferon alpha A2 (IFNα), CpG oligodeoxynucleotides, and polyinosinic:polycytidylic acid (Poly I:C).

9. 2. The method of claim 1, wherein the SIRPα inhibitor comprises an SHP-1 inhibitor.

10. The method of claim 1, further comprising contacting the macrophage with an SHP-1 inhibitor.

11. The SHP-1 inhibitor is selected from the group consisting of TPI-1 (2-(2,5-dichlorophenyl)-1,4-benzoquinone), TPI-1a1 (2-(2,5-dichlorophenyl)-2,4-benzoquinone), TPI-1a2 (2-(3-chlorophenyl)-1,4-benzoquinone), TPI-1a3 (2-phenylnaphthoquinone), TPI-1a4 (2-(4-ethoxyphenyl)-1,4-benzoquinone), TPI-1a5 (2-(4-methoxyphenyl)-1,4-benzoquinone), 11. The method of claim 9 or 10, wherein the inhibitor is selected from the group consisting of SSG (sodium stibogluconate), PTP inhibitor I (2-bromo-1-(4-hydroxyphenyl)-ethanone), PTP inhibitor II (2-bromo-1-(4-methoxyphenyl)-ethanone), PTP inhibitor III (2-[4-(2-bromoacetyl)phenoxy]-acetic acid), PTP inhibitor IV (N,N'-[1,4-phenylenebis[(1-methylethylidene)-4,1-phenylene]]bis[1,1,1-trifluoro-methanesulfonamide), NSC 23922 (3-aminocholestane), and NSC 87877 (8-hydroxy-7-[2-(6-sulfo-2-naphthalenyl)diazenyl]-5-quinolinesulfonic acid).

12. 1. A method for generating in vitro expanded tumor-specific T cells from tumor-infiltrating T lymphocytes (TILs), comprising: (a) isolating tumor-infiltrating T lymphocyte (TIL) cells from a tumor biopsy; (b) co-culturing the activated macrophages produced by the method of claim 1 with tumor cells from the tumor biopsy in vitro to produce tumor-feeding macrophages; (c) co-culturing the tumor-feeding macrophages with isolated TIL cells in vitro to expand the number of tumor-specific T cells, thereby producing in vitro expanded tumor-specific T cells from the TILs.

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