Xenogeneic antigen presenting cells and uses thereof

Abstract

The present disclosure relates to methods for treating a tumor in a subject via administering (e.g., by intratumoral injection) a composition comprising antigen presenting cells obtained from a species that is different than the subject, i.e., xenogeneic antigen presenting cells. The source of such xenogeneic antigen presenting cells could be one or more swine (e.g, a swine that expresses alpha- 1,3 galactosyltransferase). The present disclosure also relates to methods of treating tumors via administering the xenogeneic antigen presenting cells in combination with other anti-cancer therapies. Furthermore, the present disclosure relates to pharmaceutical compositions comprising xenogeneic antigen presenting cells.

Description

Attorney Docket No.14648-051-228 XENOGENEIC ANTIGEN PRESENTING CELLS AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The application claims priority to U.S. Provisional Patent Application No. 63 / 707,984, filed October 16, 2024, the content of which is incorporated by reference in its entirety herein, and to which priority is claimed. 1. FIELD OF THE DISCLOSURE

[0002] The present disclosure is in the field of compositions comprising xenogeneic antigen presenting cells (e.g., from one or more swine that express alpha-1,3 galactosyltransferase), methods of producing such compositions, and uses of such compositions for treating tumors. 2. BACKGROUND OF THE DISCLOSURE

[0003] Traditional cancer therapies, such as chemotherapy, surgery, radiation, etc. are often inadequate in treating cancer patients, and are usually associated with severe side effects. In this regard, cancer immunotherapy has offered several advantages over traditional cancer therapies. Numerous different immunotherapy approaches have been investigated. One such immunotherapy strategies has involved intratumoral administration of allogeneic inflammatory dendritic cells, typically in combination with another anticancer therapy, for treatment of certain cancers (see, e.g., Jin et al. (2022), Oncoimmunology, 11(1):e2099642; Fröbom et al. (2020), Cancer Immunol. Immunother., 69(11):2393-2401; Karlsson-Parra et al. (2018), Pharm. Res., 35:156). For example, ilixadencel, a human monocyte-derived allogeneic dendritic cell-based product stimulated with potent activators, has been investigated in human clinical trials (see, e.g., Fröbom et al. (2020), Cancer Immunol. Immunother., 69(11):2393-2401). There still remains a need for cancer immunotherapies that demonstrate a robust immune response.

[0004] Citation of a reference herein shall not be construed as an admission that such is prior art to the present disclosure. 3. SUMMARY OF THE DISCLOSURE

[0005] In one aspect, provided herein are methods for treating a tumor in a subject in need thereof comprising administering a composition comprising antigen presenting cells to the -1- NAI-5004557346v1Attorney Docket No.14648-051-228 subject, wherein the antigen presenting cells are obtained from a species that is different than the subject, and wherein the species is a swine that expresses alpha-1,3 galactosyltransferase.

[0006] In one embodiment, the tumor is a solid tumor, and the administering is into the tumor of the subject via an intratumoral injection.

[0007] In one embodiment, the antigen presenting cells are obtained from a swine that expresses alpha-1,3 galactosyltransferase, and wherein the swine is a miniature swine. In one embodiment, the subject is a human. In one embodiment, the swine that expresses alpha-1,3 galactosyltransferase is a swine leukocyte antigen (SLA)‐inbred swine. In one embodiment, the miniature swine that expresses alpha-1,3 galactosyltransferase is a swine leukocyte antigen (SLA)‐inbred swine.

[0008] In one embodiment, method triggers an immune response specific to the tumor. In one embodiment, the method yields an abscopal effect.

[0009] In one embodiment, the tumor is a solid cancerous tumor. In one embodiment, the tumor is selected from the group consisting of sarcomas, carcinomas, lymphomas, breast tumors, prostate tumors, head and neck tumors, glioblastomas, bladder tumors, pancreatic tumors, liver tumors, colon tumors, ovarian tumors, colorectal tumors, pulmonary tumors, cutaneous tumors, lymphoid tumors, gastrointestinal tumors, gastrointestinal stromal tumors, cervical tumors, hepatocellular carcinomas, renal cell carcinomas, melanomas, colorectal carcinomas, esophageal carcinomas, brain tumors, kidney tumors, lung tumors (including non-small cell lung cancer), gastric tumors, bile-duct tumors, uterine tumors, and childhood (pediatric) tumors. In one embodiment, the tumor is selected from the group consisting of brain tumor, breast tumor, liver tumor, lung tumor, melanoma, pancreatic tumor, prostate tumor, sarcoma, kidney tumor, bladder tumor, and colon tumor. In one embodiment, the tumor is selected from the group consisting of brain tumor, breast tumor, liver tumor, lung tumor, melanoma, and pancreatic tumor. In one embodiment, the tumor is selected from the group consisting of prostate tumor, sarcoma, kidney tumor, bladder tumor, and colon tumor. In one embodiment, the tumor is resistant to treatment to chemotherapy and / or treatment with an immunotherapy.

[0010] In one embodiment, the antigen presenting cells are derived from one or more swine or miniature swine using a leukapheresis procedure, wherein the leukapheresis procedure generates a leukopak containing peripheral blood mononuclear cells, and the leukopak is further fractionated by counterflow elutriation. -2- NAI-5004557346v1Attorney Docket No.14648-051-228

[0011] In one embodiment, the composition comprising antigen presenting cells is substantially free of pathogens.

[0012] In one embodiment, the antigen presenting cells are obtained from swine or miniature swine of different genotypes.

[0013] In one embodiment, the composition comprising antigen presenting cells is administered in single or multiple doses. In one embodiment, the composition comprising antigen presenting cells is administered via an intratumoral injection of at least about 1 x 106antigen presenting cells per dose. In one embodiment, the composition comprising antigen presenting cells is administered via an intratumoral injection of about 1 x 106, about 5 x 106, about 10 x 106, about 15 x 106, about 20 x 106, about 25 x 106, about 30 x 106, about 35 x 106, about 40 x 106, about 45 x 106, about 50 x 106, about 55 x 106, about 60 x 106, about 65 x 106, about 70 x 106, about 75 x 106, about 80 x 106, about 85 x 106, about 90 x 106, about 95 x 106, about 10 x 107, about 15 x 107, about 20 x 107, about 25 x 107, about 30 x 107, about 35 x 107, about 40 x 107, about 45 x 107, or about 50 x 107antigen presenting cells per dose.

[0014] In one embodiment, the antigen presenting cells are substantially mature antigen presenting cells.

[0015] In one embodiment, the antigen presenting cells are not activated or stimulated.

[0016] In one embodiment, the composition comprises peripheral blood mononuclear cells (PBMC).

[0017] In one embodiment, the composition comprises monocytes.

[0018] In one embodiment, the composition comprises dendritic cells, macrophages, granulocytes, T-cells, B-cells, and / or NK cells.

[0019] In one embodiment, the composition comprises at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or greater than about 95% PBMCs, wherein the PBMCs are mature, immature, or a combination of mature and immature PBMCs.

[0020] In one embodiment, the composition comprises at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or greater than about 95% monocytes, wherein the monocytes are mature, immature, or a combination of mature and immature monocytes. -3- NAI-5004557346v1Attorney Docket No.14648-051-228

[0021] In one embodiment, the composition comprises a mixture of at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or greater than about 95% monocytes and dendritic cells, wherein the monocytes and / or dendritic cells in said mixture may be mature, immature, or a combination of mature and immature monocytes and / or dendritic cells.

[0022] In one embodiment, the mixture comprising monocytes and dendritic cells comprises from 10% to 95%, 10% to 90%, 10% to 80%, 10% to 70%, 10% to 60%, 10% to 50%, 10% to 40%, 10% to 30%, 10% to 20%, 20% to 95%, 20% to 90%, 20% to 80%, 20% to 70%, 20% to 60%, 20% to 50%, 20% to 40%, 20% to 30%, 30% to 95%, 30% to 90%, 30% to 80%, 30% to 70%, 30% to 60%, 30% to 50%, 30% to 40%, 40% to 95%, 40% to 90%, 40% to 80%, 40% to 70%, 40% to 60%, 40% to 50%, 50% to 95%, 50% to 90%, 50% to 80%, 50% to 70%, 50% to 60%, 60% to 95%, 60% to 90%, 60% to 80%, 60% to 70%, 70% to 95%, 70% to 90%, 70% to 80%, 80% to 95%, 80% to 90%, 90% to 95%, or greater than 95% monocytes. In one embodiment, the mixture comprising monocytes and dendritic cells comprises from 10% to 30%, 35% to 55%, 60% to 80%, or 85% to 95% monocytes. In one embodiment, the mixture comprises at least 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 75%, about 80%, about 85%, about 90%, about 95%, or greater than about 95% monocytes.

[0023] In one embodiment, the mixture comprising monocytes and dendritic cells comprises from 10% to 95%, 10% to 90%, 10% to 80%, 10% to 70%, 10% to 60%, 10% to 50%, 10% to 40%, 10% to 30%, 10% to 20%, 20% to 95%, 20% to 90%, 20% to 80%, 20% to 70%, 20% to 60%, 20% to 50%, 20% to 40%, 20% to 30%, 30% to 95%, 30% to 90%, 30% to 80%, 30% to 70%, 30% to 60%, 30% to 50%, 30% to 40%, 40% to 95%, 40% to 90%, 40% to 80%, 40% to 70%, 40% to 60%, 40% to 50%, 50% to 95%, 50% to 90%, 50% to 80%, 50% to 70%, 50% to 60%, 60% to 95%, 60% to 90%, 60% to 80%, 60% to 70%, 70% to 95%, 70% to 90%, 70% to 80%, 80% to 95%, 80% to 90%, 90% to 95%, or greater than 95% dendritic cells. In one embodiment, the mixture comprising monocytes and dendritic cells comprises from 10% to 30%, 35% to 55%, 60% to 80%, or 85% to 95% dendritic cells. In one embodiment, the mixture comprising monocytes and dendritic cells comprises at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45% about 50%, about 55%, about 60%, -4- NAI-5004557346v1Attorney Docket No.14648-051-228 about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or greater than about 95% dendritic cells.

[0024] In one embodiment, the subject is receiving another anti-cancer therapy. In one embodiment, the other anti-cancer therapy comprises treatment with one or more immune checkpoint inhibitors. In one embodiment, the other anti-cancer therapy is an anti-CTLA4 therapy, anti-PD1 therapy, anti-PDL1 therapy, anti-LAG-3 therapy, tumor-treating fields (TTFs), cell-based therapy, a tyrosine kinase inhibitor, a VEGF inhibitor, or any combination thereof. In one embodiment, the other anti-cancer therapy comprises treatment with imatinib, sunitinib, regorafenib, pazopanib, nilotinib, avapritinib, ripretinib, sorafenib, pimitespib, ipilimumab, tremelimumab, nivolumab, pembrolizumab, cemiplimab, atelizumab, avelumab, durvalumab, relatlimab, or any combination thereof.

[0025] In one embodiment, the subject does not respond to the other anti-cancer therapy in the absence of administration of the composition comprising antigen presenting cells.

[0026] In another aspect, provided herein is a pharmaceutical composition suitable for intratumoral injection, wherein the pharmaceutical composition comprises antigen presenting cells obtained from one or more swine that express alpha-1,3 galactosyltransferase. 4. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIG.1 shows a general leukapheresis procedure to generate a leukopak containing antigen presenting cells from porcine blood.

[0028] FIG.2 shows a comparison of tumor size between the treatment group (administration of a composition comprising human PBMCs) and the control in a murine melanoma model at Day 12.

[0029] FIG.3 shows a comparison of tumor size between the treatment group (administration of a composition comprising human PBMCs) and the control in a murine bladder cancer model at Day 7.

[0030] FIGS.4A-4C: FIG.4A shows human CD4 T cells count at Day 7 in an in vitro study evaluating (a) porcine PBMCs plated with human PBMCs at varying ratios of human to porcine cells (h:p), (b) human PBMCs alone, and (c) allogeneic human PBMCs. FIG.4B shows human CD8 T cells count at Day 7 in an in vitro study evaluating (a) porcine PBMCs plated with human PBMCs at varying ratios of human to porcine cells (h:p), (b) human PBMCs alone, and (c) -5- NAI-5004557346v1Attorney Docket No.14648-051-228 allogeneic human PBMCs. FIG.4C shows human NK cells count at Day 7 in an in vitro study evaluating (a) porcine PBMCs plated with human PBMCs at varying ratios of human to porcine cells (h:p), (b) human PBMCs alone, and (c) allogeneic human PBMCs.

[0031] FIGS.5A-5G: FIG.5A shows human CD4 T cells count at Day 7 in an in vitro study evaluating (a) porcine monocytes plated with human PBMCs at varying ratios of human to porcine cells (h:p), (b) human PBMCs alone, and (c) allogeneic human PBMCs. FIG.5B shows human CD8 T cells count at Day 7 in an in vitro study evaluating (a) porcine monocytes plated with human PBMCs at varying ratios of human to porcine cells (h:p), (b) human PBMCs alone, and (c) allogeneic human PBMCs. FIG.5C shows proliferated human CD4 T cells count at Day 7 in an in vitro study evaluating (a) porcine monocytes plated with human PBMCs at varying ratios of human to porcine cells (h:p), (b) human PBMCs alone, and (c) allogeneic human PBMCs. FIG.5D shows proliferated human CD8 T cells count at Day 7 in an in vitro study evaluating (a) porcine monocytes plated with human PBMCs at varying ratios of human to porcine cells (h:p), (b) human PBMCs alone, and (c) allogeneic human PBMCs. FIG.5E shows HLA-DR on human CD4 T cells, in terms of mean fluorescence intensity (MFI), evaluated at Day 7 in an in vitro study evaluating (a) porcine monocytes plated with human PBMCs at varying ratios of human to porcine cells (h:p), (b) human PBMCs alone, and (c) allogeneic human PBMCs. FIG.5F shows HLA-DR on human CD8 T cells, in terms of mean fluorescence intensity (MFI), evaluated at Day 7 in an in vitro study evaluating (a) porcine monocytes plated with human PBMCs at varying ratios of human to porcine cells (h:p), (b) human PBMCs alone, and (c) allogeneic human PBMCs. FIG.5G shows human NK cells count at Day 7 in an in vitro study evaluating (a) porcine monocytes plated with human PBMCs at varying ratios of human to porcine cells (h:p), (b) human PBMCs alone, and (c) allogeneic human PBMCs.

[0032] FIG.6 shows proliferation of CD4+T cell and CD8+T cell from human PBMCs in MLR assays. MLR assays were performed at 3 different E:T ratios for Composition 1 from R&D runs and human PBMSc.

[0033] FIG.7 shows HLA-DR expression on CD4+and CD8+T cells in MLR assays. MLR assays were performed at 3 different E:T ratios for Composition 1 from R&D runs and human PBMSc. -6- NAI-5004557346v1Attorney Docket No.14648-051-228

[0034] FIG.8 shows levels of IFN-γ, IL-8, TNF-α, and GM-CSF in the supernatants of co- cultured Composition 1 and hPBMCs. MLRs were performed at 3 different E:T ratios for Composition 1 from R&D runs and PBMCs.

[0035] FIG.9 shows proliferation of CD4+T cell and CD8+T cell from human PBMCs in MLR assays. MLR assays were performed at 3 different E:T ratios for Composition 1 from PQ runs and human PBMSc.

[0036] FIG.10 shows HLA-DR expression on CD4+and CD8+T cells in MLR assays. MLR assays were performed at 3 different E:T ratios for Composition 1 from PQ runs and human PBMSc.

[0037] FIG.11 shows levels of IFN-γ, and TNF-α in the supernatants of co-cultured Composition 1 and hPBMCs. MLRs were performed at 3 different E:T ratios for Composition 1 from PQ runs and PBMCs.

[0038] FIG.12 shows Kaplan-Meier Survival Curves of mice bearing a JC syngeneic model of breast cancer.

[0039] FIG.13 shows tumor volume in the right and left flanks of breast cancer (JC) mouse model.

[0040] FIGs.14A-14B show Kaplan-Meier Survival Curves of mice bearing S180 syngeneic tumor model.

[0041] FIGs.15A-15B show tumor volume after 3 doses (FIG.15A) or 6 doses (FIG.15B) of vehicle or test article in the right and left flanks of a S180 syngeneic tumor model.

[0042] FIG.16 shows Kaplan-Meier Survival Curves of mice bearing a MB49 syngeneic tumor model.

[0043] FIGs.17A-17B show tumor volume after 3 doses (FIG.17A) or 6 doses (FIG.17B) of vehicle or test article in the right and left flanks of a MB49 syngeneic tumor model.

[0044] FIGs.18A-18B show right flank (FIG.18A) and left flank (FIG.18B) tumor volume in male and female animals in control group and Composition 1 treatment group via intratumoral injection.

[0045] FIG.19 shows schematic study design of the Phase 1 dose-escalation study. -7- NAI-5004557346v1Attorney Docket No.14648-051-228 5. DETAILED DESCRIPTION OF THE DISCLOSURE

[0046] Provided herein are methods for treating a tumor in a subject via administering a composition comprising antigen presenting cells obtained from a species that is different than the subject, i.e., xenogeneic antigen presenting cells (also referred to herein as XPCs). The source of such xenogeneic antigen presenting cells, which could be one or more swine, is described in Section 5.1. Also described in Section 5.1 are characteristics of the swine, and compositions comprising antigen presenting cells, including swine-derived antigen presenting cells. The methods of isolating antigen presenting cells (e.g., from one or more swine) are described in Section 5.2. The methods of treating tumors (e.g., a cancerous solid tumor) comprising administering the antigen presenting cells described herein (e.g., via intratumoral injection), optionally in combination with other anti-cancer therapies, is described in Section 5.3. Furthermore, pharmaceutical compositions comprising the antigen presenting cells disclosed herein are described in Section 5.4. 5.1 Xenogeneic Compositions

[0047] Provided herein are compositions for treating a tumor in a subject, wherein the compositions comprise antigen presenting cells derived from a species that is different than the subject. As used herein, the term “subject” includes animals, such as mammals, including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, monkeys, chickens, turkeys, or quails, and the like. In one embodiment, the subject is a mammal. In one embodiment, the subject is a human. In certain embodiments, provided herein are compositions comprising antigen presenting cells xenogeneic to the subject to be treated. Such compositions are referred to herein as xenogeneic compositions. The xenogeneic compositions of the present invention may be administered (e.g., by intratumoral injection) either alone or upon formulating into a pharmaceutical composition as described in Section 5.4.

[0048] The antigen presenting cells in the xenogeneic composition may be any cell that displays antigen bound by major histocompatibility complex (MHC) proteins on its surface. In one embodiment, the MHC proteins are MHC Class II molecules. The antigen presenting cells in the xenogeneic composition may include, but are not limited to, dendritic cells, macrophages, B cells, monocytes, mononuclear phagocytes, endothelial cells, thymic epithelial cells, granulocytes, or any combination thereof. -8- NAI-5004557346v1Attorney Docket No.14648-051-228

[0049] The xenogeneic compositions of the present invention may comprise antigen presenting cells from any nonhuman species, including, but not limited to, nonhuman primates, domestic animals, and rodents. The nonhuman species may be genetically modified. Domestic animals include pigs, rabbits, dogs, cats, horses, sheep, goats, and cows. In one embodiment, the nonhuman species is a pig or porcine or swine. As provided herein, the terms pig, porcine, or swine may be used interchangeably.

[0050] The antigen presenting cells in the xenogeneic compositions of the present invention may be obtained by the methods described in Section 5.2. In certain embodiments, the xenogeneic composition comprises antigen presenting cells derived from one or more pigs. The one or more pigs used for generating the antigen presenting cells could be genetically modified swine, for example, to enhance safety and / or efficacy of the antigen presenting cells. In some embodiments, the swine is an alpha-1,3 galactosyltransferase-deficient swine. In certain embodiments, the antigen presenting cells are derived from one or more recombinant swine. In certain embodiments, the antigen presenting cells are derived from recombinant swine of different genotypes. In certain embodiments, the antigen presenting cells are derived from one or more miniature swine. The one or more miniature swine used for generating the antigen presenting cells could be genetically modified miniature swine, for example, to enhance safety and / or efficacy of the antigen presenting cells. In some embodiments, the miniature swine is an alpha-1,3 galactosyltransferase-deficient miniature swine (see, e.g., U.S. Patent Nos.6,153,428, 6,413,769, and 7,547,816). In certain embodiments, the antigen presenting cells are derived from one or more recombinant miniature swine. In certain embodiments, the antigen presenting cells are derived from recombinant miniature swine of different genotypes. In some embodiments, the alpha-1,3 galactosyltransferase-deficient swine or miniature swine is a swine leukocyte antigen (SLA) inbred swine. In certain embodiments, the one or more swine or miniature swine described herein may contain knockout genes or transgenes in the genes of porcine antigen presenting cells.

[0051] In certain embodiments, the one or more pigs used for generating the antigen presenting cells are not genetically modified swine. In certain embodiments, the one or more pigs used for generating the antigen presenting cells are wild-type swine. In certain embodiments, the swine is not an alpha-1,3 galactosyltransferase-deficient swine. In certain embodiments, the swine that is not an alpha-1,3 galactosyltransferase-deficient swine is referred -9- NAI-5004557346v1Attorney Docket No.14648-051-228 to as a wild-type or Gal+ swine or pig. In certain embodiments, the antigen presenting cells are derived from one or more swine that express alpha-1,3 galactosyltransferase. In certain embodiments, the one or more swine that express alpha-1,3 galactosyltransferase are swine leukocyte antigen (SLA) inbred swine.

[0052] In certain embodiments, the antigen presenting cells are derived from one or more miniature swine, wherein the miniature swine is not genetically modified swine. In certain embodiments, the antigen presenting cells are derived from one or more miniature swine, wherein the miniature swine is a wild-type swine. In certain embodiments, the antigen presenting cells are derived from one or more miniature swine, wherein the miniature swine is not an alpha-1,3 galactosyltransferase-deficient swine. In certain embodiments, the miniature swine that is not an alpha-1,3 galactosyltransferase-deficient miniature swine is referred to as a wild-type or Gal+ swine or pig. In certain embodiments, the antigen presenting cells are derived from one or more miniature swine that express alpha-1,3 galactosyltransferase. In certain embodiments, the one or more miniature swine that express alpha-1,3 galactosyltransferase are swine leukocyte antigen (SLA) inbred swine.

[0053] In certain embodiments, the one or more pigs described herein have a high level of genetic homogeneity. In certain embodiments, the one or more pigs have genetic homogeneity of about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5%.

[0054] The donors for the antigen presenting cells used in the xenogeneic compositions described herein can be tested for infectious diseases using methods generally known in the art. In certain embodiments, the donors for the antigen presenting cells used in the xenogeneic compositions described herein tested negative for syphilis, HIV, hepatitis A, hepatitis B, and / or hepatitis C. In certain embodiments, the xenogeneic compositions comprising antigen presenting cells are substantially free of pathogens.

[0055] In certain embodiments, the xenogeneic composition comprises antigen presenting cells derived from peripheral blood. In certain embodiments, the xenogeneic composition comprises antigen presenting cells derived from porcine peripheral blood. In certain embodiments, the xenogeneic composition comprises antigen presenting cells derived from bone marrow. In certain embodiments, the xenogeneic composition comprises antigen presenting cells derived from porcine bone marrow. -10- NAI-5004557346v1Attorney Docket No.14648-051-228

[0056] In certain embodiments, the xenogeneic composition comprises peripheral blood mononuclear cells (PBMCs). In certain embodiments, the xenogeneic composition comprises monocytes. In certain embodiments, the xenogeneic composition comprises porcine PBMCs. In certain embodiments, the xenogeneic composition comprises porcine monocytes. In certain embodiments, the xenogeneic composition comprises monocytes derived from porcine peripheral blood. In certain embodiments, the xenogeneic composition comprises monocytes derived from porcine bone marrow.

[0057] In certain embodiments, the xenogeneic composition comprises at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or greater than about 95% PBMCs. The PBMCS may be mature, immature, or a combination of mature and immature PBMCs.

[0058] In certain embodiments, the xenogeneic composition comprises from 50% to 95%, 50% to 90%, 50% to 80%, 50% to 70%, 50% to 60%, 60% to 95%, 60% to 90%, 60% to 80%, 60% to 70%, 70% to 95%, 70% to 90%, 70% to 80%, 80% to 95%, 80% to 90%, 90% to 95%, or greater than 95% PBMCs.

[0059] In certain embodiments, the PBMCs present in the xenogeneic composition are at least from 10% to 95%, 10% to 90%, 10% to 80%, 10% to 70%, 10% to 60%, 10% to 50%, 10% to 40%, 10% to 30%, 10% to 20%, 20% to 95%, 20% to 90%, 20% to 80%, 20% to 70%, 20% to 60%, 20% to 50%, 20% to 40%, 20% to 30%, 30% to 95%, 30% to 90%, 30% to 80%, 30% to 70%, 30% to 60%, 30% to 50%, 30% to 40%, 40% to 95%, 40% to 90%, 40% to 80%, 40% to 70%, 40% to 60%, 40% to 50%, 50% to 95%, 50% to 90%, 50% to 80%, 50% to 70%, 50% to 60%, 60% to 95%, 60% to 90%, 60% to 80%, 60% to 70%, 70% to 95%, 70% to 90%, 70% to 80%, 80% to 95%, 80% to 90%, 90% to 95%, or greater than 95% mature PBMCs.

[0060] In certain embodiments, the PBMCs present in the xenogeneic composition are at least from 10% to 30%, 35% to 55%, 60% to 80%, or 85% to 95% mature PBMCs.

[0061] In certain embodiments, the PBMCs present in the xenogeneic composition are at least 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 75%, about 80%, about 85%, about 90%, or about 95% mature PBMCs. In certain embodiments, the PBMCs present in the xenogeneic composition are greater than about 95% mature PBMCs. In certain -11- NAI-5004557346v1Attorney Docket No.14648-051-228 embodiments, the PBMCs present in the xenogeneic composition are about 100% mature PBMCs.

[0062] In certain embodiments, the xenogeneic composition comprises at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or greater than about 95% monocytes. The monocytes may be mature, immature, or a combination of mature and immature monocytes.

[0063] In certain embodiments, the xenogeneic composition comprises from 50% to 95%, 50% to 90%, 50% to 80%, 50% to 70%, 50% to 60%, 60% to 95%, 60% to 90%, 60% to 80%, 60% to 70%, 70% to 95%, 70% to 90%, 70% to 80%, 80% to 95%, 80% to 90%, 90% to 95%, or greater than 95% monocytes.

[0064] In certain embodiments, the monocytes present in the xenogeneic composition are at least from 10% to 95%, 10% to 90%, 10% to 80%, 10% to 70%, 10% to 60%, 10% to 50%, 10% to 40%, 10% to 30%, 10% to 20%, 20% to 95%, 20% to 90%, 20% to 80%, 20% to 70%, 20% to 60%, 20% to 50%, 20% to 40%, 20% to 30%, 30% to 95%, 30% to 90%, 30% to 80%, 30% to 70%, 30% to 60%, 30% to 50%, 30% to 40%, 40% to 95%, 40% to 90%, 40% to 80%, 40% to 70%, 40% to 60%, 40% to 50%, 50% to 95%, 50% to 90%, 50% to 80%, 50% to 70%, 50% to 60%, 60% to 95%, 60% to 90%, 60% to 80%, 60% to 70%, 70% to 95%, 70% to 90%, 70% to 80%, 80% to 95%, 80% to 90%, 90% to 95%, or greater than 95% mature monocytes.

[0065] In certain embodiments, the monocytes present in the xenogeneic composition are at least from 10% to 30%, 35% to 55%, 60% to 80%, or 85% to 95% mature monocytes.

[0066] In certain embodiments, the monocytes present in the xenogeneic composition are at least 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 75%, about 80%, about 85%, about 90%, or about 95% mature monocytes. In certain embodiments, the monocytes present in the xenogeneic composition are greater than about 95% mature monocytes. In certain embodiments, the monocytes present in the xenogeneic composition are about 100% mature monocytes.

[0067] In certain embodiments, the xenogeneic composition comprises a mixture of monocytes and dendritic cells. In certain embodiments, the xenogeneic composition comprises a mixture of monocytes and dendritic cells derived from one or more pigs. The composition may further comprise macrophages, granulocytes, B cells, T cells, NK cells, or any combination -12- NAI-5004557346v1Attorney Docket No.14648-051-228 thereof. The macrophages, granulocytes, B cells, T cells, NK cells, or any combination thereof may be derived from one or more pigs, or other non-human species.

[0068] In certain embodiments, the xenogeneic composition comprises a mixture of at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or greater than about 95% monocytes and dendritic cells. The monocytes and / or dendritic cells in such mixtures may be mature, immature, or a combination of mature and immature monocytes and / or dendritic cells.

[0069] In certain embodiments, the xenogeneic composition comprises a mixture of from 50% to 95%, 50% to 90%, 50% to 80%, 50% to 70%, 50% to 60%, 60% to 95%, 60% to 90%, 60% to 80%, 60% to 70%, 70% to 95%, 70% to 90%, 70% to 80%, 80% to 95%, 80% to 90%, 90% to 95%, or greater than 95% monocytes and dendritic cells.

[0070] The mixture of monocytes and dendritic cells may comprise from 10% to 95%, 10% to 90%, 10% to 80%, 10% to 70%, 10% to 60%, 10% to 50%, 10% to 40%, 10% to 30%, 10% to 20%, 20% to 95%, 20% to 90%, 20% to 80%, 20% to 70%, 20% to 60%, 20% to 50%, 20% to 40%, 20% to 30%, 30% to 95%, 30% to 90%, 30% to 80%, 30% to 70%, 30% to 60%, 30% to 50%, 30% to 40%, 40% to 95%, 40% to 90%, 40% to 80%, 40% to 70%, 40% to 60%, 40% to 50%, 50% to 95%, 50% to 90%, 50% to 80%, 50% to 70%, 50% to 60%, 60% to 95%, 60% to 90%, 60% to 80%, 60% to 70%, 70% to 95%, 70% to 90%, 70% to 80%, 80% to 95%, 80% to 90%, 90% to 95%, or greater than 95% monocytes.

[0071] In certain embodiments, the mixture of monocytes and dendritic cells comprises from 10% to 30%, 35% to 55%, 60% to 80%, or 85% to 95% monocytes.

[0072] In certain embodiments, the mixture of monocytes and dendritic cells comprises at least 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 75%, about 80%, about 85%, about 90%, about 95%, or greater than about 95% monocytes.

[0073] In certain embodiments, the monocytes present in the mixture of monocytes and dendritic cells are at least from 10% to 95%, 10% to 90%, 10% to 80%, 10% to 70%, 10% to 60%, 10% to 50%, 10% to 40%, 10% to 30%, 10% to 20%, 20% to 95%, 20% to 90%, 20% to 80%, 20% to 70%, 20% to 60%, 20% to 50%, 20% to 40%, 20% to 30%, 30% to 95%, 30% to 90%, 30% to 80%, 30% to 70%, 30% to 60%, 30% to 50%, 30% to 40%, 40% to 95%, 40% to 90%, 40% to 80%, 40% to 70%, 40% to 60%, 40% to 50%, 50% to 95%, 50% to 90%, 50% to -13- NAI-5004557346v1Attorney Docket No.14648-051-228 80%, 50% to 70%, 50% to 60%, 60% to 95%, 60% to 90%, 60% to 80%, 60% to 70%, 70% to 95%, 70% to 90%, 70% to 80%, 80% to 95%, 80% to 90%, 90% to 95%, or greater than 95% mature monocytes.

[0074] In certain embodiments, the monocytes present in the mixture of monocytes and dendritic cells are at least from 10% to 30%, 35% to 55%, 60% to 80%, or 85% to 95% mature monocytes.

[0075] In certain embodiments, the monocytes present in the mixture of monocytes and dendritic cells are at least 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 75%, about 80%, about 85%, about 90%, or about 95% mature monocytes. In certain embodiments, the monocytes present in the mixture of monocytes and dendritic cells are greater than about 95% mature monocytes. In certain embodiments, the monocytes present in the mixture of monocytes and dendritic cells are about 100% mature monocytes.

[0076] The mixture of monocytes and dendritic cells may comprise from 10% to 95%, 10% to 90%, 10% to 80%, 10% to 70%, 10% to 60%, 10% to 50%, 10% to 40%, 10% to 30%, 10% to 20%, 20% to 95%, 20% to 90%, 20% to 80%, 20% to 70%, 20% to 60%, 20% to 50%, 20% to 40%, 20% to 30%, 30% to 95%, 30% to 90%, 30% to 80%, 30% to 70%, 30% to 60%, 30% to 50%, 30% to 40%, 40% to 95%, 40% to 90%, 40% to 80%, 40% to 70%, 40% to 60%, 40% to 50%, 50% to 95%, 50% to 90%, 50% to 80%, 50% to 70%, 50% to 60%, 60% to 95%, 60% to 90%, 60% to 80%, 60% to 70%, 70% to 95%, 70% to 90%, 70% to 80%, 80% to 95%, 80% to 90%, 90% to 95%, or greater than 95% dendritic cells.

[0077] In certain embodiments, the mixture of monocytes and dendritic cells comprises from 10% to 30%, 35% to 55%, 60% to 80%, or 85% to 95% dendritic cells.

[0078] In certain embodiments, the mixture of monocytes and dendritic cells comprises at least 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 75%, about 80%, about 85%, about 90%, or about 95% dendritic cells. In certain embodiments, the mixture of monocytes and dendritic cells comprises greater than about 95% dendritic cells.

[0079] In certain embodiments, the dendritic cells present in the mixture of monocytes and dendritic cells are at least from 10% to 95%, 10% to 90%, 10% to 80%, 10% to 70%, 10% to 60%, 10% to 50%, 10% to 40%, 10% to 30%, 10% to 20%, 20% to 95%, 20% to 90%, 20% to -14- NAI-5004557346v1Attorney Docket No.14648-051-228 80%, 20% to 70%, 20% to 60%, 20% to 50%, 20% to 40%, 20% to 30%, 30% to 95%, 30% to 90%, 30% to 80%, 30% to 70%, 30% to 60%, 30% to 50%, 30% to 40%, 40% to 95%, 40% to 90%, 40% to 80%, 40% to 70%, 40% to 60%, 40% to 50%, 50% to 95%, 50% to 90%, 50% to 80%, 50% to 70%, 50% to 60%, 60% to 95%, 60% to 90%, 60% to 80%, 60% to 70%, 70% to 95%, 70% to 90%, 70% to 80%, 80% to 95%, 80% to 90%, 90% to 95%, or greater than 95% mature dendritic cells.

[0080] In certain embodiments, the dendritic cells present in the mixture of monocytes and dendritic cells are at least from 10% to 30%, 35% to 55%, 60% to 80%, or 85% to 95% mature dendritic cells.

[0081] In certain embodiments, the dendritic cells present in the mixture of monocytes and dendritic cells are at least 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 75%, about 80%, about 85%, about 90%, or about 95% mature dendritic cells. In certain embodiments, the dendritic cells present in the mixture of monocytes and dendritic cells are greater than about 95% mature dendritic cells. In certain embodiments, the dendritic cells present in the mixture of monocytes and dendritic cells are about 100% mature dendritic cells.

[0082] In certain embodiments, the antigen presenting cells in the xenogeneic composition are not activated or stimulated. In certain embodiments, the antigen presenting cells in the xenogeneic composition are activated or stimulated. In certain embodiments, the antigen presenting cells are stimulated with one or more of pro-inflammatory factors (e.g., granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin 4 (IL-4), etc.). For example, the porcine monocytes can be cultured into dendritic cells by using GM-CSF and IL-4 for 7 days.

[0083] In certain embodiments, the PBMCs in the xenogeneic composition are not activated or stimulated. In certain embodiments, the PBMCs in the xenogeneic composition are activated or stimulated. In certain embodiments, the PBMCs are stimulated with one or more of pro- inflammatory factors.

[0084] In certain embodiments, the monocytes and / or dendritic cells in the xenogeneic composition are not activated or stimulated. In certain embodiments, the monocytes and / or dendritic cells in the xenogeneic composition are activated or stimulated. In certain embodiments, the monocytes and / or dendritic cells are stimulated with one or more of pro- inflammatory factors. -15- NAI-5004557346v1Attorney Docket No.14648-051-228

[0085] In certain embodiments, the antigen presenting cells in the xenogeneic composition are activated or stimulated using porcine B cells as stimulators. In certain embodiments, the antigen presenting cells in the xenogeneic composition are activated or stimulated using porcine T cells as stimulators. In certain embodiments, the antigen presenting cells in the xenogeneic composition are activated or stimulated using porcine monocyte-derived macrophages as stimulators. 5.2 Methods of obtaining antigen presenting cells

[0086] Provided herein are methods of obtaining the antigen presenting cells in the xenogeneic compositions described in Section 5.1.

[0087] The antigen presenting cells may be derived from tissue or blood of any nonhuman species (e.g., one or more pigs). Such antigen presenting cells may be obtained through methods generally known in the art. The antigen presenting cells may be obtained using a leukapheresis procedure. Such leukapheresis procedure may be used to generate a leukopak, containing peripheral blood mononuclear cells. The leukopak may be further fractionated by counterflow elutriation to obtain a composition comprising the desired antigen presenting cells. In some embodiments, the antigen presenting cells are obtained using fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS) techniques. 5.3 Methods of treatment

[0088] Provided herein are methods for treating a tumor in a subject in need thereof, comprising administering to the subject (e.g., by intratumoral injection) a xenogeneic composition described in Section 5.1. In certain embodiments, the administering is done via one or more intratumoral injections as described in Section 5.3.1. As used herein, the term “subject” includes animals, such as mammals, including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, monkeys, chickens, turkeys, or quails, and the like. In one embodiment, the subject is a mammal. In one embodiment, the subject is a human.

[0089] In certain embodiments, provided herein are methods for treating a tumor in a subject in need thereof, comprising administering (e.g., by intratumoral injection) a xenogeneic composition in combination with a composition comprising human antigen presenting cells. The antigen presenting cells in the xenogeneic composition and the composition comprising human antigen presenting cells may or may not be activated or stimulated. -16- NAI-5004557346v1Attorney Docket No.14648-051-228

[0090] The methods of treatment provided herein can be used for treating a wide variety of tumors. In one embodiment, the tumor is a solid tumor. In one embodiment, the tumor is a cancerous tumor. In a specific embodiment, the tumor is a solid cancerous tumor. In certain embodiments, the tumor is resistant to treatment to chemotherapy and / or treatment with another immunotherapy. In certain embodiments, the xenogeneic composition is administered (e.g., by intratumoral injection) as a frontline therapy for treating one or more tumors. In certain other embodiments, the xenogeneic composition is administered to subjects (e.g., by intratumoral injection) who are receiving or may have previously received another anticancer therapy. In one embodiment, the antigen presenting cells in the xenogeneic composition are as disparate as possible from the cells in the tumor tissue. Thus, in one embodiment, the antigen presenting cells in the xenogeneic composition are distinctly different from the cells in the tumor tissue. For example, in one embodiment, a method of treating human liver cancer comprises administration of a porcine xenogeneic composition that does not comprise porcine liver tissue.

[0091] Examples of tumors that can be treated with the methods of the present invention include, but are not limited to, sarcomas, carcinomas, lymphomas, breast tumors, prostate tumors, head and neck tumors, glioblastomas, bladder tumors, pancreatic tumors, liver tumors, colon tumors, ovarian tumors, colorectal tumors, pulmonary tumors, cutaneous tumors, lymphoid tumors, gastrointestinal tumors, gastrointestinal stromal tumors, cervical tumors, hepatocellular carcinomas, renal cell carcinomas, melanomas, colorectal carcinomas, esophageal carcinomas, brain tumors, kidney tumors, lung tumors (including non-small cell lung cancer), gastric tumors, bile-duct tumors, uterine tumors, and childhood (pediatric) tumors.

[0092] In certain embodiments, provided herein are methods for treating a neoplastic disease in a subject in need thereof, the method comprising administering (e.g., by intratumoral injection) a xenogeneic composition described in Section 5.1.

[0093] The xenogeneic compositions as described herein, may be administered to a subject by any route of administration, including, but not limited to, intratumoral, intravenous, intradermal, subcutaneous, intramuscular, or intranodal administration. In one embodiment, the administering is via parenteral administration. In another embodiment, the administering is via intratumoral administration. In such intratumoral administration, the xenogeneic composition is injected directly into a tumor. In yet another embodiment, the administering is via intracerebral administration. -17- NAI-5004557346v1Attorney Docket No.14648-051-228 5.3.1 Intratumoral Administration

[0094] In certain embodiments, the xenogeneic composition as described herein is administered to a subject by one or more intratumoral injections directly into a tumor. Such direct delivery of the therapeutic composition into target tumor lesions offers several advantages over systemic delivery, including increased local concentrations and potentially diminished systemic toxicities.

[0095] In one embodiment, the injection is into a single tumor. In another embodiment, the injection is into different sections of the same tumor. In another embodiment, the injection is into multiple tumors. The ability to inject multiple sites may lead to a more robust adaptive immune response (e.g., in patients with polyclonal metastases). In some embodiments, the injection is into a primary tumor. In other embodiments, the injection is into one or more metastatic tumors.

[0096] In one embodiment, the intratumoral injection is an image-guided intratumoral injection. The intratumoral injection may be performed under imaging techniques, including, but not limited to, ultrasound, fluoroscopy, and CT scan.

[0097] The injection technique, including the design and placement of the injection needle used and the rate of delivery of the therapeutic composition, used for carrying out the intratumoral injection may have an influence on the therapeutic effect (e.g., immune response). In certain embodiments, the intratumoral injection is carried out by placing the needle (e.g., using image guidance) into the target tumor, ensuring that the therapeutic composition is distributed throughout the target tumor without leakage into the surrounding tissue (i.e., without off-target leakage). In one embodiment, the intratumoral injection is carried out by placing the tip of the needle (e.g., using image guidance) within the center of the target tumor.

[0098] The intratumoral injection may be performed using a needle having an appropriate needle gauge. The appropriate needle gauge depends on a variety of factors, including, but not limited to, the type of target tumor and its location and size. In certain embodiments, the intratumoral injection is performed using an 18-gauge, 19-gauge, 20-gauge, 21-gauge, 22-gauge, 23-gauge, 24-gauge, 25-gauge, 26-gauge, 27-gauge, 28-gauge, 29-gauge, or 30-gauge needle. In one embodiment, the intratumoral injection is performed using a 20-gauge needle. In one embodiment, the intratumoral injection is performed using a 21-gauge needle. In one embodiment, the intratumoral injection is performed using a 22-gauge needle. -18- NAI-5004557346v1Attorney Docket No.14648-051-228

[0099] In certain embodiments, the intratumoral injection is performed using an end hole needle (EHN) having an appropriate needle gauge. In one embodiment, the intratumoral injection is performed using a 20-gauge, 21-gauge, or 22-gauge end hole needle (EHN) (e.g., commercialized by Becton Dickinson).

[0100] In certain embodiments, the intratumoral injection is performed using a multiside hole needle (MSHN) having an appropriate needle gauge. Placement of such may be performed under image guidance, ensuring that all of the side holes are positioned within the tumor. In one embodiment, the intratumoral injection is performed whereby there is no off-target leakage of the therapeutic composition. In one embodiment, the intratumoral injection is performed using a 20- gauge, 21-gauge, or 22-gauge multiside hole needle (MSHN). In one embodiment, the intratumoral injection is performed using a 21-gauge multiside hole needle (MSHN) with no end hole (e.g., ProFusion™ Therapeutic Infusion Needle commercialized by Cook Regentec). 5.3.2 Dosages and Dosing Frequency

[0101] The xenogeneic compositions of the present invention may be administered (e.g., by intratumoral injection) in a manner appropriate to the disease to be treated. The dosage amounts and frequency of administration will be determined by such factors as the route of administration, the condition of the patient, and the type and severity of the patient’s disease, although appropriate dosages may be determined based on clinical trials.

[0102] In certain embodiments, the xenogeneic compositions comprising antigen presenting cells as described herein may be administered (e.g., by intratumoral injection) in single or multiple doses. In one embodiment, the composition is administered as a priming dose. In another embodiment, the composition is administered under a booster regimen (i.e., repeated treatment). In yet another embodiment, the composition is administered under a booster regimen (i.e., repeated treatment), but in a different tumor site.

[0103] In certain embodiments, the multiple doses may be administered on the same day at essentially the same time, immediately following the previous dose (i.e., back-to-back administration). Alternatively, the multiple doses may be administered on the same day but not at the same time (i.e., not back-to-back administration). In such instances, the multiple doses may be 2 doses, 3 doses, 4 doses, 5 doses, 6 doses, 7 doses, 8 doses, 9 doses, or 10 doses.

[0104] In certain embodiments, the administration of the xenogeneic composition as described herein may be repeated and separated by at least 1 day, 2 days, 3 days, 4 days, 5 days, -19- NAI-5004557346v1Attorney Docket No.14648-051-228 7 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or at least 6 months. In some embodiments, dosing can be discontinued for a predetermined number of days.

[0105] In certain embodiments, the composition as described herein is administered once a week for a predetermined number of weeks. In one embodiment, the scheduled number of weeks is 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks. In some embodiments, dosing can be discontinued for at least one week. In certain embodiments, the composition is administered as a single dose followed by a second dose one to six weeks later. In certain embodiments, the composition may be administered at six to twelve months intervals.

[0106] When the composition as described herein is administered multiple times, the individual doses may be essentially of the same dosage amount or different dosage amounts. Furthermore, the composition may be administered (e.g., via intratumoral injection) at the same tumor site or different tumor sites.

[0107] The xenogeneic compositions of the present invention may be administered in varying dosage amounts, depending on, inter alia, the route of administration, the condition of the patient, and the type and severity of the patient’s disease, although appropriate dosages may be determined based on clinical trials.

[0108] In certain embodiments, the xenogeneic composition is administered via one or more (e.g., intratumoral) injections of from 1 x 106to 10 x 106, 10 x 106to 20 x 106, 20 x 106to 30 x 106, 30 x 106to 40 x 106, 40 x 106to 50 x 106, 50 x 106to 60 x 106, 60 x 106to 70 x 106, 70 x 106to 80 x 106, 80 x 106to 90 x 106, 90 x 106to 10 x 107, 10 x 107to 20 x 107, 20 x 107to 30 x 107, 30 x 107to 40 x 107, 40 x 107to 50 x 107, 50 x 107to 60 x 107, 60 x 107to 70 x 107, 70 x 107to 80 x 107, 80 x 107to 90 x 107, 90 x 107to 10 x 108, 10 x 108to 20 x 108, 20 x 108to 30 x 108, 30 x 108to 40 x 108, 40 x 108to 50 x 108antigen presenting cells per dose.

[0109] In certain embodiments, the xenogeneic composition is administered via one or more (e.g., intratumoral) injections of about 1 x 106, about 5 x 106, about 10 x 106, about 15 x 106, about 20 x 106, about 25 x 106, about 30 x 106, about 35 x 106, about 40 x 106, about 45 x 106, about 50 x 106, about 55 x 106, about 60 x 106, about 65 x 106, about 70 x 106, about 75 x 106, about 80 x 106, about 85 x 106, about 90 x 106, about 95 x 106, about 10 x 107, about 15 x 107, about 20 x 107, about 25 x 107, about 30 x 107, about 35 x 107, about 40 x 107, about 45 x 107, about 50 x 107, about 55 x 107, about 60 x 107, about 65 x 107, about 70 x 107, about 75 x 107, -20- NAI-5004557346v1Attorney Docket No.14648-051-228 about 80 x 107, about 85 x 107, about 90 x 107, about 95 x 107, about 10 x 108, about 15 x 108, about 20 x 108, about 25 x 108, about 30 x 108, about 35 x 108, about 40 x 108, about 45 x 108, or about 50 x 108antigen presenting cells per dose. In certain embodiments, the xenogeneic composition is administered via one or more (e.g., intratumoral) injections of about 100,000 and / or about 500,000 antigen presenting cells per dose. In certain embodiments, the xenogeneic composition is administered via one or more (e.g., intratumoral) injections of about 1 million, about 5 million, about 10 million, about 15 million, about 25 million, about 30 million, about 35 million, about 40 million, about 45 million, about 50 million, about 55 million, about 60 million, about 65 million, about 70 million, about 75 million, about 80 million, about 85 million, about 90 million, about 95 million, or about 100 million antigen presenting cells per dose. In one embodiment, the xenogeneic composition is administered via one or more (e.g., intratumoral) injections of about 100 million antigen presenting cells per dose. In some embodiments, the antigen presenting cells in such compositions are mature antigen presenting cells. In some embodiments, the antigen presenting cells in such compositions are mature antigen presenting cells in combination with immature antigen presenting cells.

[0110] A single dose of antigen presenting cells may be administered via one or more (e.g., intratumoral) injections. In certain embodiments, the xenogeneic composition is administered via one or more (e.g., intratumoral) injections of from 1 x 106to 10 x 106, 10 x 106to 20 x 106, 20 x 106to 30 x 106, 30 x 106to 40 x 106, 40 x 106to 50 x 106, 50 x 106to 60 x 106, 60 x 106to 70 x 106, 70 x 106to 80 x 106, 80 x 106to 90 x 106, 90 x 106to 10 x 107, 10 x 107to 20 x 107, 20 x 107to 30 x 107, 30 x 107to 40 x 107, 40 x 107to 50 x 107, 50 x 107to 60 x 107, 60 x 107to 70 x 107, 70 x 107to 80 x 107, 80 x 107to 90 x 107, 90 x 107to 10 x 108, 10 x 108to 20 x 108, 20 x 108to 30 x 108, 30 x 108to 40 x 108, 40 x 108to 50 x 108antigen presenting cells per injection. In certain embodiments, the xenogeneic composition is administered via one or more (e.g., intratumoral) injections of about 1 x 106, about 5 x 106, about 10 x 106, about 15 x 106, about 20 x 106, about 25 x 106, about 30 x 106, about 35 x 106, about 40 x 106, about 45 x 106, about 50 x 106, about 55 x 106, about 60 x 106, about 65 x 106, about 70 x 106, about 75 x 106, about 80 x 106, about 85 x 106, about 90 x 106, about 95 x 106, about 10 x 107, about 15 x 107, about 20 x 107, about 25 x 107, about 30 x 107, about 35 x 107, about 40 x 107, about 45 x 107, about 50 x 107, about 55 x 107, about 60 x 107, about 65 x 107, about 70 x 107, about 75 x 107, about 80 x 107, about 85 x 107, about 90 x 107, about 95 x 107, about 10 x 108, about 15 x 108, about 20 x -21- NAI-5004557346v1Attorney Docket No.14648-051-228 108, about 25 x 108, about 30 x 108, about 35 x 108, about 40 x 108, about 45 x 108, or about 50 x 108antigen presenting cells per injection. In certain embodiments, the xenogeneic composition is administered via one or more (e.g., intratumoral) injections of about 100,000 and / or about 500,000 antigen presenting cells per injections. In certain embodiments, the xenogeneic composition is administered via one or more (e.g., intratumoral) injections of about 1 million, about 5 million, about 10 million, about 15 million, about 25 million, about 30 million, about 35 million, about 40 million, about 45 million, about 50 million, about 55 million, about 60 million, about 65 million, about 70 million, about 75 million, about 80 million, about 85 million, about 90 million, about 95 million, or about 100 million antigen presenting cells per injection. In one embodiment, the xenogeneic composition is administered via one or more (e.g., intratumoral) injections of about 100 million antigen presenting cells per injection. In certain embodiments, the xenogeneic composition comprises at least 40% alive antigen presenting cells. 5.3.3 Clinical Outcomes

[0111] The therapeutic effect observed upon administration of the xenogeneic composition of the present invention, individually or in combination with another anticancer therapy, may be assessed by any of the methods commonly used in anticancer therapies. In certain embodiments, the administration of the xenogeneic composition triggers an immune response specific to the targeted tumor.

[0112] Furthermore, treatment with the xenogeneic composition described herein, individually or in combination with another anticancer therapy, may shrink not only the targeted tumor but also lead to shrinkage of untreated tumors elsewhere in the body. Such an outcome is known as abscopal effect. Thus, treatment with the xenogeneic composition described herein, individually or in combination with another anticancer therapy, may induce a systemic anti- tumor immune response. In certain embodiments, administration of the xenogeneic composition described herein via a targeted intratumoral injection leads to an abscopal effect. In certain embodiments, administration of the xenogeneic composition described herein via a targeted intratumoral injection in combination with another anticancer therapy (e.g., tumor-treating fields (TTFs) or radiation) leads to an abscopal effect.

[0113] In certain embodiments, the therapeutic effect observed upon administration of the xenogeneic composition of the present invention, individually or in combination with another anticancer therapy, is assessed by measuring immunologic response. In certain embodiments, -22- NAI-5004557346v1Attorney Docket No.14648-051-228 the immunologic response is evaluated by measuring immunologic markers in blood. In certain embodiments, tumor-specific immunological response is evaluated. In certain embodiments, systemic immunological response is evaluated. The immunological response may be evaluated via • cell tracking (which may involve staining PBMCs with specific antibodies, for example, for one HLA class I or one HLA-class II antigen that is selectively expressed on donor vaccine cells); • evaluating dendritic cell-induced alloimmunization (which may involve screening of alloantibodies against, for example, HLA-A, B, C (MHC-class I) and HLA-DR, DQ, DP (MHC-class II) antigens); • monitoring autoimmune events (which may involve screening of autoantibodies against autoantigens, including nuclear antigens (e.g., ANA, SSA, SSB, Sm, RNP, Scl-70, Centromeres, and Jo-1) and liver parenchyma-associated autoantigens (e.g., liver-kidney microsomal antigens and mitochondrial antigens) • assessing complement activation and / or classical / alternative complement function; and / or • evaluating immune cell occurrence and activation state (e.g., CD3+ , CD3+4+ and CD3+8+ T cells, CD19+ B-cells CD3-16+56+ NK-cells, CD3-16+56+69+ NK cells, CD3+16+56+ NKT-cells, CD3+16+56+69+ NKT-cells and CD3+HLA-DR+ T cells).

[0114] In certain embodiments, the therapeutic effect observed upon administration of the xenogeneic composition of the present invention, individually or in combination with another anticancer therapy, is assessed by evaluating the size of the tumor / tumors. This may be done after 3 and / or 6 months from administration of the treatment, or as deemed necessary based on tumor progression.

[0115] In certain embodiments, the therapeutic effect observed upon administration of the xenogeneic composition of the present invention, individually or in combination with another anticancer therapy, is assessed by evaluating tumor control. In some embodiments, tumor control is evaluated by CT and / or MRI scans, measuring number of tumor specific T cells, measuring AFP (alpha-feto protein) levels in blood, measuring the level of circulating tumor cell, or any combination thereof. -23- NAI-5004557346v1Attorney Docket No.14648-051-228

[0116] In certain embodiments, the therapeutic effect observed upon administration of the xenogeneic composition of the present invention, individually or in combination with another anticancer therapy, is assessed by evaluating systemic inflammatory response. This may be done by evaluating, for example, systemic release of relevant cytokines, chemokines, and other inflammatory parameters in blood (e.g., IL-1R, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL- 12p70, IL-13, IL-17A, G-CSF, GM-CSF, IFN-gamma, MCP-1, MIP-1 beta and TNF-alpha).

[0117] In certain embodiments, the therapeutic effect is assessed by evaluating long term changes in Eastern Cooperative Oncology Group (ECOG) and / or Karnofsky performance status (KPS) scores. In certain embodiments, the therapeutic effect is assessed by evaluating long term changes in Quality-of-Life scores.

[0118] In certain embodiments, the therapeutic effect is assessed in terms of partial response and / or complete response. In certain embodiments, the therapeutic effect is assessed in terms of progression free survival and / or overall survival. In certain embodiments, progression‐free survival and / or overall survival is evaluated by measuring blood parameters, for example, by measuring levels of lactate dehydrogenase (LDH) and derived neutrophil‐to‐lymphocyte ratio (dNLR). Elevated LDH and dNLR may be associated with poorer survival outcomes in patients treated with immunotherapy. In certain embodiments, administration of the xenogeneic composition of the present invention, individually or in combination with another anticancer therapy, results in reduced LDH and dNLR levels relative to baseline.

[0119] In certain embodiments, the therapeutic effect is assessed in terms of adverse events, registered as a measure of safety and tolerability, which may include changes in vital signs from baseline (e.g., heart rate, blood pressure, body temperature), changes in laboratory parameters from baseline, etc. In one embodiment, administration of the xenogeneic composition of the present invention, individually or in combination with another anticancer therapy, is not associated with severe adverse events. 5.3.4 Combination Treatment

[0120] When used in treating cancer, the xenogeneic compositions as described herein may be combined with other therapies. In some embodiments, the xenogeneic composition is administered in combination with therapies decreasing the immunosuppresive tumor environment or activating the immune system. -24- NAI-5004557346v1Attorney Docket No.14648-051-228

[0121] In certain embodiments, the xenogeneic composition is administered (e.g., by intratumoral injection) in combination with one or more other anticancer therapies. Examples of such other anti-cancer therapies include, but are not limited to, anti-CTLA4 therapy, anti-PD1 therapy, anti-PDL1 therapy, anti-LAG-3 therapy, tumor-treating fields (TTFs), cell-based therapy, a tyrosine kinase inhibitor, a VEGF inhibitor, or any combination thereof. In one embodiment, the other anti-cancer therapy comprises treatment with one or more biologics. In one embodiment, the other anti-cancer therapy comprises treatment with cellular therapy products or gene therapy products. In one embodiment, the other anti-cancer therapy is CAR T- cell based therapy. In one embodiment, the other anti-cancer therapy comprises treatment with T-cell engagers. In one embodiment, the other anti-cancer therapy comprises treatment with one or more immune checkpoint inhibitors. In one embodiment, the other anti-cancer therapy comprises treatment with one or more small-molecule drugs. In one embodiment, the other anti- cancer therapy comprises treatment with one or more protein kinase inhibitors. In one embodiment, the other anti-cancer therapy comprises treatment with one or more tyrosine kinase inhibitors. In one embodiment, the other anti-cancer therapy comprises treatment with one or more alkylating agents, antimetabolites, natural products, or hormones. In one embodiment, the other anti-cancer therapy comprises treatment with gemcitabine or 5-fluorouracil. In one embodiment, the other anti-cancer therapy comprises surgery or radiation therapy. In one embodiment, the other anti-cancer therapy comprises tumor-treating fields.

[0122] In certain embodiments, the other anti-cancer therapy comprises treatment with imatinib, sunitinib, regorafenib, pazopanib, nilotinib, avapritinib, ripretinib, sorafenib, pimitespib, ipilimumab, tremelimumab, nivolumab, pembrolizumab, cemiplimab, atelizumab, avelumab, durvalumab, relatlimab, or any combination thereof.

[0123] The combination treatments as described herein may provide “synergy” and prove “synergistic,” i.e., the therapeutic effect achieved following administration of two or more individual therapies used together is greater than the sum of the therapeutic effects achieved following administration of the individual therapies separately. Such synergy may be determined by methods commonly known in the art. In certain embodiments, the combination treatment (i.e., administration of the xenogeneic composition of the present invention in combination with another anticancer therapy) provides a synergistic antitumor effect. -25- NAI-5004557346v1Attorney Docket No.14648-051-228

[0124] Suitable dosages of the individual therapies and / or compositions may be lowered due to the combined action (synergy), so as to increase the therapeutic index or mitigate toxicity or other side-effects or consequences. 5.4 Pharmaceutical Compositions

[0125] Provided herein are pharmaceutical compositions comprising the xenogeneic compositions described in Section 5.1.

[0126] In certain embodiments, provided herein are pharmaceutical compositions comprising the xenogeneic compositions in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, and / or excipients. Such pharmaceutical compositions can be formulated according to standard procedures in the art. In one embodiment, the pharmaceutical composition is in the form of an aqueous solution.

[0127] In certain embodiments, the pharmaceutical composition comprises one or more pharmaceutically acceptable carriers. In some embodiments, the pharmaceutically acceptable carrier is a phosphate buffered saline solution, water, or an emulsion, such as an oil / water or water / oil emulsion. In certain embodiments, the pharmaceutical composition is formulated as an aqueous solution, for example, in physiologically compatible buffers such as Hanks’s solution, Ringer’s solution, or physiological saline buffer.

[0128] In certain embodiments, the pharmaceutical composition comprises pharmaceutically acceptable adjuvants, excipients, stabilizers, preservatives, and / or other components known in the art. In one embodiment, the pharmaceutical composition comprises a wetting agent. In some embodiments, the pharmaceutical composition comprises one or more suspending, stabilizing, and / or dispersing agents. In some embodiments, the pharmaceutical composition comprises one or more emulsifiers. In certain embodiments, the pharmaceutical composition is sterilized. In one embodiment, the pharmaceutical composition comprises one or more preservatives. In another embodiment, the pharmaceutical composition does not contain a preservative.

[0129] In certain embodiments, the pharmaceutical compositions provided herein may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and / or preservatives. -26- NAI-5004557346v1Attorney Docket No.14648-051-228

[0130] In certain embodiments, the pharmaceutical composition comprising the antigen presenting cells as described herein is in the form of a suspension or dispersion. Such a suspension or dispersion may be prepared by means of conventional dispersing and suspending processes, and may contain one or more pharmaceutically acceptable excipients. In one embodiment, the pharmaceutical composition is an isotonic aqueous suspension or dispersion. In one embodiment, the suspension or dispersion comprises one or more viscosity-regulating agents.

[0131] In certain embodiments, the pharmaceutical composition comprising the xenogeneic composition as described herein is formulated in the form of a hydrogel. In some embodiments, the hydrogel is a multidomain peptide-based hydrogel. Such multidomain peptide-based hydrogels may be prepared by methods known in the art. In certain embodiments, the antigen presenting cells embedded within a multidomain peptide-based hydrogel exhibit significantly improved delivery and retention within tumors. In certain embodiments, the pharmaceutical composition is a hydrogel-based sustained release composition.

[0132] In certain embodiments, the xenogeneic compositions described herein are formulated into a pharmaceutical composition with diluents and / or with other components such as IL-2 or other cytokines or cell populations.

[0133] The pharmaceutical compositions described herein may be kept at temperatures around 2-8 °C, or may be frozen and then thawed shortly before use. In certain embodiments, the pharmaceutically acceptable carrier may be a medium preserving frozen cells. For example, the xenogeneic compositions comprising antigen presenting cells described herein may be frozen in heat inactivated universal donor plasma comprising dimethyl sulfoxide (DMSO) to allow for storage thereof. Such a medium comprising the antigen presenting cells may be used directly, e.g., injected intratumorally, once thawed. Alternatively, cells frozen in such a medium may be thawed, washed, and re-suspended in an appropriately buffered saline solution or a saline solution comprising human serum albumin before being administered, e.g., injected intratumorally. In one embodiment, the pharmaceutically acceptable carrier is a saline solution comprising human serum albumin. In another embodiment, the pharmaceutically acceptable carrier is a physiological sodium chloride solution comprising 2% human serum albumin.

[0134] The pharmaceutical compositions provided herein may be formulated for any route of administration, including, but not limited to, intratumoral, intravenous, intradermal, -27- NAI-5004557346v1Attorney Docket No.14648-051-228 subcutaneous, intramuscular, or intranodal administration. In one embodiment, the pharmaceutical composition is formulated for parenteral administration. In another embodiment, the pharmaceutical composition is formulated for intratumoral administration. In intratumoral administration, the pharmaceutical composition is injected directly into a tumor. In a specific embodiment, the pharmaceutical composition as described herein is administered by one or more intratumoral injections directly into a tumor. The injection may be to a single tumor or more than one tumor.

[0135] The pharmaceutical compositions of the present invention may be administered in a manner appropriate to the disease to be treated. The dosage amounts and frequency of administration will be determined by such factors as the route of administration, the condition of the patient, and the type and severity of the patient’s disease, although appropriate dosages may be determined based on clinical trials.

[0136] The following examples are offered by way of illustration, and not by way of limitation. 6. EXAMPLES 6.1 Example 1: Xenogeneic compositions comprising porcine antigen presenting cells 6.1.1 Isolation of Peripheral Blood Mononuclear Cells

[0137] Porcine blood (~100ml total volume) collected in EDTA vacutainers (BD Biosciences, San Diego, USA) was diluted 1:1 with Phosphate Buffered Saline (PBS).30ml of the diluted blood was carefully layered on 20ml of Ficoll Paque Plus (Cytiva, Uppsala, Sweden) in a 50ml Falcon tube. Density gradient centrifugation was performed at 600xg for 20min with the centrifuge brakes turned off. After centrifugation, Peripheral Blood Mononuclear Cells (PBMC) were collected from the interphase and washed three times with PBS. Each wash consisted of a 5min centrifugation step at 350xg followed by replacement of supernatant with fresh PBS. After the final wash, PBMC were resuspended in 5ml of ACK Lysing Buffer (Gibco, Thermo Fisher Scientific Inc., Waltham, USA) and incubated for 5min. After lysis of red blood cells, PBMC were centrifuged and resuspended in AIM V Medium (Gibco, Thermo Fisher Scientific Inc., Waltham, USA) supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS; Gibco, Thermo Fisher Scientific Inc., Waltham, USA). Human PBMC were similarly -28- NAI-5004557346v1Attorney Docket No.14648-051-228 purified from anonymous healthy donor buffy coats obtained from the Karolinska University Hospital Blood Bank (Stockholm, Sweden). 6.1.2 Isolation of Porcine CD14+ Cells

[0138] Porcine CD14+ cells were isolated via positive magnetic bead selection using a cross- reactive anti-human CD14 MicroBeads isolation kit (Miltenyi, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. In two separate purifications starting from 1x108porcine PBMC, the positively selected CD14+ yield amounted to 20% of starting cell count. Cell viability of both PBMC and CD14+ fraction after positive selection was >93% when analyzed using Trypan Blue exclusion (Gibco, Thermo Fisher Scientific Inc., Waltham, USA). 6.2 Example 2: Mouse in vivo experiment using human PBMCs – murine melanoma model

[0139] Human peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood using the Ficoll density gradient centrifugation method.

[0140] PBMCs were then labeled with CFSE (carboxyfluorescein succinimidyl ester). CFSE-labeled PBMCs were intratumorally injected at a dose of approximately 1 x 106cells into C57BL / 6J mice expressing B16 melanoma cell line.

[0141] Comparison of the tumor size between the treatment group and the control was conducted by measuring tumor volume at day 12. The results are shown in the table below, and in FIG.2. Mean tumor size6.3 Example 3: Mouse in vivo experiment using human PBMCs – murine bladder cancer model

[0142] Human PBMCs were isolated from peripheral blood using the Ficoll density gradient centrifugation method.

[0143] PBMCs were then labeled with CFSE (carboxyfluorescein succinimidyl ester). CFSE-labeled PBMCs were intratumorally injected at a dose of approximately 1 x 106cells into C57BL / 6J mice expressing MB49 carcinogen induced bladder cancer cell line. -29- NAI-5004557346v1Attorney Docket No.14648-051-228

[0144] Comparison of the tumor size between the treatment group and the control was conducted by measuring tumor volume at day 7. The results are shown in the table below, and in FIG.3. Mean tumor size Treatment (n = 10) 1320 mm36.4Example 4: Mixed Lymphocyte Reaction

[0145] Human PBMC were stained with Violet Proliferation Dye 450 (VPD450; BD Biosciences, San Diego, USA) following the manufacturer’s instructions. In the first xenogeneic Mixed Lymphocyte Reaction (MLR) study, VPD450-stained human PBMC were mixed with unstained porcine PBMC at various ratios. Specifically, starting from a 10:10 human to porcine cell ratio, and proceeding with a fixed number of human cells combined with gradually decreasing numbers of porcine cells to reach a 10:1 human to porcine cell ratio. The 10:10 ratio wells contained a combination of 2x105human PBMC and 2x105porcine PBMC, while the 10:1 human to porcine cell ratio wells contained a combination of 2x105human PBMC and 2x104porcine PBMC. In the second xenogeneic study, VPD450-stained human PBMC were mixed with unstained porcine CD14+ cells at various ratios, as described above. All samples were resuspended into round-bottom 96-well cell culture plates in 200µl AIM V Medium supplemented with 10% heat-inactivated FBS and incubated at 37°C and 5% CO2 for seven days. On day five, 100µL fresh culture medium was added to each well. Both xenogeneic MLR studies included two (n=2) PBMC donors separately combined with a single porcine donor. Cells from neither donor / species were inactivated via irradiation / chemical treatment, resulting in a two-way xenogeneic MLR, however, only human cells were labeled with the proliferation dye. Control wells included allogeneic MLR consisting of 2x104human PBMC combined with 2x104human PBMC from an unrelated donor. Additional control wells received 2x104human PBMC from a single donor.

[0146] On day seven of the MLR, cells were washed twice in saline solution and incubated with Fc-receptor Binding Inhibitor (Invitrogen; Thermo Fisher Scientific Inc., Waltham, USA) followed by staining anti-CD3 VioGreen (Miltenyi, Bergisch Gladbach, Germany; Clone -30- NAI-5004557346v1Attorney Docket No.14648-051-228 REA613), anti-CD45RA BV650 (BD Biosciences, San Diego, USA; Clone HI100), anti-CD8 BB515 (BD Biosciences, San Diego, USA; Clone RPA-T8), anti-CCR7 PE (Miltenyi, Bergisch Gladbach, Germany; Clone REA546), anti-HLA-DR PerCP-Cy5.5 (BD Biosciences, San Diego, USA; Clone G46-6), anti-CD4 APC-Vio770 (Miltenyi, Bergisch Gladbach, Germany; Clone REA623), and anti-CD56 APC (Miltenyi, Bergisch Gladbach, Germany; Clone REA196). Samples were stained while resuspended in Stain Buffer (FBS) (BD Biosciences, San Diego, USA), incubated in the dark at 4°C, and, after two washing steps, were acquired using a FACS Celesta flow cytometer (BD Biosciences, San Diego, USA). Post-acquisition data analysis was conducted using FlowJo 10.8.1 software (FlowJo LLC, Ashland, USA). 6.5 Example 5: In vitro study comparison: porcine PBMC vs human PBMC

[0147] Porcine PBMCs were plated with human PBMCs at varying ratios of human to porcine cells (h:p) as described in the MLR study above. The ratios of human to porcine cells (h:p) were h:p = 10:10, 10:9, 10:8, 10:7, 10:6, 10:5, 10:4, 10:3, 10:2, and 10:1. Separately, human PBMCs alone and allogeneic human PBMCs were evaluated as reference samples.

[0148] Human CD4 T cells count and human CD8 T cells count were evaluated on Day 7 for all samples, including the reference samples. The results are shown in FIG.4A and FIG.4B, respectively.

[0149] Human NK cells count was evaluated on Day 7 for all samples, including the reference samples. The results are shown in FIG.4C. 6.6 Example 6: In vitro study comparison: porcine monocytes (CD14+ cells) vs human PBMC

[0150] Porcine monocytes (containing CD14+ cells) were plated with human PBMCs at varying ratios of human to porcine cells (h:p) as described in the MLR study above. The ratios of human to porcine cells (h:p) were h:p = 10:10, 10:9, 10:8, 10:7, 10:6, 10:5, 10:4, 10:3, 10:2, and 10:1. Separately, human PBMCs alone and allogeneic human PBMCs were evaluated as reference samples.

[0151] Human CD4 T cells count and human CD8 T cells count were evaluated on Day 7 for all samples, including the reference samples. The results are shown in FIG.5A and FIG.5B, respectively. -31- NAI-5004557346v1Attorney Docket No.14648-051-228

[0152] Proliferated human CD4 T cells count and proliferated human CD8 T cells count were also evaluated on Day 7 for all samples, including the reference samples. The results are shown in FIG.5C and FIG.5D, respectively.

[0153] HLA-DR on human CD4 T cells and human CD8 T cells, in terms of mean fluorescence intensity (MFI), was evaluated on Day 7 for all samples, including the reference samples. The results are shown in FIG.5E and FIG.5F, respectively.

[0154] Human NK cells count was evaluated on Day 7 for all samples, including the reference samples. The results are shown in FIG.5G. 6.7 Example 7: Study design for evaluation of Gal+ porcine XPCs in syngeneic mouse models

[0155] A total of approximately 22 billion mononuclear cells were acquired from Gal+ pigs’ leukopaks. Approximately 1.8 billion cells were recovered at harvest for Gal+ pigs (>80.0% monocytes). Harvested cells were washed with 2.5% HSA in PBS before final formulation (HSA + Plasmalyte A + CryoStor 10). Contents and purity of the Gal+ porcine xenogeneic antigen presenting cells (XPCs) used for in vivo animal studies are depicted in Tables 1 and 2 below. Study design for evaluation of Gal+ porcine XPCs in syngeneic mouse models of different tumor types / cell lines is depicted in Table 3 below.

[0156] Table 1. Contents of Gal+ porcine XPCs Leukopheresis n [. y p Rotea n

[0158] Table 3. In Vivo Study Design -32- NAI-5004557346v1Attorney Docket No.14648-051-228 Group ID#Cancer Treatmente e e e e e e e e e e e e e e6.8 Example 8: In vivo evaluation of Gal+ porcine XPCs in mouse breast cancer EMT-6 syngeneic model -33- NAI-5004557346v1Attorney Docket No.14648-051-228

[0159] In vivo evaluation of Gal+ porcine XPCs is conducted in a EMT-6 syngeneic model in female BALB / c mice, as per the study design outlined in Example 7. The syngeneic model is established by injecting tumor cells into both flanks. The experimental drug (Gal+ XPCs) is injected only in one flank (right flank). Gal+ porcine XPCs are injected intratumorally (30 µL) at a low dose of approximately 1 x 106cells per dose and at a high dose of approximately 5 x 106cells per dose. Animals are administered 3 doses (Day 0, 3, and 6) or 6 doses (Day 0, 3, 6, 9, 12, and 15). 6.9 Example 9: In vivo evaluation of Gal+ porcine XPCs in mouse lung cancer KLN-205 syngeneic model

[0160] In vivo evaluation of Gal+ porcine XPCs is conducted in a KLN-205 syngeneic model in female DBA / 2 mice, as per the study design outlined in Example 7. The syngeneic model is established by injecting tumor cells into both flanks. The experimental drug (Gal+ XPCs) is injected only in one flank (right flank). Gal+ porcine XPCs are injected intratumorally (30 µL) at a low dose of approximately 1 x 106cells per dose and at a high dose of approximately 5 x 106cells per dose. Animals are administered 3 doses (Day 0, 3, and 6) or 6 doses (Day 0, 3, 6, 9, 12, and 15). 6.10 Example 10: In vivo evaluation of Gal+ porcine XPCs in mouse pancreatic cancer KPC syngeneic model

[0161] In vivo evaluation of Gal+ porcine XPCs is conducted in a KPC syngeneic model in female C57BL / 6 mice, as per the study design outlined in Example 7. The syngeneic model is established by injecting tumor cells into both flanks. The experimental drug (Gal+ XPCs) is injected only in one flank (right flank). Gal+ porcine XPCs are injected intratumorally (30 µL) at a low dose of approximately 1 x 106cells per dose and at a high dose of approximately 5 x 106cells per dose. Animals are administered 3 doses (Day 0, 3, and 6) or 6 doses (Day 0, 3, 6, 9, 12, and 15). 6.11 Example 11: In vivo evaluation of Gal+ porcine XPCs in mouse prostate cancer RM-1 syngeneic model

[0162] In vivo evaluation of Gal+ porcine XPCs is conducted in a RM-1 syngeneic model in female C57BL / 6 mice, as per the study design outlined in Example 7. The syngeneic model is established by injecting tumor cells into both flanks. The experimental drug (Gal+ XPCs) is injected only in one flank (right flank). Gal+ porcine XPCs are injected intratumorally (30 µL) -34- NAI-5004557346v1Attorney Docket No.14648-051-228 at a low dose of approximately 1 x 106cells per dose and at a high dose of approximately 5 x 106cells per dose. Animals are administered 3 doses (Day 0, 3, and 6) or 6 doses (Day 0, 3, 6, 9, 12, and 15). 6.12 Example 12: In vivo evaluation of Gal+ porcine XPCs in mouse sarcoma WEHI-164 syngeneic model

[0163] In vivo evaluation of Gal+ porcine XPCs is conducted in a WEHI-164 syngeneic model in female BALB / c mice, as per the study design outlined in Example 7. The syngeneic model is established by injecting tumor cells into both flanks. The experimental drug (Gal+ XPCs) is injected only in one flank (right flank). Gal+ porcine XPCs are injected intratumorally (30 µL) at a low dose of approximately 1 x 106cells per dose and at a high dose of approximately 5 x 106cells per dose. Animals are administered 3 doses (Day 0, 3, and 6) or 6 doses (Day 0, 3, 6, 9, 12, and 15). 6.13 Example 13: In vivo evaluation of Gal+ porcine XPCs in mouse sarcoma S- 180 syngeneic model

[0164] In vivo evaluation of Gal+ porcine XPCs is conducted in a S-180 syngeneic model in female BALB / c mice, as per the study design outlined in Example 7. The syngeneic model is established by injecting tumor cells into both flanks. The experimental drug (Gal+ XPCs) is injected only in one flank (right flank). Gal+ porcine XPCs are injected intratumorally (30 µL) at a low dose of approximately 1 x 106cells per dose and at a high dose of approximately 5 x 106cells per dose. Animals are administered 3 doses (Day 0, 3, and 6) or 6 doses (Day 0, 3, 6, 9, 12, and 15). 6.14 Example 14: In vivo evaluation of Gal+ porcine XPCs in mouse renal cancer RENCA syngeneic model

[0165] In vivo evaluation of Gal+ porcine XPCs is conducted in a RENCA syngeneic model in female BALB / c mice, as per the study design outlined in Example 7. The syngeneic model is established by injecting tumor cells into both flanks. The experimental drug (Gal+ XPCs) is injected only in one flank (right flank). Gal+ porcine XPCs are injected intratumorally (30 µL) at a low dose of approximately 1 x 106cells per dose and at a high dose of approximately 5 x 106cells per dose. Animals are administered 3 doses (Day 0, 3, and 6) or 6 doses (Day 0, 3, 6, 9, 12, and 15). 6.15 Example 15: In vivo evaluation of Gal+ porcine XPCs in mouse urinary bladder cancer MB49 syngeneic model -35- NAI-5004557346v1Attorney Docket No.14648-051-228

[0166] In vivo evaluation of Gal+ porcine XPCs is conducted in a MB49 syngeneic model in female C57BL / 6 mice, as per the study design outlined in Example 7. The syngeneic model is established by injecting tumor cells into both flanks. The experimental drug (Gal+ XPCs) is injected only in one flank (right flank). Gal+ porcine XPCs are injected intratumorally (30 µL) at a low dose of approximately 1 x 106cells per dose and at a high dose of approximately 5 x 106cells per dose. Animals are administered 3 doses (Day 0, 3, and 6) or 6 doses (Day 0, 3, 6, 9, 12, and 15). 6.16 Example 16: In vivo evaluation of Gal+ porcine XPCs in mouse colon cancer MC-38 syngeneic model

[0167] In vivo evaluation of Gal+ porcine XPCs is conducted in a MC-38 syngeneic model in female C57BL / 6 mice, as per the study design outlined in Example 7. The syngeneic model is established by injecting tumor cells into both flanks. The experimental drug (Gal+ XPCs) is injected only in one flank (right flank). Gal+ porcine XPCs are injected intratumorally (30 µL) at a low dose of approximately 1 x 106cells per dose and at a high dose of approximately 5 x 106cells per dose. Animals are administered 3 doses (Day 0, 3, and 6) or 6 doses (Day 0, 3, 6, 9, 12, and 15). 6.17 Example 17: In vivo evaluation of Gal+ porcine XPCs in mouse brain Neuro- 2a syngeneic model

[0168] In vivo evaluation of Gal+ porcine XPCs is conducted in a Neuro-2a syngeneic model in female A / JGpt mice, as per the study design outlined in Example 7. The syngeneic model is established by injecting tumor cells into both flanks. The experimental drug (Gal+ XPCs) is injected only in one flank (right flank). Gal+ porcine XPCs are injected intratumorally (30 µL) at a low dose of approximately 1 x 106cells per dose and at a high dose of approximately 5 x 106cells per dose. Animals are administered 3 doses (Day 0, 3, and 6) or 6 doses (Day 0, 3, 6, 9, 12, and 15). 6.18 Example 18: In vivo evaluation of Gal+ porcine XPCs in mouse breast JC-6 syngeneic model

[0169] In vivo evaluation of Gal+ porcine XPCs is conducted in a JC syngeneic model in female BALB / c mice, as per the study design outlined in Example 7. The syngeneic model is established by injecting tumor cells into both flanks. The experimental drug (Gal+ XPCs) is injected only in one flank (right flank). Gal+ porcine XPCs are injected intratumorally (30 µL) -36- NAI-5004557346v1Attorney Docket No.14648-051-228 at a low dose of approximately 1 x 106cells per dose and at a high dose of approximately 5 x 106cells per dose. Animals are administered 3 doses (Day 0, 3, and 6) or 6 doses (Day 0, 3, 6, 9, 12, and 15). 6.19 Example 19: In vivo evaluation of Gal+ porcine XPCs in mouse liver H-22 syngeneic model

[0170] In vivo evaluation of Gal+ porcine XPCs is conducted in a H22 syngeneic model in female BALB / c mice, as per the study design outlined in Example 7. The syngeneic model is established by injecting tumor cells into both flanks. The experimental drug (Gal+ XPCs) is injected only in one flank (right flank). Gal+ porcine XPCs are injected intratumorally (30 µL) at a low dose of approximately 1 x 106cells per dose and at a high dose of approximately 5 x 106cells per dose. Animals are administered 3 doses (Day 0, 3, and 6) or 6 doses (Day 0, 3, 6, 9, 12, and 15). 6.20 Example 20: In vivo evaluation of Gal+ porcine XPCs in mouse lung LLC-1 syngeneic model

[0171] In vivo evaluation of Gal+ porcine XPCs is conducted in a LLC1 syngeneic model in female C57BL / 6 mice, as per the study design outlined in Example 7. The syngeneic model is established by injecting tumor cells into both flanks. The experimental drug (Gal+ XPCs) is injected only in one flank (right flank). Gal+ porcine XPCs are injected intratumorally (30 µL) at a low dose of approximately 1 x 106cells per dose and at a high dose of approximately 5 x 106cells per dose. Animals are administered 3 doses (Day 0, 3, and 6) or 6 doses (Day 0, 3, 6, 9, 12, and 15). 6.21 Example 21: In vivo evaluation of Gal+ porcine XPCs in mouse melanoma B16-F10 syngeneic model

[0172] In vivo evaluation of Gal+ porcine XPCs is conducted in a B16-F10 syngeneic model in female C57BL / 6 mice, as per the study design outlined in Example 7. The syngeneic model is established by injecting tumor cells into both flanks. The experimental drug (Gal+ XPCs) is injected only in one flank (right flank). Gal+ porcine XPCs are injected intratumorally (30 µL) at a low dose of approximately 1 x 106cells per dose and at a high dose of approximately 5 x 106cells per dose. Animals are administered 3 doses (Day 0, 3, and 6) or 6 doses (Day 0, 3, 6, 9, 12, and 15). 6.22 Example 22: In vivo evaluation of Gal+ porcine XPCs in mouse pancreatic Panc-02 syngeneic model -37- NAI-5004557346v1Attorney Docket No.14648-051-228

[0173] In vivo evaluation of Gal+ porcine XPCs is conducted in a Panc02 syngeneic model in female C57BL / 6 mice, as per the study design outlined in Example 7. The syngeneic model is established by injecting tumor cells into both flanks. The experimental drug (Gal+ XPCs) is injected only in one flank (right flank). Gal+ porcine XPCs are injected intratumorally (30 µL) at a low dose of approximately 1 x 106cells per dose and at a high dose of approximately 5 x 106cells per dose. Animals are administered 3 doses (Day 0, 3, and 6) or 6 doses (Day 0, 3, 6, 9, 12, and 15). 6.23 Example 23: Intratumoral administration of porcine PBMCs to human patients

[0174] This example evaluates the safety and / or efficacy of a composition comprising porcine antigen presenting cells administered intratumorally as single agent or in combination with another anticancer therapy in cancer patients.

[0175] Patients are divided in two treatment groups. The first group of patients will receive the treatment composition as monotherapy, whereas the second group of patients will receive the treatment composition in combination with a checkpoint inhibitor or a tyrosine kinase inhibitor. Patients will be treated with the treatment composition at an increasing dose and / or frequency, starting with a dose of 10 x 106cells per injection. Patients may or may not have previously been treated with an anticancer therapy. Certain outcome measures are provided below.

[0176] Adverse events will be registered as a measure of safety and tolerability, which will include changes in vital signs from baseline (e.g., heart rate, blood pressure, body temperature), changes in laboratory parameters from baseline, etc.

[0177] Immunologic response will be evaluated by measuring immunologic markers in blood. The size of the tumor / tumors will be evaluated after 3 and 6 months or as deemed necessary based on tumor progression.

[0178] Systemic inflammatory response will be evaluated, including potential systemic release of relevant cytokines, chemokines and other inflammatory parameters in blood (e.g., IL- 1R, IL-2,IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p70, IL-13, IL-17A, G-CSF, GM-CSF, IFN- gamma, MCP-1, MIP-1 beta and TNF-alpha).

[0179] Tumor control will be evaluated, which will include CT / MRI scans, measuring number of tumor specific T cells, measuring AFP (alpha-feto protein) levels in blood, and / or measuring the level of circulating tumor cell. -38- NAI-5004557346v1Attorney Docket No.14648-051-228

[0180] Tumor-specific immunological responses and systemic immunological response will be evaluated.

[0181] Long term changes in Eastern Cooperative Oncology Group (ECOG) and / or Karnofsky performance status (KPS) scores will be evaluated. Further, long term changes in Quality of Life scores will be evaluated.

[0182] Therapeutic effect will be assessed in terms of partial response, complete response, progression free survival, and / or overall survival. 6.24 Example 24: Manufacture of Xenogeneic Composition 1

[0183] Xenogeneic Composition 1 (hereafter “Composition 1”) comprises non-cultured, enriched, and unmanipulated xenogeneic-antigen presenting cells (XPCs) from a highly inbred wild-type swine source.

[0184] The manufacturing of the Composition 1 consists of isolation and enrichment of XPCs from fresh leukopak (LPK) extracted from donor blood. Upon receipt of LPK at the manufacturing site, cell viability and cell count were evaluated by automated cell counter, and the percentage of XPC was evaluated by flow cytometry. The Rotea™ Counterflow Centrifugation System was used to concentrate the XPC fraction while removing lighter, less dense material such as T cells. After centrifugation, the concentrated XPCs were assessed for viability and cell count. They were then centrifuged again and washed in a solution of PlasmaLyte A (PLA), and human serum albumin (HSA). Following washing, the XPCs were formulated in cryopreservation media containing PLA, HSA, and CryoStor™ CS10 (CS10). The washed XPCs, formulated with CS10 were then filled into primary containers, cryopreserved and stored in liquid nitrogen until use. 6.25 Example 25: Impact of Composition 1 from Research and Development runs on T cell activation and proliferation of human PBMCs

[0185] The objective of this study was to elucidate the dose-dependent effect of Composition 1 from Research and Development (R&D) runs on T-cell activation and proliferation using healthy hPBMCs.

[0186] Two R&D lots of Composition 1 were used for the MLR assays in this study. One of the lots (R&D Lot 10) was derived from a Gal- KO pig. Descriptions of the Composition 1 lots are provided in Table 4. -39- NAI-5004557346v1Attorney Docket No.14648-051-228 Table 4. Descriptions of Composition 1 Lots from R&D Runs Used in MLR Assays Composition 1 Lot Number Viability (%) R&D Lot 11 89.4

[0187] Comlthy donors to measure the proliferation and activation of human T cells in the presence of XPCs. Neither xenogeneic cells nor hPBMCs were inactivated via irradiation / chemical treatment, resulting in a 2-way xenogeneic MLR. Human PBMCs were stained with Violet Proliferation Dye 450 (VPD450; BD Biosciences) following the manufacturer’s instructions. Unstained porcine XPCs or other cells were mixed with VPD450-stained hPBMCs in 1 of 3 effector to target (E:T) ratios: 0.1:1, 0.01:1, or 0.001:1 (Table 7). All samples were resuspended into round-bottom 96-well cell culture plates in 200 µL AIM V™ Medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and incubated at 37°C and 5% CO2 for 6 days. On day 5, 100 µL fresh culture medium was added to each well.

[0188] Human PBMCs were attained from STEMCELL Technologies and are described in Table 5. Table 5. Description of hPBMCs Used in All MLR Assays Type of Cells Donor ID Batch Number Viability (%)

[0189] T (HLA)haploidentical (Table 6) and were expected to result in a robust allogeneic immune response. Table 6. HLA Information for Human PBMC Donors hPBMCs Batch number Allo PBMCs Batch number-40- NAI-5004557346v1Attorney Docket No.14648-051-228 hPBMCs Batch number Allo PBMCs Batch number (2307427012) (2307931001)Table 7. Study Design of MLR Assays MLR Co-culture Composition 1 hPBMC (number of cells) E:T Ratio (number of cells)aNbA.

[0190] Flow cytometry was used to assess T-cell proliferation in the MLR assays. On Day 6 of MLR cell culture, cells were washed twice in Cell Staining buffer (CSB) and incubated with a Fc-receptor blocking reagent to decrease non-specific binding. Then, cells were stained with anti-CD3 VioGreen, anti-HLA-DR PerCP-Cy5.5, anti-CD8 BB515, and anti-CD4 APC-Vio770. Samples were incubated for 20 ± 5 minutes in the dark at 4°C, and, after 2 washing steps, cells were resuspended in CSB and acquired using a Northern Lights®flow cytometer. Post- acquisition data analysis was conducted using FSC Express software. -41- NAI-5004557346v1Attorney Docket No.14648-051-228

[0191] MLRs were not replicated, therefore, statistical analyses were not performed.

[0192] As shown in FIG.6, co-culture of Composition 1 and hPBMCs from a healthy human donor resulted in dose-dependent increases in CD4+and CD8+T cells proliferation. Proliferation of CD4+and CD8+T cells in the MLR using Gal-negative Composition 1 was less than that found in the same E:T ratio MLR assay with Gal-positive lots of Composition 1. The results of this study indicate that Composition 1 XPCs stimulate the human immune system and result in proliferation of cytotoxic (CD8+) and helper (CD4+) T cells. 6.26 Example 26: Impact of Composition 1 from R&D runs on HLA-DR expression on human PBMCs

[0193] The objective of this study was to elucidate the dose-dependent effect of Composition 1 from R&D runs on HLA-DR expression on T-cells using healthy human PBMCs.

[0194] MLR assays were conducted as described in Example 25 (see Section 6.25) and the study design is described in Table 7. The hPBMCs used in this study are described in Table 5 and 6. The R&D lots of Composition 1 used for the MLR assays in this study are described in Table 4. One of the lots (R&D Lot 10) was derived from a Gal- KO pig.

[0195] Flow cytometry was used to assess T-cell proliferation and HLA-DR expression in the MLR assays. On Day 6 of MLR cell culture, cells were washed twice in CSB and incubated with a Fc-receptor blocking reagent to decrease non-specific binding. Then, cells were stained with anti-CD3 VioGreen, anti-HLA-DR PerCP-Cy5.5, anti-CD8 BB515, and anti-CD4 APC- Vio770. Samples were incubated for 20 ± 5 minutes in the dark at 4°C, and, after 2 washing steps, cells were resuspended in CSB and acquired using a Northern Lights® flow cytometer. Post-acquisition data analysis was conducted using FSC Express software. MLRs were not replicated, therefore, statistical analysis was not performed.

[0196] As shown in FIG.7, co-culture of R&D lots of Composition 1 and hPBMCs from healthy human donors resulted in dose-dependent increases in HLA-DR expression on CD4+and CD8+T cells. Assays using Gal-negative Composition 1 had lower expression of HLA-DR than that found at the same E:T ratio in MLR assays with Gal-positive lots of Composition 1. The expression of the surface protein HLA-DR is associated with T-cell activation and cytokine production. These results provide further support that Composition 1 activates T cells in the human immune system. -42- NAI-5004557346v1Attorney Docket No.14648-051-228 6.27 Example 27: Anti-tumor functionality: impact of Composition 1 from R&D runs on secretion of cytokines

[0197] The objective of this study was to elucidate the dose-dependent effect of Composition 1 on secretion of the cytokines GM-CSF, IFN-γ, IL-8, and TNF-α in a 2-way MLR.

[0198] Two R&D lots of Composition-1 were used for the MLR assays in this study. One of the lots (Lot 10) was derived from a Gal- KO pig. Descriptions of the Composition 1 lots are provided in Table 4.

[0199] The MLR assays were conducted as described in Example 25 (see Section 6.25) and the study design is described in Table 7. The hPBMCs used in this study are described in Table 5 and Table 6.

[0200] Levels of the targeted cytokines were measured with the Multiplexing fluorescent Bead Assay. Supernatants from the MLR assays were retained for cytokine analysis. The human cytokines IFN-γ, IL-8, TNF-α, and GM-CSF were measured with Luminex® Multiplex Technology.

[0201] The Luminex multiplexing technology is based on color-coded polystyrene beads. Bead coloration is achieved using different concentrations of red and infrared fluorophore dyes to create 100 uniquely colored bead sets. Each bead set contains a unique color / fluorophore signature, which is identified by the bead analyzer. Bead sets can be combined so that multiple analytes can be quantified within the same assay.

[0202] The bead analyzer (Bio-Plex 200) includes a dual-laser system and a flow-cytometry system. One laser activates the fluorescent dye within the beads, which identifies the specific analyte. The second laser excites the fluorescent conjugate (streptavidin-phycoerythrin) that has been bound to the beads during the assay. The amount of the conjugate detected by the analyzer is in direct proportion to the amount of the target analyte. The results are quantified according to a standard curve.

[0203] Each sample was run in duplicate, and mean cytokine levels were calculated for each sample. No statistical analysis was carried out.

[0204] As shown in FIG.8, a dose-dependent increase in the human IFN-γ, IL-8, and TNF- α, and GM-CSF was observed when T cells were activated by co-culture with Composition 1. In allogeneic MLR assays with 2 human PBMC donors, IFN-γ, IL-8, TNF-α, and GM-CSF levels -43- NAI-5004557346v1Attorney Docket No.14648-051-228 were generally in a range similar to those found at MLRs with hPBMCs and Composition 1 at E:T ratios of 0.1:1. 6.28 Example 28: Impact of Composition 1 from Process Qualification runs on T cell activation and proliferation of human PBMCs

[0205] The objective of this study was to elucidate the dose-dependent effect of Composition 1 from Process Qualification (PQ) runs on T-cell activation and proliferation using healthy hPBMCs.

[0206] Three PQ lots of Composition 1 were used for the MLR assays in this study. Descriptions of the Composition 1 lots are provided in Table 8. PBMCs from 2 healthy human donors were used in the MLR assays. Donor material was obtained from STEMCELL Technologies and is described in Table 5 and Table 6. Table 8. Descriptions of Composition 1 Lots from PQ Runs Used in MLR Assays Composition 1 Lot Number Viability (%) P L t 1 933 06

[0207] Compos althy donors to measure the proliferation and activation of human T cells in the presence of XPCs. Neither xenogeneic cells nor hPBMCs were inactivated via irradiation / chemical treatment, resulting in a 2-way xenogeneic MLR. Human PBMC were stained with Violet Proliferation Dye 450 (VPD450; BD Biosciences) following the manufacturer’s instructions. VPD450-stained hPBMCs were mixed with unstained porcine XPCs or hPBMCs at the indicated cell ratio. All samples were resuspended into round-bottom 96-well cell culture plates in 200 µL AIM VTMMedium supplemented with 10% heat-inactivated FBS and incubated at 37°C and 5% CO2 for 6 days. On day 5, 100 µL fresh culture medium was added to each well.

[0208] The MLR studies were conducted with hPBMCs from a single healthy donor combined separately with 3 PQ lots of Composition 1 from 3 different porcine donors. Composition 1 was combined with Human PBMCs in E:T ratios of 0.1:1, 0.01:1, and 0.001:1 (Table 9). The hPBMCs (Donor ID CE0009297) were also used in assays with another human PBMC donor (Allo hPBMC, Donor ID SCT3840411), a R&D lot of Gal-negative Composition 1 (R&D Lot 10), and the positive control, PHA. The negative control consisted of hPBMCs from -44- NAI-5004557346v1Attorney Docket No.14648-051-228 the single human donor (Donor ID CE0009297). Each combination of cells was run in triplicate on different days. Table 9. Study Design of MLR Assays with PQ Lots of Composition 1 MLR Co-culture Composition 1 Donor hPBMC E:T Ratio (number of cells) (number of cells)ab

[0209] Flow cytometry was used to assess T-cell proliferation in the MLR assays. On Day 6 of MLR cell culture, cells were washed twice in CSB and incubated with a Fc-receptor blocking reagent to decrease non-specific binding. Then, cells were stained with anti-CD3 VioGreen, anti-HLA-DR PerCP-Cy5.5, anti-CD8 BB515, and anti-CD4 APC-Vio770. Samples were incubated for 20 ± 5 minutes in the dark at 4°C, and, after 2 washing steps with phosphate- buffered saline (PBS), samples were stained by a live / dead (Far red) dye. Followed by two washes with CSB, cells were resuspended in CSB and acquired using a Northern Lights® flow cytometer. Post-acquisition data analysis was conducted using FSC Express software.

[0210] For each MLR assay, mean and standard deviation (SD) were calculated for CD4+and CD8+T-cell proliferation. T-tests were used to compare CD4+and CD8+T-cell proliferation between the following assays: each PQ lot of Composition 1 and donor hPBMCs at the E:T ratios of 0.1:1 or 0.01:1 compared with donor hPBMCs alone. Equal variance was assumed between the groups compared. One-way analysis of variance (ANOVA) was used to compare the 3 PQ lots at the E:T of 0.1:1. A P-value of <0.05 was considered significantly different.

[0211] As shown in FIG.9, co-culture of Composition 1 from PQ runs and hPBMCs from healthy human donors resulted in dose-dependent increases in human T-cell proliferation. Both CD4+and CD8+T cells exhibited a dose-dependent increase in proliferation. In all comparisons, -45- NAI-5004557346v1Attorney Docket No.14648-051-228 CD4+and CD8+T-cell proliferation was significantly higher at the higher E:T ratio (0.1:1 and 0.01:1) when compared with the negative control (P<0.001 and P<0.05, respectively). Among the 3 PQ lots at an E:T ratio of 0.1:1, an equivalent effect was found on CD4+and CD8+T cell proliferation (p>0.05). In addition, T-cell proliferation in co-cultures of Gal-negative Composition 1 and hPBMCs was lower than that in Gal-positive lots of Composition 1 at the same E:T ratio. Allogeneic co-culture of PBMCs from two human donors also resulted in lower T-cell proliferation than that found at the highest E:T ratio of Composition 1 and hPBMCs. The results of this study indicate that Composition 1 stimulates the human immune system and result in proliferation of cytotoxic (CD8+) and helper (CD4+) T cells. 6.29 Example 29: Impact of Composition 1 from PQ runs on HLA-DR expression on human PBMCs

[0212] The objective of this study was to elucidate the dose-dependent effect of Composition 1 from PQ lots on HLA-DR expression on T-cells derived from healthy human PBMCs.

[0213] MLR assays were conducted as described in Example 28 (see Section 6.28) and the study design is described in Table 9. The 3 lots of PQ Composition 1 used for the MLR assays in this study are described in Table 8. The hPBMCs used in this study are the same as those in Example 28 (see Section 6.28) and are described in Table 5 and Table 6.

[0214] Flow cytometry was used to assess and HLA-DR expression in the MLR assays. On Day 6 of MLR cell culture, cells were washed twice in CSB and incubated with a Fc-receptor blocking reagent to decrease non-specific binding. Then, cells were stained with anti-CD3 VioGreen, anti-HLA-DR PerCP-Cy5.5, anti-CD8 BB515, and anti-CD4 APC-Vio770. Samples were incubated for 20 ± 5 minutes in the dark at 4°C, and, after 2 washing steps with PBS, samples were stained by a live / dead (Far red) dye. Followed by 2 washes with CSB, cells were resuspended in CSB and acquired using a Northern Lights® flow cytometer. Post-acquisition data analysis was conducted using FSC Express software.

[0215] For each MLR assay, mean and SD were calculated for HLA-DR expression on CD4+and CD8+T cells. T-tests were used to compare HLA-DR expression on CD4+and CD8+T cells between the following assays: each PQ lot of Composition 1 and donor hPBMCs at the E:T ratios of 0.1:1 or 0.01:1 compared with donor hPBMCs alone. Equal variance was assumed between the groups compared. One-way ANOVAs were used to compare the 3 PQ lots at the E:T of 0.1:1. A P-value of <0.05 was considered significantly different. -46- NAI-5004557346v1Attorney Docket No.14648-051-228

[0216] As shown in FIG.10, co-culture of Composition 1 from PQ runs and hPBMCs from healthy human donors resulted in dose-dependent increases in HLA-DR expression on CD4+and CD8+T cells. In all comparisons, HLA-DR expression on CD4+and CD8+T-cells was significantly higher at the higher E:T ratios (0.1:1 and 0.01:1) when compared with the negative control (all P<0.05). At an E:T ratio of 0.1:1, HLA-DR expression on T cells was lower in allogeneic MLR assays and assays using Gal-negative Composition 1 than that found at the same E:T ratio in MLR assays with Gal-positive lots of Composition 1. At an E:T ratio of 0.1:1, HLA- DR expression was not significantly different among the 3 PQ lots (p>0.05). Thus, the XPCs from the 3 different lots / donors exhibited generally consistent effects on HLA-DR. The expression of the surface protein HLA-DR is associated with T-cell activation and cytokine production. These results provide further support that Composition 1 activates T cells in the human immune system. 6.30 Example 30. Anti-tumoral functionality of Composition 1 from PQ runs

[0217] The objective of this study was to elucidate the dose-dependent effect of Composition 1 from 3 PQ lots on IFN-γ and TNF-α release from human PBMCs.

[0218] MLR assays were conducted as described in Example 28 (see Section 6.28) and the study design is described in Table 9. The 3 lots of PQ Composition 1 used for the MLR assays in this study are described in Table 8. The hPBMCs used in this study are the same as those in Example 28 (see Section 6.28) and are described in Table 5 and Table 6.

[0219] On Day 6 of MLR cell culture, cell suspensions were centrifuged, and the supernatants were collected and stored at -80°C until the day that cytokine ELISA assays were performed. The levels of TNF-α and IFN-γ were measured using the Human IFN-gamma Quantikine™ ELISA kit and Human TNF-alpha Quantikine™ ELISA from R&D systems, respectively. This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific to a human cytokine – either IFN- γ and TNF-α – was pre-coated onto microplates. Standards and samples were pipetted into the wells and any of the specific cytokine present was bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked monoclonal antibody specific to the human cytokine was added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution was added to each well. Color development is proportional to the amount of cytokine bound in the initial step and is halted with a stop solution. Optical density in each well was then -47- NAI-5004557346v1Attorney Docket No.14648-051-228 read with a microplate reader (SpectraMax iD3), and cytokine level was calculated using the resulting standard curve.

[0220] For each MLR assay, mean and SD were calculated for TNF-α and IFN-γ levels. One-way ANOVAs were used to compare the 3 lots at the E:T of 0.1:1. A P-value of <0.05 was considered significantly different.

[0221] As shown in FIG.11, co-culture of Composition 1 and hPBMCs from healthy human donors resulted in dose-dependent secretion of IFN-γ and TNF-α from T cells. Among the 3 PQ lots at an E:T ratio of 0.1:1, there were equivalent levels of IFN-γ and TNF-α (p > 0.05). At an E:T ratio of 0.1:1 in both allogeneic MLR assays and assays using Gal-negative Composition 1, IFN-γ level was lower than that found at the same E:T ratio in MLR assays with Gal-positive lots of Composition 1. However, this was not observed for TNF-α level. 6.31 Example 31: Bioactivity and efficacy study of Composition 1 in a breast cancer model (JC) in tumor bearing mice

[0222] This study is an update of Example 18. Breast cancer may be particularly amenable to Composition 1 treatment because tumors are accessible to intratumoral (IT) injections. The objective of this study was to assess the in vivo antitumor activity of Composition 1 using the mouse breast cancer JC syngeneic model. 6.31.1 Materials and Methods (i) Test Articles

[0223] Two dosages – 5×106or 1×106cells / mouse – of the test article, Composition 1, were analyzed in the syngeneic JC breast cancer model in BALB / c mice (Table 10).

[0224] Porcine cells were cryopreserved in CryoStor10, PlasmaLyte A (PLA), and human serum albumin (HSA) and stored in liquid nitrogen. After thawing in a 37 °C water bath, cell counts and viability were measured. The dosing solutions were stored at 4 °C and used within 3 hours without any adjustment to cell concentration. Administration of PBS to the primary tumor was used as a negative control for the study. Table 10. Test Articles for Breast Cancer (JC) Mouse Model Lot Dosage Dose Cell Viability (%) Viable Cell Concentration-48- NAI-5004557346v1Attorney Docket No.14648-051-228

[0225] Thirty-five female BALB / c mice 6-8 weeks old obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. were used in this study.

[0226] JC cells were purchased from American Type Culture Collection (ATCC). (ii) Study design

[0227] Mice were randomized to 1 of 3 groups – a control group, a high-dose Composition 1 group, or a low-dose Composition 1 group – and received treatment every 3 days (Table 11). The 8 animals in the Composition 1 groups were equally divided into a group that received 6 doses of Composition 1 and a group that received 3 doses of Composition 1. The control group received a total of 6 doses. Table 11. Study Design for Breast Cancer (JC) Mouse Model Group Mode Number of Dosage Dose Dose Route of Dosing l Individuals (cells / mouse) Concentration volume Administration Schedule(iii) Establishment of Tumor Model

[0228] JC cells were maintained in RPMI 1640 medium with 10% FBS. Cells were incubated at 37℃ in a humidified incubator with 5% CO2. JC cells in the logarithmic phase of growth (4×105cells / mouse) were subcutaneously injected into the right flank of 35 mice under sterile conditions to establish a primary tumor. To establish a secondary tumor, an equivalent number of JC cells were injected into the left flank 3 days later. The secondary tumor inoculation was performed before Composition 1 treatment.

[0229] When the mean tumor volume reached 40-60 mm3, 24 mice were randomized to the 3 treatment groups (8 mice per group) according to the randomized grouping method. The tumor size and body weight were considered and balanced among groups. Variance of the mean tumor -49- NAI-5004557346v1Attorney Docket No.14648-051-228 burdens in each group was not to exceed ±10% of the overall mean. The day of grouping was designated as Day 0, and dosing was started. Mice were dosed individually with 30 μL of Composition 1 (Groups 1 and 2) or PBS (Control Group). Only tumors on the right flank received IT injection. 6.31.2 Assessments (i) Tumor Volume and Growth

[0230] Tumor size was measured with calipers 3 times per week. Tumor volumes were estimated from measurements of the two diameters of the individual tumors.

[0231] Tumor volume (TV, mm3) was calculated as TV = (a×b2) / 2, with a equal to the tumor length and b equal to the tumor width. The %TGITV and was calculated as %TGITV = (1-TVT / TVC) ×100%, where TVTis the mean TV of a treatment group on the day of tumor measurement and TVCis the mean TV of the control group on the day of tumor measurement. Relative tumor volume (RTV) was calculated as RTV = Vt / V0, where Vtequals the tumor volume on measurement day and V0is the tumor volume on randomization day. The percent treated to control ratio based on RTV (%T / CRTV) was calculated as %T / CRTV= TRTV / CRTV×100%, where TRTVis the mean RTV of the treatment group and CRTVis the mean RTV of control group.

[0232] Descriptive statistics of tumor parameters were calculated for pooled dosage groups. All animals treated with high-dose or low-dose Composition 1 were combined in calculations, regardless of the number of treatments received. For example, the mean TV for the high-dose group was calculated for animals given 3 or 6 doses of Composition 1. (ii) Clinical Symptoms and Mortality

[0233] Clinical signs were recorded daily. Animal survival also was recorded. Observations included general health, body weight, behavior, and any adverse effects related to the dose administration. (iii) Body Weight

[0234] Animal body weights were measured 3 times per week. Percent body weight change (%BWC) was calculated as %BWC = (BWt-BW0) / BW0× 100%, where BWtis the body weight on the day of measurement and BW0is the body weight on the day of randomization. (iv) Hematology and Blood Biochemistry -50- NAI-5004557346v1Attorney Docket No.14648-051-228

[0235] At the end of the study, animals were euthanatized by CO2 asphyxiation. A minimum of 0.5 mL blood was collected from each animal by cardiac puncture. Whole blood from 4 animals in each treatment group was placed in EDTA-K2 anticoagulative tubes for hematology analysis of white blood cell (WBC) count, red blood cell (RBC) count, hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelets (PLT), red blood cell distribution width-standard deviation (RDW-SD), red blood cell distribution width-coefficient of variation (RDW-CV), platelet distribution width (PDW), mean platelet volume (MPV), platelet- large cell ratio (P-LCR), procalcitonin (PCT), nucleated red blood cells (NRBC), neutrophils (NEUT), lymphocytes (LYMPH), monocytes (MONO), eosinophils (EO), basophils (BASO), reticulocytes (RET), immature reticulocyte fraction (IRF), low fluorescence reticulocyte ratio (LFR), medium fluorescence reticulocyte ratio (MFR), high fluorescence reticulocyte ratio (HFR), reticulocyte hemoglobin equivalent (RET-He), immature platelet fraction (IPF), white blood cell count in body fluid (WBC-BF), red blood cell in body fluid (RBC-BF), mononuclear cells (MN), polymorphonuclear leukocytes (PMN), total nucleated cell count (TC-BF#).

[0236] Blood from the other 4 mice in each group was placed in pro-coagulation tubes for blood biochemistry analysis of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), total protein (TP), albumin (ALB), total bilirubin (TBIL), urea (UREA), creatinine (CRE), glucose (GLU), creatine kinase (CK), total cholesterol (TCHO), and triglycerides (TG). (v) Flow Cytometry

[0237] Flow cytometry was used to phenotype peripheral blood lymphocytes obtained from study animals. Peripheral blood samples were collected on Days 0, 3, and 7 and at the end of the study and placed in tubes containing K2-EDTA. A volume of 50 μL from each sample was used for further processing. Red blood cells were lysed, and the resulting single cell suspensions were stained for murine CD45, CD3, CD4, CD8, and CD69 and porcine SWC3a. The stained cell suspensions were analyzed on a LSRFortessaTMinstrument from BD and the resulting flow cytometry data were analyzed with FLOWJOTM_V10.8.1 software. (vi) Histology of Tumors

[0238] At the end of the study, tumors were collected for hematoxylin and eosin (H&E) staining and immunohistochemical (IHC) analysis, which was conducted on 2 mice from the -51- NAI-5004557346v1Attorney Docket No.14648-051-228 control group and each treatment group. The H&E staining protocol for tumor tissue involves deparaffinizing and rehydrating slides through xylene and graded ethanol, followed by staining nuclei with hematoxylin for 3–5 minutes. Excess hematoxylin is removed by rinsing, differentiation occurs in acid alcohol, and bluing is achieved with ammonia water or tap water. The cytoplasm and extracellular components are stained with eosin for 30 seconds to 1 minute, then slides are dehydrated using increasing ethanol concentrations and cleared with xylene. Finally, a mounting medium is applied, and a coverslip is placed. This process highlights nuclei (purple / blue) and cytoplasm (pink / red), aiding in morphological assessment of tumor tissue. The percentage tumor necrosis was measured with H&E staining.

[0239] IHC was used to analyze the expression of the surface proteins CD3, CD4, CD8, CD11b, and SWC3a. For IHC analysis, tumor sections were fixed in acetone, dried, and then washed with PBS. Then, sections were blocked with normal mouse serum. Once the blocking solution was removed, sections were incubated with primary antibodies for rat anti-mouse antibodies to CD3, CD4, and CD8 and mouse anti-pig antibodies for CD11b and FITC- conjugated SWC3a. Sections were rinsed with PBS and incubated with rabbit anti-rat and anti- FITC antibodies. Hematoxylin was used as a counterstain. All stained sections were analyzed using a Leica DMRX-e microscope and representative images were captured using a 10x objective lens. Tissues were scored in a range from 0 to 4+, with 0 indicating no positive cells and 4+ indicating a massive number of positive cells (Table 12). Table 12. Scoring of Tumor Cell Surface Markers for IHC Analysis Score Description-52- NAI-5004557346v1Attorney Docket No.14648-051-228 (vii) FFPE

[0240] At the end of the study, heart, liver, spleen, breast cancer, kidney, and brain were collected from each animal, fixed in fixed in 10% neutral buffered formalin for 24 hours at room temperature (RT) and then prepared into FFPE for potential future analysis. (viii) Statistical Analysis

[0241] All quantitative data in the study were expressed as mean ± standard error of the mean (SEM). All animals within a treatment group were analyzed together regardless of the number of doses the animals received. The tumor growth curves were plotted with the observation time on the X-axis and corresponding tumor volume (geometric mean) on the Y- axis. The body weight change was plotted with the observation time on the X-axis and corresponding body weight (geometric mean) on the Y-axis. A 2-tailed t-test and additional, 2- way ANOVAs were used to analyze the statistical significance of differences between the treatment groups and the control group. A P-value of <0.05 was considered significantly difference. Median survival was calculated with GraphPad Prism version 10.4.1. 6.31.3 Results (i) Survival

[0242] By Day 33 all animals in the control group had died, whereas 3 animals in the high- dose group and 4 animals in the low-dose group remained alive on this day (FIG.12). All animals in the high-dose group had died by Day 40, but 1 animal in the low-dose group was still alive on Day 42 and subsequently euthanized on Day 45, as per protocol. Median survival increased from 26 days in the vehicle group to 31 and 32 days in the high and low dose Composition 1 groups, respectively (survival curves statistically significant by Mantel-Cox test, p<0.01). (ii) Body Weight

[0243] On Day 26, the mean body weights of the control group, high-dose group, and low- dose group increased between 11.89% - 17.65% (2.69 g - 3.85 g) compared with Day 0 (Table 13). -53- NAI-5004557346v1Attorney Docket No.14648-051-228 Table 13. Change in Body Weights of Mice Bearing a JC Syngeneic Model of Breast Cancer Body Weight DrugGroup # of%BWC Related AniTreatment(g, Mean±SEM) malsDay 26 vs Day 0 Death Day 0 Day 26Day 2618 PBS, Q3D×6 21.98 ± 0.71 25.83 ± 0.51 17.65%(3.85 g)0 / 8Composition 1- High, 2 8 Q3D×6 (n=4)22.22 ± 0.27 25.03 ± 0.42 12.61%0 / 8(2.81 g)Q3D×3 (n=4) Composition 1- Low, 11.89%Q3D×6 (n=4) 3 822.71 ± 0.42 25.40 ± 0.73 0 / 8(2.69 g)Q3D×3 (n=4) (iii) Tumor Volume

[0244] On Study Day 26, tumor volumes were compared between the control group and all animals in each treatment group (regardless of number of doses an animal received) when all animals in all groups were still alive (Table 14, FIG.13). Mean right tumor volume of the high- dose group and low-dose group were 48.95% and 38.51% smaller than the control group, respectively. The mean tumor volume in both the high-dose and low-dose groups were significantly lower than the controls on Day 26. In addition, left flank tumor volume exhibited a trend toward smaller tumor volume in the Composition 1 treated groups, indicating that Composition 1 may also have a systemic effect on distant tumor cells. -54- NAI-5004557346v1Attorney Docket No.14648-051-228 Table 14. Antitumor Activity of Test Article in JC Syngeneic Tumor Model (Both Flanks) Tumor Volume Tumor Volume # of (mm3, %T / C T RT (mm3, Treatme Group Ani Mean±SEM) %TGITV R V V Mean±SEM) %TGITV nt mals (Right Tumor) Day 26 Day 26 Day 26 (Left Tumor) Day 26 Day 0 Day 26 Day 0 Day 26 2580.1 1685.37 18 PBS,41.62 ± 3.90 ± Q3D×66 ± / 66.25 / ± / 4.03± 8.272.61 190.93 134.69ion 1- High 1317.1 34.53 1290.29 41.25 ± 8 ± ± 2 8 Q3D×6 48.954.94 ±4.80 176.31*6.05**,#52.123.26± 23.44 (n=4)**,### ##220.21 Q3D×3 (n=4) Composit ion 1- Low 1586.4 40.51 1066.56 41.55 ± 2 ± ± 3 8 Q3D×6 38.516.02 ±3.33 232.86*8.34*,##61.154.02± 36.72 (n=4)*,### #301.43##Q3D×3 (n=4) T tests, treatment compared with vehicle: *P<0.05; **P<0.01; ***P<0.001 2-way ANOVA, treatment compared with vehicle:##P<0.01;###P<0.001 (iv) Hematology and blood biochemistry

[0245] At the end of the study, there was sufficient whole blood collected for hematology assessments from 3 animals in the control group, 2 animals in the high-dose group, and 4 animals in the low-dose group. There were no significant differences among groups, although, 1 outlier in the high-dose group had a much higher total WBC count.

[0246] Sufficient blood was collected from 4 animals per group for most clinical chemistry assessments. There were no significant differences among the groups and all values were generally within the expected range for mice. (v) Peripheral blood immunophenotyping

[0247] Overall, there were no major differences between treatment groups in the levels of CD45+cells, T cell subsets, or activated T cells. No detection of porcine cells above background was detected at any timepoint in the peripheral blood. -55- NAI-5004557346v1Attorney Docket No.14648-051-228 (vi) Tumor histology

[0248] Tumor necrosis ranged from 0% to 90% and was generally greater in mice treated with Composition 1, with a mean of 45% and 65% in high- and low-dose treatment groups, respectively, compared with a mean of 17.5% in the control group (Table 15). All tumor samples were positive for CD3, CD4, CD8, and CD11b, whereas SWC3a was not detected in any of the tumor samples. The expression of CD3, CD4, and CD8 was higher in the tumors of mice treated with Composition 1 compared to those administered vehicle (Table 15). CD11b expression was higher in the tumors of mice in the control group than in those in the Composition 1 groups. Table 15. Scoring of Necrosis and Cell Surface Markers in Left and Right JC Tumors Group Mouse Flank Necrosis CD3 CD4 CD8 CD11b (%) l

[0249] Overall, this study demonstrated an anti-tumor effect of Composition 1 in primary and distant, secondary tumors after multiple IT administrations in a murine JC breast cancer cell model. Median survival time was significantly longer in mice treated with Composition 1. Treatment with Composition 1 at either high-dose or low-dose (pooled 3- and 6-dose treatments) exhibited statistically significant antitumor activity in the right flank (primary tumor) in BALB / c mice. There was also a trend towards smaller tumor volume in the left flank (secondary tumor) with Composition 1 treatment. Among the groups, no change in detection of T cells or activated T cells was observed, and no porcine cells were detected in the peripheral blood. There was an -56- NAI-5004557346v1Attorney Docket No.14648-051-228 increase in necrosis in both treatment groups compared to control group. There were no porcine cells detected in the tumors at the end of the study. 6.32 Example 32: Bioactivity and efficacy study of Composition 1 in a sarcoma model (S180) in tumor-bearing mice

[0250] This study is an update of Example 13. The objective of this study was to assess the in vivo antitumor activity of Composition 1 using the mouse sarcoma S180 syngeneic model 6.32.1 Materials and Methods (i) Test article

[0251] Two dosages – 5×106or 1×106cells / mouse – of the test article, Composition 1, were analyzed in the syngeneic S180 sarcoma model in BALB / c mice (Table 16).

[0252] Porcine cells were cryopreserved in formulation buffer consisting of CryoStor10, PLA, and HSA and stored in liquid nitrogen. After thawing in a 37°C water bath, cell counts and viability were measured. The dosing solutions were stored at 4°C and used within 3 hours without adjustment to cell concentration. Administration of formulation buffer (CryoStor10, PlasmaLyte A, and HSA) to the primary tumor was used as a negative control for the study. Table 16. Test Articles for Sarcoma (S180) Mouse Model Lot Dosage Dose Cell Viability (%) Viable Cell Concentration Number (cells / mouse) (cells / mL)

[0254] S180 cells were purchased from Type Culture Collection of the Chinese Academy of Sciences. (ii) Study design

[0255] Mice were randomized to 1 of 3 groups – a control group, a high-dose Composition 1 group, or a low-dose Composition 1 group – and received treatment every 3 days (Q3D) (Table 17). The 8 animals in each group were equally divided into a group that received 6 doses and a group that received 3 doses. -57- NAI-5004557346v1Attorney Docket No.14648-051-228 Table 17. Study Design for Sarcoma (S180) Mouse Model Group Model Number of Dosage Dose Dose Route of Dosing Individuals (cells / mouse) Concentration volume Administration Schedule ll L Ls a s o u o o e

[0256] S180 cells were maintained in RPMI 1640 medium with 10% FBS. Cells were incubated at 37℃ in a humidified incubator with 5% CO2. S180 cells in the logarithmic phase of growth (2×105cells / mouse) were subcutaneously injected into the right flank of 35 mice under sterile conditions to establish a primary tumor. To establish a secondary tumor, an equivalent number of S180 cells were injected into the left flank 3 days later. The secondary tumor inoculation occurred before treatment began.

[0257] When the mean tumor volume reached 40-60 mm3, 24 mice were randomized to the 3 treatment groups (8 mice per group) according to the randomized grouping method. The tumor size and body weight were considered and balanced among groups. Variance of the mean tumor burdens in each group was not to exceed ±10% of the overall mean. The day of grouping was designated as Day 0, and dosing was started. Mice were dosed individually with 30 μL of Composition 1 (Groups 1 and 2) or formulation buffer containing CryoStor10, PlasmaLyte A, and HSA (Control Group). Only tumors on the right flank were IT injected. 6.32.2 Assessments

[0258] Assessments of tumor growth, clinical symptoms and mortality, body weight, hematology and blood biochemistry, flow cytometry, histology, FFPE and statistical analysis were done as described in Example 31 (see Section 6.31.2). -58- NAI-5004557346v1Attorney Docket No.14648-051-228 6.32.3 Results (i) Survival

[0259] For animals that received 3 doses of Composition 1, there were 7 animals (4 in the control group, 2 in the high-dose group, and 1 in the low-dose group) that reached a humane endpoint on Day 16. Therefore, data from Day 16 were analyzed for animals administered 3 doses. For the animals that received 6 doses of Composition 1 and starting on Day 12, 4 animals (1 in the control group, 1 in the high-dose group, and 2 in the low-dose group) did not survive to scheduled termination day so data from Day 12 were analyzed.

[0260] In the groups treated with 3 injections, all animals in the control group died by Day 16, whereas 4 animals in both the high-dose group and the low-dose group remained alive on this day. All animals in the low-dose group had died by Day 23, but 1 animal in the high-dose group was still alive on Day 23. In the groups treated with 6 injections, all animals in the high-dose group died by Day 16, all animals in the low-dose group died by Day 21, and 1 animal in the control group remained alive until Day 23. Animals alive on Day 23 were subsequently euthanized, as per protocol.

[0261] Median survival in the Composition 1 groups that received 3 injections was not significantly longer than in the control group. For the animals that received 3 injections, the median survival control group, high-dose group, and low-dose group was 16, 17.5, and 19 days, respectively (FIG.14A). For animals receiving 6 doses, median survival was 18.5, 15, and 13.5 days for the control group, high-dose group, and low-dose group, respectively (FIG.14B). (ii) Body Weight

[0262] On Day 16, for groups that received 3 doses of vehicle or test article, the mean body weights increased 7.33% - 23.78% (1.62g - 5.06 g) compared with Day 0 (Table 18). On Day 12, for groups that received 6 doses of vehicle or test article, the mean body weights increased 0.52% - 7.05% (0.07 g - 1.46 g) compared with Day 0 (Table 19). -59- NAI-5004557346v1Attorney Docket No.14648-051-228 Table 18. Effects of Test Articles on Body Weight of Animals Bearing S180 Syngeneic Model (Q3D×3) Body Weight Drug Related u#(g, Mean±SEM)%BWCGro p ofAnimalsTreatmentDay16 vs Death Day 0 DayDay 0Day16161-1 4Vehicle21.50 ± 0.73 26.56 ± 0.6123.78% Q3D×3 (5.06 g)0 / 42-1 4Composition 1-7.33% 21.90 ± 0.44 23.52 ± 1.440 / 4High Q3D×3 (1.62 g)18.12%Composition 1- Low3-1 4 21.49 ± 0.61 25.37 ± 0.960 / 4Q3D×3 (3.88 g)Table 19. Effects of Test Articles on Body Weight of Animals Bearing S180 Syngeneic Model (Q3D×6) Body Weight Drug Related%BWC(g, Mean±SEM)# ofDay12 vs DeathGroup TreatmentAnimalsDay 0Day14Day 0 Day 127.05% (1.46Vehicle1-2 4 21.15 ± 0.53 22.61 ± 0.821 / 4Q3D×6 g)0.52%Composition 1-2-2 4 21.23 ± 0.72 21.30 ± 0.634 / 4Q3D×6 (0.07 g)5.47%Composition 1- Low3-2 4 21.63 ± 0.40 22.78 ± 0.243 / 4Q3D×6 (1.15 g)(iii) Tumor Volume

[0263] On Day 16 in animals treated with 3 doses of vehicle or test article, the mean volume of the right flank tumor in the high-dose group and low-dose group were 29.61% and 41.62% smaller than the control group, respectively (Table 20, FIG.15A). The mean tumor volume in both the high-dose and low-dose groups were significantly smaller than the control. -60- NAI-5004557346v1Attorney Docket No.14648-051-228 Additionally, the left flank tumor volume in the high-dose group was significantly smaller than that of the control. Table 20. Antitumor Activity of 3 Doses of Test Article in S180 Syngeneic Tumor Model (Both Flanks) Tumor Volume Tumor Volume (mm3, Mean±SEM) %TG %T / C (mm3, %T # of Gro (Right Tumor) I RTV Mean±SEM) GI Ani TreatmentTV RTV TVup Day Day 16 Day (Left Tumor) Day mals Day 0Day 1616 16DayDay 161601-1 4Vehicle,47.63 ± 2424.22 ± 61.22 ± Q3D×310.44181.61 / 17 / 0 ± 1247.59 ± .010191.93 / Compositio 47.99 ± 1706.35 ± 41.41 ± 0 ± 807.61 ± n 1- High,29.61 67.64 35.276.66 Q367.78## 15.980 3D×3162.89##Compositio 47.27 ± 1415.27 ± 28.83 ± 0 ± 1138.62 ± 3-1 4 n 1- Low,41.62 47.09 8.736.38398.48### 5.03##0292.97Q3D×3 ## ###2-way ANOVA, treatment compared with vehicle: P<0.01; P<0.001

[0264] In animals treated with 6 doses of vehicle or test article, the mean volume of the right flank tumor in the high-dose group and low-dose group were 37.81% and 22.26% smaller than the control group, respectively, but this difference was not statistically significant (Table 21, FIG.15B). The left flank tumor volume in the high-dose group was significantly smaller than that of the control. The smaller size of the left flank tumor with both 3 and 6 doses of high-dose test article may indicate that Composition 1 also has systemic effects on distant tumor cells. Table 21. Antitumor Activity of 6 Doses of Test Article in S180 Syngeneic Tumor Model (Both Flanks) Tumor Volume Tumor Volume RT %T / %T3 3# of %TG (mm , Mean±SEM) (mm , Mean±SEM) Gro V C GIRTV TVAni Treatment ITV (Right Tumor) (Left Tumor) up Day Day1 Day1 Day1212 Day 1212 22 Day 025.1 47.40 ± 950.95 ± 8 ± 357.77 ±Vehicle, 1.12 ±1-2 4 / / / Q3D×67.97241.4711.61.12 113.372Composition 12.3 47.60 ± 591.44 ± 131.11 ±1.33 ±2-2 4 1- High, 8 ±49.1737.81 63.357.11124.90 1.33 79.82#Q3D×6 2.36 Composition 15.9 47.14 ± 739.28 ± 209.08 ±1.38 ±3-2 4 1- Low, 8 ±63.4622.26 41.565.89147.45 1.38 84.28Q3D×6 3.06 -61- NAI-5004557346v1Attorney Docket No.14648-051-228 (iv) Hematology and blood biochemistry

[0265] At the end of the study for groups in which 3 doses were administered, sufficient whole blood was collected for hematology assessments from only 1 animal in the vehicle group and 2 animals each in the groups treated with Composition 1. No significant differences were found among groups, and all values fell within the normal expected range for mice. Sufficient blood was collected from 2 animals per group for most clinical chemistry assessments. There were no significant differences among the groups, and values were generally within the expected range for mice with the exception of 1 outlier in the low-dose group that had a higher ALT levels than other mice without any other correlating findings.

[0266] In the experiment in which 6 doses were administered, only 1 hematology sample was available for the vehicle group and low-dose group. The hematology values were similar for these samples. There was not enough sample collected for clinical chemistry tests from the mice that received 6 doses of Composition 1. (v) Peripheral blood immunophenotyping

[0267] No significant differences in peripheral blood immunophenotypes were found among groups treated with 3 doses of control or Composition 1. In animals treated with 6 doses of control or high-dose Composition 1, T cells and CD8+T cells were significantly higher in the Composition 1 groups compared with the control group (p< 0.01 and p<0.05, respectively) on Day 3. In animals with 6 doses of control or low-dose Composition 1, CD8+T cells were significantly higher in the Composition 1 groups compared with the control group (p<0.05) on Day 3. No significant detection of porcine cells above background was observed at any timepoint in the peripheral blood. (vi) Tumor histology

[0268] Tumor necrosis ranged from 10% to 90%, with control groups exhibiting relatively larger necrotic areas (Table 22). All tumor samples were positive for CD3, CD4, CD8, and CD11b, whereas SWC3a was not detected in any of the tumor samples. In animals administered 3 doses, slight increases in the level of CD3+cells were observed in the high-dose group compared with the control group. In animals administered 6 doses, the low-dose group had higher levels of CD3+cells compared with the control group. These data suggest a potential treatment-related effect on T-cell infiltration. CD4+and CD8+cells showed similar trends, with a -62- NAI-5004557346v1Attorney Docket No.14648-051-228 slight increase in Composition 1 groups treated with 3 doses relative to the control group. However, no significant differences were noted among groups treated with 6 doses. Large numbers of CD11b+cells were detected in all groups, with a further increase observed following Composition 1 treatment, which suggests an impact on myeloid cell recruitment. (Table 22). Table 22. Scoring of Necrosis and Cell Surface Markers in Left and Right S180 Tumors Group Mouse Flank Necrosis CD3 CD4 CD8 CD11b (%)

[0269] Overall, this study demonstrated that administration of 3 doses of Composition 1 resulted in statistically significant lower tumor volumes than controls on Day 16. Median survival time was not significantly longer in mice treated with Composition 1. Treatment with Composition 1 at either high-dose or low-dose exhibited statistically significant antitumor activity in the right flank (primary tumor) in BALB / c mice. There was also a trend towards smaller tumor volume in the left flank (secondary tumor) with Composition 1 treatment.

[0270] In conclusion, Composition 1 demonstrated significant antitumor activity in female BALB / c mice inoculated with S180 cells, with both high and low doses effectively reducing -63- NAI-5004557346v1Attorney Docket No.14648-051-228 tumor volume in the right flank by Day 16 and high-dose treatment showing efficacy in the left flank. In the 6-dose regimen, significant antitumor activity in the left flank was observed by Day 12 in the high-dose treatment group. Body weight analysis indicated overall weight gain across groups, although, the 6-dose regimen showed a lower increase indicating the safety of the drug. Among the groups, no change in the detection of T cells or activated T cells was observed, and no porcine cells were detected in the peripheral blood. In addition, no porcine cells were detected in the tumors at the end of the study.

[0271] Administration of more than 3 doses of Composition 1 resulted in lower average tumor volumes, but 1 animal in the vehicle group, 1 animal in the high-dose group, and 2 animals in the low-dose group died after the fourth dose. Three animals in the high-dose group and 1 animal in the low-dose group died after the fifth dose. The S180 tumor model is a highly aggressive form of sarcoma that is 100% fatal in control animals, so deaths in these animals are likely related to tumor progression. 6.33 Example 33: Bioactivity and efficacy study Composition 1 in a bladder cancer model (MB49) in tumor-bearing mice

[0272] This study is an update of Example 15. The objective of this study was to assess the in vivo antitumor activity of Composition 1 using the mouse bladder cancer MB49 syngeneic model. 6.33.1 Materials and Methods (i) Test articles

[0273] Two dosages – 5×106or 1×106cells / mouse – of the test article, Composition 1, were analyzed in the syngeneic MB49 bladder cancer model in BALB / c mice (Table 23). Table 23. Test Articles for Bladder Cancer (MB49) Mouse Model Lot Number Dosage Dose Cell Viability Viable Cell ( ll / m ) (%) C n ntr ti nLaboratory Animal Technology Co., Ltd. were used in this study.

[0275] MB49 cells were purchased from Meisen Chinese Tissue Culture Collections. -64- NAI-5004557346v1Attorney Docket No.14648-051-228 (ii) Study design

[0276] Mice were randomized to 1 of 3 groups – a control group, a high-dose Composition 1 group, or a low-dose Composition 1 group – and received treatment every 3 days (Q3D) (Table 24). The 8 animals in each group were equally divided into a subgroup that received 6 doses and a subgroup that received 3 doses. Table 24. Study Design for Bladder Cancer (MB49) Mouse Model Group Model Number of Dosage Dose Dose Route of Dosing Individuals (cells / mouse) Concentration volume Administration Schedule ( ll / mL) ( L)

[0277] MB49 cells were maintained in RPMI 1640 medium with 10% FBS. Cells were incubated at 37°C in a humidified incubator with 5% CO2. MB49 cells in the logarithmic phase of growth (1×106cells / mouse) were subcutaneously injected into the right flank of 35 mice under sterile conditions to establish a primary tumor. To establish a secondary tumor, an equivalent number of MB49 cells were injected into the left flank 3 days later. The secondary tumor inoculation occurred before treatment began.

[0278] When the mean tumor volume reached 40-60 mm3, 24 mice were randomized to the 3 treatment groups (8 mice per group) according to the randomized grouping method. The tumor size and body weight were considered and balanced among groups. Variance of the mean tumor burdens in each group was not to exceed ±10% of the overall mean. The day of grouping was designated as Day 0, and dosing was started. Mice were dosed individually with 30 μL of Composition 1 (Groups 1 and 2) or formulation buffer (CryoStor10, PlasmaLyte A, and HSA) (Control Group). Only tumors on the right flank were IT injected. -65- NAI-5004557346v1Attorney Docket No.14648-051-228 6.33.2 Assessments

[0279] Assessments of tumor growth, clinical symptoms and mortality, body weight, hematology and blood biochemistry, flow cytometry, histology, FFPE and statistical analysis were done as described in Example 31 (see Section 6.31.2). 6.33.3 Results (i) Survival

[0280] Beginning on Day 28, 1 animal each in the 3-injection control group and high-dose Composition 1 group reached the humane endpoint for tumor size and were euthanized. Beginning on Day 26, 2 animals in the 6-injection vehicle group reached the humane endpoint for tumor size and were euthanized. Therefore, data from Day 28 were analyzed for animals administered 3 doses and data from Day 26 for animals administered 6 doses.

[0281] In both the 3-dose and 6-dose control groups, all animals had died by Day 37. In the high-dose groups, 1 animal administered 3 injections, and 2 animals administered 6 injections were alive at the end of the study. In the low-dose group, all animals given 3 injections had died by Day 42, and those given 6 injections had died by Day 63.

[0282] Median survival for animals administered 3 doses of Composition 1 were higher than the control, and median survival for animals administered 6 doses of Composition 1 were significantly higher than the control. The median survival of animals treated with 3 doses of vehicle, high-dose Composition 1, and low-dose Composition 1 were 31.5, 37.5, and 33 days, respectively, while those treated with 6 doses of vehicle, high-dose Composition 1, and low-dose Composition 1 were 27, >41, and 41 days, respectively (FIG.16). (ii) Body weight

[0283] By Day 28, the groups that received 3 doses of vehicle or Composition 1 had increased mean body weight by 13.22% -16.68% (3.18 g – 3.84 g) compared with Day 0 (Table 25). On Day 26, the groups that received 6 doses of vehicle or Composition 1 had increased mean body weight by similar percentages, 5.32% - 18.57% (1.25g – 4.16 g), compared with Day 0 (Table 26). -66- NAI-5004557346v1Attorney Docket No.14648-051-228 Table 25. Effects of Test Articles on Body Weight of Animals Bearing MB49 Syngeneic Model (Q3D×3) Body WeightFound #of(g, Mean±SEM)%BWCGroupAnimalTreatmentDay 28 vsAnimals Death sTable 26. Effects of Test Articles on Body Weight of Animals Bearing MB49 Syngeneic Model (Q3D×6) Body Weight%BWC Found #of(g, Mean±SEM) Day 26vs Day Animals DeathGroup TreatmentAnimals0Day 26Day 0 26 Day 7222.55± 18.57% (4.16Vehicle1 426.71± 0.59 / 0 / 4Q3D×6 0.63 g)Composition 1- 27.67 ± 23.57 ± 24.82 ± 5.32% 2 4 High 0.390 / 40.46 0.69(1.25 g)Q3D×6 (N=2) 23.36 ± 26.22 ± 12.53%Composition 1- Low3 4 / 0 / 4Q3D×60.770.24 (2.86 g)(iii) Tumor volume

[0284] In animals treated with 3 doses of either vehicle or Composition 1, tumor volume in the right flank decreased significantly in the high-dose Composition 1 group compared with the vehicle group on Day 28 (P<0.05) (Table 27). Tumors in the right flank of the high- and low- dose groups were 40.36% and 24.37% smaller, respectively, than those in the vehicle group on Day 28 (Table 27, FIG.17A). -67- NAI-5004557346v1Attorney Docket No.14648-051-228 Table 27. Effects of Test Articles on Tumor Volume of Animals Bearing MB49 Syngeneic Model (Q3D×3) Tumor Volume Tumor Volume 33Grou # of (mm , Mean±SEM) %TGIT(Ri h T RTV %T / CR(mm , Mean±SEM) (Left %TGITAnima Treatment g t umor)V TVTumor)V

[0285] In animals treated with 6 doses of either vehicle or Composition 1, tumor volume in the right flank and left flank decreased significantly in both the high- and low-dose Composition 1 groups compared with the vehicle group on Day 26 (P<0.05 for all) (Table 28). On Day 28, tumors in the right flank of the high- and low-dose groups were 75.33% and 53.01% smaller, respectively, than those in the vehicle group, while those in the left flank were 91.49% and 78.58% smaller, respectively, than in the vehicle group (Table 28, FIG.17B) Table 1. Effects of Test Articles on Tumor Volume of Animals Bearing MB49 Syngeneic Model (Q3D×6) Tumor Volume Tumor Volume %T / 3 3(mm , Mean±SEM) (mm , Mean±SEM) (Left # of %TGI %TGIT T

[0286] At the end of the experiment in animals administered 3 doses, sufficient whole blood was collected for hematology assessments from 2 animals in the control group, 1 animal in high- -68- NAI-5004557346v1Attorney Docket No.14648-051-228 dose group, and 2 animals in the low-dose group. There were no significant differences among groups, and values were similar across samples. Sufficient blood was collected for clinical chemistry assessments from 2 animals per group for most tests. There were no significant differences among groups, and all values were generally within the expected range for mice.

[0287] In animals administered 6 doses, 2 samples per group were used for hematology assessments. The assessments were similar across all animals with no significant difference among groups. For clinical chemistry, 2 samples per group were used for clinical chemistry assessments. The results were similar across all animals with no significant difference among groups. (v) Peripheral blood immunophenotyping

[0288] On Day 3 in animals treated with 3 doses of Composition 1 or vehicle, peripheral blood T-cell levels in the low-dose group were significantly higher compared with the control group (P<0.05). No other significant differences were found in immunophenotype subpopulations among the groups treated with either 3 or 6 doses. Porcine cells in the peripheral blood were not significantly higher than background levels at any timepoint. (vi) Tumor histology

[0289] Tumor necrosis ranged from 10% to 90%, with treatment groups exhibiting relatively larger necrotic areas (Table 29, Table 30). All tumor samples were positive for CD3, CD4, CD8, and CD11b, whereas SWC3a was not detected in any of the tumor samples.

[0290] In the animals administered 3 doses, all groups exhibit moderate levels of CD3+and CD4+cells whereas group 3 (low dose) shows the highest CD8+presence followed by group 1 (control) and group 2 (high dose) (Table 29). Group 3 (low dose group) has the highest CD11b+expression followed by group 1 and group 2. Control groups have the lowest necrosis level whereas treated groups have higher necrosis level.

[0291] In the animals administered 6 doses, the low-dose group had higher levels of CD3+cells compared with the control group (Table 30). These data suggest a potential treatment- related effect on T-cell infiltration. CD4+expression is low in all groups and CD8+cells showed moderate expression in all groups, with a slight increase in Composition 1 low dose group. However, no significant differences were noted among groups treated with 6 doses. Large numbers of CD11b+cells were detected in all groups. The higher necrosis levels are observed in -69- NAI-5004557346v1Attorney Docket No.14648-051-228 the Composition 1-treated groups. Group 2 (high dose) and group 3 (low dose) both treated groups show an increase in tumor necrosis. Table 29. Scoring of Necrosis and Cell Surface Markers in Left and Right MB49 Tumors for mice treated with 3 doses Group Mouse Flank Necrosis CD3 CD4 CD8 CD11b (%)-70- NAI-5004557346v1Attorney Docket No.14648-051-228 Table 30. Scoring of Necrosis and Cell Surface Markers in Left and Right MB49 Tumors for mice treated with 6 doses Group Mouse Flank Necrosis CD3 CD4 CD8 CD11b (%)

[0292] Composition 1 demonstrated significant antitumor activity in both flanks of female C57BL / 6 mice inoculated with MB49 cells, with greater effects observed in the high-dose groups. On Day 28 in the 3-dose regimen and Day 26 in the 6-dose regimen, treatment with high- and low-dose Composition 1 significantly reduced tumor volume compared to the vehicle group. Additionally, body weight analysis indicated overall weight gain across all groups, suggesting that Composition 1 treatment was well tolerated. In the peripheral blood, T cells and activated T cells were similar among groups, and porcine cells were not detected above background levels in the peripheral blood. There were no porcine cells detected in any of the tumors at the end of the study. These findings support the potential efficacy and safety of Composition 1 in treating MB49 tumors in this model. -71- NAI-5004557346v1Attorney Docket No.14648-051-228 6.34 Example 34: A single dose or repeated dose pilot safety of Composition 1 in C57BL / 6 mice via intramuscular or intravenous injection

[0293] The objective of this study was to observe preliminary reactions, bioactivity, biodistribution, and elimination of Composition 1 and to characterize the immune response after intramuscular (IM) or intravenous (IV) injection once or three times in C57BL / 6 mice. 6.34.1 Materials and Methods (i) Test article

[0294] The test article, Composition 1, was administered at a single dosage (5×106) on Day 1 or on Days 1, 4, and 7 depending on the study group. A description of the test article used in this study is provided in Table 31. The vehicle control consisted of the formulation buffer (CryoStor10 + PlasmaLyte A, 833 µL per 1000 µL) and HSA (167 µL per 1000 µL). Table 31. Test Article for Mouse Safety Study Lot Dosing Day Dose Cell Cell Number (cells / mouse) Concentration Viability

[0295] Atotal of 97 healthy, 9-week-old C57BL / 6 mice (SPF grade), 49 males and 48 females, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. were included in this study. Male animals weighed within 20.24-25.72 g, and female animals weighed within 19.32-22.03 g. (ii) Study design

[0296] The mice were randomized to 8 groups: 4 groups received the vehicle control (formulation buffer), and 4 groups received 5×106cells per mouse of XPCs (Table 32). Each set of 4 groups receiving the control or Composition 1 was further divided into a subgroup that was administered a single dose intramuscularly or intravenously or a subgroup that received 3 doses (Days 1, 4, and 7) either intramuscularly or intravenously. The test article was administered via IM (biceps femoris, left leg) or IV (tail vein) injection. The IM route was used to simulate an intratumoral injection, and the IV route was used to simulate an accidental intravenous injection.

[0297] Animals were euthanized by first anesthetizing with CO2 inhalation followed by cardiac puncture blood sampling to ensure animal death. For each of the single-dose groups, 1 -72- NAI-5004557346v1Attorney Docket No.14648-051-228 male and 1 female mouse were sacrificed on Days 2, 4, and 8, whereas in the groups that received 3 doses, 1 male and 1 female mouse were sacrificed on Day 8, 10, and 14. Table 32. Study Design of Mouse Safety Study Group Test Article RoA Number of Dosage Dosing Regimen Dose volume Individuals (cells / mouse) (μL) 6 males Single dose(i) Cage-side observations

[0298] During the acclimation and experimental periods, cage-side observations were conducted twice daily on all animals. An additional observation was performed on Day 14 in surviving animals. Observations included morbidity, mortality, injury, and availability of food and water. (ii) Detailed clinical observation

[0299] During the acclimation period, detailed clinical observations were conducted once on all animals on Day -1. During the experimental period, detailed clinical observations were conducted on all surviving animals once daily. Detailed clinical observations included evaluation of the dosing sites, skin, fur, eyes, ears, nose, oral cavity, thorax, abdomen, external genitalia, limbs and feet, respiratory and circulatory effects, autonomic effects such as salivation, nervous system effects including tremors, convulsions, reactivity to handling, and unusual behavior. -73- NAI-5004557346v1Attorney Docket No.14648-051-228 (iii) Body weight

[0300] All animals were weighed once on Day -1 during the acclimation period for grouping. (iv) Food consumption

[0301] Food consumption (24±1 h) of all surviving animals was measured once daily during the experimental period, unless animals were being fasted. The mean food consumption per animal equaled the sum of food consumption for all animals in a single cage of a group divided by the sum of the actual number of surviving animals. Male and female food consumption were calculated separately. (v) Clinical pathology

[0302] During the experimental period, hematology examinations were conducted on all surviving animals on the day of scheduled necropsy (before necropsy), and clinical chemistry examination was conducted only 3 animals on Day 14 due to limited blood samples. Animals were fasted overnight (≥10 h) but had free access to drinking water before blood sample collection. Blood samples were collected via cardiac puncture after euthanasia. Approximately 0.3 mL of whole blood was placed in an anticoagulation tube containing K2EDTA for hematology analysis, and approximately 0.3 mL of whole blood was placed in separation gel collection tubes (without anticoagulant) for clinical chemistry analysis. Clinical chemistry samples were centrifuged at 3500 rpm at room temperature for 10 minutes. (vi) Hematology and blood biochemistry

[0303] Hematology analysis included WBC, RBC, HGB, HCT, MCV, MCH, MCHC, PLT, NEUT, percentage of neutrophils (NEUT%), LYMPH, percentage of lymphocytes (LYMPH%), MONO, percentage of monocytes (MONO%), EO, percentage of eosinophils (EO%) BASO, percentage of basophils (BASO%), RET, and percentage of reticulocytes (RET%). Blood biochemistry analysis included ALT, AST, ALP, TP, ALB, UREA, and globulin (GLO). (vii) Necroscopy

[0304] Complete necropsy examinations included detailed examination of external abnormalities and internal abnormalities of the abdominal, thoracic, and cranial cavities. -74- NAI-5004557346v1Attorney Docket No.14648-051-228 6.34.3 Results (i) Toxicological evaluations

[0305] There were no unscheduled deaths, and all animals survived until the scheduled necropsy day. During the study, none of the surviving animals exhibited abnormalities in behavior, body weight, or food consumption. (ii) Clinical pathology

[0306] Fluctuations in leukocyte values were noted in individual animals administrated with Composition 1. However, these changes were not considered adverse effects because the changes were small and lacked temporal or dose-response relationships. Clinical chemistry examination was conducted in only 3 animals on Day 14 due to limited blood samples, but no test article-related clinical chemistry abnormalities were noted in these animals (iii) Pathological examination

[0307] No test article related gross pathological changes were observed during the study. No tissue lesions were observed during external or internal necroscopy. 6.34.4 Conclusions

[0308] Under the conditions of this study, C57BL / 6 mice were administered vehicle control or Composition 1 via IM or IV injection as a single dose or in 3 doses given every 3 days. The animals in each group survived to the scheduled necropsy. No abnormalities related to the test article were noted in detailed clinical observation, body weight, food consumption, hematology examination, clinical chemistry examination, and gross observations. 6.35 Example 35: A Pivotal Study of Composition 1 in Tumor-bearing C57BL / 6 Mice via Intratumoral Injection

[0309] This study assessed the antitumor activity and safety of intratumoral (IT) administration of Composition 1 in murine MB49 bladder cancer.

[0310] A syngeneic model of bladder cancer was established in C57BL / 6 mice using the murine bladder cancer cell line, MB49. Cancer cells (1×106cells / mouse) were injected subcutaneously under sterile conditions in the right flank of male and female mice to establish a primary tumor. An equivalent number of cells was injected subcutaneously under sterile conditions into the left flank of mice 3 days after the right flank tumor injection to establish a distant secondary tumor. -75- NAI-5004557346v1Attorney Docket No.14648-051-228

[0311] The study design is presented in Table 33. A total of 92 animals (46 males; 46 females) were randomized to 6 groups. Groups 1 and 2 had a total of 6 animals each and were injected with Composition 1 (5×106cells / animal / dose) in a single dose (Group 1) or 3 doses (Group 2). Animals in each group were sacrificed on Day 4 or Day 8, respectively. Groups 3-6 each had a total of 20 animals that were given 3 injections of Composition 1 either subcutaneously or intratumorally. For these groups, sacrifice occurred on Day 25. Table 33. Study Design for Bladder Cancer (MB49) Mouse Model Pivotal Study Group Test Article RoA Dosage Dose volume Dosing Regimen Number of Animals (cells / mouse) (μL / animal) Male Female i l i i

[0312] Parameters evaluated in this study included cage-side observations, detailed clinical observations, body weights, tumor volume, clinical pathology (hematology and clinical chemistry), lymphocyte phenotype analysis, cytokines analysis, gross pathology, organ weight and co-efficient, and histopathology. Survival, tumor volume, lymphocyte phenotype, and cytokine analysis are reported here.

[0313] During the study period, 2 animals in Group 4 (SC injection, Composition 1) were found dead on Day 8, while 1 animal in the same group had to be euthanized on the same day due to its deteriorated condition. -76- NAI-5004557346v1Attorney Docket No.14648-051-228

[0314] The tumor volume in treatment Group 6 was smaller than the tumor volume of in vehicle Group 5, which is considered to be a test article-related pharmacological effect. Tumor volumes in male and female mice for the right and left flank are shown in FIGs 18A and 18B.

[0315] From cytokine analysis data, no test article-related abnormal changes of IFN-γ, IL-2, IL-10, IL-4 and TNF-α were noted on tumor-bearing or non-tumor-bearing group animals during the study periods. On Day 7 (1 hour after Composition 1 administration), IL-6 increased in 3 animals in Group 4 compared with the vehicle tumor-bearing group. This increase was considered test article-related but not deemed adverse due to the lack of clinical pathology and histopathology findings. 6.36 Example 36: First-in-Human, Open-Label, Single-Arm, Phase 1 Dose- Escalation Study to Evaluate Safety, Tolerability, Feasibility, and Preliminary Efficacy of Intra-tumoral Injection of Composition 1 in Relapsed / Refractory Advanced / Metastatic Solid Tumors

[0316] This is a Phase 1, first-in-human (FIH), open-label dose-escalation study to evaluate the safety, tolerability, feasibility, and preliminary efficacy of intra-tumoral injection of Composition 1 in approximately 27 participants with refractory advanced or metastatic solid tumors, including sarcoma and carcinoma of the bladder or breast.

[0317] The primary objective of this study is to characterize the safety and tolerability of escalating doses of Composition 1 administered intratumorally in select advanced / metastatic solid tumors and to determine the optimal biologic dose (OBD) and the recommended Phase 2 dose (RP2D) for subsequent studies of Composition 1 administered intratumorally alone in select advanced / metastatic solid tumors. The secondary objective of this study is to evaluate the preliminary antitumor activity of Composition 1 in select advanced / metastatic solid tumors. The exploratory objective of this study is to evaluate immunogenicity (xenoimmunization) and exploratory measures of tumor response.

[0318] The primary endpoints of this study include adverse events (AEs) according to National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) v 5.0, including number of participants with treatment-emergent adverse events (TEAEs) and serious adverse events (SAEs), and dose limiting toxicities (DLTs) assessed up to 7 days after each treatment administration. The RP2D is determined from safety, tolerability, and preliminary antitumor activity, and any other relevant data that are obtained during dose- escalation. The secondary endpoints of this study include preliminary anti-tumor activity -77- NAI-5004557346v1Attorney Docket No.14648-051-228 assessed through the objective response rate (ORR), duration of response (DOR), time to response (TTR), progression-free survival (PFS), as assessed by the investigator per Response Evaluation Criteria in Solid Tumors (RECIST) v1.1, and overall survival (OS) at 3, 6, and 12 months. The exploratory endpoints include analyzing immune activation in participant samples using the in vitro mixed lymphocyte reaction (MLR) system developed for Composition 1 pharmacology studies, evaluation of anti-drug antibodies (ADA) to porcine antigens, and evaluation of anti-tumor activity using Response Criteria for Intratumoral Immunotherapy in Solid Tumors (itRECIST). 6.36.1 Study design

[0319] Two dose levels of Composition 1 (low and high) are evaluated in 2 separate cohorts of participants (FIG.19). In each cohort, delivered cell number and injection volume are administered based on tumor size. Participants in the low dose cohort (Cohort 1) receive 5 to 20 million cells in an injection volume of 0.1 to 0.4 mL, and participants in the high dose cohort (Cohort 2) receive 25 to 100 million cells in injection volumes of 0.5 to 2 mL, depending upon tumor size (see Table 34). Table 34. Dose Levels of Composition 1 by Cohort Low Dose (Cohort 1) High Dose (Cohort 2) T Si1Injection Injection Cell Number

[0320] Each participant receives a total of 3 intra-tumoral injections of study treatment. Pre- medications including acetaminophen, antihistamine, and low-dose glucocorticoid are administered 30 minutes to 2 hours prior to each injection. Participants must fast for at least 4 hours prior to each injection administration, following the direction of the investigative site’s anesthesia guidelines and approach to patient preparation, anesthetic administration, and post- operative care. Intra-tumoral injections are administered directly (palpable subcutaneous tumors) by a qualified investigator or under computed tomography (CT) or ultrasound guidance (based on size, location, and visibility) by an interventional radiologist. The first injection is administered on Day 0 and subsequent injections are administered approximately every 14 days (±2 days) thereafter. Participants are monitored (vital signs and AEs) for a minimum of 4 hours -78- NAI-5004557346v1Attorney Docket No.14648-051-228 after each injection. All participants are evaluated for dose-limiting toxicities (DLTs) for a period of 7 days after each injection. 6.36.2 Study population

[0321] Participants who meet all the following criteria are eligible for participation in the study: 1) Male and female participants at least 18 years of age who have provided written informed consent. 2) Histologically or cytologically confirmed diagnosis of advanced or metastatic solid tumors, including sarcoma or carcinoma of the bladder or breast. 3) Participants who have failed (progressed or intolerant to) one or more lines of standard of care therapy for their condition: a. Unresectable / metastatic soft tissue sarcoma that has progressed following prior therapy with doxorubicin ± ifosfamide. b. Metastatic bladder cancer that has progressed following treatment with enfortumab vedotin plus pembrolizumab. c. Metastatic triple negative breast cancer that has progressed on a taxane and / or anthracycline-based therapy and programmed cell death protein 1 (PD1) inhibitor (if programmed cell death ligand 1 [PD-L1] positive). 4) Radiologically measurable tumor(s), i.e., at least 1 cm in longest unidimensional diameter as measured by CT. The target lesion must be at least 1 cm3in volume and feasible for injection either directly or under CT or ultrasound guidance. Injected tumor must not be in immediate contact with vital structures in the injection path, such as major nerves or blood vessels. 5) Participants must have adequate organ function as indicated by the following laboratory values during screening or ≤ 14 days before the first dose of study treatment(s): a. Creatinine clearance ≥ 60 mL / min, calculated using the Cockcroft-Gault formula. b. Serum total bilirubin ≤ 1.5 × the upper limit of normal (ULN) (< 3 × ULN for participants with Gilbert’s syndrome). c. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) ≤ 3 × ULN (or < 5 × ULN if hepatic metastases are present), and gamma-glutamyl transferase (GGT) ≤ 3 x ULN. d. Participants must not have required blood transfusion or growth factor support ≤ 14 days before sample collection for the following: i. Leukocyte count > 2.5 x109 / L -79- NAI-5004557346v1Attorney Docket No.14648-051-228 ii. Platelet count > 75 x109 / L iii. Hemoglobin > 90 g / L iv. Prothrombin time ≤ 1.4 seconds v. Activated partial thromboplastin time (aPTT) outside normal limit 6) Female who has been post-menopausal for more than one (1) year or female of childbearing potential using a highly efficient method of contraception (i.e., a method with less than 1% failure rate) during the study participation. Male partners of female participants must agree to use condoms throughout the duration of study participation. Female of childbearing potential must have a negative pregnancy test at screening (blood) and prior to the first dose of Composition 1 (blood or urine). 7) All participants (male and female) must agree that male partners will use condoms during sexual intercourse.

[0322] Participants who meet any of these criteria are not eligible for participation in the study: 1) Eastern Cooperative Oncology Group (ECOG) performance status > 2. 2) Known major reaction / AE in connection with previous vaccination (e.g., anaphylaxis or other serious reaction). 3) Participants with New York Heart Association (NYHA) Class 3 or 4 heart failure or any uncontrolled cardiac condition (e.g., arrhythmia, angina, pulmonary hypertension). 4) Target lesion is in immediate contact with vital neurovascular structures or in a location that puts the participant at risk of airway compromise in the event of post-injection swelling or inflammation. 5) Participants receiving anticoagulants. 6) Known major active viral disease (e.g., hepatitis B virus [HBV], hepatitis C virus [HCV], human immunodeficiency virus [HIV]). 7) Ongoing, active infection that requires treatment with parenteral antibiotics or antiviral medication. 8) Pregnant or lactating women. 9) Life expectancy less than 3 months. 10) Investigational treatment (within 28 days) prior to the first injection of Composition 1. 11) Any reason that, in the opinion of the investigator, contraindicates patient participation in the study. -80- NAI-5004557346v1Attorney Docket No.14648-051-228 6.36.3 Dose limiting toxicity (DLT)

[0323] All toxicities or AEs will be graded according to the NCI CTCAE v5.0. The occurrence of any of the following toxicities is considered a DLT during dosing, if it occurs ≤ 7 days after a dose of study treatment, unless clearly attributed to the underlying disease or an extraneous event. 1) Any death (Grade 5 AE) not clearly due to underlying disease (i.e., disease progression) or extraneous causes. 2) Any Grade 4 drug-related toxicity. 3) Anaphylaxis reaction (Grade 3 or higher) within 24 hours of drug administration. 4) Three or more of the same Grade 3 hematological or non-hematological toxicity, with the following exceptions: a. Grade 3 nausea, vomiting, or diarrhea for < 72 hours with adequate antiemetic and other supportive care b. Grade 3 fatigue < 7 days c. Grade ≥ 3 electrolyte abnormality lasting up to 72 hours, not clinically complicated, and resolving spontaneously or responding to conventional medical interventions d. Grade ≥ 3 amylase or lipase elevation not associated with symptoms or clinical manifestations of pancreatitis 5) Grade 3 aspartate aminotransferase (AST) or alanine aminotransferase (ALT) elevation >5 × upper limit of normal (ULN), and Grade 3 gamma-glutamyl transferase (GGT) elevation >5 × ULN, or Grade 3 bilirubin elevation >3 × ULN that persists for greater than 2 weeks (regardless of the presence or absence of liver metastasis). 6) Any death possibly related to the study product and not directly attributed to disease progression or other unrelated cause. 7) Any Grade 4 injection-related reaction / hypersensitivity. 8) Two or more of the same Grade 4 AE. 9) Detection of any assessed porcine viruses that leads to a product-related medical event for which the SRC recommends pause of the clinical trial until further assessment is completed, including: a. Porcine circovirus (including PCV1, PCV2, PCV3) b. Porcine lymphotropic herpesvirus (PLHV1, PLHV2) -81- NAI-5004557346v1Attorney Docket No.14648-051-228 c. Porcine cytomegalovirus (PCMV). 6.36.4 Dose limiting toxicity (DLT)

[0324] The imaging evaluation (unenhanced and enhanced magnetic resonance imaging [MRI]or CT or other modality, as appropriate, the specific examination site determined by the investigator), tumor marker examination within 28 days before infusion are used as the baseline. Imaging evaluation is then performed at the scheduled visits until disease progression, lost to follow-up, withdrawal of informed consent, or death (whichever occurs first). RECIST v1.1 criteria is used to evaluate the preliminary anti-tumor efficacy, including ORR, DOR, and PFS. Tumor response is evaluated using itRECIST criteria as an exploratory measure. 7. EQUIVALENTS

[0325] Although the disclosure is described in detail with reference to specific embodiments thereof, it will be understood that variations that are functionally equivalent are within the scope of this disclosure. Indeed, various modifications of the disclosure in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims. 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 disclosure described herein. Such equivalents are intended to be encompassed by the following claims.

[0326] All publications, patents and patent applications mentioned in this specification are herein incorporated by reference into the specification to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference in their entireties. -82- NAI-5004557346v1

Claims

Attorney Docket No.14648-051-228 WHAT IS CLAIMED IS:

1. A method for treating a tumor in a subject in need thereof, the method comprising: administering a composition comprising antigen presenting cells to the subject, wherein the antigen presenting cells are obtained from a species that is different than the subject, and wherein the species is a swine that expresses alpha-1,3 galactosyltransferase.

2. The method of claim 1, wherein the tumor is a solid tumor, and the administering is into the tumor of the subject via an intratumoral injection.

3. The method of any one of the preceding claims, wherein the swine that expresses alpha-1,3 galactosyltransferase is a miniature swine.

4. The method of any one of the preceding claims, wherein the subject is a human.

5. The method of any one of the preceding claims, wherein the swine is a swine leukocyte antigen (SLA)‐inbred swine.

6. The method of any one of the preceding claims, wherein the method triggers an immune response specific to the tumor.

7. The method of any one of the preceding claims, wherein the method yields an abscopal effect.

8. The method of any one of the preceding claims, wherein the tumor is a solid cancerous tumor.

9. The method of claim 8, wherein the tumor is selected from the group consisting of sarcomas, carcinomas, lymphomas, breast tumors, prostate tumors, head and neck tumors, glioblastomas, bladder tumors, pancreatic tumors, liver tumors, colon tumors, ovarian tumors, colorectal tumors, pulmonary tumors, cutaneous tumors, lymphoid tumors, gastrointestinal tumors, gastrointestinal stromal tumors, cervical tumors, hepatocellular carcinomas, renal cell -83- NAI-5004557346v1Attorney Docket No.14648-051-228 carcinomas, melanomas, colorectal carcinomas, esophageal carcinomas, brain tumors, kidney tumors, lung tumors (including non-small cell lung cancer), gastric tumors, bile-duct tumors, uterine tumors, and childhood (pediatric) tumors.

10. The method of claim 8, wherein the tumor is selected from the group consisting of brain tumor, breast tumor, liver tumor, lung tumor, melanoma, pancreatic tumor, prostate tumor, sarcoma, kidney tumor, bladder tumor, and colon tumor.

11. The method of claim 8, wherein the tumor is selected from the group consisting of brain tumor, breast tumor, liver tumor, lung tumor, melanoma, and pancreatic tumor 12. The method of claim 8, wherein the tumor is selected from the group consisting of prostate tumor, sarcoma, kidney tumor, bladder tumor, and colon tumor.

13. The method of any one of the preceding claims, wherein the tumor is resistant to treatment to chemotherapy and / or treatment with an immunotherapy.

14. The method of any one of the preceding claims, wherein the antigen presenting cells are derived from one or more swine or miniature swine using a leukapheresis procedure, wherein the leukapheresis procedure generates a leukopak containing peripheral blood mononuclear cells, and the leukopak is further fractionated by counterflow elutriation.

15. The method of any one of the preceding claims, wherein the composition comprising antigen presenting cells is substantially free of pathogens.

16. The method of any one of the preceding claims, wherein the antigen presenting cells are obtained from swine or miniature swine of different genotypes.

17. The method of any one of the preceding claims, wherein the composition comprising antigen presenting cells is administered in single or multiple doses. -84- NAI-5004557346v1Attorney Docket No.14648-051-228 18. The method of any one of the preceding claims, wherein the composition comprising antigen presenting cells is administered via an intratumoral injection of at least about 1 x 106antigen presenting cells per dose.

19. The method of any one of the preceding claims, wherein the composition comprising antigen presenting cells is administered via an intratumoral injection of about 1 x 106, about 5 x 106, about 10 x 106, about 15 x 106, about 20 x 106, about 25 x 106, about 30 x 106, about 35 x 106, about 40 x 106, about 45 x 106, about 50 x 106, about 55 x 106, about 60 x 106, about 65 x 106, about 70 x 106, about 75 x 106, about 80 x 106, about 85 x 106, about 90 x 106, about 95 x 106, about 10 x 107, about 15 x 107, about 20 x 107, about 25 x 107, about 30 x 107, about 35 x 107, about 40 x 107, about 45 x 107, or about 50 x 107antigen presenting cells per dose.

20. The method of any one of the preceding claims, wherein the antigen presenting cells are substantially mature antigen presenting cells.

21. The method of any one of the preceding claims, wherein the antigen presenting cells are not activated or stimulated.

22. The method of any one of the preceding claims, wherein the composition comprises peripheral blood mononuclear cells (PBMC).

23. The method of any one of the preceding claims, wherein the composition comprises monocytes.

24. The method of any one of the preceding claims, wherein the composition comprises dendritic cells, macrophages, granulocytes, T-cells, B-cells, and / or NK cells.

25. The method of any one of the preceding claims, wherein the composition comprises at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or greater than about 95% PBMCs, wherein the PBMCs are mature, immature, or a combination of mature and immature PBMCs. -85- NAI-5004557346v1Attorney Docket No.14648-051-228 26. The method of any one of the preceding claims, wherein the composition comprises at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or greater than about 95% monocytes, wherein the monocytes are mature, immature, or a combination of mature and immature monocytes.

27. The method of claim 26, wherein the composition comprises a mixture of at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or greater than about 95% monocytes and dendritic cells, wherein the monocytes and / or dendritic cells in said mixture may be mature, immature, or a combination of mature and immature monocytes and / or dendritic cells.

28. The method of claim 27, wherein the mixture comprises from 10% to 95%, 10% to 90%, 10% to 80%, 10% to 70%, 10% to 60%, 10% to 50%, 10% to 40%, 10% to 30%, 10% to 20%, 20% to 95%, 20% to 90%, 20% to 80%, 20% to 70%, 20% to 60%, 20% to 50%, 20% to 40%, 20% to 30%, 30% to 95%, 30% to 90%, 30% to 80%, 30% to 70%, 30% to 60%, 30% to 50%, 30% to 40%, 40% to 95%, 40% to 90%, 40% to 80%, 40% to 70%, 40% to 60%, 40% to 50%, 50% to 95%, 50% to 90%, 50% to 80%, 50% to 70%, 50% to 60%, 60% to 95%, 60% to 90%, 60% to 80%, 60% to 70%, 70% to 95%, 70% to 90%, 70% to 80%, 80% to 95%, 80% to 90%, 90% to 95%, or greater than 95% monocytes.

29. The method of claim 27, wherein the mixture comprises from 10% to 30%, 35% to 55%, 60% to 80%, or 85% to 95% monocytes.

30. The method of claim 27, wherein the mixture comprises at least 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 75%, about 80%, about 85%, about 90%, about 95%, or greater than about 95% monocytes.

31. The method of claim 27, wherein the mixture comprises from 10% to 95%, 10% to 90%, 10% to 80%, 10% to 70%, 10% to 60%, 10% to 50%, 10% to 40%, 10% to 30%, 10% to 20%, 20% to 95%, 20% to 90%, 20% to 80%, 20% to 70%, 20% to 60%, 20% to 50%, 20% to 40%, 20% to 30%, 30% to 95%, 30% to 90%, 30% to 80%, 30% to 70%, 30% to 60%, 30% to -86- NAI-5004557346v1Attorney Docket No.14648-051-228 50%, 30% to 40%, 40% to 95%, 40% to 90%, 40% to 80%, 40% to 70%, 40% to 60%, 40% to 50%, 50% to 95%, 50% to 90%, 50% to 80%, 50% to 70%, 50% to 60%, 60% to 95%, 60% to 90%, 60% to 80%, 60% to 70%, 70% to 95%, 70% to 90%, 70% to 80%, 80% to 95%, 80% to 90%, 90% to 95%, or greater than 95% dendritic cells.

32. The method of claim 27, wherein the mixture comprises from 10% to 30%, 35% to 55%, 60% to 80%, or 85% to 95% dendritic cells.

33. The method of claim 27, wherein the mixture comprises at least 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 75%, about 80%, about 85%, about 90%, about 95%, or greater than about 95% dendritic cells.

34. The method of any one of the preceding claims, wherein the subject is receiving another anti-cancer therapy.

35. The method of claim 34, wherein the other anti-cancer therapy comprises treatment with one or more immune checkpoint inhibitors.

36. The method of claim 34, wherein the other anti-cancer therapy is an anti-CTLA4 therapy, anti-PD1 therapy, anti-PDL1 therapy, anti-LAG-3 therapy, tumor-treating fields (TTFs), cell-based therapy, a tyrosine kinase inhibitor, a VEGF inhibitor, or any combination thereof.

37. The method of claim 34, wherein the other anti-cancer therapy comprises treatment with imatinib, sunitinib, regorafenib, pazopanib, nilotinib, avapritinib, ripretinib, sorafenib, pimitespib, ipilimumab, tremelimumab, nivolumab, pembrolizumab, cemiplimab, atelizumab, avelumab, durvalumab, relatlimab, or any combination thereof.

38. The method of any one of claims 34 to 37, wherein the subject does not respond to the other anti-cancer therapy in the absence of administration of the composition comprising antigen presenting cells. -87- NAI-5004557346v1Attorney Docket No.14648-051-228 39. The method of any one of the preceding claims, wherein the composition comprising antigen presenting cells is administered at a dose level based on tumor size, as shown in table below, wherein the tumor size is the maximum longitudinal axis of the tumor. Low Dose High Dose Tumor Size InjectionCell NumberInjection Cell Number40. The method of claim 39, wherein the cell number is the total number of cells of the composition, and wherein the composition comprises at least 40% antigen presenting cells.

41. The method of claim 40, wherein the antigen presenting cells are alive cells.

42. The method of claim 40, wherein the cell number is determined by using the Chemometec NucleoCounter®NC-202™ cell counter.

43. The method of any one of the preceding claims, wherein the tumor is a metastatic tumor.

44. A pharmaceutical composition suitable for intratumoral injection, wherein the pharmaceutical composition comprises antigen presenting cells obtained from one or more swine that express alpha-1,3 galactosyltransferase. -88- NAI-5004557346v1

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