Heterogeneous antigen-presenting cells and their use

Abstract

This disclosure relates to a method for treating a tumor in a subject, comprising administering (for example, via intratumoral injection) a composition containing antigen-presenting cells obtained from a different species than the subject, i.e., heterogeneous antigen-presenting cells. The source of such heterogeneous antigen-presenting cells may be one or more pigs. This disclosure also relates to a method for treating a tumor by administering heterogeneous antigen-presenting cells in combination with other anticancer therapies. Furthermore, this disclosure relates to a pharmaceutical composition containing heterogeneous antigen-presenting cells.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit of U.S. Provisional Application No. 63,458,843, filed on April 12, 2023, which is incorporated herein by reference in its entirety.

[0002] 1. Field of this disclosure This disclosure is in the art of compositions comprising heterologous antigen-presenting cells (e.g., derived from one or more pigs), methods for producing such compositions, and the use of such compositions for treating tumors. [Background technology]

[0003] 2. Background of this Disclosure Conventional cancer therapies such as chemotherapy, surgery, and radiation are often insufficient to treat cancer patients and usually come with severe side effects. In this respect, cancer immunotherapy offers several advantages over conventional cancer therapies. Many different immunotherapy approaches are being investigated. One such immunotherapy strategy involves intratumoral administration of allogeneic inflammatory dendritic cells, usually in combination with other anticancer therapies, for the treatment of specific cancers (see, for example, Jin et al. (2022), Oncoimmunology, 11(1):e2099642; Frobom et al. (2020), Cancer Immunol.Immunother., 69(11):2393-2401; Karlsson-Parra et al. (2018), Pharm.Res., 35:156). For example, ilixadenes, a product based on allogeneic dendritic cells derived from human monocytes stimulated by potent activators, is being investigated in human clinical trials (see, for example, Frobom et al. (2020), Cancer Immunol. Immunother., 69(11):2393-2401). There remains a need for cancer immunotherapies that demonstrate a robust immune response.

[0004] The references made herein shall not be construed as an admission that they constitute prior art to this disclosure. [Overview of the project]

[0005] 3. Summary of this Disclosure In one embodiment, the Specified herein provides a method for treating a tumor in a subject requiring treatment, comprising administering a composition comprising antigen-presenting cells to the subject, wherein the antigen-presenting cells are obtained from a species different from that of the subject.

[0006] In one embodiment, the tumor is a solid tumor, and administration involves introducing it into the target tumor via intratumor injection.

[0007] In one embodiment, antigen-presenting cells are obtained from a certain breed of pig. In one embodiment, antigen-presenting cells are obtained from a certain breed of miniature pig. In one embodiment, the subject is human. In one embodiment, the pig is an alpha-1,3-galactosyltransferase-deficient pig. In one embodiment, the alpha-1,3-galactosyltransferase-deficient pig is a porcine leukocyte antigen (SLA) syngeneic pig. In one embodiment, the miniature pig is an alpha-1,3-galactosyltransferase-deficient miniature pig. In one embodiment, the alpha-1,3-galactosyltransferase-deficient miniature pig is a porcine leukocyte antigen (SLA) syngeneic pig.

[0008] In one embodiment, the method induces a tumor-specific immune response. In another embodiment, the method produces an abscopal effect.

[0009] In one embodiment, the tumor is a solid cancer-like tumor. In one embodiment, the tumor is selected from the group consisting of sarcoma, carcinoma, lymphoma, breast tumor, prostate tumor, head and neck tumor, glioblastoma, bladder tumor, pancreatic tumor, liver tumor, ovarian tumor, colorectal tumor, lung tumor, skin tumor, lymphatic system tumor, gastrointestinal tumor, gastrointestinal stromal tumor, cervical tumor, hepatocellular carcinoma, renal cell carcinoma, melanoma, colorectal cancer, esophageal cancer, brain tumor, kidney tumor, lung tumor (including non-small cell lung cancer), gastric tumor, bile duct tumor, uterine tumor, and pediatric (child) tumor. In one embodiment, the tumor is resistant to treatment by chemotherapy and / or treatment by immunotherapy.

[0010] In one embodiment, the antigen-presenting cells are derived from one or more pigs or minipigs using a leukapheresis procedure, and the leukapheresis procedure generates a leukopak containing peripheral blood mononuclear cells, and the leukopak is further fractionated by countercurrent centrifugal fractionation.

[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 pigs or minipigs of different genotypes.

[0013] In one embodiment, the composition comprising antigen-presenting cells is administered in a single or multiple doses. In one embodiment, the composition comprising antigen-presenting cells is administered via intratumoral injection of at least about 1×10 6 antigen-presenting cells per dose. In one embodiment, the composition comprising antigen-presenting cells is about 1×10 6 , about 5×10 6 , about 10×10 6 , about 15×10 6 , about 20×10 6 , about 25×10 6 , about 30×10 6 , about 35×10 6 , about 40×10 6 , about 45×10 6 , about 50×10 6 , about 55×10 6 , about 60×10 6, about 65×10 6 , about 70×10 6 , about 75×10 6 , about 80×10 6 , about 85×10 6 , about 90×10 6 , about 95×10 6 , about 10×10 7 , about 15×10 7 , about 20×10 7 , about 25×10 7 , about 30×10 7 , about 35×10 7 , about 40×10 7 , about 45×10 7 , or approximately 50 x 10 7 It is administered via intratumoral injection of antigen-presenting cells.

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

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

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

[0017] In one embodiment, the composition includes a monocyte.

[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 more than about 95% of PBMC, where the PBMC is mature PBMC, immature PBMC, or a combination of mature PBMC and immature PBMC.

[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 more than about 95% of monocytes, wherein the monocytes are mature monocytes, immature monocytes, or a combination of mature and immature monocytes.

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

[0022] In one embodiment, the mixture containing monocytes and dendritic cells is comprised of 10%-95%, 10%-90%, 10%-80%, 10%-70%, 10%-60%, 10%-50%, 10%-40%, 10%-30%, 10%-20%, 20%-95%, 20%-90%, 20%-80%, 20%-70%, 20%-60%, 20%-50%, 20%-40%, 20%-30%, 30%-95%, 30%-90%, 30%-80%, 30%-70%, and 30%-60%. , 30%~50%, 30%~40%, 40%~95%, 40%~90%, 40%~80%, 40%~70%, 40%~60%, 40%~50%, 50%~95%, 50%~90%, 50%~80%, 50%~70%, 50%~60%, 60%~95%, 60%~90%, 60%~80%, 60%~70%, 70%~95%, 70%~90%, 70%~80%, 80%~95%, 80%~90%, 90%~95%, or more than 95% monocytes. In one embodiment, a mixture containing monocytes and dendritic cells contains 10%~30%, 35%~55%, 60%~80%, or 85%~95% monocytes. In one embodiment, the mixture contains approximately 10%, approximately 15%, approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, approximately 95%, or more than approximately 95% monocytes.

[0023] In one embodiment, the mixture containing monocytes and dendritic cells is comprised of 10%-95%, 10%-90%, 10%-80%, 10%-70%, 10%-60%, 10%-50%, 10%-40%, 10%-30%, 10%-20%, 20%-95%, 20%-90%, 20%-80%, 20%-70%, 20%-60%, 20%-50%, 20%-40%, 20%-30%, 30%-95%, 30%-90%, 30%-80%, 30%-70%, 30%-60%, The mixture contains dendritic cells in the following proportions: 30%-50%, 30%-40%, 40%-95%, 40%-90%, 40%-80%, 40%-70%, 40%-60%, 40%-50%, 50%-95%, 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%-95%, 60%-90%, 60%-80%, 60%-70%, 70%-95%, 70%-90%, 70%-80%, 80%-95%, 80%-90%, 90%-95%, or more than 95%. In one embodiment, the mixture containing monocytes and dendritic cells contains 10%-30%, 35%-55%, 60%-80%, or 85%-95% dendritic cells. In one embodiment, the mixture containing monocytes and dendritic cells contains 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 more than about 95% dendritic cells.

[0024] In one embodiment, the subject is receiving another anticancer therapy. In one embodiment, the other anticancer therapy includes treatment with one or more immune checkpoint inhibitors. In one embodiment, the other anticancer therapy is anti-CTLA4 therapy, anti-PD1 therapy, anti-PDL1 therapy, anti-LAG-3 therapy, tumor therapeutic field therapy (TTF), cell-based therapy, tyrosine kinase inhibitors, VEGF inhibitors, or any combination thereof. In one embodiment, the other anticancer therapy includes treatment with imatinib, sunitinib, regorafenib, pazopanib, nilotinib, avapritinib, lipretinib, sorafenib, pimitespib, ipilimumab, tremelimumab, nivolumab, pembrolizumab, semiprimab, atelizumab, avelumab, durvalumab, leratrimab, or any combination thereof.

[0025] In one embodiment, the subject does not respond to other anticancer therapies unless administered a composition containing antigen-presenting cells.

[0026] In another embodiment, provided herein are pharmaceutical compositions suitable for intratumor injection, wherein the pharmaceutical composition comprises antigen-presenting cells obtained from one or more pigs. [Brief explanation of the drawing]

[0027] 4. Brief explanation of the drawing [Figure 1] This describes a general leukocyte apheresis procedure for generating a Leukopack containing antigen-presenting cells derived from porcine blood.

[0028] [Figure 2] This shows a comparison of tumor size between the treatment group (administration of a composition containing human PBMCs) and the control group in a mouse melanoma model on day 12.

[0029] [Figure 3] This shows a comparison of tumor size between the treatment group (administration of a composition containing human PBMCs) and the control group in a mouse bladder cancer model on day 7.

[0030] [Figure 4A] (a) Pig PBMCs seeded with human PBMCs at various human cell-to-porcine cell ratios (h:p), (b) human PBMCs alone, and (c) human CD4 T cell counts on day 7 in in vitro studies evaluating allogeneic human PBMCs. [Figure 4B] (a) Porcine PBMCs seeded with human PBMCs at various human cell-to-porcine cell ratios (h:p), (b) human PBMCs alone, and (c) human CD8 T cell counts on day 7 in in vitro studies evaluating allogeneic human PBMCs. [Figure 4C](a) Pig PBMCs seeded with human PBMCs at various human cell-to-porcine cell ratios (h:p), (b) human PBMCs alone, and (c) human NK cell counts on day 7 in in vitro studies evaluating allogeneic human PBMCs.

[0031] [Figure 5A] (a) Porcine monocytes seeded with human PBMCs at various human cell-to-porcine cell ratios (h:p), (b) human PBMCs alone, and (c) human CD4 T cell counts on day 7 in in vitro studies evaluating allogeneic human PBMCs. [Figure 5B] (a) Porcine monocytes seeded with human PBMCs at various human cell-to-porcine cell ratios (h:p), (b) human PBMCs alone, and (c) human CD8 T cell counts on day 7 in in vitro studies evaluating allogeneic human PBMCs. [Figure 5C] (a) Porcine monocytes seeded with human PBMCs at various human cell-to-porcine cell ratios (h:p), (b) human PBMCs alone, and (c) the number of proliferated human CD4 T cells on day 7 in in vitro studies evaluating allogeneic human PBMCs. [Figure 5D] (a) Porcine monocytes seeded with human PBMCs at various human cell-to-porcine cell ratios (h:p), (b) human PBMCs alone, and (c) the number of proliferated human CD8 T cells on day 7 in in vitro studies evaluating allogeneic human PBMCs. [Figure 5E] (a) Porcine monocytes seeded with human PBMCs at various human cell-to-porcine cell ratios (h:p), (b) human PBMCs alone, and (c) HLA-DR on human CD4 T cell count at day 7 in in vitro studies evaluating allogeneic human PBMCs, as expressed in terms of mean fluorescence intensity (MFI). [Figure 5F] (a) Porcine monocytes seeded with human PBMCs at various human cell-to-porcine cell ratios (h:p), (b) human PBMCs alone, and (c) HLA-DR on human CD8 T cells at day 7 in in vitro studies evaluating allogeneic human PBMCs, as measured by mean fluorescence intensity (MFI). [Figure 5G](a) Porcine monocytes seeded with human PBMCs at various human cell-to-porcine cell ratios (h:p), (b) human PBMCs alone, and (c) human NK cell counts on day 7 in in vitro studies evaluating allogeneic human PBMCs. [Modes for carrying out the invention]

[0032] 5. Detailed explanation of this disclosure Provided herein are methods for treating tumors in a subject by administering compositions comprising antigen-presenting cells obtained from a species different from the subject, i.e., heterologous antigen-presenting cells. Sources of such heterologous antigen-presenting cells, which may be one or more pigs, are described in Section 5.1. Also described in Section 5.1 are porcine characteristics and compositions comprising antigen-presenting cells, including antigen-presenting cells derived from pigs. Methods for isolating antigen-presenting cells (e.g., from one or more pigs) are described in Section 5.2. Optionally, methods for treating tumors (e.g., cancerous solid tumors) comprising administering the antigen-presenting cells described herein (e.g., via intratumoral injection) in combination with other anticancer therapies are described in Section 5.3. Furthermore, pharmaceutical compositions comprising antigen-presenting cells disclosed herein are described in Section 5.4.

[0033] 5.1 Heterogeneous compositions Provided herein are compositions for treating tumors in human subjects, wherein the compositions include antigen-presenting cells derived from a species different from that of the subject. In certain embodiments, provided herein are compositions comprising antigen-presenting cells that are heterologous to the human subject being treated. Such compositions are referred to herein as heterologous compositions. The heterologous compositions of the present invention may be administered alone or formulated into pharmaceutical compositions such as those described in Section 5.4 (for example, by intratumor injection).

[0034] Antigen-presenting cells in the heterologous composition may be any cells that present an antigen bound to their surface by a major histocompatibility complex (MHC) protein. In one embodiment, the MHC protein is an MHC class II molecule. Antigen-presenting cells in the heterologous 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.

[0035] The heterogeneous compositions of the present invention may include, but are not limited to, antigen-presenting cells derived from any non-human species, including non-human primates, livestock, and rodents. Non-human species may be genetically engineered. Examples of livestock include pigs, rabbits, dogs, cats, horses, sheep, goats, and cattle. In one embodiment, the non-human species is a pig, or swine, or pig. As provided herein, the terms pig, swine, or pig may be used interchangeably.

[0036] Antigen-presenting cells in the heterologous composition of the present invention may be obtained by the method described in Section 5.2. In certain embodiments, the heterologous composition comprises antigen-presenting cells derived from one or more pigs. The one or more pigs used to produce the antigen-presenting cells may be, for example, genetically modified pigs to enhance the safety and / or efficacy of the antigen-presenting cells. In some embodiments, the pigs are alpha-1,3-galactosyltransferase-deficient pigs. In certain embodiments, the antigen-presenting cells are derived from one or more recombinant pigs. In certain embodiments, the antigen-presenting cells are derived from recombinant pigs of different genotypes. In certain embodiments, the antigen-presenting cells are derived from one or more miniature pigs. The one or more miniature pigs used to produce the antigen-presenting cells may be, for example, genetically modified miniature pigs to enhance the safety and / or efficacy of the antigen-presenting cells. In some embodiments, the miniature pigs are alpha-1,3-galactosyltransferase-deficient miniature pigs (see, for example, U.S. Patents 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 pigs. In certain embodiments, the antigen-presenting cells are derived from recombinant miniature pigs of different genotypes. In some embodiments, the alpha-1,3-galactosyltransferase-deficient pigs or miniature pigs are synbred pigs of porcine leukocyte antigen (SLA). In certain embodiments, one or more pigs or miniature pigs described herein may contain knockout genes or transgenes in the genes of the porcine antigen-presenting cells.

[0037] Donors of antigen-presenting cells used in the heterologous compositions described herein may be screened for infectious diseases using methods commonly known in the art. In certain embodiments, donors of antigen-presenting cells used in the heterologous compositions described herein are negative for syphilis, HIV, hepatitis A, hepatitis B, and / or hepatitis C. In certain embodiments, the heterologous composition comprising antigen-presenting cells is substantially pathogen-free.

[0038] In certain embodiments, the heterologous composition includes antigen-presenting cells derived from peripheral blood. In certain embodiments, the heterologous composition includes antigen-presenting cells derived from porcine peripheral blood. In certain embodiments, the heterologous composition includes antigen-presenting cells derived from bone marrow. In certain embodiments, the heterologous composition includes antigen-presenting cells derived from porcine bone marrow.

[0039] In certain embodiments, the heterologous composition includes peripheral blood mononuclear cells (PBMCs). In certain embodiments, the heterologous composition includes monocytes. In certain embodiments, the heterologous composition includes porcine PBMCs. In certain embodiments, the heterologous composition includes porcine monocytes. In certain embodiments, the heterologous composition includes monocytes derived from porcine peripheral blood. In certain embodiments, the heterologous composition includes monocytes derived from porcine bone marrow.

[0040] In certain embodiments, the heterogeneous composition contains at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more than about 95% of PBMCs. The PBMCs may be mature PBMCs, immature PBMCs, or a combination of mature and immature PBMCs.

[0041] In certain embodiments, the heterogeneous composition includes 50%-95%, 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%-95%, 60%-90%, 60%-80%, 60%-70%, 70%-95%, 70%-90%, 70%-80%, 80%-95%, 80%-90%, 90%-95%, or more than 95% PBMC.

[0042] In certain embodiments, the PBMCs present in the dissimilar compositions are at least 10%~95%, 10%~90%, 10%~80%, 10%~70%, 10%~60%, 10%~50%, 10%~40%, 10%~30%, 10%~20%, 20%~95%, 20%~90%, 20%~80%, 20%~70%, 20%~60%, 20%~50%, 20%~40%, 20%~30%, 30%~95%, 30%~90%, 30%~80%, 30%~70%, and 30%~6 Includes mature PBMCs with 0%, 30%-50%, 30%-40%, 40%-95%, 40%-90%, 40%-80%, 40%-70%, 40%-60%, 40%-50%, 50%-95%, 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%-95%, 60%-90%, 60%-80%, 60%-70%, 70%-95%, 70%-90%, 70%-80%, 80%-95%, 80%-90%, 90%-95%, or over 95%.

[0043] In certain embodiments, the PBMCs present in the heterogeneous composition are at least 10% to 30%, 35% to 55%, 60% to 80%, or 85% to 95% mature PBMCs.

[0044] In certain embodiments, the PBMCs present in the heterogeneous 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%, about 95%, or more than about 95% mature PBMCs. In certain embodiments, the PBMCs present in the heterogeneous composition are more than about 95% mature PBMCs. In certain embodiments, the PBMCs present in the heterogeneous composition are about 100% mature PBMCs.

[0045] In certain embodiments, the heterogeneous composition contains at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more than about 95% monocytes. The monocytes may be mature monocytes, immature monocytes, or a combination of mature and immature monocytes.

[0046] In certain embodiments, the heterogeneous composition includes monocytes in the following proportions: 50%-95%, 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%-95%, 60%-90%, 60%-80%, 60%-70%, 70%-95%, 70%-90%, 70%-80%, 80%-95%, 80%-90%, 90%-95%, or more than 95%.

[0047] In certain embodiments, the monocytes present in the heterogeneous composition are at least 10%-95%, 10%-90%, 10%-80%, 10%-70%, 10%-60%, 10%-50%, 10%-40%, 10%-30%, 10%-20%, 20%-95%, 20%-90%, 20%-80%, 20%-70%, 20%-60%, 20%-50%, 20%-40%, 20%-30%, 30%-95%, 30%-90%, 30%-80%, 30%-70%, and 30%-60%. These are mature monocytes with a percentage of 0%, 30%-50%, 30%-40%, 40%-95%, 40%-90%, 40%-80%, 40%-70%, 40%-60%, 40%-50%, 50%-95%, 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%-95%, 60%-90%, 60%-80%, 60%-70%, 70%-95%, 70%-90%, 70%-80%, 80%-95%, 80%-90%, 90%-95%, or over 95%.

[0048] In certain embodiments, the monocytes present in the heterogeneous composition are at least 10% to 30%, 35% to 55%, 60% to 80%, or 85% to 95% mature monocytes.

[0049] In certain embodiments, the monocytes present in the heterologous 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%, about 95%, or more than about 95% mature monocytes. In certain embodiments, the monocytes present in the heterologous composition are more than about 95% mature monocytes. In certain embodiments, the monocytes present in the heterologous composition are about 100% mature monocytes.

[0050] In certain embodiments, the heterologous composition comprises a mixture of monocytes and dendritic cells. In certain embodiments, the heterologous 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 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.

[0051] In certain embodiments, the heterogeneous composition comprises a mixture of monocytes and dendritic cells comprising at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more than about 95%. The monocytes and / or dendritic cells in such a mixture may be mature, immature, or a combination of mature monocytes and / or dendritic cells and immature monocytes and / or dendritic cells.

[0052] In certain embodiments, the heterogeneous composition includes a mixture of monocytes and dendritic cells in the following proportions: 50%-95%, 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%-95%, 60%-90%, 60%-80%, 60%-70%, 70%-95%, 70%-90%, 70%-80%, 80%-95%, 80%-90%, 90%-95%, or more than 95%.

[0053] In one embodiment, the mixture of monocytes and dendritic cells is composed of 10%-95%, 10%-90%, 10%-80%, 10%-70%, 10%-60%, 10%-50%, 10%-40%, 10%-30%, 10%-20%, 20%-95%, 20%-90%, 20%-80%, 20%-70%, 20%-60%, 20%-50%, 20%-40%, 20%-30%, 30%-95%, 30%-90%, 30%-80%, 30%-70%, 30%-60%, and 30% It may include monocytes with a percentage of 50%, 30%–40%, 40%–95%, 40%–90%, 40%–80%, 40%–70%, 40%–60%, 40%–50%, 50%–95%, 50%–90%, 50%–80%, 50%–70%, 50%–60%, 60%–95%, 60%–90%, 60%–80%, 60%–70%, 70%–95%, 70%–90%, 70%–80%, 80%–95%, 80%–90%, 90%–95%, or more than 95%.

[0054] In certain embodiments, the mixture of monocytes and dendritic cells contains 10% to 30%, 35% to 55%, 60% to 80%, or 85% to 95% monocytes.

[0055] In certain embodiments, the mixture of monocytes and dendritic cells contains 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 more than about 95% monocytes.

[0056] In a particular embodiment, the monocytes present in the mixture of monocytes and dendritic cells make up at least 10%-95%, 10%-90%, 10%-80%, 10%-70%, 10%-60%, 10%-50%, 10%-40%, 10%-30%, 10%-20%, 20%-95%, 20%-90%, 20%-80%, 20%-70%, 20%-60%, 20%-50%, 20%-40%, 20%-30%, 30%-95%, 30%-90%, 30%-80%, 30%-70%, and 30% These are mature monocytes with a percentage of 60%, 30%~50%, 30%~40%, 40%~95%, 40%~90%, 40%~80%, 40%~70%, 40%~60%, 40%~50%, 50%~95%, 50%~90%, 50%~80%, 50%~70%, 50%~60%, 60%~95%, 60%~90%, 60%~80%, 60%~70%, 70%~95%, 70%~90%, 70%~80%, 80%~95%, 80%~90%, 90%~95%, or more than 95%.

[0057] In certain embodiments, the monocytes present in the mixture of monocytes and dendritic cells include at least 10%-30%, 35%-55%, 60%-80%, or 85%-95% mature monocytes.

[0058] In certain embodiments, the monocytes present in a 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%, about 95%, or more than about 95% mature monocytes. In certain embodiments, the monocytes present in a mixture of monocytes and dendritic cells are more than 95% monocytes. In certain embodiments, the monocytes present in a mixture of monocytes and dendritic cells are about 100% mature monocytes.

[0059] The mixture of monocytes and dendritic cells consists of 10%-95%, 10%-90%, 10%-80%, 10%-70%, 10%-60%, 10%-50%, 10%-40%, 10%-30%, 10%-20%, 20%-95%, 20%-90%, 20%-80%, 20%-70%, 20%-60%, 20%-50%, 20%-40%, 20%-30%, 30%-95%, 30%-90%, 30%-80%, 30%-70%, 30%-60%, and 30%-50%. It may also contain dendritic cells making up 30%-40%, 40%-95%, 40%-90%, 40%-80%, 40%-70%, 40%-60%, 40%-50%, 50%-95%, 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%-95%, 60%-90%, 60%-80%, 60%-70%, 70%-95%, 70%-90%, 70%-80%, 80%-95%, 80%-90%, 90%-95%, or more than 95%.

[0060] In certain embodiments, the mixture of monocytes and dendritic cells contains 10% to 30%, 35% to 55%, 60% to 80%, or 85% to 95% dendritic cells.

[0061] In certain embodiments, the mixture of monocytes and dendritic cells contains 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 more than about 95% dendritic cells. In certain embodiments, the mixture of monocytes and dendritic cells contains more than about 95% dendritic cells.

[0062] In certain embodiments, the dendritic cells present in the mixture of monocytes and dendritic cells constitute at least 10%-95%, 10%-90%, 10%-80%, 10%-70%, 10%-60%, 10%-50%, 10%-40%, 10%-30%, 10%-20%, 20%-95%, 20%-90%, 20%-80%, 20%-70%, 20%-60%, 20%-50%, 20%-40%, 20%-30%, 30%-95%, 30%-90%, 30%-80%, 30%-70%, and 30% These are mature dendritic cells with percentages of %~60%, 30%~50%, 30%~40%, 40%~95%, 40%~90%, 40%~80%, 40%~70%, 40%~60%, 40%~50%, 50%~95%, 50%~90%, 50%~80%, 50%~70%, 50%~60%, 60%~95%, 60%~90%, 60%~80%, 60%~70%, 70%~95%, 70%~90%, 70%~80%, 80%~95%, 80%~90%, 90%~95%, or more than 95%.

[0063] In certain embodiments, the dendritic cells present in the mixture of monocytes and dendritic cells consist of at least 10%–30%, 35%–55%, 60%–80%, or 85%–95% mature dendritic cells.

[0064] In certain embodiments, the dendritic cells present in a mixture of monocytes and dendritic cells constitute 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 more than about 95% mature dendritic cells. In certain embodiments, the dendritic cells present in a mixture of monocytes and dendritic cells constitute more than 95% mature dendritic cells. In certain embodiments, the dendritic cells present in a mixture of monocytes and dendritic cells constitute about 100% mature dendritic cells.

[0065] In certain embodiments, antigen-presenting cells in heterologous compositions are not activated or stimulated. In certain embodiments, antigen-presenting cells in heterologous compositions are activated or stimulated. In certain embodiments, antigen-presenting cells are stimulated with one or more pro-inflammatory factors (e.g., granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-4 (IL-4), etc.). For example, porcine monocytes can be cultured into dendritic cells by using GM-CSF and IL-4 for 7 days.

[0066] In certain embodiments, PBMCs in heterogeneous compositions are not activated or stimulated. In certain embodiments, PBMCs in heterogeneous compositions are activated or stimulated. In certain embodiments, PBMCs are stimulated by one or more pro-inflammatory factors.

[0067] In certain embodiments, monocytes and / or dendritic cells in the heterologous composition are not activated or stimulated. In certain embodiments, monocytes and / or dendritic cells in the heterologous composition are activated or stimulated. In certain embodiments, monocytes and / or dendritic cells are stimulated by one or more pro-inflammatory factors.

[0068] In certain embodiments, antigen-presenting cells in a heterogeneous composition are activated or stimulated using porcine B cells as a stimulating factor. In certain embodiments, antigen-presenting cells in a heterogeneous composition are activated or stimulated using porcine T cells as a stimulating factor. In certain embodiments, antigen-presenting cells in a heterogeneous composition are activated or stimulated using porcine monocyte-derived macrophages as a stimulating factor.

[0069] 5.2 Method for obtaining antigen-presenting cells Provided herein are methods for obtaining antigen-presenting cells in heterologous compositions as described in Section 5.1.

[0070] Antigen-presenting cells may be derived from the tissue or blood of any non-human species (e.g., one or more pigs). Such antigen-presenting cells may be obtained by methods commonly known in the art. Antigen-presenting cells may be obtained using a leukocyte apheresis procedure. Such a leukocyte apheresis procedure may be used to produce a leukopak containing peripheral blood mononuclear cells. The leukopak may be further fractionated by countercurrent centrifugation to obtain a composition containing the desired antigen-presenting cells. In some embodiments, antigen-presenting cells are obtained using fluorescence-activated cell sorting (FACS) or magnetically activated cell sorting (MACS) techniques.

[0071] 5.3 Treatment method Provided herein are methods for treating tumors in subjects requiring treatment, comprising administering heterogeneous compositions described in Section 5.1 to the subject (e.g., by intratumor injection). In certain embodiments, administration is carried out via one or more intratumor injections as described in Section 5.3.1.

[0072] In certain embodiments, provided herein is a method for treating a tumor in a subject requiring treatment of the tumor, comprising administering a heterologous composition (for example, by intratumoral injection) in combination with a composition comprising human antigen-presenting cells. The antigen-presenting cells in the heterologous composition and the composition comprising human antigen-presenting cells may be activated or deactivated, or stimulated or deactivated.

[0073] The therapeutic methods provided herein can be used to treat 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 a particular embodiment, the tumor is resistant to treatment with chemotherapy and / or treatment with another immunotherapy. In a particular embodiment, the heterologous composition is administered as a first-line treatment for treating one or more tumors (e.g., by intratumor injection). In a particular other embodiment, the heterologous composition is administered (e.g., by intratumor injection) to a subject that is receiving or may have previously received another anticancer therapy. In one embodiment, the antigen-presenting cells in the heterologous composition are as different as possible from the cells in the tumor tissue. Thus, in one embodiment, the antigen-presenting cells in the heterologous composition are distinctly different from the cells in the tumor tissue. For example, in one embodiment, a method for treating human liver cancer includes the administration of a porcine heterologous composition that does not contain porcine liver tissue.

[0074] Tumors that can be treated by the method 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, ovarian tumors, colorectal tumors, lung tumors, skin tumors, lymphoid tumors, gastrointestinal tumors, gastrointestinal stromal tumors, cervical tumors, hepatocellular carcinoma, renal cell carcinoma, melanoma, colorectal cancer, esophageal cancer, brain tumors, kidney tumors, lung tumors (including non-small cell lung cancer), gastric tumors, bile duct tumors, uterine tumors, and pediatric (childhood) tumors.

[0075] In certain embodiments, provided herein is a method for treating a neoplastic disease in a subject requiring treatment, the method comprising administering a heterogeneous composition described in Section 5.1 (for example, by intratumor injection).

[0076] The heterologous compositions described herein may be administered to a subject by any route of administration, including but not limited to intratumor, intravenous, intradermal, subcutaneous, intramuscular, or lymph node administration. In one embodiment, administration is by parenteral administration. In another embodiment, administration is by intratumor administration, in which the heterologous composition is injected directly into the tumor. In yet another embodiment, administration is by intracerebral administration.

[0077] 5.3.1 Intratumor administration In certain embodiments, the heterogeneous compositions described herein are administered to a target by one or more direct intratumoral injections into the tumor. Such direct delivery of therapeutic compositions to the target tumor lesion offers several advantages over systemic delivery, including the potential for increased local concentrations and reduced systemic toxicity.

[0078] In one embodiment, the injection is within a single tumor. In another embodiment, the injection is within different parts of the same tumor. In yet another embodiment, the injection is within multiple tumors. The ability to inject into multiple sites may lead to a more robust adaptive immune response (e.g., in patients with polyclonal metastases). In some embodiments, the injection is within a primary tumor. In other embodiments, the injection is within one or more metastatic tumors.

[0079] In one embodiment, the intratumor injection is an image-guided intratumor injection. The intratumor injection may be performed using imaging techniques including, but not limited to, ultrasound, fluoroscopy, and CT scans.

[0080] The injection technique used to perform intratumor injection, including the design and placement of the injection needle used and the delivery rate of the therapeutic composition, may affect the therapeutic effect (e.g., immune response). In certain embodiments, intratumor injection is performed by positioning the needle (e.g., using image guidance) in the target tumor, thereby ensuring that the therapeutic composition is distributed throughout the target tumor without leakage into surrounding tissue (i.e., without misdirected leakage). In one embodiment, intratumor injection is performed by positioning the tip of the needle (e.g., using image guidance) in the center of the target tumor.

[0081] Intratumor injection may be performed using a needle having an appropriate needle gauge. The appropriate needle gauge depends on various factors, including, but not limited to, the type, location, and size of the target tumor. In certain embodiments, intratumor 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, intratumor injection is performed using a 20-gauge needle. In one embodiment, intratumor injection is performed using a 21-gauge needle. In one embodiment, intratumor injection is performed using a 22-gauge needle.

[0082] In certain embodiments, intratumor injection is performed using an end-hole needle (EHN) having an appropriate needle gauge. In one embodiment, intratumor injection is performed using a 20-gauge, 21-gauge, or 22-gauge end-hole needle (EHN) (e.g., commercialized by Becton Dickinson).

[0083] In certain embodiments, intratumor injection is performed using a multi-side-hole needle (MSHN) having an appropriate needle gauge. The placement of such a needle may be performed under image guidance to ensure that all side holes are positioned within the tumor. In one embodiment, intratumor injection is performed, thereby preventing unintended leakage of the therapeutic composition. In one embodiment, intratumor injection is performed using a 20-gauge, 21-gauge, or 22-gauge multi-side-hole needle (MEHN). In one embodiment, intratumor injection is performed using a 21-gauge multi-side-hole needle (MSHN) without end holes (e.g., ProFusion® Therapeutic Infusion Needle, commercialized by Cook Regentec).

[0084] 5.3.2 Dosage and frequency of administration The heterogeneous compositions of the present invention may be administered in a manner appropriate to the disease being treated (for example, by intratumoral injection). The dosage and frequency of administration are determined by factors such as the route of administration, the patient's condition, and the type and severity of the patient's disease, although the appropriate dosage may be determined based on clinical trials.

[0085] In certain embodiments, the heterologous composition comprising antigen-presenting cells as described herein may be administered in single or multiple doses (e.g., by intratumor injection). In one embodiment, the composition is administered as an initial dose. In another embodiment, the composition is administered under an additional dosing regimen (i.e., repeated treatment). In yet another embodiment, the composition is administered under an additional dosing regimen (i.e., repeated treatment), but to a different tumor site.

[0086] In certain embodiments, multiple doses may be administered immediately after the previous dose, essentially simultaneously, on the same day (i.e., consecutive doses). Alternatively, multiple doses may be administered on the same day, but not simultaneously (i.e., not consecutive doses). In such cases, multiple doses may consist of two, three, four, five, six, seven, eight, nine, or ten doses.

[0087] In certain embodiments, administration of the heterologous compositions described herein may be repeated, with intervals of at least 1, 2, 3, 4, 5, 7, 10, 15, 30, 45 days, 2 months, 75 days, 3 months, or at least 6 months. In some embodiments, administration may be discontinued for a predetermined number of days.

[0088] In certain embodiments, the compositions described herein are administered once a week for a predetermined number of weeks. In one embodiment, the planned number of weeks is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks. In some embodiments, administration may be interrupted for at least one week. In certain embodiments, the composition is administered as a single dose, followed by a second dose 1 to 6 weeks later. In certain embodiments, the composition may be administered at intervals of 6 to 12 months.

[0089] When the compositions described herein are administered multiple times, the individual doses may be essentially the same or different. Furthermore, the compositions may be administered to the same tumor site or to different tumor sites (for example, via intratumor injection).

[0090] The heterogeneous compositions of the present invention may be administered in various doses depending on the route of administration, the patient's condition, and the type and severity of the patient's disease, but the appropriate dose may be determined based on clinical trials.

[0091] In a particular embodiment, the heterogeneous composition is 1 × 10 per dose. 6 ~10×10 6 , 10×10 6 ~20×10 6 , 20×10 6 ~30×10 6 , 30×10 6 ~40×10 6 , 40×10 6 ~50×10 6 , 50×10 6 ~60×10 6, 60×10 6 ~70×10 6 , 70×10 6 ~80×10 6 , 80×10 6 ~90×10 6 , 90×10 6 ~10×10 7 , 10×10 7 ~20×10 7 , 20×10 7 ~30×10 7 , 30×10 7 ~40×10 7 , 40×10 7 ~50×10 7 , 50×10 7 ~60×10 7 , 60×10 7 ~70×10 7 , 70×10 7 ~80×10 7 , 80×10 7 ~90×10 7 , 90×10 7 ~10×10 8 , 10×10 8 ~20×10 8 , 20×10 8 ~30×10 8 , 30×10 8 ~40×10 8 , 40×10 8 ~50×10 8 It is administered via one or more (e.g., intratumor) injections of antigen-presenting cells.

[0092] In certain embodiments, the heterogeneous composition is approximately 1 × 10 per dose. 6 , about 5×10 6 , about 10×10 6 , about 15×10 6 , about 20×10 6 , about 25×10 6 , about 30×10 6 , about 35×10 6 , about 40×10 6 , about 45×10 6 , about 50×10 6 , about 55×10 6 , about 60×10 6, approximately 65 × 10 6 , approximately 70 × 10 6 , approximately 75 × 10 6 , approximately 80 × 10 6 , approximately 85 × 10 6 , approximately 90 × 10 6 , approximately 95 × 1 — 0 6 , approximately 10 × 10 7 , approximately 15 × 10 7 , approximately 20 × 10 7 , approximately 25 × 10 7 , approximately 30 × 10 7 , approximately 35 × 10 7 , approximately 40 × 10 7 , approximately 45 × 10 7 , approximately 50 × 10 7 , approximately 55 × 10 7 , approximately 60 × 10 7 , approximately 65 × 10 7 , approximately — 0 × 10 7 , approximately 75 × 10 7 , approximately 80 × 10[[ID=^3]] 7 , approximately 85 × 10 7 , approximately 90 × 10 7 , approximately 95 × 10 7 , approximately 10 × 10 8 , approximately 15 × 10 8 , approximately 20 × 10 8 , approximately 25 × 10 8 , approximately 30 × 10 8 , approximately 35 × 10 8 , approximately 40 × 10 8 , approximately 45 × 10 8 , or approximately 50 × 10 8 is administered via one or more (e.g., intratumoral) injections of antigen - presenting cells. In certain embodiments, the heterologous composition is administered via one or more (e.g., intratumoral) injections of approximately 100,000 and / or approximately 500,000 antigen - presenting cells per dose. In some embodiments, the antigen - presenting cells in such a composition are mature antigen - presenting cells. In some embodiments, the antigen - presenting cells in such a composition are mature antigen - presenting cells combined with immature antigen - presenting cells.

[0093] A single dose of antigen-presenting cells may be administered via one or more (e.g., intratumor) injections. In certain embodiments, the heterologous composition is 1 × 10¹⁶ per injection. 6 ~10×10 6 , 10×10 6 ~20×10 6 , 20×10 6 ~30×10 6 , 30×10 6 ~40×10 6 , 40×10 6 ~50×10 6 , 50×10 6 ~60×10 6 , 60×10 6 ~70×10 6 , 70×10 6 ~80×10 6 , 80×10 6 ~90×10 6 , 90×10 6 ~10×10 7 , 10×10 7 ~20×10 7 , 20×10 7 ~30×10 7 , 30×10 7 ~40×10 7 , 40×10 7 ~50×10 7 , 50×10 7 ~60×10 7 , 60×10 7 ~70×10 7 , 70×10 7 ~80×10 7 , 80×10 7 ~90×10 7 , 90×10 7 ~10×10 8 , 10×10 8 ~20×10 8 , 20×10 8 ~30×10 8 , 30×10 8 ~40×10 8 , 40×10 8 ~50×10 8 The antigen-presenting cells are administered via one or more (e.g., intratumor) injections. In certain embodiments, the heterologous composition is administered at approximately 1 × 10 per injection.6 , about 5×10 6 , about 10×10 6 , about 15×10 6 , about 20×10 6 , about 25×10 6 , about 30×10 6 , about 35×10 6 , about 40×10 6 , about 45×10 6 , about 50×10 6 , about 55×10 6 , about 60×10 6 , about 65×10 6 , about 70×10 6 , about 75×10 6 , about 80×10 6 , about 85×10 6 , about 90×10 6 , about 95×10 6 , about 10×10 7 , about 15×10 7 , about 20×10 7 , about 25×10 7 , about 30×10 7 , about 35×10 7 , about 40×10 7 , about 45×10 7 , about 50×10 7 , about 55×10 7 , about 60×10 7 , about 65×10 7 , about 70×10 7 , about 75×10 7 , about 80×10 7 , about 85×10 7 , about 90×10 7 , about 95×10 7 , about 10×10 8 , about 15×10 8 , about 20×10 8 , about 25×10 8 , about 30×10 8 , about 35×10 8 , about 40×10 8 , about 45×10 8 , or approximately 50 x 10 8The antigen-presenting cells are administered via one or more (e.g., intratumor) injections. In certain embodiments, the heterologous composition is administered via one or more (e.g., intratumor) injections of about 100,000 and / or about 500,000 antigen-presenting cells per injection.

[0094] 5.3.3 Clinical Outcomes The therapeutic effects observed upon administration of the heterologous compositions of the present invention, either alone or in combination with other anticancer therapies, may be evaluated by any method commonly used in anticancer therapy. In certain embodiments, administration of the heterologous compositions induces an immune response specific to the targeted tumor.

[0095] Furthermore, treatment with the heterogeneous compositions described herein, either alone or in combination with other anticancer therapies, may not only reduce the targeted tumor but also lead to the reduction of untreated tumors elsewhere in the body. Such results are known as the abscopal effect. Therefore, treatment with the heterogeneous compositions described herein, either alone or in combination with other anticancer therapies, may induce a systemic antitumor immune response. In certain embodiments, administration of the heterogeneous compositions described herein via intratumor injection results in the abscopal effect. In certain embodiments, administration of the heterogeneous compositions described herein via intratumor injection in combination with another anticancer therapy (e.g., tumor therapeutic field therapy (TTF) or radiation) results in the abscopal effect.

[0096] In certain embodiments, the therapeutic effect observed when heterologous compositions of the present invention are administered alone or in combination with other anticancer therapies is evaluated by measuring the immune response. In certain embodiments, the immune response is evaluated by measuring immune markers in the blood. In certain embodiments, tumor-specific immune responses are evaluated. In certain embodiments, systemic immune responses are evaluated. The immune response may be evaluated by: • Tracking cells (which may include, for example, staining PBMCs with a specific antibody against one HLA class I antigen or one HLA class II antigen selectively expressed in donor vaccine cells); • Evaluate dendritic cell-induced alloimmunity (which may include screening for alloantibodies against HLA-A, B, C (MHC class I) and HLA-DR, DQ, DP (MHC class II) antigens); • Monitor autoimmune events (nuclear antigens (e.g., ANA, SSA, SSB, Sm, RNP, Scl-70, kinetochore, and Jo-1) and hepatic parenchymal-associated autoantigens (e.g., hepatic-renal microsomal antigens and mitochondrial antigens); • To evaluate complement activation and / or classical / alternative complement function; and / or • Evaluate the presence and activation status of immune cells (e.g., CD3+, CD3+4+, and CD3+8+ T cells, CD19+ B cells, CD3-16β+56+ NK cells, CD3-16β+56+69+ NK cells, CD3+16β+56+ NKT cells, CD3+16β+56+69+ NKT cells, CD3+HLA-DR+ T cells).

[0097] In certain embodiments, the therapeutic effect observed when a heterogeneous composition of the present invention is administered alone or in combination with other anticancer therapies is evaluated by assessing the size of the tumor(single) / tumor(multiple). This may be done 3 months and / or 6 months after the administration of treatment, or as deemed necessary based on tumor progression.

[0098] In certain embodiments, the therapeutic effect observed when a heterogeneous composition of the present invention is administered alone or in combination with another anticancer therapy is evaluated by assessing tumor control. In some embodiments, tumor control is evaluated by CT scans and / or MRI scans, measurement of tumor-specific T cell counts, measurement of AFP (alpha-fetoprotein) levels in the blood, measurement of circulating tumor cell levels, or any combination thereof.

[0099] In certain embodiments, the therapeutic effect observed when heterologous compositions of the present invention are administered alone or in combination with other anticancer therapies is evaluated by assessing the systemic inflammatory response. This may be done, for example, by evaluating the systemic release of relevant cytokines, chemokines, and other inflammatory parameters in the 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).

[0100] In certain embodiments, treatment efficacy is assessed by assessing long-term changes in the East Coast Cancer Group (ECOG) and / or Karnofsky Performance Status (KPS) scores. In certain embodiments, treatment efficacy is assessed by assessing long-term changes in quality of life scores.

[0101] In certain embodiments, therapeutic efficacy is evaluated in terms of partial response and / or complete response. In certain embodiments, therapeutic efficacy is evaluated 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 lactate dehydrogenase (LDH) levels and the resulting neutrophil-to-lymphocyte ratio (dNLR). Elevated LDH and dNLR may be associated with a reduced survival outcome in patients treated with immunotherapy. In certain embodiments, administration of the heterologous composition of the present invention alone or in combination with another anticancer therapy results in a decrease in LDH and dNLR levels compared to baseline.

[0102] In certain embodiments, therapeutic effects are evaluated in terms of adverse events and recorded as indicators of safety and tolerability, which may include changes in vital signs from baseline (e.g., heart rate, blood pressure, body temperature) and changes in clinical laboratory parameters from baseline. In one embodiment, administration of heterogeneous compositions of the present invention alone or in combination with other anticancer therapies is not associated with serious adverse events.

[0103] 5.3.4 Combination Therapy When used in the treatment of cancer, the heterogeneous compositions described herein may be used in combination with other therapies. In some embodiments, the heterogeneous compositions are administered in combination with therapies that reduce the immunosuppressive tumor environment or therapies that activate the immune system.

[0104] In certain embodiments, heterologous compositions are administered in combination with one or more other anticancer therapies (e.g., by intratumoral injection). Examples of such other anticancer therapies include, but are not limited to, anti-CTLA4 therapy, anti-PD1 therapy, anti-PDL1 therapy, anti-LAG-3 therapy, tumor therapeutic field therapy (TTF), cell-based therapies, tyrosine kinase inhibitors, VEGF inhibitors, or any combination thereof. In one embodiment, the other anticancer therapy includes treatment with one or more biologics. In one embodiment, the other anticancer therapy includes treatment with cell therapy formulations or gene therapy formulations. In one embodiment, the other anticancer therapy is a CAR T cell-based therapy. In one embodiment, the other anticancer therapy includes treatment with T cell engagers. In one embodiment, the other anticancer therapy includes treatment with one or more immune checkpoint inhibitors. In one embodiment, the other anticancer therapy includes treatment with one or more small molecule drugs. In one embodiment, the other anticancer therapy includes treatment with one or more protein kinase inhibitors. In one embodiment, the other anticancer therapy includes treatment with one or more tyrosine kinase inhibitors. In one embodiment, the other anticancer therapy includes treatment with one or more alkylating agents, antimetabolites, natural products, or hormones. In one embodiment, the other anticancer therapy includes treatment with gemcitabine or 5-fluorouracil. In one embodiment, the other anticancer therapy includes surgery or radiotherapy. In one embodiment, the other anticancer therapy includes tumor-treating electric field therapy.

[0105] In certain embodiments, other anticancer therapies include treatment with imatinib, sunitinib, regorafenib, pazopanib, nilotinib, avapritinib, lipretinib, sorafenib, pimitespib, ipilimumab, tremelimumab, nivolumab, pembrolizumab, semiprimab, atelizumab, avelumab, durvalumab, leratrimab, or any combination thereof.

[0106] The combination therapies described herein may provide a “synergistic effect” and may be proven to be “synergistic,” that is, the therapeutic effect achieved after administration of two or more individual therapies used together may be proven to be greater than the sum of the therapeutic effects achieved after separate administrations of the individual therapies. Such synergistic effects may be determined by methods commonly known in the art. In certain embodiments, the combination therapy (i.e., administration of heterogeneous compositions of the present invention in combination with another anticancer therapy) results in a synergistic antitumor effect.

[0107] The preferred dosage of individual treatments and / or compositions may be reduced by combined action (synergistic effect) to increase the therapeutic index or reduce toxicity or other side effects or consequences.

[0108] 5.4 Pharmaceutical Compositions Provided herein are pharmaceutical compositions comprising heterogeneous compositions as described in Section 5.1.

[0109] In certain embodiments, provided herein are pharmaceutical compositions comprising heterogeneous compositions in combination with one or more pharmaceutically acceptable 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.

[0110] In certain embodiments, the pharmaceutical composition comprises one or more pharmaceutically acceptable carriers. In some embodiments, the pharmaceutically acceptable carriers are phosphate-buffered saline, water, or emulsions such as oil / water or water / oil emulsions. In certain embodiments, the pharmaceutical composition is formulated as an aqueous solution in a physiologically compatible buffer, such as Hanks' solution, Ringer's solution, or physiological saline buffer.

[0111] 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 agents, stabilizers, and / or dispersants. 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 preservatives.

[0112] In certain embodiments, the pharmaceutical compositions provided herein may include buffers such as neutral buffered saline or phosphate-buffered saline; carbohydrates such as glucose, mannose, sucrose or dextran, or 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.

[0113] In certain embodiments, the pharmaceutical composition comprising antigen-presenting cells described herein is in the form of a suspension or dispersion. Such a suspension or dispersion may be prepared by conventional dispersion and suspension 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 dispersion or suspension contains one or more viscosity modifiers.

[0114] In certain embodiments, the pharmaceutical composition comprising the heterogeneous compositions described herein is formulated in the form of a hydrogel. In some embodiments, the hydrogel is a hydrogel based on a multidomain peptide. Such a hydrogel based on a multidomain peptide may be prepared by methods known in the art. In certain embodiments, antigen-presenting cells embedded in a hydrogel based on a multidomain peptide exhibit significant improvement in delivery and retention within tumors. In certain embodiments, the pharmaceutical composition is a sustained-release composition based on a hydrogel.

[0115] In certain embodiments, the heterogeneous compositions described herein are formulated into pharmaceutical compositions together with diluents and / or other components such as IL-2 or other cytokines or cell populations.

[0116] The pharmaceutical compositions described herein may be maintained at a temperature of about 2–8°C, or they may be frozen and thawed immediately before use. In certain embodiments, the pharmaceutically acceptable carrier may be a culture medium for storing frozen cells. For example, a heterologous composition containing antigen-presenting cells described herein may be frozen in thermoactivated universal donor plasma containing dimethyl sulfoxide (DMSO) to enable its storage. Such a culture medium containing antigen-presenting cells may be used as is after thawing, for example, by injection into a tumor. Alternatively, the cells frozen in such a culture medium may be thawed, washed, and resuspended in appropriately buffered saline or saline containing human serum albumin before administration, for example, by intratumoral injection. In one embodiment, the pharmaceutically acceptable carrier is saline containing human serum albumin. In another embodiment, the pharmaceutically acceptable carrier is a sodium chloride solution containing 2% human serum albumin.

[0117] The pharmaceutical compositions provided herein may be formulated for any route of administration, including but not limited to intratumor, intravenous, intradermal, subcutaneous, intramuscular, or intralymph node administration. In one embodiment, the pharmaceutical composition is formulated for parenteral administration. In another embodiment, the pharmaceutical composition is formulated for intratumor administration. In intratumor administration, the pharmaceutical composition is injected directly into the tumor. In specific embodiments, the pharmaceutical compositions described herein are administered by one or more direct intratumor injections into the tumor. The injection may be directed at a single tumor or multiple tumors.

[0118] The pharmaceutical composition of the present invention may be administered in a manner appropriate to the disease being treated. The dosage and frequency of administration are determined by factors such as the route of administration, the patient's condition, and the type and severity of the patient's disease, although the appropriate dosage may be determined based on clinical trials.

[0119] The following examples are provided for illustrative purposes only and are not intended to be limiting. [Examples]

[0120] 6. Examples 6.1 Example 1: Heterogeneous composition containing porcine antigen-presenting cells 6.1.1 Isolation of Peripheral Blood Mononuclear Cells Pig blood (approximately 100 ml total) collected in an EDTA vacuum tanker (BD Biosciences, San Diego, USA) was diluted 1:1 with phosphate-buffered saline (PBS). 30 ml of the diluted blood was carefully placed on top of 20 ml of Ficoll Paque Plus (Cytiva, Uppsala, Sweden) in a 50 ml Falcon tube. Density gradient centrifugation was performed at 600 × g for 20 minutes with the centrifugal brake off. After centrifugation, peripheral blood mononuclear cells (PBMCs) were collected from the interface and washed three times with PBS. Each wash consisted of a 5 minute centrifugation at 350 × g followed by replacement of the supernatant with fresh PBS. After the final wash, the PBMCs were resuspended in 5 ml of ACK lysis buffer (Gibco, Thermo Fisher Scientific Inc., Waltham, USA) and incubated for 5 minutes. After erythrocyte lysis, PBMCs were centrifuged and resuspended in AIM V medium (Gibco, Thermo Fisher Scientific Inc., Waltham, USA) supplemented with 10% heat-immobilized fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific Inc., Waltham, USA). Human PBMCs were similarly purified from the buffy coat of anonymous healthy donors obtained from the Karolinska University Hospital Blood Bank (Stockholm, Sweden).

[0121] 6.1.2 Isolation of porcine CD14+ cells Porcine CD14+ cells were isolated by positive magnetic bead sorting using a cross-reactive anti-human CD14 microbead isolation kit (Miltenyi, Bergisch Gladbach, Germany) according to the manufacturer's instructions. 1 × 10⁻⁶ 8 In two separate purifications starting from porcine PBMCs, the yield of positively selected CD14+ cells reached 20% of the starting cell count. Cell viability in both the PBMC and CD14+ fractions after positive selection was over 93%, as analyzed using Trypan Blue exclusion (Gibco, Thermo Fisher Scientific Inc., Waltham, USA).

[0122] 6.2 Example 2: In vivo experiments in mice using human PBMCs - Mouse melanoma model Human peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood using Ficoll density gradient centrifugation.

[0123] Next, the PBMCs were labeled with CFSE (carboxyfluorescein succinimimidyl ester). Approximately 1 × 10⁻⁶ CFSE-labeled PBMCs were collected. 6 The cells were administered via intratumoral injection into C57BL / 6J mice expressing the B16 melanoma cell line.

[0124] Tumor size was compared between the treatment group and the control group by measuring tumor volume on day 12. The results are shown in the table and Figure 2 below. [Table 1]

[0125] 6.3 Example 3: In vivo mouse experiments using human PBMCs - Mouse bladder cancer model Human PBMCs were isolated from peripheral blood using Ficoll density gradient centrifugation.

[0126] Next, the PBMCs were labeled with CFSE (carboxyfluorescein succinimimidyl ester). Approximately 1 × 10⁻⁶ CFSE-labeled PBMCs were collected. 6 The MB49 carcinogen-induced bladder cancer cell line was injected intratumorically into C57BL / 6J mice expressing the MB49 carcinogen.

[0127] Tumor size was compared between the treatment group and the control group by measuring tumor volume on day 7. The results are shown in the table and Figure 3 below. [Table 2]

[0128] 6.4 Example 4: Mixed Lymphocyte Reaction Human PBMCs were stained with Violet Proliferation Dye 450 (VPD450; BD Biosciences, San Diego, USA) according to the manufacturer's instructions. In the first heterologous mixed lymphocyte reaction (MLR) test, human PBMCs stained with VPD450 were mixed with unstained porcine PBMCs in various ratios. Specifically, the ratio of human cells to porcine cells was started at 10:10, and a fixed number of human cells was combined with a gradually decreasing number of porcine cells until the ratio of human cells to porcine cells reached 10:1. The wells with a 10:10 ratio contained 2 × 10⁶ human PBMCs. 5 and Pig PBMC2 x 10 5 While containing the combination of human cells and pig cells, the well with a human cell to pig cell ratio of 10:1 contains human PBMC2 × 10 5 and Pig PBMC2 x 10 4The combination included the following. In the second heterologous test, VPD450-stained human PBMCs were mixed with unstained porcine CD14+ cells in various ratios as described above. All samples were resuspended in 200 μl of AIM V medium supplemented with 10% heat-inactivated FBS in a round-bottom 96-well cell culture plate and incubated at 37°C and 5% CO2 for 7 days. On day 5, 100 μL of fresh culture medium was added to each well. Both heterologous MLR tests included one porcine donor and two (n=2) PBMC donors paired separately. Cells from either donor / species were not inactivated by irradiation / chemical treatment and resulted in bidirectional heterologous MLR, but only human cells were labeled with growth dye. The control wells were 2 × 10 4 Human PBMCs and 2 x 10⁻¹⁰ donor-derived cells 4 The study included allogeneic MLRs consisting of a combination of human PBMCs and other materials. Additional control wells were obtained from a single donor, 2 × 10⁶ 4 I received human PBMC.

[0129] On day 7 of MLR, cells were washed twice with saline and incubated with an Fc receptor binding inhibitor (Invitrogen; Thermo Fisher Scientific Inc., Waltham, USA). Following incubation, the cells were treated with anti-CD3 VioGreen (Miltenyi, Bergisch Gladbach, Germany; clone REA613), anti-CD45RA BV650 (BD Biosciences, San Diego, USA; clone HI100), anti-CD8 antibody BB515 (BD Biosciences, San Diego, USA; clone RPA-T8), anti-CCR7 antibody PE (Miltenyi, Bergisch Gladbach, Germany; clone REA546), anti-HLA-DR antibody PerCP-Cy5.5 (BD Biosciences, San Diego, USA; clone G46-6), and anti-CD4 APC-Vio770 (Miltenyi, Bergisch The cells were stained with Gladbach, Germany (clone REA623) and anti-CD56 APC (Miltenyi, Bergisch Gladbach, Germany (clone REA196)). The samples were stained while resuspended in staining buffer (FBS) (BD Biosciences, San Diego, USA), incubated at 4°C in the dark, and after two washing steps, cells were acquired using a FACS Celesta flow cytometer (BD Biosciences, San Diego, USA). Post-acquisition data analysis was performed using FlowJo 10.8.1 software (FlowJo LLC, Ashland, USA).

[0130] 6.5 Example 5: Comparison of in vitro tests: Pig PBMC vs. Human PBMC As described in the MLR study above, porcine PBMCs were seeded on plates with human PBMCs at various human cell-to-porcine cell ratios (h:p). The human cell-to-porcine cell ratios (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. Human PBMCs alone and allogeneic human PBMCs were evaluated separately as reference samples.

[0131] For all samples, including the reference sample, the number of human CD4 T cells and human CD8 T cells was evaluated on day 7. The results are shown in Figures 4A and 4B, respectively.

[0132] The number of human NK cells was evaluated on day 7 for all samples, including the reference sample. The results are shown in Figure 4C.

[0133] 6.6 Example 6: Comparison of in vitro tests: Porcine monocytes (CD14+ cells) vs. human PBMCs As described in the MLR study above, porcine monocytes (containing CD14+ cells) were seeded on plates with human PBMCs at various human cell-to-porcine cell ratios (h:p). The human cell-to-porcine cell ratios (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. Human PBMCs alone and allogeneic human PBMCs were evaluated separately as reference samples.

[0134] For all samples, including the reference sample, the number of human CD4 T cells and human CD8 T cells was evaluated on day 7. The results are shown in Figures 5A and 5B, respectively.

[0135] For all samples, including the reference sample, the number of proliferated human CD4 T cells and the number of proliferated human CD8 T cells were also evaluated on day 7. The results are shown in Figures 5C and 5D, respectively.

[0136] HLA-DR on human CD4 T cells and human CD8 T cells was evaluated on day 7 for all samples, including the reference sample, in terms of mean fluorescence intensity (MFI). The results are shown in Figures 5E and 5F, respectively.

[0137] The number of human NK cells was evaluated on day 7 for all samples, including the reference sample. The results are shown in Figure 5G.

[0138] 6.7 Example 7: In vivo experiments with mice using porcine PBMCs Pig PBMC 1 x 10 6 The B16 melanoma cell line was injected intratumorally into C57BL / 6J mice at a cellular dose. Tumor size was compared between the treatment group and the control group by measuring tumor volume from day 7 onward.

[0139] We will perform the same experiment using C57BL / 6J mice that express MB49 carcinogen-induced bladder cancer cell lines.

[0140] 6.8 Example 8: Intratumoral administration of porcine PBMCs to human patients This example evaluates the safety and / or efficacy of a composition containing porcine antigen-presenting cells administered intratumorally as a monotherapy or in combination with another anticancer therapy to cancer patients.

[0141] Patients were divided into two treatment groups. The first patient group received the therapeutic composition as monotherapy, while the second patient group received the therapeutic composition in combination with a checkpoint inhibitor or tyrosine kinase inhibitor. Patients received 10 × 10 per injection. 6 Treatment begins with a cellular dose and is increased in dose and / or frequency with the therapeutic composition. Patients may or may not have previously been treated with anticancer therapy. Specific outcome indicators are provided below.

[0142] Adverse events are registered as measures of safety and tolerability, and include changes in vital signs (e.g., heart rate, blood pressure, body temperature) from baseline, and changes in clinical laboratory parameters from baseline.

[0143] The immune response is assessed by measuring immune markers in the blood. Tumor size is assessed at 3 months and 6 months, or as deemed necessary based on tumor progression.

[0144] The systemic inflammatory response is evaluated, including the potential for systemic release of relevant cytokines, chemokines, and other inflammatory parameters in the 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-γ, MCP-1, MIP-1β, TNF-α).

[0145] Tumor control is assessed by including CT / MRI scans, measurement of tumor-specific T cell counts, measurement of blood AFP (alpha-fetoprotein) levels, and / or measurement of circulating tumor cell levels.

[0146] To evaluate tumor-specific immune responses and systemic immune responses.

[0147] This study assesses long-term changes in the East Coast Cancer Group (ECOG) and / or Karnofsky Performance Status (KPS) scores. Furthermore, it assesses long-term changes in quality of life scores.

[0148] Treatment efficacy is evaluated in terms of partial response, complete response, progression-free survival, and / or overall survival.

[0149] 7. Equal parts While this disclosure is described in detail with reference to its specific embodiments, it will be understood that functionally equivalent variations fall within the scope of this disclosure. Indeed, various modifications of this disclosure, in addition to those shown and described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are intended to be included within the appended claims. Those skilled in the art will be able to recognize or confirm many equivalents to the specific embodiments of this disclosure described herein by means of routine experimentation alone. Such equivalents are intended to be covered by the following claims.

[0150] All publications, patents, and patent applications referenced herein are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application had been specifically and individually directed to be incorporated herein by reference as a whole.

Claims

1. A method for treating a tumor in a person requiring treatment for a tumor, wherein the method is The method comprising administering a composition containing antigen-presenting cells to the subject, wherein the antigen-presenting cells are obtained from a species different from the subject.

2. The method according to claim 1, wherein the tumor is a solid tumor, and the administration is to introduce it into the tumor of the target via intratumor injection.

3. The method according to any one of the prior claims, wherein the antigen-presenting cells are obtained from a species of pig.

4. The method according to any one of the prior claims, wherein the antigen-presenting cells are obtained from a species of miniature pig.

5. The method according to any one of the prior claims, wherein the subject is a human.

6. The method according to claim 3, wherein the pig is an alpha-1,3-galactosyltransferase-deficient pig.

7. The method according to claim 6, wherein the alpha-1,3-galactosyltransferase-deficient pig is a pig from a porcine leukocyte antigen (SLA) syngeneic cross.

8. The method according to claim 4, wherein the miniature pig is an alpha-1,3-galactosyltransferase-deficient miniature pig.

9. The method according to claim 8, wherein the alpha-1,3-galactosyltransferase-deficient miniature pig is a porcine leukocyte antigen (SLA) syngeneic pig.

10. The method according to any one of the prior claims, wherein the method induces an immune response specific to the tumor.

11. The method according to any one of the prior claims, wherein the abscopal effect is obtained by the method described above.

12. The method according to any one of the prior claims, wherein the tumor is a solid cancerous tumor.

13. The method according to claim 12, wherein the tumor is selected from the group consisting of sarcoma, carcinoma, lymphoma, breast tumor, prostate tumor, head and neck tumor, glioblastoma, bladder tumor, pancreatic tumor, liver tumor, ovarian tumor, colorectal tumor, lung tumor, skin tumor, lymphoid tumor, gastrointestinal tumor, gastrointestinal stromal tumor, cervical tumor, hepatocellular carcinoma, renal cell carcinoma, melanoma, colorectal cancer, esophageal cancer, brain tumor, kidney tumor, lung tumor (including non-small cell lung cancer), gastric tumor, bile duct tumor, uterine tumor, and pediatric (childhood) tumor.

14. The method according to any one of the prior claims, wherein the tumor is resistant to treatment with chemotherapy and / or immunotherapy.

15. The method according to any one of the prior claims, wherein the antigen-presenting cells are derived from one or more pigs or miniature pigs using a leukocyte apheresis procedure, the leukocyte apheresis procedure generates a leukopak containing peripheral blood mononuclear cells, and the leukopak is further fractionated by countercurrent centrifugation.

16. The method according to any one of the prior claims, wherein the composition comprising antigen-presenting cells is substantially free of pathogens.

17. The method according to any one of the prior claims, wherein the antigen-presenting cells are obtained from pigs or miniature pigs of different genotypes.

18. The method according to any one of the prior claims, wherein the composition comprising antigen-presenting cells is administered in a single dose or multiple doses.

19. The composition containing antigen-presenting cells contains at least about 1 × 10 per dose. ６ The method according to any one of the prior claims, administered via intratumoral injection of antigen-presenting cells.

20. The composition comprising the antigen-presenting cells is administered via intratumoral injection of about 1×10 ６ , about 5×10 ６ , about 10×10 ６ , about 15×10 ６ , about 20×10 ６ , about 25×10 ６ , about 30×10 ６ , about 35×10 ６ , about 40×10 ６ , about 45×10 ６ , about 50×10 ６ , about 55×10 ６ , about 60×10 ６ , about 65×10 ６ , about 70×10 ６ , about 75×10 ６ , about 80×10 ６ , about 85×10 ６ , about 90×10 ６ , about 95×10 ６ , about 10×10 ７ , about 15×10 ７ , about 20×10 ７ , about 25×10 ７ , about 30×10 ７ , about 35×10 ７ , about 40×10 ７ , about 4-5×10 ７ , or about 50×10 ７ of antigen-presenting cells, according to any one of the preceding claims.

21. The method according to any one of the prior claims, wherein the antigen-presenting cells are substantially mature antigen-presenting cells.

22. The method according to any one of the prior claims, wherein the antigen-presenting cells are not activated or stimulated.

23. The method according to any one of the prior claims, wherein the composition comprises peripheral blood mononuclear cells (PBMCs).

24. The method according to any one of the prior claims, wherein the composition comprises a monocyte.

25. The method according to any one of the prior claims, wherein the composition comprises dendritic cells, macrophages, granulocytes, T cells, B cells, and / or NK cells.

26. The method according to any one of the prior 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 more than about 95%, wherein the PBMC is mature PBMC, immature PBMC, or a combination of mature PBMC and immature PBMC.

27. The method according to any one of the prior 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 more than about 95%, wherein the monocytes are mature monocytes, immature monocytes, or a combination of mature and immature monocytes.

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

29. The aforementioned mixture is 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 4 The method according to claim 28, comprising 0%, 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 more than 95% monocytes.

30. The method according to claim 28, wherein the mixture contains 10% to 30%, 35% to 55%, 60% to 80%, or 85% to 95% monocytes.

31. The method according to claim 28, wherein the mixture contains 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 more than about 95% monocytes.

32. The aforementioned mixture is 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% The method according to claim 28, comprising dendritic cells in the following proportions: %, 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 more than 95%.

33. The method according to claim 28, wherein the mixture comprises 10% to 30%, 35% to 55%, 60% to 80%, or 85% to 95% dendritic cells.

34. The method according to claim 28, 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 more than about 95% dendritic cells.

35. The method according to any one of the prior claims, wherein the subject is receiving another anti-cancer therapy.

36. The method according to claim 35, wherein the other anticancer therapy includes treatment with one or more immune checkpoint inhibitors.

37. The method according to claim 35, wherein the other anticancer therapy is anti-CTLA4 therapy, anti-PD1 therapy, anti-PDL1 therapy, anti-LAG-3 therapy, tumor treatment field therapy (TTF), cell-based therapy, tyrosine kinase inhibitors, VEGF inhibitors, or any combination thereof.

38. The method according to claim 35, wherein the other anticancer therapy includes treatment with imatinib, sunitinib, regorafenib, pazopanib, nilotinib, avapritinib, lipretinib, sorafenib, pimitespib, ipilimumab, tremelimumab, nivolumab, pembrolizumab, semiprimab, atelizumab, avelumab, durvalumab, leratrimab, or any combination thereof.

39. The method according to claims 35 to 38, wherein the subject does not respond to the other anticancer therapies if the composition containing antigen-presenting cells is not administered.

40. A pharmaceutical composition suitable for intratumor injection, wherein the pharmaceutical composition comprises antigen-presenting cells obtained from one or more pigs.

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