Immunotherapy compositions and methods of use

Abstract

The present disclosure takes advantage of the therapeutic potential of autologous and allogeneic cells, particularly cancer cells and T cells, which have been genetically modified or contacted with specific recombinant proteins, peptides or nucleic acids encoding these entities. These modified cells are prepared to elicit various immune and inflammatory responses in the tumor microenvironment, thereby enhancing the natural ability of the organism to recognize and disrupt tumor cells.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of priority of U.S. Provisional Application No. 63 / 421,050, filed Oct. 31, 2022, and 63 / 437,196, filed Jan. 5, 2023, under 35 U.S.C. § 119(e). The disclosure of the prior applications is considered part of the disclosure of this application and is hereby incorporated by reference in its entirety into the disclosure of this application.

[0003] Incorporation of Sequence Listing

[0004] The material in the accompanying sequence listing is hereby incorporated by reference into this application. The accompanying sequence listing xml file is named PYRO1100 - 3WO_SL, created on Oct. 20, 2023, and is 48 KB in size. Technical Field

[0005] This disclosure generally relates to the field of immunotherapy and, more particularly, to the development of compositions and methods for cell and viral immunotherapies that confer protective immunity against human cancers. Background Art

[0006] Although cancer vaccines are effective in preventing disease, they have limited therapeutic potential for four main reasons. First, central and peripheral tolerance of the immune system. The immune system detects “self” antigens expressed by cancer cells and becomes tolerant to them rather than killing them. Second, tumor cell heterogeneity. Tumor cells lacking antigens or having defective antigen presentation evade detection and killing by antigen - specific immune cells. Third, the immune - privileged TIME, the tumor immune microenvironment. Immune - suppressive molecules and cells in the TIME limit anti - tumor immune responses. Fourth, the immune function of most patients with advanced cancer is compromised, which makes treating established malignancies challenging.

[0007] This disclosure describes genetically and chemically modified compositions for generating autologous or allogeneic cell immunotherapies or viral immunotherapies that stimulate a patient's own immune system to detect a broad repertoire of cancer - specific antigens and kill heterogeneous cancer cells. Patient - derived cells, whether cancer cells or immune cells, are genetically and / or chemically modified ex vivo to produce cell immunotherapies that overcome central and peripheral immune tolerance, increase tumor immunogenicity, reshape the immunosuppressive tumor immune microenvironment, and redirect the existing repertoire of immune cells to kill cancer cells.

[0008] Cancer vaccine platforms include cell vaccines, viral or bacterial vaccines, and molecular vaccines using DNA, RNA, or peptides.

[0009] Autologous APCs, antigen-presenting cells, particularly dendritic cells, are loaded with peptide antigens or transfected with antigen genes to generate DC vaccines. The first FDA-approved cancer vaccine, sipuleucel-T (Provenge), is used for the treatment of metastatic castration-resistant prostate cancer (mCRPC). This vaccine is generated by leukapheresis to enrich DCs and ex vivo activation with a chimeric protein GM-CSF fused to the antigen PAP, and is safe but has limited efficacy. Several other phase I and II trials of DC vaccine-based therapies using autologous DCs pulsed with cancer antigen peptides or transduced with adenoviruses encoding antigens are ongoing.

[0010] Whole-cell vaccines. Autologous and allogeneic whole-cell vaccines have been investigated in phase II and III trials but have not been approved by the FDA. Autologous tumor vaccines were clinically evaluated in the 1970s by administering patient-derived tumor cells together with adjuvants or viruses to stimulate a polyclonal immune response against TAAs (tumor-associated antigens). More recently, GVAX whole-tumor cell vaccines genetically modified to secrete the immunostimulatory cytokine, GM-CSF, granulocyte-macrophage colony-stimulating factor, have been evaluated in autologous and allogeneic settings. GVAX enhances the recruitment and maturation of hematopoietic precursors into professional antigen-presenting (APC) dendritic cells (DCs). However, even though GVAX induces immune responses and tumor regression in murine tumor models, its clinical efficacy in prostate, melanoma, lung, and pancreatic cancers has been limited. Overall, GM-CSF has been widely used in cancer vaccine trials but with limited effects.

[0011] Microorganisms to stimulate immune responses or deliver tumor antigens. Heat-inactivated bacteria were first used by Coley to elicit significant anti-tumor immune responses in cancer patients. The attenuated live strain of Mycobacterium bovis, Bacillus Calmette-Guérin (BCG), has been used to treat bladder cancer for over 35 years. Although some reports have rationalized how BCG induces anti-tumor immune responses, the exact cellular and molecular mechanisms remain unclear. Other bacterial species such as Listeria, Salmonella, Lactococcus, and Shigella have also been used as effective vaccine carriers. In preclinical models, treatment with an attenuated strain of Listeria monocytogenes results in the internalization of bacteria by APCs and the delivery of DNA or RNA-encoded tumor antigens to induce effective anti-tumor immunity. In clinical trials, Listeria-based attenuated cancer vaccines have good safety but limited efficacy.

[0012] In most cancers, tumors express self-proteins that are tolerant to the immune system. Tumor antigen-specific vaccines generally exhibit poor antigenicity due to immune tolerance and fail to activate a strong, clinically meaningful anti-tumor immune response. Not surprisingly, vaccines are effective when used in a preventive setting but have limited efficacy when the disease is well established (therapeutic setting). Thus, to be effective, cancer vaccines need to overcome tolerance by stimulating the remaining low-affinity or rare TAA-reactive T cells or by stimulating new TAA-reactive T cells. Adjuvants, antigen presentation activators, and sequential vaccinations that promote the expansion of tumor antigen-reactive T cells, particularly low-affinity T cells, are needed. Importantly, the complete repertoire of tumor antigens needs to be presented to the immune system to hedge against tolerogenic epitopes.

[0013] The heterogeneity of cancer cells confers them a significant survival advantage. Tumor cell subsets evade baseline or therapy-induced immune surveillance, which is the basis for recurrence and poor patient survival outcomes. Thus, a strategy is needed to enhance the antigenicity of heterogeneous tumor cells and to stimulate an immune response against the entire available tumor antigen repertoire.

[0014] Non-whole-cell TAA-specific vaccines, such as peptide or whole-protein vaccines, or antigen-specific DNA or mRNA vaccines need to know the expression patterns of targetable tumor antigens and their immunogenicity in cancer patients. The efficacy of these TAA-specific approaches largely depends on the expression patterns and manifestations of the target antigens in the patient's tumor. Problematically, the expression patterns of antigens are generally heterogeneous, and the immunogenicity of antigens is variable. In addition, most cancer vaccines are designed to stimulate an immune response against one or a limited set of tumor antigens. For various reasons, stimulating an immune response against a limited set of predicted neoantigens is insufficient. The expression of the entire tumor antigen repertoire is essential for activating an immune response against a broad repertoire of cancer antigens to target heterogeneous cancers.

[0015] TIME immunosuppressive cells and molecules suppress anti-tumor immunity. Primarily, the cytokine TGF-β, the transforming growth factor β ligands TGF-β1, -β2, -β3 are the cause of significant immunosuppression at that time. TGF-β is ubiquitously expressed in human cancers and is associated with disease progression and poor patient prognosis. It induces anergy in immune cells in the TIME, inhibits the cytotoxic potential of CD8 + and CD4 + T cells, and converts naive T cells into immunosuppressive regulatory T cells. TGF-β also effectively blocks the maturation of DCs through GM-CSF, the expression of co-stimulatory molecules, and MHC class II, which is important for presenting antigens to CD4 + T cells. TGF-β directly inhibits the antigen-presenting ability of macrophages and dendritic cells and restricts the cytotoxic CD8 +T cells and CD4 + The cytotoxic potential of T cells. Thus, timely overcoming TGF-β-induced immunosuppression is crucial for sustaining an effective anti-tumor immune response. Neutralizing anti-TGF-β antibodies, RNAi, mutant TGF-β1 precursors, or dominant-negative expression have been used to neutralize TGF-β activity.

[0016] Tertiary lymphoid structures are highly differentiated compartments responsible for coordinating and guiding the differentiation and proliferation of lymphocytes. The bone marrow and thymus are primary lymphoid organs where naive B and T lymphocytes mature from immature hematopoietic precursors. Secondary lymphoid organs (SLOs) include the spleen, lymph nodes, and mucosal and associated lymphoid tissues (MALT), which coordinate lymphocyte trafficking and maintain immune tolerance. Tertiary lymphoid organs (TLOs) or tertiary lymphoid structures (TLSs) are highly organized lymphocyte aggregates that accompany chronic inflammation, persistent infection, autoimmune transplant rejection, and cancer. TLSs are characterized by (i) a unique T cell- and lymphocyte-rich area with a closed center similar to germinal centers that is rich in B cells; (ii) follicular dendritic cells (FDCs) and activated stromal mesenchymal cells (fibroblasts) (iii) plasmablasts and (iv) high endothelial venules (HEVs); blood vessels that facilitate the migration of naive lymphocytes to SLOs. TLSs express lymphoid-related chemokines and lymphotoxin (LT), contain germinal centers (GCs), which are sites of in situ B cell differentiation, somatic hypermutation, oligoclonal expansion, and antibody production.

[0017] Chemokines are small (7-12 kDa) chemotactic polypeptides that direct lymphocyte recruitment and organize the architecture of lymphoid organs. Chemokines CXCL12, CXCL13, CCL19, and CCL21 are constitutively expressed in lymphoid organs where they regulate lymphocyte migration, as well as the segregation of B and T cells into T cell zones and germinal centers. Lymphoid chemokines and LT cooperate with many cytokines to shape the cellular microenvironment during tertiary lymphoid neogenesis. These can include Th17-cytokines (IL-17, IL&22, IL-23), IL-21, IL-36 agonists, and cytokines of the IL-1 family, IL-1α, IL-1β, IL-18, and IL-33.

[0018] Cancer is antigenically distinct from foreign bacteria or viruses. While vaccines against viruses typically result in >5% antigen-specific CD8 + T cell activation and proliferation relative to total circulating CD8 T cells, vaccines against cancer antigens result in <1% antigen-specific CD8 + T cells. For example, yellow fever and smallpox vaccines stimulate activated antiviral CD8 +T cells were expanded to 12.5% and 40% of total peripheral CD8 T cells. In contrast, PROSTVAC-VF, a metastatic prostate cancer vaccine targeting PSA, induced antigen-specific T cell expansion to only approximately 0.03% of the total CD8 + T cell population and was discontinued from phase III due to lack of efficacy. The absolute T cell number and quality thresholds required for tumor control remain unclear and appear to depend on antigen type, T cell receptor (TCR) affinity, tumor type, and tumor microenvironment. Collectively, these data suggest that the quantity and quality of antigen-specific T cells must exceed a critical threshold to confer clinical benefit; the mere presence of peripheral antigen-specific T cells is insufficient to predict efficacy. SUMMARY OF THE INVENTION

[0019] The present disclosure relates to the field of cancer immunotherapy and provides a series of compositions and methods for treating cancer and benign tumors, including leiomyomas.

[0020] In one embodiment, the present disclosure provides a composition comprising autologous cancer cells from a subject, wherein the autologous cancer cells comprise: (a) a recombinant protein or peptide that induces an immune response, or an mRNA encoding the recombinant protein or peptide; (b) a recombinant protein or peptide that induces an inflammatory cell death response, or an mRNA encoding the recombinant protein or peptide; (c) a recombinant protein or peptide that induces the activation and persistence of antigen-presenting cells and cytotoxic T cells within the tumor microenvironment, or an mRNA encoding the recombinant protein or peptide; or (d) a recombinant protein or peptide that induces the formation of tumor-associated tertiary lymphoid structures, or an mRNA encoding the recombinant protein or peptide. In one aspect, the recombinant protein or peptide that induces an immune response is selected from: (a) SARS-Cov-2 spike (S); (b) SARS-Cov-2 envelope (E); (c) SARS-Cov-2 membrane (M); (d) SARS-Cov-2 nucleocapsid (N); (e) SARS-CoV-2 spike S-2P and RBD antigens; (f) SARS-Cov-2 ORF3a; (g) SARS-Cov-2 ORF7a; (h) SARS-Cov-2 ORF8; (i) SARS-Cov-2 replicase 1AB; (j) enhanced green fluorescent protein (EGFP); or (k) influenza hemagglutinin (HA).

[0021] In one aspect, the recombinant protein or peptide that induces an inflammatory cell death response is selected from: (a) Gasdermin D (GSDMD); (b) Gasdermin E (GSDME); (c) absent in melanoma 2 (AIM2); (d) interleukin 33 (IL33); (e) thioredoxin interacting protein (TXNIP); (f) interleukin-1 receptor-associated kinase 1 (IRAK1); or (g) NLR family pyrin domain-containing protein 3 (NLRP3).

[0022] In one aspect, the recombinant protein or peptide that induces the activation and persistence of antigen-presenting cells and cytotoxic T cells within the tumor microenvironment, and the recombinant protein or peptide that induces the formation of tumor-associated tertiary lymphoid structures is selected from: (a) chemokine ligand 9 (CXCL9); (b) chemokine ligand 10 (CXCL10); (c) chemokine ligand 12 (CXCL12); (d) chemokine ligand 13 (CXCL13); (e) C-C motif chemokine ligand 19 (CCL19); (f) C-C motif chemokine ligand 21 (CCL21); (g) interleukin-1β (IL1β); (h) interleukin-4 (IL4); (i) interleukin-4 receptor (IL4R); (j) interleukin-21 (IL21); (k) interleukin-22 (IL22); (l) interleukin-22 receptor (IL22R); (m) interleukin-23; (n) interleukin-13 (IL13); (o) lymphotoxin β receptor (LTβR); (p) β-2-microglobulin (B2M); (q) programmed death-ligand 1 (PD-L1); (r) integrin-associated protein (CD47); (s) transforming growth factor β receptor I (TGFβRI)-extracellular domain, soluble; (t) transforming growth factor β receptor II (TGFβRII)-extracellular domain, soluble; (u) transforming growth factor β receptor III (TGFβRIII)-extracellular domain, soluble; (v) a protein consisting of the extracellular domain of at least one protein selected from transforming growth factor β receptor I (TGFβRI), transforming growth factor β receptor II (TGFβRII), and transforming growth factor β receptor III (TGFβRIII); (w) mothers against decapentaplegic homolog 2 (SMAD2); or (x) mothers against decapentaplegic homolog 3 (SMAD3).

[0023] In one aspect, autologous cancer cells are contacted with a mixture of cytokines and chemokines. In one aspect, at least one of the cytokines is tumor necrosis factor α (TNFα) or interferon γ (IFNγ).

[0024] In one embodiment, the present disclosure provides a pharmaceutical composition formulated for delivery using lipid nanoparticles, electroporation, or other delivery mechanisms and comprising autologous cancer cells.

[0025] In another embodiment, the present disclosure provides a method for treating cancer and benign tumors (including leiomyomas) in a subject, comprising the above composition.

[0026] In a further embodiment, the present disclosure provides a composition comprising allogeneic cells, the allogeneic cells comprising: (a) a recombinant protein or peptide that induces an immune response, or an mRNA encoding the recombinant protein or peptide; (b) a recombinant protein or peptide that induces an inflammatory cell death response, or an mRNA encoding the recombinant protein or peptide; (c) a recombinant protein or peptide that induces the activation and persistence of antigen-presenting cells and cytotoxic T cells within the tumor microenvironment, or an mRNA encoding the recombinant protein or peptide; or (d) a recombinant protein or peptide that induces the formation of tumor-associated tertiary lymphoid structures, or an mRNA encoding the recombinant protein or peptide.

[0027] In one aspect, the recombinant protein or peptide that induces an immune response is selected from: (a) SARS-Cov-2 spike (S); (b) SARS-Cov-2 envelope (E); (c) SARS-Cov-2 membrane (M); (d) SARS-Cov-2 nucleocapsid (N); (e) SARS-CoV-2 spike S-2P and RBD antigens; (f) SARS-Cov-2 ORF3a; (g) SARS-Cov-2 ORF7a; (h) SARS-Cov-2 ORF8; (i) SARS-Cov-2 replicase 1AB; (j) enhanced green fluorescent protein (EGFP); or (k) influenza hemagglutinin (HA).

[0028] In one aspect, the recombinant protein or peptide that induces an inflammatory cell death response is selected from: (a) Gasdermin D (GSDMD); (b) Gasdermin E (GSDME); (c) absent in melanoma 2 (AIM2); (d) interleukin 33 (IL33); (e) thioredoxin-interacting protein (TXNIP); (f) interleukin 1 receptor-associated kinase 1 (IRAK1); or (g) NLR family pyrin domain-containing protein 3 (NLRP3).

[0029] In one aspect, the recombinant proteins or peptides that induce the activation and persistence of antigen-presenting cells and cytotoxic T cells within the tumor-inducing microenvironment, and the recombinant proteins or peptides that induce the formation of tumor-associated tertiary lymphoid structures are selected from one of the following proteins: (a) chemokine ligand 9 (CXCL9); (b) chemokine ligand 10 (CXCL10); (c) chemokine ligand 12 (CXCL12); (d) chemokine ligand 13 (CXCL13); (e) C-C motif chemokine ligand 19 (CCL19); (f) C-C motif chemokine ligand 21 (CCL21); (g) interleukin 1β (IL1β); (h) interleukin 4 (IL4); (i) interleukin 4 receptor (IL4R); (j) interleukin 21 (IL21); (k) interleukin 22 (IL22); (l) interleukin 22 receptor (IL22R); (m) interleukin 23; (n) interleukin 13 (IL13); (o) lymphotoxin β receptor (LTβR); (p) β-2-microglobulin (B2M); (q) programmed death-ligand 1 (PD-L1); (r) integrin-associated protein (CD47); (s) transforming growth factor β receptor I (TGFβRI)-extracellular domain, soluble; (t) transforming growth factor β receptor II (TGFβRII)-extracellular domain, soluble; (u) transforming growth factor β receptor III (TGFβRIII)-extracellular domain, soluble; (v) a protein consisting of the extracellular domain of at least one protein selected from transforming growth factor β receptor I (TGFβRI), transforming growth factor β receptor II (TGFβRII), and transforming growth factor β receptor III (TGFβRIII); (w) mothers against decapentaplegic homolog 2 (SMAD2); or (x) mothers against decapentaplegic homolog 3 (SMAD3).

[0030] In one aspect, allogeneic cells are contacted with autologous cancer cells from a subject, wherein the autologous cancer cells are first contacted with a mixture of cytokines and chemokines.

[0031] In one embodiment, the present disclosure provides a pharmaceutical composition formulated for delivery using lipid nanoparticles, electroporation, or other suitable delivery mechanisms, comprising the above allogeneic cells.

[0032] In another embodiment, the present disclosure provides a method for treating cancer and benign tumors (including leiomyomas) in a subject, comprising administering the above pharmaceutical composition to the subject.

[0033] In a further embodiment, the present disclosure provides a composition comprising a recombinant virus, the recombinant virus comprising: (a) a recombinant protein or peptide that induces an immune response, or an mRNA encoding the recombinant protein or peptide; (b) a recombinant protein or peptide that induces an inflammatory cell death response, or an mRNA encoding the recombinant protein or peptide; (c) a recombinant protein or peptide that induces the activation and persistence of antigen-presenting cells and cytotoxic T cells within the tumor microenvironment, or an mRNA encoding the recombinant protein or peptide; or (d) a recombinant protein or peptide that induces the formation of tumor-associated tertiary lymphoid structures, or an mRNA encoding the recombinant protein or peptide.

[0034] In one aspect, the recombinant nucleic acid encoding the recombinant protein or peptide that induces an immune response is selected from the genes encoding one of the following proteins: (a) SARS-Cov-2 spike (S); (b) SARS-Cov-2 envelope (E); (c) SARS-Cov-2 membrane (M); (d) SARS-Cov-2 nucleocapsid (N); (e) SARS-CoV-2 spike S-2P and RBD antigens; (f) SARS-Cov-2 ORF3a; (g) SARS-Cov-2 ORF7a; (h) SARS-Cov-2 ORF8; (i) SARS-Cov-2 replicase 1AB; (j) enhanced green fluorescent protein (EGFP); or (k) influenza hemagglutinin (HA).

[0035] In another aspect, the recombinant nucleic acid encoding the recombinant protein or peptide that induces an inflammatory cell death response is selected from the genes encoding one of the following proteins: (a) Gasdermin D (GSDMD); (b) Gasdermin E (GSDME); (c) absent in melanoma 2 (AIM2); (d) interleukin 33 (IL33); (e) thioredoxin-interacting protein (TXNIP); (f) interleukin-1 receptor-associated kinase 1 (IRAK1); or (g) NLR family pyrin domain-containing protein 3 (NLRP3).

[0036] In a further aspect, the recombinant proteins or peptides that induce activation and persistence of antigen-presenting cells and cytotoxic T cells within the tumor-inducing microenvironment, and that induce formation of tumor-associated tertiary lymphoid structures, are selected from one of the following proteins: (a) chemokine ligand 9 (CXCL9); (b) chemokine ligand 10 (CXCL10); (c) chemokine ligand 12 (CXCL12); (d) chemokine ligand 13 (CXCL13); (e) C-C motif chemokine ligand 19 (CCL19); (f) C-C motif chemokine ligand 21 (CCL21); (g) interleukin 1β (IL1β); (h) interleukin 4 (IL4); (i) interleukin 4 receptor (IL4R); (j) interleukin 21 (IL21); (k) interleukin 22 (IL22); (l) interleukin 22 receptor (IL22R); (m) interleukin 23; (n) interleukin 13 (IL13); (o) lymphotoxin β receptor (LTβR); (p) β-2-microglobulin (B2M); (q) programmed death-ligand 1 (PD-L1); (r) integrin-associated protein (CD47); (s) transforming growth factor β receptor I (TGFβRI)-extracellular domain, soluble; (t) transforming growth factor β receptor II (TGFβRII)-extracellular domain, soluble; (u) transforming growth factor β receptor III (TGFβRIII)-extracellular domain, soluble; (v) a protein consisting of the extracellular domain of at least one protein selected from transforming growth factor β receptor I (TGFβRI), transforming growth factor β receptor II (TGFβRII), and transforming growth factor β receptor III (TGFβRIII); (w) mothers against decapentaplegic homolog 2 (SMAD2); or (x) mothers against decapentaplegic homolog 3 (SMAD3).

[0037] In one aspect, the present disclosure provides a pharmaceutical composition formulated for delivery using lipid nanoparticles, electroporation, or other suitable delivery mechanisms, comprising the recombinant virus or delivery system described above.

[0038] In another aspect, the present disclosure provides a method for treating cancer and benign tumors (including leiomyomas) in a subject, comprising administering the pharmaceutical composition described above to the subject.

[0039] In another aspect, the present disclosure provides a composition comprising allogeneic T cells, wherein the allogeneic T cells comprise: (a) a chimeric antigen receptor (CAR) or an antigen-specific T cell receptor (TCR) that confers antigen specificity to the T cells; (b) a recombinant protein or peptide, or an mRNA encoding the recombinant protein or peptide, that induces activation and persistence of antigen-presenting cells and cytotoxic T cells within the tumor microenvironment; (c) a recombinant protein or peptide, or an mRNA encoding the recombinant protein or peptide, that induces the formation of tumor-associated tertiary lymphoid structures.

[0040] In still a further aspect, the chimeric antigen receptor (CAR) or the antigen-specific T cell receptor (TCR) is specific for one of the following proteins: (a) SARS-Cov-2 spike (S); (b) SARS-Cov-2 envelope (E); (c) SARS-Cov-2 membrane (M); (d) SARS-Cov-2 nucleocapsid (N); (e) SARS-CoV-2 spike S-2P and RBD antigens; (f) SARS-Cov-2 ORF3a; (g) SARS-Cov-2 ORF7a; (h) SARS-Cov-2 ORF8; (i) SARS-Cov-2 replicase 1AB; (j) enhanced green fluorescent protein (EGFP) or (k) influenza hemagglutinin (HA).

[0041] In one aspect, the recombinant protein or peptide that activates and persists in the tumor microenvironment for the induced antigen-presenting cells and cytotoxic T cells, and the recombinant protein or peptide that induces the formation of tumor-associated tertiary lymphoid structures are selected from one of the following proteins: (a) chemokine ligand 9 (CXCL9); (b) chemokine ligand 10 (CXCL10); (c) chemokine ligand 12 (CXCL12); (d) chemokine ligand 13 (CXCL13); (e) C-C motif chemokine ligand 19 (CCL19); (f) C-C motif chemokine ligand 21 (CCL21); (g) interleukin 1β (IL1β); (h) interleukin 4 (IL4); (i) interleukin 4 receptor (IL4R); (j) interleukin 21 (IL21); (k) interleukin 22 (IL22); (l) interleukin 22 receptor (IL22R); (m) interleukin 23; (n) interleukin 13 (IL13); (o) lymphotoxin β receptor (LTβR); (p) β-2-microglobulin (B2M); (q) programmed death-ligand 1 (PD-L1); (r) integrin-associated protein (CD47); (s) transforming growth factor β receptor I (TGFβRI)-extracellular domain, soluble; (t) transforming growth factor β receptor II (TGFβRII)-extracellular domain, soluble; (u) transforming growth factor β receptor III (TGFβRIII)-extracellular domain, soluble; (v) a protein consisting of the extracellular domain of at least one protein selected from transforming growth factor β receptor I (TGFβRI), transforming growth factor β receptor II (TGFβRII), and transforming growth factor β receptor III (TGFβRIII); (w) mothers against decapentaplegic homolog 2 (SMAD2); or (x) mothers against decapentaplegic homolog 3 (SMAD3).

[0042] In one aspect, the present disclosure provides a pharmaceutical composition adapted to be delivered to a subject using lipid nanoparticles, electroporation, or other suitable delivery mechanisms, comprising the above-mentioned allogeneic cells.

[0043] In another aspect, the present disclosure provides a method for treating cancer and benign tumors (including leiomyomas) in a subject, comprising administering the pharmaceutical composition to the subject.

[0044] In another aspect, the present disclosure provides a composition comprising recombinant DNA, mRNA, lipid nanoparticles, or an electroporation system, wherein the recombinant DNA or mRNA encodes a recombinant protein or peptide that induces an immune response.

[0045] In one aspect, the recombinant nucleic acid encoding a recombinant protein or peptide that induces an immune response is selected from genes encoding one of the following proteins: (a) SARS-Cov-2 spike (S); (b) SARS-Cov-2 envelope (E); (c) SARS-Cov-2 membrane (M); (d) SARS-Cov-2 nucleocapsid (N); (e) SARS-CoV-2 spike S-2P and RBD antigens; (f) SARS-Cov-2 ORF3a; (g) SARS-Cov-2 ORF7a; (h) SARS-Cov-2 ORF8; (i) SARS-Cov-2 replicase 1AB; (j) enhanced green fluorescent protein (EGFP) or (k) influenza hemagglutinin (HA).

[0046] In another aspect, the present disclosure provides a pharmaceutical composition for delivery to a target subject using lipid nanoparticles, electroporation, or other delivery mechanisms, comprising the above recombinant DNA, mRNA, or delivery system.

[0047] In a further aspect, the present disclosure provides a method for treating cancer and benign tumors (including leiomyomas) in a subject, comprising administering the above pharmaceutical composition to the subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1A is a schematic diagram showing the process and design of generating PyroCells TM for adoptive cell immunotherapy.

[0049] Figure 1B is a schematic diagram illustrating the process and design of generating Allo-PyroCell TM for adoptive immunotherapy.

[0050] Figure 1C is a schematic diagram illustrating the process and design of generating PyroVir TM for viral immunotherapy.

[0051] Figure 2 is a schematic diagram (right) illustrating the challenges associated with therapeutic cancer vaccines and how the PyroVax TM vector encoding PyroHIM TM 、PyroACT TM and PyroTIME TM can be used to overcome some of these challenges.

[0052] Figure 3 is a schematic diagram showing the PyroVax TM components PyroHIM TM 、PyroACT TM and PyroTIME in a non-viral expression plasmidTM Schematic diagram, including PyroVax in a lentiviral vector TM .

[0053] Figure 4 Illustrates PyroVax in a non-viral expression plasmid TM Component PyroHIM TM , PyroACT TM and PyroTIME TM Schematic diagram, including PyroVax in an AAV vector TM .

[0054] Figure 5 Illustrates PyroVax in a non-viral expression plasmid TM Component PyroHIM TM , PyroACT TM and PyroTIME TM Schematic diagram, including PyroVax in an AAV vector TM .

[0055] Figure 6 Illustrates PyroVax in a non-viral expression plasmid TM Component PyroHIM TM , PyroACT TM and PyroTIME TM Schematic diagram, including the backbone

[0056] Figure 7A Is a figure showing a contour plot illustrating the expression of an indicator protein in human TNBC cells transduced with a lentiviral vector encoding the indicator gene

[0057] Figure 7B Shows from Figure 7A Quantification of the data in a curve

[0058] Figure 7C Is a set of graphs showing contour plots of the expression levels of an indicator construct using non-viral methods

[0059] Figure 7D Is a set of graphs showing a flow diagram of the tetramer-positive CD8 + T cells present in the donor blood described on the left

[0060] Figure 7E Is a graph describing the certificate of analysis highlighting key information about the donor from whom the Figure 7D and Figure 7E T cells shown in were isolated

[0061] Figure 7FA set of figures illustrating the IFNγ assay (upper) and cytotoxicity assay (lower) of cellular responses to peptides encoding T cell-specific reactive peptides.

[0062] Figure 8A A set of bioluminescence images showing PyroCells over time TM immunological rejection. By three weeks, no detectable PyroCells TM were present (left). On day 22, vaccinated mice were challenged with parental tumor (red) or without parental tumor (blue) and compared to unvaccinated controls that also received the same parental tumor. When vaccinated mice were protected from tumor challenge, all unvaccinated mice died of disease and expired. Day 28 was 7 days after tumor challenge.

[0063] Figure 8B is a line graph quantifying Figure 8A the bioluminescence signal in. Data are represented as mean and standard deviation.

[0064] Figure 9A and Figure 9B are schematic diagrams of the PyroTIMER construct highlighting the fusion of the extracellular domains of TGFβRI and TGFβRII integrated into Jurkat T cells.

[0065] Figure 9C A set of figures illustrating a co-culture killing assay using PyroTIMER CD19 CAR-T cells and Raji lymphoma cells expressing the CD19 antigen. Increased cytotoxicity of PyroTIMER CD19 CAR-T cells in the presence of 100 pM TGF-β1 compared to the control group is shown.

[0066] Figure 9D A set of graphs showing the durable stability assay of PyroTIMER and CD19 CAR expression over time in Jurkat T cells after transduction.

[0067] Figure 9E is a diagram of the effector-to-target ratio used in the co-culture killing assay highlighting the superior killing efficacy of PyroTIMER CD19 CAR-T cells at all ratios.

[0068] Figure 10A A set of figures illustrating the generation and functional characterization of PyroTIMER CD19 CAR-T cells using primary human CD3 + T cells, including a contour flow diagram showing the purity of CD3 + T cell enrichment from human donor PBMCs.

[0069] Figure 10B is a quantified curve graph showing Figure 10A .

[0070] Figure 10C is a set of graphs showing the contour plots of the frequencies of CD4 + (upper) and CD8 + (lower) PyroTIMER CD19 CAR-T cells after enrichment by cell sorting.

[0071] Figure 10D is a set of graphs showing the contour flow plots of the frequencies of CD4 + in CD3 + T cells and CD8 + .

[0072] Figure 10E is a quantified curve graph showing Figure 10D .

[0073] Figure 10F is a set of graphs showing the contour flow plots of the high-efficiency generation of CD19 CAR-T cells after chemical selection (puromycin) and cell sorting (RFP).

[0074] Figure 10G is a graph showing the number of viable CD19-expressing Raji lymphoma cells after co-culturing for 24 h at a specified effector-to-target (E:T) ratio with CD19 CAR-T cells (CAR) or PyroTIMER CD19 CAR-T cells (PyroTIMER CAR) in the presence of TGFβ1.

[0075] Figure 10H is a graph showing Figure 10G the percentage of CD69 + activated T cells in. Untransduced T cells were used as controls. The experiment was repeated twice and performed in triplicate. Data are percentages of the total, expressed as mean + / − S.E.M. Unpaired two-tailed student t-tests were used to compare killing (in G) and CD69+ cells (in H) in any co-culture (CAR vs PyroTIMER CAR) at a specific E:T ratio. p < 0.05 was considered statistically significant.

[0076] Figure 11A and Figure 11Bis a set of images and graphs that illustrate the results of in vivo studies using NOD.Cg-PrkdcscidIL2Rgtm1Wjl / Sz mice, which illustrate the anti-CD19+ Raji lymphoma disease burden of PyroTIMER CAR-T cells. Comparative tumor growth curves and survival rates between mice treated with PyroTIMER CAR-T cells and traditional CD19 CAR-T cells are shown. Detailed Description

[0077] The present disclosure relates to the field of cancer immunotherapy and provides a series of compositions and methods for treating cancer and benign tumors, including leiomyomas. The present disclosure exploits the therapeutic potential of autologous and allogeneic cells, particularly cancer cells and T cells, which have been genetically modified or contacted with specific recombinant proteins or nucleic acids encoding recombinant proteins or peptides. These modified cells are prepared to elicit various immune and inflammatory responses in the tumor microenvironment, thereby enhancing the body's natural ability to recognize and destroy tumor cells.

[0078] Before describing the compositions and methods of the present invention, it is to be understood that the invention is not limited to the specific compositions, methods, and experimental conditions described, as such compositions, methods, and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the invention will be defined only in the appended claims.

[0079] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "the method" includes one or more methods of the type described herein, which will become apparent to those skilled in the art upon reading the present disclosure.

[0080] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0081] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, it is to be understood that modifications and variations are encompassed within the spirit and scope of the present disclosure. Preferred methods and materials are now described.

[0082] The present disclosure encompasses autologous cancer cells that are modified to express an entity or are in contact with an entity that induces an immune response, pyroptosis, activation and persistence of antigen-presenting cells and cytotoxic T cells within the tumor microenvironment, and formation of tumor-associated tertiary lymphoid structures. Such autologous cells include cells in which the immune response is triggered by proteins produced by viruses such as SARS-CoV-2 or other entities such as EGFP and influenza hemagglutinin. The autologous cells of the present disclosure also include those that induce pyroptosis by targeting or inhibiting proteins such as Gasdermins, AIM2, and IL33. Other autologous cells include those that enhance cells using proteins or inhibitors such as CXCL9, CCL21, IL1β, and PD-L1 to support the activation of immune cells and the formation of lymphoid structures in the tumor environment.

[0083] Allogeneic cells, including T cells, are modified similarly to autologous cells, allowing for broader applicability in therapy. Recombinant viruses are engineered to deliver the same therapeutic entities directly to the tumor. Recombinant DNAs, mRNAs, lipid nanoparticles, or electroporation systems encoding specific proteins or peptides, particularly those that induce an immune response, are described in the present disclosure. Additionally, the present disclosure provides pharmaceutical compositions suitable for delivery to a subject using mechanisms such as lipid nanoparticles and electroporation, thereby ensuring efficient uptake and functionality of the therapeutic entity. Finally, the present disclosure provides methods of treating malignant and benign tumors in a subject using the compositions. Overall, the present disclosure provides innovative, targeted, and personalized treatment methods in the fight against cancer by harnessing the power and specificity of the body's immune system.

[0084] Immune cells constantly survey the body for pathogens such as bacteria and viruses, or normal cells that may be becoming cancerous, and kill them. However, in immunocompromised subjects, the heterogeneity of tumor cells allows subsets of cells to evade detection and killing by immune cells and establish disease. These immune-evasive tumor cells either do not present antigens vulnerable to immune cell detection or completely stop antigen presentation. Since these cancer cells express most of the proteins and antigens expressed by normal cells, the immune system recognizes the cells as "self" and becomes "tolerant" to the cancer cells. These cancer cells disguise themselves as "self", avoid expressing vulnerable antigens, and secrete immunosuppressive molecules into the microenvironment, which all impair the ability of immune cells to infiltrate the tumor and kill the cancer cells. This immunosuppressive tumor immune microenvironment recruits immunosuppressive cells, turning the TIME into an immune-privileged site that allows the tumor to grow uncontrollably.

[0085] The present disclosure describes a novel autologous and allogeneic cellular immunotherapy, as well as a viral immunotherapy, which breaks central and peripheral immune tolerance against tumor antigens, enhances the immunogenicity of cancer cells, and remodels the immunosuppressive TIME. The method emphasizes presenting the entire available repertoire of a subject's tumor-specific antigens to activate an anti-tumor immune response against a broad repertoire of cancer-specific antigens. Notably, this method redirects the existing antiviral immune cell repertoire to treat cancer. This method is important because subjects with cancer are generally immunosuppressed and do not contain a robust naïve immune cell repertoire to generate a significant anti-tumor immune response.

[0086] The term "isolated" means that a cell or cells, or a peptide or nucleic acid molecule, have been removed from their normal physiological environment (e.g., natural source), or the peptide or nucleic acid has been synthesized. The use of the term "isolated" indicates that a naturally occurring sequence has been removed from its normal cellular (i.e., chromosomal) environment. Thus, the sequence can be in a cell-free solution or placed in a different cellular environment. One or more isolated cells can, for example, be contained in a medium different from that initially provided, such as an aqueous solution, or placed in a different physiological environment. Generally, an isolated cell, peptide, or nucleic acid molecule constitutes a higher portion of the total cell, peptide, or nucleic acid molecule present in their environment, e.g., in a solution / suspension when applicable.

[0087] As used herein, the terms "treatment" and "therapy" refer to prophylactic or preventive measures that have a therapeutic effect and slow down, or at least partially alleviate or eliminate, an undesirable condition in the organism of a subject. Those in need of treatment include those who already have the condition, as well as those who are predisposed to having the condition or are to be protected (guarded) against the condition. Generally, treatment reduces, stabilizes, or inhibits the progression of symptoms associated with the presence and / or progression of an undesirable condition. The term "therapeutic effect" refers to inhibiting or activating a factor that causes or contributes to an undesirable condition. The therapeutic effect alleviates, to some extent, one or more symptoms of an undesirable condition or disease. The term "undesirable condition" refers to a function in a cell or tissue of an organism that deviates from the optimal function in that organism.

[0088] As used herein, the term "administer" refers to any manner of giving a treatment to a patient or subject. "Administer" means transferring, delivering, introducing, or transporting a substance (such as a compound, e.g., a pharmaceutical compound, or other reagent, such as an antisense oligonucleotide) to a subject in any manner. Administration can be accomplished by local, intravenous, intramuscular, systemic, oral, or parenteral methods.

[0089] The present disclosure provides an autologous cellular immunotherapy, namely PyroCells TMCompositions and methods for developing autologous cellular immunotherapies. These consist of patient-derived (autologous) tumor cells that are modified ex vivo to express PyroVax TM vector and chemically modified with PyroStim TM When re-administered to the same patient, PyroCells TM activate an immune response against a broad repertoire of tumor-specific antigens, resulting in long-term recurrence-free survival in patients with advanced solid and hematological cancers.

[0090] Another embodiment of the present disclosure provides compositions and methods for developing allogeneic cellular immunotherapies, namely, Allo-PyroCell TM , which is generated by combining autologous patient tumor-derived cells treated ex vivo with PyroStim TM with allogeneic cells (normal or transformed) that have been modified ex vivo to express the PyroVax TM vector. Allo-PyroCell TM activates a polyclonal immune response against a broad repertoire of tumor-specific antigens, conferring long-term recurrence-free survival. Notably, this allogeneic approach overcomes several manufacturing, technical, and cost challenges associated with the autologous approach.

[0091] Another embodiment of the present disclosure provides compositions for viral immunotherapy and methods for developing viral immunotherapy, namely PyroVir TM . The virus is delivered intratumorally and introduces components of PyroVax TM such as PyroHIM TM , PyroAct TM , PyroTIME TM , and PyroStim TM into the tumor to activate a polyclonal immune response against multiple tumor-specific antigens, thereby conferring long-term recurrence-free survival.

[0092] The following section summarizes the three main components of the PyroVax TM vector, namely PyroHIM TM , PyroAct TM , and PyroTIME TM , as well as the composition of the chemicals with associated dosages that make up PyroStim TM . It also summarizes the methods of using these components to generate PyroCells TM , Allo-PyroCells TM , and PyroVir TM .

[0093] The present disclosure provides compositions for generating nucleotide sequences of the PyroVax TM vector. The PyroVax TM vector is composed of nucleic acids encoding three essential ORFs, open reading frames, and various other regulatory components. These include: i) PyroHIM TM , a highly immunogenic molecule expressed at supra-physiological levels, which is non-self and has high antigenic potential; ii) PyroAct TM , an activator of inflammatory immunogenic cell death; and iii) PyroTIME TM , a remodeller of the immunosuppressive TIME tumor immune microenvironment. These components of PyroVax TM can be operably linked together or not in various arrangements on the same or different vectors and under the control of the same or different promoters.

[0094] Table 1 shows the PyroVax TM components:

[0095]

[0096]

[0097] The present disclosure describes a method of manufacturing an autologous cell immunotherapy (PyroCell TM ), comprising:

[0098] a) Harvesting surgically resected tumor tissue in a sterile container and transporting it to PyroLabs TM .

[0099] b) Mechanically and chemically dissociating the tumor into a single cell suspension.

[0100] c) Enriching tumor cells using antibody-mediated negative selection.

[0101] d) Electroporation or viral transduction of autologous tumor cells to introduce the PyroVax TM vector.

[0102] e) Chemical selection to enrich PyroCells TM expressing PyroVax TM .

[0103] f) Enhancing antigen presentation with PyroStim TM .

[0104] g) Treating a subject or freezing the immunotherapy at -80 °C for 5 years.

[0105] The present disclosure describes a method of manufacturing a combination of autologous and allogeneic cellular immunotherapies (Allo-PyroCell TM ), comprising:

[0106] a) Harvesting surgically resected autologous tumor tissue in a sterile container.

[0107] b) Mechanically and chemically dissociating the tumor into a single-cell suspension.

[0108] c) Enriching tumor cells using antibody-mediated negative selection.

[0109] d) Stimulating antigen doses, and presenting via autologous tumor cells, using a combination dose of PyroStim TM , IFNγ, and TNFα to stimulate MHC-I and MHC-II-dependent antigen presentation.

[0110] h) Mixing autologous tumor cells from (4) above with allogeneic cells that have been previously electroporated or virus-transduced to introduce the PyroVax TM vector.

[0111] i) Freezing the immunotherapy at -80 °C.

[0112] The present disclosure describes a method of preparing a cancer-specific viral immunotherapy (PyroVir TM ), comprising:

[0113] a) Generating AAV and / or tumor-specific virus (HSV) containing the PyroVax TM vector for intratumoral delivery.

[0114] b) Chemically selecting genetically modified viruses expressing PyroVax TM capable of infecting and proliferating in cancer cells in vivo.

[0115] c) Freezing the immunotherapy at -80 °C.

[0116] Central and peripheral immune tolerance limit anti-tumor immunity

[0117] The present disclosure describes a method of redirecting an immune response generated in a prophylactic setting to cancer cells in a therapeutic setting. Specifically, the present disclosure describes genetically and chemically modified compositions and methods for generating autologous or allogeneic cellular immunotherapies that redirect immune cells that detect SARS-Cov-2 antigens and control viral infection to detect cancer antigens and kill cancer cells.

[0118] This method takes advantage of the fact that most people have been and will be exposed to, infected with, recovered from, or vaccinated against SARS-Cov-2. Therefore, the available pool of antiviral immune cells includes CD8 + T cells, CD4 + T cells, and B cells, which can be redirected to detect cancer antigens and kill cancer cells.

[0119] Heterogeneity allows immune escape of tumor cells

[0120] The present disclosure describes a method of presenting the entire available subject-specific neoantigen repertoire by using intact tumor cells from a subject. Autologous or allogeneic cells are modified ex vivo such that when introduced into the subject, these cell immunotherapies stimulate an immune response or redirect existing immune reservoirs to detect cancer-specific antigens and kill cancer cells.

[0121] In other embodiments of the present disclosure, the cells genetically modified to produce cell immunotherapies can be autologous, an expansion of cells from a xenograft of autologous tumor cells, allogeneic tumor cells, allogeneic tumor cells expanded in a xenograft, or a combination of one or more thereof.

[0122] In other embodiments of the present disclosure, allogeneic cells are established from normal or transformed cell lines, or normal fibroblasts, or endothelial cells, or immortalized cell lines.

[0123] Lack of immunogenicity of tumors

[0124] The present disclosure describes a method of significantly increasing tumor immunogenicity by introducing PyroHIM TM , a highly immunogenic molecule.

[0125] In one aspect, HIM includes full-length proteins or immunogenic regions selected from proteins including but not limited to enhanced green fluorescent protein (eGFP), full-length proteins or peptide derivatives of SARS-CoV-2 (accession number NC_045512.2) structural proteins, including spike glycoprotein (S), envelope protein (E), membrane protein (M), and nucleocapsid phosphoprotein (N), etc. (see Table 1).

[0126] Patients with cancer are immunocompromised for various reasons (1) and do not have a robust reservoir of available immune cells to control aggressive solid human cancers. However, cancer patients who have recovered from SARS-CoV-2 infection or have been vaccinated with any approved anti-SARS-CoV-2 vaccine contain a powerful reservoir of SARS-CoV-2 antigen-specific immune cells. The disclosure herein redirects or reorients the immune response against SARS-CoV-2 to kill cancer cells.

[0127] In one embodiment of PyroHIM TM a full-length protein or immunogenic peptide produced by the SARS-Cov-2 virus is encoded as PyroHIMs TM in the PyroVax TM construct, and the PyroVax TM construct is operably linked to an activator of pyroptotic cell death (PyroAct TM ) and a remodeler of the tumor immune microenvironment at that time (PyroTIME TM ).

[0128] In various aspects, PyroHIMs TM encode full-length proteins or peptides of SARS-CoV-2 (accession number NC_045512.2) structural proteins, including spike glycoprotein (S), envelope protein (E), membrane protein (M), and nucleocapsid phosphoprotein (N). Notably, immunogenic peptides are used alone or in combination with other peptide fragments with or without linkers to enhance immunogenicity.

[0129] In one aspect of the present disclosure, epitopes used by Moderna and Pfizer and other vaccine manufacturers for the development of FDA-approved vaccinations are used. These include the SARS-CoV-2 spike S-2P antigen and the SARS-CoV-2 RBD antigen.

[0130] In one aspect of the present disclosure, the SARS-CoV-2 antigens ORF3a and envelope protein (E) are used to activate pyroptosis, and the SARS-CoV-2 antigens ORF3a and envelope protein (E) are direct inducers of pyroptosis.

[0131] PyroVax TM vectors containing SARS-Cov-2-specific epitopes are used as PyroHIMs TM of PyroCells TM to redirect the immune response generated against SARS-Cov-2 to treat cancer. PyroCells TMCellular immunotherapy is effective in both preventive and therapeutic settings, capable of controlling the disease and significantly prolonging the overall survival.

[0132] PyroCells containing the PyroVax TM vector encoding SARS-Cov-2 specific epitopes as PyroHIMs TM activate the pre-existing antiviral cytotoxic CD8 TM and CD4 + T cell repertoire and redirect it against cancer. In one aspect, the PyroHIM + epitopes are linked to a reporter molecule to enable supra-physiological expression, detection, and enrichment of autologous or allogeneic PyroCells TM expressing the PyroVax TM vector. TM

[0133] PyroCells containing the PyroVax TM vector encoding SARS-Cov-2 specific epitopes as PyroHIMs TM are operably linked together or not, originating from the most prevalent reactive T and B cell epitopes in people exposed to SARS-Cov-2 or vaccinated with SARS-Cov-2 vaccines. TM

[0134] EGFP, enhanced green fluorescent protein

[0135] AAB02572:239aa, Accession No.: AAB02572, Version: AAB02572.1 (SEQ ID NO:1)

[0136] 1mvskgeelftgvvpilveldgdvnghkfsvsgegegdatygkltlkficttgklpvpwpt

[0137] 61lvttltygvqcfsrypdhmkqhdffksampegyvqertiffkddgnyktraevkfegdtl

[0138] 121vnrielkgidfkedgnilghkleynynshnvyimadkqkngikvnfkirhniedgsvqla

[0139] 181dhyqqntpigdgpvllpdnhylstqsalskdpnekrdhmvllefvtaagitlgmdelyk

[0140] In PyroHIM TM In one embodiment, a full-length protein or immunogenic peptide such as EGFP or a portion thereof is encoded as an antigen in the PyroVax TM construct and is operably linked to an inflammasome activator (PyroAct TM ) and a TIME tumor immune microenvironment remodeler (PyroTIME TM ).

[0141] PyroCells TM The cellular immunotherapy contains supra-physiological concentrations of EGFP as PyroHIM TM and stimulates an immune response against the immunodominant antigen GFP, particularly against several less dominant cancer cell-specific antigens. This results in the activation of a broad repertoire of cancer antigen-specific T cells that recognize different sets of tumor antigens and control established heterogeneous diseases. GFP is a model non-self antigen used to test the utility of PyroHIMs TM in PyroVax TM since mature in vitro and in vivo tools and model systems using GFP as an antigen exist and are well validated (4, 5).

[0142] Immunosuppressive molecules and cells in the tumor immune microenvironment

[0143] PyroTIME TM is a component of the PyroVax TM vector that encodes molecules that remodel the immunosuppressive TIME by promoting tumor antigen presentation by APCs and killing by cytotoxic immune cells. PyroTIME TM acts by inhibiting immunosuppressive molecules and cells and promoting an environment that gives rise to TS-TLS (tumor-specific tertiary lymphoid structures). Specifically, PyroTIME TM inactivates the expression of specific genes and / or increases the expression of specific cytokines, chemokines, and soluble receptor antagonists that promote the recruitment of T cells (organized into T cell zones), B cells (organized into germinal centers), and follicular dendritic cells, thereby giving rise to tumor-specific tertiary lymphoid structures.

[0144] The present disclosure describes compositions and methods for remodeling immunosuppressive TIME by inhibiting the levels of the immunosuppressive cytokine TGF-β in the TIME. PyroTIME TM encodes competitive antagonists of TGFβ, including soluble TGF-β receptors (TGFβRII or TGFβRIII) or dominant negative receptors.

[0145] In another aspect, PyroTIME TM encodes guide RNAs that inactivate genes for specific TGFβ signaling, said specific TGFβ signaling genes including SMAD2 / 3, TGFβRII, TGFβRIII, etc. (see Table 1, PyroTIME TM ). In one embodiment of the present disclosure, the gRNA (crRNA + trRNA tracer RNA) and Cas9 are complexed as an RNP and electroporated or nucleofected into host cells. In another embodiment of the present disclosure, the gRNA and Cas9 are encoded in an expression vector.

[0146] In another aspect, PyroTIME TM consists of a nucleotide sequence encoding a gene that expresses a soluble or dominant-negative TGFβRII and / or TGFβRIII to inhibit TGFβ levels.

[0147] Another embodiment of the present disclosure describes compositions and methods for using PyroTIME TM to reshape the immunosuppressive TIME to promote increased intratumoral infiltration and persistence of immune cells. Specifically, the present disclosure describes PyroTIME TM and compositions of DNA sequences encoding the molecules CXCL13 and CCL19, said molecules CXCL13 and CCL19 promoting the recruitment of B cells, dendritic cells, cytotoxic T cells, the formation of germinal centers, T cell zones, and ultimately tertiary lymphoid structures containing follicular dendritic cells.

[0148] The present disclosure describes PyroTIME TM , a component of PyroVax TM that reshapes the immunosuppressive TIME and promotes the formation of tumor-specific TLS, a tertiary lymphoid structure for persistent anti-tumor immune surveillance.

[0149] PyroTIME TM encodes molecules important for the recruitment and maturation of dendritic cells, particularly follicular dendritic cells, said dendritic cells having a significant potential for cross-presenting tumor antigens. These include, but are not limited to, any of the following used alone or in combination with one or more additional molecules: CXCL13, CXCL12, CCL19, CCL21, soluble TGFβRI / II / III, IL-13, IL-4, IL-4R, IL-21, IL-22, IL-22R, IL-23. Molecules inactivated to promote antigen presentation by APCs and tumor cell uptake include CD274, CD47, TGFβR, SMAD2 / 3, etc. (see Table 1).

[0150] In another embodiment of the present disclosure, PyroTIMETM The construct is expressed in normal (fibroblasts or stromal cells), and / or transformed cells to generate PyroTIMECells TM . These stromal fibroblast-like cells are mixed with tumor cells or tumor lysates to generate tumor-specific TLS, TS-TLS in vivo. Notably, PyroTIMECells TM generate a sustained anti-tumor immune response against a broad variety of tumor-specific antigens, conferring significant anti-tumor immunity

[0151] In one aspect of the present disclosure, PyroTIMECells TM are developed from normal cells derived from autologous or allogeneic settings, while in another aspect, are developed from transformed cells derived from autologous or allogeneic settings.

[0152] The present disclosure describes compositions and methods of using PyroStim TM chemicals that stimulate antigen presentation and type I and type II interferon signaling in tumor cells.

[0153] The present disclosure describes compositions and methods of stimulating antigen presentation in tumor cells by expressing B2M, the beta-2-microglobulin gene, using PyroStim TM .

[0154] Limited diversity of anti-tumor immune cells

[0155] The present disclosure describes compositions and methods of activating inflammatory tumor cell death to elicit an endogenous T cell response to antigens that are activated and diversified to non-cross-reactive epitopes selected from the same antigen (intramolecular spreading) or other antigens (intermolecular spreading), using PyroAct TM .

[0156] In one aspect of the present disclosure, the activation signal of PyroAct TM is functionally linked to PyroHIM TM such that the antigens encoded in PyroHIM TM stimulate an immune response that drives selective inflammatory cell death. This antigen-restricted pyroptosis activation method allows for targeted activation of epitopes spreading in tumors.

[0157] The present disclosure describes compositions and methods of developing autologous or allogeneic PyroCell TM that redirect the existing immune repertoire previously generated against SARS-CoV-2 spike S-2P and RBD antigens or influenza antigens in vaccinated patients or patients recovering from disease to treat cancer.

[0158] The attenuation of live vaccines negatively affects antigen presentation

[0159] The present disclosure describes compositions and methods of developing live cancer vaccines containing the construct PyroKill TM that allow killing of any injected tumor cells not rejected by the immune system.

[0160] The attenuation of live vaccines negatively affects cytokine production

[0161] The present disclosure describes compositions and methods of developing live cancer vaccines containing the construct PyroKill TM that allow killing of any injected tumor cells not rejected by the immune system.

[0162] Limited expansion of autologous cells

[0163] Although whole cell vaccines have been shown to have clinical activity, manufacturing this type of vaccine requires surgical removal of the patient's tumor and ex vivo processing of the cells. It is often difficult to obtain sufficient cells for therapy, and it may take one to several weeks.

[0164] The present disclosure describes a composition and method for developing Allo-PyroCells TM composed of a mixture of autologous tumor cells derived from a patient and allogeneic cell lines (Allo) expressing PyroHIM TM , PyroAct TM and PyroTIME TM . The autologous cells are treated with PyroStim TM before mixing with Allo-PyroCells TM .

[0165] In one aspect of the present disclosure, the source of Allo-PyroCells TM will be tumor cells derived from allogeneic tumor cells, allogeneic tumor cells expanded in xenografts, normal cells including fibroblasts, macrophages or dendritic cells, or a combination of one or more. In another aspect, B2M-deficient Allo-PyroCells TM will be generated and used to avoid cross-reactivity with allogeneic epitopes.

[0166] Limited editing efficiency of autologous cells

[0167] The present disclosure describes a composition and method for developing Allo-PyroCells composed of autologous tumor cells (Auto) derived from a patient and expressing PyroHIM TM , PyroAct TM and PyroTIME TMAuto-Allo-PyroCell TM .

[0168] Detailed timeline for creating personalized vaccines

[0169] Vaccine manufacturing timelines are long, stretching to approximately 4 months, and rely on computationally derived predictions of TAAs. Advanced and metastatic human cancers are actively growing, with no time horizon and poor predictive power.

[0170] The present disclosure describes the generation of PyroCells in 2 weeks TM method.

[0171] The present disclosure describes the generation of Allo-PyroCells in 1 week TM method.

[0172] In vitro culture related modifications

[0173] The present disclosure describes a composition and method to develop PyroVir TM , which is the expression of PyroHIM TM 、PyroAct TM 、PyroTIME TM and PyroStim TM Intratumoral delivery of viral constructs avoids the need for ex vivo modification.

[0174] Two oncolytic HSV2 vectors were developed from the HG52 (6) strain. Modifications included deletion of the ICP47 and ICP34.5 genes and expression of PyroHIM TM 、PyroAct TM 、PyroTIME TM and PyroStim TM Insertion of the GFP expression cassette.

[0175] Patients with advanced cancer are often immunocompromised

[0176] Patients with advanced solid human cancers do not contain a robust reservoir of cytotoxic immune cells due to a variety of reasons, including central and peripheral immune tolerance, age, immunosuppressive conditioning, chemotherapy, etc. (1).

[0177] The present disclosure describes the development of autologous or allogeneic PyroCell TM Compositions and methods of the autologous or allogeneic PyroCell TM Redirecting existing immune reservoirs against SARS-CoV-2 or influenza antigens to treat cancer.

[0178] In other embodiments of the present disclosure, the genetically modified cell therapy can be administered as a standalone therapy or in combination with other therapies. In one aspect, PyroVax TM vaccine can be combined with another therapeutic agent, which includes but is not limited to IFNγ, checkpoint inhibitors (anti-PD1, anti-CTLA-4), and adoptive T cell therapy. The present disclosure's PyroCell TM can also be used in combination with monoclonal antibodies against targets, which include but are not limited to PD-1, PD-L1, CTLA-4, LAG3, TIM3, TIGIT, CD40, OX40, GITR, BCL-2, or cytokines such as IL-2, IFN-γ, TNF-α, and TLR agonists.

[0179] The present disclosure describes genetic and chemical modifications for the composition of autologous or allogeneic cellular immunotherapy or viral immunotherapy. These immunotherapies stimulate the patient's own immune system to detect a broad repertoire of cancer-specific antigens and kill heterogeneous cancer cells, thereby conferring long-term immunity to the patient against their cancer.

[0180] The present disclosure describes PyroCells TM cellular immunotherapy, which is generated by genetically and chemically modifying the patient's own tumor cells. When PyroCells TM are reintroduced into the same patient, they stimulate an anti-tumor immune response against a broad repertoire of cancer-specific antigens. PyroCells TM immunotherapy is effective in both preventive and therapeutic settings, helping to control disseminated and advanced heterogeneous human cancers.

[0181] PyroVax TM DNA or mRNA encoding vectors are used to genetically modify autologous or allogeneic tumor and normal cells and transform them into cellular immunotherapy, which activates a significant anti-tumor immune response against a broad variety of cancer-specific antigens.

[0182] PyroVax TM has three components: PyroHIM TM 、PyroAct TM and PyroTIME TM . These components together help break immune tolerance against tumor-specific antigens and activate a polyclonal immune response against a broad repertoire of tumor-specific antigens. PyroHIM TM encodes highly immunogenic molecules, which are non-self and expressed at supra-physiological levels. PyroAc TM encodes an inflammatory cell death activator, which triggers immune epitope spreading and diversification of the anti-tumor T cell repertoire. PyroTIMETM Molecules encoding the remodeling of the immunosuppressive TIME, where the tumor immune microenvironment allows cytotoxic immune cells to persist and kill tumor cells.

[0183] The following section describes PyroCells TM The design, manufacture, safety, and efficacy of cellular immunotherapy in preclinical cell and animal models.

[0184] PyroVax TM and PyroStim TM composition

[0185] PyroVax TM composition is a DNA plasmid or mRNA vector encoding: i) PyroHIMs TM , highly immunogenic molecules; ii) PyroActs TM , molecules that stimulate inflammatory cell death, and PyroTIME TM , molecules that remodel the immunosuppressive TIME and support the persistence of activated and anti-tumor immune cells. ( Figure 1A shows a schematic diagram of the PyroVax TM vector, and the following table shows examples of each of the three components).

[0186]

[0187]

[0188] The SARS-Cov-2 epitopes shown below are representative examples of peptide sequences of partial-length or full-length proteins that will be used as model antigens in human and murine models (see Table 1 and other claims).

[0189] Human epitopes

[0190]

[0191]

[0192] SARS-CoV-2 specific T cell epitopes in BALB / c mice and C57BL / 6 mice

[0193]

[0194]

[0195]

[0196] In another aspect of the present invention, full-length proteins or peptide derivatives of influenza A structural proteins, including the HA antigen (HA) and epitopes for immunization, are used as antigens. The immunogenic epitopes can be used alone, or in combination with linkers, or without enhancing immunogenicity.

[0197] Identification of MHC-I restricted cytotoxic T lymphocyte (CTL) epitopes. To predict peptides selected from SARS-Cov-2 proteins, including the spike glycoprotein (S), envelope protein (E), membrane protein (M), and nucleocapsid phosphoprotein (N) that induce CTL responses, the MHC-I binding tool of the Immune Epitope Database and Analysis Resource (IEDB; http: / / tools.iedb.org / mhci) was used.

[0198] Identification of MHC-II restricted T lymphocyte epitopes. IEDB (http: / / www.iedb.org) was also used to predict MHC-II binding of 15-mer epitopes to human HLA, including proteins, using the NN-align 2.3 method. High, medium, and low affinity epitopes were selected.

[0199] Identification of B cell epitope prediction. 16-mer linear B lymphocyte (LBL) epitopes were identified using a threshold of 0.5 (ABCpreds server). The ElliPro tool of IEDB was used to predict linear and conformational B cell epitopes.

[0200] Polypeptide structure verification. The ProtParam server was used to query the physical and chemical properties of the construct, such as amino acid composition, molecular weight, theoretical isoelectric point (pI), total average value of hydrophilicity (GRAVY), aliphatic and instability indices, and half-life. The SOPMA server was used to analyze the secondary structure characteristics of the polypeptide, and the GalaxyWEB server was used to model and refine the 3D structure model. The RAMPAGE server and ProSA-web tool were used to verify the improved 3D model.

[0201] PyroVax TM Vector (PyroHIM TM 、PyroAct TM and PyroTIME TM ) Details of each component

[0202] PyroHIM TM (Highly immunogenic molecule)

[0203] PyroHIM TMEncoding highly immunogenic molecules. These molecules are designed to elicit a strong immune response when introduced into a patient. They act as antigens, triggering the immune system to recognize them as foreign and initiate an immune response. PyroHIM TM Enables cancer cells to be more visible to the immune system. Cancer cells typically evade the immune system by looking similar to healthy cells. By introducing highly immunogenic molecules, PyroHIM TM Helps the immune system to more effectively recognize and attack cancer cells.

[0204] PyroHIM TM Can include epitopes (small protein fragments) from various sources, such as SARS-CoV-2 proteins, influenza A antigens, or other tumor-specific antigens. These epitopes are selected because they are known to stimulate strong immune responses. By encoding these epitopes in the PyroVax TM vector, the immune system is primed to target cancer cells that display these antigens.

[0205] PyroAct TM (Activator of inflammatory cell death)

[0206] PyroAct TM Encodes molecules that trigger inflammatory cell death, which is a process called pyroptosis. Pyroptosis is a form of programmed cell death that results in the release of pro-inflammatory signals, which further stimulate the immune system. PyroAct TM Amplifies the immune response. When cancer cells undergo pyroptosis, they release signals that attract immune cells to the site of cell death. This not only eliminates the cancer cells but also recruits more immune cells to the tumor microenvironment. PyroAct TM Encodes molecules like Gasdermin E (GSDME) or other proteins associated with pyroptosis. When these molecules are expressed in cancer cells, they cause pyroptosis when exposed to specific triggers. This leads to the release of danger signals, such as cytokines, alerting the immune system to the presence of cancer cells. PyroTIME TM (Tumor immune microenvironment remolder)

[0207] PyroTIME TM Encodes molecules that remodel the tumor immune microenvironment (TIME). Its role is to reduce immunosuppressive factors over a period of time and promote pro-immune responses. PyroTIME TM Creates a more favorable environment for immune cells to function effectively. Many tumors create an immunosuppressive environment that hinders the activity of immune cells. PyroTIME TMThis is intended to counteract this by inhibiting immunosuppressive molecules and promoting the recruitment and maturation of immune cells within the tumor. PyroTIME TM encodes various molecules, including cytokines, chemokines, soluble receptor antagonists, and gene editing tools such as guide RNA (gRNA). These components can manipulate the expression of specific genes or signaling pathways to enhance immune cell recruitment and reduce immunosuppressive factors such as TGF-β (transforming growth factor-β).

[0208] In summary, PyroVax TM vectors are complex tools for cancer immunotherapy. PyroHIM TM makes cancer cells more visible to the immune system, PyroAct TM amplifies the immune response and recruits immune cells, PyroTIME TM reshapes the tumor microenvironment, creating a more immune-supportive environment. These components work together to effectively target and eliminate cancer cells by harnessing the power of the patient's immune system, thus overcoming the challenges associated with cancer immunotherapy.

[0209] PyroHIM TM : Molecular selection

[0210] PyroHIM TM is designed to enhance the immunogenicity of the PyroCells vaccine. It acts as an adjuvant, stimulating an effective immune response against tumor cells. PyroHIM TM selects SARS-CoV-2-derived molecules based on several scientific principles, including high immunogenicity, broad pre-existing immunity, epitope spreading potential, molecular selection criteria, spike protein, safety, and strategic synergy.

[0211] SARS-CoV-2 has demonstrated an exceptional ability to elicit a strong immune response in infected individuals. This enhanced immunogenicity is attributed to the virus's unique structure and the presence of highly antigenic epitopes such as the spike protein. By introducing elements from SARS-CoV-2 into PyroHIM TM the present disclosure harnesses this inherent immunogenicity to amplify the immune response against tumor cells.

[0212] Due to the global impact of the COVID-19 pandemic, a significant proportion of the world's population has been exposed to SARS-CoV-2 through infection or vaccination. This exposure has led to the generation of memory T cells and SARS-CoV-2 antigen-specific antibodies. The presence of pre-existing immunity in a large portion of potential patients is strategically advantageous. PyroHIM TMAct as a lighthouse, leveraging these pre-existing immune cells and redirecting them to target tumor cells, effectively reusing the body's existing cancer immunotherapy defenses.

[0213] SARS-CoV-2-derived molecules, including the spike protein, contain multiple immunogenic epitopes. When presented to the immune system, these epitopes can trigger epitope spreading, a process by which the immune response broadens to recognize and attack multiple antigens. This disclosure discusses methods of leveraging this phenomenon to diversify the anti-tumor immune response. Since PyroHIM TM directs immune cells to tumor cells while introducing SARS-CoV-2 epitopes, it encourages the immune system to recognize and target a broader range of tumor-specific antigens. This strategy mitigates the risk of tumor escape due to antigen variation, a common challenge in cancer immunotherapy.

[0214] For PyroHIM TM specific molecules selected from SARS-CoV-2 are chosen based on their immunogenicity, safety, and strategic suitability for cancer immunotherapy:

[0215] The spike protein of SARS-CoV-2 is a key antigenic target of the immune response during natural infection and vaccination. Its prominent role in eliciting a strong immune response makes it an ideal choice for PyroHIM TM . Additionally, the spike protein is widely recognized by the immune system, enhancing the likelihood of recruiting memory T cells and antibody targeting of tumor cells.

[0216] The selected SARS-CoV-2 molecules have favorable safety profiles in extensive studies during the COVID-19 pandemic. This safety record is crucial to ensure that PyroHIM TM does not introduce unexpected adverse reactions or immune responses.

[0217] The selection of SARS-CoV-2 molecules is strategically aligned with the broader goals of this disclosure. By reusing immune responses developed against SARS-CoV-2 for cancer treatment, this disclosure describes methods and compounds for increasing the utility of existing immunity, making pyro-cells an innovative and effective therapy.

[0218] PyroHIM TM The scientific rationale for selecting specific molecules, particularly those related to SARS-CoV-2, is rooted in their proven immunogenicity, safety, and potential to induce epitope spreading. This unique approach leverages the global experience with SARS-CoV-2 to drive the field of cancer immunotherapy, offering a promising solution to address the unmet needs of cancer patients.

[0219] Using several strategies and considering multiple pathways to design PyroHIM TM , including selective immune cell recruitment, enhanced antigen presentation, T cell activation and expansion, epitope spreading, and inflammatory cell death (pyroptosis). PyroHIM TM is designed to attract and recruit specific populations of immune cells to the tumor site. The presence of SARS-CoV-2-derived epitopes in PyroHIM TM aids in this recruitment, acting as molecular "baits" to attract immune cells. These epitopes have a dual purpose: they serve as targets for pre-existing memory T cells generated against SARS-CoV-2, and they stimulate the maturation and activation of antigen-presenting cells (APCs) at the tumor site.

[0220] As PyroHIM TM recruits immune cells, it also promotes the presentation of tumor-specific antigens by APCs. The presence of SARS-CoV-2 epitopes within PyroHIM TM triggers APCs to phagocytose tumor cells and present multiple tumor antigens, including patient-specific neoantigens. This process, known as cross-presentation, is crucial for the activation of cytotoxic T cells capable of targeting and eliminating tumor cells.

[0221] Upon encountering tumor-derived antigens presented by APCs, T cells are activated. PyroHIM TM enhances this activation by providing abundant co-stimulatory signals and cytokines, such as the source of GM-CSF (granulocyte-macrophage colony-stimulating factor). GM-CSF further stimulates the maturation of dendritic cells, promoting the expansion and activation of effector T cells, particularly cytotoxic CD8+ T cells.

[0222] PyroAct TM introduces SARS-CoV-2 epitopes known to be highly immunogenic, inducing epitope spreading. This phenomenon broadens the immune response beyond the initially targeted tumor-specific antigens. Consequently, the immune system becomes coordinated with a broader spectrum of tumor antigens, reducing the risk of tumor immune escape due to antigen variation.

[0223] One of the unique features of the methods described in this disclosure is the induction of pyroptosis in pyroptotic cells. Pyroptosis is a highly inflammatory form of cell death that releases numerous cellular contents, including additional tumor antigens and inflammatory signals. This event amplifies the immune response, attracts more immune cells, and enhances the anti-tumor effect.

[0224] PyroTIME TM : Remodeling the tumor immune microenvironment

[0225] By modifying the microenvironment, the present disclosure discusses enhancing the anti-tumor immune response. Such modifications include disruption of the immunosuppressive microenvironment, reprogramming of immune cells, enhanced antigen presentation, and anti-angiogenic effects.

[0226] PyroTIME TM is strategically designed to disrupt the immunosuppressive microenvironment that typically surrounds tumors. It does this by targeting the release of cytokines and chemokines such as IFN-γ (interferon-γ) and CXCL9 / 10. These signaling molecules attract immune cells, particularly cytotoxic T cells and natural killer (NK) cells, into the tumor microenvironment.

[0227] PyroTIME TM not only attracts immune cells; it also reprograms them to adopt a more aggressive anti-tumor phenotype. PyroTIME TM releases IFN-γ, which enhances the cytotoxic activity of T cells and NK cells, making them more effective at recognizing and clearing tumor cells. Additionally, it can polarize tumor-associated macrophages (TAMs) from the M2 immunosuppressive phenotype to the M1 pro-inflammatory phenotype, further aiding in tumor clearance.

[0228] PyroTIME TM supplements the antigen presentation process initiated by PyroHIM TM By creating an inflamed tumor microenvironment, it encourages APCs to more effectively phagocytose tumor material, process it, and present it to T cells. PyroHIM TM and PyroTIME TM This synergy between PyroHIM and PyroTIME results in a robust and sustained anti-tumor immune response.

[0229] PyroTIME TM also exerts an anti-angiogenic effect by inhibiting the recruitment of new blood vessels to the tumor site. This behavior limits the tumor's nutrient supply, creating an unfavorable environment for its growth and survival.

[0230] PyroHIM TM and PyroTIME TM synergize to enhance tumor immunogenicity and reshape the tumor immune microenvironment (TIME). PyroHIM TM recruits and activates immune cells, induces epitope spreading, and amplifies the immune response, while PyroTIME TM disrupts the immunosuppressive TIME, reprograms immune cells for more aggressive anti-tumor functions, and enhances antigen presentation. This multi-faceted approach holds great promise for revolutionizing cancer immunotherapy by making the TIME more permissive to anti-tumor immune responses and ensuring sustained tumor control.

[0231] PyroHIM TM and PyroTIME TM interaction between

[0232] The present disclosure discusses the use of various techniques and cells to enhance anti-tumor immune responses, including immune cell recruitment and activation, remodeling of the tumor microenvironment, reduction of immunosuppressive cells, increase of inflammatory cytokines, enhancement of antigen presentation, and synergistic effects of modification of various techniques and materials.

[0233] PyroHIM TM is designed to recruit and activate immune cells within the tumor immune microenvironment (TIME), including dendritic cells, T cells, and natural killer (NK) cells. These immune cells are crucial for recognizing and targeting tumor cells.

[0234] PyroTIME TM supplements PyroHIM by changing the tumor microenvironment (TME) to be more immunogenic TM . It can potentially reduce immunosuppressive factors and cells within the TME. This remodeling includes:

[0235] PyroTIME TM can reduce the presence of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs), which are known to inhibit anti-tumor immune responses. This reduction helps create a more permissive immune environment.

[0236] PyroTIME TM can increase the production of pro-inflammatory cytokines (e.g., IL-2, IFN-γ) within the TME. These cytokines stimulate effector T cells and enhance their cytotoxic activity.

[0237] By promoting the maturation of dendritic cells and increasing the expression of major histocompatibility complex (MHC) molecules, PyroTIME TM improves antigen presentation. This allows T cells to better recognize tumor antigens.

[0238] PyroHIM TM and PyroTIME TM synergistically act to produce an immune-favorable TME. PyroHIM TM recruits immune cells, while PyroTIME TM helps create an environment that activates these recruited cells. This combined action amplifies the anti-tumor immune response, potentially overcoming the immunosuppression commonly seen in the TME.

[0239] PyroHIM TM 、PyroTIME TMand PyroStim TM Interaction with:

[0240] The further technologies and materials described in this disclosure enhance anti-tumor immune responses, immune cell activation, antigen presentation, immune checkpoint blockade, epitope spreading, and the precision and personalization of treatment.

[0241] PyroHIM TM and PyroTIME TM both contribute to immune cell activation. PyroHIM TM recruits immune cells and promotes their infiltration into the tumor site, while PyroTIME TM reshapes the TME to enhance immune cell activation. PyroStim TM further promotes this activation by incorporating immune checkpoint inhibitors and immune-stimulatory cytokines.

[0242] PyroHIM TM enhances antigen presentation by promoting the maturation of dendritic cells. PyroStim TM complements this by including tumor-specific antigens, ensuring the presentation of multiple tumor antigens to immune cells. This combination enhances the recognition of tumor cells by the immune system.

[0243] PyroStim TM contains immune checkpoint inhibitors, such as anti-PD-L1 antibodies, which block the PD-L1 / PD-1 interaction. This prevents T cell exhaustion and enhances their cytotoxic activity. When combined with PyroHIM TM and PyroTIME TM the immune checkpoint blocker is applied in an environment where immune cells are actively involved, maximizing its effectiveness.

[0244] PyroHIM TM and PyroTIME TM potentially promote epitope spreading by creating conditions for the release of multiple tumor antigens from dying tumor cells. PyroStim TM enhances this process by enhancing the immune response against these released antigens. This synergy can lead to a more extensive and effective anti-tumor immune response.

[0245] All three components can be personalized based on the subject's tumor profile. This ensures that the treatment is customized to the unique characteristics of the subject's cancer, thereby maximizing its efficacy.

[0246] In summary, PyroHIM TM , PyroTIME TM and PyroStimTM Work in a coordinated manner to create a highly immunogenic and pro-inflammatory tumor microenvironment. They recruit immune cells, reshape immunosuppressive elements within the tumor, activate the immune response, and enhance antigen presentation. This comprehensive approach addresses all aspects of the anti-tumor immune response and has the potential to lead to more effective and durable therapeutic outcomes.

[0247] PyroCells TM technology

[0248] The present disclosure provides mechanisms, technologies, and materials to redirect the immune response, modify immune memory, increase the functionality of epitope cross-reactivity, activate immune cells, and modify supra-physiological expression.

[0249] Redirecting the immune response is an aspect of the present disclosure, initially generated against SARS-CoV-2, towards the targeted elimination of cancer cells. This redirection is achieved through the PyroCell TM immunotherapy system and the PyroVax TM vector, which are carefully designed to harness the immune memory and responses induced by SARS-CoV-2 exposure or vaccination. This section elaborates on the mechanisms, rationale, and preliminary evidence supporting this redirection.

[0250] The memory of the immune system is a key component of this redirection. Patients exposed to SARS-CoV-2, recovered from infection, or vaccinated possess a repertoire of immune cells specific for SARS-CoV-2 antigens, including CD8+ T cells, CD4+ T cells, and B cells. These memory cells can be used to recognize cancer-specific antigens.

[0251] PyroVax TM vector introduces highly immunogenic epitopes from SARS-CoV-2 antigens, such as spike glycoprotein (S), envelope protein (E), membrane protein (M), nucleocapsid phosphoprotein (N), and other immunodominant antigens. These epitopes have been strategically selected based on their propensity for cross-reactivity with cancer-specific antigens.

[0252] PyroVax TM includes PyroHIMs TM that encode highly immunogenic molecules. These molecules serve as effective activation signals for immune cells, triggering their effector functions, including cytokine release and cytotoxic activity. By linking PyroHIMs TM to SARS-CoV-2-derived epitopes, PyroVax TM ensures the activation of memory immune cells specific for SARS-CoV-2 antigens.

[0253] In some embodiments, PyroHIMs TM are linked to a reporter molecule, enabling supra - physiological expression, detection, and enrichment of PyroCells TM expressing the PyroVax TM vector. This helps selectively amplify PyroCells TM with high immunogenicity, which are then targeted to cancer cells. The effectiveness of redirected immune responses. Pre - clinical models come from preliminary evidence of pre - clinical studies using PyroCell TM and PyroVax TM that have demonstrated: Efficacy: substantial regression of established tumors in mice. Diversity of immune responses: activation and diversification of the T - cell repertoire against a range of tumor - specific antigens. Long - term immunity: durable protection against cancer recurrence.

[0254] The pyroptosis mechanism in pyroptotic cells

[0255] Many mechanisms are involved in the activity of pyroptotic cells, including mechanisms such as T - cell activation: granzyme B release, gasdermin E activation, pore formation, inflammatory responses, and cell lysis. Possible challenges and side effects include inflammatory responses, off - target effects, and immunosuppression, while mitigation strategies include personalization, dose optimization, monitoring, and combination therapies.

[0256] Pyroptotic cells are designed to stimulate a robust immune response against cancer cells. T - cells are a key component of the immune system and play a central role in this process. After activation, cytotoxic T - cells, in particular, release molecules such as granzyme B.

[0257] Granzyme B is a protease released by cytotoxic T - cells. It is a key mediator of the immune response against infections or cancer cells. Granzyme B can enter target cells, including cancer cells.

[0258] Inside cancer cells targeted by pyroptotic cells, granzyme B initiates a cascade of events that ultimately leads to the activation of gasdermin E. Gasdermin E is a key protein in pyroptosis; it forms pores in the cell membrane.

[0259] The gasdermin E pores generated in the cell membrane disrupt its integrity. This leads to the release of cell contents, including pro - inflammatory cytokines and danger - associated molecular patterns (DAMPs).

[0260] The release of pro - inflammatory cytokines and DAMPs signals to neighboring immune cells, particularly macrophages and dendritic cells, to initiate an immune response. This response includes recruiting more immune cells to the site and activating the adaptive immune response against cancer cells.

[0261] The disruption of the cell membrane integrity by gasdermin E pores leads to cell lysis or cell rupture. These release additional pro-inflammatory molecules and cell debris.

[0262] Although the pyroptosis mechanism is a powerful tool in cancer immunotherapy, there are potential challenges and side effects that need to be considered: The hallmark of pyroptosis is a rapid and robust inflammatory response. While this is desirable for the immune system to recognize and attack cancer cells, excessive inflammation can lead to adverse reactions, including flu-like symptoms, fever, and, in severe cases, cytokine storms. Granzyme B, although highly specific for target cells such as cancer cells, may enter and activate gasdermin E in unintended cells. This off-target effect can cause damage to healthy tissues and organs. In some cases, the inflammatory response may be counteracted by regulatory immune mechanisms, leading to immunosuppression. This can hinder the overall effectiveness of the treatment.

[0263] To address these challenges and minimize the potential side effects associated with the pyroptosis mechanism, the present disclosure describes several strategies: Personalizing pyroptotic cells for each patient to target their specific cancer cells. This approach minimizes off-target effects as the vaccine is tailored to the individual's unique cancer characteristics. Conducting careful dose optimization to balance a strong immune response with safety. This includes determining the appropriate amount of pyroptotic cells to minimize excessive inflammation while ensuring efficacy. Rigorous monitoring of patients during clinical trials can detect adverse reactions early, enabling timely intervention and adjustment of the treatment regimen. The present disclosure explores combination therapies, including immunosuppressants or anti-inflammatory drugs, to modulate the immune response and mitigate potential side effects.

[0264] In summary, the pyroptosis mechanism involving granzyme B and gasdermin E secreted by T cells is one aspect of the therapeutic action of pyroptotic cells against cancer. While this mechanism has challenges and potential side effects, personalized treatment, dose optimization, vigilant monitoring, and strategic combination therapies help mitigate these risks. These efforts aim to increase the benefits of cell pyroptosis while ensuring patient safety and treatment efficacy.

[0265] Allogeneic mechanism of action

[0266] The mechanism of action of the proposed therapy involving the use of allogeneic cells includes complex but highly coordinated processes that harness the power of the immune system to target and eliminate cancer cells.

[0267] Enhanced antigen presentation: PyroCells TM Are designed to enhance antigen presentation within the tumor microenvironment. This is achieved through multiple mechanisms:

[0268] Upregulation of MHC class I molecules: PyroCells TMIncrease the expression of major histocompatibility complex (MHC) class I molecules on the surface of tumor cells. MHC class I molecules play a crucial role in presenting tumor antigens to CD8+ T cells.

[0269] Activation of dendritic cells: PyroCells TM Stimulate the maturation and activation of dendritic cells within the tumor microenvironment. Dendritic cells are professional antigen-presenting cells that can capture, process tumor antigens and present them to CD8+ T cells, initiating an immune response.

[0270] Activation of CD8+ T cells: CD8+ T cells recruited to the tumor site encounter PyroCells TM and tumor-specific antigens presented by MHC class I molecules on dendritic cells. This interaction activates CD8+ T cells, transforming them into cytotoxic effector T cells.

[0271] Tumor cell killing: Activated CD8+ T cells recognize and specifically target tumor cells expressing the presented antigen. They release cytotoxic molecules such as perforin and granzyme to induce apoptosis (cell death) of tumor cells.

[0272] Epitope spreading: As tumor cells are killed, they release additional tumor antigens into the tumor microenvironment. This process, known as epitope spreading, diversifies the antigen pool available for immune recognition. CD8+ T cells can recognize and target a broader range of tumor epitopes, reducing the risk of tumor escape due to antigen variation.

[0273] PyroStim TM Elaboration: Composition, mechanism of action, advantages

[0274] PyroStim TM is a carefully composed therapeutic composition with the potential to significantly enhance the anti-tumor immune response. Its multi-faceted mechanism of action, including immune checkpoint blockade, cytokine-mediated enhancement, and antigen presentation, makes it a promising addition to Pyrojas' anti-cancer arsenal. Its advantages include enhanced specificity, synergy with other therapies, and the ability to overcome immune escape strategies employed by tumors.

[0275] Composition: PyroStim TM is a proprietary composition designed to enhance the effectiveness of Pyrojas' cancer immunotherapy. It consists of a carefully selected combination of molecules and factors that together enhance the anti-tumor immune response. The composition includes:

[0276] Immune checkpoint inhibitors: PyroStim TMImmune checkpoint inhibitors (ICIs) are introduced, such as anti-PD-L1 (programmed death-ligand 1) antibodies. These ICIs block the PD-L1 / PD-1 interaction and prevent tumors from evading the immune system by inhibiting T cell activity.

[0277] Cytokines: PyroStim TM Contains specific cytokines, including interleukins (e.g., IL-2 and IL-12), interferons (e.g., IFN-γ), and TNFα family members, which are known for their role in enhancing immune responses. These cytokines stimulate the activation and proliferation of cytotoxic T cells, natural killer (NK) cells, and other immune effector cells.

[0278] Mechanism of action: PyroStim TM Acts through multiple mechanisms to enhance the immune response against tumors:

[0279] Immune checkpoint blockade: In PyroStim TM Contains immune checkpoint inhibitors, such as anti-PD-L1 antibodies, which play a central role. By blocking the PD-L1 / PD-1 interaction, PyroStim TM Prevents the inhibition of T cell activity, enabling activated T cells to more effectively target and eliminate tumor cells.

[0280] Cytokine-mediated enhancement: PyroStim TM The cytokines within, such as IL-2, IL-12, and IFN-γ, act as effective immune stimulants. They activate immune effector cells, particularly cytotoxic T cells and NK cells, which are crucial for tumor cell recognition and destruction.

[0281] Antigen presentation: In PyroStim TM Contains tumor-specific antigens that enhance antigen presentation. This helps direct the immune response towards tumor-specific targets, further increasing the specificity of the anti-tumor immune response.

[0282] Synergistic effect with PyroHIM TM and PyroTIME TM Synergistic effect: PyroStim TM Synergizes with PyroHIM TM and PyroTIME TM to recruit and activate immune cells and reshape the tumor immune microenvironment (TIME). The combined effect amplifies the anti-tumor response, potentially overcoming immune suppression within the tumor and promoting durable tumor control.

[0283] PyroStim TM Advantages of PyroStim

[0284] Enhanced Immune Response: PyroStim TM Acts as a powerful immune enhancer. By blocking immune checkpoints and providing immune - stimulating cytokines, it ensures that the immune system is primed to attack tumors.

[0285] Increased Specificity: PyroStim TM The inclusion of tumor - specific antigens in PyroStim enhances the specificity of the immune response. This reduces the risk of off - target effects and ensures that the immune system mainly targets tumor cells.

[0286] Synergy with Existing Therapies: PyroStim TM Is designed to work synergistically with other therapeutic components of Pyrojas, including PyroHIM TM and PyroTIME TM 。This combination approach increases the chances of a robust and sustained anti - tumor response.

[0287] Overcoming Immune Evasion: PyroStim TM The immune checkpoint inhibitors within PyroStim counteract the mechanisms that tumors use to evade the immune system. This may make tumors more vulnerable to immune attack.

[0288] Method for Creating PyroCell TM The method

[0289] Isolation of Target Cells

[0290] Techniques and Equipment: Target cells are cultured in a controlled environment using aseptic techniques, which include a laminar flow hood, a CO2 incubator, and sterile culture containers. The selection of a specific target cell line is based on the intended application and tumor type. Authentication is performed using short tandem repeat (STR) profiling. Potential Challenges and Optimizations: Ensuring the purity of the target cell line is crucial. Regular STR analysis and mycoplasma testing are performed to maintain cell line integrity. Strict aseptic procedures are followed to prevent contamination during cell culture.

[0291] Transfection with PyroVax TM Vector (DNA or mRNA)

[0292] Techniques and Equipment: Transfection Reagents: Lipofection or electroporation based on cell type. Commercial transfection reagents or custom formulations are used. PyroVax TM Vector Preparation: Purify the PyroVax TM vector and quantify it using established molecular biology techniques. Potential Challenges and Optimizations: Transfection Efficiency: Optimization of transfection conditions (such as voltage, reagent concentration) is crucial for increasing vector uptake by target cells. Vector Quality Control: Strict quality control of the PyroVax TM vector ensures consistent results.

[0293] Selection and Expansion of Transfected Cells

[0294] Techniques and Equipment: Selection Markers: Cells are typically transfected with selection marker genes, such as antibiotic resistance or fluorescent proteins. Cell Expansion: Transfected cells are expanded in culture to obtain sufficient cells for downstream applications. Potential Challenges and Optimizations: Selection Pressure: Optimizing the concentration of the selection agent is necessary to balance cell survival and marker expression. Cell Growth Optimization: Modify the medium composition, seeding density, and passage interval to promote cell growth while maintaining transgene expression.

[0295] PyroCell TM Production

[0296] Techniques and Equipment: Cell Harvesting: Harvest transfected cells at the desired growth stage. Quality Control: Evaluate cell viability, transgene expression, and other relevant parameters. Potential Challenges and Optimizations: Cell Viability: Carefully scheduling cell harvesting is crucial for improving cell viability. Consistency: Maintain batch - to - batch consistency through strict quality control measures.

[0297] Cryopreservation

[0298] Techniques and Equipment: Cryoprotectant: Use a validated cryoprotectant solution to cryopreserve cells. Cryopreservation Containers: Use vials or cryobags for storage.

[0299] Potential Challenges and Optimizations: Cell Recovery Rate: Optimizing the cryoprotectant composition and freezing rate ensures high cell recovery upon thawing. Storage Conditions: Maintain strict storage conditions (-80 °C or liquid nitrogen) for long - term storage.

[0300] Manufacture of Therapeutic Cellular Immunotherapy

[0301] For the manufacture of autologous PyroCells TM , surgical resected primary and / or metastatic patient tumors are dissociated into single cells using a cell dissociation buffer that prevents the disruption of antigen - loaded MHC - I and MHC - II molecules on the cell surface. Stromal cells, including immune cells, endothelial cells, and fibroblasts, are removed by antibody - mediated negative selection to enrich for tumor cells. PyroVax, composed of DNA vectors or mRNA constructs encoding components of PyroHIM TM , PyroAct TM and PyroTIME TM , is introduced into the tumor cells using viral transduction or electroporation. Cells expressing PyroVax TM , called PyroCells TM , are chemically enriched and stimulated with PyroStim TM , and TMTreatment, PyroStim TM is a mixture of cytokines and chemokines that increases tumor antigen presentation and the gene expression levels of ISGs, interferon-stimulated genes. After comprehensive gene editing, QA / QC, and in vitro functional validation studies, GMP-compliant immunotherapeutic tumor cells are expanded to reach therapeutic doses (0.1, 0.5, 1×10^6), rendered non-replicating, cryopreserved in single doses, and shipped to physicians for patient treatment. The entire manufacturing process complies with GMP standards and is completed within two weeks.

[0302] To manufacture Allo-PyroCell TM : Briefly, patient-derived autologous tumor cells are chemically modified using PyroStim TM and mixed with Allo-PyroCell TM which is an allogeneic cell line modified with a DNA vector or mRNA construct encoding the PyroVax TM components, which includes the sequences of PyroHIM TM , PyroAct TM and PyroTIME TM at any of the ratios: 1:1, 1:2, 1:5, 1:10, 1:100. The mixture is then shipped to physicians for patient treatment. The entire manufacturing process complies with GMP standards and is completed within two weeks. Compared to the autologous method, allogeneic cell therapy has a large number of available TAAs, is standardized, scalable, less variable, and cost-effective. The cell line can be developed from solid or liquid cancers, metastatic or circulating cancer cells, and can be derived from one or more individuals. TM

[0303] Manufacture of PyroVir TM for intratumoral delivery of PyroVax TM . The present disclosure describes compositions and methods for generating PyroVir TM carrying an expression vector of PyroVax TM , AAV, HSV1 / 2, or other cancer-specific viruses. When administered intratumorally, the virus infects tumor cells and stimulates an immune response against PyroHIMs TM and a broad repertoire of cancer-specific antigens. PyroVir TM overcomes various challenges associated with ex vivo modification of autologous or allogeneic cells.

[0304] Functional validation of the PyroVax TM constructs used to prepare PyroCells TM or PyroVir TM ​​

[0305] Verification of PyroVax TM Vectors. PyroVir TM AAV virus, lentivirus or non-viral expression plasmids are used to deliver the PyroVax TM constructs into normal and transformed human and murine cells. After chemical selection and flow cytometry-based enrichment, the transcript and protein expression levels of PyroVax TM components are quantified using RT-PCR and immunoblotting or flow cytometry: PyroHIMs TM , PyroActs TM and PyroTIME TM . Studies based on immunoblotting, flow cytometry and ELISA confirmed the efficient delivery of PyroVax TM into normal or transformed cells.

[0306] PyroCells TM cells and PyroVir TM viral immunotherapy induces pyroptotic tumor cell death

[0307] Next, the effects of PyroVax TM expression in normal and transformed human and murine cells were functionally verified. When linked to PyroHIM TM , the expression of activators of inflammasomes and pyroptotic cell death such as GSDME, GSDMD, AIM2 innate immune sensors or cytokine IL33, etc. (see Table 1 and claims) promotes immunity against different tumor antigens by activating immune epitope spreading and diversifying the anti-tumor T cell repertoire.

[0308] When killed by PyroHIM TM antigen-specific T cells, the PyroAct TM molecule acts as a trigger for the inflammatory cell death of PyroCells TM . Therefore, when co-cultured with cytotoxic CD8+ T cells that recognize PyroHIM TM antigens, the frequency of death of PyroCells TM or PyroVir TM -infected cells due to apoptosis (Annexin V+) was quantified, rather than pyroptosis (PI+). PyroAct TM containing PyroCells TM or PyroVir TM only undergoes pyroptotic cell death rather than apoptotic cell death. Consistently, inhibitors of pyroptosis limit PyroAct TMThe inhibitor of other forms of cell death had no significant effect. Collectively, these data indicate that PyroHIM TM and PyroAct TM combination triggers preferential pyroptotic cell death rather than apoptotic cell death.

[0309] To demonstrate that PyroAct TM induces epitope spreading from immunodominant PyroHIM TM epitopes to less dominant cancer antigens, PyroCells TM or PyroAct TM deficient PyroCells TM were co-cultured with PyroHIM TM antigen-specific CTLs in the presence of antigen-presenting cells (APCs), dendritic cells or macrophages. PyroCells TM and PyroAct TM deficient PyroCells TM were both killed by PyroHIM TM antigen-specific CTLs, although there were significant differences. First, although PyroCells TM lacking PyroAct TM mainly died by caspase-1-mediated apoptosis, PyroCells TM expressing PyroAct TM preferentially died by pyroptosis. These observations indicate that PyroCells TM were killed by inflammasome- or Gasdermin E-mediated pyroptotic cell death.

[0310] To evaluate whether the observed pyroptotic cell death stimulates immune epitope spreading, using co-culture assays and ELISPOT cytokine release assays, the reactivity of CTLs recovered from tumor-CTL-APC co-cultures was examined compared to previously unresponsive cancer-specific antigens. CTLs derived from co-cultures containing PyroCells TM killed parental PyroVax TM deficient tumor cells, but CTLs derived from PyroAct TM deficient PyroCells TM had no reactivity to parental cells. Collectively, these data indicate that PyroCell TM cellular immunotherapy is sensitive to CTL killing and PyroAct TM triggers pyroptotic inflammatory tumor cell death.

[0311] LDH enzyme release is used to monitor pyroptosis (7), and other markers include cytokines released due to inflammasome activation: IL-1β and IL-1RA. Activation of the inflammasome induces the formation of Gasdermin-D pores on the cell membrane, leading to the secretion of IL-1β and IL-18, and the influx of water molecules resulting in cell swelling and subsequent rupture (pyroptosis).

[0312] Interestingly, the SARS-Cov-2 antigen itself has been shown to have a significant intrinsic potential to activate the inflammasome and pyroptosis. PyroHIMs encoding the SARS-CoV-2 proteins E, M, and specific ORFs TM induce K + ion efflux, creating an imbalance that leads to oxidative stress, mitochondrial damage, and NLRP3 inflammasome activation (8,9). Interestingly, ORF3a and ORF8b directly activate the inflammasome (8,9).

[0313] Immunization and validation in mice against SARS-Cov-2

[0314] Construction, expression, and purification of the recombinant spike protein. The recombinant DNA with optimized code encoding the spike protein expressed by the SARS-Cov-2 virus was introduced into a mammalian gene expression lentiviral vector (PLV[Exp]-Puro-CMV) vector (VectorBuilder, USA). The DNA sequence of the assembled vector was verified by DNA sequencing. The sequences of the insert fragment and the vector are provided.

[0315] Female BALB / c mice were immunized subcutaneously with two doses of the spike protein or saline control. Two weeks after the booster immunization, high levels of SARS-CoV-2-specific IgG antibodies and neutralizing antibodies were elicited in the immunized mice. The immunized mice were challenged intranasally with the mouse-adapted strain MASCp6 (1.6×10 4 PFU) at the 6th generation, and lung tissues were collected 5 days after the challenge for virological and histopathological analysis as described previously (10). All PBS-treated mice maintained a high viral RNA load in the lungs 5 days after the challenge. In contrast, the immunized mice showed a significant reduction in the viral RNA load. Immunofluorescence staining of the SARS-CoV-2 S protein was used to detect viral proteins in the lungs of immunized mice and PBS-immunized mice. No pathological damage was observed in the lungs of the immunized mice, while inflammatory lung injury was present in the lungs of the control mice, with focal perivascular and peribronchiolar inflammation, and thickened alveolar septa.

[0316] ELISA. ELISA was performed to detect SARS-CoV-2 specific IgG antibodies. Briefly, ELISA plates were pre-coated with SARS-CoV-2 spike protein (1 μg / ml) overnight at 4 °C and blocked with 2% milk in PBST for 2 hours at 37 °C. Serial dilutions of sera were added to the plates and incubated for 2 hours at 37 °C. After washing four times, the bound antibodies were detected by incubation with anti-mouse IgG antibody conjugated with horseradish peroxidase (HRP) (Thermo Fisher, USA, 1:5000) for 1 hour at 37 °C. The reaction was developed by adding the substrate 3,3’,5,5’-tetramethylbenzidine (TMB) (Sigma, USA) and terminated by adding H2SO4 (1N). Absorbance at 450 nm was measured by an ELISA plate reader.

[0317] SARS-CoV-2 neutralization assay. As described previously, a micro-neutralization assay was performed to detect neutralizing antibodies against SARS-CoV-2 (11, 12). Briefly, two-fold serial dilutions of mouse sera were incubated with SARS-CoV-2 (100 TCID50) for 1 hour at 37 °C and added to Vero cells. Cells were observed daily for the presence of virus-induced cytopathic effect (CPE) and recorded at 3 dpi. The neutralizing antibody titer was expressed as the reciprocal of the highest serum dilution that completely blocked Vero cell CPE.

[0318] PyroCells TM and PyroVir TM Viral immunotherapy is safe and tolerable

[0319] using PyroCells TM cells or PyroVir TM Mice immunized against SARS-Cov-2 were treated with viral immunotherapy using PyroCells cells or PyroVir virus, and acute safety issues and long-term adverse outcomes were monitored. Throughout the treatment process and long-term follow-up, the mice maintained their body weight and did not show signs of morbidity. All major organ systems were grossly and histologically evaluated by a pathologist to assess any signs of toxicity or unexpected adverse outcomes including autoimmune reactions. The effect of the therapeutic vaccine in combination with the current standard of care (chemotherapy) or with immune checkpoint inhibitors was also tested.

[0320] PyroCells TM cells and PyroVir TM Viral immunotherapy controls disease burden and improves survival outcomes

[0321] Next, mouse models of human cancers of entities (TNBC, melanoma, ovarian cancer) and hematology (leukemia, lymphoma, and myeloma) were used to determine the anti-tumor efficacy of immunotherapy. The previously determined MTD was used to test PyroCells TM In vivo anti-tumor efficacy of immunotherapy.

[0322] Treatment with PyroCells TM of mice bearing established disease with immunotherapy alone or in combination with immune checkpoint inhibitors controlled the disease burden and increased the overall survival of the mice. Additionally, rechallenge of complete responders with parental tumor led to complete rejection of the tumor, highlighting the persistence of the ongoing anti-tumor immune memory response. Notably, when used in a prophylactic setting, allo-PyroCells TM immunotherapy inhibited the emergence and establishment of the disease burden of parental tumor cells that do not express PyroHIMs TM of mice bearing established disease with immunotherapy alone or in combination with immune checkpoint inhibitors controlled the disease burden and increased the overall survival of the mice. Additionally, rechallenge of complete responders with parental tumor led to complete rejection of the tumor, highlighting the persistence of the ongoing anti-tumor immune memory response. Notably, when used in a prophylactic setting, allo-PyroCells

[0323] Immune escape of tumor cells is driven by loss of antigen-presenting molecules such as MHC-I or B2M. Therefore, the effect of the vaccine on tumors that are predominantly B2M or MHC-I deficient was evaluated in vivo. Interestingly, PyroCells TM rendered MHC-I or B2M-deficient cancer cells sensitive to killing by CD8 + T cells, while PyroAct TM expanded sensitivity to different other tumor epitopes in vivo. These findings demonstrated the activation of antigen-independent killing.

[0324] PyroCells TM cells and PyroVir TM virus immunotherapy redirected the anti-SARS-Cov-2 immune response against a broad repertoire of cancer-specific antigens

[0325] A comprehensive evaluation of intratumoral immune infiltration was performed. Antigen-specific cytotoxic CD8 + T cells, CD4 + T cells, and B cells were significantly increased in infiltration in tumors derived from mice treated with PyroCells TM cell immunotherapy compared to control counterparts. Notably, combining PyroCells TM immunotherapy with immune checkpoint inhibitors significantly enhanced the intratumoral infiltration and persistence of anti-tumor cytotoxic T cells.

[0326] Patients with cancer are immunocompromised for various reasons (1) and do not have a robust reservoir of immune cells available to control invasive solid human cancers. However, cancer patients who have recovered from SARS-CoV-2 infection or have received any approved SARS-CoV-2 vaccine contain a robust reservoir of SARS-CoV-2 antigen-specific immune cells. The disclosure described herein redirects or reprograms the immune response against SARS-CoV-2 to kill cancer cells.

[0327] Highly immunogenic SARS-Cov-2 epitopes are used as PyroHIMs operably linked to an inflammasome activator TM , to redirect the patient's pre-existing antiviral immune response against SARS-Cov-2 (see Table 1 containing examples of PyroHIMs TM ) to cancer-specific antigens. ELISPOT and tetramer-based flow cytometry were used to confirm the presence of PyroHIM TM antigen-specific CD4 + and CD8 + T cells, SARS-Cov-2-specific T cells, particularly several novel TAA-specific CD8 + and CD4 + T cells. Paired scRNA-seq and TCRa / b sequencing were used to test the clonal expansion of polyvalent T cells against multiple cancer-specific antigens other than the immunodominant PyroHIM TM epitopes. Immunized mice generated cross-primed T cells against known cancer antigens and importantly against emerging TAAs, i.e., tumor-associated antigens. Transcriptomic analysis of tumors was used to demonstrate that the expression of target antigens and / or TAAs was significantly reduced in tumors from mice receiving PyroCells TM immunotherapy compared to control counterparts. No adverse autoimmune reactions occurred in humans or mice.

[0328] To evaluate whether the observed pyroptotic cell death stimulates immune epitope spreading, T cell and tumor cell co-culture killing assays and ELISPOT cytokine release assays were used to examine the antigen reactivity of CTLs recovered from the spleens of vaccinated or unvaccinated mice compared to previously non-responsive cancer-specific antigens. CTLs derived from mice treated with PyroCells TM killed parental PyroVax TM deficient tumor cells, but CTLs derived from PyroAct TM deficient PyroCells TM were non-reactive to parental cells. These data indicate that treatment with PyroCells TMThe treated mice generate antigen-specific T cells that recognize not only the immunodominant PyroHIM TM antigen but also significantly recognize a variety of other tumor-associated antigens.

[0329] Treatment of mice bearing established disease with PyroCells TM immunotherapy alone or in combination with immune checkpoint inhibitors controls the disease burden and increases the overall survival of the mice. Importantly, rechallenge of complete responders with epitope-free parental tumor cells results in complete rejection of the parental tumor, highlighting the diversification or spreading of epitopes and the presence of an immune memory response. Notably, when used in a preventive setting, allogeneic PyroHIMs TM immunotherapy inhibits the emergence and establishment of the disease burden of parental tumor cells that do not express PyroHIMs TM The immune escape of tumor cells is driven by the loss of antigen-presenting molecules such as MHC-I or B2M. Therefore, the effects of the vaccine on tumors that are mainly B2M- or MHC-I-deficient were evaluated in vivo. Interestingly, PyroCells

[0330] render MHC-I- or B2M-deficient cancer cells sensitive to killing by CD8 TM T cells, while PyroAct + extends the sensitivity to different other tumor epitopes in vivo. These findings demonstrate the activation of antigen-independent killing. TM These findings demonstrate the activation of antigen-independent killing.

[0331] PyroTherapies TM redirect the anti-SARS-Cov-2 immune response against uterine leiomyomas

[0332] The safety and efficacy of using PyroTherapies TM To treat benign tumors, including uterine leiomyomas or fibroids, a test called fibrotreatment TM was used. An immunocompetent Eker rat model of spontaneous uterine fibroids and an immunodeficient mouse model of human uterine fibroid xenografts were used. Briefly, immunocompetent Eker rats with spontaneous uterine fibroids or immunocompromised Balb / c mice with transplanted human patient-derived uterine fibroid xenografts were prophylactically vaccinated against the immunogen. As a proof of concept, the M-spike protein expressed by the SARS-Cov-2 virus was used as derived from PyroHIMs TMThe model immunogen can also use other immunogens. All rats generated a strong humoral and adaptive immune response against the immunogenic epitope weeks after immunization. Subsequently, a luciferase-labeled episomal expression vector (EEV) or a viral vector containing DNA encoding the immunogen for prophylactic immunization was directly injected into the fibroids. For the Eker model, intratumoral injection into the fibroids was performed laparoscopically under MRI guidance, while for the human PDX model, direct injection was used as the xenograft was accessible. Bioluminescence imaging was used to confirm the fibroid-specific expression of the DNA vector in both models. Next, antigen-specific cellular therapy targeting the immunogenic antigen, TCR-T cells or CAR-T / NK cells, was administered. After a few weeks, complete immune rejection of the rat uterine fibroids and human uterine fibroid xenografts was observed in all animals without any side effects. Notably, while prophylactic immunization alone was sufficient to induce a robust anti-fibroid CD4 + and CD8 + T cell response in Eker rats, antigen-specific allogeneic T or NK cell therapy eliminated large established fibroids. These preclinical safety and efficacy data rationalize the progression of FibroTherapy TM to human clinical trials.

[0333] Presented below are examples considered for the applications discussed Cells, protein, and nucleic acid sequences The following examples are provided to further illustrate the embodiments of the present invention and not to limit the scope of the present invention. While they are typical and can be used, other procedures, methods, or techniques known to those skilled in the art can be alternatively used.

[0334] Examples

[0335] Example 1

[0336] PyroCells Vaccine Data

[0337] Expression and Characterization of Designated Proteins in Human Breast Cancer Cells

[0338] Lentiviral Vector Transduction and Protein Expression: Using a lentiviral vector, successful transduction of human TNBC cells with the gene of interest was achieved. Contour plots ( Figure 7A ) demonstrated different expression levels of the designated protein in these cells after transduction. The comprehensive quantification of this data ( Figure 7B ) emphasized the efficiency of the lentiviral transduction process, showing significant protein expression in the transduced cells compared to control cells. Support: Demonstrating effective transduction and protein expression in human TNBC cells strengthened the feasibility of the proposed method. Successful expression in challenging cell lines such as TNBC ensured the efficacy and adaptability of the proposed construct and transduction method.

[0339] Non-viral methods for protein expression: Exploring alternative methods and also using non-viral methods to achieve protein expression in breast cancer cells. The contour plot ( Figure 7C ) showing expression levels comparable to those of the lentiviral method, indicating the potential versatility of the construct for different delivery mechanisms. Support: Demonstrating protein expression achieved through non-viral means provides a less risky, potentially safer, and more general alternative for therapeutic delivery. This broadens the therapeutic window, enhances adaptability, and makes the project more attractive for funding.

[0340] Example 2

[0341] Tumor burden analysis in murine models

[0342] Initial assessment of tumor burden: In murine models, a comprehensive assessment of tumor burden was conducted. The mean radiation rate and total flux were measured, providing key indicators for understanding tumor progression. These indicators are presented in bar charts ( Figure 8A ) and line charts ( Figure 8B ) for clear visual representation and easy interpretation of the data over time. Support: Demonstrating tangible evidence of tumor burden metrics (mean radiation rate and total flux) in murine models validates the applicability of the proposed therapy in the real world. The effective reduction in tumor burden provides a strong rationale for the potential clinical efficacy of the construct.

[0343] Re-challenge experiment: To understand the protective capacity of the therapeutic construct, mice carrying a specific tumor burden were imaged at the indicated time intervals ( Figure 8A ). After a 21-day span, a subset of these mice underwent re-challenge with the parental tumor. Interestingly, the data ( Figure 8B ) showed significant protection in the previously vaccinated mice, indicating potential not only in reducing tumor burden but also in providing long-term protection against cancer recurrence. Support: The re-challenge experiment highlights the long-term benefits and potential protective capacity of the therapeutic construct. By demonstrating resistance to cancer recurrence, it emphasizes not only treatment but also the potential preventive aspect.

[0344] Example 3

[0345] Extended tumor burden analysis

[0346] Long-Term Effects and Re-Challenge Experiments: Conducted a detailed time-course study in which tumor-bearing mice were imaged at specific intervals (Figure 8C). After 21 days of labeling, the mice were re-challenged with the parental tumor or not. The data reinforced earlier findings, highlighting the consistent therapeutic potential of the construct in controlling cancer growth and potentially preventing recurrence upon re-challenge. The results provided a comprehensive account of the in vitro and in vivo efficacy of the therapeutic construct. From efficient protein expression in human TNBC cells to a significant reduction in tumor burden in murine models, the findings paved the way for the potential clinical application of these constructs in cancer therapy. Support: The extended time-course study further validated the long-term therapeutic potential of the construct. Demonstrating control over cancer growth and recurrence prevention in an extended setting provided a compelling rationale for this application as it promises long-term patient benefit.

[0347] Example 4

[0348] PyroTIMER CAR-T Cell Data

[0349] PyroTIMER: TGF RI-II Fusion Protein, SEQ ID NO: 50

[0350]

[0351] PyroTIMER is a new class of synthetic proteins that is used to reshape the tumor immune microenvironment and promote the anti-tumor activity of CAR-T cells. Biochemical and functional characterization of the lead PyroTIMER, a recombinant fusion protein that includes the extracellular domains of TGFβRI and TGFβRII, demonstrates its sustained ability to inhibit all isoforms of TGF-β. Promising results suggest that PyroTIMER is an effective therapeutic agent to enhance CAR-T cell therapy, and further studies are needed to refine the design and evaluate the safety and efficacy in preclinical models. In a series of preliminary studies, PyroTIMER CD19-CAR T cells were generated using Jurkat T cells ( Figure 9A , Figure 9B and Figure 9C ) and CD3+ T lymphocytes derived from peripheral human blood ( Figures 10A to 10D ). CD3+ T lymphocytes were enriched, activated using CD3 / CD28 Dynabeads, and expanded in IL-2 medium. After transduction with the PyroTIMER construct and a second-generation anti-human CD19 CAR, chemical selection and flow-based cell sorting were used to select and enrich single-positive and double-positive cells to achieve stable expression of both PyroTIMER and CAR over time ( Figure 9C , Figure 10C and Figure 10F ). Appropriate controls, including untransfected cells and cells transfected with a non-functional version of PyroTIMER, were used to rule out non-specific effects.

[0352] To evaluate the efficacy of PyroTIMER CD19 CAR-T cells, a co-culture killing assay was performed using Raji lymphoma cells expressing the CD19 antigen in the presence of 100 pM TGF-β1. PyroTIMER CD19 CAR-T cells or CD19 CAR-T cells were co-cultured with Raji cells at increasing effector-to-target ratios ( Figure 9D , Figure 9E and Figure 10G ). The results showed that PyroTIMER CD19 CAR-T cells remained activated and exhibited significantly greater killing of target cells compared to CD19 CAR-T cells alone at all tested ratios ( Figure 9D , Figure 9E , Figure 10G ). Notably, Figure 10G demonstrated the superior cytotoxic ability of PyroTIMER CD19 CAR-T cells, and Figure 10H highlighted the enhanced activation, which was evident from the surge in CD69+ activated T cells.

[0353] Advantages in a TGF-β-rich environment: The data highlight the unparalleled performance of PyroTIMER CAR-T cells, especially in a TGF-β-rich environment. Figure 9D , Figure 9E , Figure 10E and Figure 10F The co-culture killing assay of CD19-expressing Raji lymphoma cells as shown in and was conducted in the presence of 100 pM TGF-β1, replicating the immunosuppressive environment of tumors replete with TGF-β. In this environment, PyroTIMER CD19 CAR-T cells had superior killing effects compared to conventional CD19 CAR-T cells, providing incontrovertible evidence for their enhanced functionality in a TGF-β-rich environment. These findings indicate that the PyroTIMER technology enhances the cytotoxicity of CD19 CAR-T cells against CD19-expressing cells in a TGF-β-rich microenvironment. Notably, inhibiting the TGF-β pathway in hematological malignancies using CAR-T cells is particularly important as TGF-β plays a key role in generating immunosuppressive times in CD19 malignancies.

[0354] In vivo efficacy verification: Notably, PyroTIMER CAR-T cells were significantly more effective in controlling the CD19+ Raji lymphoma disease burden in NOD.Cg-PrkdcscidIL2Rgtm1Wjl / Sz mice compared to conventional CD19 CAR-T cells (n = 4, per group) (Figure 11). The NOD.Cg-PrkdcscidIL2Rgtm1Wjl / Sz mouse model, as shown in Figure 11, was used to bring laboratory findings into a real-world context. The superior efficacy of PyroTIMER CAR-T cells in controlling CD19+ Raji lymphoma compared to their conventional counterparts not only reinforces their higher therapeutic potential but also underscores the translational feasibility of the technology in a practical therapeutic setting.

[0355] Based on the cumulative data, it is clear that PyroTIMER CAR-T cells are not only the epitome of a novel approach to CAR-T cell therapy but also provide robust evidence to support this disclosure. By effectively blocking the inhibitory effects of TGF-β in the tumor microenvironment, PyroTIMER CAR-T cells mark a transformative step in cancer immunotherapy, ensuring that Claim 22 stands on a solid scientific and technological foundation and is ready for clinical application and commercial success.

[0356] Example 5

[0357] Selection of uterine leiomyoma (fibroid) as a model:

[0358] 1. Prevalence and clinical significance: Uterine leiomyomas, commonly known as fibroids, are non-cancerous growths of the uterus that often occur in women of reproductive age. They are very common, and it is estimated that up to 70 - 80% of women may develop fibroids during their lifetime. The size, number, and location of fibroids within the uterus can vary, leading to a range of clinical symptoms, including heavy menstrual bleeding, pelvic pain, and reproductive problems. Due to their prevalence and clinical significance, fibroids pose a significant burden on women's health and the healthcare system.

[0359] 2. Limited treatment options: Traditional treatments for fibroids include medical therapy, surgery (myomectomy or hysterectomy), and minimally invasive procedures. However, these treatments are not always suitable for all subjects, and they may carry significant side effects, risks, or long recovery times. Additionally, they do not address the underlying causes of fibroid development.

[0360] 3. Unmet medical need: Given the limitations of current treatments and the high prevalence of fibroids, there is a significant unmet medical need for effective, minimally invasive, and targeted therapies that can specifically shrink or eliminate fibroids while preserving the uterus. FibroTherapy TM is designed to address this unmet need.

[0361] Implications and extensions to other benign tumors:

[0362] Although FibroTherapy TM was initially developed for uterine leiomyomas (fibroids), its underlying principles and mechanisms of action hold promise for extension to other benign tumors. Here are some potential implications and extensions:

[0363] Tissue - specific targeting: The key to FibroTherapy TM is its ability to selectively target and remodel fibrotic tissue within the uterus. This tissue - specific targeting is achieved through the use of PyroCells TM which are TM genetically engineered to express specific molecules that interact with fibrotic tissue. This targeting strategy may be applicable to other benign tumors involving fibrosis or excessive extracellular matrix deposition. Conditions such as keloids, hypertrophic scars, and desmoid tumors, which are characterized by fibrotic tissue growth, may benefit from similar approaches.

[0364] Customization: FibroTherapy TM demonstrates the potential of personalized medicine in the treatment of benign tumors. By engineering PyroCells TMTo express molecules customized for specific characteristics of the target tissue, it is possible to customize therapies for different types of benign tumors. This approach allows for a high degree of specificity and precision in treatment.

[0365] Minimally invasive nature: FibroTherapy TM Is designed to be delivered via minimally invasive techniques, such as laparoscopy or hysteroscopy. This minimizes the need for open surgery and reduces the associated risks and recovery time. The minimally invasive nature of this approach may be beneficial for treating various benign tumors located in accessible anatomical sites.

[0366] Potential safety features: Due to FibroTherapy TM Relies on PyroCells TM Targeting within the tumor microenvironment, it may have favorable safety features compared to more systemic therapies. This aspect can be explored in the context of other benign tumors, where minimizing off-target effects is crucial.

[0367] Clinical studies: To explore the expansion of FibroTherapy TM To other benign tumors, further research and clinical trials will be necessary. This research may involve adapting the therapy to the specific characteristics of different tumor types and evaluating its safety and efficacy in different patient populations.

[0368] In summary, while FibroTherapy TM Was initially developed for uterine leiomyomas (fibroids), its tissue-specific targeting and minimally invasive nature offer the possibility of potential expansion to other benign tumors characterized by fibrosis or similar pathological features. These expansions can provide new treatment options for subjects with a range of benign tumor diseases, addressing unmet medical needs in multiple clinical settings.

[0369] As described herein, the present disclosure provides compositions and methods for treating cancer. In certain aspects, cancers include acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, AIDS-related cancers, Kaposi sarcoma, AIDS-related lymphoma, primary CNS lymphoma, anal cancer, astrocytoma, atypical teratoid / rhabdoid tumor, central nervous system cancer, basal cell carcinoma of the skin, skin cancer, cholangiocarcinoma, bladder cancer, bone cancer (including Ewing sarcoma, osteosarcoma, and malignant fibrous histiocytoma), brain tumor, breast cancer, bronchial tumor, non-Hodgkin lymphoma (including Burkitt lymphoma), carcinoid tumor, carcinoma of unknown primary origin, cardiac tumor, medulloblastoma and other CNS embryonal tumors, germ cell tumor, cervical cancer, childhood cancer, cholangiocarcinoma (including cholangiocarcinoma), chordoma, (bone cancer), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloproliferative neoplasms, colorectal cancer, craniopharyngioma, lymphoma, cutaneous T-cell lymphoma, mycosis fungoides, and Sézary syndrome, breast cancer, ductal carcinoma in situ (DCIS), embryonal tumor, medulloblastoma, endometrial cancer, ependymoma, esophageal cancer, anesthetic neuroblastoma, Ewing sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, eye cancer, intraocular melanoma, retinoblastoma, fallopian tube cancer, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), glioma, glioblastoma multiforme, childhood central nervous system germ cell tumor, childhood extracranial germ cell tumor, extragonadal germ cell tumor, ovarian germ cell tumor, testicular cancer, gestational trophoblastic disease, hairy cell leukemia, head and neck cancer, cardiac tumor, hepatocellular carcinoma, histiocytosis, Langerhans cell, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor, pancreatic neuroendocrine tumor, kidney (renal cell) cancer, Langerhans cell histiocytosis, laryngeal cancer, head and neck cancer, leukemia, lip and oral cavity cancer, liver cancer, lung cancer (including non-small cell, small cell, pleuropulmonary blastoma, and / or tracheobronchial tumor), lymphoma, male breast cancer, melanoma, intraocular melanoma, Merkel cell carcinoma, malignant mesothelioma, metastatic carcinoma, metastatic squamous neck carcinoma with occult primary, midline tract carcinoma with NUT gene alteration, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell tumor, mycosis fungoides, myelodysplastic syndrome, myelonasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cancer, lip and oral cavity cancer, oropharyngeal cancer, osteosarcoma, undifferentiated pleomorphic sarcoma of bone, ovarian cancer, pancreatic cancer, pancreatic neuroendocrine tumor, islet cell tumor, papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid carcinoma, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, plasma cell tumor / multiple myeloma, pleuropulmonary blastoma, pregnancy and breast cancer, primary central nervous system (CNS) lymphoma, primary peritoneal cancer, prostate cancer, rare childhood cancers,Rectal cancer, recurrent cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, uterine sarcoma, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, cutaneous squamous cell carcinoma, occult primary squamous cervical cancer, gastric (stomach) cancer, T cell lymphoma, testicular cancer, throat cancer, nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer, thymoma and thymic carcinoma, thyroid cancer, tracheobronchial tumors, transitional cell carcinoma of the renal pelvis and ureter, cancer of unknown primary, ureter and renal pelvis, transitional cell carcinoma, urethral cancer, endometrial uterine cancer, uterine sarcoma, vaginal cancer, vascular tumors, vulvar cancer, Wilms' tumor and other childhood renal tumors or cancers in young adults.

[0370] In certain aspects, methods for treating, ameliorating, achieving remission of, and / or reducing the risk of the cancers disclosed herein include administering to a subject an immunotherapeutic composition according to the present disclosure. In additional aspects, the immunotherapeutic composition can be administered as a pharmaceutical composition according to any suitable route and regimen. In further aspects, the route or regimen is one associated with a positive therapeutic benefit.

[0371] In additional aspects, the exact amount of the immunotherapeutic composition administered can vary from patient to patient, depending on one or more factors known in the medical art. Such factors can include, but are not limited to, one or more of the following factors, such as species, age, general condition of the patient, the particular composition to be administered, its mode of administration, its mode of activity, the severity of the disease, the activity of the particular immunotherapeutic composition employed, the particular pharmaceutical composition administered, the half-life of the composition after administration, the weight of the patient, sex, diet, time of administration, route of administration, the excretion rate of the particular immunotherapeutic composition employed, the duration of treatment, and any other therapeutic agent combined with or used concurrently with the particular immunotherapeutic composition to be administered.

[0372] In certain aspects, the compositions of the present disclosure are administered to a subject by any suitable route known and / or employed by those skilled in the art. In some aspects, the compositions of the present disclosure are administered orally (PO), intravenously (IV), intramuscularly (IM), intra-arterially, intramedullarily, intrathecally, subcutaneously (SQ), intraventricularly, transdermally, intradermally, dermally, rectally (PR), vaginally, intraperitoneally (IP), intragastrically (IG), topically (e.g., by powder, ointment, cream, gel, lotion, and / or drops), mucosally, intranasally, orally, enterally, intravitreally, sublingually, by endotracheal instillation, bronchial instillation, and / or inhalation, as an oral spray, nasal spray, aerosol, and / or by portal vein catheter.

[0373] In some aspects, the immunotherapeutic composition and / or its pharmaceutical composition according to the present disclosure can be administered intravenously, for example, by intravenous infusion. In additional aspects, the immunotherapeutic composition and / or its pharmaceutical composition according to the present disclosure can be administered by intramuscular injection. In further aspects, the immunotherapeutic composition and / or its pharmaceutical composition according to the present disclosure can be administered by intratumoral injection. In some aspects, the immunotherapeutic composition and / or its pharmaceutical composition according to the present disclosure can be administered by subcutaneous injection. In additional aspects, the immunotherapeutic composition and / or its pharmaceutical composition according to the present disclosure can be administered via a portal vein catheter. In further aspects, considering possible advancements in the field of drug delivery, the present disclosure encompasses delivering the immunotherapeutic composition and / or its pharmaceutical composition according to the present disclosure by any suitable route.

[0374] In some aspects, the required dose can be delivered only once. In some aspects, the required dose can be delivered once a day, more than once a day, once every other day, once every third day, once a week, once every two weeks, once every three weeks, once every four weeks, once every two months, once every six months, or once every twelve months. In some aspects, multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations) can be used to deliver the required dose. In some aspects, the required dose can be delivered using one or more administrations during an initial time period, followed by a time period without dose administration.

[0375] In additional aspects, the immunotherapeutic composition according to the present disclosure can be used for prophylactic applications. In further aspects, prophylactic applications involve systems and methods for inhibiting cancer progression and / or delaying cancer onset in individuals susceptible to cancer and / or showing cancer symptoms.

[0376] In some aspects, the immunotherapeutic composition according to the present disclosure is administered to target cells in vivo. In other aspects, the immunotherapeutic composition according to the present disclosure is administered ex vivo to target cells. In additional aspects, the immunotherapeutic composition according to the present disclosure is administered ex vivo to target cells, and then the target cells are reintroduced into the organism. In some such aspects, the target cells are cultured ex vivo into a plurality of daughter cells before being reintroduced into the organism. In further aspects, the organism is a human. In some aspects, the target cells are initially derived from the organism into which they are reintroduced. In other aspects, the target cells are initially derived from a different organism into which they are reintroduced.

[0377] In some aspects, the immunotherapeutic composition and / or its pharmaceutical composition according to the present disclosure are used in combination therapies for treating cancer or reducing the risk of cancer. In such aspects, the administration can be combined with one or more additional therapeutic agents. As used herein, the phrases "combination therapy", "in combination with", "in conjunction with", etc. refer to the use of more than one drug or treatment simultaneously to increase the response. In some aspects, the immunotherapeutic composition and / or its pharmaceutical composition according to the present disclosure are administered simultaneously with, before, or after one or more other desired therapeutic agents or medical procedures. In some aspects, the immunotherapeutic composition and / or its pharmaceutical composition according to the present disclosure are administered together in a single composition or separately in different compositions.

[0378] In some aspects, the specific combination of therapies to be employed in a combination regimen generally takes into account the compatibility of the desired therapeutic agents and / or procedures, as well as the desired therapeutic effect to be achieved. In other aspects, the therapies employed can achieve the desired effect for the same purpose (e.g., an immunotherapeutic composition according to the present disclosure that can be used to treat cancer and / or delay the onset of cancer can be administered simultaneously with another therapeutic agent that can also be used to treat cancer and / or delay the onset of cancer), or they can achieve different effects. In additional aspects, the combination of therapies employed can achieve the same or substantially similar desired effect for the same cancer; the same or substantially similar desired effect for one or more different cancers; different desired effects for the same cancer; or different desired effects for one or more different cancers.

[0379] In additional aspects, the delivery of the immunotherapeutic composition according to the present disclosure as a pharmaceutical composition is combined with one or more additional components that can improve the bioavailability of the immunotherapeutic composition, reduce and / or alter its metabolism, inhibit its excretion, and / or alter its distribution in the body.

[0380] In some aspects, the combination therapy can involve the administration of multiple immunotherapeutic compositions according to the present disclosure. In additional aspects, the combination therapy can involve the administration of multiple immunotherapeutic compositions that treat, improve, achieve remission of, and / or reduce the risk of a single type of cancer. In further aspects, the combination therapy can be multiple immunotherapeutic compositions that treat, improve multiple types of cancer, achieve remission of multiple types of cancer, and / or reduce the risk of multiple types of cancer.

[0381] In some aspects, the immunotherapeutic compositions according to the present disclosure are combined with at least one pharmaceutically acceptable excipient in the form of a pharmaceutical composition. As used herein, "pharmaceutical composition" refers to a preparation containing an active ingredient and optionally a pharmaceutically acceptable carrier, diluent, or excipient. The term "active ingredient" may be interchangeably referred to as "active agent" and means any agent capable of inducing a desired effect upon administration. Examples of active ingredients include, but are not limited to, compounds, drugs, therapeutic agents, small molecules, etc. In some aspects of the present disclosure, the active ingredient is an immunotherapeutic composition as disclosed herein.

[0382] "Pharmaceutically acceptable" means that the carrier, diluent, or excipient must be compatible with the other ingredients of the preparation and not harmful to its recipient or to the activity of the active ingredient of the preparation. The pharmaceutical compositions described herein can be prepared by any method known in the pharmacological arts or developed hereafter. Pharmaceutically acceptable carriers, excipients, or stabilizers are known in the art, for example, as described in J.P. Remington & A. Osol, Remington's Pharmaceutical Sciences , 16th edition (1980) or J.P. Remington & P. Beringer, Remington:The Science and Practice of Pharmacy , 21st Edition (2006), with respect to the pharmaceutically acceptable carriers, excipients, and stabilizers provided therein, and both of these references are incorporated herein by reference.

[0383] In some aspects, the pharmaceutically acceptable carrier, excipient, or stabilizer is non-toxic to the recipient at the dosages and concentrations employed and may include buffering agents such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butanol or benzyl alcohol, alkyl parabens such as methyl paraben or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight peptides (fewer than about 10 amino acid residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, dextrin, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN TM 、PLURONICS TMor polyethylene glycol (PEG). Examples of carriers include, but are not limited to, liposomes, nanoparticles, ointments, micelles, microspheres, microparticles, creams, emulsions, and gels. Examples of excipients include, but are not limited to, anti-adhesives such as magnesium stearate, binders such as sugars and their derivatives (sucrose, lactose, starch, cellulose, sugar alcohols, etc.), proteins such as gelatin and synthetic polymers, lubricants such as talc and silica, and preservatives such as antioxidants, vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium, cysteine, methionine, citric acid, sodium sulfate, and parabens. Examples of diluents include, but are not limited to, water, alcohols, saline solutions, ethylene glycol, mineral oil, and dimethyl sulfoxide (DMSO). In additional aspects, the pharmaceutical composition comprises one or more additional therapeutic or bioactive substances.

[0384] In certain aspects, the pharmaceutical composition can be used in the manufacture of a medicament or drug. In other aspects, the pharmaceutical composition can be used for one or more of the therapeutic applications disclosed herein, for example, in an individual suffering from an autoimmune disorder. In additional aspects, the pharmaceutical composition is formulated for administration to a human subject.

[0385] In certain aspects, the pharmaceutical composition is in a sterile injectable form (e.g., a form suitable for subcutaneous injection or intravenous infusion). In further aspects, the pharmaceutical composition is a liquid dosage form suitable for injection. In other aspects, the pharmaceutical composition is a powder (e.g., lyophilized and / or sterilized), optionally under vacuum, which is reconstituted with an aqueous diluent (e.g., water; buffer; salt solution, etc.) prior to injection. In additional aspects, the pharmaceutical composition is diluted and / or reconstituted in an aqueous diluent (e.g., water, sodium chloride solution, sodium acetate solution, benzyl alcohol solution, phosphate buffered saline, etc.). In certain aspects, the pharmaceutical composition is in a form that can be refrigerated and / or frozen. In other aspects, the pharmaceutical composition is in a form that cannot be refrigerated and / or frozen. In certain aspects, the pharmaceutical composition is a reconstituted solution and / or liquid dosage form that can be stored for a period of time (e.g., 2 hours, 12 hours, 24 hours, 2 days, 5 days, 7 days, 10 days, 2 weeks, one month, two months, or longer) after reconstitution.

[0386] In some aspects, methods of preparing pharmaceutical compositions include associating an active ingredient (e.g., an immunotherapeutic composition according to the present disclosure) with one or more pharmaceutically acceptable excipients and then shaping and / or packaging the product into the desired single-dose or multi-dose units. The pharmaceutical compositions according to the present disclosure can be prepared, bulk packaged, packaged as individual unit doses, and / or packaged as multiple individual unit doses. As used herein, "unit dose" refers to a discrete amount of a pharmaceutical composition that contains a predetermined amount of the active ingredient. The amount of the active ingredient is typically equal to the dose to be administered to a subject and / or a convenient fraction of that dose, such as one-half or one-third of that dose. The relative amounts of the active ingredient, pharmaceutically acceptable excipients, and / or any additional ingredients in the pharmaceutical compositions according to the present disclosure can vary depending on the identity, size, and / or condition of the subject being treated and / or according to the route of administration of the composition. In some aspects, for example, the composition can contain from about 0.1% to 100% (w / w) of the active ingredient.

[0387] In another aspect, the present disclosure includes kits that can be used to implement the methods of the present disclosure. The components included in the kits depend on a number of factors, including the specific application (e.g., the specific route of administration to be employed, or the specific cancer to be treated). In some aspects, the present disclosure provides a kit for administering an immunotherapeutic composition according to the present disclosure to treat the types of cancers disclosed herein. In some such aspects, the kit further includes instructions for administration. In some aspects, the kit contains one or more immunotherapeutic compositions. In some aspects, the kit includes multiple unit doses of a pharmaceutical composition containing an immunotherapeutic composition. In additional aspects, the kits used according to the present disclosure include instructions (e.g., for administration, for storage, etc.), buffers, and / or other reagents. In some such aspects, the kit includes (i) at least one immunotherapeutic composition, (ii) a syringe, needle, applicator, etc. for administering the at least one immunotherapeutic composition to a subject, and (iii) instructions for use. In additional aspects, the kit includes a treatment schedule specifying when to administer the unit doses. In further aspects, placebo doses in forms similar or different from the doses of the pharmaceutical composition are included. In some aspects, the kit includes one or more containers such that some of the individual components or reagents can be separately housed. In some aspects, the kit can include means for enclosing the individual containers in a relatively tight confinement for commercial sale, such as a plastic box in which the instructions, packaging materials such as styrofoam, etc. can be enclosed.

[0388] Chen DS, Mellman I. Oncology meets immunology: the cancer-immunity cycle. Immunity. 2013 Jul 25; 39(1): 1-10. doi: 10.1016 / j.immuni.2013.07.012. PMID: 23890059.

[0389] Zheng M, Williams EP, Malireddi RKS, Karki R, Banoth B, Burton A, Webby R, Channappanavar R, Jonsson CB, Kanneganti TD. Impaired NLRP3 inflammasome activation / pyroptosis leads to robust inflammatory cell death via caspase-8 / RIPK3 during coronavirus infection. J Biol Chem. 2020 Oct 9; 295(41): 14040-14052. doi: 10.1074 / jbc.RA120.015036. Epub 2020 Aug 6. PMID: 32763970; PMCID: PMC7549031.

[0390] Lee S, Channappanavar R, Kanneganti TD. Coronaviruses: Innate Immunity, Inflammasome Activation, Inflammatory Cell Death, and Cytokines. Trends Immunol. 2020 Dec; 41(12): 1083-1099. doi: 10.1016 / j.it.2020.10.005. Epub 2020 Oct 15. PMID: 33153908; PMCID: PMC7561287.

[0391] Agudo J, Ruzo A, Park ES, Sweeney R, Kana V, Wu M, Zhao Y, Egli D, Merad M, Brown BD. GFP-specific CD8 T cells enable targeted cell depletion and visualization of T-cell interactions. Nat Biotechnol. 2015 Dec; 33(12): 1287-1292. doi: 10.1038 / nbt.3386. Epub 2015 Nov 2. PMID: 26524661; PMCID: PMC4675673.

[0392] Hoffman RM. Application of GFP imaging in cancer. Lab Invest. 2015 Apr; 95(4): 432-52. doi: 10.1038 / labinvest.2014.154. Epub 2015 Feb 16. PMID: 25686095; PMCID: PMC4383682.

[0393] Liu BL, Robinson M, Han ZQ, Branston RH, English C, Reay P, McGrath Y, Thomas SK, Thornton M, Bullock P, Love CA, Coffin RS. ICP34.5 deleted herpes simplex virus with enhanced oncolytic, immune stimulating, and anti-tumour properties. Gene Ther. 2003 Feb; 10(4): 292-303. doi: 10.1038 / sj.gt.3301885. PMID: 12595888.

[0394] Rayamajhi M, Zhang Y, Miao EA. Detection of pyroptosis by measuring released lactate dehydrogenase activity. Methods Mol Biol. 2013;1040:85-90. doi:10.1007 / 978-1-62703-523-1_7. PMID:23852598; PMCID:PMC3756820.

[0395] Huanzhou Xu, Siddhi A. Chitre, Ibukun A. Akinyemi, Julia C. Loeb, John A. Lednicky, Michael T. McIntosh, Sumita Bhaduri-McIntosh. SARS-CoV-2 viroporin triggers the NLRP3 inflammatory pathway. bioRxiv 2020.10.27.357731; doi:https: / / doi.org / 10.1101 / 2020.10.27.357731.

[0396] Shah A. Novel Coronavirus-Induced NLRP3 Inflammasome Activation: A Potential Drug Target in the Treatment of COVID-19. Front Immunol. 2020 May 19;11:1021. doi:10.3389 / fimmu.2020.01021. PMID:32574259; PMCID:PMC7248552.

[0397] Gu H, Chen Q, Yang G, He L, Fan H, Deng YQ, Wang Y, Teng Y, Zhao Z, Cui Y, Li Y, Li XF, Li J, Zhang NN, Yang X, Chen S, Guo Y, Zhao G, Wang X, Luo DY, Wang H, Yang X, Li Y, Han G, He Y, Zhou X, Geng S, Sheng X, Jiang S, Sun S, Qin CF, Zhou Y. Adaptation of SARS-CoV-2 in BALB / c mice for testing vaccine efficacy. Science. 2020 Sep 25;369(6511):1603-1607. doi:10.1126 / science.abc4730. Epub 2020 Jul 30. PMID:32732280; PMCID:PMC7574913.

[0398] Bewley KR, Coombes NS, Gagnon L, McInroy L, Baker N, Shaik I, St-Jean JR, St-Amant N, Buttigieg KR, Humphries HE, Godwin KJ, Brunt E, Allen L, Leung S, Brown PJ, Penn EJ, Thomas K, Kulnis G, Hallis B, Carroll M, Funnell S, Charlton S. Quantification of SARS-CoV-2 neutralizing antibody by wild-type plaque reduction neutralization, microneutralization and pseudotyped virus neutralization assays. Nat Protoc. 2021 Jun;16(6):3114-3140. doi:10.1038 / s41596-021-00536-y. Epub 2021 Apr 23. PMID:33893470.

[0399] Nie J, Li Q, Wu J, Zhao C, Hao H, Liu H, Zhang L, Nie L, Qin H, Wang M, Lu Q, Li X, Sun Q, Liu J, Fan C, Huang W, Xu M, Wang Y. Quantification of SARS-CoV-2 neutralizing antibody by a pseudotyped virus-based assay. Nat Protoc. 2020 Nov;15(11):3699-3715. doi:10.1038 / s41596-020-0394-5. Epub 2020 Sep 25. PMID:32978602.

[0400] It should be understood that the present disclosure is not limited to the specific compositions, methods, and experimental conditions described, as such compositions, methods, and conditions may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the present disclosure will be limited only by the appended claims.

[0401] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication, patent, or patent application is specifically and individually indicated to be incorporated by reference.

Claims

1. A pharmaceutical composition comprising autologous cancer cells isolated from a subject, wherein the autologous cancer cells comprise: a) a recombinant protein or peptide that induces an immune response, or a nucleic acid encoding the recombinant protein or peptide; b) a recombinant protein or peptide that induces an inflammatory cell death response, or a nucleic acid encoding the recombinant protein or peptide; c) a recombinant protein or peptide that induces the activation and persistence of antigen-presenting cells and cytotoxic T cells within the tumor microenvironment, or a nucleic acid encoding the recombinant protein or peptide; or d) a recombinant protein or peptide that induces the formation of tumor-associated tertiary lymphoid structures, or a nucleic acid encoding the recombinant protein or peptide.

2. The pharmaceutical composition according to claim 1, wherein the recombinant protein or peptide that induces an immune response is selected from: a) SARS-Cov-2 spike (S); b) SARS-Cov-2 envelope (E); c) SARS-Cov-2 membrane (M); d) SARS-Cov-2 nucleocapsid (N); e) SARS-CoV-2 spike S-2P and RBD antigens; f) SARS-Cov-2 ORF3a; g) SARS-Cov-2 ORF7a; h) SARS-Cov-2 ORF8; i) SARS-Cov-2 replicase 1AB; j) enhanced green fluorescent protein (EGFP); and k) influenza hemagglutinin (HA).

3. The pharmaceutical composition according to claim 1, wherein the recombinant protein or peptide that induces an inflammatory cell death response is selected from: a) Gasdermin D (GSDMD); b) Gasdermin E (GSDME); c) absent in melanoma 2 (AIM2); d) interleukin 33 (IL33); e) thioredoxin-interacting protein (TXNIP); f) interleukin-1 receptor-associated kinase 1 (IRAK1); and g) NLR family pyrin domain-containing protein 3 (NLRP3).

4. The pharmaceutical composition according to claim 1, wherein the recombinant protein or peptide that induces the activation and persistence of antigen-presenting cells and cytotoxic T cells within the tumor microenvironment, and the recombinant protein or peptide that induces the formation of tumor-associated tertiary lymphoid structures are selected from: a) chemokine ligand 9 (CXCL9); b) chemokine ligand 10 (CXCL10); c) chemokine ligand 12 (CXCL12); d) chemokine ligand 13 (CXCL13); e) C-C motif chemokine ligand 19 (CCL19); f) C-C motif chemokine ligand 21 (CCL21); g) interleukin-1β (IL1β); h) interleukin-4 (IL4); i) interleukin-4 receptor (IL4R); j) interleukin-21 (IL21); k) interleukin-22 (IL22); l) interleukin-22 receptor (IL22R); m) interleukin-23; n) interleukin-13 (IL13); o) lymphotoxin β receptor (LTβR); p) β-2-microglobulin (B2M); q) Programmed death-ligand 1 (PD-L1); r) Integrin-associated protein (CD47); s) Transforming growth factor β receptor I (TGFβRI) - extracellular domain, soluble; t) Transforming growth factor β receptor II (TGFβRII) - extracellular domain, soluble; u) Transforming growth factor β receptor III (TGFβRIII) - extracellular domain, soluble; v) A protein consisting of the extracellular domain of at least one protein selected from transforming growth factor β receptor I (TGFβRI), transforming growth factor β receptor II (TGFβRII), and transforming growth factor β receptor III (TGFβRIII); w) Mothers against decapentaplegic homolog 2 (SMAD2); x) Mothers against decapentaplegic homolog 3 (SMAD3); y) Tumor necrosis factor α (TNFα); and z) Interferon γ (IFNγ).

5. The pharmaceutical composition according to claim 1, wherein the autologous cancer cells are contacted with a mixture of cytokines and chemokines.

6. The pharmaceutical composition according to claim 4, wherein at least one of the recombinant proteins or peptides is tumor necrosis factor α (TNFα) or interferon γ (IFNγ).

7. A pharmaceutical composition formulated for delivery using lipid nanoparticles, electroporation, or other delivery mechanisms, comprising the autologous cancer cells according to claim 1.

8. A method of treating cancer in a subject, comprising administering to the subject the pharmaceutical composition according to claim 1.

9. A method of treating a benign tumor in a subject, comprising administering to the subject the pharmaceutical composition according to claim 1.

10. A pharmaceutical composition comprising allogeneic cells, the allogeneic cells comprising: a) A recombinant protein or peptide that induces an immune response, or a nucleic acid encoding the recombinant protein or peptide; b) A recombinant protein or peptide that induces an inflammatory cell death response, or a nucleic acid encoding the recombinant protein or peptide; c) A recombinant protein or peptide that induces the activation and persistence of antigen-presenting cells and cytotoxic T cells within the tumor microenvironment, or a nucleic acid encoding the recombinant protein or peptide; or d) A recombinant protein or peptide that induces the formation of tumor-associated tertiary lymphoid structures, or a nucleic acid encoding the recombinant protein or peptide.

11. The pharmaceutical composition according to claim 10, wherein the recombinant protein or peptide that induces an immune response is selected from: a) SARS-Cov-2 spike (S); b) SARS-Cov-2 envelope (E); c) SARS-Cov-2 membrane (M); d) SARS-Cov-2 nucleocapsid (N); e) SARS-CoV-2 spike S-2P and RBD antigens; f) SARS-Cov-2 ORF3a; g) SARS-Cov-2 ORF7a; h) SARS-Cov-2 ORF8; i) SARS-Cov-2 replicase 1AB; j) Enhanced green fluorescent protein (EGFP); and k) Influenza hemagglutinin (HA).

12. The pharmaceutical composition according to claim 10, wherein the recombinant protein or peptide that induces an inflammatory cell death response is selected from: a) Gasdermin D (GSDMD); b) Gasdermin E (GSDME); c) Absent in melanoma 2 (AIM2); d) Interleukin 33 (IL33); e) Thioredoxin-interacting protein (TXNIP); f) Interleukin-1 receptor-associated kinase 1 (IRAK1); and g) NLR family pyrin domain-containing protein 3 (NLRP3).

13. The pharmaceutical composition according to claim 10, wherein the recombinant protein or peptide that induces the activation and persistence of antigen-presenting cells and cytotoxic T cells within the tumor microenvironment, and the recombinant protein or peptide that induces the formation of tumor-associated tertiary lymphoid structures, or the nucleic acid encoding the recombinant protein or peptide is selected from: a) Chemokine ligand 9 (CXCL9); b) Chemokine ligand 10 (CXCL10); c) Chemokine ligand 12 (CXCL12); d) Chemokine ligand 13 (CXCL13); e) C-C motif chemokine ligand 19 (CCL19); f) C-C motif chemokine ligand 21 (CCL21); g) Interleukin 1β (IL1β); h) Interleukin 4 (IL4); i) Interleukin 4 receptor (IL4R); j) Interleukin 21 (IL21); k) Interleukin 22 (IL22); l) Interleukin 22 receptor (IL22R); m) Interleukin 23; n) Interleukin 13 (IL13); o) Lymphotoxin β receptor (LTβR); p) β-2-microglobulin (B2M); q) Programmed death-ligand 1 (PD-L1); r) Integrin-associated protein (CD47); s) Transforming growth factor β receptor I (TGFβRI)-extracellular domain, soluble; t) Transforming growth factor β receptor II (TGFβRII)-extracellular domain, soluble; u) Transforming growth factor β receptor III (TGFβRIII)-extracellular domain, soluble; v) A protein consisting of the extracellular domain of at least one protein selected from transforming growth factor β receptor I (TGFβRI), transforming growth factor β receptor II (TGFβRII), and transforming growth factor β receptor III (TGFβRIII); w) Mothers against decapentaplegic homolog 2 (SMAD2); and x) Mothers against decapentaplegic homolog 3 (SMAD3).

14. The pharmaceutical composition according to claim 10, wherein the allogeneic cells are contacted with autologous cancer cells from a subject, wherein the autologous cancer cells are first contacted with a mixture of cytokines and chemokines.

15. A pharmaceutical composition formulated for delivery using lipid nanoparticles, electroporation, or other suitable delivery mechanisms, comprising the allogeneic cells according to claim 10.

16. A method of treating cancer in a subject, comprising administering to the subject the pharmaceutical composition of claim 10.

17. A method of treating a benign tumor in a subject, comprising administering to the subject the pharmaceutical composition of claim 10.

18. A pharmaceutical composition comprising a recombinant virus selected from: a) a recombinant protein or peptide that induces an immune response, or a nucleic acid encoding said recombinant protein or peptide; b) a recombinant protein or peptide that induces an inflammatory cell death response, or a nucleic acid encoding said recombinant protein or peptide; c) a recombinant protein or peptide that induces the activation and persistence of antigen-presenting cells and cytotoxic T cells within the tumor microenvironment, and d) a recombinant protein or peptide that induces the formation of tumor-associated tertiary lymphoid structures.

19. The pharmaceutical composition according to claim 18, wherein the nucleic acid encoding the recombinant protein or peptide that induces an immune response encodes a protein or peptide selected from: a) SARS-Cov-2 spike (S); b) SARS-Cov-2 envelope (E); c) SARS-Cov-2 membrane (M); d) SARS-Cov-2 nucleocapsid (N); e) SARS-CoV-2 spike S-2P and RBD antigens; f) SARS-Cov-2 ORF3a; g) SARS-Cov-2 ORF7a; h) SARS-Cov-2 ORF8; i) SARS-Cov-2 replicase 1AB; j) enhanced green fluorescent protein (EGFP); and k) influenza hemagglutinin (HA).

20. The pharmaceutical composition according to claim 18, wherein the nucleic acid encoding the recombinant protein or peptide that induces an inflammatory cell death response encodes a protein or peptide selected from: a) Gasdermin D (GSDMD); b) Gasdermin E (GSDME); c) absent in melanoma 2 (AIM2); d) interleukin 33 (IL33); e) thioredoxin-interacting protein (TXNIP); f) interleukin-1 receptor-associated kinase 1 (IRAK1); and g) NLR family pyrin domain-containing protein 3 (NLRP3).

21. The pharmaceutical composition according to claim 18, wherein the recombinant protein or peptide that induces the activation and persistence of antigen-presenting cells and cytotoxic T cells within the tumor microenvironment, and the recombinant protein or peptide that induces the formation of tumor-associated tertiary lymphoid structures are selected from: a) chemokine ligand 9 (CXCL9); b) chemokine ligand 10 (CXCL10); c) chemokine ligand 12 (CXCL12); d) chemokine ligand 13 (CXCL13); e) C-C motif chemokine ligand 19 (CCL19); f) C-C motif chemokine ligand 21 (CCL21); g) interleukin-1β (IL1β); h) interleukin-4 (IL4); i) interleukin-4 receptor (IL4R); j) Interleukin-21 (IL21); k) Interleukin-22 (IL22); l) Interleukin-22 receptor (IL22R); m) Interleukin-23; n) Interleukin-13 (IL13); o) Lymphotoxin β receptor (LTβR); p) β-2-microglobulin (B2M); q) Programmed death-ligand 1 (PD-L1); r) Integrin-associated protein (CD47); s) Transforming growth factor β receptor I (TGFβRI)-extracellular domain, soluble; t) Transforming growth factor β receptor II (TGFβRII)-extracellular domain, soluble; u) Transforming growth factor β receptor III (TGFβRIII)-extracellular domain, soluble; v) A protein consisting of the extracellular domain of at least one protein selected from transforming growth factor β receptor I (TGFβRI), transforming growth factor β receptor II (TGFβRII), and transforming growth factor β receptor III (TGFβRIII); w) Mothers against decapentaplegic homolog 2 (SMAD2); and x) Mothers against decapentaplegic homolog 3 (SMAD3).

22. A pharmaceutical composition for delivery to a subject using lipid nanoparticles, electroporation, or other suitable delivery mechanisms, comprising the recombinant virus of claim 18.

23. A method of treating cancer in a subject, comprising administering to the subject the pharmaceutical composition of claim 18.

24. A method of treating a benign tumor in a subject, comprising administering to the subject the pharmaceutical composition of claim 18.

25. A pharmaceutical composition comprising allogeneic T cells, wherein the allogeneic T cells comprise: a) A chimeric antigen receptor (CAR) or antigen-specific T cell receptor (TCR) that confers antigen specificity to the T cells; b) A recombinant protein or peptide, or mRNA encoding the recombinant protein or peptide, that induces the activation and persistence of antigen-presenting cells and cytotoxic T cells within the tumor microenvironment; and c) A recombinant protein or peptide, or mRNA encoding the recombinant protein or peptide, that induces the formation of tumor-associated tertiary lymphoid structures.

26. The pharmaceutical composition according to claim 25, wherein the chimeric antigen receptor (CAR) or antigen-specific T cell receptor (TCR) is specific for a protein selected from: a) SARS-Cov-2 spike (S); b) SARS-Cov-2 envelope (E); c) SARS-Cov-2 membrane (M); d) SARS-Cov-2 nucleocapsid (N); e) SARS-CoV-2 spike S-2P and RBD antigens; f) SARS-Cov-2 ORF3a; g) SARS-Cov-2 ORF7a; h) SARS-Cov-2 ORF8; i) SARS-Cov-2 replicase 1AB; j) Enhanced green fluorescent protein (EGFP); and k) Influenza hemagglutinin (HA).

27. The pharmaceutical composition according to claim 25, wherein the recombinant protein or peptide that activates and persists in the tumor microenvironment for antigen-presenting cells and cytotoxic T cells, and the recombinant protein or peptide that induces the formation of tumor-associated tertiary lymphoid structures are selected from: a) Chemokine ligand 9 (CXCL9); b) Chemokine ligand 10 (CXCL10); c) Chemokine ligand 12 (CXCL12); d) Chemokine ligand 13 (CXCL13); e) C-C motif chemokine ligand 19 (CCL19); f) C-C motif chemokine ligand 21 (CCL21); g) Interleukin 1β (IL1β); h) Interleukin 4 (IL4); i) Interleukin 4 receptor (IL4R); j) Interleukin 21 (IL21); k) Interleukin 22 (IL22); l) Interleukin 22 receptor (IL22R); m) Interleukin 23; n) Interleukin 13 (IL13); o) Lymphotoxin β receptor (LTβR); p) β-2-microglobulin (B2M); q) Programmed death-ligand 1 (PD-L1); r) Integrin-associated protein (CD47); s) Transforming growth factor β receptor I (TGFβRI)-extracellular domain, soluble; t) Transforming growth factor β receptor II (TGFβRII)-extracellular domain, soluble; u) Transforming growth factor β receptor III (TGFβRIII)-extracellular domain, soluble; v) A protein composed of the extracellular domain of at least one protein selected from transforming growth factor β receptor I (TGFβRI), transforming growth factor β receptor II (TGFβRII), and transforming growth factor β receptor III (TGFβRIII); w) Mothers against decapentaplegic homolog 2 (SMAD2); and x) Mothers against decapentaplegic homolog 3 (SMAD3).

28. A pharmaceutical composition suitable for delivery to a subject using lipid nanoparticles, electroporation, or other suitable delivery mechanisms, comprising the allogeneic cells according to claim 25.

29. A method of treating cancer in a subject, comprising administering to the subject the pharmaceutical composition according to claim 25.

30. A method of treating a benign tumor in a subject, comprising administering to the subject the pharmaceutical composition according to claim 25.

31. A pharmaceutical composition comprising a recombinant nucleic acid, lipid nanoparticles, or an electroporation system, wherein the recombinant nucleic acid encodes a recombinant protein or peptide that induces an immune response.

32. The pharmaceutical composition according to claim 31, wherein the recombinant protein or peptide that induces an immune response is selected from: a) SARS-Cov-2 spike (S); b) SARS-Cov-2 envelope (E); c) SARS-Cov-2 membrane (M); d) SARS-Cov-2 nucleocapsid (N); e) SARS-CoV-2 spike S-2P and RBD antigens; f) SARS-Cov-2 ORF3a; g) SARS-Cov-2 ORF7a; h) SARS-Cov-2 ORF8; i) SARS-Cov-2 replicase 1AB; j) Enhanced green fluorescent protein (EGFP); and k) Influenza hemagglutinin (HA).

33. A pharmaceutical composition for delivery to a subject using lipid nanoparticles, electroporation, or other delivery mechanisms, comprising the recombinant nucleic acid or delivery system of claim 31.

34. A method of treating cancer in a subject, comprising administering to the subject the pharmaceutical composition of claim 31.

35. A method of treating a benign tumor in a subject, comprising administering to the subject the pharmaceutical composition of claim 31.

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