Major histocompatibility complex (MHC) composition and method of use thereof

Abstract

Providing major histocompatibility complex (MHC) compositions and methods for using the same. [Solution] An immunotherapy composition comprising a class I MHC component, a non-classical MHC class I component, or a class II MHC component, and a method of using the same are described. The class I MHC, non-classical class I MHC, and class II MHC components may be MHC components that do not exist in nature. Furthermore, an immunotherapy composition comprising a nucleic acid encoding an inactivated CRISPR-related nuclease fused to a TET enzyme, and a gRNA that targets a methylated region of a genetic element that controls the expression of MHC genes, and a method of using the same are described. The compositions and methods described herein may further include the administration of an immune checkpoint inhibitor and the immunotherapy composition.

Description

[Technical Field]

[0001] Related applications This application claims the benefits of U.S. Provisional Application No. 62 / 609,589, filed on 22 December 2017. This Provisional Application is incorporated herein by reference in its entirety for all purposes. [Background technology]

[0002] Background of Disclosure Major histocompatibility complex (MHC) molecules are crucial in the body's immune response because they bind to antigens derived from pathogens or tumors and display these antigens on the cell surface for recognition by T cells. In humans, MHC genes, often referred to as human leukocyte antigen (HLA) genes, include class I, class II MHC, non-classical MHC I, and non-classical MHC II genes. Class I MHC molecules are ubiquitously expressed on the surface of adult somatic cells and typically present cytosolic peptides, but can also present extracellular antigens through cross-presentation mechanisms. Non-classical MHC I molecules are associated with natural killer (NK) cells and CD8 + Class II MHC molecules can be recognized by T cells. While their expression is typically restricted to professional antigen-presenting cells (APCs) such as dendritic cells, macrophages, and B cells, the expression of MHC class II molecules can be induced in other cell types, such as tumor cells. Non-classical MHC II molecules are generally not exposed on the cell surface but are exposed on the internal membrane of lysosomes.

[0003] One way tumor cells evade recognition by T cells is by expressing immune checkpoints, masking their identity as cancerous cells and escaping attack from the immune system. Immune checkpoint inhibitors are used to block this mechanism and allow T cells to recognize such cells as cancerous. However, such therapies have been found to be ineffective in some cancers.

[0004] Immune checkpoint inhibitors can only be effective if T cells can, firstly, recognize tumor cells. Some cancers have been shown to lack or significantly reduce the expression of MHC molecules, which can interfere with this tumor recognition, and this may be a way for tumor cells to evade detection. Therefore, it is desirable to develop methods to increase MHC expression in cancer cells, as this can not only increase the body's innate immune response in the absence of any additional treatments, but also function as a way to enhance the effectiveness of therapeutic agents such as immune checkpoint inhibitors in cancers that were previously unresponsive. [Overview of the project] [Means for solving the problem]

[0005] Summary of Disclosure Immunotherapy compositions comprising nucleic acid molecules encoding MHC components or fragments thereof are provided herein. The MHC components can be formulated with at least one, two, three, four or more different excipients for delivery to a subject or organism. The MHC components may be naturally occurring MHC components, or alternatively, the MHC components may not be naturally occurring. In some embodiments, the MHC components are not naturally occurring and exhibit enhanced recognition by T cells compared to naturally occurring MHC components. In some embodiments, the MHC components are naturally occurring, and cells expressing heterologous MHC components have enhanced recognition by T cells compared to similar cells that have not been modified to express heterologous MHC components. In some examples, the modified cells are cancer cells. Such cancer cells may be solid tumor cancer cells. Such cancer cells may be breast cancer cells, prostate cancer cells, lung cancer cells, pancreatic cancer cells, ovarian cancer cells, liver cancer cells, colon cancer cells, or any other cancer cells.

[0006] In some embodiments, the nucleic acid molecules of this disclosure encode MHC components that do not exist in nature. These non-naturally occurring MHC components may be modified MHC components that have high sequence homology to naturally occurring MHC components.

[0007] In some embodiments, the compositions herein contain homologs of naturally occurring MHC components that do not exist in nature. Such homologs may include at least one variant in comparison to a nucleic acid molecule encoding a naturally occurring MHC component. In some embodiments, the variant is a mutation, insertion, deletion, or duplication. The MHC homologs herein preferably have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% amino acid sequence homology to a naturally occurring MHC component. In some embodiments, the nucleic acid molecule is at least 80%, 90%, 95%, 98%, or 99% similar to, or has at least 80%, 90%, 95%, 98%, or 99% sequence homology to, a nucleic acid sequence encoding a naturally occurring MHC component. In some embodiments, the nucleic acid molecule encodes an MHC component that is at least 80%, 90%, 95%, 98%, or 99% similar to a naturally occurring MHC component, or has at least 80%, 90%, 95%, 98%, or 99% sequence homology to it. In some embodiments, the nucleic acid molecule is at least 80%, 90%, 95%, 98%, or 99% similar to a nucleic acid sequence encoding a naturally occurring MHC component. In some embodiments, the nucleic acid encodes an MHC component that is at least 80%, 90%, 95%, 98%, or 99% similar to a naturally occurring MHC component.

[0008] In some embodiments, the MHC components are genes selected from the list consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-G, HLA-F, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA-DQB1, HLA-DOA, HLA-DOB, HLA-DMA, HLA-DMB, HLA-DPA1, and HLA-DPB1. The MHC components may be class I MHC components. In some embodiments, the class I MHC components are heavy (α) chains, light chains (β2 microglobulin), or a combination thereof.

[0009] In some embodiments, the immunotherapy composition further comprises a second nucleic acid molecule encoding a second class I MHC component or a functional (e.g., antigenic) fragment thereof. In some embodiments, the second class I MHC component is a heavy (α) chain, a light chain (β2-microglobulin), or a combination thereof. In some embodiments, the second class I MHC component is a naturally occurring or non-naturally occurring MHC component. In some embodiments, the naturally occurring or non-naturally occurring MHC component is a class II MHC component. In some embodiments, the class II MHC component includes an alpha (α) chain, a beta (β) chain, or a combination thereof. In some embodiments, the immunotherapy composition further comprises a second nucleic acid molecule encoding a second class II MHC component or a functional fragment thereof. In some embodiments, the second class II MHC component includes an alpha (α) chain, a beta (β) chain, or a combination thereof. In some embodiments, the second class II MHC component is a naturally occurring or non-naturally occurring MHC component. In some embodiments, the nucleic acid molecule is DNA or RNA. In some embodiments, the nucleic acid is a plasmid. In some embodiments, the nucleic acid is a viral vector. In some embodiments, the viral vector is an alphavirus, retrovirus, or adenovirus. These include herpesviruses, poxviruses, lentiviruses, oncolytic viruses, reoviruses, or adeno-associated viruses (AAVs). In some embodiments, the nucleic acids are formulated for targeted delivery to tumor cells. In some embodiments, the nucleic acids are formulated in vesicles such as liposomes, exosomes, lipid nanoparticles, or biomaterials. In some embodiments, the liposomes contain additional therapeutic compounds, polyethylene glycol (PEG), cell membrane permeable peptides, ligands, aptamers, antibodies, or combinations thereof. In some embodiments, the liposomes are formulated for targeted delivery to cancer cells. In some embodiments, the method further comprises at least one pharmaceutically acceptable excipient, diluent, or carrier. In some embodiments, the method further comprises a unit dose of the nucleic acids disclosed herein, between about 0.01 μg and about 100 μg. In other embodiments, the method further comprises a unit dose (does) of an MHC molecule encoded by the nucleic acids disclosed herein, between about 0.01 μg and about 100 μg.

[0010] Methods for treating cancer in an individual, comprising the step of administering to the individual a nucleic acid molecule encoding an MHC component or a functional fragment thereof, are also provided herein. In some embodiments, the MHC component may not be naturally occurring. In other embodiments, the MHC component is naturally occurring. In some embodiments, the naturally occurring MHC component exhibits enhanced recognition by T cells compared to the naturally occurring MHC component. In some embodiments, the cancer is ovarian cancer, pancreatic cancer, or colon cancer. In some embodiments, the cancer has reduced MHC expression. In some embodiments, the method further comprises the step of determining the sequence of the individual's native MHC component. In some embodiments, the method further comprises the step of diagnosing cancer as having reduced MHC expression, comprising the steps of (a) obtaining a biological sample from an individual, (b) isolating cancerous cells from the biological sample, and (c) detecting whether the MHC expression in the isolated cancerous cells is reduced compared to a control. In some embodiments, the individual has been previously administered an additional therapeutic compound selected from the group consisting of immune checkpoint inhibitors, immune checkpoint stimulants, cancer vaccines, small molecule therapies, monoclonal antibodies, cytokines, cell therapies, or combinations thereof. In some embodiments, the method further includes the step of administering the additional therapeutic compound to the individual. In some embodiments, the additional therapeutic compound is an immune checkpoint inhibitor, immune checkpoint stimulant, cancer vaccine, small molecule therapy, monoclonal antibody, cytokine, or cell therapy. In some embodiments, the immune checkpoint inhibitor is A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM-3, VISTA, or a molecule that binds to their ligands. In some embodiments, the immune checkpoint stimulant is CD27, CD28, CD40, CD122, CD137, OX40, GITR, ICOS, or a molecule that binds to their ligands. In some embodiments, small molecule therapies include proteasome inhibitors, tyrosine kinase inhibitors, cyclin-dependent kinase inhibitors, or poly-ADP-ribose polymerase (PARP) inhibitors.In some embodiments, the cytokine is INFα, INFβ, IFNγ, or TNF. In some embodiments, the cell therapy is adoptive T cell transfer (ACT) therapy. Alternatively, the cell therapy may be chimeric antigen receptor (CAR) T cell therapy or T cell antigen coupler (TAC) T cell therapy.

[0011] In some embodiments, administration of a nucleic acid molecule to an individual results in cancer that exhibits increased sensitivity to at least one additional therapeutic compound. In some embodiments, the nucleic acid molecule is a non-naturally occurring MHC component containing at least one variant compared to a nucleic acid molecule encoding a naturally occurring MHC component. In some embodiments, the variant is a mutation, insertion, deletion, or duplication. In some embodiments, the MHC component is a gene selected from the list consisting of HLA-A, HLA-B, HLA-C, HLA-DRA, HLA-E, HLA-G, HLA-F, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA-DQB1, HLA-DOA, HLA-DOB, HLA-DMA, HLA-DMB, HLA-DPA1, and HLA-DPB1. In some embodiments, the nucleic acid molecule is at least 95% similar to a nucleic acid sequence encoding a naturally occurring MHC component. In some embodiments, the nucleic acid molecule is at least 80% similar to a nucleic acid sequence encoding a naturally occurring MHC component. In some embodiments, the MHC component is a class I MHC component. In some embodiments, the class I MHC component is a heavy (α) chain, a light chain (β2 microglobulin), or a combination thereof. In some embodiments, the immunotherapy composition further comprises a second nucleic acid molecule encoding a second class I MHC component or a fragment thereof. In some embodiments, the second class I MHC component is a heavy (α) chain, a light chain (β2 microglobulin), or a combination thereof. In some embodiments, the second class I MHC component is a naturally occurring or non-naturally occurring MHC component. In some embodiments, the MHC component is a class II MHC component. In some embodiments, the class II MHC component includes an alpha (α) chain, a beta (β) chain, or a combination thereof. In some embodiments, the immunotherapy composition further comprises a second nucleic acid molecule encoding a second class II MHC component or a fragment thereof. In some embodiments, the second class II MHC component includes an alpha (α) chain, a beta (β) chain, or a combination thereof.In some embodiments, the second class II MHC component is a naturally occurring or non-naturally occurring MHC component. In some embodiments, the nucleic acid molecule is DNA or RNA. In some embodiments, the nucleic acid is a plasmid. In some embodiments, the nucleic acid is a viral vector. In some embodiments, the viral vector is an alphavirus, retrovirus, adenovirus, herpesvirus, poxvirus, lentivirus, oncolytic virus, reovirus, or adeno-associated virus (AAV). In some embodiments, the nucleic acid is formulated for targeted delivery to tumor cells. In some embodiments, the nucleic acid is formulated in liposomes. In some embodiments, the liposomes contain additional therapeutic compounds, polyethylene glycol (PEG), cell membrane permeable peptides, ligands, aptamers, antibodies, or combinations thereof. In some embodiments, the liposomes are formulated for targeted delivery to cancer cells.

[0012] Immunotherapy compositions comprising a nucleic acid encoding an inactivated CRISPR-related nuclease fused to an enzyme that modifies nucleic acid molecules (e.g., a TET enzyme) and a guide RNA (gRNA) having a region complementary to the transcription factor or promoter of an MHC gene are also provided herein. In some embodiments, the MHC genes are HLA-A, HLA-B, HLA-C, HLA-E, HLA-G, HLA-F, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA-DQB1, HLA-DOA, HLA-DOB, HLA-DMA, HLA-DMB, HLA-DPA1, and HLA-DPB1. In some embodiments, the inactivated CRISPR-related nuclease is an inactivated Cas9 (dCas9). In some embodiments, the TET enzyme is TET1, TET2, TET3, or their catalytic domains. In some embodiments, the nucleic acid molecule is DNA or RNA. In some embodiments, the nucleic acid is a plasmid. In some embodiments, the nucleic acid is a viral vector. In some embodiments, the viral vector is an alphavirus, retrovirus, adenovirus, herpesvirus, poxvirus, lentivirus, oncolytic virus, reovirus, or adeno-associated virus (AAV). In some embodiments, the nucleic acid is formulated for targeted delivery to tumor cells. In some embodiments, the nucleic acid is formulated in liposomes. In some embodiments, the liposomes contain additional therapeutic compounds, polyethylene glycol (PEG), cell membrane permeable peptides, ligands, aptamers, antibodies, or combinations thereof. In some embodiments, the liposomes are formulated for targeted delivery to cancer cells. In some embodiments, the composition further comprises at least one pharmaceutically acceptable excipient, diluent, or carrier.

[0013] Also provided herein is a method for increasing the expression of MHC genes in cancer in an individual, comprising the step of administering to the individual an immunotherapy composition comprising a nucleic acid encoding a deactivated CRISPR-related nuclease fused to a TET enzyme and a guide RNA (gRNA) having a region complementary to the transcription factor or promoter of an MHC gene. In some embodiments, the MHC genes are HLA-A, HLA-B, HLA-C, HLA-E, HLA-G, HLA-F, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA-DQB1, HLA-DOA, HLA-DOB, HLA-DMA, HLA-DMB, HLA-DPA1, and HLA-DPB1. In some embodiments, the cancer is ovarian cancer, pancreatic cancer, or colon cancer. In some embodiments, the cancer has reduced MHC expression. In some embodiments, the method further includes a step of diagnosing cancer as having reduced MHC expression, comprising the steps of (a) obtaining a biological sample from an individual, (b) isolating cancerous cells from the biological sample, and (c) detecting whether or not MHC expression is reduced in the isolated cancerous cells. In some embodiments, the individual has been previously administered an additional therapeutic compound selected from the group consisting of immune checkpoint inhibitors, immune checkpoint stimulants, cancer vaccines, small molecule therapies, monoclonal antibodies, cytokines, cell therapies, or combinations thereof. In some embodiments, the method further includes a step of administering the additional therapeutic compound to the individual. In some embodiments, the additional therapeutic compound is an immune checkpoint inhibitor, immune checkpoint stimulant, cancer vaccine, small molecule therapy, monoclonal antibody, cytokine, or cell therapy. In some embodiments, the immune checkpoint inhibitor is A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM-3, VISTA, or a molecule that binds to their ligands. In some embodiments, the immune checkpoint stimulator is a molecule that binds to CD27, CD28, CD40, CD122, CD137, OX40, GITR, ICOS, or their ligands.In some embodiments, the small molecule therapy is a proteasome inhibitor, tyrosine kinase inhibitor, cyclin-dependent kinase inhibitor, or poly-ADP-ribose polymerase (PARP) inhibitor. In some embodiments, the cytokine is INFα, INFβ, IFNγ, or TNF. In some embodiments, the cell therapy is adoptive T cell transfer (ACT) therapy. Alternatively, the cell therapy may be chimeric antigen receptor (CAR) T cell therapy or T cell antigen coupler (TAC) T cell therapy.

[0014] In some embodiments, the expression of nucleic acid molecules by cancer results in cancer showing increased sensitivity to at least one additional therapeutic compound. In some embodiments, the inactivated CRISPR-related nuclease is inactivated Cas9 (dCas9). In some embodiments, the TET enzyme is TET1, TET2, TET3 or their catalytic domains. In some embodiments, the nucleic acid molecule is DNA or RNA. In some embodiments, the nucleic acid is a plasmid. In some embodiments, the nucleic acid is a viral vector. In some embodiments, the viral vector is an alphavirus, retrovirus, adenovirus, herpesvirus, poxvirus, lentivirus, oncolytic virus, reovirus, or adeno-associated virus (AAV). In some embodiments, the nucleic acid is formulated for targeted delivery to tumor cells. In some embodiments, the nucleic acid is formulated in liposomes. In some embodiments, the liposomes contain an additional therapeutic compound, polyethylene glycol (PEG), cell membrane permeable peptides, ligands, aptamers, antibodies, or combinations thereof. In some embodiments, liposomes are formulated for targeted delivery to cancer cells.

[0015] Furthermore, immunotherapy compositions comprising nucleic acid molecules encoding regulators of MHC molecules are provided herein. In some embodiments, the regulators of MHC molecules are selected from the group consisting of transactivators, transcription factors, acetyltransferases, methyltransferases, elongation factors, and any combination thereof. In some embodiments, the transactivators are selected from the group consisting of class II, major histocompatibility complex, transactivator (CIITA), and NOD-like receptor family CARD domain-containing 5 (NLRC5). In some embodiments, the transcription factors are selected from the group consisting of nuclear transcription factor Y (NF-Y), cAMP response element-binding protein (CREB), regulatory factor X (RFX), interferon regulatory factor (IRF), signaling and transcription activator (STAT), ubiquitous transcription factor (USF), and activated B cell nuclear factor copper light chain enhancer (NF-κB). In some embodiments, NF-Y is selected from the group consisting of NF-Ya, NF-Yb, and NF-Yc. In some embodiments, RFX is selected from the group consisting of RFXANK / RFXB, RFX5, and RFXAP. In some embodiments, IRF is selected from the group consisting of IRF-1, IRF-2, IRF-3, IRF-4, IRF-5, IRF-6, IRF-7, IRF-8, and IRF-9. In some embodiments, STAT is selected from the group consisting of STAT-1, STAT-2, STAT-3, STAT-4, STAT-5, and STAT-6. In some embodiments, USF is selected from the group consisting of USF-1 and USF-2. In some embodiments, acetyltransferase is selected from the group consisting of CREB-binding protein (CBP), p300, and p300 / CBP-related factor (pCAF). In some embodiments, methyltransferase is Zeste homolog 2 enhancer (EZH2), protein arginine N-methyltransferase 1 (PRMT1), and coactivator-bound arginine methyltransferase 1 (CARM1). In some embodiments, the elongation factor is a positive transcription elongation factor (pTEF). bIn some embodiments, the nucleic acid molecule is DNA or RNA. In some embodiments, the nucleic acid is a plasmid. In some embodiments, the nucleic acid is a viral vector. In some embodiments, the viral vector is an alphavirus, retrovirus, adenovirus, herpesvirus, poxvirus, lentivirus, oncolytic virus, reovirus, or adeno-associated virus (AAV). In some embodiments, the nucleic acid is formulated for targeted delivery to tumor cells. In some embodiments, the nucleic acid is formulated in liposomes. In some embodiments, the liposomes contain additional therapeutic compounds, polyethylene glycol (PEG), cell membrane permeable peptides, ligands, aptamers, antibodies, or combinations thereof. In some embodiments, the liposomes are formulated for targeted delivery to cancer cells. In some embodiments, the immunotherapy composition further comprises at least one pharmaceutically acceptable excipient, diluent, or carrier.

[0016] Furthermore, the present invention provides a method for treating cancer in an individual, comprising the step of administering to the individual a nucleic acid molecule encoding a regulator of MHC molecules. In some embodiments, the cancer is ovarian cancer, pancreatic cancer, or colon cancer. In some embodiments, the cancer has reduced MHC expression. In some embodiments, the method further comprises the step of diagnosing the cancer as having reduced MHC expression, comprising the steps of (a) obtaining a biological sample from an individual, (b) isolating cancerous cells from the biological sample, and (c) detecting whether the MHC expression in the isolated cancerous cells is reduced compared to a control. In some embodiments, the individual has been previously administered an additional therapeutic compound selected from the group consisting of immune checkpoint inhibitors, immune checkpoint stimulants, cancer vaccines, small molecule therapies, monoclonal antibodies, cytokines, cell therapies, or combinations thereof. In some embodiments, the method further comprises the step of administering the additional therapeutic compound to the individual. In some embodiments, the additional therapeutic compound is an immune checkpoint inhibitor, immune checkpoint stimulant, cancer vaccine, small molecule therapy, monoclonal antibody, cytokine, or cell therapy. In some embodiments, the immune checkpoint inhibitor is A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM-3, VISTA, or a molecule that binds to their ligands. In some embodiments, the immune checkpoint stimulator is CD27, CD28, CD40, CD122, CD137, OX40, GITR, ICOS, or a molecule that binds to their ligands. In some embodiments, the small molecule therapy is a proteasome inhibitor, tyrosine kinase inhibitor, cyclin-dependent kinase inhibitor, or poly-ADP-ribose polymerase (PARP) inhibitor. In some embodiments, the cytokine is INFα, INFβ, IFNγ, or TNF. In some embodiments, the cell therapy is adoptive T cell transfer (ACT) therapy. Alternatively, the cell therapy may be chimeric antigen receptor (CAR) T cell therapy or T cell antigen coupler (TAC) T cell therapy.

[0017] In some embodiments, administration of nucleic acid molecules to an individual results in cancer showing increased sensitivity to at least one additional therapeutic compound. In some embodiments, the regulators of MHC molecules are selected from the group consisting of transactivators, transcription factors, acetyltransferases, methyltransferases, elongation factors, and any combination thereof. In some embodiments, the transactivators are selected from the group consisting of class II, major histocompatibility complex, transactivator (CIITA), and NOD-like receptor family CARD domain-containing 5 (NLRC5). In some embodiments, the transcription factors are selected from the group consisting of nuclear transcription factor Y (NF-Y), cAMP response element-binding protein (CREB), regulatory factor X (RFX), interferon regulatory factor (IRF), signaling and transcription activator (STAT), ubiquitous transcription factor (USF), and activated B cell nuclear factor copper light chain enhancer (NF-κB). In some embodiments, NF-Y is selected from the group consisting of NF-Ya, NF-Yb, and NF-Yc. In some embodiments, RFX is selected from the group consisting of RFXANK / RFXB, RFX5, and RFXAP. In some embodiments, IRF is selected from the group consisting of IRF-1, IRF-2, IRF-3, IRF-4, IRF-5, IRF-6, IRF-7, IRF-8, and IRF-9. In some embodiments, STAT is selected from the group consisting of STAT-1, STAT-2, STAT-3, STAT-4, STAT-5, and STAT-6. In some embodiments, USF is selected from the group consisting of USF-1 and USF-2. In some embodiments, acetyltransferase is selected from the group consisting of CREB-binding protein (CBP), p300, and p300 / CBP-related factor (pCAF). In some embodiments, methyltransferase is Zeste homolog 2 enhancer (EZH2), protein arginine N-methyltransferase 1 (PRMT1), and coactivator-bound arginine methyltransferase 1 (CARM1). In some embodiments, the elongation factor is a positive transcription elongation factor (pTEF). bIn some embodiments, the nucleic acid molecule is DNA or RNA. In some embodiments, the nucleic acid is a plasmid. In some embodiments, the nucleic acid is a viral vector. In some embodiments, the viral vector is an alphavirus, retrovirus, adenovirus, herpesvirus, poxvirus, lentivirus, oncolytic virus, reovirus, or adeno-associated virus (AAV). In some embodiments, the nucleic acid is formulated for targeted delivery to tumor cells. In some embodiments, the nucleic acid is formulated in liposomes. In some embodiments, the liposomes contain additional therapeutic compounds, polyethylene glycol (PEG), cell membrane permeable peptides, ligands, aptamers, antibodies, or combinations thereof. In some embodiments, the liposomes are formulated for targeted delivery to cancer cells.

[0018] Reference Any publications, patents, and patent applications referenced herein are incorporated herein by reference to the same extent as each individual publication, patent, or patent application is specifically and individually indicated as being incorporated herein by reference.

[0019] The features of this disclosure are described in detail in the attached claims. A more complete understanding of the features and advantages of this disclosure will be obtained by referring to the following detailed description, which specifies the explanatory embodiments in which the principles of this disclosure are utilized, and to the attached drawings. The present invention provides, for example, the following items. (Item 1) An immunotherapy composition comprising a nucleic acid molecule encoding a first MHC component or a fragment thereof, and at least one pharmaceutically acceptable excipient, diluent, or carrier. (Item 2) The immunotherapy composition according to item 1, wherein the nucleic acid molecule is a nucleic acid molecule that does not exist in nature, and the first MHC component is found in nature. (Item 3) The immunotherapy composition according to item 1, wherein the first MHC component is a non-naturally occurring protein or polypeptide. (Item 4) The immunotherapy composition according to item 3, wherein the non-naturally occurring MHC component exhibits enhanced recognition by T cells compared to a naturally occurring MHC component. (Item 5) The immunotherapy composition according to item 1, wherein the first MHC component is HLA-A, HLA-B, HLA-C, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA-DQB1, HLA-DOA, HLA-DOB, HLA-DMA, HLA-DMB, HLA-DPA1, HLA-DPB1, or a functional fragment thereof. (Item 6) The immunotherapy composition according to item 1, further comprising an immune checkpoint inhibitor, an immune checkpoint stimulator, a cancer vaccine, a small molecule therapy, a monoclonal antibody, a cytokine, a cell therapy, or a combination thereof. (Item 7) The immunotherapy composition according to item 2, wherein the nucleic acid molecule is at least 80% identical to a nucleic acid sequence encoding a naturally occurring MHC component. (Item 8) The immunotherapy composition according to item 1, wherein the MHC component is a class I MHC component. (Item 9) The immunotherapy composition according to item 8, wherein the class I MHC component is (a) a heavy (α) chain and a light chain (β2 microglobulin), or (b) comprises an allele represented by Table 3. (Item 10) The immunotherapy composition according to item 1, further comprising a second nucleic acid molecule encoding a second class I MHC component or a fragment thereof, wherein the first MHC component and the second MHC component are different. (Item 11) The immunotherapy composition according to item 10, wherein the second class I MHC component is a heavy (α) chain and a light chain (β2 microglobulin). (Item 12) The immunotherapy composition according to item 11, wherein the second class I MHC component is a naturally occurring MHC component. (Item 13) The immunotherapy composition according to item 1, wherein the first MHC component is a class II MHC component. (Item 14) The immunotherapy composition according to item 13, wherein the class II MHC component comprises an alpha (α) chain, a beta (β) chain, or a combination thereof. (Item 15) The immunotherapy composition according to item 13, further comprising a second nucleic acid molecule encoding a second class II MHC component or a fragment thereof. (Item 16) The immunotherapy composition according to item 15, wherein the second class II MHC component comprises an alpha (α) chain, a beta (β) chain, or a combination thereof. (Item 17) The immunotherapy composition according to item 16, wherein the second class II MHC component is a naturally occurring component. (Item 18) The immunotherapy composition according to item 2, wherein the nucleic acid encoding the MHC component is DNA or RNA. (Item 19) The immunotherapy composition according to item 2, wherein the nucleic acid encoding the MHC component is part of a plasmid. (Item 20) The immunotherapy composition according to item 2, wherein the nucleic acid encoding the MHC component is part of a viral vector. (Item 21) The immunotherapy composition according to item 20, wherein the viral vector is an alphavirus, retrovirus, adenovirus, herpesvirus, poxvirus, lentivirus, oncolytic virus, reovirus, or adeno-associated virus (AAV). (Item 22) The immunotherapy composition according to item 2, wherein the nucleic acid encoding the MHC component is formulated for targeted delivery to tumor cells. (Item 23) The immunotherapy composition according to item 2, wherein the nucleic acid is formulated in liposomes, exosomes, lipid nanoparticles, or biomaterials. (Item 24) The immunotherapy composition according to item 23, wherein the liposome comprises an additional therapeutic compound, polyethylene glycol (PEG), a cell membrane permeable peptide, a ligand, an aptamer, an antibody, or a combination thereof. (Item 25) The immunotherapy composition according to item 23, wherein the liposomes are formulated for targeted delivery to cancer cells. (Item 26) The immunotherapy composition according to item 1, wherein the first MHC component is an HLA having the alleles shown in Table 3. (Item 27) A method for treating cancer in an organism, comprising the step of administering to the organism a therapeutically effective amount of nucleic acid molecules encoding major histocompatibility complex (MHC) components or functional fragments thereof. (Item 28) The method according to item 26, wherein the non-MHC component increases T cell activation or enhances T cell recognition of cancer cells. (Item 29) The method according to item 26, wherein the cancer is ovarian cancer, pancreatic cancer, or colon cancer. (Item 30) The method according to item 26, wherein the cancer has reduced MHC expression. (Item 31) The method according to item 26, further comprising the step of determining the sequence of the native MHC components of the individual prior to the administration step. (Item 32) The method of item 26, further comprising the step of diagnosing cancer as having reduced MHC expression, the step of (a) obtaining a biological sample from the individual; (b) isolating cancer cells from the biological sample; and (c) detecting whether the MHC expression in the isolated cancer cells is reduced compared to a control. (Item 33) The method according to item 26, wherein the individual has previously been administered an additional therapeutic compound selected from the group consisting of immune checkpoint inhibitors, immune checkpoint stimulants, cancer vaccines, small molecule therapies, monoclonal antibodies, cytokines, cell therapies, or combinations thereof. (Item 34) The method according to item 26, further comprising the step of administering an additional therapeutic compound to the individual. (Item 35) The method according to item 34, wherein the additional therapeutic compound is an immune checkpoint inhibitor, an immune checkpoint stimulant, a cancer vaccine, a small molecule therapy, a monoclonal antibody, a cytokine, or a cell therapy. (Item 36) The method according to item 35, wherein the immune checkpoint inhibitor is A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM-3, VISTA, or a molecule that binds to one of these ligands. (Item 37) The method according to item 35, wherein the immune checkpoint stimulating factor is a molecule that binds to CD27, CD28, CD40, CD122, CD137, OX40, GITR, ICOS, or their ligands. (Item 38) The method according to item 35, wherein the small molecule therapy is a proteasome inhibitor, a tyrosine kinase inhibitor, a cyclin-dependent kinase inhibitor, or a poly-ADP-ribose polymerase (PARP) inhibitor. (Item 39) The method according to item 35, wherein the cytokine is INFα, INFβ, IFNγ, or TNF. (Item 40) The method described in item 35, wherein the cell therapy is adoptive T cell transfer (ACT) therapy. (Item 41) The method described in item 40, wherein the ACT therapy utilizes multiple chimeric antigen receptor (CAR) T cells. (Item 42) The ACT therapy described above is the method described in item 40, which utilizes multiple T cell antigen coupler (TAC) T cells. (Item 43) The method according to item 34, wherein administration of the nucleic acid molecule to the individual results in the cancer exhibiting increased sensitivity to at least one additional therapeutic compound. (Item 44) The method according to item 26, wherein the nucleic acid molecule encoding the naturally occurring MHC component includes at least one variant in comparison with a nucleic acid molecule encoding a naturally occurring MHC component. (Item 45) The method according to item 44, wherein the variant is a mutation, insertion, deletion, or duplication. (Item 46) The method according to item 44, wherein the MHC component is a gene selected from the list consisting of HLA-A, HLA-B, HLA-C, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA-DQB1, HLA-DOA, HLA-DOB, HLA-DMA, HLA-DMB, HLA-DPA1, and HLA-DPB1. (Item 47) The method according to item 44, wherein the nucleic acid molecule is at least 95% similar to the nucleic acid sequence encoding the naturally occurring MHC component. (Item 48) The method according to item 44, wherein the nucleic acid molecule is at least 80% similar to the nucleic acid sequence encoding the naturally occurring MHC component. (Item 49) The method according to item 26, wherein the MHC component that does not exist in nature is a class I MHC component. (Item 50) The method according to item 49, wherein the class I MHC component is a heavy (α) chain, a light chain (β2 microglobulin), or a combination thereof. (Item 51) The method according to item 49, wherein the immunotherapy composition further comprises a second nucleic acid molecule encoding a second class I MHC component or a fragment thereof. (Item 52) The method according to item 51, wherein the second class I MHC component is a heavy (α) chain, a light chain (β2 microglobulin), or a combination thereof. (Item 53) The method according to item 52, wherein the second class I MHC component is a naturally occurring or non-naturally occurring MHC component. (Item 54) The method according to item 26, wherein the MHC component that does not exist in nature is a class II MHC component. (Item 55) The method according to item 54, wherein the class II MHC component comprises an alpha (α) chain, a beta (β) chain, or a combination thereof. (Item 56) The method according to item 54, wherein the immunotherapy composition further comprises a second nucleic acid molecule encoding a second class II MHC component or a fragment thereof. (Item 57) The method according to item 56, wherein the second class II MHC component comprises an alpha (α) chain, a beta (β) chain, or a combination thereof. (Item 58) The method according to item 57, wherein the second class II MHC component is a naturally occurring or non-naturally occurring MHC component. (Item 59) The method according to item 26, wherein the nucleic acid molecule is DNA or RNA. (Item 60) The method according to item 26, wherein the nucleic acid is a plasmid. (Item 61) The method according to item 26, wherein the nucleic acid is a viral vector. (Item 62) The method according to item 61, wherein the viral vector is an alphavirus, retrovirus, adenovirus, herpesvirus, poxvirus, lentivirus, oncolytic virus, reovirus, or adeno-associated virus (AAV). (Item 63) The method according to item 26, wherein the nucleic acid is formulated for targeted delivery to tumor cells. (Item 64) The method according to item 26, wherein the nucleic acid is formulated in liposomes, exosomes, lipid nanoparticles, or biomaterials. (Item 65) The method according to item 64, wherein the liposome comprises an additional therapeutic compound, polyethylene glycol (PEG), a cell membrane permeable peptide, a ligand, an aptamer, an antibody, or a combination thereof. (Item 66) The method according to item 64, wherein the liposomes are formulated for targeted delivery to cancer cells. (Item 67) An immunotherapy composition comprising an inactivated CRISPR-related nuclease fused to a TET enzyme and a nucleic acid encoding a guide RNA (gRNA) having a region complementary to the transcription factor or promoter of an MHC gene. (Item 68) The immunotherapy composition according to item 67, wherein the MHC gene is HLA-A, HLA-B, HLA-C, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA-DQB1, HLA-DOA, HLA-DOB, HLA-DMA, HLA-DMB, HLA-DPA1, and HLA-DPB1. (Item 69) The immunotherapy composition according to item 67, wherein the inactivated CRISPR-related nuclease is inactivated Cas9 (dCas9). (Item 70) The immunotherapy composition according to item 67, wherein the TET enzyme is TET1, TET2, TET3, or their catalytic domains. (Item 71) The immunotherapy composition according to item 67, wherein the nucleic acid molecule is DNA or RNA. (Item 72) The immunotherapy composition according to item 67, wherein the nucleic acid is a plasmid. (Item 73) The immunotherapy composition according to item 67, wherein the nucleic acid is a viral vector. (Item 74) The immunotherapy composition according to item 73, wherein the viral vector is an alphavirus, retrovirus, adenovirus, herpesvirus, poxvirus, lentivirus, oncolytic virus, reovirus, or adeno-associated virus (AAV). (Item 75) The immunotherapy composition according to item 67, wherein the nucleic acid is formulated for targeted delivery to tumor cells. (Item 76) The immunotherapy composition according to item 67, wherein the nucleic acid is formulated in liposomes. (Item 77) The immunotherapy composition according to item 76, wherein the liposome comprises an additional therapeutic compound, polyethylene glycol (PEG), a cell membrane permeable peptide, a ligand, an aptamer, an antibody, or a combination thereof. (Item 78) The immunotherapy composition according to item 76, wherein the liposomes are formulated for targeted delivery to cancer cells. (Item 79) The immunotherapy composition according to item 67, further comprising at least one pharmaceutically acceptable excipient, diluent, or carrier. (Item 80) A method for increasing the expression of MHC genes in cancer in an individual, comprising the step of administering to the individual an immunotherapy composition comprising a nucleic acid encoding an inactivated CRISPR-related nuclease fused to a TET enzyme and a guide RNA (gRNA) having a region complementary to the transcription factor or promoter of the MHC gene. (Item 81) The method according to item 80, wherein the MHC genes are HLA-A, HLA-B, HLA-C, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA-DQB1, HLA-DOA, HLA-DOB, HLA-DMA, HLA-DMB, HLA-DPA1, and HLA-DPB1. (Item 82) The method according to item 80, wherein the cancer is ovarian cancer, pancreatic cancer, or colon cancer. (Item 83) The method according to item 80, wherein the cancer has reduced MHC expression. (Item 84) The method according to item 80, further comprising the step of diagnosing cancer as having reduced MHC expression, the step of (a) obtaining a biological sample from the individual; (b) isolating cancer cells from the biological sample; and (c) detecting whether or not MHC expression has decreased in the isolated cancer cells. (Item 85) The method according to item 80, wherein the individual has previously been administered an additional therapeutic compound selected from the group consisting of immune checkpoint inhibitors, immune checkpoint stimulants, cancer vaccines, small molecule therapies, monoclonal antibodies, cytokines, cell therapies, or combinations thereof. (Item 86) The method according to item 80, further comprising the step of administering an additional therapeutic compound to the individual. (Item 87) The method according to item 86, wherein the additional therapeutic compound is an immune checkpoint inhibitor, an immune checkpoint stimulant, a cancer vaccine, a small molecule therapy, a monoclonal antibody, a cytokine, or a cell therapy. (Item 88) The method according to item 87, wherein the immune checkpoint inhibitor is A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM-3, VISTA, or a molecule that binds to one of these ligands. (Item 89) The method according to item 87, wherein the immune checkpoint stimulating factor is a molecule that binds to CD27, CD28, CD40, CD122, CD137, OX40, GITR, ICOS, or their ligands. (Item 90) The method according to item 87, wherein the small molecule therapy is a proteasome inhibitor, a tyrosine kinase inhibitor, a cyclin-dependent kinase inhibitor, or a poly-ADP-ribose polymerase (PARP) inhibitor. (Item 91) The method according to item 87, wherein the cytokine is INFα, INFβ, IFNγ, or TNF. (Item 92) The method described in item 87, wherein the cell therapy is adoptive T cell transfer (ACT) therapy. (Item 93) The method described in item 92, wherein the ACT therapy utilizes multiple chimeric antigen receptor (CAR) T cells. (Item 94) The method described in item 92, wherein the ACT therapy utilizes multiple T cell antigen coupler (TAC) T cells. (Item 95) The method according to item 86, wherein the expression of the nucleic acid molecule by the cancer results in the cancer exhibiting increased sensitivity to the at least one additional therapeutic compound. (Item 96) The method according to item 80, wherein the inactivated CRISPR-related nuclease is inactivated Cas9 (dCas9). (Item 97) The method according to item 80, wherein the TET enzyme is TET1, TET2, TET3, or their catalytic domains. (Item 98) The method according to item 80, wherein the nucleic acid molecule is DNA or RNA. (Item 99) The method according to item 80, wherein the nucleic acid is a plasmid. (Item 100) The method according to item 80, wherein the nucleic acid is a viral vector. (Item 101) The method according to item 100, wherein the viral vector is an alphavirus, retrovirus, adenovirus, herpesvirus, poxvirus, lentivirus, oncolytic virus, reovirus, or adeno-associated virus (AAV). (Item 102) The method according to item 80, wherein the nucleic acid is formulated for targeted delivery to tumor cells. (Item 103) The method according to item 80, wherein the nucleic acid is formulated in liposomes. (Item 104) The method according to item 103, wherein the liposome comprises an additional therapeutic compound, polyethylene glycol (PEG), a cell membrane permeable peptide, a ligand, an aptamer, an antibody, or a combination thereof. (Item 105) The method according to item 103, wherein the liposomes are formulated for targeted delivery to cancer. (Item 106) An immunotherapy composition containing nucleic acid molecules that encode regulators of MHC molecules. (Item 107) The immunotherapy composition according to item 106, wherein the regulatory factor of the MHC molecule is selected from the group consisting of a transactivator, a transcription factor, an acetyltransferase, a methyltransferase, an elongation factor, and any combination thereof. (Item 108) The immunotherapy composition according to item 107, wherein the transactivator is selected from the group consisting of Class II, major histocompatibility complex, transactivator (CIITA), and NOD-like receptor family CARD domain-containing 5 (NLRC5). (Item 109) The immunotherapy composition according to item 107, wherein the transcription factor is selected from the group consisting of nuclear transcription factor Y (NF-Y), cAMP response element binding protein (CREB), regulatory factor X (RFX), interferon regulatory factor (IRF), signaling and transcriptional activator (STAT), ubiquitous transcription factor (USF), and activated B cell nuclear factor copper light chain enhancer (NF-κB). (Item 110) The immunotherapy composition according to item 109, wherein the NF-Y is selected from the group consisting of NF-Ya, NF-Yb, and NF-Yc. (Item 111) The immunotherapy composition according to item 109, wherein the RFX is selected from the group consisting of RFXANK / RFXB, RFX5, and RFXAP. (Item 112) The immunotherapy composition according to item 109, wherein the IRF is selected from the group consisting of IRF-1, IRF-2, IRF-3, IRF-4, IRF-5, IRF-6, IRF-7, IRF-8, and IRF-9. (Item 113) The immunotherapy composition according to item 109, wherein the STAT is selected from the group consisting of STAT-1, STAT-2, STAT-3, STAT-4, STAT-5, and STAT-6. (Item 114) The immunotherapy composition according to item 109, wherein the USF is selected from the group consisting of USF-1 and USF-2. (Item 115) The immunotherapy composition according to item 107, wherein the acetyltransferase is selected from the group consisting of CREB-binding protein (CBP), p300, and p300 / CBP-related factor (pCAF). (Item 116) The immunotherapy composition according to item 107, wherein the methyltransferase is Zeste homolog 2 enhancer (EZH2), protein arginine N-methyltransferase 1 (PRMT1), and coactivator-bound arginine methyltransferase 1 (CARM1). (Item 117) The aforementioned elongation factor is a positive transcription elongation factor (pTEF). b The immunotherapy composition described in item 107. (Item 118) The immunotherapy composition according to item 106, wherein the nucleic acid molecule is DNA or RNA. (Item 119) The immunotherapy composition according to item 106, wherein the nucleic acid is a plasmid. (Item 120) The immunotherapy composition according to item 106, wherein the nucleic acid is a viral vector. (Item 121) The immunotherapy composition according to item 120, wherein the viral vector is an alphavirus, retrovirus, adenovirus, herpesvirus, poxvirus, lentivirus, oncolytic virus, reovirus, or adeno-associated virus (AAV). (Item 122) The immunotherapy composition according to item 106, wherein the nucleic acid is formulated for targeted delivery to tumor cells. (Item 123) The immunotherapy composition according to item 106, wherein the nucleic acid is formulated in liposomes. (Item 124) The immunotherapy composition according to item 123, wherein the liposome comprises an additional therapeutic compound, polyethylene glycol (PEG), a cell membrane permeable peptide, a ligand, an aptamer, an antibody, or a combination thereof. (Item 125) The immunotherapy composition according to item 123, wherein the liposomes are formulated for targeted delivery to cancer cells. (Item 126) The immunotherapy composition according to item 106, further comprising at least one pharmaceutically acceptable excipient, diluent, or carrier. (Item 127) A method for treating cancer in an individual, comprising the step of administering to the individual a nucleic acid molecule encoding a regulator of MHC molecules. (Item 128) The method according to item 127, wherein the cancer is ovarian cancer, pancreatic cancer, or colon cancer. (Item 129) The method according to item 127, wherein the cancer has reduced MHC expression. (Item 130) The method according to item 127, further comprising the steps of (a) obtaining a biological sample from the individual, (b) isolating cancer cells from the biological sample, and (c) detecting whether the MHC expression in the isolated cancer cells is reduced compared to a control, wherein the cancer is diagnosed as having reduced MHC expression. (Item 131) The method according to item 127, wherein the individual has previously been administered an additional therapeutic compound selected from the group consisting of immune checkpoint inhibitors, immune checkpoint stimulants, cancer vaccines, small molecule therapies, monoclonal antibodies, cytokines, cell therapies, or combinations thereof. (Item 132) The method according to item 127, further comprising the step of administering an additional therapeutic compound to the individual. (Item 133) The method according to item 132, wherein the additional therapeutic compound is an immune checkpoint inhibitor, an immune checkpoint stimulant, a cancer vaccine, a small molecule therapy, a monoclonal antibody, a cytokine, or a cell therapy. (Item 134) The method according to item 133, wherein the immune checkpoint inhibitor is A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM-3, VISTA, or a molecule that binds to one of these ligands. (Item 135) The method according to item 133, wherein the immune checkpoint stimulating factor is a molecule that binds to CD27, CD28, CD40, CD122, CD137, OX40, GITR, ICOS, or their ligands. (Item 136) The method according to item 133, wherein the small molecule therapy is a proteasome inhibitor, a tyrosine kinase inhibitor, a cyclin-dependent kinase inhibitor, or a poly-ADP-ribose polymerase (PARP) inhibitor. (Item 137) The method according to item 133, wherein the cytokine is INFα, INFβ, IFNγ, or TNF. (Item 138) The method described in item 133, wherein the cell therapy is adoptive T cell transfer (ACT) therapy. (Item 139) The ACT therapy described above is the method described in item 133, which utilizes multiple chimeric antigen receptor (CAR) T cells. (Item 140) The ACT therapy described above is the method described in item 133, which utilizes multiple T cell antigen coupler (TAC) T cells. (Item 141) The method according to item 132, wherein administration of the nucleic acid molecule to the individual results in the cancer exhibiting increased sensitivity to the at least one additional therapeutic compound. (Item 142) The method according to item 127, wherein the regulatory factor of the MHC molecule is selected from the group consisting of a transactivator, a transcription factor, an acetyltransferase, a methyltransferase, an elongation factor, and any combination thereof. (Item 143) The method according to item 142, wherein the transactivator is selected from the group consisting of Class II, major histocompatibility complex, transactivator (CIITA), and NOD-like receptor family CARD domain-containing 5 (NLRC5). (Item 144) The method according to item 142, wherein the transcription factor is selected from the group consisting of nuclear transcription factor Y (NF-Y), cAMP response element binding protein (CREB), regulatory factor X (RFX), interferon regulatory factor (IRF), signaling and transcriptional activator (STAT), ubiquitous transcription factor (USF), and nuclear factor copper light chain enhancer of activated B cells (NF-κB). (Item 145) The method according to item 144, wherein the NF-Y is selected from the group consisting of NF-Ya, NF-Yb, and NF-Yc. (Item 146) The method according to item 144, wherein the RFX is selected from the group consisting of RFXANK / RFXB, RFX5, and RFXAP. (Item 147) The method according to item 144, wherein the IRF is selected from the group consisting of IRF-1, IRF-2, IRF-3, IRF-4, IRF-5, IRF-6, IRF-7, IRF-8, and IRF-9. (Item 148) The method according to item 144, wherein the STAT is selected from the group consisting of STAT-1, STAT-2, STAT-3, STAT-4, STAT-5, and STAT-6. (Item 149) The method according to item 144, wherein the USF is selected from the group consisting of USF-1 and USF-2. (Item 150) The method according to item 142, wherein the acetyltransferase is selected from the group consisting of CREB-binding protein (CBP), p300, and p300 / CBP-related factor (pCAF). (Item 151) The method according to item 142, wherein the methyltransferase is Zeste homolog 2 enhancer (EZH2), protein arginine N-methyltransferase 1 (PRMT1), and coactivator-bound arginine methyltransferase 1 (CARM1). (Item 152) The aforementioned elongation factor is a positive transcription elongation factor (pTEF). b ) The method described in item 142. (Item 153) The method according to item 127, wherein the nucleic acid molecule is DNA or RNA. (Item 154) The method according to item 127, wherein the nucleic acid is a plasmid. (Item 155) The method according to item 127, wherein the nucleic acid is a viral vector. (Item 156) The method according to item 155, wherein the viral vector is an alphavirus, retrovirus, adenovirus, herpesvirus, poxvirus, lentivirus, oncolytic virus, reovirus, or adeno-associated virus (AAV). (Item 157) The method according to item 127, wherein the nucleic acid is formulated for targeted delivery to tumor cells. (Item 158) The method according to item 127, wherein the nucleic acid is formulated in liposomes. (Item 159) The method according to item 158, wherein the liposome comprises an additional therapeutic compound, polyethylene glycol (PEG), a cell membrane permeable peptide, a ligand, an aptamer, an antibody, or a combination thereof. (Item 160) The method according to item 158, wherein the liposomes are formulated for targeted delivery to cancer cells. [Brief explanation of the drawing]

[0020] [Figure 1] Figures 1A to 1D illustrate the transfection of HLA-DR alleles in RKO colon cancer cell lines. Figure 1A shows no surface expression of either HLA receptor in parental RKO cells. Figure 1B shows no HLA-DR expression detected on the cell surface in RKO cells transfected with HLADR A alone. However, intracellular expression of the Myc-DKK tag (not shown in the data) indicated successful transfection. Figure 1C shows no HLA-DR surface expression in RKO cell lines transfected with HLADR B1 alone. However, GFP expression indicated successful transfection. Figure 1D shows high and moderate GFP expression due to surface expression of both alpha and beta chains in cells co-transfected with HLA-DR A and B.

[0021] [Figure 2]Figures 2A-2C illustrate HLA-DR allele transfection in RKO colon cancer and SKOV3 cell lines. Figure 2A shows flow cytometry analysis of parental RKO cells. Figure 2B shows flow cytometry analysis of GFP HLA-DRAB1*15 RKO cells. Figure 2C shows green fluorescent cytoplasm in co-transfected RKO cells when only punctate GFP vs. HLA-DR B is transfected.

[0022] [Figure 3] Figures 3A to 3D illustrate fluorescence images of stably cotransfected RKO and SKOV3 cells, as follows: RKO HLA-DR AB1 (Figure 3A); SKOV3 HLA-DR AB1 (Figure 3B); RKO HLA-DR AB3 (Figure 3C); and SKOV3 HLA-DR AB3 (Figure 3D).

[0023] [Figure 4A-B] Figure 4A illustrates the vector structure of HLA-DR B3.

[0024] Figure 4B illustrates the vector structure of HLA-DR B4.

[0025] [Figure 4C-D] Figure 4C illustrates the vector structure of HLA-DR B5.

[0026] Figure 4D illustrates the vector structure of HLA-DR alpha-a.

[0027] [Figure 4E] Figure 4E illustrates the vector structure of HLA-DR B1*15.

[0028] [Figure 5] Figure 5A illustrates two representative dendritic cells prepared from two different donors expressing high levels of HLA-DR and PD-L1.

[0029] Figure 5B illustrates primary T cells prepared for a mixed lymphocyte (MLR) reaction assay from two different donors genotyped as HLA-DR1.

[0030] Figure 5C illustrates RKO cells expressing high levels of PD-L1.

[0031] [Figure 6A-D] Figures 6A to 6F illustrate T cell proliferation when cultured with HLA-DR transfected RKO cells along with anti-PD-1 antibodies.

[0032] [Figure 6E-H] Figures 6A to 6F illustrate T cell proliferation when cultured with HLA-DR transfected RKO cells along with anti-PD-1 antibodies. Figure 6G illustrates that T cells did not proliferate when cultured with RKO parent cells.

[0033] Figure 6H illustrates that T cells did not proliferate without any treatment.

[0034] [Figure 7] Figure 7A illustrates T cell proliferation when cultured with parental RKO cells along with an anti-PD-1 antibody.

[0035] Figure 7B illustrates T cell proliferation when cultured with HLA-DR transfected RKO cells along with an anti-PD-1 antibody.

[0036] Figure 7C illustrates T cell proliferation when cultured with HLA-DR transfected RKO cells.

[0037] Figure 7D illustrates T cell proliferation when cultured with HLA-DR transfected RKO cells along with an anti-PD-1 antibody.

[0038] [Figure 8A-B]Figures 8A to 8C illustrate that HLA-DR transfected RKO cells increased T cell proliferation and inflammatory cytokine secretion. [Figure 8C] Figures 8A to 8C illustrate that HLA-DR transfected RKO cells increased T cell proliferation and inflammatory cytokine secretion. [Modes for carrying out the invention]

[0039] Detailed explanation of disclosure This specification discloses immunotherapy compositions and methods of using them for treating or preventing conditions such as cancer. The immunotherapy compositions described herein may include nucleic acid molecules encoding MHC components or functional fragments thereof, or regulators of nucleic acid molecules encoding MHC components or functional fragments thereof. Furthermore, immunotherapy compositions comprising MHC component polypeptides or functional fragments thereof, or regulators of nucleic acid molecules encoding MHC components or functional fragments thereof are disclosed herein. MHC components

[0040] As used herein, “MHC component” or “MHC molecule” refers to a nucleic acid encoding an MHC gene, a polypeptide encoded by an MHC gene, a gene or gene product associated with MHC, or a regulator of MHC or a regulator of a nucleic acid encoding an MHC component, or a functional fragment thereof. Thus, unless otherwise limited by the sentence, the term MHC molecule should encompass both nucleic acid sequences encoding MHC proteins and the proteins themselves. Furthermore, a functional fragment refers to a fragment of a protein or nucleic acid molecule that provides substantially the same function as the complete sequence. In some embodiments, the functional fragment is a nucleic acid sequence encoding the extracellular portion of a molecule or the extracellular portion of a protein described herein. In other examples, the functional fragment includes both the extracellular and transmembrane domains of a molecule (or the nucleic acid encoding them).

[0041] In this specification, MHC components may refer to mammalian MHC components, or more specifically, human MHC components, which may instead be referred to as human leukocyte antigens (HLA). For example, HLA genes that are MHC components include HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-H, HLA-J, HLA-K. , HLA-N, HLA-P, HLA-S, HLA-T, HLA-U, HLA-V, HLA-W, HLA-X, HLA-Y, HLA-Z, HLA-DRA, HLA-DR B1, HLA-DRB2, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DRB6, HLA-DRB7, HLA-DRB8, HLA-DRB9, HLA-DQA1, HLA-DQB1, HLA-DQA2, HLA-DQB2, HLA-DQB3, HLA-DOA, HLA-DOB, HLA-DMA, HLA-DMB This includes HLA-DPA1, HLA-DPB1, HLA-DPA2, HLA-DPB2, and HLA-DPA3. The gene or gene product associated with the MHC component may be β2-microglobulin (B2M). The MHC component can be used to describe the entire MHC molecule, or a part thereof or a functional fragment. The MHC molecule as used herein may be an MHC class I molecule, a non-classical MHC molecule, or an MHC class II molecule, or a homolog or functional fragment of any of the above.

[0042] Class I MHC molecules can induce an immune response by presenting peptides derived from cytosolic proteins to cytotoxic T cells. Class I MHC molecules can also present exogenous peptides through cross-presentation. Class I MHC molecules may contain two domains: a heavy (α) chain and a light chain (β2 microglobulin), which are linked by non-covalent bonds. The heavy (α) chain may further contain three extracellular domains: an α1 domain, an α2 domain, and an α3 domain, where the α2 and α3 domains form grooves to which the peptide presented by the class I MHC molecule binds. The non-classical MHC I molecules of this disclosure are used on natural killer (NK) cells and CD8 +HLA-E, HLA-F, and HLA-G can be recognized by T cells. HLA-E, HLA-F, and HLA-G are non-classical MHC I molecules encoded at the MHC I locus, exhibiting lower levels of heterogeneity compared to classical MHC I molecules. HLA-E expression is IFN-γ-inducible, while HLA-G expression can be induced by interferon-inducible transcription factors such as IRF-1 and other stimuli.

[0043] In this specification, MHC components may be class I MHC components or functional fragments thereof. Examples of functional fragments include any of the domains described above, but do not include entire MHC genes. For example, in one example, an MHC component includes a heavy (α) chain without a light chain (β2 microglobulin). In another example, an MHC component includes a light chain (β2 microglobulin) without a heavy (α) chain. In yet another example, a class I MHC component may include a heavy (α) chain, a light chain (β2 microglobulin), or a combination thereof. In some examples, an MHC component includes one or two of the α1, α2, and α3 domains, but not all three domains.

[0044] Class I MHC components may be human HLA-A genes, HLA-B genes, HLA-C genes, their polypeptide products, their homologs, or functional fragments thereof. Class I MHC components may be molecules encoded by any suitable HLA-A allele derived from the human genome. Class I MHC components may be molecules encoded by any suitable HLA-B allele derived from the human genome. MHC components can be molecules encoded by any suitable HLA-C allele derived from the human genome. Class I MHC components can be molecules encoded by any suitable β2 microglobulin allele derived from the human genome. In some cases, class I MHC components are fragments of class I MHC components. For example, class I MHC components can be exons or specific domains of class I MHC components, such as the α2 and α3 domains of the heavy chain. In some cases, class I MHC components are polypeptides encoded by class I MHC genes. Thus, this disclosure intends both the MHV and HLA polypeptide products and fragments (domains) described herein, as well as the nucleic acid molecules encoding them.

[0045] The heavy chains of class I MHC components can be functionally variable, and multiple different gene products can be produced by a single gene. Functionally variable products of class I MHC genes can be referred to as class I MHC serotypes. There may be at least 25 serotypes of HLA-A, at least 50 serotypes of HLA-B, and at least 12 serotypes of HLA-C. A class I MHC component can be any suitable class I MHC serotype. A class I MHC serotype may be HLA-A2, HLA-A3, or HLA-B8. Alleles representing these different serotypes can be selected from Table 3 attached herein. In some embodiments, the compositions described herein include one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more different MHC components, HLA alleles, or HLA alleles listed in Table 3, or nucleic acids encoding functional fragments thereof.

[0046] Nucleic acids encoding class I MHC components may include nucleic acids encoding class I MHC component polypeptides. For example, nucleic acids encoding class I MHC components may include nucleic acids encoding HLA-A2, HLA-A3, or HLA-B8 alleles.

[0047] In some cases, the nucleic acid sequences encoding MHC components are identical to naturally occurring class I MHC nucleic acid sequences. In other cases, the nucleic acid sequences encoding MHC components are codon-optimized or manipulated for more efficient transfection or expression in target cells. For example, in one case, all intron sequences are removed. In some cases, the nucleic acid molecules encoding MHC components are not naturally occurring, but the MHC components encoded by them have naturally occurring amino acid sequences. This is true for all MHC components described herein. In some cases, the nucleic acid sequences are different from naturally occurring class I MHC nucleic acid sequences, but encode the same polypeptide as a class I MHC polypeptide due to codon degeneracy. For example, a class I MHC nucleic acid sequence may be a codon-optimized class I MHC nucleic acid sequence. In some cases, the nucleic acids encoding class I MHC components include nucleic acids optimized to improve the expression of class I MHC components. In some cases, nucleic acid sequences encoding class I MHC components differ from naturally occurring class I MHC nucleic acid sequences, but they encode the same polypeptides as class I MHC polypeptides and show increased expression compared to naturally occurring class I MHC nucleic acid sequences.

[0048] Furthermore, MHC components may be non-classical MHC I components or fragments thereof. Non-classical MHC-I molecules are typically non-polymorphic and tend to exhibit more restricted expression patterns than their MHC class I counterparts. Non-classical MHC I components may be heavy (α) chains, light chains (β2 microglobulin), or combinations thereof. Non-classical MHC components may be HLA-E genes, HLA-G genes, HLA-F genes, or polypeptide products thereof. Non-classical MHC components may be molecules encoded by any suitable HLA-E allele from the human genome. Non-classical MHC components may be molecules encoded by any suitable HLA-G allele from the human genome. Non-classical MHC components may be molecules encoded by any suitable HLA-F allele from the human genome. Non-classical MHC components may be molecules encoded by any suitable β2 microglobulin allele from the human genome. In some cases, non-classical MHC components are functional fragments of non-classical MHC components. For example, non-classical MHC components may be exons or specific domains of non-classical MHC components, such as the α2 and α3 domains of the heavy chain. In some cases, class I MHC components are polypeptides encoded by non-classical MHC genes. Different alleles representing HLA-E, HLA-G, and HLA-F can be selected from Table 3.

[0049] Nucleic acids encoding non-classical MHC I components may include nucleic acids encoding non-classical MHC I components. In some cases, the nucleic acid sequence is identical to a naturally occurring non-classical MHC I nucleic acid sequence. In some cases, the nucleic acid sequence differs from a naturally occurring non-classical MHC I nucleic acid sequence but encodes the same polypeptide as a non-classical MHC I polypeptide due to codon degeneracy. For example, a non-classical MHC I nucleic acid sequence may be a codon-optimized non-classical MHC I nucleic acid sequence. In some cases, nucleic acids encoding non-classical MHC I components include nucleic acids optimized to improve the expression of non-classical MHC I components. In some cases, nucleic acid sequences encoding non-classical MHC I components differ from a naturally occurring non-classical MHC I nucleic acid sequence but encode the same polypeptide as a non-classical MHC I polypeptide and show increased expression compared to the expression of a naturally occurring non-classical MHC I nucleic acid sequence.

[0050] Class II MHC molecules can present peptides derived from extracellular proteins. Such class II molecules are typically found on the surface of antigen-presenting cells (APCs) such as dendritic cells, macrophages, and B cells, but their expression can be induced in non-antigen-presenting cells such as tumor cells. Class II MHC molecules can contain alpha (α) and beta (β) chains. The alpha chain may contain α1 and α2 domains, while the beta chain may contain β1 and β2 domains, with the α1 and β1 domains forming grooves to which the peptide presented by the class II MHC molecule binds. In some cases, the MHC components contain less than the entirety of the class II MHC molecule.

[0051] An MHC component may be a class II MHC component or a fragment thereof. A class II MHC component may be an alpha (α) chain, a beta (β) chain, or a combination thereof. A class II MHC component may be an HLA-DM gene, an HLA-DO gene, an HLA-DP, an HLA-DQ gene, an HLA-DR gene, or their polypeptide products. The alpha and beta chains of HLA-DM, HLA-DO, HLA-DP, and HLA-DQ, respectively, are listed in Table 1. A class II MHC component may be a molecule encoded by any suitable HLA-DM, HLA-DO, HLA-DP, or HLA-DQ allele from the human genome. In some cases, a class II MHC component is a fragment of a class II MHC component. For example, a class II MHC component may be an exon or specific domain of a class II MHC component, such as the α1 domain of the alpha chain and the β1 domain of the beta chain. In some cases, a class II MHC component is a polypeptide encoded by a class II MHC gene in Table 1. In some cases, the class II MHC components are polypeptides encoded by HLA-DR4 or HLA-DR15. Table 1. Genes encoding alpha and beta strands of class II MHC molecules [Table 1-1] [Table 1-2]

[0052] Class II MHC components can be class II MHC molecules such as HLA-DM, HLA-DO, HLA-DP, HLA-DQ, or HLA-DR. Each MHC molecule may contain alpha and beta chains encoded by the genes in Table 1. The alpha and beta chain genes in Table 1 may be functionally variable, and multiple different gene products may be produced by a single gene. For example, different gene products may be produced by a single gene through alternative splicing of exons. The functionally variable alpha and beta chain products shown in Table 1 can be referred to as class II MHC serotypes. There may be at least 21 serotypes of HLA-DR and at least 8 serotypes of HLA-DQ. A class II MHC component can be any suitable class II MHC serotype. A class II MHC component may be HLA-DR4 or HLA-DR15. The alleles representing these different serotypes can be selected from Table 3 attached herein.

[0053] Nucleic acids encoding class II MHC components may include nucleic acids encoding class II MHC components. In some examples, nucleic acid sequences are naturally occurring class II It is identical to the MHC nucleic acid sequence. In some cases, the nucleic acid sequence is naturally occurring Class II. Although different from the MHC nucleic acid sequence, it encodes the same polypeptide as the class II MHC polypeptide due to codon degeneracy. For example, a class II MHC nucleic acid sequence can be a codon-optimized class II MHC nucleic acid sequence. In some cases, nucleic acid sequences encoding class II MHC components are different from naturally occurring class II MHC nucleic acid sequences, but encode the same polypeptide as the class II MHC polypeptide and show increased expression compared to the expression of the naturally occurring class II MHC nucleic acid sequence. In some cases, nucleic acids encoding class II MHC components include nucleic acids optimized to improve the expression of class II MHC components. In some cases, nucleic acid sequences encoding class II MHC components are different from naturally occurring class II MHC nucleic acid sequences, but encode the same polypeptide as the class II MHC polypeptide. It encodes the same polypeptide as the MHC polypeptide and shows increased expression compared to the expression of naturally occurring class II MHC nucleic acid sequences.

[0054] In certain embodiments, MHC components or fragments that do not exist in nature are disclosed herein. In some examples, the MHC components that do not exist in nature are homologs of either class I MHC components or class II MHC components. A homolog is a sequence that does not exist in nature but has high sequence similarity or sequence identity to a sequence that exists in nature.

[0055] Generally, the interchangeable terms “sequence similarity,” “sequence identity,” or “sequence homology” refer to the exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence between two polynucleotide or polypeptide sequences, respectively. Typically, techniques for determining sequence identity involve the steps of determining the nucleotide sequence of a polynucleotide and / or the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. Two or more sequences (polynucleotides or amino acids) can be compared by determining their “percent identity,” also referred to as “percent homology.” Percent identity to a reference sequence (e.g., a nucleic acid or amino acid sequence), which may be a longer intramolecular sequence (e.g., a polynucleotide or polypeptide), can be calculated as the number of exact matches between the two optimally aligned sequences, divided by the length of the reference sequence and multiplied by 100. Percent identity can also be determined by comparing sequence information using advanced BLAST computer programs, including version 2.2.9, available from the National Institutes of Health. The BLAST program is based on the alignment methods described in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990), as well as Altschul, et al., J. Mol. Biol. 215:403-410 (1990); Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993); and Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997). In short, the BLAST program defines identity as the number of identical aligned symbols divided by the total number of symbols (i.e., nucleotides or amino acids) in the shorter of the two sequences. The program can be used to determine the percentage identity over the entire length of the sequences being compared.Default parameters are provided to optimize searches using short query sequences, for example, with the blastp program. This program was developed by Wootton and Federhen. As determined by the SEG program in Computers and Chemistry 17: 149-163 (1993), the use of SEG filters to remove the masking of segments in the query sequence is also permitted. High sequence identity between the disclosed sequence and the claimed sequence intends to be at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%. In some cases, the reference to percent sequence identity refers to sequence identity measured using BLAST (Basic Local Alignment Search Tool). As used herein, percent sequence identity or homology can be determined by any one or more of the conventional methods. Methods for analyzing sequence homology include, but are not limited to, pairwise sequence alignment, which is used to identify regions of similarity that can show functional, structural, and / or evolutionary relationships between two biological sequences (proteins or nucleic acids); and multiple sequence alignment (MSA), which is the alignment of three or more biological sequences of similar length. Various software and analysis tools are available for determining sequence homology based on global alignment, local alignment, or genome alignment.Examples include, but are not limited to: EMBOSS Needle, which uses the Needleman-Wunsch algorithm to provide optimal global alignment of two sequences; EMBOSS Stretcher, which uses a modified version of the Needleman-Wunsch algorithm that allows larger sequences to be globally aligned; EMBOSS Water, which uses the Smith-Waterman algorithm to calculate local alignment of two sequences; EMBOSS Matcher, which uses a rigorous algorithm based on the LALIGN application to provide local similarity between two sequences; LALIGN identifies internal overlaps by calculating non-cross-local alignment of protein or DNA sequences; and Wise2DBA (DNA Block Aligner aligns two sequences based on the assumption that the sequences share many conserved collinear blocks separated by DNA of potentially large and varied lengths in the two sequences; GeneWise compares a protein sequence to a genomic DNA sequence, allowing for intron and frameshift errors; PromoterWise compares two DNA sequences, allowing for promoter-ideal inversions and translocations; BLAST provides localized searches using a fast k-tuple discovery method; FASTA provides localized searches using a fast k-tuple discovery method, which is faster than BLAST but less sensitive; ClustalW provides localized or global incremental alignment. In some cases, ClustalW can be used for multiple sequence alignment. In some cases, homologous sequences can be found using Smith-Waterman and / or BLAST by searching and comparing query sequences with sequences in a database. In some cases, the Smith-Waterman algorithm compares segments of any possible length and optimizes the similarity measure; therefore, the Smith-Waterman algorithm is preferably used for determining sequence identity within a domain, or for local sequence alignment instead of comparing the entire length or the entire sequence.In some cases, the Needleman-Wunsch algorithm is used, preferably for the alignment of the entire protein or nucleotide sequence, to determine global or overall sequence identity. The EMBOSS Needle and Stretcher tools use the Needleman-Wunsch algorithm for global alignment. The EMBOSS Water tool uses the Smith-Waterman algorithm for local alignment. In various embodiments disclosed herein, overall or local sequence identity is preferably determined using BLAST.

[0056] MHC components that do not exist naturally can be expressed in cells that do not normally express the corresponding naturally occurring MHC components. MHC components that do not exist naturally can show enhanced expression by cells compared to naturally occurring MHC components. Cellular expression of MHC components that do not exist naturally can lead to enhanced recognition by T cells compared to naturally occurring MHC components. Expression of MHC components that do not exist naturally can lead to increased apoptosis in cells expressing these components. These cells may be tumor cells.

[0057] Nucleic acids encoding MHC components that do not exist in nature may contain at least one variant in comparison to nucleic acid molecules encoding naturally occurring MHC components. Variants may be mutations, insertions, deletions, or duplications. Mutations may result in substitutions that further encode synonymous or non-synonymous mutations, frameshift mutations, or nonsense mutations. In some cases, mutations reside in the protein-coding portion of the gene encoding the naturally occurring MHC component. In other cases, mutations reside in the promoter region of the gene encoding the naturally occurring MHC component.

[0058] Nucleic acid molecules of MHC components that do not exist in nature may be at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% similar to the nucleic acid sequence encoding the corresponding naturally occurring MHC component. In some cases, the nucleic acid molecule is at least 20% similar to the nucleic acid sequence encoding the naturally occurring MHC component. In some cases, the nucleic acid molecule is at least 80% similar to the nucleic acid sequence encoding the naturally occurring MHC component. In some cases, the nucleic acid molecule is at least 95% similar to the nucleic acid sequence encoding the naturally occurring MHC component.

[0059] Polypeptides of naturally occurring MHC components may be at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% similar to naturally occurring MHC component polypeptides. In some cases, the polypeptide is at least 80% similar to a naturally occurring MHC component polypeptide. In some cases, the polypeptide is at least 95% similar to a naturally occurring MHC component polypeptide.

[0060] A regulator of an MHC molecule may be a regulator of a class I MHC molecule or a class II MHC molecule. The regulator can regulate the transcription of nucleic acids encoding MHC molecules. Regulation of the transcription of nucleic acids encoding MHC molecules may include an increase in the level of MHC molecule transcription. Regulation of the transcription of nucleic acids encoding MHC molecules may include a decrease in the level of MHC molecule transcription. The regulator may be a transactivator, transcription factor, acetyltransferase, methyltransferase, elongation factor, or a combination of these.

[0061] The transactivator may be a class II, major histocompatibility complex, transactivator (CIITA) or a NOD-like receptor family CARD domain-containing 5 (NLRC5). In some cases, CIITA is a transactivator of class II MHC molecules. In some cases, NLRC5 is a transactivator of class I MHC molecules.

[0062] Transcription factors may be nuclear transcription factor Y (NF-Y), cAMP response element-binding protein (CREB), regulatory factor X (RFX), interferon regulatory factor (IRF), signaling and transcriptional activator (STAT), ubiquitous transcription factor (USF), or nuclear factor copper light chain enhancer (NF-κB) of activated B cells. NF-Y may be NF-Ya, NF-Yb, or NF-Yc. RFX may be RFXANK / RFXB, RFX5, or RFXAP. IRF may be IRF-1, IRF-2, IRF-3, IRF-4, IRF-5, IRF-6, IRF-7, IRF-8, or IRF-9. STAT may be STAT-1, STAT-2, STAT-3, STAT-4, STAT-5, or STAT-6. USF may be USF-1 or USF-2.

[0063] The acetyltransferase may be a histone acetyltransferase (HAT). The HAT may be a CREB-binding protein (CBP), p300, or a p300 / CBP-related factor (pCAF). In some embodiments, the regulator is a histone deacetylase inhibitor (DAI).

[0064] Methyltransferases can be histone methyltransferases (HMTases), DNA / RNA methyltransferases, or arginine methyltransferases. HMTases can be Zeste homolog 2 enhancers (EZH2). Arginine methyltransferases can be protein arginine N-methyltransferase 1 (PRMT1) or coactivator-bound arginine methyltransferase 1 (CARM1). For example, reduced expression of EZH2 can increase CIITA expression.

[0065] Elongation factors are positive transcription elongation factors (pTEF). b ) is possible.

[0066] In some embodiments, the regulators of MHC molecules are upregulated by additional factors. These additional factors that upregulate MHC molecules may be IFN-γ, lipopolysaccharide (LPS), or IL-4. In other embodiments, the regulators of MHC molecules are downregulated by additional factors. These additional factors that downregulate MHC molecules may be IFN-β, IL-10, nitric oxide (NO), or TGFβ. The regulators of MHC molecules that are upregulated or downregulated by additional factors may be CIITA or NLRC5.

[0067] Regulators of MHC molecules can be ligands for costimulatory molecules. Costimulatory molecules can be molecules required for T cell activation. Costimulatory molecules can be CD40. Regulators of MHC molecules can be ligands for CD40. immunotherapy composition

[0068] In certain embodiments, immunotherapy compositions comprising nucleic acid molecules encoding MHC components or fragments thereof are disclosed herein. In certain embodiments, the immunotherapy composition comprises polypeptides of MHC components or fragments thereof. In certain embodiments, immunotherapy compositions comprising nucleic acid molecules encoding regulators of MHC components or fragments thereof, or polypeptides of regulators of MHC components or fragments thereof, are further disclosed herein. The nucleic acid molecules may be DNA or RNA. Any of the MHC components herein may be used as an immunotherapy composition.

[0069] The immunotherapy composition may contain nucleic acid molecules encoding a class I MHC component, such as a class I MHC heavy (α) chain. The nucleic acid molecule may further encode a second class I MHC component, such as a class I MHC light chain (β2 microglobulin). For example, the immunotherapy composition may contain nucleic acid molecules encoding a class I MHC heavy (α) chain and a class I MHC light chain (β2 microglobulin). In some examples, the immunotherapy composition may further contain a second nucleic acid molecule encoding a second class I MHC component. For example, the immunotherapy composition may contain a first nucleic acid molecule encoding a class I MHC heavy (α) chain and a second nucleic acid molecule encoding a class I MHC light chain (β2 microglobulin).

[0070] The immunotherapy composition may contain nucleic acid molecules encoding a class II MHC component, such as a class II MHC alpha (α) chain. The nucleic acid molecule may further encode a second class II MHC component, such as a class II MHC beta (β) chain. For example, the immunotherapy composition may contain nucleic acid molecules encoding both a class II MHC alpha (α) chain and a class II MHC beta (β) chain. In some examples, the immunotherapy composition may further include a second nucleic acid molecule encoding a second class II MHC component. For example, the immunotherapy composition may contain a first nucleic acid molecule encoding a class II MHC alpha (α) chain and a second nucleic acid molecule encoding a class II MHC beta (β) chain.

[0071] The immunotherapy composition may contain nucleic acids encoding regulators of MHC components or fragments thereof. The immunotherapy composition may contain polypeptides of regulators of MHC components or fragments thereof. The regulators may be transactivators, transcription factors, acetyltransferases, methyltransferases, elongation factors, or any combination thereof, as previously described herein. The immunotherapy composition may contain additional factors that modulate the regulators of MHC components or fragments thereof. Additional factors that modulate the regulators of MHC components may be IFN-γ, lipopolysaccharide (LPS), IL-4, IFN-β, IL-10, nitric oxide (NO), or TGFβ. The additional factors may be administered as polypeptides or small molecules (e.g., NO).

[0072] Additional factors can be administered simultaneously with nucleic acids encoding regulators of MHC components or their fragments. Additional factors can be administered sequentially following the administration of nucleic acids encoding regulators of MHC components or their fragments. Nucleic acids encoding regulators of MHC components or their fragments can be administered sequentially following the administration of additional factors.

[0073] The immunotherapy composition may contain a ligand for a co-stimulatory molecule. The co-stimulatory molecule may be CD40.

[0074] An immunotherapy composition may contain a nucleic acid encoding an inactivated CRISPR-related nuclease fused to a TET enzyme. The nucleic acid encoding the inactivated CRISPR-related nuclease fused to the TET enzyme may further encode at least one guide RNA (gRNA). An immunotherapy composition containing a nucleic acid encoding an inactivated CRISPR-related nuclease fused to a TET enzyme may further contain a second nucleic acid encoding a gRNA. The gRNA may include a transcription factor, a regulator of MHC components, or a region complementary to the promoter of an MHC gene. The inactivated CRISPR-related nuclease may be inactivated Cas9 (dCas9) or inactivated Cpf1 (dCfp1). The TET enzyme may be TET1, TET2, TET3, or their catalytic domains. In some examples, the TET enzyme is the TET1 enzyme, or the catalytic domain of the TET1 enzyme. Demethylation of promoters, regulators of MHC components, or transcription factors associated with MHC genes can be achieved by administering an immunotherapy composition containing a nucleic acid encoding an inactivated CRISPR-related nuclease fused to a TET enzyme. Demethylation of promoters, regulators of MHC components, or transcription factors associated with MHC genes can result in increased expression of MHC genes.

[0075] The immunotherapy composition may further comprise at least a second nucleic acid encoding a second inactivated CRISPR-related nuclease fused to the TET enzyme. The second nucleic acid may further encode at least one second guide RNA. In some examples, the immunotherapy composition comprises multiple nucleic acids encoding inactivated CRISPR-related nucleases fused to the TET enzyme, and multiple guide RNAs. In some examples, the immunotherapy composition comprises a single nucleic acid encoding an inactivated CRISPR-related nuclease fused to the TET enzyme, and multiple nucleic acids encoding multiple guide RNAs. In some examples, the gRNA is designed to target a single methylated CpG site. In other examples, the gRNA is designed to target at least two methylated CpG sites.

[0076] The immunotherapy composition can be formulated as an aqueous solution. The immunotherapy composition can also be formulated as a powder, for example, a dry powder nucleic acid composition containing a lipid-DNA complex. The powder formulation can further be suspended in an aqueous solution. The immunotherapy composition can be freeze-dried, sterilized, or a combination of these methods.

[0077] The immunotherapy composition may further include at least pharmaceutically acceptable excipients. The term “pharmaceutically acceptable” is used herein to mean, within reasonable medical judgment, a compound, material, composition and / or dosage form suitable for use in contact with human and animal tissues, without excessive toxicity, irritation, allergic response or other problems or complications, and with a reasonable benefit / risk ratio.

[0078] Any suitable pharmaceutically acceptable excipient can be used. Excipients may be carriers, diluents, detergents, buffers, salts, peptides, surfactants, oligosaccharides, amino acids, carbohydrates, or adjuvants. In some examples, hydrophilic excipients are used; for example, a dry powder immunotherapy composition contains nucleic acids dispersed within a hydrophilic excipient. Examples of excipients include, but are not limited to, human serum albumin, collagen, gelatin, hyaluronic acid, glucose, lactose, sucrose, xylose, ribose, trehalose, mannitol, raffinose, stachyose, dextran, maltodextrin, cyclodextrin (cylcodextrin), cellulose, methylcellulose, glycine, alanine, glutamate, ascorbic acid, ascorbate, citric acid, citrate, NaCl, NaHCO3, NH4HCO3, MgSO4, and Na2SO4.

[0079] In some cases, excipients are used to stabilize immunological compositions. Excipients may be salts dissolved in a buffer solution (which can also provide pH control or maintenance), including but not limited to phosphate-buffered saline. In some cases, excipients increase the volume of the immunological composition. Excipients can increase or decrease the absorption of the immunological composition by an individual.

[0080] The compositions described herein can be formulated for oral delivery, or for delivery via intravenous, intramuscular, subcutaneous, subdermal, sublingual, and other routes.

[0081] Suitable solid dosage forms for oral administration according to this instruction include, but are not limited to, capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is sodium citrate or dicalcium phosphate. At least one inert and pharmaceutically acceptable excipient or carrier, such as phosphate, and / or (a) fillers or bulking agents such as starch, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia; (c) water-retaining agents such as glycerol; (d) disintegrants such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (e) dissolution retarders such as paraffin; (f) absorption enhancers such as quaternary ammonium compounds; (g) wetting agents such as acetyl alcohol and glycerol monostearate; (h) absorbents such as kaolin and bentonite clay, and (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, and sodium lauryl sulfate, and mixtures thereof, which are mixed together. In the case of capsules, tablets, and pills, the dosage form may also include a buffering agent.

[0082] The active compound may be in microencapsulated form, along with one or more of the excipients listed above. Encapsulation may include the use of liposomes, exosomes, lipid nanoparticles, or biomaterials.

[0083] Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs.

[0084] Preparations for injection (e.g., sterile aqueous or oily suspensions for injection) can be formulated according to known techniques using suitable dispersants or wetting agents and suspending agents.

[0085] Any formulation or composition described herein is preferably designed to specifically target cancer cells. For example, in some cases, MHC components are formulated in exosomes that selectively target cancer cells. An example of such an exosome is described in Gomari et al., Onco Targets (2018) 11: 5753-5762 “Targeted cancer therapy using engineered exosome as a natural drug delivery vehicle”. In some cases, the MHC components or the vesicles encapsulating them contain aptamers that selectively target the MHC components or the vesicles encapsulating them to cancer cells. An example of aptamers that selectively target cancer cells is described in Cerchia et al, Trends Biotechnol. (2010) Oct 28(10): 517-25 “Targeting cancer cells with nucleic acid aptamers”. In another case, the MHC components or the vesicles encapsulating them are coupled to nanomaterials that selectively target cancer cells, such as cancer stem cells. An example of such nanomaterials is the nanomaterial described in Qin et al. (2017) Front. Pharmacol. “Nanomaterials in targeting cancer stem cells for cancer therapy”. In another example, MHC components or vesicles encapsulating them are coupled to antibodies that selectively target cancer stem cells. This can form a drug-antibody conjugate. Alternatively, the antibody may be present on the surface of a vesicle that directs the encapsulated MHC components toward cancer cells.Examples of drug-antibody conjugations are described in Thomas et al, (2016) Lancet Oncol., June 17(6), “Antibody-drug conjugates for cancer therapy” and Dan et al., (2018) Pharmaceutical (Basel) (2018) June; 11(2):32, “Antibody-drug conjugates for cancer therapy: chemistry to clinical implications”.

[0086] Nucleic acids encoding MHC components, nucleic acids encoding regulators of MHC components, or nucleic acids encoding inactivated CRISPR-related nucleases fused to TET enzymes can be delivered to cells by vectors. The nucleic acid may be RNA or DNA. The cells may be tumor cells. The vector may be a viral vector or a non-viral vector. Non-viral vector delivery systems include DNA plasmids, RNA (e.g., transcripts of vectors described herein), naked nucleic acids, and nucleic acids complexed with a delivery medium such as lipids or liposomes.

[0087] Lipids may be cationic lipids, anionic lipids, or neutral lipids. Lipids may be liposomes, small monolayer vesicles (SUVs), lipid envelopes, lipidoids, or lipid nanoparticles (LNPs). Lipids can be mixed with nucleic acids to form lipoplexes (nucleic acid-liposome complexes). Lipids can be conjugated to nucleic acids. Lipids may be non-pH sensitive or pH sensitive lipids. Lipids may further contain polyethylene glycol (PEG).

[0088] The cationic lipid may be a monovalent cationic lipid such as N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), [1,2-bis(oleoyloxy)-3-(trimethylammonio)propane] (DOTAP) or 3β[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol). The cationic lipid may be a polyvalent cationic lipid such as di-octadecyl-amido-glycyl-spermine (DOGS) or {2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-l-propanaminium trifluoroacetate} (DOSPA).

[0089] The anionic lipid may be a phospholipid or dioleoylphosphatidylglycerol (DOPG). Examples of phospholipids include, but are not limited to, phosphatidic acid, phosphatidylglycerol or phosphatidylserine. In some examples, the anionic lipid is Ca 2 +, Mg 2 +, Mn2+ and Ba 2 + and other divalent cations.

[0090] The cationic lipid or anionic lipid may further comprise a neutral lipid. The neutral lipid may be dioleoylphosphatidylethanolamine (DOPE) or dioleoylphosphatidylcholine (DOPC). In some examples, use of a helper lipid in combination with a charged lipid results in higher transfection efficiency.

[0091] Liposomes may further include polymers, lipids, peptides, magnetic nanoparticles (MNPs), additional compounds, or combinations thereof. Polymers, lipids, or magnetic nanoparticles can be attached to liposomes or integrated into the liposome membrane. The polymer may be polyethylene glycol (PEG). The polymer may be N-[2-hydroxypropyl]methacrylamide (HPMA), poly(2-(dimethylamino)ethyl methacrylate) (pDMAEMA), or arginine-grafted bioreducible polymer (ABP). The peptide may be a cell membrane permeable peptide, a cell adhesion peptide, or a peptide that binds to a cell surface receptor. The cell may be a tumor cell. Any suitable cell membrane permeable peptide can be used. Examples of cell membrane permeable peptides include, but are not limited to, polylysine peptides and polyarginine peptides. The cell adhesion peptide may be arginylglycylaspartate (RGD) peptide. The additional compound may be a compound that binds to a cell surface receptor, such as folic acid.

[0092] The vector may be a viral vector. The viral vector may be a viral vector with replication ability or a viral vector without replication ability. The viral vector may be an oncolytic virus. Examples of viral vectors include, but are not limited to, alphaviruses, retroviruses, adenoviruses, herpesviruses, poxviruses, lentiviruses, oncolytic viruses, reoviruses, or adeno-associated viruses (AAVs). Alphaviruses may be Semryki forest virus (SFV), Sindbisvirus (SIN), or Venezuelan encephalitis virus (VEE). Poxviruses may be vaccinia viruses. Herpesviruses may be herpes simplex virus (HSV) or Epstein-Barr virus (EBV). Adeno-associated viruses may be AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, or AAV8.

[0093] Viral vectors may be modified viral vectors. Modified viral vectors may exhibit reduced immunogenicity, increased persistence of the vector in the bloodstream, or impaired uptake of the vector by macrophages and antigen-presenting cells.

[0094] The modified viral vector may further include polymers, lipids, peptides, magnetic nanoparticles (MNPs), additional compounds, or combinations thereof. Polymers, lipids, or magnetic nanoparticles can be attached to the capsid of the viral vector. The polymer may be polyethylene glycol (PEG). The polymer may be N-[2-hydroxypropyl]methacrylamide (HPMA), poly(2-(dimethylamino)ethyl methacrylate) (pDMAEMA), or arginine-grafted bioreducible polymer (ABP). The peptide may be a cell membrane permeable peptide, a cell adhesion peptide, or a peptide that binds to a cell surface receptor. The cell may be a tumor cell. Any suitable cell membrane permeable peptide can be used. Examples of cell membrane permeable peptides include, but are not limited to, polylysine peptides and polyarginine peptides. The cell adhesion peptide may be arginylglycylaspartate (RGD) peptide. The additional compound may be a compound that binds to a cell surface receptor, such as folic acid.

[0095] Magnetic nanoparticles can be superparamagnetic nanoparticles. In some cases, binding of MNPs can result in lower viral vector doses for optimal transgene delivery. In some cases, binding of MNPs improves transduction efficiency.

[0096] In some cases, a modified viral vector is a genetically modified vector. A genetically modified vector may have reduced immunogenicity, reduced genotoxicity, increased loading capacity, increased transgene expression, or a combination thereof. In some cases, a genetically modified viral vector is a pseudotyped viral vector. A pseudotyped viral vector may have at least one exogenous viral envelope protein. The exogenous viral envelope protein may be an envelope protein derived from lyssavirus, arenavirus, hepadnavirus, flavivirus, paramyxovirus, baculovirus, filovirus, or alphavirus. The exogenous viral envelope protein may be glycoprotein G of vesicular stomatitis virus (VSV). In some cases, the exogenous viral envelope protein is a genetically modified viral envelope protein. A genetically modified viral envelope protein may be a viral envelope protein that does not exist in nature.

[0097] In some cases, the capsid of a viral vector is conjugated with a bispecific antibody. The bispecific antibody can be targeted to bind to target cells, which may be tumor cells.

[0098] Any of the compositions and immunotherapies described herein may further comprise one or more therapeutic portions. Such therapeutic portions may include immune checkpoint inhibitors, immune checkpoint stimulants, cancer vaccines, small molecule therapies, monoclonal antibodies, cytokines, cell therapies, or combinations thereof. How to use

[0099] The compositions described herein can be used to increase T cell activation and / or cytokine release. This can occur in vivo or in vitro. Such methods can be further used to treat immune-evading conditions, such as cancer. Thus, in certain embodiments, methods for activating the immune system and / or enhancing T cell activity and / or increasing cytokine-mediated responses in a subject are described herein. Such cytokine release may be the release of interferon-gamma and TNF-alpha. In certain embodiments, methods for treating cancer in an individual are also described herein, comprising the step of administering to the individual an immunotherapy composition comprising a nucleic acid molecule or polypeptide encoding an MHC component. In certain embodiments, methods for treating cancer in an individual are further described herein, comprising the step of administering to the individual an immunotherapy composition comprising a nucleic acid molecule or polypeptide encoding a regulator of an MHC component. In certain embodiments, methods for treating cancer in an individual are further described herein, comprising the step of administering to the individual an immunotherapy composition comprising a nucleic acid molecule encoding a deactivated CRISPR-related nuclease fused to a TET enzyme. In some cases, a method for treating cancer in an individual comprises the step of administering to the individual an immunotherapy composition comprising at least one nucleic acid molecule encoding at least two of the following: MHC components, modifiers of MHC components, additional factors that modulate the modifiers of MHC molecules, and an inactivated CRISPR-related nuclease fused to a TET enzyme. The at least one nucleic acid molecule may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleic acid molecules. Thus, the compositions herein may contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different nucleic acid molecules operably linked to each other or present in separate plasmids, each of which may include a nucleic acid molecule encoding an MHC component.

[0100] Cancer can be treated using the compositions described herein. Cancer may include solid tumor cancers, hematological cancers, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, non-Hodgkin lymphoma, Hodgkin lymphoma, multiple myeloma, bladder cancer, pancreatic cancer, cervical cancer, endometrial cancer, lung cancer, bronchial cancer, liver cancer, ovarian cancer, colon and rectal cancer, stomach cancer, gastric cancer, gallbladder cancer, gastrointestinal stromal tumor cancer, thyroid cancer, head and neck cancer, oropharyngeal cancer, esophageal cancer, melanoma, non-melanoma skin cancer, Merkel cell carcinoma, virus-induced cancers, neuroblastoma, breast cancer, prostate cancer, kidney cancer, renal cell carcinoma, renal pelvis cancer, leukemia, lymphoma, sarcoma, glioma, brain tumor, and carcinoma. In some examples, cancer may be ovarian cancer, pancreatic cancer, or colon cancer. Cancer may not express MHC molecules. Cancer may show reduced expression of MHC molecules. MHC molecules may be class I or class II MHC molecules. In some cases, cancer is cancer that does not respond to immune checkpoint inhibitor therapy.

[0101] In some cases, the method further includes a step of diagnosing cancer as having no or reduced MHC molecule expression. The step of diagnosing cancer as having no or reduced MHC molecule expression may include (a) obtaining a biological sample from an individual, (b) isolating cancerous cells from the biological sample, and (c) detecting whether MHC molecule expression in the isolated cancerous cells is reduced or eliminated compared to a control. The control may be a predetermined level, the level of MHC expression in the individual's non-cancerous tissue, or the level of MHC molecule expression in the non-cancerous tissue of a different subject.

[0102] In some cases, the method further includes the step of sequencing the MHC components of an individual. The MHC component sequences may include exons and introns of MHC genes, as well as their promoters, 5'UTR and 3'UTR regions. The individual's MHC components may be sequences of the individual's native or endogenous MHC components. Sequencing of the individual's MHC components may include Sanger sequencing or next-generation sequencing (NGS). Sequencing of the MHC components may further include an initial step of treating the individual's nucleic acids with bisulfite prior to sequencing. Methylated CpG sites can be identified by comparing the nucleic acid sequence to the bisulfite-treated nucleic acid sequence. In some cases, sequencing of the individual's MHC components has informational value regarding the desired sequence of an immunotherapy composition. For example, if the promoter of an MHC component derived from cancer cells is hypermethylated compared to an MHC component derived from non-cancerous cells, the immunotherapy composition can be designed to demethylate at least one methylated CpG site of the promoter. In another example, sequencing an individual's MHC components allows for the design of naturally occurring, non-naturally occurring MHC components that would be immunologically compatible with the individual.

[0103] The method may further include a step of administering an additional therapeutic compound to the individual. The additional therapeutic compound may be a therapeutic agent that binds to an immune checkpoint gene or its ligand, a cancer vaccine, a small molecule therapy, a monoclonal antibody, a cytokine, a cell therapy, or a combination thereof. The therapeutic agent that binds to an immune checkpoint molecule or its ligand may be an immune checkpoint inhibitor or an immune checkpoint agonist. Examples of immune checkpoint molecules include, but are not limited to, CD27, CD28, CD40, CD122, OX40, ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM-3, VISTA, 4-1BB, and GITR. Examples of immune checkpoint inhibitors include, but are not limited to, ipilimumab, tremelimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, and lirilumab. Small molecule therapies may include proteasome inhibitors, tyrosine kinase inhibitors, cyclin-dependent kinase inhibitors, or poly-ADP-ribose polymerase (PARP) inhibitors. Cytokines may include INFα, INFβ, IFNγ, or TNF. Cell therapy may include adoptive T cell transfer (ACT) therapy. Alternatively, cell therapy may include chimeric antigen receptor (CAR) T cell therapy or T cell antigen coupler (TAC) T cell therapy. The TAC receptor acts via the native T cell receptor (TCR). Furthermore, the TAC includes (1) an antigen-binding domain, (2) a TCR recruitment domain, and (3) a co-receptor domain (hinge, transmembrane, and cytosolic regions).

[0104] Additional therapeutic compounds can be administered concurrently with the administration of the immunotherapy compound, or before or after the administration of the immunotherapy compound. In some cases, administration of the immunotherapy composition results in cancer that shows increased sensitivity to at least one additional therapeutic compound.

[0105] In some cases, immunotherapy compositions are delivered by various routes. Exemplary delivery routes include oral (including buccal and sublingual), rectal, nasal, topical, percutaneous patch, lung, vaginal, suppository, or parenteral (including intramuscular, intra-arterial, subarachnoid, intradermal, intraperitoneal, subcutaneous, and intravenous) administration, or administration in a form suitable for aerosolization, inhalation, or gas injection. In some cases, the immunotherapy compositions described herein may be administered intramuscularly, or percutaneously by intradermal or subcutaneous injection or by iontophoresis. In some cases, epithelial administration of the immunotherapy composition is used.

[0106] Immunotherapy compositions can be administered to the subject in need, for example, daily, weekly, monthly, semi-annually, annually, or once or multiple times (e.g., 1 to 10 times or more) as medically required. Dosages can be provided in one or multiple dosing regimens. The timing between administrations can be reduced as the medical condition improves or increased as the patient's health deteriorates.

[0107] The dosage of the pharmaceutical compositions of this disclosure depends on factors including the route of administration, the disease to be treated, and the physical characteristics of the subject, such as age, weight, and overall health. Typically, the amount of pharmaceutical composition contained in a single dose may be an amount that effectively prevents, delays, or treats a disease without inducing significant toxicity. An effective dose for use in humans can be determined from animal models. For example, a dose for humans may be formulated to achieve circulating, hepatic, topical, and / or gastrointestinal concentrations that have been found to be effective in animals. Based on animal data and other types of similar data, those skilled in the art can determine an effective dose of a vaccine composition suitable for humans. The dosage can be adapted by a physician according to conventional factors such as the severity of the disease and various parameters of the subject.

[0108] Immunotherapy compositions can be administered before, during, or after the onset of symptoms associated with a disease or condition (e.g., cancer). In some cases, immunotherapy compositions are administered for the treatment of cancer. In some cases, immunotherapy compositions are administered for prevention, such as in prophylactic treatment for cancer. In some cases, immunotherapy compositions are administered to induce an immune response from the patient.

[0109] In some embodiments, the immunotherapy compositions and kits described herein are stored between 2°C and 8°C. In some examples, the immunotherapy compositions are not stored frozen. In some examples, the immunotherapy compositions are stored at temperatures such as -20°C or -80°C. In some examples, the immunotherapy compositions are stored away from sunlight. kit

[0110] In certain embodiments, kits and products for use in one or more of the methods described herein are disclosed herein. The kit may include an immunotherapy composition described herein, formulated in a suitable pharmaceutical excipient and placed in a suitable container.

[0111] This kit may include a carrier, package, or container partitioned to receive one or more containers such as vials or tubes, and each container(s) may contain one of the separate elements to be used in the method described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. Containers may be formed from a variety of materials such as glass or plastic.

[0112] This kit may include identification statements, labels, or accompanying documents. Labels or accompanying documents may contain the contents of the kit or immunological composition, instructions for use in the manner described herein, or a combination thereof. Labels may be present on or attached to the container. Labels may be present on the container if the letters, numbers, or other characters forming the label are attached to, molded into, or etched onto the container itself. Labels may be attached to the container, for example, if they are present in a receptacle or carrier that similarly holds the container, as accompanying documents. In some examples, labels are used to indicate that the contents should be used for a specific therapeutic application.

[0113] The kits described herein may further comprise one or more reagents, such as site-specific primers or probes, for extracting, enriching, and / or determining the sequences of an individual's HLA alleles. The kits may further comprise one or more different HLA alleles. The therapeutic treatment comprises administering to the individual an MHC component having the same HLA allele as found in the individual being treated.

[0114] This kit may further include one or more other therapeutic agents, such as immune checkpoint inhibitors, immune checkpoint stimulants, cancer vaccines, small molecule therapies, monoclonal antibodies, cytokines, cell therapies, or combinations thereof. A certain terminology

[0115] The terminology used herein is intended to describe specific cases only and not to limit them. The terms described below are provided to illustrate the meaning of the terms used herein, in addition to the understanding of such terms by those skilled in the art. Where used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly indicates otherwise. It should be further noted that the claims may be drafted to exclude elements as needed. Therefore, this expression is intended to function as an antecedent for the use of such exclusive terminology as “only,” “solely,” or other “negative” limitation in relation to the enumeration of elements of the claims.

[0116] Certain ranges are presented herein by numerical values ​​preceded by the term “approximately.” The term “approximately” is used herein to provide literal support for the exact number preceded by the term, and for numbers near or before or after the number preceded by the term. In determining whether a number is near or before or after a particularly enumerated number, any near or before / after number that is not enumerated may be a number that provides a substantial equivalent to the particularly enumerated number in the context in which it is presented. Where a range of values ​​is provided, unless the context otherwise clearly indicates otherwise, it is understood that each intervening value, between the upper and lower limits of the range, and any other described or intervening values ​​within the described range, up to one-tenth of the lower limit unit, are encompassed within the methods and compositions described herein. The upper and lower limits of these smaller ranges may independently be contained within smaller ranges and also encompassed within the methods and compositions described herein, and are subject to any specifically excluded limits within the described range. If the described scope includes one or both of the limits, the scope excluding either or both of these included limits is also included in the methods and compositions described herein.

[0117] The terms “individual,” “patient,” or “subject” are used interchangeably. None of these terms require, or are limited to, a situation characterized by supervision (e.g., continuous or intermittent) by a healthcare professional (e.g., physician, registered nurse, nurse practitioner, physician’s assistant, orderly, or hospice staff). Furthermore, these terms refer to human or animal subjects.

[0118] "Treating" or "treatment" refers to both therapeutic treatments and preventive or protective measures, the goal of which is to prevent or slow (reduce) the targeted pathological condition or disability. Those requiring treatment include those who already have a disability, and those who are prone to developing a disability, or for whom the disability should be prevented. For example, if, after receiving a therapeutic dose of a target oligonucleotide conjugate according to the method of this disclosure, the subject or mammal has successfully "treated" cancer: a reduction in the number of cancer cells or the absence of cancer cells; a reduction in tumor size; inhibition of cancer cell infiltration into peripheral organs, including the spread of cancer to soft tissues and bone (i.e., slowing to some extent, preferably stopping); inhibition of tumor metastasis (i.e., slowing to some extent, preferably stopping); inhibition of tumor growth to some extent; and / or mitigation to some extent of one or more specific cancer-related symptoms; reduced morbidity and / or mortality, and improvement in quality of life issues.

[0119] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art in the field to which the methods and compositions described herein belong. Any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of the methods and compositions described herein, but representative methods and materials for illustrative purposes are described herein. [Examples]

[0120] (Example 1)

[0121] Cotransfection of complete alpha and beta HLA-DR chains into cell lines that do not natively express HLA-DR resulted in HLA-DR expression on the cell surface.

[0122] RKO colon cancer cell lines lacking HLA-DR expression (ATCC CRL-2577) were stably transfected with either the HLA-DR A plasmid (alpha, cat#RC209920(NM_019111)) (Figure 4D) or the HLADRB1*15 plasmid (beta, cat#RG218764(NM_002124)) (Figure 4F), obtained from OriGene Technologies Inc. RKO cells were also co-transfected with both plasmids. All transfections were performed using electroporation (Mirus Bio LLC kit) according to the manufacturer's protocol. Transfected cells were subjected to selective pressure with the antibiotic Geneticin® (G418-ThermoFisher) for at least two weeks. Transfected cells were tested by FACS using antibodies against HLA-DR A and B (ThermoFisher). For flow cytometry testing, cells were detached from the flask and stained with anti-HLA-DR alpha (LN3, APC) or HLA-DR beta (UT36, PE) for 30 minutes at 4 degrees Celsius. The transfected cells were then washed twice with FACS buffer (PBS containing 2% FBS). Next, the cells were passed through a FACS analyzer (CytoFlex S), and the data were analyzed using Flowjo software version 10.2.

[0123] Referring to Figure 1A, the parental RKO cells showed no surface expression of either HLA receptor. Figure 1B shows successful transfection of RKO cells with HLA-DR A (evidence of intracellular expression of Myc-DKK tag; data not shown). However, no HLA-DR expression was detected on the cell surface. Furthermore, Figure 1C shows that in RKO cell lines transfected with HLADR B1 alone, no HLA-DR surface expression was detected even when GFP expression indicated successful transfection. Moreover, Figure 1D shows surface expression of both alpha and beta chains in cells co-transfected with HLA-DR A and B (transfection confirmed by high and moderate GFP expression). This data supports the conclusion that the HLA-DR gene is silent in RKO cells, and that HLA-DR surface expression occurs only when both A and B1 chains are expressed simultaneously.

[0124] Furthermore, according to the left column of the FACS plots in Figures 1A–1D, the large squares represent GFP-positive gated populations (i.e., GFP expression), and the small squares represent cells expressing moderate (dark green overlays shown in the circles in Figures 1C and 1D) and high (light green overlays shown in the squares in Figures 1C and 1D) GFP expression. The middle column of the FACS plots shows the surface expression of alpha chains (X axis) and beta chains (Y axis). In cotransfected cells, both moderate and high-expression GFP populations exhibit surface expression of HLA-DR A and B, as can be seen from the right column of the FACS plots. The overlays and dark green represent moderate GFP expression populations expressing moderate intensity HLA-DR A and B, while the light green represents high GFP expression populations with high HLA-DR A and B expression.

[0125] Referring to Figures 2A and 2B, high-GFP HLA-DRAB1*15 RKO-transfected cell lines were sorted (using Sony Sorter, Sony Biotech) and re-evaluated using flow cytometry analysis (Figure 2B) compared with the parental RKO cell line (Figure 2A). Figure 2C shows co-transfected GFP HLA-DRAB1*15 RKO cells in the left column (GFP / bright-field) versus GFP in the right column. A representative fluorescence image (20x magnification) of HLA-DR B1*15 alone is shown. Cells co-transfected with both alpha and beta units show punctate GFP, while cells transfected with only HLA-DR B show scattered green fluorescent protein in the cytoplasm. This result indicates that the alpha and beta chains associate and migrate to the cell surface.

[0126] Figures 3A–3D show representative fluorescence images of stably cotransfected RKO and SKOV3 cells, as follows: RKO HLA-DR AB1, SKOV3 HLA-DR AB1, RKO HLA-DR AB3, and SKOV3 HLA-DR AB3. The RKO parent cell line was also cotransfected with HLA-DR A in combination with B3 (RG210732, NM_022555), B4 (RG202743, NM_021983), or B5 (RG203646, NM_002125), all obtained from OriGene. As described above, the data were verified using flow cytometry (data not shown). SKOV3 is an ovarian adenocarcinoma cell line (HTB-7, ATCC) and is the second cell line lacking HLA-DR expression due to the absence of A and B chain expression. It was co-transfected with HLA-DR A B1, HLA-DR A B3, HLA-DR A B4, and HLA-DR A B5. Transfected SKOV3 cells were sorted. GFP and HLA-DR expression in the RKO cell line and the pancreatic adenocarcinoma BxPC3 cell line (CRL-1687, ATCC) are not shown. Plasmids with different beta chains are shown in Figures 4A-4C. Fluorescence images of RKO HLA-AB1 and RKO HLA-AB3 are shown in Figures 6A and 6C, and fluorescence images of SKOV3 HLA-AB1 and SKOV3 HLA-AB3 are shown in Figures 6B and 6D. White errors indicate punctate GFP vesicular expression, which indicates the transfer of MHC molecules to the cell surface.

[0127] (Example 2) T cell proliferation is dependent on HLA expression.

[0128] Functional mixed lymphocyte response, T cell proliferation, and cytokine release assays will be used to test the effect of HLA-DR-expressing tumor cell lines on T cell activation compared to non-expressing tumor cells.

[0129] Human mixed lymphocyte reaction assay

[0130] To test whether cotransfected HLA-DR RKO cells could activate T cells with similar and different HLA-DRs, dendritic cells (DCs) were used as positive controls for the MLR assay. These DCs were generated according to the protocol described below: Human buffy coat was purchased from Stanford Blood Center (Stanford, CA), diluted with PBS, and overlaid onto buffy coat for the isolation of human PBMCs. Human PBMCs were washed four times with PBS, and surface antigen classification 14 (CD14+) monocytes were isolated using a human-specific CD14+ cell isolation kit with positive selection, as described in the manufacturer's protocol (Miltenyi Biotec, San Diego, CA). Complete Roswell cells were then supplemented with 10% fetal bovine serum (FBS). In Park Memorial Institute (RPMI) 1640 medium, 5 × 10⁶ days. 5 CD14+ cells were seeded at 1000 cells / mL. On days 0, 2, and 5, the cultures were supplemented with recombinant human (rh-)IL-4 (1000 U / mL) (R&D Systems, Minneapolis, MN) and rh granulocyte-macrophage colony-stimulating factor (GM-CSF) (rh-GMCSF) (500 U / mL) (R&D Systems, Minneapolis, MN). On day 7, immature DCs were collected, washed, and counted.

[0131] These DCs expressed high levels of HLA-DR and PD-L1, as shown in Figure 5A of two representative DCs prepared from two different donors (D1 and D2). Flow cytometry using a Cytoflex analyzer (Beckman Culture) was used to test each preparation sample for PD-L1 expression with r-phycoerythrin (RPE)-labeled anti-hu-PD-L1 (eBioscience / Affymatrix, Santa Clara, CA). Furthermore, HLA-DR alpha (APC) and HLA-DR beta-PE (eBioscience / Affymatrix, Santa Clara, CA) expression were also evaluated in cells isolated from D1 and D2.

[0132] Referring to Figure 5B, human T lymphocytes were isolated from Buffycote (Stanford Blood Center, CA), diluted in phosphate-buffered saline (PBS), and layered onto Ficoll for PBMC isolation. Human PBMCs were washed four times with PBS, and T lymphocytes were isolated using a human-specific pan-T cell isolation kit with negative selection, as described in the manufacturer's protocol (Miltenyi Biotec, San Diego, CA). As shown in Figure 5B, the DCs expressed minimal levels of co-inhibitory receptors, such as LAG3 and PD-1, as expected from quiescent T cells. Furthermore, referring to Figure 5C, the parental RKO cell line and HLA-DR AB1*15 co-transfected cells were grown in Eagle's Minimum Essential Medium (MEM) (Corning, Fisher Scientific) containing 10% FBS. G418 was added as a selective antibiotic for stably transfected RKO cells. Both parental and HLA-DR AB-transfected RKO cells expressed high levels of PD-L1.

[0133] T-cell proliferation assay & cytokine release assay

[0134] The MLR protocol was adapted from Kruisbeek et al, 2004, with several modifications. Primary human DC-derived, HLA-DR AB1-transfected RKO cells and RKO parents were collected on the day of the experiment for optimal antigen-presenting cell status and validated by flow cytometry for high levels of PD-L1 expression, necessary for T cell activation, as well as co-stimulatory markers such as CD80 and CD86 (data not shown). Cells were counted and treated with a low dose of 50 ug / mL mitomycin C (sigma Aldrich, Saint Louis, MO) to prevent cell secretion but to ensure that the cells functioned solely as support for antigen presentation to T cells. Therefore, assay results were induced by T cells only.

[0135] Human T cells were collected freshly isolated from allogeneic donors according to the same protocol described above. T cells were seeded with irradiated DCs in a 10:1 ratio (T:DC or RKO- for optimal assay conditions) in the presence of different concentrations of anti-PD-1 antibodies (nivolumab and pembrolizumab), anti-LAG3 antibodies, negative and positive control antibodies, or medium alone (to assess baseline response). All conditions were seeded in 96-well flat-bottom tissue culture treatment plates (Fisher Scientific Pittsburg, PA). Cells were cultured using serum-free X-vivo15 medium (Lonza, Walkersville, MD) to prevent inter-experimental human serum variability. Cultures were incubated at 37°C with 5% CO2 for 5–8 days, depending on the donor. T cell aggregate formation was monitored under a light microscope to capture any signs of T cell proliferation (examples in Figures 6A–6F, 7A–7D, and 8A). On the day of collection, supernatant was collected and cytokine concentrations were measured for IFN-gamma and TNF-alpha using the Meso Scale Discovery (MSD LLC., Maryland, MD) kit according to the manufacturer's protocol. For T cell proliferation measurement from the MLR assay, T cells were treated with the Violet CellTrace® Violet Cell Growth Kit (ThermoFisher, San Diego, CA). On the day of collection, cells were stained with anti-CD3 antibody PE (ThermoFisher, San Diego, CA). Dead cells (live and dead cell staining eFlour510, ThermoFisher, San Diego (stained with CA) and GFP-positive cells were removed from the gate. CD3-positive cells were placed back into the gate and analyzed for violet trace staining.

[0136] As shown in the central histogram of Figure 8A, RKO-transfected cells were able to activate and proliferate T cells. Proliferated T cells lost their pigment due to equal partitioning of the pigment in each proliferation cycle and appeared negative. As shown in both the left and right panels of the histogram in Figure 8A, T cells that did not proliferate retained their pigment. DCs showed similar results compared to RKO HLA-DR AB (data not shown). Since RKO and DCs express high levels of PD-L1, PD-L1 expression inhibited T cell proliferation. As shown in Figure 8B, the addition of anti-PD-L1 antibody increased T cell proliferation. Data were acquired using flow cytometry (CytofLEX S analyzer, Beckman Coulter) and data analysis was performed using Flowjo software version 10.2.

[0137] Figures 6A–6F and 7A–7D show representative photographs of proliferating T cells obtained from donors 1 and 2 at different magnifications. Figure 7A demonstrates that donor 1 (D1) T cells did not proliferate when cultured with RKO parental cells. Figure 6A shows that D1 T cells proliferated after treatment with anti-PD-1 antibody, and Figure 6E shows that donor 2 (D2) T cells proliferated after treatment with anti-PD-1 antibody. Figures 6B and 6C show that D1 T cells proliferated when cultured with HLA-DR (containing both alpha and beta units) transfected RKO cells. Similarly, Figures 6D and 6F show that D2 T cells proliferated when cultured with HLA-DR (containing both alpha and beta units) transfected RKO cells. In contrast, T cells did not proliferate when cultured with RKO parental cells (Figure 6G) or without any treatment (Figure 6H).

[0138] Furthermore, referring to Figures 7B–7D, T cells proliferated when cultured with RKO cells transfected with HLA-DR A+B (containing both alpha and beta units). T cell blasts and clusters are shown in solid circles, and RKO cells are enclosed in dashed circles.

[0139] Cytokines were measured from the supernatant of the cultures described above using the MSD U-Plex kit (Meso Scale Discovery LLC (Maryland MD)). The results were passed through an MSD MESO QuickPlex SQ 120 analyzer and analyzed using MSD software and GraphPad Prism. A two-way Anova was used for statistical analysis. Levels of IFN-gamma, TNF-alpha, IL-1 beta, and IL-6 were measured. Referring to Figure 8C, IFN-gamma and TNF-alpha were increased from T cells incubated with RKO HLA-DR cells or DCs (not shown, positive control used only as a positive control) compared to the RKO parent lineage, and treatment with checkpoint inhibitors increased cytokine secretion in these cultures. IL-1 beta and IL-6 were not detected or detected at low levels, indicating that cytokines were secreted by T cell activation rather than from innate cells such as DCs or tumor RKO cells. Two sets of data are presented with SEM. Data for RKO or RKO HLA-DR1 are shown in Figure 8C and the table below. Table 2. Cytokine secretion from T cells activated in the MLR by parental RKO cell lines versus HLA-DR AB cotransfected RKO cell lines. [Table 2]

[0140] (Example 3) Administration of naturally occurring Class I MHC components

[0141] Individuals with ovarian cancer are found to exhibit reduced HLA-A expression in ovarian tissue compared to baseline HLA-A expression levels. Patients are administered an adenovirus vector containing a naturally occurring, non-existent HLA-A gene modified for enhanced expression in ovarian tissue. Expression of the naturally occurring non-existent HLA-A gene is restored in the individual.

[0142] (Example 4) Targeted demethylation of hypermethylated HLA promoter regions in colon cancer

[0143] Tumor biopsies were performed on individuals with colon cancer previously shown to be unresponsive to immune checkpoint inhibitor therapy. First, the expression of each class I HLA and class II HLA gene was determined. Each class I HLA gene was shown to have severely reduced expression compared to normal class I HLA expression. DNA from non-cancerous tissue of the same individual was extracted along with the tumor-derived DNA. Aliquots of each DNA sample were sequenced for each of the HLA-A, HLA-B, and HLA-C genes. The remaining DNA samples were treated with bisulfite, and the same genes were subsequently sequenced. Comparison of bisulfite-treated DNA with non-bisulfite-treated sequences revealed that the promoters of each of the three HLA class I genes were methylated in relation to non-cancerous HLA class I genes at two different CpG sites per promoter.

[0144] An immunotherapy composition containing seven different nucleic acid molecules was prepared. These consisted of one nucleic acid molecule encoding an inactivated CRISPR-related nuclease fused to a TET enzyme (demethylase), and the remaining six nucleic acid molecules encoding guide RNAs (gRNAs). Each gRNA targeted one of six methylated CpG sites identified in its promoter. This composition was administered to an individual. After one day, the expression of class I HLA molecules in the individual was assessed and shown to be elevated. Next, the individual was administered immune checkpoint inhibitor therapy.

[0145] (Example 5) Administration of Class II MHC components

[0146] Individuals with pancreatic cancer are administered liposomes containing plasmids encoding the HLA-DQA1 and HLA-DQB1 genes.

[0147] While preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided as examples only. Therefore, those skilled in the art will anticipate numerous variations, changes, and substitutions without departing from the present disclosure. It should be understood that in practicing the present disclosure, various alternatives to the embodiments of the present disclosure described herein may be used. The following claims define the scope of the present disclosure and are intended to encompass the methods and structures of such claims, as well as their equivalents. Table 3. HLA alleles [Table 3-1] [Table 3-2] [Table 3-3]

Claims

[Claim 1] An immunotherapy composition comprising a nucleic acid molecule encoding a first MHC component or a fragment thereof, and at least one pharmaceutically acceptable excipient, diluent, or carrier.

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