Treatment of MHC-I negative tumors by NK and T cells

Abstract

A method and composition are provided herein, wherein a therapeutic agent comprising NK cells is administered to a cancer patient to induce the expression of MHC-I in a tumor, and subsequent treatment with T cells effectively targets tumor-associated antigens and neoepitopes presented by the newly expressed MHC-I in said tumor cells.

Description

Technical Field

[0001] This application claims the benefit of priority based on U.S. Provisional Application No. 63 / 391,912, filed on July 25, 2022. This case and all other external references cited herein are incorporated by reference in their entirety.

[0002] The field of the present invention is combinatorial immunotherapy technology.

Background Art

[0003] Major histocompatibility complex (MHC) proteins are found on the surface of all nucleated cells. It is involved in the presentation of intracellular digestion peptides on the cell surface, whereby screening by T cells for non-self peptide sequences is carried out. Peptide sequences from non-self sources such as cancer and infectious pathogens are bound to MHC and presented on the cell surface, whereby T cells bind to such occupied MHC proteins and initiate various immune defense programs including cytokine production and direct cytotoxicity. Intracellular proteins are presented on the cell surface by MHC-I, while extracellular proteins are ingested and processed by phagocytosis and presented by MHC-II. MHC-I is expressed on the surface of all nucleated cells and is involved in the presentation of mutated (non-self) peptides of cellular origin.

[0004] Alternatively, natural killer (NK) cell-mediated cytotoxicity is inhibited by the presence of MHC-I expressed on the surface of target cells. Infectious agents have a mechanism to reduce MHC expression in infected cells, thereby reducing the host's ability to initiate T cell-mediated immunological defenses, while NK cell-mediated defenses are enabled by the reduction of MHC expression in infected cells.

[0005] Loss of MHC proteins has been described in cancer cells, whereby T cell activity is diminished. Typically, loss of MHC is associated with a decrease in the amount of tumor infiltrating lymphocytes. As a result, patients are unable to initiate an effective immune response directed against non-self peptides in cancerous or infected tissues. MHC-I expression can be induced by various agents with well-characterized inductions, notably interferon-g. T cell therapy following induction of MHC-I can be an effective therapeutic approach.

Summary of the Invention

Problems to be Solved by the Invention

[0006] Accordingly, there remains a need for an effective therapy that enables and increases the host immune response against cancerous tissue.

Means for Solving the Problems

[0007] The subject of the present invention provides compositions, methods, and kits that enable or enhance an immune response against cancer or infectious diseases.

[0008] In one embodiment, the subject matter of the present invention includes a method for treating cancer, the method including determining the MHC-I expression level in a tumor. If it is determined that the MHC-I level is low compared to at least control non-cancerous tissue, a plurality of NK cells are administered to the patient, where the NK cells include at least one of aNK cells, haNK cells, t-haNK cells, or primary ceNK or memory-like ceNK (m-ceNK) cells. The NK cells can be administered intravenously or intratumorally. After a certain time, a plurality of T cells are administered to the patient. The T cells can be autologous or allogeneic. The T cells can further be genetically modified to express a targeting agent, and in that case, the agent includes a CAR or a TCR. By way of example and not limitation, the T cells can be administered to the patient up to 1 hour after administration of the NK cells, or up to 2 hours after administration of the NK cells, or up to 5 hours after administration of the NK cells, or up to 10 hours after administration of the NK cells, or up to 1 day after administration of the NK cells, or up to 5 days after administration of the NK cells, or up to 1 week after administration of the NK cells, or up to 2 weeks after administration of the NK cells, or up to 1 month after administration of the NK cells, or up to 2 months after administration of the NK cells, or up to 6 months after administration of the NK cells, or up to 1 year after administration of the NK cells, or up to 2 years after administration of the NK cells.

[0009] In one embodiment, the subject matter of the present invention includes a method for treating an infectious disease, the method including determining the MHC-I expression level in the affected tissue. If it is determined that the MHC-I level is low compared to control healthy normal tissue, a plurality of NK cells are administered to the patient, where the NK cells include at least one of aNK cells, haNK cells, t-haNK cells, or primary ceNK or memory-like ceNK (m-ceNK) cells. The NK cells can be administered intravenously or intratumorally. After a certain time, a plurality of T cells are administered to the patient. The T cells can be autologous or allogeneic. The T cells can further be genetically modified to express a targeting agent, and in that case, the agent includes a CAR or a TCR.

[0010] In one embodiment, the subject matter of the present invention includes an immunotherapy composition comprising a plurality of NK cells, where the NK cells include at least one of aNK cells, haNK cells, t-haNK cells, ceNK cells, or m-ceNK cells. The NK cells can be formulated as a plurality of whole cells for intravenous administration or as a cell lysate for intratumoral administration. The composition further includes a plurality of T cells, where the T cells are formulated for intravenous administration. The T cells can be autologous or allogeneic. The T cells can further be genetically modified to express a targeting agent, where in that case the agent includes a CAR or a TCR.

[0011] In one embodiment, the subject matter of the present invention includes the use of an immunotherapy composition in the treatment of cancer or an infectious disease, the use including determining the MHC-I expression level in the affected tissue, and the composition comprising a plurality of NK cells, where the NK cells include at least one of aNK cells, haNK cells, t-haNK cells, ceNK cells, or m-ceNK cells. The NK cells can be formulated as a plurality of whole cells for intravenous administration or as a cell lysate for intratumoral administration. The composition further includes a plurality of T cells, where the T cells are formulated for intravenous administration. The T cells can be autologous or allogeneic. The T cells can further be genetically modified to express a targeting agent, where in that case the agent includes a CAR or a TCR.

[0012] The treatment method for cancer or infectious diseases may further include the administration of a secondary agent administered in parallel with NK cells. The agent may include at least one therapeutic agent selected from the group consisting of gemcitabine, IFNγ, HDAC inhibitor, 5-aza-2'-deoxycytidine, DNA methyltransferase inhibitor, hydralazine, valproic acid, microRNA (miRNA), or inhibitors of ALK, RET, or MAPK kinases, glycosyltransferase inhibitor, thymidylate synthase inhibitor, dexamethasone, SMAC mimetic, IL-15, N-803, IL-15 derivatives, and autophagy inhibitor.

[0013] The immunotherapy composition may further include a secondary agent administered in parallel with NK cells. The agent may include at least one therapeutic agent selected from the group consisting of gemcitabine, IFNγ, HDAC inhibitor, 5-aza-2'-deoxycytidine, DNA methyltransferase inhibitor, hydralazine, valproic acid, microRNA (miRNA), or inhibitors of ALK, RET, or MAPK kinases, glycosyltransferase inhibitor, thymidylate synthase inhibitor, dexamethasone, SMAC mimetic, IL-15, N-803, IL-15 derivatives, and autophagy inhibitor.

[0014] The immunotherapy composition for use in the treatment of cancer or infectious diseases may further include a secondary agent administered in parallel with NK cells. The agent may include at least one therapeutic agent selected from the group consisting of gemcitabine, IFNγ, HDAC inhibitor, 5-aza-2'-deoxycytidine, DNA methyltransferase inhibitor, hydralazine, valproic acid, microRNA (miRNA), or inhibitors of ALK, RET, or MAPK kinases, glycosyltransferase inhibitor, thymidylate synthase inhibitor, dexamethasone, SMAC mimetic, IL-15, N-803, IL-15 derivatives, and autophagy inhibitor.

[0015] In one embodiment, the subject matter of the present invention includes a method for inducing MHC-I in cancer, the method including determining the MHC-I expression level in a tumor. If it is determined that the MHC-I level is lower compared to a control non-cancerous tissue, a plurality of NK cells are administered to the patient, where the NK cells include at least one of aNK cells, haNK cells, t-haNK cells, ceNK cells, or m-ceNK cells. The NK cells can be administered intravenously or intratumorally.

[0016] In one embodiment, the subject matter of the present invention includes a method for increasing the level of IFNγ in cancer, the method including determining the IFNγ expression level in a tumor. If it is determined that the IFNγ level is lower compared to a control non-cancerous tissue, a plurality of NK cells are administered to the patient, where the NK cells include at least one of aNK cells, haNK cells, t-haNK cells, ceNK cells, or m-ceNK cells. The NK cells can be administered intravenously or intratumorally.

[0017] In one embodiment, the subject matter of the present invention includes a method for determining the MHC-I inducibility of a tumor. In one aspect of the present invention, a patient biopsy sample is dissociated enzymatically and / or mechanically ex vivo, and individual aliquots are seeded into individual wells of a multi-well microplate. The sample is then exposed to an agent known to induce MHC-I, where the agent includes gemcitabine, IFNγ, HDAC inhibitor, 5-aza-2'-deoxycytidine, DNA methyltransferase inhibitor, hydralazine, valproic acid, microRNA (miRNA), or an inhibitor of ALK, RET, or MAPK kinase, a glycosyltransferase inhibitor, a thymidylate synthase inhibitor, dexamethasone, a SMAC mimetic, IL-15, N-803, an IL-15 derivative, and an autophagy inhibitor. After a certain time, the sample is assayed for surface expression of MHC-I.

[0018] Various objects, features, aspects, and advantages of the subject matter of the present invention will become apparent from the following detailed description of the preferred embodiments in conjunction with the accompanying drawings, in which like numerals represent like elements. **DETAILED DESCRIPTION OF THE INVENTION**

[0019] The subject matter of the present invention provides compositions, methods, and kits that enable or enhance an immune response against cancer or infectious diseases. An established biomarker for an immune response that can be used against exogenous or endogenous non-self immune stimulants is MHC-I expression. MHC-I is intended to mean MHC class I molecules, which are one of two major classes of major histocompatibility complex (MHC) molecules (the others being MHC class II), and are found on the cell surface of all nucleated cells in the vertebrate body. Its function is the external presentation on the cell surface of peptide fragments of proteins from within the cell to cytotoxic T cells, which will trigger an immediate response from the immune system against cells presenting specific non-self antigens by MHC class I proteins. Since MHC class I molecules present peptides derived from cytosolic proteins, the pathway of MHC class I presentation is often called the cytosolic or endogenous pathway. In humans, the HLAs corresponding to MHC class I are HLA-A, HLA-B, and HLA-C.

[0020] Regarding NK cells, all NK cells are considered suitable for the uses described herein, so it should be noted that they include primary NK cells (preserved, expanded, and / or fresh cells), immortalized secondary NK cells, autologous or heterologous NK cells (banks, preserved, fresh, etc.), and modified NK cells as described in more detail below. In some embodiments, the NK cells are preferably NK-92 cells. The NK-92 cell line is a unique cell line that has been found to grow in the presence of interleukin 2 (IL-2) (see, for example, Gong et al., Leukemia 8:652-658 (1994)). NK-92 cells are cancerous NK cells that have broad anti-tumor cytotoxicity and a predictable yield after expansion in a suitable culture medium. Advantageously, NK-92 cells have high cytolytic activity against various cancers.

[0021] The original NK-92 cell line expressed the CD56bright, CD2, CD7, CD11a, CD28, CD45, and CD54 surface markers and did not present the CD1, CD3, CD4, CD5, CD8, CD10, CD14, CD16, CD19, CD20, CD23, and CD34 markers. The growth of such NK-92 cells in culture depends on the presence of interleukin 2 (e.g., rIL-2) at a low dose of about 1 IU / mL, which is sufficient to maintain proliferation. IL-7 and IL-12 do not support long-term growth, nor do the various other cytokines tested, including IL-1a, IL-6, tumor necrosis factor α, interferon α, and interferon γ. Compared to primary NK cells, NK-92 typically has high cytotoxicity even at a relatively low effector:target (E:T) ratio (e.g., 1:1). Representative NK-92 cells are deposited with the American Type Culture Collection (ATCC) under the name CRL-2407. U.S. Patent No. 7,618,817, U.S. Patent No. 8,034,332, U.S. Patent No. 8,313,943, U.S. Patent No. 9,150,636, U.S. Patent No. 9,181,322, U.S. Patent No. 10,138,462, and U.S. Patent No. 10,258,649 are hereby incorporated by reference in their entirety, as are all other external references.

[0022] In another aspect of the subject matter of the present invention, the genetically engineered NK cells can also be NK-92 derivatives modified to express the high-affinity Fcγ receptor (CD16). Sequences of high-affinity variants of the Fcγ receptor are well known in the art (see, e.g., Blood 2009 113:3716-3725), and all methods of production and expression are considered suitable for the uses described herein. Expression of such receptors is thought to enable specific targeting of tumor cells using antibodies specific for the patient's tumor cells (e.g., neoepitopes), specific tumor types (e.g., her2neu, PSA, PSMA, etc.), or cancer-associated ones (e.g., CEA-CAM). Advantageously, such antibodies are commercially available and can be used in combination with the cells (e.g., by binding to the Fcγ receptor). Alternatively, such cells can also be obtained commercially from NantKwest as haNK cells. Such cells can then be further genetically modified to be CARs as described in more detail below. U.S. Patent No. 10,738,279, U.S. Patent No. 10,456,420, U.S. Patent No. 10,736,921, U.S. Patent No. 11,000,550, U.S. Patent No. 10,801,013, and U.S. Patent No. 10,774,310.

[0023] The genetic modification of NK cells contemplated herein can be carried out in many ways, and all known methods are considered suitable for use herein. Further, it should be recognized that NK cells are transfectable with DNA or RNA, and the specific choice of transfection will depend, at least in part, on the desired recombinant cell type and transfection efficiency. For example, if it is desired to stably transfect NK cells, linearized DNA can be introduced into the cells for integration into the genome. On the other hand, if transient transfection is desired, circular DNA or linear RNA (e.g., mRNA having a polyA+ tail) can be used.

[0024] For example, when the NK cells are autologous NK cells or NK-92 cells, the recombinant nucleic acid comprises a segment encoding a CAR comprising the FcεRIγ signaling domain and preferably a segment encoding a cytokine that provides autocrine growth stimulation (e.g., IL-2, IL-2 modified with an ER retention sequence, IL-15, or IL-15 modified with an ER retention sequence) and / or a segment encoding CD16 or high-affinity CD16 158V and is contemplated to be included. As will be readily appreciated, the incorporation of a cytokine that provides autocrine growth stimulation will provide a modified recombinant that is independent of exogenous cytokine addition, thereby enabling the large-scale production of such cells to be economically feasible. Similarly, when the modified recombinant expresses CD16 or high-affinity CD16 158V such cells will have further enhanced ADCC properties and thereby improved targeted cytotoxicity.

[0025] The recombinant nucleic acid encoding the cytokine and / or CD16 or high-affinity CD16 158V should be recognized as being integratable into the genome of the NK cells or as being supplyable as an extrachromosomal unit (which can be linear or circular DNA or linear RNA delivered by viral delivery or via chemical, mechanical, or electrical transfection). For example, recombinant NK-92 cells expressing IL-2ER and CD16158V are known as haNK cells (Oncotarget 2016 Dec 27;7(52):86359-86373) and are transfectable with a recombinant nucleic acid comprising a segment encoding a CAR comprising the FcεRIγ signaling domain. Again, such recombinant nucleic acids can include additional segments that can encode additional immunotherapy proteins such as N-803, TxM-type compounds, IL-8 trap, TGF-β trap, etc. Similarly, NK-92 cells can already be transfected with cDNA encoding IL-2 (e.g., NK-92MI, ATCC CRL-2408). Such cells can then be transfected with a segment encoding CD16 or high-affinity CD16158V It is further transfectable with a recombinant nucleic acid comprising a segment encoding 158V and a segment encoding a CAR comprising an FcεRIγ signaling domain.

[0026] On the other hand, (e.g., autologous, fresh, cultured, or pre-frozen) NK cells or NK-92 cells also include a segment encoding a CAR having an FcεRIγ signaling domain, a segment encoding a cytokine that provides autocrine growth stimulation (e.g., IL-2, IL-2 modified with an ER retention sequence, IL-15, or IL-15 modified with an ER retention sequence), and CD16 (SEQ ID NO: 34) or high-affinity CD16 158VA recombinant nucleic acid comprising a segment encoding (Array No. 35 encoded by Array No. 36), which is further disclosed in PCT / US2019 / 033407, the entire content of which is incorporated herein by reference. Most typically, such a recombinant nucleic acid will be arranged as a tricistronic construct. As described above, such constructs can be extrachromosomal circular plasmids, linear DNA (which can integrate into the genome of NK cells), or linear RNA. Such nucleic acids will typically be transfected into cells by methods well known in the art (e.g., electroporation, lipofection, biolistic gene transfer, etc.). Similarly, nucleic acids can be delivered to cells via recombinant viruses. Accordingly, NK cells suitable for use herein include NK-92 cells (which can be transfected with a tricistronic construct encoding a CAR, CD16 or a variant thereof, and a cytokine or a variant thereof), genetically modified NK cells or NK-92 cells expressing CD16 or a variant thereof or a cytokine or a variant thereof (which can be transfected with a nucleic acid encoding a CAR, CD16 or a variant thereof, or a cytokine or a variant thereof), and genetically modified NK cells or NK-92 cells expressing CD16 or a variant thereof and a cytokine or a variant thereof (which can be transfected with a nucleic acid encoding a CAR). U.S. Patent No. 17 / 056,385, U.S. Patent No. 11,077,143, U.S. Patent No. 10738279, and U.S. Patent No. 16 / 969,152 are incorporated herein by reference.

[0027] Thus, in preferred embodiments, it should be noted that genetically modified NK cells (especially when the cells express a CAR and CD16 or a variant thereof) will exhibit three distinguishable modes of cell death, namely, general cytotoxicity mediated by activation of receptors (e.g., NKG2D receptor), ADCC mediated by antibodies bound to target cells, and CAR-mediated cytotoxicity.

[0028] Therefore, it should be recognized that the method of transfection will likely depend, at least in part, on the type of nucleic acid being utilized. Accordingly, methods of viral transfection, chemical transfection, and mechanical transfection are all considered suitable for the uses described herein. For example, in one embodiment, the vectors described herein are transient expression vectors. Since the exogenous transgene introduced using such vectors is not integrated into the nuclear genome of the cell, in the absence of vector replication, the foreign transgene will degrade or dilute over time.

[0029] In another embodiment, the vectors described herein enable stable transfection of cells. In one embodiment, the vector enables the incorporation of a transgene into the genome of the cell. Preferably, such a vector has a positive selection marker, and suitable positive selection markers include any gene that enables cell growth under conditions that can kill cells that do not express the gene. Examples include, but are not limited to, antibiotic resistance such as geneticin (Neo gene derived from Tn5). Alternatively or additionally, the vector is a plasmid vector. In one embodiment, the vector is a viral vector. As will be understood by those skilled in the art, any suitable vector can be used, and suitable vectors are well known in the art.

[0030] In yet other embodiments, the cells are transfected with mRNA encoding the target protein (e.g., CAR). Transfection with mRNA results in transient expression of the protein. In one embodiment, transfection of the mRNA into NK-92 cells is performed immediately prior to administration of the cells. In one embodiment, "immediately prior" to administration of the cells means from about 15 minutes to about 48 hours prior to administration. Preferably, transfection with the mRNA is performed from about 5 hours to about 24 hours prior to administration. As described in more detail below, in at least some embodiments, transfection of NK cells with mRNA resulted in unexpectedly consistent and strong expression of CAR in a high fraction of transfected cells. Furthermore, such transfected cells also exhibited high specific cytotoxicity at a relatively low effector cell to target cell ratio.

[0031] It should be noted that with respect to the CARs contemplated, NK or NK-92 cells will be genetically modified to express the CAR as a membrane-bound protein by exposing a portion of the CAR on the cell surface while maintaining the signaling domain in the intracellular space. Most typically, the CAR will comprise at least the following elements, namely, an extracellular binding domain, a hinge domain, a transmembrane domain, and an FcεRIγ signaling domain (in that order).

[0032] In preferred embodiments, the cytoplasmic domain of the CAR comprises or consists of the signaling domain of FcεRIγ. Notably, as described in more detail below, the FcεRIγ signaling domain provides a substantially increased expression level of the CAR, along with significantly long-term cytotoxicity over time. In some embodiments, the FcεRIγ cytoplasmic domain is the sole signaling domain. However, it should be recognized that additional elements, such as other signaling domains (e.g., CD28 signaling domain, CD3ζ signaling domain, 4-1BB signaling domain, etc.) may also be included. These additional signaling domains may be located downstream and / or upstream of the FcεRIγ cytoplasmic domain. In alternative embodiments, the cytoplasmic domain of the CAR may also include the signaling domain of CD3 zeta (CD3ζ). In one embodiment, the cytoplasmic domain of the CAR consists of the signaling domain of CD3 zeta.

[0033] Accordingly, the CARs contemplated will include the general structure of a desired antigen-binding domain attached to a hinge domain attached to a transmembrane domain attached to a signaling domain. Viewed from another perspective, the CARs contemplated may have a desired binding domain, which is then attached to a hybrid protein that comprises or consists of or consists essentially of a hinge domain attached to a transmembrane domain attached to a signaling domain.

[0034] Although not necessarily, most typically, the extracellular binding domain of a CAR will be a scFv or other natural or synthetic binding moiety that specifically binds to a target antigen. Particularly suitable binding moieties include small antibody fragments with single, dual, or multiple target specificities, β-barrel main binders, phage display fusion proteins, and the like. Among the suitable extracellular binding domains, particularly preferred domains will specifically bind to tumor-specific antigens, tumor-associated antigens, or antigens specific to the patient and tumor. Tumor-specific antigens include, but are not limited to, NKG2D ligands, CS1, GD2, CD138, EpCAM, EBNA3C, GPA7, CD244, CA-125, ETA, MAGE, CAGE, BAGE, HAGE, LAGE, PAGE, NY-SEO-1, GAGE, CEA, CD52, CD30, MUC5AC, c-Met, EGFR, FAP, WT-1, PSMA, NY-ESO1, AFP, CEA, CTAG1B, and CD33. Additional tumor-associated antigens and related malignancies, although not limited, can be found in Table 1. Additional tumor-specific antigens include, but are not limited to, for example, those described in U.S. Patent Application Publication No. 2013 / 0189268, International Publication No. 1999024566A1, U.S. Patent No. 7,098,008, and International Publication No. 2000020460 (each incorporated herein by reference in its entirety). Similarly, other preferred domains will specifically bind to (pathogenic) virus-specific antigens, such as antigens of the HIV virus (e.g., gp120), HPV virus, RSV virus, influenza virus, Ebola virus, or HCV virus.

[0035] Therefore, the intended CARs will target antigens associated with specific cancer types. For example, target cancers include leukemia (including acute leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia (including promyelocytic, myeloblastic, myelomonocytic, monocytic, and erythroleukemia)), and chronic leukemia (e.g., chronic myelogenous (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphoma (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenström macroglobulinemia, heavy chain disease, solid tumors, e.g., but not limited to sarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0036] In one embodiment, the subject matter of the present invention includes a method of treating cancer, the method comprising determining the MHC-I expression level in a tumor. If a determination is made that the MHC-I level is low compared to a control non-cancerous tissue, a plurality of NK cells are administered to the patient, where the NK cells include at least one of aNK cells, haNK cells, t-haNK cells, or primary ceNK or memory-like ceNK (m-ceNK) cells. The NK cells can be autologous or allogeneic. The NK cells can be administered intravenously or intratumorally. After a period of time, a plurality of T cells are administered to the patient. The T cells can be autologous or allogeneic. The T cells can further be genetically modified to express a targeting agent, where the agent includes a CAR or a TCR.

[0037] Suitable methods for determining MHC-I levels in tumor and normal tissue samples involve measuring cell surface expression of MHC-I. Enzymatic or mechanical dissociation of tissue in which viable whole cells are purified and isolated from primary tissue is required for Ab staining and flow cytometric determination of MHC-I surface expression. Alternative methods may include transcriptomic analysis, proteomics, Western blotting, and surface plasmon resonance (SPR).

[0038] As used herein, t-haNK cells are NK cells that express a genetically modified CAR. Without being bound by any particular theory, the targeting moiety on the CAR is understood to serve a dual purpose. First, the targeting moiety facilitates delivery of NK cells to tumor tissue by directing the NK cells to the site of antigen expression in a patient. Second, the targeting moiety may be immunogenic and thereby facilitate delivery of antibodies and T cells to the tumor.

[0039] Primary NK cells can also be enriched and expanded from whole cells or cord blood mononuclear cells via standard methods including exposure of the primary NK cells to CD16 antibodies, dexamethasone, and / or IL-15. Stabilized IL-15 can be used, in which case the stabilized IL-15 includes IL-15 superagonists such as n803 (Alt-803, N-803, Vesanktiva™), and further stabilized IL-15 / IL15-Ra fusion proteins. U.S. Patent Application No. 16 / 985,728, U.S. Patent Application No. 16 / 505,528, U.S. Patent No. 11,351,196, U.S. Patent Application No. 63 / 156,269, U.S. Patent No. 8,163,879, U.S. Patent No. 8,507,222, and U.S. Patent No. 10,537,615 are incorporated herein by reference.

[0040] The cytokine-enhanced NK (ceNK) cells disclosed herein refer to NK cells whose cytotoxic activity is enhanced by cytokine stimulation. ceNK cells are prepared by inducing NK cells with corticosteroids and optionally a cytokine composition containing IL-15, IL-15:IL-15Rα, or an agonist derivative thereof such as N-803. The cytokine composition may contain a fusion protein, and in that case, the fusion protein contains IL-15 or an agonist derivative thereof. A fusion protein containing IL-15, which is a fusion protein with increased stability compared to IL-15, is preferred. Without limiting the subject matter of the present invention, it is generally preferred that the corticosteroid is hydrocortisone and the optional cytokine is N-803.

[0041] The memory-like cytokine-enhanced NK cells (m-ceNK) disclosed herein include enriched and expanded NK cells obtained from a donor's peripheral blood using apheresis technology to generate NK cells with a memory-like phenotype. m-ceNK cells exhibit both high cytotoxicity and increased interferon-gamma production. Such m-ceNK cells can be generated from individual donors for autologous cell therapy or can be generated from umbilical cord blood as an allogeneic product. In addition to enhanced efficacy, m-ceNK cells can be easily injected in an outpatient setting.

[0042] Although not limited thereto, for example, m-ceNK cells can be generated by obtaining a plurality of mononuclear cells and contacting the plurality of mononuclear cells with corticosteroids and optionally cytokines. In another step, the plurality of mononuclear cells are incubated in the presence of corticosteroids and optional cytokines to enrich the NK cells of the mononuclear cells, and then the enriched NK cells are induced with a cytokine composition containing IL-15, IL-12, and IL-18 or agonist derivatives thereof. The composition can include one or more fusion proteins, and in that case, the fusion protein includes at least one of the IL-15, IL-12, and IL-18 cytokines or agonist derivatives thereof. The cytokine composition can include a TxM fusion protein for generating m-ceNK cells, and in that case, the TxM fusion protein includes a protein portion having IL-12 activity, a protein portion having IL-15 activity, and a protein portion having IL-18 activity.

[0043] Further description of making m-ceNK cells and their advantageous properties is described in PCT / US2022 / 018290 (which is incorporated by reference in its entirety). US Patent Application No. 17 / 375,985 and US Patent No. 11,453,862 provide additional alternative methods for inducing enrichment and expansion of NK cells. Each of the above references is incorporated by reference in its entirety.

[0044] Activated cytotoxic cell therapy "T cell receptor" or "TCR" typically refers to a dimeric polypeptide found on the surface of T cells. Each peptide chain of the TCR generally includes an extracellular domain containing a variable region and a constant region, a transmembrane domain, and an intracellular domain. The variable region is the part of the TCR that interacts with the antigen presented by MHC. The constant region is the region of each of the two peptides, where the two peptide chains are covalently linked by a disulfide bond. The intracellular domain generally includes CD3z, which contains one or more immunoreceptor tyrosine-based activation motifs (ITAMs). The ITAMs mediate the binding of the variable region to the appropriate intracellular signaling pathway.

[0045] The intracellular signaling domain of the CAR may also include an FcεRIγ moiety. The specification of U.S. Patent Application No. 17 / 341098 is incorporated herein by reference.

[0046] T cells may optionally contain a modified TCR, which is associated with a dimeric polypeptide based on the TCR structure. Specifically, the modified TCR includes two peptide chains, each of which includes an extracellular domain (including a variable region, a constant region, and a connecting peptide), a transmembrane domain, and an intracellular domain. In a specific embodiment, the variable region and the constant region are attached via a linker. In another specific embodiment, the connecting peptide is located between the constant region and the transmembrane domain. In a further specific embodiment, the two peptide chains are connected to each other by a disulfide bond between the connecting peptides of each peptide chain. The modified TCR does not interact with the endogenous TCR produced by the T cell. The content of U.S. Patent Application No. 63 / 227,195 is incorporated herein by reference.

[0047] Once a CAR-based therapeutic agent (e.g., an antigen-binding domain conjugated to a CAR scaffold) binds to an antigen expressed by cancer cells, cytotoxic cells can trigger the destruction of the cancer cells. All cytotoxic cells are generally contemplated to be suitable for the uses described herein, and among the particularly preferred cytotoxic cells are those modified to recombinantly express a CAR-based therapeutic agent among NK cells, activated NK cells, high-affinity NK cells, CD8+ T cells, and CD4+ T cells (which may all be of different origins). Cytotoxic cells are engineered to express a TCR that recognizes MHC-I presented peptides.

[0048] The therapeutic T cells used herein can be patient-derived (autologous) or donor-derived (allogeneic). T cells are typically obtained via leukapheresis and further separated according to surface marker (CD4, CD8) expression. Purified T cells can be activated by exposure to CD3 and / or CD28 Ab or by exposure to antigen-presenting cells (APCs). Cells can be expanded by exposure to a cytokine cocktail containing one or more of IL-2, IL-15, and IL-7. In a preferred embodiment, T cells or primary NK cells are expanded on an automated platform and can be transfected by microfluidic through-electroporation as described in U.S. Patent No. 11,377,652, the content of which is incorporated herein by reference.

[0049] Since T cells can be purified from tumors, they are tumor-infiltrating lymphocytes (TILs). TILs can be purified from tumor tissue and expanded ex-vivo. TILs can be re-introduced into a patient as autologous cells or administered to a different subject as donor cells. Stimulation of MHC-I expression by NK cells is expected to result in enhanced cytotoxic efficacy by TILs.

[0050] In one embodiment of the present invention, TILs are isolated from a patient's tumor by standard techniques. The TILs are then exposed to the patient's tumor tissue, where the patient has been treated with NK cells, thereby inducing the expression of MHC-I. NK exposure can be by intravenous injection or intratumoral injection. Exposure of the tumor tissue to NK can be performed ex vivo. TILs exposed to the tumor tissue ex vivo are thereby activated and expanded. The expanded TILs, including CD4 and CD8 cytotoxic T cells, are administered to the patient.

[0051] T cells can be transfected to express one or more CARs. Transfection techniques include, but are not limited to, viral transduction, mRNA transfection, and the Sleeping Beauty transposon system. Following transfection, the CAR T cells can be expanded in a bioreactor until a clinically effective cell number is obtained.

[0052] However, in other aspects, it should be recognized that the cytotoxic cells can also be macrophages, monocytes, neutrophils, basophils, or eosinophils. Thus, from different perspectives, the cells contemplated herein can exert cytotoxic effects, such as by triggering the TNF or fas-mediated cell death pathway through phagocytosis, pore formation, induction of antibody-dependent cell-mediated cytotoxicity (ADCC), etc.

[0053] Cytotoxic cells can release various types of cytotoxic granules (e.g., granulysin, perforin, granzyme) as part of the cytotoxic anti-tumor process. To monitor cell-mediated cytotoxicity, various assays, including flow cytometric assays, are available based on the presence of lytic granules such as perforin, granzyme, or the production of TNF family members such as TNF-α, FasL, TRAIL (Zaritskaya 2010, Clay, T. et al., Clin. Cancer Res. (2001) &: 1127 - 1135).

[0054] In one embodiment, the body fluid is obtained after treatment with NK cells, in which case the body fluid contains cellular components, e.g., tumorigenic cells or cancer cells that present antigens for binding by CAR-expressing cytotoxic cells as described herein and for contact of cytotoxic cells expressing antigen-binding moieties with cells. Then, an assay is performed, for example, to detect an immune response suggesting that an ADCC response or an ADCP response has been triggered by the patient's own immune cells.

[0055] Assays for detecting immune responses are known in the art and are described herein. For example, assays for detecting such responses can detect the release of cytotoxic granules (e.g., granzyme, perforin, granzyme), or phagocytosis, or receptor-ligand mediated cell lysis (e.g., mediated by the Fas / APO pathway). To monitor cell-mediated cytotoxicity, various flow cytometric assays are available based on the presence of lytic granules such as perforin, granzyme, etc. or the production of TNF family members such as TNF-α, FasL, TRAIL, etc. (Zaritskaya 2010, Clay, T. et al., Clin. Cancer Res. (2001) 7:1127-1135).

[0056] In other embodiments, to promote or trigger an immune response, an immunostimulatory cytokine is administered to the patient in combination with cytotoxic cells expressing a CAR-based therapeutic agent (e.g., an antigen-binding domain conjugated to a CAR scaffold). Cytokines include, but are not limited to, IL2, IL4, IL7, IL11, IL15, IL21, TNF-alpha, IFN-gamma, etc. In some embodiments, the cytokine can reactivate exhausted T cells. In other cases, immune competent cells can be engineered to recombinantly express one or more cytokines.

[0057] Other cancer treatment techniques include surgery, radiation therapy, chemotherapy, immunosuppressive agents (e.g., azathioprine, cyclosporine, methotrexate, mycophenolate, etc.), immunotherapy, targeted therapy, hormone therapy, stem cell transplantation, or other precision methods. Any of these techniques can be combined with embodiments of the present invention for treating cancer.

[0058] Embodiments of the present invention are understood to be administrable to patients using appropriate formulations, indications, and dosing regimens as deemed suitable by government regulatory authorities such as the Food and Drug Administration (FDA) in the United States.

[0059] In some embodiments, cytotoxic cells expressing a TCR, modified TCR, or CAR-based therapeutic agent (e.g., an antigen-binding domain conjugated to a CAR scaffold) are administered to a patient as a pharmaceutical composition. In another embodiment, a method of treating cancer by administering cytotoxic cells to a subject is contemplated. In yet another embodiment, a method of inhibiting or reducing the growth of cells expressing a corresponding antigen (to which the antigen-binding region binds) on the surface of cells by administering cytotoxic cells to a subject is contemplated.

[0060] In one embodiment, the patient can be lymphodepleted, thereby reducing the number of endogenous lymphocytes, increasing the availability of essential endogenous cytokines, and promoting the survival of infused T cells.

[0061] In some embodiments, cytotoxic cells expressing a TCR, modified TCR, or CAR-based therapeutic agent (e.g., an antigen-binding domain conjugated to a CAR scaffold) reduce the amount (e.g., cell number, mass size, etc.) by at least 25%, at least 50%, at least 75%, at least 90%, at least 95%, or at least 99% in a subject having cancer associated with the expression of the corresponding antigen on the surface of the cells compared to a negative control.

[0062] Examples of cancers that can be treated by the cytotoxic cells contemplated herein include any cancer that expresses or overexpresses a cancer - associated antigen on the cell surface. Examples of cancers that can be treated with cytotoxic cells expressing a TCR, modified TCR, or CAR - based therapeutic (e.g., an antigen - binding domain conjugated to a CAR scaffold) include, but are not limited to, breast cancer, colon cancer, leukemia, lung cancer, melanoma, neuroblastoma, pancreatic cancer, pediatric intracranial ependymoma, and prostate cancer.

[0063] The pharmaceutical composition can comprise cytotoxic cells comprising an antigen - binding domain conjugated or linked to a CAR scaffold as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Additionally, the pharmaceutical composition can include one or more adjuvants (e.g., aluminum hydroxide), antioxidants, bacteriostatic agents, buffers, carbohydrates, chelating agents such as EDTA or glutathione, coloring substances, flavoring substances, and / or aromatic substances, emulsifying agents, excipients, lubricants, pH buffers, preservatives, salts that affect osmotic pressure, polypeptides (e.g., glycine), proteins, solubilizing agents, stabilizing agents, wetting agents, etc., that do not react detrimentally with the active compound (e.g., an antigen - binding domain conjugated to a CAR scaffold, etc.) or otherwise interfere with its activity. Buffers include, but are not limited to, neutral buffered saline, phosphate - buffered saline, etc. Carbohydrates include, but are not limited to, dextran, glucose, mannose, mannitol, sucrose, etc.

[0064] The pharmaceutical composition can be formulated for a particular mode of administration. Modes of administration include, but are not limited to, the following: intra - articular, intradermal, intranasal, intraperitoneal, intrathecal, intratumoral, intravenous, intraventricular / intracerebroventricular, subcutaneous, transdermal, transmucosal, or topical routes.

[0065] In a preferred embodiment, the cytotoxic cells are administered by intravenous injection. Such formulations can be prepared according to standard techniques known to those skilled in the art. For example, a composition for intravenous administration can have one or more components (e.g., diluents, suspension buffers, saline or dextrose / water, other components such as cytokines, etc.) prior to injection into the patient.

[0066] Many such techniques for formulating and administering pharmaceutical compositions are known in the art and can be found, for example, in U.S. Patent Application Publication No. 2014 / 0242025, and all such references are hereby incorporated by reference in their entirety.

[0067] In some embodiments, the cytotoxic cells proliferate in vivo and thereby provide a sustained mechanism for inhibiting tumor growth or recurrence by persisting in the patient for several months or even years after administration. In some aspects, the cytotoxic cells persist for at least 3 months, 6 months, 9 months, 12 months, 15 months, 18 months, 2 years, 3 years, 4 years, or 5 years after administration of the cytotoxic cells to the patient.

[0068] The cytotoxic cells can be obtained from any of a variety of sources (e.g., from humans, from commercially available cytotoxic cells, cells isolated from a repository, etc.). Ex vivo expansion procedures for NK cells, T cells, or other types of cytotoxic cells are known in the art (e.g., Smith et al., Clinical & Translational Immunology (2015) 4:e31). The examples presented herein are not intended to be limited to any particular ex vivo expansion method for cytotoxic cells.

[0069] The pharmaceutical composition containing cytotoxic cells described in this specification can be administered at a dosage of 10^4 to 10^9 cells / kg body weight, 10^5 to 10^6 cells / kg body weight, or any integer value within these ranges. The cytotoxic cell composition can be administered once or sequentially (over several days or weeks or months) at these dosages. Injection techniques for cytotoxic cells such as T cells are known in the art (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988).

[0070] In other embodiments, the pharmaceutical composition is administered in a therapeutically effective amount that is effective to treat a specific indication. Administration can be as a single dose or based on an interval. As used herein, "interval" implies that the therapeutically effective amount is administered regularly (distinguished from a single dose). The dosing interval for an individual need not be a fixed interval and can vary over time. The terms "in combination with" or "co-administered" imply that the composition can be administered immediately before, simultaneously or nearly simultaneously with, or immediately after another composition.

Examples

[0071] Example 1 aNK, haNK, HER2 t-haNK, and CD19 t-haNK cells are administered to immunodeficient mice bearing HER2 or CD19-expressing tumor xenografts. The cells are administered as whole cell preparations or lysates. The whole cells and lysates are administered either intratumorally or intravenously. The tumors are then monitored for the expression of MHC-I, MHC-II, B2M, and CIITA. MHC-I and MHC-II are monitored by flow cytometry for cell surface expression. Media-treated mice are used as controls.

[0072] Example 2 Assay a cancer patient biopsy containing cancer cells for MHC-I expression. The assay preferably includes MHC-I expression in non-cancerous tissue from the same tissue source as the biopsy. The expression of MHC-I in cancerous cells should be at least statistically significantly (p>0.05) lower in three cancerous samples compared to three control samples. Preferably, the expression of MHC-I in carcinoma cells should be 80%, 60%, 40%, 20%, or <10% of the expression of MHC-I in normal cells. Preferably, the expression of MHC-I in carcinoma cells is <10% of the expression in control cells.

[0073] Example 3 Treat a cancer patient having a tumor determined to have low MHC-I expression with NK cells or NK cell lysate. The treatment is by intratumoral injection. Re-evaluate the tumor for MHC-I expression within 1 - 7 days. An increase in expression suggests that the patient is currently a suitable candidate for T cell therapy. T cells are derived from 1) the tumor as TIL, 2) lymph nodes, or 3) peripheral blood. T cells can be combined. Grow them in vitro to a clinically effective amount. Administer the T cells to the patient by intravenous or intratumoral injection.

[0074] The foregoing considerations provide many embodiments of the subject matter of the present invention. Each embodiment represents a single combination of elements of the present invention, but the subject matter of the present invention is considered to include all possible combinations of the disclosed elements. Thus, if one embodiment includes elements A, B, and C, and a second embodiment includes elements B and D, the subject matter of the present invention is also considered to include other remaining combinations of A, B, C, or D, even if not explicitly disclosed herein.

[0075] It should be apparent to those skilled in the art that many more changes are possible without departing from the concept of the invention described herein than those already described. Accordingly, the subject matter of the present invention should not be limited except within the spirit of the appended claims. Further, when interpreting both this specification and the claims, all terms should be interpreted in the broadest possible sense consistent with the context. Specifically, the terms "comprises" and "comprising" should be interpreted as referring non-exclusively to elements, components, or steps, indicating that the referenced elements, components, or steps may exist, or that they may be utilized, or that they may be combined with other elements, components, or steps not explicitly referenced. When this specification or the claims refer to at least any one selected from the group consisting of A, B, C.... and N, the language should be interpreted as requiring only one element of the group, not A+N, B+N, etc.

[0076] All publications mentioned in this specification are hereby incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Where the definition or use of a term in an incorporated reference conflicts or contradicts the definition of that term provided in this specification, the definition of that term provided in this specification applies and the definition of that term in the reference does not apply.

[0077] In some embodiments, the numbers representing the amounts, properties, such as concentrations, reaction conditions, etc., of the components used to describe and claim certain embodiments of the subject matter of the present invention should be understood to be modified in some instances by the term "about." Therefore, in some embodiments, the numerical parameters recited in the written description and the appended claims are approximate values that can vary depending on the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the reported significant digits and by applying ordinary rounding techniques. Although the numerical ranges and parameters indicating the broad scope of some embodiments of the subject matter of the present invention are approximations, the numerical values set forth in the specific examples are reported as accurately as possible. The numerical values presented in some embodiments of the subject matter of the present invention may contain certain errors that necessarily result from the standard deviation found in their respective test measurements.

[0078] In some embodiments, the numbers representing the amounts, properties, such as concentrations, reaction conditions, etc., of the components used to describe and claim certain embodiments of the subject matter of the present invention should be understood to be modified in some instances by the term "about." Therefore, in some embodiments, the numerical parameters recited in the written description and the appended claims are approximate values that can vary depending on the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the reported significant digits and by applying ordinary rounding techniques. Although the numerical ranges and parameters indicating the broad scope of some embodiments of the subject matter of the present invention are approximations, the numerical values set forth in the specific examples are reported as accurately as possible. The numerical values presented in some embodiments of the subject matter of the present invention may contain certain errors that necessarily result from the standard deviation found in their respective test measurements.

[0079] Unless otherwise defined in context, all ranges set forth in this specification are to be construed as including their endpoints, and open-ended ranges are to be construed as including only commercially practical values. Similarly, all lists of values are to be considered as including intermediate values, unless otherwise suggested by context.

[0080] As used throughout this specification and the following claims, the meanings of "a", "an", and "the" include references to plural forms, unless the context clearly dictates otherwise. Also, as used in this specification, the meaning of "in" includes "in" and "on", unless the context clearly dictates otherwise.

[0081] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise specified herein, each individual value is incorporated herein as if it were individually recited herein. All methods described herein are capable of being performed in any suitable order, unless otherwise specified herein or otherwise clearly contradicted by context. Any examples or exemplary representations provided for a particular embodiment herein (e.g., "such as") are merely intended to better enable an understanding of the subject matter of the invention and are not intended to limit the scope of the subject matter of the invention as otherwise claimed. No representation herein is to be construed as suggesting that any non-claimed element is essential to the practice of the subject matter of the invention.

[0082] The grouping of alternative elements or alternative embodiments of the subject matter of the invention disclosed in this specification should not be construed as limiting. Each group member can be referenced and claimed individually or in any combination with other members of that group or other elements found in this specification. One or more members of the group can be included or excluded for reasons of convenience and / or patentability. When making any such inclusion or exclusion, the designation is considered in this specification to contain the group as amended, so as to satisfy the description of all Markush groups used in the appended claims.

Claims

**Claim 1** A method for treating a patient having MHC-I negative tumor tissue, the method comprising the sequential administration of an effective amount of NK cells and then an effective amount of T cells. **Claim 2** The method of claim 1, wherein the NK cells are NK-92 cells or genetically modified derivatives thereof, activated cytokine-enriched NK (ceNK) cells, or memory-like ceNK (m-ceNK) cells. **Claim 3** The method of claim 2, wherein the NK cells are induced or genetically modified to overexpress interferon gamma (IFNγ). **Claim 4** The method of claim 3, wherein the NK cells comprise at least one of a chimeric antigen receptor (CAR) and an Fc receptor. **Claim 5** The method of claim 4, wherein the CAR targets MHC-I negative tumor tissue. **Claim 6** The method of claim 4, wherein the CAR targets a tumor-associated antigen or a tumor-specific antigen. **Claim 7** The method of claim 4, wherein the NK cells release IFNγ upon CAR-mediated engagement of the NK cells and the tumor tissue. **Claim 8** The method of claim 2, further comprising administering an antibody in parallel with the NK cells, the antibody targeting MHC-I negative tumor tissue. **Claim 9** The method of claim 8, wherein the antibody targets a tumor-associated antigen or a tumor-specific antigen. **Claim 10** The method of claim 9, wherein the NK cells release IFNγ upon Ab-mediated engagement of the NK cells and the tumor tissue. **Claim 11** The method of claim 1, wherein administering the NK cells increases the expression of MHC-I in the tumor. **Claim 12** The method of claim 1, further comprising administering in parallel with the NK cells at least one therapeutic agent selected from the group consisting of gemcitabine, IFNγ, HDAC inhibitor, 5-aza-2'-deoxycytidine, DNA methyltransferase inhibitor, hydralazine, valproic acid, microRNA (miRNA), or an inhibitor of ALK, RET, or MAPK kinase, a glycosyltransferase inhibitor, a thymidylate synthase inhibitor, dexamethasone, a SMAC mimetic, and an autophagy inhibitor. **Claim 13** The method of claim 1, further comprising administering IFNγ in parallel with the NK cells. **Claim 14** The method according to claim 1, further comprising administering IL-15, an agonist derivative thereof, or N-803 (nagapentacine alpha inbaxcept) in parallel with the NK cells.

15. The method according to claim 1, wherein the T cells are genetically modified to express at least one of a chimeric antigen receptor (CAR) and a T cell receptor (TCR).

16. The method according to claim 15, wherein the at least one CAR and TCR are specific for a tumor-associated antigen, a tumor-specific antigen, or a neoepitope.

17. A pharmaceutical composition for use in a patient having MHC-I negative tumor tissue, the composition comprising an effective amount of NK cells and an effective amount of T cells, wherein the NK cells and the T cells are administered sequentially.

18. The composition according to claim 17, wherein the NK cells are NK-92 cells or activated cytokine-enriched NK (ceNK) cells or memory-like ceNK (m-ceNK) cells.

19. The composition according to claim 17, wherein the NK cells are induced or genetically modified to overexpress interferon gamma (IFNγ).

20. The composition according to claim 18, wherein the NK cells comprise at least one of a chimeric antigen receptor (CAR) and an Fc receptor.

21. The composition according to claim 20, wherein the CAR is specific for MHC-I negative tumor tissue.

22. The composition according to claim 20, wherein the CAR is specific for a tumor-associated antigen or a tumor-specific antigen.

23. The composition according to claim 17, further comprising an antibody administered in parallel with the NK cells, wherein the antibody is specific for MHC-I negative tumor tissue.

24. The composition according to claim 23, wherein the antibody is specific for a tumor-associated antigen or a tumor-specific antigen.

25. The composition according to claim 17, wherein administering the NK cells increases the expression of MHC-I in the tumor.

26. The composition according to claim 17, further comprising at least one therapeutic agent selected from the group consisting of gemcitabine, IFNγ, HDAC inhibitor, 5-aza-2'-deoxycytidine, DNA methyltransferase inhibitor, hydralazine, valproic acid, microRNA (miRNA), or an inhibitor of ALK, RET, or MAPK kinase, a glycosyltransferase inhibitor, a thymidylate synthase inhibitor, dexamethasone, and an autophagy inhibitor, wherein the at least one therapeutic agent is administered in parallel with the NK cells.

27. The composition according to claim 17, further comprising IFNγ, wherein the IFNγ is administered in parallel with the NK cells.

28. The composition according to claim 17, wherein the T cells are genetically modified to express at least one of a chimeric antigen receptor (CAR) and a T cell receptor (TCR).

29. The composition according to claim 28, wherein the at least one CAR and TCR are specific for a tumor-associated antigen, a tumor-specific antigen, or a neoepitope.

30. The composition according to claim 17, further comprising IL-15, an agonist derivative thereof, or N-803 (nagapentidekin alpha inbaxcept).

31. A method for determining MHC-I induction in a tumor, the method comprising: a. Quantifying ex vivo MHC-1 expression in a patient's tumor tissue; b. Ex vivo treating the tumor tissue with at least one agent selected from the group consisting of gemcitabine, IFNγ, HDAC inhibitor, 5-aza-2'-deoxycytidine, DNA methyltransferase inhibitor, hydralazine, valproic acid, microRNA (miRNA), or an inhibitor of ALK, RET, or MAPK kinase, a glycosyltransferase inhibitor, or a thymidylate synthase inhibitor; c. Quantifying MHC-I expression in the ex vivo treated tumor tissue; d. Identifying at least one agent that significantly increases the expression level of MHC-I by the treatment; and comprising.

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