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

Abstract

Provided herein are methods and compositions whereby a treatment comprising NK cells is administered to a patient suffering from cancer to induce expression of MHC-I in a tumor, and wherein subsequent treatment with T cells effectively targets tumor-associated antigens and neoepitopes presented by newly expressed MHC-I in the tumor cells.

Description

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

[0002] The field of the invention is combinatorial immunotherapy techniques. Background Art

[0003] Major histocompatibility complex (MHC) proteins are present on the surface of all nucleated cells. They are responsible for presenting peptides digested in cells on the cell surface, thus screening their non-self peptide sequences by T cells. Peptide sequences from non-self sources (such as cancer and infectious pathogens) are presented on the cell surface combined with MHC, thus T cells combine with such occupied MHC proteins and start various immune defense programs, including cytokine production and direct cytotoxicity. Intracellular proteins are presented on the cell surface by MHC-I, and extracellular proteins are taken in by phagocytosis and processed for presentation by MHC-II. MHC-I is expressed on the surface of all nucleated cells, and is responsible for presenting mutations (non-self) peptides of cell origin.

[0004] Alternatively, natural killer (NK) cell-mediated cytotoxicity is inhibited by the presence of MHC-I expressed on the surface of target cells. Since infectious agents have mechanisms to reduce MHC expression in infected cells, thereby alleviating the host's ability to mount a T cell-mediated immune defense, NK cell-mediated defense is achieved by reducing MHC expression in infected cells.

[0005] Loss of MHC proteins in cancer cells has been described, whereby T cell activity is reduced. Typically, loss of MHC is associated with lower amounts of tumor infiltrating lymphocytes. As a result, patients are unable to mount an effective immune response against non-self peptides in cancerous or infected tissues. MHC-I expression is inducible and can be well characterized by a variety of agents, among which interferon-g is prominent. Induction of MHC-I followed by T cell therapy may be an effective treatment approach.

[0006] Therefore, there remains a need for effective therapies that can generate and increase the host immune response against cancer tissues. Summary of the invention

[0007] The present subject matter provides compositions, methods, and kits by which an immune response to cancer or infectious disease can be generated or enhanced.

[0008] In an 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. When it is determined that the MHC-I level is low relative to at least a control non-cancerous tissue, a variety of NK cells are administered to the patient, wherein these NK cells include at least one of aNK cells, haNK cells, t-haNK cells, or primary ceNK or memory-like ceNK (m-ceNK) cells. NK cells can be administered intravenously or intratumorally. After a period of time, a variety of T cells are administered to the patient. T cells can be autologous or heterologous. T cells can be further genetically engineered to express a targeting agent, wherein the agent comprises CAR or TCR. In a non-limiting example, T cells can be administered to a patient at most 1 hour after administration of the NK cells, or at most 2 hours after administration of the NK cells, or at most 5 hours after administration of the NK cells, or at most 10 hours after administration of the NK cells, or at most 1 day after administration of the NK cells, or at most 5 days after administration of the NK cells, or at most 1 week after administration of the NK cells, or at most 2 weeks after administration of the NK cells, or at most 1 month after administration of the NK cells, or at most 2 months after administration of the NK cells, or at most 6 months after administration of the NK cells, or at most 1 year after administration of the NK cells, or at most 2 years after administration of the NK cells.

[0009] In an 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. When it is determined that the MHC-I level is low relative to a control healthy normal tissue, a variety of NK cells are administered to the patient, wherein these NK cells include at least one of aNK cells, haNK cells, t-haNK cells, or primary ceNK or memory-like ceNK (m-ceNK) cells. NK cells can be administered intravenously or intratumorally. After a period of time, a variety of T cells are administered to the patient. T cells can be autologous or heterologous. T cells can be further genetically engineered to express a targeting agent, wherein the agent comprises CAR or TCR.

[0010] In an embodiment, the subject matter of the present invention includes an immunotherapy composition comprising a variety of NK cells, wherein these NK cells include at least one of aNK cells, haNK cells, t-haNK cells, ceNK cells or m-ceNK cells. NK cells can be formulated into a plurality of whole cells for intravenous administration, or they can be formulated into cell lysates for intratumoral administration. The composition further includes a variety of T cells, wherein these T cells are formulated for intravenous administration. T cells can be autologous or heterologous. T cells can be further genetically engineered to express a targeting agent, wherein the agent includes CAR or TCR.

[0011] In an 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 includes a variety of NK cells, wherein these NK cells include at least one of aNK cells, haNK cells, t-haNK cells, ceNK cells or m-ceNK cells. NK cells can be formulated into a plurality of whole cells for intravenous administration, or they can be formulated into cell lysates for intratumoral administration. The composition further includes a variety of T cells, wherein these T cells are formulated for intravenous administration. T cells can be autologous or allogeneic. T cells can be further genetically engineered to express a targeting agent, wherein the agent includes CAR or TCR.

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

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

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

[0015] In an embodiment, the subject matter of the present invention includes a method for inducing MHC-I in cancer, the method comprising determining the level of MHC-I expression in a tumor. When it is determined that the MHC-I level is low relative to a control non-cancerous tissue, a plurality of NK cells are administered to the patient, wherein 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 an embodiment, the subject matter of the present invention includes a method for increasing IFNg levels in cancer, the method comprising determining the IFNg expression level in a tumor. When it is determined that the IFNg level is low relative to a control non-cancerous tissue, a plurality of NK cells are administered to the patient, wherein 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 an 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 invention, a patient biopsy sample is enzymatically and / or mechanically dissociated in vitro, and each aliquot is inoculated into each hole of a multi-wall microplate. The sample is then exposed to an agent known to induce MHC-I, wherein these agents include gemcitabine, IFNg, HDAC inhibitors, 5-aza-2'-deoxycytidine, DNA methyltransferase inhibitors, hydralazine, valproic acid, microRNA (miRNA), or inhibitors of ALK, RET or MAPK kinases, glycosyltransferase inhibitors, thymidylate synthase inhibitors, dexamethasone, SMAC mimetics, IL-15, N-803, IL-15 derivatives and autophagy inhibitors. After a period of time, the surface expression of the MHC-I of the sample is determined.

[0018] Various objects, features, aspects and advantages of the present subject matter will become more apparent from the following detailed description of preferred embodiments and the accompanying drawings, in which like numerals represent like components. DETAILED DESCRIPTION

[0019] The subject matter of the present invention provides compositions, methods and kits, by which immune responses to cancer or infectious diseases can be generated or enhanced. A determined biomarker capable of generating immune responses to exogenous or endogenous non-autoimmune stimulants is MHC-I expression. MHC-I is intended to refer to MHC class I molecules, which are one of the two main categories of major histocompatibility complex (MHC) molecules (the other is MHC class II), and are present on the cell surface of all nucleated cells in vertebrates. Their function is to display peptide fragments from the cell to the protein of cytotoxic T cells on the cell surface; this will trigger the immune system to respond immediately to cells displaying specific non-self antigens through MHC class I proteins. Because MHC class I molecules present peptides derived from cytoplasmic proteins, the pathway presented by MHC class I is generally referred to as cytoplasmic or endogenous pathways. In humans, the HLA corresponding to MHC class I is HLA-A, HLA-B and HLA-C.

[0020] With regard to NK cells, it should be noted that all NK cells are considered suitable for use herein, thus including primary NK cells (preserved, expanded and / or fresh cells), immortalized secondary NK cells, autologous or allogeneic NK cells (banked, preserved, fresh, etc.), and modified NK cells, as described in more detail below. In some embodiments, preferably, the NK cells are NK-92 cells. The NK-92 cell line is a unique cell line that was found to proliferate in the presence of interleukin 2 (IL-2) (see, e.g., Gong et al., Leukemia 8: 652-658 (1994)). NK-92 cells are cancerous NK cells that have a wide range of anti-tumor cytotoxicity and predictable yields after expansion in a suitable culture medium. Advantageously, NK-92 cells have high cytolytic activity against a variety of cancers.

[0021] The original NK-92 cell line expresses CD56bright, CD2, CD7, CD11a, CD28, CD45 and CD54 surface markers, but does not display 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), and a dose as low as 1IU / mL is sufficient to maintain proliferation. IL-7 and IL-12 do not support long-term growth, and the other various cytokines tested (including IL-1a, IL-6, tumor necrosis factor α, interferon α and interferon γ) do not support it. Compared with primary NK cells, NK-92 typically has higher cytotoxicity even at relatively low effector cells: target (E: T) ratios (e.g., 1: 1). Representative NK-92 cells are deposited in the American Type Culture Collection (ATCC) and designated as 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 and all other external references are incorporated herein by reference in their entirety.

[0022] In another aspect of the subject matter of the present invention, genetically engineered NK cells can also be NK-92 derivatives modified to express high-affinity Fcγ receptors (CD16). The sequences of high-affinity variants of Fcγ receptors are well known in the art (see, e.g., Blood [blood] 2009113: 3716-3725), and all methods of production and expression are considered to be applicable herein. It is believed that the expression of such receptors allows the use of antibodies specific to the patient's tumor cells (e.g., new epitopes), specific tumor types (e.g., her2neu, PSA, PSMA, etc.), or antibodies associated with cancer (e.g., CEA-CAM) to specifically target tumor cells. Advantageously, such antibodies are commercially available and can be used in conjunction with cells (e.g., in combination with Fcγ receptors). Alternatively, such cells can also be commercially available from NantKwest as haNK cells. Such cells can then be modified to CARs by additional genes, 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 the NK cells envisioned herein can be carried out in a variety of ways, and all known methods are considered to be applicable to this. Moreover, it should be appreciated that NK cells can be transfected by DNA or RNA, and the specific selection of transfection will depend at least in part on the type and transfection efficiency of the expected recombinant cells. For example, in the case of expected stable transfection of NK cells, linearized DNA can be introduced into the cell to be integrated into the genome. On the other hand, in the case of expected transient transfection, circular DNA or linear RNA (e.g., mRNA with poly A+ tail) can be used.

[0024] For example, in the case where the NK cells are autologous NK cells or NK-92 cells, it is envisioned that the recombinant nucleic acid will include a fragment encoding a CAR, the CAR including an FcεRIγ signaling domain, and preferably also includes a fragment encoding a cytokine to provide 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 fragment encoding CD16 or high-affinity CD16158V. As will be readily understood, including cytokines that provide autocrine growth stimulation will make the modified recombinant independent of the addition of exogenous cytokines, which will make the large-scale production of such cells economically feasible. Similarly, in the case where the modified recombinant also expresses CD16 or high-affinity CD16158V, such cells will have further enhanced ADCC properties and have further improved targeted cytotoxicity.

[0025] It should be understood that the recombinant nucleic acid encoding cytokines and / or CD16 or high affinity CD16158V can be integrated into the genome of NK cells, or can be provided as an extrachromosomal unit (which can be a linear or circular DNA or linear RNA delivered by virus or chemical, mechanical or electrotransfected). For example, recombinant NK-92 cells expressing IL-2ER and CD16158V are referred to as haNK cells (Oncotarget [Tumor Targeting] December 27, 2016; 7 (52): 86359-86373), and can be transfected by recombinant nucleic acid, which includes a fragment encoding a CAR including an FcεRIγ signaling domain. Once again, such recombinant nucleic acids may further include fragments that can encode additional immunotherapy proteins, such as N-803, TxM-type compounds, IL-8 traps, TGF-β traps, etc. Similarly, NK-92 cells may have been transfected with cDNA encoding IL-2 (e.g., NK-92MI, ATCC CRL-2408). Such cells can then be further transfected with a recombinant nucleic acid comprising a fragment encoding a CAR comprising an FcεRIγ signaling domain and a fragment encoding CD16 or high affinity CD16158V.

[0026] On the other hand, as further disclosed in PCT / US2019 / 033407 (incorporated herein by reference in its entirety), (e.g., autologous, fresh, cultured, or previously frozen) NK cells or NK-92 cells can also be transfected with a recombinant nucleic acid comprising a fragment encoding a CAR having an FcεRIγ signaling domain, a fragment encoding a cytokine to provide 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 a fragment encoding CD16 (SEQ ID NO: 34) or high affinity CD16158V (SEQ ID NO: 35, encoded by SEQ ID NO: 36). Most typically, such recombinant nucleic acids will be configured as tricistronic constructs. As previously described, such constructs can be extrachromosomal circular plasmids, linear DNA (which can be integrated into the genome of NK cells), or linear RNA. Such nucleic acids are usually transfected into cells in a manner well known in the art (e.g., electroporation, lipofection, ballistic gene transfer, etc.). Similarly, nucleic acids can be delivered to cells by recombinant viruses. Therefore, NK cells suitable for use herein include NK-92 cells (which can be transfected with a tricistronic construct encoding CAR, CD16 or its variants, cytokines or their variants), genetically modified NK cells or NK-92 cells expressing CD16 or its variants or cytokines or their variants (which can be transfected with nucleic acids encoding CAR and CD16 or its variants or cytokines or their variants), and genetically modified NK cells or NK-92 cells expressing CD16 or its variants and cytokines or their variants (which can be transfected with nucleic acids encoding CAR). U.S. Patent Application No. 17 / 056,385, U.S. Patent No. 11,077,143, U.S. Patent No. 10738279 and U.S. Patent Application No. 16 / 969,152 are incorporated herein by reference.

[0027] Therefore, in preferred embodiments, it should be noted that genetically modified NK cells (particularly cells expressing CAR and CD16 or its variants) will exhibit three different modes of cell killing: general cytotoxicity mediated by activating receptors (e.g., NKG2D receptor), ADCC mediated by antibodies bound to target cells, and CAR-mediated cytotoxicity.

[0028] Therefore, it should be understood that the mode of transfection will depend at least in part on the type of nucleic acid used. Therefore, viral transfection, chemical transfection, and mechanical transfection methods are all considered suitable for use herein. For example, in one embodiment, the vectors described herein are transient expression vectors. The exogenous transgene introduced using such vectors is not integrated into the nuclear genome of the cell; therefore, in the absence of vector replication, the exogenous transgene will degrade or dilute over time.

[0029] In another embodiment, the vectors described herein allow stable transfection of cells. In one embodiment, the vector allows one or more transgenics to be incorporated into the genome of the cell. Preferably, such vectors have positive selection markers, and suitable positive selection markers include any gene that allows cells to grow under conditions that kill cells that do not express the gene. Non-limiting examples include antibiotic resistance, such as geneticin (Neo gene from Tn5). Alternatively, or additionally, the vector is a plasmid vector. In one embodiment, the vector is a viral vector. As will be appreciated by those skilled in the art, any suitable vector can be used, and suitable vectors are well known in the art.

[0030] In other embodiments, cells are transfected with mRNA encoding a protein of interest (e.g., CAR). Transfection of mRNA results in transient expression of protein. In one embodiment, mRNA is transfected into NK-92 cells immediately before the cells are administered. In one embodiment, "before" the cells are to be administered refers to between about 15 minutes and about 48 hours before administration. Preferably, mRNA transfection is performed about 5 hours to about 24 hours before administration. In at least some embodiments as described in more detail below, NK cells transfected with mRNA result in unexpectedly consistent and strong expression of CAR on a high proportion of transfected cells. Moreover, such transfected cells also show high specific cytotoxicity when the ratio of effector cells to target cells is quite low.

[0031] Regarding the envisioned CAR, it is noted that NK or NK-92 cells will be genetically modified to express CAR as a membrane-bound protein, thereby exposing a portion of the CAR on the cell surface while maintaining the signaling domain in the intracellular space. Most typically, CAR will include at least the following elements (in order): an extracellular binding domain, a hinge domain, a transmembrane domain, and an FcεRIγ signaling domain.

[0032] In a preferred embodiment, the cytoplasmic domain of CAR comprises or consists of a signaling domain of FcεRIγ. It is noteworthy that, and as described in more detail below, the FcεRIγ signaling domain provides a significant increase in the expression level of CAR and a significant extension of cytotoxicity over time. In some embodiments, the FcεRIγ cytoplasmic domain is the only signaling domain. However, it should be understood 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 of the FcεRIγ cytoplasmic domain and / or upstream of the FcεRIγ cytoplasmic domain. In an alternative embodiment, the cytoplasmic domain of CAR may also include a signaling domain of CD3 zeta (CD3ζ). In one embodiment, the cytoplasmic domain of CAR consists of a signaling domain of CD3ζ.

[0033] Thus, the envisioned CAR will include the general structure of an expected antigen binding domain coupled to a hinge domain, which is coupled to a transmembrane domain, which is coupled to a signaling domain. From another perspective, the envisioned CAR may have an expected binding domain, which is then coupled to a hybrid protein comprising, consisting of, or consisting essentially of a hinge domain, which is coupled to a transmembrane domain, which is coupled to a signaling domain.

[0034] Most typically, but not necessarily, the extracellular binding domain of CAR will be a scFv or other natural or synthetic binding portion that specifically binds to the target antigen. Particularly suitable binding portions include small antibody fragments with single, dual or multiple target specificities, β barrel domain binders, phage display fusion proteins, etc. Among other suitable extracellular binding domains, preferred domains will specifically bind to tumor-specific antigens, tumor-associated antigens, or patient-specific antigens and tumor-specific antigens. 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, CTAGIB and CD33. Other non-limiting tumor-associated antigens, and malignancies associated therewith, are listed in Table 1. As non-limiting examples, still further tumor-specific antigens are described in US2013 / 0189268; WO1999024566A1; U.S. Patent No. 7,098,008: and WO2000020460, each of which is incorporated herein by reference in its entirety. Similarly, other preferred domains will specifically bind to (pathogenic) virus-specific antigens such as antigens of HIV virus (e.g., gp120), HPV virus, RSV virus, influenza virus, Ebola virus or HCV.

[0035] Therefore, the envisioned CAR will target antigens associated with specific cancer types. For example, targeted cancers include leukemias (including acute leukemias (e.g., acute lymphocytic leukemia, acute myeloid leukemia (including myeloid, promyelocytic, myelomonocytic, monocytic and erythroleukemia)) and chronic leukemias (e.g., chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, solid tumors, including but not limited to sarcomas and cancers, such as fibrosarcomas, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphatic cavity Endotheliosarcoma, 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, hepatocellular carcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumors, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0036] In an 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. When it is determined that the MHC-I level is low relative to a control non-cancerous tissue, a variety of NK cells are administered to the patient, wherein these NK cells include at least one of aNK cells, haNK cells, t-haNK cells, or primary ceNK or memory-like ceNK (m-ceNK) cells. NK cells can be autologous or allogeneic. NK cells can be administered intravenously or intratumorally. After a period of time, a variety of T cells are administered to the patient. T cells can be autologous or allogeneic. T cells can be further genetically engineered to express a targeting agent, wherein the agent comprises 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 and / or mechanical dissociation of tissues (thereby separating and purifying living whole cells from primary tissues) is necessary for Ab staining and flow cytometry to measure 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 expressing genetically engineered CARs. Without being bound by any particular theory, it should be understood that the targeting portion on CAR is a dual purpose. First, the targeting portion guides NK cells to the expression site of the antigen in the patient, thereby facilitating the delivery of NK cells to tumor tissue. Second, the targeting portion can be immunogenic, thereby facilitating the delivery of antibodies and T cells to tumors.

[0039] Primary NK cells can also be enriched and expanded from whole blood or cord blood mononuclear cells via standard methods, including exposure of primary NK cells to CD16 antibodies, dexamethasone and / or IL-15. Stabilized IL-15 can be used, wherein the stabilized IL-15 includes IL-15 superagonists, such as nogapendenkin alpha imbakicept (Alt-803, N-803, Vesanktiva TM ) and stabilized IL-15 / IL-15Ra 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 in which cytotoxic activity is enhanced by cytokine stimulation. ceNK cells are prepared by inducing NK cells with corticosteroids and optionally a cytokine composition, the cytokine composition comprising IL-15, IL-15:IL-15Rα or an agonist derivative thereof, such as N-803. The cytokine composition may comprise a fusion protein, wherein the fusion protein comprises IL-15 or an agonist derivative thereof. Preferably, a fusion protein comprising IL-15 is included, wherein the fusion protein has increased stability relative to IL-15. Although not limiting the subject matter of the present invention, it is generally preferred that the corticosteroid is hydroxycortisone and the optional cytokine is N-803.

[0041] Memory-like cytokine enhanced NK cells (m-ceNK) disclosed herein include enriched and amplified NK cells, which are obtained from the peripheral blood of the donor using apheresis technology to produce NK cells with memory-like phenotype. M-ceNK cells show high cytotoxicity and increased interferon-γ production. These m-ceNK cells can be produced from individual donors for autologous cell therapy, or can be produced as allogeneic products from umbilical cord blood. In addition to enhanced efficacy, m-ceNK cells can be easily infused in an outpatient setting.

[0042] As a non-limiting example, m-ceNK cells can be produced by the following steps: obtaining a plurality of monocytes and contacting the plurality of monocytes with corticosteroids and optionally cytokines. In another step, a plurality of monocytes are incubated in the presence of corticosteroids and optional cytokines to enrich the monocytes in NK cells, and then the enriched NK cells are induced with a cytokine composition comprising IL-15, IL-12 and IL-18 or its agonist derivatives. The composition may comprise one or more fusion proteins, wherein these fusion proteins comprise at least one of IL-15, IL-12 and IL-18 cytokines or their agonist derivatives. The cytokine composition may comprise a TxM fusion protein to produce m-ceNK cells, wherein the TxM fusion protein comprises 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 the preparation of m-ceNK cells and their advantageous properties is described in PCT / US2022 / 018290, which is incorporated by reference in its entirety. U.S. Patent Application 17 / 375,985 and U.S. Patent 11,453,862 provide additional alternative methods for inducing NK cell enrichment and expansion. Each of the above references is incorporated by reference in its entirety.

[0044] Activated cytotoxic cell therapy

[0045] "T cell receptor" or "TCR" refers to a dimeric polypeptide typically present on the surface of a T cell. Each peptide chain of a TCR generally comprises an extracellular domain, a transmembrane domain and an intracellular domain containing a variable region and a constant region. The variable region is the part of the TCR that interacts with the antigen presented by MHC. The constant region is the region in each of the two peptides, wherein the two peptide chains are covalently linked by a disulfide bond. The intracellular domain generally comprises CD3z, which comprises one or more immunoreceptor tyrosine-based activation motifs (ITAMs). ITAMs mediate the combination of the variable region with appropriate intracellular signal transduction pathways.

[0046] The intracellular signaling domain of the CAR may also comprise an FcεRIγ portion. U.S. Patent Application No. 17 / 341,098 is incorporated herein by reference.

[0047] T cells may optionally include modified TCRs, which relate to dimeric polypeptides based on TCR structures. In particular, modified TCRs include 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 joint. In another specific embodiment, the connecting peptide is located between the constant region and the transmembrane domain. In another specific embodiment, the two peptide chains are connected to each other by a disulfide bond between the connecting peptides of each peptide chain. Modified TCR does not interact with endogenous TCRs produced by T cells. The contents of U.S. Patent Application No. 63 / 227,195 are incorporated herein by reference.

[0048] Once a CAR-based therapeutic agent (e.g., an antigen binding domain coupled to a CAR scaffold) binds to an antigen expressed by a cancer cell, a cytotoxic cell can trigger the destruction of the cancer cell. Although it is generally expected that all cytotoxic cells are considered to be applicable herein, particularly preferred cytotoxic cells include NK cells, activated NK cells, high-affinity NK cells, CD8+T cells, and CD4+T cells, which have been modified to recombinantly express CAR-based therapeutic agents, any of which may have different sources. Cytotoxic cells are engineered to express TCRs that recognize peptides presented by MHC-I.

[0049] As used herein, therapeutic T cells can be patient-derived (autologous) or donor-derived (allogeneic). T cells are usually obtained by leukocyte apheresis, 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 (APC). Cells can be expanded by exposure to a cytokine mixture comprising 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 electroporation microfluidics as described in U.S. Patent No. 11,377,652, the content of which is incorporated herein by reference.

[0050] T cells can be purified from tumors and are therefore tumor infiltrating lymphocytes (TIL). TIL can be purified from tumor tissue and amplified ex vivo. TIL can be reintroduced into the patient as autologous cells, or can be administered to different subjects as donor cells. It is expected that stimulation of MHC-I expression by NK cells will lead to enhanced cytotoxic efficacy of TIL.

[0051] In an embodiment of the present invention, TILs are isolated from patient tumors by standard techniques. TILs are then exposed to patient tumor tissues where the patient has been treated with NK cells to induce the expression of MHC-I. NK exposure can be performed by IV injection or by intratumoral injection. NK exposure to tumor tissues can be performed ex vivo. Thus, TILs exposed to tumor tissues ex vivo are activated and amplified. Amplified TILs containing CD4 and CD8 cytotoxic T cells are administered to patients.

[0052] 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. After transfection, CART cells can be amplified in a bioreactor until a clinically effective number of cells is obtained.

[0053] However, it should be understood that in other aspects, the cytotoxic cell may also be a macrophage, a monocyte, a neutrophil, a basophil or an eosinophil. Therefore, and from a different perspective, the cells contemplated herein may achieve cytotoxic effects by phagocytosis, pore formation, induction of antibody-dependent cell-mediated cytotoxicity (ADCC), by initiating TNF or fas-mediated killing pathways, etc.

[0054] As part of the cytotoxic anti-tumor process, cytotoxic cells can release various types of cytotoxic granules (e.g., granulysins, perforins, granzymes). A variety of assays can be used to monitor cell-mediated cytotoxicity, including flow cytometry assays (e.g., based on the presence of lytic granules such as perforins, granzymes, or the production of TNF family members such as TNF-α, FasL, or TRAIL) (Zaritskaya 2010, Clay, T. et al., Clin. Cancer Res. [Clinical Cancer Research] (2001) &: 1127-1135).

[0055] In one embodiment, a body fluid is obtained after treatment with NK cells, wherein the body fluid comprises a cellular component, such as a tumorigenic cell or cancer cell that displays an antigen bound to a cytotoxic cell expressing CAR as described herein, and a cytotoxic cell expressing an antigen binding moiety is contacted with these cells. Then an assay is performed to detect an immune response, such as an indication that an ADCC response or an ADCP response has been elicited by the patient's own immune cells.

[0056] Assays for detecting immune responses are known in the art and described herein. For example, assays for detecting such responses can detect the release of cytotoxic granules (e.g., granulysin, perforin, granzymes), or phagocytosis, or receptor-ligand mediated cell lysis (e.g., mediated by the Fas / APO pathway). Various flow cytometry assays can be used to monitor cell-mediated cytotoxicity, for example based on the presence of lytic granules such as perforin, granzymes, or the production of TNF family members such as TNF-α, FasL or TRAIL (Zaritskaya 2010, Clay, T, et al., Clin. Cancer Res. [Clinical Cancer Research] (2001) &: 1127-1135).

[0057] In other embodiments, immunostimulatory cytokines are administered to patients in combination with cytotoxic cells (expressing CAR-based therapeutic agents (e.g., antigen binding domains coupled to CAR scaffolds)) to promote or induce an immune response. Cytokines include but are not limited to IL2, IL4, IL7, IL11, IL15, IL21, TNF-α, IFN-γ, etc. In some embodiments, cytokines can reactivate exhausted T cells. In other cases, immune competent cells can be engineered to recombinantly express one or more cytokines.

[0058] Other techniques for treating cancer include surgery, radiotherapy, chemotherapy, immunosuppressants (e.g., azathioprine, cyclosporine, methotrexate, mycophenolate mofetil, etc.), immunotherapy, targeted therapy, hormone therapy, stem cell transplantation, or other precise methods. Any of these techniques can be combined with embodiments of the present invention to treat cancer.

[0059] It will be understood that embodiments of the present invention can be administered to patients using appropriate formulations, indications, and dosing regimens that are applicable to government regulatory agencies such as the U.S. Food and Drug Administration (FDA).

[0060] In some embodiments, cytotoxic cells expressing TCR, modified TCR or CAR-based therapeutic agents (e.g., antigen binding domains coupled to CAR scaffolds) are administered to patients as pharmaceutical compositions. In another embodiment, a method for treating cancer by administering cytotoxic cells to a subject is contemplated. In yet another embodiment, a method for inhibiting the proliferation of cells expressing corresponding antigens (antigen binding regions are bound thereto) on their cell surfaces or reducing their proliferation is contemplated by administering cytotoxic cells to a subject.

[0061] In embodiments, the patient may be lymphodepleted, whereby the number of endogenous lymphocytes is reduced, thereby increasing the availability of essential endogenous cytokines and promoting survival of the infused T cells.

[0062] In some embodiments, the cytotoxic cells expressing a TCR, a modified TCR, or a CAR-based therapeutic (e.g., an antigen binding domain coupled to a CAR scaffold) reduce the amount of subjects with a cancer associated with expression of a corresponding antigen on the cell surface (e.g., cell number, tumor size, etc.) by at least 25%, at least 50%, at least 75%, at least 90%, at least 95%, or at least 99%, relative to a negative control.

[0063] Examples of cancers that can be treated by cytotoxic cells considered herein include any cancer that expresses or overexpresses cancer-associated antigens on its cell surface. Examples of cancers that can be treated by cytotoxic cells expressing TCR, modified TCR, or CAR-based therapeutic agents (e.g., antigen-binding domains coupled to CAR scaffolds) include, but are not limited to, breast cancer, colon cancer, leukemia, lung cancer, melanoma, neuroblastoma, pancreatic cancer, pediatric intracranial ependymoma, and prostate cancer.

[0064] The pharmaceutical composition may include cytotoxic cells (these cytotoxic cells include antigen binding domains coupled or connected to the CAR scaffold as described herein), and one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. In addition, the pharmaceutical composition may include one or more adjuvants (eg, aluminum hydroxide), antioxidants, antibacterial agents, buffers, carbohydrates, chelating agents such as EDTA or glutathione; colorants, flavoring agents and / or aromatic substances, emulsifiers, excipients, lubricants, pH buffers, preservatives, salts for affecting osmotic pressure, polypeptides (eg, glycine), proteins, solubilizers, stabilizers, wetting agents, etc., which do not react harmfully with active compounds (eg, antigen binding domains coupled to CAR scaffolds, etc.) or otherwise interfere with their 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.

[0065] The pharmaceutical composition can be formulated for a particular mode of administration. The mode of administration may include, but is not limited to, intraarticular, intradermal, intranasal, intraperitoneal, intrathecal, intratumoral, intravenous, intraventricular, subcutaneous, transdermal, transmucosal or topical routes.

[0066] In a preferred embodiment, the cytotoxic cells are administered by intravenous infusion. Such formulations can be prepared according to standard techniques known to those of ordinary skill in the art. For example, a composition to be administered intravenously may have one or more ingredients (e.g., diluents, suspension buffers, saline or glucose / water, other components such as cytokines, etc.) prior to infusion into a patient.

[0067] Many such techniques for formulating and administering pharmaceutical compositions are known in the art, for example, U.S. Patent Application Publication No. 2014 / 0242025, and all such references are incorporated herein by reference in their entireties.

[0068] In some embodiments, the cytotoxic cells proliferate in vivo, thereby persisting in the patient for months or even years after administration to provide a persistent mechanism for inhibiting tumor growth or recurrence. In some aspects, the cytotoxic cells persist for at least three months, six months, nine months, twelve months, fifteen months, eighteen months, two years, three years, four years, or five years after administration to the patient.

[0069] Cytotoxic cells can be obtained from any of a variety of sources (e.g., isolated from humans, isolated from commercially available cytotoxic cells, isolated from cell banks, etc.). Procedures for ex vivo expansion of 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 method for ex vivo expansion of cytotoxic cells.

[0070] Pharmaceutical compositions comprising cytotoxic cells as described herein can be administered at a dose of 104 to 109 cells / kg body weight, 105 to 106 cells / kg body weight, or any integer value within these ranges. Cytotoxic cell compositions can be administered at these doses once or continuously (over the course of days, weeks, or months). Infusion techniques for cytotoxic cells (such as T cells) are known in the art (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988).

[0071] In other embodiments, the pharmaceutical composition is administered in a therapeutically effective amount, which is an amount that effectively treats a specific indication. Administration can be performed as a one-time dose or based on an interval. As used herein, an "interval" indicates that a therapeutically effective amount (different from a one-time dose) is administered regularly. The administration interval for a single individual does not need to be performed at fixed intervals, but can vary over time. The term "in combination with" or "co-administered" indicates that a composition can be administered shortly before, at the same time, at about the same time, or shortly thereafter, another composition.

[0072] Examples

[0073] Example 1

[0074] ANK, haNK, HER2t-haNK and CD19t-haNK cells were administered to immunodeficient mice carrying tumor xenografts expressing HER2 or CD19. Cells were administered as whole cell preparations or lysates. Whole cells and lysates were administered intratumorally or intravenously. The expression of MHC-I, MHC-II, B2M and CIITA of the tumor was then monitored. The cell surface expression of MHC-I and MHC-II was monitored by flow cytometry. Vehicle-treated mice were used as controls.

[0075] Example 2

[0076] The expression of MHC-I of a cancer patient biopsy sample containing cancer cells is determined. The determination includes the expression of MHC-I in non-cancerous tissues, preferably from the same tissue source as the biopsy sample. Compared with 3 control samples, in three cancer samples, the expression of MHC-I in cancer cells should be at least statistically significantly (p>0.05) lower. Preferably, the expression of MHC-I in cancer 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 cancer cells is <10% of the expression of MHC-I in control cells.

[0077] Example 3

[0078] Cancer patients with tumors that have been determined to have low MHC-I expression are treated with NK cells or NK cell lysates. Treatment is performed by intratumoral injection. Within 1-7 days, the MHC-I expression of the tumor is reassessed. Increased expression indicates that the patient is now a suitable candidate for T cell therapy. T cells are derived from 1) tumors (such as TILs), 2) lymph nodes, or 3) peripheral blood. T cells can be combined. They grow in vitro to a clinically effective amount. T cells are administered to patients by intravenous or intratumoral injection.

[0079] The above discussion provides many exemplary embodiments of the subject matter of the present invention. Although each embodiment represents a single combination of inventive elements, 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.

[0080] It will be apparent to those skilled in the art that, without departing from the inventive concepts herein, more modifications are possible in addition to those modifications that have been described. Therefore, the subject matter of the present invention is not limited except in the spirit of the appended claims. In addition, when interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms "comprises" and "comprising" should be interpreted as referring to elements, components or steps in a non-exclusive manner, indicating that the referenced elements, components or steps may exist or be utilized, or be combined with other elements, components or steps that are not explicitly referenced. When the specification or claim refers to at least one selected from the group consisting of A, B, C... to N, the text should be interpreted as requiring only one element in the group, rather than A plus N, or B plus N, etc.

[0081] All publications identified herein are 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. If a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

[0082] In some embodiments, the numerals used to describe and claim the amount of the expression composition, characteristic (such as concentration), reaction conditions, etc. of certain embodiments of the present invention should be understood to be modified by the term "about" in some cases. Therefore, in some embodiments, the numerical parameters set forth in the written description and the appended claims are approximate values, which can vary according to the desired properties sought to be obtained in a particular embodiment. In some embodiments, the numerical parameters should be interpreted according to the number of significant figures reported and by applying common inclusion techniques. Although the wide range of numerical ranges and parameters of some embodiments of the present invention are approximate values, the numerical values ​​set forth in the specific examples are reported as accurately as possible. The numerical values ​​presented in some embodiments of the present invention may include certain errors caused by the standard deviation found in their corresponding test measurements.

[0083] In certain embodiments, the numerals for describing and claiming the amount of the expression composition, characteristic (such as concentration), reaction conditions, etc. of certain embodiments of the present invention should be understood to be modified by the term "about" in some cases.Therefore, in certain embodiments, the numerical parameters set forth in the written description and the appended claims are approximate values, which can change according to the desired properties sought to be obtained in certain embodiments.In certain embodiments, the numerical parameters should be explained according to the number of significant figures reported and by applying common rounding techniques.Although the wide range of numerical ranges and parameters of certain embodiments of the present invention are approximate values, the numerical values ​​set forth in the specific examples are reported as accurately as possible.The numerical values ​​presented in certain embodiments of the present invention may include certain errors caused by the standard deviation found in their corresponding test measurements.

[0084] Unless the context indicates otherwise, all ranges set forth herein should be interpreted as including their endpoints, and open-ended ranges should be interpreted as including only commercially practical values. Likewise, all lists of values ​​should be interpreted as including intermediate values, unless the context indicates otherwise.

[0085] As used in the specification herein and throughout the claims, the meanings of “a”, “an”, and “the” include plural referents unless the context clearly indicates otherwise. Similarly, as used in the specification herein, the meaning of “in” includes “in” and “on” unless the context clearly indicates otherwise.

[0086] The description of value ranges herein is intended only to be used as a shorthand method for individually referring to each individual value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually described herein. All methods described herein can be performed in any suitable order, unless otherwise indicated herein or otherwise clearly contradictory with context. The use of any and all examples or exemplary language (e.g., "such as") provided with respect to some embodiments herein is intended only to better illustrate the subject matter of the present invention, and is not intended to limit the scope of the subject matter of the present invention claimed in addition. Any language in the specification should not be interpreted as indicating that any unclaimed element is essential for the practice of the subject matter of the present invention.

[0087] The grouping of the alternative elements or embodiments of the subject matter of the present invention disclosed herein should not be construed as limiting. Each group member may be mentioned and claimed individually, or mentioned and claimed in any combination with other members of the group or other elements found herein. For convenience and / or patentability reasons, one or more members in the group may be included in the group or deleted from the group. When any such inclusion or deletion occurs, this specification is considered to contain the modified group in this article, thereby satisfying the written description of all Markush groups used in the appended claims.

Claims

1. A method for treating a patient with MHC-I negative tumor tissue, the method comprising sequentially administering an effective amount of NK cells and then administering an effective amount of T cells.

2. The method of claim 1, wherein the NK cell is a NK-92 cell or a genetically modified derivative thereof, an activated cytokine-enriched NK (ceNK) cell, or a memory-like ceNK (m-ceNK) cell.

3. The method of claim 2, wherein the NK cells are induced or genetically engineered to overexpress interferon gamma (IFNγ).

4. The method of claim 3, wherein the NK cell comprises at least one of a chimeric antigen receptor (CAR) and an Fc receptor.

5. The method of claim 4, wherein the CAR targets the MHC-I negative tumor tissue.

6. The method of claim 4, wherein the CAR targets a tumor-associated antigen or a tumor-specific antigen.

7. The method of claim 4, wherein the NK cell releases IFNγ following CAR-mediated engagement of the NK cell with the tumor tissue.

8. The method of claim 2, further comprising administering an antibody simultaneously with the NK cell, wherein the antibody targets the MHC-I negative tumor tissue.

9. The method of claim 8, wherein the antibody targets a tumor-associated antigen or a tumor-specific antigen.

10. The method of claim 9, wherein the NK cell releases IFNγ following Ab-mediated engagement of the NK cell with the tumor tissue.

11. The method of claim 1, wherein administration of the NK cells increases the expression of MHC-I in the tumor.

12. The method of claim 1, further comprising administering at least one therapeutic agent selected from the group consisting of gemcitabine, IFNγ, HDAC inhibitors, 5-aza-2'-deoxycytidine, DNA methyltransferase inhibitors, hydralazine, valproic acid, microRNA (miRNA), or inhibitors of ALK, RET or MAPK kinases, glycosyltransferase inhibitors, thymidylate synthase inhibitors, dexamethasone, SMAC mimetics and autophagy inhibitors simultaneously with the NK cells.

13. The method of claim 1, further comprising administering IFNγ concurrently with the NK cells.

14. The method of claim 1, further comprising administering IL-15, an agonist derivative thereof, or N-803 (nogapendenkin alpha imbakicept) simultaneously with the NK cells.

15. The method of claim 1, wherein the T cell is genetically engineered to express at least one of a CAR and a T cell receptor (TCR).

16. The method of claim 15, wherein the at least one CAR and TCR is specific for a tumor-associated antigen, a tumor-specific antigen, or a neo-epitope.

17. A pharmaceutical composition for use in a patient with 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 of 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 of claim 17, wherein the NK cells are induced or genetically engineered to overexpress interferon gamma (IFNγ).

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

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

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

23. The composition of claim 17, further comprising an antibody to be administered simultaneously with the NK cell, wherein the antibody is specific for the MHC-I negative tumor tissue.

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

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

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

27. The composition of claim 17, further comprising IFNγ, wherein the IFNγ is administered simultaneously with the NK cells.

28. The composition of claim 17, wherein the T cell is genetically engineered to express at least one of a CAR and a T cell receptor (TCR).

29. The composition of claim 28, wherein the at least one CAR and TCR is specific for a tumor-associated antigen, a tumor-specific antigen, or a neo-epitope.

30. The composition of claim 17, further comprising IL-15, an agonist derivative thereof, or N-803 (nogapendenkin alpha imbakicept).

31. A method for determining MHC-I induction in a tumor, the method comprising: a. Quantification of MHC-I expression in ex vivo patient tumor tissue; b. treating the tumor tissue ex vivo with at least one agent selected from the group consisting of gemcitabine, IFNγ, HDAC inhibitors, 5-aza-2'-deoxycytidine, DNA methyltransferase inhibitors, hydralazine, valproic acid, microRNA (miRNA), or inhibitors of ALK, RET or MAPK kinases, glycosyltransferase inhibitors or thymidylate synthase inhibitors; c. quantifying the MHC-I expression in the ex vivo treated tumor tissue; d. Identifying at least one agent in which the expression level of MHC-I is significantly increased by the treatment.