Chimeric antigen receptor-modified NK-92 cells

Abstract

To provide CAR-expressing NK-92 cells that express a high-activity CAR in significant quantities, and that can be readily cultivated in a simple and effective manner.SOLUTION: Provided are NK-92 cells expressing a chimeric antigen receptor (CAR). The CAR can comprise an intracellular domain of FcεRIγ. Also described are methods for treating a patient having or suspected of having a disease that is treatable with NK-92 cells, such as cancer or a viral infection, comprising administering to the patient NK-92-CAR cells.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] This application claims priority to our co-pending U.S. provisional patent applications having serial numbers U.S. Provisional Patent Application No. 62 / 756,395 and U.S. Provisional Patent Application No. 62 / 756,402 (both filed November 6, 2018).

[0002] Sequence Listing The contents of the ASCII text file of the Sequence Listing entitled 104077.0003PCT5 Sequence Listing_ST25, 134kb in size, created on May 20, 2019, was submitted electronically via EFS-Web with this application and is incorporated by reference in its entirety.

[0003] The field of the invention is genetically engineered immunocompetent cells expressing chimeric antigen receptors (CARs), in particular modified NK-92 cells expressing CARs with an Fc epsilon receptor gamma (FcεRIγ) signaling domain. [Background technology]

[0004] Natural killer (NK) cells are cytotoxic lymphocytes that constitute a significant component of the innate immune system. In most cases, NK cells represent approximately 10–15% of circulating lymphocytes and bind to and kill targeted cells, such as virus-infected cells and numerous malignant cells. NK cell killing is nonspecific with respect to a particular antigen and can occur without prior immunization. Targeted cell killing is typically mediated by cytolytic proteins, including perforin, granzymes, and granulysin.

[0005] Autologous NK cells have been used as therapeutic entities. To this end, NK cells are isolated from the peripheral lymphocyte fraction of whole blood, expanded in cell culture to obtain sufficient numbers of cells, and then reinfused into the subject. Autologous NK cells have shown moderate efficacy in both ex vivo and in vivo treatments, at least in some cases. However, the isolation and expansion of autologous NK cells is time- and cost-intensive. Furthermore, autologous NK cell therapy is further limited by the fact that not all NK cells are cytolytic.

[0006] At least some of these difficulties can be overcome by the use of NK-92 cells, a cytolytic cancer cell line discovered in the blood of subjects suffering from non-Hodgkin's lymphoma and immortalized in vitro (Gong et al., Leukemia 8:652-658 (1994)). While NK-92 cells are NK cell derivatives, they lack most of the inhibitory receptors normally displayed by normal NK cells and retain most of the activating receptors. However, NK-92 cells do not attack normal cells in humans, nor do they induce unacceptable immune rejection responses. Due to these desirable characteristics, NK-92 cells have been extensively characterized and investigated as therapeutic agents in the treatment of certain cancers, as described, for example, in International Publication No. WO 1998 / 049268 or U.S. Patent Application Publication No. 2002 / 068044.

[0007] Phenotypic changes that distinguish tumor cells from normal cells of the same tissue origin are often associated with one or more changes in the expression of specific gene products, including a decrease in normal cell surface components or an increase in other (i.e., antigens undetectable in the corresponding normal, non-cancerous tissue). Antigens that are expressed in neoplastic or tumor cells but not in normal cells, or that are expressed in neoplastic cells at levels substantially above those found in normal cells, have been termed "tumor-specific antigens" or "tumor-associated antigens." Such tumor-specific antigens can serve as markers for tumor phenotype. Tumor-specific antigens include cancer / testis-specific antigens (e.g., MAGE, BAGE, GAGE, PRAME, and NY-ESO-1), melanocyte differentiation antigens (e.g., tyrosinase, MelanA / MART, gpl00, TRP-1, and TRP-2), mutated or aberrantly expressed antigens (e.g., MUM-1, CDK4, beta-catenin, gp100-in4, p15, and N-acetylglucosaminyltransferase V), and antigens expressed at higher levels in tumors (e.g., CD19 and CD20).

[0008] Tumor-specific antigens have been used as targets for cancer immunotherapy. One such treatment uses chimeric antigen receptors (CARs) expressed on the surface of immune cells, including T cells and NK cells, to improve cytotoxicity against cancer cells. CARs contain a single-chain variable fragment (scFv) linked to at least one intracellular signaling domain. The scFv recognizes and binds to antigens on target cells (e.g., cancer cells) and induces effector cell activation. The signaling domain contains an immunoreceptor tyrosine-based activation domain (ITAM), which is important for intracellular signaling by the receptor.

[0009] The first generation of CARs used in T cells had one cytoplasmic signaling domain. For example, one version of the first generation CAR in T cells contained a signaling domain from Fc epsilon receptor gamma (FcεRIγ) with one ITAM, while another version contained a signaling domain from CD3ζ with three ITAMs. In vivo and in vitro studies have shown that CD3ζ CAR T cells are more efficient at eradicating tumors than FcεRIγ CAR T cells (e.g., Haynes, et al. 2001, J. Immunology 166:182-187; Cartellieri, et al. 2010, J. Biomed and Biotech, Vol. 2010, Article ID 956304). Further studies then demonstrated that specific costimulatory signals are required for sufficient activation and proliferation of such recombinant T cells, and that combining second- and third-generation CARs with multiple signaling domains into a single CAR enhanced the efficacy of recombinant CAR T cells. Due to their largely undesirable pharmacokinetic effects in tested T cells, first-generation CARs and the FcεRIγ signaling domain were largely discarded in favor of new, more efficient CARs that use CD3ζ in combination with one or more additional signaling domains (e.g., Hermanson and Kaufman 2015, Frontiers in Immunol., Vol. 6, Article 195).

[0010] More recently, select CARs have also been expressed in NK cells. For example, CAR-modified NK-92 cells use a first-generation CAR containing only the CD3ζ intracellular signaling domain. Several antigens have been targeted by these first-generation CAR-NK cells, including CD19 and CD20 for B-cell lymphoma, ErbB2 for breast cancer, ovarian cancer, and squamous cell carcinoma, GD2 for neuroblastoma, and CD138 for multiple myeloma. Second-generation CAR-NK cells from the NK-92 line have also been engineered against several antigens, including EpCAM for multiple cancers, HLA-A2 EBNA3 complex for Epstein-Barr virus, CS1 for multiple myeloma, and ErbB2 for HER2-positive epithelial cancers. The most common intracellular costimulatory domain used in conjunction with CD3ζ in second-generation NK-92 CARs is CD28. However, the potential effect of the CD28 domain is unclear, as NK cells do not naturally express CD28. Additional second-generation CARs incorporate the 4-1BB intracellular signaling domain in conjunction with CD3ζ to improve NK cell persistence. Elsewhere, the functionality of different intracellular domains was compared using ErbB2 scFv fused to CD3ζ alone, CD28 and CD3ζ, or 4-1BB and CD3ζ tested against breast cancer cells. All second-generation constructs were found to improve killing compared to first-generation CARs, with CD28 and CD3ζ providing 65% target lysis, 4-1BB and CD3ζ lysing 62%, and CD3ζ alone killing 51% of targets. In a recent study, the 4-1BB and CD28 intracellular domains were also compared using an anti-CD19 CAR expressed on NK-92 cells against B-cell malignancies. Furthermore, it was found that the CD3ζ / 4-1BB construct was less effective than CD3ζ / CD28 in cell killing and cytokine production, highlighting the differential effects of the CD28 and 4-1BB costimulatory domains.

[0011] Third-generation NK-92 CARs are composed of CD3ζ, CD28, and 4-1BB intracellular signaling domains along with an anti-CD5 scFv and have shown specific and potent anti-tumor activity against various T-cell leukemia and lymphoma cell lines and primary tumor cells. These cells were also able to inhibit disease progression in xenograft mouse models of T-cell acute lymphoblastic leukemia (ALL) cell lines and primary tumor cells (Transl Res. 2017 September;187:32-43). In further examples, International Publication Nos. WO 2016 / 201304 and WO 2018 / 076391 teach the use of third-generation CD3ζ CARs expressed in NK cells and NK-92 cells. Summary of the Invention [Problem to be solved by the invention]

[0012] However, NK cells (and NK-92 cells in particular) are often difficult to genetically modify, as evidenced by the numerous failures to engineer NK-92 cells to express Fc receptors. Such difficulties are further complicated when NK-92 cells are transfected with multiple recombinant genes or relatively large recombinant nucleic acid payloads for heterologous expression. In addition, NK-92 cells also exhibit a significant lack of predictability with respect to the recombinant expression of foreign proteins (e.g., CD16). On a functional level, while often exhibiting targeted cytotoxicity, nearly all CAR NK-92 cells require a high effector-to-target cell ratio.

[0013] Thus, even though numerous recombinant NK-92 cells are known in the art, all or nearly all of them face various challenges. Consequently, there remains a need for CAR-expressing NK-92 cells that express significant amounts of highly active CARs and that can be easily cultured in a simple and effective manner. [Means for solving the problem]

[0014] The inventors have unexpectedly discovered that NK-92 cells expressing FcεRIγ-bearing CARs exhibit excellent cytolytic activity, typically at relatively low effector-to-target cell ratios compared to other constructs, and exhibit high levels of expression of FcεRIγ-bearing CARs. Furthermore, such recombinant cells also express CD16 at desirable levels, and when further modified to express stimulatory cytokines, the recombinant NK-92 cells were also readily cultured without the need for exogenous IL-2.

[0015] Thus, in one aspect of the present subject matter, the inventors contemplate a genetically engineered NK cell bearing a membrane-bound recombinant chimeric antigen receptor (CAR) comprising, in a single polypeptide chain, (i) an extracellular binding domain, (ii) a hinge domain, (iii) a transmembrane domain, and (iv) an FcεRIγ signaling domain. Most typically, although not necessarily, the NK cell is an NK-92 cell.

[0016] In some embodiments, the extracellular binding domain comprises an scFv that may specifically bind to a tumor-specific antigen (e.g., CD19, CD20, GD2, HER-2, CD30, EGFR, FAP, CD33, CD123, PD-L1, IGF1R, CSPG4, or B7-H4), a tumor-associated antigen (e.g., MUC-2, Brachyury, CEA), or a patient- and tumor-specific antigen (e.g., a neoepitope with high affinity for the patient's MHC I and / or MHC II). Alternatively, the extracellular binding domain may also specifically bind to a virus-specific antigen; exemplary viruses discussed herein include HIV, HPV, RSV, influenza, Ebola, or HCV. For example, a suitable viral antigen comprises gp120 of the HIV virus.

[0017] In a further embodiment, the hinge domain and / or transmembrane domain comprises a CD8 hinge domain and / or a CD28 transmembrane domain, and / or the FcεRIγ signaling domain has the amino acid sequence of SEQ ID NO:1.

[0018] Additionally, it is contemplated that the genetically modified NK cells may further comprise membrane-bound recombinant CD16 (and particularly high-affinity variants of CD16) and / or that the genetically modified NK cells may express a recombinant cytokine having an endoplasmic reticulum retention sequence.

[0019] Therefore, from a different perspective, the inventors also contemplate genetically engineered NK cells comprising a recombinant nucleic acid encoding a chimeric antigen receptor (CAR), wherein the CAR comprises, in a single polypeptide chain, (i) an extracellular binding domain, (ii) a hinge domain, (iii) a transmembrane domain, and (iv) an FcεRIγ signaling domain. As mentioned above, it is generally preferred that the NK cells are NK-92 cells. In some embodiments, the recombinant nucleic acid is RNA, which may be a polycistronic RNA further encoding CD16 and / or a cytokine having an endoplasmic reticulum retention sequence. The same considerations as above apply with respect to the various domains.

[0020] In a further aspect of the present subject matter, the inventors also contemplate a method of treating cancer in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of the genetically modified NK cells provided herein, thereby treating the cancer. As will be readily understood, the contemplated methods will further comprise administering at least one additional therapeutic entity, e.g., a viral cancer vaccine, a bacterial cancer vaccine, a yeast cancer vaccine, N-803, an antibody, a stem cell transplant, and / or a tumor-targeting cytokine.

[0021] For example, cancers that may be treated by the contemplated methods include leukemia, acute lymphocytic leukemia, acute myeloid leukemia, chronic leukemia, chronic myeloid (granulocytic) leukemia, chronic lymphocytic leukemia, polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's disease, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, solid tumors, including, but not limited to, sarcomas and carcinomas, such as fibrosarcoma, myxoid sarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordal sarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovial sarcoma, and the like. tumor, mesothelial tumor, 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, liver tumor, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytic tumor, medulloblastic tumor, craniopharyngeal tumor, ependymal tumor, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0022] Similarly, the inventors contemplate a method of treating a viral infection in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of the genetically modified NK cells presented herein (having an extracellular binding domain that may specifically bind to a virus-specific antigen), thereby treating the viral infection. Of course, the contemplated methods may further comprise the step of administering an antiviral agent.

[0023] Regardless of the type of treatment, 1 m of the patient's body surface area 2 Approximately 1 x 10 8 ~Approx. 1×10 11 It is contemplated that the cells are administered to a patient.

[0024] Therefore, the inventors also contemplate the use of genetically modified NK cells as presented herein in the treatment of cancer or viral infections.

[0025] Various objects, features, aspects and advantages of the present subject matter will become more apparent from the following detailed description of preferred embodiments, taken in conjunction with the accompanying drawings in which like numerals represent like components. [Brief explanation of the drawings]

[0026] [Figure 1]

[0039] Figure 1 shows a schematic representation of representative CD19-CARs tested. All CD19-CAR variants had an extracellular domain containing an anti-CD19 scFv region (αCD19-scFv), a hinge region from CD8 (CD8 hinge), and a transmembrane domain from CD28 (CD28 TM). The intracellular domain of the CD19CAR was altered as indicated. [Figure 2A] Representative results of the percentage of NK-92 cells expressing the CD19-CAR of Figure 1 after transfection with CD19-CAR mRNA, as measured by flow cytometry using an anti-scFv antibody labeled with eF660. [Figure 2B] Representative results of median fluorescence intensity (MFI) vs. background in NK-92 cells expressing CD19-CAR labeled with eF660-labeled anti-scFv antibody. [Figure 3A] Representative results are shown for the percentage of NK-92 cell-sensitive target cancer cells (K562) killed by NK-92 cells (effectors) expressing CD19CAR at effector:target ratios ranging from 5:1 to 0.3:1. [Figure 3B] Representative results are shown for the percentage of NK-92 cell-resistant CD19-positive target cancer cells (SUP-B15) killed by CD19CAR-expressing NK-92 cells (effectors) at effector:target ratios ranging from 5:1 to 0.3:1. [Figure 4]Representative results are shown for MFI of NK-92 cells (effectors) expressing CD19-CAR, labeled with anti-CD107a antibody, in a degranulation assay using SUP-B15 target cells at effector:target ratios of 2:1 to 0.25:1. [Figure 5] Representative survival curves of IV Rag tumor-bearing animals are shown, as described in the Examples. Statistical analysis was by log-rank (Mantel-Cox) test. ****, P<0.0001. [Figure 6] Representative results of animal weight changes in the IV large tumor model are shown. Data are mean ± standard error of the mean. The standard error of the mean was calculated as the standard deviation divided by the square root of N. [Figure 7] Representative tumor growth curves in the SC Large model are shown. Data are means ± standard error of the mean. Statistical analysis was performed using two-way analysis of variance followed by Tukey's test for multiple comparisons; ***, P<0.001; ****, P<0.0001. [Figure 8] Representative data are shown demonstrating that CD19 t-haNK reduced metastatic disease burden in the livers of SC Raji tumor-bearing mice. Panel a: Whole liver images of animals from the indicated treatment groups on day 13. Arrows indicate metastatic lesions. Livers were fixed in 10% formalin for at least 24 hours before photography. Panel b: Quantification of the percentage of tumor cell lesions in the liver (assessed by H&E staining) on ​​the indicated days. Day 13: *, P=0.0257 (by unpaired two-tailed t-test). Statistical analysis on days 11 and 15 was not possible due to limited sample size. See Table 4 for raw data. [Figure 9] Representative results of animal weight changes in the SC Large tumor model are shown. Data are means ± standard errors of the mean. [Figure 10] Representative Kaplan-Meier survival curves are shown for mice injected with L1210-Luc tumor cells following intratumoral treatment with mCD19-CAR NK-92 cells versus vehicle control, as described in the Examples. [Figure 11]Representative results are shown for tumor size of complete responders versus naive controls re-challenged with L1210-Luc tumor cells as described in the Examples. [Figure 12] Representative Kaplan-Meier survival curves are shown for mice injected with A20 tumor cells following intratumoral treatment with mCD19-CAR NK-92 cells versus vehicle control, as described in the Examples. [Figure 13] Representative results are shown for tumor size in complete responders versus naive controls re-challenged with A20 tumor cells as described in the Examples. [Figure 14] Representative results are shown for the cytotoxicity of HER2.CAR-t-haNK cells against BT-474 cells. [Figure 15] Representative results are shown for the cytotoxicity of CD33.CAR-t-haNK cells against THP-1 cells. [Figure 16] Representative results are shown for the cytotoxicity of PD-L1.CAR-t-haNK cells against SUP-B15.PD-L1+ cells. [Figure 17] Representative results are shown for the cytotoxicity of PD-L1.CAR-t-haNK cells against U251 cells. [Figure 18] Representative results are shown for the cytotoxicity of EGFR.CAR-t-haNK cells against A-549 cells. [Figure 19] Representative results are shown for the cytotoxicity of CD19.CAR-t-haNK cells against K562 cells. [Figure 20] Representative results are shown for the cytotoxicity of CD19.CAR-t-haNK cells against SUP-B15 cells. [Figure 21] Representative results are shown for ADCC of CD19.CAR-t-haNK cells against SKBr3 cells. [Figure 22] Representative results are shown for the cytotoxicity of IGF1R.CAR-t-haNK cells against MDA-MB-231 cells. [Figure 23]Representative results of the cytotoxicity of PD-L1.CAR-t-haNK cells against various cancer cells are shown. [Figure 24] Representative comparative results of the cytotoxicity of PD-L1.CAR-t-haNK cells against MDA-MB-231 cells are shown. [Figure 25] Representative results for CD16 and CD19.CAR expression are shown. [Figure 26] Representative results for the natural cytotoxicity of CD19.CAR-t-haNK cells are shown. [Figure 27] Representative results for CAR-mediated cytotoxicity of CD19.CAR-t-haNK cells are shown. [Figure 28] Representative results for ADCC of CD19.CAR-t-haNK cells are shown. [Figure 29] Representative comparative results for CD16 and CD20.CAR expression are shown. [Figure 30] Representative results for the natural cytotoxicity of CD20.CAR-t-haNK cells are shown. [Figure 31] Representative results for CD16 and CD33.CAR expression are shown. [Figure 32] Representative results for the natural cytotoxicity of CD33.CAR-t-haNK cells are shown. [Figure 33] Representative results for CAR-mediated cytotoxicity of CD33.CAR-t-haNK cells are shown. [Figure 34] Representative results for ADCC of CD33.CAR-t-haNK cells are shown. [Figure 35] Representative results for CD16 and EGFR.CAR expression are shown. [Figure 36] Representative results for the natural cytotoxicity of EGFR.CAR-t-haNK cells are shown. [Figure 37] Representative results for CAR-mediated cytotoxicity of EGFR.CAR-t-haNK cells are shown. [Figure 38]Representative results for CAR-mediated cytotoxicity of EGFR.CAR-t-haNK cells are shown. [Figure 39] Representative results for ADCC of EGFR.CAR-t-haNK cells are shown. [Figure 40] Representative results for CD16 and HER2.CAR expression are shown. [Figure 41] Representative results for the natural cytotoxicity of HER2.CAR-t-haNK cells are shown. [Figure 42] Representative results for CAR-mediated cytotoxicity of HER2.CAR-t-haNK cells are shown. [Figure 43] Representative results for ADCC of HER2.CAR-t-haNK cells are shown. [Figure 44] Representative results for CD16 and PD-L1.CAR expression are shown. [Figure 45] Representative results for the natural cytotoxicity of PD-L1.CAR-t-haNK cells are shown. [Figure 46] Representative results for CAR-mediated cytotoxicity of PD-L1.CAR-t-haNK cells are shown. [Figure 47] Representative results for ADCC of PD-L1.CAR-t-haNK cells are shown. [Figure 48] Representative results for CAR-mediated cytotoxicity of CD123.CAR-t-haNK cells are shown. [Figure 49] Representative results for ADCC of CD123.CAR-t-haNK cells are shown. [Figure 50] Representative results for CD16 and CD30.CAR expression are shown. [Figure 51] Representative results for the natural cytotoxicity of CD30.CAR-t-haNK cells are shown. [Figure 52] Representative results for CAR-mediated cytotoxicity of CD30.CAR-t-haNK cells are shown. [Figure 53] Representative results for ADCC of CD30.CAR-t-haNK cells are shown. [Figure 54] Representative results for CD16 and BCMA.CAR expression are shown. [Figure 55] Representative results for CAR-mediated cytotoxicity of BCMA.CAR-t-haNK cells are shown. [Figure 56] Representative results for ADCC of BCMA.CAR-t-haNK cells are shown. [Figure 57] Representative results for CD16 and gp120.CAR expression are shown. [Figure 58] Representative results for GP120 binding of gp120.CAR-t-haNK cells are shown. [Figure 59] Representative results for the natural cytotoxicity of gp120.CAR-t-haNK cells are shown. [Figure 60] Representative results for ADCC of gp120.CAR-t-haNK cells are shown. [Figure 61] Representative results for CD16 and FAP.CAR expression are shown. [Figure 62] Representative results for CAR-mediated cytotoxicity of FAP.CAR-t-haNK cells are shown. [Figure 63] CSPG4. Representative results for the expression of CSPG4 in CAR-t-haNK cells are shown. [Figure 64] Representative results for CAR-mediated cytotoxicity of CSPG4.CAR-t-haNK cells are shown. [Figure 65] A representative tricistronic construct encoding IGF1R-CAR, CD16, and IL-2ER is depicted. DETAILED DESCRIPTION OF THE INVENTION

[0027] To date, CARs containing FcεRIγ as a signaling domain (e.g., CD3ζ) have not been utilized in NK-92 cells, other NK cell lines, or endogenous NK cells because they appear to be more effective when combined with additional signaling domains (e.g., in second- and third-generation CARs). The inventors have now made the unexpected and surprising discovery that NK-92 cells expressing first-generation CARs containing the intracellular domain from FcεRIγ, which has only one ITAM domain, have equal or greater cytotoxic activity against cancer cells expressing the antigen recognized by the CAR than NK-92 cells expressing CARs containing the CD3ζ signaling domain, which has three ITAM domains, even when these ITAM domains are combined with other signaling domains (i.e., second- or third-generation CARs). Notably, the IgE receptor (FcεRI), in its native context, contains two γ chains coupled to each other via disulfide bonds and is normally expressed exclusively in eosinophils, basophils, and epidermal Langerhans cells. The inventors also made the unexpected discovery that CARs containing the intracellular domain from FcεRIγ were expressed at higher levels on the surface of NK-92 cells than other CARs, particularly CARs containing the CD3ζ signaling domain.

[0028] Thus, the present subject matter is directed to genetically engineered NK-92 cells or NK cell lines that have been engineered to express a chimeric antigen receptor (CAR) on the cell surface. Most typically, the CAR comprises the intracellular domain from the Fc epsilon receptor gamma (FcεRIγ), although in other embodiments, the CAR may also comprise the T cell receptor (TCR) CD3 zeta (CD3ζ) intracellular domain. As will be readily appreciated, the CAR may be transiently or stably expressed by the NK-92 cells from recombinant DNA or RNA molecules.

[0029] Consequently, in one embodiment of the present subject matter, NK cells, NK-92 cells, or NK / NK-92 cell lines express a chimeric antigen receptor (CAR) on the surface of NK-92 cells that comprises the cytoplasmic domain of FcεRIγ (e.g., having the amino acid sequence of SEQ ID NO: 1). Alternatively, or additionally, the CAR may also comprise the cytoplasmic domain of CD3 zeta (e.g., having the amino acid sequence of SEQ ID NO: 10 and may be encoded by the nucleic acid of SEQ ID NO: 11 (codon-optimized) or SEQ ID NO: 12 (non-codon-optimized); the full-length sequence is set forth in SEQ ID NO: 47). In another embodiment, it is contemplated that the NK or NK-92 cell line is transformed with a nucleic acid encoding the chimeric antigen receptor (CAR). For example, a preferred nucleic acid encodes the cytoplasmic domain of FcεRIγ (e.g., comprising or consisting of SEQ ID NO: 2). Alternatively, or additionally, the nucleic acid encodes the cytoplasmic domain of CD3 zeta (e.g., comprising or consisting of SEQ ID NO: 11 (human, codon-optimized) or SEQ ID NO: 12 (human)). As will be readily appreciated, a CAR may target a cancer-associated or virus-associated antigen via its extracellular binding domain, as described in more detail below.

[0030] In further contemplated embodiments, NK or NK-92 cells can be modified to express at least one cytokine or variant thereof. For example, the cytokine may be transiently or stably expressed by the recombinant cell, and the cytokine may include an endoplasmic reticulum retention signal. If desired, the NK or NK-92 cells can also be modified to express a suicide gene (e.g., the suicide gene is thymidine kinase). Without being bound by any theory, it is believed that expression of the suicide gene may prevent uncontrolled proliferation of NK-92 cells by providing a mechanism for selectively killing the cells upon introduction of an appropriate stimulus.

[0031] In another aspect of the present subject matter, the inventors also contemplate a method of treating cancer in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of modified NK / NK-92 cells or NK / NK-92 cell lines engineered to express a chimeric antigen receptor (CAR) as described herein. From a different perspective, the inventors also contemplate modified NK / NK-92 cells or NK / NK-92 cell lines expressing a chimeric antigen receptor (CAR), preferably comprising the cytoplasmic domain of FcεRIγ, intended for use in treating a tumor in a subject. In some embodiments, the use comprises administering to a subject an effective amount of the modified cells or cell lines described herein to treat the tumor. In yet other embodiments, an in vitro method for killing tumor cells is contemplated, which may comprise contacting the tumor cells with the modified NK-92 cells or NK-92 cell lines described herein. In some embodiments, the modified NK-92 cells or NK-92 cell lines express a CAR that binds to an antigen on the tumor cells. In some embodiments, the CAR preferably comprises the intracellular domain from the Fc epsilon receptor gamma (FcεRIγ). Alternatively, or additionally, the CAR comprises the T cell receptor (TCR) CD3 zeta (CD3ζ) intracellular domain.

[0032] In yet other embodiments, methods of treating a viral infection in a patient in need thereof are described, comprising administering to the patient a therapeutically effective amount of NK-92 cells expressing a CAR as described herein.

[0033] After reading this description, it will be apparent to one skilled in the art how to implement the invention in various alternative embodiments and alternative applications. However, not all embodiments of the invention are described herein. It will be understood that the embodiments presented herein are presented by way of example only, and not by way of limitation. As such, this detailed description of various alternative embodiments should not be construed as limiting the scope or breadth of the invention, as set forth below.

[0034] Before the present invention is disclosed and described, it is to be understood that the following embodiments are not limited to particular compositions, methods of preparing such compositions, or their uses as such, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0035] Titles or subtitles may be used herein for the convenience of the reader and are not intended to affect the scope of the invention. In addition, some terms used herein are more specifically defined below.

[0036] With respect to suitable NK cells, it should be noted that all NK cells are believed to be suitable for use herein, and therefore include primary NK cells (stored, expanded, and / or fresh), immortalized secondary NK cells, autologous or heterologous NK cells (banked, stored, fresh, etc.), and modified NK cells as described in more detail below. In some embodiments, it is preferred that the NK cells are NK-92 cells. The NK-92 cell line is the only cell line discovered 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 cancer NK cells with broad antitumor cytotoxicity and predictable yields after expansion in suitable media. Advantageously, NK-92 cells have high cytolytic activity against a variety of cancers.

[0037] The original NK-92 cell line was CD56 bright, CD2, CD7, CD11a, CD28, CD45, and CD54 surface markers, and did not display CD1, CD3, CD4, CD5, CD8, CD10, CD14, CD16, CD19, CD20, CD23, or CD34 markers. Proliferation of such NK-92 cells in culture is dependent on the presence of interleukin-2 (e.g., rIL-2), where a dose of as little as 1 IU / mL is sufficient to maintain proliferation. IL-7 and IL-12 do not support long-term proliferation, and various other cytokines, including IL-1α, IL-6, tumor necrosis factor α, interferon α, and interferon γ, have not been tested. Compared to primary NK cells, NK-92 typically have high cytotoxicity even at relatively low effector:target (E:T) ratios, e.g., 1:1. Representative NK-92 cells have been deposited with the American Type Culture Collection (ATCC) under the designation CRL-2407.

[0038] Thus, suitable NK cells may have one or more modified KIRs, mutated, for example, to reduce or eliminate interaction with MHC class I molecules. Of course, it should be noted that one or more KIRs may be deleted or their expression may be suppressed (e.g., via miRNA, siRNA, etc.). Most typically, two or more KIRs will be mutated, deleted, or silenced; specifically contemplated KIRs include those with short or long cytoplasmic tails and two or three domains. From a different perspective, modified, silenced, or deleted KIRs may include KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DL2, KIR3DL3, and KIR3DS1. Such modified cells may be prepared using protocols well known in the art. Alternatively, such cells may also be commercially obtained as NK cells ('activated natural killer cells') from NantKwest (see URL: www.nantkwest.com). Such cells may then be further genetically engineered for a CAR, as described in further more detail below.

[0039] In another embodiment of the present subject matter, the genetically engineered NK cells may also be NK-92 derivatives modified to express a high-affinity Fcγ receptor (CD16). Sequences for high-affinity mutant Fcγ receptors are well known in the art (see, e.g., Blood 2009 113:3716-3725; SEQ ID NOs: 43 and 44), and all modes of production and expression are deemed suitable for use herein. Expression of such receptors may allow for specific targeting of tumor cells using antibodies specific to the patient's tumor cells (e.g., neoepitopes), specific tumor types (e.g., her2neu, PSA, PSMA, etc.), or cancer-associated antibodies (e.g., CEA-CAM). Advantageously, such antibodies are commercially available and can be used in combination with the cells (e.g., coupled to the Fcγ receptor). Alternatively, such cells may also be commercially obtained as haNK cells from NantKwest. Such cells may then be further genetically engineered with a CAR, as described in further detail below.

[0040] Accordingly, NK cells suitable for use herein include NK-92 cells (which may be transfected with a tricistronic construct encoding a CAR, CD16 or a variant thereof, and a cytokine or variant thereof), genetically modified NK cells or NK-92 cells expressing CD16 or a variant thereof or a cytokine or variant thereof (which may be transfected with a nucleic acid encoding a CAR and CD16 or a variant thereof or a cytokine or variant thereof), and genetically modified NK cells or NK-92 cells expressing CD16 or a variant thereof and a cytokine or variant thereof (which may be transfected with a nucleic acid encoding a CAR).

[0041] The genetic modification of NK cells discussed herein can be performed in numerous ways, and all known ways are deemed suitable for use herein. Furthermore, it should be understood that NK cells can be transfected with DNA or RNA, with the particular choice of transfection depending, at least in part, on the type of recombinant cell desired and the transfection efficiency. For example, if it is desired to stably transfect NK cells, linearized DNA may 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., polyA + A tailed mRNA may also be used.

[0042] Similarly, it should be understood that the mode of transfection will depend, at least in part, on the type of nucleic acid used. Thus, 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. Exogenous transgenes introduced using such vectors are not integrated into the nuclear genome of the cell; therefore, in the absence of vector replication, the exogenous transgene will be degraded or diluted over time.

[0043] In another embodiment, the vectors described herein allow for stable transfection of cells. In one embodiment, the vector allows for integration of the transgene into the genome of the cell. Preferably, such vectors have a positive selection marker, and suitable positive selection markers include any gene that allows cells to grow under conditions that would kill cells that do not express the gene. Non-limiting examples include antibiotic resistance, such as geneticin (the neo gene from Tn5).

[0044] 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.

[0045] In yet other embodiments, cells are transfected with mRNA encoding a protein of interest (e.g., a CAR). Transfection of the 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 refers to between about 15 minutes and about 48 hours prior to administration. Preferably, transfection of the mRNA is performed between about 5 hours and about 24 hours prior to administration. In at least some embodiments, as described in more detail below, transfection of the mRNA into NK cells resulted in consistent and robust expression of the CAR in a surprisingly high percentage of transfected cells. Furthermore, such transfected cells also exhibited high specific cytotoxicity at relatively low effector-to-target cell ratios.

[0046] With regard to the contemplated CARs, it is recognized that NK / NK-92 cells are genetically engineered to express the CAR as a membrane-bound protein that exposes a portion of the CAR on the cell surface while maintaining the signaling domain within the intracellular space. Most typically, the 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.

[0047] In preferred embodiments, the cytoplasmic domain of the CAR comprises or consists of the signaling domain of FcεRIγ. For example, the FcεRIγ signaling domain comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 1. 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.

[0048] In some embodiments, the FcεRIγ signaling domain comprises, consists of, or consists essentially of an amino acid sequence having at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:1.

[0049] As noted above, in some embodiments, the cytoplasmic domain of the CAR comprises the signaling domain of CD3 zeta (CD3ζ). In one embodiment, the cytoplasmic domain of the CAR consists of the signaling domain of CD3 zeta. In one embodiment, the CD3 zeta signaling domain comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 10. In some embodiments, the CD3 zeta signaling domain comprises, consists of, or consists essentially of an amino acid sequence having at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 10.

[0050] The CAR may comprise any suitable transmembrane domain. In one aspect, the CAR comprises a CD28 transmembrane domain. In one embodiment, the CD28 transmembrane domain comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 7. In one embodiment, the CD28 transmembrane domain comprises, consists of, or consists essentially of an amino acid sequence having at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology to the amino acid sequence of SEQ ID NO: 7. In one embodiment, the transmembrane domain is selected from a CD28 transmembrane domain, a 4-1BB transmembrane domain, or an FcεRIγ transmembrane domain.

[0051] The CAR may comprise any suitable hinge region. In one aspect, the CAR comprises the hinge region of CD8. In one embodiment, the CD8 hinge region comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 6. In one embodiment, the CD8 hinge region comprises, consists of, or consists essentially of an amino acid sequence having at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 6.

[0052] Most typically, although not necessarily, the extracellular binding domain of the CAR will be an scFv or other natural or synthetic binding moiety that specifically binds to an antigen of interest. Particularly suitable binding moieties include small antibody fragments with single, dual, or multiple target specificities, beta barrel domain binders, page display fusion proteins, and the like. Among other suitable extracellular binding domains, preferred domains will specifically bind to tumor-specific, tumor-associated, or patient- and tumor-specific antigens. For example, contemplated antigens include CD19, CD20, GD2, HER-2, CD30, EGFR, FAP, CD33, CD123, PD-L1, IGF1R, CSPG4, or B7-H4. Additional tumor-specific antigens are described, by way of non-limiting example, in U.S. Patent Application Publication No. 2013 / 0189268; WO 1999024566A1; U.S. Patent No. 7,098,008; and WO 2000020460, 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 the HIV virus (e.g., gp120), HPV, RSV, influenza, Ebola, or HCV viruses.

[0053] With regard to the construction of contemplated CARs, it should be understood that CARs can be modified in numerous ways, as described, for example, in WO 2014 / 039523; U.S. Patent Application Publication No. 2014 / 0242701; U.S. Patent Application Publication No. 2014 / 0274909; U.S. Patent Application Publication No. 2013 / 0280285, and WO 2014 / 099671, each of which is incorporated herein by reference in its entirety.

[0054] Therefore, and from a different perspective, contemplated CARs target antigens associated with specific cancer types. In one embodiment, the cancer is leukemia (including acute leukemia (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemia (e.g., chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphoma (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 carcinomas, such as fibrosarcoma, myxoid sarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordal sarcoma, angiosarcoma, endothelial sarcoma, lymphatic sarcoma, lymphovascular sarcoma, lymphovascular endothelial tumors, and the like. sarcoma, synovial tumor, mesothelial tumor, 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 lung cancer, renal cell carcinoma, liver tumor, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytic tumor, medulloblastic tumor, craniopharyngeal tumor, ependymal tumor, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, or retinoblastoma.

[0055] Thus, contemplated CARs will generally have the structure of an extracellular binding domain coupled (directly) to a hinge domain, coupled (directly) to a transmembrane domain, and coupled (directly) to an FcεRIγ signaling domain. In further contemplated embodiments, contemplated CARs may also include one or more signaling domains in addition to or instead of the FcεRIγ signaling domain; particularly contemplated signaling domains include a CD3ζ signaling domain, a 4-1BB signaling domain, and a CD28 signaling domain. Thus, for example, a contemplated CAR may include any one of the binding domains having SEQ ID NOs: 4, 23-42, and 48-59 coupled to a hinge domain (e.g., a CD8 hinge such as SEQ ID NO: 6), which in turn is coupled to a transmembrane domain (e.g., a CD28 TM such as SEQ ID NO: 7), and coupled to a signaling domain (e.g., an FcεRIγ signaling domain such as SEQ ID NO: 1, a CD28 signaling domain such as SEQ ID NO: 8, a 4-1BB signaling domain such as SEQ ID NO: 9, or a CD3ζ signaling domain such as SEQ ID NO: 10).

[0056] In further contemplated aspects, the NK cells may be further genetically engineered to express one or more cytokines, thereby providing a selectable marker when the cytokine and CAR are encoded on the same recombinant nucleic acid and / or rendering the recombinant cells independent of exogenous IL-2. Thus, in some embodiments, NK-92 cells are modified to express at least one cytokine. In particular, the at least one cytokine is IL-2, IL-12, IL-15, IL-18, IL-21, or a variant thereof. In preferred embodiments, the cytokine is IL-2 or a variant thereof, with particularly preferred variants including an endoplasmic reticulum retention signal (e.g., human IL-2, such as SEQ ID NO: 18, or an endoplasmic reticulum retention signal, such as SEQ ID NO: 19). For example, the IL-2 gene is cloned and expressed with a signal sequence that directs IL-2 to the endoplasmic reticulum. This allows expression at levels sufficient for autocrine activation of IL-2 without releasing IL-2 extracellularly (e.g., Exp Hematol. 2005 Feb;33(2):159-64). Alternatively, expression of a cytokine (particularly IL-15) may also be such that the cytokine is expressed in an amount sufficient to not only provide an autocrine growth signal to the recombinant cell but also to allow at least a portion of the expressed IL-15 to be released from the cell, thereby providing an immunostimulatory signal. For example, such expression may be achieved using a human IL-15 sequence that includes both a signal peptide and an endoplasmic reticulum retention sequence. Representative DNA and protein sequences for endoplasmic reticulum-retained IL-15 are set forth in SEQ ID NO:72 and SEQ ID NO:73, respectively.

[0057] If desired, the cells under study may also express a suicide gene. The term "suicide gene" refers to a transgene that allows for negative selection of cells expressing the suicide gene. A suicide gene is used as a safety system that allows cells expressing the gene to be killed by the introduction of a selective drug. This is desirable when the recombinant gene causes a mutation that leads to uncontrolled cell proliferation, i.e., the cell itself is capable of such proliferation. Several suicide gene systems have been identified, including the herpes simplex virus thymidine kinase (TK) gene, the cytosine deaminase gene, the varicella-zoster virus thymidine kinase gene, the nitroreductase gene, the Escherichia coli gpt gene, and the E. coli Deo gene. Typically, a suicide gene encodes a protein that has no deleterious effect on the cell, but will kill the cell in the presence of a specific compound. Thus, a suicide gene is typically part of the system.

[0058] In one embodiment, the suicide gene is active in NK-92 cells. In one embodiment, the suicide gene is a thymidine kinase (TK) gene. The TK gene may be a wild-type or mutant TK gene (e.g., tk30, tk75, sr39tk). Cells expressing the TK protein can be killed using ganciclovir. In another embodiment, the suicide gene is cytosine deaminase, which is toxic to cells in the presence of 5-fluorocytosine. Garcia-Sanchez et al. "Cytosine deaminase adenoviral vector and 5-fluorocytosine selectively reduce breast cancer cells 1 million-fold when they contaminate hematopoietic cells: a potential purging method for autologous transplantation." Blood. 1998 Jul 15;92(2):672-82. In a further embodiment, the suicide gene is a cytochrome P450, which is toxic in the presence of ifosfamide or cyclophosphamide. See, e.g., Touati et al. "A suicide gene therapy combining the improvement of cyclophosphamide tumor cytotoxicity and the development of an anti-tumor immune response." Curr Gene Ther. 2014;14(3):236-46. In yet another embodiment, the suicide gene is iCasp9. Di Stasi, (2011) "Inducible apoptosis as a safety switch for adoptive cell therapy." N Engl J Med 365:1673-1683. See also Morgan, "Live and Let Die: A New Suicide Gene Therapy Moves to the Clinic" Molecular Therapy (2012);20:11-13.iCasp9 induces apoptosis in the presence of the small molecule AP1903, a biologically inert small molecule that has been shown to be well tolerated in clinical trials and has been used in the context of adoptive cell therapy.

[0059] It should be noted, of course, that all of the recombinant proteins can be expressed from separate recombinant sequences. However, when multiple recombinant sequences are expressed (e.g., CAR, CD16, cytokines), it is generally preferred that the coding regions be arranged in a polycistronic unit having at least two or at least three coding regions encoding the recombinant proteins. Thus, transgenes can be engineered into expression vectors by any mechanism known to those of skill in the art. When multiple transgenes are to be inserted into cells, the transgenes can be engineered into the same or different expression vectors. In some embodiments, the cells are transfected with mRNA encoding the transgenic proteins to be expressed. In some embodiments, the cells are transfected with DNA encoding the transgenic proteins to be expressed. The transgenes, mRNA, and DNA can be introduced into NK-92 cells using any transfection method known in the art, including, but not limited to, infection, viral vectors, electroporation, lipofection, nucleofection, or "gene guns."

[0060] It should be noted, therefore, that in preferred embodiments, genetically modified NK cells (especially when the cells express a CAR and CD16 or a variant thereof) exhibit three distinct modes of cell death: general cytotoxicity mediated by activating receptors (e.g., NKG2D receptors), ADCC mediated by antibodies bound to target cells, and CAR-mediated cytotoxicity. As will be readily appreciated, contemplated genetically modified cells can be used in the treatment of a variety of diseases, particularly various cancers and viral infections where abnormal cells present disease-specific or disease-associated antigens. Consequently, the inventors contemplate methods of treating patients using modified NK or NK-92 cells as described herein. In one embodiment, the patient is suffering from cancer (e.g., a tumor), and the modified NK-92 cells or cell line express a CAR specific for an antigen expressed on the surface of cells from the cancer or tumor. In one embodiment, the patient is suffering from a viral infection, and the modified NK-92 cells or cell line express a CAR specific for an antigen expressed on the surface of cells infected by the virus. In one embodiment, the patient is suffering from a bacterial infection and the modified NK-92 cells or cell line express a CAR specific for an antigen expressed on the surface of the bacterial cells causing the infection.

[0061] In some embodiments, the cancer is leukemia (including acute leukemia (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemia (e.g., chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphoma (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 carcinomas, such as fibrosarcoma, myxoid sarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordal sarcoma, angiosarcoma, endothelial sarcoma, lymphatic sarcoma, lymphatic endothelial sarcoma, tumor, synovial tumor, mesothelial tumor, 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, liver tumor, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytic tumor, medulloblastic tumor, craniopharyngeal tumor, ependymal tumor, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0062] Contemplated modified NK or NK-92 cells can be administered to an individual in absolute numbers of cells, e.g., from about 1000 cells / injection up to about 10 billion cells / injection, e.g., about, at least about, or up to about 1 x 10 cells per injection. 8 , 1×10 7 , 5×10 7 , 1×10 6 , 5×10 6 , 1×10 5 , 5×10 5 , 1×10 4 , 5×10 4 , 1×10 3 , 5×10 3(etc.), or any range between any two of those numbers (endpoints included). In other embodiments, the modified NK-92 cells may be administered to an individual in relative numbers of cells, e.g., from about 1,000 cells up to about 10 billion cells / kg of the individual, e.g., about, at least about, or up to about 1 x 10 cells per kg of the individual. 8 , 1×10 7 , 5×10 7 , 1×10 6 , 5×10 6 , 1×10 5 , 5×10 5 , 1×10 4 , 5×10 4 , 1×10 3 , 5×10 3 (etc.), or any range between any two of those numbers (endpoints included), of modified NK-92 cells may be administered. In other embodiments, the total dose is calculated based on m of body surface area. 2 , for example, about 1 x 10 11 / m 2 , 1×10 10 / m 2 , 1×10 9 / m 2 , 1×10 8 / m 2 , 1×10 7 / m 2 , or any range between any two of those numbers (endpoints included). The average for an individual is approximately 1.6 m 2 ~approx. 1.8m 2 In a preferred embodiment, between about 1 billion and about 3 billion NK-92 cells are administered to a patient.

[0063] The modified NK-92 cells, and optionally other anti-cancer or antiviral agents, can be administered once to a patient with cancer or infected with a virus, or multiple times, for example, once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours, or once every 1, 2, 3, 4, 5, 6, or 7 days, or once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more weeks, or any range between any two of these numbers (endpoints inclusive), during the course of treatment.

[0064] In one embodiment, if the modified NK-92 cells express the suicide gene, an agent is administered to the patient to induce death of the modified NK-92 cells, hi one embodiment, the agent is administered at a time after administration of the modified NK-92 cells that is sufficient for the NK-92 cells to kill the target cells.

[0065] In one embodiment, the modified NK-92 cells are irradiated prior to administration to a patient. Irradiation of NK-92 cells is described, for example, in U.S. Patent No. 8,034,332, the entire contents of which are incorporated herein by reference. In one embodiment, modified NK-92 cells that have not been modified to express a suicide gene are irradiated.

[0066] Furthermore, it should be understood that contemplated treatments will also include the administration of other immunotherapeutic entities, with particularly preferred immunotherapeutic entities including viral cancer vaccines (e.g., adenoviral vectors encoding cancer-specific antigens), bacterial cancer vaccines (e.g., non-thermophilic E. coli expressing one or more cancer-specific antigens), yeast cancer vaccines, N-803 (also known as ALT-803, ALTOR Biosciences), antibodies (e.g., binding to tumor-associated antigens or patient-specific tumor neo-antigens), stem cell transplants (e.g., allogeneic or autologous), and tumor-targeted cytokines (e.g., NHS-IL12, IL-12 conjugated to a tumor-targeted antibody or fragment thereof). [Example]

[0067] The following examples are for illustrative purposes only and should not be construed as limiting the claimed invention. Various alternative techniques and methods available to those skilled in the art will similarly enable one to fully practice the intended invention.

[0068] Example 1: Preparation of CAR mRNA DNA sequences encoding each variant of CD19CAR, represented schematically in Figure 1, were designed in silico, synthesized de novo, and subcloned into the mRNA expression vector pXT7 (GeneArt, Life Technologies). Ten micrograms (μg) of plasmid were linearized by digestion with SalI restriction enzyme (New England Biolabs) and purified using a QIAgen gel purification kit (QIAgen) according to the manufacturer's instructions.

[0069] Linearized DNA was used as a template for in vitro synthesis of mRNA using the T7 mMessage mMachine Ultra transcription kit (ThermoFisher Scientific, Waltham, MA) according to the manufacturer's instructions, which includes a polyadenylation extension step that increases the length of the mRNA's poly(A) tail, thereby enhancing its stability in vivo.

[0070] mRNA for six CD19-CAR variants was prepared, along with green fluorescent protein (GFP) mRNA as a negative control. All of the CD19-CAR polypeptide variants had an extracellular domain containing an anti-CD19 scFv region (αCD19-scFv) (SEQ ID NO: 4), a hinge region from CD8 (SEQ ID NO: 6), and a transmembrane domain from CD28 (SEQ ID NO: 7). The intracellular domains of the CD19CAR are as follows and are shown schematically in Figure 1: CAR 3z has a CD3ζ signaling domain; CAR FcRe has an FcεRIγ signaling domain (SEQ ID NO: 1); CAR 28_3z has a CD28 signaling domain fused to a CD3ζ signaling domain; CAR BB_3z has a 4-1BB signaling domain fused to a CD3ζ signaling domain; CAR 28_BB_3z has a CD28 signaling domain fused to a 4-1BB signaling domain fused to a CD3ζ signaling domain; CAR BB_3z_28 has a 4-1BB signaling domain fused to a CD3ζ signaling domain fused to a CD28 signaling domain.

[0071] More specifically, the first-generation CAR with the CD3ζ signaling domain in Figure 1 had the nucleic acid sequence of SEQ ID NO: 13 (human) and SEQ ID NO: 21 (mouse), which translated to the amino acid sequence of SEQ ID NO: 22. The first-generation CAR with the FcεRIγ signaling domain had the nucleic acid sequence of SEQ ID NO: 5 and the amino acid sequence of SEQ ID NO: 3. The second-generation CAR with the CD28 / CD3ζ signaling domain had the nucleic acid sequence of SEQ ID NO: 14, and the second-generation CAR with the 4-1BB / CD3ζ signaling domain had the nucleic acid sequence of SEQ ID NO: 15. The third-generation CAR with the CD28 / 4-1BB / CD3ζ signaling domain had the nucleic acid sequence of SEQ ID NO: 16, and the third-generation CAR with the 4-1BB / CD3ζ / CD28 signaling domain had the nucleic acid sequence of SEQ ID NO: 17.

[0072] An additional first generation CAR with an FcεRIγ signaling domain was prepared as described in more detail below, where the hinge region was a CD8 hinge (SEQ ID NO: 6 or SEQ ID NO: 45 (human), encoded by SEQ ID NO: 46), the transmembrane domain was a CD28 transmembrane domain (SEQ ID NO: 7), and the signaling domain was an FcεRIγ signaling domain (SEQ ID NO: 1, encoded by nucleic acid SEQ ID NO: 2).

[0073] Next, scFv portions selected as follows were used to target various tumor-associated targets: CD19 (using the anti-CD19 scFv of SEQ ID NO: 4 or SEQ ID NO: 24, encoded by codon-optimized SEQ ID NO: 23), CD20 (using the anti-CD20 scFv of SEQ ID NO: 26, encoded by codon-optimized SEQ ID NO: 25), CD33 (using the anti-CD33 scFv of SEQ ID NO: 28, encoded by codon-optimized SEQ ID NO: 27), CSPG4 (using the anti-CSPG4 scFv of SEQ ID NO: 30, encoded by codon-optimized SEQ ID NO: 29), EGFR (using the anti-EGFR scFv of SEQ ID NO: 32, encoded by codon-optimized SEQ ID NO: 31), IGF1R (using the anti-IGF1R scFv of SEQ ID NO: 34, encoded by codon-optimized SEQ ID NO: 33), CD30 (using the anti-CD30 scFv of SEQ ID NO: 36, encoded by codon-optimized SEQ ID NO: 35), HER2 / neu (using the anti-HER2 / neu scFv of SEQ ID NO: 38, encoded by codon-optimized SEQ ID NO: 37). scFv), GD2 (using anti-GD2 scFv of SEQ ID NO:40 or SEQ ID NO:42, encoded by codon-optimized SEQ ID NO:39 or SEQ ID NO:41), CD123 (using anti-CD123 scFv of SEQ ID NO:49, encoded by codon-optimized SEQ ID NO:48), PD-L1 (using anti-PD-L1 scFv of SEQ ID NO:51, encoded by codon-optimized SEQ ID NO:50), B7-H4 (using anti-B7-H4 scFv of SEQ ID NO:53, encoded by codon-optimized SEQ ID NO:52), and FAP (using anti-FAP scFv of SEQ ID NO:58 or SEQ ID NO:59, encoded by codon-optimized SEQ ID NO:56 or SEQ ID NO:57) (all in a sequential configuration as shown for the CAR FcRe in Figure 1).

[0074] Similarly, the following selected scFv moieties (all in a sequential arrangement as shown in the CAR FcRe in Figure 1) were used to confer target specificity for a variety of virus-related targets: HIV gp120 (using the anti-gp120 scFv of SEQ ID NO: 55, encoded by codon-optimized SEQ ID NO: 54).

[0075] All of the constructs prepared above were fully expressed in NK-92 cells, and representative results demonstrate the physiological activity of such modified NK-92 cells.

[0076] Example 2: Electroporation of CD19CAR mRNA into NK-92 cells NK-92 cells were grown in X-Vivo10 medium (Lonza, Basel, Switzerland) supplemented with 5% human AB serum (Valley Biomedical, Winchester, VA) and 500 IU / mL IL-2 (Prospec, Rehovot, Israel). Cells were electroporated using a Neon™ electroporator (Life Technologies, Carlsbad, CA) according to the manufacturer's parameters for NK-92 cells (1250 V, 10 ms, 3 pulses) and 5 μg of mRNA / 10 μg in a volume of 100 μl. 6 Cells were electroporated with mRNA. Electroporated cells were maintained in the same medium (as above) for 20 hours (h).

[0077] Expression of CD19CAR on the surface of NK-92 cells was measured by flow cytometry using an anti-scFv antibody labeled with eF660 (eBioscience, San Diego, CA). Figure 2A shows the % expression of the indicated CD19CAR within the NK-92 cell population. Figure 2B shows the median fluorescence intensity (MFI, minus background) of cells electroporated with the indicated CD19CAR. As can be interpreted from Figures 2A and 2B, CAR FcRe surprisingly had the highest MFI (amount of CAR expressed on recombinant cells) along with the highest percentage of cells expressing CD19CAR on the cell surface (75.2%), followed by 28_3z (61.7%).

[0078] Example 3: Cytotoxicity of NK-92 cells expressing CD19CAR against cancer cell lines The efficacy of CAR-expressing NK-92 cells against target cancer cells in vitro was tested 20 hours after electroporation using a flow-based in vitro cytotoxicity assay. Effector cells (NK-92 expressing CD19CAR or GFP) were electrophoresed against PKHGL67-labeled (Sigma-Aldrich, St. Louis, MO) target cells (K562; or SUPB15, B-ALL, CD19 + ) were mixed in 96-well plates at different effector-to-target ratios (5:1 to 0.3:1) and incubated at 37°C for 4 hours. Propidium iodide (PI) (Sigma Aldrich, St. Louis, MO) was added to the cells, and samples were analyzed within 2 hours using an Attune flow cytometer (Life Technologies, Carlsbad, CA). Cytotoxicity was determined by the percentage of PI-positive cells within the PKH-positive target population.

[0079] Representative results are presented in Figures 3A and 3B. As can be seen from Figure 3A, NK-92 cells are effective in killing K562 cells regardless of the expression of the CD19CAR. Therefore, it should be noted that the recombinant cells do not lose their cytotoxicity. In contrast, NK-92 cells expressing GFP were inefficient in killing the cancer cell line SUP-B15. SUP-B15 is a CD19-positive acute lymphoblastic leukemia cell line that is resistant to cytotoxicity. As can be easily interpreted from Figure 3B, expression of any of the CD19CARs tested resulted in enhanced cytotoxic activity against the SUP-B15 cell line compared to the control (NK-92 cells expressing GFP). Surprisingly, the CAR FcRe exhibited similar or superior cytotoxicity against second- and third-generation CARs. This finding is particularly unexpected because the FcεRIγ signaling domain was present only as a single entity and was not combined with other signaling domains. Such a configuration did not result in the desired targeted cytotoxicity when used in CAR T cells. Advantageously, the tricistronic mRNA construct was able to produce substantial amounts of the desired CAR with excellent functional activity. Such a construct is particularly advantageous when CAR expression needs to be transient.

[0080] Degranulation is a critical step required for the release of soluble proteins (e.g., perforin and granzymes) from secretory granules in NK-92 cells. Degranulation is initiated by target cell recognition by NK-92. To test degranulation in the constructs, effector cells (NK-92) were mixed with unlabeled target cells (SUP-B15) at different effector-to-target ratios (5:1 to 0.3:1) in a 96-well plate, and anti-CD107a (FITC-conjugated, BD Pharmingen, San Jose, CA) was added to each well. The plate was incubated at 37°C in a CO2 incubator. After 1 hour, monensin (Golgi arrest) was added to the wells. The plate was incubated for an additional 3 hours at 37°C, and samples were analyzed by flow cytometry (Attune, Life Technologies, Carlsbad, CA). The percentage of degranulation was determined by subtracting the %CD107a positivity in NK-92 cells alone from the %CD107a positivity in effector + target samples, and representative results are presented in Figure 4.

[0081] Example 4: CD19t-haNK cells significantly improved animal survival in a large tumor xenograft model. CD19t-haNK cells (clone 19.6) contain the Fcε intracellular signaling domain. CD19t-haNK cells were cultured in X-VIVO™ 10 medium supplemented with 5% heat-inactivated human AB serum.

[0082] Test animals: Animal strain / species: NOD.Cg-PrkdcscidIl2rgtm1Wjl / SzJ (NSG) mice; age: 9-10 weeks (after isolation) at the start of the study; sex: female; weight: 20-27 grams at the start of the study. Number of animals: 20 for the IV tumor model; 12 for the SC tumor model. Supplier: The Jackson Laboratory (610 Main Street, Bar Harbor, ME 04609 US).

[0083] Raji Tumor Model: Raji cancer cell line: Raji cells were initially purchased from ATCC (Cat. No. CCL-86™; Lot No. 61723871) and then expanded and prepared for administration.

[0084] Cell culture medium: ATCC formulated RPMI-1640 medium supplemented with 10% fetal bovine serum with penicillin (100 U / mL), streptomycin (100 μg / mL).

[0085] Cell harvest: Logarithmic-phase Raji cells (passage 12) were harvested by centrifugation. Cells were washed and diluted to 5 x 10 for IV inoculation. 5 in serum-free medium at a concentration of 2.5 × 10 viable cells / mL for SC injection. 6 The cells were resuspended in medium / Matrigel (1:1 v / v) at a concentration of viable cells / mL. Cells were stored on ice before animal injection. Cells used in in vivo studies had a viability of 96%.

[0086] Raji cell inoculation: Raji IV model. Twenty animals were injected IV with 0.2 mL of Raji cell suspension (1 × 10 cells) via a lateral tail vein with a 27-gauge needle. 5 Raji SC model. Twelve animals were injected SC into both flanks with 0.1 mL of Raji cell suspension (2.5 × 10 cells) using a 25-gauge needle. 5 inoculation of cells).

[0087] Other reagents: RPMI-1640 medium, X-VIVO™ 10 medium; heat-inactivated human male AB serum (Access Cell Culture (Access Biologicals LLC); fetal bovine serum (FBS); Pen Strep glutamine (100X) (Life Technologies, catalog number 10378, lot number 1881463, expiration date: May 2018); Matrigel basement membrane matrix; Pluronic® F-68, 10% solution.

[0088] Experimental procedure IV Large Model - Randomization: Within 24 hours after cancer cell inoculation, defined as day 1, 20 animals were pseudo-randomized into two groups of 10 based on body weight to achieve similar mean body weights between groups.

[0089] Test article administration: On days 2, 5, 8, 10, 12, and 17, logarithmic-phase expanded CD19t-haNK cells were collected by centrifugation and injected in a volume of 200 μL at 1 × 10 7 Dose of cells / mouse: 5 x 10 for IV administration 7 The cells were formulated in X-VIVO™ 10 at a concentration of 1000 cells / mL. Animals in group A received a vehicle control, while animals in group C received CD19t-haNK cells.

[0090] Body weight: Animals were weighed before tumor cell injection and twice weekly.

[0091] Clinical Observations: Animals were observed daily for mortality / morbidity (G0-G4) and clinical signs of toxicity. Paralyzed or moribund animals were euthanized.

[0092] Euthanasia: Animals were euthanized by CO2 inhalation followed by cervical dislocation. Mortality events (euthanasia or spontaneous) were recorded in the mortality register (Appendix 6), tabulated, and survival curves calculated.

[0093] SC Large Model - Tumor Volume Measurement: After SC tumor injection, animals were examined for tumor establishment at least twice weekly. When tumors became palpable, tumor volume (TV) was measured once or twice weekly with a handheld digital caliper and calculated using the formula: TV = length × width 2 / 2, where length is the maximum diameter of the tumor and width is the minimum diameter of the tumor.

[0094] Randomization: When the mean tumor volume reached an injectable size (in this case 195 mm 3 On day 24 after implantation, 12 tumor-bearing animals were pseudo-randomized into two groups of six to achieve similar tumor volumes between groups. This was defined as day 0.

[0095] Test article administration: On days 1, 4, 7, 9, 11, and 13, logarithmic phase expanded CD19t-haNK cells were collected by centrifugation, γ-irradiated at 1000 cGy, and injected in a volume of 200 μL with 1 × 10 7 Dose of cells / mouse: 5 x 10 for IV administration 7 The cells were formulated in X-VIVO™ 10 medium at a concentration of 1000 cells / mL. As shown in Table 1, animals in group D received a vehicle control, while animals in group F received CD19t-haNK cells.

[0096] Body weight. Animals were weighed before tumor cell injection and then twice weekly.

[0097] Clinical Observations. Animals were observed daily for mortality / morbidity (G0-G4) and clinical signs of toxicity (T1-T12). Paralyzed or moribund animals were euthanized.

[0098] Endpoints and Euthanasia. Moribund animals were euthanized as soon as they showed signs of morbidity, while surviving animals were euthanized as scheduled for tissue collection. Specifically, on Day 13, 6 hours after administration of the final dose of test article, half of the surviving animals (maximum of 3 mice / group) were euthanized. On Day 15, 48 hours after the final dose, the remaining animals were euthanized.

[0099] Necropsy and Tumor and Tissue Collection. At termination, necropsies were performed and organs with visible gross lesions were collected, fixed in 10% formalin, and submitted to a pathology center (Seventh Wave Laboratories) for histological evaluation of tumor / metastatic disease burden.

[0100] [Table 1]

[0101] Data analysis Calculation of tumor volume: tumor volume = length × width 2 / 2 (length and width are the maximum and minimum diameters of the tumor, respectively); Calculation of tumor growth inhibition (TGI): TGI = (TC - Tt) / ΔTC × 100% (where TC and Tt are the mean tumor volumes in the control and treatment groups, respectively, at the end of the study, and ΔTC is the change in mean tumor volume in the control group).

[0102] Statistical Analysis - Tumor growth curves: Tumor growth curves were analyzed by two-way analysis of variance followed by Tukey's test for multiple comparisons. Survival curves: Survival curves were analyzed by the log-rank (Mantel-Cox) test.

[0103] Liver metastasis assessment: Differences in liver metastatic disease burden for individual days were analyzed by unpaired two-tailed t-test. Statistical significance: P<0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism version 7.

[0104] result IV Large Model: The primary readout in the IV tumor model was animal survival. Mortality events were counted when animals were found dead or euthanized due to disease-related morbidity and / or paralysis. As shown in Figure 5, CD19t-haNK cell treatment significantly improved animal survival compared with vehicle control, resulting in a median survival of 27 days compared with 21.5 days in the vehicle control group (P<0.0001).

[0105] Animal weight changes were also monitored throughout the study. As shown in Figure 6, CD19t-haNK-treated animals showed moderate (less than 10%) and short-term weight loss at the initial start of treatment, which is not an uncommon phenomenon in animals receiving IV infusions of NK cells and is not specific to CD19t-haNK cells (reference study: LABC-TX01701). Their weight was able to recover after the first week of treatment before decreasing again due to disease progression.

[0106] SC Large Model: The primary readout in the SC tumor model was tumor growth. As shown in Figure 7, from day 7 onwards, CD19thaNK cells demonstrated clear and statistically significant tumor growth inhibition compared to the vehicle control group, with a TGI of 49% at the end of the study (day 13).

[0107] Furthermore, as Raji is an aggressive lymphoma model, even when inoculated SC, cancer cells were able to disseminate and generate multiple sites of metastasis, ultimately leading to morbidity and / or mortality in the animals. In the vehicle group, a total of three animals (50%) were euthanized due to moribundity between days 11 and 13. In contrast, no unplanned deaths occurred in the CD19t-haNK cell group (Table 3).

[0108] Furthermore, during necropsy, a qualitative reduction in liver metastases was observed in CD19t-haNK-treated animals (Figure 8A). Semiquantitative assessment of disease burden was performed by pathology service (Seventh Wave Laboratories) on representative H&E-stained liver sections. As summarized in Figure 8B and Table 4, a clear trend toward increasing disease burden was observed as the study progressed. The livers of CD19t-haNK-treated animals displayed a significantly lower percentage of cancer infiltrated area compared with vehicle controls. Due to the small sample size and unplanned early deaths in the control group, statistical analysis could only be performed on the data from day 13. This analysis demonstrated a significant difference in disease burden, with mean infiltration of 10% in CD19t-haNK-treated animals compared with 30% in the control group.

[0109] Body weight changes were monitored throughout the study and as can be interpreted from Figure 9, similar to the IV Large model, CD19t-haNK treated animals showed a moderate (less than 10%) and transient weight loss early in the treatment regimen.

[0110] [Table 2]

[0111] [Table 3]

[0112] To evaluate the antitumor efficacy of CD19t-haNK cells in a repeated IV administration regimen, two variations of the large xenograft model with IV and SC tumor inoculation, respectively, were utilized in this study.

[0113] In the IV tumor model, CD19t-haNK cells significantly improved animal survival, extending median survival by 5.5 days (a 26% increase) compared with the vehicle control group. In the SC tumor model, CD19t-haNK cells significantly suppressed tumor growth, resulting in a 49% TGI at the end of the study. Furthermore, CD19t-haNK treatment reduced the number of animal morbidity / mortality events (3 / 6 in the control group vs. 0 / 6 in CD19t-haNK-treated animals) and significantly reduced the metastatic disease burden in the livers of SC large tumor-bearing animals.

[0114] As can be seen from the data above, in both variations of the large xenograft model, CD19t-haNK cells demonstrated significant therapeutic efficacy compared to vehicle controls.

[0115] Example 5. Treatment of L1210 tumor-bearing mice with CD19-CAR-NK-92 cells enhanced survival, and mice that responded completely to treatment rejected the L1210 tumor allograft upon rechallenge. Experimental Design: Thirty male DBA / 2J mice (Jackson Laboratories), 6-8 weeks old, were enrolled on Day 0 after randomization. All animals were housed under standard environmental conditions, maintained on irradiated rodent chow LabDiet 5053, and provided with sterile water ad libitum. Upon arrival, animals were identified by ear punch, housed in cages of 10, and allowed to acclimate to the site for a minimum of 3 days before the start of the study. After acclimatization, the injection area of ​​each mouse was shaved and washed with sterile EtOH swabs. On Day PR0 (Day 0, before randomization), animals were anesthetized with isoflurane for tumor cell injection. On Day PR0, all animals received 2 x 105 L1210-Luc tumor cells were injected subcutaneously (sc) into the right flank in a volume of 0.1 mL of serum-free DMEM. Starting on day 7 of PR, tumors were measured daily with digital calipers in all animals. Approximately 50–150 mm 3 The tumor volume was measured and was approximately 100 mm 3 The mean tumor volume was measured around day 7 of PR, approximately 100 mm 3 Twenty animals with tumors closest to the tumor site were selected; these animals were randomized into two groups of 10 animals each. The randomization date was considered day 0 of the study, and treatment administration began on this day. Animals not enrolled in the study were immediately euthanized by CO2 overdose. Animals in group 1 received vehicle (serum-free DMEM) as an intratumoral (it) injection of 50 μl. Animals in group 2 received 2 × 10 6 mCD19-CAR-aNK cells were administered it in a volume of 50 μl. Identical treatments were administered on days 0, 2, and 4 of the study.

[0116] Animals were weighed daily and monitored for general health. After randomization, tumors were measured with digital calipers three times each week (3x / week). Tumors >2500mm 3 Any animals with tumors that were ulcerating; tumors that had lost >30% of their initial body weight (day 0); or that were found to be moribund, distressed, or paralyzed were euthanized by CO2 overdose and the cause of death / sacrifice was recorded. On day 30, five additional approximately 10-week-old naive male DBA / 2J mice (Jackson Laboratories; Barrier), including complete responders and group 4, received a rechallenge tumor cell inoculum of 2 x 10 5 L1210-Luc tumor cells were administered subcutaneously (sc) in the left flank in a volume of 0.1 mL of serum-free DMEM. All animals continued to be weighed and monitored daily, with tumor measurements taken 3 times per week until day 60.

[0117] result Animal survival to Welfare Threshold - Initial tumor burden: Animals were monitored daily for survival. 3 Animals requiring euthanasia in response to animal health and welfare thresholds, including tumors exceeding 100 μg / kg, inability to obtain food / water, or being found moribund, were included in the survival analysis. Animals requiring euthanasia in response to tumor ulceration were not included in the survival analysis.

[0118] Cumulative survival of animals over time to the welfare threshold is shown in Figure 10. L1210 is an extremely rapidly growing, aggressive tumor cell line, and 0% of vehicle-treated control animals survived longer than 23 days after tumor challenge. In contrast, treatment with CD19-CAR-aNK cells enhanced survival compared to vehicle treatment. Not surprisingly, 25% (2 / 8) of animals treated with CD19-CAR-aNK cells survived from tumor xenograft challenge to the end of the study on day 61.

[0119] The statistical significance of the observed survival enhancement conferred by the test treatment was assessed by log-rank (Mantel-Cox) and Gehan-Breslow-Wilcoxon tests. Treatment with mCD19-CAR-aNK cells resulted in a statistically significant enhancement of survival (p=0.05 (Mantel-Cox); p=0.04 (Gehan-Breslow-Wilcoxon)). These results indicate that in this preclinical subcutaneous model of murine lymphocytic leukemia, treatment with CD19-CAR-aNK resulted in a statistically significant improvement in survival compared to vehicle at the welfare threshold.

[0120] Tumor rechallenge in complete responders: On day 33, two complete responder animals from group 2, along with five age-matched naive animals, received a second inoculum of 2 x 10 5 The mice were challenged / rechallenged with 1000 L1210-Luc cells and injected into the opposite (left) flank (primary tumors were inoculated on the right flank). Animals were monitored daily for survival. 3Animals requiring euthanasia in response to animal health and welfare thresholds, including tumors exceeding 100 μg / kg, inability to obtain food / water, or being found moribund, were included in the survival analysis. Animals requiring euthanasia in response to tumor ulceration were not included in the survival analysis.

[0121] All naive animals eligible for survival analysis (5 of 5) required euthanasia due to tumor volume by day 52; in contrast, all complete responder animals pre-treated with 2 million CD19-CAR-aNK (N=2) cells survived through the end of the study (day 62). The statistical significance of the observed survival enhancement conferred by the study treatment was assessed by log-rank (Mantel-Cox) and Gehan-Breslow-Wilcoxon tests, but the enhancement in survival was not statistically discernible, most likely due to the small sample size.

[0122] Tumors were measured three times weekly (3x / week) during the rechallenge phase. The mean tumor volume + standard error of the mean in each group from administration of L1210-Luc cells for challenge / rechallenge until 0% survival in the control group (day 52) is shown in Figure 11.

[0123] Tumors in naive animals were first detectable approximately 7 days after dosing (study day 40) and grew steadily and rapidly. In contrast, after rechallenge of complete responder animals previously treated with 2 million CD19-CAR-aNK cells, no tumors were detected at any time point throughout the entire rechallenge phase (days 33-61).

[0124] The data presented in this example suggest that complete responder animals pre-treated with 2 million CD19-CAR-aNK cells are able to mount an effective immune response against L1210 tumor cells.

[0125] Example 6: Treatment of A20 tumor-bearing mice with mCD19-CAR-NK-92 cells enhanced survival, and mice that achieved a complete response to treatment rejected the A20 tumor allograft when rechallenged. Experimental design Part A: Forty 5-7 week old BALB / c mice (20 males and 20 females) were provided by Taconic Biosciences to serve in Part A. On pre-randomization (PR) day 0, animals were inoculated with 2.5 × 10 mice in a volume of 100 μL of serum-free medium. 6 A20 mice were injected subcutaneously (sc) with lymphoma cells into the left flank. Tumors were measured daily starting on PR day 7. Ten days after tumor cell implantation (PR day 10; day 0), mice were randomized into treatment groups, with each group containing animals with tumors of similar volume and extent. The day of randomization was considered day 0 of the study. Tumors were measured three times per week (3x / week) with digital calipers until the end of Part A on day 26 to monitor tumor growth.

[0126] On days 0, 3, and 5, mice were injected intratumorally (it) into the tumor mass of each animal with test cells or vehicle in a volume of 50 μl of serum-free medium according to a pre-established it procedure (see Experimental Procedures). 6 mCD19-CAR-NK-92 was administered. Day 26, volume >40mm 3 Animals that did not develop tumors were not enrolled in the study and were euthanized by CO2 asphyxiation; animals that showed a complete response to treatment (CR; tumors >40 mm over several days) were 3 It became undetectable due to the disappearance of 3 Enrolled animals demonstrating a ≥ 1% recurrence rate and no relapse before day 26 were enrolled in Part B.

[0127] Part B: Part B began on day 26. Tumor-free animals from Part A were enrolled in Part B along with 12 naive animals (6 males and 6 females). All animals in Part B received 2.5 x 10 6 A20 cells were administered into the right flank. Tumors were measured twice weekly. Animals were euthanized on day 57.

[0128] Results: Part A - Animal Survival: Animals were monitored daily for general health and survival. 3Animals requiring euthanasia in response to animal health and welfare thresholds, including tumors exceeding 1500 mm, inability to obtain food / water, or being found moribund, were included in the survival analysis. Animals requiring euthanasia in response to tumor ulceration were not included in the survival analysis. In this study, all animals considered in the survival analysis were euthanized within 1500 mm. 3 Animals were euthanized depending on tumor burden exceeding 100 mg / kg / day. Because the subcutaneous tumor burden threshold represents an arbitrary cutoff point, the analysis of "survival" in this case should be viewed solely as an indicator of relative tumor growth. Cumulative survival over time for all animals considered is shown in Figure 12.

[0129] Control animals received vehicle intratumorally (it) on days 1, 3, and 5; 0 of 15 animals (0%) survived to the end of Part A on day 26. Survival was enhanced in all treated animals through day 26: 9 of 18 animals (50%) received 5 million mCD19-CAR-NK92 cells. All groups were compared to each other using the log-rank (Mantel-Cox) test. A statistically significant increase in survival was observed in animals receiving 5 million mCD19-CAR-NK92 cells compared to animals receiving vehicle (p=<0.0001). These results suggest that all treatments improved survival through day 26 compared to vehicle treatment.

[0130] Part B - Tumor rechallenge in complete responders: Animals that have achieved a complete response to treatment (tumors >40 mm responding to treatment over the course of days 0-26 (Part A)) 3 and tumor volume was 0.00 mm by day 26. 3 On day 27, 2.5 × 10 cells were detected in 0.1 mL of serum-free RPMI-1640 medium. 6A20 tumor cells were rechallenged in a second subcutaneous inoculation into the flank (contralateral to the first implant); the rechallenge portion of the study was designated Part B. Twelve additional animals were enrolled in Part B of the study to serve as naive controls; six male and six female age-matched BALB / c mice received at the same time and vendor as the Part A mice received 2.5 x 10 tumor cells on day 27. 6 A20 tumor cells were administered. Tumors were measured three times per week for all animals through day 57. The mean tumor volume + standard error of the mean for each treatment group and naive control in Part A is shown in Figure 13. Tumors from cell inoculation into naive animals grew steadily, as expected; whereas inoculation of complete responder animals with rechallenge tumor cells resulted in viable tumors (>40 mm 3 ) was not brought about.

[0131] In summary, the data presented in this example demonstrate that, in contrast to naive mice, pre-treated mice that fully responded to treatment were able to reject A20 tumor allografts applied as a rechallenge independently of treatment, and suggest that these animals developed a memory response to the tumor antigen.

[0132] The following examples of targeted CAR constructs and associated functional data were derived from vector constructions of linearized DNA that allowed transfected cells to integrate the linearized DNA into their genome, thereby providing a means for non-transient expression of a specific CAR.

[0133] Example 7: HER2-CAR with FcεRIγ signaling domain In this example, the inventors constructed a first-generation CAR with an FcεRIγ signaling domain, comprising an anti-HER2 scFv conjugated to a CD8 hinge, which in turn was conjugated to a CD28 transmembrane domain and conjugated to an FcεRIγ signaling domain. The HER2-CAR thus constructed had the nucleic acid sequence of SEQ ID NO: 60.

[0134] The functionality of the thus constructed HER2.CAR-t-haNK cells against BT-474 cells was tested using a standard cytotoxicity assay, and representative results are shown in Figure 14. As can be readily seen from the data, HER2.CAR-t-haNK cells expressing a CAR with an FcεRIγ signaling domain exhibited significant cytotoxicity against BT-474 target cells.

[0135] In further experiments, the inventors demonstrated expression of HER2.CAR in HER2.CAR-t-haNK cells, as illustrated in Figure 40. The natural cytotoxicity of HER2.CAR-t-haNK cells is shown in the results of Figure 41, while the results for CAR-mediated cytotoxicity are shown in Figure 42. Representative data for ADCC of HER2.CAR-t-haNK cells are shown in the graph of Figure 43.

[0136] Example 8: CD30-CAR with FcεRIγ signaling domain In this example, the inventors constructed a first-generation CAR with an FcεRIγ signaling domain, comprising an anti-CD30 scFv conjugated to a CD8 hinge, which in turn was conjugated to a CD28 transmembrane domain and conjugated to an FcεRIγ signaling domain. The CD30-CAR thus constructed had the nucleic acid sequence of SEQ ID NO: 61.

[0137] Expression of CD30-CAR is shown in the results of Figure 50, while results for natural cytotoxicity of the recombinant cells are shown in Figure 51. CAR-mediated cytotoxicity is shown in the results of Figure 52, while representative results for ADCC are shown in the data of Figure 53.

[0138] Example 9: EGFR-CAR with FcεRIγ signaling domain In this example, the inventors constructed a first-generation CAR with an FcεRIγ signaling domain, comprising an anti-EGFR scFv conjugated to a CD8 hinge, which in turn was conjugated to a CD28 transmembrane domain and conjugated to an FcεRIγ signaling domain. The EGFR-CAR thus constructed had the nucleic acid sequence of SEQ ID NO: 62.

[0139] The functionality of the thus constructed EGFR.CAR-t-haNK cells against A-549 cells was tested using a standard cytotoxicity assay, and representative results are shown in Figure 17. As can be readily seen from the data, EGFR.CAR-t-haNK cells expressing a CAR with an FcεRIγ signaling domain exhibited significant cytotoxicity against A-549 target cells. Expression of EGFR-CAR in EGFR.CAR-t-haNK cells is shown in Figure 35, while the results of natural cytotoxicity are shown in Figure 36. Representative results of CAR-mediated cytotoxicity of EGFR.CAR-t-haNK cells are shown in Figures 37 and 38, while the results of ADCC of EGFR.CAR-t-haNK cells are shown in Figure 39.

[0140] Example 10: IGF1R-CAR with FcεRIγ signaling domain In this example, the inventors constructed a first-generation CAR with an FcεRIγ signaling domain, comprising an anti-IGF1R scFv conjugated to a CD8 hinge, which in turn is conjugated to a CD28 transmembrane domain and conjugated to an FcεRIγ signaling domain. The IGF1R-CAR thus constructed has the nucleic acid sequence of SEQ ID NO: 63 and is capable of binding to IGF1R-CAR, CD16, and IL-2. ER The tricistronic construct encoding has the nucleic acid sequence of SEQ ID NO: 76, which is also depicted diagrammatically in FIG.

[0141] The functionality of the thus constructed IGF1R.CAR-t-haNK cells against MDA-MB-231 cells was tested in comparison with a second-generation CAR (CD28 / CD3z) using a standard cytotoxicity assay, and representative results are shown in Figure 22. As can be readily seen from the data, IGF1R.CAR-t-haNK cells expressing a CAR with an FcεRIγ signaling domain exhibited significant target-specific cytotoxicity against MDA-MB-231 target cells, which was comparable to the cytotoxicity of the second-generation CAR.

[0142] Example 11: CD123-CAR with FcεRIγ signaling domain In this example, the inventors constructed a first-generation CAR with an FcεRIγ signaling domain comprising an anti-CD123 scFv conjugated to a CD8 hinge, which in turn was conjugated to a CD28 transmembrane domain and conjugated to an FcεRIγ signaling domain. The CD123-CAR thus constructed had the nucleic acid sequence of SEQ ID NO: 64. Data on CAR-mediated cytotoxicity of recombinant NK cells expressing CD123-CAR are shown in Figure 48, and representative data on ADCC of recombinant NK cells expressing CD123-CAR are shown in Figure 49.

[0143] Example 12: PD-L1-CAR with FcεRIγ signaling domain In this example, the inventors constructed a first-generation CAR with an FcεRIγ signaling domain, comprising an anti-PD-L1 scFv conjugated to a CD8 hinge, which in turn was conjugated to a CD28 transmembrane domain and conjugated to an FcεRIγ signaling domain. The PD-L1-CAR thus constructed had the nucleic acid sequence of SEQ ID NO:65.

[0144] SUP-B15.PD-L1 in the constructed PD-L1.CAR-t-haNK cells + Functionality against SUP-B15.PD-L1 cells was tested using a standard cytotoxicity assay, and representative results are shown in Figure 16. As can be readily seen from the data, PD-L1.CAR-t-haNK cells expressing a CAR with an FcεRIγ signaling domain were significantly more potent than SUP-B15.PD-L1. + It showed significant cytotoxicity against target cells.

[0145] The functionality of the PD-L1.CAR-t-haNK cells so constructed was further tested against U251 cells using a standard cytotoxicity assay, with representative results shown alongside non-transfected haNK cells in Figure 17. As can be readily seen from the data, PD-L1.CAR-t-haNK cells expressing a CAR with an FcεRIγ signaling domain exhibited significant target-specific cytotoxicity against U251 target cells, while the haNK control cells had virtually no cytotoxicity against the same U251 cells.

[0146] In further experiments on target cell specificity for PD-L1, the inventors tested several PD-L1-positive tumor cell lines using PD-L1.CAR-t-haNK cells, along with haNK cells as a control for general cytotoxicity. As can be easily seen from Figure 24, PD-L1.CAR-t-haNK cells had excellent cytotoxicity across a wide variety of tumor cells (lung, breast, and genitus tumor cells, as well as head and neck small cell carcinoma and chordoma). Notably, PD-L1.CAR-t-haNK cells required less than 4 hours to kill the majority (>85%) of the cells, while control haNK cells required more than 12 hours.

[0147] Figure 24 further illustrates the cytotoxicity of PD-L1.CAR-t-haNK cells against MDA-MB-231 cells compared to various other control cells (haNK cells as indicated). As can be interpreted from the data, lysis of MDA-MB-231 by PD-L1.t-haNK was improved by cetuximab at a 5:1 E:T ratio, and haNK activity was improved by the addition of cetuximab and a-PD-L1. Plain PD-L1.thank had improved cytotoxic activity compared to haNK and haNK + cetuximab, and while plain PD-L1.thank killing was comparable to haNK + PD-L1 antibody, PD-L1.thank + cetuximab was superior to haNK + cetuximab and haNK + PD-L1. At an E:T ratio of 1:1, PD-L1.thaNK activity was similar in the presence or absence of cetuximab, PD-L1.thaNK was significantly superior to endogenous hank.haNK activity, and ADCC-mediated killing by hank.haNK activity was improved by the addition of cetuximab and a-PD-L1.

[0148] In further experiments, the inventors demonstrated expression of PD-L1.CAR in PD-L1.CAR-t-haNK cells, as depicted in Figure 44. The natural cytotoxicity of PD-L1.CAR-t-haNK cells is shown in the results in Figure 45, while the results for CAR-mediated cytotoxicity are shown in Figure 46. Representative data for ADCC of PD-L1.CAR-t-haNK cells are shown in the graph in Figure 47.

[0149] Example 13: CD33-CAR with FcεRIγ signaling domain In this example, the inventors constructed a first-generation CAR with an FcεRIγ signaling domain, comprising an anti-HER2 scFv conjugated to a CD8 hinge, which in turn was conjugated to a CD28 transmembrane domain and conjugated to an FcεRIγ signaling domain. The CD33.CAR thus constructed had the nucleic acid sequence of SEQ ID NO: 66.

[0150] The functionality of the constructed CD33.CAR-t-haNK cells against THP-1 cells was tested using a standard cytotoxicity assay, and representative results are shown in Figure 15. As can be readily seen from the data, CD33.CAR-t-haNK cells expressing a CAR with an FcεRIγ signaling domain exhibited significant cytotoxicity against THP-1 target cells. Further data demonstrating the strong expression of CD33CAR in NK-92 cells is presented in Figure 31. The natural cytotoxicity of CD33.CAR-t-haNK cells against K562 cells is shown in Figure 32, and Figure 33 presents the results for CAR-mediated cytotoxicity against THP-1 cells. Figure 34 shows the results of the SUP-B15 CD19 cytotoxicity with rituximab. KO / CD20 + Further results are shown for ADCC of CD33.CAR-t-haNK cells against IgG4-associated leukemia virus ( ...

[0151] Example 14: gp120-CAR with FcεRIγ signaling domain In this example, the inventors constructed a first-generation CAR with an FcεRIγ signaling domain comprising an anti-gp120 scFv conjugated to a CD8 hinge, which in turn was conjugated to a CD28 transmembrane domain and conjugated to an FcεRIγ signaling domain. The gp120-CAR thus constructed had the nucleic acid sequence of SEQ ID NO: 67.

[0152] The inventors further demonstrated that the cells so generated express significant amounts of CD16 and gp120CAR, as can be seen in Figure 57. Binding of GP120 to gp120CAR was demonstrated against non-recombinant aNK cells as a negative control, as shown in Figure 58. The natural cytotoxicity of the cells so generated is shown in Figure 59, while the corresponding ADCC data is shown in Figure 60.

[0153] Example 15: B7-H4-CAR with FcεRIγ signaling domain In this example, the inventors constructed a first-generation CAR with an FcεRIγ signaling domain, comprising an anti-B7-H4 scFv conjugated to a CD8 hinge, which in turn was conjugated to a CD28 transmembrane domain and conjugated to an FcεRIγ signaling domain. The B7-H4-CAR thus constructed had the nucleic acid sequence of SEQ ID NO: 68.

[0154] Example 16: BCMA-CAR with FcεRIγ signaling domain In this example, the inventors constructed a first generation CAR with an FcεRIγ signaling domain comprising an anti-BCMA scFv conjugated to a CD8 hinge, which in turn was conjugated to a CD28 transmembrane domain, and conjugated to an FcεRIγ signaling domain. The BCMA-CAR so constructed had the nucleic acid sequence of SEQ ID NO: 69.

[0155] BCMA expression was confirmed as shown in the representative results in Figure 54, and CAR-mediated cytotoxicity against target cells was demonstrated as shown in Figure 55. Similarly, as can be seen from the results in Figure 56, the recombinant cells had significant ADCC using rituximab as the antibody against the target cells.

[0156] Example 17: GD2-CAR with FcεRIγ signaling domain In this example, the inventors constructed a first-generation CAR with an FcεRIγ signaling domain, comprising an anti-GD2 scFv conjugated to a CD8 hinge, which in turn was conjugated to a CD28 transmembrane domain and conjugated to an FcεRIγ signaling domain. The GD2-CAR thus constructed had the nucleic acid sequence of SEQ ID NO: 70.

[0157] Example 18: FAP-CAR with FcεRIγ signaling domain In this example, the inventors constructed a first-generation CAR with an FcεRIγ signaling domain, comprising an anti-FAP scFv conjugated to a CD8 hinge, which in turn was conjugated to a CD28 transmembrane domain and conjugated to an FcεRIγ signaling domain. The FAP-CAR constructed in this manner had the nucleic acid sequence of SEQ ID NO: 71. The expression of the FAP-CAR is shown in the data in Figure 61, and the cytotoxicity of the FAP.CAR against target cells is shown in the results in Figure 62.

[0158] Example 19: CD20-CAR with FcεRIγ signaling domain In this example, the inventors constructed a first-generation CAR with an FcεRIγ signaling domain comprising an anti-CD20 scFv conjugated to a CD8 hinge, which in turn was conjugated to a CD28 transmembrane domain and conjugated to an FcεRIγ signaling domain. The CD20-CAR thus constructed had the nucleic acid sequence of SEQ ID NO:74.

[0159] The expression of CD20 CAR in NK-92 cells is shown in the results in Figure 29. As can be readily seen, CD20.CAR (along with CD16 from linearized DNA as described above) is strongly expressed in the majority of recombinant cells. Figure 30 shows the expression of CD20 + Representative results are shown for the cytotoxicity of CD20.CAR NK cells against target cells.

[0160] Example 20: CSPG-4-CAR with FcεRIγ signaling domain In this example, the inventors constructed a first-generation CAR with an FcεRIγ signaling domain, comprising an anti-CSPG-4 scFv conjugated to a CD8 hinge, which in turn was conjugated to a CD28 transmembrane domain and conjugated to an FcεRIγ signaling domain. The CSPG-4-CAR constructed in this way had the nucleic acid sequence of SEQ ID NO: 75. Expression of the CSPG-4-CAR was confirmed by FACS analysis, and representative results are shown in Figure 63. As shown in the representative data in Figure 64, the cells constructed in this way also exhibited significant cytotoxicity.

[0161] Example 21: CD19-CAR with FcεRIγ signaling domain In this example, the inventors used a first generation CAR as described above with an FcεRIγ signaling domain comprising an anti-CD19 scFv coupled to a CD8 hinge, which in turn is coupled to a CD28 transmembrane domain, and coupled to an FcεRIγ signaling domain, and transfected linearized DNA into NK-92 cells for functional testing.

[0162] To determine general cytotoxicity, the functionality of the constructed CD19.CAR-t-haNK cells against K562 cells was tested using a standard cytotoxicity assay. Representative results are shown in Figure 19. As can be seen, CD19.CAR-t-haNK cells expressing a CAR with an FcεRIγ signaling domain exhibited significant cytotoxicity against K562 target cells. In a further set of experiments, target-specific cytotoxicity was assessed using SUP-B15 cells compared with aNK cells as a control. Representative results are shown in Figure 20. Again, CD19.CAR-t-haNK cells expressing a CAR with an FcεRIγ signaling domain exhibited significant target-specific cytotoxicity. In yet another set of experiments, target-specific ADCC was assessed using SKBr3 cells with Herceptin and Rituxan as antibodies. Representative results are shown in Figure 21. Again, CD19.CAR-t-haNK cells expressing a CAR with an FcεRIγ signaling domain exhibited significant antibody- and target-specific ADCC. Notably, the doubling time of the recombinant NK cells was virtually identical to that of aNK cells.

[0163] Figure 25 shows the CD16 and IL-2 expression in NK-92 cells relative to controls. ERFigure 25 exemplarily illustrates the expression of CD19.CAR from linearized DNA containing a segment encoding the CAR. As can be seen from Figure 25, the expression was very strong across the majority of cells. Further results on the natural cytotoxicity of CD19.CAR t-haNK cells against K562 cells and targeted cytotoxicity against SUP-B15 cells are presented in Figures 26 and 27. SUP-B15CD19 of CD19.CAR t-haNK cells KO / CD20 + Further representative results for ADCC against cells are shown in FIG.

[0164] Example 22: Antitumor activity of PD-L1-targeted t-haNK cells in a human xenograft model in NSG mice MDA-MB-231 and HCC827 were used as validated PD-L1-positive xenograft models to evaluate the efficacy, dose levels, and routes of administration (IV and IT) of PD-L1 t-haNK cells in different formulations.

[0165] Animals: Animal type: NSG mice (JAX), female, 9-10 weeks old; number of animals in the MDA-MB-231 model: 24 (fresh cells), and number of animals in the HCC827 model: 24 (fresh cells) + 6 (cryopreserved cells). The tumor models used cell lines: MDA-MB-231 (human breast adenocarcinoma) and HCC827 (human lung adenocarcinoma). The inoculation route was subcutaneous in both flanks. The average tumor volume at the start of treatment was approximately 100 mm for MDA-MB-231. 3 , HCC827: about 75-80 mm 3 It was.

[0166] Treatment: Freshly prepared, irradiated anti-PD-L1 t-haNK at a concentration of 5E7 cells / mL or 2E7 cells / mL; vehicle control was X-VIVO™ 10 medium; administration methods were IV and IT as described above. The dose for IV NK administration was 1E7 cells / dose (freshly prepared cells) in 200 μL or 4E6 cells / dose (cryopreserved cells) in 200 μL; the dose for IT NK administration (fresh cells only) was 2.5E6 cells / tumor / dose in 50 μL. Dosing frequency was twice weekly for 4 consecutive weeks (M / Th or T / F), with the first day of administration defined as day 1.

[0167] The study design for MDA-MB-231 is shown in Table 4 below (this study was performed with some animals in groups A, C, and D growing >2000 mm 3 The treatment was terminated on day 27 when the combined tumor volume had reached 100 mg / kg.

[0168] [Table 4]

[0169] The study design for HCC827 is shown in Table 5 below (the study was terminated on day 29 when surviving animals were repurposed and transferred to another study).

[0170] [Table 5]

[0171] Results: Freshly prepared PD-L1 t-haNK cells (1E7 cells / dose) produced significant and long-lasting tumor growth inhibition in both MDA-MB-231 and HCC827 models.

[0172] MDA-MB-231: Tumor stagnation: TGI on day 16: 84% (peak); TGI on day 26: 79% (final measurement).

[0173] HCC827: Tumor regression: TGI at day 16: 120% (peak); TGI at day 29: 84% (end of study).

[0174] Cryopreserved PD-L1 t-haNK cells (4E6 cells / dose) also demonstrated statistically significant efficacy in inhibiting tumor growth compared to X-VIVO™ 10 medium: TGI on day 26: 60% (peak) and TGI on day 29: 40% (end of study).

[0175] Freshly prepared PD-L1 t-haNK cells (1E7 cells / dose) also resulted in a significant reduction in metastatic disease burden in the MDA-MB-231 model, as shown in Table 6 below.

[0176] [Table 6]

[0177] Number of visible nodules in the liver present in vehicle: 29±9, in PD-L1 t-haNK group: 0 (P=0.0116 by unpaired two-tailed t-test).

[0178] Based on the experiments performed, IV administration of freshly prepared PD-L1 t-haNK cells at a dose level of 1E7 cells / dose twice weekly for 4 weeks demonstrated significant anti-tumor efficacy in both subcutaneous xenograft models tested: treatment led to tumor stasis in MDA-MB-231 tumor-bearing mice, with a peak TGI of 84% on day 16 and a TGI of 79% at the end of the study (P<0.0001 for both time points by two-way ANOVA followed by Tukey's test for multiple comparisons), and tumor regression in the HCC827 model, with a peak TGI of 120% on day 16 and a TGI of 84% at the end of the study (P<0.0001). Cryopreserved PD-L1 t-haNK cells administered IV twice weekly for 4 weeks at a dose level of 4E6 cells / dose also demonstrated significant therapeutic efficacy in the HCC827 tumor model, achieving a peak TGI of 60% (P<0.0001) and an end-of-study TGI of 40% (P<0.01). Freshly prepared PD-L1 t-haNK cells administered IT twice weekly for 4 weeks at a dose level of 2.5E6 cells / dose / tumor effectively suppressed HCC827 tumor growth, resulting in a peak TGI of 70% at day 20 and an end-of-study TGI of 49% (P<0.001).

[0179] Significant adverse reactions were observed in animals receiving IV administration of freshly prepared PD-L1 t-haNK cells (1E7 cells / dose). In contrast to freshly prepared PD-L1 t-haNK cells, cryopreserved cells (administered at a lower level of 4E6 cells / dose) were found to be safe for animals after IV administration. PD-L1 t-haNK cells demonstrated remarkable efficacy in two subcutaneous tumor models. Cryopreserved cells administered at the lower level of 4E6 cells / dose also demonstrated significant efficacy in inhibiting tumor growth and were found to be safe for animals.

[0180] It should, of course, be understood that for every nucleic acid sequence provided herein, the corresponding encoded protein is also expressly contemplated herein, and similarly, for every amino acid sequence, the corresponding nucleic acid sequence (with any codon usage) is also contemplated herein.

[0181] All patent applications, publications, references, and sequence accession numbers cited herein are hereby incorporated by reference in their entirety.

[0182] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0183] In this specification and in the claims that follow, reference will be made to a number of terms which shall be defined to have the following meanings.

[0184] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0185] It is understood that all numerical values ​​(including ranges, e.g., pH, temperature, time, concentration, amount, and molecular weight) set forth herein include normal variations in measurements encountered by those of ordinary skill in the art. Thus, numerical values ​​set forth herein include variations of + / - 0.1 to 10%, e.g., + / - 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. It is to be understood, although not always explicitly stated, that all numerical designations may be preceded by the term "about." Thus, the term about includes variations of + / - 0.1 to 10%, e.g., + / - 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the numerical value. It is also to be understood, although not always explicitly stated, that the reagents set forth herein are exemplary only and that equivalents of such are known in the art.

[0186] As will be understood by those of skill in the art, for all purposes, particularly in terms of presenting a specification, all ranges disclosed herein include the endpoints of the range and include all values ​​between the endpoints. All ranges disclosed herein also encompass any and all possible subranges and combinations thereof. Any recited range can be readily understood as fully descriptive and allowing for the range to be broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third, upper third, etc. As will further be understood by those of skill in the art, any term such as "up to," "at least," etc., refers to a range that is inclusive of the recited numbers and can be subsequently broken down into subranges as discussed above. Finally, as will be understood by those of skill in the art, a range includes each individual member. Thus, for example, a group having 1 to 3 cells refers to a group having 1, 2, or 3 cells. Similarly, for example, a group having 1 to 5 cells refers to a group having 1, 2, 3, 4, or 5 cells.

[0187] It is also to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0188] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes examples when the event or circumstance occurs and examples when it does not occur.

[0189] The term "comprising" is intended to mean that compositions and methods include the recited elements, but do not exclude others. "Consisting essentially of," when used to define compositions and methods, is intended to mean excluding other elements of any essential significance to the combination. For example, a composition consisting essentially of elements as defined herein would not exclude other elements that do not materially affect the basic and novel characteristics of the claimed invention. "Consisting of" is intended to mean excluding more than trace amounts of other ingredients, and that substantial method steps are recited. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0190] As used herein, "immunotherapy" refers to the use of NK-92 cells (modified or unmodified), naturally occurring or modified NK cells, or T cells, either alone or in combination, that are capable of inducing cytotoxicity when contacted with target cells.

[0191] As used herein, "natural killer (NK) cells" are cells of the immune system that are not restricted by major histocompatibility complex (MHC) class and kill target cells in the absence of specific antigenic stimulation. Target cells may be tumor cells or virus-carrying cells. NK cells are characterized by the presence of CD56 and the absence of CD3 surface markers.

[0192] The term "endogenous NK cells" is used to refer to NK cells derived from a donor (or patient), as distinguished from the NK-92 cell line. Endogenous NK cells are generally a heterogeneous population of cells that are enriched in NK cells. Endogenous NK cells may be intended for autologous or allogeneic therapy of a patient.

[0193] The term "NK-92" refers to natural killer cells derived from a highly potent, unique cell line (the rights of which are owned by NantKwest) described by Gong et al. (1994) (hereinafter "NK-92™ cells"). The immortal NK cell line was originally obtained from a patient with non-Hodgkin's lymphoma. Unless otherwise specified, the term "NK-92™" is intended to refer to the original NK-92 cell line, as well as NK-92 cell lines that have been modified (e.g., by the introduction of an exogenous gene). NK-92™ cells, and representative and non-limiting modifications thereof, are described in U.S. Patent Nos. 7,618,817; 8,034,332; 8,313,943; 9,181,322; 9,150,636; and published U.S. patent application Ser. No. 10 / 008,955 (all of which are incorporated by reference in their entirety), and include wild-type NK-92™, NK-92™-CD16, NK-92™-CD16-γ, NK-92™-CD16-ζ, NK-92™-CD16(F176V), NK-92™ MI, and NK-92™ CI. NK-92 cells are known to those of skill in the art, and such cells are readily available from NantKwest, Inc.

[0194] The term "aNK" refers to unmodified natural killer cells derived from the highly potent and unique cell line described in Gong et al. (1994), the rights of which are owned by NantKwest (hereinafter "aNK™ cells"). The term "haNK" refers to natural killer cells derived from the highly potent and unique cell line described in Gong et al. (1994), the rights of which are owned by NantKwest (hereinafter "CD16+NK-92™ cells" or "haNK® cells"). In some embodiments, CD16+NK-92™ cells comprise the high-affinity CD16 receptor on the cell surface. The term "taNK" refers to natural killer cells derived from the highly potent and unique cell line described in Gong et al. (1994) that have been modified to express a chimeric antigen receptor, the rights of which are owned by NantKwest (hereinafter "CAR-modified NK-92™ cells" or "taNK® cells"). The term "t-haNK" refers to natural killer cells derived from the highly potent and unique cell line described in Gong et al. (1994) that have been modified to express a chimeric antigen receptor on the cell surface, the rights of which are owned by NantKwest (hereinafter "CAR-modified CD16+ NK-92™ cells" or "t-haNK™ cells"). In some embodiments, t-haNK™ cells express the high-affinity CD16 receptor on the cell surface.

[0195] "Modified NK-92 cells" refer to NK-92 cells that express an exogenous gene or protein, e.g., an Fc receptor, a CAR, a cytokine (such as IL-2 or IL-12), and / or a suicide gene. In some embodiments, the modified NK-92 cells comprise a vector encoding a transgene, e.g., an Fc receptor, a CAR, a cytokine (such as IL-2 or IL-12), and / or a suicide gene. In one embodiment, the modified NK-92 cells express at least one transgenic protein.

[0196] As used herein, "non-irradiated NK-92 cells" are NK-92 cells that have not been irradiated. Irradiation renders the cells incapable of growth and proliferation. Because the time between irradiation and infusion must be no more than four hours for optimal activity, it is contemplated that the NK-92 cells may be irradiated at a treatment facility or some other location prior to patient treatment. Alternatively, the NK-92 cells may be prevented from proliferating by another mechanism.

[0197] As used herein, "inactivation" of NK-92 cells renders the cells incapable of proliferation. Inactivation can also refer to the death of NK-92 cells. It is envisioned that NK-92 cells may be inactivated after effectively purging an ex vivo sample of cells associated with a pathology in therapeutic applications, or after they have resided within a mammalian body for a sufficient period of time to effectively kill many or all target cells present within the body. Inactivation may be induced, by way of a non-limiting example, by administering an inactivating agent to which the NK-92 cells are sensitive.

[0198] As used herein, the terms "cytotoxic" and "cytolytic" are intended to be synonymous when used to describe the activity of effector cells, such as NK-92 cells. Generally, cytotoxic activity relates to the killing of target cells by any of a variety of biological, biochemical, or biophysical mechanisms. Cytolysis more specifically relates to the activity of an effector cell to lyse the plasma membrane of a target cell, thereby disrupting its physical integrity, resulting in the killing of the target cell. Without wishing to be bound by theory, it is believed that the cytotoxic effect of NK-92 cells is due to cytolysis.

[0199] The term "killing" in relation to a cell / cell population is intended to include any type of manipulation that will result in the death of the cell / cell population.

[0200] The term "Fc receptor" refers to a protein found on the surface of certain cells (e.g., natural killer cells) that contributes to the protective function of immune cells by binding to a portion of an antibody known as the Fc region. Binding of the Fc region of an antibody to a cell's Fc receptor (FcR) stimulates the cell's phagocytic or cytotoxic activity via antibody-mediated phagocytosis or antibody-dependent cell-mediated cytotoxicity (ADCC). FcRs are classified based on the type of antibody they recognize. For example, Fc-γ receptors (FCγR) bind to the IgG class of antibodies. FcγRIII-A (also known as CD16; SEQ ID NO: 20) is a low-affinity Fc receptor that binds to IgG antibodies and activates ADCC. FcγRIII-A is typically found on NK cells. NK-92 cells do not express FcγRIII-A. Fc-ε receptors (FcεR) bind to the Fc region of IgE antibodies.

[0201] The term "chimeric antigen receptor" (CAR), as used herein, refers to an extracellular antigen-binding domain fused to an intracellular signaling domain. CARs can be expressed in T cells or NK cells to enhance cytotoxicity. Typically, the extracellular antigen-binding domain is an scFv specific for an antigen found on the target cell. NK-92 cells expressing CARs target cells expressing a specific antigen on their cell surface based on the specificity of the scFv domain. The scFv domain can be engineered to recognize any antigen, including tumor-specific antigens and virus-specific antigens. For example, CD19CAR recognizes CD19, a cell surface marker expressed by some cancers.

[0202] The term "tumor-specific antigen," as used herein, refers to an antigen that is presented on cancer or neoplastic cells but is undetectable on normal cells derived from the same tissue or lineage as the cancer cells. Tumor-specific antigen, as used herein, also refers to tumor-associated antigens, i.e., antigens that are expressed at higher levels on cancer cells compared to normal cells derived from the same tissue or lineage as the cancer cells.

[0203] The term "virus-specific antigen" as used herein refers to an antigen that is presented on a virus-infected cell but is undetectable on a normal cell derived from the same tissue or lineage as the virus-infected cell. In one embodiment, the virus-specific antigen is a viral protein expressed on the surface of the infected cell.

[0204] The terms "polynucleotide," "nucleic acid," and "oligonucleotide" are used interchangeably and refer to polymeric forms of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: genes or gene fragments (e.g., probes, primers, ESTs, or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polynucleotide. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides may be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to both double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention that is a polynucleotide encompasses both the double-stranded form and each of the two complementary single-stranded forms that are known or predicted to form the double-stranded form.

[0205] A polynucleotide consists of a specific sequence of the four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U), which corresponds to thymine when the polynucleotide is RNA. Thus, the term "polynucleotide sequence" is the alphabetical representation of a polynucleotide molecule.

[0206] "Homology" or "identity" or "similarity" refers to sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing a position within each sequence, which may be aligned for purposes of comparison. When a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences.

[0207] As used herein, "percent identity" refers to the sequence identity between two peptides or two nucleic acid molecules. Percent identity can be determined by comparing a position within each sequence, which may be aligned for purposes of comparison. When a position within the compared sequences is occupied by the same base or amino acid, the molecules are identical at that position. Homologous nucleotide sequences include sequences that encode naturally occurring allelic variants and mutations of the nucleotide sequences set forth herein. Homologous nucleotide sequences include nucleotide sequences that encode proteins of mammalian species other than humans. Homologous amino acid sequences include amino acid sequences with conservative amino acid substitutions, where the polypeptide has the same binding and / or activity. In some embodiments, homologous amino acid sequences have no more than 15, no more than 10, no more than 5, or no more than 3 conservative amino acid substitutions. In some embodiments, a nucleotide or amino acid sequence has at least 60%, at least 65%, at least 70%, at least 80%, or at least 85%, or more, percent identity to a sequence set forth herein. In some embodiments, the nucleotide or amino acid sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a sequence described herein. Percent identity can be determined, for example, using the Smith and Waterman algorithm with default settings in the Gap program (Wisconsin Sequence Analysis Package, Version 8 for UNIX, Genetics Computer Group, University Research Park, Madison, Wis.) (Adv. Appl. Math., 1981, 2, 482-489). Suitable algorithms for determining percent sequence identity include the BLAST and BLAST 2.0 algorithms described by Altschul et al. (Nuc. Acids Res. 25:3389-402, 1977) and Altschul et al. (J. Mol. Biol. 215:403-10, 1990), respectively.Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (see internet ncbi.nlm.nih.gov). The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length of 3, and an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989) of alignments (B) of 50, an expectation (E) of 10, M=5, N=-4.

[0208] In some embodiments, nucleic acid sequences are codon-optimized for expression in a particular species, for example, a mouse sequence can be codon-optimized for expression in humans (expression of the protein encoded by the codon-optimized nucleic acid sequence). Thus, in some embodiments, the codon-optimized nucleic acid sequence has at least 60%, at least 65%, at least 70%, at least 80%, or at least 85% or greater percent identity to a nucleic acid sequence described herein. In some embodiments, the codon-optimized nucleic acid sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a sequence described herein.

[0209] The term "express" refers to the production of a gene product (e.g., a protein). The term "transient," when referring to expression, means that a polynucleotide is not integrated into the genome of the cell. The term "stable," when referring to expression, means that a polynucleotide is integrated into the genome of the cell or that a positive selection marker (i.e., an exogenous gene expressed by the cell that benefits under certain growth conditions) is utilized to maintain expression of the transgene.

[0210] The term "cytokine" or "cytokines" refers to a general class of biomolecules that affect cells of the immune system. Exemplary cytokines include, but are not limited to, interferons and interleukins (IL), particularly IL-2, IL-12, IL-15, IL-18, and IL-21. In a preferred embodiment, the cytokine is IL-2.

[0211] As used herein, the term "vector" refers to a non-chromosomal nucleic acid containing an intact replicon so that the vector can replicate when placed into a permissive cell, for example, by the process of transformation. A vector may replicate in one cell type, such as bacteria, but have little or no ability to replicate in another cell, such as mammalian cells. A vector may be viral or non-viral. Representative non-viral vectors for delivering nucleic acids include naked DNA; DNA complexed with cationic lipids alone or in combination with cationic polymers; anionic and cationic liposomes; DNA-protein complexes and particles containing DNA condensed with cationic polymers such as heterogeneous polylysine, defined-length oligopeptides, and polyethyleneimine, optionally contained in liposomes; and ternary complexes containing virus and polylysine-DNA. In one embodiment, the vector is a viral vector, such as an adenovirus. Viral vectors are well known in the art.

[0212] As used herein, the term "targeted," when referring to protein expression, is intended to include, but is not limited to, directing a protein or polypeptide to its appropriate intracellular or extracellular destination. Targeting is typically achieved through a signal peptide or targeting peptide, which is a stretch of amino acid residues within the polypeptide chain. These signal peptides can be located anywhere within the polypeptide sequence but are often located at the N-terminus. Polypeptides can also be modified to have a signal peptide at the C-terminus. A signal peptide can direct a polypeptide to an extracellular area located relative to the plasma membrane, Golgi, endosome, endoplasmic reticulum, and other cellular compartments. For example, polypeptides with a particular amino acid sequence (e.g., KDEL) at the C-terminus are retained in or re-transported back to the ER lumen.

[0213] As used herein, the term "target," when referring to tumor targeting, refers to the ability of NK-92 cells to recognize and kill tumor cells (i.e., target cells). The related term "targeted," for example, refers to the ability of a CAR expressed by an NK-92 cell to recognize and bind to a cell surface antigen expressed by a tumor.

[0214] As used herein, the term "transfect" refers to the insertion of a nucleic acid into a cell. Transfection may be performed using any means that allows the nucleic acid to enter the cell. DNA and / or mRNA may be transfected into the cell. Preferably, the transfected cell expresses the gene product (i.e., protein) encoded by the nucleic acid.

[0215] The term "suicide gene" refers to a transgene that allows for negative selection of cells expressing the transgene. Suicide genes are used as a safety system that allows cells expressing the gene to be killed by the introduction of a selective drug. Several suicide gene systems have been identified, including the herpes simplex virus thymidine kinase (TK) gene, cytosine deaminase gene, varicella-zoster virus thymidine kinase gene, nitroreductase gene, Escherichia coli gpt gene, and E. coli Deo gene (see, e.g., Yazawa K, Fisher WE, Brunicardi FC: Current progress in suicide gene therapy for cancer. World J. Surg. 2002 July;26(7):783-9). In one embodiment, the suicide gene is a thymidine kinase (TK) gene. The TK gene may be a wild-type or mutant TK gene (e.g., tk30, tk75, sr39tk). Cells expressing the TK protein can be killed using ganciclovir.

Claims

1. Encoded on the transfected recombinant plasmid and in a single polypeptide chain, Extracellular binding domain, hinge domain, transmembrane domain, and FcεRIγ signaling domain A genetically modified NK cell, which is an NK-92 cell, having a membrane-bound recombinant chimeric antigen receptor (CAR) comprising:

2. The genetically modified NK cell of claim 1 , wherein the extracellular binding domain comprises an scFv.

3. The genetically modified NK cell according to claim 1 or 2, wherein the extracellular binding domain specifically binds to a tumor-specific antigen, a tumor-associated antigen, or a patient- and tumor-specific antigen.

4. The genetically modified NK cell according to claim 3, wherein the tumor-specific antigen is CD19, CD20, GD2, HER-2, CD30, EGFR, FAP, CD33, CD123, PD-L1, IGF1R, CSPG4, or B7-H4.

5. The genetically modified NK cell according to claim 1 or 2, wherein the extracellular binding domain specifically binds to a virus-specific antigen.

6. The genetically modified NK cell according to claim 5 , wherein the virus-specific antigen is an antigen of HIV virus, HPV virus, RSV virus, influenza virus, Ebola virus, or HCV virus.

7. The genetically modified NK cell according to claim 5, wherein the virus-specific antigen is gp120 of the HIV virus.

8. The genetically modified NK cell according to any one of claims 1 to 7, wherein the hinge domain and / or the transmembrane domain comprises a CD8 hinge domain and / or a CD28 transmembrane domain.

9. The genetically modified NK cell of any one of claims 1 to 8, wherein the FcεRIγ signaling domain has the amino acid sequence of SEQ ID NO:

1.

10. The genetically modified NK cell according to any one of claims 1 to 9, further comprising membrane-bound recombinant CD16.

11. The genetically modified NK cell according to any one of claims 1 to 10, further comprising a recombinant cytokine having an endoplasmic reticulum retention sequence.

12. comprising a recombinant nucleic acid encoding a chimeric antigen receptor (CAR), wherein the recombinant nucleic acid is a transfecting plasmid; wherein the CAR comprises an extracellular binding domain, a hinge domain, a transmembrane domain, and an FcεRIγ signaling domain in a single polypeptide chain. NK-92 cells, genetically modified NK cells.

13. The genetically modified NK cell of claim 12 , wherein the recombinant nucleic acid is RNA.

14. The genetically modified NK cell of claim 13, wherein the RNA is a polycistronic RNA further encoding CD16 and / or a cytokine having an endoplasmic reticulum retention sequence.

15. The genetically modified NK cell according to any one of claims 12 to 14, wherein the extracellular binding domain comprises an scFv.

16. The genetically modified NK cell according to any one of claims 12 to 15, wherein the extracellular binding domain specifically binds to a tumor-specific antigen, a tumor-associated antigen, or a patient- and tumor-specific antigen.

17. The genetically modified NK cell of claim 16, wherein the tumor-specific antigen is CD19, CD20, GD2, HER-2, CD30, EGFR, FAP, CD33, CD123, PD-L1, IGF1R, CSPG4, or B7-H4.

18. The genetically modified NK cell according to any one of claims 12 to 15, wherein the extracellular binding domain specifically binds to a virus-specific antigen.

19. The genetically modified NK cell according to claim 18, wherein the virus-specific antigen is an antigen of HIV virus, HPV virus, RSV virus, influenza virus, Ebola virus, or HCV virus.

20. The genetically modified NK cell according to claim 18, wherein the virus-specific antigen is gp120 of the HIV virus.

21. The genetically modified NK cell according to any one of claims 12 to 20, wherein the hinge domain and / or the transmembrane domain comprises a CD8 hinge domain and / or a CD28 transmembrane domain.

22. The genetically modified NK cell of any one of claims 12 to 21, wherein the FcεRIγ signaling domain has the nucleic acid sequence of SEQ ID NO:

2.

23. 23. A method of treating cancer in a patient in need thereof, comprising the step of administering to said patient a therapeutically effective amount of any one of the genetically modified NK cells of claims 1 to 22, thereby treating said cancer.

24. 24. The method of claim 23, further comprising administering at least one additional therapeutic entity selected from the group consisting of a viral cancer vaccine, a bacterial cancer vaccine, a yeast cancer vaccine, N-803, an antibody, a stem cell transplant, and a tumor-targeting cytokine.

25. The cancer may be leukemia, acute lymphocytic leukemia, acute myeloid leukemia, chronic leukemia, chronic myeloid (granulocytic) leukemia, chronic lymphocytic leukemia, polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's disease, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, solid tumors, including, but not limited to, sarcomas and carcinomas, such as fibrosarcoma, myxoid sarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordal sarcoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovial sarcoma, mesothelial sarcoma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, 25. The method of claim 23 or 24, wherein the cancer is selected from 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, liver tumor, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytic tumor, medulloblastic tumor, craniopharyngeal tumor, ependymal tumor, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

26. 21. A method of treating a viral infection in a patient in need thereof, comprising administering to said patient a therapeutically effective amount of any one of the genetically modified NK cells of claims 1, 5, 6, 7, 12, 18, 19, or 20, thereby treating said viral infection.

27. 27. The method of claim 26, further comprising administering an antiviral agent.

28. 1 m of the patient's body surface area ２ Approximately 1 x 10 ８ ~Approx. 1×10 １１ 28. The method of any one of claims 23 to 27, wherein cells are administered to the patient.

29. Use of the genetically modified NK cells according to any one of claims 12 to 22 in the treatment of cancer or viral infection.

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