Chimeric antigen receptor modified NK-92 cell

Abstract

The invention relates to an NK-92 cell modified by a chimeric antigen receptor. NK-92 cells that express a chimeric antigen receptor (CAR) are provided. The CAR may comprise an intracellular domain of Fc [epsilon] RI [gamma]. Also described are methods for treating a patient suffering from or suspected of suffering from a disease that can be treated with NK-92 cells, such as cancer or a viral infection, comprising administering to the patient NK-92-CAR cells.

Description

[0001] This application claims priority to our co-pending U.S. provisional applications Serial Nos. 62 / 756,395 and 62 / 756,402, both filed on November 6, 2018.

[0002] Sequence Listing

[0003] The ASCII text file of the sequence listing is named 104077.0003PCT5 Sequence Listing_ST25, is 134kb in size, was created on May 20, 2019, and was submitted electronically with the application through EFS-Web, the contents of which are incorporated by reference in their entirety. Technical Field

[0004] The field of the invention is genetically modified immune competent cells expressing a chimeric antigen receptor (CAR), in particular modified NK-92 cells expressing a CAR having an Fcε receptor γ (FcεRIγ) signaling domain. Background Art

[0005] Natural killer (NK) cells are cytotoxic lymphocytes that constitute an important component of the innate immune system. In most cases, NK cells comprise approximately 10%-15% of circulating lymphocytes and bind to and kill target cells, which include virally infected cells and many malignant cells. NK cell killing is not specific for particular antigens and can occur in the absence of prior immune sensitization. Killing of targeted cells is often mediated by cytolytic proteins, including perforins, granzymes, and granulysins.

[0006] Autologous NK cells have been used as therapeutic entities. To this end, NK cells are isolated from the peripheral blood lymphocyte fraction of whole blood, expanded in cell culture to obtain a sufficient number of cells, and then re-infused into the subject. Autologous NK cells have shown moderate effectiveness in ex vivo and in vivo treatments at least in some cases. However, the separation and growth of autologous NK cells requires a lot of time and cost. In addition, not all NK cells have cytolytic effects, which further limits autologous NK cell therapy.

[0007] At least some of these difficulties can be overcome by using NK-92 cells, which are a cytolytic cancer cell line that is found in the blood of subjects with non-Hodgkin's lymphoma and then propagated indefinitely in vitro (Gong et al., Leukemia 8: 652-658 (1994)). Although NK-92 cells are derivatives of NK cells, NK-92 cells lack the main inhibitory receptors possessed by normal NK cells, while retaining most of the activating receptors. However, NK-92 cells do not attack normal cells, nor do they induce immune rejection reactions that are unacceptable to the human body. Due to these expected properties, NK-92 cells have been characterized in detail and explored as therapeutic agents for treating certain cancers, for example, as described in WO 1998 / 049268 or US2002 / 068044.

[0008] Phenotypic changes that distinguish tumor cells from normal cells derived from the same tissue are usually associated with one or more changes in the expression of specific gene products, including the loss of normal cell surface components or the acquisition of other components (i.e., antigens that cannot be detected in the corresponding normal non-cancerous tissue). Antigens expressed in tumors or tumor cells but not in normal cells, or antigens expressed at levels in tumor cells that are much higher than those found in normal cells, are referred to as "tumor-specific antigens" or "tumor-associated antigens". Such tumor-specific antigens can be used as markers of tumor phenotypes. Tumor-specific antigens include cancer / testis-specific antigens (e.g., MAGE, BAGE, GAGE, PRAME, and NY-ESO-1), melanocyte differentiation antigens (e.g., tyrosinase, Melan-A / MART, gpl00, TRP-1, and TRP-2), mutated or abnormally expressed antigens (e.g., MUM-1, CDK4, β-catenin, gp100-in4, p15, and N-acetylglucosaminyltransferase V), and antigens expressed at higher levels in tumors (e.g., CD19 and CD20).

[0009] Tumor-specific antigens have been used as targets for cancer immunotherapy. One such therapy is to improve the cytotoxicity of cancer cells using chimeric antigen receptors (CARs) expressed on the surface of immune cells (including T cells and NK cells). CAR includes a single-chain variable fragment (scFv) connected to at least one intracellular signaling domain. The scFv recognizes and binds to antigens on target cells (e.g., cancer cells) and triggers effector cell activation. The signaling domain includes an immunoreceptor tyrosine-based activation domain (ITAM), which is very important for the intracellular signaling of the receptor.

[0010] The first generation CAR for T cells includes a cytoplasmic signaling domain. For example, one version of the first generation CAR in T cells includes a signaling domain from Fcε receptor γ (FcεRIγ), which includes an ITAM, while another version includes a signaling domain from CD3ζ, wherein CD3ζ includes three ITAMs. In vivo and in vitro studies have shown that CD3ζCAR T cells are more effective than FcεRIγCAR T cells in eradicating tumors (e.g., Haynes et al., 2001, J.Immunology [Journal of Immunology] 166: 182-187; Cartellieri et al., 2010, J.BiomedandBiotech [Biomedicine and Biotechnology], 2010 volume, article ID 956304). Further studies then showed that the full activation and proliferation of such recombinant T cells requires certain costimulatory signals, and that the second and third generation CARs combine multiple signaling domains into a single CAR to enhance the efficacy of recombinant CAR T cells. Because of their less than ideal effects reported in the literature on the T cells tested, first-generation CARs and the FcεRIγ signaling domain were largely abandoned in favor of using CD3ζ in combination with one or more additional signaling domains (e.g., Hermanson and Kaufman 2015, Frontiers in Immunol., vol. 6, article 195).

[0011] Recently, selected CARs have also been expressed in NK cells. For example, CAR-modified NK-92 cells have used first-generation CARs with only CD3ζ intracellular signaling domains. These first-generation CAR-NK cells have targeted a variety of antigens, 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 constructed for several antigens, including EpCAM for a variety of cancers, HLA-A2 EBNA3 complex for EB (Epstein-Barr) virus, CS1 for multiple myeloma, and ErbB2 for HER2-positive epithelial cancer. In the second-generation NK-92CAR, the most common intracellular co-stimulatory domain used with CD3ζ is CD28. However, since NK cells do not naturally express CD28, the potential role of the CD28 domain is unclear. Additional second-generation CARs have combined the 4-1BB intracellular signaling domain with CD3ζ to improve NK cell persistence. Others have compared the functionality of different intracellular domains against breast cancer cells using ErbB2 scFv fused to CD3ζ alone, CD28 and CD3ζ, or 4-1BB and CD3ζ. They found that both second-generation constructs had better killing than the first-generation CARs, with 65% target lysis with CD28 and CD3ζ, 62% lysis with 4-1BB and CD3ζ, and 51% target killing with CD3ζ alone. In a recent study, 4-1BB and CD28 intracellular domains were also compared for B-cell malignancies using an anti-CD19 CAR expressed on NK-92 cells. Still others have found that the CD3ζ / 4-1BB construct was less effective than CD3ζ / CD28 in terms of cell killing and cytokine production, highlighting the different roles of the CD28 and 4-1BB costimulatory domains.

[0012] The third generation NK-92CAR is constructed by anti-CD5 scFv with CD3ζ, CD28 and 4-1BB intracellular signaling domains, and has been shown to have specific and potent anti-tumor activity for a variety of T cell leukemia and lymphoma cell lines and primary tumor cells. Such cells can also inhibit disease progression in xenograft mouse models of T cell acute lymphoblastic leukemia (ALL) cell lines and primary tumor cells (TranslRes. [Translational Medicine Research] September 2017; 187: 32-43). In a further example, WO 2016 / 201304 and WO 2018 / 076391 teach the use of the third generation CD3ζCAR expressed in NK cells and NK-92 cells.

[0013] However, as demonstrated by the repeated failures of engineering NK-92 cells to express Fc receptors, NK cells (especially NK-92 cells) are generally difficult to be genetically modified. These difficulties are further exacerbated when NK-92 cells are transfected with a variety of recombinant genes or relatively large recombinant nucleic acid payloads for heterologous expression. In addition, NK-92 cells also show a clear lack of predictability in terms of recombinant expression of exogenous proteins (e.g., CD16). At the functional level, although targeted cytotoxicity is shown in most cases, most (such as not all) CAR NK-92 cells require an efficient target cell ratio.

[0014] Therefore, although a large number of recombinant NK-92 cells are known in the art, all or almost all of them encounter various difficulties. Therefore, there is still a need for NK-92 cells expressing CAR, which express highly active CAR in large quantities and are easy to culture in a simple and effective manner. Summary of the invention

[0015] The inventors found that compared with other constructs, NK-92 cells expressing FcεRIγ-containing CAR unexpectedly exhibited excellent cytolytic activity (usually at relatively low effector-target cell ratios), as well as high levels of expression of FcεRIγ-containing CAR. In addition, such recombinant cells also expressed CD16 at expected levels, and while further modified to express stimulatory cytokines, recombinant NK-92 cells were also easy to culture without exogenous IL-2.

[0016] Thus, in one aspect of the inventive subject matter, the inventors contemplate a genetically modified NK cell carrying a membrane-bound recombinant chimeric antigen receptor (CAR) comprising (i) an extracellular binding domain, (ii) a hinge domain, (iii) a transmembrane domain, and (iv) an FcεRIγ signaling domain as a single polypeptide chain. Most typically, but not necessarily, the NK cell is a NK-92 cell.

[0017] In some embodiments, the extracellular binding domain comprises a scFv that can 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-specific antigen and a tumor-specific antigen (e.g., a new epitope with high affinity to the patient's MHC I and / or MHC II). Alternatively, the extracellular binding domain can also specifically bind to a virus-specific antigen, and typical viruses contemplated herein include HIV, HPV, RSV, influenza, Ebola, or HCV. For example, suitable viral antigens include gp120 of HIV.

[0018] In a further embodiment, the hinge domain and / or the transmembrane domain comprises a CD8 hinge domain and / or a CD28 transmembrane domain, and / or the FcεRIγ signaling domain has the amino acid sequence shown in SEQ ID NO:1.

[0019] In addition, it is envisioned that the genetically modified NK cells may further carry membrane-bound recombinant CD16 (particularly a high-affinity variant of CD16), and / or the genetically modified NK cells may express recombinant cytokines with endosomal retention sequences.

[0020] Therefore, from a different perspective, the inventors also envision a genetically modified NK cell comprising a recombinant nucleic acid encoding a chimeric antigen receptor (CAR), wherein the CAR comprises (i) an extracellular binding domain, (ii) a hinge domain, (iii) a transmembrane domain, and (iv) an FcεRIγ signaling domain in a single polypeptide chain. As previously described, preferably, the NK cell is usually a NK-92 cell. In some embodiments, the recombinant nucleic acid is RNA, which may be a polycistronic RNA, which further encodes CD16 and / or a cytokine with an endoplasmic retention sequence. With respect to various domains, the same considerations as above apply.

[0021] In another aspect of the subject matter of the present invention, the inventors also contemplate a method of treating cancer in a patient in need thereof, the method comprising the steps of administering to the patient a therapeutically effective amount of the genetically modified NK cells described herein, thereby treating the cancer. As will be readily appreciated, the contemplated method will further include the step of administering at least one additional therapeutic entity, the additional therapeutic entity comprising a viral cancer vaccine, a bacterial cancer vaccine, a yeast cancer vaccine, N-803, an antibody, a stem cell transplant, and / or a tumor-targeted cytokine.

[0022] For example, cancers treated by the contemplated methods include leukemias, acute lymphocytic leukemias, acute myeloid leukemias, chronic leukemias, chronic myeloid (granulocytic) leukemias, chronic lymphocytic leukemias, polycythemia vera, lymphomas, 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 fibrosarcomas, myxosarcoma , liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, bile duct cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumors, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma and retinoblastoma.

[0023] Similarly, the inventors have envisioned a method for treating a viral infection in a patient in need, the method comprising the following steps: administering to the patient a therapeutically effective amount of the genetically modified NK cells described herein (having an extracellular binding domain that can also specifically bind to a virus-specific antigen), thereby treating the viral infection. Of course, the envisioned method may further include the step of administering an antiviral drug.

[0024] Regardless of the type of treatment, it is envisioned that approximately 1 x 10 8 About 1x 10 11 Cells / m 2 The patient's body surface area.

[0025] Therefore, the inventors also envision the use of genetically modified NK cells as described herein in the treatment of cancer or viral infection.

[0026] Various objects, features, aspects and advantages of the present subject matter will become more apparent from the following detailed description of preferred embodiments and the accompanying drawings, in which like reference numerals represent like components. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1It is a schematic diagram of an exemplary detected CD19-CAR. All CD19-CAR variants include an extracellular domain, which includes an anti-CD19 scFv region (αCD19-scFv), a hinge region (CD8 hinge) from CD8, and a transmembrane domain (CD28 TM) from CD28. The intracellular domain of CD19CAR changes as shown.

[0028] Figure 2A The expression of CD19-CAR mRNA was confirmed by flow cytometry using anti-scFv antibody labeled with eF660. Figure 1 Exemplary percentage results of CD19-CAR NK-92 cells.

[0029] Figure 2B Figure 2 is an exemplary result of background-subtracted median fluorescence intensity (MFI) of NK-92 cells expressing CD19-CAR labeled with anti-scFv antibody labeled with eF660.

[0030] Figure 3A Exemplary results are shown for the percentage of NK-92 cell-sensitive target cancer cells (K562) killed by NK-92 cells expressing CD19CAR (effector) at effector-target ratios of 5:1 to 0.3:1.

[0031] Figure 3B Exemplary results are shown for the percentage of CD19-positive target cancer cells (SUP-B15) tolerated by NK-92 cells (effectors) expressing CD19CAR at effector-target ratios of 5:1 to 0.3:1.

[0032] Figure 4 Shown are exemplary results of the MFI of NK-92 expressing CD19-CAR (effector) 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.

[0033] Figure 5 An exemplary survival curve of animals bearing IV Raji tumors as described in the Examples is shown. Statistical analysis was performed using the log-rank (Mantel-Cox) test. ****, P<0.0001.

[0034] Figure 6 Exemplary results of animal body weight changes in the IV Raji tumor model are shown. Data are mean ± SEM. SEM is calculated as the standard deviation divided by the square root of N.

[0035] Figure 7An exemplary tumor growth curve of the SC Raji model is shown. Data are mean ± SEM. Statistical analysis was performed using two-way ANOVA followed by Tukey's test for multiple comparisons; ***, P < 0.001; ****, P < 0.0001.

[0036] Figure 8 shows exemplary data demonstrating that CD19 t-haNK reduces the burden of metastatic disease in the liver of SC Raji tumor-bearing mice. Panel a: Whole liver images of animals in the designated treatment groups on day 13. Arrows indicate metastatic lesions. The livers were fixed in 10% formalin for at least 24 hours prior to photography. Panel b: Quantification of the percentage of tumor cell involvement in the liver (assessed by H&E staining) on ​​the designated days. On day 13: *, P = 0.0257, by unpaired two-tailed t-test. Due to limited sample size, statistical analysis on days 11 and 15 could not be performed. See Table 4 for raw data.

[0037] Fig. 9 Exemplary results of animal body weight changes in the SC Raji tumor model are shown. Data are mean ± SEM.

[0038] Fig.10 Shown are exemplary Kaplan-Meier survival curves of mice injected with L1210-Luc tumor cells following intratumoral treatment with mCD19-CAR NK-92 cells versus vehicle control as described in the Examples.

[0039] Fig.11 Shown are exemplary results of tumor size in complete remission animals re-challenged with L1210-Luc tumor cells versus naive control animals as described in the Examples.

[0040] Fig.12 Shown are exemplary Kaplan-Meier survival curves of mice injected with A20 tumor cells following intratumoral treatment with mCD19-CAR NK-92 cells as described in the Examples.

[0041] Fig.13 Shown are exemplary results of tumor size in complete remission animals rechallenged with A20 tumor cells versus naive control animals as described in the Examples.

[0042] Fig.14 Exemplary results of cytotoxicity of HER2.CAR-t-haNK cells against BT-474 cells are shown.

[0043] Fig.15 Exemplary results of the cytotoxicity of CD33.CAR-t-haNK cells against THP-1 cells are shown.

[0044] Fig.16 Exemplary results of cytotoxicity of PD-L1.CAR-t-haNK cells against SUP-B15.PD-L1+ cells are shown.

[0045] Fig.17 Exemplary results of cytotoxicity of PD-L1.CAR-t-haNK cells against U251 cells are shown.

[0046] Fig.18 Exemplary results of cytotoxicity of EGFR.CAR-t-haNK cells against A-549 cells are shown.

[0047] Fig.19 Exemplary results of the cytotoxicity of CD19.CAR-t-haNK cells against K562 cells are shown.

[0048] Fig. 20 Exemplary results of the cytotoxicity of CD19.CAR-t-haNK cells against SUP-B15 cells are shown.

[0049] Fig.21 Exemplary results of ADCC of CD19.CAR-t-haNK cells against SKBr3 cells are shown.

[0050] Fig. 22 Exemplary results of the cytotoxicity of IGF1R.CAR-t-haNK cells against MDA-MB-231 cells are shown.

[0051] Fig.23 Exemplary results of the cytotoxicity of PD-L1.CAR-t-haNK cells against various cancer cells are shown.

[0052] Fig.24 Exemplary comparative results of the cytotoxicity of PD-L1.CAR-t-haNK cells against MDA-MB-231 cells are shown.

[0053] Fig.25 Exemplary results of CD16 and CD19.CAR expression are shown.

[0054] Fig.26 Exemplary results of natural cytotoxicity of CD19.CAR-t-haNK cells are shown.

[0055] Fig. 27 Exemplary results of CAR-mediated cytotoxicity of CD19.CAR-t-haNK cells are shown.

[0056] Fig.28Exemplary results of ADCC of CD19.CAR-t-haNK cells are shown.

[0057] Fig.29 Exemplary comparison results of CD16 and CD20.CAR expression are shown.

[0058] Fig.30 Exemplary results of natural cytotoxicity of CD20.CAR-t-haNK cells are shown.

[0059] Fig.31 Exemplary results of CD16 and CD33.CAR expression are shown.

[0060] Fig.32 Exemplary results of natural cytotoxicity of CD33.CAR-t-haNK cells are shown.

[0061] Fig.33 Exemplary results of CAR-mediated cytotoxicity of CD33.CAR-t-haNK cells are shown.

[0062] Fig.34 Exemplary results of ADCC of CD33.CAR-t-haNK cells are shown.

[0063] Fig.35 Exemplary results of CD16 and EGFR.CAR expression are shown.

[0064] Fig.36 Exemplary results of natural cytotoxicity of EGFR.CAR-t-haNK cells are shown.

[0065] Fig.37 Exemplary results of CAR-mediated cytotoxicity of EGFR.CAR-t-haNK cells are shown.

[0066] Fig.38 Exemplary results of CAR-mediated cytotoxicity of EGFR.CAR-t-haNK cells are shown.

[0067] Fig.39 Exemplary results of ADCC of EGFR.CAR-t-haNK cells are shown.

[0068] Fig.40 Exemplary results of CD16 and HER2.CAR expression are shown.

[0069] Fig.41 Exemplary results of natural cytotoxicity of HER2.CAR-t-haNK cells are shown.

[0070] Fig.42Exemplary results of CAR-mediated cytotoxicity of HER2.CAR-t-haNK cells are shown.

[0071] Fig.43 Exemplary results of ADCC of HER2.CAR-t-haNK cells are shown.

[0072] Fig.44 Exemplary results of CD16 and PD-L1.CAR expression are shown.

[0073] Fig.45 Exemplary results of natural cytotoxicity of PD-L1.CAR-t-haNK cells are shown.

[0074] Fig.46 Exemplary results of CAR-mediated cytotoxicity of PD-L1.CAR-t-haNK cells are shown.

[0075] Fig.47 Exemplary results of ADCC of PD-L1.CAR-t-haNK cells are shown.

[0076] Fig.48 Exemplary results of CAR-mediated cytotoxicity of CD123.CAR-t-haNK cells are shown.

[0077] Fig.49 Exemplary results of ADCC of CD123.CAR-t-haNK cells are shown.

[0078] Fig.50 Exemplary results of CD16 and CD30.CAR expression are shown.

[0079] Fig.51 Exemplary results of natural cytotoxicity of CD30.CAR-t-haNK cells are shown.

[0080] Fig.52 Exemplary results of CAR-mediated cytotoxicity of CD30.CAR-t-haNK cells are shown.

[0081] Fig.53 Exemplary results of ADCC of CD30.CAR-t-haNK cells are shown.

[0082] Fig.54 Exemplary results of CD16 and BCMA.CAR expression are shown.

[0083] Fig.55 Exemplary results of CAR-mediated cytotoxicity of BCMA.CAR-t-haNK cells are shown.

[0084] Fig.56 Exemplary results of ADCC of BCMA.CAR-t-haNK cells are shown.

[0085] Fig.57 Exemplary results of CD16 and gp120.CAR expression are shown.

[0086] Fig.58 Exemplary results of GP120 binding of gp120.CAR-t-haNK cells are shown.

[0087] Fig.59 Exemplary results of natural cytotoxicity of gp120.CAR-t-haNK cells are shown.

[0088] Fig.60 Exemplary results of ADCC of gp120.CAR-t-haNK cells are shown.

[0089] Fig.61 Exemplary results of CD16 and FAP.CAR expression are shown.

[0090] Fig.62 Exemplary results of CAR-mediated cytotoxicity of FAP.CAR-t-haNK cells are shown.

[0091] Fig.63 Exemplary results of CSPG4 expression in CSPG4.CAR-t-haNK cells are shown.

[0092] Fig.64 Exemplary results of CAR-mediated cytotoxicity of CSPG4.CAR-t-haNK cells are shown.

[0093] Fig.65 Describes a gene encoding IGF1R-CAR, CD16, and IL-2 ER Exemplary tricistronic constructs of. DETAILED DESCRIPTION

[0094] To date, CARs containing FcεRIγ have not been used in NK-92 cells, other NK cell lines, or endogenous NK cells because it is believed that the signaling domain (e.g., CD3ζ) is more effective, especially when combined with other signaling domains (in second and third generation CARs). The inventors have now made an unexpected and unexpected discovery that NK-92 cells expressing a first generation CAR containing an intracellular domain from FcεRIγ (having only one ITAM domain) have the same or higher cytotoxic activity against cancer cells expressing an antigen recognized by the CAR, compared to NK-92 cells expressing a CAR with a CD3ζ signaling domain (which has 3 ITAM domains, even if these ITAM domains are combined with other signaling domains (i.e., second or third generation CARs). It is noteworthy that in its natural environment, the IgE receptor (FcεRI) includes two γ chains interconnected by disulfide bonds and is usually expressed only in eosinophils, basophils, and epidermal Langerhans cells. The inventors also made the unexpected discovery that CARs comprising the intracellular domain from FcεRIγ are expressed on the surface of NK-92 cells at higher levels than other CARs, particularly CARs comprising the CD3ζ signaling domain.

[0095] Therefore, the subject matter of the present invention relates to a genetically modified NK-92 cell or NK cell line, which is engineered to express a chimeric antigen receptor (CAR) on the cell surface. Most typically, the CAR comprises an intracellular domain from Fcε receptor γ (FcεRIγ), however, in other embodiments, the CAR may also comprise a T cell receptor (TCR) CD3ζ (CD3zeta) intracellular domain. As will be readily understood, the CAR can be transiently or stably expressed by NK-92 cells from recombinant DNA or RNA molecules.

[0096] Therefore, in one aspect of the subject matter of the present invention, NK cells, NK-92 cells or NK / NK-92 cell lines express chimeric antigen receptors (CAR) on the surface of NK-92 cells comprising an FcεRIγ cytoplasmic domain (e.g., having an amino acid sequence as shown in SEQ ID NO: 1). Alternatively, or in addition, the CAR may also comprise a cytoplasmic domain of CD3ζ (e.g., having an amino acid sequence as shown in SEQ ID NO: 10, which may be encoded by a nucleic acid as shown in SEQ ID NO: 11 (codon optimized) or SEQ ID NO: 12 (non-codon optimized); the full-length sequence is as shown in SEQ ID NO: 47). On the other hand, a NK or NK-92 cell line is envisioned, which is transformed with a nucleic acid encoding a chimeric antigen receptor (CAR). For example, a preferred nucleic acid encodes a cytoplasmic domain of FcεRIγ (e.g., comprising or consisting of SEQ ID NO: 2). Alternatively, or in addition, the nucleic acid encodes the cytoplasmic domain of CD3ζ (e.g., comprising or consisting of SEQ ID NO: 11 (human, codon optimized) or SEQ ID NO: 12 (human). As will be readily appreciated, the CAR can target cancer-associated or virus-associated antigens through its extracellular binding domain, as described in more detail below.

[0097] In further contemplated embodiments, NK or NK-92 cells can be modified to express at least one cytokine or its variant. For example, cytokines can be transiently or stably expressed by recombinant cells, and the cytokine can include endoplasmic retention signals. As expected, NK or NK-92 cells can also be modified to express suicide genes (for example, suicide genes are thymidine kinases). Not subject to any theoretical constraints, it is believed that the expression of suicide genes can prevent the uncontrolled proliferation of NK-92 cells by providing a mechanism for selectively killing cells after introducing suitable stimulation.

[0098] In another aspect of the subject matter of the present invention, the inventors also envision a method for treating cancer in a patient in need, the method comprising the following steps: administering to the patient a therapeutically effective amount of modified NK / NK-92 cells or NK / NK-92 cell lines, which are engineered to express a chimeric antigen receptor (CAR) as described herein. From a different perspective, the inventors also envision a modified NK / NK-92 cell or NK / NK-92 cell line expressing a chimeric antigen receptor (CAR), which preferably comprises a cytoplasmic domain of FcεRIγ, for treating cancer in a subject. In some embodiments, the use includes administering to a subject an effective amount of modified cells or cell lines as described herein to treat tumors. In yet other embodiments, an in vitro method for killing tumor cells is envisioned, and the method may include the step of contacting tumor cells with modified NK-92 cells or NK-92 cell lines as described herein. In some embodiments, the modified NK-92 cells or NK-92 cell lines express CARs that bind to antigens on tumor cells. In some embodiments, the CAR preferably comprises an intracellular domain from Fcε receptor γ (FcεRIγ). Alternatively, or additionally, the CAR comprises a T cell receptor (TCR) CD3ζ (CD3 zeta) intracellular domain.

[0099] In yet other embodiments, a method of treating a viral infection in a patient in need thereof is described, the method comprising administering to the patient a therapeutically effective amount of the CAR-expressing NK-92 cells described herein.

[0100] After reading this description, it will become apparent to those skilled in the art how to implement the present invention in various alternative embodiments and alternative applications. However, all embodiments of the present invention are not described herein. It will be understood that the embodiments presented here are presented only by way of example and not limitation. Likewise, this detailed description of various alternative embodiments should not be construed as limiting the scope or breadth of the present invention, as described below.

[0101] Before disclosing and describing the present invention, it should be understood that the aspects described below are not limited to specific compositions, methods for preparing such compositions, or uses thereof, which can of course vary. It should also be understood that the terminology used herein is only for the purpose of describing specific aspects and is not intended to be limiting thereof.

[0102] For the convenience of the reader, titles or subtitles may be used in the specification, but this is not intended to affect the scope of the present invention. In addition, some terms used in this specification are defined in more detail below.

[0103] About suitable NK cells, it should be noted that all NK cells are considered suitable for the present invention, thus including primary NK cells (preserved, expanded and / or fresh cells), immortalized secondary NK cells, autologous or xenogeneic NK cells (stock, preserved, fresh, etc.) and modified NK cells, as described in more detail below. In some embodiments, preferably, the NK cell is a NK-92 cell. The NK-92 cell line is a unique cell line that was found to proliferate in the presence of interleukin 2 (IL-2) (see, e.g., Gong et al., Leukemia [leukemia] 8: 652-658 (1994)). NK-92 cells are cancerous NK cells that have a wide range of anti-tumor cytotoxicity and predictable yields after amplification in a suitable culture medium. Advantageously, NK-92 cells have high cytolytic activity against a variety of cancers.

[0104] The original NK-92 cell line expresses CD56 bright , CD2, CD7, CD11a, CD28, CD45 and CD54 surface markers, but CD1, CD3, CD4, CD5, CD8, CD10, CD14, CD16, CD19, CD20, CD23 and CD34 markers are not displayed. The growth of such NK-92 cells in culture depends on the presence of interleukin 2 (e.g., rIL-2), and a dose as low as 1IU / mL is sufficient to maintain proliferation. IL-7 and IL-12 do not support long-term growth, and other multiple cytokines have not been detected, including IL-1α, IL-6, tumor necrosis factor α, interferon α and interferon γ. Compared with primary NK cells, NK-92 usually has higher cytotoxicity even at a relatively low effect-target (E:T) ratio (e.g., 1:1). Representative NK-92 cells are preserved in the American Type Culture Collection (ATCC) and named CRL-2407.

[0105] Therefore, suitable NK cells can have one or more modified KIRs, which are mutated to reduce or eliminate the interaction with MHC class I molecules. Of course, it should be noted that one or more KIRs can also be deleted or their expression can be inhibited (e.g., by miRNA, siRNA, etc.). Most typically, more than one KIR will be mutated, deleted or silenced, and the KIRs expected in particular include those with two or three domains, with short or long cytoplasmic tails. From different perspectives, modified, silent or missing KIRs will include KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DL2, KIR3DL3, and KIR3DS1. Such modified cells can be prepared using protocols well known in the art. Alternatively, such cells can also be purchased commercially as aNK cells ('activated natural killer cells) from NantKwest (see URL www.nantkwest.com). These cells can then be modified with additional genes to become CARs, as described in more detail below.

[0106] In another aspect of the subject matter of the present invention, the genetically engineered NK cells can also be NK-92 derivatives modified to express high-affinity Fcγ receptors (CD16). The sequences of high-affinity variants of Fcγ receptors are well known in the art (see, e.g., Blood [blood] 2009113: 3716-3725; SEQ ID NO: 43 and 44), and all methods of production and expression are considered applicable herein. It is believed that the expression of this receptor allows the use of antibodies specific to the patient's tumor cells (e.g., new epitopes), specific tumor types (e.g., her2neu, PSA, PSMA, etc.), or antibodies associated with cancer (e.g., CEA-CAM) to specifically target tumor cells. Advantageously, such antibodies are commercially available and can be used in conjunction with cells (e.g., in combination with Fcγ receptors). Alternatively, such cells can also be commercially available from Nantquist as haNK cells. These cells can then be modified to CARs by additional genes, as described in more detail below.

[0107] Therefore, NK cells suitable for use herein include NK-92 cells (which can be transfected with a tricistronic construct encoding CAR, CD16 or a variant thereof, a cytokine or a variant thereof), genetically modified NK cells or NK-92 cells expressing CD16 or a variant thereof or a cytokine or a variant thereof (which can be transfected with a nucleic acid encoding CAR and CD16 or a variant thereof or a cytokine or a variant thereof), and genetically modified NK cells or NK-92 cells expressing CD16 or a variant thereof and a cytokine or a variant thereof (which can be transfected with a nucleic acid encoding CAR)

[0108] The genetic modification of NK cells contemplated herein can be performed in a variety of ways, and all known methods are considered applicable hereto. Moreover, it should be recognized that NK cells can be transfected with DNA or RNA, and the specific choice of transfection will depend at least in part on the type of recombinant cells expected and the transfection efficiency. For example, in the case of expected stable transfection of NK cells, linearized DNA can be introduced into the cells for integration into the genome. On the other hand, in the case of expected transient transfection, circular DNA or linear RNA (e.g., with polyA + tail of mRNA).

[0109] Similarly, it should be understood that the mode of transfection will depend at least in part on the type of nucleic acid used. Therefore, viral transfection, chemical transfection, and mechanical transfection methods are all considered suitable for use herein. For example, in one embodiment, the vectors described herein are transient expression vectors. The exogenous transgene introduced using such vectors is not integrated into the nuclear genome of the cell; therefore, in the absence of vector replication, the exogenous transgene will degrade or dilute over time.

[0110] In another embodiment, the vectors described herein allow stable transfection of cells. In one embodiment, the vector allows transgenic incorporation into the genome of the cell. Preferably, such vectors have positive selection markers, and suitable positive selection markers include any gene that allows cells to grow under conditions that kill cells that do not express the gene. Non-limiting examples include antibiotic resistance, for example, geneticin (Neo gene from Tn5).

[0111] 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 may be used, and suitable vectors are well known in the art.

[0112] In other embodiments, cells are transfected with mRNA encoding a protein of interest (e.g., CAR). mRNA transfection results in transient expression of protein. In one embodiment, mRNA is transfected into NK-92 cells immediately before the cells are administered. In one embodiment, "immediately before" the cells are administered refers to about 15 minutes to about 48 hours before administration. Preferably, mRNA transfection is performed about 5 hours to about 24 hours before administration. In at least some embodiments described in more detail below, NK cells transfected with mRNA result in unexpectedly consistent and strong expression of CAR on a high proportion of transfected cells. Moreover, such transfected cells also show high specific cytotoxicity when the effect-target cell ratio is quite low.

[0113] Regarding the envisioned CAR, it is noted that NK / NK-92 cells will be genetically modified to express CAR as a membrane-bound protein, thereby exposing a portion of the CAR on the cell surface while maintaining the signaling domain in the intracellular space. Most typically, CAR will include at least the following elements (in order): an extracellular binding domain, a hinge domain, a transmembrane domain, and an FcεRIγ signaling domain.

[0114] In a preferred embodiment, the cytoplasmic domain of CAR comprises or consists of a signaling domain of FcεRIγ. For example, the FcεRIγ signaling domain comprises or consists of an amino acid sequence shown in SEQ ID NO: 1 or consists of or consists essentially of it. 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.

[0115] 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 homology to the amino acid sequence shown in SEQ ID NO:1.

[0116] As described above, in some embodiments, the cytoplasmic domain of the CAR comprises a signaling domain of CD3 ζ (CD3zeta). In one embodiment, the cytoplasmic domain of the CAR consists of a signaling domain of CD3 ζ. In one embodiment, the CD3 ζ signaling domain comprises, consists of, or consists essentially of an amino acid sequence as shown in SEQ ID NO: 10. In some embodiments, the CD3 ζ 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 homology to the amino acid sequence as shown in SEQ ID NO: 10.

[0117] The CAR may comprise any suitable transmembrane domain. In one aspect, the CAR comprises a transmembrane domain of CD28. In one embodiment, the CD28 transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:7 or consists of it or consists essentially of it. In some embodiments, the CD28 transmembrane domain comprises an amino acid sequence having at least about 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology to the amino acid sequence shown in SEQ ID NO:7 or consists of it or consists essentially of it. In one embodiment, the transmembrane domain is selected from a CD28 transmembrane domain, a 4-1BB transmembrane domain or a FcεRIγ transmembrane domain.

[0118] The CAR may include any suitable hinge region. In one aspect, the CAR comprises a hinge region of CD8. In one embodiment, the CD8 hinge region comprises or consists of or consists essentially of the amino acid sequence shown in SEQ ID NO:6. In one embodiment, the CD8 hinge region comprises or 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 shown in SEQ ID NO:6.

[0119] Most typically, but not necessarily, the extracellular binding domain of CAR will be a scFv or other natural or synthetic binding portion that specifically binds to the antigen of interest. Particularly suitable binding moieties include small antibody fragments with single, dual or multiple target specificities, beta barrel domain binders, phage display fusion proteins, etc. In other suitable extracellular binding domains, preferred domains will specifically bind to tumor-specific antigens, tumor-associated antigens, or patient-specific antigens and tumor-specific antigens. For example, the envisioned antigens include CD19, CD20, GD2, HER-2, CD30, EGFR, FAP, CD33, CD123, PD-L1, IGF1R, CSPG4 or B7-H4. As non-limiting examples, further tumor-specific antigens are described in US2013 / 0189268; WO1999024566A1; US ​​7098008; and WO 2000020460, each of which is incorporated herein by reference in its entirety. Likewise, other preferred domains will specifically bind to (pathogenic) virus-specific antigens such as antigens of HIV virus (eg, gp120), HPV virus, RSV virus, influenza virus, Ebola virus or HCV.

[0120] With respect to the construction of contemplated CARs, it should be appreciated that CARs can be engineered in a variety of ways as described, for example, in WO 2014 / 039523; US 2014 / 0242701; US ​​2014 / 0274909; US 2013 / 0280285 and WO 2014 / 099671, each of which is incorporated herein by reference in its entirety.

[0121] Therefore, and from a different perspective, the envisioned CAR targets antigens associated with specific cancer types. In one embodiment, the cancer is a leukemia (including acute leukemia (e.g., acute lymphocytic leukemia, acute myeloid leukemia (including myeloid, 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 cancers, such as fibrosarcomas, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangiosarcoma, lymphangiosarcoma, Endothelioma, synovialoma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, or retinoblastoma.

[0122] Therefore, the envisioned CAR will generally have a structure of an extracellular binding domain (directly) coupled to a hinge domain, which is (directly) coupled to a transmembrane domain, which in turn is (directly) coupled to an FcεRIγ signaling domain. In further envisioned aspects, in addition to or in place of the FcεRIγ signaling domain, the envisioned CAR may also include one or more signaling domains, and in particular, the envisioned signaling domains include a CD3ζ signaling domain, a 4-1BB signaling domain, and a CD28 signaling domain. For example, a contemplated CAR can therefore include any one of the binding domains of SEQ ID NOs: 4, 23-42, and 48-59, coupled to a hinge domain (e.g., a CD8 hinge as set forth in SEQ ID NO: 6), which in turn is coupled to a transmembrane domain (e.g., a CD28 TM as set forth in SEQ ID NO: 7), which is coupled to a signaling domain (e.g., an FcεRIγ signaling domain as set forth in SEQ ID NO: 1, a CD28 signaling domain as set forth in SEQ ID NO: 8, a 4-1BB signaling domain as set forth in SEQ ID NO: 9, a CD3ζ signaling domain as set forth in SEQ ID NO: 10).

[0123] In further contemplated aspects, NK cells can be further genetically modified to express one or more cytokines, thereby providing a selection marker, wherein the cytokine and CAR are encoded with the same recombinant nucleic acid and / or the recombinant cells are independent of exogenous IL-2. Therefore, in some embodiments, NK-92 cells are modified to express at least one cytokine. In particular, at least one cytokine is IL-2, IL-12, IL-15, IL-18, IL-21 or a variant thereof. In a preferred embodiment, the cytokine is IL-2 or a variant thereof, and a particularly preferred variant includes an endoplasmic retention signal (e.g., human IL-2 shown in SEQ ID NO: 18, or with an ER retention signal shown in 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 the expression of IL-2 to be sufficient for autocrine activation, but does not release IL-2 outside the cell (e.g., Exp Hematol. [Experimental Hematology] February 2005; 33 (2): 159-64). Alternatively, the expression of cytokines (and in particular IL-15) can also be such that the expression of the cytokines is sufficient to provide an autocrine growth signal to the recombinant cells, but also allows at least some of the expressed IL-15 to be released from the cells, thereby providing an immunostimulatory signal. For example, such expression can be achieved using a human IL-15 sequence that includes both a signal peptide and an endoplasmic retention sequence. Exemplary DNA and protein sequences of endoplasmic retention IL-15 are shown in SEQ ID NO: 72 and SEQ ID NO: 73, respectively.

[0124] As expected, the envisioned cells can also express suicide genes. The term "suicide gene" refers to a transgene that allows negative selection of cells expressing suicide genes. Suicide genes are used as a safety system to allow cells expressing the gene to be killed by introducing a selection agent. If the recombinant gene causes a mutation that causes uncontrolled cell growth, or the cell itself is capable of such growth, this is in line with expectations. A variety of suicide gene systems have been identified, including herpes simplex virus thymidine kinase (TK) gene, cytosine deaminase gene, varicella zoster virus thymidine kinase gene, nitroreductase gene, Escherichia coli gpt gene and Escherichia coli Deo gene. Typically, the protein encoded by the suicide gene has no adverse effects on cells, but will kill cells in the presence of a specific compound. Therefore, suicide genes are usually part of the system.

[0125] 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 can be a wild-type or mutant TK gene (e.g., tk30, tk75, sr39tk). Cells expressing TK protein can be killed using ganciclovir. In another embodiment, the suicide gene is a 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 July 15; 92(2): 672-82. In a further embodiment, the suicide gene is a cytochrome P450 that 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." NEngl 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. AP1903 is a biologically inert small molecule that has been shown to be well tolerated in clinical studies and has been used in adoptive cell therapy.

[0126] Of course, it should be noted that all recombinant proteins can be expressed from a single recombinant sequence. However, it is generally preferred that in the case of expressing multiple recombinant sequences (e.g., CAR, CD16, cytokines), the coding region can be arranged in a polycistronic unit with at least two or at least three coding regions encoding recombinant proteins. Therefore, transgenic engineering can be converted into an expression vector by any mechanism known to those skilled in the art. When multiple transgenics are to be inserted into cells, transgenics can be engineered into the same expression vector or different expression vectors. In some embodiments, cells are transfected with mRNA encoding transgenic proteins to be expressed. In some embodiments, cells are transfected with DNA encoding transgenic proteins to be expressed. Transgenics, 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, nuclear transfection or "gene gun".

[0127] Therefore, in a preferred embodiment, it should be noted that genetically modified NK cells (particularly cells expressing CAR and CD16 or variants thereof) will exhibit three different cell killing modes: general cytotoxicity mediated by an activating receptor (e.g., NKG2D receptor), ADCC mediated by an antibody bound to a target cell, and CAR-mediated cytotoxicity. It is apparent that the envisioned genetically modified cells can be used to treat a variety of diseases, and are a variety of cancers and viral infections in which diseased cells present disease-specific or disease-associated antigens. Therefore, the inventors have contemplated methods for treating patients with modified NK or NK-92 cells as described herein. In one embodiment, the patient suffers from cancer (e.g., a tumor), and the modified NK-92 cells or cell lines express CARs specific to antigens expressed on the cell surface from cancer or a tumor. In one embodiment, the patient suffers from a viral infection, and the modified NK-92 cells or cell lines express CARs specific to antigens expressed on the surface of cells infected by the virus. In one embodiment, the patient suffers from a bacterial infection, and the modified NK-92 cells or cell lines express CARs specific to antigens expressed on the surface of bacterial cells causing the infection.

[0128] In some embodiments, the cancer is selected from the group consisting of leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myeloid leukemia (including myeloid, promyelocytic, myelomonocytic, monocytic and erythroleukemia)) and chronic leukemias (e.g., chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, 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, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma , lymphangiomatous endothelioma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumors, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0129] Contemplated modified NK or NK-92 cells can be administered to an individual in absolute numbers of cells. For example, about 1000 cells / injection up to about 10 billion cells / injection can be administered to an individual, such as about, at least about, or at most about 1×10 cells / 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.) modified NK-92 cells, or any range (including endpoints) between any two values. In other embodiments, the modified NK-92 cells can be administered to an individual in a relative number of cells, for example, about 1000 cells to up to about 10 billion cells per kilogram of the individual can be administered to the individual, such as about, at least about, or at most about 1×10 per kilogram of the individual. 8 , 1×10 7 , 5×10 7 , 1×10 6 , 5×106 , 1×10 5 , 5×10 5 , 1×10 4 , 5×10 4 , 1×10 3 , 5×10 3 (etc.) modified NK-92 cells, or any range (including endpoints) between any two values. In other embodiments, the total dose can be m 2 The body surface area is calculated, including the 2 About 1×10 11 , 1×10 10 , 1×10 9 , 1×10 8 , 1×10 7 , or any range between any two values ​​(including endpoints). The average human body is about 1.6 to about 1.8m 2 In a preferred embodiment, about 1 billion to about 3 billion NK-92 cells are administered to the patient.

[0130] The modified NK-92 cells, and optionally other anti-cancer or anti-viral agents, can be administered to a patient suffering from cancer or a viral infection once, or can be administered 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 values ​​(including endpoints).

[0131] In one embodiment, in the case where the modified NK-92 cells express a suicide gene, an agent is administered to the patient that triggers the death of the modified NK-92 cells. In one embodiment, the agent is administered at a time point after administration of the modified NK-92 cells sufficient to allow the NK-92 cells to kill target cells.

[0132] In one embodiment, the modified NK-92 cells are irradiated prior to administration to a patient. The irradiation of NK-92 cells is, for example, as described in U.S. Pat. No. 8,034,332, which is incorporated herein by reference in its entirety. In one embodiment, the modified NK-92 cells that have not been engineered to express a suicide gene are irradiated.

[0133] In addition, it should be understood that the contemplated treatment methods will also include the administration of other immunotherapeutic entities, with particular preference for immunotherapeutic entities, including viral cancer vaccines (e.g., adenoviral vectors encoding cancer-specific antigens), bacterial cancer vaccines (e.g., non-pyrogenic Escherichia coli expressing one or more cancer-specific antigens), yeast cancer vaccines, N-803 (also known as ALT-803, ALTOR Biosciences), and antibodies (e.g., binding to tumor-associated antigens or patient-specific tumor neoantigens), stem cell transplants (e.g., allogeneic or autologous), and tumor-targeted cytokines (e.g., NHS-IL12, IL-12 conjugated to tumor-targeted antibodies or fragments thereof).

[0134] Examples

[0135] The following examples are for illustrative purposes only and should not be construed as limiting the rights and interests of the present invention. A variety of alternative techniques and procedures may be used by those skilled in the art that will similarly allow one to successfully perform the intended invention.

[0136] Example 1: CAR mRNA preparation

[0137] Designing codes using computer methods Figure 1 The DNA sequence of each variant of CD19CAR schematically depicted in , de novo synthesis, and then subcloned into the mRNA expression vector pXT7 (GeneArt, Life Technologies). 10 micrograms (μg) of plasmid were linearized by digestion with SalI restriction enzyme (New England Biolabs) and purified using QIAgen gel purification kit (QIAgen company) according to the manufacturer's instructions.

[0138] The linearized DNA was used as a template for in vitro synthesis of mRNA using the T7 mMessage mMachine Ultra Transcription Kit (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's instructions. The kit includes a polyadenylation extension step that increases the length of the polyA tail of the mRNA, thereby enhancing its in vivo stability.

[0139] The mRNA of six CD19-CAR variants was prepared, and green fluorescent protein (GFP) mRNA was prepared as a negative control. All CD19-CAR polypeptide variants include an extracellular domain, which includes 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 domain of CD19CAR is shown below, and its schematic diagram is as follows Figure 1As shown: CAR 3z comprises a CD3ζ signaling domain; CARFcRe contains an FcεRIγ signaling domain (SEQ ID NO: 1); CAR 28_3z comprises a CD28 signaling domain fused to a CD3ζ signaling domain; CARBB_3z comprises a 4-1BB signaling domain fused to a CD3ζ signaling domain; CAR28_BB_3z comprises a CD28 signaling domain fused to a 4-1BB signaling domain, wherein the 4-1BB signaling domain is fused to a CD3ζ signaling domain; CARBB_3z_28 comprises a 4-1BB signaling domain fused to a CD3ζ signaling domain, wherein the CD3ζ signaling domain is fused to a CD28 signaling domain.

[0140] More specifically, Figure 1 The first generation CAR with CD3 ζ signaling domain has the nucleic acid sequence shown in SEQ ID NO: 13 (human) and SEQ ID NO: 21 (mouse), which is translated into the amino acid sequence shown in SEQ ID NO: 22. The first generation CAR with FcεRIγ signaling domain nucleic acid has the nucleic acid sequence shown in SEQ ID NO: 5 and the amino acid sequence shown in SEQ ID NO: 3. The second generation CAR with CD28 / CD3 ζ signaling domain has the nucleic acid sequence shown in SEQ ID NO: 14, and the second generation CAR with 4-1BB / CD3 ζ signaling domain has the nucleic acid sequence shown in SEQ ID NO: 15. The nucleic acid sequence of the third generation CAR with CD28 / 4-1BB / CD3 ζ signaling domain has the nucleic acid sequence shown in SEQ ID NO: 16, and the third generation CAR with 4-1BB / CD3 ζ / CD28 signaling domain has the nucleic acid sequence shown in SEQ ID NO: 17.

[0141] As described in more detail below, a first generation CAR having an FcεRIγ signaling domain is further prepared, wherein the hinge region is a CD8 hinge (SEQ ID NO:6 or SEQ ID NO:45 (human), encoded by SEQ ID NO:46), wherein the transmembrane domain is a CD28 transmembrane domain (SEQ ID NO:7), and wherein the signaling domain is an FcεRIγ signaling domain (SEQ ID NO:1, encoded by the nucleic acid set forth in SEQ ID NO:2).

[0142] The selected scFv portions were then used as described below (all in Figure 1, CAR FcRe) confers target specificity to a variety of tumor-related targets: CD19 (using the anti-CD19 scFv set forth in SEQ ID NO:4 or SEQ ID NO:24, encoded by codon-optimized SEQ ID NO:23), CD20 (using the anti-CD20 scFv set forth in SEQ ID NO:26, encoded by codon-optimized SEQ ID NO:25), CD33 (using the anti-CD33 scFv set forth in SEQ ID NO:28, encoded by codon-optimized SEQ ID NO:27), CSPG4 (using the anti-CSPG4 scFv set forth in SEQ ID NO:30, encoded by codon-optimized SEQ ID NO:29), EGFR (using the anti-EGFR scFv set forth in SEQ ID NO:32, encoded by codon-optimized SEQ ID NO:31), IGF1R (using the anti-IGF1R scFv set forth in SEQ ID NO:34, encoded by codon-optimized SEQ ID NO:33), CD30 (using the anti-CD33 scFv set forth in SEQ ID NO:29), CSPG4 (using the anti-CSPG4 scFv set forth in SEQ ID NO:30, encoded by codon-optimized SEQ ID NO:31), NO:36, encoded by codon-optimized SEQ ID NO:35), HER2 / neu (using anti-HER2 / neu scFv as set forth in SEQ ID NO:38, encoded by codon-optimized SEQ ID NO:37), GD2 (using anti-GD2 scFv as set forth in 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 as set forth in SEQ ID NO:49, encoded by codon-optimized SEQ ID NO:48), PD-L1 (using anti-PD-L1 scFv as set forth in SEQ ID NO:51, encoded by codon-optimized SEQ ID NO:50), B7-H4 (using anti-B7-H4 scFv as set forth in SEQ ID NO:53, encoded by codon-optimized SEQ ID NO:52), and FAP (using anti-FAP as set forth in SEQ ID NO:58 or SEQ ID NO:59). scFv, encoded by codon-optimized SEQ ID NO:56 or SEQ ID NO:57).

[0143] Likewise, selected scFv portions were used as follows (to confer target specificity to all virus-related targets) (all expressed as Figure 1 Sequence arrangement as shown, CARFcRe): HIV gp120 (using the anti-gp120 scFv shown in SEQ ID NO:55, encoded by the codon-optimized SEQ ID NO:54).

[0144] All constructs prepared as above were well expressed in NK-92 cells, and exemplary results of the physiological activities of the NK-92 cells thus modified were shown.

[0145] Example 2: Electroporation of NK-92 cells with CD19CAR mRNA

[0146] NK-92 cells were grown in X-Vivo 10 medium (Lonza, Basel, Switzerland) supplemented with 5% human AB serum (Valley Biomedical, Winchester, VA) and 500 IU / mL IL-2 (Prospec, Rehovot, Israel). TM Electroporation was performed using an electroporation apparatus (Life Technologies, Carlsbad, CA) according to the manufacturer's parameters for NK-92 cells (1250 V, 10 ms, 3 pulses) and 10 cells were added in a volume of 100 μl. 6 The cells were electroporated with mRNA at 5 μg mRNA per cell. The electroporated cells were maintained in the culture medium (same as above) for 20 hours (h).

[0147] CD19CAR expression on the surface of NK-92 cells was determined by flow cytometry using an anti-scFv antibody labeled with eF660 (eBioscience, San Diego, CA). Figure 2A The % expression of the indicated CD19CARs in NK-92 cell populations is shown. Figure 2B The median fluorescence intensity (MFI, background subtracted) of cells electroporated with the indicated CD19 CARs is shown. Figure 2A and 2B It can be seen that CARFcRe unexpectedly had the highest percentage of cells expressing CD19CAR on the cell surface (75.2%), as well as the highest MFI (amount of CAR expressed on recombinant cells), followed by 28_3z (61.7%).

[0148] Example 3: Cytotoxicity of NK-92 cells expressing CD19CAR against cancer cell lines

[0149] The efficacy of CAR-expressing NK-92 cells in targeting cancer cells in vitro was tested 20 hours after electroporation using a flow cytometry-based in vitro cytotoxicity assay. Effector cells (NK-92 expressing CD19CAR or GFP) were co-cultured with PKHGL67-labeled (Sigma-Aldrich, St. Louis, MO) target cells (K562; or SUPB15, B-ALL, CD19 +) were mixed in 96-well plates at different effector-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 % of PI-positive cells in the PKH-positive target cell population.

[0150] Figure 3A and 3B Exemplary results are provided in Figure 3A As shown, NK-92 cells effectively killed K562 cells regardless of CD19CAR expression. Therefore, it should be noted that the recombinant cells did not lose cytotoxicity. In contrast, NK-92 cells expressing GFP were inefficient in killing the cancer cell line SUP-B15. SUP-B15 is an acute lymphoblastic leukemia cell line that is CD19 positive and resistant to NK-92-mediated cytotoxicity. Compared to the control (NK-92 cells expressing GFP), the expression of any of the CD19CARs tested provided increased cytotoxic activity against the SUP-B15 cell line, which can be easily seen from Figure 3B Unexpectedly, CARFcRe exhibited similar or better cytotoxicity than second- and third-generation CARs. This finding was particularly unexpected because the FcεRIγ signaling domain acts only as a single unit and is not combined with other signaling domains. When used in CART cells, such an arrangement does not provide the expected targeted cytotoxicity. Advantageously, the tricistronic mRNA construct is able to produce a large number of expected CARs with excellent functional activity. Such constructs are particularly beneficial in cases where CAR expression should be transient.

[0151] Degranulation is a key step required for releasing lytic proteins (e.g., perforin and granzymes) from secretory granules in NK-92 cells. Target cells are identified by NK-92 to initiate degranulation. In order to detect degranulation in the construct, effector cells (NK-92) and unlabeled target cells (SUP-B15) were mixed in 96-well plates with different effect-target ratios (5: 1 to 0.3: 1), and anti-CD107a (FITC-conjugated, BD Pharmingen, San Jose, California) was added to each well. The culture plate was incubated at 37°C in a CO2 incubator, and monensin (Golgi-stop) was added to the well after 1 hour. The culture plate was incubated for another 3h at 37°C, and the sample was analyzed by flow cytometry (Attune, Life Technologies, Carlsbad, California). The percentage of degranulation was determined by subtracting the % CD107a positivity in NK-92 cells from the % CD107a positivity in the effector+target samples alone. Figure 4 Exemplary results are provided.

[0152] Example 4: CD19 t-haNK cells significantly improved animal survival in the Raji tumor xenograft model.

[0153] CD19 t-haNK cells containing the Fcε intracellular signaling domain (clone 19.6) CD19 t-haNK cells were cultured in X-VIVOTM 10 medium supplemented with 5% heat-inactivated human AB serum.

[0154] Experimental animals: Animal strain / species: NOD.Cg-PrkdcscidIl2rgtm1Wjl / SzJ (NSG) mice; Age: 9-10 weeks at the start of the study (after isolation); Sex: female; Weight: 20-27 g at the start of the study. Number of animals: 20 for IV tumor model; 12 for SC tumor model. Supplier: Jackson Laboratory (610 Main Street Bar Harbor, ME04609).

[0155] Raji Tumor Model: Raji Cancer Cell Line: Raji cells were originally purchased from ATCC (Catalog No. CCL-86TM; Lot No. 61723871), then expanded and prepared for administration.

[0156] Cell culture medium: RPMI-1640 medium prepared by ATCC, supplemented with 10% fetal bovine serum, penicillin (100 U / mL), and streptomycin (100 μg / mL).

[0157] Cell harvesting: Raji cells in the exponential phase (passage 12) were collected by centrifugation. The cells were washed and plated at 5 × 10 5 They were resuspended in serum-free medium at a concentration of 10 viable cells / mL for IV inoculation and then inoculated at 2.5×10 6 The cells were resuspended in culture medium / Matrigel (1:1 v / v) at a concentration of 10 viable cells / mL for SC implantation. The cells were kept on ice before animal injection. The cells used in the in vivo study had a viability of 96%.

[0158] Raji cell inoculation: Raji IV model. Use a 27-gauge needle (1×10 5 20 animals were injected IV with 0.2 mL of Raji cell suspension via the lateral tail vein. Raji SC model. A 25-gauge needle (2.5 × 10 5 0.1 mL of Raji cell suspension was implanted SC into the bilateral axillae of 12 animals (cell inoculum).

[0159] Other reagents: RPMI-1640 medium, X-VIVO TM10 culture 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 base membrane matrix; Pluronic (R) F-68, 10% solution.

[0160] Experimental procedures

[0161] IVRaji Model - Randomization: Within 24 hours after cancer cell inoculation defined as day 1, 20 animals were pseudo-randomly divided into 2 groups of 10 animals each based on body weight to achieve similar mean body weights between groups.

[0162] Test Article: CD19 t-haNK cells growing in exponential phase were harvested by centrifugation on days 2, 5, 8, 10, 12, and 17 and cultured in X-VIVO TM 10 is prepared as 5×10 7 The concentration of cells / mL was 1×10 per mouse. 7 The dose of 10 cells was administered IV in a volume of 200 μL. Animals in Group A received vehicle control, while animals in Group C received CD19t-haNK cells.

[0163] Body Weight: Animals were weighed prior to tumor cell injection and twice weekly.

[0164] Clinical Observations: Animals were observed daily for mortality / morbidity (G0 to G4) and clinical signs of toxicity. Paralyzed or moribund animals were euthanized.

[0165] Euthanasia: Animals were euthanized by CO2 inhalation followed by cervical dislocation. Mortality events (euthanasia or spontaneous) were recorded in a death log (Appendix 6) and survival curves were calculated.

[0166] SC Raji Model - Tumor Volume Measurement: After implantation of SC tumors, animals were examined at least twice a week for tumor formation. When tumors became evident, tumor volume (TV) was measured once or twice a week using a digital handheld caliper and calculated using the following formula: TV = length × width 2 / 2 [length is the largest diameter of the tumor, width is the smallest diameter of the tumor].

[0167] Randomization: When the mean tumor volume reached an injectable size (in this case 195 mm3; 24 days after implantation), 12 tumor-bearing animals were pseudo-randomized into 2 groups of 6 to obtain similar tumor volumes between groups. This was defined as day 0.

[0168] Test article: On days 1, 4, 7, 9, 11, and 13, CD19 t-haNK cells growing in the exponential phase were collected by centrifugation, irradiated with 1000 cGy of gamma rays, and then analyzed by X-VIVO. TM 5×10 7 The concentration was 1×10 cells / mL for each mouse. 7 The dose of 10 cells was administered IV in a volume of 200 μL. As shown in Table 1, animals in group D received vehicle solution, while animals in group F received CD19 t-haNK cells.

[0169] Body Weight. Animals were weighed prior to tumor cell injection and then twice weekly.

[0170] Clinical Observations. Animals were observed daily for mortality / morbidity (G0 to G4) and clinical signs of toxicity (T1 to T12). Paralyzed or moribund animals were euthanized.

[0171] Endpoint and euthanasia. Moribund animals were euthanized as soon as they showed signs of morbidity, but surviving animals were euthanized regularly for tissue collection. Specifically, half of the surviving animals (maximum 3 mice / group) were euthanized on day 13, 6 hours after the last administration of the test article. The remaining animals were euthanized on day 15, 48 hours after the last administration.

[0172] Necropsy and Tumor and Tissue Collection. Following termination, necropsy was performed and organs with macroscopic lesions were collected, fixed in 10% formalin, and sent to a contract pathology laboratory (Seventh Wave Laboratories) for histological evaluation of tumor / metastatic disease burden.

[0173] Table 1

[0174]

[0175] IR, irradiation (1000 cGy); non-IR, non-irradiation; IV, intravenous; SC, subcutaneous; Tx, treatment.

[0176] Data analysis

[0177] Tumor volume calculation: tumor volume = length × width 2 / 2 (length and width are the longest and shortest diameters of the tumor, respectively); tumor growth inhibition (TGI) calculation: TGI = (TC-Tt) / ΔTC×100%, where TC and Tt are the average tumor volumes of the control group and the treatment group at the end of the study, respectively, and ΔTC is the average tumor volume of the control group.

[0178] Statistical analysis-Tumor growth curves: Tumor growth curves were analyzed by two-way ANOVA followed by Tukey's test for multiple comparisons.Survival curves: Survival curves were analyzed by the log-rank (Mantel-Cox) test.

[0179] Liver metastasis estimation: Differences in liver metastatic disease burden on each day 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.

[0180] result

[0181] IV Raji Model: The primary readout in the IV tumor model is animal survival. Death events are counted when an animal is found dead or euthanized due to disease-related morbidity and / or paralysis. Figure 5 As shown, CD19 t-haNK cell treatment was able to significantly improve the survival rate of animals compared with the vehicle control group, resulting in a median survival of 27 days compared with 21.5 days in the vehicle control group (P<0.0001).

[0182] Animal body weight changes were also monitored throughout the study. Figure 6 As shown, when treatment was first initiated, animals treated with CD19 t-haNK exhibited moderate (less than 10%) and short-term weight loss, which is common in animals receiving IV NK infusions and is not specific to CD19 t-haNK cells (reference study: LABC-TX01701). After the first week of treatment, animals regained weight and then lost it again due to disease progression.

[0183] SC Raji Model: The primary readout in the SC tumor model is tumor growth. Figure 7 As shown, CD19 t haNK cells exhibited a clear and statistically significant tumor growth inhibition compared to the vehicle control group on day 7 and thereafter, with a TGI of 49% at the end of the study (day 13).

[0184] In addition, since Raji is an aggressive lymphoma model, even with SC inoculation, cancer cells are able to spread and form multiple metastatic sites, ultimately leading to animal morbidity and / or death. A total of 3 animals (50%) in the vehicle group became moribund between days 11 and 13 and were therefore euthanized. In contrast, there were no unplanned deaths in the CD19 t-haNK cell group (Table 3).

[0185] Furthermore, a qualitative reduction in liver metastases was observed in CD19 t-haNK treated animals during necropsy ( Fig. 8ASemiquantitative estimates of disease burden were performed by a contract pathology laboratory (Seventh Wave Laboratories) on representatively sampled H&E-stained liver sections. Figure 8B As shown in Table 4, there was a clear trend toward increasing disease burden as the study progressed. The livers of CD19 t-haNK treated animals showed a significantly lower percentage of cancer infiltrated areas compared to vehicle controls. Due to the small sample size and excessive unplanned mortality in the control group, statistical analysis was only possible for data through day 13. This analysis showed a significant difference in disease burden, with an average infiltration rate of 10% in CD19 t-haNK treated animals compared to 30% in the control group.

[0186] Body weight changes were monitored throughout the study and, similar to the IV Raji model, CD19t-haNK treated animals showed moderate (less than 10%) and transient weight loss at the start of the treatment regimen. Fig. 9 get.

[0187] Table 2

[0188]

[0189] Plan: Plan euthanasia for tissue collection.

[0190] Table 3

[0191]

[0192] To evaluate the antitumor efficacy of CD19 t-haNK cells in a repeated IV dosing regimen, 2 variations of the Raji xenograft model with IV and SC tumor inoculation, respectively, were used in this study.

[0193] In the IV tumor model, CD19 t-haNK cells significantly improved animal survival, with a median survival extension of 5.5 days (26% increase) compared to the vehicle control group. In the SC tumor model, CD19 t-haNK cells significantly inhibited tumor growth, resulting in a 49% TGI at the end of the study. In addition, CD19 t-haNK treatment reduced the number of animal morbidity / mortality events (0 / 6 animals treated with CD19 t-haNK vs. 3 / 6 controls) and significantly reduced the burden of metastatic disease in the liver of SC Raji tumor-bearing animals.

[0194] As can be seen from the above data, CD19t-haNK cells showed significant therapeutic efficacy compared to vehicle controls in both variants of the Raji xenograft model.

[0195] Example 5. Treatment of mice bearing L1210 tumors with CD19-CAR-NK-92 cells improves survival and has an Mice that achieved complete remission rejected L1210 tumor allografts upon rechallenge.

[0196] Experimental Design: Thirty (30) male DBA / 2J mice (Jackson Laboratories) aged 6-8 weeks were enrolled after randomization on day 0. All animals were housed under standard environmental conditions and fed LabDiet 5053 irradiated rodent chow, with sterile water provided ad libitum. Upon arrival, animals were identified by ear tags, housed in ten (10) cages, and acclimated for at least three days prior to the start of the study. After acclimation, the injection area of ​​each mouse was shaved and cleaned with a sterile EtOH swab. On day PR0 (day 0 before randomization), animals were anesthetized with isoflurane for injection of tumor cells. On day PR0, all animals were injected subcutaneously (sc) with 2×10 5 L1210-Luc tumor cells were added to the 100 mL serum-free DMEM. All animals were measured daily for tumor size using a digital caliper starting on PR day 7. On PR day 7, when tumor size was approximately 50–150 mm 3 The average tumor volume is about 100 mm 3 Select twenty (20) tumors closest to 100 mm 3 Animals were selected for the study; these animals were randomly divided into two (2) groups of ten (10) animals each. The day of randomization was considered as day 0 of the study and treatment began on this day. Animals not selected for the study were immediately euthanized by overdose of CO2. Animals in group 1 were administered vehicle (serum-free DMEM) by intratumoral (it) injection of 50 μl. Animals in group 2 were injected it with 2×10 6 mCD19-CAR-aNK cells. The same treatment was performed on days 0, 2, and 4 of the study.

[0197] Animals were weighed daily and their general health was monitored. After randomization, tumors were measured 3 times per week (3x / week) using digital calipers. 3 or tumor ulceration; lost more than 30% of its initial body weight (on day 0); or was euthanized by overdose of CO2 for the indicated cause of death / sacrifice, found moribund, distressed, or paralyzed. On day 30, animals in complete remission and five (5) additional virgin male DBA / 2J mice (Jackson Laboratories; Barrier) at 10 weeks of age (comprising Group 4) were injected subcutaneously (sc) into the left axilla with a re-stimulated tumor cell inoculum of 2x105 L1210-Luc tumor cells in a volume of 0.1 mL serum-free DMEM. All animals continued to be weighed and monitored daily until day 60, with tumors measured 3x / week.

[0198] result

[0199] Animal survival to welfare thresholds - initial tumor challenge: Animal survival was monitored daily. Animals that required euthanasia based on animal health and welfare thresholds, including weight loss greater than 30% of their initial body weight, tumors greater than 2500 mm, and 3 Animals that were unable to obtain food / water or were moribund were included. Animals that required euthanasia due to ulcerated tumors were not included in the survival analysis.

[0200] Cumulative survival of animal welfare thresholds over time Fig.10 As shown. L1210 is a rapidly growing, invasive tumor cell line, 0% of vehicle-treated control animals were alive twenty-three (23) days after tumor challenge. In contrast, CD19-CAR-aNK cell treatment increased survival compared to vehicle treatment. In fact, 25% (2 / 8) of animals treated with CD19-CAR-aNK cells survived until completion of the study with tumor graft challenge at day 61.

[0201] The observed survival increase provided by the experimental treatment was evaluated by log-rank sum (Mantel-Cox) and Gehan-BreslowWilcoxon test with statistical significance. Treatment with mCD19-CAR-aNK cells can statistically significantly improve survival (p=0.05 (Mantel-Cox); p=0.04 (Gehan-Breslow-Wilcoxon). These results show that in the preclinical subcutaneous model of mouse lymphocytic leukemia, treatment with CD19-CAR-aNK statistically significantly improves survival to the welfare threshold compared with the vehicle.

[0202] Tumor Rechallenge in Complete Remission Animals: On Day 33, two (2) complete remission animals from Group 2 and five (5) age-matched naive animals were rechallenged with a second 2×10 5 The animals were challenged / re-challenged with an inoculum of L1210-Luc cells and then injected into the contralateral (left) axilla (primary tumor inoculated on the right side). Animals were monitored daily for survival. Animals that required euthanasia based on animal health and welfare thresholds, including weight loss greater than 30% of their initial body weight, tumors greater than 2500 mm 3 Animals that were unable to obtain food / water or were moribund were included. Animals that required euthanasia due to ulcerated tumors were not included in the survival analysis.

[0203] Before day 52, all original animals (5 of 5) that met the requirements for survival analysis required euthanasia due to tumor volume; in contrast, all animals with complete remission previously treated with 2MCD19-CAR-aNK (N=2) cells survived until study completion (day 62). The observed increase in survival provided by the experimental treatment was statistically significant as assessed by log-rank sum (Mantel-Cox) and Gehan-Breslow Wilcoxon tests, but the increase in survival was not statistically different, most likely due to the small sample size.

[0204] During the rechallenge phase, tumors were measured 3 times per week (3x / week). The mean tumor volume + SEM of each group from the administration of the priming / rechallenge L1210-Luc cells to 0% control survival (day 52) is shown as Fig.11 shown.

[0205] Tumors were first detected in the original animals about seven days after administration (study day 40) and increased steadily and rapidly. In contrast, no tumors were detected at any time during the entire re-challenge phase (days 33-61) until re-challenge to animals in complete remission that had been pre-treated with 2M CD19-CAR-aNK cells.

[0206] The data presented in this example suggest that animals in complete remission that had been previously treated with 2M CD19-CAR-aNK cells may have mounted an effective immune response against L1210 tumor cells.

[0207] Example 6: Treatment of A20 tumor-bearing mice with mCD19-CAR-NK-92 cells improves survival and has a positive effect on Mice in complete remission rejected A20 tumor allografts upon rechallenge.

[0208] Experimental design

[0209] Part A: Forty (40) 5-7 week old BALB / c mice (20 males and 20 females) were obtained from Taconic Biosciences for use in Part A. On day 0 of randomization (PR), 2.5×10 6 Animals were injected subcutaneously (sc) with A20 murine lymphoma cells. Tumors were measured once a day starting on day PR7. Ten (10) days after tumor cell implantation (day PR10; day 0), mice were randomized to treatment groups so that each group contained animals with tumors of similar volume and extent. The day of randomization was considered day 0 of the study. Tumors were measured 3 times per week (3x / week) with a digital caliper to monitor tumor growth until Part A was completed on day 26.

[0210] On days 0, 3, and 5, mice were injected intratumorally (it) with test cells or vehicle in 50 μl of serum-free medium according to a predefined it procedure (see Experimental Procedures). Briefly, animals were administered vehicle alone or 5 × 10 6 mCD19-CAR-NK-92. On day 26, no cells larger than 40 mm 3 Animals with tumors were removed from the study and euthanized by CO2 asphyxiation; enrolled animals that showed complete response (CR; tumors >40 mm 3 Subsided and was not detected for many days before day 26 (0 mm 3 ) without recurrence) were selected into Part B.

[0211] Part B: Part B started on day 26. Tumor-free animals from Part A and twelve (12) naive animals (6 males and 6 females) were enrolled in Part B. 2.5×10 6 A20 cells were administered to the right axilla of all animals in Part B. Tumors were measured twice a week. Animals were euthanized on day 57.

[0212] Results: Part A - Survival: The overall health and survival of the animals were monitored daily. Animals were euthanized based on animal health and welfare thresholds, including weight loss greater than 30% of their initial body weight, tumors greater than 1500 mm, and 3 Animals that were unable to survive, had no access to food / water, or were moribund were included. Animals that required euthanasia due to ulcerated tumors were not included in the survival analysis. 3 , all animals considered in the survival analysis were euthanized. Because the subcutaneous tumor burden threshold represents an arbitrary cutoff point, the analysis of “survival” in this context should be considered only as an indicator of relative tumor growth. Fig.12 Cumulative survival over time for all animals considered is shown.

[0213] In the control animals administered intratumorally (it) on days 1, 3, and 5, 0 of 15 animals (0%) survived to complete Part A on day 26. For all treated animals, the survival of the animals on day 26 increased: 9 of 18 animals (50%) were administered 5M mCD19-CAR-NK92 cells. All groups were compared by the log-rank sum (Mantel-Cox) test. A statistically significant increase in the survival of animals administered 5MmCD19-CAR-NK92 cells was observed compared to animals administered the vehicle (p=<0.0001). These results indicate that all treatments improved survival to day 26 compared to vehicle treatment.

[0214] Part B - Tumor Rechallenge in Animals with Complete Remission: Animals with complete remission (those bearing tumors > 40 mm in response to treatment during the 0-26 days (Part A) schedule) will be rechallenged. 3 , so that the tumor volume measured on day 26 was 0.00 mm 3 , no regrowth or recurrence) were re-challenged on day 27 with a second subcutaneous inoculum into the axilla (opposite to the first graft) containing 2.5 × 10 6 A20 tumor cells; the rechallenge portion of the study was designated Part B. Twelve additional animals were enrolled in Part B as primary controls; on Day 27, six (6) male and six (6) female age-matched BALB / c mice supplied at the same time as the mice in Part A were administered 2.5×10 6 A20 tumor cells. Tumors were measured 3 times a week in all animals until day 57. The mean tumor volume + SEM of each A-treated group and the original control group is shown in Fig.13 As shown. Tumors generated after cell inoculation into the original animals grew steadily as expected; however, no active tumors (>40mm) were generated in animals that were re-stimulated with tumor cell inoculation to complete remission. 3 ).

[0215] In summary, the data presented in this example demonstrate that previously treated mice that were in complete remission to treatment were able to reject A20 tumor allografts that were rechallenged unrelated to treatment compared to naive mice and suggest that these animals developed memory remissions to tumor antigens.

[0216] The following examples of targeted CAR constructs and associated functional data are from linearized DNA vector constructs that allow transfection of cells to integrate the linearized DNA into the genome, providing a pathway for non-transient expression of a specific CAR.

[0217] Example 7: HER2-CAR with FcεRIγ signaling domain

[0218] In this example, the inventors constructed a first generation CAR with an FcεRIγ signaling domain, which includes an anti-HER2 scFv coupled to a CD8 hinge, which in turn is coupled to a CD28 transmembrane domain, which is coupled to an FcεRIγ signaling domain. The HER2-CAR thus constructed has a nucleic acid sequence shown in SEQ ID NO: 60.

[0219] The function of the HER2.CAR-t-haNK cells constructed in this way on BT-474 cells was tested using a standard cytotoxicity assay, and the exemplary results are shown in Fig.14As shown. It is easy to see from the data that HER2.CAR-t-haNK cells expressing CAR with FcεRIγ signaling domain show significant cytotoxicity against BT-474 target cells.

[0220] In further experiments, the present inventors demonstrated the expression of HER2.CAR in HER2.CAR-t-haNK cells, such as Fig.40 The natural cytotoxicity of HER2.CAR-t-haNK cells is shown in Fig.41 As shown in Figure 2, CAR-mediated cytotoxicity results are shown in Figure 2. Fig.42 shown. Fig.43 The figure shows exemplary data of ADCC of HER2.CAR-t-haNK cells.

[0221] Example 8: CD30-CAR with FcεRIγ signaling domain

[0222] In this example, the inventors constructed a first generation CAR with an FcεRIγ signaling domain, which includes an anti-CD30 scFv coupled to a CD8 hinge, which in turn is coupled to a CD28 transmembrane domain, which is coupled to an FcεRIγ signaling domain. The CD30-CAR thus constructed has a nucleic acid sequence shown in SEQ ID NO: 61.

[0223] Fig.50 The results demonstrated the expression of CD30-CAR, while Fig.51 The natural cytotoxicity results of the recombinant cells are shown. Fig.52 The results demonstrated CAR-mediated cytotoxicity, while Fig.53 Exemplary results for ADCC are shown in the data of FIG.

[0224] Example 9: EGFR-CAR with FcεRIγ signaling domain

[0225] In this example, the inventors constructed a first generation CAR with an FcεRIγ signaling domain, which includes an anti-EGFR scFv coupled to a CD8 hinge, which in turn is coupled to a CD28 transmembrane domain, which is coupled to an FcεRIγ signaling domain. The EGFR-CAR thus constructed has a nucleic acid sequence shown in SEQ ID NO: 62.

[0226] The function of the EGFR.CAR-t-haNK cells constructed in this way on A-549 cells was tested using a standard cytotoxicity assay, and the exemplary results are shown in Fig.17As shown. It is easy to see from the data that EGFR.CAR-t-haNK cells expressing CAR with FcεRIγ signaling domain showed significant cytotoxicity against A-549 target cells. Fig.35 The expression of EGFR-CAR in EGFR.CAR-t-haNK cells is shown, while the natural cytotoxicity results are shown in Fig.36 The exemplary results of CAR-mediated EGFR.CAR-t-haNK cell cytotoxicity are shown in Fig.37 and Fig.38 As shown in Figure 2, while the ADCC results of EGFR.CAR-t-haNK cells are shown in Figure 2 Fig.39 shown.

[0227] Example 10: IGF1R-CAR with FcεRIγ signaling domain

[0228] In this example, the inventors constructed a first generation CAR with an FcεRIγ signaling domain, which includes an anti-IGF1R scFv coupled to a CD8 hinge, which in turn is coupled to a CD28 transmembrane domain, which is coupled to an FcεRIγ signaling domain. The IGF1R-CAR thus constructed has a nucleic acid sequence shown in SEQ ID NO: 63, and encodes IGF1R-CAR, CD16 and IL-2. ER The tricistronic construct has the nucleic acid sequence shown in SEQ ID NO: 76, which is also Fig.65 As shown in FIG.

[0229] Compared with the second generation CAR (CD28 / CD3z), the function of IGF1R.CAR-t-haNK cells constructed in this way on MDA-MB-231 cells was evaluated using a standard cytotoxicity assay, and exemplary results are shown in Fig. 22 As shown in the data, it can be easily seen that IGF1R.CAR-t-haNK cells expressing CAR with 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.

[0230] Example 11: CD123-CAR with FcεRIγ signaling domain

[0231] In this example, the inventors constructed a first generation CAR with an FcεRIγ signaling domain, which includes an anti-CD123 scFv coupled to a CD8 hinge, which in turn is coupled to a CD28 transmembrane domain, which is coupled to an FcεRIγ signaling domain. The CD123-CAR thus constructed has a nucleic acid sequence shown in SEQ ID NO: 64. Fig.48 CAR-mediated cytotoxicity data of recombinant NK cells expressing CD123-CAR are shown. Fig.49 Exemplary data of ADCC of recombinant NK cells expressing CD123-CAR are shown.

[0232] Example 12: PD-L1-CAR with FcεRIγ signaling domain

[0233] In this example, the inventors constructed a first generation CAR with an FcεRIγ signaling domain, which includes an anti-PD-L1 scFv coupled to a CD8 hinge, which in turn is coupled to a CD28 transmembrane domain, which is coupled to an FcεRIγ signaling domain. The PD-L1-CAR thus constructed has a nucleic acid sequence shown in SEQ ID NO: 65.

[0234] The cytotoxicity of PD-L1.CAR-t-haNK cells constructed in this way to SUP-B15.PD-L1 was detected using a standard cytotoxicity assay. + Cell function, exemplary results such as Fig.16 As shown. It is easy to see from the data that PD-L1.CAR-t-haNK cells expressing CAR with FcεRIγ signaling domain show inhibition of SUP-B15.PD-L1 + The target cells have significant cytotoxicity.

[0235] The function of the PD-L1.CAR-t-haNK cells thus constructed on U251 cells was also tested using a standard cytotoxicity assay, and the exemplary results are shown together with untransfected haNK cells. Fig.17 As shown. It is easy to see from the data that PD-L1.CAR-t-haNK cells expressing CAR with FcεRIγ signaling domain exhibit target-specific and significant cytotoxicity against U251 target cells, while haNK control cells have essentially no cytotoxicity against the same U251 cells.

[0236] In further experiments on the target cell specificity of PD-L1, the inventors tested several PD-L1 positive tumor cell lines using PD-L1.CAR-t-haNK cells as well as haNK cells as controls for general cytotoxicity. Fig.24 It can be easily seen that PD-L1.CAR-t-haNK cells are effective against a variety of tumor cells (lung cancer, breast cancer, genital tumor cells, small cell carcinoma of the head and neck, chordoma). It is worth noting that PD-L1.CAR-t-haNK cells take less than 4 hours to kill most (>85%) cells, while control haNK cells take more than 12 hours.

[0237] Fig.24 The cytotoxicity of PD-L1.CAR-t-haNK cells to MDA-MB-231 cells compared to various other control cells (such as haNK cells as shown) is further illustrated. As can be seen from the data, at an E:T ratio of 5:1, cetuximab improves the dissolution of MDA-MB-231 by PD-L1.thaNK, and haNK activity can be improved by adding cetuximab and a-PD-L1. Compared with haNK and haNK+cetuximab, ordinary PD-L1.thank has better cytotoxic activity, and the killing of ordinary PD-L1.thank is comparable to that of haNK+PD-L1 antibodies, but PD-L1.thank+cetuximab outperforms haNK+cetuximab and haNK+PD-L1. At an E:T ratio of 1:1, the activity of PD-L1.thaNK with or without cetuximab is the same, and PD-L1.thaNK is significantly superior to intrinsic and ADCC-mediated killing by hank. HaNK activity was improved by the addition of cetuximab and a-PD-L1.

[0238] In further experiments, the inventors demonstrated the expression of PD-L1.CAR in PD-L1.CAR-t-haNK cells, such as Fig.44 As shown. The natural cytotoxicity of PD-L1.CAR-t-haNK cells is shown Fig.45 The results of CAR-mediated cytotoxicity are shown in Fig.46 Exemplary data of ADCC of PD-L1.CAR-t-haNK cells are shown in Fig.47 shown.

[0239] Example 13: CD33-CAR with FcεRIγ signaling domain

[0240] In this example, the inventors constructed a first generation CAR with an FcεRIγ signaling domain, which includes an anti-HER2 scFv coupled to a CD8 hinge, which in turn is coupled to a CD28 transmembrane domain, which is coupled to an FcεRIγ signaling domain. The CD33.CAR thus constructed has a nucleic acid sequence shown in SEQ ID NO: 66.

[0241] The function of the CD33.CAR-t-haNK cells constructed in this way on THP-1 cells was tested using a standard cytotoxicity assay, and the exemplary results are shown in Fig.15 As shown. It is easy to see from the data that CD33.CAR-t-haNK cells expressing CAR with FcεRIγ signaling domain show significant cytotoxicity against THP-1 target cells. Further data describing the strong expression of CD33CAR in NK-92 cells are shown in Fig.31 shown. Fig.32 The natural cytotoxicity of CD33.CAR-t-haNK cells to K562 cells is shown. Fig.33 The results of CAR-mediated cytotoxicity against THP-1 cells are described. Fig.34 It further shows that CD33.CAR-t-haNK cells combined with rituximab have an inhibitory effect on SUP-B15 CD19 KO / CD20 + The results of ADCC.

[0242] Example 14: gp120-CAR with FcεRIγ signaling domain

[0243] In this example, the inventors constructed a first generation CAR with an FcεRIγ signaling domain, which includes an anti-gp120 scFv coupled to a CD8 hinge, which in turn is coupled to a CD28 transmembrane domain, which is coupled to an FcεRIγ signaling domain. The gp120-CAR thus constructed has a nucleic acid sequence as shown in SEQ ID NO: 67.

[0244] The inventors further demonstrated that the cells thus generated expressed large amounts of CD16 and gp120 CAR, such as Fig.57 As shown. The binding of GP120 to gp120CAR is shown Fig.58 As shown, compared to non-recombinant aNK cells as a negative control. The natural cytotoxicity of the cells thus generated was Fig.59 The corresponding ADCC data are shown in Fig.60 shown.

[0245] Example 15: B7-H4-CAR with FcεRIγ signaling domain

[0246] In this example, the inventors constructed a first generation CAR with an FcεRIγ signaling domain, which includes an anti-B7-H4 scFv coupled to a CD8 hinge, which in turn is coupled to a CD28 transmembrane domain, which is coupled to an FcεRIγ signaling domain. The B7-H4-CAR thus constructed has a nucleic acid sequence as shown in SEQ ID NO: 68.

[0247] Example 16: BCMA-CAR with FcεRIγ signaling domain

[0248] In this example, the inventors constructed a first generation CAR with an FcεRIγ signaling domain, which includes an anti-BCMA scFv coupled to a CD8 hinge, which in turn is coupled to a CD28 transmembrane domain, which is coupled to an FcεRIγ signaling domain. The BCMA-CAR thus constructed has a nucleic acid sequence shown in SEQ ID NO: 69.

[0249] like Fig.54 As shown in the exemplary results of Fig.55 As shown, CAR-mediated cytotoxicity against target cells was demonstrated. Fig.56 As can be seen from the results, using rituximab as the antibody against target cells, the recombinant cells have significant ADCC.

[0250] Example 17: GD2-CAR with FcεRIγ signaling domain

[0251] In this example, the inventors constructed a first generation CAR with an FcεRIγ signaling domain, which includes an anti-GD2 scFv coupled to a CD8 hinge, which in turn is coupled to a CD28 transmembrane domain, which is coupled to an FcεRIγ signaling domain. The GD2-CAR thus constructed has a nucleic acid sequence shown in SEQ ID NO: 70.

[0252] Example 18: FAP-CAR with FcεRIγ signaling domain

[0253] In this example, the inventors constructed a first generation CAR with an FcεRIγ signaling domain, which includes an anti-FAP scFv coupled to a CD8 hinge, which in turn is coupled to a CD28 transmembrane domain, which is coupled to an FcεRIγ signaling domain. The FAP-CAR thus constructed has a nucleic acid sequence as shown in SEQ ID NO: 71. The expression of FAP-CAR is as follows Fig.61 The data show that FAP.CAR has a cytotoxic effect on target cells. Fig.62 This is proved in the results.

[0254] Example 19: CD20-CAR with FcεRIγ signaling domain

[0255] In this example, the inventors constructed a first generation CAR with an FcεRIγ signaling domain, which includes an anti-CD20 scFv coupled to a CD8 hinge, which in turn is coupled to a CD28 transmembrane domain, which is coupled to an FcεRIγ signaling domain. The CD20-CAR thus constructed has a nucleic acid sequence shown in SEQ ID NO: 74.

[0256] Expression of CD20 CAR in NK-92 cells Fig.29 As shown in the results. It can be easily seen that CD20.CAR is strongly expressed in the vast majority of recombinant cells (as described above, together with CD16 in linearized DNA). Fig.30 Describes the CD20.CARNK cell response to CD20 + Exemplary results of target cell cytotoxicity.

[0257] Example 20: CSPG-4-CAR with FcεRIγ signaling domain

[0258] In this example, the inventors constructed a first generation CAR with an FcεRIγ signaling domain, which includes an anti-CSPG-4 scFv coupled to a CD8 hinge, which in turn is coupled to a CD28 transmembrane domain, which is coupled to an FcεRIγ signaling domain. The CSPG-4-CAR thus constructed has a nucleic acid sequence as shown in SEQ ID NO: 75. The expression of CSPG-4-CAR was confirmed by FACS analysis, and the exemplary results are shown in FIG. Fig.63 Therefore, the constructed cells also showed significant cytotoxicity, as shown in Fig.64 The exemplary data are shown in FIG.

[0259] Example 21: CD19-CAR with FcεRIγ signaling domain

[0260] In this example, the inventors used the first generation CAR with an FcεRIγ signaling domain as described above, which included an anti-CD19 scFv coupled to a CD8 hinge, which in turn was coupled to a CD28 transmembrane domain, which was coupled to FcεRIγ signaling, and transfected NK-92 cells with linearized DNA for functional testing.

[0261] The function of the CD19.CAR-t-haNK cells constructed in this way on K562 cells was tested using a standard cytotoxicity assay to determine general cytotoxicity, and exemplary results are shown in Fig.19 As shown. It can be easily seen that CD19.CAR-t-haNK cells expressing CAR with FcεRIγ signaling domain showed significant cytotoxicity against K562 target cells. In another set of experiments, target-specific cytotoxicity was determined using SUP-B15 cells compared with aNK cells as a control, and the exemplary results are shown in Fig. 20 Again, CD19.CAR-t-haNK cells expressing CAR with FcεRIγ signaling domain showed significant target-specific cytotoxicity. In another set of experiments, Herceptin and Rituximab were used as antibodies and SKBr3 cells were used to determine target-specific ADCC. The results are shown in Fig.21 As shown. Similarly, CD19.CAR-t-haNK cells expressing CAR with FcεRIγ signaling domain exhibited significant antibody and target-specific ADCC. Notably, the doubling time of recombinant NK cells was essentially the same as that of aNK cells.

[0262] Fig.25 The results are shown in an exemplary manner in NK-92 cells relative to the control from linearized DNA (which includes DNA encoding CD16 and IL-2 ER CD19.CAR expression of fragments of Fig.25 It can be seen that the expression is very strong in most cells. Other results of CD19.CARt-haNK cells' natural cytotoxicity against K562 cells and targeted cytotoxicity against SUP-B15 cells are shown in Figure 2. Fig.26 and Fig. 27 As shown. CD19.CAR t-haNK cells against SUP-B15CD19 KO / CD20 + Exemplary other results of ADCC of cells are as follows Fig.28 shown.

[0263] Example 22: Antitumor activity of t-haNK cells targeting PD-L1 in NSG mouse xenograft model

[0264] MDA-MB-231 and HCC827 were used as validated xenograft models for PD-L1 positivity, and the efficacy of PD-L1t-haNK cells was evaluated in different formulations, dosing levels, and routes of administration (IV and IT).

[0265] Animals: Animal type: NSG mice (JAX), female, 9-10 weeks old; number of animals for MDA-MB-231 model: 24 (fresh cells), and number of animals for HCC827 model: 24 (fresh cells) + 6 (cryopreserved cells). The tumor models used the following cell lines: MDA-MB-231 (human breast adenocarcinoma) and HCC827 (human lung adenocarcinoma), the inoculation route was subcutaneous inoculation into the bilateral axilla, and the average tumor load of MDA-MB-231 was approximately 100 mm 3 , HCC827 is about 75-80mm 3 .

[0266] Treatments: Freshly prepared anti-PD-L1 t-haNK, irradiated, at 5E7 cells / mL or 2E7 cells / mL; vehicle control was X-VIVO TM 10 culture medium; as described above, the method of administration was IV and IT as described. The dose for IV NK administration was 1E7 cells / dose in 200 μL (freshly prepared cells) and 4E6 cells / dose in 200 μL (cryopreserved cells); the dose for IT NK administration (fresh cells only) was 2.5E6 cells / tumor in 50 μL. The dosing frequency (M / Th or T / F) was twice a week for 4 consecutive weeks, and the first day of dosing was defined as Day 1.

[0267] The study design for MDA-MB-231 is in Table 4 below (the study ended on Day 27 when some animals in Groups A, C, and D had total tumor volumes >2000 mm 3 )

[0268] Table 4

[0269]

[0270] The study design for HCC827 is in Table 5 below (the study ended at Day 29 when surviving animals were rescheduled and transferred to another study).

[0271] Table 5

[0272]

[0273] Results: Freshly prepared PD-L1 t-haNK cells (1E7 cells / dose) resulted in significant and durable tumor growth inhibition in both MDA-MB-231 and HCC827 models MDA-MB-231: tumor stasis: TGI on day 16: 84% (peak); TGI on day 26: 79% (last measurement).

[0274] HCC827: Tumor regression: TGI on day 16: 120% (peak); TGI on day 29: 84% (end of study).

[0275] Cryopreserved PD-L1 t-haNK cells (4E6 cells / dose) also demonstrated statistically significant efficacy in inhibiting tumor growth compared to X-VIVOTM 10 medium: TGI on Day 26: 60% (peak), TGI on Day 29: 40% (end of study).

[0276] 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.

[0277] Table 6

[0278]

[0279] The number of visible nodules in the liver in the vehicle group was: 29±9, and in the PD-L1 t-haNK group: 0 (P=0.0116 by unpaired two-tailed t-test).

[0280] According to the experiments performed, IV administration of freshly prepared PD-L1 t-haNK cells at a level of 1E7 cells / dose twice a week for 4 weeks showed significant antitumor efficacy in both subcutaneous xenograft models tested: the treatment resulted in 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 multiple comparisons by Tukey's test), as well as 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). IV administration of cryopreserved PD-L1 t-haNK cells at a dosing level of 4E6 cells / dose twice a week for 4 consecutive weeks also showed significant therapeutic efficacy in the HCC827 tumor model, reaching a peak TGI of 60% (P<0.0001) and a TGI of 40% at the end of the study (P<0.01). IT administration of freshly prepared PD-L1 t-haNK cells at a dosing level of 2.5E6 cells / dose / tumor twice a week for 4 consecutive weeks effectively inhibited the growth of HCC827 tumors, resulting in a peak TGI of 70% on day 20 and a TGI of 49% at the end of the study (P<0.001).

[0281] Significant adverse reactions were observed in animals that received 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 safe in animals after IV administration. PD-L1 t-haNK cells showed significant efficacy in two subcutaneous tumor models. Cryopreserved cells administered at a lower level of 4E6 cells / dose also showed significant efficacy in inhibiting tumor growth and were proven to be safe in animals.

[0282] Of course, it should be appreciated that for all nucleic acid sequences provided herein, the corresponding encoded proteins are also within the clear scope of envision herein. Likewise, for all amino acid sequences, the corresponding nucleic acid sequences are also within the scope of envision herein (using any codon).

[0283] All patent applications, publications, references, and sequence accession numbers cited in this specification are incorporated herein by reference in their entirety.

[0284] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0285] Throughout this specification and the claims that follow, reference will be made to a number of terms which shall be defined to have the following meanings:

[0286] The terminology used is for the purpose of describing particular embodiments only and is not intended to limit the present 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.

[0287] It should be understood that all numerical values ​​described herein (e.g., pH, temperature, time, concentration, quantity and molecular weight, including ranges) include normal variations of measured values ​​encountered by those of ordinary skill in the art. Therefore, the numerical values ​​described include variations of + / -0.1% to 10%, for example, + / -0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%. It should be understood that, although not always clearly stated, all numerical markings may be preceded by the term "about". Therefore, the term approximately includes variations of + / -0.1% to 10%, for example, + / -0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%. It should also be understood that, although not always clearly stated, the reagents are exemplary only, and their equivalents are known in the art.

[0288] As will be understood by those skilled in the art, for any and all purposes, particularly in providing a written description, all ranges disclosed include the endpoints of the range, and include all values ​​between the endpoints of the range. All ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily considered to be fully described, and the same range can be decomposed into at least equal half, two-thirds, one-quarter, one-fifth, a few tenths, etc. As a non-limiting example, each range discussed herein can be easily decomposed into a lower third, a middle third, and an upper third, etc. As will be understood by those skilled in the art, all languages, such as "at most", "at least", etc., include the listed numerical values, and refer to the ranges that can be subsequently subdivided into subranges as described above. Finally, as will be understood by those skilled in the art, the range includes each individual member. Therefore, for example, a group having 1-3 cells refers to a group having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to a group having 1, 2, 3, 4, or 5 cells, etc.

[0289] It is also understood, although not always explicitly stated, that the agents described are exemplary only and that equivalents thereof are known in the art.

[0290] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not occur.

[0291] The term "comprising" is intended to indicate that the compositions and methods include the recited elements, but not excluding other elements. When used to define compositions and methods, "consisting essentially of shall mean excluding other elements of any substantial significance to the combination. For example, a composition consisting essentially of the elements defined herein would not exclude other elements that do not materially affect the basic and novel characteristics of the claimed invention. "Consisting of" means excluding more than trace amounts of other ingredients and the recited substantial method steps. Embodiments defined by each of these transition terms are within the scope of the present disclosure.

[0292] As used herein, "immunotherapy" refers to the use of NK-92 cells, modified or unmodified, naturally occurring or modified NK cells or T cells, alone or in combination, whether used alone or in combination, and capable of inducing cytotoxicity when in contact with target cells.

[0293] As used herein, "natural killer (NK) cells" are cells of the immune system that kill target cells in the absence of specific antigenic stimulation and are not limited according to major histocompatibility complex (MHC) classes. The target cell can be a tumor cell or a cell carrying a virus. NK cells are characterized by the presence of CD56 and the absence of CD3 surface markers.

[0294] The term "endogenous NK cells" is used to refer to NK cells derived from a donor (or patient), which is different from the NK-92 cell line. Endogenous NK cells are generally a heterogeneous cell population in which NK cells have been enriched. Endogenous NK cells can be used for autologous or allogeneic treatment of patients.

[0295] The term "NK-92" refers to natural killer cells derived from a highly potent unique cell line described by Gong et al. (1994), the rights to which are owned by Nantquist Corporation (hereinafter referred to as "NK-92 TM The immortalized NK cell line was originally obtained from a patient with non-Hodgkin lymphoma. Unless otherwise specified, the term “NK-92 TM " refers to the original NK-92 cell line as well as the NK-92 cell line that has been modified (e.g., by the introduction of foreign genes). NK-92 TMCells and exemplary and non-limiting modifications thereof are described in U.S. Pat. Nos. 7,618,817; 8,034,332; 8,313,943; 9,181,322; 9,150,636; and published U.S. Application No. 10 / 008,955, all of which are incorporated herein by reference in their entirety, and include wild-type NK-92 TM 、NK-92 TM -CD16, NK-92 TM -CD16-γ, NK-92 TM -CD16-ζ、NK-92 TM -CD16(F176V), NK-92 TM MI and NK-92 TM CI. NK-92 cells are known to those of ordinary skill in the art and can be readily obtained from Nantquist.

[0296] The term "aNK" refers to unmodified natural killer cells derived from a unique highly potent cell line described by Gong et al. (1994), the rights to which are owned by Nantquist Corporation (hereinafter referred to as "aNK TM The term "haNK" refers to unmodified natural killer cells derived from a highly potent unique cell line described by Gong et al. (1994), the rights of which are owned by Nantquist, Inc., which have been modified to express CD16 on the cell surface (hereinafter "CD16+NK-92 TM Cells" or " Cells"). In some embodiments, CD16+NK-92 TM The cells contain a high affinity CD16 receptor on the cell surface. The term "taNK" refers to unmodified natural killer cells derived from a highly potent unique cell line described by Gong et al. (1994), the rights of which are owned by Nantquist, which have been modified to express a chimeric antigen receptor (hereinafter "CAR-modified NK-92 TM Cells" or " The term "t-haNK" refers to unmodified natural killer cells derived from a highly potent unique cell line described by Gong et al. (1994), the rights of which are owned by Nantquist, which have been modified to express CD16 on the cell surface and to express a chimeric antigen receptor (hereinafter "CAR-modified CD16+NK-92 TM Cells" or "t-haNK TM In some embodiments, t-haNK TM Cells express the high affinity CD16 receptor on the cell surface.

[0297] "Modified NK-92 cells" refer to NK-92 cells expressing exogenous genes or proteins, such as Fc receptors, CARs, cytokines (such as IL-2 or IL-12), and / or suicide genes. In some embodiments, the modified NK-92 cells contain vectors encoding transgenes, such as Fc receptors, CARs, cytokines (such as IL-2 or IL-12), and / or suicide genes. In one embodiment, the modified NK-92 cells express at least one transgenic protein.

[0298] 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. It is envisioned that NK-92 cells will be irradiated at other time points prior to treatment of a therapeutic device or patient, as the time between irradiation and infusion should not exceed four hours to maintain optimal activity. Alternatively, NK-92 cell proliferation may be prevented by another mechanism.

[0299] As used herein, "inactivation" of NK-92 cells renders them incapable of growth. Inactivation may also be associated with the death of NK-92 cells. It is contemplated that NK-92 cells may be inactivated after an ex vivo sample of cells associated with pathology has been effectively cleared in a therapeutic application, or after having remained in a mammal for a sufficient period of time to effectively kill many or all target cells in the body. As a non-limiting example, inactivation may be induced by administering an inactivating agent to which NK-92 cells are sensitive.

[0300] 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). In general, cytotoxic activity involves killing target cells by any of a variety of biological, biochemical, or biophysical mechanisms. Cytolysis more specifically refers to the activity of effectors that dissolve the plasma membrane of target cells, thereby destroying their physical integrity. This results in killing of target cells. Without wishing to be bound by theory, it is believed that the cytotoxic effect of NK-92 cells is due to cytolysis.

[0301] The term "killing" with respect to a cell / cell population is intended to include any type of manipulation that will result in the death of that cell / cell population.

[0302] 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 part of an antibody called the Fc region. The binding of the Fc region of an antibody to the Fc receptor (FcR) of a cell stimulates the phagocytic or cytotoxic activity of the cell through antibody-mediated phagocytosis or antibody-dependent cell-mediated cytotoxicity (ADCC). FcRs are classified according to the type of antibody they recognize. For example, Fc-γ receptors (FCγRs) bind to IgG class 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 (FcεRs) bind to the Fc region of IgE antibodies.

[0303] As used herein, the term "chimeric antigen receptor" (CAR) refers to an extracellular antigen binding domain fused to an intracellular signaling domain. CAR can be expressed in T cells or NK cells to increase cytotoxicity. Typically, the extracellular antigen binding domain is a scFv that is specific for an antigen found on the target cell. Based on the specificity of the scFv domain, NK-92 cells expressing CAR are targeted to cells that express certain antigens on the cell surface. 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 certain cancers.

[0304] As used herein, the term "tumor-specific antigen" refers to an antigen present on cancer cells or neoplastic cells but not detectable on normal cells derived from the same tissue or lineage as the cancer cells. As used herein, a tumor-specific antigen also refers to an antigen associated with a tumor, i.e., an antigen expressed at a higher level on cancer cells than on normal cells derived from the same tissue or lineage as the cancer cells.

[0305] As used herein, the term "virus-specific antigen" refers to an antigen present on virus-infected cells but not detectable on normal cells derived from the same tissue or lineage as the virus-infected cells. In one embodiment, the virus-specific antigen is a viral protein expressed on the surface of infected cells.

[0306] The terms "polynucleotide", "nucleic acid" and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, i.e., deoxyribonucleotides or ribonucleotides or their analogs. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. The following are non-limiting examples of polynucleotides: genes or gene fragments (e.g., probes, primers, EST 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 can include modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, nucleotide structure modification can be performed before or after polynucleotide assembly. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to double-stranded and single-stranded molecules. Unless otherwise stated or required, any embodiment of a polynucleotide of the invention includes both a double-stranded form and each of the two complementary single-stranded forms known or predicted to comprise the double-stranded form.

[0307] A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and, when the polynucleotide is RNA, thymine is uracil (U). Thus, the term "polynucleotide sequence" is an alphabetic representation of a polynucleotide molecule.

[0308] "Homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing positions in each sequence that can be aligned for the purpose of comparison. When a position in the compared sequences is occupied by the same base or amino acid, then 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.

[0309] As used herein, "percent identity" refers to the sequence identity between two peptides or between two nucleic acid molecules. The percent identity can be determined by comparing the position in each sequence, which can be compared for the purpose of comparison. When the position in the comparison sequence is occupied by the same base or amino acid, the molecule has identity at that position. Homologous nucleotide sequences include natural allelic variants and mutated sequences encoding the nucleotide sequences described herein. Homologous nucleotide sequences include nucleotide sequences encoding proteins of mammalian species other than humans. Homologous amino acid sequences include amino acid sequences containing conservative amino acid substitutions and polypeptides having 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, the nucleotide or amino acid sequence has at least 60%, at least 65%, at least 70%, at least 80% or at least 85% or greater identity with the sequence described 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 by using the Gap program (Wisconsin Sequence Analysis Package, Version 8 for UNIX, Genetic Computer Group, University Research Park, Madison) using the default settings, which uses the Smith and Waterman algorithm (Adv. Appl. Math., 1981, 2, 482-489). Suitable algorithms for determining percent sequence identity include BLAST and BLAST 2.0 algorithms, which are 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 the ncbi.nlm.nih.gov website). 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 wordlength of 3, and an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989) with an alignment (B) of 50, an expectation (E) of 10, M=5, and N=-4.

[0310] In certain embodiments, for expressing in particular species, nucleotide sequence is codon optimized, for example, can be for expressing in people, mouse sequence is codon optimized (expression of the protein encoded by codon optimized nucleotide sequence).Therefore, in certain embodiments, codon optimized nucleotide sequence has at least 60%, at least 65%, at least 70%, at least 80% or at least 85% or greater homogeneity with nucleotide sequence as described herein.In certain embodiments, codon optimized nucleotide sequence acid sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homogeneity with sequence described herein.

[0311] The term "expression" refers to the production of a gene product (e.g., a protein). When referring to expression, the term "transient" refers to the fact that the polynucleotide is not incorporated into the genome of the cell. The term "stable" when referring to expression refers to the incorporation of the polynucleotide into the genome of the cell, or the use of a positive selection marker (i.e., an exogenous gene expressed by the cell that has a benefit under certain growth conditions) to maintain the expression of the transgene.

[0312] The term "cytokine" or "cytokines" refers to a general class of biological molecules 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.

[0313] As used herein, the term "vector" refers to a non-chromosomal nucleic acid containing a complete replicon so that when placed in a permissive cell, for example, by a transformation process, the vector can be replicated. The vector can replicate in one cell type (such as bacteria), but has limited or no ability to replicate in another cell (such as a mammalian cell). The vector can be viral or non-viral. Exemplary 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, comprising DNA condensed with cationic polymers, such as heterogeneous polylysine, oligopeptides of defined length, and polyethyleneimine, contained in liposomes in some cases; and the use of ternary complexes comprising viruses and polylysine-DNA. In one embodiment, the vector is a viral vector, for example, an adenovirus. Viral vectors are well known in the art.

[0314] As used herein, the term "targeted" is intended to include, but is not limited to, directing a protein or polypeptide to a suitable destination within or outside a cell when it comes to protein expression. Targeting is usually achieved by a signal peptide or targeting peptide, which is a segment of amino acid residues in a polypeptide chain. These signal peptides can be located anywhere within the polypeptide sequence, but are typically located at the N-terminus. The polypeptide can also be engineered to have a signal peptide at the C-terminus. The signal peptide can direct the polypeptide to be cleaved extracellularly and localized to the plasma membrane, Golgi apparatus, endosomes, endoplasmic reticulum, and other cell compartments. For example, a polypeptide having a specific amino acid sequence at its C-terminus (e.g., KDEL) is retained in the ER lumen or transported back to the ER lumen.

[0315] As used herein, the term "targeting" refers to the ability of NK-92 cells to recognize and kill tumor cells (i.e., target cells) when referring to the target of a tumor. In this article, the term "targeted" refers to the ability of, for example, CAR expressed by NK-92 cells to recognize and bind to cell surface antigens expressed by tumors.

[0316] As used herein, the term "transfection" refers to the insertion of a nucleic acid into a cell. Transfection can be performed using any method that allows nucleic acid to enter a cell. DNA and / or mRNA can be transfected into a cell. Preferably, the transfected cell expresses a gene product (i.e., a protein) encoded by the nucleic acid.

[0317] The term "suicide gene" refers to a transgene that allows negative selection of cells expressing the transgene. Suicide genes are used as a safety system to allow cells expressing the gene to be killed by the introduction of a selection agent. A variety of 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 Escherichia 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 can 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. A genetically modified NK cell, the genetically modified NK cell carrying a membrane-bound recombinant chimeric antigen receptor (CAR), the chimeric antigen receptor comprising: The extracellular binding domain, hinge domain, transmembrane domain, and FcεRIγ signaling domain; wherein the extracellular binding domain comprises a scFv portion, and the scFv portion specifically binds to a tumor-specific antigen, a tumor-associated antigen, a patient-specific antigen and a tumor-specific antigen, or a virus-specific antigen; and The genetically modified NK cells further comprise a recombinant nucleic acid encoding a suicide gene to allow negative selection of cells expressing the suicide gene.

2. The genetically modified NK cell of claim 1, wherein the suicide gene is selected from the group consisting of thymidine kinase (TK) gene, cytosine deaminase (CD) gene, nitroreductase (NR) gene, hypoxanthine-guanine phosphorylation transferase (E.coligpt) gene, purine nucleoside phosphorylase (E.coli Deo) gene, cytochrome P450 gene or inducible caspase 9 (iCasp9) gene.

3. The genetically modified NK cell of claim 1, wherein the recombinant nucleic acid further encodes at least a second suicide gene, the second suicide gene being selected from a thymidine kinase (TK) gene, a cytosine deaminase (CD) gene, a nitroreductase (NR) gene, a hypoxanthine-guanine phosphorylation nucleoside transferase (E.coligpt) gene, a purine nucleoside phosphorylase (E.coliDeo) gene, a cytochrome P450 gene or an inducible caspase 9 (iCasp9) gene.

4. The genetically modified NK cell of claim 3, wherein the recombinant nucleic acid encodes a thymidine kinase (TK) gene and a cytosine deaminase (CD) gene.

5. The genetically modified NK cell of claim 3, wherein the recombinant nucleic acid encodes a thymidine kinase (TK) gene and an inducible caspase 9 (iCasp9) gene.

6. The genetically modified NK cell of claim 1, wherein the tumor-specific antigen is CD19, CD20, GD2, HER-2, CD30, EGFR, FAP, CD33, CD123, PD-L1, IGF1R, CSPG4 or B7-H4.

7. The genetically modified NK cell of claim 6, wherein the extracellular binding domain comprises a scFv portion having a polypeptide sequence selected from the group consisting of a CD19 binding polypeptide sequence having at least 90% homology to SEQ ID NO:4 or SEQ ID NO:24, a CD20 binding polypeptide sequence having at least 90% homology to SEQ ID NO:26, a GD2 binding polypeptide sequence having at least 90% homology to SEQ ID NO:40 or SEQ ID NO:42, a HER-2 binding polypeptide sequence having at least 90% homology to SEQ ID NO:38, a CD30 binding polypeptide sequence having at least 90% homology to SEQ ID NO:36, an EGFR binding polypeptide sequence having at least 90% homology to SEQ ID NO:32, a FAP binding polypeptide sequence having at least 90% homology to SEQ ID NO:58, a CD33 binding polypeptide sequence having at least 90% homology to SEQ ID NO:28, a CD123 binding polypeptide sequence having at least 90% homology to SEQ ID NO:49, a CD123 binding polypeptide sequence having at least 90% homology to SEQ ID NO:49, a CD123 binding polypeptide sequence having at least 90% homology to SEQ ID NO:40 NO:51 has a PD-L1 binding polypeptide sequence with at least 90% homology, an IGF1R binding polypeptide sequence with at least 90% homology to SEQ ID NO:34, a CSPG4 binding polypeptide sequence with at least 90% homology to SEQ ID NO:30, and a B7-H4 binding polypeptide sequence with at least 90% homology to SEQ ID NO:

53.

8. The genetically modified NK cell of claim 1, wherein the virus-specific antigen is an antigen of HIV virus, HPV virus, RSV virus, influenza virus, Ebola virus or HCV virus.

9. The genetically modified NK cell of claim 8, wherein the virus-specific antigen is gp120 of HIV virus.

10. The genetically modified NK cell of any one of claims 1-9, wherein the hinge domain and / or the transmembrane domain comprises a CD8 hinge domain and / or a CD28 transmembrane domain, and optionally wherein the FcεRIγ signaling domain has the amino acid sequence shown in SEQ ID NO:

1.

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

1.

12. The genetically modified NK cell of claim 1, further carrying membrane-bound recombinant CD16.

13. The genetically modified NK cell of claim 12, wherein the membrane-bound recombinant CD16 is a high-affinity variant of CD16.

14. The genetically modified NK cell of claim 1, further comprising a recombinant cytokine having an endoplasmic retention sequence.

15. The genetically modified NK cell of claim 1, wherein the NK cell is a NK-92 cell.

16. A genetically modified NK cell comprising a first recombinant nucleic acid encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an extracellular binding domain, a hinge domain, a transmembrane domain, and an FcεRIγ signaling domain as a single polypeptide chain; wherein the extracellular binding domain comprises a scFv portion, and the scFv portion specifically binds to a tumor-specific antigen, a tumor-associated antigen, a patient-specific antigen and a tumor-specific antigen, or a virus-specific antigen; and A second recombinant nucleic acid encodes a suicide gene that allows for negative selection of cells expressing the suicide gene.

17. The genetically modified NK cell of claim 16, wherein the suicide gene is selected from the group consisting of thymidine kinase (TK) gene, cytosine deaminase (CD) gene, nitroreductase (NR) gene, hypoxanthine-guanine phosphorylation transferase (E.coli gpt) gene, purine nucleoside phosphorylase (E.coli Deo) gene, cytochrome P450 gene or inducible caspase 9 (iCasp9) gene.

18. The genetically modified NK cell of claim 17, wherein the second recombinant nucleic acid further encodes at least a second suicide gene, the second suicide gene being selected from a thymidine kinase (TK) gene, a cytosine deaminase (CD) gene, a nitroreductase (NR) gene, a hypoxanthine-guanine phosphorylation transferase (E. coli gpt) gene, a purine nucleoside phosphorylase (E. coli Deo) gene, a cytochrome P450 gene or an inducible caspase 9 (iCasp9) gene.

19. The genetically modified NK cell of claim 18, wherein the second recombinant nucleic acid encodes a thymidine kinase (TK) gene and a cytosine deaminase (CD) gene.

20. The genetically modified NK cell of claim 18, wherein the second recombinant nucleic acid encodes a thymidine kinase (TK) gene and an inducible caspase 9 (iCasp9) gene.

21. The genetically modified NK cell of any one of claims 16-20, wherein the first and / or second recombinant nucleic acid is RNA.

22. The genetically modified NK cell of claim 21, wherein the RNA is a polycistronic RNA that also encodes CD16 and / or a cytokine with an endosomal retention sequence.

23. 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.

24. The genetically modified NK cell of claim 16, wherein the virus-specific antigen is an antigen of HIV virus, HPV virus, RSV virus, influenza virus, Ebola virus or HCV virus.

25. The genetically modified NK cell of claim 16, wherein the virus-specific antigen is gp120 of HIV virus.

26. The genetically modified NK cell of claim 16, wherein the hinge domain and / or the transmembrane domain comprises a CD8 hinge domain and / or a CD28 transmembrane domain, and optionally wherein the FcεRIγ signaling domain has the amino acid sequence shown in SEQ ID NO:

2.

27. The genetically modified NK cell of claim 16, wherein the FcεRIγ signaling domain has the amino acid sequence shown in SEQ ID NO:

2.

28. Use of the genetically modified NK cells of any one of claims 1-7, 10-23 and 26-27 in the preparation of a medicament for treating cancer in a patient in need thereof.

29. The use of claim 28, wherein the treatment of cancer comprises administering at least one additional therapeutic entity selected from the group consisting of viral cancer vaccines, bacterial cancer vaccines, yeast cancer vaccines, N-803, antibodies, stem cell transplants, and tumor-targeted cytokines.

30. The method of claim 28 or 29, wherein the cancer is selected from 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, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, smooth muscle cell carcinoma, ... smooth muscle cell carcinoma, smooth muscle cell carcinoma, smooth muscle cell carcinoma, smooth muscle cell carcinoma, smooth muscle cell carcinoma, smooth muscle cell carcinoma, smooth muscle cell carcinoma, smooth muscle cell carcinoma, smooth muscle cell carcinoma, smooth muscle cell carcinoma, smooth muscle cell carcinoma, smooth muscle cell carcinoma, Myoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma and retinoblastoma.

31. Use of the genetically modified NK cells of any one of claims 1-5, 8-22 and 24-27 in the preparation of a medicament for treating a viral infection in a patient in need thereof.

32. The use of claim 31, wherein the treatment of viral infection comprises treatment with an antiviral drug.

33. The method of any one of claims 25-29, wherein about 1 x 10 8 About 1x 10 11 Cells / m 2 The patient's body surface area.

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