Fc Epsilon CAR

Abstract

Recombinant NK cells, particularly recombinant NK-92 cells, express a chimeric antigen receptor (CAR) containing the intracellular domain of FcεRIγ. Notably, CAR constructs containing the intracellular domain of FcεRIγ had substantially extended expression duration and significantly prolonged cytotoxicity over time. CARs can be expressed from RNA and DNA, preferably as tricistronic constructs that further encode CD16 and cytokines that confer autocrine growth support. Advantageously, such constructs also allow for high-level transfection and recombinant protein expression while providing convenient selection markers that facilitate rapid generation of recombinant NK / NK-92 cells.

Description

[Technical Field]

[0001] This application claims the benefit of priority to U.S. patent application Ser. No. 62 / 674,936, filed May 22, 2018.

[0002] Array List The contents of the ASCII text file of the Sequence Listing designated 104077.0004PCT_ST25, which is 106kb in size, created on May 21, 2019, and submitted electronically via EFS-Web herewith, are incorporated by reference in their entirety.

[0003] The field of this invention is recombinant nucleic acids for generating genetically engineered cells that express chimeric antigen receptors (CARs) and cells containing same, particularly engineered NK and NK-92 cells that express CARs with an Fc epsilon receptor gamma (FcεRIγ) signaling domain. [Background technology]

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

[0005] Autologous NK cells have been used as a therapeutic entity. For this purpose, NK cells are isolated from the peripheral lymphocyte fraction of whole blood, expanded in cell culture to obtain sufficient numbers of cells, and then reinfused into the subject. Autologous NK cells have shown modest efficacy in both ex vivo and in vivo therapy, at least in some cases. However, the isolation and growth of autologous NK cells is time-consuming and costly. Furthermore, autologous NK cell therapy is further limited by the fact that not all NK cells are cytolytic.

[0006] At least some of these difficulties can be overcome by the use of NK-92 cells, a cytolytic cancer cell line that was immortalized in vitro after being discovered in the blood of a subject suffering from non-Hodgkin's lymphoma (Gong et al., Leukemia 8:652-658 (1994)). NK-92 cells are NK cell derivatives, but they lack most of the inhibitory receptors present on normal NK cells and retain most of the activating receptors. However, NK-92 cells do not attack normal cells or induce unacceptable immune rejection responses in humans. Due to these desirable properties, NK-92 cells have been extensively characterized and explored as therapeutic agents in the treatment of certain cancers, as described, for example, in International Publication WO 1998 / 049268 or U.S. Patent Application Publication No. 2002 / 068044.

[0007] Phenotypic changes that distinguish normal cells from tumor cells derived from the same tissue are often associated with one or more changes in the expression of specific gene products, such as the loss of normal cell surface components or the gain of other components (i.e., antigens undetectable in the corresponding normal, non-cancerous tissue). Antigens that are expressed in neoplastic or tumor cells but not in normal cells, or that are expressed in neoplastic cells at levels substantially exceeding those found in normal cells, have been termed "tumor-specific antigens" or "tumor-associated antigens." Such tumor-specific antigens can serve as markers of the tumor phenotype. Tumor-specific antigens include cancer / testis-specific antigens (e.g., MAGE, BAGE, GAGE, PRAME, and NY-ESO-1), melanocyte differentiation antigens (e.g., tyrosinase, Melan-A / MART, gp100, TRP-1, and TRP-2), mutated or aberrantly 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).

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

[0009] The first generation of CARs used in T cells contained a single cytoplasmic signaling domain. For example, one form of first-generation T cell CAR contained a signaling domain derived from Fc epsilon receptor gamma (FcεRIγ), which contains one ITAM, while another form contained a signaling domain derived from CD3ζ, which contains three ITAMs. In vivo and in vitro studies have shown that CD3ζ CART cells are more efficient at eradicating tumors than FcεRIγ CART cells (e.g., Haynes, et al. 2001, J. Immunology 166:182-187; Cartellieri, et al. 2010, J. Biomed and Biotech, Vol. 2010, Article ID 956304). Subsequently, additional studies revealed that certain costimulatory signals were required for the full activation and proliferation of such recombinant T cells, and second- and third-generation CARs combined multiple signaling domains into a single CAR to enhance the efficacy of the recombinant CAR cells. Because the clinical efficacy of the tested T cells was less than desirable, first-generation CARs and the FcεRIγ signaling domain were largely discarded in favor of new, more efficient CARs that use CD3ζ in combination with one or more additional signaling domains (e.g., Hermanson and Kaufman 2015, Frontiers in Immunol., Vol. 6, Article 195).

[0010] More recently, select CARs have also been expressed on NK cells. For example, CAR-modified NK-92 cells use a first-generation CAR containing only the CD3ζ intracellular signaling domain. Several antigens have been targeted by these first-generation CAR-NK cells, including CD19 and CD20 for B-cell lymphoma, ErbB2 for breast cancer, ovarian cancer, and squamous cell carcinoma, GD2 for neuroblastoma, and CD138 for multiple myeloma. Second-generation CAR-NK cells derived from the NK-92 line have also been generated against several antigens, including EpCAM for multiple carcinomas, the HLA-A2EBNA3 complex for Epstein-Barr virus, CS1 for multiple myeloma, and ErbB2 for HER2-positive epithelial carcinomas. The most common intracellular costimulatory domain used in conjunction with CD3ζ in second-generation NK-92 CARs is CD28. However, because NK cells do not naturally express CD28, the potential effect of the CD28 domain is unknown. Other second-generation CARs incorporate the 4-1BB intracellular signaling domain in conjunction with CD3ζ to improve NK cell persistence. Others have tested and compared the function of various intracellular domains against breast cancer cells using ErbB2 scFv fused to CD3ζ alone, CD28 and CD3ζ, or 4-1BB and CD3ζ. All second-generation constructs were found to improve killing compared to first-generation CARs, with CD28 and CD3ζ providing 65% target lysis, 4-1BB and CD3ζ providing 62% lysis, and CD3ζ alone killing 51% of targets. The 4-1BB and CD28 intracellular domains were also compared in a recent study using an anti-CD19 CAR expressed on NK-92 cells against B-cell malignancies. Still others have found that the CD3ζ / 4-1BB construct is less effective than CD3ζ / CD28 at cell killing and cytokine production, and have noted differential effects of the CD28 and 4-1BB costimulatory domains.

[0011] A third-generation NK-92 CAR was constructed with an anti-CD5 scFv bearing CD3ζ, CD28, and 4-1BB intracellular signaling domains and demonstrated specific and potent anti-tumor activity against various T-cell leukemia and lymphoma cell lines and primary tumor cells. Such cells were also able to inhibit disease progression in T-cell acute lymphoblastic leukemia (ALL) cell lines as well as primary tumor cell xenograft mouse models (Transl Res. 2017 September;187:32-43). In further examples, WO 2016 / 201304 and WO 2018 / 076391 teach the use of third-generation CD3ζ CARs expressed in NK cells and NK-92 cells.

[0012] Autologous NK cells and NK-92 cells require exogenous IL-2 as a survival factor and cytotoxicity enhancer. Unfortunately, systemic administration of IL-2 is often associated with significant undesirable side effects and toxicity. To overcome these challenges, cells can be cultured and expanded in vitro before administration to patients. While IL-2 may enable the generation of sufficient quantities of NK cells or NK-92 cells, the use of exogenous IL-2 in large-scale production of NK cells is typically limited by cost. The requirement for exogenous IL-2 has been resolved by confining recombinant expression of IL-2 from retroviral vectors to the endoplasmic reticulum (see Exp Hematol. 2005 Feb;33(2):159-64). Such an approach eliminated the requirement for exogenous IL-2. However, retroviral transfection efficiency is often less than desirable and may become even less efficient when expressing multiple recombinant genes.

[0013] Additionally, NK cells, particularly NK-92 cells, are often difficult to genetically modify, as evidenced by the numerous failed attempts to engineer NK-92 cells to express Fc receptors. These difficulties are further compounded when transfecting NK-92 cells with multiple recombinant genes or relatively large recombinant nucleic acid payloads for heterologous expression. Furthermore, NK-92 cells also exhibit a significant lack of predictability with respect to the recombinant expression of exogenous proteins (e.g., CD16). At the functional level, most, if not all, CARNK-92 cells require a relatively high effector-to-target cell ratio, likely due to the relatively low expression of the CAR construct. Furthermore, such CARNK-92 cells also experience a rapid decline in cytotoxicity over time, thereby reducing their clinical appeal.

[0014] Thus, although many recombinant NK-92 cells are known in the art, all or nearly all of them suffer from various difficulties. Therefore, there remains a need for CAR-expressing NK-92 cells that express highly active CARs in significant amounts with associated sustained cytotoxicity, and that are easily cultured in a simple and effective manner. Summary of the Invention

[0015] The present inventors have discovered that NK-92 cells can be efficiently transfected with recombinant nucleic acids to express FcεRIγ-containing CARs. Unexpectedly, CARs with an FcεRIγ signaling domain significantly increase CAR expression levels and also deliver prolonged cytotoxicity over time. Contemplated recombinant nucleic acids encoding CARs are preferably tricistronic, also including sequence portions encoding CD16 or a CD16 variant and / or IL-2 or an IL-2 variant. Advantageously, such recombinant nucleic acids not only provide an efficient method for selecting transfected cells (because IL-2 not only confers an autocrine growth stimulus but also acts as a selectable marker for co-expressed proteins), but also result in CARNK cells with superior cytolytic activity (e.g., at a relatively low effector-to-target cell ratio compared to other constructs) and high expression levels of CD16- and FcεRIγ-containing CARs.

[0016] Thus, in one aspect of the inventive subject matter, the inventors contemplate genetically modified NK cells recombinantly expressing a cytokine, CD16, and a membrane-bound chimeric antigen receptor (CAR). The CAR will typically comprise (i) an extracellular binding domain, (ii) a hinge domain, (iii) a transmembrane domain, and (iv) an FcεRIγ signaling domain in a single polypeptide chain (e.g., having the amino acid sequence of SEQ ID NO: 1).

[0017] In many embodiments, the NK cells are NK-92 cells and / or the recombinantly expressed cytokine is or comprises IL-2 or IL-15 (which may further comprise an endoplasmic retention sequence). In further embodiments, the CD16 is a high affinity CD16 variant (e.g., CD16 158V ) can be.

[0018] Preferably, but not necessarily, the extracellular binding domain will comprise an scFv capable of specifically binding to a tumor-specific antigen (e.g., CD19, CD20, NKG2D ligand, CS1, GD2, CD138, EpCAM, HER-2, EBNA3C, GPA7, CD244, CA-125, MUC-1, ETA, MAGE, CEA, CD52, CD30, MUC5AC, c-Met, EGFR, FAP, WT-1, PSMA, NY-ESO1, CSPG-4, IGF1-R, Flt-3, CD276, CD123, PD-L1, BCMA, or CD33), a tumor-associated antigen, or a patient- and tumor-specific antigen, or a virus-specific antigen (e.g., an antigen of the HIV virus, HPV virus, RSV virus, influenza virus, Ebola virus, or HCV virus).

[0019] In some embodiments, the cytokine, CD16, and CAR are expressed from a tricistronic recombinant nucleic acid, while in other embodiments, the cytokine and / or CD16 are expressed from a recombinant nucleic acid integrated into the genome of the NK cell.

[0020] Thus, the present inventors also contemplate a recombinant nucleic acid comprising a first sequence portion encoding a cytokine, a second sequence portion encoding CD16, and a third sequence portion encoding a chimeric antigen receptor (CAR) comprising an extracellular binding domain, a hinge domain, a transmembrane domain, and an FcεRIγ signaling domain in a single polypeptide chain. Most typically, the first, second, and third sequence portions are on the same nucleic acid.

[0021] In some embodiments, the nucleic acid is tricistronic RNA, while in other embodiments, the nucleic acid is tricistronic DNA.

[0022] Furthermore, typically, the cytokine is IL-2 or IL15 (which may or may not contain an endoplasmic retention sequence), the CD16 is a high-affinity CD16 variant having a 158V mutation, and / or the extracellular binding domain preferably comprises an scFv. As mentioned above, the extracellular binding domain may specifically bind to a tumor-specific antigen, a tumor-associated antigen, or a patient- and tumor-specific antigen, or the extracellular binding domain may specifically bind to a virus-specific antigen.

[0023] In further contemplated embodiments, the hinge domain and / or transmembrane domain can comprise a CD8 hinge domain and / or a CD28 transmembrane domain, while the FcεRIγ signaling domain can have the nucleic acid sequence of SEQ ID NO:2.

[0024] In yet further aspects of the inventive subject matter, the inventors also contemplate recombinant cells comprising the recombinant nucleic acids described hereinabove. Where nucleic acids are prepared and / or amplified, the recombinant cells may be bacterial cells. On the other hand, where recombinant nucleic acids are expressed, the cells will typically be autologous NK cells or NK cells (optionally genetically modified NK-92 cells).

[0025] Therefore, the present inventors also contemplate a method for treating cancer in a patient in need thereof, in which a therapeutically effective amount of any one of the genetically modified NK cells is administered to the patient to treat the cancer. Optionally, the contemplated method may further comprise the step of administering at least one additional therapeutic entity selected from the group consisting of a viral cancer vaccine, a bacterial cancer vaccine, a yeast cancer vaccine, N-803, an antibody, a stem cell transplant, and a tumor-targeted cytokine.

[0026] Among cancers, particularly contemplated cancers include leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, chronic leukemia, chronic myelocytic (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, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovium, and mesothelioma. , Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0027] Similarly, the present inventors also contemplate methods for treating a viral infection in a patient in need thereof, in which the viral infection is treated by administering to the patient a therapeutically effective amount of any one of the genetically modified NK cells. If desired or required, an antiviral agent may also be administered.

[0028] Regardless of the type of treatment, generally, 1 × 10 8 ~Approx. 1×10 11 It is contemplated that the cells will be administered to a patient at a dose of 1000 cells / m2 of patient body surface area. In various respects, the use of the genetically modified NK cells provided herein is contemplated for the treatment of cancer or viral infections.

[0029] Various objects, features, aspects and advantages of the present subject matter will become apparent from the following detailed description of preferred embodiments taken in conjunction with the accompanying drawings, in which like numerals represent like elements. [Brief explanation of the drawings]

[0030] [Figure 1]

[0039] Figure 1 shows a schematic diagram of exemplary CD19-CARs tested. All CD19-CAR variants contained an extracellular domain comprising an anti-CD19 scFv region (αCD19-scFv) and a CD8-derived hinge region (CD8 hinge), and a CD28-derived transmembrane domain (CD28™). The intracellular domains of the CD19CARs were varied as indicated. [Figure 2A] 2 is an exemplary result of the percentage of NK-92 cells expressing the CD19-CAR of FIG. 1 after transfection with CD19-CAR mRNA as determined by flow cytometry using an anti-scFv antibody labeled with eF660. [Figure 2B] Exemplary results of median fluorescence intensity (MFI)-background of CD19-CAR-expressing NK-92 cells labeled with eF660-labeled anti-scFv antibody. [Figure 3A] Exemplary results are shown for the percentage of NK-92 cell-sensitive target cancer cells (K562) killed by CD19CAR-expressing NK-92 cells (effectors) at effector:target ratios ranging from 5:1 to 0.3:1. [Figure 3B] Exemplary results are shown for the percentage of NK-92 cell-resistant CD19-positive target cancer cells (SUP-B15) killed by CD19CAR-expressing NK-92 cells (effectors) at effector:target ratios ranging from 5:1 to 0.3:1. [Figure 4] Shown are exemplary results of MFI of CD19-CAR-expressing NK-92 cells (effectors) labeled with anti-CD107a antibody in a degranulation assay using SUP-B15 target cells at effector:target ratios of 2:1 to 0.25:1. [Figure 5] Exemplary results are shown for surface expression of CD19CAR on haNK cells transfected with CD19CAR mRNA constructs at various time points. All CAR constructs tested showed detectable expression for up to 72 hours under the conditions used, with the CD19 / CD28-Fc-epsilon CAR having the longest duration of expression. [Figure 6]Exemplary results of CD19.taNK cytotoxicity against SUPB15CD19+ cells (aNK-resistant cell line) are shown. All tested CAR constructs show similar (maximal) cytotoxicity at 24 h. However, at 48 h, CD19 / CD3 zeta shows a marked decrease in cytotoxicity, while Fc epsilon-based CAR shows only a slight decrease 48 h after electroporation. [Figure 7] FIG. 1 is an exemplary schematic diagram of a recombinant tricistronic DNA construct and the corresponding protein product. [Figure 8] An exemplary linearized form of the plasmid shown in FIG. 8 is shown. [Figure 9A] Exemplary results are shown in vitro data demonstrating that CD33-positive (CD33+) THP-1 cells are relatively resistant to cytotoxicity (specific lysis) by control NK-92 (aNK) cells, but that there is a high percentage of specific lysis when THP-1 cells are cultured with NK-92 cells expressing a CAR that specifically binds to CD33 (CD33-CAR / NK-92 cells). [Figure 9B] 1 shows exemplary results of in vitro data demonstrating that K562 cells are killed by both control aNK cells and CD33-CAR / NK-92 cells. [Figure 10] 1 shows exemplary results of HER2.CAR-t-haNK cell cytotoxicity against BT-474 cells. [Figure 11] 1 shows exemplary results of cytotoxicity of CD33.CAR-t-haNK cells against THP-1 cells. [Figure 12] 1 shows exemplary results of cytotoxicity of PD-L1.CAR-t-haNK cells against SUP-B15.PD-L1+ cells. [Figure 13] 1 shows exemplary results of cytotoxicity of PD-L1.CAR-t-haNK cells against U251 cells. [Figure 14] 1 shows exemplary results of EGFR.CAR-t-haNK cell cytotoxicity against A-549 cells. [Figure 15]1 shows exemplary results of cytotoxicity of CD19.CAR-t-haNK cells against K562 cells. [Figure 16] 1 shows exemplary results of cytotoxicity of CD19.CAR-t-haNK cells against SUP-B15 cells. [Figure 17] 1 shows exemplary results of ADCC of CD19.CAR-t-haNK cells against SKBr3 cells. [Figure 18] 1 shows exemplary results of cytotoxicity of IGF1R.CAR-t-haNK cells against MDA-MB-231 cells. [Figure 19] Exemplary results of cytotoxicity of PD-L1.CAR-t-haNK cells against various cancer cells are shown. [Figure 20] Exemplary comparative results of the cytotoxicity of PD-L1.CAR-t-haNK cells against MDA-MB-231 cells are shown. [Figure 21] Exemplary results of CD16 and CD19.CAR expression are shown. [Figure 22] 1 shows exemplary results of natural cytotoxicity of CD19.CAR-t-haNK cells against K562 cells. [Figure 23] 1 shows exemplary results of CAR-mediated cytotoxicity of CD19.CAR-t-haNK cells against SUP-B15 cells. [Figure 24] 1 shows exemplary results of ADCC of CD19.CAR-t-haNK cells. [Figure 25] An exemplary comparison of CD16 and CD20.CAR expression is shown. [Figure 26] 1 shows exemplary results of the natural cytotoxicity of CD20.CAR-t-haNK cells. [Figure 27] Exemplary results of CD16 and CD33.CAR expression are shown. [Figure 28] 1 shows exemplary results of natural cytotoxicity of CD33.CAR-t-haNK cells against K562 cells. [Figure 29] 1 shows exemplary results of CAR-mediated cytotoxicity of CD33.CAR-t-haNK cells against THP-1 cells. [Figure 30] Exemplary results of ADCC of CD33.CAR-t-haNK cells are shown. [Figure 31] Exemplary results of CD16 and EGFR.CAR expression are shown. [Figure 32] 1 shows exemplary results of natural cytotoxicity of EGFR.CAR-t-haNK cells against K562 cells. [Figure 33] 1 shows exemplary results of CAR-mediated cytotoxicity of EGFR.CAR-t-haNK cells against A549 cells. [Figure 34] 1 shows exemplary results of CAR-mediated cytotoxicity of EGFR.CAR-t-haNK cells against HCT116 cells. [Figure 35] Exemplary results of ADCC of EGFR.CAR-t-haNK cells are shown. [Figure 36] Exemplary results of CD16 and HER2.CAR expression are shown. [Figure 37] 1 shows exemplary results of natural cytotoxicity of HER2.CAR-t-haNK cells against K562 cells. [Figure 38] 1 shows exemplary results of CAR-mediated cytotoxicity of HER2.CAR-t-haNK cells against SKBR-3 cells. [Figure 39] 1 shows exemplary results of ADCC of HER2.CAR-t-haNK cells. [Figure 40] Exemplary results of CD16 and PD-L1.CAR expression are shown. [Figure 41] 1 shows exemplary results of natural cytotoxicity of PD-L1.CAR-t-haNK cells against K562 cells. [Figure 42] 1 shows exemplary results of CAR-mediated cytotoxicity of PD-L1.CAR-t-haNK cells. [Figure 43] Exemplary results of ADCC of PD-L1.CAR-t-haNK cells are shown. [Figure 44] 1 shows exemplary results of CAR-mediated cytotoxicity of CD123.CAR-t-haNK cells. [Figure 45]1 shows exemplary results of ADCC of CD123.CAR-t-haNK cells. [Figure 46] Exemplary results for CD16 and CD30.CAR expression are shown. [Figure 47] 1 shows exemplary results of natural cytotoxicity of CD30.CAR-t-haNK cells against K562 cells. [Figure 48] 1 shows exemplary results of CAR-mediated cytotoxicity of CD30.CAR-t-haNK cells against THP-1 cells. [Figure 49] 1 shows exemplary results of ADCC of CD30.CAR-t-haNK cells. [Figure 50] Exemplary results for CD16 and BCMA.CAR expression are shown. [Figure 51] 1 shows exemplary results of CAR-mediated cytotoxicity of BCMA.CAR-t-haNK cells. [Figure 52] 1 shows exemplary results of ADCC of BCMA.CAR-t-haNK cells. [Figure 53] Exemplary results for CD16 and gp120.CAR expression are shown. [Figure 54] 1 shows exemplary results of GP120 binding of CAR-t-haNK cells. [Figure 55] 1 shows exemplary results of natural cytotoxicity of CAR-t-haNK cells against K562 cells. [Figure 56] 1 shows exemplary results of ADCC of CAR-t-haNK cells. [Figure 57] Exemplary results for CD16 and FAP.CAR expression are shown. [Figure 58] 1 shows exemplary results of CAR-mediated cytotoxicity of FAP.CAR-t-haNK cells. [Figure 59] CSPG4. Exemplary results of CSPG4 expression in CAR-t-haNK cells are shown. [Figure 60] 1 shows exemplary results of CAR-mediated cytotoxicity of CSPG4.CAR-t-haNK cells against SK-MEL-28 cells. [Figure 61]An exemplary tricistronic construct encoding IGF1R-CAR, CD16, and IL-2ER is depicted. DETAILED DESCRIPTION OF THE INVENTION

[0031] As described in more detail below, the inventors unexpectedly discovered that CAR-mediated cytotoxicity and CAR expression in recombinant NK cells (e.g., NK-92 cells) are substantially increased when the recombinant CAR comprises an FcεRIγ signaling domain. The finding that CARs with an FcεRIγ signaling domain have superior properties in NK cells is particularly unexpected, as such CARs in T cells exhibit relatively poor performance compared to CARs with CD3ζ, 4-1BB, or CD28 signaling domains, optionally with additional signaling domains typically found in second- and third-generation CARs.

[0032] Thus, in some embodiments, recombinant nucleic acids are contemplated that encode a CAR having an FcεRIγ signaling domain, preferably, but not necessarily, in a tricistronic arrangement that also includes sequence portions encoding CD16 or a CD16 variant and / or IL-2 or an IL-2 variant. In yet another advantageous aspect of the inventive subject matter, such recombinant nucleic acids not only provide an efficient method for selecting transfected cells (since IL-2 does not only confer an autocrine growth stimulus), but also act as a selectable marker for the co-expressed protein.

[0033] Thus, the present invention relates to genetically engineered NK cells, NK-92 cells, and derivatives thereof that express a chimeric antigen receptor (CAR) on the cell surface, wherein the CAR preferably comprises an intracellular signaling domain derived from Fc epsilon receptor gamma (FcεRIγ). For example, the cytoplasmic domain of FcεRIγ can have an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1, or comprise, consist of, or consist essentially of an amino acid sequence having the sequence set forth in SEQ ID NO: 1. In some embodiments, the cytoplasmic domain of FcεRIγ is encoded by a nucleic acid having at least 95% sequence identity to SEQ ID NO: 2. Contemplated recombinant cells may further express one or more cytokines and various other proteins, including CD16. As will be readily apparent, the CAR and / or other proteins may be transiently or stably expressed by the recombinant cells.

[0034] In some embodiments, the CAR comprises a hinge region from CD8, and / or in some embodiments, the CAR comprises a transmembrane domain from CD28 having the amino acid sequence of SEQ ID NO:6 (encoded by the nucleic acid of SEQ ID NO:7). The full-length amino acid sequence of CD28 is set forth in SEQ ID NO:23. In further embodiments, recombinant cells are genetically modified with a nucleic acid having the sequence of SEQ ID NO:9, which encodes a hybrid protein having the sequence of SEQ ID NO:8 comprising a CD8 hinge region linked to a CD28 transmembrane domain linked to an FcεRIγ signaling domain. As will be appreciated, addition of a binding domain to the hinge region will form a functional CAR. For example, the binding domain can target or specifically bind to a tumor-associated antigen. Suitable antigens also include CD19, CD20, NKG2D ligand, CS1, GD2, CD138, EpCAM, HER-2, EBNA3C, GPA7, CD244, CA-125, MUC-1, ETA, MAGE, CEA, CD52, CD30, MUC5AC, c-Met, EGFR, FAP, WT-1, PSMA, NY-ESO1, CSPG-4, IGF1-R, Flt-3, CD276, CD123, PD-L1, BCMA, and CD33.

[0035] In some embodiments, the nucleic acid construct further comprises an (inducible) promoter that facilitates transcription of the nucleic acid sequence. Preferably, but not necessarily, the nucleic acid construct is a multicistronic vector or RNA containing one or more internal ribosome entry sites (IRES) that allow initiation of translation from an internal region of the mRNA transcribed from the nucleic acid sequence. Alternatively or additionally, the nucleic acid construct comprises a sequence encoding a 2A peptide, such as a T2A, P2A, E2A, or F2A peptide, to generate equimolar levels of polypeptides encoded by the same mRNA. In some embodiments, the nucleic acid construct further comprises a nucleic acid sequence encoding an antigen binding protein (ABP). In some embodiments, the ABP is an scFv or a codon-optimized scFv. In some embodiments, the ABP specifically binds to an antigen expressed by a tumor cell. In some embodiments, the ABP is part of a chimeric antigen receptor (CAR). In further embodiments, the construct comprises a nucleic acid encoding a cytokine, such as IL-2 or IL-15, that can be targeted to the endoplasmic reticulum. In some embodiments, the NK-92 cells or cell lines are also genetically modified to express CD16 on the cell surface. In one embodiment, the NK-92 cells or cell line are genetically modified to express high affinity CD16(F158V) on the cell surface.

[0036] With respect to suitable NK cells, it should be noted that all NK cells are considered suitable for the uses described herein, including primary NK cells (preserved, expanded, and / or fresh cells), immortalized secondary NK cells, autologous or heterologous NK cells (banked, preserved, fresh, etc.), as well as modified NK cells, which are described in more detail below. In some embodiments, the NK cells are preferably NK-92 cells. The NK-92 cell line is a unique cell line that was discovered to proliferate in the presence of interleukin 2 (IL-2) (see, e.g., Gong et al., Leukemia 8:652-658 (1994)). NK-92 cells are cancerous NK cells with broad antitumor cytotoxicity and predictable yields after expansion in a suitable culture medium. Advantageously, NK-92 cells have high cytolytic activity against a variety of cancers.

[0037] The original NK-92 cell line expressed CD56bright, CD2, CD7, CD11a, CD28, CD45, and CD54 surface markers, and did not display CD1, CD3, CD4, CD5, CD8, CD10, CD14, CD16, CD19, CD20, CD23, and CD34 markers. Growth of such NK-92 cells in culture is dependent on the presence of sufficient interleukin-2 (e.g., rIL-2) to maintain proliferation even at doses as low as 1 IU / mL. IL-7 and IL-12 do not support long-term growth, as do various other cytokines tested, including IL-1α, IL-6, tumor necrosis factor α, interferon α, and interferon γ. Compared to primary NK cells, NK-92 typically possess high cytotoxicity even at relatively low effector:target (E:T) ratios (e.g., 1:1). Representative NK-92 cells have been deposited with the American Type Culture Collection (ATCC) under the designation CRL-2407.

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

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

[0040] Genetic modification of NK cells as contemplated herein can be accomplished in a number of ways, and all known methods are deemed suitable for use herein. Furthermore, 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 desired recombinant cell type and transfection efficiency. For example, if it is desired to stably transfect NK cells, linearized DNA may be introduced into the cells for integration into the genome. On the other hand, if transient transfection is desired, circular DNA or linear RNA (e.g., mRNA with a polyA+ tail) may be used.

[0041] For example, if the NK cells are autologous NK cells or NK-92 cells, the recombinant nucleic acid may comprise a segment encoding a CAR comprising an FcεRIγ signaling domain and, preferably, a segment encoding a cytokine that provides autocrine growth stimulation (e.g., IL-2, IL-2 modified with an ER retention sequence, IL-15, or IL-15 modified with an ER retention sequence) and / or CD16 or high-affinity CD16. 158V and a segment encoding a cytokine that provides an autocrine growth stimulus. As will be readily apparent, the incorporation of a cytokine that provides an autocrine growth stimulus will render the modified recombinant independent of exogenous cytokine addition, thereby making large-scale production of such cells economically feasible. Similarly, the incorporation of a cytokine that provides an autocrine growth stimulus will render the modified recombinant independent of exogenous cytokine addition, thereby making large-scale production of such cells economically feasible. 158V If such cells express the .gtoreq.1, they will have further enhanced ADCC properties and thereby improved targeted cytotoxicity.

[0042] Of course, cytokines and / or CD16 or high affinity CD16 158VIt should be recognized that the recombinant nucleic acid encoding can be integrated into the genome of the NK cell or can be supplied as an extrachromosomal unit (which can be linear or circular DNA or linear RNA delivered virally or via chemical, mechanical, or electrical transfection). For example, recombinant NK-92 cells expressing IL-2ER and CD16158V, known as haNK cells (Oncotarget 2016 Dec 27;7(52):86359-86373), can be transfected with a recombinant nucleic acid comprising a segment encoding a CAR comprising an FcεRIγ signaling domain. Again, such recombinant nucleic acids can include additional segments that can encode additional immunotherapeutic proteins, e.g., N-803, TxM-type compounds, IL-8 trap, TGF-β trap, etc. Similarly, NK-92 cells can be already transfected with a cDNA encoding IL-2 (e.g., NK-92MI, ATCC CRL-2408). Such cells are then transfected with CD16 or high affinity CD16 158V The subject can further be transfected with a recombinant nucleic acid comprising a segment encoding a CAR comprising the FcεRIγ signaling domain, together with a segment encoding a CAR comprising the FcεRIγ signaling domain.

[0043] On the other hand, (autologous, fresh, cultured, or pre-frozen) NK cells or NK-92 cells can also be expressed as a segment encoding a CAR having an FcεRIγ signaling domain, a segment encoding a cytokine that provides autocrine growth stimulation (e.g., IL-2, IL-2 modified with an ER retention sequence, IL-15, or IL-15 modified with an ER retention sequence), and a segment encoding CD16 (SEQ ID NO: 34) or high-affinity CD16. 158VThe NK cell may be transfected with a recombinant nucleic acid comprising a segment encoding SEQ ID NO:35 (encoded by SEQ ID NO:36). Most typically, such recombinant nucleic acid will be configured as a tricistronic construct. As noted above, such constructs can be extrachromosomal circular plasmids, linear DNA (which can integrate into the genome of the NK cell), or linear RNA. Such nucleic acids will typically be transfected into cells by methods well known in the art (e.g., electroporation, lipofection, ballistic gene transfer, etc.). Similarly, nucleic acids can be delivered to cells via recombinant viruses. Accordingly, NK cells suitable for use as described herein include NK-92 cells (which may be transfected with a tricistronic construct encoding a CAR, CD16 or a variant thereof, and a cytokine or variant thereof), genetically modified NK cells or NK-92 cells expressing CD16 or a variant thereof, or a cytokine or variant thereof (which may be transfected with a nucleic acid encoding a CAR, and CD16 or a variant thereof, or a cytokine or variant thereof), and genetically modified NK cells or NK-92 cells expressing CD16 or a variant thereof, and a cytokine or variant thereof (which may be transfected with a nucleic acid encoding a CAR).

[0044] It should be noted, therefore, that in preferred embodiments, genetically modified NK cells (especially when the cells express a CAR and CD16 or a variant thereof) will exhibit three distinguishable modes of cell killing: general cytotoxicity mediated by activation of a receptor (e.g., the NKG2D receptor), ADCC mediated by antibodies bound to target cells, and CAR-mediated cytotoxicity.

[0045] It should be recognized that the transfection method will depend, at least in part, on the type of nucleic acid utilized. Accordingly, viral, chemical, and mechanical transfection methods are all considered suitable for use as described herein. For example, in one embodiment, the vectors described herein are transient expression vectors. Because exogenous transgenes introduced using such vectors are not integrated into the nuclear genome of the cell, in the absence of vector replication, the exogenous transgene will degrade or be diluted over time.

[0046] In another embodiment, the vectors described herein allow for stable transfection of cells. In one embodiment, the vector allows for integration of a transgene into the genome of a cell. Preferably, such vectors have a positive selection marker; suitable positive selection markers include any gene that allows cells to grow under conditions that would kill cells that do not express the gene. Examples include, but are not limited to, antibiotic resistance, such as geneticin (Neo gene from Tn5).

[0047] Alternatively or additionally, the vector is a plasmid vector. In one embodiment, the vector is a viral vector. As will be appreciated by those skilled in the art, any suitable vector can be used, and suitable vectors are well known in the art.

[0048] In yet other embodiments, cells are transfected with mRNA encoding a protein of interest (e.g., a CAR). Transfection of the mRNA results in transient expression of the protein. In one embodiment, transfection of the mRNA into NK-92 cells is performed immediately prior to administration of the cells. In one embodiment, "immediately prior to" administration to the cells means about 15 minutes to about 48 hours prior to administration. Preferably, transfection of the mRNA is performed about 5 hours to about 24 hours prior to administration. As described in more detail below, in at least some embodiments, NK cell transfection with mRNA resulted in unexpectedly consistent and strong expression of the CAR in a high fraction of transfected cells. Furthermore, such transfected cells also exhibited high specific cytotoxicity at relatively low effector-to-target cell ratios.

[0049] Note that with regard to contemplated CARs, NK or NK-92 cells will be genetically modified to express the CAR as a membrane-bound protein, exposing a portion of the CAR on the cell surface while maintaining the signaling domain in the intracellular space. Most typically, the CAR will contain at least the following elements (in order): an extracellular binding domain, a hinge domain, a transmembrane domain, and an FcεRIγ signaling domain.

[0050] In preferred embodiments, the cytoplasmic domain of the CAR comprises or consists of the signaling domain of FcεRIγ. Notably, as described in more detail below, the FcεRIγ signaling domain provides substantially increased expression levels of the CAR while simultaneously providing significantly prolonged cytotoxicity over time. For example, the FcεRIγ signaling domain comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 1. In some embodiments, the FcεRIγ cytoplasmic domain is the only signaling domain. However, it should be recognized that additional elements, such as other signaling domains (e.g., CD28 signaling domain, CD3ζ signaling domain, 4-1BB signaling domain, etc.), may also be included. These additional signaling domains may be located downstream of the FcεRIγ cytoplasmic domain and / or upstream of the FcεRIγ cytoplasmic domain.

[0051] In some embodiments, the FcεRIγ signaling domain comprises, consists of, or consists essentially of an amino acid sequence having at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:1.

[0052] In alternative embodiments, the cytoplasmic domain of the CAR may also comprise the signaling domain of CD3 zeta (CD3ζ). In one embodiment, the cytoplasmic domain of the CAR consists of the signaling domain of CD3 zeta. In one embodiment, the CD3 zeta signaling domain comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 15. In some embodiments, the CD3 zeta signaling domain comprises, consists of, or consists essentially of an amino acid sequence having at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 15.

[0053] The CAR can comprise any suitable transmembrane domain. In one aspect, the CAR comprises a CD28 transmembrane domain. In one embodiment, the CD28 transmembrane domain comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 6 (encoded by a nucleic acid having SEQ ID NO: 7). In one embodiment, the CD28 transmembrane domain comprises, consists of, or consists essentially of an amino acid sequence having at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 6. In other embodiments, the transmembrane domain may be a 4-1BB transmembrane domain.

[0054] The CAR can comprise any suitable hinge region. In one aspect, the CAR comprises the hinge region of CD8. In one embodiment, the CD8 hinge region comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4. In one embodiment, the CD8 hinge region comprises, consists of, or consists essentially of an amino acid sequence having at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4. Such a region can be encoded by a nucleic acid having the sequence of SEQ ID NO:5.

[0055] Thus, a contemplated CAR will comprise the general structure of a desired antigen-binding domain linked to a hinge domain linked to a transmembrane domain linked to a signaling domain. Viewed from another perspective, a contemplated CAR can have a desired binding domain, which is then linked to a hybrid protein comprising, consisting of, or consisting essentially of a hinge domain linked to a transmembrane domain linked to a signaling domain. For example, such a hybrid protein can have an amino acid sequence having at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:8 (encoded by the nucleic acid sequence SEQ ID NO:9).

[0056] Most typically, but not necessarily, the extracellular binding domain of the CAR will be an scFv or other natural or synthetic binding moiety that specifically binds to an antigen of interest. Particularly suitable binding moieties include small antibody fragments, beta-barrel domain binders, phage display fusion proteins, and the like, with single, dual, or multiple target specificities. Among suitable extracellular binding domains, particularly preferred domains will specifically bind to tumor-specific, tumor-associated, or patient- and tumor-specific antigens. Tumor-specific antigens include, but are not limited to, NKG2D ligand, CS1, GD2, CD138, EpCAM, EBNA3C, GPA7, CD244, CA-125, ETA, MAGE, CAGE, BAGE, HAGE, LAGE, PAGE, NY-SEO-1, GAGE, CEA, CD52, CD30, MUC5AC, c-Met, EGFR, FAP, WT-1, PSMA, NY-ESO1, AFP, CEA, CTAG1B, and CD33. Non-limiting examples of additional tumor-associated antigens and their associated malignancies can be found in Table 1. Other additional tumor-specific antigens are described, for example and without limitation, in U.S. Patent Application Publication No. 2013 / 0189268, WO 1999024566 A1, U.S. Patent No. 7,098,008, and WO 2000020460 (each incorporated by reference in its entirety). Similarly, other preferred domains will specifically bind to (pathogenic) virus-specific antigens, such as antigens of the HIV virus (e.g., gp120), HPV virus, RSV virus, influenza virus, Ebola virus, or HCV virus.

[0057] [Table 1]

[0058] [Table 2]

[0059] [Table 3]

[0060] [Table 4]

[0061] For example, the CAR can comprise an anti-CD19 extracellular domain. In one embodiment, the anti-CD19 extracellular domain comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 11. In one embodiment, the anti-CD19 extracellular domain comprises, consists of, or consists essentially of an amino acid sequence having at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 11.

[0062] As such, contemplated CARs will target antigens associated with specific cancer types. For example, target cancers include leukemias (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including promyelocytic, myeloblastic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, solid tumors, including, but not limited to, sarcomas and carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, and angiosarcoma. , endothelial cell carcinoma, lymphangiosarcoma, lymphangioendothelial cell carcinoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0063] Thus, contemplated CARs will generally have the structure of an extracellular binding domain (directly) linked to a hinge domain (directly) linked to a transmembrane domain (directly) linked to an FcεRIγ signaling domain. In other further contemplated embodiments, contemplated CARs may also include one or more signaling domains in addition to or instead of an FcεRIγ signaling domain, with particularly contemplated signaling domains including the CD3ζ signaling domain, the 4-1BB signaling domain, and the CD28 signaling domain. Thus, for example, a contemplated CAR may include any binding domain (e.g., having SEQ ID NO: 11) coupled to a hinge domain (e.g., the CD8 hinge of SEQ ID NO: 3 or SEQ ID NO: 4 encoded by SEQ ID NO: 5), which is in turn coupled to a transmembrane domain (e.g., the CD28TM of SEQ ID NO: 6 encoded by SEQ ID NO: 7) coupled to a signaling domain (e.g., the FcεRIγ signaling domain of SEQ ID NO: 1 encoded by SEQ ID NO: 1, or the CD28 signaling domain of SEQ ID NO: 13, or the 4-1BB signaling domain of SEQ ID NO: 14, or the CD3ζ signaling domain of SEQ ID NO: 15).

[0064] With regard to the construction of contemplated CARs, it should be recognized that CARs can be engineered by many methods, for example, as described in WO 2014 / 039523, U.S. Patent Application Publication No. 2014 / 0242701, U.S. Patent Application Publication No. 2014 / 0274909, U.S. Patent Application Publication No. 2013 / 0280285, and WO 2014 / 099671 (each of which is incorporated by reference in its entirety).

[0065] In other further contemplated aspects, as discussed above, NK cells may be further genetically modified to express one or more cytokines to provide a selectable marker, with the cytokine and CAR encoded on the same recombinant nucleic acid, and / or to render the recombinant cells independent of exogenous IL-2. Thus, in some aspects of the inventive subject matter, NK-92 cells are modified to express at least one cytokine. Specifically, the at least one cytokine is IL-2, IL-12, IL-15, IL-18, IL-21, or a variant thereof. In preferred embodiments, the cytokine is IL-2 or a variant thereof, with particularly preferred variants including an endoplasmic reticulum retention signal (e.g., human IL-2 of SEQ ID NO:21, or one having an ER retention signal of SEQ ID NO:22, SEQ ID NO:30, or SEQ ID NO:33). For example, the IL-2 gene is cloned and expressed with a signal sequence that directs IL-2 to the endoplasmic reticulum. This allows for expression of IL-2 at levels sufficient for autocrine activation without releasing the IL-2 extracellularly (e.g., Exp Hematol. 2005 Feb;33(2):159-64). Alternatively, cytokines (especially IL-15) can be expressed in amounts sufficient to not only provide an autocrine growth signal to the recombinant cells, but also to release at least some of the expressed IL-15 from the cells to provide an immunostimulatory signal. For example, such expression can be achieved using a human IL-15 sequence that includes both a signal peptide and an endogenous retention sequence. Exemplary DNA and protein sequences for endogenous retention IL-15 are set forth in SEQ ID NO:49 and SEQ ID NO:50, respectively.

[0066] Optionally, the contemplated cells may also express a suicide gene. The term "suicide gene" refers to a transgene that allows for negative selection of cells expressing the suicide gene. Suicide genes are used as a safety system, allowing the death of cells expressing the gene by the introduction of a selection agent. This is desirable when the recombinant gene mutates, resulting in uncontrolled cell growth or when the cells themselves are susceptible to such growth. Several suicide gene systems have been identified, including the herpes simplex virus thymidine kinase (TK) gene, the cytosine deaminase gene, the varicella-zoster virus thymidine kinase gene, the nitroreductase gene, the Escherichia coli gpt gene, and the E. coli Deo gene. Typically, suicide genes encode proteins that have no adverse effects on the cell but that will kill the cell in the presence of a specific compound. Therefore, suicide genes are typically part of the system.

[0067] 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 the 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 Jul 15;92(2):672-82. In a further embodiment, the suicide gene is a cytochrome P450, which is toxic in the presence of ifosfamide or cyclophosphamide. See, e.g., Touati et al. "A suicide gene therapy combining the improvement of cyclophosphamide tumor cytotoxicity and the development of an anti-tumor immune response." Curr Gene Ther. 2014;14(3):236-46. In yet another embodiment, the suicide gene is iCasp9. Di Stasi, (2011) "Inducible apoptosis as a safety switch for adoptive cell therapy." N Engl J Med 365:1673-1683. See also Morgan, "Live and Let Die: A New Suicide Gene Therapy Moves to the Clinic" Molecular Therapy (2012);20:11-13.iCasp9 induces apoptosis in the presence of the small molecule AP1903, a biologically inert small molecule that has been shown to be well tolerated in clinical trials and has been used in adoptive cell therapy settings.

[0068] It should be noted, of course, that any recombinant protein can be expressed from an individual recombinant sequence. However, when multiple recombinant sequences are to be expressed (e.g., CAR, CD16, cytokines), it is generally preferred that the coding regions can be arranged in a polycistronic unit having at least two or at least three coding regions encoding recombinant proteins. For example, a tricistronic DNA or RNA construct (e.g., FcεRIγ signaling domain and CD16 158V and IL-2 ER or IL15 ER and encoding a CAR having the following structure: a transgene may be transfected into NK or NK-92 cells. Thus, transgenes can be engineered into expression vectors by any mechanism known to those of skill in the art. When multiple transgenes are inserted into a cell, the transgenes may be engineered into the same expression vector or different expression vectors. In some embodiments, cells are transfected with mRNA encoding the transgenic protein to be expressed. In some embodiments, cells are transfected with DNA encoding the transgenic protein to be expressed. Transgene mRNA and DNA can be introduced into NK-92 cells using any transfection method known in the art, for example, but not limited to, infection, viral vectors, electroporation, lipofection, nucleofection, or "gene gun."

[0069] As will be apparent, contemplated genetically modified cells can be used to treat a variety of diseases, particularly various cancers and viral infections in which diseased cells present disease-specific or disease-associated antigens. Accordingly, the inventors contemplate methods of treating patients with the modified NK or NK-92 cells described herein. In one embodiment, the patient is afflicted with cancer (e.g., a tumor), and the modified NK-92 cells or cell line express a CAR specific for an antigen expressed on the surface of cancer or tumor cells. In one embodiment, the patient is afflicted with a viral infection, and the modified NK-92 cells or cell line express a CAR specific for an antigen expressed on the surface of virally infected cells. In one embodiment, the patient is afflicted with a bacterial infection, and the modified NK-92 cells or cell line express a CAR specific for an antigen expressed on the surface of bacterial cells causing the infection.

[0070] Contemplated modified NK or NK-92 cells can be administered to an individual in absolute cell numbers, e.g., from about 1,000 cells / injection to about 10 billion cells / injection, e.g., about, at least about, or at most about 1 x 10 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 / injection, or any range between any two of these numbers inclusive of the endpoints. In other embodiments, the modified NK-92 cells can be administered to an individual in relative numbers of cells, e.g., from about 1,000 cells to about 10 billion cells / kilogram of individual, e.g., about, at least about, or at most about 1 x 10 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 (and so on) modified NK-92 cells / kilogram individual, or any range between any two of these numbers inclusive of the endpoints. In other embodiments, the total dose is m 2 Body surface area, e.g., approximately 1 × 10 11 , 1×10 10 , 1×10 9 , 1×10 8 , 1×10 7 / m 2 , or any range between any two of these numbers, inclusive of the endpoints. The average person is about 1.6 to 1.8 m 2 In a preferred embodiment, about 1 billion to about 3 billion NK-92 cells are administered to a patient.

[0071] The modified NK-92 cells and optionally other anti-cancer or antiviral agents can be administered once to a cancer or virally infected patient, or multiple times, for example, once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours, or once every 1, 2, 3, 4, 5, 6, or 7 days, or once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks or more, or any range between any two of these numbers inclusive of the endpoints, during the course of treatment.

[0072] In one embodiment, an agent that triggers the death of the modified NK-92 cells when the modified NK-92 cells express the suicide gene is administered to the patient. In one embodiment, the agent is administered at a time after administration of the modified NK-92 cells sufficient for the NK-92 cells to kill the target cells.

[0073] In one embodiment, the modified NK-92 cells are irradiated prior to administration to a patient. Irradiation of NK-92 cells is described, for example, in U.S. Patent No. 8,034,332, which is incorporated herein by reference in its entirety. In one embodiment, modified NK-92 cells that have not been engineered to express a suicide gene are irradiated.

[0074] Furthermore, it should be recognized that contemplated treatments will also include the administration of other immunotherapeutic entities, among which preferred immunotherapeutic entities include viral cancer vaccines (e.g., adenoviral vectors encoding cancer-specific antigens), bacterial cancer vaccines (e.g., non-pyrogenic E. coli expressing one or more cancer-specific antigens), yeast cancer vaccines, N-803 (also known as ALT-803 from ALTOR Biosciences), antibodies (e.g., those that bind to tumor-associated antigens or patient-specific tumor neo-antigens), stem cell transplants (e.g., allogeneic or autologous), and tumor-targeted cytokines (e.g., NHS-IL12 or fragments thereof, which is IL-12 conjugated to a tumor-targeted antibody). [Example]

[0075] The following examples are for illustrative purposes only and should not be construed as limiting the claimed invention. There are a variety of alternative techniques and procedures available to those skilled in the art that may similarly enable successful practice of the intended invention.

[0076] Example 1: Preparation of CAR mRNA DNA sequences encoding each variant of CD19CAR, represented schematically in Figure 1, were designed in silico, synthesized de novo, and subcloned into the mRNA expression vector pXT7 (GeneArt, Life Technologies). Ten micrograms (μg) of plasmid were linearized by digestion with SalI restriction enzyme (New England Biolabs) and purified using a QIAgen gel purification kit (QIAgen) according to the manufacturer's instructions.

[0077] Linearized DNA was used as a template for in vitro synthesis of mRNA using the T7 mMessage mMachine Ultra transcription kit (ThermoFisher Scientific, Waltham, MA) according to the manufacturer's instructions. This kit includes a polyadenylation extension step that increases the length of the mRNA's poly(A) tail, thereby enhancing its stability in vivo.

[0078] As a negative control, green fluorescent protein (GFP) mRNA was prepared, and mRNAs of six CD19CAR variants were also prepared. All CD19CAR variants contained an extracellular domain comprising an anti-CD19 scFv region (αCD19-scFv) (SEQ ID NO: 11) and a CD8-derived hinge region (SEQ ID NO: 3 or 4), and a CD28-derived transmembrane domain (SEQ ID NO: 6, encoded by SEQ ID NO: 7). The intracellular domains of CD19CAR are as follows, and are shown schematically in Figure 1. That is, CAR3z contained a CD3ζ signaling domain, CARFcRe contained an FcεRIγ signaling domain (sequence number 1), CAR28_3z contained a CD28 signaling domain fused to a CD3ζ signaling domain, CARBB_3z contained a 4-1BB signaling domain fused to a CD3ζ signaling domain, CAR28_BB_3z contained a CD28 signaling domain fused to a 4-1BB signaling domain fused to a CD3ζ signaling domain, and CARBB_3z_28 contained a 4-1BB signaling domain fused to a CD3ζ signaling domain fused to a CD28 signaling domain.

[0079] More specifically, the first-generation CAR with the CD3ζ signaling domain in Figure 1 had the nucleic acid sequence of SEQ ID NO: 16 (human). The first-generation CAR with the FcεRIγ signaling domain had the nucleic acid sequence of SEQ ID NO: 12 and the amino acid sequence of SEQ ID NO: 10. The second-generation CAR with the CD28 / CD3ζ signaling domain had the nucleic acid sequence of SEQ ID NO: 17, and the second-generation CAR with the 4-1BB / CD3ζ signaling domain had the nucleic acid sequence of SEQ ID NO: 18. The third-generation CAR with the CD28 / 4-1BB / CD3ζ signaling domain had the nucleic acid sequence of SEQ ID NO: 19, and the third-generation CAR with the 4-1BB / CD3ζ / CD28 signaling domain had the nucleic acid sequence of SEQ ID NO: 20. A further first-generation CAR with the FcεRIγ signaling domain had the amino acid sequence of SEQ ID NO: 25.

[0080] Example 2: Electroporation of CD19CAR mRNA into NK-92 cells NK-92 cells were grown in X-Vivo10 medium (Lonza, Basel, Switzerland) supplemented with 5% human AB serum (Valley Biomedical, Winchester, VA) and 500 IU / mL IL-2 (Prospec, Rehovot, Israel) using a Neon™ electroporation device (Life Technologies, Carlsbad, CA) according to the manufacturer's parameters for NK-92 cells (1250 V, 10 ms, 3 pulses) and 5 μg of mRNA / 10 in a volume of 100 μl. 6 The cells were electroporated with mRNA using the following method: The electroporated cells were maintained in the medium (same as above) for 20 hours (h).

[0081] CD19CAR expression on the surface of NK-92 cells was determined by flow cytometry using an eF660-labeled anti-scFv antibody (eBioscience, San Diego, CA). Figure 2A shows the % expression of the indicated CD19CAR in the NK-92 cell population. Figure 2B shows the median fluorescence intensity (MFI, minus background) of cells electroporated with the indicated CD19CAR. As can be seen from Figures 2A and 2B, 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 the recombinant cells), followed by 28_3z (61.7%).

[0082] Example 3: Cytotoxicity of CD19CAR-expressing NK-92 cells against cancer cell lines Twenty hours after electroporation, we tested the efficacy of CAR-expressing NK-92 cells against target cancer cells in vitro using a flow-based in vitro cytotoxicity assay. Effector cells (NK-92 expressing CD19CAR or GFP) were cultured in 96-well plates at various effector-to-target ratios (5:1 to 0.3:1) with PKHGL67-labeled (Sigma-Aldrich, St. Louis, MO) target cells (K562 or SUPB15, B-ALL, CD19). + ) and PKH-positive target cells were mixed and incubated at 37°C for 4 h. Propidium iodide (PI) (Sigma Aldrich, St. Louis, MO) was added to the cells, and samples were analyzed within 2 h using an Attune flow cytometer (Life Technologies, Carlsbad, CA). Cytotoxicity was determined by the percentage of PI-positive cells within the PKH-positive target population.

[0083] Exemplary results are provided in Figures 3A and 3B. As can be seen from Figure 3A, NK-92 cells are effective at killing K562 cells regardless of CD19CAR expression. Therefore, it should be noted that the recombinant cells would not lose their cytotoxicity. In contrast, GFP-expressing NK-92 cells were less efficient at killing the cancer cell line SUP-B15. SUP-B15 is a CD19-positive acute lymphoblastic leukemia cell line that is resistant to NK-92-mediated cytotoxicity. As can be readily seen from Figure 3B, expression of any of the CD19CARs tested resulted in increased cytotoxic activity against the SUP-B15 cell line compared to the control (GFP-expressing NK-92 cells). Surprisingly, CARs with an FcεRIγ signaling domain exhibited cytotoxicity similar to, and even superior to, second- and third-generation CARs. This finding is particularly unexpected because the FcεRIγ signaling domain was present only as a single unit rather than in combination with other signaling domains, an arrangement that failed to provide the desired targeted cytotoxicity when used in CART cells.

[0084] Degranulation is a critical step required for the release of soluble proteins (e.g., perforin and granzymes) from secretory granules in NK-92 cells. Degranulation is initiated by target cell recognition by NK-92. To test degranulation of the constructs, effector cells (NK-92) and unlabeled target cells (SUP-B15) were mixed at various effector-to-target ratios (5:1 to 0.3:1) in a 96-well plate, and anti-CD107a (FITC-conjugated, BD Pharmingen, San Jose, CA) was added to each well. The plate was incubated at 37°C in a CO2 incubator, and after 1 hour, monensin (Golgi-stop) was added to the wells. The plate was incubated at 37°C for an additional 3 hours, and samples were analyzed by flow cytometry (Attune, Life Technologies, Carlsbad, CA). Percent degranulation was determined by subtracting the %CD107a positivity of NK-92 cells alone from the %CD107a positivity of the effector + target sample. Exemplary results are provided in Figure 4.

[0085] Example 4. Surface expression of CD19CAR-expressing NK-92 cells and cytotoxicity against cancer cell lines. The inventors quantified the expression levels of various CAR constructs and examined the durability of expression over time. As can be seen from the results in Figure 5, NK-92 cells transfected with various CD19CAR constructs expressed detectable levels of each CAR on the cell surface for up to 72 hours. Unexpectedly, as can be readily seen from Figure 5, CAR constructs containing an Fc epsilon cytoplasmic signaling domain had a substantially longer duration of expression. Also noteworthy, it was observed that adding one or more signaling domains in addition to the FcεRIγ signaling domain (e.g., the CD28 signaling domain in the example presented herein) did not adversely affect the duration of expression. Indeed, for CARs containing the FcεRIγ signaling domain and the CD28 signaling domain, the duration of expression further increased over time, while CAR constructs containing the CD3 zeta signaling domain had a dramatic reduction in expression at and even before 72 hours.

[0086] Furthermore, as can also be seen from the results in Figure 5, the expression levels of the CAR constructs with the FcεRIγ signaling domain were also initially significantly higher than the corresponding constructs with the CD3 zeta signaling domain.

[0087] We then began to test whether prolonged and stronger expression of a CAR construct with the FcεRIγ signaling domain would lead to higher cytotoxicity rates. +Exemplary cell test results are presented in Figure 6. As can be seen from the results, all tested CAR constructs exhibited similar (maximal) cytotoxicity at 24 hours. However, at 48 hours, CD19 / CD3 zeta showed a marked decrease in cytotoxicity. Notably, the Fc epsilon-based CAR showed only a slight decrease in cytotoxic activity 48 hours after electroporation, corresponding to the long-term expression results in Figure 5. Therefore, it should be noted that the CAR construct with the FcεRIγ signaling domain exhibited long-term cytotoxicity that is believed to have substantial clinical efficacy.

[0088] Advantageously, tricistronic RNA constructs can produce substantial amounts of the desired CAR with excellent functional activity. Such constructs are particularly useful when CAR expression is to be transient. In contrast, the following examples of targeted CAR constructs and related functional data are derived from linearized DNA vector constructs, which allow transfected cells to integrate the linearized DNA into the genome, providing a means for non-transient expression of specific CARs.

[0089] Example 5. Map of the tricistronic expression cassette. Figure 7 shows a schematic of the DNA and protein products produced by a representative tricistronic expression cassette, and Figure 8 shows the linearized form of the plasmid carrying the expression cassette.

[0090] SEQ ID NO: 28 is an exemplary nucleic acid sequence of a portion of the pNEUKv1_CD19CAR_CD16(158V)_ERIL-2 vector, a construct similar to Figure 8. SEQ ID NO: 29 is an exemplary tricistronic protein (similar to Figure 7) representing the CD19CAR_P2A_CD16(158V) protein. Similarly, SEQ ID NO: 31 is an exemplary nucleic acid sequence of a codon-optimized CD33ScfV-P2A-CD16-IRES-erIL2 tricistronic sequence, while SEQ ID NO: 32 represents the CD33CAR-P2A-CD16 peptide.

[0091] Other additional constructs that were produced include SEQ ID NO: 24, an exemplary amino acid sequence of the CD19K_transmembrane and signaling domain, SEQ ID NO: 26, an exemplary nucleic acid sequence of 15AD23HC_1805843_CD19K_Eps (879-1319), and SEQ ID NO: 27, an exemplary nucleic acid sequence of 15AD23HC_1805843_CD19K_Eps that did not include the CD28 transmembrane domain.

[0092] Example 6. Cytotoxicity of CD33-CAR-expressing NK-92 cells against cancer cell lines The following example is provided to demonstrate that cells that are resistant to specific lysis (cytotoxicity) by control (unmodified) NK-92 cells can be efficiently killed by CAR-expressing NK-92 cells. In this example, the cells were THP-1 cells expressing CD33. The NK-92 cells were modified to express a CAR having an extracellular binding domain that specifically binds to CD33 and an FcεRIγ signaling domain, as shown in Figures 8 and 9.

[0093] Figure 9A provides in vitro data showing that CD33-positive (CD33+) THP-1 cells are relatively resistant to cytotoxicity (specific lysis) by control NK-92 (aNK) cells, but a high percentage of specific lysis is present when THP-1 cells are cultured with NK-92 cells expressing a CAR that specifically binds to CD33 (CD33-CAR / NK-92 cells). Furthermore, it should be noted that the CAR-expressing modified NK-92 cells exhibited killing at a relatively low effector:target ratio. Figure 9B provides in vitro data showing that K562 cells are efficiently killed by both control aNK cells and CD33-CAR / NK-92 cells.

[0094] Example 7: HER2-CAR with FcεRIγ signaling domain In this example, we constructed a first-generation CAR with an FcεRIγ signaling domain, which contained an anti-HER2 scFv linked to a CD8 hinge, which in turn was linked to a CD28 transmembrane domain linked to the FcεRIγ signaling domain. The HER2-CAR thus constructed had the nucleic acid sequence of SEQ ID NO: 37.

[0095] The functionality of the thus constructed HER2.CAR-t-haNK cells was tested against BT-474 cells using a standard calcein AM-based cytotoxicity assay. Exemplary results are shown in Figure 10. As can be readily seen from the data, HER2.CAR-t-haNK cells expressing a CAR with an FcεRIγ signaling domain exhibited significant cytotoxicity against BT-474 target cells.

[0096] In further experiments, the inventors demonstrated expression of HER2.CAR in HER2.CAR-t-haNK cells, as illustrated in Figure 36. The natural cytotoxicity of HER2.CAR-t-haNK cells is shown in the results of Figure 37, while the results of CAR-mediated cytotoxicity are shown in Figure 38. Exemplary data for ADCC of HER2.CAR-t-haNK cells are shown in the graph of Figure 39.

[0097] Example 8: CD30-CAR with FcεRIγ signaling domain In this example, we constructed a first-generation CAR with an FcεRIγ signaling domain, which contained an anti-CD30 scFv linked to a CD8 hinge, which in turn was linked to a CD28 transmembrane domain linked to the FcεRIγ signaling domain. The CD30-CAR thus constructed had the nucleic acid sequence of SEQ ID NO: 38.

[0098] Expression of CD30-CAR is demonstrated in the results in Figure 46, while the natural cytotoxicity results of the recombinant cells are shown in Figure 47. CAR-mediated cytotoxicity is demonstrated in the results in Figure 48, while exemplary results of ADCC are shown in the data in Figure 49.

[0099] Example 9: EGFR-CAR with FcεRIγ signaling domain In this example, we constructed a first-generation CAR with an FcεRIγ signaling domain, which contained an anti-EGFR scFv linked to a CD8 hinge, which in turn was linked to a CD28 transmembrane domain linked to the FcεRIγ signaling domain. The EGFR-CAR thus constructed had the nucleic acid sequence of SEQ ID NO: 39.

[0100] The functionality of the thus constructed EGFR.CAR-t-haNK cells was tested against A-549 cells using a standard cytotoxicity assay. Exemplary results are shown in Figure 14. As can be readily seen from the data, EGFR.CAR-t-haNK cells expressing a CAR with an FcεRIγ signaling domain exhibited significant cytotoxicity against A-549 target cells. Expression of EGFR-CAR on EGFR.CAR-t-haNK cells is shown in Figure 31, while the results of natural cytotoxicity are shown in Figure 32. Exemplary results of CAR-mediated cytotoxicity of EGFR.CAR-t-haNK cells are shown in Figures 33 and 34, while the results of ADCC of EGFR.CAR-t-haNK cells are shown in Figure 35.

[0101] Example 10: IGF1R-CAR with FcεRIγ signaling domain In this example, the inventors constructed a first-generation CAR with an FcεRIγ signaling domain, which contained an anti-IGF1R scFv linked to a CD8 hinge, and the CD8 hinge was linked to a CD28 transmembrane domain linked to the FcεRIγ signaling domain. The IGF1R-CAR thus constructed had the nucleic acid sequence of SEQ ID NO: 40, and was composed of IGF1R-CAR, CD16, and IL-2. ER The tricistronic construct encoding the nucleotide sequences of SEQ ID NO: 53, which is also illustrated diagrammatically in FIG.

[0102] The functionality of the thus constructed IGF1R.CAR-t-haNK cells was tested against MDA-MB-231 cells using a standard cytotoxicity assay in comparison with a second-generation CAR (CD28 / CD3z). Exemplary results are shown in Figure 18. As can be readily seen from the data, IGF1R.CAR-t-haNK cells expressing a CAR with an FcεRIγ signaling domain exhibited significant target-specific cytotoxicity against MDA-MB-231 target cells that was comparable to the cytotoxicity of the second-generation CAR.

[0103] Example 11: CD123-CAR with FcεRIγ signaling domain In this example, the inventors constructed a first-generation CAR with an FcεRIγ signaling domain. This comprised an anti-CD123 scFv linked to a CD8 hinge, which in turn was linked to a CD28 transmembrane domain linked to the FcεRIγ signaling domain. The CD123-CAR thus constructed had the nucleic acid sequence of SEQ ID NO: 41. Data on CAR-mediated cytotoxicity of CD123-CAR expressing recombinant NK cells is shown in Figure 44, and Figure 45 shows exemplary data on ADCC of CD123-CAR expressing recombinant NK cells.

[0104] Example 12: PD-L1-CAR with FcεRIγ signaling domain In this example, we constructed a first-generation CAR with an FcεRIγ signaling domain, which comprised an anti-PD-L1 scFv linked to a CD8 hinge, which in turn was linked to a CD28 transmembrane domain linked to the FcεRIγ signaling domain. The PD-L1-CAR thus constructed had the nucleic acid sequence of SEQ ID NO:42.

[0105] The functionality of the constructed PD-L1.CAR-t-haNK cells was evaluated using standard cytotoxicity assays to assess the efficacy of SUP-B15.PD-L1 +The exemplary results are shown in Figure 12. As can be readily seen from the data, PD-L1.CAR-t-haNK cells expressing a CAR with an FcεRIγ signaling domain were tested against SUP-B15.PD-L1 + It exhibited significant cytotoxicity against target cells.

[0106] The functionality of the thus constructed PD-L1.CAR-t-haNK cells was further tested against U251 cells using a standard cytotoxicity assay. Exemplary results are shown in Figure 13 along with non-transfected haNK cells. As can be readily seen from the data, PD-L1.CAR-t-haNK cells expressing a CAR with an FcεRIγ signaling domain exhibited target-specific and significant cytotoxicity against U251 target cells, whereas the haNK control cells had virtually no cytotoxicity against the same U251 cells.

[0107] In further experiments regarding target cell specificity for PD-L1, the inventors tested several PD-L1-positive tumor cell lines using PD-L1.CAR-t-haNK cells along with haNK cells as a general cytotoxicity control. As can be readily seen from Figure 19, PD-L1.CAR-t-haNK cells had excellent cytotoxicity across a wide variety of tumor cells (lung, breast, genitourinary tumor cells, as well as head and neck small cell carcinoma and chordoma). Notably, PD-L1.CAR-t-haNK cells required less than 4 hours to kill the majority of cells (>85%), while control haNK cells required more than 12 hours.

[0108] Figure 20 further illustrates the cytotoxicity of PD-L1.CAR-t-haNK cells against MDA-MB-231 cells compared to various other control cells (haNK cells, as indicated). As can be seen from the data, at an E:T ratio of 5:1, MDA-MB-231 lysis by PD-L1.t-haNK was improved by cetuximab, and haNK activity was improved by the addition of cetuximab and a-PD-L1. Plain PD-L1.thank improved cytotoxic activity compared to haNK and haNK + cetuximab, and killing by plain PD-L1.thank was comparable to haNK + PD-L1 antibody, while PD-L1.thank + cetuximab outperformed haNK + cetuximab and haNK + PD-L1. At a 1:1 E:T ratio, PD-L1.thaNK activity was similar with or without cetuximab, and PD-L1.thaNK significantly outperformed endogenous and ADCC-mediated killing by hank. hank activity was improved by the addition of cetuximab and a-PD-L1.

[0109] In further experiments, the inventors demonstrated expression of PD-L1.CAR on PD-L1.CAR-t-haNK cells, as illustrated in Figure 40. The natural cytotoxicity of PD-L1.CAR-t-haNK cells is shown in the results in Figure 41, while the results of CAR-mediated cytotoxicity are shown in Figure 42. Exemplary data for ADCC of PD-L1.CAR-t-haNK cells are shown in the graph in Figure 43.

[0110] Example 13: CD33-CAR with FcεRIγ signaling domain In this example, we constructed a first-generation CAR with an FcεRIγ signaling domain, which contained an anti-HER2 scFv linked to a CD8 hinge, which in turn was linked to a CD28 transmembrane domain linked to the FcεRIγ signaling domain. The CD33-CAR thus constructed had the nucleic acid sequence of SEQ ID NO: 43.

[0111] The functionality of the constructed CD33.CAR-t-haNK cells was tested against THP-1 cells using a standard cytotoxicity assay. Exemplary results are shown in Figure 11. As can be readily seen from the data, CD33.CAR-t-haNK cells expressing a CAR with an FcεRIγ signaling domain exhibited significant cytotoxicity against THP-1 target cells. Further data demonstrating strong expression of CD33CAR on NK-92 cells is presented in Figure 27. The natural cytotoxicity of CD33.CAR-t-haNK cells against K562 cells is shown in Figure 28, and Figure 29 presents the results of CAR-mediated cytotoxicity against THP-1 cells. Figure 30 shows the results of SUP-B15CD19 in combination with rituximab. KO / CD20 + Further results of ADCC of CD33.CAR-t-haNK cells against IgG4-associated leukemia virus (IgG4-associated leukemia virus) are shown.

[0112] Example 14: gp120-CAR with FcεRIγ signaling domain In this example, we constructed a first-generation CAR with an FcεRIγ signaling domain, which contained an anti-gp120 scFv linked to a CD8 hinge, which in turn was linked to a CD28 transmembrane domain linked to the FcεRIγ signaling domain. The gp120-CAR thus constructed had the nucleic acid sequence of SEQ ID NO: 44.

[0113] The inventors further demonstrated that the cells so generated express significant amounts of CD16 and gp120CAR as can be seen in Figure 53. Binding of GP120 to gp120CAR was shown as demonstrated in Figure 54 versus non-modified aNK cells as a negative control. Correspondingly, the natural cytotoxicity of the cells so generated is shown in Figure 55, while the corresponding ADCC data is shown in Figure 56.

[0114] Example 15: B7-H4-CAR with FcεRIγ signaling domain In this example, we constructed a first-generation CAR with an FcεRIγ signaling domain, which contained an anti-B7-H4 scFv linked to a CD8 hinge, which in turn was linked to a CD28 transmembrane domain linked to the FcεRIγ signaling domain. The B7-H4-CAR thus constructed had the nucleic acid sequence of SEQ ID NO: 45.

[0115] Example 16: BCMA-CAR with FcεRIγ signaling domain In this example, we constructed a first-generation CAR with an FcεRIγ signaling domain, which comprised an anti-BCMA scFv linked to a CD8 hinge, which in turn was linked to a CD28 transmembrane domain linked to the FcεRIγ signaling domain. The BCMA-CAR thus constructed had the nucleic acid sequence of SEQ ID NO: 46.

[0116] BCMA expression was confirmed as shown in the exemplary results in Figure 50, and CAR-mediated cytotoxicity was demonstrated against target cells as shown in Figure 51. Similarly, as can be seen from the results in Figure 52, the recombinant cells had significant ADCC using rituximab as the antibody against the target cells.

[0117] Example 17: GD2-CAR with FcεRIγ signaling domain In this example, we constructed a first-generation CAR with an FcεRIγ signaling domain, which contained an anti-GD2 scFv linked to a CD8 hinge, which in turn was linked to a CD28 transmembrane domain linked to the FcεRIγ signaling domain. The GD2-CAR thus constructed had the nucleic acid sequence of SEQ ID NO: 47.

[0118] Example 18: FAP-CAR with FcεRIγ signaling domain In this example, the inventors constructed a first-generation CAR with an FcεRIγ signaling domain. This comprised an anti-FAP scFv linked to a CD8 hinge, which in turn was linked to a CD28 transmembrane domain linked to the FcεRIγ signaling domain. The FAP-CAR thus constructed had the nucleic acid sequence of SEQ ID NO: 48. Expression of the FAP-CAR is shown in the data in Figure 57, and FAP.CAR cytotoxicity against target cells was demonstrated in the results in Figure 58.

[0119] Example 19: CSPG-4-CAR with FcεRIγ signaling domain In this example, the inventors constructed a first-generation CAR with an FcεRIγ signaling domain. This comprised an anti-CSPG-4 scFv linked to a CD8 hinge, which in turn was linked to a CD28 transmembrane domain linked to the FcεRIγ signaling domain. The CSPG-4-CAR thus constructed had the nucleic acid sequence of SEQ ID NO: 52. Expression of the CSPG-4-CAR was confirmed by FACS analysis, and exemplary results are shown in Figure 59. Cells constructed in this manner also exhibited significant cytotoxicity, as shown in the exemplary data in Figure 60.

[0120] Example 20: CD20-CAR with FcεRIγ signaling domain In this example, we constructed a first-generation CAR with an FcεRIγ signaling domain, which contained an anti-CD20 scFv linked to a CD8 hinge, and the CD8 hinge was linked to a CD28 transmembrane domain linked to the FcεRIγ signaling domain. The CD20-CAR thus constructed had the nucleic acid sequence of SEQ ID NO: 51.

[0121] The expression of CD20CAR in NK-92 cells is shown in the results of Figure 25. As can be easily seen, CD20.CAR is strongly expressed in the majority of recombinant cells (along with CD16 in the linearized DNA described above). Figure 26 shows the expression of CD20 +1 shows exemplary results of CD20.CARNK cell cytotoxicity against target cells.

[0122] Example 21: CD19-CAR with FcεRIγ signaling domain In this example, we used the first-generation CAR described above with an FcεRIγ signaling domain, which contains an anti-CD19 scFv linked to a CD8 hinge, which in turn is linked to a CD28 transmembrane domain linked to the FcεRIγ signaling domain, and transfected NK-92 cells with the linearized DNA for functional testing.

[0123] The functionality of the constructed CD19.CAR-t-haNK cells was tested against K562 cells to determine general cytotoxicity using a standard cytotoxicity assay. Exemplary results are shown in Figure 15. As can be seen, CD19.CAR-t-haNK cells expressing a CAR with an FcεRIγ signaling domain exhibited significant cytotoxicity against K562 target cells. In a further set of experiments, target-specific cytotoxicity was determined using SUP-B15 cells compared with aNK cells as a control. Exemplary results are shown in Figure 16. Again, CD19.CAR-t-haNK cells expressing a CAR with an FcεRIγ signaling domain exhibited significant target-specific cytotoxicity. Furthermore, in yet another set of experiments, target-specific ADCC was determined using SKBr3 cells and Herceptin and Rituxan as antibodies. Exemplary results are shown in Figure 17. Again, CD19.CAR-t-haNK cells expressing a CAR with an FcεRIγ signaling domain exhibited significant antibody and target-specific ADCC.

[0124] Figure 21 shows the expression of CD16 and IL-2 in NK-92 cells compared to controls. ERFigure 25 exemplarily illustrates CD19.CAR expression from linearized DNA containing segments encoding the SUP-B15 and SUP-B15CD19. As can be seen from Figure 25, expression was very strong across the majority of cells. Additional results of the natural cytotoxicity of CD19.CARt-haNK cells against K562 cells and targeted cytotoxicity against SUP-B15 cells are presented in Figures 22 and 23. KO / CD20 + Further exemplary results of ADCC of CD19.CARt-haNK cells against IgG4-associated leukemia cells are shown in Figure 24.

[0125] Example 22: Antitumor activity of PD-L1-targeted t-haNK cells in a human xenograft model in NSG mice Using MDA-MB-231 and HCC827 as validated PDL1-positive xenograft models, we evaluated the efficacy of PDL1t-haNK cells at different formulations, dose levels, and routes of administration (IV and IT).

[0126] Animals: Animal type: NSG mice (JAX), female, 9-10 weeks old. Number of animals for the MDA-MB-231 model: 24 (fresh cells), and for the HCC827 model: 24 (fresh cells) + 6 (cryopreserved cells). The tumor models used were the following cell lines: MDA-MB-231 (human breast adenocarcinoma) and HCC827 (human lung adenocarcinoma). The inoculation route was subcutaneous in both flanks. The mean tumor burden at the start of treatment was approximately 100 mm3 for MDA-MB-231 and approximately 75-80 mm3 for HCC827.

[0127] Treatment: Anti-PD-L1t-haNK was freshly prepared and irradiated at a concentration of 5E7 cells / mL or 2E7 cells / mL. The vehicle control was X-VIVO™ 10 medium. Administration was IV and IT as indicated. The dose for IV NK administration was 1E7 cells / administration in 200 μL (freshly prepared cells) or 4E6 cells / administration in 200 μL (cryopreserved cells). For IT NK administration (fresh cells only), the dose was 2.5E6 cells / tumor / administration in 50 μL. Administration frequency was twice weekly for 4 consecutive weeks (M / Th or T / F), with the first day of administration defined as day 1.

[0128] The MDA-MB-231 study design is set forth below in Table 3 (the study was terminated on day 27, when some animals in groups A, C, and D reached a total tumor volume of >2000 mm3).

[0129] [Table 5]

[0130] The study design for HCC827 is set forth below in Table 4 (the study was terminated on day 29, at which time surviving animals were repurposed and transferred to other studies).

[0131] [Table 6]

[0132] Results: Freshly prepared PD-L1t-haNK cells (1E7 cells / dose) produced significant and long-lasting tumor growth inhibition in both MDA-MB-231 and HCC827 models.

[0133] MDA-MB-231: Tumor stasis: TGI on day 16: 84% (peak), TGI on day 26: 79% (last measurement).

[0134] HCC827: Tumor regression: TGI on day 16: 120% (peak), TGI on day 29: 84% (end of study).

[0135] Cryopreserved PDL1t-haNK cells (4E6 cells / dose) also demonstrated statistically significant efficacy in tumor growth inhibition compared to X-VIVO™ 10 medium: TGI on day 26: 60% (peak) and TGI on day 29: 40% (end of study).

[0136] Freshly prepared PDL1t-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 5 below.

[0137] [Table 7]

[0138] Number of visible liver nodules present in the vehicle: 29±9, in the PD-L1t-haNK group: 0 (P=0.0116 by unpaired two-tailed t-test).

[0139] Based on the experiments performed, twice-weekly IV administration of freshly prepared PD-L1t-haNK cells at a dose level of 1E7 cells / dose for 4 weeks demonstrated remarkable anti-tumor efficacy in both subcutaneous xenograft models tested: treatment resulted in tumor stasis in MDA-MB-231 tumor-bearing mice, with a peak TGI of 84% at day 16 and a TGI of 79% at study end (P<0.0001 by two-way ANOVA for both time points, followed by Tukey's test for multiple comparisons), and tumor regression in the HCC827 model, with a peak TGI of 120% at day 16 and a TGI of 84% at study end (P<0.0001). IV administration of cryopreserved PD-L1t-haNK cells at a dose level of 4E6 cells / dose twice weekly for 4 weeks further demonstrated significant therapeutic efficacy in the HCC827 tumor model, reaching a peak TGI of 60% (P<0.0001) and an end-of-study TGI of 40% (P<0.01). IT administration of freshly prepared PD-L1t-haNK cells at a dose level of 2.5E6 cells / dose / tumor twice weekly for 4 weeks effectively inhibited HCC827 tumor growth, resulting in a peak TGI of 70% at day 20 and a TGI of 49% at the end of the study (p<0.001).

[0140] Significant adverse reactions were observed in animals receiving IV administration (1E7 cells / dose) of freshly prepared PD-L1t-haNK cells. In contrast to freshly prepared PD-L1t-haNK cells, cryopreserved cells (administered at a lower level of 4E6 cells / dose) were found to be safe in animals after IV administration. PD-L1t-haNK cells demonstrated significant efficacy in two subcutaneous tumor models. Cryopreserved cells administered at the lower level of 4E6 cells / dose also showed significant efficacy in tumor growth inhibition and were found to be safe in animals.

[0141] Of course, it should be recognized that for all nucleic acid sequences provided herein, the corresponding encoded proteins are also expressly contemplated herein. Similarly, for all amino acid sequences, the corresponding nucleic acid sequences are also contemplated herein (in any codon usage).

[0142] All patent applications, publications, references, and sequence accession numbers cited herein are hereby incorporated by reference in their entirety.

[0143] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0144] In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings.

[0145] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0146] All numerical values ​​(e.g., pH, temperature, time, concentration, amount, and molecular weight, including ranges) set forth herein are understood to include normal variations in measurement encountered by those of ordinary skill in the art. Thus, numerical values ​​set forth herein include variations of ±0.1 to 10%, e.g., ±0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. It should be understood, although not always explicitly stated, that all numerical designations may be preceded by the term "about." Thus, the term "about" includes variations of ±0.1 to 10%, e.g., ±0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of that numerical value. It should also be understood, although not always explicitly stated, that the reagents set forth herein are merely exemplary and that equivalents of such are known in the art.

[0147] Those skilled in the art will understand that for all purposes, particularly in connection with providing a written description, all ranges disclosed herein include the endpoints of the range and all values ​​between the endpoints. Also, all ranges disclosed herein encompass any possible subranges and combinations of subranges. Any recited range is readily recognizable as being fully descriptive and capable of being divided into at least two, three, four, five, ten, etc. For example, and without limitation, each range discussed herein can be readily broken down into a lower third, middle third, and upper third. Those skilled in the art will also understand that all expressions such as "up to," "at least," and the like, refer to ranges that are inclusive of the recited numbers and that can subsequently be broken down into subranges as discussed above. Finally, those skilled in the art will understand that a range includes each individual member. Thus, for example, a group having 1 to 3 cells refers to a group having 1, 2, or 3 cells. Similarly, a group having 1 to 5 cells means a group having 1, 2, 3, 4, or 5 cells, and so forth.

[0148] It is also to be understood, although not necessarily expressly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0149] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes cases where the event or circumstance occurs and cases where it does not occur.

[0150] The term "comprising" is intended to mean that the compositions and methods include the recited elements, but do not exclude others. When used in defining compositions and methods, "Consisting essentially of" shall mean excluding other elements that are essentially significant 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" shall mean excluding more than trace amounts of other ingredients and substantial method steps other than those recited. Embodiments defined by each of these transitional phrases are within the scope of this disclosure.

[0151] As used herein, "immunotherapy" refers to the use of modified or unmodified NK-92 cells, naturally occurring or modified NK cells, or T cells, either alone or in combination, that are capable of inducing cytotoxicity upon contact with target cells.

[0152] As used herein, "natural killer (NK) cells" refer to cells of the immune system that kill target cells in the absence of specific antigenic stimulation and without major histocompatibility complex (MHC) class restriction. Target cells may be tumor cells or virus-bearing cells. NK cells are characterized by the presence of CD56 and the absence of CD3 surface markers.

[0153] The term "endogenous NK cells" is used to refer to NK cells derived from a donor (or patient) that are distinguished from the NK-92 cell line. Endogenous NK cells are generally a heterogeneous cell population enriched for NK cells. Endogenous NK cells may be intended for autologous or allogeneic therapy of a patient.

[0154] The term "NK-92" refers to natural killer cells derived from a highly potent, unique cell line described by Gong et al. (1994) and owned by NantKwest (hereinafter "NK-92™ cells"). The immortal NK cell line was originally obtained from a patient with non-Hodgkin's lymphoma. Unless otherwise specified, the term "NK-92™" is intended to refer to the original NK-92 cell line as well as modified NK-92 cell lines (e.g., by introduction of an exogenous gene). NK-92™ cells and exemplary, non-limiting variants thereof are described in U.S. Patent Nos. 7,618,817, 8,034,332, 8,313,943, 9,181,322, and 9,150,636, and U.S. Application No. 10 / 008,955, all of which are incorporated by reference in their entireties, and include, for example, wild-type NK-92™, NK-92™-CD16, NK-92™-CD16-γ, NK-92™-CD16-ζ, NK-92™-CD16(F176V), NK-92™ MI, and NK-92™ CI. NK-92 cells are known to those of skill in the art, and such cells are readily available from NantKwest, Inc.

[0155] The term "aNK" refers to natural killer cells derived from a highly potent, unique cell line described in Gong et al. (1994), the property of NantKwest (hereinafter "aNK™ cells"). The term "haNK" refers to natural killer cells derived from a highly potent, unique cell line described in Gong et al. (1994), the property of NantKwest, that have been modified to express CD16 on the cell surface (hereinafter "CD16+ NK-92™ cells" or "haNK® cells"). In some embodiments, CD16+ NK-92™ cells contain the high-affinity CD16 receptor on the cell surface. The term "taNK" refers to natural killer cells derived from a highly potent, unique cell line described in Gong et al. (1994), the property of NantKwest, that have been modified to express a chimeric antigen receptor (hereinafter "CAR-modified NK-92™ cells" or "taNK® cells"). The term "t-haNK" refers to natural killer cells derived from a highly potent and unique cell line described in Gong et al. (1994), the rights of which are owned by NantkWest, that have been modified to express CD16 and a chimeric antigen receptor on the cell surface (hereinafter "CAR-modified CD16+ NK-92™ cells" or "t-haNK™ cells"). In some embodiments, t-haNK™ cells express the high-affinity CD16 receptor on the cell surface.

[0156] By "modified NK-92 cells" is meant NK-92 cells that express an exogenous gene or protein, e.g., an Fc receptor, a CAR, a cytokine (e.g., IL-2 or IL-15), and / or a suicide gene. In some embodiments, the modified NK-92 cells comprise a vector encoding a transgene, such as an Fc receptor, a CAR, a cytokine (e.g., IL-2 or IL-15), and / or a suicide gene. In one embodiment, the modified NK-92 cells express at least one transgenic protein.

[0157] As used herein, "non-irradiated NK-92 cells" refer to NK-92 cells that have not been irradiated. Irradiation renders the cells incapable of viability and proliferation. Because the time between irradiation and infusion should be four hours or less to maintain optimal activity, it is contemplated that the NK-92 cells will be irradiated at the treatment facility or elsewhere prior to patient treatment. Alternatively, NK-92 cells may be prevented from proliferating by other mechanisms.

[0158] As used herein, "inactivation" of NK-92 cells refers to rendering them unable to grow. Inactivation can also refer to the death of NK-92 cells. It is contemplated that NK-92 cells can be inactivated after effectively purging ex vivo samples of cells relevant to pathological diagnosis in therapeutic applications, or after they have been present in a mammalian body for a time sufficient to effectively kill many or all target cells present in the body. Inactivation can be induced by, but is not limited to, administering an inactivating agent to which NK-92 cells are sensitive.

[0159] As used herein, the terms "cytotoxic" and "cytolytic" are intended to be synonymous when used to describe the activity of effector cells, such as NK-92 cells. Generally, cytotoxic activity refers to the killing of target cells by any of a variety of biological, biochemical, or biophysical mechanisms. Cytolysis more specifically refers to the activity of an effector cell to lyse the plasma membrane of a target cell, disrupting its physical integrity, resulting in the death of the target cell. Without wishing to be bound by theory, it is believed that the cytotoxic effect of NK-92 cells is due to cytolysis.

[0160] The term "killing" a cell / cell population is meant to include any type of manipulation that can result in the death of that cell / cell population.

[0161] The term "Fc receptor" refers to a protein found on the surface of certain cells (e.g., natural killer cells) that contributes to the protective function of immune cells by binding to a portion of an antibody known as the Fc region. Binding of the Fc region of an antibody to a cell's Fc receptor (FcR) stimulates the cell's phagocytic or cytotoxic activity through antibody-mediated phagocytosis or antibody-dependent cell-mediated cytotoxicity (ADCC). FcRs are classified based on the type of antibody they recognize. For example, Fcγ receptors (FCγR) bind to the IgG class of antibodies. FcγRIII-A (also known as CD16, SEQ ID NO: 34) 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 epsilon receptors (FcεR) bind to the Fc region of IgE antibodies.

[0162] As used herein, the term "chimeric antigen receptor" (CAR) refers to an extracellular antigen-binding domain fused to an intracellular signaling domain. CARs can be expressed in T cells or NK cells to increase cytotoxicity. Typically, the extracellular antigen-binding domain is an scFv specific for an antigen found on the target cell. CAR-expressing NK-92 cells target cells expressing a particular antigen on their cell surface based on the specificity of the scFv domain. The scFv domain can be engineered to recognize any antigen, including tumor-specific and virus-specific antigens. For example, CD19CAR recognizes the cell surface marker CD19, which is expressed by some cancers.

[0163] As used herein, the term "tumor-specific antigen" refers to an antigen that is present in cancer or neoplastic cells but is undetectable in normal cells derived from the same tissue or lineage as the cancer cells. As used herein, tumor-specific antigen also refers to a tumor-associated antigen, i.e., an antigen that is expressed at a higher level in cancer cells compared to normal cells derived from the same tissue or lineage as the cancer cells.

[0164] As used herein, the term "virus-specific antigen" refers to an antigen that is present in virus-infected cells but is undetectable in 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.

[0165] The terms "polynucleotide," "nucleic acid," and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure and may perform any known or unknown function. Examples of polynucleotides include, but are not limited to, genes or gene fragments (e.g., probes, primers, ESTs, or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may contain modified nucleotides, such as methylated nucleotides or nucleotide analogs. If present, modifications to nucleotide structure can be imparted before or after assembly of the polynucleotide. The nucleotide sequence can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation of a labeling component. The term also refers to both double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention that is a polynucleotide encompasses both the double-stranded form and each of the single-stranded forms of the two complementary portions that are known or predicted to make up the double-stranded form.

[0166] A polynucleotide is composed of a specific sequence of the four nucleotide bases: adenine (A), cytosine (C), guanine (G), and thymine (T), with uracil (U) substituted for thymine when the polynucleotide is RNA. Thus, the term "polynucleotide sequence" is the alphabetical representation of a polynucleotide molecule.

[0167] "Homology" or "identity" or "similarity" refers to sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing a position in each sequence that can be aligned for purposes of comparison. When a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences.

[0168] As used herein, "percent identity" refers to the sequence identity between two peptides or two nucleic acid molecules. Percent identity can be determined by comparing a position in each sequence that can be aligned for purposes of comparison. When a position in the compared sequences is occupied by the same base or amino acid, the molecules are identical at that position. Homologous nucleotide sequences include sequences that encode naturally occurring allelic variants and mutations of the nucleotide sequences set forth herein. Homologous nucleotide sequences include nucleotide sequences that encode proteins from mammalian species other than humans. Homologous amino acid sequences include amino acid sequences that contain conservative amino acid substitutions and where the polypeptide has the same binding and / or activity. In some embodiments, homologous amino acid sequences have no more than 15, no more than 10, no more than 5, or no more than 3 conservative amino acid substitutions. In some embodiments, a nucleotide sequence or amino acid sequence has at least 60%, at least 65%, at least 70%, at least 80%, or at least 85% or more percent identity to a sequence set forth herein. In some embodiments, the nucleotide or amino acid sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence described herein. Percent identity can be determined, for example, using the default settings of the Gap program (Wisconsin Sequence Analysis Package, Version 8 for UNIX, Genetics Computer Group, University Research Park, Madison, Wis.) using the Smith-Waterman algorithm (Adv. Appl. Math., 1981, 2, 482-489). Suitable algorithms for determining percent sequence identity include the BLAST and BLAST 2.0 algorithms, described by Altschul et al. (Nuc. Acids Res. 25:3389-402, 1977) and Altschul et al. (J. Mol. Biol. 215:403-10, 1990), respectively.Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (see the internet at ncbi.nlm.nih.gov). The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length of 3 and an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989). Alignment (B) of 50, an expectation (E) of 10, M=5, N=-4.

[0169] In some embodiments, nucleic acid sequences are codon-optimized for expression in a particular species, e.g., a mouse sequence can be codon-optimized for expression in a human (expression of the protein encoded by the codon-optimized nucleic acid sequence). Thus, in some embodiments, the codon-optimized nucleic acid sequence has at least 60%, at least 65%, at least 70%, at least 80%, or at least 85% or more percent identity to a nucleic acid sequence described herein. In some embodiments, the codon-optimized nucleic acid sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a sequence described herein.

[0170] The term "express" refers to the production of a gene product (e.g., a protein). The term "transient" when referring to expression means that the polynucleotide is not introduced into the genome of the cell. The term "stable" when referring to expression means that the polynucleotide is introduced into the genome of the cell or a positive selection marker is utilized to maintain expression of the transgene (i.e., an exogenous gene expressed by a beneficial cell under certain growth conditions).

[0171] The terms "cytokine" or "cytokines" refer 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.

[0172] As used herein, the term "vector" refers to a non-chromosomal nucleic acid that contains an intact replicon such that the vector can replicate when placed into a permissive cell, for example, by the process of transformation. Vectors can replicate in some cell types, such as bacteria, but have limited or no replication capacity in other cells, such as mammalian cells. Vectors can be viral or non-viral. Exemplary non-viral vectors for delivering nucleic acids include the use of naked DNA, DNA complexed with cationic lipids, alone or in combination with cationic polymers, anionic and cationic liposomes, DNA-protein complexes and particles (in some cases contained in liposomes) containing DNA condensed with cationic polymers such as heterogeneous polylysine, defined-length oligopeptides, and polyethyleneimine, and ternary complexes containing a virus and polylysine-DNA. In one embodiment, the vector is a viral vector, e.g., an adenovirus. Viral vectors are well known in the art.

[0173] As used herein, the term "targeting" when referring to protein expression is intended to include, but is not limited to, directing a protein or polypeptide to an appropriate destination within or outside a cell. Targeting is typically achieved via a signal peptide or targeting peptide, which is a stretch of amino acid residues in the polypeptide chain. These signal peptides can be located anywhere within the polypeptide sequence but are often located at the N-terminus. Polypeptides can also be engineered to have a signal peptide at their C-terminus. Signal peptides can direct polypeptides to extracellular locations such as the plasma membrane, Golgi, endosomes, endoplasmic reticulum, and other cellular compartments. For example, polypeptides with a specific amino acid sequence at their C-terminus (e.g., KDEL) are retained in the ER lumen or transported back to the ER lumen.

[0174] As used herein, the term "targeting" when referring to tumor targeting refers to the ability of NK-92 cells to recognize and kill tumor cells (i.e., target cells). In this context, the term "targeted" refers, for example, to the ability of a CAR expressed by an NK-92 cell to recognize and bind to a cell surface antigen expressed by the tumor.

[0175] As used herein, the term "transfect" refers to the insertion of a nucleic acid into a cell. Transfection may be performed using any means that allows entry of a nucleic acid into a cell. DNA and / or mRNA may be transfected into a cell. Preferably, the transfected cell expresses the gene product (i.e., protein) encoded by the nucleic acid.

[0176] The term "suicide gene" refers to a transgene that allows for negative selection of cells expressing the transgene. Suicide genes are used as safety systems, allowing the death of cells expressing the gene by the introduction of a selection agent. Several suicide gene systems have been identified, including the herpes simplex virus thymidine kinase (TK) gene, cytosine deaminase gene, varicella-zoster virus thymidine kinase gene, nitroreductase gene, Escherichia coli gpt gene, and E. coli Deo gene (see, e.g., Yazawa K, Fisher WE, Brunicardi FC: Current progress in suicide gene therapy for cancer. World J. Surg. 2002 July;26(7):783-9). In one embodiment, the suicide gene is a thymidine kinase (TK) gene. The TK gene 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 recombinantly expressed cytokine; recombinantly expressed CD16; a membrane-bound recombinantly expressed chimeric antigen receptor (CAR) comprising an extracellular binding domain, a hinge domain, a transmembrane domain, and an FcεRIγ signaling domain in this specific order in a single polypeptide chain; A genetically modified NK cell comprising: the NK cells are NK-92 cells; the recombinantly expressed cytokine is IL-2; The FcεRIγ signaling domain has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:

1. Genetically modified NK cells.

2. 2. The genetically modified NK cell of claim 1, wherein the recombinantly expressed cytokine comprises an endogenous retention sequence.

3. The genetically modified NK cell of claim 1 or 2, wherein the recombinantly expressed CD16 is a high-affinity CD16 mutant having a 158V mutation.

4. The genetically modified NK cell of any one of claims 1 to 3, wherein the extracellular binding domain comprises an scFv.

5. The genetically modified NK cell of any one of claims 1 to 4, wherein the extracellular binding domain specifically binds to a tumor-specific antigen, a tumor-associated antigen, or a patient- and tumor-specific antigen.

6. 6. The genetically modified NK cell of claim 5, wherein the tumor-specific antigen is CD19, CD20, NKG2D ligand, CS1, GD2, CD138, EpCAM, HER-2, EBNA3C, GPA7, CD244, CA-125, MUC-1, B7-H4, ETA, MAGE, CEA, CD52, CD30, MUC5AC, c-Met, EGFR, FAP, WT-1, PSMA, NY-ESO1, CSPG-4, IGF1-R, Flt-3, CD276, CD123, PD-L1, BCMA, or CD33.

7. The genetically modified NK cell according to any one of claims 1 to 4, wherein the extracellular binding domain specifically binds to a virus-specific antigen.

8. The genetically modified NK cell of claim 7 , 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 any one of claims 1 to 8, wherein the FcεRIγ signaling domain has the amino acid sequence of SEQ ID NO:

1.

10. The genetically modified NK cell of any one of claims 1 to 9, wherein the recombinantly expressed cytokine, the recombinantly expressed CD16, and the recombinantly expressed CAR are expressed from a tricistronic recombinant nucleic acid.

11. The genetically modified NK cell of any one of claims 1 to 10, wherein the recombinantly expressed cytokine and / or the recombinantly expressed CD16 is expressed from a recombinant nucleic acid integrated into the genome of the NK cell.

12. a first sequence portion encoding a cytokine; a second sequence portion encoding CD16; and a third sequence portion encoding a chimeric antigen receptor (CAR) comprising an extracellular binding domain, a hinge domain, a transmembrane domain, and an FcεRIγ signaling domain in a single polypeptide chain; and Including, A recombinant nucleic acid for producing a genetically modified NK cell, wherein the first, second, and third sequence portions are on the same nucleic acid, the NK cells are NK-92 cells; the cytokine is IL-2; The FcεRIγ signaling domain has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:

1. Recombinant nucleic acid.

13. The recombinant nucleic acid of claim 12, wherein the nucleic acid is a tricistronic RNA.

14. The recombinant nucleic acid of claim 12, wherein the nucleic acid is a tricistronic DNA.

15. The recombinant nucleic acid of any one of claims 12 to 14, wherein the cytokine comprises an endogenous retention sequence.

16. The recombinant nucleic acid according to any one of claims 12 to 15, wherein the CD16 is a high-affinity CD16 mutant having a 158V mutation.

17. The recombinant nucleic acid of any one of claims 12 to 16, wherein the extracellular binding domain comprises an scFv.

18. 18. The recombinant nucleic acid of claim 17, wherein the extracellular binding domain specifically binds to a tumor-specific antigen, a tumor-associated antigen, or a patient- and tumor-specific antigen.

19. 18. The recombinant nucleic acid of claim 17, wherein the extracellular binding domain specifically binds to a virus-specific antigen.

20. 20. The recombinant nucleic acid of any one of claims 12 to 19, wherein the hinge domain and / or the transmembrane domain comprises a CD8 hinge domain and / or a CD28 transmembrane domain.

21. A recombinant cell comprising the recombinant nucleic acid of any one of claims 12 to 20, wherein the cell is an NK-92 cell.

22. The genetically modified NK cell of any one of claims 1 to 11 for treating cancer in a patient in need thereof.

23. 23. The genetically modified NK cells of claim 22 in combination with at least one additional therapeutic entity selected from the group consisting of a viral cancer vaccine, a bacterial cancer vaccine, a yeast cancer vaccine, N-803, an antibody, a stem cell transplant, and a tumor-targeted cytokine.

24. 24. The genetically modified NK cell of claim 22 or 23, wherein the cancer is selected from leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, chronic leukemia, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's disease, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors.

25. 24. The genetically modified NK cell of claim 22 or 23, wherein the cancer is selected from sarcoma and carcinoma.

26. The cancer may be fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, 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, or the like.

24. The genetically modified NK cell of claim 22 or 23, selected from carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung carcinoma, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

27. The genetically modified NK cell of any one of claims 1 to 4 and 7 to 10 for treating a viral infection in a patient in need thereof.

28. 28. The genetically modified NK cell of claim 27 in combination with an antiviral agent.

29. A pharmaceutical composition comprising the genetically modified NK cells of any one of claims 1 to 11 for treating cancer or viral infections.

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