FC-epsilon CAR

Abstract

A recombinant NK cell, in particular a recombinant NK-92 cell, expresses a chimeric antigen receptor (CAR) having an intracellular domain of Fc [epsilon] RI [gamma]. Notably, the CAR constructs having the intracellular domain of Fc [epsilon] RI [gamma] have a significantly prolonged expression duration and a significantly prolonged cytotoxicity over time. The CARs can be expressed from RNA and DNA, preferably as tricistronic constructs that further encode CD16 and cytokines to confer autocrine growth support. Advantageously, such constructs also enable high levels of transfection and expression of recombinant proteins and provide convenient selection markers to promote rapid production of recombinant NK / NK-92 cells.

Description

[0001] This application is a divisional application of the invention patent application with application date of May 21, 2019, application number 201980034416.6, and invention name “FC-εCAR”. CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. patent application serial number 62 / 674,936 filed on May 22, 2018. Technical Field

[0003] The field of the invention is recombinant nucleic acids and cells comprising the same to generate genetically modified cells expressing a chimeric antigen receptor (CAR), particularly modified NK and NK-92 cells expressing a CAR having an Fcε receptor γ (FcεRIγ) signaling domain. Background Art

[0004] Natural killer (NK) cells are cytotoxic lymphocytes that constitute an important component of the innate immune system. In most cases, NK cells comprise approximately 10%-15% of circulating lymphocytes and bind to and kill target cells, which include virally infected cells and many malignant cells. NK cell killing is not specific for particular antigens and can occur in the absence of prior immune sensitization. Killing of targeted cells is often mediated by cytolytic proteins, including perforins, granzymes, and granulysins.

[0005] Autologous NK cells have been used as therapeutic entities. To this end, NK cells are isolated from the peripheral blood lymphocyte fraction of whole blood, expanded in cell culture to obtain a sufficient number of cells, and then re-infused into the subject. Autologous NK cells have shown moderate effectiveness in ex vivo and in vivo treatments at least in some cases. However, the separation and growth of autologous NK cells requires a lot of time and cost. In addition, not all NK cells have cytolytic effects, which further limits autologous NK cell therapy.

[0006] At least some of these difficulties can be overcome by using NK-92 cells, which are a cytolytic cancer cell line that is found in the blood of subjects with non-Hodgkin's lymphoma and then propagated indefinitely in vitro (Gong et al., Leukemia 8: 652-658 (1994)). Although NK-92 cells are derivatives of NK cells, NK-92 cells lack the major inhibitory receptors possessed by normal NK cells, while retaining most of the activating receptors. However, NK-92 cells do not attack normal cells, nor do they induce immune rejection reactions that are unacceptable to the human body. Due to these expected properties, NK-92 cells have been characterized in detail and explored as therapeutic agents for treating certain cancers, for example, as described in WO 1998 / 049268 or US 2002 / 068044.

[0007] Phenotypic changes that distinguish tumor cells from normal cells derived from the same tissue are usually associated with one or more changes in the expression of specific gene products, including the loss of normal cell surface components or the gain of other cell surface components (antigens that are not detected in the corresponding normal non-cancerous tissue). Antigens expressed in neoplastic or tumor cells but not in normal cells or antigens expressed at levels in neoplastic cells that are much higher than those found in normal cells are called "tumor-specific antigens" or "tumor-associated antigens". Such tumor-specific antigens can be used as markers of 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, gpl00, TRP-1, and TRP-2), mutated or abnormally expressed antigens (e.g., MUM-1, CDK4, β-catenin, gp100-in4, p15, and N-acetylglucosaminyltransferase V), and antigens expressed at higher levels in tumors (e.g., CD19 and CD20).

[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, such as cancer cells, and triggers effector cell activation. The signaling domain contains an immunoreceptor tyrosine-based activation domain (ITAM), which is important for intracellular signaling through the receptor.

[0009] The first generation of CARs for T cells contained a cytoplasmic signaling domain. For example, one version of the first generation CAR in T cells included a signaling domain from Fcε receptor γ (FcεRIγ), which contained one ITAM, while another version contained a signaling domain from CD3ζ, which contained three ITAMs. In vivo and in vitro studies have shown that CD3ζCAR T cells are more effective than FcεRIγ CAR T cells in eradicating tumors (e.g., Haynes et al., 2001, J. Immunology 166: 182-187; Cartellieri et al., 2010, J. Biomed and Biotech, Vol. 2010, Article ID 956304). Further studies then showed that full activation and proliferation of such recombinant T cells required certain co-stimulatory signals, and second and third generation CARs combined multiple signaling domains into a single CAR to enhance the efficacy of recombinant CAR T cells. Because of their less than ideal effects reported in the literature on the T cells tested, first-generation CARs and the FcεRIγ signaling domain were largely abandoned in favor of using CD3ζ in combination with one or more additional signaling domains (e.g., Hermanson and Kaufman 2015, Frontiers in Immunol., vol. 6, article 195).

[0010] Recently, selected CARs have also been expressed in NK cells. For example, CAR-modified NK-92 cells have used first-generation CARs with only the CD3ζ intracellular signaling domain. These first-generation CAR-NK cells have targeted a variety of antigens, including CD19 and CD20 for B-cell lymphomas, ErbB2 for breast cancer, ovarian cancer, and squamous cell carcinomas, GD2 for neuroblastoma, and CD138 for multiple myeloma. Second-generation CAR-NK cells from the NK-92 line have also been constructed against several antigens, including EpCAM for a variety of cancers, HLA-A2 EBNA3 complex for Epstein-Barr virus, CS1 for multiple myeloma, and ErbB2 for HER2-positive epithelial cancers. In the second-generation NK-92 CAR, the most common intracellular co-stimulatory domain used with CD3ζ is CD28. However, because NK cells do not naturally express CD28, the potential role of the CD28 domain is unclear. Additional second-generation CARs have combined the 4-1BB intracellular signaling domain with CD3ζ to improve NK cell persistence. Others have compared the functionality of different intracellular domains against breast cancer cells using ErbB2 scFv fused to CD3ζ alone, CD28 and CD3ζ, or 4-1BB and CD3ζ. They found that both second-generation constructs had better killing than the first-generation CARs, with 65% target lysis with CD28 and CD3ζ, 62% lysis with 4-1BB and CD3ζ, and 51% target killing with CD3ζ alone. In a recent study, the 4-1BB and CD28 intracellular domains were also compared for B-cell malignancies using an anti-CD19 CAR expressed on NK-92 cells. Still others have found that the CD3ζ / 4-1BB construct was less effective than CD3ζ / CD28 in terms of cell killing and cytokine production, highlighting the different roles of the CD28 and 4-1BB costimulatory domains.

[0011] The third-generation NK-92 CAR is constructed by anti-CD5 scFv with CD3ζ, CD28 and 4-1BB intracellular signaling domains, and has been shown to have specific and potent anti-tumor activity against a variety of T-cell leukemia and lymphoma cell lines and primary tumor cells. Such cells are also able to inhibit disease progression in xenograft mouse models of T-cell acute lymphoblastic leukemia (ALL) cell lines and primary tumor cells (Transl Res. [Translational Medicine Research] September 2017; 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 potentiator of cytotoxic potential. Unfortunately, systemic administration of IL-2 is often associated with significant undesirable adverse side effects and toxicity. To overcome these problems, cells can be cultured and expanded in vitro before administration to patients. Although IL-2 will 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 often costly. The need for exogenous IL-2 can be addressed by recombinant expression of IL-2 confined to the endoplasmic reticulum in a retroviral vector (see Exp Hematol [Experimental Hematology]. 2005 Feb;33(2):159-64). Such approaches eliminate the need for exogenous IL-2. However, retroviral transfection efficiencies are often suboptimal, and are even less efficient when multiple recombinant genes are to be expressed.

[0013] In addition, as demonstrated by the repeated failures of engineering NK-92 cells to express Fc receptors, NK cells, especially NK-92 cells, are generally difficult to genetically modify. These difficulties are further exacerbated when NK-92 cells are transfected with multiple recombinant genes or relatively large recombinant nucleic acid payloads for heterologous expression. In addition, NK-92 cells also show a clear lack of predictability in the recombinant expression of exogenous proteins (e.g., CD16). At the functional level, most (if not all) CARNK-92 cells require relatively efficient target ratios, which may be due to the relatively low expression of CAR constructs. Moreover, the cytotoxicity of such CARNK-92 cells will also decline rapidly over time, making such cells less attractive clinically.

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

[0015] The inventors found that NK-92 cells can be effectively transfected with recombinant nucleic acids to express CARs containing FcεRIγ. Unexpectedly, CARs with FcεRIγ signaling domains significantly increase the expression level of CARs, and further transmit cytotoxicity over time. The envisioned recombinant nucleic acid encoding CAR is preferably a tricistronic arrangement, which also includes a sequence portion encoding CD16 or CD16 variants, and / or IL-2 or IL-2 variants. Advantageously, such recombinant nucleic acids not only provide an effective way to select transfected cells (because IL-2 not only provides autocrine growth stimulation, but also acts as a selection marker for co-expressed proteins), but also produce CAR NK cells with excellent cytolytic activity (e.g., a relatively low effect-target ratio compared to other constructs), and CD16 and FcεRIγ-containing CARs have high expression levels.

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

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

[0018] Preferably, but not necessarily, the extracellular binding domain will comprise a scFv that can specifically bind 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 can bind to a virus-specific antigen (e.g., an antigen of HIV virus, HPV virus, RSV virus, influenza virus, Ebola virus, or HCV virus).

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

[0020] Therefore, the 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), wherein the CAR comprises 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 located on the same nucleic acid.

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

[0022] Moreover, it is generally preferred that the cytokine is IL-2 or IL15 (which may or may not include an endosomal retention sequence), CD16 is a high affinity CD16 variant having a 158V mutation, and / or the extracellular binding domain comprises a scFv. As previously described, 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 aspects, the hinge domain and / or the transmembrane domain comprises a CD8 hinge domain and / or a CD28 transmembrane domain, and the FcεRIγ signaling domain may have the nucleic acid sequence shown in SEQ ID NO:2.

[0024] In other aspects of the subject matter of the present invention, the inventors also envision a recombinant cell comprising a recombinant nucleic acid as described above and herein. In the case of preparing and / or amplifying nucleic acids, the recombinant cell can be a bacterial cell. On the other hand, in the case of recombinant nucleic acids to be expressed, the cell is typically an autologous NK cell or NK cell (which can also be an optionally genetically modified NK-92 cell).

[0025] Therefore, the inventors also envision a method for treating cancer in patients in need. In such methods, a therapeutically effective amount of any genetically modified NK cell is administered to the patient to treat cancer. In addition, and in the case of expectation, the envisioned method may include a further step of administering at least one additional therapeutic entity, the additional therapeutic entity being selected from the group consisting of: viral cancer vaccines, bacterial cancer vaccines, yeast cancer vaccines, N-803, antibodies, stem cell transplants, and tumor-targeted cytokines.

[0026] Among other cancers, contemplated cancers include leukemias, acute lymphocytic leukemias, acute myeloid leukemias, chronic leukemias, chronic myeloid (granulocytic) leukemias, chronic lymphocytic leukemias, polycythemia vera, lymphomas, Hodgkin's disease, non-Hodgkin's disease, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, solid tumors including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, Myoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma and retinoblastoma.

[0027] Likewise, the inventors have also contemplated a method for treating a viral infection in a patient in need thereof. In such methods, a therapeutically effective amount of any genetically modified NK cell is administered to the patient to treat the viral infection. Antiviral drugs may also be administered where desired or necessary.

[0028] Regardless of the type of treatment, it is generally envisaged that approximately 1 x 10 8 About 1 x 10 11 From a different perspective, the use of genetically modified NK cells as described herein in the treatment of cancer or viral infection is envisioned.

[0029] Various objects, features, aspects and advantages of the present subject matter will become more apparent from the following detailed description of preferred embodiments and the accompanying drawings, in which like reference numerals represent like components. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram of an exemplary CD19-CAR assay. All CD19-CAR variants comprise an extracellular domain comprising an anti-CD19 scFv region (αCD19-scFv), a hinge region from CD8 (CD8 hinge), and a transmembrane domain from CD28 (CD28 TM). The intracellular domain of CD19CAR varies as shown.

[0031] Figure 2AThe expression of CD19-CAR mRNA was confirmed by flow cytometry using anti-scFv antibody labeled with eF660. Figure 1 Exemplary percentage results of CD19-CAR NK-92 cells.

[0032] Figure 2B Figure 2 is an exemplary result of background-subtracted median fluorescence intensity (MFI) of NK-92 cells expressing CD19-CAR labeled with anti-scFv antibody labeled with eF660.

[0033] Figure 3A Exemplary results are shown for the percentage of NK-92 cell-sensitive target cancer cells (K562) killed by NK-92 cells expressing CD19CAR (effector) at effector-to-target ratios of 5:1 to 0.3:1.

[0034] Figure 3B Exemplary results are shown for the percentage of CD19-positive target cancer cells (SUP-B15) tolerated by NK-92 cells (effectors) expressing CD19CAR at effector-target ratios of 5:1 to 0.3:1.

[0035] Figure 4 Shown are exemplary results of the MFI of NK-92 expressing CD19-CAR (effector) labeled with anti-CD107a antibody in a degranulation assay using SUP-B15 target cells at effector:target ratios ranging from 2:1 to 0.25:1.

[0036] Figure 5 Exemplary results of CD19 CAR surface expression on haNK cells transfected with CD19 CAR mRNA constructs at different time points are shown. Under the conditions used for the CD19 / CD28-Fc-ε-CAR with the longest expression duration, all tested CAR constructs showed detectable expression for up to 72 h.

[0037] Figure 6 Exemplary results of the cytotoxicity of CD19.taNK against SUPB15 CD19+ cells (aNK-resistant cell line) are shown. All CAR constructs tested showed comparable (maximum) cytotoxicity at 24 h. However, at 48 h, CD19 / CD3-ζ showed a significant decrease in cytotoxicity, while Fc-ε-based CARs showed only a minimal decrease 48 hours after electroporation.

[0038] Figure 7 is an exemplary schematic diagram of a recombinant tricistronic DNA construct and the corresponding protein product.

[0039] Figure 8Shows Figure 8 Exemplary linearized forms of the indicated plasmids.

[0040] Fig. 9A Exemplary results of in vitro data are shown, showing that CD33-positive (CD33+) THP-1 cells are relatively resistant to cytotoxicity (specific lysis) by control NK-92 (aNK) cells, while there is a high percentage of specific lysis when THP-1 cells are cultured with NK-92 cells expressing a CAR that specifically binds CD33 (CD33-CAR / NK-92 cells).

[0041] Fig. 9B Exemplary results of in vitro data are shown, showing that both control aNK cells and CD33-CAR / NK-92 cells can kill K562 cells.

[0042] Fig.10 Exemplary results of cytotoxicity of HER2.CAR-t-haNK cells against BT-474 cells are shown.

[0043] Fig.11 Exemplary results of the cytotoxicity of CD33.CAR-t-haNK cells against THP-1 cells are shown.

[0044] Fig.12 The expression of PD-L1.CAR-t-haNK cells to SUP-B15.PD-L1 is shown. + Exemplary results of cellular cytotoxicity.

[0045] Fig.13 Exemplary results of cytotoxicity of PD-L1.CAR-t-haNK cells against U251 cells are shown.

[0046] Fig.14 Exemplary results of cytotoxicity of EGFR.CAR-t-haNK cells against A-549 cells are shown.

[0047] Fig.15 Exemplary results of the cytotoxicity of CD19.CAR-t-haNK cells against K562 cells are shown.

[0048] Fig.16 Exemplary results of the cytotoxicity of CD19.CAR-t-haNK cells against SUP-B15 cells are shown.

[0049] Fig.17 Exemplary results of ADCC of CD19.CAR-t-haNK cells against SKBr3 cells are shown.

[0050] Fig.18Exemplary results of the cytotoxicity of IGF1R.CAR-t-haNK cells against MDA-MB-231 cells are shown.

[0051] Fig.19 Exemplary results of the cytotoxicity of PD-L1.CAR-t-haNK cells against various cancer cells are shown.

[0052] Fig. 20 Exemplary comparative results of the cytotoxicity of PD-L1.CAR-t-haNK cells against MDA-MB-231 cells are shown.

[0053] Fig.21 Exemplary results of CD16 and CD19.CAR expression are shown.

[0054] Fig. 22 Exemplary results of natural cytotoxicity of CD19.CAR-t-haNK cells against K562 cells are shown.

[0055] Fig.23 Exemplary results of CAR-mediated cytotoxicity of CD19.CAR-t-haNK cells against SUP-B15 cells are shown.

[0056] Fig.24 Exemplary results of ADCC of CD19.CAR-t-haNK cells are shown.

[0057] Fig.25 Exemplary comparison results of CD16 and CD20.CAR expression are shown.

[0058] Fig.26 Exemplary results of natural cytotoxicity of CD20.CAR-t-haNK cells are shown.

[0059] Fig. 27 Exemplary results of CD16 and CD33.CAR expression are shown.

[0060] Fig.28 Exemplary results of natural cytotoxicity of CD33.CAR-t-haNK cells against K562 cells are shown.

[0061] Fig.29 Exemplary results of CAR-mediated cytotoxicity of CD33.CAR-t-haNK cells against THP-1 cells are shown.

[0062] Fig.30 Exemplary results of ADCC of CD33.CAR-t-haNK cells are shown.

[0063] Fig.31Exemplary results of CD16 and EGFR.CAR expression are shown.

[0064] Fig.32 Exemplary results of natural cytotoxicity of EGFR.CAR-t-haNK cells against K562 cells are shown.

[0065] Fig.33 Exemplary results of CAR-mediated cytotoxicity of EGFR.CAR-t-haNK cells against A549 cells are shown.

[0066] Fig.34 Exemplary results of CAR-mediated cytotoxicity of EGFR.CAR-t-haNK cells against HCT116 cells are shown.

[0067] Fig.35 Exemplary results of ADCC of EGFR.CAR-t-haNK cells are shown.

[0068] Fig.36 Exemplary results of CD16 and HER2.CAR expression are shown.

[0069] Fig.37 Exemplary results of natural cytotoxicity of HER2.CAR-t-haNK cells against K562 cells are shown.

[0070] Fig.38 Exemplary results of CAR-mediated cytotoxicity of HER2.CAR-t-haNK cells against SKBR-3 cells are shown.

[0071] Fig.39 Exemplary results of ADCC of HER2.CAR-t-haNK cells are shown.

[0072] Fig.40 Exemplary results of CD16 and PD-L1.CAR expression are shown.

[0073] Fig.41 Exemplary results of natural cytotoxicity of PD-L1.CAR-t-haNK cells against K562 cells are shown.

[0074] Fig.42 Exemplary results of CAR-mediated cytotoxicity of PD-L1.CAR-t-haNK cells are shown.

[0075] Fig.43 Exemplary results of ADCC of PD-L1.CAR-t-haNK cells are shown.

[0076] Fig.44Exemplary results of CAR-mediated cytotoxicity of CD123.CAR-t-haNK cells are shown.

[0077] Fig.45 Exemplary results of ADCC of CD123.CAR-t-haNK cells are shown.

[0078] Fig.46 Exemplary results of CD16 and CD30.CAR expression are shown.

[0079] Fig.47 Exemplary results of natural cytotoxicity of CD30.CAR-t-haNK cells against K562 cells are shown.

[0080] Fig.48 Exemplary results of CAR-mediated cytotoxicity of CD30.CAR-t-haNK cells against THP-1 cells are shown.

[0081] Fig.49 Exemplary results of ADCC of CD30.CAR-t-haNK cells are shown.

[0082] Fig.50 Exemplary results of CD16 and BCMA.CAR expression are shown.

[0083] Fig.51 Exemplary results of CAR-mediated cytotoxicity of BCMA.CAR-t-haNK cells are shown.

[0084] Fig.52 Exemplary results of ADCC of BCMA.CAR-t-haNK cells are shown.

[0085] Fig.53 Exemplary results of CD16 and gp120.CAR expression are shown.

[0086] Fig.54 Exemplary results of GP120 binding of gp120.CAR-t-haNK cells are shown.

[0087] Fig.55 Exemplary results of natural cytotoxicity of gp120.CAR-t-haNK cells against K562 cells are shown.

[0088] Fig.56 Exemplary results of ADCC of gp120.CAR-t-haNK cells are shown.

[0089] Fig.57 Exemplary results of CD16 and FAP.CAR expression are shown.

[0090] Fig.58 Exemplary results of CAR-mediated cytotoxicity of FAP.CAR-t-haNK cells are shown.

[0091] Fig.59 Exemplary results of CSPG4 expression in CSPG4.CAR-t-haNK cells are shown.

[0092] Fig.60 Exemplary results of CAR-mediated cytotoxicity of CSPG4.CAR-t-haNK cells against SK-MEL-28 cells are shown.

[0093] Fig.61 Describes a gene encoding IGF1R-CAR, CD16, and IL-2 ER Exemplary tricistronic constructs of. DETAILED DESCRIPTION

[0094] The inventors unexpectedly discovered that, when the recombinant CAR includes an FcεRIγ signaling domain, CAR-mediated cytotoxicity and CAR expression in recombinant NK cells (e.g., NK-92 cells) are significantly increased, as described in more detail below. The discovery that CARs with FcεRIγ signaling domains have superior properties in NK cells is particularly unexpected, because CARs in such T cells perform relatively poorly compared to CARs with CD3ζ, 4-1BB or CD28 signaling domains and other signaling domains commonly found in second- and third-generation CARs.

[0095] 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 sequence, which also includes a sequence portion 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 will not only provide an efficient means of selecting transfected cells (since IL-2 not only provides autocrine growth stimulation), but also serve as a selection marker for co-expressed proteins.

[0096] Therefore, the subject matter of the present invention relates to genetically modified 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 from Fcε receptor γ (FcεRIγ). For example, the cytoplasmic domain of FcεRIγ may have an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 1, or may comprise, consist of or consist essentially of an amino acid sequence having a sequence as shown 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 with SEQ ID NO: 2. The envisioned recombinant cells may further express various other proteins, including one or more cytokines and CD16. As will be readily appreciated, CAR and / or other proteins may be transiently expressed or stably expressed by recombinant cells.

[0097] In some embodiments, the CAR comprises a hinge region of CD8 and / or in some embodiments, the CAR comprises a transmembrane domain of CD28 having an amino acid sequence as shown in SEQ ID NO:6 (encoded by a nucleic acid as shown in SEQ ID NO:7). The full-length amino acid sequence of CD28 is shown in SEQ ID NO:23. In a further embodiment, the recombinant cell is genetically modified with a nucleic acid having a sequence as set forth in SEQ ID NO:9, which encodes a hybrid protein having a sequence as set forth in SEQ ID NO:8, the hybrid protein comprising a CD8 hinge region coupled to a CD28 transmembrane domain coupled to an FcεRIγ signaling domain. It should be understood that adding a binding domain to the hinge region will form a functional CAR. For example, the binding domain targets or specifically can bind to a tumor-associated antigen, and suitable antigens 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.

[0098] In some embodiments, the nucleic acid construct further comprises an (inducible) promoter that promotes transcription of the nucleic acid sequence. Preferably, but not necessarily, the nucleic acid construct is a polycistronic vector or RNA comprising one or more internal ribosome entry sites (IRES) to achieve translation initiation from the 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 produce an equimolar level of polypeptide 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 a 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 a further embodiment, the construct comprises a nucleic acid encoding a cytokine such as IL-2 or IL-15 that can target the endoplasmic reticulum. In some embodiments, NK-92 cells or cell lines are also genetically modified to express CD16 on the cell surface. In one embodiment, NK-92 cells or cell lines are genetically modified to express high affinity CD16 (F158V) on the cell surface.

[0099] About suitable NK cells, it should be noted that all NK cells are considered suitable for the present invention, thus including primary NK cells (preserved, expanded and / or fresh cells), immortalized secondary NK cells, autologous or xenogeneic NK cells (stock, preserved, fresh, etc.) and modified NK cells, as described in more detail below. In some embodiments, preferably, the NK cells are NK-92 cells. The NK-92 cell line is a unique cell line that was found to proliferate in the presence of interleukin 2 (IL-2) (see, e.g., Gong et al., Leukemia 8: 652-658 (1994)). NK-92 cells are cancerous NK cells that have a wide range of anti-tumor cytotoxicity and predictable yields after amplification in a suitable culture medium. Advantageously, NK-92 cells have high cytolytic activity against a variety of cancers.

[0100] The original NK-92 cell line expresses CD56bright, CD2, CD7, CD11a, CD28, CD45, and CD54 surface markers, but does not display CD1, CD3, CD4, CD5, CD8, CD10, CD14, CD16, CD19, CD20, CD23, and CD34 markers. The growth of these NK-92 cells in culture is dependent on the presence of interleukin 2 (e.g., rIL-2), with doses as low as 1 IU / mL sufficient to sustain proliferation. IL-7 and IL-12 do not support long-term growth, and a variety of other cytokines have not been tested, including IL-1α, IL-6, tumor necrosis factor α, interferon α, and interferon γ. Compared to primary NK cells, NK-92 generally has higher cytotoxicity even at relatively low effector-target (E:T) ratios (e.g., 1:1). Representative NK-92 cells are deposited with the American Type Culture Collection (ATCC) and designated CRL-2407.

[0101] Therefore, suitable NK cells can have one or more modified KIRs that are mutated to reduce or eliminate interactions with MHC class I molecules. Of course, it should be noted that one or more KIRs can also be deleted or their expression can be inhibited (e.g., via miRNA, siRNA, etc.). Most typically, more than one KIR will be mutated, deleted or silenced, and KIRs particularly contemplated include those with two or three domains, with short or long cytoplasmic tails. From a different perspective, modified, silent or deleted KIRs will include KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DL2, KIR3DL3, and KIR3DS1. Such modified cells can be prepared using protocols well known in the art. Alternatively, such cells can also be purchased commercially as aNK cells ('activated natural killer cells) from NantKwest (see URL www.nantkwest.com). These cells can then be further genetically modified to become CARs, as described in more detail below.

[0102] In another aspect of the subject matter of the present invention, the genetically engineered NK cells can also be NK-92 derivatives modified to express high-affinity Fcγ receptors (CD16). The sequences of high-affinity variants of Fcγ receptors are well known in the art (see, e.g., Blood 2009 113: 3716-3725), and all methods of production and expression are considered to be applicable herein. The expression of such receptors is considered to achieve specific targeting of tumor cells using antibodies specific to the patient's tumor cells (e.g., new epitopes), specific tumor types (e.g., her2neu, PSA, PSMA, etc.), or antibodies associated with cancer (e.g., CEA-CAM). Advantageously, such antibodies are commercially available and can be used in conjunction with cells (e.g., in combination with Fcγ receptors). Alternatively, such cells can also be commercially available from Nantquist as haNK cells. These cells can then be modified to CARs through additional genes, as described in more detail below.

[0103] The genetic modification of NK cells envisioned herein can be carried out in a variety of ways, and all known methods are considered applicable here. Moreover, it should be recognized that NK cells can be transfected with DNA or RNA, and the specific selection of transfection will depend at least in part on the type and transfection efficiency of the expected recombinant cells. For example, in the case of expected stable transfection of NK cells, linearized DNA can be introduced into the cell to be integrated into the genome. On the other hand, in the case of expected transient transfection, circular DNA or linear RNA (e.g., mRNA with polyA+ tail) can be used.

[0104] For example, where the NK cells are autologous NK cells or NK-92 cells, it is contemplated that the recombinant nucleic acid will include a segment encoding a CAR that includes an FcεRIγ signaling domain and preferably also includes a segment encoding a cytokine to provide autocrine growth stimulation (e.g., IL-2, IL-2 modified with an ER retention sequence, IL-15, or IL-15 modified with an ER retention sequence) and / or a segment encoding CD16 or a high affinity CD16 158V As will be readily appreciated, the inclusion of cytokines that provide autocrine growth stimulation will render the modified recombinant independent of the addition of exogenous cytokines, which will make large-scale production of such cells economically feasible. Similarly, in the modified recombinant also expressing CD16 or high affinity CD16 158V In this case, such cells will have further enhanced ADCC properties and have further improved targeted cytotoxicity.

[0105] Of course, it will be appreciated that proteins encoding cytokines and / or CD16 or high affinity CD16 158VThe recombinant nucleic acid can be integrated into the genome of the NK cell, or can be an extrachromosomal unit (which can be a linear or circular DNA or linear RNA delivered by virus or chemically, mechanically or electrically transfected). For example, recombinant NK-92 cells expressing IL-2ER and CD16158V are called haNK cells (Oncotarget [Tumor Targeting] 2016 Dec 27; 7(52): 86359-86373) and can be transfected with a recombinant nucleic acid that includes a fragment encoding a CAR that includes an FcεRIγ signaling domain. Once again, such recombinant nucleic acids can further include fragments that can encode additional immunotherapeutic proteins, such as N-803, TxM-type compounds, IL-8 trap, TGF-β trap, etc. Similarly, NK-92 cells may have been transfected with a cDNA encoding IL-2 (e.g., NK-92MI, ATCC CRL-2408). Such cells can then be further transfected with a recombinant nucleic acid comprising a fragment encoding a CAR comprising an FcεRIγ signaling domain and a fragment encoding CD16 or a high affinity CD16 158V Fragment of .

[0106] In another aspect, NK cells or NK-92 cells (autologous, fresh, cultured or previously frozen) can also be transfected with a recombinant nucleic acid comprising a fragment encoding a CAR having an FcεRIγ signaling domain, a fragment encoding a cytokine to provide autocrine growth stimulation (e.g., IL-2, IL-2 modified with an ER retention sequence, IL-15, or IL-15 modified with an ER retention sequence), and a fragment encoding CD16 (SEQ ID NO: 34) or a high affinity CD16 158V (SEQ ID NO:35, encoded by SEQ ID NO:36). Most typically, such recombinant nucleic acids will be configured as tricistronic constructs. As previously described, such constructs can be extrachromosomal circular plasmids, linear DNA (which can be integrated into the genome of NK cells) or linear RNA. Such nucleic acids will typically be transfected into cells in a manner well known in the art (e.g., electroporation, lipofection, ballistic gene transfer, etc.). Similarly, nucleic acids can be delivered to cells by recombinant viruses. Therefore, NK cells suitable for use herein include NK-92 cells (which can be transfected with tricistronic constructs encoding CAR, CD16 or variants thereof, cytokines or variants thereof), genetically modified NK cells or NK-92 cells expressing CD16 or variants thereof or cytokines or variants thereof (which can be transfected with nucleic acids encoding CAR and CD16 or variants thereof or cytokines or variants thereof), and genetically modified NK cells or NK-92 cells expressing CD16 or variants thereof and cytokines or variants thereof (which can be transfected with nucleic acids encoding CAR).

[0107] Therefore, in preferred embodiments, it should be noted that genetically modified NK cells (particularly cells expressing CAR and CD16 or its variants) will exhibit three different modes of cell killing: general cytotoxicity mediated by activating receptors (e.g., NKG2D receptor), ADCC mediated by antibodies bound to target cells, and CAR-mediated cytotoxicity.

[0108] Therefore, it should be understood that the mode of transfection will depend at least in part on the type of nucleic acid used. Therefore, viral transfection, chemical transfection, and mechanical transfection methods are all considered suitable for use herein. For example, in one embodiment, the vectors described herein are transient expression vectors. The exogenous transgene introduced using such vectors is not integrated into the nuclear genome of the cell; therefore, in the absence of vector replication, the exogenous transgene will degrade or dilute over time.

[0109] In another embodiment, the vectors described herein allow stable transfection of cells. In one embodiment, the vector allows one or more transgenes to be incorporated into the genome of the cell. Preferably, such vectors have positive selection markers, and suitable positive selection markers include any gene that allows cells to grow under conditions that kill cells that do not express the gene. Non-limiting examples include antibiotic resistance, for example, geneticin (Neo gene from Tn5).

[0110] Alternatively, or additionally, the vector is a plasmid vector. In one embodiment, the vector is a viral vector. As will be appreciated by those skilled in the art, any suitable vector may be used, and suitable vectors are well known in the art.

[0111] In yet other embodiments, cells are transfected with mRNA encoding a protein of interest (e.g., CAR). Transfection of mRNA results in transient expression of protein. In one embodiment, mRNA is transfected into NK-92 cells immediately before the cells are administered. In one embodiment, "before" the cells are to be administered refers to between about 15 minutes and about 48 hours before administration. Preferably, mRNA transfection is performed about 5 hours to about 24 hours before administration. In at least some embodiments described in more detail below, NK cells transfected with mRNA result in unexpectedly consistent and strong expression of CAR on a high proportion of transfected cells. Moreover, such transfected cells also exhibit high specific cytotoxicity when the effect-target cell ratio is quite low.

[0112] With respect to the envisioned CAR, it is noted that the NK or NK-92 cells will be genetically modified to express the CAR as a membrane-bound protein, thereby exposing a portion of the CAR on the cell surface while maintaining the signaling domain in the intracellular space. Most typically, the CAR will include at least the following elements (in order): an extracellular binding domain, a hinge domain, a transmembrane domain, and an FcεRIγ signaling domain.

[0113] In a preferred embodiment, the cytoplasmic domain of the CAR comprises or consists of a signaling domain of FcεRIγ. It is noteworthy that, and as described in more detail below, the FcεRIγ signaling domain provides a significant increase in the expression level of the CAR and a significant extension of cytotoxicity over time. For example, the FcεRIγ signaling domain comprises or consists of or consists essentially of the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the FcεRIγ cytoplasmic domain is the only signaling domain. However, it should be understood that additional elements, such as other signaling domains (e.g., CD28 signaling domain, CD3ζ signaling domain, 4-1BB signaling domain, etc.) may also be included. These additional signaling domains may be located downstream of the FcεRIγ cytoplasmic domain and / or upstream of the FcεRIγ cytoplasmic domain.

[0114] In some embodiments, the FcεRIγ signaling domain comprises, consists of, or consists essentially of an amino acid sequence having at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology to the amino acid sequence set forth in SEQ ID NO:1.

[0115] In an optional embodiment, the cytoplasmic domain of the CAR may also include a signaling domain of CD3 zeta (CD3ζ). In one embodiment, the cytoplasmic domain of the CAR consists of a signaling domain of CD3ζ. In one embodiment, the CD3ζ signaling domain comprises, consists of, or consists essentially of an amino acid sequence as shown in SEQ ID NO: 15. In some embodiments, the CD3ζ signaling domain comprises, consists of, or consists essentially of an amino acid sequence having at least about 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology to the amino acid sequence as shown in SEQ ID NO: 15.

[0116] The CAR may comprise any suitable transmembrane domain. In one aspect, the CAR comprises a transmembrane domain of CD28. In one embodiment, the CD28 transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:6 (encoded by the nucleic acid shown in SEQ ID NO:7) or consists of or consists essentially of it. In some embodiments, the CD28 transmembrane domain comprises an amino acid sequence having at least about 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology to the amino acid sequence shown in SEQ ID NO:6 or consists of or consists essentially of it. In other embodiments, the transmembrane domain may also be a 4-1BB transmembrane domain.

[0117] The CAR may include any suitable hinge region. In one aspect, the CAR comprises a hinge region of CD8. In one embodiment, the CD8 hinge region comprises or consists of or consists essentially of an amino acid sequence as shown in SEQ ID NO:3 or SEQ ID NO:4. In one embodiment, the CD8 hinge region comprises or consists of or consists essentially of an amino acid sequence having at least about 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology to an amino acid sequence as shown in SEQ ID NO:3 or SEQ ID NO:4. Such regions may be encoded by a nucleic acid having a sequence as shown in SEQ ID NO:5.

[0118] Thus, the envisioned CAR will include the general structure of an expected antigen binding domain coupled to a hinge domain, which is coupled to a transmembrane domain, which is coupled to a signaling domain. From another perspective, the envisioned CAR may have an expected binding domain, which is then coupled to a hybrid protein, which comprises or consists of a hinge domain or consists essentially of a hinge domain, which is coupled to a transmembrane domain, which is coupled to a signaling domain. For example, such a hybrid protein may have an amino acid sequence having at least about 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology to the amino acid sequence shown in SEQ ID NO: 8 (encoded by the nucleic acid sequence shown in SEQ ID NO: 9).

[0119] Most typically, but not necessarily, the extracellular binding domain of CAR will be a scFv or other natural or synthetic binding portion that specifically binds to the target antigen. Particularly suitable binding portions include small antibody fragments with single, dual or multiple target specificities, beta barrel domain binders, phage display fusion proteins, etc. Among other suitable extracellular binding domains, preferred domains will specifically bind to tumor-specific antigens, tumor-associated antigens, or patient-specific antigens and tumor-specific antigens. Tumor-specific antigens include, but are not limited to, NKG2D ligands, CS1, GD2, CD138, EpCAM, EBNA3C, GPA7, CD244, CA-125, ETA, MAGE, CAGE, BAGE, HAGE, LAGE, PAGE, NY-SEO-1, GAGE, CEA, CD52, CD30, MUC5AC, c-Met, EGFR, FAP, WT-1, PSMA, NY-ESO1, AFP, CEA, CTAG1B and CD33. Other non-limiting tumor-associated antigens, and malignancies associated therewith, are listed in Table 1. As non-limiting examples, still further tumor-specific antigens are described in US2013 / 0189268; WO 1999024566 A1; US ​​7098008; and WO 2000020460, each of which is incorporated herein by reference in its entirety. Likewise, other preferred domains will specifically bind to (pathogenic) virus-specific antigens, such as antigens of HIV virus (e.g., gp120), HPV virus, RSV virus, influenza virus, Ebola virus, or HCV.

[0120]

[0121]

[0122]

[0123] .

[0124] For example, the CAR may comprise an anti-CD19 extracellular domain. In one embodiment, the anti-CD19 extracellular domain comprises, consists of, or is substantially composed of the amino acid sequence shown in SEQ ID NO: 11. In some embodiments, the anti-CD19 extracellular domain comprises, consists of, or is substantially composed of an amino acid sequence having at least about 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology to the amino acid sequence shown in SEQ ID NO: 11.

[0125] Thus, the envisioned CAR will target antigens associated with a particular cancer type. For example, targeted cancers include leukemias (including acute leukemias (e.g., acute lymphocytic leukemia, acute myeloid leukemia (including myeloid, promyelocytic, myelomonocytic, monocytic and erythroleukemia)) and chronic leukemias (e.g., chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, solid tumors, including but not limited to sarcomas and carcinomas, such as fibrosarcomas, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphovascular cavity Endotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumors, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0126] Thus, the envisioned CAR will generally have a structure of an extracellular binding domain (directly) coupled to a hinge domain, which is (directly) coupled to a transmembrane domain, which in turn is (directly) coupled to an FcεRIγ signaling domain. In yet further envisioned aspects, in addition to or in place of the FcεRIγ signaling domain, the envisioned CAR may also include one or more signaling domains, and in particular, the envisioned signaling domains include a CD3ζ signaling domain, a 4-1BB signaling domain, and a CD28 signaling domain. For example, a contemplated CAR can therefore include any binding domain (e.g., having SEQ ID NO: 11) coupled to a hinge domain (e.g., a CD8 hinge set forth in SEQ ID NO: 3 or SEQ ID NO: 4, which is encoded by SEQ ID NO: 5), which in turn is coupled to a transmembrane domain (e.g., a CD28 TM set forth in SEQ ID NO: 6, which is encoded by SEQ ID NO: 7), which is coupled to a signaling domain (e.g., an FcεRIγ signaling domain set forth in SEQ ID NO: 1, which is encoded by SEQ ID NO: 1, or a CD28 signaling domain set forth in SEQ ID NO: 13, or a 4-1BB signaling domain set forth in SEQ ID NO: 14, or a CD3ζ signaling domain set forth in SEQ ID NO: 15).

[0127] With regard to the construction of the contemplated CARs, it will be appreciated that the CARs may be engineered in a variety of ways as described, for example, in WO 2014 / 039523; US 2014 / 0242701; US ​​2014 / 0274909; US 2013 / 0280285 and WO 2014 / 099671, each of which is incorporated herein by reference in its entirety.

[0128] In further contemplated aspects, and as shown above, NK cells can be further genetically modified to express one or more cytokines, thereby providing a selection marker, wherein the cytokine and CAR are encoded with the same recombinant nucleic acid and / or the recombinant cells are independent of exogenous IL-2. Therefore, in some aspects of the subject matter of the present invention, NK-92 cells are modified to express at least one cytokine. In particular, at least one cytokine is IL-2, IL-12, IL-15, IL-18, IL-21, or a variant thereof. In a preferred embodiment, the cytokine is IL-2 or a variant thereof, and a particularly preferred variant includes an endoplasmic retention signal (e.g., human IL-2 shown in SEQ ID NO: 21, or having an ER retention signal shown in 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 expression of IL-2 sufficient for autocrine activation but without extracellular release of IL-2 (e.g., Exp Hematol. 2005 Feb;33(2):159-64). Alternatively, expression of cytokines (and particularly IL-15) can also be such that the cytokine is expressed in an amount sufficient to provide an autocrine growth signal to the recombinant cells, but also allows at least some of the expressed IL-15 to be released from the cells, thereby providing an immunostimulatory signal. For example, a human IL-15 sequence comprising both a signal peptide and an endoplasmic retention sequence can be used to achieve such expression. Exemplary DNA and protein sequences of endoplasmic retained IL-15 are shown in SEQ ID NO:49 and SEQ ID NO:50, respectively.

[0129] As expected, the envisioned cells can also express suicide genes. The term "suicide gene" refers to a transgene that allows negative selection of cells expressing suicide genes. Suicide genes are used as a safety system to allow cells expressing the gene to be killed by introducing a selection agent. If the recombinant gene causes a mutation that leads to uncontrolled cell growth, or the cell itself is capable of such growth, this is in line with expectations. Many suicide gene systems have been identified, including herpes simplex virus thymidine kinase (TK) gene, cytosine deaminase gene, varicella zoster virus thymidine kinase gene, nitroreductase gene, Escherichia coli gpt gene and Escherichia coli Deo gene. Typically, the protein encoded by the suicide gene has no adverse effects on the cell, but will kill the cell in the presence of a specific compound. Therefore, suicide genes are typically part of the system.

[0130] 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 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 that is toxic in the presence of ifosfamide or cyclophosphamide. See, e.g., Touati et al. "A suicide gene therapy combining the improvement of cyclophosphamide tumor cytotoxicity and the development of an anti-tumor immune response." Curr Gene Ther. 2014; 14(3): 236-46. In yet another embodiment, the suicide gene is iCasp9. Di Stasi, (2011) "Inducible apoptosis as a safety switch for adoptive cell therapy." 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 studies and has been used in the adoptive cell therapy setting.

[0131] Of course, it should be noted that all recombinant proteins can be expressed from a single recombinant sequence. However, it is generally preferred that in the case of expressing multiple recombinant sequences (e.g., CAR, CD16, cytokine), the coding regions can be arranged in a polycistronic unit with at least two or at least three coding regions encoding recombinant proteins. For example, a tricistronic DNA or RNA construct (e.g., with FcεRIγ signaling domain, CD16 158V and IL-2 ER or IL15 ER The transgenic protein (encoding CAR) is transfected into NK or NK-92 cells. Therefore, the transgenic engineering can be converted into an expression vector by any mechanism known to those skilled in the art. In the case where multiple transgenes are inserted into cells, the transgenic engineering can be transformed into the same expression vector or different expression vectors. In some embodiments, the cells are transfected with mRNA encoding the transgenic protein to be expressed. In some embodiments, the cells are transfected with DNA encoding the transgenic protein to be expressed. Transgenes, mRNA and DNA can be introduced into NK-92 cells using any transfection method known in the art, including but not limited to infection, viral vectors, electroporation, lipofection, nucleofection or "gene gun".

[0132] It is obvious that the envisioned genetically modified cells can be used to treat a variety of diseases, and are a variety of cancers and viral infections in which the diseased cells present disease-specific or disease-associated antigens. Therefore, the inventors envision a method of treating a patient using the modified NK or NK-92 cells described herein. In one embodiment, the patient has cancer (e.g., a tumor), and the modified NK-92 cells or cell lines express a CAR specific for an antigen expressed on the surface of a cell from the cancer or tumor. In one embodiment, the patient has a viral infection, and the modified NK-92 cells or cell lines express a CAR specific for an antigen expressed on the surface of a cell infected with the virus. In one embodiment, the patient has a bacterial infection, and the modified NK-92 cells or cell lines express a CAR specific for an antigen expressed on the surface of a bacterial cell causing the infection.

[0133] Contemplated modified NK or NK-92 cells can be administered to an individual in absolute numbers of cells. For example, about 1000 cells / injection up to about 10 billion cells / injection can be administered to an individual, such as about, at least about, or at most about 1×10 cells / injection. 8 , 1 × 10 7 , 5 × 10 7 , 1 × 10 6 , 5 × 10 6 , 1 × 10 5 , 5 × 10 5 , 1 × 10 4 , 5 × 10 4 , 1 × 10 3 , 5 × 10 3 (etc.) modified NK-92 cells, or any range (including endpoints) between any two values. In other embodiments, the modified NK-92 cells can be administered to an individual in a relative number of cells, for example, about 1000 cells to up to about 10 billion cells per kilogram of the individual can be administered to the individual, such as about, at least about, or at most about 1 × 10 per kilogram of the individual. 8 , 1 × 10 7 , 5 × 10 7 , 1 × 10 6 , 5 × 10 6 , 1 × 10 5 , 5 × 10 5 , 1 × 10 4 , 5 × 10 4 , 1 × 10 3 , 5 × 10 3 (etc.) modified NK-92 cells, or any range (including endpoints) between any two values. In other embodiments, the total dose can be m 2 The body surface area is calculated, including the 2 About 1 × 10 11 , 1 × 10 10 , 1 × 10 9 , 1 × 10 8 , 1 × 10 7 , or any range between any two values ​​(including endpoints). The average human body is about 1.6 to about 1.8 m 2 In a preferred embodiment, about 1 billion to about 3 billion NK-92 cells are administered to the patient.

[0134] The modified NK-92 cells, and optionally other anti-cancer or anti-viral agents, can be administered to a patient suffering from cancer or a viral infection once, or can be administered multiple times, for example, once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours, or once every 1, 2, 3, 4, 5, 6, or 7 days, or once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more weeks, or any range between any two values ​​(including endpoints).

[0135] In one embodiment, in the case where the modified NK-92 cells express a suicide gene, an agent is administered to the patient that triggers the death of the modified NK-92 cells. In one embodiment, the agent is administered at a time point after administration of the modified NK-92 cells sufficient to allow the NK-92 cells to kill target cells.

[0136] In one embodiment, the modified NK-92 cells are irradiated prior to administration to a patient. The irradiation of NK-92 cells is, for example, as described in U.S. Pat. No. 8,034,332, which is incorporated herein by reference in its entirety. In one embodiment, the modified NK-92 cells that have not been engineered to express a suicide gene are irradiated.

[0137] In addition, it should be understood that the contemplated treatment methods will also include the administration of other immunotherapeutic entities, with particular preference for immunotherapeutic entities, including viral cancer vaccines (e.g., adenoviral vectors encoding cancer-specific antigens), bacterial cancer vaccines (e.g., non-pyrogenic Escherichia coli expressing one or more cancer-specific antigens), yeast cancer vaccines, N-803 (also known as ALT-803, ALTOR Biosciences), and antibodies (e.g., binding to tumor-associated antigens or patient-specific tumor neoantigens), stem cell transplants (e.g., allogeneic or autologous), and tumor-targeted cytokines (e.g., NHS-IL12, IL-12 conjugated to tumor-targeted antibodies or fragments thereof). Example

[0138] The following examples are for illustrative purposes only and should not be construed as limiting the rights and interests of the present invention. A variety of alternative techniques and procedures may be used by those skilled in the art that will similarly allow one to successfully perform the intended invention. Example 1: CAR mRNA preparation

[0139] Designing codes using computer methods Figure 1The DNA sequence of each variant of CD19CAR, schematically depicted in Figure , was synthesized de novo and then subcloned into the mRNA expression vector pXT7 (GeneArt, Life Technologies). 10 micrograms (µg) of plasmid were linearized by digestion with SalI restriction enzyme (New England Biolabs) and purified using QIAgen gel purification kit (QIAgen) according to the manufacturer's instructions.

[0140] The linearized DNA was used as a template for in vitro synthesis of mRNA using the T7 mMessage mMachine Ultra Transcription Kit (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s instructions. The kit includes a polyadenylation extension step that increases the length of the polyA tail of the mRNA, thereby enhancing its in vivo stability.

[0141] The mRNA of six CD19CAR variants was prepared, and green fluorescent protein (GFP) mRNA was prepared as a negative control. All CD19CAR polypeptide variants comprise an extracellular domain comprising an anti-CD19 scFv region (αCD19-scFv) (SEQ ID NO: 11), a hinge region from CD8 (SEQ ID NO: 3 or NO: 4), and a transmembrane domain from CD28 (SEQ ID NO: 6, encoded by SEQ ID NO: 7). The intracellular domain of CD19CAR is as follows, and as Figure 1 Schematic representation: CAR 3z comprises a CD3ζ signaling domain; CAR FcRe contains an FcεRIγ signaling domain (SEQ ID NO: 1); CAR 28_3z comprises a CD28 signaling domain fused to a CD3ζ signaling domain; CAR BB_3z comprises a 4-1BB signaling domain fused to a CD3ζ signaling domain; CAR 28_BB_3z comprises a CD28 signaling domain fused to a 4-1BB signaling domain, which is fused to a CD3ζ signaling domain; CAR BB_3z_28 comprises a 4-1BB signaling domain fused to a CD3ζ signaling domain, which is fused to a CD28 signaling domain.

[0142] More specifically, Figure 1The first generation CAR with CD3 ζ signaling domain has the nucleic acid sequence shown in SEQ ID NO: 16 (human). The first generation CAR with FcεRIγ signaling domain nucleic acid has the nucleic acid sequence shown in SEQ ID NO: 12 and the amino acid sequence shown in SEQ ID NO: 10. The second generation CAR with CD28 / CD3 ζ signaling domain has the nucleic acid sequence shown in SEQ ID NO: 17, and the second generation CAR with 4-1BB / CD3 ζ signaling domain has the nucleic acid sequence shown in SEQ ID NO: 18. The nucleic acid sequence of the third generation CAR with CD28 / 4-1BB / CD3 ζ signaling domain has the nucleic acid sequence shown in SEQ ID NO: 19, and the third generation CAR with 4-1BB / CD3 ζ / CD28 signaling domain has the nucleic acid sequence shown in SEQ ID NO: 20. A further first generation CAR with FcεRIγ signaling domain nucleic acid has the amino acid sequence shown in SEQ ID NO: 25. Example 2: Electroporation of NK-92 cells with CD19CAR mRNA

[0143] 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). TM Electroporation was performed using an electroporation apparatus (Life Technologies, Carlsbad, CA) according to the manufacturer's parameters for NK-92 cells (1250 V, 10 ms, 3 pulses) and 10 cells were added in a 100 µl volume. 6 The cells were electroporated with mRNA at 5 µg mRNA per cell. The electroporated cells were maintained in the same culture medium (same as above) for 20 hours (h).

[0144] CD19CAR expression on the surface of NK-92 cells was determined by flow cytometry using an anti-scFv antibody labeled with eF660 (eBioscience, San Diego, CA). Figure 2A The % expression of the indicated CD19CAR in NK-92 cell populations is shown. Figure 2B Shown are the median fluorescence intensity (MFI, background subtracted) of cells electroporated with the indicated CD19CARs. Figure 2A and 2B As can be seen, CAR FcRe unexpectedly had the highest percentage of cells expressing CD19CAR on the cell surface (75.2%), as well as the highest MFI (amount of CAR expressed on recombinant cells), followed by 28_3z (61.7%). Example 3: Cytotoxicity of NK-92 cells expressing CD19CAR against cancer cell lines

[0145] The efficacy of CAR-expressing NK-92 cells in targeting cancer cells in vitro was examined 20 h after electroporation using a flow cytometry-based in vitro cytotoxicity assay. Effector cells (NK-92 expressing CD19CAR or GFP) were co-administered with PKHGL67-labeled (Sigma-Aldrich, St. Louis, MO) target cells (K562; or SUPB15, B-ALL, CD19 + ) were mixed at different effector:target ratios (5:1 to 0.3:1) in 96-well plates 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 % of PI-positive cells in the PKH-positive target cell population.

[0146] Figure 3A and 3B Exemplary results are provided in Figure 3A As shown, NK-92 cells efficiently killed K562 cells regardless of CD19CAR expression. Therefore, it should be noted that the recombinant cells did not lose cytotoxicity. In contrast, NK-92 cells expressing GFP were inefficient in killing the cancer cell line SUP-B15. SUP-B15 is an acute lymphoblastic leukemia cell line that is CD19 positive and resistant to NK-92-mediated cytotoxicity. Expression of any of the CD19CARs tested provided increased cytotoxic activity against the SUP-B15 cell line compared to the control (NK-92 cells expressing GFP), which can be easily seen from the Figure 3B Unexpectedly, CARs with FcεRIγ signaling domains exhibited similar or even better cytotoxicity than second- and third-generation CARs. This finding was particularly unexpected because the FcεRIγ signaling domain only acts as a single unit and is not combined with other signaling domains. When used in CAR T cells, such an arrangement does not provide the expected targeted cytotoxicity.

[0147] Degranulation is a key step required for the release of lytic proteins (e.g., perforin and granzymes) from secretory granules in NK-92 cells. Degranulation is initiated by recognition of target cells by NK-92. To examine degranulation in constructs, effector cells (NK-92) were mixed with unlabeled target cells (SUP-B15) in 96-well plates at different effector:target ratios (5:1 to 0.3:1), and anti-CD107a (FITC-conjugated, BD Pharmingen, San Jose, CA) was added to each well. The plates were incubated at 37°C in a CO2 incubator, and monensin (Golgi-stop) was added to the wells after 1 h. The plates were incubated at 37°C for an additional 3 h, and the samples were analyzed by flow cytometry (Attune, Life Technologies, Carlsbad, CA). The percentage of degranulation was determined by subtracting the % CD107a positivity in NK-92 cells from the % CD107a positivity in the effector + target samples alone. Figure 4 Exemplary results are provided. Example 4. Surface expression of NK-92 cells expressing CD19CAR and cytotoxicity against cancer cell lines

[0148] The inventors quantified the expression levels of various CAR constructs to study the persistence of expression over time. Figure 5 As can be seen from the results, NK-92 cells transfected with different CD19 CAR constructs expressed detectable levels of each CAR on the cell surface for up to 72 hours. Figure 5 It is easy to see that the CAR construct containing the Fc-ε cytoplasmic signaling domain has a significantly higher duration of expression. Notably, it was also observed that the addition of one or more signaling domains (e.g., in the examples presented herein, the CD28 signaling domain) in addition to the FcεRIγ signaling domain did not adversely affect the duration of expression. In fact, in CARs with FcεRIγ signaling domains and CD28 signaling domains, the duration of expression increased even further over time, while CAR constructs with CD3-ζ signaling domains showed a significant decrease in expression at 72 hours or even earlier.

[0149] Moreover, from Figure 5 It can also be seen from the results that the expression level of the CAR construct with the FcεRIγ signaling domain was initially significantly higher than that of the corresponding construct with the CD3-ζ signaling domain.

[0150] The inventors then set out to examine whether the prolonged and more robust expression of CAR constructs with the FcεRIγ signaling domain would also translate into higher rates of cytotoxicity. Figure 6Figure 3 shows the expression of SUPB15 CD19 at 24 and 48 hours. + Figure 2 shows an exemplary result of testing cells. As can be seen from the results, all tested CAR constructs showed comparable (maximum) cytotoxicity at 24 hours. However, at 48 hours, CD19 / CD3-ζ showed a significant decrease in cytotoxicity. It is worth noting that the Fc-ε-based CAR showed only a minimal decrease in cytotoxic activity 48 hours after electroporation, which is consistent with the Figure 5 The results of prolonged expression of CAR constructs with FcεRIγ signaling domains were similar. Therefore, it should be recognized that CAR constructs with FcεRIγ signaling domains exhibited prolonged cytotoxicity, which is considered to have substantial clinical benefit.

[0151] Advantageously, the tricistronic mRNA construct is capable of producing a large number of expected CARs with excellent functional activity. Such constructs are particularly beneficial in cases where CAR expression should be transient. On the contrary, the following embodiments for targeted CAR constructs and related functional data are from linearized DNA vector constructs, which allow transfected cells to integrate linearized DNA into the genome, thereby providing a way for non-transient expression of specific CARs. Example 5. Schematic diagram of tricistronic expression cassette

[0152] Figure 7 The DNA and protein products produced by representative tricistronic expression cassettes are schematically shown. Figure 8 A linearized version of the plasmid with the expression cassette is shown.

[0153] SEQ ID NO:28 is an exemplary nucleic acid sequence of a portion of the pNEUKv1-CD19CAR_CD16(158V)_ERIL-2 vector, which is similar to Figure 8 SEQ ID NO: 29 is an exemplary tricistronic protein representing the CD19CAR_P2A_CD16(158V) protein (similar to Figure 7 ). Similarly, SEQ ID NO:31 is an exemplary nucleic acid sequence of a codon-optimized CD33ScfV-P2A-CD16-IRES-erIL2 tricistronic sequence, and SEQ ID NO:32 shows a CD33 CAR-P2A-CD16 peptide.

[0154] Still further constructs prepared included SEQ ID NO:24, which is an exemplary amino acid sequence of CD19K_transmembrane and signaling domains, while SEQ ID NO:26 is an exemplary nucleic acid sequence of 15AD23HC_1805843_CD19K_Eps (879-1319), and SEQ ID NO:27 is an exemplary nucleic acid sequence of 15AD23HC_1805843_CD19K_Eps, which does not include the CD28 transmembrane domain. Example 6. Cytotoxicity of NK-92 cells expressing CD33-CAR against cancer cell lines

[0155] The following example is provided to demonstrate that cells resistant to specific lysis (cytotoxicity) by control (unmodified) NK-92 cells can be effectively killed by NK-92 cells expressing a CAR. In this example, the cells are THP-1 cells expressing CD33. NK-92 cells are modified to express a CAR having an extracellular binding domain that specifically binds to CD33 and an FcεRIγ signaling domain, such as Figure 8 and 9.

[0156] Fig. 9A In vitro data were provided showing that CD33-positive (CD33+) THP-1 cells were relatively resistant to cytotoxicity (specific lysis) by control NK-92 (aNK) cells, whereas a high percentage of specific lysis was present when THP-1 cells were 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 modified NK-92 cells expressing the CAR exhibited killing at a relatively low effector-target ratio. Fig. 9B In vitro data are provided showing that both control aNK cells and CD33-CAR / NK-92 cells effectively kill K562 cells. Example 7: HER2-CAR with FcεRIγ signaling domain

[0157] In this embodiment, the inventors constructed a first generation CAR with an FcεRIγ signaling domain, which includes an anti-HER2 scFv coupled to a CD8 hinge, which in turn is coupled to a CD28 transmembrane domain, which is coupled to an FcεRIγ signaling domain. The HER2-CAR thus constructed has a nucleic acid sequence shown in SEQ ID NO: 37.

[0158] The function of the HER2.CAR-t-haNK cells constructed in this way on BT-474 cells was tested using a standard CalceinAM-based cytotoxicity assay, and the exemplary results are shown in Fig.10As shown. It is easy to see from the data that HER2.CAR-t-haNK cells expressing CAR with FcεRIγ signaling domain show significant cytotoxicity against BT-474 target cells.

[0159] In further experiments, the present inventors demonstrated the expression of HER2.CAR in HER2.CAR-t-haNK cells, such as Fig.36 The natural cytotoxicity of HER2.CAR-t-haNK cells is shown in Fig.37 As shown in Figure 2, CAR-mediated cytotoxicity results are shown in Figure 2. Fig.38 shown. Fig.39 The figure shows exemplary data of ADCC of HER2.CAR-t-haNK cells. Example 8: CD30-CAR with FcεRIγ signaling domain

[0160] In this embodiment, the inventors constructed a first generation CAR with an FcεRIγ signaling domain, which includes an anti-CD30 scFv coupled to a CD8 hinge, which in turn is coupled to a CD28 transmembrane domain, which is coupled to an FcεRIγ signaling domain. The CD30-CAR thus constructed has a nucleic acid sequence shown in SEQ ID NO: 38.

[0161] Fig.46 The results demonstrated the expression of CD30-CAR, while Fig.47 The natural cytotoxicity results of the recombinant cells are shown. Fig.48 The results demonstrated CAR-mediated cytotoxicity, while Fig.49 Exemplary results for ADCC are shown in the data of FIG. Example 9: EGFR-CAR with FcεRIγ signaling domain

[0162] In this embodiment, the inventors constructed a first generation CAR with an FcεRIγ signaling domain, which includes an anti-EGFR scFv coupled to a CD8 hinge, which in turn is coupled to a CD28 transmembrane domain, which is coupled to an FcεRIγ signaling domain. The EGFR-CAR thus constructed has a nucleic acid sequence shown in SEQ ID NO: 39.

[0163] The function of the EGFR.CAR-t-haNK cells constructed in this way on A-549 cells was tested using a standard cytotoxicity assay, and the exemplary results are shown in Fig.14As shown. It is easy to see from the data that EGFR.CAR-t-haNK cells expressing CAR with FcεRIγ signaling domain showed significant cytotoxicity against A-549 target cells. Fig.31 The expression of EGFR-CAR in EGFR.CAR-t-haNK cells is shown, and the natural cytotoxicity results are shown in Fig.32 The exemplary results of CAR-mediated EGFR.CAR-t-haNK cell cytotoxicity are shown in Fig.33 and Fig.34 As shown in Figure 2, while the ADCC results of EGFR.CAR-t-haNK cells are shown in Figure 2 Fig.35 shown. Example 10: IGF1R-CAR with FcεRIγ signaling domain

[0164] In this embodiment, the inventors constructed a first generation CAR with an FcεRIγ signaling domain, which includes an anti-IGF1R scFv coupled to a CD8 hinge, which in turn is coupled to a CD28 transmembrane domain, which is coupled to an FcεRIγ signaling domain. The IGF1R-CAR thus constructed has a nucleic acid sequence shown in SEQ ID NO: 40, and encodes IGF1R-CAR, CD16 and IL-2. ER The tricistronic construct has the nucleic acid sequence shown in SEQ ID NO:53, which is also Fig.61 As shown in FIG.

[0165] The function of IGF1R.CAR-t-haNK cells spiked in this way was evaluated against MDA-MB-231 cells using a standard cytotoxicity assay compared to the second generation CAR (CD28 / CD3z), and exemplary results are shown in Fig.18 As shown in the data, it can be easily seen that IGF1R.CAR-t-haNK cells expressing CAR with FcεRIγ signaling domain exhibited significant target-specific cytotoxicity against MDA-MB-231 target cells, which was comparable to the cytotoxicity of the second-generation CAR. Example 11: CD123-CAR with FcεRIγ signaling domain

[0166] In this embodiment, the inventors constructed a first generation CAR with an FcεRIγ signaling domain, which includes an anti-CD123 scFv coupled to a CD8 hinge, which in turn is coupled to a CD28 transmembrane domain, which is coupled to an FcεRIγ signaling domain. The CD123-CAR thus constructed has a nucleic acid sequence shown in SEQ ID NO: 41. Fig.44CAR-mediated cytotoxicity data of recombinant NK cells expressing CD123-CAR are shown. Fig.45 Exemplary data of ADCC of recombinant NK cells expressing CD123-CAR are shown. Example 12: PD-L1-CAR with FcεRIγ signaling domain

[0167] In this embodiment, the inventors constructed a first generation CAR with an FcεRIγ signaling domain, which includes an anti-PD-L1 scFv coupled to a CD8 hinge, which in turn is coupled to a CD28 transmembrane domain, which is coupled to an FcεRIγ signaling domain. The PD-L1-CAR thus constructed has a nucleic acid sequence shown in SEQ ID NO: 42.

[0168] The cytotoxicity of PD-L1.CAR-t-haNK cells constructed in this way to SUP-B15.PD-L1 was detected using a standard cytotoxicity assay. + Cell function, exemplary results such as Fig.12 As shown. It is easy to see from the data that PD-L1.CAR-t-haNK cells expressing CAR with FcεRIγ signaling domain show inhibition of SUP-B15.PD-L1 + The target cells have significant cytotoxicity.

[0169] The function of the PD-L1.CAR-t-haNK cells thus constructed on U251 cells was also tested using a standard cytotoxicity assay, and the exemplary results are shown together with untransfected haNK cells. Fig.13 As shown. It is easy to see from the data that PD-L1.CAR-t-haNK cells expressing CAR with FcεRIγ signaling domain exhibit target-specific and significant cytotoxicity against U251 target cells, while haNK control cells have essentially no cytotoxicity against the same U251 cells.

[0170] In further experiments on the target cell specificity of PD-L1, the inventors tested several PD-L1 positive tumor cell lines using PD-L1.CAR-t-haNK cells as well as haNK cells as controls for general cytotoxicity. Fig.19 It can be easily seen that PD-L1.CAR-t-haNK cells have strong effects on a variety of tumor cells (lung cancer, breast cancer, urogenital system tumor cells, head and neck small cell carcinoma, chordoma). It is worth noting that PD-L1.CAR-t-haNK cells take less than 4 hours to kill most (> 85%) cells, while control haNK cells take more than 12 hours.

[0171] Fig. 20 The cytotoxicity of PD-L1.CAR-t-haNK cells against MDA-MB-231 cells compared to various other control cells (such as haNK cells as shown) is further illustrated. As can be seen from the data, at an E: T ratio of 5: 1, cetuximab improves the dissolution of PD-L1.thaNK against MDA-MB-231, and haNK activity can be improved by adding cetuximab and a-PD-L1. Compared with haNK and haNK + cetuximab, ordinary PD-L1.thank has better cytotoxic activity, and the killing of ordinary PD-L1.thank is comparable to that of haNK + PD-L1 antibodies, but PD-L1.thank + cetuximab performs better than haNK + cetuximab and haNK + PD-L1. At an E:T ratio of 1:1, PD-L1.thaNK activity was equivalent with or without cetuximab, and PD-L1.thaNK was significantly superior to both intrinsic and ADCC-mediated killing by hank. hank.haNK activity was improved by the addition of cetuximab and a-PD-L1.

[0172] In further experiments, the inventors demonstrated the expression of PD-L1.CAR in PD-L1.CAR-t-haNK cells, such as Fig.40 As shown. The natural cytotoxicity of PD-L1.CAR-t-haNK cells is shown Fig.41 The results of CAR-mediated cytotoxicity are shown in Fig.42 Exemplary data of ADCC of PD-L1.CAR-t-haNK cells are shown in Fig.43 shown. Example 13: CD33-CAR with FcεRIγ signaling domain

[0173] In this embodiment, the inventors constructed a first generation CAR with an FcεRIγ signaling domain, which includes an anti-HER2 scFv coupled to a CD8 hinge, which in turn is coupled to a CD28 transmembrane domain, which is coupled to an FcεRIγ signaling domain. The CD33-CAR thus constructed has a nucleic acid sequence shown in SEQ ID NO: 43.

[0174] The function of the CD33.CAR-t-haNK cells constructed in this way on THP-1 cells was tested using a standard cytotoxicity assay, and the exemplary results are shown in Fig.11As shown. It is easy to see from the data that CD33.CAR-t-haNK cells expressing CAR with FcεRIγ signaling domain show significant cytotoxicity against THP-1 target cells. Further data describing the strong expression of CD33CAR in NK-92 cells are shown in Fig. 27 shown. Fig.28 The natural cytotoxicity of CD33.CAR-t-haNK cells to K562 cells is shown. Fig.29 The results of CAR-mediated cytotoxicity against THP-1 cells are described. Fig.30 It further shows that CD33.CAR-t-haNK cells combined with rituximab have an inhibitory effect on SUP-B15 CD19 KO / CD20 + The results of ADCC. Example 14: gp120-CAR with FcεRIγ signaling domain

[0175] In this embodiment, the inventors constructed a first generation CAR with an FcεRIγ signaling domain, which includes an anti-gp120 scFv coupled to a CD8 hinge, which in turn is coupled to a CD28 transmembrane domain, which is coupled to an FcεRIγ signaling domain. The gp120-CAR thus constructed has a nucleic acid sequence shown in SEQ ID NO: 44.

[0176] The inventors further demonstrated that the cells thus generated expressed large amounts of CD16 and gp120 CAR, such as Fig.53 As shown. The binding of GP120 to gp120CAR is shown Fig.54 As shown, compared to non-recombinant aNK cells as a negative control. The natural cytotoxicity of the cells thus generated was Fig.55 The corresponding ADCC data are shown in Fig.56 shown. Example 15: B7-H4-CAR with FcεRIγ signaling domain

[0177] In this embodiment, the inventors constructed a first generation CAR with an FcεRIγ signaling domain, which includes an anti-B7-H4 scFv coupled to a CD8 hinge, which in turn is coupled to a CD28 transmembrane domain, which is coupled to an FcεRIγ signaling domain. The B7-H4-CAR thus constructed has a nucleic acid sequence as shown in SEQ ID NO: 45. Example 16: BCMA-CAR with FcεRIγ signaling domain

[0178] In this embodiment, the inventors constructed a first generation CAR with an FcεRIγ signaling domain, which includes an anti-BCMA scFv coupled to a CD8 hinge, which in turn is coupled to a CD28 transmembrane domain, which is coupled to an FcεRIγ signaling domain. The BCMA-CAR thus constructed has a nucleic acid sequence shown in SEQ ID NO: 46.

[0179] like Fig.50 As shown in the exemplary results of Fig.51 As shown, CAR-mediated cytotoxicity against target cells was demonstrated. Fig.52 As can be seen from the results, using rituximab as the antibody against target cells, the recombinant cells have significant ADCC. Example 17: GD2-CAR with FcεRIγ signaling domain

[0180] In this embodiment, the inventors constructed a first generation CAR with an FcεRIγ signaling domain, which includes an anti-GD2 scFv coupled to a CD8 hinge, which in turn is coupled to a CD28 transmembrane domain, which is coupled to an FcεRIγ signaling domain. The GD2-CAR thus constructed has a nucleic acid sequence shown in SEQ ID NO: 47. Example 18: FAP-CAR with FcεRIγ signaling domain

[0181] In this embodiment, the inventors constructed a first generation CAR with an FcεRIγ signaling domain, which includes an anti-FAP scFv coupled to a CD8 hinge, which in turn is coupled to a CD28 transmembrane domain, which is coupled to an FcεRIγ signaling domain. The FAP-CAR thus constructed has a nucleic acid sequence as shown in SEQ ID NO: 48. The expression of FAP-CAR is as follows Fig.57 The data show that FAP.CAR has a cytotoxic effect on target cells. Fig.58 This is proved in the results. Example 19: CSPG-4-CAR with FcεRIγ signaling domain

[0182] In this embodiment, the inventors constructed a first generation CAR with an FcεRIγ signaling domain, which includes an anti-CSPG-4 scFv coupled to a CD8 hinge, which in turn is coupled to a CD28 transmembrane domain, which is coupled to an FcεRIγ signaling domain. The CSPG-4-CAR thus constructed has a nucleic acid sequence as shown in SEQ ID NO: 52. The expression of CSPG-4-CAR was confirmed by FACS analysis, and the exemplary results are shown in FIG. Fig.59 Therefore, the constructed cells also showed significant cytotoxicity, as shown in Fig.60 The exemplary data are shown in FIG. Example 20: CD20-CAR with FcεRIγ signaling domain

[0183] In this embodiment, the inventors constructed a first generation CAR with an FcεRIγ signaling domain, which includes an anti-CD20 scFv coupled to a CD8 hinge, which in turn is coupled to a CD28 transmembrane domain, which is coupled to an FcεRIγ signaling domain. The CD20-CAR thus constructed has a nucleic acid sequence shown in SEQ ID NO: 51.

[0184] Expression of CD20 CAR in NK-92 cells Fig.25 As can be easily seen, CD20.CAR is strongly expressed in the vast majority of recombinant cells (as described above, together with CD16 in linearized DNA). Fig.26 Describes the CD20.CARNK cell response to CD20 + Exemplary results of target cell cytotoxicity. Example 21: CD19-CAR with FcεRIγ signaling domain

[0185] In this example, the inventors used a first generation CAR with an FcεRIγ signaling domain as described above, which included an anti-CD19 scFv coupled to a CD8 hinge, which in turn was coupled to a CD28 transmembrane domain, which was coupled to FcεRIγ signaling, and transfected NK-92 cells with linearized DNA for functional testing.

[0186] The function of the CD19.CAR-t-haNK cells constructed in this way on K562 cells was tested using a standard cytotoxicity assay to determine general cytotoxicity, and exemplary results are shown in Fig.15As shown. It can be easily seen that CD19.CAR-t-haNK cells expressing CAR with FcεRIγ signaling domain showed significant cytotoxicity against K562 target cells. In another set of experiments, target-specific cytotoxicity was determined using SUP-B15 cells compared with aNK cells as a control, and the exemplary results are shown in Fig.16 Again, CD19.CAR-t-haNK cells expressing CAR with FcεRIγ signaling domain showed significant target-specific cytotoxicity. In another set of experiments, Herceptin and Rituximab were used as antibodies and SKBr3 cells were used to determine target-specific ADCC. The results are shown in Fig.17 Likewise, CD19.CAR-t-haNK cells expressing CARs with an FcεRIγ signaling domain exhibited significant antibody- and target-specific ADCC.

[0187] Fig.21 The results are shown in an exemplary manner in NK-92 cells compared to controls from linearized DNA (including DNA encoding CD16 and IL-2 ER CD19.CAR expression of fragments of Fig.25 It can be seen that the expression is very strong in most cells. Other results of CD19.CAR t-haNK cell natural cytotoxicity against K562 cells and targeted cytotoxicity against SUP-B15 cells are shown in Figure 2. Fig. 22 and Fig.23 As shown. CD19.CAR t-haNK cells against SUP-B15CD19 KO / CD20 + Other exemplary results of ADCC of cells are as follows Fig.24 shown. Example 22: Anti-tumor activity of t-haNK cells targeting PD-L1 in NSG mouse xenograft model

[0188] MDA-MB-231 and HCC827 were used as validated xenograft models for PDL1 positivity and the efficacy of PDL1 t-haNK cells was evaluated in different formulations, dosing levels, and routes of administration (IV and IT).

[0189] Animals: Animal type: NSG mice (JAX), female, 9-10 weeks old; Number of animals for MDA-MB-231 model: 24 (fresh cells), and for HCC827 model: 24 (fresh cells) + 6 (cryopreserved cells). The tumor models used the following cell lines: MDA-MB-231 (human breast adenocarcinoma) and HCC827 (human lung adenocarcinoma), and the inoculation route was subcutaneous inoculation into the bilateral axilla, with an average tumor load of approximately 100 mm3 for MDA-MB-231 and 75-80 mm3 for HCC827.

[0190] Treatments: Freshly prepared anti-PD-L1 t-haNK, irradiated, at 5E7 cells / mL or 2E7 cells / mL; vehicle control was X-VIVO TM 10 culture medium; administration was IV and IT as described above. The dose for IV NK administration was 1E7 cells / dose in 200 µL (freshly prepared cells) and 4E6 cells / dose in 200 µL (cryopreserved cells); for IT NK administration (fresh cells only), the dose was 2.5E6 cells / tumor in 50 µL. The dosing frequency (M / Th or T / F) was twice weekly for 4 consecutive weeks, with the first day of dosing defined as Day 1.

[0191] The study design for MDA-MB-231 is in Table 3 below (the study ended on Day 27 when some animals in Groups A, C, and D had total tumor volumes > 2000 mm3)

[0192] .

[0193] The study design for HCC827 is in Table 4 below (the study ended at Day 29 when surviving animals were rescheduled and transferred to another study).

[0194]

[0195] .

[0196] Results: Freshly prepared PD-L1 t-haNK cells (1E7 cells / dose) resulted in significant and durable tumor growth inhibition in both MDA-MB-231 and HCC827 models.

[0197] MDA-MB-231: Tumor stasis: TGI on day 16: 84% (peak); TGI on day 26: 79% (last measurement).

[0198] HCC827: Tumor regression: TGI on Day 16: 120% (peak); TGI on Day 29: 84% (end of study).

[0199] With X-VIVO TM Cryopreserved PDL1 t-haNK cells (4E6 cells / dose) also showed statistically significant efficacy in inhibiting tumor growth compared to 10 culture medium: TGI on day 26: 60% (peak), TGI on day 29: 40% (end of study).

[0200] Freshly prepared PDL1 t-haNK cells (1E7 cells / dose) also resulted in a significant reduction in metastatic disease burden in the MDA-MB-231 model, as shown in Table 5 below.

[0201]

[0202] .

[0203] The number of visible nodules in the liver in the vehicle group was: 29 ± 9, and in the PD-L1 t-haNK group: 0 (P = 0.0116 by unpaired two-tailed t-test).

[0204] According to the experiments performed, IV administration of freshly prepared PD-L1 t-haNK cells at a level of 1E7 cells / dose twice a week for 4 weeks showed significant antitumor efficacy in both subcutaneous xenograft models tested: the treatment resulted in tumor stasis in MDA-MB-231 tumor-bearing mice, with a peak TGI of 84% on day 16 and a TGI of 79% at the end of the study (P < 0.0001 for both time points by 2-way ANOVA followed by Tukey's test for multiple comparisons), and caused tumor regression in the HCC827 model, with a peak TGI of 120% on day 16 and a TGI of 84% at the end of the study (P < 0.0001). Cryopreserved PD-L1 t-haNK cells administered intravenously (IV) twice weekly for 4 weeks at a dosing level of 4E6 cells / dose also demonstrated significant therapeutic efficacy in the HCC827 tumor model, achieving a peak TGI of 60% (P < 0.0001) and a TGI of 40% at the end of the study (P < 0.01). Freshly prepared PD-L1 t-haNK cells administered intrathecally (IT) twice weekly for 4 weeks at a dosing level of 2.5E6 cells / dose / tumor effectively inhibited the growth of HCC827 tumors, resulting in a peak TGI of 70% on day 20 and a TGI of 49% at the end of the study (P < 0.001).

[0205] Significant adverse reactions were observed in animals that received IV administration of freshly prepared PD-L1 t-haNK cells (1E7 cells / dose). In contrast to freshly prepared PD-L1 t-haNK cells, cryopreserved cells (administered at a lower level of 4E6 cells / dose) were safe in animals after IV administration. PD-L1 t-haNK cells showed significant efficacy in two subcutaneous tumor models. Cryopreserved cells administered at a lower level of 4E6 cells / dose also showed significant efficacy in inhibiting tumor growth and were shown to be safe in animals.

[0206] Of course, it should be appreciated that for all nucleic acid sequences provided herein, the corresponding encoded proteins are also within the clear scope of envision herein. Likewise, for all amino acid sequences, the corresponding nucleic acid sequences are also within the scope of envision herein (using any codon).

[0207] All patent applications, publications, references, and sequence accession numbers cited in this specification are incorporated herein by reference in their entirety.

[0208] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0209] Throughout this specification and the claims that follow, reference will be made to a number of terms which shall be defined to have the following meanings:

[0210] The terminology used is for the purpose of describing particular embodiments only and is not intended to be limiting of the present invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0211] It should be understood that all numerical values ​​described herein (e.g., pH, temperature, time, concentration, quantity and molecular weight, including ranges) include normal variations in measured values ​​encountered by those of ordinary skill in the art. Thus, the numerical values ​​described include variations of + / -0.1% to 10%, for example, + / -0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%. It should be understood that, although not always explicitly stated, all numerical designations may be preceded by the term "about". Thus, the term approximately includes variations of + / -0.1% to 10%, for example, + / -0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%. It should also be understood that, although not always explicitly stated, the agents described herein are exemplary only, and their equivalents are known in the art.

[0212] As will be understood by those skilled in the art, for any and all purposes, particularly in providing a written description, all ranges disclosed include the endpoints of the range, and include all values ​​between the endpoints of the range. All ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily considered to be fully described, and the same range can be decomposed into at least equal half, two-thirds, one-quarter, one-fifth, a few tenths, etc. As a non-limiting example, each range discussed herein can be easily decomposed into a lower third, a middle third, and an upper third, etc. As will be understood by those skilled in the art, all languages, such as "at most", "at least", etc., include the listed numerical values, and refer to the ranges that can be subsequently subdivided into subranges as described above. Finally, as will be understood by those skilled in the art, the range includes each individual member. Therefore, for example, a group having 1-3 cells refers to a group having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to a group having 1, 2, 3, 4, or 5 cells, etc.

[0213] It is also understood, although not always explicitly stated, that the reagents described herein are exemplary only and that equivalents thereof are known in the art.

[0214] "Optional" or "optionally" means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not occur.

[0215] The term "comprising" is intended to indicate that compositions and methods include the listed elements, but do not exclude other elements. When used to define compositions and methods, "consisting essentially of shall mean excluding other elements of any significance to the combination. For example, a composition consisting essentially of the elements defined herein will not exclude other elements that do not materially affect the basic and novel characteristics of the claimed invention. "Consisting of" means excluding more than trace amounts of other ingredients and substantial method steps listed. Embodiments defined by each of these transition terms are within the scope of the present disclosure.

[0216] As used herein, "immunotherapy" refers to the use of NK-92 cells, modified or unmodified, naturally occurring or modified NK cells or T cells, alone or in combination, whether used alone or in combination, and capable of inducing cytotoxicity when in contact with target cells.

[0217] As used herein, "natural killer (NK) cells" are cells of the immune system that kill target cells in the absence of specific antigenic stimulation and without restriction according to major histocompatibility complex (MHC) class. The target cell can be a tumor cell or a cell carrying a virus. NK cells are characterized by the presence of CD56 and the absence of CD3 surface markers.

[0218] The term "endogenous NK cells" is used to refer to NK cells derived from a donor (or patient), as distinct from the NK-92 cell line. Endogenous NK cells are typically a heterogeneous cell population in which NK cells have been enriched. Endogenous NK cells can be used for autologous or allogeneic treatment of a patient.

[0219] The term "NK-92" refers to natural killer cells derived from a highly potent unique cell line described by Gong et al. (1994), the rights to which are owned by Nantquist Corporation (hereinafter "NK-92™ cells"). The immortalized NK cell line was originally obtained from a patient with non-Hodgkin's lymphoma. Unless otherwise indicated, the term "NK-92™" refers to the original NK-92 cell line as well as NK-92 cell lines that have been modified (e.g., by the introduction of foreign genes). NK-92™ cells and exemplary and non-limiting modifications thereof are described in U.S. Pat. Nos. 7,618,817; 8,034,332; 8,313,943; 9,181,322; 9,150,636; and published U.S. Application No. 10 / 008,955, all of which are incorporated herein by reference in their entirety, and include wild-type NK-92™, 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 ordinary skill in the art and are readily available from Nantquist Corporation.

[0220] The term "aNK" refers to unmodified natural killer cells derived from the highly potent and unique cell line described by Gong et al. (1994), whose rights are owned by Nantquist Corporation (hereinafter "aNK™ cells"). The term "haNK" refers to unmodified natural killer cells derived from the highly potent and unique cell line described by Gong et al. (1994), whose rights are owned by Nantquist Corporation, which are modified to express CD16 on the cell surface (hereinafter "CD16+ NK-92™ cells" or "haNK® cells"). In some embodiments, CD16+ NK-92™ cells contain a high affinity CD16 receptor on the cell surface. The term "taNK" refers to unmodified natural killer cells derived from the highly potent and unique cell line described by Gong et al. (1994), whose rights are owned by Nantquist Corporation, which are modified to express a chimeric antigen receptor (hereinafter "CAR-modified NK-92™ cells" or "taNK® cells"). The term "t-haNK" refers to unmodified natural killer cells derived from a highly potent unique cell line described by Gong et al. (1994), whose rights are owned by Nantquist, Inc., which have been modified to express CD16 on the cell surface and to express a chimeric antigen receptor (hereinafter "CAR-modified CD16+ NK-92™ cells" or "t-haNK™ cells"). In some embodiments, t-haNK™ cells express a high affinity CD16 receptor on the cell surface.

[0221] "Modified NK-92 cells" refer to NK-92 cells that express exogenous genes or proteins, such as Fc receptors, CARs, cytokines (such as IL-2 or IL-15) and / or suicide genes. In some embodiments, the modified NK-92 cells contain vectors encoding transgenes, such as Fc receptors, CARs, cytokines (such as IL-2 or IL-15) and / or suicide genes. In one embodiment, the modified NK-92 cells express at least one transgenic protein.

[0222] As used herein, "non-irradiated NK-92 cells" are NK-92 cells that have not been irradiated. Irradiation renders the cells incapable of growth and proliferation. It is envisioned that NK-92 cells will be irradiated at other time points prior to treatment of a therapeutic device or patient, as the time between irradiation and infusion should not exceed four hours to maintain optimal activity. Alternatively, NK-92 cell proliferation may be prevented by another mechanism.

[0223] As used herein, "inactivation" of NK-92 cells renders them incapable of growth. Inactivation may also be associated with the death of NK-92 cells. It is contemplated that NK-92 cells may be inactivated after an ex vivo sample of cells associated with pathology has been effectively cleared in a therapeutic application, or after having remained in a mammal for a sufficient period of time to effectively kill many or all target cells in the body. As a non-limiting example, inactivation may be induced by administering an inactivating agent to which NK-92 cells are sensitive.

[0224] 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 involves killing target cells by any of a variety of biological, biochemical, or biophysical mechanisms. Cytolysis more specifically refers to the activity of effectors that lyse the plasma membrane of target cells, thereby destroying their physical integrity. This results in the killing of target cells. Without wishing to be bound by theory, it is believed that the cytotoxic effect of NK-92 cells is due to cytolysis.

[0225] The term "killing" with respect to a cell / cell population is intended to include any type of manipulation that will result in the death of that cell / cell population.

[0226] The term "Fc receptor" refers to a protein found on the surface of certain cells (e.g., natural killer cells) that contributes to the protective function of immune cells by binding to a part of an antibody called the Fc region. Binding of the Fc region of an antibody to the 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 according to the type of antibody they recognize. For example, Fc-gamma receptors (FCγRs) bind to IgG class 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 commonly found on NK cells. NK-92 cells do not express FCγRIII-A. Fc-ε receptors (FcεRs) bind to the Fc region of IgE antibodies.

[0227] As used herein, the term "chimeric antigen receptor" (CAR) refers to an extracellular antigen binding domain fused to an intracellular signaling domain. CAR can be expressed in T cells or NK cells to increase cytotoxicity. Typically, the extracellular antigen binding domain is a scFv that is specific for an antigen found on the target cell. Based on the specificity of the scFv domain, NK-92 cells expressing CAR are targeted to cells that express certain antigens on the cell surface. The scFv domain can be engineered to recognize any antigen, including tumor-specific antigens and virus-specific antigens. For example, CD19CAR recognizes CD19, a cell surface marker expressed by certain cancers.

[0228] As used herein, the term "tumor-specific antigen" refers to an antigen present on cancer cells or neoplastic cells but not detectable on normal cells derived from the same tissue or lineage as the cancer cells. As used herein, tumor-specific antigens also refer to tumor-associated antigens, i.e., antigens expressed at higher levels on cancer cells than on normal cells derived from the same tissue or lineage as the cancer cells.

[0229] As used herein, the term "virus-specific antigen" refers to an antigen present on virus-infected cells but not detectable on normal cells derived from the same tissue or lineage as the virus-infected cells. In one embodiment, the virus-specific antigen is a viral protein expressed on the surface of infected cells.

[0230] The terms "polynucleotide", "nucleic acid" and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, i.e., deoxyribonucleotides or ribonucleotides or their analogs. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. The following are non-limiting examples of polynucleotides: genes or gene fragments (e.g., probes, primers, EST or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, nucleotide structural modifications may be performed before or after polynucleotide assembly. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides may be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to double-stranded and single-stranded molecules. Unless otherwise stated or required, any embodiment of a polynucleotide of the invention includes both a double-stranded form and each of the two complementary single-stranded forms known or predicted to comprise the double-stranded form.

[0231] A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U), which is thymine when the polynucleotide is RNA. Thus, the term "polynucleotide sequence" is an alphabetic representation of a polynucleotide molecule.

[0232] "Homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing positions in each sequence that can be aligned for the purpose of comparison. When a position in the compared sequences is occupied by the same base or amino acid, then the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences.

[0233] As used herein, "percent identity" refers to the sequence identity between two peptides or between two nucleic acid molecules. The percent identity can be determined by comparing the position in each sequence, which can be compared for the purpose of comparison. When the position in the comparison sequence is occupied by the same base or amino acid, the molecule has identity at that position. Homologous nucleotide sequences include sequences encoding natural allelic variants and mutations of the nucleotide sequences described herein. Homologous nucleotide sequences include nucleotide sequences encoding proteins of mammalian species other than humans. Homologous amino acid sequences include amino acid sequences containing conservative amino acid substitutions and polypeptides having the same binding and / or activity. In some embodiments, homologous amino acid sequences have no more than 15, no more than 10, no more than 5 or no more than 3 conservative amino acid substitutions. In some embodiments, the nucleotide or amino acid sequence has at least 60%, at least 65%, at least 70%, at least 80% or at least 85% or greater identity with the sequence described herein. In some embodiments, the nucleotide or amino acid sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the sequence described herein. Percent identity can be determined by using the Gap program (Wisconsin Sequence Analysis Package, Version 8 for UNIX, Genetic Computer Group, University Research Park, Madison) with default settings, which uses the Smith and Waterman algorithm (Adv. Appl. Math., 1981, 2, 482-489). Algorithms suitable for determining percent sequence identity include BLAST and BLAST 2.0 algorithms, which are described by Altschul et al. (Nuc. Acids Res. 25: 3389-402, 1977), and Altschul et al. (J. Mol. Biol. 215: 403-10, 1990), respectively. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (see ncbi.nlm.nih.gov website). The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M = 5, N = -4, and a comparison of both strands.For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915, 1989) with alignments (B) of 50, expectation (E) of 10, M = 5, and N = -4.

[0234] In certain embodiments, for expressing in a particular species, codon optimized is carried out to nucleotide sequence, for example, codon optimized (expression of the protein encoded by codon optimized nucleotide sequence) can be carried out to mouse sequence for expressing in people.Therefore, in certain embodiments, codon optimized nucleotide sequence has at least 60%, at least 65%, at least 70%, at least 80% or at least 85% or greater identity with nucleotide sequence as described herein.In certain embodiments, codon optimized nucleotide sequence acid sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with sequence described herein.

[0235] The term "expression" refers to the production of a gene product (e.g., a protein). When referring to expression, the term "transient" refers to the polynucleotide not being incorporated into the genome of the cell. The term "stable" when referring to expression refers to the incorporation of the polynucleotide into the genome of the cell, or the use of a positive selection marker (i.e., an exogenous gene expressed by the cell that has a benefit under certain growth conditions) to maintain expression of the transgene.

[0236] The term "cytokine" or "cytokines" refers to a general class of biological molecules that affect cells of the immune system. Exemplary cytokines include, but are not limited to, interferons and interleukins (IL), particularly IL-2, IL-12, IL-15, IL-18, and IL-21. In a preferred embodiment, the cytokine is IL-2.

[0237] As used herein, the term "vector" refers to a non-chromosomal nucleic acid containing an intact replicon so that when placed in a permissive cell, for example, by a transformation process, the vector can be replicated. A vector can replicate in one cell type (such as bacteria), but has limited or no ability to replicate in another cell (such as a mammalian cell). A vector can be viral or non-viral. Exemplary non-viral vectors for delivering nucleic acids include naked DNA; DNA complexed with cationic lipids, alone or in combination with cationic polymers; anionic and cationic liposomes; DNA-protein complexes and particles, comprising DNA condensed with cationic polymers, such as heterogeneous polylysine, oligopeptides of defined length, and polyethyleneimine, in some cases contained in liposomes; and the use of ternary complexes comprising viruses and polylysine-DNA. In one embodiment, the vector is a viral vector, for example, an adenovirus. Viral vectors are well known in the art.

[0238] As used herein, the term "targeted" is intended to include, but is not limited to, directing a protein or polypeptide to a suitable destination within or outside a cell when it comes to protein expression. Targeting is usually achieved by a signal peptide or targeting peptide, which is a segment of amino acid residues in a polypeptide chain. These signal peptides can be located anywhere within the polypeptide sequence, but are typically located at the N-terminus. The polypeptide can also be engineered to have a signal peptide at the C-terminus. The signal peptide can direct the polypeptide to be cleaved extracellularly and localized to the plasma membrane, Golgi apparatus, endosomes, endoplasmic reticulum, and other cellular compartments. For example, a polypeptide having a specific amino acid sequence at its C-terminus (e.g., KDEL) is retained in the ER lumen or transported back to the ER lumen.

[0239] As used herein, the term "targeting" refers to the ability of NK-92 cells to recognize and kill tumor cells (i.e., target cells) when referring to a target of a tumor. In this article, the term "targeted" refers to the ability of, for example, a CAR expressed by a NK-92 cell to recognize and bind to a cell surface antigen expressed by a tumor.

[0240] As used herein, the term "transfection" refers to the insertion of a nucleic acid into a cell. Transfection can be performed using any means that allow nucleic acid to enter a cell. DNA and / or mRNA can be transfected into a cell. Preferably, the transfected cell expresses a gene product (i.e., a protein) encoded by the nucleic acid.

[0241] The term "suicide gene" refers to a transgene that allows negative selection of cells expressing the transgene. Suicide genes are used as a safety system to allow cells expressing the gene to be killed by the introduction of a selection agent. A variety of suicide gene systems have been identified, including the herpes simplex virus thymidine kinase (TK) gene, the cytosine deaminase gene, the varicella zoster virus thymidine kinase gene, the nitroreductase gene, the Escherichia coli gpt gene, and the Escherichia coli Deo gene (see also, e.g., Yazawa K, Fisher WE, Brunicardi FC: Current progress in suicide gene therapy for cancer. World J. Surg. 2002 Jul;26(7):783-9). In one embodiment, the suicide gene is a thymidine kinase (TK) gene. The TK gene can be a wild-type or mutant TK gene (e.g., tk30, tk75, sr39tk). Cells expressing the TK protein can be killed using ganciclovir.

Claims

1. A genetically modified NK cell, comprising: recombinantly expressed cytokines; Recombinantly expressed CD16; and A membrane-bound, recombinantly expressed EGFR chimeric antigen receptor (EGFR-CAR), the EGFR-CAR consisting of (i) an extracellular binding domain comprising an anti-EGFR single-chain variable fragment (scFv), (ii) a hinge domain, (iii) a transmembrane domain, and (iv) an FcεRIγ signaling domain in a single polypeptide chain, wherein the FcεRIγ signaling domain has an amino acid sequence as shown in SEQ ID NO: 1; and Wherein the EGFR-CAR is encoded by a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO:

39.

2. The genetically modified NK cell according to claim 1, wherein The NK cells are NK-92 cells.

3. A genetically modified NK cell as claimed in any one of the preceding claims, wherein The recombinantly expressed cytokine is IL-2.

4. The genetically modified NK cell according to claim 1, wherein The recombinantly expressed cytokine comprises an endosomal retention sequence.

5. The genetically modified NK cell according to claim 1, wherein The recombinantly expressed CD16 is a high-affinity CD16 variant having an amino acid sequence shown in SEQ ID NO: 35 and a 158V mutation.

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

7. The genetically modified NK cell according to claim 1, wherein The extracellular binding domain specifically binds to virus-specific antigens.

8. The genetically modified NK cell according to 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 according to claim 1, wherein The genetically modified NK cell comprises a tricistronic nucleic acid sequence, which comprises a sequence encoding the recombinantly expressed cytokine, a sequence encoding the recombinantly expressed CD16, and a sequence encoding the recombinantly expressed CAR.

10. The genetically modified NK cell according to claim 9, wherein The tricistronic nucleic acid sequence is integrated into the genome of the NK cell.

11. The genetically modified NK cell of claim 1, for use in treating cancer or viral infection.

12. The genetically modified NK cell according to claim 1, wherein The hinge domain comprises a polypeptide having an amino acid sequence shown in SEQ ID NO:

3.

13. The genetically modified NK cell according to claim 1, wherein The hinge domain comprises a polypeptide having an amino acid sequence shown in SEQ ID NO:

4.

14. The genetically modified NK cell according to claim 1, wherein The transmembrane domain comprises a polypeptide having an amino acid sequence shown in SEQ ID NO:

6.

15. The genetically modified NK cell according to claim 1, wherein The extracellular binding domain has the amino acid sequence shown in SEQ ID NO:11, the hinge domain has the amino acid sequence shown in SEQ ID NO:3 or SEQ ID NO:4, and the transmembrane domain has the amino acid sequence shown in SEQ ID NO:

6.

16. The genetically modified NK cell according to claim 1, wherein The recombinantly expressed cytokine is IL-15.

17. The genetically modified NK cell according to claim 5, wherein The recombinantly expressed CD16 has the amino acid sequence shown in SEQ ID NO:

35.

18. A recombinant nucleic acid comprising: The first sequence portion encoding a cytokine; A second sequence portion encoding CD16; The third sequence portion encoding EGFR-CAR, wherein, The EGFR-CAR consists of (i) an extracellular binding domain comprising an anti-EGFR single-chain variable fragment (scFv), (ii) a hinge domain, (iii) a transmembrane domain, and (iv) an FcεRIγ signaling domain in a single polypeptide chain, wherein the FcεRIγ signaling domain has an amino acid sequence as shown in SEQ ID NO: 1; and in, The first, second, and third sequence portions are located on the same nucleic acid; and Wherein the EGFR-CAR is encoded by a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO:

39.

19. The recombinant nucleic acid according to claim 18, wherein The nucleic acid is a tricistronic RNA.

20. The recombinant nucleic acid according to claim 18, wherein The nucleic acid is a tricistronic DNA.

21. The recombinant nucleic acid according to claim 18, wherein The cytokine is IL-2.

22. The recombinant nucleic acid according to claim 18, wherein This cytokine contains an endosomal retention sequence.

23. The recombinant nucleic acid according to claim 18, wherein The CD16 is a high-affinity CD16 variant having an amino acid sequence shown in SEQ ID NO: 35 and a 158V mutation.

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

25. The recombinant nucleic acid of claim 18, wherein The extracellular binding domain specifically binds to virus-specific antigens.

26. The recombinant nucleic acid of claim 18, wherein The CAR comprises a CD8 hinge domain or a CD28 transmembrane domain, or wherein the CAR comprises a CD8 hinge domain and a CD28 transmembrane domain.

27. A recombinant cell comprising the recombinant nucleic acid of claim 18.

28. The recombinant cell of claim 27, wherein The cell is a bacterial cell.

29. The recombinant cell of claim 27, wherein The cells are autologous NK cells.

30. The recombinant cell of claim 29, wherein The NK cells are genetically modified NK-92 cells.

31. Use of the genetically modified NK cells of claim 1 in the preparation of a medicament for treating cancer in a patient in need thereof, wherein a therapeutically effective amount of the genetically modified NK cells of claim 1 is administered to the patient, thereby treating the cancer.

32. The use of claim 31, wherein at least one additional therapeutic entity is administered, the additional therapeutic entity selected from the group consisting of: viral cancer vaccines, bacterial cancer vaccines, yeast cancer vaccines, N-803, antibodies, stem cell transplants, and tumor-targeted cytokines.

33. The use according to claim 31 or 32, wherein The cancer is selected from the group consisting of leukemia, polycythemia vera, lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors.

34. The use according to claim 33, wherein Leukemia includes acute lymphoblastic leukemia, acute myeloid leukemia and chronic leukemia.

35. The use according to claim 34, wherein Chronic leukemias include chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia.

36. The use according to claim 33, wherein Lymphomas include Hodgkin's disease and non-Hodgkin's disease.

37. The use according to claim 33, wherein Solid tumors include sarcomas and carcinomas.

38. The use according to claim 37, wherein Sarcomas and carcinomas include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, endotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, Medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms tumor, cervical cancer, testicular tumors, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinomas, gliomas, astrocytomas, medulloblastomas, craniopharyngiomas, ependymomas, pinealomas, hemangioblastomas, acoustic neuromas, oligodendrogliomas, meningiomas, melanomas, neuroblastomas, and retinoblastomas.

39. Use of the genetically modified NK cells as described in claim 1 in the preparation of a medicament for treating a viral infection in a patient in need thereof, wherein a therapeutically effective amount of the genetically modified NK cells as described in claim 1 is administered to the patient to treat the viral infection.

40. The use of claim 39, wherein an antiviral drug is administered.

41. The use according to claim 31 or 39, wherein The patient was given 1 x 10 8 Up to 1 x 10 11 Cells / m 2 The genetically modified NK cells are administered to the patient's body surface area.

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