Inhibitory Chimeric Antigen Receptors Prevent On-Target and Off-Tumor Effects of Adoptive Cell Therapy

Abstract

Embodiments of the present disclosure encompass methods and compositions that enhance adoptive cell therapy by preventing or at least reducing on-target-off-tumor effects of adoptive cell therapy. The present disclosure relates to immune effector cells of any type that are engineered to express two separate chimeric molecules, an activating chimeric antigen receptor that activates immune effector cells via a costimulatory domain following binding to a first antigen, and an inhibitory chimeric antigen receptor that inhibits cell-mediated activation upon binding to a second antigen. In a specific instance, the inhibitory chimeric antigen receptor prevents fratricide and exhaustion by inhibiting activation of cells via the activating chimeric antigen receptor when the inhibitory chimeric antigen receptor binds to a specific antigen, including one taken up by trogocytosis by a sister engineered immune effector cell.

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 193,363, filed May 26, 2021, and also claims priority to U.S. Provisional Patent Application No. 63 / 296,785, filed January 5, 2022, both of which are incorporated herein by reference.

[0002] This application contains a Sequence Listing that has been submitted electronically in ASCII format, which is incorporated by reference in its entirety. The ASCII copy, created on May 13, 2022, is named MDACP1275WO_ST25.txt and is 13,050 bytes in size. Technical Field

[0003] The present disclosure relates generally to at least the fields of immunology, cell biology, molecular biology, and medicine. [Background technology]

[0004] CAR-engineered immune effector cells have demonstrated their revolutionary therapeutic potential against intractable malignancies (see, for example, Rohaan et al., 2019, June & Sadelain, 2018, and Rafiq et al., 2020). CAR molecules redirect immune effector cytotoxicity toward target cells expressing the cognate antigen. By endowing CAR-T cells and CAR-NK cells with distinct antigen specificities, these cells can mediate cytotoxicity against various tumor types. The most promising clinical data have been reported for CD19 CART cells (see, for example, June & Sadelain, 2018, and Rafiq et al., 2020) and CD19 CAR NK cells (see, for example, Liu et al., 2020). In addition to CD19, CAR antigen specificity can also be directed against other surface molecules (see, for example, Sadelain et al., 2013), such as HER-2, ERBB-2, and mesothelin (MSLN) (see, for example, Rohaan et al., 2019, Sadelain 2015, and Morgan et al., 2010). Ideal tumor target antigens should be expressed exclusively on tumor cells. However, most tumor antigens are also expressed, often at low levels, on normal tissues. As a result, the therapeutic benefits of using CAR-T or CAR-NK cell therapy in clinical settings are hindered by the induction of on-target and off-tumor effects (see, for example, Kalos et al., 2011, Brentjens et al., 2011, Brentjens et al., 2013, and Parker et al., 2020). Indeed, infusion of CAR-T cells against HER-2, ERBB-2, or MSLN was thought to contribute to acute severe side effects, such as acute respiratory failure as a result of cross-reactivity with the lung epithelium (see, e.g., Morgan et al., 2010; Hegde et al., 2020; and Haas et al., 2019). While the use of immunosuppressive regimens, such as corticosteroids, can mitigate some of these deleterious toxicities, it also undermines the therapeutic benefit of the cells (see, e.g., Lupo-Stanghellini et al., 2010; Vogler et al., 2010; and Kieback et al., 2008).Recently, it has been reported that CAR-mediated trogocytosis (trogo) transfers target antigens to CAR-T cells (see, for example, Hamieh et al., 2019) and CAR-NK cells, resulting in fratricide of trogo-positive CAR T cells and CAR-NK cells, thereby limiting their in vivo persistence and their potential for clinical antitumor activity. Therefore, these on-target and off-tumor effects hinder the clinical use of CAR-T cells and CAR-NK cells. Summary of the Invention

[0005] Embodiments of the present disclosure include methods and compositions for enhancing adoptive cell therapy. In certain embodiments, the methods and compositions enhance adoptive cell therapy by increasing the persistence of adoptive cell therapy cells and / or by protecting cells not targeted by the cell therapy. The present disclosure provides improved efficacy of cell therapies for cancers, including solid tumors, while minimizing their side effects. In specific embodiments, the improved efficacy of cell therapy is achieved because the engineered cells of the therapy enable the cell therapy to distinguish and spare "off-target" normal or other cells from "on-target" cancer cells without neutralizing the cell therapy's function in tumor rejection.

[0006] The present disclosure relates to any type of genetically engineered immune effector cell that has an inhibitory chimeric antigen receptor (iCAR) in addition to being engineered to have one or more other engineered antigen receptors, including chimeric antigen receptors (activating CARs, or aCARs) that contain at least one activation signaling domain. The iCAR is configured to contain one or more intracellular inhibitory domains (e.g., inhibitory signaling endodomains) that prevent cytotoxicity against cells that are not intended to be targeted, such as normal tissues and / or its own sibling cells (other engineered immune effector cells). In specific embodiments, the iCAR utilizes the natural inhibitory signaling of NK cell inhibitory receptors, such as KIRs and other NK cell inhibitory receptors. Thus, upon binding of the iCAR to the antigen targeted by the iCAR, the iCAR signals the cell expressing the iCAR to avoid killing the cell expressing the antigen targeted by the iCAR. In a specific embodiment, this dual system includes an NK self-recognition iCAR that, upon binding to a cell expressing the antigen targeted by the iCAR, transmits a "don't kill me" signal to the NK cell expressing the iCAR, thereby stopping the engineered NK cell expressing the iCAR from killing the cell targeted by the iCAR (by the aCAR). Thus, when cells expressing both an iCAR and an aCAR engage with the antigen to which the iCAR binds, they do not kill the cell expressing the antigen targeted by the iCAR because the iCAR inhibits the aCAR from killing the cell expressing the antigen targeted by the iCAR. In some cases, the antigen targeted by the iCAR may be expressed by a normal cell or by a sister cell of the engineered immune effector cell (another engineered immune effector cell of the same type), preventing the iCAR from fratriciding them.

[0007] As described above, in specific embodiments, the iCAR of a particular engineered immune effector cell prevents other engineered immune effector cells of the same type from being killed by the aCAR. In specific embodiments, immune effector cells that express both an iCAR and an aCAR express the antigen targeted by the iCAR due to trogocytosis (a process by which lymphocytes bound to antigen-expressing cells detach surface molecules from those antigen-expressing cells and then express them on their own surface). That is, in some cases, the antigen targeted by an engineered immune effector cell is internalized by the engineered immune effector cell itself, but the iCAR prevents one engineered immune effector cell from killing another immune effector cell that happens to express that antigen as a result of trogocytosis.

[0008] In specific embodiments, the methods of the present disclosure enhance the persistence and anti-tumor activity of CAR NK cells while preventing fratricide, off-target toxicity, and exhaustion, thereby enhancing or improving anti-tumor cell therapy.

[0009] Embodiments of the present disclosure include compositions comprising engineered immune effector cells comprising: (a) at least one inhibitory chimeric antigen receptor (iCAR), comprising (1) at least one extracellular antigen-binding domain, where the first extracellular antigen-binding domain binds to a first antigen, (2) a first transmembrane domain, and (3) at least one natural killer (NK) cell inhibitory signaling domain and / or at least one co-inhibitory domain; and (b) at least one activating chimeric antigen receptor (aCAR), comprising (1) at least one extracellular antigen-binding domain, where the second extracellular binding domain binds to a second antigen, (2) a second transmembrane domain, and (3) at least one activating endodomain, with or without a co-stimulatory signaling domain. The cells may be NK cells or T cells. The first and second antigens may be different antigens. In a specific example, the first extracellular antigen-binding domain of (a)(1) binds to an antigen on an NK cell. The cell can be an NK cell, and the first extracellular antigen-binding domain of (a)(1) binds to an antigen on the NK cell, e.g., KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL5, KIR3DL1, KIR3DL2, KIR3DL3, KIR2DL4, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DS1, DAP10, DAP12 , CD56, CD57, CD25, CD122, NKP30, NKP44, NKP46, NKG2C, NKG2D, NKG2A, CRTAM, TIGIT, CD96, 2B4, CD16, CD27, CD100, CD160, ILT2, ILT4, KLRG1, LAIR1, CD161, CS1, (natural cytotoxicity receptor) NCR, KIR, and / or other NK-associated antigens. In some cases, the iCAR has two antigen-binding domains, each binding to a different antigen. The (b)(1) second extracellular antigen-binding domain can bind to a cancer antigen or a pathogen antigen. The (b)(1) second extracellular antigen-binding domain can bind to a cancer antigen on a solid tumor or a hematological malignancy. In specific embodiments, the NK cell inhibitory signaling domain and / or co-inhibitory domain are derived from an NK cell inhibitory receptor,The NK cell inhibitory signaling domain and / or co-inhibitory domain can be derived from leukocyte immunoglobulin-like receptor (LIR-1), CD300A, NKG2A, Siglec-7, CD96, TIM3, TIGIT, LAIR-1, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, KIR3DL3, KIR2DL5A, and / or KIR2DL5B. The first transmembrane domain and the inhibitory signaling domain can or can not be derived from the same molecule, e.g., the first transmembrane domain and the inhibitory signaling domain can be derived from LIR-1 or KIR2DL1. In specific embodiments, the iCAR comprises at least one co-inhibitory domain comprising a co-inhibitory domain from LAIR-1, NKG2A, CD300A, or a combination thereof. The first and / or second extracellular antigen-binding domains may comprise scFvs or natural ligands. In a specific embodiment, the second extracellular antigen-binding domain of (b)(1) is selected from the group consisting of CD19, EBNA, CD123, HER2, CA-125, TRAIL / DR4, CD20, CD70, HLA-G, CD38, CD123, CLL1, carcinoembryonic antigen, alpha-fetoprotein, CD56, AKT, Her3, epithelial tumor antigen, CD319 (CS1), ROR1, folate-binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, CD5, CD23, CD30, HERV-K, IL-11R alpha, kappa chain, lambda chain, CSPG4, CD33, CD47, CLL-1, U5s nRNP200, CD200, BAFF-R, BCMA, CD99, p53, mutant p53, Ras, mutant ras, c-Myc, cytoplasmic serine / threonine kinase, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, MART-1, melanoma-associated antigen, BAGE, DAM-6, -10, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7A, GAGE-7B, GAGE-8, NA88-A, MC1R, MDA-7, gp75, Gp100, PSA, PSM, tyrosinase, tyrosinase-related protein,TRP-1, TRP-2, ART-4, CAMEL, CEA, Cyp-B, hTERT, hTRT, iCE, MUC1, MUC2, phosphoinositide 3-kinase, TRK receptor, PRAME, P15, RU1, RU2, SART-1, SART-3, Wilms tumor antigen (WT1), AFP, -catenin / m, caspase-8 / m, CDK-4 / m, ELF2M, GnT-V, G250, HAGE, HSP70-2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, myosin / m, RAGE, SART-2, TRP-2 / INT2, 707-AP, annexin II, CDC27 / m, TPI / mbc r-abl, BCR-ABL, interferon regulatory factor 4 (IRF4), ETV6 / AML, LDLR / FUT, Pml / RAR, tumor-associated calcium signaling factor 1 (TACSTD1), TACSTD2, receptor tyrosine kinases, epidermal growth factor receptor (EGFR), EGFRvIII, platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR), VEGFR2, cytoplasmic tyrosine kinase, integrin-linked kinase (ILK), signal transduction and activator of transcription STAT3, STATS, and STATE, HIF-1, HIF-2, nuclear factor kappa BB (NF-B), Notch receptor NY ESO1, c-Met, mammalian target of rapamycin (mTOR), WNT, extracellular signal-regulated kinase (ERK), PMSA, PR-3, MDM2, mesothelin, renal cell carcinoma-5T4, SM22-alpha, carbonic anhydrase I (CAI), CAIX, STEAD, TEL / AML1, GD2, proteinase 3, hTERT, sarcoma translocation breakpoint, EphA2, ML-IAP, EpCAM, TMPRSS2 ETS fusion gene, ERG, NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, GD3, fucosyl GM1, mesothelian, PSCA, sLe, PLAC1, GM3, BORIS, Tn, GLoboH, NY-BR-1, RGsS, SAGE, SART3, STn, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE1,The scFvs bind to an antigen selected from the group consisting of B7H3, legumain, TIE2, Page4, MAD-CT-1, FAP, MAD-CT-2, fos-related antigen 1, CBX2, CLDN6, SPANX, TPTE, ACTL8, ANKRD30A, CDKN2A, MAD2L1, CTAG1B, SUNC1, LRRN1, and combinations thereof.

[0010] In certain embodiments, the composition of the iCAR comprises an (a)(1) first extracellular antigen-binding domain comprising an scFv that binds to CS1, an IgG1 hinge, an (a)(2) first transmembrane domain from KIR2DL1, and an (a)(3) inhibitory signaling domain from KIR2DL1.

[0011] In certain embodiments, the composition of the iCAR comprises an (a)(1) first extracellular antigen-binding domain comprising an scFv that binds to CD19, an IgG1 hinge, an (a)(2) first transmembrane domain from KIR2DL1, and an (a)(3) inhibitory signaling domain from KIR2DL1.

[0012] In certain embodiments, the composition of the iCAR comprises an (a)(1) first extracellular antigen-binding domain comprising an scFv that binds to CD19, an IgG1 hinge, an (a)(2) first transmembrane domain from LIR-1, and an (a)(3) inhibitory signaling domain from LIR-1.

[0013] In certain embodiments, the composition of the iCAR comprises an (a)(1) first extracellular antigen-binding domain comprising an scFv that binds to CD19, an IgG1 hinge, an (a)(2) first transmembrane domain from KIR2DL1, an (a)(3) inhibitory signaling domain from KIR2DL1, and a co-inhibitory domain from LAIR-1.

[0014] In certain embodiments, the composition of the iCAR comprises an (a)(1) first extracellular antigen-binding domain comprising an scFv that binds to CD19, an IgG1 hinge, an (a)(2) first transmembrane domain from KIR2DL1, an (a)(3) inhibitory signaling domain from KIR2DL1, and a co-inhibitory domain from NKG2A.

[0015] In certain embodiments, the composition of the iCAR comprises an (a)(1) first extracellular antigen-binding domain comprising an scFv that binds to CD19, an IgG1 hinge, an (a)(2) first transmembrane domain from KIR2DL1, an (a)(3) inhibitory signaling domain from KIR2DL1, and a co-inhibitory domain from CD300A.

[0016] In specific embodiments, the iCAR comprises SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10.

[0017] In specific embodiments, at least a portion of the iCAR is encoded by the sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9.

[0018] Compositions comprising a plurality of any of the cells encompassed herein are encompassed, including any composition contained in a pharmaceutically acceptable carrier.

[0019] An embodiment of the present disclosure is a method of enhancing adoptive cell therapy for an individual in need of adoptive cell therapy, the method comprising administering to the individual a therapeutically effective amount of engineered immune effector cells, each comprising: (a) at least one inhibitory chimeric antigen receptor (iCAR), wherein a first extracellular antigen-binding domain comprises at least one extracellular antigen-binding domain that binds a first antigen, and at least one natural killer (NK) cell inhibitory signaling domain and / or at least one co-inhibitory domain; and (b) at least one activating chimeric antigen receptor (aCAR), wherein a second antigen-binding domain comprises at least one extracellular antigen-binding domain that binds a second antigen, and an activating endodomain and at least one co-stimulatory signaling domain; wherein (I) the first antigen and the second antigen are the same, and the engineered immune effector cells deliver the second extracellular antigen-binding domain to cells expressing the antigens. or (II) the first and second antigens are non-identical and are both expressed on fellow engineered immune effector cells, or on non-engineered immune effector cells of the same type, or on non-diseased cells, and the iCAR inhibits killing of the fellow engineered immune effector cells, or on non-engineered immune effector cells of the same type, or on non-diseased cells, respectively, when the engineered immune effector cells bind to the second antigen on the fellow engineered immune effector cells, or on non-engineered immune effector cells of the same type, or on non-diseased cells, respectively, via the second extracellular antigen-binding domain. In certain cases, in (I), the cells expressing the antigens are non-cancerous cells. In some cases, in (I), the cells expressing the antigen are engineered immune effector cells of the same species.In certain cases, in (II), the engineered immune effector cell binds to the second antigen on the fellow engineered immune effector cell via the second extracellular antigen-binding domain, which may or may not be expressed by the fellow engineered immune effector cell as a result of trogocytosis.

[0020] In any of the methods of the present disclosure, the engineered immune effector cells may be obtained from a reservoir or may be produced without reservoir. The engineered immune effector cells may or may not be engineered to express an iCAR before being engineered to express an aCAR. The engineered immune effector cells may or may not be engineered to express an iCAR after being engineered to express an aCAR. In a specific embodiment, the engineered immune effector cells are NK cells and are engineered to express an iCAR that targets an NK cell autoantigen. The engineered immune effector cells can be engineered to express an iCAR that targets an NK cell autoantigen, and then engineered to express an aCAR that targets an antigen on an individual's cancer cells.

[0021] In any of the methods of the present disclosure, the individual has cancer (including metastasis) and the individual is administered a therapeutically effective amount of a second cancer treatment, such as surgery, radiation, chemotherapy, drug therapy, hormone therapy, immunotherapy, or a combination thereof. The second cancer treatment can be delivered before, during, or after the delivery of the engineered immune effector cells.

[0022] In certain embodiments, the engineered immune effector cells are derived from cells that are autologous with respect to the individual or cells that are allogeneic with respect to the individual. Any of the engineered immune effector cells can be NK cells or T cells.

[0023] In a specific embodiment, the first extracellular antigen-binding domain binds to an antigen on an NK cell. The engineered immune effector cell can be an NK cell, and the first extracellular antigen-binding domain binds to an antigen on an NK cell, such as KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL5, KIR3DL1, KIR3DL2, KIR3DL3, KIR2DL4, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DS1, DAP10, Binds to DAP12, CD56, CD57, CD25, CD122, NKP30, NKP44, NKP46, NKG2C, NKG2D, NKG2A, CRTAM, TIGIT, CD96, 2B4, CD16, CD27, CD100, CD160, ILT2, ILT4, KLRG1, LAIR1, CD161, CS1, (natural cytotoxicity receptor) NCR, KIR, and / or other NK-associated antigens.

[0024] In a specific embodiment of this method, the iCAR has two antigen-binding domains, each targeting a different antigen. The second extracellular antigen-binding domain may or may not bind to a cancer antigen or a pathogen antigen. The second extracellular antigen-binding domain may bind to a cancer antigen on a solid tumor or a hematological malignancy. In a specific example, the NK cell inhibitory signaling domain and / or co-inhibitory domain are derived from an NK cell inhibitory receptor. The first transmembrane domain and the inhibitory signaling domain may or may not be derived from the same molecule. In a specific example, the first transmembrane domain and the inhibitory signaling domain are derived from LIR-1 or KIR2DL1. The iCAR may include at least one co-inhibitory domain, including a co-inhibitory domain from LAIR-1, NKG2A, CD300A, or a combination thereof.

[0025] In certain embodiments, the first and / or second extracellular antigen-binding domain comprises an scFv or a natural ligand, and the second extracellular antigen-binding domain is selected from the group consisting of CD19, EBNA, CD123, HER2, CA-125, TRAIL / DR4, CD20, CD70, HLA-G, CD38, CD123, CLL1, carcinoembryonic antigen, alpha-fetoprotein, CD56, AKT, Her3, epithelial tumor antigen, CD319 (CS1), ROR1, folate binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp120, and HIV-1 envelope glycoprotein gp120. 41, CD5, CD23, CD30, HERV-K, IL-11Ralpha, kappa chain, lambda chain, CSPG4, CD33, CD47, CLL-1, U5snRNP200, CD200, BAFF-R, BCMA, CD99, p53, mutant p53, Ras, mutant ras, c-Myc, cytoplasmic serine / threonine kinase, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, MAGE-A1, melanoma-associated antigen (MAA), GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9, GAGE-10, GAGE-11, GAGE-12, GAGE-13, GAGE-14, GAGE-15, GAGE-16, GAGE-17, GAGE-18, GAGE-19, GAGE-20, GAGE-21, GAGE-22, GAGE-23, GAGE-24, GAGE-25, GAGE-26, GAGE-27, GAGE-28, GAGE-29, GAGE-30, GAGE-31, GAGE-32, GAGE-33, GAGE-34, GAGE-35, GAGE-36, GAGE-37, GAGE-38, GAGE-39 ... AGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7A, GAGE-7B, GAGE-8, NA88-A, MC1R, MDA-7, gp75, Gp100, PSA, PSM, tyrosinase, tyrosinase-related protein, TRP-1, TRP-2, ART-4, CAMEL, CEA, Cyp-B, hTERT, hTRT, iCE, MUC1, MUC2, phosphoinositide 3-kinase, TRK receptor, PRAME, P15, RU1, RU2, SART-1, SART-3, Wilms tumor antigen (WT1), AFP, -catenin / m, CAMEL Ssase-8 / m, CDK-4 / m, ELF2M, GnT-V, G250, HAGE, HSP70-2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, myosin / m, RAGE, SART-2, TRP-2 / INT2, 707-AP, annexin II, CDC27 / m, TPI / mbcr-abl, BCR-ABL, interferon regulatory factor 4 (IRF4), ETV6 / AML, LDLR / FUT, Pml / RAR, tumor-associated calcium signaling factor 1 (TACSTD1), TACSTD2, receptor tyrosine kinase,Epidermal growth factor receptor (EGFR), EGFRvIII, platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR), VEGFR2, cytoplasmic tyrosine kinase, integrin-linked kinase (ILK), signal transducer and activator of transcription STAT3, STATS, and STATE, HIF-1, HIF-2, nuclear factor kappa B (NF-B), Notch receptor NY ESO1, c-Met, mammalian target of rapamycin (mTOR), WNT, extracellular signal-regulated kinase (ERK), PMSA, PR-3, MDM2, mesothelin, renal cell carcinoma-5T4, SM22-alpha, carbonic anhydrase I (CAI), CAIX, STEAD, TEL / AML1, GD2, proteinase 3, hTERT, sarcoma translocation breakpoints, EphA2, ML-IAP, EpCAM, TMPRSS2 The scFv may comprise an scFv that binds to an antigen selected from the group consisting of ETS fusion genes, ERG, NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, GD3, fucosyl GM1, mesothelial, PSCA, sLe, PLAC1, GM3, BORIS, Tn, GLoboH, NY-BR-1, RGsS, SAGE, SART3, STn, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE1, B7H3, legumain, TIE2, Page4, MAD-CT-1, FAP, MAD-CT-2, fos-related antigen 1, CBX2, CLDN6, SPANX, TPTE, ACTL8, ANKRD30A, CDKN2A, MAD2L1, CTAG1B, SUNC1, LRRN1, and combinations thereof. ,

[0026] In certain instances of this method, the iCAR comprises an (a)(1) first extracellular antigen-binding domain comprising an scFv that binds to CS1, an IgG1 hinge, an (a)(2) first transmembrane domain from KIR2DL1, and an (a)(3) inhibitory signaling domain from KIR2DL1.

[0027] In certain instances of this method, the iCAR comprises an (a)(1) first extracellular antigen-binding domain comprising an scFv that binds to CD19, an IgG1 hinge, an (a)(2) first transmembrane domain from KIR2DL1, and an (a)(3) inhibitory signaling domain from KIR2DL1.

[0028] In particular instances of this method, the iCAR comprises an (a)(1) first extracellular antigen-binding domain comprising an scFv that binds to CD19, an IgG1 hinge, an (a)(2) first transmembrane domain from LIR-1, and an (a)(3) inhibitory signaling domain from LIR-1.

[0029] In particular instances of this method, the iCAR comprises an (a)(1) first extracellular antigen-binding domain comprising an scFv that binds to CD19, an IgG1 hinge, an (a)(2) first transmembrane domain from KIR2DL1, an (a)(3) inhibitory signaling domain from KIR2DL1, and a co-inhibitory domain from LAIR-1.

[0030] In particular instances of this method, the iCAR comprises an (a)(1) first extracellular antigen-binding domain comprising an scFv that binds to CD19, an IgG1 hinge, an (a)(2) first transmembrane domain from KIR2DL1, an (a)(3) inhibitory signaling domain from KIR2DL1, and a co-inhibitory domain from NKG2A.

[0031] In certain instances of this method, the iCAR comprises an (a)(1) first extracellular antigen-binding domain comprising an scFv that binds to CD19, an IgG1 hinge, an (a)(2) first transmembrane domain from KIR2DL1, an (a)(3) inhibitory signaling domain from KIR2DL1, and a co-inhibitory domain from CD300A.

[0032] In some instances of the method, the iCAR comprises SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10. In specific instances of the method, at least a portion of the iCAR is encoded by the sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9.

[0033] Other objects, features, and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating particular embodiments of the present disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawings]

[0034] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0035] [Figure 1] CAR expression in NK cells. Representative FACS plots (n=5 donors) showing the percentage (%) of CAR+ transduced primary human NK cells expressing CAR19, 19scFv, or any of the five different iCAR19 constructs. The inset numbers are the percentage (%) of CAR-NK cells within the indicated gated region.

[0036] [Figure 2]Dual CAR expression in NK cells. Schematic of retroviral vectors encoding aCAR19 and / or iCAR1-CS1. Following expression of aCAR19 and iCAR1-CS1 in engineered NK cells, surface expression was assessed using tag-tandem protein CD19 and CS1 flow cytometry analysis. Inset numbers are the percentage (%) of cells within the indicated gated quadrants. TM: transmembrane; SE: signaling endodomain.

[0037] [Figure 3A-3B] CAR-mediated phosphorylation in NK cells. Representative flow cytometry histograms (left) and pooled data (right) summarizing mean fluorescence intensity (MFI) of phospho-SHP1 (pSHP1) (A) and phospho-Syk / Zap70 (pSyk / pZap70) (B) in NK cells (n=5 donors) expressing CAR19, 19scFv, or different iCAR19 constructs after coculture with Raji CD19+ cells. P values ​​were determined by two-tailed one-way ANOVA. ***P<0.0001. Data were assessed by flow cytometry and presented as mean + sem. Each dot represents an individual donor.

[0038] [Figures 4A-4D] iCAR19 NK cells exhibit reduced cytotoxicity against CD19+ targets. Plots summarizing flow cytometry data for cytokine production (IFN-γ and TNF-α) and degranulation (CD107a) by CAR19 cells and individual iCAR19 NK cells after coculture with K562gCD19+ cells (A), Raji cells (B), K562 cells (C), or RajiCD19KO cells (D) (n=5 donors per condition). Statistical significance is expressed as *P<0.01, **P<0.001, ***P<0.0001. Bars represent mean + sem. Each dot represents an individual donor.

[0039] [Figure 5A-5B]iCAR-mediated NK cytotoxicity. Cytotoxicity of NK cells transduced with CAR19, 19scFv, or different iCAR19 constructs against RajiCD19+ cells (A) and autoNKgCD19+ target cells (B) over a 30-hour period measured by Incucyte live imaging cell killing assay (Incucyte analysis of percentage (%) caspase-3 / 7+ events) (representative example of 3 donors). ***P<0.0001. Bars represent mean + sem. P values ​​were determined by two-tailed two-way ANOVA.

[0040] [Figure 6] Dual CAR-mediated NK cytotoxicity against MM1S cells. Cytotoxicity (caspase-3 / 7+) of CAR19 / iCAR-CS1 NK cells ((ii); bottom) and CAR19 / CS1scFv NK cells ((i); top) against MM1SCD19- / CS1+ targets over 30 hours as measured by Incucyte live imaging cell killing assay (n=3). **P<0.0001. Bars represent mean + sem.

[0041] [Figures 7A-7E]iCAR- and aCAR-expressing NK cells recognize targets in vitro. (A-C) RajiCD19+ / CS1- cells, SKOV3gCD19+ / CS1- cells, RajiCD19- / CS1- (e.g., CD19KO / CS1-) cells, and SKOV3CD19- / CS1- cells (A), autologous target NKCD19+ / CS+ cells overexpressing CD19 on their surface (B), and normal CD1 at different time points over a 30-hour period, as measured by an Incucyte live imaging cell killing assay. Incucyte analysis (n=3) of the percentage (%) cytotoxicity (caspase-3 / 7+ events) of NK cells (representative of 3 donors) expressing 19scFv / CS1scFv (scFv; (i)), 19scFv / iCARCS1 (iCAR1; (ii)), aCAR19 / CS1scFv (aCAR19; (iii)), or aCAR19 / iCARCS1 (aCAR / iCAR; (iv)) against 9+ / CS1 low B cells (C). (D-E) Production of CD107a (left), IFN-γ (center), and TNF-α (right) by NK cells expressing each CAR in response to 6 hours of stimulation with Raji cells (D) or K562 target cells (E) (n=3 donors). P values ​​were determined by two-sided two-way ANOVA for Figures 7A, 7B, and 7C, and by two-sided one-way ANOVA for Figures 7D and 7E. *P<0.01, **P<0.001, ***P<0.0001, ns: not significant. Data are presented as mean + sem.

[0042] [Figure 8A-8B] Effect of iCAR signaling on NK cell expansion and proliferation. Graphs showing cumulative population doublings (PD) of NK cells expressing CAR19, 19scFv, or different iCAR19 constructs over 70 days of culture with IL-2 alone (A) or IL-2 plus weekly uAPC stimulation (B) (n=5 donors). Bars represent mean + sem.

[0043] [Figure 9]Effect of iCAR signaling on CAR-NK proliferation. Graph showing cumulative population doublings (PD) of NK cells with dual CAR expression ((i) CAR19 / CS1scFv-NK cells, (ii) CAR19 / iCAR1-CS1-NK cells) cultured with IL-2 alone (n=5 donors for each). Bars represent mean + sem.

[0044] [Figures 10A-10G]CAR19-mediated trogocytosis in NK cells cocultured with CD19+ tumor targets. (A) Representative flow cytometry analysis (FACS) (representative of three donors) showing CD19 expression on CAR NK cell products gated on CAR-positive and CAR-negative fractions and on NK cells expressing 19scFv (without the intracellular signaling domain) cocultured with Raji cells for 5 minutes following pretreatment with latrunculin A (LatA) or vehicle control. All NK cell populations were pre-gated on live single cells characterized as GFP-CD56+CD3-. The CD19+ gate was determined based on a fluorescent minus one (FMO) control and by reference to a negative control of NK cells cultured alone. The inset indicates the percentage of cells within the indicated gated region. (B) Trogocytic (TROG) CD19 (tCD19) expression on CAR19-NK ((i) or (iv) with LatA), 19scFv-NK ((ii) or (v) with LatA), and NT-NK ((iii) or (vi) with LatA) singlets at different time points after coculture with Raji cells under latrunculin A (LatA) pretreatment or vehicle control (n = 3 donors per condition). The TROG fraction contained CAR+CD19+-expressing NK cells (Figure 10A, from Q2), while the TROG- fraction contained CAR+CD19--expressing NK cells (Figure 10A, from Q1). (C) tCD19 expression on singlet CAR19-NK cells (light gray symbols; top) or 19scFv-NK cells (black symbols; bottom) shown as the ratio of TROG+ / TROG- cell populations at different time points after coculture with patient-derived B-CLL cells (n=4) (circle, square, triangle, and diamond symbols) or patient-derived B-ALL cells (n=4) (x, plus, vertical line, or star symbols), respectively. Each symbol represents a different patient. (D) Flow cytometry analysis showing CD107a and IFN-γ expression in the TROG+ and TROG- fractions of CAR19-NK cells after 6 hours of stimulation with Raji cells (representative of 3 donors).Inset numbers indicate the percentage of cells within the indicated gated region. Bar graphs show the percentage (%) of CD107a+ and IFN-γ+ cells in each fraction, normalized to expression levels in CAR19-NK cells cultured alone (unstimulated; n = 5 donors per condition). (E) Schematic of the engineering for CD19-mCherry fusion protein expression on CD19 knockout Raji (RajiCD19-KO) cells, compared with Raji cells genetically modified for intracellular mCherry expression (RajimCherry) as a control. (F) Incucyte analysis (representative of 3 donors) showing the percentage (%) of CAR19-NK or 19scFv-NK cells acquiring mCherry expression after coculture with engineered CD19-KO RajiCD19-mCherry cells or RajimCherry under LatA pretreatment or vehicle control (%NKTROG+ cells). (G) Incucyte analysis showing the percentage (%) of caspase 3 / 7 events in the TROG+ fraction of CAR19-NK cells and 19scFv-NK cells cultured alone or in coculture with autologous fresh CAR19-NK cells. For CAR19-NKTROG+ cells, an anti-human CD19 blocking antibody (αCD19) was added to the culture medium during incubation to block the CD19 antigen; an antigen-mismatched scFv antibody served as a control (representative example from three donors). P values ​​were determined by two-tailed, two-way ANOVA for Figures 10B, 10C, 10F, and 10G, and by two-tailed Student's paired t-test for Figure 10D. ***P<0.0001. ns: not significant. Data were evaluated by flow cytometry and presented as mean + sem for Figures 10A-10F. Each circle represents an individual NK cell donor or experimental replicate of Raji cells.

[0045] [Figures 11A-11H]The impact of antigen-induced self-association on CAR-NK effector cell phenotype and function. (A) Schematic illustrating a rechallenge assay in which CAR19-NK cells were repeatedly challenged (chlg) with autologous NK cells genetically engineered to express the CD19 antigen (autoNKgCD19+) at an E:T ratio of 1:3 and cocultured with autoNK cells (lacking CD19 expression) as a control. Both autoNKgCD19+ and autoNK cells were genetically modified to express intracellular GFP to facilitate their identification when cocultured with effector CAR19-NK cells (GFP-negative). (B) tSNE analysis of live hCD45+GFP-CD56+CD3-CAR19-NK cells 4 days after a second round of antigen challenge with autoNKgCD19+ / gGFP+ cells. Controls included CAR19-NK cells cultured alone or after 4 days of coculture with autoNKgGFP+ cells. Cells from each condition were evaluated by mass cytometry, and their phenotypic signatures were merged to create a single t-SNE map (10,000 cells from three pooled donors per condition). Each t-SNE cluster (EC1–EC5) is represented by a different color, and the frequency of each cluster is shown in a contour plot generated for each condition. (C) Heatmap of mass cytometry data showing the expression levels of key NK cell phenotypic and functional markers in each cluster. The expression level of each marker is represented by color, ranging from light gray (low) to orange (high; positive Z-scores are indicated by a plus sign in the upper right corner of each circle, while negative Z-scores remain unmarked). The size of the circle indicates marker expression; larger circles indicate a higher proportion of cells expressing the indicated marker. TF: transcription factor; Grm: granzyme. (D) Schematic diagram illustrating the single-cell time-lapse imaging cytotoxicity assay. Time was recorded over a 6-hour period (T0-T360 min) from the start of co-culture, during which a single CAR-NK cell was incubated with a single target tumor cell. The amount of time required to detect Annexin V influx in tumor cells during the assay period was determined as the time required to induce cell apoptosis.(E and F) Kaplan-Meier curves showing the percentage (%) of apoptosis in target cells when CAR-NK effector cells ((i): fresh CAR-NK cells; (ii): CAR-NK cells isolated after 4 days of co-culture with autoNK cells (CD19 negative); (iii): CAR-NK cells isolated after a second round of antigen challenge with autoNKgCD19+ / GFP+ cells) were co-cultured with (E) K562 cells (Compare: top (i) vs. (ii) P=0.4359; middle (i) vs. (iii) P<0.0001; bottom (ii) vs. (iii) P<0.0001) or (F) Raji cells (Compare: top (i) vs. (ii) P=0.1090; middle (i) vs. (iii) P<0.0001; bottom (ii) vs. (iii) P<0.0001). (G) Ex vivo analysis of glycolytic fitness of CAR-NK cells measured by ECAR (extracellular acidification rate). The adjacent bar graphs show their basal glycolytic ECAR levels (left) determined by culturing cells in basal medium containing 10 mM glucose and their maximum ECAR glycolytic capacity (right) after exposure to 1 mM oligomycin (a mitochondrial ATP synthase inhibitor). (H) Oxidative metabolism (OXPHOS) of CAR-NK cells measured by OCR (oxygen consumption rate). The adjacent bar graphs show their basal OCR levels (left) determined at steady state and their maximum OCR (right) occurring in response to exposure to 0.5 mM FCCP, which uncouples the mitochondrial OXPHOS chain. P values ​​were determined by the log-rank test in Figures 11E and 11F and by a two-tailed Student's t-test in Figures 11G and 11H. Data are shown as mean + sem. Each symbol represents an individual donor-derived CAR-NK cell sample.

[0046] [Figures 12A-12L]The impact of TROG antigen acquisition on CAR-NK cell phenotype and function in vivo. (A) tSNE analysis of live hCD45+GFP-CD56+CD3- NK cells collected from different organs (blood, bone marrow, spleen, and liver) of mice at different time points during the treatment course. The phenotypic signatures of all collected NK cells were assessed by mass cytometry and merged to create a single tSNE map. Here, the analysis generated four distinct color-coded clusters (C1–C4). (B) Contour plots showing the abundance of tSNE clusters at pre-infusion, 2 weeks post-infusion (days 13–15), 3–4 weeks post-infusion (days 20–27), or endpoint (days 29–34). The number of cell objects is shown for each condition. (C) The frequency of NK cells expressing the TROG antigen (tCD19) in the CAR19-positive (top, CAR-NK cells) or CAR-negative (bottom, NT-NK cells) fractions at different time points during treatment is shown, relative to their counterparts in the pre-infusion product. (D) Heatmap showing the expression levels of phenotypic and functional markers on CAR-NK cells within each cluster. The expression level of each marker is represented by color, ranging from gray (low) to orange (high; positive Z-scores are indicated by a plus sign in the upper right corner of each circle, while negative Z-scores remain unmarked). The size of the circle indicates marker expression; the larger the circle, the higher the proportion of cells expressing the mentioned marker. (E) FlowSOM analysis of the post-infusion NK cell population, in which each metacluster is mapped using a self-organizing mapping strategy. Each colored area corresponds to one metacluster. The inset pie charts represent the frequency of NK cells expressing CAR and TROG antigens (tCD19) on clustered cells, and the size of each chart represents the number of clustered cells. (F and G) Violin plots showing the expression of (F) CAR19 and (G) tCD19 on CAR19-NK cells in each cluster, determined based on their levels in pre-infusion NT-NK cells, shown as gray lines.(H) Violin plots showing cisplatin levels in CAR19-NKTROG+ cells (those expressing CD19) or their CAR19-NKTROG- counterparts after infusion for each cluster. Cisplatin levels represent cell viability in each population. (I-L) Violin plots showing the expression of (I) c-Kit, EOMES, and Tbet; (J) ZAP70, Syk, and 2B4; (K) granzyme (Gr) A, GrB, and perforin; and (L) PD-1, TIM3, and TIGIT in the TROG+ and TROG- fractions of CAR19-NK cells. The median expression intensity of each marker (in C1) in CAR19-NK cells before fusion is indicated by a gray line. P values ​​were determined by a two-tailed Wilcoxon matched-pairs test. *P<0.001, **P<0.0001, ***P<0.00001.

[0047] [Figures 13A-13D]Low levels of CAR-mediated TROG antigen expression were associated with improved clinical responses to CAR-NK cell-based immunotherapy. (A) tCD19 expression on donor-derived CAR-expressing NK cells (NKCAR+), donor-derived non-CAR-expressing NK cells (NKCAR-), and patient-derived NK cell singlets at different time points after CAR19-NK cell immunotherapy. The geometric mean fluorescence intensity (gMFI) of tCD19 expression was assessed by flow cytometry. Samples from individual patients at different time points after CAR-NK cell infusion are shown. (B) Nonlinear regression analysis using a polynomial model shows tCD19 expression on donor-derived NKCAR+ cells over time after CAR-NK cell infusion. The normalized mean tCD19 gMFI on CAR19-NK cells for the entire patient population was 6.29 (range, 0.61–35.77). Patients with high (>mean) normalized tCD19-gMFI and patients with low (≤mean) normalized tCD19-gMFI at two or more time points were defined as the TROGhigh group (ii) (n = 4 patients) and the TROGlow group (i) (n = 7 patients), respectively. (C) CD19 expression (top) and cell count (bottom) on singlet CD19+ B cells in the TROGlow and TROGhigh patient groups at different time points after CAR-NK cell infusion. (D) Pie charts showing the number of responders (res, top) and non-responders (non-res, bottom) in the TROGlow and TROGhigh groups after CAR19-NK cell infusion. P values ​​were determined by two-sided two-way ANOVA in Figure 13B, two-sided one-way ANOVA in Figure 13C, and two-sided Fisher's exact test in Figure 13D. *P<0.1. Data were assessed by mass cytometry and presented as mean + sem. Each circle represents an individual patient.

[0048] [Figures 14A-14I]Genetic modification of NK cells to express iCAR reduced aCAR-NK cell-induced fratricide and exhaustion. (A) Schematic illustrating the association of AI-CAR-expressing NK cells with potential targets. The "-" symbol indicates an inhibitory signal, and the "+" symbol indicates an activating signal. (B) Flow cytometry analysis showing the expression of phos-CD3ζ (pCD3z, left) and phos-Syk / Zap70 (right) in NK cells expressing 19scFv / CS1scFv (scFv only - no intracellular signaling; (i)), 19scFv / iCAR1-CS1 (iCAR / scFv; (ii)), aCAR19 / CS1scFv (aCAR / scFv; (iii)), or aCAR19 / iCAR1-CS1 (aCAR / iCAR; (iv)) after stimulation with RajiCD19+ / CS1- cells. The adjacent bar graph shows their expression as determined by the fold change (FC) of their gMFI after normalization to isotopic controls (n=5 donors per condition). (C) Incucyte analysis showing the percentage (%) of caspase 3 / 7 events in CD19+CS1- primary tumor cells from CLL patients after co-culture with CAR-expressing NK cells, with scFv-expressing NK cells as a control (representative of 3 donors). (D) Flow cytometry analysis showing the expression of phos-CD3ζ (pCD3z, left) and phos-Syk / Zap70 (right) in NK cells expressing 19scFv / CS1scFv (scFv only - no intracellular signaling; (i)), 19scFv / iCAR1-CS1 (iCAR / scFv; (ii)), aCAR19 / CS1scFv (aCAR / scFv; (iii)), or aCAR19 / iCAR1-CS1 (aCAR / iCAR; (iv)) after stimulation with genetically modified CD19+autoNKCS1+ cells. The adjacent bar graphs show their expression as determined by FC of their gMFI after normalization to isotope controls (n = 5 donors per condition).(E) Incucyte analysis showing the percentage (%) of caspase 3 / 7 events in gCD19+CS1+ autoNK cells after co-culture with CAR-expressing NK cells (representative of 3 donors) versus scFv-expressing NK cells: 19scFv / CS1scFv (scFv only - no intracellular signaling; (i)), 19scFv / iCAR1-CS1 (iCAR / scFv; (ii)), aCAR19 / CS1scFv (aCAR / scFv; (iii)), or aCAR19 / iCAR1-CS1 (aCAR / iCAR; (iv)). (F and G) Co-expression of PD1, TIM3, and TIGIT, and (G) expression of TF, expressed as the ratio of EOMES expression to Tbet expression, in singlet CAR-expressing NK cell populations after a second round of antigen challenge with autoNKgCD19+ / CS1+ / GFP+ cells (n=5 donors per condition). Representative flow cytometry histograms of EOMES and Tbet expression are shown (left panel). (H) Cumulative population doublings (PD) of each CAR-expressing NK cells (n=3 donors per condition) over 70 days of culture with IL-2: 19scFv / CS1scFv ((i)), 19scFv / iCAR1-CS1 ((ii)), aCAR19 / CS1scFv ((iii)), or aCAR19 / iCAR1-CS1 ((iv)). (I) tCD19 expression on singlet CAR-expressing NK cells (n=3 donors per condition) shown as the ratio of TROG+ / TROG- cell populations at different time points after coculture with Raji cells, with scFv-expressing NK cells as a control (representative example of 3 donors): 19scFv / CS1scFv ((i)), 19scFv / iCAR1-CS1 ((ii)), aCAR19 / CS1scFv ((iii)), or aCAR19 / iCAR1-CS1 ((iv)). P values ​​were determined by two-sided two-way ANOVA for Figures 14C, 14E, 14H, and 14I, and by two-sided one-way ANOVA for Figures 14B, 14D, 14F, and 14G. ***P<0.0001; ns: not significant. Data were assessed by flow cytometry and are shown as mean±sem in Figures 14B, 14D, 14F, 14G, and 14I. Each circle represents an individual donor.

[0049] [Figures 15A-15Q]AI-CAR-expressing NK cells demonstrated excellent in vivo antitumor activity. (A) Schematic diagram of the timeline using a mouse model implanted with Raji cells. Mice were injected with luciferase / GFP-expressing CD19+ Raji cells (0.2 × 105 cells) followed by a single injection of NK cells expressing 19scFv / CS1scFv, 19scFv / iCAR-CS1, aCAR19 / CS1scFv, or aCAR19 / iCAR-CS1. trt.: treatment, n = 5 mice per group. (B.1, B.2, and C) Tumor burden was assessed weekly by bioluminescence imaging (BLI). (B.1 and B.2) Representative images for specific time points (days 0, 7, 14, and 21) are shown for Raji cells only (left columns in B.1 and B.2), 19scFv / CS1scFv-treated animals (B.1, middle column), 19scFv / iCAR-CS1-treated animals (B.1, right column), aCAR19 / CS1scFv-treated animals (B.2, middle column), or aCAR19 / iCAR-CS1-treated animals (B.2, right column). trt.: treatment, n=5 mice per group, and (C) normalized BLI intensity in mice after infusion with different NK cell groups. Untreated mice were used as controls. Dashed lines indicate data for each mouse. (D) Kaplan-Meier curves showing the percent survival of mice after infusion with different NK cell treatment groups. (E) CD19 expression on Raji cells, shown as counts of molecules per cell, in peripheral blood (left) and bone marrow (BM, right) harvested from mice at different time points after infusion of CAR-expressing NK cells, with untreated tumor-only group as a control. (F) tCD19 expression on singlet NKCAR19+ cells, shown as TROG+ / TROG- ratio cell population (left graph), and percentage (%) viability in the TROG+ fraction (NKtCD19+, right graph) of NK cells expressing aCAR19 / CS1scFv ((i)) or aCAR19 / iCAR-CS1 ((ii)) harvested from the peripheral blood of mice at different time points after infusion of aCAR19-expressing NK cells (n=5 mice per group).(G) Percentage (%) of live GFP-CD3-CD56+ aCAR19+ NK cells in the peripheral blood of mice at different time points after infusion of cells expressing aCAR19 / iCAR1-CS1 ((i), left) and aCAR19 / CS1scFv ((ii), right) (n=5 mice per group). (H) Cell counts of live NCaCAR19+ cells in the blood and spleen at days 3, 10, and 20 after infusion; cells expressing aCAR19 / iCAR1-CS1 ((ii); upper cluster) and cells expressing aCAR19 / CS1scFv ((i); lower cluster) are shown (n=5 mice per group). (I) Schematic diagram of the timeline (n = 5 mice per group) using mice implanted with SKOV3ROR1+ ovarian cancer cells and treated with a single injection of NK cells expressing aCAR-ROR1 / CS1scFv or aCAR-ROR1 / iCAR-CS1, with an untreated tumor-implanted group as a control. (J-L) Tumor burden in SKOV3-implanted mice without NK cell treatment (left column; (i)), with aCAR-ROR1 / CS1scFv NK cell treatment (middle column; (ii)), or with aCAR-ROR1 / iCAR-CS1 NK cell treatment (right column; (iii)) was determined weekly by BLI. (J) Representative images at specific time points are shown (days 0, 7, 21, and 28). (K) Graph showing normalized BLI intensity over time during the treatment course. Dashed lines indicate data for each mouse. (L) BLI intensity for each group at day 28. (M and N) tROR1 expression on singlet NKaCAR-ROR1+ cells shown as the ratio of TROG+ / TROG- cell populations, and (N) percentage (%) viability in the TROG+ fraction (NKtROR1+) of NK cells expressing aCAR-ROR1 / CS1scFv ((i), left bar) or aCAR-ROR1 / iCAR-CS1 ((ii), right bar) harvested from the periphery of mice at different time points after NK cell infusion (n=5 mice per group).(O) Percentage (%) of live GFP-CD3-CD56+aCAR-ROR1+ NK cells in the peripheral blood of mice at different time points after infusion of NK cells expressing aCAR-ROR1 / CS1scFv ((i), left) or aCAR-ROR1 / iCAR-CS1 ((ii), right) (n=5 mice per group). (P) Cell counts of live NK aCAR-ROR1+ cells in the blood on days 5, 15, and 30 after infusion of NK cells expressing aCAR-ROR1 / CS1scFv ((i), lower cluster) or aCAR-ROR1 / iCAR-CS1 ((ii), upper cluster) (n=5 mice per group). (Q) Levels of IFN-γ and TNF-α in the serum of mice at days 3, 10, and 20 after infusion of CAR-expressing NK cells: Raji only ((i)), Raji + 19scFv / iCAR-CS1-NK ((ii)), Raji + 19scFv / iCAR-CS1-NK ((iii)), Raji + aCAR19 / CS1scFv-NK ((iv)), and Raji + aCAR19 / iCAR-CS1-NK ((v)); (n = 5 mice per group). P values ​​were determined by two-tailed two-way ANOVA in Figures 14C, E, K, and I; by two-tailed one-way ANOVA in Figures G, O, and L; by log-rank test in Figure 14D; and by two-tailed Student's t-test in Figures 14F, H, M, N, P, and Q. ***P<0.0001. Data from two independent experiments were pooled in Figures 14C, D, Q, and L, where NK cells were derived from different donors, and data were evaluated by flow cytometry in Figures 14E, F, G, H, M, N, O, and P, where data were presented as mean + sem. Each symbol represents an individual mouse sample.

[0050] [Figures 16A-16G]CAR-mediated CD19 trogocytosis in NK cells in vitro. (A) Gating strategy for distinguishing NK cells from Raji cells in coculture experiments. GFP, CD56, and CAR expression were used to identify CAR-NK cells (GFP-CD56+CAR+), Raji cells genetically modified to express GFP (CD19+GFP+CD56-CAR-), and CAR-NK / Raji doublets (CD19+GFP+CD56+CAR+). CD19 expression on CAR-NK cells after coculture with Raji cells was compared to that of control CAR-NK cells cultured alone. The inset numbers indicate the percentage of cells within the indicated gated region. (B) Representative AMNIS® images showing surface protein expression of CD56, CAR, and CD19, as well as intracellular expression of F-actin and GFP, in CAR-NK cells cultured alone, Raji cells cultured alone, or CAR-NK / Raji doublets (CAR-NK cells associated with Raji cells). Cells were identified by nuclear staining with DAPI. Scale bar indicates 7 μm. Representative images show trogocytic CD19 (tCD19) expression on CAR-NK cells after engagement with Raji tumor targets. (C) AMNIS® Imaging flow cytometry analysis of the surface of singlet CAR-NK cells cultured alone (negative control), CAR-NK cells associated with Raji cells (d:CAR-NK / Raji doublet), or singlet CAR-NK cells after incubation with Raji cells for 5 minutes. The geometric mean fluorescence intensity (gMFI) of CD19 is shown for each condition. The inserted images show representative cells for each culture condition.(D) Quantification of AMNIS® Imaging flow cytometry analysis (n = 25 objects per culture condition) showing the percentage (%) of tCD19 expression on the tCD19 fraction of singlet CD56+ CAR-NK cells cultured alone, CAR-NK cells associated with Raji cells (CAR-NK / Raji doublets), or singlet CAR-NK cells after 5 minutes of coculture with Raji cells (left), and the percentage (%) of colocalized tCD19 and CAR molecules on singlet NK cells (right). (E-F) Flow cytometry analysis (representative of 3 donors) showing CD19, CD20, and CD22 expression at the (E) protein and (F) mRNA levels on CAR-NK cells cultured alone, on Raji cells cultured alone, and on CAR-NK cells after 5 minutes of coculture with Raji cells. The inset numbers indicate the percentage of cells within the indicated gated regions. (G) Bar graphs showing summary data for each of the markers shown in Figures 16E and 16F. P values ​​were determined by two-tailed one-way ANOVA in Figure 16D and by two-tailed Student's t-test in Figure 16G. **P<0.001, ***P<0.0001. Data are presented as mean + sem. Each circle represents an individual cell.

[0051] [Figures 17A-17F]CAR19-mediated TROG-CD19 transfer on NK cells is associated with compensatory CD19 antigen reduction on tumor targets. (A) tCD19 expression on singlet CAR19-NK ((i)), NT-NK ((ii)), CAR19-T ((iii)), or T ((iv)) cells, shown as the ratio of TROG+ / TROG- cell populations, at different time points after coculture with Raji CD19+ cells (n = 3 donors). (B) Flow cytometry histograms (left) showing different CD19 expression levels on Raji clones after CD19 gene knockout using CRISPR-Cas9. Five clones were selected that expressed CD19 at high (H; (i)), moderately high (MH; (ii)), medium (M; (iii)), moderately low (ML; (iv)), or low (L; (v)) levels. The gMFI for CD19 is shown for each Raji cell clone. The acquisition of tCD19 expression on singlet CAR19-NK cells cocultured with different Raji clones is shown as the ratio of TROG+ / TROG- cell populations (right graph) (n=3 donors). (C) CD19 expression, determined as the number of molecules per cell, on Raji singlet cells at different time points after coculture with CAR19-NK cells or 19scFv-NK cells under LatA pretreatment or vehicle control conditions (n=3 donors). (D) tCD19 expression on CAR19-NK, 19scFv-NK, or NT-NK singlet cells, shown as the ratio of TROG+ / TROG- cell populations at different time points after coculture with CD19+ target cells, including NALM-6, Ramos, healthy B cells, or SKOV3 genetically modified to express CD19 (SKOV3gCD19+) (n=3 NK cell donors per tumor condition). (E) CD19 expression, determined as the number of molecules per cell, on NALM-6, Ramos, healthy B cells, or SKOV3gCD19+ singlet cells at different time points after coculture with CAR19-NK cells or 19scFv-NK cells, respectively (n=3 donors per coculture condition).(F) CD19 expression, determined as the number of molecules per cell, on singlet CD19+ primary B cells derived from either CLL (left panel) or ALL (right panel) patients (n=5 patients per experimental condition) at different time points after coculture with CAR19-NK cells or 19scFv-NK cells, respectively. P values ​​were determined by two-tailed, two-way ANOVA. ***P<0.0001. Data were evaluated by flow cytometry and presented as mean + sem.

[0052] [Figures 18A-18E] CAR-NK cell-mediated trogocytosis occurs in several targets and tumor cell types. Cognate TROG antigen expression on transduced singlet NK cells in (A) CAR5-NK cells cocultured with CD5+ CCRF tumor targets, (B) CAR70-NK cells cocultured with CD70+ THP-1 tumor targets, (C) CAR123-NK cells cocultured with CD123+ MOLM14 tumor targets, (D) CAR-BCMA-NK cells cocultured with BCMA+ MM1-S tumor targets, or (E) CAR-ROR1-NK cells cocultured with ROR1+ SKOV3 tumor targets. TROG antigen expression is shown as the ratio of TROG+ / TROG- NK cell populations at different time points during coculture (left panels in Figures 18A-E). Expression levels of cognate antigens, based on the number of surface molecules per cell, are shown for tumor cell lines before and after coculture with relevant CAR-NK cells (right panels in Figures 18A-18E). n = 3 NK cell donors per tumor condition. CAR19-NK cells (antigen-mismatched) and NT-NK cells were used as controls. P values ​​were determined by two-sided, two-way ANOVA. ***P < 0.0001. Data were assessed by flow cytometry and presented as mean + sem.

[0053] [Figures 19A-19I]NK cell-mediated trogocytosis of CAR-NK cells is influenced by the affinity of the CAR for its cognate antigen and is determined by different activation signaling endodomains. (A) Binding affinity of NK cells expressing CARs with different NK-binding affinities for CD70, containing the extracellular domain of CD27 and scFv70 derived from two different antibody clones (ARGX-110 or LB#14) (left panel), and TROG antigen expression on singlet CD70-targeted NK cells shown as the ratio of TROG+ / TROG- cell populations at different time points during coculture with RajiCD70+ cells (right panel) (n=3 donors per condition). (B-I) TROG antigen expression, shown as the ratio of TROG+ / TROG- cell populations, on singlet NK cells expressing anti-CD5 CARs bearing different intracellular signaling endodomains: (B) CD28 / CD3ζ, (C) CD3ζ, (D) DAP10 / CD3ζ, (E) DAP10 only (without CD3ζ), (F) NKG2D / CD3ζ, (G) 4-1BB / CD3ζ, (H) DAP12 / CD3ζ, or (I) DAP12 only (without CD3ζ) at different time points during coculture with Jurkat CD5+ cells (n = 3 donors per condition). NT-NK cells were used as a control. P values ​​were determined by two-way ANOVA. ***P < 0.0001. Data were evaluated by flow cytometry and presented as mean + sem.

[0054] [Figures 20A-20E]CAR19-mediated acquisition of tCD19 on NK cells from targeted Raji cells was associated with reduced antitumor cytotoxicity. (A) UMAP analysis of CAR-NK cells collected after coculture with RajiCD19+ cells. The phenotypic signature of all collected CAR-NK cells was assessed by mass cytometry, and data from 10,000 cells from three donors was merged to generate a single UMAP map. This analysis generated seven distinct color-coded clusters representing different NK cell subsets: (i) CD2+KIR+NKG2A-NKG2C+CD94-, (ii) CD2-NKG2A-NKG2C+CD94-, (iii) CD2-KIR+NKG2A+CD94+, (iv) CD2+KIR-NKG2A+NKG2C-CD94+, (v) CD2+NKG2A+NKG2C-CD94+KLRG1+, (vi) CD2+KIR+NKG2A+CD94+CD16-, and (vii) CD2+KIR+NKG2A+CD94+CD16+. Marker expression for each NK cell subset is shown in the UMAP plot (2B4, CD2, CD94, DAP12, KIR, KLRG-1, TRAIL, NKG2D, NKG2A, NKG2C, NKp30, NKp44, NKp46, and CD16, respectively). (B) Contour plots showing UMAP cluster abundance after 30 min, 1 h, 3 h, or 6 h of co-culture of CAR-NK cells alone or with Raji cells. The percentage of TROG+CAR-NK cells and tCD19 expression are shown for each subset for the different conditions. (C) Real-time images showing co-culture of CAR19-NK cells (green) with RajiCD19-mCherry cells (red). Black arrows (top row) indicate cell apoptosis events, yellow arrows (second and bottom rows, 4 and 20 min (top two arrows) conditions) indicate immune synapse-like structures, and white arrows (second and bottom rows, 12 min, 20 min (bottom left arrow), 34 min, 44 min, and 54 min conditions) indicate CAR19-NK cells with evidence of mCherry translocation. Scale bar indicates 10 µm.(D) Flow cytometry analysis showing co-expression of CD19 and mCherry on singlet RajiCD19-mCherry cells cultured alone ((i), representative of 3 samples) or on CAR19-NK cells after only 5 minutes of co-culture with RajiCD19-mCherry cells ((ii), representative of 5 donors). The adjacent graph shows the correlation between mCherry expression (determined as gMFI) and CD19 expression (determined as number of molecules per cell) for each singlet cell. (E) CD19 expression, determined as number of molecules per cell, and gMFI of mCherry expression on singlet RajiCD19-mCherry cells at different time points during co-culture with CAR19-NK cells (n = 3 donors). P values ​​were determined by Pearson's correlation coefficient in Figure 20D and by two-tailed Student's t-test in Figure 20E. ***P<0.0001. Data were evaluated by flow cytometry and presented as mean + sem in Figures 20D and 20E.

[0055] [Figures 21A-21J]Effect of repeated antigen-induced CAR activation on CAR-NK cell phenotype. (A) Tumor rechallenge assay in which CAR-19 NK cells were repeatedly challenged with RajiCD19-mCherry at different E:T ratios of 3:1 ((i)), 1:1 ((ii)), or 1:3 ((iii)). Tumor cells were added every 2 days (n = 5 donors), and tumor cell killing was measured by tumor cell index and the percentage (%) of caspase 3 / 7 events in RajiCD19-mCherry (B-D). (B-D) Incucyte analysis showing the percentage (%) of caspase 3 / 7 events in RajiCD19-mCherry after co-culture with CAR19-NK cells at E:T ratios of (B) 3:1, (C) 1:1, or (D) 1:3. Tumor cells were added every 2 days, and data were normalized to tumor cells alone. (E) tCD19-mCherry expression on singlet CAR19-NK cells is shown as the ratio of TROG+ / TROG- cells at different time points after re-challenge with RajiCD19-mCherry cells. (F) Incucyte analysis showing the percentage (%) of caspase 3 / 7 events in RajiCD19-mCherry cells after co-culture with CAR19-NK cells 6 days after re-challenge at E:T ratios of 3:1, 1:1, or 1:3, respectively, compared to co-culture with fresh CAR-NK cells. (G) Schematic illustrating the re-challenge assay in which CAR19-NK cells were repeatedly challenged with CD19-expressing Raji cells or autologous NK cells genetically modified to express CD19 (autoNKgCD19+ / GFP) at an E:T ratio of 1:3. Raji cells and autoNKgCD19+ target cells were modified to express GFP to facilitate their identification when cocultured with effector CAR19-NK cells (GFP-negative). (H) Flow cytometry analysis of CD19 expression on CAR19-NK cells cultured alone, on the TROG+ (tCD19+) and TROG- fractions of CAR19-NK cells after 1 hour of coculture with Raji cells, and on autoNKgCD19+ cells cultured alone.(I) Number of CD19 molecules per cell for CAR19-NK cells cultured alone, the TROG+ (tCD19+) and TROG- fractions of CAR19-NK cells after coculture with Raji cells, and autoNKgCD19+ cells cultured alone (n = 3 donors). (J) Percentage (%) of PD1, TIM3, and TIGIT coexpression (lower left panel) and the ratio of EOMES to Tbet (lower right panel) in CAR19-NK cells after several rounds of antigen challenge with RajiCD19+ / gGFP+ cells (circles: tumor targets) or autologous NK cells genetically modified to express both CD19 and intracellular GFP (NKgCD19+ / GFP+ cells; squares: autologous targets). CAR19-NK cells were evaluated after several days (D) of coculture with several rounds (R) of antigen challenge (e.g., D2R1 is day 2 after one round of antigen challenge). Representative histograms are shown for each marker expression. TT: tumor target (RajiCD19+), ST: autologous target (autoNKgCD19+) (n=5 donors per condition). P values ​​were determined by two-tailed two-way ANOVA for Figures 21A-F and by Student's t-test for Figures 21I and 21J. *P<0.01, **P<0.001, ***P<0.0001, ns not significant. Data were evaluated by flow cytometry and presented as mean + sem. Each symbol represents an individual donor.

[0056] [Figures 22A-22F]Repeated challenge of CAR19-NK cells with autoNKCD19+ cells results in hyporesponsiveness of CAR-NK cells. (A) tSNE analysis of live hCD45+GFP-CD56+CD3-CAR19-NK cells 4 days after a second round of antigen challenge with autoNKgCD19+gGFP+ cells. Controls included CAR19-NK cells cultured alone for 4 days or cocultured with autoNKgGFP+ cells (lacking CD19 expression) for 4 days. Phenotypic cell signatures for each condition were assessed by mass cytometry and merged to create a single t-SNE map (10,000 cells from 3 pooled donors per condition). Total cells were labeled (blue (i)), and CAR19 (green; (ii)) and tCD19 (orange; (iii)) expression was determined based on their expression on NT-NK cell controls. The numbers indicate the percentage (%) of CAR and tCD19 on CAR19-NK cells for each condition. (B) Kaplan-Meier plot showing the percentage (%) of apoptotic Raji cells after coculture with CAR-NK cells isolated after the first (day 2, left) or third (day 6, right) round of rechallenge with autoNKgCD19+ / GFP+ cells compared to fresh CAR-NK cells. Assays were performed in microwells at a 1:1 E:T ratio. Data from two donors were pooled. (C) Schematic of a single-cell time-lapse imaging cytotoxicity assay in which a single CAR-NK cell was cocultured with two Raji cells. Cell apoptosis was defined by Annexin V influx in the Raji cells. The bar graph on the right shows the percentage of CAR-NK cells that successfully lysed one (light) or two (dark) Raji cells isolated at different time points after rechallenge with autoNKgCD19+ / GFP+ cells. Fresh CAR-NK cells (pre) were used as a control (n = 2–4 donors).(D-F) Incucyte analysis showing the percentage (%) of caspase 3 / 7 events in Raji cells cocultured with CAR19-NK cells isolated after (D) the first, (E) the second, or (F) the third round of rechallenge of autoNKgCD19+ / GFP+ cells. Fresh CAR-NK cells and CAR-NK cells isolated after each round of rechallenge and cultured for 24 hours in complete medium supplemented with 100 U / mL IL-2 (referred to as "rested" cells) were used as controls (representative of three donors). P values ​​were determined by the log-rank test in Figure 22B, by a two-tailed paired Student's t-test in Figure 22C, and by two-tailed two-way ANOVA in Figures 22D, 22E, and 22F. *P<0.01, **P<0.001, ***P<0.0001. Data are presented as mean + sem.

[0057] [Figures 23A-23E]Trogocytosis of CAR19-NK cells in vivo and compensatory reduction of CD19 antigen expression on tumor cells. (A) Schematic diagram of the timeline using a mouse model implanted with Raji cells. Mice received three dose levels of Luc / GFP-expressing CD19+ Raji cells (0.2 × 10, 1 × 10, or 5 × 10 cells), followed by a single injection of CAR19-NK cells (1 × 10 cells) or NT-NK cells alone (1 × 10 cells) as a control. (B) Graph showing bioluminescence imaging (BLI) intensity over time for Raji cells alone (black), Raji cells + NT-NK cells (blue or light gray dashed lines), and Raji cells + CAR19-NK cells (green or dark gray solid lines). BLI is shown for days 0–7, 7–14, and 14–21. (C) tCD19 expression (shown as the TROG+ / TROG- ratio) on singlet hCD45+GFP-CD56+CD3- cells for CAR-NK cell products gated on the CAR19 fraction (dark gray; left bar) and the CAR-negative fraction (light gray; right bar) in peripheral blood samples collected at different time points after infusion (n = 15 mice per group). (D) CD19 expression on Raji cells, shown as molecule counts per cell, in the peripheral blood (left, n = 15 mice) and organs [spleen, liver, bone marrow (BM), and blood] (right, n = 24 mice) of mice at the end of CAR19-NK cell infusion. (E) CD19 expression on Raji cells, shown as molecule counts per cell, in organs harvested at the end of NT-NK cell infusion (n = 10 mice). P values ​​were determined by two-tailed one-way ANOVA for analysis. Data were evaluated by flow cytometry in samples with a count of >20 cell objects of interest and are shown as mean + sem. Each circle represents an individual mouse sample, and outliers are indicated by dark dots.

[0058] [Figures 24A-24B]CD19 expression on Raji cells recovered from Raji-bearing mice treated with CAR19-NK cells was reversible after short-term in vitro culture. (A) CD19 expression on Raji cells, shown as counts of CD19 molecules per cell, was serially performed on ex vivo cultured Raji cells harvested from the livers of Raji-bearing mice treated with CAR19-NK cells (n = 10 mice). Raji cells from untreated mice were used as a control. (B) Incucyte analysis (representative of 3 experiments) showing the percentage (%) of caspase 3 / 7 events in Raji cells harvested from CAR19-treated Raji-bearing mice and cocultured with fresh CAR19-NK cells or NT-NK cells either immediately after harvest (D0) or after 3 days of in vitro culture (D3). Coculture with fresh Raji cells serves as a control. P values ​​were determined by two-tailed one-way ANOVA in Figure 24A and by two-tailed two-way ANOVA in Figure 24B. ***P<0.0001. Data were evaluated by flow cytometry and presented as mean + sem. Each dot represents an individual mouse-derived Raji cell sample.

[0059] [Figures 25A-25D]In vivo trogocytosis was associated with limited persistence of CAR-NK cells. (A) Ratio of live CAR19-expressing NK cells to live CAR-negative NK cells in the peripheral blood of mice at different time points after CAR-NK cell infusion (left, n = 15 mice) and in different organs at the end point (right, n = 24 mice). (B.1 and B.2) tCD19 expression on NK cells and their viability in the CAR19-expressing and CAR-negative NK cell fractions based on TROG positivity (Q2: CAR19-NKTROG+, Q3: CAR-negative NKTROG+) in cells harvested from different organs (B.1 includes results from the spleen and liver, and B.2 includes results from the BM and blood) (representative example from n = 24 mice). The inset numbers indicate the percentage of cells within the indicated gated region. (C) tCD19 expression (shown as TROG+ / TROG- ratio) on singlet hCD45+GFP-CD56+CD3-CAR19-NK cells (dark gray, left bars) and CAR-negative NK cells (light gray, right bars) harvested from mouse organs at the end time point after CAR-NK cell infusion (left graph, n=24 mice), and in mice treated with NT-NK cells only (right graph, n=10 mice). (D) Percentage (%) of viable NKTROG+ (tCD19+, left graphs, (i) left bar or (ii) right bar) and NKTROG− cells (middle graphs, (iii) left bar or (iv) right bar) among CAR19-NK cells ((i) or (iii)) and CAR-negative NK cells ((ii) or (iv)) harvested from organs of mice (n=24 mice) at the end time point after CAR-NK cell infusion, or in mice treated with NT-NK cells (right graph: dark gray (v), n=10 mice). P values ​​were determined by two-tailed one-way ANOVA in Figure 25A (right panel) and by two-tailed Student's paired t-test in Figure 25A (left panel), Figure 25C, and Figure 25D. Data were evaluated by flow cytometry in samples with >20 counts of cell objects of interest and are presented as mean + sem. Each circle represents an individual mouse sample, and outliers are indicated by dark dots.

[0060] [Figures 26A-26F] CAR5-NK cell trogocytosis in vivo and compensatory reduction of CD5 antigen on CCRFCD5+ tumor cells. (A) Tumor burden was assessed weekly by BLI (gray) after CAR5-NK cell infusion. Mice implanted with CCRF tumors alone served as controls (black). Data were pooled from two independent experiments (n = 5 mice per group). (B) CD5 expression, expressed as gMFI, on implanted CCRF cells in the peripheral blood of mice at different time points after CAR5-NK cell infusion (left) and in organs harvested from mice at the end of the study (right). (C-D) Ratio of live CAR5-expressing NK cells to live non-CAR-expressing NK cells in the peripheral blood of mice at different time points after CAR5-NK cell infusion (C) and in organs harvested from mice at the end of the study (D). Data were pooled from two independent experiments (n = 5 mice per group). (E) tCD5 expression, shown as the TROG+ / TROG− ratio, on singlet hCD45+GFP-CD56+CD3- NK cells in the CAR5-expressing NK cell fraction ((i), left bar) and non-CAR-expressing NK cell fraction ((ii), right bar) in organs of mice harvested at the end time point after CAR5-NK cell infusion. (F) Percentage (%) of viable NKTROG+ (tCD5+; left), NKTROG− (right), and cells in the CAR5-expressing NK cell fraction (dark gray) and non-CAR-expressing NK cell fraction (light gray) harvested from organs of mice at the end time point after CAR5 NK cell infusion. P values ​​were determined by two-tailed one-way ANOVA for Figures 26B and 26D and by two-tailed Student's t-test for Figures 26C, 26E, and 26F. Data were evaluated by flow cytometry in samples with >20 counts of cell objects of interest, and are shown as mean + sem, pooled data from two independent experiments in which NK cells were derived from different donors. Each circle represents an individual mouse sample, and outliers are indicated by dark dots.

[0061] [Figures 27A-27E]CAR123-NK cell trogocytosis in vivo and compensatory reduction of CD123 antigen on MOLM-14 CD123+ tumor cells. (A) Kaplan-Meier curves showing the percent survival of MOLM-14-implanted mice after CAR123-NK cell infusion and without treatment (n=5 mice per group). Data from two independent experiments were pooled. (B) CD123 expression on implanted MOLM-14 cells in the peripheral blood of mice at different time points after CAR123-NK cell infusion (left) and in organs harvested from mice at the end of the study (right). (C) CAR123 expression on live hCD45+GFP-CD56+CD3- NK cells, shown as the ratio of CAR123-expressing NK cells to non-CAR-expressing NK cells, in the peripheral blood of mice at different time points after CAR123-NK cell infusion (left) and in organs harvested from mice at the end of the study (right). (D) tCD123 expression, shown as the TROG+ / TROG- ratio, on singlet hCD45+GFP-CD56+CD3- NK cells gated on CAR123-expressing NK cells (dark gray, left bar) and non-CAR-expressing NK cells (light gray, right bar) in organs of mice harvested at termination. (E) Percentage (%) of viable NKTROG+ cells (tCD123+; left) and NKTROG- cells (right) in the CAR123-expressing NK cell fraction (dark gray, left bar) and non-CAR-expressing NK cell fraction (light gray, right bar). P values ​​were determined by the log-rank test in Figure 27A, by two-tailed one-way ANOVA in Figures 27B and 27C, and by two-tailed Student's t-test in Figures 27D and 27E. Data were evaluated by flow cytometry in samples with >20 counts of cell objects of interest, and are shown as mean + sem, pooled from two independent experiments in which NK cells were derived from different donors. Each circle represents an individual mouse sample.

[0062] [Figures 28A-28G]In vivo trogocytosis was associated with poor survival of CAR-NK cells. (A) Schematic diagram of the timeline using a mouse model of lymphoma implanted with 0.2 × 10 luc / GFP-expressing CD19+ Raji cells and treated with a single injection of CAR19-NK cells or NT-NK cells as a control. Two weeks (days 13–15), three to four weeks (days 20–27), or termination (days 29–34) after injection, blood, BM, spleen, and liver were harvested for analysis. (B) Tumor burden was assessed weekly by BLI. BLI intensity is shown for each mouse after injection with CAR-NK cells (green or dark gray solid line) or NT-NK cells (blue or light gray dashed line). Untreated mice were used as controls (black). (C) Heatmaps depicting the expression levels of phenotypic and functional markers on the TROG+ (tCD19+) and TROG- hCD45+GFP-CD56+CD3- NK cell fractions at different time points after infusion. The expression level of each marker is represented by color, ranging from gray (low) to orange (high; positive Z-scores are indicated by a plus sign in the upper right corner of each circle, while negative Z-scores remain unmarked). The size of the circle indicates marker expression; the larger the circle, the higher the proportion of cells expressing the mentioned marker. (D) Phenotypic cell signatures for each condition were assessed by mass cytometry and merged to create a single t-SNE map. Expression of tCD19 (orange, (ii)) and CAR19 (green, (iii)) on hCD45+GFP-CD56+CD3- NK cells was determined based on their expression on NT-NK cell controls. (E) Violin plots showing tCD19 expression on NK cells within each cluster harvested from mice treated with CAR19-NK cells (left). Cisplatin levels within the TROG+ and TROG- fractions are shown for each cluster (right). (F) Violin plots showing tCD19 expression on NT-NK cells within each cluster (left). Cisplatin levels within the TROG+ and TROG- fractions are shown for each cluster (right). (G) Gene signatures for total hCD45+ cells at different time points during the treatment course.t-SNE maps generated with the Seurat package in R show color-coded expression levels of CD19 and MS4A1 (Raji cells), NKG7 and FCGR3A (NK cells) for each cluster (NK-C1, NK-C2, NK-C3, and Raji cells). P values ​​were determined by two-tailed Wilcoxon matched-pairs test in Figures 28E and 28F. **P<0.001, ***P<0.0001. Data were evaluated by mass cytometry, and median values ​​are displayed in the violin graph.

[0063] [Figure 29] Gating strategy for immunophenotyping of human PBMCs after CAR19-NK cell-based immunotherapy. PBMCs were isolated from patients receiving CAR-NK cell therapy and prepared for flow cytometry analysis as described in the "Methods" section. Single live cells were determined based on FSC / SSC selection and live / dead separation. Hematopoietic cells within the live population were then selected by gating on hCD45+CD33-CD14- cells. Differential expression of CD56, CD3, CD16, and CD19 was used to identify NK cell populations (CD56+CD16+CD3-), T cell populations (CD56-CD16-CD3+), or B cell populations (CD19+CD56-CD16-CD3-). Within the CD3-CD56+ subset, donor NK cells derived from umbilical cord blood were identified based on the expression of donor-specific HLA antigens. CAR expression on donor NK cells was further determined by CAR expression determined using an antibody against the CH2-CH3 domain of the human IgG hinge (109606088 / Jackson Immuno Rsch).

[0064] [Figure 30A-30B]iCAR design and effect on primary human NK cell trogocytosis. (A) Schematic illustration of viral vectors encoding different anti-CD19 iCARs. TM: transmembrane; SE: signaling endodomain. (B) tCD19 expression on NK cells transduced with CAR19, 19scFv, or different iCAR19 constructs, shown as the ratio of TROG+ cells / TROG- cells at different time points during coculture with RajiCD19+ cells (n=5 donors). P values ​​were determined by two-tailed, two-way ANOVA. *P<0.01, **P<0.001, ***P<0.0001. Data were evaluated by flow cytometry and presented as mean+sem.

[0065] [Figures 31A-31F]CS1 expression on Raji cells and NK cell populations. (A) CS1 expression on healthy human B cells (hCD45+CD56-CD3-CD19+CD20+), primary human NK cells (hCD45+CD56+CD3-CD19-CD20-), resting T cells (hCD45+CD56-CD3+CD19-CD20-), or CD3 / CD28 bead-activated T cells (n=3 donors). (B) Flow cytometry analysis of CS1 expression on Raji cells and primary human NK cells from five individual donors. (C) CS1 expression on Raji cells cultured alone, on Raji cells cocultured with CAR19-NK cells for 1 hour, and on the TROG+ (tCD19+) and TROG- fractions of CAR19- and non-CAR-expressing NK cells after coculture with Raji cells for 1 hour (n=5 donors). (D) Tumor burden in mice implanted with Raji cells was assessed weekly by BLI. BLI intensity is shown for each mouse after injection of CAR19-NK cells (light gray (ii)). Untreated mice were used as controls (black (i); n = 5 mice). (E) CS1 expression on Raji cells (i), CAR-negative NK cells (ii), or CAR19-NK cells (iii) in the blood of mice at different time points after injection (n = 10 mice). (F) CS1 expression on Raji cells harvested from mouse organs at the end of CAR19 NK cell infusion, as well as on the TROG+ (tCD19+) and TROG- fractions of CAR19-NK-expressing (left) or non-CAR-expressing (right) NK cell fractions; Raji (i), NKTROG- (ii), NKTROG+ (iii), CAR19-NKTROG- (iv), and CAR19-NKTROG+ (v); (n = 10 mice). CS1 expression was assessed by flow cytometry, determined by gMFI, and normalized to isotope control. Fold change 1 is indicated by a gray dashed line. P values ​​were determined by two-tailed one-way ANOVA. Data are shown as mean + sem. Each dot represents an individual mouse sample.

[0066] [Figures 32A-32F]AI-CAR-expressing NK cells exhibit excellent anti-tumor activity in vivo. (A and B) tCD19 expression (shown as TROG+ / TROG- populations) and viability of singlet NK cells harvested from the (A) peripheral blood and (B) BM of mice at different time points after injection of (A) 19scFv / CS1scFv NK cells (dark gray; left bar) or 19scFv / iCAR-CS1 NK cells (light gray; right bar) (n=5 mice per group). (C) tCD19 expression (shown as TROG+ / TROG- populations) and viability of singlet NK cells harvested from the BM of mice at different time points after injection of aCAR19 / CS1scFv NK cells (dark gray; left bar) or aCAR19 / iCAR-CS1 NK cells (light gray; right bar) (n=5 mice per group). (D) Live NK cell counts in blood, BM, spleen, and liver collected from mice on days 3, 10, and 20 after infusion of 19scFv / CS1scFv NK cells (light gray) or 19scFv / iCAR-CS1 NK cells (black, mean dark line) (n=5 mice per group). (E) Live NK cell counts collected from BM and liver of mice on days 3, 10, and 20 after infusion of aCAR19 / CS1scFv NK cells (dark gray; (i)) or aCAR19 / iCAR-CS1 NK cells (light gray; (ii)) (n=5 mice per group). (F) Levels of IL-15, granzyme A (GrA), GrB, and perforin in serum collected from mice on days 3, 10, and 20 after infusion of CAR-expressing NK cells: Raji only ((i)), Raji + 19scFv / CS1-scFv-NK ((ii)), Raji + 19scFv / iCAR-CS1-NK ((iii)), Raji + aCAR19 / CS1scFv-NK ((iv)), and Raji + aCAR19 / iCAR-CS1-NK ((v)); (n=5 mice per group). P values ​​were determined by two-tailed Student's t-test in Figures 32A-E and by two-tailed two-way ANOVA in Figure 32F. **P<0.001, ***P<0.0001, nd: not detected.Data were assessed by flow cytometry and presented as mean + sem in Figures 32A-E, with each circle representing an individual mouse sample.

[0067] [Figures 33A-33I]AI-CAR-expressing NK cells exhibit excellent in vivo antitumor activity in the SKOV3gCD19+ ovarian cancer model. (A) Schematic diagram of the timeline using a mouse model of ovarian cancer implanted with 0.5 × 106 SKOV3 tumor cells genetically modified to express CD19 (SKOV3gCD19+). Seven days later, mice were injected once with 1 × 107 NK cells expressing aCAR19 / CS1scFv (dark gray, center column) or aCAR19 / iCAR-CS1 (light gray, right column), or no NK cells were injected (black; left column, tumor-only control group) (n = 5 mice per group). (B and C) Tumor burden was determined weekly by BLI. (B) Representative images at selected time points are shown. (C) Normalized BLI intensity for each treatment group over the course of treatment; dashed lines represent data for each mouse. (D) Kaplan-Meier curves showing mouse survival rates after NK cell injection. (E and F) Viability of (E) tCD19 (shown as TROG+ / TROG- populations) and (F) TROG+ fraction (NKtCD19+) of NK cells in peripheral blood of mice at days 5, 15, and 30 after infusion of aCAR19 / CS1scFv NK cells ((i)) or aCAR19 / iCAR-CS1 NK cells ((ii)) (n=5 mice per group). (G) Percentage (%) of live GFP-CD3-CD56+CAR19+ NK cells in peripheral blood of mice at days 5, 15, and 30 after infusion of aCAR19 / CS1scFv NK cells ((i)), left) or aCAR19 / iCAR-CS1 NK cells ((ii)), right) (n=5 mice per group). (H) Live NKCAR19+ NK cell counts in the peripheral blood of mice on days 5, 15, and 30 after infusion of aCAR19 / CS1scFv NK cells ((i)) or aCAR19 / iCAR-CS1 NK cells ((ii)) (n=5 mice per group). (I) Representative images showing H&E staining and IHC staining with anti-luciferase, anti-hCD45, or anti-hROR1 antibodies on sections from mesenteric tissue of SKOV3ROR1+ transplanted mice treated with aCAR-ROR1 / CS1-scFv NK cells or aCAR-ROR1 / iCAR-CS1 NK cells.The numbers indicate hCD45+ cell counts per 0.1 mm. The black arrow (right panel) indicates ROR1 expression on tumor cells, and the light gray arrow (right panel) indicates ROR1 expression on NK cells. The scale bar indicates 100 μm. P values ​​were determined by two-tailed one-way ANOVA in Figure 33C, by log-rank test in Figure 33D, by two-tailed Student's t-test in Figures 33E, 33F, 33H, and 33I, and by two-tailed one-way ANOVA in Figure 33G. *P<0.01, **P<0.001, ***P<0.0001, ns: not significant. In Figures 33C and 33D, data from two independent experiments in which NK cells were derived from different donors were pooled. In Figures 33E, 33F, 33G, and 33H, data were evaluated by flow cytometry and are presented as mean + sem. Each circle represents an individual mouse sample.

[0068] [Figure 34A-34B] Model of AI-CAR NK cell function. (A) aCAR-NK cell-mediated trogocytosis results in a decrease in antigen density on tumor cells, promoting CAR-NK cell fratricide and hyporesponsiveness. (B) Engineering NK cells to express both an aCAR against a tumor antigen and a KIR-based inhibitory CAR (iCAR) against an NK self-antigen prevents TROG-induced self-recognition and TROG antigen-mediated fratricide of CAR-NK cells while retaining their on-target tumor recognition and cytotoxicity.

[0069] [Figures 35A-35D]Self-association of TROG+ CAR-NK cells resulted in NK cell fratricide. (A and B) Schematic diagram (left panel) illustrating the single-cell time-lapse imaging cytotoxicity / viability assay (right panel). Time was recorded over 5 hours (T0–T300 min) from the start of co-culture, in which (A) one single cell or (B) two cells of non-TROG antigen-expressing fresh CAR-NK cells (control; gray (i)) or CAR-NK TROG+ cells (dark gray (ii)) were incubated in each nanowell. The amount of time required to detect Annexin V influx in sorted CAR-NK cells during the assay period was determined as the time required to induce cell apoptosis. Kaplan-Meier curves (right panel) show the percent (%) of apoptosis in CAR-NK cells during incubation. (C) Schematic diagram of the experimental design (top left panel). TROG+CAR19-NK cells were purified, and their phenotypic signatures were assessed by mass cytometry before and after 5 hours of culture. Data from 10,000 cells from three donors per condition were merged to create a single UMAP map containing distinct color-coded clusters representing different CAR-NK cell subsets. Marker expression (NKp30, NKp44, TRAIL, CD94, CD2, CD16, 2B4, NKp46, DAP12, KIR, KLRG-1, NKG2A, NKG2C, and NKG2D) for each NK cell subset is shown. (D) UMAP plot showing the expression of the TROG antigen (tCD19) on CAR-NK cells before (left) and after 5 hours of culture with sister cells (right). Contour plots show the abundance of each CAR-NK cell subset before and after 5 hours of culture, with the percentage of each subset also shown. Changes in the CD2+CD16+NKG2A+NKG2C+CD94+, CD2-KIR+NKG2C+CD94+, and CD2-KIR+NKG2A+NKG2C-CD94+ cell populations were observed. P values ​​were determined by the log-rank test in Figures 35A and 35B. Detailed Description of the Invention

[0070] Trogocytosis is a well-known phenomenon in which lymphocytes shed plasma membrane fragments from their targets after immune synapse formation (see, e.g., Joly & Hundrisier 2003, Dance 2019, and Ahmed et al., 2008). Throughout this disclosure, evidence is provided for a unique mechanism of tumor escape following CAR-effector cell therapy, specifically evidence that activation of activated CARs (aCARs) drives the transfer of target antigens from target cells engaged but not killed by effector cells to effector cells. In some embodiments, effector cells that acquire target antigens (referred to herein as TROG antigens) by trogocytosis (TROG) in turn become target cells (antigen-induced self-recognition), and such cells are lysed by other aCAR-effector cells, and those not killed by fratricide become hyporesponsive, similar to reports for CAR-T cells (see, e.g., Hamieh et al., 2019, and Wang et al., 2021). In some embodiments, this phenomenon is accompanied by the concomitant loss of target antigens on cancer cells, reducing their susceptibility to aCAR-mediated killing and thus increasing the risk of tumor recurrence in subjects. Also described and demonstrated herein are novel inhibitory CAR (iCAR) systems that combine at least one extracellular domain targeting an effector cell-specific antigen with one or more signaling endodomains from inhibitory killer Ig-like receptors (KIRs). These novel iCAR systems successfully achieve aCAR-mediated antigen-specific suppression of effector cell fratricide and hyporesponsiveness while preserving the on-target antitumor activity of effector cells and demonstrating improved persistence and / or activity in multiple in vitro and in vivo models.

[0071] Intercellular protein transfer is mediated by multiple pathways (see, e.g., Dance 2019, Griffiths & Tsun 2010, Hochreiter-Hufford & Ravichandran 2013, and Kalluri & LeBleu 2020). Among these pathways, trogocytosis is a well-known mechanism for the exchange of surface proteins between NK cells and their targets (see, e.g., Miyake & Karasuyama 2021, Tabiasco et al. 2002, and Tabiasco et al. 2003). In addition, trogocytosis has been shown to significantly affect NK cell function (see, e.g., Caumartin et al. 2007, Nakamura et al. 2013(b), Domaica et al. 2009, and Nakayama et al. 2011). For example, NKG2D-mediated trogocytosis of NKG2D ligands has been associated with NK cell hyporesponsiveness and fratricide (see, e.g., Nakamura et al., 2013(a), Miner et al., 2015, and Nakamura et al., 2013(b)). Similarly, trogocytosis triggered by monoclonal antibody engagement of the CD16 receptor on NK cells leads to target antigen modulation and impaired therapeutic efficacy (see, e.g., Carlsten et al., 2016, Taylor & Lindorfer 2015, and Beum et al., 2008). Recent studies have reported how trogocytosis promotes reduced antigen density and T cell exhaustion and fratricide after CAR-T cell therapy (see, e.g., Hamieh et al., 2019). This disclosure is the first to demonstrate that human CAR-NK cells acquire their cognate antigen targets from tumor cells by antigen-specific trogocytosis, which requires CAR activation and signaling. This phenomenon has been observed with CARs that express different CAR signaling endodomains and target multiple antigens across multiple cancer types. In some embodiments, the extent of trogocytosis is influenced by the affinity of the CAR for its cognate ligand and / or the density of the antigen on the tumor cell.Given that TROG antigen-expressing NK cells are susceptible to aCAR-NK cell cytotoxicity due to induced self-recognition, in some embodiments, preventing trogocytosis may be a beneficial approach to improving the potency and / or in vivo persistence of aCAR-NK cells.

[0072] To date, there are no therapeutically applicable strategies to specifically control trogocytosis. Herein, we demonstrate aCAR-mediated recognition of TROG antigen-expressing NK cells (TROG) while preserving aCAR activity against tumor targets. + We describe and demonstrate a novel engineering approach that harnesses NK cell biology and utilizes ITIM-containing iCARs to suppress NK cell on-target / off-tumor recognition. In some embodiments, by combining the activities of two chimeric receptors—one that generates a dominant negative signal upon recognition of an NK-specific antigen and one that induces an activating signal upon engagement with a tumor target—we can successfully switch off the countervailing aCAR activation response to the TROG antigen on NK cells while preserving effector cell activity against tumor targets. In some embodiments, NK cells transduced with this AI-CAR system were less susceptible to TROG antigen-mediated fratricide and exhaustion and mediated superior antitumor responses in both in vitro and multiple in vivo models (see Figures 34A and 34B for graphical representations).

[0073] This disclosure describes and demonstrates how aCAR-mediated trogocytosis, which contributes to reduced target antigen density and effector cell fratricide and hyporesponsiveness, contributes as a novel mechanism for disease recurrence after aCAR cell therapy. Multiple examples and proofs of concept are provided herein suitable for use in rescuing aCAR effector cell (e.g., aCAR-NK cell) function from the consequences of TROG antigen-induced immunomodulation using antigen-specific iCARs that successfully inhibit aCAR-mediated TROG antigen-induced effector cell fratricide and exhaustion while preserving essential effector function against tumor cells expressing the same antigen. This dynamic modulation of iCAR signaling can be applied to at least improve the in vivo persistence and therapeutic efficacy of a range of adoptive effector cell therapies for various diseases, including, but not limited to, cancer (e.g., hematological or solid tumors), infectious diseases (e.g., viral, bacterial, fungal, or parasitic infections), autoimmune disorders, and genetic disorders. I. Definition

[0074] As used herein, "a" or "an" can mean one or more. In the claims, when the word "a" is used with the word "comprising," it can mean one or more. As used herein, "another" can mean at least a second, or more. Furthermore, those of skill in the art will recognize that the terms "having," "including," "containing," and "comprising" are interchangeable and are open-ended terms. In specific embodiments, aspects of the present disclosure can, for example, "consist essentially of" or "consist of" one or more sequences of the present disclosure. Some embodiments of the present invention consist of, or consist essentially of, one or more elements, method steps, and / or methods of the present disclosure. It is contemplated that any method and composition described herein can be implemented with respect to any other method or composition described herein. The scope of the present application is not limited to the particular embodiments of the process, machine, manufacture, composition of matter, means, methods, and steps described in the specification. As used herein, the terms "or" and "and / or" are used to describe multiple elements in combination or mutually exclusive. For example, "x, y, and / or z" can refer to "x" alone, "y" alone, "z" alone, "x, y, and z," "(x and y) or z," "x or (y and z)," or "x or y or z." It is specifically contemplated that x, y, or z may be specifically excluded from an embodiment.

[0075] Use of the term "or" in the claims is used to mean "and / or" unless expressly stated to refer to alternatives only or the alternatives are mutually exclusive. However, this disclosure supports definitions that refer to alternatives only and to "and / or." As used herein, "another" can mean at least a second, or more. The terms "about," "substantially," and "approximately" generally mean plus or minus 5% of the stated value.

[0076] Throughout this specification, references to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "certain embodiments," "additional embodiments," or "further embodiments," or combinations thereof, mean that the particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the disclosure. Thus, the appearances of such phrases in various places throughout the specification are not necessarily all referring to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0077] As used herein, the term "engineered" refers to an entity created by the hand of man, including cells, nucleic acids, polypeptides, vectors, combinations thereof, and the like. In at least some cases, an engineered entity is synthetic and includes elements that do not occur in nature or are not organized in the manner utilized in this disclosure. With respect to cells, the cells may be engineered because they express one or more heterologous genes (such as synthetic antigen receptors and / or cytokines) and / or because they have reduced expression of one or more endogenous genes, in which case their engineering was entirely by the hand of man. With respect to antigen receptors, an antigen receptor may be considered engineered because it contains multiple components that have been genetically engineered to be organized in a manner not found in nature, for example, in the form of a fusion protein of components not found in such an organization in nature.

[0078] "Treating" a disease or condition or treatment of a disease or condition refers to the implementation of a protocol in an attempt to alleviate the signs or symptoms of the disease, which may include administering one or more drugs to the patient. Desirable effects of treatment include reducing the rate of disease progression, ameliorating or alleviating the disease state, and remission or improved prognosis. Alleviation can occur before or after the signs or symptoms of the disease or condition appear. Thus, "treating" or "treatment" can include "preventing" or "preventing" a disease or undesirable condition. In addition, "treating" or "treatment" does not require complete alleviation of signs or symptoms, does not require a cure, and specifically includes protocols that have only a marginal effect on the patient.

[0079] As used throughout this application, the term "therapeutic benefit" or "therapeutically effective" refers to anything that promotes or enhances the well-being of a subject with respect to medical treatment of that condition. This includes, but is not limited to, reducing the frequency or severity of signs or symptoms of a disease. For example, treating cancer can involve, for example, reducing tumor size, reducing tumor invasiveness, reducing the rate of cancer growth, or preventing metastasis. Treating cancer can also refer to increasing the survival time of a subject with cancer.

[0080] "Subject" and "patient" or "individual" refers to either a human or non-human, e.g., primates, mammals, and vertebrates. In certain embodiments, the subject is a human.

[0081] As used herein, a "mammal" is a suitable subject for the methods of the present invention. A mammal may be any member of the higher vertebrate class Mammalia, including humans, and is characterized by production, hair, and mammary glands in females that secrete milk to feed their young. In addition, mammals are characterized by their ability to maintain a constant body temperature despite changing climatic conditions. Examples of mammals are humans, cats, dogs, cows, mice, rats, horses, goats, sheep, and chimpanzees. A mammal may also be referred to as a "patient" or "subject" or "individual."

[0082] The phrases "pharmaceutically or pharmacologically acceptable" refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal, e.g., a human, as appropriate. The preparation of pharmaceutical compositions containing an antibody or additional active ingredient will be known to those of skill in the art in light of the present disclosure. Furthermore, it will be understood that if administered to an animal (e.g., a human), preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by the FDA's Office of Biological Standards.

[0083] As used herein, "pharmaceutically acceptable carriers" include any and all aqueous solvents (e.g., water, alcoholic / aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters such as ethyl oleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, and inert gases), isotonicity agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, liquid and nutritional supplements, other materials, and combinations thereof, as would be known to one of ordinary skill in the art. The pH and precise concentrations of the various components in a pharmaceutical composition are adjusted according to well-known parameters.

[0084] As used herein, the term "disruption" of a gene refers to the elimination or reduction of the expression of one or more gene products encoded by a gene of interest in a cell, compared to the level of expression of the gene product in the absence of the disruption. Exemplary gene products include mRNA and protein products encoded by the gene. The disruption may be transient or reversible in some cases, and permanent in other cases. The disruption may be of a functional or full-length protein or mRNA, despite the fact that in some cases, a truncated or non-functional product may be produced. In some embodiments herein, the activity or function of a gene, rather than its expression, is disrupted. Gene disruption is generally induced by artificial means, i.e., by the addition or introduction of a compound, molecule, complex, or composition, and / or by disrupting the nucleic acid of the gene or nucleic acid associated with the gene, for example, at the DNA level. Exemplary methods for gene disruption include gene disruption techniques such as gene silencing, knockdown, knockout, and / or gene editing. Examples include antisense technologies, such as RNAi, siRNA, shRNA, and / or ribozymes, which generally result in a transient reduction in expression, and gene editing techniques that result in the inactivation or targeted disruption of a target gene, for example, by inducing cleavage and / or homologous recombination. Examples include insertions, mutations, and deletions. Disruptions typically result in the suppression and / or complete absence of expression of the normal or "wild-type" product encoded by the gene. Examples of such gene disruptions include insertions, frameshift and missense mutations, deletions, knock-in of a gene or part of a gene, and knockouts, including the deletion of the entire gene. Such disruptions can occur within the coding region, for example, in one or more exons, and can prevent the production of a full-length product, a functional product, or any product, for example, by inserting a stop codon. Such disruptions can also occur through disruptions in promoters or enhancers or other regions that affect transcriptional activation to prevent gene transcription.Gene disruption involves gene targeting, such as inactivation of a targeted gene by homologous recombination.

[0085] As used herein, the term "xenogeneic" refers to a cell type different from that of the recipient or derived from a different species. In specific instances, it refers to a gene or protein that is synthetic and / or not derived from an NK cell. The term also refers to a synthetically derived gene or gene construct. The term also refers to a synthetically derived gene or gene construct. For example, a cytokine may be considered xenogeneic to an NK cell because it is synthetically derived, such as by genetic recombination, including providing the NK cell with a vector containing a nucleic acid sequence encoding the cytokine, even if the cytokine is naturally produced by the NK cell.

[0086] The present disclosure provides methods and compositions for cell therapy that are cytotoxic to cells that need to be killed, e.g., cancer cells. The cells of the cell therapy contain a mechanism that acts as an inhibitory signal for the cell therapy when cytotoxicity of the cells needs to be prevented. In certain embodiments, the inhibitory signal acts under conditions in which the cytotoxic cell therapy kills cells that are not the intended target, e.g., cells that are not desired to be killed. In specific embodiments, the cells that are not the intended target are non-cancerous cells. In specific embodiments, the cells that are not the intended target have acquired, by trogocytosis, an antigen that would not otherwise be expressed by the cell, at least to a detectable extent. In some cases, the cells of the cell therapy have acquired an antigen by trogocytosis, and the antigen becomes a target for destruction by other cells of the cell therapy (which may or may not have also acquired the antigen by trogocytosis). The present disclosure provides methods and compositions that use this inhibitory signal to prevent hyporesponsiveness induced by fratricide and / or self-association between cells of the cell therapy. In certain embodiments, the inhibitory signal is an inhibitory CAR (iCAR).

[0087] Trogocytosis is an active cellular process involving the transfer of surface materials from one cell to another, mediated by constitutive ligand-induced and receptor-mediated endocytosis and recycling processes. CAR-mediated trogocytosis has been reported to suppress the antitumor cytotoxicity of CAR-T cells by mediating fratricide and exhaustion (see, e.g., Hamieh et al., 2019). The present disclosure provides CAR-NK cells (e.g., as an example of cell therapy) engineered to express a dual CAR system, including at least one activating CAR (aCAR) and at least one iCAR that recognizes an antigen on another cell and, as a result of that antigen recognition, directs the iCAR to inhibit the cytotoxic activity of the antigen-expressing cell through the activity of the CAR.

[0088] This technology can be applied to reduce on-target and off-tumor toxicity of CAR T cells or CAR NK cells targeting solid tumor antigens that are also expressed on normal tissues (e.g., mesothelin, which is also expressed on lung epithelium). That is, because iCARs exert their inhibitory function in an antigen-specific manner, iCARs can target antigens specifically expressed on normal tissues. In combination with iCAR engineering, CAR-expressing immune effector cells can distinguish off-tumor targets from on-tumor targets, which would significantly improve their therapeutic efficacy and / or reduce unwanted side effects. II. Inhibitory CARs and their constructions and uses

[0089] In some embodiments, an iCAR of the present disclosure encompasses a single polypeptide comprising one or more extracellular antigen-binding domains, a transmembrane domain, and one or more intracellular inhibitory signaling domains. In some embodiments, the inhibitory signaling domain, upon binding of the antigen targeted by the iCAR, signals inhibition of the cell expressing the iCAR, including by inhibiting an activated CAR also expressed by the cell. In some embodiments, the inhibitory signaling domain is derived from the natural inhibitory signaling of NK cells. That is, in some cases, the inhibitory signaling domain is an inhibitory signaling domain found in natural NK cell inhibitory receptors (which act via an immunoreceptor tyrosine-based inhibitory motif). In some embodiments, upon binding of the iCAR to the antigen targeted by the iCAR, the inhibitory signaling domain from the NK cell receptor inhibits the activity of the corresponding activated CAR (aCAR), regardless of whether the dual CAR system is in an NK cell or another type of cell, such as a T cell. Examples of inhibitory signaling domains include, but are not limited to, any of the signaling domains of killer Ig-like receptors (KIR), leukocyte immunoglobulin-like receptors (LIR-1; also known as LILRB1), CD300A, NKG2A, Siglec-7, CD96, TIM3, TIGIT, and / or LAIR-1. Examples of KIR include at least KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, KIR3DL3, KIR2DL5A, and KIR2DL5B. In some embodiments, one or more co-inhibitory domains are utilized in addition to or in place of one or more inhibitory signaling domains. In specific embodiments, the co-inhibitory domain is derived from LAIR-1, NKG2A, CD300A, or a combination thereof.In some embodiments, when two or more inhibitory signaling domains are utilized, the order from N-terminus to C-terminus can be in any order.

[0090] In specific embodiments, an iCAR targets one or more antigens. In some embodiments, when an iCAR targets two or more antigens, the two or more antigens can be non-identical. In specific embodiments, the choice of antigen targeted by the antigen-binding domain of an iCAR depends on the cells that require protection from immune effector cells expressing the iCAR and aCAR. In some embodiments, the cells requiring protection may or may not be the same type of cell as the immune effector cells (e.g., both NK cells, both T cells, or one type of each). In specific embodiments, the antigen is selected because it is self-antigen to NK cells, such that the iCAR becomes an NK self-recognition inhibitory iCAR that recognizes self-antigens and transmits a "don't kill me" signal to NK immune effector cells, for example, when associated with their sister cells. Such an inhibitory mechanism prevents or reduces fratricide and / or exhaustion, while still allowing the aCAR to target and kill cancer cells that express tumor antigens. Examples of NK autoantigens include, but are not limited to, at least KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL5, KIR3DL1, KIR3DL2, KIR3DL3, KIR2DL4, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DS1, DAP10, DAP12, CD56, CD57, CD25, CD122, NKP30, NKP44, NKP46, NKG2C, NKG2D, NKG2A, CRTAM, TIGIT, CD96, 2B4, CD16, CD27, CD100, CD160, ILT2, ILT4, KLRG1, LAIR1, CD161, CS1, (natural cytotoxicity receptor) NCR, KIR, and / or other NK-associated antigens.

[0091] In one specific example, an iCAR comprises two antigen-binding domains, each specific for a different NK cell autoantigen, such as an antigen-binding domain targeting CS1 and an antigen-binding domain targeting CD56. In any case where two antigen-binding domains are used in an iCAR, the order of the first and second antigen-binding domains can be in any order from N-terminus to C-terminus. In a specific embodiment, when two antigen-binding domains are used in an iCAR, a spacer of appropriate length and content can be present between the two antigen-binding domains. In either case, the antigen-binding domain of an iCAR can comprise an antibody or functional fragment thereof (e.g., an scFv or single-domain antibody) that binds to the antigen, or the antigen-binding domain can be a natural ligand in some cases. The extracellular antigen-binding domain can be associated with any type of hinge, such as a hinge derived from IgG1, CD28, CD8 alpha, etc.

[0092] With respect to the transmembrane domain (TM) of iCAR, the TM can be of any suitable type. In specific embodiments, the TM may or may not be derived from the same molecule as the inhibitory signaling domain or co-inhibitory domain. In specific embodiments, the TM is derived from KIR2DL1 or LIR-1, but can also be derived from the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS / CD278, GITR / CD357, NKG2D, and DAP molecules.

[0093] Example of iCAR signaling construct: By utilizing the natural inhibitory pathway that prevents NK cells from inflicting cytotoxicity against normal antigens, the inventors designed a dual CAR system containing at least one iCAR. In a specific embodiment, an scFv against a normal antigen or an autoantigen is selectively conjugated to the transmembrane domain (TM or TMD), intracellular inhibitory domain (ID; also referred to as inhibitory signaling domain), and intracellular co-inhibitory domain (CID) of KIR2DL1, LIR1, LAIR-1, NKG2A, and CD300A. Examples of combinations of TMD and intracellular domain (ICD, which may also include ID and CID) are as follows:

[0094] KIR2DL1 (TMD)- KIR2DL1 (ICD)

[0095] KIR2DL (TMD)- KIR2DL1 (ICD)- Siglec-7 (ICD)

[0096] KIR2DL1 (TMD)- KIR2DL1 (ICD)- LAIR-1 (ICD)

[0097] KIR2DL1 (TMD)- KIR2DL1 (ICD)- NKG2A(ICD)

[0098] KIR2DL1 (TMD)- KIR2DL1 (ICD)-CD300A(ICD)

[0099] KIR2DL2 (TMD)- KIR2DL2 (ICD)

[0100] KIR2DL2 (TMD)- KIR2DL2 (ICD)- Siglec-7 (ICD)

[0101] KIR2DL2 (TMD)- KIR2DL2 (ICD)- LAIR-1 (ICD)

[0102] KIR2DL2 (TMD)- KIR2DL2 (ICD)- NKG2A(ICD)

[0103] KIR2DL2 (TMD)- KIR2DL2 (ICD)-CD300A(ICD)

[0104] KIR2DL3 (TMD)- KIR2DL3 (ICD)

[0105] KIR2DL3 (TMD)- KIR2DL3 (ICD)- Siglec-7 (ICD)

[0106] KIR2DL3 (TMD)- KIR2DL3 (ICD)- LAIR-1 (ICD)

[0107] KIR2DL3 (TMD)- KIR2DL3 (ICD)- NKG2A(ICD)

[0108] KIR2DL3 (TMD)- KIR2DL3 (ICD)-CD300A(ICD)

[0109] KIR3DL1 (TMD)- KIR3DL1 (ICD)

[0110] KIR3DL1 (TMD)- KIR3DL1 (ICD)- Siglec-7 (ICD)

[0111] KIR3DL1 (TMD)- KIR3DL1 (ICD)- LAIR-1 (ICD)

[0112] KIR3DL1 (TMD)- KIR3DL1 (ICD)- NKG2A(ICD)

[0113] KIR3DL1 (TMD)- KIR3DL1 (ICD)-CD300A(ICD)

[0114] KIR3DL2 (TMD)- KIR3DL2 (ICD)

[0115] KIR3DL2 (TMD)- KIR3DL2 (ICD)- Siglec-7 (ICD)

[0116] KIR3DL2 (TMD)- KIR3DL2 (ICD)- LAIR-1 (ICD)

[0117] KIR3DL2 (TMD)- KIR3DL2 (ICD)- NKG2A(ICD)

[0118] KIR3DL2 (TMD)- KIR3DL2 (ICD)-CD300A(ICD)

[0119] KIR3DL3 (TMD)- KIR3DL3 (ICD)

[0120] KIR3DL3 (TMD)- KIR3DL3 (ICD)- Siglec-7 (ICD)

[0121] KIR3DL3 (TMD)- KIR3DL3 (ICD)- LAIR-1 (ICD)

[0122] KIR3DL3 (TMD)- KIR3DL3 (ICD)- NKG2A(ICD)

[0123] KIR3DL3 (TMD)- KIR3DL3 (ICD)-CD300A(ICD)

[0124] KIR2DL4 (TMD)- KIR2DL4 (ICD)

[0125] KIR2DL4 (TMD)- KIR2DL4 (ICD)- Siglec-7 (ICD)

[0126] KIR2DL4 (TMD)- KIR2DL4 (ICD)- LAIR-1 (ICD)

[0127] KIR2DL4 (TMD)- KIR2DL4 (ICD)- NKG2A(ICD)

[0128] KIR2DL4 (TMD)- KIR2DL4 (ICD)-CD300A(ICD)

[0129] KIR2DL5 (TMD)- KIR2DL5 (ICD)

[0130] KIR2DL5 (TMD)- KIR2DL5 (ICD)- Siglec-7 (ICD)

[0131] KIR2DL5 (TMD)- KIR2DL5 (ICD)- LAIR-1 (ICD)

[0132] KIR2DL5 (TMD)- KIR2DL5 (ICD)- NKG2A(ICD)

[0133] KIR2DL5 (TMD)- KIR2DL5 (ICD)-CD300A(ICD)

[0134] NKG2A (TMD)-NKG2A(ICD)

[0135] CD300A (TMD)-CD300A(ICD)

[0136] LAIR-1 (TMD)-LAIR-1 (ICD)

[0137] Siglec-7(TMD)- Siglec-7 (ICD)

[0138] CD96(TMD)- CD96 (ICD)

[0139] TIM-3(TMD)- TIM3(ICD)

[0140] TIGIT(TMD)- TIGIT(ICD)

[0141] NKG2A (TMD)-NKG2A(ICD)- LAIR-1 (ICD)

[0142] NKG2A (TMD)-NKG2A(ICD)- CD300A(ICD)

[0143] NKG2A (TMD)-NKG2A(ICD)- LAIR-1 (ICD)

[0144] NKG2A (TMD)-NKG2A(ICD)- Siglec-7 (ICD)

[0145] NKG2A (TMD)-NKG2A(ICD)- CD96 (ICD)

[0146] NKG2A (TMD)-NKG2A(ICD)-TIM-3(ICD)

[0147] NKG2A (TMD)-NKG2A(ICD)- TIGIT (ICD)

[0148] LAIR-1 (TMD)- Siglec-7 (ICD)

[0149] LAIR-1 (TMD)- CD96 (ICD)

[0150] LAIR-1 (TMD)- TIM-3 (ICD)

[0151] LAIR-1 (TMD)- TIGIT (ICD)

[0152] Siglec-7(TMD)- CD96 (ICD)

[0153] Siglec-7(TMD)- TIM-3 (ICD)

[0154] CD96(TMD)- TIM3 (ICD)

[0155] CD96(TMD)- TIM3 (ICD)

[0156] CD96 (TMD)- TIGIT(ICD)

[0157] The sequences of each part of an example iCAR are shown below. SEQ ID NO: 1 - iCAR1: KIR2DL1 (TM)- KIR2DL1 (ID) nucleotide sequence: attctgattgggacctcagtggtcatcatcctcttcatcctcctcttcttttctccttcatcgctggtgctccaacaaaaaaaatgctgcggtaatggaccaagagtctgcaggaaacagaacagcgaatagcgaggactctgatgaacaagacc ctcaggaggtgacatacacacagttgaatcactgcgttttcacacagagaaaaatcactcgcccttctcagaggcccaagacacccccaacagatatcatcgtgtacacggaacttccaaatgctgagtccagatccaaagttgtctcctgccca (SEQ ID NO:1) SEQ ID NO: 2 - iCAR1: KIR2DL1 (TM)- KIR2DL1 (ID) peptide sequence: ILIGTSVVIILFILLFFLLHRWCSNKKNAAVMDQESAGNRTANSEDSDEQDPQEVTYTQLNHCVFTQRKITRPSQRPKTPPTDIIVYTELPNAESRSKVVSCP (SEQ ID NO:2) SEQ ID NO: 3 - iCAR2: LIR-1 (TM)- LIR-1 (ID) nucleotide sequence: gttgtgatcggcatcttggtggccgtcatcctactgctcctcctcctcctcctcctcttcctcatcctccgacatcgacgtcagggcaaacactggacatcgacccagagaaaggctgatttccaacatcctgcaggggctgtggggccagagcccacagacagaggcctgcagtggaggtccagcccagctgccgatgcccaggaagaaaacctctatgctgccgtgaagcacacacagcctgaggatggggtggagatggacactcggcagagcccacacgatgaagacccccaggcagtgacgtatgccgaggtgaaacactccagacctaggagagaaatggcctctcctccttccccactgtctggggaattcctggacacaaaggacagacaggcggaagaggacaggcagatggacactgaggctgctgcatctgaagccccccaggatgtgacctacgcccagctgcacagcttgaccctcagacgggaggcaactgagcctcctccatcccaggaagggccctctccagctgtgcccagcatctacgccactctggccatccac (SEQ ID NO:3) SEQ ID NO: 4 - iCAR2: LIR-1 (TM)- LIR-1 (ID) peptide sequence: VIGILVAVILLLLLLLLLFLILRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKHTQPEDGVEMDTRSPHDEDPQAVTYAEVKHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSLTLRREATEPPPSQEGPSPAVPSIYATLAIH (SEQ ID NO:4) SEQ ID NO: 5 - iCAR3: KIR2DL1 (TM)- KIR2DL1 (ID)- LAIR-1 (CID) nucleotide sequence: attctgattgggacctcagtggtcatcatcctcttcatcctcctcttctttctccttcatcgctggtgctccaacaaaaaaaatgctgcggtaatggaccaagagtctgcaggaaacagaacagcgaatagcgaggactctgatgaacaagaccctcaggaggtgacatacacacagttgaatcactgcgttttcacacagagaaaaatcactcgcccttctcagaggcccaagacacccccaacagatatcatcgtgtacacggaacttccaaatgctgagtccagatccaaagttgtctcctgcccacatcgccagaatcagataaagcaggggccccccagaagcaaggacgaggagcagaagccacagcagaggcctgacctggctgttgatgttctagagaggacagcagacaaggccacagtcaatggacttcctgagaaggacagagagacggacacctcggccctggctgcagggagttcccaggaggtgacgtatgctcagctggaccactgggccctcacacagaggacagcccgggctgtgtccccacagtccacaaagcccatggccgagtccatcacgtatgcagccgttgccagacac (SEQ ID NO:5) SEQ ID NO: 6 - iCAR3: KIR2DL1 (TM)- KIR2DL1 (ID)- LAIR-1 (CID) peptide sequence: ILIGTSVVIILFILLFFLLHRWCSNKKNAAVMDQESAGNRTANSEDSDEQDPQEVTYTQLNHCVFTQRKITRPSQRPKTPPTDIIVYTELPNAESRSKVVSCPHRQNQIKQGPPRSKDEEQKPQQRPDLAVDVLERTADKATVNGLPEKDRETDTSALAAGSSQEVTYAQLDHWALTQRTARAVSPQSTKPMAESITYAAVARH (SEQ ID NO:6) SEQ ID NO: 7 - iCAR4: KIR2DL1 (TM)- KIR2DL1 (ID)- NKG2A(CID) nucleotide sequence: attctgattgggacctcagtggtcatcatcctcttcatcctcctcttctttctccttcatcgctggtgctccaacaaaaaaaatgctgcggtaatggaccaagagtctgcaggaaacagaacagcgaatagcgaggactctgatgaacaagaccctcaggaggtgacatacacacagttgaatcactgcgttttcacacagagaaaaatcactcgcccttctcagaggcccaagacacccccaacagatatcatcgtgtacacggaacttccaaatgctgagtccagatccaaagttgtctcctgcccaaaagaaccggcgagcccgctggataaatgccattataccaaagataacggccagtttgatcagagcgcgaaacagctgaacctggaagcgtataccattgaacaggaaaccgcgctgattagcaacaaaaacggcaaaccgaaacgccagcagcgcaaaccgaacccgccgctgaacctggatagctatattgtgggccagaacgatatg (SEQ ID NO:7) SEQ ID NO: 8 - iCAR4: KIR2DL1 (TM)- KIR2DL1 (ID)- NKG2A(CID) peptide sequence: ILIGTSVVIILFILLFFLLHRWCSNKKNAAVMDQESAGNRTANSEDSDEQDPQEVTYTQLNHCVFTQRKITRPSQRPKTPPTDIIVYTELPNAESRSKVVSCPKEPASPLDKCHYTKDNGQFDQSAKQLNLEAYTIEQETALISNKNGKPKRQQRKPNPPLNLDSYIVGQNDM (SEQ ID NO:8) SEQ ID NO: 9 - iCAR5: KIR2DL1 (TM)- KIR2DL1 (ID)-CD300A(CID) nucleotide sequence: attctgattgggacctcagtggtcatcatcctcttcatcctcctcttctttctccttcatcgctggtgctccaacaaaaaaaatgctgcggtaatggaccaagagtctgcaggaaacagaacagcgaatagcgaggactctgatgaacaagaccctcaggaggtgacatacacacagttgaatcactgcgttttcacacagagaaaaatcactcgcccttctcagaggcccaagacacccccaacagatatcatcgtgtacacggaacttccaaatgctgagtccagatccaaagttgtctcctgcccaaggatgtttcagaaatggatcaaagctggtgaccattcagagctgtcccagaaccccaagcaggctgccacgcagagtgagctgcactacgcaaatctggagctgctgatgtggcctctgcaggaaaagccagcaccaccaagggaggtggaggtggaatacagcactgtggcctcccccagggaagaacttcactatgcctcggtggtgtttgattctaacaccaacaggatagctgctcagaggcctcgggaggaggaaccagattcagattacagtgtgataaggaagaca (SEQ ID NO:9) SEQ ID NO: 10 - iCAR5: KIR2DL1 (TM)- KIR2DL1 (ID)-CD300A(CID) peptide sequence: ILIGTSVVIILFILLFFLLHRWCSNKKNAAVMDQESAGNRTANSEDSDEQDPQEVTYTQLNHCVFTQRKITRPSQRPKTPPTDIIVYTELPNAESRSKVVSCPRMFQKWIKAGDHSELSQNPKQAATQSELHYANLELLMWPLQEKPAPPREVEVEYSTVASPREELHYASVVFDSNTNRIAAQRPREEEPDSDYSVIRKT (SEQ ID NO:10)

[0158] In some embodiments, the present disclosure demonstrates the use of CD19-targeted iCARs to prevent the recognition and killing of CD19-expressing cells. This approach can also be used to target other antigens that are common between healthy cells and cancer cells. In some embodiments, this approach is useful for solid tumors, where antigens are often common to normal cells. Thus, in certain embodiments, iCARs signal to prevent cell killing when antigens are common to normal and healthy cells. While iCARs and aCARs can target the same antigen, in certain embodiments, iCARs and aCARs target different antigens.

[0159] In certain embodiments, a single immune effector cell expresses one or more iCARs and one or more aCARs. In situations where two or more iCARs are used per cell, each antigen-binding domain of a different iCAR can target a different antigen, such as a different NK autoantigen. In specific embodiments, when cells are engineered to express multiple engineered receptors of any type, they may or may not be expressed from the same vector. If expressed from the same vector, they can ultimately be produced as separate polypeptides, such as by separation at a cleavage site, 2A element (e.g., T2A, P2A, E2A, F2A, etc.), or IRES element contained in the vector.

[0160] In certain embodiments, aCARs and / or iCARs can be adapted to meet specific needs. For example, immune effector cells can be engineered to express a specific iCAR and then tailored to express a specific aCAR. In other cases, immune effector cells can be engineered to express a specific aCAR and then tailored to express a specific iCAR. Between such modifications, the immune effector cells may or may not be stored, e.g., cryogenically stored. In some embodiments, NK cells are engineered to express a specific iCAR, e.g., an iCAR targeting an NK autoantigen (e.g., CS1), and then (e.g., following cryogenic storage) are engineered to express a certain aCAR based on the needs of the individual, e.g., the type of cancer of the individual. In some embodiments, NK cells are engineered to express a specific aCAR, e.g., an aCAR targeting a cancer antigen, and then (e.g., following cryogenic storage) are engineered to express a certain iCAR.

[0161] Embodiments of the present disclosure include the use of any of the engineered immune effector cells encompassed herein. The methods include enhancing cell therapy, including adoptive cell therapy, for an individual in need thereof, for example, for an individual with cancer, in which engineered immune effector cells are used to treat the cancer. In specific embodiments, the cell therapy uses an aCAR that targets one or more antigens present on cancer cells. In specific instances, the iCAR inhibits the death of cells expressing the antigen to which the aCAR is directed unless the antigen is present on the cancer cell.

[0162] Embodiments of the present disclosure include a method of administering to an individual a therapeutically effective amount of engineered immune effector cells, each comprising: (a) at least one inhibitory chimeric antigen receptor (iCAR), wherein the iCAR comprises at least one extracellular antigen-binding domain, wherein the first extracellular antigen-binding domain binds a first antigen, and at least one natural killer (NK) cell inhibitory signaling domain and / or at least one co-inhibitory domain; and (b) at least one activating chimeric antigen receptor (aCAR), wherein the aCAR comprises at least one extracellular antigen-binding domain, wherein the second antigen-binding domain binds a second antigen, and an activating endodomain and at least one co-stimulatory signaling domain, comprising:

[0163] (I) when the first antigen and the second antigen are the same and the engineered immune effector cell binds to a cell expressing the antigen via the second extracellular antigen-binding domain, the iCAR inhibits killing of the cell expressing the antigen by the engineered immune effector cell; or

[0164] (II) the first antigen and the second antigen are non-identical and are both expressed on fellow engineered immune effector cells, or on non-engineered immune effector cells of the same type, or on non-disease cells, and when the engineered immune effector cells bind to the second antigen on the fellow engineered immune effector cells, or on non-engineered immune effector cells of the same type, or on non-disease cells, respectively, via the second extracellular antigen-binding domain, the iCAR inhibits killing of the fellow engineered immune effector cells, or on non-engineered immune effector cells of the same type, or on non-disease cells, respectively, by the engineered immune effector cells. The method includes:

[0165] In a specific embodiment, the antigen-expressing cell is a fellow engineered immune effector cell. In a specific embodiment, the engineered immune effector cell binds to a second antigen on the fellow engineered immune effector cell via the second extracellular antigen-binding domain. In a specific embodiment, the second antigen is expressed by the fellow engineered immune effector cell as a result of trogocytosis. III.NK cells

[0166] While it is contemplated that the methods and compositions of the present disclosure may utilize any type of immune effector cell, including any type of T cell, in certain embodiments, the present disclosure relates to a process for modifying NK cells rather than other types of immune cells. NK cells are a subpopulation of lymphocytes that possess spontaneous cytotoxicity against various tumor cells, virus-infected cells, and some normal cells in the bone marrow and thymus. NK cells differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus. NK cells can be detected by specific surface markers, such as CD16 and / or CD56 in humans. NK cells do not express T cell antigen receptors, the pan-T marker CD3, or surface immunoglobulin B cell receptors.

[0167] In certain embodiments, NK cells can be derived from human peripheral blood mononuclear cells (PBMCs), unstimulated leukapheresis products (PBSCs), human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs), human hematopoietic stem cells, bone marrow, or umbilical cord blood, or NK cell lines by methods well known in the art. In particular, umbilical CBs can be used to derive NK cells. In certain embodiments, progenitor cells are engineered as described herein prior to differentiation and / or induction into effector cells such as NK cells. In certain aspects, NK cells are isolated and expanded by previously described methods of ex vivo expansion of NK cells (see, e.g., Spanholtz et al., 2011 and Shah et al., 2013). In this method, CB mononuclear cells are isolated by Ficoll density gradient centrifugation and cultured in a bioreactor with IL-2 and artificial antigen-presenting cells (aAPCs). After 7 days, the cell culture can be depleted of all cells expressing CD3 and re-cultured for another 7 days. + / CD3 - Alternatively, CD34 cells can be characterized to determine the percentage of CD34 or NK cells. + CD56 by isolating cells and culturing them in medium containing SCF, IL-7, IL-15, and / or IL-2 + / CD3 - Umbilical CBs are used to elicit NK cells by differentiation into NK cells.

[0168] As described elsewhere herein, in some embodiments, NK cells and / or their progenitor cells are expanded once or twice during their preparation. As noted above, in specific instances, NK cell expansion involves stimulating mononuclear cells (MNCs) from umbilical cord blood in the presence of antigen-presenting cells (APCs) and IL-2, and restimulating the cells with APCs at some point during the process to produce expanded NK cells. In at least some instances, the method is carried out in a bioreactor. Multiple steps of this process can be carried out in the same vessel, e.g., in the same bioreactor. This stimulation step can direct MNCs toward NK cells. The restimulation step may or may not include the presence of IL-2. In certain embodiments, the method does not remove or add any media components during the stimulation step. In certain embodiments, the method is carried out within a certain time frame, e.g., less than 15 days, e.g., 14 days.

[0169] As described in detail elsewhere herein, in certain embodiments, NK cells are expanded by an ex vivo expansion method comprising (a) obtaining a starting population of mononuclear cells (MNCs) from umbilical cord blood, (b) stimulating the MNCs in the presence of antigen-presenting cells (APCs) and IL-2, and (c) restimulating the cells with APCs to produce expanded NK cells, the method being carried out in a bioreactor and in compliance with Good Manufacturing Practice (GMP). The stimulation in step (b) can direct MNCs toward NK cells. Step (c) may or may not, in some cases, include the presence of IL-2. In certain embodiments, the method does not remove or add any media components during step (b). In certain embodiments, the method is carried out in less than 15 days, e.g., 14 days.

[0170] As described above, in certain embodiments, the method further comprises depleting cells that are positive for one or more specific markers, such as, for example, CD3, CD14, and / or CD19. In certain embodiments, this depletion step is performed before transduction or transfection and / or after gene editing. In some embodiments, cells are removed from the bioreactor for CD3, CD14, and / or CD19 depletion and then placed into the bioreactor for subsequent steps.

[0171] In certain embodiments, obtaining a starting population of MNCs from umbilical cord blood comprises thawing the cord blood in the presence of dextran, human serum albumin (HSA), DNAse, and / or magnesium chloride. In particular embodiments, obtaining a starting population of MNCs from umbilical cord blood comprises thawing the cord blood in the presence of dextran and / or DNase. In specific embodiments, the cord blood is washed in the presence of 5-20%, e.g., 10%, dextran. In certain embodiments, the cord blood is washed in the presence of 5-20%, e.g., 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% dextran. In certain embodiments, the cord blood is suspended in the presence of magnesium chloride, e.g., at a concentration of 100-300 mM, particularly 200 mM magnesium chloride. In certain embodiments, the cord blood is suspended in the presence of magnesium chloride, e.g., at a concentration of 100-300 mM, e.g., 100-120 mM, 120-140 mM, 140-160 mM, 160-180 mM, 180-200 mM, 190-210 mM, 200-220 mM, 220-240 mM, 240-260 mM, 260-280 mM, or 280-300 mM. In some embodiments, obtaining includes Ficoll density gradient centrifugation to obtain mononuclear cells (MNCs). In certain embodiments, the cord blood from which the NK cells are derived is frozen cord blood. In certain embodiments, the frozen cord blood has been tested for one or more infectious diseases, such as Hepatitis A, Hepatitis B, Hepatitis C, Trypanosoma cruzi, HIV, coronavirus, human T-lymphotropic virus, syphilis, Zika virus, etc. In some embodiments, the cord blood is pooled cord blood from 3, 4, 5, 6, 7, or 8 individual cord blood units.

[0172] In certain embodiments, the method does not involve human leukocyte antigen (HLA) matching. In some embodiments, the starting population of NK cells is not obtained from a haploidentical donor.

[0173] In some embodiments, the expanded NK cells produced by this process comprise a clinically relevant dose. In some embodiments, the NK cells are autologous with respect to the recipient individual. In certain embodiments, the NK cells are allogeneic with respect to the recipient individual.

[0174] In some embodiments, the NK cells express one or more heterologous antigen receptors, one or more antibodies, and / or one or more bispecific, trispecific, or multispecific engager / antibodies.

[0175] In some embodiments, NK cells can be characterized by their cellular phenotype. In some embodiments, NK cells can be described as hyporesponsive and / or exhausted if they express certain cellular markers described herein (see, e.g., Figures 11B-11C and 12A-12D). In some embodiments, the techniques provided herein reduce the rate of NK cell hyporesponsiveness and / or exhaustion. For example, in some embodiments, the techniques provided herein reduce the percentage of hyporesponsive and / or exhausted NK cells in a population by 5% to 100% when compared to an appropriate control. This includes, for example, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, Including a 9%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% reduction.

[0176] In some embodiments, NK cells can be characterized by their propensity to engage in fratricide. In some embodiments, the likelihood of an NK cell engaging in fratricide can be described as a function of the percentage of fraternal cells that have acquired the target antigen in addition to the presence of any NK cell-specific negative inhibitor, such as an iCAR described herein, and can be measured by assays known in the art and / or described herein. In some embodiments, NK cells can be characterized by their propensity to become victims of fratricide. In some embodiments, the likelihood of an NK cell engaging in fratricide can be described as a function of the percentage of NK cells that have acquired the target antigen in addition to the presence of any sibling cell-specific negative inhibitor, such as an iCAR described herein. In some embodiments, the techniques provided herein reduce the rate of NK cell fratricide, which can be measured by assays known in the art and / or described herein. For example, in some embodiments, the techniques provided herein reduce the percentage of fratricide in an NK cell population by 5% to 100% when compared to an appropriate control.This includes, for example, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, Including a 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% reduction.

[0177] In some embodiments, NK cells can be characterized by their in vivo viability. In some embodiments, the in vivo viability of NK cells can be determined by analyzing checkpoint markers such as TIGIT, PD1, and / or TIM3. In some embodiments, the in vivo viability of NK cells can be increased using the techniques provided herein. In some embodiments, the in vivo viability can be increased by 1.1 to 10-fold (e.g., 1.1× to 10×) when compared to an appropriate control. In some embodiments, in vivo viability can be increased by up to 1.1×, 1.2×, 1.3×, 1.4×, 1.5×, 1.6×, 1.7×, 1.8×, 1.9×, 2×, 2.5×, 3×, 3.5×, 4×, 4.5×, 5×, 5.5×, 6×, 6.5×, 7×, 7.5×, 8×, 8.5×, 9×, 9.5×, 10×, 15×, 20×, 25×, 30×, 40×, 50×, 60×, 70×, 80×, 90×, 100×, 1000×, or 10,000× when compared to an appropriate control.

[0178] In some embodiments, NK cells can be characterized by their effector function. In some embodiments, effector function can be measured by determining the cytotoxicity of NK cells against target cells as compared to an appropriate control. In some embodiments, NK cell effector function can be increased using the techniques provided herein. In some embodiments, NK cell effector function can be increased. In some embodiments, NK cell effector function can be increased by 1.1 to 10 fold (e.g., 1.1× to 10×) as compared to an appropriate control. In some embodiments, NK cell effector function can be increased by up to 1.1x, 1.2x, 1.3x, 1.4x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2x, 2.5x, 3x, 3.5x, 4x, 4.5x, 5x, 5.5x, 6x, 6.5x, 7x, 7.5x, 8x, 8.5x, 9x, 9.5x, 10x, 15x, 20x, 25x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 1000x, or 10000x when compared to an appropriate control.

[0179] In some embodiments, NK cells can be characterized by their in vivo persistence. In some embodiments, the in vivo persistence of NK cells can be determined by analyzing cell levels at appropriate time points after administration. In some embodiments, the in vivo persistence of NK cells can be increased using the techniques provided herein. In some embodiments, the in vivo persistence can be increased by 1.1 to 10-fold (e.g., 1.1× to 10×) when compared to an appropriate control. In some embodiments, in vivo persistence can be increased by up to 1.1x, 1.2x, 1.3x, 1.4x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2x, 2.5x, 3x, 3.5x, 4x, 4.5x, 5x, 5.5x, 6x, 6.5x, 7x, 7.5x, 8x, 8.5x, 9x, 9.5x, 10x, 15x, 20x, 25x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 1000x, or 10000x when compared to an appropriate control.

[0180] In some embodiments, NK cells can be characterized by their ability to reduce and / or inhibit the growth of a target cell population (e.g., a tumor cell population). In some embodiments, the ability of NK cells to reduce and / or inhibit the growth of a target cell population can be measured using known methods, such as ultrasound, x-ray, CT scan, MRI, biopsy, etc. In some embodiments, the ability of NK cells to reduce and / or inhibit the growth of a target cell population can be increased using the techniques provided herein. In some embodiments, the ability of NK cells to reduce and / or inhibit the growth of a target cell population can be increased by 1.1 to 10 fold (e.g., 1.1× to 10×) when compared to an appropriate control. In some embodiments, the ability of NK cells to reduce and / or inhibit the growth of a target cell population can be increased by 1.1x, 1.2x, 1.3x, 1.4x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2x, 2.5x, 3x, 3.5x, 4x, 4.5x, 5x, 5.5x, 6x, 6.5x, 7x, 7.5x, 8x, 8.5x, 9x, 9.5x, 10x, 15x, 20x, 25x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 1000x, or 10000x when compared to an appropriate control. IV. Xenoantigen Receptors

[0181] The immune effector cells of the present disclosure are genetically engineered to express one or more iCARs and one or more heterologous antigen receptors, such as one or more engineered TCRs, one or more CARs, or a combination thereof. The heterologous antigen receptors are synthetically produced by human hands. In certain embodiments, the immune effector cells are modified to express one or more CARs and / or one or more TCRs, each with antigen specificity for a different cancer antigen, in addition to the iCAR. In some embodiments, the immune effector cells are engineered to express a CAR and / or a TCR by knocking in the CAR or TCR at a specific locus, such as by using CRISPR.

[0182] In certain cases, NK cells are optionally edited using CRISPR, although alternative suitable modification methods are known in the art. See, e.g., Sambrook and Ausubel, supra. For example, cells can be transduced to express a CAR or TCR with antigen specificity for a cancer antigen using the transduction techniques described in Heemskerk et al., 2008 and Johnson et al., 2009. In some embodiments, cells contain one or more genetically engineered nucleic acids encoding one or more antigen receptors and genetically engineered products of such nucleic acids. In some embodiments, the nucleic acids are heterologous; that is, the nucleic acids are not normally found in a given cell or sample obtained from the cell, e.g., obtained from another organism or cell not normally found in the engineered cell and / or the organism from which the cell is derived. In some embodiments, the nucleic acids are not naturally occurring, e.g., nucleic acids not found in nature (e.g., chimeras).

[0183] In some embodiments, CAR contains an extracellular antigen recognition domain that specifically binds to an antigen.In some embodiments, the antigen is a protein expressed on the surface of a cell.In some embodiments, CAR is a TCR-like CAR, and the antigen is a processed peptide antigen, such as a peptide antigen of an intracellular protein, that is recognized on the cell surface in association with a major histocompatibility complex (MHC) molecule, just like TCR.

[0184] Exemplary antigen receptors, including CARs and recombinant TCRs, and methods for engineering receptors and introducing them into cells are described, for example, in International Patent Application Publication Nos. WO200014257, WO2013126726, WO2012 / 129514, WO2014031687, WO2013 / 166321, WO2013 / 071154, WO2013 / 123061, U.S. Patent Application Publication Nos. US2002131960, US2013287748, US20130149337, U.S. Patent Nos. 6,451,995, 7,446,190, 8,252,5 92, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, and European Patent Application No. EP 2537416, and / or those described in Sadelain et al., 2013, Davila et al., 2013, Turtle et al., 2012, and / or Wu et al., 2012. In some embodiments, engineered antigen receptors include CARs described in U.S. Patent No. 7,446,190 and those described in International Patent Application Publication No. WO / 2014055668A1. A. Chimeric Antigen Receptor

[0185] In some embodiments, the CAR comprises a) one or more intracellular signaling domains, b) a transmembrane domain, and c) an extracellular domain comprising one or more antigen-binding domains. The extracellular antigen-binding domain can be associated with any type of hinge, such as a hinge derived from IgG1, CD28, CD8 alpha, etc.

[0186] In some embodiments, engineered antigen receptors include CARs, including activating or stimulatory CARs, costimulatory CARs (see WO 2014 / 055668), and / or inhibitory CARs (iCARs, see e.g., Fedorov et al., 2013). CARs generally comprise an extracellular antigen (or ligand) binding domain linked (in some embodiments via a linker and / or transmembrane domain) to one or more intracellular signaling components. Such molecules typically mimic or mimic the signaling of natural antigen receptors, such receptors in combination with costimulatory receptors, and / or costimulatory receptors alone.

[0187] Certain embodiments of the present disclosure relate to the use of nucleic acids, such as nucleic acids encoding antigen-specific CAR polypeptides (including CARs that have been humanized to reduce immunogenicity (hCARs)) that include an intracellular signaling domain, a transmembrane domain, and an extracellular domain and include one or more signaling motifs. In certain embodiments, the CAR can recognize an epitope that includes a space shared between one or more antigens. In certain embodiments, the binding region can include a complementarity-determining region of a monoclonal antibody, a variable region of a monoclonal antibody, and / or an antigen-binding fragment thereof. In another embodiment, the specificity is derived from a peptide that binds to a receptor (e.g., a cytokine).

[0188] It is envisioned that the human CAR nucleic acid may be a human gene used to enhance cellular immunotherapy in human patients. In a specific embodiment, the present invention comprises a full-length CAR cDNA or coding region. The antigen-binding region or domain is the V of a single-chain variable fragment (scFv) derived from a particular human monoclonal antibody. H and V L The fragments can include fragments of the chains, such as those described in U.S. Patent No. 7,109,304, which is incorporated herein by reference. The fragments can also be any number of different antigen-binding domains of a human antigen-specific antibody. In a more specific embodiment, the fragment is an antigen-specific scFv encoded by a sequence optimized for human codon usage for expression in human cells.

[0189] In some embodiments, the antigen-specific CAR polypeptide may comprise one or more epitope recognition domains that do not comprise an scFv. In some embodiments, the antigen-specific CAR polypeptide may comprise an antigen-binding domain derived from one or more proteins selected from adnectins, affibodies, affillins, anticalins, atrimers, avimers, bicyclic peptides, centyrins, cys-knots, DARPins, FN3s, fynomers, Kunitz domains, obodies, pronectins, and Tn3s. In some embodiments, such antigen-binding domains may be modified and / or optimized for human codon usage and / or expression in human cells.

[0190] In some embodiments, the CAR arrangement can include polypeptides and / or proteins that can be in multimeric form, such as diabodies or multimers. Multimers are often formed by cross-pairing the variable portions of light and heavy chains into diabodies. The hinge portion of the construct can have several alternatives, ranging from complete deletion to retaining the first cysteine, substituting proline for serine, or truncating up to the first cysteine. The Fc portion can be deleted. Any stable and / or dimerizing protein can serve this purpose. Only one of the Fc domains from a human immunoglobulin can be used, such as either the CH2 or CH3 domain. The hinge, CH2, and CH3 regions of a human immunoglobulin that have been modified to improve dimerization can also be used. Only the hinge portion of an immunoglobulin can also be used. Portions of CD8 alpha can also be used.

[0191] In some embodiments, the CAR nucleic acid comprises a sequence encoding another costimulatory receptor, such as a transmembrane domain and a modified CD28 intracellular signaling domain. Other costimulatory receptors include, but are not limited to, one or more of CD28, CD27, OX-40 (CD134), DAP10, DAP12, 4-1BB (CD137), or combinations thereof. In addition to the primary signal initiated by CD3ζ, additional signals provided by the human costimulatory receptor inserted into the human CAR are important for full activation of NK cells and may contribute to improved in vivo persistence and therapeutic success of adoptive immunotherapy.

[0192] In some embodiments, CARs are constructed with specificity for a particular antigen (or marker or ligand), such as an antigen expressed in a specific cell targeted by adoptive therapy, e.g., a cancer marker, and / or an antigen against which an attenuated response is intended, e.g., an antigen expressed on a normal or non-diseased cell. Thus, CARs typically comprise, in their extracellular portion, one or more antigen-binding molecules, e.g., one or more antigen-binding fragments, domains, or portions, or one or more antibody variable domains, and / or antibody molecules. In some embodiments, the CAR comprises one or more antigen-binding portions of an antibody molecule, e.g., a single-chain antibody fragment (scFv) derived from the variable heavy chain (VH) and variable light chain (VL) of a monoclonal antibody (mAb).

[0193] In certain embodiments of chimeric antigen receptors, the antigen-specific portion of the receptor (which can also be referred to as the extracellular domain containing the antigen-binding region) comprises a tumor-associated antigen-specific or pathogen-specific antigen-binding domain. Antigens include carbohydrate antigens recognized by pattern recognition receptors such as Dectin-1. The tumor-associated antigen can be of any type, as long as it is expressed on the cell surface of tumor cells. Exemplary embodiments of antigens include CD19, CD70, HLA-G, CD38, CD123, CLL1, EBNA, CD123, HER2, CA-125, TRAIL / DR4, CD20, carcinoembryonic antigen, alpha-fetoprotein, CD56, AKT, Her3, epithelial tumor antigen, CD319 (CS1), ROR1, folate-binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, CD5, CD 23, CD30, HERV-K, IL-11R alpha, kappa chain, lambda chain, CSPG4, CD33, CD47, CLL-1, U5snRNP200, CD200, BAFF-R, BCMA, CD99, p53, mutant p53, Ras, mutant ras, c-Myc, cytoplasmic serine / threonine kinases (e.g., A-Raf, B-Raf, and C-Raf, cyclin-dependent kinases), MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4 , MAGE-A6, MAGE-A10, MAGE-A12, MART-1, melanoma-associated antigen, BAGE, DAM-6, -10, GAGE-1, -2, -8, GAGE-3, -4, -5, -6, -7B, NA88-A, MC1R, mda-7, gp75, Gp100, PSA, PSM, tyrosinase, tyrosinase-related protein, TRP-1, TRP-2, ART-4, CAMEL, CEA, Cyp-B, hTERT, hTRT, iCE, MUC1, MUC2 , phosphoinositide 3 kinase (PI3K), TRK receptor, PRAME, P15, RU1, RU2, SART-1, SART-3, Wilms tumor antigen (WT1), AFP, -catenin / m, caspase-8 / m, CDK-4 / m, ELF2M, GnT-V, G250, HAGE, HSP70-2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, myosin / m, RAGE, SART-2, TRP-2 / INT2, 707-AP,Annexin II, CDC27 / m, TPI / mbcr-abl, BCR-ABL, interferon regulatory factor 4 (IRF4), ETV6 / AML, LDLR / FUT, Pml / RAR, tumor-associated calcium signaling transduction factor 1 (TACSTD1) and TACSTD2, receptor tyrosine kinases (e.g., epidermal growth factor receptor (EGFR) (especially EGFRvIII), platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR)), VEGFR2, cytoplasmic tyrosine kinases (e.g., src family, syk-ZAP70 family), integrin-linked kinase (ILK), signal transduction and activator of transcription factors STAT3, STATS, and STATE, hypoxia-inducible factors (e.g., HIF-1 and HIF-2), nuclear factor kappa B (NF-B), Notch receptors (e.g., Notch1-4), NY ESO1, c-Met, mammalian target of rapamycin (mTOR), WNT, extracellular signal-regulated kinase (ERK) and their regulatory subunits, PMSA, PR-3, MDM2, mesothelin, renal cell carcinoma-5T4, SM22-alpha, carbonic anhydrase I (CAI) and IX (CAIX) (also known as G250), STEAD, TEL / AML1, GD2, proteinase 3, hTERT, sarcoma translocation breakpoints, EphA2, ML-IAP, EpCAM, ERG (TMPRSS2) ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, GD3, fucosyl-GM1, mesothelial, PSCA, sLe, PLAC1, GM3, BORIS, Tn, GLoboH, NY-BR-1, RGsS, SAGE, SART3, STn, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE1, B7H3, legumain, TIE2, Page4, MAD-CT-1, FAP, MAD-CT-2, fos-related antigen 1, CBX2, CLDN6, SPANX, TPTE, ACTL8, ANKRD30A, CDKN2A, MAD2L1, CTAG1B, SUNC1, and LRRN1, or a combination thereof.

[0194] In certain embodiments, when the tumor-associated antigen is in low abundance, the CAR may be co-expressed with one or more cytokines, for example, the CAR may be co-expressed with one or more cytokines, such as IL-7, IL-2, IL-15, IL-12, IL-18, IL-21, or a combination thereof.

[0195] The sequence of the open reading frame encoding the chimeric receptor can be obtained from a genomic DNA source, a cDNA source, or synthesized (e.g., by PCR), or a combination thereof. Depending on the size of the genomic DNA and the number of introns, it may be desirable to use cDNA, or a combination thereof, since introns are known to stabilize mRNA. It may also be advantageous to use endogenous or exogenous non-coding regions to stabilize mRNA.

[0196] It is assumed that the chimeric construct can be introduced into immune cells as naked DNA or in a suitable vector.Methods for stable cell transfection by naked cell electroporation are known in the art.See, for example, U.S. Patent No. 6,410,319.Naked DNA generally refers to the DNA encoding the chimeric receptor contained in a plasmid expression vector in the appropriate orientation for expression.

[0197] Alternatively, viral vectors (such as retroviral vectors, adenoviral vectors, adeno-associated viral vectors, or lentiviral vectors) can be used to introduce chimeric constructs into immune cells.The vectors suitable for use in the method of the present disclosure are non-replicative in immune cells.Many virus-based vectors are known, and the copy number of the virus that is maintained in cells is low enough to maintain cell viability, such as the vectors based on HIV, SV40, EBV, HSV, or BPV.

[0198] In some embodiments, antigen-specific binding or recognition component is linked to one or more transmembrane domains and intracellular signaling domains.In some embodiments, CAR comprises a transmembrane domain fused to the extracellular domain of CAR.In one embodiment, the transmembrane domain that is naturally associated with one of the domains in CAR is used.In some cases, to minimize interaction with other members of receptor complex, the transmembrane domain is selected or modified by amino acid substitution to avoid such domain from binding to the transmembrane domain of the same or different surface membrane protein.

[0199] In some embodiments, the transmembrane domain is derived from a natural or synthetic source. If the source is natural, the domain in some aspects is derived from any membrane-bound or transmembrane protein. Transmembrane regions include those derived from (i.e., comprising at least the transmembrane region of) the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS / CD278, GITR / CD357, NKG2D, and DAP molecules. Alternatively, the transmembrane domain in some embodiments is synthetic. In some aspects, synthetic transmembrane domains primarily comprise hydrophobic residues such as leucine and valine. In some embodiments, a triplet of phenylalanine, tryptophan, and valine will be found at each end of the synthetic transmembrane domain.

[0200] In certain embodiments, the platform technology disclosed herein for genetically modifying immune cells, such as NK cells, includes: (i) non-viral gene transfer using an electroporation device (e.g., a nucleofector); (ii) CARs that signal through an endodomain (e.g., CD28 / CD3-ζ, CD137 / CD3-ζ, or other combinations); (iii) CARs with extracellular domains of various lengths that tether antigen recognition domains to the cell surface; and, in some cases, (iv) CARs + To enable robust and numerical expansion of immune cells, K562-derived artificial antigen-presenting cells (aAPCs) are included (see, e.g., Singh et al., 2008, and Singh et al., 2011). BT cell receptor (TCR)

[0201] In some embodiments, the engineered antigen receptor comprises a recombinant TCR and / or a TCR cloned from a naturally occurring T cell. "T cell receptor" or "TCR" refers to a molecule that contains a variable a chain and a variable β chain (also known as TCRα and TCRβ, respectively) or a variable γ chain and a variable δ chain (also known as TCRγ and TCRδ, respectively), and is capable of specifically binding to an antigenic peptide bound to an MHC receptor. In some embodiments, the TCR is of the αβ type.

[0202] TCRs, typically present in αβ and γδ types, are generally structurally similar, although the T cells expressing them may have distinct anatomical locations or functions. TCRs can be found on the surface of cells or in soluble form. Generally, TCRs are found on the surface of T cells or T lymphocytes, where they are generally responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules. In some embodiments, TCRs can contain a constant domain, a transmembrane domain, and / or a short cytoplasmic tail. For example, in some embodiments, each chain of a TCR can have an N-terminal immunoglobulin variable domain, an immunoglobulin constant domain, a transmembrane region, and a C-terminal short cytoplasmic tail (see, e.g., Janeway et al., 1997). In some embodiments, TCRs associate with invariant proteins of the CD3 complex, which are involved in mediating signal transduction. Unless otherwise stated, the term "TCR" should be understood to encompass functional TCR fragments thereof. The term also encompasses intact or full-length TCRs, including TCRs of the αβ or γδ types.

[0203] Thus, as used herein, reference to a TCR encompasses any TCR or functional fragment thereof, for example, any antigen-binding portion of a TCR that binds to a specific antigen peptide bound in an MHC molecule, i.e., bound in an MHC-peptide complex. The terms "antigen-binding portion" or "antigen-binding fragment" of a TCR can be used interchangeably and refer to a molecule that contains the structural domains of a TCR that bind to the antigen (e.g., an MHC-peptide complex) to which the entire TCR binds. In some cases, the antigen-binding portion contains sufficient variable domains of the TCR, such as the variable a chain and variable β chain of the TCR, where each chain typically contains three complementarity-determining regions, to form a binding moiety for binding to a specific MHC-peptide complex.

[0204] In some embodiments, the variable domains of TCR chains associate to form immunoglobulin-like loops or complementarity-determining regions (CDRs), which form the binding site of the TCR molecule, thereby conferring antigen recognition and determining peptide specificity. Typically, like immunoglobulins, CDRs are separated by framework regions (FRs) (see, e.g., Jores et al., 1990, Chothia et al., 1988, and Lefranc et al., 2003). In some embodiments, CDR3 is the primary CDR responsible for recognizing processed antigens, but CDR1 of the alpha chain has also been shown to interact with the N-terminal portion of antigenic peptides, while CDR1 of the beta chain interacts with the C-terminal portion of peptides. CDR2 is thought to recognize MHC molecules. In some embodiments, the variable region of the beta chain can contain an additional hypervariable (HV4) region.

[0205] In some embodiments, a TCR chain contains a constant domain. For example, like an immunoglobulin, the extracellular portion of a TCR chain (e.g., an a chain, a β chain) contains two immunoglobulin domains: an N-terminal variable domain (e.g., a V a or Vp; typically amino acids 1-116 according to Kabat numbering, Kabat et al., "Sequences of Proteins of Immunological Interest," U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, 1991, 5th ed.), and a constant domain adjacent to the cell membrane (e.g., an a-chain constant domain or C aThe TCR may contain a α-chain constant domain or Cp, typically amino acids 117-259 according to Kabat, and a β-chain constant domain or Cp, typically amino acids 117-295 according to Kabat. For example, in some cases, the extracellular portion of the TCR formed by the two chains contains two membrane-proximal constant domains and two membrane-distal variable domains containing the CDRs. The constant domains of the TCR domains contain a short connective sequence in which cysteine ​​residues form disulfide bonds to link the two chains. In some embodiments, the TCR may have an additional cysteine ​​residue in each of the α-chain and β-chain such that the TCR contains two disulfide bonds in the constant domains.

[0206] In some embodiments, the TCR chain can contain a transmembrane domain. In some embodiments, the transmembrane domain is positively charged. In some cases, the TCR chain contains a cytoplasmic tail. In some cases, this structure allows the TCR to associate with other molecules, such as CD3. For example, a TCR containing a constant domain with a transmembrane region can anchor the protein to the cell membrane and associate with the invariant subunit of the CD3 signaling apparatus or complex.

[0207] Generally, CD3 is a multiprotein complex that can have three distinct chains (γ, δ, and ε) and a ζ chain in mammals. For example, in mammals, this complex can contain a homodimer of the CD3γ chain, the CD3δ chain, two CD3ε chains, and the CD3ζ chain. The CD3γ, CD3δ, and CD3ε chains are highly related cell surface proteins of the immunoglobulin superfamily that contain a single immunoglobulin domain. The transmembrane regions of the CD3γ, CD3δ, and CD3ε chains are negatively charged, a feature that allows these chains to associate with the positively charged T cell receptor chains. The intracellular tails of the CD3γ, CD3δ, and CD3ε chains each contain one conserved motif known as an immunoreceptor tyrosine-based activation motif (ITAM), while the CD3ζ chain has three. Generally, ITAMs are involved in the signaling function of the TCR complex. These accessory molecules contain negatively charged transmembrane regions and play a role in transmitting signals from the TCR into the cell. Together with the TCR, the CD3 and ζ chains form what is known as the T cell receptor complex.

[0208] In some embodiments, the TCR can be a heterodimer of two chains, α and β (and optionally γ and δ), or a single-chain TCR construct. In some embodiments, the TCR is a heterodimer containing two separate chains (α and β or γ and δ) linked, such as by one or more disulfide bonds. In some embodiments, a TCR against a target antigen (e.g., a cancer antigen) is identified and introduced into a cell. In some embodiments, nucleic acids encoding the TCR can be obtained from various sources, such as polymerase chain reaction (PCR) amplification of published TCR DNA sequences. In some embodiments, the TCR is obtained from a biological source, such as from a cell, for example, from a T cell (e.g., a cytotoxic T cell), a T cell hybridoma, or other publicly available source. In some embodiments, the T cell can be obtained from an in vivo isolated cell. In some embodiments, a high-affinity T cell clone can be isolated from a patient and the TCR isolated. In some embodiments, the T cell can be a cultured T cell hybridoma or a cultured T cell clone. In some embodiments, TCR clones against target antigens are generated in transgenic mice engineered with human immune system genes (e.g., human leukocyte antigen system or HLA). See, for example, tumor antigens (see, e.g., Parkhurst et al., 2009, and Cohen et al., 2005). In some embodiments, TCRs against target antigens are isolated using phage display (see, e.g., Varela-Rohena et al., 2008, and Li et al., 2005). In some embodiments, TCRs or antigen-binding portions thereof can be synthetically generated from knowledge of the TCR sequence. C. Antigen

[0209] Antigens targeted by engineered iCARs and / or aCARs include those expressed in association with diseases, conditions, syndromes, or cell types targeted by adoptive cell therapy. Diseases and conditions include proliferative, neoplastic, and malignant diseases and disorders, including cancers and tumors, including hematological cancers, cancers of the immune system, lymphomas, leukemias, and / or myelomas, such as B, T, and myeloid leukemias, lymphomas, and multiple myelomas. In some embodiments, the antigen is selectively expressed or overexpressed on cells of a disease or condition, for example, on tumor cells or pathogenic cells, compared to normal cells or tissues or non-target cells or tissues. In another embodiment, the antigen is expressed on normal cells and / or on engineered cells.

[0210] Any suitable antigen can be targeted in this method. In some cases, the antigen may be associated with certain cancer cells but not with non-cancerous cells. Exemplary antigens include, but are not limited to, antigenic molecules from infectious agents, autologous / self-antigens, tumor / cancer-associated antigens, and tumor neoantigens (see, for example, Linnemann et al., 2015). In certain embodiments, antigens include CD19, EBNA, CD123, HER2, CA-125, TRAIL / DR4, CD20, CD70, HLA-G, CD38, CD123, CLL1, carcinoembryonic antigen, alpha-fetoprotein, CD56, AKT, Her3, epithelial tumor antigen, CD319 (CS1), ROR1, folate-binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, CD5, CD23, CD30, HERV-K, IL-11R alpha, kappa chain, lambda chain, CSPG4, CD33, CD47, CLL-1, U5snRNP200, CD200, BAFF-R, BCMA, CD99, p53, mutant p53, Ras, mutant ras, c-Myc, cytoplasmic serine / threonine kinases (e.g., A-Raf, B-Raf, and C-Raf, cyclin-dependent kinases), MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-A13, MAGE-A14, MAGE-A15, MAGE-A16, MAGE-A17, MAGE-A18, MAGE-A19 ... -A6, MAGE-A10, MAGE-A12, MART-1, melanoma-associated antigen, BAGE, DAM-6, -10, GAGE-1, -2, -8, GAGE-3, -4, -5, -6, -7B, NA88-A, MC1R, mda-7, gp75, Gp100, PSA, PSM, tyrosinase, tyrosinase-related protein, TRP-1, TRP-2, ART-4, CAMEL, CEA, Cyp-B, hTERT, hTRT, iCE, MUC1, MUC2, phosphoinositide Cytide 3 kinase (PI3K), TRK receptor, PRAME, P15, RU1, RU2, SART-1, SART-3, Wilms tumor antigen (WT1), AFP, -catenin / m, caspase-8 / m, CDK-4 / m, ELF2M, GnT-V, G250, HAGE, HSP70-2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, myosin / m, RAGE, SART-2, TRP-2 / INT2, 707-AP, annexin II,CDC27 / m, TPI / mbcr-abl, BCR-ABL, interferon regulatory factor 4 (IRF4), ETV6 / AML, LDLR / FUT, Pml / RAR, tumor-associated calcium signaling transduction factor 1 (TACSTD1) TACSTD2, receptor tyrosine kinases (e.g., epidermal growth factor receptor (EGFR) (especially EGFRvIII), platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR)), VEGFR2, cytoplasmic tyrosine kinases (e.g., src family, syk-ZAP70 family), integrin-linked kinase (ILK), signal transduction and activator of transcription factors STAT3, STATS, and STATE, hypoxia-inducible factors (e.g., HIF-1 and HIF-2), nuclear factor kappa B (NF-B), Notch receptors (e.g., Notch1-4), NY ESO1, c-Met, mammalian target of rapamycin (mTOR), WNT, extracellular signal-regulated kinase (ERK) and their regulatory subunits, PMSA, PR-3, MDM2, mesothelin, renal cell carcinoma-5T4, SM22-alpha, carbonic anhydrase I (CAI) and IX (CAIX) (also known as G250), STEAD, TEL / AML1, GD2, proteinase 3, hTERT, sarcoma translocation breakpoints, EphA2, ML-IAP, EpCAM, ERG (TMPRSS2) ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, GD3, fucosyl-GM1, mesothelial, PSCA, sLe, PLAC1, GM3, BORIS, Tn, GLoboH, NY-BR-1, RGsS, SAGE, SART3, STn, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE1, B7H3, legumain, TIE2, Page4, MAD-CT-1, FAP, MAD-CT-2, fos-related antigen 1, CBX2, CLDN6, SPANX, TPTE, ACTL8, ANKRD30A, CDKN2A, MAD2L1, CTAG1B, SUNC1, and LRRN1. Examples of antigen sequences are known in the art, for example in the GENBANK® database: CD19 (accession number NG_007275.1),EBNA (accession number NG_002392.2), WT1 (accession number NG_009272.1), CD123 (accession number NC_000023.11), NY-ESO (accession number NC_000023.11), EGFRvIII (accession number NG_007726.3), MUC1 (accession number NG_029383.1), HER2 (accession number NG_007503.1), CA-125 (accession number NG_007726.3), and HER2 (accession number NG_007503.1). accession number NG_055257.1), WT1 (accession number NG_009272.1), Mage-A3 (accession number NG_013244.1), Mage-A4 (accession number NG_013245.1), Mage-A10 (accession number NC_000023.11), TRAIL / DR4 (accession number NC_000003.12), and / or CEA (accession number NC_000019.10).

[0211] Tumor-associated antigens can be derived from, for example, prostate cancer, breast cancer, colorectal cancer, lung cancer, pancreatic cancer, kidney cancer, mesothelioma, ovarian cancer, liver cancer, brain cancer, bone cancer, stomach cancer, spleen cancer, testicular cancer, cervical cancer, anal cancer, gallbladder cancer, thyroid cancer, or melanoma cancer. Exemplary tumor-associated antigens or tumor cell-derived antigens include MAGE1, 3, and MAGE4 (or other MAGE antigens, such as those disclosed in International Patent Publication No. WO99 / 40188); PRAME; BAGE; RAGE, LAGE (also known as NY ESO 1); SAGE, and HAGE or GAGE. These non-limiting examples of tumor antigens are expressed in a wide range of major types of cancer, including melanoma, lung cancer, sarcoma, and bladder cancer. See, for example, U.S. Patent No. 6,544,518. Prostate cancer tumor-associated antigens include, for example, prostate-specific membrane antigen (PSMA), prostate-specific antigen (PSA), prostatic acid phosphates, NKX3.1, and six-transmembrane epithelial antigen of the prostate (STEAP).

[0212] Other tumor-associated antigens include Plu-1, HASH-1, HasH-2, Cripto, and Criptin. In addition, tumor antigens may be self-peptide hormones, such as full-length gonadotropin-releasing hormone (GnRH), a short 10-amino acid peptide that is useful in the treatment of many cancers.

[0213] Antigens may include epitope regions or epitope peptides derived from genes that are mutated or differentially transcribed in tumor cells compared to normal cells, such as telomerase enzyme, survivin, mesothelin, mutant ras, bcr / abl rearrangements, Her2 / neu, mutant or wild-type p53, cytochrome P450 1B1, and aberrantly expressed intronic sequences, such as N-acetylglucosaminyltransferase-V; clonal rearrangements of immunoglobulin genes that generate unique idiotypes in myeloma and B-cell lymphoma; tumor antigens that include epitope regions or epitope peptides derived from oncogenic viral processes, such as human papillomavirus proteins E6 and E7; Epstein-Barr virus protein LMP2; and non-mutated oncofetal proteins with tumor-selective expression, such as carcinoembryonic antigen and alpha-fetoprotein.

[0214] In another embodiment, instead of a cancer antigen (tumor antigen), the antigen is obtained or derived from a pathogenic or opportunistic pathogenic microorganism (also referred to herein as an infectious disease microorganism), such as a virus, fungus, parasite, or bacterium. In certain embodiments, antigens derived from such microorganisms include full-length proteins.

[0215] Illustrative examples of pathogenic organisms with antigens contemplated for use in the methods described herein include human immunodeficiency virus (HIV), herpes simplex virus (HSV), respiratory syncytial virus (RSV), coronavirus, cytomegalovirus (CMV), Epstein-Barr virus (EBV), influenza A, B, and C, vesicular stomatitis virus (VSV), polyomaviruses (e.g., BK virus and JC virus), adenovirus, Staphylococcus species, including methicillin-resistant Staphylococcus aureus (MRSA), and Streptococcus species, including Streptococcus pneumoniae. As will be appreciated by those skilled in the art, proteins from these and other pathogenic microorganisms used as antigens described herein, and the nucleotide sequences encoding those proteins, can be found in the literature and in public databases such as GENBANK®, SWISS-PROT®, and TREMBL®.

[0216] Antigens derived from human immunodeficiency virus (HIV) include HIV virion structural proteins (e.g., gp120, gp41, p17, p24), protease, reverse transcriptase, or any of the HIV proteins encoded by tat, rev, nef, vif, vpr, and vpu.

[0217] Antigens from herpes simplex viruses (e.g., HSV1 and HSV2) include, but are not limited to, proteins expressed from the HSV late genes. The late gene cluster primarily encodes proteins that form virion particles. Such proteins include five proteins from the (UL) that form the viral capsid: UL6, UL18, UL35, UL38, and the major capsid proteins UL19, UL45, and UL27, each of which may be used as antigens as described herein. Illustrative examples of other HSV proteins contemplated for use as antigens herein include ICP27 (H1, H2), glycoprotein B (gB), and glycoprotein D (gD) proteins. The HSV genome contains at least 74 genes, each encoding a protein that could potentially be used as an antigen.

[0218] Antigens from cytomegalovirus (CMV) include CMV structural proteins, viral antigens expressed in immediate early and early stages of viral replication, glycoproteins I and III, capsid protein, coat protein, lower matrix protein pp65 (ppUL83), p52 (ppUL44), IE1 and 1E2 (UL123 and UL122), protein products from the UL128-UL150 gene cluster (see, e.g., Ryckman et al., 2006), envelope glycoproteins B (gB), gH, gN, and pp150. As will be appreciated by those of skill in the art, CMV proteins for use as antigens described herein may be identified in public databases such as GENBANK®, SWISS-PROT®, and TREMBL® (see, e.g., Bennekov et al., 2004; Loewendorf & Benedict 2010; and Marschall et al., 2009).

[0219] Antigens derived from Epstein-Barr virus (EBV) contemplated for use in certain embodiments include the EBV lytic phase proteins gp350 and gp110, EBV proteins produced during latent cycle infection, such as Epstein-Barr nuclear antigen (EBNA)-1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C, EBNA-leader protein (EBNA-LP), and latent membrane protein (LMP)-1, LMP-2A, and LMP-2B (see, e.g., Lockey et al., 2008).

[0220] Antigens derived from respiratory syncytial virus (RSV) contemplated for use herein include any of 11 proteins or antigenic fragments thereof encoded by the RSV genome: NS1, NS2, N (nucleocapsid protein), M (matrix protein) SH, G and F (viral coat proteins), M2 (second matrix protein), M2-1 (elongation factor), M2-2 (transcriptional regulator), RNA polymerase, and phosphoprotein P.

[0221] Antigens derived from vesicular stomatitis virus (VSV) that are contemplated for use include any of the five major proteins and their antigenic fragments encoded by the VSV genome: large protein (L), glycoprotein (G), nucleoprotein (N), phosphoprotein (P), and matrix protein (M) (see, e.g., Rieder et al., 2009).

[0222] Antigens derived from influenza virus contemplated for use in certain embodiments include hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix proteins M1 and M2, NS1, NS2 (NEP), PA, PB1, PB1-F2, and PB2.

[0223] Antigens derived from coronaviruses (e.g., SARS-CoV-2) contemplated for use in certain embodiments include membrane (M) protein, envelope (E) protein, spike (S) protein, nucleocapsid (N) protein, coronavirus RNA, nonstructural proteins Nsp1 through Nsp16, and / or accessory proteins (3a, 3b, 6, 7a, 7b, 8, 9b, 9c, and / or 10). In a preferred embodiment, the coronavirus-derived antigen is an antigen from the S protein.

[0224] Exemplary viral antigens include adenovirus polypeptides, alphavirus polypeptides, calicivirus polypeptides (e.g., calicivirus capsid antigen), coronavirus polypeptides, distemper virus polypeptides, Ebola virus polypeptides, enterovirus polypeptides, flavivirus polypeptides, hepatitis virus (AE) polypeptides (hepatitis B core or surface antigen, hepatitis C virus E1 or E2 glycoprotein, core, or nonstructural proteins), herpesvirus polypeptides (including glycoproteins of herpes simplex virus or varicella zoster virus), infectious peritonitis virus polypeptides, leukemia virus polypeptides, marbuviral polypeptides, and the like. Also included are, but are not limited to, cuvirus polypeptides, orthomyxovirus polypeptides, papillomavirus polypeptides, parainfluenza virus polypeptides (e.g., hemagglutinin and neuraminidase polypeptides), paramyxovirus polypeptides, parvovirus polypeptides, pestivirus polypeptides, picornavirus polypeptides (e.g., poliovirus capsid polypeptides), poxvirus polypeptides (e.g., vaccinia virus polypeptides), rabies virus polypeptides (e.g., rabies virus glycoprotein G), reovirus polypeptides, retrovirus polypeptides, and rotavirus polypeptides.

[0225] In certain embodiments, the antigen may be a bacterial antigen. In certain embodiments, the bacterial antigen of interest may be a secreted polypeptide. In other certain embodiments, the bacterial antigen includes an antigen having one or more portions of the polypeptide exposed on the extracellular surface of the bacterium.

[0226] Antigens derived from Staphylococcus species, including methicillin-resistant Staphylococcus aureus (MRSA), that are contemplated for use include virulence regulators such as the Agr system, Sar and Sae, the Arl system, Sar homologs (Rot, MgrA, SarS, SarR, SarT, SarU, SarV, SarX, SarZ, and TcaR), the Srr system, and TRAP. Other staphylococcal proteins that serve as antigens include Clp proteins, HtrA, MsrR, aconitase, CcpA, SvrA, Msa, CfvA, and CfvB (see, e.g., "Staphylococcus: Molecular Genetics," 2008 Caister Academic Press, edited by Jodi Lindsay). The genomes of two species of Staphylococcus aureus (N315 and Mu50) have been sequenced and published, for example, in PATRIC (PATRIC: The VBI PathoSystems Resource Integration Center, see, for example, Snyder et al., 2009, and Snyder et al., 2010). As will be appreciated by those skilled in the art, staphylococcal proteins for use as antigens may also be found in other public databases, such as GENBANK®, SWISS-PROT®, and TREMBL®.

[0227] Antigens from Streptococcus pneumoniae contemplated for use in certain embodiments described herein include pneumolysin, PspA, choline-binding protein A (CbpA), NanA, NanB, SpnHL, PavA, LytA, Pht, and pilin proteins (RrgA; RrgB; RrgC). Antigenic proteins of Streptococcus pneumoniae are also known in the art and may be used as antigens in some embodiments (see, e.g., Zysk et al., 2000). The entire genome sequence of virulent strains of Streptococcus pneumoniae has been sequenced, and as will be appreciated by those of skill in the art, S. pneumoniae proteins for use herein may also be found in other public databases, such as GENBANK®, SWISS-PROT®, and TREMBL®. Proteins of particular interest for antigens according to the present disclosure include virulence factors and proteins predicted to be exposed on the surface of Streptococcus pneumoniae (see, e.g., Frolet et al., 2010).

[0228] Examples of bacterial antigens that can be used as antigens include Actinomyces polypeptides, Bacillus polypeptides, Bacteroides polypeptides, Bordetella polypeptides, Bartonella polypeptides, Borrelia polypeptides (e.g., B. burgdorferi OspA), Brucella polypeptides, Campylobacter polypeptides, Capnocytophaga polypeptides, Chlamydia polypeptides, and the like. ) polypeptides, Corynebacterium polypeptides, Coxiella polypeptides, Dermatophilus polypeptides, Enterococcus polypeptides, Ehrlichia polypeptides, Escherichia polypeptides, Francisella polypeptides, Fusobacterium polypeptides, Haemobartonella polypeptides, Haemophilus polypeptides (e.g., H. influenzae).influenza type b outer membrane protein), Helicobacter polypeptide, Klebsiella polypeptide, L-form polypeptide, Leptospira polypeptide, Listeria polypeptide, Mycobacteria polypeptide, Mycoplasma polypeptide, Neisseria polypeptide, Neorickettsia polypeptide, Nocardia polypeptide, Pasteurella polypeptide, Peptococcus polypeptide, Peptostreptococcus polypeptide These polypeptides include, but are not limited to, Pneumococcus polypeptides (i.e., Streptococcus pneumoniae polypeptides), Proteus polypeptides, Pseudomonas polypeptides, Rickettsia polypeptides, Rochalimaea polypeptides, Salmonella polypeptides, Shigella polypeptides, Staphylococcus polypeptides, Group A Streptococcus polypeptides (e.g., S. pyogenes M protein), Group B Streptococcus (S. agalactiae) polypeptides, Treponema polypeptides, and Yersinia polypeptides (e.g., Y. pestis F1 and V antigens).

[0229] Examples of fungal antigens include Absidia polypeptides, Acremonium polypeptides, Alternaria polypeptides, Aspergillus polypeptides, Basidiobolus polypeptides, Bipolaris polypeptides, Blastomyces polypeptides, Candida polypeptides, Coccidioides polypeptides, Conidiobolus polypeptides, Cryptococcus polypeptides, Curvalaria polypeptides, Epidermophyton polypeptides, Exophiala polypeptides, Geotrichum polypeptides, Histoplasma polypeptides, Madurella polypeptides, Malassezia polypeptides, Microsporum polypeptides, Peptides, Moniliella polypeptides, Mortierella polypeptides, Mucor polypeptides, Paecilomyces polypeptides, Penicillium polypeptides, Phialemonium polypeptides, Phialophora polypeptides, Prototheca polypeptides, Pseudallescheria polypeptides, Pseudomicrodochium polypeptides, Pythium polypeptides, Rhinosporidium polypeptides, Rhizopus polypeptides, Scolecobasidium polypeptides, Sporothrix polypeptides, Stemphylium polypeptides, Trichophyton polypeptides, Trichosporon polypeptides,and Xylohypha polypeptides, but are not limited to them.

[0230] Examples of protozoan parasitic antigens include, but are not limited to, Babesia polypeptides, Balantidium polypeptides, Besnoitia polypeptides, Cryptosporidium polypeptides, Eimeria polypeptides, Encephalitozoon polypeptides, Entamoeba polypeptides, Giardia polypeptides, Hammondia polypeptides, Hepatozoon polypeptides, Isospora polypeptides, Leishmania polypeptides, Microsporidia polypeptides, Neospora polypeptides, Nosema polypeptides, Pentatrichomonas polypeptides, and Plasmodium polypeptides. Examples of helminth parasite antigens include Acanthocheilonema polypeptides, Aelurostrongylus polypeptides, Ancylostoma polypeptides, Angiostrongylus polypeptides, Ascaris polypeptides, Brugia polypeptides, Bunostomum polypeptides, Capillaria polypeptides, Chabertia polypeptides, Cooperia polypeptides, Crenoso polypeptides, Crenosoma polypeptides, Dictyocaulus polypeptides, Dioctophyme polypeptides, Dipetalonema polypeptides, Diphyllobothrium polypeptides, Diplydium polypeptides, Dirofilaria polypeptides, Dracunculus polypeptides, Enterobius polypeptides, Filaroides polypeptides,Haemonchus Polypeptide, Lagochilascaris Polypeptide, Loa Polypeptide, Mansonella Polypeptide, Muellerius Polypeptide, Nanophyetus Polypeptide, Necator Polypeptide, Nematodirus Polypeptide, Oesophagostomum Polypeptide, Onchocerca Polypeptide, Opisthorchis Polypeptide, Ostertagia Polypeptide, Parafilaria Polypeptide, Paragonimus Polypeptide, Parascaris Polypeptide, Physaloptera Polypeptide Peptides include, but are not limited to, Protostrongylus polypeptides, Setaria polypeptides, Spirocerca polypeptides, Spirometra polypeptides, Stephanofilaria polypeptides, Strongyloides polypeptides, Strongylus polypeptides, Thelazia polypeptides, Toxascaris polypeptides, Toxocara polypeptides, Trichinella polypeptides, Trichostrongylus polypeptides, Trichuris polypeptides, Uncinaria polypeptides, and Wuchereria polypeptides. (e.g., P. falciparum circumsporozoite (PfCSP)), sporozoite surface protein 2 (PfSSP2), carboxyl terminus of liver state antigen (PfLSA1 c-term), and exported protein 1 (PfExp-1),Pneumocystis polypeptides, Sarcocystis polypeptides, Schistosoma polypeptides, Theileria polypeptides, Toxoplasma polypeptides, and Trypanosoma polypeptides.

[0231] Examples of ectoparasite antigens include, but are not limited to, polypeptides (including antigens and allergens) from fleas; ticks, including hard mites and ulcerative colitis; flies, such as midges, mosquitoes, sand flies, black flies, bot flies, horn flies, deer flies, tsetse flies, stable flies, myiasis-causing flies and midges; ants; spiders, lice; mites, and hemipteran insects, such as bed bugs and assassin bugs. D. Suicide gene

[0232] In some cases, any cell of the present disclosure is modified to produce one or more agents other than heterologous cytokines, engineered receptors, etc. In specific embodiments, cells such as NK cells are engineered to harbor one or more suicide genes, the term "suicide gene" as used herein being defined as a gene that converts a gene product into a compound that kills the host cell when a prodrug is administered. In some cases, NK cell therapy may be contingent on the use of one or more suicide genes of any type if the patient exhibits one or more symptoms of, or is deemed to be at risk (including imminent risk) of, one or more adverse events, such as cytokine release syndrome, neurotoxicity, anaphylaxis / allergy, and / or on-target / off-tumor toxicity. The use of a suicide gene may be part of a planned protocol of treatment or may be used only when its use is recognized as necessary. In some cases, cell therapy is terminated by the use of an agent targeting a suicide gene or a gene product therefrom because treatment is no longer necessary.

[0233] Examples of suicide genes include engineered non-secreted (including membrane-bound) tumor necrosis factor (TNF) alpha mutant polypeptides (see PCT / US19 / 62009, incorporated herein by reference in its entirety), which can be targeted by antibodies that bind to TNF alpha mutants. Examples of suicide gene / prodrug combinations include herpes simplex virus thymidine kinase (HSV-tk) and ganciclovir, acyclovir, or FIAU; oxidoreductase and cycloheximide; cytosine deaminase and 5-fluorocytosine; thymidine kinase-thymidylate kinase (Tdk::Tmk) and AZT; and deoxycytidine kinase and cytosine arabinoside. The so-called suicide gene, Escherichia coli (E. coli) purine nucleoside phosphorylase, which converts the prodrug 6-methylpurine deoxyriboside to the toxic purine 6-methylpurine, may also be utilized. Other suicide genes include, for example, CD20, CD52, inducible caspase 9, purine nucleoside phosphorylase (PNP), cytochrome p450 enzymes (CYP), carboxypeptidase (CP), carboxylesterase (CE), nitroreductase (NTR), guanine ribosyltransferase (XGRTP), glycosidase enzymes, methionine-α,γ-lyase (MET), and thymidine phosphorylase (TP). E. Delivery method

[0234] In specific embodiments, any of the compositions can be delivered to recipient immune effector cells by any suitable method. The compositions can be delivered to cells, for example, by electroporation or by a vector. In specific embodiments, for example, one or more compositions for introducing at least one or more heterologous antigen receptors are delivered to immune effector cells in a vector. In some embodiments, one or more compositions for gene editing are delivered to cells in a vector. Those skilled in the art will be familiar with the knowledge to construct vectors using standard recombinant techniques (see, for example, Sambrook et al., 2001 and Ausubel et al., 1996) to express the antigen receptors of the present disclosure. Vectors include plasmids, cosmids, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs), such as retroviral vectors (e.g., derived from Moloney murine leukemia virus vector (MoMLV), MSCV, SFFV, MPSV, SNV, etc.), lentiviral vectors (e.g., derived from HIV-1, HIV-2, SIV, BIV, FIV, etc.), adenoviral (Ad) vectors (including replication-competent, replication-deficient, and gutless forms thereof), adeno-associated virus (AAV) vectors, simian virus 40 (SV-40) vectors, bovine papillomavirus vectors, Epstein-Barr virus vectors, herpesvirus vectors, vaccinia virus vectors, Harvey murine sarcoma virus vectors, mouse mammary tumor virus vectors, Rous sarcoma virus vectors, parvovirus vectors, poliovirus vectors, vesicular stomatitis virus vectors, Maraba virus vectors, and the like. Examples of vectors that can be used include, but are not limited to, adenovirus vectors and group B adenovirus enadenotucirev vectors.

[0235] In specific embodiments, the vector is a polycistronic vector, such as those described in PCT / US19 / 62014, which is incorporated herein by reference in its entirety. In such cases, a single vector may encode one or more CARs and / or TCRs (and the expression construct may be configured in a modular manner, allowing portions of the CAR or TCR to be interchangeable), a suicide gene, and / or one or more cytokines. In some embodiments, at least one activating CAR and at least one inhibitory CAR are contained in a single vector. 1. Viral Vectors

[0236] In certain embodiments of the present disclosure, a viral vector encoding an antigen receptor may be provided. When generating a recombinant viral vector, typically, non-essential genes are replaced with genes or coding sequences for heterologous (or non-native) proteins. A viral vector is a type of expression construct that utilizes viral sequences to introduce nucleic acids and, optionally, proteins into cells. The ability of certain viruses to infect cells or enter cells by receptor-mediated endocytosis and integrate into the host cell genome to stably and efficiently express viral genes makes them attractive candidates for introducing foreign nucleic acids into cells (e.g., mammalian cells). Non-limiting examples of viral vectors that can be used to deliver nucleic acids of certain embodiments of the present invention are described below.

[0237] Lentiviruses are complex retroviruses that contain the common retroviral genes gag, pol, and env, as well as other genes with regulatory or structural functions. Lentiviral vectors are well known in the art (see, e.g., U.S. Patent Nos. 6,013,516 and 5,994,136).

[0238] Recombinant lentiviral vectors have the ability to infect non-dividing cells and can be used for gene transfer and expression of nucleic acid sequences both in vivo and ex vivo. For example, recombinant lentiviruses capable of infecting non-dividing cells, in which suitable host cells are transfected with two or more vectors carrying packaging functions, i.e., gag, pol, and env, and rev and tat, are described in U.S. Patent No. 5,994,136, incorporated herein by reference. a. Control Elements

[0239] The expression cassette contained in the vector useful in the present disclosure contains, inter alia, a eukaryotic transcriptional promoter operably linked (5' to 3') to the protein-coding sequence, a splice signal including intervening sequences, and a transcription termination / polyadenylation sequence. The promoters and enhancers that control the transcription of protein-coding genes in eukaryotic cells are composed of multiple genetic elements. The cellular machinery is able to collect and integrate the regulatory information carried by each element, thereby allowing different genes to develop distinct, often complex, patterns of transcriptional regulation. Promoters used in the context of the present disclosure include constitutive, inducible, and tissue-specific promoters. b. promoter / enhancer

[0240] The expression constructs provided herein include a promoter that drives expression of an antigen receptor. Promoters generally contain sequences that function to position the start site for RNA synthesis. The most well-known example is the TATA box, but in some promoters lacking a TATA box, such as the promoters of the mammalian terminal deoxynucleotidyl transferase gene and the SV40 late gene promoter, discrete elements surrounding the start site themselves help to fix the start location. Additional promoter elements control the frequency of transcription initiation. Typically, these are located 30–110 bp upstream from the start site, although some promoters have been shown to contain functional elements downstream of the start site. To place a coding sequence "under the control" of a promoter, the 5' end of the transcription initiation site of the open reading frame is positioned "downstream" (i.e., 3') of the chosen promoter. This "upstream" promoter stimulates DNA transcription and promotes expression of the encoded RNA.

[0241] Spacing between promoter elements is often flexible, so elements can be inverted or moved relative to one another while still retaining promoter function. In the tk promoter, increasing the spacing between promoter elements by up to 50 bp does not begin to reduce activity. Depending on the promoter, individual elements appear to be able to function either cooperatively or independently to activate transcription. Promoters may or may not be used in conjunction with "enhancers," which refer to cis-acting regulatory sequences involved in the transcriptional activation of a nucleic acid sequence.

[0242] A promoter may be one naturally associated with a nucleic acid sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of a coding segment and / or exon. Such a promoter may be referred to as "endogenous." Similarly, an enhancer may be one naturally associated with a nucleic acid sequence, located either downstream or upstream of the sequence. In some embodiments, the promoter and / or enhancer may be that of an endogenous gene. In some embodiments, the promoter and / or enhancer may be associated with a "safe harbor" locus known in the art. Alternatively, in certain aspects, certain advantages may be achieved by placing a coding nucleic acid segment under the control of a recombinant or heterologous promoter (which refers to a promoter not normally associated with a nucleic acid sequence in its natural environment). A recombinant or heterologous enhancer also refers to an enhancer not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers can include promoters or enhancers from other genes, promoters or enhancers isolated from any other viruses or prokaryotic or eukaryotic cells, and promoters or enhancers that are not "naturally occurring," i.e., contain different elements of different transcriptional control regions and / or mutations that alter expression. For example, promoters most commonly used in recombinant DNA construction include the β-lactamase (penicillinase), lactose, and tryptophan (trp) promoter systems. For the compositions disclosed herein, in addition to synthetically producing promoter and enhancer nucleic acid sequences, sequences may also be generated using recombinant cloning and / or nucleic acid amplification techniques, including PCR™. Furthermore, it is contemplated that control sequences that direct transcription and / or expression of sequences in organelles other than the nucleus, such as mitochondria and chloroplasts, may also be used.

[0243] Of course, it will be important to use a promoter and / or enhancer that effectively directs expression of the DNA segment in the organelle, cell type, tissue, organ, or organism chosen for expression. Those skilled in the art of molecular biology are generally aware of the use of promoter, enhancer, and cell type combinations for protein expression (see, e.g., Sambrook et al. 2001). The promoter used may be constitutive, tissue-specific, inducible, and / or useful under appropriate conditions for directing high-level expression of the introduced DNA segment, such as would be advantageous for large-scale production of recombinant proteins and / or peptides. The promoter may be heterologous or endogenous.

[0244] Additionally, any promoter / enhancer combination could be used to drive expression (e.g., according to the Eukaryotic Promoter Database EPDB, available on the World Wide Web at epd.isb-sib.ch / ). Use of the T3, T7, or SP6 cytoplasmic expression systems is another possible embodiment. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional gene expression construct.

[0245] Non-limiting examples of promoters include early or late viral promoters, such as the SV40 early or late promoter, the cytomegalovirus (CMV) immediate early promoter, and the Rous sarcoma virus (RSV) early promoter; eukaryotic promoters, such as the beta-actin promoter, the GAPDH promoter, the metallothionein promoter, and linked response element promoters, such as the cyclic AMP response element promoter (cre), the serum response element promoter (sre), the phorbol ester promoter (TPA), and the minimal TATA box-proximal response element promoter (tre). Human growth hormone promoter sequences (e.g., the human growth hormone minimal promoter described in Genbank, accession number X05244, nucleotides 283-341) or the mouse mammary tumor promoter (available from ATCC catalog number ATCC 45007) can also be used. In certain embodiments, the promoter is a CMV IE, Dectin-1, Dectin-2, human CD11c, F4 / 80, SM22, RSV, SV40, AdMLP, beta-actin, MHC class I, or MHC class II promoter, although any other promoter useful for driving expression of a therapeutic gene can be applied in the practice of the present disclosure.

[0246] In certain embodiments, the methods of the present disclosure also relate to enhancer sequences, i.e., nucleic acid sequences that increase the activity of a promoter and have the potential to act in cis, regardless of their orientation, even over relatively long distances (up to several kilobases away from the target promoter), although enhancer function is not necessarily limited to such long distances, as they can also function in the vicinity of a given promoter. c. Initiation signal and cascading expression

[0247] Specific initiation signals may also be used in the expression constructs provided in this disclosure for efficient translation of the coding sequence. These signals include the ATG initiation codon or adjacent sequences. It may be necessary to provide exogenous translational control signals, including the ATG initiation codon. One of ordinary skill in the art would be readily able to determine this and provide the necessary signals. It is well known that the initiation site must be "in-frame" with the reading frame of the desired coding sequence to ensure translation of the entire insert. Exogenous translational control signals and initiation codons can be either natural or synthetic. The efficiency of expression may be enhanced by including appropriate transcriptional enhancer elements.

[0248] In certain embodiments, the use of internal ribosome entry site (IRES) elements is used to create multigene or polycistronic messages. IRES elements can bypass the ribosome scanning model of 5' methylated cap-dependent translation and initiate translation at an internal site. IRES elements from two members of the picornavirus family (polio and encephalomyocarditis) have been described, along with IRESs from mammalian messages. IRES elements can be linked to heterologous open reading frames. Polycistronic messages can be created by transcribing multiple open reading frames together, each separated by an IRES. Thanks to the IRES element, each open reading frame can be utilized by the ribosome for efficient translation. Transcription of a single message using a single promoter / enhancer allows efficient expression of multiple genes.

[0249] Additionally, certain 2A sequence elements could be used to induce linked or co-expression of genes in the constructs provided herein. For example, cleavage sequences could be used to link open reading frames to form a single cistron, thereby co-expressing genes. Exemplary cleavage sequences are F2A (foot-and-mouth disease virus 2A) or "2A-like" sequences (e.g., Thosea asigna virus 2A; T2A). d. Origin of replication

[0250] To propagate a vector in a host cell, the vector may contain one or more origin of replication sites (often referred to as "ori"), which are specific nucleic acid sequences that serve as the starting point of replication, such as a nucleic acid sequence corresponding to the EBV oriP described above, or a genetically engineered oriP with a similar or improved function in programming. Alternatively, the replication origins or autonomously replicating sequences (ARS) of other extrachromosomally replicating viruses described above can be used. e. Selectable and screenable markers

[0251] In some embodiments, cells containing a construct of the present disclosure can be identified in vitro or in vivo by including a marker in the expression vector. Such a marker would confer an identifiable change to the cell, facilitating identification of cells containing the expression vector. Generally, a selectable marker confers a property that allows for selection. A positive selectable marker is one in which the presence of the marker allows for its selection, while a negative selectable marker is one in which its presence prevents its selection. One example of a positive selectable marker is a drug resistance marker.

[0252] Typically, the inclusion of a drug selection marker aids in the cloning and identification of transformants; for example, genes conferring resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, and histidinol are useful selection markers. In addition to markers that confer a phenotype that allows for the identification of transformants based on conditionality, other types of markers are contemplated, including colorimetrically based screenable markers such as GFP. Alternatively, screenable enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be utilized as negative selection markers. Those skilled in the art will also know how to use immunological markers, possibly in conjunction with FACS analysis. The marker used is not believed to be critical, so long as it can be expressed simultaneously with the nucleic acid encoding the gene product. Further examples of selection and screenable markers are well known to those skilled in the art. 2. Other nucleic acid delivery methods

[0253] In addition to viral delivery of nucleic acids encoding antigen receptors, the following methods are also methods of recombinant gene delivery into a given host cell and are therefore contemplated in this disclosure.

[0254] The introduction of nucleic acids such as DNA or RNA into immune cells of the present disclosure can be carried out using any suitable nucleic acid delivery method for transforming cells, as described herein or as would be known to those of ordinary skill in the art. Such methods include, but are not limited to, direct delivery of DNA, such as by ex vivo transfection, injection, including microinjection; calcium phosphate precipitation; DEAE-dextran followed by polyethylene glycol; direct sonic loading; liposome-mediated transfection and receptor-mediated transfection; microprojectile bombardment; agitation with silicon carbide fibers; Agrobacterium-mediated transformation; desiccation / inhibition-mediated DNA uptake, and any combination of such methods. By applying such techniques, organelles, cells, tissues, or organisms can be stably or transiently transformed. V. Gene Editing and CRISPR

[0255] The NK cell production process of the present disclosure can include gene editing of NK cells to remove 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more endogenous genes in the NK cells. In some cases, gene editing is performed in NK cells that express one or more xenogeneic antigen receptors, while in other cases, gene editing is performed in NK cells that do not express xenogeneic antigen receptors, but that will eventually express one or more xenogeneic antigen receptors, at least in some cases. In certain embodiments, the gene-edited NK cells are expanded NK cells. In certain embodiments, the gene-edited NK cells are derived from progenitor cells that have previously been gene-edited.

[0256] In certain cases, one or more endogenous genes of NK cells are modified, e.g., expression is disrupted, resulting in partial or complete reduction of expression. In specific cases, one or more genes are knocked down or knocked out using the processes of the present disclosure. In specific cases, multiple genes are knocked down or knocked out in the same step of the processes of the present disclosure. The genes edited in NK cells can be of any type, but in specific embodiments, the genes are genes whose gene products inhibit the activity and / or proliferation of NK cells. In specific cases, the genes edited in NK cells enable the NK cells to function more effectively in the tumor microenvironment. In a specific embodiment, the gene is one or more of NKG2A, SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, ADORA2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1, ADAM17, RPS6, 4EBP1, CD25, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, TDAG8, CD5, CD7, SLAMF7, CD38, LAG3, TCR, beta2-microglobulin, HLA, CD73, and CD39. In a specific embodiment, the TGFBR2 gene is knocked out or down in NK cells. In a specific embodiment, the CISH gene is knocked out or down in NK cells. In a specific embodiment, the CD38 gene is knocked out or down in NK cells. In a specific embodiment, the CISH gene and the CD38 gene are knocked out or knocked down in the cells.

[0257] In some embodiments, gene editing is performed using one or more DNA-binding nucleic acids, for example, by RNA-guided endonuclease (RGEN)-mediated modification. For example, modification can be performed using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins. Generally, the term "CRISPR system" refers to the transcripts and other elements involved in the expression or directing the activity of CRISPR-associated ("Cas") genes, including sequences encoding Cas genes, tracr (transactivating CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr mate sequences (including "direct repeats" and partial direct repeats processed by tracrRNA in the context of endogenous CRISPR systems), guide sequences (also referred to as "spacers" in the context of endogenous CRISPR systems), and / or other sequences and transcripts from CRISPR loci.

[0258] A CRISPR / Cas nuclease or CRISPR / Cas nuclease system can include a non-coding RNA molecule (guide) RNA that binds to DNA in a sequence-specific manner and a Cas protein (e.g., Cas9) with nuclease functionality (e.g., two nuclease domains). One or more elements of a CRISPR system can be derived from a Type I, Type II, or Type III CRISPR system derived from a particular organism that contains an endogenous CRISPR system, such as, for example, Streptococcus pyogenes.

[0259] In some embodiments, a Cas nuclease and a gRNA (comprising a fusion of a target sequence-specific crRNA and a fixed tracrRNA) are introduced into cells. Generally, a target site at the 5' end of the gRNA targets the Cas nuclease to the target site, e.g., a gene, using complementary base pairing. The target site can be selected based on its location immediately 5' to a protospacer adjacent motif (PAM) sequence, typically NGG or NAG. In this regard, the gRNA is targeted to a desired sequence by modifying the first 20, 19, 18, 17, 16, 15, 14, 14, 12, 11, or 10 nucleotides of the guide RNA to correspond to the target DNA sequence. Generally, CRISPR systems are characterized by elements that promote the formation of a CRISPR complex at the site of the target sequence. Generally, "target sequence" refers broadly to a sequence to which a guide sequence is designed to be complementary, and hybridization between the target sequence and the guide sequence promotes the formation of a CRISPR complex. Absolute complementarity is not required, as long as there is sufficient complementarity to cause hybridization and promote the formation of a CRISPR complex.

[0260] As described herein, the CRISPR system can induce a double-stranded break (DSB) at a target site followed by damage or remodeling. In another embodiment, a Cas9 variant, considered a "nickase," is used to nick a single strand at the target site. For example, to improve specificity, a pair of nickases can be used, each directed by a pair of different gRNA targeting sequences, such that a 5' overhang is introduced when the nicks are introduced simultaneously. In another embodiment, a catalytically inactive Cas9 is fused to a heterologous effector domain, such as a transcriptional repressor or activator, to affect gene expression.

[0261] The target sequence can comprise any polynucleotide, such as a DNA polynucleotide or an RNA polynucleotide. The target sequence can be located in the nucleus or cytoplasm of a cell, for example, within an organelle of a cell. Generally, a sequence or template for recombination into a target locus that includes a target sequence is referred to as an "editing template" or an "editing polynucleotide" or an "editing sequence." In some embodiments, the exogenous template polynucleotide can be referred to as an editing template. In some embodiments, the recombination is homologous recombination.

[0262] Typically, in the context of an endogenous CRISPR system, formation of a CRISPR complex (including a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands at or near the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 base pairs or more from the target sequence). A tracr sequence that includes or consists of all or a portion of a wild-type tracr sequence (e.g., about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of the wild-type tracr sequence, or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of the wild-type tracr sequence) can also form part of a CRISPR complex, for example, by hybridization along at least a portion of the tracr sequence to all or a portion of a tracr mate sequence operably linked to a guide sequence. The tracr sequence has sufficient complementarity to the tracr mate sequence to hybridize and participate in the formation of a CRISPR complex, e.g., at least 50%, 60%, 70%, 80%, 90%, 95% or 99% sequence complementarity over the entire length of the tracr mate sequence when optimally aligned.

[0263] One or more vectors driving the expression of one or more elements of a CRISPR system can be introduced into a cell such that expression of those CRISPR system elements directs the formation of CRISPR complexes at one or more target sites. Components can also be delivered to a cell as protein and / or RNA. For example, a Cas enzyme, a guide sequence linked to a tracr mate sequence, and a tracr sequence could each be operably linked to separate control elements on separate vectors. Alternatively, two or more elements expressed from the same or different control elements can be combined in a single vector, with one or more additional vectors providing any components of the CRISPR system not included in the initial vector. A vector can contain one or more insertion sites (also referred to as "cloning sites"), such as restriction endonuclease recognition sequences. In some embodiments, the one or more insertion sites are located upstream and / or downstream of one or more sequence elements in one or more vectors. When multiple different guide sequences are used, a single expression construct can be used to target CRISPR activity to multiple different corresponding target sequences within a cell.

[0264] The vector can include regulatory elements operably linked to an enzyme coding sequence that encodes a CRISPR enzyme, such as a Cas protein. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Csel, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof. These enzymes are known, for example the amino acid sequence of the Streptococcus pyogenes Cas9 protein can be found in the SwissProt database under accession number Q99ZW2.

[0265] The CRISPR enzyme can be Cas9 (e.g., from Streptococcus pyogenes or Streptococcus pneumoniae). The CRISPR enzyme can direct cleavage of one or both strands at the location of a target sequence, e.g., within the target sequence and / or within the complementary strand of the target sequence. The vector can encode a CRISPR enzyme that is mutated relative to the corresponding wild-type enzyme such that the mutant CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing the target sequence. For example, an aspartic acid to alanine substitution (D10A) in the RuvCI catalytic domain of Cas9 from Streptococcus pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaving a single strand). In some embodiments, Cas9 nickase can be used in combination with guide sequences, e.g., two guide sequences, that target the sense and antisense strands of a DNA target, respectively. This combination allows nicking of both strands, which can then be used to induce NHEJ or HDR.

[0266] In some embodiments, the enzyme coding sequence encoding the CRISPR enzyme is codon-optimized for expression in a specific cell, such as a eukaryotic cell. The eukaryotic cell may be a cell of a specific organism, such as a mammal, including but not limited to a human, mouse, rat, rabbit, dog, or non-human primate, or may be derived from such a specific animal. Generally, codon optimization refers to the process of modifying a nucleic acid sequence to enhance expression in a host cell of interest by replacing at least one codon of the native sequence with a codon that is more frequently or most frequently used in the host cell's genes while maintaining the native amino acid sequence. Different species exhibit specific biases toward certain codons for certain amino acids. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of messenger RNA (mRNA) translation, which is thought to depend, among other things, on the nature of the codon being translated and the availability of specific transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a given cell generally reflects the codons most frequently used in peptide synthesis, and thus codon optimization can be used to tailor genes for optimal gene expression in a given organism.

[0267] Generally, a guide sequence is any polynucleotide sequence that has sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of a CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or more, or greater than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or more.

[0268] Optimal alignment may be determined using any suitable algorithm for aligning sequences, non-limiting examples of such algorithms include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler transformation (e.g., the Burrows Wheeler aligner), Clustal W, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).

[0269] CRISPR enzymes may be part of a fusion protein containing one or more heterologous protein domains. CRISPR enzyme fusion proteins may contain any additional protein sequences and, optionally, a linker sequence between any two domains. Examples of protein domains that can be fused to CRISPR enzymes include, but are not limited to, epitope tags, reporter gene sequences, and protein domains with one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporter genes include, but are not limited to, glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP). CRISPR enzymes can be fused to gene sequences encoding proteins or protein fragments that bind to DNA molecules or other cellular molecules, such as, but not limited to, maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GAL4A DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. Additional domains that can form part of fusion proteins containing CRISPR enzymes are described in US20110059502. VI. Treatment Method

[0270] In some embodiments, immune effector cells produced by the methods of the present disclosure are used in methods of treating individuals in need of treatment. Embodiments of the present disclosure include methods of treating individuals for, for example, cancer, any type of infectious disease, and / or any immune disorder. The individual may use the treatment methods of the present disclosure as a first-line treatment or after (and / or in conjunction with) another treatment. In cancer embodiments, immunotherapy can be tailored to the needs of the individual with cancer based on the type and / or stage of the cancer, and in at least some cases, immunotherapy can be modified during the course of the individual's treatment.

[0271] In specific cases, exemplary treatment methods are: 1) adoptive cell therapy with engineered immune effector cells (ex vivo expanded or expressing a CAR or TCR) to treat cancer patients with any type of hematological malignancy; (2) adoptive cell therapy with engineered immune effector cells (ex vivo expanded or expressing a CAR or TCR) to treat cancer patients with any type of solid tumor; and (3) adoptive cell therapy with engineered immune effector cells (ex vivo expanded or expressing a CAR or TCR) to treat patients with infectious diseases and / or immune disorders.

[0272] In some embodiments, the present disclosure provides a method for immunotherapy, comprising administering an effective amount of immune effector cells produced by the methods of the present disclosure. In one embodiment, at least in certain cases, a medical disease or disorder is treated by one or more introductions of immune response-inducing immune effector cell populations produced by the methods of the present disclosure. In certain embodiments of the present disclosure, cancer or infectious diseases are treated by delivery of one or more immune response-inducing immune effector cell populations produced by the methods of the present disclosure. Provided herein is a method for treating cancer or delaying the progression of cancer in an individual, comprising administering an effective amount of antigen-specific cell therapy. The method may be applicable to the treatment of immune disorders, solid cancers, hematological cancers, and / or viral infections.

[0273] The tumors that this treatment method is useful for include any malignant cell type, such as those found in solid tumors or blood tumors.Exemplary solid tumors include, but are not limited to, tumors of organs selected from the group consisting of pancreas, colon, appendix, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and breast.Exemplary blood tumors include bone marrow tumors, malignant diseases of T-cell or B-cell, leukemia, lymphoma, blastoma, myeloma, etc. Further examples of cancers that may be treated using the methods provided herein include, but are not limited to, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), cancer of the peritoneum, stomach or gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, various types of head and neck cancer, and melanoma.

[0274] Cancer may specifically be of the following histological types, but is not limited to: neoplasia, malignant; carcinoma; carcinoma, undifferentiated; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; mixed hepatocellular and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; adenocarcinoma, familial adenomatous polyposis; solid tumor; carcinoid tumor, malignant; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophilic carcinoma; acidophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; condylar carcinoma Granular cell carcinoma; Follicular adenocarcinoma; Papillary follicular gland carcinoma; Non-encapsulated sclerosing carcinoma; Adrenal cortical carcinoma; Endometrioid carcinoma; Adnexal carcinoma; Apocrine gland carcinoma; Sebaceous gland carcinoma; Ceruminous gland carcinoma; Mucoepidermoid carcinoma; Cystadenocarcinoma; Papillary cystadenocarcinoma; Papillary serous cystadenocarcinoma; Mucinous cystadenocarcinoma; Mucinous adenocarcinoma; Signet ring cell carcinoma; Invasive ductal carcinoma; Medullary carcinoma; Lobular carcinoma; Inflammatory carcinoma; Paget's disease of the breast; Acinic cell carcinoma; Adenosquamous carcinoma; Adenocarcinoma with squamous metaplasia; Thymoma, malignant; Ovarian stromal tumor, malignant; Theca cell tumor, malignant; Granulosa cell tumor, malignant; Androblastoma, malignant; Sertoli cell carcinoma; Leydig cell tumor, malignant; Lipidocytoma, malignant ; Paraganglioma, malignant; Extramammary paraganglioma, malignant; Pheochromocytoma; Glomus angiosarcoma; Malignant melanoma; Amelanotic melanoma; Superficial spreading melanoma; Lentigo maligna melanoma; Acral lentigo melanoma; Nodular melanoma; Malignant melanoma in giant pigmented nevus; Epithelioid cell melanoma; Blue nevus, malignant; Sarcoma; Fibrosarcoma; Fibrous histiocytoma, malignant; Myxosarcoma; Liposarcoma; Leiomyosarcoma; Rhabdomyosarcoma; Embryonic rhabdomyosarcoma; Alveolar rhabdomyosarcoma; Stromal sarcoma; Mixed tumor, malignant; Mixed Müllerian tumor; Nephroblastoma; Hepatoblastoma; Carcinosarcoma; Mesenchymoma, malignant; Brenner tumor, malignant; Phyllodes tumor, malignant; Synovial sarcoma; Mesothelioma, Malignant; Dysgerminoma; Embryonal carcinoma; Teratoma, malignant; Ovarian goiter, malignant; Choriocarcinoma; Mesonephroma, malignant; Angiosarcoma; Hemangioendothelioma, malignant; Kaposi's sarcoma; Hemangiopericytoma, malignant; Lymphangiosarcoma; Osteosarcoma; Parosteal osteosarcoma; Chondrosarcoma; Chondroblastoma, malignant; Mesenchymal chondrosarcoma; Giant cell tumor of bone; Ewing's sarcoma; Odontogenic tumor, malignant; Ameloblastic odontosarcoma; Ameloblastoma, malignant; Ameloblastic fibrosarcoma; Pinealoma, malignant; Chordoma; Glioma, malignant; Ependymoma; Astrocytoma; Protoplasmic astrocytoma; Fibrillar astrocytoma; Astroblastoma; Glioblastoma; Oligodendroglioma; Oligodendroglioma;Primitive neuroectodermal; Cerebellar sarcoma; Ganglioneuroblastoma; Neuroblastoma; Retinoblastoma; Olfactory neurogenic tumor; Meningioma, malignant; Neurofibrosarcoma; Schwannoma, malignant; Granular cell tumor, malignant; Malignant lymphoma; Hodgkin's disease; Hodgkin's; Lateral granuloma; Malignant lymphoma, small lymphocytic; Malignant lymphoma, large cell, diffuse; Malignant lymphoma, follicular; Mycosis fungoides; Other specified non-Hodgkin's lymphoma; B-cell lymphoma; Low-grade / follicular non-Hodgkin's lymphoma (NHL); Small lymphocytic (SL) NHL; Intermediate-grade / follicular NHL; Intermediate-grade diffuse NHL; High-grade immunoblastic NHL; High-grade lymphoblastic NHL; High-grade small non-cleaved cell NHL; giant mass disease NHL; mantle cell lymphoma; AIDS-related lymphoma; Waldenstrom's macroglobulinemia; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; hairy cell leukemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML), and chronic myeloblastic leukemia.

[0275] Certain embodiments relate to methods for treating hematological malignancies, such as lymphoma or leukemia. Leukemia is a cancer of the blood or bone marrow, characterized by the abnormal proliferation (production by multiplication) of blood cells, usually white blood cells (leukocytes). It is part of a broad group of diseases called hematological neoplasms. Leukemia is a broad term that covers a range of diseases. Clinically and pathologically, leukemia is divided into acute and chronic forms.

[0276] In certain embodiments of the present disclosure, immune cells are delivered to an individual in need thereof, for example, an individual with cancer or an infectious disease. These cells then strengthen the individual's immune system to attack the respective cancer or pathogenic cells. In some cases, the individual is given immune cells one or more times. In cases where an individual is given immune cells more than once, the interval between administrations should be sufficient to allow time for proliferation in the individual, and in specific embodiments, the interval between administrations is 1, 2, 3, 4, 5, 6, 7 days or more, or 1, 2, 3, or 4 weeks or more, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more.

[0277] Certain embodiments of the present disclosure provide methods for treating or preventing immune-mediated disorders. In one embodiment, the subject has an autoimmune disease. Non-limiting examples of autoimmune diseases include alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune disease of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac disease, and rheumatoid arthritis. spate-dermatitis, chronic fatigue and immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Guillain-Barré, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, lupus erthematosus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, type 1 or immune-mediated diabetes mellitus, myasthenia gravis, nephrotic syndrome (minimal change disease), focal glomerular sclerosis, or membranous nephropathy nephropathy), pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, stiff-man syndrome, systemic lupus erythematosus, ulcerative colitis, uveitis, vasculitis (such as polyarteritis nodosa, Takayasu's arteritis, temporal arteritis / giant cell arteritis, or dermatitis herpetiformis vasculitis), vitiligo, and Wegener's granulomatosis. Thus, some examples of autoimmune diseases that can be treated using the methods disclosed herein include, but are not limited to, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, type I diabetes, Crohn's disease, ulcerative colitis, myasthenia gravis, glomerulonephritis, ankylosing spondylitis, vasculitis, or psoriasis. The subject may also have an allergic disorder, such as asthma.

[0278] In yet another embodiment, the subject is a recipient of a transplanted organ or transplanted stem cells, and the immune cells are used to prevent and / or treat rejection. In certain embodiments, the subject has or is at risk of developing graft-versus-host disease. GVHD is a potential complication of any transplant that uses or contains stem cells from either related or unrelated donors. There are two types of GVHD: acute and chronic. Acute GVHD appears within the first three months after transplant. Symptoms of acute GVHD include a reddish rash on the hands and feet, which can spread and become more severe, accompanied by peeling or blistering of the skin. Acute GVHD can also affect the stomach and intestines, causing cramps, nausea, and diarrhea. Yellowing of the skin and eyes (jaundice) indicates that acute GVHD is affecting the liver. Chronic GVHD is classified based on its severity: Stage / Grade 1 is mild, and Stage / Grade 4 is severe. Chronic GVHD develops three months or more after transplantation. Symptoms of chronic GVHD are similar to those of acute GVHD, but in addition, chronic GVHD can affect the mucous glands in the eyes, the salivary glands in the mouth, and the glands that lubricate the stomach lining and intestines. Any of the populations of immune cells disclosed herein can be utilized. Examples of transplanted organs include solid organ transplants, such as kidney, liver, skin, pancreas, lung, and / or heart, or cell transplants, such as islets, hepatocytes, myoblasts, bone marrow, or hematopoietic or other stem cells. The transplant can be a composite transplant, such as facial tissue. The immune cells can be administered prior to, concurrently with, or after transplantation. In some embodiments, the immune cells are administered prior to transplant, e.g., at least 1 hour, at least 12 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, or at least 1 month prior to transplant. In one specific, non-limiting example, administration of a therapeutically effective amount of immune cells occurs 3-5 days prior to transplant.

[0279] In some embodiments, the subject may be administered a non-myeloablative lymphodepleting chemotherapy prior to immune cell therapy. The non-myeloablative lymphodepleting chemotherapy may be any suitable such treatment, and may be administered by any suitable route. The non-myeloablative lymphodepleting chemotherapy may include, for example, administration of cyclophosphamide and fludarabine, particularly when the cancer is melanoma and may be metastatic. An exemplary administration route for cyclophosphamide and fludarabine is intravenous. Similarly, any suitable dose of cyclophosphamide and fludarabine may be administered. In certain embodiments, around 60 mg / kg of cyclophosphamide is administered for two days, followed by 25 mg / m 2 Fludarabine is administered for 5 days before and after.

[0280] In certain embodiments, one or more growth factors that promote the proliferation and activation of NK cells are administered to a subject simultaneously with or after the NK cells. The growth factors can be any suitable growth factor that promotes the growth and activation of NK cells. Examples of suitable immune cell growth factors include interleukin (IL)-2, IL-7, IL-12, IL-15, IL-18, and IL-21, which can be used alone or in various combinations, such as IL-2 and IL-7, IL-2 and IL-15, IL-7 and IL-15, IL-2, IL-7 and IL-15, IL-12 and IL-7, IL-12 and IL-15, or IL-12 and IL-2.

[0281] Therapeutically effective amounts of the generated NK cells can be administered by several routes, including parenteral administration, for example, intravenous, intraperitoneal, intramuscular, intrasternal, intratumoral, intrathecal, intraventricular, via a reservoir, intraarticular injection, or infusion.

[0282] The therapeutically effective amount of the immune effector cells produced for use in adoptive cell therapy is the amount that achieves the desired effect in the treated subject.For example, it can be the amount of NK cells that is required to inhibit the progression or cause the regression of autoimmune disease or alloimmune disease, or the amount of NK cells that can alleviate the symptoms caused by autoimmune disease, such as pain and inflammation.It can be the amount that is required to alleviate the symptoms associated with inflammation, such as pain, edema, and elevated body temperature.It can also be the amount that is required to reduce or prevent the rejection of transplanted organs.

[0283] The generated immune effector cell populations can be administered in a treatment regimen consistent with the disease, e.g., single or multiple doses over one to several days to ameliorate the disease state, or intermittently over an extended period to inhibit disease progression and prevent disease recurrence. The precise dose to be employed in the formulation will also depend on the route of administration and the severity of the disease or disorder, and should be determined according to the judgment of the practitioner and each patient's circumstances. The therapeutically effective amount of immune effector cells will depend on the subject being treated, the severity and type of affliction, and the manner of administration. In some embodiments, doses that can be used in treating human subjects are at least 3.8 x 10 4 , at least 3.8 × 10 5 , at least 3.8 × 10 6 , at least 3.8 × 10 7 , at least 3.8 × 10 8 , at least 3.8 × 10 9 , or at least 3.8 × 10 10 immune effector cells / m 2 In certain embodiments, the dose used in treating a human subject ranges from about 3.8×10 9 ~Approx. 3.8×10 10 immune effector cells / m 2 In a further embodiment, the therapeutically effective amount of immune effector cells is in the range of about 5 x 10 per kg of body weight. 6 Approximately 7.5 x 10 cells per kg of body weight8 up to approximately 2 x 10 cells per kg of body weight 7 Approximately 5 × 10 cells 8 cells, or approximately 5 x 10 per kg of body weight 7 Approximately 2 x 10 cells 8 The amount of immune effector cells can vary from 0.01 to 0.01. The exact amount of immune effector cells can be easily determined by those skilled in the art based on the age, weight, sex, and physiological condition of the subject. Effective amounts can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0284] The immune effector cells can be administered in combination with one or more other therapeutic agents for the treatment of an immune-mediated disorder. Combination therapy can include, but is not limited to, one or more antimicrobial agents (e.g., antibiotics, antivirals, and antifungals), antitumor agents (e.g., fluorouracil, methotrexate, paclitaxel, fludarabine, etoposide, doxorubicin, or vincristine), immunodepleting agents (e.g., fludarabine, etoposide, doxorubicin, or vincristine), immunosuppressants (e.g., azathioprine, or glucocorticoids such as dexamethasone or prednisone), anti-inflammatory agents (e.g., glucocorticoids such as hydrocortisone, dexamethasone, or prednisone, or nonsteroidal anti-inflammatory agents such as acetylsalicylic acid, ibuprofen, or naproxen sodium), cytokines (e.g., interleukin-10 or transforming growth factor-beta), hormones (e.g., estrogen), or vaccines. In addition, immunosuppressants or tolerogenic agents can be administered, including, but not limited to, calcineurin inhibitors (e.g., cyclosporine and tacrolimus); mTOR inhibitors (e.g., rapamycin); mycophenolate mofetil; antibodies (e.g., those that recognize CD3, CD4, CD40, CD154, CD45, IVIG, or B cells); chemotherapeutic agents (e.g., methotrexate, treosulfan, busulfan); irradiation; or chemokines, interleukins, or their inhibitors (e.g., BAFF, IL-2, anti-IL-2R, IL-4, JAK kinase inhibitors). Such additional pharmaceutical agents can be administered before, during, or after administration of immune cells, depending on the desired effect. This administration of cells and agents can be by the same route or by different administrations, and can be administered at the same or different sites.

[0285] In some embodiments, provided herein are methods for increasing the viability of effector cells (e.g., CAR-NK cells) in a subject. For example, in some embodiments, use of an iCAR described herein can increase the viability of effector cells in a subject by 1.1 to 10 fold (e.g., 1.1× to 10×) when compared to a suitable control. In some embodiments, the viability of effector cells in a subject can be increased by 1.1×, 1.2×, 1.3×, 1.4×, 1.5×, 1.6×, 1.7×, 1.8×, 1.9×, 2×, 2.5×, 3×, 3.5×, 4×, 4.5×, 5×, 5.5×, 6×, 6.5×, 7×, 7.5×, 8×, 8.5×, 9×, 9.5×, 10×, 15×, 20×, 25×, 30×, 40×, 50×, 60×, 70×, 80×, 90×, 100×, 1000×, or up to 10,000× when compared to an appropriate control.

[0286] In some embodiments, provided herein are methods for increasing the persistence of effector cells (e.g., CAR-NK cells) in a subject. For example, in some embodiments, use of an iCAR described herein can increase the persistence of effector cells in a subject by 1.1 to 10-fold (e.g., 1.1× to 10×) when compared to a suitable control. In some embodiments, effector cell persistence in a subject can be increased by 1.1x, 1.2x, 1.3x, 1.4x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2x, 2.5x, 3x, 3.5x, 4x, 4.5x, 5x, 5.5x, 6x, 6.5x, 7x, 7.5x, 8x, 8.5x, 9x, 9.5x, 10x, 15x, 20x, 25x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 1000x, or up to 10000x, when compared to an appropriate control.

[0287] In some embodiments, provided herein are methods for increasing the potency of effector cells (e.g., CAR-NK cells) in a subject. For example, in some embodiments, use of an iCAR described herein can increase the potency of effector cells in a subject by 1.1 to 10-fold (e.g., 1.1× to 10×) when compared to a suitable control. In some embodiments, the efficacy of the effector cells in a subject can be increased by 1.1x, 1.2x, 1.3x, 1.4x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2x, 2.5x, 3x, 3.5x, 4x, 4.5x, 5x, 5.5x, 6x, 6.5x, 7x, 7.5x, 8x, 8.5x, 9x, 9.5x, 10x, 15x, 20x, 25x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 1000x, or up to 10000x when compared to a suitable control.

[0288] In some embodiments, provided herein are methods for increasing the survival time of a subject when compared to a suitable control subject or control population. For example, in some embodiments, the use of effector cells comprising an iCAR described herein can increase the survival time of a subject by 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years, 5 years, 5.5 years, 6 years, 6.5 years, 7 years, 7.5 years, 8 years, 8.5 years, 9 years, 9.5 years, or 10 years or more when compared to a suitable control subject or control population.

[0289] In some embodiments, provided herein are methods for inhibiting tumor growth and / or initiating tumor shrinkage. For example, in some embodiments, use of effector cells comprising an iCAR described herein can inhibit tumor growth by 5% to 100% when compared to an appropriate control. For example, in some embodiments, the techniques provided herein can inhibit tumor growth by 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 11 8%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% inhibition. In some embodiments, the use of effector cells comprising an iCAR described herein can shrink tumors by 5% to 100% when compared to an appropriate control.For example, in some embodiments, the techniques provided herein may reduce tumor size by 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, Shrink by 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0290] In some embodiments, provided herein are methods suitable for increasing the rate of subjects classified as responding to effector cell-based therapy. For example, in some embodiments, use of effector cells comprising an iCAR described herein can increase the percentage of subjects classified as responding to effector cell-based therapy by 5% to 100% when compared to a suitable control. For example, in some embodiments, the technology provided herein may be used to improve or decrease the percentage of subjects classified as responsive to an effector cell-based therapy by 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, or more than 5% of subjects classified as responsive to an effector cell-based therapy compared to a suitable control. , 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% increase.

[0291] In some embodiments, provided herein are methods for increasing circulating serum levels of effector cell-associated proteins, such as, but not limited to, GrA, GrB, perforin, IFNγ, TNFα, or combinations thereof. In some embodiments, use of effector cells comprising an iCAR described herein can increase the circulating levels of one or more effector cell-associated proteins by 1.1 to 10 fold (e.g., 1.1× to 10×) when compared to a suitable control. In some embodiments, circulating levels of one or more effector cell-associated proteins can be increased by 1.1×, 1.2×, 1.3×, 1.4×, 1.5×, 1.6×, 1.7×, 1.8×, 1.9×, 2×, 2.5×, 3×, 3.5×, 4×, 4.5×, 5×, 5.5×, 6×, 6.5×, 7×, 7.5×, 8×, 8.5×, 9×, 9.5×, 10×, 15×, 20×, 25×, 30×, 40×, 50×, 60×, 70×, 80×, 90×, 100×, 1000×, or up to 10,000× when compared to an appropriate control. A. Pharmaceutical Compositions

[0292] Also provided herein are pharmaceutical compositions and formulations comprising immune effector cells produced by the processes encompassed herein and a pharmaceutically acceptable carrier.

[0293] The pharmaceutical compositions and formulations described herein can be prepared in the form of a lyophilized formulation or an aqueous solution by mixing an active ingredient (e.g., a cell described herein) having the desired purity with one or more optional pharmaceutically acceptable carriers (see, e.g., "Remington's Pharmaceutical Sciences," 22nd Edition, 2012). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations used and include buffers such as phosphate, citric acid, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol, and m-cresol); low molecular weight (less than about 10 residues) polypeptides; Examples of suitable pharmaceutically acceptable carriers include, but are not limited to, proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersion agents, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs, including rHuPH20, and methods of use are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968.In one embodiment, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases. B. Combination Treatment

[0294] In certain embodiments, the compositions and methods of the present embodiments involve immune effector cell populations combined with at least one additional therapy. In cancer embodiments, the additional therapy can be radiation therapy, surgery (e.g., lumpectomy and mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplant, nanotherapy, monoclonal antibody therapy, or a combination thereof. The additional therapy can be in the form of adjuvant or neoadjuvant therapy. In the case of pathogenic conditions, the additional therapy can include one or more antibiotics, antivirals, etc.

[0295] In some cancer embodiments, the additional treatment is a small molecule enzyme inhibitor or an anti-metastatic agent. In some embodiments, the additional treatment is administration of a side effect limiting agent (e.g., an agent intended to reduce the occurrence and / or severity of treatment side effects, such as an anti-nausea agent). In some embodiments, the additional treatment is radiation therapy. In some embodiments, the additional treatment is surgery. In some embodiments, the additional treatment is a combination of radiation therapy and surgery. In some embodiments, the additional treatment is gamma irradiation. In some embodiments, the additional treatment is a therapy targeting the PBK / AKT / mTOR pathway, an HSP90 inhibitor, a tubulin inhibitor, an apoptosis inhibitor, and / or a chemopreventive agent. The additional treatment may be one or more of chemotherapeutic agents known in the art.

[0296] The immune effector cell therapy of the present disclosure may be administered prior to, during, or after an additional cancer therapy, such as an immune checkpoint therapy, or in various combinations. Administration may occur simultaneously, or at intervals ranging from minutes to days to weeks. In embodiments in which the immune cell therapy is administered to a patient separately from the additional therapeutic agent, one will generally ensure that no significant time elapses between each delivery so that the two compounds can still exert their beneficial combined effect on the patient. In such instances, it is contemplated that the antibody therapy and anti-cancer therapy will be administered to the patient within about 12-24 or 72 hours of each other, more specifically, within about 6-12 hours of each other. In some circumstances, it may be desirable to significantly extend the duration of treatment, such that several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) elapse between each administration.

[0297] Various combinations may be used. In the examples below, the immune cell therapy is designated "A" and the anti-cancer treatment is designated "B." A / B / AB / A / BB / B / AA / A / BA / B / BB / A / AA / B / B / BB / A / B / B B / B / B / AB / B / A / BA / A / B / BA / B / A / BA / B / B / AB / B / A / A B / A / B / AB / A / A / BA / A / A / BB / A / A / AA / B / A / AA / A / B / A

[0298] Administration of any compound or treatment of the present embodiments to a patient will follow standard protocols for the administration of such compounds, taking into account the toxicity, if any, of the agent. Thus, in some embodiments, a step is provided to monitor for toxicity that may result from the combination treatment. 1.Chemotherapy

[0299] A wide variety of chemotherapeutic agents may be used in embodiments of the present invention. The term "chemotherapeutic" refers to the use of drugs to treat cancer. "Chemotherapeutic agent" is used to refer to a compound or composition administered in the treatment of cancer. These agents or drugs are classified according to their mode of activity within cells, for example, whether they affect the cell cycle and at what stage of the cell cycle they affect. Alternatively, agents can be characterized based on their ability to directly crosslink DNA, their ability to intercalate into DNA, or their ability to induce chromosomal and mitotic abnormalities by affecting nucleic acid synthesis.

[0300] Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylamelamines such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); camptothecin (including the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); cryptophycins (especially cryptophycin 1 and and cryptophycins 8); dolastatins; duocarmycins (including synthetic analogs KW-2189 and CB1-TM1); eluterobin; pancratistatin; sarcodictyins; spongistatins; nitrogen mustards, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, fenesterol nitril, prednimustine, trofosfamide, and uracil mustard; nitrosureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma 1I and calicheamicin omega 1I); dynemicins, including dynemicin A; bisphosphonates, such as clodronate; esperamicins;and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomycins, actinomycin, authrarnycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6 -diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins, such as mitomycin C, mycophenolic acid, nogalarnycin, olivomycin, peplomycin, potfilomycin cin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, pteropterin, and trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiazolinone, and thiazolinone; amiprine, and thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calsterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; antiadrenal drugs such as mitotane and trilostane; folic acid supplements such as furoic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatraxate; defofamine; demecolcine; diaziconazole; elformithine; elliptinium acetate; epothilones; etoglucide;Gallium nitrate; Hydroxyurea; Lentinan; Lonidainine; Maytansinoids, such as maytansine and ansamitocins; Mitoguazone; Mitoxantrone; Mopidanmol; Nitraerine; Pentostatin; Fenamet; Pirarubicin; Rosoxantrone; Podophyllic acid; 2-Ethylhydrazide; Procarbazine; PSK polysaccharide complex; Razoxane; Rhizoxin; Schizophyte orchids; spirogermanium; tenuazonic acid; triazicon; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; taxoids, such as paclitaxel cisplatin and docetaxel-gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitors anti-inflammatory drug RFS2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabien, navelbine, farnesyl-protein transferase inhibitors, transplatin, and pharmaceutically acceptable salts, acids, or derivatives of any of the above; 2. Radiation therapy

[0301] Other widely used agents that cause DNA damage include what are commonly known as gamma rays, X-rays, and / or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging agents are also contemplated, such as microwaves, proton beam radiation, and UV radiation. All of these agents likely affect a wide range of damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. X-ray doses range from daily doses of 50-200 roentgens over prolonged periods (3-4 weeks) to single doses of 2000-6000 roentgens. Dosage ranges for radioisotopes vary widely and depend on the half-life of the isotope, the strength and type of radiation emitted, and uptake by neoplastic cells. 3.Immunotherapy

[0302] Those skilled in the art will understand that additional immunotherapy may be used in combination with or with the method of this embodiment. In the context of cancer treatment, immunotherapy generally relies on the use of immune effector cells and molecules to target and kill cancer cells. Rituximab (RITUXAN®) is one such example. The immune effector may be, for example, an antibody specific to some marker on the surface of tumor cells. The antibody may serve alone as the therapeutic effector, or the antibody may recruit other cells to actually affect cell death. The antibody may also be conjugated to a drug or toxin (such as a chemotherapeutic agent, a radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) to serve as a targeting agent. Alternatively, the effector may be a lymphocyte bearing a surface molecule that interacts directly or indirectly with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

[0303] Antibody-drug conjugates (ADCs) comprise monoclonal antibodies (mAbs) covalently linked to cytotoxic drugs and can be used in combination therapy. This approach combines the high specificity of MAbs for their antigen targets with highly potent cytotoxic drugs, resulting in "armed" MAbs that deliver the payload (drug) to tumor cells where antigen levels are elevated. Targeted delivery of the drug also minimizes its exposure in normal tissues, resulting in reduced toxicity and an improved therapeutic index. Exemplary ADC drugs include ADCETRIS® (brentuximab vedotin) and KADCYLA® (trastuzumab emtansine or T-DM1).

[0304] In one aspect of immunotherapy, tumor cells must possess some marker suitable for targeting (i.e., not present on the majority of other cells). Many tumor markers exist, any of which may be suitable for targeting in the context of this embodiment. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, laminin receptor, erbB, and p155. An alternative aspect of immunotherapy is to combine anti-cancer effects with immunostimulatory effects. Immune stimulatory molecules also exist, including cytokines such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, chemokines such as MIP-1, MCP-1, and IL-8, and growth factors such as FLT3 ligand.

[0305] Examples of immunotherapies include immunoadjuvants such as Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds; cytokine therapy such as interferon alpha, beta, and gamma, IL-1, GM-CSF, and TNF; gene therapy such as TNF, IL-1, IL-2, and p53; and monoclonal antibodies such as anti-CD20, anti-ganglioside GM2, and anti-p185. It is contemplated that one or more anti-cancer therapies may be used in conjunction with the antibody therapies described herein.

[0306] In some embodiments, the immunotherapy can be an immune checkpoint inhibitor. Immune checkpoints either enhance or attenuate signals (e.g., costimulatory molecules). Inhibitory immune checkpoints that can be targeted by immune checkpoint blockade include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B-lymphocyte and T-lymphocyte attenuator (BTLA), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin (KIR), lymphocyte activation gene 3 (LAG3), programmed death 1 (PD-1), T-cell immunoglobulin domain and mucin domain 3 (TIM-3), and V-domain Ig suppressor of T-cell activation (VISTA). In particular, immune checkpoint inhibitors target the PD-1 axis and / or CTLA-4.

[0307] The immune checkpoint inhibitor can be a drug, such as a small molecule, a recombinant ligand or receptor, or, in particular, an antibody, for example, a human antibody. Known inhibitors of immune checkpoint proteins or their analogs can be used, particularly chimeric, humanized, or human antibodies. As will be appreciated by those skilled in the art, alternative and / or equivalent names may be used for certain antibodies referred to in this disclosure. Such alternative and / or equivalent names are interchangeable in the context of this disclosure. For example, it is known that lambrolizumab is also referred to as MK-3475 and pembrolizumab, which are equivalent alternative names.

[0308] In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In one specific embodiment, the PD-1 ligand binding partner is PDL1 and / or PDL2. In another embodiment, the PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partner. In one specific embodiment, the PDL1 binding partner is PD-1 and / or B7-1. In another embodiment, the PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partner. In one specific embodiment, the PDL2 binding partner is PD-1. The antagonist can be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.

[0309] In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011. In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising the extracellular portion or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some embodiments, the PD-1 binding antagonist is AMP-224. Nivolumab is also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO ( Pembrolizumab, also known as MK-3475, Merck3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an exemplary anti-PD-1 antibody. CT-011, also known as hBAT or hBAT-1, is also an anti-PD-1 antibody. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor.

[0310] Another immune checkpoint that can be targeted in the methods provided herein is cytotoxic T lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The GenBank accession number for the entire cDNA sequence of human CTLA-4 is L15006. CTLA-4 is found on the surface of T cells and acts as an "off" switch upon binding to CD80 or CD86 on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of helper T cells and transmits inhibitory signals to T cells. CTLA4 is similar to the T cell costimulatory protein CD28; both molecules bind to CD80 and CD86 (also known as B7-1 and B7-2, respectively) on antigen-presenting cells. CTLA4 transmits inhibitory signals to T cells, while CD28 transmits stimulatory signals. Intracellular CTLA4 is also found on regulatory T cells and may be important for their function. T cell activation via the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for B7 molecules.

[0311] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.

[0312] Anti-human CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art-recognized anti-CTLA-4 antibodies can be used. An exemplary anti-CTLA-4 antibody is ipilimumab (also known as 10D1, MDX-010, MDX-101, and YERVOY®) or antigen-binding fragments and variants thereof. In another embodiment, the antibody comprises the heavy and light chain CDRs or VRs of ipilimumab. Thus, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of ipilimumab and the CDR1, CDR2, and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes for binding with and / or binds to the same epitope on CTLA-4 as the above-mentioned antibodies. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity to the above-described antibody (eg, at least about 90%, 95%, or 99% variable region identity to ipilimumab). 4.Surgery

[0313] Approximately 60% of people with cancer will undergo some type of surgery, including preventative surgery, diagnostic or staging surgery, curative surgery, and palliative surgery. Curative surgery includes resection, in which all or part of the cancerous tissue is physically removed, excised, and / or destroyed, and may be used in conjunction with other treatments, such as the treatment of the present embodiments, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies. Tumor resection refers to the physical removal of at least part of the tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microsurgery (e.g., Mohs surgery).

[0314] Removal of part or all of the cancerous cells, tissue, or tumor can result in the formation of a cavity in the body. Treatment can be achieved by perfusion, direct injection, or local application of additional anti-cancer treatment to the area. Such treatment can be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments can be administered at varying dosages. 5. Other agents

[0315] It is contemplated that other agents may be used in combination with certain aspects of the present embodiments to improve the therapeutic efficacy of treatment. These additional agents include agents that affect the upregulation of cell surface receptors and gap junctions, cytostatic and differentiating agents, inhibitors of cell adhesion, agents that increase the sensitivity of hyperproliferative cells to apoptosis inducers, or other biologics. Increasing intercellular signaling by increasing the number of gap junctions will increase the anti-hyperproliferative effect on adjacent hyperproliferative cell populations. In another embodiment, cytostatic or differentiating agents may be used in combination with certain aspects of the present embodiments to improve the anti-hyperproliferative efficacy of treatment. It is contemplated that inhibitors of cell adhesion will improve the efficacy of the present embodiments. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. It is further contemplated that other agents that increase the sensitivity of hyperproliferative cells to apoptosis, such as the antibody c225, could be used in combination with certain aspects of the present embodiments to improve the efficacy of treatment. VII. Manufactured Articles or Kits

[0316] Articles of manufacture or kits are provided that include immune effector cells and / or one or more reagents for producing them. The immune effector cells may be derived from any source, and in specific embodiments, the immune effector cells are produced by the methods encompassed herein. In specific embodiments, the immune effector cells have been gene-edited and can be provided in a kit so that they can be further modified to express one or more iCARs and one or more heterologous antigen receptors. In specific embodiments, the immune effector cells have been modified to express one or more iCARs and one or more heterologous antigen receptors and can be provided in a kit so that they can be further modified for gene editing. In specific embodiments, the kits include one or more reagents for producing immune effector cells, such as an iCAR-encoding vector or reagents for producing it, a CAR-encoding vector or reagents for producing it; reagents targeting specific NK cell genes, or a combination thereof. In general embodiments, the reagents can include nucleic acids, including DNA or RNA, primers, proteins, media, buffers, salts, cofactors, etc. In particular instances, the kit includes one or more CRISPR-related reagents, including reagents for targeting specific NK cell genes of interest.

[0317] The article of manufacture or kit can further include a package insert containing instructions for using the immune cells to treat or delay the progression of cancer in an individual or to enhance the immune function of an individual with cancer. Any of the antigen-specific immune cells described herein can be included in the article of manufacture or kit. Suitable containers include, for example, bottles, vials, bags, and syringes. The containers can be formed from a variety of materials, such as glass, plastic (e.g., polyvinyl chloride or polyolefin), or alloys (e.g., stainless steel or Hastelloy). In some embodiments, the container holds the formulation, and a label on or associated with the container can indicate usage. The article of manufacture or kit can further include other materials from a commercial and user perspective, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. In some embodiments, the article of manufacture further includes one or more additional agents (e.g., chemotherapeutic agents and anti-neoplastic agents). Suitable containers for the one or more agents include, for example, bottles, vials, bags, and syringes. VIII. Working Examples

[0318] The following examples are included to demonstrate preferred embodiments of the invention. Those of skill in the art should understand that the techniques disclosed in the examples which follow represent techniques found by the inventors to function well in the practice of the invention, and therefore can be considered to constitute preferred modes for its practice. However, those of skill in the art should understand in light of this disclosure that numerous changes can be made in the specific embodiments which are disclosed and still obtain like or similar results without departing from the spirit and scope of the invention. method

[0319] Unless otherwise stated, the assays and experiments described in the following examples were performed as described herein. Cell lines, primary cells and culture conditions

[0320] CD19 in Raji (CCL-86), NALM-6 (CRL-3273), and Ramos (CRL-1596) + cell line, CD5 + Cell line CCRF (CRM-CCL-119), CD70 + Cell line THP-1 (TIB-202), CD123 + Cell line MOLM-14 (ACC777), BCMA + The cell lines MM1S (CRL-2974), SKOV3 (HTB-77), K562 (CRL-3344), and 293T (CRL-3216) were obtained from the American Type Culture Collection (ATCC). Raji, NALM-6, Ramos, CCRF, MOLM-14, and K562 cells were cultured in RPMI-1640 (Invitrogen) supplemented with 10% fetal bovine serum (FBS; Hyclone), 1% penicillin-streptomycin, and 1% GLUTAMAX™; THP-1 and 293T cells were cultured in DMEM (Invitrogen) supplemented with 10% FBS, 1% penicillin-streptomycin, and 1% GLUTAMAX™; and SKOV3 cells were cultured in McCoy's 5a medium (Invitrogen) supplemented with 10% FBS, 1% penicillin-streptomycin, and 1% GLUTAMAX™. K562 cells were retrovirally transduced to co-express 4-1BBL, CD48, and membrane-bound interleukin (IL)-21, and used as universal antigen-presenting cells (uAPCs) for in vitro NK cell expansion (see, e.g., Liu et al., 2021). To model trogocytosis detection using a fluorescently traceable marker, the CRISPR-Cas9 system was used to delete the CD19 gene in Raji cells (crRNA1:CTAGGTCCGAAACATTCCAC-CGG (SEQ ID NO:11), crRNA2:CGAGGAACCTCTAGTGGTGA-AGG (SEQ ID NO:12)). CD19-KO) Cells were purified by MoFlo Astrios (Beckmen Coulter) and then retrovirally transduced (Raji) to express CD19-mCherry fusion protein with or without GFP co-expression. CD19-mtCherry / GFP and Raji CD19-mCherry ) Raji cells were transduced with firefly luciferase-GFP to allow for in vivo tumor burden testing using an IVIS Spectrum imaging system (Caliper). To model solid tumors in vivo, CD19 (SKOV3 gCD19+ SKOV3 cells were retrovirally transduced to express GFP and firefly luciferase. All cells were maintained in a 37°C incubator with 5% CO and routinely tested for mycoplasma contamination using the MycoAlert Mycoplasma Detection Kit (Lonza). PBMCs from patients treated with CAR19 / IL-15 NK cells

[0321] The clinical samples used in this study were collected from patients treated in a previously reported clinical trial of iC9 / CAR19 / IL-15 (CAR19 / IL-15)-transduced umbilical cord blood (CB)-NK cells (NCT03056339; see, e.g., Liu et al., 2020). Primary peripheral blood monocytes (PBMCs) were collected from 11 patients with chronic lymphocytic leukemia (CLL) or non-Hodgkin's lymphoma (NHL) at different time points during their respective treatments at MD Anderson Cancer Center. The mean normalized TROG-CD19 (tCD19) gMFI on CAR19-NK cells for the entire patient cohort was 6.29 (range, 0.61–35.77). Patients with high (>mean) normalized tCD19-gMFI and patients with low (≤mean) normalized tCD19-gMFI at two or more time points were classified as TROG hi (n=4 patients) and TROG low(n=7 patients). In addition, circulating leukemia cells from four patients with CLL and four patients with B-cell acute lymphoblastic leukemia (ALL) enrolled in the study protocol were isolated after density gradient centrifugation for in vitro studies of trogocytosis. Informed consent was obtained from all patients in accordance with the Institutional Review Board (IRB). All studies were conducted in accordance with the Declaration of Helsinki. Vector constructs and retrovirus production

[0322] Retroviral vectors encoding iCas9, 19scFv, CD28, zeta, 2A, IL-15 (CAR19 / IL15) were kindly provided by Dr. Gianpietro Dotti (University of North Carolina at Chapel Hill) (see, e.g., Hoyos, 2010). Other vectors encoding iCas9, 19scFv, 2A, IL-15 (19scFv / IL15), iCas9, CD5scFv (XZ-CD5 (see, e.g., Przepiorka et al., 1994)), CD28, zeta, 2A, IL-15 (CAR5 / IL15), iCas9, CD5scFv, DAP10, zeta, 2A, IL-15 (CAR(DAP10z) / IL15), and iCas9, CD5scFv, zeta, 2A, IL-15 (CAR(3z)). ) / IL15), iCas9.CD5scFv.DAP12.zeta.2A.IL-15(CAR(DAP12z) / IL15), iCas9.CD5scFv.NKG2D.zeta.2A.IL-15(CAR( 2Dz) / IL15), iCas9.CD5scFv.41BB.zeta.2A.IL-15(CAR(BBz) / IL15), iCas9.CD5scFv.DAP10.2A.IL-15(CAR(DAP10) / IL15), iCas9.CD5scFv.DAP12.2A.IL-15 (CAR(DAP12) / IL15), iCas9.CD70scFv (ARGX-110 or LB#14 (see, e.g., Silence et al., 2014)).CD28.Zeta.2A.IL-15 (CAR70 / IL15), iCas9,CD27(ECD).CD28.Zeta.2A.IL-15 (CAR27s / IL15), iCas9.CD12 Additional viral vectors were generated by cloning the 3scFv (26292 (see, e.g., Du et al., 2007)).CD28.zeta.2A.IL-15 (CAR123 / IL15) and iCas9.BCMAscFv (huc11D5.3-Luc90 (see, e.g., Zah et al., 2020)).CD28.zeta.2A.IL-15 (CAR-BCMA / IL15) constructs into the SFG retroviral backbone.To construct inhibitory CARs (iCARs), the transmembrane domains of KIR2DL1 and LIR-1, and the cytoplasmic signaling domains of KIR2DL1, LIR-1, LAIR-1, NKG2A, and CD300A were used as inhibitory signals. The extracellular domains of either 19scFv or CS1scFv (HuLuc63 (see, e.g., Tai et al., 2008)) were fused to an IgG hinge to generate iCAR19 or iCAR-CS1 constructs, respectively. CS1-scFv, iCAR-CS1, and iCAR19 / IL15 constructs were then cloned into the SFG retroviral backbone. The CD19-mCherry construct was generated by fusing the entire CD19 coding sequence to the mCherry reporter gene at its 3' end. The 2A peptide was then used to link CD19-mCherry to GFP, creating a bicistronic CD19-mCherry / GFP construct. mCherry, CD19-mCherry, and CD19-mCherry / GFP were also cloned into the SFG retroviral backbone, respectively. All construct synthesis and molecular cloning were performed by GeneArt Gene Synthesis (Thermo Fisher Scientific). Transient retroviral supernatants were produced from transfected 293T cells as previously described (see, e.g., Vera et al., 2006). Transduction and expansion of cord blood NK cells

[0323] Umbilical cord blood (CB) units were provided by the MD Anderson Cancer Center Cord Blood Bank. CB-derived NK cells (CB-NK) were isolated and expanded as previously described (see, e.g., Liu et al., 2018). Briefly, lymphocytes were collected by density gradient centrifugation using Ficoll-Histopaque solution (Sigma-Aldrich). Then, CD56 NK cells were isolated using an NK negative isolation kit (Miltenyi Biotec). + CD3 -Purified NK cells were cocultured with irradiated (100 Gy) uAPCs at a 2:1 ratio in complete stem cell growth medium (SCGM) supplemented with 200 U / ml recombinant human IL-2 (Proleukin). On day 4 after uAPC stimulation, fresh NK cells were purified again and transduced with a retroviral vector expressing the CAR construct. A second retroviral transduction of the iCAR construct was then performed on day 6 to generate AI-CAR-expressing NK cells. The same approach was followed to prepare CD19-mCherry or CD19-mCherry / GFP-expressing cells (both primary NK cells and tumor cells). CAR transduction efficiency was measured by flow cytometry. Irradiated uAPCs were added weekly to NK cell cultures to support NK cell expansion. Flow cytometry

[0324] CAR expression was measured by IgG hinge detection using conjugated goat anti-human IgG (H+L) (Jackson ImmunoResearch). For AI-CAR detection, anti-CD19 aCAR expression was measured using CD19-CAR detection reagent (Miltenyi Biotec). Anti-CS1 iCAR expression was measured by CAR binding to CS1 his-tag fusion protein (ACRO Biosystems). GHOST DYE™ Violet 450 (TONBO Biosciences) was used to determine viability, and if a fixation protocol was applied, aqua fixable viability dye (eBioscience) was used to assess viability. Non-target-specific staining was minimized by blocking Fc receptors using human Fc receptor blocking solution (Miltenyi Biotec). For intracellular staining, cells were fixed and permeabilized using an intracellular fixation and permeabilization buffer kit (eBioscience) according to the manufacturer's protocol. For phosphoflow staining, cells were prepared and fixed using a Perfix exposure kit from Beckman Coulter according to the manufacturer's protocol. The number of CD19, CD5, CD70, CD123, and BCMA molecules per cell was determined using a phycoerythrin fluorometric quantification kit (BD Biosciences) according to the manufacturer's protocol. AccuCheck counting beads (ThermoFisher) were used to determine the cell concentration in each test population. Images were captured using an Amnis ImageStream-X Mark II (Millipore) at 60x magnification and 0.1 μm resolution. 2 Fixed cells were visualized at a pixel size of 1000 x 1000 and data were analyzed using IDEAS (Millipore). Flow cytometry analysis was performed on an LSRFORTESSA™ X-20 (BD Bioscience) and data were analyzed using FlowJo (BD Bioscience). Cell sorting was performed using a MoFlo XDP cell sorter (Beckman Coulter). Trogocytosis assay

[0325] NK cells were designated GFP + Target cells were co-cultured at a 1:1 effector:target (E:T) ratio. The co-cultured cells were washed with FACS buffer and then subjected to surface staining with anti-hCD56 (Biolegend, HCD56) and anti-hCD3 (Biolegend, SK7) antibodies for 20 minutes at 4°C in the dark. Following staining, the cells were washed and evaluated by flow cytometry. + The population was singlet NK (CD56 + CD3 - GFP - ) were defined by the detection of TROG antigen on the surface of the cells and co-cultured with tumor cells (CD56 - GFP + Cognate antigen expression on Raji was also assessed. CD19-mCherry Cells or Raji mCherry For mCherry-based trogocytosis assays, cells were co-cultured with CFSE-labeled NK cells (ThermoFisher) at a 1:1 ratio using an IncuCyte Live Cell Analysis System (ESSEN Bioscience). Image scanning of the mCherry signal in NK cells was recorded in real time, and cells that showed both mCherry and CFSE signals were classified as NK cells. TROG+ To block trogocytosis, NK cells were pretreated with 1 μM latrunculin A (Sigma-Aldrich) for 20 min at 37°C before co-culture with target cells. NK activation assay

[0326] NK cells were stimulated with target cells at a 1:1 E:T ratio for 6 hours. To inhibit protein transport, GolgiStop and GolgiPlug (BD Bioscience) were added to the cultures 2 hours after co-culture according to the manufacturer's protocol. Anti-CD107a (Biolegend, H4A3) was also added at this time to capture CD107a as a marker of NK cell degranulation. NK TROG+For cell populations examined, GolgiStop and GolgiPlug were not added to allow trogocytosis. After incubation, cells were washed with FACS buffer (BD Bioscience) and stained with anti-hCD56 and anti-hCD3. The viability of the NK population was determined using GHOST DYE™ Violet 450 (Tonbo Biology). Next, intracellular staining with interferon-gamma (IFN-γ) antibody (BD Bioscience, B27) and tumor necrosis factor alpha (TNF-α) antibody (BD Bioscience), MAb11, was applied. Expression of CD107a, IFN-γ, and TNF-α was measured, and the CD56 expression was compared to unstimulated NK cells. + CD3 - It was expressed as a percentage of NK cells. Cytotoxicity assay in the IncuCyte system

[0327] NK cells were co-cultured with tumor cells labeled with Vybrant DyeCycle Ruby Stain (ThermoFisher) or expressing mCherry signals at a 1:1 E:T ratio. IncuCyte Caspase-3 / 7 Green Apoptosis Assay Reagent (SAETORIUS) was added to each well to label apoptotic cells. Images of each well were captured in real time over a period of 6 to 30 hours after addition. Data were analyzed using an IncuCyte Live Cell Analysis System, which evaluates the number of apoptotic cells (green) and target cells (red) in real time. The percentage (%) of caspase-3 / 7 expression in cells showing both green and red signals was measured and calculated as the expression of total target cells (red) detected. To block CD19 antigen exposure, CD19-scFv antibody (200 ng / ml, Invivogen) was added to NK cells. TROG+ Anti-β-Gal scFv antibody (Invivogen), preincubated with the population for 30 min, was used as a negative control. Single-cell cytotoxicity assay

[0328] To examine NK-mediated cytotoxicity at the single-cell scale, we used time-lapse imaging microscopy in nanowell grids (TIMING) as previously described (see, e.g., Liadi et al., 2015). Briefly, NK cell populations and target cells (K562 or Raji) were labeled with lipophilic PKH dyes and loaded into nanowell arrays. The arrays were incubated with medium premixed with Annexin V (BD Bioscience) and monitored in real time over 5–6 h using a Carl Zeiss Axio Observer equipped with a Hamamatsu Orca-Flash sCMOS camera with a 20x 0.8 NA objective. Images of approximately 5,000 wells were collected and processed using in-house algorithms for cell tracking and segmentation (see, e.g., Merouane et al., 2015). NK population doubling assay

[0329] After initial transduction and expansion, CB-NK cells were subcultured weekly with or without uAPC feeder cells. Weekly population doublings (PD) were measured using the equation: PD = log10[(A / B) / 2] (where A is the number of harvested cells and B is the number of plated cells from each subculture). The sum of each PD over time was then determined as cumulative PD. The assay was terminated 3 weeks after the cell count of cells harvested from the subculture failed to reach at least an amount equal to the plated cells. Data were obtained from three different CB-derived NK populations for each condition. Mass cytometry (CyTOF)

[0330] Mass cytometry was performed as previously described (see, for example, Li et al., 2019 and Daher et al., 2021(b)). Primary antibodies were conjugated in-house with the corresponding metal tags using the MaxparX8 polymer antibody labeling kit according to the manufacturer's protocol (Fludigm). NK cells were washed with cell staining buffer (0.5% bovine serum albumin / PBS) and incubated with human Fc receptor blocking solution (Miltenyi Biotec) before the addition of the antibody mixture. Next, cells were incubated with 2.5 μM cisplatin (Sigma-Aldrich) before fixation and permeabilization using BD CYTOFIX / CYTOPERM™ solution according to the manufacturer's protocol. For intracellular staining, cells were washed twice with perm / wash buffer and immediately incubated with an antibody master mix against intracellular markers. The cells were then stored overnight in 500 μl of 1.6% paraformaldehyde (EMD Millipore) / PBS containing 125 nM iridium nucleic acid intercalator (Fluidigm). On the day of cell evaluation, they were washed in 1 ml of MilliQ dH2O and filtered through a 35 μm nylon mesh (cell strainer cap tube, BD Bioscience). The cells were then resuspended in MilliQ dH2O supplemented with EQ™ 4-element calibration beads and then acquired at 300 events / second on a Helios instrument (Fluidigm). Antibodies used in in vitro experiments with the corresponding metal tag isotopes: CD45 (Fluidigm, HI30, 89 Y), GFP(Bilegend, FM264G, 144 Nd), DAP12(R&D, 406288, 146 Nd), NKG2C(Bilegend, 134591, 147 Sm), TRAIL (Miltenyi, REA1113, 148 Nd), CD25 (Miltenyi, REA570, 149 Sm), CD69(Biolegend, FN50, 150 Nd), CD2 (Miltenyi, REA972, 151Eu)、CAR(Jackson immune research、ポリクローナル、 152 Sm)、TIGIT(ThermoFisher、MBSA43、 154 Sm)、OX40(Miltenyi、REA621、 158 Gd)、パーフォリン(Miltenyi、REA1061、 159 Tb)、PD1(Miltenyi、PD1.3.1.3、 160 Gd)、Tbet(Miltenyi、4B10、 161 Dy)、EOMES(ThermoFisher、WD1928、 162 Dy)、c-Kit(Miltenyi、REA787、 163 Dy)、SAP(Biolegend、1A9、 164 Dy)、TIM3(R&D、344823、 165 Ho)、NKG2D(Miltenyi、REA797、 166 Er)、2B4(ThermoFisher、C1.7、 167 Er)、Ki67(Biolegend、Ki67、 168 Er)、NKG2A(Miltenyi、REA110、 169 Tm)、DNAM-1(Miltenyi、REA1040、 170 Er)、CS1(Biolegend、162.1、 172 Yb)、グランザイムB(Miltenyi、REA226、 173 Yb)、CD94(Miltenyi、REA113、 174 Yb)、LAG3(Miltenyi、REA351、 175 Lu)、ICOS(Miltenyi、REA192、 176 Yb)、CD16(Fluidigm、3G8、 209 Bi)、CD3(Biolegend、UCHT1、 194 Pt)、シスプラチンL / D(Fluidigm、 198 Pt)、CD56(BD Bioscience、NCAM16.2、 106 Cd)、CD19(Biolegend、HIB19、 110Cd), granzyme A (Miltenyi, REA162, 111 Cd), Syk(Biolegend, 4D10.2, 112 Cd), NKp30 (Miltenyi, AF29-4D12, 113 Cd), NKp46 (Miltenyi, REA808, 114 Cd), NKp44 (Miltenyi, REA1163, 116 Cd).

[0331] Antibodies used with the corresponding metal tag isotopes in in vivo experiments: CD45 (Biolegend, HI30, 89 Y), CD2 (Biolegend, TS1 / 8, 14 1Pr), CD62L (BD Bioscience, DREG-56, 143 Nd), CD27(Biolegend, M-T271, 144 Nd), CD56 (Biolegend, HCD56, 146 Nd), NKG2C (R&D, MAB138, 147 Sm), CXCR6 (R&D, MAB699, 148 Nd), CXCR3 (R&D, MAB160, 149 Sm), Granzyme B (R&D, polyclonal, 150 Nd), Tbet(Biolegend, 4B10, 151 Eu), TIGIT (Biolegend, A15153G, 152 Sm), granzyme A (Biolegend, CB9, 154 Sm), NKG2A (R&D, MAB1059, 155 Gd), TIM3(Biolegend, F38-2E2, 156 Gd), 2B4(Biolegend, 2-69, 158 Gd), CLA (Bioledend, KPL-1, 159 Tb), CD20 (Biolegend, RIK-2, 160 Gd), DNAM-1 (Miltenyi, DX11, 161 Dy), EOMES (Thermo Fisher, WD1928, 162Dy), NKp30 (Biolegend, P30-15, 163 Dy), c-Kit (BD Bioscience, YB5.B8, 164 Dy), CD25 (BD Bioscience, 2A3, 165 Ho), NKG2D (R&D, MAB139, 166 Er), perforin (BD Bioscience, δG9, 167 Er), ZAP70(ThomoFisher, 1E7.2, 168 Er), CCR5(Biolegend, J418F1, 169 Tm), CAR (Jackson immune research, polyclonal, 170 Er), CX3CR1(Bioledend, 2A9-1, 171 Yb), CXCR1(Bilegend, 8f1, 172 Yb), PD1(Bilegend, EH12.2H7, 173 Yb), Syk(Bioledend, 4D10.2, 174 Yb), NKp46 (R&D, MAB1850, 175 Lu), KLRG1(ThermoFisher, 13F12F2, 176 Yb), CD57(Biolegend, HNK-1, 194 Pt), cisplatin L / D (Fluidigm, 198 Pt), CD16 (Fluidigm, 3G8, 209 Bi). Mass cytometry data analysis

[0332] Mass cytometry data was analyzed using Cytobank. Pt195 (cisplatin) low hCD45 + CD56 + CD3 - The NK cell population was identified by using a gating strategy for singlets in CAR and applied to all files. + and CD19 +Expression was determined based on either an isotype control or NK cell monocultures. Data from 10,000 identified NK cells per in vitro sample were randomly subsampled in FlowJo. Normalized data from each sample were pooled and analyzed to obtain their signal variability. t-SNE maps were generated using t-SNE analysis, which performs pairwise comparisons of cell phenotypes to optimally plot clusters and reduce dimensionality from multiple parameters. Next, FlowSOM analysis was performed to determine metaclusters with empirically predicted optimized grouping distances between nodes, and a minimum spanning tree was constructed by hierarchically connecting the nodes. After local transformation and normalization, the expression of each marker was hierarchically clustered and plotted as a heatmap using Morpheus matrix visualization and analysis software (Broad Institute). Metabolic assays

[0333] Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were measured in GFP-negative CAR-NK effector cells using the Seahorse XF Cellular Mitostress Assay Kit (Agilent) and the Seahorse XF Glycolysis Stress Assay Kit (Agilent) on an Agilent Seahorse XFe96 analyzer according to the manufacturer's protocol. Assays were performed in phenol red / carbonate-free RPMI medium (Agilent) containing 2 nM L-glutamine (Agilent), 25 mM glucose, and 2 mM pyruvate (Agilent, but not in the glycolysis assay). Cellular mitostress assays were performed by examining the OCR after administration of 1.5 μM oligomycin, 0.5 μM fluorocarbonyl cyanide phenylhydrazone (FCCP), 0.5 μM rotenone, and antimycin A. Glycolysis tests were measured as ECAR after injection of 10 mM glucose, 1 μM oligomycin, and 50 mM 2-deoxy-D-glucose (2-DG). CAR-NK cell affinity experiments

[0334] Experiments were performed using poly-L-lysine (Sigma-Aldrich) coated z-MOVI® chips. CD70+ Cells were seeded onto a Z-MOVI® chip to form a monolayer. The Z-MOVI® chip was then sealed and incubated in a dry incubator for 30 minutes. Effector cells were stained with Cell Trace Far Red (ThermoFisher) and their flow onto the monolayer was measured at 200–500 cells per cell. The effectors were then incubated with the target cell monolayer for 5 minutes before initiating a force ramp. Each force ramp was set to 1000 pN for 90 seconds. Affinity measurements were performed on the Z-MOVI® Cell Avidity Analyzer using Ocean software. Luminex assay

[0335] Human granzyme A, granzyme B, perforin, TNF-α, and IFN-γ were measured in serum collected from mice at different time points after NK cell infusion using MILLIPLEX® MAP magnetic beads (Millipore) kits according to the manufacturer's protocol. Measurements were performed on a Luminex 200 system. Xenograft mouse model

[0336] As previously described, we investigated the antitumor activity of different NK populations in NOD / SCID IL-2Rγ null (NSG) mice implanted with aggressive NK-resistant tumor cells (see, e.g., Liu et al., 2018, and Daher et al., 2021(b)). + Raji lymphoma, CD5 + CCRF T-ALL, CD123 +The tumors included MOML14 acute myeloid leukemia (AML) and SKOV3 ovarian cancer. All experiments were performed under protocols approved by the Institutional Animal Care and Use Committee and in accordance with the recommendations of the American Veterinary Medical Association (AVMA) and the NIH. Seven-week-old female NSG mice (Jackson Laboratories) were irradiated (300 cGy) on day -1. On day 0, firefly luciferase-GFP-labeled Raji cells were injected into the mice at three increasing dose levels (0.2 × 10 5 pieces, 1×10 5 pieces, or 5 x 10 5 For other hematologic tumor models, one dose of CCRF was injected intravenously (iv). CD5+ / Luci+ / GFP+ cells (0.5×10 5 pcs) or MOML14 CD123+ / Luci+ / GFP+ cells (0.5×10 5 Mice were then treated sequentially with the indicated NK cell populations. For the ovarian cancer model, 7-week-old female NSG mice (Jackson Laboratories) were injected with luciferase-GFP-labeled SKOV3 cells (1 × 10 cells) 7 days before treatment (day -7). 6 SKOV3 ROR1+ or 0.5 × 10 6 SKOV3 gCD19+ ) were injected intraperitoneally (i.p.), and the mice were irradiated (300 cGy) on day -1, followed by injection of AI-CAR-expressing NK cells (1–1.5 × 10 7 (1000 cells) were given by i.p. injection. Bioluminescence imaging (Xenogen-IVIS200 imaging system; Caliper) was performed periodically to examine the engraftment of Raji and SKOV3 cells. Signal quantification in photons / second was measured using IVIS Living Image software (Caliper Life Sciences). statistics

[0337] Statistical analyses were performed and plotted using Prism7 software (GraphPad). Student's t-test was used to test for significance; one-way ANOVA was applied to determine between-group comparisons within a given condition, and two-way ANOVA was applied to determine between-group comparisons over time. P values ​​for pairwise comparisons were conservatively adjusted for multiple comparisons using the Bonferroni correction. Mean + s.e.m. values ​​are shown. A nonlinear regression model with the least trimmed sum of squares was selected for robust fit (see, e.g., Andreas et al., 2013). Overall survival (OS) analysis was calculated using the Kaplan-Meier method and compared with treatment groups using the log-rank test with 95% confidence intervals (CI). Example 1 Inhibitory chimeric antigen receptors (iCARs) prevent on-target and off-tumor effects of antitumor cell therapy using CAR-T cells and CAR-NK cells

[0338] We engineered five iCAR constructs [KIR2DL1™-KIR2DL1 (CDs); LIR-1™-LIR-1 (CDs); KIR2DL1™-KIR2DL1 (CDs)-LAIR-1 (CD); KIR2DL1™-KIR2DL1 (CDs)-NKG2A (CDs); KIR2DL1™-KIR2DL1 (CDs)-CD300A (CD)] and successfully expressed them in natural killer (NK) cells derived from umbilical cord blood stored in a cord blood bank. When the iCARs engage their cognate ligands, inhibitory signaling is induced by each iCAR, resulting in increased ITIM phosphorylation but not activating signals such as Syk and Zap70. The association of iCARs with their cognate ligands functions to inhibit NK-mediated cytokine secretion (IFN-gamma, TNF-alpha), degranulation (CD107a), and cytotoxicity in an antigen-specific manner. NK cells transduced with iCARs recognizing the NK autoantigen CS1 fused to an inhibitory signaling endodomain prevent CAR-NK cell fratricide without impairing their cytotoxicity against "on-target" tumors. Furthermore, expression of iCARs did not negatively affect the proliferation and expansion of CAR-NK cells. Thus, engineering immune effectors using a dual CAR system containing an NK self-recognition inhibitory CAR (iCAR) that delivers a "don't kill me" signal to NK cells upon association with normal cells prevents the off-target on-tumor activity of activating CARs (aCARs) against tumor antigens while preserving their on-target on-tumor signaling. Example 2 Expression of iCAR on NK cells derived from umbilical cord blood

[0339] We first designed iCARs against CD19 as a well-studied model. Anti-CD19 (IgG1 hinge-attached single-chain fragment variable (19scFv)) was ligated with the inhibitory KIR signals listed in Example 1. Each construct was then cloned into an SFG retroviral backbone, allowing for the engineering of NK cells by retroviral-mediated transduction. Surface expression of the iCARs on NK cells was confirmed by flow cytometry using a goat anti-mouse IgG(H+L) antibody. As shown in Figure 1, after transduction, stable engineering of NK cells with each of the five CD19 iCARs (19scFv-iCAR1, 19scFv-iCAR2, 19scFv-iCAR3, 19scFv-iCAR4, and 19scFv-iCAR5) was successfully achieved with an efficiency of 75%.

[0340] Alternatively, iCAR1 was fused to an anti-CS1 scFv (fused to an IgG1 hinge) that recognizes CS1, which is expressed on normal NK cells and T cells (Tai et al., 2008), and then cloned into an SFG retroviral backbone. Anti-CS1-iCAR1 (iCAR-CS1) was successfully introduced into NK cells along with an activating anti-CD19 CAR (CAR19-CD3 zeta) in NK cells (Figure 2). Fluorescently labeled immunogens (CS1 and CD19 peptides) were used to assess the expression levels of iCAR-CS1 and aCAR19, respectively. More than 80% of NK cells expressed iCAR-CS1, and more than 70% expressed aCAR19. Importantly, approximately 70% of the engineered NK cells co-expressed iCAR-CS1 and aCAR19. Example 3 Effect of iCAR signaling in inhibiting NK-activated phosphorylation

[0341] NK cell activation is strictly determined by the phosphorylation state of signaling molecules, not by transcriptional regulation (see, e.g., Bryceson & Long 2008; Vivier et al., 2004, and Long et al., 2013). Engagement of a cognate ligand by an inhibitory receptor results in phosphorylation of the ITIM domain, which competes with the activating signal. We combined the iCAR signaling endodomain with a CD19-recognition ectodomain, which allows the transmission of inhibitory signals upon engagement with CD19-expressing cells, thus demonstrating Raji CD19+ After co-culture testing, iCAR mediated phosphorylation signaling at the ITIM-enriched adaptor (SHP1) but not at the ITAM-enriched adaptor (Syk / Zap70). aCAR NK cells (CD19ScFv linked to CD3 zeta) that deliver activation signals and NK cells expressing only the CD19 recognition domain (scFv) (no signaling endodomain) were included as controls. Co-culture of iCAR-expressing NK cells with Raji cells resulted in a rapid increase in pSHP1, which also limited the phosphorylation of Syk and ZAP70 (Figure 3), confirming the strong inhibitory function of iCAR signaling in preventing NK cell activation. Example 4 Negative effect of iCAR signaling on NK-mediated cytotoxicity

[0342] After transduction of NK cells with iCAR19, their function against CD19+ targets was examined. For each iCAR19-NK cell, CD19-expressing targets (K562 CD19+ Stimulation with iCAR19-CD3 zeta NK cells (K562 and Raji, Figures 4A and 4B) resulted in reduced degranulation levels (CD107a) and cytokine secretion (TNF-alpha, IFN-gamma) when compared to control 19scFv-NK cells or CAR19-CD3 zeta NK cells. In contrast, this inhibitory effect of iCAR19 on NK cell cytotoxicity was not observed when the cells were stimulated with non-CD19 target antigen-expressing cells (K562 and Raji). CD19- (Raji CD19-KOThis was not observed when the iCAR was stimulated with NK cells containing iCAR (also referred to as iCAR, Figures 4C and 4D). These findings demonstrate that iCAR inhibits NK cell activation in an antigen-specific manner.

[0343] Because iCAR signaling significantly inhibits NK cell activation, we investigated the effect of iCAR on NK cell-mediated cytotoxicity against cognate antigen-expressing tumor targets. iCAR-expressing NK cells were cultured in Raji cells or NK CD19+ These cells were co-cultured with NK cells (e.g., NK cells engineered to express CD19), both of which express the cognate antigen CD19 (Figure 5). CD19+ When tested against CD3 zeta-NK cells, CAR19-CD3 zeta-NK cells exhibited stronger cytotoxicity against targets than 19scFv-NK cells (without signaling endodomain) or iCAR (inhibitory signaling endodomain) NK cells (Figure 5A). CD19+ When tested against NK cells, only CAR19-CD3 zeta-NK cells demonstrated CD19+ induced fratricide and apoptosis of NK target cells (Figure 5B). Overall, these data demonstrate that iCAR signaling inhibits NK cell effector function and cytotoxicity in an antigen-dependent manner. Example 5 The role of iCAR signaling in regulating CAR-NK cell-mediated cytotoxicity

[0344] Since we confirmed that iCAR signaling can inhibit NK effector function in an antigen-dependent manner, we designed a dual CAR system that combines an activating CAR against a tumor antigen (e.g., CD19) with an inhibitory signal against a "self-antigen" expressed on normal cells, such as CS1, which is expressed on all NK cells. Specifically, we investigated whether iCARs could suppress aCAR signaling.

[0345] First, we transduced NK cells with a CAR19-CD3 zeta (aCAR)-expressing construct, along with iCAR1-CS1 or CS1scFv (without signaling endodomain) as a control, and then cocultured them with the MM1S myeloma cell line, which expresses CS1 but not CD19 (Figure 6). NK cells expressing aCAR19 / iCAR-CS1 killed significantly fewer MM1S cells than aCAR19 / CS1scFv-NK cells, indicating that the inhibitory signaling of iCAR1 suppresses NK cell-mediated cytotoxicity after engagement with the self-surface antigen CS1 on target cells.

[0346] Next, we investigated whether iCAR could suppress aCAR19-mediated cytotoxicity. Dual-CAR NK cells expressing both CAR19 and iCAR1-CS1, along with CAR19 / CS1scFv-NK cells, were transfected with CD19 + Target (e.g. Raji CD19+ / CS1- cells, SKOV3 gCD19+ / CS1- cells), CD19-targets (e.g., Raji CD19- / CS1- cells, SKOV3 CD19- / CS1- cells), and autologous CS1-expressing targets (e.g., NK CD19+ / CS1+ In the presence of the cognate antigen CD19, aCAR19 inhibited Raji CD19+ / CS1- cells and SKOV3 gCD19+ / CS1-CAR19-NK mediated strong cytotoxicity, resulting in rapid cell death after coculture with CAR19 cells. In contrast, no significant difference was observed compared to NK cells that also expressed iCAR1-CS1 or CS1scFv (Figure 7A). Furthermore, when tested against tumor targets that did not express the cognate autoantigen CS1, coexpression of iCAR1-CS1 or CS1scFv had little effect on CAR19-NK-mediated cytotoxicity, again demonstrating the antigen specificity of inhibitory CAR function (Figure 7A). Importantly, NK cells expressing activating CAR19 mediated strong cytotoxicity (fratricide) against their CD19+ sister cells, whereas coexpression of iCAR1-CS1 on NK cells prevented and reduced the magnitude of self-killing (Figure 7B), suggesting that the association of iCAR-CS1 with the autoantigen CS1 on NK cells provides an inhibitory signal that can overcome NK cell activation mediated by the activating CAR19 signal. These data support that additional expression of iCAR1-CS1 in NK cells can suppress anti-self activation signals and prevent their on-target and off-tumor activity. Example 6 Effect of iCAR signaling on CAR-NK and NK c...

Claims

1. (a) (1) At least one extracellular antigen-binding domain, wherein the first extracellular antigen-binding domain binds to a first antigen, (2) A first transmembrane domain, and (3) At least one natural killer (NK) cell inhibitory signaling domain and / or at least one co-inhibitory domain Comprising at least one inhibitory chimeric antigen receptor (iCAR), and (b) (1) At least one extracellular antigen-binding domain, wherein the second extracellular binding domain binds to a second antigen, (2) A second transmembrane domain, and (3) At least one activation end domain, with or without a co-stimulatory signaling domain Comprising at least one activating chimeric antigen receptor (aCAR) A composition comprising engineered immune effector cells.

2. The composition according to claim 1, wherein the cells are NK cells or T cells.

3. The composition according to claim 1 or 2, wherein the first antigen and the second antigen are different antigens.

4. The composition according to claim 1, 2 or 3, wherein the first extracellular antigen-binding domain of (a)(1) binds to an antigen on NK cells.

5. The composition according to claim 4, wherein the cells are NK cells, and the first extracellular antigen-binding domain of (a)(1) binds to an antigen on NK cells.

6. The composition according to claim 5, wherein the iCAR has two antigen-binding domains targeting different antigens.

7. The composition according to any one of claims 1 to 6, wherein the second extracellular antigen-binding domain of (b)(1) binds to a cancer antigen or a pathogen antigen.

8. The composition according to any one of claims 1 to 7, wherein the NK cell inhibitory signaling domain and / or the co-inhibitory domain is derived from an NK cell inhibitory receptor.

9. The composition according to any one of claims 1 to 8, wherein the NK cell inhibitory signaling domain and / or the co-inhibitory domain is derived from leukocyte immunoglobulin-like receptor (LIR-1), CD300A, NKG2A, Siglec-7, CD96, T cell immunoglobulin and mucin domain-containing 3 (TIM3), TIGIT, LAIR-1, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, KIR3DL3, KIR2DL5A, and / or KIR2DL5B.

10. The composition according to any one of claims 1 to 9, wherein the first transmembrane domain and the inhibitory signal transduction domain are derived from the same molecule.

11. The composition according to claim 10, wherein the first transmembrane domain and the inhibitory signal transduction domain are derived from LIR-1 or KIR2DL1.

12. The composition according to any one of claims 1 to 11, wherein the iCAR comprises at least one co-inhibitory domain.

13. The composition according to claim 12, wherein the co-inhibitory domain is derived from LAIR-1, NKG2A, CD300A, or a combination thereof.

14. The composition according to any one of claims 1 to 13, wherein the first and / or second extracellular antigen-binding domain comprises an scFv or a natural ligand.

15. The iCAR is comprising an scFv that binds to CS1 as the first extracellular antigen-binding domain of (a)(1), an IgG1 hinge, the first transmembrane domain of (a)(2) from KIR2DL1, and the inhibitory signal transduction domain of (a)(3) from KIR2DL1 The composition according to any one of claims 1 to 14.

16. A method for enhancing adoptive cell therapy for an individual in need thereof, the method comprising (a) at least one extracellular antigen-binding domain in which the first extracellular antigen-binding domain binds to a first antigen, and at least one natural killer (NK) cell inhibitory signal transduction domain and / or at least one co-inhibitory domain comprising at least one inhibitory chimeric antigen receptor (iCAR), (b) at least one extracellular antigen-binding domain in which the second antigen-binding domain binds to a second antigen, and an activation endodomain and at least one co-stimulatory signal transduction domain comprising at least one activating chimeric antigen receptor (aCAR) administering to the individual a therapeutically effective amount of engineered immune effector cells, each comprising (I) when the first antigen and the second antigen are the same and the engineered immune effector cells bind to the cells expressing the antigen via the second extracellular antigen-binding domain, the iCAR inhibits the killing of the cells expressing the antigen by the engineered immune effector cells, or (II) The first antigen and the second antigen are non-identical, and both are expressed on engineered immune effector cells of the same kind, or on the same type of non-engineered immune effector cells, or on non-diseased cells. When the engineered immune effector cells bind to the second antigen on the same kind of engineered immune effector cells, or the same type of non-engineered immune effector cells, or the non-diseased cells via the second extracellular antigen-binding domain, the iCAR inhibits the killing of the engineered immune effector cells by the same kind of engineered immune effector cells, or the same type of non-engineered immune effector cells, or the non-diseased cells. Method.

17. The method according to claim 16, wherein in (I), the cells expressing the antigen are non-cancerous cells.

18. The method according to claim 16, wherein in (I), the cells expressing the antigen are engineered immune effector cells of the same kind.

19. The method according to claim 16, wherein in (II), the engineered immune effector cells bind to the second antigen on the same kind of engineered immune effector cells via the second extracellular antigen-binding domain.

20. The method according to claim 19, wherein the second antigen is expressed as a result of trogocytosis by the same kind of engineered immune effector cells.

21. The method according to any one of claims 16 to 20, wherein the engineered immune effector cells are NK cells and are engineered to express an iCAR targeting an NK cell self-antigen.

22. The method according to any one of claims 16 to 21, wherein the engineered immune effector cells are engineered to express an iCAR targeting an NK cell self-antigen and then are engineered to express an aCAR targeting an antigen on the cancer cells of the individual.

23. The method according to any one of claims 16 to 22, wherein the first extracellular antigen-binding domain binds to an antigen on NK cells.

24. The method according to any one of claims 16 to 23, wherein the engineered immune effector cell is a NK cell and the first extracellular antigen-binding domain binds to an antigen on the NK cell.

25. The method according to any one of claims 16 to 24, wherein the iCAR has two antigen-binding domains each targeting a different antigen.

26. The method according to any one of claims 16 to 25, wherein the second extracellular antigen-binding domain binds to a cancer antigen or a pathogen antigen.

27. The method according to any one of claims 16 to 26, wherein the NK cell inhibitory signaling domain and / or the co-inhibitory domain is derived from a NK cell inhibitory receptor.

28. The method according to any one of claims 16 to 27, wherein the first transmembrane domain and the inhibitory signaling domain are derived from the same molecule.

29. The method according to claim 28, wherein the first transmembrane domain and the inhibitory signaling domain are derived from LIR-1 or KIR2DL1.

30. The method according to any one of claims 16 to 29, wherein the iCAR comprises at least one co-inhibitory domain.

31. The method according to claim 30, wherein the co-inhibitory domain is derived from LAIR-1, NKG2A, CD300A, or a combination thereof.

32. The method according to any one of claims 16 to 31, wherein the first and / or second extracellular antigen-binding domain comprises a scFv or a natural ligand.

33. The iCAR is comprising a scFv that binds to CS1 as the first extracellular antigen-binding domain of (a)(1), an IgG1 hinge, the first transmembrane domain of (a)(2) from KIR2DL1, and the inhibitory signaling domain of (a)(3) from KIR2DL1 The method according to any one of claims 16 to 32.

34. A method for increasing the likelihood that an individual is classified as responding to immune effector cell therapy when compared to a control population of individuals receiving immune effector cell therapy, comprising: (a) at least one extracellular antigen-binding domain wherein the first extracellular antigen-binding domain binds to a first antigen, and at least one natural killer (NK) cell inhibitory signaling domain and / or at least one co-inhibitory domain comprising at least one inhibitory chimeric antigen receptor (iCAR). (b) At least one extracellular antigen-binding domain in which the second antigen-binding domain binds to a second antigen, and An activation endodomain and at least one co-stimulatory signaling domain At least one activating chimeric antigen receptor (aCAR) comprising A method comprising administering to the individual a therapeutically effective amount of engineered immune effector cells, each comprising **Claim 35** A method for increasing at least one of the in vivo persistence, survival rate, or efficacy of effector cells included in an immune effector cell therapy when compared to control effector cells included in a control immune effector cell therapy, comprising (a) At least one extracellular antigen-binding domain in which the first antigen-binding domain binds to a first antigen, and At least one natural killer (NK) cell inhibitory signaling domain and / or at least one co-inhibitory domain At least one inhibitory chimeric antigen receptor (iCAR) comprising (b) At least one extracellular antigen-binding domain in which the second antigen-binding domain binds to a second antigen, and An activation endodomain and at least one co-stimulatory signaling domain At least one activating chimeric antigen receptor (aCAR) comprising A method comprising administering to the individual a therapeutically effective amount of engineered immune effector cells, each comprising **Claim 36** A method for increasing the viability of an individual after treatment with an immune effector cell therapy when compared to a control population of individuals receiving the immune effector cell therapy, comprising (a) At least one extracellular antigen-binding domain in which the first antigen-binding domain binds to a first antigen, and At least one natural killer (NK) cell inhibitory signaling domain and / or at least one co-inhibitory domain At least one inhibitory chimeric antigen receptor (iCAR) comprising (b) At least one extracellular antigen-binding domain in which the second antigen-binding domain binds to a second antigen, and An activation endodomain and at least one co-stimulatory signaling domain At least one activating chimeric antigen receptor (aCAR) comprising A method comprising administering to the individual a therapeutically effective amount of engineered immune effector cells, each comprising **Claim 37** A method for increasing the circulating serum level of at least one effector cell-related protein in an individual after treatment with immune effector cell therapy, when compared to a control population of individuals receiving immune effector cell therapy, the method comprising: (a) At least one extracellular antigen-binding domain, wherein the first extracellular antigen-binding domain binds to a first antigen, and At least one natural killer (NK) cell inhibitory signaling domain and / or at least one co-inhibitory domain Comprising at least one inhibitory chimeric antigen receptor (iCAR), and (b) At least one extracellular antigen-binding domain, wherein the second antigen-binding domain binds to a second antigen, and An activation endodomain and at least one co-stimulatory signaling domain Comprising at least one activating chimeric antigen receptor (aCAR) Administering to the individual a therapeutically effective amount of the engineered immune effector cells, each comprising, Wherein the at least one effector cell-related protein is selected from granzyme A (GrA), granzyme B (GrB), perforin, interferon gamma (IFN-γ), and tumor necrosis factor alpha (TNF-α). Method.

38. (I) When the first antigen and the second antigen are the same and the engineered immune effector cell binds to the cell expressing the antigen via the second extracellular antigen-binding domain, the iCAR inhibits the killing of the cell expressing the antigen by the engineered immune effector cell, or (II) The first antigen and the second antigen are non-identical, and both are expressed on engineered immune effector cells of the same kind, or on the same type of non-engineered immune effector cells, or on non-diseased cells. When the engineered immune effector cells bind to the second antigen on the same kind of engineered immune effector cells or the same type of non-engineered immune effector cells or the non-diseased cells via the second extracellular antigen-binding domain, the iCAR inhibits the killing of the engineered immune effector cells by the same kind of engineered immune effector cells or the same type of non-engineered immune effector cells or the non-diseased cells. The method according to any one of claims 34 to 37.

39. The method according to claim 38, wherein in (I), the cell expressing the antigen is a non-cancerous cell.

40. The method according to claim 38, wherein in (I), the cell expressing the antigen is an engineered immune effector cell of the same kind.

41. The method according to claim 38, wherein in (II), the engineered immune effector cell binds to the second antigen on the same kind of engineered immune effector cells via the second extracellular antigen-binding domain.

42. The method according to claim 41, wherein the second antigen is expressed as a result of trogocytosis by the engineered immune effector cells of the same kind.

43. The method according to any one of claims 38 to 42, wherein the engineered immune effector cell is an NK cell and is engineered to express an iCAR targeting an NK cell self-antigen.

44. The method according to any one of claims 38 to 43, wherein the engineered immune effector cell is engineered to express an iCAR targeting an NK cell self-antigen and then is engineered to express an aCAR targeting an antigen on the cancer cells of the individual.

45. The method according to any one of claims 38 to 44, wherein the first extracellular antigen-binding domain binds to an antigen on an NK cell.

46. The method according to any one of claims 38 to 45, wherein the engineered immune effector cell is an NK cell and the first extracellular antigen-binding domain binds to an antigen on the NK cell.

47. The method according to any one of claims 38 to 46, wherein the NK cell inhibitory signaling domain and / or the co-inhibitory domain is derived from an NK cell inhibitory receptor.

48. The method according to any one of claims 38 to 47, wherein the first transmembrane domain and the inhibitory signaling domain are derived from the same molecule.

49. The method according to claim 48, wherein the first transmembrane domain and the inhibitory signaling domain are derived from LIR-1 or KIR2DL1.

50. The method according to any one of claims 38 to 49, wherein the iCAR comprises at least one co-inhibitory domain.

51. The method according to claim 50, wherein the co-inhibitory domain is derived from LAIR-1, NKG2A, CD300A, or a combination thereof.

52. The method according to any one of claims 38 to 51, wherein the first and / or second extracellular antigen-binding domain comprises an scFv or a natural ligand.

53. The iCAR is comprising an scFv that binds to CS1 as the first extracellular antigen-binding domain of (a)(1), an IgG1 hinge, the first transmembrane domain of (a)(2) from KIR2DL1, and the inhibitory signaling domain of (a)(3) from KIR2DL1 The method according to any one of claims 38 to 52.

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