Combinations of engineered natural killer (NK) cells with antibody therapy and related methods

Abstract

Provided herein are methods of treatment and uses involving the administration of a composition comprising an NK cell (g-NK cell) that lacks expression of the FcR gamma chain, engineered with a recombinant chimeric antigen receptor (CAR), in combination with a monoclonal antibody. The provided methods and uses include methods and uses for treating cancer, such as multiple myeloma or lymphoma.

Description

Cross - Reference to Related Applications

[0001] This application claims priority to U.S. Provisional Application No. 63 / 357,637, filed Jun. 30, 2022, entitled "COMBINATION OF ENGINEERED NATURAL KILLER (NK) CELLS AND ANTIBODY THERAPY AND RELATED METHODS", the contents of which are incorporated herein by reference in their entirety. Incorporation - by - Reference of Sequence Listing

[0002] This application is accompanied by a Sequence Listing submitted in electronic format. The Sequence Listing is provided as a file named "776032001440SeqList.xml", created on Jun. 30, 2023, and is 125,042 bytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0003] The present disclosure provides methods of treatment and uses that relate to administering a combination of a composition comprising NK cells lacking expression of the FcRγ chain (g-NK cells) and a monoclonal antibody, wherein the NK cells are engineered with a recombinant chimeric antigen receptor (CAR). Embodiments of the present disclosure include methods of treatment and uses for treating cancers such as multiple myeloma or lymphoma. BACKGROUND OF THE DISCLOSURE

[0004] Antibody-based therapies have been widely used to treat cancer and other diseases. Responses to antibody therapies typically focus on the direct inhibitory effects of these antibodies on tumor cells (e.g., inhibiting growth factor receptors and subsequently inducing apoptosis), but the in vivo effects of these antibodies are more complex and involve the host immune system. Natural killer (NK) cells are immune effector cells that mediate antibody-dependent cellular cytotoxicity when the Fc receptor (CD16; FcγRIII) binds to the Fc portion of an antibody bound to an antigen-carrying cell. NK cells, including their specific specialized subsets, can be used in therapeutic methods, including for improving responses to antibody therapies. Improved methods are needed for the therapeutic uses involving NK cells. The embodiments provided herein can meet such needs. SUMMARY OF THE INVENTION

[0005] In some aspects, the present disclosure provides methods of inducing cytolytic killing of target cells, the methods comprising contacting a target cell known or suspected of expressing a first antigen and a second antigen with: (a) a composition comprising natural killer (NK) cells lacking expression of the FcRγ chain (g-NK cells), wherein the g-NK cells express a chimeric antigen receptor (CAR) comprising an extracellular binding domain that binds to the first antigen; and (b) a monoclonal antibody that binds to the second antigen. In any of the foregoing embodiments, the first and second antigens can be different. In any of the foregoing embodiments, the first and second antigens can be the same. In any of the foregoing embodiments, the monoclonal antibody can be a full-length antibody. In any of the foregoing embodiments, the monoclonal antibody can be an IgG1 antibody. In any of the foregoing embodiments, the CAR and the monoclonal antibody can bind to different epitopes of the same antigen.

[0006] In any of the foregoing embodiments, the target cell can be a tumor cell. In any of the foregoing embodiments, the tumor cell can be a hematological malignancy cell. In any of the foregoing embodiments, the target cell can be a B cell. In any of the foregoing embodiments, the first and second antigens can be selected from the group consisting of CD30, CD19, CD20, CD22, ROR1, Igk, CD38, CD138, BCMA, CD33, CD70, CD79b, CD123, SLAMF7, GPRC5D, FCRH5, FLT3, CLEC12, and Lewis Y antigen.

[0007] In some embodiments, the hematological malignancy can be multiple myeloma. In some embodiments, the first and second antigens are selected from the group consisting of CD38, SLAMF7, CD138, FCRH5, GPRC5D, and BCMA. In some embodiments, the CAR can be an anti-BMCA CAR, and the monoclonal antibody can be an anti-CD38 antibody. In some embodiments, the anti-CD38 antibody can be daratumumab or isatuximab.

[0008] In some embodiments, the hematologic malignancy can be lymphoma. In some embodiments, the lymphoma can be non-Hodgkin lymphoma (NHL). In some embodiments, the first and second antigens can be selected from the group consisting of CD19, CD20, CD22, ROR1, CD30, CD38, and CD79b. In some embodiments, the first and second antigens can be selected from the group consisting of CD19, CD20, CD22, ROR1, and CD30. In some embodiments, the CAR can be an anti-CD19 CAR, and the antibody can be an anti-CD20 antibody. In some embodiments, the anti-CD20 antibody can be rituximab, obinutuzumab, or ofatumumab.

[0009] In some embodiments, the CAR can be an anti-CD19 CAR, and the antibody is an anti-CD38 antibody. In some embodiments, the CAR can be an anti-CD20 CAR, and the antibody is an anti-CD38 antibody. In some embodiments, the anti-CD38 antibody can be daratumumab or isatuximab.

[0010] In some embodiments, the hematologic malignancy can be leukemia. In some embodiments, the leukemia can be acute myeloid leukemia (AML). In some embodiments, the first and second antigens can be selected from the group consisting of CD123, Flt3, CD70, CD33, CLEC12A, and CD38.

[0011] In some embodiments, the tumor cells can be cells of a solid malignancy. In some embodiments, the first antigen and the second antigen can be selected from the group consisting of GPC3, HER2, GD2, EGFR mutant III (EGFR vIII), EGFR, CEA, PSMA, FRα, FAP, glypican-3, EPCAM, MUC1, ROR1, MUCI16eto, VEGFR2, CD171, PSCA, EphA2, survivin, mesothelin, TROP2, B7H3, CCR4, PDGFRα, laminin 4, tissue factor, CLDN6, FGFR2b, and IL-13α.

[0012] In some of any of the foregoing embodiments, the monoclonal antibody can be contacted separately with the cells from the composition comprising g-NK cells. In some embodiments, at least a portion of the contact with the composition comprising g-NK cells and the contact with the monoclonal antibody can be simultaneous. In some embodiments, the contact with the composition comprising g-NK cells can be simultaneous with the contact with the monoclonal antibody.

[0013] In some of any of the foregoing embodiments, the monoclonal antibody can be secreted from g-NK cells.

[0014] In some of any of the foregoing embodiments, the contacting can be carried out in the body of a subject.

[0015] In some aspects, provided herein is a method of treating cancer in a subject, the method can include: (a) administering to a subject having cancer an NK cell therapy, the therapy comprising a dose of a composition comprising natural killer (NK) cells lacking expression of the FcRγ chain (g-NK cells), wherein the g-NK cells express a chimeric antigen receptor (CAR) that comprises an extracellular binding domain that binds to a first antigen expressed by cancer cells; and (b) administering to the subject a dose of a monoclonal antibody that binds to a second antigen expressed by cancer cells.

[0016] In some aspects, provided herein is a method of treating cancer in a subject, the method can include administering to a subject having cancer an NK cell therapy, the NK cell therapy comprising a dose of a composition comprising natural killer (NK) cells lacking expression of the FcRγ chain (g-NK cells), wherein: the g-NK cells express a chimeric antigen receptor (CAR) that comprises an extracellular binding domain that binds to a first antigen expressed by cancer cells; and the g-NK cells express a secretable monoclonal antibody that binds to a second antigen expressed by cancer cells.

[0017] In some of any of the foregoing embodiments, the first and second antigens can be different. In some of any of the foregoing embodiments, the first and second antigens can be the same. In some of any of the foregoing embodiments, the monoclonal antibody is a full-length antibody. In some of any of the foregoing embodiments, the monoclonal antibody can be an IgG1 antibody. In some of any of the foregoing embodiments, the CAR and the monoclonal antibody can bind to different epitopes of the same antigen. In some of any of the foregoing embodiments, the first and second antigens can be expressed by the same cancer cells.

[0018] In some of any of the foregoing embodiments, the cancer is a hematological malignancy. In some of any of the foregoing embodiments, the cancer cells are B cells, and the cancer is a B cell cancer. In some embodiments, the first antigen and the second antigen may be selected from the group consisting of CD30, CD19, CD20, CD22, ROR1, Igk, CD38, CD138, BCMA, CD33, CD70, CD79b, CD123, SLAMF7, GPRC5D, FCRH5, FLT3, CLEC12, and Lewis Y antigen.

[0019] In some embodiments, the cancer may be multiple myeloma. In some embodiments, the multiple myeloma may be relapsed / refractory multiple myeloma. In some embodiments, the first antigen and the second antigen may be selected from the group consisting of CD38, SLAMF7, CD138, FCRH5, GPRC5D, and BCMA. In some embodiments, the CAR may be an anti-BMCA CAR, and the monoclonal antibody may be an anti-CD38 antibody. In some embodiments, the anti-CD38 antibody may be daratumumab or isatuximab.

[0020] In some embodiments, the cancer may be lymphoma. In some embodiments, the lymphoma may be non-Hodgkin lymphoma (NHL). In some embodiments, the NHL may be relapsed / refractory multiple NHL. In some embodiments, the first and second antigens are selected from the group consisting of CD19, CD20, CD22, ROR1, CD30, CD38, and CD79b. In some embodiments, the first and second antigens may be selected from the group consisting of CD19, CD20, CD22, ROR1, and CD30. In some embodiments, the CAR may be an anti-CD19 CAR, and the antibody may be an anti-CD20 antibody. In some embodiments, the anti-CD20 antibody may be rituximab, obinutuzumab, or ofatumumab.

[0021] In some embodiments, the CAR may be an anti-CD19 CAR, and the antibody is an anti-CD38 antibody. In some embodiments, the CAR may be an anti-CD20 CAR, and the antibody is an anti-CD38 antibody. In some embodiments, the anti-CD38 antibody may be daratumumab or isatuximab.

[0022] In some embodiments, the cancer can be leukemia. In some embodiments, the leukemia can be acute myeloid leukemia (AML). In some embodiments, the AML can be relapsed / refractory AML. In some embodiments, the first and second antigens can be selected from the group consisting of CD123, Flt3, CD70, CD33, CLECL12A, and CD38.

[0023] In some embodiments, the cancer is a solid malignancy. In some embodiments, the first and second antigens can be GPC3, HER2, GD2, EGFR mutant III (EGFR vIII), EGFR, CEA, PSMA, FRα, FAP, glypican-3, EPCAM, MUC1, ROR1, MUCI16 etc., VEGFR2, CD171, PSCA, EphA2, survivin, mesothelin, TROP2, B7H3, CCR4, PDGFRα, laminin 4, tissue factor, CLDN6, FGFR2b, and IL-13α.

[0024] In some of any of the foregoing embodiments, a certain dose of the g-NK cell composition can comprise a plurality of doses. In some of any of the foregoing embodiments, the NK cell therapy can comprise the administration of 1 - 8 doses of a composition comprising g-NK cells. In some of any of the foregoing embodiments, each dose of the g-NK cell composition can be administered once a week. In some of any of the foregoing embodiments, the NK cell therapy is administered in two doses of a composition comprising g-NK cells within a 14-day cycle, wherein the 14-day cycle can be repeated one to three times. In some of any of the foregoing embodiments, the NK cell therapy is administered in three doses of a composition comprising g-NK cells within a 21-day cycle, wherein the 21-day cycle can be repeated one to three times.

[0025] In some of any of the foregoing embodiments, the subject has received lymphodepletion therapy prior to the administration of the g-NK cell dose. In some of any of the foregoing embodiments, the method can further comprise administering lymphodepletion therapy to the subject prior to the administration of g-NK cells. In some of any of the foregoing embodiments, the administration of a certain dose of g-NK cells can be initiated within two weeks or at or about two weeks after the start of lymphodepletion therapy. In some of any of the foregoing embodiments, the administration of a certain dose of g-NK cells is initiated within 7 days or at or about 7 days after the start of lymphodepletion therapy. In some of any of the foregoing embodiments, the subject can be administered lymphodepletion therapy prior to repeating subsequent cycles. In some of any of the foregoing embodiments, the lymphodepletion therapy can comprise fludarabine and / or cyclophosphamide. In some of any of the foregoing embodiments, the lymphodepletion therapy can comprise the administration of at or about 20 - 40 mg / m2 Fludarabine and / or body surface area of ​​the subject is or is about 200-400 mg / m 2 In some embodiments, fludarabine is at or about 30 mg / m 2 Administer once daily for 2-4 days. In some embodiments, cyclophosphamide is at or about 300 mg / m 2 , once daily for 2-4 days. In some of any of the foregoing embodiments, the lymphodepleting therapy may include administering at or about 30 mg / m 2 Fludarabine once daily and at or about 300 mg / m 2 The subject's body surface area is treated with cyclophosphamide once daily for 2-4 days, optionally for 3 days.

[0026] In some of any of the foregoing embodiments, the administration of at least one dose of monoclonal antibody can start within one month before the administration of NK cell therapy. In some of any of the foregoing embodiments, the administration of at least one dose of monoclonal antibody can start within three weeks before the administration of NK cell therapy. In some of any of the foregoing embodiments, the administration of at least one dose of monoclonal antibody can start within two weeks before the administration of NK cell therapy. In some of any of the foregoing embodiments, the monoclonal antibody can be administered intravenously. In some of any of the foregoing embodiments, the monoclonal antibody can be administered subcutaneously. In some of any of the foregoing embodiments, the monoclonal antibody of the loading dose can be administered intravenously before subcutaneous administration. In some of any of the foregoing embodiments, a dose of monoclonal antibody can include multiple numbers of doses. In some of any of the foregoing embodiments, the monoclonal antibody can be administered once every four weeks, once every three weeks, once every two weeks, once a week, or twice a week. In some of any of the foregoing embodiments, each dose of monoclonal antibody can be administered once a week. In some of any of the foregoing embodiments, the monoclonal antibody can be administered in 4 to 16 doses, optionally for or about 4 doses or for or about 8 doses.

[0027] In some of any of the foregoing embodiments, the CAR can comprise: 1) an antigen-binding domain that binds to a first antigen; 2) a spacer; 3) a transmembrane region; and 4) an intracellular signaling domain. In some of any of the foregoing embodiments, the antigen-binding domain can be a single-chain variable fragment (scFv). In some of any of the foregoing embodiments, the intracellular signaling domain can comprise one or more signaling domains of CD3ζ, DAP10, DAP12, CD28, 4-1BB, or OX40. In some of any of the foregoing embodiments, the intracellular signaling domain can comprise two or more signaling domains of CD3ζ, DAP10, DAP12, CD28, 4-1BB, or OX40. In some of any of the foregoing embodiments, the intracellular signaling domain can comprise a primary signaling domain containing the signaling domain of CD3ζ. In some of any of the foregoing embodiments, wherein the intracellular signaling domain can further comprise a co-stimulatory signaling domain. In some embodiments, the co-stimulatory signaling domain is the signaling domain of CD28. In some embodiments, the co-stimulatory signaling domain is the signaling domain of 4-1BB.

[0028] In some of any of the foregoing embodiments, the heterologous nucleic acid encoding the CAR is capable of stably integrating into the genome of the cell. In some of any of the foregoing embodiments, the heterologous nucleic acid encoding the CAR can be transiently expressed. In some of any of the foregoing embodiments, the g-NK cell can further comprise a heterologous nucleic acid encoding an immunomodulatory protein. In some of any of the foregoing embodiments, the immunomodulatory protein can be a cytokine. In some of any of the foregoing embodiments, the cytokine can be secreted from the g-NK cell. In some of any of the foregoing embodiments, the secreted cytokine can be IL-2 or a biological moiety thereof; IL-15 or a biological moiety thereof; or IL-21 or a biological moiety thereof; or a combination thereof. In some of any of the foregoing embodiments, the cytokine can be membrane-bound. In some of any of the foregoing embodiments, the membrane-bound cytokine is membrane-bound IL-2 (mbIL-2); membrane-bound IL-15 (mbIL-15); membrane-bound IL-21 (mbIL-21); or a combination thereof. In some of any of the foregoing embodiments, the heterologous nucleic acid encoding the immunomodulator can be stably integrated into the genome of the cell. In some of any of the foregoing embodiments, the heterologous nucleic acid encoding the immunomodulator can be transiently expressed.

[0029] In some of any of the foregoing embodiments, the method can further comprise administering an exogenous cytokine to promote the expansion or persistence of g-NK cells in a subject. In some embodiments, the exogenous cytokine is or comprises IL-15.

[0030] In some of any of the foregoing embodiments, the FcRγ chain in the g-NK cells may not be detectable by immunoblotting.

[0031] In some of any of the foregoing embodiments, in the cells of the g-NK cell composition, greater than or about 60% of the cells are g-NK cells, greater than or about 70% of the cells are g-NK cells, greater than or about 80% of the cells are g-NK cells, greater than or about 90% of the cells are g-NK cells, or greater than or about 95% of the cells are g-NK cells. In some of any of the foregoing embodiments, at least or about 50% of the cells in the g-NK cell composition are FcRγ-deficient (FcRγ 阴性 ) NK cells (g-NK), wherein greater than or about 70% of the g-NK cells can be perforin-positive, and greater than or about 70% of the g-NK cells can be granzyme B-positive. In some of any of the foregoing embodiments, (i) greater than or about 80% of the g-NK cells can be perforin-positive, greater than or about 80% of the g-NK cells are granzyme B-positive, (ii) greater than or about 90% of the g-NK cells are perforin-positive, greater than or about 90% of the g-NK cells can be granzyme B-positive, or (iii) greater than or about 95% of the g-NK cells can be perforin-positive, greater than or about 95% of the g-NK cells can be granzyme B-positive. In some of any of the foregoing embodiments, in the perforin-positive cells, the average level of perforin that the cells can express, as measured by intracellular flow cytometry, according to the mean fluorescence intensity (MFI), is at least or about twice the average level of perforin expressed by FcRγ 阳性 cells. In some of any of the foregoing embodiments, in the granzyme B-positive cells, the average level of granzyme B that the cells can express, as measured by intracellular flow cytometry, according to the mean fluorescence intensity (MFI), is at least or about twice the average level of granzyme B expressed by FcRγ 阳性 cells.

[0032] In some of any of the foregoing embodiments, greater than 10% of the cells in the g-NK cell composition can be capable of degranulating tumor target cells. In some embodiments, the g-NK cells capable of degranulating are measured by CD107a expression. In some embodiments, degranulation is measured in the absence of an antibody against the tumor target cells.

[0033] In some of any of the foregoing embodiments, in the cells of the g-NK cell composition, greater than or about 15%, greater than or about 20%, greater than or about 30%, greater than or about 40%, or greater than or about 50% exhibit degranulation. In some embodiments, in the presence of cells expressing a target antigen (target cells) and an antibody directed against the target antigen (anti-target antibody), the g-NK cells capable of degranulation are measured by CD107a expression. In some embodiments, the g-NK cells capable of degranulation can be measured by CD107a expression. In some embodiments, degranulation is measured in the presence of cells expressing a target antigen (target cells) and an antibody directed against the target antigen (anti-target antibody).

[0034] In some of any of the foregoing embodiments, greater than 10% of the cells in the g-NK cell composition may be capable of producing interferon-γ or TNF-α against tumor target cells. In some embodiments, interferon-γ or TNF-α is measured in the absence of an antibody against the tumor target cells.

[0035] In some of any of the foregoing embodiments, in the cells of the g-NK cell composition, greater than or about 15%, greater than or about 20%, greater than or about 30%, greater than or about 40%, or greater than or about 50% produce effector cytokines in the presence of cells expressing a target antigen (target cells) and an antibody directed against the target antigen (anti-target antibody). In some of any of the foregoing embodiments, the effector cytokine can be IFN-γ or TNF-α. In some of any of the foregoing embodiments, the effector cytokine can be IFN-γ and TNF-α.

[0036] In some of any of the foregoing embodiments, the g-NK cell composition has been produced by in vitro expansion of CD3− / CD57+ cells or CD3− / CD56+ cells cultured with irradiated HLA-E+ feeder cells, wherein the CD3− / CD57+ cells or CD3− / CD55+ cells can be enriched from a biological sample of a donor subject. In some of any of the foregoing embodiments, the donor subject can be CMV seropositive. In some of any of the foregoing embodiments, the donor subject can have a CD16 158V / V NK cell genotype. In some of any of the foregoing embodiments, the donor subject can have a CD16 158V / F NK cell genotype. In some embodiments, the biological sample can be from a human subject selected for the CD16 158V / V NK cell genotype. In some embodiments, the biological sample can be from a human subject selected for the CD16 158V / F NK cell genotype.

[0037] In some of any of the foregoing embodiments, at least or about 20% of the natural killer (NK) cells in the peripheral blood sample from the donor subject can be positive for NKG2C (NKG2C-positive), and at least 70% of the NK cells in the peripheral blood sample can be negative or low NKG2A for NKG2A (NKG2A-negative). In some of any of the foregoing embodiments, the irradiated feeder cells can be deficient in HLA class I and HLA class II. In some of any of the foregoing embodiments, the irradiated feeder cells can be 221.AEH cells. In some of any of the foregoing embodiments, the culture can be carried out in the presence of two or more recombinant cytokines, wherein at least one recombinant cytokine can be interleukin (IL)-2, and at least one recombinant cytokine can be IL-21. In some of any of the foregoing embodiments, the recombinant cytokines can be IL-21 and IL-2. In some of any of the foregoing embodiments, the recombinant cytokines are IL-21, IL-2, and IL-15.

[0038] In some of any of the foregoing embodiments, the g-NK cells can be genetically engineered to knockout the gene encoding the FcRγ chain. In some of any of the foregoing embodiments, the gene knockout can be the introduction of a gene disruption of the gene, wherein the gene disruption results in a deletion, insertion, or mutation in the gene. In some of any of the foregoing embodiments, both alleles of the gene encoding the FcRγ chain can be disrupted in the engineered cells. In some of any of the foregoing embodiments, the gene disruption can be achieved by an endonuclease. In some of any of the foregoing embodiments, the endonuclease can be a TAL nuclease, a meganuclease, a zinc finger nuclease, an Argonaute nuclease, or a combination of a CRISPR enzyme and a guide RNA. In some of any of the foregoing embodiments, wherein the endonuclease can be a combination of CRISPR / Cas9 and a guide RNA.

[0039] In some of any of the foregoing embodiments, the g-NK cells can further comprise a nucleic acid encoding a heterologous CD16. In some of any of the foregoing embodiments, the heterologous CD16 can comprise an activating mutation of CD16, wherein the mutation results in a higher affinity for IgG1. In some of any of the foregoing embodiments, the heterologous CD16 can comprise the 158V mutation. In some of any of the foregoing embodiments, the engineered g-NK cells can be derived from primary cells obtained from a human subject.

[0040] In some of any of the foregoing embodiments, the g-NK cell composition can be formulated in a serum-free cryopreservation medium containing a cryoprotectant. In some embodiments, the cryoprotectant can be DMSO, and the cryopreservation medium can be 5%-10% DMSO (v / v). In some of any of the foregoing embodiments, each dose of g-NK cells can be from or about from or about 1x10 8 cells to a g-NK cell composition of or about 50x10 9 cells. In some embodiments, each dose of g-NK cells can be a g-NK cell composition of or about 5x10 8 cells. In some embodiments, each dose of g-NK cells can be a g-NK cell composition of or about 5x10 9 cells. In some embodiments, each dose of g-NK cells can be a g-NK cell composition of or about 10x10 9 cells. In some of any of the foregoing embodiments, the subject is a human subject. In some of any of the foregoing embodiments, the NK cells in the composition can be allogeneic to the subject.

[0041] In some aspects, provided is an engineered natural killer (NK) cell, wherein the NK cell can be deficient in the expression of the FcRγ chain (g-NK cell), and wherein the g-NK cell can comprise: a heterologous nucleic acid encoding a chimeric antigen receptor (CAR), the chimeric antigen receptor comprising an extracellular binding domain that binds to a first antigen; and a heterologous nucleic acid encoding a secreted monoclonal antibody that binds to a second antigen. In some of any of the foregoing embodiments, the first and second antigens can be different. In some of any of the foregoing embodiments, the first and second antigens can be the same. In some of any of the foregoing embodiments, the monoclonal antibody can be a full-length antibody. In some of any of the foregoing embodiments, the monoclonal antibody can be an IgG1 antibody. In some of any of the foregoing embodiments, the CAR and the monoclonal antibody can bind to different epitopes of the same antigen. In some of any of the foregoing embodiments, the first and second antigens can be expressed by the same target cell. In any of the foregoing embodiments, the target cell can be a tumor cell.

[0042] Also provided is a pharmaceutical composition comprising any of the engineered NK cells and a pharmaceutically acceptable carrier. In some of any of the foregoing embodiments, the pharmaceutical composition can comprise a cryoprotectant. In some of any of the foregoing embodiments, the pharmaceutical composition can be formulated in a serum-free cryopreservation medium containing a cryoprotectant. In some of any of the foregoing embodiments, the cryoprotectant is DMSO. In some embodiments, the cryopreservation medium can be 5%-10% DMSO (v / v).

[0043] The present invention also provides a method for treating cancer in a subject, which comprises administering the pharmaceutical composition to a subject suffering from cancer. Brief Description of the Drawings

[0044] Figure 1A and Figure 1B The expansion of g-NK cells in the presence of 221.AEH or K562-mbIL15-41BBL feeder cells and in NK cell medium with or without IL-21 is described. Figure 1A The total NK cell count is shown. Figure 1B The n-fold expansion at day 21 of expansion is shown.

[0045] Figure 2A and Figure 2B The daratumumab- and elotuzumab-mediated cytotoxic activities of g-NK cells expanded in the presence of 221.AEH or K562-mbIL15-41BBL feeder cells and in NK cell medium with or without IL-21 after 21 days of expansion are described. Figure 2A The cytotoxicity of g-NK cells against the LP1 cell line is shown. Figure 2B The cytotoxicity of g-NK cells against the MM.1S cell line is shown.

[0046] Figure 3A and Figure 3D The daratumumab- and elotuzumab-mediated degranulation levels (CD107a 阳性 ) of g-NK cells expanded in the presence of 221.AEH or K562-mbIL15-41BBL feeder cells and in NK cell medium with or without IL-21 are described. Figure 3A The degranulation level of g-NK cells against the LP1 cell line at 13 days after expansion is shown. Figure 3B The degranulation level of g-NK cells against the MM.1S cell line at 13 days after expansion is shown. Figure 3C The degranulation level of g-NK cells against the LP1 cell line at 21 days after expansion is shown. Figure 3D The degranulation level of g-NK cells against the MM.1S cell line at 21 days after expansion is shown.

[0047] Figures 4A - 4D The levels of perforin and granzyme B expression of g-NK cells expanded in the presence of 221.AEH or K562-mbIL15-41BBL feeder cells and in NK cell medium with or without IL-21 are described. Figure 4AShows the expression of perforin and granzyme B in cells 13 days after expansion, expressed as a percentage of g-NK. Figure 4B Shows the total perforin and granzyme B expression 13 days after expansion. Figure 4C Shows the expression of perforin and granzyme B in cells 21 days after expansion, expressed as a percentage of g-NK. Figure 4D Shows the total perforin and granzyme B expression 21 days after expansion.

[0048] Figures 5A - 5D Describes the daratumumab- and elotuzumab-mediated interferon-γ expression levels of g-NK cells expanded in the presence of 221.AEH or K562-mbIL15-41BBL feeder cells and with or without IL-21 in the NK cell medium. Figure 5A Shows the interferon-γ expression level of g-NK cells against the LP1 cell line 13 days after expansion. Figure 5B Shows the interferon-γ expression level of g-NK cells against the MM.1S cell line 13 days after expansion. Figure 5C Shows the interferon-γ expression level of g-NK cells against the LP1 cell line 21 days after expansion. Figure 5D Shows the interferon-γ expression level of g-NK cells against the MM.1S cell line 21 days after expansion.

[0049] Figures 6A - 6D Describes the daratumumab- and elotuzumab-mediated TNF-α expression levels of g-NK cells expanded in the presence of 221.AEH or K562-mbIL15-41BBL feeder cells and with or without IL-21 in the NK cell medium. Figure 6A Shows the TNF-α expression level of g-NK cells against the LP1 cell line 13 days after expansion. Figure 6B Shows the TNF-α expression level of g-NK cells against the MM.1S cell line 13 days after expansion. Figure 6C Shows the TNF-α expression level of g-NK cells against the LP1 cell line 21 days after expansion. Figure 6D Shows the TNF-α expression level of g-NK cells against the MM.1S cell line 21 days after expansion.

[0050] Figure 7 Describes the g-NK cell expansion of NK cells expanded for 15 days in the presence of multiple cytokine mixtures and concentrations.

[0051] Figures 8A - 8J Shows the cell effector functions of g-NK cells expanded in the presence of multiple cytokine mixtures and concentrations.

[0052] Figure 8A andFigure 8B Describes the daratumumab - and elotuzumab - mediated cytotoxic activity of g - NK cells expanded in the presence of multiple cytokine mixtures and concentrations. Figure 8A Shows the cytotoxicity of g - NK cells against the LP1 cell line. Figure 8B Shows the cytotoxicity of g - NK cells against the MM.1S cell line.

[0053] Figure 8C and Figure 8D Describes the daratumumab - and elotuzumab - mediated degranulation levels (CD107a 阳性 ) of g - NK cells expanded in the presence of multiple cytokine mixtures and concentrations. Figure 8C Shows the degranulation levels of g - NK cells against the LP1 cell line. Figure 8D Shows the degranulation levels of g - NK cells against the MM.1S cell line.

[0054] Figure 8E and Figure 8F Shows the levels of perforin and granzyme B expression of g - NK cells expanded in the presence of multiple cytokine mixtures and concentrations. Figure 8E Shows the cell perforin and granzyme B expression, expressed as a percentage of g - NK cells. Figure 8F Shows the total perforin and granzyme B expression levels.

[0055] Figure 8G and Figure 8H Describes the daratumumab - and elotuzumab - mediated interferon - γ expression levels of g - NK cells expanded in the presence of multiple cytokine mixtures and concentrations. Figure 8G Shows the interferon - γ expression levels of g - NK cells against the LP1 cell line. Figure 8H Shows the interferon - γ expression levels of g - NK cells against the MM.1S cell line.

[0056] Figure 8I and Figure 8J Describes the daratumumab - and elotuzumab - mediated TNF - α expression levels of g - NK cells expanded in the presence of multiple cytokine mixtures and concentrations. Figure 8I Shows the TNF - α expression levels of g - NK cells against the LP1 cell line. Figure 8J Shows the TNF - α expression levels of g - NK cells against the MM.1S cell line.

[0057] Figure 9A and Figure 9BDescribes the expansion of g-NK cells expanded in the presence of IL-21 compared to g-NK cells expanded in the absence of IL-21. Figure 9A Shows the percentage of g-NK cells before and after expansion. Figure 9B Shows the number of g-NK cells expanded per 10 million NK cells. Values are mean ± SE. CD3 阴性 / CD57 阳性 +IL-21 expansion compared to CD3 阴性 / CD57 阳性 expansion without IL-21, #p < 0.001. CD3 阴性 / CD57 阳性 expansion compared to other CMV 阳性 expansion, ^p < 0.05. CMV 阳性 expansion compared to CMV 阴性 CD3 阴性 expansion, *p < 0.001.

[0058] Figure 9C Describes the comparison of the ratios of g-NK before and after expansion (percentage of the total number of NK cells from CMV+ (n = 8) and CMV- donors (n = 6)). Figure 9D Describes the n-fold expansion rates of g-NK from CMV+ and CMV- donors. Figure 9E Provides a representative flow plot of FcεR1γ vs. CD56 for CMV+ donors. Figure 9F Provides representative histograms of FcεR1γ expression on CD3- / CD56+ NK cells for CMV+ and CMV- donors. Differences between CMV+ and CMV- donors before and after expansion were determined using an independent samples t-test ( Figure 9C and Figure 9D ). Values are mean ± SE. *P < 0.05, **P < 0.01, and ***P < 0.001.

[0059] Figure 9G and Figure 9H Describes the daratumumab- and elotuzumab-mediated cytotoxic activity 14 days after expansion of g-NK cells expanded in the presence of IL-21 compared to g-NK expanded in the absence of IL-21. Figure 9G Shows the cytotoxicity of g-NK cells against the LP1 cell line. Figure 9H Shows the cytotoxicity of g-NK cells against the MM.1S cell line. Values are mean ± SE. CD3 阴性 / CD57 阳性 +IL-21 expansion compared to CD3 阴性 / CD57 阳性 Comparison of expansion, *p < 0.05, **p < 0.01, and ***p < 0.001.

[0060] Figure 9I and Figure 9J Describes the levels of daratumumab - and elotuzumab - mediated degranulation (CD107a 阳性 ) of g - NK cells expanded in the presence of IL - 21 compared to g - NK expanded in the absence of IL - 21. Figure 9I Shows the levels of g - NK cell degranulation against the LP1 cell line 14 days after expansion. Figure 9J Shows the levels of g - NK cell degranulation against the MM.1S cell line 14 days after expansion. Values are mean ± SE. CD3 阴性 / CD57 阳性 Expansion with IL - 21 compared to CD3 阴性 / CD57 阳性 Expansion without IL - 21, *p < 0.05, **p < 0.01, and ***p < 0.001.

[0061] Figure 9K and Figure 9L Shows the levels of perforin and granzyme B expression in g - NK cells expanded in the presence of IL - 21 compared to g - NK expanded in the absence of IL - 21. Figure 9K Shows perforin and granzyme B expression 14 days after expansion as a percentage of NK cells. Figure 9L Shows total perforin and granzyme B expression 14 days after expansion. Values are mean ± SE. CD3 阴性 / CD57 阳性 Expansion with IL - 21 compared to CD3 阴性 / CD57 阳性 Expansion without IL - 21, *p < 0.05, **p < 0.01, and ***p < 0.001.

[0062] Figure 9M Describes the baseline expression of perforin (left) and granzyme B (right) in expanded g - NK cells compared to cNK cells (n = 5). To compare the expression of effector perforin and granzyme B between g - NK and cNK, an independent samples t - test was used. Values are mean ± SE. Statistical differences from cNK cells are indicated by ***p < 0.001.

[0063] Figure 9N Describes representative histograms of perforin and granzyme B expression in g - NK and cNK cells.

[0064] Figure 9O and Figure 9P describe the daratumumab- and elotuzumab-mediated interferon-γ expression levels of g-NK cells expanded in the presence of IL-21 compared to g-NK expanded in the absence of IL-21. Figure 9O Show the interferon-γ expression levels of g-NK cells against the LP1 cell line 14 days after expansion. Figure 9P Show the interferon-γ expression levels of g-NK cells against the MM.1S cell line 14 days after expansion. Values are mean ± SE. CD3 阴性 / CD57 阳性 +IL-21 expansion versus CD3 阴性 / CD57 阳性 expansion without IL-21, *p<0.05, **p<0.01, and ***p<0.001.

[0065] Figure 9Q and Figure 9R describe the daratumumab- and elotuzumab-mediated TNF-α expression levels of g-NK cells expanded in the presence of IL-21 compared to g-NK expanded in the absence of IL-21. Figure 9Q Show the TNF-α expression levels of g-NK cells against the LP1 cell line 14 days after expansion. Figure 9R Show the TNF-α expression levels of g-NK cells against the MM.1S cell line 14 days after expansion. Values are mean ± SE. CD3 阴性 / CD57 阳性 +IL-21 expansion versus CD3 阴性 / CD57 阳性 expansion without IL-21, *p<0.05, **p<0.01, and ***p<0.001.

[0066] Figure 9S Describe the daratumumab- and elotuzumab-mediated interferon-γ expression levels of expanded g-NK cells against the MM.1S cell line in different donors compared to cNK cells. Figure 9T Describe the daratumumab- and elotuzumab-mediated TNF-α expression levels of expanded g-NK cells against the MM.1S cell line in different donors compared to cNK cells.

[0067] Figure 10 Describe the expansion of g-NK in the presence of IL-21 / anti-IL-21 complex (n = 4). Values are mean ± SE. Comparison of expansion with IL-21 versus expansion with IL-21 / anti-IL-21 complex, #p<0.001.

[0068] Figures 11A - 11H Shows the comparison of NK cell effector functions between previously cryopreserved g-NK cells and freshly enriched g-NK cells (n = 4). Values are mean ± SE. #p < 0.05 when freshly enriched g-NK cells were compared with previously cryopreserved g-NK cells.

[0069] Figure 11A and Figure 11B Describes the levels of daratumumab- and elotuzumab-mediated degranulation (CD107a 阳性 ) of previously cryopreserved g-NK cells compared with freshly enriched g-NK cells. Figure 11A Shows the degranulation levels of g-NK cells against the LP1 cell line. Figure 11B Shows the degranulation levels of g-NK cells against the MM.1S cell line.

[0070] Figure 11C and Figure 11D Describes the expression levels of perforin and granzyme B of previously cryopreserved g-NK cells compared with freshly enriched g-NK cells. Figure 11C Shows the total perforin expression of g-NK cells. Figure 11D Shows the total granzyme B expression of g-NK.

[0071] Figure 11E and Figure 11F Describes the levels of daratumumab- and elotuzumab-mediated interferon-γ expression of previously cryopreserved g-NK cells compared with freshly enriched g-NK cells. Figure 11E Shows the interferon-γ expression levels of g-NK cells against the LP1 cell line. Figure 11F Shows the interferon-γ expression levels of g-NK cells against the MM.1S cell line.

[0072] Figure 11G and Figure 11H Describes the levels of daratumumab- and elotuzumab-mediated TNF-α expression of previously cryopreserved g-NK cells compared with freshly enriched g-NK cells. Figure 11G Shows the TNF-α expression levels of g-NK cells against the LP1 cell line. Figure 11H Shows the TNF-α expression levels of g-NK cells against the MM.1S cell line.

[0073] Figures 12A - 12C Describes the persistence of cNK (cryopreserved) and g-NK (cryopreserved or fresh) cells in NSG mice after infusion of a single dose of 1x10 7 expanded cells. Figure 12AShows the numbers of cNK and g-NK cells in peripheral blood collected on days 6, 16, 26, and 31 after infusion. Figure 12B Shows the number of NK cells present in the spleen on day 31 after infusion, i.e., at sacrifice. Figure 12C Shows the number of NK cells present in the bone marrow at sacrifice. All three groups had N = 3. Values are mean ± SE. Frozen cNK cells were compared with fresh or frozen g-NK cells, *p < 0.05, ***p < 0.001.

[0074] Figures 13A - 13D Describes the expression of CD20 (target of rituximab), CD38 (target of daratumumab), and SLAMF7 (target of elotuzumab) on g-NK and cNK. Figure 13A Shows the percentages of expanded g-NK cells, unexpanded NK cells (CD3 阴性 / CD56 阳性 ) and Raji cells expressing CD20. Figure 13B Shows the percentages of expanded g-NK cells, unexpanded NK cells (CD3 阴性 / CD56 阳性 ) and MM.1S cells expressing CD38. Figure 13C Shows the percentages of expanded g-NK cells, unexpanded NK cells (CD3 阴性 / CD56 阳性 ) and MM.1S cells expressing SLAMF7. Figure 13D Shows the percentages of cNK and g-NK expressing CD38 before and after expansion. All groups had N = 3.

[0075] Figure 13E Describes the mean fluorescence intensity (MFI) of CD38 阳性 NK cells before and after expansion (n = 4). Figure 13F Provides representative histograms depicting the reduced CD38 expression of g-NK cells relative to cNK and MM.1S cells. Values are mean ± SE. g-NK cells were compared with all other cells, #p < 0.001. Figure 13G Describes the comparison of daratumumab-induced allogeneic killing of expanded g-NK cells and cNK cells.

[0076] Figure 14A -F shows the therapeutic effects of cNK and daratumumab ("cNK + Dara" or "cNK + daratumumab") or g-NK and daratumumab ("g-NK + Dara" or "g-NK + daratumumab") on tumor burden and survival in a murine model of multiple myeloma. 5 x 10 5Luciferase-labeled MM.1S human myeloma cells were intravenously injected (I.V.) into the tail vein of female NSG mice. Weekly for 5 weeks, expanded NK cells were administered I.V. (6.0 x 10 6 cells per mouse) and daratumumab was injected I.P. (10 μg per mouse). Figure 14A BLI imaging of the mice was performed twice weekly at days 20, 27, 37, 41, 48, and 57 after tumor inoculation (left). The corresponding number of days post-treatment is shown on the right side of the figure. Figure 14B Shows the change in tumor BLI (photons / sec) over time for the g-NK+Dara group relative to the control and cNK+Dara groups. *p < 0.05 for comparison of the g-NK group with the control or cNK groups. Figure 14C Shows the percentage of survival over time, and the arrows indicate the administration of cNK+Dara or g-NK+Dara therapy. Figure 14D Shows the change in body weight over time for the mice in the control, cNK+Dara, and g-NK+Dara groups. Figure 14E Describes the number of CD138 + tumor cells in the bone marrow of mice sacrificed after treatment with cNK+Dara and g-NK+Dara. ***p < 0.001 for comparison of g-NK and cNK cells. Values are mean ± SE. Figure 14F Shows representative flow cytometry plots using a sorting strategy to resolve the presence of NK cells and tumor cells in control and mice treated with cNK+Dara or g-NK+Dara. N = 8 for the control group, N = 7 for the g-NK or cNK groups.

[0077] Figure 14G Shows all BLI images of control, cNK+Dara- and g-NK+Dara-treated mice collected throughout the study. Figure 14H Describes X-ray images obtained from all mice in the control, cNK+Dara, and g-NK+Dara groups before sacrifice. Arrows indicate fractures and deformities. The sacrifice date is marked below each mouse.

[0078] Figure 15A -C shows comparative data of persistent NK cells in NSG mice treated with cNK+Dara or g-NK+Dara. All data are the number of cells detected by flow cytometry at the time of sacrifice. Figure 15A Shows the number of cNK and g-NK cells in the blood. Figure 15B Shows the number of NK cells in the spleen. Figure 15C Shows the number of NK cells in the bone marrow. Values are mean ± SE. ***p < 0.001 for comparison of g-NK and cNK cells.

[0079] Figure 16 Describes a cell subset with CD45 阳性 / CD3 阴性 / CD56 阳性 / CD16 阳性 / CD57 阳性 / CD7 微弱 / 阴性 / CD161 阴性 or CD45 阳性 / CD3 阴性 / CD56 阳性 / NKG2A 阴性 / CD161 阴性 Percentage of g-NK (CD45 阳性 / CD3 阴性 / CD56 阳性 / FcRγ 阴性 ) in alternative extracellular surface phenotype cell subsets. Values are mean ± standard error.

[0080] Figure 17 Describes GFP and CD20-CAR expression after transduction of g-NK cells in two independent experiments, each using a different donor.

[0081] Figure 18 Describes the potency of g-NK cells with or without CD20-CAR against Raji lymphoma cells in the presence or absence of rituximab (anti-CD20 monoclonal antibody).

[0082] Figure 19 describes the percentage of viable g-NK cells expressing CD20 CAR after electroporation.

[0083] Figures 20A and 20B show the expression of CD19, CD20, and CD38 in Raji lymphoma cells. Figure 20A identifies Raji cells by expression of CD19. Figure 20B confirms the expression of CD20 and CD38 in Raji cells.

[0084] Figures 21A and 21B demonstrate that g-NK cells exhibit antibody-dependent cell-mediated cytotoxicity (ADCC) against Raji lymphoma cells in the presence or absence of CD20 CAR expression, and in the presence or absence of daratumumab (anti-CD38 monoclonal antibody). Figure 21A describes the percentage of Raji cell death under each condition at an effector-to-target ratio of 0.05:1. Figure 21B describes the number of Raji cells killed per NK cell under each condition at an effector-to-target ratio of 0.05:1. The percentage of Raji cell death is calculated excluding spontaneously dying Raji cells. Detailed implementation manners

[0085] The present invention provides methods of administering a combination of engineered natural killer (NK) cells lacking expression of the FcRγ chain (g-NK cells) and an antibody (e.g., a monoclonal antibody), wherein the g-NK cells comprise a recombinant chimeric antigen receptor (CAR). FcRγ is also known as FcεR1γ and is used interchangeably herein. In some embodiments, the antibody is administered separately from the g-NK cells. In some embodiments, the antibody can be secreted from the g-NK cells. Natural killer (NK) cells are a type of innate lymphocyte that play an important role in mediating antiviral and anti-cancer immune responses by secreting cytokines and chemokines, and releasing cytotoxic granules (Vivier et al., Science 331(6013):44-49 (2011); Caligiuri, Blood 112(3):461-469 (2008); Roda et al., Cancer Res. 66(1):517-526 (2006)). NK cells are effector cells that comprise the third largest lymphocyte population and are important for host immune surveillance of tumor and pathogen-infected cells. However, unlike T lymphocytes and B lymphocytes, NK cells use germline-encoded activating receptors and are thought to have only limited target recognition capabilities (Bottino et al., Curr Top Microbiol Immunol. 298:175-182 (2006); Stewart et al., Curr Top Microbiol Immunol. 298:1-21 (2006)).

[0086] Activation of NK cells can occur through direct binding of NK cell receptors to ligands on target cells, such as direct tumor cell killing, or through crosslinking of Fc receptors (CD16; also known as CD16a or FcγRIIIa) that bind to the Fc portion of antibodies bound to antigen-bearing cells. Upon activation, NK cells produce large amounts of cytokines and chemokines and simultaneously exhibit potent cytolytic activity. NK cells are capable of killing tumor cells through antibody-dependent cell-mediated cytotoxicity (ADCC). In some cases, ADCC is triggered when receptors on the surface of NK cells, such as CD16, recognize cell surface-bound IgG1 or IgG3 antibodies. This triggers the release of cytoplasmic granules containing perforin and granzymes, leading to the death of the target cell. Since NK cells express the activating Fc receptor CD16, which recognizes IgG-coated target cells, target recognition is expanded (Ravetch & Bolland, Annu Rev Immunol. 19:275-290 (2001); Lanier Nat. Immunol. 9(5):495-502 (2008); Bryceson & Long, Curr Opin Immunol. 20(3):344-352 (2008)). ADCC and antibody-dependent cytokine / chemokine production are mediated primarily by NK cells.

[0087] CD16 also exists in a glycosylphosphatidylinositol-anchored form (also known as FcγRIIIB or CD16B). It should be understood that CD16 as referred to herein is the CD16a form expressed on NK cells and involved in antibody-dependent responses such as NK cell-mediated ADCC, and this is not the glycosylphosphatidylinositol-anchored form.

[0088] The CD16 receptor is capable of binding to aptamers, the ζ chain of the TCR-CD3 complex (CD3ζ), and / or the FcRγ chain and transmitting signals through immunoreceptor tyrosine-based activation motifs (ITAMs). In some aspects, CD16 engagement (CD16 crosslinking) initiates an NK cell response through intracellular signals generated by one or both of the CD16-associated adaptor chains FcRγ or CD3ζ. Triggering CD16 results in phosphorylation of the γ or ζ chain, which in turn recruits tyrosine kinases, syk, and ZAP-70, initiating a signal transduction cascade that generates rapid and potent effector functions. The best-known effector function is the release of cytoplasmic granules carrying toxic proteins, killing nearby target cells through the process of antibody-dependent cytotoxicity. CD16 crosslinking also leads to the production of cytokines and chemokines, which in turn activate and coordinate a series of immune responses.

[0089] The release of such cytokines and chemokines can play a role in the in vivo anti-cancer activity of NK cells. NK cells also have small granules in their cytoplasm, which contain perforin and proteases (granzyme). After release from NK cells, perforin forms pores in the cell membrane of the targeted cells, through which granzyme and related molecules can enter, inducing apoptosis. The fact that NK cells induce apoptosis rather than necrosis in target cells is significant - necrosis of virus-infected cells releases virions, while apoptosis leads to the destruction of the virus within the cell.

[0090] A specialized subset of NK cells lacking the FcRγ adaptor protein, also known as g-NK cells, is capable of mediating robust ADCC responses (see, for example, published patent application No. US2013 / 0295044). The mechanism for increased responsiveness may be due to changes in epigenetic modifications that affect the expression of FcRγ. g-NK cells express high levels of the signaling adaptor ζ chain but lack the expression of the signaling adaptor γ chain. Compared to conventional NK cells, the activity of these γ-deficient g-NK cells is significantly enhanced when activated by antibodies. For example, g-NK cells can be activated by antibody-mediated CD16 cross-linking or antibody-coated tumor cells. In some aspects, compared to conventional NK cells expressing the γ chain, g-NK cells produce large amounts of cytokines (such as IFN-γ or TNF-α) and chemokines (such as MIP-1α, MIP-1β, and RANTES) and / or exhibit a higher degranulation response. g-NK cells provide high expression of granzyme B, which is a component of the natural killer cell cytotoxicity mechanism. In addition, compared to conventional NK cells, g-NK cells have a longer lifespan and their persistence lasts for a long time. In some embodiments, g-NK cells are functionally and phenotypically stable.

[0091] In some embodiments, compared to conventional NK cells (such as NK cells that do not lack the γ chain), g-NK cells are more effective in eliciting ADCC responses. In some embodiments, compared to conventional NK cells, g-NK cells are more effective in eliciting cell-mediated cytotoxicity, even in the absence of antibodies. In some cases, ADCC is a mechanism of action of therapeutic antibodies (including anti-cancer antibodies). In some aspects, cell therapy by administering NK cells can be used together with antibodies for therapeutic and related purposes.

[0092] For example, certain therapeutic monoclonal antibodies, such as daratumumab targeting CD38 and elotuzumab targeting SLAMF7, have been approved by the FDA for the treatment of diseases such as multiple myeloma (MM). Although the clinical responses of therapeutic antibodies are promising, they are often not ideal. For example, although the initial clinical responses are usually encouraging, especially for daratumumab, essentially all patients eventually develop progressive disease. Therefore, there is an urgent need for new strategies to drive deeper remissions or overcome resistance to these agents. The provided embodiments, including the compositions, address these needs.

[0093] Provided herein is an engineered natural killer (NK) cell (g-NK cell) lacking expression of the FcRγ chain, which further comprises a recombinant chimeric antigen receptor (CAR) and a composition comprising the g-NK cell. Also provided are methods of engineering the g-NK cells. In some embodiments, CAR-dependent antigen targeting of the engineered g-NK cells results in improved patient outcomes due to improved avidity, cytotoxicity, and / or cytokine-mediated effector functions of g-NK cell subsets. It has been found herein that CAR-dependent antigen targeting can be combined with antibody-directed targeting of g-NK cells through CD16 engagement and ADCC activity. In other words, the results herein demonstrate that antibody-directed targeting via ADCC in CAR-engineered T cells is not affected even though signal transduction occurs through the same CD3ζ signaling pathway. These results suggest that the two antigen-directed killing mechanisms of g-NK cells can be used as a combination therapy strategy to further enhance the killing of target cells.

[0094] These methods are improvements over traditional NK cells. Traditional NK cells are typically activated when the Fc portion of an antibody binds to its Fc receptor (FcγRIIIa or CD16a) and trigger activation and degranulation through a process involving the adaptor proteins CD3ζ and FcεR1γ. Binding and crosslinking of the Fc receptor CD16 on traditional NK cells initiates signaling through both CD3ζ and FcεR1γ, which can lead to variability in signaling depending on the expression of signaling adaptors in the NK cells. Finally, the activity of NK cell activity typically requires the support of cytokines, such as through IL-15, to enhance cytotoxic activity; thus, the lack of sufficient supportive cytokines may limit the persistence of the response. Each of these factors, either alone or in combination, poses an obstacle to the application of certain NK cell therapies.

[0095] The engineered NK cells and compositions containing these cells provided herein, such as cells produced by the provided methods, provide an improved cell therapy in several aspects. First, the provided g-NK cells and compositions containing these cells, such as cells produced by the provided methods, are engineered to express a chimeric antigen receptor (CAR). Expression of the CAR enables the g-NK cells to target target cells or tissues in a diseased subject or individual in an antibody-independent manner. In addition, combination therapy with monoclonal antibodies enables the g-NK cells to target target cells or tissues in a diseased subject or individual in a potent, ADCC-mediated, antibody-directed manner. The provided cells and compositions produced by such methods are particularly powerful in their ability to target g-NK cells to the appropriate location in a subject or individual. Surprisingly, the g-NK cells have potent ADCC activity, and co-expression of the CAR in the engineered g-NK cells does not attenuate this activity, thus enabling these achievements to be realized.

[0096] These two antigen-driven mechanisms for targeted killing of target cells (such as cancer cells) have led to improvements in strategies for targeting certain cancers. Although clinical trial results have shown that the cell CAR T cell therapy has encouraging clinical efficacy for some hematological malignancies, relapse with diminished or complete loss of cell surface antigen expression has been observed in approximately 30-50% of patients who achieved remission after receiving anti-CD19 CAR T cell therapy, typically within one year of treatment. Relapses associated with antigen loss have also been reported in CARs directed against other targets such as CD22 and B cell maturation antigen, highlighting antigen escape as a major and common impediment to the success of CAR-T cell therapy. In addition to blood cancers, antigen escape may be an even greater challenge in solid tumors, which typically consist of cells with varying antigen expression profiles. Therefore, targeting a single antigen poses a risk of immune escape, and this risk can be overcome by targeting multiple desired antigens, especially in solid tumors with higher tumor heterogeneity. Therefore, there is still a need to improve chimeric antigen receptor cell-based therapies to enable more effective, safe, and efficient targeting of various cancers such as B cell-related malignancies (ALL, CLL, and NHL), multiple myeloma, AML, lymphoma, and many other solid tumors.

[0097] The provided embodiments include methods involving combination therapies that bring target cells into contact with g-NK cells engineered with a chimeric antigen receptor (CAR) and a monoclonal antibody, where the CAR contains an extracellular antigen-binding domain (such as a scFv) that binds a first antigen, and the monoclonal antibody binds a second antigen. The first and second antigens can be the same or different. Generally, if the antigens are the same, the epitopes recognized by the CAR and the monoclonal antibody are different. In certain aspects, the first and second antigens are different and both are antigens known or suspected to be expressed on target cells in a disease or disorder, such as cancer. In some embodiments, the first and second antigens are expressed on the same target cells. In some embodiments, the first and second antigens are expressed on different target cells, both of which are associated with the disease or disorder, for example due to tumor heterogeneity. In some embodiments, the monoclonal antibody is a recombinant molecule that is administered alone to a subject. In some embodiments, the g-NK cells are engineered with a secreted monoclonal antibody. In some embodiments, the methods involve administering to a subject having a disease or disorder (such as cancer) a composition of g-NK cells engineered to express a CAR for targeting a first antigen and a monoclonal antibody for targeting a second antigen. In some embodiments, the methods involve administering to a subject having a disease or disorder (such as cancer) a composition of g-NK cells engineered to express a CAR for targeting a first antigen and engineered with a secreted monoclonal antibody that targets a second antigen.

[0098] Specifically, the provided embodiments relate to NK cell compositions that are enriched for a particular subset of g-NK cells (i.e., NK cells lacking FceRIγ), which have a number of advantages compared to conventional NK cells or NK cells enriched for other subsets. g-NK cells are a relatively rare subset, as they are detected at levels of only about 3%-10% of total NK cells in only 25-30% of individuals who are CMV-seropositive. As described herein, methods can be used to provide particularly robust g-NK cell expansion and enrichment, enabling sufficient expansion for in vivo use, while also engineering the enriched g-NK cells with a CAR before, during, or after their expansion. The provided cells and compositions generated by this method are particularly potent in their ability to target g-NK cells to appropriate locations in a subject or individual.

[0099] The proportion of g-NK cells among peripheral blood NK cells is relatively small, thus limiting the ability to use these cells in therapeutic methods. Specifically, in order to clinically utilize g-NK cells, a high priority amplification rate must be achieved because g-NK cells are typically a rare population. Other methods for amplifying NK cells can achieve a thousand-fold NK cell amplification rate in 14 days, but they harvest low-differentiated NKG2C 阴性 , FceRIγ 阳性 (FcRγ 阳性 ) NK cells (Fujisaki et al. (2009) Cancer Res., 69:4010 - 4017; Shah et al. (2013) PLoS One, 8:e76781). Additionally, it has been found that amplification optimized for expanding NK cells phenotypically overlapping with g-NK cells does not preferentially amplify g-NK cells to a number supportive of therapeutic use. Specifically, it has previously been reported that those NKG2C 阳性 NK cells that phenotypically overlap with g-NK cells can be preferentially amplified by using HLA-E transfected 221.AEH cells and adding IL-15 to the culture medium (Bigley et al. (2016) Clin. Exp. Immunol., 185:239 - 251). Culturing with these HLA-expressing cells that constitutively express HLA-E drives NK cells towards the NKG2C 阳性 / NKG2A 阴性 phenotype direction (NKG2C is an activating receptor for HLA-E, while NKG2A is an inhibitory receptor for HLA-E). It was thought that since g-NK is included among such cells, this method would be sufficient to amplify g-NK cells. However, this method fails to achieve potent amplification of g-NK cells.

[0100] The amplification method described herein is capable of generating an NK cell composition enriched in g-NK cells, overcoming these limitations. Compared to previous methods, the provided method utilizes a greater proportion of HLA-E+ feeder cells (such as 221.AEH cells) and NK cells that lack HLA class I and HLA class II. In particular, previous methods used a lower proportion of 221.AEH cells, for example, a ratio of 10:1 of NK cells to 221.AEH cells. It has been found herein that a greater proportion of feeder cells expressing HLA-E (such as 221.AEH cells) results in a greater overall amplification and a greater bias towards the g-NK phenotype. In some embodiments, the proportion of HLA-E+ feeder cells (such as 221.AEH cells) can be increased by irradiating the feeder cells. In some aspects, using an irradiated feeder cell line also has the advantage that it provides a GMP-compatible method. The addition of any one or a combination of recombinant IL-2, IL-7, IL-15, IL-12, IL-18, IL-21, IL-27 during amplification has also been found to support robust amplification. In a particular embodiment of the provided method, at least one recombinant cytokine is IL-2. In some embodiments, there are two or more recombinant cytokines, wherein at least one recombinant cytokine is IL-2 and at least one recombinant cytokine is IL-21.

[0101] The method for expanding g-NK cells is based on the discovery that expansion culture of NK cells in the presence of IL-21 enhances NK cell activity to produce cytokines or effector molecules such as perforin and granzyme B. The compositions containing NK cells produced during the expansion process herein are highly functional, exhibiting robust proliferative capacity and functioning well even without resuscitation after they are frozen. For example, when expanded in the presence of IL-21, the NK cells produced by the provided process not only exhibit strong ADCC activity but also exhibit antibody-independent cytotoxic activity. This robust activity, including antibody-independent cytotoxic activity, is particularly applicable to the strategies described herein, in which the cells are further engineered with CAR and immunomodulators, because after the target antigen engages with the CAR, the NK cells are activated and ready to exert effector activity. For example, effector molecules such as perforin and granzyme are spontaneously present in the NK cells expanded by the provided method, thus providing cells with high cytotoxic potential. As shown herein, NK cell compositions produced by the method provided herein that includes IL-21 (such as IL-2, IL-15, and IL-21) not only exhibit a higher proportion of perforin- or granzyme B-positive NK cells compared to NK cell compositions produced by a method that includes only IL-2 without the addition of IL-21, but they also exhibit a higher average expression level or degree of these molecules in these cells. In addition, NK cell compositions produced by the method provided herein that includes IL-21 (such as IL-2, IL-15, and IL-12) also produce g-NK cell compositions that exhibit substantial effector activity in response to target cells, including the ability to degranulate and express more IFN-γ and TNF-α. This functional activity is highly retained even when the expanded NK cells are cryopreserved and thawed. The significant increase in lytic enzymes and the more robust activation phenotype underlie the enhanced ability of the expanded g-NK cells to induce apoptosis in tumor targets. Although many of these activities are exemplified in the examples herein upon binding with an antibody via CD16 crosslinking, similar activities are also generated when the CAR binds to the target antigen because signal transduction is also mediated by CD3ζ. This prominent antibody-independent effector phenotype, combined with cellular engineering with CAR and immunomodulators such as cytokines, also supports the potential application of g-NK cells as a single therapy.

[0102] In addition, in some embodiments, the amount of cytokines produced by g-NK cells is significantly greater than that of natural killer cells expressing FcRγ. In another embodiment, the cytokine is interferon γ (IFN-γ), tumor necrosis factor-α (TNF-α), or a combination thereof. In one embodiment, g-NK cells produce a significantly greater amount of chemokines. In one embodiment, the chemokine is MIP-1α, MIP-1β, or a combination thereof. In another embodiment, g-NK cells produce cytokines or chemokines upon CD3ζ signaling, such as by engagement of a CAR or, in some cases, by stimulation of the Fc receptor CD16.

[0103] In addition, the findings herein also demonstrate that the provided NK cells expanded in the presence of IL-21 have the potential to persist and proliferate well over a longer period of time, which is superior to, for example, cells expanded in the presence of only IL-2 without the addition of IL-21. Furthermore, the results indicate that the persistence level of cryopreserved g-NK cells is comparable to that of fresh g-NK cells. This significantly improved persistence highlights the potential application of fresh or cryopreserved g-NK as an off-the-shelf cell therapy to enhance targeted cytotoxicity. This finding of improved persistence is advantageous because the clinical utility of many NK cell therapies is affected by the limited persistence of NK cells.

[0104] The study also found that enriching NK cells from a cell sample prior to the expansion method, such as enriching CD16 or CD57 cells prior to expansion, further significantly increases the expansion amount of g-NK cells compared to the method of initially enriching NK cells based solely on CD3 depletion. In another embodiment, another enrichment that can be performed prior to expansion is to enrich NK cells by positive selection of CD56 and negative selection or depletion of CD38. In another embodiment, another enrichment that can be performed prior to expansion is to enrich NK cells by positive selection of CD56, followed by negative selection or depletion of NKG2A 阴性 and negative selection or depletion of CD161 阴性 to enrich NK cells. In another embodiment, another enrichment that can be performed prior to expansion is to enrich NK cells by positive selection of CD57, followed by negative selection or depletion of NKG2A and / or positive selection of NKG2C. In another embodiment, another enrichment that can be performed prior to expansion is to enrich NK cells by positive selection of CD56, followed by negative selection or depletion of NKG2A and / or positive selection of NKG2C. In any such embodiment, NKG2C 阳性 and / or NKG2A 阴性 NK cells can be enriched after expansion.

[0105] In any such embodiment, the enriched NK cells can be enriched from a cell sample containing NK cells, such as from peripheral blood mononuclear cells (PBMC). In some embodiments, T cells can be removed by negative selection or depletion of CD3 before enriching NK cells from the cell sample. In any such embodiment, the enriched NK cells can be enriched from a biological sample (such as PBMC) containing NK cells and having a relatively high proportion of g-NK cells from a human subject, for example, can be enriched from a biological sample of a human subject selected for having a relatively high proportion of g-NK cells in NK cells. In any such embodiment, the enriched NK cells can be enriched from a biological sample containing NK cells from a human subject, such as PBMC, wherein the sample contains a relatively high proportion of NKG2C 阳性 NK cells (e.g., being or about or greater than about 20% of NKG2C 阳性 NK cells) and / or NKG2A 阴性 NK cells (e.g., being or about or greater than about 70% of NKG2A 阴性 NK cells). In any such embodiment, the enriched NK cells can be enriched from a biological sample containing NK cells from a human subject, such as PBMC, wherein the sample contains a relatively high proportion of NKG2C 阳性 NK cells (e.g., being or about or greater than about 20% of NKG2C 阳性 NK cells) and NKG2A 阴性 NK cells (e.g., being or about or greater than about 70% of NKG2A 阴性 NK cells). In a particular embodiment, the subject from whom the sample is derived is CMV seropositive because more g-NK cells can be detected in the peripheral blood of such subjects.

[0106] In summary, the provided method for expanding g-NK cells enables the expansion of NK cells, starting from 10 million enriched NK cells at the beginning of culturing, to over 1 billion cells, and in some cases even reaching 8 billion cells or more. In particular, the provided method enables a high-yield (>1000-fold) expansion rate and maintains or in some cases enhances the function of g-NK cells after expansion. In some embodiments, the provided method can result in a population of g-NK cells that express high levels of perforin and granzyme B. Additionally, it has been found that the provided method is sufficient to expand previously cryopreserved NK cells, which is generally not achievable with many existing methods that require rescue treatment of thawed NK cells. In some embodiments, this is achieved by increasing the duration of the expansion protocol. In some embodiments, this is achieved by reducing the ratio of HLA-E+ feeder cells to NK cells, such as reducing the ratio of HLA-E+ feeder cells to NK cells to approximately 1:1 for 221.AEH to NK cells. In some embodiments, this is achieved by adding recombinant IL-2, IL-7, IL-15, IL-12, IL-18, IL-21, IL-27, or a combination thereof. In certain embodiments, at least one recombinant cytokine is IL-2. In some embodiments, the expansion is carried out in the presence of two or more recombinant cytokines, wherein at least one is recombinant IL-21 and at least one is recombinant IL-2.

[0107] As shown herein, the engineered g-NK cells and compositions containing these cells provided herein, such as those produced by the provided method, can be used in cancer therapy. In some embodiments, the adoptive transfer of NK cells does not result in severe graft-versus-host (GVHD), and thus this cell therapy can be used in clinical applications in an "off-the-shelf" manner. In some aspects, the NK cells can be further engineered to reduce or eliminate individual HLA molecules in the NK cells, thereby enhancing the allogeneic potential of the provided cell therapy.

[0108] All references cited herein, including patent applications, patent publications, scientific literature, and databases, are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual reference had been specifically and individually indicated to be incorporated by reference.

[0109] For clarity of disclosure, and not by way of limitation, the detailed description is divided into the following subsections. The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. I. Definitions

[0110] Unless otherwise defined, all technical terms, notations and other technical and scientific terms or phrases used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some instances, terms with commonly understood meanings are defined herein for clarity and / or for the purpose of convenient reference, and the inclusion of such definitions herein should not be construed to represent a substantial difference from what is commonly understood in the art.

[0111] As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a molecule" optionally includes a combination of two or more such molecules, and the like.

[0112] As used herein, the term "about" refers to the usual error range of the respective values well known to those skilled in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments that refer to the value or parameter itself.

[0113] It is to be understood that the various aspects and embodiments of the invention described herein include "comprising", "consisting of" and "consisting essentially of".

[0114] As used herein, "optionally" or "optionally" means that the subsequent described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not occur. For example, an optionally substituted group means that the group is either unsubstituted or substituted.

[0115] As used herein, "antibody" refers to immunoglobulins and immunoglobulin fragments, whether natural or partially or fully synthetic (such as recombinantly produced), including any fragment containing at least a portion of the variable heavy chain region and / or light chain region of an immunoglobulin molecule, which portion is sufficient to form an antigen-binding site and which, when assembled, is sufficient to specifically bind an antigen. Thus, an antibody includes any protein having a binding domain that is homologous or substantially homologous to an immunoglobulin antigen-binding domain ((antibody binding site). Generally, an antibody minimally includes all or at least a portion of the variable heavy ((VH) chain and / or variable light ((VL) chain. Generally, the pairing of VH and VL together forms an antigen-binding site, but in some cases, a single VH or VL domain is sufficient for antigen binding. An antibody may also include all or a portion of the constant region. Reference herein to an antibody includes full-length antibodies and antigen-binding fragments. The term "immunoglobulin" ((Ig) is used interchangeably with "antibody" herein.

[0116] The terms "full-length antibody", "intact antibody", or "whole antibody" are used interchangeably to refer to an antibody in its substantially intact form, as opposed to antibody fragments. A full-length antibody is an antibody that generally has two full-length heavy chains (e.g., VH-CH1-CH2-CH3 or VH-CH1-CH2-CH3-CH4) and two full-length light chains (VL-CL) and a hinge region, such as an antibody produced by a B cell secreting the antibody from a mammalian species (e.g., human, mouse, rat, rabbit, non-human primate, etc.) and an antibody synthetically produced having the same domains. Specifically, a whole antibody includes an antibody having a heavy chain and a light chain including an Fc region. The constant domain can be a native sequence constant domain (e.g., a human native sequence constant domain) or an amino acid sequence variant thereof. In some cases, an intact antibody can have one or more effector functions.

[0117] "Antibody fragment" includes a portion of an intact antibody, i.e., the antigen-binding region and / or variable region of an intact antibody. Antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab') 2 fragments, Fv fragments, disulfide-linked Fv (dsFv), Fd fragments, Fd' fragments; diabodies; linear antibodies (see U.S. Patent No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10) :1057-1062

[1995] ); single-chain antibody molecules, including single-chain Fv (scFv) or single-chain Fab (scFab); antigen-binding fragments of any of the foregoing and multispecific antibodies from antibody fragments. For the purposes herein, an antibody fragment generally includes an antibody fragment sufficient to engage or crosslink CD16 on the surface of an NK cell.

[0118] The term "autologous" refers to cells or tissues derived from within or taken from an individual's own tissue. For example, in an autologous transfer or transplantation of NK cells, the donor and recipient are the same individual.

[0119] The term "allogeneic" refers to cells or tissues that are of or obtained from the same species but are genetically different and are thus immunologically incompatible in some cases. Generally, the term "allogeneic" is used to define cells transplanted from a donor to a recipient of the same species.

[0120] The term "enriched" with respect to a cell composition refers to a composition in which the number or percentage of a cell type or population is increased compared to the number or percentage of cell types in the same volume of a starting composition (such as a starting composition directly obtained from or isolated from a subject). The term does not require the complete removal of other cells, cell types, or populations from the composition, nor does it require that the cells so enriched be present at or even close to 100% in the enriched composition.

[0121] The term "expression" refers to the process of transcription of a polynucleotide from a DNA template (such as transcription into mRNA or other RNA transcripts) and / or the subsequent translation of the transcribed mRNA into a peptide, polypeptide, or protein. The transcript and the encoded polypeptide may be collectively referred to as "gene product". If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0122] The term "heterologous" with respect to a protein or nucleic acid refers to a protein or nucleic acid that has been transformed or introduced into a cell. In some cases, a heterologous protein or nucleic acid is exogenous to the cell, for example because it is derived from a different organism or individual than the cell in which it is expressed. It should be understood that the reference to "heterologous" does not exclude that the protein or nucleic acid may also be naturally expressed by the cell into which it is introduced. A heterologous nucleic acid or the encoded heterologous protein can be introduced into NK cells, for example, by any of a variety of methods capable of introducing or transforming a nucleic acid (such as a nucleic acid encoding a heterologous protein) into a cell, including virus-based methods, such as by transduction, or non-viral delivery methods, such as electroporation or lipid nanoparticle delivery. An NK cell that has been introduced or transformed may carry an exogenous or heterologous nucleic acid that is extrachromosomal or integrated into the chromosome. Integration into the cell genome and self-replicating vectors generally results in the transformed nucleic acid molecule having a genetically stable inheritance. An NK cell containing the transformed nucleic acid is referred to as "genetically engineered", but may also be referred to interchangeably as "recombinant" or "transformed".

[0123] As used herein, the term "introducing" encompasses a variety of methods for introducing DNA into a cell in vitro or in vivo, such methods including transformation, transduction, transfection (such as electroporation), lipid delivery, and infection. A vector can be used to introduce DNA encoding a molecule into a cell. Possible vectors include plasmid vectors and viral vectors. Viral vectors include retroviral vectors, lentiviral vectors, or other vectors, such as adenoviral vectors or adeno-associated vectors. Lipid nanoparticles can also be used to introduce nucleic acids (DNA or mRNA) into a cell.

[0124] The terms "polynucleotide", "nucleotide sequence", "nucleic acid", "nucleic acid molecule", "nucleic acid sequence", and "oligonucleotide" refer to a series of nucleobases (also called "nucleotides") in DNA and RNA, and refer to any chain of two or more nucleotides. Polynucleotides, nucleotide sequences, nucleic acids, etc. can be single-stranded or double-stranded chimeric mixtures or their derivatives or modified forms. For example, they can be modified at the base moiety, sugar moiety, or phosphate backbone to improve the stability of the molecule, its hybridization parameters, etc. Nucleotide sequences usually carry genetic information, including but not limited to information for the cellular machinery to make proteins and enzymes. These terms include double-stranded or single-stranded genomic DNA, RNA, any synthetic and genetically engineered polynucleotide, and sense and antisense polynucleotides. These terms also include nucleic acids containing modified bases.

[0125] The terms "protein", "peptide", and "polypeptide" are used interchangeably and refer to an ordered chain of amino acids linked together by peptide bonds. These terms include individual proteins, groups or complexes of proteins associated together, and fragments or portions, variants, derivatives, and analogs of such proteins. Peptide sequences are presented in this document using conventional symbols, starting from the amino or N-terminus on the left and continuing to the carboxyl or C-terminus on the right. Standard single-letter or three-letter abbreviations can be used.

[0126] As used herein in the context of nucleic acids (e.g., genes, genomic regions encoding proteins, promoters), the term "endogenous" refers to a native nucleic acid or protein in its native location (e.g., within the genome of a cell). In contrast, as used herein in the context of nucleic acids (e.g., expression constructs, cDNA, indels, and nucleic acid vectors), the term "exogenous" refers to a nucleic acid that has been artificially introduced into the genome of a cell using, for example, genetic engineering techniques (such as transformation of heterologous nucleic acids or gene editing, e.g., CRISPR-based editing techniques).

[0127] The term "composition" refers to any mixture of two or more products, substances, or compounds (including cells or antibodies). It can be a solution, suspension, liquid, powder, paste, aqueous solution, non-aqueous solution, or any combination thereof. Formulations are generally in a form that allows the biological activity of the active ingredient (e.g., an antibody) to take effect.

[0128] "Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation that is non-toxic to a subject in addition to the active ingredient. Pharmaceutically acceptable carriers include but are not limited to buffers, excipients, stabilizers, or preservatives.

[0129] As used herein, a combination refers to any association between or among two or more items. The combination can be two or more separate items (such as two compositions or two collections), can be a mixture thereof (such as a single mixture of two or more items), or any variation thereof. The elements of the combination are generally functionally associated or related.

[0130] As used herein, a kit is a packaged combination that optionally includes other elements, such as additional agents and instructions for using the combination or its elements, for purposes including but not limited to therapeutic use.

[0131] As used herein, the term "treatment" refers to a clinical intervention designed to alter the natural course of a treated individual or cell during a clinical pathological process. Desired therapeutic effects include reducing the rate of disease progression, ameliorating or relieving the disease state, and alleviating or improving the prognosis. For example, an individual is successfully "treated" if one or more symptoms associated with a disorder (e.g., an eosinophil-mediated disease) are reduced or eliminated. For example, an individual is successfully "treated" if the treatment results in an improvement in the quality of life of a person suffering from a disease, a reduction in the dosage of other medications required to treat the disease, a reduction in the frequency of disease recurrence, a reduction in the severity of the disease, a delay in the development or progression of the disease, and / or an increase in the survival of the individual.

[0132] "Effective amount" means an amount that is at least effective, at a necessary dosage and for a necessary period of time, to achieve a desired or indicated effect (including a therapeutic or prophylactic outcome). The effective amount can be provided in one or more administrations. A "therapeutically effective amount" is at least the minimum cellular dose required to achieve a measurable improvement in a particular disorder. In some embodiments, a therapeutically effective amount is an amount of a composition that reduces the severity, duration, and / or symptoms associated with cancer, viral infection, microbial infection, or septic shock in an animal. The therapeutically effective amount herein can vary depending on factors such as the disease state, age, sex, and weight of the patient. A therapeutically effective amount can also be an amount where the therapeutic beneficial effects exceed any toxic or detrimental effects of the antibody. A "prophylactically effective amount" means an amount that is effective, at a necessary dosage and for a necessary period of time, to achieve a desired prophylactic outcome. Typically but not necessarily, since prophylactic doses are used in subjects before or at an early stage of disease, a prophylactically effective amount can be less than a therapeutically effective amount.

[0133] As used herein, "individual" or "subject" is a mammal. "Mammal" for therapeutic purposes includes humans, domestic and farm animals, and zoo, sports, or pet animals such as dogs, horses, rabbits, cattle, pigs, hamsters, gerbils, mice, ferrets, rats, cats, etc. In some embodiments, the individual or subject is a human. II. Methods of Cytolytic Killing and Treatment

[0134] The present disclosure provides methods of lysing and killing target cells, which involve a combination of a g-NK cell composition and antibody therapy, the g-NK cell composition comprising engineered g-NK cells, the engineered g-NK cells comprising a heterologous nucleic acid encoding an antigen receptor (such as a CAR). In some embodiments, the methods provided herein involve contacting target cells known or suspected of expressing a first antigen and a second antigen with: (a) a composition comprising natural killer (NK) cells lacking expression of the FcRγ chain (g-NK cells), wherein the g-NK cells express a chimeric antigen receptor (CAR) comprising an extracellular binding domain that binds to the first antigen; and (b) an antibody that binds to the second antigen. In some embodiments, the lysing and killing of the target cells occurs in a subject. In some embodiments, the target cells are associated with a disease or disorder, and the lysing and killing of the target cells is a treatment for the disease or disorder. In some embodiments, the target cells are cancer cells, and the method can be used to treat cancer.

[0135] The present disclosure also provides methods and uses for combination therapies, said combination therapies involving the combination of a g-NK cell composition with an antibody therapy for treating a disease or disorder, said g-NK cell composition comprising engineered g-NK cells comprising a heterologous nucleic acid encoding an antigen receptor (such as a CAR). In such methods, the CAR binds to a first antigen expressed by the cells of the disease or disorder, and the antibody therapy binds to a second antigen expressed by the cells of the disease or disorder. In some embodiments, the cells are the same cells. In some embodiments, the antibody is administered to a subject known or suspected of having a disease or disorder and is administered separately from the g-NK cells. In some embodiments, the disease or disorder is cancer. In some embodiments, the method comprises: (a) administering to a subject having cancer an NK cell therapy comprising a dose of a composition comprising natural killer (NK) cells lacking expression of the FcRγ chain (g-NK cells), wherein the g-NK cells express a chimeric antigen receptor (CAR) comprising an extracellular binding domain that binds to a first antigen expressed by the cells of the cancer; and (b) administering to the subject a dose of an antibody that binds to a second antigen expressed by the cancer cells. In some embodiments, the antibody is secreted from the g-NK cells. In some embodiments, the method comprises administering to a subject having cancer an NK cell therapy comprising a dose of a composition comprising natural killer (NK) cells lacking expression of the FcRγ chain (g-NK cells), wherein: the g-NK cells express a chimeric antigen receptor (CAR) comprising an extracellular binding domain that binds to a first antigen expressed by the cancer cells; and the g-NK cells express a secreted antibody that binds to a second antigen expressed by the cancer cells.

[0136] In the methods provided herein, compositions containing engineered g-NK cells as provided herein exhibit ADCC-mediated activity when activated or contacted with an antibody or an Fc-containing protein. In some embodiments, the g-NK cells provided herein exhibit unique enhanced ADCC activity, e.g., compared to conventional NK cells. For example, the g-NK cells can be activated by antibody-mediated crosslinking of CD16. In some embodiments, the present disclosure provides a method of treating a disorder in an individual, the method comprising administering to the subject engineered g-NK cells or a composition thereof and an antibody. In some embodiments, the antibody is capable of binding to and engaging CD16 on the surface of the NK cells. In some embodiments, the antibody comprises an Fc domain. In some embodiments, the antibody is an IgG1 Fc antibody. In some embodiments, the antibody is a full-length antibody. In specific embodiments, any such antibody in the provided methods is a monoclonal antibody.

[0137] In some aspects, the methods provided herein can provide a dual-targeting strategy for killing cancer cells. In some embodiments, the dual-targeting strategy enhances the killing of cancer cells, thereby treating a disease or disorder, such as by increasing the specificity for targeting cancer cells or providing a compensatory strategy for target cell killing in the case of antigen escape. In some embodiments, the methods provided herein increase the likelihood that cancer cells are killed, such as by providing different lytic killing mechanisms for NK cells through the additive effect of two therapies.

[0138] Such methods and uses include therapeutic methods and uses, such as those involving administering g-NK to a subject having a disease, disorder, or condition. In some cases, the disease or disorder is a tumor or cancer. In some embodiments, the disease or disorder is a viral infection. In some embodiments, the cells and antibodies or their pharmaceutical compositions are administered in an effective amount to effect the treatment of the disease or disorder. Uses include the use of the cells, antibodies, or their pharmaceutical compositions in such methods and treatments, and the use in the preparation of a medicament for practicing such therapeutic methods. In some embodiments, the method thereby treats the disease, condition, or disorder of the subject.

[0139] In some embodiments, any method and use provided herein that involves an NK cell composition comprising engineered g-NK cells can include the methods and uses described in PCT Publication No. WO2020 / 107002 or PCT Application No. PCT / US2021 / 028504.

[0140] The engineered g-NK cell compositions provided herein can be used in methods for treating an individual having a tumor or a hyperproliferative disorder. The engineered g-NK cell compositions provided herein can be administered for treating an animal, such as a mammal, e.g., a human subject. In some instances, the methods include treating a hyperproliferative disorder, such as a hematologic malignancy or a solid tumor. Examples of types of cancers and hyperproliferative disorders that can be treated with the compositions described herein include, but are not limited to, multiple myeloma, leukemia (e.g., myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, erythroleukemia, chronic myelocytic (granulocytic) leukemia, and chronic lymphocytic leukemia), lymphoma (e.g., Hodgkin's disease and non-Hodgkin's disease), fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, angiosarcoma, endotheliosarcoma, Ewing's tumor, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, renal cell carcinoma, liver cancer, Wilm's tumor, cervical cancer, uterine cancer, testicular tumors, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, oligodendroglioma, melanoma, neuroblastoma, retinoblastoma, dysplasia, and hyperplasia. Treatment and / or prevention of cancer includes, but is not limited to, alleviating one or more symptoms associated with cancer, inhibiting or reducing cancer development, promoting cancer regression, and / or promoting an immune response.

[0141] In some embodiments, the first and second antigens are cancer-related. In some embodiments, the first and second antigens are expressed on the same cancer target cell. In some embodiments, the first antigen is expressed on a first target cell of the cancer, and the second antigen is expressed on a second target cell of the cancer.

[0142] In some embodiments, the cancer is a hematologic malignancy. In some embodiments, the hematologic malignancy is a B cell malignancy. In some embodiments, the cancer is lymphoma, leukemia, or multiple myeloma. In some embodiments, any one of such cancers is a relapsed / refractory cancer. In some embodiments, the subject has non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), or multiple myeloma.

[0143] In some embodiments, the first antigen and the second antigen are selected from the group consisting of CD30, CD19, CD20, CD22, ROR1, Igk, CD38, CD138, BCMA, CD33, CD70, CD79b, CD123, SLAMF7, GPRC5D, FCRH5, FLT3, CLEC12, and LewisY antigen.

[0144] In some embodiments, the hematologic malignancy is multiple myeloma. In some embodiments, the multiple myeloma is relapsed / refractory. In some embodiments, the first antigen and the second antigen are selected from the group consisting of CD38, SLAMF7, CD138, FCRH5, GPRC5D, and BCMA. Those skilled in the art are familiar with any of the various chimeric antigen receptors (CARs) or monoclonal antibodies against such antigens. Exemplary CARs and antibodies are described herein.

[0145] In some embodiments, the CAR is an anti-BCMA CAR and the monoclonal antibody is an anti-CD38 antibody. Those skilled in the art are aware of many anti-BCMA CARs. Exemplary anti-BCMA CARs are described in Section III.A. In some embodiments, the anti-CD38 antibody is daratumumab (Darzalex TM ). In some embodiments, the anti-CD38 antibody is isatuximab.

[0146] In some embodiments, the anti-CD38 antibody can be administered subcutaneously. In some embodiments, the anti-CD38 antibody (such as daratumumab) can be administered in an anti-CD38 antibody composition comprising hyaluronidase. For example, the antibody can be administered as an anti-CD38 antibody composition comprising daratumumab and recombinant human hyaluronidase PH20 (such as hyaluronidase-fihj). Examples of such compositions are described in published U.S. Patent Publication No. US20170121414. In some embodiments, each dose of the anti-CD38 antibody composition comprises from or about 1200 mg to about 2400 mg of the anti-CD38 antibody (such as daratumumab) and from or about 15,000 units (U) to about 45,000 U of hyaluronidase (such as hyaluronidase-fihj). In some embodiments, each dose of the anti-CD38 antibody composition comprises about 1800 mg of the anti-CD38 antibody (such as daratumumab) and about 30,000 U of hyaluronidase (such as hyaluronidase-fihj).

[0147] In some embodiments, the CAR is an anti-BCMA CAR and the monoclonal antibody is an anti-SLAMF7 antibody. Those skilled in the art are aware of many anti-BCMA CARs. Exemplary anti-BCMA CARs are described in Section III.A. In some embodiments, the antibody is elotuzumab (such as ).

[0148] In some embodiments, the CAR binds a first antigen that is CD38, SLAMF7, CD138, FCRH5, or GPRC5D, and the monoclonal antibody binds BCMA. CARs directed against such antigens are well known to those skilled in the art. Exemplary CARs are described in Section III.A. In some embodiments, the antibody is melflufen (such as Blenrep).

[0149] In some embodiments, the hematological malignancy is lymphoma. In some embodiments, the lymphoma is non-Hodgkin lymphoma (NHL). In some embodiments, the lymphoma is relapsed / refractory lymphoma, such as relapsed / refractory NHL. In some embodiments, the first and second antigens are selected from the group consisting of CD19, CD20, CD22, ROR1, CD30, CD38, and CD79b. In some embodiments, the first and second antigens are selected from the group consisting of CD19, CD20, CD22, ROR1, and CD30. In some embodiments, one of the first and second antigens may also be CD38. Those skilled in the art are aware of any of the various CARs or monoclonal antibodies directed against such antigens. Exemplary CARs and antibodies are described herein.

[0150] In some embodiments, the CAR is an anti-CD19 CAR and the antibody is an anti-CD20 antibody. Those skilled in the art are aware of many anti-CD19 CARs. Exemplary anti-CD19 CARs are described in Section III.A. In some embodiments, the antibody is rituximab (such as ). In some embodiments, the antibody is obinutuzumab. In some embodiments, the antibody is ofatumumab. In some embodiments, the antibody is ibritumomab. In some embodiments, the antibody is tositumomab. In some embodiments, the antibody is ublituximab.

[0151] In some embodiments, the anti-CD20 antibody can be administered subcutaneously. In some embodiments, the anti-CD20 antibody (such as rituximab) can be administered in an anti-CD20 antibody composition comprising hyaluronidase. For example, the antibody can be administered as an anti-CD20 antibody composition comprising rituximab and recombinant human hyaluronidase PH20. Exemplary examples of such compositions are described in the published PCT publication number WO2011029892.

[0152] In some embodiments, each dose of the anti-CD20 antibody composition comprises from or about 1200 mg to about 2400 mg of an anti-CD20 antibody (such as rituximab) and from or about 15000 units (U) to about 45000 U of hyaluronidase (such as hyaluronidase). In some embodiments, each dose of the anti-CD20 antibody composition comprises about 1400 mg of an anti-CD20 antibody (such as rituximab) and about 23400 U of hyaluronidase. In some embodiments, each dose of the anti-CD20 antibody composition comprises about 1600 mg of an anti-CD20 antibody (such as rituximab) and about 26800 U of hyaluronidase.

[0153] In some embodiments, the CAR is an anti-CD19 CAR and the antibody is an anti-CD30 antibody. Those skilled in the art are aware of many anti-CD19 CARs. Exemplary anti-CD19 CARs are described in Section III.A. In some embodiments, the antibody is an anti-CD30 antibody. In some embodiments, the antibody is brentuximab, )

[0154] In some embodiments, the CAR is an anti-CD20 CAR and the antibody is an antibody directed against CD19, CD20, CD22, ROR1 or CD30. Those skilled in the art are aware of many anti-CD20 CARs. Exemplary anti-CD20 CARs are described in Section III.A. Those skilled in the art are aware of any of the various monoclonal antibodies against such antigens. In some embodiments, the antibody is an anti-CD19 antibody. In some embodiments, the anti-CD19 antibody is tafasitamab (such as ) In other embodiments, the anti-CD19 antibody is loncastuximab (such as ) In some embodiments, the anti-CD19 antibody is blinatumomab. In some embodiments, the anti-CD19 antibody is denintuzumab. In some embodiments, the antibody is an anti-CD30 antibody. In some embodiments, the anti-CD30 antibody is brentuximab The present disclosure describes exemplary antibodies.

[0155] In some embodiments, the CAR is an anti-CD20 CAR and the antibody is an antibody directed against CD38. Those skilled in the art are aware of many anti-CD20 CARs. Exemplary anti-CD20 CARs are described in Section III.A. In some embodiments, the CAR is an anti-CD19 CAR and the antibody is an antibody directed against CD38. Those skilled in the art are aware of many anti-CD19 CARs. Exemplary anti-CD19 CARs are described in Section III.A. In some embodiments, the anti-CD38 antibody is daratumumab (Darzalex TM ). In some embodiments, the anti-CD38 antibody is isatuximab. In some embodiments, the anti-CD38 antibody can be administered subcutaneously. In some embodiments, the anti-CD38 antibody (such as daratumumab) can be administered in an anti-CD38 antibody composition comprising hyaluronidase. For example, the antibody can be administered as an anti-CD38 antibody composition comprising daratumumab and recombinant human hyaluronidase PH20 (such as hyaluronidase-fihj). Examples of such compositions are described in published U.S. Patent Publication No. US20170121414. In some embodiments, each dose of the anti-CD38 antibody composition comprises from or about 1200 mg to about 2400 mg of the anti-CD38 antibody (such as daratumumab) and from or about 15,000 units (U) to about 45,000 U of hyaluronidase (such as hyaluronidase-fihj). In some embodiments, each dose of the anti-CD38 antibody composition comprises about 1800 mg of the anti-CD38 antibody (such as daratumumab) and about 30,000 U of hyaluronidase (such as hyaluronidase-fihj).

[0156] In some embodiments, the hematological malignancy is leukemia. In some embodiments, the leukemia is relapsed / refractory leukemia, such as relapsed / refractory AML. In some embodiments, the leukemia is acute myeloid leukemia (AML). In some embodiments, the first and second antigens are selected from the group consisting of CD123, Flt3, CD70, CD33, CLEC12A, and CD38. Those skilled in the art are aware of any of the various monoclonal antibodies and CARs directed against such antigens.

[0157] In some embodiments, g-NK cells have low or no CD38 expression, such as where fewer than 25% of the cells in a g-NK cell composition are positive for surface CD38. In some embodiments, the cells in the g-NK cell composition are not engineered to reduce or eliminate CD38 expression. This is because it has been found that g-NK cells do not express CD38. In some embodiments, the g-NK cell composition exhibits minimal anti-CD38-induced fratricide, optionally where fewer than 10% of the cells in the g-NK cell composition exhibit anti-CD38-induced fratricide.

[0158] In some embodiments, the cancer is a solid malignancy. In some embodiments, solid tumors include, but are not limited to, lung cancer, colorectal cancer, prostate cancer, pancreatic cancer, and breast cancer, including triple-negative breast cancer. For example, the indications include bone disease or bone metastasis in cancer, regardless of the origin of the primary tumor; breast cancer, including non-limiting examples, ER / PR+ breast cancer, Her2+ breast cancer, triple-negative breast cancer; colorectal cancer; endometrial cancer; gastric cancer; glioblastoma; head and neck cancer, such as esophageal cancer; lung cancer, such as non-limiting examples, non-small cell lung cancer; multiple myeloma ovarian cancer; pancreatic cancer; prostate cancer; sarcoma, such as osteosarcoma; kidney cancer, such as non-limiting examples, renal cell carcinoma; and / or skin cancer, such as non-limiting examples, squamous cell carcinoma, basal cell carcinoma, or melanoma. In some embodiments, the cancer is squamous cell carcinoma. In some embodiments, the cancer is cutaneous squamous cell carcinoma. In some embodiments, the cancer is esophageal squamous cell carcinoma. In some embodiments, the cancer is head and neck squamous cell carcinoma. In some embodiments, the cancer is lung squamous cell carcinoma. In some embodiments, the first antigen and the second antigen are selected from the group consisting of GPC3, HER2, GD2, EGFR mutant III (EGFR vIII), EGFR, CEA, PSMA, FRα, FAP, glypican-3, EPCAM, MUC1, ROR1, MUCI16eto, VEGFR2, CD171, PSCA, EphA2, survivin, mesothelin, TROP2, B7H3, CCR4, PDGFRα, nectin-4, tissue factor, CLDN6, FGFR2b, and IL-13α. Those skilled in the art are aware of any of the various monoclonal antibodies and CARs directed against such antigens.

[0159] In some embodiments, the method or use of treatment involves administering to an individual an effective amount of the cells of a g-NK cell composition as provided herein, such as a composition containing engineered g-NK cells as provided herein, including any such composition of expanded NK cells produced by the methods provided herein. In some embodiments, it is or is about 10 5 to about 10 12, or about 10 5 and about 10 8 , or about 10 6 and about 10 12 , or about 10 8 and about 10 11 , or about 10 9阿 and about 10 10 such g-NK cell compositions provided herein, such as compositions comprising engineered NK cells provided herein, including any compositions prepared by the methods provided herein, are administered to an individual subject. In some embodiments, a dose of cells is administered to the individual, the dose of cells comprising at least or about 10 5 , at least or about 10 6 , at least or about 10 7 , at least or about 10 8 , at least or about 10 9 , at least or about 10 10 , at least or about 10 11 or at least or about 10 12 cells from such g-NK cell compositions provided herein, such as compositions containing engineered NK cells provided herein, including any compositions produced by the methods provided.

[0160] In some embodiments, the method or use of treatment involves administering to an individual an effective amount of cells from any of the provided NK cell compositions, including any engineered g-NK cell compositions as described herein. In some embodiments, from about 10 5 to about 10 12 , or about 10 5 and about 10 8 , or about 10 6 and about 10 12 , or about 10 8 to about 10 11 , or about 10 9 and about 10 10 cells from any of the provided compositions containing engineered g-NK cells are administered to an individual subject. In some embodiments, a dose of cells is administered to the individual, the dose of cells comprising at least or about 10 5 , at least or about 10 6 , at least or about 10 7 , at least or about 10 8 , at least or about 109 and is or greater than or about 10 10 and is or greater than or about 10 11 or is or greater than or about 10 12 cells from the provided composition comprising engineered g-NK cells. In some embodiments, per kg is or is about 10 6 to 10 10 such cells in the provided composition comprising engineered g-NK cells are administered to the subject.

[0161] In some embodiments, the composition comprising engineered g-NK cells is administered once a week for a pre-determined number of doses.

[0162] In some embodiments, the pre-determined number of once-weekly doses is one dose, two doses, three doses, four doses, five doses, six doses, seven doses, eight doses, nine doses, ten doses, eleven doses or twelve doses. In some embodiments, the once-weekly dose is administered for 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 16 weeks, 20 weeks, 24 weeks, 28 weeks, 32 weeks, 36 weeks or longer. In some embodiments, six (6) once-weekly doses of the g-NK cell composition are administered. In some embodiments, the once-weekly dose is administered over consecutive weeks..

[0163] In some embodiments, the once-weekly dose is administered in a cycling regimen. In some embodiments, the cycling regimen is a 14-day cycle. In some embodiments, two once-weekly doses are administered in the 14-day cycle. In some embodiments, the 14-day cycle is repeated twice. In some embodiments, the 14-day cycle is repeated three times..

[0164] In some embodiments, the once-weekly dose is administered in a cycling regimen. In some embodiments, the cycling regimen is a 21-day cycle. In some embodiments, three once-weekly doses are administered in the 21-day cycle. In some embodiments, the 21-day cycle is repeated twice. In some embodiments, the 21-day cycle is repeated three times.

[0165] In some embodiments, an effective amount of any of the disclosed cells or a composition comprising the engineered g-NK cells disclosed herein is administered to the subject once a week for five weeks.

[0166] In some embodiments, the cells in the g-NK cell composition comprising engineered g-NK cells per dose can be or about or about 1x10 8 cells up to or about 50x10 9The cells in the g-NK cell composition. In some embodiments, the cells in each dose of the g-NK cell composition containing engineered g-NK cells can be or can be 5x10 8 The cells in the g-NK cell composition. In some embodiments, the cells in each dose of the g-NK cell composition containing engineered g-NK cells can be or can be about 5x10 9 The cells in the g-NK cell composition. In some embodiments, the cells in each dose of the g-NK cell composition containing engineered g-NK cells can be or can be about 10x10 9 The cells in the g-NK cell composition.

[0167] In some embodiments, the administration dose according to any one of the provided treatment methods or uses is or is about 1×10 5 cells / kg to is or is about 1×10 7 cells / kg, such as is or is about 1×10 5 cells / kg to is or is about 7.5×10 6 cells / kg, is or is 1×10 5 cells / kg to is or is about 5×10 6 cells / kg, is or is about 1×10 5 cells / kg to is or is about 2.5×10 6 cells / kg, is or is about 1×10 5 cells / kg to is or is about 1×10 6 cells / kg, is or is about 1×10 5 cells / kg to is or is about 7.5×10 5 cells / kg, is or is about 1×10 5 cells / kg to is or is about 5×10 5 cells / kg, is or is about 1×10 5 cells / kg to is or is about 2.5×10 5 cells / kg, is or is about 2.5×10 5 cells / kg to is or is about 1×10 7 cells / kg, is or is about 2.5×10 5 cells / kg to is or is about 7.5×10 6 cells / kg, is or is about 2.5×10 5 cells / kg to is or is about 5×10 6 cells / kg, is or is about 2.5×10 5 cells / kg to is or is about 2.5×10 6cells / kg, being or being about 2.5×10 5 cells / kg to being or being about 1×10 6 cells / kg, being or being about 2.5×10 5 cells / kg to being or being about 7.5×10 5 cells / kg, being or being about 2.5×10 5 cells / kg to being or being about 5×10 5 cells / kg, being or being about 5×10 5 cells / kg to being or being about 1×10 7 cells / kg, being or being about 5×10 5 cells / kg to being or being about 7.5×10 6 cells / kg, being or being about 5×10 5 cells / kg to being or being about 5×10 6 cells / kg, being or being about 5×10 5 cells / kg to being or being about 2.5×10 6 cells / kg, being or being about 5×10 5 cells / kg to being or being about 1×10 6 cells / kg, being or being about 5×10 5 cells / kg to being or being about 7.5×10 5 cells / kg, being or being about 1×10 6 cells / kg to being or being about 1×10 7 cells / kg, being or being about 1×10 6 cells / kg to being or being about 7.5×10 6 cells / kg, being or being about 1×10 6 cells / kg to being or being about 5×10 6 cells / kg, being or being about 1×10 6 cells / kg to being or being about 2.5×10 6 cells / kg, being or being about 2.5×10 6 cells / kg to being or being about 1×10 7 cells / kg, being or being about 2.5×10 6 cells / kg to being or being about 7.5×10 6 cells / kg, being or being about 2.5×10 6 cells / kg to being or being about 5×10 6 cells / kg, being or being about 5×10 6 cells / kg to being or being about 1×10 7 cells / kg, being or being about 5×106 cells / kg to or about 7.5×10 6 cells / kg, or to or about 7.5×10 6 cells / kg to or about 1×10 7 cells / kg. In some embodiments, the administered dose is to or about 1×10 5 cells / kg to or about 1×10 8 cells / kg, such as to or about 2.5×10 5 cells / kg to or about 1×10 8 cells / kg, to or about 5×10 5 cells / kg to or about 1×10 8 cells / kg, to or about 7.5×10 5 cells / kg to or about 1×10 8 cells / kg, to or about 1×10 6 cells / kg to or about 1×10 8 cells / kg, to or about 2.5×10 6 cells / kg to or about 1×10 8 cells / kg, to or about 5×10 6 cells / kg to or about 1×10 8 cells / kg, to or about 7.5×10 6 cells / kg to or about 1×10 8 cells / kg, to or about 1×10 7 cells / kg to or about 1×10 8 cells / kg, to or about 2.5×10 7 cells / kg to or about 1×10 8 cells / kg, to or about 5×10 7 cells / kg to or about 1×10 8 cells / kg, or to or about 7.5×10 7 cells / kg to or about 1×10 8 cells / kg.

[0168] In some embodiments, the dose is given in terms of the number of g-NK cells or NK cell subsets (such as any NK cell subset described herein) in the composition that are associated with or include a surrogate marker for the g-NK cells (or the number of any of the foregoing viable cells). In any of the foregoing embodiments, the dose is given in terms of the number of cells in the composition of engineered cells (e.g., produced by the provided method) or the number of any of the foregoing viable cells.

[0169] In some embodiments, the administration dosage according to any treatment method or use is or is about 5×10 7 to or is about 10×10 9 , such as being or being about 5×10 7 to or is about 5×10 9 , being or being about 5×10 7 to or is about 1×10 9 , being or being about 5×10 7 to or is about 5×10 8 , being or being about 5×10 7 to or is about 1×10 8 , 1×10 8 to or is about 10×10 9 , being or being about 1×10 8 to or is about 5×10 9 , being or being about 1×10 8 to or is about 1×10 9 , being or being about 1×10 8 to or is about 5×10 8 , being or being about 5×10 8 to or is about 10×10 9 , being or being about 5×10 8 to or is about 5×10 9 , being or being about 5×10 8 to or is about 1×10 9 , being or being about 1×10 9 to or is about 10×10 9 , being or being about 1×10 9 to or is about 5×10 9 , or being or being about 5×10 9 to or is about 10×10 9 . In some embodiments, the administration dosage is or is about 5×10 8 cells. In some embodiments, the administration dosage is or is about 1×10 9 cells. In some embodiments, the administration dosage is or is about 5×10 9 cells. In some embodiments, the administration dosage is or is about 1×10 10cells. In some embodiments, the dose is given as the number of g-NK cells or NK cell subsets (such as any NK cell subset described herein) associated with or including a surrogate marker for g-NK cells (or the number of any of the foregoing viable cells). In any of the foregoing embodiments, the dose is given as the number of cells in the composition of the expanded cells produced by the provided method or the number of any of the foregoing viable cells.

[0170] In some embodiments, the cells of a composition containing engineered g-NK cells are administered to an individual shortly after being expanded and / or engineered according to the provided method. In other embodiments, the g-NK cell composition containing engineered g-NK cells is stored prior to administration (e.g., by the foregoing method). For example, NK cells can be stored for greater than 6 months, 12 months, 18 months, or 24 months prior to administration to an individual.

[0171] In some embodiments, the provided compositions containing NK cells and their subsets (such as g-NK cells) can be administered to a subject by any convenient route, including parenteral routes such as subcutaneous, intramuscular, intravenous, and / or epidural administration routes.

[0172] In a specific embodiment, the provided composition is administered by intravenous infusion. In some embodiments, the intravenous infusion is administered in a volume of 1 mL to 100 mL as or about 10×10 6 cells to as or about 10×10 9 cells. In some embodiments, the administration is as or about 50×10 6 cells. In some embodiments, the administration is as or about 1×10 9 cells. In some embodiments, the administration is as or about 5×10 9 cells. In some embodiments, the administration is as or about 10×10 9 cells. The volume of cells for infusion is determined such that the number of administered cells is within the level of those skilled in the art. In one example, 0.5×10 7 cells are administered by intravenous infusion of a composition (such as a thawed cryopreserved composition) having a volume of about 20 mL formulated at a concentration of as or about 2.5×10 9 cells / mL (e.g., 5×10 9 cells in 200 mL).

[0173] In some aspects, once the cells are administered to a subject (e.g., a human), the bioactivity of the engineered cell population can be measured by any of a number of known methods.

[0174] In some embodiments, the antibody is a therapeutic monoclonal antibody, such as an anti-tumor antigen or anti-cancer antibody. One of ordinary skill in the art can select an appropriate therapeutic (e.g., anti-cancer) monoclonal antibody to administer to a subject using the engineered g-NK cells and compositions provided herein, e.g., according to the individual's particular disease or condition. Suitable antibodies can include polyclonal antibodies, monoclonal antibodies, fragment antibodies (such as Fab fragments), single-chain antibodies, and other forms of specific binding molecules.

[0175] In some embodiments, the antibody may further comprise a humanized or human antibody. A humanized form of a non-human antibody is a chimeric Ig, Ig chain, or fragment (such as Fv, Fab, Fab′, F(ab’)2, or other antigen-binding sequences of an antibody) that contains minimal sequences derived from a non-human Ig. In some embodiments, the antibody comprises an Fc domain.

[0176] Typically, a humanized antibody has one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are usually referred to as "imported" residues, which are usually taken from the "imported" variable domain. Humanization is achieved by replacing the CDR or CDR sequences of a rodent antibody with the corresponding sequences of a human antibody (Jones et al., 1986; Riechmann et al., 1988; Verhoeyen et al., 1988). Such "humanized" antibodies are chimeric antibodies (1989), in which substantially less than the complete human variable domain has been replaced by the corresponding sequences from a non-human species. In fact, a humanized antibody is usually a human antibody in which some CDR residues and possibly some Fc residues have been replaced by residues at similar sites in a rodent antibody. Humanized antibodies include human antibodies (recipient antibodies) in which the residues of the complementarity-determining regions (CDRs) of the recipient have been replaced by the residues of the CDRs of a non-human species (donor antibody) such as a mouse, rat, or rabbit (having the desired specificity, affinity, and capacity). In some cases, the corresponding non-human residues replace the Fv framework residues of the human antibody. A humanized antibody can contain residues that are neither present in the recipient antibody nor in the imported CDR or framework sequences. Generally, a humanized antibody substantially comprises all of at least one (usually two) variable domains, where most (if not all) of the CDR regions correspond to regions of a non-human Ig, and most (if not all) of the FR regions correspond to those of the human antibody consensus sequence. A humanized antibody preferably also comprises at least a portion of the antibody constant region (Fc), usually the constant region of a human antibody (Jones et al., 1986; Presta, 1992; Riechmann et al., 1988).

[0177] Human antibodies can also be generated by various techniques, including phage display libraries (Hoogenboom et al., 1991; Marks et al., 1991) and the preparation of human mAbs (Boerner et al., 1991; Reisfeld and Sell, 1985). Similarly, transgenic animals in which the endogenous antibody genes are partially or completely inactivated and into which human Ig genes are introduced can also be used to synthesize human Abs. After challenge, the production of human antibodies has been observed, which are very similar to humans in all aspects, including gene rearrangement, assembly, and antibody repertoire (1997a; 1997b; 1997c; 1997d; 1997; 1997; Fishwild et al., 1996; 1997; 1997; 2001; 1996; 1997; 1997; 1997; Lonberg and Huszar, 1995; Lonberg et al., 1994; Marks et al., 1992; 1997; 1997; 1997).

[0178] Those skilled in the art will appreciate that the engineered g-NK cells of the present application are suitable for use with a variety of antibodies that recognize tumor-associated antigens. Non-limiting examples of tumor-associated antigens include CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD40, CD52, CD56, CD70, CD74, CD140, EpCAM, CEA, gpA33, mesothelin, alpha-fetoprotein, mucin, PDGFR-alpha, TAG-72, CAIX, PSMA, folate-binding protein, scatter factor receptor kinase, ganglioside, cytokeratin, frizzled receptor, VEGF, VEGFR, integrin alphaVbeta3, integrin alpha5beta1, EGFR, EGFL7, ERBB2 (HER2), ERBB3, fibronectin, HGF, HER3, LOXL2, MET, IGF1R, IGLF2, EPHA3, FR-alpha, phosphatidylserine, syndecan-1, SLAMF7 (CD319), TRAILR1, TRAILR2, RANKL, FAP, vimentin, or tenascin. In some cases, the antibody is an anti-CD20 antibody (such as rituximab), an anti-HER2 antibody (such as cetuximab), an anti-CD52 antibody, an anti-EGFR antibody, and an anti-CD38 antibody (such as daratumumab), an anti-SLAMF7 antibody (such as elotuzumab).

[0179] Non-limiting antibodies that can be used in the provided methods for combination therapy with cell compositions including g-NK cells include Trastuzumab Ramucirumab Atezolizumab (Tecentriq TM ), Nivolumab Durvalumab (Imfinzi TM ), Avelumab Pembrolizumab Bevacizumab Everolimus Pertuzumab ado-Trastuzumab emtansine Cetuximab Denosumab Rituximab Alemtuzumab Ofatumumab Obinutuzumab Necitumab (Portrazza TM ), Ibritumomab tiuxetan Brentuximab vedotin Siltuximab Bortezomib Darzalex TM ), Empliciti TM ), Dinutuximab (Unituxin TM ), Olaratumab (Lartruvo TM) Ocrelizumab, Isatuximab, Truxima, Blitzima, Ritemvia, Rituzena, Herzuma, Ruxience, ABP 798, Kanjinti, Ogivry, BI 695500, Novex (RTXM83), Tositumomab, or Ontruzant or a biosimilar thereof. Exemplary antibodies include rituximab, trastuzumab, alemtuzumab, cetuximab, daratumumab, veltuzumab, ofatumumab, ulixertinib, ocrelizumab, or elotuzumab.

[0180] In some embodiments, the antibody can be an anti-PD-1 or anti-PD-L1 antibody. Antibodies targeting PD-1 or PD-L1 include, but are not limited to, nivolumab, pembrolizumab, or atezolizumab.

[0181] An antibody specific for a selected cancer type can be chosen, and the antibody includes any antibody approved for the treatment of cancer. Examples include trastuzumab (Herceptin) for breast cancer, rituximab for lymphoma and cetuximab (Erbitux) for squamous cell carcinoma of the head and neck. Those skilled in the art are familiar with monoclonal antibodies approved by the FDA that can bind to specific tumor or disease antigens, and any one of these antibodies can be used for the treatment of tumors or diseases according to the provided methods.

[0182] In some embodiments, the method is for treating adenocarcinoma of the stomach or gastroesophageal junction, and the antibody is trastuzumab or ramucirumab

[0183] In some embodiments, the method is for treating bladder cancer, and the antibody is atezolizumab (Tecentriq TM ) or nivolumab durvalumab (Imfinzi TM ) or avelumab or pembrolizumab

[0184] In some embodiments, the method is for treating brain cancer, and the antibody is bevacizumab

[0185] In some embodiments, the method is for treating breast cancer, and the antibody is trastuzumab

[0186] In some embodiments, the method is used to treat cervical cancer, and the antibody is bevacizumab

[0187] In some embodiments, the method is used to treat colorectal cancer, and the antibody is cetuximab Panitumumab Bevacizumab or ramucirumab

[0188] In some embodiments, the method is used to treat endocrine / neuroendocrine tumors, and the antibody is avelumab

[0189] In some embodiments, the method is used to treat head and neck cancer, and the antibody is cetuximab Pembrolizumab Nivolumab Trastuzumab or ramucirumab.

[0190] In some embodiments, the method is used to treat bone cancer, and the antibody is denosumab

[0191] In some embodiments, the method is used to treat renal cancer, and the antibody is bevacizumab or nivolumab

[0192] In some embodiments, the method is used to treat leukemia, and the antibody is rituximab Alemtuzumab Ofatumumab Obinutuzumab or blinatumomab

[0193] In some embodiments, the method is used to treat lung cancer, and the antibody is bevacizumab Ramucirumab Nivolumab Necitumumab (Portrazza TM ), pembrolizumab or atezolizumab (Tecentriq TM ).

[0194] In some embodiments, the method is used to treat lymphoma, and the antibody is ibritumomab Brentuximab vedotin Rituximab Siltuximab Obinutuzumab Nivolumab or pembrolizumab

[0195] In some embodiments, the method is used to treat multiple myeloma, and the antibody is bortezomib Daratumumab (Darzalex TM ) or elotuzumab (Empliciti TM ).

[0196] In some embodiments, the method is used to treat neuroblastoma, and the antibody is dinutuximab (Unituxin TM ).

[0197] In some embodiments, the method is used to treat ovarian epithelial cancer / fallopian tube cancer / primary peritoneal cancer, and the antibody is bevacizumab

[0198] In some embodiments, the method is used to treat pancreatic cancer, and the antibody is cetuximab or bevacizumab

[0199] In some embodiments, the method is used to treat skin cancer, and the antibody is ipilimumab pembrolizumab avelumab or nivolumab

[0200] In some embodiments, the method is used to treat soft tissue sarcoma, and the antibody is olaratumab (Lartruvo TM ).

[0201] Table 1 lists exemplary first and second antigens and combinations of CARs and antibodies according to the provided method. Table 1. Exemplary first and second antigens and combinations of CARs and antibodies

[0202] In certain instances, a subject is administered an effective dose of an antibody before, after, or substantially simultaneously with administration of a population of engineered g-NK cells. In some instances, an antibody is administered to the subject at about 0.1 mg / kg to about 100 mg / kg (such as about 0.5 - 10 mg / kg, about 1 - 20 mg / kg, about 10 - 50 mg / kg, about 20 - 100 mg / kg, for example about 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 8 mg / kg, about 10 mg / kg, about 16 mg / kg, about 20 mg / kg, about 24 mg / kg, about 36 mg / kg, about 48 mg / kg, about 60 mg / kg, about 75 mg / kg, or about 100 mg / kg). The effective amount of the antibody can be determined by a skilled clinician considering the particular antibody, the particular disease or disorder (such as a tumor or other disease), the general condition of the subject, any other treatments the subject is receiving or has previously received, and other relevant factors. The subject is also administered a population of engineered g-NK cells as described herein. Both the antibody and the population of engineered g-NK cells are typically administered parenterally, such as by intravenous; but injection or infusion into or near the tumor (local administration), or intraperitoneal administration can also be used. A person skilled in the art can determine the appropriate route of administration.

[0203] In some embodiments, administration of at least one dose of the antibody can be initiated within one month prior to administration of the composition of g-NK cells. In some embodiments, administration of at least one dose of the antibody can be initiated within three weeks prior to administration of the composition of g-NK cells. In some embodiments, administration of at least one dose of the antibody can be initiated within two weeks prior to administration of the composition of g-NK cells.

[0204] In certain instances, a subject is administered an effective dose of an antibody before, after, or substantially simultaneously with administration of a population of g-NK cells. The effective amount of the antibody can be determined by a skilled clinician considering the particular antibody, the particular disease or disorder (such as a tumor or other disease), the general condition of the subject, any other treatments the subject is receiving or has previously received, and other relevant factors. The subject is also administered a population of g-NK cells as described herein. Both the antibody and the population of g-NK cells are typically administered parenterally, such as by intravenous; but injection or infusion into or near the tumor (local administration), or intraperitoneal administration can also be used. A person skilled in the art can determine the appropriate route of administration.

[0205] In some embodiments, the antibody can be administered at a once-weekly dose. In some embodiments, the antibody can be administered according to a cycle regimen. In some embodiments, the antibody is administered in 28-day cycles. In some embodiments, the antibody is administered for one or two 28-day cycles. In some embodiments, the antibody is administered once a week for at least one cycle (such as each cycle). In some embodiments, the antibody is administered once a week for 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 16 weeks, 20 weeks, 24 weeks, 28 weeks, 32 weeks, 36 weeks or longer. In some embodiments, eight (8) once-weekly doses of the antibody are administered. In some embodiments, the once-weekly doses are administered over consecutive weeks.

[0206] In some embodiments, the antibody can be administered intravenously.

[0207] In some embodiments, the antibody is daratumumab, and each dose of the antibody can be administered in an amount that can be or is about 8 mg / kg to about 32 mg / kg. In some embodiments, each dose is or is about 16 mg / kg.

[0208] In some embodiments, the anti-SLAMF7 antibody (such as elotuzumab) can be administered at a dose that can be or is about 10 mg / kg per week for two cycles, and then once every 2 weeks thereafter. In some embodiments, the anti-SLAMF7 antibody is administered in combination with lenalidomide and dexamethasone. In some embodiments, the anti-SLAMF7 antibody is administered after dexamethasone, diphenhydramine, ranitidine, and acetaminophen.

[0209] In some embodiments, the anti-BCMA antibody (such as Blenrep) can be administered by intravenous infusion at a dose that can be or is about 2.5 mg / kg for a duration that can be or is about 30 minutes. In some embodiments, the anti-BCMA antibody (such as Blenrep) is administered once every three weeks.

[0210] In some embodiments, each dose of the anti-CD20 antibody can be administered in an amount that can be or is about 250 mg / m 2 to an amount that can be or is about 500 mg / m 2 In some embodiments, each dose is or is about 375 mg / m 2 administered.

[0211] In some embodiments, the anti-CD20 antibody composition can be administered in a once-weekly dose. In some embodiments, the anti-CD20 antibody is administered in 4 or 8 doses. In some embodiments, the antibody is administered subcutaneously in 3 or 7 doses after an intravenous once-weekly dose of the anti-CD20 antibody. In some embodiments, the method comprises administering the anti-CD20 antibody once a week for 8 total doses, and administering the g-NK cell composition once a week for 6 total doses, wherein one or two doses of the anti-CD20 antibody are administered before administering the composition comprising the g-NK cells.

[0212] In some embodiments, the anti-CD19 antibody (such as tafasitamab) is administered at or about 12 mg / kg. In some embodiments, the anti-CD19 antibody (such as tafasitamab) is administered in four cycles. In some embodiments, the first cycle comprises administering on days 1, 4, 8, 15, and 22 of a 28-day cycle. In some embodiments, the second and third cycles comprise administering on days 1, 8, 15, and 22 of a 28-day cycle. In some embodiments, the fourth cycle and subsequent cycles comprise administering on days 1 and 15 of a 28-day cycle. In some embodiments, the anti-CD19 antibody (such as tafasitamab) is administered in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 cycles.

[0213] In some embodiments, the anti-CD19 antibody (such as loncastuximab) is administered at or about 0.15 mg / kg once every 3 weeks for 2 cycles. In some embodiments, for subsequent cycles, the anti-CD19 antibody (such as loncastuximab) is administered at or about 0.075 mg / kg once every 3 weeks. In some embodiments, dexamethasone is administered before administering the anti-CD19 antibody (such as loncastuximab).

[0214] In some embodiments, the anti-CD30 antibody (such as brentuximab vedotin) can be administered at or about 1.8 mg / kg. In some embodiments, the anti-CD30 antibody (such as brentuximab vedotin) is administered at a maximum of 180 mg. In some embodiments, anti-CD30 (such as brentuximab vedotin) is administered once every three weeks.

[0215] In some embodiments, the antibody is a secretable antibody. A. Combination Therapy

[0216] In some embodiments, the provided methods can be practiced as combination therapies with one or more other agents. In such embodiments, a composition containing engineered g-NK cells as provided herein can be administered before, simultaneously with, or after the administration of one or more other agents. For example, a dose of engineered g-NK cells can be administered concurrently with or sequentially with antimicrobial, antiviral, and other therapeutic agents. In some embodiments, the methods are practiced in combination with the administration of chemotherapeutic agents, cytotoxic agents, or immunomodulatory agents to a subject. Exemplary combination therapies are described in the sections below.

[0217] Engineered g-NK cells and additional agents can be administered sequentially or simultaneously. In some embodiments, the additional agent is administered before the administration of g-NK cells. In some embodiments, the additional agent is administered after the administration of engineered g-NK cells. For example, engineered g-NK cells can be administered concurrently with an antibody specific for a selected cancer type. Alternatively, engineered g-NK cells can be administered at a selected time that is different from the time of administration of an antibody specific for a selected cancer type. 1. Cytokines and Growth Factors

[0218] In some embodiments provided herein, engineered g-NK cells or a composition containing such cells can be administered to an individual in combination with cytokines and / or growth factors. In some embodiments provided herein, engineered g-NK cells or a composition containing such cells can be administered to an individual in combination with other exogenously administered cytokines and / or growth factors. Since cytokines are required for NK cell activity, a common approach involves co-administering exogenous cytokines with NK cell therapy as a support for the exogenous cytokines to a subject.

[0219] According to some embodiments, at least one growth factor or cytokine comprises a growth factor selected from the group consisting of SCF, FLT3, IL-2, IL-7, IL-15, IL-12, IL-21, and IL-27. In certain embodiments, recombinant IL-2 is administered to a subject. In other certain embodiments, recombinant IL-15 is administered to a subject. In other certain embodiments, recombinant IL-21 is administered to a subject.

[0220] In some embodiments, at least one cytokine is administered to a subject in combination with engineered g-NK cells or a composition thereof.

[0221] Cytokines are a large class of proteins that play important roles in cell signaling, particularly in the context of the immune system. Cytokines have been shown to function as immunomodulators in autocrine, paracrine, and endocrine signaling. Cytokines can act as immune activators, thereby stimulating immune-mediated responses, or as immune inhibitors, thereby attenuating immune-mediated responses. Cytokines include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors, but generally do not include hormones or growth factor interleukins.

[0222] In some embodiments, the cytokine is an interleukin. Interleukins are a group of cytokines that are typically secreted proteins and signaling molecules that mediate a wide range of immune responses. For example, interleukin (IL)-2 plays a role in regulating the activity of white blood cells, while interleukin (IL)-15 plays a major role in the development of inflammatory and protective immune responses to microbial invaders and parasites by regulating the activity of cells of the innate and adaptive immune systems. In some embodiments, one or more activities of NK cells (including the provided g-NK cells) are regulated by IL-2, IL-21, and / or IL-15 or another cytokine described herein.

[0223] In some embodiments, interleukins include cytokines produced by immune cells such as lymphocytes, monocytes, or macrophages. In some embodiments, the cytokine is an immune activating cytokine that can be used to induce NK cells to, for example, promote NK cell survival, activation, and / or proliferation. For example, certain cytokines (such as IL-15 or IL-21) can prevent or reduce NK cells from undergoing senescence, such as by enhancing their ability to expand in vitro or in vivo. In some embodiments, the interleukin or a functional portion thereof is a partial or full peptide of one or more of IL-2, IL-4, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-15, IL-18, or IL-21. In some embodiments, the cytokine is IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, Flt3-L, SCF, or IL-7. In some embodiments, the cytokine is IL-2. In some embodiments, the cytokine is IL-12. In some embodiments, the cytokine is IL-15. In some embodiments, the cytokine is IL-21. In some embodiments, the cytokine can be administered together with the corresponding receptor of the cytokine. In some embodiments, the step of administering the cytokine together with engineered g-NK cells allows cytokine signaling, thereby maintaining or improving the cell growth, proliferation, expansion, and / or effector function of NK cells.

[0224] In certain embodiments, recombinant IL-2 is administered to a subject. In other certain embodiments, recombinant IL-15 is administered to a subject. In other certain embodiments, recombinant IL-21 is administered to a subject.

[0225] In some embodiments, the cytokine is IL-15 or a functional portion thereof. IL-15 is a cytokine that regulates NK cell activation and proliferation. In some cases, IL-15 and IL-12 share similar biological activities. For example, IL-15 and IL-2 bind to common receptor subunits and can compete for the same receptor. In some embodiments, IL-15 induces the activation of JAK kinases and the phosphorylation and activation of the transcriptional activators STAT3, STAT5, and STAT6. In some embodiments, IL-15 promotes or regulates one or more functional activities of NK cells, such as promoting NK cell survival, regulating the activation and proliferation of NK cells and T cells, and supporting the development of NK cells from hematopoietic stem cells. In some embodiments, the functional portion is a part (e.g., a truncated contiguous amino acid sequence containing full-length IL-15) that retains all or one or more functions of full-length or mature IL-15 (such as promoting NK cell survival, regulating the activation and proliferation of NK cells and T cells, and supporting the development of NK cells from hematopoietic stem cells). All or a functional portion of IL-15 can be administered to a subject.

[0226] As will be appreciated by those skilled in the art, the sequences of a variety of IL-15 molecules are known in the art. In one aspect, IL-15 is wild-type IL-15. In some aspects, IL-15 is mammalian IL-15 (e.g., Homo sapiens interleukin 15 (IL15), transcript variant 3, mRNA, NCBI reference sequence: NM_000585.4; Canis lupus familiaris interleukin 15 (IL15), mRNA, NCBI reference sequence: NM_001197188.1; Felis catus interleukin 15 (IL15), mRNA, NCBI reference sequence: NM_001009207.1). Examples of "mammal" or "mammalian" include primates (e.g., humans), canines, felines, rodents, pigs, ruminants, etc. Specific examples include humans, dogs, cats, horses, cows, sheep, goats, rabbits, guinea pigs, rats, and mice. In a specific aspect, mammalian IL-15 is human IL-15. The human IL-15 amino acid sequence includes, for example, Genbank accession numbers: NR_751915.1, NP_000576.l, AAI00963.1, AAI00964.1, AAI00962.1, CAA71044.1, AAH18149.1, AAB97518.1, CAA63914.1, and CAA63913.1 interleukin interleukin interleukin.

[0227] In some embodiments, the IL-15 nucleotide sequence is as set forth in SEQ ID NO:9, or is a sequence having at least or at least about 85%, at least or at least about 90%, at least or at least about 95%, or at least or at least about 98% sequence identity to SEQ ID NO:9. In some embodiments, IL-15 is the mature form lacking the signal peptide sequence and in some cases also lacking the propeptide sequence. In some embodiments, IL-15 has the amino acid sequence as set forth in SEQ ID NO:2 or a sequence having at least or at least about 85%, at least or at least about 90%, at least or at least about 95%, or at least or at least about 98% sequence identity to SEQ ID NO:2.

[0228] In some embodiments, the IL-15 molecule is a variant of human IL-5, for example having one or more amino acid alterations, such as substitutions, to the human IL-15 amino acid sequence. In some embodiments, the IL-15 variant comprises or consists of mutations at positions 45, 51, 52 or 72, such as those described in US2016 / 0184399. In some embodiments, the IL-15 variant comprises or consists of a substitution of N, S or L for one of D, E, A, Y or P. In some embodiments, the mutations are selected from L45D, L45E, S51D, L52D, N72D, N72E, N72A, N72S, N72Y or N72P (with respect to the sequence of human IL-15, SEQ ID NO:2).

[0229] In an embodiment, the IL-15 molecule comprises an IL-15 variant, such as a human IL-15 polypeptide having one or more amino acid substitutions. In some embodiments, the IL-15 molecule comprises a substitution at position 72, such as a substitution of N for D. In one embodiment, the IL-15 molecule is the IL-15N72D polypeptide of SEQ ID NO:2 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto (which has IL-15Ra binding activity).

[0230] In some embodiments, IL-15 is administered together with IL-15 receptor α (IL15RA), such as in a complex therewith or as a fusion with it. IL15RA binds IL-15 specifically with very high affinity and is capable of binding IL-15 independently of other subunits. In some aspects, this property allows IL-15 to be produced by one cell, endocytosed by another cell, and then presented to a third cell. In some embodiments, IL-15 / IL-15Ra is administered to a subject. In some embodiments, an IL-15 / IL-15R fusion protein is administered to a subject. In some embodiments, a single-chain IL-15 / IL-15R fusion protein is administered to a subject. In some embodiments, IL-15 / IL-15Ra is a soluble IL15Ra.IL15 complex (e.g., Mortier E et al., JBC, 2006; Bessard A, Mol. Cancer Ther., 2009; and Desbois M, J. Immunol., 2016).

[0231] In some embodiments, the cytokine is IL-2 or a functional portion thereof. In some embodiments, IL-2 is a member of the cytokine family that also includes IL-4, IL-7, IL-9, IL-15, and IL-21. IL-2 signals through a receptor complex composed of three chains (referred to as α, β, and γ). All members of this cytokine receptor family share the γ chain. Similar to IL-15, IL-2 promotes immunoglobulin production by B cells and induces the differentiation and proliferation of NK cells. The major difference between IL-2 and IL-15 is found in the adaptive immune response. For example, IL-2 is essential for the adaptive immunity to foreign pathogens as it is the basis for the development of immune memory. On the other hand, IL-15 is necessary for maintaining a highly specific T cell response by supporting the survival of CD8 memory T cells. The whole or a functional portion of IL-2 can be expressed as a membrane-bound polypeptide and / or a secreted polypeptide. As understood by those skilled in the art, the sequences of various IL-2 molecules are known in the art. In one aspect, IL-2 is wild-type IL-2. In some aspects, IL-2 is mammalian IL-2. In some embodiments, IL-2 is human IL-2.

[0232] In some embodiments, IL-2 is a mature form that lacks the signal peptide sequence and in some cases also lacks the propeptide sequence. In some embodiments, IL-2 has the amino acid sequence shown in SEQ ID NO:1 or a sequence having at least or at least about 85%, at least or at least about 90%, at least or at least about 95%, or at least or at least about 98% sequence identity with SEQ ID NO:1.

[0233] In some embodiments, the cytokine is IL-21 or a functional portion thereof. IL-21 binds to the IL-21 receptor (IL-21R) and a co-receptor (common gamma chain (CD132)). The IL-21 receptor has been identified on NK cells, T cells, and B cells, indicating that IL-21 acts on hematopoietic lineage cells, particularly lymphoid progenitors and lymphocytes. IL-21 has been shown to be an effective regulator of cytotoxic T cells and NK cells. (Parrish-Novak, et al. Nature 408:57-63, 2000; Parrish-Novak, et al., J. Leuk. Bio. 72:856-863, 2002; Collins et al., Immunol. Res. 28:131-140, 2003; Brady, et al. J. Immunol. 172:2048-58, 2004).

[0234] As will be appreciated by those skilled in the art, the sequences of a variety of IL-21 molecules are known in the art. In one aspect, IL-21 is wild-type IL-21. In some aspects, IL-21 is mammalian IL-21. In one embodiment, the IL-21 sequence is a human IL-21 sequence. Human IL-21 amino acid sequences include, for example, Genbank accession numbers: AAU88182.1, EAX05226.1, CAI94500.1, CAJ47524.1, CAL81203.1, CAN87399.1, CAS03522.1, CAV33288.1, CBE74752.1, CBI70418.1, CBI85469.1, CBI85472.1, CBL93962.1, CCA63962.1, AAG29348.1, AAH66258.1, AAH66259.1, AAH66260.1, AAH66261.1, AAH66262.1, AAH69124.1, and ABG36529.1.

[0235] In some embodiments, IL-21 is a mature form that lacks a signal peptide sequence and, in some cases, also lacks a propeptide sequence. In some embodiments, IL-21 has the amino acid sequence shown in SEQ ID NO:3 or a sequence having at least or at least about 85%, at least or at least about 90%, at least or at least about 95%, or at least or at least about 98% sequence identity to SEQ ID NO:3. In some embodiments, IL-21 has the amino acid sequence shown in SEQ ID NO:4 or a sequence having at least or at least about 85%, at least or at least about 90%, at least or at least about 95%, or at least or at least about 98% sequence identity to SEQ ID NO:4.

[0236] The amino acid sequence of a cytokine (such as IL-2, IL-15, or IL-21) can include any functional portion of the mature cytokine, such as any functional portion of mature IL-2, mature IL-15, or mature IL-15. A functional portion can be any portion that contains contiguous amino acids of the interleukin to which it belongs, provided that the functional portion specifically binds to the corresponding interleukin receptor. When used with respect to an interleukin, the term "functional portion" refers to any portion or fragment of the interleukin that retains the biological activity of the interleukin (parental interleukin) to which it belongs. Functional portions encompass, for example, interleukin portions that retain the ability to specifically bind to the corresponding interleukin receptor, activate downstream targets of the interleukin, and / or induce the differentiation, proliferation (or death), and activity of immune cells (such as NK cells), to an extent similar to, the same as, or higher than that of the parental interleukin. The biological activity of a functional portion of an interleukin can be measured using assays known in the art. With respect to the parental interleukin, a functional portion can include, for example, an amino acid sequence that is about 60%, about 70%, about 80%, about 90%, about 95%, or more of the amino acid sequence of the parental mature interleukin.

[0237] Functional variants of the interleukins described herein are included within the scope of the cytokines or functional portions according to the provided embodiments. As used herein, the term "functional variant" refers to an interleukin that has substantial or significant sequence identity or similarity to the parental interleukin and that retains the biological activity of its native interleukin. Functional variants encompass, for example, those variants of the interleukins described herein (parental interleukins) that retain the ability to specifically bind to the corresponding interleukin receptor, activate downstream targets of the interleukin, and / or induce the differentiation, proliferation (or death), and activity of immune cells (such as NK cells), to an extent similar to, the same as, or higher than that of the parental interleukin. With respect to the parental interleukin, a functional variant can, for example, be at least about 80%, about 90%, about 95%, about 99%, or more identical to the parental interleukin in amino acid sequence.

[0238] A functional variant can, for example, contain the amino acid sequence of the parental interleukin with at least one conservative amino acid substitution. Alternatively or additionally, a functional variant can contain the amino acid sequence of the parental interleukin with at least one non-conservative amino acid substitution. In some embodiments, the amino acid substitution (such as a conservative or non-conservative amino acid substitution) does not interfere with or inhibit the biological activity of the functional variant compared to the parental interleukin sequence. In some embodiments, the amino acid substitution (such as a conservative or non-conservative amino acid substitution) can enhance the biological activity of the functional variant such that the biological activity of the functional variant is increased compared to the parental interleukin.

[0239] In some embodiments, one or more amino acid substitutions of the interleukin are conservative amino acid substitutions. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid having certain physical and / or chemical properties is replaced with another amino acid having the same or similar chemical or physical properties. For example, conservative amino acid substitutions may be: replacing one acidic / negatively charged polar amino acid (e.g., Asp or Glu) with another acidic / negatively charged polar amino acid, replacing one amino acid having a nonpolar side chain with another amino acid having a nonpolar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), replacing one basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.) with another basic / positively charged polar amino acid, replacing one uncharged amino acid having a polar side chain with another uncharged amino acid having a polar side chain (e.g., Asn, Gln, Ser, Thr, Tyr, etc.), replacing one amino acid having a β-branched side chain with another amino acid having a β-branched side chain (e.g., Ile, Thr, and Val), replacing one amino acid having an aromatic side chain with another amino acid having an aromatic side chain (e.g., His, Phe, Trp, and Tyr), etc. for the interleukin.

[0240] In some embodiments, one or more cytokines (such as IL-2, IL-15, IL-21, IL-27, and / or IL-12) are administered to a subject to support the survival and / or growth of NK cells. The one or more cytokines may be administered before, after, or substantially simultaneously with the NK cells. In some instances, the one or more cytokines may be administered after the NK cells. In one specific instance, the cytokine is administered to the subject within about 1 hour to 8 hours (such as within about 1 hour to 4 hours, within about 2 hours to 6 hours, within about 4 hours to 6 hours, or within about 5 hours to 8 hours) after the administration of the NK cells. In some embodiments, a dose of the provided engineered g-NK cell composition and a cytokine or growth factor are administered sequentially. For example, the g-NK cells may be administered first, followed by the cytokine and / or growth factor. In some embodiments, a dose of the cells containing the engineered g-NK cells is administered simultaneously with the cytokine or growth factor. 2. Cytotoxic agent or lymphodepletion therapy

[0241] In some embodiments, the provided method may further include administering a dose of the cells containing the engineered g-NK cells with another treatment, such as with a chemotherapeutic agent or a cytotoxic agent or other treatment.

[0242] In some aspects, the provided methods may further include administering one or more lymphodepletion therapies, such as before or concurrently with initiation of administration of a g-NK cell composition comprising engineered g-NK cells. In some embodiments, the lymphodepletion therapy includes administering phosphoramide, such as cyclophosphamide. In some embodiments, the lymphodepletion therapy may include administering fludarabine.

[0243] In some aspects, pre-treating a subject with an immune depletion (e.g., lymphodepletion) therapy may improve the efficacy of adoptive cell therapy (ACT). In some embodiments, the lymphodepletion therapy includes a combination of cyclosporine and fludarabine.

[0244] Such pre-treatment may be targeted at reducing the risk of one or more of various outcomes that may inhibit the efficacy of the therapy. These phenomena include a phenomenon called "cytokine sink", by which T cells, B cells, NK cells compete with TILs for homeostatic and activating cytokines such as IL-2, IL-7, and / or IL-15; inhibition of TILs by regulatory T cells, NK cells, or other cells of the immune system; the influence of negative regulators in the tumor microenvironment. Muranski et al., Nat Clin Pract Oncol. December; 3(12):668–681 (2006).

[0245] Thus, in some embodiments, the provided methods further involve administering a lymphodepletion therapy to a subject. In some embodiments, the method includes administering a lymphocyte depletion therapy to the subject prior to administering the dose of cells. In some embodiments, the lymphodepletion therapy comprises chemotherapeutic agents such as fludarabine and / or cyclophosphamide. In some embodiments, the administration of the cells and / or the lymphodepletion therapy is performed via outpatient delivery.

[0246] In some embodiments, these methods include administering a pre-treatment agent, such as a lymphodepleting agent or a chemotherapeutic agent such as cyclophosphamide, fludarabine, or a combination thereof, to the subject prior to administering the dose of cells. For example, the pre-treatment agent, such as a lymphodepleting agent or a chemotherapeutic agent such as cyclophosphamide, fludarabine, or a combination thereof, may be administered to the subject at least 2 days, such as at least 3, 4, 5, 6, or 7 days, prior to the first or subsequent dose. In some embodiments, the pre-treatment agent, such as a lymphodepleting agent or a chemotherapeutic agent such as cyclophosphamide, fludarabine, or a combination thereof, is administered to the subject no more than 7 days, such as no more than 6, 5, 4, 3, or 2 days, prior to administering the dose of cells. In some embodiments, the pre-treatment agent, such as a lymphodepleting agent or a chemotherapeutic agent such as cyclophosphamide, fludarabine, or a combination thereof, is administered to the subject no more than 14 days, such as no more than 13, 12, 11, 10, 9, or 8 days, prior to administering the dose of cells.

[0247] In some embodiments, the subject is pre-treated with cyclophosphamide at a dose between 20 mg / kg and 100 mg / kg or between about 20 mg / kg and about 100 mg / kg, such as between 40 mg / kg and 80 mg / kg or between about 40 mg / kg and about 80 mg / kg. In some aspects, the subject is pre-treated with or with about 60 mg / kg of cyclophosphamide. In some embodiments, fludarabine can be administered as a single dose or can be administered in multiple doses, such as daily, every other day, or every three days. In some embodiments, cyclophosphamide is administered once daily for one or two days.

[0248] In some embodiments, when the lymphodepleting agent comprises fludarabine, it is administered to the subject at a dose between 1 mg / m 2 and 100 mg / m 2 or between about 1 mg / m 2 and about 100 mg / m 2 such as between the following doses or between about the following doses: 10 mg / m 2 and 75 mg / m 2 15 mg / m 2 and 50 mg / m 2 20 mg / m 2 and 30 mg / m 2 or 24 mg / m 2 and 26 mg / m 2 of fludarabine. In some cases, 25 mg / m2 of fludarabine is administered to the subject. In some embodiments, fludarabine can be administered as a single dose or can be administered in multiple doses, such as daily, every other day, or every three days. In some embodiments, fludarabine is administered daily, such as for 1 to 5 days, for example for 3 to 5 days.

[0249] In some embodiments, the lymphodepleting agent comprises a combination of agents, such as a combination of cyclophosphamide and fludarabine. Thus, the combination of agents can include cyclophosphamide at any dose or dosing regimen, such as those described above, and fludarabine at any dose or dosing regimen, such as those described above. For example, in some aspects, 60 mg / kg (about 2 g / m 2 ) of cyclophosphamide and 3 to 5 doses of 25 mg / m 2 of fludarabine are administered to the subject prior to administering the dose of the cells.

[0250] In some embodiments, prior to administering the dose of g-NK cells, the subject has received lymphodepletion therapy. In some embodiments, the lymphodepletion therapy comprises fludarabine and / or cyclophosphamide. In some embodiments, the lymphodepletion comprises from 20 mg / m 2 to 40 mg / m2 or about 20 mg / m 2 to about 40 mg / m 2 of the subject's body surface area, optionally or about 30 mg / m 2 , fludarabine is administered daily for 2 to 4 days, and / or at 200 mg / m 2 to 400 mg / m 2 or about 200 mg / m 2 to about 400 mg / m 2 of the subject's body surface area, optionally or about 300 mg / m 2 , cyclophosphamide is administered daily for 2 to 4 days.

[0251] In some embodiments, lymphodepletion therapy includes fludarabine and cyclophosphamide. In some embodiments, lymphodepletion therapy includes administering fludarabine at 30 mg / m 2 or about 30 mg / m 2 of the subject's body surface area daily, and cyclophosphamide at 300 mg / m 2 or about 300 mg / m 2 of the subject's body surface area daily, each for 2 to 4 days, optionally for 3 days.

[0252] In some embodiments, administering a conditioning agent prior to infusion of the dose of cells improves the outcome of the treatment. For example, in some aspects, conditioning, such as a lymphodepleting agent or chemotherapeutic agent, such as cyclophosphamide, fludarabine, or a combination thereof, improves the efficacy of treatment with the dose or increases the persistence of NK cells in the subject. In some embodiments, the conditioning treatment increases the disease-free survival rate, such as the percentage of subjects who survive and do not exhibit minimal residual or molecularly detectable disease after a given period of time after administration of the dose of cells. In some embodiments, the time to median disease-free survival is increased.

[0253] Once the cells are administered to a subject (e.g., a human), the bioactivity of the engineered cell population is measured in some aspects by any of a number of known methods. Evaluation parameters include specific binding of engineered or natural T cells or other immune cells to an antigen, in vivo, e.g., by imaging, or in vitro, e.g., by ELISA or flow cytometry. In certain embodiments, the ability of NK cells to lyse target cells can be measured using any suitable method known in the art, such as the cytotoxicity assays described, for example, in Kochenderfer et al., J. Immunotherapy, 32(7):689-702 (2009); and Herman et al., J. Immunological Methods, 285(1):25-40 (2004). In certain embodiments, the bioactivity of the cells can also be measured by analyzing the expression and / or secretion of certain cytokines or other effector molecules, such as CD107a, IFNγ, and TNF. In some aspects, the bioactivity is measured by evaluating clinical outcomes, such as a reduction in tumor burden or load. In some aspects, toxicity outcomes, persistence and / or expansion of the cells, and / or the presence or absence of a host immune response are evaluated. III. Engineered Fc receptor γ-deficient natural killer cells (γ-NK cells)

[0254] The provided embodiments relate to methods and uses of engineered natural killer (NK) cells (γ-NK cells) that express a chimeric antigen receptor (CAR) and lack FcRγ expression. In some embodiments, the engineered NK cells are γ-NK cells that lack FcRγ expression. In some embodiments, a γ-NK cell subset of NK cells can be detected by observing whether an NK cell or population of NK cells expresses FcRγ, wherein the cells lacking FcRγ are γ-NK. The FcRγ protein is an intracellular protein. Thus, in some aspects, the presence or absence of FcRγ can be detected, for example, after treating the cells by fixation and permeabilization to allow detection of intracellular proteins.

[0255] In some cases, γ-NK cells can also be identified by surface markers that serve as surrogate markers for γ-NK cells. As further described below, certain combinations of cell surface markers have also been found to be associated with the γ-NK cell phenotype (i.e., cells lacking intracellular FcRγ expression or defective in intracellular FcRγ expression), thus providing a surrogate marker profile for identifying or detecting γ-NK cells in a non-damaging manner to the cells. In some embodiments, the surrogate marker profile for the γ-NK cells provided herein is based on one or more markers CD16 (CD16 阳性 ), NKG2C (NKG2C 阳性) or positive surface expression of CD57 (CD57 positive) and / or low or negative surface expression based on one or more markers CD7 (CD7 微弱 / 阴性 ), CD161 (CD161 阴性 ), and / or NKG2A (NKG2A 阴性 ). In some embodiments, one or more surface markers of NK cells, such as CD45, CD3, and / or CD56, are further evaluated on the cells. In some embodiments, alternative marker profiles CD45 阳性 / CD3 阴性 / CD56 阳性 / CD16 阳性 / CD57 阳性 / CD7 微弱 / 阴性 / CD161 阴性 can be used to identify, detect, enrich, and / or isolate g-NK cells. In some embodiments, alternative marker profiles CD45 阳性 / CD3 阴性 / CD56 阳性 / NKG2A 阴性 / CD161 阴性 are used to identify, detect, enrich, and / or isolate g-NK cells. In some embodiments, NKG2C 阳性 and / or NKG2A 阴性 g-NK cells are identified, detected, enriched, and / or isolated. In some embodiments, g-NK cells have a surface phenotype of CD16 阳性 / CD57 阳性 / CD7 微弱 / 阴性 / CD161 阴性 . In some embodiments, g-NK cells also have a surface phenotype of NKG2A 阴性 / CD161 阴性 . In some embodiments, g-NK cells also have a surface phenotype of CD38 阴性 . In some embodiments, g-NK cells also have a surface phenotype of CD45 阳性 / CD3 阴性 / CD56 阳性 .

[0256] In some embodiments, g-NK cells are engineered to express a CAR. In some embodiments, the CAR is a fusion protein that generally includes an extracellular domain (which includes an antigen recognition region), a transmembrane domain, and an intracellular domain. The extracellular domain (i.e., the antigen recognition region or antigen-binding domain) and the transmembrane domain may be linked by a flexible linker. The intracellular domain may include an intracellular signaling domain that propagates an external cellular stimulus intracellularly. In some embodiments, the CAR includes 1) an antigen-binding domain; 2) a flexible linker; 3) a transmembrane region; and 4) an intracellular signaling domain. In some embodiments, the CAR binds to a target antigen and induces cytotoxicity upon antigen binding.

[0257] In some embodiments, the engineered g-NK cells may further express one or more additional heterologous protein agents. In some embodiments, the engineered g-NK cells also express an immunomodulator, such as a cytokine. In some embodiments, the engineered g-NK cells also express a secreted antibody.

[0258] In some embodiments, the immunomodulator is an agent capable of modulating the immune function of NK cells. In some embodiments, the immunomodulator may be an immune activator. In other embodiments, the immunomodulator may be an immune inhibitor. In some embodiments, the immunomodulator is an exogenous cytokine, such as interleukin or a functional portion thereof. Exemplary features of the CAR and immunomodulator are described further below.

[0259] In some embodiments, the g-NK cells may be further engineered by gene editing as described in Section IV. A. Chimeric Antigen Receptor

[0260] In the provided embodiments, the g-NK cells are genetically engineered to express an antigen receptor that binds to an antigen of interest. In certain embodiments, the antigen receptor is a chimeric antigen receptor (CAR). The antigen receptor can bind to, for example, a tumor-specific or tumor-associated antigen or a pathogen antigen. Thus, the engineered antigen receptor (e.g., CAR) is a recombinant antigen receptor that is designed to introduce a certain antigen specificity into NK cells. In some embodiments, the antigen receptor (such as CAR) is stably integrated into the g-NK cells. In other embodiments, the antigen receptor (e.g., CAR) is transiently expressed by the g-NK cells. For example, the g-NK cells include a CAR having a defined polypeptide sequence that is expressed by an exogenous polynucleotide that has been introduced into the immune effector cell (either transiently or integrated into the genome). In the provided embodiments, the engineered NK cells provided herein that include an antigen receptor (e.g., CAR) can be used in immunotherapy to target and destroy cells (such as cancer cells) associated with a disease or disorder that express a target antigen recognized by the antigen receptor (e.g., CAR).

[0261] In some embodiments, the antigen receptor is a chimeric antigen receptor (CAR). A CAR is typically encoded by a nucleic acid sequence (polynucleotide) that includes a leader sequence, an extracellular targeting domain (also referred to as an extracellular domain); for example, an antigen-binding domain (such as an scFv), a transmembrane domain, and one or more intracellular signaling domains. In some embodiments, the CAR is a fusion protein that includes: an extracellular targeting domain (extracellular domain) that includes an antigen recognition or antigen-binding domain; a transmembrane domain; and an intracellular signaling domain. The extracellular domain and the transmembrane domain can be linked by a flexible linker (also referred to as a spacer). In some embodiments, the antigen-binding domain (such as a single-chain variable fragment (scFv) derived from a monoclonal antibody) recognizes a target antigen. In some embodiments, the antigen-binding domain (e.g., scFv) is linked or fused to the transmembrane domain via a spacer. In some embodiments, the intracellular signaling domain includes an immunoreceptor tyrosine-based activation motif (ITAM). Activation of the CAR fusion protein results in cell activation in response to recognition of its target by the scFv (or other antigen-binding domain). When a cell expresses such a CAR, it can recognize and kill target cells that express the target antigen. This property makes cells expressing CARs particularly attractive agents for specifically targeting cell activity to abnormal cells, including but not limited to cancer cells. Various CARs have been developed against target antigens, including tumor-associated antigens, for expression in various immune cells, including T lymphocytes and natural killer (NK) cells, to mediate cytotoxic activity against target cells expressing that antigen, and the immune cells can be the engineered g-NK cells disclosed herein.

[0262] In some embodiments, the leader sequence can be any of the signal peptide sequences described herein. Exemplary CD8α signal peptides are shown in SEQ ID NO:12. Exemplary GM-CSFRα signal peptides are shown in SEQ ID NO:13. Exemplary IgK signal peptides are shown in SEQ ID NO:14. Exemplary IgK signal peptides are shown in SEQ ID NO:43.

[0263] Any kind of chimeric antigen receptor can be expressed in engineered NK cells, including those described in international PCT applications PCT / US2018 / 024650, PCT / IB2019 / 000141, PCT / IB2019 / 000181, and / or PCT / US2020 / 020824, PCT / US2020 / 035752.

[0264] In certain embodiments, the extracellular antigen-binding domain specifically binds to an antigen. In some embodiments, the extracellular antigen-binding domain or targeting domain is derived from an antibody molecule and includes one or more complementarity-determining regions (CDRs) from the antibody molecule that confer antigen specificity to the CAR. In certain embodiments, the extracellular antigen-binding domain is a single-chain variable fragment (scFv). In certain embodiments, the scFv is a human scFv. In certain embodiments, the scFv is a humanized scFv. In certain embodiments, the extracellular antigen-binding domain is a Fab (optionally cross-linked). In certain embodiments, the extracellular binding domain is F(ab') 2 . In certain embodiments, any of the foregoing molecules can be included in a fusion protein with a heterologous sequence to form an extracellular antigen-binding domain. In certain embodiments, the scFv is identified by screening an scFv phage library with an antigen-Fc fusion protein.

[0265] In some embodiments, the scFv comprises variable chain portions of an immunoglobulin light chain and an immunoglobulin heavy chain molecule separated by a flexible linker polypeptide. The order of the heavy and light chains is not restricted and can be reversed. The flexible polypeptide linker allows the heavy and light chains to associate with each other and reconstitute the immunoglobulin antigen-binding domain. In some embodiments, the flexible linker is a GS linker, such as that shown in SEQ ID NO:56. In some embodiments, the flexible linker is a Whitlow linker, such as that shown in SEQ ID NO:55. Suitably, the light chain variable region comprises three CDRs, and the heavy chain variable region comprises three CDRs. Suitably, the CDRs for the antigen-binding targeting domain are derived from antibody molecules of any species (e.g., human, mouse, rat, rabbit, goat, sheep), and the framework regions between the CDRs are humanized or comprise a sequence that is at least 85%, 90%, 95%, or 99% identical to a human framework region.

[0266] When the targeting domain of the CAR comprises an scFv, the immunoglobulin light chain and the immunoglobulin heavy chain are linked by polypeptide linkers of various lengths. Suitably, the polypeptide linker comprises a length of more than or equal to 10 amino acids. Suitably, the polypeptide linker comprises a length of more than 10, 15, 20, or 25 amino acids. Suitably, the polypeptide linker comprises a length of less than or equal to 30 amino acids. Suitably, the polypeptide linker comprises a length of less than 15, 20, 25, or 30 amino acids. Suitably, the polypeptide linker comprises between 10 and 30 amino acids in length. Suitably, the polypeptide linker comprises between 10 and 25 amino acids in length. Suitably, the polypeptide linker comprises between 10 and 20 amino acids in length. Suitably, the polypeptide linker comprises between 10 and 15 amino acids in length. Suitably, the polypeptide linker comprises between 15 and 30 amino acids in length. Suitably, the polypeptide linker comprises between 20 and 30 amino acids in length. Suitably, the polypeptide linker comprises between 25 and 30 amino acids in length. Suitably, the polypeptide linker comprises hydrophilic amino acids. Suitably, the polypeptide linker consists of hydrophilic amino acids. Suitably, the polypeptide linker comprises a G 4 S sequence (GGGGS). G 4 S linkers allow the linker to have flexibility and protease resistance. Suitably, G 4 S linkers are repeated continuously 1, 2, 3, 4, 5, 6, 7, or 8 times in the polypeptide linker.

[0267] In certain embodiments, the antigen is a tumor antigen. In certain embodiments, the antigen is a pathogen antigen, including, for example, a viral antigen or a bacterial antigen.

[0268] Binding of the extracellular antigen-binding domain (e.g., scFv or analog thereof) of the CAR targeting an antigen can be confirmed by, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, a biological assay (e.g., growth inhibition), or a Western blot assay. Each of these assays generally detects the presence of a protein-antibody complex of particular interest by using a labeled reagent (e.g., an antibody or scFv) specific for the complex of interest. For example, an scFv can be radiolabeled and used in a radioimmunoassay (RIA) (see, e.g., Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March 1986, which is incorporated herein by reference). The radioisotope can be detected by means such as using a gamma counter or a scintillation counter or by autoradiography. In certain embodiments, the extracellular antigen-binding domain of the CAR is labeled with a fluorescent label. Non-limiting examples of fluorescent labels include green fluorescent protein (GFP), blue fluorescent proteins (e.g., EBFP, EBFP2, Azurite, and mKalamal), cyan fluorescent proteins (e.g., ECFP, Cerulean, and CyPet), and yellow fluorescent proteins (e.g., YFP, Citrine, Venus, and YPet).

[0269] In certain embodiments, the antigen recognition receptor binds to a tumor-associated antigen or a tumor-specific antigen. Any suitable tumor-associated antigen or tumor-specific antigen (e.g., an antigenic peptide) can be used in the embodiments described herein. The antigen can be, but is not limited to, a protein, a non-protein, a neoantigen, a post-translationally modified antigen, a peptide-MHC antigen, and / or an overexpressed antigen.

[0270] For example, tumor targets include, but are not limited to, CD38 (multiple myeloma); CD20 (lymphoma); epidermal growth factor receptor (EGFR; non-small cell lung cancer, epithelial cancer, and glioma); type III variant of epidermal growth factor receptor (EGFRvIII; glioblastoma); human epidermal growth factor receptor 2 (HER2; ovarian cancer, breast cancer, glioblastoma, colon cancer, osteosarcoma, and medulloblastoma); mesothelin (mesothelioma, ovarian cancer, and pancreatic cancer); prostate-specific membrane antigen (PSMA; prostate cancer); carcinoembryonic antigen (CEA; pancreatic cancer, breast cancer, and colorectal cancer); disialoganglioside 2 (GD2; neuroblastoma and melanoma); interleukin-13 receptor alpha 2 (IL-13Ra2; glioma); glypican-3 (hepatocellular carcinoma); carbonic anhydrase IX (CAIX; renal cell carcinoma); L1 cell adhesion molecule (L1-CAM; neuroblastoma, melanoma, and ovarian cancer); cancer antigen 125 (CA 125; epithelial ovarian cancer); CD133 (glioblastoma and cholangiocarcinoma); fibroblast activation protein (FAP; malignant pleural mesothelioma); cancer / testis antigen 1B (CTAG1B; melanoma and ovarian cancer); mucin 1 (seminal vesicle carcinoma); and folate receptor-alpha (FR-alpha; ovarian cancer).

[0271] Other non-limiting examples of tumor antigens include, but are not limited to, carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CD8, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CLL1, CD34, CD38, CD41, CD44, CD49c, CD49f, CD56, CD66c, CD73, CD74, CD104, CD133, CD138, CD123, CD142, CD44V6, antigens of cytomegalovirus (CMV)-infected cells (e.g., cell surface antigens), cutaneous lymphocyte-associated antigen (CLA; a specialized glycoform of P-selectin glycoprotein ligand-1 (PSGL-1)), epithelial glycoprotein-2 (EGP2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine protein kinases erb-B2,3,4 (erb-B2,3,4), folate-binding protein (EBP), fetal acetylcholine receptor (AChR), folate receptor-α, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER2), human telomerase reverse transcriptase (hTERT), interleukin-13 receptor subunit alpha-2 (IL-13Rα2), kappa-light chain, kinase insert domain receptor (KDR), Lewis Y (LeY), LI cell adhesion molecule (L1CAM), melanoma antigen family A,1 (MAGE-A1), mucin 16 (MUC16), mucin 1 (MUC1), mesothelin (MSLN), ERBB2, MAGEA3, p53, MARTI, GP100, proteinase 3 (PR1), tyrosinase, survivin, hTERT, EphA2, NKG2D ligand, cancer-testis antigen NY-ESO-1, carcinoembryonic antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), ROR1, tetraspanin 8 (TSPAN8), tumor-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), cytokine receptor-like factor 2 (CRLF2), BCMA, GPC3, NKCS1, EGF1R, EGFR-VIII, and ERBB.

[0272] In some embodiments, the tumor antigen is CD19, ROR1, Her2, PSMA, PSCA, mesothelin (MSLN) or CD20. In some embodiments, the tumor antigen is CD19, CD20, CD33, MSLN or cytokine receptor-like factor 2 (CRLF2), which are expressed on leukemia or lymphoma. In some embodiments, the CAR binds to a target antigen selected from Her2, EGFR, α-folate receptor, CEA, cMET, MUC2, mesothelin or ROR1. In one embodiment, the target antigen is CD38, CD319 / SLAMF-7, TNFRSF 17 / BCMA, SYND1 / CD138, CD229, CD47, Her2 / Neu, epidermal growth factor receptor (EGFR), CD123 / IL3-RA, CD19, CD20, CD22, mesothelin, EpCAM, MUC1, MUC 16, Tn antigen, NEU5GC, NeuGcGM3, GD2, CLL-1 or HERV-K. In some embodiments, the target antigen is a blood cancer-related antigen. For example, the target antigen can be CD38, CD319 / SLAMF-7, TNFRSF 17 / BCMA, SYND1 / CD138, CD229, CD47, CD123 / IL3-RA, CD19, CD20, CD22 or CLL-1.

[0273] A variety of antigen-binding domains for incorporation into CARs are known. In one non-limiting example, g-NK cells are engineered with a CD38-specific CAR (see, e.g., WO2018 / 104562).

[0274] In some embodiments, g-NK cells are engineered with bispecific CARs or multiple different CARs, where the CARs are affinity for two different ligands / antigens. Bispecific CAR-NKs can be used to increase the number of potential binding sites on cancer cells or, alternatively, to target cancer cells to other immune effector cells that express ligands specific for the NK-CAR. For use in cancer therapy, the bispecific CAR can bind to target tumor cells and effector cells, such as T cells, NK cells, or macrophages. Thus, for example, in the case of multiple myeloma, the bispecific CAR can bind to a T cell antigen (such as CD3, etc.) and a tumor cell marker (such as CD38, etc.). The bispecific CAR can alternatively bind to two different tumor cell markers, thereby increasing the overall binding affinity of NK cells for target tumor cells. This can reduce the risk of cancer cells developing resistance by downregulating one of the target antigens. In this case, in multiple myeloma, an example is a CAR that binds to both CD38 and CS-1 / SLAMF7 simultaneously. Another tumor cell marker that CARs are suitable for targeting is the "don't eat me" type marker on tumors, such as CD47.

[0275] In some embodiments, engineered g-NK cells can include a bispecific CAR or multiple CARs expressed by the same NK cell. This allows the NK cell to target two different antigens simultaneously. Suitably, the bispecific CAR is specific for any two of the following antigens: CD38, CD319 / SLAMF-7, TNFRSF 17 / BCMA, CD123 / IL3-RA, SYND1 / CD138, CD229, CD47, Her2 / Neu, epidermal growth factor receptor (EGFR), CD19, CD20, CD22, mesothelin, EpCAM, MUC1, MUC16, Tn antigen, NEU5GC, NeuGcGM3, GD2, CLL-1, CD 123, HERV-K. Suitably, the bispecific nature of the CAR NK cells can allow binding to a tumor antigen and another immune cell, such as a T cell or a dendritic cell. Suitably, the bispecific nature of the CAR NK cells can allow binding to a checkpoint inhibitor, such as PDL-1 or CD47. Suitably, the first CAR has CD38 specificity, and the second CAR is specific for any one of SLAMF-7, BCMA, CD138, CD229, PDL-1 or CD47. Suitably, the first CAR is specific for CD38, and the second CAR is specific for SLAMF-7, BCMA, CD138, CD229. Suitably, the first CAR is specific for CD38, and the second CAR is specific for SLAMF-7. Suitably, the first CAR is specific for CD38, and the second CAR is specific for BCMA. Suitably, the first CAR is specific for CD38, and the second CAR is specific for CD 138. Suitably, the first CAR is specific for CD38, and the second CAR is specific for CD229.

[0276] In some embodiments, the transmembrane domain of the CAR comprises hydrophobic amino acid residues and permits anchoring of the CAR into the cell membrane of the engineered NK cell. Suitably, the transmembrane domain comprises an amino acid sequence derived from a transmembrane protein. Suitably, the transmembrane domain comprises an amino acid sequence derived from the alpha, beta, or zeta chain of the T cell receptor, CD27, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. Suitably, the CAR comprises a transmembrane domain having an amino acid sequence derived from the transmembrane domain of CD8. Suitably, the CAR comprises a transmembrane domain having an amino acid sequence derived from human CD8α. In some embodiments, the CAR contains the transmembrane domain of CD8α having the amino acid sequence shown in SEQ ID NO: 61 or an amino acid sequence exhibiting at least 85%, 90%, or 95% sequence identity with SEQ ID NO: 61. In some embodiments, the transmembrane domain is shown in SEQ ID NO: 61. In some embodiments, the CAR comprises the transmembrane domain of CD8α having the amino acid sequence shown in SEQ ID NO: 73, or an amino acid sequence exhibiting at least 85%, 90%, or 95% sequence identity with SEQ ID NO: 73. In some embodiments, the transmembrane domain is shown in SEQ ID NO: 73.

[0277] In some embodiments, suitably, the CAR comprises a transmembrane domain having an amino acid sequence derived from the transmembrane domain of CD28. Suitably, the CAR comprises a transmembrane domain having an amino acid sequence derived from the transmembrane domain of human CD28. In some embodiments, the CAR contains a hinge domain and the transmembrane domain of CD28 having the amino acid sequence shown in SEQ ID NO:39 or an amino acid sequence exhibiting at least 85%, 90%, or 95% sequence identity with SEQ ID NO:39. In some embodiments, the transmembrane domain is shown in SEQ ID NO:39. In some embodiments, the transmembrane domain of CD28 has the amino acid sequence shown in SEQ ID NO:74 or an amino acid sequence exhibiting at least 85%, 90%, or 95% sequence identity with SEQ ID NO:74. In some embodiments, the transmembrane domain is shown in SEQ ID NO:74. In some embodiments, the CAR comprises a CD28 hinge domain and a CD28 transmembrane domain. In some embodiments, the CD28 hinge domain and the transmembrane domain are shown with the amino acid sequence shown in SEQ ID NO:10 or an amino acid sequence exhibiting at least 85%, 90%, or 95% sequence identity with SEQ ID NO:10. In some embodiments, the CD28 hinge domain and the transmembrane domain are shown with the amino acid sequence shown in SEQ ID NO:10.

[0278] In some embodiments, the CAR may further include a spacer located between the antigen-binding targeting domain and the transmembrane domain. In some embodiments, the spacer comprises hydrophilic amino acids and allows the targeting domain to have flexibility relative to the cell surface. Suitably, the spacer comprises more than 5, 10, 15, 20, 25, or 30 amino acids. Suitably, the spacer comprises less than 10, 15, 20, 25, 30, or 35 amino acids. In some embodiments, the spacer is a hinge region and comprises the hinge sequence of CD8 or an immunoglobulin molecule.

[0279] In some embodiments, the spacer is or comprises a CD8 hinge. In some embodiments, the spacer is the hinge region of human CD8. In some embodiments, the CAR contains a CD8 hinge spacer sequence having the amino acid sequence shown in SEQ ID NO:60 or an amino acid sequence that exhibits at least 85%, 90%, or 95% sequence identity with SEQ ID NO:60. In some embodiments, the sequence of the spacer is shown in SEQ ID NO:60. In some embodiments, the CAR contains a CD8 hinge spacer sequence having the amino acid sequence shown in SEQ ID NO:71 or an amino acid sequence that exhibits at least 85%, 90%, or 95% sequence identity with SEQ ID NO:71. In some embodiments, the spacer sequence is shown in SEQ ID NO:71.

[0280] In some embodiments, the spacer is or comprises a CD28 hinge. In some embodiments, the spacer region is the hinge region of human CD28. In some embodiments, the CAR contains a CD28 hinge spacer sequence having the amino acid sequence shown in SEQ ID NO:72 or an amino acid sequence that exhibits at least 85%, 90%, or 95% sequence identity with SEQ ID NO:72. In some embodiments, the sequence of the spacer is shown in SEQ ID NO:72.

[0281] In some embodiments, the spacer comprises all or a portion of a hinge domain containing an IgG1 Fc or IgG4 Fc. In some embodiments, the spacer is an IgG4 Fc spacer. In some embodiments, the CAR contains an IgG4Fc spacer having the amino acid sequence shown in SEQ ID NO:38 or an amino acid sequence that exhibits at least 85%, 90%, or 95% sequence identity with SEQ ID NO:38. In some embodiments, the sequence of the spacer is shown in SEQ ID NO:38. In some embodiments, the sequence of the spacer is the hinge portion of an IgG1 Fc or IgG4 Fc. In some embodiments, the CAR contains an IgG4 hinge spacer. In some embodiments, the IgG4 hinge spacer has the amino acid sequence shown in SEQ ID NO:59 or an amino acid sequence that exhibits at least 85%, 90%, or 95% sequence identity with SEQ ID NO:59. In some embodiments, the sequence of the spacer is shown in SEQ ID NO:59. In some embodiments, the IgG4 hinge spacer has the amino acid sequence shown in SEQ ID NO:75 or an amino acid sequence that exhibits at least 85%, 90%, or 95% sequence identity with SEQ ID NO:75. In some embodiments, the sequence of the spacer is shown in SEQ ID NO:75.

[0282] In some embodiments, the intracellular signaling domain of the CAR increases the potency of the CAR and comprises an intracellular signaling domain derived from a protein involved in immune cell signaling. Suitably, one or more intracellular signaling domains comprise an intracellular signaling domain derived from CD3ζCD28, OX-40, 4-1BB, DAP10, DAP 12, 2B4 (CD244), or any combination thereof. Suitably, one or more intracellular signaling domains comprise an intracellular signaling domain derived from any two of CD3ζCD28, OX-40, 4-lBB, DAP10, DAP 12, 2B4 (CD244), or any combination thereof.

[0283] In some embodiments, the intracellular domain of the CAR may comprise two additional signaling domains. For example, the CAR may comprise a primary intracellular signaling domain (such as the CD3ζ intracellular signaling domain), and an intracellular signaling domain from a co-stimulatory molecule to provide an additional signal to the cell, such as to further enhance the potency of the immune cell expressing the CAR. Thus, in some embodiments, the chimeric antigen receptor (CAR) comprises: 1) an antigen-binding domain; 2) a flexible linker; 3) a transmembrane region; and 4) an intracellular signaling region that comprises a first primary intracellular signaling domain (such as the CD3ζ intracellular signaling domain) and a second co-stimulatory intracellular signaling domain. In some embodiments, the co-stimulatory domain may be the CD27, CD28, 4-1BB (CD137), 0X40 (CD134), CD30, CD40, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and / or B7-H3 co-stimulatory domain. In some embodiments, the co-stimulatory domain may be CD27, CD28, 4-1BB (CD137), 0X40 (CD134), DAP10, DAP12, ICOS, and / or 2B4. In some embodiments, the co-stimulatory domain may be CD27, CD28, 4-1BB, 2B4, DAP10, DAP12, 0X40, CD30, CD40, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and / or B7-H3 co-stimulatory domain. In some embodiments, the co-stimulatory signaling domain is the signaling domain of CD28. In some embodiments, the co-stimulatory signaling domain is the signaling domain of 4-1BB.

[0284] In some embodiments, the CAR contains an intracellular signaling domain that contains the signaling domain of CD3ζ, which has the amino acid sequence shown in SEQ ID NO: 41 or an amino acid sequence that exhibits at least 85%, 90%, or 95% sequence identity with SEQ ID NO: 41. In some embodiments, the CAR contains an intracellular signaling domain that contains the signaling domain of CD3ζ, which has the amino acid sequence shown in SEQ ID NO: 41. In some embodiments, the CAR contains an intracellular signaling domain that contains the signaling domain of CD3ζ, which has the amino acid sequence shown in SEQ ID NO: 50 or an amino acid sequence that exhibits at least 85%, 90%, or 95% sequence identity with SEQ ID NO: 50. In some embodiments, the CAR contains an intracellular signaling domain that contains the signaling domain of CD3ζ, which has the amino acid sequence shown in SEQ ID NO: 50.

[0285] In some embodiments, the CAR contains an intracellular signaling domain that contains the co-stimulatory signaling domain of CD28, which has the amino acid sequence shown in SEQ ID NO: 40 or an amino acid sequence that exhibits at least 85%, 90%, or 95% sequence identity with SEQ ID NO: 40. In some embodiments, the CAR contains an intracellular signaling domain, and the co-stimulatory signaling domain contained in the intracellular signaling domain has the amino acid sequence shown in SEQ ID NO: 40. In some embodiments, the CAR contains an intracellular signaling domain that contains the co-stimulatory signaling domain of CD28, which has the amino acid sequence shown in SEQ ID NO: 52 or an amino acid sequence that exhibits at least 85%, 90%, or 95% sequence identity with SEQ ID NO: 52. In some embodiments, the CAR contains an intracellular signaling domain that contains the co-stimulatory signaling domain of CD28, which has the amino acid sequence shown in SEQ ID NO: 52.

[0286] In some embodiments, the CAR contains an intracellular signaling domain that contains a 4-1BB co-stimulatory signaling domain having the amino acid sequence shown in SEQ ID NO: 51 or an amino acid sequence that exhibits at least 85%, 90%, or 95% sequence identity with SEQ ID NO: 51. In some embodiments, the CAR contains an intracellular signaling domain that contains a 4-1BB co-stimulatory signaling domain having the amino acid sequence shown in SEQ ID NO: 51.

[0287] In some embodiments, the intracellular signaling domain can be a domain of CD3ζ, CD28, and / or 4-1BB. In some embodiments, the intracellular signaling domain contains a 4-1BB co-stimulatory signaling domain (e.g., SEQ ID NO: 51 or a sequence that exhibits at least 85%, 90%, or 95% sequence identity with SEQ ID NO: 51) and a CD3ζ signaling domain (e.g., SEQ ID NO: 41 or 50 or a sequence that exhibits at least 85%, 90%, or 95% sequence identity with SEQ ID NO: 41 or 50). In some embodiments, the intracellular signaling domain contains a CD28 co-stimulatory signaling domain (e.g., SEQ ID NO: 52 or a sequence that exhibits at least 85%, 90%, or 95% sequence identity with SEQ ID NO: 52) and a CD3ζ signaling domain (e.g., SEQ ID NO: 41 or 50 or a sequence that exhibits at least 85%, 90%, or 95% sequence identity with SEQ ID NO: 41 or 50).

[0288] Suitably, the CAR comprises at least two intracellular signaling domains derived from CD3ζ and 4-1BB. In some embodiments, the CAR contains an intracellular signaling domain that contains the sequences shown in SEQ ID NO: 41 and SEQ ID NO: 51. In some embodiments, the CAR contains an intracellular signaling domain that contains the sequences shown in SEQ ID NO: 50 and SEQ ID NO: 51.

[0289] In other embodiments, suitably, the CAR comprises at least two intracellular signaling domains derived from CD3ζ and CD28. In some embodiments, the CAR comprises an intracellular signaling domain comprising the sequences shown in SEQ ID NO:41 and SEQ ID NO:40. In some embodiments, the CAR comprises an intracellular signaling domain comprising the sequences shown in SEQ ID NO:41 and SEQ ID NO:52. In some embodiments, the CAR comprises an intracellular signaling domain comprising the sequences shown in SEQ ID NO:50 and SEQ ID NO:40. In some embodiments, the CAR comprises an intracellular signaling domain comprising the sequences shown in SEQ ID NO:50 and SEQ ID NO:52.

[0290] In some embodiments, the antigen receptor (e.g., CAR) is encoded by a polynucleotide encoding a CAR having an NH 2 terminal leader sequence. The leader sequence (also referred to as a signal peptide) allows the expressed CAR construct to enter the endoplasmic reticulum (ER) and target the cell surface. The leader sequence is cleaved in the ER, and the mature cell surface CAR does not have the leader sequence. Generally, the leader sequence length will be in the range of 5 to 30 amino acids and includes a stretch of hydrophobic amino acids. Suitably, the leader sequence includes more than 5, 10, 15, 20, or 25 amino acids in length. Suitably, the leader sequence includes less than 10, 15, 20, 25, or 30 amino acids in length. Suitably, the leader sequence includes a sequence derived from any secreted protein. Suitably, the leader sequence includes a sequence derived from the CD8α leader sequence. In some embodiments, suitably, the leader sequence includes a sequence derived from the IgK leader sequence. In some embodiments, the leader sequence is shown in SEQ ID NO:43.

[0291] In some embodiments, the CAR is a CAR present in any of a variety of known engineered cell products. The CAR can include, but is not limited to, a CAR engineered into a cell: JCARH125, CARVYKTI TM(NJ-68284528; Janssen / Legend), P-BCMA-101 (Poseida), PBCAR269A (Poseida), P-BCMA-Allo1 (Poseida), Allo-715 (Pfizer / Allogene), CT053 (Carsgen), Descartes-08 (Cartesian), PHE885 (Novartis), CTX120 (CRISPR Therapeutics); or

[0292] In some embodiments, the CAR comprises the CARs of commercially available CAR cell therapies. Non-limiting examples of CARs in commercially available cell-based therapies include CARs engineered in cells: brexucabtagene autoleucel axicabtagene ciloleucel idecabtagene vicleucel ciltacabtagene autoleucel (CARVYKTI TM )、lisocabtagene maraleucel tisagenlecleucel

[0293] In some embodiments, g-NK cells are engineered with a chimeric antigen receptor (CAR) that binds to CD19. Cluster of differentiation 19 (CD19) is an antigenic determinant that can be detected on pre-leukemic cells. The amino acid and nucleic acid sequences for human and murine CD19 can be found in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequence of human CD19 can be found under UniProt / Swiss-Prot accession number P15391, and the nucleotide sequence encoding human CD19 can be found under accession number NM_001178098. CD19 is expressed in most B-lineage cancers, including, for example, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin lymphoma. It is also an early marker for B cell progenitors. See, for example, Nicholson et al., Mol. Immunol. 34(16-17):1157-1165 (1997). The antigen-binding extracellular domain in the CAR polypeptides disclosed herein is specific for CD19 (e.g., human CD19). In some instances, the antigen-binding extracellular domain can include a single-chain variable fragment (scFv) extracellular domain capable of binding to CD19. In some embodiments, the anti-CD19 CAR can include an anti-CD19 single-chain variable fragment (scFv) specific for CD19, followed by a spacer and transmembrane domain fused to an intracellular co-signaling domain (e.g., CD28 or 4-1BB) and a CD3ζ signaling domain.

[0294] In some embodiments, the extracellular binding domain of the CD19 CAR can include the heavy chain variable region (V H ) shown in SEQ ID NO:54 and the light chain variable region (V L)。In some embodiments, the linker separating VH and VL in the scFv is a GS linker, such as that shown in SEQ ID NO:56. In some embodiments, the linker separating VH and VL in the scFv is the Whitlow linker shown in SEQ ID NO:55. In some embodiments, the scFv has the amino acid sequence shown in SEQ ID NO:57. In some embodiments, the scFv has the amino acid sequence shown in SEQ ID NO:58. In some embodiments, the spacer is a CD8 hinge, such as that shown in SEQ ID NO:60. In some embodiments, the spacer is an IgG4 hinge, such as that shown in SEQ ID NO:59. In some embodiments, the intracellular signaling domain contains a 4-1BB co-stimulatory signaling domain and a CD3ζ signaling domain, such as any of those described herein. In some embodiments, the intracellular signaling domain contains a CD28 co-stimulatory signaling domain and a CD3ζ signaling domain, such as any of those described herein. In some embodiments, it should be understood that the CAR includes any of the following sequences: those that exhibit some sequence variation from any of the above or described SEQ ID NOs, such as having at least 85%, 90%, 95% or higher sequence identity therewith, and retaining binding to CD19 as well as intracellular signaling and cytotoxic activity.

[0295] In some embodiments, the CAR includes an anti-CD19 CAR of a commercial CAR cell therapy. Non-limiting examples of anti-CD19 CARs in commercially available cell-based therapies include anti-CD19 CARs engineered in cells: or

[0296] In some embodiments, the CAR is an anti-CD19 CAR having the amino acid sequence shown in SEQ ID NO:76 or an amino acid sequence having at least 85%, 90% or 95% sequence identity with SEQ ID NO:76. In some embodiments, the CAR is a CD19 CAR having the amino acid sequence shown in SEQ ID NO:76. In some embodiments, the anti-CD19 CAR is encoded by a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:76 or an amino acid sequence having at least 85%, 90% or 95% sequence identity with SEQ ID NO:76. In some embodiments, the anti-CD19 CAR is encoded by a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:76.

[0297] In some embodiments, the CAR is an anti-CD19 CAR having the amino acid sequence shown in SEQ ID NO:77 or an amino acid sequence exhibiting at least 85%, 90%, or 95% sequence identity to SEQ ID NO:77. In some embodiments, the CAR is a CD19 CAR having the amino acid sequence shown in SEQ ID NO:77. In some embodiments, the anti-CD19 CAR is encoded by a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:77 or an amino acid sequence exhibiting at least 85%, 90%, or 95% sequence identity to SEQ ID NO:77. In some embodiments, the anti-CD19 CAR is encoded by a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:77.

[0298] In some embodiments, the CAR is an anti-CD19 CAR having the amino acid sequence shown in SEQ ID NO:78 or an amino acid sequence exhibiting at least 85%, 90%, or 95% sequence identity to SEQ ID NO:78. In some embodiments, the CAR is a CD19 CAR having the amino acid sequence shown in SEQ ID NO:78. In some embodiments, the anti-CD19 CAR is encoded by a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:78 or an amino acid sequence exhibiting at least 85%, 90%, or 95% sequence identity to SEQ ID NO:78. In some embodiments, the anti-CD19 CAR is encoded by a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:78.

[0299] In some embodiments, the CAR is an anti-CD19 CAR having the amino acid sequence shown in SEQ ID NO:79 or an amino acid sequence exhibiting at least 85%, 90%, or 95% sequence identity to SEQ ID NO:79. In some embodiments, the CAR is a CD19 CAR having the amino acid sequence shown in SEQ ID NO:79. In some embodiments, the anti-CD19 CAR is encoded by a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:79 or an amino acid sequence exhibiting at least 85%, 90%, or 95% sequence identity to SEQ ID NO:79. In some embodiments, the anti-CD19 CAR is encoded by a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:79.

[0300] CD20 has been demonstrated to be a therapeutic target for hematological malignancies such as B-NHL and is supported by approved and widely used monoclonal antibody therapies. In addition, the ubiquitous presence of CD19, CD20, and CD22 antigens on malignant B cells makes them perfect targets for cell therapies. In some embodiments, the CAR contains an extracellular antigen-binding domain that binds to CD20. In a specific embodiment, the CD20 CAR comprises a CAR directed against CD20, wherein the CAR directed against CD20 comprises a single-chain Fv antibody or antibody fragment (scFv). In some embodiments, the anti-CD20 CAR may comprise an anti-CD20 single-chain variable fragment (scFv) specific for CD20, followed by a spacer and transmembrane domain fused to an intracellular co-signaling domain (e.g., CD28 or 4-1BB) and the CD3ζ signaling domain. In some embodiments, the CAR contains an anti-CD20 scFv, followed by an IgG4-Fc spacer, a CD28 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3ζ signaling domain. In some embodiments, the CAR is the Leu16 CAR, as described by Rufener et al. Cancer Immunol. Res. 2016 4:509-519. See also GenBank accession number #KX055828.

[0301] In some embodiments, the extracellular binding domain of the CD20 CAR may comprise the heavy chain variable region (V H ) shown in SEQ ID NO:36 and the light chain variable region (V L)。In some embodiments, the linker separating VH and VL in the scFv is a GS linker, such as that shown in SEQ ID NO:56. In some embodiments, the linker separating VH and VL in the scFv is the Whitlow linker shown in SEQ ID NO:55. In some embodiments, the anti-CD20 scFv is shown in SEQ ID NO:37. In some embodiments, the intracellular signaling domain contains a 4-1BB co-stimulatory signaling domain and a CD3ζ signaling domain, such as any of those described herein. In some embodiments, the intracellular signaling domain contains a CD28 co-stimulatory signaling domain and a CD3ζ signaling domain, such as any of those described herein. In some embodiments, it should be understood that the CAR includes any of the following sequences: those that exhibit some sequence variation to any of the above or described SEQ ID NOs, such as having at least 85%, 90%, 95% or higher sequence identity thereto, and retaining binding to CD20 as well as intracellular signaling and cytotoxic activity. In some embodiments, the anti-CD20 CAR contains the scFv shown in SEQ ID NO:37 and an IgG4 Fc spacer (e.g., SEQ ID NO:38), a CD28 transmembrane domain (e.g., SEQ ID NO:39), a CD28 co-stimulatory signaling domain (e.g., SEQ ID NO:40) and a CD3ζ signaling domain (e.g., SEQ ID NO:41). In some embodiments, the CD20 CAR has the amino acid sequence shown in SEQ ID NO:42 or a sequence that exhibits at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO:42. In some embodiments, the CD20 CAR has the sequence shown in SEQ ID NO:42. In some embodiments, the CAR is encoded by the polynucleotide (e.g., mRNA) shown in SEQ ID NO:45.

[0302] In some embodiments, the anti-CD20 CAR comprises the scFv shown in SEQ ID NO:37, a CD8 hinge spacer (such as SEQ ID NO:71), a CD8 transmembrane domain (such as SEQ ID NO:73), a 4-1BB co-stimulatory signaling domain (such as SEQ ID NO:51), and a CD3ζ signaling domain (such as SEQ ID NO:41). In some embodiments, the anti-CD20 CAR comprises the scFv shown in SEQ ID NO:37, a CD28 hinge spacer (such as SEQ ID NO:72), a CD8 transmembrane domain (such as SEQ ID NO:73), a 4-1BB co-stimulatory signaling domain (such as SEQ ID NO:51), and a CD3ζ signaling domain (such as SEQ ID NO:41). In some embodiments, the anti-CD20 CAR comprises the scFv shown in SEQ ID NO:37, an IgG4 hinge spacer (such as SEQ ID NO:59 or 75), a CD8 transmembrane domain (such as SEQ ID NO:73), a 4-1BB co-stimulatory signaling domain (such as SEQ ID NO:51), and a CD3ζ signaling domain (such as SEQ ID NO:41). In some embodiments, the anti-CD20 CAR comprises the scFv shown in SEQ ID NO:37, a CD8 hinge spacer (such as SEQ ID NO:71), a CD28 transmembrane domain (such as SEQ ID NO:39), a 4-1BB co-stimulatory signaling domain (such as SEQ ID NO:51), and a CD3ζ signaling domain (such as SEQ ID NO:41). In some embodiments, the anti-CD20 CAR comprises the scFv shown in SEQ ID NO:37, a CD28 hinge spacer (such as SEQ ID NO:72), a CD28 transmembrane domain (such as SEQ ID NO:39), a 4-1BB co-stimulatory signaling domain (such as SEQ ID NO:51), and a CD3ζ signaling domain (such as SEQ ID NO:41). In some embodiments, the anti-CD20 CAR comprises the scFv shown in SEQ ID NO:37, an IgG4 hinge spacer (such as SEQ ID NO:59 or 75), a CD28 transmembrane domain (such as SEQ ID NO:39), a 4-1BB co-stimulatory signaling domain (such as SEQ ID NO:51), and a CD3ζ signaling domain (such as SEQ ID NO:41). In some embodiments, it should be understood that the CAR includes any of the following sequences: those that exhibit some sequence variation to any of the above or described SEQ ID NOs, such as having at least 85%, 90%, 95% or more sequence identity thereto.

[0303] In some embodiments, the extracellular binding domain of the CD20 CAR can comprise the heavy chain variable region (V H ) shown in SEQ ID NO:81 and the light chain variable region (V L ) shown in SEQ ID NO:80. In some embodiments, the linker separating VH and VL in the scFv is a GS linker, such as that shown in SEQ ID NO:56. In some embodiments, the linker separating VH and VL in the scFv is a Whitlow linker, such as that shown in SEQ ID NO:55. In some embodiments, the anti-CD20 scFv is as shown in SEQ ID NO:82. In some embodiments, as described herein, for example, the intracellular signaling domain contains a 4-1BB co-stimulatory signaling domain and a CD3ζ signaling domain. In some embodiments, as described herein, for example, the intracellular signaling domain contains a CD28 co-stimulatory signaling domain and a CD3ζ signaling domain. In some embodiments, it should be understood that any of the following sequences of the CAR: which exhibits some sequence variation with respect to any of the above or described SEQ ID NOs, such as having at least 85%, 90%, 95% or more sequence identity therewith, and retains binding to CD20 as well as intracellular signal transduction and cytotoxic activity.

[0304] In certain embodiments, the anti-CD20 CAR comprises the scFv shown in SEQ ID NO:82, an IgG4 Fc spacer (such as SEQ ID NO:38), a CD28 transmembrane domain (such as SEQ ID NO:39), a CD28 co-stimulatory signaling domain (such as SEQ ID NO:40), and a CD3ζ signaling domain (such as SEQ ID NO:41). In some embodiments, the anti-CD20 CAR comprises the scFv shown in SEQ ID NO:82, a CD8 hinge spacer (such as SEQ ID NO:71), a CD8 transmembrane domain (such as SEQ ID NO:73), a 4-1BB co-stimulatory signaling domain (such as SEQ ID NO:51), and a CD3ζ signaling domain (such as SEQ ID NO:41). In some embodiments, the anti-CD20 CAR comprises the scFv shown in SEQ ID NO:82, a CD28 hinge spacer (such as SEQ ID NO:72), a CD8 transmembrane domain (such as SEQ ID NO:73), a 4-1BB co-stimulatory signaling domain (such as SEQ ID NO:51), and a CD3ζ signaling domain (such as SEQ ID NO:41). In some embodiments, the anti-CD20 CAR comprises the scFv shown in SEQ ID NO:82, an IgG4 hinge spacer (such as SEQ ID NO:59 or 75), a CD8 transmembrane domain (such as SEQ ID NO:73), a 4-1BB co-stimulatory signaling domain (such as SEQ ID NO:51), and a CD3ζ signaling domain (such as SEQ ID NO:41). In some embodiments, the anti-CD20 CAR comprises the scFv shown in SEQ ID NO:82, a CD8 hinge spacer (such as SEQ ID NO:71), a CD28 transmembrane domain (such as SEQ ID NO:39), a 4-1BB co-stimulatory signaling domain (such as SEQ ID NO:51), and a CD3ζ signaling domain (such as SEQ ID NO:41). In some embodiments, the anti-CD20 CAR comprises the scFv shown in SEQ ID NO:82, a CD28 hinge spacer (such as SEQ ID NO:72), a CD28 transmembrane domain (such as SEQ ID NO:39), a 4-1BB co-stimulatory signaling domain (such as SEQ ID NO:51), and a CD3ζ signaling domain (such as SEQ ID NO:41).In some embodiments, the anti-CD20 CAR comprises the scFv shown in SEQ ID NO:82, an IgG4 hinge spacer (such as SEQ ID NO:59 or 75), a CD28 transmembrane domain (such as SEQ ID NO:39), a 4-1BB co-stimulatory signaling domain (such as SEQ ID NO:51), and a CD3ζ signaling domain (such as SEQ ID NO:41). In some embodiments, it should be understood that any of the following sequences of the CAR: which exhibits some sequence variation with respect to any of the above or described SEQ ID NOs, such as having at least 85%, 90%, 95% or more sequence identity therewith.

[0305] In some embodiments, the CAR comprises an extracellular antigen-binding domain that binds to CD22. In one specific embodiment, the CD22 CAR includes a CAR directed to CD22, wherein the CAR directed to CD22 comprises a single-chain Fv antibody or antibody fragment (scFv). In some embodiments, the extracellular antigen-binding domain of the CD22 CAR is derived from an antibody specific for CD22, such as m971, SM03, inotuzumab, epratuzumab, moxetumomab, and pinatuzumab. In any of these embodiments, the extracellular binding domain of the CD22 CAR may comprise the V H , V L and / or one or more CDRs or consist thereof. In some embodiments, the extracellular binding domain of the CD22 CAR comprises the heavy chain variable region (V H ) shown in SEQ ID NO:84 and the light chain variable region (V L ) shown in SEQ ID NO:85. In some embodiments, the linker separating VH and VL in the scFv is a GS linker, such as that shown in SEQ ID NO:56. In some embodiments, the linker separating VH and VL in the scFv is a Whitlow linker, such as that shown in SEQ ID NO:55. In some embodiments, the anti-CD22 scFv is shown in SEQ ID NO:86. In some embodiments, the extracellular binding domain of the CD22 CAR comprises the heavy chain variable region (V H ) shown in SEQ ID NO:87 and the light chain variable region (V L)。In some embodiments, the linker separating VH and VL in the scFv is a GS linker, such as that shown in SEQ ID NO: 56. In some embodiments, the linker separating VH and VL in the scFv is a Whitlow linker, such as that shown in SEQ ID NO: 55. In some embodiments, the anti-CD22 scFv is shown in SEQ ID NO: 89.

[0306] In some embodiments, the anti-CD22 CAR comprises the scFv shown in SEQ ID NO:86, an IgG4 Fc spacer (such as SEQ ID NO:38), a CD28 transmembrane domain (such as SEQ ID NO:39), a CD28 co-stimulatory signaling domain (such as SEQ ID NO:40), and a CD3ζ signaling domain (such as SEQ ID NO:41). In some embodiments, the anti-CD22 CAR comprises the scFv shown in SEQ ID NO:86, a CD8 hinge spacer (such as SEQ ID NO:71), a CD8 transmembrane domain (such as SEQ ID NO:73), a 4-1BB co-stimulatory signaling domain (such as SEQ ID NO:51), and a CD3ζ signaling domain (such as SEQ ID NO:41). In some embodiments, the anti-CD22 CAR comprises the scFv shown in SEQ ID NO:86, a CD28 hinge spacer (such as SEQ ID NO:72), a CD8 transmembrane domain (such as SEQ ID NO:73), a 4-1BB co-stimulatory signaling domain (such as SEQ ID NO:51), and a CD3ζ signaling domain (such as SEQ ID NO:41). In some embodiments, the anti-CD22 CAR comprises the scFv shown in SEQ ID NO:86, an IgG4 hinge spacer (such as SEQ ID NO:59 or 75), a CD8 transmembrane domain (such as SEQ ID NO:73), a 4-1BB co-stimulatory signaling domain (such as SEQ ID NO:51), and a CD3ζ signaling domain (such as SEQ ID NO:41). In some embodiments, the anti-CD22 CAR comprises the scFv shown in SEQ ID NO:86, a CD8 hinge spacer (such as SEQ ID NO:71), a CD28 transmembrane domain (such as SEQ ID NO:39), a 4-1BB co-stimulatory signaling domain (such as SEQ ID NO:51), and a CD3ζ signaling domain (such as SEQ ID NO:41). In some embodiments, the anti-CD22 CAR comprises the scFv shown in SEQ ID NO:86, a CD28 hinge spacer (such as SEQ ID NO:72), a CD28 transmembrane domain (such as SEQ ID NO:39), a 4-1BB co-stimulatory signaling domain (such as SEQ ID NO:51), and a CD3ζ signaling domain (such as SEQ ID NO:41).In some embodiments, the anti-CD22 CAR comprises the scFv shown in SEQ ID NO:86, an IgG4 hinge spacer (such as SEQ ID NO:59 or 75), a CD28 transmembrane domain (such as SEQ ID NO:39), a 4-1BB co-stimulatory signaling domain (such as SEQ ID NO:51), and a CD3ζ signaling domain (such as SEQ ID NO:41). In some embodiments, it should be understood that any of the following sequences of the CAR: which exhibits some sequence variation with respect to any of the above or described SEQ ID NOs, such as having at least 85%, 90%, 95% or more sequence identity therewith.

[0307] In some embodiments, the anti-CD22 CAR comprises the scFv shown in SEQ ID NO:89, an IgG4 Fc spacer (such as SEQ ID NO:38), a CD28 transmembrane domain (such as SEQ ID NO:39), a CD28 co-stimulatory signaling domain (such as SEQ ID NO:40), and a CD3ζ signaling domain (such as SEQ ID NO:41). In some embodiments, the anti-CD22 CAR comprises the scFv shown in SEQ ID NO:89, a CD8 hinge spacer (such as SEQ ID NO:71), a CD8 transmembrane domain (such as SEQ ID NO:73), a 4-1BB co-stimulatory signaling domain (such as SEQ ID NO:51), and a CD3ζ signaling domain (such as SEQ ID NO:41). In some embodiments, the anti-CD22 CAR comprises the scFv shown in SEQ ID NO:89, a CD28 hinge spacer (such as SEQ ID NO:72), a CD8 transmembrane domain (such as SEQ ID NO:73), a 4-1BB co-stimulatory signaling domain (such as SEQ ID NO:51), and a CD3ζ signaling domain (such as SEQ ID NO:41). In some embodiments, the anti-CD22 CAR comprises the scFv shown in SEQ ID NO:89, an IgG4 hinge spacer (such as SEQ ID NO:59 or 75), a CD8 transmembrane domain (such as SEQ ID NO:73), a 4-1BB co-stimulatory signaling domain (such as SEQ ID NO:51), and a CD3ζ signaling domain (such as SEQ ID NO:41). In some embodiments, the anti-CD22 CAR comprises the scFv shown in SEQ ID NO:89, a CD8 hinge spacer (such as SEQ ID NO:71), a CD28 transmembrane domain (such as SEQ ID NO:39), a 4-1BB co-stimulatory signaling domain (such as SEQ ID NO:51), and a CD3ζ signaling domain (such as SEQ ID NO:41). In some embodiments, the anti-CD22 CAR comprises the scFv shown in SEQ ID NO:89, a CD28 hinge spacer (such as SEQ ID NO:72), a CD28 transmembrane domain (such as SEQ ID NO:39), a 4-1BB co-stimulatory signaling domain (such as SEQ ID NO:51), and a CD3ζ signaling domain (such as SEQ ID NO:41).In some embodiments, the anti-CD22 CAR comprises the scFv shown in SEQ ID NO:89, an IgG4 hinge spacer (such as SEQ ID NO:59 or 75), a CD28 transmembrane domain (such as SEQ ID NO:39), a 4-1BB co-stimulatory signaling domain (such as SEQ ID NO:51), and a CD3ζ signaling domain (such as SEQ ID NO:41). In some embodiments, it should be understood that any of the following sequences of the CAR: which exhibits some sequence variation with respect to any of the above or described SEQ ID NOs, such as having at least 85%, 90%, 95% or more sequence identity therewith.

[0308] In some embodiments, the anti-CD22 CAR may comprise an anti-CD22 single-chain variable fragment (scFv) specific for CD22, a subsequent spacer and transmembrane domain, which transmembrane domain is fused to an intracellular co-signaling domain (such as CD28 or 4-1BB) and a CD3ζ signaling domain. In some embodiments, the CAR comprises an anti-CD22 scFv, a subsequent IgG4-Fc spacer, a CD28 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3ζ signaling domain.

[0309] In some embodiments, g-NK cells are engineered with a chimeric antigen receptor (CAR) that binds to BCMA. BCMA RNA has been ubiquitously detected in multiple myeloma cells and other lymphomas, and several investigators have detected BCMA protein on the surface of plasma cells from multiple myeloma patients (see, e.g., Novak et al., Blood, 103(2):689-694, 2004; Neri et al., Clinical Cancer Research, 73(19):5903-5909, 2007; Bellucci et al., Blood, 105(10):3945-3950, 2005; and Moreaux et al., Blood, 703(8):3148-3157, 2004). CARs for targeting BCMA are known and include, but are not limited to, those described in U.S. Patent No. 10,934,363 or WO 2018 / 028647. In some embodiments, the CAR contains an extracellular antigen-binding domain that binds to BCMA. In a specific embodiment, the BCMA CAR comprises a CAR against BCMA, wherein the CAR against BCMA comprises a single-chain Fv antibody or antibody fragment (scFv). In some embodiments, the anti-BCMA CAR may comprise an anti-BCMA single-chain variable fragment (scFv) that is specific for BCMA, followed by a spacer and a transmembrane domain fused to an intracellular co-signaling domain (e.g., CD28 or 4-1BB) and a CD3ζ signaling domain.

[0310] In some embodiments, the extracellular binding domain of the BCMA CAR comprises an scFv derived from C11D5.3, a murine monoclonal antibody, as described in Carpenter et al., Clin. Cancer Res. 19(8):2048-2060 (2013). See also PCT Application Publication No. WO2010 / 104949. The scFv derived from C11D5.3 may comprise the heavy-chain variable region (V H ) and the light-chain variable region (V L)。In some embodiments, VH has the amino acid sequence shown in SEQ ID NO:63, and VL has the amino acid sequence shown in SEQ ID NO:62. In some embodiments, the linker separating VH and VL in the scFv is a GS linker, such as that shown in SEQ ID NO:56. In some embodiments, the linker separating VH and VL in the scFv is the Whitlow linker shown in SEQ ID NO:55. In some embodiments, the scFv has the amino acid sequence shown in SEQ ID NO:65. In some embodiments, the intracellular signaling domain contains a 4-1BB co-stimulatory signaling domain and a CD3ζ signaling domain, such as any of those described herein. In some embodiments, the intracellular signaling domain contains a CD28 co-stimulatory signaling domain and a CD3ζ signaling domain, such as any of those described herein. In some embodiments, it should be understood that the CAR includes any of the following sequences: those that exhibit some sequence variation with any of the above or described SEQ ID NOs, such as having at least 85%, 90%, 95% or higher sequence identity therewith, and retaining binding to BCMA as well as intracellular signaling and cytotoxic activity.

[0311] In some embodiments, the extracellular binding domain of BCMACAR comprises an scFv derived from another murine monoclonal antibody, C12A3.2, as described in Carpenter et al., Clin. Cancer Res. 19(8):2048-2060 (2013) and PCT application publication number WO2010 / 104949. In some embodiments, VH has the amino acid sequence shown in SEQ ID NO:66, and VL has the amino acid sequence shown in SEQ ID NO:64. In some embodiments, the linker separating VH and VL in the scFv is a GS linker, such as that shown in SEQ ID NO:56. In some embodiments, the linker separating VH and VL in the scFv is the Whitlow linker shown in SEQ ID NO:55. In some embodiments, the scFv has the amino acid sequence shown in SEQ ID NO:67. In some embodiments, the intracellular signaling domain contains a 4-1BB co-stimulatory signaling domain and a CD3ζ signaling domain, such as any of those described herein. In some embodiments, the intracellular signaling domain contains a CD28 co-stimulatory signaling domain and a CD3ζ signaling domain, such as any of those described herein. In some embodiments, it should be understood that the CAR includes any of the following sequences: those that exhibit some sequence variation with any of the above or described SEQ ID NOs, such as having at least 85%, 90%, 95% or higher sequence identity therewith, and retaining binding to BCMA as well as intracellular signaling and cytotoxic activity.

[0312] In some embodiments, the extracellular binding domain of BCMACAR comprises a murine monoclonal antibody that is highly specific for human BCMA and is designated BB2121 in Friedman et al., Hum. Gene Ther. 29(5):585-601 (2018). See also PCT application publication no. WO2012163805. BB2121 is also designated anti-BCMA02 CAR. In some embodiments, VH has the amino acid sequence shown in SEQ ID NO:68, and VL has the amino acid sequence shown in SEQ ID NO:69. In some embodiments, the linker separating VH and VL in the scFv is a GS linker, such as that shown in SEQ ID NO:56. In some embodiments, the linker separating VH and VL in the scFv is the Whitlow linker shown in SEQ ID NO:55. In some embodiments, the scFv has the amino acid sequence shown in SEQ ID NO:70. In some embodiments, the intracellular signaling domain contains a 4-1BB co-stimulatory signaling domain and a CD3ζ signaling domain, such as any of those described herein. In some embodiments, the intracellular signaling domain contains a CD28 co-stimulatory signaling domain and a CD3ζ signaling domain, such as any of those described herein. In some embodiments, it should be understood that the CAR includes any of the following sequences: those that exhibit some sequence variation from any of the above or described SEQ ID NOs, such as having at least 85%, 90%, 95% or higher sequence identity therewith, and retaining binding to BCMA as well as intracellular signaling and cytotoxic activity.

[0313] In some embodiments, the extracellular binding domain of BCMACAR comprises a single variable fragment of two heavy chains (VHH) that can bind to two epitopes of BCMA, as described in Zhao et al., J. Hematol. Oncol., 11(1):141 (2018), and is also designated LCAR-B38M. See also PCT application publication no. WO2018 / 028647. In some embodiments, the intracellular signaling domain contains a 4-1BB co-stimulatory signaling domain and a CD3ζ signaling domain, such as any of those described herein. In some embodiments, the intracellular signaling domain contains a CD28 co-stimulatory signaling domain and a CD3ζ signaling domain, such as any of those described herein. In some embodiments, it should be understood that the CAR includes any of the following sequences: those that exhibit some sequence variation from any of the above or described SEQ ID NOs, such as having at least 85%, 90%, 95% or higher sequence identity therewith, and retaining binding to BCMA as well as intracellular signaling and cytotoxic activity.

[0314] In some embodiments, the extracellular binding domain of BCMACAR comprises a fully human heavy chain variable domain (FHVH), as described in Lam et al., Nat. Commun. 11(1):283 (2020), which is also referred to as FHVH33. In some embodiments, the intracellular signaling domain contains a 4-1BB co-stimulatory signaling domain and a CD3ζ signaling domain, such as any of those described herein. In some embodiments, the intracellular signaling domain contains a CD28 co-stimulatory signaling domain and a CD3ζ signaling domain, such as any of those described herein. In some embodiments, it should be understood that the CAR includes any of the following sequences: those that exhibit some sequence variation from any of the above or described SEQ ID NOs, such as having at least 85%, 90%, 95% or higher sequence identity therewith, and retaining binding to BCMA as well as intracellular signaling and cytotoxic activity.

[0315] In some embodiments, the CAR is an anti-BCMACAR having the amino acid sequence shown in SEQ ID NO:83 or an amino acid sequence exhibiting at least 85%, 90% or 95% sequence identity with SEQ ID NO:83. In some embodiments, the CAR is a BCMACAR having the amino acid sequence shown in SEQ ID NO:83. In some embodiments, the anti-BCMACAR is encoded by a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:83 or an amino acid sequence exhibiting at least 85%, 90% or 95% sequence identity with SEQ ID NO:83. In some embodiments, the anti-BCMACAR is encoded by a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:83.

[0316] In some embodiments, the CAR includes an anti-BCMACAR of a commercial CAR cell therapy. Non-limiting examples of anti-BCMACARs in commercially available cell-based therapies include anti-BCMACARs engineered in cells: idecabtagene vicleucel or ciltacabtagene autoleucel (CARVYKTI TM ).

[0317] In some embodiments, the antigen is GPRC5D. In some embodiments, the scFv contains V H and V L, which is derived from an antibody or antibody fragment specific for GPRC5D. In some embodiments, the antibody or antibody fragment that binds GPRC5D is or comprises the V of the antibody or antibody fragment shown in International Patent Application Publication Nos. WO 2016 / 090329, WO 2016 / 090312, and WO 2020 / 092854 H and V L , each of which is incorporated herein by reference in its entirety.

[0318] In some embodiments, the antigen is FcRL5. In some embodiments, the scFv comprises V H and V L , which is derived from an antibody or antibody fragment specific for FcRL5. In some embodiments, the antibody or antibody fragment that binds FcRL5 is or comprises the V of the antibody or antibody fragment shown in International Patent Application Publication Nos. WO 2016 / 090337 and WO 2017 / 096120 H and V L , each of which is incorporated herein by reference in its entirety.

[0319] CD38 (Cluster of Differentiation 38), also known as cyclic ADP-ribose hydrolase, is a glycoprotein found on the surface of many immune cells (white blood cells), particularly T cells, including CD4+, CD8+, B lymphocytes, and natural killer cells. CD38 also plays a role in cell adhesion, signal transduction, and calcium signaling. Structural information on this protein can be found in the UniProtKB / Swiss-Prot database under the reference number P28907. In the human body, the CD38 protein is encoded by the CD38 gene located on chromosome 4. CD38 is a multifunctional extracellular enzyme that catalyzes the synthesis and hydrolysis of cyclic ADP-ribose (cADPR) from NAD+ to ADP-ribose. These reaction products are thought to be essential for intracellular Ca2+ regulation. Moreover, loss of CD38 function has been associated with impaired immune responses and metabolic disorders (Malavasi F., et al. (2008). “Evolution and function of the ADP ribosyl cyclase / CD38 gene family in physiology and pathology”. Physiol. Rev. 88(3):841-86). The CD38 protein is a marker for HIV infection, leukemia, myeloma, solid tumors, type II diabetes, and bone metabolism. CD38 expression is an important prognostic factor in B-cell chronic lymphocytic leukemia. Blood 98:181-186). In some embodiments, the anti-CD38 CAR may include an anti-CD38 single-chain variable fragment (scFv) specific for CD38, followed by a spacer and transmembrane domain fused to an intracellular co-signaling domain (e.g., CD28 or 4-1BB) and the CD3ζ signaling domain.

[0320] In some embodiments, the extracellular binding domain of the CD38 CAR may include the heavy chain variable region (V H ) shown in SEQ ID NO:46 or SEQ ID NO:47 and the light chain variable region (V L)。In some embodiments, the linker separating VH and VL in the scFv is a GS linker, such as that shown in SEQ ID NO:56. In some embodiments, the linker separating VH and VL in the scFv is the Whitlow linker shown in SEQ ID NO:55. In some embodiments, the intracellular signaling domain contains a 4-1BB costimulatory signaling domain and a CD3ζ signaling domain, such as any of those described herein. In some embodiments, the intracellular signaling domain contains a CD28 costimulatory signaling domain and a CD3ζ signaling domain, such as any of those described herein. In some embodiments, it should be understood that the CAR includes any of the following sequences: those that exhibit some sequence variation with any of the above or described SEQ ID NOs, such as having at least 85%, 90%, 95% or higher sequence identity therewith, and retaining binding to CD38 as well as intracellular signaling and cytotoxic activity. B. Immunomodulators (such as cytokines)

[0321] In the provided embodiments, engineered g-NK cells or populations of g-NK cells are engineered to express a heterologous immunomodulator, such as an exogenous cytokine, for example, an interleukin. In some embodiments, the heterologous nucleic acid encoding the immunomodulator is stably integrated into the genome of the g-NK cells. In other embodiments, the heterologous nucleic acid encoding the immunomodulator is transiently expressed. In some embodiments, the immunomodulator is an immunosuppressant. In other embodiments, the immunomodulator is an immunostimulant. In some embodiments, the immunostimulant is a cytokine.

[0322] In the provided embodiments, engineered NK cells express a heterologous cytokine or a functional portion thereof. According to the provided embodiments, in some embodiments, the NK cells are engineered to express the cytokine in a secreted form, while in some embodiments, the cytokine is membrane-bound. In some embodiments, the heterologous cytokine or a functional portion thereof can be secreted from the cell. In some embodiments, the heterologous cytokine or a functional portion thereof is expressed as a membrane-bound protein on the cell surface.

[0323] Cytokines are a large class of proteins that play important roles in cell signaling, particularly in the context of the immune system. Cytokines have been shown to function as immunomodulators in autocrine, paracrine, and endocrine signaling. Cytokines can act as immunostimulants, thereby stimulating immune-mediated responses, or as immunosuppressants, thereby dampening immune-mediated responses. Cytokines include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors, but generally do not include hormones or growth factors.

[0324] In some embodiments, the cytokine is an interleukin. Interleukins are a group of cytokines, typically secreted proteins and signaling molecules that mediate a wide range of immune responses. For example, interleukin (IL)-2 plays a role in regulating the activity of white blood cells, while interleukin (IL)-15 plays a major role in the development of inflammatory and protective immune responses against microbial invaders and parasites by regulating the activity of cells of the innate and adaptive immune systems. In some embodiments, one or more activities of NK cells (including the provided g-NK cells) are regulated by IL-2, IL-21, and / or IL-15 or another cytokine described.

[0325] Since cytokines are required for NK cell activity, typical methods involve administering exogenous cytokines as an exogenous cytokine support in combination with NK cell therapy to a subject. However, in some respects, the administration of exogenous cytokines can lead to a risk of systemic toxicity, particularly when certain cytokines are administered at high doses. In the provided embodiments, engineering NK cells with a cytokine-secreting or membrane-bound cytokine provides a local source of the cytokine to the NK cells while avoiding or reducing the risk of systemic toxicity.

[0326] In some embodiments of the provided engineered cells, interleukin or a functional portion thereof is introduced into g-NK cells or a population of g-NK cells. In some embodiments, the interleukin comprises a cytokine produced by immune cells such as lymphocytes, monocytes, or macrophages. In some embodiments, the cytokine is an immune activating cytokine (also referred to as an immune activator) that can be used to induce NK cells to, for example, promote NK cell survival, activation, and / or proliferation. For example, certain cytokines (such as IL-15 or IL-21) can prevent or reduce NK cells from undergoing senescence, such as by enhancing their ability to expand in vitro or in vivo. In some embodiments, the interleukin or a functional portion thereof is a partial or full peptide of one or more of IL-2, IL-4, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-15, IL-18, or IL-21. In some embodiments, the cytokine is IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, Flt3-L, SCF, or IL-7. In some embodiments, the cytokine is IL-2 or a functional portion thereof. In some embodiments, the cytokine is IL-12 or a functional portion thereof. In some embodiments, the cytokine is IL-15 or a functional portion thereof. In some embodiments, the cytokine is IL-21 or a functional portion thereof. In some embodiments, the cytokine can be introduced together with the corresponding receptor of the cytokine. In some embodiments, the step of engineering a heterologous cytokine into the engineered cells allows cytokine signaling, thereby maintaining or improving the cell growth, proliferation, expansion, and / or effector function of NK cells, but reducing the risk of cytokine toxicity. In some embodiments, the introduced cytokine or in some cases also its corresponding cytokine receptor is expressed on the cell surface. In some embodiments, the cytokine signaling is constitutively activated. In some embodiments, the activation of cytokine signaling is inducible. In some embodiments, the activation of cytokine signaling is transient or temporary.

[0327] Exemplary secreted and membrane-bound (mb) cytokines are known, as described, for example, in the following: patent publication numbers US2017 / 0073638; US2020 / 0199532, US2021 / 0024959; and PCT patent publication numbers WO2015174928, WO 2019 / 126748, WO 2019 / 191495, WO2020056045, WO2021021907, WO 2021 / 011919, WO 2021 / 062281, any of which can be used in the provided engineered cells.

[0328] In some embodiments, the cytokine is IL-15 or a functional portion thereof. IL-15 is a cytokine that regulates NK cell activation and proliferation. In some cases, IL-15 and IL-12 share similar biological activities. For example, IL-15 and IL-2 bind to common receptor subunits and can compete for the same receptor. In some embodiments, IL-15 induces the activation of JAK kinases and the phosphorylation and activation of the transcriptional activators STAT3, STAT5, and STAT6. In some embodiments, IL-15 promotes or regulates one or more functional activities of NK cells, such as promoting NK cell survival, regulating the activation and proliferation of NK cells and T cells, and supporting the development of NK cells from hematopoietic stem cells. In some embodiments, the functional portion is a part of IL-15 that retains one or more functions of the full-length or mature IL-15 (such as promoting NK cell survival, regulating the activation and proliferation of NK cells and T cells, and supporting the development of NK cells from hematopoietic stem cells) (e.g., a truncated contiguous amino acid sequence containing full-length IL-15). The whole or functional portion of IL-15 can be expressed as a membrane-bound polypeptide and / or a secreted polypeptide.

[0329] As will be appreciated by those skilled in the art, the sequences of various IL-15 molecules are known in the art. In one aspect, IL-15 is wild-type IL-15. In some aspects, IL-15 is mammalian IL-15 (e.g., Homo sapiens interleukin 15 (IL15), transcript variant 3, mRNA, NCBI reference sequence: NM_000585.4; Canis lupus familiaris interleukin 15 (IL15), mRNA, NCBI reference sequence: NM_001197188.1; Felis catus interleukin 15 (IL15), mRNA, NCBI reference sequence: NM_001009207.1). Examples of "mammal" or "mammalian" include primates (e.g., humans), canines, felines, rodents, pigs, ruminants, etc. Specific examples include humans, dogs, cats, horses, cows, sheep, goats, rabbits, guinea pigs, rats, and mice. In a specific aspect, mammalian IL-15 is human IL-15. The human IL-15 amino acid sequences include, for example, Genbank accession numbers: NR_751915.1, NP_000576.l, AAI00963.1, AAI00964.1, AAI00962.1, CAA71044.1, AAH18149.1, AAB97518.1, CAA63914.1, and CAA63913.1.

[0330] In some embodiments, the engineered NK cells comprise a heterologous nucleotide sequence encoding IL-15. In some embodiments, the IL-15 nucleotide sequence is as set forth in SEQ ID NO:9, or is a sequence having at least or at least about 85%, at least or at least about 90%, at least or at least about 95%, or at least or at least about 98% sequence identity to SEQ ID NO:9. In some embodiments, IL-15 is expressed by the cell in a mature form lacking a signal peptide sequence and in some cases also lacking a propeptide sequence. In some embodiments, IL-15 has the amino acid sequence as set forth in SEQ ID NO:2 or a sequence having at least or at least about 85%, at least or at least about 90%, at least or at least about 95%, or at least or at least about 98% sequence identity to SEQ ID NO:2.

[0331] In some embodiments, the IL-15 molecule is a variant of human IL-5, e.g., having one or more amino acid alterations, e.g., substitutions, to the human IL-15 amino acid sequence. In some embodiments, the IL-15 variant comprises or consists of mutations at positions 45, 51, 52, or 72, e.g., as described in US2016 / 0184399. In some embodiments, the IL-15 variant comprises or consists of a substitution of N, S, or L for one of D, E, A, Y, or P. In some embodiments, the mutations are selected from L45D, L45E, S51D, L52D, N72D, N72E, N72A, N72S, N72Y, or N72P (with respect to the sequence of human IL-15, SEQ ID NO:2).

[0332] In an embodiment, the IL-15 molecule comprises an IL-15 variant, e.g., a human IL-15 polypeptide having one or more amino acid substitutions. In some embodiments, the IL-15 molecule comprises a substitution at position 72, e.g., a substitution of N for D. In one embodiment, the IL-15 molecule is the IL-15 polypeptide of SEQ ID NO:2 (which contains the amino acid substitution N72D), or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto (which has IL-15Ra binding activity).

[0333] In some embodiments, the cytokine is IL-2 or a functional portion thereof. In some embodiments, IL-2 is a member of the cytokine family that also includes IL-4, IL-7, IL-9, IL-15, and IL-21. IL-2 signals through a receptor complex composed of three chains, called alpha, beta, and gamma. All members of this cytokine receptor family share the gamma chain. Similar to IL-15, IL-2 promotes immunoglobulin production by B cells and induces the differentiation and proliferation of NK cells. A major difference between IL-2 and IL-15 is found in the adaptive immune response. For example, IL-2 is required for the adaptive immunity to foreign pathogens as it is the basis for the development of immune memory. On the other hand, IL-15 is required to maintain highly specific T cell responses by supporting the survival of CD8 memory T cells. The whole or a functional portion of IL-2 can be expressed as a membrane-bound polypeptide and / or a secreted polypeptide. As understood by those skilled in the art, the sequences of various IL-2 molecules are known in the art. In one aspect, IL-2 is wild-type IL-2. In some aspects, IL-2 is mammalian IL-2. In some embodiments, IL-2 is human IL-2..

[0334] In some embodiments, the engineered NK cells comprise a heterologous nucleotide sequence encoding IL-2. In some embodiments, IL-2 is expressed by the cells in a mature form that lacks a signal peptide sequence and in some cases also lacks a propeptide sequence. In some embodiments, IL-2 has the amino acid sequence shown in SEQ ID NO:1 or a sequence having at least or at least about 85%, at least or at least about 90%, at least or at least about 95%, or at least or at least about 98% sequence identity to SEQ ID NO:1.

[0335] In some embodiments, the cytokine is IL-21 or a functional portion thereof. IL-21 binds to the IL-21 receptor (IL-21R) and a co-receptor (common γ chain (CD132)). The IL-21 receptor has been identified on NK cells, T cells, and B cells, indicating that IL-21 acts on hematopoietic lineage cells, particularly lymphoid progenitors and lymphocytes. IL-21 has been shown to be an effective regulator of cytotoxic T cells and NK cells. (Parrish-Novak, et al. Nature 408:57-63, 2000; Parrish-Novak, et al., J. Leuk. Bio. 72:856-863, 2022; Collins et al., Immunol. Res. 28:131-140, 2003; Brady, et al. J. Immunol. 172:2048-58, 2004.). In murine studies, IL-21 enhanced the maturation and effector function of NK cells (Kasaian et al., Immunity 16:559-569, 2002).

[0336] As will be appreciated by those skilled in the art, the sequences of a variety of IL-21 molecules are known in the art. In one aspect, IL-21 is wild-type IL-21. In some aspects, IL-21 is mammalian IL-21. In one embodiment, the IL-21 sequence is a human IL-21 sequence. Human IL-21 amino acid sequences include, for example, Genbank accession numbers: AAU88182.1, EAX05226.1, CAI94500.1, CAJ47524.1, CAL81203.1, CAN87399.1, CAS03522.1, CAV33288.1, CBE74752.1, CBI70418.1, CBI85469.1, CBI85472.1, CBL93962.1, CCA63962.1, AAG29348.1, AAH66258.1, AAH66259.1, AAH66260.1, AAH66261.1, AAH66262.1, AAH69124.1, and ABG36529.1.

[0337] In some embodiments, the engineered NK cells comprise a heterologous nucleotide sequence encoding IL-21. In some embodiments, IL-21 is expressed by the cell in a mature form lacking a signal peptide sequence and in some cases also lacking a propeptide sequence. In some embodiments, IL-21 has the amino acid sequence shown in SEQ ID NO:3 or a sequence having at least or at least about 85%, at least or at least about 90%, at least or at least about 95%, or at least or at least about 98% sequence identity to SEQ ID NO:3. In some embodiments, IL-21 has the amino acid sequence shown in SEQ ID NO:4 or a sequence having at least or at least about 85%, at least or at least about 90%, at least or at least about 95%, or at least or at least about 98% sequence identity to SEQ ID NO:4.

[0338] The cytokine (e.g., IL-2, IL-15 or IL-21) amino acid sequence can include any functional portion of the mature cytokine, e.g., any functional portion of mature IL-2, mature IL-15 or mature IL-21. A functional portion can be any portion that contains contiguous amino acids of the interleukin to which it belongs, provided that the functional portion specifically binds to the corresponding interleukin receptor. When used with respect to an interleukin, the term "functional portion" refers to any portion or fragment of the interleukin that retains the biological activity of the interleukin to which it belongs (the parental interleukin). Functional portions encompass, for example, interleukin portions that retain the ability to specifically bind to the corresponding interleukin receptor, activate downstream targets of the interleukin, and / or induce one or more of the differentiation, proliferation (or death), and activity of immune cells (e.g., NK cells), to an extent similar to, the same as, or higher than that of the parental interleukin. The biological activity of a functional portion of an interleukin can be measured using assays known in the art. With respect to the parental interleukin, a functional portion can include, for example, about 60%, about 70%, about 80%, about 90%, about 95% or more of the amino acid sequence of the parental mature interleukin.

[0339] Functional variants of the interleukins described herein are included within the scope of the cytokines or functional moieties according to the provided embodiments. As used herein, the term "functional variant" refers to an interleukin that has substantial or significant sequence identity or similarity to a parental interleukin and that retains the biological activity of its native interleukin. Functional variants encompass, for example, those variants of the interleukins (parental interleukins) described herein that retain the ability to specifically bind to the corresponding interleukin receptor, activate downstream targets of the interleukin, and / or induce the differentiation, proliferation (or death), and activity of immune cells (such as NK cells), to an extent similar to, the same as, or higher than that of the parental interleukin. With respect to the parental interleukin, the functional variant may, for example, be at least about 80%, about 90%, about 95%, about 99%, or more identical to the parental interleukin in amino acid sequence.

[0340] Functional variants may, for example, include the amino acid sequence of a parental interleukin having at least one conservative amino acid substitution. Alternatively or additionally, functional variants may include the amino acid sequence of a parental interleukin having at least one non-conservative amino acid substitution. In some embodiments, the amino acid substitution (such as a conservative or non-conservative amino acid substitution) does not interfere with or inhibit the biological activity of the functional variant compared to the parental interleukin sequence. In some embodiments, the amino acid substitution (such as a conservative or non-conservative amino acid substitution) may enhance the biological activity of the functional variant such that the biological activity of the functional variant is increased compared to the parental interleukin.

[0341] In some embodiments, the amino acid substitution of the interleukin is a conservative amino acid substitution. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid having certain physical and / or chemical properties is replaced with another amino acid having the same or similar chemical or physical properties. For example, a conservative amino acid substitution may be: replacing one acidic / negatively charged polar amino acid with another acidic / negatively charged polar amino acid (e.g., Asp or Glu), replacing one amino acid having a nonpolar side chain with another amino acid having a nonpolar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), replacing one basic / positively charged polar amino acid with another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), replacing one uncharged amino acid having a polar side chain with another uncharged amino acid having a polar side chain (e.g., Asn, Gln, Ser, Thr, Tyr, etc.), replacing one amino acid having a β-branched side chain with another amino acid having a β-branched side chain (e.g., Ile, Thr, and Val), replacing one amino acid having an aromatic side chain with another amino acid having an aromatic side chain (e.g., His, Phe, Trp, and Tyr), etc.

[0342] In some embodiments, all or a functional portion of a cytokine (e.g., IL-2, IL-15, IL-21, or a functional portion of any of the foregoing) can be expressed as a secreted polypeptide by g-NK cells in a variety of ways. For example, all or a functional portion of the cytokine can be expressed within the NK cell and secreted from the NK cell. In some embodiments, the secreted cytokine does not contain a transmembrane domain.

[0343] In some embodiments, the cytokine can be secreted from engineered g-NK cells. In some embodiments, the secreted cytokine is constitutively expressed. In other embodiments, the secreted cytokine is transiently expressed. In some embodiments, the secreted cytokine is under an inducible promoter. In some embodiments, the secreted cytokine is IL-2 or a functional portion thereof. In some embodiments, the amino acid sequence of IL-2 is or comprises SEQ ID NO:1. In some embodiments, the secreted cytokine is IL-15 or a functional portion thereof. In some embodiments, the amino acid sequence of IL-15 is or comprises SEQ ID NO:2. In some embodiments, the secreted cytokine is IL-21 or a functional portion thereof. In some embodiments, the amino acid sequence of IL-21 is or comprises SEQ ID NO:3. In some embodiments, g-NK cells are engineered with two or more secreted cytokines, such as a combination of two or more of IL-2, IL-15, and IL-21.

[0344] Although interleukins and other cytokines are typically secreted, they can also be membrane-bound. When co-expressed with a CAR fusion protein, it is possible to concentrate the immunocyte-activating cytokine and the CAR fusion protein near the target cell. When co-expressed with a CAR fusion protein in g-NK cells, the g-NK cells show enhanced targeting and killing capabilities and thus represent an attractive and effective therapeutic agent.

[0345] In other embodiments, the cytokine is membrane-bound (mb). In some embodiments, the membrane-bound cytokine is constitutively expressed. In other embodiments, the membrane-bound cytokine is transiently expressed. In some embodiments, the membrane-bound cytokine is under an inducible promoter. In some embodiments, the membrane-bound cytokine is membrane-bound IL-2 (mbIL-2). In some embodiments, the membrane-bound cytokine is membrane-bound IL-15 (mbIL-15). In some embodiments, the membrane-bound cytokine is membrane-bound IL-21 (mbIL-21). In some embodiments, g-NK cells are engineered with two or more membrane-bound cytokines (such as a combination of two or more of mbIL-2, mbIL-15, and mbIL-21). The membrane-bound cytokine can include any form of interleukin cytokine formatted in a membrane-bound form (e.g., IL-2, IL-15, or IL-21), such as any of those described herein.

[0346] In some embodiments, all or a functional portion of the cytokine (e.g., IL-2, IL-15, IL-21, or a functional portion of any of the foregoing) can be expressed as a membrane-bound cytokine by g-NK cells in a variety of ways. In some embodiments, any one of a variety of linkers known in the art can be used to directly or indirectly (e.g., ionically, non-ionically, covalently) link (e.g., conjugate or fuse) the cytokine or its functional portion to the surface of the g-NK cell (e.g., at or within the membrane of the NK cell) (Hermanson, G., Bioconjugate Techniques, Academic Press, 1996). In some aspects, all or a portion of the cytokine is linked to all or a portion of a transmembrane protein. In one aspect, the NK cell expresses a fusion protein that includes all or a portion of the cytokine fused to all or a portion of a transmembrane protein. In some embodiments, the linker can be a peptide linker, such as a flexible linker. In some embodiments, the flexible linker primarily includes glycine and serine residues. For example, the flexible linker can include one or more repeats of either G4S and G3S (e.g., about 3 to about 15 or about 5 to about 12 repeats of G4S and G3S). In some embodiments, the linker is a cleavable linker, such as a furin-cleavable sequence. Exemplary furin cleavage sequences are described in Duckert et al., Protein Engineering, Design & Selection, 17(1):107-112 (2004) and U.S. Patent 8,871,906, each of which is incorporated herein by reference.

[0347] In a specific aspect, the portion of the transmembrane protein includes all or a portion of the transmembrane domain of the transmembrane protein. In some embodiments, the transmembrane protein can be any protein located on and / or within a membrane, which is a phospholipid bilayer such as a biological membrane (e.g., a biological membrane such as a cell membrane). In some embodiments, the transmembrane domain is a domain of a transmembrane protein that is typically present within a membrane, particularly those that form channels and pores. In some embodiments, the transmembrane domain is a three-dimensional protein structure that is thermodynamically stable within a membrane (e.g., the membrane of a vesicle, such as the membrane of a cell). Examples of transmembrane domains include a single α-helix, a stable complex of several transmembrane α-helices, a transmembrane β-barrel, the β-helix of gramicidin A, or any other structure. Transmembrane helices are typically about 20 amino acids in length.

[0348] Examples of transmembrane proteins include receptors, ligands, immunoglobulins, blood group glycoproteins, or combinations thereof. Specific examples of transmembrane proteins include, but are not limited to, CD8α, CD4, CD3ε, CD3γ, CD3δ, CD3ζ, CD28, CD137, FcεRIγ, T cell receptor (TCR, such as TCRα and / or TCRβ), nicotinic acetylcholine receptor, GABA receptor, or combinations thereof. Specific examples of immunoglobulins include IgG, IgA, IgM, IgE, IgD, or combinations thereof. Specific examples of blood group glycoproteins include blood group glycoprotein A, blood group glycoprotein D, or combinations thereof.

[0349] In some embodiments, the transmembrane domain is the CD28 transmembrane domain. An exemplary sequence of the CD28 transmembrane domain together with the CD28 hinge domain is shown in SEQ ID NO:10. IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLL VTVAFIIFWVR(SEQ ID NO:10)

[0350] In some embodiments, the transmembrane domain is the CD8 transmembrane domain. An exemplary sequence of the CD8 transmembrane domain together with the CD8 hinge domain is shown in SEQ ID NO:11. TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC(SEQ ID NO:11)

[0351] In some embodiments, the transmembrane domain is the CD4 transmembrane domain. An exemplary sequence of the CD4 transmembrane domain is shown in SEQ ID NO:15. MALIVLGGVAGLLLFIGLGIFF(SEQ ID NO:15)

[0352] In some embodiments, all or a functional portion of a cytokine (e.g., IL-2, IL-15, IL-21, or a functional portion of any of the foregoing) can be linked to other components, such as a signal peptide, a leader sequence, a secretion signal, a tag (e.g., a reporter gene), or any combination thereof.

[0353] In some embodiments, the nucleic acid sequence encoding all or a functional portion of a cytokine (e.g., IL-2, IL-15, IL-21, or a functional portion of any of the foregoing) is replaced by a nucleic acid sequence encoding a signal peptide from a heterologous protein. The heterologous protein can be, for example, CD8α, CD28, tissue plasminogen activator (tPA), growth hormone, granulocyte-macrophage colony-stimulating factor (GM-CSF), GM-CSF receptor (GM-CSFRa), or an immunoglobulin (e.g., IgE or IgK).

[0354] In some embodiments, all or a functional portion of a cytokine (e.g., IL-2, IL-15, IL-21, or a functional portion of any of the foregoing) is fused to the signal peptide of CD8α. An exemplary CD8α signal peptide is shown in SEQ ID NO:12. In some embodiments, all or a functional portion of a cytokine (e.g., IL-15 or a functional portion thereof, IL-2 or a functional portion thereof, or IL-21 or a functional portion thereof) is fused to the signal peptide of GM-CSFRa (SEQ ID NO:13). An exemplary GM-CSFRa signal peptide is shown in SEQ ID NO:13. An exemplary IgK signal peptide is shown in SEQ ID NO:14. An exemplary IgK signal peptide is shown in SEQ ID NO:43.

[0355] In some embodiments, all or a functional portion of a cytokine (e.g., IL-2, IL-15, IL-21, or a functional portion of any of the foregoing) is fused to the signal peptide of CD8α and all or a portion of the transmembrane domain of CD8α. In some embodiments, the heterologous cytokine is the membrane-bound IL-15 shown in SEQ ID NO:7 or a sequence having at least about 85%, at least about 90%, at least about 95%, or at least about 98% sequence identity to SEQ ID NO:7. In some embodiments, the heterologous cytokine is the membrane-bound IL-15 shown in SEQ ID NO:8 or a sequence having at least about 85%, at least about 90%, at least about 95%, or at least about 98% sequence identity to SEQ ID NO:8.

[0356] In some embodiments, all or a functional portion of a cytokine (e.g., IL-2, IL-15, IL-21, or a functional portion of any of the foregoing) is fused to the Fc region of an immunoglobulin to generate a bivalent cytokine. In some embodiments, the cytokine-Fc fusion protein may be further linked to a transmembrane domain for expression as a membrane-bound cytokine.

[0357] In some embodiments, the heterologous cytokine is the membrane-bound IL-15 shown in SEQ ID NO:5 or a sequence having at least or at least about 85%, at least or at least about 90%, at least or at least about 95%, or at least or at least about 98% sequence identity to SEQ ID NO:5.

[0358] In some embodiments, the heterologous cytokine is the membrane-bound IL-21 shown in SEQ ID NO:6 or a sequence having at least or at least about 85%, at least or at least about 90%, at least or at least about 95%, or at least or at least about 98% sequence identity to SEQ ID NO:6.

[0359] In some embodiments, IL-15 and IL-15 receptor alpha (IL15RA) are engineered together into cells. IL15RA binds IL-15 specifically with very high affinity and is able to bind IL-15 independently of other subunits. In some aspects, this property allows IL-15 to be produced by one cell, endocytosed by another cell, and then presented to a third cell. In some embodiments, g-NK cells express a heterologous (e.g., exogenous) IL-15 / IL-15Ra. In some embodiments, g-NK cells are engineered with an IL-15 / IL-15R fusion protein. In some embodiments, g-NK cells are engineered with a single-chain IL-15 / IL-15R fusion protein. In some embodiments, IL-15 / IL-15Ra is expressed as a membrane-bound IL-15.IL15Ra complex (e.g., Imamura et al., Blood, 2014 124(7):108 and Hurton LV et al., PNAS, 2016). In some embodiments, the exogenous IL-15 / IL-15Ra is secretable and is expressed as a soluble IL15Ra.IL15 complex (e.g., Mortier E et al., JBC, 2006; Bessard A, Mol. Cancer Ther., 2009; and Desbois M, J. Immunol., 2016). In some embodiments, the provided engineered g-NK cells express both a membrane-bound IL15 / IL15Ra complex and a soluble (secretable) IL15Ra / IL15 complex. In some embodiments, the engineered g-NK cells express a membrane-bound form of the IL15.IL15Ra complex with a cleavable linker. C. Polynucleotide

[0360] In some embodiments, provided herein are polynucleotides having a nucleic acid sequence encoding an antigen receptor, such as a chimeric antigen receptor, including any of the chimeric antigen receptors provided herein. In some embodiments, provided herein are polynucleotides having a nucleic acid sequence encoding any of the immunomodulators provided herein, such as a cytokine, including a secretable cytokine or a membrane-bound cytokine.

[0361] In some embodiments, the nucleic acid encoding an antigen receptor, such as a chimeric antigen receptor, and the nucleic acid encoding an immunomodulator, such as a cytokine, including a secretable cytokine or a membrane-bound cytokine, are provided as separate polynucleotides.

[0362] In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding an antigen receptor, such as a chimeric antigen receptor, and a nucleic acid encoding an immunomodulator, such as a cytokine, including a secreted cytokine or a membrane-bound cytokine. Thus, in some aspects, the nucleic acid sequences are provided as part of the same polynucleotide. For example, the provided embodiments include such polynucleotides, wherein the engineered components are encoded by a polynucleotide comprising one or more protease cleavage sites, such as self-cleaving peptides, such as T2A, P2A, E2A, or F2A. Such sites are recognized and cleaved by proteases, which can result in the separation (and separate expression) of various components (e.g., cytokines and CARs) encoded by the polynucleotide engineered into the NK cells. Thus, according to the embodiments, the various components of the engineered components can be delivered to the NK cells in a single vector or by multiple vectors.

[0363] Also provided herein are mediators encoding any of the provided polynucleotides, such as for delivering the polynucleotide to a cell, such as a g-NK cell. In some embodiments, the mediator is a vector, such as a viral vector or a non-viral vector. In some embodiments, the mediator is a viral vector that is a lentiviral vector. In some embodiments, the mediator is a liposome. In some embodiments, the mediator is a lipid nanoparticle. Other mediators (including vector or non-vector delivery mediators) include those known to those of skill in the art, including any of the mediators described below.

[0364] In some embodiments, according to the provided methods, the polynucleotide is engineered into a g-NK cell or a composition comprising a plurality of g-NK cells. Exemplary methods for engineering NK cells are described below. D. Methods of Delivery of Heterologous Agents

[0365] In some embodiments, the engineered g-NK cells provided herein, including the engineered g-NK cells for the provided methods, can be generated by engineering the CAR gene into the g-NK cells. In some embodiments, the method of genetic engineering includes introducing a nucleic acid encoding the CAR into the g-NK cells. In some embodiments, one or more other heterologous protein agents, such as cytokine immunomodulators, can be engineered into the cells, which can be performed simultaneously with or in any order sequentially to engineering the CAR into the g-NK cells. The nucleic acid introduced into the g-NK cells can be introduced for stable integration into the genome or for transient expression. Stable integration or transient expression can be selected based on a variety of factors, including but not limited to the ability of a particular nucleic acid to integrate efficiently into the host genome or the amount and half-life of the nucleic acid.

[0366] In some embodiments, introduction of a heterologous agent (such as a CAR) into g-NK cells can be performed in a method of enriching a g-NK cell subset from a starting sample of NK cells. Thus, it should be understood that the provided methods do not require engineering of g-NK cells specifically selected for FcRγ-chain deficiency (or g-NK cells selected or identified only by a g-NK surrogate marker profile), but can involve engineering of cells of an NK cell composition that will or have preferentially expanded g-NK cells or are enriched in g-NK cells. Thus, the final cell composition enriched in g-NK cells includes g-NK cells into which a heterologous antigen receptor (such as a CAR) and an immunomodulator (such as a cytokine) (such as a secreted or membrane-bound interleukin, such as IL-15 or IL-21) have been introduced. Exemplary methods for preparing and expanding compositions enriched in g-NK cells are provided in Section VI.

[0367] In some embodiments, introduction of a heterologous agent (such as a CAR) can be performed at any suitable time during a method of expanding g-NK cells, such as as described in Section VI. In some embodiments, introduction is performed after selection of cells from a subject (such as selection or enrichment of CD3 阴性 CD57 阳性 or CD3 阴性 CD56 阳性 cells) and before incubation or culture of the selected or enriched cells with feeder cells (such as feeder cells expressing HLA-E) for NK cell proliferation or expansion. In some embodiments, introduction is performed after incubation or culture with feeder cells (such as feeder cells expressing HLA-E) and thus after the selected or enriched cells have proliferated or expanded. In some embodiments, introduction is performed in any order sequentially with the methods for gene editing described herein.

[0368] In some embodiments, the expansion phase of the cells (such as that described in Section VI) is divided into a first expansion and a second expansion. In some embodiments, the selected cells from the biological sample are cultured for a first period of time under expansion conditions, such as for the following period of time or more than the following period of time, before introduction (e.g., viral transduction): about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, or any time between the times listed (including the endpoints). In some embodiments, after the first expansion phase, an engineered construct encoding one or more heterologous agents (such as the chimeric antigen receptor described above) is introduced (e.g., transduced into) the expanded cells (such as NK cells). After introduction (e.g., viral transduction), the engineered cells are cultured for a second period of time, such as for the following period of time or more than the following period of time: about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, or any time between the times listed (including the endpoints).

[0369] The culture medium can be supplemented with feeder cells expressing HLA-E and / or one or more stimulators (such as IL12 and / or IL21) at any time during the culture process. For example, one or more stimulators can be added at the start of the culture, such as at time point 0 (e.g., at the start of the culture). One or more agents can be added a second, third, fourth, fifth, or more times. Subsequent additions can be at the same or different concentrations as the previous addition. The intervals between multiple additions can vary, such as intervals of about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, or longer time intervals, and any time therebetween (including the endpoints). If the stimulator is added multiple times, the concentration of the first supplementary addition can be the same or different from the concentration of the second (and / or any supplementary addition). For example, in several embodiments, the concentrations of the stimulator added at multiple time points can gradually increase, gradually decrease, remain constant, or vary between multiple non-identical concentrations.

[0370] In some embodiments, a nucleic acid encoding a heterologous agent (such as a CAR) is introduced under conditions of transient expression in g-NK cells. In some embodiments, the methods for introducing nucleic acids for transient expression include any method that will produce a nucleic acid that can express its encoded content for a short period of time before degradation.

[0371] In some embodiments, nucleic acids encoding heterologous agents such as CARs are introduced under conditions of stable expression in g-NK cells. In some embodiments, methods for introducing nucleic acids for stable expression in cells involve any such method that results in stable integration of the nucleic acid into the genome of the cell such that if the cell into which the nucleic acid integrates divides, the nucleic acid can proliferate.

[0372] Methods for delivering polynucleotides and compositions containing such polynucleotides are known to those of skill in the art. Those of skill in the art are able to select appropriate methods for transient or stable expression in cells.

[0373] In some embodiments, engineering of NK cells can be accomplished by transducing a cell composition with a polynucleotide encoding a heterologous agent such as a CAR or a vector comprising the polynucleotide. The vector can be a viral vector such as a lentiviral vector, a gamma-retroviral vector, a recombinant AAV, an adenoviral vector, or an oncolytic viral vector. In other aspects, non-viral vectors such as nanoparticles and liposomes can also be used to introduce and deliver a polynucleotide encoding a heterologous agent such as a CAR into NK cells.

[0374] In some embodiments, vectors packaging polynucleotides encoding heterologous agents can be used to deliver the packaged polynucleotides to g-NK cells or a cell composition or population enriched in g-NK cells. These vectors can be of any type, including DNA vectors, RNA vectors, plasmids, viral vectors, and particles. Viral vector technology is well known and is described in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). Viruses that can be used as vectors include, but are not limited to, lentiviral vectors, adenoviral vectors, adeno-associated virus (AAV) vectors, herpes simplex virus vectors, retroviral vectors, oncolytic viruses, and the like.

[0375] Generally, a vector contains at least one origin of replication function in an organism, a promoter sequence, suitable restriction endonuclease sites, and one or more selectable markers such as drug resistance genes.

[0376] A promoter may include any DNA sequence that is required to initiate specific transcription of a polynucleotide sequence and is recognized by the transcriptional machinery of a cell. A vector may include a native or non-native promoter operably linked to a polynucleotide. The promoter selected may be strong, weak, constitutive, inducible, tissue-specific, development stage-specific, and / or organism-specific. An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high-level expression of a polynucleotide sequence operably linked thereto. Another example of a promoter is elongation growth factor-1α (EF-1α). Other constitutive promoters may also be used, including but not limited to simian virus 40 (SV40), mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, which include but are not limited to phosphoglycerate kinase (PGK) promoter, actin promoter, myosin promoter, hemoglobin promoter, ubiquitin C (Ubc) promoter, human U6 small nuclear ribonucleoprotein promoter, and creatine kinase promoter. In some cases, inducible promoters such as but not limited to metallothionein promoter, glucocorticoid promoter, progesterone promoter, and tetracycline promoter may be used.

[0377] Additional promoter elements (e.g., enhancers) may be used to regulate the frequency of transcriptional initiation. Such regions may be located 10 to 100 base pairs upstream or downstream of the start site. In some cases, two or more promoter elements may be used to activate transcription cooperatively or independently.

[0378] In some embodiments, the polynucleotide may be packaged into a viral vector or integrated into a viral genome, allowing for transient or stable expression of the polynucleotide. Viral vectors may include retroviral vectors, which include lentiviral vectors. To construct a retroviral vector, a polynucleotide molecule encoding a heterologous agent is inserted into the viral genome to replace certain viral sequences, resulting in a replication-defective virus. The recombinant viral vector is then introduced into a packaging cell line containing the gag, pol, and env genes but lacking the LTR and packaging components. Recombinant retroviral particles are secreted into the culture medium, then collected, optionally concentrated, and used for gene transfer. Lentiviral vectors are particularly preferred because they are capable of infecting both dividing and non-dividing cells.

[0379] In some embodiments, a polynucleotide encoding a heterologous agent (such as a CAR) is incorporated into a viral vector for delivery by transduction. Viral transduction is the process of deliberately introducing nucleic acids into eukaryotic cells by virus-mediated means.

[0380] In some embodiments, the viral vector is a lentiviral vector. Lentiviral vectors are particularly suitable means for successful viral transduction because they allow for stable expression of the genes contained within the delivered nucleic acid transcript. Lentiviral vectors express reverse transcriptase and integrase, two enzymes required for stable expression of the genes contained within the delivered nucleic acid transcript. Reverse transcriptase converts the RNA transcript into DNA, while integrase inserts and integrates the DNA into the genome of the target cell. Once the DNA has been stably integrated into the genome, it divides with the host. The gene of interest contained within the integrated DNA can be constitutively expressed, or it can be inducible. As part of the host cell genome, it may be subject to cellular regulation, including activation or inhibition, depending on many factors in the target cell.

[0381] Lentiviruses are a subgroup of retroviridae viruses, so named because they require the viral RNA genome to be reverse transcribed into DNA before integration into the host genome. Thus, the most important feature of lentiviral vectors / particles is the integration of their genetic material into the genome of the target / host cell. Some examples of lentiviruses include human immunodeficiency virus: HIV-1 and HIV-2, simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), Jembrana Disease Virus (JDV), equine infectious anemia virus (EIAV), equine infectious anemia virus, visna-maedi virus, and caprine arthritis encephalitis virus (CAEV).

[0382] Typically, the lentiviral particles that make up the gene delivery vector are themselves replication-defective (also known as "self-inactivating"). Due to the mechanism of entry through the intact host nuclear envelope, lentiviruses are able to infect both dividing and non-dividing cells (Naldini L et al., Curr. Opin. Biotechnol, 1998, Vol. 9: pp. 457-463). Recombinant lentiviral vectors / particles have been generated by multiple attenuation of HIV virulence genes, e.g., deletion of the genes Env, Vif, Vpr, Vpu, Nef, and Tat, making the vector biologically safe. Correspondingly, lentiviral vectors, such as those derived from HIV-1 / HIV-2, can mediate efficient delivery, integration, and long-term expression of transgenes into non-dividing cells.

[0383] Lentiviral particles can be generated by co-expressing viral packaging elements and the vector genome itself in producer cells such as human HEK293T cells. These elements are typically provided as three separate plasmids (in second-generation lentiviral systems) or four separate plasmids (in third-generation lentiviral systems). The producer cells are co-transfected with plasmids encoding lentiviral components, which include the core (i.e., structural proteins) and enzymatic components of the virus as well as the envelope protein (referred to as the packaging system), and also with a plasmid encoding the genome (which includes the exogenous transgene to be transferred to the target cells), which is the vector itself (also referred to as the transfer vector). Generally, the plasmids or vectors are included in the producer cell line. The plasmids / vectors are introduced into the producer cell line via transfection, transduction, or infection. Methods of transfection, transduction, or infection are well known to those skilled in the art. As non-limiting examples, the packaging and transfer constructs can be introduced into the producer cell line by calcium phosphate transfection, lipofection, or electroporation, typically together with a dominant selectable marker such as neomycin (neo), dihydrofolate reductase (DHFR), glutamine synthetase, or adenosine deaminase (ADA), and then selection and isolation of clones are carried out in the presence of the appropriate drug.

[0384] The producer cells generate recombinant virus particles containing an exogenous gene, which is, for example, a polynucleotide encoding a heterologous agent. The recombinant virus particles are recovered from the culture medium and titrated by standard methods used by those skilled in the art. The recombinant lentiviral vector can be used to infect target cells such as γ-NK cells or cell compositions or populations enriched in γ-NK cells.

[0385] Cells that can be used to produce high-titer lentiviral particles can include, but are not limited to, HEK293T cells, 293G cells, STAR cells (Relander et al., Mol Ther. 2005, 11:452-459), FreeStyle TM 293 Expression System (ThermoFisher, Waltham, MA), and other HEK293T-based producer cell lines (e.g., Stewart et al., Hum Gene Ther. 2011, 22(3):357-369; Lee et al., Biotechnol Bioeng, 2012, 109(6):1551-1560; Throm et al., Blood. 2009, 113(21):5104-5110).

[0386] In some aspects, the envelope protein can be a heterologous envelope protein from another virus, such as the G protein of Vesicular Stomatitis Virus (VSV G) or the baculovirus gp64 envelope protein. The VSV-G glycoprotein can particularly be selected from species classified in the genus Vesiculovirus: Carajas virus (CJSV), Chandipura virus (CHPV), Cocal virus (COCV), Isfahan virus (ISFV), Maraba virus (MARAV), Piry virus (PIRYV), Vesicular Stomatitis Aiagoas virus (VSAV), Vesicular Stomatitis Indiana virus (VSTV), and Vesicular Stomatitis New Jersey virus (VSNJV), and / or species temporarily classified in the genus Vesiculovirus, such as snakehead rhabdovirus, BeAn 157575 virus (BeAn 157575), Boteke virus (BTKV), Calchaqui virus (CQFV), American eel virus (EVA), Gray Lodge virus (GLOV), Jurona virus (JURY), Klamath virus (KLAV), Kwatta virus (KWAV), La Joya virus (LJV), Malpais Spring virus (MSPV), Mount Elgon bat virus (MEB V), Ferine t virus (PERV), pike fry rhabdovirus (PFRV), Porton virus (PORV), Radi virus (RADIV), spring viraemia of carp virus (SVCV), Tupaia virus (TUPV), ulcerative disease rhabdovirus (UDRV), and Yug Bogdanovac virus (YBV). The gp64 or other baculovirus env protein can be derived from Autographa californica multiple nucleopolyhedrovirus (AcMNPV), Laphygma exigua multiple nucleopolyhedrovirus, Bombyx mori multiple nucleopolyhedrovirus, Choristoneura fumiferana multiple nucleopolyhedrovirus, Orgyia pseudotsugata single nucleocapsid multiple nucleopolyhedrovirus, Epiphyas postvittana multiple nucleopolyhedrovirus, Hyphantria cunea multiple nucleopolyhedrovirus, Galleria mellonella multiple nucleopolyhedrovirus, Dhori virus, Thogoto virus, Antheraea pernyi multiple nucleopolyhedrovirus, or Batken virus.

[0387] The additional elements provided in the lentiviral particles may include retroviral LTRs (long terminal repeats) located at the 5' or 3' ends, retroviral export elements, an optional lentiviral Rev response element (RRE), a promoter or an active portion thereof, and a locus control region (LCR) or an active portion thereof. Other elements include a central polypurine tract (cPPT) sequence that enhances transduction efficiency in non-dividing cells, and a woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE) that enhances the expression of the transgene and increases the titer.

[0388] Methods for generating recombinant lentiviral particles are known to those skilled in the art and are described, for example, in U.S. Patent Nos. 8,846,385, 7,745,179, 7,629,153, 7,575,924, 7,179,903, and 6,808,905. The lentiviral vectors used may be selected from, but are not limited to, pLVX, pLenti, pLenti6, pLJM1, FUGW, pWPXL, pWPI, pLenti CMV puroDEST, pLJM1-EGFP, pULTRA, pInducer2Q, pHIV-EGFP, pCW57.1, pTRPE, pELPS, pRRL, and pLionII. Any known lentiviral vector may also be used (see U.S. Patent Nos. 9,260,725, 9,068,199, 9,023,646, 8,900,858, 8,748,169, 8,709,799, 8,420,104, 8,329,462, 8,076,106, 6,013,516, and 5,994,136; International Patent Publication No.: WO2012079000).

[0389] Other retroviral vectors may also be used to package nucleic acids encoding heterologous agents for delivery to γ-NK cells or cell compositions or populations enriched in γ-NK cells. Retroviral vectors (RVs) allow for the permanent integration of the transgene into the target cells. In addition to complex HIV-1 / 2-based lentiviral vectors, simple γ-retrovirus-based retroviral vectors have also been widely used for delivering therapeutic genes and have been shown clinically to be one of the most effective and powerful gene delivery systems capable of transducing a wide range of cell types. Examples of γ-retroviruses include murine leukemia virus (MLV) and feline leukemia virus (FeLV).

[0390] In some embodiments, γ-retroviral vectors derived from mammalian γ-retroviruses such as murine leukemia virus (MLV) are recombinant. The γ-retroviruses of the MLV family include ecotropic, amphotropic, xenotropic, and polytropic subfamilies. Ecotropic viruses are only able to infect murine cells using the mCAT-1 receptor. Examples of ecotropic viruses are Moloney MLV and AKV. Amphotropic viruses infect murine, human, and other species via the Pit-2 receptor. An example of an amphotropic virus is the 4070A virus. Xenotropic and polytropic viruses utilize the same (Xpr1) receptor, but they have different species tropisms. Xenotropic viruses such as NZB-9-1 infect humans and other species, but not murine species, while polytropic viruses such as mink cell focus-forming virus (MCF) infect murine, human, and other species.

[0391] γ-retroviral vectors can be produced in packaging cells by co-transfecting the cells with several plasmids, including a plasmid encoding the retroviral structural polyprotein and enzymatic polyprotein (gag-pol), a plasmid encoding the envelope (env) protein, and a plasmid encoding the vector mRNA, which includes a polynucleotide encoding a heterologous agent to be packaged in newly formed viral particles.

[0392] In some aspects, recombinant γ-retroviral vectors are pseudotyped with envelope proteins from other viruses. The envelope glycoproteins are incorporated into the outer lipid layer of the viral particle, which can increase / alter cell tropism. Exemplary envelope proteins include gibbon ape leukemia virus envelope protein (GALV) or vesicular stomatitis virus G protein (VSV-G), or simian endogenous retrovirus envelope protein, or measles virus H and F proteins, or human immunodeficiency virus gp120 envelope protein, or cocal vesicular stomatitis virus envelope protein (see, for example, U.S. Patent Application Publication No.: 2012 / 164118). In other aspects, the envelope glycoproteins can be genetically modified to incorporate targeting / binding ligands into the γ-retroviral vector, and the binding ligands include, but are not limited to, peptide ligands, single-chain antibodies, and growth factors (Waehier et al., Nat. Rev. Genet. 2007, 8(8):573-587). These engineered glycoproteins can re-target the vector to cells expressing their corresponding target moieties. In other aspects, a "molecular bridge" can be introduced to direct the vector to specific cells. The molecular bridge has dual specificity: one end can recognize the viral glycoprotein, while the other end can bind to a molecular determinant on the target cell. Such molecular bridges (e.g., ligand-receptor, avidin-biotin, and chemical conjugates, monoclonal antibodies, and engineered membrane fusion proteins) can direct the attachment of the viral vector to the target cell for transduction (Yang et al., Biotechnol Bioeng., 2008, 101(2):357-368; and Maetzig et al., Viruses, 2011, 3, 677-713).

[0393] In some embodiments, the recombinant γ-retroviral vector is a self-inactivating (SIN) γ-retroviral vector. The vector can be replication-incompetent. The SIN vector can have deletions in the 3'U3 region that originally includes enhancer / promoter activity. Additionally, the 5'U3 region can be replaced with a strong promoter from cytomegalovirus or RSV (required in the packaging cell line) or a selected internal promoter and / or enhancer element. The selection of the internal promoter can be made according to the specific requirements of gene expression needed for a particular purpose.

[0394] In some embodiments, a polynucleotide encoding a heterologous agent is inserted into the recombinant viral genome. Other components of the viral mRNA of the recombinant γ-retroviral vector can be modified by inserting or removing naturally occurring sequences (e.g., inserting an IRES, inserting a heterologous polynucleotide encoding a polypeptide or inhibitory nucleic acid of interest, replacing the wild-type promoter with a more efficient promoter from a different retrovirus or viral shuffling, etc.). In some instances, the recombinant γ-retroviral vector can include a modified packaging signal, and / or a primer binding site (PBS), and / or a 5'-enhancer / promoter element in the U3 region of the 5'-long terminal repeat (LTR), and / or a modified 3'-SIN element in the US region of the 3-LTR. These modifications can increase the titer and infectivity. The γ-retroviral vectors suitable for delivering heterologous agents are selected from those disclosed in U.S. Patent Nos.: 8,828,718, 7,585,676, 7,351,585; U.S. Patent Application Publication No.: US2007 / 048285; PCT Application Publication Nos.: WO2010 / 113037, WO2014 / 121005, WO2015 / 056014; and EP Patent Nos.: EP1757702, EP1757703.

[0395] In some embodiments, polynucleotides encoding heterologous agents can be packaged into recombinant adeno-associated virus (rAAV) vectors. Such vectors or viral particles can be designed to utilize any known serotype capsid or combination of serotype capsids. Serotype capsids can include capsids from any identified AAV serotype and variants thereof, such as AAV1, AAV2, AAV2G9, AAV3, AAV4, AAV4-4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and AAVrh10. In some embodiments, the AAV serotype can be or have the sequences described in the following documents: U.S. Publication No. US20030138772; Pulicherla et al., Molecular Therapy, 2011, Vol. 19, No. 6: pp. 1070-1078; U.S. Patent Nos.: 6,156,303; 7,198,951; U.S. Patent Publication Nos.: US2015 / 0159173 and US2014 / 0359799; and International Patent Publication Nos.: WO1998 / 011244, WO2005 / 033321, and WO2014 / 14422.

[0396] AAV vectors include not only single-stranded vectors but also self-complementary AAV vectors (scAAV). ScAAV vectors contain DNA that anneals together to form a double-stranded vector genome. By skipping second-strand synthesis, scAAV allows for rapid expression in cells. rAAV vectors can be manufactured by standard methods in the art, such as by triple transfection in sf9 insect cells or in suspension cell cultures of human cells such as HEK293 cells.

[0397] In some embodiments, non-viral-based methods can be used. For example, in some aspects, vectors comprising polynucleotides can be transferred into cells by non-viral methods using: physical methods such as needles, electroporation, sonoporation, hydrodynamic poration; chemical carriers such as inorganic particles (e.g., calcium phosphate, silica, gold) and / or chemical methods. In other aspects, synthetic or natural biodegradable agents can be used for delivery, such as cationic lipids, lipid nanoemulsions, nanoparticles, peptide-based carriers, or polymer-based carriers.

[0398] In some embodiments, polynucleotides encoding heterologous agents (such as CAR) are designed as messenger RNA (mRNA) for delivery.

[0399] In some embodiments, a polynucleotide encoding a heterologous agent (such as mRNA) is incorporated into a lipid nanoparticle. In some embodiments, the formulation is a nanoparticle that may comprise at least one lipid. The lipid may be selected from, but not limited to, DLin-DMA, DLin-K-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG, and pegylated lipids. In another aspect, the lipid may be a cationic lipid, such as, but not limited to, DLin-DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, and DODMA.

[0400] Lipid nanoparticles can be used to deliver encapsulated or associated (e.g., complexed) therapeutic agents, including mRNA. In particular, some nanoparticle compositions are particularly suitable for delivering nucleic acids, including messenger RNA (mRNA), antisense oligonucleotides, plasmid DNA, microRNA (miRNA), miRNA inhibitors (antagonists / enantiomers), complementary RNA that interferes with messenger RNA (micRNA), DNA, multivalent RNA, dicer substrate RNA, complementary DNA (cDNA), and self-amplifying RNA (saRNA). See, e.g., U.S. Patent No. 10,723,692 B2.

[0401] Accordingly, the methods provided herein include methods for delivering a nucleic acid (including DNA, RNA, mRNA, and self-amplifying RNA (saRNA)) encoding a heterologous agent (such as a CAR) to g-NK cells or a composition or a cell population enriched in g-NK cells. In some embodiments, the heterologous agent is packaged or incorporated into a lipid nanoparticle for delivery of the nucleic acid, such as DNA, RNA, mRNA, and self-amplifying RNA (saRNA). In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is RNA. In some embodiments, the nucleic acid is mRNA. In some embodiments, the nucleic acid is self-amplifying RNA (saRNA).

[0402] In some embodiments, the mRNA is self-amplifying mRNA. Self-amplifying RNA (saRNA) is capable of self-amplification through the presence of 5' and 3' conserved sequence elements (CSEs) and the nsP1-4 genes, as well as subgenomic promoters. See, for example, Bloom, vanden Berg, and Arbuthnot, Gene Therapy, 2021. After in situ translation, the nsP1-4 proteins form an RdRP complex that recognizes the panhandle CSE sequence and amplifies the sequence contained within the RNA. Introduction of the saRNA into target cells can be carried out via lipid nanoparticle delivery. In some embodiments, such self-amplifying RNAs can have the structural features or components of any of those taught in International Patent Application Publication No. WO201105799.

[0403] In some embodiments, the provided methods involve using lipid nanoparticles (LNPs) comprising mRNA encoding a heterologous agent, such as a CAR. In some embodiments, the mRNA encoding the heterologous agent can be produced using methods known in the art, such as in vitro transcription. In some embodiments of the method, the mRNA comprises a 5' cap. In some embodiments, the 5' cap is an altered nucleotide at the 5' end of a primary transcript, such as a messenger RNA. In some aspects, the 5' cap of the mRNA improves one or more of RNA stability and processing, mRNA metabolism, processing and maturation of the RNA transcript in the nucleus, transport of the mRNA from the nucleus to the cytoplasm, mRNA stability, and efficient translation of the mRNA into protein. In some embodiments, the 5' cap can be a naturally occurring 5' cap or a cap different from the naturally occurring cap of the mRNA. The 5' cap can be any 5' cap known to those of skill in the art. In certain embodiments, the 5' cap is selected from the group consisting of anti-reverse cap analog (ARCA) caps, 7-methyl-guanosine (7mG) caps, analogs, vaccinia caps, and analogs thereof. For example, the 5' cap can include, but is not limited to, an anti-reverse cap analog (ARCA) (US7074596), 7-methyl-guanosine, analogs, such as a Cap 1 analog (Trilink, San Diego, CA), or enzymatic capping using, for example, a vaccinia capping enzyme. In some embodiments, the mRNA can be polyadenylated. The mRNA can contain various 5' and 3' untranslated sequence elements to enhance the expression of the encoded engineered heterologous agent and / or the stability of the mRNA itself. Such elements can include, for example, post-transcriptional regulatory elements, such as the woodchuck hepatitis virus post-transcriptional regulatory element.

[0404] In some embodiments, the mRNA includes at least one nucleoside modification. The mRNA can contain modifications from naturally occurring nucleosides to nucleoside analogs. Any nucleoside analog known in the art is contemplated. Such nucleoside analogs can include, for example, those described in US 8,278,036. In certain embodiments of the method, the nucleoside modification is selected from the group consisting of modifications from uridine to pseudouridine and from uridine to N1-methylpseudouridine. In a specific embodiment of the method, the nucleoside modification is the modification from uridine to pseudouridine.

[0405] LNPs particularly suitable for the methods herein include cationic lipids selected from the group consisting of DLin-DMA (1,2-dilinoleyloxy-3-dimethylaminopropane), DLin-MC3-DMA (dilinoleylmethyl-4-dimethylaminobutyrate), DLin-KC2-DMA (2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane), DODMA (1,2-dioleyloxy-N,N-dimethyl-3-aminopropane), SS-OP (bis[2-(4-{2-[4-(cis-9-octadecenyl-oxy)phenylacetoxy]ethyl}piperidinyl)ethyl] disulfide), and derivatives thereof. DLin-MC3-DMA and its derivatives are described, for example, in WO2010144740. DODMA and its derivatives are described, for example, in US 7,745,651 and Mok et al., 1999, Biochimica et Biophysica Acta, Vol. 1419, No. 2: pp. 137-150. DLin-DMA and its derivatives are described, for example, in US7,799,565. DLin-KC2-DMA and its derivatives are described, for example, in US 9,139,554. SS-OP (NOF America Corporation, White Plains, NY) is described, for example, at www.nofamerica.com / store / index.php?dispatch=products.view&product_id=962. Additional and non-limiting examples of cationic lipids include methylpyridinyl-dialkanoic acid (MPDACA), palmitoyl-oleoyl-nor-arginine (PONA), guanidino-dialkanoic acid (GUADACA), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), bis{2-[N-methyl-N-(α-D-tocopheryl succinate propyl)amino]ethyl} disulfide (SS-33 / 3AP05), bis{2-[4-(α-D-tocopheryl succinate ethyl)piperidinyl]ethyl} disulfide (SS33 / 4PE15), bis{2-[4-(cis-9-octadecenoate ethyl)-1-piperidinyl]ethyl} disulfide (SS18 / 4PE16), and bis{2-[4-(cis,cis-9,12-octadecadienoate ethyl)-1-piperidinyl]ethyl} disulfide (SS18 / 4PE13). In other embodiments, the lipid nanoparticles further include one or more non-cationic lipids and lipid conjugates.

[0406] In some embodiments, the molar concentration of the cationic lipid is about 20% to about 80%, about 30% to about 70%, about 40% to about 60%, about 45% to about 55%, or about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, or about 80% of the total lipid molar concentration, wherein the total lipid molar concentration is the sum of the molar concentrations of the cationic lipid, the non-cationic lipid, and the lipid conjugate. In certain embodiments, the molar ratio of the cationic lipid of the lipid nanoparticle to the mRNA is about 1 to about 20, about 2 to about 16, about 4 to about 12, about 6 to about 10, or about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20.

[0407] In some embodiments, the lipid nanoparticles utilized in the methods disclosed herein may include at least one non-cationic lipid. In specific embodiments, the molar concentration of the non-cationic lipid is about 20% to about 80%, about 30% to about 70%, about 40% to about 70%, about 40% to about 60%, about 46% to about 50%, or about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 48.5%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, or about 80% of the total lipid molar concentration. In some embodiments, the non-cationic lipid includes phospholipids and steroids.

[0408] In some embodiments, the phospholipids useful for the lipid nanoparticles described herein include, but are not limited to, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-didecanoyl-sn-glycero-3-phosphocholine (DDPC), 1,2-dierucoyl-sn-glycero-3-phosphate (sodium salt) (DEPA-NA), 1,2-dierucoyl-sn-glycero-3-phosphocholine (DEPC), 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE), 1,2-dierucoyl-sn-glycero-3[phospho-rac-(1-glycerol)(sodium salt) (DEPG-NA), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLOPC), 1,2-dilauroyl-sn-glycero-3-phosphate (sodium salt) (DLPA-NA), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,2-dilauroyl-sn-glycero-3[phospho-rac-(1-glycerol...)(sodium salt) (DLPA-NA), 1,2-dilauroyl-sn-glycero-3[phospho-rac-(1-glycerol)(ammonium salt) (DLPG-NH4), 1,2-dilauroyl-sn-glycero-3-phosphoserine (sodium salt) (DLPS-NA), 1,2-dimyristoyl-sn-glycero-3-phosphate (sodium salt) (DMPA-NA), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dimyristoyl-sn-glycero-3[phospho-rac-(1-glycerol)(sodium salt) (DMPG-NA), 1,2-dimyristoyl-sn-glycero-3[phospho-rac-(1-glycerol)(ammonium salt) (DMPG-NH4), 1,2-dimyristoyl-sn-glycero-3[phospho-rac-(1-glycerol)(sodium / ammonium salt) (DMPG-NH4 / NA), 1,2-dimyristoyl-sn-glycero-3-phosphoserine (sodium salt) (DMPS-NA), 1,2-dioleoyl-sn-glycero-3-phosphate (sodium salt) (DOPA-NA), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3[phospho-rac-(1-glycerol)(sodium salt) (DOPG-NA), 1,2-dioleoyl-sn-glycero-3-phosphoserine (sodium salt) (DOPS-NA), 1,2-dipalmitoyl-sn-glycero-3-phosphate (sodium salt) (DPPA-NA), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dipalmitoyl-sn-glycero-3[phospho-rac-(1-glycerol)(sodium salt)] (DPPG-NA), 1,2-dipalmitoyl-sn-glycero-3[phospho-rac-(1-glycerol)(ammonium salt)] (DPPG-NH4), 1,2-dipalmitoyl-sn-glycero-3-phosphoserine (sodium salt) (DPPS-NA), 1,2-distearoyl-sn-glycero-3-phosphate (sodium salt) (DSPA-NA), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-distearoyl-sn-glycero-3[phospho-rac-(1-glycerol)(sodium salt)] (DSPG-NA), 1,2-distearoyl-sn-glycero-3[phospho-rac-(1-glycerol)(ammonium salt)] (DSPG-NH4), 1,2-distearoyl-sn-glycero-3-phosphoserine ...

Claims

1. A method of inducing cytolytic killing of target cells, the method comprising contacting target cells known or suspected of expressing a first antigen and a second antigen with: (a) a composition comprising natural killer (NK) cells lacking expression of the FcRγ chain (g-NK cells), wherein the g-NK cells express a chimeric antigen receptor (CAR) that comprises an extracellular binding domain that binds to the first antigen; and (b) a monoclonal antibody that binds to the second antigen.

2. The method according to claim 1, wherein the first and second antigens are different.

3. The method according to claim 1, wherein the first and second antigens are the same.

4. The method according to any one of claims 1-3, wherein the monoclonal antibody is a full-length antibody.

5. The method according to any one of claims 1-4, wherein the monoclonal antibody is an IgG1 antibody.

6. The method according to any one of claims 1-5, wherein the CAR and the monoclonal antibody bind to different epitopes of the same antigen.

7. The method according to any one of claims 1-6, wherein the target cells are tumor cells.

8. The method according to any one of claims 1-7, wherein the tumor cells are hematological malignancy cells.

9. The method according to any one of claims 1-8, wherein the target cells are B cells.

10. The method according to any one of claims 1-9, wherein the first antigen and the second antigen are selected from the group consisting of CD30, CD19, CD20, CD22, ROR1, Igk, CD38, CD138, BCMA, CD33, CD70, CD79b, CD123, SLAMF7, GPRC5D, FCRH5, FLT3, CLEC12, and Lewis Y antigen.

11. The method according to any one of claims 8-10, wherein the hematological malignancy cells are multiple myeloma.

12. The method according to any one of claims 1-11, wherein the first antigen and the second antigen are selected from the group consisting of CD38, SLAMF7, CD138, FCRH5, GPRC5D, and BCMA.

13. The method according to any one of claims 1-12, wherein the CAR is an anti-BCMA CAR and the monoclonal antibody is an anti-CD38 antibody.

14. The method according to claim 13, wherein the anti-CD38 antibody is daratumumab or isatuximab.

15. The method according to any one of claims 8-10, wherein the hematological malignancy cells are lymphoma.

16. The method according to claim 15, wherein the lymphoma is non-Hodgkin lymphoma (NHL).

17. The method according to any one of claims 1-10 and 15-16, wherein the first and second antigens are selected from the group consisting of CD19, CD20, CD22, ROR1, CD30, CD38, and CD79b.

18. The method according to any one of claims 1-10 and 15-17, wherein the first and second antigens are selected from the group consisting of CD19, CD20, CD22, ROR1, and CD30.

19. The method according to any one of claims 1-10 and 15-18, wherein the CAR is an anti-CD19 CAR, and the antibody is an anti-CD20 antibody.

20. The method according to claim 19, wherein the anti-CD20 antibody is rituximab, ofatumumab, or obinutuzumab.

21. The method according to any one of claims 1-10 and 15-18, wherein the CAR is an anti-CD19 CAR, and the antibody is an anti-CD38 antibody.

22. The method according to any one of claims 1-10 and 15-18, wherein the CAR is an anti-CD20 CAR, and the antibody is an anti-CD38 antibody.

23. The method according to claim 21 or claim 22, wherein the anti-CD38 antibody is daratumumab or isatuximab.

24. The method according to any one of claims 8-10, wherein the hematological malignancy cells are leukemia.

25. The method according to claim 24, wherein the leukemia is acute myeloid leukemia (AML).

26. The method according to any one of claims 1-10 and 24-25, wherein the first and second antigens are selected from the group consisting of CD123, Flt3, CD70, CD33, CLEC12A, and CD38.

27. The method according to any one of claims 1-7, wherein the tumor cells are solid malignant tumor cells.

28. The method according to any one of claims 1-7 and 27, wherein the first and second antigens are selected from the group consisting of GPC3, HER2, GD2, EGFR mutant III (EGFR vIII), EGFR, CEA, PSMA, FRα, FAP, glypican-3, EPCAM, MUC1, ROR1, MUCI16eto, VEGFR2, CD171, PSCA, EphA2, survivin, mesothelin, TROP2, B7H3, CCR4, PDGFRα, laminin 4, tissue factor, CLDN6, FGFR2b, and IL-13α.

29. The method according to any one of claims 1-28, wherein the monoclonal antibody is contacted separately with cells from the composition comprising the g-NK cells.

30. The method according to any one of claims 1-29, wherein at least a portion of the contact with the composition comprising the g-NK cells and the contact with the monoclonal antibody are carried out simultaneously.

31. The method according to any one of claims 1-30, wherein the contact with the composition comprising g-NK cells and the contact with the monoclonal antibody are carried out simultaneously.

32. The method according to any one of claims 1-31, wherein the monoclonal antibody can be secreted from the g-NK cells.

33. The method according to any one of claims 1-32, wherein the contact is carried out in a subject.

34. A method of treating cancer in a subject, the method comprising: (a) administering to a subject having cancer an NK cell therapy comprising a dose of a composition comprising natural killer (NK) cells lacking expression of the FcRγ chain (g-NK cells), wherein the g-NK cells express a chimeric antigen receptor (CAR) comprising an extracellular binding domain that binds to a first antigen expressed by the cells of the cancer; and (b) administering to the subject a dose of a monoclonal antibody that binds to a second antigen expressed by the cells of the cancer.

35. A method of treating cancer in a subject, the method comprising administering to a subject having cancer an NK cell therapy comprising a dose of a composition comprising natural killer (NK) cells lacking expression of the FcRγ chain (g-NK cells), wherein: the g-NK cells express a chimeric antigen receptor (CAR) comprising an extracellular binding domain that binds to a first antigen expressed by the cells of the cancer; and the g-NK cells express a secretable monoclonal antibody that binds to a second antigen expressed by the cells of the cancer.

36. The method according to claim 34 or claim 35, wherein the first and second antigens are different.

37. The method according to claim 34 or claim 35, wherein the first and second antigens are the same.

38. The method according to any one of claims 34-37, wherein the monoclonal antibody is a full-length antibody.

39. The method according to any one of claims 34-38, wherein the monoclonal antibody is an IgG1 antibody.

40. The method according to any one of claims 34-39, wherein the CAR and the monoclonal antibody bind to different epitopes of the same antigen.

41. The method according to any one of claims 34-40, wherein the first and second antigens are expressed by the same cells of the cancer.

42. The method according to any one of claims 34-41, wherein the cancer is a hematological malignancy.

43. The method according to any one of claims 34-42, wherein the cells of the cancer are B cells and the cancer is a B cell cancer.

44. The method according to any one of claims 34 - 43, wherein the first antigen and the second antigen are selected from the group consisting of CD30, CD19, CD20, CD22, ROR1, Igk, CD38, CD138, BCMA, CD33, CD70, CD79b, CD123, SLAMF7, GPRC5D, FCRH5, FLT3, CLEC12, and Lewis Y antigen.

45. The method according to any one of claims 34 - 44, wherein the cancer is multiple myeloma.

46. The method according to claim 45, wherein the multiple myeloma is relapsed / refractory multiple myeloma.

47. The method according to any one of claims 34 - 46, wherein the first antigen and the second antigen are selected from the group consisting of CD38, SLAMF7, CD138, FCRH5, GPRC5D, and BCMA.

48. The method according to any one of claims 34 - 47, wherein the CAR is an anti - BCMA CAR, and the monoclonal antibody is an anti - CD38 antibody.

49. The method according to claim 48, wherein the anti - CD38 antibody is daratumumab or isatuximab.

50. The method according to any one of claims 34 - 44, wherein the cancer is lymphoma.

51. The method according to claim 50, wherein the lymphoma is non - Hodgkin lymphoma (NHL).

52. The method according to claim 51, wherein the NHL is relapsed / refractory multiple NHL.

53. The method according to any one of claims 34 - 44 and 50 - 52, wherein the first and second antigens are selected from the group consisting of CD19, CD20, CD22, ROR1, CD30, CD38, and CD79b.

54. The method according to any one of claims 34 - 44 and 50 - 53, wherein the first and second antigens are selected from the group consisting of CD19, CD20, CD22, ROR1, and CD30.

55. The method according to any one of claims 34 - 44 and 50 - 54, wherein the CAR is an anti - CD19 CAR, and the monoclonal antibody is an anti - CD20 antibody.

56. The method according to claim 55, wherein the anti - CD20 antibody is rituximab, ofatumumab, and obinutuzumab.

57. The method according to any one of claims 34 - 44 and 50 - 54, wherein the CAR is an anti - CD19 CAR, and the antibody is an anti - CD38 antibody.

58. The method according to any one of claims 34 - 44 and 50 - 54, wherein the CAR is an anti - CD20 CAR, and the antibody is an anti - CD38 antibody.

59. The method according to claim 57 or claim 58, wherein the anti - CD38 antibody is daratumumab or isatuximab.

60. The method according to any one of claims 34 - 44, wherein the cancer is leukemia.

61. The method according to claim 60, wherein the leukemia is acute myeloid leukemia (AML).

62. The method according to claim 61, wherein the AML is relapsed / refractory AML.

63. The method according to any one of claims 34 - 44 and 60 - 62, wherein the first and second antigens are selected from the group consisting of CD123, Flt3, CD70, CD33, CLEC12A, CD38.

64. The method according to any one of claims 34 - 41, wherein the cancer is a solid malignant tumor.

65. The method according to any one of claims 34 - 41 and 64, wherein the first and second antigens are GPC3, HER2, GD2, EGFR mutant III (EGFR vIII), EGFR, CEA, PSMA, FRα, FAP, glypican - 3, EPCAM, MUC1, ROR1, MUCI16 etc., VEGFR2, CD171, PSCA, EphA2, survivin, mesothelin, TROP2, B7H3, CCR4, PDGFRα, laminin 4, tissue factor, CLDN6, FGFR2b, and IL - 13α.

66. The method according to any one of claims 34 - 65, wherein the composition of a certain dose of g - NK cells comprises a plurality of doses.

67. The method according to any one of claims 34 - 66, wherein the NK cell therapy comprises administering 1 - 8 doses of the composition comprising g - NK cells.

68. The method according to any one of claims 34 - 67, wherein each dose of the composition comprising g - NK cells is administered once a week.

69. The method according to any one of claims 34 - 68, wherein the NK cell therapy is administered with two doses of the composition comprising g - NK cells within a 14 - day cycle, and the 14 - day cycle is repeated one to three times.

70. The method according to any one of claims 34 - 68, wherein the NK cell therapy is administered with two doses of the composition comprising g - NK cells within a 21 - day cycle, and the 21 - day cycle is repeated one to three times.

71. The method according to any one of claims 34 - 70, wherein: Before administering the certain dose of g - NK cells, the subject has received lymphodepletion therapy; or The method further comprises administering lymphodepletion therapy to the subject before administering the g - NK cells.

72. The method according to claim 71, wherein the administration of the certain dose of g - NK cells starts within two weeks or at two weeks or about two weeks after the start of the lymphodepletion therapy.

73. The method according to claim 71 or claim 72, wherein the administration of a certain dose of g-NK cells starts within 7 days or at 7 days or at about 7 days after the start of the lymphodepletion therapy.

74. The method according to claim 69 or claim 70, wherein the lymphodepletion therapy is administered to the subject before repeating subsequent cycles.

75. The method according to any one of claims 71 - 74, wherein the lymphodepletion therapy comprises fludarabine and / or cyclophosphamide.

76. The method according to any one of claims 71 - 74, wherein the lymphodepletion therapy comprises fludarabine and cyclophosphamide.

77. The method according to any one of claims 71-76, wherein said lymph node clearance comprises administering fludarabine at or about 20-40 mg / m 2 of the body surface area of the subject, optionally at or about 30 mg / m 2 , once daily for 2-4 days; and / or cyclophosphamide at or about 200-400 mg / m 2 of the body surface area of the subject, optionally at or about 300 mg / m 2 , once daily for 2-4 days.

78. The method according to any one of claims 71 - 77, wherein the lymphodepletion therapy comprises administering fludarabine at or about 30 mg / m 2 of the body surface area of the subject, once daily, and cyclophosphamide at or about 300 mg / m 2 of the body surface area of the subject, once daily, each for 2 - 4 days, optionally 3 days.

79. The method according to claim 34 and any one of claims 36 - 78, wherein the administration of at least one dose of the monoclonal antibody starts within one month before the administration of the NK cell therapy.

80. The method according to claim 34 and any one of claims 36 - 79, wherein the administration of at least one dose of the monoclonal antibody starts within three weeks before the administration of the NK cell therapy.

81. The method according to claim 34 and any one of claims 36 - 80, wherein the administration of at least one dose of the monoclonal antibody starts within two weeks before the administration of the NK cell therapy.

82. The method according to any one of claims 34 and 36 - 81, wherein the monoclonal antibody is administered intravenously.

83. The method according to any one of claims 34 and 36 - 81, wherein the monoclonal antibody is administered subcutaneously.

84. The method according to claim 83, wherein a loading dose of the monoclonal antibody is administered intravenously before subcutaneous administration.

85. The method according to any one of claims 34 and 36 - 84, wherein the certain dose of the monoclonal antibody comprises a plurality of doses.

86. The method according to any one of claims 34 and 36 - 85, wherein the monoclonal antibody is administered once every four weeks, once every three weeks, once every two weeks, once a week, or twice a week.

87. The method according to any one of claims 34 and 36 - 86, wherein each dose of the monoclonal antibody is administered once a week.

88. The method according to any one of claims 34 and 36 - 87, wherein the monoclonal antibody is administered in 4 to 16 doses, optionally 4 doses or about 4 doses or 8 doses or about 8 doses.

89. The method according to any one of claims 1 - 88, wherein the CAR comprises 1) an antigen-binding domain that binds to the first antigen; 2) a spacer; 3) a transmembrane region; and 4) an intracellular signaling domain.

90. The method according to claim 89, wherein the antigen-binding domain is a single-chain variable fragment (scFv).

91. The method according to claim 89 or claim 90, wherein the intracellular signaling domain comprises one or more signaling domains of CD3ζ, DAP10, DAP12, CD28, 4 - 1BB, or OX40.

92. The engineered NK cells according to claim 89 or claim 90, wherein the intracellular signaling domain comprises two or more signaling domains selected from CD3ζ, DAP10, DAP12, CD28, 4-1BB, or OX40.

93. The method according to any one of claims 89-92, wherein the intracellular signaling domain comprises a primary signaling domain containing the signaling domain of CD3ζ.

94. The method according to claim 93, wherein the intracellular signaling domain further comprises a co-stimulatory signaling domain, optionally wherein the co-stimulatory signaling domain is the signaling domain of CD28 or 4-1BB.

95. The method according to any one of claims 1-94, wherein the heterologous nucleic acid encoding the CAR is stably integrated into the genome of the cell.

96. The method according to any one of claims 1-94, wherein the heterologous nucleic acid encoding the CAR is transiently expressed.

97. The method according to any one of claims 1-96, wherein the g-NK cells further comprise a heterologous nucleic acid encoding an immunomodulatory protein.

98. The method according to claim 97, wherein the immunomodulatory protein is a cytokine.

99. The method according to claim 98, wherein the cytokine is secreted from the g-NK cells.

100. The method according to claim 99, wherein the secreted cytokine is IL-2 or a biological moiety thereof; IL-15 or a biological moiety thereof; or IL-21 or a biological moiety thereof; or a combination thereof.

101. The method according to claim 98, wherein the cytokine is membrane-bound.

102. The method according to claim 101, wherein the membrane-bound cytokine is membrane-bound IL-2 (mbIL-2); membrane-bound IL-15 (mbIL-15); membrane-bound IL-21 (mbIL-21); or a combination thereof.

103. The method according to any one of claims 97-102, wherein the heterologous nucleic acid encoding the immunomodulator is stably integrated into the genome of the cell.

104. The method according to any one of claims 97-102, wherein the heterologous nucleic acid encoding the immunomodulator is transiently expressed.

105. The method according to any one of claims 1-104, further comprising administering an exogenous cytokine to promote the expansion or persistence of the g-NK cells in the subject, optionally wherein the exogenous cytokine is or comprises IL-15.

106. The method according to any one of claims 1-105, wherein the FcRγ chain in the g-NK cells is undetectable by immunoblotting.

107. The method according to any one of claims 1-106, wherein in the cells of the g-NK cell composition, greater than or about 60% of the cells are g-NK cells, greater than or about 70% of the cells are g-NK cells, greater than or about 80% of the cells are g-NK cells, greater than or about 90% of the cells are g-NK cells, or greater than or about 95% of the cells are g-NK cells.

108. The method according to any one of claims 1-107, wherein at least or about 50% of the cells in the g-NK cell composition are FcRγ-deficient (FcRγ 阴性 ) NK cells (g-NK), wherein greater than or about 70% of the g-NK cells are positive for perforin, and greater than or about 70% of the g-NK cells are positive for granzyme B.

109. The method according to claim 107 or claim 108, wherein (i) greater than or about 80% of the g-NK cells are positive for perforin and greater than or about 80% of the g-NK cells are positive for granzyme B, (ii) greater than or about 90% of the g-NK cells are positive for perforin and greater than or about 90% of the g-NK cells are positive for granzyme B, or (iii) greater than or about 95% of the g-NK cells are positive for perforin and greater than or about 95% of the g-NK cells are positive for granzyme B.

110. The method according to claim 108 or claim 109, wherein: In the cells positive for perforin, the average level of perforin expressed by the cells, as measured by intracellular flow cytometry, is at least about twice, according to the mean fluorescence intensity (MFI), that of the perforin expressed by cells that are 阳性 阳性 FcRγ; and / or In the cells that are positive for granzyme B, the average level of granzyme B expressed by the cells, as measured by intracellular flow cytometry, is at least about two-fold, according to the mean fluorescence intensity (MFI), of the average level of granzyme B expressed by cells of FcRγ 阳性 and is at least about two-fold of that expressed by cells of FcRγ.

111. The method according to any one of claims 1-110, wherein greater than 10% of the cells in the g-NK cell composition are capable of degranulating tumor target cells, optionally as measured by CD107a expression, optionally wherein degranulation is measured in the absence of an antibody against the tumor target cells.

112. The method according to any one of claims 1-111, wherein, in the presence of cells expressing a target antigen (target cells) and an antibody against the target antigen (anti-target antibody), in the cells of the g-NK cell composition, greater than or about 15%, greater than or about 20%, greater than or about 30%, greater than or about 40% or greater than or about 50% of the cells exhibit degranulation, optionally as measured by CD107a expression.

113. The method according to any one of claims 1-112, wherein greater than 10% of the cells in the g-NK cell composition are capable of producing interferon-γ or TNF-α against tumor target cells, optionally, wherein interferon-γ or TNF-α is measured in the absence of an antibody against the tumor target cells.

114. The method according to any one of claims 1-113, wherein, in the presence of cells expressing a target antigen (target cells) and an antibody against the target antigen, in the cells of the g-NK cell composition, greater than or about 15%, greater than or about 20%, greater than or about 30%, greater than or about 40% or greater than or about 50% produce effector cytokines.

115. The method according to claim 114, wherein the effector cytokine is IFN-γ or TNF-α.

116. The method according to claim 114 or claim 115, wherein the effector cytokine is IFN-γ and TNF-α.

117. The method according to any one of claims 1-116, wherein the g-NK cell composition has been produced by in vitro expansion of CD3- / CD57+ cells or CD3- / CD56+ cells cultured with irradiated HLA-E+ feeder cells, wherein the CD3- / CD57+ cells or CD3- / CD55+ cells are enriched from a biological sample of a donor subject.

118. The method according to claim 117, wherein the donor subject is CMV seropositive.

119. The method according to claim 117 or claim 118, wherein the donor subject has a CD16 158V / V NK cell genotype or a CD16 158V / F NK cell genotype, optionally wherein the biological sample is from a human subject selected for the CD16 158V / V NK cell genotype or the CD16 158V / F NK cell genotype.

120. The method according to any one of claims 117-119, wherein at least or about 20% of the natural killer (NK) cells in a peripheral blood sample from the donor subject are positive for NKG2C (NKG2C positive), and at least 70% of the NK cells in the peripheral blood sample are negative or low for NKG2A (NKG2A negative).

121. The method according to any one of claims 117-120, wherein the irradiated feeder cells are deficient in HLA class I and HLA class II.

122. The method according to any one of claims 117-121, wherein the irradiated feeder cells are 221.AEH cells.

123. The method according to any one of claims 117-122, wherein the culturing is carried out in the presence of two or more recombinant cytokines, wherein at least one recombinant cytokine is interleukin (IL)-2, and at least one recombinant cytokine is IL-21.

124. The method according to claim 123, wherein the recombinant cytokines are IL-21 and IL-2.

125. The method according to claim 123, wherein the recombinant cytokines are IL-21, IL-2, and IL-15.

126. The method according to any one of claims 1-116, wherein the g-NK cells are genetically engineered to knockout the gene encoding the FcRγ chain.

127. The method according to claim 126, wherein the knockout is the introduction of a gene disruption in the gene, wherein the gene disruption results in a deletion, insertion, or mutation in the gene.

128. The method according to claim 126 or claim 127, wherein both alleles of the gene encoding the FcRγ chain are disrupted in the engineered cells.

129. The method according to any one of claims 126-128, wherein the gene disruption is by an endonuclease.

130. The method according to claim 129, wherein the endonuclease is a TAL nuclease, a meganuclease, a zinc finger nuclease, an Argonaute nuclease or a CRISPR enzyme in combination with a guide RNA.

131. The method according to claim 130, wherein the endonuclease is CRISPR / Cas9 in combination with a guide RNA.

132. The method according to any one of claims 126-131, wherein the g-NK cells further comprise a nucleic acid encoding a heterologous CD16.

133. The method according to claim 132, wherein the heterologous CD16 comprises an activating mutation of CD16, wherein the mutation results in a higher affinity for IgG1.

134. The method according to claim 133, wherein the heterologous CD16 comprises the 158V mutation.

135. The method according to any one of claims 126-134, wherein the engineered g-NK cells are derived from primary cells obtained from a human subject.

136. The method according to any one of claims 1-135, wherein the g-NK cell composition is formulated in a serum-free cryopreservation medium, the cryopreservation medium comprising a cryoprotectant, optionally wherein the cryoprotectant is DMSO, and the cryopreservation medium is 5% to 10% DMSO (v / v).

137. The method according to any one of claims 1-136, wherein each dose of g-NK cells is or is about or about 1×10 8 cells to is or is about 50×10 9 cells of g-NK cell composition, optionally wherein each dose of g-NK cells is or is about 5×10 8 cells of g-NK cell composition, is or is about 5×10 9 cells of g-NK cell composition, or is or is about 10×10 9 cells of g-NK cell composition.

138. The method according to any one of claims 34-137, wherein the subject is a human subject.

139. The method according to any one of claims 1-138, wherein the NK cells in the composition are allogeneic to the subject.

140. An engineered natural killer (NK) cell, wherein the NK cell lacks expression of the FcRγ chain (g-NK cell), and wherein the g-NK cell comprises: a heterologous nucleic acid encoding a chimeric antigen receptor (CAR), the CAR comprising an extracellular binding domain that binds to the first antigen; and a heterologous nucleic acid encoding a secreted monoclonal antibody that binds to a second antigen.

141. The engineered NK cell according to claim 140, wherein the first and second antigens are different.

142. The engineered NK cell according to claim 141, wherein the first and second antigens are the same.

143. The engineered NK cell according to any one of claims 140-142, wherein the monoclonal antibody is a full-length antibody.

144. The engineered NK cell according to any one of claims 140-143, wherein the monoclonal antibody is an IgG1 antibody.

145. The engineered NK cell according to any one of claims 140-144, wherein the CAR and the monoclonal antibody bind to different epitopes of the same antigen.

146. The engineered NK cell according to any one of claims 140-145, wherein the first and second antigens are expressed by the same target cell.

147. The engineered NK cells according to claim 146, wherein the target cells are tumor cells.

148. A pharmaceutical composition comprising the engineered NK cells according to any one of claims 140 - 147 and a pharmaceutically acceptable carrier.

149. The pharmaceutical composition according to claim 148, which comprises a cryoprotectant.

150. The pharmaceutical composition according to claim 148 or claim 149, wherein the composition is formulated in a serum - free cryopreservation medium comprising a cryoprotectant.

151. The pharmaceutical composition according to claim 149 or claim 150, wherein the cryoprotectant is DMSO, and the cryopreservation medium is 5% to 10% DMSO (v / v).

152. A method of treating cancer in a subject, the method comprising administering to a subject suffering from cancer the pharmaceutical composition according to any one of claims 148 - 151.

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