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

Abstract

Provided herein are treatment methods and uses, including administering a composition containing recombinant chimeric antigen receptor (CAR)-engineered NK cells (g-NK cells) lacking FcRγ chain expression in combination with a monoclonal antibody. Among the provided methods and uses are those for treating cancers such as multiple myeloma or lymphoma.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent No. 63 / 357,637, entitled "COMBINATION OF ENGINEERED NATURAL KILLER (NK) CELLS AND ANTIBODY THERAPY AND RELATED METHODS," filed June 30, 2022, the entire contents of which are incorporated by reference.

[0002] INCORPORATION-BY-REFERENCE TO SEQUENCE LISTING This application is filed with an electronic Sequence Listing, which is provided as a file entitled 776032001440SeqList.xml, created on June 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.

[0003] Field The present disclosure provides methods of treatment and uses, including administering a composition containing NK cells (g-NK cells) engineered with a recombinant chimeric antigen receptor (CAR) and lacking expression of the FcRγ chain in combination with a monoclonal antibody. Among the embodiments of the present disclosure are methods and uses for treating cancers such as multiple myeloma or lymphoma. [Background technology]

[0004] background Antibody-based therapies are becoming increasingly used to treat cancer and other diseases. While responses to antibody therapy have typically focused on the direct inhibitory effects of these antibodies on tumor cells (e.g., inhibition of growth factor receptors and subsequent induction of apoptosis), the in vivo effects of these antibodies can be more complex and involve the host immune system. Natural killer (NK) cells are immune effector cells that mediate antibody-dependent cellular cytotoxicity when Fc receptors (CD16; FcγRIII) bind to the Fc portion of antibodies bound to antigen-bearing cells. NK cells, including certain specialized subsets thereof, can be used in therapeutic methods, including to improve responses to antibody therapy. Improved methods for therapeutic uses involving NK cells are needed. Provided herein are embodiments that meet this need. Summary of the Invention

[0005] overview In some aspects, target cells known or suspected to express the first antigen and the second antigen are (a) a composition comprising natural killer (NK) cells (g-NK cells) lacking expression of an FcRγ chain, 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. Provided herein is a method for inducing cytolytic killing of a target cell, which can include contacting the target cell with a CAR. In any of the above-mentioned embodiments, the first antigen and the second antigen can be different. In any of the above-mentioned embodiments, the first antigen and the second antigen can be the same. In any of the above-mentioned embodiments, the monoclonal antibody can be a full-length antibody. In any of the above-mentioned embodiments, the monoclonal antibody can be an IgG1 antibody. In any of the above-mentioned 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 cell of a hematological malignancy. In any of the foregoing embodiments, the target cell can be a B cell. In any of the foregoing embodiments, the first antigen and the second antigen 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 antigens.

[0007] In some embodiments, the hematological malignancy may be 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.

[0008] In some embodiments, the hematological malignancy can be lymphoma. In some embodiments, the lymphoma can be non-Hodgkin's 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 hematological 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 may be cells of a solid malignant tumor. In some embodiments, the first antigen and the second antigen may be selected from the group consisting of GPC3, HER2, GD2, EGFR variant 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.

[0012] In some of the foregoing embodiments, the monoclonal antibody may contact the cells separately from the composition comprising g-NK cells. In some embodiments, at least some of the steps of contacting with the composition comprising g-NK cells and contacting with the monoclonal antibody may be performed simultaneously. In some embodiments, the step of contacting with the composition comprising g-NK cells may be performed simultaneously with the step of contacting with the monoclonal antibody.

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

[0014] In some of any of the foregoing embodiments, the contacting step can be performed in vivo in a subject.

[0015] In some aspects, provided herein are methods of treating cancer in a subject that can include: (a) administering to a subject having cancer a natural killer (NK) cell therapy comprising a dose of a composition comprising NK cells that lack 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 cells of the cancer; and (b) administering to the subject a dose of a monoclonal antibody that binds to a second antigen expressed by cells of the cancer.

[0016] In some aspects, provided herein are methods of treating cancer in a subject that can include administering to a subject having cancer a natural killer (NK) cell therapy comprising a dose of a composition comprising NK cells that lack 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 cells of the cancer, and the g-NK cells express a secretable monoclonal antibody that binds to a second antigen expressed by cells of the cancer.

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

[0018] In some of the foregoing embodiments, the cancer may be a hematological malignancy. In some of the foregoing embodiments, the cancer cells may be 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's lymphoma (NHL). In some embodiments, the NHL may be relapsed / refractory multifocal 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, 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 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.

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

[0023] In some embodiments, the cancer may be a solid malignant tumor. In some embodiments, the first and second antigens may be GPC3, HER2, GD2, EGFR variant 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.

[0024] In some of the foregoing embodiments, the dose of the composition of g-NK cells can include multiple doses. In some of the foregoing embodiments, the NK cell therapy can include administration of 1 to 8 doses of the composition comprising g-NK cells. In some of the foregoing embodiments, each dose of the composition of g-NK cells can be administered once a week. In some of the foregoing embodiments, the NK cell therapy can be administered as two doses of the composition comprising g-NK cells in a 14-day cycle, which can be repeated 1 to 3 times. In some of the foregoing embodiments, the NK cell therapy can be administered as three doses of the composition comprising g-NK cells in a 21-day cycle, which can be repeated 1 to 3 times.

[0025] In some of any of the foregoing embodiments, the subject has undergone lymphodepletion therapy prior to administration of the dose of g-NK cells. In some of any of the foregoing embodiments, the method can further include administering lymphodepletion therapy to the subject prior to administering the g-NK cells. In some of the foregoing embodiments, administration of the dose of g-NK cells can be initiated within two weeks, or at or about the second week, after initiation of lymphodepletion therapy. In some of the foregoing embodiments, administration of the dose of g-NK cells can be initiated within seven days, or at or about the seventh day, after initiation of lymphodepletion therapy. In some of the foregoing embodiments, before repeating the subsequent cycle, the subject can be administered lymphodepletion therapy. In some of the foregoing embodiments, the lymphodepletion therapy can include fludarabine and / or cyclophosphamide. In some of the foregoing embodiments, the lymphodepletion therapy can be initiated at a dose of 100 mg / mL of the subject's body surface area. 2 20-40 mg per m2 of body surface area 2 Approximately 20-40 mg of fludarabine per m2 of body surface area of the subject 2 200-400 mg per m2 of body surface area 2 In some embodiments, fludarabine can be administered at a dose of about 200-400 mg / m. 2 or approximately 30 mg / m 2 In some embodiments, cyclophosphamide is administered at a dose of 300 mg / m 2 or approximately 300 mg / m 2 In some of the above embodiments, the lymphodepletion therapy is administered at a dose of 100 mg / mL per 1 m of the subject's body surface area. 2 30 mg per m2 of body surface area of the subject 2 Approximately 30 mg of fludarabine per square meter of body surface area was administered daily. 2 300 mg per m2 of body surface area of the subject 2 This may include administering approximately 300 mg of cyclophosphamide per day for 2 to 4 days, optionally 3 days.

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

[0027] In some of the aforementioned embodiments, the CAR may 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 the aforementioned embodiments, the antigen-binding domain may be a single-chain variable fragment (scFv). In some of the aforementioned embodiments, the intracellular signaling domain may comprise one or more signaling domains of CD3ζ, DAP10, DAP12, CD28, 4-1BB, or OX40. In some of the aforementioned embodiments, the intracellular signaling domain may comprise two or more signaling domains of CD3ζ, DAP10, DAP12, CD28, 4-1BB, or OX40. In some of the aforementioned embodiments, the intracellular signaling domain may comprise a primary signaling domain comprising the signaling domain of CD3ζ. In some of the aforementioned embodiments, the intracellular signaling domain may further comprise a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is the signaling domain of CD28. In some embodiments, the costimulatory signaling domain is the signaling domain of 4-1BB.

[0028] In some of the foregoing embodiments, the heterologous nucleic acid encoding the CAR may be stably integrated into the genome of the cell. In some of the foregoing embodiments, the heterologous nucleic acid encoding the CAR may be transiently expressed. In some of the foregoing embodiments, the g-NK cells may further comprise a heterologous nucleic acid encoding an immunomodulatory protein. In some of the foregoing embodiments, the immunomodulatory protein may be a cytokine. In some of the foregoing embodiments, the cytokine may be secretable from the g-NK cells. In some of the foregoing embodiments, the secretable cytokine may be IL-2 or a biological portion thereof; IL-15 or a biological portion thereof; or IL-21 or a biological portion thereof; or a combination thereof. In some of the foregoing embodiments, the cytokine may be membrane-bound. In some of the foregoing embodiments, the membrane-bound cytokine may be 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 the foregoing embodiments, the heterologous nucleic acid encoding the immunomodulatory agent may be stably integrated into the genome of the cell. In some of the foregoing embodiments, the heterologous nucleic acid encoding the immunomodulatory agent may be transiently expressed.

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

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

[0031] In some of any of the foregoing embodiments, of the cells in 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 50% or at least about 50% of the cells in the g-NK cell composition are FcRγ deficient (FcRγ neg ) NK cells (g-NK), and greater than or about 70% of the g-NK cells may be positive for perforin, and greater than or about 70% of the g-NK cells may be positive for granzyme B. In some of any of the foregoing embodiments, (i) greater than or about 80% of the g-NK cells may be positive for perforin, and greater than or about 80% of the g-NK cells may be positive for granzyme B, (ii) greater than or about 90% of the g-NK cells may be positive for perforin, and greater than or about 90% of the g-NK cells may be positive for granzyme B, or (iii) greater than or about 95% of the g-NK cells may be positive for perforin, and greater than or about 95% of the g-NK cells may be positive for granzyme B. In some of any of the foregoing embodiments, in cells that are positive for perforin, the cells are positive for FcRγ based on mean fluorescence intensity (MFI) as measured by intracellular flow cytometry. pos In some of the above embodiments, in cells that are positive for granzyme B, the cells can express an average level of perforin that is at least two-fold or at least about two-fold the average level of perforin expressed by cells that are positive for granzyme B. In some of the above embodiments, in cells that are positive for granzyme B, the cells can express an average level of perforin that is at least two-fold or at least about two-fold the average level of perforin expressed by cells that are positive for FcRγ based on mean fluorescence intensity (MFI) as measured by intracellular flow cytometry. pos The cells may express an average level of granzyme B that is at least twice or at least about twice the average level of granzyme B expressed by cells that are

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

[0033] In some of any of the foregoing embodiments, 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 in the g-NK cell composition exhibit degranulation. In some embodiments, g-NK cells capable of degranulation can be measured by CD107a expression in the presence of cells expressing a target antigen (target cells) and an antibody against the target antigen (anti-target antibody). In some embodiments, g-NK cells capable of degranulation are measured by CD107a expression. In some embodiments, degranulation is measured in the presence of cells expressing a target antigen (target cells) and an antibody against the target antigen (anti-target antibody).

[0034] In 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 the tumor target cells. In some embodiments, interferon-γ or TNF-α may be measured in the absence of antibodies against the tumor target cells.

[0035] In any of the foregoing embodiments, 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 in the g-NK cell composition produce effector cytokines in the presence of cells expressing a target antigen (target cells) and antibodies against the target antigen (anti-target antibodies). In some of any of the foregoing embodiments, the effector cytokines can be IFN-γ or TNF-α. In some of the foregoing embodiments, the effector cytokines can be IFN-γ and TNF-α.

[0036] In some of the foregoing embodiments, the g-NK cell composition is generated by ex vivo expansion of CD3- / CD57+ cells or CD3- / CD56+ cells cultured with irradiated HLA-E+ feeder cells, and the CD3- / CD57+ cells or CD3- / CD55+ cells can be enriched from a biological sample from a donor subject. In some of the foregoing embodiments, the donor subject can be CMV-seropositive. In some of the foregoing embodiments, the donor subject can have a CD16 158V / V NK cell genotype. In some 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 the foregoing embodiments, at least 20% or at least about 20% of natural killer (NK) cells in a peripheral blood sample from the donor subject may be positive for NKG2C (NKG2Cpos), and at least 70% of the NK cells in the peripheral blood sample may be negative or have low levels of NKG2A (NKG2Aneg). In some of the foregoing embodiments, the irradiated feeder cells may be HLA class I and HLA class II deficient. In some of the foregoing embodiments, the irradiated feeder cells may be 221.AEH cells. In some of the foregoing embodiments, the culture may be performed in the presence of two or more recombinant cytokines, where at least one recombinant cytokine may be interleukin (IL)-2 and at least one recombinant cytokine may be IL-21. In some of the foregoing embodiments, the recombinant cytokines may be IL-21 and IL-2. In some of the foregoing embodiments, the recombinant cytokines may be IL-21, IL-2, and IL-15.

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

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

[0040] In some of the foregoing embodiments, the g-NK cell composition may be formulated in a serum-free cryopreservation medium containing a cryoprotectant. In some embodiments, the cryoprotectant may be DMSO, and the cryopreservation medium may be 5%-10% DMSO (v / v). In some of the foregoing embodiments, each dose of g-NK cells may be 1 x 10 8 or approximately 1 x 10 8 From cells, 50 x 10 9 or approximately 50 x 10 9 In some embodiments, each dose of g-NK cells may be 5×108 The g-NK cell composition may be approximately 5 x 10 cells. 8 In some embodiments, each dose of g-NK cells may be 5×10 9 The g-NK cell composition may be approximately 5 x 10 cells. 9 In some embodiments, each dose of g-NK cells may be 10 x 10 9 The g-NK cell composition may be approximately 10 x 10 cells. 9 In some of the foregoing embodiments, the subject may be a human subject. In some of the foregoing embodiments, the NK cells in the composition may be allogeneic to the subject.

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

[0042] Pharmaceutical compositions comprising any of the engineered NK cells and a pharmaceutically acceptable carrier are also provided. In some of the aforementioned embodiments, the pharmaceutical composition may include a cryoprotectant. In some of the aforementioned embodiments, the pharmaceutical composition may be formulated in a serum-free cryopreservation medium containing the cryoprotectant. In some of the aforementioned embodiments, the cryoprotectant is DMSO. In some embodiments, the cryopreservation medium may be 5% to 10% DMSO (v / v).

[0043] Also provided herein is a method of treating cancer in a subject, comprising administering to a subject having cancer the pharmaceutical composition. [Brief explanation of the drawings]

[0044] [Figure 1] Figures 1A and 1B show the expansion of g-NK cells expanded in the presence of 221.AEH or K562-mbIL15-41BBL feeder cells with or without IL-21 in the NK cell culture medium. Figure 1A shows the total NK cell count. Figure 1B shows the fold expansion at day 21. [Figure 2] Figures 2A and 2B show daratumumab- and elotuzumab-mediated cytotoxic activity after 21 days of expansion of g-NK cells expanded in the presence of 221.AEH or K562-mbIL15-41BBL feeder cells with or without IL-21 in the NK cell culture medium. Figure 2A shows the cytotoxicity of g-NK cells against the LP1 cell line. Figure 2B shows the cytotoxicity of g-NK cells against the MM.1S cell line. [Figure 3-1]Figures 3A-3D show daratumumab- and elotuzumab-mediated degranulation levels (CD107apos) of g-NK cells expanded in the presence of 221.AEH or K562-mbIL15-41BBL feeder cells with or without IL-21 in the NK cell culture medium. Figure 3A shows the g-NK cell degranulation levels after 13 days of expansion for the LP1 cell line. Figure 3B shows the g-NK cell degranulation levels after 13 days of expansion for the MM.1S cell line. Figure 3C shows the g-NK cell degranulation levels after 21 days of expansion for the LP1 cell line. Figure 3D shows the g-NK cell degranulation levels after 21 days of expansion for the MM.1S cell line. [Figure 3-2] See description of Figure 3-1. [Figure 4-1] Figures 4A-4D show the levels of perforin and granzyme B expression in g-NK cells expanded in the presence of 221.AEH or K562-mbIL15-41BBL feeder cells with or without IL-21 in the NK cell culture medium. Figure 4A shows perforin and granzyme B expression as a percentage of g-NK cells after 13 days of expansion. Figure 4B shows the total expression of perforin and granzyme B after 13 days of expansion. Figure 4C shows perforin and granzyme B expression as a percentage of g-NK cells after 21 days of expansion. Figure 4D shows the total expression of perforin and granzyme B after 21 days of expansion. [Figure 4-2] See description of Figure 4-1. [Figure 5-1]Figures 5A-5D show 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 with or without IL-21 in the NK cell culture medium. Figure 5A shows the interferon-γ expression levels of g-NK cells after 13 days of expansion against the LP1 cell line. Figure 5B shows the interferon-γ expression levels of g-NK cells after 13 days of expansion against the MM.1S cell line. Figure 5C shows the interferon-γ expression levels of g-NK cells after 21 days of expansion against the LP1 cell line. Figure 5D shows the interferon-γ expression levels of g-NK cells after 21 days of expansion against the MM.1S cell line. [Figure 5-2] See description of Figure 5-1. [Figure 6-1] Figures 6A-6D show 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 with or without IL-21 in the NK cell culture medium. Figure 6A shows the TNF-α expression levels of g-NK cells after 13 days of expansion against the LP1 cell line. Figure 6B shows the TNF-α expression levels of g-NK cells after 13 days of expansion against the MM.1S cell line. Figure 6C shows the TNF-α expression levels of g-NK cells after 21 days of expansion against the LP1 cell line. Figure 6D shows the TNF-α expression levels of g-NK cells after 21 days of expansion against the MM.1S cell line. [Figure 6-2] See description of Figure 6-1. [Figure 7] Figure 1 depicts g-NK cell expansion of NK cells expanded for 15 days in the presence of various cytokine mixtures and cytokine concentrations. [Figure 8-1]Figures 8A-8J show the cellular effector function of g-NK cells expanded in the presence of various cytokine mixtures and concentrations. Figures 8A and 8B show the daratumumab- and elotuzumab-mediated cytotoxic activity of g-NK cells expanded in the presence of various 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. Figures 8C and 8D show the daratumumab- and elotuzumab-mediated degranulation levels (CD107apos) of g-NK cells expanded in the presence of various cytokine mixtures and concentrations. Figure 8C shows the degranulation level of g-NK cells against the LP1 cell line. Figure 8D shows the degranulation level of g-NK cells against the MM.1S cell line. Figures 8E and 8F show the expression levels of perforin and granzyme B in g-NK cells expanded in the presence of various cytokine mixtures and concentrations. Figure 8E shows the expression of perforin and granzyme B as a percentage of g-NK cells. Figure 8F shows the total expression of perforin and granzyme B. Figures 8G and 8H show the daratumumab- and elotuzumab-mediated interferon-γ expression levels of g-NK cells expanded in the presence of various cytokine mixtures and concentrations. Figure 8G shows the interferon-γ expression levels of g-NK cells for the LP1 cell line. Figure 8H shows the interferon-γ expression levels of g-NK cells for the MM.1S cell line. Figures 8I and 8J show the daratumumab- and elotuzumab-mediated TNF-α expression levels of g-NK cells expanded in the presence of various cytokine mixtures and concentrations. Figure 8I shows the TNF-α expression levels of g-NK cells for the LP1 cell line. Figure 8J shows the TNF-α expression levels of g-NK cells for the MM.1S cell line. [Figure 8-2] See description of Figure 8-1. [Figure 8-3] See description of Figure 8-1. [Figure 8-4] See description of Figure 8-1. [Figure 8-5] See description of Figure 8-1. [Figure 9-1]Figures 9A and 9B show the expansion of g-NK cells expanded in the presence of IL-21 compared to g-NK cells expanded without IL-21. Figure 9A shows the percentage of g-NK cells before and after expansion. Figure 9B shows the number of expanded g-NK cells per 10 million NK cells. Values are mean ± SE. #p<0.001 for expansion of CD3neg / CD57pos+IL-21 vs. expansion of CD3neg / CD57pos without IL-21. ^p<0.05 for expansion of CD3neg / CD57pos vs. expansion of other CMVpos. *p<0.001 for expansion of CMVpos vs. expansion of CMVnegCD3neg. Figure 9C shows a comparison of the g-NK proportion (% of total NK cells from CMV+ donors (n=8) and CMV- donors (n=6)) before and after expansion. Figure 9D shows a comparison of the n-fold expansion rate of g-NK cells from CMV+ and CMV- donors. Figure 9E shows a representative flow plot of FcεR1γ versus CD56 for CMV+ donors. Figure 9F shows a representative histogram of FcεR1γ expression on CD3- / CD56+ NK cells for CMV+ and CMV- donors. An independent-samples t-test was used to determine differences between CMV+ and CMV- donors before and after expansion (Figures 9C and 9D). Values are means ± SE. *p<0.05, **p<0.01, and ***p<0.001. Figures 9G and 9H show 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 cells expanded without 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. *p<0.05, **p<0.01, and ***p<0.001 for the comparison of expansion of CD3neg / CD57pos+IL-21 versus expansion of CD3neg / CD57pos without IL-21.Figures 9I and 9J show the daratumumab- and elotuzumab-mediated degranulation levels (CD107apos) of g-NK cells expanded in the presence of IL-21 compared to g-NK cells expanded without IL-21. Figure 9I shows the degranulation levels of g-NK cells after 14 days of expansion for the LP1 cell line. Figure 9J shows the degranulation levels of g-NK cells after 14 days of expansion for the MM.1S cell line. Values are mean ± SE. *p<0.05, **p<0.01, and ***p<0.001 for the comparison of expansion of CD3neg / CD57pos+IL-21 versus expansion of CD3neg / CD57pos without IL-21. Figures 9K and 9L show the levels of perforin and granzyme B expression in g-NK cells expanded in the presence of IL-21 compared to g-NK cells expanded without IL-21. Figure 9K shows the expression of perforin and granzyme B as a percentage of NK cells after 14 days of expansion. Figure 9L shows the total expression of perforin and granzyme B after 14 days of expansion. Values are means ± SE. *p<0.05, **p<0.01, and ***p<0.001 for the comparison of expansion of CD3neg / CD57pos + IL-21 versus expansion of CD3neg / CD57pos without IL-21. Figure 9M shows the baseline expression of perforin (left) and granzyme B (right) in expanded g-NK cells compared with cNK cells (n=5). An independent-samples t-test was used to compare the expression of the effectors perforin and granzyme B between g-NK and cNK. Values are means ± SE. Statistically significant differences from cNK cells are indicated by ***p<0.001. Figure 9N shows representative histograms of perforin and granzyme B expression for g-NK cells and cNK cells. Figures 9O and 9P show daratumumab- and elotuzumab-mediated interferon-γ expression levels in g-NK cells expanded in the presence of IL-21 compared to g-NK cells expanded without IL-21. Figure 9O shows interferon-γ expression levels in g-NK cells after 14 days of expansion for the LP1 cell line.Figure 9P shows interferon-γ expression levels of g-NK cells after 14 days of expansion against the MM.1S cell line. Values are means ± SE. *p<0.05, **p<0.01, and ***p<0.001 for the comparison of CD3 / CD57+IL-21 expansion versus CD3 / CD57 expansion without IL-21. Figures 9Q and 9R show daratumumab- and elotuzumab-mediated TNF-α expression levels of g-NK cells expanded in the presence of IL-21 compared to g-NK cells expanded without IL-21. Figure 9Q shows TNF-α expression levels of g-NK cells after 14 days of expansion against the LP1 cell line. Figure 9R shows TNF-α expression levels of g-NK cells after 14 days of expansion against the MM.1S cell line. Values are means ± SE. *p<0.05, **p<0.01, and ***p<0.001 for the comparison of CD3neg / CD57pos+IL-21 expansion versus CD3neg / CD57pos expansion without IL-21. Figure 9S shows the daratumumab- and elotuzumab-mediated interferon-γ expression levels of expanded g-NK cells compared to cNK cells against the MM.1S cell line among different donors. Figure 9T shows the daratumumab- and elotuzumab-mediated TNF-α expression levels of expanded g-NK cells compared to cNK cells against the MM.1S cell line among different donors. [Figure 9-2] See description of Figure 9-1. [Figure 9-3] See description of Figure 9-1. [Figure 9-4] See description of Figure 9-1. [Figure 9-5] See description of Figure 9-1. [Figure 9-6] See description of Figure 9-1. [Figure 9-7] See description of Figure 9-1. [Figure 10] Figures represent the expansion of g-NK cells expanded in the presence of IL-21 / anti-IL-21 complexes (n=4). Values are means ± SE. #p<0.001 for the comparison of expansion by IL-21 vs. expansion by IL-21 / anti-IL-21 complexes. [Figure 11-1] Figures 11A-11H show the NK cell effector function of previously cryopreserved g-NK cells compared with that of freshly enriched g-NK cells (n=4). Values are means ± SE. #p<0.05 for the comparison of freshly enriched g-NK cells vs. previously cryopreserved g-NK cells. Figures 11A and 11B show the daratumumab- and elotuzumab-mediated degranulation levels (CD107apos) 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. Figures 11C and 11D show the levels of perforin and granzyme B expression in 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 levels of g-NK cells. Figures 11E and 11F show the daratumumab- and elotuzumab-mediated interferon-γ expression levels 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 for the LP1 cell line. Figure 11F shows the interferon-γ expression levels of g-NK cells for the MM.1S cell line. Figures 11G and 11H show the daratumumab- and elotuzumab-mediated TNF-α expression levels 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 for the LP1 cell line. Figure 11H shows the TNF-α expression levels of g-NK cells for the MM.1S cell line. [Figure 11-2] See description of Figure 11-1. [Figure 12]Figures 12A-C show the persistence of cNK (cryopreserved) and g-NK (cryopreserved or fresh) cells in NSG mice after a single infusion of 1 x 107 expanded cells. Figure 12A shows the number 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 at the time of sacrifice, 31 days after infusion. Figure 12C shows the number of NK cells present in the bone marrow at the time of sacrifice. N = 3 for all three arms. Values are mean ± SE. *p<0.05 and ***p<0.001 for the comparison of cryopreserved cNK cells with fresh or cryopreserved g-NK cells. [Figure 13-1] Figures 13A-13D show the expression of CD20 (the target of rituximab), CD38 (the target of daratumumab), and SLAMF7 (the target of elotuzumab) in g-NK and cNK. Figure 13A shows the percentage of expanded g-NK cells, non-expanded NK cells (CD3 / CD56), and Raji cells that express CD20. Figure 13B shows the percentage of expanded g-NK cells, non-expanded NK cells (CD3 / CD56), and MM.1S cells that express CD38. Figure 13C shows the percentage of expanded g-NK cells, non-expanded NK cells (CD3 / CD56), and MM.1S cells that express SLAMF7. Figure 13D shows the percentage of cNK and g-NK cells that express CD38 before and after expansion. N=3 for each arm. Figure 13E shows the mean fluorescence intensity (MFI) for CD38 NK cells (n=4) before and after expansion. Figure 13F provides a representative histogram showing reduced CD38 expression on g-NK cells compared to cNK cells and MM.1S cells. Values are mean ± SE. #p<0.001 for g-NK cells vs. all other cells. Figure 13G shows a comparison of daratumumab-induced fratricide by expanded g-NK cells and cNK cells. [Figure 13-2] See description of Figure 13-1. [Figure 13-3] See description of Figure 13-1. [Figure 13-4] See description of Figure 13-1. [Figure 14-1]Figures 14A-F show the effects of treatment with 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 mouse model of multiple myeloma. 5 x 10 luciferase-labeled MM.1S human myeloma cells were injected intravenously (IV) into the tail vein of female NSG mice. Every week for 5 weeks, NSG mice received IV expanded NK cells (6.0 x 10 cells per mouse) and IP injections of daratumumab (10 μg per mouse). Figure 14A shows BLI imaging of mice twice weekly at days 20, 27, 37, 41, 48, and 57 after tumor inoculation (left). The corresponding number of days after treatment is indicated on the right side of the figure. Figure 14B shows tumor BLI (photons / second) in g-NK+Dara compared with control and cNK+Dara groups over time. *p<0.05 for g-NK vs. control or cNK groups. Figure 14C shows percent survival over time, with arrows indicating administration of treatment with either cNK+Dara or g-NK+Dara. Figure 14D shows the change in body weight of mice over time in the control, cNK+Dara, and g-NK+Dara groups. Figure 14E shows the number of CD138+ tumor cells present in the bone marrow at the time of sacrifice in cNK+Dara- and g-NK+Dara-treated mice. ***p<0.001 for g-NK vs. cNK cells. Values are mean ± SE. Figure 14F shows representative flow plots using the gating strategy to analyze the presence of NK cells and tumor cells in the control group and in mice treated with either cNK+Dara or g-NK+Dara. N=8 for the control group and N=7 for the g-NK or cNK groups. Figure 14G shows all BLI images collected throughout the study for all control, cNK+Dara, and g-NK+Dara-treated mice. Figure 14H shows X-ray images obtained for all mice in the control, cNK+Dara, and g-NK+Dara groups prior to sacrifice. Arrows indicate fractures and bone deformations. The date of sacrifice is indicated below each mouse. [Figure 14-2] See description of Figure 14-1. [Figure 14-3] See description of Figure 14-1. [Figure 14-4] See description of Figure 14-1. [Figure 14-5] See description of Figure 14-1. [Figure 15] Figures 15A-C present comparative data on persistent NK cells in NSG mice after treatment with cNK+Dara or g-NK+Dara. All data present the amount of cells detected using 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 present in the spleen. Figure 15C shows the number of NK cells present in the bone marrow. Values are mean ± SE. ***p<0.001 for g-NK cells compared to cNK cells. [Figure 16] The percentage of g-NK (CD45 / CD3 / CD56 / FcRγ) cells within cell subsets with either the surrogate extracellular surface phenotype CD45 / CD3 / CD56 / CD16 / CD57 / CD7dim / neg / CD161neg or CD45 / CD3 / CD56 / NKG2Aneg / CD161neg is shown. Values are means ± standard error. [Figure 17] Represents post-transduction expression of GFP and CD20-CAR by g-NK cells in two separate experiments, each using a different donor. [Figure 18] Figure 1 depicts the efficacy of g-NK cells with or without CD20-CAR in the presence or absence of rituximab (anti-CD20 monoclonal antibody) against Raji lymphoma cells. [Figure 19] Represents the percentage of viable g-NK cells expressing CD20 CAR after electroporation. [Figure 20]Figures 20A and 20B show the expression of CD19, CD20, and CD38 by Raji lymphoma cells. Figure 20A identifies Raji cells by their expression of CD19. Figure 20B confirms the expression of CD20 and CD38 by Raji cells. [Figure 21] Figures 21A and 21B demonstrate antibody-dependent cell-mediated cytotoxicity (ADCC) exhibited by g-NK cells with or without CD20 CAR expression and in the presence or absence of daratumumab (anti-CD38 monoclonal antibody) against Raji lymphoma cells. Figure 21A represents the percentage of Raji cell death within each condition at an effector-to-target ratio of 0.05:1. Figure 21B instead represents the number of Raji cells killed per NK cell within each condition at an effector-to-target ratio of 0.05:1. The percentage of Raji cell death is calculated without including spontaneous Raji cell death. DETAILED DESCRIPTION OF THE INVENTION

[0045] Detailed Description Provided herein are methods for administering engineered natural killer (NK) cells (g-NK cells) lacking expression of the FcRγ chain, comprising a recombinant chimeric antigen receptor (CAR), in combination with an antibody (e.g., a monoclonal antibody). 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 is secretable from the g-NK cells. Natural killer (NK) cells are innate lymphocytes important for mediating antiviral and anticancer immunity through the secretion of cytokines and chemokines and the release of 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 constitute the third largest population of lymphocytes and are important for host immune surveillance against tumor cells and pathogen-infected cells. However, unlike T and B lymphocytes, NK cells use germline-encoded activating receptors and are thought to have 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)).

[0046] NK cell activation can occur either through direct binding of the NK cell receptor to a ligand on the target cell, as seen in direct tumor cell killing, or through cross-linking of the Fc receptor (also known as CD16, CD16a, or FcγRIIIa) by binding to the Fc portion of an antibody bound to an antigen-bearing cell. Upon activation, NK cells abundantly produce cytokines and chemokines and simultaneously exhibit potent cytolytic activity. NK cells can kill tumor cells by antibody-dependent cell-mediated cytotoxicity (ADCC). In some cases, ADCC is triggered when receptors on the NK cell surface (such as CD16) recognize IgG1 or IgG3 antibodies bound to the cell surface. This triggers the release of cytoplasmic granules containing perforin and granzymes, which leads to the death of the target cell. NK cells express the activating Fc receptor CD16, which recognizes IgG-coated target cells, thus broadening target recognition (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 primarily mediated by NK cells.

[0047] CD16 also exists in a glycosylphosphatidylinositol-anchored form (also known as FcγRIIIB or CD16B). As used herein, CD16 refers to the CD16a form, which is expressed on NK cells and is involved in antibody-dependent responses (such as NK cell-mediated ADCC), and does not refer to the glycosylphosphatidylinositol-anchored form.

[0048] The CD16 receptor associates with the adaptor ζ chain of the TCR-CD3 complex (CD3ζ) and / or the FcRγ chain, enabling signaling via immunoreceptor tyrosine-based activation motifs (ITAMs). In certain contexts, CD16 engagement (CD16 cross-linking) initiates NK cell responses through intracellular signals generated by one or both of the CD16-associated adaptor chains, FcRγ and CD3ζ. CD16 triggering leads to phosphorylation of the γ or ζ chain, which then recruits the tyrosine kinases Syk and ZAP-70, initiating a signaling cascade leading to rapid and potent effector functions. The most well-known effector function is the release of cytoplasmic granules carrying toxic proteins for killing nearby target cells by the process of antibody-dependent cellular cytotoxicity. CD16 cross-linking also leads to the production of cytokines and chemokines, which then activate and orchestrate a range of immune responses.

[0049] This release of cytokines and chemokines may play a role in the anticancer activity of NK cells in vivo. NK cells also have small granules in their cytoplasm that contain perforin and proteases (granzymes). Upon release from NK cells, perforin forms small pores in the plasma membrane of target cells, through which granzymes and related molecules can enter and induce apoptosis. The fact that NK cells induce apoptosis rather than necrosis in target cells is important—necrosis of virus-infected cells would result in the release of virions, whereas apoptosis leads to the destruction of the virus within the cell.

[0050] A specialized subset of NK cells lacking the FcRγ adaptor protein, also known as g-NK cells, can mediate robust ADCC responses (see, e.g., Patent Application No. US2013 / 0295044). The mechanism of enhanced responses may be due to altered epigenetic modifications affecting FcRγ expression. g-NK cells abundantly express the signaling adaptor ζ chain but lack expression of the signaling adaptor γ chain. Compared to conventional NK cells, these gamma-deficient g-NK cells exhibit dramatically enhanced activity when activated by antibodies. For example, g-NK cells can be activated by antibody-mediated crosslinking of CD16 or by antibody-coated tumor cells. In some aspects, g-NK cells produce greater amounts of cytokines (e.g., IFN-γ or TNF-α) and chemokines (e.g., MIP-1α, MIP-1β, and RANTES) and / or exhibit a more extensive degranulation response than gamma-chain-expressing conventional NK cells. g-NK cells express elevated levels of granzyme B, a component of the cytotoxic machinery of natural killer cells. Furthermore, g-NK cells have a longer lifespan than conventional NK cells, and their existence is maintained for extended periods of time. In some embodiments, g-NK cells are functionally and phenotypically stable.

[0051] In some embodiments, g-NK cells are more effective at eliciting an ADCC response than conventional NK cells, e.g., NK cells that are not gamma chain deficient. In some embodiments, g-NK cells are more effective at eliciting cell-mediated cytotoxicity than conventional NK cells, even in the absence of antibodies. In some cases, ADCC is the mechanism of action of therapeutic antibodies, including anti-cancer antibodies. In some aspects, cell therapy by administering NK cells can be used in conjunction with antibodies for therapeutic and related purposes.

[0052] For example, certain therapeutic monoclonal antibodies, such as daratumumab, which targets CD38, and elotuzumab, which targets SLAMF7, have been approved by the FDA for the treatment of diseases such as multiple myeloma (MM). While clinical responses to therapeutic antibodies have been promising, they are often not ideal. For example, although initial clinical responses have generally been encouraging, particularly for daratumumab, essentially all patients eventually develop progressive disease. Thus, there is a great need for new strategies to elicit deeper remissions or overcome resistance to these agents. The provided embodiments, including compositions, address these needs.

[0053] Provided herein are engineered natural killer (NK) cells (g-NK cells) lacking expression of the FcRγ chain and further comprising a recombinant chimeric antigen receptor (CAR), and compositions containing the same. Methods for engineering g-NK cells are also provided. In some embodiments, CAR-dependent antigen targeting by engineered g-NK cells leads to improved patient outcomes due to improved affinity, cytotoxicity, and / or cytokine-mediated effector function of g-NK cell subsets. It is discovered herein that CAR-dependent antigen targeting can be combined with antibody-mediated targeting of g-NK cells via CD16 engagement and ADCC activity. In other words, the results herein demonstrate that antibody-mediated targeting via ADCC is not impaired in CAR-engineered T cells, even though both signal through the same CD3ζ signaling pathway. These results indicate that these two mechanisms of antigen-mediated killing by g-NK cells can be employed as a combined therapeutic strategy to further improve target cell killing.

[0054] These methods offered improvements over conventional NK cells. Conventional NK cells are typically activated when the Fc portion of an antibody binds the NK cell's Fc receptor (FcγRIIIa or CD16a), triggering activation and degranulation through a process involving the adaptor proteins CD3ζ and FcεR1γ. Binding and cross-linking of the Fc receptor CD16 on conventional NK cells activates signaling through both CD3ζ and FcεR1γ, which can result in signaling variations depending on the expression of signaling adaptors on the NK cell. Finally, NK cell activity often requires cytokine support, such as IL-15, to enhance cytotoxic activity; therefore, the absence of sufficient supportive cytokines can limit the durability of the response. Each of the above factors, alone and together, has hindered the usefulness of certain NK cell therapies.

[0055] The engineered NK cells and compositions containing the same provided herein, such as those produced by the provided methods, offer improved cell therapy in several respects. First, the provided g-NK cells and compositions containing the same, such as those produced by the provided methods, are engineered to express a chimeric antigen receptor (CAR). Expression of a CAR enables g-NK cells to target cells or tissues in a diseased subject or individual in an antibody-independent manner. Furthermore, combination therapy with a monoclonal antibody enables potent ADCC-mediated antibody-mediated targeting of g-NK cells to target cells or tissues in a diseased subject or individual. The provided cells and compositions produced by such methods are particularly robust in their ability to target g-NK cells to the appropriate location in a subject or individual. Surprisingly, this result is made possible by the potent ADCC activity of g-NK cells, which is not impaired by co-expression of a CAR in the engineered g-NK cells.

[0056] These two mechanisms of antigen-driven targeted killing of target cells, such as cancer cells, enable improved strategies for targeting certain cancers. While clinical trial results have shown promising clinical efficacy of CAR T-cell therapy for certain hematological malignancies, relapse with reduced or complete loss of cell surface antigen expression is observed in approximately 30–50% of patients who achieve remission after treatment with anti-CD19 CAR T cells, usually within one year of treatment. Relapse with antigen loss has also been reported with CARs directed against other targets, such as CD22 and B-cell maturation antigens, highlighting antigen escape as a significant and common obstacle to the success of CAR T-cell therapy. Beyond hematological cancers, antigen escape can pose an even greater challenge in solid tumors, which are generally composed of cells with heterogeneous antigen expression. Therefore, targeting a single antigen carries the risk of immune escape, which can be overcome by targeting multiple desired antigens, especially in solid tumors with higher tumor heterogeneity. Therefore, there remains a need for improved chimeric antigen receptor-cell-based therapies that allow for more effective, safe, and efficient targeting of various cancers, such as B-cell-related malignancies (ALL, CLL, and NHL), multiple myeloma, AML, lymphoma, as well as many other solid tumors.

[0057] Among the provided embodiments are methods relating to combination therapy, in which target cells are contacted with g-NK cells engineered with a CAR containing an extracellular antigen-binding domain (e.g., scFv) that binds to a first antigen and a monoclonal antibody that binds to a second antigen. The first and second antigens may be the same or different. Typically, when 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 of a disease or condition, such as cancer. In some embodiments, the first and second antigens are expressed on the same target cell. In some embodiments, the first and second antigens are both expressed on different target cells associated with the disease or condition, e.g., due to tumor heterogeneity. In some embodiments, the monoclonal antibody is a recombinant molecule that is administered separately to a subject. In some embodiments, the g-NK cells are engineered with a secretable monoclonal antibody. In some embodiments, the method comprises administering to a subject having a disease or condition, such as cancer, a composition of g-NK cells engineered to express a CAR to target a first antigen and a monoclonal antibody to target a second antigen. In some embodiments, the method comprises administering to a subject having a disease or condition, such as cancer, a composition of g-NK cells engineered to express a CAR to target a first antigen and engineered with a secretable monoclonal antibody that targets a second antigen.

[0058] In particular, provided embodiments relate to NK cell compositions enriched in a specialized subset of g-NK cells (i.e., NK cells lacking FceRIγ), which offer numerous advantages over conventional NK cells or NK cells enriched in other subsets. Because g-NK cells are detectable at levels of approximately 3-10% of total NK cells in only 25-30% of CMV-seropositive individuals, g-NK cells are a relatively rare subset. Methods such as those described herein can be used to provide particularly robust expansion and enrichment of g-NK cells, thus enabling sufficient expansion required for in vivo use while also being suitable for manipulation of enriched g-NK cells with CARs before, during, or after g-NK cell expansion. The provided cells and compositions produced by such methods are particularly robust in their ability to target g-NK cells to appropriate locations within a subject or individual.

[0059] g-NK cells represent a relatively small percentage of NK cells in peripheral blood, which limits the availability of these cells for therapeutic use. Specifically, clinical use of g-NK cells requires a high preferential expansion rate, as g-NK cells are generally a rare population. Other methods for expanding NK cells can achieve a 1000-fold expansion rate of NK cells in 14 days, but they are limited to the poorly differentiated NKG2C neg , FceRIγ pos (FcRγ pos ) NK cells (Fujisaki et al. (2009) Cancer Res., 69:4010-4017; Shah et al. (2013) PLoS One, 8:e76781). Furthermore, optimized expansion of NK cells with phenotypes overlapping with g-NK cells has been found not to preferentially expand g-NK cells to therapeutically viable levels. Specifically, using HLA-E-transfected 221.AEH cells and incorporating IL-15 into the culture medium, NKG2C cells with phenotypes overlapping with g-NK cells were found to be phenotypically ... posIt has been previously reported that culturing with such HLA-expressing cells, which constitutively express HLA-E, can preferentially expand NK cells (Bigley et al. (2016) Clin. Exp. Immunol., 185:239-251). NK cells are differentiated into NKG2C pos / NKG2A neg (NKG2C is an activating receptor for HLA-E, while NKG2A is an inhibitory receptor for HLA-E.) It was thought that such a method would be sufficient to expand g-NK cells, since such cells contain g-NK within them. However, this method does not achieve robust expansion of g-NK cells.

[0060] The expansion methods described herein overcome these limitations and can generate NK cell compositions enriched for g-NK cells. The provided methods utilize a greater ratio of HLA-E+ feeder cells, e.g., 221.AEH cells, lacking HLA class I and HLA class II, to NK cells compared to previous methods. Specifically, previous methods used lower ratios of 221.AEH cells, e.g., NK cells to 221.AEH cells, e.g., a 10:1 ratio. It is demonstrated herein that a greater ratio of HLA-E-expressing feeder cells, e.g., 221.AEH cells, results in greater overall expansion, more strongly biased toward the g-NK phenotype. In some embodiments, a greater ratio of HLA-E+ feeder cells, e.g., 221.AEH cells, is achieved by irradiating the feeder cells. In some aspects, the use of irradiated feeder cell lines is advantageous because it provides a method that is GMP-compliant. During expansion, incorporation of any of recombinant IL-2, IL-7, IL-15, IL-12, IL-18, IL-21, IL-27, or a combination thereof has also been shown to support robust expansion. In certain embodiments of the provided methods, at least one recombinant cytokine is IL-2. In some embodiments, two or more recombinant cytokines are present, wherein at least one recombinant cytokine is IL-2 and at least one recombinant cytokine is IL-21.

[0061] The method for expanding g-NK cells is based on the finding that culturing NK cells for expansion in the presence of IL-21 further enhances the production of cytokines or effector molecules, such as perforin and granzyme B, by the NK cells. Compositions containing NK cells produced by the expanded process herein are highly functional, exhibit robust proliferation, and function well without rescue even after being frozen. For example, NK cells produced by the provided process not only exhibit strong ADCC activity when expanded in the presence of IL-21, but also antibody-independent cytotoxicity. Because the NK cells are primed and ready to exhibit effector activity immediately after CAR engagement with the target antigen, robust activity, including antibody-independent cytotoxicity, is particularly suited to the strategy described herein in which the cells are further engineered with a CAR and immunomodulatory factors. For example, effector molecules (e.g., perforin and granzyme) are naturally present in NK cells expanded by the provided methods, thereby providing cells with enhanced cytotoxic potential. As shown herein, NK cell compositions produced by the provided processes that include IL-21 (e.g., IL-2, IL-15, and IL-21) not only have a higher percentage of NK cells positive for perforin or granzyme B than NK cell compositions produced by processes that include only IL-2 without added IL-21, but also have a higher average level or degree of expression of these molecules in the cells. Furthermore, NK cell compositions produced by the methods provided herein that include IL-21 (e.g., IL-2, IL-15, and IL-12) also result in g-NK cell compositions that exhibit substantial effector activity, including the ability to degranulate and express greater amounts of IFN-γ and TNF-α in response to target cells. This functional activity is highly preserved even after cryopreservation and thawing of the expanded NK cells. The significant increase in cytolytic enzymes, along with a more robust activation phenotype, reinforces the enhanced ability of the expanded g-NK cells to induce apoptosis of tumor targets.Many of these activities are exemplified in the Examples herein upon engagement with an antibody via CD16 cross-linking, but signaling is also mediated via CD3ζ, so similar activities occur upon engagement of the CAR with a target antigen. The prominent antibody-independent effector phenotype, coupled with manipulation of the cells with CARs and immune modulators (e.g., cytokines), also supports the potential utility of g-NK cells as monotherapy.

[0062] Furthermore, in some embodiments, g-NK cells produce significantly more cytokines than natural killer cells that express 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 significantly more 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 signaling via CD3ζ, which can occur, for example, via CAR engagement or, in some cases, stimulation through the Fc receptor CD16.

[0063] Furthermore, the findings herein demonstrate that donated NK cells expanded in the presence of IL-21 have the potential to persist and proliferate for longer periods than, for example, cells expanded in the presence of IL-2 alone without the addition of IL-21. Furthermore, the results also show that cryopreserved g-NK cells persist at levels comparable to fresh g-NK cells. This significantly improved persistence highlights the potential utility of fresh or cryopreserved g-NK as an off-the-shelf cell therapy for enhancing targeted cytotoxicity. This finding of improved persistence is advantageous, as the clinical utility of many NK cell therapies has been compromised by the limited persistence of NK cells.

[0064] It has also been found that enriching NK cells from a cell sample prior to expansion methods, for example by enrichment for CD16 cells or CD57 cells prior to expansion, further substantially increases the amount of g-NK cell expansion that can be achieved compared to methods that initially enrich for NK cells based solely on CD3 depletion. In another embodiment, another enrichment that can be performed prior to expansion is enriching for NK cells by positive selection for CD56 and negative selection or depletion for CD38. In a further embodiment, another enrichment that can be performed prior to expansion is enriching for NK cells by positive selection for CD56 followed by NKG2A depletion. neg Negative selection or depletion for and CD161 neg In another embodiment, prior to expansion, another enrichment that can be performed is to enrich for NK cells by positive selection for CD57 followed by negative selection or depletion for NKG2A and / or positive selection for NKG2C. In another embodiment, prior to expansion, another enrichment that can be performed is to enrich for NK cells by positive selection for CD56 followed by negative selection or depletion for NKG2A and / or positive selection for NKG2C. In any of such embodiments, NKG2C pos and / or NKG2A neg Enrichment for NK cells can be performed after expansion.

[0065] In any of such embodiments, the enriched NK cells can be enriched from a cell sample containing NK cells, e.g., from peripheral blood mononuclear cells (PBMCs). In some embodiments, prior to enriching NK cells from the cell sample, T cells can be removed by negative selection or depletion for CD3. In any of such embodiments, the enriched NK cells can be enriched from a biological sample (e.g., PBMCs) from a human subject containing NK cells, with a relatively high proportion of g-NK cells, e.g., from a human subject selected for a high percentage of g-NK cells among their NK cells. In any of such embodiments, the enriched NK cells can be enriched from a biological sample (e.g., PBMCs) from a human subject containing NK cells, with a relatively high proportion of NKG2C cells. pos NK cells (e.g., 20% or about 20% or more than 20% NKG2C pos NK cells) and / or NKG2A neg NK cells (e.g., 70% or about 70% or more than 70% NKG2A neg The enriched NK cells can be enriched from a biological sample from a human subject containing NK cells, e.g., PBMCs, containing NK cells. In any of such embodiments, the enriched NK cells can be determined by determining whether the sample contains a relatively high proportion of NKG2C pos NK cells (e.g., 20% or about 20% or more than 20% NKG2C pos NK cells) and NKG2A neg NK cells (e.g., 70% or about 70% or more than 70% NKG2A neg The NK cells can be enriched from a biological sample from a human subject containing NK cells, such as PBMCs, containing g-NK cells. In certain embodiments, the subject from which the sample is derived is CMV seropositive, such as a subject with more detectable g-NK cells in their peripheral blood.

[0066] Taken together, the provided approaches for expanding g-NK cells can achieve an expansion of NK cells from an initial 10 million enriched NK cells at the initiation of culture to over 1 billion cells, and in some cases up to 8 billion or more cells. In particular, the provided methods can result in high-yield (>1000-fold) expansion rates, with g-NK cell functionality maintained and in some cases increased after expansion. In some embodiments, the provided methods can result in g-NK cell populations that express high levels of perforin and granzyme B. Furthermore, the provided methods prove sufficient to expand previously frozen NK cells, which is generally not achieved by many existing methods involving the rescue 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 decreasing the ratio of HLA-E+ feeder cells to NK cells, for example, to about 1:1 221.AEH to NK cells. In some embodiments, this is accomplished by incorporating any of recombinant IL-2, IL-7, IL-15, IL-12, IL-18, IL-21, IL-27, or combinations thereof during expansion. In particular embodiments, at least one recombinant cytokine is IL-2. In some embodiments, expansion is carried out in the presence of two or more recombinant cytokines, where at least one is recombinant IL-21 and at least one is recombinant IL-2.

[0067] As demonstrated herein, the engineered g-NK cells provided and compositions containing the same, produced by the provided methods, can be used for cancer treatment. In some embodiments, adoptive transfer of NK cells does not result in severe graft-versus-host disease (GVHD), and thus, such cell therapy can be administered in an "off-the-shelf" manner for clinical use. In some aspects, NK cells can be further engineered to reduce or eliminate individual HLA molecules in the NK cells, thereby improving the potential for allogeneic use of the provided cell therapy.

[0068] All references mentioned in this specification, including patent applications, patent publications, and scientific literature and databases, are incorporated by reference in their entirety for all purposes to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference.

[0069] 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 are not to be construed as limiting the subject matter described.

[0070] I. Definition Unless otherwise defined, all terminology, notations, and other technical and scientific terms and terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. In some cases, terms with commonly understood meanings are defined herein for the sake of clarity and / or ready reference, but the inclusion of such definitions herein should not necessarily be construed as representing a substantial departure from what is commonly understood in the art.

[0071] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a molecule" optionally includes a combination of two or more such molecules.

[0072] The term "about" as used herein refers to a normal error range for each value that is readily apparent to one skilled in the art. When "about" is used herein with respect to a value or parameter, the embodiment directed to the value or parameter itself is included (and described).

[0073] Aspects and embodiments of the invention described herein are understood to encompass "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0074] As used herein, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description encompasses both 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 unsubstituted or substituted.

[0075] As used herein, "antibody" refers to immunoglobulins and immunoglobulin fragments, whether natural or partially or wholly synthetically, e.g., recombinantly produced, including any fragment thereof that contains at least a portion of the variable heavy and / or variable light chain region of the immunoglobulin molecule sufficient to form an antigen-binding site and, when assembled, to specifically bind to an antigen. Thus, an antibody encompasses any protein having a binding domain that is homologous or substantially homologous to an immunoglobulin antigen-binding domain (antibody combining site). Typically, an antibody will contain, at a minimum, a variable heavy (V H ) chain and / or variable light (V L ) chain. Generally, V H and V L pair with each other to form the antigen-binding site, but in some cases, a single V H Domain or V L A single domain is sufficient for antigen binding. An antibody can also include all or a portion of a constant region. As used herein, antibodies include full-length antibodies and antigen-binding fragments. The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein.

[0076] The terms "full-length antibody," "intact antibody," or "whole antibody" are used interchangeably to refer to antibodies in a substantially intact form, rather than antibody fragments. Full-length antibodies typically include antibodies having 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 those produced by antibody-secreting B cells from mammalian species (e.g., humans, mice, rats, rabbits, non-human primates, etc.), as well as synthetically produced antibodies having the same domains. In particular, whole antibodies include heavy and light chains, including an Fc region. The constant domains can be native-sequence constant domains (e.g., human native-sequence constant domains) or amino acid sequence variants thereof. In some cases, intact antibodies can have one or more effector functions.

[0077] An "antibody fragment" includes a portion of an intact antibody, the antigen-binding and / or variable region of the intact antibody. Antibody fragments include Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fvs (dsFv), Fd fragments, Fd' fragments, diabodies, linear antibodies (see Example 2 of U.S. Pat. No. 5,641,870; Zapata et al., Protein Eng. 8(10) :1057-1062

[1995] ), single-chain antibody molecules such as single-chain Fv (scFv) or single-chain Fab (scFab), antigen-binding fragments of any of the above, and multispecific antibodies formed from antibody fragments. For purposes of this specification, antibody fragments typically include those sufficient to engage or cross-link CD16 on the surface of NK cells.

[0078] The term "autologous" refers to cells or tissues that originate within or are taken from an individual's own tissues. For example, in autologous transfer or transplantation of NK cells, the donor and recipient are the same person.

[0079] The term "allogeneic" refers to cells or tissues that belong to or are obtained from the same species but are genetically distinct and, in some cases, therefore immunologically incompatible. Typically, the term "allogeneic" is used to define cells that are transplanted from a donor into a recipient of the same species.

[0080] The term "enriched," with respect to a cell composition, refers to a composition in which the number or percentage of a cell type or cell population is increased compared to the number or percentage of that cell type in the same volume of starting composition, e.g., a starting composition obtained or isolated directly from a subject. The term does not require the complete removal of other cells, cell types, or cell populations from the composition, nor does it require that such enriched cells be 100% or even close to 100% present in the enriched composition.

[0081] The term "expression" refers to the process by which a polynucleotide is transcribed from a DNA template (e.g., into mRNA or other RNA transcript) and / or the process by which the transcribed mRNA is then translated into a peptide, polypeptide, or protein. Collectively, the transcript and the encoded polypeptide may be referred to as the "gene product." If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0082] 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 a cell, for example, because it originates from an organism or individual other than the cell in which it is expressed. It is understood that reference to "heterologous" does not exclude that the protein or nucleic acid may be naturally expressed by the cell into which it is introduced. A heterologous nucleic acid or encoded protein can be introduced into an NK cell by any of a variety of methods by which a nucleic acid (e.g., encoding a heterologous protein) can be introduced or transformed into a cell, including viral-based methods such as transduction or non-viral delivery methods such as electroporation or lipid nanoparticle delivery. Transduced or transformed NK cells can harbor exogenous or heterologous nucleic acid integrated extrachromosomally or intrachromosomally. Integration into the cellular genome and a self-replicating vector generally results in genetically stable inheritance of the transformed nucleic acid molecule. NK cells containing transformed nucleic acid are referred to as "genetically engineered" but may also be interchangeably referred to as "recombinant" or "transformed."

[0083] As used herein, the term "introduce" encompasses various methods of introducing DNA into cells either in vitro or in vivo, including transformation, transduction, transfection (e.g., electroporation), lipid delivery, and infection. Vectors are useful for introducing DNA encoding molecules into cells. 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, either DNA or mRNA, into cells.

[0084] The terms "polynucleotide," "nucleotide sequence," "nucleic acid," "nucleic acid molecule," "nucleic acid sequence," and "oligonucleotide" refer to a series of nucleotide bases (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 derivatives, or modified versions thereof. They can be modified at the base moiety, sugar moiety, or phosphate backbone to improve, for example, the molecule's stability, its hybridization parameters, etc. Nucleotide sequences typically carry genetic information, including, but not limited to, information used by cellular machinery to make proteins and enzymes. These terms include double-stranded or single-stranded genomic DNA, RNA, any synthetic and genetically engineered polynucleotides, and both sense and antisense polynucleotides. These terms also include nucleic acids containing modified bases.

[0085] The terms "protein," "peptide," and "polypeptide" are used interchangeably to refer to a continuous chain of amino acids linked together via peptide bonds. These terms include individual proteins, groups or complexes of proteins associated together, as well as fragments or portions, variants, derivatives, and analogs of such proteins. Peptide sequences are represented herein using conventional notation, beginning with the amino or N-terminus on the left and proceeding to the carboxyl or C-terminus on the right. Standard one-letter or three-letter abbreviations can be used.

[0086] The term "endogenous" as used herein with respect to a nucleic acid (e.g., a gene, a genomic region encoding a protein, a promoter) refers to a naturally occurring nucleic acid or protein in its natural location, e.g., within the genome of a cell. In contrast, the term "exogenous" as used herein with respect to a nucleic acid, e.g., an expression construct, cDNA, indel, and nucleic acid vector, refers to a nucleic acid that has been artificially introduced into the genome of a cell using genetic engineering techniques, such as, for example, transformation of a heterologous nucleic acid or gene editing, e.g., CRISPR-based editing techniques.

[0087] 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, non-aqueous, or any combination thereof. The preparation is generally in a form that allows the biological activity of the active ingredient (e.g., antibody) to be effective.

[0088] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0089] As used herein, a combination refers to any association between two or more items. The combination can be two or more separate items, such as two compositions or two collections, or a mixture thereof, such as a single mixture of two or more items, or any variation thereof. The elements of a combination are generally functionally related or associated.

[0090] As used herein, a kit is a packaged combination, optionally including other elements, such as additional agents, and instructions for use of the combination or elements thereof, for example, but not limited to, therapeutic use.

[0091] As used herein, the term "treatment" or "treating" refers to a clinical intervention designed to alter the natural course of the treated individual or cell during the course of clinical pathology. Desirable effects of treatment include reducing the rate of disease progression, ameliorating or alleviating the disease state, and remission or improved prognosis. An individual is successfully "treated," for example, if one or more symptoms associated with a disorder (e.g., an eosinophil-mediated disease) are alleviated or eliminated. For example, an individual is successfully "treated" if the treatment improves the quality of life of the person suffering from the disease, reduces the dosage of other medications required to treat the disease, reduces the frequency of disease recurrence, reduces the severity of the disease, delays the onset or progression of the disease, and / or prolongs the individual's survival.

[0092] An "effective amount" refers to at least an amount effective, at dosages and for periods of time necessary, to achieve a desired or indicated effect, including a therapeutic or preventative result. An effective amount can be provided one or more times. A "therapeutically effective amount" is at least the minimum dose of cells necessary 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 may vary depending on factors such as the patient's disease state, age, sex, and weight. A therapeutically effective amount may also be an amount in which the therapeutically beneficial effects of the antibody outweigh any toxic or detrimental effects of the antibody. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired preventative result. Because a prophylactic dose is used in pre-diseased subjects or subjects at an early stage of disease, typically, but not necessarily, the prophylactically effective amount may be less than the therapeutically effective amount.

[0093] As used herein, an "individual" or "subject" is a mammal. With respect to treatment, "mammals" includes humans, domestic and farm animals, as well as zoo, sport, or pet animals, such as dogs, horses, rabbits, cows, pigs, hamsters, gerbils, mice, ferrets, rats, cats, etc. In some embodiments, the individual or subject is a human.

[0094] II. Methods of Cytolytic Killing and Treatment Provided herein are methods for cytolytic killing of target cells, comprising a combination of antibody therapy and a g-NK cell composition comprising engineered g-NK cells comprising heterologous nucleic acid encoding an antigen receptor (e.g., CAR). In some embodiments, the provided methods comprise: (a) a composition comprising natural killer (NK) cells (g-NK cells) lacking expression of an FcRγ chain, 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 cytolytic killing of the target cells occurs in vivo in a subject. In some embodiments, the target cells are associated with a disease or condition, and the cytolytic killing of the target cells is a treatment for the disease or condition. In some embodiments, the target cells are cancer cells, and the method can be used to treat cancer.

[0095] Also provided herein are methods and uses for combination therapy comprising g-NK cell compositions comprising engineered g-NK cells comprising heterologous nucleic acid encoding an antigen receptor (e.g., CAR) in combination with antibody therapy for use in treating a disease or condition. In such methods, the CAR binds to a first antigen expressed by cells of the disease or condition, and the antibody therapy binds to a second antigen expressed by cells of the disease or condition. In some embodiments, the cells are the same cells. In some embodiments, the antibody is administered to a subject known or suspected to have a disease or condition distinct from the g-NK cells. In some embodiments, the disease or condition is cancer. In some embodiments, a method comprises: (a) administering to a subject having cancer an NK cell therapy comprising a dose of a composition comprising natural killer (NK) cells (g-NK cells) that lack expression of the FcRγ chain, wherein the g-NK cells express a chimeric antigen receptor (CAR) comprising an extracellular binding domain that binds to a first antigen expressed by cells of the cancer; and (b) administering to the subject a dose of an antibody that binds to a second antigen expressed by cells of the cancer. In some embodiments, the antibody is secretable from the g-NK cells. In some embodiments, a method comprises administering to a subject having cancer an NK cell therapy comprising a dose of a composition comprising natural killer (NK) cells (g-NK cells) that lack expression of the FcRγ chain, wherein the g-NK cells express a chimeric antigen receptor (CAR) comprising an extracellular binding domain that binds to a first antigen expressed by cells of the cancer; and the g-NK cells express a secretable antibody that binds to a second antigen expressed by cells of the cancer.

[0096] In the provided methods, compositions containing the engineered g-NK cells provided herein exhibit ADCC-mediated activity when activated or contacted with an antibody or Fc-containing protein. In some embodiments, the provided g-NK cells exhibit uniquely enhanced ADCC activity, e.g., compared to conventional NK cells. For example, g-NK cells can be activated by antibody-mediated crosslinking of CD16. In some embodiments, provided herein are methods for treating a condition in an individual, comprising administering engineered g-NK cells, or compositions thereof, and an antibody to a subject. In some embodiments, the antibody is capable of binding to and engaging CD16 on the surface of NK cells. In some embodiments, the antibody contains an Fc domain. In some embodiments, the antibody is an IgG1 Fc antibody. In some embodiments, the antibody is a full-length antibody. In certain embodiments, any of such antibodies in the provided methods is a monoclonal antibody.

[0097] In some aspects, provided methods can provide a dual targeting strategy for killing cancer cells.In some embodiments, the dual targeting strategy improves the killing of cancer cells, thereby treating disease or condition, for example, by increasing the specificity of targeting cancer cells or by providing a compensatory strategy for target cell killing in the case of antigen escape.In some embodiments, provided methods increase the possibility of killing cancer cells, for example, by the additive effect of two therapies (CAR and antibody) that provide NK cells with different cytolytic killing mechanisms.

[0098] Such methods and uses include, for example, therapeutic methods and uses comprising administering g-NK cells and antibodies to a subject having a disease, condition, or disorder. 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 pharmaceutical compositions thereof are administered in an amount effective to treat the disease or disorder. Uses include the use of the cells and antibodies or pharmaceutical compositions thereof in such methods and treatments, and in the preparation of medicaments for carrying out such therapeutic methods. In some embodiments, the method thereby treats a disease, condition, or disorder in a subject.

[0099] In some embodiments, the provided methods and uses may be any of the provided NK cell compositions comprising engineered g-NK cells, and may include methods and uses as described in PCT Publication No. WO2020 / 107002 or PCT Application No. PCT / US2021 / 028504.

[0100] The provided engineered g-NK cell compositions can be used in methods for treating individuals with tumors or hyperproliferative disorders. The provided engineered g-NK cell compositions can be administered to treat animals, such as mammals, e.g., human subjects. In some examples, the method includes treating a hyperproliferative disorder, such as a hematological malignancy or a solid tumor. Examples of types of cancers and proliferative disorders that can be treated with the compositions described herein include, but are not limited to, multiple myeloma, leukemia (e.g., myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia, chronic myelocytic (granulocytic) leukemia, and chronic lymphocytic leukemia), lymphoma (e.g., Hodgkin's disease and non-Hodgkin's disease), fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, angiosarcoma, endothelial carcinoma, Ewing's tumor, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, renal cell carcinoma, liver cancer, Wilms' tumor, cervical cancer, uterine cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, oligodendroglioma, melanoma, neuroblastoma, retinoblastoma, dysplasia, and hyperplasia. Treating and / or preventing cancer includes, but is not limited to, alleviating one or more symptoms associated with cancer, inhibiting or reducing the progression of cancer, promoting regression of cancer, and / or promoting an immune response.

[0101] In some embodiments, the first and second antigens are associated with cancer. In some embodiments, the first antigen and the second antigen are expressed on the same target cell of cancer. In some embodiments, the first antigen is expressed on the first target cell of cancer, and the second antigen is expressed on the second target cell of cancer.

[0102] In some embodiments, the cancer is a hematological malignancy. In some embodiments, the hematological malignancy is a B-cell malignancy. In some embodiments, the cancer is lymphoma, leukemia, or multiple myeloma. In some embodiments, any of these cancers is a relapsed / refractory cancer. In some embodiments, the subject has non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), or multiple myeloma.

[0103] In some embodiments, the first and second antigens 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 antigens.

[0104] In some embodiments, the hematological malignancy is multiple myeloma. In some embodiments, the multiple myeloma can be relapsed / refractory multiple. In some embodiments, the first and second antigens are selected from the group consisting of CD38, SLAMF7, CD138, FCRH5, GPRC5D, and BCMA. Various CARs or monoclonal antibodies against such antigens are known to those skilled in the art. Exemplary CARs and antibodies are described herein.

[0105] In some embodiments, the CAR is an anti-BCMA CAR and the monoclonal antibody is an anti-CD38 antibody. Many anti-BCMA CARs are known to those skilled in the art. Exemplary anti-BCMA CARs are described in Section III.A. In some embodiments, the anti-CD38 antibody is daratumumab (Darzalex™). In some embodiments, the anti-CD38 antibody is isatuximab.

[0106] In some embodiments, the anti-CD38 antibody may be administered subcutaneously. In some embodiments, the anti-CD38 antibody (e.g., daratumumab) may be administered in an anti-CD38 antibody composition containing hyaluronidase. For example, the antibody may be administered as an anti-CD38 antibody composition containing daratumumab and recombinant human hyaluronidase PH20 (e.g., hyaluronidase-fihj). An example of such a composition is described in published U.S. Patent Application Publication No. 20170121414. In some embodiments, each dose of the anti-CD38 antibody composition contains, at or about 1200 mg to about 2400 mg of an anti-CD38 antibody (e.g., daratumumab) and, at or about 15,000 units (U) to about 45,000 U of hyaluronidase (e.g., hyaluronidase-fihj). In some embodiments, each dose of the anti-CD38 antibody composition comprises about 1800 mg of an anti-CD38 antibody (e.g., daratumumab) and about 30,000 U of hyaluronidase (e.g., hyaluronidase-fihj).

[0107] In some embodiments, the CAR is an anti-BCMA CAR and the monoclonal antibody is an anti-SLAMF7 antibody. Many anti-BCMA CARs are known to those skilled in the art. Exemplary anti-BCMA CARs are described in Section III.A. In some embodiments, the antibody is elotuzumab (e.g., EMPLICITI®).

[0108] In some embodiments, the CAR binds a first antigen that is CD38, SLAMF7, CD138, FCRH5, or GPRC5D, and the monoclonal antibody binds BCMA. CARs for 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 belantamab (e.g., Blenrep).

[0109] In some embodiments, the hematological malignancy is lymphoma. In some embodiments, the lymphoma is non-Hodgkin's lymphoma (NHL). In some embodiments, the lymphoma can be a 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 can also be CD38. Various CARs or monoclonal antibodies against such antigens are known to those skilled in the art. Exemplary CARs and antibodies are described herein.

[0110] In some embodiments, the CAR is an anti-CD19 CAR and the antibody is an anti-CD20 antibody. Many anti-CD19 CARs are known to those skilled in the art. Exemplary anti-CD19 CARs are described in Section III.A. In some embodiments, the antibody is rituximab (e.g., Rituxan®). 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.

[0111] In some embodiments, the anti-CD20 antibody can be administered subcutaneously. In some embodiments, the anti-CD20 antibody (e.g., rituximab) can be administered in an anti-CD20 antibody composition containing hyaluronidase. For example, the antibody can be administered as an anti-CD20 antibody composition containing rituximab and recombinant human hyaluronidase PH20. An illustrative example of such a composition is described in published PCT Publication No. WO2011029892.

[0112] In some embodiments, each dose of the anti-CD20 antibody composition comprises at or about 1200 mg to about 2400 mg of an anti-CD20 antibody (e.g., rituximab) and at or about 15,000 Units (U) to about 45,000 U of hyaluronidase. In some embodiments, each dose of the anti-CD20 antibody composition comprises at or about 1400 mg of an anti-CD20 antibody (e.g., rituximab) and about 23,400 U of hyaluronidase. In some embodiments, each dose of the anti-CD20 antibody composition comprises at or about 1600 mg of an anti-CD20 antibody (e.g., rituximab) and about 26,800 U of hyaluronidase.

[0113] In some embodiments, the CAR is an anti-CD19 CAR and the antibody is an anti-CD30 antibody. Many anti-CD19 CARs are known to those skilled in the art. 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 (ADCETRIS®).

[0114] In some embodiments, the CAR is an anti-CD20 CAR and the antibody is an antibody to CD19, CD20, CD22, ROR1, or CD30. Many anti-CD20 CARs are known to those of skill in the art. Exemplary anti-CD20 CARs are described in Section III.A. Any of a variety of monoclonal antibodies to such antigens are known to those of skill in the art. In some embodiments, the antibody is anti-CD19. In some embodiments, the anti-CD19 antibody is tafasitamab (e.g., MONJUVI®). In other embodiments, the anti-CD19 antibody is loncastuximab (e.g., ZYNLONTA®). 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 (ADCETRIS®). Exemplary antibodies are described herein.

[0115] In some embodiments, the CAR is an anti-CD20 CAR and the antibody is an antibody to CD38. Many anti-CD20 CARs are known to those of skill in the art. 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 to CD38. Many anti-CD19 CARs are known to those of skill in the art. Exemplary anti-CD19 CARs are described in Section III.A. In some embodiments, the anti-CD38 antibody is daratumumab (Darzalex™). 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 (e.g., daratumumab) can be administered in an anti-CD38 antibody composition that includes hyaluronidase. For example, the antibody can be administered as an anti-CD38 antibody composition comprising daratumumab and recombinant human hyaluronidase PH20 (e.g., hyaluronidase-fihj). Examples of such compositions are described in published U.S. Patent Application Publication No. 20170121414. In some embodiments, each dose of the anti-CD38 antibody composition comprises, at or about 1200 mg to about 2400 mg of an anti-CD38 antibody (e.g., daratumumab) and, at or about 15,000 Units (U) to about 45,000 U of hyaluronidase (e.g., hyaluronidase-fihj). In some embodiments, each dose of the anti-CD38 antibody composition comprises about 1800 mg of an anti-CD38 antibody (e.g., daratumumab) and about 30,000 U of hyaluronidase (e.g., hyaluronidase-fihj).

[0116] In some embodiments, the hematological malignancy is leukemia. In some embodiments, the leukemia can be 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. A variety of monoclonal antibodies and CARs against such antigens are known to those skilled in the art.

[0117] In some embodiments, the g-NK cells have low or no expression of CD38, such as less than 25% of the cells in the g-NK cell composition being positive for surface CD38. In some embodiments, the cells in the g-NK cell composition have not been engineered to reduce or eliminate CD38 expression, as g-NK cells have been found to uniquely not express CD38. In some embodiments, the g-NK cell composition exhibits minimal anti-CD38-induced fratricide, optionally less than 10% of the cells in the g-NK cell composition exhibit anti-CD38-induced fratricide.

[0118] In some embodiments, the cancer is a solid malignant tumor. In some embodiments, solid tumors include, but are not limited to, lung, colorectal, prostate, pancreatic cancer, and breast cancer, including triple-negative breast cancer. For example, indications include bone disease or metastasis in cancer, regardless of the origin of the primary tumor; breast cancer, including, but not limited to, ER / PR+ breast cancer, Her2+ breast cancer, and triple-negative breast cancer; colorectal cancer; endometrial cancer; gastric cancer; glioblastoma; head and neck cancer, such as esophageal cancer; lung cancer, including but not limited to, non-small cell lung cancer; multiple myeloma; ovarian cancer; pancreatic cancer; prostate cancer; sarcoma, such as osteosarcoma; renal cancer, including but not limited to, renal cell carcinoma; and / or skin cancer, including but not limited to, 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 variant 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. A variety of monoclonal antibodies and CARs against such antigens are known to those skilled in the art.

[0119] In some embodiments, the method of treatment or use comprises administering to an individual an effective amount of cells, such as the g-NK cell compositions provided herein, e.g., compositions containing engineered g-NK cells provided herein, including any such compositions comprising expanded NK cells produced by the methods provided. 5 Or about 10 5 From, 10 12 Or about 10 12, or 10 5 Or about 10 5 From, 10 8 Or about 10 8 , or 10 6 Or about 10 6 From, 10 12 Or about 10 12 , or 10 8 Or about 10 8 From, 10 11 Or about 10 11 , or 10 9 Or about 10 9 From, 10 10 Or about 10 10 10 of such g-NK cell compositions provided herein, e.g., compositions containing engineered NK cells provided herein, including any compositions produced by the methods provided, are administered to an individual subject. In some embodiments, 10 of such g-NK cell compositions provided herein, e.g., compositions containing engineered NK cells provided herein, including any compositions produced by the methods provided, are administered to an individual subject. 5 Pieces or 10 5 More than or about 10 5 Over 10 pieces 6 Pieces or 10 6 More than or about 10 6 Over 10 pieces 7 Pieces or 10 7 More than or about 10 7 Over 10 pieces 8 Pieces or 10 8 More than or about 10 8 Over 10 pieces 9 Pieces or 10 9 More than or about 10 9 Over 10 pieces 10 Pieces or 10 10 More than or about 10 10 Over 10 pieces 11 Pieces or 10 11 More than or about 10 11 More than 10 12 Pieces or 10 12 More than or about 10 12A dose of cells containing more than 100 cells is administered to an individual.

[0120] In some embodiments, the method of treatment or use comprises administering to an individual an effective amount of cells from any of the provided NK cell compositions, including any of the engineered g-NK cell compositions described herein. In some embodiments, the method comprises administering to an individual an effective amount of cells from any of the provided compositions containing engineered g-NK cells. 5 pieces or about 10 5 From 10 12 pieces or about 10 12 pieces or 10 5 pieces or about 10 5 From 10 8 pieces or about 10 8 pieces or 10 6 pieces or about 10 6 From 10 12 pieces or about 10 12 pieces or 10 8 pieces or about 10 8 From 10 11 pieces or about 10 11 pieces or 10 9 pieces or about 10 9 From 10 10 pieces or about 10 10 In some embodiments, 10 cells from any of the provided compositions containing engineered g-NK cells are administered to an individual subject. 5 Pieces or 10 5 More than or about 10 5 Over 10 pieces 6 Pieces or 10 6 More than or about 10 6 Over 10 pieces 7 Pieces or 10 7 More than or about 10 7 Over 10 pieces 8 Pieces or 10 8 More than or about 10 8 Over 10 pieces 9 Pieces or 10 9 More than or about 10 9 Over 10 pieces 10 Pieces or 10 10More than or about 10 10 Over 10 pieces 11 Pieces or 10 11 More than or about 10 11 More than 10 12 Pieces or 10 12 More than or about 10 12 A dose of cells containing more than 10 cells is administered to an individual. In some embodiments, a dose of any of the provided compositions containing engineered g-NK cells is administered per kg. 6 pieces or about 10 6 pcs to 10 10 A number of such cells are administered to a subject.

[0121] In some embodiments, the composition containing the engineered g-NK cells may be administered once a week for a predetermined number of doses.

[0122] In some embodiments, the predetermined number of weekly doses is 1 dose, 2 doses, 3 doses, 4 doses, 5 doses, 6 doses, 7 doses, 8 doses, 9 doses, 10 doses, 11 doses, or 12 doses. In some embodiments, the weekly doses are 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 more. In some embodiments, 6 weekly doses of the g-NK cell composition are administered. In some embodiments, the weekly doses are administered in consecutive weeks.

[0123] In some embodiments, the once-weekly dose is administered in a cyclical regimen. In some embodiments, the cyclical regimen is a 14-day cycle. In some embodiments, the once-weekly dose is administered twice in a 14-day cycle. In some embodiments, the 14-day cycle is repeated twice. In some embodiments, the 14-day cycle is repeated three times.

[0124] In some embodiments, the weekly dose is administered in a cyclical regimen. In some embodiments, the cyclical regimen is a 21-day cycle. In some embodiments, the weekly dose is administered three times in a 21-day cycle. In some embodiments, the 21-day cycle is repeated twice. In some embodiments, the 21-day cycle is repeated three times.

[0125] In some embodiments, an effective amount of any of the disclosed cells or compositions containing engineered g-NK cells disclosed herein is administered to a subject once a week for a period of five weeks.

[0126] In some embodiments, each dose of cells of the g-NK cell composition containing engineered g-NK cells comprises 1 x 10 8 pieces or approximately 1 x 10 8 From cells, 50 x 10 9 pieces or approximately 50 x 10 9 In some embodiments, each dose of cells of the g-NK cell composition containing engineered g-NK cells is 5×10 8 pieces or approximately 5 x 10 8 In some embodiments, each dose of cells of the g-NK cell composition containing engineered g-NK cells is 5×10 9 pieces or approximately 5 x 10 9 In some embodiments, each dose of cells of the g-NK cell composition containing engineered g-NK cells can be 10 x 10 9 pieces or approximately 10 x 10 9 The g-NK cell composition may be a single cell.

[0127] In some embodiments, the dose for administration according to any of the provided methods of treatment or use is 1 x 10 5 cells / kg or approximately 1 x 10 5 cells / kg to 1 x 10 7 cells / kg or approximately 1 x 10 7cells / kg, e.g., 1 x 10 5 cells / kg or approximately 1 x 10 5 cells / kg to 7.5 x 10 6 cells / kg or approximately 7.5 x 10 6 cells / kg, 1 x 10 5 cells / kg or approximately 1 x 10 5 cells / kg to 5 x 10 6 cells / kg or approximately 5 x 10 6 cells / kg, 1 x 10 5 cells / kg or approximately 1 x 10 5 cells / kg to 2.5 x 10 6 cells / kg or approximately 2.5 x 10 6 cells / kg, 1 x 10 5 cells / kg or approximately 1 x 10 5 cells / kg to 1 x 10 6 cells / kg or approximately 1 x 10 6 cells / kg, 1 x 10 5 cells / kg or approximately 1 x 10 5 cells / kg to 7.5 x 10 5 cells / kg or approximately 7.5 x 10 5 cells / kg, 1 x 10 5 cells / kg or approximately 1 x 10 5 cells / kg to 5 x 10 5 cells / kg or approximately 5 x 10 5 cells / kg, 1 x 10 5 cells / kg or approximately 1 x 10 5 cells / kg to 2.5 x 10 5 cells / kg or approximately 2.5 x 10 5 cells / kg, 2.5 x 10 5 cells / kg or approximately 2.5 x 10 5 cells / kg to 1 x 10 7 cells / kg or approximately 1 x 10 7 cells / kg, 2.5 x 10 5 cells / kg or approximately 2.5 x 10 5 cells / kg to 7.5 x 10 6cells / kg or approximately 7.5 x 10 6 cells / kg, 2.5 x 10 5 cells / kg or approximately 2.5 x 10 5 cells / kg to 5 x 10 6 cells / kg or approximately 5 x 10 6 cells / kg, 2.5 x 10 5 cells / kg or approximately 2.5 x 10 5 cells / kg to 2.5 x 10 6 cells / kg or approximately 2.5 x 10 6 cells / kg, 2.5 x 10 5 cells / kg or approximately 2.5 x 10 5 cells / kg to 1 x 10 6 cells / kg or approximately 1 x 10 6 cells / kg, 2.5 x 10 5 cells / kg or approximately 2.5 x 10 5 cells / kg to 7.5 x 10 5 cells / kg or approximately 7.5 x 10 5 cells / kg, 2.5 x 10 5 cells / kg or approximately 2.5 x 10 5 cells / kg to 5 x 10 5 cells / kg or approximately 5 x 10 5 5 x 10 cells / kg 5 cells / kg or approximately 5 x 10 5 cells / kg to 1 x 10 7 cells / kg or approximately 1 x 10 7 5 x 10 cells / kg 5 cells / kg or approximately 5 x 10 5 cells / kg to 7.5 x 10 6 cells / kg or approximately 7.5 x 10 6 5 x 10 cells / kg 5 cells / kg or approximately 5 x 10 5 cells / kg to 5 x 10 6 cells / kg or approximately 5 x 10 6 5 x 10 cells / kg 5 cells / kg or approximately 5 x 10 5cells / kg to 2.5 x 10 6 cells / kg or approximately 2.5 x 10 6 5 x 10 cells / kg 5 cells / kg or approximately 5 x 10 5 cells / kg to 1 x 10 6 cells / kg or approximately 1 x 10 6 5 x 10 cells / kg 5 cells / kg or approximately 5 x 10 5 cells / kg to 7.5 x 10 5 cells / kg or approximately 7.5 x 10 5 cells / kg, 1 x 10 6 cells / kg or approximately 1 x 10 6 cells / kg to 1 x 10 7 cells / kg or approximately 1 x 10 7 cells / kg, 1 x 10 6 cells / kg or approximately 1 x 10 6 cells / kg to 7.5 x 10 6 cells / kg or approximately 7.5 x 10 6 cells / kg, 1 x 10 6 cells / kg or approximately 1 x 10 6 cells / kg to 5 x 10 6 cells / kg or approximately 5 x 10 6 cells / kg, 1 x 10 6 cells / kg or approximately 1 x 10 6 cells / kg to 2.5 x 10 6 cells / kg or approximately 2.5 x 10 6 cells / kg, 2.5 x 10 6 cells / kg or approximately 2.5 x 10 6 cells / kg to 1 x 10 7 cells / kg or approximately 1 x 10 7 cells / kg, 2.5 x 10 6 cells / kg or approximately 2.5 x 10 6 cells / kg to 7.5 x 10 6 cells / kg or approximately 7.5 x 10 6 cells / kg, 2.5 x 10 6cells / kg or approximately 2.5 x 10 6 cells / kg to 5 x 10 6 cells / kg or approximately 5 x 10 6 5 x 10 cells / kg 6 cells / kg or approximately 5 x 10 6 cells / kg to 1 x 10 7 cells / kg or approximately 1 x 10 7 5 x 10 cells / kg 6 cells / kg or approximately 5 x 10 6 cells / kg to 7.5 x 10 6 cells / kg or approximately 7.5 x 10 6 cells / kg, or 7.5 x 10 6 cells / kg or approximately 7.5 x 10 6 cells / kg to 1 x 10 7 cells / kg or approximately 1 x 10 7 In some embodiments, the dose for administration is 1 x 10 cells / kg. 5 cells / kg or approximately 1 x 10 5 cells / kg to 1 x 10 8 cells / kg or approximately 1 x 10 8 cells / kg, e.g., 2.5 x 10 5 cells / kg or approximately 2.5 x 10 5 cells / kg to 1 x 10 8 cells / kg or approximately 1 x 10 8 5 x 10 cells / kg 5 cells / kg or approximately 5 x 10 5 cells / kg to 1 x 10 8 cells / kg or approximately 1 x 10 8 cells / kg, 7.5 x 10 5 cells / kg or approximately 7.5 x 10 5 cells / kg to 1 x 10 8 cells / kg or approximately 1 x 10 8 cells / kg, 1 x 10 6 cells / kg or approximately 1 x 10 6 cells / kg to 1 x 10 8cells / kg or approximately 1 x 10 8 cells / kg, 2.5 x 10 6 cells / kg or approximately 2.5 x 10 6 cells / kg to 1 x 10 8 cells / kg or approximately 1 x 10 8 5 x 10 cells / kg 6 cells / kg or approximately 5 x 10 6 cells / kg to 1 x 10 8 cells / kg or approximately 1 x 10 8 cells / kg, 7.5 x 10 6 cells / kg or approximately 7.5 x 10 6 cells / kg to 1 x 10 8 cells / kg or approximately 1 x 10 8 cells / kg, 1 x 10 7 cells / kg or approximately 1 x 10 7 cells / kg to 1 x 10 8 cells / kg or approximately 1 x 10 8 cells / kg, 2.5 x 10 7 cells / kg or approximately 2.5 x 10 7 cells / kg to 1 x 10 8 cells / kg or approximately 1 x 10 8 5 x 10 cells / kg 7 cells / kg or approximately 5 x 10 7 cells / kg to 1 x 10 8 cells / kg or approximately 1 x 10 8 cells / kg, or 7.5 x 10 7 cells / kg or approximately 7.5 x 10 7 cells / kg to 1 x 10 8 cells / kg or approximately 1 x 10 8 cells / kg.

[0128] In some embodiments, the dose is given as the number of g-NK cells, or NK cell subsets with or comprising surrogate markers for g-NK cells, e.g., any of the NK cell subsets described herein, or the number of viable cells of any of the foregoing. In any of the above embodiments, the dose is given as the number of cells in a composition of engineered cells provided, e.g., produced by a provided method, or the number of viable cells of any of the foregoing.

[0129] In some embodiments, the dose for administration according to any of the methods of treatment or use is 5×10 7 Or about 5 x 10 7 From 10 x 10 9 Or about 10 x 10 9 , e.g., 5 x 10 7 Or about 5 x 10 7 From 5 x 10 9 Or about 5 x 10 9 , about 5×10 7 Or about 5 x 10 7 From 1×10 9 Or about 1 x 10 9 , 5×10 7 Or about 5 x 10 7 From 5 x 10 8 Or about 5 x 10 8 , about 5×10 7 Or about 5 x 10 7 From 1×10 8 Or about 1 x 10 8 , 1×10 8 From 10 x 10 9 Or about 10 x 10 9 , 1×10 8 Or about 1 x 10 8 From 5 x 10 9 Or about 5 x 10 9 , about 1×10 8 Or about 1 x 10 8 From 1×10 9 Or about 1 x 10 9 , 1×10 8 Or about 1 x 10 8 From 5 x 10 8Or about 5 x 10 8 , 5×10 8 Or about 5 x 10 8 From 10 x 10 9 Or about 10 x 10 9 , 5×10 8 Or about 5 x 10 8 From 5 x 10 9 Or about 5 x 10 9 , about 5×10 8 Or about 5 x 10 8 From 1×10 9 Or about 1 x 10 9 , 1×10 9 Or about 1 x 10 9 From 10 x 10 9 Or about 10 x 10 9 , 1×10 9 Or about 1 x 10 9 From 5 x 10 9 Or about 5 x 10 9 , or 5 × 10 9 Or about 5 x 10 9 From 10 x 10 9 Or about 10 x 10 9 In some embodiments, the dose for administration is 5×10 containing engineered g-NK cells. 8 pieces or approximately 5 x 10 8 In some embodiments, the dose for administration is 1 x 10 g-NK cell composition containing engineered g-NK cells. 9 pieces or approximately 1 x 10 9 In some embodiments, the dose for administration is 5 x 10 cells containing engineered g-NK cells. 9 pieces or approximately 5 x 10 9 In some embodiments, the dose for administration is 1 x 10 g-NK cell composition containing engineered g-NK cells. 10 pieces or approximately 1 x 10 10In some embodiments, the dose is a number of g-NK cells or an NK cell subset with or including a surrogate marker for g-NK cells, e.g., any of the NK cell subsets described herein, or a number of viable cells of any of the foregoing. In any of the above embodiments, the dose is given as the number of cells in the expanded cell composition produced by the provided methods, or the number of viable cells of any of the foregoing. In some embodiments, the dose is given as the number of g-NK cells or an NK cell subset with or including a surrogate marker for g-NK cells, e.g., any of the NK cell subsets described herein, or the number of viable cells of any of the foregoing.

[0130] In some embodiments, the dose of cells of the composition containing engineered g-NK cells is administered to an individual immediately after expansion and / or manipulation according to the provided methods. In other embodiments, the composition of g-NK cells containing engineered g-NK cells is stored prior to administration, such as by the methods described above. For example, the NK cells can be stored for more than 6, 12, 18, or 24 months prior to administration to an individual.

[0131] In some embodiments, provided compositions containing NK cells and subsets thereof, 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.

[0132] In certain embodiments, provided compositions are administered by intravenous infusion. 6 pieces or approximately 10 x 10 6 From cells, 10 x 10 9 In some embodiments, 50 x 10 cells are administered by intravenous infusion in a volume of 1 mL to 100 mL. 6 pieces or approximately 50 x 10 6 In some embodiments, 1 x 10 cells are administered. 9 pieces or approximately 1 x 10 9 In some embodiments, 5 x 10 cells are administered. 9 pieces or approximately 5 x 10 9 In some embodiments, 10 x 10 cells are administered. 9pieces or approximately 10 x 10 9 Determining the volume of cells for injection that will administer this number of cells is within the level of one skilled in the art. In one example, 2.5 x 10 cells are administered. 7 pieces or approximately 2.5 x 10 7 cells / mL (e.g., 5 x 10 in 200 mL) 9 pieces or approximately 5 x 10 9 0.5 x 10 cells) by intravenous infusion of a volume of about 20 mL, e.g., from a thawed cryopreserved composition. 9 cells are administered.

[0133] Once the cells are administered to a subject (eg, a human), some aspect of the biological activity of the engineered cell population is measured by any of a number of known methods.

[0134] In some embodiments, the antibody is a therapeutic monoclonal antibody, such as an anti-tumor antigen or anti-cancer antibody. Those skilled in the art can select an appropriate therapeutic (e.g., anti-cancer) monoclonal antibody to administer to a subject with the engineered g-NK cells and compositions described herein, for example, depending on the individual's specific disease or condition. Suitable antibodies can include polyclonal, monoclonal, fragments (such as Fab fragments), single-chain antibodies, and other forms of specific binding molecules.

[0135] In some embodiments, the antibody may further comprise a humanized or human antibody. Humanized forms of non-human antibodies are chimeric Igs, Ig chains, or fragments (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human Igs. In some embodiments, the antibody comprises an Fc domain.

[0136] Generally, humanized antibodies have one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as "import" residues and are typically taken from an "import" variable domain. Humanization is achieved by substituting rodent CDR or CDR sequences for 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 in which significantly less of the intact human variable domain has been replaced by the corresponding sequence from a non-human species (1989). In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some Fc residues are substituted by residues from analogous sites in rodent antibodies. Humanized antibodies include human antibodies (recipient antibodies) in which residues from the recipient's complementarity-determining regions (CDRs) are replaced by residues from the CDRs of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some instances, corresponding non-human residues replace Fv framework residues of the human antibody. Humanized antibodies may contain residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. Generally, humanized antibodies comprise substantially all of at least one, and typically two, variable domains, with most, if not all, of the CDR regions corresponding to those of a non-human Ig, and most, if not all, of the FR regions being those of a human antibody consensus sequence. Humanized antibodies optimally also comprise at least a portion of an antibody constant region (Fc), typically that of a human antibody (Jones et al., 1986; Presta, 1992; Riechmann et al., 1988).

[0137] Human antibodies can also be produced using a variety of techniques, including phage display libraries (Hoogenboom et al., 1991; Marks et al., 1991) and preparation of human mAbs (Boerner et al., 1991; Reisfeld and Sell, 1985). Similarly, introduction of human Ig genes into transgenic animals in which the endogenous antibody genes have been partially or completely inactivated can be used to synthesize human Abs. Upon antigen exposure, human antibody production is observed that closely resembles that seen in humans in all respects, 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).

[0138] Those skilled in the art will appreciate that the engineered g-NK cells of the present invention are suitable for use with a wide 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-α, TAG-72, CAIX, PSMA, folate-binding protein, scatter factor receptor kinase, gangliosides, cytokeratin, frizzled receptor agonists, and the like. receptor, VEGF, VEGFR, integrin αVβ3, integrin α5β1, EGFR, EGFL7, ERBB2 (HER2), ERBB3, fibronectin, HGF, HER3, LOXL2, MET, IGF1R, IGLF2, EPHA3, FR-α, phosphatidylserine, syndecan 1, SLAMF7 (CD319), TRAILR1, TRAILR2, RANKL, FAP, vimentin, or tenascin. In some cases, the antibody is an anti-CD20 antibody (e.g., rituximab), an anti-HER2 antibody (e.g., cetuximab), an anti-CD52 antibody, an anti-EGFR antibody, and an anti-CD38 antibody (e.g., daratumumab), an anti-SLAMF7 antibody (e.g., elotuzumab).

[0139] Non-limiting antibodies that can be used in the provided methods in combination therapy with cell compositions comprising g-NK cells include trastuzumab (Herceptin®), ramucirumab (Cyramza®), atezolizumab (Tecentriq™), nivolumab (Opdivo®), durvalumab (Imfinzi™), avelumab (Bavencio®), pembrolizumab (Keytruda®), bevacizumab (Avastin®), everolimus (Afinitor®), pertuzumab (Perjeta®), ado-trastuzumab emtansine (Kadcyla®), cetuximab (Erbitux®), denosumab (Xgeva®), rituximab (Rituxan®). , alemtuzumab (Campath®), ofatumumab (Arzerra®), obinutuzumab (Gazyva®), necitumumab (Portrazza™), ibritumomab tiuxetan (Zevalin®), brentuximab vedotin (Adcetris®), siltuximab (Sylvant®), bortezomib (Velcade®), (R), daratumumab (Darzalex™), elotuzumab (Empliciti™), dinutuximab (Unituxin™), olaratumumab (Lartruvo™), 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, ublituximab, ocaratuzumab, or elotuzumab.

[0140] In some embodiments, the antibody can be an anti-PD-1 antibody or an anti-PD-L1 antibody. Antibodies that target PD-1 or PD-L1 include, but are not limited to, nivolumab, pembrolizumab, or atezolizumab.

[0141] Antibodies specific to the selected cancer type can be selected, including any antibody approved for the treatment of cancer. Examples include trastuzumab (Herceptin) for breast cancer, rituximab (Rituxan®) for lymphoma, and cetuximab (Erbitux) for head and neck squamous cell carcinoma. Those skilled in the art are familiar with FDA-approved monoclonal antibodies capable of binding specific tumor or disease antigens, any of which can be used in accordance with the provided methods for treating tumors or diseases.

[0142] In some embodiments, the method is for treating adenocarcinoma of the stomach or gastroesophageal junction and the antibody is trastuzumab (Herceptin®) or ramucirumab (Cyramza®).

[0143] In some embodiments, the method is for treating bladder cancer and the antibody is atezolizumab (Tecentriq™), nivolumab (Opdivo®), durvalumab (Imfinzi™), avelumab (Bavencio®), or pembrolizumab (Keytruda®).

[0144] In some embodiments, the method is for treating brain cancer and the antibody is bevacizumab (Avastin®).

[0145] In some embodiments, the method is for treating breast cancer and the antibody is trastuzumab (Herceptin®).

[0146] In some embodiments, the method is for treating cervical cancer and the antibody is bevacizumab (Avastin®).

[0147] In some embodiments, the method is for treating colorectal cancer and the antibody is cetuximab (Erbitux®), panitumumab (Vectibix®), bevacizumab (Avastin®), or ramucirumab (Cyramza®).

[0148] In some embodiments, the method is for treating endocrine / neuroendocrine tumors and the antibody is avelumab (Bavencio®).

[0149] In some embodiments, the method is for treating head and neck cancer and the antibody is cetuximab (Erbitux®), pembrolizumab (Keytruda®), nivolumab (Opdivo®), trastuzumab, or ramucirumab.

[0150] In some embodiments, the method is for treating bone cancer and the antibody is denosumab (Xgeva®).

[0151] In some embodiments, the method is for treating kidney cancer and the antibody is bevacizumab (Avastin®) or nivolumab (Opdivo®).

[0152] In some embodiments, the method is for treating leukemia and the antibody is rituximab (Rituxan®), alemtuzumab (Campath®), ofatumumab (Arzerra®), obinutuzumab (Gazyva®), or blinatumomab (Blincyto®).

[0153] In some embodiments, the method is for treating lung cancer and the antibody is bevacizumab (Avastin®), ramucirumab (Cyramza®), nivolumab (Opdivo®), necitumumab (Portrazza™), pembrolizumab (Keytruda®), or atezolizumab (Tecentriq™).

[0154] In some embodiments, the method is for treating lymphoma and the antibody is ibritumomab tiuxetan (Zevalin®), brentuximab vedotin (Adcetris®), rituximab (Rituxan®), siltuximab (Sylvant®), obinutuzumab (Gazyva®), nivolumab (Opdivo®), or pembrolizumab (Keytruda®).

[0155] In some embodiments, the method is for treating multiple myeloma and the antibody is bortezomib (Velcade®), daratumumab (Darzalex™), or elotuzumab (Empliciti™).

[0156] In some embodiments, the method is for treating neuroblastoma and the antibody is dinutuximab (Unituxin™).

[0157] In some embodiments, the method is for treating ovarian epithelial / fallopian tube / primary peritoneal cancer and the antibody is bevacizumab (Avastin®).

[0158] In some embodiments, the method is for treating pancreatic cancer and the antibody is cetuximab (Erbitux®) or bevacizumab (Avastin®).

[0159] In some embodiments, the method is for treating skin cancer and the antibody is ipilimumab (Yervoy®), pembrolizumab (Keytruda®), avelumab (Bavencio®), or nivolumab (Opdivo®).

[0160] In some embodiments, the method is for treating soft tissue sarcoma and the antibody is olaratumab (Lartruvo™).

[0161] Table 1 shows exemplary first and second antigen and CAR and antibody combinations according to the provided methods.

[0162] Table 1. Exemplary first and second antigen and CAR and antibody combinations TIFF2025525439000001.tif216165TIFF2025525439000002.tif94165

[0163] In certain instances, the subject is administered an effective dose of an antibody before, after, or substantially simultaneously with the population containing engineered g-NK cells. In some instances, the subject is administered about 0.1 mg / kg to about 100 mg / kg of the antibody (e.g., about 0.5 to 10 mg / kg, about 1 to 20 mg / kg, about 10 to 50 mg / kg, or about 20 to 100 mg / kg, e.g., 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 selected by one skilled in the art taking into consideration the particular antibody, the particular disease or condition (e.g., tumor or other disorder), the subject's general condition, any additional treatments the subject is undergoing or has previously undergone, and other relevant factors. The subject is also administered a population containing the engineered g-NK cells described herein. Both the antibody and the population of engineered g-NK cells are typically administered parenterally, for example, intravenously, although injection or infusion into or near the tumor (local administration) or administration into the peritoneal cavity can also be used. Those skilled in the art can determine the appropriate administration route.

[0164] In some embodiments, administration of at least one dose of the antibody may be initiated within one month prior to administration of the g-NK cell composition. In some embodiments, administration of at least one dose of the antibody may be initiated within three weeks prior to administration of the g-NK cell composition. In some embodiments, administration of at least one dose of the antibody may be initiated within two weeks prior to administration of the g-NK cell composition.

[0165] In certain examples, the subject is administered an effective dose of an antibody before, after, or substantially simultaneously with the population of g-NK cells. The effective amount of the antibody can be selected by one of skill in the art taking into consideration the particular antibody, the particular disease or condition (e.g., tumor or other disorder), the subject's general condition, any additional treatments the subject is receiving or has previously received, and other relevant factors. The subject is also administered a population of g-NK cells described herein. Both the antibody and the population of g-NK cells are typically administered parenterally, for example, intravenously, although injection or infusion into or near a tumor (local administration) or administration into the peritoneal cavity can also be used. One of skill in the art can determine the appropriate route of administration.

[0166] In some embodiments, the antibody can be administered as a weekly dose. In some embodiments, the antibody can be administered in a cyclical regimen. In some embodiments, the antibody is administered in a 28-day cycle. In some embodiments, the antibody is administered over one or two 28-day cycles. In some embodiments, the antibody is administered once a week for at least one cycle, for example, in each cycle. In some embodiments, the antibody is administered once a week for 4, 6, 8, 10, 12, 16, 20, 24, 28, 32, 36 or more weeks. In some embodiments, eight weekly doses of the antibody are administered. In some embodiments, the weekly doses are administered on consecutive weeks.

[0167] In some embodiments, the antibody may be administered intravenously.

[0168] In some embodiments, the antibody is daratumumab, and each dose of the antibody is administered in an amount that can be at or about 8 mg / kg to about 32 mg / kg. In some embodiments, each dose is at or about 16 mg / kg.

[0169] In some embodiments, the anti-SLAMF7 antibody (e.g., elotuzumab) can be administered weekly for two cycles and then every two weeks in an amount that can be at or about 10 mg / kg. In some embodiments, the anti-SLAMF7 antibody is administered together with lenalidomide and dexamethasone. In some embodiments, the anti-SLAMF7 antibody is administered after dexamethasone, diphenhydramine, ranitidine, and acetaminophen.

[0170] In some embodiments, the anti-BCMA antibody (e.g., Blenrep) may be administered at or about 2.5 mg / kg as an intravenous infusion over 30 minutes or about 30 minutes. In some embodiments, the anti-BCMA antibody (e.g., Blenrep) is administered once every three weeks.

[0171] In some embodiments, each dose of anti-CD20 antibody is at or about 250 mg / m to 500 mg / m 2 In some embodiments, each dose may be 375 mg / m 2 or approximately 375 mg / m 2 It is administered at .

[0172] In some embodiments, the anti-CD20 antibody composition may be administered as a single weekly dose. In some embodiments, the anti-CD20 antibody is administered as four or eight doses. In some embodiments, the antibody is administered subcutaneously in three or seven doses, followed by a single weekly dose of the anti-CD20 antibody intravenously. In some embodiments, the method comprises administering the anti-CD20 antibody once per week for a total of eight doses and administering the g-NK cell composition once per week for a total of six doses, wherein one or two doses of the anti-CD20 antibody may be administered prior to administration of the composition comprising g-NK cells.

[0173] In some embodiments, the anti-CD19 antibody (e.g., tafasitamab) is administered at or about 12 mg / kg. In some embodiments, the anti-CD19 antibody (e.g., tafasitamab) is administered for four cycles. In some embodiments, the first cycle comprises administration on days 1, 4, 8, 15, and 22 of a 28-day cycle. In some embodiments, the second and third cycles comprise administration on days 1, 8, 15, and 22 of a 28-day cycle. In some embodiments, the fourth cycle and beyond comprise administration on days 1 and 15 of a 28-day cycle. In some embodiments, the anti-CD19 antibody (e.g., tafasitamab) is administered for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 cycles.

[0174] In some embodiments, the anti-CD19 antibody (e.g., loncastuximab) is administered at or about 0.15 mg / kg every three weeks for two cycles. In some embodiments, the anti-CD19 antibody (e.g., loncastuximab) is administered at or about 0.075 mg / kg every three weeks for the subsequent cycle. In some embodiments, dexamethasone is administered prior to administration of the anti-CD19 antibody (e.g., loncastuximab).

[0175] In some embodiments, the anti-CD30 antibody (e.g., brentuximab) may be administered at or about 1.8 mg / kg. In some embodiments, the anti-CD30 antibody (e.g., brentuximab) may be administered up to 180 mg. In some embodiments, the anti-CD30 antibody (e.g., brentuximab) may be administered every three weeks.

[0176] In some embodiments, the antibody is a secretable antibody.

[0177] A. Combination Therapy In some embodiments, the provided methods can be practiced as a combination therapy with one or more other additional agents. In such embodiments, the compositions containing the engineered g-NK cells provided herein can be administered prior to, simultaneously with, or subsequently to (after) the administration of one or more other agents. For example, a dose of engineered g-NK cells can be administered simultaneously or sequentially with antibacterial agents, antiviral agents, and other therapeutic agents. In some embodiments, the methods are performed in combination with administering to the subject a chemotherapeutic agent, a cytotoxic agent, or an immunomodulatory agent. Exemplary combination therapies are described in the following subsections.

[0178] The engineered g-NK cells and the additional agent may be administered sequentially or simultaneously. In some embodiments, the additional agent may be administered before the administration of the g-NK cells. In some embodiments, the additional agent may be administered after the administration of the engineered g-NK cells. For example, the engineered g-NK cells may be administered simultaneously with an antibody specific for a selected cancer type. Alternatively, the engineered g-NK cells may be administered at a selected time that is different from the time that the antibody specific for a selected cancer type is administered.

[0179] 1. Cytokines and growth factors In some embodiments provided herein, the engineered g-NK cells or compositions containing them can be administered to an individual in combination with cytokines and / or growth factors. In some embodiments provided herein, the engineered g-NK cells or compositions containing them can be administered to an individual in combination with additional exogenously administered cytokines and / or growth factors. Because cytokines are necessary for NK cell activity, a typical method involves administering exogenous cytokines to a subject in combination with NK cell therapy as exogenous cytokine supplementation.

[0180] According to some embodiments, the 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 the subject. In other certain embodiments, recombinant IL-15 is administered to the subject. In other certain embodiments, recombinant IL-21 is administered to the subject.

[0181] In some embodiments, at least one cytokine is administered to the subject in combination with administration of the engineered g-NK cells or composition thereof.

[0182] Cytokines are a broad class of proteins that play an important role in cell signaling, particularly in the context of the immune system. Cytokines have been shown to play a role as immunomodulators in autocrine, paracrine, and endocrine signaling. Cytokines can function as immunostimulators that stimulate immune-mediated responses or as immunosuppressants that attenuate immune-mediated responses. Cytokines include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors, but generally do not include hormones or growth factors.

[0183] In some embodiments, the cytokine is an interleukin. Interleukins are a group of cytokines that are generally 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 both 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 as described.

[0184] In some embodiments, the interleukin includes a cytokine produced by an immune cell, such as a lymphocyte, monocyte, or macrophage. In some embodiments, the cytokine is an immunostimulatory cytokine that can be used to induce NK cells, e.g., to promote the survival, activation, and / or proliferation of NK cells. For example, certain cytokines, such as IL-15 or IL-21, can prevent or reduce NK cells from undergoing senescence, e.g., by improving their ability to expand ex vivo or in vivo. In some embodiments, the interleukin or functional portion thereof is a partial or complete 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 may be administered along with the respective receptor for the cytokine. In some embodiments, administering the cytokine along with the engineered g-NK cells allows cytokine signaling, thereby maintaining or improving cell growth, proliferation, expansion and / or effector function of the NK cells.

[0185] In certain embodiments, recombinant IL-2 is administered to the subject. In other certain embodiments, recombinant IL-15 is administered to the subject. In other certain embodiments, recombinant IL-21 is administered to the subject.

[0186] In some embodiments, the cytokine is IL-15 or a functional portion thereof. IL-15 is a cytokine that regulates the activation and proliferation of NK cells. In some cases, IL-15 and IL-12 share similar biological activities. For example, IL-15 and IL-2 may bind a common receptor subunit and compete for the same receptor. In some embodiments, IL-15 induces the activation of JAK kinases and the phosphorylation and activation of 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 NK cell development from hematopoietic stem cells. In some embodiments, the functional portion is a portion of IL-15 (e.g., containing a truncated contiguous sequence of amino acids of full-length IL-15) that retains 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 NK cell development from hematopoietic stem cells. All or a functional portion of IL-15 can be administered to a subject.

[0187] As will be appreciated by those skilled in the art, various IL-15 molecule sequences are known in the art. In one aspect, the IL-15 is wild-type IL-15. In some aspects, the 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 "mammalian" or "mammals" 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 certain aspects, the mammalian IL-15 is human IL-15. Human IL-15 amino acid sequences include, for example, Genbank Accession Nos. NR_751915.1, NP_000576.1, AAI00963.1, AAI00964.1, AAI00962.1, CAA71044.1, AAH18149.1, AAB97518.1, CAA63914.1, and CAA63913.1.

[0188] In some embodiments, the IL-15 nucleotide sequence is set forth in SEQ ID NO:9 or a sequence having at least 85%, or at least about 85%, at least 90%, or at least about 90%, at least 95%, or at least about 95%, or at least 98%, or at least about 98% sequence identity to SEQ ID NO:9. In some embodiments, the IL-15 is a mature form lacking a signal peptide sequence, and in some cases, a propeptide sequence. In some embodiments, the IL-15 has a sequence of amino acids set forth in SEQ ID NO:2 or a sequence having at least 85%, or at least about 85%, at least 90%, or at least about 90%, at least 95%, or at least about 95%, or at least 98%, or at least about 98% sequence identity to SEQ ID NO:2.

[0189] In some embodiments, the IL-15 molecule is a variant of human IL-5, e.g., having one or more amino acid changes, e.g., substitutions, relative to the amino acid sequence of human IL-15. In some embodiments, the IL-15 variant comprises or consists of a mutation at position 45, 51, 52, or 72, e.g., as described in U.S. Patent Application Publication No. 2016 / 0184399. In some embodiments, the IL-15 variant comprises or consists of a substitution of N, S, or L with one of D, E, A, Y, or P. In some embodiments, the mutation is selected from L45D, L45E, S51D, L52D, N72D, N72E, N72A, N72S, N72Y, or N72P (with reference to the sequence of human IL-15, SEQ ID NO:2).

[0190] In embodiments, 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., an N to D substitution. 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 to the IL-15N72D polypeptide of SEQ ID NO:2, which has IL-15Ra binding activity.

[0191] In some embodiments, IL-15 is administered in conjunction with IL-15 receptor alpha (IL15RA), e.g., in a complex with IL-15 receptor alpha (IL15RA) or as a fusion with IL-15 receptor alpha (IL15RA). IL15RA specifically binds IL-15 with extremely high affinity and can bind IL1-5 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, a subject is administered IL-15 / IL-15Ra. In some embodiments, a subject is administered an IL-15 / IL-15R fusion protein. In some embodiments, a subject is administered a single-chain IL-15 / IL-15R fusion protein. 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).

[0192] 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 consisting of three chains, designated alpha, beta, and gamma. The gamma chain is shared by all members of this family of cytokine receptors. Like IL-15, IL-2 promotes immunoglobulin production by B cells and induces NK cell differentiation and proliferation. The primary difference between IL-2 and IL-15 is in the adaptive immune response. For example, IL-2 is necessary for adaptive immunity against foreign pathogens because it is the basis for the development of immunological memory. On the other hand, IL-15 is necessary to maintain highly specific T cell responses by supporting the survival of CD8 memory T cells. All or a functional portion of IL-2 can be expressed as a membrane-bound and / or secreted polypeptide. As will be appreciated by those skilled in the art, various IL-2 molecule sequences are known in the art. In one aspect, the IL-2 is wild-type IL-2. In some aspects, the IL-2 is mammalian IL-2. In some embodiments, the IL-2 is human IL-2.

[0193] In some embodiments, the IL-2 is a mature form that lacks a signal peptide sequence, and in some cases also lacks a propeptide sequence. In some embodiments, the IL-2 has the sequence of amino acids shown in SEQ ID NO:1 or a sequence having at least 85%, at least about 85%, at least 90%, at least about 90%, at least 95%, or at least about 95%, or at least 98% or at least about 98% sequence identity to SEQ ID NO:1.

[0194] In some embodiments, the cytokine is IL-21 or a functional portion thereof. IL-21 binds to the IL-21 receptor (IL-21R) and co-receptor, the common gamma chain (CD132). IL-21 receptors have been identified on NK cells, T cells, and B cells, indicating that IL-21 acts on hematopoietic lineage cells, particularly lymphoid progenitor cells and lymphoid cells. IL-21 has been shown to be a potent 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, 202; Collins et al., Immunol. Res. 28:131-140, 2003; Brady, et al. J. Immunol. 172:2048-58, 2004.) In mouse studies, IL-21 enhances NK cell maturation and effector function (Kasaian et al., Immunity 16:559-569, 2002).

[0195] As will be appreciated by those skilled in the art, various IL-21 molecule sequences are known in the art. In one aspect, the IL-21 is wild-type IL-21. In some aspects, the IL-21 is mammalian IL-21. In one embodiment, the IL-21 sequence is a human IL-21 sequence. Examples of 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.

[0196] 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 a sequence of amino acids set forth in SEQ ID NO:3 or a sequence having at least 85%, or at least about 85%, at least 90%, or at least about 90%, at least 95%, or at least about 95%, or at least 98%, or at least about 98% sequence identity to SEQ ID NO:3. In some embodiments, IL-21 has a sequence of amino acids set forth in SEQ ID NO:4 or a sequence having at least 85%, or at least about 85%, at least 90%, or at least about 90%, at least 95%, or at least about 95%, or at least 98%, or at least about 98% sequence identity to SEQ ID NO:4.

[0197] A cytokine (e.g., IL-2, IL-15, or IL-21) amino acid sequence can include any functional portion of a mature cytokine, e.g., any functional portion of mature IL-2, mature IL-15, or mature IL-15. A functional portion can be any portion comprising consecutive amino acids of the interleukin of which it is a part, so long as the functional portion specifically binds to the respective interleukin receptor. The term "functional portion," when used with respect to an interleukin, refers to any portion or fragment of an interleukin that retains the biological activity of the interleukin of which it is a part (parent interleukin). Functional portions include, for example, portions of interleukins that retain the ability to specifically bind to the respective 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 a similar, equal, or greater extent than the parent interleukin. The biological activity of a functional portion of an interleukin can be measured using assays known in the art. With respect to a parent interleukin, a functional portion can comprise, for example, about 60%, about 70%, about 80%, about 90%, about 95% or more of the amino acid sequence of the parent mature interleukin.

[0198] The scope of cytokines or functional portions according to the provided embodiments includes functional variants of the interleukins described herein. As used herein, the term "functional variant" refers to an interleukin having substantial or significant sequence identity or similarity with a parent interleukin, where the functional variant retains the biological activity of the interleukin of which it is a variant. Functional variants include, for example, variants of the interleukins described herein (parent interleukins) that retain the ability to specifically bind to their respective interleukin receptors, 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 a similar, equal, or greater extent than the parent interleukin. With respect to parent interleukins, functional variants can be, for example, at least about 80%, about 90%, about 95%, about 99%, or more identical in amino acid sequence to the parent interleukin.

[0199] A functional variant can, for example, comprise the amino acid sequence of a parent interleukin with at least one conservative amino acid substitution. Alternatively or additionally, a functional variant can comprise the amino acid sequence of a parent interleukin with at least one non-conservative amino acid substitution. In some embodiments, the amino acid substitution, e.g., a conservative or non-conservative amino acid substitution, does not interfere with or inhibit the biological activity of the functional variant compared to the parent interleukin sequence. In some embodiments, the amino acid substitution, e.g., 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 parent interleukin.

[0200] In some embodiments, the amino acid substitutions in the interleukins 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, a conservative amino acid substitution can be an acidic / negatively charged polar amino acid (e.g., Asp or Glu) substituted for another acidic / negatively charged polar amino acid; an amino acid having a nonpolar side chain substituted for another amino acid having a nonpolar side chain (e.g., Ala, Gly, Val, Li, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.); a basic / positively charged polar amino acid substituted for another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.); an uncharged amino acid having a polar side chain substituted for another uncharged amino acid having a polar side chain (e.g., Asn, Gin, Ser, Thr, Tyr, etc.); an amino acid having a beta-branched side chain substituted for another amino acid having a beta-branched side chain (e.g., Li, Thr, and Val); an amino acid having an aromatic side chain substituted for another amino acid having an aromatic side chain (e.g., His, Phe, Trp, and Tyr), etc.

[0201] In some embodiments, the subject is administered one or more cytokines (such as IL-2, IL-15, IL-21, IL-27, and / or IL-12) to support the survival and / or growth of the NK cells. The cytokines can be administered before, after, or substantially simultaneously with the NK cells. In some examples, the cytokines can be administered after the NK cells. In one specific example, the cytokines are administered to the subject within about 1 to 8 hours (e.g., within about 1 to 4 hours, about 2 to 6 hours, about 4 to 6 hours, or about 5 to 8 hours) of the administration of the NK cells. In some embodiments, the dose of a provided engineered g-NK cell composition and the cytokine or growth factor are administered sequentially. For example, the g-NK cells can be administered first, followed by the cytokine and / or growth factor. In some embodiments, the dose of cells containing engineered g-NK cells is administered simultaneously with the cytokine or growth factor.

[0202] 2. Cytotoxic or lymphodepleting therapy In some embodiments, the provided methods can also include administering a dose of cells containing engineered g-NK cells in conjunction with another treatment, e.g., in conjunction with a chemotherapeutic or cytotoxic agent or other treatment.

[0203] In some aspects, the provided methods can further include administering one or more lymphodepleting therapies, for example, prior to or simultaneously with the initiation of administration of the g-NK cell composition containing engineered g-NK cells. In some embodiments, the lymphodepleting therapy includes administration of a phosphamide, such as cyclophosphamide. In some embodiments, the lymphodepleting therapy can include administration of fludarabine.

[0204] In some aspects, preconditioning a subject with immunodepleting (e.g., lymphodepleting) therapy can improve the efficacy of adoptive cell therapy (ACT). In some embodiments, the lymphodepleting therapy comprises a combination of cyclosporine and fludarabine.

[0205] Such preconditioning can be performed with the ultimate goal of reducing the risk of one or more of a variety of outcomes that may weaken the effectiveness of treatment. These include the phenomenon known as "cytokine sink," in which T cells, B cells, and NK cells compete with TILs for homeostatic and activating cytokines such as IL-2, IL-7, and / or IL-15; suppression of TILs by regulatory T cells, NK cells, or other cells of the immune system; and the influence of negative regulators in the tumor microenvironment. Muranski et al., Nat Clin Pract Oncol. December; 3(12): 668-681 (2006).

[0206] Thus, in some embodiments, the provided methods further comprise administering lymphodepleting therapy to the subject. In some embodiments, the methods comprise administering lymphodepleting therapy to the subject prior to administering the dose of cells. In some embodiments, the lymphodepleting therapy comprises a chemotherapeutic agent such as fludarabine and / or cyclophosphamide. In some embodiments, administering the cells and / or lymphodepleting therapy is performed via outpatient delivery.

[0207] In some embodiments, the method comprises administering to the subject a preconditioning agent, such as lymphodepleting agent or chemotherapy agent, for example, cyclophosphamide, fludarabine or a combination thereof, before the administration of a dose of cells.For example, the subject can be administered a preconditioning agent, such as lymphodepleting agent or chemotherapy agent, for example, cyclophosphamide, fludarabine or a combination thereof, at least 2 days before the first or subsequent dose, for example, at least 3, 4, 5, 6 or 7 days before.In some embodiments, the subject is administered a preconditioning agent, such as lymphodepleting agent or chemotherapy agent, for example, cyclophosphamide, fludarabine or a combination thereof, at least 7 days before the administration of a dose of cells, for example, at least 6 days, 5 days, 4 days, 3 days or 2 days before. In some embodiments, the subject is administered a preconditioning agent, such as a lymphodepleting agent or chemotherapeutic agent, such as cyclophosphamide, fludarabine, or a combination thereof, up to 14 days prior to administration of the dose of cells, e.g., up to 13, 12, 11, 10, 9, or 8 days prior to administration of the dose of cells.

[0208] In some embodiments, subjects are preconditioned with cyclophosphamide at a dose of 20 mg / kg to 100 mg / kg or about 20 mg / kg to 100 mg / kg, for example, 40 mg / kg to 80 mg / kg or about 40 mg / kg to 80 mg / kg. In some aspects, subjects are preconditioned with cyclophosphamide at 60 mg / kg or about 60 mg / kg. In some embodiments, fludarabine can be administered in a single dose, or 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.

[0209] In some embodiments, when the lymphodepleting agent comprises fludarabine, the subject receives 1 mg / m 2 ~100mg / m 2 or approximately 1 mg / m 2 ~100mg / m 2, e.g., 10 mg / m 2 ~75mg / m 2 or approximately 10 mg / m 2 ~75mg / m 2 , 15 mg / m 2 ~50mg / m 2 or approximately 15 mg / m 2 ~50mg / m 2 , 20 mg / m 2 ~30mg / m 2 or approximately 20 mg / m 2 ~30mg / m 2 , or 24 mg / m 2 ~26mg / m 2 or approximately 24 mg / m 2 ~26mg / m 2 In some examples, the subject is administered fludarabine at a dose of 25 mg / m 2 In some embodiments, fludarabine is administered in a single dose, or in multiple doses, such as given daily, every other day, or every three days. In some embodiments, fludarabine is administered daily, for example, for 1 to 5 days, for example, for 3 to 5 days.

[0210] 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 administration schedule, such as those described above, and fludarabine at any dose or administration schedule, such as those described above. For example, in some aspects, the subject receives 60 mg / kg (about 2 g / m) of cyclophosphamide prior to the dose of cells. 2 ) cyclophosphamide and 3 to 5 doses of 25 mg / m 2 The patient will be given fludarabine.

[0211] In some embodiments, prior to administration of the dose of g-NK cells, the subject has undergone lymphodepletion therapy. In some embodiments, the lymphodepletion therapy comprises fludarabine and / or cyclophosphamide. In some embodiments, lymphodepletion is administered to a subject with a body surface area of 1 m 2 or less. 2 20-40 mg per m2 of body surface area 2 Approximately 20-40 mg per unit, optionally 30 mg / m 2 or approximately 30 mg / m 2 of fludarabine daily for 2-4 days and / or 1 m2 of body surface area of the subject 2 200-400 mg per m2 of body surface area 2 Approximately 200-400 mg per unit, optionally 300 mg / m 2 or approximately 300 mg / m 2 of cyclophosphamide given daily for 2 to 4 days.

[0212] In some embodiments, the lymphodepleting therapy comprises fludarabine and cyclophosphamide. In some embodiments, the lymphodepleting therapy comprises 100 mg / mL of lymphocytes per 1 m of body surface area of the subject. 2 30 mg per m2 of body surface area of the subject 2 Approximately 30 mg of fludarabine per square meter of body surface area was administered daily. 2 300 mg per m2 of body surface area of the subject 2 This involves administering approximately 300 mg of cyclophosphamide per day, daily for 2 to 4 days, or optionally for 3 days.

[0213] In some embodiments, administering a preconditioning agent prior to the injection of a dose of cells improves the outcome of treatment.For example, in some aspects, preconditioning, such as lymphodepleting agents or chemotherapeutic agents, such as cyclophosphamide, fludarabine, or a combination thereof, improves the efficacy of treatment with the dose or increases the persistence of NK cells in subjects.In some embodiments, preconditioning treatment increases disease-free survival, such as the percentage of subjects who survive a predetermined period after the injection of cells and do not show minimal residual disease or molecularly detectable disease.In some embodiments, it increases the time to median disease-free survival.

[0214] Once the cells are administered to a subject (e.g., a human), the biological activity of the engineered cell population in some aspects is measured by any of a number of known methods. Parameters to be evaluated include specific binding of engineered or natural T cells or other immune cells to antigens in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry. In certain embodiments, the ability of NK cells to destroy target cells can be measured using any suitable method known in the art, such as the cytotoxicity assays described 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 biological activity of the cells can also be measured by assaying the expression and / or secretion of certain cytokines or other effector molecules, such as CD107a, IFNγ, and TNF. In some aspects, biological activity is measured by assessing clinical outcomes such as reduction in tumor burden or tumor burden. In some aspects, toxicity outcomes, cell persistence and / or expansion, and / or the presence or absence of a host immune response are assessed.

[0215] III. Engineered FC receptor gamma-deficient natural killer cells (G-NK cells) The provided embodiments relate to methods and uses of engineered natural killer (NK) cells that lack expression of FcRγ (g-NK cells) and express a chimeric antigen receptor (CAR). In some embodiments, the engineered NK cells are g-NK cells that lack expression of FcRγ. In some embodiments, the g-NK cell subset of NK cells can be detected by observing whether FcRγ is expressed by an NK cell or a population of NK cells; if FcRγ is absent, the cells are g-NK. FcRγ protein is an intracellular protein. Thus, in some aspects, the presence or absence of FcRγ can be detected after processing of the cells, for example, by fixation and permeabilization, to enable detection of intracellular proteins.

[0216] In some cases, g-NK cells can also be identified by surface markers that are surrogate markers for g-NK cells. As described further below, certain combinations of cell surface markers have also been found to correlate with the g-NK cell phenotype, i.e., cells lacking or defective intracellular expression of FcRγ, thereby providing a surrogate marker profile for identifying or detecting g-NK cells in a non-damaging manner. In some embodiments, the surrogate marker profile for g-NK cells provided herein includes one or more markers CD16 (CD16 pos ), NKG2C(NKG2C pos ) or CD57 (CD57pos), and / or based on positive surface expression of one or more markers CD7 (CD7 dim / neg ), CD161(CD161 neg ) and / or NKG2A (NKG2A negIn some embodiments, the cells are further evaluated for one or more surface markers of NK cells, such as CD45, CD3, and / or CD56. In some embodiments, g-NK cells are characterized by a surrogate marker profile of CD45 pos / CD3 neg / CD56 pos / CD16 pos / CD57 pos / CD7 dim / neg / CD161 neg In some embodiments, g-NK cells can be identified, detected, enriched, and / or isolated using the surrogate marker profile CD45 pos / CD3 neg / CD56 pos / NKG2A neg / CD161 neg In some embodiments, the NKG2C is identified, detected, enriched, and / or isolated using pos and / or NKG2A neg g-NK cells are identified, detected, enriched and / or isolated.

[0217] In some embodiments, the g-NK cells are CD16 pos / CD57 pos / CD7 dim / neg / CD161 neg In some embodiments, the g-NK cells have a surface phenotype that is NKG2A neg / CD161 neg In some embodiments, the g-NK cells further have a surface phenotype that is CD38 neg In some embodiments, the g-NK cells further have a surface phenotype that is CD45 pos / CD3 neg / CD56 pos The surface phenotype is

[0218] In some embodiments, g-NK cells are engineered to express a CAR. In some embodiments, a CAR is generally a fusion protein comprising an ectodomain containing an antigen recognition region, a transmembrane domain, and an endodomain. The ectodomain (i.e., the antigen recognition region or antigen-binding domain) and the transmembrane domain may be connected by a flexible linker. The endodomain may comprise an intracellular signaling domain that propagates an external cellular stimulus into the cell. In some embodiments, the CAR comprises: 1) an antigen-binding domain; 2) a flexible linker; 3) a transmembrane domain; and 4) an intracellular signaling domain. In some embodiments, the CAR binds to a target antigen and induces cytotoxicity upon antigen binding.

[0219] In some embodiments, the engineered g-NK cells may further express one or more other additional heterologous protein agents. In some embodiments, the engineered g-NK cells also express immunomodulatory factors such as cytokines. In some embodiments, the engineered g-NK cells also express secretable antibodies.

[0220] In some embodiments, the immunomodulator is an agent that can regulate the immune function of NK cells. In some embodiments, the immunomodulator can be an immunoactivator. In other embodiments, the immunomodulator can be an immunosuppressant. In some embodiments, the immunomodulator is an exogenous cytokine, such as an interleukin, or a functional portion thereof. Exemplary features of CARs and immunomodulators are further described in the following subsections.

[0221] In some embodiments, the g-NK cells can be further engineered by gene editing, as described in Section IV.

[0222] A. Chimeric Antigen Receptor In provided embodiments, 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, for example, to a tumor-specific or tumor-associated antigen or a pathogen antigen. Thus, an engineered antigen receptor, e.g., a CAR, is a recombinant antigen receptor intended to introduce a certain antigen specificity into NK cells. In some embodiments, the antigen receptor, such as a CAR, is stably integrated into g-NK cells. In other embodiments, the antigen receptor, e.g., a CAR, is transiently expressed by g-NK cells. For example, g-NK cells comprise a CAR having a defined polypeptide sequence that is expressed from an exogenous polynucleotide introduced into immune effector cells, either transiently or integrated into the genome. In provided embodiments, the engineered NK cells provided herein that comprise an antigen receptor (e.g., a CAR) can be used for immunotherapy to target and destroy cells associated with a disease or disorder, e.g., cancer cells, that express the target antigen recognized by the antigen receptor (e.g., CAR).

[0223] In some embodiments, the antigen receptor is a chimeric antigen receptor (CAR). CARs are typically encoded by a nucleic acid sequence (polynucleotide) comprising a leader sequence, an extracellular targeting domain (also called an ectodomain; e.g., 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 comprising an extracellular targeting domain (ectodomain) comprising an antigen-recognition domain or antigen-binding domain; a transmembrane domain; and an intracellular signaling domain. The ectodomain and transmembrane domain may be linked by a flexible linker (also called a spacer). In some embodiments, an antigen-binding domain, such as a single-chain variable fragment (scFv) derived from a monoclonal antibody, recognizes the target antigen. In some embodiments, the antigen-binding domain, e.g., the scFv, is linked or fused to the transmembrane domain via a spacer. In some embodiments, the intracellular signaling domain comprises 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, the cell can recognize and kill target cells that express the target antigen. This property makes CAR-expressing cells particularly attractive agents for specifically targeting cellular activity to abnormal cells, including but not limited to cancer cells. A variety of CARs against target antigens, including tumor-associated antigens, have been developed for expression in various immune cells, including T lymphocytes and natural killer (NK) cells, to mediate cytotoxic activity against target cells that express the antigen, and may be the engineered g-NK cells disclosed herein.

[0224] In some embodiments, the leader sequence can be any of the signal peptide sequences described herein. An exemplary CD8α signal peptide is set forth in SEQ ID NO:12. An exemplary GM-CSFRα signal peptide is set forth in SEQ ID NO:13. An exemplary IgK signal peptide is set forth in SEQ ID NO:14. An exemplary IgK signal peptide is set forth in SEQ ID NO:43.

[0225] Any of a variety of chimeric antigen receptors can be expressed in the engineered NK cells, including those described in International PCT Application Nos. PCT / US2018 / 024650, PCT / IB2019 / 000141, PCT / IB2019 / 000181, and / or PCT / US2020 / 020824, PCT / US2020,035752.

[0226] 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 comprises one or more complementarity-determining regions (CDRs) from the antibody molecule, which 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 an optionally cross-linked Fab. 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 the extracellular antigen-binding domain. In certain embodiments, the scFv is identified by screening an scFv phage library with an antigen-Fc fusion protein.

[0227] In some embodiments, the scFv comprises a variable chain portion 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 limited 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 use in 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 sequences at least 85%, 90%, 95%, or 99% identical to human framework regions.

[0228] When the targeting domain of the CAR comprises an scFv, the immunoglobulin light chain and the immunoglobulin heavy chain are linked by a polypeptide linker of various lengths. Suitably, the polypeptide linker comprises a length of 10 amino acids or more. 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 30 amino acids or less. Suitably, the polypeptide linker comprises a length of less than 15, 20, 25, or 30 amino acids. Suitably, the polypeptide linker comprises a length of 10 to 30 amino acids. Suitably, the polypeptide linker comprises a length of 10 to 25 amino acids. Suitably, the polypeptide linker comprises a length of 10 to 20 amino acids. Suitably, the polypeptide linker comprises a length of 10 to 15 amino acids. Suitably, the polypeptide linker comprises a length of 15 to 30 amino acids. Suitably, the polypeptide linker comprises a length of 20 to 30 amino acids. Suitably, the polypeptide linker comprises a length of 25 to 30 amino acids. Suitably, the polypeptide linker comprises a hydrophilic amino acid. Suitably, the polypeptide linker consists of a hydrophilic amino acid. Suitably, the polypeptide linker comprises a G4S sequence (GGGGS). The G4S linker allows the linker flexibility and protease resistance. Suitably, the G4S linker is repeated 1, 2, 3, 4, 5, 6, 7 or 8 times consecutively in the polypeptide linker.

[0229] 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.

[0230] The binding of the extracellular antigen-binding domain (e.g., scFv or its analog) of antigen-targeting CAR can be confirmed, for example, by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition) or Western blot assay. Each of these assays generally uses a labeled reagent (e.g., antibody or scFv) specific to the complex of interest to detect the presence of a protein-antibody complex of interest. For example, scFv can be radiolabeled and used in radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March 1986, which is incorporated herein by reference). Radioisotopes can be detected by means such as using a gamma counter or scintillation counter, or by autoradiography. In certain embodiments, the extracellular antigen-binding domain of CAR is labeled with a fluorescent marker. Non-limiting examples of fluorescent markers include green fluorescent protein (GFP), blue fluorescent protein (e.g., EBFP, EBFP2, Azurite, and mKalamal), cyan fluorescent protein (e.g., ECFP, Cerulean, and CyPet), and yellow fluorescent protein (e.g., YFP, Citrine, Venus, and YPet).

[0231] In certain embodiments, the antigen-recognizing receptor binds to a tumor-associated antigen or a tumor-specific antigen. In the embodiments described herein, any suitable tumor-associated antigen or tumor-specific antigen (e.g., antigenic peptide) can be used. 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.

[0232] For example, tumor targets include CD38 (multiple myeloma); CD20 (lymphoma); epidermal growth factor receptor (EGFR; non-small cell lung cancer, epithelial carcinoma, and glioma); epidermal growth factor receptor variant III (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 adenocarcinoma); prostate-specific membrane antigen (PSMA; prostate cancer); carcinoembryonic antigen (CEA; pancreatic adenocarcinoma, breast cancer, and colorectal carcinoma); disialoganglioside 2 (GD2; These include, but are not limited to, interleukin-13Ra2 (glioma); glypican-3 (hepatocellular carcinoma); carbonic anhydrase IX (CAIX; renal cell carcinoma); L1 cell adhesion molecule (L1-CAM; neuroblastoma, melanoma, and ovarian adenocarcinoma); cancer antigen 125 (CA125; 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-a (FR-a; ovarian cancer).

[0233] Further non-limiting examples of tumor antigens include 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 special glycoform of P-selectin glycoprotein ligand-1 (PSGL-1)), epithelial glycoprotein 2 (EGP-2), epithelial glycoprotein 40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine-protein kinase erb-B2,3,4 (erb-B2,3,4), folate-binding protein (EBP), fetal acetylcholine receptor (AChR), folate receptor-alpha, 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, MAGE These include, but are not limited to, A3, 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.

[0234] 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 in leukemia or lymphoma. In some embodiments, the CAR binds a target antigen selected from Her2, EGFR, alpha-folate receptor, CEA, cMET, MUC2, mesothelin, or ROR1. In certain embodiments, the target antigen is CD38, CD319 / SLAMF-7, TNFRSF17 / BCMA, SYND1 / CD138, CD229, CD47, Her2 / Neu, epidermal growth factor receptor (EGFR), CD123 / IL3-RA, CD19, CD20, CD22, mesothelin, EpCAM, MUC1, MUC16, 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, TNFRSF17 / BCMA, SYND1 / CD138, CD229, CD47, CD123 / IL3-RA, CD19, CD20, CD22, or CLL-1.

[0235] Various antigen-binding domains are known for incorporation into CARs. In one non-limiting example, g-NK cells are engineered with a CD38-specific CAR (see, e.g., WO 2018 / 104562).

[0236] In some embodiments, g-NK cells are engineered with bispecific CARs or multiple different CARs, whose affinities are directed against two different ligands / antigens. Bispecific CAR-NKs can be used to increase the number of potential binding sites on cancer cells or to localize cancer cells to other immune effector cells that express ligands specific for the NK-CAR. For use in cancer therapy, bispecific CARs 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, bispecific CARs can bind to T cell antigens (e.g., CD3) and tumor cell markers (e.g., CD38). Alternatively, bispecific CARs can bind to two distinct tumor cell markers, increasing the overall binding affinity of NK cells to target tumor cells. This can reduce the risk of cancer cells developing resistance by downregulating one of the target antigens. An example in this case would be CAR binding to both CD38 and CS-1 / SLAMF7 in multiple myeloma. Another tumor cell marker that is well targeted by CARs is the "don't eat me" type marker on tumors, exemplified by CD47.

[0237] In some embodiments, engineered g-NK cells can contain bispecific CARs or multiple CARs expressed by the same NK cell. This allows the NK cell to simultaneously target two different antigens. Suitably, the bispecific CAR has specificity for any two of the following antigens: CD38, CD319 / SLAMF-7, TNFRSF17 / 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, CD123, HERV-K. Suitably, the bispecificity of the CAR NK cell can allow it to bind to a tumor antigen and another immune cell, such as a T cell or dendritic cell. Suitably, the bispecificity of the CAR NK cells can allow them to bind to checkpoint inhibitors such as PDL-1 or CD47. Suitably, the first CAR has specificity for CD38, and the second CAR has specificity for any one of SLAMF-7, BCMA, CD138, CD229, PDL-1, or CD47. Suitably, the first CAR has specificity for CD38, and the second CAR has specificity for SLAMF-7, BCMA, CD138, or CD229. Suitably, the first CAR has specificity for CD38, and the second CAR has specificity for SLAMF-7. Suitably, the first CAR has specificity for CD38, and the second CAR has specificity for BCMA. Suitably, the first CAR has specificity for CD38, and the second CAR has specificity for CD138. Suitably, the first CAR has specificity for CD38, and the second CAR has specificity for CD229.

[0238] In some embodiments, the transmembrane domain of the CAR comprises hydrophobic amino acid residues, allowing the CAR to be anchored in the cell membrane of engineered NK cells. 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 transmembrane domain of the alpha, beta or zeta chain of 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 the transmembrane domain of human CD8 alpha. In some embodiments, the CAR contains a transmembrane domain of CD8 alpha having a sequence of amino acids set forth in SEQ ID NO:61, or a sequence of amino acids that exhibits at least 85%, 90%, or 95% sequence identity to SEQ ID NO:61. In some embodiments, the transmembrane domain is set forth in SEQ ID NO:61. In some embodiments, the CAR contains a transmembrane domain of CD8 alpha having a sequence of amino acids set forth in SEQ ID NO:73, or a sequence of amino acids that exhibits at least 85%, 90%, or 95% sequence identity to SEQ ID NO:73. In some embodiments, the transmembrane domain is set forth in SEQ ID NO:73.

[0239] 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 a transmembrane domain of CD28 having a sequence of amino acids set forth in SEQ ID NO:39, or a sequence of amino acids that exhibits at least 85%, 90%, or 95% sequence identity to SEQ ID NO:39. In some embodiments, the transmembrane domain is set forth in SEQ ID NO:39. In some embodiments, the transmembrane domain of CD28 has a sequence of amino acids set forth in SEQ ID NO:74, or a sequence of amino acids that exhibits at least 85%, 90%, or 95% sequence identity to SEQ ID NO:74. In some embodiments, the transmembrane domain is set forth 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 transmembrane domain are represented by the sequence of amino acids set forth in SEQ ID NO: 10 or a sequence of amino acids exhibiting at least 85%, 90%, or 95% sequence identity to SEQ ID NO: 10. In some embodiments, the CD28 hinge domain and transmembrane domain are represented by the sequence of amino acids set forth in SEQ ID NO: 10.

[0240] In some embodiments, the CAR can also include a spacer region located between the antigen-binding targeting domain and the transmembrane domain. In some embodiments, the spacer region comprises hydrophilic amino acids, allowing flexibility of the targeting domain relative to the cell surface. Suitably, the spacer region comprises more than 5, 10, 15, 20, 25, or 30 amino acids. Suitably, the spacer region comprises less than 10, 15, 20, 25, 30, or 35 amino acids. In some embodiments, the spacer region is a hinge region and comprises the hinge sequence of CD8 or an immunoglobulin molecule.

[0241] In some embodiments, the spacer region 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 a sequence of amino acids set forth in SEQ ID NO:60, or a sequence of amino acids that exhibits at least 85%, 90%, or 95% sequence identity to SEQ ID NO:60. In some embodiments, the sequence of the spacer is set forth in SEQ ID NO:60. In some embodiments, the CAR contains a CD8 hinge spacer sequence having a sequence of amino acids set forth in SEQ ID NO:71, or a sequence of amino acids that exhibits at least 85%, 90%, or 95% sequence identity to SEQ ID NO:71. In some embodiments, the sequence of the spacer is set forth in SEQ ID NO:71.

[0242] In some embodiments, the spacer region is or comprises a CD28 hinge. In some embodiments, the spacer is the hinge region of human CD28. In some embodiments, the CAR contains a CD28 hinge spacer sequence having a sequence of amino acids set forth in SEQ ID NO:72 or a sequence of amino acids exhibiting at least 85%, 90%, or 95% sequence identity to SEQ ID NO:72. In some embodiments, the sequence of the spacer is set forth in SEQ ID NO:72.

[0243] In some embodiments, the spacer region comprises all or a portion of the hinge domain of an IgG1 Fc or an IgG4 Fc. In some embodiments, the spacer is an IgG4 Fc spacer. In some embodiments, the CAR contains an IgG4 Fc spacer having a sequence of amino acids set forth in SEQ ID NO:38, or a sequence of amino acids exhibiting at least 85%, 90%, or 95% sequence identity to SEQ ID NO:38. In some embodiments, the sequence of the spacer is the hinge portion of an IgG1 Fc or an IgG4 Fc. In some embodiments, the CAR contains an IgG4 hinge spacer. In some embodiments, the IgG4 hinge spacer has a sequence of amino acids set forth in SEQ ID NO:59, or a sequence of amino acids exhibiting at least 85%, 90%, or 95% sequence identity to SEQ ID NO:59. In some embodiments, the sequence of the spacer is set forth in SEQ ID NO:59. In some embodiments, the IgG4 hinge spacer has the sequence of amino acids set forth in SEQ ID NO:75 or a sequence of amino acids that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO:75. In some embodiments, the sequence of the spacer is set forth in SEQ ID NO:75.

[0244] In some embodiments, the intracellular signaling domain of the CAR increases the efficacy 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, DAP12, 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, 4-1BB, DAP10, DAP12, 2B4 (CD244), or any combination thereof.

[0245] In some embodiments, the endodomain of the CAR can comprise two additional signaling domains. For example, the CAR can comprise a primary intracellular signaling domain, such as a CD3ζ intracellular signaling domain, and an intracellular signaling domain from a costimulatory molecule to provide an additional signal to the cell, for example, to further enhance the efficacy of immune cells 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 comprising a first primary intracellular signaling domain, such as a CD3ζ intracellular signaling domain, and a second costimulatory intracellular signaling domain. In some embodiments, the costimulatory domain can be a CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and / or B7-H3 costimulatory domain. In some embodiments, the costimulatory domain can be CD27, CD28, 4-1BB (CD137), 0X40 (CD134), DAP10, DAP12, ICOS, and / or 2B4. In some embodiments, the costimulatory domain can 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 costimulatory domain. In some embodiments, the costimulatory signaling domain is the signaling domain of CD28. In some embodiments, the costimulatory signaling domain is the signaling domain of 4-1BB.

[0246] In some embodiments, the CAR contains an intracellular signaling domain containing the signaling domain of CD3ζ having a sequence of amino acids set forth in SEQ ID NO:41, or a sequence of amino acids that exhibits at least 85%, 90%, or 95% sequence identity to SEQ ID NO:41. In some embodiments, the CAR contains an intracellular signaling domain containing the signaling domain of CD3ζ having a sequence of amino acids set forth in SEQ ID NO:41. In some embodiments, the CAR contains an intracellular signaling domain containing the signaling domain of CD3ζ having a sequence of amino acids set forth in SEQ ID NO:50, or a sequence of amino acids that exhibits at least 85%, 90%, or 95% sequence identity to SEQ ID NO:50. In some embodiments, the CAR contains an intracellular signaling domain containing the signaling domain of CD3ζ having the sequence of amino acids set forth in SEQ ID NO:50.

[0247] In some embodiments, the CAR contains an intracellular signaling domain containing a costimulatory signaling domain of CD28 having a sequence of amino acids set forth in SEQ ID NO:40, or a sequence of amino acids that exhibits at least 85%, 90%, or 95% sequence identity to SEQ ID NO:40. In some embodiments, the CAR contains an intracellular signaling domain containing a costimulatory signaling domain of CD28 having a sequence of amino acids set forth in SEQ ID NO:40. In some embodiments, the CAR contains an intracellular signaling domain containing a costimulatory signaling domain of CD28 having a sequence of amino acids set forth in SEQ ID NO:52, or a sequence of amino acids that exhibits at least 85%, 90%, or 95% sequence identity to SEQ ID NO:52. In some embodiments, the CAR contains an intracellular signaling domain containing a costimulatory signaling domain of CD28 having a sequence of amino acids set forth in SEQ ID NO:52.

[0248] In some embodiments, the CAR contains an intracellular signaling domain that contains a costimulatory signaling domain of 4-1BB having a sequence of amino acids set forth in SEQ ID NO:51, or a sequence of amino acids that exhibits at least 85%, 90%, or 95% sequence identity to SEQ ID NO:51. In some embodiments, the CAR contains an intracellular signaling domain that contains a costimulatory signaling domain of 4-1BB having a sequence of amino acids set forth in SEQ ID NO:51.

[0249] In some embodiments, the intracellular signaling domain can be a CD3ζ, CD28, and / or 4-1BB domain. In some embodiments, the intracellular signaling domain contains a 4-1BB costimulatory signaling domain (e.g., SEQ ID NO:51 or a sequence that exhibits at least 85%, 90%, or 95% sequence identity to 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 to SEQ ID NO:41 or 50). In some embodiments, the intracellular signaling domain contains a CD28 costimulatory signaling domain (e.g., SEQ ID NO:52 or a sequence that exhibits at least 85%, 90%, or 95% sequence identity to 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 to SEQ ID NO:41 or 50).

[0250] Suitably, the CAR comprises at least two intracellular signaling domains derived from CD3ζ and 4-1BB. In some embodiments, the CAR comprises an intracellular signaling domain comprising the sequence set forth in SEQ ID NO:41 and SEQ ID NO:51. In some embodiments, the CAR comprises an intracellular signaling domain comprising the sequence set forth in SEQ ID NO:50 and SEQ ID NO:51.

[0251] 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 sequence set forth in SEQ ID NO:41 and SEQ ID NO:40. In some embodiments, the CAR comprises an intracellular signaling domain comprising the sequence set forth in SEQ ID NO:41 and SEQ ID NO:52. In some embodiments, the CAR comprises an intracellular signaling domain comprising the sequence set forth in SEQ ID NO:50 and SEQ ID NO:40. In some embodiments, the CAR comprises an intracellular signaling domain comprising the sequence set forth in SEQ ID NO:50 and SEQ ID NO:52.

[0252] In some embodiments, the antigen receptor (e.g., CAR) is encoded by a CAR-encoding polynucleotide having an NH2-terminal leader sequence. The leader sequence (also known as a signal peptide) enables the expressed CAR construct to enter the endoplasmic reticulum (ER) and target to the cell surface. The leader sequence is cleaved in the ER, and the mature cell surface CAR lacks the leader sequence. Generally, the leader sequence is in the range of 5 to 30 amino acids in length and comprises a stretch of hydrophobic amino acids. Suitably, the leader sequence comprises more than 5, 10, 15, 20, or 25 amino acids in length. Suitably, the leader sequence comprises less than 10, 15, 20, 25, or 30 amino acids in length. Suitably, the leader sequence comprises a sequence derived from any secreted protein. Suitably, the leader sequence comprises a sequence derived from the CD8α leader sequence. In some embodiments, suitably, the leader sequence comprises a sequence derived from an IgK leader sequence. In some embodiments, the leader sequence is set forth in SEQ ID NO:43.

[0253] In some embodiments, the CAR is a CAR present in any of a variety of known engineered cell products. CARs may include, but are not limited to, CARs engineered in ABECMA®, JCARH125, CARVYKTI™ (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); YESCARTA®, KYMRIAH®, TECARTUS®, or BREYANZI® cells.

[0254] In some embodiments, the CAR comprises a CAR from a commercially available CAR cell therapy. Non-limiting examples of CARs in commercially available cell-based therapies include CARs engineered in the cells of brexcavtagene autolucel (TECARTUS®), axicavtagene siloleucel (YESCARTA®), idecavtagene biculeucel (ABECMA®), siltacavtagene autolucel (CARVYKTI™), lysocabtagene maraleucel (BREYANZI®), and tisagenleucel (KYMRIAH®).

[0255] In some embodiments, g-NK cells are engineered with a CAR that binds to CD19. Cluster of differentiation 19 (CD19) is an antigenic determinant detectable on leukemia progenitor cells. Human and mouse amino acid and nucleic acid sequences 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's lymphoma. CD19 is also an early marker of B-cell precursors. See, for example, Nicholson et al. Mol. Immun. 34(16-17):1157-1165(1997). The antigen-binding extracellular domain in the CAR polypeptide disclosed herein is specific for CD19 (e.g., human CD19). In some examples, the antigen-binding extracellular domain can comprise an scFv extracellular domain capable of binding to CD19. In some embodiments, an anti-CD19 CAR can comprise an anti-CD19 single-chain variable fragment (scFv) specific for CD19, 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.

[0256] In some embodiments, the extracellular binding domain of the CD19 CAR comprises the heavy chain variable region (V) as shown in SEQ ID NO:54. H ) and the light chain variable region (V) shown in SEQ ID NO:53 L) In some embodiments, the linker separating the VH and VL in the scFv is a GS linker as shown in SEQ ID NO:56. In some embodiments, the linker separating the VH and VL in the scFv is a Whitlow linker as shown in SEQ ID NO:55. In some embodiments, the scFv has a sequence of amino acids as shown in SEQ ID NO:57. In some embodiments, the scFv has a sequence of amino acids as shown in SEQ ID NO:58. In some embodiments, the spacer is a CD8 hinge as shown in SEQ ID NO:60. In some embodiments, the spacer is an IgG4 hinge as shown in SEQ ID NO:59. In some embodiments, the intracellular signaling domain contains a 4-1BB costimulatory signaling domain and a CD3 zeta signaling domain, such as any described herein. In some embodiments, the intracellular signaling domain contains a CD28 costimulatory signaling domain and a CD3 zeta signaling domain, such as any described herein. In some embodiments, a CAR is understood to include any sequence that exhibits some sequence variation, such as, for example, at least 85%, 90%, 95% or more sequence identity to any of the above or described SEQ ID NOs, and retains binding to CD19 and intracellular signaling and cytotoxic activity.

[0257] In some embodiments, the CAR comprises an anti-CD19 CAR from a commercially available CAR cell therapy. Non-limiting examples of anti-CD19 CARs in commercially available cell-based therapies include anti-CD19 CARs engineered in YESCARTA®, KYMRIAH®, TECARTUS®, or BREYANZI® cells.

[0258] In some embodiments, the CAR is an anti-CD19 CAR having a sequence of amino acids set forth in SEQ ID NO:76, or a sequence of amino acids that exhibits at least 85%, 90%, or 95% sequence identity to SEQ ID NO:76. In some embodiments, the CAR is an anti-CD19 CAR having a sequence of amino acids set forth in SEQ ID NO:76. In some embodiments, the anti-CD19 CAR is encoded by a sequence of nucleotides that encodes the sequence of amino acids set forth in SEQ ID NO:76, or a sequence of amino acids that exhibits at least 85%, 90%, or 95% sequence identity to SEQ ID NO:76. In some embodiments, the anti-CD19 CAR is encoded by a sequence of nucleotides that encodes the sequence of amino acids set forth in SEQ ID NO:76.

[0259] In some embodiments, the CAR is an anti-CD19 CAR having a sequence of amino acids set forth in SEQ ID NO:77 or a sequence of amino acids that exhibits at least 85%, 90%, or 95% sequence identity to SEQ ID NO:77. In some embodiments, the CAR is an anti-CD19 CAR having a sequence of amino acids set forth in SEQ ID NO:77. In some embodiments, the anti-CD19 CAR is encoded by a sequence of nucleotides that encodes the sequence of amino acids set forth in SEQ ID NO:77 or a sequence of amino acids that exhibits at least 85%, 90%, or 95% sequence identity to SEQ ID NO:77. In some embodiments, the anti-CD19 CAR is encoded by a sequence of nucleotides that encodes the sequence of amino acids set forth in SEQ ID NO:77.

[0260] In some embodiments, the CAR is an anti-CD19 CAR having a sequence of amino acids set forth in SEQ ID NO:78, or a sequence of amino acids that exhibits at least 85%, 90%, or 95% sequence identity to SEQ ID NO:78. In some embodiments, the CAR is an anti-CD19 CAR having a sequence of amino acids set forth in SEQ ID NO:78. In some embodiments, the anti-CD19 CAR is encoded by a sequence of nucleotides that encodes the sequence of amino acids set forth in SEQ ID NO:78, or a sequence of amino acids that exhibits at least 85%, 90%, or 95% sequence identity to SEQ ID NO:78. In some embodiments, the anti-CD19 CAR is encoded by a sequence of nucleotides that encodes the sequence of amino acids set forth in SEQ ID NO:78.

[0261] In some embodiments, the CAR is an anti-CD19 CAR having a sequence of amino acids set forth in SEQ ID NO:79 or a sequence of amino acids that exhibits at least 85%, 90%, or 95% sequence identity to SEQ ID NO:79. In some embodiments, the CAR is an anti-CD19 CAR having a sequence of amino acids set forth in SEQ ID NO:79. In some embodiments, the anti-CD19 CAR is encoded by a sequence of nucleotides that encodes the sequence of amino acids set forth in SEQ ID NO:79 or a sequence of amino acids that exhibits at least 85%, 90%, or 95% sequence identity to SEQ ID NO:79. In some embodiments, the anti-CD19 CAR is encoded by a sequence of nucleotides that encodes the sequence of amino acids set forth in SEQ ID NO:79.

[0262] CD20 is a proven therapeutic target for hematological malignancies such as B-NHL, supported by approved and widely used monoclonal antibody therapy. Furthermore, CD19, CD20, and CD22 antigens are ubiquitously present on malignant B cells, making them perfect targets for cell therapy. 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 against CD20, which 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 a transmembrane domain fused to an intracellular co-signaling domain (e.g., CD28 or 4-1BB) and a 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 costimulatory domain, and a CD3ζ signaling domain. In some embodiments, the CAR is the Leu16 CAR described in Rufener et al. Cancer Immunol. Res. 2016 4:509-519. See also GenBank Accession No. KX055828.

[0263] In some embodiments, the extracellular binding domain of the CD20 CAR comprises the heavy chain variable region (V) as shown in SEQ ID NO:36. H ) and the light chain variable region (V) shown in SEQ ID NO:35 L) In some embodiments, the linker separating the VH and VL in the scFv is a GS linker as shown in SEQ ID NO:56. In some embodiments, the linker separating the VH and VL in the scFv is a Whitlow linker as 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 costimulatory signaling domain and a CD3ζ signaling domain, such as any described herein. In some embodiments, the intracellular signaling domain contains a CD28 costimulatory signaling domain and a CD3ζ signaling domain, such as any described herein. In some embodiments, a CAR is understood to include any sequence that exhibits some sequence variation, e.g., at least 85%, 90%, 95% or more sequence identity to any of the above or described SEQ ID NOs, and retains binding to CD20 and intracellular signaling and cytotoxic activity. In some embodiments, the anti-CD20 CAR contains the scFv set forth in SEQ ID NO:37, as well as an IgG4 Fc spacer (e.g., SEQ ID NO:38), a CD28 transmembrane domain (e.g., SEQ ID NO:39), a CD28 costimulatory signaling domain (e.g., SEQ ID NO:40), and a CD3 zeta signaling domain (e.g., SEQ ID NO:41). In some embodiments, the CD20 CAR has the sequence of amino acids set forth 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 set forth in SEQ ID NO:42. In some embodiments, the CAR is encoded by a polynucleotide (e.g., mRNA) set forth in SEQ ID NO:45.

[0264] In some embodiments, the anti-CD20 CAR contains an scFv shown in SEQ ID NO:37, a CD8 hinge spacer (e.g., SEQ ID NO:71), a CD8 transmembrane domain (e.g., SEQ ID NO:73), a 4-1BB costimulatory signaling domain (e.g., SEQ ID NO:51), and a CD3 zeta signaling domain (e.g., SEQ ID NO:41). In some embodiments, the anti-CD20 CAR contains an scFv shown in SEQ ID NO:37, a CD8 hinge spacer (e.g., SEQ ID NO:72), a CD8 transmembrane domain (e.g., SEQ ID NO:73), a 4-1BB costimulatory signaling domain (e.g., SEQ ID NO:51), and a CD3 zeta signaling domain (e.g., SEQ ID NO:41). In some embodiments, the anti-CD20 CAR contains an scFv shown in SEQ ID NO:37, an IgG4 hinge spacer (e.g., SEQ ID NO:59 or 75), a CD8 transmembrane domain (e.g., SEQ ID NO:73), a 4-1BB costimulatory signaling domain (e.g., SEQ ID NO:51), and a CD3 zeta signaling domain (e.g., SEQ ID NO:41). In some embodiments, the anti-CD20 CAR contains an scFv shown in SEQ ID NO:37, a CD8 hinge spacer (e.g., SEQ ID NO:71), a CD28 transmembrane domain (e.g., SEQ ID NO:39), a 4-1BB costimulatory signaling domain (e.g., SEQ ID NO:51), and a CD3 zeta signaling domain (e.g., SEQ ID NO:41). In some embodiments, the anti-CD20 CAR contains the scFv shown in SEQ ID NO:37, a CD28 hinge spacer (e.g., SEQ ID NO:72), a CD28 transmembrane domain (e.g., SEQ ID NO:39), a 4-1BB costimulatory signaling domain (e.g., SEQ ID NO:51), and a CD3ζ signaling domain (e.g., SEQ ID NO:41).In some embodiments, the anti-CD20 CAR contains the scFv shown in SEQ ID NO:37, an IgG4 hinge spacer (e.g., SEQ ID NO:59 or 75), a CD28 transmembrane domain (e.g., SEQ ID NO:39), a 4-1BB costimulatory signaling domain (e.g., SEQ ID NO:51), and a CD3ζ signaling domain (e.g., SEQ ID NO:41). In some embodiments, the CAR is understood to include any sequence that exhibits some sequence variation, such as at least 85%, 90%, 95% or more sequence identity to any of the above or described SEQ ID NOs.

[0265] In some embodiments, the extracellular binding domain of the CD20 CAR comprises the heavy chain variable region (V) as shown in SEQ ID NO:81. H ) and the light chain variable region (V L ) In some embodiments, the linker separating the VH and VL in the scFv is a GS linker as shown in SEQ ID NO:56. In some embodiments, the linker separating the VH and VL in the scFv is a Whitlow linker as shown in SEQ ID NO:55. In some embodiments, the anti-CD20 scFv is shown in SEQ ID NO:82. In some embodiments, the intracellular signaling domain contains a 4-1BB costimulatory signaling domain and a CD3 zeta signaling domain, such as any described herein. In some embodiments, the intracellular signaling domain contains a CD28 costimulatory signaling domain and a CD3 zeta signaling domain, such as any described herein. In some embodiments, a CAR is understood to include any sequence that exhibits some sequence variation, e.g., at least 85%, 90%, 95% or more sequence identity to any of the above or described SEQ ID NOs, and retains binding to CD20 and intracellular signaling and cytotoxic activity.

[0266] In some embodiments, the anti-CD20 CAR contains the scFv shown in SEQ ID NO:82, as well as an IgG4 Fc spacer (e.g., SEQ ID NO:38), a CD28 transmembrane domain (e.g., SEQ ID NO:39), a CD28 costimulatory signaling domain (e.g., SEQ ID NO:40), and a CD3 zeta signaling domain (e.g., SEQ ID NO:41). In some embodiments, the anti-CD20 CAR contains the scFv shown in SEQ ID NO:82, a CD8 hinge spacer (e.g., SEQ ID NO:71), a CD8 transmembrane domain (e.g., SEQ ID NO:73), a 4-1BB costimulatory signaling domain (e.g., SEQ ID NO:51), and a CD3 zeta signaling domain (e.g., SEQ ID NO:41). In some embodiments, the anti-CD20 CAR contains an scFv shown in SEQ ID NO:82, a CD28 hinge spacer (e.g., SEQ ID NO:72), a CD8 transmembrane domain (e.g., SEQ ID NO:73), a 4-1BB costimulatory signaling domain (e.g., SEQ ID NO:51), and a CD3 zeta signaling domain (e.g., SEQ ID NO:41). In some embodiments, the anti-CD20 CAR contains an scFv shown in SEQ ID NO:82, an IgG4 hinge spacer (e.g., SEQ ID NO:59 or 75), a CD8 transmembrane domain (e.g., SEQ ID NO:73), a 4-1BB costimulatory signaling domain (e.g., SEQ ID NO:51), and a CD3 zeta signaling domain (e.g., SEQ ID NO:41). In some embodiments, the anti-CD20 CAR contains the scFv shown in SEQ ID NO:82, a CD8 hinge spacer (e.g., SEQ ID NO:71), a CD28 transmembrane domain (e.g., SEQ ID NO:39), a 4-1BB costimulatory signaling domain (e.g., SEQ ID NO:51), and a CD3 zeta signaling domain (e.g., SEQ ID NO:41).In some embodiments, the anti-CD20 CAR contains an scFv shown in SEQ ID NO:82, a CD28 hinge spacer (e.g., SEQ ID NO:72), a CD28 transmembrane domain (e.g., SEQ ID NO:39), a 4-1BB costimulatory signaling domain (e.g., SEQ ID NO:51), and a CD3 zeta signaling domain (e.g., SEQ ID NO:41). In some embodiments, the anti-CD20 CAR contains an scFv shown in SEQ ID NO:82, an IgG4 hinge spacer (e.g., SEQ ID NO:59 or 75), a CD28 transmembrane domain (e.g., SEQ ID NO:39), a 4-1BB costimulatory signaling domain (e.g., SEQ ID NO:51), and a CD3 zeta signaling domain (e.g., SEQ ID NO:41). In some embodiments, it is understood that a CAR comprises any sequence that exhibits some sequence variation, such as, for example, at least 85%, 90%, 95% or more sequence identity to any of the above or described SEQ ID NOs.

[0267] In some embodiments, the CAR contains an extracellular antigen-binding domain that binds to CD22. In a specific embodiment, the CD22 CAR comprises a CAR against CD22, and the CAR against CD20 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 is derived from the V of any antibody. H , V L and / or one or more CDRs. In some embodiments, the extracellular binding domain of the CD22 CAR comprises a heavy chain variable region (V) as set forth in SEQ ID NO:84. H ) and the light chain variable region (V LIn some embodiments, the linker separating the VH and VL in the scFv is a GS linker as shown in SEQ ID NO:56. In some embodiments, the linker separating the VH and VL in the scFv is a Whitlow linker as 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 a heavy chain variable region (VL) as shown in SEQ ID NO:87. H ) and the light chain variable region (V) shown in SEQ ID NO:88 L In some embodiments, the linker separating the VH and VL in the scFv is a GS linker as shown in SEQ ID NO:56. In some embodiments, the linker separating the VH and VL in the scFv is a Whitlow linker as shown in SEQ ID NO:55. In some embodiments, the anti-CD22 scFv is as shown in SEQ ID NO:89.

[0268] In some embodiments, the anti-CD22 CAR contains the scFv shown in SEQ ID NO:86, as well as an IgG4 Fc spacer (e.g., SEQ ID NO:38), a CD28 transmembrane domain (e.g., SEQ ID NO:39), a CD28 costimulatory signaling domain (e.g., SEQ ID NO:40), and a CD3 zeta signaling domain (e.g., SEQ ID NO:41). In some embodiments, the anti-CD22 CAR contains the scFv shown in SEQ ID NO:86, a CD8 hinge spacer (e.g., SEQ ID NO:71), a CD8 transmembrane domain (e.g., SEQ ID NO:73), a 4-1BB costimulatory signaling domain (e.g., SEQ ID NO:51), and a CD3 zeta signaling domain (e.g., SEQ ID NO:41). In some embodiments, the anti-CD22 CAR contains an scFv shown in SEQ ID NO:86, a CD28 hinge spacer (e.g., SEQ ID NO:72), a CD8 transmembrane domain (e.g., SEQ ID NO:73), a 4-1BB costimulatory signaling domain (e.g., SEQ ID NO:51), and a CD3 zeta signaling domain (e.g., SEQ ID NO:41). In some embodiments, the anti-CD22 CAR contains an scFv shown in SEQ ID NO:86, an IgG4 hinge spacer (e.g., SEQ ID NO:59 or 75), a CD8 transmembrane domain (e.g., SEQ ID NO:73), a 4-1BB costimulatory signaling domain (e.g., SEQ ID NO:51), and a CD3 zeta signaling domain (e.g., SEQ ID NO:41). In some embodiments, the anti-CD22 CAR contains the scFv shown in SEQ ID NO:86, a CD8 hinge spacer (e.g., SEQ ID NO:71), a CD28 transmembrane domain (e.g., SEQ ID NO:39), a 4-1BB costimulatory signaling domain (e.g., SEQ ID NO:51), and a CD3 zeta signaling domain (e.g., SEQ ID NO:41).In some embodiments, the anti-CD22 CAR contains an scFv shown in SEQ ID NO:86, a CD28 hinge spacer (e.g., SEQ ID NO:72), a CD28 transmembrane domain (e.g., SEQ ID NO:39), a 4-1BB costimulatory signaling domain (e.g., SEQ ID NO:51), and a CD3 zeta signaling domain (e.g., SEQ ID NO:41). In some embodiments, the anti-CD22 CAR contains an scFv shown in SEQ ID NO:86, an IgG4 hinge spacer (e.g., SEQ ID NO:59 or 75), a CD28 transmembrane domain (e.g., SEQ ID NO:39), a 4-1BB costimulatory signaling domain (e.g., SEQ ID NO:51), and a CD3 zeta signaling domain (e.g., SEQ ID NO:41). In some embodiments, it is understood that a CAR comprises any sequence that exhibits some sequence variation, such as, for example, at least 85%, 90%, 95% or more sequence identity to any of the above or described SEQ ID NOs.

[0269] In some embodiments, the anti-CD22 CAR contains the scFv shown in SEQ ID NO:89, as well as an IgG4 Fc spacer (e.g., SEQ ID NO:38), a CD28 transmembrane domain (e.g., SEQ ID NO:39), a CD28 costimulatory signaling domain (e.g., SEQ ID NO:40), and a CD3 zeta signaling domain (e.g., SEQ ID NO:41). In some embodiments, the anti-CD22 CAR contains the scFv shown in SEQ ID NO:89, a CD8 hinge spacer (e.g., SEQ ID NO:71), a CD8 transmembrane domain (e.g., SEQ ID NO:73), a 4-1BB costimulatory signaling domain (e.g., SEQ ID NO:51), and a CD3 zeta signaling domain (e.g., SEQ ID NO:41). In some embodiments, the anti-CD22 CAR contains an scFv shown in SEQ ID NO:89, a CD28 hinge spacer (e.g., SEQ ID NO:72), a CD8 transmembrane domain (e.g., SEQ ID NO:73), a 4-1BB costimulatory signaling domain (e.g., SEQ ID NO:51), and a CD3 zeta signaling domain (e.g., SEQ ID NO:41). In some embodiments, the anti-CD22 CAR contains an scFv shown in SEQ ID NO:89, an IgG4 hinge spacer (e.g., SEQ ID NO:59 or 75), a CD8 transmembrane domain (e.g., SEQ ID NO:73), a 4-1BB costimulatory signaling domain (e.g., SEQ ID NO:51), and a CD3 zeta signaling domain (e.g., SEQ ID NO:41). In some embodiments, the anti-CD22 CAR contains the scFv shown in SEQ ID NO:89, a CD8 hinge spacer (e.g., SEQ ID NO:71), a CD28 transmembrane domain (e.g., SEQ ID NO:39), a 4-1BB costimulatory signaling domain (e.g., SEQ ID NO:51), and a CD3 zeta signaling domain (e.g., SEQ ID NO:41).In some embodiments, the anti-CD22 CAR contains an scFv shown in SEQ ID NO:89, a CD28 hinge spacer (e.g., SEQ ID NO:72), a CD28 transmembrane domain (e.g., SEQ ID NO:39), a 4-1BB costimulatory signaling domain (e.g., SEQ ID NO:51), and a CD3 zeta signaling domain (e.g., SEQ ID NO:41). In some embodiments, the anti-CD22 CAR contains an scFv shown in SEQ ID NO:89, an IgG4 hinge spacer (e.g., SEQ ID NO:59 or 75), a CD28 transmembrane domain (e.g., SEQ ID NO:39), a 4-1BB costimulatory signaling domain (e.g., SEQ ID NO:51), and a CD3 zeta signaling domain (e.g., SEQ ID NO:41). In some embodiments, it is understood that a CAR comprises any sequence that exhibits some sequence variation, such as, for example, at least 85%, 90%, 95% or more sequence identity to any of the above or described SEQ ID NOs.

[0270] In some embodiments, an anti-CD22 CAR can comprise an anti-CD22 single-chain variable fragment (scFv) specific for CD22, 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. In some embodiments, the CAR contains an anti-CD22 scFv, followed by an IgG4-Fc spacer, a CD28 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3ζ signaling domain.

[0271] In some embodiments, g-NK cells are engineered with CARs that bind to BCMA. BCMA RNA is commonly detected in multiple myeloma cells and other lymphomas, and BCMA protein has been detected on the surface of plasma cells from multiple myeloma patients by several researchers (see, for example, 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, including, but 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) 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.

[0272] In some embodiments, the extracellular binding domain of the BCMA CAR comprises an scFv derived from C11D5.3, a murine monoclonal antibody 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 comprises the heavy chain variable region (V) of C11D5.3. H ) and the light chain variable region (V L) In some embodiments, the VH has the sequence of amino acids set forth in SEQ ID NO:63, and the VL has the sequence of amino acids set forth in SEQ ID NO:62. In some embodiments, the linker separating the VH and VL in the scFv is a GS linker as set forth in SEQ ID NO:56. In some embodiments, the linker separating the VH and VL in the scFv is a Whitlow linker as set forth in SEQ ID NO:55. In some embodiments, the scFv has the sequence of amino acids set forth in SEQ ID NO:65. In some embodiments, the intracellular signaling domain contains a 4-1BB costimulatory signaling domain and a CD3 zeta signaling domain, such as any described herein. In some embodiments, the intracellular signaling domain contains a CD28 costimulatory signaling domain and a CD3 zeta signaling domain, such as any described herein. In some embodiments, a CAR is understood to include any sequence that exhibits some sequence variation, such as, for example, at least 85%, 90%, 95% or more sequence identity to any of the above or described SEQ ID NOs, and retains binding to BCMA and intracellular signaling and cytotoxic activity.

[0273] In some embodiments, the extracellular binding domain of the BCMA CAR comprises an scFv derived from C12A3.2, another murine monoclonal antibody, described in Carpenter et al., Clin. Cancer Res. 19(8):2048-2060 (2013) and PCT Application Publication No. WO2010 / 104949. In some embodiments, the VH has the sequence of amino acids set forth in SEQ ID NO:66, and the VL has the sequence of amino acids set forth in SEQ ID NO:64. In some embodiments, the linker separating the VH and VL in the scFv is a GS linker as set forth in SEQ ID NO:56. In some embodiments, the linker separating the VH and VL in the scFv is a Whitlow linker as set forth in SEQ ID NO:55. In some embodiments, the scFv has the sequence of amino acids set forth in SEQ ID NO:67. 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, a CAR is understood to include any sequence that exhibits some sequence variation, such as at least 85%, 90%, 95% or more sequence identity to any of the above or described SEQ ID NOs, and that retains binding to BCMA and intracellular signaling and cytotoxic activity.

[0274] In some embodiments, the extracellular binding domain of the BCMA CAR comprises a murine monoclonal antibody with high specificity for human BCMA, designated BB2121 in Friedman et al., Hum. Gene Ther. 29(5):585-601 (2018)). See also PCT Application Publication No. WO2012163805. BB2121 is also known as anti-BCMA02 CAR. In some embodiments, the VH has the sequence of amino acids set forth in SEQ ID NO:68, and the VL has the sequence of amino acids set forth in SEQ ID NO:69. In some embodiments, the linker separating the VH and VL in the scFv is a GS linker as set forth in SEQ ID NO:56. In some embodiments, the linker separating the VH and VL in the scFv is a Whitlow linker as set forth in SEQ ID NO:55. In some embodiments, the scFv has the sequence of amino acids set forth in SEQ ID NO:70. 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, a CAR is understood to include any sequence that exhibits some sequence variation, such as at least 85%, 90%, 95% or more sequence identity to any of the above or described SEQ ID NOs, and that retains binding to BCMA and intracellular signaling and cytotoxic activity.

[0275] In some embodiments, the extracellular binding domain of the BCMA CAR comprises two heavy chain single variable fragments (VHHs) capable of binding to two epitopes of BCMA, as described in Zhao et al., J. Hematol. Oncol. 11(1):141 (2018), also referred to as LCAR-B38M. See also PCT Application Publication No. WO2018 / 028647. 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 is understood that a CAR includes any sequence that exhibits some sequence variation, e.g., at least 85%, 90%, 95% or more sequence identity to any of the above or described SEQ ID NOs, and retains binding to BCMA and intracellular signaling and cytotoxic activity.

[0276] In some embodiments, the extracellular binding domain of the BCMA CAR comprises a fully human heavy chain variable domain (FHVH) as described in Lam et al., Nat. Commun. 11(1):283 (2020), also referred to as FHVH33. In some embodiments, the intracellular signaling domain contains a 4-1BB costimulatory signaling domain and a CD3ζ signaling domain, such as any described herein. In some embodiments, the intracellular signaling domain contains a CD28 costimulatory signaling domain and a CD3ζ signaling domain, such as any described herein. In some embodiments, a CAR is understood to include any sequence that exhibits some sequence variation, e.g., at least 85%, 90%, 95% or more sequence identity to any of the above or described SEQ ID NOs, and retains binding to BCMA and intracellular signaling and cytotoxic activity.

[0277] In some embodiments, the CAR is an anti-BCMA CAR having a sequence of amino acids set forth in SEQ ID NO:83 or a sequence of amino acids that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO:83. In some embodiments, the CAR is an anti-BCMA CAR having a sequence of amino acids set forth in SEQ ID NO:83. In some embodiments, the anti-BCMA CAR is encoded by a sequence of nucleotides that encodes the sequence of amino acids set forth in SEQ ID NO:83 or a sequence of amino acids that exhibits at least 85%, 90% or 95% sequence identity to SEQ ID NO:83. In some embodiments, the anti-BCMA CAR is encoded by a sequence of nucleotides that encodes the sequence of amino acids set forth in SEQ ID NO:83.

[0278] In some embodiments, the CAR comprises an anti-BCMA CAR from a commercially available CAR cell therapy. Non-limiting examples of anti-BCMA CARs in commercially available cell-based therapies include anti-BCMA CARs engineered in idecactogen bicleucel (ABECMA®) or siltacactogen autocleucel (CARVYKTI™) cells.

[0279] In some embodiments, the antigen is GPRC5D. In some embodiments, the scFv is a V derived from an antibody or antibody fragment specific for GPRC5D. H and V L In some embodiments, the antibody or antibody fragment that binds GPRC5D comprises a V from the antibodies or antibody fragments described in International Patent Application Publication Nos. WO2016 / 090329, WO2016 / 090312, and WO2020 / 092854, the contents of each of which are incorporated by reference in their entirety. H and V L is or contains

[0280] In some embodiments, the antigen is FcRL5. In some embodiments, the scFv is a V derived from an antibody or antibody fragment specific for FcRL5. H and V L In some embodiments, the antibody or antibody fragment that binds FcRL5 comprises a V from the antibodies or antibody fragments described in International Patent Application Publication Nos. WO2016 / 090337 and WO2017 / 096120, the contents of each of which are incorporated by reference in their entirety. H and V L is or contains

[0281] CD38 (cluster of differentiation 38), also known as cyclic ADP-ribose hydrolase, is a glycoprotein found on the surface of many immune cells (leukocytes), particularly CD4+, CD8+, and T cells, including B lymphocytes and natural killer cells. CD38 also functions in cell adhesion, signal transduction, and calcium signaling. Structural information about this protein can be found in the UniProtKB / Swiss-Prot database under reference P28907. In humans, the CD38 protein is encoded by the CD38 gene located on chromosome 4. CD38 is a multifunctional surface 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 the regulation of intracellular Ca2+. Loss of CD38 function has also 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). CD38 protein is a marker for HIV infection, leukemia, myeloma, solid tumors, type II diabetes, and bone metabolism. CD38 expression as an important prognostic factor in B-cell chronic lymphocytic leukemia. Blood 98:181-186). In some embodiments, an anti-CD38 CAR may comprise an anti-CD38 single-chain variable fragment (scFv) specific for CD38, 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.

[0282] In some embodiments, the extracellular binding domain of the CD38 CAR comprises the heavy chain variable region (V) as set forth in SEQ ID NO:46 or SEQ ID NO:47. H) and the light chain variable region (V) as shown in SEQ ID NO:48 or SEQ ID NO:49 L ) In some embodiments, the linker separating the VH and VL in the scFv is a GS linker as shown in SEQ ID NO:56. In some embodiments, the linker separating the VH and VL in the scFv is a Whitlow linker as 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 described herein. In some embodiments, the intracellular signaling domain contains a CD28 costimulatory signaling domain and a CD3ζ signaling domain, such as any described herein. In some embodiments, a CAR is understood to include any sequence that exhibits some sequence variation, e.g., at least 85%, 90%, 95% or more sequence identity to any of the above or described SEQ ID NOs, and retains binding to CD38 and intracellular signaling and cytotoxic activity.

[0283] B. Immunomodulators (e.g., cytokines) In provided embodiments, the engineered g-NK cell or cells are engineered to express a heterologous immunomodulatory agent, such as an exogenous cytokine, e.g., an interleukin. In some embodiments, the heterologous nucleic acid encoding the immunomodulator is stably integrated into the genome of the g-NK cell. 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 immunoactivator. In some embodiments, the immunoactivator is a cytokine.

[0284] In provided embodiments, the engineered NK cells express a heterologous cytokine or a functional portion thereof. According to provided embodiments, the NK cells are engineered to express the cytokine in some embodiments in a secreted form, while in some embodiments, the cytokine is membrane-bound. In some embodiments, the heterologous cytokine or a functional portion thereof is secretable from the cell. In some embodiments, the heterologous cytokine or a functional portion thereof is expressed as a membrane-bound protein on the surface of the cell.

[0285] Cytokines are a broad class of proteins that play an important role in cell signaling, particularly in the context of the immune system. Cytokines have been shown to play a role as immunomodulators in autocrine, paracrine, and endocrine signaling. Cytokines can function as immunostimulators that stimulate immune-mediated responses or as immunosuppressants that attenuate immune-mediated responses. Cytokines include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors, but generally do not include hormones or growth factors.

[0286] In some embodiments, the cytokine is an interleukin. Interleukins are a group of cytokines that are generally 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 both 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 as described.

[0287] Because cytokines are necessary for NK cell activity, typical methods include administering exogenous cytokines to a subject in combination with NK cell therapy as exogenous cytokine supplementation. However, in some aspects, administration of exogenous cytokines can pose a risk of systemic toxicity, particularly as can occur with high-dose administration of certain cytokines. In provided embodiments, engineering NK cells with secretable or membrane-bound cytokines provides NK cells with a local source of cytokines while avoiding or reducing the risk of systemic toxicity.

[0288] In some embodiments of the engineered cells provided, an 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 immunostimulatory cytokine (also called an immunoactivator) that can be used to induce NK cells, e.g., to promote the survival, activation, and / or proliferation of NK cells. For example, certain cytokines, such as IL-15 or IL-21, can prevent or reduce NK cells from undergoing senescence, e.g., by improving their ability to expand ex vivo or in vivo. In some embodiments, the interleukin or a functional portion thereof is a partial or complete 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 may be introduced along with the respective receptor for the cytokine. In some embodiments, engineering a heterologous cytokine into the engineered cells allows cytokine signaling, thereby maintaining or improving NK cell growth, proliferation, expansion, and / or effector function, while reducing the risk of cytokine toxicity. In some embodiments, the introduced cytokine, or in some cases its respective cytokine receptor, is expressed on the cell surface. In some embodiments, cytokine signaling is constitutively activated. In some embodiments, activation of cytokine signaling is inducible.In some embodiments, activation of cytokine signaling is transient or temporary.

[0289] Exemplary secretable and membrane-bound (mb) cytokines are known, as described, for example, in U.S. Patent Application Publication No. 2017 / 0073638; U.S. Patent Application Publication No. 2020 / 0199532, U.S. Patent Application Publication No. 2021 / 0024959; WO 2015174928, WO 2019 / 126748, WO 2019 / 191495, WO 2020056045, WO 2021021907, WO 2021 / 011919, WO 2021 / 062281, any of which may be used in the engineered cells provided.

[0290] In some embodiments, the cytokine is IL-15 or a functional portion thereof. IL-15 is a cytokine that regulates the activation and proliferation of NK cells. In some cases, IL-15 and IL-12 share similar biological activities. For example, IL-15 and IL-2 may bind a common receptor subunit and compete for the same receptor. In some embodiments, IL-15 induces the activation of JAK kinases and the phosphorylation and activation of 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 NK cell development from hematopoietic stem cells. In some embodiments, the functional portion is a portion of IL-15 (e.g., containing a truncated contiguous sequence of amino acids of full-length IL-15) that retains 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 NK cell development from hematopoietic stem cells. All or a functional portion of IL-15 can be expressed as a membrane-bound and / or secreted polypeptide.

[0291] As will be appreciated by those skilled in the art, various IL-15 molecule sequences are known in the art. In one aspect, the IL-15 is wild-type IL-15. In some aspects, the 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 "mammalian" or "mammals" 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 certain aspects, the mammalian IL-15 is human IL-15. Human IL-15 amino acid sequences include, for example, Genbank Accession Nos. NR_751915.1, NP_000576.1, AAI00963.1, AAI00964.1, AAI00962.1, CAA71044.1, AAH18149.1, AAB97518.1, CAA63914.1, and CAA63913.1.

[0292] In some embodiments, the engineered NK cells comprise a heterologous nucleotide sequence encoding IL-15. In some embodiments, the IL-15 nucleotide sequence is set forth in SEQ ID NO:9 or a sequence having at least 85%, or at least about 85%, at least 90%, or at least about 90%, at least 95%, or at least about 95%, or at least 98%, or at least about 98% sequence identity to SEQ ID NO:9. In some embodiments, the IL-15 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, the IL-15 has a sequence of amino acids set forth in SEQ ID NO:2 or a sequence having at least 85%, or at least about 85%, at least 90%, or at least about 90%, at least 95%, or at least about 95%, or at least 98%, or at least about 98% sequence identity to SEQ ID NO:2.

[0293] In some embodiments, the IL-15 molecule is a variant of human IL-5, e.g., having one or more amino acid changes, e.g., substitutions, relative to the amino acid sequence of human IL-15. In some embodiments, the IL-15 variant comprises or consists of a mutation at position 45, 51, 52, or 72, e.g., as described in U.S. Patent Application Publication No. 2016 / 0184399. In some embodiments, the IL-15 variant comprises or consists of a substitution of N, S, or L with one of D, E, A, Y, or P. In some embodiments, the mutation is selected from L45D, L45E, S51D, L52D, N72D, N72E, N72A, N72S, N72Y, or N72P (with reference to the sequence of human IL-15, SEQ ID NO:2).

[0294] In embodiments, 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., an N to D substitution. In one embodiment, the IL-15 molecule is an IL-15 polypeptide of SEQ ID NO:2 containing the amino acid substitution N72D therein, 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.

[0295] 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 consisting of three chains, designated alpha, beta, and gamma. The gamma chain is shared by all members of this family of cytokine receptors. Like IL-15, IL-2 promotes immunoglobulin production by B cells and induces NK cell differentiation and proliferation. The primary difference between IL-2 and IL-15 is in the adaptive immune response. For example, IL-2 is necessary for adaptive immunity against foreign pathogens because it is the basis for the development of immunological memory. On the other hand, IL-15 is necessary to maintain highly specific T cell responses by supporting the survival of CD8 memory T cells. All or a functional portion of IL-2 can be expressed as a membrane-bound and / or secreted polypeptide. As will be appreciated by those skilled in the art, various IL-2 molecule sequences are known in the art. In one aspect, the IL-2 is wild-type IL-2. In some aspects, the IL-2 is mammalian IL-2. In some embodiments, the IL-2 is human IL-2.

[0296] In some embodiments, the engineered NK cells comprise a heterologous nucleotide sequence encoding IL-2. In some embodiments, the 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, the IL-2 has the sequence of amino acids set forth in SEQ ID NO:1 or a sequence having at least 85%, or at least about 85%, at least 90%, or at least about 90%, at least 95%, or at least about 95%, or at least 98%, or at least about 98% sequence identity to SEQ ID NO:1.

[0297] In some embodiments, the cytokine is IL-21 or a functional portion thereof. IL-21 binds to the IL-21 receptor (IL-21R) and co-receptor, the common gamma chain (CD132). IL-21 receptors have been identified on NK cells, T cells, and B cells, indicating that IL-21 acts on hematopoietic lineage cells, particularly lymphoid progenitor cells and lymphoid cells. IL-21 has been shown to be a potent 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, 202; Collins et al., Immunol. Res. 28:131-140, 2003; Brady, et al. J. Immunol. 172:2048-58, 2004.) In mouse studies, IL-21 enhances NK cell maturation and effector function (Kasaian et al., Immunity 16:559-569, 2002).

[0298] As will be appreciated by those skilled in the art, various IL-21 molecule sequences are known in the art. In one aspect, the IL-21 is wild-type IL-21. In some aspects, the IL-21 is mammalian IL-21. In one embodiment, the IL-21 sequence is a human IL-21 sequence. Examples of 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.

[0299] In some embodiments, the engineered NK cells comprise a heterologous nucleotide sequence encoding IL-21. In some embodiments, IL-21 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-21 has a sequence of amino acids set forth in SEQ ID NO:3 or a sequence having at least 85%, or at least about 85%, at least 90%, or at least about 90%, at least 95%, or at least about 95%, or at least 98%, or at least about 98% sequence identity to SEQ ID NO:3. In some embodiments, IL-21 has a sequence of amino acids set forth in SEQ ID NO:4 or a sequence having at least 85%, or at least about 85%, at least 90%, or at least about 90%, at least 95%, or at least about 95%, or at least 98%, or at least about 98% sequence identity to SEQ ID NO:4.

[0300] A cytokine (e.g., IL-2, IL-15, or IL-21) amino acid sequence can include any functional portion of a 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 comprising consecutive amino acids of the interleukin of which it is a part, so long as the functional portion specifically binds to the respective interleukin receptor. The term "functional portion," when used in reference to an interleukin, refers to any portion or fragment of an interleukin that retains the biological activity of the interleukin of which it is a part (parent interleukin). Functional portions include, for example, portions of interleukins that retain the ability to specifically bind to the respective 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 a similar, equal, or greater extent than the parent interleukin. The biological activity of a functional portion of an interleukin can be measured using assays known in the art. With respect to a parent interleukin, a functional portion can comprise, for example, about 60%, about 70%, about 80%, about 90%, about 95% or more of the amino acid sequence of the parent mature interleukin.

[0301] The scope of cytokines or functional portions according to the provided embodiments includes functional variants of the interleukins described herein. As used herein, the term "functional variant" refers to an interleukin having substantial or significant sequence identity or similarity with a parent interleukin, where the functional variant retains the biological activity of the interleukin of which it is a variant. Functional variants include, for example, variants of the interleukins described herein (parent interleukins) that retain the ability to specifically bind to their respective interleukin receptors, 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 a similar, equal, or greater extent than the parent interleukin. With respect to parent interleukins, functional variants can be, for example, at least about 80%, about 90%, about 95%, about 99%, or more identical in amino acid sequence to the parent interleukin.

[0302] A functional variant can, for example, comprise the amino acid sequence of a parent interleukin with at least one conservative amino acid substitution. Alternatively or additionally, a functional variant can comprise the amino acid sequence of a parent interleukin with at least one non-conservative amino acid substitution. In some embodiments, the amino acid substitution, e.g., a conservative or non-conservative amino acid substitution, does not interfere with or inhibit the biological activity of the functional variant compared to the parent interleukin sequence. In some embodiments, the amino acid substitution, e.g., 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 parent interleukin.

[0303] In some embodiments, the amino acid substitutions in the interleukins 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, a conservative amino acid substitution can be an acidic / negatively charged polar amino acid (e.g., Asp or Glu) substituted for another acidic / negatively charged polar amino acid; an amino acid having a nonpolar side chain substituted for another amino acid having a nonpolar side chain (e.g., Ala, Gly, Val, Li, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.); a basic / positively charged polar amino acid substituted for another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.); an uncharged amino acid having a polar side chain substituted for another uncharged amino acid having a polar side chain (e.g., Asn, Gin, Ser, Thr, Tyr, etc.); an amino acid having a beta-branched side chain substituted for another amino acid having a beta-branched side chain (e.g., Li, Thr, and Val); an amino acid having an aromatic side chain substituted for another amino acid having an aromatic side chain (e.g., His, Phe, Trp, and Tyr), etc.

[0304] 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 by g-NK cells as a secreted polypeptide in a variety of ways. For example, all or a functional portion of a cytokine can be expressed within and secreted from NK cells. In some embodiments, the secretable cytokine does not contain a transmembrane domain.

[0305] In some embodiments, the cytokine is secretable from the engineered g-NK cells. In some embodiments, the secretable cytokine is constitutively expressed. In other embodiments, the secretable cytokine is transiently expressed. In some embodiments, the secretable cytokine is under an inducible promoter. In some embodiments, the secretable 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 secretable 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 secretable 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, the g-NK cells are engineered with two or more secretable cytokines, such as a combination of two or more of IL-2, IL-15, and IL-21.

[0306] Interleukins and other cytokines are generally secreted, but can also be membrane-bound. When co-expressed with a CAR fusion protein, immune cell-activating cytokines and the CAR fusion protein can be concentrated in the vicinity of target cells. When co-expressed with a CAR fusion protein in g-NK cells, the g-NK cells exhibit increased targeting and killing capabilities, making them attractive and effective therapeutic agents.

[0307] 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, the 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 format of interleukin-cytokine (e.g., IL-2, IL-15, or IL-21) formatted in membrane-bound form, such as any described herein.

[0308] 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 by g-NK cells as a membrane-bound cytokine in a variety of ways. In some embodiments, the cytokine or a functional portion thereof can be directly or indirectly (e.g., ionic, non-ionic, covalently) linked (e.g., conjugated or fused) to the surface of g-NK cells (e.g., on the surface or within the membrane of the NK cell) using any of a variety of linkers known in the art (Hermanson, G., Bioconjugate Techniques, Academic Press 1996). In some aspects, all or a functional portion of the cytokine is linked to all or a portion of a transmembrane protein. In one aspect, NK cells express a fusion protein comprising 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 comprises primarily glycine and serine residues. For example, the flexible linker can include one or more repeats of one or both of 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 No. 8,871,906, each of which is incorporated herein by reference.

[0309] In certain aspects, a portion of a transmembrane protein comprises all or part of the transmembrane domain of a transmembrane protein. In some embodiments, a transmembrane protein can be any protein located in and / or within a membrane, such as the phospholipid bilayer of a biological membrane (e.g., a biological membrane such as a cell membrane). In some embodiments, a transmembrane domain is a domain of a transmembrane protein that is normally present within a membrane, particularly one that forms a channel or pore. In some embodiments, a transmembrane domain is a thermodynamically stable three-dimensional protein structure in a membrane (e.g., a membrane of a vesicle such as a cell). Examples of transmembrane domains include a single alpha helix, a stable complex of several transmembrane alpha helices, a transmembrane beta barrel, a beta helix of gramicidin A, or any other structure. A transmembrane helix is usually about 20 amino acids in length.

[0310] Examples of transmembrane proteins include receptors, ligands, immunoglobulins, glycophorins, 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 receptors (TCRs, such as TCRα and / or TCRβ), nicotinic acetylcholine receptors, GABA receptors, or combinations thereof. Specific examples of immunoglobulins include IgG, IgA, IgM, IgE, IgD, or combinations thereof. Specific examples of glycophorins include glycophorin A, glycophorin D, or combinations thereof.

[0311] In some embodiments, the transmembrane domain is a CD28 transmembrane domain. An exemplary sequence of a CD28 transmembrane domain together with a CD28 hinge domain is shown in SEQ ID NO:10. TIFF2025525439000003.tif11158

[0312] In some embodiments, the transmembrane domain is a CD8 transmembrane domain. An exemplary sequence of a CD8 transmembrane domain together with a CD8 hinge domain is shown in SEQ ID NO:11. TIFF2025525439000004.tif11158

[0313] In some embodiments, the transmembrane domain is a CD4 transmembrane domain. An exemplary sequence of a CD4 transmembrane domain is shown in SEQ ID NO:15. TIFF2025525439000005.tif4128

[0314] 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) may be linked to other components such as a signal peptide, a leader sequence, a secretion signal, a label (e.g., a reporter gene), or any combination thereof.

[0315] 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 with 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-CSFRα), or an immunoglobulin (e.g., IgE or IgK).

[0316] 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 set forth 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-CSFRα (SEQ ID NO:13). An exemplary GM-CSFRα signal peptide is set forth in SEQ ID NO:13. An exemplary IgK signal peptide is set forth in SEQ ID NO:14. An exemplary IgK signal peptide is set forth in SEQ ID NO:43.

[0317] 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 membrane-bound IL-15 as set forth in SEQ ID NO:7 or a sequence having at least 85%, or at least about 85%, at least 90%, or at least about 90%, at least 95%, or at least about 95%, or at least 98% or at least about 98% sequence identity to SEQ ID NO:7. In some embodiments, the heterologous cytokine is membrane-bound IL-15 as set forth in SEQ ID NO:8 or a sequence having at least 85%, or at least about 85%, at least 90%, or at least about 90%, at least 95%, or at least about 95%, or at least 98% or at least about 98% sequence identity to SEQ ID NO:8.

[0318] 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.

[0319] In some embodiments, the heterologous cytokine is membrane-bound IL-15 as set forth in SEQ ID NO:5 or a sequence having at least 85%, or at least about 85%, at least 90%, or at least about 90%, at least 95%, or at least about 95%, or at least 98%, or at least about 98% sequence identity to SEQ ID NO:5.

[0320] In some embodiments, the heterologous cytokine is membrane-bound IL-21 as set forth in SEQ ID NO:6 or a sequence having at least 85%, or at least about 85%, at least 90%, or at least about 90%, at least 95%, or at least about 95%, or at least 98%, or at least about 98% sequence identity to SEQ ID NO:6.

[0321] In some embodiments, IL-15 is engineered into cells that have IL-15 receptor alpha (IL15RA). IL15RA specifically binds IL-15 with extremely high affinity and can 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 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, exogenous IL-15 / IL-15Ra is secretable and 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 engineered g-NK cells provided express a membrane-bound IL15 / IL15Ra complex and a soluble (secretable) IL15Ra / IL15 complex. In some embodiments, the engineered g-NK cells express membrane-bound from an IL15.IL15Ra complex with a cleavable linker.

[0322] C. Polynucleotides In some embodiments, provided herein is a polynucleotide having a nucleic acid sequence encoding an antigen receptor, such as a chimeric antigen receptor, including any of the provided chimeric antigen receptors. In some embodiments, provided herein is a polynucleotide having a nucleic acid sequence encoding any of the provided immunomodulators, such as cytokines, including secretable or membrane-bound cytokines.

[0323] In some embodiments, the nucleic acid encoding the antigen receptor, such as a chimeric antigen receptor, and the nucleic acid encoding the immunomodulatory agent, such as a cytokine, including secretable or membrane-bound cytokines, are provided as separate polynucleotides.

[0324] 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 secretable or membrane-bound cytokine. Thus, in some aspects, the nucleic acid sequences are provided as part of the same polynucleotide. For example, provided embodiments include a polynucleotide in which the engineered component is encoded by a polynucleotide containing one or more protease cleavage sites, e.g., a self-cleaving peptide such as T2A, P2A, E2A, or F2A. Such sites can be recognized and cleaved by a proteinase, resulting in the separation (and separate expression) of the various components (e.g., cytokine and CAR) encoded by the engineered polynucleotide in NK cells. Consequently, depending on the embodiment, the various components of the engineered component can be delivered to NK cells in a single vector or by multiple vectors.

[0325] Also provided herein is a vehicle encoding any of the provided polynucleotides, such as for delivering the polynucleotide to cells, for example, g-NK cells. In some embodiments, the vehicle is a vector, such as a viral vector or a non-viral vector. In some embodiments, the vehicle is a viral vector, such as a lentiviral vector. In some embodiments, the vehicle is a liposome. In some embodiments, the vehicle is a lipid nanoparticle. Other vehicles, including vectors or non-vector delivery vehicles, include those known to those skilled in the art, including any of the following:

[0326] In some embodiments, the polynucleotide is engineered into a g-NK cell or a composition containing a plurality of g-NK cells according to the methods provided. Exemplary methods for engineering NK cells are described below.

[0327] D. Methods of Delivery of Heterologous Agents In some embodiments, the engineered g-NK cells provided herein, including for use in the provided methods, can be generated by genetically engineering a CAR into g-NK cells. In some embodiments, the genetic engineering method includes introducing a nucleic acid encoding a CAR into 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 done simultaneously with the engineering of the CAR into the g-NK cells or sequentially in any order. The nucleic acid introduced into the g-NK cells can be introduced for stable integration into the genome or for transient expression. Stable integration versus transient expression can be selected based on various factors, including, but not limited to, the ability of a particular nucleic acid to be efficiently integrated into the host genome or the content of the nucleic acid and its half-life.

[0328] In some embodiments, introducing a heterologous agent such as a CAR into g-NK cells can be performed in a method for enriching a g-NK cell subset from a starting sample of NK cells. Thus, it is understood that the provided methods do not require specific manipulation of only g-NK cells selected for NK cells lacking the FcRγ chain (or only those selected or identified by a g-NK surrogate marker profile), but can include manipulation of the NK cells to be preferentially expanded or enriched in g-NK cells, or the cells of a composition of NK cells preferentially expanded or enriched in g-NK cells. Thus, the final composition of g-NK cell-enriched cells contains g-NK cells into which a heterologous antigen receptor (e.g., a CAR) and an immunomodulatory factor such as a cytokine (e.g., a secretable or membrane-bound interleukin such as IL-15 or IL-21) have been introduced. Exemplary methods for preparing and expanding a g-NK cell-enriched composition are provided in Section VI.

[0329] In some embodiments, the introduction of a heterologous agent, such as a CAR, can occur at any appropriate time during the method of expanding g-NK cells, as described in Section VI. In some embodiments, the introduction occurs after selection of cells from the subject (e.g., CD3 neg CD57 pos or CD3 neg CD56 pos In some embodiments, the introduction is performed after incubation or culture with feeder cells (e.g., HLA-E-expressing feeder cells), and before incubation or culture of the selected or enriched cells with feeder cells (e.g., HLA-E-expressing feeder cells) for the proliferation or expansion of NK cells. In some embodiments, the introduction is performed after incubation or culture with feeder cells (e.g., HLA-E-expressing feeder cells), thus expanding or expanding the selected or enriched cells. In some embodiments, the introduction is performed sequentially in any order using methods for gene editing as described herein.

[0330] In some embodiments, the period for cell expansion as described in Section VI is divided into a first expansion and a second expansion. In some embodiments, prior to introduction (e.g., viral transduction), selected cells from a biological sample are cultured for a first period, e.g., about or greater than 6 hours, about or greater than 12 hours, about or greater than 18 hours, about or greater than 24 hours, about or greater than 2 days, about or greater than 3 days, about or greater than 4 days, about or greater than 5 days, about or greater than 6 days, about or greater than 7 days, about or greater than 8 days, about or greater than 9 days. The cells are cultured under expansion conditions for about or greater than 10 days, about or greater than 11 days, about or greater than 12 days, about or greater than 13 days, about or greater than 14 days, about or greater than 15 days, about or greater than 16 days, about or greater than 17 days, about or greater than 18 days, about or greater than 19 days, about or greater than 20 days, about or greater than 21 days, or any time in between those listed, including the endpoint. In some embodiments, after the first expansion period, the expanded cells (e.g., NK cells) are introduced (e.g., transduced) with an engineered construct encoding one or more heterologous agents, such as a chimeric antigen receptor as described.After transduction (e.g., viral transduction), the engineered cells are allowed to stand for a second period of time, e.g., about or greater than 6 hours, about or greater than 12 hours, about or greater than 18 hours, about or greater than 24 hours, about or greater than 2 days, about or greater than 3 days, about or greater than 4 days, about or greater than 5 days, about or greater than 6 days, about or greater than 7 days, about or greater than 8 days, about or greater than 9 days, about or greater than 10 days. or for about 10 days or more, at or above 11 days, at or above 12 days, at or above 13 days, at or above 14 days, at or above 15 days, at or above 16 days, at or above 17 days, at or above 18 days, at or above 19 days, at or above 20 days, at or above 21 days, or any time in between those recited, including the endpoint.

[0331] Supplementing the medium with HLA-E-expressing feeder cells and / or one or more stimulating agents, such as IL12 and / or IL21, can be performed at any time during the culture process. For example, one or more stimulating agents can be added at the beginning of culturing, for example, at time 0 (e.g., the beginning of culture). One or more agents can be added two, three, four, five, or more times. Subsequent additions may or may not be at the same concentration as the previous addition. The interval between multiple additions can vary, for example, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, or longer, and any time therebetween, including the end point. When multiple additions of a stimulating agent are used, the concentration of the first supplementary addition can be the same or different from the second supplementary addition (and / or any supplementary addition). For example, in some embodiments, the addition of stimulant over multiple time points can be stepped up, stepped down, held constant, or varied over multiple non-equivalent concentrations.

[0332] In some embodiments, a nucleic acid encoding a heterologous agent, such as a CAR, is introduced under conditions for transient expression in g-NK cells. In some embodiments, methods for introducing a nucleic acid for transient expression include any method that results in a nucleic acid that can express its encoded contents for a short period of time before being degraded.

[0333] In some embodiments, a nucleic acid encoding a heterologous protein agent, such as a CAR, is introduced under conditions for stable expression in g-NK cells. In some embodiments, methods for introducing a nucleic acid for stable expression in a cell include any method that results in stable integration of the nucleic acid into the genome of the cell so that the nucleic acid can be propagated when the cell into which the nucleic acid has been incorporated divides.

[0334] Methods for delivering polynucleotides and compositions containing polynucleotides are known to those skilled in the art. Choosing an appropriate method for transient or stable expression in cells is within the level of one of ordinary skill in the art.

[0335] In some embodiments, the manipulation of NK cells can be achieved by transducing a cell composition with a polynucleotide encoding a heterologous agent such as a CAR or a vector containing 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.

[0336] In some embodiments, vectors that package polynucleotides encoding heterologous agents can be used to deliver the packaged polynucleotides to g-NK cells or to compositions or populations of cells 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 useful as vectors include, but are not limited to, lentiviral vectors, adenoviral vectors, adeno-associated viral (AAV) vectors, herpes simplex viral vectors, retroviral vectors, oncolytic viruses, and the like.

[0337] Generally, a vector contains an origin of replication functional in at least one organism, a promoter sequence and convenient restriction endonuclease sites, and one or more selectable markers, such as drug resistance genes.

[0338] A promoter can include any DNA sequence recognized by the cell's transcriptional machinery, which is required to initiate specific transcription of a polynucleotide sequence. A vector can include a native or non-native promoter operably linked to the polynucleotide. The selected promoter can be strong, weak, constitutive, inducible, tissue-specific, developmental stage-specific, and / or organism-specific. One example of a suitable promoter is the immediate-early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of inducing high-level expression of a polynucleotide sequence operably linked to it. Another example of a promoter is elongation growth factor-1 alpha (EF-1 alpha). Other constitutive promoters may also be used, including, but not limited to, the simian virus (vims) 40 (SV40), mouse mammary tumor virus (MMTV), human immunodeficiency virus (vims) (HIV) long terminal repeat (LTR) promoter, avian leukosis virus (vims) promoter, Epstein-Barr virus (vims) immediate early promoter, Rous sarcoma virus (vims) promoter, and human gene promoters, including, but not limited to, the phosphoglycerate kinase (PGK) promoter, actin promoter, myosin promoter, hemoglobin promoter, ubiquitin C (Ubc) promoter, human U6 small nuclear protein promoter, and creatine kinase promoter. In some examples, inducible promoters may be used, such as, but not limited to, the metallothionine promoter, glucocorticoid promoter, progesterone promoter, and tetracycline promoter.

[0339] Additional promoter elements, such as enhancers, can be used to control the frequency of transcription initiation. Such regions can be located 10 to 100 base pairs upstream or downstream of the initiation site. In some instances, two or more promoter elements can be used to activate transcription cooperatively or independently.

[0340] In some embodiments, polynucleotides can be packaged into viral vectors or integrated into viral genomes, allowing for transient or stable expression of polynucleotides. Viral vectors can include retroviral vectors, including lentiviral vectors. To construct retroviral vectors, a polynucleotide molecule encoding a heterologous agent is inserted into the viral genome in place of a specific viral sequence to create a replication-deficient virus. The recombinant viral vector is then introduced into a packaging cell line that contains gag, pol, and env genes but lacks LTR and packaging components. The 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 can infect both dividing and non-dividing cells.

[0341] In some embodiments, the polynucleotide encoding one or more heterologous agents, such as CAR, is incorporated into a viral vector for delivery by transduction. Viral transduction is the process of intentionally introducing nucleic acid into eukaryotic cells via virus-mediated means.

[0342] In some embodiments, the viral vector is a lentiviral vector.Lentiviral vectors are particularly useful for successful viral transduction because they allow the stable expression of the gene contained in the nucleic acid transcript delivered.Lentiviral vectors express two enzymes, reverse transcriptase and integrase, that are required for the stable expression of the gene contained in the nucleic acid transcript delivered.Reverse transcriptase converts RNA transcripts into DNA, while integrase inserts and integrates DNA into the genome of target cells.Once DNA is stably integrated into the genome, it will divide with the host.The gene of interest contained in the integrated DNA can be constitutively expressed or inducible.As part of the host cell genome, the gene of interest contained in the integrated DNA can be subject to cellular control, including activation or repression, depending on a number of factors in target cells.

[0343] Lentiviruses are a subgroup of the retroviridae family of viruses, named for the need for reverse transcription of the viral RNA genome into DNA prior to integration into the host genome. Therefore, the most important feature of lentiviral vehicles / particles is the integration of their genetic material into the genome of target / host cells. Some examples of lentiviruses include human immunodeficiency viruses (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 maedii, and caprine arthritis-encephalitis virus (CAEV).

[0344] Typically, lentiviral particles constituting gene delivery vehicles are themselves replication-deficient (also referred to as "self-inactivating"). Lentiviruses can infect both dividing and non-dividing cells by entering through the intact host nuclear membrane (Naldini L et al., Curr. Opin. Biol. 1998, 9:457-463). Recombinant lentiviral vehicles / particles have been generated by multiple attenuation of HIV pathogenic genes, for example, deletion of the Env, Vif, Vpr, Vpu, Nef, and Tat genes, making the vector biologically safe. Correspondingly, lentiviral vehicles derived from, for example, HIV-1 / HIV-2 can mediate efficient delivery, integration, and long-term expression of transgenes in non-dividing cells.

[0345] Lentiviral particles can be produced by co-expressing viral packaging elements and the vector genome itself in producer cells such as human HEK293T cells. These elements are usually provided in three (second-generation lentiviral systems) or four (third-generation lentiviral systems) separate plasmids. The producer cells are co-transfected with a plasmid (called a packaging system) encoding lentiviral components, including the viral core (i.e., structural proteins) and enzyme components, and envelope proteins, as well as a plasmid encoding the genome containing the foreign transgene to be introduced into target cells, i.e., the vehicle itself (also called a transfer vector). Generally, the plasmid or vector is contained in a producer cell line. The plasmid / vector is introduced into the producer cell line via transfection, transduction, or infection. Methods for transfection, transduction, or infection are well known to those skilled in the art. By way of non-limiting example, packaging and transfer constructs can be introduced into producer cell lines by calcium phosphate transfection, lipofection, or electroporation, typically along with a dominant selectable marker such as neomyosin (neo), dihydrofolate reductase (DHFR), glutamine synthetase, or adenosine deaminase (ADA), followed by selection in the presence of the appropriate drug and isolation of clones.

[0346] The producing cell produces recombinant viral particles that contain the polynucleotide encoding foreign gene, for example, heterologous agent.Recombinant viral particles are recovered from culture medium and titered by the standard method used by those skilled in the art.Recombinant lentivirus vehicle can be used to infect target cells, such as g-NK cells or g-NK cell-rich cell composition or population.

[0347] 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), the FreeStyle™ 293 Expression System (ThermoFisher, Waltham, MA) and other HEK293T-based producer cell lines (e.g., Stewart et al., Hum Gene Ther. 2011, 2, 2.(3):357-369; Lee et al., Biotechnol Bioeng, 2012, 10996):1551-1560; Throm et al., Blood. 2009, 113(21):5104-5110).

[0348] 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 gp64 envelope protein of baculovirus. The VSV-G glycoprotein is particularly effective against the following species of the genus Vesiculovirus: Carajas virus (CJSV), Chandipura virus (CHPV), Cocal virus (COCV), Isfahan virus (ISFV), Maraba virus (MARAV), Piry virus (PIRYV), Vesicular stomatitis Iagoas virus (VSAV), Vesicular stomatitis Indiana virus (VSTV) and Vesicular stomatitis New Jersey virus (VSNJV) and / or grass carp rhabdovirus, BeAn 157575 virus (BeAn 157575), Boteke virus (BTKV), Calchaqui virus (CQFV), Eel virus American (EVA), Gray Lodge virus (Gray Lodge virus), and / or the following viruses: 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 virus (PERV), Pike fry rhabdovirus (PFRV), Porton virus (PORV), Radi virus (RADIV), Spring viremia of carp virus (SVCV), Tupaia virus (TUPV), ulcerative disease rhabdovirus (UDRV), and Yugbogdanova virus (Yugbogdanova). They may in particular be selected from among the strains provisionally classified in the genus Vesiculovims, such as Bogdanovac virus (YBV).gp64 or other baculovirus env proteins are expressed in Autographa californica nuclear polyhedrosis virus (AcMNPV), Anagrapha falcifera nuclear polyhedrosis virus, Bombyx mori nuclear polyhedrosis virus, Choristoneura fiimiferana nuclear polyhedrosis virus, Orgyia pseudotsugata single capsid nuclear polyhedrosis virus, Epiphyas postvittana nuclear polyhedrosis virus, Hypharitria cunea nuclear polyhedrosis virus, Galleria mellonella nuclear polyhedrosis virus, and others. The virus may be derived from Bacillus mellonella nuclear polyhedrosis virus, Dhori virus, Thogoto virus, Antheraea pemyi nuclear polyhedrosis virus or Batken virus.

[0349] Additional elements provided in the lentiviral particle may include a retroviral LTR (long terminal repeat) at either the 5' or 3' end, a retroviral export element, optionally a lentiviral reverse 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 to improve transduction efficiency in non-dividing cells, and a woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE) to enhance transgene expression and increase titer.

[0350] Methods for producing 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 vector used can be selected from, but is not limited to, pLVX, pLenti, pLenti6, pLJM1, FUGW, pWPXL, pWPI, pLenti CMV puro DEST, pLJM1-EGFP, pULTRA, pInducer2Q, pHIV-EGFP, pCW57.1, pTRPE, pELPS, pRRL and pLionII. Any known lentiviral vehicle may 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; WO 2012079000).

[0351] Other retroviral vectors can also be used to package nucleic acids encoding heterologous agents for delivery into g-NK cells or compositions or populations of cells enriched in g-NK cells. Retroviral vectors (RVs) allow for permanent integration of transgenes in target cells. In addition to complex HIV-1 / 2-based lentiviral vectors, simple gammaretrovirus-based retroviral vectors have been widely used to deliver therapeutic genes and have been clinically proven as one of the most efficient and powerful gene delivery systems capable of transducing a wide range of cell types. Exemplary species of gammaretroviruses include murine leukemia virus (MLV) and feline leukemia virus (FeLV).

[0352] In some embodiments, gammaretroviral vectors derived from mammalian gammaretroviruses, such as murine leukemia viruses (MLVs), are recombinant. The MLV family of gammaretroviruses includes the subfamilies ecotropic, amphotropic, xenotropic, and polytropic. Ecotropic viruses can infect only mouse cells using the mCAT-1 receptor. Examples of ecotropic viruses are Moloney MLV and AKV. Amphotropic viruses infect mice, humans, and other species via the Pit-2 receptor. An example of an amphotropic virus is the 4070A virus. Xenotropic and polytropic viruses use the same (Xpr1) receptor but differ in their species tropism. Xenotropic viruses, such as NZB-9-1, can infect humans and other species but not mice, while polytropic viruses, such as focus-forming virus (MCF), can infect mice, humans, and other species.

[0353] Gammaretroviral vectors can be produced in packaging cells by co-transfecting the cells with several plasmids, including a plasmid encoding the retroviral structural and enzymatic (gag-pol) polyprotein, a plasmid encoding the envelope (env) protein, and a plasmid encoding a vector mRNA containing a polynucleotide encoding a heterologous agent to be packaged into newly formed viral particles.

[0354] In some aspects, recombinant gammaretroviral vectors are pseudotyped with envelope proteins from other viruses. Envelope glycoproteins are incorporated into the outer lipid layer of viral particles, which can increase / change cellular tropism. Exemplary envelope proteins include gibbon ape leukemia virus (vims) 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 vesiculovirus envelope protein (see, for example, U.S. Patent Application Publication No. 2012 / 164118). In other aspects, envelope glycoproteins can be genetically modified to incorporate targeting / binding ligands into gammaretroviral vectors, including but 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 retarget the vector to cells expressing their corresponding targeting 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, and the other end can bind to a molecular determinant on the target cell. Such molecular bridges, such as ligand-receptor, avidin-biotin and chemical conjugation, monoclonal antibodies and engineered fusogenic proteins, can direct the attachment of viral vectors to target cells for transduction (Yang et al, Biotechnol Bioeng., 2008, 101(2):357-368; and Maetzig et al, Viruses, 2011, 3, 677-713).

[0355] In some embodiments, the recombinant gammaretroviral vector is a self-inactivating (SIN) gammaretroviral vector. The vector may be replication-incompetent. The SIN vector may have a deletion in the 3'U3 region, which originally contains enhancer / promoter activity. Furthermore, the 5'U3 region may be replaced with a strong promoter from cytomegalovirus or RSV (required in packaging cell lines), or a selected internal promoter and / or enhancer element. The selection of the internal promoter may be made according to the specific requirements of gene expression required for a particular purpose.

[0356] In some embodiments, the polynucleotide encoding a heterologous agent is inserted into the recombinant virus genome.Other components of the viral mRNA of recombinant gammaretroviral vector can be modified by inserting or removing naturally occurring sequences (for example, inserting IRES, inserting heterologous polynucleotide encoding a polypeptide or inhibitory nucleic acid of interest, shuffling a more effective promoter from a different retrovirus or virus instead of a wild-type promoter, etc.).In some examples, recombinant gammaretroviral vector can contain 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 titer and infectivity. Suitable gammaretroviral vectors for delivering heterologous agents can be selected from those disclosed in U.S. Pat. Nos. 8,828,718; 7,585,676; 7,351,585; U.S. Patent Application Publication No. 2007 / 048285; WO 2010 / 113037; WO 2014 / 121005; WO 2015 / 056014; EP 1757702; and EP 1757703.

[0357] In some embodiments, the polynucleotide encoding the heterologous agent can be packaged in a recombinant adeno-associated virus (rAAV) vector.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 specified AAV serotype and its variants, 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 a sequence described in U.S. Patent Application Publication No. 20030138772; Pulicherla et al. Molecular Therapy, 2011, 19(6):1070-1078; U.S. Patent Nos. 6,156,303; 7,198,951; U.S. Patent Application Publication Nos. 2015 / 0159173 and 2014 / 0359799, and WO 1998 / 011244, WO 2005 / 033321 and WO 2014 / 14422.

[0358] AAV vectors include not only single-stranded vectors, but also self-complementary AAV vectors (scAAV).scAAV vectors contain DNA that anneals together to form double-stranded vector genomes.By skipping second-strand synthesis, scAAV allows rapid expression in cells.rAAV vectors can be produced by standard methods in the art, for example, by triple transfection, in sf9 insect cells or in suspension cell cultures of human cells such as HEK293 cells.

[0359] In some embodiments, non-viral-based methods can be used.For example, in some aspects, the vector containing polynucleotide can be introduced into cells by non-viral methods such as needle, electroporation, sonoporation, hydroporation, etc. physical methods; such as inorganic particles (e.g., calcium phosphate, silica, gold) and / or chemical carriers, etc. chemical methods.In other aspects, synthetic or natural biodegradable agents can be used for delivery, such as cationic lipids, lipid nanoemulsions, nanoparticles, peptide-based vectors or polymer-based vectors.

[0360] In some embodiments, a polynucleotide encoding a heterologous agent, such as a CAR, is designed as a messenger RNA (mRNA) for delivery.

[0361] In some embodiments, the polynucleotide such as mRNA encoding heterologous agent is incorporated into lipid nanoparticles.In some embodiments, the formulation is nanoparticles that can contain at least one lipid.The lipid can 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 lipid.In another aspect, the lipid can be cationic lipid such as but not limited to DLin-DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA and DODMA.

[0362] Lipid nanoparticles can be used to deliver encapsulated or associated (e.g., complexed) therapeutic agents, including mRNA. In particular, some nanoparticle compositions are particularly useful for delivering nucleic acids, including messenger RNA (mRNA), antisense oligonucleotides, plasmid DNA, microRNA (miRNA), miRNA inhibitors (antagomir / antimers), messenger RNA interference complementary RNA (micRNA), DNA, polyvalent RNA, Dicer substrate RNA, complementary DNA (cDNA), and self-amplifying RNA (saRNA). For example, see U.S. Patent No. 10,723,692.

[0363] Therefore, among the methods provided herein are methods for the delivery of nucleic acids, including DNA, RNA, mRNA, and self-amplifying RNA (saRNA), encoding heterologous agents such as CAR, for delivery into g-NK cells or a composition or population of cells enriched in g-NK cells. In some embodiments, the heterologous agents are packaged or incorporated into lipid nanoparticles for the delivery of nucleic acids, 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).

[0364] In some embodiments, the mRNA is a self-amplifying mRNA. Self-amplifying RNA (saRNA) can self-amplify itself through the presence of 5' and 3' conserved sequence elements (CSEs) and nsP1-4 genes along with a subgenomic promoter. See, for example, Bloom, van den Berg, and Arbuthnot, Gene Therapy, 2021. After in situ translation, nsP1-4 proteins recognize the flaking CSE sequence and form an RdRP complex that amplifies the sequence contained within the RNA. Introduction of saRNA into target cells can be achieved via lipid nanoparticle delivery. In some embodiments, such self-amplifying RNA can have any structural features or components taught in WO201105799.

[0365] In some embodiments, the provided methods involve the use of lipid nanoparticles (LNPs) containing mRNA encoding a heterologous agent, such as a CAR. In some embodiments, the mRNA encoding the heterologous agent can be generated using methods known in the art, such as in vitro transcription. In some embodiments of the methods, the mRNA comprises a 5' cap. In some embodiments, the 5' cap is an altered nucleotide on the 5' end of a primary transcript, such as a messenger RNA. In some aspects, the 5' cap of an mRNA improves one or more of RNA stability and processing, mRNA metabolism, processing and maturation of RNA transcripts in the nucleus, transport of mRNA from the nucleus to the cytoplasm, mRNA stability, and efficient translation of mRNA into protein. In some embodiments, the 5' cap can be different from the naturally occurring 5' cap or 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 an Anti-Reverse Cap Analog (ARCA) cap, a 7-methyl-guanosine (7mG) cap, a CleanCap® analog, a vaccinia cap, and analogs thereof. For example, the 5' cap may include, but is not limited to, an Anti-Reverse Cap Analog (ARCA) (U.S. Patent No. 7,074,596), a 7-methyl-guanosine cap, a CleanCap® analog, such as a Cap1 analog (Trilink; San Diego, CA), or an enzymatic cap, such as with vaccinia capping enzyme. In some embodiments, the mRNA may be polyadenylated. The mRNA may contain various 5' and 3' untranslated sequence elements to enhance expression of the encoded engineered heterologous agent and / or the stability of the mRNA itself. Such elements may include post-translational regulatory elements, such as woodchuck hepatitis virus (VIMs) post-translational regulatory elements.

[0366] In some embodiments, the mRNA comprises at least one nucleoside modification. The mRNA may contain naturally occurring nucleosides modified into nucleoside analogs. Any nucleoside analog known in the art is contemplated. Such nucleoside analogs may include, for example, those described in U.S. Patent No. 8,278,036. In certain embodiments of the method, the nucleoside modification is selected from the group consisting of a uridine-to-pseudouridine modification and a uridine-to-N1-methylpseudouridine modification. In certain embodiments of the method, the nucleoside modification is a uridine-to-pseudouridine modification.

[0367] Particularly useful LNPs for the present method include cationic lipids selected from 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 octadecenoyloxy)phenylacetoxy]ethyl}piperidinyl)ethyl]disulfide), and derivatives thereof. DLin-MC3-DMA and its derivatives are described, for example, in WO 2010144740. DODMA and its derivatives are described, for example, in U.S. Patent No. 7,745,651 and Mok et al. (1999), Biochimica et Biophysica Acta, 1419(2):137-150. DLin-DMA and its derivatives are described, for example, in U.S. Patent No. 7,799,565. DLin-KC2-DMA and its derivatives are described, for example, in U.S. Patent No. 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.Further non-limiting examples of cationic lipids include methylpyridyl-dialkyl acids (MPDACA), palmitoyl-oleoyl-nor-arginine (PONA), guanidino-dialkyl acids (GUADACA), 1,2-di-0-octadecenyl-3-trimethylammonium propane (DOTMA), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), bis{2-[N-methyl-N-(α-D-tocopherol hemisuccinatopro...

Claims

1. A composition comprising a monoclonal antibody and / or natural killer (NK) cells (g-NK cells) lacking expression of the FcεR1γ chain for use in a method for inducing cytolytic killing of target cells, wherein the method Target cells known or suspected to express the first and second antigens, (a) A composition comprising natural killer (NK) cells (g-NK cells) lacking expression of the FcεR1γ chain, wherein the g-NK cells express a chimeric antigen receptor (CAR) comprising an extracellular binding domain that binds to the first antigen, and (b) Monoclonal antibody that binds to the second antigen A composition comprising a monoclonal antibody and / or natural killer (NK) cells (g-NK cells) lacking expression of the FcεR1γ chain, comprising the step of contacting with a monoclonal antibody.

2. A composition comprising a monoclonal antibody and / or natural killer (NK) cells for use by the method according to claim 1, wherein the first antigen and the second antigen are different.

3. A composition for use by the method of claim 1, comprising a monoclonal antibody and / or natural killer (NK) cells, wherein the first antigen and the second antigen are the same, and optionally, the CAR and the monoclonal antibody bind to different epitopes of the same antigen.

4. A composition comprising a monoclonal antibody and / or natural killer (NK) cells for use by the method of claim 1, 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.

5. A composition for use by the method of claim 1, comprising a monoclonal antibody and / or natural killer (NK) cells, wherein the target cells are tumor cells or B cells, optionally the tumor cells are cells of a hematological malignancy, and further optionally the hematological malignancy is multiple myeloma, or lymphoma, or leukemia, optionally the lymphoma is non-Hodgkin lymphoma (NHL), or the leukemia is acute myeloid leukemia (AML).

6. A composition comprising a monoclonal antibody and / or natural killer (NK) cells for use by the method of claim 1, wherein the first antigen and the second antigen are selected from the group consisting of GPC3, HER2, GD2, EGFR variant 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.

7. (a) The CAR is an anti-BCMA CAR, and the monoclonal antibody is an anti-CD38 antibody, optionally the anti-CD38 antibody is daratumumab or isatuximab, or (b) The CAR is an anti-CD19 CAR and the antibody is an anti-CD20 antibody, optionally the anti-CD20 antibody is rituximab, obinutuzumab, or ofatumumab, (c) The CAR is an anti-CD20 CAR, and the antibody is an anti-CD38 antibody, optionally the anti-CD38 antibody is daratumumab or isatuximab, (d) The CAR is an anti-CD19 CAR, and the antibody is an anti-CD38 antibody, optionally the anti-CD38 antibody is daratumumab or isatuximab. A composition comprising a monoclonal antibody and / or natural killer (NK) cells for use by the method described in claim 1.

8. A composition comprising a monoclonal antibody and / or natural killer (NK) cells for use by the method of claim 1, wherein the contact step is performed in vivo in a human subject.

9. NK cell therapy and / or monoclonal antibodies for use in a method of treating cancer in a subject, wherein the method is (a) A step of administering NK cell therapy to a subject having cancer, comprising a dose of a composition comprising natural killer (NK) cells (g-NK cells) lacking expression of FcεR1γ chain, 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 (b) The step of administering to the subject a dose of a monoclonal antibody that binds to a second antigen expressed by the cancer cells. NK cell therapy and / or monoclonal antibodies, including.

10. An NK cell therapy for use in a method of treating cancer in a subject, comprising the step of administering an NK cell therapy to a subject having cancer, comprising administering an NK cell therapy comprising a dose of a composition comprising natural killer (NK) cells (g-NK cells) lacking expression of FcεR1γ chain, The g-NK cells express a chimeric antigen receptor (CAR) that includes an extracellular binding domain that binds to a first antigen expressed by the cancer cells, and The g-NK cells express secretible monoclonal antibodies that bind to a second antigen expressed by the cancer cells. NK cell therapy.

11. 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, optionally, (i) The antigen-binding domain is a single-stranded variable fragment (scFv), and / or (ii) The intracellular signaling domain comprises one or more signaling domains selected from CD3ζ, DAP10, DAP12, CD28, 4-1BB, or OX40, and / or the intracellular signaling domain comprises a primary signaling domain comprising a CD3ζ signaling domain, optionally further comprising a co-stimulatory signaling domain, optionally being a CD28 or 4-1BB signaling domain. NK cell therapy and / or monoclonal antibodies for use by any one of claims 1 to 10.

12. NK cell therapy and / or monoclonal antibody for use by any one of claims 1 to 10, wherein the g-NK cells further comprise heterologous nucleic acids encoding an immunomodulatory protein, and optionally the immunomodulatory protein is a cytokine.

13. (A) The cytokine is secretible from the g-NK cells, and optionally the secretible cytokine is IL-2 or a biological part thereof, IL-15 or a biological part thereof, or IL-21 or a biological part thereof, or a combination thereof. (B) The cytokine is membrane-bound, and optionally 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. NK cell therapy and / or monoclonal antibodies for use by the method described in claim 12.

14. NK cell therapy and / or monoclonal antibody for use by the method according to any one of claims 1 to 10, further comprising the step of administering an exogenous cytokine in vivo to promote the growth or persistence of the g-NK cells in the subject, wherein the exogenous cytokine is or comprises IL-15.

15. In the g-NK cell composition, more than 60% or approximately 60% of the cells are g-NK cells, more than 70% or approximately 70% of the cells are g-NK cells, more than 80% or approximately 80% of the cells are g-NK cells, more than 90% or approximately 90% of the cells are g-NK cells, or more than 95% or approximately 95% of the cells are g-NK cells, and optionally, (i) More than 80% or approximately more than 80% of the g-NK cells are positive for perforin, and more than 80% or approximately more than 80% of the g-NK cells are positive for granzyme B, (ii) More than 90% or approximately more than 90% of the g-NK cells are positive for perforin, and more than 90% or approximately more than 90% of the g-NK cells are positive for granzyme B, (iii) More than 95% or about 95% of the g-NK cells are positive for perforin, and more than 95% or about 95% of the g-NK cells are positive for granzyme B, and / or (iv) At least 50% or at least about 50% of the cells in the g-NK cell composition are FcεR1γ deficient (FcεR1γ neg ) NK cells (g-NK), wherein more than 70% or approximately more than 70% of the g-NK cells are positive for perforin, and more than 70% or approximately more than 70% of the g-NK cells are positive for granzyme B, NK cell therapy and / or monoclonal antibodies for use by any one of claims 1 to 10.

16. NK cell therapy and / or monoclonal antibody for use by any one of claims 1 to 10, wherein more than 15% or about 15%, more than 20% or about 20%, more than 30% or about 30%, more than 40% or about 40%, or more than 50% or about 50% of the cells in the g-NK cell composition produce effector cytokines in the presence of cells expressing a target antigen (target cells) and antibodies against the target antigen (anti-target antibodies), and optionally, the effector cytokines are IFN-γ or TNF-α.

17. NK cell therapy and / or monoclonal antibody for use by the method according to any one of claims 1 to 10, wherein the g-NK cell composition is produced by ex vivo amplification of CD3- / CD57+ cells or CD3- / CD56+ cells cultured with irradiated HLA-E+ feeder cells, wherein the CD3- / CD57+ cells or CD3- / CD56+ cells are enriched from a biological sample derived from a donor subject, optionally the donor subject is serologically positive and / or the donor subject has a CD16 158V / V NK cell genotype or a CD16 158V / F NK cell genotype, and optionally the biological sample is derived from a human subject selected for the CD16 158V / V NK cell genotype or a CD16 158V / F NK cell genotype.

18. NK cell therapy and / or monoclonal antibody for use by any one of claims 1 to 10, wherein the g-NK cells are genetically engineered to have a knockout of the gene encoding the FcεR1γ chain, and optionally, one or more of the following: (i) the knockout is the introduction of a gene disruption of the gene, the gene disruption resulting in a deletion, insertion, or mutation in the gene; (ii) both alleles of the gene encoding the FcεR1γ chain are disrupted in the engineered cells; and (iii) the engineered g-NK cells are derived from primary cells obtained from a human subject.

19. Manipulated natural killer (NK) cells (g-NK cells) lacking expression of FcεR1γ chain, A heterogeneous nucleic acid encoding a chimeric antigen receptor (CAR) containing an extracellular binding domain that binds to a first antigen, and Heteronucleotide encoding a secretible monoclonal antibody that binds to a second antigen. Manipulated NK cells, including those mentioned above.

20. A pharmaceutical composition comprising any of the manipulated NK cells described in claim 19 and a pharmaceutically acceptable carrier.

21. A pharmaceutical composition according to claim 20 for use in a method for treating cancer in a subject, wherein the method comprises the step of administering the pharmaceutical composition to a subject having cancer.

Images