Methods for activating and expanding engineered natural killer cells in combination with antibodies

Abstract

The embodiments of the present disclosure relate to methods and compositions related to the preparation and use of combinatorial immunotherapy. In certain embodiments, compositions comprising engineered NK cells prepared by certain methods also include certain antibodies. These compositions are utilized in treatments, such as cancer treatments. In certain embodiments, the compositions include a complex of engineered NK cells and an antibody, where the antibody binds to the engineered NK cells and may also bind to another antigen, such as on a cancer cell.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to International Application No. PCT / US2022 / 023920, filed April 7, 2022, U.S. Provisional Application No. 63 / 348,259, filed June 2, 2022, and U.S. Provisional Application No. 63 / 415,517, filed October 12, 2022, each of which is incorporated by reference in its entirety herein.

[0002] Sequence Listing This application contains a Sequence Listing submitted in ST26 format, which is incorporated by reference in its entirety. The ST26 copy, created on April 3, 2023, is named MDAC_1330WO_Sequence_Listing.xml and is 26,915 bytes in size.

[0003] Technical Field Embodiments of the present disclosure include the fields of medicine including at least cell biology, molecular biology, immunology, and cancer medicine. [Background technology]

[0004] background In the field of natural killer (NK) cells as a therapy, certain barriers hinder their use, including the need for ex vivo expansion due to limited numbers, in addition to their poor persistence that affects their efficacy as an anti-cancer treatment. Cytokine stimulation is a key signal that enhances the functional capacity of NK cells to respond to tumor target cells. In addition, it has been shown that preactivating NK cells overnight with a combination of IL-18, IL-15, and IL-12 enhances cytokine production upon restimulation and generates long-lived memory-like NK cells (Leong et al., 2014). However, the procedure of using preactivated NK cells from peripheral blood (PB) without ex vivo expansion requires cumbersome procedures to generate sufficient quantities for clinical use. In addition, there are other obstacles related to the lack of cancer recognition and poor activation of NK cells. Thus, there is an unmet need for improved strategies to generate highly functional NK cells for therapeutic applications, especially NK cells that are efficient in cancer antigen targeting. Summary of the Invention [Means for solving the problem]

[0005] overview Embodiments of the present disclosure relate to methods and compositions related to immunotherapy for disease conditions. Immunotherapy may be utilized for any disease condition where targeting of cells in need of destruction is clinically useful and where there is a specific antigen on the targeted cells for which the antibody and / or cell may be specific. In specific embodiments, immunotherapy comprises, consists of, or consists essentially of adoptive cell therapy and an antibody configured to bind to the cell.

[0006] In some embodiments, provided herein are compositions comprising: (i) one or more engineered natural killer (NK) cells comprising one or more transgenes; and (ii) one or more antibody molecules comprising amivantamab, margetuximab, and / or imgatuzumab. In some embodiments, the engineered NK cells are derived from an expanded or non-expanded population. In some embodiments, the engineered NK cells are derived from an expanded population. In some embodiments, the engineered NK cells are derived from a pre-activated or non-pre-activated population. In some embodiments, the engineered NK cells are derived from a pre-activated population.

[0007] In some embodiments, the compositions provided herein are further defined as a complex between an engineered NK cell and margetuximab, amibamab, and / or imgatuzumab via binding of the Fc region of the antibody to the engineered NK cell. In some embodiments, the complex further comprises one or more antigen binding domains of one or more antibodies bound to a target antigen.

[0008] In some embodiments, the one or more antibody target antigens are epidermal growth factor receptor (EGFR), epidermal growth factor receptor-2 (HER2 / EGFR-2), and / or tyrosine protein kinase Met (c-Met). In some embodiments, the engineered NK cells are derived from umbilical cord blood (CB) mononuclear cells, CB hematopoietic stem cells, peripheral blood (PB) NK cells, NK cells or NK cell precursors obtained from bone marrow, stem cells, iPSCs, NK cell lines, or combinations thereof. In some embodiments, the engineered NK cells are derived from primary NK cells and are not derived from stem cells and / or induced pluripotent stem cells (iPSCs). In some embodiments, the NK cells are sourced from umbilical cord blood and the source of CB mononuclear cells and / or CB hematopoietic stem cells is CB from one donor or CB pooled from two or more individual CB units. In some embodiments, the engineered NK cells are modified to contain one or more transgenes for expression of one or more heterologous proteins.

[0009] In some embodiments, the one or more heterologous proteins comprise one or more engineered receptors and / or one or more cytokines. In some embodiments, the one or more engineered receptors are chimeric antigen receptors (CARs), T cell receptors (TCRs), receptors that recognize the Fc portion of Ig (FC-recognizing receptors), chemokine receptors, homing receptors, chimeric cytokine receptors, or any combination thereof. In some embodiments, the Fc-recognizing receptors comprise CD16, CD32, and / or CD64, and / or the engineered NK cells express one or more transgenes encoding one or more receptors to enhance binding to antibodies. In some embodiments, the engineered receptors comprise TCRs and / or CARs. In some embodiments, the engineered receptor is CD19, CD319 (CS1), ROR1, CD20, CD22, CD70, carcinoembryonic antigen, alpha fetoprotein, CA-125, MUC-1, EGFR, epithelial tumor antigen, melanoma associated antigen, mutated p53, mutated ras, EGFR-2 / HER2 / Neu, ERBB2, folate binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, C The target is a stem cell antigen, an autoantigen, or a cancer antigen selected from the group consisting of D5, CD123, CD23, CD30, CD38, CD56, CD70, c-Met, mesothelin, GD3, HERV-K, IL-11Rα, kappa chain, lambda chain, CSPG4, ERBB2, WT-1, TRAIL / DR4, VEGFR2, CD33, CD47, CLL-1, U5snRNP200, CD200, BAFF-R, BCMA, CD99, HLA-G, Trop2, and combinations thereof.

[0010] In some embodiments, the engineered antigen receptor targets CD70 and / or TROP2. In some embodiments, the antigen receptor that targets CD70 comprises, consists of, or consists essentially of a sequence at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:2 or 5. In some embodiments, the engineered NK cells comprise a construct comprising, consisting of, or consisting essentially of a sequence at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:1, 3, or 4. In some embodiments, the antigen receptor targeting TROP2 comprises, consists of, or consists essentially of a sequence at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:7 or 9. In some embodiments, the engineered NK cells comprise a construct comprising, consisting of, or consisting essentially of a sequence at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:6, 8, or 10.

[0011] In some embodiments, the engineered NK cells comprise one or more heterologous cytokines, including IL-2, IL-4, IL-7, IL-12, IL-15, IL-18, IL-21 and / or IL-23. In some embodiments, the cytokine comprises, consists of, or consists essentially of, encoded by and / or a sequence that is at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:11 or 12, comprises, consists of, or consists essentially of IL-15. In some embodiments, the cytokine comprises, consists of, or consists essentially of IL-21 encoded by and / or comprising, consisting of, or consisting essentially of a sequence that is at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs:13-16.

[0012] In some embodiments, the engineered NK cells express one or more transgenes encoding one or more suicide genes. In some embodiments, the engineered NK cells comprise one or more engineered mutations in endogenous genes. In some embodiments, the engineered mutations comprise CISH, CD38, glucocorticoid receptor, and / or TGFBR2 gene mutations.

[0013] In some embodiments, conjugating the one or more antibody molecules to the NK cells comprises incubating the NK cells with the one or more antibodies for at least or about 1 hour before washing the composition to remove unbound antibodies. In some embodiments, washing comprises washing at least twice with a suitable solution. In some embodiments, the suitable solution comprises PBS. In some embodiments, washing comprises washing for at least or about 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, or more than 10 minutes. In some embodiments, washing comprises washing for at least or about 5 minutes.

[0014] In some embodiments, the one or more antibody molecules are incubated with the NK cells for complex formation at a final concentration of about 1 to about 1000 μg / ml, about 1 μg / ml, about 10 μg / ml, about 50 μg / ml, or about 100 μg / ml. In some embodiments, the one or more antibodies and the NK cells are incubated in Click / RPMI medium. In some embodiments, the incubation and washing conditions for conjugation of the engineered NK cells with the antibody are such that at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the engineered NK cells are conjugated with the antibody for at least 1 hour, 4 hours, 8 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or 12 days after incubation and washing. In some embodiments, greater than about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, or 12% of the antibody is bound to the NK cell surface after at least 3 days of incubation and washing, relative to the amount of antibody complexed to the engineered NK cells measured about 1 hour after incubation and washing. In some embodiments, greater than about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or 10% of the antibody is bound to the NK cell surface after at least 5 days of incubation and washing, relative to the amount of antibody complexed to the engineered NK cells measured about 1 hour after incubation and washing. In some embodiments, relative to the amount of antibody complexed to the engineered NK cells measured about 1 hour after incubation and washing, at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, or greater than 8% of the antibody is bound to the NK cell surface after at least 7 days of incubation and washing.In some embodiments, relative to the amount of antibody complexed to the engineered NK cells measured about 1 hour after incubation and washing, at least about 0.5%, 1%, 2%, 3%, 4%, 5%, or greater than 5% of the antibody is bound to the NK cell surface after at least 12 days of incubation and washing.

[0015] In some embodiments, the composition comprising the engineered NK cells and the antibody is non-frozen, cryopreserved, or thawed from cryopreservation. In some embodiments, the composition comprising the engineered NK cells and the antibody is cryopreserved. In some embodiments, the composition is thawed from cryopreservation and at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the engineered NK cells are complexed with the antibody. In some embodiments, at least about 5%, 10%, 20%, 30%, 40%, 50%, or 60%, or any range derivable therein, of the amount of antibody complexed to non-cryopreserved engineered NK cells, measured about 1 hour after incubation and washing, is bound to the NK cell surface after thawing from cryopreservation. In some embodiments, the engineered NK cells are inactivated prior to cryopreservation. In some embodiments, the NK cell inactivator comprises a kinase inhibitor. In some embodiments, the NK cell inactivator comprises, consists of, or consists essentially of dasatinib.

[0016] In some embodiments, the composition comprising the engineered NK cells and the antibody is produced in vivo. In some embodiments, producing the composition in vivo comprises loading the engineered NK cells with the antibody in vivo by administering the engineered NK cells within about 1 hour of administering the antibody. In some embodiments, the one or more antibodies comprise or consist of amivantamab. In some embodiments, the one or more antibodies comprise or consist of margetuximab. In some embodiments, the one or more antibodies comprise or consist of imgatuzumab.

[0017] Also provided herein is a method of producing the compositions described herein. In some embodiments, the method of producing the compositions includes (i) optionally expanding engineered NK cells in a culture comprising an effective amount of: (a) a cytokine selected from the group consisting of IL-2, IL-15, IL-18, IL-21, and combinations thereof; and (b) an antigen-presenting cell / feeder, a fragment of an antigen-presenting cell / feeder, or an NK cell activation bead, and (ii) providing one or more antibody molecules, including amivantamab, margetuximab, and / or imgatuzumab, to the engineered NK cells.

[0018] In some embodiments, the method includes a preactivation step before and / or after the expansion step, in which the engineered NK cells are preactivated in a culture comprising one or more of IL-2, IL-12, IL-15, and IL-18 at effective concentrations. In some embodiments, the culture comprises effective concentrations of IL-12, IL-15, and IL-18. In some embodiments, the providing step is further defined as incubating the engineered NK cells with the antibody molecule for a particular duration, combining the engineered NK cells with the antibody molecule immediately prior to injection, or combining the engineered NK cells with the antibody in vivo in the subject through timed administration. In some embodiments, the duration is from about 5 minutes to about 24 hours or more. In some embodiments, the duration is at most, at least, or about 1 hour. In some embodiments, the culture for the preactivation step comprises IL-18 and / or IL-15 at a concentration of 0.1 to 1000 ng / mL, 1 to 1000 ng / mL, or about 10 ng / mL. In some embodiments, the culture for the preactivation step comprises IL-12 at a concentration of 0.1-1000 ng / mL, 1-1000 ng / mL, or about 10 ng / mL. In some embodiments, the method further comprises one or more washing steps of the preactivated engineered NK cells before and / or after the expansion step. In some embodiments, the engineered NK cells are activated at least twice or more during the expansion step with IL-12, IL-15, IL-18, IL-2, or any combination thereof. In some embodiments, the NK cell expansion step is for 5-60 days, 12-16 days, or 18-24 days. In some embodiments, the expansion culture further comprises IL-2. In some embodiments, IL-2 is present at a concentration of 10-500 U / mL, 100-300 U / mL, or about 200 U / mL. In some embodiments, IL-2 is replenished in the expansion culture every 2-3 days.

[0019] In some embodiments, the methods described herein include incubation conditions for conjugation of engineered NK cells with one or more antibodies such that at least or about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, or any range derivable therein, of the engineered NK cells are conjugated to the antibody for at least 1 hour, 4 hours, 8 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or 12 days, or any range derivable therein, after incubation and washing. In some embodiments, greater than about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, or 12% of the antibody is bound to the NK cell surface after at least 3 days of incubation and washing, relative to the amount of antibody complexed to the engineered NK cells measured about 1 hour after incubation and washing. In some embodiments, greater than about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or 10% of the antibody is bound to the NK cell surface after at least 5 days of incubation and washing, relative to the amount of antibody complexed to the engineered NK cells measured about 1 hour after incubation and washing. In some embodiments, greater than about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, or 8% of the antibody is bound to the NK cell surface after at least 7 days of incubation and washing, relative to the amount of antibody complexed to the engineered NK cells measured about 1 hour after incubation and washing. In some embodiments, greater than about 0.5%, 1%, 2%, 3%, 4%, 5%, or 5% of the antibody is bound to the NK cell surface after at least 12 days of incubation and washing, relative to the amount of antibody complexed to the engineered NK cells measured about 1 hour after incubation and washing.

[0020] In some embodiments, the method of preparing the composition includes not freezing the composition, or instead cryopreserving and / or thawing from cryopreservation. In some embodiments, the NK cells comprising the one or more antibodies are not frozen, cryopreserved, or thawed from cryopreservation. In some embodiments, the NK cells comprising the one or more antibodies are thawed from cryopreservation and at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the engineered NK cells are complexed with an antibody. In some embodiments, at least about 5%, 10%, 20%, 30%, 40%, 50%, or 60%, or greater than 60%, of the antibody is bound to the NK cell surface after thawing from cryopreservation relative to the amount of antibody complexed to non-cryopreserved engineered NK cells measured after about 1 hour of incubation and washing.

[0021] Also provided herein is a method of treating a disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of any one or more of the compositions described herein. In some embodiments, the disease or disorder is cancer, inflammation, graft-versus-host disease, transplant rejection, autoimmune disease, immunodeficiency disease, B-cell malignancy, or infectious disease. In some embodiments, the cancer comprises a hematological cancer or a solid tumor. In some embodiments, the cancer comprises pancreatic cancer, colon cancer, renal cancer, brain cancer, breast cancer, renal cancer, and / or myeloma. In some embodiments, the engineered NK cells are allogeneic with respect to the subject. In some embodiments, the engineered NK cells are autologous with respect to the subject. In some embodiments, the subject is a human. In some embodiments, the method of treatment further comprises administering at least a second therapeutic agent to the subject. In some embodiments, the at least a second therapeutic agent is a therapeutically effective amount of one or more anti-cancer agents, one or more immunomodulatory agents, and / or one or more immunosuppressive agents. In some embodiments, the anti-cancer agent is chemotherapy, radiation therapy, gene therapy, surgery, hormone therapy, anti-angiogenic therapy, or immunotherapy. In some embodiments, the immunosuppressant is a calcineurin inhibitor, an mTOR inhibitor, an antibody, a chemotherapeutic agent, irradiation, a chemokine, an interleukin, or an inhibitor of a chemokine or an interleukin. In some embodiments, the composition and / or at least the second therapeutic agent is administered intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intrainvasively, percutaneously, subcutaneously, topically, or by direct injection or perfusion. In some embodiments, the second therapeutic agent is an antibody.

[0022] In some embodiments, provided herein is a method of treating a subject having cancer with engineered NK cells, comprising: a) optionally stimulating a population of engineered NK cells by preactivation with a cytokine cocktail; b) optionally expanding the engineered NK cells; and ci) loading the engineered NK cells with one or more antibodies ex vivo and then administering the loaded engineered NK cells to a subject, and / or cii) loading the engineered NK cells with one or more antibodies in vivo by administering the engineered NK cells within one hour of administration of the antibodies; wherein the engineered NK cells comprise one or more transgenes, and the loading comprises exposure of the engineered NK cells to the one or more antibodies for a time effective for binding of the antibodies to the engineered NK cell surface, and wherein the one or more antibodies comprise or consist of amivantamab, margetuximab, and / or imgatuzumab. In some embodiments, the method comprises administering a composition comprising the engineered NK cells loaded ex vivo, followed by administering additional antibodies to the subject at a later time to additionally load the NK cells with antibodies in vivo. In some embodiments, loading the engineered NK cells comprises exposing the engineered NK cells to one or more antibodies for about 1 hour or more, or about 1 hour or less, or about 1 hour. In some embodiments, the engineered NK cells remain loaded with antibodies for at least about 1 hour, 4 hours, 8 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or 12 days. In some embodiments, the one or more antibodies are bound to the engineered NK cells via interaction with the CD16 surface protein. In some embodiments, the engineered NK cells are derived from umbilical cord blood. In some embodiments, the engineered NK cells are activated ex vivo. In some embodiments, the engineered NK cells are preactivated with IL-18, IL-15, and IL-12.

[0023] In some embodiments, the therapeutic method includes the use of NK cells that include one or more transgenes that include an engineered receptor. In some embodiments, the engineered receptor includes a chimeric antigen receptor (CAR), a T cell receptor, a chemokine receptor, a chimeric cytokine receptor, or any combination thereof. In some embodiments, the engineered receptor includes a CAR or a TCR. In some embodiments, the engineered receptor includes CD19, CD319 (CS1), ROR1, CD20, CD22, CD70, carcinoembryonic antigen, alpha fetoprotein, CA-125, MUC-1, EGFR, epithelial tumor antigen, melanoma associated antigen, mutated p53, mutated ras, EGFR-2 / HER2 / Neu, ERBB2, folate binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, C In some embodiments, the engineered antigen receptor targets a stem cell antigen, autoantigen, or cancer antigen selected from the group consisting of D5, CD123, CD23, CD30, CD38, CD56, CD70, c-Met, mesothelin, GD3, HERV-K, IL-11Rα, kappa chain, lambda chain, CSPG4, ERBB2, WT-1, TRAIL / DR4, VEGFR2, CD33, CD47, CLL-1, U5snRNP200, CD200, BAFF-R, BCMA, CD99, HLA-G, Trop2, and combinations thereof. In some embodiments, the engineered antigen receptor targets CD70 and / or TROP2. In some embodiments, the antigen receptor that targets CD70 comprises, consists of, or consists essentially of a sequence at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:2 or 5. In some embodiments, the engineered NK cells comprise a construct that comprises, consists of, or consists essentially of a sequence at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:1, 3, or 4.In some embodiments, the antigen receptor targeting TROP2 comprises, consists of, or consists essentially of a sequence at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:7 or 9. In some embodiments, the engineered NK cells comprise a construct comprising, consists of, or consists essentially of a sequence at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:6, 8, or 10. In some embodiments, the NK cells comprise one or more transgenes comprising a cytokine (e.g., a heterologous cytokine). In some embodiments, the one or more heterologous cytokines comprise IL-2, IL-4, IL-7, IL-12, IL-15, IL-18, IL-21 and / or IL-23. In some embodiments, the heterologous cytokines comprise IL-2, IL-12, IL-15, IL-18 and / or IL-21. In some embodiments, the cytokine comprises, consists of, or consists essentially of, IL-15, encoded by and / or comprising a sequence that is at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:11 or 12. In some embodiments, the cytokine comprises, consists of, or consists essentially of IL-21 encoded by and / or comprising a sequence that is at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 13-16. In some embodiments, the engineered NK cells comprise one or more engineered mutations in an endogenous gene.In some embodiments, the one or more engineered mutations in endogenous genes include CISH, CD38, glucocorticoid receptor, and / or TGFBR2 gene mutations.

[0024] Also provided herein are compositions comprising: (i) one or more engineered natural killer (NK) cells comprising one or more transgenes; and (ii) one or more antibodies, including the antibody IPH61. In some embodiments, the one or more antibodies target at least CD123 and NKp46. In some embodiments, the composition is further defined as a complex between the engineered NK cell IPH61 via binding of the Fc region of the antibody to the engineered NK cell and / or binding of the NKp46 targeting region of the antibody to the engineered NK cell. In some embodiments, the complex further comprises one or more antigen binding domains of the one or more antibodies bound to its target antigen. In some embodiments, the engineered NK cell is derived from NK cells or NK cell precursors obtained from cord blood (CB) mononuclear cells, CB hematopoietic stem cells, peripheral blood (PB) NK cells, bone marrow, stem cells, iPSCs, NK cell lines, or combinations thereof. In some embodiments, the engineered NK cell is a primary NK cell (e.g., an NK cell not derived from a stem cell, iPSC, etc.).

[0025] In some embodiments, the composition comprises engineered NK cells modified to include one or more transgenes for expression of one or more heterologous proteins. In some embodiments, the one or more heterologous proteins include one or more engineered receptors and / or one or more cytokines. In some embodiments, the one or more engineered receptors are chimeric antigen receptors (CARs), T cell receptors (TCRs), receptors that recognize the Fc portion of Ig (FC-recognizing receptors), chemokine receptors, homing receptors, chimeric cytokine receptors, or any combination thereof. In some embodiments, the Fc-recognizing receptors include CD16, CD32, and / or CD64, and / or the engineered NK cells express one or more transgenes encoding one or more receptors and / or NK cell surface antigens to enhance binding of the NK cells to antibodies. In some embodiments, the surface antigen and / or receptor of the engineered NK cells is CD16, CD32, CS1, CD56, CD64, NKG2D, NKG2C, DNAM, 2B4, CD2, NCR, NKp30, NKp44, NKp46, or KIR. In some embodiments, the engineered receptor comprises a TCR and / or a CAR. In some embodiments, the engineered receptor comprises CD19, CD319 (CS1), ROR1, CD20, CD22, CD70, carcinoembryonic antigen, alpha fetoprotein, CA-125, MUC-1, EGFR, epithelial tumor antigen, melanoma associated antigen, mutated p53, mutated ras, EGFR-2 / HER2 / Neu, ERBB2, folate binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, C The target is a stem cell antigen, an autoantigen, or a cancer antigen selected from the group consisting of D5, CD123, CD23, CD30, CD38, CD56, CD70, c-Met, mesothelin, GD3, HERV-K, IL-11Rα, kappa chain, lambda chain, CSPG4, ERBB2, WT-1, TRAIL / DR4, VEGFR2, CD33, CD47, CLL-1, U5snRNP200, CD200, BAFF-R, BCMA, CD99, HLA-G, Trop2, and combinations thereof.In some embodiments, the engineered antigen receptor targets CD70 and / or TROP2. In some embodiments, the antigen receptor that targets CD70 comprises, consists of, or consists essentially of a sequence at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:2 or 5. In some embodiments, the engineered NK cells comprise a construct comprising, consisting of, or consisting essentially of a sequence at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:1, 3, or 4. In some embodiments, the antigen receptor targeting TROP2 comprises, consists of, or consists essentially of a sequence at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:7 or 9. In some embodiments, the engineered NK cells comprise a construct comprising, consisting of, or consisting essentially of a sequence at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:6, 8, or 10. In some embodiments, the one or more heterologous cytokines are IL-2, IL-4, IL-7, IL-12, IL-15, IL-18, IL-21, and / or IL-23.In some embodiments, the cytokine comprises, consists of, or consists essentially of IL-15, encoded by and / or comprising, consisting of, or consisting essentially of a sequence that comprises, consists of, or consists of a sequence at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:11 or 12. In some embodiments, the cytokine comprises, consists of, or consists essentially of IL-21, encoded by a sequence comprising, consisting of, or essentially consisting of a sequence that is at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:13-16. In some embodiments, the engineered NK cells express one or more transgenes encoding one or more suicide genes. In some embodiments, the engineered NK cells comprise one or more engineered mutations in endogenous genes. In some embodiments, the one or more engineered mutations comprise CISH, CD38, glucocorticoid receptor, and / or TGFBR2 gene mutations. In some embodiments, complexing one or more antibodies, including IPH61, with the NK cells comprises incubating the NK cells with the one or more antibodies for at least or about 1 hour before washing the composition to remove unbound antibodies.

[0026] In some embodiments, the composition comprises one or more antibody molecules that are incubated with NK cells to form a complex at a final concentration of about 1 to about 1000 μg / ml, about 1 μg / ml, about 10 μg / ml, about 50 μg / ml, or about 100 μg / ml. In some embodiments, the antibody and NK cells are incubated in Click / RPMI medium. In some embodiments, the incubation and washing conditions for complexing the engineered NK cells with the antibody are such that at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the engineered NK cells are complexed with the antibody for at least 1 hour, 4 hours, 8 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or 12 days after incubation and washing. In some embodiments, the composition is unfrozen, cryopreserved, or thawed from cryopreservation. In some embodiments, the composition is thawed from cryopreservation and at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the engineered NK cells are complexed with the antibody. In some embodiments, at least about 30%, 40%, 50%, or 60% of the antibody is bound to the NK cell surface after thawing from cryopreservation relative to the amount of antibody complexed to the non-cryopreserved engineered NK cells measured about 1 hour after incubation and washing. In some embodiments, the engineered NK cells are inactivated prior to cryopreservation. In some embodiments, the inactivation agent comprises a kinase inhibitor. In some embodiments, the inactivation agent comprises dasatinib. In some embodiments, the composition is produced in vivo.In some embodiments, in vivo production of the composition comprises loading the engineered NK cells with one or more antibodies in vivo by administering the engineered NK cells within about 1 hour of administration of the antibodies.

[0027] Also provided herein is a method of producing a composition described herein, comprising: (i) optionally expanding the engineered NK cells in a culture comprising an effective amount of: (a) a cytokine selected from the group consisting of IL-2, IL-15, IL-18, IL-21, and combinations thereof; and (b) an antigen-presenting cell / feeder, a fragment of an antigen-presenting cell / feeder, or an NK cell activation bead; and (ii) providing the engineered NK cells with one or more antibody molecules, including IPH61. In some embodiments, the method comprises a preactivation step before and / or after the expansion step, in which the engineered NK cells are preactivated in a culture comprising an effective concentration of one or more of IL-2, IL-12, IL-15, and IL-18. In some embodiments, the culture comprises effective concentrations of IL-12, IL-15, and IL-18. In some embodiments, the providing step is further defined as incubating the engineered NK cells with the antibody molecule for a specific duration, combining the engineered NK cells with the antibody molecule immediately prior to injection, or combining the engineered NK cells with the antibody in vivo in the subject through time-coordinated administration. In some embodiments, the duration is from about 5 minutes to about 24 hours or more. In some embodiments, the duration is about 1 hour. In some embodiments, the culture for the preactivation step comprises IL-18 and / or IL-15 at a concentration of 0.1-1000 ng / mL, 1-1000 ng / mL, or about 10 ng / mL. In some embodiments, the culture for the preactivation step comprises IL-12 at a concentration of 0.1-1000 ng / mL, 1-1000 ng / mL, or about 10 ng / mL. In some embodiments, the expansion is for 5-60 days, 12-16 days, or 18-24 days. In some embodiments, the expansion culture further comprises IL-2. In some embodiments, IL-2 is present at a concentration of 10-500 U / mL, 100-300 U / mL, or about 200 U / mL.

[0028] In some embodiments, incubation conditions for conjugation of the engineered NK cells with the antibody are such that at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the engineered NK cells are conjugated with the antibody for at least 1 hour, 4 hours, 8 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or 12 days after incubation and washing. In some embodiments, the NK cells with the antibody are unfrozen, cryopreserved, or thawed from cryopreservation. In some embodiments, the NK cells comprising the antibody are thawed from cryopreservation and at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the engineered NK cells are complexed with the antibody. In some embodiments, at least about 30%, 40%, 50%, or 60% of the antibody is bound to the NK cell surface after thawing from cryopreservation relative to the amount of antibody complexed to non-cryopreserved engineered NK cells measured after about 1 hour of incubation and washing.

[0029] Also provided herein are methods of treating a disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of a composition comprising NK cells and one or more antibodies, including IPH61. In some embodiments, the disease or disorder is cancer, inflammation, graft-versus-host disease, transplant rejection, autoimmune disease, immunodeficiency, B-cell malignancy, or infectious disease. In some embodiments, the cancer comprises a hematological cancer or a solid tumor. In some embodiments, the cancer comprises acute myeloid leukemia, blastic plasmacytoid dendritic cell neoplasm, acute lymphoblastic leukemia, hairy cell leukemia, and / or systemic mastocytosis. In some embodiments, the engineered NK cells are allogeneic with respect to the subject. In some embodiments, the engineered NK cells are autologous with respect to the subject. In some embodiments, the subject is human. In some embodiments, the method of treatment further comprises administering to the subject at least a second therapeutic agent. In some embodiments, the at least a second therapeutic agent is a therapeutically effective amount of one or more anti-cancer agents, one or more immunomodulatory agents, and / or one or more immunosuppressive agents.

[0030] Also provided herein is a composition comprising: (i) one or more engineered natural killer (NK) cells comprising one or more transgenes; and (ii) one or more antibody molecules, wherein the one or more transgenes comprise a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:2 or 5. Compositions are provided comprising a CD70-targeted CAR consisting of, or consisting essentially of, and / or a TROP2-targeted CAR, wherein one or more transgenes comprise a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:7 or 9. In some embodiments, the engineered NK cells are derived from an expanded or non-expanded population. In some embodiments, the engineered NK cells are derived from an expanded population. In some embodiments, the engineered NK cells are derived from a pre-activated or non-pre-activated population. In some embodiments, the engineered NK cells are derived from a pre-activated population.

[0031] In some embodiments, the composition is further defined as a complex between the engineered NK cell and one or more antibodies. In some embodiments, the complex further comprises one or more antigen binding domains of the one or more antibodies bound to their target antigens. In some embodiments, the antibodies are CD19, CD319 (CS1), ROR1, CD20, CD22, CD70, carcinoembryonic antigen, alpha fetoprotein, CA-125, MUC-1, EGFR, epithelial tumor antigen, melanoma associated antigen, mutated p53, mutated ras, EGFR-2 / HER2 / Neu, ERBB2 folate binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, CD5, The antibody targets a cellular, autoantigen, or cancer antigen selected from the group consisting of CD123, CD23, CD30, CD38, CD56, CD70, c-Met, mesothelin, GD3, HERV-K, IL-11Rα, kappa chain, lambda chain, CSPG4, ERBB2, WT-1, TRAIL / DR4, VEGFR2, CD33, CD47, CLL-1, U5snRNP200, CD200, BAFF-R, BCMA, CD99, HLA-G, Trop2, and combinations thereof. In some embodiments, the one or more antibodies target epidermal growth factor receptor (EGFR), epidermal growth factor receptor-2 (HER2 / EGFR-2), NKp46, CD123, and / or tyrosine protein kinase Met (c-Met).

[0032] In some embodiments, the engineered NK cells are derived from umbilical cord blood (CB) mononuclear cells, CB hematopoietic stem cells, peripheral blood (PB) NK cells, NK cells or NK cell precursors obtained from bone marrow, stem cells, iPSCs, NK cell lines, or combinations thereof. In some embodiments, the engineered NK cells are derived from primary NK cells and are not derived from stem cells and / or induced pluripotent stem cells (iPSCs). In some embodiments, the NK cells are sourced from umbilical cord blood and the source of CB mononuclear cells and / or CB hematopoietic stem cells is CB from a single donor or CB pooled from two or more individual CB units.

[0033] In some embodiments, the engineered NK cells are further modified to include one or more additional transgenes for expression of one or more heterologous proteins. In some embodiments, the one or more heterologous proteins include one or more engineered receptors and / or one or more cytokines. In some embodiments, the one or more engineered receptors are chimeric antigen receptors (CARs), T cell receptors (TCRs), receptors that recognize the Fc portion of Ig (FC-recognizing receptors), chemokine receptors, homing receptors, chimeric cytokine receptors, or any combination thereof. In some embodiments, the Fc-recognizing receptors include CD16, CD32, and / or CD64, and / or the engineered NK cells express one or more transgenes encoding one or more receptors to enhance binding to antibodies. In some embodiments, the engineered receptors include TCRs and / or CARs. In some embodiments, the engineered receptor is CD19, CD319 (CS1), ROR1, CD20, CD22, CD70, carcinoembryonic antigen, alpha fetoprotein, CA-125, MUC-1, EGFR, epithelial tumor antigen, melanoma associated antigen, mutated p53, mutated ras, EGFR-2 / HER2 / Neu, ERBB2, folate binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, C The target is a stem cell antigen, an autoantigen, or a cancer antigen selected from the group consisting of D5, CD123, CD23, CD30, CD38, CD56, CD70, c-Met, mesothelin, GD3, HERV-K, IL-11Rα, kappa chain, lambda chain, CSPG4, ERBB2, WT-1, TRAIL / DR4, VEGFR2, CD33, CD47, CLL-1, U5snRNP200, CD200, BAFF-R, BCMA, CD99, HLA-G, Trop2, and combinations thereof.

[0034] In some embodiments, the engineered NK cells include constructs comprising, consisting of, or consisting essentially of a sequence at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:1, 3, or 4. In some embodiments, the engineered NK cells include constructs comprising, consisting of, or consisting essentially of a sequence at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:6, 8, or 10.

[0035] In some embodiments, the NK cells are engineered to express one or more heterologous cytokines. In some embodiments, the one or more heterologous cytokines include IL-2, IL-4, IL-7, IL-12, IL-15, IL-18, IL-21 and / or IL-23. In some embodiments, the cytokine comprises, consists of, or consists essentially of SEQ ID NO:11 or 12, and / or is encoded by a sequence comprising, consisting of, or essentially consisting of a sequence that is at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:11 or 12, comprises, consists of, or consists essentially of IL-15. In some embodiments, the cytokine comprises, consists of, or consists essentially of SEQ ID NOs:13-16, and / or is encoded by, and / or comprises, consists of, or consists essentially of a sequence that is at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs:13-16, comprises, consists of, or consists essentially of IL-21. In some embodiments, the engineered NK cells express one or more transgenes encoding one or more suicide genes. In some embodiments, the engineered NK cells comprise one or more engineered mutations in endogenous genes. In some embodiments, the engineered mutations comprise CISH, CD38, glucocorticoid receptor, and / or TGFBR2 gene mutations.

[0036] In some embodiments, complexing the one or more antibody molecules with the NK cells comprises incubating the NK cells with the one or more antibodies for at least or about 1 hour before washing the composition to remove unbound antibodies. In some embodiments, the one or more antibody molecules are incubated with the NK cells to form complexes at a concentration of about 1 to about 1000 μg / ml final concentration, about 1 μg / ml final concentration, about 10 μg / ml final concentration, about 50 μg / ml final concentration, or about 100 μg / ml final concentration. In some embodiments, the antibody and NK cells are incubated in Click / RPMI medium. In some embodiments, the incubation and washing conditions for conjugation of the engineered NK cells with the antibody are such that at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the engineered NK cells are conjugated with the antibody for at least 1 hour, 4 hours, 8 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or 12 days after incubation and washing. In some embodiments, for the amount of antibody complexed to the engineered NK cells measured about 1 hour after incubation and washing, at least about 9% of the antibody is bound to the NK cell surface after at least 3 days after incubation and washing. In some embodiments, for the amount of antibody complexed to the engineered NK cells measured about 1 hour after incubation and washing, at least about 6% of the antibody is bound to the NK cell surface after at least 5 days after incubation and washing. In some embodiments, for the amount of antibody complexed to the engineered NK cells measured about 1 hour after incubation and washing, at least about 4% of the antibody is bound to the NK cell surface after at least 7 days after incubation and washing.In some embodiments, at least about 3% of the antibody is bound to the surface of the NK cells at least 12 days after incubation and washing, relative to the amount of antibody complexed to the engineered NK cells measured about 1 hour after incubation and washing. In some embodiments, the composition is not frozen, cryopreserved, or thawed from cryopreservation. In some embodiments, the composition is thawed from cryopreservation and at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the engineered NK cells are complexed to the antibody. In some embodiments, at least about 30%, 40%, 50%, or 60% of the antibody is bound to the NK cell surface after thawing from cryopreservation relative to the amount of antibody complexed to non-cryopreserved engineered NK cells measured after about 1 hour of incubation and washing. In some embodiments, the engineered NK cells are inactivated prior to cryopreservation. In some embodiments, the inactivating agent comprises a kinase inhibitor. In some embodiments, the inactivating agent comprises dasatinib. In some embodiments, the composition is produced in vivo. In some embodiments, producing the composition in vivo comprises loading the engineered NK cells with the antibody in vivo by administering the engineered NK cells within about 1 hour of administering the antibody. In some embodiments, the one or more antibodies comprise or consist of amivantamab. In some embodiments, the one or more antibodies comprise or consist of margetuximab. In some embodiments, the one or more antibodies comprise or consist of imgatuzumab. In some embodiments, the one or more antibodies comprise or consist of IPH61.

[0037] Also provided herein are methods for producing a composition comprising: (i) optionally expanding engineered NK cells in a culture comprising an effective amount of: (a) a cytokine selected from the group consisting of IL-2, IL-15, IL-18, IL-21, and combinations thereof; and (b) an antigen presenting cell / feeder, a fragment of an antigen presenting cell / feeder, or an NK cell activation bead; and (ii) providing one or more antibody molecules to the engineered NK cells. In some embodiments, the method comprises a preactivation step before and / or after the expansion step, wherein the engineered NK cells are preactivated in a culture comprising an effective concentration of one or more of IL-2, IL-12, IL-15, and IL-18. In some embodiments, the culture comprises an effective concentration of IL-12, IL-15, and IL-18. In some embodiments, the providing step is further defined as incubating the engineered NK cells with the antibody molecule for a particular duration, combining the engineered NK cells with the antibody molecule immediately prior to injection, or combining the engineered NK cells with the antibody in vivo in the subject through time-coordinated administration. In some embodiments, the duration is from about 5 minutes to about 24 hours or more. In some embodiments, the duration is about 1 hour. In some embodiments, the culture for the preactivation step comprises IL-18 and / or IL-15 at a concentration of 0.1-1000 ng / mL, 1-1000 ng / mL, or about 10 ng / mL. In some embodiments, the culture for the preactivation step comprises IL-12 at a concentration of 0.1-1000 ng / mL, 1-1000 ng / mL, or about 10 ng / mL. In some embodiments, the method further comprises one or more washing steps of the preactivated engineered NK cells before and / or after the expansion step. In some embodiments, the engineered NK cells are activated with IL-12, IL-15, IL-18, IL-2, or any combination thereof at least two or more times during the expansion process. In some embodiments, expansion is for 5-60 days, 12-16 days, or 18-24 days. In some embodiments, the expansion culture further comprises IL-2.In some embodiments, IL-2 is present at a concentration of 10-500 U / mL, 100-300 U / mL, or about 200 U / mL, hi some embodiments, IL-2 is replenished in expansion cultures every 2-3 days.

[0038] In some embodiments, incubation conditions for complexing the engineered NK cells with one or more antibodies are such that at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the engineered NK cells are complexed with the antibody for at least 1 hour, 4 hours, 8 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or 12 days after incubation and washing. In some embodiments, for the amount of antibody complexed to the engineered NK cells measured after about 1 hour of incubation and washing, at least about 9% of the antibody is bound to the NK cell surface after at least 3 days of incubation and washing. In some embodiments, for the amount of antibody complexed to the engineered NK cells measured after about 1 hour of incubation and washing, at least about 6% of the antibody is bound to the NK cell surface after at least 5 days of incubation and washing. In some embodiments, for the amount of antibody complexed to the engineered NK cells measured after about 1 hour of incubation and washing, at least about 4% of the antibody is bound to the NK cell surface after at least 7 days of incubation and washing. In some embodiments, for the amount of antibody complexed to the engineered NK cells measured after about 1 hour of incubation and washing, at least about 3% of the antibody is bound to the NK cell surface after at least 12 days of incubation and washing. In some embodiments, the NK cells comprising one or more antibodies are unfrozen, cryopreserved, or thawed from cryopreservation. In some embodiments, the NK cells comprising one or more antibodies are thawed from cryopreservation and at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the engineered NK cells are complexed with an antibody.In some embodiments, after thawing from cryopreservation, at least about 30%, 40%, 50%, or 60% of the antibody is bound to the NK cell surface relative to the amount of antibody complexed to non-cryopreserved engineered NK cells measured after about 1 hour of incubation and washing.

[0039] Also provided herein is a method of treating a disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of any one or more of the compositions provided herein. In some embodiments, the disease or disorder is cancer, inflammation, graft-versus-host disease, transplant rejection, autoimmune disease, immunodeficiency disease, B-cell malignancy, or infectious disease. In some embodiments, the cancer comprises a hematological cancer or a solid tumor. In some embodiments, the cancer comprises pancreatic cancer, colon cancer, renal cancer, brain cancer, breast cancer, renal cancer, and / or myeloma. In some embodiments, the cancer comprises acute myeloid leukemia, blastic plasmacytoid dendritic cell neoplasm, acute lymphoblastic leukemia, hairy cell leukemia, and / or systemic mastocytosis. In some embodiments, the engineered NK cells are allogeneic with respect to the subject. In some embodiments, the engineered NK cells are autologous with respect to the subject. In some embodiments, the subject is a human. In some embodiments, the method of treatment further comprises administering to the subject at least a second therapeutic agent. In some embodiments, the at least second therapeutic agent is a therapeutically effective amount of one or more anti-cancer agents, one or more immunomodulatory agents, and / or one or more immunosuppressive agents. In some embodiments, the at least second therapeutic agent is the same antibody and / or targets the same antigen as the antibody previously provided in the composition comprising antibody-loaded NK cells. In some embodiments, the anti-cancer agent is chemotherapy, radiation therapy, gene therapy, surgery, hormonal therapy, anti-angiogenic therapy, or immunotherapy. In some embodiments, the immunosuppressive agent is a calcineurin inhibitor, an mTOR inhibitor, an antibody, a chemotherapeutic agent, irradiation, a chemokine, an interleukin, or an inhibitor of a chemokine or an interleukin. In some embodiments, the composition and / or the at least second therapeutic agent is administered intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intrainvasively, percutaneously, subcutaneously, topically, or by direct injection or perfusion. In some embodiments, the second therapeutic agent is an antibody.

[0040] Also provided herein is a method of treating a subject having cancer with engineered NK cells, the method comprising: a) optionally stimulating a population of engineered NK cells by preactivation with a cytokine cocktail; b) optionally expanding the engineered NK cells; and ci) loading the engineered NK cells with one or more antibodies ex vivo and then administering the loaded engineered NK cells to a subject, or cii) loading the engineered NK cells with one or more antibodies in vivo by administering the engineered NK cells within one hour of administration of the antibodies; wherein the engineered NK cells comprise one or more transgenes, and the loading is performed for a time effective for binding of the antibodies to the engineered NK cell surface. and exposing the engineered NK cells to one or more antibodies, wherein the one or more transgenes comprise a CD70-targeted CAR comprising a sequence at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:2 or 5, and / or a TROP2-targeted CAR comprising a sequence at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:7 or 9. In some embodiments, loading of the engineered NK cells comprises exposing the engineered NK cells to one or more antibodies for about 1 hour or more, or about 1 hour or less, or about 1 hour. In some embodiments, the engineered NK cells remain loaded with the antibodies for at least or about 1 hour, 4 hours, 8 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or 12 days. In some embodiments, the one or more antibodies are bound to the engineered NK cells via interaction with the CD16 surface protein. In some embodiments, the engineered NK cells are derived from umbilical cord blood. In some embodiments, the engineered NK cells are activated ex vivo. In some embodiments, the engineered NK cells are pre-activated with IL-18, IL-15, and IL-12.In some embodiments, the engineered NK cells include one or more additional transgenes, and optionally, an engineered receptor. In some embodiments, the engineered receptor includes a chimeric antigen receptor (CAR), a T cell receptor, a chemokine receptor, a chimeric cytokine receptor, or any combination thereof. In some embodiments, the engineered receptor includes a CAR or a TCR. In some embodiments, the engineered receptor includes CD19, CD319 (CS1), ROR1, CD20, CD22, CD70, carcinoembryonic antigen, alpha fetoprotein, CA-125, MUC-1, EGFR, epithelial tumor antigen, melanoma associated antigen, mutated p53, mutated ras, EGFR-2 / HER2 / Neu.ERBB2, folate binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, C The target is a stem cell antigen, an autoantigen, or a cancer antigen selected from the group consisting of D5, CD123, CD23, CD30, CD38, CD56, CD70, c-Met, mesothelin, GD3, HERV-K, IL-11Rα, kappa chain, lambda chain, CSPG4, ERBB2, WT-1, TRAIL / DR4, VEGFR2, CD33, CD47, CLL-1, U5snRNP200, CD200, BAFF-R, BCMA, CD99, HLA-G, Trop2, and combinations thereof. In some embodiments, the engineered NK cells comprise a construct that is at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1, 3, or 4. In some embodiments, the engineered NK cells comprise a construct that is at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 6, 8, or 10. In some embodiments, the one or more transgenes comprise a cytokine.In some embodiments, the one or more heterologous cytokines comprise IL-2, IL-4, IL-7, IL-12, IL-15, IL-18, IL-21 and / or IL-23. In some embodiments, the cytokines comprise IL-2, IL-12, IL-15, IL-18 and / or IL-21. In some embodiments, the cytokine comprises, consists of, or consists essentially of IL-15 encoded by and / or comprising, consisting of, or consisting essentially of a sequence comprising, consisting of, or consisting of a sequence at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:11 or 12. In some embodiments, the cytokine comprises, consists of, or consists essentially of IL-21 encoded by and / or comprising, consisting of, or consisting essentially of a sequence that comprises, consists of, or consists of at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 13-16. In some embodiments, the engineered NK cells comprise one or more engineered mutations in endogenous genes. In some embodiments, the one or more engineered mutations in endogenous genes comprise CISH, CD38, glucocorticoid receptor, and / or TGFBR2 gene mutations.

[0041] In certain embodiments, the efficacy of a composition comprising a complex of engineered NK cells and an antibody is enhanced compared to when used separately. In some embodiments, the efficacy of the engineered NK cells and the antibody when used as a complex is synergistic. In some embodiments, the efficacy of the engineered NK cells and the antibody is additive.

[0042] In either method, the composition (and / or at least the second therapeutic agent) may be administered intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intrainvasively, percutaneously, subcutaneously, topically, or by direct injection or perfusion or infusion.

[0043] The foregoing has outlined, rather broadly, the features and technical advantages of the present disclosure in order that the detailed description that follows may be better understood. Additional features and advantages will now be described which form the subject matter of the claims appended hereto. It should be understood by those skilled in the art that the conception and specific embodiments disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present designs. It should also be understood by those skilled in the art that such equivalent constructions do not depart from the spirit and scope as defined by the appended claims. The novel features believed characteristic of the designs disclosed herein, both as to organization and method of operation, together with further objects and advantages, will be better understood from the following description. It is to be expressly understood, however, that this disclosure is provided for purposes of illustration and description only, and is not intended as a definition of the limits of the present disclosure.

[0044] BRIEF DESCRIPTION OF THE DRAWINGS For a more complete understanding of the present disclosure, reference is now made to the following descriptions taken in conjunction with the accompanying drawings, in which: [Brief description of the drawings]

[0045] [Figure 1] FIG. 1. Schematic detailing one embodiment of the experimental design for expanding and preloading cord blood-derived NK cells with antibodies.

[0046] [Diagram 2] Figure 2. Margetuximab bound to pre-activated and proliferating (PE) NK cells with higher affinity compared to regular proliferating NK cells.

[0047] [Diagram 3]Figure 3. Margetuximab-loaded NK cells showed enhanced cytotoxicity (Y-axis) against HER2+ (e.g., SKOV3) tumor cells in a long-term Xcelligence killing assay (X-axis tracks time in hours). NK cells were either normally expanded (NE) or pre-activated and expanded (P+E).

[0048] [Figure 4-1] Figures 4A-4C. Margetuximab-loaded NK cells enhanced tumor control in NSG mouse models of HER2+ ovarian cancer (e.g., SKOV3). [Figure 4-2] Same as above. [Figure 4-3] Same as above.

[0049] [Diagram 5] Figure 5. Amivantamab bound with higher affinity to preactivated and expanded (PE) NK cells compared to normal expanded NK cells (NE).

[0050] [Figure 6] Figure 6. Amivantamab-loaded NK cells showed enhanced cytotoxicity against EGFR+ / cMET+ tumor cells (e.g., SKOV3, UMRC3, PATC148) in short-term 51Cr-release killing assays (X-axis tracks effector-to-target (E:T) ratios of 20:1, 10:1, 5:1, or 1:1). NK cells were either normally expanded (NE) or preactivated and expanded (PE).

[0051] [Figure 7-1] Figure 7A-7B. Amivantamab-loaded NK cells showed enhanced cytotoxicity against EGFR+ / cMET+ tumor cells (e.g., SKOV3 and PATC148) in a long-term Xcelligence killing assay (X-axis tracks time in hours). NK cells were either normally expanded (NE) or pre-activated and expanded (PE). [Figure 7-2] Same as above.

[0052] [Figure 8-1] Figures 8A-8C. Amivantamab-loaded NK cells enhanced tumor control in NSG mouse models of EGFR+ / c-MET+ ovarian cancer (e.g., SKOV3). [Figure 8-2] Same as above. [Figure 8-3] Same as above.

[0053] [Figure 9] Figure 9. Imgatuzumab bound with higher affinity to preactivated and expanded (PE) NK cells compared to normally expanded (NE) NK cells.

[0054] [Figure 10] Figure 10. Imugatuzumab-loaded NK cells showed enhanced cytotoxicity against EGFR+ tumor cells (e.g., SKOV3, UMRC3, PATC148) in short-term 51Cr-release killing assays. NK cells were either normally expanded (NE) or pre-activated and expanded (P+E).

[0055] [Figure 11-1] Figures 11A-11C. Imgatuzumab-loaded NK cells showed enhanced cytotoxicity (Y-axis, normalized cell index) against EGFR+ tumor cells (e.g., SKOV3, PATC148, UMRC3) in a long-term Xcelligence killing assay (X-axis tracks time in hours). NK cells were either normally expanded (NE) or pre-activated and expanded (P+E). [Figure 11-2] Same as above. [Figure 11-3] Same as above.

[0056] [Figure 12-1] Figures 12A-12C. Imgatuzumab-loaded NK cells enhanced tumor control in NSG mouse models of EGFR+ ovarian cancer (eg, SKOV3). [Figure 12-2] Same as above. [Figure 12-3] Same as above.

[0057] [Figure 13] Figure 13. A series of Western blots showing that EGFR was expressed in various tumor cell lines, including pancreatic (e.g., Capan-1, Capan-2, MIA-Capa2, CFPAC-1, PANC-1, BcPC3), colon (e.g., WiDr, HCT15, RKO, LOVO, SW403, LS174T, HCT116, SW116), kidney (e.g., 786-0, UMRC3, A498, 769-P, ACHN, Caki-1, Caki-2, A704), glioblastoma multiforme (GBM) (e.g., GSC272, GSC8-11, GSC20, GSC267), breast cancer (MDA-231, BCX010, etc.), and renal cancer (SN12C, etc.) tumor cell lines. β-actin was used as a loading control.

[0058] [Figure 14] Figure 14. Imgatuzumab bound to NK cells and EGFR+ tumor cell lines when added to the cells at 1 μg or 10 μg as analyzed by flow cytometry.

[0059] [Figure 15] Figure 15. Imgatuzumab bound to non-transduced (NT) or transduced (e.g., with a chimeric antigen receptor (CAR)) NK cells. Furthermore, imgatuzumab remained bound to NT or CAR NK cells (e.g., engineered NK cells) and persisted on the cell surface for at least 5 days.

[0060] [Figure 16] Figure 16. Imgatuzumab-loaded NK cells demonstrated enhanced killing of breast (e.g., MDA-231 cell line), pancreatic (e.g., pancreatic ductal adenocarcinoma) (PDAC) (e.g., PATC-148 cell line), and colorectal (CRC) (e.g., WiDR cell line) cell lines in vitro compared to non-loaded NK cells.

[0061] [Figure 17] Figure 17. Engineered NK cells loaded with imgatuzumab (e.g., CAR NK+Imga 1 μg) exhibited enhanced killing of UMRC3 (RCC) cells in vitro compared to Imga-treated tumor cells alone, unloaded non-transduced (NT) (e.g., non-engineered) NK cells, imgatuzumab-loaded NT-NK cells (e.g., NT-NK+Imga 1 μg), or engineered non-loaded NK (CAR NK) cells.

[0062] [Figure 18] Figure 18. Engineered NK cells loaded with Imgatuzumab (e.g., CAR NK+Imga 1 μg) showed enhanced killing of SKOV3 (ovarian cancer) cells in vitro compared to Imga-treated tumor cells alone, unloaded non-transduced (NT) (e.g., non-engineered) NK cells, imgatuzumab-loaded NT-NK cells (e.g., NT-NK+Imga 1 μg), or engineered non-loaded NK (CAR NK) cells.

[0063] [Figure 19] Figure 19. Engineered NK cells loaded with Imgatuzumab (e.g., CAR NK+Imga 1 μg) showed enhanced killing of PATC-148 (PDAC) cells in vitro compared to Imga-treated tumor cells alone, unloaded non-transduced (NT) (e.g., non-engineered) NK cells, imgatuzumab-loaded NT-NK cells (e.g., NT-NK+Imga 1 μg), or engineered non-loaded NK (CAR NK) cells.

[0064] [Figure 20]Figure 20. Antibodies (e.g., Imgatuzumab) were retained on the NK cell surface for at least 12 days after antibody loading. NK cells were left unloaded (-Imga) or loaded with Imgatuzumab (final concentration 100 μg / ml) (+Imga) in Click / RPMI medium for 1 h at 37°C and washed twice with PBS (5 min each wash) before verifying Imgatuzumab binding by flow cytometry. NK cells were stained with Alexa-Fluor647 affinity purified F(ab')2 fragment goat anti-human IgG(H+L) antibody at different time points (1 h, 3 days, 5 days, 7 days, 12 days) and analyzed by flow cytometry.

[0065] [Figure 21-1] Figures 21A-21B. Imgatuzumab alone did not cause cytotoxicity against ovarian cancer or PDAC cell lines. Xcelligence assays were performed to evaluate the cytotoxicity of imgatuzumab against ovarian cell lines (e.g., SKOV3, Figure 21A) and pancreatic ductal adenocarcinoma (PDAC) cell lines (e.g., PATC148, Figure 21B). Graphs plot normalized cell index (Y-axis) against time (X-axis, hours). Cell lines cultured alone served as negative controls and cell lines lysed with SDS served as positive controls. Statistical analysis consisted of two-way ANOVA. [Figure 21-2] Same as above.

[0066] [Figure 22-1]Figures 22A-22B. Imgatuzumab-loaded NK cells showed enhanced killing of ovarian cancer (e.g., SKOV3) cell lines and PDAC (e.g., PATC148) cells compared to unloaded NK cells in a long-term Xcelligence cytotoxicity assay. NK cells were loaded with imgatuzumab (100 μg imgatuzumab, e.g., 100 μg / ml final concentration in 1 ml of NK cells) in Click / RPMI medium for 1 hour at 37°C, washed twice with PBS (5 min each wash), and then co-cultured with tumor cells at a 1:1 effector-to-target ratio. Compared to unloaded NK cells, imgatuzumab-loaded NK cells showed increased cytotoxic activity against SKOV3 (Figure 22A) and PATC148 (Figure 22B) cells. Cell lines cultured alone served as negative controls and cell lines lysed with SDS served as positive controls, and statistical analysis consisted of two-way ANOVA. [Figure 22-2] Same as above.

[0067] [Diagram 23] Figure 23. Imgatuzumab-loaded NK cells showed enhanced killing of colorectal cancer (CRC) cells (e.g., WiDR) compared to non-loaded NK cells in a long-term Xcelligence cytotoxicity assay. NK cells were loaded with imgatuzumab (final concentration 100 μg / ml) in Click / RPMI medium for 1 h at 37°C, washed twice with PBS (5 min each wash), and then co-cultured with tumor cells at an effector-to-target ratio of 1:1. Statistical analysis consisted of two-way ANOVA.

[0068] [Figure 24-1]Figures 24A-24B. Imgatuzumab-loaded NK cells showed enhanced killing of lung cancer cells (e.g., CRL-5922 and HTB-58) compared to unloaded NK cells in the Xcelligence long-term cytotoxicity assay. NK cells were loaded with imgatuzumab (final concentration 100 μg / ml) in Click / RPMI medium for 1 h at 37°C, washed twice with PBS (5 min each wash) and then co-cultured with tumor cells at a 1:1 effector-to-target ratio. Compared to unloaded NK cells, imgatuzumab-loaded NK cells showed increased cytotoxic activity against CRL-5922 (Figure 24A) and HTB-58 (Figure 24B) lung cancer cell lines. Cell lines cultured alone served as negative controls and cell lines lysed with SDS served as positive controls. Statistical analysis consisted of two-way ANOVA. [Figure 24-2] Same as above.

[0069] [Figure 25-1] Figures 25A-25B. Imgatuzumab-loaded NK cells showed enhanced killing of breast cancer cells (e.g., MDA 468 and SKBR3) compared to unloaded NK cells in the Xcelligence long-term cytotoxicity assay. NK cells were loaded with Imgatuzumab (final concentration 100 μg / ml) in Click / RPMI medium for 1 h at 37°C, washed twice with PBS (5 min each wash) and then co-cultured with tumor cells at a 1:1 effector-to-target ratio. Compared to unloaded NK cells, Imgatuzumab-loaded NK cells showed increased cytotoxic activity against CRL-5922 (Figure 25A) and HTB-58 (Figure 25B) lung cancer cell lines. Cell lines cultured alone served as negative controls and cell lines lysed with SDS served as positive controls. Statistical analysis consisted of two-way ANOVA. [Figure 25-2] Same as above.

[0070] [Figure 26-1]Figures 26A-26B. Imgatuzumab-loaded NK cells demonstrated enhanced killing of PDAC (e.g., PATC148) tumor spheroids compared to unloaded NK cells in an Incucyte live cell imaging assay. Figure 26A shows representative images of PATC148 spheroids (20,000 tumor cells transduced with GFP and allowed to form spheroids for 48 hours) left alone or treated with 50,000 unloaded NK cells or 50,000 NK cells preloaded with Imgatuzumab (final concentration 100 μg / ml) at different time points. Data showed a significant decrease in spheroid size in wells treated with Imgatuzumab-loaded NK cells. Figure 26B is a quantification of total integrated green intensity over time. Statistical analysis consisted of a two-way ANOVA. [Figure 26-2] Same as above.

[0071] [Figure 27-1] Figures 27A-27B. Imgatuzumab-loaded NK cells showed enhanced cytotoxicity against CRC (e.g., WiDR) tumor spheroids compared to unloaded NK cells in an Incucyte live cell imaging assay. Figure 27A shows representative images of WiDR spheroids (20,000 tumor cells transduced with GFP and allowed to form spheroids for 48 hours) left alone or treated with 50,000 unloaded NK cells or 50,000 NK cells preloaded with Imgatuzumab (final concentration 100 μg / ml) at different time points. Data showed a significant decrease in spheroid size in wells treated with Imgatuzumab-loaded NK cells. Figure 27B is a quantification of total integrated green intensity over time. Statistical analysis consisted of a two-way ANOVA. [Figure 27-2] Same as above.

[0072] [Figure 28-1]Figures 28A-28B. Imgatuzumab-loaded NK cells showed enhanced cytotoxicity against UMRC3 (RCC) tumor spheroids compared to unloaded NK cells in an Incucyte live cell imaging assay. Figure 28A shows representative images of UMRC3 spheroids (20,000 tumor cells transduced with GFP and allowed to form spheroids for 48 hours) left alone or treated with 50,000 unloaded NK cells or 50,000 NK cells preloaded with Imgatuzumab (final concentration 100 μg / ml) at different time points. Data showed a significant decrease in spheroid size in wells treated with Imgatuzumab-loaded NK cells. Figure 28B is a quantification of total integrated green intensity over time. Statistical analysis consisted of a two-way ANOVA. [Figure 28-2] Same as above.

[0073] [Figure 29-1] Figures 29A-29B. Imgatuzumab-loaded NK cells showed enhanced cytotoxicity against GBM (e.g., GSC20) tumor spheroids compared to unloaded NK cells in an Incucyte live cell imaging assay. Figure 29A shows representative images of GSC20 spheroids (20,000 tumor cells transduced with mCherry and allowed to form spheroids for 48 hours) left alone or treated with 50,000 unloaded NK cells or 50,000 NK cells preloaded with Imgatuzumab (final concentration 100 μg / ml) at different time points. The data showed that wells treated with Imgatuzumab-loaded NK cells had significantly reduced spheroid size compared to unloaded NK cells. Figure 29B is a quantification of total integrated red intensity over time. Statistical analysis consisted of a two-way ANOVA. [Figure 29-2] Same as above.

[0074] [Figure 30-1]Figures 30A-30D. Imgatuzumab-loaded NK cells led to enhanced tumor control in NSG mouse models of EGFR+ PDAC (e.g., PATC148). Figure 30A is a scheme of the experimental design of the PATC148 mouse model. 10-week-old female NSG mice were intravenously injected with 0.3x106 firefly luciferase (FFluc)-transduced PATC148 cells on day -7, BLI was performed on day -2 to record engraftment rates, mice were irradiated with 300cGy on day -1, and on day 0, 107 fresh NK cells alone or 107 fresh NK cells preloaded with imgatuzumab (final concentration 100μg / ml) were washed twice with saline (5 min each wash) and suspended in saline and injected intravenously into each mouse. Test mice were then injected weekly with 5mg / kg imgatuzumab for 3 weeks. Figure 30B is a bioluminescence imaging showing tumor growth over time in mice implanted with PATC148 transduced with firefly luciferase (Ffluc) and left untreated, or treated with NK cells alone, or with NK cells preloaded with imgatuzumab. Figure 30C is a graph showing the mean brightness over time for the three groups of mice described in Figure 30C. Figure 30D is a graph showing survival curves for the three groups of mice described in Figure 30B. Statistical analysis consisted of a two-way ANOVA. [Figure 30-2] Same as above. [Figure 30-3] Same as above. [Figure 30-4] Same as above.

[0075] [Diagram 31]Figure 31. Imgatuzumab-loaded NT NK cells (shown in Figure 20) and TROP2 CAR-NK cells retained antibody on the cell surface for at least 12 days. NK cells were derived from umbilical cord blood and expanded on irradiated (100 Gy) UAPC feeder cells (2:1 feeder cell:NK ratio) and recombinant human IL-2 (200 U / ml) in complete NK cell growth medium (Click / RPMI) and were left unmodified and untransduced (NT) or transduced with TROP2 CAR and IL-15 (TROP2) on day 6. NT or TROP2 CAR NK cells were loaded with imgatuzumab (final concentration 100 μg / ml) in Click / RPMI medium for 1 hour at 37°C and washed twice with PBS (5 min each wash) before verifying imgatuzumab binding by flow cytometry. NK cells were stained with Alexa-Fluor647 affinity purified F(ab')2 fragment goat anti-human IgG(H+L) antibody at different time points (1 hr, 3 days, 5 days, 7 days, 12 days) and analyzed by flow cytometry. The results showed that Imgatuzumab was retained on the surface of NT NK cells (Figure 20) and TROP2 CAR NK cells (Figure 31) for at least 12 days.

[0076] [Figure 32-1] Figures 32A-32B. TROP2 CAR-NK cells loaded with imgatuzumab showed enhanced killing of ovarian cancer (e.g., SKOV3) and gastrointestinal stromal tumor (GIST) (e.g., OVCAR5) cells compared to unloaded NK cells in the Xcelligence long-term cytotoxicity assay. Non-transduced (NT) or TROP2 CAR and IL-15 transduced (TROP2 / IL15) NK cells were loaded with imgatuzumab (final concentration 100 μg / ml) in Click medium for 1 h at 37°C, washed twice with PBS (5 min each wash), and then co-cultured with tumor cells at a 1:1 effector-to-target ratio. Compared to unloaded NK cells, imgatuzumab-loaded NK cells showed increased cytotoxic activity against target cells. Statistical analysis consisted of two-way ANOVA. [Figure 32-2] Same as above.

[0077] [Diagram 33] Figure 33. Imgatuzumab-loaded NK cells maintained the antibody on their surface after freezing and thawing. NK cells were derived from umbilical cord blood and expanded in complete NK cell growth medium (Click / RPMI) with irradiated (100 Gy) UAPC feeder cells (2:1 feeder:NK ratio) and recombinant human IL-2 (200 U / ml) and were left unmodified and untransduced (NT) or transduced with TROP2 CAR and IL-15 (TROP2CAR / IL-15) on day 6. NT NK cells or TROP2CAR / IL-15 cells were loaded with imgatuzumab (final concentration 100 μg / ml) (+Imga) in Click / RPMI medium for 1 hour at 37°C, washed twice with PBS (5 minutes each wash), and then inactivated with dasatinib before cryopreservation. NK cells were then thawed, stained with Alexa-Fluor647 affinity-purified F(ab')2 fragment goat anti-human IgG(H+L) antibody, and analyzed by flow cytometry.

[0078] [Figure 34-1] Figures 34A-34D. Imgatuzumab-loaded NT and TROP2 CAR-NK cells showed enhanced cytotoxicity against multiple cancer cell lines after freeze-thawing compared to unloaded NK cells. Non-transduced (NT) or chimeric antigen receptor TROP2 CAR and IL-15-transduced (TROP2 / IL15) NK cells were loaded with imgatuzumab (final concentration 100 μg / ml) and cultured in Click / RPMI medium at 37°C for 1 h, then washed twice with PBS (5 min each wash) and inactivated with dasatinib before cryopreservation. Cells were thawed and used in Xcelligence cytotoxicity assays against multiple cancer cell lines (e.g., PDAC tumor cell line PATC148 (Figure 34A), CRC cell line WiDR (Figure 34B), GIST cell line OVCAR5 (Figure 34C), and ovarian cancer cell line SKOV3 (Figure 34D)). Statistical analysis consisted of two-way analysis of variance. [Figure 34-2] Same as above. [Figure 34-3] Same as above. [Figure 34-4] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0079] Detailed Description Following long-standing patent law practice, the words "a" and "an" herein, when used in conjunction with the word comprising, including in the claims, refer to "one or more." Some embodiments of the present disclosure may consist of, or consist essentially of, one or more elements, method steps, and / or methods of the present disclosure. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein, and that different embodiments may be combined.

[0080] Throughout this specification, unless the context dictates otherwise, the words "comprise", "comprises" and "comprising" are understood to mean the inclusion of the recited step or element or steps or elements, but not the exclusion of other steps or elements or steps or elements. "Consisting of" means including and limited to what follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the recited elements are required or essential, and other elements may not be present. "Consisting essentially of" means including the elements recited after the phrase, and is limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the recited elements. Thus, the phrase "consisting essentially of" indicates that the recited elements are required or essential, but other elements are optional and may or may not be present depending on whether they affect the activity or action of the recited elements.

[0081] Throughout this specification, reference to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "an embodiment," "an additional embodiment," or "a further embodiment," or combinations thereof, means that the particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Thus, the appearances of such phrases in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0082] As used herein, the terms "or" and "and / or" are used to describe multiple components in combination or mutually exclusive. For example, "x, y, and / or z" can refer to "x" alone, "y" alone, "z" alone, "x, y, and z," "(x and y) or z," "x or (y and z)," or "x or y or z." It is specifically contemplated that x, y, or z may be specifically excluded from an embodiment.

[0083] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the invention, and vice versa. Further, a composition of the invention can be used to achieve a method of the invention.

[0084] Throughout this application, the term "about" is used according to its plain and ordinary meaning within the art of cell and molecular biology to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0085] The term "engineered" as used herein refers to an entity produced by the hand of man, including cells, nucleic acids, polypeptides, vectors, and the like. In at least some cases, an engineered entity is synthetic and comprises elements that do not occur in nature or are not constructed by the methods utilized in this disclosure. In specific embodiments, vectors are engineered by recombinant nucleic acid techniques and cells are engineered by transfection or transduction of the engineered vector. In specific embodiments, engineered cells are cells that contain modifications of one or more exogenous antigen receptors (e.g., chimeric antigen receptors, T cell receptors, etc.), suicide genes, cytokines and / or cytokine receptors (e.g., IL-15, IL-15R, etc.), chemokine / homing receptors, and / or one or more endogenous genes.

[0086] As used herein, the terms "load," "loaded," or "loading" refer to adoptive cell therapy cells having one or more antibodies bound to the cells at their surface.

[0087] The phrase "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other adverse reactions when administered to animals, such as humans, as appropriate. Preparation of pharmaceutical compositions containing antibodies or additional active ingredients will be known to those skilled in the art in light of the present disclosure. Furthermore, it will be understood that when administered to animals (e.g., humans), preparations should meet the standards of sterility, pyrogenicity, general safety, and purity required by the FDA Office of Biological Standards.

[0088] As used herein, "pharmaceutical acceptable carriers" include any and all aqueous solvents (e.g., water, alcoholic / aqueous solutions, saline, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters such as ethyloleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, and inert gases), isotonicity agents, absorption retardants, salts, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, fluid and nutrient replenishers, such materials and combinations thereof will be known to those skilled in the art. The pH and exact concentration of the various components in the pharmaceutical composition are adjusted according to well-known parameters.

[0089] As used herein, the term "preactivation" or "preactivated" refers to exposure of NK cells to IL-12, IL-15 or IL-2, and / or IL-18, resulting in an increase in signaling pathways associated with NK cell effector function, such as IFN-γ response, TNF signaling, IL-2 / STAT5 signaling, IL-6 / JAK / STAT3 signaling, mTOR pathway, and / or enrichment of genes associated with inflammatory immune responses. In certain cases, expression of TRAIL, NKp44, and / or CD69 is increased.

[0090] As used herein, the terms "pre-load," "pre-loaded," or "pre-loading" refer to adoptive cell therapy cells that have one or more antibodies bound to their cell surface prior to using the cells for any reason.

[0091] As used herein, "prevent" and similar terms such as "prevented," "preventing," and the like refer to an approach to prevent, inhibit, or reduce the likelihood of occurrence or recurrence of a disease or condition, such as cancer. It also refers to delaying the onset or recurrence of a disease or condition, or delaying the onset or recurrence of symptoms of a disease or condition. As used herein, "prevention" and similar terms also include reducing the intensity, impact, symptoms, and / or burden of a disease or condition prior to the onset or recurrence of the disease or condition.

[0092] The term "subject" as used herein generally refers to an individual who has or is suspected of having cancer. A subject may be any organism or animal subject that is the subject of a method or material, including mammals, e.g., humans, laboratory animals (e.g., primates, rats, mice, rabbits), livestock (e.g., cows, sheep, goats, pigs, turkeys, and chickens), household pets (e.g., dogs, cats, and rodents), horses, and transgenic non-human animals. A subject may be, for example, a patient who has or is suspected of having a disease (sometimes called a pathology), such as a benign or malignant neoplasm, or cancer. A subject may be undergoing or having been treated. A subject may be asymptomatic. A subject may be a healthy individual, but may wish to prevent cancer. The terms "individual" are used interchangeably, at least in some cases. A "subject" or "individual" as used herein may or may not be housed in a medical facility, or may be treated as an outpatient in a medical facility. An individual may receive "one or more medical compositions" via the Internet. Individuals may include humans or non-human animals of any age, and thus include both adults and juveniles (i.e., children) and infants, including individuals in utero. The term does not imply the need for medical treatment, and thus individuals may participate in experiments, whether clinical or in support of basic science research, voluntarily or involuntarily.

[0093] As used herein, "treatment" or "treatment" includes any beneficial or desired effect on the symptoms or pathology of a disease or pathological condition, and may include even a minimal reduction in one or more measurable markers of the disease or condition being treated, such as cancer. Treatment may optionally include either the alleviation or amelioration of one or more symptoms of a disease or condition, or the delay in the progression of a disease or condition. "Treatment" does not necessarily indicate a complete eradication or cure of a disease or condition, or symptoms associated therewith.

[0094] I. Natural Killer (NK) Cells and Methods of Production NK cells have emerged as a promising source of cellular immunotherapy for patients with hematological malignancies and solid cancers. However, most studies with adoptively transferred NK cells have been limited by poor persistence, poor in vivo expansion, and disappointing antitumor activity of the infused cells. Thus, a barrier to overcome in the field of NK immunotherapy is the need for a biologically driven approach to enhance the antitumor function of NK cells, such as by manipulating the cells before administering them as a therapy. Thus, in certain embodiments, the present disclosure provides methods for the production of NK cells with any type of enhanced potency compared to NK cells that have not been so engineered. Some embodiments of the present disclosure relate to the isolation, activation, and expansion of NK cells, including for cancer immunotherapy.

[0095] In certain embodiments, the present disclosure encompasses loaded, (optionally) pre-activated, and (optionally) expanded NK cells produced by the present methods that exhibit enhanced anti-tumor functionality against cancer. In certain embodiments, the pre-activated and expanded NK cells also exhibit enhanced antibody-dependent cellular cytotoxicity (ADCC).

[0096] In certain embodiments, the present disclosure encompasses engineered, loaded, (optionally) preactivated, (optionally) expanded NK cells produced by the present methods that exhibit enhanced anti-tumor functionality against cancer. In specific embodiments, the loaded and / or engineered NK cells exhibit enhanced antibody-dependent cellular cytotoxicity (ADCC) compared to unloaded and / or unengineered NK cells. In certain embodiments, the loaded and / or engineered NK cells exhibit enhanced long-term cytotoxicity compared to unloaded and / or unengineered NK cells. In certain embodiments, the loaded and / or engineered NK cells exhibit enhanced long-term cytotoxicity against solid tumors and / or tumor spheroids compared to unloaded and / or unengineered NK cells. In certain embodiments, the loaded and / or engineered NK cells exhibit enhanced synergistic long-term cytotoxicity compared to unloaded and / or unengineered NK cells. In certain embodiments, the loaded and / or engineered NK cells exhibit enhanced synergistic long-term cytotoxicity against solid tumors and / or tumor spheroids compared to unloaded and / or unengineered NK cells. In certain embodiments, the imgatuzumab-loaded and engineered NK cells exhibit enhanced synergistic long-term cytotoxicity against solid tumors and / or tumor spheroids compared to unloaded and / or unengineered NK cells.

[0097] A.NK cells, general In certain embodiments, the NK cells (e.g., engineered NK cells) utilized in the present methods may be derived from any suitable source, such as umbilical cord blood (CB) containing human CB. Notably, the NK cells are not derived from umbilical cord tissue (the insulating material surrounding the blood vessels of the umbilical cord (i.e., Wharton's jelly)). In alternative embodiments, the NK cells are derived from human peripheral blood mononuclear cells (PBMCs), unstimulated leukocyte products (PBSCs), human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs), and / or bone marrow, or from patient-derived NK cell lines such as NK-92, by methods well known in the art. In certain embodiments, the NK cells are isolated from pooled CB. The CB may be pooled from 2, 3, 4, 5, 6, 7, 8, 9, 10, or more units. The NK cells may be autologous or allogeneic with respect to the recipient individual. The isolated NK cells may or may not be haplotype matched to the subject to whom the cell therapy is administered. The NK cells may or may not be detected by specific surface markers, such as CD16 and / or CD56 in humans. In some cases, the NK cells may or may not be depleted for the presence of one or more surface markers, such as depleted for CD3+, CD14+ and / or CD19+ cells. In certain embodiments, the NK cells are CD3- CD56+.

[0098] In certain embodiments, NK cells are isolated by the previously described ex vivo expansion method of NK cells (Spanholtz et al., 2011; Shah et al., 2013). In this method, CB mononuclear cells are isolated by Ficoll density gradient centrifugation. This cell culture may be depleted of cells expressing CD3 and may be characterized by determining the percentage of CD56+ / CD3- cells or NK cells. In other methods, umbilical cord CB is used to isolate CD34+ cells and induce NK cells.

[0099] B. NK cell loading In certain embodiments, the NK cells are loaded with the antibody prior to use. The NK cells can be loaded in any particular manner, including, for example, immediately prior to culture (e.g., incubation) or injection, to generate a complex between the NK cells and the antibody. The culture (e.g., incubation) conditions are sufficient to allow an effective amount of the antibody to bind to the surface of the NK cells. When a monospecific antibody is used, the Fc region of the monospecific antibody binds to the NK cell, while the antigen-binding domain of the monospecific antibody is free to bind to the target antigen. In certain embodiments when a multispecific antibody is used, one or more antigen-binding domains of the antibody can bind to the surface of the NK cell, such as through an antigen on the surface of the NK cell (e.g., but not limited to, NKp30, NKp44, NKp46, CD16, CD32, CD64, KIR, etc.), and the other antigen-binding domain is free to bind to its target antigen. In certain embodiments when a multispecific antibody is used, one or more antigen-binding domains of the antibody can bind to one or more target antigens.

[0100] The culture conditions under which the NK cells are loaded may or may not be of a specific type having one or more specific parameters. In a specific embodiment, the loading of the NK cells occurs during culture at a specific temperature, such as 37° C., while in alternative embodiments, the temperature is 36° C. or 38° C., or lower or higher. The duration of the loading step may be any suitable time, such as ranging from 1 minute to 24 hours or more. For example, it may range from 1 minute to 24 hours, 1 minute to 18 hours, 1 minute to 12 hours, 1 minute to 6 hours, 1 minute to 1 hour, 30 minutes to 24 hours, 30 minutes to 18 hours, 30 minutes to 12 hours, 30 minutes to 6 hours, 30 minutes to 1 hour, 1 to 24 hours, 1 to 18 hours, 1 to 12 hours, 1 to 6 hours, 6 to 24 hours, 6 to 18 hours, 6 to 12 hours, 12 to 24 hours, 12 to 18 hours, or 18 to 24 hours. In some embodiments, the duration of the loading step can be about 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48 hours or more, or any range derivable therein. In certain embodiments, the cell culture medium is a basal medium or a complex medium. In some cases, the culture may or may not include one or more reagents utilized during the preactivation and / or expansion steps. In certain embodiments, the culture includes one or more cytokines, including, for example, one or more of IL-12, IL-15, IL-2, and IL-18. In some embodiments, the culture includes any type of APC.

[0101] In some embodiments, the loading of the NK cells is performed such that the antibody remains bound to the NK cell surface after washing of the NK cells. In some embodiments, the NK cells are loaded with the antibody under conditions suitable for retention of at least a detectable fraction of the antibody on the NK cell surface for at least or exactly 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 84 hours, 96 hours, 108 hours, 120 hours, 132 hours, 144 hours, 156 hours, 168 hours, 180 hours, 192 hours, 204 hours, 216 hours, 228 hours, 240 hours, 252 hours, 264 hours, 276 hours, 288 hours, 300 hours, 312 hours, or more than 312 hours, or any range derivable therein. In some embodiments, the NK cells are loaded with the antibody under conditions suitable for retaining the antibody on the NK cell surface for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days, or for a period longer than 15 days, or any range derivable therein. In some embodiments, the NK cells are loaded with the antibody under conditions suitable for retaining the antibody on the NK cell surface prior to cryopreservation and / or thawing. In some embodiments, confirmation of antibody bearing on the NK cell surface can be measured using flow cytometry.

[0102] In some embodiments, the NK cells are at least, exactly or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 μg / ml final concentration or a final concentration of greater than 100 μg / ml, or any range derivable therein. In some embodiments, the NK cells are at least, exactly or about 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, Loaded with antibody to a final concentration of 560, 580, 600, 620, 640, 660, 680, 700, 720, 740, 760, 780, 800, 820, 840, 860, 880, 900, 920, 940, 960, 980, or 1000 μg / ml, or to a final concentration of greater than 1000 μg / ml, or any range derivable therein.

[0103] In some embodiments, the NK cells and antibodies are at least, exactly or about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 109%, 109%, 109%, 108%, 109%, 109%, 100%, 101%, 102%, 103%, 104%, 1 3%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, are incubated such that NK cells are complexed with the antibody.

[0104] In some embodiments, the NK cells and antibodies are at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 109%, 109%, 108%. ,37%,38%,39%,40%,41%,42%,43%,44%,45%,46%,47%,48%,49%,50%,51%,52%,53%,54%,55%,56%,57%,58%,59%,60%,61%,62%,63%,64%,65%,66%,67%,68%,69%,70%,71%,72%,73%,74%,75%,76%,77%,78% , 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, binding of the antibody to NK cells for at least 1 hour, 2 hours, 3 hours, 4 hours, or 5 hours after incubation of the NK cells with the antibody. Incubate for a period of time such as 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, or any range of time derivable therein.

[0105] In certain embodiments, at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, or greater than 20%, or any range derivable therein, of the antibody is bound to the NK cell surface relative to the amount of antibody complexed to the engineered NK cells measured about 1 hour after incubation and washing, at least 3 days after incubation and washing. In certain embodiments, at least about 9% of the antibody is bound to the NK cell surface relative to the amount of antibody complexed to the engineered NK cells measured about 1 hour after incubation and washing, at least 3 days after incubation and washing.

[0106] In certain embodiments, at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, or greater than 20%, or any range derivable therein, of the antibody is bound to the NK cell surface relative to the amount of antibody complexed to the engineered NK cells measured about 1 hour after incubation and washing, at least 5 days after incubation and washing. In certain embodiments, at least about 6% of the antibody is bound to the NK cell surface relative to the amount of antibody complexed to the engineered NK cells measured about 1 hour after incubation and washing, at least 5 days after incubation and washing.

[0107] In certain embodiments, at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, or greater than 20%, or any range derivable therein, of the antibody is bound to the NK cell surface relative to the amount of antibody complexed to the engineered NK cells measured about 1 hour after incubation and washing, at least 7 days after incubation and washing. In certain embodiments, at least about 4% of the antibody is bound to the NK cell surface relative to the amount of antibody complexed to the engineered NK cells measured about 1 hour after incubation and washing, at least 7 days after incubation and washing.

[0108] In certain embodiments, at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, or greater than 20%, or any range derivable therein, of the antibody is bound to the NK cell surface relative to the amount of antibody complexed to the engineered NK cells measured about 1 hour after incubation and washing, at least 12 days after incubation and washing. In certain embodiments, at least about 3% of the antibody is bound to the NK cell surface relative to the amount of antibody complexed to the engineered NK cells measured about 1 hour after incubation and washing, at least 12 days after incubation and washing.

[0109] In certain embodiments, the composition comprising the engineered NK cells and the antibody is cryopreserved. In certain embodiments, the composition comprising the engineered NK cells and the antibody is thawed from cryopreservation and at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the engineered NK cells are complexed with the antibody.

[0110] In certain embodiments, at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater than 90%, or any range derivable therein, of the antibody is bound to the NK cell surface after thawing from cryopreservation relative to the amount of antibody complexed to non-cryopreserved engineered NK cells measured about 1 hour after incubation and washing. In certain embodiments, at least about 30% of the antibody is bound to the NK cell surface after thawing from cryopreservation relative to the amount of antibody complexed to non-cryopreserved engineered NK cells measured about 1 hour after incubation and washing. In certain embodiments, at least about 40% of the antibody is bound to the NK cell surface after thawing from cryopreservation relative to the amount of antibody complexed to non-cryopreserved engineered NK cells measured after about 1 hour of incubation and washing. In certain embodiments, at least about 50% of the antibody is bound to the NK cell surface after thawing from cryopreservation relative to the amount of antibody complexed to non-cryopreserved engineered NK cells measured after about 1 hour of incubation and washing. In certain embodiments, at least about 60% of the antibody is bound to the NK cell surface after thawing from cryopreservation relative to the amount of antibody complexed to non-cryopreserved engineered NK cells measured after about 1 hour of incubation and washing.

[0111] In some embodiments, the incubation of the NK cells and the antibody is performed in any suitable NK cell medium known to one of skill in the art. In certain embodiments, the incubation of the NK cells and the antibody is performed in a medium comprising, consisting of, or consisting essentially of Click / RPMI medium. In certain embodiments, the incubation of the NK cells and the antibody is performed in a medium comprising, consisting of, or consisting essentially of Click medium. In certain embodiments, the incubation of the NK cells and the antibody is performed in a medium comprising, consisting of, or consisting essentially of RPMI medium. In certain embodiments, the incubation of the NK cells and the antibody is performed in a medium comprising, consisting of, or consisting essentially of SCGM medium.

[0112] The antibodies of the composition are provided in an effective amount to an NK cell of the present disclosure, thereby producing a "chimeric antigen receptor-like" complex. In particular, the antigen-binding domain of the antibody binds to the NK cell, such as through an antigen that is a cell surface protein. Multiple antibodies may be provided to multiple NK cells, such that there are multiple cell / antibody complexes. The antibody may be monospecific, bispecific, or multispecific, and in certain cases, the antibody engages both the NK cell and the target antigen through the antigen-binding domain of the antibody (such as engagers in the art, which are fusion proteins consisting of two single-chain variable fragments (scFv) of different antibodies). In an example where the antibody is monospecific, the antigen-binding domain of the antibody binds to a target antigen, such as a cancer antigen, and another portion of the antibody (such as the Fc region of the antibody) binds to the NK cell. When the antibody is multispecific, one or more antigen-binding domains of the antibody bind to the NK cell (such as through an NK cell surface antigen) and one or more antigen-binding domains of the antibody bind to one or more target antigens. Multispecific antibodies may be, for example, bispecific, trispecific, or tetraspecific. If the antibody is trispecific or tetraspecific, the additional antigen-binding domain may bind to other cells, such as stem cells.

[0113] In certain embodiments, the antibody can bind to any NK cell surface antigen (which may or may not be a receptor) on NK cells, such as CD16 (including CD16a or CD16b), CD32, CD56, CD64, c-type lectins such as NKG2D, NKG2C, costimulatory molecules such as CS1, DNAM, 2B4, CD2, NCR, NKp30, NKp44, NKp46, or KIR, and can target NK cells and increase their reactivity and specificity against different tumors.

[0114] In some embodiments, the antibody may bind to any suitable antigen (e.g., an antigen described herein, such as those described as targets of the CAR and / or TCR). In certain embodiments, the antibody targets CD123. In certain embodiments, the antibody targets EGFR. In certain embodiments, the antibody targets EGFR2. In certain embodiments, the antibody is bispecific and targets EGFR and c-MET. In certain embodiments, the antibody is imgatuzumab, amivantamab, and / or margetuximab.

[0115] The formation of the complex can be performed by any suitable means that provides sufficient conditions for the appropriate region of the antibody to bind to the appropriate surface region of the NK cell. In some instances, a specific culture medium can be utilized. In certain cases, Plasma-Lyte A and / or human serum albumin are utilized, but not in other cases. Once the complex is formed in culture, it may or may not be washed before administration to a subject, such as by infusion. In another embodiment, the NK cells and the antibody are administered separately, and the complex is formed in vivo. In certain embodiments, the composition comprising the NK cells and the antibody is washed with PBS to remove unbound antibody. In certain embodiments, the composition comprising the NK cells and the antibody is washed at least 1, 2, 3, 4, or 5 times, or more than 5 times, to remove unbound antibody. In certain embodiments, the composition comprising the NK cells and the antibody is washed for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes, or more than 10 minutes, to remove unbound antibody. In certain embodiments, the composition comprising NK cells and an antibody is washed twice to remove unbound antibody. In certain embodiments, the washing is for exactly or about 5 minutes. In certain embodiments, the washing includes agitating the composition, for example, using a cell shaker.

[0116] C. Preactivation In certain embodiments, the NK cells are preactivated prior to administration to the recipient individual. The preactivation step may or may not occur prior to any expansion step. In specific embodiments, the NK cells are preactivated with one or more cytokines, and in specific embodiments, the NK cells are preactivated with one or more of IL-12, IL-15, IL-2, and IL-18, including two, three, or more. If all three of IL-12, IL-15, IL-2, and IL-18 are not utilized, it can be IL-12 and IL-15 but not IL-18; or IL-12 and IL-18 but not IL-15; or IL-15 and IL-18 but not IL-12. IL-2 may or may not be used in place of IL-15.

[0117] In certain embodiments, the preactivation cytokines may be IL-12, IL-15, and IL-18. One or more additional cytokines may be used in the preactivation step. Preactivation may be for a short period of time, such as 5-72 hours, e.g. 10-50 hours, in particular 10-20 hours, e.g. 12, 13, 14, 15, 16, 17, 18, 19, or 20 hours, in particular about 16 hours in some cases. The preactivation culture may contain IL-18 and / or IL-15 at a concentration of 10-100 ng / mL, e.g. 40-60 ng / mL, in particular 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 ng / mL, in particular about 50 ng / mL. In some cases, the pre-activation culture comprises IL-12 at a concentration of 0.1-150 ng / mL, including a concentration of 1-20 ng / mL, such as a concentration of 10 ng / mL. In alternative embodiments, the NK cells may be stimulated with IL-2, or other cytokines that bind to the common gamma chain (e.g., IL-7, IL-21, and others), which may be in addition to, or as a replacement for, one or more of IL-12, IL-15, and IL-18. In such cases, the pre-activation culture may comprise IL-12 at a concentration of 0.1-150 ng / mL, such as 0.5-50 ng / mL, particularly 1-20 ng / mL, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 ng / mL, particularly about 10 ng / mL.

[0118] D. Proliferation In certain embodiments, the NK cells are expanded to increase their quantity before administration to an individual in need thereof. The expanded cells may or may not be derived from preactivated NK cells, such that a preactivation step may occur prior to the expansion step. The NK cell expansion step may be any suitable one in which the NK cell population is expanded, but in certain aspects, the expansion step utilizes a specific one or more reagents, such as in culture, to enhance their expansion. In certain aspects, the NK cells may not be expanded. IL-2 or IL-15 or IL-18 or any combination of these cytokines may be added to the expansion culture before or during expansion. In specific embodiments, the NK cells may be expanded ex vivo in flasks or in one of several different bioreactor configurations with continuous perfusion of media / additives.

[0119] In a specific aspect, the NK cells (whether pre-activated or not) may be washed (e.g., with PBS or Plasma Lyte or human serum albumin or culture medium or a combination thereof) before and / or after expansion, such as 2, 3, 4, or 5 times, particularly 3 times. In a specific embodiment, the NK cells are expanded in the presence of artificial antigen presenting cells (aAPCs). In a specific embodiment, the NK cells are expanded in the presence of a fragment of an aAPC. The aAPCs can be engineered to express CD137 ligand and / or membrane-bound cytokines. The membrane-bound cytokines may be membrane-bound IL-21 (mIL-21) or membrane-bound IL-15 (mIL-15). In a specific embodiment, the aAPCs are engineered to express CD137 ligand and mIL-21. The aAPCs may be derived from cancer cells, such as leukemia cells. The aAPCs may not express endogenous HLA class I, II, or CD1d molecules. They may express ICAM-1 (CD54) and LFA-3 (CD58) or CD48. In particular, the aAPCs may be K562 cells, such as K562 cells engineered to express CD137 ligand and mIL-21. Engineering may use methods known in the art, such as retroviral transduction, but any viral or non-viral vector may be utilized. The aAPCs may or may not be irradiated. Expansion may be for a specific period of time, such as about 2-30 days, such as 3-20 days, in particular 12-16 days, such as 12, 13, 14, 15, 16, 17, 18 or 19 days, in particular about 14 days. The preactivated NK cells and the aAPCs may be present in a ratio of about 3:1-1:3, such as 2:1, 1:1, 1:2, in particular about 1:2. The expansion culture may further include one or more cytokines to promote proliferation, such as IL-2. IL-2 may be present at a concentration of about 10-500 U / mL, for example 100-300 U / mL, particularly about 200 U / mL. IL-2 can be replenished in the expansion cultures at regular intervals, such as every 2-3 days. aAPCs can be added to the cultures at least a second time, for example on about day 7.The cytokines used in the preactivation and / or expansion steps may be recombinant human cytokines.

[0120] After expansion, the NK cells may be used immediately in any manner, such as by conjugation to one or more antibodies, or may be preserved, such as by cryopreservation, In certain embodiments, the cells may be expanded ex vivo as a bulk population within about 1, 2, 3, 4, 5 days, or for days, weeks, or months.

[0121] Activated and / or expanded NK cells secrete type I cytokines, such as interferon-γ, tumor necrosis factor-α, and granulocyte-macrophage colony-stimulating factor (GM-CSF), which activate both innate and adaptive immune cells, as well as other cytokines and chemokines. Measuring these cytokines can provide information about the activation state of NK cells. Additionally, other methods known in the art for determining NK cell activation can be used to characterize the NK cells of the present disclosure.

[0122] Thus, with regard to certain preactivation and expansion aspects of the present disclosure, in certain embodiments, NK cells preactivated with any combination of IL-12, IL15, and / or IL-18 and then expanded with aAPCs, such as K562 cells, expressing mIL-21 and CD137 ligand, provide a highly potent cell product. Thus, methods of using the NK cells of the present invention for the treatment of various diseases, such as immunotherapy of cancer patients, are provided. In an exemplary method, isolated NK cells can be subjected to a short period of preactivation, such as about 16 hours, with a combination of cytokines, such as interleukin-12 (IL-12), IL-15, and / or IL-18, and then subjected to expansion with artificial antigen presenting cells (aAPCs), such as K562 feeder cells, expressing membrane-bound IL-21 and CD137 ligand, and / or exogenous IL-2. IL-2, IL-15, IL-18, or a combination of these cytokines can be added to the expansion culture at least a second time.

[0123] E. Manipulation of NK cells As described herein, in some embodiments, the NK cells are modified (e.g., engineered) in one or more ways compared to natural NK cells. The engineered NK cells may be engineered to express one or more transgenes. The engineered NK cells may be engineered to express one or more heterologous proteins. Alternatively, or in addition, the engineered NK cells may be modified to reduce or inhibit expression of one or more endogenous genes. Alternatively, or in addition, the engineered NK cells may be modified to express one or more chemokines, chemokine receptors, cytokines, cytokine receptors, and / or suicide genes. In certain embodiments, the NK cells are engineered to express CD16, CD32, CD64, or other FCR binding regions. In some embodiments, the engineered NK cells do not include a non-naturally occurring CD16 gene. In some embodiments, the engineered NK cells do not include a transgene encoding a CD16 gene. In certain embodiments, the NK cells are engineered to express the CD16 gene. In certain embodiments, the NK cells are engineered to express a wild-type CD16 gene. In certain embodiments, the NK cells are engineered to express a CD32 gene. In certain embodiments, the NK cells are engineered to express a CD64 gene. In certain embodiments, the NK cells are engineered to express a transgenic receptor that is the target of a multispecific antibody.

[0124] 1. Engineered antigen receptors The NK cells may be genetically modified to express one or more engineered antigen receptors, including at least one or more chimeric antigen receptors (CARs) and / or one or more TCRs. In a specific embodiment, the engineered antigen receptors are directed to target one or more cancer antigens, which may or may not be the same as the antigens to which the multispecific antibodies (e.g., engagers) are directed.

[0125] The NK cells may be modified to encode at least one CAR, which may be, for example, first, second, third or later generation. The CAR may or may not be bispecific for two or more different antigens. The CAR may include one or more costimulatory domains. The NK cells may also be modified to express receptors for enhancing binding to antibodies, such as CD16, CD32 and / or CD64 receptors. Each costimulatory domain may include, for example, one or more costimulatory domains of any of the following members of the TNFR superfamily: CD28, CD137 (4-1BB), CD134 (OX40), DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1 (CD11a / CD18), Lck, TNFR-I, TNFR-II, Fas, CD30, CD27, NKG2D, 2B4M, CD40, or combinations thereof. In certain embodiments, the CAR comprises CD3 zeta. In certain embodiments, the CAR lacks one or more specific costimulatory domains; for example, the CAR can lack 4-1BB and / or lack CD28.

[0126] In certain embodiments, the intracellular CAR polypeptide comprises an extracellular spacer domain that links the antigen-binding domain and the transmembrane domain, which may be referred to as a hinge. The extracellular spacer domain includes, but is not limited to, an Fc fragment of an antibody or a fragment or derivative thereof, a hinge region of an antibody or a fragment or derivative thereof, an antibody CH2 region, an antibody CH3 region, an artificial spacer sequence, or a combination thereof. Examples of extracellular spacer domains include, but are not limited to, artificial spacers made from polypeptides such as CD8α hinge, CD28, Gly3, or the CH1, CH3 domains of IgG (such as human IgG1 or IgG4). In certain embodiments, the extracellular spacer domain may comprise (i) an IgG4 hinge, CH2 and CH3 region, (ii) an IgG4 hinge region, (iii) an IgG4 hinge and CH2, (iv) a CD8α or CD4 hinge region, (v) an IgG1 hinge, CH2 and CH3 region, (vi) an IgG1 hinge region or (vii) an IgG1 hinge and CH2, (viii) a CD28 hinge region, or a combination thereof. In certain embodiments, the hinge is derived from IgG1, and in certain embodiments, the CAR polypeptide comprises a particular IgG1 hinge amino acid sequence or is encoded by a particular IgG1 hinge nucleic acid sequence.

[0127] The transmembrane domain of the CAR may be of natural or synthetic origin. If of natural origin, in some embodiments, a domain derived from a membrane-bound or transmembrane protein is used. Transmembrane regions include those derived from (i.e., at least the transmembrane region of) the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD30, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS / CD278, GITR / CD357, NKG2D, and DAP molecules (such as DAP10 or DAP12). Alternatively, the transmembrane domain in some embodiments is synthetic. In some embodiments, the synthetic transmembrane domain mainly comprises hydrophobic residues such as leucine and valine. In some embodiments, a phenylalanine, tryptophan, valine triplet may be found on either side of the synthetic transmembrane domain.

[0128] In certain embodiments, the CAR can be co-expressed with one or more cytokines to improve persistence when the amount of tumor-associated antigen is low. For example, the CAR can be co-expressed with one or more cytokines, such as IL-7, IL-2, IL-15, IL-12, IL-23, IL-18, IL-21, IL-7, GMCSF, or combinations thereof. In some embodiments, the NK cells expressing the CAR are engineered to express one or more heterologous cytokines and / or to upregulate the normal expression of one or more heterologous cytokines. The cells may or may not be transduced or transfected with one or more cytokines on the same vector as other genes.

[0129] In certain embodiments, the CD27 CAR targets CD70. In some embodiments, the CAR comprises a transmembrane domain from CD28. In some embodiments, the CAR comprises a CD3 zeta intracellular stimulatory domain. In some embodiments, the CAR is co-expressed with IL-15.

[0130] In certain embodiments, the NK cells are engineered to express a polynucleotide comprising a sequence having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:1, 3, 4, 6, 8, 10, 11, 13, or 15, or any range derivable therein. In certain embodiments, the NK cells are engineered to express a polynucleotide comprising a sequence encoding a polypeptide having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, sequence identity to SEQ ID NO: 2, 5, 7, 9, 12, 14, or 16. SEQ ID NO:1 - Exemplary polynucleotide sequence comprising a CD27 CAR construct targeting the CD70 antigen. (SEQ ID NO:1) SEQ ID NO:2 - Exemplary amino acid sequence comprising a CD27 CAR construct targeting the CD70 antigen. MARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRFWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 2) SEQ ID NO:3 - Exemplary polynucleotide sequence comprising a CD27 CAR construct targeting CD70 antigen. SEQ ID NO:4 - Exemplary polynucleotide sequence comprising a CD27 CAR construct targeting CD70 antigen and IL-15. SEQ ID NO:5 - Exemplary amino acid sequence comprising a CD27 CAR construct targeting the CD70 antigen. MARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 5) SEQ ID NO:6 - Exemplary polynucleotide sequence comprising a TROP2 CAR construct. SEQ ID NO:7 - An exemplary amino acid sequence comprising a TROP2 CAR construct. MEFGLSWLFLVAILKGVQCSREDIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTK VEIKRLEIKGSTSGSGKPGSGEGSTQVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGF GSSYWYFDVWGQGSLVTVSSPYAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLPGPSKPKFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRG (SEQ ID NO: 7) SEQ ID NO:8 - Exemplary polynucleotide sequence comprising TROP2 CAR scFv. ATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTGGCTATTTTAAAAGGTGTCCAGTGCTCTAGAGAGGACATCCAGCTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCAGCATCACCTGCAAGGCCAGTCAGGATGTGAGTATTGCTGTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTACTCGGCATCCTACCGGTACACTGGAGTCCCTGATAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAGTTTATTACTGTCAGCAACATTATATTACTCCGCTCACGTTCGGTGCTGGGACCAAGGTGGAGATCAAACGTTTGGAAATAAAGGGCTCTACAAGCGGCTCAGGAAAACCTGGATCAGGCGAAGGGTCTACGCAGGTCCAACTGCAGCAATCTGGGTCTGAGTTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCCTGCAAGGCTTCTGGATACACCTTCACAAACTATGGAATGAACTGGGTGAAGCAGGCCCCTGGACAAGGGCTTAAATGGATGGGCTGGATAAACACCTACACTGGAGAGCCAACATATACTGATGACTTCAAGGGACGGTTTGCCTTCTCCTTGGACACCTCTGTCAGCACGGCATATCTCCAGATCAGCAGCCTAAAGGCTGACGACACTGCCGTGTATTTCTGTGCAAGAGGGGGGTTCGGTAGTAGCTACTGGTACTTCGATGTCTGGGGCCAAGGGTCCCTGGTCACCGTCTCCTCA(SEQ ID NO: 8) Exemplary amino acid sequence containing SEQ ID NO: 9 - TROP2 CAR scFv. MEFGLSWLFLVAILKGVQCSREDIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKRLEIKGSTSGSGKPGSGEGSTQVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSS (SEQ ID NO: 9) SEQ ID NO:10 - Exemplary vector sequence comprising iCas9, TROP2 CAR, and IL-15.

[0131] The sequence of the open reading frame encoding the chimeric receptor can be obtained from genomic DNA sources, cDNA sources, or synthetic (e.g., PCR), or a combination thereof. Depending on the size of the genomic DNA and the number of introns, it may be preferable to use cDNA or a combination thereof, since introns have been shown to stabilize mRNA. It may also be advantageous to use endogenous or exogenous non-coding regions to stabilize the mRNA.

[0132] It is contemplated that chimeric constructs can be introduced into any kind of immune cell as naked DNA or in a suitable vector.Methods for stably transfecting cells with naked DNA by electroporation are known in the art.See, for example, U.S. Patent No. 6,410,319.Naked DNA generally refers to the DNA encoding chimeric receptor contained in a plasmid expression vector in the proper orientation for expression.

[0133] Alternatively, a viral vector (e.g., retroviral vector, adenoviral vector, adeno-associated viral vector, or lentiviral vector) can be used to introduce the chimeric construct into immune cells. The vector suitable for use according to the method of the present disclosure is non-replicative in immune cells. Many vectors are known that are based on viruses, such as, for example, vectors based on HIV, SV40, EBV, HSV, or BPV, whose copy number of the virus maintained in the cell is low enough to maintain the viability of the cell. Non-viral vectors include plasmids, transposons, nanoparticles, liposomes, lipids, metals, or combinations thereof.

[0134] In some embodiments, the engineered antigen receptor comprises a recombinant TCR and / or a TCR cloned from a naturally occurring T cell. "T cell receptor" or "TCR" refers to a molecule that comprises a variable a chain and a variable β chain (also known as TCRα and TCRβ, respectively) or a variable γ chain and a variable δ chain (also known as TCRγ and TCRδ, respectively) and is capable of specifically binding to an antigenic peptide bound to an MHC receptor. In some embodiments, the TCR is of the αβ type.

[0135] TCRs, which are usually present in αβ and γδ types, are generally structurally similar, although the T cells expressing them may differ in anatomical location and function. TCRs may be present on the cell surface or in a soluble form. Generally, TCRs are present on the surface of T cells (or T lymphocytes) and are generally responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules. In some embodiments, TCRs may also include a constant domain, a transmembrane domain, and / or a short cytoplasmic tail (see, e.g., Janeway et al., 1997). For example, in some aspects, each chain of the TCR may have one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region, and a short C-terminal cytoplasmic tail. In some embodiments, the TCR is associated with an invariant protein of the CD3 complex, which is involved in mediating signal transduction. Unless otherwise specified, the term "TCR" should be understood to include functional TCR fragments thereof. The term also includes intact or full-length TCRs, including αβ or γδ TCRs. In some embodiments, the TCR and / or CD3 complex is as described in International Application PCT / US2022 / 074062, filed July 22, 2022, and published as WO2023004425A2 on January 26, 2023, which is hereby incorporated by reference in its entirety for purposes described herein.

[0136] Thus, as used herein, TCR refers to any TCR that binds to a specific antigen peptide bound to an MHC molecule, i.e., in an MHC-peptide complex, or a functional fragment, such as an antigen-binding portion of a TCR. An "antigen-binding portion" or "antigen-binding fragment" of a TCR may be used interchangeably and refers to a molecule that includes a portion of the structural domain of a TCR, but that binds to the antigen (e.g., an MHC-peptide complex) that the complete TCR binds. In some cases, the antigen-binding portion includes a variable domain of the TCR, such as the variable a chain and variable β chain of the TCR, sufficient to form a binding site for binding to a specific MHC-peptide complex, such that each chain typically includes three complementarity determining regions.

[0137] In some embodiments, the variable domains of the TCR chains associate to form loops, or complementarity determining regions (CDRs) similar to immunoglobulins, which form the binding site of the TCR molecule, thereby conferring antigen recognition and determining peptide specificity. Typically, like immunoglobulins, the CDRs are separated by framework regions (FRs) (see, for example, Jores et al., 1990; Chothia et al., 1988; Lefranc et al., 2003). In some embodiments, CDR3 is the main CDR that recognizes processed antigens, but CDR1 of the α chain has also been shown to interact with the N-terminal portion of antigenic peptides, and CDR1 of the β chain interacts with the C-terminal portion of peptides. CDR2 is believed to recognize MHC molecules. In some embodiments, the variable region of the β chain can include an additional hypervariable (HV4) region.

[0138] In some embodiments, the TCR chain comprises a constant domain. For example, similar to an immunoglobulin, the extracellular portion of a TCR chain (e.g., a chain, β chain) comprises two immunoglobulin domains, a variable domain (e.g., V aor Vp; typically, an N-terminus of amino acids 1-116 according to Kabat numbering in Kabat et al., "Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5th ed.), and one constant domain adjacent to the cell membrane (e.g., an a-chain constant domain or C a The constant domains of the TCR domains may comprise a β chain constant domain or Cp, typically amino acids 117 to 259 based on Kabat, and a β chain constant domain or Cp, typically amino acids 117 to 295 based on Kabat). For example, in some cases, the extracellular portion of the TCR formed by the two chains comprises two membrane proximal constant domains and two membrane distal variable domains that comprise the CDRs. The constant domains of the TCR domains comprise a short linking sequence in which cysteine ​​residues form disulfide bonds, making the connection between the two chains. In some embodiments, the TCR may have additional cysteine ​​residues in each of the α and β chains, such that the TCR comprises two disulfide bonds in the constant domains.

[0139] In some embodiments, the TCR chain can include a transmembrane domain. In some embodiments, the transmembrane domain is positively charged. In some cases, the TCR chain includes a cytoplasmic tail. In some cases, this structure allows the TCR to associate with other molecules, such as CD3. For example, a TCR that includes a constant domain with a transmembrane region can anchor the protein to the cell membrane and associate with the invariant subunit of the CD3 signaling apparatus or complex.

[0140] In general, CD3 is a multiprotein complex, which in mammals can have three different chains (γ, δ, ε) and a ζ chain. For example, in mammals, it can contain a homodimer of CD3γ chain, CD3δ chain, two CD3ε chains, and CD3ζ chain. CD3γ, CD3δ, and CD3ε chains are highly related cell surface proteins of the immunoglobulin superfamily that contain a single immunoglobulin domain. The transmembrane regions of CD3γ, CD3δ, and CD3ε chains are negatively charged, a feature that allows these chains to associate with the positively charged T cell receptor chains. The intracellular tails of CD3γ, CD3δ, and CD3ε chains each contain one conserved motif known as an immunoreceptor tyrosine-based activation motif or ITAM, while the CD3ζ chain has three. In general, ITAMs are involved in the signaling capacity of the TCR complex. These accessory molecules have negatively charged transmembrane regions and play a role in propagating signals from the TCR into the cell. The CD3- and ζ-chains together with the TCR form what is called the T cell receptor complex.

[0141] In some embodiments, the TCR may be a heterodimer of two chains, α and β (or optionally γ and δ), or may be a single chain TCR construct. In some embodiments, the TCR is a heterodimer comprising two separate chains (α and β, or γ and δ) linked by a disulfide bond or disulfide bond or the like. In some embodiments, a TCR against a target antigen (e.g., a cancer antigen) is identified and introduced into a cell. In some embodiments, a nucleic acid encoding the TCR can be obtained from a variety of sources, such as by polymerase chain reaction (PCR) amplification of publicly available TCR DNA sequences. In some embodiments, the TCR is obtained from a biological source, such as from cells, such as T cells (e.g., cytotoxic T cells), T cell hybridomas, or other publicly available sources. In some embodiments, the T cells can be obtained from cells isolated in vivo. In some embodiments, a high affinity T cell clone can be isolated from a patient and the TCR isolated. In some embodiments, the T cells may be cultured T cell hybridomas or clones. In some embodiments, TCR clones against a target antigen have been generated in transgenic mice engineered with human immune system genes (e.g., human leukocyte antigen system, or HLA). See, e.g., tumor antigens (see, e.g., Parkhurst et al., 2009 and Cohen et al., 2005). In some embodiments, phage display is used to isolate TCRs against a target antigen (see, e.g., Varela-Rohena et al., 2008 and Li, 2005). In some embodiments, the TCR or antigen-binding portion thereof may be synthetically generated from knowledge of the sequence of the TCR.

[0142] The CAR and TCR of the present disclosure target one or more specific antigens. Some of the antigens targeted by the engineered antigen receptors are expressed in the context of the disease, condition, or cell type targeted via adoptive cell therapy. Among the diseases and conditions are proliferative, neoplastic, and malignant diseases and disorders, including hematological cancers, lymphomas, leukemias, and / or cancers of the immune system, such as B, T, and myeloid leukemias, lymphomas, and myelomas, such as multiple myeloma. In some embodiments, the antigen is selectively expressed or overexpressed on disease or condition cells, such as tumor or pathogenic cells, compared to normal or non-target cells or tissues. In other embodiments, the antigen is expressed on normal cells and / or expressed on engineered cells.

[0143] Any suitable antigen can be targeted in the method. In some cases, the antigen may be associated with a particular cancer cell but not with a non-cancer cell. Exemplary antigens include, but are not limited to, antigenic molecules from infectious agents, self / autoantigens, tumor / cancer-associated antigens, and tumor neoantigens (Linnemann et al., 2015). In certain embodiments, antigens include CD19, EBNA, HER2, CA-125, TRAIL / DR4, CD20, CD70, CD38, trop2, HLA-G, CD123, CLL1, carcinoembryonic antigens, alpha fetoprotein, CD56, AKT, Her3, epithelial tumor antigen, CD319 (CS1), ROR1, folate binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, CD5, CD23, CD30, HERV-K, IL-11Rα, κ chain, λ chain, CSPG4, CD33, CD47, CLL-1, U5snRNP200, CD200, BAFF-R, BCMA, CD99, p53, mutant p53, Ras, mutant ras, c-Myc, cytoplasmic serine / threonine kinases (e.g., A-Raf, B-Raf, and C-Raf, cyclin-dependent kinases), MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-A13, MAGE-A14, MAGE-A15, MAGE-A16, MAGE-A17, MAGE-A18, MAGE-A19, MAGE-A21, MAGE-A22, MAGE-A23, MAGE-A24, MAGE-A25, MAGE-A26, MAGE-A27, MAGE-A28, MAGE-A29, MAGE-A30, MAGE-A31, MAGE-A32, MAGE-A33, MAGE-A34, MAGE-A35, MAGE-A36, MAGE-A37, MAGE-A38, MAGE-A3 ...9, MAGE-A39, MAGE-A30, MAGE-A31, MAGE-A32, MAGE-A33, MAGE-A34 AGE-A10, MAGE-A12, MART-1, melanoma-associated antigen, BAGE, DAM-6, -10, GAGE-1, -2, -8, GAGE-3, -4, -5, -6, -7B, NA88-A, MC1R, mda-7, gp75, Gp100, PSA, PSM, tyrosinase, tyrosinase-related protein, TRP-1, TRP-2, ART-4, CAMEL, CEA, Cyp-B, hTERT, hTRT, iCE, MUC1, MUC2, phosphoinositide 3kinase PI3K, TRK receptor, PRAME, P15, RU1, RU2, SART-1, SART-3, Wilms tumor antigen (WT1), AFP, -catenin / m, caspase-8 / m, CDK-4 / m, ELF2M, GnT-V, G250, HAGE, HSP70-2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, myosin / m, RAGE, SART-2, TRP-2 / INT2, 707-AP, annexin II, CDC27 / m,TPI / mbcr-abl, BCR-ABL, interferon regulatory factor 4 (IRF4), ETV6 / AML, LDLR / FUT, Pml / RAR, tumor-associated calcium signal transducer 1 (TACSTD1) TACSTD2, receptor tyrosine kinases (e.g., epidermal growth factor receptor (EGFR) (especially EGFRvIII), platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR)), VEGFR2, cytoplasmic tyrosine kinases (e.g., src family, syk-ZAP70 family), integrin-linked kinase (ILK), signal transducer and activator of transcription STAT3, STATS, and STATE, hypoxia-inducible factors (e.g., HIF-1 and HIF-2), nuclear factor-κB (NF-B), notch receptors (e.g., Notch1-4), NY ESO 1, c-Met, mammalian target of rapamycin (mTOR), WNT, extracellular signal-regulated kinase (ERK) and their regulatory subunits, PMSA, PR-3, MDM2, mesothelin, renal cell carcinoma-5T4, SM22α, carbonic anhydrase I (CAI) and IX (CAIX) (also known as G250), STEAD, TEL / AML1, GD2, proteinase 3, hTERT, sarcoma translocation breakpoints, EphA2, ML-IAP, EpCAM, ERG (TMPRSS2), ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, GD3, fucosyl GM1, mesothelial, PSCA, sLe, PLAC1, GM3, BORIS, Tn, GLoboH, NY-BR-1, RGsS, SAGE, SART3, STn, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE1, B7H3, legumain, TIE2, Page4, MAD-CT-1, FAP, MAD-CT-2, fos-related antigen 1, CBX2, CLDN6, SPANX, TPTE, ACTL8, ANKRD30A, CDKN2A, MAD2L1, CTAG1B, SUNC1, and LRRN1. Examples of antigen sequences can be found, for example, in GenBank, 登録商標In the database: CD19 (accession number NG_007275.1), EBNA (accession number NG_002392.2), WT1 (accession number NG_009272.1), CD123 (accession number NC_000023.11:1336785-1382689), NY-ESO (accession number: NC_000023.11), EGFRvIII (accession number: NG_007726.3), MUC1 (accession number: NG_029383.1), HER2 (accession number: NG_007503.1), CA-125 (accession number NG_055257.1), WT1 (accession number NG_009272.1), Mage-A3 (accession number NG_013244.1), Mage-A4 (Accession No. NG_013245.1), Mage-A10 (Accession No. NC_000023.11), TRAIL / DR4 (Accession No. NC_000003.12), and / or CEA (Accession No. NC_000019.10) are known in the art.

[0144] Tumor-associated antigens can be derived from, for example, prostate cancer, breast cancer, colon cancer, lung cancer, pancreatic cancer, kidney cancer, mesothelioma, ovarian cancer, liver cancer, brain cancer, bone cancer, stomach cancer, spleen cancer, testicular cancer, cervical cancer, anal cancer, gallbladder cancer, thyroid cancer, or melanoma cancer. Exemplary tumor-associated antigens or tumor cell-derived antigens include MAGE1, 3, and MAGE4 (or other MAGE antigens as disclosed in International Patent Publication No. WO99 / 40188); PRAME; BAGE; RAGE, Lage (also known as NY ESO 1); SAGE; and HAGE or GAGE. These non-limiting examples of tumor antigens are expressed in a wide range of tumor types, such as melanoma, lung cancer, sarcoma, and bladder cancer. See, for example, U.S. Patent No. 6,544,518. Prostate cancer tumor associated antigens include, for example, prostate specific membrane antigen (PSMA), prostate specific antigen (PSA), prostatic acid phosphate, NKX3.1, and six-stage membrane epithelial antigen of the prostate (STEAP).

[0145] Other tumor-associated antigens include Plu-1, HASH-1, HasH-2, Cripto, Criptin, etc. In addition, tumor antigens may be self-peptide hormones, such as gonadotrophin-releasing hormone (GnRH), a short peptide with a full length of 10 amino acids, which is useful in the treatment of many cancers.

[0146] Antigens can include genes mutated in tumor cells, such as telomerase enzyme, survivin, mesothelin, mutant ras, bcr / abl rearrangements, Her2 / neu, mutant or wild-type p53, cytochrome P450 1B1, aberrantly expressed intronic sequences such as N-acetylglucosaminyltransferase-V; clonal rearrangements of immunoglobulin genes that generate unique idiotypes in myelomas and B-cell lymphomas; tumor antigens containing epitopic regions or epitopic peptides derived from oncoviral processes, such as human papilloma virus proteins E6 and E7; Epstein-Barr virus protein LMP2; epitopic regions or epitopic peptides derived from genes transcribed at different levels in tumor cells compared to normal cells, such as unmutated oncophetel proteins with tumor-selective expression, such as carcinoembryonic antigens and alpha-fetoprotein.

[0147] 2. The suicide gene In certain embodiments, suicide genes are utilized with antibody combination therapy to control their use and allow for the termination of cell therapy at a desired event and / or time. Suicide genes are employed in transduced cells to induce the death of the transduced cells when necessary. The cells of the present disclosure modified to carry the vectors encompassed by the present disclosure may contain one or more suicide genes. In some embodiments, the term "suicide gene" as used herein is defined as a gene that, upon administration of a prodrug or other drug, results in the transfer of the gene product to a compound that kills the host cell. In other embodiments, the suicide gene encodes a gene product that is optionally targeted by a drug (such as an antibody) that targets the suicide gene product.

[0148] In some cases, cell therapy may be subject to the use of one or more suicide genes of any kind when the individual undergoing cell therapy and / or the individual receiving cell therapy is considered to be at risk of having one or more symptoms, including imminent symptoms, of one or more adverse events such as cytokine release syndrome, neurotoxicity, anaphylaxis / allergy, and / or on-target / off-tumor toxicity (as examples). The use of suicide genes may be part of a planned protocol for treatment or may be used only if the need for its use is recognized. In some cases, cell therapy may be terminated using an agent targeting the suicide gene or its gene product because treatment is no longer necessary.

[0149] The use of suicide genes can be initiated upon the onset of at least one adverse event for the individual, which can be recognized by any means, including during regular monitoring, which may or may not be continuous from the start of cell therapy. The adverse event can be detected by examination and / or testing. If the individual is experiencing cytokine release syndrome (sometimes referred to as cytokine storm), the individual will experience elevated inflammatory cytokine(s) (by way of example only: interferon-γ, granulocyte macrophage colony stimulating factor, IL-10, IL-6, and TNF-α), fever, fatigue, hypotension, hypoxia, tachycardia, nausea, capillary leakage, cardiac / renal / liver dysfunction, or a combination thereof. If the individual is experiencing neurotoxicity, the individual may experience confusion, delirium, agenesis, and / or seizures. In some cases, the individual is tested for markers associated with the onset and / or severity of cytokine release syndrome, such as C-reactive protein, IL-6, TNF-α, and / or ferritin.

[0150] Examples of suicide genes include engineered non-secreted (including membrane-bound) tumor necrosis factor (TNF)-α mutant polypeptides (see PCT / US19 / 62009, incorporated herein by reference in its entirety), which may be affected by delivery of antibodies that bind to TNF-α mutants. Examples of suicide gene / prodrug combinations that may be used are herpes simplex virus-thymidine kinase (HSV-tk) and ganciclovir, acyclovir, or FIAU; oxidoreductase and cycloheximide; cytosine deaminase and 5-fluorocytosine; thymidine kinase-thymidylate kinase (Tdk::Tmk) and AZT; and deoxycytidine kinase and cytosine arabinoside. E. coli purine nucleoside phosphorylase is a so-called suicide gene that converts the prodrug 6-methylpurine deoxyriboside to the toxic purine 6-methylpurine, and may be used. Other examples of suicide genes include CD20, CD52, inducible caspase 9, purine nucleoside phosphorylase (PNP), cytochrome p450 enzymes (CYP), carboxypeptidase (CP), carboxylesterase (CE), nitroreductase (NTR), guanine ribosyltransferase (XGRTP), glycosidase enzymes, methionine-α,γ-lyase (MET), and thymidine phosphorylase (TP).

[0151] In certain embodiments, the vector encoding the CAR or any vector in the NK cell encompassed herein comprises one or more suicide genes.The suicide gene may or may not be on the same vector as the CAR.When the suicide gene is on the same vector as the CAR, the suicide gene and the CAR may be separated, for example, by an IRES or 2A element.

[0152] 3. Cytokines In some embodiments, the NK cell expressing cells are engineered to express one or more heterologous cytokines and / or to upregulate the normal expression of one or more heterologous cytokines. The cells may or may not be transduced or transfected with the one or more cytokines on the same vector as other genes.

[0153] One or more cytokines may be co-expressed from vectors containing polypeptides separate from the engineered antigen receptor and / or suicide gene. For example, interleukin-15 (IL-15) is tissue-restricted and is only observed at any level in serum or systemically under pathological conditions. IL-15 has several properties that are desirable for adoptive therapy. IL-15 is a homeostatic cytokine that promotes the eradication of established tumors by inducing natural killer cell development and cell proliferation, relieving functional suppression of tumor-resident cells, and inhibiting activation-induced cell death (AICD). In addition to IL-15, other cytokines are also envisioned. These include, but are not limited to, cytokines (e.g., IL-2, IL-12, IL-18, and / or IL-21), chemokines, and other molecules that contribute to the activation and proliferation of cells used in human applications. In certain embodiments, NK cells expressing IL-15 allow for sustained supportive cytokine signaling, which is useful for survival after infusion. In certain embodiments, NK cells expressing IL-21 are capable of sustained symptomatic cytokine signaling, which is beneficial for survival after infusion. In certain embodiments, the cytokine is expressed as part of a multicistronic construct with one or more functional and / or marker proteins.

[0154] In some embodiments, a specific sequence of IL-15 is utilized, such as that shown below (the underline indicates the signal peptide sequence, which may be included or omitted): SEQ ID NO:11 - Exemplary polynucleotide sequence constituting IL-15 (signal peptide underlined). ATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCC GGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGTG ATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGC (SEQ ID NO: 11) SEQ ID NO:12 - An exemplary amino acid sequence that constitutes IL-15 (signal peptide underlined). MRSSPGNMERIVICLMVIFLGTLV HKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 12)

[0155] In some embodiments, specific sequences of IL-21 are utilized, such as those shown below (underline denotes signal peptide sequence, which may be included or omitted): SEQ ID NO:13 - An exemplary codon optimized polynucleotide sequence constituting IL-21 (signal peptide underlined). ATGAGGAGCAGTCCAGGCAATATGGAGCGGATAGTAATTTGTCTCATGGTAATATTCCTCGGTACTCTGGTACATAAATCTTCCTCTCAAGGTCAGGACCGCCATATGATTCGAATGCGGCAGCTGATTGACATAGTCGATCAACTGAAGAACTATGTGAATGATCTTGTGCCCGAGTTTTTGCCAGCCCCTGAAGACGTAGAAACTAATTGTGAGTGGAGTGCCTTTTCCTGCTTTCAAAAGGCACAGCTGAAATCCGCCAACACGGGCAATAACGAACGGA TAATTAACGTATCCATTAAGAAGCTGAAGCGGAAGCCGCCCTCAACCAATGCGGGACGGCGGCAAAAGCATCGCTTGACCTGTCCGTCATGCGACAGCTACGAGAAAAAAGCCCCCGAAGGAGTTCTTGGAACGCTTCAAGAGTCTCCTTCAGAAAATGATTCACCAGCACCTGTCCTCACGGACGCACGGAAGCGAGGACAGT (SEQ ID NO: 13) SEQ ID NO:14 - An exemplary codon-optimized amino acid sequence constituting IL-21 (signal peptide underlined). MRSSPGNMERIVICLMVIFLGTLV HKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFCQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 14) SEQ ID NO:15 - An exemplary polynucleotide sequence constituting IL-21. GGCCAGGACCGGCACATGATCCGGATGAGACAGCTGATCGACATCGTGGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTCCTGCCTGCCCCCGAGGACGTGGAAACAAACTGCGAGTGGAGCGCCTTCAGCTGCTTCCAGAAGGCCCAGCTGAAAAGCGCCAACACCGGCAACAACGAGCGGATCATCAACG TGTCCATCAAGAAGCTGAAGCGGAAGCCCCCCAGCACCAACGCCGGAAGAAGGCAGAAGCACAGACTGACCTGCCCCAGCTGCGACAGCTACGAGAAGAAGCCCCCTAAAGAGTTCCTGGAACGGTTCAAGAGCCTGCTGCAGAAGATGATCCACCAGCACCTGAGCAGCCGGACCCACGGCTCTGAGGACAGC (SEQ ID NO: 15) SEQ ID NO:16 - An example of the amino acid sequence that makes up IL-21. GQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFCQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 16)

[0156] In a specific embodiment, the cells express one or more exogenously supplied cytokines. In one example, the cytokines are IL-15, IL-12, IL-2, IL-18, IL-21, IL-23, GMCSF, or a combination thereof. The cytokines may be exogenously supplied to the NK cells as they are expressed from an expression vector in the cells. In another case, the endogenous cytokine in the cells is upregulated by manipulation of the expression control of the endogenous cytokine, such as genetic modification at the promoter site of the cytokine. When the cytokine is provided to the cells on an expression construct, the cytokine may be encoded from the same vector as the suicide gene and / or CAR. In some embodiments, the present disclosure relates to the co-use of IL-15 and a CAR and, optionally, a suicide gene.

[0157] In some embodiments, the cells express one or more exogenously provided engineered receptors, where the engineered receptors include a chemokine receptor and / or a cytokine receptor. In some embodiments, the cytokine receptor is an IL-15 receptor. In some embodiments, the cytokine receptor is a non-naturally occurring variant of a cytokine receptor. In some embodiments, the cytokine receptor is an IL-15, IL-12, IL-2, IL-18, IL-21, IL-23, or GMCSF receptor, or a combination thereof.

[0158] 4. Knockout or knockdown of endogenous genes The NK cell production process of the present disclosure can include gene editing of NK cells to remove one, two, three, four, five, six, seven, eight, nine, ten, or more endogenous genes in the NK cells. In some cases, gene editing is performed in NK cells that express one or more xenogeneic antigen receptors, while in other cases, gene editing is performed in at least some cases in NK cells that do not express xenogeneic antigen receptors, but will eventually express one or more xenogeneic antigen receptors. In certain embodiments, the NK cells that are gene edited are expanded NK cells.

[0159] In certain embodiments, one or more endogenous genes of NK cells are modified, such as expression disruption that reduces some or all of the expression.In certain cases, one or more genes are knocked down or knocked out using the process of the present disclosure.In certain cases, multiple genes are knocked down or knocked out in the same process of the present disclosure.The gene that is edited in NK cells can be any, but in a specific embodiment, the gene is a gene whose gene product inhibits the activity and / or proliferation of NK cells.In certain cases, the gene that is edited in NK cells allows NK cells to work more effectively in tumor microenvironment. In a specific case, the gene is one or more of NKG2A, SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, GR, TIGIT, CD96, ADORA2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1, ADAM17, RPS6, 4EBP1, CD25, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, TDAG8, CD5, CD7, SLAMF7, CD38, LAG3, TCR, β2-microglobulin, HLA, CD73, CREB, CREM, ICER, and CD39.In a specific embodiment, the TGFBR2 gene is knocked out or down in NK cells.In a specific embodiment, the CISH gene is knocked out or down in NK cells. In a specific embodiment, the CD38 gene is knocked out or down in the NK cells. In a specific embodiment, the glucocorticoid receptor (GR) gene is knocked out or down in the NK cells.

[0160] In some embodiments, gene editing is performed using one or more DNA-binding nucleic acids, such as modification via RNA-guided endonuclease (RGEN). For example, modification can be performed using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins. In general, "CRISPR system" refers collectively to the transcripts and other elements involved in directing the expression or activity of CRISPR-associated ("Cas") genes, including sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active portion tracrRNA), tracr-mate sequences (including "direct repeats" and tracrRNA-processing portion direct repeats in the context of endogenous CRISPR systems), guide sequences (also referred to as "spacers" in the context of endogenous CRISPR systems), and / or other sequences and transcripts from CRISPR loci. Methods of utilizing CRISPR systems are well known in the art.

[0161] F. Cryopreservation In certain cases, the NK cells and / or antibodies of the present disclosure are preserved in a cryopreservation medium composition comprising at least one cryoprotectant, serum (human or animal serum) or a non-serum alternative to serum (not human or animal serum), and at least one cytokine and / or at least one growth factor. In some cases, the cryoprotectant is dimethyl sulfoxide (DMSO), glycerin, glycerol, hydroxyethyl starch, or a combination thereof. The non-serum alternative can be of any type, including at least platelet lysate and / or blood product lysate (e.g., human serum albumin). In embodiments of the composition in which one or more (including two or more) cytokines are utilized, the cytokines can be natural or recombinant or synthetic proteins. At least one of the cytokines can be a Food and Drug Administration (FDA) approved cytokine. Examples of cytokines and growth factors include at least IL-1, IL-2, IL-3, IL-4, IL-6, IL-7, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL-22, interferons, tumor necrosis factor, stem cell factor, FLT3-ligand, APRIL, thrombopoietin, erythropoietin, or combinations thereof. In serum embodiments, the serum may be human serum (including human AB serum) or animal derived serum such as bovine serum. DMSO and other cryoprotectants, if utilized, may comprise 4-10%, 4-6%, 4-8%, 5-10%, 5-8%, 6-10%, 6-8%, 8-10%, etc. of the composition.For embodiments in which serum is employed, the serum may be present in an amount of 5-99%, 5-95%, 5-90%, 5-85%, 5-80%, 5-75%, 5-70%, 5-65%, 5-60%, 5-55%, 5-50%, 5-45%, 5-40%, 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, 5-10%, 10-99%, 10-95%, 10-90%, 10-85%, 10-80%, 10-75%, 10-70%, 10-65%, 10-60%, 10-75%, 10-70%, 10-65%, 10-60%, 10-85%, 10-8 ... %, 10~55%, 10~50%, 10~45%, 10~40%, 10~35%, 10~30%, 10~25%, 10~20%, 10~15%, 20~99%, 20~95%, 20~90%, 20~85%, 20~80%, 20~75%, 20~70%, 20~65%, 20~60%, 20~55%, 20~50%, 20~45%, 20~40%, 20~35%, 20~30%, 20~25%, 30~99%, 30~95%, 30~90 %, 30~85%, 30~80%, 30~75%, 30~70%, 30~65%, 30~60%, 30~55%, 30~50%, 30~45%, 30~40%, 30~35%, 40~99%, 40~95%, 40~90%, 40~85%, 40~80%, 40~75%, 40~70%, 40~65%, 40~60%, 40~55%, 40~50%, 40~45%, 50~99%, 50~95%, 50~90%, 50~85%, 50~80 %, 50-75%, 50-70%, 50-65%, 50-60%, 50-55%, 60-99%, 60-95%, 60-90%, 60-85%, 60-80%, 60-75%, 60-70%, 60-65%, 70-99%, 70-95%, 70-90%, 70-85%, 70-80%, 70-75%, 80-99%, 80-95%, 80-90%, 80-85%, 90-99%, 90-95%, or 95-99% of the composition. The composition may comprise at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or less serum.In certain embodiments, the composition comprises platelet lysate which may be at any concentration in the composition, however in certain embodiments, the platelet lysate is at any concentration between 5-99%, 5-95%, 5-90%, 5-85%, 5-80%, 5-75%, 5-70%, 5-65%, 5-60%, 5-55%, 5-50%, 5-45%, 5-40%, 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, 5-10%, 10-99%, 10-95%, 10-90%, 10-85% , 10~80%, 10~75%, 10~70%, 10~65%, 10~60%, 10~55%, 10~50%, 10~45%, 10~40%, 10~35%, 10~30%, 10~25%, 10~20%, 10~15%, 20~99%, 20~95%, 20~90%, 20~85%, 20~80%, 20~75%, 20~70%, 20~65%, 20~60%, 20~55%, 20~50%, 20~45%, 20~40%, 20~35%, 20~30%, 20~ 25%, 30~99%, 30~95%, 30~90%, 30~85%, 30~80%, 30~75%, 30~70%, 30~65%, 30~60%, 30~55%, 30~50%, 30~45%, 30~40%, 30~35%, 40~99%, 40~95%, 40~90%, 40~85%, 40~80%, 40~75%, 40~70%, 40~65%, 40~60%, 40~55%, 40~50%, 40~45%, 50~99%, 50~95%, 50~90% , 50-85%, 50-80%, 50-75%, 50-70%, 50-65%, 50-60%, 50-55%, 60-99%, 60-95%, 60-90%, 60-85%, 60-80%, 60-75%, 60-70%, 60-65%, 70-99%, 70-95%, 70-90%, 70-85%, 70-80%, 70-75%, 80-99%, 80-95%, 80-90%, 80-85%, 90-99%, 90-95%, or 95-99% composition. The composition may contain at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or less platelet lysate.

[0162] The composition may have a particular concentration of components, including cytokines and / or growth factors. In particular cases, any cytokine, including, for example, IL-2, IL-21, and / or IL-15, is present in the composition at a particular concentration. IL-2 may be present, for example, at a concentration of 1-5000, 1-1000, 1-500, 1-100, 100-5000, 100-500, 500-5000, 500-1000, or 1000-5000 U / mL. In particular cases, IL-2 is present in the composition at a concentration of at least 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 U / mL, or less. In certain embodiments, IL-21 is present in the composition at a concentration of 10-3000, 10-2000, 10-1000, 10-500, 10-100, 100-3000, 100-2000, 100-1000, 500-3000, 500-2000, 500-1000, 1000-3000, 1000-2000, or 2000-3000 ng / mL. IL-21 may be present in the composition at a concentration of at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, or 3000 ng / mL or less. IL-15 may be present in the composition at a concentration of 1-2000, 1-1000, 1-500, 1-100, 100-2000, 100-1000, 100-500, 500-2000, 500-1000, or 1000-2000 ng / mL. IL-15 may be present in the composition at a concentration of at least 10, 50, 100, 500, 1000, 1500, or 2000 ng / mL, or less.

[0163] Compositions as encompassed herein that include at least one cryoprotectant, serum or a non-serum alternative to serum, and at least one cytokine and / or at least one growth factor may each further include any type of multiple immune cells and / or stem cells. In specific embodiments, the cells are NK cells, T cells, B cells, NKT cells from mature bone marrow or peripheral blood cells; cell lines such as tumor cell lines (e.g., NK92 or other NK lines), which may be derived from bone marrow, peripheral blood, skin, adipose tissue, or combinations thereof, hematopoietic stem cells, induced pluripotent stem cells, MSCs (cell populations also referred to in the literature as "mesenchymal stem cells" and "mesenchymal stromal cells"), or mixtures thereof. In embodiments in which NK cells are utilized, the NK cells may or may not be expanded NK cells. Embodiments of the present disclosure also encompass pharmaceutical compositions that include any of the compositions of the present disclosure and a suitable pharma- ceutically acceptable carrier.

[0164] In certain embodiments, the cells and / or antibodies are treated with one or more inactivating agents (eg, kinase inhibitors such as dasatinib, nilotinib, rapamycin, etc.) prior to cryopreservation.

[0165] In some embodiments, the techniques described herein include inactivating NK cells, which includes treating NK cells with an effective amount of one or more inactivating agents under conditions that generate inactivated NK cells. In some embodiments, the inactivating agent is a kinase inhibitor. In some embodiments, the inactivating agent is a mechanistic target of rapamycin (mTOR) inhibitor. In some embodiments, the mTOR inhibitor is rapamycin, everolimus, and / or temsirolimus. In some embodiments, the mTOR inhibitor is rapamycin. In some embodiments, the inactivating agent is a tyrosine kinase (TK) inhibitor. In some embodiments, the TK inhibitor is selected from the group consisting of lorlatinib, brigatinib, ceritinib, alectinib, crizotinib, bosutinib, ponatinib, nilotinib, dasatinib, imatinib, zanubrutinib, acalabrutinib, ibrutinib, capmatinib, pexidartinib, dacomitinib, osimertinib, erlotinib, gefitinib, lapatinib, afatinib, pemigatinib, erdafitinib, nintedanib, gilteritinib, midosumab, sirolimus ... In some embodiments, the TK inhibitor is a BCR-Abl inhibitor. In some embodiments, the TK inhibitor is a bosutinib, ponatinib, nilotinib, dasatinib, and / or imatinib ... In some embodiments, the TK inhibitor is dasatinib and / or nilotinib. In some embodiments, the TK inhibitor is dasatinib.

[0166] In some embodiments, the treatment with the inactivating agent is at any time during the culture of the NK cells. In some embodiments, the treatment is for about 24 hours to about 96 hours, about 36 hours to about 84 hours, or about 48 hours to about 72 hours. In some embodiments, the treatment is for about 24 hours, about 48 hours, or about 72 hours. In some embodiments, the NK cells are treated with the inactivating agent at a concentration of about 1 to about 1000 nM. In some embodiments, the NK cells are treated with the inactivating agent at a concentration of about 5 to about 500 nM. In some embodiments, the NK cells are treated with the inactivating agent at a concentration of about 20 to about 200 nM. In some embodiments, the NK cells are treated with the inactivating agent at a concentration of about 30 to about 100 nM. In some embodiments, inactivated NK cells have increased expression of one or more of C-kit, CCR-5, CD62L, and / or CXCR4, and / or decreased expression of one or more of NKG2D, DNAM, OX-40, TRAIL, HLA-DR, CD2, CD25, ICOS, and / or CD95, compared to activated NK cells.

[0167] In some embodiments, the techniques described herein include methods of maintaining the viability of a population of cells at least 50% or greater after cryopreservation of the population, the methods including subjecting the population to an effective amount of one or more inactivating agents (e.g., tyrosine kinase inhibitors) to inactivate the cells prior to cryopreservation, cryopreserving the cells, and thawing the population, wherein upon thawing, the viability of the population is at least 50% or greater. In some cases, upon thawing of the cells, the viability of the population of cells is at least 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or greater after cryopreservation of the population.

[0168] In a specific embodiment, cells of the present disclosure at any stage are preserved in the following specific formulations for cryopreservation medium (see, for example, Tables 1 and / or 2). Table 1 - Exemplary cryopreservation media [Table 1] Table 2 - Exemplary cryopreservation media with specific concentrations [Table 2]

[0169] II.How to use An embodiment of the present disclosure relates to a method of using a composition comprising NK cells (e.g., engineered NK cells) and an antibody provided herein to treat or prevent a medical disease or disorder. The method includes administering to a subject a therapeutically effective amount of loaded, optionally pre-activated, and optionally expanded NK cells and an antibody, thereby treating or preventing a disease in the subject, including reducing the risk of the disease, reducing the severity of the disease, and / or delaying the onset of the disease. In certain embodiments of the present disclosure, a cancer or infectious disease is treated by transfer of a composition comprising a population of NK cells and an antibody. In at least some cases, NK cells, due to their release of inflammatory cytokines, may augment the adaptive immune response by reversing the anti-inflammatory tumor microenvironment and promoting the differentiation, activation, and / or recruitment of accessory immune cells to the site of the malignant tumor.

[0170] The cancers for which the therapeutic method of the present invention is useful include any malignant cell type, such as those found in solid tumors or blood tumors.Exemplary solid tumors can include, but are not limited to, tumors of organs selected from the group consisting of pancreas, colon, appendix, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and breast.Exemplary blood tumors include bone marrow tumors, T-cell or B-cell malignancies, leukemia, lymphoma, blastoma, myeloma, etc. Further examples of cancers that may be treated using the methods provided herein include, but are not limited to, lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), cancer of the peritoneum, gastric or stomach cancer (including gastrointestinal and gastrointestinal stromal cancer), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, renal or kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, various types of head and neck cancer, and melanoma.

[0171] In certain embodiments, cancers for which the compositions and methods described herein are useful for treating, preventing, and / or ameliorating symptoms include at least EGFR, EGFR2, and / or c-MET expressing cancers. In certain embodiments, cancers for which the compositions and methods described herein are useful for treating, preventing, and / or ameliorating symptoms include at least CD70 and / or TROP2 expressing cancers. In certain embodiments, the compositions and methods described herein are utilized for the treatment, prevention, and / or amelioration of symptoms associated with PDAC. In certain embodiments, the compositions and methods described herein are utilized for the treatment, prevention, and / or amelioration of symptoms associated with CRC. In certain embodiments, the compositions and methods described herein are utilized for the treatment, prevention, and / or amelioration of symptoms associated with kidney cancer. In certain embodiments, the compositions and methods described herein are utilized for the treatment, prevention, and / or amelioration of symptoms associated with glioblastoma. In certain embodiments, the compositions and methods described herein are utilized for the treatment, prevention, and / or amelioration of symptoms associated with breast cancer. In certain embodiments, the compositions and methods described herein are utilized for the treatment, prevention, and / or amelioration of symptoms associated with RCC. In certain embodiments, the compositions and methods described herein are utilized for the treatment, prevention, and / or amelioration of symptoms associated with myeloma.

[0172] Cancer may be of the following histological types, specifically but not limited to: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilonidal carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; cavernous adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma of adenomatous polyps; adenocarcinoma, familial polyposis coli; solid tumors; malignant carcinoid tumor; lobular-alveolar adenocarcinoma; papillary adenocarcinoma; chromatophore carcinoma; Eosinophilic carcinoma;Eosinophilic adenocarcinoma;Basophilic carcinoma;Clear cell adenocarcinoma;Granular cell carcinoma;Follicle adenocarcinoma;Papillary and follicular adenocarcinoma;Nonencapsulated sclerosing carcinoma;Adrenal cortical carcinoma;Endometrial carcinoma;Cutaneous adnexal carcinoma;Apocrine adenocarcinoma;Sebaceous gland carcinoma;Keratin adenocarcinoma;Mucoepidermoid carcinoma;Cystadenocarcinoma;Papillary cystadenocarcinoma;Papillary serous cystadenocarcinoma;Mucinous cystadenocarcinoma;Mucinous adenocarcinoma;Signet ring cell carcinoma;Invasive ductal carcinoma;Medullary carcinoma;Lobular carcinoma;Inflammatory carcinoma;Paget's disease, breast;Acinic cell carcinoma;Adenosquamous carcinoma;Adenocarcinoma with squamous metaplasia;Thymoma, malignant;Ovarian stromal tumor, malignant;Sarcoma, malignant;Granulosa cell Tumor, malignant;Androblastoma, malignant;Sertoli cell carcinoma;Leydig cell tumor, malignant;Lipocytoma, malignant;Paraganoneuroma, malignant;Extramammary paraganglioma, malignant;Pheochromocytoma;Angiosarcoma;Malignant melanoma;Amelanotic melanoma;Superficial spreading melanoma;Lentigo maligna melanoma;Lentigo acuminata melanoma;Nodular melanoma;Malignant melanoma of giant pigmented nevus;Epithelioid cell melanoma;Blue nevus, malignant;Sarcoma;Fibrosarcoma;Malignant fibrous histiocytoma;Myxosarcoma;Liposarcoma;Leiomyosarcoma;Rhabdomyosarcoma;Embryonic rhabdomyosarcoma;Alveolar rhabdomyosarcoma;Stromatous sarcoma;Mixed tumor, malignant;Müllerian mixed tumor;Nephroblastoma;Hepatoblastoma ;Carcinosarcoma;Mesenchymoma, malignant;Brenner tumor, malignant;Philodes tumor, malignant;Synovial sarcoma;Mesothelioma, malignant;Germinal dysplasia;Embryonal carcinoma;Teratoma, malignant;Ovarian goiter, malignant;Choriocarcinoma;Mesostosis, malignant;Angiosarcoma;Hemangioendothelioma, malignant;Kaposi's sarcoma;Hemangiopericytoma, malignant;Lymphangiosarcoma;Osteosarcoma;Soft cortical osteosarcoma;Chondrosarcoma;Chondrosarcoma, malignant;Mesenchymal chondrosarcoma;Giant cell tumor of bone;Ewing's sarcoma;Odontogenic tumor, malignant;Ameloblastoma, malignant;Ameloblastoma, malignant;Ameloblastic fibrosarcoma;Pinealoma, malignant;Chordoma;Glioma, malignant;Ependymoma;Astrocytoma;Protoplasmic astrocytoma;Fibrous astrocytoma;Astroblastoma;Glioblastoma;Oligodendroglioma;Oligodendroglioma;Primitive neuroectodermal tumor;Cerebellar sarcoma;Ganglioneuroblastoma;Neuroblastoma;Retinoblastoma;Olfactory neurogenic tumor;Meningioma, malignant;Neurofibrosarcoma;Schwannoma, malignant;Granular cell tumor, malignant;Malignant lymphoma;Hodgkin's disease;Hodgkin;Paragranuloma;Malignant lymphoma, small lymphocytic;Malignant lymphoma, large cell, diffuse;Malignant lymphoma, follicular;Mycosis fungoides;Other specified non-Hodgkin lymphoma;B-cell lymphoma;Low-grade / follicular non-Hodgkin lymphoma (NHL);Small lymphocytic (SL) NHL;Intermediate-grade / follicular NHL;Intermediate-grade diffuse NHL;High-grade immunoblastic NHL;High-grade lymphoblastic NHL;High-grade small non-necrotic cell NHL;Bulky disease NHL;Mantle cell lymphoma;AIDS-related lymphoma;Waldenstrom's macroglobulinemia;Malignant histiocytosis;Multiple myeloma;Mast cell sarcoma;Immunoproliferative small intestinal disease;Leukemia;Lymphocytic leukemia;Plasma cell leukemia;Erythroid leukemia;Lymphosarcoma cell leukemia;Myeloid leukemia;Basophilic leukemia;Eosinophilic leukemia;Monocytic leukemia;Mast cell leukemia;Megakaryocytic leukemia;Myeloid sarcoma;Hairy cell leukemia;Chronic lymphocytic leukemia (CLL);Acute lymphoblastic leukemia (ALL);Acute myeloid leukemia (AML);And chronic myeloblastic leukemia. ;

[0173] Certain embodiments relate to methods of treating leukemia. Leukemia is a cancer of the blood or bone marrow, characterized by abnormal proliferation (production by multiplication) of blood cells, usually white blood cells (leukocytes). It is part of a broad group of diseases called hematological neoplasms. Leukemia is a broad term that covers a spectrum of diseases. Leukemia is divided clinically and pathologically into acute and chronic forms.

[0174] Acute leukemia is characterized by the rapid proliferation of immature blood cells. This crowding causes the bone marrow to be unable to produce healthy blood cells. Acute leukemia can affect children and young adults. In fact, it is the leading cause of death in children in the United States more than any other type of malignant disease. Acute leukemia requires immediate treatment due to the rapid progression and accumulation of malignant cells, which then leak into the bloodstream and spread to other organs in the body. Central nervous system (CNS) involvement is rare, but sometimes cranial nerve palsies can occur. Chronic leukemia is distinguished by the excessive accumulation of relatively mature but still abnormal blood cells. It usually progresses over months to years, and they are produced at a much higher rate than normal cells, resulting in many abnormal white blood cells in the blood. Chronic leukemia primarily affects older people, but theoretically can occur at any age. While acute leukemia must be treated immediately, chronic leukemia may be observed for a period of time before treatment to maximize the effectiveness of treatment.

[0175] The disease is further classified as lymphocytic or lymphoblastic, which indicates that cancerous changes occur in the types of bone marrow cells that normally form lymphocytes, and myeloid or myeloid, which indicates that cancerous changes occur in the types of bone marrow cells that normally form red blood cells, certain white blood cells, and platelets (see Comparison of lymphocytic and myeloid cells).

[0176] Acute lymphocytic leukemia (also known as acute lymphoblastic leukemia, ALL) is a type of leukemia that is most common in young children. The disease can also affect adults, especially those over 65 years of age. Chronic lymphocytic leukemia (CLL) most commonly affects adults over 55 years of age. It can affect younger adults, but is rare in children. Acute myeloid leukemia (also known as acute myeloid leukemia or AML) is more common in adults than in children. This type of leukemia was previously called "acute nonlymphocytic leukemia." Chronic myeloid leukemia (CML) affects mostly adults, although a small number of children also develop the disease.

[0177] Lymphoma is a type of cancer that begins in lymphocytes (a type of white blood cell that is part of the immune system of vertebrates). There are many types of lymphoma. According to the National Institutes of Health, lymphoma accounts for about 5% of all cancer cases in the United States, with Hodgkin's lymphoma accounting for less than 1% of all cancer cases in the United States. Because the lymphatic system is part of the body's immune system, patients with weakened immune systems, such as those with HIV infection or taking certain drugs or medications, also have a higher incidence of lymphoma.

[0178] In certain embodiments of the present disclosure, a composition comprising NK cells and antibodies is delivered to an individual in need thereof, such as an individual suffering from cancer or an infectious disease. In at least some cases, the cells can boost the individual's immune system to attack the respective cancer or pathogenic cells. In some cases, the individual is provided with one or more doses of the composition comprising NK cells and antibodies. If more than one dose of NK cells / antibodies is administered to an individual, the period between doses should be sufficient to allow time for proliferation in the individual, and in specific embodiments, the period between doses is 1, 2, 3, 4, 5, 6, 7, or more days.

[0179] The source of the (optionally) pre-activated and expanded NK cells can be any source, but in specific embodiments, the cells are obtained, for example, from umbilical cord blood, peripheral blood, human embryonic stem cells, or a bank of induced pluripotent stem cells. A suitable dosage for therapeutic effect is, for example, at least 10 5 , or about 10 5 ~about 10 12 The cells are preferably administered in a series of cycles. An exemplary administration regimen includes at least about 10 5 Starting with cells, for example, within a few weeks of initiating an intrapatient dose escalation scheme, 12 The treatment consists of four weekly dose-escalating cycles, gradually increasing the dose to a target dose of cells. Suitable modes of administration include intravenous, subcutaneous, intracavitary (e.g., via a reservoir access device), intraperitoneal, or direct injection into the tumor mass.

[0180] Compositions comprising NK cells and antibodies produced according to the present methods have many potential uses, including experimental and therapeutic uses. In particular, it is envisioned that such cell populations will be useful in suppressing unwanted or inappropriate immune responses. In such methods, small numbers of NK cells are removed from a patient, manipulated and expanded ex vivo, and then re-inoculated into the patient. Examples of diseases that may be treated in such a manner include autoimmune diseases and conditions in which suppression of immune activity is desirable, such as allograft tolerance. Methods of treatment may include obtaining NK cells from a mammal; expanding the NK cells ex vivo according to the present methods as described herein; exposing the NK cells to an antibody under sufficient conditions; and administering a composition comprising the expanded NK cells / antibodies to the mammal to be treated.

[0181] The pharmaceutical compositions of the present disclosure can be used alone or in combination with other well-established agents useful in the treatment of cancer. Whether delivered alone or in combination with other agents, the pharmaceutical compositions of the present disclosure can be delivered to various sites in the mammalian, particularly human, body via various routes to achieve a particular effect. Those skilled in the art will recognize that, although multiple routes of administration can be used, certain routes may provide a more immediate and effective response than other routes. For example, intradermal administration may be advantageously used rather than inhalation for the treatment of melanoma. Local or systemic delivery can be achieved by application or injection of the formulation into a body cavity, inhalation or insufflation of an aerosol, or parenteral introduction, including intramuscular, intravenous, intraportal, intrahepatic, intraperitoneal, subcutaneous, or intradermal administration.

[0182] In one embodiment, the subject has an autoimmune disease. Non-limiting examples of autoimmune diseases include: alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune disease of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behçet's disease, bullous pemphigoid, cardiomyopathy, celiac hidrodactyly, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, Systemic lupus erythematosus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, type 1 or immune-mediated diabetes mellitus, myasthenia gravis, nephrotic syndrome (including minimal change disease, focal glomerulosclerosis, membranous nephropathy), pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary cholesteatoma, Autoimmune diseases include, but are not limited to, cirrhosis of the liver, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff man syndrome, systemic lupus erythematosus, lupus erythematosus, ulcerative colitis, uveitis, vasculitis (such as polyarteritis nodosa, Takayasu's arteritis, temporal arteritis / giant cell arteritis, or dermatitis herpetiformis vasculitis), vitiligo, and Wegener's granulomatosis.Thus, some examples of autoimmune diseases that can be treated using the methods disclosed herein include, but are not limited to, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, type I diabetes, Crohn's disease; ulcerative colitis, myasthenia gravis, glomerulonephritis, ankylosing spondylitis, vasculitis, or psoriasis.Subjects can also have allergic diseases, such as asthma.

[0183] In yet another embodiment, the subject is a recipient of a transplanted organ or stem cells, and the expanded NK cells / antibodies of the present disclosure are used to prevent and / or treat rejection. In certain embodiments, the subject has or is at risk of developing graft-versus-host disease. GVHD can be a complication of transplants that use or include stem cells from either related or unrelated donors. There are two types of GVHD: acute and chronic. Acute GVHD appears within three months of transplant. Signs of acute GVHD include a reddish rash on the hands and feet that spreads and becomes severe, resulting in peeling and blisters. Acute GVHD can also affect the stomach and intestines, causing cramps, nausea, and diarrhea. Yellowing of the skin and eyes (jaundice) indicates that acute GVHD is affecting the liver. Chronic GVHD is graded by its severity: stage / grade 1 is mild, and stage / grade 4 is severe. Chronic GVHD develops after 3 months post-transplant. Symptoms of chronic GVHD are similar to acute GVHD, but in addition chronic GVHD can affect the mucosal glands of the eye, the salivary glands in the mouth, the mucosa of the stomach, and the glands that lubricate the intestines. Any of the populations of NK cells disclosed herein can be utilized. Examples of transplanted organs include solid organ transplants such as kidney, liver, skin, pancreas, lung, and / or heart, or cell transplants such as pancreatic islets, hepatocytes, myoblasts, bone marrow, or hematopoietic or other stem cells. Transplants can also be composite transplants, such as facial tissue. NK cells, such as immunosuppressive CD19+ cells, can be administered with antibodies prior to, concurrently with, and / or after transplantation. In some embodiments, the composition comprising the NK cells and the antibody is administered prior to transplantation, e.g., at least 1 hour, at least 12 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, or at least 1 month prior to transplantation. In certain non-limiting examples, administration of a therapeutically effective amount of the composition comprising the NK cells and the antibody occurs 3-5 days prior to transplantation.

[0184] The NK cells / antibodies administered to the transplant patient can be sensitized with antigens specific to the transplant material prior to administration. According to this embodiment, the transplant recipient will have a reduced immune / inflammatory response to the transplanted material, thereby minimizing the possibility of rejection of the transplanted tissue. Similarly, for the treatment of graft-versus-host disease, the NK cells can be sensitized with antigens specific to the host. According to this embodiment, the recipient will have a reduced immune / inflammatory response to self-antigens.

[0185] In further embodiments, administering to a subject a therapeutically effective amount of a composition comprising NK cells and an antibody treats or suppresses inflammation in the subject. Thus, the method includes administering to a subject a therapeutically effective amount of a composition comprising NK cells and an antibody to inhibit an inflammatory process. Examples of inflammatory diseases include, but are not limited to, asthma, encephalitis, inflammatory bowel disease, chronic obstructive pulmonary disease (COPD), allergic disease, septic shock, pulmonary fibrosis, undifferentiated spondyloarthropathy, undifferentiated arthropathy, arthritis, inflammatory osteolysis, and chronic inflammation resulting from chronic viral or bacterial infection. The methods disclosed herein can also be used to treat allergic diseases.

[0186] Administration of compositions comprising NK cells and antibodies can be used whenever immunosuppression or anti-inflammatory suppression is desired, for example, at the first signs or symptoms of disease or inflammation. These may be general, such as pain, edema, elevated temperature, or specific signs and symptoms associated with dysfunction of the affected organ, such as elevated serum creatinine in kidney transplant rejection, rash in GVHD, and shortness of breath and wheezing in asthma.

[0187] Administration of a composition comprising NK cells and an antibody can also be used to prevent immune-mediated diseases in a subject of interest. For example, a composition comprising NK cells and an antibody can be administered to a subject who will be a transplant recipient prior to transplant. In another example, a composition comprising NK cells and an antibody is administered to a subject undergoing an allogeneic bone marrow transplant without T cell depletion. In a further example, a composition comprising NK cells and an antibody can be administered to a subject with a family history of diabetes. In another example, a composition comprising NK cells and an antibody is administered to a subject with asthma to prevent asthma attacks. In some embodiments, a therapeutically effective amount of a composition comprising NK cells and an antibody is administered to a subject prior to symptoms. Administration of a composition comprising NK cells and an antibody results in a reduction in the incidence or severity of a subsequent immunological event or symptom (such as an asthma attack) or improved survival of the patient compared to patients receiving other therapies that do not include a composition comprising NK cells and an antibody.

[0188] In certain embodiments, the composition comprising NK cells and antibodies is administered in combination with a second therapeutic agent. For example, the second therapeutic agent may include T cells, immunomodulatory agents, monoclonal antibodies, chemotherapeutic agents, hormone(s), any type of drug, surgery, radiation, etc. In a non-limiting example, the immunomodulatory agent is lenalidomide, the monoclonal antibody is rituximab, ofatumumab, or lumiliximab, and the chemotherapeutic agent is fludarabine or cyclophosphamide.

[0189] The composition of the present disclosure can be provided in unit dosage form, and each dosage unit, such as an injection, contains a predetermined amount of the composition, alone or in appropriate combination with other active agents.The term unit dosage form as used herein refers to a physically discrete unit suitable as a unitary dose for human and animal subjects, each unit contains a predetermined amount of the composition of the present disclosure, alone or in combination with other active agents, calculated in an amount sufficient to produce a desired effect, in association with a pharma- ceutically acceptable diluent, carrier, or vehicle as appropriate.The specification of the unit dosage form of the present disclosure depends on the specific pharmacodynamics associated with the pharmaceutical composition in a specific subject.

[0190] Desirably, an effective amount or sufficient number of isolated transduced NK cells are present in the composition and introduced into the subject such that a specific anti-tumor response is established that lasts longer than in the absence of such treatment to reduce tumor size or eliminate tumor growth or regrowth. Desirably, the amount of NK cells reintroduced into the subject causes a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 100% reduction in tumor size compared to otherwise identical conditions in which the NK cells are not present.

[0191] Thus, the amount of the composition comprising NK cells and antibodies administered should take into account the route of administration and should ensure that a sufficient number of the composition comprising NK cells and antibodies is introduced to achieve the desired therapeutic response. Additionally, the amount of each active agent included in the compositions described herein (e.g., the amount per each cell contacted or the amount per specific body weight) may vary for different applications. In general, the concentration of NK cells is desirably at least about 1×10 6 From about 1×10 12 NK cells, more preferably about 1×10 7 From about 5 × 10 10 The amount should be sufficient to provide the subject with 5×10 NK cells, but any suitable amount may be used, such as above, for example, 5×10 8 More than or less than 1 x 10 cells 7 Administration schedules can be based on established cell-based therapies (see, e.g., U.S. Pat. No. 4,690,915) or alternative continuous infusion strategies can be employed.

[0192] These values ​​provide a general guideline for the range of compositions comprising NK cells and antibodies that a person skilled in the art should utilize when optimizing the disclosed method for carrying out the disclosed method. The description of such ranges herein in no way precludes the use of higher or lower amounts of components as may be warranted in a particular application. For example, the actual dose and schedule may vary depending on whether the composition is administered in combination with other pharmaceutical compositions, or depending on inter-individual differences in pharmacokinetics, drug disposition, and metabolism. A person skilled in the art can easily make the necessary adjustments depending on the exigencies of a particular situation.

[0193] III. Antibodies Aspects of the present disclosure relate to the use of antibodies or functional fragments thereof in compositions that also include certain NK cells. The term "antibody" refers to an intact immunoglobulin of any isotype, or a fragment thereof that can compete with the intact antibody for specific binding to a target antigen, such as through an antigen-binding domain, and includes chimeric antibodies, humanized antibodies, fully human antibodies, monospecific antibodies, and multispecific antibodies (including at least bispecific and trispecific and even more specific). As used herein, the terms "antibody" or "immunoglobulin" are used interchangeably and refer to any of several classes of structurally related proteins that function as part of an animal's immune response, including IgG, IgD, IgE, IgA, IgM, and related proteins, as well as polypeptides that contain antibody CDR domains that retain antigen-binding activity. In a specific embodiment, the antibody comprises an scFv.

[0194] The term "antigen" refers to a molecule or a portion of a molecule capable of binding to a selective binding agent such as an antibody. An antigen can have one or more epitopes capable of interacting with different antibodies.

[0195] The term "epitope" includes a region or portion of a molecule that can elicit an immune response by binding to an immunoglobulin or T-cell receptor. Epitopic determinants include chemically active surface groups such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and may have specific three-dimensional structural characteristics and / or specific charge characteristics. In general, an antibody specific for a particular target antigen will preferentially recognize an epitope on the target antigen in a complex.

[0196] Epitope regions of a given polypeptide can be identified using many different epitope mapping techniques known in the art, including X-ray crystallography, nuclear magnetic resonance spectroscopy, site-directed mutagenesis mapping, and protein display arrays, see, for example, Epitope Mapping Protocols (Johan Rockberg and Johan Nilvebrant, Ed., 2018) Humana Press, New York, NY. Such techniques are well known in the art, see, for example, U.S. Patent No. 4,708,871; Geysen et al. Proc. Natl. Acad. Sci. USA 82:178-182 (1985); Geysen et al. Molec. Immunol. 23:709-715 (1986), see, for example, Epitope Mapping Protocols (supra). Additionally, antigenic regions of proteins can also be predicted and identified using standard antigenicity and hydropathy plots.

[0197] Intact antibodies are generally composed of two full-length heavy chains and two full-length light chains, but may contain fewer chains, such as antibodies naturally occurring in camelids, which may contain only heavy chains. The antibodies disclosed herein may be from only a single source, or may be "chimeric" in which different portions of the antibody are derived from two different antibodies. For example, the variable or CDR regions may be from rat or mouse, while the constant regions may be from different animals, such as humans. Antibodies or binding fragments may be produced by hybridomas, recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Unless otherwise specified, the term "antibody" includes derivatives, variants, fragments, and muteins thereof, examples of which are described below (Sela-Culang et al. Front Immunol. 2013; 4: 302; 2013).

[0198] The term "light chain" includes full-length light chains and fragments thereof that contain sufficient variable region sequence to confer binding specificity. Full-length light chains have a molecular weight of about 25,000 daltons and contain a variable region domain (abbreviated herein as VL) and a constant region domain (abbreviated herein as CL). There are two classes of light chains, called kappa (κ) and lambda (λ). A "VL fragment" refers to a fragment of the light chain of a monoclonal antibody that contains all or a portion of the light chain variable region, including the CDRs. A VL fragment may further contain light chain constant region sequence. The light chain variable region domain is at the amino terminus of the polypeptide.

[0199] The term "heavy chain" includes full-length heavy chains and fragments thereof having sufficient variable region sequence to confer binding specificity. A full-length heavy chain has a molecular weight of about 50,000 daltons and contains a variable region domain (abbreviated herein as VH) and three constant region domains (abbreviated herein as CH1, CH2, and CH3). A "VH fragment" refers to a fragment of a heavy chain of a monoclonal antibody that contains all or a portion of the heavy chain variable region, including the CDRs. A VH fragment may further contain heavy chain constant region sequence. The number of heavy chain constant region domains varies depending on the isotype. The VH domain is at the amino terminus of the polypeptide, the CH domain is at the carboxy terminus, and CH3 is closest to the -COOH terminus. An antibody isotype is either IgM, IgD, IgG, IgA, or IgE, which are defined by the presence of five heavy chains, classified as mu (μ), delta (δ), gamma (γ), alpha (α), and epsilon (ε) chains, respectively. There are several subtypes of IgG, including but not limited to IgG1, IgG2, IgG3, and IgG4. Subtypes of IgM include IgM1 and IgM2. Subtypes of IgA include IgA1 and IgA2.

[0200] Antibodies can be whole immunoglobulins of any isotype or classification, chimeric antibodies, or hybrid antibodies having specificity for more than one antigen. They can also be fragments, including hybrid fragments (e.g., F(ab')2, Fab', Fab, Fv, etc.). Immunoglobulins also include natural, synthetic, or genetically engineered proteins that bind to a specific antigen to form a complex and act like an antibody. The term antibody includes genetically engineered or otherwise modified forms of immunoglobulins, such as:

[0201] The term "monomer" refers to an antibody that contains only one Ig unit. A monomer is the basic functional unit of an antibody. The term "dimer" refers to an antibody that contains two Ig units bound to each other via the constant domain (Fc, or fragment crystallizable region) of the antibody heavy chain. This complex may be stabilized by a joining (J) chain protein. "Multimer" refers to an antibody that contains two or more Ig units bound to each other via the constant domain (Fc region) of the antibody heavy chain. This complex may be stabilized by a joining (J) chain protein.

[0202] The term "bivalent antibody" refers to an antibody that contains two antigen-binding sites. The two binding sites may have the same antigen specificity or may be bispecific, meaning that the two antigen-binding sites have different antigen specificities.

[0203] Bispecific antibodies are a class of antibodies that have two paratopes with different binding sites for two or more different epitopes. In some embodiments, bispecific antibodies can be dual paratopic, where the bispecific antibody can specifically recognize different epitopes from the same antigen. In some embodiments, bispecific antibodies can be constructed from a pair of different single domain antibodies called "nanobodies." Single domain antibodies have been sourced and engineered from cartilaginous fish and camelids. Nanobodies can be joined by linkers using techniques typical to those of skill in the art. Such methods for the selection and conjugation of nanobodies are described in PCT Publication Nos. WO2015044386A1, WO2010037838A2, and Bever et al., Anal Chem. 86:7875-7882 (2014), each of which is specifically incorporated herein by reference in its entirety.

[0204] Bispecific antibodies can be constructed as whole IgG, Fab'2, Fab'PEG, diabodies, or scFv. Diabodies and scFv lack the Fc region and can be constructed using only variable domains, potentially reducing the effects of anti-idiotypic reaction. Bispecific antibodies can be produced by a variety of methods, including, but not limited to, fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai and Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148:1547-1553 (1992), each of which is specifically incorporated herein by reference in its entirety.

[0205] In certain embodiments, the antigen-binding domain may be multispecific or heterospecific by multimerizing with pairs of VH and VL regions that bind different antigens. For example, the antibody may bind to or interact with (a) a cell surface antigen, (b) an Fc receptor on the surface of an effector cell, or (c) at least one other component. Thus, embodiments include, but are not limited to, bispecific, trispecific, tetraspecific, and other multispecific antibodies or antigen-binding fragments thereof that are directed to other targets, such as epitopes or Fc receptors on effector cells.

[0206] In some embodiments, multispecific antibodies can be used and directly linked via short flexible polypeptide chains using routine methods known in the art. Such examples include diabodies, which are bivalent bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, utilizing a linker that is too short to allow pairing between the domains on the same chain, thereby forcing the domains to pair with complementary domains on another chain, forming two antigen binding sites. The functionality of the linker is applicable to triabodies, tetrabodies, and higher order antibody multimer embodiments. (See, e.g., Hollinger et al., Proc Natl. Acad. Sci. USA 90:6444-6448 (1993); Polijak et al., Structure 2:1121-1123 (1994); Todorovska et al., J. Immunol. Methods 248:47-66 (2001)).

[0207] Bispecific diabodies are advantageous because, in contrast to bispecific whole antibodies, they can be easily constructed and expressed in E. coli. Diabodies (and other polypeptides, such as antibody fragments) with appropriate binding specificity can be easily selected from libraries using phage display (WO94 / 13804). If one arm of the diabody is kept constant, e.g., has specificity for a protein, a library can be created in which the other arm is varied, and antibodies with appropriate specificity can be selected. Bispecific whole antibodies can be produced by alternative engineering methods, as described in Ridgeway et al., (Protein Eng., 9:616-621, 1996) and Krah et al., (N Biotechnol. 39:167-173, 2017), each of which is incorporated herein by reference in its entirety.

[0208] Heteroconjugate antibodies are two covalently joined monoclonal antibodies with different specificities, see, e.g., U.S. Patent No. 6,010,902, incorporated herein by reference in its entirety.

[0209] The portion of the Fv fragment of an antibody molecule that binds to an epitope of an antigen with high specificity is referred to herein as a "paratope". The paratope consists of amino acid residues that contact the epitope of an antigen and promote antigen recognition. The two Fv fragments of an antibody each consist of two dimerized variable domains, VH and VL. The primary structure of each variable domain includes three hypervariable loops flanked by framework regions (FR). The hypervariable loops are the regions with the highest primary sequence variability in antibody molecules from any mammal. The term hypervariable loop is sometimes used interchangeably with the term "complementarity determining region (CDR)". The length of the hypervariable loops (or CDRs) varies from one antibody molecule to another. The framework regions of all antibody molecules from a mammal share a high degree of primary sequence similarity / consensus. The consensus of the framework regions can be used by one skilled in the art to identify both the framework regions and the hypervariable loops (or CDRs) that are interspersed between the framework regions. The hypervariable loops are given identifiers that distinguish their location in the polypeptide and the domain in which they are found. The CDRs of the VL domain are identified as L1, L2, and L3, with L1 being the most distal and L3 being closest to the CL domain. The CDRs are sometimes named CDR-1, CDR-2, and CDR-3. L3 (CDR-3) is generally the most variable region of all antibody molecules produced by an organism. The CDRs are linearly arranged regions in the primary structure of the polypeptide chain and are separated from each other by framework regions. The amino-terminus (N-terminus) of the VL chain is named FR1. The region identified as FR2 is between the hypervariable loops of L1 and L2. FR3 is between the hypervariable loops of L2 and L3, and the FR4 region is closest to the CL domain. This structure and nomenclature is repeated in the VH chain, which contains three CDRs identified as H1, H2, and H3. The majority of the amino acid residues in the variable domains, or Fv fragments (VH and VL), are part of the framework regions (about 85%). The three-dimensional structure (tertiary structure) of an antibody molecule is such that the framework regions are more prevalent inside the molecule and occupy the majority of the structure, while the CDRs are present on the outer surface of the molecule.

[0210] Several methods have been developed and are available to those skilled in the art to identify the exact amino acids that make up each of these regions. This can be done using any of a number of multiple sequence alignment methods and algorithms that identify the conserved amino acid residues that make up the framework regions and thus identify the CDRs that are different in length but are located between the framework regions. Three common methods have been developed to identify the CDRs of antibodies: Kabat (described in T. T. Wu and E. A. Kabat, “AN ANALYSIS OF THE SEQUENCES OF THE VARIABLE REGIONS OF BENCE JONES PROTEINS AND MYELOMA LIGHT CHAINS AND THEIR IMPLICATIONS FOR ANTIBODY COMPLEMENTARITY,” J Exp Med, vol. 132, no. 2, pp. 211-250, Aug. 1970); Chothia (described in C. Chothia et al., “Conformations of immunoglobulin hypervariable regions,” Nature, vol. 342, no. 6252, pp. 877-883, Dec. 1989); and IMGT (described in M.-P. Lefranc et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Developmental & Comparative (See, for example, Immunology, vol. 27, no. 1, pp. 55-77, Jan. 2003). Each of these methods includes a unique numbering system for identifying the amino acid residues that make up the variable region. In most antibody molecules, the amino acid residues that actually contact the antigen epitope are located in the CDRs, although in some cases residues in the framework regions may also contribute to antigen binding.

[0211] One skilled in the art can use any of several methods to determine the paratope of an antibody. These methods include: 1) computational prediction of the tertiary structure of the antibody / epitope binding interaction based on the chemical nature of the amino acid sequence of the antibody variable region and the composition of the epitope; 2) hydrogen-deuterium exchange and mass spectrometry; 3) polypeptide fragmentation and peptide mapping approaches, which generate multiple overlapping peptide fragments from the full length of the polypeptide and evaluate the binding affinity of these peptides to the epitope; 4) antibody phage display library analysis, in which genes encoding mammalian antibody Fab fragments are expressed by bacteriophage so that they are incorporated into the phage coat. This population of Fab-expressing phages is allowed to interact with immobilized antigen or expressed in another exogenous expression system. Non-binding Fab fragments are washed away, leaving only the specific binding Fab fragments bound to the antigen. The binding Fab fragments can be easily isolated and their encoding genes determined. This approach can also be used appropriately for Fv fragments and smaller regions of Fab fragments containing specific VH and VL domains.

[0212] In certain embodiments, affinity matured antibodies are enhanced by one or more modifications in one or more CDRs thereof, so that the affinity of the antibody to the target antigen is improved compared to the parent antibody without those modifications. Some affinity matured antibodies have nanomolar or picomolar affinities to the target antigen. Affinity matured antibodies are generated by procedures known in the art. For example, Marks et al., Bio / Technology 10:779 (1992) describes affinity maturation by shuffling VH and VL domains, and random mutagenesis of CDR and / or framework residues used in phage display is described in Rajpal et al., PNAS. 24: 8466-8471 (2005) and Thie et al., Methods Mol Biol. 525:309-22 (2009), in combination with the computational method demonstrated in Tiller et al., Front. Immunol. 8:986 (2017).

[0213] Chimeric immunoglobulins are the product of the fusion of genes from different species. "Humanized" chimeras generally have framework regions (FR) derived from a human immunoglobulin and one or more CDRs are of non-human origin.

[0214] In certain embodiments, a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence from another specific species or belonging to a specific antibody class or subclass, and the remaining portion of the chain is identical or homologous to the corresponding sequence in the antibody from another species or belonging to another antibody class or subclass, and the fragment of such an antibody, so long as it shows the desired biological activity. U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851 (1984). For methods related to chimeric antibodies, see, for example, U.S. Patent No. 4,816,567, Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1985), each of which is specifically incorporated herein by reference in its entirety. CDR grafting is described, for example, in US Pat. Nos. 6,180,370, 5,693,762, 5,693,761, 5,585,089, and 5,530,101, all of which are incorporated by reference herein for all purposes.

[0215] In some embodiments, the antibody polypeptide sequence from the non-human species is minimized to optimize the function of the chimeric antibody and reduce immunogenicity. Certain amino acid residues in the non-antigen recognition regions of the non-human antibody are modified to become homologous to corresponding residues in a human antibody or isotype. One example is a "CDR-grafted antibody," which contains one or more CDRs from a particular species or belonging to a particular antibody class or subclass, with the remainder of the antibody chains being identical or homologous to corresponding sequences in antibodies from other species or belonging to other antibody classes or subclasses. For use in humans, the V regions, consisting of CDR1, CDR2, and partial CDR3 of the light and heavy chain dispersion regions from a non-human immunoglobulin, are grafted into the framework regions of a human antibody, replacing the naturally occurring antigen receptor of the human antibody with the non-human CDRs. In some examples, the corresponding non-human residues replace the framework region residues of the human immunoglobulin. Additionally, humanized antibodies may contain residues not found in the recipient antibody or donor antibody to further refine performance. The humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. See, e.g., Jones et al., Nature 321:522 (1986); Riechmann et al., Nature 332:323 (1988); Presta, Curr. Op. Struct. Biol. 2:593 (1992); Vaswani and Hamilton, Ann. Allergy, Asthma and Immunol. 1:105 (1998); Harris, Biochem. Soc. Transactions 23; 1035 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428 (1994); Verhoeyen et al., Science 239:1534-36 (1988).

[0216] Intrabodies are immunoglobulins that are localized within cells and bind to intracellular antigens, in contrast to secreted antibodies, which bind to extracellular antigens.

[0217] Polyclonal antibody preparations usually contain different antibodies against different determinants (epitopes). To produce polyclonal antibodies, a host such as a rabbit or goat is immunized with an antigen or an antigen fragment, typically with an adjuvant, and optionally coupled to a carrier. Antibodies against the antigen are then collected from the host's serum. Polyclonal antibodies are affinity purified against the antigen and exhibit a monospecificity.

[0218] Monoclonal antibody or "mAb" refers to an antibody obtained from a homogeneous population of antibodies obtained from a single parent cell, i.e., the population is identical except for possible minor naturally occurring mutations. Each monoclonal antibody is directed against a single antigenic determinant.

[0219] Functional antibody fragments and antigen-binding fragments may also be utilized. Certain aspects relate to antibody fragments, such as antibody fragments that bind and / or neutralize inflammatory mediators. The term functional antibody fragment includes antigen-binding fragments of antibodies that retain the ability to specifically bind to an antigen. These fragments are composed of various sequences of variable region heavy chain (VH) and / or light chain (VL); in some embodiments, they contain constant region heavy chain 1 (CH1) and light chain (CL). In some embodiments, they lack the Fc region, which is composed of heavy chain 2 (CH2) and 3 (CH3) domains. Embodiments of antigen-binding fragments and variants thereof may include: (i) a Fab fragment type composed of VL, VH, CL, and CH1 domains; (ii) an Fd fragment type composed of VH and CH1 domains; (iii) an Fv fragment type composed of VH and VL domains; (iv) a single domain fragment type, a dAb (Ward, 1989; McCafferty et al., 1990; Holt et al., 2003), composed of a single VH or VL domain; (v) isolated complementarity determining region (CDR) regions. Such terms are described, for example, in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, NY (1989); Molec. Biology and Biotechnology: A Comprehensive Desk Reference (Myers, RA (ed.), New York: VCH Publisher, Inc.); Huston et al., Cell Biophysics, 22:189-224 (1993); Pluckthun and Skerra, Meth. Enzymol., 178:497-515 (1989) and in Day, ED, Advanced Immunochemistry, 2d ed., Wiley-Liss, Inc. New York, NY (1990); Antibodies, 4:259-277 (2015). All citations in this paragraph are incorporated by reference.

[0220] Antigen-binding fragments also include fragments of antibodies that retain exactly at least or at most one, two, or three of the complementarity determining regions (CDRs) from the light chain variable region. Fusions of CDR-containing sequences to an Fc region (or its CH2 or CH3 regions) are included within this definition, including, for example, an scFv fused directly or indirectly to an Fc region.

[0221] The term Fab fragment refers to a monovalent antigen-binding fragment of an antibody that contains the VL, VH, CL and CH1 domains. The term Fab' fragment refers to a monovalent antigen-binding fragment of a monoclonal antibody that is larger than the Fab fragment. For example, the Fab' fragment contains the VL, VH, CL and CH1 domains and all or part of the hinge region. The term F(ab')2 fragment refers to a bivalent antigen-binding fragment of a monoclonal antibody that contains two Fab' fragments linked by a disulfide bridge at the hinge region. The F(ab')2 fragment, for example, contains all or part of two VH and VL domains and may further contain all or part of two CL and CH1 domains.

[0222] The term Fd fragment refers to a fragment of the heavy chain of a monoclonal antibody that contains all or a portion of the VH including the CDRs. The Fd fragment may further contain sequences of the CH1 region.

[0223] The term Fv fragment refers to a monovalent antigen-binding fragment of a monoclonal antibody that includes all or part of the VL and VH, but does not include the CL and CH1 domains. The VL and VH include, for example, the CDRs. A single-chain antibody (sFv or scFv) is an Fv molecule in which the VL and VH regions are linked by a flexible linker to form a single polypeptide chain that forms the antigen-binding fragment. Single-chain antibodies are discussed in detail in International Patent Application Publication No. WO88 / 01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203, the disclosures of which are incorporated herein by reference. The term (scFv)2 refers to a bivalent or bispecific sFv polypeptide chain that includes an oligomerization domain at the C-terminus separated from the sFv by a hinge region (Pack et al. 1992). The oligomerization domain consists of self-associating a-helices, such as leucine zippers, and is further stabilized by additional disulfide bonds. (scFv)2 fragments are also known as "miniantibodies" or "minibodies".

[0224] A single domain antibody is an antigen-binding fragment that contains only a VH or VL domain. In some cases, two or more VH regions are covalently linked with a peptide linker to form a bivalent domain antibody. The two VH regions of a bivalent domain antibody may target the same antigen or different antigens.

[0225] In some cases, a fragment crystallizable region, Fc, is utilized. The Fc region comprises two heavy chain fragments, including the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and hydrophobic interactions of the CH3 domain. As used herein, the term "Fc polypeptide" includes native and mutein forms of polypeptides derived from the Fc region of an antibody. Also included are truncated forms of such polypeptides, including the hinge region that promotes dimerization. In some embodiments, antibodies incorporating cytokines, including those mentioned herein, can be utilized (such as TRIKE).

[0226] In some embodiments, the antibody is an antibody-drug conjugate. In some embodiments, the antibody may specifically be one or more of the following, but is not limited to: [fam]-trastuzumab deruxtecan, abciximab, adalimumab, ado-trastuzumab emtansine, aducanumab, alemtuzumab, alirocumab, amivantamab, anifrolumab, ansuvimab, atezolizumab, atortivimab and maftivimab, and odesivimab-ebgn (also known as inmazeb), avelumab, basiliximab, belantamab mafodotin, belimumab, benralizumab, bevac ... Zlotoxumab, bimekizumab, blinatumomab, brentuximab vedotin, brodalumab, brolucizumab, burosumab, canakinumab, caplacizumab, casirivimab + imdevimab, catumaxomab, cemiplimab, certolizumab pegol, cetuximab, crizanlizumab, daclizumab, daratumumab, denosumab, dinutuximab, donanemab, dostallimab, dupilumab, durvalumab, eculizumab, edrecolomab, efalizumab, elotuzumab, emapalumab, emicizumab, enfortumab vedotin, Petitezumab, erenumab, evinacumab, evolocumab, faricimab, fremanezumab, galcanezumab, gemtuzumab, golimumab, guselkumab, ibalizumab, ibritumomab tiuxetan, idarucizumab, inebilizumab, infliximab, inolimomab, inotuzumab, ipilimumab, isatuximab, ixekizumab, lanadelumab, lecanemab, loncastuximab tesirin, margetuximab, mepolizumab, mirvetuximab soravtansine, mogamulizumab, mosunetuzumab, moxetumomab pasudotox, mucositis Romonab-CD3, narsoplimab, natalizumab, naxitamab, nebacumab, necitumumab, nirsevimab, nivolumab, obiltoxaximab, obinutuzumab, ocrelizumab, ofatumumab, olaratumab, omalizumab, omburtamab, oportuzumab monatox, palivizumab, panitumumab, pembrolizumab, pemplimab, pertuzumab, polatuzumab vedotin, ramucirumab, ranibizumab, ravulizumab, raxibacumab, regdanvimab, relatorimab, reslizumab, retifanlimab, risankizumab,Rituximab, romosozumab, sacituzumab govitecan, sarilumab, satralizumab, secukinumab, siltuximab, sintilimab, sotrovimab, spesolimab, stimulimab, tafasitamab, tebentafsp, teclistamab, teplizumab, teprotumumab, tezepelumab, tildrakizumab, tislelizumab, tisotumab vedotin, tixagevimab, silgavimab, tocilizumab, toripalimab, tositumomab-I131, tralokinumab, trastuzumab, tremelimumab, ublituximab, ustekinumab, vedolizumab, or any combination thereof. In some embodiments, the antibody is imgatuzumab. In some embodiments, the antibody is margetuximab. In some embodiments, the antibody is amivantamab. In some embodiments, the antibody is obinutuzumab. In some embodiments, the antibody is IPH61 (also known as IPH6101 or SAR443579).

[0227] IV. Pharmaceutical Compositions The pharmaceutical composition of the present disclosure comprises an effective amount of a composition comprising NK cells and antibodies dispersed in a pharma- ceutically acceptable carrier. The phrase "pharmaceutical or pharmacologically acceptable" refers, as appropriate, to molecular entities and compositions that do not produce adverse, allergic or other adverse reactions when administered to an animal, such as a human. Preparation of pharmaceutical compositions comprising the present composition will be known to those skilled in the art in light of the present disclosure, as exemplified in Remington: The Science and Practice of Pharmacy, 21st Ed. Lippincott Williams and Wilkins, 2005, which is incorporated herein by reference. Furthermore, it will be understood that for animal (e.g., human) administration, the preparation should meet the sterility, pyrogenicity, general safety and purity standards required by the FDA Office of Biological Standards.

[0228] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents, isotonicity agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, such materials, and combinations thereof, which would be known to one of ordinary skill in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the pharmaceutical compositions is contemplated.

[0229] Pharmaceutical compositions can contain different types of carriers depending on whether they are administered in solid, liquid or aerosol form and whether they need to be sterile for routes of administration such as injection. The presently disclosed compositions can be administered intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, topically, intramuscularly, subcutaneously, mucosally, orally, topically, by inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, localized perfusion, direct immersion in target cells, via catheter, via lavage, in creams, in lipid compositions (e.g., liposomes), or by any other method or any combination thereof that would be known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, which is incorporated herein by reference).

[0230] The composition comprising NK cells and antibodies can be formulated into a composition in free base, neutral or salt form. Where appropriate, pharma-ceutically acceptable salts include acid addition salts, for example, those formed with free amino groups of the proteinaceous composition, or those formed with inorganic acids, for example, hydrochloric acid or phosphoric acid, or organic acids, for example, acetic acid, oxalic acid, tartaric acid or mandelic acid. Salts formed with free carboxyl groups can also be derived from inorganic bases, for example, sodium, potassium, ammonium, calcium or ferric hydroxide; or organic bases, for example, isopropylamine, trimethylamine, histidine or procaine. The formulated solution is administered in a therapeutically effective amount in a manner compatible with the dosage form. The formulation is easily administered in a variety of dosage forms, including those formulated for parenteral administration, such as injection solutions, aerosols for delivery to the lungs, or those formulated for oral administration, such as drug release capsules.

[0231] Further in accordance with the present disclosure, compositions of the present disclosure suitable for administration are provided in a pharma- ceutically acceptable carrier, with or without an inert diluent. The carrier should be absorbable, and includes liquid, semi-solid, i.e., paste, or solid carriers. Except where any conventional vehicle, agent, diluent or carrier is deleterious to the recipient or to the therapeutic effect of the composition contained therein, its use in administrable compositions for use in carrying out the methods of the present invention is appropriate. Examples of carriers or diluents include fats, oils, water, saline, lipids, liposomes, resins, binders, fillers, and the like, or combinations thereof. The compositions may also include various antioxidants to retard oxidation of one or more components. In addition, prevention of the action of microorganisms can be brought about by preservatives, such as various antibacterial and antifungal agents, including, but not limited to, parabens (e.g., methylparaben, propylparaben), chlorobutanol, phenol, sorbic acid, thimerosal, or combinations thereof.

[0232] In accordance with the present disclosure, the compositions are combined with the carrier by any convenient and practical method, i.e., solution, suspension, emulsion, mixing, encapsulation, absorption, etc. Such procedures are routine to those of ordinary skill in the art.

[0233] In a specific embodiment of the present disclosure, the composition is combined or thoroughly mixed with semi-solid or solid carrier.Mixing can be carried out in any convenient way, such as grinding.Stabilizers can also be added in the mixing step to protect the composition from loss of therapeutic activity, i.e., denaturation in the stomach.Examples of stabilizers for use in the composition include buffers, amino acids such as glycine and lysine, carbohydrates such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.

[0234] In further embodiments, the disclosure may relate to the use of pharmaceutical lipid vehicle compositions comprising compositions comprising NK cells and antibodies, and optionally an aqueous solvent. As used herein, the term "lipid" is defined to include any of a wide variety of substances that are characteristically insoluble in water and extractable with an organic solvent. This broad class of compounds is well known to those of skill in the art, and the term "lipid" as used herein is not limited to a particular structure. Examples include compounds that contain long chain aliphatic hydrocarbons and their derivatives. Lipids may be naturally occurring or synthetic (i.e., artificially designed or produced). However, lipids are typically biological substances. Biological lipids are well known in the art, and include, for example, neutral lipids, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulfatides, lipids with ether and ester-linked fatty acids, polymerizable lipids, and combinations thereof. Of course, compounds other than those specifically described herein that would be understood by those of skill in the art to be lipids are also encompassed by the compositions and methods of the present invention.

[0235] Those skilled in the art will be familiar with the various techniques that can be employed to disperse the composition in nanoparticles or lipid vehicles. For example, the composition comprising NK cells and antibodies can be dispersed in a solution comprising lipids, dissolved in lipids, emulsified in lipids, mixed with lipids, bound to lipids, covalently bound to lipids, contained as a suspension in lipids, contained or complexed with micelles or liposomes, or otherwise associated with lipids or lipid structures by any means known to those skilled in the art. The dispersion may or may not form liposomes.

[0236] The actual dosage of the composition of the present disclosure administered to an animal patient can be determined by physical and physiological factors such as body weight, severity of condition, type of disease to be treated, previous or concurrent therapeutic intervention, characteristics of the patient, and route of administration. Depending on the dosage and route of administration, the preferred dosage and / or the number of administrations of an effective amount can vary depending on the subject's response. The physician responsible for administration will in any case determine the concentration of active ingredient in the composition and the appropriate dosage for each individual subject.

[0237] In certain embodiments, the pharmaceutical composition may contain, for example, at least about 0.1% of the active compound. In other embodiments, the active compound may contain, for example, about 2% to about 75% of the weight of the unit, or about 25% to about 60%, and any range derivable therein. Of course, the amount of active compound in each therapeutically useful composition may be prepared so that an appropriate dosage is obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations are contemplated by those skilled in the art of preparing such pharmaceutical formulations, and as such, various dosages and treatment regimens may be desirable.

[0238] In other non-limiting examples, dosages can also include about 1 microgram / kg / body weight, about 5 micrograms / kg / body weight, about 10 micrograms / kg / body weight, about 50 micrograms / kg / body weight, about 100 micrograms / kg / body weight, about 200 micrograms / kg / body weight, about 350 micrograms / kg / body weight, about 500 micrograms / kg / body weight, about 1 milligram / kg / body weight, about 5 milligrams / kg / body weight, about 10 milligrams / kg / body weight, about 50 milligrams / kg / body weight, about 100 milligrams / kg / body weight, about 200 milligrams / kg / body weight, about 350 milligrams / kg / body weight, about 500 milligrams / kg / body weight, up to about 1000 mg / kg / body weight or more per administration, and any range derivable therein. Non-limiting examples of ranges that can be derived from the numerical values ​​described herein include ranges based on the above numerical values, such as about 5 mg / kg / body weight to about 100 mg / kg / body weight, about 5 micrograms / kg / body weight to about 500 milligrams / kg / body weight, etc.

[0239] Therapeutic compositions comprising NK cells and antibodies of the present disclosure may be administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In some embodiments, antibiotics are administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. Appropriate dosages may be determined based on the type of disease being treated, the severity and course of the disease, the clinical condition of the individual, the individual's clinical history and response to treatment, and the discretion of the attending physician.

[0240] The therapeutic agent may include various "unit doses". A unit dose is defined as containing a predetermined amount of the therapeutic composition. The amount to be administered, as well as the specific route and formulation, are within the judgement of those skilled in the art of clinical medicine. A unit dose need not be administered as a single injection, but may include continuous infusion over a period of time. In some embodiments, a unit dose includes a single administrable dose.

[0241] The dosage depends on the desired therapeutic effect, both in number of treatments and unit dose. Effective amount is understood to refer to the amount required to achieve a specific effect. In practice, it is contemplated that in certain embodiments, a dosage ranging from 10mg / kg to 200mg / kg can affect the protective capacity of these agents. Thus, dosages include about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200, 300, 400, 500, 1000 μg / kg, mg / kg, μg / day, or mg / day, or any range derivable therein. Furthermore, such dosages can be administered multiple times during the day and / or on multiple days, weeks, or months.

[0242] In certain embodiments, an effective amount of the pharmaceutical composition is one that can provide a blood level of about 1 μM to 150 μM. In other embodiments, an effective amount provides a blood level of about 4 μM to 100 μM; or about 1 μM to 100 μM; or about 1 μM to 50 μM; or about 1 μM to 40 μM; or about 1 μM to 30 μM; or about 1 μM to 20 μM; or about 1 μM to 10 μM; or about 10 μM to 150 μM; or about 10 μM to 100 μM; or about 10 μM to about 50 μM; or about 25 μM to about 150 μM; or about 25 μM to about 100 μM; or about 25 μM to about 50 μM; or about 50 μM to about 150 μM; or about 50 μM to about 100 μM (or any range derivable therein). In other embodiments, the dose is about, at least, or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, The blood concentration may be 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 μM, or any range derivable therein. In certain embodiments, the therapeutic agent administered to the subject is metabolized in the body to become a metabolized therapeutic agent, in which case the blood concentration may refer to the amount of the therapeutic agent. Alternatively, to the extent that the therapeutic agent is not metabolized by the subject, the blood concentration discussed herein may refer to the therapeutic agent that is not metabolized.

[0243] A. Nutritional Compositions and Formulations In certain embodiments of the present disclosure, the compositions comprising NK cells and antibodies are formulated to be administered via the gastrointestinal route. The gastrointestinal route includes all possible routes of administration in which the composition comes into direct contact with the gastrointestinal tract. Specifically, the pharmaceutical compositions disclosed herein can be administered orally, bucally, rectally, or sublingually. As such, these compositions can be formulated with an inert diluent or with an assimilable edible carrier, or can be enclosed in hard or soft shell gelatin capsules, or can be compressed into tablets, or can be directly incorporated into the diet.

[0244] In certain embodiments, the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like (Mathiowitz et al., 1997; Hwang et al., 1998; U.S. Pat. Nos. 5,641,515; 5,580,579 and 5,792,451, each of which is specifically incorporated herein by reference in its entirety). Tablets, troches, pills, capsules, etc. may also contain: binders, such as gum tragacanth, acacia, corn starch, gelatin, or combinations thereof; excipients, such as dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, or combinations thereof; disintegrating agents, such as corn starch, potato starch, alginic acid, or combinations thereof; lubricants, such as magnesium stearate; sweeteners, such as sucrose, lactose, saccharin, or combinations thereof; flavoring agents, such as peppermint, oil of wintergreen, cherry flavor, orange flavor, etc. When the dosage unit form is a capsule, it may contain a liquid carrier in addition to the above types of materials. Various other materials may be present as coatings or to modify the physical form of the dosage unit. For example, tablets, pills, or capsules may be coated with shellac, sugar, or both. When the dosage form is a capsule, it may contain a carrier, such as a liquid carrier, in addition to the above types of materials. Gelatin capsules, tablets, or pills may be enteric coated. Enteric coating prevents the composition from denaturing in the stomach or upper intestine where the pH is acidic. For example, see U.S. Patent No. 5,629,001. Upon reaching the small intestine, the basic pH there dissolves the coating, allowing the composition to be released and absorbed by specialized cells, such as epithelial intestinal cells and Peyer's patch M cells. Syrup of elixir may contain the active compound sucrose as a sweetener, methyl and propyl parabens as preservatives, dyes and flavors, such as cherry or orange flavor.Of course, any material used in preparing any dosage form should be pharma- ceutically pure and substantially non-toxic in the amounts used.Furthermore, the active compound may be incorporated into sustained-release preparations and formulations.

[0245] For oral administration, the composition of the present disclosure may alternatively be incorporated with one or more excipients in the form of a mouthwash, dentifrice, buccal tablet, oral spray, or sublingual oral administration formulation. For example, a mouthwash can be prepared by mixing the required amount of active ingredient in a suitable solvent, such as sodium borate solution (Dobell's solution). Alternatively, the active ingredient can be mixed into oral fluid, such as a solution containing sodium borate, glycerin, potassium bicarbonate, dispersed in a dentifrice, or added in a therapeutically effective amount to a composition containing water, binder, abrasive, flavoring agent, foaming agent, and humectant. Alternatively, the composition can be in the form of a tablet or solution and placed under the tongue or dissolved in the mouth.

[0246] Other suitable formulations for gastrointestinal administration include suppositories. Suppositories are solid dosage forms of various weights and shapes, usually medicated, for insertion into the rectum. After insertion, the suppository softens, melts or dissolves in the cavity fluid. In general, for suppositories, conventional carriers include, for example, polyalkylene glycols, triglycerides or combinations thereof. In certain embodiments, suppositories can be formed from mixtures containing, for example, about 0.5% to about 10%, preferably about 1% to about 2% of the active ingredient.

[0247] B. Parenteral Compositions and Formulations In further embodiments, the composition can be administered via parenteral route.As used herein, the term "parenteral" includes the route that bypasses the digestive tract.Specifically, the pharmaceutical composition disclosed herein can be administered, for example, intravenously, intradermally, intramuscularly, intraarterially, intravenously, intrathecally, intraventricularly, intratumorally, subcutaneously, or intraperitoneally, including, but not limited to, U.S. Patent No. 6,613,308; No. 5,466,468; No. 5,543,158; No. 5,641,515; and No. 5,399,363 (each of which is specifically incorporated herein in its entirety by reference).

[0248] Solutions of the active compounds as free bases or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. Pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Patent No. 5,466,468, specifically incorporated herein by reference in its entirety). In all cases, the form must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (i.e., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and / or vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferable to include an isotonic agent, for example, sugar or sodium chloride. Prolonged absorption of the injectable composition can be achieved by using an absorption-delaying agent, for example, aluminum monostearate or gelatin, in the composition.

[0249] For example, when administered parenterally in an aqueous solution, the solution should be suitably buffered, if necessary, and the liquid diluent should first be rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, the sterile aqueous media that can be employed will be known to those skilled in the art in light of this disclosure. For example, one dose can be dissolved in an isotonic NaCl solution and added to a subcutaneous injection solution or injected at the intended site of injection (see, for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Dosages will necessarily vary somewhat depending on the condition of the subject being treated. In any event, the person responsible for administration will determine the appropriate dose for the individual subject. Furthermore, when administered to humans, the formulations must meet the sterility, pyrogenicity, general safety and purity standards required by FDA Office of Biologics standards.

[0250] Sterile injectable solutions are prepared by incorporating the required amount of active compound into a suitable solvent, with various other ingredients as listed above as necessary, and then sterilizing by filtration.Generally, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle that contains a basic dispersion medium and other ingredients as listed above.For the preparation of sterile powders for sterile injectable solutions, the preferred preparation method is vacuum drying and freeze-drying technology, which allows the powder of active ingredients to be added to the desired additional ingredients from a previously sterile-filtered solution.Powder compositions are combined with liquid carriers, such as water or saline, with or without stabilizers.

[0251] C. Other Pharmaceutical Compositions and Formulations In other specific embodiments of the present disclosure, the active compound compositions comprising NK cells and antibodies may be formulated for administration via a variety of miscellaneous routes, for example, topical (i.e., transdermal), mucosal (intranasal, intravaginal, etc.) and / or inhalation.

[0252] Pharmaceutical compositions for topical administration may include active compounds formulated for medicinal use such as ointments, pastes, creams, or powders. Ointments include all oleaginous, adsorbent, emulsion, and water-soluble based compositions for topical application, while creams and lotions are compositions that contain only emulsion bases. Topical drugs may include penetration enhancers to promote adsorption of the active ingredient from the skin. Suitable penetration enhancers include glycerin, alcohol, alkyl methyl sulfoxides, pyrrolidones, and laurocapram. Bases that may be used for topical application include polyethylene glycol, lanolin, cold cream, petrolatum, and other suitable absorbent, emulsion, and water-soluble ointment bases. Topical formulations may also include emulsifiers, gelling agents, and antimicrobial preservatives as necessary to preserve the active ingredient and provide a homogenous mixture. Transdermal administration of the present invention may include the use of a "patch." For example, a patch may deliver one or more active agents continuously at a predetermined rate and for a period of time.

[0253] In certain embodiments, pharmaceutical compositions can be delivered by eye drops, intranasal sprays, inhalation, and / or other aerosol delivery vehicles.Methods of delivering compositions directly to the lungs via nasal aerosol sprays are described, for example, in U.S. Patent Nos. 5,756,353 and 5,804,212, each of which is specifically incorporated herein by reference in its entirety.Similarly, nasal microparticle resins (Takenaga et al., 1998) and lysophosphatidylglycerol compounds (U.S. Patent No. 5,725,871, specifically incorporated herein by reference in its entirety) are also well known in the pharmaceutical art.Similarly, transmucosal drug delivery in the form of polytetrafluoroethylene support matrix is ​​described in U.S. Patent No. 5,780,045, which is specifically incorporated herein by reference in its entirety.

[0254] The term aerosol refers to a colloidal system of finely divided solid or liquid particles dispersed in a liquefied or pressurized gas propellant. A typical aerosol of the present invention for inhalation may consist of a suspension of the active ingredient in a liquid propellant or a mixture of a liquid propellant and a suitable solvent. Suitable propellants include hydrocarbons and hydrocarbon ethers. Suitable containers may vary depending on the pressure requirements of the propellant. The administration of the aerosol may vary depending on the age, weight, severity of symptoms and response of the subject.

[0255] V. Combination Therapy In certain embodiments, the compositions and methods of the present embodiments include a cancer therapy in addition to the composition comprising the NK cells and the antibody. The additional therapy may be radiation therapy, surgery (e.g., lumpectomy and mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplant, nanotherapy, monoclonal antibody therapy, hormonal therapy, or a combination thereof. The additional therapy may take the form of adjuvant or neoadjuvant therapy.

[0256] In some embodiments, the additional therapy is administration of a small molecule enzyme inhibitor(s) or an anti-metastatic agent(s). In some embodiments, the additional therapy is administration of a side effect limiting agent (e.g., an agent intended to reduce the occurrence and / or severity of side effects of treatment, such as an anti-nausea agent). In some embodiments, the additional therapy is radiation therapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiation therapy and surgery. In some embodiments, the additional therapy is gamma irradiation. In some embodiments, the additional therapy is a therapy targeting the PBK / ACT / mTOR pathway, an HSP90 inhibitor, a tubulin inhibitor, an apoptosis inhibitor, and / or a chemopreventive agent. The additional therapy may be one or more chemotherapeutic agents known in the art.

[0257] Immune cell therapy (in addition to the disclosed compositions) can be administered before, during, after, or in various combinations with additional cancer therapy, such as immune checkpoint therapy. Administration can occur simultaneously, minutes, days, or weeks apart. In embodiments where immune cell therapy is provided to a patient separately from the disclosed composition(s), one generally ensures that no significant period of time passes between the times of each administration, so that the two compounds can still exert their beneficially combined effect on the patient. In such cases, it is contemplated that the immunotherapy and the disclosed compositions will be provided to the patient within about 12-24 hours or 72 hours of each other, more particularly within about 6-12 hours of each other. In some circumstances, it may be desirable to significantly extend the time period for treatment, such that days (2, 3, 4, 5, 6, or 7) to weeks (1, 2, 3, 4, 5, 6, 7, or 8) pass between the respective administrations.

[0258] Administration of any compound or treatment of the present embodiments to a patient may follow general protocols for administration of such compounds, taking into account the toxicity, if any, of the agent. Thus, in some embodiments, there is a step of monitoring for toxicity resulting from the combination therapy.

[0259] A. Chemotherapy A wide variety of chemotherapeutic agents can be used in accordance with this embodiment. The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapeutic agent" is used to refer to a compound or composition administered in the treatment of cancer. These agents or drugs are classified according to their mode of activity within the cell, for example, whether and at what stage they affect the cell cycle. Alternatively, agents can be characterized based on their ability to directly crosslink DNA, to intercalate into DNA, or to induce chromosomal or mitotic abnormalities by affecting nucleic acid synthesis.

[0260] Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa and uredopa; ethylenimines and methylamines such as altretamine, triethylenemelamine, triethylenephosphoramide, triethyleneethylethiophosphoramide and trimethylolmelamine; acetogenins (particularly bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin and bizelesin); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycins (synthetic analogs , KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictine; spongiostatins; nitrogen mustards such as chlorambucil, chlornaphazine, colofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembitine, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimnustine; antibiotics such as the enediyne antibiotics (e.g. calicheamicin, especially calicheamicin gamma II and calicheamicin omega II); dynemicins such as dynemicin A; bisphosphonates such as clodronate; esperamicin;and neocarzinostatin chromophore and related chromoprotein enediine antibiotic chromophores, aclacinomycin, actinomycin, ausularisin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, deoxydoxorubicin). ), mitomycins such as epirubicin, esorubicin, idarubicin, marcelomycin, and mitomycin C, mycophenolic acid, nogalarnisin, olivomycin, peplomycin, potfilomycin, puromycin, keramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; metabolic inhibitors such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, pteropterin, and trimetrexate; fludarabine, 6-mercapto Purine analogues such as toprine, thiamiprine, and thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; antiadrenergic drugs such as mitotane and trilostane; folic acid supplements such as floric acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; enyluracil; amsacrine ;Bestravcil;Bisantrene;Edatraxate;Defofamine;Demecolcine;Diazicon;Elformitin;Elliptinium acetate;Epothilone;Etoglucide;Gallium nitrate;Hydroxyurea;Lentinan;Lonidynin;Maytansinoids such as maytansine and ansamitocin;Mitoguazone;Mitoxantrone;Mopidammol;Nitraerin;Pentostatin;Fenamet;Pirarubicin;Rosoxantrone;Podophyllic acid;2-Ethylhydrazide;Procarbazine;PSK polysaccharide complex;Razoxane;Rhizoxin;Schizofiran;Spirogermanium;Tenuazonic acid;Triazicon;2,2',2''-Trichlorotriethylamine;Tricothecenes (especially T-2 toxin, veraculin A, roridin A and anguidine);Urethane;Vindesine;Dacarbazine;Mannomustine;Mitobronitol;Mitolactol;Pipobroman;Gasitosine;Arabinoside ("Ara-C");Cyclophosphamide;Taxoids such as paclitaxel and docetaxel;Gemcitabine;6-Thioguanine;Mercaptopurine;Platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin;Vinblastine;Platinum;Etoposide (VP -16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitors RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabine, navelbine, farnesyl-protein transferase inhibitors, transplatinum, and pharmaceutically acceptable salts, acids, or derivatives of any of the above.;

[0261] B. Radiation therapy Other agents that cause DNA damage and have been used extensively include what are commonly known as gamma radiation, X-rays, and / or the directed delivery of radioisotopes to tumor cells. Other DNA damaging agents are also possible, such as microwaves, proton beam irradiation (U.S. Patents Nos. 5,760,395 and 4,870,287), and ultraviolet radiation. All of these agents likely cause widespread damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. X-ray doses range from daily doses of 50-200 roentgens over prolonged periods (3-4 weeks) to single doses of 2000-6000 roentgens. Dose ranges for radioisotopes vary and depend on the half-life of the isotope, the strength and type of radiation, and uptake by tumor cells.

[0262] C. Immunotherapy One of skill in the art will appreciate that additional immunotherapies (other than the disclosed cellular therapies) may be used in combination or in conjunction with the methods of the embodiments.

[0263] In certain embodiments, the additional immunotherapy comprises administering one or more antibodies to the subject. In certain embodiments, the additional one or more antibodies administered to the subject may be the same one or more antibodies that are loaded onto the NK cells. In some embodiments, the additional one or more antibodies administered to the subject may target one or more of the same antigens as the one or more antibodies that are loaded onto the NK cells.

[0264] In the context of cancer therapy, immunotherapeutics generally rely on the use of immune effector cells or molecules to target and destroy cancer cells. Rituximab (RITUXAN®) is an example. Immune effectors are, for example, antibodies specific for tumor cell surface markers. Antibodies may function alone as therapeutic effectors or may recruit other cells to actually affect cell killing. Antibodies may also be conjugated to drugs or toxins (chemotherapeutic agents, radionuclides, ricin A chain, cholera toxin, pertussis toxin, etc.) and function as targeting agents. Alternatively, effectors may be lymphocytes with surface molecules that directly or indirectly interact with tumor cell targets. Various effector cells include cytotoxic T cells and NK cells, other than cells with knockdown or knockout of TGF-βR2.

[0265] Antibody-drug conjugates have emerged as a breakthrough approach in the development of cancer therapeutics. Antibody-drug conjugates (ADCs) are monoclonal antibodies (MAbs) covalently linked to cytotoxic drugs. This approach combines the high specificity of MAbs for antigen targets with potent cytotoxic agents, resulting in “armed” MAbs that deliver the payload (drug) to tumor cells where the antigen concentration is enriched. Targeted delivery of drugs also minimizes drug exposure in normal tissues, reducing toxicity and improving the therapeutic index. The FDA approval of two ADC drugs, ADCETRIS® (brentuximab vedotin) in 2011 and KADCYLA® (trastuzumab emtansine or T-DM1) in 2013, has proven this approach effective. Currently, there are more than 30 ADC drug candidates in various stages of clinical trials for cancer treatment (Leal et al., 2014). As antibody engineering and linker-payload optimization become increasingly mature, the discovery and development of new ADCs increasingly relies on the identification and validation of new targets amenable to this approach and the generation of targeted MAbs. Two criteria for ADC targets are increased / high levels of expression in tumor cells and robust internalization.

[0266] In one aspect of immunotherapy, the tumor cells must have some marker suitable for targeting, i.e., not present on the majority of other cells. Many tumor markers exist, any of which are suitable for targeting in the context of this embodiment. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, laminin receptor, erb B, p155, and the like. Another aspect of immunotherapy is to combine anti-cancer and immune stimulatory effects. There are also immune stimulatory molecules, including cytokines such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, chemokines such as MIP-1, MCP-1, IL-8, growth factors such as FLT3 ligand, and the like.

[0267] Examples of immunotherapies currently under investigation or in use include immune adjuvants, such as Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds (U.S. Pat. Nos. 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998); cytokine therapy, such as all types of interferon, IL-1, GM-CSF, and TNF (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998); gene therapy, such as TNF, IL-1, IL-2, and p53 (Qin et al., 1998; Austin-Ward and Villaseca, 1998; U.S. Patent Nos. 5,830,880 and 5,846,945); and monoclonal antibodies, such as anti-CD20, anti-ganglioside GM2, and anti-p185 (Hollander, 2012; Hanibuchi et al., 1998; U.S. Patent No. 5,824,311). It is contemplated that one or more anti-cancer drug therapies may be employed in conjunction with the antibody therapies described herein.

[0268] In some embodiments, the immunotherapy is an immune checkpoint inhibitor. Immune checkpoints either raise or lower signals (e.g., costimulatory molecules). Inhibitory immune checkpoints that can be targeted by immune checkpoint inhibitors include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuating factor (BTLA), cytotoxic T lymphocyte-associated protein 4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin (KIR), lymphocyte activation gene-3 (LAG3), programmed death 1 (PD-1), T cell immunoglobulin domain and mucin domain 3 (TIM-3), and V domain Ig suppressor of T cell activation (VISTA). In particular, immune checkpoint inhibitors target the PD-1 axis and / or CTLA-4.

[0269] D.Surgery Approximately 60% of cancer patients undergo some type of surgery, including preventive surgery, diagnostic or staging surgery, curative surgery, and palliative surgery. Curative surgery includes resection surgery, which physically removes, excises, and / or destroys all or part of the cancerous tissue, and may be combined with other therapies, such as the present treatment, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies. Tumor resection refers to the physical removal of at least a portion of the tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs' surgery).

[0270] When cancer cells, tissue, or tumors are partially or completely removed, a cavity may form in the body. Treatment can be by perfusion, direct injection, or local application of anticancer therapy to the site. Such treatments can be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, or 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments are also available in a variety of dosages.

[0271] E. Other drugs It is contemplated that other agents may be used in combination with certain aspects of the present embodiment to improve the efficacy of the treatment. These additional agents include agents that affect the upregulation of cell surface receptors and GAP junctions, cell activators and differentiation promoters, cell adhesion inhibitors, agents that increase the sensitivity of hyperproliferative cells to apoptosis inducers, or other biological agents. Increasing intercellular signaling by increasing the number of GAP junctions will increase the anti-hyperproliferative effect on adjacent hyperproliferative cell populations. In other embodiments, cell activators or differentiation promoters may be used in combination with certain aspects of the present embodiment to improve the anti-hyperproliferative effect of the treatment. Cell adhesion inhibitors are contemplated to improve the efficacy of the present embodiment. Examples of cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and lovastatin. It is further contemplated that other agents that increase the sensitivity of hyperproliferative cells to apoptosis, such as the antibody c225, may be used in combination with certain aspects of the present embodiment to improve the efficacy of the treatment.

[0272] VI. Kits of the Disclosure In some embodiments, kits are provided that may include, for example, NK cells, optionally one or more media and components for producing NK cells, one or more antibodies or reagent(s) for producing antibodies, and the like. In some embodiments, the formulation may include a cocktail of factors including in a form suitable for combination with the NK cells. The reagent system or any kit components may be packaged, as appropriate, either in aqueous medium or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means into which the components may be placed, and preferably suitably dispensed. Where more than one component is included in the kit, the kit will generally also include a second, third or other additional container into which the additional components may be placed separately. However, combinations of various components may also be included in a vial. The components of the kit may also be provided as a dry powder. Where reagents and / or components are provided as a dry powder, the powder may be reconstituted by the addition of a suitable solvent. It is also contemplated that the solvent may be provided in another container means. The kits will also typically include a means for containing the kit components in close confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are retained. The kit may also include instructions for use, such as in printed or electronic, such as digital, format.

[0273] In specific embodiments, the kit may include one or more cytokines, including at least IL-12, IL-15, IL-18, and / or IL-2, at specific concentrations as described elsewhere herein. The kit may include any type of culture medium, any components of cryopreservation medium, as described elsewhere herein. The kit may include cord blood (including pooled cord blood), any type of antigen presenting cells, beads for depleting specific NK cells (as described herein), vectors encoding one or more proteins described herein, NK cells, antibodies or reagents for generating antibodies, etc.

[0274] Individual components may also be provided in the kit in concentrated amounts. In some embodiments, components are provided individually at the same concentration as they are in solution with other components. The concentrations of components may be provided as 1x, 2x, 5x, 10x, or 20x or more.

[0275] VII. Aspects The following embodiments describe certain inventions described herein. Embodiment 1. A composition comprising: (i) one or more engineered natural killer (NK) cells comprising one or more transgenes; and (ii) one or more antibody molecules, wherein (a) the antibody is monospecific, such that an Fc region of the monospecific antibody binds to the engineered NK cell and an antigen binding domain of the monospecific antibody is capable of binding to a target antigen; or (b) the antibody is multispecific, such that one or more antigen binding domains of the antibody bind to a target antigen and other antigen binding domain(s) bind to other target antigens or other antigen binding domain(s) of the antibody are capable of binding to an NK cell surface antigen.

[0276] Embodiment 2. The composition of embodiment 1, wherein the engineered NK cells are expanded or not expanded.

[0277] Embodiment 3. The composition according to embodiment 1 or 2, wherein the engineered NK cells are pre-activated or not pre-activated.

[0278] Embodiment 4. The composition according to any one of embodiments 1 to 3, wherein the multispecific antibody is bispecific, trispecific, or multispecific.

[0279] The composition according to any one of aspects 1 to 4, wherein in aspect 5.a), the composition is further defined as a complex between the engineered NK cell and the monospecific antibody via binding between an Fc region of the monospecific antibody and the engineered NK cell.

[0280] Embodiment 6. The composition according to any one of embodiments 1 to 5, wherein the complex further comprises an antigen-binding domain of a monospecific antibody bound to the target antigen.

[0281] In embodiment 7.b), the composition according to any one of embodiments 1 to 4, wherein the composition is further defined as a complex of the engineered NK cell with a multispecific antibody via binding of antigen binding domain(s) of the multispecific antibody which binds to surface antigen(s) on the engineered NK cell.

[0282] Embodiment 8. The composition according to any one of embodiments 1 to 4 and 7, wherein one or more antigen-binding domain(s) of the antibody binds to a target antigen.

[0283] Aspect 9. The target antigen is CD19, CD319 (CS1), ROR1, CD20, CD22, CD70, carcinoembryonic antigen, alpha fetoprotein, CA-125, MUC-1, epidermal growth factor receptor (EGFR), epithelial tumor antigen, melanoma associated antigen, mutated p53, mutated ras, HER2 / Neu, ERBB2, folate binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, CD5, CD123, CD2 3. The composition of any one of aspects 1-6, wherein the stem cell antigen, autoantigen, or cancer antigen is selected from the group consisting of CD30, CD38, CD56, CD70, c-Met, mesothelin, GD3, HERV-K, IL-11Rα, κ chain, λ chain, CSPG4, ERBB2, WT-1, TRAIL / DR4, VEGFR2, CD33, CD47, CLL-1, U5snRNP200, CD200, BAFF-R, BCMA, CD99, HLA-G, Trop2, and combinations thereof.

[0284] Embodiment 10. The composition according to any one of embodiments 1 to 9, wherein the engineered NK cell surface antigen is CD16, CD32, CS1, CD56, CD64, NKG2D, NKG2C, DNAM, 2B4, CD2, NCR, NKp30, NKp44, NKp46, or KIR.

[0285] Embodiment 11. The composition according to any one of embodiments 1 to 9, wherein the NK cells are sourced from umbilical cord blood, and the source of the cord blood is cord blood from one donor or cord blood pooled from two or more individual cord blood units.

[0286] Embodiment 12. The composition of embodiment 11, wherein the CB is pooled from 3, 4, 5, 6, 7, or 8 individual cord blood units.

[0287] Embodiment 13. The composition according to any one of embodiments 1 to 12, wherein the engineered NK cells are derived from umbilical cord blood mononuclear cells, from umbilical cord blood hematopoietic stem cells, from iPSCs, from peripheral blood NK cells, or from an NK cell line such as NK-92.

[0288] Embodiment 14. The composition according to any one of embodiments 1 to 13, wherein the engineered NK cells are CD56+, CD3-, or both.

[0289] Aspect 15. The composition according to any one of aspects 1 to 14, wherein the composition is used fresh or is cryopreserved.

[0290] Embodiment 16. The composition according to any one of embodiments 1 to 15, wherein the source of the engineered NK cells is a fresh source or a frozen storage repository.

[0291] Embodiment 17. The composition according to embodiment 15 or 16, wherein if the engineered NK cells are provided from cryopreservation, the engineered NK cells have been cryopreserved in a medium comprising at least one cryoprotectant, at least one serum or a non-serum alternative to serum, and optionally at least one cytokine and / or at least one growth factor.

[0292] Embodiment 18. The composition according to embodiment 17, wherein the cryoprotectant is dimethyl sulfoxide (DMSO), glycerin, glycerol, hydroxyethyl starch, dextran trehalose, or a combination thereof.

[0293] Embodiment 19. The composition according to embodiment 17 or 18, wherein the non-serum substitute comprises a platelet lysate and / or a blood product lysate, or human or animal serum albumin.

[0294] Embodiment 20. The composition according to any one of embodiments 17 to 19, wherein at least one cytokine is a natural protein, a recombinant protein, a synthetic protein, or a mixture thereof.

[0295] Embodiment 21. The composition according to any one of embodiments 17 to 20, wherein the at least one cytokine is interleukin (IL)-1, IL-2, IL-3, IL-4, IL-6, IL-7, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL-22, IL-23, interferon, tumor necrosis factor, stem cell factor, FLT3-ligand, APRIL, thrombopoietin, erythropoietin, or a combination thereof.

[0296] Embodiment 22. The composition according to any one of embodiments 1 to 21, wherein the engineered NK cells comprise one or more transgenes encoding one or more engineered receptors.

[0297] Embodiment 23. The composition according to embodiment 22, wherein the one or more engineered receptors are chimeric antigen receptors, T cell receptors, receptors that recognize the Fc portion of Ig (e.g., CD16, CD32, CD64, etc.), chemokine receptors, homing receptors, cytokine genes, chimeric cytokine receptors, or any combination thereof.

[0298] Embodiment 24. The composition according to any one of embodiments 1 to 23, wherein the engineered NK cells express one or more transgenes encoding one or more heterologous cytokines and / or cytokine receptors.

[0299] Embodiment 25. The composition according to embodiment 24, wherein the one or more heterologous cytokines are IL-2, IL-4, IL-7, IL-12, IL-15, IL-18, IL-21 and / or IL-23.

[0300] Embodiment 26. The composition according to any one of embodiments 1 to 25, wherein the engineered NK cells express one or more transgenes encoding one or more receptors for enhancing binding to the antibody.

[0301] Embodiment 27. The composition according to embodiment 26, wherein the one or more receptors comprise or are Fc receptors.

[0302] Embodiment 28. The composition according to embodiment 26, wherein the receptor is CD16, CD32, CD64, or a combination thereof.

[0303] Embodiment 29. The composition according to any one of embodiments 1 to 28, wherein the engineered NK cells express one or more transgenes encoding one or more suicide genes.

[0304] Embodiment 30. The composition according to any one of embodiments 1 to 29, wherein the composition is contained in a solution or solid comprising one or more cryoprotectants.

[0305] Embodiment 31. The composition according to any one of embodiments 1 to 30, wherein the composition is contained in a pharma- ceutically acceptable carrier.

[0306] Embodiment 32. A method of producing a composition of any one of embodiments 1 to 31, comprising: (i) optionally expanding the engineered NK cells in a culture comprising an effective amount of: (a) a cytokine selected from the group consisting of IL-2, IL-15, IL-18, IL-21, and combinations thereof; and (b) an antigen presenting cell / feeder, a fragment of an antigen presenting cell / feeder, or an NK cell activating bead; and (ii) providing an antibody molecule to the engineered NK cells, whereby if expanded, the antibody molecule is provided to the engineered NK cells before and / or after expansion.

[0307] Embodiment 33. The method according to embodiment 32, wherein the method comprises a preactivation step before and / or after the expansion step, in which the engineered NK cells are preactivated in a culture comprising one or more of IL-2, IL-12, IL-15, and IL-18 at effective concentrations.

[0308] Embodiment 34. The method of embodiment 33, wherein the culture comprises effective concentrations of two or more of IL-2, IL-12, IL-15, and IL-18.

[0309] Embodiment 35. The method of embodiment 33 or 34, wherein the culture comprises effective concentrations of three or more of IL-2, IL-12, IL-15, and IL-18.

[0310] Embodiment 36 The method according to any one of embodiments 32 to 35, wherein the culture comprises effective concentrations of IL-12, IL-15, and IL-18.

[0311] Embodiment 37. The method according to any one of embodiments 32 to 36, wherein IL-12 is utilized instead of IL-15 in the culture.

[0312] Embodiment 38. The method according to any one of embodiments 32 to 37, wherein the providing step is further defined as culturing the engineered NK cells and the antibody molecule for a specific duration or combining the engineered NK cells and the antibody molecule immediately prior to infusion.

[0313] Aspect 39. The method of aspect 38, wherein the duration is from about 5 minutes to about 24 hours or more.

[0314] Embodiment 40. The method according to embodiment 38 or 39, wherein the culture comprises Plasma-Lyte A and / or human serum albumin.

[0315] Embodiment 41. The method according to any one of embodiments 32 to 40, wherein after culturing, the composition is injected into the recipient subject without first washing.

[0316] Embodiment 42. The method according to any one of embodiments 32 to 40, wherein after culturing, the composition is injected into the recipient subject after one or more washes.

[0317] Embodiment 43. The method according to any one of embodiments 32 to 42, wherein the engineered NK cells deplete CD3+, CD14+ and / or CD19+ cells.

[0318] Embodiment 44. A method according to embodiment 43, wherein the depletion step is performed before the pre-activation step, and / or before expansion with feeder cells and / or NK cell activation beads, and / or before culture with one or more cytokines, and / or before infusion.

[0319] Embodiment 45. The method according to any one of embodiments 32 to 44, further comprising the step of obtaining NK cells from umbilical cord blood, wherein the umbilical cord blood does not contain umbilical cord tissue.

[0320] Embodiment 46. The method according to any one of embodiments 32 to 45, wherein the antigen presenting cell is artificial (aAPC).

[0321] The method of embodiment 46, wherein the aAPC expresses CD137 ligand.

[0322] Embodiment 48 The method according to embodiment 46 or 47, wherein the aAPC further expresses a membrane-bound cytokine.

[0323] Embodiment 49. The method of embodiment 48, wherein the membrane-bound cytokine is membrane-bound IL-21 (mIL-21) or membrane-bound IL-15 (mIL-15).

[0324] Embodiment 50. A method according to any one of embodiments 46 to 49, wherein the aAPCs have essentially no expression of endogenous HLA class I, II, or CD1d molecules.

[0325] Embodiment 51. A method according to any one of embodiments 46 to 50, wherein the aAPC expresses ICAM-1 (CD54) and / or LFA-3 (CD58) or CD48.

[0326] Embodiment 52. The method according to any one of embodiments 46 to 51, wherein the aAPCs are further defined as leukemic cell-derived aAPCs.

[0327] Embodiment 53. The method of embodiment 52, wherein the leukemia cell-derived aAPCs are K562 cells engineered to express CD137 ligand and / or mIL-21.

[0328] Embodiment 54 The method of embodiment 53, wherein the K562 cells are engineered to express CD137 ligand and mIL-21.

[0329] Embodiment 55. A method according to any one of embodiments 46 to 54, wherein the aAPCs are engineered by retroviral transduction.

[0330] Embodiment 56. A method according to any one of embodiments 46 to 55, wherein the aAPCs are irradiated.

[0331] Embodiment 57. The method according to any one of embodiments 32 to 56, wherein the preactivation step is for 10 to 20 hours.

[0332] Embodiment 58. The method according to any one of embodiments 32 to 57, wherein the preactivation step is for 14 to 18 hours.

[0333] Embodiment 59. The method according to any one of embodiments 32 to 58, wherein the preactivation step is for 16 hours.

[0334] Embodiment 60. The method according to any one of embodiments 32 to 59, wherein the culture for the preactivation step comprises IL-18 and / or IL-15 at a concentration of 1 to 1000 ng / mL.

[0335] Embodiment 61. The method according to any one of embodiments 32 to 60, wherein the culture for the preactivation step comprises IL-18 and / or IL-15 at a concentration of 1 to 1000 ng / mL.

[0336] Embodiment 62. The method according to any one of embodiments 32 to 61, wherein the culture for the preactivation step comprises IL-18 and / or IL-15 at a concentration of 1 to 1000 ng / mL.

[0337] Embodiment 63. A method according to any one of embodiments 32 to 62, wherein the culture for the preactivation step contains IL-12 at a concentration of 0.1 to 1000 ng / mL.

[0338] Embodiment 64. The method according to any one of embodiments 32 to 63, wherein the culture for the preactivation step contains IL-12 at a concentration of 1 to 1000 ng / mL.

[0339] Embodiment 65. The method according to any one of embodiments 32 to 64, wherein the culture for the preactivation step contains IL-12 at a concentration of 10 ng / mL.

[0340] Embodiment 66. The method according to any one of embodiments 32 to 65, further comprising washing the preactivated engineered NK cells before and / or after the expansion step.

[0341] Embodiment 67. The method according to any one of embodiments 32 to 66, wherein the engineered NK cells are activated with IL-12, IL-15, IL-18, IL-2, or any combination thereof at least two or more times during the expansion step.

[0342] Embodiment 68. The method according to embodiment 67, wherein washing is performed multiple times.

[0343] Embodiment 69. The method according to any one of embodiments 34 to 68, wherein the growth is for 5 to 60 days.

[0344] Embodiment 70. The method according to any one of embodiments 32 to 69, wherein the growth is for 12 to 16 days.

[0345] Embodiment 71. The method according to any one of embodiments 32 to 69, wherein the growth is for 18 to 24 days.

[0346] Embodiment 72. The method according to any one of embodiments 32 to 71, wherein the preactivated engineered NK cells and aAPCs are present in the expansion culture in a ratio of 3:1 to 1:3.

[0347] Embodiment 73. The method of embodiment 72, wherein the preactivated engineered NK cells and aAPCs are present in the expansion culture in a ratio of 1:2.

[0348] Embodiment 74. The method according to any one of embodiments 32 to 73, wherein the expansion culture further comprises IL-2.

[0349] Embodiment 75. The method according to embodiment 74, wherein IL-2 is present at a concentration of 10 to 500 U / mL.

[0350] Embodiment 76. The method according to embodiment 75, wherein IL-2 is present at a concentration of 100 to 300 U / mL.

[0351] Embodiment 77. The method of embodiment 76, wherein IL-2 is present at a concentration of 200 U / mL.

[0352] Embodiment 78. A method according to any one of embodiments 32 to 77, wherein the IL-12, IL-18, IL-15, and / or IL-2 are recombinant.

[0353] Embodiment 79. A method according to any one of embodiments 32 to 78, wherein IL-2 is replenished in the expansion culture every 2 to 3 days.

[0354] Embodiment 80. A method according to any one of embodiments 32 to 79, wherein APCs are added to the expansion culture at least a second time.

[0355] Embodiment 81. The method according to any one of embodiments 32 to 80, wherein one or more steps of the method are carried out in serum-free medium.

[0356] Embodiment 82. A method for treating a disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of a composition according to any one of embodiments 1 to 31.

[0357] Embodiment 83. The method of embodiment 82, wherein the disease or disorder is cancer, inflammation, graft-versus-host disease, transplant rejection, an autoimmune disease, an immunodeficiency disease, a B-cell malignancy, or an infectious disease.

[0358] Embodiment 84. The method according to embodiment 82 or 83, wherein the cancer is a blood cancer or a solid cancer.

[0359] Embodiment 85. The method of embodiment 84, wherein the hematological cancer is a leukemia selected from the group consisting of acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), multiple myeloma, acute myelogenous leukemia (AML), and chronic myelogenous leukemia (CML).

[0360] Embodiment 86 The method according to any one of embodiments 82 to 85, wherein the engineered NK cells are allogeneic with respect to the subject.

[0361] Embodiment 87. The method according to any one of embodiments 82 to 86, wherein the engineered NK cells are autologous with respect to the subject.

[0362] Embodiment 88. The method according to embodiment 82, wherein the disorder is graft-versus-host disease (GVHD).

[0363] Embodiment 89. The method of embodiment 82, wherein the disorder is multiple sclerosis, inflammatory bowel disease, rheumatoid arthritis, type I diabetes, systemic lupus erythematosus, contact hypersensitivity, asthma or Sjogren's syndrome.

[0364] Embodiment 90. The method according to any one of embodiments 82 to 89, wherein the subject is a human.

[0365] Embodiment 91. The method of any one of embodiments 82 to 90, further comprising administering to the subject at least a second therapeutic agent.

[0366] Embodiment 92. The method of embodiment 91, wherein the at least second therapeutic agent is a therapeutically effective amount of one or more anti-cancer agents, one or more immunomodulatory agents, and / or one or more immunosuppressive agents.

[0367] Aspect 93. The method according to aspect 92, wherein the anti-cancer agent is chemotherapy, radiation therapy, gene therapy, surgery, hormonal therapy, anti-angiogenic therapy or immunotherapy.

[0368] Embodiment 94. The method of embodiment 92, wherein the immunosuppressant is a calcineurin inhibitor, an mTOR inhibitor, an antibody, a chemotherapeutic agent, radiation, a chemokine, an interleukin, or an inhibitor of a chemokine or an interleukin.

[0369] Embodiment 95. The method according to any one of embodiments 91 to 94, wherein the composition and / or at least the second therapeutic agent is administered intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intrainvasively, percutaneously, subcutaneously, topically, or by direct injection or perfusion.

[0370] Embodiment 96. The method according to any one of embodiments 91 to 95, wherein the second therapeutic agent is an antibody.

[0371] Embodiment 97. The method according to embodiment 96, wherein the antibody is a monoclonal antibody, a bispecific antibody, or a multispecific antibody.

[0372] Embodiment 98. A method of treating a subject having cancer with engineered NK cells comprising: a) optionally stimulating a population of engineered NK cells by pre-activation with a cytokine cocktail; b) optionally expanding the engineered NK cells; and ci) loading the engineered NK cells with an antibody ex vivo and then administering the loaded engineered NK cells to the subject, or cii) loading the engineered NK cells with an antibody in vivo by administering the engineered NK cells within one hour of administration of the antibody; wherein the engineered NK cells comprise one or more transgenes, and wherein the loading comprises exposure of the engineered NK cells to the antibody for a time effective for binding of the antibody to the surface of the engineered NK cells.

[0373] Embodiment 99. The method of embodiment 98, wherein loading of the engineered NK cells comprises exposure of the engineered NK cells to the antibody for about 1 hour or more, or about 1 hour or less.

[0374] Embodiment 100. The method of embodiment 98 or 99, wherein the plurality of engineered NK cells remains loaded with the antibody for 5 days or more.

[0375] Embodiment 101. A method according to any one of embodiments 98 to 100, wherein the antibody is bound to the artificial NK cell via interaction with the CD16 surface protein.

[0376] Embodiment 102. The method according to any one of embodiments 98 to 101, wherein the engineered NK cells are derived from umbilical cord blood.

[0377] Embodiment 103. The method according to any one of embodiments 98 to 102, wherein the engineered NK cells are activated ex vivo.

[0378] Embodiment 104. The method according to any one of embodiments 98 to 103, wherein the engineered NK cells are preactivated with IL-18, IL-15, and IL-12.

[0379] Embodiment 105. A method according to any one of embodiments 98 to 104, wherein the antibody is an anti-EGFR antibody.

[0380] Embodiment 106. A method according to any one of embodiments 98 to 104, wherein the antibody is an anti-EGFR2 antibody.

[0381] Embodiment 107. The method according to any one of embodiments 98 to 104, wherein the antibody is a monospecific antibody.

[0382] Embodiment 108. The method according to embodiment 105, wherein the antibody is imgatuzumab.

[0383] Embodiment 109. The method according to embodiment 106, wherein the antibody is margetuximab.

[0384] Embodiment 110. The method according to any one of embodiments 98 to 104, wherein the antibody is a bispecific antibody.

[0385] Embodiment 111. The method according to embodiment 110, wherein the antibody is amivantamab.

[0386] Embodiment 112. A method according to any one of embodiments 98 to 104, wherein the antibody is an anti-CD123 antibody.

[0387] Embodiment 113. The method according to any one of embodiments 98 to 104, wherein the antibody is a bispecific antibody.

[0388] Embodiment 114. The method according to any one of embodiments 98 to 104, wherein the antibody comprises an antigen-binding domain targeting CD123 and an antigen-binding domain targeting NKp46.

[0389] Embodiment 115. The method according to embodiment 105, wherein the antibody is IPH61.

[0390] Embodiment 116. The method according to any one of embodiments 98 to 115, wherein the one or more transgenes comprises an engineered receptor.

[0391] Embodiment 117. The method of embodiment 116, wherein the engineered receptor comprises a chimeric antigen receptor (CAR), a T cell receptor, a chemokine receptor, a chimeric cytokine receptor, or any combination thereof.

[0392] Embodiment 118. The method according to embodiment 116 or 117, wherein the engineered receptor comprises a CAR.

[0393] Embodiment 119. The method according to any one of embodiments 98 to 118, wherein the one or more transgenes comprises a cytokine.

[0394] Embodiment 120. The method of embodiment 119, wherein the cytokine is IL-2, IL-12, IL-15, IL-18, and / or IL-21.

[0395] Embodiment 121 The method according to any one of embodiments 98 to 120, wherein the engineered NK cell comprises an engineered mutation in an endogenous gene.

[0396] Embodiment 122. The method of embodiment 121, wherein the engineered mutation in the endogenous gene is in the CISH, CD38, glucocorticoid receptor, and / or TGFBR2 gene. EXAMPLES

[0397] The following examples are included to demonstrate preferred embodiments of the invention. Those skilled in the art should understand that the techniques disclosed in the examples that follow represent techniques that the inventors have discovered to work well in the practice of the invention, and therefore can be considered to constitute preferred modes for its practice. However, those skilled in the art should understand in light of this disclosure that many changes can be made in the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the invention.

[0398] Example 1 - Preparation and Use of Compositions A composition comprising specific engineered NK cells and also an antibody capable of binding to the engineered NK cells is prepared and, in certain cases, administered in an effective amount to an individual in need of treatment. In certain embodiments, an effective amount of engineered NK cells that are autologous or allogeneic with respect to the individual is administered to the individual, and the engineered NK cells are particularly umbilical cord blood cells. The engineered NK cells may or may not be sourced from a cryopreservation repository, and in some cases have been cryopreserved in a specific cryopreservation medium. The engineered NK cells are optionally preactivated and optionally expanded before exposing an effective amount of the engineered NK cells to an antibody under conditions in which the antibody can bind to the engineered NK cells via a suitable antigen-binding fragment and / or Fc domain. An effective amount of the composition is then provided to the individual in need thereof.

[0399] Example 2 - Monoclonal antibodies and NK cells This example includes the preparation and use of NK cells preloaded with antibodies. Figure 1 shows an example of an experimental procedure for NK cell expansion and antibody loading. NK cells can be obtained from processed blood, and in a particular embodiment, specific NK cells are isolated, such as those that are CD3- CD56+. Optionally, the NK cells are expanded, for example, in the presence of IL-2 and universal antigen presenting cells or other feeder cells, and the expansion step can be for any suitable period of time, such as up to about 14 days in a particular case. In another optional embodiment, the NK cells are preactivated prior to expansion. Any preactivation step can be utilized, but in a particular embodiment, the preactivation step includes exposure to one or more cytokines, such as one or more of IL-12, IL-15, and / or IL-18. The preactivation step can be for any suitable length of time, but in a particular embodiment, is for up to about 16 hours. After this step, the NK cells can be optionally expanded, including, for example, in the presence of universal antigen presenting cells and IL-2. The cultured cells may be reactivated during proliferation with IL2, IL-18, or IL-15, or any combination of these cytokines. After proliferation, the NK cells are exposed to an appropriate amount of antibody under sufficient conditions such that the specific antigen against which the antibody is directed binds to the outside of the NK cells. Specifically, the antibody is a monoclonal antibody. The antibody loading conditions for the NK cells may be at a specific temperature for a specific time, for example, 37° C. for 1 hour.

[0400] Figure 2 examines the binding of margetuximab (an anti-HER2 monoclonal antibody) to NK cells. NK cells were derived from umbilical cord blood and were either grown normally (NE) with irradiated (100 Gy) uAPC feeder cells (2:1 feeder:NK ratio) and recombinant human IL-2 (200 U / ml) in 50:50 Click / RPMI medium, or preactivated with IL-12 (10 ng / ml), IL-15 (50 ng / ml) and IL-18 (50 ng / ml) for 16 hours, then washed and grown (PE) with irradiated uAPC and IL-2 (200 U / ml). NK cells were loaded with 1 μg / ml margetuximab in Click / RPMI medium for 1 hour at 37°C and washed before examining the binding of margetuximab to NK cells by flow cytometry. Unloaded NE and PE NK cells were used as negative controls. Margetuximab binding was detected by flow cytometry staining with Alexa-Fluor 647 affinity purified F(ab')2 fragment goat anti-human IgG(H+L) antibody. The graph shows that PE NK cells exhibit higher levels of margetuximab binding compared to NE NK cells. The histograms are from three different cord blood NK cell donors.

[0401] Margetuximab-loaded NK cells show enhanced cytotoxicity against HER2+ tumor cells (Figure 3). Xcelligence cytotoxicity assay shows normalized cell index of HER2+ SKOV3 (ovarian cancer) cells cultured alone or with NK cells at an effector to target (E:T) ratio of 2:1. NK cells were either normally expanded (NE) or preactivated and expanded (P+E). NE NK cells were cultured in 50:50 Click / RPMI medium with irradiated (100Gy) uAPC feeder cells (feeder:NK ratio 2:1) and recombinant human IL-2 (200U / ml), while PE cells were preactivated with IL-12 (10ng / ml), IL-15 (50ng / ml) and IL-18 (50ng / ml) for 16 hours, then washed and expanded with irradiated uAPC and IL-2 (200U / ml). NK cells were either unloaded or loaded with 1 μg / ml margetuximab in Click / RPMI medium for 1 h at 37°C and then washed prior to assay. Data show that margetuximab loading enhances NE and PE NK cell cytotoxicity against tumor cells.

[0402] Margetuximab-loaded NK cells enhanced tumor control in a NSG mouse model (SKOV3) of HER2+ ovarian cancer (Figure 4). In Figure 4A, an example of a scheme of experimental design for the SKOV3 mouse model is shown. In Figure 4B, bioluminescence imaging showing tumor growth over time in mice implanted with firefly luciferase (FFluc)-transduced SKOV3 and left untreated, treated with NK cells alone, margetuximab alone, or NK cells preloaded with margetuximab is shown. Figure 4C is a graph showing the mean radiance over time for the four groups of mice described in panel 4A.

[0403] In Figure 5, validation of amivantamab (EGFR-MET bispecific antibody) binding to NK cells is shown. NK cells were derived from umbilical cord blood and were either grown normally (NE) in 50:50 Click / RPMI medium with irradiated (100 Gy) uAPC feeder cells (2:1 feeder:NK ratio) and recombinant human IL-2 (200 U / ml) or preactivated for 16 h with IL-12 (10 ng / ml), IL-15 (50 ng / ml), and IL-18 (50 ng / ml), then washed and grown (PE) with irradiated uAPC and IL-2 (200 U / ml). NK cells were loaded with 1 μg / ml amivantamab for 1 h at 37°C in Click / RPMI medium and washed before validating amivantamab binding to NK cells by flow cytometry. Unloaded NE and PE NK cells were used as negative controls. Amivantamab binding was detected by flow cytometry staining with Alexa-Fluor647 affinity purified F(ab')2 fragment goat anti-human IgG(H+L) antibody. Graph shows that PE NK cells have higher levels of amivantamab binding compared to NE NK cells. Histogram shows data from three different cord blood NK cell donors.

[0404] Amivantamab-loaded NK cells show enhanced cytotoxicity against EGFR+ / cMET+ tumor cells (Figure 6). Chromium release assays are provided showing specific lysis of three EGFR+ / c-MET+ cancer cell lines, SKOV3 (ovarian cancer), UMRC3 (renal cell carcinoma), and PATC-148 (pancreatic ductal adenocarcinoma), using three different cord blood NK cells (N=3 per assay) at various effector / target (E:T) ratios (20:1, 10:1, 5:1, 1:1). NK cells were either conventionally expanded (NE) or pre-activated (P+E) expanded. NE NK cells were cultured in 50:50 Click / RPMI medium with irradiated (100 Gy) uAPC feeder cells (2:1 feeder:NK ratio) and recombinant human IL-2 (200 U / ml), whereas PE cells were preactivated with IL-12 (10 ng / ml), IL-15 (50 ng / ml), and IL-18 (50 ng / ml) for 16 h, washed, and expanded with irradiated uAPC and IL-2 (200 U / ml). NK cells were either unloaded or loaded with amivantamab 1 μg / ml in Click / RPMI medium for 1 h at 37°C and then washed before assay. Loading with amivantamab enhanced the cytotoxicity of NE and PE NK cells against tumor cells.

[0405] In Figures 7A and 7B, NK cells loaded with amivantamab show enhanced cytotoxicity against EGFR+ / cMET+ tumor cells. Xcelligence cytotoxicity assays show normalized cell index for two EGFR+ / c-MET+ cancer cell lines, SKOV3 (ovarian cancer; Figure 7A) and PATC-148 (pancreatic ductal adenocarcinoma; Figure 7B), using three different cord blood NK cells (N=3 for each assa...

Claims

1. (a) Below: (ai) A nucleic acid sequence encoding a CD70-targeted CAR, comprising a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 2 or 5. (aii) A nucleic acid sequence encoding a TROP2-targeted CAR, comprising a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 7 or 9, or (aiiii)(ai) and (aiii) One or more manipulated natural killer (NK) cells, including, and (b) One or more antibody molecules, A composition containing the following:

2. The above one or more antibodies are as follows: Epidermal growth factor receptor-2 (EGFR-2), tyrosine protein kinase MET (c-MET), HER2, epidermal growth factor receptor (EGFR), CD123, NKp46, CD16, CD19, CD20, GD2, CD319 (CS1), ROR1, CD22, CD70, carcinombryonic antigen, alpha-fetoprotein, CA-125, MUC-1, epithelial tumor antigen, melanoma-associated antigen, variant p53, variant ras, Neu, ERBB2, folate-binding protein, HIV-1 envelope glycoprotein gp120, The composition according to claim 1, which binds to an antigen, autoantigen, or cancer antigen selected from the group consisting of HIV-1 envelope glycoprotein gp41, CD5, CD23, CD30, CD38, CD56, CD70, c-Met, mesoserine, GD3, HERV-K, IL-11Rα, κ chain, λ chain, CSPG4, WT-1, TRAIL / DR4, VEGFR2, CD33, CD47, CLL-1, U5snRNP200, CD200, BAFF-R, BCMA, CD99, HLA-G, Trop2, and combinations thereof.

3. The above one or more antibodies are as follows: The composition according to claim 1, selected from the group consisting of amivantamab, margetuximab, imugatuzumab, IPH61, tafacitamab, cetuximab, avelumab, trastuzumab, rituximab, obinutuzumab, pertuzumab, dinutuximab, mogamulizumab, or a combination thereof.

4. The composition according to claim 3, wherein the manipulated NK cells are further modified to include one or more additional transgenes for expressing one or more heterologous proteins.

5. The composition according to claim 4, wherein the one or more heterologous proteins comprise one or more manipulated receptors and / or one or more cytokines.

6. The composition according to claim 5, wherein the one or more manipulated receptors are a chimeric antigen receptor (CAR), a T cell receptor (TCR), a receptor that recognizes the FC portion of Ig (FC-recognizing receptor), a chemokine receptor, a homing receptor, a chimeric cytokine receptor, or any combination thereof.

7. The manipulated NK cells include a construct that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 1, 3, or 4. or The manipulated NK cells include a construct that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 6, 8, or 10. The composition according to claim 1.

8. The composition according to claim 5, wherein the one or more heterologous cytokines include IL-15, IL-21, IL-2, IL-4, IL-7, IL-12, IL-18, and / or IL-23.

9. The composition according to claim 8, wherein the one or more cytokines are encoded by a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 11 or 12, and / or comprise IL-15 comprising the same.

10. The composition according to claim 3, wherein one or more antibody molecules are incubated with NK cells at a final concentration of about 1 to about 1000 μg / mL, about 1 μg / mL, about 10 μg / mL, about 50 μg / mL, or about 100 μg / mL for complex formation.

11. The composition according to claim 3, wherein at least about 9% of the antibody bound to the surface of the manipulated NK cells at least 3 days after incubation and washing is measured approximately 1 hour after incubation and washing.

12. The composition according to claim 3, wherein the composition is not frozen, has been stored frozen, or has been thawed from frozen storage.

13. The composition according to claim 3, wherein the manipulated NK cells are inactivated before cryopreservation by contact with an inactivator containing a kinase inhibitor.

14. A method for producing a manipulated NK cell-antibody complex, (a) In one or more NK cells, (ai) A nucleic acid sequence encoding a CD70-targeted CAR, comprising a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 2 or 5. (aii) A nucleic acid sequence encoding a TROP2-targeted CAR, comprising a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 7 or 9, or (aiiii)(ai) and (aiii) The process of introducing; and (b) The process of contacting the manipulated NK cells with one or more antibodies selected from the group consisting of amivantamab, margetuximab, imugatuzumab, IPH61, tafacitamab, cetuximab, avelumab, trastuzumab, rituximab, obinutuzumab, pertuzumab, dinutuximab, mogamulizumab, or a combination thereof. Methods that include...

15. The above method further, (i) A step of growing and / or pre-activating the manipulated NK cells prior to step (a), and / or (ii) A step of inactivating and / or freezing after step (b), Includes, Here, the proliferation includes culturing the manipulated NK cells with cytokines selected from the group consisting of IL-2, IL-15, IL-18, IL-21 and combinations thereof. The aforementioned pre-activation includes culturing the manipulated NK cells at one or more effective concentrations of IL-2, IL-12, IL-15, and IL-18. The method according to claim 14.

16. The aforementioned contact step (b) (i) The manipulated NK cells are brought into contact with one or more antibodies in vitro for about one hour, wherein at least about 9% of the antibodies bind to the manipulated NK cells, or (ii) Contact the manipulated NK cells with one or more antibodies in vivo. The method according to claim 15, including the method described in claim 15.

17. (a) Below: (ai) A nucleic acid sequence encoding a CD70-targeted CAR, comprising a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 2 or 5. (aii) A nucleic acid sequence encoding a TROP2-targeted CAR, comprising a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 7 or 9, or (aiiii)(ai) and (aiii) A composition comprising one or more manipulated natural killer (NK) cells, (b) One or more antibody molecules, A composition for use in combination with [another substance].

18. The above one or more antibodies are as follows: Epidermal growth factor receptor-2 (EGFR-2), tyrosine protein kinase MET (c-MET), HER2, epidermal growth factor receptor (EGFR), CD123, NKp46, CD16, CD19, CD20, GD2, CD319 (CS1), ROR1, CD22, CD70, carcinombryonic antigen, alpha-fetoprotein, CA-125, MUC-1, epithelial tumor antigen, melanoma-associated antigen, variant p53, variant ras, Neu, ERBB2, folate-binding protein, HIV-1 envelope glycoprotein gp120, The composition according to claim 17, which binds to an antigen, autoantigen, or cancer antigen selected from the group consisting of HIV-1 envelope glycoprotein gp41, CD5, CD23, CD30, CD38, CD56, CD70, c-Met, mesoserine, GD3, HERV-K, IL-11Rα, κ chain, λ chain, CSPG4, WT-1, TRAIL / DR4, VEGFR2, CD33, CD47, CLL-1, U5snRNP200, CD200, BAFF-R, BCMA, CD99, HLA-G, Trop2, and combinations thereof.

19. The above one or more antibodies are as follows: The composition according to claim 18, selected from the group consisting of amivantamab, margetuximab, imugatuzumab, IPH61, tafacitamab, cetuximab, avelumab, trastuzumab, rituximab, obinutuzumab, pertuzumab, dinutuximab, mogamulizumab, or a combination thereof.

20. The composition according to claim 18, wherein the composition and the one or more antibody molecules are administered simultaneously or sequentially in any order.

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