Methods of activation and expansion of engineered natural killer cells and combinations with antibodies

Abstract

Embodiments of the present disclosure relate to methods and compositions related to the preparation and use of combination immunotherapy. In particular embodiments, compositions comprising engineered NK cells prepared in particular ways also include certain antibodies. These compositions are useful in therapy, for example in the treatment of cancer. In particular embodiments, compositions comprise a complex of engineered NK cells and an antibody, where the antibody binds to engineered NK cells and may also bind to another antigen, such as an antigen on cancer cells.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to International Application Serial No. PCT / US2022 / 023920 filed on April 7, 2022, U.S. Provisional Patent Application Serial No. 63 / 348,259 filed on June 2, 2022, and U.S. Provisional Patent Application Serial No. 63 / 415,517 filed on October 12, 2022, each of which is incorporated herein by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which has been submitted in ST26 format and is hereby incorporated by reference in its entirety. The ST26 copy was created on April 3, 2023, named MDAC_1330WO_Sequence_Listing.xml, and is 26,915 bytes in size. Technical Field

[0005] Embodiments of the present disclosure include at least the fields of cell biology, molecular biology, immunology, and medicine, including cancer medicine.

[0006] background

[0007] In the field of natural killer (NK) cells as a treatment, certain obstacles hinder their use, including the need for ex vivo expansion due to limited numbers, and their lack of persistence affects their efficacy as an anti-cancer treatment. Cytokine stimulation is an important signal that enhances the functional ability of NK cells to respond to tumor target cells. In addition, pre-activation of NK cells overnight with a combination of IL-18, IL-15 and IL-12 has been shown to produce long-lived memory-like NK cells with enhanced cytokine production after restimulation (Leong et al., 2014). However, the procedure of using pre-activated NK cells from peripheral blood (PB) without ex vivo expansion requires cumbersome procedures to produce sufficient quantities for clinical use. In addition, there are other obstacles related to cancer recognition defects and poor NK cell activation. Therefore, there is an unmet need for improved strategies to produce highly functional NK cells for therapeutic applications and particularly effective in cancer antigen targeting. SUMMARY OF THE INVENTION

[0009] Embodiments of the present disclosure relate to methods and compositions related to immunotherapy for medical conditions. Immunotherapy can be used for any medical condition where targeting cells that need to be destroyed is clinically useful and there are antibodies on the target cells and / or specific antigens for which the cells may have specificity. In specific embodiments, the immunotherapy comprises, consists of, or consists essentially of adoptive cell therapy and antibodies configured to bind to cells.

[0010] In some embodiments, provided herein is a composition comprising (i) one or more engineered natural killer (NK) cells comprising one or more transgenics; and (ii) one or more antibody molecules including Amivantamab, Margetuximab and / or Imgatuzumab. In some embodiments, engineered NK cells are from a population that has been expanded or not expanded. In some embodiments, engineered NK cells are from a population that has been expanded. In some embodiments, engineered NK cells are from a pre-activated or non-pre-activated population. In some embodiments, engineered NK cells are from a pre-activated population.

[0011] In some embodiments, the compositions provided herein are further defined as a complex formed between engineered NK cells and magetuximab, ervantumab and / or imatrazumab through the binding of the Fc region of the antibody to the engineered NK cells. In some embodiments, the complex further includes one or more antigen binding domains of one or more antibodies that bind to its target antigen.

[0012] In some embodiments, 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, engineered NK cells are derived from NK cells or NK cell precursors obtained from cord blood (CB) monocytes, CB hematopoietic stem cells, peripheral blood (PB) NK cells, bone marrow, stem cells, iPSC, NK cell lines, or a combination thereof. In some embodiments, engineered NK cells are derived from primary NK cells, rather than stem cells and / or induced pluripotent stem cells (iPSC). In some embodiments, NK cells are derived from cord blood, and the source of the CB monocytes and / or CB hematopoietic stem cells is CB from one donor or from a collection of 2 or more individual CB units. In some embodiments, engineered NK cells are modified to include one or more transgenes for expression of one or more heterologous proteins.

[0013] In some embodiments, one or more heterologous proteins include one or more engineered receptors and / or one or more cytokines. In some embodiments, one or more engineered receptors are chimeric antigen receptors (CAR), T cell receptors (TCR), receptors (FC recognition receptors) that recognize the FC portion of Ig, chemokine receptors, homing receptors, chimeric cytokine receptors, or any combination thereof. In some embodiments, FC recognition receptors include CD16, CD32, and / or CD64, and / or wherein engineered NK cells express one or more transgenics encoding one or more receptors to enhance their binding to antibodies. In some embodiments, engineered receptors include TCR and / or CAR. In some embodiments, the engineered receptor targets a stem cell antigen, a self antigen, or a cancer antigen selected from the group consisting of CD19, CD319 (CS1), ROR1, CD20, CD22, CD70, carcinoembryonic antigen, alpha-fetoprotein, CA-125, MUC-1, EGFR, epithelial tumor antigen, melanoma-associated antigen, mutant p53, mutant ras, EGFR-2 / HER2 / Neu, ERBB2, folate binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope Membrane glycoproteins gp41, GD2, CD5, 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.

[0014] In some embodiments, the engineered antigen receptor targets CD70 and / or TROP2. In some embodiments, the antigen receptor targeting CD70 comprises, consists of, or consists essentially of a sequence that is at least or about 80%, 81%, 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 that is at least or about 80%, 81%, 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 that is at least or about 80%, 81%, 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 that is at least or about 80%, 81%, 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.

[0015] In some embodiments, the engineered NK cells comprise one or more heterologous cytokines, comprising 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, which is encoded by and / or comprises, consists of, or consists essentially of a sequence that is at least or about 80%, 81%, 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, which is encoded by and / or comprises, consists of, or consists essentially of a sequence that is at least or about 80%, 81%, 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.

[0016] In some embodiments, engineered NK cells express one or more transgenes encoding one or more suicide genes. In some embodiments, engineered NK cells comprise one or more engineered mutations in endogenous genes. In some embodiments, engineered mutations include CISH, CD38, glucocorticoid receptor and / or TGFBR2 gene mutations.

[0017] In some embodiments, the compound of one or more antibody molecules and NK cells includes incubating NK cells with one or more antibodies for at least or about 1 hour and then washing the composition to remove unbound antibodies. In some embodiments, washing includes washing at least twice with a suitable solution. In some embodiments, a suitable solution includes PBS. In some embodiments, washing includes 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 longer than 10 minutes. In some embodiments, washing includes washing for at least or about 5 minutes.

[0018] In some embodiments, one or more antibody molecules are incubated with NK cells to form a complex 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, one or more antibodies and NK cells are incubated in Click's / RPMI medium. In some embodiments, the incubation and wash 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, relative to the amount of antibodies compounded with engineered NK cells measured about 1 hour after incubation and washing, at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12% or more than 12% of the antibodies are bound to the NK cell surface for at least 3 days after incubation and washing. In some embodiments, relative to the amount of antibodies compounded with engineered NK cells measured about 1 hour after incubation and washing, at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or more than 10% of the antibodies are bound to the NK cell surface for at least 5 days after incubation and washing. In some embodiments, relative to the amount of antibodies compounded with engineered NK cells measured about 1 hour after incubation and washing, at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% 10% or more than 10% of the antibodies are bound to the NK cell surface for at least 7 days after incubation and washing. In some embodiments, at least about 0.5%, 1%, 2%, 3%, 4%, 5% or greater than 5% of the antibody is bound to the NK cell surface for at least 12 days after incubation and washing, relative to the amount of antibody complexed with the engineered NK cells measured at about 1 hour after incubation and washing.

[0019] In some embodiments, the composition comprising engineered NK cells and antibodies is not frozen, cryopreserved or thawed from cryopreservation. In some embodiments, the composition comprising engineered NK cells and antibodies 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 compounded with antibodies. In some embodiments, relative to the amount of antibodies compounded with non-cryopreserved engineered NK cells measured about 1 hour after incubation and washing, after thawing from cryopreservation, at least about 5%, 10%, 20%, 30%, 40%, 50% or 60% or any range of antibodies derived therein bind to the NK cell surface. In some embodiments, engineered NK cells are deactivated before cryopreservation. In some embodiments, NK cell deactivators include kinase inhibitors. In some embodiments, NK cell deactivators include, consist of, or are substantially comprised of dasatinib.

[0020] In some embodiments, a composition comprising engineered NK cells and antibodies is produced in vivo. In some embodiments, producing a composition in vivo includes loading engineered NK cells with antibodies in vivo by administering engineered NK cells within about 1 hour after administering the antibodies. In some embodiments, one or more antibodies include or consist of ervantumab. In some embodiments, one or more antibodies include or consist of magetuximab. In some embodiments, one or more antibodies include or consist of imametuzumab.

[0021] Also provided herein is a method for making the compositions described herein. In some embodiments, the method for producing the compositions comprises the following steps: (i) optionally expanding engineered NK cells in a culture containing an effective amount of the following substances: (a) a cytokine selected from IL-2, IL-15, IL-18, IL-21, and a combination thereof; and (b) antigen presenting cells / feeder cells, fragments of antigen presenting cells / feeder cells, or NK cell activation beads; and (ii) providing one or more antibody molecules including ivantazumab, magetuximab, and / or imatuzumab to the engineered NK cells.

[0022] In some embodiments, the method includes a pre-activation step before and / or after the amplification step, wherein the engineered NK cells are pre-activated in a culture comprising one or more of IL-2, IL-12, IL-15 and IL-18 at an effective concentration. In some embodiments, the culture comprises an effective concentration of IL-12, IL-15 and IL-18. In some embodiments, the step of providing is further defined as incubating the engineered NK cells with antibody molecules for a specific duration, immediately combining the engineered NK cells and antibody molecules before infusion, or coordinating administration of the engineered NK cells and antibodies in the subject by time. In some embodiments, the duration is about 5 minutes to about 24 hours or longer. In some embodiments, the duration is at most, at least or about 1 hour. In some embodiments, the culture for the pre-activation step comprises IL-18 and / or IL-15 at a concentration of 0.1-1000ng / mL, 1-1000ng / mL or about 10ng / mL. In some embodiments, the culture for the pre-activation step comprises IL-12 at a concentration of 0.1-1000ng / mL, 1-1000ng / mL or about 10ng / mL. In some embodiments, the method also includes one or more washing steps for pre-activated engineered NK cells before and / or after the amplification step. In some embodiments, the engineered NK cells are activated at least twice or more with IL-12, IL-15, IL-18, IL-2 or any combination thereof during the amplification step. In some embodiments, the NK cell amplification step lasts for 5-60 days, 12-16 days or 18-24 days. In some embodiments, the amplification culture also comprises IL-2. In some embodiments, IL-2 is present at a concentration of 10-500U / mL, 100-300U / mL or about 200U / mL. In some embodiments, IL-2 is supplemented in the amplification culture every 2-3 days.

[0023] In some embodiments, the methods described herein include incubation conditions for complexing engineered NK cells with one or more antibodies such that after incubation and washing, 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 complexed with the antibodies 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. In some embodiments, relative to the amount of antibodies compounded with engineered NK cells measured about 1 hour after incubation and washing, at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12% or more than 12% of the antibodies are bound to the NK cell surface for at least 3 days after incubation and washing. In some embodiments, relative to the amount of antibodies compounded with engineered NK cells measured about 1 hour after incubation and washing, at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or more than 10% of the antibodies are bound to the NK cell surface for at least 5 days after incubation and washing. In some embodiments, relative to the amount of antibodies compounded with engineered NK cells measured about 1 hour after incubation and washing, at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% 10% or more than 10% of the antibodies are bound to the NK cell surface for at least 7 days after incubation and washing. In some embodiments, at least about 0.5%, 1%, 2%, 3%, 4%, 5% or greater than 5% of the antibody is bound to the NK cell surface for at least 12 days after incubation and washing, relative to the amount of antibody complexed with the engineered NK cells measured at about 1 hour after incubation and washing.

[0024] In some embodiments, the method for preparing the composition includes not freezing or selectively freezing and / or thawing the composition from freezing. In some embodiments, the NK cells provided together with one or more antibodies are not frozen, frozen or thawed from freezing. In some embodiments, the NK cells provided together with one or more antibodies are thawed from freezing, 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 compounded with antibodies. 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 surface of NK cells following thawing from cryopreservation relative to the amount of antibody complexed with non-cryopreserved engineered NK cells measured about 1 hour after incubation and washing.

[0025] Also provided herein is a method for treating a disease or condition of a subject, the method comprising administering to the subject a therapeutically effective amount of any one or more compositions described herein. In some embodiments, the disease or condition is cancer, inflammation, graft-versus-host disease, transplant rejection, autoimmune disease, immunodeficiency disease, B cell malignancy or infection. In some embodiments, cancer includes hematological cancer or solid tumors. In some embodiments, cancer includes pancreatic cancer, colorectal cancer, kidney cancer, brain cancer, breast cancer, renal cancer and / or myeloma. In some embodiments, engineered NK cells are allogeneic relative to the subject. In some embodiments, engineered NK cells are autologous relative to the subject. In some embodiments, the subject is human. In some embodiments, the method of treatment also includes administering to the subject at least one second therapeutic agent. In some embodiments, at least one second therapeutic agent is a therapeutically effective amount of one or more anticancer agents, one or more immunomodulators and / or one or more immunosuppressants. In some embodiments, the anticancer agent is chemotherapy, radiotherapy, gene therapy, surgery, hormone therapy, anti-angiogenesis therapy or immunotherapy. In some embodiments, 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. In some embodiments, the composition and / or at least one second therapeutic agent is administered intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, transdermally, subcutaneously, regionally, or by direct injection or infusion. In some embodiments, the second therapeutic agent is an antibody.

[0026] In some embodiments, provided herein is a method for treating a cancer subject using engineered NK cells, the method comprising: a) optionally stimulating an engineered NK cell population by pre-activation with a cytokine mixture; b) optionally expanding engineered NK cells; and ci) loading engineered NK cells ex vivo with one or more antibodies, then administering the loaded engineered NK cells to a subject, and / or cii) loading engineered NK cells in vivo with one or more antibodies by administering engineered NK cells within 1 hour after administering the antibodies; wherein the engineered NK cells comprise one or more transgenes, wherein loading comprises exposing the engineered NK cells to one or more antibodies for a time effective to bind the antibodies to the surface of the engineered NK cells, and wherein the one or more antibodies comprise or consist of ervantumab, magetuximab and / or imatrazumab. In some embodiments, the method comprises administering a composition comprising engineered NK cells loaded ex vivo, and then administering additional antibodies to the subject at a later time point to additionally load NK cells with antibodies in vivo. In some embodiments, loading engineered NK cells comprises exposing the engineered NK cells to one or more antibodies for greater than or equal to about 1 hour or less than or equal to about 1 hour. In some embodiments, the engineered NK cells maintain loading antibodies for at least about 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, one or more antibodies are combined with engineered NK cells by interacting with CD16 surface proteins. In some embodiments, engineered NK cells are derived from cord blood. In some embodiments, engineered NK cells are activated ex vivo. In some embodiments, engineered NK cells are pre-activated with IL-18, IL-15 and IL-12.

[0027] In some embodiments, the treatment method includes the use of NK cells comprising one or more transgenes, wherein the transgene includes 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 targets a stem cell antigen, an autoantigen, or a cancer antigen selected from the following: 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, 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 targeting CD70 comprises, consists of, or consists essentially of a sequence that is at least or about 80%, 81%, 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 that is at least or about 80%, 81%, 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 that is at least or about 80%, 81%, 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 that is at least or about 80%, 81%, 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, which is encoded by and / or comprises, consists of, or consists essentially of a sequence that is at least or about 80%, 81%, 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, which is encoded by a sequence that is at least or about 80%, 81%, 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 and / or comprises, consists of, or consists essentially of the sequence. 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.

[0028] Also provided herein is a composition comprising (i) one or more engineered natural killer (NK) cells comprising one or more transgenics; and (ii) one or more antibodies including antibody IPH61. In some embodiments, one or more antibodies target at least CD123 and NKp46. In some embodiments, the composition is further defined as a complex formed by the combination of the Fc region of the antibody with the engineered NK cells and / or the NKp46 targeting region of the antibody with the engineered NK cells between the engineered NK cells IPH61. In some embodiments, the complex further includes one or more antigen binding domains of one or more antibodies bound to its target antigen. In some embodiments, the engineered NK cells are 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, iPSC, NK cell lines, or a combination thereof. In some embodiments, the engineered NK cells are primary NK cells (e.g., NK cells not derived from stem cells, iPSCs, etc.).

[0029] In some embodiments, the composition comprises an engineered NK cell modified to include one or more transgenics for expressing one or more heterologous proteins. In some embodiments, one or more heterologous proteins include one or more engineered receptors and / or one or more cytokines. In some embodiments, one or more engineered receptors are chimeric antigen receptors (CAR), T cell receptors (TCR), receptors (FC recognition receptors) for the FC portion of Ig recognition, chemokine receptors, homing receptors, chimeric cytokine receptors, or any combination thereof. In some embodiments, FC recognition receptors include CD16, CD32, and / or CD64, and / or wherein engineered NK cells express one or more transgenics encoding one or more receptors and / or NK cell surface antigens to enhance the binding of NK cells to antibodies. In some embodiments, engineered NK cell surface antigens and / or receptors are CD16, CD32, CS1, CD56, CD64, NKG2D, NKG2C, DNAM, 2B4, CD2, NCR, NKp30, NKp44, NKp46, or KIR. In some embodiments, engineered receptors include TCR and / or CAR. In some embodiments, the engineered receptor targets a stem cell antigen, a self antigen, or a cancer antigen selected from the group consisting of CD19, CD319 (CS1), ROR1, CD20, CD22, CD70, carcinoembryonic antigen, alpha-fetoprotein, CA-125, MUC-1, EGFR, epithelial tumor antigen, melanoma-associated antigen, mutant p53, mutant ras, EGFR-2 / HER2 / Neu, ERBB2, folate binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope Glycoprotein gp41, GD2, CD5, 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 targeting CD70 comprises, consists of, or consists essentially of a sequence that is at least or about 80%, 81%, 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 that is at least or about 80%, 81%, 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 that is at least or about 80%, 81%, 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, consist of, or consist essentially of a construct comprising, consisting of, or consisting essentially of a sequence that is at least or about 80%, 81%, 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, which is encoded by a sequence that comprises, consists of, or consists essentially of the following sequence: a sequence that is at least or about 80%, 81%, 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, and / or comprises, consists of, or consists essentially of said sequence. In some embodiments, the cytokine comprises, consists of, or consists essentially of IL-21, which is encoded by a sequence that comprises, consists of, or consists essentially of a sequence that is at least or about 80%, 81%, 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, and / or comprises, consists of, or consists essentially of the sequence. In some embodiments, the engineered NK cells express one or more transgenes encoding one or more suicide genes.In some embodiments, engineered NK cells include one or more engineered mutations in endogenous genes. In some embodiments, one or more engineered mutations include CISH, CD38, glucocorticoid receptor and / or TGFBR2 gene mutations. In some embodiments, the composite of one or more antibodies including IPH61 and NK cells includes incubating NK cells with one or more antibodies for at least or about 1 hour and then washing the composition to remove unbound antibodies.

[0030] In some embodiments, the composition comprises one or more antibody molecules that are incubated with NK cells to form a complex 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's / RPMI medium. In some embodiments, the incubation and washing conditions for the complexing of engineered NK cells with 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 antibodies 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 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 compounded with antibodies. In some embodiments, relative to the amount of antibodies compounded with non-cryopreserved engineered NK cells measured about 1 hour after incubation and washing, after thawing from cryopreservation, at least about 30%, 40%, 50% or 60% of the antibodies are bound to the NK cell surface. In some embodiments, the engineered NK cells are deactivated before cryopreservation. In some embodiments, the deactivator includes a kinase inhibitor. In some embodiments, the deactivator includes 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 one or more antibodies in vivo by administering the engineered NK cells within about 1 hour after administration of the antibody.

[0031] Also provided herein is a method for producing a composition as described herein, the method comprising the following steps: (i) optionally expanding engineered NK cells in a culture containing an effective amount of the following substances: (a) a cytokine selected from IL-2, IL-15, IL-18, IL-21 and a combination thereof; and (b) antigen presenting cells / feeder cells, fragments of antigen presenting cells / feeder cells or NK cell activation beads; and (ii) providing one or more antibody molecules including IPH61 to engineered NK cells. In some embodiments, the method includes a pre-activation step before and / or after the amplification step, wherein the engineered NK cells are pre-activated in a culture containing one or more of IL-2, IL-12, IL-15 and IL-18 at an effective concentration. In some embodiments, the culture contains an effective concentration of IL-12, IL-15 and IL-18. In some embodiments, the providing step is further defined as incubating engineered NK cells with antibody molecules for a specific duration, combining engineered NK cells and antibody molecules immediately before infusion, or combining engineered NK cells and antibodies in a subject by time-coordinated administration. In some embodiments, the duration is about 5 minutes to about 24 hours or longer. In some embodiments, the duration is about 1 hour. In some embodiments, the culture for the pre-activation step comprises IL-18 and / or IL-15 at a concentration of 0.1-1000ng / mL, 1-1000ng / mL or about 10ng / mL. In some embodiments, the culture for the pre-activation step comprises IL-12 at a concentration of 0.1-1000ng / mL, 1-1000ng / mL or about 10ng / mL. In some embodiments, the expansion continues for 5-60 days, 12-16 days or 18-24 days. In some embodiments, the expansion culture also comprises IL-2. In some embodiments, IL-2 is present at a concentration of 10-500U / mL, 100-300U / mL or about 200U / mL.

[0032] In some embodiments, the incubation conditions for the complexing of engineered NK cells with 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 antibodies 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 provided with the antibody are not frozen, cryopreserved or thawed from cryopreservation. In some embodiments, the NK cells provided with 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 compounded with the antibody. In some embodiments, relative to the amount of the antibody compounded with the non-cryopreserved engineered NK cells measured about 1 hour after incubation and washing, after thawing from cryopreservation, at least about 30%, 40%, 50% or 60% of the antibody is bound to the NK cell surface.

[0033] Also provided herein is a method for treating a disease or condition of a subject, the method comprising administering to the subject a therapeutically effective amount of a composition comprising NK cells and one or more antibodies comprising IPH61. In some embodiments, the disease or condition is cancer, inflammation, graft-versus-host disease, transplant rejection, autoimmune disease, immunodeficiency disease, B cell malignancy or infection. In some embodiments, cancer includes hematological cancer or solid tumors. In some embodiments, cancer includes acute myeloid leukemia, blastic plasmacytoid dendritic cell tumors, acute lymphocytic leukemia, hairy cell leukemia and / or systemic mastocytosis. In some embodiments, engineered NK cells are allogeneic relative to the subject. In some embodiments, engineered NK cells are autologous relative to the subject. In some embodiments, the subject is human. In some embodiments, the method of treatment also includes administering to the subject at least one second therapeutic agent. In some embodiments, at least one second therapeutic agent is a therapeutically effective amount of one or more anticancer agents, one or more immunomodulators and / or one or more immunosuppressants.

[0034] 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 antibody molecules, wherein the one or more transgenes comprise a CAR targeting CD70, the CAR targeting CD70 comprising, consisting of, or consisting essentially of a sequence that is at least or about 80%, 81%, 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 wherein the one or more transgenes include a CAR targeting TROP2, the CAR targeting TROP2 comprising a sequence that is at least or about 80%, 81%, 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. NO: 7 or 9 is at least or about 80%, 81%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical sequence, consisting of or consisting essentially of it. In some embodiments, the engineered NK cells are from an expanded or unamplified population. In some embodiments, the engineered NK cells are from an expanded population. In some embodiments, the engineered NK cells are from a pre-activated or non-pre-activated population. In some embodiments, the engineered NK cells are from a pre-activated population.

[0035] In some embodiments, the composition is further defined as a complex between an engineered NK cell and one or more antibodies. In some embodiments, the complex further includes one or more antigen binding domains of one or more antibodies that bind to its target antigen. In some embodiments, the antibody targets a cellular antigen, a self antigen, or a cancer antigen selected from the following: CD19, CD319 (CS1), ROR1, CD20, CD22, CD70, carcinoembryonic antigen, alpha-fetoprotein, CA-125, MUC-1, EGFR, epithelial tumor antigen, melanoma-associated antigen, mutant p53, mutant ras, EGFR-2 / HER2 / Neu, ERBB2, folate binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, CD5, CD123, CD23, 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. In some embodiments, 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).

[0036] In some embodiments, engineered NK cells are 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, iPSC, NK cell lines, or a combination thereof. In some embodiments, engineered NK cells are derived from primary NK cells, rather than stem cells and / or induced pluripotent stem cells (iPSC). In some embodiments, NK cells are derived from cord blood, and the source of CB mononuclear cells and / or CB hematopoietic stem cells is CB from one donor or from the collection of 2 or more individual CB units.

[0037] In some embodiments, engineered NK cells are further modified to include one or more additional transgenes for expressing one or more heterologous proteins. In some embodiments, one or more heterologous proteins include one or more engineered receptors, and / or one or more cytokines. In some embodiments, one or more engineered receptors are chimeric antigen receptors (CAR), T cell receptors (TCR), receptors (FC recognition receptors) for the FC portion of Ig recognition, chemokine receptors, homing receptors, chimeric cytokine receptors, or any combination thereof. In some embodiments, FC recognition receptors include CD16, CD32, and / or CD64, and / or wherein engineered NK cells express one or more transgenes encoding one or more receptors to enhance their binding to antibodies. In some embodiments, engineered receptors include TCR and / or CAR. In some embodiments, the engineered receptor targets a stem cell antigen, a self antigen, or a cancer antigen or a cancer antigen selected from the group consisting of CD19, CD319 (CS1), ROR1, CD20, CD22, CD70, carcinoembryonic antigen, alpha-fetoprotein, CA-125, MUC-1, EGFR, epithelial tumor antigen, melanoma-associated antigen, mutant p53, mutant ras, EGFR-2 / HER2 / Neu, ERBB2, folate binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 Envelope glycoproteins gp41, GD2, CD5, 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.

[0038] In some embodiments, the engineered NK cells comprise a construct comprising, consisting of, or consisting essentially of a sequence that is at least or about 80%, 81%, 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 comprising, consisting of, or consisting essentially of a sequence that is at least or about 80%, 81%, 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.

[0039] In some embodiments, NK cells are engineered to express one or more heterologous cytokines. In some embodiments, 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 includes, consists of, or is substantially composed of IL-15, which is encoded by a sequence comprising, consisting of, or substantially consisting of the following sequence: a sequence that is at least or about 80%, 81%, 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, and / or comprises, consists of, or is substantially composed of the sequence. In some embodiments, the cytokine comprises, consists of, or consists essentially of IL-21, which is encoded by a sequence comprising, consisting of, or consisting essentially of ... a sequence comprising, a sequence comprising, a sequence comprising, a sequence comprising, a sequence comprising, a sequence comprising, a sequence comprising, a sequence comprising, a sequence comprising, a sequence comprising, a sequence comprising, a sequence comprising, a sequence comprising, a sequence comprising, a sequence comprising, a sequence comprising, a sequence comprising, a sequence comprising, a sequence comprising, a sequence comprising, a sequence

[0040] In some embodiments, the complexing of one or more antibody molecules with NK cells comprises incubating the NK cells with the one or more antibodies for at least or about 1 hour and then washing the composition to remove unbound antibodies. In some embodiments, the one or more antibody molecules are incubated with NK cells to form a complex 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 antibodies and NK cells are incubated in Click's / RPMI medium. In some embodiments, the incubation and washing conditions for the complexing of engineered NK cells with 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 antibodies 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, at least about 9% of the antibodies are bound to the NK cell surface at least 3 days after incubation and washing relative to the amount of antibodies complexed with engineered NK cells measured at about 1 hour after incubation and washing. In some embodiments, at least about 6% of the antibodies bind to the NK cell surface for at least 5 days after incubation and washing, relative to the amount of antibodies compounded with the engineered NK cells measured about 1 hour after incubation and washing. In some embodiments, at least about 4% of the antibodies bind to the NK cell surface for at least 7 days after incubation and washing, relative to the amount of antibodies compounded with the engineered NK cells measured about 1 hour after incubation and washing. In some embodiments, at least about 3% of the antibodies bind to the NK cell surface for at least 12 days after incubation and washing, relative to the amount of antibodies compounded with 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 compounded with antibodies. In some embodiments, relative to the amount of antibodies compounded with non-cryopreserved engineered NK cells measured about 1 hour after incubation and washing, after thawing from cryopreservation, at least about 30%, 40%, 50% or 60% of the antibodies are bound to the NK cell surface. In some embodiments, the engineered NK cells are deactivated before cryopreservation.In some embodiments, the deactivator includes a kinase inhibitor. In some embodiments, the deactivator includes dasatinib. In some embodiments, the composition is produced in vivo. In some embodiments, producing the composition in vivo includes loading engineered NK cells with antibodies in vivo by administering engineered NK cells within about 1 hour after administering the antibody. In some embodiments, one or more antibodies include or consist of ervantumab. In some embodiments, one or more antibodies include or consist of magetuximab. In some embodiments, one or more antibodies include or consist of imametuzumab. In some embodiments, one or more antibodies include or consist of IPH61.

[0041] Also provided herein is a method for producing a composition, the method comprising the following steps: (i) optionally expanding engineered NK cells in a culture containing an effective amount of the following substances: (a) a cytokine selected from IL-2, IL-15, IL-18, IL-21 and a combination thereof; and (b) antigen presenting cells / feeder cells, fragments of antigen presenting cells / feeder cells or NK cell activation beads; and (ii) providing one or more antibody molecules to engineered NK cells. In some embodiments, the method includes a pre-activation step before and / or after the amplification step, wherein the engineered NK cells are pre-activated in a culture containing one or more of IL-2, IL-12, IL-15 and IL-18 at an effective concentration. In some embodiments, the culture contains an effective concentration of IL-12, IL-15 and IL-18. In some embodiments, the providing step is further defined as incubating engineered NK cells with antibody molecules for a specific duration, immediately combining engineered NK cells and antibody molecules before infusion, or combining engineered NK cells and antibodies in a subject by time coordination. In some embodiments, the duration is from about 5 minutes to about 24 hours or longer. In some embodiments, the duration is about 1 hour. In some embodiments, the culture for the pre-activation step comprises IL-18 and / or IL-15 at a concentration of 0.1-1000ng / mL, 1-1000ng / mL or about 10ng / mL. In some embodiments, the culture for the pre-activation step comprises IL-12 at a concentration of 0.1-1000ng / mL, 1-1000ng / mL or about 10ng / mL. In some embodiments, the method also includes one or more washing steps for pre-activated engineered NK cells before and / or after the amplification step. In some embodiments, the engineered NK cells are activated at least twice or more with IL-12, IL-15, IL-18, IL-2 or any combination thereof during the amplification step. In some embodiments, the amplification continues for 5-60 days, 12-16 days or 18-24 days. In some embodiments, the amplification culture also 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 supplemented in the expansion culture every 2-3 days.

[0042] In some embodiments, the incubation conditions for the complexing of 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 antibodies 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, relative to the amount of antibodies complexed with engineered NK cells measured at about 1 hour after incubation and washing, at least about 9% of the antibodies are bound to the NK cell surface at least 3 days after incubation and washing. In some embodiments, at least about 6% of the antibodies are bound to the NK cell surface at least 5 days after incubation and washing, relative to the amount of antibodies compounded with the engineered NK cells measured at about 1 hour after incubation and washing. In some embodiments, at least about 4% of the antibodies are bound to the NK cell surface at least 7 days after incubation and washing, relative to the amount of antibodies compounded with the engineered NK cells measured at about 1 hour after incubation and washing. In some embodiments, at least about 3% of the antibodies are bound to the NK cell surface at least 12 days after incubation and washing, relative to the amount of antibodies compounded with the engineered NK cells measured at about 1 hour after incubation and washing. In some embodiments, the NK cells provided with one or more antibodies are not frozen, cryopreserved, or thawed from cryopreservation. In some embodiments, NK cells provided with 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 compounded with antibodies. In some embodiments, relative to the amount of antibodies compounded with non-cryopreserved engineered NK cells measured about 1 hour after incubation and washing, after thawing from cryopreservation, at least about 30%, 40%, 50% or 60% of the antibodies are bound to the NK cell surface.

[0043] Also provided herein is a method for treating a disease or condition of a subject, the method comprising administering to the subject any one or more compositions provided herein of a therapeutically effective amount. In some embodiments, the disease or condition is cancer, inflammation, graft-versus-host disease, transplant rejection, autoimmune disease, immunodeficiency disease, B cell malignancy or infection. In some embodiments, cancer includes hematological cancer or solid tumors. In some embodiments, cancer includes pancreatic cancer, colorectal cancer, kidney cancer, brain cancer, breast cancer, renal cancer and / or myeloma. In some embodiments, cancer includes acute myeloid leukemia, blastic plasmacytoid dendritic cell tumor, acute lymphocytic leukemia, hairy cell leukemia and / or systemic mastocytosis. In some embodiments, engineered NK cells are allogeneic relative to the subject. In some embodiments, engineered NK cells are autologous relative to the subject. In some embodiments, the subject is human. In some embodiments, the method of treatment also includes administering to the subject at least one second therapeutic agent. In some embodiments, at least one second therapeutic agent is a therapeutically effective amount of one or more anticancer agents, one or more immunomodulators and / or one or more immunosuppressants. In some embodiments, at least one second therapeutic agent is an antibody identical to the antibody previously provided in the composition of the NK cells comprising antibody loading and / or targets the same antigen. In some embodiments, the anticancer agent is chemotherapy, radiotherapy, gene therapy, surgery, hormone therapy, anti-angiogenesis therapy or immunotherapy. In some embodiments, the immunosuppressant is a calcineurin inhibitor, an mTOR inhibitor, an antibody, chemotherapeutic radiation, a chemokine, an interleukin or an inhibitor of a chemokine or an interleukin. In some embodiments, the composition and / or at least one second therapeutic agent is administered intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, transdermally, subcutaneously, regionally or by direct injection or infusion. In some embodiments, the second therapeutic agent is an antibody.

[0044] Also provided herein is a method of treating a subject with cancer using engineered NK cells, the method 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 ex vivo with one or more antibodies and then administering the loaded engineered NK cells to the subject, or cii) loading the engineered NK cells in vivo with one or more antibodies by administering the engineered NK cells within 1 hour after administration of the antibodies; wherein the engineered NK cells comprise one or more transgenes, wherein the loading comprises exposing the engineered NK cells to the one or more antibodies for a time effective to allow the antibodies to bind to the surface of the engineered NK cells, and wherein the one or more transgenes comprise a CAR targeting CD70, wherein the CAR targeting CD70 comprises a polypeptide having the same sequence as SEQ ID NO: NO: 2 or 5 is at least or about 80%, 81%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence, and / or a CAR targeting TROP2, wherein the CAR targeting TROP2 comprises a sequence that is at least or about 80%, 81%, 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 engineered NK cells includes exposing engineered NK cells to one or more antibodies greater than or equal to about 1 hour or less than or equal to about 1 hour. In some embodiments, engineered NK cells maintain loading antibodies for at least about 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, one or more antibodies are combined with engineered NK cells by interacting with CD16 surface proteins. In some embodiments, engineered NK cells are derived from cord blood. In some embodiments, engineered NK cells are activated ex vivo. In some embodiments, engineered NK cells are pre-activated with IL-18, IL-15 and IL-12. In some embodiments, engineered NK cells include one or more additional transgenics, optionally including engineered receptors. In some embodiments, engineered receptors include chimeric antigen receptors (CAR), T cell receptors, chemokine receptors, chimeric cytokine receptors, or any combination thereof. In some embodiments, engineered receptors include CAR or TCR.In some embodiments, the engineered receptor targets a stem cell antigen, a self antigen, or a cancer antigen or a cancer antigen selected from the group consisting of CD19, CD319 (CS1), ROR1, CD20, CD22, CD70, carcinoembryonic antigen, alpha-fetoprotein, CA-125, MUC-1, EGFR, epithelial tumor antigen, melanoma-associated antigen, mutant p53, mutant ras, EGFR-2 / HER2 / Neu, ERBB2, folate binding protein, HIV-1 envelope glycoprotein gp120, HIV- 1Envelope glycoproteins gp41, GD2, CD5, CD123, CD23, 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. In some embodiments, the engineered NK cells comprise a construct that is at least or about 80%, 81%, 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%, 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 include IL-2, IL-4, IL-7, IL-12, IL-15, IL-18, IL-21 and / or IL-23. In some embodiments, the cytokines include 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, which is encoded by a sequence that comprises, consists of, or consists essentially of a sequence that is at least or about 80%, 81%, 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, and / or comprises, consists of, or consists essentially of the sequence. In some embodiments, the cytokine comprises, consists of, or consists essentially of IL-21, which is encoded by a sequence comprising, consisting of, or consisting essentially of ...

[0045] In certain embodiments, the efficacy of the composition comprising a complex of an engineered NK cell and an antibody is enhanced compared to its use alone. In some embodiments, when used as a complex, the efficacy of the engineered NK cell and the antibody has a synergistic effect. In some embodiments, the efficacy of the engineered NK cell and the antibody has an additive effect.

[0046] In any of the methods, the composition (and / or at least one second therapeutic agent) can be administered intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, transdermally, subcutaneously, regionally, or by direct injection or infusion or infusion.

[0047] The foregoing has outlined rather broadly the features and technical advantages of the present disclosure so that the following detailed description may be better understood. Additional features and advantages that constitute the subject matter of the claims herein will be described hereinafter. It will be appreciated by those skilled in the art that the disclosed concepts and specific embodiments may be readily used as a basis for modifying or designing other structures for achieving the same purposes as the present design. It will also be appreciated by those skilled in the art that such equivalent constructions do not depart from the spirit and scope set forth in the appended claims. From the following description, the novel features, both in organization and in method of operation, which are believed to be features of the designs disclosed herein, as well as further objects and advantages, may be better understood. However, it should be expressly understood that the present disclosure is for illustration and description purposes only and is not intended to be a definition of limitations of the present disclosure.

[0048] BRIEF DESCRIPTION OF THE DRAWINGS

[0049] For a more complete understanding of the present disclosure, reference is now made to the following description taken in conjunction with the accompanying drawings.

[0050] Figure 1 . Schematic diagram detailing one embodiment of an experimental plan to expand umbilical cord blood-derived NK cells and preload them with antibodies.

[0051] Figure 2 .Magetuximab binds with higher affinity to pre-activated and expanded (PE) NK cells compared to normally expanded (NE) NK cells.

[0052] Figure 3 In a long-term Xcelligence killing assay, NK cells loaded with magetuximab exhibited enhanced cytotoxicity (Y-axis) against HER2+ (e.g., SKOV3) tumor cells (X-axis tracks time in hours). NK cells expanded normally (NE) or were pre-activated and expanded (P+E).

[0053] Figure 4 A-4C. NK cells loaded with magetuximab resulted in enhanced tumor control in NSG mouse models of HER2+ ovarian cancer (eg, SKOV3).

[0054] Figure 5 .Evantumab binds with higher affinity to pre-activated and expanded (PE) NK cells compared to normally expanded (NE) NK cells.

[0055] Figure 6In a short-term 51Cr release killing assay, NK cells loaded with ervantumab exhibited enhanced cytotoxicity against EGFR+ / cMET+ tumor cells (e.g., SKOV3, UMRC3, and PATC148) (X-axis tracks effector to target (E:T) ratios of 20:1, 10:1, 5:1, or 1:1). NK cells expanded normally (NE) or were preactivated and expanded (PE).

[0056] Figure 7 A-7B. In long-term Xcelligence killing assays, NK cells loaded with entuzumab exhibited enhanced cytotoxicity against EGFR+ / cMET+ tumor cells (e.g., SKOV3 and PATC148) (X-axis tracks time in hours). NK cells expanded normally (NE) or were pre-activated and expanded (PE).

[0057] Figure 8 A-8C. NK cells loaded with ivanovumab resulted in enhanced tumor control in the NSG mouse model of EGFR+ / c-MET+ ovarian cancer (eg, SKOV3).

[0058] Fig. 9 Imatrazumab binds with higher affinity to pre-activated and expanded (PE) NK cells compared to normally expanded (NE) NK cells.

[0059] Fig.10 In the short term 51 In the Cr release killing assay, NK cells loaded with imatinib showed enhanced cytotoxicity against EGFR+ tumor cells (e.g., SKOV3, UMRC3, and PATC148). NK cells expanded normally (NE) or were preactivated and expanded (P+E).

[0060] Fig.11 A-11C. NK cells loaded with imatinib exhibit enhanced cytotoxicity (Y-axis, Normalized Cell Index) against EGFR+ tumor cells (e.g., SKOV3, PATC148, and UMRC3) in long-term Xcelligence killing assays (X-axis tracks time in hours). NK cells expanded normally (NE) or were pre-activated and expanded (P+E).

[0061] Fig.12 A-12C. NK cells loaded with imatinib result in enhanced tumor control in NSG mouse models of EGFR+ ovarian cancer (eg, SKOV3).

[0062] Fig.13A series of western blots demonstrated that EGFR is expressed on various tumor cell lines, including pancreatic cancer (e.g., Capan-1, Capan-2, MIA-Capa2, CFPAC-1, PANC-1, BcPC3), colorectal cancer (e.g., WiDr, HCT15, RKO, LOVO, SW403, LS174T, HCT116, SW116), renal cancer (e.g., 786-0, UMRC3, A498, 769-P, ACHN, Caki-1, Caki-2, A704), glioblastoma multiforme (GBM) (e.g., GSC272, GSC8-11, GSC20, and GSC267), breast cancer (e.g., MDA-231, BCX010), and renal cancer (e.g., SN12C) tumor cell lines. β-actin was used as a loading control.

[0063] Fig.14 When added to cells at 1 μg or 10 μg, imatinib bound to NK cells and EGFR+ tumor cell lines as analyzed by flow cytometry.

[0064] Fig.15 .Imacolomab binds to NK cells that are not transduced (NT) or transduced (e.g., using a chimeric antigen receptor (CAR)). In addition, imacolomab remains bound to NT or CAR NK cells (e.g., engineered NK cells) and remains on their cell surface for at least 5 days.

[0065] Fig.16 NK cells loaded with imatinib exhibited enhanced killing of breast cancer (e.g., MDA-231 cell line), pancreatic cancer (e.g., pancreatic ductal adenocarcinoma) (PDAC) (e.g., PATC-148 cell line), and colorectal cancer (CRC) (e.g., WiDR cell line) cell lines in vitro compared to unloaded NK cells.

[0066] Fig.17 . Engineered NK cells loaded with imatumomab (e.g., CAR NK + imatumomab 1 μg) exhibited enhanced killing of UMRC3 (RCC) cells in vitro compared to tumor cells treated with imatumomab alone, unloaded non-transduced (NT) (e.g., non-engineered) NK cells, NT-NK cells loaded with imatumomab (e.g., NT-NK + imatumomab 1 μg), or engineered unloaded NK (CARNK) cells.

[0067] Fig.18. Engineered NK cells loaded with imatumomab (e.g., CAR NK + imatumomab 1 μg) exhibited enhanced killing of SKOV3 (ovarian cancer) cells in vitro compared to tumor cells treated with imatumomab alone, unloaded non-transduced (NT) (e.g., non-engineered) NK cells, NT-NK cells loaded with imatumomab (e.g., NT-NK + imatumomab 1 μg), or engineered unloaded NK (CARNK) cells.

[0068] Fig.19 . Engineered NK cells loaded with imatumomab (e.g., CAR NK + imatumomab 1 μg) exhibited enhanced killing of PATC-148 (PDAC) cells in vitro compared to tumor cells treated with imatumomab alone, unloaded non-transduced (NT) (e.g., non-engineered) NK cells, NT-NK cells loaded with imatumomab (e.g., NT-NK + imatumomab 1 μg), or engineered unloaded NK (CARNK) cells.

[0069] Fig. 20 .Antibodies (e.g., imatrazumab) remain on the surface of NK cells for at least 12 days after antibody loading. NK cells were not loaded (-imatrazumab) or loaded with imatrazumab (100 μg / ml final concentration) (+imatrazumab) in Click's / RPMI medium at 37°C for 1 hour and washed twice with PBS (5 minutes each wash), and then imatrazumab binding was verified 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 hour, 3 days, 5 days, 7 days, and 12 days) and analyzed by flow cytometry.

[0070] Fig.21 A-21B. Imacolomab alone is not cytotoxic to ovarian cancer or PDAC cell lines. Xcelligence assays were performed to evaluate the effects of imacolomab on ovarian cell lines (e.g., SKOV3, Fig.21 A) and pancreatic ductal adenocarcinoma (PDAC) cell lines (e.g., PATC148, Fig.21 B) Cytotoxicity. The graph shows the normalized cell index (Y axis) plotted over time (X axis, hours). Cell lines cultured alone served as negative controls, and cell lines lysed with SDS served as positive controls. Statistical analysis included two-way ANOVA.

[0071] Fig. 22A-22B. NK cells loaded with imabrutinib exhibited enhanced killing of ovarian cancer (e.g., SKOV3) cell lines and PDAC (e.g., PATC148) cells in a long-term Xcelligence cytotoxicity assay compared to unloaded NK cells. NK cells were loaded with imabrutinib (100 μg imabrutinib, e.g., a final concentration of 100 μg / ml imabrutinib in 1 ml NK cells) in Click's / RPMI medium at 37°C for one hour, then washed twice with PBS (5 minutes each wash), and then co-cultured with tumor cells at a 1:1 effector cell to target cell ratio. Compared to unloaded NK cells, NK cells loaded with imabrutinib showed enhanced killing of SKOV3 ( Fig. 22 A) and PATC148( Fig. 22 B) Enhanced cytotoxic activity of cells. Cell lines cultured alone were used as negative controls, and cell lines lysed with SDS were used as positive controls. Statistical analysis included two-way ANOVA.

[0072] Fig.23 In a long-term Xcelligence cytotoxicity assay, NK cells loaded with imatumomab exhibited enhanced killing of colorectal cancer (CRC) cells (e.g., WiDR) compared to unloaded NK cells. NK cells were loaded with imatumomab (100 μg / ml final concentration) in Click's / RPMI medium for one hour at 37°C, then washed twice with PBS (5 minutes each wash), and then co-cultured with tumor cells at a 1:1 effector to target ratio. Statistical analysis included two-way ANOVA.

[0073] Fig.24 A-24B. In the Xcelligence long-term cytotoxicity assay, NK cells loaded with imatumomab exhibited enhanced killing of lung cancer cells (e.g., CRL-5922 and HTB-58) compared to unloaded NK cells. NK cells were loaded with imatumomab (final concentration of 100 μg / ml) in Click's / RPMI medium for 1 hour at 37°C, then washed twice with PBS (5 minutes each wash), and then co-cultured with tumor cells at a 1:1 effector to target ratio. Compared to unloaded NK cells, NK cells loaded with imatumomab exhibited enhanced killing of CRL-5922 ( Fig.24 A) and HTB-58( Fig.24 B) Increased cytotoxic activity of lung cancer cell lines. Cell lines cultured alone served as negative controls, and cell lines lysed with SDS served as positive controls. Statistical analysis included two-way ANOVA.

[0074] Fig.25A-25B. In the Xcelligence long-term cytotoxicity assay, NK cells loaded with imatumomab showed enhanced killing of breast cancer cells (e.g., MDA468 and SKBR3) compared to unloaded NK cells. NK cells were loaded with imatumomab (final concentration of 100 μg / ml) in Click's / RPMI medium for one hour at 37°C, then washed twice with PBS (5 minutes each wash), and then co-cultured with tumor cells at a 1:1 effector to target ratio. Compared to unloaded NK cells, NK cells loaded with imatumomab showed enhanced killing of CRL-5922 ( Fig.25 A) and HTB-58( Fig.25 B) Increased cytotoxic activity of lung cancer cell lines. Cell lines cultured alone served as negative controls, and cell lines lysed with SDS served as positive controls. Statistical analysis included two-way ANOVA.

[0075] Fig.26 A-26B. In the Incucyte live cell imaging assay, NK cells loaded with imatumomab exhibited enhanced killing of PDAC (eg, PATC148) tumor spheroids compared to unloaded NK cells. Fig.26 A shows representative images of PATC148 spheroids (20,000 tumor cells transduced with GFP and allowed to form spheroids for 48 hours) placed alone or treated with 50,000 unloaded NK cells or 50,000 NK cells pre-loaded with imatumumab (100 μg / ml final concentration) at different time points. The data show that the spheroid size was significantly reduced in wells treated with imatumumab-loaded NK cells. Fig.26 B is quantification of the integrated total green intensity over time. Statistical analysis included two-way ANOVA.

[0076] Fig. 27 A-27B. In the Incucyte live cell imaging assay, NK cells loaded with imatumomab exhibited enhanced cytotoxicity against CRC (e.g., WiDR) tumor spheroids compared to unloaded NK cells. Fig. 27 A shows representative images of WiDR spheroids (20,000 tumor cells transduced with GFP and allowed to form spheroids for 48 hours) placed alone or treated with 50,000 unloaded NK cells or 50,000 NK cells pre-loaded with imatulumab (100 μg / ml final concentration) at different time points. The data show that the spheroid size was significantly reduced in wells treated with imatulumab-loaded NK cells. Fig. 27 B is quantification of the integrated total green intensity over time. Statistical analysis included two-way ANOVA.

[0077] Fig.28 A-28B. In the Incucyte live cell imaging assay, NK cells loaded with imatumomab exhibited enhanced cytotoxicity against UMRC3 (RCC) tumor spheroids compared to unloaded NK cells. Fig.28 A shows representative images of UMRC3 spheroids (20,000 tumor cells transduced with GFP and allowed to form spheroids for 48 hours) placed alone or treated with 50,000 unloaded NK cells or 50,000 NK cells pre-loaded with imatulumab (100 μg / ml final concentration) at different time points. The data show that the spheroid size was significantly reduced in wells treated with imatulumab-loaded NK cells. Fig.28 B is quantification of the integrated total green intensity over time. Statistical analysis included two-way ANOVA.

[0078] Fig.29 A-29B. In the Incucyte live cell imaging assay, NK cells loaded with imatumomab exhibited enhanced cytotoxicity against GBM (e.g., GSC20) tumor spheroids compared to unloaded NK cells. Fig.29 A shows representative images of GSC20 spheroids (20,000 tumor cells were transduced with mCherry and allowed to form spheroids for 48 hours) placed alone or treated with 50,000 unloaded NK cells or 50,000 NK cells pre-loaded with imatumumab (100 μg / ml final concentration) at different time points. The data show that the spheroid size in wells treated with imatumumab-loaded NK cells was significantly reduced compared to unloaded NK cells. Fig.29 B is quantification of the integrated total red intensity over time. Statistical analysis included two-way ANOVA.

[0079] Fig.30 A-30D. NK cells loaded with imatumomab resulted in enhanced tumor control in the NSG mouse model of EGFR+ PDAC (eg, PATC148). Fig.30 A is a schematic diagram of the experimental plan of the PATC148 mouse model. On day -7, 10-week-old female NSG mice were injected intravenously (IV) with 0.3x10 6 PATC148 cells transduced with firefly luciferase (FFluc) were used for BLI on day -2 to record engraftment. Mice were irradiated with 300 cGy on day -1. On day 0, each mouse was injected intravenously with 10 7 Individual fresh NK cells or 10 7Fresh NK cells preloaded with imatumomab (100 μg / ml final concentration) were washed twice with saline (5 minutes each wash) and then suspended in saline. Test mice were then injected with 5 mg / kg imatumomab weekly for three weeks. Fig.30 B Bioluminescence imaging shows tumor growth over time in mice transplanted with firefly luciferase (Ffluc)-transduced PATC148 that were either untreated or treated with NK cells alone or NK cells preloaded with imatumomab. Fig.30 C, chart display Fig.30 C shows the average irradiance of the three groups of mice over time. Fig.30 D, Fig.30 Survival curves of the three groups of mice are depicted in B. Statistical analysis included two-way ANOVA.

[0080] Fig.31 NT NK cells loaded with imatinib (eg Fig. 20 ) and TROP2 CAR-NK cells retained the antibodies on their cell surface for at least 12 days. NK cells were derived from umbilical cord blood, expanded with 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's / RPMI), and either remained unmodified and untransduced (NT) or transduced with TROP2 CAR and IL-15 (TROP2) on day 6. NT NK cells or TROP2CAR NK cells were loaded with imatumomab (final concentration of 100 μg / ml) in Click's / RPMI medium for 1 hour at 37°C, washed twice with PBS (5 minutes each wash), and then imatumomab binding was verified by flow cytometry. NK cells were stained with Alexa-Fluor 647 affinity-purified F(ab')2 fragment goat anti-human IgG (H+L) antibody at different time points (1 hour, 3 days, 5 days, 7 days, 12 days) and analyzed by flow cytometry. The results showed that imatumomab binding was significantly inhibited in NT cells. NK cells ( Fig. 20 ) and TROP2 CAR NK cells ( Fig.31 ) surface for at least 12 days.

[0081] Fig.32A-32B. In the Xcelligence long-term cytotoxicity assay, TROP2 CAR-NK cells loaded with imatumomab exhibited enhanced killing of ovarian cancer (e.g., SKOV3) cells and gastrointestinal stromal tumor (GIST) (e.g., OVCAR5) cells compared to unloaded NK cells. NK cells that were not transduced (NT) or transduced with TROP2 CAR and IL-15 (TROP2 / IL15) were loaded with imatumomab (100 μg / ml final concentration) in click medium at 37°C for one hour, then washed twice with PBS (5 minutes each wash), and then co-cultured with tumor cells at a 1:1 effector to target ratio. NK cells loaded with imatumomab exhibited increased cytotoxic activity against target cells compared to unloaded NK cells. Statistical analysis included two-way ANOVA.

[0082] Fig.33 NK cells loaded with imatumomab retain the antibody on their surface after freezing and thawing. NK cells were derived from umbilical cord blood and expanded with 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's / RPMI) and kept unmodified and untransduced (NT) or transduced with TROP2 CAR and IL-15 (TROP2 CAR / IL-15) on day 6. NT NK cells or TROP2 CAR / IL-15 cells were loaded with imatumomab (100 μg / ml final concentration) (+imatumomab) in Click's / RPMI medium for 1 h at 37°C, washed twice with PBS (5 min each wash), and then deactivated with dasatinib before cryopreservation. NK cells were then thawed, stained with Alexa-Fluor 647 affinity-purified F(ab')2 fragment goat anti-human IgG (H+L) antibody, and analyzed by flow cytometry.

[0083] Fig.34 A-34D. After freezing / thawing, NT and TROP2CAR-NK cells loaded with imatumomab exhibited enhanced cytotoxicity against multiple cancer cell lines compared to unloaded NK cells. NK cells that were not transduced (NT) or transduced with the chimeric antigen receptor TROP2CAR and IL-15 (TROP2 / IL15) were loaded with imatumomab (100 μg / ml final concentration) in Click's / RPMI medium for one hour at 37°C, then washed twice with PBS (5 minutes each wash), and then deactivated with dasatinib before cryopreservation. The cells were then thawed and used to target multiple cancer cell lines (e.g., PDAC tumor cell line PATC148 ( Fig.34A), CRC cell line WiDR ( Fig.34 B) GIST cell line OVCAR5 ( Fig.34 C) and ovarian cancer cell line SKOV3 ( Fig.34 D)) Xcelligence cytotoxicity assay. Statistical analysis included two-way ANOVA. DETAILED DESCRIPTION OF THE INVENTION

[0085] Consistent with long-standing patent law practice, the words "a" and "an" when used with the word "comprising" in this specification, including the claims, mean "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 may be implemented with respect to any other method or composition described herein, and that different embodiments may be combined.

[0086] Throughout this specification, unless the context requires otherwise, the words "comprises," "comprising," and "containing" will be understood to imply the inclusion of the stated steps or elements or groups of steps or elements, but not the exclusion of any other steps or elements or groups of steps or elements. "Consisting of" means including and limited to whatever follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may be present. "Consisting essentially of" means including any elements listed after the phrase, and is limited to other elements that do not interfere with or contribute to the activity or action specified for the listed elements in the present disclosure. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but no other elements are optional and may or may not be present, depending on whether they affect the activity or action of the listed elements.

[0087] Reference throughout this specification to "one embodiment," "an embodiment," "a specific embodiment," "a related embodiment," "an embodiment," "an additional embodiment," or "a further embodiment," or combinations thereof, means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the foregoing phrases in various places throughout the specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0088] As used herein, the terms "or" and "and / or" are used to describe multiple components that are combined with or mutually exclusive of each other. 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 can be explicitly excluded from an embodiment.

[0089] 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. In addition, the compositions of the invention can be used to implement the methods of the invention.

[0090] Throughout this application, the term "about" is used according to its plain and ordinary meaning in the arts of cellular and molecular biology to indicate that a value includes the standard deviation of error for the device or method employed to determine the value.

[0091] The term "engineering" as used herein refers to an artificially produced entity, including cells, nucleic acids, polypeptides, vectors, etc. In at least some cases, the engineered entity is synthetic and includes elements that are not naturally present or configured in the manner used in the present disclosure. In a specific embodiment, the vector is engineered by recombinant nucleic acid technology, and the cell is engineered by transfection or transduction of the engineered vector. In a specific embodiment, the engineered cell is a cell comprising 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.), chemokines / homing receptors and / or modifications of one or more endogenous genes.

[0092] As used herein, the terms "loaded," "loaded," or "loaded" refer to adoptive cell therapy cells having one or more antibodies on the cell surface that bind to the cells.

[0093] The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce side effects, allergic reactions or other adverse reactions when administered to animals (e.g., humans) as appropriate. The preparation of pharmaceutical compositions comprising antibodies or additional active ingredients is known to those skilled in the art in light of the present disclosure. In addition, for animal (e.g., human) administration, it should be understood that the formulation should meet the sterility, pyrogenicity, general safety and purity standards required by the FDA Office of Biological Standards.

[0094] As used herein, "pharmaceutically acceptable carriers" include any and all aqueous solvents (e.g., water, alcohol / aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters such as ethyl oleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, and inert gases), isotonic agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, fluids, and nutritional supplements, materials such as these, and combinations thereof, as known to those of ordinary skill in the art. The pH and exact concentrations of the various components in the pharmaceutical composition are adjusted according to well-known parameters.

[0095] As used herein, the term "preactivated" or "preactivation" 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 functions, such as enrichment of genes involved in IFN-γ response, TNF signaling, IL-2 / STAT5 signaling, IL-6 / JAK / STAT3 signaling, mTOR pathways, and / or genes associated with inflammatory immune responses. In certain cases, the expression of TRAIL, NKp44, and / or CD69 is increased.

[0096] As used herein, the terms "preloaded," "preloaded," or "pre-charged" refer to adoptive cell therapy cells that already have one or more antibodies bound to the cells on the cell surface prior to use of the cells for any reason.

[0097] As used herein, "prevention" and similar words such as "prevent", "prevent", etc. refer to methods for preventing, inhibiting, or reducing the likelihood of the 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 words also include reducing the intensity, effects, symptoms, and / or burden of a disease or condition before the onset or recurrence of the disease or condition.

[0098] The term "subject" as used herein generally refers to an individual suffering from or suspected of having cancer. Subjects can be any organism or animal subject as the object of the method or material, including mammals, such as humans, experimental animals (such as primates, rats, mice, rabbits), livestock (such as cattle, sheep, goats, pigs, turkeys and chickens), household pets (such as dogs, cats and rodents), horses and transgenic non-human animals. Subjects can be patients, such as suffering from or suspected of having a disease (which can be referred to as a medical condition), such as benign or malignant neoplasia or cancer. Subjects can be receiving or have been treated. Subjects may have no symptoms. Subjects can be healthy individuals but wish to prevent cancer. At least in some cases, the term "individual" can be used interchangeably. "Subjects" or "individuals" as used herein may or may not be placed in a medical institution and may be treated as outpatients of a medical institution. Individuals can receive one or more pharmaceutical compositions via the Internet. Individuals can include humans or non-human animals of any age, therefore including adults and adolescents (that is, children) and infants, and including individuals in the uterus. The term does not imply a need for medical treatment; therefore, individuals may voluntarily or involuntarily participate in clinical trials or support basic scientific research.

[0099] As used herein, "treatment" or "treating" 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 a reduction or amelioration of one or more symptoms of a disease or condition, or a delay in the progression of a disease or condition. "Treatment" does not necessarily mean complete eradication or cure of a disease or condition or its associated symptoms.

[0100] I. Natural Killer (NK) Cells and Production Methods

[0101] NK cells are becoming an exciting source of cellular immunotherapy for patients with hematological malignancies as well as solid tumors; however, most studies using adoptively transferred NK cells are limited by insufficient persistence, poor in vivo expansion, and disappointing anti-tumor activity of the infused cells. Therefore, one obstacle that needs to be overcome in the field of NK immunotherapy is the need for biologically driven methods to increase the anti-tumor function of NK cells, such as by manipulating the cells prior to administration as a treatment. Therefore, in certain embodiments, the present disclosure provides methods for producing NK cells having any kind of enhanced efficacy compared to NK cells that have not been so manipulated. Some embodiments of the present disclosure relate to the separation, activation and expansion of NK cells, including for use in cancer immunotherapy.

[0102] In specific embodiments, the present disclosure encompasses loaded, pre-activated (optionally) and expanded (optionally) NK cells produced by the methods of the present invention that exhibit enhanced anti-tumor function against cancer. In specific embodiments, pre-activated and expanded NK cells also exhibit enhanced antibody-dependent cellular cytotoxicity (ADCC).

[0103] In specific embodiments, the present disclosure encompasses engineered, loaded, pre-activated (optionally) and amplified (optionally) NK cells produced by the present method, which exhibit enhanced anti-tumor function for cancer. In specific embodiments, loaded and / or engineered NK cells exhibit enhanced antibody-dependent cellular cytotoxicity (ADCC) relative to unloaded and / or non-engineered NK cells. In specific embodiments, loaded and / or engineered NK cells exhibit enhanced long-term cytotoxicity relative to unloaded and / or non-engineered NK cells. In specific embodiments, loaded and / or engineered NK cells exhibit enhanced long-term cytotoxicity for solid tumors and / or tumor spheroids relative to unloaded and / or non-engineered NK cells. In specific embodiments, loaded and / or engineered NK cells exhibit enhanced synergistic long-term cytotoxicity relative to unloaded and / or non-engineered NK cells. In specific embodiments, loaded and / or engineered NK cells show enhanced synergistic long-term cytotoxicity to solid tumors and / or tumor spheroids compared to unloaded and / or unengineered NK cells. In certain embodiments, imatumomab-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.

[0104] A. NK cells, an overview

[0105] In certain embodiments, the NK cells used in the method (e.g., the NK cells to be engineered) can be derived from any suitable source, such as umbilical cord blood (CB), including human CB. In special cases, NK cells are not derived from umbilical cord tissue (insulating material around umbilical cord blood vessels (i.e., Wharton's jelly)). In alternative embodiments, NK cells are derived from human peripheral blood mononuclear cells (PBMC), unstimulated leukocyte separation products (PBSC), human embryonic stem cells (hESC), induced pluripotent stem cells (iPSC) and / or bone marrow, or derived from the patient's NK cell line, such as NK-92, by methods well known in the art. In a specific embodiment, NK cells are separated from the collected CB. CB can be collected from 2, 3, 4, 5, 6, 7, 8, 9, 10 or more units. NK cells can be autologous or allogeneic for the recipient individual. The separated NK cells may or may not match the haplotype of the subject receiving cell therapy. NK cells may or may not be detected by specific surface markers (e.g., CD16 and / or CD56 in humans). In some cases, NK cells are depleted for the presence of one or more surface markers, such as CD3+, CD14+ and / or CD19+ cells. In specific embodiments, NK cells are CD3-CD56+.

[0106] In certain aspects, NK cells are isolated by a previously described NK cell ex vivo expansion method (Spanholtz et al., 2011; Shah et al., 2013). In this method, CB monocytes are isolated by Ficoll density gradient centrifugation. Cell cultures can be depleted of any cells expressing CD3 and can be characterized to determine CD56. + / CD3 - In other methods, umbilical cord CB is used to isolate CD34 + Cells are used to derive NK cells.

[0107] B. Loading of NK cells

[0108] In certain embodiments, NK cells are loaded with antibodies before use. NK cells can be loaded in any particular manner, including in culture (e.g., incubation) or immediately before infusion, for example, to produce a complex of NK cells and antibodies. Cultivation (e.g., incubation) conditions are sufficient to allow an effective amount of antibodies to bind to the NK cell surface. In the case of using a monospecific antibody, the Fc region of the monospecific antibody binds to NK cells, and the antigen-binding domains of the monospecific antibody freely bind to its target antigen. In certain embodiments using multispecific antibodies, one or more antigen-binding domains of the antibody can bind to the surface of NK cells, such as by antigens on the NK cell surface (e.g., but not limited to NKp30, NKp44, NKp46, CD16, CD32, CD64, KIR, etc.), and another antigen-binding domain can freely bind to its target antigen. In certain embodiments using multispecific antibodies, one or more antigen-binding domains of the antibody can bind to one or more target antigens.

[0109] The culture conditions of NK cell loading may or may not be a specific type with one or more specific parameters. In a specific embodiment, the loading of NK cells occurs in culture at a specific temperature, such as 37 ° C, but in an alternative embodiment, the temperature is 36 ° C or 38 ° C, or lower or higher. The duration of the loading step can be any suitable amount of time, such as in the range of 1 minute to 24 hours or longer. For example, the range can be 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, 1-24 hours, 1-18 hours, 1-12 hours, 1-6 hours, 6-24 hours, 6-18 hours, 6-12 hours, 12-24 hours, 12-18 hours or 18-24 hours in the range of hours. In some embodiments, the duration of the loading step can be greater than or equal to 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 any range derivable therein. In specific embodiments, the cell culture medium is a basal medium or a complex medium. In some cases, the culture contains one or more reagents used during the pre-activation and / or expansion steps, while in other cases the culture does not contain these reagents. In specific embodiments, the culture contains one or more cytokines, for example, including one or more of IL-12, IL-15, IL-2 and IL-18. In some embodiments, the culture contains any type of APC.

[0110] In some embodiments, the loading of NK cells is such that the antibody is still bound to the NK cell surface after the NK cells are washed. In some embodiments, the NK cells are loaded with antibodies under conditions suitable for at least a detectable portion of the antibody to remain on the NK cell surface for at least or just 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 greater than 312 hours, or any range that can be derived therein. In some embodiments, NK cells are loaded with antibodies under conditions suitable for the antibodies to remain on the surface of NK cells for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days or more than 15 days or any range that can be derived therein. In some embodiments, NK cells are loaded with antibodies under conditions suitable for the antibodies to remain on the surface of NK cells after cryopreservation and / or thawing. In some embodiments, flow cytometry can be used to determine confirmation that the antibodies are loaded on the surface of NK cells.

[0111] In some embodiments, the NK cells are loaded with 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 95, 96, 97, 98, 99, or an antibody at or greater than 100 μg / ml final concentration or any range derivable therein. In some embodiments, the NK cells are loaded with 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, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800 0, 540, 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 final concentration or greater than 1000 μg / ml final concentration or any range derivable therein.

[0112] In some embodiments, the NK cells and the antibody are incubated so 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%, 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%, 1%, 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 of NK cells complexed with the antibody.

[0113] In some embodiments, the NK cells and the antibody are incubated 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%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31% after the NK cells and the antibody are incubated. , 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%, or any range derivable therein, of the antibody remaining bound to NK cells for at least 1 hour , 2 hours, 3 hours, 4 hours, 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 derived therein.

[0114] 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 more than 20%, or any range of antibodies derived therein, bind to the NK cell surface for at least 3 days after incubation and washing, relative to the amount of antibodies complexed with engineered NK cells measured about 1 hour after incubation and washing. In certain embodiments, at least about 9% of the antibodies bind to the NK cell surface for at least 3 days after incubation and washing, relative to the amount of antibodies complexed with engineered NK cells measured about 1 hour after incubation and washing.

[0115] 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 more than 20%, or any range of antibodies derived therein, bind to the NK cell surface for at least 5 days after incubation and washing, relative to the amount of antibodies complexed with engineered NK cells measured about 1 hour after incubation and washing. In certain embodiments, at least about 6% of the antibodies bind to the NK cell surface for at least 5 days after incubation and washing, relative to the amount of antibodies complexed with engineered NK cells measured about 1 hour after incubation and washing.

[0116] 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 more than 20%, or any range of antibodies derived therein, bind to the NK cell surface for at least 7 days after incubation and washing, relative to the amount of antibodies complexed with engineered NK cells measured about 1 hour after incubation and washing. In certain embodiments, at least about 4% of the antibodies bind to the NK cell surface for at least 7 days after incubation and washing, relative to the amount of antibodies complexed with engineered NK cells measured about 1 hour after incubation and washing.

[0117] 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 more than 20%, or any range of antibodies derived therein, bind to the NK cell surface for at least 12 days after incubation and washing, relative to the amount of antibodies complexed with engineered NK cells measured about 1 hour after incubation and washing. In certain embodiments, at least about 3% of the antibodies bind to the NK cell surface for at least 12 days after incubation and washing, relative to the amount of antibodies complexed with engineered NK cells measured about 1 hour after incubation and washing.

[0118] In certain embodiments, the composition comprising engineered NK cells and antibodies is cryopreserved. In certain embodiments, the composition comprising engineered NK cells and antibodies 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 compounded with the antibodies.

[0119] In certain embodiments, relative to the amount of antibody complexed with non-cryopreserved engineered NK cells measured about 1 hour after incubation and washing, after thawing from cryopreservation, 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 more than 90% or any range of antibodies derived therein bind to the NK cell surface. In certain embodiments, relative to the amount of antibody complexed with non-cryopreserved engineered NK cells measured about 1 hour after incubation and washing, after thawing from cryopreservation, at least about 30% of the antibody binds to the NK cell surface. In certain embodiments, at least about 40% of the antibodies are bound to the NK cell surface after thawing from cryopreservation, relative to the amount of antibodies complexed with non-cryopreserved engineered NK cells measured about 1 hour after incubation and washing. In certain embodiments, at least about 50% of the antibodies are bound to the NK cell surface after thawing from cryopreservation, relative to the amount of antibodies complexed with non-cryopreserved engineered NK cells measured about 1 hour after incubation and washing. In certain embodiments, at least about 60% of the antibodies are bound to the NK cell surface after thawing from cryopreservation, relative to the amount of antibodies complexed with non-cryopreserved engineered NK cells measured about 1 hour after incubation and washing.

[0120] In some embodiments, the incubation of NK cells and antibodies occurs in any suitable NK cell culture medium known to those skilled in the art. In certain embodiments, the incubation of NK cells and antibodies is carried out in a culture medium comprising, consisting of, or consisting essentially of Click's / RPMI culture medium. In certain embodiments, the incubation of NK cells and antibodies is carried out in a culture medium comprising, consisting of, or consisting essentially of Click's culture medium. In certain embodiments, the incubation of NK cells and antibodies is carried out in a culture medium comprising, consisting of, or consisting essentially of RPMI culture medium. In certain embodiments, the incubation of NK cells and antibodies is carried out in a culture medium comprising, consisting of, or consisting essentially of SCGM culture medium.

[0121] An effective amount of the antibody of the composition is exposed to an effective amount of the NK cells of the present disclosure, thereby producing a "chimeric antigen receptor-like" complex. Specifically, the antigen binding domain of the antibody is, for example, bound to the NK cell by an antigen as a cell surface protein. A variety of antibodies can undergo a variety of NK cells, thereby there are a variety of cell / antibody complexes. The antibody can be any time, including monospecific, bispecific or multispecific, and in certain cases, the antibody is engaged by the antigen binding domain of the antibody (for example, using a conjugate in the art, which is a fusion protein composed of two single-chain variable fragments (scFv) of different antibodies) NK cells and target antigens. In the example where the antibody is monospecific, the antigen binding domain of the antibody binds to the target antigen, such as a cancer antigen, and another part of the antibody binds to the NK cell, such as the Fc region of the antibody. In the case where the antibody is multispecific, one or more antigen binding domains of the antibody bind to NK cells (for example, through NK cell surface antigens) and one or more antigen binding domains of the antibody bind to one or more target antigens. Multispecific antibodies can be, for example, bispecific, trispecific or tetraspecific. Where the antibody is trispecific or tetraspecific, the additional antigen binding domains may bind other cells, such as stem cells.

[0122] 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, co-stimulatory molecules such as CS1, DNAM, 2B4, CD2, NCR, NKp30, NKp44, NKp46 or KIR, and redirect NK cells to the target, thereby increasing the response and specificity against different tumors.

[0123] In some embodiments, the antibody can be combined with any suitable antigen (e.g., an antigen described herein, such as an antigen described as a target of a 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 imatinib, ervantumab, and / or magetuximab.

[0124] The generation of the complex can be carried out in any suitable manner so that the conditions are sufficient to allow the appropriate region of the antibody to bind to the appropriate surface region of the NK cell. In some cases, any specific culture medium can be used. In certain cases, Plasma-Lyte A and / or human serum albumin are used, and in other cases, they are not used. Once the complex is formed in the culture, they may or may not be washed before being administered to the subject (e.g., by infusion). In alternative embodiments, the NK cells and antibodies are administered separately, and the complex is formed in vivo. In certain embodiments, the composition comprising the NK cells and antibodies is washed with PBS to remove unbound antibodies. In certain embodiments, the composition comprising the NK cells and antibodies is washed at least 1, 2, 3, 4 or 5 times or more than 5 times to remove unbound antibodies. In certain embodiments, the composition comprising the NK cells and antibodies is washed at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 minutes or more than 10 minutes to remove unbound antibodies. In certain embodiments, the composition comprising the NK cells and antibodies is washed twice to remove unbound antibodies. In certain embodiments, the washing time is just or about 5 minutes. In certain embodiments, washing comprises agitating the composition, for example using a cell shaker.

[0125] C. Pre-activation

[0126] In some embodiments, NK cells are preactivated before being administered to a recipient individual. The preactivation step may or may not occur before any amplification step. In a specific embodiment, NK cells are preactivated with one or more cytokines, and in a specific embodiment, NK cells are preactivated with one or more of IL-12, IL-15, IL-2, and IL-18 and include two, three, or more preactivations. In the case of using less than all three of IL-12, IL-15, IL-2, and IL-18, it may 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 replace IL-15.

[0127] In a specific embodiment, the pre-activation cytokine can be IL-12, IL-15 and IL-18. One or more other cytokines can be used for the pre-activation step. Pre-activation can last for a shorter time, such as 5-72 hours, such as 10-50 hours, particularly 10-20 hours, such as 12, 13, 14, 15, 16, 17, 18, 19 or 20 hours, and particularly about 16 hours in some cases. The pre-activation culture can include a concentration of 10-100ng / mL, such as 40-60ng / mL, particularly 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 or 55ng / mL, particularly about 50ng / mL of IL-18 and / or IL-15. In some cases, the pre-activation culture includes a concentration of 0.1-150ng / mL, including a concentration of 1-20ng / mL, such as a concentration of 10ng / mL of IL-12. In alternative embodiments, NK cells can be stimulated with IL-2 or other cytokines (e.g., IL-7, IL-21, etc.) that bind to a common γ chain, and this can be in addition to IL-12, IL-15, and IL-18 or as a substitute for one or more thereof. In this case, the pre-activated culture can include 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.

[0128] D. Amplification

[0129] In a specific embodiment, before being applied to an individual in need, NK cells are expanded to increase their number. The expanded cells may or may not be derived from pre-activated NK cells, so that the pre-activation step can occur before the expansion step. The NK cell expansion step can be any suitable, so that the NK cell population is expanded, but in certain cases, the expansion step utilizes specific one or more reagents, such as in culture, to enhance their expansion. In some cases, NK cells may not be expanded. IL-2 or IL-15 or IL-18 or any combination of cytokines can be added to the expansion culture before or during expansion. In a specific embodiment, NK cells can be expanded in vitro in a flask or using continuous perfusion of culture medium / additives in one of several different bioreactor configurations.

[0130] In certain cases, 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, for example 2, 3, 4 or 5 times, in particular 3 times. In a specific embodiment, NK cells are expanded in the presence of artificial antigen presenting cells (aAPC). In a specific embodiment, NK cells are expanded in the presence of aAPC fragments. aAPCs may be engineered to express CD137 ligands and / or membrane-bound cytokines. The membrane-bound cytokine may be membrane-bound IL-21 (mIL-21) or membrane-bound IL-15 (mIL-15). In a specific embodiment, aAPCs are engineered to express CD137 ligands and mIL-21. aAPCs may be derived from cancer cells, such as leukemia cells. aAPCs may not express endogenous class I HLA, class II HLA or CD1d molecules. They may express ICAM-1 (CD54) and LFA-3 (CD58) or CD48. Specifically, aAPC can be K562 cells, for example, K562 cells engineered to express CD137 ligands and mIL-21. Engineering can be performed by any method known in the art, such as retroviral transduction, although any viral or non-viral vector can be used. aAPC may be irradiated or may not be irradiated. Amplification can last for a specific duration, such as about 2-30 days, such as 3-20 days, particularly 12-16 days, such as 12, 13, 14, 15, 16, 17, 18 or 19 days, particularly about 14 days. Preactivated NK cells and aAPC can be present in a ratio of about 3: 1-1: 3, such as 2: 1, 1: 1, 1: 2, particularly about 1: 2. The amplification culture may also include one or more cytokines that promote amplification, such as IL-2. IL-2 can be present in a concentration of about 10-500U / mL, such as 100-300U / mL, particularly about 200U / mL. IL-2 can be supplemented during expansion culture, including at a certain frequency, such as every 2-3 days. aAPC can be added to the culture at least a second time, such as at about 7 days of expansion. Any cytokine used in the pre-activation and / or expansion steps can be a recombinant human cytokine.

[0131] After expansion, NK cells can be used immediately in any manner, such as complexed with one or more antibodies, or they can be stored, such as by cryopreservation. In certain aspects, the cells can be proliferated ex vivo as a large population for days, weeks or months within about 1, 2, 3, 4 or 5 days.

[0132] Activated and / or amplified NK cells can secrete type I cytokines, such as interferon-γ, tumor necrosis factor-α, and granulocyte-macrophage colony stimulating factor (GM-CSF), which activate innate and adaptive immune cells and other cytokines and chemokines. Measurement of these cytokines can be used to determine the activation state of NK cells. In addition, other methods known in the art for determining NK cell activation can be used to characterize the NK cells of the present disclosure.

[0133] Therefore, with respect to the specific pre-activation and expansion aspects of the present disclosure, in a specific embodiment, NK cells pre-activated with any combination of IL-12, IL15 and / or IL-18, and then amplified with aAPC (e.g., K562 cells expressing mIL-21 and CD137 ligands) provide efficient cell products. Therefore, methods for treating various diseases (e.g., immunotherapy of cancer patients) using NK cells of the present invention are provided. In an exemplary method, the separated NK cells can be pre-activated for a short period of time (e.g., about 16 hours) with a combination of cytokines (e.g., interleukin-12 (IL-12), IL-15 and / or IL-18), and then amplified using artificial antigen presenting cells (aAPC) (e.g., K562 feeder cells expressing membrane-bound IL-21 and CD137 ligands) and / or exogenous IL-2. IL-2 or IL-15 or IL-18 or any combination of cytokines can be added to the amplified culture at least a second time.

[0134] E. Engineering of NK cells

[0135] As described herein, in some embodiments, compared with natural NK cells, NK cells are modified (e.g., engineered) in one or more ways. Engineered NK cells can be artificially manipulated to be engineered to express one or more transgenics. Engineered NK cells can be artificially manipulated to be engineered to express one or more heterologous proteins. Alternatively or additionally, engineered NK cells can be modified to reduce or inhibit the expression of one or more endogenous genes. Alternatively or additionally, engineered NK cells can be modified to express one or more chemokines, chemokine receptors, cytokines, cytokine receptors and / or suicide genes. In some embodiments, NK cells are engineered to express CD16, CD32, CD64 or other FCR binding regions. In some embodiments, engineered NK cells do not include non-naturally occurring CD16 genes. In some embodiments, engineered NK cells do not include transgenics encoding CD16 genes. In certain embodiments, NK cells are engineered to express CD16 genes. In certain embodiments, NK cells are engineered to express wild-type CD16 genes. In certain embodiments, NK cells are engineered to express CD32 genes. In certain embodiments, the NK cells are engineered to express the CD64 gene. In certain embodiments, the NK cells are engineered to express a transgenic receptor that is a target of a multispecific antibody.

[0136] 1. Engineered Antigen Receptors

[0137] NK cells can 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 receptor is directed against one or more cancer antigens, and the antigen may or may not be the same antigen to which a multispecific antibody (e.g., conjugate) is directed.

[0138] NK cells can be modified to encode at least one CAR, and the CAR can be, for example, the first generation, the second generation, or the third generation or subsequent generations. CAR may or may not be bispecific for two or more different antigens. CAR may include one or more costimulatory domains. NK cells may also be modified to express receptors (e.g., CD16, CD32, and / or CD64 receptors) to enhance their binding to antibodies. Each costimulatory domain may, for example, include, for example, TNFR superfamily members, 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 any one or more of its combinations. In a specific embodiment, CAR includes CD3ζ. In certain embodiments, the CAR lacks one or more specific co-stimulatory domains; for example, the CAR may lack 4-1BB and / or lack CD28.

[0139] In a specific embodiment, the CAR polypeptide in the cell comprises an extracellular spacer domain connecting the antigen binding domain and the transmembrane domain, and this can be referred to as a hinge. The extracellular spacer domain may include, but is not limited to, an Fc fragment of an antibody or its fragment or derivative, a hinge region of an antibody or its fragment or derivative, 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, CD8-α hinges, CD28, artificial spacers made of polypeptides such as Gly3, or CH1, CH3 domains of IgG (e.g., human IgG1 or IgG4). In a specific case, the extracellular spacer domain may include (i) hinge, CH2 and CH3 regions of IgG4, (ii) hinge region of IgG4, (iii) hinge and CH2 of IgG4, (iv) hinge region of CD8-α or CD4, (v) hinge, CH2 and CH3 regions of IgG1, (vi) hinge region of IgG1 or (vii) hinge and CH2 of IgG1, (viii) hinge region of CD28, or a combination thereof. In a specific embodiment, the hinge is from IgG1, and in certain aspects, the CAR polypeptide includes a specific IgG1 hinge amino acid sequence or is encoded by a specific IgG1 hinge nucleic acid sequence.

[0140] The membrane-spanning domain in CAR can be derived from natural or synthetic sources.When the source is natural, the domain is derived from any membrane-bound protein or membrane-spanning protein in some aspects.The membrane-spanning region includes α, β or ζ chains derived from T cell receptors, CD28, CD3ζ, CD3ε, CD3γ, CD3δ, 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 (i.e., at least including its membrane-spanning region). Alternatively, the membrane-spanning domain in some embodiments is synthetic.In some aspects, the synthetic membrane-spanning domain mainly includes hydrophobic residues, such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan, and valine can be present at each end of the synthetic transmembrane domain.

[0141] In certain embodiments, CAR can be co-expressed with one or more cytokines to improve persistence in the presence of low amounts of tumor-associated antigens. For example, 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 a combination thereof. In some embodiments, NK cells expressing CAR are engineered to express one or more heterologous cytokines and / or engineered to upregulate the normal expression of one or more heterologous cytokines. One or more cytokines may or may not be transduced or transfected to cells on the same vector as other genes.

[0142] In certain embodiments, the CD27 CAR targets CD70. In some embodiments, the CAR comprises a CD28-derived transmembrane domain. In some embodiments, the CAR comprises a CD3ζ intracellular stimulatory domain. In some embodiments, the CAR is co-expressed with IL-15.

[0143] In certain embodiments, 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, 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% sequence identity to SEQ ID NO: 2, 5, 7, 9, 12, 14 or 16, or any range derivable therein.

[0144] SEQ ID NO: 1—Exemplary polynucleotide sequence comprising a CD27 CAR construct targeting the CD70 antigen.

[0145] ATGGCACGGCCACATCCCTGGTGGCTGTGCGTTCTGGGGACCCTGGTGGGGCTCTCAGCTACTCCAGCCCCCAAGAGCTGCCCAGAGAGGCACTACTGGGCTCAGGGAAAGCTGTGCTGCCAGATGTGTGAGCCAGGAACATTCCTCGTGAAGGACTGTGACCAGCATAGAAAGGCTGCTCAGTGTGATCCTTGCATACCGGGGGTCTCCTTCTCTCCTGACCACCACACCCGGCCCCACTGTGAGAGCTGTCGGCACTGTAACTCTGGTCTTCTCGTTCGCAACTGCACCATCACTGCCAATGCTGAGTGTGCCTGTCGCAATGGCTGGCAGTGCAGGGACAAGGAGTGCACCGAGTGTGATCCTCTTCCAAACCCTTCGCTGACCGCTCGGTCGTCTCAGGCCCTGAGCCCACACCCTCAGCCCACCCACTTACCTTAT GTCAGTGAGATGCTGGAGGCCAGGACAGCTGGGCACATGCAGACTCTGGCTGACTTCAGGCAGCTGCCTGCCCGGACTCTCTCTACCCACTGGCCACCCCAAAGATCCCTGTGCAGCTCCGATTTTATTCGCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTG(SEQ ID NO:1)

[0146] SEQ ID NO:2 – Exemplary amino acid sequence of a CD27 CAR construct targeting the CD70 antigen.

[0147] MARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRFWVLVVVGGVLACYSLLVTVAFIIFWV(SEQ ID NO:2)

[0148] SEQ ID NO:3—Exemplary polynucleotide sequence comprising a CD27 CAR construct targeting the CD70 antigen.

[0149] ATGGCACGGCCACATCCCTGGTGGCTGTGCGTTCTGGGGACCCTGGTGGGGCTCTCAGCTACTCCAGCCCCCAAGAGCTGCCCAGAGAGGCACTACTGGGCTCAGGGAAAGCTGTGCTGCCAGATGTGTGAGCCAGGAACATTCCTCGTGAAGGACTGTGACCAGCATAGAAAGGCTGCTCAGTGTGATCCTTGCATACCGGGGGTCTCCTTCTCTCCTGACCACCACACCCGGCCCCACTGTGAGAGCTGTCGGCACTGTAACTCTGGTCTTCTCGTTCGCAACTGCACCATCACTGCCAATGCTGAGTGTGCCTGTCGCAATGGCTGGCAGTGCAGGGACAAGGAGTGCACCGAGTGTGATCCTCTTCCAAACCCTTCGCTGACCGCTCGGTCGTCTCAGGCCCTGAGCCCACACCCTCAGCCCACCCACTTACCTTATGTCAGTGAGATGCTGGAGGCCAGGACAGCTGGGCACATGCAGACTCTGGCTGACTTCAGGCAGCTGCCTGCCCGGACTCTCTCTACCCACTGGCCACCCCAAAGATCCCTGTGCAGCTCCGATTTTATTCGCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCACGCGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAAAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO:3)

[0150] SEQ ID NO:4 - Exemplary polynucleotide sequences comprising a CD27 CAR construct targeting the CD70 antigen and IL-15.

[0151] ATGGCACGGCCACATCCCTGGTGGCTGTGCGTTCTGGGGACCCTGGTGGGGCTCTCAGCTACTCCAGCCCCCAAGAGCTGCCCAGAGAGGCACTACTGGGCTCAGGGAAAGCTGTGCTGCCAGATGTGTGAGCCAGGAACATTCCTCGTGAAGGACTGTGACCAGCATAGAAAGGCTGCTCAGTGTGATCCTTGCATACCGGGGGTCTCCTTCTCTCCTGACCACCACACCCGGCCCCACTGTGAGAGCTGTCGGCACTGTAACTCTGGTCTTCTCGTTCGCAACTGCACCATCACTGCCAATGCTGAGTGTGCCTGTCGCAATGGCTGGCAGTGCAGGGACAAGGAGTGCACCGAGTGTGATCCTCTTCCAAACCCTTCGCTGACCGCTCGGTCGTCTCAGGCCCTGAGCCCACACCCTCAGCCCACCCACTTACCTTATGTCAGTGAGATGCTGGAGGCCAGGACAGCTGGGCACATGCAGACTCTGGCTGACTTCAGGCAGCTGCCTGCCCGGACTCTCTCTACCCACTGGCCACCCCAAAGATCCCTGTGCAGCTCCGATTTTATTCGCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCACGCGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAAAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGACCGCAGTGTACTAATTATGCTCTCTTGAAATTGGCTGGAGATGTTGAGAGCAATCCCGGGCCCATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGCTGA(SEQ ID NO:4)

[0152] SEQ ID NO:5—Exemplary amino acid sequence of a CD27 CAR construct targeting the CD70 antigen.

[0153] MARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRD KECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHS DYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:5)

[0154] SEQ ID NO:6—Exemplary polynucleotide sequence comprising a TROP2 CAR construct.

[0155]

[0156] SEQ ID NO: 7 - Exemplary amino acid sequence comprising a TROP2 CAR construct.

[0157] MEFGLSWLFLVAILKGVQCSREDIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKRLEIKGSTSGSGKPGSGEGSTQVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSSPYAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPKFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRG (SEQ ID NO: 7)

[0158] SEQ ID NO: 8 - Exemplary polynucleotide sequence comprising a TROP2 CAR scFv.

[0159] ATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTGGCTATTTTAAAAGGTGTCCAGTGCTCTAGAGAGGACATCCAGCTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCAGCATCACCTGCAAGGCCAGTCAGGATGTGAGTATTGCTGTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTACTCGGCATCCTACCGGTACACTGGAGTCCCTGATAGGTTCAGTGGCAGTGGA TCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAGTTTATTACTGTCAGCAACATTATATTACTCCGCTCACGTTCGGTGCTGGGACCAAGGTGGAGATCAAACGTTTGGAAATAAAGGGCTCTACAAGCGGCTCAGGAAAACCTGGATCAGGCGAAGGGTCTACGCAGGTCCAACTGCAGCAATCTGGGTCTGAGTTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCCTGCAAGGCTTCTGGATACACCTTCACAAACTATGGAATGAACTGGGTGAAGCAGGCCCCTGGACAAGGGCTTAAATGGATGGGCTGGATAAACACCTACACTGGAGAGCCAACATATACTGATGACTTCAAGGGACGGTTTGCCTTCTCCTTGGACACCTCTGTCAGCACGGCATATCTCCAGATCAGCAGCCTAAAGGCTGACGACACTGCCGTGTATTTCTGTGCAAGAGGGGGGTTCGGTAGTAGCTACTGGTACTTCGATGTCTGGGGCCAAGGGTCCCTGGTCACCGTCTCCTCA(SEQ ID NO:8)

[0160] SEQ ID NO:9 – Exemplary amino acid sequence containing the TROP2 CAR scFv.

[0161] MEFGLSWLFLVAILKGVQCSREDIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKRLEIKGSTSGSGKPGSGEGSTQVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSS(SEQ ID NO:9)

[0162] SEQ ID NO:10 - Exemplary vector sequence containing iCas9, TROP2 CAR, and IL-15.

[0163]

[0164] The sequence of the open reading frame encoding the chimeric receptor can be obtained from a genomic DNA source, a cDNA source, or can be synthesized (e.g., by PCR), or a combination thereof. Depending on the size of the genomic DNA and the number of introns, it may be desirable to use cDNA or a combination thereof, as introns have been found to stabilize mRNA. Furthermore, it may be further advantageous to use endogenous or exogenous non-coding regions to stabilize mRNA.

[0165] It is contemplated that the chimeric construct can be introduced into any type of immune cell as naked DNA or in a suitable vector. Methods for stably transfecting cells using naked DNA by electroporation are known in the art. See, for example, U.S. Patent No. 6,410,319. Naked DNA generally refers to DNA encoding a chimeric receptor contained in a plasmid expression vector in an appropriate expression orientation.

[0166] Alternatively, viral vectors (e.g., retroviral vectors, adenoviral vectors, adeno-associated viral vectors, or lentiviral vectors) can be used to introduce the chimeric construct into immune cells. Suitable vectors for use according to the methods of the present disclosure are non-replicative in immune cells. A large number of viral-based vectors are known, in which the viral copy number maintained in the cell is low enough to maintain the viability of the cell, such as vectors based on HIV, SV40, EBV, HSV, or BPV. Non-viral vectors include plasmids, transposons, nanoparticles, liposomes, lipids, metals, or combinations thereof.

[0167] In some embodiments, the genetically engineered antigen receptor includes a recombinant TCR and / or a TCR cloned from a naturally occurring T cell."T cell receptor" or "TCR" refers to a molecule containing a variable α chain and a β chain (also referred to as TCRα and TCRβ, respectively) or a variable γ chain and a δ chain (also referred to as TCRγ and TCRδ, respectively), and capable of specifically binding to an antigenic peptide bound to an MHC receptor. In some embodiments, the TCR is an αβ form.

[0168] Generally, TCRs in the form of αβ and γδ are usually similar in structure, but the T cells expressing them may have different anatomical locations or functions. TCRs may be present on the cell surface or in a soluble form. Generally speaking, TCRs are present on the surface of T cells (or T lymphocytes), where they are generally responsible for identifying antigens bound to major histocompatibility complex (MHC) molecules. In some embodiments, TCRs may also contain constant domains, transmembrane domains and / or short cytoplasmic tails (see, for example, Janeway et al., 1997). For example, in some aspects, each chain of TCR may have an N-terminal immunoglobulin variable domain, an immunoglobulin constant domain, a transmembrane region and a short cytoplasmic tail at the C-terminus. In some embodiments, TCRs are associated with the invariant proteins of the CD3 complexes involved in mediating signal transduction. Unless otherwise indicated, the term "TCR" should be understood to encompass its functional TCR fragments. The term also encompasses complete or full-length TCRs, including TCRs in the form of αβ or γδ. In some embodiments, the TCR and / or CD3 complex is as described in international application PCT / US2022 / 074062, filed on July 22, 2022, and published as WO2023004425A2 on January 26, 2023, which is incorporated herein in its entirety for the purposes described herein.

[0169] Therefore, for the purposes of this article, reference to a TCR includes any TCR or functional fragment, such as an antigen binding portion of a TCR that binds to a specific antigenic peptide (i.e., an MHC-peptide complex) bound to an MHC molecule. "Antigen binding portion" or "antigen binding fragment" of a TCR can be used interchangeably and refers to a molecule that contains a portion of a TCR domain but binds to an antigen (e.g., an MHC-peptide complex) bound by a full-length TCR. In some cases, the antigen binding portion comprises a variable domain of a TCR, such as a variable α chain and a variable β chain of a TCR, which is sufficient to form a binding site for binding to a specific MHC-peptide complex, such as typically when each chain comprises three complementary determining regions.

[0170] In some embodiments, the variable domains of the TCR chains associate to form a loop or complementary determining region (CDR) similar to an immunoglobulin, which confers antigen recognition and determines peptide specificity by forming a binding site for the TCR molecule. Typically, like immunoglobulins, CDRs are separated by framework regions (FRs) (see, e.g., Jores et al., 1990; Chothia et al., 1988; Lefranc et al., 2003). In some embodiments, CDR3 is the main CDR responsible for identifying processed antigens, although the CDR1 of the α chain has also been shown to interact with the N-terminal portion of the antigenic peptide, while the CDR1 of the β chain interacts with the C-terminal portion of the peptide. CDR2 is considered to recognize MHC molecules. In some embodiments, the variable region of the β-chain may contain additional hypervariable (HV4) regions.

[0171] In some embodiments, the TCR chain contains a constant domain. For example, like immunoglobulins, the extracellular portion of a TCR chain (e.g., an α chain, a β chain) can contain two immunoglobulin domains, a variable domain (e.g., a V chain) at the N-terminus and a V chain at the N-terminus. a or Vp; usually amino acids 1 to 116 based on Kabat numbering (Kabat et al., "Sequences of Proteins of Immunological Interest, USDept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5th ed.), and a constant domain adjacent to the cell membrane (e.g., the alpha chain constant domain or Ca, usually amino acids 117 to 259 based on Kabat, the beta-chain constant domain or Cp, usually 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 containing CDRs. The constant domain of the TCR domain comprises a short linker sequence in which cysteine ​​residues form a disulfide bond, constituting a connection between the two chains. In some embodiments, the TCR may have additional cysteine ​​residues in each of the alpha chain and the beta chain, such that the TCR contains two disulfide bonds in the constant domain.

[0172] In some embodiments, the TCR chain may contain a transmembrane domain. In some embodiments, the transmembrane domain is positively charged. In some cases, the TCR chain comprises a cytoplasmic tail. In some cases, this structure allows TCR to bind to other molecules such as CD3. For example, a TCR containing a constant domain with a transmembrane region can anchor the protein in the cell membrane and associate with the invariant subunit of the CD3 signaling device or complex.

[0173] In general, CD3 is a multiprotein complex that can have three different chains (γ, δ and ε) and a ζ chain in mammals. For example, in mammals, the complex can contain a homodimer of a CD3γ chain, a CD3δ chain, two CD3ε chains and a 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 property that enables these chains to bind to positively charged T cell receptor chains. The intracellular tails of CD3γ, CD3δ and CD3ε chains each contain a conserved motif, called an immunoreceptor tyrosine activation motif or ITAM, and each CD3δ chain has three. In general, ITAMs participate in the signal transduction ability of the TCR complex. These auxiliary molecules have a negatively charged transmembrane region and play a role in propagating signals from TCR to cells. CD3- and ζ- chains form the so-called T cell receptor complex together with TCR.

[0174] In some embodiments, TCR can be a heterodimer of two chains α and β (or optionally γ and δ), or it can be a single-chain TCR construct. In some embodiments, TCR is a heterodimer containing two separate chains (α and β chains or γ and δ chains) connected, for example, by one or more disulfide bonds. In some embodiments, the TCR of the target antigen (e.g., cancer antigen) is identified and introduced into the cell. In some embodiments, the nucleic acid encoding 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, TCR is obtained from a biological source, such as from a cell, such as from a T cell (e.g., a cytotoxic T cell), a T cell hybridoma, or other publicly available sources. In some embodiments, T cells can be obtained from cells separated from the body. In some embodiments, high-affinity T cell clones can be separated from patients, and TCR can be separated. In some embodiments, T cells can be cultured T cell hybridomas or clones. In some embodiments, the TCR clones of the target antigen have been produced 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 directed against a target antigen (see, e.g., Varela-Rohena et al., 2008 and Li, 2005). In some embodiments, a TCR or antigen binding portion thereof can be produced synthetically based on knowledge of the TCR sequence.

[0175] The CAR and TCR of the present disclosure target one or more specific antigens.The antigen targeted by the genetically engineered antigen receptor is an antigen expressed in the context of a disease, condition or cell type targeted by adoptive cell therapy.Diseases and conditions include proliferative, neoplastic and malignant diseases and disorders, including cancer and tumors, including blood cancers, immune system cancers, such as lymphomas, leukemias and / or myeloma, such as B, T and myeloid leukemia, lymphoma and multiple myeloma.In some embodiments, compared with normal or non-targeted cells or tissues, antigens are selectively expressed or overexpressed on cells (such as tumors or pathogenic cells) of a disease or condition.In other embodiments, antigens are expressed on normal cells and / or expressed on engineered cells.

[0176] Any suitable antigen can be targeted in the present method. In some cases, the antigen may be associated with certain cancer cells, but not with non-cancerous cells. Exemplary antigens include, but are not limited to, antigen molecules from infectious agents, self / autologous antigens, tumor / cancer-associated antigens, and tumor neoantigens (Linnemann et al., 2015). In particular aspects, antigens include CD19, EBNA, HER2, CA-125, TRAIL / DR4, CD20, CD70, CD38, trop2, HLA-G, CD123, CLL1, carcinoembryonic antigen, alpha-fetoprotein, CD56, AKT, Her3, epithelial tumor antigen, CD319 (CS1), ROR1, folate binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, CD5, CD23, CD30, HERV-K, IL-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-A10, MAGE-A12, MART-1, melanoma-associated antigen, BAGE, DAM-6, -10, GAGE-1, -2, -8, G AGE-3, -4, -5, -6, -7B, NA88-A, MC1R, mda-7, gp75, Gp100, PSA, PSM, tyrosinase, tyrosinase-related protein, TRP-1, TRP-2, ART-4, CAMEL, CEA, Cyp-B, hTERT, hTRT, iCE, MUC1, MUC2, phosphoinositide 3-kinase (PI3K), TRK receptor, PRAME, P15, RU1, RU2, SART-1, SART-3, Wilms tumor antigen (WT1), AFP, -catenin / m, caspase-8 / m, CDK-4 / m, ELF2M, GnT-V, G250, HAGE, HSP70-2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, myosin / m, RAGE, SART-2, TRP-2 / INT2, 707-AP, annexin II, CDC27 / m, TPI / mbcr-abl, BCR-ABL, interferon regulatory factor 4 (IRF4), ETV6 / AML, LDLR / FUT, Pml / RAR, tumor-associated calcium 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 its regulatory subunits, PMSA, PR-3, MDM2, mesothelin, renal cell carcinoma-5T4, SM22-α, 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, mesothelin, 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, legumin, TIE2, Page4, MAD-CT-1, FAP, MAD-CT-2, fos-related antigen 1, CBX2, CLDN6, SPANX, TPTE, ACTL8, ANKRD30A, CDKN2A, MAD2L1, CTAG1B, SUNC1 and LRRN1. Examples of antigen sequences are known in the art, for example, in, 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_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_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_009272 ... 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 number NG_013245.1), Mage-A10 (Accession number NC_000023.11), TRAIL / DR4 (Accession number NC_000003.12) and / or CEA (Accession number NC_000019.10).

[0177] Tumor-associated antigens may be derived from, for example, prostate, breast, colorectal, lung, pancreas, kidney, mesothelioma, ovary, liver, brain, bone, stomach, spleen, testis, cervix, anus, gallbladder, thyroid or melanoma cancer. Exemplary tumor-associated antigens or tumor cell-derived antigens include MAGE1, 3 and MAGE4 (or other MAGE antigens, such as those disclosed in International Patent Publication No. WO99 / 40188); PRAME; BAGE; RAGE, Lage (also known as NY ESO 1); SAGE; and HAGE or GAGE. These non-limiting examples of tumor antigens are expressed in a variety of tumor types, such as melanoma, lung cancer, sarcoma and bladder cancer. See, for example, U.S. Pat. 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 transmembrane epithelial antigens of the prostate (STEAP).

[0178] Other tumor-associated antigens include Plu-1, HASH-1, HasH-2, Cripto, and Criptin. In addition, tumor antigens can be self-peptide hormones, such as full-length gonadotropin-releasing hormone (GnRH), a short 10-amino acid long peptide that can be used to treat many cancers.

[0179] Antigens may include epitope regions or epitope peptides derived from genes that are mutated in tumor cells or from genes that are transcribed at different levels in tumor cells compared to normal cells, such as telomerase, survivin, mesothelin, mutated ras, bcr / abl rearrangement, Her2 / neu, mutant or wild-type p53, cytochrome P450 1B1, and abnormally expressed intronic sequences, such as N-acetylglucosaminyltransferase-V; clonal rearrangements of immunoglobulin genes that produce unique idiotypes in myeloma and B-cell lymphoma; tumor antigens, which include epitope regions or epitope peptides derived from tumor viral processes, such as human papillomavirus proteins E6 and E7; Epstein bar virus protein LMP2; non-mutated oncofetal proteins with tumor-selective expression, such as carcinoembryonic antigen and alpha-fetoprotein.

[0180] 2. Suicide Gene

[0181] In specific embodiments, suicide genes are used in combination with antibody combination therapies to control their use and allow cell therapy to be terminated at desired events and / or times. The purpose of using suicide genes in transduced cells is to trigger transduced cell death when needed. Cells of the present disclosure that have been modified to carry vectors encompassed by the present disclosure may include one or more suicide genes. In some embodiments, the term "suicide gene" as used herein is defined as a gene that achieves conversion of a gene product into a compound that kills its host cell after administration of a prodrug or other agent. In other embodiments, the suicide gene encodes a gene product that, when needed, is targeted by an agent (e.g., an antibody) that targets the suicide gene product.

[0182] In some cases, cell therapy may undergo the use of one or more suicide genes of any kind when the individual receiving the cell therapy and / or who has received the cell therapy exhibits one or more symptoms of one or more adverse events (e.g., cytokine release syndrome, neurotoxicity, allergic reaction / allergy, and / or on-target / off-target tumor toxicity (as examples)) or is considered at risk of developing one or more symptoms (including imminent symptoms). The use of suicide genes may be part of a planned treatment regimen, or may be used only when it is recognized that they are needed. In some cases, cell therapy is terminated by using an agent that targets a suicide gene or its gene product because treatment is no longer needed.

[0183] The use of suicide genes can be initiated when at least one adverse event occurs in an individual, and the adverse event can be identified by any means, including identification based on routine monitoring that may or may not be continuous from the start of cell therapy. Adverse events can be detected by inspection and / or testing. In the case where an individual suffers from cytokine release syndrome (which may also be referred to as a cytokine storm), the individual may, for example, have elevated inflammatory cytokines (for example only: interferon-γ, granulocyte macrophage colony stimulating factor, IL-10, IL-6, and TNF-α); fever; fatigue; hypotension; hypoxia, tachycardia; nausea; capillary leakage; heart / kidney / liver dysfunction; or a combination thereof. In the case where an individual has neurotoxicity, the individual may suffer from mental confusion, delirium, dysplasia, and / or epileptic seizures. In some cases, the individual is tested for markers (e.g., C-reactive protein, IL-6, TNF-α, and / or ferritin) associated with the onset and / or severity of cytokine release syndrome.

[0184] Examples of suicide genes include engineered non-secretable (including membrane-bound) tumor necrosis factor (TNF)-α mutant polypeptides (see PCT / US19 / 62009, which is incorporated herein by reference in its entirety), and they may be affected by the delivery of antibodies that bind to TNF-α mutants. Examples of suicide gene / prodrug combinations that can 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. Escherichia coli purine nucleoside phosphorylase, a so-called suicide gene, can be utilized, which converts the prodrug 6-methylpurine deoxynucleoside to the toxic purine 6-methylpurine. Other suicide genes include, for example, CD20, CD52, inducible caspase 9, purine nucleoside phosphorylase (PNP), cytochrome p450 enzymes (CYP), carboxypeptidase (CP), carboxylesterase (CE), nitroreductase (NTR), guanine ribosyltransferase (XGRTP), glycosidase, methionine-α,γ-lyase (MET), and thymidine phosphorylase (TP).

[0185] In a specific embodiment, any vector in the NK cell encoding CAR or covered herein includes one or more suicide genes. The suicide gene may be located on the same vector as the CAR, or it may not be on the same vector. In the case where the suicide gene and CAR are present on the same vector, the suicide gene and CAR can be separated, for example, by an IRES or 2A element.

[0186] 3. Cytokines

[0187] In some embodiments, cells expressing NK cells are engineered to express one or more heterologous cytokines and / or are engineered to upregulate 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.

[0188] One or more cytokines can be co-expressed from a vector, including as a polypeptide separated from an engineered antigen receptor and / or suicide gene. For example, interleukin 15 (IL-15) is tissue-restricted, and any level of it can only be observed in serum or systemically under pathological conditions. IL-15 has a variety of properties suitable for adoptive therapy. IL-15 is a homeostatic cytokine that induces the development and cell proliferation of natural killer cells, promotes the eradication of established tumors by alleviating the functional inhibition of tumor-resident cells, and inhibits 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 for human applications. In certain embodiments, NK cells expressing IL-15 can continuously support cytokine signaling, which is useful for their survival after infusion. In certain embodiments, NK cells expressing IL-15 can continuously support cytokine signaling, which is useful for their survival after infusion. In certain embodiments, NK cells expressing IL-21 are able to sustain cytokine signaling, which is useful for their survival after infusion. In certain embodiments, the cytokine is expressed as part of a polycistronic construct with one or more functional proteins and / or marker proteins.

[0189] In some embodiments, a specific sequence of IL-15 is utilized, such as the following sequence (the signal peptide sequence is underlined and may be included or omitted):

[0190] SEQ ID NO: 11—Exemplary polynucleotide sequence comprising IL-15 (signal peptide is underlined).

[0191] ATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAG CCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAG GTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGC(SEQ ID NO:11)

[0192] SEQ ID NO: 12—comprising an exemplary amino acid sequence of IL-15 (signal peptide is underlined).

[0193] MRSSPGNMERIVICLMVIFLGTLV HKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS(SEQ ID NO:12)

[0194] In some embodiments, a specific sequence of IL-21 is utilized, such as the following sequence (the underline indicates the signal peptide sequence, which may be included or omitted):

[0195] SEQ ID NO: 13—Comprising an exemplary codon-optimized polynucleotide sequence of IL-21 (signal peptide is underlined).

[0196] ATGAGGAGCAGTCCAGGCAATATGGAGCGGATAGTAATTTGTCTCATGGTAATATTCCTCGGTACTCT GGTACATAAATCTTTCCTCTCAAGGTCAGGACCGCCATTGATTCGAATGCGGCAGCTGATTGACATAGTCGATCAACTGAAGAACTATGTGAATGATCTTGTGCCCGAGTTTTTGCCAGCCCCTGAAGACGTAGAAACTAATTGTGAGTGGAGTGCCTTTTCCTGCTTTCAAAAGGCACAGCTGAAATCCGCCAACACGGGCAATAACGAACG GATAATTAACGTATCCATTAAGAAGCTGAAGCGGAAGCCGCCCTCAACCAATGCGGGACGGCGGCAAAAGCATCGCTTGACCTGTCCGTCATGCGACAGCTACGAGAAAAAGCCCCCGAAGGAGTTCTTGGAACGCTTCAAGAGTCTCCTTCAGAAAATGATTCACCAGCACCTGTCCTCACGGACGCACGGAAGCGAGGACAGT(SEQ ID NO:13)

[0197] SEQ ID NO: 14—encompasses an exemplary codon-optimized amino acid sequence of IL-21 (signal peptide is underlined).

[0198] MRSSPGNMERIVICLMVIFLGTLV HKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS(SEQ ID NO:14)

[0199] SEQ ID NO: 15—Exemplary polynucleotide sequence comprising IL-21.

[0200] GGCCAGGACCGGCACATGATCCGGATGAGACAGCTGATCGA CATCGTGGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTCCTGCCTGCCCCCGAGGACGTGGAAACAAACTGCGAGTGGAGCGCCTTCAGCTGCTTCCAGAAGGCCCAGCTGAAAAGCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGTCCATCAAGAAGCTGA AGCGGAAGCCCCCCAGCACCAACGCCGGAAGAAGGCAGAAGCACAGACTGACCTGCCCCAGCTGCGACAGCTACGAGAAGAAGCCCCCTAAAGAGTTCCTGGAACGGTTCAAGAGCCTGCTGCAGAAGATGATCCACCAGCACCTGAGCAGCCGGACCCACGGCTCTGAGGACAGC(SEQ ID NO:15)

[0201] SEQ ID NO: 16—Comprising an exemplary amino acid sequence of IL-21.

[0202] GQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEW SAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRL TCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS(SEQ ID NO:16)

[0203] In a specific embodiment, the cell expresses one or more exogenously provided cytokines. As an example, the cytokine is IL-15, IL-12, IL-2, IL-18, IL-21, IL-23, GMCSF or a combination thereof. The cytokine can be provided to NK cells exogenously because it is expressed by an expression vector in the cell. In another case, the endogenous cytokine in the cell is upregulated after the regulation of the expression of the endogenous cytokine (e.g., gene recombination at the promoter site of the cytokine). In the case where the cytokine is provided to the cell on the expression construct, the cytokine can be encoded by the same vector as the suicide gene and / or CAR. In some embodiments, the present disclosure relates to the common use of CAR and IL-15 and an optional suicide gene.

[0204] In some embodiments, the cell expresses one or more exogenously provided engineered receptors, wherein the engineered receptors include chemokine receptors and / or cytokine receptors. 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.

[0205] 4. Knockout or knockdown of endogenous genes

[0206] The NK cell production method of the present disclosure may include gene editing of NK cells to remove 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more endogenous genes in NK cells. In some cases, gene editing occurs in NK cells expressing one or more heterologous antigen receptors, while in other cases, gene editing occurs in NK cells that do not express heterologous antigen receptors but will eventually express one or more heterologous antigen receptors in at least some cases. In a specific embodiment, the gene-edited NK cells are amplified NK cells.

[0207] In certain cases, one or more endogenous genes of NK cells are modified, such as expression is disrupted, wherein expression is partially or completely reduced. In certain cases, one or more genes are knocked down or knocked out using the methods of the present disclosure. In certain cases, multiple genes are knocked down or knocked out in the same steps as the methods of the present disclosure. The gene edited in the NK cell can be of any kind, 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 edited in the NK cell enables the NK cell to work more effectively in the tumor microenvironment. In certain cases, 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 knocked down in NK cells. In a specific embodiment, the CISH gene is knocked out or knocked down in NK cells. In a specific embodiment, the CD38 gene is knocked out or knocked down in NK cells. In specific embodiments, the glucocorticoid receptor (GR) gene is knocked out or knocked down in NK cells.

[0208] In some embodiments, gene editing is performed using one or more DNA binding nucleic acids, for example, by RNA-guided endonucleases (RGENs). For example, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-related (Cas) proteins can be used to change. In general, "CRISPR systems" refer to transcripts and other elements related to the expression of CRISPR-related ("Cas") genes or instructing their activity, including sequences encoding Cas genes, tracr (trans-activated CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr pairing sequences (including "direct repeat sequences" and partial direct repeat sequences processed by tracrRNA in the context of endogenous CRISPR systems), guide sequences (also referred to as "spacers" in the context of endogenous CRISPR systems), and / or other sequences and transcripts from CRISPR loci. The method of utilizing the CRISPR system is well known in the art.

[0209] F. Cryopreservation

[0210] In certain cases, the NK cells and / or antibodies of the present disclosure are stored in a cryopreservation medium composition comprising at least one cryoprotectant, serum (human or animal serum) or a non-serum substitute for serum (not human serum or animal serum), and at least one cytokine and / or at least one growth factor. In some cases, the cryoprotectant is dimethyl sulfoxide (DMSO), glycerol, glycerol, hydroxyethanol starch or a combination thereof. The non-serum substitute can be of any kind, including at least platelet lysate and / or blood product lysate (e.g., human serum albumin). In embodiments in which a composition of one or more (including two or more) cytokines is used, the cytokine can be a natural or recombinant or synthetic protein. At least one cytokine can be a cytokine approved by the Food and Drug Administration (FDA). 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, interferon, tumor necrosis factor, stem cell factor, FLT3-ligand, APRIL, thrombopoietin, erythropoietin or a combination thereof. For serum embodiments, serum can be serum of animal origin, such as human serum (including human AB serum) or bovine serum. When used, DMSO and other cryoprotectants can constitute 4-10%, 4-6%, 4-8%, 5-10%, 5-8%, 6-10%, 6-8%, 8-10% and the like compositions.For embodiments in which serum is employed, the serum may constitute 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%, 10-6 0%, 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 a composition. The composition may comprise at least or no more than 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% serum.In specific embodiments, the compositions comprise platelet lysate which may be at any concentration in the composition, but in certain embodiments, the platelet lysate comprises 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%, 50-9 0%, 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 or no more than 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% platelet lysate.

[0211] Compositions can have a certain 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 with a specific concentration. IL-2 can 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 a particular case, IL-2 is present in the composition with a concentration of at least or no more than 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 U / mL. In specific 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 can be present in the composition at a concentration of at least or no more than 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. IL-15 can 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 or no more than 10, 50, 100, 500, 1000, 1500 or 2000 ng / mL.

[0212] Compositions encompassed herein comprising at least one cryoprotectant, serum or non-serum substitute for serum and at least one cytokine and / or at least one growth factor may also include a variety of immune cells and / or stem cells, each of which is a cell of any kind. In a specific embodiment, the cell is a NK cell, a T cell, a B cell, a NKT cell derived from a mature bone marrow or peripheral blood cell, a cell line such as a tumor cell line (e.g., NK92 or other NK lines), a hematopoietic stem cell, an induced pluripotent stem cell, MSC (also referred to as a cell group of "mesenchymal stem cells" and "mesenchymal stromal cells" in the literature) or a mixture thereof, which may be derived from bone marrow, peripheral blood, skin, adipose tissue or a combination thereof. In an embodiment utilizing NK cells, NK cells may or may not be amplified NK cells. Embodiments of the present disclosure also encompass pharmaceutical compositions comprising any composition of the present disclosure and a suitable pharmaceutically acceptable carrier.

[0213] In certain embodiments, cells and / or antibodies are treated with one or more inactivating agents (eg, kinase inhibitors, eg, dasatinib, nilotinib, rapamycin, etc.) prior to cryopreservation.

[0214] In some embodiments, the technology described herein includes deactivating NK cells, including treating NK cells with an effective amount of one or more deactivators under certain conditions to produce deactivated NK cells. In some embodiments, the deactivator is a kinase inhibitor. In some embodiments, the deactivator 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 deactivator is a tyrosine kinase (TK) inhibitor. In some embodiments, the TK inhibitor is lorlatinib, brigatinib, ceritinib, alectinib, crizotinib, bosutinib, ponatinib, nilotinib, dasatinib, imatinib, zanubrutinib, acalabrutinib, ibrutinib, capmatinib, pesidatinib, dacomitinib, osimertinib, erlotinib, gefitinib, lapatinib, afatinib, pemigatinib, erdatinib, nintedanib, gilteritinib, midostaurinib, tucatinib, neratinib, In some embodiments, the TK inhibitor is a BCR-Abl inhibitor. In some embodiments, the TK inhibitor is 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 and / or nilotinib. In some embodiments, the TK inhibitor is dasatinib.

[0215] In some embodiments, the treatment with a deactivator is any time point during the NK cell culture. In some embodiments, the treatment lasts about 24 to about 96 hours, about 36 to about 84 hours, or about 48 to about 72 hours. In some embodiments, the treatment lasts about 24 hours, about 48 hours, or about 72 hours. In some embodiments, the NK cells are treated with a deactivator at a concentration of about 1 to about 1000 nM. In some embodiments, the NK cells are treated with a deactivator at a concentration of about 5 to about 500 nM. In some embodiments, the NK cells are treated with a deactivator at a concentration of about 20 to about 200 nM. In some embodiments, the NK cells are treated with a deactivator at a concentration of about 30 to about 100 nM. In some embodiments, relative to activated NK cells, deactivated 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.

[0216] In some embodiments, the technology described herein includes a method for maintaining the viability of a cell population greater than at least 50% after cryopreservation of the cell population, the method comprising the steps of subjecting the cell population to an effective amount of one or more deactivating agents (e.g., tyrosine kinase inhibitors) to deactivate the cells before cryopreservation, cryopreserving the cells, and thawing the cell population, wherein after thawing, the viability of the cell population exceeds at least 50%. In some cases, after the cell population is cryopreserved, the viability of the cell population after the cells are thawed is at least greater than 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0217] In specific embodiments, the cells of the present disclosure at any step are maintained in the following specific formulation for cryopreservation medium (see, e.g., Table 1 and / or Table 2).

[0218] Table 1. Exemplary cryopreservation media

[0219]

[0220]

[0221] Table 2. Exemplary cryopreservation media with specific concentrations:

[0222]

[0223]

[0224] II. How to use

[0225] Embodiments of the present disclosure relate to methods for treating or preventing medical diseases or conditions using compositions comprising NK cells (e.g., engineered NK cells) and antibodies provided herein. The method comprises administering to a subject a therapeutically effective amount of loaded, optionally pre-activated and optionally amplified NK cells and antibodies, thereby treating or preventing the subject's disease, 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, cancer or infection is treated by transferring a composition comprising an NK cell group and an antibody. At least in some cases, due to the release of proinflammatory cytokines by NK cells, NK cells can reverse the anti-inflammatory tumor microenvironment, and enhance adaptive immune responses by promoting differentiation, activation and / or recruiting auxiliary immune cells to the site of malignant tumors.

[0226] The cancer that the treatment method of the present invention can be used for includes any malignant cell type, such as those found in solid tumors or hematological tumors.Exemplary solid tumors can include, but are not limited to, tumors of organs selected from the group consisting of pancreas, colon, cecum, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate and breast.Exemplary hematological tumors include myeloid tumors, T or B cell malignancies, leukemia, lymphoma, blastoma, myeloma, etc. Further examples of cancers that can 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), peritoneal cancer, gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, various types of head and neck cancer and melanoma.

[0227] In certain embodiments, the compositions of the present invention and the methods described herein can be used to treat, prevent and / or improve symptoms of cancers including cancers that express at least EGFR, EGFR2 and / or c-MET. In certain embodiments, the compositions of the present invention and the methods described herein can be used to treat, prevent and / or improve symptoms of cancers including cancers that express at least CD70 and / or TROP2. In certain embodiments, the compositions and methods described herein are used to treat, prevent and / or improve symptoms associated with PDAC. In certain embodiments, the compositions and methods described herein are used to treat, prevent and / or improve symptoms associated with CRC. In certain embodiments, the compositions and methods described herein are used to treat, prevent and / or improve symptoms associated with kidney cancer. In certain embodiments, the compositions and methods described herein are used to treat, prevent and / or improve symptoms associated with glioblastoma. In certain embodiments, the compositions and methods described herein are used to treat, prevent and / or improve symptoms associated with breast cancer. In certain embodiments, the compositions and methods described herein are used to treat, prevent and / or improve symptoms associated with RCC. In certain embodiments, the compositions and methods described herein are used to treat, prevent and / or improve symptoms associated with myeloma.

[0228] The cancer may specifically be of the following histological types, but is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatricoma carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; bronchoalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe cell carcinoma; oncocytic carcinoma; aerobic adenocarcinoma; basophilic cell carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; non-encapsulated sclerosing carcinoma; adrenal cortex carcinoma; endometrioid carcinoma; carcinoma of skin appendages; apocrine carcinoma; sebaceous carcinoma; cervical adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, breast; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; theca cell tumor, malignant; granulosa cell tumor, malignant; male cell tumor, malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extramammary paraganglioma, malignant; pheochromocytoma; glomus sarcoma; malignant melanoma; amelanotic melanoma melanoma; superficial spreading melanoma; lentigo maligna melanoma; acral lentigo melanoma; nodular melanoma; malignant melanoma in giant nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrohistiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mixed Müllerian tumor; Wilms tumor; hepatoblastoma; carcinosarcoma; stromal tumor, malignant; Brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; ovarian goiter, malignant; choriocarcinoma; mesonephroblastoma, malignant; angiosarcoma; hemangioendothelioma, malignant; Kaposi sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic odontosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrous astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroglioma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemoma, malignant;Granular cell tumor, malignant; Lymphoma, malignant; Hodgkin's disease; Hodgkin's; Paragranuloma; Lymphoma, malignant, small lymphocytic; Lymphoma, malignant, large cell, diffuse; Lymphoma, malignant, follicular; Mycosis fungoides; Other specified non-Hodgkin's lymphoma; B-cell lymphoma; Low-grade / follicular non-Hodgkin's lymphoma (NHL); Small lymphocytic (SL) NHL; Intermediate-grade / follicular NHL; Intermediate-grade diffuse NHL; High-grade immunoblastic NHL; High-grade lymphoblastic NHL; High-grade small non-lytic cell NHL; Bulky disease disease)NHL; mantle cell lymphoma; AIDS-related lymphoma; Waldenstrom's macroglobulinemia; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; hairy cell leukemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); and chronic myeloblastic leukemia. ;

[0229] Particular 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 known as blood neoplasms. Leukemia is a broad term covering a range of diseases. Leukemia is clinically and pathologically divided into acute and chronic forms.

[0230] Acute leukemia is characterized by the rapid proliferation of immature blood cells. This crowding makes it impossible for the bone marrow to produce healthy blood cells. Acute leukemia can occur in children and young adults. In fact, it is the most common cause of death in children in the United States than any other type of malignant disease. Acute leukemia requires immediate treatment because the malignant cells grow and accumulate rapidly, then spill into the bloodstream and spread to other organs in the body. Although the disease occasionally causes cranial nerve palsies, involvement of the central nervous system (CNS) is uncommon. Chronic leukemia is characterized by an excessive accumulation of relatively mature but still abnormal blood cells. It usually takes months to years to progress, with the cells being produced at a much higher rate than normal cells, resulting in many abnormal white blood cells in the blood. Chronic leukemia occurs primarily in older people, but can theoretically occur in any age group. Acute leukemia must be treated immediately, while chronic leukemia is sometimes monitored for a period of time before treatment to ensure maximum effectiveness of the treatment.

[0231] Additionally, the disorders are classified as lymphocytic or lymphoblastic, indicating that the cancer occurs in the type of bone marrow cell that normally gives rise to lymphocytes, and myeloid or myeloid, indicating that the cancer occurs in the type of bone marrow cell that normally gives rise to red blood cells, certain types of white blood cells, and platelets (see Lymphocytes and Myeloid Cells).

[0232] Acute lymphocytic leukemia (also called acute lymphoblastic leukemia or ALL) is the most common type of leukemia in young children. This disease can also affect adults, especially those aged 65 and older. Chronic lymphocytic leukemia (CLL) most often affects adults over the age of 55. It sometimes occurs in younger adults but almost never affects children. Acute myeloid leukemia (also called acute myeloid leukemia or AML) occurs more often in adults than in children. This type of leukemia was previously called "acute nonlymphocytic leukemia." Chronic myeloid leukemia (CML) occurs mainly in adults. Very rarely, children can also develop this disease.

[0233] Lymphoma is a type of cancer that begins in lymphocytes, a type of white blood cell in the vertebrate immune system. 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, and Hodgkin lymphoma in particular accounts 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 caused by HIV infection or certain medications or drug treatments) also have a higher incidence of lymphoma.

[0234] In certain embodiments of the present disclosure, a composition comprising NK cells and antibodies is delivered to an individual in need, such as an individual suffering from cancer or infection. At least in some cases, these cells can enhance the immune system of an individual to attack the corresponding cancer or pathogenic cells. In some cases, one or more doses of a composition comprising NK cells and antibodies are provided to an individual. In the case of providing two or more doses of NK cells / antibodies to an individual, the duration between administrations should be sufficient to allow the time for reproduction in the individual, and in a specific embodiment, the duration between doses is 1, 2, 3, 4, 5, 6, 7 or more days.

[0235] The source of pre-activated (optionally) and expanded NK cells can be of any kind, but in specific embodiments, the cells are obtained from, for example, umbilical cord blood, peripheral blood, human embryonic stem cell banks, or induced pluripotent stem cells. A suitable dose for therapeutic effect would be, for example, at least 10 5 or about 10 5 About 10 12cells, preferably in a series of dosing cycles. An exemplary dosing regimen consists of four weekly dosing cycles of increasing doses, starting with at least about 10 cells on day 0. 5 Starting with 10 cells, for example, gradually increasing to about 10 within a few weeks of starting an intra-patient dose escalation regimen. 12 Suitable modes of administration include intravenous, subcutaneous, intracavitary (e.g., via a reservoir access device), intraperitoneal, and direct injection into the tumor mass.

[0236] Compositions comprising NK cells and antibodies produced according to the methods of the present invention have many potential uses, including experimental and therapeutic uses. In particular, it is envisioned that such cell populations can be used to suppress undesirable or inappropriate immune responses. In such methods, a small amount of NK cells are removed from the patient's body and then manipulated and amplified in vitro before they are reinfused into the patient's body. Examples of diseases that can be treated in this manner are autoimmune diseases and conditions in which immune activity needs to be suppressed, such as for allogeneic transplant tolerance. The treatment method may include obtaining NK cells from a mammal; amplifying NK cells in vitro according to the methods of the present method described herein; exposing NK cells to antibodies under sufficient conditions; and administering a composition comprising amplified NK cells / antibodies to the mammal to be treated.

[0237] The pharmaceutical composition of the present disclosure can be used alone or in combination with other recognized medicaments that can be used to treat cancer.Whether it is delivered alone or in combination with other medicaments, the pharmaceutical composition of the present disclosure can be delivered to various parts in mammals (particularly humans) to achieve specific effects through various approaches.Though more than one approach can be used for administration, a specific approach can provide a more direct and more effective reaction than another approach.For example, intradermal delivery can be more advantageously used to treat melanoma than inhalation.Local or systemic delivery can be completed by administration, which includes applying or dripping the preparation into the body cavity, inhaling or blowing into an aerosol, or introducing parenterally, including intramuscular, intravenous, intraportal, intrahepatic, peritoneal, subcutaneous or intradermal administration.

[0238] In one embodiment, the subject suffers from an autoimmune disease. Non-limiting examples of autoimmune diseases include: alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, adrenal autoimmune disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac dermatitis, spate-dermatitis, 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, idiopathic mixed cryoglobulinemia, fibromyalgia-fibromyositis; glomerulonephritis, Graves' disease, Guillain-Barre, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, lupus erythematosus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, type 1 or immune-mediated diabetes mellitus, myasthenia gravis, nephrotic syndrome (e.g., mild change disease, focal glomerulosclerosis, or membranous nephropathy), pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Rett syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, stiffness syndrome, systemic lupus erythematosus, lupus erythematosus, ulcerative colitis, uveitis, vasculitis (e.g., polyarteritis nodosa, Takayasu's arteritis, temporal arteritis / giant cell arteritis, or dermatitis herpetiformis vasculitis), vitiligo, and Wegener's granulomatosis. Thus, some examples of autoimmune diseases that can be treated using the methods disclosed herein include, but are not limited to, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, type I diabetes, Crohn's disease, ulcerative colitis, myasthenia gravis, glomerulonephritis, ankylosing spondylitis, vasculitis, or psoriasis. The subject may also suffer from an allergic condition, such as asthma.

[0239] In another embodiment, the subject is a recipient of a transplanted organ or stem cell, and the expanded NK cells / antibodies of the present disclosure are used to prevent and / or treat rejection. In a specific embodiment, the subject suffers from graft-versus-host disease or is at risk of developing graft-versus-host disease. GVHD is a possible complication of any transplant using or containing stem cells from related or unrelated donors. There are two types of GVHD, acute and chronic. Acute GVHD appears within the first three months after transplantation. Signs of acute GVHD include a red rash on the hands and feet that may spread and become more severe, with peeling or blistering of the skin. Acute GVHD can also affect the stomach and intestines, in which case cramps, nausea, and diarrhea can occur. Yellowing of the skin and eyes (jaundice) indicates that acute GVHD has affected the liver. Chronic GVHD is graded according to its severity: stage / grade 1 is mild; stage / grade 4 is severe. Chronic GVHD develops three months or more after transplantation. The symptoms of chronic GVHD are similar to those of acute GVHD, but in addition, chronic GVHD may also affect the mucous glands of the eyes, the salivary glands of the mouth, and the glands that lubricate the gastric mucosa and the intestines. Any NK cell population disclosed herein can be utilized. Examples of transplanted organs include solid organ transplants, such as kidneys, livers, skin, pancreas, lungs, and / or hearts, or cell transplants, such as islets, hepatocytes, myoblasts, bone marrow, or hematopoietic or other stem cells. The transplant can be a composite transplant, such as facial tissue. NK cells, such as immunosuppressive CD19+ cells, can be administered with antibodies before, during, and / or after transplantation. In some embodiments, a composition comprising NK cells and antibodies is administered before transplantation, such as 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 before transplantation. In a specific, non-limiting example, administration of a therapeutically effective amount of a composition comprising NK cells and antibodies is performed 3-5 days prior to transplantation.

[0240] NK cells / antibodies administered to patients undergoing transplantation can be sensitized with antigens specific to the transplant material prior to administration. According to this embodiment, the immune / inflammatory response of the transplant recipient to the transplant material will be reduced, and therefore, the possibility of transplant tissue rejection is minimized. Similarly, for the treatment of graft-versus-host disease, NK cells can be sensitized with host-specific antigens. According to this embodiment, the immune / inflammatory response of the recipient to self-antigens will be reduced.

[0241] In a further embodiment, a composition comprising NK cells and antibodies is administered to a subject in a therapeutically effective amount to treat or suppress inflammation in the subject. Therefore, the method includes administering to a subject a composition comprising NK cells and antibodies in a therapeutically effective amount to suppress the inflammatory process. Examples of inflammatory conditions include, but are not limited to, asthma, encephalitis, inflammatory bowel disease, chronic obstructive pulmonary disease (COPD), allergic diseases, septic shock, pulmonary fibrosis, undifferentiated spondyloarthropathy, undifferentiated arthropathy, arthritis, inflammatory osteolysis, and chronic inflammation caused by chronic viral or bacterial infections. The method disclosed herein can also be used to treat allergic diseases.

[0242] Administration of a composition comprising NK cells and antibodies can be used whenever immunosuppression or suppression of inflammation is needed, such as at the first signs or symptoms of disease or inflammation. These symptoms may be general, such as pain, edema, elevated body temperature, or may be specific signs or symptoms associated with dysfunction of the affected organ. For example, in renal transplant rejection, elevated serum creatinine levels may occur, while in GVHD, a rash may occur, and in asthma, shortness of breath and wheezing may occur.

[0243] The administration of the composition comprising NK cells and antibodies can also be used to prevent immune-mediated diseases in the subject of interest. For example, the composition comprising NK cells and antibodies can be applied to the subject who will become the transplant recipient before transplantation. In another example, the composition comprising NK cells and antibodies is applied to the subject who receives allogeneic bone marrow transplantation but does not have T cell exhaustion. In a further example, the composition comprising NK cells and antibodies can be applied to the subject with a family history of diabetes. In other examples, the composition comprising NK cells and antibodies is applied to the subject with asthma to prevent asthma attacks. In some embodiments, the composition comprising NK cells and antibodies is applied to the subject before the onset of symptoms. Compared with patients receiving other treatments that do not include the composition comprising NK cells and antibodies, the administration of the composition comprising NK cells and antibodies results in a reduction in the incidence or severity of subsequent immune events or symptoms (such as asthma attacks), or improved patient survival rates.

[0244] 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, immunomodulators, monoclonal antibodies, chemotherapeutic agents, hormones, any type of drug, surgery, radiation, etc. In a non-limiting example, the immunomodulator is lenalidomide, the monoclonal antibody is rituximab, ofatumumab or lumiliximab, and the chemotherapeutic agent is fludarabine or cyclophosphamide.

[0245] The compositions of the present disclosure may be provided in unit dosage form, wherein each dosage unit (e.g., each 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 unit dosage for human and animal subjects, each unit containing 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 the desired effect, in combination with a pharmaceutically acceptable diluent, carrier, or vehicle, as appropriate. The specifications of the unit dosage form of the present disclosure depend on the specific pharmacodynamics associated with the pharmaceutical composition in a specific subject.

[0246] Ideally, an effective amount or sufficient number of isolated transduced NK cells are present in the composition and introduced into a subject to establish a long-term, specific anti-tumor response to reduce the size of the tumor or eliminate tumor growth or regrowth better than the results without such treatment. 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 without the presence of NK cells.

[0247] Therefore, the amount of the composition comprising NK cells and antibodies administered should take into account the route of administration and should be such that a sufficient number of compositions comprising NK cells and antibodies will be introduced to achieve the desired therapeutic response. In addition, the amount of each active agent contained in the compositions described herein (e.g., the amount of each cell to be contacted or the amount per specific body weight) can vary in different applications. In general, the concentration of NK cells should ideally be sufficient to provide at least about 1×10 6 About 1×10 12 NK cells, or even more ideally, about 1×10 7 About 5×10 10 NK cells, although higher numbers such as greater than 5 × 10 can be used. 8 cells or lower, for example, less than 1×10 7 Any suitable amount of cells may be administered. The dosing regimen may be based on well-established cell-based therapies (see, e.g., U.S. Pat. No. 4,690,915), or an alternative continuous infusion strategy may be employed.

[0248] These values ​​provide general guidance for the range of compositions comprising NK cells and antibodies used by implementers in optimizing the methods of the present disclosure to implement the methods of the present invention. Such ranges listed herein by no means exclude the use of higher or lower amounts of components, as may be warranted in a particular application. For example, actual dosages and schedules may vary depending on whether the composition is administered in conjunction with other pharmaceutical compositions, or according to individual differences in pharmacokinetics, drug distribution, and metabolism. Those skilled in the art can easily make any necessary adjustments according to the urgency of a particular situation.

[0249] III. Antibodies

[0250] Aspects of the present disclosure relate to the use of antibodies or their functional fragments in compositions that also include certain NK cells. The term "antibody" refers to a complete immunoglobulin of any isotype, or a fragment thereof, which can compete with a complete antibody for specific binding to a target antigen, such as through an antigen binding domain, and includes chimeric, humanized, fully human, monospecific and multispecific (including at least bispecific and trispecific, etc.) antibodies. 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 a part of an animal's immune response, including IgG, IgD, IgE, IgA, IgM and related proteins, and polypeptides comprising antibody CDR domains that retain antigen binding activity. In a specific embodiment, the antibody comprises scFv.

[0251] The term "antigen" refers to a molecule or a portion of a molecule that can be bound by a selective binding agent (eg, an antibody). An antigen can possess one or more epitopes that can interact with different antibodies.

[0252] The term "epitope" includes any region or portion of a molecule that can elicit an immune response by binding to an immunoglobulin or T cell receptor. Epitope determinants may include chemically active surface groups such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and may have specific three-dimensional structural characteristics and / or specific charge characteristics. In general, antibodies specific for a particular target antigen will preferentially recognize an epitope on the target antigen within a complex mixture.

[0253] The epitope region of a given polypeptide can be identified using a number of different epitope mapping techniques well known in the art, including: X-ray crystallography, nuclear magnetic resonance spectroscopy, site-directed mutagenesis mapping, protein display arrays, see, e.g., Epitope Mapping Protocols, (Johan Rockberg and Johan Nilvebrant, Ed., 2018) Humana Press, New York, NY. Such techniques are known in the art and are described, for example, in U.S. Pat. No. 4,708,871; Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1984); Geysen et al., Proc. Natl. Acad. Sci. USA 82:178-182 (1985); Geysen et al., Molec. Immunol. 23:709-715 (1986). See, e.g., Epitope Mapping Protocols, supra. In addition, standard antigenicity and hydropathy plots can be used to predict and identify antigenic regions of proteins.

[0254] A complete antibody is usually composed of two full-length heavy chains and two full-length light chains, but in some cases fewer chains may be included, for example, antibodies naturally present in camelids may only contain heavy chains. Antibodies disclosed herein may be derived from a single source only or may be "chimeric", i.e., different parts of the antibody may be derived from two different antibodies. For example, the variable region or CDR region may be derived from rat or mouse sources, while the constant region may be derived from different animal sources, such as humans. Antibodies or binding fragments may be produced in hybridomas by recombinant DNA technology or by enzymatic or chemical cleavage of complete antibodies. Unless otherwise indicated, the term "antibody" includes derivatives, variants, fragments and mutant proteins thereof, examples of which are described below (Sela-Culang et al., Front Immunol. 2013; 4: 302; 2013).

[0255] The term "light chain" includes full-length light chains and fragments thereof having sufficient variable region sequences to confer binding specificity. Full-length light chains have a molecular weight of about 25,000 daltons and include variable region domains (abbreviated herein as VL) and constant region domains (abbreviated herein as CL). Light chains are classified in two ways, kappa (κ) and lambda (λ). The term "VL fragment" refers to a fragment of the light chain of a monoclonal antibody, which includes all or part of the light chain variable region, including CDRs. The VL fragment may also include a light chain constant region sequence. The variable region domain of the light chain is located at the amino terminus of the polypeptide.

[0256] The term "heavy chain" includes a full-length heavy chain and a fragment 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 includes a variable region domain (abbreviated herein as VH) and three constant region domains (abbreviated herein as CH1, CH2, and CH3). The term "VH fragment" refers to a fragment of the heavy chain of a monoclonal antibody, which includes all or part of the heavy chain variable region, including CDR. The VH fragment may also include a heavy chain constant region sequence. The number of heavy chain constant region domains will depend on the isotype. The VH domain is located at the amino terminus of the polypeptide, and the CH domain is located at the carboxyl terminus, with CH3 closest to the -COOH end. The isotype of an antibody may be IgM, IgD, IgG, IgA, or IgE, and is defined by a heavy chain, of which there are five classifications: mu (μ), delta (δ), gamma (γ), alpha (α), or epsilon (ε) chains, respectively. IgG has a variety of subtypes, including but not limited to IgG1, IgG2, IgG3, and IgG4. IgM subtypes include IgM1 and IgM2. IgA subtypes include IgA1 and IgA2.

[0257] Antibodies can be whole immunoglobulins of any isotype or class, chimeric antibodies, or hybrid antibodies specific for two or more antigens. They can also be fragments (e.g., F(ab')2, Fab', Fab, Fv, etc.), including hybrid fragments. Immunoglobulins also include natural, synthetic, or genetically engineered proteins that act like antibodies by binding to specific antigens to form complexes. The term antibody includes genetically engineered or otherwise modified forms of immunoglobulins, such as the following:

[0258] The term "monomer" refers to an antibody containing only one Ig unit. A monomer is the basic functional unit of an antibody. The term "dimer" refers to an antibody containing two Ig units connected to each other by an antibody heavy chain constant domain (Fc or fragment crystallizable region). The complex can be stabilized by connecting (J) chain proteins. The term "multimer" refers to an antibody containing more than two Ig units connected to each other by an antibody heavy chain constant domain (Fc region). The complex can be stabilized by connecting (J) chain proteins.

[0259] The term "bivalent antibody" means an antibody that contains two antigen binding sites. The two binding sites can have the same antigen specificity, or they can be bispecific, meaning that the two antigen binding sites have different antigen specificities.

[0260] Bispecific antibodies are a class of antibodies with two paratopes, and the two paratopes have different binding sites for two or more different epitopes. In some embodiments, bispecific antibodies can be biparatopic, wherein bispecific antibodies can specifically recognize different epitopes from the same antigen. In some embodiments, bispecific antibodies can be constructed by a pair of different single-domain antibodies referred to as "nanoantibodies". Single-domain antibodies are derived from cartilaginous fish and camelids and are modified. Nanoantibodies can be connected together by joints using typical techniques for those skilled in the art; such methods for selecting and connecting nanoantibodies are described in PCT Publication Nos. WO2015044386A1, WO2010037838A2 and Bever et al., Anal Chem.86:7875-7882 (2014), each of which is incorporated herein by reference in its entirety.

[0261] Bispecific antibodies can be constructed as: complete IgG, Fab'2, Fab'PEG, diabodies, or alternatively as scFv. Diabodies and scFv can be constructed without an Fc region, using only the variable domains, thereby potentially reducing the impact of anti-idiotypic responses. Bispecific antibodies can be produced by a variety of methods, including but not limited to fusion of hybridomas or connection of Fab' fragments. See, for example, 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 by reference in its entirety.

[0262] In certain aspects, antigen binding domains can be multispecific or heterospecific by multimerization with VH and VL regions that bind to different antigens. For example, an antibody can 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, various aspects may include, but are not limited to, bispecific, trispecific, tetraspecific, and other multispecific antibodies or antigen binding fragments thereof directed against epitopes and other targets (e.g., Fc receptors on effector cells).

[0263] In some embodiments, multispecific antibodies can be used and directly connected by short flexible polypeptide chains using conventional methods known in the art. One such example is a diabody, which is a bivalent, bispecific antibody in which the VH and VL domains are expressed on a single polypeptide chain and a linker that is too short to allow pairing between domains on the same chain is used, thereby forcing the domain to pair with the complementary domain of another chain to form two antigen binding sites. The linker functionality is applicable to embodiments of tri-antibodies, tetra-antibodies, and higher-order antibody multimers. (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)).

[0264] Compared with bispecific complete antibodies, bispecific diabodies may also be advantageous because they can be easily constructed and expressed in Escherichia coli. Using phage display (WO94 / 13804), diabodies (and other polypeptides, such as antibody fragments) with appropriate binding specificity can be easily selected from a library. If one arm of a diabody remains constant, for example, with specificity for protein, a library can be prepared in which the other arm is changed and antibodies with appropriate specificity are selected. Bispecific complete antibodies can be prepared 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.

[0265] Heteroconjugate antibodies consist of two covalently linked monoclonal antibodies of different specificities. See, e.g., U.S. Patent 6,010,902, the entire contents of which are incorporated herein by reference.

[0266] The part of the Fv fragment of an antibody molecule that binds to an antigenic epitope with high specificity is referred to herein as a "paratope". The paratope is composed of amino acid residues that contact the antigenic epitope to promote antigen recognition. Each of the two Fv fragments of an antibody is composed of two variable domains VH and VL in a dimerization configuration. The primary structure of each variable domain includes three hypervariable loops separated and flanked by a framework region (FR). The hypervariable loop is the region with the highest primary sequence variability in the antibody molecule from any mammal. The term hypervariable loop is sometimes used interchangeably with the term "complementarity determining region (CDR)". The length of the hypervariable loop (or CDR) varies from antibody molecule to antibody molecule. The framework region of all antibody molecules from a given mammal has a high degree of primary sequence similarity / commonality. A person skilled in the art can use the common framework region to identify the framework region and the hypervariable loops (or CDRs) interspersed between the framework regions. The hypervariable loops are given identification names to distinguish their positions within the polypeptide and on which domain they appear. The CDRs in the VL domain are identified as L1, L2, and L3, with L1 occurring at the most distal end and L3 occurring closest to the CL domain. CDRs may also be designated as CDR-1, CDR-2, and CDR-3. L3 (CDR-3) is generally the region with the highest variability among all antibody molecules produced by a given organism. CDRs are regions of the polypeptide chain that are arranged linearly in the primary structure and are separated from each other by framework regions. The amino terminus (N-terminus) of the VL chain is designated as FR1. The region identified as FR2 occurs between the L1 and L2 hypervariable loops. FR3 occurs between the L2 and L3 hypervariable loops, and the FR4 region is closest to the CL domain. The VH chain repeats this structure and nomenclature, including three CDRs identified as H1, H2, and H3. Most of the amino acid residues in the variable domains or Fv fragments (VH and VL) are part of the framework region (approximately 85%). The three-dimensional or tertiary structure of the antibody molecule causes the framework region to be located further inside the molecule and provide most of the structure, while the CDRs are located on the outer surface of the molecule.

[0267] Several methods have been developed and can be used by 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 variety of multiple sequence alignment methods and algorithms that identify the conserved amino acid residues that make up the framework regions, thereby identifying CDRs that may vary in length but are located between the framework regions. Three commonly used methods have been developed to identify the CDRs of antibodies: Kabat (as described in TT Wu and EA Kabat, "AN ANALYSIS OF THE SEQUENCES OF THE VARIABLE REGIONS OF BENCE JONES PROTEINS AND MY ELOMA LIGHT CHAINS AND THEIR IMPLICATIONS FOR ANTIBODY COMPLEMENTARITY," J Exp Med, vol. 132, no. 2, pp. 211-250, Aug. 1970); Chothia (as described in C. Chothia et al., "Conformations of immunoglobulin hypervariable regions," Nature, vol. 342, no. 6252, pp. 877-883, Dec. 1989); and IMGT (as 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 Biology, vol. 1996). 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 appear in the CDR, although in some cases, residues within the framework region contribute to antigen binding.

[0268] Those skilled in the art can use any of a variety of methods to determine the paratope of an antibody. These methods include: 1) based on the chemical properties of the variable region amino acid sequence of the antibody and the composition of the epitope, the tertiary structure of the antibody / epitope binding interaction is calculated and predicted; 2) hydrogen deuterium exchange and mass spectrometry; 3) polypeptide fragmentation and peptide mapping methods, in which multiple overlapping peptide fragments are generated from the full-length polypeptide and the binding affinity of these peptides to the epitope is evaluated; 4) antibody phage display library analysis, in which mammalian antibody Fab fragment encoding genes are expressed by phages and thus incorporated into the phage shell. Then, the Fab-expressing phage population is allowed to interact with an antigen that has been fixed or can be expressed by different exogenous expression systems. The non-binding Fab fragments are washed away, leaving only the specific binding Fab fragments attached to the antigen. The bound Fab fragments can be easily separated and the genes encoding them can be determined. This method can also be used for smaller regions of Fab fragments, including Fv fragments or specific VH and VL domains (as appropriate).

[0269] In some aspects, affinity-matured antibodies are enhanced by one or more modifications in one or more CDRs, and compared with parent antibodies without these changes, the modifications cause the antibody to have an increased affinity for the target antigen. Some affinity-matured antibodies have nanomolar or picomolar affinity for the target antigen. Affinity-matured antibodies are produced by procedures known in the art, such as Marks et al., Bio / Technology 10: 779 (1992) describes the use of VH and VL domains to achieve affinity maturation, and random mutagenesis of CDRs 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), which is combined with computational methods such as Tiller et al., Front.Immunol.8: 986 (2017).

[0270] Chimeric immunoglobulins are the product of fused genes derived from different species; a "humanized" chimera typically has framework regions (FRs) from a human immunoglobulin and one or more CDRs from a non-human source.

[0271] In certain aspects, portions of the heavy and / or light chains are identical or homologous to corresponding sequences from another particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired biological activity. U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851 (1984). For methods related to chimeric antibodies, see, e.g., U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1985), each of which is incorporated herein by reference in its entirety. CDR grafting is described in, e.g., U.S. Patent Nos. 6,180,370, 5,693,762, 5,693,761, 5,585,089, and 5,530,101, which are incorporated herein by reference in their entirety for all purposes.

[0272] In some embodiments, minimizing the antibody polypeptide sequence from non-human species optimizes chimeric antibody function and reduces immunogenicity.Specific amino acid residues from the non-antigen recognition region of non-human antibodies are modified to be homologous to the corresponding residues in human antibodies or isotypes.An example is a "CDR transplantation" antibody, in which the antibody comprises one or more CDRs from a specific species or belonging to a specific antibody class or subclass, and the remainder of the antibody chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass.In order to be used for humans, the V region composed of CDR1, CDR2 and part of CDR3 of the light chain and heavy chain variable region of non-human immunoglobulin is transplanted with human antibody framework region, replacing the naturally occurring antigen receptor of the human antibody with non-human CDR.In some cases, corresponding non-human residues replace the framework region residues of human immunoglobulin.In addition, humanized antibodies can be included in residues not found in receptor antibodies or donor antibodies to further improve performance.Humanized antibodies can also include at least a portion of immunoglobulin constant region (Fc) (usually 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).

[0273] Intrabodies are intracellularly localized immunoglobulins that bind intracellular antigens, as opposed to secreted antibodies that bind antigens in the extracellular space.

[0274] Polyclonal antibody preparations usually include different antibodies for different determinants (epitopes). In order to produce polyclonal antibodies, an antigen or antigen fragment is used to immunize a host, such as a rabbit or goat, usually together with an adjuvant, and if necessary, coupled to a carrier. Antibodies for the antigen are subsequently collected from host serum. Polyclonal antibodies can be affinity purified for the antigen to make it have monospecificity.

[0275] A monoclonal antibody or "mAb" refers to an antibody obtained from a homogeneous population of antibodies from exclusive parental cells, eg, the population is identical except for possible naturally occurring mutations that may be present in minor amounts. Each monoclonal antibody is directed against a single antigenic determinant.

[0276] Functional antibody fragments and antigen binding fragments can be used. 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 antigens. These fragments are composed of various arrangements of variable region heavy chains (VH) and / or light chains (VL); in some embodiments, they include constant region heavy chain 1 (CH1) and light chain (CL). In some embodiments, they lack the Fc region composed of heavy chain 2 (CH2) and 3 (CH3) domains. Embodiments of antigen-binding fragments and modifications thereof may include: (i) Fab fragment types consisting of VL, VH, CL and CH1 domains; (ii) Fd fragment types consisting of VH and CH1 domains; (iii) Fv fragment types consisting of VH and VL domains; (iv) single domain fragment type dAb consisting of a single VH or VL domain (Ward, 1989; McCafferty et al., 1990; Holt et al., 2003); (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.

[0277] Antigen binding fragments also include antibody fragments that accurately retain at least or at most 1, 2 or 3 complementary determining regions (CDRs) from the light chain variable region. Fusions of sequences containing CDRs with Fc regions (or their CH2 or CH3 regions) are included within the scope of this definition, including, for example, scFvs fused directly or indirectly to Fc regions are also included herein.

[0278] The term Fab fragment means a monovalent antigen-binding fragment of an antibody containing VL, VH, CL and CH1 domains. The term Fab' fragment means a monovalent antigen-binding fragment of a monoclonal antibody that is larger than a Fab fragment. For example, a Fab' fragment includes VL, VH, CL and CH1 domains and all or part of the hinge region. The term F(ab')2 fragment means a divalent antigen-binding fragment of a monoclonal antibody comprising two Fab' fragments connected by a disulfide bridge at the hinge region. The F(ab')2 fragment includes, for example, all or part of two VH and VL domains, and may further include all or part of two CL and CH1 domains.

[0279] The term Fd fragment refers to a heavy chain fragment of a monoclonal antibody, which includes all or part of VH, including CDR. The Fd fragment may also include a CH1 region sequence.

[0280] The term Fv fragment means a monovalent antigen-binding fragment of a monoclonal antibody, including all or part of VL and VH, and without CL and CH1 domains. VL and VH include, for example, CDRs. Single-chain antibodies (sFv or scFv) are Fv molecules in which the VL region and the VH region are connected by a flexible linker to form a single polypeptide chain, thereby forming an antigen-binding fragment. Single-chain antibodies are discussed in detail in International Patent Application Publication No. WO88 / 01649 and U.S. Patent Nos. 4,946,778 and 5,260,203, the disclosures of which are incorporated herein by reference. The term (scFv) 2 means a bivalent or bispecific sFv polypeptide chain, which includes an oligomerization domain at its C-terminus, separated from the sFv by a hinge region (Pack et al. 1992). The oligomerization domain comprises a self-associated α-helix, such as a leucine zipper, which can be further stabilized by additional disulfide bonds. The (scFv) 2 fragment is also referred to as a "mini antibody" or "minibody".

[0281] Single domain antibodies are antigen binding fragments containing only a VH or VL domain. In some cases, two or more VH regions are covalently linked with a peptide linker to produce a bivalent domain antibody. The two VH regions of a bivalent domain antibody can target the same or different antigens.

[0282] In some cases, a fragment crystallizable region Fc is utilized. The Fc region contains two heavy chain fragments, which comprise the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains. The term "Fc polypeptide" used herein includes native and mutant protein forms of polypeptides derived from the Fc region of an antibody. Including truncated forms of such polypeptides containing hinge regions that promote dimerization. In some embodiments, antibodies (e.g., together with TRIKE) incorporating cytokines (including those mentioned herein) can be utilized.

[0283] In some embodiments, the antibody is an antibody-drug conjugate. In some embodiments, the antibody can specifically be one or more of the following, although the antibody is not limited to these: [fam]-trastuzumab deruxtecan, Abciximab, Adalimumab, Ado-trastuzumab emtansine), Aducanumab, Alemtuzumab, Alirocumab, Amivantamab, Anifrolumab, Ansuvimab, Atezolizumab, Atoltivimab together with Maftivimab and Odesivimab-ebgn (also known as Inmazeb), Avelumab, Basiliximab, Belantamab mafodotin), Belimumab, Benralizumab, Bevacizumab, Bezlotoxumab, Bimekizumab, Blinatumomab, Brentuximabvedotin, Brodalumab, Brolucizumab, Burosumab, Canakinumab, Caplacizumab, Casirivimab+imdevimab, Catumaxomab, Cemiplimab, Certolizumabpegol), Cetuximab, Crizanlizumab, Daclizumab, Daratumumab, Denosumab, Dinutuximab, Donanemab, Dostarlimab, Dupilumab, Durvalumab, Eculizumab, Edrecolomab, Efalizumab, Elotuzumab, Emapalumab, Emicizumab, Enfortumab vedotin), Eptinezumab, Erenumab, Evinacumab, Evolocumab, Faricimab, Fremanezumab, Galcanezumab, Gemtuzumab, Golimumab, Guselkumab, Ibalizumab, Ibritumomab tiuxetan), Idarucizumab, Inebilizumab, Infliximab, Inolimomab, Inotuzumab, Ipilimumab, Isatuximab, Ixekizumab, Lanadelumab, Lecanemab, Loncastuximab tesirine, Margetuximab, Mepolizumab, Mirvetuximab soravtansine, Mogamulizumab, Mosunetuzumab, Moxetumomabpasudotox, Muromonab-CD3, Narsoplimab, Natalizumab, Naxitamab, Nebacumab, Necitumumab, Nirsevimab, Nivolumab, Obiltoxaximab, Obinutuzumab, Ocrelizumab, Ofatumumab, Olaratumab, Omalizumab, Omburtamab, Oportuzumab monatox), Palivizumab, Panitumumab, Pembrolizumab, Penpulimab, Pertuzumab, Polatuzumab vedotin, Ramucirumab, Ranibizumab, Ravulizumab, Raxibacumab, Regdanvimab, Relatlimab, Reslizumab, Retifanlimab, Risankizumab, Rituximab, Romosozumab, Sacituzumabgovitecan, Sarilumab, Satralizumab, Secukinumab cukinumab), Siltuximab, Sintilimab, Sotrovimab, Spesolimab, Sutimlimab, Tafasitamab, Tebentafusp, Teclistamab, Teplizumab, Teprotumumab, Tezepelumab, Tildrakizumab, Tislelizumab, TisotumabVedotin, Tixagevimab, cilgavimab, Tocilizumab, Toripalimab, Tositumomab-I131, Tralokinumab, Trastuzuma, Tremelimumab, Ublituximab, Ustekinumab, Vedolizumab, or any combination thereof. In some embodiments, the antibody is Imatrazumab. In some embodiments, the antibody is Margetuximab. In some embodiments, the antibody is Evantuzumab. In some embodiments, the antibody is Otuzumab. In some embodiments, the antibody is IPH61 (also known as IPH6101 or SAR443579).

[0284] IV. Pharmaceutical Compositions

[0285] The pharmaceutical composition of the present disclosure comprises an effective amount of a composition comprising NK cells and antibodies dispersed in a pharmaceutically acceptable carrier. The phrase "pharmaceutically or pharmacologically acceptable" refers to a molecular entity and composition that does not produce side effects, allergic reactions or other adverse reactions when applied to an animal such as a human being when appropriate. According to the present disclosure, the preparation of the pharmaceutical composition comprising the composition will be known to those skilled in the art, as illustrated by Remington: The Science and Practice of Pharmacy, 21st Ed. Lippincott Williams and Wilkins, 2005, which is incorporated herein by reference. In addition, for animal (e.g., human) use, it should be understood that the preparation should meet the sterility, pyrogenicity, general safety and purity standards required by the FDA Office of Biological Standards.

[0286] As used herein, "pharmaceutically acceptable carriers" include any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes and the like materials and combinations thereof, as known to those of ordinary skill in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, which is incorporated herein by reference). Unless any conventional carrier is incompatible with the active ingredient, its use in the pharmaceutical composition is contemplated.

[0287] Pharmaceutical composition can comprise different types of carriers, depending on whether it is used in solid, liquid or aerosol form, and whether sterility is required for routes of administration such as injection. Compositions of the present disclosure can be administered intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, locally, intramuscularly, subcutaneously, mucosally, orally, locally, regionally, inhaled (e.g., aerosol inhalation), injected, infused, continuously infused, directly localized perfusion bathing target cells, by catheter, by lavage, in frost, in lipid compositions (e.g., liposomes), or by other methods known to those of ordinary skill or any combination of the aforementioned methods (see, e.g., Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, which is incorporated herein by reference).

[0288] Compositions comprising NK cells and antibodies can be formulated as compositions in free base, neutral or salt form. Where appropriate, pharmaceutically acceptable salts include acid addition salts, such as salts formed with free amino groups of protein compositions, or salts formed with inorganic acids such as hydrochloric acid or phosphoric acid or organic acids such as acetic acid, oxalic acid, tartaric acid or mandelic acid. Salts formed with free carboxyl groups can also be derived from inorganic bases, such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide; or organic bases, such as isopropylamine, trimethylamine, histidine or procaine. After preparation, the solution will be administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The preparation is easily administered in a variety of dosage forms, such as formulated for parenteral administration, such as injectable solutions or aerosols for delivery to the lungs, or formulated for food administration, such as drug release capsules, etc.

[0289] Further according to the present disclosure, the composition of the present disclosure that is suitable for use is provided in a pharmaceutically acceptable carrier with or without an inert diluent. The carrier should be assimilable and include liquid, semisolid, i.e., paste or solid carrier. Except that any conventional medium, reagent, diluent or carrier is harmful to the therapeutic effect of the recipient or the composition contained therein, its use in the composition that can be used for practicing the method of the present invention is suitable. The example of carrier or diluent includes fat, oil, water, saline solution, lipid, liposome, resin, adhesive, filler, etc., or a combination thereof. The composition can also include various antioxidants to delay the oxidation of one or more components. In addition, the effect of microorganisms can be prevented by preservatives such as various antibacterial agents and antifungal agents, and the preservatives include but are not limited to parabens (such as methyl paraben, propyl paraben), chlorobutanol, phenol, sorbic acid, thimerosal or a combination thereof.

[0290] According to the present disclosure, the composition is combined with the carrier in any convenient and practical manner, ie, by solution, suspension, emulsification, mixing, encapsulation, absorption, etc. Such procedures are routine to those skilled in the art.

[0291] In a specific embodiment of the present disclosure, the composition is combined or fully mixed with a semisolid or solid carrier. Mixing can be carried out in any convenient manner, such as grinding. Stabilizers can also be added during the mixing process to protect the composition from losing therapeutic activity, i.e., denaturation in the stomach. Examples of stabilizers for the composition include buffers, amino acids such as glycine and lysine, carbohydrates such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.

[0292] In a further embodiment, the disclosure may relate to the use of a pharmaceutical lipid vehicle composition, which includes a composition containing NK cells and antibodies and optionally an aqueous solvent. As used herein, the term "lipid" will be defined as including any of a variety of substances that are characteristically insoluble in water and can be extracted with an organic solvent. This large class of compounds is well known to those skilled in the art, and when the term "lipid" is used herein, it is not limited to any specific structure. Examples include compounds containing long-chain aliphatic hydrocarbons and derivatives thereof. Lipids can be naturally occurring or synthetic (i.e., designed or produced by people). However, lipids are generally biological substances. Biological lipids are well known in the art, and include, for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, hemosoluble lipids, glycosphingolipids, glycolipids, sulfatides, lipids and polymerizable lipids with ether and ester-connected fatty acids, and combinations thereof. Of course, compounds that are understood by those skilled in the art as lipids except for the compounds specifically described herein are also included in the compositions and methods of the present invention.

[0293] Those of ordinary skill in the art will be familiar with a range of techniques that can be used to disperse compositions in nanoparticles or lipid vehicles. For example, compositions comprising NK cells and antibodies can be dispersed in a solution containing lipids, dissolved with lipids, emulsified with lipids, mixed with lipids, combined with lipids, covalently bonded with lipids, contained in lipids as a suspension, contained in micelles or liposomes, or compounded with micelles or liposomes, or associated with lipids or lipid structures by any means known to those of ordinary skill in the art. Dispersions may or may not result in the formation of liposomes.

[0294] 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 the condition, type of disease being treated, previous or concurrent therapeutic interventions, idiopathic conditions of the patient, and route of administration. Depending on the dosage and route of administration, the preferred dosage and / or number of administrations of the effective amount can vary according to the subject's response. In any case, the practitioner responsible for administration will determine the concentration of the active ingredient in the composition and the appropriate dosage for an individual subject.

[0295] In certain embodiments, the pharmaceutical composition may include, for example, at least about 0.1% of an active compound. In other embodiments, the active compound may, for example, account for about 2% to about 75% of a unit weight, or about 25% to about 60%, and any range that may be derived therefrom. Of course, the amount of the active compound in each therapeutically useful composition may be prepared in such a way that a suitable dosage will be obtained in any given compound unit dose. The technicians in the preparation of such pharmaceutical formulations will consider factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations, and therefore, multiple doses and treatment regimens may be desirable.

[0296] In other non-limiting examples, dosages may also include about 1 microgram / kg / body weight, about 5 microgram / kg / body weight, about 10 microgram / kg / body weight, about 50 microgram / kg / body weight, about 100 microgram / kg / body weight, about 200 microgram / kg / body weight, about 350 microgram / kg / body weight, about 500 microgram / kg / body weight, about 1 milligram / kg / body weight, about 5 milligram / kg / body weight, about 10 milligram / kg / body weight, about 50 milligram / kg / body weight, about 100 milligram / kg / body weight, about 200 milligram / kg / body weight, about 350 milligram / kg / body weight, about 500 milligram / kg / body weight, to about 1000 milligram / kg / body weight or more, and any range derivable therein, per administration. In non-limiting examples of ranges derivable from the numbers listed herein, ranges from about 5 mg / kg / body weight to about 100 mg / kg / body weight, about 5 micrograms / kg / body weight to about 500 mg / kg / body weight, etc. can be administered based on the above numbers.

[0297] The therapeutic composition comprising the NK cells and antibodies of the present invention can be administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly or intranasally. In some embodiments, the antibiotic is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly or intranasally. The appropriate dosage can be determined according to the type of disease to be treated, the severity and course of the disease, the clinical condition of the individual, the clinical history of the individual and the response to treatment, and the judgment of the attending physician.

[0298] Treatment may include various "unit doses". A unit dose is defined as a therapeutic composition containing a predetermined amount. The amount to be administered, as well as the specific route and formulation, is determined within the skill of a person skilled in the clinical art. A unit dose need not be administered as a single injection, but may include continuous infusion over a set period of time. In some embodiments, a unit dose includes a single administrable dose.

[0299] The amount to be administered according to the number of treatments and the unit dose depends on the desired therapeutic effect. An effective dose is understood to mean the amount required to achieve a specific effect. In practice, in certain embodiments, it is expected that doses within the range of 10 mg / kg to 200 mg / kg may affect the protective ability of these agents. Thus, contemplated dosages include dosages of 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. In addition, such dosages may be administered multiple times a day and / or over several days, weeks, or months.

[0300] In certain embodiments, the effective dose of the pharmaceutical composition is a dose that can provide a blood level of about 1 μM to 150 μM. In another embodiment, the effective dose 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 50 μM; or about 25 μM to 150 μM; or about 25 μM to 100 μM; or about 25 μM to 50 μM; or about 50 μM to 150 μM; or about 50 μM to 100 μM (or any range derivable therein). In other embodiments, the dose can provide a blood level of the therapeutic agent administered to a subject of about, at least about, 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, 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 vivo to a metabolized therapeutic agent, in which case the blood level may refer to the amount of the agent. Alternatively, to the extent that the therapeutic agent is not metabolized by the subject, the blood levels discussed herein may refer to the unmetabolized therapeutic agent.

[0301] A. Food Compositions and Formulations

[0302] In a specific embodiment of the present disclosure, the composition comprising NK cells and antibodies is formulated to be administered by the food route. The food route includes all possible routes of administration in which the composition is in direct contact with the digestive tract. Specifically, the pharmaceutical compositions disclosed herein can be administered orally, buccally, rectally or sublingually. Therefore, these compositions can be formulated with an inert diluent or an assimilable edible carrier, or they can be encapsulated in a hard or soft shell gelatin capsule, or they can be compressed into tablets, or they can be directly incorporated into the food in the diet.

[0303] In certain embodiments, the active compound can be combined with an excipient and used in the form of an ingestible tablet, lozenge, troche, capsule, elixir, suspension, syrup, wafer, etc. (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 incorporated herein by reference in its entirety). Tablets, troches, pills, capsules, etc. may also contain the following substances: a binder such as gum tragacanth, gum arabic, corn starch, gelatin, or a combination thereof; an excipient such as dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, or a combination thereof; a disintegrant such as corn starch, potato starch, alginic acid, or a combination thereof; a lubricant such as magnesium stearate; a sweetener such as sucrose, lactose, saccharin, or a combination thereof; a flavoring such as mint, oil of wintergreen, cherry flavoring, orange flavoring, etc. When the dosage unit form is a capsule, in addition to the above-mentioned types of materials, it can also contain a liquid carrier. Various other materials can exist as coatings or otherwise change the physical form of the dosage unit. For example, tablets, pills or capsules can be coated with shellac, sugar or both. When the dosage form is a capsule, in addition to the above-mentioned types of materials, it can also contain a carrier, such as a liquid carrier. Gelatin capsules, tablets or pills can be enteric coated. Enteric coatings prevent the composition from denaturing in the stomach or upper intestine where the pH is acidic. See, for example, U.S. Patent No. 5,629,001. When arriving at the small intestine, the alkaline pH therein dissolves the coating and allows the composition to be released and absorbed by special cells such as epithelial enterocytes and Peyer's patch M cells. The syrup of the elixir can contain the active compound sucrose as a sweetener, methylparaben and propylparaben as preservatives, dyes and flavorings, such as cherry or orange flavorings. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and formulations.

[0304] For oral administration, the compositions of the present disclosure may be optionally combined with one or more excipients in the form of a mouthwash, a dentifrice, an oral tablet, an oral spray, or a sublingual oral administration formulation. For example, a mouthwash may be prepared by incorporating the desired amount of the active ingredient into an appropriate solvent such as a sodium borate solution (Dobell's solution). Alternatively, the active ingredient may be incorporated into an oral solution, such as one containing sodium borate, glycerol, and potassium bicarbonate, or dispersed in a dentifrice, or added to a composition that may include water, a binder, an abrasive, a flavoring agent, a foaming agent, and a humectant in a therapeutically effective amount. Alternatively, the composition may be prepared in tablet or solution form, which may be placed under the tongue or otherwise dissolved in the oral cavity.

[0305] Other formulations suitable for other modes of administration of food include suppositories. Suppositories are solid dosage forms of various weights and shapes for insertion into the rectum, usually containing medicine. After insertion, the suppository softens, melts or dissolves in the cavity fluid. In general, for suppositories, traditional carriers may include, for example, polyalkylene glycols, triglycerides or combinations thereof. In certain embodiments, suppositories may be formed by a mixture containing, for example, an active ingredient in the range of about 0.5% to about 10%, preferably about 1% to about 2%.

[0306] B. Parenteral Compositions and Formulations

[0307] In a further embodiment, the composition can be administered by a parenteral route. As used herein, the term "parenteral" includes routes that bypass the digestive tract. Specifically, the pharmaceutical compositions disclosed herein can be administered, for example but not limited to, intravenously, intradermally, intramuscularly, intraarterially, intravenously, intrathecally, intraventricularly, intratumorally, subcutaneously, or intraperitoneally. U.S. Patent Nos. 6,613,308; 5,466,468; 5,543,158; 5,641,515; and 5,399,363 (each specifically incorporated herein by reference in its entirety).

[0308] The solution of the active compound in the form of a free alkali or pharmacologically acceptable salt can be prepared in water appropriately mixed with a surfactant (e.g., hydroxypropylcellulose). Dispersions can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, and in oil. Under normal conditions of storage and use, these preparations contain preservatives to prevent microbial growth. Suitable drug forms for injection include sterile aqueous solutions or dispersions and sterile powders (U.S. Patent No. 5,466,468, which is particularly incorporated herein by reference in its entirety) for the temporary preparation of sterile injection solutions or dispersions. In all cases, the form must be sterile and must have fluidity to the extent that it is easy to inject. It must be stable under manufacturing and storage conditions, and must prevent the contamination of microorganisms (e.g., bacteria and fungi). The carrier can be a solvent or a dispersion medium containing, for example, water, ethanol, a polyol (i.e., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), a suitable mixture thereof, and / or a vegetable oil. Suitable fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the desired particle size in the case of a dispersion, and by using a surfactant. The prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferred to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be achieved by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0309] For example, for parenteral administration in aqueous solution, solution should be properly buffered if necessary, and first make liquid diluent isotonic with enough saline or glucose.These specific aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.In this regard, according to the present disclosure, those skilled in the art will know the sterile aqueous medium that can be adopted.For example, a dose can be dissolved in isotonic NaCl solution, and subcutaneous injection is added or injected at the proposed infusion site (see, for example, " Remington's Pharmaceutical Sciences " the 15th edition, pp. 1035-1038 and pp. 1570-1580).Depending on the condition of the subject being treated, some changes will inevitably occur in dosage.In any case, the personnel responsible for administration will determine appropriate dosage for individual subjects.In addition, for human administration, preparation should meet the sterility, pyrogenicity, general safety and purity standards required by the FDA Office of Biological Products standards.

[0310] Sterile injection solutions are prepared by mixing the required amount of active compound with various other ingredients listed above (as required) in a suitable solvent, and then filtering and sterilizing. Generally speaking, dispersions are prepared by mixing various sterile active ingredients into sterile vehicles, which contain basic dispersion media and other required ingredients from those listed above. In the case of sterile powders for the preparation of sterile injection solutions, preferred preparation methods are vacuum drying and freeze drying techniques, which produce active ingredients plus powders of any other required ingredients from their previously sterile filtered solutions. Powdered compositions are combined with liquid carriers (e.g., water or saline) with or without stabilizers.

[0311] C. Various pharmaceutical compositions and preparations

[0312] In other specific embodiments of the present disclosure, active compound compositions comprising NK cells and antibodies can be formulated for administration by a variety of different routes, such as topical (ie, transdermal) administration, mucosal administration (intranasal, vaginal, etc.) and / or inhalation.

[0313] The pharmaceutical composition for topical application may include an active compound formulated for pharmaceutical application, such as an ointment, paste, cream or powder. Ointments include all oily, absorbent, emulsion and water-soluble base compositions for topical application, while creams and lotions are those compositions that only contain emulsion bases. Topical drugs may contain penetration enhancers to promote the absorption of active ingredients through the skin. Suitable penetration enhancers include glycerol, alcohol, alkyl methyl sulfoxides, pyrrolidones and laurocapram. Possible bases for topical compositions include polyethylene glycol, lanolin, cold cream and vaseline and any other suitable absorbent, emulsion or water-soluble ointment bases. Topical preparations may also include necessary emulsifiers, gelling agents and antimicrobial preservatives to preserve active ingredients and provide a uniform mixture. Transdermal application of the present invention may also include the use of "patch". For example, a patch may provide one or more active substances at a predetermined rate and in a continuous manner over a fixed time period.

[0314] In certain embodiments, pharmaceutical compositions can be delivered by eye drops, intranasal sprays, inhalants and / or other aerosol delivery vehicles. The method for delivering the composition directly to the lungs by nasal aerosol sprays has been described in, for example, U.S. Patent Nos. 5,756,353 and 5,804,212 (each incorporated herein by reference in its entirety). Similarly, the delivery of drugs using intranasal microparticle resins (Takenaga et al., 1998) and lysophosphatidylglycerol compounds (U.S. Patent No. 5,725,871, particularly incorporated herein by reference in its entirety) is also well known in the pharmaceutical field. Similarly, transmucosal drug delivery in the form of a polytetrafluoroethylene support matrix is ​​described in U.S. Patent No. 5,780,045 (particularly incorporated herein by reference in its entirety).

[0315] The term aerosol refers to a colloidal system of finely divided solids of liquid particles dispersed in a liquefied or pressurized gas propellant. Typical inhalation aerosols of the present invention are composed of a suspension of active ingredients in a liquid propellant or a mixture of a liquid propellant and a suitable solvent. Suitable propellants include hydrocarbons and hydrocarbon ethers. Suitable containers will vary according to the pressure requirements of the propellant. The use of aerosols will vary according to the age, weight, and severity and response of the symptoms of the experimenter.

[0316] V. Combination Therapy

[0317] In certain embodiments, the compositions and methods of the embodiments of the present invention relate to cancer therapies other than compositions comprising NK cells and antibodies. Additional therapies may be radiotherapy, surgery (e.g., lumpectomy and mastectomy), chemotherapy, gene therapy, DNA therapy, virotherapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, hormone therapy, or a combination of the foregoing. Additional therapies may be in the form of adjuvant therapy or neoadjuvant therapy.

[0318] In some embodiments, additional therapy is the administration of small molecule enzyme inhibitors or anti-metastatic agents. In some embodiments, additional therapy is the administration of side effect limiters (e.g., agents intended to reduce the occurrence and / or severity of the side effects of treatment, such as anti-nausea agents, etc.). In some embodiments, additional therapy is radiotherapy. In some embodiments, additional therapy is surgery. In some embodiments, additional therapy is a combination of radiotherapy and surgery. In some embodiments, additional therapy is gamma irradiation. In some embodiments, additional therapy is targeted PBK / AKT / mTOR pathway therapy, HSP90 inhibitors, tubulin inhibitors, apoptosis inhibitors and / or chemopreventive agents. Additional therapy can be one or more chemotherapeutic agents known in the art.

[0319] Can be relative to other cancer therapy (such as immune checkpoint therapy) before, during, after or in various combinations to apply immune cell therapy (except the composition of the present disclosure).Application interval can be from simultaneously to several minutes to several days to several weeks.In the embodiment in which immune cell therapy and the composition of the present disclosure are separately provided to the patient, it is generally ensured that there is no significant time period between the time of each delivery, so that the two compounds will still be able to play a joint effect that is beneficial to the patient.In such a case, it is expected that immunotherapy and disclosed compositions can be provided to patients within about 12 to 24 or 72 hours each other, more specifically within about 6-12 hours each other.In some cases, it may be expected that the treatment time will be significantly extended, wherein a few days (2,3,4,5,6 or 7) to several weeks (1,2,3,4,5,6,7 or 8) pass between each application.

[0320] Administration of any compound or therapy of the embodiments of the invention to a patient will follow the general regimen 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 toxicity due to the combination therapy.

[0321] A. Chemotherapy

[0322] According to an embodiment of the present invention, a variety of chemotherapeutic agents can be used. The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapeutic agent" is used to represent a compound or composition used in the treatment of cancer. These agents or drugs are classified according to their intracellular activity mode (e.g., whether they affect the cell cycle and at what stage the cell cycle is affected). Alternatively, agents can be characterized based on their ability to directly cross-link DNA, insert DNA, or induce chromosome and mitotic aberrations by affecting nucleic acid synthesis.

[0323] Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclophosphamide; alkyl sulfonates, such as busulfan, improsulfan and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa and uredopa; ethyleneimines and methylamelamines, including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (particularly bullatacin and bullatacinone); camptothecins (including the synthetic analogs topotecan); bryostatin; callystatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin and bizelesin); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycins (including synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards, for example chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine hydrochloride; oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, and uracil mustard;Nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimnustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicins, particularly calicheamicin gamma II and calicheamicin omega II); dynemicins, including dynemicin A; bisphosphonates, such as clodronate; esperamicin; and neocarcinogen chromophores and related chromoprotein enediyne antibiotics. Chromophores, aclacinomysin, actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, quercetin, lamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folate analogs such as leucovorin, pteropterin, and trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiopurine, and thioguanine;Pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; adrenal antistagnation drugs such as mitotane and trilostane; folic acid supplements such as folinic acid; aceglatone; aldophosphamide glycosides; androgens such as dapoxetine, dapoxetine, and dapoxetine. glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid acid; 2-ethylhydrazines; methylbenzylhydrazine; PSK polysaccharide complex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triazinon; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, mucin A, baculosporin A, and anguidine); urethan; vindesine; dacarbazine; mannomustine; dibromomannitol; dibromodulanol; pipobroman;gacytosine; cytarabine ("Ara-C"); cyclophosphamide; taxanes 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; daunorubicin; aminopterin; xeloda; ibandronic acid salts; irinotecan (e.g. CPT-11); topoisomerase inhibitor 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. ;

[0324] B. Radiation therapy

[0325] Other factors that cause DNA damage and have been widely used include the directional delivery of commonly known gamma rays, X-rays and / or radioisotopes to tumor cells. Other forms of DNA damaging factors may also be considered, such as microwaves, proton beam irradiation (USPs 5,760,395 and 4,870,287) and UV irradiation. Most likely, all of these factors affect extensive damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. The dosage range of X-rays ranges from daily doses of 50 to 200 roentgens for a long time (3-4 weeks) to single doses of 2000 to 6000 roentgens. The dosage range of radioisotopes varies widely and depends on the half-life of the isotope, the intensity and type of the irradiation emitted, and the absorption of tumor cells.

[0326] C. Immunotherapy

[0327] Those skilled in the art will appreciate that other immunotherapies (outside of the disclosed cell therapies) may be combined or used in conjunction with the methods of the embodiments.

[0328] In certain embodiments, other immunotherapies include administering one or more antibodies to the subject. In certain embodiments, the other one or more antibodies administered to the subject may be the same one or more antibodies loaded onto NK cells. In some embodiments, the other one or more antibodies administered to the subject may target one or more antigens that are the same as the antigens targeted by the one or more antibodies loaded onto NK cells.

[0329] In the context of cancer treatment, immunotherapy often relies on the use of immune effector cells and molecules to target and destroy cancer cells. is such an example. Immune effectors can be, for example, antibodies that are specific for certain markers on the surface of tumor cells. A single antibody can act as an effector of therapy, or it can recruit other cells to actually affect cell killing. Antibodies can also be conjugated to drugs or toxins (chemotherapeutic agents, radionuclides, ricin A chain, cholera toxin, pertussis toxin, etc.) and used as targeting agents. Alternatively, effectors can be lymphocytes that carry surface molecules that interact directly or indirectly with tumor cell targets. Various effector cells include cytotoxic T cells and NK cells, in addition to cells with knockdown or knockout of TGF-βR2.

[0330] Antibody-drug conjugates have emerged as a breakthrough approach to the development of cancer therapeutics. Antibody-drug conjugates (ADCs) comprise a monoclonal antibody (MAb) covalently linked to a cytocidal drug. This approach combines the high specificity of a MAb for its antigenic target with a highly potent cytotoxic drug, resulting in an “armed” MAb that delivers its payload (drug) to tumor cells that have abundant levels of antigen. Targeted delivery of the drug also minimizes its exposure to normal tissues, thereby reducing toxicity and improving the therapeutic index. Two ADC drugs approved by the FDA, (brentuximab vedotin) and the 2013 (trastuzumab emtansine or T-DM1) validated this approach. There are currently 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 more sophisticated, the discovery and development of new ADCs increasingly rely on the identification and validation of new targets suitable for this approach and the generation of targeted MAbs. Two criteria for ADC targets are upregulation / high-level expression in tumor cells and robust internalization.

[0331] In one aspect of immunotherapy, tumor cells must carry some markers that are easy to target, i.e., they are not present on most other cells. There are many tumor markers, and in the context of embodiments of the present invention, any of these tumor markers may be suitable for targeting. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialylated Lewis antigens, MucA, MucB, PLAP, laminin receptor, erbB and p155. An optional aspect of immunotherapy is to combine anticancer effects with immunostimulatory effects. There are also immunostimulatory molecules, including: cytokines, such as IL-2, IL-4, IL-12, GM-CSF, γ-IFN, chemokines, such as MIP-1, MCP-1, IL-8 and growth factors, such as FLT3 ligand.

[0332] Examples of immunotherapies currently under investigation or in use are 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 any type of interferon, IL-1, GM-CSF, and TNF (Bukowski et al., 1998; Davidson et al., 1998; H ellstrand 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. Patents 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 5,824,311). It is contemplated that one or more anti-cancer therapies may be used with the antibody therapies described herein.

[0333] In some embodiments, the immunotherapy may be an immune checkpoint inhibitor. Immune checkpoints turn up signals (e.g., co-stimulatory molecules) or turn down signals. Inhibitory immune checkpoints that may be targeted by immune checkpoint blockade include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), cytotoxic T lymphocyte-associated protein 4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin (KIR), lymphocyte activation gene-3 (LAG3), programmed death 1 (PD-1), T cell immunoglobulin domain and mucin domain 3 (TIM-3), and V-domain Ig inhibitor of T cell activation (VISTA). In particular, immune checkpoint inhibitors target the PD-1 axis and / or CTLA-4.

[0334] D. Surgery

[0335] Approximately 60% of cancer patients will undergo some type of surgery, including preventive, diagnostic or staging, curative, and palliative surgery. Curative surgery includes resection (in which all or part of the cancerous tissue is physically removed, excised, and / or destroyed) and may be used in conjunction with other therapies (e.g., treatment of embodiments of the present invention, 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 a tumor. In addition to tumor resection, surgical treatments also include laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs surgery).

[0336] After removing part or all of the cancer cells, tissues or tumors, a cavity may be formed in the body. Treatment may be accomplished by perfusing, directly injecting or topically applying other anti-cancer therapies to the area. For example, this treatment may be repeated every 1, 2, 3, 4, 5, 6 or 7 days, or every 1, 2, 3, 4 and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months. These treatments may also have different dosages.

[0337] E. Other medicines

[0338] It is expected that other agents can be used in combination with certain aspects of the embodiments of the present invention to improve the efficacy of treatment. These additional agents include agents that affect the upregulation of cell surface receptors and GAP connections, cell growth inhibitors and differentiation agents, cell adhesion inhibitors, agents that increase the sensitivity of overproliferation cells to apoptosis inducing agents or other biological agents. Increasing intercellular signal transduction by increasing the number of GAP connections will increase the anti-overproliferation effect on neighboring overproliferation cell groups. In other embodiments, cell growth inhibitors or differentiation agents can be used in combination with certain aspects of the embodiments of the present invention to improve the anti-overproliferation efficacy of treatment. It is expected that inhibitors of cell adhesion can improve the efficacy of the embodiments of the present invention. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. It is further considered that other agents (such as antibody c225) that increase the sensitivity of overproliferation cells to apoptosis can be used in combination with certain aspects of the embodiments of the present invention to improve the therapeutic efficacy.

[0339] VI. Kits of the Disclosure

[0340] In some embodiments, a kit is provided that may include, for example, NK cells, optionally one or more culture media and components for producing NK cells, one or more antibodies or reagents for producing antibodies, etc. In some embodiments, the preparation may include a mixture of factors, including a form suitable for combination with NK cells. Where appropriate, the reagent system or any kit component may be packaged in an aqueous medium or in a lyophilized form. The container device of the kit generally includes at least one vial, test tube, flask, bottle, syringe or other container device, into which the components may be placed and preferably appropriately aliquoted. When there is more than one component in the kit, the kit will generally also include a second, third or other additional container, in which the additional components may be placed separately. However, a combination of various components may be included in the vial. The components of the kit may be provided in the form of a dry powder. When the reagent and / or component is provided in the form of a dry powder, the powder may be reconstituted by adding a suitable solvent. It is conceivable that the solvent may also be provided in another container device. The kit generally also includes a device for enclosing the kit components for commercial sale. Such a container may include an injection-molded or blow-molded plastic container in which the desired vial is retained. The kit may also include instructions for use, eg, in printed or electronic format, eg, digital format.

[0341] In a specific embodiment, the kit may include one or more cytokines, including at least IL-12, IL-15, IL-18 and / or IL-2, including specific concentrations described elsewhere herein. The kit may include any type of medium, any component of a cryopreservation medium, as described elsewhere herein. The kit may include cord blood (including pooled cord blood), any type of antigen presenting cells, beads for eliminating specific NK cells (as described herein), vectors encoding one or more proteins as described herein, NK cells, antibodies, or reagents for producing antibodies, etc.

[0342] Individual components may also be provided in a concentrated amount in the kit; in some embodiments, a component is provided alone at the same concentration as in solution with the other components. The concentration of a component may be provided at 1x, 2x, 5x, 10x, or 20x or higher.

[0343] VII. Aspects

[0344] The following aspects describe certain inventions disclosed herein.

[0345] Aspect 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, wherein the Fc region of the monospecific antibody binds to the engineered NK cells and the antigen binding domain of the monospecific antibody is capable of binding to the target antigen; or (b) the antibody is multispecific, and one or more antigen binding domains of the antibody bind to the target antigen, another one or more antigen binding domains bind to another target antigen, or another antigen binding domain of the antibody is capable of binding to an NK cell surface antigen.

[0346] Aspect 2. The composition of Aspect 1, wherein the engineered NK cells are expanded or not expanded.

[0347] Aspect 3. The composition of Aspect 1 or 2, wherein the engineered NK cells are or are not pre-activated.

[0348] Aspect 4. The composition of any one of Aspects 1-3, wherein the multispecific antibody is bispecific, trispecific or multispecific.

[0349] Aspect 5. The composition of any one of Aspects 1-4, wherein in (a), the composition is further defined as a complex between the engineered NK cells and the monospecific antibody formed by binding of the Fc region of the monospecific antibody to the engineered NK cells.

[0350] Aspect 6. The composition of any one of Aspects 1-5, wherein the complex further comprises an antigen binding domain of a monospecific antibody that binds to its target antigen.

[0351] Aspect 7. The composition of any of Aspects 1-4, wherein in (b), the composition is further defined as a complex between the engineered NK cell and the multispecific antibody formed by the binding of one or more antigen binding domains of the multispecific antibody that binds to one or more surface antigens on the engineered NK cell.

[0352] Aspect 8. The composition of any one of Aspects 1-4 and 7, wherein one or more antigen binding domains of the antibody bind to its target antigen.

[0353] Aspect 9. The composition of any one of aspects 1-6, wherein the target antigen is a stem cell antigen, an autoantigen, or a cancer antigen selected from the group consisting of 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, mutant p53, mutant ras, HER2 / Neu, ERBB2, folate binding protein, HIV-1 envelope glycoprotein gp120, H IV-1 envelope glycoproteins gp41, GD2, CD5, CD123, CD23, 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.

[0354] Aspect 10. The composition of any one of Aspects 1-9, wherein the engineered NK cell surface antigen is CD16, CD32, CS1, CD56, CD64, NKG2D, NKG2C, DNAM, 2B4, CD2, NCR, NKp30, NKp44, NKp46 or KIR.

[0355] Aspect 11. The composition of any one of Aspects 1-9, wherein the NK cells are derived from umbilical cord blood, and wherein the source of the umbilical cord blood is umbilical cord blood from one donor or a pool of umbilical cord blood units from two or more individuals.

[0356] Aspect 12. The composition of aspect 11, wherein the CB is pooled from 3, 4, 5, 6, 7 or 8 individual cord blood units.

[0357] Aspect 13. The composition of any one of Aspects 1-12, wherein the engineered NK cells are derived from umbilical cord blood mononuclear cells, umbilical cord blood hematopoietic stem cells, iPSCs, peripheral blood NK cells, or NK cell lines (eg, NK-92).

[0358] Aspect 14. The composition of any one of Aspects 1-13, wherein the engineered NK cells are CD56+, CD3-, or both.

[0359] Aspect 15. The composition of any one of Aspects 1-14, wherein the composition is used fresh or stored frozen.

[0360] Aspect 16. The composition of any one of Aspects 1-15, wherein the source of the engineered NK cells is a fresh source or a cryopreserved bank.

[0361] Aspect 17. The composition of Aspect 15 or 16, wherein when the engineered NK cells are derived from cryopreservation, the engineered NK cells are cryopreserved in a culture medium comprising at least one cryoprotectant, at least one serum or a non-serum substitute for serum, and optionally at least one cytokine and / or at least one growth factor.

[0362] Aspect 18. The composition of aspect 17, wherein the cryoprotectant is dimethyl sulfoxide (DMSO), glycerol, glycerol, hydroxyethyl starch, dextran trehalose, or a combination thereof.

[0363] Aspect 19. The composition of aspect 17 or 18, wherein the non-serum substitute comprises platelet lysate and / or blood product lysate or human or animal serum albumin.

[0364] Aspect 20. The composition of any one of Aspects 17-19, wherein at least one cytokine is a natural protein, a recombinant protein, a synthetic protein, or a mixture thereof.

[0365] Aspect 21. The composition of any one of Aspects 17-20, wherein 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.

[0366] Aspect 22. The composition of any one of Aspects 1-21, wherein the engineered NK cells comprise one or more transgenes encoding one or more engineered receptors.

[0367] Aspect 23. The composition of Aspect 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.

[0368] Aspect 24. The composition of any one of Aspects 1-23, wherein the engineered NK cells express one or more transgenes encoding one or more heterologous cytokines and / or cytokine receptors.

[0369] Aspect 25. The composition of Aspect 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.

[0370] Aspect 26. The composition of any one of Aspects 1-25, wherein the engineered NK cells express one or more transgenes encoding one or more receptors to enhance their binding to antibodies.

[0371] Aspect 27. The composition of Aspect 26, wherein the one or more receptors include or are Fc receptors.

[0372] Aspect 28. The composition of Aspect 26, wherein the receptor is CD16, CD32, CD64 or a combination thereof.

[0373] Aspect 29. The composition of any one of Aspects 1-28, wherein the engineered NK cells express one or more transgenes encoding one or more suicide genes.

[0374] Aspect 30. The composition of any one of Aspects 1-29, wherein the composition is contained in a solution or solid comprising one or more cryoprotectants.

[0375] Aspect 31. The composition of any one of Aspects 1-30, wherein the composition is contained in a pharmaceutically acceptable carrier.

[0376] Aspect 32. A method for producing a composition of any one of Aspects 1-31, the method comprising the following steps: (i) optionally expanding engineered NK cells in a culture containing effective amounts of: (a) cytokines selected from IL-2, IL-15, IL-18, IL-21 and combinations thereof; and (b) antigen presenting cells / feeder cells, fragments of antigen presenting cells / feeder cells or NK cell activation beads; and (ii) providing antibody molecules to the engineered NK cells, and providing the antibody molecules to the engineered NK cells before and / or after expansion.

[0377] Aspect 33. The method of Aspect 32, wherein the method comprises a pre-activation step before and / or after the expansion step, wherein the engineered NK cells are pre-activated in a culture comprising an effective concentration of one or more of IL-2, IL-12, IL-15 and IL-18.

[0378] Aspect 34. The method of Aspect 33, wherein the culture comprises two or more of IL-2, IL-12, IL-15 and IL-18 at effective concentrations.

[0379] Aspect 35. The method of Aspect 33 or 34, wherein the culture comprises effective concentrations of three or more of IL-2, IL-12, IL-15 and IL-18.

[0380] Aspect 36. The method of any one of Aspects 32-35, wherein the culture comprises effective concentrations of IL-12, IL-15 and IL-18.

[0381] Aspect 37. The method of any one of Aspects 32-36, wherein IL-12 is used in the culture instead of IL-15.

[0382] Aspect 38. The method of any one of Aspects 32-37, wherein the providing step is further defined as culturing the engineered NK cells with the antibody molecule for a specific duration or combining the engineered NK cells and the antibody molecule immediately prior to infusion.

[0383] Aspect 39. The method of Aspect 38, wherein the duration is from about 5 minutes to about 24 hours or more.

[0384] Aspect 40. The method of Aspect 38 or 39, wherein the culture comprises Plasma-Lyte A and / or human serum albumin.

[0385] Aspect 41. The method of any one of Aspects 32-40, wherein after incubation, the composition is infused into a recipient subject without prior washing.

[0386] Aspect 42. The method of any one of Aspects 32-40, wherein after culturing, the composition is infused into a recipient subject after one or more washes.

[0387] Aspect 43. The method of any one of Aspects 32-42, wherein the engineered NK cells are depleted of CD3+, CD14+ and / or CD19+ cells.

[0388] Aspect 44. The method of Aspect 43, wherein the depletion step occurs prior to the pre-activation step, and / or prior to expansion with feeder cells and / or NK cell activation beads, and / or prior to culturing with one or more cytokines, and / or prior to infusion.

[0389] Aspect 45. The method of any one of Aspects 32-44, further comprising the step of obtaining NK cells from umbilical cord blood, wherein the umbilical cord blood does not include umbilical cord tissue.

[0390] Aspect 46. The method of any one of Aspects 32-45, wherein the antigen presenting cell is an artificial (aAPC).

[0391] Aspect 47. The method of Aspect 46, wherein the aAPC expresses CD137 ligand.

[0392] Aspect 48. The method of Aspect 46 or 47, wherein the aAPCs also express membrane-bound cytokines.

[0393] Aspect 49. The method of Aspect 48, wherein the membrane-bound cytokine is membrane-bound IL-21 (mIL-21) or membrane-bound IL-15 (mIL-15).

[0394] Aspect 50. The method of any one of Aspects 46-49, wherein the aAPCs do not substantially express endogenous HLA class I, II or CD1d molecules.

[0395] Aspect 51. The method of any one of Aspects 46-50, wherein the aAPC expresses ICAM-1 (CD54) and / or LFA-3 (CD58) or CD48.

[0396] Aspect 52. The method of any one of Aspects 46-51, wherein the aAPC is further defined as a leukemia cell-derived aAPC.

[0397] Aspect 53. The method of Aspect 52, wherein the leukemia cell-derived aAPCs are K562 cells engineered to express CD137 ligand and / or mIL-21.

[0398] Aspect 54. The method of Aspect 53, wherein the K562 cells are engineered to express CD137 ligand and mIL-21.

[0399] Aspect 55. The method of any one of Aspects 46-54, wherein the aAPCs have been engineered by retroviral transduction.

[0400] Aspect 56. The method of any one of Aspects 46-55, wherein the aAPCs are irradiated.

[0401] Aspect 57. The method of any one of Aspects 32-56, wherein the pre-activation step lasts for 10-20 hours.

[0402] Aspect 58. The method of any one of Aspects 32-57, wherein the pre-activation step lasts for 14-18 hours.

[0403] Aspect 59. The method of any one of Aspects 32-58, wherein the pre-activation step lasts for 16 hours.

[0404] Aspect 60. The method of any one of Aspects 32-59, wherein the culture used in the pre-activation step comprises IL-18 and / or IL-15 at a concentration of 1-1000 ng / mL.

[0405] Aspect 61. The method of any one of Aspects 32-60, wherein the culture used in the pre-activation step comprises IL-18 and / or IL-15 at a concentration of 1-1000 ng / mL.

[0406] Aspect 62. The method of any one of Aspects 32-61, wherein the culture used in the pre-activation step comprises IL-18 and / or Il-15 at a concentration of 1-1000 ng / mL.

[0407] Aspect 63. The method of any one of Aspects 32-62, wherein the culture used in the pre-activation step comprises IL-12 at a concentration of 0.1-1000 ng / mL.

[0408] Aspect 64. The method of any one of Aspects 32-63, wherein the culture of the pre-activation step comprises IL-12 at a concentration of 1-1000 ng / mL.

[0409] Aspect 65. The method of any one of Aspects 32-64, wherein the culture of the pre-activation step comprises IL-12 at a concentration of 10 ng / mL.

[0410] Aspect 66. The method of any one of Aspects 32-65, further comprising washing the pre-activated engineered NK cells before and / or after the expansion step.

[0411] Aspect 67. The method of any one of Aspects 32-66, wherein the engineered NK cells are activated at least twice or more with IL-12, IL-15, IL-18, IL-2, or any combination thereof during the expansion step.

[0412] Aspect 68. The method of Aspect 67, wherein washing is performed multiple times.

[0413] Aspect 69. The method of any one of Aspects 34-68, wherein the expansion is continued for 5-60 days.

[0414] Aspect 70. The method of any one of Aspects 32-69, wherein the expansion is continued for 12-16 days.

[0415] Aspect 71. The method of any one of Aspects 32-69, wherein the expansion is continued for 18-24 days.

[0416] Aspect 72. The method of any one of Aspects 32-71, wherein the pre-activated engineered NK cells and aAPCs are present in the expansion culture at a ratio of 3:1 to 1:3.

[0417] Aspect 73. The method of Aspect 72, wherein the pre-activated engineered NK cells and aAPCs are present in the expansion culture at a ratio of 1:2.

[0418] Aspect 74. The method of any one of Aspects 32-73, wherein the expansion culture further comprises IL-2.

[0419] Aspect 75. The method of Aspect 74, wherein IL-2 is present at a concentration of 10-500 U / mL.

[0420] Aspect 76. The method of Aspect 75, wherein IL-2 is present at a concentration of 100-300 U / mL.

[0421] Aspect 77. The method of Aspect 76, wherein IL-2 is present at a concentration of 200 U / mL.

[0422] Aspect 78. The method of any one of Aspects 32-77, wherein IL-12, IL-18, IL-15 and / or IL-2 are recombinant.

[0423] Aspect 79. The method of any one of Aspects 32-78, wherein IL-2 is supplemented in the expansion culture every 2-3 days.

[0424] Aspect 80. The method of any one of Aspects 32-79, wherein the APCs are added to the expansion culture at least a second time.

[0425] Aspect 81. The method of any one of Aspects 32-80, wherein one or more steps of the method are performed in serum-free medium.

[0426] Aspect 82. A method of treating a disease or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of the composition of any one of Aspects 1-31.

[0427] Aspect 83. The method of Aspect 82, wherein the disease or condition is cancer, inflammation, graft-versus-host disease, transplant rejection, an autoimmune disease, an immunodeficiency disease, a B-cell malignancy, or an infection.

[0428] Aspect 84. The method of Aspect 82 or 83, wherein the cancer is a hematological cancer or a solid tumor.

[0429] Aspect 85. The method of Aspect 84, wherein the blood cancer is a leukemia selected from acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), multiple myeloma, acute myeloid leukemia (AML) and chronic myeloid leukemia (CML).

[0430] Aspect 86. The method of any one of Aspects 82-85, wherein the engineered NK cells are allogeneic to the subject.

[0431] Aspect 87. The method of any one of Aspects 82-86, wherein the engineered NK cells are autologous to the subject.

[0432] Aspect 88. The method of Aspect 82, wherein the disease is graft-versus-host disease (GVHD).

[0433] Aspect 89. The method of Aspect 82, wherein the disease is multiple sclerosis, inflammatory bowel disease, rheumatoid arthritis, type I diabetes, systemic lupus erythematosus, contact hypersensitivity, asthma, or Sjögren's syndrome.

[0434] Aspect 90. The method of any one of Aspects 82-89, wherein the subject is a human.

[0435] Aspect 91. The method of any one of Aspects 82-90, further comprising administering to the subject at least one second therapeutic agent.

[0436] Aspect 92. The method of Aspect 91, wherein the at least one 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.

[0437] Aspect 93. The method of Aspect 92, wherein the anticancer agent is chemotherapy, radiotherapy, gene therapy, surgery, hormone therapy, anti-angiogenic therapy or immunotherapy.

[0438] Aspect 94. The method of Aspect 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.

[0439] Aspect 95. The method of any one of Aspects 91-94, wherein the composition and / or at least one second therapeutic agent is administered intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, transdermally, subcutaneously, regionally, or by direct injection or infusion.

[0440] Aspect 96. The method of any one of Aspects 91-95, wherein the second therapeutic agent is an antibody.

[0441] Aspect 97. The method of Aspect 96, wherein the antibody is a monoclonal antibody, a bispecific antibody or a multispecific antibody.

[0442] Aspect 98. A method for treating a subject with cancer using engineered NK cells, the method 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 ex vivo with an antibody and then administering the loaded engineered NK cells to the subject, or cii) loading the engineered NK cells in vivo with the antibody by administering the engineered NK cells within 1 hour of administering the antibody; wherein the engineered NK cells comprise one or more transgenes, and wherein the loading comprises exposing the engineered NK cells to the antibody for a time effective to allow the antibody to bind to the surface of the engineered NK cells.

[0443] Aspect 99. The method of Aspect 98, wherein loading the engineered NK cells comprises exposing the engineered NK cells to the antibody for greater than or equal to about 1 hour or less than or equal to about 1 hour.

[0444] Aspect 100. The method of Aspect 98 or 99, wherein the plurality of engineered NK cells remain loaded with antibody for equal to or greater than 5 days.

[0445] Aspect 101. The method of any one of Aspects 98-100, wherein the antibody binds to the engineered NK cell via interaction with the CD16 surface protein.

[0446] Aspect 102. The method of any one of Aspects 98-101, wherein the engineered NK cells are derived from umbilical cord blood.

[0447] Aspect 103. The method of any one of Aspects 98-102, wherein the engineered NK cells are activated ex vivo.

[0448] Aspect 104. The method of any one of Aspects 98-103, wherein the engineered NK cells are pre-activated with IL-18, IL-15 and IL-12.

[0449] Aspect 105. The method of any one of Aspects 98-104, wherein the antibody is an anti-EGFR antibody.

[0450] Aspect 106. The method of any one of Aspects 98-104, wherein the antibody is an anti-EGFR2 antibody.

[0451] Aspect 107. The method according to any one of Aspects 98 to 104, wherein the antibody is a monospecific antibody.

[0452] Aspect 108. The method of Aspect 105, wherein the antibody is imatinib.

[0453] Aspect 109. The method of Aspect 106, wherein the antibody is magetuximab.

[0454] Aspect 110. The method of any one of Aspects 98-105, wherein the antibody is a bispecific antibody.

[0455] Aspect 111. The method of Aspect 110, wherein the antibody is ivanovumab.

[0456] Aspect 112. The method of any one of Aspects 98-104, wherein the antibody is an anti-CD123 antibody.

[0457] Aspect 113. The method of any one of Aspects 98-104, wherein the antibody is a bispecific antibody.

[0458] Aspect 114. The method of any one of Aspects 98-104, wherein the antibody comprises an antigen binding domain that targets CD123 and an antigen binding domain that targets NKp46.

[0459] Aspect 115. The method of Aspect 105, wherein the antibody is IPH61.

[0460] Aspect 116. The method of any one of Aspects 98-115, wherein one or more transgenes comprise an engineered receptor.

[0461] Aspect 117. The method of Aspect 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.

[0462] Aspect 118. The method of Aspect 116 or 117, wherein the engineered receptor comprises a CAR.

[0463] Aspect 119. The method of any one of Aspects 98-118, wherein the one or more transgenes comprise a cytokine.

[0464] Aspect 120. The method of Aspect 119, wherein the cytokine is IL-2, IL-12, IL-15, IL-18 and / or IL-21.

[0465] Aspect 121. The method of any one of Aspects 98-120, wherein the engineered NK cells comprise engineered mutations in endogenous genes.

[0466] Aspect 122. The method of Aspect 121, wherein the engineered mutation in the endogenous gene is in the CISH, CD38, glucocorticoid receptor and / or TGFBR2 gene. Example

[0467] The following examples are included to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques that the inventors have discovered to work well in practicing the present invention and therefore can be considered to constitute preferred modes for its practice. However, based on this disclosure, it will be appreciated by those skilled in the art that many changes may be made in the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the present invention.

[0468] Example 1 - Preparation and Use of Compositions

[0469] The composition comprising specific engineered NK cells and antibodies capable of binding engineered NK cells is prepared, and in certain cases, an individual in need of treatment is given in an effective amount. In a specific embodiment, an individual is administered an engineered NK cell that is autologous or allogeneic relative to the individual in an effective amount, and the engineered NK cell is particularly a cord blood cell. The engineered NK cell may or may not be derived from a cryopreservation bank, and is cryopreserved in a specific cryopreservation medium in some cases. Before an effective amount of engineered NK cells is exposed to an antibody under conditions that allow the antibody to bind to the engineered NK cell by an appropriate antigen binding fragment and / or Fc domain, the engineered NK cell is optionally preactivated and optionally amplified. Thereafter, an effective amount of the composition is provided to an individual in need thereof.

[0470] Example 2-Monoclonal Antibodies and NK Cells

[0471] This example includes the preparation and use of NK cells preloaded with antibodies. Figure 1An example of an experimental procedure for NK cell expansion and antibody loading is shown.NK cells can be obtained from processed blood, and in a specific embodiment, certain NK cells are separated, such as NK cells of CD3-CD56+ in a specific embodiment.Optionally, NK cells are expanded, such as in the presence of IL-2 and universal antigen presenting cells or other feeder cells, and the expansion step can be any suitable duration, such as up to about 14 days in a specific case. In another optional embodiment, NK cells are pre-activated before expansion. Although any pre-activation step can be used, in a specific embodiment, the pre-activation step includes exposure to one or more cytokines, such as one or more of IL-12, IL-15 and / or IL-18. The pre-activation step can last for any suitable length of time, but in a specific embodiment, the pre-activation step is up to about 16 hours. After this step, NK cells can be selectively expanded, including expansion in the presence of universal antigen presenting cells and IL-2. During expansion, the cultured cells can be activated again with IL2, IL-18 or IL-15 or any combination of these cytokines. After expansion, the NK cells are exposed to an appropriate amount of the antibody under conditions sufficient to allow the antibody to bind to the exterior of the NK cell via the specific antigen to which the antibody is directed. In certain instances, the antibody is a monoclonal antibody. The conditions for loading the antibody onto the NK cells may be at a specific temperature for a specific duration, such as 37° C. for one hour.

[0472] Figure 2 Validation of the binding of magetuximab (anti-HER2 monoclonal antibody) to NK cells is provided. NK cells were derived from umbilical cord blood and were expanded normally (NE) with irradiated (100Gy) uAPC feeder cells (2:1 feeder cell:NK ratio) and recombinant human IL-2 (200U / ml) in 50:50Click's / RPMI medium, or pre-activated for 16 hours with IL-12 (10ng / ml), IL-15 (50ng / ml) and IL-18 (50ng / ml), then washed and expanded with irradiated uAPC and IL-2 (200U / ml) (PE). NK cells were loaded with 1μg / ml magetuximab for one hour at 37°C in Click's / RPMI medium and then washed before verifying the binding of magetuximab to NK cells by flow cytometry. Unloaded NE and PE NK cells were used as negative controls. Magetuximab binding was detected by flow cytometry using Alexa-Fluor 647 affinity purified F(ab')2 fragment goat anti-human IgG (H+L) antibody staining. The graph shows that PE NK cells showed higher magetuximab binding levels compared to NE NK cells. The histogram provides data from 3 different cord blood NK cell donors.

[0473] NK cells loaded with magetuximab showed enhanced cytotoxicity against HER2+ tumor cells ( Figure 3 ). Provided herein is an Xcelligence cytotoxicity assay showing the normalized cell index of HER2+SKOV3 (ovarian cancer) cultured alone or with NK cells at a 2:1 effector to target (E:T) ratio. NK cells were expanded normally (NE) or pre-activated and expanded (P+E). NE NK cells were cultured with irradiated (100 Gy) uAPC feeder cells (2:1 feeder cell:NK ratio) and recombinant human IL-2 (200 U / ml) in 50:50 Click's / RPMI medium, and PE cells were pre-activated with IL-12 (10 ng / ml), IL-15 (50 ng / ml) and IL-18 (50 ng / ml) for 16 hours, then washed and expanded with irradiated uAPC and IL-2 (200 U / ml). NK cells were either kept unloaded or loaded with 1 μg / ml magetuximab in Click's / RPMI medium at 37°C for one hour and then washed prior to the assay. The data demonstrate that magetuximab loading enhances the cytotoxicity of NE and PE NK cells against tumor cells.

[0474] NK cells loaded with magetuximab resulted in enhanced tumor control in the NSG mouse model of HER2+ ovarian cancer (SKOV3) Figure 4 ).exist Figure 4 In A, there is an example of a schematic diagram of an experimental plan for the SKOV3 mouse model. Figure 4 In B, bioluminescence imaging shows tumor growth over time in mice transplanted with firefly luciferase (FFluc)-transduced SKOV3 that were either untreated or treated with NK cells alone, magetuximab alone, or NK cells preloaded with magetuximab. Figure 4 C provides display Figure 4 A is a graph of the mean radiance over time for the four groups of mice depicted.

[0475] exist Figure 5In the figure, the validation of the binding of efantuzumab (EGFR-MET bispecific antibody) to NK cells is shown. NK cells were derived from umbilical cord blood and expanded normally (NE) with irradiated (100Gy) uAPC feeder cells (2:1 feeder cell:NK ratio) and recombinant human IL-2 (200U / ml) in 50:50Click's / RPMI medium, or pre-activated 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) (PE). NK cells were loaded with 1μg / ml efantuzumab for 1 hour at 37°C in Click's / RPMI medium, washed, and then the binding of efantuzumab to NK cells was verified by flow cytometry. Unloaded NE and PE NK cells were used as negative controls. Evantamab binding was detected by flow cytometry using Alexa-Fluor647 affinity purified F(ab')2 fragment goat anti-human IgG (H+L) antibody staining. The graph shows that PE NK cells exhibited higher levels of Evantamab binding compared to NE NK cells. The histogram shows data from 3 different cord blood NK cell donors.

[0476] NK cells loaded with ivantacin showed enhanced cytotoxicity against EGFR+ / cMET+ tumor cells ( Figure 6 Chromium release assays showed specific lysis of 3 EGFR+ / c-MET+ cancer cell lines SKOV3 (ovarian cancer), UMRC3 (renal cell carcinoma), PATC-148 (pancreatic ductal adenocarcinoma) using 3 different cord blood NK cells (N=3 ...

Claims

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 comprising ervantumab, magetuximab and / or imatinib.

2. The composition of claim 1, wherein the engineered NK cells are from an expanded or unexpanded population.

3. The composition of claim 1 or 2, wherein the engineered NK cells are from a pre-activated or non-pre-activated population.

4. The composition of any one of claims 1-3, wherein in (i), the composition is further defined as a complex formed between the engineered NK cells and magetuximab, ervantumab and / or imaretuzumab through the binding of the Fc region of the antibody to the engineered NK cells.

5. The composition of any one of claims 1-4, wherein the complex further comprises one or more antigen binding domains of one or more antibodies that bind to their target antigen.

6. The composition of any one of claims 1-5, wherein 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).

7. The composition of any one of claims 1-6, wherein the engineered NK cells are 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 a combination thereof.

8. The composition of any one of claims 1-7, wherein the engineered NK cells are derived from primary NK cells rather than from stem cells and / or induced pluripotent stem cells (iPSCs).

9. The composition of any one of claims 1-8, wherein the NK cells are derived from umbilical cord blood, and wherein the source of the CB monocytes and / or CB hematopoietic stem cells is CB from one donor or from a pool of 2 or more individual CB units.

10. The composition of any one of claims 1-9, wherein the engineered NK cells are modified to contain one or more transgenes for expressing one or more heterologous proteins.

11. The composition of claim 10, wherein the one or more heterologous proteins comprise one or more engineered receptors and / or one or more cytokines.

12. The composition of claim 11, wherein the one or more engineered receptors are chimeric antigen receptors (CARs), T cell receptors (TCRs), receptors that recognize the FC portion of Ig (FC recognition receptors), chemokine receptors, homing receptors, chimeric cytokine receptors, or any combination thereof.

13. The composition of claim 12, wherein the FC recognition receptors include CD16, CD32 and / or CD64, and / or wherein the engineered NK cells express one or more transgenes encoding one or more receptors to enhance their binding to antibodies.

14. The composition of any one of claims 11-13, wherein the engineered receptor comprises a TCR and / or a CAR.

15. The composition of any one of claims 11-14, wherein the engineered receptor targets a stem cell antigen, a self antigen, or a cancer antigen selected from the group consisting of 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, CD123, CD23, 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.

16. The composition of any one of claims 11-15, wherein the engineered antigen receptor targets CD70 and / or TROP2.

17. The composition of claim 16, wherein the antigen receptor targeting CD70 comprises a sequence that is at least or exactly 80%, 81%, 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.

18. The composition of claim 16 or 17, wherein the engineered NK cells comprise a construct that is at least or exactly 80%, 81%, 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.

19. The composition of any one of claims 16-18, wherein the antigen receptor targeting TROP2 comprises a sequence that is at least or exactly 80%, 81%, 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.

20. The composition of any one of claims 16-19, wherein the engineered NK cells comprise a construct that is at least or exactly 80%, 81%, 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.

21. The composition of any one of claims 11-20, wherein 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.

22. The composition of claim 21, wherein the cytokine comprises IL-15 encoded by and / or comprising a sequence that is at least or exactly 80%, 81%, 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.

23. The composition of claim 21 or 22, wherein the cytokine comprises IL-21 encoded by and / or comprising a sequence that is at least or exactly 80%, 81%, 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.

24. The composition of any one of claims 1-23, wherein the engineered NK cells express one or more transgenes encoding one or more suicide genes.

25. The composition of any one of claims 1-24, wherein the engineered NK cells comprise one or more engineered mutations in endogenous genes.

26. The composition of claim 25, wherein the engineered mutations comprise mutations in CISH, CD38, glucocorticoid receptor and / or TGFBR2 genes.

27. The composition of any one of claims 4-26, wherein 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 exactly about 1 hour and then washing the composition to remove unbound antibodies.

28. The composition of any one of claims 4-27, wherein one or more antibody molecules are incubated with NK cells to form a complex 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.

29. The composition of claim 27 or 28, wherein the antibody and NK cells are incubated in Click's / RPMI medium.

30. The composition of any one of claims 27-29, wherein the incubation and wash 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.

31. The composition of claim 30, wherein at least about 9% of the antibody is bound to the surface of the NK cells at least 3 days after incubation and washing relative to the amount of antibody complexed with the engineered NK cells measured at about 1 hour after incubation and washing.

32. The composition of claim 30, wherein at least about 6% of the antibody is bound to the surface of the NK cells for at least 5 days after incubation and washing relative to the amount of antibody complexed with the engineered NK cells measured at about 1 hour after incubation and washing.

33. The composition of claim 30, wherein at least about 4% of the antibody is bound to the surface of the NK cells for at least 7 days after incubation and washing relative to the amount of antibody complexed with the engineered NK cells measured at about 1 hour after incubation and washing.

34. The composition of claim 30, wherein 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 with the engineered NK cells measured at about 1 hour after incubation and washing.

35. The composition of any one of claims 1-34, wherein the composition is not frozen, is cryopreserved, or is thawed from cryopreservation.

36. The composition of claim 35, wherein 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.

37. The composition of claim 35 or 36, wherein at least about 30%, 40%, 50% or 60% of the antibody is bound to the surface of NK cells after thawing from cryopreservation relative to the amount of antibody complexed with non-cryopreserved engineered NK cells measured about 1 hour after incubation and washing.

38. The composition of any one of claims 35-36, wherein the engineered NK cells are deactivated prior to cryopreservation.

39. The composition of claim 38, wherein the deactivating agent comprises a kinase inhibitor.

40. The composition of claim 39, wherein the deactivating agent comprises dasatinib.

41. The composition of any one of claims 1-26, wherein the composition is produced in vivo.

42. The composition of claim 41, wherein 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 after administration of the antibody.

43. The composition of any one of claims 1-42, wherein the one or more antibodies comprises or consists of evitumomab.

44. The composition of any one of claims 1-42, wherein the one or more antibodies comprises or consists of magetuximab.

45. The composition of any one of claims 1-42, wherein the one or more antibodies comprises or consists of imatumomab.

46. ​​A method of producing a composition according to any one of claims 1 to 45, comprising the steps of: (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) antigen presenting cells / feeder cells, fragments of antigen presenting cells / feeder cells, or NK cell activating beads; and (ii) providing one or more antibody molecules including ervantumab, magetuximab and / or imatinib to the engineered NK cells.

47. The method of claim 46, wherein the method comprises a pre-activation step before and / or after the expansion step, wherein the engineered NK cells are pre-activated in a culture comprising an effective concentration of one or more of IL-2, IL-12, IL-15 and IL-18.

48. The method of claim 47, wherein the culture comprises effective concentrations of IL-12, IL-15, and IL-18.

49. The method of any one of claims 46-48, wherein 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 and the antibody molecule immediately prior to infusion, or combining the engineered NK cells and the antibody in the subject by time-coordinated administration.

50. The method of claim 49, wherein the duration is from about 5 minutes to about 24 hours or more.

51. The method of claim 50, wherein the duration is about 1 hour.

52. The method of any one of claims 46-51, wherein the culture used in the pre-activation 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.

53. The method of any one of claims 46-52, wherein the culture used in the pre-activation step comprises IL-12 at a concentration of 0.1-1000 ng / mL, 1-1000 ng / mL, or about 10 ng / mL.

54. The method of any one of claims 46-53, further comprising one or more washing steps of the pre-activated engineered NK cells before and / or after the expansion step.

55. The method of any one of claims 46-54, wherein the engineered NK cells are activated at least two or more times with IL-12, IL-15, IL-18, IL-2, or any combination thereof during the expansion step.

56. The method of any one of claims 46-55, wherein the expansion continues for 5-60 days, 12-16 days, or 18-24 days.

57. The method of any one of claims 46-56, wherein the expansion culture further comprises IL-2.

58. The method of claim 57, wherein IL-2 is present at a concentration of 10-500 U / mL, 100-300 U / mL, or about 200 U / mL.

59. The method of any one of claims 46-58, wherein IL-2 is supplemented in the expansion culture every 2-3 days.

60. The method of any one of claims 46-59, wherein the 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 antibodies 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.

61. The method of any one of claims 46-60, wherein at least about 9% of the antibody is bound to the NK cell surface at least 3 days after incubation and washing relative to the amount of antibody complexed with the engineered NK cells measured at about 1 hour after incubation and washing.

62. The method of any one of claims 46-61, wherein at least about 6% of the antibody is bound to the NK cell surface at least 5 days after incubation and washing relative to the amount of antibody complexed with the engineered NK cells measured at about 1 hour after incubation and washing.

63. The method of any one of claims 46-62, wherein at least about 4% of the antibody is bound to the NK cell surface at least 7 days after incubation and washing relative to the amount of antibody complexed with the engineered NK cells measured at about 1 hour after incubation and washing.

64. The method of any one of claims 46-63, wherein at least about 3% of the antibody is bound to the NK cell surface at least 12 days after incubation and washing relative to the amount of antibody complexed with the engineered NK cells measured at about 1 hour after incubation and washing.

65. The method of any one of claims 46-64, wherein the NK cells provided with the one or more antibodies are not frozen, are cryopreserved, or are thawed from cryopreservation.

66. The method of claim 65, wherein the NK cells provided with 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 the antibodies.

67. The method of claim 65 or 66, wherein 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 with non-cryopreserved engineered NK cells measured about 1 hour after incubation and washing.

68. A method of treating a disease or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of a composition of any one of claims 1-45.

69. The method of claim 68, wherein the disease or condition is cancer, inflammation, graft-versus-host disease, transplant rejection, an autoimmune disease, an immunodeficiency disease, a B-cell malignancy, or an infection.

70. The method of claim 68 or 69, wherein the cancer comprises a hematological cancer or a solid tumor.

71. The method of claim 70, wherein the cancer comprises pancreatic cancer, colorectal cancer, kidney cancer, brain cancer, breast cancer, renal cancer, and / or myeloma.

72. The method of any one of claims 68-71, wherein the engineered NK cells are allogeneic with respect to the subject.

73. The method of any one of claims 68-71, wherein the engineered NK cells are autologous to the subject.

74. The method of any one of claims 68-73, wherein the subject is a human.

75. The method of any one of claims 68-74, further comprising administering to the subject at least one second therapeutic agent.

76. The method of claim 75, wherein the at least one 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.

77. The method of claim 76, wherein the anticancer agent is chemotherapy, radiation therapy, gene therapy, surgery, hormone therapy, anti-angiogenic therapy, or immunotherapy.

78. The method of claim 76, 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.

79. The method of any one of claims 75-78, wherein the composition and / or the at least one second therapeutic agent is administered intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, transdermally, subcutaneously, regionally, or by direct injection or infusion.

80. The method of any one of claims 75-79, wherein the second therapeutic agent is an antibody.

81. A method of treating a cancer patient using engineered NK cells, the method comprising: a) stimulating the engineered NK cell population, optionally by pre-activation with a cytokine cocktail; b) optionally expanding the engineered NK cells; and ci) loading the engineered NK cells ex vivo with one or more antibodies and then administering the loaded engineered NK cells to a subject, or cii) loading the engineered NK cells in vivo with one or more antibodies by administering the engineered NK cells within 1 hour of administering the antibodies; wherein the engineered NK cells comprise one or more transgenes, wherein loading comprises exposing the engineered NK cells to one or more antibodies for a period of time effective to allow the antibodies to bind to the surface of the engineered NK cells, and The one or more antibodies include or consist of ervantumab, magetuximab and / or imatumomab.

82. The method of claim 81, wherein loading the engineered NK cells comprises exposing the engineered NK cells to one or more antibodies for greater than or equal to about 1 hour or less than or equal to about 1 hour.

83. The method of claim 81 or 82, wherein the engineered NK cells remain loaded with antibody for at least about 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.

84. The method of any one of claims 81-83, wherein the one or more antibodies bind to the engineered NK cells by interacting with CD16 surface protein.

85. The method of any one of claims 81-84, wherein the engineered NK cells are derived from umbilical cord blood.

86. The method of any one of claims 81-85, wherein the engineered NK cells are activated ex vivo.

87. The method of any one of claims 81-86, wherein the engineered NK cells are pre-activated with IL-18, IL-15, and IL-12.

88. The method of any one of claims 81-87, wherein the one or more transgenes comprise an engineered receptor.

89. The method of claim 88, 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.

90. The method of claim 88 or 89, wherein the engineered receptor comprises a CAR or a TCR.

91. The method of any one of claims 88-90, wherein the engineered receptor targets a stem cell antigen, a self antigen, or a cancer antigen selected from the group consisting of 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, CD123, CD23, 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.

92. The method of claim 91, wherein the engineered antigen receptor targets CD70 and / or TROP2.

93. The method of claim 91 or 92, wherein the antigen receptor targeting CD70 comprises a sequence that is at least or exactly 80%, 81%, 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.

94. The method of claim 92 or 93, wherein the engineered NK cells comprise a construct that is at least or exactly 80%, 81%, 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.

95. The method of any one of claims 91-94, wherein the antigen receptor targeting TROP2 comprises a sequence that is at least or exactly 80%, 81%, 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.

96. The method of any one of claims 91-95, wherein the engineered NK cells comprise a construct that is at least or exactly 80%, 81%, 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.

97. The method of any one of claims 81-96, wherein the one or more transgenes comprises a cytokine.

98. The method of claim 97, wherein 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.

99. The method of claim 98, wherein the cytokines comprise IL-2, IL-12, IL-15, IL-18 and / or IL-21.

100. The method of claim 99, wherein the cytokine comprises or consists of IL-15 encoded by and / or comprises a sequence that is at least or exactly 80%, 81%, 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.

101. The method of claim 99 or 100, wherein the cytokine comprises or consists of IL-21 encoded by and / or comprising a sequence that is at least or exactly 80%, 81%, 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.

102. The method of any one of claims 81-101, wherein the engineered NK cells comprise one or more engineered mutations in endogenous genes.

103. The method of claim 102, wherein the one or more engineered mutations in the endogenous gene include CISH, CD38, glucocorticoid receptor and / or TGFBR2 gene mutations.

104. A composition comprising: (i) one or more engineered natural killer (NK) cells comprising one or more transgenes; and (ii) one or more antibodies including antibody IPH61.

105. The composition of claim 104 or 105, wherein the antibody targets at least CD123 and NKp46.

106. The composition of any one of claims 104-105, wherein the composition is further defined as a complex formed between engineered NK cells IPH61 through binding of the Fc region of the antibody to the engineered NK cells and / or binding of the NKp46 targeting region of the antibody to the engineered NK cells.

107. The composition of any one of claims 104-106, wherein the complex further comprises one or more antigen binding domains of one or more antibodies that bind to their target antigen.

108. The composition of any one of claims 104-107, wherein the engineered NK cells are 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 a combination thereof.

109. The composition of any one of claims 104-108, wherein the engineered NK cells are modified to contain one or more transgenes for expressing one or more heterologous proteins.

110. The composition of claim 109, wherein the one or more heterologous proteins comprise one or more engineered receptors and / or one or more cytokines.

111. The composition of claim 110, wherein the one or more engineered receptors are chimeric antigen receptors (CARs), T cell receptors (TCRs), receptors that recognize the FC portion of Ig (FC recognition receptors), chemokine receptors, homing receptors, chimeric cytokine receptors, or any combination thereof.

112. The composition of claim 111, wherein the FC recognition receptors include CD16, CD32 and / or CD64, and / or wherein the engineered NK cells express one or more transgenes encoding one or more receptors and / or NK cell surface antigens to enhance binding of NK cells to antibodies.

113. The composition of claim 110, wherein the engineered NK cell surface antigen and / or receptor is CD16, CD32, CS1, CD56, CD64, NKG2D, NKG2C, DNAM, 2B4, CD2, NCR, NKp30, NKp44, NKp46 or KIR.

114. The composition of any one of claims 110-113, wherein the engineered receptor comprises a TCR and / or a CAR.

115. The composition of any one of claims 110-114, wherein the engineered receptor targets a stem cell antigen, a self antigen, or a cancer antigen selected from the group consisting of 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 gp1 20. HIV-1 envelope glycoprotein gp41, GD2, CD5, CD123, CD23, 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.

116. The composition of any one of claims 110-115, wherein the engineered antigen receptor targets CD70 and / or TROP2.

117. The composition of claim 116, wherein the antigen receptor targeting CD70 comprises a sequence that is at least or exactly 80%, 81%, 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.

118. The composition of claim 116 or 117, wherein the engineered NK cells comprise a construct that is at least or exactly 80%, 81%, 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.

119. The composition of any one of claims 116-118, wherein the antigen receptor targeting TROP2 comprises a sequence that is at least or exactly 80%, 81%, 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.

120. The composition of any one of claims 116-119, wherein the engineered NK cells comprise a construct that is at least or exactly 80%, 81%, 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.

121. The composition of any one of claims 116-120, 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.

122. The composition of claim 121, wherein the cytokine comprises IL-15 encoded by and / or comprising a sequence that is at least or exactly 80%, 81%, 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.

123. The composition of claim 121 or 122, wherein the cytokine comprises IL-21 encoded by and / or comprising a sequence that is at least or exactly 80%, 81%, 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.

124. The composition of any one of claims 104-123, wherein the engineered NK cells express one or more transgenes encoding one or more suicide genes.

125. The composition of any one of claims 104-124, wherein the engineered NK cells comprise one or more engineered mutations in endogenous genes.

126. The composition of claim 125, wherein the one or more engineered mutations include CISH, CD38, glucocorticoid receptor and / or TGFBR2 gene mutations.

127. The composition of any one of claims 106-126, wherein the complexing of one or more antibodies to IPH61 with NK cells comprises incubating the NK cells with the one or more antibodies for at least or exactly about 1 hour and then washing the composition to remove unbound antibodies.

128. The composition of any one of claims 106-127, wherein one or more antibody molecules are incubated with NK cells to form a complex 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.

129. The composition of claim 127 or 128, wherein the antibody and NK cells are incubated in Click's / RPMI medium.

130. The composition of any one of claims 127-129, wherein the incubation and wash 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.

131. The composition of any one of claims 104-130, wherein the composition is not frozen, is cryopreserved, or is thawed from cryopreservation.

132. The composition of claim 131, wherein 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.

133. The composition of claim 131 or 132, wherein at least about 30%, 40%, 50% or 60% of the antibody is bound to the surface of NK cells after thawing from cryopreservation relative to the amount of antibody complexed with non-cryopreserved engineered NK cells measured at about 1 hour after incubation and washing.

134. The composition of any one of claims 131-133, wherein the engineered NK cells are deactivated prior to cryopreservation.

135. The composition of claim 134, wherein the deactivating agent comprises a kinase inhibitor.

136. The composition of claim 135, wherein the deactivating agent comprises dasatinib.

137. The composition of any one of claims 104-136, wherein the composition is produced in vivo.

138. The composition of claim 137, wherein producing the composition in vivo comprises loading the engineered NK cells with one or more antibodies in vivo by administering the engineered NK cells within about 1 hour after administration of the antibodies.

139. A method of producing the composition of any one of claims 104-138, the method comprising the steps of: (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) antigen presenting cells / feeder cells, fragments of antigen presenting cells / feeder cells, or NK cell activating beads; and (ii) providing one or more antibody molecules including IPH61 to the engineered NK cells.

140. The method of claim 139, wherein the method comprises a pre-activation step before and / or after the expansion step, wherein the engineered NK cells are pre-activated in a culture comprising an effective concentration of one or more of IL-2, IL-12, IL-15 and IL-18.

141. The method of claim 140, wherein the culture comprises effective concentrations of IL-12, IL-15, and IL-18.

142. The method of any one of claims 139-141, wherein 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 and the antibody molecule immediately prior to infusion, or combining the engineered NK cells and the antibody in the subject by time-coordinated administration.

143. The method of claim 142, wherein the duration is from about 5 minutes to about 24 hours or more.

144. The method of claim 143, wherein the duration is about 1 hour.

145. The method of any one of claims 139-144, wherein the culture used in the pre-activation 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.

146. The method of any one of claims 139-145, wherein the culture used in the pre-activation step comprises IL-12 at a concentration of 0.1-1000 ng / mL, 1-1000 ng / mL, or about 10 ng / mL.

147. The method of any one of claims 139-146, wherein the expansion continues for 5-60 days, 12-16 days, or 18-24 days.

148. The method of any one of claims 139-147, wherein the expansion culture further comprises IL-2.

149. The method of claim 148, wherein the IL-2 is present at a concentration of 10-500 U / mL, 100-300 U / mL, or about 200 U / mL.

150. The method of any one of claims 139-149, wherein the incubation 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.

151. The method of any one of claims 139-150, wherein the NK cells provided with the antibody are not frozen, are cryopreserved, or are thawed from cryopreservation.

152. The method of claim 151, wherein the NK cells provided with 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.

153. The method of claim 151 or 152, wherein at least about 30%, 40%, 50% or 60% of the antibody is bound to the surface of NK cells after thawing from cryopreservation relative to the amount of antibody complexed with non-cryopreserved engineered NK cells measured about 1 hour after incubation and washing.

154. A method of treating a disease or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of the composition of any one of claims 104-138.

155. The method of claim 154, wherein the disease or condition is cancer, inflammation, graft-versus-host disease, transplant rejection, an autoimmune disease, an immunodeficiency disease, a B-cell malignancy, or an infection.

156. The method of claim 154 or 155, wherein the cancer comprises a hematological cancer or a solid tumor.

157. The method of claim 156, wherein the cancer comprises acute myeloid leukemia, blastic plasmacytoid dendritic cell neoplasm, acute lymphoblastic leukemia, hairy cell leukemia, and / or systemic mastocytosis.

158. The method of any one of claims 154-158, wherein the engineered NK cells are allogeneic to the subject.

159. The method of any one of claims 154-158, wherein the engineered NK cells are autologous to the subject.

160. The method of any one of claims 154-159, wherein the subject is a human.

161. The method of any one of claims 154-160, further comprising administering to the subject at least one second therapeutic agent.

162. The method of claim 161, wherein the at least one 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.

163. 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 CAR targeting CD70 comprising a sequence that is at least or exactly 80%, 81%, 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 wherein the one or more transgenes comprise a CAR targeting TROP2, the CAR targeting TROP2 comprising a sequence that is at least or exactly 80%, 81%, 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.

164. The composition of claim 163, wherein the engineered NK cells are from an expanded or unexpanded population.

165. The composition of claim 163 or 164, wherein the engineered NK cells are from a pre-activated or non-pre-activated population.

166. The composition of any one of claims 163-165, wherein the composition is further defined as a complex between the engineered NK cells and one or more antibodies.

167. The composition of any one of claims 163-166, wherein the complex further comprises one or more antigen binding domains of one or more antibodies that bind to their target antigen.

168. The composition of any one of claims 163-167, wherein the antibody targets a cellular antigen, a self antigen, or a cancer antigen selected from the group consisting of 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, CD123, CD23, 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.

169. The composition of any one of claims 163-168, wherein 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).

170. The composition of any one of claims 163-169, wherein the engineered NK cells are 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 a combination thereof.

171. The composition of any one of claims 163-170, wherein the engineered NK cells are derived from primary NK cells rather than from stem cells and / or induced pluripotent stem cells (iPSCs).

172. The composition of any one of claims 163-171, wherein the NK cells are derived from umbilical cord blood, and wherein the source of CB monocytes and / or CB hematopoietic stem cells is CB from one donor or from a pool of 2 or more individual CB units.

173. The composition of any one of claims 163-172, wherein the engineered NK cells are further modified to comprise one or more additional transgenes for expressing one or more heterologous proteins.

174. The composition of claim 173, wherein the one or more heterologous proteins comprise one or more engineered receptors, and / or one or more cytokines.

175. The composition of claim 174, wherein the one or more engineered receptors are chimeric antigen receptors (CARs), T cell receptors (TCRs), receptors that recognize the FC portion of Ig (FC recognition receptors), chemokine receptors, homing receptors, chimeric cytokine receptors, or any combination thereof.

176. The composition of claim 175, wherein the FC recognition receptors include CD16, CD32 and / or CD64, and / or wherein the engineered NK cells express one or more transgenes encoding one or more receptors to enhance their binding to antibodies.

177. The composition of any one of claims 174-176, wherein the engineered receptor comprises a TCR and / or a CAR.

178. The composition of any one of claims 174-177, wherein the engineered receptor targets a stem cell antigen, a self antigen, or a cancer antigen selected from the group consisting of 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 gp1 20. HIV-1 envelope glycoprotein gp41, GD2, CD5, CD123, CD23, 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.

179. The composition of any one of claims 163-178, wherein the engineered NK cells comprise a construct that is at least or exactly 80%, 81%, 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.

180. The composition of any one of claims 163-179, wherein the engineered NK cells comprise a construct that is at least or exactly 80%, 81%, 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.

181. The composition of any one of claims 174-180, wherein 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.

182. The composition of claim 181, wherein the cytokine comprises IL-15 encoded by and / or comprising a sequence that is at least or exactly 80%, 81%, 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.

183. The composition of claim 181 or 182, wherein the cytokine comprises IL-21 encoded by and / or comprising a sequence that is at least or exactly 80%, 81%, 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.

184. The composition of any one of claims 163-183, wherein the engineered NK cells express one or more transgenes encoding one or more suicide genes.

185. The composition of any one of claims 163-184, wherein the engineered NK cells comprise one or more engineered mutations in endogenous genes.

186. The composition of claim 185, wherein the engineered mutations comprise mutations in CISH, CD38, glucocorticoid receptor and / or TGFBR2 genes.

187. The composition of any one of claims 166-186, wherein the complexing of one or more antibody molecules with NK cells comprises incubating the NK cells with the one or more antibodies for at least or exactly about 1 hour and then washing the composition to remove unbound antibodies.

188. The composition of any one of claims 166-187, wherein one or more antibody molecules are incubated with NK cells to form a complex 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.

189. The composition of claim 187 or 188, wherein the antibody and NK cells are incubated in Click's / RPMI medium.

190. The composition of any one of claims 187-189, wherein the incubation and wash 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.

191. The composition of claim 190, wherein at least about 9% of the antibody is bound to the NK cell surface for at least 3 days after incubation and washing relative to the amount of antibody complexed with the engineered NK cells measured at about 1 hour after incubation and washing.

192. The composition of claim 190, wherein at least about 6% of the antibody is bound to the NK cell surface for at least 5 days after incubation and washing relative to the amount of antibody complexed with the engineered NK cells measured at about 1 hour after incubation and washing.

193. The composition of claim 190, wherein at least about 4% of the antibody is bound to the NK cell surface for at least 7 days after incubation and washing relative to the amount of antibody complexed with the engineered NK cells measured at about 1 hour after incubation and washing.

194. The composition of claim 190, wherein at least about 3% of the antibody is bound to the NK cell surface for at least 12 days after incubation and washing relative to the amount of antibody complexed with the engineered NK cells measured at about 1 hour after incubation and washing.

195. The composition of any one of claims 163-194, wherein the composition is not frozen, is cryopreserved, or is thawed from cryopreservation.

196. The composition of claim 195, wherein 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.

197. The composition of claim 195 or 196, wherein at least about 30%, 40%, 50% or 60% of the antibody is bound to the surface of NK cells after thawing from cryopreservation relative to the amount of antibody complexed with non-cryopreserved engineered NK cells measured about 1 hour after incubation and washing.

198. The composition of any one of claims 195-196, wherein the engineered NK cells are deactivated prior to cryopreservation.

199. The composition of claim 198, wherein the deactivator comprises a kinase inhibitor.

200. The composition of claim 199, wherein the deactivating agent comprises dasatinib.

201. The composition of any one of claims 163-186, wherein the composition is produced in vivo.

202. The composition of claim 201, wherein 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 after administration of the antibody.

203. The composition of any one of claims 163-202, wherein the one or more antibodies comprises or consists of ivanovumab.

204. The composition of any one of claims 163-203, wherein the one or more antibodies comprises or consists of magetuximab.

205. The composition of any one of claims 163-204, wherein the one or more antibodies comprises or consists of imatumomab.

206. The composition of any one of claims 163-205, wherein the one or more antibodies comprises or consists of IPH61.

207. A method of producing the composition of any one of claims 163-206, the method comprising the steps of: (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) antigen presenting cells / feeder cells, fragments of antigen presenting cells / feeder cells, or NK cell activating beads; and (ii) providing one or more antibody molecules to the engineered NK cells.

208. The method of claim 207, wherein the method comprises a pre-activation step before and / or after the expansion step, wherein the engineered NK cells are pre-activated in a culture comprising an effective concentration of one or more of IL-2, IL-12, IL-15 and IL-18.

209. The method of claim 208, wherein the culture comprises effective concentrations of IL-12, IL-15, and IL-18.

210. The method of any one of claims 207-209, wherein 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 and the antibody molecule immediately prior to infusion, or combining the engineered NK cells and the antibody in the subject by time-coordinated administration.

211. The method of claim 210, wherein the duration is from about 5 minutes to about 24 hours or longer.

212. The method of claim 211, wherein the duration is about 1 hour.

213. The method of any one of claims 207-212, wherein the culture used in the pre-activation 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.

214. The method of any one of claims 207-213, wherein the culture used in the pre-activation step comprises IL-12 at a concentration of 0.1-1000 ng / mL, 1-1000 ng / mL, or about 10 ng / mL.

215. The method of any one of claims 207-214, further comprising one or more washing steps of the pre-activated engineered NK cells before and / or after the expansion step.

216. The method of any one of claims 207-215, wherein the engineered NK cells are activated at least twice or more with IL-12, IL-15, IL-18, IL-2, or any combination thereof during the expansion step.

217. The method of any one of claims 207-216, wherein the expansion lasts for 5-60 days, 12-16 days, or 18-24 days.

218. The method of any one of claims 207-217, wherein the expansion culture further comprises IL-2.

219. The method of claim 218, wherein IL-2 is present at a concentration of 10-500 U / mL, 100-300 U / mL, or about 200 U / mL.

220. The method of any one of claims 207-219, wherein IL-2 is supplemented in the expansion culture every 2-3 days.

221. The method of any one of claims 207-220, wherein the 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 antibodies 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.

222. The method of any one of claims 207-221, wherein at least about 9% of the antibody is bound to the NK cell surface at least 3 days after incubation and washing relative to the amount of antibody complexed with the engineered NK cells measured at about 1 hour after incubation and washing.

223. The method of any one of claims 207-222, wherein at least about 6% of the antibody is bound to the NK cell surface for at least 5 days after incubation and washing relative to the amount of antibody complexed with the engineered NK cells measured at about 1 hour after incubation and washing.

224. The method of any one of claims 207-223, wherein at least about 4% of the antibody is bound to the NK cell surface at least 7 days after incubation and washing relative to the amount of antibody complexed with the engineered NK cells measured at about 1 hour after incubation and washing.

225. The method of any one of claims 207-224, wherein at least about 3% of the antibody is bound to the NK cell surface at least 12 days after incubation and washing relative to the amount of antibody complexed with the engineered NK cells measured about 1 hour after incubation and washing.

226. The method of any one of claims 207-225, wherein the NK cells provided with the one or more antibodies are not frozen, are cryopreserved, or are thawed from cryopreservation.

227. The method of claim 226, wherein the NK cells provided with 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 the antibodies.

228. The method of claim 226 or 227, wherein 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 with non-cryopreserved engineered NK cells measured about 1 hour after incubation and washing.

229. A method of treating a disease or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of a composition of any one of claims 163-205.

230. The method of claim 229, wherein the disease or condition is cancer, inflammation, graft-versus-host disease, transplant rejection, an autoimmune disease, an immunodeficiency disease, a B-cell malignancy, or an infection.

231. The method of claim 229 or 230, wherein the cancer comprises a hematological cancer or a solid tumor.

232. The method of claim 231, wherein the cancer comprises pancreatic cancer, colorectal cancer, kidney cancer, brain cancer, breast cancer, renal cancer, and / or myeloma.

233. The method of claim 231, wherein the cancer comprises acute myeloid leukemia, blastic plasmacytoid dendritic cell neoplasm, acute lymphoblastic leukemia, hairy cell leukemia, and / or systemic mastocytosis.

234. The method of any one of claims 229-232, wherein the engineered NK cells are allogeneic to the subject.

235. The method of any one of claims 229-232, wherein the engineered NK cells are autologous to the subject.

236. The method of any one of claims 229-235, wherein the subject is a human.

237. The method of any one of claims 229-236, further comprising administering to the subject at least one second therapeutic agent.

238. The method of claim 237, wherein the at least one 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.

239. The method of claim 238, wherein the anticancer agent is chemotherapy, radiotherapy, gene therapy, surgery, hormone therapy, anti-angiogenic therapy, or immunotherapy.

240. The method of claim 238, 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.

241. The method of any one of claims 237-240, wherein the composition and / or at least one second therapeutic agent is administered intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, transdermally, subcutaneously, regionally, or by direct injection or infusion.

242. The method of any one of claims 237-241, wherein the second therapeutic agent is an antibody.

243. A method of treating a subject with cancer using engineered NK cells, the method comprising: a) stimulating the engineered NK cell population, optionally by pre-activation with a cytokine cocktail; b) optionally expanding the engineered NK cells; and ci) loading the engineered NK cells ex vivo with one or more antibodies and then administering the loaded engineered NK cells to a subject, or cii) loading the engineered NK cells in vivo with one or more antibodies by administering the engineered NK cells within 1 hour of administering the antibodies; wherein the engineered NK cells comprise one or more transgenes, wherein loading comprises exposing the engineered NK cells to one or more antibodies for a period of time effective to allow the antibodies to bind to the surface of the engineered NK cells, and wherein the one or more transgenes comprise a CAR targeting CD70 comprising a sequence that is at least or exactly 80%, 81%, 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 CAR targeting TROP2 comprising a sequence that is at least or exactly 80%, 81%, 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.

244. The method of claim 243, wherein loading the engineered NK cells comprises exposing the engineered NK cells to one or more antibodies for greater than or equal to about 1 hour or less than or equal to about 1 hour.

245. The method of claim 243 or 244, wherein the engineered NK cells remain loaded with antibody for at least about 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.

246. The method of any one of claims 243-245, wherein the one or more antibodies bind to the engineered NK cells by interacting with CD16 surface protein.

247. The method of any one of claims 243-246, wherein the engineered NK cells are derived from umbilical cord blood.

248. The method of any one of claims 243-247, wherein the engineered NK cells are activated ex vivo.

249. The method of any one of claims 243-248, wherein the engineered NK cells are pre-activated with IL-18, IL-15, and IL-12.

250. The method of any one of claims 243-249, wherein the engineered NK cells comprise one or more additional transgenes, optionally including an engineered receptor.

251. The method of claim 250, 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.

252. The method of claim 250 or 251, wherein the engineered receptor comprises a CAR or a TCR.

253. The method of any one of claims 250-252, wherein the engineered receptor targets a stem cell antigen, a self antigen, or a cancer antigen selected from the group consisting of 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 gp12 0, HIV-1 envelope glycoprotein gp41, GD2, CD5, CD123, CD23, 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.

254. The method of any one of claims 243-253, wherein the engineered NK cells comprise a construct that is at least or exactly 80%, 81%, 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.

255. The method of any one of claims 243-254, wherein the engineered NK cells comprise a construct that is at least or exactly 80%, 81%, 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.

256. The method of any one of claims 243-255, wherein the one or more transgenes comprises a cytokine.

257. The method of claim 256, wherein 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.

258. The method of claim 257, wherein the cytokines comprise IL-2, IL-12, IL-15, IL-18 and / or IL-21.

259. The method of claim 258, wherein the cytokine comprises or consists of IL-15 encoded by and / or comprising a sequence that is at least or exactly 80%, 81%, 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.

260. The method of claim 258 or 259, wherein the cytokine comprises or consists of IL-21 encoded by and / or comprises a sequence that is at least or exactly 80%, 81%, 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.

261. The method of any one of claims 243-260, wherein the engineered NK cells comprise one or more engineered mutations in an endogenous gene.

262. The method of claim 261, wherein the one or more engineered mutations in the endogenous gene include mutations in the CISH, CD38, glucocorticoid receptor and / or TGFBR2 genes.

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