Therapeutic preparations of gamma-delta t cells and natural killer cells and methods for making and using them

Abstract

To provide a method for preparing natural immune cell compositions comprising gamma-delta (γδ) T cells and / or natural killer (NK) cells.SOLUTION: Provided is a method for producing a composition comprising 70% or more γδ T cells derived from a sample comprising cells derived from one or more subjects, the method comprising: a) depleting αβ T cells from the under a condition that generates a depleted cell population comprising NK cells and γδ T cells; b) exposing the depleted cell population to an activation condition comprising contacting the depleted cell population with: (a) an antibody or antigen binding fragment thereof that immunospecifically binds to cell adhesion molecule polypeptide CD2; and (b) an antibody or antigen binding fragment thereof that immunospecifically binds to NKp46; and c) exposing the depleted cell population to an expansion condition comprising contacting the depleted cell population with IL-2, thereby generating the composition comprising 70-75% γδ T cells.SELECTED DRAWING: None

Description

[Technical field]

[0001] Related Patent Applications This patent application claims the benefit of U.S. Provisional Patent Application No. 62 / 703,654, entitled "THERAPEUTIC PREPARATONS OF GAMMA-DELTA T CELLS AND NATURAL KILLER CELLS AND METHODS FOR MAKING AND USING THEM," filed July 26, 2018, with Concetta QUINTARELLI et al. as inventors, and designated by Attorney Docket No. 6474-131400. The entire contents of the aforementioned application, including all text, tables, and drawings, are incorporated herein by reference for all purposes.

[0002] Field The present technology relates in part to therapeutic innate immune cell compositions, and methods of making and using such compositions. [Background technology]

[0003] Immune responses to antigen-presenting pathogens and other foreign antigen-presenting entities include innate and adaptive defenses. Innate immune responses are the first-line immune defenses that are active and persistently functional in the host. Innate immune cells, such as NK cells and gamma delta (γδ) T cells, do not recognize classical HLA antigens. Adaptive immune responses are responses that specifically match antigen-presenting substances, such as foreign bodies, cells, or microorganisms, and often can take several days to mature. Immunotherapy uses natural or modified (e.g., CAR-modified) adaptive immune cells (e.g., alpha beta (αβ) T cells, dendritic cells, macrophages). For example, αβ T cells expand upon binding to ligands on antigen-presenting substances in vivo, which can take several days or even weeks. Similarly, engagement of αβ T cells occurs through one or more T cell surface molecules or complexes of molecules including CD4, CD8, and the T cell receptor (TCR) that recognize MHC class I and class II ligands encoded by the HLA gene complex (Miceli et al., Semin. Immunol., 3(3):133-141 (1991). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Miceli et al., Semin. Immunol., 3(3):133-141 (1991) Summary of the Invention [Means for solving the problem]

[0005] overview Provided herein are compositions related to immunology and medicine, methods of making such compositions, and methods of treatment using such compositions. In certain embodiments, compositions and methods, including products and kits, are provided that include gamma delta T cells (γδ), natural killer cells (NK), or a combination of the two, for various cell therapies. Also provided are methods of making gamma delta T cells (γδ) and natural killer cells (NK) that can be genetically modified for use in these therapies. In alternative embodiments, genetically modified gamma delta T cells (γδ) and natural killer cells (NK) can be modified to express chimeric antigen receptors (CARs) or exogenous or heterologous T cell receptors (TCRs), which can be used to directly or indirectly target any cell surface molecule, such as markers on cancer cells or infected cells.

[0006] In certain embodiments, methods are provided for producing compositions containing populations of cells enriched for NK cells and gamma.delta T cells. In certain embodiments of the methods provided herein, a sample obtained from a donor (e.g., a tissue, organ, or blood sample from a subject who is a healthy subject or a patient treated with a population of cells) is exposed to activation conditions comprising (a) at least one exogenous polypeptide that immunospecifically binds to a cell adhesion polypeptide, and (b) at least one exogenous polypeptide that immunospecifically binds to a polypeptide different from the cell adhesion polypeptide and is expressed on the surface of one or more cells of the sample, and is exposed to expansion conditions comprising contacting the sample with at least one auxiliary polypeptide, thereby producing a composition containing a population of cells enriched for NK cells and gamma.delta T cells. The sample is sometimes exposed sequentially to the activation conditions and then the expansion conditions. The methods provided herein can include methods for detecting between about 30% and about 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more of the cells in a population, or at least about 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%, 85%, 90%, 95% or more of the cells in a population. %, 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 more, up to 100% high levels of activation.

[0007] The exogenous and auxiliary polypeptides used in the methods provided herein can be easily identified and isolated, synthesized, or otherwise obtained, including from commercial sources, based in part on available nucleic acid and amino acid sequences and other knowledge about cell adhesion molecules and other immune cell molecules and receptors. In certain aspects, the exogenous polypeptide is human. In some aspects, the exogenous polypeptide is isolated. In certain aspects, the auxiliary polypeptide is human. In some aspects, the auxiliary polypeptide is isolated.

[0008] In certain embodiments of the methods provided herein, the sample is exposed to activation and expansion conditions after depleting alpha.beta T cells, and the resulting depleted cell population is then sometimes exposed to activation and expansion conditions. In certain embodiments, the exogenous polypeptide of (a), the exogenous polypeptide of (b), or both the exogenous polypeptide of (a) and the exogenous polypeptide of (b) are soluble. In some embodiments, the exogenous polypeptide of (a) or the exogenous polypeptide of (b) is bound to a solid phase substrate.

[0009] In some embodiments of the methods provided herein, the at least one auxiliary polypeptide is selected such that the amount of NK cells relative to the amount of gamma.delta T cells in the population is dependent on the amount and / or type of the at least one auxiliary polypeptide. In certain embodiments, the auxiliary polypeptide increases or decreases the amount of NK cells relative to gamma.delta T cells in the population of cells after contacting the depleted cell population with the at least one auxiliary polypeptide. In certain embodiments, the period of exposure of the sample or alpha.beta T cell depleted sample to the expansion conditions is selected to obtain a desired ratio of NK cells to gamma.delta T cells.

[0010] In certain aspects of the methods provided herein, the activation conditions do not include serum from a non-human animal. In some aspects of the methods provided herein, the expansion conditions do not include serum from a non-human animal.

[0011] In the methods provided herein, a high percentage of cells are activated, often without the use of feeder cells. Sometimes, at least about 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 109%, 109%, 108%, 109%, 109%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109 ...9%, 109%, 109%, 10 2%, 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%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, up to 100%, are activated without using feeder cells. Thus, in certain embodiments of the methods provided herein, the activation conditions do not include feeder cells. In some embodiments of the methods provided herein, the expansion conditions do not include feeder cells, and sometimes both the activation conditions and the expansion conditions do not include feeder cells.

[0012] Any source of immune cells can be used as a sample in the methods provided herein. In certain embodiments, the sample is selected from bone marrow, peripheral blood, liver tissue, epithelial tissue, and umbilical cord blood. In some embodiments of the methods provided herein, the sample is not derived from an embryonic source. In certain embodiments of the methods provided herein, the sample is peripheral blood, and in some embodiments, the peripheral blood sample is a processed sample that is processed before being subjected to alpha.beta T cell depletion in the methods provided herein. For example, the peripheral blood sample can be processed by density gradient centrifugation to separate and / or isolate the buffy coat containing white blood cells, platelets, granulocytes, etc., which can then be subjected to alpha.beta T cell depletion according to the methods provided herein. In certain embodiments, the buffy coat can be further subjected to Ficoll density gradient separation to obtain mononuclear cells (PBMCs), which can then be subjected to alpha.beta T cell depletion in the methods provided herein. In some embodiments, a peripheral blood sample can be subjected to apheresis to separate plasma from cells, and the cells are then sometimes subjected to alpha.beta T cell depletion in the methods provided herein. In certain embodiments of the methods provided herein, the sample is umbilical cord blood, and sometimes the umbilical cord blood is processed cord blood that is processed before being subjected to alpha.beta T cell depletion in the methods provided herein.

[0013] In certain embodiments of the methods provided herein, the exogenous polypeptide of (b) immunospecifically binds to an NK cell activating receptor, a gamma.delta T cell activating receptor, or both. Such receptors include, but are not limited to, CD2, CD3, CD56, NKp30, NKp44, NKp46, NKG2A, NKG2C, NKG2D, KAR receptors, KIR receptors, SIGLEC-7, KIR3DS1, KIR3D51, KIR2DL1 (antibody: 11PB6), DNAM1, NTBA, HLA-DR, and the like. In some embodiments, the receptor is NKp46. In certain embodiments of the methods provided herein, the exogenous polypeptide of (a) immunospecifically binds to CD2. In some embodiments, the exogenous polypeptide of (a) or (b), or (a) and (b), is an antibody or an antigen-binding fragment thereof. For purposes herein, the recitation of "antibody" includes full-length antibodies and portions thereof, including antibody fragments. Antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fvs (dsFvs), Fd fragments, Fd' fragments, single-chain Fvs (scFvs), single-chain Fabs (scFabs), diabodies, anti-idiotypic (anti-Id) antibodies, or antigen-binding fragments of any of the above. Antibodies also include synthetic antibodies, recombinantly produced antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, and intrabodies. Antibodies can include members of any immunoglobulin type (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass (e.g., IgG2a and IgG2b).

[0014] In certain embodiments of the methods provided herein, the activating conditions include contacting the sample or depleted cell population with at least two exogenous polypeptides. In some embodiments, a first exogenous polypeptide immunospecifically binds to CD2, and a second exogenous polypeptide immunospecifically binds to NKp46. In some embodiments, the activating conditions consist of or consist essentially of a first exogenous polypeptide immunospecifically binds to CD2 and a second exogenous polypeptide immunospecifically binds to NKp46. In certain embodiments, the exogenous polypeptide immunospecifically binds to CD2, the exogenous polypeptide immunospecifically binds to NKp46, or both the exogenous polypeptide immunospecifically binds to CD2 and the exogenous polypeptide immunospecifically binds to NKp46 are soluble. In certain embodiments, the first exogenous polypeptide and / or the second exogenous polypeptide is an antibody or an antigen-binding fragment thereof.

[0015] In certain aspects of the methods provided herein, the expansion conditions comprise, consist of, or consist essentially of at least one auxiliary polypeptide that is a cytokine, and / or a polypeptide that immunospecifically binds to a receptor on a gamma.delta T cell, and / or a portion thereof that immunospecifically binds to a receptor on a gamma.delta T cell. In certain embodiments, the cytokine is an interleukin, such as IL-1 (see, e.g., GenBank Accession No. BC008678.1), IL-2 (see, e.g., GenBank Accession No. S77834.1), IL-4 (see, e.g., GenBank Accession No. BC070123.1), IL-7 (see, e.g., GenBank Accession No. BC047698.1), IL-9 (see, e.g., GenBank Accession No. BC066285.1), IL-15 (see, e.g., GenBank Accession Nos. BC100962.1; 100963.1; 100961.1), IL-21 (see, e.g., GenBank Accession No. LC133256.1), or any combination thereof. In some embodiments of the methods provided herein, the cytokine is IL-2, IL-15, or a combination thereof. In certain embodiments, the expansion conditions comprises, consists of, or consists essentially of a polypeptide that immunospecifically binds to IL-2, IL-15, and a receptor on gamma.delta T cells. In certain embodiments, the receptor on gamma.delta T cells is CD3. In some embodiments, the polypeptide that immunospecifically binds to the CD3 receptor on gamma.delta T cells is an antibody or an antigen-binding fragment thereof, and in certain embodiments, the antibody is OKT3. In certain embodiments, the expansion conditions include contacting the sample or depleted cell population with: (a) an IL-2 polypeptide; (b) an IL-15 polypeptide; (c) an IL-2 polypeptide and an IL-15 polypeptide; (d) an antibody that immunospecifically binds to an IL-2 polypeptide and CD3; or (e) an antibody that immunospecifically binds to an IL-2 polypeptide, an IL-15 polypeptide, and CD3. In some embodiments, the antibody that immunospecifically binds to CD3 is OKT3.

[0016] In certain embodiments, the expansion conditions include sequentially exposing a sample (e.g., a source sample or a sample cell population depleted of alpha.beta T cells) to more than one set of conditions. In some embodiments, the sample is exposed to two sets of expansion conditions. In some embodiments, the population of cells exposed to a first set of expansion conditions is washed and then exposed to a second set of expansion conditions. In certain embodiments, (a) the first set of conditions includes IL-2 and the second set of conditions includes IL-15; (b) the first set of conditions includes IL-15 and the second set of conditions includes IL-2; (c) the first set of conditions includes an antibody that immunospecifically binds to IL-2 and CD3 and the second set of conditions includes IL-15; or (d) the first set of conditions includes an antibody that immunospecifically binds to IL-15 and CD3 and the second set of conditions includes an antibody that immunospecifically binds to IL-2 and CD3.

[0017] In certain aspects of the methods provided herein, a first set of expansion conditions comprising one or more auxiliary polypeptides often results in a first cell population comprising a first ratio of NK cells to gamma.delta T cells, which can then be fine-tuned to a desired final ratio of NK cells to gamma.delta T cells using a second set of expansion conditions, the first set of conditions being different from the second set of conditions.

[0018] In the methods provided herein, the cell population can be exposed to activation and expansion conditions simultaneously or sequentially in any order. Additionally, the exogenous polypeptide(s) can function as auxiliary polypeptide(s) and / or vice versa.

[0019] The immune cell composition obtained by the method provided herein is enriched for the innate immune cells NK cells and gamma delta T cells compared to compositions of immune cells in nature (e.g., biological fluids or tissues). In nature, the adaptive immune cells alpha beta T cells are found in much greater amounts than NK cells and gamma delta T cells. In contrast, in the compositions provided herein, alpha beta T cells are absent or present in negligible to low amounts, and NK cells and gamma delta T cells are the major immune cell components.

[0020] Depending on the expansion conditions, the compositions enriched for innate immune cells obtained by the methods provided herein can contain different amounts of NK cells compared to gamma delta T cells. In certain non-limiting examples, (i) when the auxiliary polypeptide is IL-2, the resulting population of cells enriched for NK cells and gamma delta T cells often contains about 25-30% NK cells and about 70-75% gamma delta T cells; (ii) when the auxiliary polypeptide is IL-15, the resulting population of cells enriched for NK cells and gamma delta T cells often contains about 80-99% NK cells and about 1-20% gamma delta T cells; (iii) when the auxiliary polypeptide is IL-2 and an antibody that immunospecifically binds to CD3, e.g., OKT3, the resulting population of cells enriched for NK cells and gamma delta T cells often contains about 40-50% NK cells and about 50-60% gamma delta T cells. (iv) when the supplemental polypeptide is IL-2 until day 20 of the expansion conditions and then switched to IL-15 until day 30, the percentage of gamma.delta T cells often increases from about 50% to about 70%, often with a corresponding decrease in the percentage of NK cells, compared to treatment with IL-2 alone; and (v) when the supplemental polypeptide is IL-15 until day 20 of the expansion conditions and then switched to IL-2 until day 30, the percentage of NK cells often increases from about 80% to about 90%, often with a corresponding decrease in the percentage of gamma.delta T cells, compared to treatment with IL-15 alone.

[0021] In certain embodiments of the methods provided herein, the sample or depleted cell population is not exposed to conditions that select for NK cells or gamma.delta T cells. In some embodiments, the sample or depleted cell population is not exposed to conditions that deplete cells other than alpha-beta T cells.

[0022] The cells of the compositions prepared by the methods provided herein can be further genetically modified to express an exogenous polynucleotide, such as a tumor necrosis factor receptor, a chimeric antigen receptor (CAR), a myeloid differentiation primary response protein, or an innate immune signaling adaptor, etc. The cells can also be modified to mutate one or more polypeptides or to deplete one or more polypeptides.

[0023] In certain embodiments of the methods provided herein, the composition containing gamma.delta cells and NK cells can be subjected to a treatment that results in a composition consisting of or consisting essentially of NK cells or gamma.delta cells. The treatment can be depletion, e.g., obtaining NK cells by depleting all gamma.delta (CD3+) cells using anti-CD3 antibodies, or positive selection, e.g., selecting for gamma.delta T cells using anti-CD3 antibodies. In some embodiments, anti-CD3 antibodies, e.g., OKT3 antibodies, can be bound to a solid phase.

[0024] In embodiments of the methods provided herein, the activation conditions, expansion conditions, or activation and expansion conditions comprise incubation of the sample or depleted cell population in feeder cell free medium for about 1 hour, 2 hours, 5 hours, 10 hours, 12 hours, 15 hours, 20 hours, or for several days, such as 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 25 days, 30 days, 35 days, 40 days, 45 days, 50 days, 55 days, or 60 days, or more days or weeks, such as about 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks. In certain embodiments, the activation conditions are for a period of about 1 hour, 5 hours, 10 hours, 12 hours, 15 hours, or 20 hours to about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 1 week. In some embodiments, the activation conditions are for a period of about 12 hours, 24 hours, 36 hours, or 2 days to about 3 days, 4 days, 5 days, 6 days, or 1 week, or for a period of about 2 days to about 4 days or 5 days, or for a period of about 3 days to about 4 days. In certain embodiments, the expansion conditions are for a period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 1 week. In certain embodiments, the expansion conditions are performed in successive cycles, each cycle being independently performed for a period of about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or for a period of 1 week, 2 weeks, 3 weeks, or longer. In some embodiments, the number of expansion cycles is greater than 1, such as 2, 3, 4, 5, 6, 7, 8, 9, or even 10 cycles. In certain embodiments, each expansion cycle is about 7 days. In some embodiments, the number of expansion cycles is 3.

[0025] In some embodiments, the population of expanded cells enriched for expanded NK cells and gamma.delta T cells does not contain exhausted cells, hi certain embodiments, the population of expanded cells enriched for expanded NK cells and gamma.delta T cells does not contain exhausted cells after 60 days of expansion conditions.

[0026] In certain embodiments, the population of cells enriched for NK cells and gamma.delta T cells obtained by the methods provided herein contains 80% or more innate immune cells. In some embodiments, between about 80% and about 100%, or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, up to 100%, are innate immune cells.

[0027] In some embodiments of the methods provided herein, the activation conditions, the expansion conditions, or both the activation conditions and the expansion conditions do not include a bisphosphonate. Exemplary bisphosphonates include, but are not limited to, clodronate, etidronate, alendronate, pamidronate, zoledronate (zoledronic acid), neridronate, and the like.

[0028] In certain embodiments of the methods provided herein, the compositions obtained by the methods provided herein contain gamma.delta T cells that are polyclonal for V.delta.1 and V.delta.2 expression. The relative amounts of V.delta.1 and V.delta.2 cells can be adjusted by one or more of the expansion conditions, such as the absence of bisphosphonates, the selection of auxiliary polypeptides, and the duration of time that the sample or depleted cell population is subjected to the expansion conditions. In certain non-limiting examples, when the expansion conditions include IL-2 and a polypeptide that immunospecifically binds to CD3 (e.g., OKT3), (i) V.delta.1 cells can be between about 75% and about 95% of gamma.delta T cells, sometimes between about 80% and about 90% of gamma.delta T cells, and (ii) V.delta.2 cells can be between about 10% and about 25% of gamma.delta T cells, sometimes between about 10% and about 15%, or 20% of gamma.delta T cells. In certain embodiments, when the expansion conditions include IL-2 (e.g., do not include IL-15 and do not include a polypeptide that immunospecifically binds to CD3), (i) V.delta.1 cells can be between about 30% and about 60% of gamma.delta T cells, sometimes between about 35% and about 55% of gamma.delta T cells, and (ii) V.delta.2 cells can be between about 35% and about 60% of gamma.delta T cells, sometimes between about 40% and about 50%, or 55% of gamma.delta T cells. In certain embodiments, when the expansion conditions include IL-15 (e.g., do not include IL-2 and do not include a polypeptide that immunospecifically binds to CD3), (i) V.delta.1 cells can be between about 10% and about 30% of gamma.delta T cells, sometimes between about 20% and about 25% of gamma.delta T cells, and (ii) V.delta.2 cells can be between about 65% and about 80% of gamma.delta T cells, sometimes between about 70% and about 75%, or 80% of gamma.delta T cells.

[0029] Provided herein in certain embodiments is a composition comprising a cell population comprising a plurality of NK cells and a plurality of gamma.delta T cells, and depleted of alpha.beta T cells. In some embodiments, the composition does not comprise feeder cells. Also provided herein in certain embodiments is a composition comprising a cell population comprising a plurality of NK cells and a plurality of gamma.delta T cells, and depleted of alpha.beta T cells, and does not comprise feeder cells. In certain embodiments, the population of cells is a population of modified peripheral blood cells. In some embodiments, the compositions provided herein contain (i) between about 25% and about 45% NK cells and between about 55% and about 75% gamma.delta T cells; (ii) between about 25% and about 30% NK cells and between about 70% and about 75% gamma.delta T cells; (iii) between about 80% and about 99% NK cells and between about 1% and about 20% gamma.delta T cells; or (iv) between about 40% and about 45% NK cells and between about 55% and about 60% gamma.delta T cells.

[0030] In certain embodiments, 30% or more of the cells in the compositions provided herein are activated. In some embodiments, between about 30% and about 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more of the cells in the population, or at least about 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%, 85%, 86%, 87%, 88%, 89%, 90%, 95%, or more of the cells in the population are activated. 0%, 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%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more up to 100%.

[0031] In some embodiments, the compositions provided herein contain a population of cells comprising one or more of the following activation markers as a percentage of the total number of cells in the population: (a) 90% or more of the cells in the population express KIR5; (b) 10% or more of the cells in the population express SIGLEC-7; (c) 60% or more of the cells in the population express KIR3D51; (d) 10% or more of the cells in the population express KIR2DL1; (e) 25% or more of the cells in the population express NKp30, NKp44, and / or NKp46; (f) 35% or more of the cells in the population express NKG2D; (g) 90% or more of the cells in the population express DNAM1; (h) 85% or more of the cells in the population express NTBA; and (i) 95% or more of the cells in the population express CD2.

[0032] In certain embodiments, the compositions provided herein contain 80% or more innate immune cells. In some embodiments, the compositions are enriched for activated cytotoxic cells that are CD56+; in certain embodiments, between about 80% and about 100%, or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the cells are CD56+. In some embodiments, the compositions provided herein are enriched for activated cytotoxic cells that are CD57-. In certain embodiments, the compositions provided herein are enriched for activated cytotoxic cells that are CD56+CD57-. In some embodiments, between about 10% and about 40%, or at least about 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%, or 40% of the cells are CD16+. In certain embodiments, less than 5%, less than 4%, less than 3%, or less than 2% of the cells in the compositions provided herein are CD57+.

[0033] In certain embodiments, the compositions provided herein are substantially free of cells other than NK cells and gamma delta T cells. In some embodiments, the compositions provided herein are substantially free of about 5% or less, about 4% or less, about 3% or less, about 2% or less, about 1% or less, about 0.9% or less, about 0.8% or less, about 0.7% or less, about 0.6% or less, about 0.5% or less, about 0.4% or less, about 0.3% or less, about 0.2% or less, about 0.1% or less, or less than NKT cells. The composition contains about 5% or less, about 4% or less, about 3% or less, about 2% or less, about 1% or less, about 0.9% or less, about 0.8% or less, about 0.7% or less, about 0.6% or less, about 0.5% or less, about 0.4% or less, about 0.3% or less, about 0.2% or less, about 0.1% or less, or less than alpha.beta T cells. In certain embodiments, a subset of NK cells in the composition are CD16+ cells. In some embodiments, a majority of gamma.delta T cells, or a majority of NK cells, or a majority of both gamma.delta T cells and NK cells are CD57- cells.

[0034] In certain embodiments, the gamma.delta T cells of the compositions provided herein are polyclonal with respect to V.delta.1 and V.delta.2 expression. In some embodiments, the gamma.delta T cell population of the compositions provided herein is: (i) between about 75% and about 95% V.delta.1 cells, e.g., between about 80% and about 90% V.delta.1 cells; and between about 5% and about 25% V.delta.2 cells, e.g., between about 10% and about 15% or 20% V.delta.2 cells; or (ii) between about 30% and about 60% V.delta.1 cells, e.g., between about 35% and about 55% or (iii) between about 10% and about 30% V.delta.1 cells, such as between about 20% and about 25% V.delta.1 cells, and between about 65% and about 80% V.delta.2 cells, such as between about 70% and about 75% or 80% V.delta.2 cells.

[0035] In certain embodiments, between about 50% to about 99% or more of the NK cells and / or gamma.delta T cells of the compositions provided herein are between about 50% to about 99% or more, or greater than or equal to about 50%, greater than or equal to about 51%, greater than or equal to about 52%, greater than or equal to about 53%, greater than or equal to about 54%, greater than or equal to about 55%, greater than or equal to about 56%, greater than or equal to about 57%, greater than or equal to about 58%, greater than or equal to about 59%, greater than or equal to about 60%, greater than or equal to about 61%, greater than or equal to about 62%, greater than or equal to about 63%, greater than or equal to about 64%, greater than or equal to about 65%, greater than or equal to about 66%, greater than or equal to about 67%, greater than or equal to about 68%, greater than or equal to about 69%, greater than or equal to about 70%, greater than or equal to about 71%. more than or equal to about 72%, more than or equal to about 73%, more than or equal to about 74%, more than or equal to about 75%, more than or equal to about 76%, more than or equal to about 77%, more than or equal to about 78%, more than or equal to about 79%, more than or equal to about 80%, more than or equal to about 81%, more than or equal to about 82%, more than or equal to about 83%, more than or equal to about 84%, more than or equal to about 85%, more than or equal to about 86%, more than or equal to about 87%, more than or equal to about 88%, more than or equal to about 89%, more than or equal to about 90%, more than or equal to about 91%, more than or equal to about 92%, more than or equal to about 93%, more than or equal to about 94%, more than or equal to about 95%,More than or equal to about 96%, more than or equal to about 97%, more than or equal to about 98%, more than or equal to about 99%, more than or equal to about 80%, more than or equal to about 81%, more than or equal to about 82%, more than or equal to about 83%, more than or equal to about 84%, more than or equal to about 85%, more than or equal to about 86%, more than or equal to about 87%, more than or equal to about 88%, more than or equal to about 89%, more than or equal to about 90%, more than or equal to about 91%, more than or equal to about 92%, more than or equal to about 93%, more than or equal to about 94%, more than or equal to about 95%, more than or equal to about 96%, more than or equal to about 97%, more than or equal to about 98%, more than or equal to about 99%, or fractions thereof, up to 100% are CD8+. In some embodiments, less than 2% of the NK cells and / or gamma delta T cells are CD4+. In some embodiments, less than 2% of the NK cells and / or gamma delta T cells are CD8+CD4+. In certain embodiments, a fraction of between about 15% and about 30% of the NK cells and / or between about 55% and 85% of the gamma delta T cells are CD8-CD4-.

[0036] In certain embodiments of the compositions provided herein, between about 30% and about 99% or more of the cells in the population, or at least about 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%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 109%, 109%, 102%, 104%, 105%, 106%, 107%, 108%, 109%, 109%, 109%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 109%, 109%, 109%, 102%, 103%, 104%, 105%, 106%, 107%, 10 %, 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 fractions thereof, up to 100%, further comprises a genetic modification comprising an exogenous polynucleotide, a mutated polynucleotide, a deleted polynucleotide, or a combination thereof. In some embodiments, the genetic modification comprises an exogenous polynucleotide. The exogenous polynucleotide sometimes is in a retroviral or lentiviral vector, and sometimes the exogenous polynucleotide is integrated into the genome of one or more cells of the modified cell population.

[0037] The exogenous polynucleotide may in certain embodiments encode an exogenous or heterologous T cell receptor, a tumor necrosis factor receptor, a chimeric antigen receptor (CAR), a myeloid differentiation primary response protein, and an innate immune signaling adaptor, or other protein or polypeptide of interest, and in some embodiments may include a promoter or other regulator of gene expression. In some embodiments, the exogenous polynucleotide is a regulatory sequence, such as a promoter or enhancer.

[0038] In certain embodiments, the exogenous polynucleotide encodes a chimeric antigen receptor (CAR) and the cells in the composition contain a CAR. A CAR is a recombinant receptor that provides both antigen binding and T cell activation functions (see, e.g., Sadelain et al., Cancer Discov., 2003). 3(4):388-398 (2013)). When an immune cell, such as a T cell, is engineered (genetically modified) to express a CAR, this provides the immune cell with a new and / or improved ability to target a protein or antigen of interest. The target protein or antigen of interest may, in certain embodiments, be a cancer antigen or an infectious disease antigen, some of which are known and / or identifiable in the art. In some embodiments, the CAR is a CAR-specific antigen, such as CD19 (see, e.g., GenBank Accession No. AH005421.2), GD2 (disialoganglioside; see, e.g., Schulz et al., Cancer Res., 44(12):5914-5920 (1984)), HER3 (see, e.g., GenBank Accession No. ... No. M34309.1), B7H3 (see, e.g., GenBank Accession No. BC062581.1), CD123 (see, e.g., GenBank Accession Nos. BC035407.1; BX296563.3), or CD30 (see, e.g., GenBank Accession Nos. M83554.1; AY498860.1).

[0039] Any of the compositions provided herein can be further treated to remove either gamma.delta T cells or NK cells, thereby generating a composition containing substantially all NK cells or substantially all gamma.delta T cells. For example, the compositions provided herein can be treated with an anti-CD3 antibody to deplete gamma.delta T cells from a mixture of NK cells and gamma.delta T cells, leaving a composition containing substantially all or all NK cells, or alternatively, an anti-CD3 antibody can be used to isolate a substantially pure gamma.delta T cell population from the mixture.

[0040] In certain embodiments, therapeutic compositions (or therapeutic combinations) of cells are provided that include a plurality of gamma delta T cells (γδ); a plurality of natural killer cells (NK); or a combination of γδ and NK cells. In certain embodiments, the γδ and / or NK cells are recombinantly engineered or genetically modified, where optionally the γδ and / or NK cells are recombinantly engineered or genetically modified to extracellularly express an exogenous or heterologous protein, where optionally the exogenous, heterologous, or chimeric protein is a chimeric antigen receptor (CAR) or an exogenous or heterologous T cell receptor (TCR), where optionally the exogenous, heterologous, or chimeric protein or CAR is specific for (can specifically bind to) a cancer cell or a tumor marker or an infected cell, or the exogenous, heterologous, or chimeric protein or CAR is specific for (can specifically bind to) an antibody that can specifically target and bind to a cancer cell or a tumor marker or an infected cell, or any disease-associated antigen.

[0041] In some embodiments, the γδ and / or NK cells are human or animal cells. In certain embodiments, the therapeutic composition (or therapeutic combination) is formulated for intravenous (IV), intrathecal, intramuscular (IM), intraperitoneal (IP), or intratumoral (IT) administration, into a joint cavity, or injected or implanted at or near the site of the cancer or infection, or administered intravenously within about 10 minutes, as needed. 2 From 10 12 and formulated in a unit dosage form containing between about 10 and 15 cells,

[0042] or formulated for intramuscular (IM) administration, or about 10 2 From 10 12 The cell is formulated in a unit dosage form comprising between 1 and 2 cells.

[0043] In some embodiments, γδ and / or NK cells are isolated from an in vivo source. In some embodiments, γδ and / or NK cells are expanded in culture or isolated from an in vivo source and expanded in culture. In certain embodiments, γδ and / or NK cells are isolated from an in vivo source and expanded in culture without the use of any feeder cells or without the use of a feeder cell layer, thereby generating a population of expanded γδ and / or NK cells that are devoid of feeder cells. In some embodiments, γδ and / or NK cells are isolated from an in vivo source and expanded in culture using feeder cells or a feeder cell layer, optionally with the feeder cells substantially removed and / or killed, generating a population of expanded γδ and / or NK cells that are substantially devoid of feeder cells.

[0044] In certain embodiments, the in vivo source of γδ and / or NK cells is derived from an autologous source (optionally from the individual who will be the recipient of the γδ and / or NK cells), or from an exogenous, xenogeneic, or allogeneic source.

[0045] Also provided herein, in certain aspects, is a pharmaceutical composition comprising any of the compositions provided herein and a pharma- ceutically acceptable carrier.

[0046] In certain embodiments, the present disclosure provides a method for making genetically modified immune cells by adding exogenous polynucleotide to the compositions provided herein, mutating polynucleotide in one or more cells of the compositions provided herein, or deleting polynucleotide in one or more cells of the compositions provided herein.In some embodiments, the genetic modification is an exogenous polynucleotide.The method used to produce the compositions provided herein often results in cells in a high activation state, which facilitates the introduction of exogenous polynucleotide, for example, by retroviral or lentiviral transduction. In certain embodiments, the methods of generating genetically modified immune cells provided herein often result in between about 30% and about 99% or more of the cells being genetically modified, or at least about 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 109%, 109%, 108%. , 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 fraction thereof, up to 100% of the compositions contain the genetic modification.

[0047] In certain embodiments, kits are provided herein that contain any of the compositions or pharmaceutical compositions provided herein, optionally with instructions for use, and optionally with cytokines. The compositions, pharmaceutical compositions, or kits provided herein can be stored at refrigerated temperatures, such as 10 degrees Celsius or lower, such as 9, 8, 7, 6, 5, 4, 3, 2, 1, minus 4 degrees Celsius or lower, or at freezing temperatures (e.g., minus 15, minus 20, minus 25, minus 30, minus 35, minus 40, minus 45, minus 50, minus 55, minus 60, minus 65, minus 70, minus 75, minus 80, minus 85 or lower), depending on storage and / or transportation needs. In certain embodiments, the kits can be used to store about 1×10 cells. 5 Approximately 1 x 10 cells 12 for example, about 1 x 10 cells 6 pieces, cells 1×10 7 pieces, cells 1×10 8 pieces, cells 1×10 9 pieces, or 1 x 10 cells 10In certain embodiments, the kits provided herein may include a cytokine. In some embodiments, the cytokine is selected from the group consisting of TNF (see, e.g., GenBank Accession No. KJ892290.1; AY214167.1), IFNγ (see, e.g., GenBank Accession No. J00219.1), interleukin IL-1β, IL-2, IL-4, IL-6, IL-7, IL-10 (see, e.g., GenBank Accession No. U16720.1), IL-12 (see, e.g., GenBank Accession No. AF404773.1 for IL-12A; see, e.g., GenBank Accession No. AF404773.1 for IL-12B), IL-1β (see, e.g., GenBank Accession No. AF404773.1 for IL-12C ... For -12B, see GenBank Accession No. AF512686.1), IL-15, IL-18 (see, e.g., GenBank Accession Nos. BC007461.1; BC007007.1), IL-21, CCL4 (see, e.g., GenBank Accession Nos. CR542119.1; KJ901727.1; KJ901726.1), RANTES (see, e.g., GenBank Accession No. GQ504011.1), and TGFβ (see, e.g., National Center for Biotechnology Information (NCBI) Accession No. NM_000660.7). In certain embodiments, the kit contains a composition or pharmaceutical composition provided herein in unit dosage form.

[0048] In certain aspects, products and kits are provided for practicing the methods provided herein, comprising the therapeutic combination of cells provided herein. In certain aspects, the products and kits further comprise instructions for practicing the methods provided herein. In some aspects, the products and kits further comprise an antibody capable of specifically binding to a cancer-associated, or tumor-associated, infectious disease-associated, or disease-associated antigen. The products provided herein can include implants comprising the therapeutic combination of cells provided herein.

[0049] Also provided herein in certain embodiments is a method of treating cancer or infectious disease by administering to a subject in need thereof any one of the compositions, pharmaceutical compositions, or kits provided herein in an amount effective for treating cancer or infectious disease. Treatment can be administered in an autologous or allogeneic setting. In some embodiments, the donor of the sample from which the composition, pharmaceutical composition, or kit is produced is the recipient of the treatment. In certain embodiments, treatment can be administered on two or more separate days, and in certain embodiments, treatment can be administered in multiple doses. In certain embodiments, treatment is administered between about 1 unit dosage and about 36 or more unit dosages, at intervals between about 2 weeks and about 4 weeks. In some embodiments, the treatment is administered as a single unit dosage once, twice, three times, four times, or up to five times daily, or once, twice, three times, four times, five times, six times, seven times, eight times, nine times, or ten times, or more times over the course of several days, weeks, or months, or every other day, or once, twice, three times, four times, five times, or six times weekly. Treatment can be administered intravenously (IV), intrathecally, or intramuscularly (IM), intraperitoneally (IP), intrapleurally, intraarticularly, or at about 10 cells per kilogram of the subject's body weight at or near the site of the cancer or infection. 4 About 10 10 Between 10 and 10 cells per subject 6 About 10 12 In certain embodiments, the unit dosage is about 10 cells per subject. 10 or approximately 10 cells per kilogram of subject's body weight 8 There are 10 pieces.

[0050] In certain aspects of the methods of treatment provided herein, the treatment is for cancer. In some aspects, the cancer is selected from lung cancer, melanoma, breast cancer, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, neuroblastoma, rhabdomyosarcoma, leukemia or lymphoma, Hodgkin's lymphoma or childhood acute lymphoblastic leukemia, non-Hodgkin's lymphoma, mast cell tumor or mast cell tumor, ovarian cancer or carcinoma, pancreatic cancer, non-small cell lung cancer, small cell lung cancer, hepatocellular carcinoma, retinoblastoma, breast tumor, colorectal cancer, leukemia, lymphoma, acute lymphoblastic leukemia (ALL) or acute lymphocytic leukemia, acute myeloid leukemia (AML), histiocytic sarcoma, brain tumor, astrocytoma, glioblastoma, neuroma, colon cancer, cervical cancer, sarcoma, bladder tumor, tumor of reticuloendothelial tissue, Wilms' tumor, bone cancer, osteosarcoma, kidney cancer, or head and neck cancer, oral cavity cancer, laryngeal cancer, metastatic disease, or oropharyngeal cancer.

[0051] In certain embodiments of the methods of treatment provided herein, a second agent is co-administered with the composition, pharmaceutical composition, or kit. In some embodiments, the second agent is an antibody that immunospecifically binds to a cancer-associated antigen. In certain embodiments, the cancer associated antigen is alpha-fetoprotein (AFP), alpha-actinin-4, A3, an antigen specific for the A33 antibody, ART-4, B7, B7-H3, Ba733, BAGE, BrE3-antigen, CA125, CAMEL, CAP-1, carbonic anhydrase IX, CASP-8 / m, CCL19, CCL21, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD65, CD66, CD67, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75, CD76, CD77, CD78, CD79, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD87, CD88, CD89, CD90, CD91, CD92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107, CD108, CD109, CD110, CD111, CD112, CD113, CD114, CD115, CD116, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD3 D66a-e, CD67, CD70, CD70L, CD74, CD79a, CD79b, CD80, CD83, CD95, CD123, CD126, CD132, CD133 , CD138, CD147, CD154, CDC27, CDK-4 / m, CDKN2A, CTLA4, CXCR4, CXCR7, CXCL12, HIF-1α, colon-specific antigen- p(CSAp), CEA(CEACAM-5), CEACAM-6, c-Met, DAM, EGFR, EGFRvIII, EGP-1(TROP-2), EGP-2, ELF 2-M, Ep-CAM, fibroblast growth factor (FGF), Flt-1, Flt-3, folate receptor, G250 antigen, GAGE, gp100, GRO-β, HLA-DR, HM1.24, human chorionic gonadotropin (HCG) and its subunits, HER2 / neu, HMGB-1, hypoxia-inducible factor (HIF-1), HSP70-2M, HST-2, Ia, IGF-1R, IFN-γ, IFN-α, IFN-β, IFN-λ, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12, IL -15, IL-17, IL-18, IL-23, IL-25, insulin-like growth factor 1 (IGF-1), KC4 antigen, KS-1 antigen, KS1-4, Le-Y, LDR / FUT, macrophage migration Inhibiting factor (MIF), GD2, MAGE, MAGE-3, MART-1, MART-2, NY-ESO-1, TRAG-3, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC 1, MUC2, MUC3, MUC4, MUC5ac, MUC13, MUC16, MUM-1 / 2, MUM-3, NCA66, NCA95, NCA90, pancreatic cancer mucin, PD1 receptor, placental growth factor, p53, PLAGL2, prostatic acid phosphatase, PSA, PRAME, PSMA, PlGF, ILGF, ILGF-R, L-6, IL-25, RS5, RANTES, T101, SAGE , S100, survivin, survivin-2B, TAC, TAG-72, tenascin, TRAIL receptor, TNF-α, Tn antigen, Thomson-Friedreich antigen, tumor necrosis antigen, VEGFR, ED-B fibronectin, WT-1, 17-1A antigen, complement factors C3, C3a, C3b, C5a, C5, angiogenesis markers, bcl-2, bcl-6, and Kras.

[0052] In certain embodiments, the cells of the compositions provided herein can be engineered to express an antibody that binds to a cancer-associated antigen. In some embodiments, the antibody is co-administered with a second agent. In certain embodiments, the cancer-associated antigen is selected from the group consisting of hR1 (anti-IGF-1R), hPAM4 (anti-mucin), KC4 (anti-mucin), hA20 (anti-CD20), hA19 (anti-CD19), hIMMU31 (anti-AFP), hLL1 (anti-CD74), hLL2 (anti-CD22), anti-CD19 / CD22 bispecific antibody, RFB4 (anti-CD22), hMu-9 (anti-CSAp), hL243 (anti-HLA-DR), hMN-14 (anti-ILA), hILA, hILB ... anti-CEACAM-5), hMN-15 (anti-CEACAM-6), hRS7 (anti-TROP-2), hMN-3 (anti-CEACAM-6), CC49 (anti-TAG-72), J591 (anti-PSMA), D2 / B (anti-PSMA), G250 (anti-carbonic anhydrase IX), dinutuximab (anti-GD2), infliximab (anti-TNF-α), certolizumab pegol (anti-TNF-α), adalimumab (anti-TNF-α), Alemtuzumab (anti-CD52), bevacizumab (anti-VEGF), cetuximab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomab tiuxetan (anti-CD20), panitumumab (anti-EGFR), rituximab (anti-CD20), tositumomab (anti-CD20), GA101 (anti-CD20), trastuzumab (anti-HER2 / neu), tocilizumab (anti-IL-6 receptor), basiliximab (anti-C D25), daclizumab (anti-CD25), efalizumab (anti-CD11a), muromonab-CD3 (anti-CD3 receptor), natalizumab (anti-alpha4 integrin), BWA-3 (anti-histone H2A / H4), LG2-1 (anti-histone H3), MRA12 (anti-histone H1), PR1-1 (anti-histone H2B), LG11-2 (anti-histone H2B), and LG2-2 (anti-histone H2B).

[0053] In certain embodiments of the methods of treatment provided herein, the treatment is for an infectious disease. In some embodiments, the infectious disease is characterized by the presence of a bacterial pathogen, a fungal pathogen, a viral pathogen, or a protozoan pathogen. In embodiments of the methods of treatment provided herein, the infectious disease is characterized by the presence of a bacterial pathogen, a fungal pathogen, a viral pathogen, or a protozoan pathogen. In some embodiments of the methods of treatment provided herein, the infectious disease is characterized by the presence of a bacterial pathogen, a fungal pathogen, a viral pathogen, or a protozoan pathogen. In some embodiments of the methods of treatment provided herein, the infectious disease is characterized by the presence of a bacterial pathogen, a fungal pathogen, a viral pathogen, or a protozoan pathogen. pylori, Klebsiella, Legionella, Meningococci, mycobacteria, Pseudomonas, Pneumonococci, Rickettsia bacteria, Salmonella, Serratia, Staphylococci, Streptococci, Tetanus, Aspergillus (A.fumigatus, A.niger, etc.), Blastomyces dermatitidis, Candida (C.albicans, C.krusei, C.glabrata, C.tropicalis, etc.), Cryptococcus neoformans, Genus Mucorales (mucor, absidia, rhizopus), Sporothrix schenkii, Paracoccidioides brasiliensis, Coccidioides immitis, Histoplasma capsulatum, Leptospirosis, Borrelia burgdorferi, helminth parasites (hookworms, tapeworms, flukes, flatworms (e.g., Schistosomia), Giardia lambia, trichinella, Dientamoeba Fragilis, Trypanosoma brucei, Trypanosoma cruzi, or Leishmania donovani.

[0054] In some aspects, methods of treating cancer, tumors, dysfunctional cells, or infected cells are provided herein, comprising: (a) administering to an individual in need thereof a therapeutically effective amount of a cellular therapeutic composition described herein; or (b) (i) providing or having provided a cellular therapeutic composition described herein; and (ii) administering to an individual in need thereof a therapeutically effective amount of a cellular therapeutic composition described herein; The present invention provides a method comprising administering or having administered to an individual in need thereof a therapeutically effective amount of a therapeutic composition of cells.

[0055] In certain embodiments of the methods of treatment provided herein, the individual in need thereof is a human or an animal. In some embodiments, the γδ and / or NK cells are isolated from an in vivo source, and optionally the in vivo source of γδ and / or NK cells is derived from a syngeneic or autologous source (optionally from the individual who will be the recipient of the γδ and / or NK cells), or from an exogenous, xenogeneic, or allogeneic source, or a combination thereof. In embodiments of the methods provided herein, the therapeutic composition or combination of cells is administered intravenously (IV), intrathecally, or intramuscularly (IM), or injected or implanted into or near (near) the cancer, tumor, dysfunction, or infected cells, and optionally the therapeutic composition of cells is delivered as an implant or gel, and optionally the gel is a hydrogel.

[0056] In some embodiments of the methods of treatment provided herein, the therapeutic composition or combination of cells is administered in a unit dosage form, and optionally the unit dosage is about 10 cells. 2 From 10 12 Between 10 or 10 cells 4 From 10 10 or a daily dose of about 10 cells 2 From 10 12 Between 10 or 10 cells 4 From 10 10In certain embodiments, a therapeutic composition or combination or unit dosage form of cells is administered to an individual in need thereof several times (multiple times), or 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times over the course of days, weeks, or months, where each of the multiple unit dosage forms is administered daily, every other day, 2, 3, 4, 5, or 6 times per week, or once per week, as appropriate.

[0057] In certain embodiments of the methods of treatment provided herein, an individual in need of treatment is induced to mount an immune response against cancer, infection, or disease by first pre-administering to the individual in need thereof an unconjugated antibody against a cancer- or tumor-associated, infection- or disease-associated antigen, followed by administration of a therapeutic composition of cells described herein, at least a portion of which express a polypeptide, optionally a CAR, that specifically binds to the unconjugated antibody on its cell surface. In some embodiments, an individual in need thereof is administered an antibody capable of specifically binding to a cancer- or tumor-associated, infection- or disease-associated antigen, followed by administration of a therapeutic composition of cells described herein, at least a portion of which express a polypeptide, optionally a CAR, that specifically binds to the antibody on its cell surface, optionally the antibody is administered before, with, or after administration of the therapeutic composition of cells.

[0058] The details of certain embodiments of the technology are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the technology will be apparent from the description and drawings, and from the claims.

[0059] All publications, patents, and patent applications cited herein are hereby expressly incorporated by reference for all purposes.

[0060] The drawings are illustrative of certain embodiments of the present technology, but not limiting. For clarity and ease of illustration, the drawings are not drawn to scale, and in some instances various aspects may be shown exaggerated or enlarged to facilitate an understanding of particular embodiments. In certain embodiments, for example, the following items are provided: (Item 1) 1. A method for producing a composition comprising a population of cells enriched for NK cells and gamma delta T cells, comprising: obtaining a cell-containing sample from one or more subjects; depleting alpha.beta T cells from said sample under conditions to produce a depleted cell population comprising NK cells and gamma.delta T cells; and exposing the depleted cell population to activation conditions comprising contacting the depleted cell population with (a) at least one exogenous polypeptide that immunospecifically binds to a cell adhesion polypeptide, and (b) at least one exogenous polypeptide that immunospecifically binds to a polypeptide distinct from the cell adhesion polypeptide and that is expressed on the surface of one or more cells of the sample population; and exposing said depleted cell population to expansion conditions comprising contacting said depleted cell population with at least one auxiliary polypeptide, thereby producing a composition comprising a population of cells enriched for NK cells and gamma delta T cells. The method includes: (Item 2) 1. A method for producing a composition comprising a population of cells enriched for NK cells and gamma delta T cells, comprising: obtaining a cell-containing sample from one or more subjects; exposing the sample to activating conditions comprising contacting the sample with (a) at least one exogenous polypeptide that immunospecifically binds to a cell adhesion polypeptide, and (b) at least one exogenous polypeptide that immunospecifically binds to a polypeptide distinct from the cell adhesion polypeptide and that is expressed on the surface of one or more cells of the sample population, where (a) or (b) are soluble, or where (a) and (b) are soluble; and exposing said sample to expansion conditions comprising contacting said sample with at least one auxiliary polypeptide, thereby producing a composition comprising a population of cells enriched for NK cells and gamma delta T cells. The method includes: (Item 3) The method of claim 1 or 2, wherein the at least one auxiliary polypeptide is selected such that the amount of NK cells relative to the amount of gamma delta T cells in the population is dependent on the amount and / or type of the at least one auxiliary polypeptide. (Item 4) The method according to any one of items 1 to 3, wherein the at least one auxiliary polypeptide increases or decreases the amount of NK cells relative to gamma delta T cells in the population of depleted cells after contacting the population of depleted cells with the at least one auxiliary polypeptide. (Item 5) 5. The method according to any one of items 1 to 4, wherein the activation conditions do not include serum derived from a non-human animal. (Item 6) 6. The method according to any of items 1 to 5, wherein the expansion conditions do not include serum from a non-human animal. (Item 7) 7. The method according to any of items 1 to 6, wherein the activation conditions do not include feeder cells. (Item 8) 8. The method according to any of items 1 to 7, wherein said expansion conditions do not include feeder cells. (Item 9) 9. The method according to any of items 1 to 8, wherein the sample is selected from peripheral blood, liver tissue, bone marrow, epithelial tissue, and umbilical cord blood. (Item 10) 10. The method of claim 9, wherein the sample is peripheral blood. (Item 11) 11. The method of claim 10, wherein the peripheral blood sample is a processed sample. (Item 12) 10. The method of claim 9, wherein the sample is umbilical cord blood. (Item 13) 13. The method of claim 12, wherein the cord blood sample is a processed sample. (Item 14) 14. The method according to any of items 1 to 13, wherein the at least one exogenous polypeptide in (b) immunospecifically binds to an NK cell activating receptor, a gamma delta T cell activating receptor, or both. (Item 15) The method of claim 14, wherein the receptor is NKp30, NKp44, or NKp46. (Item 16) The method of claim 15, wherein the receptor is NKp46. (Item 17) 17. The method according to any of items 1 to 16, wherein the exogenous polypeptide in (a) immunospecifically binds to CD2. (Item 18) 18. The method according to any of items 1 to 17, wherein the exogenous polypeptide in (a) or (b), or (a) and (b), is an antibody or an antigen-binding fragment thereof. (Item 19) 19. The method according to any of items 1 to 18, wherein at least one of (a) or (b) is soluble. (Item 20) 20. The method according to any of items 1 to 19, wherein both of the exogenous polypeptides in (a) and (b) are soluble. (Item 21) 20. The method according to any of items 1 to 19, wherein the exogenous polypeptide in (a) or the exogenous polypeptide in (b) is bound to a substrate. (Item 22) 22. The method according to any of items 1 to 21, wherein the activating conditions comprise contacting the sample or depleted cell population with at least two exogenous polypeptides. (Item 23) 23. The method of claim 22, wherein a first exogenous polypeptide immunospecifically binds to CD2 and a second exogenous polypeptide immunospecifically binds to NKp46. (Item 24) 24. The method of claim 22 or 23, wherein the first exogenous polypeptide and / or the second exogenous polypeptide is an antibody or an antigen-binding fragment thereof. (Item 25) The polypeptide component of the activating condition is (a) an exogenous polypeptide that immunospecifically binds to the cell adhesion polypeptide CD2; and (b) an exogenous polypeptide that immunospecifically binds to NKp46, different from the exogenous polypeptide in (a); 25. The method according to any of items 1 to 24, consisting essentially of or consisting of: (Item 26) 26. The method according to any of items 1 to 25, wherein the at least one auxiliary polypeptide is a cytokine and / or a polypeptide that immunospecifically binds to a receptor on a gamma delta T cell. (Item 27) 27. The method of claim 26, wherein the expansion conditions include contacting the sample or depleted cell population with at least one auxiliary polypeptide that is a cytokine, and optionally an auxiliary polypeptide that immunospecifically binds to a receptor on gamma.delta T cells. (Item 28) 28. The method of claim 27, wherein the cytokine is an interleukin (IL). (Item 29) 29. The method of any of items 26 to 28, wherein the at least one auxiliary polypeptide comprises IL-2, IL-4, IL-15, or any combination thereof. (Item 30) The expansion conditions are (a) an IL-2 polypeptide, and optionally a polypeptide that immunospecifically binds to a receptor on a gamma.delta T cell; (b) an IL-15 polypeptide; or (c) an IL-2 polypeptide and an IL-15 polypeptide, and optionally a polypeptide that immunospecifically binds to a receptor on a gamma delta T cell. 30. The method according to any one of items 26 to 29, comprising contacting the (Item 31) 31. The method according to any of items 26 to 30, wherein the receptor on the gamma delta T cells is CD3. (Item 32) 32. The method according to any of items 26 to 31, wherein the polypeptide that immunospecifically binds to a receptor on the gamma delta T cell is an antibody or an antigen-binding fragment thereof. (Item 33) The magnification conditions are (a) an IL-2 polypeptide; (b) an IL-15 polypeptide; (c) an IL-2 polypeptide and an IL-15 polypeptide; (d) an antibody that immunospecifically binds to an IL-2 polypeptide and CD3; or (e) an antibody that immunospecifically binds to an IL-2 polypeptide, an IL-15 polypeptide, and CD3; 33. The method of claim 32, comprising contacting the (Item 34) 34. The method of claim 33, wherein the antibody that immunospecifically binds to CD3 is OKT3. (Item 35) 35. The method according to any of items 1 to 34, wherein said activation and expansion conditions are carried out simultaneously or sequentially in any order. (Item 36) the at least one exogenous polypeptide can also function as an auxiliary polypeptide; or The at least one auxiliary polypeptide can also function as an exogenous polypeptide; or 36. The method according to any of items 1 to 35, wherein the at least one exogenous polypeptide can also function as an auxiliary polypeptide, and the at least one auxiliary polypeptide can also function as an exogenous polypeptide. (Item 37) (i) the expansion conditions include contacting the sample or depleted cell population with an IL-2 polypeptide; (ii) The method according to any of items 33 to 36, wherein the obtained population of cells enriched for NK cells and gamma delta T cells comprises about 25-30% NK cells and about 70-75% gamma delta T cells. (Item 38) (i) the expansion conditions comprise contacting the sample or depleted cell population with an IL-15 polypeptide; (ii) The method according to any of items 33 to 36, wherein the obtained population of cells enriched for NK cells and gamma delta T cells comprises about 80-99% NK cells and about 1-20% gamma delta T cells. (Item 39) (i) the expansion conditions include contacting the sample or depleted cell population with an IL-2 polypeptide and an antibody that immunospecifically binds CD3; (ii) The method according to any of items 33 to 36, wherein the obtained population of cells enriched for NK cells and gamma delta T cells comprises about 40-45% NK cells and about 55-60% gamma delta T cells. (Item 40) The expansion condition is contacting the sample or depleted cell population with a first set of conditions comprising one or more auxiliary polypeptides to result in a first cell population comprising a first ratio of NK cells to gamma delta T cells; and 40. The method of any of items 1-39, comprising contacting the first cell population with a second set of conditions comprising one or more auxiliary polypeptides to result in a second cell population comprising a desired final ratio of NK cells to gamma delta T cells, wherein the first set of conditions is different from the second set of conditions. (Item 41) 41. The method of claim 40, wherein the first cell population is washed prior to contact with the second set of conditions. (Item 42) the first set of conditions comprises IL-2 and the second set of conditions comprises IL-15; the first set of conditions comprises IL-15 and the second set of conditions comprises IL-2; the first set of conditions comprises an antibody that immunospecifically binds to IL-2 and CD3, and the second set of conditions comprises IL-15; or 42. The method of claim 40 or 41, wherein the first set of conditions comprises an antibody that immunospecifically binds to IL-15 and CD3, and the second set of conditions comprises an antibody that immunospecifically binds to IL-2 and CD3. (Item 43) 43. The method of claim 42, wherein the antibody that immunospecifically binds to CD3 is OKT3. (Item 44) depleting alpha.beta T cells from said sample prior to exposing said sample to said activation and expansion conditions, thereby generating a depleted cell population; and subjecting said depleted cell population to said activation and expansion conditions. Further comprising: 44. The method according to any of items 2 to 43, whereby a composition comprising a population of cells enriched for NK cells and gamma delta T cells is obtained. (Item 45) 45. The method of any of items 1 to 44, wherein prior to activation and expansion, the sample or the depleted cell population is not exposed to conditions that select for NK cells or gamma.delta T cells or that deplete cells other than the alpha-beta T cells. (Item 46) 46. ​​The method of any of items 1-45, wherein prior to activation and expansion, the sample or the depleted cell population is not exposed to conditions that deplete all CD3+ cells in the sample or the depleted cell population. (Item 47) 47. The method according to any of items 1 to 46, wherein the cells of the sample or depleted cell population are free of exogenous nucleic acid before, during or after activation and expansion. (Item 48) 47. The method of any of items 1-46, wherein the cells of the sample or depleted cell population do not contain a tumor necrosis factor receptor, a chimeric antigen receptor (CAR), a myeloid differentiation primary response protein, or an exogenous nucleic acid encoding an innate immune signaling adaptor before, during, or after activation and expansion. (Item 49) 49. The method according to any of items 1 to 48, wherein the cells of the sample or depleted cell population are not genetically modified before, during or after activation and expansion. (Item 50) 50. The method of any of items 1 to 49, further comprising subjecting the population of cells enriched for NK cells and gamma delta T cells to a treatment whereby said gamma delta cells are depleted, wherein the resulting population consists essentially of or consists of NK cells. (Item 51) 50. The method according to any of items 1 to 49, further comprising subjecting said population of cells enriched for NK cells and gamma.delta T cells to a treatment whereby said NK cells are depleted, wherein the resulting population consists essentially of or consists of gamma.delta T cells. (Item 52) 51. The method of any of items 1 to 50, further comprising subjecting said population of cells enriched for NK cells and gamma delta T cells to a positive selection for NK cells, thereby obtaining a cell population consisting essentially of or consisting of NK cells. (Item 53) 52. The method of any of items 1 to 49 and 51, further comprising subjecting the population of cells enriched for NK cells and gamma.delta T cells to positive selection for gamma.delta cells, thereby obtaining a cell population that consists essentially of or consists of gamma.delta cells. (Item 54) 54. The method of any of items 1 to 53, wherein the expansion conditions comprise incubation of the sample or depleted cell population in feeder cell free medium for about 1 week to about 10 weeks, thereby obtaining a composition comprising an expanded population of cells enriched for NK cells and gamma delta T cells. (Item 55) 55. The method of claim 54, wherein the culture conditions comprise incubation of the sample or depleted cell population in feeder cell free medium for about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 25, 30, 35, 40, 45, 50, 55, or 60 days or more, or about 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks. (Item 56) 56. The method of any of items 1 to 55, wherein the population of cells enriched for NK cells and gamma delta T cells is expanded by greater than about 2 logs in 30 days under said expansion conditions. (Item 57) 57. The method of claim 56, wherein the population of cells is expanded by greater than about 3 logs in 30 days under the expansion conditions. (Item 58) 58. The method of any of items 1 to 57, wherein the expanded population of cells enriched for NK cells and gamma delta T cells does not contain exhausted cells. (Item 59) 59. The method of any of items 1 to 58, wherein the expanded population of cells enriched for NK cells and gamma delta T cells does not contain exhausted cells after 60 days of expansion conditions. (Item 60) 60. The method of any of items 1-59, wherein less than 5%, less than 4%, less than 3%, or less than 2% of the NK cells in the expanded population of cells enriched for NK cells and gamma.delta T cells comprise the PD-1 marker, and / or about 20%, 15%, 10%, or less of the total cells in the expanded population or the gamma.delta T cells in the expanded population comprise the PD-1 marker. (Item 61) The population of cells enriched for NK cells and gamma delta T cells is enriched for the following activation markers as a percentage of the total number of cells in the population: (a) 90% or more KIR5; (b) 10% or more SIGLEC-7; (c) 60% or more KIR3D51; (d) 10% or more KIR2DL1; (e) 25% or more NKp30, NKp44, or NKp46; (f) NKG2D of 35% or more; (g) 90% or more DNAM1; (h) 85% or more NTBA; (i) 95% or greater CD2; and (j) 55% or more KIR3DS1 61. The method according to any one of items 1 to 60, comprising one or more of the following: (Item 62) 62. The method according to any of items 1 to 61, wherein the population of cells enriched for NK cells and gamma delta T cells comprises 80% or more innate immune cells. (Item 63) 63. The method of paragraph 62, wherein between about 70% and about 100%, or at least about 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% of the cells are CD56+. (Item 64) 64. The method of claim 62 or 63, wherein between about 10% and about 40%, or at least about 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%, or 40% of the cells are CD16+. (Item 65) 65. The method of any of items 62-64, wherein less than 5%, less than 4%, less than 3%, or less than 2% of the cells are CD57+. (Item 66) 66. The method according to any of items 1 to 65, wherein the sample or depleted cell population does not contain CD4+CD8+ cells during or after activation and expansion. (Item 67) 67. The method according to any of items 1 to 66, wherein the activation conditions and the expansion conditions do not comprise a bisphosphonate. (Item 68) 68. The method of claim 67, wherein the bisphosphonate is pamidronate or zoledronate. (Item 69) 69. The method of any of items 1 to 68, wherein the gamma.delta T cells are polyclonal for V.delta.1 and V.delta.2 expression. (Item 70) 70. The method of claim 69, wherein the polyclonal gamma.delta T cells comprise at least one subpopulation selected from V.delta.1+ and V.delta.1-, and at least one subpopulation selected from V.delta.2+ and V.delta.2-. (Item 71) 71. A composition obtainable or obtained by the method according to any of items 1 to 70. (Item 72) A composition comprising a population of modified peripheral blood cells, said population comprising Contains multiple NK cells and multiple gamma delta T cells; Alpha and beta T cells are depleted; The composition does not include feeder cells. (Item 73) Between about 25% and about 45% of the cells are NK cells and between about 55% and about 75% of the cells are gamma delta T cells; Between about 25% and about 30% of the cells are NK cells and between about 70% and about 75% of the cells are gamma delta T cells; Between about 80% and about 99% of the cells are NK cells and between about 1% and about 20% of the cells are gamma delta T cells; or 73. The composition of item 72, wherein between about 40% and about 45% of the cells are NK cells and between about 55% and about 60% of the cells are gamma delta T cells. (Item 74) The composition of item 72 or item 73, wherein 30% or more of the cells are activated. (Item 75) The population of modified cells has the following activation markers as a percentage of the total number of cells in the population: (a) 90% or more KIR5; (b) 10% or more SIGLEC-7; (c) 60% or more KIR3D51; (d) 10% or more KIR2DL1; (e) 25% or more NKp30, NKp44, or NKp46; (f) NKG2D of 35% or more; (g) 90% or more DNAM1; (h) 85% or more NTBA; (i) 95% or greater CD2; and (j) 55% or more KIR3DS1 75. The composition according to any of items 72 to 74, comprising one or more of: (Item 76) 76. The composition of any of items 72 to 75, wherein the modified population comprises 80% or more innate immune cells. (Item 77) 77. The composition of any of items 72-76, wherein the modified population is enriched for activated cytotoxic cells that are CD56+. (Item 78) 78. The composition according to any of items 72 to 77, wherein the modified cells are enriched for activated cytotoxic cells that are CD57-. (Item 79) 79. The composition according to any of items 72 to 78, wherein the population is enriched for activated cytotoxic cells that are CD56+CD57-. (Item 80) 80. The composition of any of items 77-79, wherein between about 80% and about 100%, or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the cells are CD56+. (Item 81) 81. The composition of any of items 77-80, wherein between about 10% and about 40%, or at least about 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%, or 40% of the cells are CD16+. (Item 82) 82. The composition of any of items 77-81, wherein less than 5%, less than 4%, less than 3%, or less than 2% of the cells are CD57+. (Item 83) 83. The composition according to any of items 72 to 82, which is substantially free of cells other than NK cells and gamma delta T cells. (Item 84) 84. The composition of any of items 72-83, comprising less than 5 percent NKT cells. (Item 85) 85. The composition of any of items 72-84, comprising less than 1 percent NKT cells. (Item 86) 86. The composition of any of items 72-85, comprising less than 0.1 percent NKT cells. (Item 87) 87. The composition of any of items 72 to 86, comprising less than 2 percent alpha.beta T cells. (Item 88) 88. The composition of any of items 72 to 87, comprising less than 1 percent alpha.beta T cells. (Item 89) 89. The composition of any of items 72 to 88, comprising less than 0.1 percent alpha.beta T cells. (Item 90) 90. The composition according to any of items 72 to 89, wherein a subset of NK cells in the population is CD16+ cells. (Item 91) 91. The composition according to any of items 72 to 90, wherein the majority of gamma delta T cells are CD57- cells. (Item 92) 92. The composition of any of items 72 to 91, wherein a majority of the NK cells are CD57- cells. (Item 93) 93. The composition of any of items 72 to 92, wherein the gamma.delta T cells are polyclonal for V.delta.1 and V.delta.2 expression. (Item 94) The composition of item 93, wherein the polyclonal gamma.delta T cells comprise at least one subpopulation selected from V.delta.1+ and V.delta.1-, and at least one subpopulation selected from V.delta.2+ and V.delta.2-. (Item 95) the majority of gamma.delta T cells express V.delta.1 and a minority of gamma.delta T cells express V.delta.2; or 95. The composition of any of items 72 to 94, wherein a minority of gamma.delta T cells express V.delta.1 and a majority of gamma.delta T cells express V.delta.2. (Item 96) A minority of cells in said population are CD3 positive cells and a majority of cells in said population are CD3 negative cells; or 96. The composition according to any of items 72 to 95, wherein a majority of cells in the population are CD3 positive cells and a minority of cells in the population are CD3 negative cells. (Item 97) 97. The composition according to any of items 72 to 96, wherein the ratio of NK cells to gamma delta T cells is greater than 1. (Item 98) 97. The composition according to any of items 72 to 96, wherein the ratio of NK cells to gamma delta T cells is less than 1. (Item 99) 98. The composition of claim 97, wherein the population of modified cells comprises about 98-99% NK cells and about 1-2% gamma delta T cells. (Item 100) The composition of item 98, wherein the population of modified cells comprises between about 25% and about 45% NK cells and between about 55% and about 75% gamma.delta T cells. (Item 101) 99. The composition of claim 98, wherein the population of modified cells comprises about 25-30% NK cells and about 70-75% gamma delta T cells. (Item 102) 99. The composition of claim 98, wherein the population of modified cells comprises about 40-45% NK cells and about 55-60% gamma delta T cells. (Item 103) Between about 50% to about 99% or more of said NK cells and / or said gamma.delta T cells, or greater than or equal to about 50%, greater than or equal to 51%, greater than or equal to 52%, greater than or equal to 53%, greater than or equal to 54%, greater than or equal to 55%, greater than or equal to 56%, greater than or equal to 57%, greater than or equal to 58%, greater than or equal to 59%, greater than or equal to 60%, greater than or equal to 6%, greater than or equal to 62%, greater than or equal to 63%, greater than or equal to 64%, greater than or equal to 65%, greater than or equal to 66%, greater than or equal to 67%, greater than or equal to 68%, greater than or equal to 69%, greater than or equal to 70%, greater than or equal to 71%, greater than or equal to 72%, greater than or equal to 73%, greater than or equal to 74%, greater than or equal to 75%, greater than or equal to 76%, greater than or equal to 77%, greater than or equal to 78%, greater than or equal to 79%, greater than or equal to 80%, greater than or equal to 81%, greater than or equal to 82%, greater than or equal to 83%, greater than or equal to 84%, greater than or equal to 85%, greater than or equal to 86%, greater than or equal to 87%, greater than or equal to 88%, greater than or equal to 89%, greater than or equal to 90%, greater than or equal to 91%, greater than or equal to 92%, greater than or equal to 93%, greater than or equal to 94%, greater than or equal to 95%, greater than or equal to 96%, greater than or equal to 97%, greater than or equal to 98%, greater than or equal to 99%, greater than or equal to 99%, greater than or equal to 99%, greater than or equal to 99%, greater than %, more than or equal to 74%, more than or equal to 75%, more than or equal to 76%, more than or equal to 77%, more than or equal to 78%, more than or equal to 79%, more than or equal to 80%, more than or equal to 81%, more than or equal to 82%, more than or equal to 83%, more than or equal to 84%, more than or equal to 85%, more than or equal to 86%, more than or equal to 87%, more than or equal to 88%, more than or equal to 89%, more than or equal to 90%, more than or equal to 91%, more than or equal to 92%, more than or equal to 93%, more than or equal to 94%, more than or equal to 95%, more than or equal to 96%, more than or equal to 97%, more than or equal to 98%,103. The composition of any of items 72-102, wherein greater than or equal to 99%, greater than or equal to 80%, greater than or equal to 81%, greater than or equal to 82%, greater than or equal to 83%, greater than or equal to 84%, greater than or equal to 85%, greater than or equal to 86%, greater than or equal to 87%, greater than or equal to 88%, greater than or equal to 89%, greater than or equal to 90%, greater than or equal to 91%, greater than or equal to 92%, greater than or equal to 93%, greater than or equal to 94%, greater than or equal to 95%, greater than or equal to 96%, greater than or equal to 97%, greater than or equal to 98%, greater than or equal to 99%, or fractions thereof, up to 100%, are CD8+. (Item 104) The composition of item 103, wherein less than 2% of the NK cells and / or gamma.delta T cells are CD4+. (Item 105) The composition of claim 103 or 104, wherein less than 2% of the NK cells and / or the gamma.delta T cells are CD8+CD4+. (Item 106) 106. The composition according to any of items 103 to 105, wherein a fraction of between about 15% and about 30% of the NK cells and / or between about 55% and 85% of the gamma.delta T cells are CD8-CD4-. (Item 107) Between about 30% and about 99% or more of the cells in the population, or at least about 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% 107. The composition of any of items 72-106, wherein 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 fractions thereof, up to 100%, further comprises a genetic modification comprising an exogenous polynucleotide, a mutated polynucleotide, a deleted polynucleotide, or a combination thereof. (Item 108) 108. The composition of claim 107, wherein at least about 95%, 96%, 97%, 98%, 99% of the cells in the population contain the genetic modification, or about 100% or 100% of the cells in the population contain the genetic modification. (Item 109) The composition of claim 107 or 108, wherein the genetic modification comprises an exogenous polynucleotide. (Item 110) 110. The composition of claim 109, wherein the exogenous polynucleotide is in a retroviral vector or a lentiviral vector. (Item 111) 110. The composition of claim 109, wherein the exogenous polynucleotide is integrated into the genome of one or more cells of the modified cell population. (Item 112) 112. The composition of any of items 107-111, wherein the cells in the population comprise a chimeric antigen receptor (CAR). (Item 113) 13. The composition of claim 112, wherein the chimeric antigen receptor comprises a binding molecule portion that immunospecifically binds to one or more of CD19, GD2, HER3, B7H3, CD123, or CD30. (Item 114) The composition according to any one of Items 72 to 113, wherein the population comprising the plurality of NK cells and the plurality of gamma delta T cells is derived from the peripheral blood cells of more than one subject. (Item 115) A pharmaceutical composition comprising the composition according to any one of embodiments 72 to 114 and a pharma- ceutically acceptable carrier. (Item 116) 1. A method for producing genetically modified immune cells, comprising: (a) adding an exogenous polynucleotide to the composition according to any of items 71 to 106; (b) mutating a polynucleotide in one or more cells of a composition according to any one of items 71 to 106; or (c) Deleting the polynucleotide in one or more cells of the composition according to any one of items 71 to 106. The method includes one or more of the following: (Item 117) 117. The method of claim 116, wherein the genetic modification is by retroviral transduction, lentiviral transduction, electroporation, transfection, CRISPR / cas9, or TALENS. (Item 118) 118. The method of claim 116 or 117, wherein the genetic modification consists or consists essentially of adding an exogenous polynucleotide as in (a). (Item 119) 119. The method according to any of items 116 to 118, wherein the genetic modification comprises adding an exogenous polynucleotide as in (a) and / or mutating a polynucleotide as in (b), and wherein the exogenous polynucleotide and / or the mutated polynucleotide is integrated into the genome of the immune cell. (Item 120) 120. The method of claim 119, wherein the integration is by electroporation, transfection, CRISPR / cas9, or TALENS. (Item 121) 122. The method of any of items 116 to 121, wherein the exogenous polynucleotide encodes a chimeric antigen receptor (CAR). (Item 122) 122. The method of claim 121, wherein the chimeric antigen receptor comprises a binding molecule portion that immunospecifically binds to one or more of CD19, GD2, HER3, B7H3, CD123, or CD30. (Item 123) Between about 30% and about 99% or more of the cells in the population, or at least about 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%, 123. The method of any of items 116 to 122, wherein 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 any fraction thereof, up to 100%, comprises the genetic modification. (Item 124) 124. The method of claim 123, wherein the genetic modification comprises an exogenous polynucleotide. (Item 125) 125. The method of claim 124, wherein about 100% or 100% of the cells in the population contain the exogenous polynucleotide. (Item 126) A kit comprising the composition according to any of items 71 to 114, or the pharmaceutical composition according to item 115, optionally with instructions for use, and optionally with a cytokine. (Item 127) Item 127. The kit of item 126, wherein the composition or pharmaceutical composition is at or below minus 4 degrees Celsius. (Item 128) Item 128. The kit of item 127, wherein the composition or pharmaceutical composition is at about minus 75 degrees Celsius to about minus 80 degrees Celsius. (Item 129) cells approximately 1 x 10 5 Approximately 1 x 10 cells 12 129. The kit according to any one of items 126 to 128, comprising: (Item 130) 130. The kit according to any of items 126 to 129, wherein the cytokine is an interleukin polypeptide. (Item 131) 131. The kit of claim 130, wherein the interleukin peptide is IL-2, IL-4, or IL-15. (Item 132) 132. The kit according to any of items 126 to 131, which is non-human serum-free and / or bovine serum-free. (Item 133) 133. The kit according to any of items 126 to 132, which is xenogeneic. (Item 134) 134. The kit according to any of items 126 to 133, which does not contain exogenous feeder cells. (Item 135) 135. The kit according to any one of items 126 to 134 in unit dosage form. (Item 136) The unit dosage form contains about 1×10 cells 6 Approximately 1 x 10 cells 12 The kit described in embodiment E10, wherein (Item 137) 136. A collection of cells derived from different donor subjects comprising a plurality of containers each containing cells derived from one or more donor subjects, each container comprising the composition according to any one of items 71 to 114, the pharmaceutical composition according to item 115, or the kit according to any of items 126 to 136. (Item 138) A method for treating cancer or an infectious disease, comprising administering to a subject in need thereof an effective amount for treating said cancer or infectious disease of the composition according to any one of items 71 to 114, the pharmaceutical composition according to item 115, or the kit according to any one of items 126 to 136, wherein the cells in the composition, the pharmaceutical composition, or the kit are allogeneic to the subject. (Item 139) A method for treating cancer or an infectious disease, comprising administering to a subject in need thereof an effective amount for treating said cancer or infectious disease of the composition according to any one of items 71 to 114, the pharmaceutical composition according to item 115, or the kit according to any one of items 126 to 136, wherein the cells in the composition, the pharmaceutical composition, or the kit are autologous to the subject. (Item 140) 139. The method of claim 138 or claim 139, comprising administering the composition to the subject on two or more separate days. (Item 141) The method of claim 139 or 140, wherein the donor of the cells is the recipient of the treatment. (Item 142) The method of claim 138 or claim 140, wherein the donor of the cells is not the recipient of the treatment. (Item 143) The method of claim 142, wherein the recipient of the treatment is predisposed to GvHD when treated with alpha.beta T cells derived from the donor. (Item 144) The method according to any one of items 138 to 143, wherein the treatment is administered in between about 1 unit dosage and about 36 or more unit dosages at intervals of between about 2 weeks and about 4 weeks. (Item 145) 144. The method according to any of items 138-143, wherein the treatment is administered as a single unit dosage once, twice, three times, four times, or up to five times daily, or once, twice, three times, four times, five times, six times, seven times, eight times, nine times, or ten times or more over the course of several days, weeks or months, or every two days, or once, twice, three times, four times, five times, or six times weekly. (Item 146) 146. The method according to any of items 138 to 145, wherein the treatment is administered intravenously (IV), intrathecally or intramuscularly (IM), intraperitoneally (IP), intrapleurally, intraarticularly, or injected or implanted at or near the site of the cancer or infection. (Item 147) The unit dosage is about 10 cells per kilogram of the subject's body weight. 4 Approximately 10 cells 10 Between 10 and 10 cells per subject 6 Approximately 10 cells 12 147. The method according to any one of items 144 to 146, comprising between 1 and 147. (Item 148) The unit dosage is about 10 cells per subject. 10 or about 10 cells per kilogram of the subject's body weight. 8 Item 148. The method according to item 147, wherein (Item 149) 149. The method according to any of items 138 to 148, wherein the treatment is for cancer. (Item 150) 150. The method of claim 149, wherein the cancer is selected from lung cancer, melanoma, breast cancer, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, neuroblastoma, rhabdomyosarcoma, leukemia or lymphoma, Hodgkin's lymphoma or childhood acute lymphoblastic leukemia, non-Hodgkin's lymphoma, mast cell tumor or mast cell tumor, ovarian cancer or carcinoma, pancreatic cancer, non-small cell lung cancer, small cell lung cancer, hepatocellular carcinoma, retinoblastoma, breast tumor, colorectal cancer, leukemia, lymphoma, acute lymphoblastic leukemia (ALL) or acute lymphocytic leukemia, acute myeloid leukemia (AML), histiocytic sarcoma, brain tumor, astrocytoma, glioblastoma, neuroma, colon cancer, cervical cancer, sarcoma, bladder tumor, tumor of reticuloendothelial tissue, Wilms' tumor, bone cancer, osteosarcoma, kidney cancer, or head and neck cancer, oral cavity cancer, laryngeal cancer, metastatic disease, or oropharyngeal cancer. (Item 151) The method of claim 149 or 150, wherein a second agent is co-administered with the composition, pharmaceutical composition, or kit. (Item 152) 152. The method of claim 151, wherein the second agent is an antibody that immunospecifically binds to a cancer associated antigen. (Item 153) The cancer-associated antigen is alpha-fetoprotein (AFP), alpha-actinin-4, A3, antigen specific for A33 antibody, ART-4, B7, B7-H3, Ba733, BAGE, BrE3 antigen, CA125, CAMEL, CAP-1, carbonic anhydrase IX, CASP-8 / m, CCL19, CCL21, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e , CD67, CD70, CD70L, CD74, CD79a, CD79b, CD80, CD83, CD95, CD123, CD126, CD132, CD133, CD13 8, CD147, CD154, CDC27, CDK-4 / m, CDKN2A, CTLA4, CXCR4, CXCR7, CXCL12, HIF-1α, colon-specific antigen-p(C SAp), CEA (CEACAM-5), CEACAM-6, c-Met, DAM, EGFR, EGFRvIII, EGP-1 (TROP-2), EGP-2, ELF2-M, Ep-CAM, fibroblast growth factor (FGF), Flt-1, Flt-3, folate receptor, G250 antigen, GAGE, gp100, GRO-β, HLA-DR, HM1.24, human chorionic gonadotropin (HCG) and its subunits, HER2 / neu, HMGB-1, hypoxia-inducible factor (HIF-1), HSP70-2M, HST-2, Ia, IGF-1R, IFN-γ, IFN-α, IFN-β, IFN-λ, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15 , IL-17, IL-18, IL-23, IL-25, insulin-like growth factor-1 (IGF-1), KC4 antigen, KS-1 antigen, KS1-4, Le-Y, LDR / FUT, macrophage migration inhibitory factor Child (MIF), GD2, MAGE, MAGE-3, MART-1, MART-2, NY-ESO-1, TRAG-3, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC 2, MUC3, MUC4, MUC5ac, MUC13, MUC16, MUM-1 / 2, MUM-3, NCA66, NCA95, NCA90, pancreatic cancer mucin, PD1 receptor, placental growth factor, p53, PLAGL2, prostatic acid phosphatase, PSA, PRAME, PSMA, PlGF, ILGF, ILGF-R, L-6, IL-25, RS5, RANTES, T101, SAGE, S100, mackerel 153. The method of claim 152, wherein the IL-16 receptor is selected from the group consisting of IL-16, survivin-2B, TAC, TAG-72, tenascin, TRAIL receptor, TNF-α, Tn antigen, Thomson-Friedreich antigen, tumor necrosis antigen, VEGFR, ED-B fibronectin, WT-1, 17-1A antigen, complement factors C3, C3a, C3b, C5a, C5, angiogenesis markers, bcl-2, bcl-6, and Kras. (Item 154) The antibodies are hR1 (anti-IGF-1R), hPAM4 (anti-mucin), KC4 (anti-mucin), hA20 (anti-CD20), hA19 (anti-CD19), hIMMU31 (anti-AFP), hLL1 (anti-CD74). , hLL2 (anti-CD22), anti-CD19 / CD22 bispecific antibody, RFB4 (anti-CD22), hMu-9 (anti-CSAp), hL243 (anti-HLA-DR), hMN-14 (anti-CEACAM-5), hMN -15 (anti-CEACAM-6), hRS7 (anti-TROP-2), hMN-3 (anti-CEACAM-6), CC49 (anti-TAG-72), J591 (anti-PSMA), D2 / B (anti-PSMA), G250 (anti-carbonic anhydrase IX), dinutuximab (anti-GD2), infliximab (anti-TNF-α), certolizumab pegol (anti-TNF-α), adalimumab (anti-TNF-α), alemtuzumab (anti-CD52), Basiliximab (anti-VEGF), cetuximab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomab tiuxetan (anti-CD20), panitumumab (anti-EGFR), rituximab (anti-CD20), tositumomab (anti-CD20), GA101 (anti-CD20), trastuzumab (anti-HER2 / neu), tocilizumab (anti-IL-6 receptor), basiliximab (anti-CD25), daclizumab (anti-CD25) , efalizumab (anti-CD11a), muromonab-CD3 (anti-CD3 receptor), natalizumab (anti-alpha4 integrin), BWA-3 (anti-histone H2A / H4), LG2-1 (anti-histone H3), MRA12 (anti-histone H1), PR1-1 (anti-histone H2B), LG11-2 (anti-histone H2B), and LG2-2 (anti-histone H2B). (Item 155) 149. The method according to any of items 138 to 148, wherein the treatment is for an infection. (Item 156) 156. The method of claim 155, wherein the infectious disease is characterized by the presence of a bacterial, fungal, viral, or protozoan pathogen. (Item 157) The infectious disease may be caused by herpes, Ebola, West Nile virus, vaccinia virus, Epstein-Barr virus, hepatitis A virus (HAV); hepatitis B virus (HBV); hepatitis C virus (HCV); herpes viruses (e.g., HSV-1, HSV-2, HHV-6, CMV), human immunodeficiency virus (HIV), vesicular stomatitis virus (VSV), Bacilli, Citrobacter, Cholera, Diphtheria, Enterobacter, Gonococci, Helicobacter pylori, Klebsiella, Legionella, Meningococci, mycobacteria, Pseudomonas, Pneumonococci, Rickettsia bacteria, Salmonella, Serratia, Staphylococci, Streptococci, Tetanus, Aspergillus (A. fumigatus, A. niger, etc.), Blastomyces dermatitidis, Candida (C.albicans, C.krusei, C.glabrata, C.tropicalis, etc.), Cryptococcus neoformans, Genus Mucorales (mucor, absidia, rhizopus), Sporothrix schenkii, Paracoccidioides brasiliensis, Coccidioides immitis, Histoplasma 157. The method of claim 156, wherein the host is selected from the group consisting of Borrelia capsulatum, Leptospirosis, Borrelia burgdorferi, helminth parasites (hookworms, tapeworms, trematodes, flatworms (e.g., Schistosomia), Giardia lambia, trichinella, Dientamoeba Fragilis, Trypanosoma brucei, Trypanosoma cruzi, or Leishmania donovani. (Item 158) A therapeutic composition of cells comprising: a plurality of gamma delta T cells (γδ); a plurality of natural killer cells (NK); or a combination of γδ cells and NK cells. (Item 159) the γδ and / or NK cells are recombinantly engineered or genetically modified; Optionally, the γδ and / or NK cells are recombinantly engineered or genetically modified to extracellularly express exogenous or heterologous proteins; Optionally, the exogenous, heterologous, or chimeric protein is a chimeric antigen receptor (CAR) or an exogenous or heterologous T cell receptor (TCR); Optionally, the exogenous, heterologous, or chimeric protein or CAR is specific (or can specifically bind) to a cancer cell or a tumor marker or an infected cell, or the exogenous, heterologous, or chimeric protein or CAR is specific (or can specifically bind) to an antibody that can specifically target and bind to a cancer cell or a tumor marker or an infected cell or any disease-associated antigen. (Item 160) The therapeutic composition of cells described in item 158 or item 159, wherein the γδ and / or NK cells are human or animal cells. (Item 161) The therapeutic composition may be formulated for intravenous (IV), intrathecal, or intramuscular (IM) administration, or may be formulated in a unit dosage form, optionally with the unit dosage being about 10 cells. 2 From 10 cells 12 161. A therapeutic composition of the cells according to any of items 158 to 160, including between 1 and 2. (Item 162) 162. The therapeutic composition of cells according to any of items 158-161, wherein said γδ and / or NK cells are isolated from an in vivo source. (Item 163) 163. The therapeutic composition of cells according to any of items 158-162, wherein said γδ and / or NK cells are expanded in culture or isolated from an in vivo source and expanded in culture. (Item 164) The therapeutic composition of cells according to paragraph 163, wherein said γδ and / or NK cells are isolated from an in vivo source and expanded in culture without the use of any feeder cells or a feeder cell layer, thereby generating a population of expanded γδ and / or NK cells that are feeder cell devoid. (Item 165) The therapeutic composition of cells according to paragraph 163, wherein said γδ and / or NK cells are isolated from an in vivo source and expanded in culture using feeder cells or a feeder cell layer, and optionally said feeder cells are substantially removed and / or killed to generate an expanded population of γδ and / or NK cells substantially devoid of feeder cells. (Item 166) 166. The therapeutic composition of cells according to any of items 158 to 165, wherein the in vivo source of said γδ and / or NK cells is derived from an autologous source (optionally from the individual who will be the recipient of said γδ and / or NK cells), or from an exogenous, xenogeneic, or allogeneic source. (Item 167) 1. A method of treating a cancer, a tumor, a dysfunctional cell, or an infected cell, comprising: (a) administering to an individual in need thereof a therapeutically effective amount of a therapeutic composition of cells according to any of the preceding paragraphs; or (b)(i) providing or having provided a therapeutic composition of cells according to any of the preceding paragraphs; and (ii) administering or having administered to an individual in need thereof a therapeutically effective amount of a therapeutic composition of said cells. The method includes: (Item 168) 168. The method of item 167, wherein the individual in need thereof is a human or an animal. (Item 169) The method of claim 167 or claim 168, wherein the γδ and / or NK cells are isolated from an in vivo source, optionally wherein the in vivo source of the γδ and / or NK cells is derived from a syngeneic or autologous source (optionally from the individual who will be the recipient of the γδ and / or NK cells), or from an exogenous, xenogeneic, or allogeneic source, or a combination thereof. (Item 170) 169. The method of any of items 167-169, wherein the cellular therapeutic composition is administered intravenously (IV), intrathecally, or intramuscularly (IM) or injected or implanted into or near the cancer, tumor, dysfunction, or infected cell, optionally wherein the cellular therapeutic composition is delivered as an implant or gel, optionally wherein the gel is a hydrogel. (Item 171) The therapeutic composition of cells is administered in a unit dosage form, optionally with the unit dosage being about 10 cells. 2 From 10 cells 12 Between 10 or 10 cells 4 From 10 cells 10 or a daily dose of about 10 cells 2 From 10 cells 12 Between 10 or 10 cells 4 From 10 cells 10 171. The method according to any one of items 167 to 170, including between 1 and 170. (Item 172) 172. The method of any of items 167-171, wherein the therapeutic composition or unit dosage form of the cells is administered to the individual in need thereof several times (multiple times), or 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times over the course of several days, weeks, or months, optionally wherein each of the multiple unit dosage forms is administered daily, every other day, twice, three, four, five, or six times per week, or once per week. (Item 173) The method according to any one of items 167 to 172, wherein the cancer or tumor is lung cancer, melanoma, breast cancer, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, neuroblastoma, rhabdomyosarcoma, leukemia or lymphoma, Hodgkin's lymphoma or childhood acute lymphoblastic leukemia, mast cell tumor or mast cell tumor, ovarian cancer or carcinoma, pancreatic cancer, non-small cell lung cancer, small cell lung cancer, hepatocellular carcinoma, retinoblastoma, breast tumor, colorectal cancer, leukemia, lymphoma, acute lymphoblastic leukemia (ALL) or acute lymphocytic leukemia, acute myeloid leukemia (AML), histiocytic sarcoma, brain tumor, astrocytoma, glioblastoma, neuroma, colon cancer, cervical cancer, sarcoma, bladder tumor, tumor of reticuloendothelial tissue, Wilms' tumor, bone cancer, osteosarcoma, kidney cancer, or head and neck cancer, oral cavity cancer, laryngeal cancer, or oropharyngeal cancer. (Item 174) 174. The method of any of items 167-173, wherein said individual in need thereof is induced to mount an immune response against cancer, infection, or disease by first pre-administering to said individual in need thereof an unconjugated antibody against a cancer-associated or tumor-associated, infection-associated or disease-associated antigen, followed by administration of a therapeutic composition of cells according to any of the preceding items, at least a portion of said therapeutic composition of cells expressing on its cell surface a polypeptide, optionally a CAR, that specifically binds to said unconjugated antibody. (Item 175) said individual in need thereof is administered an antibody capable of specifically binding to a cancer-associated or tumor-associated, infection-associated or disease-associated antigen, followed by a therapeutic composition of cells according to any of the preceding paragraphs, wherein at least a portion of said therapeutic composition of cells expresses on its cell surface a polypeptide that specifically binds to said antibody, optionally a CAR; 175. The method according to any of items 167 to 174, optionally wherein the antibody is administered prior to, together with, or after administration of the therapeutic composition of the cells. (Item 176) The cancer-associated or tumor-associated antigen is alpha-fetoprotein (AFP), alpha-actinin-4, A3, antigen specific for the A33 antibody, ART-4, B7, B7-H3, Ba733, BAGE, BrE3 antigen, CA125, CAMEL, CAP-1, carbonic anhydrase IX, CASP-8 / m, CCL19, CCL21, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66 a-e, CD67, CD70, CD70L, CD74, CD79a, CD79b, CD80, CD83, CD95, CD123, CD126, CD132, CD133, C D138, CD147, CD154, CDC27, CDK-4 / m, CDKN2A, CTLA4, CXCR4, CXCR7, CXCL12, HIF-1α, colon-specific antigen-p (CSAp), CEA(CEACAM-5), CEACAM-6, c-Met, DAM, EGFR, EGFRvIII, EGP-1(TROP-2), EGP-2, ELF2 -M, Ep-CAM, fibroblast growth factor (FGF), Flt-1, Flt-3, folate receptor, G250 antigen, GAGE, gp100, GRO-β, HLA-DR, HM1.24, human chorionic gonadotropin (HCG) and its subunits, HER2 / neu, HMGB-1, hypoxia-inducible factor (HIF-1), HSP70-2M, HST-2, Ia, IGF-1R, IFN-γ, IFN-α, IFN-β, IFN-λ, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15, IL-17, IL-1 8, IL-23, IL-25, insulin-like growth factor-1 (IGF-1), KC4 antigen, KS-1 antigen, KS1-4, Le-Y, LDR / FUT, macrophage migration inhibitory factor (MIF), GD2 (disialoganglioside expressed in tumors of neuroectodermal origin), MAGE, MAGE-3, MART-1, MART-2, NY-ESO-1, TRAG-3, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, M UC1, MUC2, MUC3, MUC4, MUC5ac, MUC13, MUC16, MUM-1 / 2, MUM-3, NCA66, NCA95, NCA90, pancreatic cancer mucin, PD1 receptor, placental growth factor, p53, PLAGL2, prostatic acid phosphatase, PSA, PRAME, PSMA, PlGF, ILGF, ILGF-R, L-6, IL-25, RS5, RANTES, T101, SAGE, S100, survivin , survivin-2B, TAC, TAG-72, tenascin, TRAIL receptor, TNF-α, Tn antigen, Thomson-Friedreich antigen, tumor necrosis antigen, VEGFR, ED-B fibronectin, WT-1, 17-1A antigen, complement factors C3, C3a, C3b, C5a, C5, angiogenesis markers, bcl-2, bcl-6, and Kras. (Item 177) The antibody or the unconjugated antibody is selected from the group consisting of hR1 (anti-IGF-1R), hPAM4 (anti-mucin), KC4 (anti-mucin), hA20 (anti-CD20), hA19 (anti-CD19), hIMMU31 (anti-AFP), hLL1 (anti-CD74), hLL2 (anti-CD22), RFB4 (anti-CD22), hMu-9 (anti-CSAp), hL243 (anti-HLA-DR), hMN-14 (anti-CEACAM-5), hM N-15 (anti-CEACAM-6), hRS7 (anti-TROP-2), hMN-3 (anti-CEACAM-6), CC49 (anti-TAG-72), J591 (anti-PSMA), D2 / B (anti-PSMA), G250 (anti-carbonic anhydrase IX), infliximab (anti-TNF-α), certolizumab pegol (anti-TNF-α), adalimumab (anti-TNF-α), alemtuzumab (anti-CD52), bevacizumab (anti-VEGF) F), cetuximab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomab tiuxetan (anti-CD20), panitumumab (anti-EGFR), rituximab (anti-CD20), tositumomab (anti-CD20), GA101 (anti-CD20), trastuzumab (anti-HER2 / neu), tocilizumab (anti-IL-6 receptor), basiliximab (anti-CD25), daclizumab (anti-CD25), efalizumab 176. The method of claim 174 or 175, wherein the antibody is selected from the group consisting of: muromonab-CD3 (anti-CD11a), muromonab-CD3 (anti-CD3 receptor), natalizumab (anti-alpha4 integrin), BWA-3 (anti-histone H2A / H4), LG2-1 (anti-histone H3), MRA12 (anti-histone H1), PR1-1 (anti-histone H2B), LG11-2 (anti-histone H2B), and LG2-2 (anti-histone H2B). [Brief description of the drawings]

[0061] [Figure 1] FIG. 1 shows the expansion of αβTCRneg mononuclear cells as a function of the number of days the cells were activated with antibody and expanded in feeder-free culture conditions, analyzed on day 10 of culture. [Diagram 2] FIG. 2 shows an analysis of the composition of activated and expanded αβTCR neg mononuclear cell populations (herein referred to as INNATE-K or BINATE cell populations). [Diagram 3] FIG. 3 shows the frequency and distribution of T cell lineage markers, CD4 (helper) and CD8 (cytotoxic), on γδ TCR+ cells in a day 10 sample of activated αβ TCRneg cells expanded in feeder-free culture as analyzed by flow cytometry. [Figure 4] FIG. 4 shows an analysis of the expansion kinetics of CAR.19 INNATE-K cells over a 10 day period. [Diagram 5] FIG. 5 shows data from a co-culture cytotoxicity assay in which INNATE-K and INNATE-CAR.19 cells were co-cultured with CD19+ leukemia (221) or CD19+ lymphoma (Daudi) cell lines. [Figure 6] FIG. 6 shows the total number of expanded INNATE-NK cell populations cultured in vitro over a long period of time in plates. [Figure 7] FIG. 7 shows a comparison of total cell numbers between plate and bioreactor expanded INNATE-NK populations. [Figure 8] FIG. 8 shows subset cell composition at different time points of INNATE-NK expansion in vitro. [Figure 9] FIG. 9 shows chimeric antigen receptor (CAR) molecule expression in INNATE-NK CAR cells during long-term in vitro culture. [Figure 10] FIG. 10 shows expression of activation and cytolytic molecules following expansion of INNATE-NK and INNATE-NK CAR populations. [Figure 11] FIG. 11 shows lack of exhaustion in feeder-free expanded INNATE-NK and INNATE-NK CAR cells. [Figure 12]Figure 12 shows a cytotoxicity co-culture assay with INNATE-NK or INNATE-NK-CAR.19 and four tumor cell lines: Column A: 221, a CD19+ leukemia cell line; Column B: Daudi, a CD19+ lymphoma cell line; Column C: BV173, a CD19+ (variably expressing) pre-B tumor cell line; and Column D: KARPAS, a CD19- tumor cell line. [Figure 13] Figures 13 and 14 show % specific lysis of primary tumor cells as a function of effector (E) to target (T) ratio for both INNATE-NK and INNATE-NK-CAR.19 cell populations in various test runs. [Figure 14] Figures 13 and 14 show % specific lysis of primary tumor cells as a function of effector (E) to target (T) ratio for both INNATE-NK and INNATE-NK-CAR.19 cell populations in various test runs. [Figure 15] FIG. 15 shows the percentage remaining primary CD19+ tumor after co-culture with effector cells (INNATE-NK and INNATE-NK-CAR.19 cell populations) compared to control conditions where primary CD19+ leukemic blasts were plated in the absence of effector cells. [Figure 16] FIG. 16 shows survival curves for animals administered INNATE-NK and INNATE-NK-CAR.19 cells. [Figure 17] FIG. 17 shows total BINATE expansion expressed by total cell number over time (i.e., expansion of activated αβTCRneg cells in BINATE medium supplemented with either IL-2 or IL-15 in flasks and expansion of activated αβTCRneg cell populations with IL-15 in bioreactors). [Figure 18]FIG. 18 shows total BINATE expansion expressed by total cell number over time (i.e., expansion of activated αβTCR cells in flasks in BINATE medium supplemented with either IL-2, IL-15, IL-2 / OKT3, IL-2 / IL-15, or IL-2 / IL-15 / OKT3). [Figure 19] FIG. 19 shows phenotypic analysis by marker type (activated, cytotoxic, and exhausted / immature) for IL-15 expansion conditions. [Figure 20] FIG. 20 shows phenotypic analysis by marker type (activated, cytotoxic, and exhausted / immature) for IL-2 expansion conditions. [Figure 21] FIG. 21 shows phenotypic analysis by marker type (activated, cytotoxic, and exhausted / immature) for the IL-2 / OKT3 expansion condition. [Figure 22] FIG. 22 shows cell numbers recorded approximately every 7 days for the BINATE and BINATE.CARGD2 populations. [Figure 23] FIG. 23 shows the in vivo maintenance of human BINATE cells in both mouse blood and mouse liver. [Figure 24] FIG. 24 shows a flow chart illustrating an example variation of a process for isolating a pure NK cell population and / or for isolating a pure γδ T(gd) cell population. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0062] In certain embodiments, provided herein are therapeutic immune cell compositions in which a significant number of immune cells or a majority of immune cells are innate immune cells. Such compositions can generate an innate immune response in a subject after administration. The innate immune system is active and constantly functional in the host, responds quickly (within minutes to hours after infection), and responds non-specifically, offering the potential to be used as an "off-the-shelf" immunotherapy for a broader patient population. Innate immune cells, such as NK cells and gamma delta (γδ) T cells, do not recognize classical HLA antigens and can therefore be used in off-the-shelf (allogeneic) situations while mitigating graft-versus-host disease (GvHD). Innate immune cells can be expanded ex vivo, thereby avoiding cytokine release syndrome (CRS). Innate immune cell compositions in which the components (e.g., NK cells, gamma delta (γδ) T cells) and amounts of the components can be adjusted can be utilized to provide a series of synergistic attacks against a variety of cancers, including solid tumors and hematological cancers, infectious diseases, and the like.

[0063] In contrast, certain immune cell compositions not generated by the methods described herein contain a majority of adaptive immune cells instead of innate immune cells. When utilizing immune cell compositions in which the majority of cells are adaptive immune cells, recognition is through HLA antigens and there is a significant risk of GvHD, especially when the adaptive immune cells used in immunotherapy are derived from an allogeneic source. Thus, the adaptive immune cells utilized in such compositions are most often derived from the patient / subject (autologous) or from a designated "matched" donor, which limits the immunotherapy repertoire, i.e., the ability to use it in an "off-the-shelf" manner in large patient populations. Most cell therapies utilizing such compositions are autologous or derived from the least specific donor. This means that each patient's dose must be individually tailored to that patient. Thus, current T cell-based therapeutic compositions, such as cell therapies using chimeric antigen receptors (CARs) or exogenous T cell receptors (TCRs) (as αβ T cells), cannot be used "off-the-shelf". The manufacturing process can take six weeks or longer, during which time the patient's disease, e.g., cancer or infection, can progress. Similarly, some of these patient-specific manufacturing runs have failed for a variety of reasons, including general manufacturing failures, but also specific failures due to the exhausted state of the patient-donor immune system after chemotherapy or radiation therapy. In addition, in vivo expansion of αβ T cells (or CAR-modified, "CAR-T" cells) sometimes causes a rapid release of large amounts of cytokines into the blood, resulting in CRS that can be severe or life-threatening.

[0064] Thus, the therapeutic immune cell compositions described herein provide advantages over cell compositions that contain a majority of adaptive immune cells. The therapeutic immune cell compositions described herein are also modified or altered compared to naturally occurring biological samples obtained from a subject. The therapeutic immune cell compositions, methods of manufacture, and therapeutic uses are described in detail below.

[0065] Methods for Producing Innate Immune Cell Compositions Provided herein are methods for producing innate immune cell compositions. The compositions produced by the methods provided herein often contain a mixture of two activated innate immune cell populations: natural killer (NK) cells and gamma delta T cells.

[0066] The immune cell composition obtained by the method provided herein is enriched for the innate immune cells NK cells and gamma delta T cells, as compared to compositions of immune cells in nature (e.g., biological fluids and tissues). In nature, the adaptive immune cells alpha beta T cells are found in much greater amounts than NK cells and gamma delta T cells. In contrast, in the compositions provided herein, alpha beta T cells are absent or present in negligible to low amounts, and NK cells and gamma delta T cells are the major immune cell components.

[0067] For example, in human peripheral blood, it has been found that in peripheral blood of healthy human donors, (i) for individuals under 40 years of age, the median percentage of NK cells in PBMCs is 5% and the median percentage of T cells (CD8+ and CD4+ populations combined, which represents alpha-beta T cells since gamma-delta T cells are typically CD8-CD4-) is 53%; and (ii) for individuals over 40 years of age, the median percentage of NK cells in PBMCs is 10% and the median percentage of T cells (CD8+ and CD4+ populations combined) is 37% (Lepone et al., J. Circ. Biomark., 5(5):1-17 (2016)). Thus, peripheral In the blood, in individuals over 40 years of age, there are approximately 4 circulating alpha.beta T cells for every circulating NK cell, and in individuals under 40 years of age, there are approximately 10 circulating alpha.beta T cells for every circulating NK cell. Thus, the ratio of circulating NK cells to alpha.beta T cells ranges from about 1:4 to about 1:10. In contrast, in the compositions provided herein, NK cells comprise 20% or more, up to 99%, of the composition, while alpha.beta T cells are virtually absent and comprise less than 2% of the composition, and generally comprise approximately 0% or less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1% of the composition. Thus, in the compositions provided herein, the ratio of NK cells to alpha.beta T cells is at least 10:1, and generally is significantly greater than 10:1, such as about 15:1 or greater, about 20:1 or greater, about 25:1 or greater, about 30:1 or greater, about 35:1 or greater, about 40:1 or greater, about 45:1 or greater, about 50:1 or greater, about 55:1 or greater, about 60:1 or greater, about 65:1 or greater, about 70:1 or greater, about 75:1 or greater, about 80:1 or greater, about 85:1 or greater, about 90:1 or greater, about 95:1 or greater, about 100:1 or greater, about 10 ... :1 or higher, about 100:1 or higher, about 150:1 or higher, about 200:1 or higher, about 250:1 or higher, about 300:1 or higher, about 350:1 or higher, about 400:1 or higher, about 450:1 or higher, about 500:1 or higher, about 550:1 or higher, about 600:1 or higher, about 650:1 or higher, about 700:1 or higher, about 750:1 or higher, about 800:1 or higher, about 850:1 or higher, about 900:1 or higher, about 950:1 or higher, or about 1000:1 or higher.Thus, while alpha.beta T cells are naturally abundant relative to NK cells, in the compositions provided herein the NK cell population is greatly expanded relative to the alpha.beta T cell population.

[0068] For example, with regard to gamma delta T cells in peripheral blood, less than about 10% of T cells, typically about 5% of T cells, are gamma delta T cells, while the rest are alpha beta T cells (Esin et al., Scand. J. Immunol., 43(5):593-596 (1996); Radestad et al., J. Immunol. Res., Article ID 578741 (2014)). Thus, circulating The ratio of gamma.delta T cells to alpha.beta T cells ranges from about 1:10 to about 1:20. Meanwhile, in the compositions provided herein, gamma.delta T cells comprise 1% or more of the composition, typically comprise between 2% or more and up to 70-75% of the composition, while alpha.beta T cells are practically absent and comprise less than 2% of the composition, typically comprise approximately 0% or less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1% of the composition. Thus, in the compositions provided herein, even assuming a composition in which gamma.delta T cells are present at 1% and alpha.beta T cells are present at 2%, the ratio of gamma.delta T cells to alpha.beta T cells is at least 0.5:1. Generally, the ratio of gamma.delta T cells to alpha.beta T cells in the compositions provided herein is fairly high, e.g., about 5:1 or higher, about 10:1 or higher, about 15:1 or higher, about 20:1 or higher, about 25:1 or higher, about 30:1 or higher, about 35:1 or higher, about 40:1 or higher, about 45:1 or higher, about 50:1 or higher, about 55:1 or higher, about 60:1 or higher, about 65:1 or higher, about 70:1 or higher, about 75:1 or higher, about 80:1 or higher, about 85:1 or higher, about 90:1 or higher, about 95:1 or higher, about 100:1 or higher, about 10 ... or higher, about 85:1 or higher, about 90:1 or higher, about 95:1 or higher, about 100:1 or higher, about 150:1 or higher, about 200:1 or higher, about 250:1 or higher, about 300:1 or higher, about 350:1 or higher, about 400:1 or higher, about 450:1 or higher, about 500:1 or higher, about 550:1 or higher, about 600:1 or higher, about 650:1 or higher, about 700:1 or higher, or about 750:1 or higher, or higher.

[0069] As used herein, the term "enriched" means that the following two ratios in the compositions provided herein: (i) the ratio of gamma.delta T cells to alpha.beta T cells, and (ii) the ratio of NK cells to alpha.beta T cells, are higher than these ratios in a biological sample, such as naturally occurring peripheral blood. As generally used herein, "enriched" means that the ratio of gamma.delta T cells to alpha.beta T cells in the compositions provided herein is increased by at least 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold, 150-fold, 200-fold, 250-fold, 300-fold, 350-fold, 400-fold, 450-fold, 500-fold, 550-fold, 600-fold, 650-fold, 700-fold, or 750-fold or more as compared to the ratio in a biological sample, e.g., tissue, umbilical cord blood, or peripheral blood. In the case of NK cells, as generally used herein, "enriched" means that the ratio of NK cells to alpha.beta T cells in the compositions provided herein is increased by at least 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold, 150-fold, 200-fold, 250-fold, 300-fold, 350-fold, 400-fold, 450-fold, 500-fold, 550-fold, 600-fold, 650-fold, 700-fold, 750-fold, 800-fold, 850-fold, 900-fold, 950-fold, or 1000-fold or more, as compared to the ratio in a biological sample, e.g., tissue, umbilical cord blood, or peripheral blood. In certain embodiments, the term "enriched" means that the compositions provided herein have a ratio of NK cells to alpha.beta T cells and a ratio of gamma.delta T cells to alpha.beta T cells that is greater than 1 (which ratio is generally less than 1 in nature).

[0070] In peripheral blood, adaptive immune cells B cells and alpha.beta T cells constitute the majority of lymphocytes. Meanwhile, the composition produced by the method provided herein is enriched for innate immune cells, namely NK cells and gamma.delta T cells. The terms alpha.beta T cells and αβ T cells are used interchangeably herein to refer to adaptive immune T cells, while the terms gamma.delta T cells and γδ T cells are used interchangeably herein to refer to innate immune T cells.

[0071] Immunotherapeutic compositions comprised of innate immune cells are advantageous compared to compositions containing adaptive immune cells for a variety of reasons. Innate immune responses are non-specific and are initiated against a target of interest (e.g., cancer or infectious disease) within minutes to hours after infection. Innate immune cells can be expanded ex vivo. In the methods provided herein, the expansion conditions can result in compositions of NK cells and gamma delta T cells that are not exhausted for as long as 60 days after expansion, allowing for long-term storage (suitable for inventory storage and re-administration if necessary) and allowing multiple doses to be administered cost-effectively. On the other hand, adaptive immune cells, upon binding to a target antigen in vivo, initiate and expand an antigen-specific immune response, which can take days or even weeks. Furthermore, unlike innate immunity, adaptive immune responses are dependent on HLA antigens, and therefore the cells must be autologous or "matched" to the recipient patient / subject in order to minimize or avoid GvHD (graft-versus-host disease). Compositions containing innate immune cells, such as NK cells and gamma delta (γδ) T cells in compositions made by the methods provided herein do not require HLA antigen recognition to mediate killing and therefore can be used in a broader range of off-the-shelf (allogeneic) settings while avoiding GvHD. Adaptive immune cells can also produce large amounts of cytokines when expanded in vivo, which can lead to cytokine release syndrome (CRS), whereas innate immune cells can be expanded ex vivo, thereby minimizing or avoiding CRS.

[0072] In the methods provided herein, a sample containing cells, for example peripheral blood or umbilical cord blood, is obtained from a subject. The subject is generally a healthy donor, but may also be a patient who needs treatment with the immunotherapy composition produced by the methods provided herein. In certain embodiments, when the sample is peripheral blood, a universal donor bank of peripheral blood can be used as the source of the sample. The sample is sometimes subjected to activation conditions, which include contacting the sample with (a) at least one exogenous polypeptide that immunospecifically binds to cell adhesion polypeptide, and (b) at least one exogenous polypeptide that immunospecifically binds to a polypeptide different from the cell adhesion polypeptide and is expressed on the surface of one or more cells of the sample population. Examples of cell adhesion polypeptides include, but are not limited to, CD2 (see, e.g., GenBank Accession Nos. KJ905161.1; KJ896558.1), LFA-1 (see, e.g., GenBank Accession No. BC005861.2), LFA-3 (see, e.g., GenBank Accession No. BC005930.1), CD8 (see, e.g., GenBank Accession Nos. AH003215.2; AY039664.1 for CD8A; see, e.g., GenBank Accession Nos. KJ896562.1; BC100911.1; BC100912.2; BC100913.1; BC100914.1 for CD8B), and CD4 (see, e.g., GenBank Accession Nos. M35160.1; DQ892052.2).Examples of polypeptides expressed on the surface of one or more cells of the sample population, e.g., NK cells and / or gamma delta cells, include CD2 (see, e.g., GenBank Accession Nos. KJ905161.1; KJ896558.1), CD3 (see, e.g., GenBank Accession No. AB583162.1 for CD3γ; see, e.g., GenBank Accession No. AB583139.1 for CD3ε; see, e.g., GenBank Accession No. AH002612.2 for CD3δ), CD 56 (see, e.g., GenBank Accession Nos. U63041.1; BC047244.1; BC029119.1), NKp30 (see, e.g., GenBank Accession No. AB055881.1), NKp44 (see, e.g., GenBank Accession No. BC166647.1), NKp46 (see, e.g., GenBank Accession Nos. BC064806.1; AY346373.1), NKG2A (see, e.g., GenBank Accession Nos. AF461812.1; BC053840.1), PD-1 ( See, e.g., GenBank Accession No. L27440.1), NKG2C (see, e.g., GenBank Accession Nos. BC093644.1; BC112039.1), NKG2D (see, e.g., GenBank Accession Nos. AF461811.1; BC039836.1), KAR receptors, KIR receptors, SIGLEC-7 (see, e.g., GenBank Accession Nos. AF193441.1; AF170485.1), KIR3DS1 (see, e.g., GenBank Accession No. EU156175.1). , KIR2DL1 (see, e.g., GenBank Accession Nos. LT984790.1; LT984791.1; Antibody: 11PB6), DNAM1 (see, e.g., GenBank Accession Nos. BC074787.2; U56102.1), NTBA (see, e.g., GenBank Accession Nos. BC114495.1; BC113893.1), HLA-DR (see, e.g., GenBank Accession Nos. AH001506.2 for alpha; AH002824.2 for beta), and the like.Activation of the cells generally initiates an innate immune response in the cells of the compositions provided herein, which can be used to target a disease, such as cancer or an infectious disease, in a subject in need of treatment of such a disease.

[0073] A polypeptide immunospecifically binds to a region (i.e., epitope) of another molecule if it reacts or associates with that epitope more frequently, more rapidly, for a longer period, and / or with a higher affinity than an alternative epitope. For example, a polypeptide (e.g., an antibody or an antigen-binding fragment thereof) that immunospecifically binds to a first epitope is an antibody that binds to this first epitope with a higher affinity, avidity, more readily, and / or for a longer period than it binds to a second epitope or other epitopes. A polypeptide that immunospecifically binds to a first target may or may not specifically or preferentially bind to a second target, and immunospecific binding is not necessarily exclusive binding.

[0074] In certain embodiments of the methods provided herein, the activating conditions include contacting the sample with at least two exogenous polypeptides. In some embodiments, the first exogenous polypeptide immunospecifically binds to CD2 and the second exogenous polypeptide immunospecifically binds to NKp46. In some embodiments, the activating conditions consist of or essentially consist of a first exogenous polypeptide that immunospecifically binds to CD2 and a second exogenous polypeptide that immunospecifically binds to NKp46. The phrase "essentially consist of" as used herein means that the presence of components other than the recited components does not substantially alter the activity of the recited components. Thus, for example, in the context of the activating conditions described above, the activating conditions may include one or more components that do not substantially alter the activity of the first exogenous polypeptide and the second exogenous polypeptide other than the first exogenous polypeptide that immunospecifically binds to CD2 and the second exogenous polypeptide that immunospecifically binds to NKp46. In certain embodiments, the first exogenous polypeptide and / or the second exogenous polypeptide is an antibody or an antigen-binding fragment thereof.

[0075] Also, the sample is often subjected to expansion conditions that include contacting the sample with at least one auxiliary polypeptide, thereby generating a composition that includes a population of cells enriched for NK cells and gamma.delta T cells. In certain embodiments of the methods provided herein, the expansion conditions include at least one auxiliary polypeptide that is a cytokine and / or a polypeptide that immunospecifically binds to a receptor on gamma.delta T cells. In certain embodiments, the cytokine is an interleukin, such as IL-1, IL-2, IL-4, IL-7, IL-9, IL-15, IL-21, or any combination thereof. In some embodiments of the methods provided herein, the cytokine is IL-2, IL-15, or any combination thereof. In some embodiments, the expansion conditions include, consist of, or consist essentially of IL-2, IL-15, and a polypeptide that immunospecifically binds to a receptor on gamma.delta T cells. In certain embodiments, the receptor on gamma.delta T cells is CD3. In some embodiments, the polypeptide that immunospecifically binds to CD3 receptor on gamma.delta T cells is an antibody or antigen-binding fragment thereof, and in certain embodiments, the antibody is OKT3. In certain embodiments, the expansion conditions comprise, consist of, or consist essentially of contacting the sample with: (a) an IL-2 polypeptide; (b) an IL-15 polypeptide; (c) an IL-2 polypeptide and an IL-15 polypeptide; (d) an antibody that immunospecifically binds to an IL-2 polypeptide and CD3; or (e) an antibody that immunospecifically binds to an IL-2 polypeptide, an IL-15 polypeptide, and CD3. In some embodiments, the antibody that immunospecifically binds to CD3 is OKT3.

[0076] In some embodiments, the activation conditions consist of, or consist essentially of, contacting the sample with a first exogenous polypeptide that immunospecifically binds CD2 and a second exogenous polypeptide that immunospecifically binds NKp46, and the expansion conditions consist of, or consist essentially of, contacting the sample with: (a) an IL-2 polypeptide; (b) an IL-15 polypeptide; (c) an IL-2 polypeptide and an IL-15 polypeptide; (d) an IL-2 polypeptide and an antibody that immunospecifically binds CD3; or (e) an IL-2 polypeptide, an IL-15 polypeptide, and an antibody that immunospecifically binds CD3. In some embodiments, the antibody that immunospecifically binds CD3 is OKT3.

[0077] In certain embodiments, the methods provided herein for producing compositions include depleting alpha.beta T cells from a sample prior to activation and expansion, thereby generating a depleted cell population, which is then activated and expanded. For example, alpha.beta T cells can be depleted from a sample using an antibody that immunospecifically binds to an alpha.beta T cell receptor, e.g., alpha.beta TCR. The term "depleted" as used herein means that substantially all of the components to be depleted (e.g., alpha.beta T cells) have been removed from the sample, e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more, up to about 100% of the components to be depleted have been removed from the sample. In general, the term "depleted cell population" as used herein refers to a population of cells derived from a sample after depleting alpha.beta T cells from the sample. In certain embodiments, the sample is further depleted of B cells, for example, using a polypeptide that immunospecifically binds to a B cell receptor, such as CD19.

[0078] For example, if the sample is a peripheral blood sample, the sample can be processed by density gradient centrifugation to separate and / or isolate the buffy coat containing white blood cells, platelets, granulocytes, etc., which can then be subjected to alpha.beta T cell depletion according to the methods provided herein. In certain embodiments, the buffy coat can be further subjected to Ficoll density gradient separation to obtain mononuclear cells (PBMCs), which can then be subjected to alpha.beta T cell depletion in the methods provided herein. In some embodiments, the peripheral blood sample can be subjected to apheresis to separate plasma from the cells (e.g., using a Terumo Optia instrument), and in certain embodiments, the cells are then subjected to alpha.beta T cell depletion in the methods provided herein. Alpha.beta T cell depletion can be performed by methods known to those of skill in the art. In certain embodiments, alpha.beta T cells can be depleted using a Miltenyi LS column. In some embodiments, the apheresis product can be subjected to alpha.beta T cell depletion using a Miltenyi Clinimacs separation device. The depleted cell populations can be cryopreserved (e.g., at minus 70, minus 75, minus 80, minus 85 degrees Celsius or lower) if necessary or desired and can be stored until activation and expansion. Non-limiting examples of culture conditions for activation and expansion include the activation and expansion conditions described in the Examples section herein for cell populations containing NK cells and gamma delta cells, or NK MACS™ medium (#130-107-879 (Miltenyi Biotec, Inc., San Diego, CA, USA) supplemented with 5% AB serum or other media used in the art (e.g., R&D Systems, CellGenix).

[0079] In certain embodiments, the cell population is not exposed to conditions that positively select for NK cells or conditions that positively select for gamma.delta T cells prior to the activation and expansion conditions described herein.

[0080] In certain embodiments of the methods provided herein, the sample or depleted cell population is subjected to activation conditions in which the exogenous polypeptide (a), the exogenous polypeptide (b), or both the exogenous polypeptide (a) and the exogenous polypeptide (b) are soluble. In some embodiments, the soluble exogenous polypeptide (a), the soluble exogenous polypeptide (b), or both the soluble exogenous polypeptides (a) and (b) are antibodies or antigen-binding fragments thereof. The term "soluble" as used herein in reference to a component, e.g., a polypeptide, means that the component is not bound to a solid phase or support and is in a homogeneous single phase or emulsion under culture conditions (e.g., activation conditions, expansion conditions). In some embodiments, the exogenous polypeptide (a) or (b), e.g., an antibody, is bound to a solid phase or support. Examples of solid supports include, but are not limited to, silica, glass (e.g., glass, controlled pore glass (CPG)), nylon, Wang resin, Merrifield resin, Sephadex, Sepharose, cellulose, magnetic beads, Dynabeads, metal surfaces (e.g., steel, gold, silver, aluminum, silicon, and copper), plastic materials (e.g., polyethylene, polypropylene, polyamide, polyester, polyvinylidene fluoride (PVDF)). The solid support can be in any desired form, including, but not limited to, beads, chips, capillaries, plates, membranes, wafers, combs, pins, substantially flat surfaces, arrays of pits or nanoliter wells, and other geometries and shapes known to those of skill in the art. Using a soluble exogenous polypeptide rather than a polypeptide bound to a solid support can reduce steric hindrance (e.g., for GMP manufacturing) while increasing the scalability of the method. In certain embodiments, the first exogenous polypeptide is a soluble anti-CD2 antibody and the second polypeptide is a soluble anti-NKp46 antibody. In certain embodiments, either the anti-CD2 antibody or the anti-NKp46 antibody is bound to a solid support.

[0081] Antibodies, e.g., polyclonal and monoclonal antibodies, can be prepared using standard methods (see, e.g., Kohler et al., Nature 256:495-497 (1975); Kohler et al., Eur. J. Immunol. 6:511-519 (1976); and WO02 / 464). 55). For example, to generate polyclonal antibodies, an immune response against the antigen of interest is elicited in a host animal. Blood is then collected from the host animal and the serum fraction containing the secreted antibodies is separated from the cellular fraction using methods known to those skilled in the art. To generate monoclonal antibodies, animals are immunized by standard methods to generate antibody-secreting somatic cells. These cells are then removed from the immunized animal and fused with myeloma cells. Somatic cells capable of producing antibodies, particularly B cells, can be used to fuse with myeloma cell lines. These somatic cells can be derived from lymph nodes, spleens, and peripheral blood of primed animals. Specialized myeloma cell lines for use in hybridoma-producing fusion techniques have been generated from lymphoid tumors (Kohler and Milstein, Eur. J. Immunol. 6:511-519 (1976); Shulman et al., Nature, 276:269-282 (1978); Volk et al., J. Virol., 42:220-227 (1982). These cell lines have three useful The hybridomas have several unique properties. The first is that they have an enzyme deficiency that renders them unable to grow in selective media that support hybridoma growth, thereby facilitating the selection of fused hybridomas from unfused, similarly self-propagating myeloma cells indefinitely. The second is that they are capable of producing antibodies and are unable to produce endogenous light or heavy immunoglobulin chains. The third property is that they fuse efficiently with other cells. Hybridomas and other methods of producing monoclonal antibodies are well known to those skilled in the art. It is routine to produce antibodies against any polypeptide, for example an antigenic marker on an immune cell population.

[0082] In certain embodiments of the methods provided herein, the activation condition, the expansion condition, or both the activation condition and the expansion condition are feeder cell-free.The term "free" as used herein (e.g., feeder cell-free or feeder cell-free, serum-free, serum-free from non-human animals, exhausted cell-free) means that the condition is substantially free of components (i.e., feeder cells, serum, exhausted cells, or other components mentioned herein), i.e., at least 80%, 85%, 90%, 95%, generally 95% or more, for example, 96%, 97%, 98%, 99% or more, and up to 100% free.In certain embodiments of the methods provided herein, the sample, the activation condition, the expansion condition, or the activation and expansion condition, or all of the above, are free of exogenous cells, free of exogenous feeder cells, free of irradiated cells, and / or free of irradiated feeder cells. Exogenous cells and exogenous feeder cells are generally cells from a different subject or from a different part of a subject, compared to the subject or part of a subject from which the sample cells are obtained and subjected to activation and / or expansion conditions. In a non-limiting example, (i) the sample cells are from a first type of subject, and the exogenous cells are from a second type of subject (e.g., the sample cells are from a human, and the exogenous cells are from a non-human animal, such as a rodent or monkey), and (ii) the sample cells are from the peripheral blood of a human subject, and the exogenous cells are from a different part of the same subject (e.g., from the umbilical cord blood or organ of the same subject, or from a different human subject). A composition is generally "free" of a particular component if the sample is not in contact with the particular component during one or all of the following: processing before activation and / or expansion, activation, and expansion.

[0083] Reliance on feeder cells, such as K562 cells, can limit where and how cells are cultured and can significantly increase the cost of culturing cells. The use of feeder cells can also be problematic due to cell culture variability caused by undefined biological factors derived from the feeder cells. In addition, feeder cells are a barrier against unwanted agents (e.g., retroviruses, other pathogens, and immunogenic non-human sialic acids, e.g., In some cases, the method may introduce an exogenous polypeptide that immunospecifically binds to a cell adhesion polypeptide, such as Neu5Gc, into the composition produced by the method provided herein, which may be disadvantageous for certain applications, such as transplantation. Without wishing to be bound by theory, the method provided herein allows for the generation of an "endogenous feeder cell-like layer" that can generate a significantly activated and expanded cell population without the need for exogenous feeder cells.

[0084] In certain embodiments of the methods provided herein, the selection of the auxiliary polypeptide(s) in the expansion conditions and / or the expansion time can be tailored to the desired relative amount of NK cells and gamma delta T cells. For example, compositions containing relatively more NK cells and relatively fewer gamma delta T cells generally have greater applicability to solid tumors, while compositions containing relatively more gamma delta T cells and relatively fewer NK cells generally have greater applicability to hematological malignancies. The auxiliary polypeptide(s) can be selected and / or the expansion reaction can be performed for a time that facilitates obtaining a composition having a desired ratio of NK cells and gamma delta T cells.The desired ratio of NK cells to gamma delta T cells can be obtained by selection of specific expansion conditions, non-limiting examples of combinations are as follows: (i) when the auxiliary polypeptide is IL-2, the resulting population of cells enriched for NK cells and gamma delta T cells often contains about 25-30% NK cells and about 70-75% gamma delta T cells; (ii) when the auxiliary polypeptide is IL-15, the resulting population of cells enriched for NK cells and gamma delta T cells often contains about 80-99% NK cells and about 1-20% gamma delta T cells; (iii) when the auxiliary polypeptide is IL-2 and an antibody that immunospecifically binds to CD3, e.g., OKT3, the resulting population of NK cells and gamma delta T cells often contains about 25-30% NK cells and about 70-75% gamma delta T cells. A population of cells enriched for gamma.delta T cells often contains about 40-45% NK cells and about 55-60% gamma.delta T cells; (iv) when the supplemental polypeptide is IL-2 until day 20 of the expansion conditions and then switched to IL-15 until day 30, the percentage of gamma.delta T cells often increases from about 50% to about 70%, with a corresponding decrease in the percentage of NK cells, compared to treatment with IL-2 alone; and (v) when the supplemental polypeptide is IL-15 until day 20 of the expansion conditions and then switched to IL-2 until day 30, the percentage of NK cells often increases from about 80% to about 90%, with a corresponding decrease in the percentage of gamma.delta T cells, compared to treatment with IL-15 alone.

[0085] The duration of the expansion conditions (e.g., 12 days vs. 25 days) can also control the relative amount of NK cells and gamma.delta T cells in the compositions produced by the methods provided herein. For example, the length of incubation of the sample or depleted cell population in feeder cell-free medium under expansion conditions can be about 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 25 days, 30 days, 35 days, 40 days, 45 days, 50 days, 55 days, or 60 days or more, or about 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks. In certain embodiments of the methods provided herein, the composition does not include exhausted cells, and in some embodiments, the composition does not include exhausted cells after at least 60 days of expansion conditions. Preparing cell compositions substantially free of exhausted cells allows for immediate treatment and multiple administration of many current immunotherapies (e.g., CAR-T using alpha.beta T cells) at a fraction of the cost due to the increased availability of expanded cytotoxic cell compositions ex vivo.

[0086] In certain embodiments of the methods provided herein, the activation, or expansion, or activation and expansion conditions do not include bisphosphonates, resulting in a polyclonal population of V.delta.1 and V.delta.2 gamma.delta T cells. The use of bisphosphonates, such as zoledronate and pamidronate, may favor one clonal population of gamma.delta T cells over other populations, but this may limit the repertoire of compositions for certain tumors and infectious diseases. In some embodiments, the absence of bisphosphonates in the methods provided herein generates compositions that are polyclonal with respect to gamma.delta T cells, thereby increasing the range of cancers and infectious diseases to which they can be administered as immunotherapy.

[0087] A non-limiting example outline of steps for carrying out the methods provided herein is as follows: (1) A sample from a donor (e.g., peripheral blood directly from a donor or from a blood bank) is thawed and cultured as needed; (2) if desired, the culture is subjected to alpha.beta T cell depletion, and optionally B cell depletion; (3) the sample or the cells remaining after depletion (the depleted cell population) are subjected to activation conditions for 3-4 days to generate activated cells; (4) if desired, the activated cells are transduced with an exogenous polynucleotide, for example using a retroviral or lentiviral vector; (5) after activation in (3) or transduction in (4), the cells are subjected to a 7 day cycle of expansion conditions, washing between cycles, typically for 2-3 cycles, thereby generating expanded cells, which are sometimes used immediately or sometimes refrigerated, kept on ice, or frozen for transport and / or storage until needed for immunotherapy. The ratio of NK cells and gamma delta T cells can be adjusted by the choice of supplemental polypeptide(s) used during expansion, including switching supplemental polypeptides during a 7 day cycle, if desired, and by controlling the expansion time.

[0088] In certain embodiments, the composition resulting from the sample cell population exposed to activation and expansion conditions has the following characteristics: (i) a ratio of greater than 1 (e.g., about 1.5 or greater, 2 or greater, 3 or greater, 4 or greater, or 5 or greater; about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.2, a ratio of NK cells to gamma delta T cells of 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100, or greater; or (ii) a ratio of NK cells to gamma-delta T cells of less than 1 (e.g., a ratio of about 0.8 or less, 0.7 or less, 0.6 or less, 0.5 or less, 0.1 or less, 0.05 or less, 0.01 or less; a ratio of about 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01, 0.009, 0.008, 0.007, 0.006, 0.005, 0.004, 0.003, 0.002, 0.001 or less); and optionally have the following characteristics: (iii) a ratio of NK cells to alpha.beta T cells greater than 2, greater than 3, greater than 4, greater than 5, greater than 6, greater than 7, greater than 8, greater than 9, greater than 10, greater than 20, greater than 30, greater than 40, greater than 50, greater than 60, greater than 70, greater than 80, greater than 90, greater than 100, greater than 200, greater than 300, greater than 400, greater than 500, greater than 600, greater than 700, greater than 800, greater than 900, greater than 1000, or greater; (iv) a ratio of gamma-delta T cells to alpha-beta T cells greater than 2, greater than 3, greater than 4, greater than 5, greater than 6, greater than 7, greater than 8, greater than 9, greater than 10, greater than 20, greater than 30, greater than 40, greater than 50, greater than 60, greater than 70, greater than 80, greater than 90, greater than 100, greater than 200, greater than 300, greater than 400, greater than 500, greater than 600, greater than 700, greater than 800, greater than 900, greater than 1000, or greater; (v) percentage of NK cells to total cells of 20% or greater; (vi) a percentage of gamma.delta T cells relative to total cells of 2%, 3%, 4%, 5%, or greater; (vii) about 75% to about 95% of the gamma.delta T cells (e.g., about 80% to about 90% (e.g., about 85%) of the gamma.delta T cells express V.delta.1; (viii) about 10% to about 25% (e.g., about 10% to about 20% (e.g., about 15%) of the gamma.delta T cells express V.delta.2; (ix) about 30% to about 60% of gamma.delta T cells (about 35% to about 55% of gamma.delta T cells) express V.delta.1; (x) about 35% to about 60% of the gamma.delta T cells (e.g., about 40% to about 50% or 55% of the gamma.delta T cells) express V.delta.2; (xi) about 10% to about 30% of gamma.delta T cells (e.g., about 20% to about 25% of gamma.delta T cells) express V.delta.1; (xii) about 65% to about 80% (e.g., about 70% to about 80% (e.g., about 75% of the gamma.delta T cells) express V.delta.2; (xiii) about 80% or more of the total cells (e.g., about 90% or more of the cells) express KIR5; (xiv) about 5% or more of the total cells (e.g., about 10% or more of the cells) express SIGLEC-7; (xv) about 50% or more of the total cells (e.g., about 60% or more of the cells) express KIR3D51; (xvi) about 5% or more of the total cells (e.g., about 10% or more of the cells) express KIR2DL1; (xvii) about 20% or more of the total cells (e.g., about 25% or more of the cells) express NKp30, NKp44, or NKp46; (xviii) about 25% or more of the total cells (e.g., 35% or more of the cells) express NKG2D; (xix) about 80% or more of the total cells (e.g., about 90% or more of the cells) express DNAM1; (xx) about 75% or more of the total cells (e.g., about 85% or more of the cells) express NTBA; (xxi) about 85% or more of the total cells (e.g., about 95% or more of the cells) express CD2; (xxii) about 80% to about 100% of the total cells (e.g., at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the cells) express CD56; (xxiii) about 51% to about 100% of the total cells (e.g., at least about 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the cells) do not express CD57; (xxiv) about 10% to about 40% of the total cells (e.g., at least about 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%, or 40%) express CD16; (xxv) about 10% or less of the total cells (e.g., about 5% or less, 4% or less, 3% or less, 2% or less of the cells) express CD57; (xxvi) about 50% or more of the total cells (e.g., about 55% or more of the cells) express KIR3DS1; (xxvii) about 5% or less of the total cells (e.g., about 4% or less, 3% or less, 2% or less, 1% or less, 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, 0.1% or less) are NKT cells; (xxviii) about 5% or less of the total cells (e.g., about 4% or less, 3% or less, 2% or less, 1% or less, 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, 0.1% or less) are alpha.beta T cells; (xxix) about 50% or more of NK cells and / or gamma delta cells (e.g., about 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 6%, 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%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 1 %, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or fractions thereof, up to 100%) express CD8; (xxx) about 5% or less of the NK cells and / or gamma.delta T cells (e.g., about 4% or less, about 3% or less, about 2% or less, or about 1% or less of the NK cells and / or gamma.delta T cells) express CD4; (xxxi) about 5% or less of the NK cells and / or gamma.delta T cells (e.g., about 4% or less, about 3% or less, about 2% or less, or about 1% or less of the NK cells and / or gamma.delta T cells) express CD8 and CD4; (xxxii) about 15% to about 35% of NK cells (e.g., 16%, 17%, 18%, 19%, 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34% of NK cells do not express CD8 and CD4; and (xxxiii) about 55% to about 85% of the gamma delta T cells (e.g., about 56%, 58%, 60%, 62%, 63%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, or 84% of the gamma delta T cells do not express either CD8 or CD4 The present invention has one or more of the following:

[0089] Innate immune cell composition Certain compositions prepared by the manufacturing methods described herein are referred to as BINATE compositions. BINATE compositions contain two natural cell types, namely NK cells and gamma delta T cells, and the relative amounts of these two cell types can be adjusted to treat solid tumors, blood cancers, or infectious diseases by selecting the appropriate supplemental polypeptide or polypeptides, including their sequence of use and duration of expansion conditions.

[0090] In some embodiments, the BINATE compositions do not contain feeder cells, and often these compositions do not include feeder cells (e.g., do not include exogenous feeder cells and / or irradiated feeder cells). In certain embodiments, the activation and expansion conditions are bisphosphonate-free, which allows the polyclonal gamma.delta T cells to have a broader repertoire of activity against tumors and infectious diseases (i.e., polyclonal for V.delta.1 and V.delta.2 gamma.delta T cells). This innate immune cell platform, interchangeably referred to herein as BINATE cells, INNATE cells, or INNATE-K cells, can comprehensively bind both tumor signals and receptors, providing a readily available and versatile therapy for solid tumors, hematological cancers, and infectious diseases. The improved safety of innate immune therapies allows for their use in community hospitals, reducing healthcare costs and bringing effective treatment to more patients.

[0091] In certain embodiments, the BINATE cell compositions provided herein can be further treated to generate compositions containing substantially all NK cells (referred to herein as "INNATE-NK") or substantially all gamma.delta T cells using markers that positively select for desired cell populations or remove undesirable cell populations.

[0092] The BINATE compositions provided herein can be developed to target both solid tumors and hematological malignancies, as: a) non-genetically modified cells in post-transplant or other settings / indications; b) non-genetically modified cells administered in combination with other therapeutic agents, such as commercially available antibodies for the treatment of cancer; or c) genetically modified cells, for example, by mutating endogenous polynucleotides, deleting endogenous polynucleotides, or adding exogenous mutant polynucleotides (wild type in non-modified cells), or by adding exogenous polynucleotides of heterologous nature, such as CAR polynucleotides (BINATE.CAR). CAR-modified INNATE-K (same as BINATE) or INNATE-NK populations are referred to herein by any of the following terms.

[0093] Generally, for immune cells modified with a CAR, the cells are referred to with a period or hyphen followed by a "CAR" suffix, e.g., INNATE-CAR, INNATE-K.CAR, INNATE-NK.CAR, and BINATE.CAR; and

[0094] In the case of immune cells engineered with a particular CAR, e.g., a CAR that targets CD19, the cells are referred to interchangeably with a period or hyphen followed by the "CD19", "CAR19", or "CAR.CD19" suffix, e.g., BINATE.CD19, BINATE.CAR19, or BINATE.CAR.CD19.

[0095] The methods of production provided herein can include methods of detecting between about 30% to about 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more of the cells in a population, or at least about 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% of the cells in a population. , 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 higher, resulting in a BINATE cell population with high levels of activation, up to 100%. In comparison, eliciting an innate immune response in vivo results in activation of 10% or less of the cells.

[0096] These activated cells are highly cytotoxic and have a significant proportion of CD56+CD16+ cells (e.g., about 40% of the NK cell population in the compositions provided herein can be CD56+CD16+). As used herein, a "+" sign, or the term "plus," in association with a description of a cellular marker on a cell indicates that the marker is expressed on (or on the cell surface), whereas a "-" sign, or the term "minus," in association with a description of a cellular marker on a cell indicates that the marker is absent or not detectable.

[0097] The BINATE cells of the compositions provided herein are highly cytotoxic but less mature (i.e., they are further from senescence) as the majority of the cells have low levels of the CD57 marker (high levels of the CD57 marker indicate cytoxicity, but also indicate that the cells are closer to senescence and therefore have a shorter life span (see Kared et al., Cancer Immunol. Immunotherap., 65(4):441-452 (2016))). As used herein, the term "majority" refers to more than 50% of the cells in a population, typically 50.5% or more, such as 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, , 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%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or fractions thereof, up to 100%. The BINATE cell compositions produced by the manufacturing methods provided herein also have low levels of exhaustion markers, such as PD-1 and TIM-3. Non-limiting examples of activation / cytotoxicity / exhaustion marker phenotypes for BINATE compositions obtained under different expansion conditions according to the manufacturing methods provided herein are summarized below. [Table A]

[0098] In certain embodiments, the cells of the BINATE composition provided herein can be genetically modified.Some specific non-limiting examples of genetic modification include: (i) adding an exogenous polynucleotide that encodes a polypeptide with desired activity; (ii) changing or adding an endogenous polynucleotide, or adding an exogenous regulatory polynucleotide (e.g., primer or enhancer) that regulates the expression of an endogenous polypeptide with desired activity; (iii) changing and / or disrupting the endogenous polynucleotide that encodes a polypeptide with desired activity (e.g., insertion mutagenesis); (iv) partially or completely deleting the regulatory polynucleotide that regulates the expression of a polypeptide with desired activity, thereby disrupting its regulation; and / or (v) partially or completely deleting the coding sequence that encodes a polypeptide with desired activity, thereby reducing or eliminating its activity (e.g., knockout mutagenesis).

[0099] In some embodiments, the BINATE composition is genetically modified by adding an exogenous (regulatory or coding sequence) polynucleotide. The highly activated cells of the BINATE composition are efficiently transformed into cells that are highly efficient, often at or above 80% of the cells in the population, and generally at least about 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 fractions thereof, up to 100%, which is considerably higher than is often observed for adaptive immune cells (e.g., about 9% for alpha.beta. T cells).

[0100] In certain embodiments, the exogenous polynucleotide encodes a chimeric antigen receptor (CAR) and the cells in the composition comprise the CAR (referred to as "BINATE.CAR cells"). In some embodiments, the CAR contains a binding molecule moiety that immunospecifically binds to one or more of CD19, GD2, HER3, B7H3, CD123, or CD30.

[0101] The BINATE and BINATE.CAR cells provided herein can be used to target a variety of cancers and infectious diseases (e.g., GD2 / HER3 / B7H3: lung / bronchus, prostate, breast, colon, pancreas, ovarian; CD123: leukemia; CD30: non-Hodgkin's lymphoma; other cancers including liver and intrahepatic cholangiocarcinoma, esophageal cancer, bladder cancer, kidney and renal pelvis cancer, uterine cancer, brain / nervous system cancer). GD2, a disialoganglioside, has also been found to be expressed on the surface of tumor cells of neuroectodermal origin. Tumors with GD2 expression have a high mortality rate (pediatric tumors-neuroblastoma, retinoblastoma, sarcoma; adult tumors-melanoma, non-small cell lung cancer, breast cancer). Currently, monoclonal antibodies tested as treatments have limitations due to toxicity in neuropathic pain. Initial testing with the BINATE.CAR.GD2 construct shows comparable high levels of transduction in both NK cells and gamma.delta T cells (approximately 80% in NK cells and 40-60% in gamma.delta T cells); expression is stable and maintained over the BINATE culture period. In addition, in vitro killing of solid tumor cells was observed (Example 18).

[0102] Pharmaceutical Compositions and Kits Any of the compositions provided herein can be formulated as a pharmaceutical composition together with a pharma- ceutically acceptable carrier.Like the compositions, the pharmaceutical compositions provided herein can be used to treat cancer and infectious diseases.Also provided herein is a kit that contains the compositions or pharmaceutical compositions provided herein and, optionally, instructions for use.In certain aspects, the kits provided herein can include cytokines.Innate immune cells control opportunistic invasion by a wide range of viral, fungal, bacterial, and parasitic pathogens, in part by releasing a plethora of cytokines and chemokines to coordinate with other cells and thereby orchestrate immune responses.

[0103] The pharmaceutical composition or kit sometimes includes a specific dosage of the therapeutic cells, and sometimes the pharmaceutical composition or kit provides a unit dosage of the therapeutic cells. In certain embodiments, the unit dosage is about 10 cells per kilogram of body weight of the intended subject. 4 About 10 10 or approximately 10 cells per subject 6 About 10 12 Between 10 and 20 cells per subject (e.g., approximately 10 cells per subject) 10 or approximately 10 cells per kilogram of intended subject body weight 8 pcs).

[0104] The pharmaceutical composition or kit may include a pharma- ceutically acceptable carrier. The term "pharmaceutical acceptable" means approved by a federal or state regulatory agency for use in animals, more particularly in humans, or listed in the United States Pharmacopeia or other generally recognized pharmacopeia. The term "carrier" refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete), excipient, or vehicle with which the therapeutic is administered. If the composition is administered by injection, it can be dispensed into an infusion bottle containing sterile pharmaceutical grade water or saline. If the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0105] The pharmaceutical composition is sometimes provided as a pharmaceutical pack or kit comprising one or more containers filled with the therapeutic composition of cells prepared by the methods described herein, alone or together with such pharma- ceutically acceptable carrier. In addition, one or more other preventive or therapeutic agents useful for disease treatment can also be included in the pharmaceutical pack or kit. The pharmaceutical pack or kit can comprise one or more containers filled with one or more components of the pharmaceutical composition described herein. If necessary, such container(s) can be associated with a notice in a form prescribed by a governmental authority regulating the manufacture, use, or sale of pharmaceuticals or biologics, which notice reflects the approval of the authority for manufacture, use, or sale for human administration. The pharmaceutical pack or kit sometimes comprises one or more other preventive and / or therapeutic agents useful for disease treatment in one or more containers.

[0106] When cells are expanded ex vivo and then administered as an immunotherapy, the cells sometimes do not produce sufficient amounts of cytokines by themselves. Cytokines that can be optionally added to the kits provided herein include, but are not limited to, TNF, IFNγ, interleukins, IL-1β, IL-4, IL-6, IL-7, IL-10, IL-12, IL-18, IL-21, CCL4 / RANTES, and TGFβ. In certain embodiments, the pharmaceutical compositions and / or kits provided herein optionally include a second agent for co-administration with the compositions provided herein. The terms "co-administer", "co-administers", "co-administration", and the like, as used herein, mean that the agent is administered before, after, or together with the natural cell compositions provided herein.

[0107] In some embodiments, the second agent is an antibody that targets a cancer cell antigen or an infectious agent.The compositions, pharmaceutical compositions, and kits provided herein can be kept at or below minus 4°C, or at about minus 75°C to about minus 80°C, as appropriate, for storage or transportation. Methods for Producing Genetically Modified Natural Cell Compositions

[0108] The natural cell compositions provided herein can be genetically modified in a suitable manner (e.g., by adding an exogenous polynucleotide that is a gene or a regulatory sequence, by mutating an endogenous gene, or by deleting an endogenous gene). Genetic modification can be performed following activation and expansion after obtaining the final composition, or can be performed after activation and before expanding the cells. Non-limiting examples of methods for making various genetic modifications are described below:

[0109] (a) CRISPR-CAS9 targeted suppression (permanent gene / locus deletion) Cells can be transfected with a DNA plasmid expressing both the CAS9 protein and a guide RNA (gRNA) specific for the gene of interest. The gRNA-CAS9-mediated break in the genome can be repaired using a donor DNA plasmid, which causes a specific deletion of the target gene and a permanent and complete loss of the gene-encoded protein. Loss of protein expression can be verified using PCR (DNA level), Northern blot / FISH (RNA level), or any protein assay, such as Western blot or flow cytometry.

[0110] (b) CRISPR-CAS9 targeted expression (insertion of permanent genes / locuses) Using this method, a gene of interest can be inserted into a specific location in the cell genome. Cells can be transfected with a DNA plasmid expressing both the CAS9 protein and a guide RNA (gRNA) specific for the specific insertion location. The gRNA-CAS9 mediated break in the genome can be repaired using a donor DNA plasmid with the inserted gene of interest flanked by sequences of the cell genome on both sides of the location of the DNA cut / double strand break, resulting in a homologous recombination mediated insertion of the gene of interest in a specific, non-random genomic location. Successful insertion and protein expression can be verified using PCR (DNA level), Northern blot / FISH (RNA level), or any suitable protein assay, such as Western blot or flow cytometry.

[0111] (c) RNA interference (permanent gene silencing) by retrovirus / lentivirus / transposon-mediated transduction of shRNA / microRNA shRNA / microRNA can be designed to target specific genes / proteins of interest and cloned in retroviral / lentiviral / transposon vectors for stable integration into cell genome. Cells can be transduced with the vector and successfully transduced cells can be selected using vector-encoded selection markers. shRNA-mediated suppression of genes of interest can be evaluated using, for example, Northern blot and protein assays.

[0112] (d) Lentivirus / γ-retrovirus-mediated random / multiple copy gene insertion Specific gene / protein of interest can be designed and / or cloned in retroviral or lentiviral vector for stable random integration into cell genome. Cells can be transduced with viral vector and successfully transduced cells can be selected using vector-encoded selection marker. shRNA-mediated suppression of gene of interest can be evaluated using Northern blot and any suitable protein assay, such as Western blot, flow cytometry, etc.

[0113] (e) Transposon-mediated random / multiple copy gene insertion A specific gene / protein of interest is designed and / or cloned in a mammalian transposon vector system, such as PiggyBac (SBI System Biosciences) or equivalent. Cells can be co-transfected with a transposon vector carrying the gene of interest (cDNA) flanked by inverted terminal repeat (ITR) sequences and a transposase vector. The transposase enzyme can mediate the transfer of the gene of interest into the TTAA chromosomal integration site. Successfully transduced cells can be selected using vector-encoded selection markers, if necessary. Successful insertion and protein expression can be verified using PCR (DNA level), Northern blot / FISH (RNA level), or any suitable protein assay, such as Western blot or flow cytometry.

[0114] (f) Direct transfection The mRNA encoding the gene / protein of interest can be directly transfected into cells. Transfection can be performed using any established methodology, such as calcium chloride transfection, lipofection, Xfect, electroporation, sonoporation, and cell squeezing (e.g., to introduce siRNA).

[0115] Treatment Method Also provided is a method of treating cancer or infectious disease by administering to a subject in need thereof any of the compositions, pharmaceutical compositions, or kits provided herein in an amount effective for treating cancer or infectious disease. Treatment can be administered in an autologous or allogeneic setting. The donor of the sample from which the composition, pharmaceutical composition, or kit is made can be the recipient of the treatment. Often, the composition, pharmaceutical composition, or kit is prepared from a sample from one subject, and the treatment is administered to a different subject. In certain embodiments, the treatment can be administered on two or more separate days, and in certain embodiments, the treatment can be administered in multiple doses. In certain embodiments, the treatment is administered between about 1 unit dosage and about 36 units or more dosages at intervals of about 2 weeks to about 4 weeks. In some embodiments, the treatment is administered as a single unit dosage once, twice, three times, four times, or up to five times daily, or once, twice, three times, four times, five times, six times, seven times, eight times, nine times, or ten times, or more, over the course of several days, weeks, or months, or every other day, or once, twice, three times, four times, five times, or six times weekly. Treatment can be administered intravenously (IV), intrathecally, or intramuscularly (IM), intraperitoneally (IP), intrapleurally, intraarticularly, or at about 10 cells per kilogram of the subject's body weight at or near the site of the cancer or infection. 4 About 10 10 Between 10 and 10 cells per subject 6 About 10 12 In certain embodiments, the unit dosage is about 10 cells per subject. 10 or approximately 10 cells per kilogram of subject's body weight 8 There are 10 pieces.

[0116] In certain embodiments of the methods of treatment provided herein, the treatment is for cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a hematological cancer. In certain embodiments, the cancer is a hematological cancer, and the ratio of gamma delta T cells to NK cells is greater than 1. In some embodiments, the cancer is a solid tumor, and the ratio of NK cells to gamma delta T cells is greater than 1. In certain embodiments of the methods of treatment provided herein, a second agent is co-administered with the composition, pharmaceutical composition, or kit. In some embodiments, the second agent is an antibody that immunospecifically binds to a cancer-associated antigen.

[0117] In certain embodiments of the methods of treatment provided herein, the treatment is for an infectious disease. In some embodiments, the infectious disease is characterized by the presence of a bacterial pathogen, a fungal pathogen, a viral pathogen, or a protozoal pathogen.

[0118] In certain aspects of the methods provided herein, the composition, pharmaceutical composition, or kit contains a polyclonal population of gamma.delta T cells (e.g., polyclonal for V.delta.1 and V.delta.2 gamma.delta T cells).

[0119] The present technology will be further described with reference to examples described herein, however, it should be understood that the present technology is not limited to such examples. EXAMPLES

[0120] The examples set forth below illustrate certain specific embodiments but do not limit the present technology.

[0121] The following materials were used in certain methods described below (eg, activation conditions, expansion conditions, phenotypic analysis).

[0122] The following monoclonal antibodies (mAbs) were generated at the Ospedale Pediatrico Bambino Gesu (OPBG), Laboratory of “Cell and Gene Therapy for Pediatric Tumor”:

[0123] c218 (IgG1, anti-CD56), c127 (IgG1, anti-CD16), AZ20 and F252 (IgG1 and IgM, anti-NKp30, respectively), BAB281 and KL247 (IgG1 and IgM, anti-NKp46, respectively), Z231 (IgG1, anti-NKp44), ECM217 and BAT221 (IgG2b and IgG1, respectively, anti-NKG2D, respectively), KRA236, GN18 and F5 (IgG1, IgG3 and IgM, respectively, anti-DNAM-1, respectively), EA4 (IgG2a, anti-CD18), MAR206, MA258 and QA196 (IgG1, IgG2b and IgM, respectively, anti-CD2, MA127 and and ON56 (IgG1 and IgG2b, respectively, anti-NTB-A), PP35, ST39 and CO54 (IgG1, IgG1a and IgM, respectively, anti-2B4), z27 (IgG1, anti-KIR3DL1 / S1), AZ158 (IgG2a, anti-KIR3DL1 / L2 / S1), z270, z199 and Y9 (IgG1, IgG2b and IgM, respectively, anti-NKG2A), 6A4 and A6 / 136 (IgG1 and IgM, respectively, anti-HLA-class I), D1 / 12 (IgG2a, anti-HLA-DR), 5A10 (IgG1, anti-PVR), L14 (IgG2a, anti-nectin-2), BAM195 (IgG1, anti-MICA).

[0124] F278 (IgG1, anti-CD85j) mAb was a kind gift from Dr. Daniela Pende, Istituto Giannina Gaslini, Genoa, Italy (see also, e.g., Costa et al., Aids, 15:965-974 (2001)). Anti-NKG2C (IgG2b, clone 134522), anti-ULBP-1 (IgG2a, clone 170818), anti-ULBP-2 (IgG2a, clone 165903), anti-ULBP-3 (IgG2a, clone 166510), anti-CD34-APC (IgG1, clone QBEnd10), IgG-APC isotype control (IgG1 clone 11711) and anti-KIR2DL1-FITC or unconjugated (IgG1, clone 143211) mAbs were purchased from R&D System Inc (Abingdon, United Kingdom). Anti-KIR2DL1 / S1-Vioblue or -PE (IgG1, 11PB6 clone), Anti-NKG2C-ViobrightFITC (REA205 clone), Anti-KIR3DL1-Biotin or -FITC (IgG1, DX9 clone), Anti-CD3-Viogreen (IgG2a, BW264 / 56 clone), Anti-CD57-Vioblue (IgM, TB03 clone), Anti-SIGLEC-7-Vioblue (REA214 clone), Anti-NKp30-PE (IgG1, AF29-4D12 clone), Anti-NKp46-PE (IgG1, 9E2 clone), Anti-NKp44-PE (IgG1, 2.29 clone), Anti-Biotin-PerCPVio700 (REA746 clone), REA control VioBright FITC (clone REA293), anti-PD-1 (IgG2b, PD1.3.1.3 clone) mAb were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany).Anti-CD34 (IgG1, QBEnd10 clone), anti-NKG2A-PC7 (z199 clone), anti-KIR3DL1 / S1-PE (z27 clone), anti-KIR2DL2 / L3 / S2-PE (GL183 clone), anti-CD19-FITC (IgG1, J3-119 clone), anti-CD56-PC7 (IgG1, N901 clone), IgG1-PC7 or -PE or -FITC isotype controls (679.1Mc7 clone) mAbs were purchased from Beckman Coulter, Immunotech (Marseille, France). Anti-KIR2DL2 / L3-S2-FITC or unconjugated (IgG2b, CHL-clone), anti-CD107-PE (IgG1, H4A3 clone), anti-CD85j (IgG2b, GHI / 75 clone), anti-CD16-PerCpCy5.5 (IgG1, 3G8 clone), anti-CD56-BV510 (IgG2b, NCAM16.2 clone), IgG1-PE isotype control (clone MOPC-21) mAbs and Brilliant Stain buffer were obtained from BD Bioscience Pharmingen (San Diego, CA). Anti-HLA-Bw6-FITC and anti-HLA-Bw4-FITC mAbs were purchased from ONE LAMBDA INC (Canoga Park, CA). Anti-human HLA-E (IgG1, 3D12 clone) and anti-human HLA-G (IgG1, MEM-G / 9 clone) mAbs were purchased from BioLegend (San Diego, CA) and Abnova (Taipei, Taiwan), respectively. Anti-NKG2D (IgG2a, 5C6 clone) and anti-HLA-C (IgG1, C-8 clone) were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA).

[0125] For expansion conditions, IL-2, IL-15, and OKT3 were obtained from Miltenyi Biotec (San Diego, Calif., USA), as were anti-CD2 and anti-NKp46 antibodies cross-linked or conjugated to beads.

[0126] Example 1 Enrichment and expansion of specific innate cytolytic immune cell (INNATE-K) populations derived from αβTCRneg cell populations This example describes a process to enrich and expand in feeder-free culture conditions a specific innate cytolytic immune cell population consisting mainly of NK and γδTCR+ T cells (herein referred to as INNATE-K or BINATE cell population) from a starting αβTCRneg cell population activated by antibodies against one or more immunoglobulin subfamily surface molecules (see FIG. 2 for an analysis of the composition of this expanded T cell population; the terms INNATE-K and BINATE may be used interchangeably herein and refer to the cell populations discussed herein that are activated by a combination of NCR antibodies (e.g., anti-NKp46) and LFA antibodies (e.g., anti-CD2) and contain a mixture of NK cells and γδT cells).

[0127] 1. Large-scale leukopheresis was performed on normal healthy donors who had been mobilized with G-CSF; alternatively, large-scale leukopheresis was performed on normal healthy donors without mobilization or using buffy coat.

[0128] 2. Ficoll™ density gradient cell separation was performed with a SEPAX™ device (Sepax Technologies, Inc., Newark Del.) to remove red blood cells, platelets, and granulocytes and obtain a mononuclear cell suspension; alternative methods of performing Ficoll™ gradient separation include MILTENYI PRODIGY™ (Miltenyi Biotec, San Diego, Calif.) or manual separation with a centrifuge.

[0129] 3. Mononuclear cells were subjected to αβ T cell depletion using clinical-scale MILTENYI CLINIMACS™ (Miltenyi Biotec, San Diego, CA) according to the manufacturer's instructions or subjected to laboratory-scale Miltenyi LS column separation.

[0130] 4. The resulting αβTCRneg cell population was placed in feeder-free culture conditions and activated with anti-CD2 and anti-NKp46 beads (NK Cell Activation / Expansion Kit™ (Miltenyi Biotec, Inc., San Diego, CA, USA)) and 500 IU / mL human interleukin 2 (IL-2) (Miltenyi Biotec) according to the manufacturer's instructions.

[0131] 5. Activated αβTCR cell population at 0.25 x 10 in 24-well plates 6 The cells were further expanded in feeder-free culture conditions using NK MACS™ medium (#130-107-879 (Miltenyi Biotec, Inc., San Diego, CA, USA)) supplemented with 5% AB serum and 500 IU / mL human interleukin-2 (IL-2) at a cell seeding concentration of 100 / ml and then transferred to T75 flasks; alternative culture vessels may include bioreactors (G-Rex™ 25 ml, Wilson Wolf Manufacturing, St. Paul MN).

[0132] 6. Media changes were performed approximately every 3 days with fresh media and fresh IL-2 at the concentrations listed in #5, with cells at 0.25 to 0.5 x 10 6 The cells were seeded at a concentration of 10 ...

[0133] 7. At any time after the initiation of expansion, the resulting enriched specific innate immune cytolytic populations (NK and γδTCR+ T cells) can be further enriched for either pure NK or γδTCR+ T cells using CD3+ or CD56+ depletion of the undesired populations, respectively, to obtain pure cell populations and further cultured as in step #5.

[0134] 8. At the end of expansion, INNATE-K cells are cryopreserved in serum-free freezing medium (CryoStor™, BioLife Solutions, Bothell WA) containing 10% DMSO solution.

[0135] FIG. 1 illustrates the expansion of αβTCRneg cells as a function of the number of days the cells were activated with antibodies and expanded in feeder-free culture conditions, as analyzed at day 10 of culture. The graph shows that the cells began to achieve logarithmic expansion in approximately one week and continued to expand, generating large numbers of cells suitable for successful off-the-shelf therapy. In FIG. 1, activated αβTCRneg cells were culture-expanded in feeder-free culture conditions. The total cell number of the culture is shown by day of expansion, with a significant increase in expansion observed after 6 days of culture.

[0136] Figure 2 shows 70% NK cells and 28% γδTCR+T(GD) cells, a ratio suitable for a therapeutic product. Further expansion of the cells may result in a shift; these two cell types were obtained in large numbers without feeder cell culture and without contamination by other cells. Figure 2 illustrates the enrichment and phenotype of specific innate immune cytolytic cells (INNATE-K), NK, and γδTCR+T cells, as well as CD3+CD56+NKT cells and adaptive immune αβTCR+T cells at various steps of the following example of the manufacturing process.

[0137] a) The frequency of cells in the mononuclear cell fraction after Ficoll density gradient separation was 8% + 12% NK cells and 0.1% + 1% γδTCR+ T cells, 6% CD3+CD56+ NKT cells and 73% αβTCR+ T cells.

[0138] b) The frequency of cells after the αβTCR+ T cell depletion step was 25% + 15% NK cells and 3.2% + 1.2% γδTCR+ T cells (of which 0.5% were CD56-CD3+ and 2.7% were CD56+CD3+) and 1% or less αβTCR+ T cells.

[0139] c) After 10 days of activation and feeder-free expansion, NK cell frequencies were substantially enriched to 68% + 23% and γδTCR+ T cells were substantially enriched to 15.7% + 5% (of which 12.2% were CD56-CD3+ and 3.5% were CD56+CD3+). Negligible levels of CD3+ adaptive immune cells were detected: αβTCR+ T cells at 0.3% + 0.5% and NKT cells at 0.2% + 0.3%.

[0140] Flow cytometric analysis (fluorescence-activated cell sorter, or FACS analysis) of cells co-expressing the CD56 (NK) surface antigen, CD16, and CD57 was performed on day 10. CD16 is an Fcγ receptor III and can thus bind the Fc portion of IgG antibodies and mediate antibody-dependent cell-mediated cytotoxicity (ADCC) of antibody-bound target cells. CD16 has a role in NK cell-mediated spontaneous cytotoxicity. The CD56+CD16+ subset is considered to be the most cytotoxic subset and constitutes the majority of NK cells (e.g., under physiological conditions). CD57 may be a marker of NK cells with poor proliferative capacity. Acquisition of CD57 on NK cells after stimulation with IL-2 or co-culture with target cells correlates with maturation of the CD56+ NK cell subset. This differentiation is accompanied by functional changes: compared with CD57- cells, CD57+ NK cells proliferate poorly in response to IL-2 and IL-15 and produce less IFN-γ in response to IL-12 and IL-18. Of CD56+ NK cells, 37% were CD16+ and 63% were CD16-; of CD56+CD16- cells, 13% were CD57+ and 87% were CD57-; and of CD56+CD16+ cells, 15% were CD57+ and 85% were CD57-. Flow cytometry phenotypic results showed that the cell composition contained enriched NK cells that were highly cytotoxic NK cells in the expanded αβTCR cells, as evidenced by the co-expression of CD56(NK) and CD16, yet also contained cells with an immature phenotype that had the potential to mature in vivo into more responsive and cytotoxic cells, as evidenced by the number of NK cells that did not express CD57 in both CD16+ and CD16- NK cells.

[0141] We measured the distribution of the frequency of different γδ T cell subsets in day 10 samples of feeder-free culture-expanded activated αβ TCR cells analyzed by flow cytometry. γδ T cells, in contrast to MHC-restricted αβ T cells, are able to recognize and lyse a variety of cancers in an MHC-unrestricted manner, highlighting their potential for off-the-shelf allogeneic immunotherapy. Human γδ T cells can be divided into three main populations based on the expression of the δ chain. The majority of circulating γδ T lymphocytes in healthy human adults, up to 50%–90%, express the γδ2 chain. γδ T cells expressing the γδ1 chain are most prominent in the epithelial lining of mucosal surfaces, where they are implicated in the maintenance of epithelial tissue integrity in the face of injury, infection, or transformation, although they can also appear in peripheral blood. γδ3 T cells constitute approximately 0.2% of circulating T cells, including the CD4+, CD8+, and CD4-CD8- subsets. Although rare in peripheral blood, γδ3 T cells are usually more highly expressed in the liver. The percentage distribution of γδTCR+ T cells at day 10 (41% γδ1, 50% γδ2, 7% other γδ) indicates that the propagated γδ T cells are predominantly Vδ1, Vδ2, and Vδ1. neg Vδ2 neg The expression of a subset of γδ T cells clearly indicates that they were polyclonal, ensuring the involvement of the expanded γδ T cells in the natural killing of tumors, altered cells, or infections.

[0142] Figure 3 illustrates the frequency and distribution of T cell lineage markers, CD4 (helper), and CD8 (cytotoxic) on γδTCR+ cells in a day 10 sample of feeder-free culture-expanded activated αβTCRneg cells as analyzed by flow cytometry. Most of the γδ1 T cells expressed neither CD4 nor CD8, whereas a small percentage (19%) expressed the cytotoxic CD8 lineage marker, which has been described for maximal tumor and infection killing and is consistent with a desirable innate cytolytic immune population. γδ2+ cells expressed small amounts of either CD4 or CD8, whereas almost half of the "other γδTCR+ cells", mainly composed of γδ3 T cells, expressed the cytotoxic CD8 lineage marker.

[0143] Further flow cytometry phenotyping data was obtained at day 8 to obtain γδ T cells from feeder-free culture-expanded activated αβ TCR cells that had been depleted by Miltenyi Biotec, Inc. αβ T cell depletion reagent at laboratory scale. For large-scale depleted cells, cultured CD3+ T cells at day 8 expressed predominantly γδ T cell receptors (αβ TCR+ cells were <0.05%). Phenotyping was performed to analyze the co-expression of markers on γδ T cells. All cells were CD3+ and 31.8% were CD16+. Co-expression of CD57 and CD16 on CD3+ γδ T cells was observed. 10.7% co-expressed CD57 and CD16 markers, thus representing the most mature cells. The majority were CD57- and CD16- (57%), thus representing immature cells. Expression of inhibitory and activating ligand receptors was analyzed. NKG2D is an activating receptor on the NK cell surface. NKG2A dimerizes with CD94 to create an inhibitory receptor. The cells lacked expression of NKG2C, and 55% of the cells co-expressed NKG2A and NKG2D. Phenotypic analysis was performed to analyze the co-expression of markers on γδ T cells in relation to the co-expression of CD56. Of all γδ cells that expressed CD3+ and CD56+, 29% co-expressed CD16. Co-expression of CD57 and CD16 was observed in CD3+CD56+ γδ T cells. 5% co-expressed CD57 and CD16 markers, thus representing the most mature cells. The majority were CD57- and CD16- (67%), thus representing immature cells. Expression of inhibitory and activating ligand receptors was analyzed. The cells lacked expression of NKG2C on CD3+CD56+ cells, the majority of cells (82%) expressed NKG2A, and 80% of CD3+CD56+ cells co-expressed NKG2A and NKG2D, indicating that these cells expressed phenotypic markers most commonly associated with NK cells and represent an active innate immune cell population.

[0144] Example 2 Expansion of specific transduced innate cytolytic immune cells (INNATE-CAR) derived from αβTCRneg cell population This example describes the expansion and production of a transduced specific innate cytolytic immune cell (INNATE-CAR) population, including NK and γδTCR+ T cells, expanded from an activated culture in feeder-free conditions of an αβTCRneg cell population.

[0145] 1. Take an aliquot of cells from the activated, culture-expanded αβTCR cell population described in Example 1, step #5 on day 4 (alternatively, an aliquot can be taken between days 5-15) and plate 0.25 x 10 cells on human fibronectin-coated plates. 6 The cells were seeded in the presence of 500 IU / mL human interleukin 2 (IL-2) or 10 ng / ml IL-15 at a concentration of 100 / mL (alternatively RetroNectin™ (Takara Bio USA, Mountain View Calif.) coated plates or VECTOFUSIN-1 (Miltenyi Biotec) transduction enhancer could be used) and transduced with gamma retroviral supernatant containing a construct of CD+19 chimeric antigen receptor containing a non-functional fragment of the CD34 surface antigen used as a marker for cell transduction.

[0146] After 3 days, cells were detached and plated at 0.25 × 10 in 24-well plates or T75 in NK MACS™ medium (#130-107-879 (Miltenyi Biotec, Inc., San Diego, CA, USA)) supplemented with 5% AB serum and 500 IU / mL human interleukin 2 (IL-2). 6 Alternative culture vessels may include bioreactors (G-Rex™ 25 ml, Wilson Wolf Manufacturing, St. Paul MN).

[0147] 3. Medium replacement was performed every 3 days with fresh medium and fresh IL-2 at the concentrations described in #6 of Example 1, and cells were cultured at 0.25 to 0.5 × 10 6When seeding the bioreactor, medium changes are performed every 7 days until the target dose is achieved.

[0148] 4. At any time after day 14, the INNATE-CAR population can be further enriched for either NK-CAR or γδ-CAR T cells using CD3+ or CD56+ depletion of the unwanted populations, respectively, to obtain pure cell populations, which were further cultured as in step #3.

[0149] 5. At the end of expansion, cells are cryopreserved in serum-free freezing medium (CryoStor, BioLife Solutions, Bothell, WA) containing 10% DMSO solution.

[0150] Figure 4 illustrates the analysis of the expansion kinetics of CAR.19 INNATE-K cells over a 10 day period. Similar to the expanded non-genetically modified populations, the cultures demonstrated logarithmic expansion beginning at approximately one week, demonstrating the robust expansion potential of off-the-shelf INNATE-CAR products. In Figure 4, activated INNATE-K cells were expanded in culture in feeder-free culture conditions and transduced on day 4 with a gammaretrovirus containing the CD19 CAR and a non-functional CD34 marker.

[0151] Transduction of INNATE-CAR populations, NK cells, and γδTCR+ T cells was compared (with three different products). Specifically, %CAR.CD19 expression was compared in each of these cell populations using the CD34 marker incorporated into the CAR molecule. Both NK and γδTCR+ T populations showed significant transduction with the CAR.CD19 retroviral vector, as evidenced by analysis of the CD34 marker in the stated cell subsets. Transduction efficiency was on average 33% and 30% for NK and γδTCR+ T cells, respectively. Flow cytometry analysis (fluorescence-activated cell sorter, or FACS analysis) was performed on feeder-free NK cells expanded for 10 days and not transduced with the retroviral vector bearing CAR.CD19, showing the specificity of CD34+ staining on CD56+ cells. Flow cytometry (FACS) analysis was performed on feeder-free NK cells genetically modified on day 4 of the activation process with a retroviral vector carrying CAR.CD19 and expanded for 10 days. The results showed that the CAR expression level in CD56+ cells was significant after transduction, both in terms of the percentage of CD34+ cells and the mean fluorescence intensity of expression. Both features are associated with highly efficient tumor recognition. The same findings were also valid for the situation of feeder-free γδTCR T cells present in the transduced and expanded INNATE population. Indeed, the γδTCR T cell component was significantly transduced as assessed by CD34 expression analysis in γδTCR T cells.

[0152] The INNATE-CAR platform is highly efficient in tumor recognition and elimination, with strong synergy between innate activity and CAR-mediated functionality. Indeed, Figure 5 graphically illustrates data from a co-culture cytotoxicity assay in which INNATE and INNATE-CAR.CD19 cells were co-cultured with CD19+ leukemia (221) or CD19+ lymphoma (Daudi) cell lines. The % remaining tumor after culture with respect to the different cellular effectors is represented in the graph above. Non-transduced NK cells (INNATE-NK) killed 40 and 29% of leukemia and lymphoma tumor cells, respectively, whereas INNATE-K cells, characterized by a combined population of NK and γδTCR T cells, killed 88.4% and 96.1% of tumor cells, respectively. Transduction of INNATE-NK cells with CAR.CD19 (INNATE-NK-CAR.CD19) enabled elimination of 93% and 84% of tumor cells, whereas the combined INNATE-CAR.CD19 population killed nearly all tumor cells (97% and 99%).

[0153] Example 3 INNATE-Alternative enrichment of NK cells This example describes the enrichment process of INNATE-NK cells:

[0154] 1. NK cells were isolated from buffy coats by NK DEPLETION KIT™ (Miltenyi Biotec, Inc., San Diego, Calif., USA).

[0155] 2. NK cells were activated by NK Cell ACTIVATION / EXPANSION KIT™ (Miltenyi Biotec, Inc., San Diego, CA, USA) and recombinant human IL-2 (500 U / ml).

[0156] On day 3.0, after activation, NK cells were cultured in 5% AB serum-enriched medium (NK MACS™ medium (#130-107-879 Miltenyi Biotec, Inc., San Diego, Calif., USA).

[0157] 4. From day 4 to day 15, aliquots of cells were plated at 0.25 x 10 6 The cells were subjected to transduction in the presence of 500 IU / mL human interleukin 2 (IL-2) at a concentration of 100 / mL.

[0158] 5. After 3 days, the cells were detached and plated at a cell seeding concentration of 0.25 × 10 in 5% AB serum-enriched medium and 500 IU / mL human interleukin 2 (IL-2). 6 Further expansion was performed at 100 / ml.

[0159] 6. After transduction, NK cells were plated in 24-well plates and then in T-flasks or bioreactors.

[0160] 7. Medium changes were performed every 3 days with fresh medium and fresh IL-2, and cells were cultured at 0.25–0.5 × 10 until the target cell number was reached. 6 The cells were seeded at a concentration of 100 / mL (if they were on a plate). Bioreactor cultures were changed every 7 days.

[0161] 8. On day +14, if there was more than 5% T cell contamination, non-transduced and CAR-transduced INNATE-NK cells were depleted again for CD3+ cells to obtain pure NK cells and further cultured as in step #7.

[0162] An exemplary highly enriched NK population with >90% innate immune cells was robustly expanded in both plates and bioreactors as illustrated graphically in Figures 6-8. Figure 6 shows the total number of INNATE-NK cell populations expanded over several days in plates. Figure 7 compares the total cell number of INNATE-NK populations expanded in plates and bioreactors. Figure 8 shows the subset cell composition at different time points of in vitro expansion, demonstrating significant purity levels of INNATE-NK represented by >95% NK cells and negligible abundance of CD3+ T cells and NKT cells.

[0163] The level of CAR transduction efficiency was demonstrated using flow cytometry (FACS) analysis evaluating the CD34 marker contained in the CAR.CD19 molecule. Non-transduced INNATE-NK cells expanded feeder-free do not express the CD34 marker. INNATE-NK cells were genetically modified on day 4 with a retroviral vector carrying CAR.CD19. INNATE-NK CAR cells were significantly transduced and the percentage of CAR+ cells in the NK population (CD56+) was high. The expression of the CAR molecule in INNATE-NK CAR cells was stable during long-term in vitro culture as shown in Figure 9.

[0164] The increased expression of activation and cytolytic molecules following expansion of INNATE-NK and INNATE-NK CAR populations is illustrated in FIG. 10, which graphically depicts data from flow cytometry analysis of INNATE-NK and INNATE-NK-CAR.19 cells, respectively. The analysis included evaluation of several NK markers associated with innate cell activation and maturation. In particular, CD16 was included as a fundamental marker for maturation analysis, as it is associated with a more mature NK subgroup with high cytolytic activity but low proliferative potential; signaling and costimulatory molecules for activation and proliferation of NK populations were included, including CD2, LFA-1, NKp44, NKp30, and NKp46, and DNAM-1. All of these markers were significantly increased in the expanded populations of both INNATE-NK and INNATE-NK.CAR.19 cells, indicating that the cells are activated compared to circulating peripheral blood NK cells.

[0165] FIG. 11 graphically illustrates the lack of exhaustion in feeder-free expanded INNATE-NK cells and INNATE-NK CAR cells. INNATE-NK and INNATE-NK-CAR.19 cells were generated and expanded as described in Example 3, and flow cytometry analysis was performed at days 20 and 60 to evaluate markers of cell maturity and exhaustion. LIR-1 is a marker of natural cell inhibitory activity and showed only a slight increase in the NK population at day 20 and an increase in both populations at day 60; NKG2c is a marker of signal-specific activity and maturation and did not change during the culture period; CD57 is a marker of maturation and is associated with a decrease in proliferative capacity and is thus expressed on circulating NK populations derived from peripheral blood of healthy donors. Feeder-free expanded INNATE-NK cells at days 20 and 60 of in vitro expansion showed a significantly lower percentage of CD57+ NK cells, suggesting that these cells still have proliferative capacity upon in vivo infusion. The programmed cell death 1 molecule (PD-1) is not only a marker of reduced cellular potential and "exhaustion" in T cells, but has also recently been described as a checkpoint inhibitor in NK cells. Neither the INNATE-NK nor INNATE-NK CAR cell populations showed the presence of a PD-1+ cell subset at either day 20 or day 60, indicating that the cells are not exhausted after prolonged in vitro culture.

[0166] FIG. 12 graphically illustrates a cytotoxicity co-culture assay with INNATE-NK or INNATE-NK-CAR.19 and four tumor cell lines: Column A: 221, a CD19+ leukemia cell line; Column B: Daudi, a CD19+ lymphoma cell line; Column C: BV173, a CD19+ (variable expression) pre-B tumor cell line; and Column D: KARPAS, a CD19- tumor cell line. Panel 1 of FIG. 12 shows the reduction of CD19+ cells after in vitro co-culture with INNATE-NK (left subpanel) or INNATE-NK.CAR.19 (right subpanel) cells. The top left section (boxed section) in the INNATE-NK panel highlights the CD19+ tumor population after co-culture with non-modified INNATE-NK cells. The top left compartment (boxed compartment) in the INNATE-NK.CAR.19 panel highlights the residual CD19+ tumor population after co-culture with INNATE-NK.CAR.19 cells. INNATE-NK.CAR.19 cells were able to exert significant tumor control against CD19+ 221 and DAUDI tumor targets, with negligible amounts of tumor remaining after 5 days of co-culture in vitro. Although BV173 showed higher resistance to recognition and elimination, genetic modification of INNATE-NK cells with CAR.CD19 was able to significantly increase anti-tumor activity compared to non-modified INNATE-NK cells. The average residual tumor observed in 10 different experiments is shown in Figure 12, panel 2, for each of the co-culture experiments. Degranulation (analyzed by expression of CD107a by NK cells after short-term co-culture in vitro for 3 hours), representing the cytolytic activity of INNATE-NK and INNATE-NK CAR effector cells, is shown in Figure 12, panel 3 (rows A-D). In all cases, INNATE-NK.CAR.19 cells expressed greater cytolytic activity than INNATE-NK cells; in the negative control represented by the KARPAS co-culture condition, neither population expressed the CD107a degranulation marker. Figure 12, panels 4 and 5 show the production of regulatory cytokines that are typically produced in the case of lymphocyte cytolytic activity.Both of these cytokines are highly elevated during CD19 CAR T cell administration and likely play a role in cytokine release syndrome. In the case of INNATE-NK and INNATE-NK CAR activity against CD19+ tumor cells, cytokine production of both IFNα and TNFα ranged from 20 to 150 pg / ml / cell. 6 The range of cytokine secretion was many times less than that of adaptive T cell killing. Even with high target killing, the extent of cytokine secretion was not as great as that observed for CAR-T (typically 100- to 1,000-fold or higher), thus potentially representing a less toxic treatment.

[0167] Figures 13-15 graphically illustrate that INNATE-NK and INNATE-NK CAR-cells can exert significant antitumor activity against primary Bcp-ALL blasts. Figures 13 and 14 graphically illustrate the % specific lysis of primary tumor cells as a function of effector (E) to target (T) ratio for both INNATE-NK cells and INNATE-NK-CAR.19 cell populations in different experimental runs; the INNATE-NK-CAR.19 cell population was more effective across the entire E:T range. Figure 15 graphically illustrates the percentage remaining primary CD19+ tumor after co-culture with effector cells (INNATE-NK cells and INNATE-NK-CAR.19 cell populations) compared to the control condition where primary CD19+ leukemic blasts were plated in the absence of effector cells. Coculture with INNATE-NK cells resulted in approximately 18% residual tumor cells, and coculture with the INNATE-NK-CAR.19 cell population resulted in approximately 5% residual tumor cells.

[0168] In one in vivo CD19+ lymphoma mouse model, each NGS mouse was injected with 0.25 × 10 tumor cells genetically modified with firefly luciferase that could be analyzed by in vivo fluorescence analysis. 6 Three days later, the injected mice showed established leukemia, and the mice were then administered 5×106 Mice were randomized into treatment cohorts receiving IV infusions of INNATE-NK or INNATE-NK-CAR.19. Animals were analyzed for the presence of luciferase+ tumor cells during the course of the experiment. Data were compared with data from another experiment of the same design, in which animals were administered 5×10 6 non-modified adaptive T cells were administered.

[0169] Example 4 INNATE-NK Study This example describes data showing that one dose of INNATE-NK cells without genetic modification was able to extend the time until an animal died compared to the same dose of adapted T cells. 6 5 × 10 6 INNATE-NK or INNATE-NK-CAR were administered IV and the data were compared with data from an experiment with 5M adaptive T cells. In mice administered INNATE-NK CAR.19, tumor control was complete and no tumor biofluorescence was detected after day 11, demonstrating that INNATE-NK-CAR19 eradicated the tumor without recurrence until day 72 (the end of the experiment). Importantly, INNATE-NK cells also did not demonstrate a xenogeneic response (which has been observed at this point in the past in the case of CAR T cells). The data also showed that one dose of INNATE-NK cells without genetic modification was able to extend the overall survival of the animals compared to the same dose of adaptive T cells (days 50 and 28, respectively). Figure 16 graphically illustrates the survival curves of animals from the above experiment administered INNATE-NK and INNATE-NK-CAR.19 cells. The data curves of INNATE-NK.CAR.19 recipients demonstrated improved survival compared to recipients administered unmodified INNATE-NK cells.

[0170] Example 5 INNATE-NK cells against solid tumors This example demonstrates that INNATE-NK cells are a suitable platform for treating solid tumors. In this example, a neuroblastoma model was examined. INNATE-NK and INNATE-NK CAR cells were generated as described in Example 3, steps 1-8. INNATE-NK cells were genetically modified on day 4 by a retroviral vector carrying a third generation CAR specific for the GD2 antigen expressed by neuroblastoma tumor cells. Results demonstrated that the INNATE-NK CAR.GD2 generation exhibited significant levels of CAR expression in CD56+ cells as assessed by flow cytometry (FACS) analysis by staining with an anti-idiotypic antibody (1A7). The purity of the NK population in INNATE-NK CAR.GD2 production was assessed in terms of a negligible percentage of CD3+ cells, while the level of CAR+ cells was assessed as 1A7+ cells. Expression of CAR.GD2 in INNATE-NK CARs was stable during long-term in vitro culture (measured on days 8, 15, 25, 31, and 35).

[0171] Example 6 Evaluating the role of innate immunity in solid tumor invasion This example evaluated the role of innate immunity in its ability to invade solid tumors. To that effect, 3-D neuroblastoma tumor cell spheroids were grown in MATRIGEL™ culture, and then adaptive T cells, INNATE-NK, or INNATE-NK-CAR.GD2 cells were introduced and evaluated for their ability to invade the spheroids. Specifically, solid 3-D tumor models were created by SH-SY5Y neuroblastoma tumor cells genetically modified to express green fluorescent protein (GFP), and then cultured in vitro to form solid 3-D tumor spheres. Adaptive peripheral blood T cells, unmodified INNATE-NK cells, and INNATE-NK.CAR.GD2 were labeled with a red fluorescent cell marker and incubated with the tumor spheres. Neurospheres were assayed by 3-D fluorescence microscopy in three planes to evaluate the location of cells in the tumor spheres and their relative ability to invade the tumor. Confocal microscopy images of adaptive T cells (red) showed that adaptive T cells were observed in all three planes, but were unable to actively invade the 3-D tumor as they were only localized on the outer surface of the tumorsphere (green) and did not migrate to the center of the tumor. Further images showed that INNATE-NK cells (red) had begun to break through the surface of the tumorsphere, as observed in all three planes; INNATE-NK-CAR.GD2 cells broke through the surface of the tumorsphere, indicating that these cells have the ability to migrate some distance into the tumor, which was observed in all three planes.

[0172] Example 7 Crosslinking or soluble activation Cell populations were generated in culture following activation with a combination of cross-linked natural cytotoxicity receptor (NCR) antibodies (e.g., anti-NKp46) and lymphocyte function-associated antigen (LFA) antibodies (e.g., anti-CD2) ("coated beads"; Miltenyi Biotec) as described in Example 1. The cell populations discussed herein that have been activated with a combination of NCR antibodies (e.g., anti-NKp46) and LFA antibodies (e.g., anti-CD2) and contain a mixture of NK cells and γδ T cells are referred to herein as "BINATE" cell populations (and in certain examples above, as "INNATE-K" cells). In this example, the resulting BINATE population was analyzed after activation with either 1) a combination of cross-linked anti-NKp46 (clone 9E2 500ng / ml; Miltenyi Biotec) and cross-linked anti-CD2 (OKT11 500ng / ml), or 2) a combination of soluble anti-NKp46 (clone 9E2 500ng / ml) and soluble anti-CD2 (clone OKT11 500ng / ml). Cross-linking was achieved by coating flasks with the antibody combination in PBS for 24 hours prior to the start of the experiment, followed by decanting the antibody solution and rinsing with PBS at the time of the experiment (referred to herein as coated plates).

[0173] 1. Buffy coats from normal healthy donors were subjected to Ficoll™ gradient cell separation, performed by manual centrifuge separation (in certain variations of the method described in this example and other examples herein, normal healthy donors are subjected to an apheresis procedure (apheresis, generally without Ficoll™ gradient cell separation), and the mononuclear cell product is collected according to standard procedures, typically a Terumo Optia machine). The mononuclear cells were subjected to αβ T cell depletion using laboratory-scale Miltenyi LS column separation (in certain variations described in this example and other examples herein, the apheresis product may be subjected to αβ T cell depletion using a CLINIMACS separation device; further depletion may include CD19 B cell depletion. An aliquot of mononuclear cells depleted of αβ T cells may be cryopreserved and used later for expansion). The resulting αβTCR-neg cell population was cultured in BINATE medium (i.e., NK MACS™ medium (#130-107-879 (Miltenyi Biotec, Inc., San Diego, CA, USA)) supplemented with 5% AB serum (alternatively, other media, e.g., Life Technologies, R&D systems' medium, CELLGENIX medium, etc., may be used with or without human serum)) and 500 IU / mL human interleukin 2 (IL-2) (Miltenyi Biotec) at a cell seeding concentration of 0.25-0.5 × 10 6 Cells were activated in feeder-free culture at 100 ng / ml without additional activation reagents in 24-well coated plates or with a combination of 500 ng / ml soluble anti-NKp46 (clone 9E2) and 500 ng / ml anti-CD2 (clone OKT11) in 24-well uncoated plates (alternatively, other concentrations of activating agents may be used or other agents may be added for activation).

[0174] 2. Activated αβTCR cell populations were cultured at a cell seeding concentration of 0.25–0.5 × 10 using BINATE medium and 500 IU / mL IL-2. 6Cells / ml in a T75 flask (alternatively cell seeding concentration can be up to 1 × 10 in culture bags, flasks, or other larger culture vessels, e.g., bioreactors (G-Rex) 6 The cells were then further expanded in feeder-free culture conditions at 100-200 cells / ml (which may range from 100-200 cells / ml). Medium changes were performed every 3 days (or less frequently in bioreactors (every 5-7 days)) with fresh BINATE medium and IL-2. In certain variations of the methods of this example and other examples herein, the cultured BINATE cells may be cryopreserved after 1, 2, 3, 4, or 5 weeks of expansion. In one example, the expanded BINATE cells are washed, concentrated, and supplemented with serum-free cryopreservation medium (BIOLIFE). The cryopreserved BINATE product may be thawed and reseeded as described herein for further culture, or thawed and infused, for example, into a patient with cancer or viral infection.

[0175] 3. At the end of 15 days of expansion, BINATE cells were analyzed in detail for NK and γδCD3+ T cell content and their subsets, e.g., CD56, with further analysis of possible contaminating cell types, e.g., αβTCR+ T cells.

[0176] The phenotype of BINATE cells, reported as a percentage of the total cell population, showed no difference between activation on coated plates or with soluble antibodies. The NK population (CD56+CD3-) was 89.1% of the final population when activated on coated plates versus 92% when activated with soluble antibodies. CD3+γδ T cells were 6.2% and 5.5% of the population in the coated plate and soluble antibody conditions, respectively. CD3+CD56+γδ T cells and CD3+CD56-γδ T cells were 2.8% and 3.4% of the population, respectively, in the activation cultures on coated plates and 3.3% and 2.2% of the population, respectively, in the activation cultures with soluble antibodies. The percentage of αβTCR+CD3+ T cells was negligible in both conditions, 0.12% for activation on coated plates and 0.41% for activation with soluble antibodies.

[0177] Previous reports have shown that using cross-linked antibodies can have greater activity, but the use of soluble antibodies is often preferred over cross-linked antibodies (e.g., when used on surfaces such as plates or beads) due to, for example, greater control over the culture. Using a combination of soluble anti-NKp46 (clone 9E2, Miltenyi) and soluble anti-CD2 (clone OKT11) showed no difference compared to cross-linked conditions, and all experiments were subsequently performed with soluble antibody activation reagents.

[0178] Example 8 Assessment of activation and expansion using various amounts of anti-NKp46 In separate experiments, different anti-NKp46 (Bab281) reagents were evaluated at three concentrations (10 ng / ml, 50 ng / ml, and 500 ng / ml) in two expansion conditions: 1) IL-2 (500 IU / mL human interleukin 2 (Miltenyi Biotec)), or 2) IL-15 (10 ng / ml human interleukin 15 (Miltenyi Biotec)).

[0179] 1. Buffy coats from normal healthy donors were subjected to Ficoll™ gradient cell separation performed by manual centrifuge separation. Mononuclear cells were subjected to αβT cell depletion using lab-scale Miltenyi LS column separation. The resulting αβTCRneg cell population was cultured at 0.25-0.5×10 in 24-well plates. 6 At a cell seeding concentration of 100 / ml, cells were placed in feeder-free culture conditions using BINATE medium and IL-2 or IL-15 with a combination of 500 ng / ml anti-CD2 (clone OKT11) and one of three concentrations of anti-NKp46 (Bab281): 10 ng / ml, 50 ng / ml, or 500 ng / ml.

[0180] 2. Activated αβTCR cell populations were cultured at a cell seeding concentration of 0.25–0.5 × 10 using BINATE medium and 500 IU / mL IL-2 or 10 ng / ml IL-15. 6 Cell seeding concentration is 1 × 10 cells / ml in a T75 flask (or 1 × 10 cells / ml in a culture bag, flask, or other large culture vessel, e.g., bioreactor (G-Rex)). 6 The cells were then further expanded in feeder-free conditions at 100-200 cells / ml (cell counts can range from 100-200 cells / ml) with medium changes every 3 days for plates or flasks (or less frequently in bioreactors (every 5-7 days)) with fresh BINATE medium and IL-2 or IL-15.

[0181] 3. At the end of expansion on day 15, BINATE cells were analyzed for NK and γδCD3+ T cell content and subsets thereof, such as CD56, and further analysis of possible contaminating cell types, such as αβTCR+ T cells.

[0182] The phenotype of BINATE cells, reported as a percentage of the total cell population, showed no significant differences between the three concentrations of anti-NKp46 (Bab281) when cultures were expanded by IL-15 expansion. The NK population (CD56+CD3-) constituted 84.5%, 87%, and 87.2% for 500ng / ml anti-NKp46 (Bab281), 50ng / ml anti-NKp46 (Bab281), and 10ng / ml anti-NKp46 (Bab281), respectively. The γδTCR CD3+ population constituted 11.4%, 8.7%, and 8.1%, respectively, whereas the CD3+56+ population constituted 7.40%, 5.40%, and 5.30%, respectively. αβTCR+ cells were virtually undetectable at all concentrations. [Table 1]

[0183] Example 9 Marker assessment of activated and expanded cells In a different experiment, the BINATE phenotype was compared in two expansion conditions (i.e., IL-2 and IL-15) after activation with a combination of Good Manufacturing Practice (GMP) quality anti-NKp46 (Bab281) antibody at 10 ng / ml and GMP quality anti-CD2 (OKT11) antibody at 500 ng / ml.

[0184] 1. Buffy coats from normal healthy donors were subjected to Ficoll™ gradient cell separation performed by manual centrifuge separation. Mononuclear cells were subjected to αβT cell depletion using lab-scale Miltenyi LS column separation. The resulting αβTCRneg cell population was cultured at 0.25-0.5×10 in 24-well plates. 6The cells were placed in feeder-free culture conditions using BINATE medium and either 500 IU / ml IL-2 or 10 ng / ml IL-15 with 500 ng / ml anti-CD2 (OKT11) and 10 ng / ml anti-NKp46 (Bab281) (alternatively, other concentrations of activating agents may be used or other agents may be added for activation) at a cell seeding concentration of 10 cells / ml.

[0185] 2. Activated αβTCR cell populations were cultured at a cell seeding concentration of 0.25–0.5 × 10 using BINATE medium and 500 IU / mL IL-2 or 10 ng / ml IL-15. 6 Cell seeding concentration is 1 × 10 cells / ml in a T75 flask (or 1 × 10 cells / ml in a culture bag, flask, or other large culture vessel, e.g., bioreactor (G-Rex)). 6 The cells were then further expanded in feeder-free conditions at 100-200 cells / ml (cell counts can range from 100-200 cells / ml) with medium changes every 3 days for plates or flasks (or less frequently in bioreactors (every 5-7 days)) with fresh BINATE medium and IL-2 or IL-15.

[0186] 3. At the end of the 20 day culture expansion, BINATE cells were extensively analyzed for the content of natural, cytotoxic, and exhaustion expressing cells and their subset markers, e.g., CD56, CD16, and CD57, with additional analysis for possible contaminating cell types, e.g., αβTCR+ T cells.

[0187] Total BINATE cell populations contained 92% and 84.3% CD56+ natural cells after expansion with IL-15 and IL-2, respectively. The populations contained 33% and 12% CD56+CD16+ cytotoxic natural cells, 76% and 59.6% CD8+ cytotoxic natural cells, and 99% and 96.5% CD56+CD57- immature natural cells after culture expansion with IL-15 and IL-2, respectively. There were no detectable abTCR+ T cells after culture expansion with either IL-15 or IL-2. The CD4+, CD8+, and CD4-CD8- populations of NK and γδ T cells are provided in Table 2 below. [Table 2-1] [Table 2-2]

[0188] Example 10 Evaluation of cells expanded under static and bioreactor culture conditions In separate experiments, BINATE proliferation in static and bioreactor culture conditions in two expansion conditions (IL-2 and IL-15) after activation with a combination of GMP quality anti-NKp46 (Bab281) antibody at 50 ng / ml and GMP quality anti-CD2 (OKT11) antibody at 50 ng / ml was compared.

[0189] 1. Buffy coats from normal healthy donors were subjected to Ficoll™ gradient cell separation performed by manual centrifuge separation. Mononuclear cells were subjected to αβT cell depletion using lab-scale Miltenyi LS column separation. The resulting αβTCRneg cell population was cultured at 0.25-0.5×10 in 24-well plates. 6The cells were placed in feeder-free culture conditions using BINATE medium and either 500 IU / ml IL-2 or 10 ng / ml IL-15 with a combination of 50 ng / ml anti-CD2 (OKT11) and 50 ng / ml anti-NKp46 (Bab281) (alternatively, other concentrations of activating agents may be used or other agents may be added for activation) at a cell seeding concentration of 10 cells / ml.

[0190] 2. Activated αβTCR cell populations were cultured at a cell seeding concentration of 0.25–0.5 × 10 using BINATE medium and 500 IU / mL IL-2 or 10 ng / ml IL-15. 6 Cell seeding concentration is 1 × 10 cells / ml in a T75 flask or G-Rex benchtop bioreactor (alternatively, cell seeding concentration is 1 × 10 cells / ml in a culture bag, flask, or other large culture vessel, e.g., 1 × 10 cells / ml in a G-Rex benchtop bioreactor (alternatively, cell seeding concentration is ... 6 The cells were then further expanded in feeder-free conditions at cell density (which can range from 0.1 to 1.5 cells / ml) with fresh BINATE medium and IL-15 every 3 days in plates or flasks (or less frequently in bioreactors (every 5-7 days)).

[0191] 3. Activated αβTCR cell populations were also cultured in GRex culture devices using BINATE medium and 10 ng / ml IL-15 at a cell seeding concentration of 1×10 6 The cells were further expanded in feeder-free conditions at 100 cells / ml, with medium changes every 7 days using fresh BINATE medium and IL-15.

[0192] Total BINATE expansion is shown as total cell number in Figure 17. Expansion of activated αβTCRneg cells in BINATE medium supplemented with either IL-2 or IL-15 in flasks was equivalent over a 41 day period. Similarly, IL-15 expansion of activated αβTCRneg cell populations in the first 21 days in bioreactors was equivalent to IL-15 expansion in flasks.

[0193] Example 11 Assessment of alloreactivity of activated and expanded cells In separate experiments, alloreactivity of activated BINATE cell populations expanded in either IL-2 or IL-15 was assessed.

[0194] 1. Buffy coats from normal healthy donors were subjected to Ficoll™ gradient cell separation performed by manual centrifuge separation. Mononuclear cells were subjected to αβT cell depletion using lab-scale Miltenyi LS column separation. The resulting αβTCRneg cell population was cultured at 0.25-0.5×10 in 24-well plates. 6 The cells were placed in feeder-free culture conditions using BINATE medium with a combination of 50 ng / ml anti-CD2 (OKT11) and 50 ng / ml anti-NKp46 (Bab281) (alternatively, other concentrations of activating agents may be used or other agents may be added for activation) at a cell seeding concentration of 100 / ml.

[0195] 2. Activated αβTCR cell populations were cultured at cell seeding concentrations of 0.25–0.5 × 10 using BINATE medium and either 500 IU / mL IL-2 or 10 ng / ml IL-15. 6 The cells were further expanded in feeder-free conditions at 100 cells / ml in T75 flasks, with medium changes every 3 days using fresh BINATE medium and IL-2 or IL-15.

[0196] 3. BINATE cells and stimulator cells derived from PBMCs of three random healthy donors (HDs) were tested in a one-way mixed lymphocyte reaction (MLR). Stimulator cells from three HDs were pooled and used after irradiation (30 Gy). BINATE cells were added at 100 μl / well (1 × 10 cells). 5 The cells were seeded in 200 μl of BINATE medium in 96-well flat-bottom microtiter plates with an equal number of stimulator cells. After 5 days of incubation, the cultures were 3H-thymidine was pulsed for 12 hours and harvested on glass fiber filters. Dried filters were counted in a Microbeta Trilux 1450 counter (Wallac, Perkin Elmer). Results were expressed as SI (cpm with antigen / cpm with background). BINATE cells without added cytokines were used as negative control, and positive control was represented by BINATE cells seeded with cytokines (IL-2 or IL-15).

[0197] Mixed lymphocyte co-cultures were analyzed using a 100% ELISA kit, which is an indicator of proliferation. 3 H thymidine incorporation (CPM) was analyzed. In the IL-15 experiments, the negative control consisted of unstimulated BINATE cells not supplemented with IL-15. 3 H thymidine incorporation was 1144, indicating essentially no proliferation. Irradiated pooled healthy donor cells (HD) 3 H thymidine incorporation was 1194, also indicating no proliferation. Positive controls included proliferation stimulation with IL-15, 3 H thymidine incorporation was 15558, a 14-fold increase compared to the negative control. Activated BINATE cells expanded in IL-15 3 3H thymidine incorporation was 942, essentially indistinguishable from background.

[0198] For IL-2 experiments, negative controls included unstimulated BINATE cells not supplemented with IL-2; 3 H thymidine incorporation was 834, indicating essentially no proliferation. Irradiated pooled human donor cells (HD) 3 H thymidine incorporation was 1194, also indicating no proliferation. Positive controls included proliferation stimulation with IL-2, 3 H thymidine incorporation was 18834, a 22-fold increase compared to the negative control. Activated BINATE cells expanded in IL-15 3 3 H thymidine incorporation was 1245, essentially not different from the background control.

[0199] The absence of MLR responses may be due to the absence of αβTCR+ T cells and confirms the lack of alloreactivity in the natural BINATE cell population, which is clinically significant as it confirms the lack of ability of the cells to mount a graft-versus-host response.

[0200] Example 12 Assessment of cell growth and phenotype under different expansion conditions In separate experiments, the proliferative potential and BINATE phenotype following activation with GMP quality anti-NKp46 (Bab281) antibody (50 ng / ml) (Caprico) and GMP quality anti-CD2 (OKT11) antibody (50 ng / ml) (Caprico) were compared in five different expansion conditions.

[0201] 1. Buffy coats from normal healthy donors were subjected to Ficoll™ gradient cell separation performed by manual centrifuge separation. Mononuclear cells were subjected to αβT cell depletion using lab-scale Miltenyi LS column separation. The resulting αβTCRneg cell population was cultured at 0.25-0.5×10 in 24-well plates. 6 A combination of 50ng / ml anti-CD2 (OKT11) and 50ng / ml anti-NKp46 (Bab281) (alternatively, other concentrations of activating agents may be used or other agents may be added for activation) at a cell seeding concentration of 1000 / ml, as well as the following expansion supplements: a.500IU / mL IL-2(Miltenyi Biotec) b.10ng / ml IL-15(Miltenyi Biotec) c. A combination of 500IU / mL IL-2 and 10ng / mL IL-15 (IL-2 / IL-15) d. Combination of 500IU / mL IL-2 and 10ng / ml OKT3 clone anti-CD3 antibody (Miltenyi Biotec) (IL-2 / OKT3) e. A combination of IL-15 10ng / mL and 500IU / mL IL-2 and 10ng / ml OKT3 (IL-2 / IL-15 / OKT3) The cells were then placed in feeder-free culture conditions using BINATE medium having one of the following:

[0202] 2. Activated αβTCR cell populations were cultured in BINATE medium at a cell seeding concentration of 0.25–0.5 × 10 6 Cell seeding concentration is 1 × 10 cells / ml in a T75 flask (or 1 × 10 cells / ml in a culture bag, flask, or other large culture vessel, e.g., bioreactor (G-Rex)). 6 The cultures were then further expanded in feeder-free conditions at 100-200 cells / ml (cell counts can range from 100-200 cells / ml) using one of the 5 expansion conditions listed above. Medium changes were performed with fresh BINATE medium every 3 days for plates or flasks (less frequently (every 5-7 days) for bioreactors), and the same expansion conditions were used during the initial culture activation and expansion phase.

[0203] Total BINATE expansion is shown as total cell number in Figure 18. Expansion of activated αβTCRneg cells in BINATE medium supplemented with either IL-2, IL-15, or IL-2 / OKT3 in flasks was equivalent over a 40-day period. Expansion of activated αβTCRneg cells in BINATE medium supplemented with either IL-2 / IL-15 or IL-2 / IL-15 / OKT3 in flasks was equivalent over a 26-day period, after which proliferation slowed and total cell numbers were lower compared to the other three groups from 26 to 41 days of culture.

[0204] At days 7, 20, and 30 of culture expansion, BINATE cells from each of the five conditions were determined to be NK or γδ T cells by flow cytometry, as shown in Table 3 below. Cells from each of the conditions at days 7, 20, and 30 were also analyzed by flow cytometry for specific markers of innate, cytotoxic, and exhaustion on cells, and immature expression, as well as specific VdTCR usage in γδ T cells, as shown in Table 4. [Table 3-1] [Table 3-2] [Table 4]

[0205] PD-1 expression, an indicator of cellular exhaustion, was shown to be low. At day 30, IL-15 expansion showed 2.7% expression in the total BINATE population, 2% expression in CD56+CD3- NK cells, 10.6% expression in CD3+CD56- γδ T cells, and 17.05% expression in CD3+CD56+ γδ T cells, demonstrating a lack of exhaustion. Under IL-2 expansion, PD-1 expression was shown to be as low as 14.2% of the total BINATE population in IL-2 expansion as determined by flow cytometry, with 2% CD56+CD3- NK cells expressing PD-1, and 62.9% and 78.3% of CD3+CD56- γδ T cells and CD3+CD56+ γδ T cells, respectively.

[0206] Example 13 CD107a cytotoxicity Day 30 cells from each of the five expansion conditions in Example 12 were co-cultured with tumor cell lines including IMR32, SH-SY5Y (a neuroblastoma cell line), and K562 (a B-cell leukemia cell line). After 3 days of culture, CD10 expression, a measurement of cytotoxic granule release, was measured. Expression of 7a was measured by flow cytometry analysis in basal BINATE cells and in BINATE cells co-cultured with each tumor cell line. Table 5 shows the difference between tumor-stimulated expression minus basal values ​​for each tumor cell line and each BINATE subset (CD56+CD3-NK cells, and CD3+CD56+γδT cells, and CD3+CD56-γδT cells) in each of the five expansion conditions (IL-2, IL-2 / OKT3, IL-15, IL-2 / IL-15, and IL-2 / IL-15 / OKT3). Statistical significance is reported as p-values ​​by T-test. [Table 5-1] [Table 5-2]

[0207] Example 14 Evaluation of phenotypic markers under different expansion conditions Day 20 cells from the three expansion conditions in Example 12 (conditions IL-2, IL-15, and IL-2 / OKT3) were analyzed for specific activation, cytotoxicity, and exhaustion markers. Figure 19 shows the phenotypic analysis by marker type (activation, cytotoxicity, and exhaustion / immature) for the IL-15 expansion condition. Figure 20 shows the phenotypic analysis by marker type (activation, cytotoxicity, and exhaustion / immature) for the IL-2 expansion condition. Figure 21 shows the phenotypic analysis by marker type (activation, cytotoxicity, and exhaustion / immature) for the IL-2 / OKT3 expansion condition. Under all conditions, BINATE cells generally showed high activation and cytotoxicity, and low exhaustion.

[0208] Example 15 In vitro tumor killing assessment In a separate experiment, the in vitro tumor killing of BINATE cells in two expansion conditions (IL-2 and IL-15) after activation with a combination of 10 ng / ml anti-NKp46 (Bab281) and 500 ng / ml anti-CD2 (OKT11) antibodies was compared.

[0209] 1. Buffy coats from normal healthy donors were subjected to Ficoll™ gradient cell separation performed by manual centrifuge separation. Mononuclear cells were subjected to αβT cell depletion using lab-scale Miltenyi LS column separation. The resulting αβTCRneg cell population was cultured at 0.25-0.5×10 in 24-well plates. 6 The cells were placed in feeder-free culture conditions using BINATE medium and either 500 IU / ml IL-2 or 10 ng / ml IL-15 with a combination of 500 ng / ml anti-CD2 (OKT11) and 10 ng / ml anti-NKp46 (Bab281) (alternatively, other concentrations of activating agents may be used or other agents may be added for activation) at a cell seeding concentration of 10 cells / ml.

[0210] 2. Activated αβTCR cell populations were cultured in BINATE medium and 500 IU / mL IL-2 or 10 ng / ml IL-15 at a cell seeding concentration of 0.25–0.5 × 10 6 Cell seeding concentration is 1 × 10 cells / ml in a T75 flask (or 1 × 10 cells / ml in a culture bag, flask, or other large culture vessel, e.g., bioreactor (G-Rex)). 6 The cells were then further expanded in feeder-free conditions at 100-200 cells / ml (cell counts can range from 100-200 cells / ml) with medium changes every 3 days for plates or flasks (or less frequently in bioreactors (every 5-7 days)) with fresh BINATE medium and IL-2 or IL-15.

[0211] 3. After completion of the 20 day culture expansion, BINATE cells were placed into tumor co-culture experiments for 5-7 days. The experiments shown in Table 6 below involved culturing leukemia cell lines in 6-well culture dishes at a 1:1 effector to target ratio (i.e., 0.5M tumor cells (target) with 0.5M BINATE cells (effector)) in 2ml medium / culture well.

[0212] The experiment shown in Table 7 below involved culturing solid tumor cell lines (neuroblastoma, sarcoma, and colon tumor lines) in 6-well culture dishes at a 2:1 effector to target ratio (i.e., 0.25M tumor cells (target) with 0.5M BINATE cells (effector)) in 2 ml medium / culture well. Co-culture of tumor and effector cells was performed in the absence of cytokines. Co-cultures were assessed on days 3-7 by microscopy and flow cytometry.

[0213] When examining residual tumor in co-culture experiments, Table 6 shows that activated BINATE cells expanded in the presence of either IL-2 or IL-15 were successful in eradicating 50% or more of the tumor cells in all cell lines, and more than 75% in four to five of the AML cell lines. In Table 6, short-term co-culture of all cell lines resulted in BINATE cells killing all 221 leukemia cells, >80% of Daudi leukemia cells, and up to 50% of the Karpas cell line, which is often considered resistant to natural killing. [Table 6]

[0214] When examining residual tumors in co-culture experiments, Table 7 shows that activated BINATE cells expanded with either IL-2 or IL-15 were successful in eradicating neuroblastoma tumors in short-term co-culture experiments. IL-2-expanded BINATE cells were not tested in co-culture with colon cancer, but IL-15-expanded BINATE cells showed activity against sarcoma and colon cancer. [Table 7]

[0215] (Example 16) Assessment of in vitro transduction efficiency In a separate experiment, to assess the stability of transduction, early and late in vitro transduction efficiencies of BINATE cells with retroviral constructs encoding third generation GD2 chimeric antigen receptors (CARs) were compared after activation with a combination of 10 ng / ml anti-NKp46 (Bab281) and 500 ng / ml anti-CD2 (OKT11) antibodies.

[0216] 1. Buffy coats from normal healthy donors were subjected to Ficoll™ gradient cell separation performed by manual centrifuge separation. Mononuclear cells were subjected to αβT cell depletion using lab-scale Miltenyi LS column separation. The resulting αβTCRneg cell population was cultured at 0.25-0.5×10 in 24-well plates. 6 The cells were placed in feeder-free culture conditions using BINATE medium and 500 IU / ml IL-2 with a combination of 500 ng / ml anti-CD2 (OKT11) and 10 ng / ml anti-NKp46 (Bab281) (alternatively, other concentrations of activating agents may be used or other agents may be added for activation) at a cell seeding concentration of 10 cells / ml.

[0217] 2. For the generation of any BINATE.CAR described herein, an aliquot of cells from the activated, culture-expanded αβTCR cell population is harvested on days 4-15 and cultured at 0.25×10 in the presence of gammaretroviral supernatant containing a construct for a specific chimeric antigen receptor. 6The cells were seeded on human fibronectin-coated plates or alternatively culture bags (Gobain) at a concentration of 10 ... For this example, the activated αβTCR cell population on day 5 was plated onto human fibronectin-coated plates (or alternatively culture bags (Gobain)) at 0.25 × 10 6 The cells were placed in the presence of gammaretroviral supernatant containing a construct for the GD2 chimeric antigen receptor (CAR) at a concentration of 1000 / ml. The GD2 construct contained a mutant fragment of the CD34 receptor as a selection and tracking marker.

[0218] 3. After 3 days, wash the transduced cells and culture at a cell seeding concentration of 0.25–0.5 × 10 using BINATE medium and 500 IU / mL IL-2. 6 Cell seeding concentration is 1 × 10 cells / ml in a T75 flask (or 1 × 10 cells / ml in a culture bag, flask, or other large culture vessel, e.g., bioreactor (G-Rex)). 6 The cells were then further expanded in feeder-free conditions at 100-200 nmol / ml (cell density can range from 100-200 nmol / ml) with medium changes every 3 days (or less frequently, every 5-7 days, in bioreactors) with fresh BINATE medium and IL-2 in plates or flasks.

[0219] 4. Three and 15 days after transduction, the BINATE cell subset as well as the VdTCR subset of γδ T cells were analyzed for expression of mutant CD34 receptors (an indicator of transduction efficiency). At days 3 and 15, the transduction efficiency of CD56+CD3- NK cells was 82.4% and 82.4%, respectively. At days 3 and 15, the transduction efficiency of CD3+56+ cells was 43.2% and 62.4%, respectively. At days 3 and 15, the transduction efficiency of CD3+CD56- γδ T cells was 37.3% and 56.8%, respectively. The genetic modification was stable over the culture period, implying a highly activated population that allows high transduction and persistent integration.

[0220] In a similar experiment, transduction of the BINATE subpopulation showed high transduction of the GD2 CAR construct as well as polyclonal transduction of both Vd1 and Vd2 TCR γδ T cells. CD56+CD3- NK cells were transduced at 69%, whereas CD3+ γδ T cells were transduced at 39%. The Vd1 TCR population had a transduction efficiency of 67% and the Vd2 TCR population had a transduction efficiency of 34%.

[0221] (Example 17) Assessment of proliferation following in vitro transduction of GD2 CAR In a separate experiment, the proliferative potential of BINATE cells following in vitro transduction with a retroviral construct encoding a third generation GD2 CAR after activation with a combination of 50 ng / ml anti-NKp46 (Bab281) and 50 ng / ml anti-CD2 (OKT11) antibodies was compared to unmodified BINATE cells in IL-2 and IL-15 expansion conditions.

[0222] 1. Buffy coats from normal healthy donors were subjected to Ficoll™ gradient cell separation performed by manual centrifuge separation. Mononuclear cells were subjected to αβT cell depletion using lab-scale Miltenyi LS column separation. The resulting αβTCRneg cell population was cultured at 0.25-0.5×10 in 24-well plates. 6 The cells were placed in feeder-free culture conditions using BINATE medium and 500 IU / ml IL-2 or 10 ng / ml IL-15 with a combination of 50 ng / ml anti-CD2 (OKT11) and 50 ng / ml anti-NKp46 (Bab281) (alternatively, other concentrations of activating agents may be used or other agents may be added for activation) at a cell seeding concentration of 10 cells / ml.

[0223] 2. Activated non-genetically modified αβTCR cell populations were cultured at a cell seeding concentration of 0.25–0.5 × 10 using BINATE medium and 500 IU / mL IL-2 or 10 ng / ml IL-15. 6 Cell seeding concentration is 1 × 10 cells / ml in a T75 flask (or 1 × 10 cells / ml in a culture bag, flask, or other large culture vessel, e.g., bioreactor (G-Rex)). 6 The cells were then further expanded in feeder-free conditions at 100-200 cells / ml (cell counts can range from 100-200 cells / ml) with medium changes every 3 days for plates or flasks (or less frequently in bioreactors (every 5-7 days)) with fresh BINATE medium and IL-2 or IL-15.

[0224] On day 3.5, the activated αβTCR cell population was cultured at 0.25 × 10 6 The cells were placed in the presence of gammaretroviral supernatant containing a construct for the GD2 chimeric antigen receptor (CAR) at a concentration of 1000 / ml. The GD2 construct contained a mutant fragment of the CD34 receptor as a selection and tracking marker.

[0225] After 4.5 days, the transduced cells were washed and recultured in BINATE medium and 500 IU / mL IL-2 or 10 ng / ml IL-15 at a cell seeding concentration of 0.25–0.5 × 10 6 Cell seeding concentration is 1 × 10 cells / ml in a T75 flask (or 1 × 10 cells / ml in a culture bag, flask, or other large culture vessel, e.g., bioreactor (G-Rex)). 6 The cells were then further expanded in feeder-free conditions at 100-200 cells / ml (cell counts can range from 100-200 cells / ml) with medium changes every 3 days for plates or flasks (or less frequently in bioreactors (every 5-7 days)) with fresh BINATE medium and IL-2 or IL-15.

[0226] 5. Cell numbers were recorded every 7 days for the BINATE and BINATE.CARGD2 populations.

[0227] Unmodified BINATE and BINATE.CARGD2 cells were expanded prior to transduction (represented by vertical lines in FIG. 22). Expansion after transduction is represented by diagonal lines in FIG. 22 for the different conditions. There was no significant difference between genetically modified or unmodified cells, nor was there any difference in expansion with IL-2 or IL-15 in terms of total cell number.

[0228] (Example 18) Assessment of in vitro killing of solid tumors In a separate experiment, we compared in vitro killing of solid tumors in two expansion conditions (IL-2 and IL-15) following transduction of activated BINATE with a retroviral construct encoding a third generation GD2 CAR.

[0229] 1. Buffy coats from normal healthy donors were subjected to Ficoll™ gradient cell separation performed by manual centrifuge separation. Mononuclear cells were subjected to αβT cell depletion using lab-scale Miltenyi LS column separation. The resulting αβTCRneg cell population was cultured at 0.25-0.5×10 in 24-well plates. 6At a cell seeding concentration of 100 / ml, the cells were placed in feeder-free culture conditions using BINATE medium and 500 IU / ml IL-2 or 10 ng / ml IL-15 with a combination of 500 ng / ml anti-CD2 (OKT11) and 10 ng / ml anti-NKp46 (Bab281) (alternatively, other concentrations of activating agents may be used or other agents may be added for activation).

[0230] On day 2.5, the activated αβTCR cell population was cultured at 0.25 × 10 6 The GD2 construct contained a mutant fragment of the CD34 receptor as a selection and tracking marker.

[0231] 3. After 3 days, the transduced cells were washed and cultured at a cell seeding concentration of 0.25–0.5 × 10 using BINATE medium and 500 IU / mL IL-2 or 10 ng / ml IL-15. 6 Cell seeding concentration is 1 × 10 cells / ml in a T75 flask (or 1 × 10 cells / ml in a culture bag, flask, or other large culture vessel, e.g., bioreactor (G-Rex)). 6 The cells were then further expanded in feeder-free conditions at 100-200 cells / ml (cell counts can range from 100-200 cells / ml) with medium changes every 3 days for plates or flasks (or less frequently, every 5-7 days for bioreactors) with fresh BINATE medium and IL-2 or IL-15.

[0232] 4. At the end of 20 days of culture expansion, BINATE cells were placed in tumor co-culture experiments for 5-7 days. Experiments involved culturing in 6-well culture dishes at a 1:1 effector to target ratio (i.e., 0.5M BINATE or BINATE.CAR.GD2 cells (effector) with 0.5M tumor cells (target)) in 2ml medium / culture well. Co-culture of tumor and effector cells was performed in the absence of cytokines. Co-cultures were assessed by microscopy and flow cytometry on days 3-7.

[0233] Table 8 shows the tumor response to unmodified BINATE IL-2 or BINATE IL-15, or CAR.GD2 modified BINATE IL-2 or CAR.GD2 modified BINATE IL-15. Data from co-cultures are shown for the percentage of remaining tumor after co-culture with either IL-15 or IL-2. Residual tumor in the control was measured as 100%. Both BINATE IL-2 and BINATE IL-15 showed significant tumor killing. CAR.GD2 modified BINATE IL-2 and BINATE IL-15 also showed significant killing. [Table 8]

[0234] (Example 19) Evaluation of in vitro killing of myeloid leukemia cell lines In separate experiments, in myeloid leukemia cell lines in long-term coculture following transduction of activated BINATE with a retroviral construct encoding a third-generation CD123CAR In vitro killing was assessed.

[0235] 1. Buffy coats from normal healthy donors were subjected to Ficoll™ gradient cell separation performed by manual centrifuge separation. Mononuclear cells were subjected to αβT cell depletion using lab-scale Miltenyi LS column separation. The resulting αβTCRneg cell population was cultured at 0.25-0.5×10 in 24-well plates.6 The cells were placed in feeder-free culture conditions using BINATE medium and 10 ng / ml IL-15 with a combination of 50 ng / ml anti-CD2 (OKT11) and 50 ng / ml anti-NKp46 (Bab281) (alternatively, other concentrations of activating agents may be used or other agents may be added for activation) at a cell seeding concentration of 10 cells / ml.

[0236] On day 2.5, the activated αβTCR cell population was cultured with 0.25 × 10 6 The CD123 construct contained a mutant fragment of the CD19 receptor as a selection and tracking marker.

[0237] 3. After 3 days, wash the transduced cells and culture at a cell seeding concentration of 0.25–0.5 × 10 using BINATE medium and 10 ng / mL IL-15. 6 Cell seeding concentration is 1 × 10 cells / ml in a T75 flask (or 1 × 10 cells / ml in a culture bag, flask, or other large culture vessel, e.g., bioreactor (G-Rex)). 6 The cells were then further expanded in feeder-free conditions at 100-200 cells / ml (cell counts can range from 100-200 cells / ml) with fresh BINATE medium and IL-15 every 3 days for plates or flasks (or less frequently, every 5-7 days for bioreactors).

[0238] 4. At the end of the 20-day culture expansion, BINATE cells and BINATE.CAR123 were placed in tumor co-culture experiments for 7 or more days. The experiments were performed in 6-well culture dishes with 1:1 effector to target ratio (i.e., 0.5×10 6 0.5 × 10 BINATE or BINATE.CAR123 cells (effectors) 6The co-culture of tumor and effector cells was performed in the absence of cytokines. Co-cultures were evaluated by microscopy and flow cytometry on days 3-7.

[0239] BINATE.CD123 cells were transduced with 65% efficiency. Myeloid leukemia tumor lines evaluated in coculture included THP1, MOLM3, and OCI AML cell lines. Data included percentage of residual tumor following coculture of tumor with either unmodified BINATE or BINATE.CAR.CD123. Residual tumor in controls was measured as 100%. BINATE cells were not successful in eradicating myeloid leukemia cells due to persistent growth of residual tumor after initial short-term killing. BINATE.CAR.CD123 cells were able to successfully kill and maintain tumor-free cultures in coculture with THP1 and MOLM3 cell lines, and OCI AML cell lines at >70% of controls.

[0240] (Example 20) Assessment of in vivo killing of leukemic cells In a separate experiment, in vivo killing of leukemic cells was evaluated following transduction of activated BINATE cells with a retroviral construct encoding a second generation CD19 CAR.

[0241] 1. Buffy coats from normal healthy donors were subjected to Ficoll™ gradient cell separation performed by manual centrifuge separation. Mononuclear cells were subjected to lab-scale Miltenyi LS column separation and NK enrichment using the Miltenyi NK Selection Kit. NK and γδ T cell populations were cultured at 0.25-0.5×10 in 24-well plates. 6 The cells were placed in feeder-free culture conditions using BINATE medium and 500 IU / ml IL-2 with a combination of 500 ng / ml anti-CD2 antibody (clone LT2, Miltenyi) and 10 ng / ml anti-NKp46 (alternatively, other concentrations of activating agents may be used or other agents may be added for activation) at a cell seeding concentration of 10 cells / ml.

[0242] On day 2.5, the activated population was cultured with 0.25×10 6 The CD19 construct contained a mutant fragment of the CD34 receptor as a selection and tracking marker.

[0243] 3. After 3 days, wash the transduced cells and culture them in BINATE medium and 500 IU / mL IL-2 at a cell seeding concentration of 0.25–0.5 × 10 6 Cell seeding concentration is 1 × 10 cells / ml in a T75 flask (or 1 × 10 cells / ml in a culture bag, flask, or other large culture vessel, e.g., bioreactor (G-Rex)). 6 The cells were then further expanded in feeder-free conditions at 100-200 cells / ml (cell counts can range from 100-200 cells / ml). Medium changes were performed every 3 days with fresh BINATE medium and IL-2 in plates or flasks (or less frequently (every 5-7 days) in bioreactors). At the end of expansion, the BINATE cells were washed and formulated in PBS solution for IV administration to mice.

[0244] Xenogeneic in vivo leukemia model NOD / SCID IL-2Rγnull (NSG) xenografted mice were injected with DAUDI cells to evaluate the antitumor efficacy of CAR-transduced cells in vivo. Mouse experiments were performed in accordance with ethical international, EU and national requirements and were approved by the Italian Ministry of Health (N°88 / 2016-PR). NGS mice (5 weeks old; The Jackson Laboratory, USA) were injected with firefly luciferase-labeled Daudi cells (FF-Daudi) (0.25 × 10 6 Mice were inoculated with 50 × 10 6 BINATE or BINATE.CAR19 cells were injected on day 0 by IV injection and subjected to weekly bioluminescence imaging (IVIS System, Perkin Elmer, USA).

[0245] Three doses of BINATE or BINATE.CD19 cells were injected into the animal model on day 0. Non-genetically modified BINATE cells were able to generate a partial response, with mice surviving until day 42. BINATE cells genetically modified with CAR.CD19 demonstrated a dose-effect response, with the lowest dose of 1 × 10 6 BINATE cells were able to maintain animals for 1 week longer than non-transduced cells, whereas 5 × 10 6 Pieces or 10x10 6 BINATE.CAR.CD19 cells were not maintained as long as animals receiving BINATE.CAR.CD19 cells. This model was supported by two weekly subcutaneous IL-2 injections. Unlike CAR-T CD19 genetically modified αβ T cells, animals survived until sacrifice at day 90, with no evidence of cytokine release syndrome (CRS) or allo- or xeno-reactivity despite IL-2 administration, supporting the improved safety of natural cells. Animals were sacrificed at day 90 and tissues were evaluated for the presence of human CD45+ cells (BINATE) in peripheral blood and liver, after which the %NK and γδT subsets were examined in each tissue, respectively. As shown in Figure 23, BINATE cells were maintained in both blood and tissues, demonstrating the viability and expansion of the BINATE cell population in an in vivo environment.

[0246] Example 21 Evaluation of in vivo killing of solid tumors (neuroblastoma) In a separate experiment, in vivo killing of a solid tumor, neuroblastoma, was evaluated following transduction of activated BINATE cells with a retroviral construct encoding the third generation GD2 CAR.

[0247] 1. Buffy coats from normal healthy donors were subjected to Ficoll™ gradient cell separation performed by manual centrifuge separation. Mononuclear cells were subjected to αβT cell depletion using lab-scale Miltenyi LS column separation. The resulting αβTCRneg cell population was cultured at 0.25-0.5×10 in 24-well plates. 6The cells were placed in feeder-free culture conditions using BINATE medium and 10 ng / ml IL-15 with a combination of 50 ng / ml anti-CD2 (OKT11) and 50 ng / ml anti-NKp46 (Bab281) (alternatively, other concentrations of activating agents may be used or other agents may be added for activation) at a cell seeding concentration of 10 cells / ml.

[0248] On day 2.5, the activated αβTCR cell population was cultured at 0.25 × 10 6 The GD2 construct contained a mutant fragment of the CD34 receptor as a selection and tracking marker.

[0249] 3. After 3 days, wash the transduced cells and culture at a cell seeding concentration of 0.25–0.5 × 10 using BINATE medium and 10 ng / mL IL-15. 6 Cell seeding concentration is 1 × 10 cells / ml in a T75 flask (or 1 × 10 cells / ml in a culture bag, flask, or other large culture vessel, e.g., bioreactor (G-Rex)). 6 The cells were further expanded in feeder-free conditions at 100-mL PBS (cells per ml) with medium changes every 3 days (less frequently in bioreactors (every 5-7 days)). On day 20, the cells were washed and formulated in PBS for administration.

[0250] Xenogeneic in vivo neuroblastoma model NOD / SCID IL-2Rγnull (NSG) xenografted mice were injected with SH-SY5Y cells to evaluate the antitumor efficacy of CAR-transduced cells in vivo. Mouse experiments were performed in accordance with ethical international, EU and national requirements and were approved by the Italian Ministry of Health (N°88 / 2016-PR). NGS mice (5 weeks old; The Jackson Laboratory, USA) were injected with firefly luciferase-labeled SH-SY5Y cells (0.75 × 10 6 Mice were inoculated intraperitoneally with 30 × 10 6BINATE or BINATE.CARGD2 cells were injected at three doses every 2 weeks by intraperitoneal injection and subjected to weekly bioluminescence imaging (IVIS System, Perkin Elmer, USA).

[0251] Non-genetically modified BINATE cells demonstrated a partial response with disappearance of peripheral tumor masses at approximately day 18, but mice subsequently showed local progression. Genetically modified BINATE cells with CAR.CDGD2 demonstrated significant elimination of peripheral tumor masses but failed to kill local lesions. The model was supported by twice weekly subcutaneous injections of IL-2.

[0252] Example 22 Evaluation of antibodies for in vitro killing of solid tumors (neuroblastoma) and ADCC by NK cells In a separate experiment, in vitro killing of a solid tumor, neuroblastoma, by natural NK cells or natural NK.CAR.GD2 cells was evaluated.

[0253] 1. Buffy coats from normal healthy donors were subjected to Ficoll™ gradient cell separation performed by manual centrifuge separation. Mononuclear cells were subjected to lab-scale Miltenyi LS column separation and NK enrichment using the Miltenyi NK Selection Kit. The resulting populations were cultured at 0.25-0.5×10 in 24-well plates. 6 The cells were placed in feeder-free culture conditions using BINATE medium and 500 IU / ml IL-2 with a combination of 500 ng / ml anti-CD2 antibody (clone LT2, Miltenyi) and 10 ng / ml anti-NKp46 (alternatively, other concentrations of activating agents may be used or other agents may be added for activation) at a cell seeding concentration of 10 cells / ml.

[0254] On day 2.5, the activated population was cultured at 0.25 × 10 6The GD2 construct contained a mutant fragment of the CD34 receptor as a selection and tracking marker.

[0255] 3. After 3 days, wash the transduced cells and culture them in BINATE medium and 500 IU / mL IL-2 at a cell seeding concentration of 0.25–0.5 × 10 6 Cell seeding concentration is 1 × 10 cells / ml in a T75 flask (or 1 × 10 cells / ml in a culture bag, flask, or other large culture vessel, e.g., bioreactor (G-Rex)). 6 The cells were then further expanded in feeder-free conditions at 100-200 nmol / ml (cell density can range from 100-200 nmol / ml) with medium changes every 3 days (or less frequently, every 5-7 days, in bioreactors) with fresh BINATE medium and IL-2 in plates or flasks.

[0256] 4. At the end of the 20 day culture expansion, BINATE and BINATE.CARGD2 cells were placed in tumor co-culture experiments for 7 or more days. The experiments were performed in 6-well culture dishes with 1:1 effector to target ratio (i.e., 0.5×10 6 0.5×10 Natural NK or Natural NK.CAR.GD2 (Effector) 6 The co-culture entailed culturing the activated cells (14.G2a monoclonal antibody) with 1000 SH-SY5Y tumor cells (targets). Anti-GD2 antibody (10 μg, 14.G2a monoclonal antibody) was added to each of the co-culture groups to evaluate the ADCC ability of the activated cells. Co-culture of tumor and effector cells was performed in the absence of cytokines. Co-culture was evaluated by microscopy and flow cytometry on days 3–7.

[0257] Data included percentage of residual tumor after co-culture of tumors with unmodified natural NK or natural NK.CAR.GD2 in the presence or absence of 14.G2a ADCC antibody. Residual SH-SY5Y tumor in the control was measured as 99.5%. Addition of 14.G2a antibody to SH-SY5Y tumor cells did not make a difference compared to the control (98.5%). Addition of unmodified activated natural NK cells to SH-SY5Y tumor cells resulted in 22% residual tumor. Addition of 14G.2a antibody to the combination of natural NK cells and SH-SY5Y tumor cells resulted in a lower percentage of residual tumor (14.5%). Addition of natural NK.CAR.GD2 cells to SH-SY5Y tumor cells resulted in a residual tumor percentage of 40.9%, and when natural NK.CAR.GD2 cells, SH-SY5Y tumor cells, and 14.G2a antibody were co-cultured, the residual tumor percentage was reduced to 8.7%. In the presence of cells with FcγIII receptor expression, e.g., activated natural cells, antibody-mediated ADCC activity enhanced tumor killing.

[0258] (Example 23) In separate experiments, the effect of switching cytokines during expansion on cell content was assessed.

[0259] 1. Buffy coats from normal healthy donors were subjected to Ficoll™ gradient cell separation performed by manual centrifuge separation.

[0260] 2. Mononuclear cells were subjected to αβ T cell depletion using laboratory scale Miltenyi LS column separation.

[0261] 3. The obtained αβTCRneg cell population was placed in feeder-free culture conditions using NK MACS™ medium (#130-107-879 (Miltenyi Biotec, Inc., San Diego, CA, USA) supplemented with 5% AB serum (alternatively, other media such as R&D systems' medium, CELLGENIX medium, etc. may be used).

[0262] 4. Activation was achieved with either a combination of 10ng / ml anti-NKp46 (clone Bab281) and 10ng / ml anti-CD2 (clone OKT11) or 50ng / ml anti-NKp46 (clone Bab281) and 50ng / ml anti-CD2 (clone OKT11) GMP purified by Caprico (alternatively, other concentrations of activating agents may be used or other agents may be added for activation). Activated αβTCRneg cell populations were cultured at 0.25×10 6 At a cell seeding concentration of 100 / ml, NK MACS™ medium (#130-107-879 (Miltenyi Biotec, Inc., San Diego, CA, USA) supplemented with 5% AB serum (alternatively, other medium such as R&D systems medium, CELLGENIX medium, etc. may be used) was used under the following conditions: a. 500IU / mL human interleukin 2 (IL-2) (Miltenyi Biotec) b. 10 ng / ml human interleukin 15 (IL-15) (Miltenyi Biotec) c. A combination of 500IU / mL IL-2 and 10ng / mL IL-15 d. Combination of 500IU / mL IL-2 and 10ng / ml OKT3 (Miltenyi Biotec) e. A combination of 10ng / mL IL-15 and 500IU / mL IL-2 and 10ng / ml OKT3 After expansion under feeder-free culture conditions using one of the following media, the cells were transferred to a T75 flask.

[0263] 5. Medium changes were performed every 3 days (or less frequently in bioreactors, every 5-7 days) using fresh medium and one of the conditions listed above.

[0264] 6. Cells were diluted to 0.25-0.5 x 10 6 Cells / ml (or cell seeding concentration is 1 × 10 for culture bags, flasks, or other large culture vessels, e.g., bioreactors (G-Rex) 6The cells were seeded at a concentration of 10 ...

[0265] 7. Additionally, cells expanded initially with IL-2 for the first 25 days were then washed and supplemented with IL-15 until the end of culture (day 30). Cells expanded initially with IL-15 were washed on day 25 and then supplemented with IL-2 until the end of culture on day 30 (referred to as cytokine switch conditions). In some cases, cells expanded initially with IL-2 for the first 20 days were then washed and supplemented with IL-15 until the end of culture (day 30). Cells expanded initially with IL-15 were washed on day 20 and then supplemented with IL-2 until the end of culture on day 30. Alternative combinations or other cytokines may be used in the switch conditions, such as IL-7, IL-12, IL-18, IL-21, OKT3, OKT11, and anti-NCR.

[0266] 8. At the end of expansion, BINATE cells were analyzed for NK and γδCD3+ T cell content and characterized by receptor expression on the cells via flow cytometry.

[0267] In this example, a single cytokine was compared to the switched conditions (IL-2 vs. switching IL-2 to IL-15) and, in the case of IL-2 vs. IL-2 / IL-15, γδ T cell numbers were increased with the addition of IL-15 compared to IL-2 alone (70% vs. 50%, respectively). In the case of IL-15 vs. IL-15 / IL-2, NK populations were increased in the switched conditions compared to IL-15 alone (90% vs. 80%, respectively). Since both IL-2 and IL-15 act through interactions with the IL-2 receptor on cells, the data indicate that expansion of populations that favor initial expansion conditions with a particular cytokine is enhanced by switching to another cytokine.

[0268] Example 24 Isolation of pure NK cells or pure γδ T cells In this example, a process for isolating a pure NK cell population and a process for isolating a pure γδ T cell population are described. A flow chart illustrating an example variation of this process is presented in FIG.

[0269] 1. Buffy coats from normal healthy donors are subjected to gradient cell separation with Ficoll™ performed by manual centrifuge separation; Percoll gradients, SEPAX, Cobe2991, or elutriation systems may also be used. Alternatively, apheresis, cord blood units, bone marrow, or body fluids such as ascites, CSF, or pleural effusions may be used as cell sources with or without gradient centrifugation.

[0270] 2. The mononuclear cells are used directly or after gradient separation or subjected to further cell selection, such as Ficoll™ MNC, antibody depletion, positive selection of NK, negative selection of NK, negative selection of γδ T cells, and / or positive selection of CD3+. In certain examples, the mononuclear cells are subjected to positive or negative selection of CD56+ NK cells using Miltenyi magnetic bead-based methods or other cell selection techniques. Alternatively, the cells may be subjected to positive selection of γδ TCR+ using Miltenyi magnetic bead-based techniques or other cell selection techniques. Alternatively, the cells may be subjected to CD3+ cell depletion using Miltenyi magnetic bead-based techniques or other cell selection techniques.

[0271] 3. The resulting cell population is then activated in feeder-free culture in the presence of soluble or cross-linked anti-LFA and anti-NCR antibodies using BINATE medium (NK MACS™ medium #130-107-879 Miltenyi Biotec, Inc., San Diego, CA, USA supplemented with 5% AB serum (alternatively, other media such as R&D systems medium, CELLGENIX medium, etc. may be used)) or other animal component-free culture media such as In Vivo or R&D Systems, or Cellgenix, with or without human serum supplementation.

[0272] 4. Cell expansion: from 0.25 to 5 x 10 cells in T75 flasks 6 Cell seeding concentration of 10 cells / ml (or cell seeding concentration is 1 × 10 cells / ml for culture bags, flasks, or other large culture vessels, e.g., bioreactors (G-Rex) 6 Medium changes are performed every 3 days (or less frequently in bioreactors (every 5-7 days)) with growth medium and cytokines.

[0273] 5. The resulting cultures at days 7, 20, 30, 45, and 60 may contain a BINATE population of a mixture of NK and γδ T cells, which are then cryopreserved for administration. Alternatively, the resulting cell population may be subjected to a further post-expansion cell selection step, such as NK CD56+ depletion, thereby obtaining a pure γδ T cell population. Alternatively, the cells may be subjected to positive depletion or negative selection of γδ T cells to obtain a pure NK population, or positive selection for CD16 to yield a highly enriched ADCC FcyIII receptor+ population. Alternatively, the cells may be subjected to NK depletion followed by CD16 selection for the ADCC γδ T cell population.

[0274] (Example 25) Retroviral constructs This example describes the retroviral vectors CARGD2, CARCD123, and CARCD19 used in certain examples above.

[0275] Vector skeleton The constructs described below contain the SFG backbone, which uses a retroviral vector based on Moloney Murine Leukemia Virus (MoMLV). All env and gag-pol sequences except the packaging sequence (psi) were removed. As a result, the vector is replication incompetent.

[0276] Production cell lines Packaging cell lines for production of the following retroviral constructs were grown in a laboratory environment at the OPBG, Institute for Cell and Gene Therapy for Pediatric Tumor, equipped with dedicated laminar flow hoods and CO2 incubators, and containing gamma-irradiated Pharmagrade FBS (EuroClone, catalog ECS017). The packaging cell lines were generated using non-animal derived materials, with the exception of the 293VEC RD114 cell line (2L, Lot EUS0131906GI). The packaging cell lines were generated from the human-based 293VEC RD114 producer line deposited under cGMP received from BioVec Pharma. In particular, 293VEC RD114 cells were selected as the packaging cell line because they are of human origin and allow for the production of high vector titers suitable for large-scale clinical-grade production.

[0277] CARGD2 The iC9-CARGD2.CD28.41bb.CD3 zeta (OPBG-91 vector) retroviral vector was constructed at the Ospedale Pediatrico Bambino Gesu (OPBG), "Cell and Gene Therapy for Pediatric Tumor" Laboratory. A bicistronic vector was used that allows simultaneous expression of two transgenes, inducible caspase 9 (iC9) and CARGD2 (iC9-CARGD2.CD28.41bb.CD3 zeta). Single cell cloning was performed and the 293VEC RD114 clone (i.e., OPBG-91-7) that produced the highest titer (using PCR analysis for the presence of vector in the supernatant) was expanded, deposited at Officina Farmaceutica OPBG, and used for retroviral production under cGMP conditions after testing for sterility and mycoplasma.

[0278] In the iC9 component, the catalytic domain of the intracellularly expressed Casp9 protein is fused to the drug-binding domain derived from the human FK506 binding protein (FKBP12) with the F36V mutation. A CAR molecule based on a single chain of the fused VH-VL region of the monoclonal antibody 14.G2A specific for the human antigen GD2, in frame with the CD28 transmembrane domain and its endodomain, the 4.1bb costimulatory domain and the CD3ζ cytoplasmic domain to transduce an activation signal after antigen binding, was cloned after the gene cassette containing the sequence of iC9 in a retroviral vector to improve the safety aspect of the approach.

[0279] Certain functional and structural components of iC9.CARGD2.CD28.41bb.CD3z expression and activity are summarized in Table 9 below and listed herein:

[0280] · 5'LTR - retroviral long terminal repeat at the 5' end of the vector (acts as a promoter sequence).

[0281] · ψ - retroviral encapsidation signal (psi; required for packaging RNA into virion particles).

[0282] · SA – splice acceptor site.

[0283] iCasp9 - an inducible caspase-9 expression cassette. iCasp9 consists of human FK506 binding protein (FKBP12) with the F36V mutation connected to a modified CARD domain-deleted human caspase-9 via a 6 amino acid Gly-Ser linker.

[0284] · FKBP12-F36V - an engineered FK506-binding protein containing the F36V mutation to optimize binding affinity for AP1903. The FKBP12-F36V protein domain serves as a drug-binding / oligomerization domain for the linked therapeutic protein. FKBP12-F36V functions as a regulator of caspase-9. In the absence of AP1903, iCasp9 has minimal activity; upon binding of AP1903 to FKBP12-F36V, it promotes dimerization and juxtaposes two caspase-9 molecules to initiate apoptosis. Thus, the FKBP12-F36V moiety functionally replaces the endogenous dimerization / activation module (caspase activation and recruitment domain; CARD) of caspase-9 that mediates Apaf-1-associated oligomerization.

[0285] Linker - A synthetic Ser-Gly-Gly-Gly-Ser-Gly peptide linker used to fuse the switch regulator sequence to caspase-9.

[0286] Caspase-9 - Human caspase-9 cDNA sequence (a key pro-apoptotic regulator) and the therapeutic component of the construct (a regulated suicide gene). The endogenous dimerization / activation module (caspase activation and recruitment domain; CARD) was deleted to reduce spontaneous Apaf1 binding and therefore background killing.

[0287] 2A - encodes a synthetic 20 amino acid peptide derived from the Thosea Asigna insect virus, which acts as a cleavable linker between the caspase-9 protein and the CAR protein.

[0288] · Signal peptide - A short amino acid sequence that enables the correct translocation of a secretory protein from the endoplasmic reticulum to the cell membrane.

[0289] · CAR - A CAR molecule based on a single chain of the fused VH-VL region of monoclonal antibody 14.G2A specific for the human antigen GD2, in frame with CD28TM and costimulatory domains, 4.1bb costimulatory domain and CD3ζ cytoplasmic domain.

[0290] · 3'LTR - retroviral long terminal repeat at the 3' end of the vector (functions as a terminator / polyadenylation sequence). [Table 9-1] [Table 9-2]

[0291] A reference electronic vector sequence was assembled by combining the DNA sequence files of each component of the vector construct. Since the retroviral genome is RNA-based, sequence analysis was performed on the plasmid DNA used for transfection into the 293VEC cell line (first step in preparing the retroviral product). Bidirectional sequencing was performed on the entire OPBG-91 vector at Ospedale Pediatrico Bambino Gesu. The sequencing run was assembled using SnapGene software. No mismatched bases were identified compared to the theoretical reference electronic sequence.

[0292] CARCD123 The ΔCD19-2A-CAR-CD123-ΔCD34.CD8.41bb.CD3 zeta (OPBG-242 vector) retroviral vector was constructed at the Ospedale Pediatrico Bambino Gesu (OPBG), "Cell and Gene Therapy for Pediatric Tumor" Laboratory. A bicistronic vector (ΔCD19-2A-CAR-CD123-ΔCD34.CD8.41bb.CD3 zeta) was used, allowing the simultaneous expression of two transgenes, namely ΔCD19 and CARCD123. Single cell cloning was performed and the clone that produced the highest titer (using PCR analysis for the presence of vector in the supernatant) was expanded and deposited as described above.

[0293] ΔCD19 represents the extracellular domain of human CD19 linked to a transmembrane portion. It has a dual function to aid in the selection and phenotypic characterization of genetically modified cells. A single chain CAR molecule based on the fusion VH-VL region of the monoclonal antibody 7G3 specific for the human antigen CD123, in frame with the CD8 transmembrane domain and its endodomain, the 4.1bb costimulatory domain and the CD3ζ cytoplasmic domain for transduction of activation signals after antigen binding, was cloned after a gene expression cassette containing the sequence of ΔCD19 through the use of the 2A sequence in a retroviral vector.

[0294] Certain functional and structural components for ΔCD19-2A-CAR-CD123-ΔCD34.CD8.41bb.CD3z expression and activity are summarized in Table 10 below and listed herein:

[0295] · 5'LTR - retroviral long terminal repeat at the 5' end of the vector (acts as a promoter sequence).

[0296] · ψ - retroviral encapsidation signal (psi; required for packaging RNA into virion particles).

[0297] · SA – splice acceptor site.

[0298] · △CD19 - contains optimized human extracellular and transmembrane domains.

[0299] 2A - codes for a synthetic 20 amino acid peptide derived from the Thosea Asigna insect virus.

[0300] · Signal peptide - A short amino acid sequence that enables the correct translocation of a secretory protein from the endoplasmic reticulum to the cell membrane.

[0301] · △CD34 - Contains a short peptide derived from human CD34 that helps detect CAR+ T cells after transduction.

[0302] · CAR - A CAR molecule based on a single chain of the fused VH-VL region of the monoclonal antibody 7G3 specific for the human antigen CD123, in frame with CD8TM and costimulatory domains, 4.1bb costimulatory domain and CD3ζ cytoplasmic domain.

[0303] · 3'LTR - retroviral long terminal repeat at the 3' end of the vector (functions as a terminator / polyadenylation sequence). [Table 10]

[0304] A reference electronic vector sequence was assembled by combining the DNA sequence files of each component of the vector construct. Since the retroviral genome is RNA-based, sequence analysis was performed on the plasmid DNA used for transfection into the 293VEC cell line (first step in preparing the retroviral product). Bidirectional sequencing was performed on the entire OPBG-242 vector at Ospedale Pediatrico Bambino Gesu. The sequencing run was assembled using SnapGene software. No mismatched bases were identified compared to the theoretical reference electronic sequence.

[0305] CARCD19 The therapeutic retroviral construct SFG-iC9-Car.CD19.41bb encodes a synthetic ligand-inducible human caspase-9 cDNA linked to a single strand of fusion VH-VL regions of a monoclonal antibody specific for the mouse antigen CD19, 4.1bb costimulatory domain. The assembly is the same as CARGD2 described above, except that it is the second generation. The retroviral vector was constructed in the EU Cell and Gene Therapy Laboratory. Single cell cloning was performed and the clone that produced the highest titer (using PCR analysis for the presence of vector in the supernatant) was expanded, deposited in a cGMP facility, and used for retroviral production under cGMP conditions after testing for sterility and mycoplasma.

[0306] Certain functional and structural components for SFG-iC9-Car.CD19.41bb expression and activity are listed below:

[0307] · 5'LTR - retroviral long terminal repeat at the 5' end of the vector (acts as a promoter sequence).

[0308] · ψ - retroviral encapsidation signal (psi; required for packaging RNA into virion particles).

[0309] · SA – splice acceptor site.

[0310] iCasp9 - an inducible caspase-9 expression cassette. iCasp9 consists of human FK506 binding protein (FKBP12) with the F36V mutation connected to a modified CARD domain-deleted human caspase-9 via a 6 amino acid Gly-Ser linker.

[0311] · FKBP12-F36V - an engineered FK506-binding protein containing the F36V mutation to optimize binding affinity for AP1903. The FKBP12-F36V protein domain serves as a drug-binding / oligomerization domain for the linked therapeutic protein. FKBP12-F36V functions as a regulator of caspase-9. In the absence of AP1903, iCasp9 has minimal activity; upon binding of AP1903 to FKBP12-F36V, it promotes dimerization and juxtaposes two caspase-9 molecules to initiate apoptosis. Thus, the FKBP12-F36V moiety functionally replaces the endogenous dimerization / activation module (caspase activation and recruitment domain; CARD) of caspase-9 that mediates Apaf-1-associated oligomerization.

[0312] Linker - A synthetic Ser-Gly-Gly-Gly-Ser-Gly peptide linker used to fuse the switch regulator sequence to caspase-9.

[0313] Caspase-9 - Human caspase-9 cDNA sequence (a key pro-apoptotic regulator) and the therapeutic component of the construct (a regulated suicide gene). The endogenous dimerization / activation module (caspase activation and recruitment domain; CARD) was deleted to reduce spontaneous Apaf1 binding and therefore background killing.

[0314] 2A - encodes a synthetic 20 amino acid peptide derived from the Thosea Asigna insect virus, which acts as a cleavable linker between the caspase-9 protein and the CAR protein.

[0315] · Signal peptide - A short amino acid sequence that enables the correct translocation of a secretory protein from the endoplasmic reticulum to the cell membrane.

[0316] · CAR - CAR molecule based on a single chain of fused VH-VL regions of a monoclonal antibody specific for the mouse antigen CD19, 4.1bb costimulatory domain.

[0317] · 3'LTR - retroviral long terminal repeat at the 3' end of the vector (functions as a terminator / polyadenylation sequence).

[0318] (Example 26) Examples of Certain Non-Limiting Embodiments Below are listed non-limiting examples of certain embodiments of the present technology.

[0319] A1. A method of producing a composition comprising a population of cells enriched for NK cells and gamma delta T cells, comprising: obtaining a cell-containing sample from one or more subjects; depleting alpha.beta T cells from the sample under conditions to produce a depleted cell population that includes NK cells and gamma.delta T cells; and exposing the depleted cell population to activation conditions comprising contacting the depleted cell population with (a) at least one exogenous polypeptide that immunospecifically binds to a cell adhesion polypeptide, and (b) at least one exogenous polypeptide that immunospecifically binds to a polypeptide different from the cell adhesion polypeptide and that is expressed on the surface of one or more cells of the sample population; and exposing the depleted cell population to expansion conditions comprising contacting the depleted cell population with at least one auxiliary polypeptide, thereby producing a composition comprising a population of cells enriched for NK cells and gamma delta T cells. The method includes:

[0320] A1.1. A method of producing a composition comprising a population of cells enriched for NK cells and gamma delta T cells, comprising: obtaining a cell-containing sample from one or more subjects; exposing the sample to activating conditions comprising contacting the sample with (a) at least one exogenous polypeptide that immunospecifically binds to a cell adhesion polypeptide, and (b) at least one exogenous polypeptide that immunospecifically binds to a polypeptide different from the cell adhesion polypeptide and that is expressed on the surface of one or more cells of the sample population, where either (a) or (b) is soluble, or where both (a) and (b) are soluble; and exposing the sample to expansion conditions comprising contacting the sample with at least one auxiliary polypeptide, thereby producing a composition comprising a population of cells enriched for NK cells and gamma delta T cells. The method includes:

[0321] A2. The method of embodiment A1 or A1.1, wherein the at least one auxiliary polypeptide is selected such that the amount of NK cells compared to the amount of gamma.delta T cells in the population is dependent on the amount and / or type of the at least one auxiliary polypeptide.

[0322] A2.1. The method of embodiment A1, A1.1 or A2, wherein at least one auxiliary polypeptide increases or decreases the amount of NK cells relative to gamma.delta T cells in the population of cells after contacting the depleted cell population with at least one auxiliary polypeptide.

[0323] A3. The method of any of embodiments A1, A1.1, A2 or A2.1, wherein the activation conditions do not include serum from a non-human animal.

[0324] A4. The method of any of embodiments A1-A3, wherein the expansion conditions do not include serum from a non-human animal.

[0325] A5. The method of any of embodiments A1-A4, wherein the activating conditions do not include feeder cells.

[0326] A6. The method of any of embodiments A1-A5, wherein the expansion conditions do not include feeder cells.

[0327] A7. The method of any of embodiments A1-A6, wherein the sample is selected from peripheral blood, liver tissue, epithelial tissue, bone marrow, and umbilical cord blood.

[0328] A8. The method of embodiment A7, wherein the sample is peripheral blood.

[0329] A9. The method of embodiment A8, wherein the peripheral blood sample is a processed sample.

[0330] A10. The method of embodiment A7, wherein the sample is cord blood.

[0331] A11. The method of embodiment A10, wherein the cord blood sample is a processed sample.

[0332] A12. The method of any of embodiments A1-A11, wherein the exogenous polypeptide in (b) immunospecifically binds to an NK cell activating receptor, a gamma delta T cell activating receptor, or both.

[0333] A13. The method of embodiment A12, wherein the receptor is NKp30, NKp44, or NKp46.

[0334] A14. The method of embodiment A13, wherein the receptor is NKp46.

[0335] A15. The method of any of embodiments A1-A14, wherein the exogenous polypeptide in (a) immunospecifically binds to CD2.

[0336] A16. The method of any of embodiments A1-A15, wherein the exogenous polypeptide in (a) or (b), or (a) and (b), is an antibody or an antigen-binding fragment thereof.

[0337] A17. The method of any of embodiments A1-A16, wherein at least one of (a) or (b) is soluble.

[0338] A17.1. The method of any of embodiments A1-A17, wherein both the exogenous polypeptides in (a) and (b) are soluble.

[0339] A18. The method of any of embodiments A1-A16, wherein the exogenous polypeptide in (a) or the exogenous polypeptide in (b) is bound to a substrate.

[0340] A19. The method of any of embodiments A1-A18, wherein the activating conditions comprise contacting the sample or depleted cell population with at least two exogenous polypeptides.

[0341] A20. The method of embodiment A19, wherein a first exogenous polypeptide immunospecifically binds to CD2 and a second exogenous polypeptide immunospecifically binds to NKp46.

[0342] A21. The method of embodiment A19 or A20, wherein the first exogenous polypeptide and / or the second exogenous polypeptide is an antibody or an antigen-binding fragment thereof.

[0343] A22. The polypeptide component of the activating condition is (a) an exogenous polypeptide that immunospecifically binds to the cell adhesion polypeptide CD2; and (b) an exogenous polypeptide that immunospecifically binds to NKp46, different from the exogenous polypeptide in (a); The method of any of embodiments A1-A21, consisting essentially of or consisting of.

[0344] A23. The method of any of embodiments A1-A22, wherein the auxiliary polypeptide is a cytokine and / or a polypeptide that immunospecifically binds to a receptor on a gamma.delta T cell.

[0345] A24. The method of embodiment A23, wherein the expansion conditions include contacting the sample or depleted cell population with at least one auxiliary polypeptide that is a cytokine, and optionally an auxiliary polypeptide that immunospecifically binds to a receptor on gamma.delta T cells.

[0346] A25. The method of embodiment A24, wherein the cytokine is an interleukin (IL).

[0347] A26. The method of any of embodiments A23-A25, wherein the at least one auxiliary polypeptide comprises IL-2, IL-4, IL-15, or any combination thereof.

[0348] A27. Expansion conditions are used to expand the sample or depleted cell population. (a) an IL-2 polypeptide, and optionally a polypeptide that immunospecifically binds to a receptor on a gamma.delta T cell; (b) an IL-15 polypeptide; or (c) an IL-2 polypeptide and an IL-15 polypeptide, and optionally a polypeptide that immunospecifically binds to a receptor on a gamma delta T cell. The method of any of embodiments A23-A26 comprising contacting with

[0349] A28. The method of any of embodiments A23-A27, wherein the receptor on gamma.delta T cells is CD3.

[0350] A29. The method of any of embodiments A23-A28, wherein the polypeptide that immunospecifically binds to a receptor on gamma.delta T cells is an antibody or an antigen-binding fragment thereof.

[0351] A30. The magnification conditions are (a) an IL-2 polypeptide; (b) an IL-15 polypeptide; (c) an IL-2 polypeptide and an IL-15 polypeptide; (d) an antibody that immunospecifically binds to an IL-2 polypeptide and CD3; or (e) an antibody that immunospecifically binds to an IL-2 polypeptide, an IL-15 polypeptide, and CD3; The method of embodiment A29, comprising contacting with

[0352] The method of embodiment A30, wherein the antibody that immunospecifically binds to CD3 is OKT3.

[0353] A32. The method of any of embodiments A1-A31, wherein activation and expansion conditions are performed simultaneously or sequentially in any order.

[0354] A33. At least one exogenous polypeptide can also function as an auxiliary polypeptide; or At least one auxiliary polypeptide can also function as an exogenous polypeptide; or The method according to any of embodiments A1 to A32, wherein at least one exogenous polypeptide can also function as an auxiliary polypeptide, and at least one auxiliary polypeptide can also function as an exogenous polypeptide.

[0355] A34.(i) the expansion conditions include contacting the sample or the depleted cell population with an IL-2 polypeptide; (ii) The method of any of embodiments A30 to A33, wherein the resulting population of cells enriched for NK cells and gamma.delta T cells comprises about 25-30% NK cells and about 70-75% gamma.delta T cells.

[0356] A35.(i) the expansion conditions include contacting the sample or the depleted cell population with an IL-15 polypeptide; (ii) The method of any of embodiments A30 to A33, wherein the resulting population of cells enriched for NK cells and gamma.delta T cells comprises about 80-99% NK cells and about 1-20% gamma.delta T cells.

[0357] A36.(i) the expansion conditions include contacting the sample or the depleted cell population with an IL-2 polypeptide and an antibody that immunospecifically binds to CD3; (ii) The method of any of embodiments A30 to A33, wherein the resulting population of cells enriched for NK cells and gamma delta T cells comprises about 40-45% NK cells and about 55-60% gamma delta T cells.

[0358] A37. The expansion conditions are: contacting the sample or depleted cell population with a first set of conditions comprising one or more auxiliary polypeptides to result in a first cell population comprising a first ratio of NK cells to gamma delta T cells; and The method of any of embodiments A1-A36, comprising contacting the first cell population with a second set of conditions comprising one or more auxiliary polypeptides to result in a second cell population comprising a desired final ratio of NK cells to gamma delta T cells, wherein the first set of conditions is different from the second set of conditions.

[0359] A38. The method of embodiment A37, wherein the first cell population is washed prior to contact with the second set of conditions.

[0360] A39. A first set of conditions includes IL-2 and a second set of conditions includes IL-15; A first set of conditions includes IL-15 and a second set of conditions includes IL-2; a first set of conditions includes an antibody that immunospecifically binds to IL-2 and CD3, and a second set of conditions includes IL-15; or The method of embodiment A37 or A38, wherein the first set of conditions comprises an antibody that immunospecifically binds to IL-15 and CD3, and the second set of conditions comprises an antibody that immunospecifically binds to IL-2 and CD3.

[0361] A40. The method of embodiment A39, wherein the antibody that immunospecifically binds to CD3 is OKT3.

[0362] A41. Depleting alpha.beta T cells from a sample prior to exposing the sample to activation and expansion conditions, thereby generating a depleted cell population; and Subjecting the depleted cell population to activation and expansion conditions Further comprising: The method according to any of embodiments A1.1 and A2 to A40, whereby a composition comprising a population of cells enriched for NK cells and gamma.delta T cells is obtained.

[0363] A41.1. The method of any of embodiments A1-A41, wherein prior to activation and expansion, the sample or depleted cell population is not exposed to conditions that select for NK cells or gamma.delta T cells, nor to conditions that deplete cells other than alpha-beta T cells.

[0364] A41.2. The method of embodiment A41.1, wherein the sample or depleted cell population is not exposed to conditions that deplete CD3+ cells prior to activation and expansion.

[0365] A42. The method of any of embodiments A1-A41.1, wherein the cells of the sample or depleted cell population do not contain exogenous nucleic acid before, during, or after activation and expansion.

[0366] A43. The method of any of embodiments A1-A41.1, wherein the cells of the sample or depleted cell population do not contain a tumor necrosis factor receptor, a chimeric antigen receptor (CAR), a myeloid differentiation primary response protein, or an exogenous nucleic acid encoding an innate immune signaling adaptor before, during, or after activation and expansion.

[0367] A44. The method of any of embodiments A1-A43, wherein the cells of the sample or depleted cell population are not genetically modified before, during, or after activation and expansion.

[0368] A45. The method of any of embodiments A1 to A44, further comprising subjecting the population of cells enriched for NK cells and gamma.delta T cells to a treatment whereby gamma.delta cells are depleted, such that the resulting population consists essentially of or consists of NK cells.

[0369] A46. The method of any of embodiments A1 to A44, further comprising subjecting the population of cells enriched for NK cells and gamma.delta T cells to a treatment whereby NK cells are depleted, such that the resulting population consists essentially of or consists of gamma.delta T cells.

[0370] A47. The method of any of embodiments A1 to A45, further comprising subjecting the population of cells enriched for NK cells and gamma.delta T cells to positive selection for NK cells, thereby obtaining a cell population consisting essentially of or consisting of NK cells.

[0371] A48. The method of any of embodiments A1 to A44 and A46, further comprising subjecting the population of cells enriched for NK cells and gamma.delta T cells to positive selection for gamma.delta cells, thereby obtaining a cell population consisting essentially of or consisting of gamma.delta cells.

[0372] A49. The method of any of embodiments A1-A48, wherein the expansion conditions comprise incubation of the sample or depleted cell population in feeder cell-free medium for about 1 week to about 10 weeks, thereby resulting in a composition comprising an expanded population of cells enriched for NK cells and gamma.delta T cells.

[0373] A50. The method of embodiment A49, wherein the culture conditions comprise incubation of the sample or depleted cell population in feeder cell-free medium for about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 25, 30, 35, 40, 45, 50, 55, or 60 days or more, or for about 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks.

[0374] A51. The method of any of embodiments A1-A50, wherein the population of cells enriched for NK cells and gamma.delta T cells is expanded by greater than about 2 logs over 30 days under expansion conditions.

[0375] A52. The method of embodiment A51, wherein the population of cells is expanded by greater than about 3 logs under expansion conditions over 30 days.

[0376] A53. The method of any of embodiments A1-A52, wherein the population of expanded cells enriched for NK cells and gamma.delta T cells does not include exhausted cells.

[0377] A54. The method of any of embodiments A1-A53, wherein the population of expanded cells enriched for NK cells and gamma.delta T cells does not contain exhausted cells after 60 days of expansion conditions.

[0378] A55. The method of any of embodiments A1-A54, wherein less than 5%, less than 4%, less than 3%, or less than 2% of the NK cells in the expanded population of cells enriched for NK cells and gamma.delta T cells comprise the PD-1 marker, and / or about 20%, 15%, 10%, or less of the total cells in the expanded population or gamma.delta T cells in the expanded population comprise the PD-1 marker.

[0379] A56. A population of cells enriched for NK cells and gamma delta T cells was assayed for the following activation markers as a percentage of the total number of cells in the population: (a) 90% or more KIR5; (b) 10% or more SIGLEC-7; (c) 60% or more KIR3D51; (d) 10% or more KIR2DL1; (e) 25% or more NKp30, NKp44, or NKp46; (f) NKG2D of 35% or more; (g) 90% or more DNAM1; (h) 85% or more NTBA; (i) 95% or greater CD2; and (j) 55% or more KIR3DS1 The method of any of embodiments A1-A55, comprising one or more of:

[0380] A57. The method of any of embodiments A1-A56, wherein the population of cells enriched for NK cells and gamma.delta T cells comprises 80% or more innate immune cells.

[0381] A58. The method of embodiment A57, wherein between about 70% and about 100%, or at least about 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% of the cells are CD56+.

[0382] A59. The method of embodiment A57 or A58, wherein between about 10% and about 40%, or at least about 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%, or 40% of the cells are CD16+.

[0383] A60. The method of any of embodiments A57-A59, wherein less than 10%, less than 5%, less than 4%, less than 3%, or less than 2% of the cells are CD57+.

[0384] A61. The method of any of embodiments A1-A60, wherein the sample or depleted cell population does not contain CD4+CD8+ cells during or after activation and expansion.

[0385] A62. The method of any of embodiments A1-A61, wherein the activation and expansion conditions do not comprise a bisphosphonate.

[0386] A63. The method of embodiment A62, wherein the bisphosphonate is pamidronate or zoledronate.

[0387] A64. The method of any of embodiments A1-A63, wherein the gamma.delta T cells are polyclonal for V.delta.1 and V.delta.2 expression.

[0388] A65. The method of embodiment A64, wherein the polyclonal gamma.delta T cells comprise at least one subpopulation selected from V.delta.1+ and V.delta.1-, and at least one subpopulation selected from V.delta.2+ and V.delta.2-.

[0389] A66. A composition obtainable or obtained by the method according to any of embodiments A1-A65.

[0390] B1. A composition comprising a population of modified peripheral blood cells, the population comprising: Contains multiple NK cells and multiple gamma delta T cells; Alpha and beta T cells are depleted; The composition does not include feeder cells.

[0391] B2. Between about 25% and about 45% of the cells are NK cells, and between about 55% and about 75% of the cells are gamma.delta T cells; Between about 25% and about 30% of the cells are NK cells and between about 70% and about 75% of the cells are gamma.delta T cells; Between about 80% and about 99% of the cells are NK cells and between about 1% and about 20% of the cells are gamma delta T cells; or The composition of embodiment B1, wherein between about 40% and about 45% of the cells are NK cells and between about 55% and about 60% of the cells are gamma.delta T cells.

[0392] B3. The composition of embodiment B1 or B2, wherein 30% or more of the cells are activated.

[0393] B4. The population of modified cells expresses the following activation markers as a percentage of the total number of cells in the population: (a) 90% or more KIR5; (b) 10% or more SIGLEC-7; (c) 60% or more KIR3D51; (d) 10% or more KIR2DL1; (e) 25% or more NKp30, NKp44, or NKp46; (f) NKG2D of 35% or more; (g) 90% or more DNAM1; (h) 85% or more NTBA...

Claims

Claim 1: A method for producing a composition comprising 70% or more γδ T cells from a sample containing cells from one or more subjects, comprising: (a) depleting αβ T cells from said sample under conditions to produce a depleted cell population comprising NK cells and γδ T cells; (b) exposing the depleted cell population to activating conditions comprising contacting the depleted cell population with (a) an antibody or antigen-binding fragment thereof that immunospecifically binds to cell adhesion molecule polypeptide CD2, and (b) an antibody or antigen-binding fragment thereof that immunospecifically binds to NKp46; and (c) exposing said depleted cell population to expansion conditions comprising contacting said depleted cell population with IL-2, thereby generating a composition comprising 70-75% γδ T cells. The method includes:

2. The method of claim 1, wherein the composition contains 30% or less NK cells.

3. The method of claim 1 or 2, wherein the expansion conditions further comprise contacting the depleted cell population with an antibody or antigen-binding fragment thereof that immunospecifically binds to CD3.

4. A method according to any one of claims 1 to 3, wherein the expansion conditions comprise incubation of the depleted cell population in the presence of IL-2 and / or anti-CD3 antibody for 1 week to 6 weeks.

5. The method of claim 1, wherein the activation conditions comprise incubation of the depleted cell population for 1 hour to 1 week in the presence of (a) an antibody or antigen-binding fragment thereof that immunospecifically binds to the cell adhesion molecule polypeptide CD2, and (b) an antibody or antigen-binding fragment thereof that immunospecifically binds to NKp46.

6. A method according to any one of claims 1 to 5, wherein 1% or less of the total cells in the composition are αβ T cells.

7. A method according to any one of claims 1 to 6, wherein depleting αβ T cells from the sample comprises contacting the sample with an antibody that immunospecifically binds to the αβ T cell receptor.

8. A method according to any one of claims 1 to 7, wherein the antibody or antigen-binding fragment thereof that immunospecifically binds to CD2 is a monoclonal anti-CD2 antibody, and / or the antibody or antigen-binding fragment thereof that immunospecifically binds to NKp46 is a monoclonal anti-NKp46 antibody.

9. The method of claim 8, wherein the anti-CD2 antibody and / or anti-NKp46 antibody is soluble.

10. The method of claim 1, wherein the activation conditions and / or expansion conditions do not include feeder cells.

11. The method of claim 1, wherein the activation and / or expansion conditions do not include a bisphosphonate.

12. The method of claim 11, further comprising subjecting the depleted cell population to genetic modification conditions prior to exposing the depleted cell population to activating conditions.

13. The method of claim 11, further comprising subjecting the depleted cell population to genetic modification conditions after exposing the depleted cell population to expansion conditions.

14. The method described in claim 12 or 13, wherein the genetic modification comprises introducing an exogenous polynucleotide encoding a chimeric antigen receptor into the cell.

15. The method of claim 1, wherein the activation conditions and the expansion conditions are carried out simultaneously.

16. The method of claim 1, wherein the activation conditions and the expansion conditions are carried out sequentially.

17. The method of any one of claims 1 to 16, wherein the sample is peripheral blood, liver tissue, bone marrow, epithelial tissue or umbilical cord blood.

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