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

Abstract

The present invention provides methods for preparing innate immune cell compositions comprising γ.δ (γδ) T cells and / or natural killer (NK) cells, as well as the resulting compositions and related manufacturing products and kits, which are used in cancer and infectious disease therapies. The methods provided herein allow for adjustment of the relative amounts of γ.δ (γδ) T cells and natural killer (NK) cells in the composition for cell therapy against a variety of cancers and infectious diseases. The resulting composition can be further used to produce a composition comprising only NK cells or only γ.δ T cells for immune cell therapy. The compositions provided herein can also be genetically altered: γδ T cells and natural killer cells are modified to express chimeric antigen receptors (CARs) or exogenous T cell receptors (TCRs), which can be used to directly or indirectly target any cell surface molecules, such as markers on cancer cells or infected cells.

Description

[0001] Related patent applications

[0002] This patent application claims the benefit of U.S. Provisional Patent Application No. 62 / 703,654, filed on July 26, 2018, entitled THERAPEUTIC PREPARATONS OF GAMMA-DELTA T CELLS AND NATURAL KILLER CELLS AND METHODS FOR MAKING AND USING THEM, which designates Concetta QUINTARELLI et al. as inventors and is assigned by attorney docket number 6474-131400. The entire contents of the foregoing application are incorporated herein by reference for all purposes, including all text, tables, and drawings. Technical Field

[0003] The present technology relates in part to innate immune cell compositions for use in therapy, and methods of making and using such compositions. Background Art

[0004] The immune response to antigen-presenting pathogens and other foreign antigen-presenting entities includes innate defense and adaptive defense. The innate immune response is the first line of immune defense, which is active and continuously active in the host. Innate immune cells, such as NK cells and γ.δ (γδ) T cells cannot recognize classical HLA antigens. The adaptive immune response is a response specifically tailored to antigen presenters (such as foreign bodies, cells or microorganisms), and it may usually take several days to mature. Immunotherapy is performed using natural or modified (e.g., CAR-modified) adaptive immune cells (e.g., α.β (αβ) T cells, dendritic cells, macrophages). For example, αβ T cells will proliferate after binding to ligands on antigen presenters in vivo, which may take days or even weeks. In addition, the binding of αβ T cells is carried out through one or more T cell surface molecules or molecular complexes (including CD4, CD8 and 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)). Summary of the invention

[0005] Provided herein are compositions related to immunology and medicine, methods for preparing these compositions, and methods for treating these compositions. In some aspects, compositions (including manufacturing products and kits) and methods for various cell therapies comprising γδT cells (γδ), natural killer cells (NK) or a combination of the two are provided. Also provided are methods for preparing γδT cells (γδ) and natural killer cells (NK) for these therapies that can be genetically altered. In alternative aspects, genetically altered γδT cells (γδ) and natural killer cells (NK) are modified to express chimeric antigen receptors (CAR) or exogenous or heterologous T cell receptors (TCR), which can be used to directly or indirectly target any cell surface molecules, such as markers on cancer cells or infected cells.

[0006] In certain aspects, methods for making a composition comprising a cell population rich in NK cells and γ.δT cells are provided. In certain aspects of the methods provided herein, a sample obtained from a donor (e.g., a tissue, organ, or blood sample from a healthy subject or a subject who is a patient to be treated with a cell population) is exposed to activation conditions, the 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 expressed on the surface of one or more cells of the sample; and exposed to expansion conditions, the expansion conditions comprising contacting the sample with at least one supplementary polypeptide, thereby producing a composition comprising a cell population rich in NK cells and γ.δT cells. The sample is sometimes exposed to activation conditions sequentially and then to expansion conditions. The methods provided herein can generate a population of cells having a high activation level, wherein the high activation level is from about 30% to 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%, 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%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 1 98%, 99%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, up to 100% of the cells.

[0007] Based in part on available nucleic acid and amino acid sequences and other knowledge of cell adhesion molecules and other immune cell molecules and receptors, it is possible to easily identify and separate, synthesize or otherwise obtain (including obtaining from commercial sources) the exogenous polypeptides and supplementary polypeptides used in the methods provided herein. In some aspects, the exogenous polypeptides are human. In some aspects, the exogenous polypeptides are separated. In some aspects, the supplementary polypeptides are human. In some aspects, the supplementary polypeptides are separated.

[0008] In certain aspects of the methods provided herein, the sample is depleted of α.β T cells prior to exposure to activation and expansion conditions, and the resulting depleted cell population is then sometimes exposed to activation and expansion conditions. In certain aspects, the exogenous polypeptide in (a) above, the exogenous polypeptide in (b) above, or both the exogenous polypeptide in (a) above and the exogenous polypeptide in (b) above are soluble. In some aspects, the exogenous polypeptide in (a) or the exogenous polypeptide in (b) is bound to a solid substrate.

[0009] In some aspects of the methods provided herein, at least one supplemental polypeptide is selected so that the amount of NK cells in the population relative to the amount of γ.δT cells depends on the amount and / or type of the at least one supplemental polypeptide. In some aspects, after the removal of the cell population is contacted with at least one supplemental polypeptide, the supplemental polypeptide increases or decreases the amount of NK cells in the cell population relative to γ.δT cells. In some aspects, the time at which the sample or the sample from which α.βT cells have been removed is exposed to the amplification conditions is selected to obtain a desired ratio of NK cells to γ.δT cells.

[0010] In certain aspects of the methods provided herein, the activation conditions are free of serum from a non-human animal. In certain aspects of the methods provided herein, the amplification conditions are free of serum from a non-human animal.

[0011] In the methods provided herein, a high percentage of cells are often activated 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%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125 In some aspects of the method provided herein, the activation conditions do not contain feeder cells. In some aspects of the method provided herein, the amplification conditions do not contain feeder cells, and sometimes the activation conditions and the amplification conditions do not contain feeder cells.

[0012] In the method provided herein, immune cells from any source can be used as sample. In some aspects, sample is selected from bone marrow, peripheral blood, liver tissue, epithelial tissue and umbilical cord blood. In some aspects of the method provided herein, sample is not derived from embryonic source. In some aspects of the method provided herein, sample is peripheral blood, and in some aspects, peripheral blood sample is a sample processed before the α.βT cells in the method provided herein are removed. For example, peripheral blood sample can be processed by density gradient centrifugation to separate and / or separate the buffy coat containing leukocytes, platelets, granulocytes, etc., and then α.βT cells can be removed according to the method provided herein. In some aspects, buffy coat can further undergo Ficoll gradient separation to obtain mononuclear cells (PBMC), and then α.βT cells can be removed in the method provided herein. In some aspects, peripheral blood sample can undergo single blood component method to separate cells from plasma, and then α.βT cells are removed to cells in the method provided herein sometimes. In certain aspects of the methods provided herein, the sample is umbilical cord blood, and sometimes the umbilical cord blood is processed umbilical cord blood that has been processed prior to being subjected to α.β T cell depletion in the methods provided herein.

[0013] In some aspects of the methods provided herein, the exogenous polypeptide in (b) immunospecifically binds to NK cell activating receptors, γ.δT cell activating receptors, or both. These 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, etc. In some aspects, the receptor is NKp46. In some aspects of the methods provided herein, the exogenous polypeptide in (a) immunospecifically binds to CD2. In some aspects, the exogenous polypeptides in (a) or (b) or (a) and (b) are antibodies or antigen-binding fragments thereof. For the purposes of this article, the description 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 Fv (dsFv), Fd fragments, Fd' fragments, single-chain Fv (scFv), single-chain Fab (scFab), bivalent antibodies, anti-idiotype (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 may 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 some aspects of the methods provided herein, the activation conditions include contacting the sample or the removed cell population with at least two exogenous polypeptides. In some aspects, the first exogenous polypeptide immunospecifically binds to CD2, and the second exogenous polypeptide immunospecifically binds to NKp46. In some aspects, the activation conditions consist of or are substantially composed of the first exogenous polypeptide immunospecifically bound to CD2 and the second exogenous polypeptide immunospecifically bound to NKp46. In some aspects, the exogenous polypeptide immunospecifically bound to CD2, the exogenous polypeptide immunospecifically bound to NKp46, or the exogenous polypeptide immunospecifically bound to CD2 and the exogenous polypeptide immunospecifically bound to NKp46 are both soluble. In some aspects, the first exogenous polypeptide and / or the second exogenous polypeptide are antibodies or antigen-binding fragments thereof.

[0015] In certain aspects of the methods provided herein, the expansion conditions include, consist of, or consist essentially of at least one supplemental polypeptide, which is a cytokine and / or a polypeptide that immunospecifically binds to a receptor on a γ.δT cell; and / or a portion thereof that immunospecifically binds to a receptor on a γ.δT cell. In certain aspects, 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 No. BC100962.1; 100963.1; 100961.1), IL-21 (see, e.g., GenBank Accession No. LC133256.1), or any combination thereof. In some aspects of the methods provided herein, the cytokine is IL-2, IL-15, or a combination thereof. In some aspects, the expansion conditions include, consist of, or consist essentially of IL-2, IL-15, and a polypeptide that immunospecifically binds to a receptor on a γ.δT cell. In some aspects, the receptor on the γ.δT cell is CD3. In some aspects, the polypeptide that immunospecifically binds to the CD3 receptor on the γ.δT cell is an antibody or an antigen-binding fragment thereof, and in some aspects, the antibody is OKT3. In some aspects, the expansion conditions include contacting the sample or removing the cell population with the following polypeptides: (a) IL-2 polypeptide; (b) IL-15 polypeptide; (c) IL-2 polypeptide and IL-15 polypeptide; (d) IL-2 polypeptide and an antibody that immunospecifically binds to CD3; or (e) IL-2 polypeptide, IL-15 polypeptide, and an antibody that immunospecifically binds to CD3. In some aspects, the antibody that immunospecifically binds to CD3 is OKT3.

[0016] In some aspects, the amplification conditions include exposing the sample (e.g., a source sample or a sample cell population from which α.βT cells have been removed) to more than one set of conditions in sequence. In some aspects, the sample is exposed to two sets of amplification conditions. In some aspects, the cell population exposed to the first set of amplification conditions is washed before being exposed to the second set of amplification conditions. In some aspects: (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 IL-2 and an antibody that immunospecifically binds to CD3 and the second set of conditions includes IL-15; or (d) the first set of conditions includes IL-15 and an antibody that immunospecifically binds to CD3 and the second set of conditions includes IL-2 and an antibody that immunospecifically binds to CD3.

[0017] In certain aspects of the methods provided herein, a first set of expansion conditions comprising one or more supplemental polypeptides typically generates a first cell population comprising a first ratio of NK cells to γ.δT cells, which can then be fine-tuned to a desired final ratio of NK cells to γ.δT cells using a second set of expansion conditions, wherein the first set of conditions is different from the second set of conditions.

[0018] In the methods provided herein, a cell population can be exposed to activation conditions and expansion conditions simultaneously or sequentially in any order. In addition, an exogenous polypeptide can function as a supplemental polypeptide, and / or vice versa.

[0019] Relative to natural immune cell compositions (e.g., biological fluids and tissues), the immune cell compositions obtained by the methods provided herein are rich in NK cells and γ.δT cells as natural immune cells. In nature, the amount of α.βT cells found as adaptive immune cells is much higher than that of NK cells and γ.δT cells. On the other hand, in the compositions provided herein, α.βT cells are not present or are present in negligible to very low amounts, while NK cells and γ.δT cells are the main immune cell components.

[0020] Depending on the expansion conditions, the composition enriched in innate immune cells obtained by the methods provided herein may contain different amounts of NK cells relative to γ.δT cells. In certain non-limiting examples: (i) when the supplementary polypeptide is IL-2, the resulting cell population enriched in NK cells and γ.δT cells generally contains about 25-30% NK cells and about 70-75% γ.δT cells; (ii) when the supplementary polypeptide is IL-15, the resulting cell population enriched in NK cells and γ.δT cells generally contains about 80-99% NK cells and about 1-20% γ.δT cells; (iii) when the supplementary polypeptide is IL-2 and an antibody that immunospecifically binds to CD3 (e.g., OKT3), the resulting cell population enriched in NK cells and γ.δT cells generally contains about 40-45% NK cells. (iv) when the polypeptide is supplemented with IL-2 until day 20 of expansion conditions and then switched to IL-15 until day 30, the percentage of γ.δ T cells generally increases from about 50% to about 70%, generally accompanied by a corresponding decrease in the percentage of NK cells, compared to treatment with IL-2 alone; and (v) when the polypeptide is supplemented with IL-15 until day 20 of expansion conditions and then switched to IL-2 until day 30, the percentage of NK cells generally increases from about 80% to about 90%, generally accompanied by a corresponding decrease in the percentage of γ.δ T cells, compared to treatment with IL-15 alone.

[0021] In certain aspects of the methods provided herein, the sample or depleted cell population is not exposed to conditions that select for NK cells or γ.δ T cells. In some aspects, the sample or depleted cell population is not exposed to conditions that deplete cells other than α-β T cells.

[0022] The cells of the compositions prepared by the methods provided herein can be further genetically modified to express exogenous polynucleotides, such as tumor necrosis factor receptors, chimeric antigen receptors (CARs), myeloid differentiation primary response proteins, or innate immune signal transduction aptamers. Cells can also be modified to mutate one or more polypeptides or to delete one or more polypeptides.

[0023] In certain aspects of the methods provided herein, a composition comprising γ.δ cells and NK cells can be treated to obtain a composition consisting of or substantially consisting of NK cells or γ.δ cells. Treatment can be removal, for example, by removing all γ.δ (CD3+) cells using an anti-CD3 antibody to obtain NK cells, or positive selection, for example, using an anti-CD3 antibody to select γ.δ T cells. In some aspects, the anti-CD3 antibody, such as an OKT3 antibody, can be bound to a solid phase.

[0024] In aspects of the methods provided herein, activation conditions, expansion conditions, or activation conditions and expansion conditions include incubating the sample or the removed cell population in a culture medium without feeder cells for about 1 hour, 2 hours, 5 hours, 10 hours, 12 hours, 15 hours, 20 hours, or several days, such as 1, 2, 3, 4, 5, 6, 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 several weeks, such as about 2, 3, 4, 5, 6, 7, 8, 9 or 10 weeks. In certain aspects, the activation conditions last for 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 aspects, activation condition is to carry out about 12 hours, 24 hours, 36 hours or 2 days to about 3 days, 4 days, 5 days, 6 days or 1 week, or about 2 days to about 4 days or 5 days, or about 3 days to about 4 days time period.In some aspects, amplification condition is to carry out 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or 1 week time period.In some aspects, amplification condition is to carry out 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or 1 week time period.In some aspects, amplification condition is carried out with continuous cycle, and each cycle independently carries out about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days or 1 week, 2 weeks, 3 weeks or longer time.In some aspects, the number of amplification cycles is greater than one, for example 2,3,4,5,6,7,8,9 or 10 cycles.In some aspects, each amplification cycle is to carry out about 7 days.In some aspects, the number of amplification cycles is 3.

[0025] In some aspects, the expanded cell population enriched for NK cells and γ.δ T cells is free of exhausted cells. In certain aspects, the expanded cell population enriched for NK cells and γ.δ T cells is free of exhausted cells after 60 days under the expansion conditions.

[0026] In some aspects, the cell colonies rich in NK cells and γ.δT cells obtained by the methods provided herein comprise 80% or more innate immune cells. In some aspects, about 80% to 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, at most 100% of the cells are innate immune cells.

[0027] In some aspects of the method provided herein, activation conditions, amplification conditions or both activation conditions and amplification conditions do not include bisphosphonates. Exemplary bisphosphonates include but are not limited to clodronate, etidronate, alendronate, pamidronate, zoledronate (zoledronic acid), neridronate etc.

[0028] In certain aspects of the methods provided herein, the compositions obtained by the methods provided herein comprise γ.δT cells that are polyclonal with respect to V.δ.1 and V.δ.2 expression. The relative amounts of V.δ.1 and V.δ.2 cells can be customized by, for example, the expansion conditions of one or more of the following: the absence of bisphosphonates, the choice of supplemental polypeptides, and the time period for which the sample or removed 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.δ.1 cells can be about 75% to about 95% of γ.δT cells, sometimes about 80% to about 90% of γ.δT cells, and (ii) V.δ.2 cells can be about 10% to about 25% of γ.δT cells, sometimes about 10% to about 15% or 20% of γ.δT cells. In certain aspects, when expansion conditions include IL-2 (e.g., without IL-15 and without a polypeptide that immunospecifically binds to CD3), (i) V.δ.1 cells can be about 30% to about 60% of γ.δ T cells, sometimes about 35% to about 55% of γ.δ T cells, and (ii) V.δ.2 cells can be about 35% to about 60% of γ.δ T cells, sometimes about 40% to about 50% or 55% of γ.δ T cells. In certain aspects, when expansion conditions include IL-15 (e.g., without IL-2 and without a polypeptide that immunospecifically binds to CD3), (i) V.δ.1 cells can be about 10% to about 30% of γ.δ T cells, sometimes about 20% to about 25% of γ.δ T cells, and (ii) V.δ.2 cells can be about 65% to about 80% of γ.δ T cells, sometimes about 70% to about 75% or 80% of γ.δ T cells.

[0029] Provided herein in certain aspects are compositions comprising a cell population, wherein the population comprises: a plurality of NK cells and a plurality of γ.δT cells; and α.βT cells are removed. In some aspects, the composition does not contain feeder cells. Also provided herein in certain aspects are compositions comprising a cell population, wherein the population comprises: a plurality of NK cells and a plurality of γ.δT cells; and α.βT cells are removed; and feeder cells are not contained. In certain aspects, the cell population is a modified population of peripheral blood cells. In certain aspects, the compositions provided herein comprise: (i) about 25% to about 45% of NK cells and about 55% to about 75% of γ.δT cells; (ii) about 25% to about 30% of NK cells and about 70% to about 75% of γ.δT cells; (iii) about 80% to about 99% of NK cells and about 1% to about 20% of γ.δT cells; or (iv) about 40% to about 45% of NK cells and about 55% to about 60% of γ.δT cells.

[0030] In some aspects, 30% or more of the cells in the compositions provided herein are activated. In some aspects, about 30% to 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%, 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%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125 %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, up to 100% of the cells.

[0031] In some aspects, the compositions provided herein comprise a cell population comprising one or more of the following activation markers, expressed 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 some aspects, the compositions provided herein include 80% or more innate immune cells. In some aspects, the compositions are rich in activated cytotoxic cells, which are CD56+; and in some aspects, about 80% to 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 aspects, the compositions provided herein are rich in activated cytotoxic cells, which are CD57-. In some aspects, the compositions provided herein are rich in activated cytotoxic cells, which are CD56+CD57-. In some aspects, about 10% to 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 aspects, 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 some aspects, the compositions provided herein are substantially free of cells other than NK cells and γ.δT cells. In some aspects, the compositions provided herein include about or less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% or less NKT cells and / or about or less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% or less α.βT cells. In some aspects, the subset of NK cells in the composition is CD16+ cells. In some aspects, most γ.δT cells, or most NK cells, or most γ.δT cells and NK cells are CD57- cells.

[0034] In certain aspects, the γ.δ T cells of the compositions provided herein are polyclonal with respect to V.δ.1 and V.δ.2 expression. In some aspects, the γ.δT cell population of the compositions provided herein comprises: (i) about 75% to about 95% V.δ.1 cells, for example, about 80% to about 90% V.δ.1 cells; and about 5% to about 25% V.δ.2 cells, for example, about 10% to about 15% or 20% V.δ.2 cells; or (ii) about 30% to about 60% V.δ.1 cells, for example, about 35% to about 55% V.δ.1 cells, and about 35% to about 60% V.δ.2 cells, for example, about 40% to about 50% or 55% V.δ.2 cells; or (iii) about 10% to about 30% V.δ.1 cells, for example, about 20% to about 25% V.δ.1 cells, and about 65% to about 80% V.δ.2 cells, for example, about 70% to about 75% or 80% V.δ.2 cells.

[0035] In certain aspects, about 50% to about 99% or more, or greater than or equal to about 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%, 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%, , 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or a portion thereof, up to 100% of the NK cells and / or the γ.δ T cells are CD8+. In some aspects, less than 2% of the NK cells and / or the γ.δ T cells are CD4+. In some aspects, less than 2% of the NK cells and / or the γ.δ T cells are CD8+CD4+. In certain aspects, a portion of about 15% to about 30% of said NK cells and / or about 55% to 85% of said γ.δ T cells are CD8-CD4-.

[0036] In some aspects of the compositions provided herein, about 30% to about 99% or more of 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%, 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 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 part thereof, up to 100% of the cells further include genetic modification, the genetic modification includes exogenous polynucleotides, mutated polynucleotides, deleted polynucleotides or a combination thereof. In some aspects, the genetic modification includes exogenous polynucleotides. The exogenous polynucleotides are sometimes in a retroviral vector or a lentiviral vector, and sometimes the exogenous polynucleotides are integrated into the genome of one or more cells of the modified cell population.

[0037] In some aspects, the exogenous polynucleotides can encode exogenous or heterologous T cell receptors, tumor necrosis factor receptors, chimeric antigen receptors (CAR), myeloid differentiation primary response proteins, innate immune signal transduction adapters or other target proteins or polypeptides, and in some aspects, promoters or other regulators of gene expression can be included. In some aspects, the exogenous polynucleotides are regulatory sequences, such as promoters or enhancers.

[0038] In some aspects, the exogenous polynucleotide encodes a chimeric antigen receptor (CAR), and the cells in the composition include CAR. CAR is a recombinant receptor that provides antigen binding and T cell activation functions (see, for example, Sadelain et al., Cancer Discov., 3 (4): 388-398 (2013)). When engineered (genetically modified) immune cells (eg, T cells) are expressed in CAR, it provides immune cells with new and / or improved capabilities for targeting target proteins or antigens. In some aspects, the target protein or antigen may be a cancer antigen or an infectious disease antigen, some of which are known in the art and / or identifiable. In some aspects, the CAR comprises a binding molecule portion that immunospecifically binds to one or more of: 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. 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 composition provided herein can be further treated to remove γ.δ T cells or NK cells, thereby producing a composition comprising substantially all NK cells or substantially all γ.δ T cells. For example, a composition provided herein can be treated with an anti-CD3 antibody to remove γ.δ T cells from a mixture of NK cells and γ.δ T cells, leaving a composition comprising substantially all or all NK cells, or an anti-CD3 antibody can be used to isolate a substantially pure population of γ.δ T cells from a mixture.

[0040] In some aspects, a therapeutic composition (or therapeutic combination) of cells is provided, comprising: a plurality of γδT cells (γδ); a plurality of natural killer cells (NK); or a combination of γδ and NK cells. In some aspects, γδ and / or NK cells are recombinantly engineered or genetically modified, wherein, optionally, γδ and / or NK cells are recombinantly engineered or genetically modified to express exogenous or heterologous proteins outside the cells, and optionally, the exogenous, heterologous or chimeric protein is a chimeric antigen receptor (CAR) or an exogenous or heterologous T cell receptor (TCR), and optionally, the exogenous, heterologous or chimeric protein or CAR has specificity for cancer cells or tumor markers or infected cells (can specifically bind to it), or the exogenous, heterologous or chimeric protein or CAR has specificity for antibodies that can specifically target and bind to cancer cells or tumor markers or infected cells or any disease-associated antigens (can specifically bind to it).

[0041] In some aspects, the γδ and / or NK cells are human cells or animal cells. In certain aspects, the therapeutic composition (or therapeutic combination) is formulated for intravenous (IV), intrathecal, intramuscular (IM), intraperitoneal (IP) or intrapleural (IT) administration, intraarticular administration, or injection or implantation at or near the site of cancer or infection, or formulated as a unit dosage form, wherein optionally, the unit dose comprises about 10 2 Up to 10 12 cells.

[0042] or intramuscular (IM) administration, or formulated into a unit dosage form, wherein optionally, the unit dose comprises about 10 2 Up to 10 12 cells.

[0043] In some aspects, γδ and / or NK cells are separated from in vivo sources. In some aspects, γδ and / or NK cells are expanded in culture or separated from in vivo sources and expanded in culture. In some aspects, γδ and / or NK cells are separated from in vivo sources and expanded in culture without using any feeder cells or without using feeder cell layers, so as to produce an amplification colony of γδ and / or NK cells without feeder cells. In some aspects, γδ and / or NK cells are separated from in vivo sources and expanded in culture using feeder cells or feeder cell layers, wherein optionally, the feeder cells are substantially removed and / or killed to produce an amplification colony of γδ and / or NK cells without feeder cells.

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

[0045] In certain aspects, also provided herein are pharmaceutical compositions comprising any of the compositions provided herein and a pharmaceutically acceptable carrier.

[0046] In some aspects, provided herein is a method for preparing genetically modified immune cells by adding exogenous polynucleotides to the compositions provided herein, mutating the polynucleotides in one or more cells of the compositions provided herein, or lacking the polynucleotides in one or more cells of the compositions provided herein. In some aspects, genetic modification is exogenous polynucleotides. The method for making the compositions provided herein generally results in cells having a high activation state, which promotes the introduction of exogenous polynucleotides, for example, by retroviral or lentiviral transduction. In certain aspects, the methods for making the genetically modified immune cells provided herein typically produce about 30% to about 99% or more, 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%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125 %, 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 a portion thereof, up to 100% of the cells include the genetically modified composition.

[0047] In some aspects, kits are provided herein, comprising any composition or pharmaceutical composition provided herein, optionally further comprising instructions for use and optionally cytokines. Compositions, pharmaceutical compositions or kits provided herein can be stored as needed at refrigerated temperatures (e.g., 10 degrees Celsius or lower, e.g., 9, 8, 7, 6, 5, 4, 3, 2, 1 to minus 4 degrees Celsius or lower) or frozen temperatures (e.g., minus 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 degrees or lower) for storage and / or transportation. In some aspects, the kit comprises about 1×10 5 cells to about 1×10 12 cells, for example, about 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 or 1×10 10Cells. In some aspects, the kits provided herein may include cytokines. In some aspects, the cytokines are selected from one or more of the following: TNF (see, e.g., GenBank Accession Nos. KJ892290.1; AY214167.1), IFNγ (see, e.g., GenBank Accession No. J00219.1), interleukins IL-1β, IL-2, IL-4, IL-6, IL-7, IL-10 (see, e.g., GenBank Accession No. U16720.1), IL-12 (for IL-12A, see, e.g., GenBank Accession No. AF404773.1; for IL-12B, see, e.g., GenBank Accession No. AF404773.1), 1), IL-15, IL-18 (see, e.g., GenBank Accession No. AF512686.1), IL-15, IL-18 (see, e.g., GenBank Accession No. BC007461.1; BC007007.1), IL-21, CCL4 (see, e.g., GenBank Accession No. 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 aspects, the kit comprises a composition or pharmaceutical composition provided herein in unit dosage form.

[0048] In some aspects, manufacturing products and kits for practicing the method provided herein are provided, including the therapeutic combination of cells provided herein. In some aspects, manufacturing products and kits also include instructions for practicing the method provided herein. In some aspects, manufacturing products and kits also include antibodies that can specifically bind to cancer-related or tumor-related, infection-related or disease-related antigens. Manufacturing products provided herein may include implants, and the implants include the therapeutic combination of cells provided herein.

[0049] In some aspects, the present invention also provides a method for treating cancer or infection by administering any of the compositions, pharmaceutical compositions or kits provided herein to a subject in need in an amount that can effectively treat cancer or infection. Treatment can be administered in an autologous environment or an allogeneic environment. In some aspects, the donor of the sample producing the composition, pharmaceutical composition or kit is the recipient of treatment. In some aspects, treatment can be administered on two or more separate days, and in some aspects, treatment can be administered in multiple doses. In some aspects, treatment is administered at intervals of about 1 unit dose to about 36 or more unit doses, from about 2 weeks to about 4 weeks. In some aspects, treatment is administered once, twice, three times, four times or up to five times a day with a single unit dose, or once, twice, three times, four times, five times, six times, seven times, eight times, nine times or ten times or more over a period of several days, weeks or months, or administered once every other day, or administered once, twice, three times, four times, five times or six times a week. Treatment can be administered at about 10 times per kilogram of subject body weight. 4 to about 10 10 A unit dose of 10 cells, or approximately 10 per subject 6 to about 10 12 The unit dose of about 10 cells is administered intravenously (IV), intrathecally or intramuscularly (IM), intraperitoneally (IP), intrapleurally, intraarticularly, or injected or implanted at or near the site of cancer or infection. In certain aspects, the unit dose is about 10 cells per subject. 10 cells, or about 10 per kilogram of subject body weight. 8 cells.

[0050] In some aspects of the methods of treatment provided herein, 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 lymphoma or childhood acute lymphoblastic leukemia, non-Hodgkin lymphoma, mastocytoma or mast cell tumor, ovarian cancer, pancreatic cancer, non-small cell lung cancer, small cell lung cancer, liver cancer, retinoblastoma, breast tumor, colorectal cancer, leukemia, lymphoma, acute lymphoblastic leukemia (ALL) or acute lymphatic leukemia, acute myeloid leukemia (AML), histiocytic sarcoma, brain tumor, astrocytoma, glioblastoma, neuroma, colon cancer, cervical cancer, sarcoma, bladder tumor, reticuloendothelial tumor, Wilm's tumor, bone cancer, osteosarcoma, kidney cancer or head and neck cancer, oral cancer, laryngeal cancer, metastatic disease or oropharyngeal cancer.

[0051] In certain aspects of the methods of treatment provided herein, a second agent is co-administered with the composition, pharmaceutical composition, or kit. In some aspects, the second agent is an antibody that immunospecifically binds to a cancer-associated antigen. In certain aspects, the cancer-associated antigen is selected from the group consisting of: alpha-fetoprotein (AFP), α-actinin-4, A3, an antigen specific for the A33 antibody, ART-4, B7, B7-H3, Ba 733, 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, CD2 2. 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, C D80, 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, 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 (MIF), GD2, MAGE, MAGE-3, MART-1, MART-2, NY-ESO-1, TRAG-3, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MU C1, 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, SA GE, S100, survivin, survivin-2B, TAC, TAG-72, tenascin, TRAIL receptor, TNF-α, Tn antigen, Thomson-Friedenreich 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 some aspects, the cells of the compositions provided herein can be engineered to express antibodies that bind to cancer-associated antigens. In some aspects, the antibodies are co-administered as a second agent. In some aspects, the cancer-associated antigens are selected from 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 antibodies, RFB4 (anti-CD22), hMu-9 (anti-CSAp), hL243 (anti-HLA-DR), hMN-14 (anti-CEAC AM-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-CD25), daclizumab (daclizumab) (anti-CD25), efalizumab (anti-CD11a), muromonab-CD3 (anti-CD3 receptor), natalizumab (anti-α4 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 some aspects of the methods of treatment provided herein, treatment is directed to infection. In some aspects, infection is characterized by the presence of bacteria, fungi, viruses or protozoan pathogens. In some aspects of the methods of treatment provided herein, infection is selected from the group consisting of: herpes, Ebola virus, West Nile virus, vaccinia virus, Epstein-Barr virus, hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), herpes virus (such as HSV-1, HSV-2, HHV-6, CMV), human immunodeficiency virus (HIV), vesicular stomatitis virus (VSV), bacillus (Bacilli), Citrobacter (Citrobacter), cholera (Cholera), diphtheria (Diphtheria), Enterobacter (Enterobacter), gonococcus (Gonococci), Helicobacter pylori (Helicobacter pylori), Klebsiella, Legionella, Meningococci, mycobacteria, Pseudomonas, Pneumococci, 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.).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 Fragilis, Trypanosoma brucei, Trypanosoma cruzi, or Leishmania donovani.

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

[0055] In some aspects of the methods of treatment provided herein, individuals in need are humans or animals. In some aspects, γδ and / or NK cells are separated from in vivo sources, and optionally, the in vivo sources of γδ and / or NK cells are from isogenic or autologous sources (optionally, from individuals who will become recipients of the γδ and / or NK cells), or exogenous, heterologous or allogeneic sources, or combinations thereof. In some aspects of the methods provided herein, the therapeutic composition or combination of cells are administered intravenously (IV), intrathecally or intramuscularly (IM), or injected or implanted in or near (adjacent) cancer cells, tumor cells, dysfunctional cells or infected cells, and optionally, the therapeutic composition of cells is delivered in the form of implants or gels, wherein optionally, the gel is a hydrogel.

[0056] In some aspects of the treatment methods provided herein, the therapeutic composition or combination of cells is administered in a unit dosage form, wherein optionally, the unit dose comprises about 10 2 Up to 10 12 cells or 10 4 Up to 10 10 cells; or a daily dose comprising about 10 2 Up to 10 12 cells or 10 4 Up to 10 10 In certain aspects, the therapeutic composition or combination or unit dosage form of cells is administered to the individual in need thereof several times (multiple times) or two, three, four, five, six, seven, eight, nine or ten times or more over the course of days, weeks or months, and optionally, each of the plurality of unit dosage forms is administered once a day, once every other day, 2, 3, 4, 5 or 6 times per week, or once a week.

[0057] In certain aspects of the treatment methods provided herein, an individual in need of treatment is first induced to initiate an immune response against cancer, infection or disease by pre-administering an unbound antibody to a cancer-related or tumor-related, infection-related or disease-related antigen to the individual in need, followed by administration of a therapeutic composition of the cells described herein, wherein at least some of the therapeutic compositions of the cells express polypeptides that specifically bind to the unbound antibody on their cell surfaces, optionally CAR. In some aspects, antibodies that can specifically bind to cancer-related or tumor-related, infection-related or disease-related antigens are administered to the individual in need, followed by administration of a therapeutic composition of the cells described herein, wherein at least some of the therapeutic compositions of the cells express polypeptides that specifically bind to the antibody on their cell surfaces, optionally CAR, and optionally, the antibodies are administered before, during or after administration of the therapeutic composition of the cells.

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

[0059] All publications, patents, and patent applications cited herein are expressly incorporated by reference for all purposes. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] The accompanying drawings illustrate certain embodiments of the present technology and are not limiting. For clarity and ease of illustration, the accompanying drawings are not drawn to scale, and in some cases, various aspects may be shown exaggerated or enlarged to facilitate understanding of specific embodiments.

[0061] Figure 1Shown is the expansion of αβTCRneg monocytes as a function of the number of days the cells were activated with antibodies and expanded in feeder-free culture conditions, as analyzed on day 10 of culture.

[0062] Figure 2 Analysis of the composition of the activated expanded αβTCRneg monocyte population (referred to herein as the INNATE-K or BINATE cell population) is shown.

[0063] Figure 3 Shown are the frequencies and distributions of T cell lineage markers CD4 (helper) and CD8 (cytotoxic) on γδTCR+ cells in day 10 samples of activated αβTCRneg cells expanded in feeder-free culture, as analyzed by flow cytometry.

[0064] Figure 4 Shown is an analysis of the expansion rate of CAR.19INNATE-K cells over 10 days.

[0065] Figure 5 Data are shown for co-culture cytotoxicity assays in which INNATE-K cells and INNATE-CAR.19 cells were co-cultured with CD19+ leukemia (221) or CD19+ lymphoma (Daudi) cell lines.

[0066] Figure 6 Shown are the total number of INNATE-NK cell populations expanded in plates during prolonged in vitro culture.

[0067] Figure 7 Comparison of total cell numbers of plate- versus bioreactor-expanded INNATE-NK populations is shown.

[0068] Figure 8 Subset cell composition during different time points of in vitro INNATE-NK expansion is shown.

[0069] Fig. 9 Shown is the expression of chimeric antigen receptor (CAR) molecules in INNATE-NK CAR cells during prolonged in vitro culture.

[0070] Fig.10 The expression of activation and cytolytic molecules after expansion of INNATE-NK and INNATE-NK CAR populations is shown.

[0071] Fig.11 Lack of depletion in feeder-free expanded INNATE-NK cells and INNATE-NK CAR cells is shown.

[0072] Fig.12Cytotoxicity co-culture assays of INNATE-NK or INNATE-NK-CAR.19 and 4 tumor cell lines are shown: Row A: 221, a CD19+ leukemia cell line; Row B: Daudi, a CD19+ lymphoma cell line; Row C: BV173, a CD19+ (variable expression) pre-B tumor cell line; and Row D: KARPAS, a CD19- tumor cell line.

[0073] Fig.13 and 14 Shown are the percentages of specific lysis of primary tumor cells in different assay runs as a function of the ratio of effector (E) to target (T) for INNATE-NK cell and INNATE-NK-CAR.19 cell populations.

[0074] Fig.15 Shown are the percentages of residual primary CD19+ tumors after co-culture with effector cells (INNATE-NK cell and INNATE-NK-CAR.19 cell populations) compared to control conditions in which primary CD19+ leukemic blasts were inoculated in the absence of effector cells.

[0075] Fig.16 Survival curves for animals receiving INNATE-NK and INNATE-NK-CAR.19 cells are shown.

[0076] Fig.17 Total BINATE expansion is shown, represented by total cell number over time (i.e., expansion of activated αβTCR neg cells in BINATE medium supplemented with IL-2 or IL-15 in flasks, and IL-15 expansion of activated αβTCR neg cell populations in bioreactors).

[0077] Fig.18 Total BINATE expansion is shown, represented by total cell number over time (i.e., expansion of activated αβTCRneg cells in a flask in BINATE medium supplemented with IL-2, IL-15, IL-2 / OKT3, IL-2 / IL-15, or IL-2 / IL-15 / OKT3).

[0078] Fig.19 Phenotypic analysis by marker type (activation, cytotoxicity, and exhaustion / immaturity) is shown for IL-15 expansion conditions.

[0079] Fig. 20 Phenotypic analysis by marker type (activation, cytotoxicity, and exhaustion / immaturity) for IL-2 expansion conditions is shown.

[0080] Fig.21 Phenotypic analysis by marker type (activation, cytotoxicity, and exhaustion / immaturity) is shown for the IL-2 / OKT3 expansion condition.

[0081] Fig. 22 Shown are the cell numbers recorded approximately every 7 days for the BINATE and BINATE.CARGD2 populations.

[0082] Fig.23 In vivo maintenance of human BINATE cells in mouse blood and mouse liver is shown.

[0083] Fig.24 Shown are flow charts illustrating example variations of methods for isolating a pure population of NK cells and / or isolating a pure population of γδ T (gd) cells. DETAILED DESCRIPTION

[0084] In certain aspects, therapeutic immune cell compositions are provided herein, wherein a large number of immune cells or most of the immune cells are innate immune cells. This composition can produce an innate immune response in a subject after administration. The innate immune system is active and continuously works in the host, reacts rapidly (within minutes to hours after infection), and reacts in a non-specific manner, thereby providing the potential for "ready-made" immunotherapy for a wider patient population. Innate immune cells (such as NK cells and γ.δ (γδ) T cells) do not recognize classical HLA antigens, so they can be used in ready-made (allogeneic) environments, while alleviating graft-versus-host disease (GvHD). Innate immune cells can be amplified in vitro, thereby avoiding cytokine release syndrome (CRS). Innate immune cell compositions (wherein components (such as NK cells, γ.δ (γδ) T cells) and the amount of components can be customized) can be used to provide a coordinated attack front for multiple cancers (including solid tumors and hematological cancers), infections, etc.

[0085] In contrast, some immune cell compositions not produced by the methods described herein include most adaptive immune cells rather than innate immune cells. When utilizing an immune cell composition in which most cells are adaptive immune cells, recognition is performed through HLA antigens, and there is a significant risk of GvHD, especially if the adaptive immune cells used in immunotherapy are from allogeneic sources. Therefore, the adaptive immune cells used in such compositions are most often derived from patients / subjects (autologous) or designated "matched" donors, which limits the full functionality of immunotherapy, i.e., the ability to be used in a "ready-made" manner for a broad patient population. Most cell therapies utilizing such compositions are autologous, or at least derived from specific donors. This means that the dose of the patient must be adjusted individually for each patient. Therefore, current T cell-based therapeutic compositions, such as cell therapies using chimeric antigen receptors (CARs) or exogenous T cell receptors (TCRs) (like in αβT cells), cannot be used "ready-made". The manufacturing process may take six weeks or more, during which the patient's disease (such as cancer or infection) may have progressed. In addition, some of these patient-specific manufacturing processes fail for a variety of reasons, including not only general manufacturing failures, but also specific failures due to the exhausted state of the patient-donor's immune system after chemotherapy or radiation therapy. In addition, the expansion of αβT cells (or CAR-modified "CAR-T" cells) in vivo sometimes leads to a large and rapid release of cytokines into the blood, resulting in severe or life-threatening CRS.

[0086] Therefore, the therapeutic immune cell compositions described herein provide advantages over cell compositions comprising most adaptive immune cells. The therapeutic immune cell compositions described herein are also modified and altered relative to biological samples obtained from naturally occurring subjects. The therapeutic immune cell compositions, manufacturing methods, and therapeutic uses are described in detail below.

[0087] Methods for producing innate immune cell compositions

[0088] Provided herein are methods of making innate immune cell compositions. The compositions made by the methods provided herein typically contain a mixture of two activated innate immune cell populations: Natural Killer (NK) cells and γ.δ T cells.

[0089] Relative to the composition of immune cells in nature (e.g., biological fluids and tissues), the immune cell composition obtained by the method provided herein is rich in NK cells and γ.δT cells as natural immune cells. In nature, the amount of α.βT cells found as adaptive immune cells is much higher than that of NK cells and γ.δT cells. On the other hand, in the composition provided herein, α.βT cells are not present or are present in negligible to very low amounts, while NK cells and γ.δT cells are the main immune cell components.

[0090] For example, in human peripheral blood, it has been found that in the peripheral blood of healthy human donors: (i) for individuals under 40 years old, the median percentage of NK cells in PBMCs is 5%, while the median percentage of T cells (CD8+ and CD4+ combined population, representing α.βT cells, because γ.δT cells are usually CD8-CD4-) is 53%; and (ii) for individuals over 40 years old, the median percentage of NK cells in PBMCs is 10%, while the median percentage of T cells (CD8+ and CD4+ combined population) is 37% (Lepone et al., J. Circ. Biomark., 5(5): 1-17 (2016)). Therefore, in peripheral blood, in individuals over 40 years old, for every 1 circulating NK cell, there are approximately almost 4 circulating α.βT cells, while in individuals under 40 years old, for every 1 circulating NK cell, there are approximately slightly more than 10 circulating α.βT cells. Thus, the ratio of NK cells to α.β T cells in the circulating blood is in the range of about 1: 4 to about 1: 10. On the other hand, in the compositions provided herein, NK cells account for 20% or more of the composition, up to 99%, while α.β T cells are almost absent and account for less than 2%, typically almost 0%, or less than 0.5%, 0.4%, 0.3%, 0.2% or 0.1% of the composition. Thus, in the compositions provided herein, the ratio of NK cells to α.β T cells is at least 10:1, and typically much higher than 10:1, for example, about or greater than 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, 100:1, 150:1, 200:1, 250:1, 300:1, 350:1, 400:1, 450:1, 500:1, 550:1, 600:1, 650:1, 700:1, 750:1, 800:1, 850:1, 900:1, 950:1 or 1000:1 or more. Thus, although α.β T cells predominate relative to NK cells in nature, in the compositions provided herein, the NK cell population is greatly increased relative to the α.β T cell population.

[0091] Regarding γ.δT cells, for example, in peripheral blood, less than about 10% of T cells, typically about 5% of T cells are γ.δT cells, and the rest are α.βT cells (Esin et al., Scand. J. Immunol., 43(5):593-596 (1996); Radestad et al., J. Immunol. Res., Article ID 578741 (2014)). Therefore, the ratio of γ.δT cells to α.βT cells in circulating blood is in the range of about 1:10 to about 1:20. On the other hand, in the compositions provided herein, γ.δT cells account for 1% or more of the composition, typically between 2% or more and 70-75%, while α.βT cells are almost absent and account for less than 2% of the composition, typically almost 0%, or less than 0.5%, 0.4%, 0.3%, 0.2% or 0.1% of the composition. Therefore, in the composition provided herein, even assuming a composition in which γ.δ T cells are present at 1% and α.β T cells are present at 2%, the ratio of γ.δ T cells to α.β T cells is at least 0.5:1. Thus, in the compositions provided herein, the ratio of γ.δ T cells to α.β T cells is much higher, for example, about or greater than 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, 100:1, 150:1, 200:1, 250:1, 300:1, 350:1, 400:1, 450:1, 500:1, 550:1, 600:1, 650:1, 700:1 or 750:1 or more.

[0092] As used herein, the term "enriched" refers to the following two ratios in the composition provided herein: (i) the ratio of γ.δ T cells to α.β T cells, and (ii) the ratio of NK cells to α.β T cells are higher than these ratios in nature (e.g., in a biological sample, such as peripheral blood). Generally, as used herein, "enriched" means that the ratio of γ.δ T cells to α.β 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 relative to that ratio in a biological sample (e.g., tissue, umbilical cord blood, or peripheral blood). About NK cells, in general, as used herein, "enriched" means that the ratio of NK cells to α.βT cells in the compositions provided herein is increased by at least 40 times, 45 times, 50 times, 55 times, 60 times, 65 times, 70 times, 75 times, 80 times, 85 times, 90 times, 95 times, 100 times, 150 times, 200 times, 250 times, 300 times, 350 times, 400 times, 450 times, 500 times, 550 times, 600 times, 650 times, 700 times, 750 times, 800 times, 850 times, 900 times, 950 times or 1000 times or more relative to the ratio in a biological sample (e.g., tissue, cord blood, or peripheral blood). In certain aspects, the term "enriched" means that the ratio of NK cells to α.βT cells and the ratio of γ.δT cells to α.βT cells of the compositions provided herein are greater than 1 (the ratio is usually less than 1 in nature).

[0093] In peripheral blood, B cells and α.βT cells, which are adaptive immune cells, constitute the majority of lymphocytes. On the other hand, the composition manufactured by the method provided herein is rich in innate immune cells, i.e., NK cells and γ.δT cells. The terms α.βT cells and αβT cells are used interchangeably herein to refer to adaptive immune T cells, while the terms γ.δT cells and γδT cells are used interchangeably herein to refer to innate immune T cells.

[0094] For a variety of reasons, compositions for immunotherapy composed of innate immune cells are superior to compositions comprising adaptive immune cells. The innate immune response is non-specific and is 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 a composition that does not exhaust NK cells and γ.δT cells for up to 60 days after expansion, which allows for long-term storage (suitable for ready storage and re-administration when necessary) and the ability to administer multiple doses cost-effectively. On the other hand, adaptive immune cells initiate antigen-specific immune responses and expand when bound to target antigens in vivo, which may take several days or even weeks. In addition, unlike innate immunity, adaptive immune responses rely on HLA antigens, which require cells to be autologous or "matched" with recipient patients / subjects to minimize or avoid GvHD (graft versus host disease). Compositions comprising innate immune cells (e.g., NK cells and γ.δ (γδ) T cells) prepared by the methods provided herein do not require HLA antigen recognition to mediate killing, and can therefore be used in a wider range of off-the-shelf (allogeneic) environments while alleviating GvHD. Adaptive immune cells also produce a large amount of cytokines when expanded in vivo, which may lead to cytokine release syndrome (CRS); innate immune cells can be expanded ex vivo, thereby minimizing or avoiding CRS.

[0095] In the methods provided herein, a sample comprising cells, such as peripheral blood or umbilical cord blood, is obtained from a subject. The subject is usually a healthy donor, but may also be a patient who needs to be treated with the immunotherapy composition prepared by the method provided herein. In some aspects, when the sample is peripheral blood, a peripheral blood universal donor pool can be used as a sample source. The sample sometimes undergoes activation conditions, including contacting the sample with the following polypeptides: (a) at least one immunospecific binding to an exogenous polypeptide of a cell adhesion polypeptide, and (b) at least one immunospecific binding to a polypeptide different from the cell adhesion polypeptide and an exogenous polypeptide expressed on the surface of one or more cells of a 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 (for CD8A, see, e.g., GenBank Accession Nos. AH003215.2; AY039664.1; for CD8B, see, e.g., GenBank Accession Nos. KJ896562.1; BC100911.1; BC100912.2; BC100913.1; BC100914.1), and CD4 (see, e.g., GenBank Accession Nos. M35160.1; DQ892052.2).Examples of polypeptides expressed on the surface of one or more cells (e.g., NK cells and / or γ.δ cells) of a sample population include, but are not limited to, CD2 (see, e.g., GenBank Accession Nos. KJ905161.1; KJ896558.1), CD3 (for CD3γ, see, e.g., GenBank Accession No. AB583162.1; for CD3ε, see, e.g., GenBank Accession No. AB583139.1; for CD3δ, see, e.g., GenBank Accession No. AH002612.2), CD56 (see, e.g., GenBank Accession No. AB583162.1), CD57 (see, e.g., GenBank Accession No. AB583163.1), CD58 (see, e.g., GenBank Accession No. AB583164.1), CD59 (see, e.g., GenBank Accession No. AB583165.2), CD61 (see, e.g., GenBank Accession No. AB583166.1), CD70 (see, e.g., GenBank Accession No. AB583167.1), CD71 (see, e.g., GenBank Accession No. AB583168.1), CD80 (see, e.g., GenBank Accession No. AB583169.1), CD81 (see, e.g., GenBank Accession No. AB583167.1), CD82 (see, e.g., GenBank Accession No. AB583168.1), CD83 (see, e.g., GenBank Accession No. AB583169.1), CD84 (see, e.g., GenBank Accession No. AB583167.1), CD85 such as 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 ( 193441.1; AF170485.1), KIR3DS1 (see, e.g., GenBank Accession No. EU156175.1), NKG2C (see, e.g., GenBank Accession No. BC093644.1; BC112039.1), NKG2D (see, e.g., GenBank Accession No. AF461811.1; BC039836.1), KAR receptors, KIR receptors, SIGLEC-7 (see, e.g., GenBank Accession No. AF ... .1), KIR2DL1 (see, e.g., GenBank Accession No. LT984790.1; LT984791.1; antibody: 11PB6), DNAM1 (see, e.g., GenBank Accession No. BC074787.2; U56102.1), NTBA (see, e.g., GenBank Accession No. BC114495.1; BC113893.1), HLA-DR (for α, see, e.g., GenBank Accession No. AH001506.2; for β, see, e.g., AH002824.2), etc. Activation of cells generally initiates an innate immune response in the cells of the compositions provided herein, which can be used to target such diseases, e.g., cancer or infectious diseases, in subjects in need of treatment of the disease.

[0096] A polypeptide immunospecifically binds to a region of another molecule (i.e., an epitope) if the polypeptide reacts or associates with the epitope more frequently, more rapidly, for a longer duration, and / or with a greater affinity relative to an alternative epitope. For example, a polypeptide (e.g., an antibody or antigen-binding fragment thereof) that immunospecifically binds to a first epitope is an antibody that binds to the first epitope with greater affinity, avidity, more readily, and / or for a longer duration 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.

[0097] In certain aspects of the methods provided herein, the activation conditions include contacting the sample with at least two exogenous polypeptides. In some aspects, the first exogenous polypeptide immunospecifically binds to CD2, and the second exogenous polypeptide immunospecifically binds to NKp46. In some aspects, the activation conditions consist of or consist essentially of a first exogenous polypeptide immunospecifically bound to CD2 and a second exogenous polypeptide immunospecifically bound to NKp46. As used herein, the phrase "consisting essentially of" means that components other than the listed components (if present) do not substantially change the activity of the listed components. Thus, for example, in the context of the above-mentioned activation conditions, the activation conditions may include one or more components other than the first exogenous polypeptide immunospecifically bound to CD2 and the second exogenous polypeptide immunospecifically bound to NKp46, which components do not substantially change the activity of the first exogenous polypeptide and the second exogenous polypeptide. In some aspects, the first exogenous polypeptide and / or the second exogenous polypeptide is an antibody or an antigen-binding fragment thereof.

[0098] The sample is also generally subjected to amplification conditions, including contacting the sample with at least one supplementary polypeptide, thereby producing a composition comprising a population rich in NK cells and γ.δT cells. In some aspects of the methods provided herein, the amplification conditions include at least one supplementary polypeptide, which is a cytokine and / or a polypeptide that immunospecifically binds to a receptor on a γ.δT cell. In some aspects, 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 aspects of the methods provided herein, the cytokine is IL-2, IL-15, or a combination thereof. In some aspects, the amplification conditions include, consist of, or are substantially comprised of IL-2, IL-15, and a polypeptide that immunospecifically binds to a receptor on a γ.δT cell. In some aspects, the receptor on a γ.δT cell is CD3. In some aspects, the polypeptide that immunospecifically binds to the CD3 receptor on a γ.δT cell is an antibody or an antigen-binding fragment thereof, and in some aspects, the antibody is OKT3. In certain aspects, the amplification 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 IL-2 polypeptide and an antibody that immunospecifically binds to CD3; or (e) an IL-2 polypeptide, an IL-15 polypeptide, and an antibody that immunospecifically binds to CD3. In some aspects, the antibody that immunospecifically binds to CD3 is OKT3.

[0099] In some aspects, the activation conditions consist or consist essentially of contacting the sample with a first exogenous polypeptide that immunospecifically binds to CD2 and a second exogenous polypeptide that immunospecifically binds to NKp46, and the expansion conditions consist or consist essentially of contacting the sample with the following polypeptides: (a) IL-2 polypeptide; (b) IL-15 polypeptide; (c) IL-2 polypeptide and IL-15 polypeptide; (d) IL-2 polypeptide and an antibody that immunospecifically binds to CD3; or (e) IL-2 polypeptide, IL-15 polypeptide and an antibody that immunospecifically binds to CD3. In some aspects, the antibody that immunospecifically binds to CD3 is OKT3.

[0100] In some aspects, the method for making a composition provided herein includes removing α.βT cells from a sample before activation and amplification, thereby producing a cell population that can then undergo activation and amplification. For example, an antibody that immunospecifically binds to an α.βT cell receptor, such as an α.βTCR, can be used to remove α.βT cells from a sample. As used herein, the term "removal" refers to substantially all of the removed components (e.g., α.βT cells) have been removed from a sample, for example, 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 higher, up to about 100% of the removed components have been removed from the sample. Typically, as used herein, the term "removal of a cell population" refers to a cell population derived from a sample after α.βT cells are removed from a sample. In some aspects, a polypeptide that immunospecifically binds to a B cell receptor, such as CD19, is used to additionally remove B cells from a sample.

[0101] For example, when the sample is a peripheral blood sample, the sample can be processed by density gradient centrifugation to separate and / or separate the buffy coat containing leukocytes, platelets, granulocytes, etc., and then α.βT cells can be removed according to the method provided herein. In some aspects, the buffy coat can be further subjected to Ficoll gradient separation to obtain mononuclear cells (PBMC), and then α.βT cells can be removed in the method provided herein. In some aspects, the peripheral blood sample can be subjected to a single blood component method to separate cells from plasma (e.g., using a Terumo Optia machine), and in some aspects, α.βT cells can then be removed from cells in the method provided herein. α.βT cell removal can be carried out by methods known to those skilled in the art. In some aspects, α.βT cells can be removed using a Miltenyi Ls column. In some aspects, α.βT cells can be removed from a single blood component method product using a Miltenyi Clinimacs separation device. If necessary or desired, the depleted cell population can be cryopreserved (e.g., at minus 70, 75, 80, 85 or lower degrees Celsius) and stored prior to 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 NK cells and γ.δ cells, or NK MACS supplemented with 5% AB serum. TM Culture medium (#130-107-879 (Miltenyi Biotec, Inc., San Diego, CA, USA)), or other culture media used in the art (eg, R&D Systems, CellGenix).

[0102] In certain aspects, the cell population is not exposed to conditions that positively select for NK cells or positively select for γ.δ T cells prior to the activation and expansion conditions described herein.

[0103] In some aspects of the methods provided herein, the sample or the removed cell population is subjected to activation conditions, wherein the exogenous polypeptide in (a), the exogenous polypeptide in (b), or the exogenous polypeptide in (a) and the exogenous polypeptide in (b) are both soluble. In some aspects, the soluble exogenous polypeptide in (a), the soluble exogenous polypeptide in (b), or the soluble exogenous polypeptide in both (a) and (b) is an antibody or an antigen-binding fragment thereof. As used herein, the term "soluble" with respect to a component such as a polypeptide refers to that the component is not bound to a solid phase or a carrier, and is a uniform single phase or emulsion under culture conditions (e.g., activation conditions, amplification conditions). In some aspects, the exogenous polypeptide in (a) or (b), such as an antibody, is bound to a solid phase or a carrier. 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 (dextran gel), Sepharose (agarose gel), cellulose, magnetic beads, Dynabeads, metal surfaces (e.g., steel, gold, silver, aluminum, silicon, and copper), plastic materials (e.g., polyethylene, polypropylene, polyamide, polyester, polyvinyl difluoride (PVDF)). The solid support can be in any desired form, including, but not limited to, beads, chips, capillaries, plates, membranes, wafers, combs, needles, substantially flat surfaces, arrays of pits or nanoliter wells, and other geometries and forms known to those skilled in the art. Using a soluble exogenous polypeptide rather than a polypeptide bound to a solid support can reduce steric hindrance while increasing the scalability of the process (e.g., for GMP manufacturing). In some aspects, the first exogenous polypeptide is a soluble anti-CD2 antibody, and the second polypeptide is a soluble anti-NKp46 antibody. In some aspects, the anti-CD2 antibody or the anti-NKp46 antibody is bound to a solid support.

[0104] Antibodies, such as polyclonal antibodies and monoclonal antibodies, can be prepared using standard methods (see, for example, Kohler et al., Nature 256: 495-497 (1975); Kohler et al., Eur. J. Immunol. 6: 511-519 (1976); and WO 02 / 46455). For example, to produce polyclonal antibodies, an immune response to the antigen of interest is induced in a host animal. Blood from the host animal is then collected using methods known to those skilled in the art, and the serum fraction containing the secreted antibodies is separated from the cell fraction. In order to produce monoclonal antibodies, animals are immunized by standard methods to produce somatic cells that secrete antibodies. These cells are then removed from the immunized animal to fuse with myeloma cells. Somatic cells that can produce antibodies, particularly B cells, can be used to fuse with myeloma cell lines. These somatic cells can be derived from lymph nodes, spleen, and peripheral blood of primary immunized animals. Specialized myeloma cell lines have been developed from lymphocytic tumors for use in fusion procedures to produce hybridomas (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 properties. The first property is that they have an enzyme defect that prevents them from growing in a selective medium that supports hybridoma growth, thereby facilitating the selection of fused hybridomas from myeloma cells that have never been fused and similarly self-propagated indefinitely. The second property is that they have the ability to produce antibodies and cannot produce endogenous immunoglobulin light or heavy chains. The third property is that they can be effectively fused with other cells. Other methods for producing hybridomas and monoclonal antibodies are well known to those skilled in the art. It is conventional for producing antibodies against any polypeptide (e.g., an antigenic marker on an immune cell population).

[0105] In some aspects of the methods provided herein, activation conditions, amplification conditions, or activation conditions and amplification conditions are both free of feeder cells. As used herein, the term "free" (e.g., free of multiple feeder cells or free of feeder cells, free of serum, free of serum from non-human animals, free of exhausted cells) refers to conditions that are substantially free of (i.e., at least 80%, 85%, 90%, 95%, typically 95% or higher, such as 96%, 97%, 98%, 99% or higher, up to 100% free of) the component (i.e., feeder cells, serum, exhausted cells or other components mentioned herein). In some aspects of the methods provided herein, samples, activation conditions, amplification conditions, or activation conditions and amplification conditions or all of the aforementioned 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 typically cells from different subjects, or cells from different parts of subjects, compared to subjects or subject parts from which sample cells are obtained and subjected to activation conditions and / or amplification conditions. In non-limiting examples, (i) the sample cells are from a subject of a first species, and the exogenous cells are from a subject of a second species (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 peripheral blood of a human subject, and the exogenous cells are from a different part of the same subject (e.g., cord blood or an organ from the same subject, or from a different human subject). A composition is typically "free of" a component when the sample has not been contacted with the component during one or all of the following processes: processing prior to activation and / or expansion, activation, and expansion.

[0106] Dependence on feeder cells (e.g., K562 cells or other cells) limits the culture location and culture method of cells, and can significantly increase the cost of cell culture. Due to the cell culture variability caused by uncertain biological factors derived from feeder cells, the use of feeder cells may also be problematic. In addition, feeder cells have the possibility of introducing unwanted reagents (e.g., retroviruses, other pathogens, and immunogenic non-human sialic acids, such as Neu5Gc) into the compositions prepared by the methods provided herein, which may be undesirable for certain applications such as transplantation. Without being bound by theory, in the methods provided herein, exogenous polypeptides that immunospecifically bind to cell adhesion polypeptides can produce an "endogenous feeder cell-like layer", thereby producing a significantly activated amplified cell population without the need for exogenous feeder cells.

[0107] In certain aspects of the methods provided herein, the selection of one or more supplementary polypeptides and / or the time of expansion under expansion conditions can be customized according to the desired relative amounts of NK cells and γ.δT cells. For example, compositions containing relatively more NK cells and relatively fewer γ.δT cells generally have greater applicability to solid tumors, while compositions containing relatively more γ.δT cells and relatively fewer NK cells generally have greater applicability to hematological malignancies. One or more supplementary polypeptides can be selected, and / or the expansion reaction can be performed for a period of time to facilitate obtaining a composition having a desired ratio of NK cells and γ.δT cells. The desired ratio of NK cells to γ.δT cells can be obtained by selecting specific expansion conditions, and non-limiting examples of combinations are as follows: (i) when the supplementary polypeptide is IL-2, the resulting cell population enriched in NK cells and γ.δT cells generally contains about 25-30% NK cells and about 70-75% γ.δT cells; (ii) when the supplementary polypeptide is IL-15, the resulting cell population enriched in NK cells and γ.δT cells generally contains about 80-99% NK cells and about 1-20% γ.δT cells; (iii) when the supplementary polypeptide is IL-2 and an antibody that immunospecifically binds to CD3 (e.g., OKT3), the resulting cell population enriched in NK cells and γ.δT cells generally contains about 10-20% NK cells and about 20-30% γ.δT cells. The cell population typically contains about 40-45% NK cells and about 55-60% γ.δT cells; (iv) when the polypeptide is supplemented with IL-2 until day 20 of the expansion conditions and then switched to IL-15 until day 30, the percentage of γ.δT cells typically increases from about 50% to about 70%, accompanied by a corresponding decrease in the percentage of NK cells, compared to treatment with IL-2 alone; and (v) when the polypeptide is supplemented with IL-15 until day 20 of the expansion conditions and then switched to IL-2 until day 30, the percentage of NK cells typically increases from about 80% to about 90%, accompanied by a corresponding decrease in the percentage of γ.δT cells, compared to treatment with IL-15 alone.

[0108] The duration of the amplification conditions (e.g., 12 days vs. 25 days) can also control the relative amount of NK cells and γ.δT cells in the compositions prepared by the methods provided herein. For example, the incubation time of the sample or the removal of the cell population in the culture medium without feeder cells under amplification conditions can be 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 longer, or about 2, 3, 4, 5, 6, 7, 8, 9 or 10 weeks. In some aspects of the methods provided herein, the composition does not contain exhausted cells, and in some aspects, after at least 60 days of amplification conditions, the composition does not contain exhausted cells. Due to the increased ex vivo availability of the amplified cytotoxic cell composition, the preparation of a cell composition substantially free of exhausted cells allows for immediate treatment and multiple administrations, and the cost is only a fraction of many current immunotherapies (e.g., CAR-T using α.βT cells).

[0109] In certain aspects of the methods provided herein, the activation conditions or expansion conditions or the activation conditions and expansion conditions do not include bisphosphonates, generating polyclonal populations of V.δ.1 and V.δ.2γ.δT cells. The use of bisphosphonates, such as zoledronate and pamidronate, may favor a certain clonal population of γ.δT cells over another clonal population, which may limit the full functionality of the composition against certain tumors and infections. In some aspects, the absence of bisphosphonates in the methods provided herein produces a composition that is polyclonal with respect to γ.δT cells, thereby increasing the range of cancers and infectious diseases for which they can be administered as immunotherapy.

[0110] A non-limiting example of an overview of steps for performing the methods provided herein is as follows:

[0111] (1) If necessary, a sample from a donor (e.g., peripheral blood directly from a donor or a blood bank) is thawed and placed in culture; (2) if necessary, the culture is depleted of α.β T cells and optionally B cells; (3) the sample or the remaining cells after depletion (the depleted cell population) are subjected to activation conditions for 3-4 days to produce activated cells; (4) if necessary, the activated cells are transduced with an exogenous polynucleotide using, for example, a retroviral vector or a lentiviral vector; (5) after activation in (3) or transduction in (4), the cells are subjected to expansion conditions in a 7-day cycle, washed between cycles, typically for 2-3 cycles, to produce expanded cells, which are sometimes used immediately or at other times refrigerated, kept on ice or frozen for transport and / or storage until needed for immunotherapy. The ratio of NK cells to γ.δ T cells can be adjusted by selecting one or more supplementary polypeptides used in the expansion process (including switching the supplementary polypeptides between the 7-day cycles if necessary) and controlling the expansion time.

[0112] In certain aspects, the composition produced by the sample cell population exposed to activation and expansion conditions has the following characteristics:

[0113] (i) a ratio of NK cells to γ.δ T cells greater than 1 (e.g., a ratio of about 1.5 or greater, 2 or greater, 3 or greater, 4 or greater, or 5 or greater; a ratio of 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, 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 more); or

[0114] (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);

[0115] And optionally have one or more of the following features:

[0116] (iii) a ratio of NK cells to α.β T cells of greater than 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more;

[0117] (iv) a ratio of gamma-delta T cells to alpha-beta T cells of greater than 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more;

[0118] (v) the percentage of NK cells relative to total cells is 20% or greater;

[0119] (vi) the percentage of γ.δ T cells relative to total cells is 2%, 3%, 4%, 5% or greater;

[0120] (vii) about 75% to about 95% of the γ.δ T cells (e.g., about 80% to about 90% (e.g., about 85%) of the γ.δ T cells) express V.δ.1;

[0121] (viii) about 10% to about 25% of the γ.δ T cells (e.g., about 10% to about 20% (e.g., about 15% of the γ.δ T cells)) express V.δ.2;

[0122] (ix) about 30% to about 60% of the γ.δ T cells (about 35% to about 55% of the γ.δ T cells) express V.δ.1;

[0123] (x) about 35% to about 60% of the γ.δ T cells (e.g., about 40% to about 50% or 55% of the γ.δ T cells) express V.δ.2;

[0124] (xi) about 10% to about 30% of the γ.δ T cells (e.g., about 20% to about 25% of the γ.δ T cells) express V.δ.1;

[0125] (xii) about 65% to about 80% (e.g., about 70% to about 80% (e.g., about 75%) of the γ.δ T cells express V.δ.2;

[0126] (xiii) about 80% or more of the total cells (e.g., about 90% or more of the cells) express KIR5;

[0127] (xiv) about 5% or more of the total cells (e.g., about 10% or more of the cells) express SIGLEC-7;

[0128] (xv) about 50% or more of the total cells (e.g., about 60% or more of the cells) express KIR3D51;

[0129] (xvi) about 5% or more of total cells (e.g., about 10% or more of cells) express KIR2DL1;

[0130] (xvii) about 20% or more of the total cells (e.g., about 25% or more of the cells) express NKp30, NKp44, or NKp46;

[0131] (xviii) about 25% or more of the total cells (e.g., 35% or more of the cells) express NKG2D;

[0132] (xix) about 80% or more of the total cells (e.g., about 90% or more of the cells) express DNAM1;

[0133] (xx) about 75% or more of the total cells (e.g., about 85% or more of the cells) express NTBA;

[0134] (xxi) about 85% or more of the total cells (e.g., about 95% or more of the cells) express CD2;

[0135] (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;

[0136] (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;

[0137] (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% of the cells) express CD 16;

[0138] (xxv) about 10% or less of total cells (e.g., about 5% or less, 4% or less, 3% or less, 2% or less of cells) express CD57;

[0139] (xxvi) about 50% or more of the total cells (e.g., about 55% or more of the cells) express KIR3DS1;

[0140] (xxvii) about 5% or less of 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;

[0141] (xxviii) about 5% or less of 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 α.β T cells;

[0142] (xxix) about 50% or more of the NK cells and / or gamma delta cells (e.g., about 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 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%, 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%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or a portion thereof, up to 100% of said NK cells and / or said γ.δ T cells) express CD8;

[0143] (xxx) about 5% or less of the NK cells and / or γ.δ 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 γ.δ T cells) express CD4;

[0144] (xxxi) about 5% or less of the NK cells and / or γ.δ 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 γ.δ T cells) express CD8 and CD4;

[0145] (xxxii) about 15% to about 35% of the NK cells (e.g., 16%, 17%, 18%, 19%, 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34% of the NK cells) do not express CD8 and CD4; and

[0146] (xxxiii) about 55% to about 85% of the γ.δ T cells (e.g., about 56%, 58%, 60%, 62%, 63%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84% of the γ.δ T cells) do not express CD8 or CD4.

[0147] Innate immune cell composition

[0148] Certain compositions prepared by the manufacturing methods described herein are referred to as BINATE compositions. The BINATE compositions contain two innate cell types, namely NK cells and γ.δ T cells, and the relative amounts of the two cell types can be adjusted by selecting the appropriate one or more supplementary polypeptides (including the order of their use) and the duration of the expansion conditions to treat solid tumors, blood cancers, or infectious diseases.

[0149] In some aspects, the BINATE compositions do not contain feeder cells, and typically these compositions do not contain feeder cells (e.g., do not contain exogenous feeder cells and / or irradiated feeder cells). In some aspects, the activation and expansion conditions do not include bisphosphonates, so that the polyclonal γ.δT cells have a wider range of active repertoires for tumors and infectious diseases (i.e., V.δ.1 and V.δ.2 γ.δT cells are polyclonal). This innate immune cell platform is interchangeably referred to herein as BINATE cells, INNATE cells, or INNATE-K cells, which can fully participate in tumor signals and receptors and provide readily available universal therapies for solid tumors, blood cancers, and infections. Innate immunotherapy with improved safety can allow use in community hospitals, thereby reducing medical costs and bringing effective treatment to more patients.

[0150] In certain aspects, the BINATE cell compositions provided herein can be further processed using markers that positively select for desired cell populations or eliminate unwanted cell populations to produce a composition comprising substantially all NK cells (referred to herein as "INNATE-NK") or substantially all γ.δ T cells.

[0151] The BINATE compositions provided herein can be developed as: a) unmodified cells after transplantation or in other environments / indications; b) unmodified cells administered in combination with other therapeutic agents (e.g., commercially available antibodies for cancer treatment); or c) genetically modified cells, such as by mutating endogenous polynucleotides, by deleting endogenous polynucleotides, or by adding exogenous mutant polynucleotides (wherein the wild-type form is present in unmodified cells) or adding heterologous exogenous polynucleotides, for example, CAR polynucleotides (BINATE.CAR) for targeting both solid tumors and hematological malignancies. CAR-modified INNATE-K (same as BINATE) or INNATE-NK populations are named herein with any of the following terms:

[0152] Typically, immune cells modified by CAR are referred to by the suffix "CAR" with a period or hyphen preceding the suffix, e.g., INNATE-CAR, INNATE-K.CAR, INNATE-NK.CAR, or BINATE.CAR; and

[0153] For immune cells modified by a particular CAR (e.g., a CAR targeting CD19), the cells are referred to interchangeably with the suffix "CD19," "CAR19," or "CAR.CD19," preceded by a period or hyphen, for example, BINATE.CD19, BINATE.CAR19, or BINATE.CAR.CD19.

[0154] The manufacturing methods provided herein produce a population of BINATE cells having a high activation level, wherein the high activation level is from about 30% to 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% of the cells in the population. %, 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%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, up to 100% of the cells. In contrast, triggering an innate immune response in vivo results in 10% or fewer of the cells being activated.

[0155] These activated cells are highly cytotoxic, with 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 "+" symbol or the word "positive" associated with a description of a cell marker on a cell indicates that the marker is expressed in the cell (or on the cell surface), while a "-" symbol or the word "negative" associated with a description of a cell marker on a cell indicates that the marker is not present or is not detected.

[0156] Although the BINATE cells of the compositions provided herein have high cytotoxicity, their maturity is low (i.e., they are further from senescence) because most of the cells have low levels of the CD57 marker (a high level of the CD57 marker indicates cytotoxicity, but also indicates that the cells are closer to senescence and therefore have a shorter lifespan (see Kared et al., Cancer Immunol. Immunotherap., 65(4):441-452 (2016)). As used herein, the term "majority" refers to greater than 50%, typically 50.5% or more of the population, 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%, 90%, 91%, 92 ... %, 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 its part, up to 100% of cells. The BINATE cell composition prepared by the preparation method provided herein also has low levels of exhaustion markers, such as PD-1 and TIM-3. The following summarizes the non-limiting examples of the activation / cytotoxicity / exhaustion marker phenotypes of the BINATE compositions obtained under different amplification conditions according to the manufacturing method provided herein:

[0157]

[0158]

[0159] In some aspects, the cells of the BINATE compositions provided herein can be genetically modified. Certain non-limiting examples of genetic modification include (i) adding exogenous polynucleotides encoding polypeptides with desired activity, (ii) changing or adding endogenous polynucleotides, or adding exogenous regulatory polynucleotides (e.g., primers or enhancers), which regulate the expression of endogenous polypeptides with desired activity; (iii) changing and / or destroying endogenous polynucleotides encoding polypeptides with desired activity (e.g., insertion mutagenesis), (iv) partially or completely deleting regulatory polynucleotides regulating the expression of polypeptides with desired activity, thereby destroying their regulation, and / or (v) partially or completely deleting the coding sequence encoding polypeptides with desired activity, thereby weakening or eliminating activity (e.g., knockout mutagenesis).

[0160] In some aspects, the BINATE compositions are genetically modified by adding exogenous (regulatory or coding sequence) polynucleotides. The highly activated cells of the BINATE compositions can be transduced at high efficiency, typically 80% or more of the population, typically 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%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125 , 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 a portion thereof, up to 100% of the cells are transduced. This transduction rate is much higher than the transduction rate typically found in adaptive immune cells (e.g., about 9% in α.β T cells).

[0161] In some aspects, the exogenous polynucleotide encodes a chimeric antigen receptor (CAR), and the cells in the composition include CAR (referred to as "BINATE.CAR cells"). In some aspects, CAR contains a binding molecule portion that immunospecifically binds to one or more of CD19, GD2, HER3, B7H3, CD123 or CD30.

[0162] BINATE cells 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, ovary; CD123: leukemia; CD30: non-Hodgkin's lymphoma; other cancers, including liver and intrahepatic bile duct, esophagus, bladder, kidney and renal pelvis, uterus, brain / nervous system). It was also found that GD2 (a disialoganglioside) is also 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 tumors, breast tumors). Due to the toxicity of neuropathic pain, monoclonal antibodies currently being tested as therapies have limitations. Preliminary studies using the BINATE.CAR.GD2 construct showed that transduction levels in both NK cells and γ.δT cells were equally high (approximately 80% in NK cells and approximately 40-60% in γ.δT cells); expression was stable and maintained during BINATE culture. In addition, in vitro killing of solid tumor cells was observed (Example 18).

[0163] Pharmaceutical compositions and kits

[0164] Any composition provided herein can be formulated into a pharmaceutical composition together with a pharmaceutically acceptable carrier. Like the composition, the pharmaceutical composition provided herein can be used to treat cancer and infectious diseases. Also provided herein is a kit comprising a composition or pharmaceutical composition provided herein and optional instructions for use. In some aspects, the kit provided herein may include cytokines. Innate immune cells control opportunistic invasions of a variety of viral, fungal, bacterial and parasitic pathogens, in part by communicating with other cells through the release of excessive cytokines and chemokines, thereby coordinating immune responses.

[0165] A pharmaceutical composition or kit sometimes includes a specific dose of therapeutic cells, and sometimes a pharmaceutical composition or kit provides a unit dose of therapeutic cells. In certain aspects, the unit dose is about 10 cells per kilogram of body weight of the intended subject. 4 to about 10 10 cells, or approximately 10 per subject 6 to about 10 12 cells (e.g., about 10 per subject). 10 cells, or about 10 per kilogram of body weight of the expected subject. 8 cells).

[0166] The pharmaceutical composition or kit may include a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or state government, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopoeia for use in animals, especially humans. The term "carrier" refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete), excipient, or vehicle administered with the therapeutic agent. When the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. In the case of administering the composition by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed before administration.

[0167] Sometimes pharmaceutical composition is provided as pharmaceutical packaging or test kit, it comprises one or more containers, and described container is filled separately or with this pharmaceutically acceptable carrier with the therapeutic cell composition prepared by the method described herein.In addition, one or more other preventive agents or therapeutic agents that can be used for treating disease also can be included in pharmaceutical packaging or test kit.Pharmaceutical packaging or test kit can comprise one or more containers that one or more compositions of pharmaceutical composition as herein described are housed.Optionally, what can be associated with this type of container is notice, it takes the form of the government agency regulation of the manufacture, use or sale of management medicine or biological product, and described notice reflects the approval of the manufacture, use or sale mechanism of human administration.Pharmaceutical packaging or test kit sometimes comprise one or more other preventive agents and / or therapeutic agents that can be used for treating disease in one or more containers.

[0168] When cells are expanded in vitro before being used as immunotherapy, sometimes cells themselves cannot produce sufficient amounts of cytokines. Cytokines that can be optionally added to the kit provided herein include but are not limited to TNF, IFNγ, interleukin IL-1β, IL-4, IL-6, IL-7, IL-10, IL-12, IL-18, IL-21, CCL4 / RANTES and TGFβ. In some aspects, the pharmaceutical composition and / or kit provided herein optionally include a second medicament for co-administering with the composition provided herein. As used herein, the words "co-administered", "co-administered", "co-administered", etc. refer to the administration of medicaments before, after or simultaneously with the innate cell composition provided herein.

[0169] In some aspects, the second agent is an antibody that targets a cancer cell antigen or an infectious pathogen. The compositions, pharmaceutical compositions, and kits provided herein may be maintained at minus 4 degrees Celsius or lower, or about minus 75 degrees Celsius to about minus 80 degrees Celsius, as appropriate, for storage or transportation.

[0170] Methods for preparing genetically modified innate cell compositions

[0171] The innate cell compositions provided herein can be genetically modified in a suitable manner (e.g., by adding exogenous polynucleotides as genes or regulatory sequences, by mutating endogenous genes, or by deleting endogenous genes). The genetic modification can be performed after the final composition is obtained, after activation and amplification, or after activation and before the cells undergo amplification. Non-limiting examples of methods for performing various genetic modifications are described below:

[0172] (a) CRISPR-CAS9 targeted inhibition (permanent gene / locus deletion)

[0173] Cells can be transfected with DNA plasmids that simultaneously express CAS9 protein and guide RNA (gRNA) specific to the target gene. Donor DNA plasmids can be used to repair gRNA-CAS9-mediated genomic cleavage, which will result in specific deletion of the target gene and permanent and complete loss of the protein encoded by the gene. PCR (DNA level), Northern blot / FISH (RNA level) or any protein assay (such as Western blot or flow cytometry) can be used to verify the loss of protein expression.

[0174] (b) CRISPR-CAS9 targeted expression (permanent gene / locus insertion)

[0175] This method can be used to insert the target gene into a specific position of the cell genome. Cells can be transfected with a DNA plasmid expressing CAS9 protein and a guide RNA (gRNA) specific to a specific insertion position. The genomic cutting mediated by gRNA-CAS9 can be repaired using a donor DNA plasmid, which has an inserted target gene, and the flank of the target gene is the sequence of the cell genome on both sides of the DNA cutting / double-strand break position, thereby causing the insertion of the target gene mediated by homologous recombination at a specific genomic position, rather than random insertion. Successful insertion and protein expression can be verified using PCR (DNA level), Northern blotting / FISH (RNA level) or any suitable protein assay (such as Western blotting or flow cytometry).

[0176] (c) RNA interference (permanent gene knockdown) via retroviral / lentiviral / transposon-mediated shRNA / microRNA transduction

[0177] shRNA / microRNA targeting a specific gene / protein of interest can be designed and cloned into a retroviral / lentiviral / transposon vector for stable integration into the cell genome. Cells can be transduced with the vector, and successfully transduced cells can be selected using a selection marker encoded by the vector. For example, Northern blotting and protein assays can be used to assess the inhibition of shRNA-mediated target genes.

[0178] (d) Lentivirus / γ-retrovirus-mediated random / multi-copy gene insertion

[0179] The specific gene / protein of interest can be designed and / or cloned into a retroviral or lentiviral vector for stable random integration into the cell genome. Cells can be transduced with viral vectors, and successfully transduced cells can be selected using vector-encoded selection markers. Northern blotting and any suitable protein assay (e.g., Western blotting, flow cytometry, etc.) can be used to assess the inhibition of shRNA-mediated target genes.

[0180] (e) Transposon-mediated random / multi-copy gene insertion

[0181] The specific gene / protein of interest can be designed and / or cloned into a mammalian transposon vector system, such as PiggyBac (SBI System Biosciences) or equivalent. Cells can be co-transfected with a transposon vector and a transposase vector, wherein the transposon vector has a target gene (cDNA) flanked by an inverted terminal repeat (ITR) sequence. Transposase can mediate the transfer of the target gene to the TTAA chromosome integration site. The cells of successful transduction can be optionally selected using a vector-encoded selection marker. Successful insertion and protein expression can be verified using PCR (DNA level), Northern blotting / FISH (RNA level) or any suitable protein assay (such as Western blotting or flow cytometry).

[0182] (f) Direct transfection

[0183] mRNA encoding the gene / protein of interest can be transfected directly into cells. Transfection can be performed using any established method, such as: calcium chloride transfection; lipofection; Xfect; electroporation; sonoporation and cell squeezing (e.g., for the introduction of siRNA).

[0184] Treatment

[0185] Also provided herein is a method for treating cancer or infection by administering any of the compositions, pharmaceutical compositions or kits provided herein to a subject in need thereof in an amount effective to treat cancer or infection. Treatment can be administered in an autologous environment or an allogeneic environment. The donor of the sample producing the composition, pharmaceutical composition or kit can be the recipient of treatment. Typically, the composition, pharmaceutical composition or kit is prepared by a sample from a subject, and the treatment is administered to different subjects. In some aspects, treatment can be administered on two or more separate days, and in some aspects, treatment can be administered in multiple doses. In some aspects, treatment is administered at intervals of about 1 unit dose to about 36 or more unit doses and about 2 weeks to about 4 weeks. In some aspects, treatment is administered once, twice, three times, four times or up to five times a day with a single unit dose, or once, twice, three times, four times, five times, six times, seven times, eight times, nine times or ten times or more over a period of days, weeks or months, or once every other day, or once, twice, three times, four times, five times or six times a week. Treatment can be administered at about 10 times per kilogram of subject body weight. 4 to about 10 10 A unit dose of 10 cells, or approximately 10 per subject 6 to about 10 12 The unit dose of about 10 cells is administered intravenously (IV), intrathecally or intramuscularly (IM), intraperitoneally (IP), intrapleurally, intraarticularly, or injected or implanted at or near the site of cancer or infection. In certain aspects, the unit dose is about 10 cells per subject. 10 cells, or about 10 per kg of subject body weight 8 cells.

[0186] In some aspects of the methods of treatment provided herein, the treatment is for cancer. In some aspects, the cancer is a solid tumor. In some aspects, the cancer is a hematological cancer. In some aspects, the cancer is a hematological cancer, and the ratio of γ.δT cells to NK cells is greater than 1. In some aspects, the cancer is a solid tumor, and the ratio of NK cells to γ.δT cells is greater than 1. In some aspects of the methods of treatment provided herein, a second agent is co-administered with the composition, pharmaceutical composition, or kit. In some aspects, the second agent is an antibody that immunospecifically binds to a cancer-associated antigen.

[0187] In certain aspects of the methods of treatment provided herein, the treatment is for infection. In some aspects, the infection is characterized by the presence of bacterial, fungal, viral or protozoan pathogens.

[0188] In certain aspects of the methods provided herein, the composition, pharmaceutical composition, or kit comprises a polyclonal population of γ.δ T cells (eg, the γ.δ T cells are polyclonal with respect to V.δ.1 and V.δ.2).

[0189] The present technology will be further described with reference to the embodiments described herein; however, it will be understood that the present technology is not limited to such embodiments.

[0190] Example

[0191] The examples set forth below illustrate certain embodiments and do not limit the present technology.

[0192] The following materials were used in some of the methods described below (e.g., activation conditions, amplification conditions, phenotypic analysis):

[0193] The following monoclonal antibodies (mAbs) were generated in the "Cell and Gene Therapy for Pediatric Tumors" laboratory of the Ospedale Pediatrico Bambino Gesù (OPBG):

[0194] c218 (IgG1, anti-CD56), c127 (IgG1, anti-CD16), AZ20 and F252 (IgG1 and IgM, respectively, anti-NKp30), BAB281 and KL247 (IgG1 and IgM, respectively, anti-NKp46), Z231 (IgG1, anti-NKp44), ECM217 and BAT221 (IgG2b and IgG1, respectively, anti-NKG2D), KRA236, GN18 and F5 (IgG1, IgG3 and IgM, respectively, anti-DNAM-1), EA4 (IgG2a, anti-CD18), MAR206, MA258 and QA196 (IgG1, IgG2b and IgM, respectively, anti-CD2), MA127 and O N56 (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-binding protein-2), and BAM195 (IgG1, anti-MICA).

[0195] F278 (IgG1, anti-CD85j) mAb was kindly provided by Dr. Daniela Pende of 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-KIR2DL / 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-PerCP Vio700 (REA746 clone), REA control VioBright FITC (clone REA293), and anti-PD-1 (IgG2b, PD1.3.1.3 clone) mAbs were purchased from Miltenyi Biotec (Bergisch Biosystems, Berlin, und Biotech). Anti-CD34 (IgG1, clone QBEnd10), anti-NKG2A-PC7 (clone z199), anti-KIR3DL1 / S1-PE (clone z27), anti-KIR2DL2 / L3 / S2-PE (clone GL183), anti-CD19-FITC (IgG1, clone J3-119), anti-CD56-PC7 (IgG1, clone N901), IgG1-PC7 or -PE or -FITC isotype control (clone 679.1Mc7) mAbs were purchased from BeckmanCoulter, 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 Brillant staining 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, China), respectively. Anti-NKG2D (IgG2a, 5C6 clone) and anti-HLA-C (IgG1, C-8 clone) were purchased from SantaCruz Biotechnology (Dallas, Texas, USA).

[0196] For amplification conditions, IL-2, IL-15 and OKT3 were obtained from Miltenyi Biotec (San Diego, CA, USA), as were anti-CD2 and anti-NKp46 antibodies cross-linked or bound to beads.

[0197] Example 1: Enrichment and expansion of a specific population of innate cytolytic immune cells (INNATE-K) from a population of αβTCRneg cells

[0198] This example describes a method for enriching and expanding a specific innate cytolytic immune cell population (referred to herein as the INNATE-K or BINATE cell population) consisting primarily of NK and γδTCR+ T cells from a starting αβTCRneg cell population (for analysis of the composition of this expanded T cell population, see Figure 2 The terms INNATE-K and BINATE are used interchangeably herein and refer to a cell population discussed herein that is activated by a combination of an NCR antibody (e.g., anti-NKp46) and an LFA antibody (e.g., anti-CD2) and comprises a mixture of NK cells and γδ T cells), in feeder-free culture conditions, activated with antibodies against one or more immunoglobulin superfamily surface molecules:

[0199] 1. Perform mass leukapheresis on normal healthy donors that have been mobilized with G-CSF; alternatively, perform mass leukapheresis on normal healthy donors without mobilization or use buffy coat.

[0200] 2. Use SEPAX TM Ficoll was used with a 10% Ficoll-MS / MS device (Sepax Technologies, Inc., Newark DE). TM Gradient cell separation to remove erythrocytes, platelets, and granulocytes, leaving a mononuclear cell suspension; Ficoll TM Alternative methods for gradient separation include the MILTENYI PRODIGY TM (Miltenyi Biotec, San Diego, CA) or manually separated using a centrifuge.

[0201] 3. Follow the manufacturer's instructions for using the clinical-scale MILTENYI CLINIMACS TM Monocytes were subjected to αβ T cell depletion or research-scale separation on Miltenyi LS columns (Miltenyi Biotec, San Diego, Ca).

[0202] 4. The resulting αβTCRneg cell population was placed in feeder-free culture conditions and was cultured using anti-CD2 and anti-NKp46 beads (NK Cell Activation / Expansion Kit) following the manufacturer's instructions. TM (Miltenyi Biotec, Inc., San Diego, CA, USA)) and 500 IU / mL human interleukin 2 (IL-2) (Miltenyi Biotec).

[0203] 5. In a 24-well plate without a feeder layer, 0.25 × 10 6 / ml cell seeding concentration, NK MACS supplemented with 5% AB serum and 500IU / mL human interleukin 2 (IL-2) TM The activated αβTCRneg cell population is further cultured and expanded in culture medium (#130-107-879 (Miltenyi Biotec, Inc., San Diego, CA, USA)) and then transferred to a T75 flask; alternative culture vessels may include bioreactors (G-Rex TM 25ml, WilsonWolf Manufacturing, St. Paul MN).

[0204] 6. Replace the medium approximately every 3 days with fresh medium and fresh IL-2 at the concentrations described in #5 and at 0.25-0.5×10 6 Cells are inoculated at a concentration of 100 μg / ml. In some variations of this method, medium changes are performed approximately every 4 days. When inoculated into a bioreactor, medium changes may occur approximately every 6 or 7 days until the target dose is reached.

[0205] 7. At any time after the start of expansion, unwanted populations can be CD3+ depleted or CD56+ depleted separately, so that the resulting enriched specific innate immune cytolytic populations (NK and γδTCR+T cells) can be further enriched against pure NK or γδTCR+T cells to obtain pure cell populations and further cultured according to step 5#.

[0206] 8. At the end of the expansion, INNATE-K cells were cryopreserved in serum-free freezing medium (CryoStor TM , BioLife Solutions, Bothell WA).

[0207] Figure 1 The expansion of αβTCRneg cells is shown as a function of the number of days that 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 begin to reach logarithmic expansion around 1 week and continue to expand, generating large numbers of cells suitable for successful off-the-shelf therapy. Figure 1 In Figure 1, activated αβTCRneg cells were cultured and expanded under feeder-free conditions. The total cell number of the culture up to the day of expansion is shown, and a significant increase in expansion was observed after day 6 of culture.

[0208] Figure 2 NK cells were shown to be 70% and γδTCR+ T(GD) cells were 28%; this ratio is suitable for therapeutic products; it may change with further expansion of the cells; both cell types were obtained in large quantities without feeder cell culture and without contamination by other cells. Figure 2 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 immunity αβTCR+ T cells are shown at various steps in the following examples of the manufacturing method.

[0209] a) The cell frequencies in the mononuclear fraction after Ficoll gradient separation were 8% + 12% NK cells and 0.1% + 1% γδTCR+ T cells, 6% CD3+CD56+ NKT cells, and 73% αβTCR+ T cells;

[0210] b) The cell frequencies after the αβTCR+T cell depletion step were 25%+15% NK cells and 3.2%+1.2% γδTCR+T cells (of which 0.5% CD56-CD3+ and 2.7% CD56+CD3+) and 1% or less αβTCR+T cells.

[0211] c) On day 10 after activation and feeder-free culture 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% CD56-CD3+ and 3.5% CD56+CD3+). Negligible levels of CD3+ adaptive immune cells were detected: αβTCR+T cells were 0.3% + 0.5%, and NKT cells were 0.2% + 0.3%.

[0212] On day 10, cells co-expressing the CD56 (NK) surface antigen, CD16, and CD57 were subjected to flow cytometry analysis (fluorescence activated cell sorter or FACS analysis). CD16 is an Fcγ receptor III and can therefore bind the Fc portion of IgG antibodies and mediate antibody-dependent cell-mediated cytotoxicity (ADCC) of antibody-bound target cells. CD16 plays a role in spontaneous cytotoxicity mediated by NK cells. 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 for the poor proliferation capacity of NK cells. After stimulation with IL-2 or co-culture with target cells, the acquisition of CD57 on NK cells is associated with the maturation of the CD56+ NK cell subset. This difference 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. For CD56+NK cells, 37% were CD16+ and 63% were CD16-; for CD56+CD16- cells, 13% were CD57+ and 87% were CD57-; and for CD56+CD16+ cells, 15% were CD57+ and 85% were CD57-. The flow cytometry phenotypic results showed that the cell composition contained enriched NK cells in the expanded αβTCRneg cells, which were highly cytotoxic NK cells as evidenced by the co-expression of CD56(NK) and CD16, and also contained cells with an immature phenotype that had the potential to mature into even more reactive and cytotoxic cells, as evidenced by the number of NK cells that had no CD57 expression on both CD16+ and CD16-NK cells.

[0213] The frequency distribution of different γδT cell subsets was measured in day 10 samples of activated αβTCRneg cells expanded in feeder-free culture, as analyzed by flow cytometry. In contrast to MHC-restricted αβT cells, γδT cells are able to recognize and lyse a variety of cancers in an MHC-independent manner, highlighting their potential for off-the-shelf allogeneic immunotherapy. Human γδT cells can be divided into three major populations based on δ chain expression. In healthy adults, the majority (up to 50%–90%) of circulating γδT lymphocytes express the γδ2 chain. γδT cells expressing the γδ1 chain are most prominent in the epithelial lining of mucosal surfaces, where they are involved in maintaining the integrity of epithelial tissues in the face of injury, infection, or transformation; however, they may also be present in the peripheral blood. γδ3T cells account for approximately 0.2% of circulating T cells and include CD4+, CD8+, and CD4-CD8- subsets. Although uncommon in peripheral blood, γδ3T cells are usually found at higher levels in the liver. The percentage distribution of γδTCR+ T cells on day 10 (41% γδ1, 50% γδ2, 7% other γδ) clearly showed that the propagated γδ T cells were polyclonal, as they expressed Vδ1, Vδ2, and Vδ1. neg Vδ2 neg This polyclonality will ensure that the propagated γδ T cells participate in the innate killing of tumors, altered cells or infections.

[0214] Figure 3 The frequency and distribution of T cell lineage markers CD4 (helper cell) and CD8 (cytotoxicity) on γδTCR+ cells in day 10 samples of activated αβTCRneg cells expanded in feeder-free culture are shown, as analyzed by flow cytometry. Most γδ1 T cells do not express CD4 or CD8, but a small fraction (19%) does express cytotoxic CD8 lineage markers, consistent with the innate cytolytic immune population described and expected for the greatest tumor and infection killing. γδ2+ cells express almost no CD4 or CD8, while almost half of the "other γδTCR+ cells", which are mainly composed of γδ3 T cells, express cytotoxic CD8 lineage markers.

[0215] Additional flow cytometry phenotyping data for day 8 were obtained, where γδT cells were obtained from activated αβTCRneg cells expanded in feeder-free culture, which had been removed by the αβT cell removal reagent of Miltenyi Biotec, Inc. at a research scale. Like the cells removed on a large scale, the CD3+T cells cultured on day 8 mainly expressed the γδT cell receptor (<0.05% αβTCR+ cells). Phenotypic analysis was performed and the co-expression of markers on γδT cells was analyzed. All cells were CD3+ and 31.8% were CD16+. Co-expression of CD57 and CD16 on CD3+γδT cells was observed. 10.7% of the cells co-expressed CD57 and CD16 markers and were therefore the most mature cells. Most were CD57- and CD16- (57%), thus representing immature cells. The expression of inhibitory and activating ligand receptors was analyzed. NKG2D is an activating receptor on the surface of NK cells. NKG2A dimerizes with CD94 to form inhibitory receptors. Cells lacked expression of NKG2C and 55% of cells co-expressed NKG2A and NKG2D. Phenotypic analysis was performed and co-expression of markers on γδ T cells was analyzed in relation to co-expression of CD56. Of all γδ cells expressing CD3+ and CD56+, 29% co-expressed CD16. Co-expression of CD57 and CD16 was observed on CD3+CD56+γδ T cells. 5% of cells co-expressed CD57 and CD16 markers and were therefore the most mature cells. The majority were CD57- and CD16- (67%) and therefore represented immature cells. Expression of inhibitory and activating ligand receptors was analyzed. The cells lacked expression of NKG2C on CD3+CD56+ cells, with the majority of cells (82%) expressing NKG2A, and 80% of CD3+CD56+ cells co-expressing NKG2A and NKG2D, indicating that these cells express phenotypic markers most commonly associated with NK cells and indicative of an active innate immune cell population.

[0216] Example 2: Expansion of transduced specific innate cytolytic immune cells (INNATE-CAR) from αβTCRneg cell population

[0217] This example describes the expansion and production of a population of transduced specific innate cytolytic immune cells (INNATE-CAR) comprising NK and γδTCR+ T cells, which were expanded from an activated culture of an αβTCRneg cell population under feeder-free culture conditions:

[0218] 1. Take an aliquot of cells from the activated culture-expanded αβTCRneg cell population as described in step #5 of Example 1 on day 4 (or an aliquot from days 5-15) and multiply them by 0.25×10 6 / ml in the presence of 500 IU / mL human interleukin 2 (IL-2) or 10 ng / ml IL-15 on plates coated with human fibronectin (alternatively, RetroNectin TM (Takara Bio USA, Mountain View CA) coated plates or VECTOFUSIN-1 (Miltenyi Biotec) transduction enhancer) and transduced with gamma retroviral supernatant containing a construct for a CD19 chimeric antigen receptor containing a non-functional fragment of the CD34 surface antigen used as a cell transduction marker.

[0219] 2. After three days, the cells were detached and plated at 0.25 × 10 6 The cells were seeded at a concentration of 100 / ml in NK MACS medium supplemented with 5% AB serum and 500 IU / mL human interleukin 2 (IL-2). TM Culture medium (#130-107-879 (Miltenyi Biotec, Inc., San Diego, CA, USA)) for further expansion; alternative culture vessels may include bioreactors (G-Rex TM 25ml, Wilson Wolf Manufacturing, St. Paul MN).

[0220] 3. Replace the medium every 3 days with fresh medium and fresh IL-2 at the concentration described in #6 of Example 1 and at 0.25-0.5×10 6 Cells were inoculated at a concentration of 100 μg / mL; when inoculated into the bioreactor, media changes were performed every 7 days until the target dose was reached.

[0221] 4. At any time after day 14, the INNATE-CAR population can be further enriched for NK-CAR or γδ-CAR T cells by CD3+ depletion or CD56+ depletion, respectively, of unwanted populations to obtain pure cell populations and further cultured as in step #3.

[0222] 5. At the end of expansion, cells were cryopreserved in serum-free freezing medium (CryoStor, BioLife Solutions, Bothell, WA) containing 10% DMSO solution.

[0223] Figure 4 Analysis of the expansion rate of CAR.19INNATE-K cells as of day 10 is shown. Similar to the expanded non-genetically modified population, the cultures began to show logarithmic expansion in about a week, representing the strong expansion potential of the off-the-shelf INNATE-CAR product. Figure 4 In this study, activated INNATE-K cells were cultured and expanded in feeder-free conditions and transduced on day 4 with a γ-retrovirus containing the CD19CAR and a non-functional CD34 marker.

[0224] (In three different productions) INNATE-CAR colonies, NK cells and γδTCR+T cell transduction were compared. Specifically, the %CAR.CD19 expression in each of these cell colonies was compared using the CD34 marker incorporated into the CAR molecule. NK and γδTCR+ T colonies both show significant transduction of CAR.CD19 retroviral vectors, as demonstrated by analyzing the CD34 markers in the declared cell subsets. The average transduction efficiency of NK and γδTCR+T cells was 33% and 30%, respectively. Flow cytometry analysis (fluorescence activated cell sorter, or FACS analysis) was performed on NK cells without feeder layers that were expanded for 10 days and not transduced with retroviral vectors carrying CAR.CD19, which shows the specificity of CD34+ staining on CD56+ cells. Flow cytometry (FACS) analysis was performed on NK cells without feeder layers that were genetically modified with retroviral vectors carrying CAR.CD19 and expanded for 10 days on the +4th day of the activation process. The results showed that after transduction, the expression level of CAR on CD56+ cells was high in terms of both the percentage of CD34+ cells and the mean fluorescence intensity of expression. Both features are associated with efficient tumor recognition. The same observation was valid in the presence of feeder-free γδTCR T cells in the transduced and expanded INNATE population. In fact, the γδTCR T cell component was significantly transduced as assessed by analysis of CD34 expression in γδTCR T cells.

[0225] The INNATE-CAR platform is highly efficient in recognizing and eliminating tumors, with strong synergy between innate activity and CAR-mediated function. Figure 5The graph illustrates data from co-culture cytotoxicity assays in which INNATE cells and INNATE-CAR.CD19 cells were co-cultured with CD19+ leukemia (221) or CD19+ lymphoma (Daudi) cell lines. The percentage of residual tumor after culture is represented in the upper graph for the different cell effectors. Non-transduced NK cells (INNATE-NK) killed 40% and 29% of leukemia and lymphoma tumor cells, respectively, while INNATE-K cells characterized by a combination of NK and γδTCR T cells killed 88.4% and 96.1% of tumor cells, respectively. INNATE-NK cells transduced with CAR.CD19 (INNATE-NK-CAR.CD19) eliminated 93% and 84% of tumor cells, while the INNATE-CAR.CD19 combination killed nearly all tumor cells (97% and 99%).

[0226] Example 3: Alternative Enrichment of INNATE-NK Cells

[0227] This example describes a method for enriching INNATE-NK cells:

[0228] 1. Use NK DEPLETION KIT TM NK cells were isolated from buffy coats (Miltenyi Biotec, Inc., San Diego, CA, USA).

[0229] 2. Use NK Cell ACTIVATION / EXPANSION KIT TM (Miltenyi Biotec, Inc., San Diego, CA, USA) and recombinant human IL-2 (500 U / ml) were used to activate NK cells.

[0230] 3. On day 0 after activation, NK cells were cultured in 5% AB serum-enriched medium (NK MACS TM Culture medium (#130-107-879 Miltenyi Biotec, Inc., San Diego, CA, USA).

[0231] 4. On days 4 to 15, 0.25×10 6 Aliquots of cells were transduced in human fibronectin-coated plates at a concentration of 1:1 / ml.

[0232] 5. After three days, the cells were separated and cultured at 0.25 × 10 6The cells were seeded at a concentration of 100 μg / ml and further expanded in 5% AB serum-enriched medium and 500 IU / mL human interleukin 2 (IL-2).

[0233] 6. After transduction, NK cells are seeded in 24-well plates and then placed in T-flasks or bioreactors.

[0234] 7. Replace the medium with fresh medium and fresh IL-2 every 3 days at a concentration of 0.25-0.5×10 6 The cells were inoculated at a concentration of 100 μL / mL (when they were in the plate) until the target cell number was reached. The bioreactor culture was replaced every seven days.

[0235] 8. On day +14, in case of T cell contamination exceeding 5%, untransduced and CAR-transduced INNATE-NK cells were re-depleted of CD3+ cells to obtain pure NK cells and further cultured as in step #7.

[0236] An exemplary highly enriched NK population of greater than 90% (>90%) of innate immune cells was robustly expanded in both plates and bioreactors, as Figure 6-8 Graphic instructions. Figure 6 The total number of INNATE-NK cell populations expanded in the plates over several days is shown. Figure 7 The total cell numbers of plate- and bioreactor-expanded INNATE-NK populations were compared. Figure 8 The cell composition of subsets at different time points of in vitro expansion is shown, demonstrating the remarkable purity of INNATE-NK (represented by more than 95% NK cells) and the negligible presence of CD3+ T cells and NKT cells.

[0237] Flow cytometry (FACS) analysis was used to evaluate the CD34 marker contained in the CAR.CD19 molecule, demonstrating the level of CAR transduction efficiency. Non-transduced INNATE-NK cells expanded without a feeder layer did not express the CD34 marker. INNATE-NK cells were genetically modified with a retroviral vector carrying CAR.CD19 on day 4. INNATE-NK CAR cells were significantly transduced, with a high percentage of CAR+ cells in the NK population (CD56+). Fig. 9 As shown, the expression of CAR molecules in INNATE-NK CAR cells is stable during long-term in vitro culture.

[0238] Fig.10Shown is the increase in the expression of activation and cytolytic molecules after the expansion of INNATE-NK and INNATE-NK CAR populations, which illustrates the data of flow cytometry analysis of INNATE-NK and INNATE-NK-CAR.19 cells, respectively. The analysis includes evaluating several NK markers associated with the activation and maturation of innate cells. In particular, CD16 is included as a basic marker for maturation analysis, which is associated with a more mature NK subgroup with higher cytolytic activity but lower proliferation capacity; signaling molecules and co-stimulatory molecules for activating and proliferating NK populations, including CD2, LFA-1, NKp44, NKp30, NKp46, and DNAM-1. All of these markers are significantly increased in the amplified populations of INNATE-NK and INNATE-NK.CAR.19 cells, indicating that these cells are activated compared to circulating peripheral blood NK cells.

[0239] Fig.11 The lack of exhaustion in INNATE-NK cells and INNATE-NK CAR cells expanded without feeder layers is illustrated. INNATE-NK and INNATE-NK-CAR.19 cells were generated and expanded as described in Example 3, and flow cytometry analysis was performed on days 20 and 60 to evaluate markers of cell maturity and exhaustion. LIR-1 is a marker of innate cell inhibitory activity and only shows a small increase in the NK population at day 20 and in both populations at day 60; NKG2c is a marker of signal-specific activity and maturity and does not change during culture; CD57 is a marker of maturity and is associated with reduced proliferation potential, so it is expressed on circulating NK populations derived from healthy donor peripheral blood. At 20 and 60 days of in vitro expansion, INNATE-NK cells expanded without feeder layers showed very low percentages of CD57+NK cells, indicating that these cells still have proliferation potential after in vivo infusion. Programmed cell death 1 (PD-1) is a marker of reduced cell potential and "exhaustion" not only in T cells, but has also recently been described as a checkpoint inhibitor in NK cells. Neither the INNATE-NK nor the INNATE-NK CAR cell populations showed the presence of a PD-1+ cell subset at 20 or 60 days, indicating that the cells were not exhausted after prolonged in vitro culture.

[0240] Fig.12Schematic illustration of cytotoxicity co-culture assays using INNATE–NK or INNATE-NK-CAR.19 and 4 tumor cell lines: Row A: 221, a CD19+ leukemia cell line; Row B: Daudi, a CD19+ lymphoma cell line; Row C: BV173, a CD19+ (variable expression) pre-B tumor cell line; and Row D: KARPAS, a CD19- tumor cell line. Fig.12 Figure 1 shows the reduction of CD19+ cells after in vitro co-culture with INNATE-NK (left panel) or INNATE-NK.CAR.19 (right panel) cells. The box in the upper left corner of the INNATE-NK image (stroked box) highlights the CD19+ tumor population after co-culture with unmodified INNATE-NK cells. The box in the upper left corner of the INNATE-NK.CAR.19 image (stroked box) highlights the residual CD19+ tumor population after co-culture with INNATE-NK.CAR.19 cells. After five days of in vitro co-culture, INNATE-NK.CAR.19 cells were able to exert significant tumor control against CD19+221 and DAUDI tumor targets, while the amount of residual tumor was negligible. BV173 showed greater resistance to recognition and elimination, although genetic modification of INNATE-NK cells with CAR.CD19 significantly improved anti-tumor activity compared to unmodified INNATE-NK cells. Fig.12 Figure 2 shows the average of the residual tumor observed in 10 different experiments for each co-culture experiment. Degranulation (analyzed by CD107a expression on NK cells after a brief in vitro co-culture for three hours) represents the cytolytic activity of INNATE-NK and INNATE-NK CAR effector cells, as shown in Figure 2. Fig.12 Figure 3 (rows AD). In all cases, INNATE-NK.CAR.19 cells expressed higher cytolytic activity than INNATE-NK cells; in the negative control represented by the KARPAS co-culture condition, neither population expressed the CD107a degranulation marker. Fig.12 Figures 4 and 5 show the production of regulatory cytokines, which are normally produced in the context of lymphocyte cytolytic activity. Levels of both cytokines were high during CD19 CAR T cell administration and are thought to potentially play a role in cytokine release syndrome. Cytokine production ranged from 20-150 pg / ml / 10 for both IFNα and TNFα in the context of INNATE-NK and INNATE-NK CAR activity against CD19+ tumor cells. 6Cells, cytokine production is many-fold lower than that of adaptive T cell killing. Even in the case of high-target killing, cytokine secretion is not as high as that seen with CAR-T (usually 100- to 1,000-fold higher), thus representing a potentially less toxic therapy.

[0241] Figure 13-15 Schematic illustration of the ability of INNATE-NK and INNATE-NK CAR cells to exert significant antitumor activity against primary Bcp-ALL blasts. Fig.13 and 14 Graph illustrating the percentage of specific lysis of primary tumor cells in different test runs as a function of the ratio of effector (E) to target (T) for INNATE-NK cell and INNATE-NK-CAR.19 cell populations; the INNATE-NK-CAR.19 cell population was more effective across all E:T ranges. Fig.15 The graph illustrates the percentage of residual primary CD19+ tumors after co-culture with effector cells (INNATE-NK cells and INNATE-NK-CAR.19 cell populations) compared to control conditions in which primary CD19+ leukemic blasts were inoculated without effector cells. Co-culture with INNATE-NK cells produced approximately 18% residual tumor cells, and co-culture with the INNATE-NK-CAR.19 cell population produced approximately 5% residual tumor cells.

[0242] In one example of an in vivo CD19+ lymphoma mouse model, each NGS mouse received 0.25×10 6 Tumor cells genetically modified with firefly luciferase were administered intravenously (IV) and thus could be analyzed by bioluminescence analysis. After 3 days, leukemia was shown to have established in the injected mice, allowing them to be treated with 5×10 6 Mice were randomized to treatment cohorts that received intravenous infusions of either INNATE-NK or INNATE-NK-CAR.19. Animals were analyzed for the presence of luciferase+ tumor cells during the experiment. Data were compared with data from another experiment with the same design, but in this experiment, animals received 5×10 6 Unmodified adaptive T cells.

[0243] Example 4: INNATE-NK Study

[0244] This example describes data showing that a single dose of unmodified INNATE-NK cells prolonged the time to death in animals compared to the same dose of adaptive T cells. 6 Tumor cells were grown and allowed to expand for 3 days. 5 × 106 INNATE-NK or INNATE-NK-CAR, and the data was compared to data from experiments using 5M adaptive T cells. In mice receiving INNATE-NK CAR.19, tumors were completely controlled and no tumor bioluminescence was detected after day 11, indicating that INNATE-NK-CAR19 eradicated the tumors, and there were no recurrences until day 72 (the end of the experiment). Importantly, INNATE-NK cells also did not show xenogeneic reactivity (which is historically seen at this time point when using CAR T cells). The data also showed that a dose of non-genetically modified INNATE-NK cells was able to prolong the overall survival of the animals compared to the same dose of adaptive T cells (day 50 vs. day 28, respectively). Fig.16 The figure illustrates the survival curves of animals receiving INNATE-NK and INNATE-NK-CAR.19 cells from the above experiment. The data curve for INNATE-NK.CAR.19 recipients shows a higher survival rate compared to recipients receiving unmodified INNATE-NK cells.

[0245] Example 5: INNATE-NK cells targeting solid tumors

[0246] This embodiment confirms that INNATE-NK cells are suitable platforms for treating solid tumors. In this embodiment, neuroblastoma models are considered. INNATE-NK and INNATE-NK CAR cells are produced as described in Example 3 Steps 1-8. INNATE-NK cells were genetically modified with a retroviral vector on the 4th day, and the retroviral vector carries a third-generation CAR specific for the GD2 antigen expressed by neuroblastoma tumor cells. The results demonstrate the production of INNATE-NK CAR.GD2, showing a significant level of CAR expression in CD56+ cells, which is evaluated by staining with anti-idiotypic antibodies (1A7) and by flow cytometry (FACS) analysis. The purity of NK populations in INNATE-NK CAR.GD2 production was evaluated with negligible CD3+ cell percentages, and the level of CAR+ cells was evaluated with 1A7+ cells. The expression of CAR.GD2 in INNATE-NK CAR was stable during prolonged in vitro culture (measured on days 8, 15, 25, 31, and 35).

[0247] Example 6: Assessing the role of innate immunity in solid tumor penetration

[0248] This example evaluates the role of innate immunity in its ability to penetrate solid tumors. TM3-D neuroblastoma tumor cell spheroids were cultured in culture, and then adaptive T cells, INNATE-NK or INNATE-NK-CAR.GD2 cells were introduced and their ability to invade the spheroids was evaluated. Specifically, a solid 3-D tumor model was created using SH-SY5Y neuroblastoma tumor cells, which were 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 red fluorescent emitting cell markers and incubated with tumor spheres. Neurospheres were analyzed in 3 planes by 3-D fluorescence microscopy to assess 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 unable to actively penetrate 3-D tumors because they were only located on the outer surface of the tumor spheres (green) as observed in all 3 planes and did not migrate to the center of the tumor. Other images show that INNATE-NK cells (red) begin to break through the surface of the tumor spheres, as observed in all 3 planes; and INNATE-NK-CAR.GD2 cells break through the surface of the tumor spheres, indicating that these cells have the ability to migrate a certain distance into the tumor, as observed in all 3 planes.

[0249] Example 7: Cross-linking activation and soluble activation

[0250] As described in Example 1, cell populations were generated in culture after activation with a combination of a cross-linked natural cytotoxicity receptor (NCR) antibody (e.g., anti-NKp46) and a lymphocyte function-associated antigen (LFA) antibody (e.g., anti-CD2) ("coated beads"; Miltenyi Biotec). 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 that contain a mixture of NK cells and γδ T cells are referred to herein as "BINATE" cell populations (and in certain of the above examples as "INNATE-K" cells). In this example, the resulting BINATE population was analyzed after activation with one of the following: 1) a combination of cross-linked anti-NKp46 (clone 9E2 500 ng / ml; Miltenyi Biotec) and cross-linked anti-CD2 (OKT11 500 ng / ml), or 2) a combination of soluble anti-NKp46 (clone 9E2 500 ng / ml) and soluble anti-CD2 (clone OKT11 500 ng / ml). Cross-linking was achieved by coating flasks with the antibody combination in PBS for 24 hours before the start of the experiment, then decanting the antibody solution and rinsing with PBS at the time of the experiment (referred to herein as coated plates).

[0251] 1. The buffy coat from a normal healthy donor was manually separated in a centrifuge and Ficoll TM Gradient cell separation (In certain variations of the methods described in this and other examples herein, a normal healthy donor undergoes an apheresis procedure (apheresis; typically without Ficoll TM Gradient cell separation) and the mononuclear cell product is typically collected on a Terumo Optia machine according to standard procedures. The monocytes are depleted of αβ T cells using a research-scale Miltenyi Ls column separation method (in certain variations of the methods described in this example and other examples herein, the apheresis product may be depleted of αβ T cells using a CLINIMACS separation device; further depletion may include CD19 B cell depletion. Aliquots of the monocytes depleted of αβ T cells can be cryopreserved and then used for expansion). In feeder-free culture, BINATE medium (i.e., NK MACS medium) supplemented with 5% AB serum is used. TM culture medium (#130-107-879 (Miltenyi Biotec, Inc., San Diego, CA, USA)) (or other culture media such as Life Technologies, R&D systems culture media, CELLGENIX culture media, etc., with or without human serum)) and 500 IU / mL human interleukin 2 (IL-2) (Miltenyi Biotec) were added at 0.25-0.5×10 6 The resulting αβTCR-neg cell population was activated at a cell seeding concentration of 200 ng / ml in 24-well coated plates without additional activating reagents, or in 24-well uncoated plates with a combination of 500 ng / ml soluble anti-NKp46 (clone 9E2) and 500 ng / ml anti-CD2 (clone OKT11) (alternatively, other concentrations of activators may be used and other reagents may be added for activation).

[0252] 2. In the feeder-free culture conditions using BINATE medium and 500 IU / mL IL-2, 0.25-0.5×10 6 The activated αβTCRneg cell population was further cultured and expanded in T75 flasks at a cell seeding concentration of 1 × 10 / ml (alternatively, the cell seeding concentration can range up to 1 × 10 in culture bags, flasks or other large culture vessels such as bioreactors (G-Rex)). 6 / ml). The medium is replaced with fresh BINATE medium and IL-2 every 3 days (for bioreactors, the replacement frequency is low (5-7 days)). In certain variations of the method in this embodiment and other embodiments herein, the cultured BINATE cells can be cryopreserved after 1 week, 2 weeks, 3 weeks, 4 weeks or 5 weeks of expansion. In one embodiment, the amplified BINATE cells are washed, then concentrated, and serum-free cryopreservation medium (BIOLIFE) is added. The cryopreserved BINATE product can be thawed and inoculated again as described herein for further cultivation, or can be thawed and infused into, for example, a patient suffering from cancer or viral infection.

[0253] 3. At the end of expansion on day 15, BINATE cells were specifically analyzed for NK and γδCD3+ T cell content and their subsets (such as CD56), and potential contaminating cell types (such as αβTCR+ T cells) were additionally analyzed.

[0254] The phenotype of BINATE cells (reported as a percentage of the total cell population) showed no difference between activation on coated plates or activation with soluble antibodies. When activated on coated plates, the NK population (CD56+CD3-) accounted for 89.1% of the final population, while it was 92% when activated with soluble antibodies. CD3+γδT cells in coated plates and soluble conditions accounted for 6.2% and 5.5% of the population, respectively. CD3+CD56+γδT cells and CD3+CD56-γδT cells accounted for 2.8% and 3.4% of the population, respectively, in cultures activated by coated plates, and 3.3% and 2.2% of the population, respectively, in soluble activated cultures. Under these two conditions, the percentage of αβTCR+CD3+T cells was negligible, being 0.12% in coated activation and 0.41% in soluble activation.

[0255] Although historical reports have suggested that the use of cross-linked antibodies may have greater activity, the use of soluble antibodies is generally preferred over the use of cross-linked antibodies (e.g., for use on surfaces such as plates or beads) due to, for example, better control of the culture. The use of soluble anti-NKp46 (clone 9E2, Miltenyi) and soluble anti-CD2 (clone OKT11) did not differ compared to cross-linking conditions, and all subsequent experiments were performed with soluble antibody activation reagents.

[0256] Example 8: Evaluation of activation and expansion using different amounts of anti-NKp46

[0257] In separate experiments, different anti-NKp46 (Bab281) reagents were evaluated at three concentrations (10 ng / ml, 50 ng / ml and 500 ng / ml) under the following two amplification conditions: 1) IL-2 (500 IU / mL human interleukin 2 (Miltenyi Biotec)) or 2) IL-15 (10 ng / ml human interleukin 15 (Miltenyi Biotec)).

[0258] 1. Ficoll separation of buffy coat from normal healthy donors by manual separation in a centrifuge TM Gradient cell separation. Monocytes were depleted of αβT cells using a research-scale Miltenyi LS column separation. The resulting αβTCRneg cell population was separated at 0.25-0.5×10 6 Cells were seeded at a concentration of 100 ng / ml in 24-well plates under 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 anti-NKp46 (Bab281) at one of the following 3 concentrations: 10 ng / ml, 50 ng / ml or 500 ng / ml.

[0259] 2. In the feeder-free culture conditions using BINATE medium and 500 IU / mL IL-2 or 10 ng / ml IL-15, 0.25-0.5×10 6 The activated αβTCRneg cell population was further cultured and expanded in T75 flasks at a cell seeding concentration of 1 × 10 / ml (alternatively, the cell seeding concentration can range up to 1 × 10 in culture bags, flasks or other large culture vessels such as bioreactors (G-Rex)). 6 For plates or flasks, the medium was changed with fresh BINATE medium and IL-2 or IL-15 every 3 days (for bioreactors, the change frequency was less (5-7 days)).

[0260] 3. At the end of expansion on day 15, BINATE cells were analyzed for NK and γδCD3+ T cell content and their subsets (e.g., CD56), and additional analysis was performed for potential contaminating cell types (e.g., αβTCR+ T cells).

[0261] The phenotype of BINATE cells (reported as a percentage of the total cell population) showed that there was no significant difference between the three concentrations of anti-NKp46 (Bab281) when culture expansion was performed by IL-15 expansion. When 500ng / ml anti-NKp46 (Bab281), 50ng / ml anti-NKp46 (Bab281) and 10ng / ml anti-NKp46 (Bab281) were used, the NK population (CD56+CD3-) accounted for 84.5%, 87% and 87.2%, respectively. The γδTCR CD3+ population accounted for 11.4%, 8.7% and 8.1%, respectively, while the CD3+56+ population accounted for 7.40%, 5.40% and 5.30%, respectively. AlphaβTCR+ cells were almost undetectable at all concentrations.

[0262] Table 1

[0263]

[0264]

[0265] Example 9: Assessment of markers of activated and expanded cells

[0266] In a separate experiment, the BINATE phenotype was compared after activation with a combination of 10 ng / ml good manufacturing practice (GMP) quality anti-NKp46 (Bab281) antibody and 500 ng / ml GMP quality anti-CD2 (OKT11) antibody under two expansion conditions (i.e., IL-2 and IL-15).

[0267] 1. Ficoll separation of buffy coat from normal healthy donors by manual separation in a centrifuge TM Gradient cell separation. Monocytes were depleted of αβT cells using a research-scale Miltenyi LS column separation. The resulting αβTCRneg cell population was separated at 0.25-0.5×10 6 Cells were seeded at a concentration of 100 μg / ml in 24-well plates under feeder-free culture conditions using BINATE medium and 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 activators may be used and other agents may be added for activation).

[0268] 2. In the feeder-free culture conditions using BINATE medium and 500 IU / mL IL-2 or 10 ng / ml IL-15, 0.25-0.5×10 6The activated αβTCRneg cell population was further cultured and expanded in T75 flasks at a cell seeding concentration of 1 × 10 / ml (alternatively, the cell seeding concentration can range up to 1 × 10 in culture bags, flasks or other large culture vessels such as bioreactors (G-Rex)). 6 For plates or flasks, the medium was changed with fresh BINATE medium and IL-2 or IL-15 every 3 days (for bioreactors, the change frequency was less (5-7 days)).

[0269] 3. At the end of culture expansion on day 20, BINATE cells were specifically analyzed for markers of innate, cytotoxic, and exhaustion-expressing cell content and their subsets (e.g., CD56, CD16, and CD57), and potential contaminating cell types (e.g., αβTCR+ T cells) were additionally analyzed.

[0270] After IL-15 and IL-2 amplification, the total BINATE cell colony contained 92% and 84.3% of CD56+ innate cells, respectively. After IL-15 and IL-2 culture amplification, the colony contained 33% and 12% of CD56+CD16+ cytotoxic innate cells, 76% and 59.6% of CD8+ cytotoxic innate cells, and 99% and 96.5% of CD56+CD57- immature innate cells, respectively. AbTCR+ T cells were not detected after IL-15 or IL-2 culture amplification. The CD4+, CD8+ and CD4-CD8- colonies of NK and γδT cells are provided in Table 2 below.

[0271] Table 2

[0272] CD4+ CD8+ CD4-CD8- NKIL-15 0 77.9 22.1 γδT cell IL-15 1.2 20.7 76.1 NKIL-2 0 72.2 27.8 γδT cell IL-2 1.5 35.17 61.36

[0273] Example 10: Evaluation of cells expanded under static and bioreactor culture conditions

[0274] In a separate experiment, BINATE proliferation was compared under static and bioreactor culture conditions following activation with a combination of 50 ng / ml GMP-quality anti-NKp46 (Bab281) antibody and 50 ng / ml GMP-quality anti-CD2 (OKT11) antibody under two expansion conditions (IL-2 and IL-15).

[0275] 1. Ficoll separation of buffy coat from normal healthy donors by manual separation in a centrifuge TM Gradient cell separation. Monocytes were depleted of αβT cells using a research-scale Miltenyi LS column separation. The resulting αβTCRneg cell population was separated at 0.25-0.5×10 6Cells were seeded at a concentration of 100 μg / ml in 24-well plates 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 activators may be used and other agents may be added for activation).

[0276] 2. In the feeder-free culture conditions using BINATE medium and 500 IU / mL IL-2 or 10 ng / ml IL-15, 0.25-0.5×10 6 The activated αβTCRneg cell population was further cultured and expanded in T75 flasks or G-Rex benchtop bioreactors at a cell seeding concentration of 1 × 10 / ml (alternatively, the cell seeding concentration can range up to 1 × 10 in culture bags, flasks or other large culture vessels such as bioreactors (G-Rex)). 6 For plates or flasks, the medium was changed with fresh BINATE medium and IL-15 every 3 days (for bioreactors, the change frequency was less (5-7 days)).

[0277] 3. The activated αβTCRneg cell population can also be cultured in a feeder-free culture condition at 1×10 in a GRex culture device with BINATE medium and 10 ng / ml IL-15. 6 The cells were inoculated at a concentration of 100 / ml and further cultured and expanded. The medium was replaced with fresh BINATE medium and IL-15 every 7 days.

[0278] Total BINATE amplification is shown as Fig.17 The total cell number in the flasks was 1.34 × 10 cells / mL. The expansion of activated αβTCRneg cells in BINATE medium supplemented with IL-2 or IL-15 was equivalent over a 41-day period. Similarly, the IL-15 expansion of the activated αβTCRneg cell population in the bioreactor was identical to that in the flasks during the first 21 days.

[0279] Example 11: Assessment of Alloreactivity of Activated and Expanded Cells

[0280] In separate experiments, the alloreactivity of BINATE-activated cell populations expanded in IL-2 or IL-15 was assessed.

[0281] 1. Ficoll separation of buffy coat from normal healthy donors by manual separation in a centrifuge TMGradient cell separation. Monocytes were depleted of αβT cells using a research-scale Miltenyi LS column separation. The resulting αβTCRneg cell population was separated at 0.25-0.5×10 6 Cells were seeded at a concentration of 50 ng / ml in 24-well plates under 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 activators may be used and other agents may be added for activation).

[0282] 2. The activated αβTCRneg cell population was cultured in BINATE medium and 500 IU / mL IL-2 or 10 ng / ml IL-15 without feeder layer at a concentration of 0.25-0.5×10 6 The cells were seeded at a concentration of 100 μg / ml and further cultured and expanded in T75 flasks. For flasks, the medium was replaced with fresh BINATE medium and IL-2 or IL-15 every 3 days.

[0283] 3. BINATE cells and stimulator cells derived from PBMCs of three random healthy donors (HD) were tested in a one-way mixed lymphocyte reaction (MLR). Stimulator cells from three HDs were pooled and irradiated (30 Gy) before use. BINATE cells were inoculated at 100 μl / well (1×10 5 Cells) were seeded in 96 wells of a flat-bottom microtiter plate in BINATE medium with 200 μl of equal number of stimulator cells. After 5 days of incubation, the cultures were treated with 18 kBq 3 H-thymidine pulse treatment 12 hours, and on glass fiber filter, gather in the crops.On Microbeta Trilux 1450 counter (Wallac, PerkinElmer), dry filter is counted.Result is expressed as SI (cpm / cpm background with antigen).BINATE cells without adding cytokine are used as negative control, and positive control is represented by BINATE cells inoculated with cytokine (IL-2 or IL-15).

[0284] Analysis of mixed lymphocyte co-cultures 3 H thymidine incorporation (CPM), an indicator of proliferation. In the IL-15 experiments, the negative control consisted of unstimulated BINATE cells not supplemented with IL-15 and 3 The H-thymidine uptake value was 1144, indicating essentially no proliferation. 3 The H thymidine uptake value was 1194, also indicating no proliferation. Positive controls included proliferation stimulation with IL-15 and3 The H thymidine uptake value was 15558, a 14-fold increase over the negative control. 3 The H-thymidine uptake value was 942, which was basically the same as the background.

[0285] In the IL-2 experiments, negative controls included unstimulated BINATE cells not supplemented with IL-2 and 3 The H-thymidine uptake value was 834, indicating essentially no proliferation. 3 The H thymidine uptake value was 1194, also indicating no proliferation. Positive controls included proliferation stimulation with IL-2 and 3 The H thymidine uptake value was 18834, a 22-fold increase over the negative control. 3 The H-thymidine uptake value was 1245, which was basically no different from the background control.

[0286] The absence of MLR responses may be due to the absence of αβTCR+ T cells and supports the lack of alloreactivity in the innate BINATE cell population. The absence of alloreactivity is important clinically because it supports the lack of potential for cells to cause graft-versus-host reactions.

[0287] Example 12: Evaluation of cell proliferation and phenotype under different expansion conditions

[0288] In separate experiments, the proliferation capacity and BINATE phenotype were compared after activation with GMP quality anti-NKp46 (Bab281) antibody (50 ng / ml) (Caprico) and GMP quality anti-CD2 (OKT11) antibody (50 ng / ml) (Caprico) under 5 different expansion conditions.

[0289] 1. Ficoll separation of buffy coat from normal healthy donors by manual separation in a centrifuge TM Gradient cell separation. Monocytes were depleted of αβT cells using a research-scale Miltenyi LS column separation. The resulting αβTCRneg cell population was separated at 0.25-0.5×10 6 Cells were seeded at a concentration of 100 μg / ml in 24-well plates 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) and one of the following expansion supplements (alternatively, other concentrations of activators may be used and other agents may be added for activation):

[0290] a.500IU / mL IL-2(Miltenyi Biotec)

[0291] b.10ng / ml IL-15(Miltenyi Biotec)

[0292] c. Combination of 500 IU / mL IL-2 and 10 ng / mL IL-15 (IL-2 / IL-15)

[0293] d. Combination of 500 IU / mL IL-2 and 10 ng / ml OKT3 cloned anti-CD3 antibody (Miltenyi Biotec) (IL-2 / OKT3)

[0294] e. Combination of IL-15 10 ng / mL with 500 IU / mL IL-2 and 10 ng / ml OKT3 (IL-2 / IL-15 / OKT3).

[0295] 2. In feeder-free culture conditions using BINATE medium and one of the five expansion conditions listed above, 0.25-0.5×10 6 The activated αβTCRneg cell population was further cultured and expanded in T75 flasks at a cell seeding concentration of 1 × 10 / ml (alternatively, the cell seeding concentration can range up to 1 × 10 in culture bags, flasks or other large culture vessels such as bioreactors (G-Rex)). 6 / ml). For plates or flasks, medium was replaced with fresh BINATE medium every 3 days (for bioreactors, the replacement frequency was less (5-7 days)), and the same expansion conditions were used in the initial culture activation and expansion phases.

[0296] Total BINATE amplification is shown as Fig.18 The total cell number in the flasks. In BINATE medium supplemented with IL-2, IL-15, or IL-2 / OKT3, the expansion of activated αβTCRneg cells in flasks was equivalent over a 40-day period. In BINATE medium supplemented with IL-2 / IL-15 or IL-2 / IL-15 / OKT3, the expansion of activated αβTCRneg cells in flasks was equivalent over a 26-day period, then the proliferation rate slowed down, and the total cell number was less than that of the other 3 groups from culture day 26 to day 41.

[0297] As shown in Table 3 below, BINATE cells from each of the five conditions were determined to be NK or γδ T cells by flow cytometry at days 7, 20, and 30 of culture expansion. Cells from each condition at days 7, 20, and 30 were also analyzed by flow cytometry for specific markers of innate, cytotoxic, and exhaustion and immaturity expression on the cells, as well as specific VdTCR usage in γδ T cells, as shown in Table 4.

[0298] Table 3

[0299] CD56+CD3- CD3+CD56+ CD3+CD56- CD3+Total NK γδ T cells γδ T cells γδ T cells IL-15 Day 7 56.25 21.65 14.55 36.2 Day 20 83.45 12.55 3.7 16.25 Day 30 99.3 0.6 0.1 1.0 IL-2 Day 7 62.4 11.85 12.55 24.4 Day 20 56.55 22.95 15.3 38.25 Day 30 85.95 12.65 1.15 13.8 IL-2 / IL-15 Day 7 52.4 24.15 15.2 39.35 Day 20 51.7 36.85 11 47.85 Day 30 49.15 49.55 1.15 50.7 IL-2 / OKT3 Day 7 29.15 9.4 42.55 51.95 Day 20 26.65 26.3 43.25 69.55 Day 30 58.9 29.2 5.55 34.75 IL-2 / IL-15 / OKT3 Day 7 29.8 26.3 43.95 70.25 Day 20 36.6 47.5 15.35 62.85 Day 30 53.6 44.15 2.15 46.3

[0300]

[0301]

[0302] PD-1 expression was shown to be very low, an indicator of cell exhaustion. On day 30, the IL-15 expansion condition showed 2.7% expression in the total BINATE population, with 2% expression on CD56+CD3-NK cells, 10.6% expression on CD3+CD56-γδT cells, and 17.05% expression on CD3+CD56+γδT cells, demonstrating that there was no exhaustion. Under IL-2 expansion conditions, PD-1 expression was as low as 14.2% of the total BINATE population, with CD56+CD3-NK cells expressing 2% PD-1, and CD3+CD56-γδT cells and CD3+CD56+γδT cells expressing 62.9% and 78.3%, respectively.

[0303] Example 13: CD107a cytotoxicity

[0304] The 30th day cells of each of the 5 amplification conditions in Example 12 were co-cultured with tumor cell lines including IMR32, SH-SY5Y (neuroblastoma cell line) and K562 (B cell leukemia cell line). After 3 days of cultivation, the expression of CD107a was measured on the BINATE cells co-cultured with the corresponding tumor cell lines by flow cytometry analysis, which is a measure of the release of cytotoxic granules. Table 5 shows the difference between the tumor stimulation expression minus the basal value for each tumor cell line and each BINATE subset (CD56+CD3-NK cells, CD3+CD56+γδT cells and CD3+CD56-γδT cells) under each of the 5 amplification conditions (IL-2, IL-2 / OKT3, IL-15, IL-2 / IL-15 and IL-2 / IL-15 / OKT3). Statistical significance is reported as the p value of the T test.

[0305] Table 5

[0306]

[0307]

[0308] Example 14: Evaluation of phenotypic markers under different amplification conditions

[0309] 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. Fig.19 Shown are phenotypic analyses by marker type (activation, cytotoxicity, and exhaustion / immaturity) for IL-15 expansion conditions. Fig. 20 Shown are phenotypic analyses by marker type (activation, cytotoxicity, and exhaustion / immaturity) for IL-2 expansion conditions. Fig.21 Phenotypic analysis by marker type (activation, cytotoxicity, and exhaustion / immaturity) for IL-2 / OKT3 expansion conditions is shown. In all conditions, BINATE cells generally showed high activation and cytotoxicity and low exhaustion.

[0310] Example 15: Evaluation of in vitro tumor killing

[0311] In a separate experiment, in vitro tumor killing of BINATE cells was compared after activation with a combination of 10 ng / ml anti-NKp46 (Bab281) antibody and 500 ng / ml anti-CD2 (OKT11) antibody under two expansion conditions (IL-2 and IL-15).

[0312] 1. Ficoll separation of buffy coat from normal healthy donors by manual separation in a centrifuge TM Gradient cell separation. Monocytes were depleted of αβT cells using a research-scale Miltenyi LS column separation. The resulting αβTCRneg cell population was separated at 0.25-0.5×10 6 Cells were seeded at a concentration of 100 μg / ml in 24-well plates under 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 activators may be used and other agents may be added for activation).

[0313] 2. In the feeder-free culture conditions using BINATE medium and 500 IU / mL IL-2 or 10 ng / ml IL-15, 0.25-0.5×10 6 The activated αβTCRneg cell population was further cultured and expanded in T75 flasks at a cell seeding concentration of 1 × 10 / ml (alternatively, the cell seeding concentration can range up to 1 × 10 in culture bags, flasks or other large culture vessels such as bioreactors (G-Rex)). 6 For plates or flasks, the medium was changed with fresh BINATE medium and IL-2 or IL-15 every 3 days (for bioreactors, the change frequency was less (5-7 days)).

[0314] 3. At the end of the 20-day culture expansion, the BINATE cells were placed in a tumor co-culture experiment for 5-7 days. The experiment shown in Table 6 below involved culturing leukemia cell lines in 6-well culture dishes with 2 ml of culture medium / culture well at a 1:1 effector to target ratio (i.e., 0.5M BINATE cells (effector) to 0.5M tumor cells (target)).

[0315] The experiment shown in Table 7 below involved culturing solid tumor cell lines (neuroblastoma, sarcoma, and colon tumor cell lines) in 6-well culture dishes with 2 ml of culture medium / well at a 2:1 effector to target ratio (i.e., 0.5M BINATE cells (effector) to 0.25M tumor cells (target)). Co-culture of tumor cells and effector cells was performed in the absence of cytokines. Co-culture was assessed by microscopy and flow cytometry on days 3-7.

[0316] When residual tumor was examined in co-culture experiments, activated BINATE cells expanded in IL-2 or IL-15 successfully eradicated 50% or more of tumor cells in all cell lines and more than 75% in 4-5 AML cell lines, as shown in Table 6. 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 generally considered resistant to innate killing.

[0317] Table 6 – Residual tumor cells after co-culture with BINATE cells

[0318]

[0319]

[0320] When residual tumors were examined in co-culture experiments, activated BINATE cells expanded in either IL-2 or IL-15 successfully eradicated neuroblastoma tumors in short-term co-culture experiments, as shown in Table 7. IL-2-expanded BINATE cells were not tested in colon cancer co-culture, whereas IL-15-expanded BINATE cells showed activity against sarcomas and colon cancers.

[0321] Table 7 – Residual tumor cells after co-culture with BINATE cells

[0322] BINATE IL-15 BINATE IL-2 Neuroblastoma SH-SY5Y 0.9 4.5 IMR32 0.2 18.7 SKNSH 18.5 27.05 CHLA 3.85 14.75 LAN-1 6.5 10.8 SKNSKBeC 6.8 35.6 sarcoma RD 1.7 15.3 A673 0.6 0.7 CT10 0.2 0.7 G401 0.2 ND* colon SW480 4.6 ND DLD1 8.3 ND

[0323] *Not determined

[0324] Example 16: Evaluation of in vitro transduction efficiency

[0325] In a separate experiment, to assess the stability of transduction, early and late in vitro transduction efficiencies of BINATE cells were compared after activation with a combination of 10 ng / ml anti-NKp46 (Bab281) antibody and 500 ng / ml anti-CD2 (OKT11) antibody and a retroviral construct encoding the third-generation GD2 chimeric antigen receptor (CAR).

[0326] 1. Ficoll separation of buffy coat from normal healthy donors by manual separation in a centrifuge TM Gradient cell separation. Monocytes were depleted of αβT cells using a research-scale Miltenyi LS column separation. The resulting αβTCRneg cell population was separated at 0.25-0.5×10 6 The cells were seeded at a concentration of 100 μg / ml in 24-well plates under 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 activators may be used and other agents may be added for activation).

[0327] 2. To produce any of the BINATE.CARs described herein, an aliquot of cells is taken from the activated culture-expanded αβTCRneg cell population on days 4-15 and cultured at 0.25×10 6 / ml concentration is inoculated on a plate or culture bag (Gobain) coated with human fibronectin. After three to four days, the cells are washed and then the untransduced cells are processed, which may include amplification or cryopreservation. Amplification conditions may include 500IU / mL human interleukin 2 (IL-2) or 10ng / ml IL-15, but may also include any combination of conditions mentioned herein. The chimeric antigen receptor (CAR) constructs used herein include CAR.GD2, CAR.123 and CAR.CD19, which contain non-functional markers, such as fragments of CD34 surface antigens or mutated CD19 fragments (see Example 25 for description of some constructs). In some variants of the method herein, VECTOFUSION-1 (Miltenyi Biotec) can be used instead of RETRONECTIN to assist retroviral transfection, and genetic modification can include genetic modification of other CAR constructs of any selected antigen, or modification with other molecules. For this example, on day 5, the activated αβTCRneg cell population was incubated at 0.25×10 6 The GD2 construct contains a mutant fragment of the CD34 receptor as a selection and tracking marker.

[0328] 3. After three days, the transduced cells were washed and cultured at 0.25-0.5 × 10 6 The cells can be inoculated at concentrations up to 1 × 10 / ml for further culture expansion in T75 flasks (alternatively, the cell inoculation concentration can range up to 1 × 10 in culture bags, flasks or other large culture vessels such as bioreactors (G-Rex)). 6 For plates or flasks, the medium was changed with fresh BINATE medium and IL-2 every 3 days (for bioreactors, the change frequency was less (5-7 days)).

[0329] 4. On days 3 and 15 after transduction, the expression of mutant CD34 receptors (an indicator of transduction efficiency) was analyzed for the BINATE cell subset of γδT cells and the VdTCR subset. The transduction efficiency of CD56+CD3-NK cells was 82.4% and 82.4% on days 3 and 15, respectively. The transduction efficiency of CD3+56+ cells was 43.2% and 62.4% on days 3 and 15, respectively. The transduction efficiency of CD3+CD56-γδT cells was 37.3% and 56.8% on days 3 and 15, respectively. The genetic modification was stable over the culture time, indicating that the highly activated population was able to achieve high transduction and permanent integration.

[0330] In similar experiments, transduction of the BINATE subset showed high transduction of the GD2 CAR construct, as well as polyclonal transduction of both Vd1 and Vd2 TCRγδ T cells. The transduction efficiency of CD56+CD3- NK cells was 69%, while the transduction efficiency of CD3+γδ T cells was 39%. The transduction efficiency of the Vd1 TCR population was 67%, while the transduction efficiency of the Vd2 TCR population was 34%.

[0331] Example 17: Evaluation of proliferation after GD2 CAR transduction in vitro

[0332] In separate experiments, the proliferation capacity of BINATE cells after in vitro transduction with a retroviral construct encoding the third-generation GD2 CAR and after activation with a combination of 50 ng / ml anti-NKp46 (Bab281) antibody and 50 ng / ml anti-CD2 (OKT11) antibody was compared with that of unmodified BINATE cells under IL-2 and IL-15 expansion conditions.

[0333] 1. Ficoll separation of buffy coat from normal healthy donors by manual separation in a centrifuge TM Gradient cell separation. Monocytes were depleted of αβ T cells using a research-scale Miltenyi LS column separation. The resulting αβTCRneg cell population was placed in a 24-well plate under feeder-free culture conditions using BINATE medium and 500 IU / ml IL-2 or 10 ng / ml IL-15 (alternatively, other concentrations of activators can be used and other reagents can be added for activation) at a cell seeding concentration of 0.25-0.5×106 / ml.

[0334] 2. In the feeder-free culture conditions using BINATE medium and 500 IU / mL IL-2 or 10 ng / ml IL-15, 0.25-0.5×106 The activated non-genetically modified αβTCRneg cell population was further cultured and expanded in T75 flasks at a cell seeding concentration of 1×10 / ml (alternatively, the cell seeding concentration can range up to 1×10 in culture bags, flasks or other large culture vessels such as bioreactors (G-Rex)). 6 For plates or flasks, the medium was changed with fresh BINATE medium and IL-2 or IL-15 every 3 days (for bioreactors, the change frequency was less (5-7 days)).

[0335] 3. On day 5, the activated αβTCRneg cell population was incubated at 0.25×10 6 The GD2 construct contains a mutant fragment of the CD34 receptor as a selection and tracking marker.

[0336] 4. After 5 days, the transduced cells were washed and cultured at 0.25-0.5 × 10 cells / mL in feeder-free culture conditions using BINATE medium and 500 IU / mL IL-2 or 10-ng / ml IL-15. 6 The cells can be inoculated at concentrations up to 1 × 10 / ml for further culture expansion in T75 flasks (alternatively, the cell inoculation concentration can range up to 1 × 10 in culture bags, flasks or other large culture vessels such as bioreactors (G-Rex)). 6 For plates or flasks, the medium was replaced with fresh BINATE medium and IL-2 or IL15 every 3 days (for bioreactors, the replacement frequency was less (5-7 days)).

[0337] 5. Record the cell numbers of BINATE and BINATE.CARGD2 populations every 7 days.

[0338] Unmodified BINATE cells and BINATE.CARGD2 cells were expanded prior to transduction (by Fig. 22 For different conditions, the amplification after transduction was Fig. 22 There were no significant differences between genetically modified or unmodified cells, nor were there differences in the total number of cells expanded by IL-2 or IL-15.

[0339] Example 18: Evaluation of in vitro killing of solid tumors

[0340] In a separate experiment, in vitro killing of solid tumors was compared following transduction of activated BINATE with a retroviral construct encoding the third-generation GD2 CAR under two expansion conditions (IL-2 and IL-15).

[0341] 1. Ficoll separation of buffy coat from normal healthy donors by manual separation in a centrifuge TM Gradient cell separation. Monocytes were depleted of αβT cells using a research-scale Miltenyi LS column separation. The resulting αβTCRneg cell population was separated at 0.25-0.5×10 6 Cells were seeded at a concentration of 100 μg / ml in 24-well plates 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 activators may be used and other agents may be added for activation).

[0342] 2. On day 5, the activated αβTCRneg cell population was incubated at 0.25 × 10 6 The GD2 construct contains a mutant fragment of the CD34 receptor as a selection and tracking marker.

[0343] 3. After three days, the transduced cells were washed and cultured at 0.25-0.5×10 in feeder-free culture conditions using BINATE medium and 500 IU / mL IL-2 or 10 ng / ml IL-15. 6 The cells can be inoculated at concentrations up to 1 × 10 / ml for further culture expansion in T75 flasks (alternatively, the cell inoculation concentration can range up to 1 × 10 in culture bags, flasks or other large culture vessels such as bioreactors (G-Rex)). 6 For plates or flasks, the medium was changed with fresh BINATE medium and IL-2 or IL-15 every 3 days (for bioreactors, the change frequency was less (5-7 days)).

[0344] 4. At the end of 20 days of culture amplification, BINATE cells were placed in a tumor co-culture experiment for 5-7 days. The experiment included culturing in a 6-well culture dish with 2 ml culture medium / culture well, with a 1:1 effector to target ratio (i.e., 0.5M BINATE cells or BINATE.CAR.GD2 cells (effectors) and 0.5M tumor cells (targets)). Co-culture of tumor cells and effector cells was performed in the absence of cytokines. Co-culture was assessed by microscopy and flow cytometry on days 3-7.

[0345] Table 8 shows the percentage data of residual tumor from co-culture after tumor co-culture with unmodified BINATE IL-2 or BINATE IL-15 or BINATE IL-2 modified with CAR.GD2 or BINATE IL-15 modified with CAR.GD2. The measurement value of residual tumor in the control is 100%. Both BINATE IL-2 and BINATE IL-15 showed significant tumor killing. BINATE IL-2 and BINATE IL-15 modified with CAR.GD2 also showed significant killing.

[0346] Table 8 – Residual tumor cells after co-culture with BINATE cells and BINATE.CAR.GD2 cells

[0347]

[0348] Example 19: Evaluation of in vitro killing of myeloid leukemia cell lines

[0349] In a separate experiment, in vitro killing of myeloid leukemia cell lines in long-term co-cultures was assessed following transduction of activated BINATE with a retroviral construct encoding a third-generation CD123 CAR.

[0350] 1. Ficoll separation of buffy coat from normal healthy donors by manual separation in a centrifuge TM Gradient cell separation. Monocytes were depleted of αβT cells using a research-scale Miltenyi LS column separation. The resulting αβTCRneg cell population was separated at 0.25-0.5×10 6 Cells were seeded at a concentration of 100 μg / ml in 24-well plates 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 activators can be used and other reagents can be added for activation).

[0351] 2. On day 5, the activated αβTCRneg cell population was incubated at 0.25×10 6 The CD123 construct contains a mutant fragment of the CD19 receptor as a selection and tracking marker.

[0352] 3. After three days, the transduced cells were washed and cultured at 0.25-0.5 × 10 cells / mL in a feeder-free culture medium with 10 ng / ml IL-15. 6 The cells can be inoculated at concentrations up to 1 × 10 / ml for further culture expansion in T75 flasks (alternatively, the cell inoculation concentration can range up to 1 × 10 in culture bags, flasks or other large culture vessels such as bioreactors (G-Rex)). 6 For plates or flasks, the medium was changed with fresh BINATE medium and IL-15 every 3 days (for bioreactors, the change frequency was less (5-7 days)).

[0353] 4. At the end of the 20-day culture expansion, BINATE cells and BINATE.CAR123 were placed in a tumor co-culture experiment for 7 or more days. The experiment consisted of 2 ml of culture medium / well in a 6-well culture dish with a 1:1 effector to target ratio (i.e., 0.5×10 6 BINATE or BINATE.CAR123 cells (effector) were incubated with 0.5 × 10 6 Tumor cells (target) were cultured. Co-culture of tumor cells and effector cells was performed in the absence of cytokines. Co-culture was assessed by microscopy and flow cytometry on days 3-7.

[0354] The transduction efficiency of BINATE.CD123 cells was 65%. The myeloid leukemia tumor cell lines evaluated in co-culture included THP1, MOLM3, and OCI AML cell lines. The data include the percentage of residual tumor after co-culture of tumors with unmodified BINATE or BINATE.CAR.CD123. The measurement of residual tumor in the control was 100%. BINATE cells failed to successfully eradicate myeloid leukemia cells due to the continued growth of residual tumors after the initial short-term killing. BINATE.CAR.123 cells were able to successfully kill tumor cells and maintain tumor-free culture when co-cultured with THP1 and MOLM3 cell lines, and were greater than 70% of the control in the OCIAML cell line.

[0355] Example 20: Evaluation of in vivo killing of leukemia cells

[0356] In a separate experiment, in vivo killing of leukemic cells was assessed following transduction of activated BINATE cells with a retroviral construct encoding a second-generation CD19 CAR.

[0357] 1. Ficoll separation of buffy coat from normal healthy donors by manual separation in a centrifuge TM Gradient cell separation. Monocytes were enriched for NK using a research-scale Miltenyi LS column separation and the Miltenyi NK selection kit. NK and γδ T cell populations were separated at 0.25-0.5 × 10 6 Cells were seeded at a concentration of 100 μg / ml in 24-well plates under 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 NKp46 (alternatively, other concentrations of activators can be used and other agents can be added for activation).

[0358] 2. On day 5, the activated population was incubated at 0.25 × 10 6 The CD19 construct contains a mutant fragment of the CD34 receptor as a selection and tracking marker.

[0359] 3. After three days, the transduced cells were washed and cultured at 0.25-0.5 × 10 6 The cells can be inoculated at concentrations up to 1 × 10 / ml for further culture expansion in T75 flasks (alternatively, the cell inoculation concentration can range up to 1 × 10 in culture bags, flasks or other large culture vessels such as bioreactors (G-Rex)). 6 / ml). For plates or flasks, the medium was replaced with fresh BINATE medium and IL-2 every 3 days (for bioreactors, the replacement frequency was less (5-7 days)). At the end of expansion, the BINATE cells were washed and formulated in PBS solution for intravenous administration to mice.

[0360] Xenogeneic in vivo leukemia models

[0361] NOD / SCID IL-2Rγnull (NSG) xenograft mice were infused with DAUDI cells to evaluate the antitumor effects of CAR-transduced cells in vivo. Mouse experiments were performed under conditions that met international, EU, and national ethical requirements and were approved by the Italian Ministry of Health (N°88 / 2016-PR). On day -3, NGS mice (5 weeks old; Jackson Laboratory, USA) were inoculated with firefly luciferase-labeled Daudi cells (FF-Daudi) (0.25×10 6 On day 0, mice were injected intravenously with 50 × 10 6 BINATE cells or BINATE.CAR19 cells were cultured and subjected to bioluminescence imaging (IVIS System, Perkin Elmer, USA) every week.

[0362] Three doses of BINATE cells or BINATE.CD19 cells were infused into the animal model on day 0. Non-genetically modified BINATE cells were able to produce a partial response, and mice survived until day 42. Genetically modified BINATE cells with CAR.CD19 showed a dose-response response, with the lowest dose of 1×10 6 BINATE cells were able to keep animals alive for one week longer than non-transduced animals, but did not result in the survival of animals that received 5 × 10 6 or 10×10 6 The animals did not survive as long as animals treated with BINATE.CAR.CD19 cells. This model received subcutaneous injections of IL-2 twice weekly. Unlike the αβT cells modified with the CAR-T CD19 gene, these animals lived until sacrifice on day 90 and there was no evidence of cytokine release syndrome (CRS) or allogeneic or xenoreactivity despite IL-2 administration, supporting the improved safety of the innate cells. Animals were sacrificed on day 90 and tissues were assessed for the presence of human CD45+ cells (BINATE) in the peripheral blood and liver, and the percentages of NK and γδT subsets were then examined in each tissue separately. As Fig.23 As shown, BINATE cells are present in both blood and tissues, demonstrating the potential for survival and expansion of BINATE cell populations in an in vivo environment.

[0363] Example 21: Evaluation of in vivo killing of solid tumors (neuroblastoma)

[0364] In a separate experiment, in vivo killing of a solid tumor (neuroblastoma) was assessed following transduction of activated BINATE cells with a retroviral construct encoding the third-generation GD2 CAR.

[0365] 1. Ficoll separation of buffy coat from normal healthy donors by manual separation in a centrifugeTM Gradient cell separation. Monocytes were depleted of αβT cells using a research-scale Miltenyi LS column separation. The resulting αβTCRneg cell population was separated at 0.25-0.5×10 6 Cells were seeded at a concentration of 100 μg / ml in 24-well plates 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 activators can be used and other reagents can be added for activation).

[0366] 2. On day 5, the activated αβTCRneg cell population was incubated at 0.25×10 6 The GD2 construct contains a mutant fragment of the CD34 receptor as a selection and tracking marker.

[0367] 3. After three days, the transduced cells were washed and cultured at 0.25-0.5 × 10 cells / mL in a feeder-free culture medium with 10 ng / ml IL-15. 6 The cells can be inoculated at concentrations up to 1 × 10 / ml for further culture expansion in T75 flasks (alternatively, the cell inoculation concentration can range up to 1 × 10 in culture bags, flasks or other large culture vessels such as bioreactors (G-Rex)). 6 / ml). The medium was changed every 3 days (for bioreactors, the change frequency was less (5-7 days)). On day 20, the cells were washed and prepared in PBS for administration.

[0368] Xenogeneic in vivo neuroblastoma model

[0369] NOD / SCID IL-2Rγnull (NSG) xenograft mice were infused with SH-SY5Y cells to evaluate the antitumor effects of CAR-transduced cells in vivo. Mouse experiments were performed under conditions that met international, EU, and national ethical requirements and were approved by the Italian Ministry of Health (N°88 / 2016-PR). On day -3, NGS mice (5 weeks old; Jackson Laboratory, USA) were inoculated intraperitoneally with firefly luciferase-labeled SH-SY5Y cells (0.75×10 6 Every other week, mice were injected intraperitoneally with 30 × 10 6 BINATE cells or BINATE.CARGD2 cells were treated with 3 doses and subjected to weekly bioluminescence imaging (IVIS System, Perkin Elmer, USA).

[0370] Non-genetically modified BINATE cells showed a partial response around day 18, clearing the surrounding tumor mass, but the mice subsequently developed local progression. Genetically modified BINATE cells carrying CAR.CDGD2 showed significant clearance of the surrounding tumor mass but were unable to kill local lesions. The model received subcutaneous injections of IL-2 twice a week.

[0371] Example 22: Evaluation of in vitro killing of solid tumors (neuroblastoma) by NK cells and ADCC antibodies

[0372] In separate experiments, in vitro killing of solid tumors (neuroblastoma) by innate NK cells or innate NK.CAR.GD2 cells was evaluated.

[0373] 1. Ficoll separation of buffy coat from normal healthy donors by manual separation in a centrifuge TM Gradient cell separation. Monocytes were enriched for NK using a research-scale Miltenyi LS column separation and the Miltenyi NK selection kit. The resulting population was isolated at 0.25-0.5 × 10 6 Cells were seeded at a concentration of 100 μg / ml in 24-well plates under 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 NKp46 (alternatively, other concentrations of activators can be used and other agents can be added for activation).

[0374] 2. On day 5, the activated population was incubated at 0.25×10 6 The GD2 construct contains a mutant fragment of the CD34 receptor as a selection and tracking marker.

[0375] 3. After three days, the transduced cells were washed and cultured at 0.25-0.5 × 10 6 The cells can be inoculated at concentrations up to 1 × 10 / ml for further culture expansion in T75 flasks (alternatively, the cell inoculation concentration can range up to 1 × 10 in culture bags, flasks or other large culture vessels such as bioreactors (G-Rex)). 6 For plates or flasks, the medium was changed with fresh BINATE medium and IL-2 every 3 days (for bioreactors, the change frequency was less (5-7 days)).

[0376] 4. At the end of the 20-day culture expansion, BINATE cells and BINATE.CARGD2 cells were placed in tumor co-culture experiments for 7 or more days. The experiment consisted of 2 ml of culture medium / well in a 6-well culture dish with a 1:1 effector to target ratio (i.e., 0.5×10 6 Innate NK or innate NK.CAR.GD2 (effector) and 0.5×10 6 SH-SY5Y tumor cells (target) were cultured. Anti-GD2 antibody (10 μg 14.G2a monoclonal antibody) was added to each co-culture group to evaluate the ADCC potential of activated cells. Co-culture of tumor cells and effector cells was performed in the absence of cytokines. Co-culture was evaluated by microscopy and flow cytometry on days 3-7.

[0377] The data include the percentage of residual tumor after the tumor was co-cultured with unmodified innate NK or innate NK.CAR.GD2 with or without 14.G2a ADCC antibody. The measurement value of residual SH-SY5Y tumor in the control was 99.5%. When 14.G2a antibody was added to SH-SY5Y tumor cells, there was no difference compared with the control (98.5%). When unmodified activated innate NK cells were added to SH-SY5Y tumor cells, the residual tumor was 22%. Adding 14G.2a antibody to the combination of innate NK cells and SH-SY5Y tumor cells resulted in a smaller residual tumor percentage (14.5%). Adding innate NK.CAR.GD2 cells to SH-SY5Y tumor cells resulted in a residual tumor percentage of 40.9%, and when innate NK.CAR.GD2 cells, SH-SY5Y tumor cells and 14.G2a antibodies were co-cultured, the residual tumor percentage dropped to 8.7%. In the presence of cells with FcγIII receptor expression (e.g., activated innate cells), ADCC activity of the antibody enhances tumor killing.

[0378] Embodiment 23:

[0379] In separate experiments, the effects of switching cytokines on cell content during expansion were evaluated.

[0380] 1. Ficoll separation of buffy coat from normal healthy donors by manual separation in a centrifuge TM Gradient cell separation.

[0381] 2. Depletion of αβ T cells from monocytes using a research-scale Miltenyi LS column separation method.

[0382] 3. The obtained αβTCRneg cell population was placed in NK MACS supplemented with 5% AB serum TM The cells were cultured under feeder-free conditions using culture medium (#130-107-879 (Miltenyi Biotec, Inc., San Diego, CA, USA)) (alternatively, other culture media, such as R&D systems medium, CELLGENIX medium, etc., may be used).

[0383] 4. Activation was achieved by a combination of 10 ng / ml anti-NKp46 (clone Bab 281) and 10 ng / ml anti-CD2 (clone OKT11) or 50 ng / ml anti-NKp46 (clone Bab 281) and 50 ng / ml anti-CD2 (clone OKT11) of Caprico purified GMP (alternatively, other concentrations of activators can be used and other agents can be added for activation). The activated αβTCRneg cell population was cultured at 0.25×10 6 The cells were seeded at a concentration of 100 / ml in a 24-well plate under one of the following conditions and incubated in an NK MACS well supplemented with 5% AB serum. TM Culture medium (#130-107-879 (Miltenyi Biotec, Inc., San Diego, CA, USA)) under feeder-free culture conditions (or other culture media, such as R&D systems culture medium, CELLGENIX culture medium, etc., can be used):

[0384] a. 500 IU / mL human interleukin 2 (IL-2) (Miltenyi Biotec)

[0385] b. 10ng / ml human interleukin 15 (IL-15) (Miltenyi Biotec)

[0386] c. Combination of 500 IU / mL IL-2 and 10 ng / mL IL-15

[0387] d. Combination of 500 IU / mL IL-2 and 10 ng / ml OKT3 (Miltenyi Biotec)

[0388] e. A combination of 10 ng / mL IL-15 and 500 IU / mL IL-2 and 10 ng / ml OKT3 was then transferred to a T75 flask.

[0389] 5. Perform medium changes every 3 days with fresh medium (for bioreactors, changes are less frequent (5-7 days)) and one of the conditions listed above.

[0390] 6. Cells were plated at 0.25-0.5×10 6 / ml (alternatively, the cell inoculation concentration can range up to 1×10 6 / ml).

[0391] 7. In addition, the cells initially expanded with IL-2 in the first 25 days are then washed and supplemented with IL-15 until the end of the culture (day 30). The cells initially expanded with IL-15 are washed on day 25 and then supplemented with IL-2 until the end of the culture on day 30 (referred to as cytokine conversion conditions). In some cases, the cells initially expanded with IL-2 in the first 20 days are then washed and supplemented with IL-15 until the end of the culture (day 30). The cells initially expanded with IL-15 are washed on day 20 and then supplemented with IL-2 until the end of the culture on day 30. Alternative combinations or other cytokines, such as IL-7, IL-12, IL-18, IL-21, OKT3, OKT11, and anti-NCR, can be used under conversion conditions.

[0392] 8. At the end of expansion, BINATE cells were analyzed for NK and γδ CD3+ T cell content and characterized by receptor expression on the cells by flow cytometry.

[0393] In this example, when a single cytokine was compared to a switch condition (IL-2 vs. IL-2 switched to IL-15), the number of γδ T cells increased when IL-15 was added compared to IL-2 alone in the case of IL-2 vs. IL-2 / IL-15 (70% vs. 50%, respectively). In the case of IL-15 vs. IL-15 / IL-2, the NK population increased in the switch condition compared to IL-15 alone (90% vs. 80%, respectively). Since both IL-2 and IL-15 work by interacting with the IL-2 receptor on cells, the data suggest that the expansion of a population that is favorable under the initial expansion conditions using a particular cytokine is enhanced by switching to other cytokines.

[0394] Example 24: Isolation of pure NK cells or pure γδT cells

[0395] In this example, methods for isolating pure NK cell populations and isolating pure γδ T cell populations are described. Fig.24 A flowchart illustrating an example variation of this approach is shown in .

[0396] 1. Ficoll separation of buffy coat from normal healthy donors by manual separation in a centrifuge TMGradient cell separation; Percoll gradients, SEPAX, Cobe 2991 or elutriation systems can also be used. Alternatively, apheresis, cord blood units, bone marrow or body fluids such as ascites, CSF or pleural fluid can be used as a cell source with or without gradient centrifugation.

[0397] 2.Mononuclear cells can be used directly or after gradient separation, or further cell selection, such as Ficoll TM MNC, antibody depletion, NK positive selection, NK negative selection, γδT cell negative selection and / or CD3+ positive selection. In some cases, monocytes are positively selected or negatively selected for CD56+NK cells using Miltenyi magnetic bead-based technology or other cell selection technology. Alternatively, cells can be positively selected for γδTCR+ using Miltenyi magnetic bead-based technology or other cell selection technology. Alternatively, cells can be CD3+ cell depleted using Miltenyi magnetic bead-based technology or other cell selection technology.

[0398] 3. The resulting cell populations were then activated in feeder-free culture using BINATE medium (NK MACS medium supplemented with 5% AB serum) in the presence of soluble or cross-linked anti-LFA and anti-NCR antibodies with or without human serum supplementation. TM Medium #130-107-879 Miltenyi Biotec, Inc., San Diego, CA, USA (alternatively, other media such as R&D systems medium, CELLGENIX medium, etc. may be used)) or other animal component-free media such as in vivo or R&D Systems or Cellgenix.

[0399] 4. In a T75 flask, 0.25-5×10 6 / ml, cell expansion occurs by supplementation with cytokines such as IL-2, IL-15, IL-12, IL-18, and combinations thereof (alternatively, cell seeding concentrations can range up to 1×10 in culture bags, flasks, or other large culture vessels such as bioreactors (G-Rex)). 6 / ml). The medium was replaced with growth medium and cytokines every 3 days (for bioreactors, the replacement frequency was less frequent (5-7 days)).

[0400] 5. The cultures obtained on days 7, 20, 30, 45 and 60 may contain a BINATE population of a mixture of NK and γδT cells, which may then be cryopreserved for administration. Alternatively, the resulting cell population may undergo further post-expansion cell selection steps, such as NK CD56+ removal to obtain a pure γδT cell population. Alternatively, the cells may be positively depleted or negatively selected for γδT cells to produce a pure NK population, or positively selected for CD16, which will produce a highly enriched ADCCFcyIII receptor + Alternatively, cells can be NK-depleted and then CD16-selected for the ADCCγδ T cell population.

[0401] Example 25: Retroviral constructs

[0402] In this example, the retroviral vectors CARGD2, CARCD123 and CARCD19 used in certain examples above are described.

[0403] Carrier skeleton

[0404] The constructs described below include the SFG backbone using a retroviral vector based on Moloney murine leukemia virus (MoMLV). All env and gag-pol have been removed except for the packaging sequence (psi). Therefore, the vector is not replication-competent.

[0405] Production cell lines

[0406] The packaging cell lines used to produce the following retroviral constructs were generated in the Pediatric Oncology Cell and Gene Therapy Laboratory at OPBG in a research environment using dedicated laminar flow hoods and CO2 incubators and materials of non-animal origin, with the exception of gamma-irradiated Pharmagrade FBS (EuroClone, catalog number ECS0172L, lot number EUS0131906GI). The packaging cell lines were generated from cGMP bank human-based 293VEC RD114 production cells obtained from BioVec Pharma. In particular, 293VEC RD114 cells were selected as packaging cell lines because they are of human origin and allow the generation of high vector titers suitable for large-scale clinical grade production.

[0407] CARGD2

[0408] The iC9-CARGD2.CD28.41bb.CD3zeta (OPBG-91 vector) retroviral vector was constructed at the "Cell and Gene Therapy in Pediatric Tumors" laboratory of the Ospedale Pediatrico Bambino Gesù (OPBG). A bicistronic vector was used, which allows the simultaneous expression of two transgenes, inducible caspase 9 (iC9) and CARGD2 (iC9-CARGD2.CD28.41bb.CD3zeta). Single cell cloning was performed, and the 293VEC RD114 clone (i.e., OPBG-91-7) that produced the highest titer (using PCR analysis of the presence of vector in the supernatant) was expanded, maintained at the Officina FarmaceuticaOPBG, and used to produce retrovirus under cGMP conditions after testing for sterility and mycoplasma.

[0409] In the iC9 component, the catalytic domain of the intracellularly expressed Casp9 protein is fused to a drug binding domain derived from a human FK506 binding protein (FKBP12) with an F36V mutation. After including the iC9 sequence gene cassette, the CAR molecule is cloned into a retroviral vector to improve the safety of the method, based on a single chain of the fused VH-VL region of the monoclonal antibody 14.G2A specific for the human antigen GD2, and in the same frame with the CD28 transmembrane domain and its internal domain, the 4.1bb co-stimulatory domain and the CD3ζ cytoplasmic domain, for transducing activation signals after antigen binding.

[0410] Certain functional and structural components for iC9.CARGD2.CD28.41bb.CD3z expression and activity are summarized in Table 9 below and are listed here:

[0411] • 5'LTR - the retroviral long terminal repeat sequence at the 5' end of the vector (functions as a promoter sequence).

[0412] ψ-Retroviral encapsidation signal (psi; required for packaging of RNA into viral particles).

[0413] SA – splice acceptor site.

[0414] · iCasp9 - Inducible caspase-9 expression cassette. iCasp9 consists of human FK506 binding protein (FKBP12) with F36V mutation, linked to a modified CARD domain-deficient human caspase-9 via a 6-amino acid Gly-Ser linker.

[0415] FKBP12-F36V – An engineered FK506 binding protein containing the F36V mutation for optimized binding affinity to AP1903. The FKBP12-F36V protein domain serves as a drug binding / oligomerization domain for attached therapeutic proteins. FKBP12-F36V acts as a modulator of caspase-9. In the absence of AP1903, iCasp9 has minimal activity; AP1903 bound to FKBP12-F36V promotes dimerization and juxtaposes two caspase-9 molecules to initiate apoptosis. Thus, the FKBP12-F36V portion functionally replaces the endogenous dimerization / activation module (caspase activation and recruitment domain; CARD) of caspase-9 that mediates Apaf-1-associated oligomerization.

[0416] • Linker - A synthetic Ser-Gly-Gly-Gly-Ser-Gly peptide linker was used to fuse the switch regulatory sequence to caspase-9.

[0417] Caspase-9 – Human caspase-9 cDNA sequence (key pro-apoptotic regulator) and therapeutic component of the construct (regulated suicide gene). The endogenous dimerization / activation module (caspase activation and recruitment domain; CARD) was deleted to reduce spontaneous Apaf1 binding and thus background killing.

[0418] • 2A—encodes a 20 amino acid synthetic peptide from a Thata Asigna insect virus that functions as a cleavable linker between the caspase-9 protein and the CAR protein.

[0419] Signal peptide – a short amino acid sequence that enables the proper transfer of secretory proteins from the endoplasmic reticulum to the cell membrane.

[0420] The CAR-CAR molecule is based on a single chain of the fused VH-VL regions of the monoclonal antibody 14.G2A specific for the human antigen GD2, in frame with the CD28 TM and co-stimulatory domains – the 4.1bb co-stimulatory domain and the CD3ζ cytoplasmic domain.

[0421] • 3'LTR - the retroviral long terminal repeat at the 3' end of the vector (functions as a terminator / polyadenylation sequence).

[0422] Table 9

[0423] Components start Finish LTR 1 590 iCasp9 Box 1880 3085 FKBP12-F36V 1880 2218 Linker peptide 2219 2236 Casp9 2237 3085 2A peptide 3086 3145 CAR 3146 4723 Signal peptide 3146 3208 scFv 3209 4000 CH2-CH3 Hinge 4001 4055 CD28TM 4056 4135 CD28 co-stimulation 4136 4258 4.1bb co-stimulation 4259 4384 CD3ζ 4385 4723 LTR 4896 5485 <![CDATA[Amp R Landmarks]]> 6780 7640

[0424] The reference electronic vector sequence was assembled by combining the DNA sequence files of the components of the vector construct. Since the retroviral genome is RNA-based, the plasmid DNA used for transfection into the 293VEC cell line was sequenced (the initial step in the preparation of the retroviral product). The entire OPBG-91 vector was bidirectionally sequenced at Ospedale Pediatrico Bambino Gesù. The sequencing run was assembled using SnapGene software. No base mismatches were identified compared to the theoretical reference electronic sequence.

[0425] CARCD123

[0426] The ΔCD19-2A-CAR-CD123-ΔCD34.CD8.41bb.CD3zeta (OPBG-242 vector) retroviral vector was constructed in the "Pediatric Tumor Cell and Gene Therapy" laboratory of Ospedale Pediatrico Bambino Gesù (OPBG). A bicistronic vector was used, which allows the simultaneous expression of two transgenes, namely ΔCD19 and CARCD123 (ΔCD19-2A-CAR-CD123-ΔCD34.CD8.41bb.CD3zeta). Single cell cloning was performed as described above, and clones with the highest titer (using PCR analysis of the presence of vectors in the supernatant) were expanded and preserved.

[0427] ΔCD19 represents the extracellular domain of human CD19 connected to the transmembrane part. It has a dual function that helps in the selection and phenotypic characterization of genetically modified cells. After the gene cassette including the sequence of ΔCD19, the CAR molecule is cloned into a retroviral vector by using the 2A sequence. The CAR molecule is a single chain based on the fusion VH-VL region of the monoclonal antibody 7G3 specific for the human antigen CD123, and is in frame with the CD8 transmembrane domain and its internal domain-4.1bb co-stimulatory domain and CD3ζ cytoplasmic domain for transducing activation signals after antigen binding.

[0428] Certain functional and structural components for ΔCD19-2A-CAR-CD123-ΔCD34.CD8.41bb.CD3z expression and activity are summarized in Table 10 below and are listed here:

[0429] • 5'LTR - the retroviral long terminal repeat sequence at the 5' end of the vector (functions as a promoter sequence).

[0430] ψ-Retroviral encapsidation signal (psi; required for packaging of RNA into viral particles).

[0431] SA – splice acceptor site.

[0432] ΔCD19 – includes optimized human extracellular and transmembrane domains.

[0433] 2A – encodes a 20 amino acid synthetic peptide from a Lycopodiella glaucoma insect virus.

[0434] Signal peptide – a short amino acid sequence that enables the proper transfer of secretory proteins from the endoplasmic reticulum to the cell membrane.

[0435] ΔCD34 – Includes a short peptide derived from human CD34 to facilitate detection of CAR+ T cells after transduction.

[0436] The CAR-CAR molecule is based on a single chain of the fused VH-VL regions of the monoclonal antibody 7G3 specific for the human antigen CD123, in frame with the CD8 TM and co-stimulatory domains – the 4.1bb co-stimulatory domain and the CD3ζ cytoplasmic domain.

[0437] • 3'LTR - the retroviral long terminal repeat sequence at the 3' end of the vector (functions as a terminator / polyadenylation sequence).

[0438] Table 10

[0439] Components start Finish LTR 397 990 dCD19 2282 3280 SvFv 7G3 3404 3745 2A peptide 3746 3769 SvFv 7G3 3770 4123 ΔCD34 4124 4183 CD8 stem 4184 4309 CD8TM 4310 4372 CD8 Cyt 4373 4420 4.1bb co-stimulation 4427 4552 CD3ζ 4553 4891 3'LTR 5069 5635 AmpR promoter 6848 6952 AmpR 6953 7813

[0440] The reference electronic vector sequence was assembled by combining the DNA sequence files of the components of the vector construct. Since the retroviral genome is RNA-based, the plasmid DNA used for transfection into the 293VEC cell line was sequenced (the initial step in the preparation of the retroviral product). The entire OPBG-242 vector was bidirectionally sequenced at Ospedale Pediatrico Bambino Gesù. The sequencing run was assembled using SnapGene software. No base mismatches were identified compared to the theoretical reference electronic sequence.

[0441] CARCD19

[0442] The therapeutic retroviral construct SFG-iC9-Car.CD19.41bb encodes a synthetic ligand-inducible human caspase-9 cDNA, which is connected to a single-stranded fusion VH-VL region of a monoclonal antibody with specificity for the mouse antigen CD19, 4.1bb co-stimulatory domain. The assembly is the same as the above-mentioned CARGD2, except that it is the second generation. Retroviral vectors were constructed in the EU Cell and Gene Therapy Laboratory. Single cell cloning was performed, and the clones with the highest titer were amplified (using PCR to analyze the presence of vectors in the supernatant), which were stored in a cGMP facility and used to produce retroviruses under cGMP conditions after testing sterility and mycoplasma.

[0443] Some of the functional and structural components for SFG-iC9-Car.CD19.41bb expression and activity are listed here:

[0444] • 5'LTR - the retroviral long terminal repeat sequence at the 5' end of the vector (functions as a promoter sequence).

[0445] ψ-Retroviral encapsidation signal (psi; required for packaging of RNA into viral particles).

[0446] SA – splice acceptor site.

[0447] · iCasp9 - Inducible caspase-9 expression cassette. iCasp9 consists of human FK506 binding protein (FKBP12) with F36V mutation, linked to a modified CARD domain-deficient human caspase-9 via a 6-amino acid Gly-Ser linker.

[0448] FKBP12-F36V – An engineered FK506 binding protein containing the F36V mutation for optimized binding affinity to AP1903. The FKBP12-F36V protein domain serves as a drug binding / oligomerization domain for attached therapeutic proteins. FKBP12-F36V acts as a modulator of caspase-9. In the absence of AP1903, iCasp9 has minimal activity; AP1903 bound to FKBP12-F36V promotes dimerization and juxtaposes two caspase-9 molecules to initiate apoptosis. Thus, the FKBP12-F36V portion functionally replaces the endogenous dimerization / activation module (caspase activation and recruitment domain; CARD) of caspase-9 that mediates Apaf-1-associated oligomerization.

[0449] • Linker - A synthetic Ser-Gly-Gly-Gly-Ser-Gly peptide linker was used to fuse the switch regulatory sequence to caspase-9.

[0450] Caspase-9 – Human caspase-9 cDNA sequence (key pro-apoptotic regulator) and therapeutic component of the construct (regulated suicide gene). The endogenous dimerization / activation module (caspase activation and recruitment domain; CARD) was deleted to reduce spontaneous Apaf1 binding and thus background killing.

[0451] • 2A—encodes a 20 amino acid synthetic peptide from the Thata Asigna insect virus that serves as a cleavable linker between the caspase-9 protein and the CAR protein.

[0452] Signal peptide – a short amino acid sequence that enables the proper transfer of secretory proteins from the endoplasmic reticulum to the cell membrane.

[0453] The CAR-CAR molecule is based on a single chain fused VH-VL region of a monoclonal antibody specific for the murine antigen CD19, 4.1bb co-stimulatory domain.

[0454] • 3'LTR - the retroviral long terminal repeat sequence at the 3' end of the vector (functions as a terminator / polyadenylation sequence).

[0455] Example 26: Examples of Certain Non-Limiting Embodiments

[0456] Listed below are non-limiting examples of certain embodiments of the present technology.

[0457] A1. A method for producing a composition comprising a cell population enriched in NK cells and γ.δ T cells, comprising:

[0458] obtaining a sample comprising cells from one or more subjects;

[0459] depleting α.β T cells from the sample under conditions that produce a depleted cell population comprising NK cells and γ.δ T cells; and

[0460] Exposing the depleted cell population to an activation condition comprises 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 expressed on the surface of one or more cells of the sample population; and

[0461] Exposing the depleted cell population to expansion conditions comprises contacting the depleted cell population with at least one supplemental polypeptide, thereby producing a composition comprising a cell population enriched for NK cells and γ.δ T cells.

[0462] A1.1. A method for producing a composition comprising a cell population enriched in NK cells and γ.δ T cells, comprising:

[0463] obtaining a sample comprising cells from one or more subjects;

[0464] Exposing the sample to an activating condition comprises 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 is expressed on the surface of one or more cells in the sample population, wherein (a) or (b) is soluble, or both (a) and (b) are soluble; and

[0465] Exposing the sample to expansion conditions includes contacting the sample with at least one supplemental polypeptide, thereby producing a composition comprising a cell population enriched for NK cells and γ.δ T cells.

[0466] A2. The method of embodiment A1 or A1.1, wherein the at least one supplementary polypeptide is selected such that the amount of NK cells relative to the amount of γ.δ T cells in the population depends on the amount and / or type of the at least one supplementary polypeptide.

[0467] A2.1. The method of embodiments A1, A1.1 or A2, wherein after contacting the depleted cell population with the at least one supplemental polypeptide, the at least one supplemental polypeptide increases or decreases the amount of NK cells relative to γ.δ T cells in the cell population.

[0468] A3. The method of any one of embodiments A1, A1.1, A2 or A2.1, wherein the activation conditions are free of serum from a non-human animal.

[0469] A4. The method of any one of embodiments A1 to A3, wherein the amplification conditions are free of serum from a non-human animal.

[0470] A5. The method of any one of embodiments A1 to A4, wherein the activation conditions are free of feeder cells.

[0471] A6. The method of any one of embodiments A1 to A5, wherein the expansion conditions are free of feeder cells.

[0472] A7. The method of any one of embodiments A1 to A6, wherein the sample is selected from the group consisting of peripheral blood, liver tissue, epithelial tissue, bone marrow, and umbilical cord blood.

[0473] A8. The method of embodiment A7, wherein the sample is peripheral blood.

[0474] A9. The method of embodiment A8, wherein the peripheral blood sample is a processed sample.

[0475] A10. The method of embodiment A7, wherein the sample is umbilical cord blood.

[0476] A11. The method of embodiment A10, wherein the cord blood sample is a processed sample.

[0477] A12. The method of any one of embodiments A1 to A11, wherein the exogenous polypeptide in (b) immunospecifically binds to a NK cell activating receptor, a γ.δ T cell activating receptor, or both.

[0478] A13. The method of embodiment A12, wherein the receptor is NKp30, NKp44 or NKp46.

[0479] A14. The method of embodiment A13, wherein the receptor is NKp46.

[0480] A15. The method of any one of embodiments A1 to A14, wherein the exogenous polypeptide in (a) immunospecifically binds to CD2.

[0481] A16. The method of any one of embodiments A1 to A15, wherein the exogenous polypeptide in (a) or (b) or (a) and (b) is an antibody or an antigen-binding fragment thereof.

[0482] A17. The method of any one of embodiments A1 to A16, wherein at least one of (a) or (b) is soluble.

[0483] A17.1. The method of any one of embodiments A1 to A17, wherein the exogenous polypeptides in (a) and (b) are both soluble.

[0484] A18. The method of any one of embodiments A1 to A16, wherein the exogenous polypeptide in (a) or the exogenous polypeptide in (b) is bound to a substrate.

[0485] A19. The method of any one of embodiments A1 to A18, wherein the activation conditions comprise contacting the sample or depleted cell population with at least two exogenous polypeptides.

[0486] A20. The method of embodiment A19, wherein the first exogenous polypeptide immunospecifically binds to CD2, and the second exogenous polypeptide immunospecifically binds to NKp46.

[0487] 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.

[0488] A22. The method of any one of embodiments A1 to A21, wherein the polypeptide component of the activation condition consists essentially of or consists of:

[0489] (a) an exogenous polypeptide that immunospecifically binds to the cell adhesion polypeptide CD2; and

[0490] (b) An exogenous polypeptide that is different from the exogenous polypeptide in (a) and that immunospecifically binds to NKp46.

[0491] A23. The method of any one of embodiments A1 to A22, wherein the supplementary polypeptide is a cytokine and / or a polypeptide that immunospecifically binds to a receptor on γ.δ T cells.

[0492] A24. The method of embodiment A23, wherein the expansion conditions include contacting the sample or depleted cell population with at least one supplementary polypeptide, which is a cytokine and optionally a supplementary polypeptide that immunospecifically binds to a receptor on γ.δ T cells.

[0493] A25. The method of embodiment A24, wherein the cytokine is interleukin (IL).

[0494] A26. The method of any one of embodiments A23 to A25, wherein the at least one supplemental polypeptide comprises IL-2, IL-4, IL-15, or any combination thereof.

[0495] A27. The method of any one of embodiments A23 to A26, wherein the amplification conditions comprise contacting the sample or depleted cell population with a polypeptide:

[0496] (a) an IL-2 polypeptide, and optionally, a polypeptide that immunospecifically binds to a receptor on a γ.δ T cell;

[0497] (b) an IL-15 polypeptide; or

[0498] (c) IL-2 polypeptide and IL-15 polypeptide, and optionally, a polypeptide that immunospecifically binds to a receptor on a γ.δ T cell.

[0499] A28. The method of any one of embodiments A23 to A27, wherein the receptor on the γ.δ T cell is CD3.

[0500] A29. The method of any one of embodiments A23 to A28, wherein the polypeptide that immunospecifically binds to a receptor on a γ.δ T cell is an antibody or an antigen-binding fragment thereof.

[0501] A30. The method of embodiment A29, wherein the amplification conditions comprise contacting the sample with the following polypeptide:

[0502] (a) IL-2 polypeptide;

[0503] (b) IL-15 polypeptide;

[0504] (c) IL-2 polypeptide and IL-15 polypeptide;

[0505] (d) an IL-2 polypeptide and an antibody that immunospecifically binds to CD3; or

[0506] (e) IL-2 polypeptide, IL-15 polypeptide and an antibody that immunospecifically binds to CD3.

[0507] A31. The method of embodiment A30, wherein the antibody that immunospecifically binds to CD3 is OKT3.

[0508] A32. The method of any one of embodiments A1 to A31, wherein the activation conditions and amplification conditions are performed simultaneously or sequentially in any order.

[0509] A33. The method of any one of embodiments A1 to A32, wherein:

[0510] The at least one exogenous polypeptide may also function as a supplemental polypeptide; or

[0511] The at least one supplemental polypeptide may also function as an exogenous polypeptide; or

[0512] The at least one exogenous polypeptide may also function as a supplementary polypeptide, and the at least one supplementary polypeptide may also function as an exogenous polypeptide.

[0513] A34. The method of any one of embodiments A30 to A33, wherein:

[0514] (i) the expansion conditions comprise contacting the sample or depleted cell population with an IL-2 polypeptide; and

[0515] (ii) The resulting cell population enriched in NK cells and γ.δ T cells contains about 25-30% NK cells and about 70-75% γ.δ T cells.

[0516] A35. The method of any one of embodiments A30 to A33, wherein:

[0517] (i) the expansion conditions comprise contacting the sample or depleted cell population with an IL-15 polypeptide; and

[0518] (ii) The resulting cell population enriched in NK cells and γ.δ T cells contains about 80-99% NK cells and about 1-20% γ.δ T cells.

[0519] A36. The method of any one of embodiments A30 to A33, wherein:

[0520] (i) the expansion conditions comprise contacting the sample or the depleted cell population with an IL-2 polypeptide and an antibody that immunospecifically binds to CD3; and

[0521] (ii) The resulting cell population enriched in NK cells and γ.δ T cells contains about 40-45% NK cells and about 55-60% γ.δ T cells.

[0522] A37. The method of any one of embodiments A1 to A36, wherein the amplification conditions comprise:

[0523] contacting the sample or depleted cell population with a first set of conditions comprising one or more supplemental polypeptides, thereby producing a first cell population comprising a first ratio of NK cells to γ.δ T cells; and

[0524] The first cell population is contacted with a second set of conditions comprising one or more supplemental polypeptides, thereby producing a second cell population comprising a desired final ratio of NK cells to γ.δ T cells, wherein the first set of conditions is different from the second set of conditions.

[0525] A38. The method of embodiment A37, wherein the first cell population is washed prior to contacting with the second set of conditions.

[0526] A39. The method of embodiment A37 or A38, wherein:

[0527] The first set of conditions includes IL-2, and the second set of conditions includes IL-15;

[0528] The first set of conditions includes IL-15, and the second set of conditions includes IL-2;

[0529] The first set of conditions includes IL-2 and an antibody that immunospecifically binds to CD3, and the second set of conditions includes IL-15; or

[0530] The first set of conditions includes IL-15 and an antibody that immunospecifically binds to CD3, and the second set of conditions includes IL-2 and an antibody that immunospecifically binds to CD3.

[0531] A40. The method of embodiment A39, wherein the antibody that immunospecifically binds to CD3 is OKT3.

[0532] A41. The method of any one of embodiments A1.1 and A2 to A40, further comprising:

[0533] Depleting α.β T cells from the sample prior to exposing the sample to activation and expansion conditions, thereby producing a depleted cell population; and

[0534] The depleted cell population is subjected to activation and expansion conditions to obtain a composition comprising a cell population enriched for NK cells and γ.δ T cells.

[0535] A41.1. The method of any one of embodiments A1 to A41, wherein prior to activation and expansion, the sample or the depleted cell population is not exposed to conditions that select for NK cells or γ.δ T cells or deplete cells other than α-β T cells.

[0536] A41.2. The method of embodiment A41.1, wherein the sample or the depleted cell population is not exposed to conditions that deplete CD3+ cells prior to activation and expansion.

[0537] A42. The method of any one of embodiments A1 to A41.1, wherein the sample or cells of the depleted cell population do not contain exogenous nucleic acid before, during or after activation and expansion.

[0538] A43. The method of any one of embodiments A1 to A41.1, wherein the sample or cells from which the cell population is removed do not contain exogenous nucleic acid encoding a tumor necrosis factor receptor, a chimeric antigen receptor (CAR), a myeloid differentiation primary response protein, or an innate immune signaling aptamer before, during, or after activation and expansion.

[0539] A44. The method of any one of embodiments A1 to A43, wherein the cells of the sample or the depleted cell population are not genetically modified before, during or after activation and expansion.

[0540] A45. The method of any one of embodiments A1 to A44, further comprising treating the cell population enriched for NK cells and γ.δ T cells to remove the γ.δ cells, and the resulting population consists essentially of or consists of NK cells.

[0541] A46. The method of any one of embodiments A1 to A44, further comprising treating the cell population enriched for NK cells and γ.δ T cells so as to remove the NK cells, and the resulting population consists essentially of or consists of γ.δ T cells.

[0542] A47. The method of any one of embodiments A1 to A45, further comprising subjecting the cell population enriched in NK cells and γ.δT cells to positive selection for NK cells, thereby obtaining a cell population essentially consisting of or consisting of NK cells.

[0543] A48. The method of any one of embodiments A1 to A44 and A46, further comprising subjecting the cell population enriched in NK cells and γ.δ T cells to positive selection against γ.δ cells, thereby obtaining a cell population essentially consisting of or consisting of γ.δ cells.

[0544] A49. The method of any one of embodiments A1 to A48, wherein the expansion conditions comprise incubating the sample or the depleted cell population in a feeder cell-free culture medium for about one week to about 10 weeks, thereby obtaining a composition comprising an expanded cell population enriched in NK cells and γ.δT cells.

[0545] A50. The method of embodiment A49, wherein the culture conditions comprise incubating the sample or the depleted cell population in a feeder cell-free culture 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 longer, or about 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks.

[0546] A51. The method of any one of embodiments A1 to A50, wherein the cell population enriched for NK cells and γ.δ T cells expands by greater than about 2 logs within 30 days under said expansion conditions.

[0547] A52. The method of embodiment A51, wherein the cell population expands by greater than about 3 logs within 30 days under the expansion conditions.

[0548] A53. The method of any one of embodiments A1 to A52, wherein the expanded cell population enriched for NK cells and γ.δ T cells is free of exhausted cells.

[0549] A54. The method of any one of embodiments A1 to A53, wherein after 60 days under the expansion conditions, the expanded cell population enriched for NK cells and γ.δ T cells is free of exhausted cells.

[0550] A55. The method of any one of embodiments A1 to A54, wherein in an expanded cell population enriched in NK cells and γ.δT cells, less than 5%, 4%, 3% or 2% of the NK cells contain the PD-1 marker, and / or about 20%, 15%, 10% or less of the total cells in the expanded population or the γ.δT cells in the expanded population contain the PD-1 marker.

[0551] A56. The method of any one of embodiments A1 to A55, wherein the cell population enriched for NK cells and γ.δ T cells comprises one or more of the following activation markers as a percentage of the total number of cells in the population:

[0552] (a) 90% or more KIR5;

[0553] (b) 10% or higher SIGLEC-7;

[0554] (c) 60% or more KIR3D51;

[0555] (d) 10% or more KIR2DL1;

[0556] (e) 25% or more of NKp30, NKp44 or NKp46;

[0557] (f) 35% or more NKG2D;

[0558] (g) 90% or more DNAM1;

[0559] (h) 85% or more of NTBA;

[0560] (i) 95% or greater CD2; and

[0561] (j) 55% or more KIR3DS1.

[0562] A57. The method of any one of embodiments A1 to A56, wherein the cell population enriched for NK cells and γ.δ T cells comprises 80% or more innate immune cells.

[0563] A58. The method of embodiment A57, wherein about 70% to 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+.

[0564] A59. The method of embodiment A57 or A58, wherein about 10% to 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+.

[0565] A60. The method of any one of embodiments A57 to A59, wherein less than 10%, less than 5%, less than 4%, less than 3% or less than 2% of the cells are CD57+.

[0566] A61. The method of any one of embodiments A1 to A60, wherein the sample or depleted cell population does not comprise CD4+CD8+ cells during or after activation and expansion.

[0567] A62. The method of any one of embodiments A1 to A61, wherein the activation conditions and the amplification conditions do not include a bisphosphonate.

[0568] A63. The method of embodiment A62, wherein the bisphosphonate is pamidronate or zoledronate.

[0569] A64. The method of any one of embodiments A1 to A63, wherein the γ.δ T cells are polyclonal with respect to V.δ.1 and V.δ.2 expression.

[0570] A65. The method of embodiment A64, wherein the polyclonal γ.δ T cells comprise at least one subpopulation selected from V.δ.1+ and V.δ.1- and at least one subpopulation selected from V.δ.2+ and V.δ.2-.

[0571] A66. A composition obtainable by or by the method of any one of embodiments A1 to A65.

[0572] B1. A composition comprising a modified peripheral blood cell population, wherein the population comprises:

[0573] Multiple NK cells and multiple γ.δT cells;

[0574] α.β T cells are removed; and

[0575] Does not contain feeder cells.

[0576] B2. The composition of embodiment B1, wherein:

[0577] about 25% to about 45% of the cells are NK cells, and about 55% to about 75% of the cells are gamma delta T cells;

[0578] about 25% to about 30% of the cells are NK cells, and about 70% to about 75% of the cells are gamma delta T cells;

[0579] about 80% to about 99% of the cells are NK cells and about 1% to about 20% of the cells are gamma delta T cells; or

[0580] About 40% to about 45% of the cells are NK cells, and about 55% to about 60% of the cells are gamma delta T cells.

[0581] B3. The composition of embodiment B1 or B2, wherein 30% or more of the cells are activated.

[0582] B4. The composition of any one of embodiments B1 to B3, wherein the modified cell population comprises one or more of the following activation markers as a percentage of the total number of cells in the population:

[0583] (a) 90% or more KIR5;

[0584] (b) 10% or higher SIGLEC-7;

[0585] (c) 60% or more KIR3D51;

[0586] (d) 10% or more KIR2DL1;

[0587] (e) 25% or more of NKp30, NKp44 or NKp46;

[0588] (f) 35% or more NKG2D;

[0589] (g) 90% or more DNAM1;

[0590] (h) 85% or more of NTBA;

[0591] (i) 95% or greater CD2; and

[0592] (j) 55% or more KIR3DS1.

[0593] B5. The composition of any one of embodiments B1 to B4, wherein the modified population comprises 80% or more innate immune cells.

[0594] B6. The composition of any one of embodiments B1 to B5, wherein the modified population is enriched in activated cytotoxic cells that are CD56+.

[0595] B7. The composition of any one of embodiments B1 to B6, wherein the modified population is enriched in activated cytotoxic cells that are CD57-.

[0596] B8. The composition of any one of embodiments B1 to B7, wherein the population is enriched for activated cytotoxic cells that are CD56+CD57-.

[0597] B9. The composition of any one of embodiments B6 to B8, wherein about 80% to 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+.

[0598] B10. The composition of any one of embodiments B6 to B9, wherein about 10% to 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+.

[0599] B11. The composition of any one of embodiments B6 to B10, wherein less than 5%, less than 4%, less than 3% or less than 2% of the cells are CD57+.

[0600] B12. The composition of any one of embodiments B1 to B11, which is substantially free of cells other than NK cells and γ.δ T cells.

[0601] B13. The composition of any one of embodiments B1 to B12, comprising less than 5% NKT cells.

[0602] B14. The composition of any one of embodiments B1 to B13, comprising less than 1% NKT cells.

[0603] B15. The composition of any one of embodiments B1 to B14, comprising less than 0.1% NKT cells.

[0604] B16. The composition of any one of embodiments B1 to B15, comprising less than 2% α.β T cells.

[0605] B17. The composition of any one of embodiments B1 to B16, comprising less than 1% α.β T cells.

[0606] B18. The composition of any one of embodiments B1 to B17, comprising less than 0.1% α.β T cells.

[0607] B19. The composition of any one of embodiments B1 to B18, wherein the subset of NK cells in the population are CD16+ cells.

[0608] B20. The composition of any one of embodiments B1 to B19, wherein the majority of the γ.δ T cells are CD57- cells.

[0609] B21. The composition of any one of embodiments B1 to B20, wherein a majority of the NK cells are CD57- cells.

[0610] B22. The composition of any one of embodiments B1 to B21, wherein the γ.δ T cells are polyclonal with respect to V.δ.1 and V.δ.2 expression.

[0611] B23. The composition of embodiment B22, wherein the polyclonal γ.δ T cells comprise at least one subpopulation selected from V.δ.1+ and V.δ.1- and at least one subpopulation selected from V.δ.2+ and V.δ.2-.

[0612] B24. The composition of any one of embodiments B1 to B23, wherein:

[0613] Most γ.δ T cells express V.δ.1, and a minority of γ.δ T cells express V.δ.2; or

[0614] A minority of γ.δ T cells express V.δ.1, and the majority of γ.δ T cells express V.δ.2.

[0615] B25. The composition of any one of embodiments B1 to B24, wherein:

[0616] A minority of cells in the population are CD3-positive cells, and a majority of cells in the population are CD3-negative cells; or

[0617] The majority of cells in the population are CD3 positive cells, and a minority of cells in the population are CD3 negative cells.

[0618] B26. The composition of any one of embodiments B1 to B25, wherein the ratio of NK cells to γ.δ T cells is greater than 1.

[0619] B27. The composition of any one of embodiments B1 to B25, wherein the ratio of NK cells to γ.δ T cells is less than 1.

[0620] B28. The composition of embodiment B26, wherein the modified cell population comprises about 98-99% NK cells and about 1-2% γ.δ T cells.

[0621] B29. The composition of embodiment B27, wherein the modified cell population comprises about 25% to about 45% NK cells and about 55% to about 75% γ.δ T cells.

[0622] B30. The composition of embodiment B27, wherein the modified cell population comprises about 25-30% NK cells and about 70-75% γ.δ T cells.

[0623] B31. The composition of embodiment B27, wherein the modified cell population comprises about 40-45% NK cells and about 55-60% γ.δ T cells.

[0624] B32. The composition of any of embodiments B1 to B31, wherein about 50% to about 99% or more, or greater than or equal to about 50%, 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%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or a portion thereof, up to 100% of the NK cells and / or the γ.δ T cells are CD8+.

[0625] B33. The composition of embodiment B32, wherein less than 2% of said NK cells and / or said γ.δ T cells are CD4+.

[0626] B34. The composition of embodiment B32 or B33, wherein less than 2% of said NK cells and / or said γ.δ T cells are CD8+CD4+.

[0627] B35. The composition of any one of embodiments B32 to B34, wherein a portion of about 15% to about 30% of said NK cells and / or about 55% to 85% of said γ.δ T cells are CD8-CD4-.

[0628] B36. The composition of any one of embodiments B1 to B35, wherein about 30% to about 99% or more of 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%, 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 , 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 a portion thereof, up to 100% of the cells further comprise a genetic modification comprising an exogenous polynucleotide, a mutated polynucleotide, a deleted polynucleotide or a combination thereof.

[0629] B36.1 The composition of embodiment B36, comprising a plurality of NK cells and a plurality of γ.δ T cells.

[0630] B37. The composition of embodiment B36 or B36.1, 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.

[0631] B38. The composition of any one of embodiments B36, B36.1 or B37, wherein the genetic modification comprises an exogenous polynucleotide.

[0632] B39. The composition of embodiment B38, wherein the exogenous polynucleotide is in a retroviral vector or a lentiviral vector.

[0633] B40. The composition of embodiment B38, wherein the exogenous polynucleotide is integrated into the genome of one or more cells of the modified cell population.

[0634] B41. The composition of any one of embodiments B36 to B40, wherein the cells in the population comprise a chimeric antigen receptor (CAR).

[0635] B42. The composition of embodiment B41, 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.

[0636] B43. The composition of any one of embodiments B1 to B42, wherein the population comprising the plurality of NK cells and the plurality of γ.δ T cells is derived from peripheral blood cells of more than one subject.

[0637] C1. A pharmaceutical composition comprising the composition of any one of embodiments B1 to B43 and a pharmaceutically acceptable carrier.

[0638] D1. A method for preparing a modified immune cell, comprising one or more of the following:

[0639] (a) adding an exogenous polynucleotide to the composition of any one of embodiments A66 and B1 to B35;

[0640] (b) mutating a polynucleotide in one or more cells of the composition of any one of embodiments A66 and B1 to B35; or

[0641] (c) deleting a polynucleotide in one or more cells of the composition of any one of embodiments A66 and B1 to B35.

[0642] D2. The method of embodiment D1, wherein the genetic modification is by retroviral transduction, lentiviral transduction, electroporation, transfection, CRISPR / cas9 or TALENS.

[0643] D3. The method of embodiment D1 or D2, wherein the genetic modification consists of or consists essentially of adding an exogenous polynucleotide as in (a).

[0644] D4. The method of any one of embodiments D1-D3, wherein the genetic modification comprises adding an exogenous polynucleotide as in (a) and / or mutating a polynucleotide as in (b), and integrating the exogenous polynucleotide and / or the mutated polynucleotide into the genome of the immune cell.

[0645] D5. The method of embodiment D4, wherein the integration is by electroporation, transfection, CRISPR / cas9 or TALENS.

[0646] D6. The method of any one of embodiments D1 to D5, wherein the exogenous polynucleotide encodes a chimeric antigen receptor (CAR).

[0647] D7. The method of embodiment D6, 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.

[0648] D8. The method of any one of embodiments D1 to D7, wherein about 30% to about 99% or more of 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%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or a portion thereof, up to 100% of the cells contain the genetic modification.

[0649] D8.1 The method of embodiment D8, wherein the cells in the population include a plurality of NK cells and a plurality of γ.δ T cells.

[0650] D9. The method of embodiment D8 or D8.1, wherein the genetic modification comprises an exogenous polynucleotide.

[0651] D10. The method of embodiment D9, wherein about 100% or 100% of the cells in the population contain the exogenous polynucleotide.

[0652] E1. A kit comprising the composition of any one of embodiments A66 and B1 to B43 or the pharmaceutical composition of embodiment C1, optionally further comprising instructions for use and optionally a cytokine.

[0653] E2. The kit of embodiment E1, wherein the composition or the pharmaceutical composition is at a temperature of minus 4 degrees Celsius or lower.

[0654] E3. The kit of embodiment E2, wherein the composition or the pharmaceutical composition is at about minus 75 degrees Celsius to about minus 80 degrees Celsius.

[0655] E4. The kit of any one of embodiments E1 to E3, comprising about 1×10 5 The number of cells is about 1×10 12 cell.

[0656] E5. The kit of any one of embodiments E1 to E4, wherein the cytokine is an interleukin polypeptide.

[0657] E6. The kit of embodiment E5, wherein the interleukin peptide is IL-2, IL-4 or IL-15.

[0658] E7. The kit according to any one of embodiments E1 to E6, which is free of non-human serum and / or free of bovine serum.

[0659] E8. The kit of any one of embodiments E1 to E7, which is free of xenogens.

[0660] E9. The kit of any one of embodiments E1 to E8, which is free of exogenous feeder cells.

[0661] E10. The kit of any one of embodiments E1 to E9, which is in unit dosage form.

[0662] E11. The kit of embodiment E10, wherein the unit dosage form is about 1×10 6 cells to about 1×10 12 cells.

[0663] F1. A collection of cells from different donor subjects, comprising a plurality of containers, each container comprising cells from one or more donor subjects, wherein each container comprises the composition of any one of embodiments A66 and B1 to B43, the pharmaceutical composition of embodiment C1, or the kit of any one of embodiments E1 to E11.

[0664] G1. A method of treating cancer or infection, comprising administering to a subject in need thereof a composition of any one of embodiments A66 and B1 to B43, a pharmaceutical composition of embodiment C1, or a kit of any one of embodiments E1 to E11 in an amount effective to treat the cancer or infection, wherein the cells in the composition, the pharmaceutical composition, or the kit are allogeneic to the subject.

[0665] G2. A method of treating cancer or infection, comprising administering to a subject in need thereof a composition of any one of embodiments A66 and B1 to B43, a pharmaceutical composition of embodiment C1, or a kit of any one of embodiments E1 to E11, in an amount effective to treat the cancer or infection, wherein the cells in the composition, the pharmaceutical composition, or the kit are autologous to the subject.

[0666] G3. The method of embodiment G1 or embodiment G2, comprising administering the composition to the subject on two or more separate days.

[0667] G4. The method of embodiment G2 or embodiment G3, wherein the donor of the cells is the recipient of the treatment.

[0668] G5. The method of embodiment G1 or embodiment G3, wherein the donor of the cells is not the recipient of the treatment.

[0669] G6. The method of embodiment G5, wherein the recipient of said treatment is susceptible to GvHD if treated with α.β T cells from a donor.

[0670] G7. The method of any one of embodiments G1 to G6, wherein the treatment is administered at intervals of about 1 unit dose to about 36 or more unit doses at intervals of about 2 weeks to about 4 weeks.

[0671] G8. A method according to any one of embodiments G1 to G6, wherein the treatment is administered as a single unit dose once, twice, three times, four times or up to five times per day, or once, twice, three times, four times, five times, six times, seven times, eight times, nine times or ten times or more over a period of days, weeks or months, or once every other day, or once, twice, three times, four times, five times or six times per week.

[0672] G9. The method of any one of embodiments G1 to G8, wherein the treatment is administered intravenously (IV), intrathecally or intramuscularly (IM), intraperitoneally (IP), intrapleurally, intraarticularly, or by injection or implantation at or near the site of cancer or infection.

[0673] G10. The method of embodiment G8 or G9, wherein the unit dose comprises about 10 4 to about 10 10 cells, or about 10 per subject 6 to about 10 12 cells.

[0674] G11. The method of embodiment G10, wherein the unit dose is about 10 per subject. 10 cells, or about 10 per kilogram of subject body weight. 8 cells.

[0675] G12. The method of any one of embodiments G1 to G11, wherein the treatment is for cancer.

[0676] G13. A method for implementing embodiment G12, 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 lymphoma or childhood acute lymphoblastic leukemia, non-Hodgkin lymphoma, mast cell tumor or mast cell tumor, ovarian cancer, pancreatic cancer, non-small cell lung cancer, small cell lung cancer, liver cancer, retinoblastoma, breast tumor, colorectal cancer, leukemia, lymphoma, acute lymphoblastic leukemia (ALL) or acute lymphoid leukemia, acute myeloid leukemia (AML), histiocytic sarcoma, brain tumor, astrocytoma, glioblastoma, neuroma, colon cancer, cervical cancer, sarcoma, bladder tumor, reticuloendothelial tumor, Wilm's tumor, bone cancer, osteosarcoma, kidney cancer or head and neck cancer, oral cancer, laryngeal cancer, metastatic disease or pharyngeal cancer.

[0677] G14. The method of embodiment G12 or G13, wherein a second agent is co-administered with the composition, pharmaceutical composition or kit.

[0678] G15. The method of embodiment G14, wherein the second agent is an antibody that immunospecifically binds to a cancer-associated antigen.

[0679] G16. The method of embodiment G15, wherein the cancer-associated antigen is selected from the group consisting of: alpha-fetoprotein (AFP), α-actinin-4, A3, an antigen specific for A33 antibody, ART-4, B7, B7-H3, Ba 733, 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, CD2 2. 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, C D80, 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, 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 (MIF), GD2, MAGE, MAGE-3, MART-1, MART-2, NY-ESO-1, TRAG-3, mCRP, MCP-1, MIP-1A, MIP-1B, M IF, MUC1, 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-Friedenreich 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. .

[0680] G17. The method of embodiment G15 or G16, wherein the antibody is selected from 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), hL24 3 (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), bevacizumab (anti-VEGF), cetuximab (anti-EGFR), gemtuzumab tuzumab (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-α4 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).

[0681] G18. The method of any one of embodiments G1 to G11, wherein the treatment is for an infection.

[0682] G19. The method of embodiment G18, wherein the infection is characterized by the presence of bacterial, fungal, viral or protozoan pathogens.

[0683] G20. The method of embodiment G19, wherein the infection is selected from the group consisting of: herpes, Ebola virus, West Nile virus, vaccinia virus, Epstein-Barr virus, hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), herpes virus (e.g., HSV-1, HSV-2, HHV-6, CMV), human immunodeficiency virus (HIV), vesicular stomatitis virus (VSV), Bacilli, Citrobacter, Cholera, Diphtheria Diphtheria, Enterobacter, Gonococci, Helicobacter pylori, Klebsiella, Legionella, Meningococci, Mycobacteria, Pseudomonas, Pneumococci, 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.).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 lamblia, Trichinella, Dientamoeba Fragilis, Trypanosoma brucei, Trypanosoma cruzi, or Leishmania donovani.

[0684] ***

[0685] The entire contents of each patent, patent application, publication and document cited herein are incorporated herein by reference. The citation of the above patents, patent applications, publications and documents does not mean that any of the above contents are relevant prior art, nor does it constitute any admission of the contents or dates of these disclosures or documents. Their citation does not represent a search for relevant disclosures. All statements about the dates or contents of documents are based on the available information and are not an admission of their accuracy or correctness.

[0686] The foregoing may be modified without departing from the basic aspects of the present technology. Although the present technology has been described in great detail with reference to one or more specific embodiments, those of ordinary skill in the art will recognize that changes may be made to the embodiments specifically disclosed in this application, but these modifications and improvements are within the scope and spirit of the present technology.

[0687] The technology of illustrative description herein can be appropriately implemented in the case of lacking any element that is not specifically disclosed herein.Therefore, for example, in each case herein, any one of the terms "include / comprise", "consisting essentially of ... " and "consisting of ... " can be replaced by any one of the other two terms.The terms and expressions adopted are used as descriptions rather than terms of limitation, and the use of such terms and expressions does not exclude any equivalents of the features shown and described or their parts, and various modifications can be made within the scope of the claimed technology.Unless it is clearly known in the context that any one of a plurality of elements or more than one of a plurality of elements is described, otherwise the term "one (a)" or "one (an)" can refer to one or more (for example, "agent" can represent one or more reagents) of the element modified by it.As used herein, the term "about" refers to a value within 10% of a basic parameter (that is, plus or minus 10%), and the term "about" is used at the beginning of a string of values ​​to modify each value (that is, "about 1, 2 and 3" refers to about 1, about 2 and about 3). For example, a weight of "about 100 grams" may include weights between 90 grams and 110 grams. In addition, when a list of values ​​is described herein (e.g., about 50%, 60%, 70%, 80%, 85%, or 86%), the list includes all intermediate values ​​and fractional values ​​thereof (e.g., 54%, 85.4%). Therefore, it should be understood that although the present technology has been specifically disclosed through representative embodiments and optional features, modifications and variations of the concepts disclosed herein may be adopted by those skilled in the art, and such modifications and variations are considered to be within the scope of the present invention.

[0688] Certain embodiments of the technology are set forth in the following claims.

Claims

1. A method for producing a composition comprising 80-99% NK cells, comprising: a) obtaining a sample comprising cells from one or more subjects; b) depleting αβ T cells from said sample under conditions that produce a depleted cell population comprising NK cells and γδ T cells; c) exposing the depleted cell population to an activation condition, comprising contacting the depleted cell population with: (a) an antibody or an antigen-binding fragment thereof that immunospecifically binds to a cell adhesion molecule polypeptide CD2, and (b) an antibody or an antigen-binding fragment thereof that immunospecifically binds to NKp46; and d) exposing the depleted cell population to expansion conditions, including contacting the depleted cell population with IL-15, thereby producing the composition comprising 80-99% NK cells and 1% to 20% γδ T cells.

2. The method of claim 1, wherein the expansion conditions comprise incubating the depleted cell population in a feeder-free medium in the presence of IL-15 for 1 week to 6 weeks.

3. A method for producing a composition comprising 70% or more γδ T cells, comprising: a) obtaining a sample comprising cells from one or more subjects; b) depleting αβ T cells from said sample under conditions that produce a depleted cell population comprising NK cells and γδ T cells; c) exposing the depleted cell population to an activation condition, comprising contacting the depleted cell population with: (a) an antibody or an antigen-binding fragment thereof that immunospecifically binds to a cell adhesion molecule polypeptide CD2, and (b) an antibody or an antigen-binding fragment thereof that immunospecifically binds to NKp46; and d) exposing the depleted cell population to expansion conditions, including contacting the depleted cell population with IL-2, thereby producing a composition comprising 70-75% γδ T cells and 25-30% NK cells.

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

5. The method of claim 3, wherein the expansion conditions comprise incubating the depleted cell population in the presence of IL-2 and / or anti-CD3 antibodies for 1 week to 6 weeks.

6. The method of claim 4, wherein the expansion conditions comprise incubating the depleted cell population in the presence of IL-2 and / or anti-CD3 antibodies for 1 week to 6 weeks.

7. The method according to any one of claims 1-6, wherein the activation conditions include incubating the removed cell population for 1 hour to 1 week in the presence of: (a) an antibody or an antigen-binding fragment thereof that immunospecifically binds to the cell adhesion molecule polypeptide CD2; and (b) an antibody or an antigen-binding fragment thereof that immunospecifically binds to NKp46.

8. The method of any one of claims 1-6, wherein 1% or less of the total cells in the composition are αβ T cells.

9. The method of claim 7, wherein 1% or less of the total cells in the composition are αβ T cells.

10. The method of any one of claims 1-6, wherein removing αβ T cells from the sample comprises contacting the sample with an antibody that immunospecifically binds to an αβ T cell receptor.

11. The method of claim 7, wherein removing αβ T cells from the sample comprises contacting the sample with an antibody that immunospecifically binds to an αβ T cell receptor.

12. The method according to any one of claims 1-6, wherein the antibody or antigen-binding fragment thereof that immunospecifically binds to CD2 is a monoclonal anti-CD2 antibody, and / or wherein the antibody or antigen-binding fragment thereof that immunospecifically binds to NKp46 is a monoclonal anti-NKp46 antibody.

13. The method of claim 7, wherein the antibody or antigen-binding fragment thereof that immunospecifically binds to CD2 is a monoclonal anti-CD2 antibody, and / or wherein the antibody or antigen-binding fragment thereof that immunospecifically binds to NKp46 is a monoclonal anti-NKp46 antibody.

14. The method of claim 12, wherein the anti-CD2 antibody and / or anti-NKp46 antibody is soluble.

15. The method according to any one of claims 1 to 6, wherein the activation conditions and / or expansion conditions are free of feeder cells.

16. The method of claim 7, wherein the activation conditions and / or expansion conditions are free of feeder cells.

17. The method of any one of claims 1-6, wherein the activation conditions and / or amplification conditions do not include bisphosphonates.

18. The method of claim 7, wherein the activation conditions and / or amplification conditions do not include bisphosphonates.

19. The method of claim 17, further comprising subjecting the depleted cell population to genetic modification conditions after exposing the depleted cell population to expansion conditions.

20. The method of claim 18, further comprising subjecting the depleted cell population to genetic modification conditions after exposing the depleted cell population to expansion conditions.

21. The method of claim 19 or 20, wherein the genetic modification comprises introducing exogenous nucleotides encoding a chimeric antigen receptor into the cell.

22. The method of any one of claims 1-6, wherein the activation conditions and amplification conditions are performed simultaneously.

23. The method of claim 7, wherein the activation conditions and the amplification conditions are performed simultaneously.

24. The method according to any one of claims 1-6, wherein the activation condition and the amplification condition are performed sequentially.

25. The method of claim 7, wherein the activation condition and the amplification condition are performed sequentially.

26. The method of any one of claims 1-6, wherein the sample is peripheral blood, liver tissue, bone marrow, epithelial tissue or cord blood.

27. The method of claim 7, wherein the sample is peripheral blood, liver tissue, bone marrow, epithelial tissue or umbilical cord blood.

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