NK cell composition and preparations for immunotherapy and methods for their production

Abstract

To provide NK cell compositions and preparations for immunotherapy, and to provide methods for producing the same.SOLUTION: The present invention provides NK cell compositions and / or preparations and methods of using such NK cell compositions and / or preparations for immunotherapy. The NK cell compositions and / or preparations can be used in therapies of a broad range of viral infections, bacterial infections, cancer and leukemia malignancies, and other diseases. The present disclosure provides natural killer (NK) cell compositions and / or preparations, and methods of making and using such NK cell compositions and / or preparations for non-autologous immunotherapy. The NK cell compositions and / or preparations can be used in therapies for a broad range of viral and bacterial infections, cancer, including solid tumors and hematologic malignancies (leukemia, lymphoma), and other diseases.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 880,044, filed July 29, 2019, U.S. Provisional Application No. 62 / 892,779, filed August 28, 2019, and U.S. Provisional Application No. 62 / 960,507, filed January 13, 2020, the disclosures of which are incorporated herein by reference in their entireties.

[0002] Sequence Listing Statement The sequence listing associated with this application is provided in text format in lieu of a paper copy and is incorporated herein by reference. The text file containing the sequence listing is named 72246_Sequence_Final_2020-07-28.txt. The text file is 144 KB, was created on July 28, 2020, and has been submitted via EFS-Web with the application herein.

[0003] FIELD OF THE INVENTION The present disclosure provides natural killer (NK) cell compositions and / or preparations, and methods for making and using such NK cell compositions and / or preparations for non-autologous immunotherapy. The NK cell compositions and / or preparations can be used in the treatment of a wide range of viral and bacterial infections, cancers including solid tumors and hematological malignancies (leukemia, lymphoma), and other diseases. [Background technology]

[0004] Background of the Invention Natural killer (NK) cells are components of the innate immune system that exhibit various cytotoxic activities against transformed target cells, such as the production of certain cytokines, direct cytotoxicity, and mediation of antibody-dependent cell-mediated cytotoxicity (ADCC). NK cells essentially play a key role in host defense against any virus-infected or cancer-transformed cells and in the elimination of these cells. In addition, NK cells have been proposed as effectors for immunotherapy of various conditions, including the elimination of transformed cancer cells. Various methods for generating NK cells have been described, for example, from induced pluripotent stem cells, peripheral blood mononuclear cells, peripheral blood stem or progenitor cells, or umbilical cord blood stem or progenitor cells. For each source, the characteristics of NK cells depend on the method of their generation. Such methods traditionally rely on many rounds of ex vivo cell division to generate adequate numbers of NK cells for therapeutic purposes. NK cells obtained from such processes tend to be more mature and / or are more likely to show attrition after administration to patients. NK cell depletion can be indicated, for example, by reduced production of interferon gamma (IFNγ), CD107a, granzyme B, and / or perforin, decreased cytolytic activity, etc. Such NK cells may be less active or persistent when administered in vivo. Thus, there remains a need for NK cell populations for therapeutic purposes and compositions comprising said NK cell populations, and methods for producing such NK cell populations and / or compositions, that exhibit and maintain higher levels of activity and / or persistence after administration to a patient. Summary of the Invention [Means for solving the problem]

[0005] Summary of disclosure The present disclosure provides compositions of NK cell preparations and methods of making and using such NK cell compositions and / or preparations for immunotherapy. NK cell compositions and / or preparations can be used in the treatment of a wide range of viral infections, malignancies, and other diseases. NK cell compositions and / or preparations contain CD56 NK cells derived from multiple human donors, typically at least two or at least four human donors, or fully matched or partially matched (mismatched) donors. + The NK cell composition and / or preparation may be composed of pooled non-HLA-matched (mismatched) CD34 (primarily NK) cells. + from hematopoietic stem and progenitor cells (HSPCs), from immunologically compatible HSPCs, or from partially immunologically compatible (or incompatible) HSPCs, followed by in vitro or ex vivo expansion of HSPCs, followed by CD56 + Surprisingly, the resulting NK cell compositions and / or preparations, whether immunologically incompatible, compatible or incompatible, contain CD56 + (mostly NK) cells and CD56 - The present invention relates to a method for producing CD56 (non-NK) cells, including a mixture of CD56 (non-NK) cells. + The non-NK (NK) cells are much more potent but less mature than comparable NK cells in the prior art, typically derived from adult human donors, and may therefore exhibit greater cytolytic activity and / or persistence in vivo. The non-NK cells present in the compositions and / or preparations described herein are typically of bone marrow origin and support NK cell differentiation and activation, eliminating the need for an exogenous feeder cell layer.

[0006] In some embodiments, the NK cell compositions and / or preparations of the present disclosure comprise between about 50% and up to about 80% CD56 + cells, and approximately 50% to approximately 20% CD56 - Bone marrow-derived cells, including dendritic cells, macrophages, and granulocytes. CD56 +CD56 generated during the generation of - The cells were CD56 + In some embodiments, the NK cell compositions and / or preparations contain from about 50% to up to about 85% CD56 + cells, and approximately 50% to 15% CD56 - In some embodiments, the NK cell compositions and / or preparations produced by the disclosed methods contain between about 55% and up to about 65% CD56 + cells, and approximately 45% to 35% CD56 - In some embodiments, the resulting NK cell compositions and / or preparations contain between about 70% and up to about 85% CD56 + cells, and approximately 30% to 15% CD56 - Bone marrow-derived cells include, for example, dendritic cells, macrophages and granulocytes.

[0007] CD56 as described above + The subpopulation is primarily composed of NK cells. In addition to CD56, + The cells may express high frequencies of NKp30, NKp46, NKp44, NKG2A and / or granzyme B, intermediate to high frequencies of perforin and / or CD107a, low to intermediate frequencies of NKG2D, and substantially no killer cell immunoglobulin-like receptors (KIRs). In some embodiments, CD56 + Cells express KIR - (As used herein, "KIR - " refers to the KIR family members KIR2DL1, KIR2DS1, KIR2DS3, KIR2DS5, KIR2DL2, KIR2DL3, KIR2DS2, KIR2DS4, KIR3DL1, and KIR3DS1.) CD16 (also known as Fc gamma receptor III (FcγRIII)) may be expressed at low or intermediate frequencies on NK cells induced to differentiate in vitro by the methods described herein. In some embodiments, CD56 -The cells express a medium to high frequency of granzyme B, a high frequency of CD107a, and a low frequency of perforin. In some embodiments, the expanded NK cell composition and / or preparation comprises cells from at least two human donors who are HLA-type mismatched with the other donors and are HLA-type mismatched with the patient receiving the NK cells. In other embodiments, the expanded NK cell composition and / or preparation comprises cells that are HLA-type immunologically compatible with the subject. In still other embodiments, the expanded NK cell composition and / or preparation comprises cells that are at least partially immunologically compatible (mismatched) with the subject.

[0008] In some embodiments, in vitro or ex vivo methods for preparing NK cell compositions and / or preparations are provided. The methods typically include the steps of selecting multiple umbilical cord blood and / or placental blood units that are immunologically incompatible with each other; lysing or otherwise depleting red blood cells; depleting T cells; and CD34 + enrichment for hematopoietic stem and progenitor cells (HSPCs); followed by CD34 + culturing the enriched HSPCs in an expansion culture medium in the absence of feeder cells for a time sufficient to generate an expanded HSPC cell population, wherein the expanded HSPCs express CD56 during expansion. + and then, the expanded HSPC cell population is cultured in a differentiation culture medium containing cytokines in the absence of feeder cells, and the population is then cultured at approximately 50 to 80% CD56 + Cells and approximately 50% to 20% endogenous CD56 - In some embodiments, the NK cell composition and / or preparation comprises about 50% to up to about 85% CD56 + cells, and approximately 50% to 15% CD56 -In some embodiments, the NK cell compositions and / or preparations contain between about 55% and up to about 65% CD56 + cells, and approximately 45% to 35% CD56 - In some embodiments, the NK cell compositions and / or preparations contain between about 70% and up to about 85% CD56 + cells, and approximately 30% to 15% CD56 - Bone marrow-derived cells, including dendritic cells, macrophages, and granulocytes. CD56 + The subpopulation is primarily composed of NK cells. In addition to CD56, + The cells express high frequencies of NKp30, NKp46, NKp44, NKG2A and granzyme B, intermediate to high frequencies of perforin and CD107a, low to intermediate frequencies of NKG2D, and substantially no KIR expression. + Cells express KIR - (As used herein, "KIR - " refers to KIR2DL1, KIR2DS1, KIR2DS3, KIR2DS5, KIR2DL2, KIR2DL3, KIR2DS2, KIR2DS4, KIR3DL1 and KIR3DS1.) CD16 is expressed at low or intermediate frequencies. In some embodiments, CD56 - The cells express moderate to high frequencies of granzyme B, high frequencies of CD107a, and low frequencies of perforin.

[0009] In other embodiments, the in vitro or ex vivo method for preparing an NK cell composition and / or preparation includes selecting a fully immunologically matched or mismatched cord blood unit and / or placental blood unit. Immunological matching can be performed by any method known in the art. After selecting the cord blood and / or placental blood unit, the method includes lysing or otherwise depleting red blood cells; depleting T cells; and / or CD34 +enrichment for hematopoietic stem and progenitor cells (HSPCs); followed by CD34 + culturing the enriched HSPCs in an expansion culture medium in the absence of feeder cells for a time sufficient to generate an expanded HSPC cell population, wherein the expanded HSPCs express CD56 during expansion. + and then, the expanded HSPC cell population is cultured in a differentiation culture medium containing cytokines in the absence of feeder cells, and the population is then cultured at approximately 50 to 80% CD56 + Cells and approximately 50% to 20% endogenous CD56 - In some embodiments, the NK cell composition and / or preparation comprises about 50% to up to about 85% CD56 + cells, and approximately 50% to 15% CD56 - In some embodiments, the NK cell compositions and / or preparations contain between about 55% and up to about 65% CD56 + cells, and approximately 45% to 35% CD56 - In other embodiments, the NK cell compositions and / or preparations contain between about 70% and up to about 85% CD56 + cells, and approximately 30% to 15% CD56 - Endogenous bone marrow-derived cells, including dendritic cells, macrophages, and granulocytes. CD56 + The subpopulation is primarily composed of NK cells. In addition to CD56, + The cells express high frequencies of NKp30, NKp46, NKp44, NKG2A and granzyme B, intermediate to high frequencies of perforin and CD107a, low to intermediate frequencies of NKG2D, and substantially no KIR expression. + Cells express KIR - (As used herein, "KIR -" refers to KIR2DL1, KIR2DS1, KIR2DS3, KIR2DS5, KIR2DL2, KIR2DL3, KIR2DS2, KIR2DS4, KIR3DL1 and KIR3DS1.) CD16 may also be expressed at low or intermediate frequencies. In some embodiments, CD56 - The cells may express moderate to high frequencies of granzyme B, high frequencies of CD107a, and low frequencies of perforin.

[0010] In some embodiments, regardless of whether the HSPCs originate from unmatched, matched, or mismatched cord blood units, the HSPC expansion culture medium comprises interleukin-3 (IL-3), interleukin-6 (IL-6), thrombopoietin (TPO), Flt-3 ligand (Flt-3L), and stem cell factor (SCF) in a solid-phase tissue culture substrate coated with a Notch ligand and fibronectin or a fragment thereof. In some embodiments, the only growth factors added to the in vitro or ex vivo expansion culture medium for HSPCs are IL-3, IL-6, TPO, Flt-3L, and SCF. In some embodiments, the Notch ligand in the HSPC expansion culture medium is Delta1. ext-IgG In some embodiments, the fibronectin or fragment thereof is recombinant human fibronectin or a fragment thereof.

[0011] Differentiation of the expanded HSPCs herein involves their isolation and in vitro or ex vivo culture of the isolated HSPCs in cell culture medium supplemented with IL-2 and IL-15 to induce differentiation. The only cytokines added to the in vivo differentiation culture medium are IL-2 and IL-15. In some embodiments, the amount of IL-2 and IL-15 in the differentiation culture medium is about 25 U / ml to about 100 U / ml of IL-2 and about 25 ng / ml to about 50 ng / ml of IL-15. In other embodiments, the amount of IL-2 and IL-15 in the differentiation culture medium can be about 50 U / ml of IL-2 and about 40 ng / ml of IL-15. In some embodiments, the cytokines in the differentiation culture include IL-2 and IL-15, and other cytokines, such as Flt-3L, fibroblast growth factor 2 (FGF-2), IL-6, IL-7, IL-12, IL-3, GM-CSF, granulocyte colony-stimulating factor (G-CSF), leukemia inhibitory factor (LIF), macrophage inhibitory protein 1 alpha (MIP-1α), SCF, IL-21, IL-18, and 4-1BBL (4-1BB ligand), are not added to the differentiation culture medium. In some embodiments, the differentiation culture medium does not contain added cytokines other than the added IL-2 and IL-15 used to induce NK cell differentiation.

[0012] In certain embodiments of the present disclosure, IL-15 is used to prime the differentiation of NK cell compositions and / or preparations using CD34 +IL-15 is added during the expansion of enriched HSPCs. IL-15 may be added during the last 4 to 7 days of the expansion phase. If the expansion phase is reduced to 7 days, pre-stimulation with IL-15 may be added during the last approximately 4 days. Differentiation of the expanded, pre-stimulated HSPCs herein involves their isolation and in vitro or ex vivo culture of the isolated HSPCs in cell culture medium supplemented with IL-2 and IL-15 to induce differentiation. In some embodiments, the only cytokines added to the in vitro or ex vivo differentiation culture medium are IL-2 and IL-15. In some embodiments, the amounts of IL-2 and IL-15 in the differentiation culture medium are about 25 U / ml to about 100 U / ml of IL-2 and about 25 ng / ml to about 50 ng / ml of IL-15. In other embodiments, the amounts of IL-2 and IL-15 in the differentiation culture medium may be about 50 U / ml of IL-2 and about 40 ng / ml of IL-15. In some embodiments, the cytokines in the differentiation culture include IL-2 and IL-15, and other cytokines, such as Flt-3L, fibroblast growth factor 2 (FGF-2), IL-6, IL-7, IL-12, IL-3, GM-CSF, granulocyte colony-stimulating factor (G-CSF), leukemia inhibitory factor (LIF), macrophage inhibitory protein 1 alpha (MIP-1α), SCF, IL-21, IL-18, and 4-1BBL (4-1BB ligand), are not added to the differentiation culture medium. In some embodiments, the differentiation culture medium does not contain added cytokines other than the added IL-2 and IL-15 used to induce NK cell differentiation.

[0013] In certain embodiments of the method, the non-animal origin protein used to supplement the differentiation medium is human AB serum, fresh frozen human plasma, or human platelet lysate. Furthermore, in certain embodiments, the HSPCs are not derived from somatic cells, embryonic stem cells, peripheral blood mononuclear cells, or induced pluripotent stem cells.

[0014] In certain embodiments of the present disclosure, the methods produce NK cell compositions and / or preparations that contain less than 2% CD3+ cells, less than 2% CD19+ cells, and / or less than 2% CD34+ cells. + The cells further express a high frequency of KIR2DL4. In some embodiments, the methods include the use of a Notch ligand that is an antibody specific for DXI or Notch.

[0015] In certain embodiments, the method includes genetic modification of the NK cell composition and / or preparation. In certain specific embodiments, the genetic modification is performed during the expansion stage, while in others, the genetic modification is performed after NK cell differentiation. The cells of the NK cell composition and / or preparation can be genetically modified to express an antigen-recognition receptor. In certain specific embodiments, the genetic modification is the introduction of a polynucleotide expressing a T cell receptor (TCR) or a chimeric antigen receptor (CAR). The TCR or CAR can be designed to specifically bind to a viral antigen, a bacterial antigen, or a tumor-specific or tumor-associated antigen.

[0016] In certain embodiments of the disclosed methods, the viral antigen is present in cytomegalovirus (CMV), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), herpes simplex virus (HSV), hepatitis virus, Zika virus, influenza virus, or coronavirus. In specific embodiments, the herpes virus is HSV 1 or HSV 2, the hepatitis virus is hepatitis A, B, or C, and the coronavirus is SARS-CoV or SARS-CoV-2.

[0017] In certain other embodiments in which the CAR is directed to a tumor-specific or tumor-associated antigen, the antigen can be CD19, ROR1, Her2, PSMA, PSCA, mesothelin, or CD20.

[0018] The polynucleotide encoding the CAR may comprise an intracellular signaling domain comprising the signaling domains of CD3 zeta, CD28, and 4-1BB; at least one costimulatory domain comprising the costimulatory domain of CD27, CD28, 4-1BB, 2B4, DAP10, DAP12, OX40, CD30, CD40, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, or B7-H3; a transmembrane domain comprising the transmembrane domain of CD8, CD28, CD3 zeta, CD4, 4-1BB, OX40, ICOS, or NKG2D; and a spacer region comprising the hinge region of IgG1, the CH2CH3 region of an immunoglobulin, a portion of CD3, a portion of CD28, or a portion of CD8. In some embodiments, the CAR comprises a single-chain Fv (scFv) having the CDRs of the monoclonal antibody FMC63.

[0019] In certain embodiments, the NK cell compositions and preparations produced by the methods disclosed herein may be formulated for infusion into a subject.

[0020] In certain embodiments disclosed herein, natural killer (NK) cell compositions are produced for use in immunotherapy. The compositions contain about 50 to about 80% CD56 + Cells and approximately 50% to 20% endogenous CD56 - cells, or approximately 50 to 85% CD56 + Cells and approximately 50% to 15% endogenous CD56 - cells, + The cells express high frequencies of NKp30, NKp46, NKp44, NKG2A, NKG2D, and granzyme B, intermediate to high frequencies of perforin and CD107a, low to intermediate frequencies of CD16, and virtually no KIR; CD56 - The cells express moderate to high frequencies of granzyme B, high frequencies of CD107a, and low frequencies of perforin, CD56 + Cellular and endogenous CD56 - In a specific embodiment, the NK cell composition does not contain exogenous feeder cells.

[0021] In certain embodiments, the NK cell composition comprises less than 2% CD3 + cells and / or <2% CD19 + cells and / or less than 2% CD34 + In a specific embodiment, the NK cell composition comprises CD56 cells that further express a high frequency of KIR2DL4. + Contains cells.

[0022] The NK cell compositions described herein may include genetically modified cells. The genetically modified cells of the composition may be genetically modified to express an antigen-recognition receptor. These antigen-recognition receptors may be encoded by an introduced polynucleotide expressing a TCR or CAR. In certain specific embodiments, the TCR or CAR may specifically bind to a viral antigen, a bacterial antigen, or a tumor-associated or tumor-specific antigen. In more specific embodiments, the viral antigen is present in cytomegalovirus (CMV), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), herpes simplex virus (HSV), hepatitis virus, Zika virus, influenza virus, or coronavirus. In more preferred embodiments, the herpes virus is HSV 1 or HSV 2, the hepatitis virus is hepatitis A, B, or C, and the coronavirus is SARS-CoV or SARS-CoV-2.

[0023] In certain embodiments of NK cell compositions and / or preparations comprising a TCR or CAR, the TCR or CAR is a specific antigen for carbonic anhydrase IX (CA1X), carcinoembryonic antigen (CEA), CD8, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CLL1, CD34, CD38, CD41, CD44, CD49c, CD49f, CD56, CD66c, CD73, CD74, CD104, CD133, CD138, CD123, CD142, CD44V6, an antigen of a cytomegalovirus (CMV)-infected cell (e.g., a cell surface antigen), cutaneous lymphocyte-associated antigen (CLA; P-selectin glycoprotein ligand-1 (PSGL-1)), or a cytomegalovirus (CMV)-infected cell (e.g., a cell surface antigen). (particular glycoforms), epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine-protein kinase erb-B2, 3, 4 (erb-B2, 3, 4), folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-alpha, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER2), human telomerase reverse transcriptase (hTERT), interleukin-13 receptor subunit alpha-2 (IL-13Ralpha2), kappa light chain, kinase insert domain receptor (KDR), Lewis Y (LeY), L1 cell adhesion molecule (L1CAM), melanoma antigen family A, 1 (MAGE-A1), mucin 16 (MUC16), mucin 1 (MUC1), mesothelin (MSLN), ERBB2, MAGEA3, p53, MART1, GP100, proteinase 3 (PR1), tyrosinase, survivin, hTERT, EphA2, NKG2D ligand, cancer-testis antigen NY-ES0-1, oncofetal antigen (h5T4) ), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), ROR1, tetraspanin 8 (TSPAN8), tumor-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), BCMA, GPC3, NKCS1, EGF1R, EGFR-VIII, CRLF2, and ERBB.In more specific embodiments, the tumor-associated or tumor-specific antigen is CD19, ROR1, Her2, PSMA, PSCA, mesothelin, CRLF2, or CD20.

[0024] In certain embodiments in which NK cells comprise an antigen-recognizing receptor, the antigen-recognizing receptor can be a CAR. In more specific embodiments, the CAR can comprise the intracellular signaling domains of CD3 zeta, CD28, and 4-1BB; at least one costimulatory domain of CD27, CD28, 4-1BB, 2B4, DAP10, DAP12, OX40, CD30, CD40, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, or B7-H3 costimulatory domain; CD8, CD28, CD3 zeta, CD4, 4-1BB, OX40, ICOS, or NKG2D transmembrane domain; and the spacer region of IgG1, the CH2CH3 region of immunoglobulin, a portion of CD3, a portion of CD28, or a portion of CD8.

[0025] In certain embodiments, the NK cell composition may further comprise a cryoprotectant, and the NK cell composition is frozen for future use. In certain specific embodiments, the NK cell composition is formulated for infusion into a subject. The formulation may include a cryoprotectant used to store the composition. The NK cell composition may comprise about 50 million to about 2 billion viable cells. In certain embodiments, the NK cell composition comprises about 50 million to about 2 billion viable CD56 cells. + It may contain cells.

[0026] The present disclosure also provides a method of treating a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the NK cell composition described above. The subject may have, for example, a cancer that expresses a tumor antigen, and the NK cells of the composition express an antigen-recognition receptor that binds to the tumor antigen. In other embodiments, the subject has a viral or bacterial infection. In certain embodiments, the NK cell composition expresses a chimeric antigen receptor specific for a tumor-specific or tumor-associated antigen, a viral antigen, or a bacterial antigen.

[0027] The foregoing aspects and many of the attendant advantages of the present methods and compositions and / or preparations will become more readily appreciated as the same become better understood by reference to the following detailed description when taken in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0028] [Figure 1] Figures 1A and 1B show results from 20 batches of NK cell compositions and / or preparations produced according to the process described in Example 1. Figure 1A shows the increase in CD56+ cells / starting CD34+ cells over 28 days of culture (stages 1 and 2). Figure 1B demonstrates the reproducibility of CD56+ cell generation over the 28-day cell culture process. [Figure 2] FIG. 2 shows that substitution of fetal bovine serum (FBS) with human AB serum (hABS) or human platelet lysate (hPL) had minimal effect on cell proliferation during differentiation. [Figure 3] FIG. 3 shows that substitution of FBS with human AB serum (hABS) or human platelet lysate (hPL) had a minimal effect on the percentage of CD56+ cells generated. [Figure 4] FIG. 4 shows the percentage of CD16+ cells compared to CD56+ NK cells at day 28 of culture, comparing different serum supplements used in stage 2 (differentiation stage) of culture. [Figure 5] FIG. 5 shows the percentage of NKp46+ cells compared to CD56+ NK cells at day 28 of culture, comparing the different serum supplements used in stage 2 of the culture. [Figure 6] FIG. 6 shows that cultures supplemented with hPL generated a larger population of cytotoxic NK cells than those supplemented with hABS. [Figure 7] FIG. 7 shows that cultures supplemented with hPL generated a larger population of cytotoxic NK cells than those supplemented with hABS. [Figure 8]FIG. 8 shows that the NK cell compositions and / or preparations (referred to as NK cell products) were active against K562 and A549 target cells with similar activity (curves indicated by filled circles and triangles in the center of the graph) and were more potent than adult NK cells activated against A549 target cells. [Figure 9] Figure 9 shows that NK cell compositions and / or preparations demonstrated cell killing against many of the cancer cell lines tested. For each cell line, the left bar indicates cytotoxicity at 4 hours, and the right bar indicates cytotoxicity at 24 hours. The K562 cell line is recognized as a standard for NK cell activity and served as a positive control. [Figure 10] FIG. 10 demonstrates that the NK cell product was able to reduce tumor burden compared to saline-injected controls. [Figure 11] Figures 11A and 11B show tumor responses in an in vivo mouse model. Figure 11A is a graph of tumor size over time for the study for the untreated control (line with circles) and for the NK cell composition and / or preparation (line with squares). The NK cell composition was able to cause a significant delay in tumor progression. Figure 11B shows that the NK cell composition persisted and was detectable 37 days after injection, as measured by detecting human CD45+ cells in the tumor. [Figure 12-1] Figures 12A-12D show that proliferation of HSPCs with Notch 1 antibody (see U.S. Patent Application Publication No. 2017 / 0107493; incorporated herein by reference) was comparable to proliferation using DXI. [Figure 12-2] Figures 12A-12D show that proliferation of HSPCs with Notch 1 antibody (see U.S. Patent Application Publication No. 2017 / 0107493; incorporated herein by reference) was comparable to proliferation using DXI. [Figure 13]Figures 13A and 13B show that NK cell compositions and / or preparations (referred to as NK cell products), K562 or A549 tumor cells were cultured alone or together for three days. The study was performed in triplicate. After three days, cytokine levels were determined by Luminex assay (the detection limit of the assay used was 1 pg / ml of cytokine). Both IFNγ (Figure 13A) and TNFα (Figure 13B) levels increased when the NK cell compositions and / or preparations were co-cultured with tumor cells. These results indicated that the NK cells in the preparations became more active in the presence of tumor cells. [Figure 14-1] Figures 14A-14E show that a more consistent distribution of transgene expression across cell lines was observed at 1, 4, 7, 14 or 21 days post-transduction. [Figure 14-2] Figures 14A-14E show that a more consistent distribution of transgene expression across cell lines was observed at 1, 4, 7, 14 or 21 days post-transduction. [Figure 14-3] Figures 14A-14E show that a more consistent distribution of transgene expression across cell lines was observed at 1, 4, 7, 14 or 21 days post-transduction. [Figure 15] Figure 15 shows that the expression construct contained tEGFR as a selectable marker. S = signal sequence from GM-CSF R; VL = variable light chain; L = linker; VH = variable heavy chain; H = hinge spacer from human IgG4; TM = CD28 or NKG2D transmembrane domain; 4-1BB = intracellular signaling domain; CD3 zeta = intracellular signaling domain; 2A = self-cleaving peptide. [Figure 16] Figure 16 shows that the expression construct contained human IL-15 for NK cell support. S = signal sequence from GM-CSF R; VL = variable light chain; L = linker; VH = variable heavy chain; H = hinge spacer from human IgG4; TM = CD28 or NKG2D transmembrane domain; 4-1BB = intracellular signaling domain; CD3 zeta = intracellular signaling domain; 2A = self-cleaving peptide. [Figure 17] Figures 17A and 17B show that IL-15 added during the expansion phase to prime NK cell differentiation results in a significant increase in CD56+ cells, and that the process is reproducible. The figures show results from four batches of NK cell preparations produced at manufacturing scale. Figure 17A shows the increase in CD56+ cells / starting CD34+ cells over 24 days of culture (stages 1 and 2). Figure 17B demonstrates the reproducibility of CD56+ cell generation over the 24-day cell culture process. [Figure 18-1] Figures 18A-18C show the ability of NK cell preparations to undergo repeated doses of target cells for continuous killing over an extended period of time. Figure 18A is a fresh NK cell preparation. Figure 18B is a cryopreserved and thawed NK cell preparation, and Figure 18C is an assay in which the fresh NK cell preparation received repeated doses of target cells every 3 days for a total of 10 days. [Figure 18-2] Figures 18A-18C show the ability of NK cell preparations to undergo repeated doses of target cells for continuous killing over an extended period of time. Figure 18A is a fresh NK cell preparation. Figure 18B is a cryopreserved and thawed NK cell preparation, and Figure 18C is an assay in which the fresh NK cell preparation received repeated doses of target cells every 3 days for a total of 10 days. [Figure 19] Figure 19 shows the activity of NK cell preparations in the Kasumi-1 AML diffuse tumor model. The model was tested in NSG mice with or without NK cell preparation treatment by measuring Kasumi-1 tumor burden via bioluminescence imaging. Compared to buffer-injected control treatment, the NK cell preparation significantly inhibited tumor progression. [Figure 20] Figure 20 shows survival of NSG mice in the Kasumi 1 AML model used in Figure 19. The NK cell preparation significantly extended median survival in mice compared to mice injected with a buffer control. [Figure 21]Figure 21 shows flow cytometry-based detection of tCD19 transgene expression as an indirect measure of CAR expression in transduced CD56+ and CD56- cells. The plot shows the frequency of CD56 and tCD19 expression in cells on day 28 of culture after transduction with a lentiviral vector expressing a mesothelin-targeted CAR and a truncated CD19 (tCD19) extracellular domain driven by the EF1 alpha promoter. Bulk cells were transduced twice, 3 hours apart, on day 7 of growth, using an MOI of 30. [Figure 22] Figure 22 shows the cytotoxic activity of MSLN CAR-NK cells or control NK cells against NOMO-1 or NOMO-1MSLN- / - AML tumor cells in a 24-hour in vitro cytotoxicity assay. There was a significant and specific increase in tumor cell killing by MSLN CAR-NK cells against MSLN-expressing NOMO-1 cells, but not against NOMO-1MSLN- / - tumor cells. No difference in killing was observed with control NK cells versus NOMO-1+ / -MSLN knockout cells. [Figure 23] Figure 23 shows flow cytometry-based detection of tCD19 transgene expression as an indirect measure of CAR expression in enriched CD56+ cells. The plot shows the frequency of CD56 and tCD19 expression (Tx+) in cells on day 27 of culture after transduction with a lentiviral vector expressing a mesothelin-targeted CAR and a truncated CD19 (tCD19) extracellular domain driven by the EF1 alpha promoter. CD56+ cells were enriched by magnetic bead separation and transduced using an MOI of 40 on day 23 of differentiation culture. [Figure 24]Figure 24 shows the cytotoxic activity of CAR-NK cells or control NK cells against NOMO-1 or NOMO-1MSLN- / - cells in a 24-hour in vitro cytotoxicity assay. There was a significant and specific increase in tumor cell killing by MSLN CAR-NK cells against MSLN-expressing NOMO-1 cells, but not against NOMO-1MSLN- / - cells. No difference in killing was observed with control NK cells versus NOMO-1+ / -MSLN knockout cells. DETAILED DESCRIPTION OF THE INVENTION

[0029] Detailed Description of Specific Embodiments Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present cell compositions and / or preparations, certain preferred methods and materials are described. For purposes of this disclosure, the following terms are defined below.

[0030] As used herein, "expanded HSPCs" refers to hematopoietic stem or stem and progenitor cells that have been subjected to techniques for expanding hematopoietic stem or stem and progenitor cells ex vivo, which have been shown to result in either (i) an increased number of hematopoietic stem or stem and progenitor cells in an aliquot of cells so expanded, or (ii) an increased number of SCID-repopulating cells as determined by limiting dilution analysis, as indicated by enhanced engraftment in non-obese diabetic (NOD) / severe combined immunodeficiency (SCID) mice injected with an aliquot of cells so expanded, relative to the number not seen in an aliquot of cells not subjected to the expansion technique (U.S. Patent Application Publication No. 2013 / 0095079; Delaney et al., Nature Med. 16(2):232-236, 2010). Typically, hematopoietic stem cells or stem and progenitor cells express CD34 +In some embodiments, the hematopoietic stem cells or hematopoietic stem and progenitor cells are derived from human umbilical cord blood and / or human placental blood. In some embodiments, the expanded stem cells are prepared using a Notch agonist expansion method. In some embodiments, the expanded stem cells are prepared using a Delta1 ext-IgG (DXI) prepared using the proliferation method.

[0031] As used herein, "chemotherapeutic regimen" refers to the regimen for chemotherapy, which defines the drugs used, their dosage, frequency and duration of treatment, and other considerations.Such regimen can combine several chemotherapeutic drugs that are combined with chemotherapy.Most of the drugs used in chemotherapy today are cytostatic or cytotoxic.

[0032] The present disclosure provides compositions comprising natural killer (NK) cell preparations and methods of using such NK cell compositions and / or preparations for immunotherapy. The NK cell compositions and / or preparations can be used in the treatment of a wide range of viral infections, malignancies, and other diseases, including hematological and non-hematological malignancies. The NK cell compositions and / or preparations can be pooled, expanded, or otherwise treated with CD34 NK cells. + They are generated from hematopoietic stem and progenitor cells (HSPCs). Typically, HSPCs from at least two or more, or up to at least four different human donors are combined or pooled before or after ex vivo expansion to generate an expanded HSPC cell population. The expanded cell population is then differentiated ex vivo to express CD56 + and CD56 - An NK cell composition and / or preparation is produced that comprises a mixture of cells. + The cells are predominantly NK cells. CD56 - CD56 cells are typically of myeloid origin and support NK cell differentiation and activation during differentiation. +CD56 generated during ex vivo generation of - The cells were CD56 + It is thought to be endogenous to the cell.

[0033] In some embodiments, the NK cell compositions and / or preparations comprise from about 50% to up to about 80% CD56 + cells, and approximately 50% to approximately 20% CD56 - In some embodiments, the NK cell compositions and / or preparations contain between about 50% and up to about 85% CD56 + cells, and approximately 50% to 15% CD56 - In some embodiments, the NK cell compositions and / or preparations contain between about 55% and up to about 65% CD56 + cells, and approximately 45% to 35% CD56 - In some embodiments, the NK cell compositions and / or preparations contain between about 70% and up to about 85% CD56 + cells, and approximately 30% to 15% CD56 - Endogenous bone marrow-derived cells include, for example, dendritic cells, macrophages, and granulocytes.

[0034] In addition to CD56, + The cells may express high frequencies of the natural killer cell proteins NKp30, NKp46, NKp44, and NKG2A, and granzyme B; intermediate to high frequencies of perforin and CD107a; low to intermediate frequencies of NKG2D, and substantially no KIR expression. In some embodiments, CD56 + Cells express KIR - (As used herein, "KIR -" refers to KIR2DL1, KIR2DS1, KIR2DS3, KIR2DS5, KIR2DL2, KIR2DL3, KIR2DS2, KIR2DS4, KIR3DL1 and KIR3DS1.) In some embodiments, CD16 is expressed at low frequency. In some embodiments, CD16 is expressed at intermediate frequency. In some embodiments, CD56 - The cells express moderate to high frequencies of granzyme B, high frequencies of CD107a, and low frequencies of perforin.

[0035] In some embodiments, the NK cell compositions and / or preparations produced by the methods described herein have the following characteristics:

[0036] The frequency of expression of various NK markers is shown in the table below. [Table 1]

[0037] High expression refers to a frequency of about 60 to about 100%. Moderate expression refers to a frequency of about 20 to about 60%. Low expression refers to a frequency of about 1 to about 20%. Substantially none refers to an expression frequency of less than 1%. As used herein, "KIR - " refers to KIR2DL1, KIR2DS1, KIR2DS3, KIR2DS5, KIR2DL2, KIR2DL3, KIR2DS2, KIR2DS4, KIR3DL1 and KIR3DS1.

[0038] In some embodiments, CD56 + Cells and CD56 - The range of markers for the cells are as listed in Tables 2 and 3. [Table 2] [Table 3]

[0039] In some embodiments, the NK cell composition and / or preparation comprises less than 2% CD3 + In some embodiments, the NK cell compositions and / or preparations contain less than 1% CD3 + In some embodiments, the NK cell composition and / or preparation contains less than 2% CD19 cells (T cells). + In some embodiments, the NK cell compositions and / or preparations contain less than 1% CD19 cells (B cells). + In some embodiments, the NK cell compositions and / or preparations contain less than 2% CD34 cells (B cells). + In some embodiments, the NK cell compositions and / or preparations contain less than 1% CD34 cells. + In some embodiments, the NK cell composition and / or preparation contains less than 2% CD19 + cells (B cells), less than 2% CD3 + cells (T cells) and less than 2% CD34 + In some embodiments, the NK cell composition and / or preparation contains less than 1% CD19 + cells, less than 1% CD3 + cells (T cells) and less than 1% CD34 + Contains HSPCs.

[0040] In humans, NK cells are regulated by clonally distributed killer immunoglobulin-like receptors (KIRs) that recognize allotypic determinants presented by distinct human leukocyte antigen (HLA) class I alleles. Inhibitory KIRs are generally dominant and prevent NK cells from killing autologous cells. NK cell compositions and / or preparations prepared by this method lack MHC class I inhibitory signals. Although expressing high levels of NKG2A, NK cell compositions and / or preparations may be more active in vitro than adult NK cells.

[0041] The receptor phenotype of the NK cell compositions and / or preparations produced by the methods disclosed herein is CD56+ The NK cell compositions and / or preparations may further comprise high expression of the natural cytotoxicity receptors NKp30, NKp44, and NKp46, while having low to moderate expression of NKG2D by the cells. + The cells can be further identified by low to moderate CD16 expression.

[0042] The differentiated NK cells of the present disclosure are further characterized by their ability to lyse a variety of tumor cell lines at levels similar to or greater than activated peripheral blood NK cells (as further described in the Examples). Thus, the cytotoxic capacity of cells in culture can be measured to validate the characteristics of NK cell compositions and / or preparations.

[0043] The preparation of ex vivo differentiated NK cells described in the methods of the present disclosure involves two separate steps, a first step involving the expansion of HSPCs to generate expanded HSPCs, followed by a second step during which the expanded HSPCs differentiate to form the NK cell composition and / or preparation. In contrast to the prior art, feeder cells are not used in either step.

[0044] Expanded HSPCs include hematopoietic stem or stem and progenitor cells that have been expanded ex vivo (e.g., with a Notch ligand) and depleted of both T cells and erythrocytes. Expanded HSPCs typically express CD34 + Hematopoietic stem cells or stem and progenitor cells, typically derived from different human sources that are not HLA-matched. In some embodiments, the expanded HSPCs are derived from different human umbilical cord blood and / or placental blood sources, CD34 + Hematopoietic stem cells or stem and progenitor cells of multiple HLA types. Hematopoietic stem cells or stem and progenitor cells are not matched to each other before the HSPCs are pooled, and therefore contain multiple HLA types. As used herein, depleted T cells refer to cells that contain less than 2% CD3 + cells (T cells), or less than 1% CD3 +cells (T cells), or less than 0.5% CD3 + cells (T cells), or less than 0.1% CD3 + This refers to cells (T cells).

[0045] In certain embodiments, the hematopoietic stem cells or hematopoietic stem and progenitor cells are immunologically compatible with the patient receiving the NK cell composition and / or preparation as immunotherapy. Immunological compatibility can be a perfect match or a partial mismatch of up to two, three, or sometimes four immune types. Most compositions containing hematopoietic stem cells or hematopoietic stem and progenitor cells are compatible with the most common HLA antigens, such as HLA A-2, HLA-B7, etc., or with frequent combinations of HLA linkages. The choice of match is selected depending on the patient population to be treated.

[0046] In some embodiments, CD34 + Hematopoietic stem cells or hematopoietic stem and progenitor cells are derived from umbilical cord blood and / or placental blood (human umbilical cord blood or human placental blood). Such blood can be obtained by methods known in the art. See, for example, U.S. Patent Nos. 5,004,681 and 7,147,626, and U.S. Patent Application Publication No. 2013 / 0095079, for a discussion of collecting umbilical cord blood and placental blood at human birth. Collection of umbilical cord blood and / or human placental blood is performed under sterile conditions. Upon collection, the umbilical cord blood and / or placental blood is treated with an anticoagulant, such as CPD (citrate-phosphate-dextrose), ACD (acid citrate dextrose), Alsever solution (Alsever et al., NY St. J. Med. 41:126, 1941), De Gowin solution (De Gowin, et al., J. Am. Med. Ass. 114:850, 1940), Edglugate- Mg (Smith, et al., J. Thorac. Cardiovasc. Surg. 38:573, 1959), Rous-Turner solution (Rous and Turner, J. Exp. Med. 23:219, 1916), and other It can be mixed with glucose mixtures, heparin, ethyl biscoumarate, etc. See generally Hurn, Storage of Blood, Academic Press, New York, pp. 26-160, 1968. In one embodiment, ACD can be used.

[0047] Umbilical cord blood can be obtained by direct drainage from the umbilical cord and / or by needle aspiration from the placenta delivered by the root and swollen veins. Preferably, the collected human umbilical cord blood and / or placental blood is free of contaminants (e.g., bacteria or viruses), particularly viral contamination.

[0048] Prior to collection of cord blood, the maternal medical history can be determined to identify risks that cord blood cells may pose, for example, transmission of genetic or infectious diseases such as cancer, leukemia, immune disorders, neurological disorders, hepatitis, or HIV / AIDS. Collected cord blood can be analyzed for, for example, cell viability, HLA typing, ABO / Rh typing, CD34 + One or more of a cell count and a total nucleated cell count may be tested.

[0049] Once umbilical cord blood and / or placental blood is collected from a human donor at birth, the blood is processed to generate enriched HSPCs. Preferably, the HSPCs are CD34 + cells or mostly CD34 + HSPCs are typically depleted of T cells and red blood cells to produce enriched HSPCs. As used herein, depletion of T cells refers to the depletion of T cells to less than about 2% of CD3 + cells, approximately less than 1% CD3 + cells, or less than about 0.5% CD3 + cells, or less than about 0.1% CD3 + Enrichment refers to the presence of HSPCs in a population of cells. Thus, enrichment refers to a process by which the percentage of HSPCs in a population of cells is increased (compared to the percentage in the population before the enrichment procedure). Purification is an example of enrichment.

[0050] Before processing for enrichment, collected umbilical cord blood and / or placental blood can be fresh or previously cryopreserved. Enrichment for HSPCs can be performed using any suitable cell separation / selection technique known in the art. Methods that rely on the differential expression of cell surface markers can be used. For example, cells expressing the cell surface marker CD34 can be positively selected using a monoclonal antibody specific to CD34, resulting in the separation of CD34-expressing cells from cells that do not express CD34. Furthermore, the separation technique used preferably maximizes the viability of the selected cells. The particular technique used depends on the efficiency of separation, cytotoxicity methodology, ease and speed of performance, and the need for sophisticated equipment and / or technical skill.

[0051] Procedures for separation may include magnetic separation using antibody-coated magnetic beads, affinity chromatography, and "panning" with antibodies bound to a solid matrix, e.g., a plate, or another convenient technique. Techniques that provide precise separation / selection include fluorescence-activated cell sorters, which may have varying degrees of sophistication, e.g., multiple color channels, low-angle and obtuse-angle light scattering detection channels, impedance channels, etc.

[0052] The antibodies used in the selection process allow for easy separation of specific cell types and can be conjugated to markers, such as magnetic beads that allow direct separation, biotin that allows removal by binding to avidin or streptavidin bound to a support, fluorescent dyes that can be used with a fluorescence activated cell sorter, etc. Any technique can be used that is not unduly detrimental to the viability of the remaining cells.

[0053] In a preferred embodiment, fresh or frozen and thawed cord blood units are subjected to CD34 isolation using anti-CD34 antibodies directly or indirectly conjugated to magnetic particles in conjunction with a magnetic cell separation device, for example, the CliniMACS® Cell Separation System (Miltenyi Biotec, Bergisch Gladbach, Germany), which uses nano-sized superparamagnetic particles composed of iron oxide and dextran coupled to specific monoclonal antibodies. + The CliniMACS® Cell Separator is a closed, sterile system equipped with a single-use disposable tubing set. The disposable tubing set contains CD34 + It can be used to process a single unit of umbilical cord blood and / or placental blood collected to enrich for HSPCs and then discarded.

[0054] In a typical embodiment, two or more, or up to at least four or more, cord blood and / or placental blood units can be pooled before enrichment for HSPCs. + Individual populations of HSPCs can be pooled after being enriched for HSPCs. In specific embodiments, the number of pooled cord blood and / or placental blood units or HSPC populations is 2, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, or 40, or at least some of the foregoing numbers. The number of individual pooled HSPC populations can depend, for example, on the number of cells in each individual population and / or the number of cells required for a particular patient and intended immunotherapy. In some embodiments, the pool contains 2 to 8, 4 to 8, 2 to 10, 4 to 10, 4 to 20, or 4 to 25 cord blood and / or placental blood units, or CD34 +The pool contains a population of HSPCs. In typical embodiments, umbilical cord blood and / or placental blood units, or hematopoietic stem or stem and progenitor cell populations, can be pooled regardless of the HLA type of the HSPCs. In some embodiments, the cells in the pool are combined regardless of race or ethnicity. In some embodiments, the cells in the pool are derived from the umbilical cord blood and / or placental blood of individuals of the same race, such as African American, Caucasian, Asian, Hispanic, Native American, Australian Aborigine, Inuit, or Pacific Islander, or from the umbilical cord blood and / or placental blood of individuals of the same ethnicity, such as Irish, Italian, Indian, Japanese, Chinese, or Russian. In less common embodiments, the cells can be derived from individual cell populations that are matched or partially matched with the patient. If there are a sufficient number of matched or partially matched units available, the units can be pooled; however, given that finding a unit that matches a specific patient is sometimes a difficult process, pooling is usually not performed for matched or mismatched units.

[0055] Typically, before enrichment for HSPCs, umbilical cord blood and / or placental blood are separated from red blood cells and white blood cells. In some embodiments, red blood cell depletion refers to the separation of red blood cells from white blood cells. After red blood cell and white blood cell separation, the red blood cell fraction is discarded, and the white blood cell fraction is separated from the red blood cell fraction by, for example, CD34 +As described above, the cells can be processed in a magnetic cell separator to enrich for HSPCs. Separation of the white blood cell and red blood cell fractions can be performed by any method known in the art, including, for example, centrifugation techniques. Other separation methods that can be used include the use of commercially available products such as FICOLL™, FICOLL-PAQUE™, or PERCOLL™ (GE Healthcare, Piscataway, New Jersey). FICOLL-PAQUE™ is usually placed at the bottom of a conical tube, and whole blood is layered on top. After centrifugation, the following layers are visible in the conical tube from top to bottom: plasma and other constituents, a layer of mononuclear cells called the buffy coat containing mononuclear cells (white blood cells), and red blood cells and granulocytes, which should be present in pellet form. This separation technique allows for easy collection of mononuclear cells.

[0056] Optionally, CD34 + Prior to cell selection, aliquots of cord blood and / or placental units were analyzed for total nucleated cell count and / or CD34 + Cell counts can be checked. In a specific embodiment, CD34 + After cell selection, CD34 + and CD34 - Both cell fractions are collected. If necessary, DNA and CD34 + Even though HLA matching of cells with other cord blood and / or placental blood cells is not typically performed, CD34 cells are used for initial HLA typing and further chimerism studies. - It can be extracted from a sample of a cellular fraction.

[0057] Then, CD34 + The enriched HSPCs can be treated prior to expansion, for example, by suspension in a cell culture medium suitable for storage or transport. In a preferred embodiment, the cell culture medium is enriched for CD34 +The cell culture medium is suitable for maintaining the viability of HSPCs. For example, the cell culture medium can be a serum-free, cytokine-free hematopoietic stem cell or stem and progenitor cell culture medium supplemented with growth factors, for example, at the following concentrations: 50-300 ng / ml stem cell factor (SCF), 50-300 ng / ml Flt-3 receptor ligand (Flt3L), 50-100 ng / ml thrombopoietin (TPO), 50-100 ng / ml interleukin-6 (IL-6), and 10 ng / ml interleukin-3 (IL-3). In more specific embodiments, the cell culture medium contains 300 ng / ml stem cell factor, 300 ng / ml Flt-3 receptor ligand, 100 ng / ml TPO, 100 ng / ml IL-6, and 10 ng / ml IL-3; or 50 ng / ml SCF, 50 ng / ml Flt-3L, 50 ng / ml TPO, 50 ng / ml IL-6, and 10 ng / ml IL-3. In another preferred embodiment, the cell culture medium comprises, or alternatively consists of, serum-free hematopoietic stem cell or stem and progenitor cell culture medium (e.g., STEMSPAN™ Serum-Free Growth Medium or STEMSPAN™ Serum-Free Growth Medium II (StemCell Technologies, Vancouver, British Columbia)) supplemented with 10 ng / ml recombinant human interleukin-3 (rhIL-3), 50 ng / ml recombinant human interleukin-6 (rhIL-6), 50 ng / ml recombinant human thrombopoietin (rhTPO), 50 ng / ml recombinant human Flt-3 Ligand (rhFlt-3L), and 50 ng / ml recombinant human stem cell factor (rhSCF). In another preferred embodiment, the cell culture medium consists of serum-free hematopoietic stem cell or stem and progenitor cell culture medium (e.g., StemSpan Serum-Free Growth Medium II (SFEM II, StemCell Technologies, Vancouver, British Columbia)) supplemented with recombinant human rhSCF, rhFlt-3L, rhTPO, rhIL-6 (each at a final concentration of 50 ng / ml), and rhIL-3 (at a final concentration of 10 ng / ml).

[0058] In a specific embodiment, the cord blood and / or placental blood unit is depleted of red blood cells and CD34 in the red blood cell-depleted fraction is detected. + In some embodiments, red blood cell depletion is performed by separating red blood cells from white blood cells or by detecting CD34 + It refers to the separation of red blood cells from cells. Preferably, more than 3.5 million CD34 + The cell-containing cord blood and / or placental blood unit is subjected to the enrichment methods described above.

[0059] After HSPCs are isolated according to the enrichment methods described above or other methods known in the art (e.g., from human umbilical cord blood and / or human placental blood collected from humans at birth), the enriched HSPCs can be expanded to identify HSPCs, e.g., CD34 + Increasing the number of HSPCs. HSPCs are cultured in an expansion culture medium under cell growth conditions (e.g., promoting mitosis), so that the HSPCs grow and divide (proliferate) to produce expanded CD34 + A population of HSPCs is obtained. During the expansion of HSPCs, minimal differentiation of HSPCs into NK cells occurs (i.e., less than 2% or less than 1% of the obtained cells are NK cells). In one embodiment, individual populations of HSPCs derived from the umbilical cord blood and / or placental blood of a single human at birth can be pooled without matching the HLA type of other HSPCs before or after expansion. In another embodiment, HSPCs are expanded before pooling. Preferably, the technique used for expansion results in a higher concentration of hematopoietic stem cells or hematopoietic stem and progenitor cells, e.g., CD34, in the expanded HSPCs compared to the non-expanded population of HSPCs. + It has been shown to result in an increase in the number of cells, where the unexpanded and expanded cell populations are derived from different aliquots of the same source of HSPCs, where the expanded HSPCs but not the unexpanded HSPCs are subjected to the expansion technique.

[0060] Propagation techniques include, but are not limited to, those described in U.S. Patent No. 7,399,633 B2; U.S. Patent Application Publication No. 2013 / 0095079; Delaney et al., Nature Med. 16(2): 232-236, 2010 (hereby incorporated by reference); and those described below.

[0061] In some embodiments, HSPCs are cultured in vitro or ex vivo in an expansion culture medium that is serum-free and suitable for culturing hematopoietic stem cells or stem and progenitor cells, in the presence of growth factors, and exposed to cell growth conditions (e.g., that promote mitosis), such that the HSPCs expand to generate an expanded population of HSPCs.

[0062] In an exemplary embodiment, a suitable growth culture medium for expanding hematopoietic stem or stem and progenitor cells is a serum-free culture medium such as Iscove's MDM containing non-animal-origin BSA, recombinant human insulin, human transferrin, 2-mercaptoethanol, and other supplements, along with Notch ligands and growth factors, as described below. In other embodiments, the hematopoietic stem or stem and progenitor cell culture medium is STEMSPAN™ Serum-Free Growth Medium (StemCell Technologies, Vancouver, British Columbia) or STEMSPAN™ Serum-Free Growth Medium II (StemCell Technologies, Vancouver, British Columbia).

[0063] In some embodiments, HSPCs are cultured in an expansion culture medium in the presence of an amount of a Notch ligand (i.e., an agonist of Notch function effective to inhibit differentiation), typically a fixed agonist of Notch function, and exposed to cell growth conditions (e.g., promoting mitosis) so that the HSPCs proliferate and generate a population of expanded HSPCs. Differentiation of HSPCs into NK cells is minimized during the culture step (i.e., less than 2% or less than 1% of the resulting cells are NK cells). In a more preferred embodiment, the expansion culture medium contains an agonist of Notch function and a growth factor effective to inhibit differentiation, and the HSPCs are exposed to cell growth conditions (e.g., promoting mitosis) so that the hematopoietic stem or stem and progenitor cells proliferate and generate a population of expanded hematopoietic stem or stem and progenitor cells. The expanded population of hematopoietic stem or stem and progenitor cells is typically transferred to a differentiation cell culture medium after expansion. Optionally, the Notch ligand is inactivated or removed from the expanded HSPC cell population (eg, by separation or dilution) prior to the differentiation step.

[0064] In some embodiments, hematopoietic stem or stem and progenitor cells are cultured for expansion for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days or longer. Alternatively, preferably, hematopoietic stem or stem and progenitor cells are cultured for expansion for at least 10 days, or for about 7 to about 14 days. In some embodiments, hematopoietic stem or stem and progenitor cells are cultured for about 7 days, about 14 days, about 13 days, or about 15 days. It should be noted that the duration of expansion will be determined by the ability to achieve either the desired or maximum cell number while maintaining the immunophenotypic properties / characteristics of stem and progenitor cells necessary for generating the desired cell preparation. Expansion is stopped when cell proliferation ceases, viability decreases, or the cells begin to substantially lose HSPC properties / characteristics.

[0065] Exemplary ex vivo culture conditions for expanding hematopoietic stem or stem and progenitor cells include culturing cells in a culture medium containing a fibronectin fragment and the extracellular domain of the Delta protein fused to the Fc domain of human IgG (Delta1). ext-IgG The method comprises culturing the cells for 7 to 14 days in a serum-free growth culture medium supplemented with the following human growth factors: SCF, Flt-3L, TPO, IL-6, and IL-3. Preferably, the growth factors are present at the following concentrations: 50 to 300 ng / ml SCF, 50 to 300 ng / ml Flt-3L, 50 to 100 ng / ml TPO, 50 to 100 ng / ml IL-6, and 10 ng / ml IL-3. In a more specific embodiment, 300 ng / ml SCF, 300 ng / ml Flt-3L, 100 ng / ml TPO, 100 ng / ml IL-6, and 10 ng / ml IL-3, or 50 ng / ml SCF, 50 ng / ml Flt-3L, 50 ng / ml TPO, 50 ng / ml IL-6, and 10 ng / ml IL-3, are used. In a more preferred embodiment, the expansion culture medium (e.g., STEMSPAN™ Serum-Free Expansion Medium (StemCell Technologies, Vancouver, British Columbia)) contains or consists of 10 ng / ml recombinant human interleukin-3 (rhIL-3), 50 ng / ml rhIL-6, 50 ng / ml rhTPO, 50 ng / ml rhFlt-3L, and 50 ng / ml rhSCF. In another more preferred embodiment, the expansion culture medium (e.g., StemSpan™ Serum-Free Expansion Medium II (SFEM II, StemCell Technologies, Vancouver, British Columbia)) contains or consists of rhSCF, rhFlt-3L, rhTPO, rhIL-6 (each at a final concentration of 50 ng / ml), and rhIL-3 (at a final concentration of 10 ng / ml).

[0066] In some embodiments, the Notch ligand is DXI (Delta1 ext-IgG ) and the propagation steps are carried out as follows: Delta1 ext-IgG(DXI) is immobilized on the surface of a cell culture dish. In a specific embodiment, the cell culture dish is immobilized with 2.5 μg / ml of Delta1 in phosphate-buffered saline prior to the addition of enriched hematopoietic stem or stem and progenitor cells. ext-IgG and 5 μg / ml RetroNectin® (a recombinant human fibronectin fragment also called rFN-CH-296) overnight at 4° C. (or at least 2 hours at 37° C.) Preferably, the growth culture medium (e.g., STEMSPAN™ Serum-Free Growth Medium or StemSpan™ Serum-Free Growth Medium II (StemCell Technologies, Vancouver, British Columbia)) is supplemented with 10 ng / ml rhIL-3, 50 ng / ml rhIL-6, 50 ng / ml rhTPO, 50 ng / ml rhFlt-3L, and 50 ng / ml rhSCF.

[0067] In some embodiments, the expansion culture medium does not contain growth factors other than rhIL-3, rhIL-6, rhTPO, rhFlt-3L, and rhSCF. In some embodiments, the expansion culture medium does not contain the following added growth factors or cytokines: IL-7, GM-CSF, G-CSF, LIF, MIP-1a, IL-2, or IL-15. In some embodiments, the expansion culture medium does not contain the following added growth factors or cytokines: IL-7, GM-CSF, G-CSF, LIF, MIP-1a, or IL-2. In some embodiments, the expansion culture medium does not contain an aryl hydrocarbon receptor antagonist, such as those described in U.S. Pat. No. 9,175,266 or U.S. Patent Application Publication No. 2018 / 0237749, both of which are incorporated herein by reference.

[0068] After hematopoietic stem or stem and progenitor cell expansion, cells and survival CD34 + The total number of cells can be determined. For example, on day 14 during expansion, a sample is taken for determination of the total viable nucleated cell count. In addition, CD34 + The total number of cells can be determined by multiparameter flow cytometry, and therefore the CD34+ The percentage of cells can be determined. Typically, + Cultures that do not result in at least a 10-fold increase in absolute cell numbers are discontinued. In a preferred embodiment, fewer than 50 million CD34 + These populations containing viable cells can be discarded.

[0069] Viability can be determined by any method known in the art, for example, by trypan blue exclusion or 7-AAD exclusion. + The percentage of cells can be assessed by flow cytometry and the use of stains that are excluded by viable cells: viable CD34 + Percentage of cells = CD34 excluding 7-AAD (or other appropriate stain) in an aliquot of sample divided by the total cell count (TNC; both viable and non-viable) in the aliquot + Number of viable CD34 cells in the sample + Cells can be calculated as follows: Viable CD34 + Cells = TNCs in sample x viable CD34 in sample + cell%. survival CD34 + The ratio increase between enrichment or proliferation in cells can be calculated as follows: Total viable CD34 after culture + Cells / Pre-culture total viable CD34 + cell.

[0070] In some embodiments, hematopoietic stem or stem and progenitor cells are expanded by culturing the cells ex vivo in a growth culture medium in the presence of an agonist of Notch function and one or more growth factors or cytokines, as described above, for a given period of time. An agonist of Notch function, also referred to as a Notch agonist or Notch ligand, is an agent that promotes, i.e., causes or increases, the activation of the function of the Notch pathway. As used herein, "Notch function" means a function mediated by the Notch signal transduction pathway, including, but not limited to, nuclear translocation of the intracellular domain of Notch, nuclear translocation of a repressor of RBP-Jκ or its hairless Drosophila homolog; activation of bHLH genes, such as the Enhancer of Mastermind Split complex; activation of the HES-1 gene or the KBF2 (also called CBF1) gene; inhibition of Drosophila neuroblast separation; and binding of Notch to Delta, Jagged / Serrate, Fringe, Deltex, or a repressor of RBP-Jκ / Hairless, or their homologs or analogs. See generally the review by Kopan et al., Cell 137:216-233, 2009 for a discussion of the Notch signal transduction pathway and its effects upon activation; see also Jarriault et al., Mol. Cell. Biol. 18:7423-7431, 1998.

[0071] Notch activation is carried out by exposing cells to Notch agonist.Notch function agonist can be, but is not limited to, a molecule immobilized on a solid phase.Exemplary Notch agonist is the extracellular binding ligand Delta and Serrate, which bind to the extracellular domain of Notch and activate Notch signal transduction, or a fragment of Delta and Serrate, which bind to the extracellular domain of Notch and activate Notch signal transduction.The nucleic acid and amino acid sequences of Delta and Serrate have been isolated from several species, including humans, and are known in the art, and are disclosed in International Patent Publication Nos. WO93 / 12141, WO96 / 27610, WO97 / 01571 and Gray et al., Am. J. Path. 154:785-794, 1999 (all of which are incorporated herein by reference in their entirety).

[0072] In a preferred embodiment, the Notch agonist is a fixed fragment of the Delta or Serrate protein consisting of the extracellular domain of the protein fused to a myc epitope tag (Delta and Serrate, respectively). ext-myc or Serrate ext-myc ), or immobilized fragments of the Delta or Serrate proteins consisting of the extracellular domain of the protein fused to the Fc portion of IgG (Delta and Serrate, respectively). ext-IgG or Serrate ext-IgG). Notch agonists include, but are not limited to, Notch proteins and their analogs and derivatives (including fragments); proteins that are other components of the Notch pathway and their analogs and derivatives (including fragments); antibodies against them and fragments or other derivatives of such antibodies containing the binding region; nucleic acids encoding the proteins, derivatives, or analogs; and proteins that bind to or otherwise interact with Notch proteins or other proteins in the Notch pathway to promote Notch pathway activity, and their derivatives and analogs. Such agonists include, but are not limited to, Notch proteins and derivatives thereof containing intracellular domains, Notch nucleic acids encoding the same, and proteins containing the Notch-interacting domain of a Notch ligand (e.g., the extracellular domain of Delta or Serrate). Other agonists include, but are not limited to, suppressor of RBPJκ / hairless or Deltex. Peripheral regions can be used to enhance Notch activity, for example, in conjunction with Delta protein. These proteins, fragments, and derivatives thereof can be recombinantly expressed and isolated, or chemically synthesized.

[0073] In another embodiment, the Notch agonist is a peptide mimetic or peptide analog or an organic molecule that binds to a member of the Notch signaling pathway. Such agonists can be assayed using assays known in the art, for example, the assays described in Rebay et al. al., Cell 67:687-699, 1991 and International Patent Publication No. WO92 / 19734 (both The antibody can be identified by a binding assay selected from the cell aggregation assays described in the US Pat. No. 6,239,999, both of which are incorporated herein by reference.

[0074] In a preferred embodiment, the agonist is a protein consisting of at least a fragment of a protein encoded by a Notch-interacting gene that mediates binding to a Notch protein or a fragment of Notch, the fragment of Notch containing the region of Notch responsible for binding to an agonist protein, for example, Notch epidermal growth factor-like repeats 11 and 12. Notch-interacting gene, as used herein, is intended to mean the genes Notch, Delta, Serrate, RBPJκ, suppressor of hairless, and Deltex, as well as other members of the Delta / Serrate or Deltex families that can be identified by virtue of sequence homology or genetic interaction, and more generally, members of the "Notch cascade" or "Notch group" of genes, which are identified by molecular interaction (e.g., in vitro binding or genetic interaction (e.g., phenotypically expressed in Drosophila)). Exemplary fragments of Notch-binding proteins containing the region responsible for binding to Notch are described in U.S. Patent Nos. 5,648,464; 5,849,869; and 5,856,441 (all incorporated herein by reference).

[0075] The Notch agonists utilized by the methods described herein can be commercially available, can be produced by recombinant expression, or can be chemically synthesized.

[0076] In a specific embodiment, the exposure of the cells to the Notch agonist is not by incubation with other cells recombinantly expressing a Notch ligand on their cell surface (e.g., a feeder layer), but rather by exposure to a cell-free Notch ligand, e.g., by incubation with a cell-free ligand of Notch, which is immobilized on a solid surface, e.g., on the surface of a tissue culture dish.

[0077] In a specific embodiment, Notch activity is promoted by the binding of a Notch ligand (e.g., Delta, Serrate) to the extracellular portion of the Notch receptor. Notch signaling is thought to be triggered by the physical interaction between the extracellular domain of Notch and its ligand, which is either membrane-bound on adjacent cells or immobilized on a solid surface. Full-length ligands are Notch agonists, since their expression on a cell causes the activation of the pathway in neighboring cells that express Notch receptors. Soluble truncated Delta or Serrate molecules containing the extracellular domain of the protein or its Notch-binding portion immobilized on a solid surface, such as a tissue culture plate, are particularly preferred agonists of the Notch pathway. Such soluble proteins can be immobilized on a solid surface by antibodies or interacting proteins, such as antibodies against the epitope tag with which Delta or Serrate is expressed as a fusion protein (e.g., the myc epitope tag, which is recognized by antibody 9E10), or proteins that interact with the epitope tag with which Delta or Serrate is expressed as a fusion protein (e.g., an immunoglobulin epitope tag, which binds to protein A).

[0078] In another specific embodiment, as described in U.S. Pat. No. 5,780,300 to Artavanis-Tsakonas et al., a Notch agonist includes an agent that promotes or activates a cellular process that mediates the maturation or processing step required for activation of Notch or a member of the Notch signaling pathway, such as a furin-like convertase required for Notch processing, Kuzbanian, a disintegrin-metalloprotease (ADAM) that is thought to be required for activation of the Notch pathway upstream of or parallel to Notch (Schlondorff and Blobel, J. Cell. Sci. 112:3603-3617, 1999), or more generally, cellular transport and processing proteins such as the rab family of GTPases required for movement between intracellular compartments (for a review on Rab GTPases, see Olkkonen and Stenmark, Int. Rev. Cytol. 176:1-185, 1997. An agonist can be any molecule that increases the activity of one of the above processes, such as a nucleic acid encoding furin, a Kuzbanian or rab protein, or a fragment or derivative or dominant-active mutant thereof, or a peptidomimetic or peptide analog or organic molecule that binds to and activates the function of the above protein.

[0079] U.S. Patent No. 5,780,300 further discloses classes of Notch agonist molecules (and particular methods thereof) that can be used to activate the Notch pathway, e.g., molecules that cause dissociation of Notch ankyrin repeats by RBP-Jκ, thereby promoting translocation of RBP-Jκ from the cytoplasm to the nucleus.

[0080] In some preferred embodiments, an expansion method involving DXI is used. The Notch agonist DXI is expressed as a fixed fragment of Delta (Delta), consisting of the extracellular domain of the protein fused to the Fc portion of IgG, as described in U.S. Patent No. 7,399,633. ext-IgG or DXI), or immobilized Notch-1 or Notch-2 specific antibodies as described in U.S. Patent No. 10,208,286. Preferably, Delta1 ext-IgG In a specific embodiment, the cell culture dish is immobilized on the surface of a cell culture dish containing 2.5 μg / ml of Delta1 in phosphate buffered saline prior to the addition of hematopoietic stem or stem and progenitor cells. ext-IgGand 5 μg / ml RetroNectin® (a recombinant human fibronectin fragment also known as rFN-CH-296) overnight at 4°C (or at least 2 hours at 37°C). Preferably, the cell culture medium is serum-free hematopoietic stem cell culture medium (e.g., STEMSPAN™ Serum-Free Growth Medium or STEMSPAN™ Serum-Free Growth Medium II (StemCell Technologies, Vancouver, British Columbia)) supplemented with 10 ng / ml rhIL-3, 50 ng / ml rhIL-6, 50 ng / ml rhTPO, 50 ng / ml rhFlt-3L, and 50 ng / ml rhSCF. Hematopoietic stem or stem and progenitor cells are cultured for 7 to 14 days in this embodiment.

[0081] In certain embodiments, the CD34 as a percentage of cells in the expanded HSPCs compared to the cell population before the enrichment procedure + The increase in cell number is at least 25, 50, 75, 100, 150, 200, 250, 300, 350, 400 or at least 350 fold, preferably 100-200 fold or 100-400 fold.

[0082] Once the expanded hematopoietic stem or stem and progenitor cells are obtained, the expanded population of stem or stem and progenitor cells (expanded HSPCs) can be collected and cryopreserved or used directly in a second stage to generate NK cell compositions and / or preparations.

[0083] NK cell compositions and / or preparations result from the differentiation of the expanded HSPCs in the second step of the process, in which the HSPCs are cultured in a differentiation culture medium containing an amount of one or more cytokines effective to induce and direct HSPC differentiation into NK cells.

[0084] In some embodiments, the cytokines in the differentiation culture medium are IL-2 and IL-15. In some embodiments, the only cytokines in the differentiation culture medium are IL-2 and IL-15. In some embodiments, the amount of IL-2 and IL-15 in the differentiation culture medium is about 25 U / ml to about 100 U / ml for IL-2 and about 25 ng / ml to about 50 ng / ml for IL-15. In some embodiments, the amount of IL-2 and IL-15 in the differentiation culture medium is about 50 U / ml for IL-2 and about 40 ng / ml for IL-15. In some embodiments, the cytokines in the differentiation culture include IL-2 and IL-15, and other cytokines, such as Flt-3L, FGF-2, IL-6, IL-7, IL-12, IL-3, GM-CSF, G-CSF, LIF, MIP-1α, SCF, IL-21, IL-18, and 4-1BBL (4-1BB ligand), are not added to the differentiation culture medium. In some embodiments, the differentiation culture medium does not contain added cytokines other than IL-2 and IL-15.

[0085] In certain embodiments, the differentiation culture medium further contains supplements such as human serum or plasma, or another proteinaceous liquid that provides carrier proteins for a wide spectrum of macromolecules, lipid substances, and trace elements, adhesion and spreading factors, hormones, and growth factors that promote cell growth and health. In very early cell culture methods for hematopoietic stem cells, fetal bovine serum (FBS) was used as this cell culture supplement; however, bovine proteins are not acceptable for human pharmaceuticals. Therefore, various human biological fluids have been used, including, for example, human serum, plasma, fresh frozen plasma, and platelet lysate. In certain embodiments of this method, human AB serum is used. In certain preferred embodiments, platelet lysate is used. In some embodiments, about 2.5% to about 10% human platelet lysate is included in the differentiation medium. For example, the differentiation medium may contain about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or more than about 9% human platelet lysate. In embodiments involving human platelet lysate, the human platelet lysate is generated from human platelets (e.g., from platelet-rich plasma (PRP), expired platelets collected via apheresis, etc.) by processes such as, but not limited to, freeze-thaw cycles (e.g., 1-6 cycles), sonication, solvent / detergent treatment, or activation with calcium or thrombin. The human platelets may be derived from autologous and / or allogeneic collections from multiple different individuals. Human platelet lysate may be used with or without regard to factors that affect composition and biological activity, such as, but not limited to, plasma content, growth factor content, donor age and sex, platelet content, production process, presence of heparin or anticoagulant, fibrinogen depletion, presence of cellular components, presence of metabolites, blood type, storage conditions (e.g., duration and / or temperature), treatments to reduce / inactivate pathogens / viruses, etc. (Bieback, K., et al., Transfusion 59:3448-3460, 2019). Additionally, human platelet lysate may be used as described. The composition may be an acceptable composition obtained from a commercial source that processes the composition as described above, or by any other method that produces a commercially acceptable human platelet lysate composition. In some embodiments, fresh frozen plasma or about 2.5% to about 10% human AB serum may be included in the differentiation medium instead of platelet lysate. In some embodiments, the differentiation culture medium does not contain a feeder layer or feeder cells. In some embodiments, the differentiation culture medium does not contain fetal bovine serum (FBS), fetal calf serum (FCS), and other animal-derived products, and does not contain feeder cells or a feeder cell layer.

[0086] Although the methods disclosed herein typically and preferably do not use a feeder cell layer, the use of such a feeder cell layer does not alter the advantages of the methods described herein. As used herein, "feeder cell layer," "feeder layer," or "feeder cells" refers to one type of exogenous cell that is co-cultured with a second type of cell (e.g., HSPC) to provide an environment in which the second type of cell can be maintained and differentiated or proliferated. Without being bound by any theory, feeder cells can provide, for example, peptides, polypeptides, electrical signals, organic molecules, nucleic acid molecules, growth factors, other factors (e.g., cytokines), and metabolic nutrients to the second type of cell.

[0087] In a preferred embodiment of the method, the NK cell composition and / or preparation does not contain exogenously added cells, e.g., exogenous antigen-presenting cells (e.g., dendritic cells). As used herein, exogenous cells refer to cells that are not derived from expanded and / or differentiated HSPCs.

[0088] HSPCs are cultured in vitro or ex vivo in a differentiation culture medium for a period of time sufficient to generate an NK cell composition and / or preparation. In some embodiments, the differentiation period is about 7-21 days, about 7-14 days, about 12-16 days, about 14-16 days, about 7 days, or about 14 days. The period selected may depend on the temperature used during culture, the concentration of cytokines used, and other factors.

[0089] The state of differentiating NK cells was monitored by flow cytometry using CD56 + The developmental determination of the cells can be monitored by staining the cells with anti-CD56 antibody.

[0090] The resulting NK cell composition and / or preparation has about 50% to about 80% CD56 + cells and approximately 50% to 20% endogenous CD56 - In some embodiments, the NK cell compositions and / or preparations may contain from about 50% to up to about 85% CD56 + cells, and approximately 50% to 15% CD56 - In some embodiments, the NK cell compositions and / or preparations contain between about 55% and up to about 65% CD56 + cells, and approximately 45% to 35% CD56 - Endogenous bone marrow-derived cells, including dendritic cells, macrophages, and granulocytes. CD56 + In some embodiments, the NK cell composition and / or preparation comprises about 70% to up to about 85% CD56 + cells, and approximately 30% to 15% CD56 - Endogenous bone marrow-derived cells, such as dendritic cells, macrophages, and granulocytes. +The cells express high frequencies of NKp30, NKp46, NKp44, NKG2A, and granzyme B; intermediate to high frequencies of perforin and CD107a; low to intermediate frequencies of NKG2D; and substantially no KIR expression. In some embodiments, CD56 + Cells express KIR - (As used herein, "KIR - " refers to KIR2DL1, KIR2DS1, KIR2DS3, KIR2DS5, KIR2DL2, KIR2DL3, KIR2DS2, KIR2DS4, KIR3DL1 and KIR3DS1.) CD16 is expressed at low to intermediate frequencies. In some embodiments, CD56 - The cells express moderate to high frequencies of granzyme B; high frequencies of CD107a and low frequencies of perforin.

[0091] After the cells have been cultured in differentiation culture medium for a time sufficient to generate an NK cell composition and / or preparation, the resulting CD56 + The (primarily NK) cells are typically immature, but functional, NK cells that resemble, but are distinct from, NK cells found naturally in the human body. In some embodiments, the CD56 + The (primarily NK) cells are not further differentiated before use or storage.

[0092] In certain embodiments, priming HSPCs with IL-15 for NK cell differentiation during the expansion phase increases the resulting CD56 expression levels in the final NK cell composition and / or preparation. + It was found that the number of CD56 cells increased. Typically, IL-15 is added during the last 4 to 7 days of the expansion phase. If the expansion phase is about 7 days, pre-stimulation is performed during the last 4 days. Using this method, CD56 cells were obtained. +The total number of cells can increase by up to 50% or more, and even up to 58% or more. The remaining steps of the proliferation and differentiation stages remain as described above. In this embodiment, IL-15 can be added in an amount of about 40 ng / ml to about 100 ng / ml or more.

[0093] The NK cells comprising the compositions and / or preparations produced by any of the methods described above may be genetically engineered to express a molecule, such as a protein, nucleic acid, or carbohydrate, or a molecule of interest. In some embodiments, the NK cells comprising the compositions and / or preparations are genetically engineered to express a protein of interest, such as a protein, polypeptide, or peptide (collectively referred to as a protein). In some embodiments, the protein is an antigen-recognition receptor, other cell surface protein, or intracellular molecule. The NK cells of the compositions and / or preparations may be genetically modified before or during the expansion stage and / or during or after the differentiation stage. In typical embodiments, the NK cells are genetically engineered during the expansion stage. In some embodiments, the NK cells are genetically engineered during or after the differentiation stage.

[0094] The NK cells containing the composition and / or preparation can be genetically engineered to express an antigen-recognizing receptor that binds to the target antigen.In certain embodiments, the antigen-recognizing receptor is a chimeric antigen receptor (CAR).In certain embodiments, the antigen-recognizing receptor is a T cell receptor (TCR).The antigen-recognizing receptor can, for example, bind to tumor-specific or tumor-associated antigens, or pathogen antigens.

[0095] In certain embodiments, the antigen-recognizing receptor binds to a tumor-associated or tumor-specific antigen. Any suitable tumor-associated or tumor-specific antigen (e.g., antigenic peptide) can be used in the embodiments described herein. Sources of antigens include, but are not limited to, proteins (tumor-associated or tumor-specific antigens) associated with cancer and / or leukemia (e.g., AML). The antigen can be expressed as a peptide or as an intact protein or a portion thereof. The intact protein or a portion thereof can be native or a variant thereof, such as a mutant form.Non-limiting examples of tumor antigens include carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CD8, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CLL1, CD34, CD38, CD41, CD44, CD49c, CD49f, CD56, CD66c, CD73, CD74, CD104, CD133, CD138, CD123, CD142, CD44V6, antigens of cytomegalovirus (CMV)-infected cells (e.g., cell surface antigens), cutaneous lymphocyte-associated antigen (CLA; a special glycoform of P-selectin glycoprotein ligand-1 (PSGL-1)), epithelial glycoprotein (EGF), and IL-1. Protein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine-protein kinase erb-B2, 3, 4 (erb-B2, 3, 4), folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-alpha, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER2), human telomerase reverse transcriptase (hTERT), interleukin-13 receptor subunit alpha-2 (IL-13Ralpha2), kappa light chain, kinase insert domain receptor (KDR), Lewis Y (LeY), L1 cell adhesion molecule (L1CAM), melanoma antigen family A, 1 (MAGE-A1), mucin 16 (MUC16), mucin 1 (MUC1), mesothelin (MSLN), ERBB2, MAGEA3, p53, MART1, GP100, proteinase 3 (PR1), tyrosinase, survivin, hTERT, EphA2, NKG2D ligand, cancer-testis antigen NY-ES0-1, oncofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), ROR1, tetraspanin 8 (TSPAN8), tumor-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), cytokine receptor-like factor 2 (CRLF2), BCMA, GPC3, NKCS1, EGF1R, EGFR-VIII, and ERBB.

[0096] In some embodiments, the tumor antigen is CD19, ROR1, Her2, PSMA, PSCA, mesothelin (MSLN), or CD20. In some embodiments, the tumor antigen is CD19, CD20, CD33, MSLN, or cytokine receptor-like factor 2 (CRLF2), which are expressed in leukemia or lymphoma.

[0097] In some embodiments, the antigen is associated with or specific to a leukemia, such as acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), or chronic myeloid leukemia (CML). In preferred embodiments, the target is associated with or specific to AML. In preferred embodiments, the antigen is associated with or specific to AML. The antigen may be, but is not limited to, a protein, a non-protein, a neoantigen, a post-translationally modified antigen, a peptide-MHC antigen, and / or an overexpressed antigen.

[0098] In certain embodiments, the antigen specific for or associated with AML is AML1-ETO, DEK-CAN, promyelocytic leukemia-retinoic acid receptor alpha (PML-RARα), Fms-like tyrosine kinase 3-internal tandem duplication (Flt3-ITD), Fms-like tyrosine kinase 3 (Flt3), nucleophosmin 1 (NPM1), Aurora A kinase (AurA), B-cell lymphoma-2 (Bcl-2), Bax inhibitor 1 (Bl-1), B-lymphoma Mo-MLV insertion region 1 homolog (BMI1), BRCA1-associated protein (BRAP), chronic myeloid leukemia (CML) 28 (CML28), CML66, cyclin B1, cyclin E, cytochrome P450 1B1 (CYP1B1), ETO / MTG8 (myeloid translocation gene at 8q), carbonic anhydrase IX (CAIX), G250 / CAIX, homeobox A9 (HOXA9), human telomerase reverse transcriptase (hTERT), myeloid cell leukemia sequence 1 (Mcl-1), mesothelin (MSLN), minor histocompatibility antigens (mHAg) (e.g., lymphoid-restricted histocompatibility antigen 1 (LRH-1)), myeloperoxidase, M-phase phosphoprotein 11 (MPP11), mucin 1 (MUC1), nucleolus and spindle-associated protein 1 (NuSAP1), oncofetal antigen-immature laminin receptor protein (OFA / iLRP), proteinase 3, regulator of G protein signaling 5 (RGS5), receptor for hyaluronan-mediated motility (RHAMM), synovial sarcoma X breakpoint 2-interacting protein (SSX2IP), survivin, Wilms' tumor 1 protein (WT1), cyclin A1, melanoma antigen (MAGE), Per ARNT Sim domain containing 1 (PASD1), antigen preferentially expressed in melanoma (PRAME), kidney antigen-1 (RAGE-1), heat shock DnaJ protein homolog 2 (HSJ2), Myc-associated zinc finger protein (MAZ), renal cell carcinoma antigen (NY-REN60), novel Cys-His protein of particular interest (PINCH), recombination signal binding protein 1 for J-κ (RBPJk), syntaxin, methyl-lysophosphatidic acid (mLPA), α-galactosylceramide (α-GalCer), LewisY antigen (LeY), isocitrate dehydrogenase 1 (IDH1(R132)), isocitrate dehydrogenase 2 (IDH2(R140)), nucleophosmin 1 mutant (NPM1 mut ), Notch signaling molecule isoforms (Notch variants), hyaluronan receptor isoforms (CD44v6), phosphopeptides, protein tyrosine phosphatase type IVa member 3 (PRL3), proteinase 3 peptide HLA-A2-restricted (PR1 / HLA-A2), 20 Wilms tumor peptide HLA-A2-restricted (WT1 / HLA-A2), interleukin-12 receptor beta 1 (IL12RB1), and / or members of the immunoglobulin superfamily (CD96) (Goswami et al., Curr. Drug Targets 18:296-303, 2017).

[0099] In certain embodiments, the antigen-recognizing receptor binds to a pathogen antigen, for example, for use in the treatment and / or prevention of a pathogen infection or other infectious disease, e.g., in an immunocompromised subject. In certain embodiments, the pathogen includes a virus, bacterium, fungus, parasite, or protozoan that can cause disease.

[0100] Non-limiting examples of viruses include Retroviridae (e.g., human immunodeficiency viruses, e.g., HIV-1, and other isolates, e.g., HIV-LP); Picornaviridae (e.g., poliovirus, hepatitis A virus, enterovirus, human coxsackievirus, rhinovirus, echovirus); Calciviridae (e.g., strains that cause gastroenteritis); Togaviridae (e.g., equine encephalitis virus, rubella virus); Flaviviridae (e.g., dengue virus, encephalitis virus, yellow fever virus, Zika virus); Coronaviridae (e.g., SARS-CoV and and coronaviruses, including SARS-CoV-2; Rhabdoviridae (e.g., vesicular stomatitis virus, rabies virus); Filoviridae (e.g., Ebola virus); Paramyxoviridae (e.g., parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenza virus); Bunyaviridae (e.g., Hantaan virus, Bunga virus, Phlebovirus, and Naira virus) viruses); Arenaviridae (hemorrhagic fever viruses); Reoviridae (e.g., reoviruses, orbiviruses, and rotaviruses); Birnaviridae; Hepadnaviridae (hepatitis B virus); Parvoviridae (parvoviruses); Papovaviridae (papillomaviruses, polyomaviruses); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex viruses (HSV) 1 and 2, varicella-zoster virus, cytomegalovirus (CMV), herpesviruses); Poxviridae (variola virus, vaccinia virus, poxviruses); and Iridoviridae (e.g., African swine fever virus); and unclassified viruses (e.g., the agent of hepatitis delta (thought to be an incomplete satellite of hepatitis B virus), non-A, non-B hepatitis pathogens (Class 1 = transmitted internally; Class 2 = transmitted parenterally (i.e., hepatitis C); Norwalk and related viruses, and astroviruses).

[0101] In certain embodiments, the pathogen antigen is a viral antigen present in cytomegalovirus (CMV), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), herpes simplex virus (HSV1 or HSV2), hepatitis virus (A, B, or C), Zika virus, influenza virus, or coronavirus (SARS-CoV or SARS-CoV-2). In preferred embodiments, the viral antigen is specific to or related to HIV, HSV 1 or 2, Zika virus, hepatitis A, B, or C, SARS-CoV, or SARS-CoV-2.

[0102] Non-limiting examples of bacteria include Pasteurella, Staphylococci, Streptococcus, Escherichia coli, Pseudomonas species, and Salmonella species.Specific examples of infectious bacteria include, but are not limited to, Helicobacter pyloris, Borrelia burgdorferi, Legionella pneumophilia, Mycobacteria species (e.g., M. tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococci), Streptococcus agalactiae (Group B Streptococci), Streptococci (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococci (anaerobic species), Streptococcus pneumoniae, pathogenic Campylobacter species, Enterococcus species, Haemophilus influenzae, Bacillus antracis, Corynebacterium diphtherias, Corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium perfringens, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leptospira, Rickettsia and Actinomyces israelli.

[0103] In certain embodiments, the antigen-recognizing receptor is a TCR. A TCR is a disulfide-linked heterodimeric protein consisting of two variable chains expressed as part of a complex with an invariant CD3 chain molecule. A TCR is found on the surface of a T cell and is responsible for recognizing antigens as peptides that bind to major histocompatibility complex (MHC) molecules. In certain embodiments, the TCR comprises an alpha chain and a beta chain (encoded by TRA and TRB, respectively). In certain embodiments, the TCR comprises a gamma chain and a delta chain (encoded by TRG and TRD, respectively).

[0104] Each chain of the TCR is composed of two extracellular domains: a variable (V) region and a constant (C) region. The constant region is proximal to the cell membrane and is followed by a transmembrane region and a short cytoplasmic tail. The variable region binds to the peptide / MHC complex. The variable domains of both chains each have three complementarity-determining regions (CDRs).

[0105] In certain embodiments, a TCR can form a receptor complex with three dimeric signaling molecules: CD3 delta / epsilon, CD3 gamma / epsilon, and CD247 zeta / zeta or zeta / eta. T cells expressing the TCR complex are activated when the TCR complex engages with its antigen and MHC (peptide / MHC).

[0106] In certain embodiments, NK cells comprising the compositions and / or preparations described are genetically modified to express recombinant TCR. In certain embodiments, the TCR is a non-naturally occurring TCR. In certain embodiments, the TCR differs from any naturally occurring TCR by at least one amino acid residue. In certain embodiments, the TCR differs from any naturally occurring TCR by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acid residues. In certain embodiments, the TCR is modified from any naturally occurring TCR by at least one amino acid residue. In certain embodiments, the TCR is modified from a naturally occurring TCR by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acid residues.

[0107] In some embodiments, the TCR specifically binds to an antigen of cytomegalovirus (CMV), adenovirus, human herpesvirus 6 (HHV6), BK virus, Epstein-Barr virus (EBV), HIV, or SARS (SARS-CoV or SARS-CoV-2).

[0108] In certain embodiments, the antigen-recognizing receptor is a chimeric antigen receptor (CAR). A CAR is an engineered receptor that confers a desired specificity on cells, for example, the cells of the NK cell compositions and / or preparations described herein. CARs can be used to transfer the specificity of monoclonal antibodies onto the NK cells of the NK cell compositions and / or preparations described herein, and the transfer of their coding sequences can be facilitated, for example, by retroviral vectors.

[0109] The previously described expanded HSPCs can be differentiated as previously described to form CAR-HSPCs and CAR-NK cells. CAR-HSPCs include HSPCs engineered to express a CAR receptor, which express the engineered CAR receptor. CAR-NK cells include NK cells engineered to express a CAR receptor, which express the engineered CAR receptor.

[0110] CAR generation includes the following: "First-generation" CARs are typically composed of an extracellular antigen-binding domain (e.g., a single-chain variable fragment (scFv)) fused to a transmembrane domain fused to the cytoplasmic / intracellular signaling domain of a T cell receptor chain. "First-generation" CARs typically have an intracellular signaling domain derived from the CD3 zeta chain, which is the primary transmitter of signals from endogenous TCRs. "First-generation" CARs can provide de novo antigen recognition and can trigger cellular activation via their CD3 zeta chain signaling domain in a single fusion molecule, independent of HLA-mediated antigen presentation. "Second-generation" CARs add intracellular signaling domains from various costimulatory molecules (e.g., CD28, 4-1BB, ICOS, OX40, or 2B4) to the cytoplasmic tail of the CAR to provide additional signals to the cell. "Second-generation" CARs include those that provide both costimulation (e.g., CD28, 4-1BB, or 2B4) and activation (CD3 zeta). Preclinical studies have shown that "second-generation" CARs can improve the antitumor activity of T cells. For example, robust efficacy of T cells modified with "second-generation" CARs was demonstrated in clinical trials targeting the CD19 molecule in patients with chronic lymphoblastic leukemia (CLL) and acute lymphoblastic leukemia (ALL). "Third-generation" CARs include those that provide multiple costimulatory (e.g., CD28 and 4-1BB) and activating (CD3 zeta) molecules.

[0111] In some embodiments, the costimulatory domain may be a CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and / or B7-H3 costimulatory domain. In some embodiments, the costimulatory domain may be a CD27, CD28, 4-1BB (CD137), OX40 (CD134), DAP10, DAP12, ICOS, and / or 2B4. In some embodiments, the costimulatory domain may be a CD27, CD28, 4-1BB, 2B4, DAP10, DAP12, OX40, CD30, CD40, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and / or B7-H3 costimulatory domain. In some embodiments, the intracellular signaling domain can be a domain of CD3 zeta, CD28 and / or 4-1BB.

[0112] In certain non-limiting embodiments, the extracellular antigen binding domain of the CAR (e.g., embodied as an scFv or analog thereof) is about 2×10 -7 M or lower dissociation constant (K d ) binds to the antigen. In certain embodiments, K d is approximately 1 x 10 -7 M or lower, approximately 5 × 10 -8 M or lower, approximately 1×10 -8 M or lower, approximately 5 × 10 -9 M or lower, or about 1 x 10 -9 M or lower.

[0113] The binding of the extracellular antigen-binding domain (e.g., scFv or its analog) of the antigen-targeting CAR can be confirmed, for example, by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), or Western blot assay. Each of these assays generally detects the presence of a specific protein-antibody complex of interest by using a labeled reagent (e.g., antibody or scFv) specific to the complex of interest. For example, scFv can be radiolabeled and used in radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March 1986, incorporated herein by reference). Radioisotopes can be detected by means such as the use of a gamma counter or scintillation counter, or by autoradiography. In certain embodiments, the extracellular antigen-binding domain of the CAR is labeled with a fluorescent marker. Non-limiting examples of fluorescent markers include green fluorescent protein (GFP), blue fluorescent protein (e.g., EBFP, EBFP2, Azurite, and mKalama1), cyan fluorescent protein (e.g., ECFP, Cerulean, and CyPet), and yellow fluorescent protein (e.g., YFP, Citrine, Venus, and YPet).

[0114] A CAR can comprise an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, where the extracellular antigen-binding domain specifically binds to an antigen, e.g., a tumor antigen, or a pathogen antigen, including, e.g., a viral or bacterial antigen.

[0115] In certain embodiments, the extracellular antigen-binding domain specifically binds to an antigen. In certain embodiments, the extracellular antigen-binding domain is an scFv. In certain embodiments, the scFv is a human scFv. In certain embodiments, the scFv is a humanized scFv. In certain embodiments, the extracellular antigen-binding domain is a Fab, which is optionally cross-linked. In certain embodiments, the extracellular binding domain is a F(ab')2. In certain embodiments, any of the foregoing molecules may be included in a fusion protein with a heterologous sequence to form the extracellular antigen-binding domain. In certain embodiments, the scFv is identified by screening an scFv phage library with an antigen-Fc fusion protein. In certain embodiments, the antigen is a tumor antigen. In certain embodiments, the antigen is a pathogen antigen, including, for example, a viral or bacterial antigen.

[0116] In certain embodiments, the extracellular binding domain is an scFv that specifically binds to CD19, such as an scFv derived from the FMC63 antibody or the 4G7 antibody. In some embodiments, the scFv comprises the CDRs of the FMC63 antibody: the CDRL1 sequence of RASQDISKYLN (SEQ ID NO: 1), the CDRL2 sequence of SRLHSGV (SEQ ID NO: 2), the CDRL3 sequence of GNTLPYTFG (SEQ ID NO: 3), the CDRH1 sequence of DYGVS (SEQ ID NO: 4), the CDRH2 sequence of VTWGSETTYYNSALKS (SEQ ID NO: 5), and the CDRH3 sequence of YAMDYWG (SEQ ID NO: 6); or the CDRL1 sequence of RASQDISKYLN (SEQ ID NO: 1), the CDRL2 sequence of SRLHSGV (SEQ ID NO: 2), the CDRL3 sequence of GNTLPYTFG (SEQ ID NO: 3), the CDRH1 sequence of DYGVS (SEQ ID NO: 4), the CDRH2 sequence of DNSKSQ (SEQ ID NO: 63), and the CDRH3 sequence of YAMDYWG (SEQ ID NO: 6). In some embodiments, the extracellular binding domain is an scFv derived from or including the heavy and light chain variable regions of antibody FMC63, which are set forth in SEQ ID NO: 64 and SEQ ID NO: 65, respectively. [ka]

[0117] In some embodiments, the scFV comprises the heavy and light chain CDRs of CD19 monoclonal antibody 4G7 set forth in SEQ ID NO:25 and SEQ ID NO:26 or SEQ ID NO:27. In some embodiments, the extracellular antigen binding fragment comprises the heavy and light chain variable region CDRs set forth in SEQ ID NO:25 and 26, or SEQ ID NO:25 and 27. In some embodiments, the scFV is derived from CD19 monoclonal antibody 4G7 and comprises a portion of the binding domain of CD19 monoclonal antibody 4G7, a portion of the variable region of the immunoglobulin gamma 1 heavy chain of CD19 monoclonal antibody 4G7 (SEQ ID NO:25), and a variable fragment of the immunoglobulin kappa light chain of CD19 monoclonal antibody 4G7 (SEQ ID NO:26 or SEQ ID NO:27), preferably linked together by a flexible linker. In certain embodiments, the flexible linker has the amino acid sequence set forth in SEQ ID NO:28. In some embodiments, the extracellular antigen binding comprises the heavy and light chain variable region CDRs set forth in SEQ ID NO:25 and SEQ ID NO:26 or SEQ ID NO:25 and SEQ ID NO:27. [ka]

[0118] In some embodiments, the scFV is derived from a CLA (cutaneous lymphocyte antigen) monoclonal antibody and comprises the heavy and light chain variable regions of the CLA monoclonal antibody set forth in SEQ ID NO:29 and SEQ ID NO:30, respectively (see U.S. Patent Application Publication No. 2019 / 0209611; incorporated herein by reference). In some embodiments, the extracellular antigen-binding domain comprises the heavy and light chain variable region CDRs set forth in SEQ ID NO:29 and SEQ ID NO:30. [ka]

[0119] In some embodiments, the scFV is derived from the CD142 monoclonal antibody and comprises the heavy and light chain variable regions of the CD142 monoclonal antibody set forth in SEQ ID NOs: 31 and 32, respectively (see U.S. Patent Application Publication No. 2019 / 0209611, incorporated herein by reference). In some embodiments, the extracellular antigen-binding domain comprises the heavy and light chain variable region CDRs set forth in SEQ ID NOs: 31 and 32. [ka]

[0120] In some embodiments, the scFV is derived from the CD73 monoclonal antibody and comprises the heavy and light chain variable regions of the CD73 monoclonal antibody set forth in SEQ ID NOs: 33 and 34, respectively (see U.S. Patent Application Publication No. 2019 / 0209611, incorporated herein by reference). In some embodiments, the extracellular antigen-binding domain comprises the heavy and light chain variable region CDRs set forth in SEQ ID NOs: 33 and 34. [ka] [ka]

[0121] In some embodiments, the scFV is derived from a CD49c monoclonal antibody and comprises the heavy and light chain variable regions of the CD49c monoclonal antibody set forth in SEQ ID NOs: 35 and 36, respectively (see U.S. Patent Application Publication No. 2019 / 0209611; incorporated herein by reference). In some embodiments, the extracellular antigen-binding domain comprises the heavy and light chain variable region CDRs set forth in SEQ ID NOs: 35 and 36. [ka]

[0122] In some embodiments, the scFV is derived from the CD66c monoclonal antibody and comprises the heavy and light chain variable regions of the CD66c monoclonal antibody set forth in SEQ ID NOs: 37 and 38, respectively (see U.S. Patent Application Publication No. 2019 / 0209611, incorporated herein by reference). In some embodiments, the extracellular antigen-binding domain comprises the heavy and light chain variable region CDRs set forth in SEQ ID NO: 37 and SEQ ID NO: 38. [ka] [ka]

[0123] In some embodiments, the scFV is derived from a CD104 monoclonal antibody and comprises the heavy and light chain variable regions of the CD104 monoclonal antibody set forth in SEQ ID NOs: 39 and 40, respectively (see U.S. Patent Application Publication No. 2019 / 0209611; incorporated herein by reference). In some embodiments, the extracellular antigen-binding domain comprises the heavy and light chain variable region CDRs set forth in SEQ ID NOs: 39 and 40. [ka]

[0124] In some embodiments, the scFV is derived from the CD318 monoclonal antibody and comprises the heavy and light chain variable regions of the CD318 monoclonal antibody set forth in SEQ ID NO:41 and SEQ ID NO:42, respectively (see U.S. Patent Application Publication No. 2019 / 0209611; incorporated herein by reference). In some embodiments, the extracellular antigen-binding domain comprises the heavy and light chain variable region CDRs set forth in SEQ ID NO:41 and SEQ ID NO:42. [ka]

[0125] In some embodiments, the scFV is derived from the TSPAN8 monoclonal antibody and comprises the heavy and light chain variable regions of the TSPAN8 monoclonal antibody set forth in SEQ ID NOs: 43 and 44, respectively (see U.S. Patent Application Publication No. 2019 / 0209611; incorporated herein by reference). In some embodiments, the extracellular antigen-binding domain comprises the heavy and light chain variable region CDRs set forth in SEQ ID NOs: 43 and 44. [ka]

[0126] In some embodiments, the scFV and CLA have the amino acid sequence set forth in SEQ ID NO: 45 or SEQ ID NO: 46 (see U.S. Patent Application Publication No. 2019 / 0209611; incorporated herein by reference). [ka]

[0127] In some embodiments, the scFV and CD142 have the amino acid sequence set forth in SEQ ID NO: 47 or SEQ ID NO: 48 (see U.S. Patent Application Publication No. 2019 / 0209611; incorporated herein by reference). [ka]

[0128] In some embodiments, the scFV and CD73 have the amino acid sequence set forth in SEQ ID NO: 49 or SEQ ID NO: 50 (see U.S. Patent Application Publication No. 2019 / 0209611; incorporated herein by reference). [ka]

[0129] In some embodiments, the scFV and CD49c have the amino acid sequence set forth in SEQ ID NO: 51 or SEQ ID NO: 52 (see U.S. Patent Application Publication No. 2019 / 0209611; incorporated herein by reference). [ka]

[0130] In some embodiments, the scFV and CD66c have the amino acid sequence set forth in SEQ ID NO: 53 or SEQ ID NO: 54 (see U.S. Patent Application Publication No. 2019 / 0209611; incorporated herein by reference). [ka]

[0131] In some embodiments, the scFV and CD104 have the amino acid sequence set forth in SEQ ID NO: 55 or SEQ ID NO: 56 (see U.S. Patent Application Publication No. 2019 / 0209611; incorporated herein by reference). [ka]

[0132] In some embodiments, the scFV and CD318 have the amino acid sequence set forth in SEQ ID NO: 57 or SEQ ID NO: 58 (see U.S. Patent Application Publication No. 2019 / 0209611; incorporated herein by reference). [ka]

[0133] In some embodiments, the scFV and TSPAN8 have the amino acid sequence set forth in SEQ ID NO: 59 or SEQ ID NO: 60 (see U.S. Patent Application Publication No. 2019 / 0209611; incorporated herein by reference). [ka]

[0134] Transmembrane domain of CAR In some embodiments, the transmembrane domain of CAR comprises a hydrophobic alpha helix that spans at least part of the membrane. Different transmembrane domains result in different receptor stabilities. After antigen recognition, receptor clusters and signals are transmitted to cells. The transmembrane domain of CAR can comprise, for example, CD8 polypeptide, CD28 polypeptide, CD3 zeta polypeptide, CD4 polypeptide, 4-1BB polypeptide, OX40 polypeptide, ICOS polypeptide, NKG2D polypeptide, synthetic peptide (not based on proteins related to immune response), or a combination thereof.

[0135] In certain embodiments, the transmembrane domain comprises a CD8 polypeptide. In certain embodiments, the CD8 polypeptide has an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the sequence having NCBI Reference Number: NP_001139345.1, SEQ ID NO: 7 (as used herein, sequence identity may be determined using standard software such as BLAST or FASTA), or a fragment thereof, and / or optionally contains at most one, at most two, or at most three conservative amino acid substitutions. As used herein, a "conservative amino acid substitution" refers to an amino acid substitution in which an amino acid is replaced with another electronically similar amino acid. For example, an amino acid having a hydrophobic side chain can be substituted with a different amino acid that also has a hydrophobic side chain (e.g., leucine is substituted with isoleucine, alanine is substituted with valine, etc.); an amino acid having an acidic side chain can be substituted with a different amino acid that also has an acidic side chain (e.g., aspartic acid is substituted with glutamic acid, etc.); an amino acid having a basic side chain can be substituted with a different amino acid that also has a basic side chain (e.g., lysine is substituted with arginine, etc.); an amino acid having a polar side chain can be substituted with a different amino acid that also has a polar side chain (e.g., serine is substituted with threonine, etc.). In certain embodiments, a CD8 polypeptide can have an amino acid sequence that is a contiguous portion of SEQ ID NO:7 that is at least 20, or at least 30, or at least 40, or at least 50, and up to 235 amino acids in length. Alternatively or additionally, in various non-limiting embodiments, the CD8 polypeptide comprises or has the amino acid sequence of amino acids 1-235, 1-50, 50-100, 100-150, 150-200, or 200-235 of SEQ ID NO: 7. In certain embodiments, the CAR comprises a transmembrane domain comprising a human CD8 polypeptide comprising the amino acid sequence of amino acids 137-209 of SEQ ID NO: 7. [ka]

[0136] In certain embodiments, a CD8 polypeptide has an amino acid sequence or fragment thereof that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the sequence having NCBI Reference Number: AAA92533.1, SEQ ID NO: 8, and / or optionally contains up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, a CD8 polypeptide may have an amino acid sequence that is a contiguous portion of SEQ ID NO: 8 that is at least about 20, or at least about 30, or at least about 40, or at least about 50, or at least about 60, or at least about 70, or at least about 100, or at least about 200, and up to 247 amino acids in length. Alternatively or additionally, in various non-limiting embodiments, the CD8 polypeptide comprises or has the amino acid sequence of amino acids 1-247, 1-50, 50-100, 100-150, 150-200, 151-219, or 200-247 of SEQ ID NO: 8. In certain embodiments, the CAR comprises a transmembrane domain comprising a murine CD8 polypeptide comprising the amino acid sequence of amino acids 151-219 of SEQ ID NO:8. [ka]

[0137] In certain embodiments, the CD8 polypeptide comprises or has the amino acid sequence set forth in SEQ ID NO:9. [ka]

[0138] In certain embodiments, the transmembrane domain of the CAR comprises a CD28 polypeptide. The CD28 polypeptide may have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the sequence having NCBI reference number: P10747 or NP_006130 (SEQ ID NO: 10), or a fragment thereof, and / or may optionally contain at most one, at most two, or at most three conservative amino acid substitutions. In certain non-limiting embodiments, the CD28 polypeptide may have an amino acid sequence that is a contiguous portion of SEQ ID NO: 10 that is at least 20, or at least 30, or at least 40, or at least 50, and at most 220 amino acids in length. Alternatively or additionally, in various non-limiting embodiments, the CD28 polypeptide has the amino acid sequence of amino acids 1-220, 1-50, 50-100, 100-150, 114-220, 150-200, or 200-220 of SEQ ID NO: 10. In certain embodiments, the CD28 polypeptide comprised in the transmembrane domain of a CAR of the present disclosure has the amino acid sequence of amino acids 153-179 of SEQ ID NO: 10. [ka]

[0139] In certain embodiments, the transmembrane domain of the CAR comprises an NKG2D polypeptide. The NKG2D polypeptide may have an amino acid sequence or fragment thereof that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the sequence having NCBI Reference Number: NP_0031386.2 (SEQ ID NO: 66), and / or may optionally contain at most one, at most two, or at most three conservative amino acid substitutions. In certain non-limiting embodiments, the NKG2D polypeptide may have an amino acid sequence that is a contiguous portion of SEQ ID NO: 66 that is at least 20, or at least 30, or at least 40, or at least 50, and at most 216 amino acids in length. In certain embodiments, the NKG2D polypeptide comprises the transmembrane domain of SEQ ID NO: 10 (e.g., amino acids 52-72). [ka]

[0140] In some embodiments, the transmembrane domain of the CAR comprises a CD3 zeta (CD3ζ) polypeptide. The transmembrane domain of the CD3 zeta polypeptide can have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the sequence of the transmembrane domain of CD3 zeta set forth in NCBI Reference No. NP_932170 (SEQ ID NO: 11), SEQ ID NO: 12, or SEQ ID NO: 13, and / or optionally contains at most one, at most two, or at most three conservative amino acid substitutions.

[0141] In some embodiments, the transmembrane domain of the CAR comprises a CD4 polypeptide. The transmembrane domain of the CD4 polypeptide may have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the sequence of the transmembrane domain of CD4 set forth in NCBI Reference Nos. NP_000607.1, NP_001181943.1, or NP_001181946.1 (each of which is incorporated herein by reference), and / or may optionally contain at most one, at most two, or at most three conservative amino acid substitutions.

[0142] In some embodiments, the transmembrane domain of the CAR comprises a 4-1BB polypeptide. The transmembrane domain of the 4-1BB polypeptide may have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the sequence of the transmembrane domain of 4-1BB set forth in NCBI Reference No. P41273 (incorporated herein by reference) or NP_001552.2 (SEQ ID NO: 18), and / or may optionally contain at most one, at most two, or at most three conservative amino acid substitutions.

[0143] In some embodiments, the transmembrane domain of the CAR comprises an OX40 polypeptide. The transmembrane domain of the OX40 polypeptide can have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the sequence of the transmembrane domain of OX40 set forth in NCBI Reference Number: NP_003318.1 (incorporated herein by reference), and / or optionally contains at most one, at most two, or at most three conservative amino acid substitutions.

[0144] In some embodiments, the transmembrane domain of the CAR comprises an ICOS polypeptide. The transmembrane domain of the ICOS polypeptide may have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the sequence of the transmembrane domain of ICOS set forth in NCBI Reference No. NP_036224.1 (incorporated herein by reference), and / or may optionally contain at most one, at most two, or at most three conservative amino acid substitutions.

[0145] Spacer region In certain non-limiting embodiments, the CAR can also include a spacer region linking the extracellular antigen-binding domain to the transmembrane domain. The spacer region can be flexible enough to allow the antigen-binding domain to orient in different directions to facilitate antigen recognition. The spacer region can be a hinge region from IgG1 (GenPept Reference No. P01857.1, incorporated herein by reference), or the CH2CH3 region of an immunoglobulin (e.g., IgG4 (GenPept Reference No. P01861.1, incorporated herein by reference), as well as a portion of CD3, a portion of a CD28 polypeptide (e.g., a portion of SEQ ID NO: 10), a portion of a CD8 polypeptide (e.g., a portion of SEQ ID NO: 7 or a portion of SEQ ID NO: 8), a variant of any of the foregoing that is at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical thereto, or a synthetic spacer sequence.

[0146] Intracellular signaling domain of CAR In certain non-limiting embodiments, the intracellular signaling domain of the CAR can comprise a CD3 zeta (CD3ζ) polypeptide, which can activate or stimulate cells (e.g., cells of the lymphoid lineage, e.g., NK cells). CD3ζ contains three immunoreceptor tyrosine-based activation motifs (ITAMs) and transmits an activation signal to cells (e.g., cells of the lymphoid lineage, e.g., NK cells) after antigen binding. In certain embodiments, the CD3 zeta polypeptide has an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the sequence having NCBI Reference Number: NP_932170 (SEQ ID NO: 11), or a fragment thereof, and / or can optionally contain at most one, or at most two, or at most three conservative amino acid substitutions. In certain non-limiting embodiments, the CD3 zeta polypeptide comprises or has an amino acid sequence that is a contiguous portion of SEQ ID NO: 11 that is at least 20, or at least 30, or at least 40, or at least 50, and up to 164 amino acids in length. Alternatively or additionally, in various non-limiting embodiments, the CD3 zeta polypeptide comprises or has the amino acid sequence of amino acids 1-164, 1-50, 50-100, 100-150, or 150-164 of SEQ ID NO: 11. In certain embodiments, the CD3 zeta polypeptide comprises or has the amino acid sequence of amino acids 52-164 of SEQ ID NO: 11. [ka]

[0147] In certain embodiments, a CD3 zeta polypeptide has an amino acid sequence or fragment thereof that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the sequence having NCBI Reference Number: NP_001106864.2 (SEQ ID NO: 12), and / or optionally contains up to one, or up to two, or up to three conservative amino acid substitutions. In certain non-limiting embodiments, a CD3 zeta polypeptide may have an amino acid sequence that is a contiguous portion of SEQ ID NO: 12 that is at least about 20, or at least about 30, or at least about 40, or at least about 50, or at least about 90, or at least about 100, and up to 188 amino acids in length. Alternatively or additionally, in various non-limiting embodiments, a CD3ζ polypeptide comprises or has the amino acid sequence of amino acids 1-164, 1-50, 50-100, 52-142, 100-150, or 150-188 of SEQ ID NO: 12. In certain embodiments, a CD3 zeta polypeptide comprises or has the amino acid sequence of amino acids 52-142 of SEQ ID NO: 12. [ka]

[0148] In certain embodiments, the CD3 zeta polypeptide comprises or has the amino acid sequence set forth in SEQ ID NO:13. [ka]

[0149] In certain non-limiting embodiments, the intracellular signaling domain of the CAR further comprises at least a costimulatory signaling region. In certain embodiments, the costimulatory region comprises at least one costimulatory molecule, which can provide optimal lymphocyte activation. As used herein, "costimulatory molecule" refers to a cell surface molecule other than an antigen receptor or its ligand, which is necessary for an efficient lymphocyte response to an antigen. The at least one costimulatory signaling region may include, for example, a CD28 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a DAP-10 polypeptide, a DAP-12 polypeptide, a 2B4 polypeptide, or a combination thereof. When a costimulatory molecule binds to its receptor, it can bind to a costimulatory ligand, which is a protein expressed on the cell surface that generates a costimulatory response, i.e., an intracellular response that provides stimulation when an antigen binds to the CAR molecule. Costimulatory ligands include, but are not limited to, CD80, CD86, CD70, OX40L, and 4-1BBL. As an example, 4-1BB ligand (i.e., 4-1BBL) can bind to 4-1BB (also known as "CD137") to provide an intracellular signal that, in combination with the CAR signal, induces the effector cell function of NK cells. CARs comprising an intracellular signaling domain comprising a costimulatory signaling region comprising 4-1BB, ICOS, or DAP-10 are disclosed in U.S. Pat. No. 7,446,190, which is incorporated herein by reference (i.e., the nucleotide sequence encoding 4-1BB is set forth in SEQ ID NO: 15, and the protein sequence is set forth in NP_001551.2; the nucleotide sequence encoding ICOS is set forth in SEQ ID NO: 16, and the protein sequence is set forth in NP_036224.1; the nucleotide sequence encoding DAP-10 is set forth in SEQ ID NO: 17, and the protein sequence is set forth in NP_055081.1), and the U.S. Pat. No. 7,446,190 and the reference protein sequences are incorporated herein by reference in their entireties. [ka] [ka]

[0150] In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising a CD28 polypeptide. The CD28 polypeptide can have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to the sequence having NCBI Reference Number: P10747 (incorporated herein by reference) or NP_006130 (SEQ ID NO: 10), or a fragment thereof, and / or optionally contains at most one, at most two, or at most three conservative amino acid substitutions. In certain non-limiting embodiments, the CD28 polypeptide has an amino acid sequence that is a contiguous portion of SEQ ID NO: 10 that is at least 20, or at least 30, or at least 40, or at least 50, and at most 220 amino acids in length. Alternatively or additionally, in various non-limiting embodiments, the CD28 polypeptide has the amino acid sequence of amino acids 1-220, 1-50, 50-100, 100-150, 114-220, 150-200, or 200-220 of SEQ ID NO: 10. In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising a CD28 polypeptide having the amino acid sequence of amino acids 180-220 of SEQ ID NO: 10.

[0151] In certain embodiments, a CD28 polypeptide has an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the sequence having NCBI Reference Number: NP_031668.3 (SEQ ID NO: 14), or a fragment thereof, and / or optionally contains up to one, or up to two, or up to three conservative amino acid substitutions. In certain non-limiting embodiments, a CD28 polypeptide has an amino acid sequence that is a contiguous portion of SEQ ID NO: 14 that is at least about 20, or at least about 30, or at least about 40, or at least about 50, and up to 218 amino acids in length. Alternatively or additionally, in various non-limiting embodiments, the CD28 polypeptide has the amino acid sequence of amino acids 1-218, 1-50, 50-100, 100-150, 114-220, 150-200, 178-218, or 200-220 of SEQ ID NO: 14. In certain embodiments, the costimulatory signaling region of a CAR of the present disclosure comprises a CD28 polypeptide that comprises or has amino acids 178-218 of SEQ ID NO: 14. [ka]

[0152] In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising two costimulatory molecules: CD28 and 4-1BB or CD28 and OX40.

[0153] 4-1BB can act as a tumor necrosis factor (TNF) ligand and can have stimulatory activity. The 4-1BB polypeptide can have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the sequence set forth in NCBI Reference No. P41273 (incorporated herein by reference) or NP_001552.2 (SEQ ID NO: 18), or a fragment thereof, and / or can optionally contain at most one, at most two, or at most three conservative amino acid substitutions. [ka]

[0154] An OX40 polypeptide may have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical to a sequence having NCBI Reference Number: P43489 (incorporated herein by reference) or NP_003318.1 (SEQ ID NO: 19), or a fragment thereof, and / or may optionally contain up to one, or up to two, or up to three conservative amino acid substitutions. [ka]

[0155] An ICOS polypeptide may have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical to the sequence having NCBI Reference Number: NP_036224.1 (SEQ ID NO: 20), or a fragment thereof, and / or may optionally contain up to one, or up to two, or up to three conservative amino acid substitutions. [ka]

[0156] A DAP-12 polypeptide may have a costimulatory region amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to a sequence having NCBI Reference Number: NP_003323.1, NP_001166986.1, NP_001166985.1, or NP_937758.1 (each of which is incorporated herein by reference), or a fragment thereof, and / or may optionally contain up to one, or up to two, or up to three conservative amino acid substitutions.

[0157] The 2B4 polypeptide may have a costimulatory region amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to a sequence having NCBI Reference Number: NP_057466.1, NP_001160135.1, or NP_001160136.1 (each of which is incorporated herein by reference), or a fragment thereof, and / or may optionally contain up to one, or up to two, or up to three conservative amino acid substitutions.

[0158] Additional genes In some embodiments, the CAR construct further comprises a gene encoding an additional gene product, such as a cytokine or transfection marker. In some embodiments, the additional gene encodes a cytokine such as human IL-15 (U.S. Pat. No. 9,931,377; SEQ ID NO: 68), IL-18, or IL-12. In some embodiments, the additional gene encodes a transfection marker or suicide gene, such as truncated EGFR (tEGFR) (see WO2011 / 056894; SEQ ID NO: 67) or iCaps9 (WO2013 / 040371), the sequences of which are incorporated herein by reference. [ka] [ka]

[0159] Exemplary CAR Constructs In certain embodiments, the CAR comprises an extracellular antigen-binding domain that binds to CD19, a transmembrane domain comprising a CD28 polypeptide, an intracellular signaling domain comprising a CD3 zeta polypeptide, and a costimulatory signaling region comprising a CD28 polypeptide. In certain embodiments, the CAR is designated 1928z. In certain embodiments, 1928z is a protein having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to the amino acid sequence set forth in SEQ ID NO: 21. The protein sequence comprises the CD8 leader sequence of amino acids 1-18 and is capable of binding to human CD19. [ka]

[0160] In another embodiment, a CAR is provided that has an extracellular antigen-binding domain that binds to CD19, a hinge spacer derived from human IgG4, a transmembrane domain comprising a CD28 transmembrane domain, a 4-1BB costimulatory signaling region, and a CD3ζ intracellular signaling domain. In certain embodiments, the CAR is designated 1928z1. In certain embodiments, 1928z1 is a protein having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to the amino acid sequence set forth in SEQ ID NO: 22. [ka] [ka]

[0161] In certain embodiments, the CAR comprises an extracellular antigen-binding domain that binds to MUC16, a transmembrane domain comprising a CD28 polypeptide, an intracellular signaling domain comprising a CD3 zeta polypeptide, and a costimulatory signaling region comprising a CD28 polypeptide. In certain embodiments, the CAR is designated 4H1128z. In certain embodiments, 4H1128z is a protein having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to the amino acid sequence set forth in SEQ ID NO: 23. The protein comprises a CD8 leader sequence from amino acids 1 to 18 and binds to the MUC-16 extracellular domain. [ka] [ka]

[0162] In certain embodiments, CAR comprises an extracellular antigen binding domain that binds to CD19, a transmembrane domain that comprises a CD8 polypeptide, an intracellular signaling domain that comprises a CD3 zeta polypeptide, and a costimulatory signaling region that comprises a 4-1BB polypeptide.In certain embodiments, CAR is 19BBz.The exemplary protein sequence of 19BBz polypeptide is set forth in SEQ ID NO:24. [ka]

[0163] In certain embodiments, a CAR is provided that has an extracellular antigen-binding domain that binds to CD19, a hinge spacer derived from human IgG4, a transmembrane domain comprising a CD28 transmembrane domain, a 4-1BB costimulatory signaling region, and a CD3 zeta intracellular signaling domain. An exemplary CD19 CAR protein sequence that also includes a truncated EGFR (tEGFR) polypeptide linked to the carboxy-terminal portion of the CAR by a self-cleaving 2A peptide is set forth in SEQ ID NO: 61. [ka]

[0164] In certain embodiments, a CAR is provided that has an extracellular antigen-binding domain that binds to CD19, a hinge spacer derived from human IgG4, a transmembrane domain of the CD28 transmembrane domain, a 4-1BB costimulatory signaling region, and a CD3ζ intracellular signaling domain. An exemplary CD19 CAR protein sequence that also contains human IL-15 linked to the carboxy-terminal portion of the CAR by a self-cleaving 2A peptide is set forth in SEQ ID NO: 62. [ka]

[0165] The corresponding nucleic acid sequence capable of encoding the construct in SEQ ID NO:62 is set forth in SEQ ID NO:69. [ka] [ka] [ka] [ka]

[0166] In certain embodiments, a CAR is provided that has an extracellular antigen-binding domain that binds to CD19, a hinge spacer derived from human IgG4, a transmembrane domain of the CD28 transmembrane domain, a 2B4 costimulatory signaling region, and a CD3 zeta intracellular signaling domain. An exemplary CD19 CAR protein sequence that also contains human IL-15 linked to the carboxy-terminal portion of the CAR by a self-cleaving 2A peptide is set forth in SEQ ID NO: 70. [ka] [ka]

[0167] The corresponding nucleic acid sequence encoding the construct of SEQ ID NO:70 is set forth in SEQ ID NO:71. [ka] [ka] [ka]

[0168] In certain embodiments, a CAR is provided that has an extracellular antigen-binding domain that binds to CD19, a hinge spacer derived from human IgG4, a transmembrane domain of the NKG2D transmembrane domain, a 4-1BB costimulatory signaling region, and a CD3 zeta intracellular signaling domain. The use of the NKG2D transmembrane domain has been used previously (Xu et al., J. Hematol. Oncol. 12:49, 2019), but some have suggested that since NKGD2 is a type II membrane protein, it should be inserted in the reverse direction. An exemplary CD19 CAR protein sequence that also includes human IL-15 linked to the carboxy-terminal portion of the CAR by a self-cleaving 2A peptide is set forth in SEQ ID NO: 72. [ka]

[0169] The corresponding nucleic acid sequence encoding the construct of SEQ ID NO:72 is set forth in SEQ ID NO:73. [ka] [ka] [ka] [ka]

[0170] In certain embodiments, a CAR is provided that has an extracellular antigen-binding domain that binds to CD19, a hinge spacer derived from human IgG4, a transmembrane domain of the NKG2D transmembrane domain, a 2B4 costimulatory signaling region, and a CD3ζ intracellular signaling domain. An exemplary CD19 CAR protein sequence that also contains human IL-15 linked to the carboxy-terminal portion of the CAR by a self-cleaving 2A peptide is set forth in SEQ ID NO: 74. [ka] [ka]

[0171] The corresponding nucleic acid sequence encoding the construct of SEQ ID NO:74 is set forth in SEQ ID NO:75. [ka] [ka]

[0172] Expression constructs In certain embodiments, the CAR can be further expressed from a nucleic acid comprising an inducible promoter to express the nucleic acid sequence in human cells. The promoter for use in expressing the CAR gene can be a constitutive promoter, such as ubiquitin C (UbiC), PGK, EF-1 alpha, MND (a synthetic viral promoter containing the U3 region of the long terminal repeat of the modified Moloney murine leukemia virus with a myeloproliferative sarcoma virus enhancer) or a promoter for chicken beta-actin.

[0173] The method for preparing genetically modified NK cell compositions and / or preparations for immunotherapy includes the step of: +This involves introducing a polynucleotide encoding an antigen-recognizing receptor, e.g., a CAR, into the (primarily NK) cells. The polynucleotide encoding the desired molecule may be introduced into the HSPCs before, during, or after expansion, or may be introduced into the HSPC or NK cell composition and / or preparation before, during, or after differentiation. Some embodiments relate to methods of manipulating NK cell compositions and / or preparations by transforming, transducing, or transfecting the HSPC or NK cell composition and / or preparation with at least one polynucleotide encoding a CAR, TCR, or other antigen-recognizing receptor, and expressing the polynucleotide in the cells. A desired polynucleotide encoding a gene can be introduced into HSPCs or other cells by any method known in the art, including transfection, electroporation, microinjection, lipofection, calcium phosphate-mediated transfection, infection with a viral or bacteriophage vector containing the gene sequence, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, using CRISPR or other rare-cutting endonucleases (e.g., TALE-nuclease or Cas9 endonuclease), etc. Numerous techniques are known in the art for the introduction of foreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol. 217:599-618, 1993; Cohen et al., Meth. Enzymol. 217:618-644, 1993; Cline, Pharmac. Ther. 29:69-92, 1985), and any of these may be used as long as the necessary physiological functions of the recipient cells are not disrupted. It can be used. The technique should provide for stable transfer of the gene into cells, so that the gene can be expressed by the cell, preferably inherited, and expressed by its cell progeny. In some embodiments, the transfer method includes transferring a selection marker or tag sequence into the cells. The cells are then placed under selection to isolate those cells that have taken up and expressed the transferred gene. In a preferred embodiment, the polynucleotide or gene is included in a lentiviral vector, allowing for stable expression in cells.

[0174] In one embodiment, an antigen-recognizing receptor (e.g., a CAR or a TCR) is introduced into HSPCs during the expansion step and before differentiation to form antigen-recognizing receptor-expressing HSPCs and / or antigen-recognizing receptor-expressing NK cells. In a preferred embodiment, an antigen-recognizing receptor CAR is introduced into HSPCs during the expansion step and before differentiation to form CAR-HSPCs and / or CAR-NK cells. In another embodiment, an antigen-recognizing receptor (e.g., a CAR or a TCR) is introduced into isolated enriched CD56 cells after expansion and during the differentiation step. + In a preferred embodiment, the antigen-recognizing receptor CAR is introduced into NK cells to form antigen-recognizing receptor-expressing HSPCs and / or antigen-recognizing receptor-expressing NK cells. In a preferred embodiment, the antigen-recognizing receptor CAR is introduced into isolated enriched CD56 cells during the post-expansion and differentiation stages. + The CAR-HSPC and / or CAR-NK cell are then transduced into NK cells to form CAR-HSPC and / or CAR-NK cells. The antigen recognition receptor transduction, CAR transduction, cell proliferation, and cell differentiation described above are carried out as described above and below, respectively.

[0175] The different methods described above include introducing CAR or another antigen-recognizing receptor into cells.As a non-limiting example, CAR or TCR can be introduced as a transgene encoded by a plasmid vector.The plasmid vector can also contain a selection marker that provides for identification and / or selection of cells that have received the vector.

[0176] A polypeptide such as a CAR or TCR can be synthesized in situ in a cell as a result of introducing into the cell a polynucleotide encoding the polypeptide.

[0177] Methods for viral-mediated introduction of polynucleotide constructs into cells are known in the art and include, by way of non-limiting example, recombinant viral vectors (eg, retroviruses, adenoviruses).

[0178] Cryopreservation of NK cell compositions and / or preparations Genetically engineered or unengineered NK cell compositions and / or preparations can be divided into one or more bags (or units) and frozen. In a preferred embodiment, about 50 million to about 500 million total cells are frozen in a single bag (or unit). In another preferred embodiment, about 100 million to about 500 million cells are frozen in a single bag (or unit). In other preferred embodiments, about 50 million, 100 million, 200 million, 300 million, or 400 million cells are frozen in a single bag (or unit). In some embodiments, a single bag (or unit) contains about 50 million to about 2 billion viable cells per dose. In some embodiments, a single bag (or unit) contains about 100 million, about 200 million, about 300 million, about 400 million, about 500 million, about 600 million, about 750 million, about 1 billion, about 1.5 billion, or about 2 billion viable cells. In some embodiments, a single bag contains about 50 million to about 2 billion viable CD56 cells per dose. + In some embodiments, a single bag (or unit) contains about 100 million, about 200 million, about 300 million, about 400 million, about 500 million, about 600 million, about 750 million, about 1 billion, about 1.5 billion, or about 2 billion viable CD56 cells. + Contains cells.

[0179] In a preferred embodiment, the NK cell composition and / or preparation is frozen or cryopreserved. In another embodiment, the NK cell composition and / or preparation is fresh, i.e., the cells have not been previously frozen prior to expansion or cryopreservation. The terms "frozen / freezing" and "cryopreserved / cryopreserved / cryopreserved" are used interchangeably in this application. Cryopreservation can be by any method known in the art that preserves cells in a viable form. Freezing cells is usually destructive because intracellular water freezes upon cooling, resulting in damage caused by osmotic effects on cell membranes, cell dehydration, solute concentration, and ice crystal formation. As ice forms outside the cells, available water is removed from solution and detached from the cells, causing osmotic dehydration and an increase in solute concentration that ultimately destroys the cells. For a discussion, see Mazur, P., Cryobiology 14:251-272, 1977.

[0180] These damaging effects can be avoided by (a) the use of cryoprotectants, (b) controlling the rate of freezing, and (c) storage at temperatures low enough to minimize degradative reactions.

[0181] Cryoprotectants that can be used include, but are not limited to, dimethyl sulfoxide (DMSO) (Lovelock and Bishop, Nature 183:1394-1395, 1959; Ashwood-Smith, Nature 190:1204-1205, 1961); glycerol, polyvinylpyrrolidine (Rinfret, Ann. NY Acad. Sci. 85:576, 1960); polyethylene glycol (Sloviter and Ravdin, Nature 196:548, 1962); albumin, dextran, sucrose, ethylene glycol, i-erythritol, D-ribitol, D-mannitol (Rowe et al., Fed. Proc. 21:157, 1962); D-sorbitol, i-inositol, D-lactose, choline chloride (Bender et al., J. Appl. Physiol. 15:520, 1960); amino acids (Phan The Tran and Bender, 1960, Exp. Cell Res. 20:651, 1960); methanol, acetamide, glycerol monoacetate (Lovelock, Biochem. J. 56:265, 1954); inorganic salts (Phan The Tran and Bender, Proc. Soc. Exp. Biol. Med. 104:388, 1960;Phan The Tran and Bender, in Radiobiology, Proceedings of the Third Australian Conference on Radiobiology, Ilbery ed., Butterworth, London, p. 59, 1961), and CryoStor® CS5 or CS10 (BioLife Solutions Inc., Bothell, WA). In a preferred embodiment, DMSO is used. For example, DMSO is used at a concentration that is non-toxic to cells. In addition, DMSO is included in compositions containing up to about 20% of the composition, up to about 15% of the composition, up to about 10% of the composition, up to about 5% of the composition, up to about 2% of the composition, up to about 1% of the composition, or up to about 0.5% of the composition. In some embodiments, the addition of plasma (e.g., up to about 20-25% concentration) can enhance the protective effect of DMSO. In some embodiments, the addition of a human protein, such as human serum albumin (e.g., up to about 2-10% concentration) can enhance the protective effect of DMSO. After addition of DMSO, cells must be kept at 0° C. until frozen, as DMSO concentrations of approximately 1% can be toxic at temperatures above 4° C.

[0182] In another embodiment, PBS containing 20% ​​DMSO and 8% human serum albumin (HSA) or other suitable cell protection medium is used.Then, this mixture is diluted with medium at 1:1, so that the final concentration of DMSO and HSA is 10% and 4%, respectively.Then, the cells in this mixture are frozen to -80°C at a rate of 1°C per minute, and stored in the vapor phase of liquid nitrogen storage tank.

[0183] A controlled, slow cooling rate may be important. Different cryoprotectants (Rapatz et al. al., Cryobiology 5(1):18-25, 1968) and different cell types have different optimal cooling rates (see, e.g., Rowe and Rinfret, Blood 20:636, 1962; Rowe, Cryobiology 3(1):12-18, 1966; Lewis, et al., Transfusion 7(1):17-32, 1967; and Mazur, Science 168:939-949, 1970, for the effect of cooling rate on the survival of bone marrow stem cells and their engraftment potential). The heat of the melting phase, where water turns to ice, is minimal. The cooling procedure can be accomplished, for example, by use of a programmable freezing device or a methanol bath procedure known in the art.

[0184] Programmable freezing devices allow for the determination of optimal cooling rates and facilitate standard, reproducible cooling. Programmable, controlled-rate freezers, such as Cryomed or Planar, allow for tuning of freezing regimens to the desired cooling rate curve. For example, for bone marrow cells in 10% DMSO and 20% plasma, the optimal rate is 1°C to 3°C per minute from 0°C to -80°C. In a preferred embodiment, this cooling rate of 1°C to 3°C per minute from 0°C to -80°C can be used. The container holding the cells must be stable at low temperatures and capable of rapid heat transfer for effective control of both freezing and thawing. Sealed plastic vials (e.g., Nunc, the Wheaton Cryule®) or glass ampoules can be used for multiple small (1-2 ml) or larger volumes (e.g., 5-30 ml), while larger volumes (20-200 ml) can be frozen in polyolefin bags (e.g., Del-Med) or ethylene vinyl acetate freezer bags (e.g., OriGen) held between metal plates for better heat transfer during cooling. As an example, bags of bone marrow cells have been successfully frozen by placing them in a -80°C freezer, which gives a cooling rate of approximately 3°C / min.

[0185] In an alternative embodiment, a methanol bath method of cooling can be used. This method is suitable for routine cryopreservation of multiple small items on a large scale. This method does not require manual control of the freezing rate or a recorder to monitor the rate. In a preferred embodiment, DMSO-treated cells are pre-chilled on ice, transferred to a tray containing chilled methanol, and placed in a mechanical refrigerator (e.g., Harris or Revco) at -80°C. Thermocouple measurements of the methanol bath and sample indicate a desired cooling rate of 1°C to 3°C per minute. After at least 2 hours, the specimens reach a temperature of -80°C and can be placed directly into liquid nitrogen (-196°C) for permanent storage.

[0186] After complete freezing, the NK cell compositions and / or preparations can be rapidly transferred to a long-term cryogenic storage container. In a preferred embodiment, samples are cryogenically stored in liquid nitrogen (-196°C) or its vapor (approximately -140°C to -180°C). In another preferred embodiment, samples are cryogenically stored in the vapor phase of liquid nitrogen (e.g., approximately -140°C to -180°C). Such storage is greatly facilitated by the availability of highly efficient liquid nitrogen cooling devices similar to large Thermos® vessels, which have an extremely low vacuum and internal super-insulation such that heat dissipation and nitrogen loss are minimized.

[0187] Suitable racking systems are commercially available and can be used for cataloging, storing and retrieving individual specimens.

[0188] Other methods of cryopreservation of viable cells, or modifications thereof, are available and are contemplated for use (e.g., the cold metal mirror technique; Livesey and Linner, Nature 327:255, 1987; Linner et al., J. Histochem. Cytochem. 34(9):1123-1135, 1986; see also U.S. Pat. No. 4,199,022 to Senkan et al., U.S. Pat. No. 3,753,357 to Schwartz, and U.S. Pat. No. 4,559,298 to Fahy).

[0189] Cryopreserved or frozen cells are preferably thawed quickly (e.g., in a water bath maintained at 37°C to 41°C) and cooled immediately upon thawing. In a specific embodiment, since thawing increases heat transfer from the warm water to the ice mass inside, the vial containing the frozen cells can be immersed up to its neck in a warm water bath with gentle rotation to ensure mixing of the cell suspension. Once the ice has completely melted, the vial is immediately placed on ice.

[0190] In certain embodiments, cryopreserved NK cell compositions and / or preparations are thawed and the entire preparation, or a portion thereof, is infused into a human or animal patient in need (e.g., having leukemia such as AML; another hematological malignancy; a viral infection (e.g., HIV, HSV1 or 1, Hepatitis A, B, or C, Zika, SARS-CoV, or SARS-CoV-2, etc.); or another infection disclosed herein). Several procedures for processing thawed cells are available and known in the art and can be used as desired.

[0191] It may be desirable to treat the cells to prevent clumping upon thawing. A variety of procedures are known in the art to prevent clumping, including, but not limited to, the addition of DNase before and / or after freezing (Spitzer et al., Cancer 45:3075-3085, 1980), low molecular weight dextran and citrate, and / or hydroxyethyl starch (Stiff et al., Cryobiology 20:17-24, 1983), etc. and can be used in the disclosed methods.

[0192] If the cryoprotectant is toxic in humans, it must be removed prior to therapeutic use of the thawed NK cell composition and / or preparation. In embodiments using DMSO as the cryoprotectant, it is preferable to omit this step to avoid cell loss. However, if removal of the cryoprotectant is desired, removal is preferably accomplished upon thawing.

[0193] For example, the cryoprotectant can be removed by dilution to achieve a low concentration of the cryoprotectant. This can be achieved by adding medium, followed by one or more cycles of centrifugation to pellet the cells as needed, removing the supernatant, and resuspending the cells. For example, the intracellular DMSO in the thawed cells can be reduced to a level (less than 1%) that does not adversely affect the recovered cells. This is preferably done slowly to minimize the adverse osmotic gradient that may occur during the removal of DMSO.

[0194] After removal of the cytoprotectant, cell counts (e.g., by use of a hemocytometer) and viability tests (e.g., by trypan blue exclusion; Kuchler, Biochemical Methods in Cell Culture and Virology, Dowden, Hutchinson & Ross, Stroudsburg, Pa., pp. 18-19, 1977; Methods in Medical Research, Eisen et al., eds., Vol. 10, Cell viability can be confirmed by performing a 7-AAD (or other suitable dye excluded by viable cells) immunofluorescence assay (Year Book Medical Publishers, Inc., Chicago, pp. 39-47, 1964). The percentage of viable antigen (e.g., CD56)-positive cells can be determined by dividing the number of antigen-positive cells that exclude 7-AAD (or other suitable dye excluded by viable cells) in an aliquot of cells by the total number of nucleated cells (TNC) (both viable and non-viable) in the aliquot of cells. The number of viable antigen-positive cells can then be determined by multiplying the percentage of viable antigen-positive cells by TNC.

[0195] Before cryopreservation and / or after thawing, the total number of nucleated cells, or in a specific embodiment, CD56 + The total number of cells can be determined. For example, a total nucleated cell count can be performed using a hemocytometer and trypan blue dye exclusion. Highly cellular specimens can be diluted to an appropriate concentration range for manual counting. The final cell count for the product is corrected by any dilution factor.

[0196] The total nucleated cell count is equal to the number of viable nucleated cells per mL times the volume of the product in milliliters (ml). + The number of positive cells can be determined, for example, by the use of flow cytometry using an anti-CD56 monoclonal antibody conjugated to a fluorescent dye.

[0197] NK cell compositions and / or preparations can be used in immunotherapy for the treatment of solid tumors, hematopoietic malignancies, viral disorders, bacterial infections, and the like. In some embodiments, the NK cell compositions and / or preparations are administered to inhibit tumor growth in a subject (also referred to as a patient) in need thereof. The method according to this aspect of the invention is effected by administering a therapeutically effective amount of the NK cell composition and / or preparation to a subject. As used herein, "treating" or "treatment" includes, but is not limited to, the administration of an NK cell composition and / or preparation to reduce or delay the onset of symptoms, complications, or biochemical signs of a disease, alleviate symptoms of, or arrest or inhibit further development of, a disease, condition, or disorder (e.g., cancer, metastatic cancer, metastatic solid tumor, viral, or bacterial condition). Treatment can be prophylactic, i.e., adjuvant (to prevent recurrence of the disease or delay the onset of the disease or prevent the manifestation of clinical or subclinical symptoms thereof), or therapeutic suppression or alleviation of symptoms after the manifestation of the disease.

[0198] In one embodiment, the NK cell composition and / or preparation is administered in an amount effective to reduce or eliminate, or prevent the occurrence or recurrence of, a cancer, such as a solid tumor, or a hematological malignancy, e.g., leukemia or lymphoma. An "effective amount to reduce or eliminate, or prevent the occurrence or recurrence of, a solid tumor or hematological malignancy" or an "effective amount to reduce or eliminate, or prevent the occurrence or recurrence of a hyperproliferative disease" refers to an amount of NK cell composition and / or preparation that improves a subject's outcome or survival after treatment for a neoplastic disease state or hyperproliferative disease, as measured by patient test data, survival data, increased or suppressed tumor marker levels, reduced susceptibility based on genetic profile, or exposure to environmental factors. "Inhibiting tumor growth" refers to reducing the size of a tumor, or the viability or number of cells in a tumor. "Cancer," "malignant tumor," "solid tumor," or "hyperproliferative disease" are used synonymously and refer to any of several diseases characterized by the uncontrolled, abnormal growth of cells; the ability of pathological cells to spread locally or to other parts of the body via the bloodstream and lymphatic system (i.e., metastasis); and any other characteristic structural and / or molecular features. "Cancerous," "malignant cell," or "solid tumor cell" is understood as a cell that has specific structural properties, lacks differentiation, and is capable of invasion and metastasis. "Cancer" refers to all types of cancer or neoplasms or malignant tumors found in mammals, including carcinomas and sarcomas. Examples are cancers of the breast, lung, non-small cell lung, stomach, brain, head and neck, medulloblastoma, bone, liver, colon, genitourinary tract, bladder, urinary tract, kidney, testis, uterus, ovary, cervix, or prostate, as well as melanoma, mesothelioma, and sarcoma (DeVita, et al., (eds.), Cancer Principles and Practice of Oncology, 6 th . Ed., Lippincott Williams & Wilkins, Philadelphia, Pa., 2001; this reference is incorporated herein by reference in its entirety for all purposes). "Hyperproliferative disease" refers to any disease or disorder in which cells proliferate more rapidly than normal tissue growth. Thus, hyperproliferative cells are cells that proliferate more rapidly than normal cells.

[0199] "Solid tumors" include, but are not limited to, sarcomas, melanomas, carcinomas, or other solid tumor cancers. "Sarcoma" refers to a tumor composed of a substance like embryonic connective tissue and generally composed of closely packed cells embedded in a fibrous or homogeneous substance. Sarcomas include, but are not limited to, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemethy's sarcoma, liposarcoma, and sarcoma. sarcoma), liposarcoma, alveolar soft tissue sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma, choriocarcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, B-cell immunoblastic sarcoma, T-cell immunoblastic sarcoma, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukemia sarcoma, malignant mesenchymal sarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serosarcoma cystic sarcoma, synovial sarcoma, and telangiectatic sarcoma.

[0200] "Melanoma" refers to tumors arising from the melanocytic system of the skin and other organs. Examples of melanoma include acral lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentiginous malignant melanoma, malignant melanoma, nodular melanoma, subungual melanoma, and superficial spreading melanoma.

[0201] "Carcinoma" refers to a malignant new growth made up of epithelial cells that tends to infiltrate surrounding tissues and give rise to metastases. Exemplary carcinomas include, for example, acinar cell carcinoma, acinar cell carcinoma, adenocystic carcinoma, adenomatous carcinoma, carcinoma of the adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchoalveolar carcinoma, bronchiolar carcinoma, bronchial carcinoma, cerebriform carcinoma, cholangiocarcinoma, choriocarcinoma, colloid carcinoma, comedocarcinoma, corpus carcinoma, cribriform carcinoma, armor carcinoma, skin carcinoma, columnar cell carcinoma, columnar cell carcinoma, ductal carcinoma, durum carcinoma, embryonal carcinoma, encephaloid carcinoma, epidermoid carcinoma, epithelial adenoid carcinoma, exophytic carcinoma, ulcer carcinoma, fibrous carcinoma, and gelatiniform carcinoma. carcinoma), gelatinous carcinoma, giant cell carcinoma, adenocarcinoma, granulosa cell carcinoma, pilomatrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hürthle cell carcinoma, hyalinous carcinoma, Grawitz tumor (hypemephroid carcinoma), childhood embryonal carcinoma, intramucosal carcinoma, table Intradermal carcinoma, intraepithelial carcinoma, Krompecher carcinoma, Kurticaysky cell carcinoma, large cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoma Epithelial carcinoma, medullary carcinoma, medullary carcinoma, melanoma, mucinous carcinoma, mucinous adenocarcinoma, mucous cell carcinoma, choriomeningeal carcinoma, mucinous carcinoma, mucosal carcinoma, myxomatous carcinoma, naspharyngeal carcinoma, oat cell carcinoma, ossifying carcinoma, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, squamous cell carcinoma, medullary carcinoma, renal cell carcinoma of the kidney, storage cell carcinoma, sarcomatoid carcinoma, scirrhous carcinoma, scrotal carcinoma, signet ring cell carcinoma, simplex carcinoma, small cell carcinoma, spheroid cell carcinoma, spindle cell carcinoma, cavernous carcinoma, squamous cell carcinoma, squamous cell carcinoma, string carcinoma, telangiectatic carcinoma, telangiectatic carcinoma, transitional cell carcinoma, nodular carcinoma, tuberous carcinoma, verrucous carcinoma and choriocarcinoma.

[0202] "Leukemia" refers to a progressive, malignant disease of the blood-forming organs, generally characterized by distorted proliferation and development of white blood cells and their precursors in the blood and bone marrow. Leukemias are generally classified clinically based on (1) the duration and character of the disease—acute or chronic; (2) the type of cells involved—myeloid (myeloid), lymphoid (lymphotropic), or monocytic; and (3) the increase or absence of an increase in the number of abnormal cells in the blood—leukemic or nonleukemic (subleukemic). Examples of leukemia include acute nonlymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, non-leukemic leukemia, leukemia, basophilic blastic leukemia, bovine leukemia, chronic myeloid leukemia, leukemia cutis, embryonic cell leukemia, eosinophilic leukemia, Gross' leukemia, hairy cell leukemia, hemoblastic leukemia, hemoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, and leukopenia. These include myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, small myeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myelogenous leukemia, myelogranulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, promyelocytic leukemia, Rieder cell leukemia, Schilling leukemia, stem cell leukemia, subleukemic leukemia and anaplastic cell leukemia.

[0203] Additional cancers include, for example, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocythemia, primary macroglobulinemia, small cell lung tumor, primary brain tumor, gastric cancer, colon cancer, malignant pancreatic insulinoma, malignant carcinoid, bladder cancer, precancerous skin lesions, testicular cancer, lymphoma, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, adrenocortical carcinoma, and prostate cancer.

[0204] In another embodiment, a method of inhibiting a viral infection in a subject in need thereof is provided. The method is affected by administering a therapeutic amount of an NK cell composition and / or preparation to the subject. Viral infections suitable for treatment with the NK cell composition and / or preparation include, but are not limited to, HIV, lymphocytic choriomeningitis virus (LCMV), cytomegalovirus (CMV), vaccinia virus, coronaviruses (SARS-CoV and SARS-CoV-2), influenza and parainfluenza viruses, Zika virus, hepatitis (including hepatitis A, hepatitis B, hepatitis C, non-A-non-B, etc.), herpes simplex virus (HSV 1 or HSV 2), varicella-zoster virus, and Theiler's virus. Other infectious diseases suitable for treatment with NK cell compositions and / or preparations include, but are not limited to, parasitic infections such as Plasmodium, Leishmania, and Toxoplasma infections, and bacterial infections such as mycobacteria and Listeria (for a review of NK cells in the treatment of viral, bacterial, and protozoan diseases, see Zucchini et al., Exp. Rev. Anti-Infect. Ther. 6:867-885, 2008, which is incorporated herein by reference).

[0205] According to some aspects of the present invention, there is provided a pharmaceutical composition comprising an NK cell preparation for the treatment of a disease, for example, the NK cell preparation is formulated together with a pharmaceutically acceptable carrier to form an NK cell composition for the treatment of, for example, metastatic cancer, solid tumor, hematological malignancy, hyperproliferative disease, any viral infection, bacterial infection, etc.

[0206] Therapeutic applications, compositions, or medicaments are administered to subjects suspected of or already suffering from such diseases in an amount sufficient to cure or partially arrest the symptoms (biochemical, histological, and / or behavioral) of the disease, including its complications and intermediate pathological phenotypes in the development of the disease. An amount adequate to achieve such therapeutic treatment is defined as a therapeutically effective dose. In a treatment regimen, NK cell compositions and / or preparations are usually administered in several dosages until a sufficient antiproliferative response is achieved. Typically, the antiproliferative response is monitored, and repeat dosages are given once the antiproliferative response begins to wane.

[0207] The effective dose of the NK cell compositions and / or preparations described herein for treating diseases, such as metastatic cancer, solid tumors, hematological malignancies, hyperproliferative diseases, any viral infection, bacterial infection, etc., will vary depending on factors including the means of administration, the target site, the physiological condition of the patient, whether the patient is human or an animal, other medications being administered, and whether the treatment is prophylactic or therapeutic. Typically, the patient is a human, although non-human mammals, including transgenic mammals, can also be treated. Treatment dosages need to be titrated to optimize safety and efficacy.

[0208] For administration with therapeutic NK cell compositions and / or preparations, dosages typically range from about 50 million to about 500 million total cells per dose. In some embodiments, dosages may be about 50 million, 100 million, 200 million, 300 million, or 400 million cells per dose. In some embodiments, doses of NK cell compositions and / or preparations may be about 50 million to about 2 billion viable cells per dose. In some embodiments, doses of NK cell compositions and / or preparations contain about 100 million, about 200 million, about 300 million, about 400 million, about 500 million, about 600 million, about 750 million, about 1 billion, about 1.5 billion, or about 2 billion viable cells. In some embodiments, doses of NK cell compositions and / or preparations contain about 50 million to about 2 billion viable CD56 cells per dose. +In some embodiments, the dose of the NK cell composition and / or preparation may be about 100 million, about 200 million, about 300 million, about 400 million, about 500 million, about 600 million, about 750 million, about 1 billion, about 1.5 billion, or about 2 billion viable CD56 cells. + Contains cells.

[0209] Exemplary treatment regimens entail administration approximately once, twice, or three times per week; once every two weeks, twice, or three times evenly; approximately once per month; approximately once every three to six months; or approximately once every six to twelve months. Multiple administrations of the NK cell composition and / or preparation may be provided. The interval between single doses may be several days, weekly, monthly, yearly, or some combination of such intervals. The intervals may also be irregular, as indicated by measuring the patient's blood levels of the NK cell composition and / or preparation. Alternatively, the NK cell composition and / or preparation may be administered as a sustained-release formulation, in which case less frequent administration is required. The dosage and frequency may vary depending on the half-life of the NK cell composition and / or preparation in the patient. The dosage and frequency of administration may vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, relatively low dosages are administered at relatively irregular intervals over an extended period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, relatively high dosages at relatively short intervals may be required until the progression of the disease is reduced or halted, preferably until the patient shows partial or complete alleviation of the symptoms or signs of the disease, after which the patient can be administered a prophylactic regimen.

[0210] The NK cell compositions and / or preparations for the treatment of diseases, such as metastatic cancer, solid tumors, hematological malignancies, hyperproliferative diseases, any viral infection, bacterial infection, etc., may be administered by intravenous, intravesicular, intraarterial, intracranial, or intraperitoneal means.

[0211] In some embodiments, the NK cell composition and / or preparation is administered after a chemotherapy regimen. The chemotherapy regimen can be a single-agent or multiple-agent regimen. In some embodiments, the chemotherapy regimen is an induction regimen or a consolidation regimen. In some embodiments, the chemotherapy regimen is a salvage regimen.

[0212] In some embodiments, a fixed dose of an NK cell composition and / or preparation may be administered after a chemotherapy regimen, such as an induction regimen, or a cycle thereof. A fixed dose of an NK cell composition and / or preparation may also be administered after a consolidation regimen or a cycle thereof. A fixed dose of an NK cell composition and / or preparation may also be administered after a salvage regimen or a cycle thereof. In some embodiments, a fixed dose of an NK cell composition and / or preparation may be administered after a second induction regimen or a cycle thereof, or after a second cycle of an induction regimen, as desired or required. In some embodiments, a fixed dose of an NK cell composition and / or preparation may be administered after a second consolidation regimen or a cycle thereof, or a second cycle of a consolidation regimen is desired or required. In some embodiments, a fixed dose of an NK cell composition and / or preparation may be administered after a second salvage regimen or a cycle thereof, or a second cycle of a salvage regimen is desired or required. [Example]

[0213] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will be apparent to those skilled in the art from the description herein and the accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

[0214] Various publications, including patents, patent application publications, and scientific articles, are cited herein, the disclosures of which are incorporated herein by reference in their entireties for all purposes. While illustrative embodiments have been shown and described, it will be apparent that various changes can be made therein without departing from the spirit and scope of the invention.

[0215] Example 1 Generation of NK cell compositions and / or preparations This example describes the manufacture and storage of an NK cell preparation and its formulation into an NK cell composition.

[0216] Umbilical cord blood / placental blood units (CBUs) were collected from human donors at birth. Typically, CBUs were frozen after collection, and selected CBUs were donor-eligible and fully qualified before use. The collected blood was then mixed with an anticoagulant to prevent clotting. The blood was stored at 4°C in an isolated, monitored refrigerator. Received units were evaluated to determine which units would be processed for expansion.

[0217] Stage 1 involved the expansion of hematopoietic stem and progenitor cells. CD34 + Enriched hematopoietic stem and progenitor cells (HSPCs) were plated onto tissue culture-treated plastic vessels (Thermo Scientific Nunc™ EasyFlask™). + HSPCs were prepared from pooled cord blood units as described in U.S. Patent Application Publication No. 2013 / 0095079 (incorporated herein by reference). Generally, CD34 HSPCs from at least four cord blood units were isolated. + Cells were transfected with CD34 + Cells were pooled either before or after enrichment.

[0218] Tissue culture-treated plastic vessels were filled with recombinant human fibronectin fragments (Takara Bio Inc.; RetroNectin® recombinant human fibronectin fragments, 5 μg / ml and 0.8 μg / cm) in DPBS (Gibco). 2 surface area) and Notch agonist (Delta1 extIgG , 2.5 μg / ml and 0.4 μg / cm 2 The cells were pre-coated with a solution of 1000 μg / ml of rhSCF (Miltenyi Biotec), Flt-3L (Miltenyi Biotec), TPO (Miltenyi Biotec), and IL-6 (Miltenyi Biotec), and 10 ng / ml of IL-3 (Miltenyi Biotec). The culture medium consisted of a suitable serum-free cell culture medium (StemSpan™ Serum-Free Growth Medium II (SFEM II; StemCell Technologies)) designed for the cultivation and expansion of hematopoietic cells, supplemented with 50 ng / ml each of rhSCF (Miltenyi Biotec), Flt-3L (Miltenyi Biotec), TPO (Miltenyi Biotec), and IL-6 (Miltenyi Biotec). Cells were cultured for 14 days to achieve a cell density of <2 × 10 6 Cells were passaged into larger volume culture vessels when appropriate to maintain a density of 10 cells / ml.

[0219] An alternative protocol for stage 1 expansion cultures that generates the same cell preparation is Delta1 as a Notch agonist. extIgG with 2.5-10 μg / ml of purified anti-human Notch 1 antibody (BioLegend, LEAF™ purified, clone MHN1-519).

[0220] Stage 2 of the culture process differentiates the expanded hematopoietic stem and progenitor cells toward an NK cell phenotype. At the end of the 14-day expansion stage (described above), all cells are harvested and cultured in a culture medium containing a Notch agonist (Delta1). extIgGThe cells were replated onto tissue-culture-treated plastic vessels without or with a RetroNectin® coating. Culture medium contained RPMI 1640 (Gibco) supplemented with 5-10% heat-inactivated fetal bovine serum (FBS; Gibco), 40 ng / ml rhIL-15 (PeproTech), and 50 U / ml rhIL-2 (PeproTech). Cells were cultured for up to 14 additional days, until the cell density reached <2 × 10 6 Cells were passaged into larger volume culture vessels when appropriate to maintain a density of 10 cells / ml.

[0221] In addition to CD56, + The cells expressed high frequencies of NKp30, NKp46, NKp44, NKG2A, and granzyme B, intermediate to high frequencies of perforin and CD107a, low to intermediate frequencies of NKG2D, and virtually no KIR. + Cells express KIR - (As used herein, "KIR" refers to a protein that is a nucleotide sequence of ....) - " refers to KIR2DL1, KIR2DS1, KIR2DS3, KIR2DS5, KIR2DL2, KIR2DL3, KIR2DS2, KIR2DS4, KIR3DL1, and KIR3DS1.) CD16 was expressed at low to intermediate frequencies. In these batches tested, CD56 - The cells expressed moderate to high frequencies of granzyme B, high frequencies of CD107a, and low frequencies of perforin. See Table 1 above. Table 2 above also shows the expression of CD56 in exemplary NK cell preparations. + It shows the phenotypic characteristics of the cells.

[0222] NK cell preparations are approximately 25%–50% CD56 - These cells are mostly derived from the bone marrow and may include dendritic cells, macrophages, and granulocytes. - Across the cells, the following cell surface markers were expressed at the indicated frequencies: Granzyme B was expressed at intermediate frequency, and CD107a was expressed at high frequency. Table 3 above shows the expression of CD56 in exemplary NK cell preparations.- It shows the phenotypic characteristics of the cells.

[0223] Figure 1A shows CD56 expression over 28 days of culture (stages 1 (proliferation) and 2 (differentiation)). + CD34 cells / starting + Figure 1B shows the increase in CD56 cells over the 28-day cell culture process. + Demonstrating the reproducibility of cell generation, Figures 1A and 1B show results from 20 batches of NK cell preparations generated according to the process described above.

[0224] Example 2 Generation of animal-derived serum-free NK cell preparations To avoid the use of animal-derived serum, an alternative protocol for stage 2 culture (differentiation) to generate NK cell preparations is to supplement fetal bovine serum (FBS) with heat-inactivated human AB serum (Valley Biomedical) or human platelet lysate (Mill Creek Biosciences). Applicants unexpectedly found that they were able to substitute heat-inactivated human AB serum or human platelet lysate in step 2 (the differentiation step) of the process described in Example 1.

[0225] Replacement of FBS with human AB serum (hABS) or human platelet lysate (hPL) did not significantly affect cell proliferation during differentiation (Figure 2) or the production of CD56 + hPL had a minimal effect on cell number (Figure 3). hPL maintained culture growth slightly better than hABS, but the resulting cell phenotypes were very similar between FBS, hABS, and hPL cultures.

[0226] Figures 4 and 5 compare the different serum supplements used in stage 2 of the culture, and show the CD16 + Cell percentage (Figure 4) and NKp46 + The cell percentages (Figure 5) were compared between both CD56 + Shown as a percentage of cells.

[0227] In vitro cytotoxicity assays using NK cell preparations and K562 target cells were performed to compare NK cell preparations cultured with various serum supplements (FBS, hABS, or hPL) in stage 2. Figures 6 and 7 show that cultures supplemented with hPL generated a larger cytotoxic NK cell population than those supplemented with hABS.

[0228] Example 3 In vitro cytotoxicity assay with NK cell preparations In vitro cytotoxicity assays were performed using day 28 NK cell preparations or K562-activated adult peripheral blood NK cells as effector cells (E). NK cell preparations were prepared as described above. Fluorescently labeled K562 cells (chronic myeloid leukemia cell line) or A549 cells (lung cancer cell line) were included as target cells (T). Cells were co-incubated at 37°C for 4 hours at the E:T ratios shown in Figure 8. Cells were then labeled with 4',6-diamidino-2-phenylindole (DAPI), and DAPI-colabeled fluorescent target cells were identified as stained and dead target cells by flow cytometry analysis.

[0229] Figure 8 shows that the NK cell preparation (referred to as the NK cell product) was active against K562 and A549 target cells with similar activity (curves indicated by filled circles and filled triangles). In contrast, adult peripheral blood NK cells activated with K562 cells (referred to as K562-activated adult PB NK cells) were active against K562 target cells (upper curve with open circles), but not against A549 target cells (lower curve with open triangles). This study demonstrated the ability of the NK cell preparation to be active against a variety of target cells.

[0230] Example 4 Additional in vitro assays with NK cell preparations The cytotoxicity of the NK cell preparation was tested in a standard in vitro cell killing assay (as described above in Example 3) against a range of myeloid leukemia (AML-like) cell lines. Figure 9 demonstrates that the NK cell preparation exhibited cell killing against many of these cancer cell lines. In Figure 9, for each cell line, the left bar shows cytotoxicity at 4 hours and the right bar shows cytotoxicity at 24 hours. The K562 cell line is accepted as a standard for NK cell activity and serves as a positive control.

[0231] Example 5 In vivo mouse xenograft assay with NK cell preparations The activity of NK cell preparations in a U-87 glioblastoma tumor model was tested by measuring U-87 tumor burden in NSG mice with or without NK cell preparation treatment. U-87 cells were injected subcutaneously (2 × 10 cells) into the hind flank of test NSG mice along with Matrigel® (a jelly-like protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells intended to resemble the complex extracellular environment found in many tissues and as a substrate for cultured cells). 6 cells / mouse), grown for 9 days, and incubated for 1 × 10 cells per mouse. 7 Measurable tumors were allowed to form prior to intratumoral injection of single-cell NK cell preparations (referred to as NK cell products or compositions). Tumor growth was assessed by caliper measurements 2-3 times per week. Figure 10 demonstrates that the NK cell products were able to reduce tumor burden compared to saline-injected controls.

[0232] Figure 11 shows a graph of tumor size over time for the untreated control (line with circles) and for the NK cell preparation (line with squares) for this study. The NK cell preparation was able to cause a significant delay in tumor progression (Figure 11). The NK cell preparation increased the expression of human CD45 +The tumor growth factor receptor 1 (TLR1) persisted and was detectable 37 days after injection, as measured by detecting cells (Figure 11).

[0233] Example 6 CD34 using Notch1 or Notch2 specific antibodies + HSPC proliferation CD34 + HSPCs were expanded in standard substrate (5 μg / ml RetroNectin® + 2.5 μg / ml DXI), or 5 μg / ml RetroNectin® + 5 μg / ml anti-Notch1 antibody (anti-Notch1 Ab), or 5 μg / ml RetroNectin® + 10 μg / ml anti-Notch1 Ab, as described above (Example 1). Figures 12A-D show that expansion of HSPCs with Notch1 or Notch2 antibodies (see U.S. Patent Application Publication No. 2017 / 0107493, incorporated herein by reference) was comparable to expansion using DXI.

[0234] Example 7 Cytokine release by NK cell preparations in the presence of tumor cells This study was conducted to determine the expression of the cytokines interferon gamma (IFNγ) or tumor necrosis factor alpha (TNFα) by NK cell preparations in the presence or absence of K562 or A549 tumor cells. Figure 13 shows that NK cell preparations generated by the above method (referred to as NK cell product) or K562 or A549 tumor cells were cultured alone or together for three days. This study was performed in triplicate. After three days, cytokine levels were determined by Luminex assay (the detection limit was 1 pg / ml of cytokine). As shown in Figure 13, the levels of both IFNγ (Figure 13A) and TNFα (Figure 13B) increased when the NK cell preparation was co-cultured with tumor cells. These results indicated that the NK cells in the preparations became more active in the presence of tumor cells.

[0235] Example 8 Transduction time course for production of genetically modified NK cell preparations This study was conducted to determine the optimal time between the proliferation and differentiation stages for genetic modification. Using GFP (green fluorescent protein) lentiviruses carrying either the envelope glycoprotein of VSVG (vesicular stomatitis virus) or Cocal pseudotyped virus, we transduced CD34 cells at different time points during the proliferation stage (days 1, 4, 7, or 14) or the NK differentiation stage (day 21) using two different multiplicities of infection (MOI). + Cord blood cells were transduced and NK cell culture continued for up to 28 days. On day 28, cells were immunophenotyped for different cell populations, including CD56, CD16, CD14, CD15, and CD7. Figure 14 shows that a more consistent distribution across cell lineages was observed at 1, 4, or 7 days post-transduction.

[0236] Example 9 Preparation of CAR constructs targeting cells expressing human CD19 CAR constructs were prepared containing anti-CD19 scFV (FMC63), a hinge region derived from human IgG4, a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain. The expression constructs contained either tEGFR (FIG. 15) or human IL-15 (FIG. 16) as a selection marker. The CAR constructs were transduced into HSPCs, generally as described in Example 8.

[0237] Example 10 Preparation and characterization of NK cells with IL-15 pre-stimulation during the expansion phase This example describes the production and characterization of an NK cell preparation with IL-15 added during the stage 1 expansion step.

[0238] Stage 1 involved the expansion of hematopoietic stem and progenitor cells. CD34 +Enriched hematopoietic stem and progenitor cells (HSPCs) were plated onto tissue culture-treated plastic vessels (Thermo Scientific Nunc™ EasyFlask™). + HSPCs were prepared as described above. Tissue culture-treated plastic vessels were filled with recombinant human fibronectin fragments (Takara Biosciences, Inc.) in DPBS (Gibco). Bio, Inc.; RetroNectin® recombinant human fibronectin fragments, 5 μg / ml and 0.8 μg / cm 2 surface area) and Notch agonist (Delta1 extIgG , 2.5 μg / ml and 0.4 μg / cm 2 The cells were pre-coated with a solution of 100 μg / ml of rhSCF (Miltenyi Biotec), Flt-3L (Miltenyi Biotec), TPO (Miltenyi Biotec), and IL-6 (Miltenyi Biotec), and a compatible cell culture medium suitable for the cultivation and expansion of hematopoietic cells (StemSpan™ Serum-Free Growth Medium II (SFEM II; StemCell Technologies)) supplemented with 50 ng / ml each of rhSCF (Miltenyi Biotec), Flt-3L (Miltenyi Biotec), TPO (Miltenyi Biotec), and IL-6 (Miltenyi Biotec), and 10 ng / ml of IL-3 (Miltenyi Biotec). Cells were cultured for 14 days to achieve a cell density of <2×10 6 Cells were passaged to larger volume culture vessels as appropriate to maintain a density of 1 cell / ml. Expansion cultures were primed on days 7–14 or 10–14 by the addition of 40 ng / ml IL-15 (Pepro Tech).

[0239] Phase 2 of the culture process differentiates the expanded hematopoietic stem and progenitor cells toward an NK cell phenotype. At the end of the 14-day expansion phase (phase 1 above), all cells are harvested and cultured at 1 x 10 in 1000 ml of culture medium. 6 At a cell density of 100 cells / ml, Notch agonist (Delta1 extIgGThe cells were then replated onto culture vessels (G-Rex100M vessels, WilsonWolf) without or without RetroNectin® coating. The culture medium contained RPMI 1640 (Gibco) supplemented with 5% human platelet lysate (hPL; Mill Creek Life Sciences), 40 ng / ml rhIL-15 (PeproTech), and 50 U / ml rhIL-2 (PeproTech). The cells were cultured for an additional 10 days, and 50% of the medium in the vessels was replaced with fresh differentiation medium on day 7 for differentiation.

[0240] In addition to CD56, + The cells expressed high frequencies of NKp30, NKp46, NKp44, and NKG2A, and intermediate frequencies of CD16, NKp44, and NKG2D.

[0241] Table 4 shows the CD56 expression in exemplary NK cell preparations generated by this method. + It shows the phenotypic characteristics of the cells. [Table 4]

[0242] NK cell preparations were approximately 26% CD56 - As in the previous method, these cells are mostly derived from bone marrow and may include dendritic cells, macrophages, and granulocytes. Table 5 shows the CD56 expression levels in exemplary NK cell preparations generated by this method. - It indicates the phenotypic characteristics of the cells. [Table 5]

[0243] Figures 17A and 17B show the results of four batches of NK cell preparations produced at a manufacturing scale according to the method described above. Figure 17A shows the CD56 + CD34 cells / starting +Figure 17B shows the increase in CD56 cells over the 24-day cell culture process. + The reproducibility of cell generation is demonstrated.

[0244] Example 11 In vitro cell killing by NK cell preparations generated by IL-15 prestimulation This example demonstrates the ability of NK cell preparations generated with IL-15 prestimulation to serially kill repeated doses of target cells. The serial killing assay was performed over an extended period of time in a modification of the standard in vitro cell killing assay described in Example 3. In this example, the cell killing assay used the Kasumi-1 myeloid leukemia cell line (similar to AML). Fresh NK cell preparations (FIG. 18A) or cryopreserved and thawed NK cell preparations (FIG. 18B) were initially plated in the assay and received repeated doses of target cells every 24 hours for a total of 3 days, with analysis of specific cell death of target cells performed 24 hours after each challenge. Additional assays were plated using fresh NK cell preparations that received repeated doses of target cells every 3 days for a total of 10 days (FIG. 18C), with analysis of specific cell death of target cells performed 24 hours after each challenge. Both fresh and cryopreserved and thawed NK cell preparations achieved dose-dependent cell killing upon repeated target additions over the duration of each experiment, demonstrating the ability of NK cells to continuously kill target cells for at least 10 days.

[0245] Example 12 In vivo murine AML xenograft assay with NK cell preparations The activity of NK cell preparations in the Kasumi-1 AML diffuse tumor model was tested by measuring Kasumi-1 tumor burden and animal survival in NSG mice with or without NK cell preparation treatment. Kasumi-1 cells expressing firefly luciferase were injected at 2 × 10 per mouse on days 2 and 6. 7 NSG mice were intravenously injected into the tail vein on day 0 prior to the intravenous injection of two doses of single-cell NK cell preparation (2 × 106 Tumor growth was assessed once or twice weekly by bioluminescence imaging. Compared to buffer-injected control treatments, the NK cell preparation inhibited tumor progression (Figure 19, p-values: *=<0.05, **=<0.01, ***=<0.001) and significantly prolonged median survival in mice (Figure 20; 83.5 days vs. 70 days, p=<0.01 by log-rank test).

[0246] Example 13 Generation of mesothelin CAR-NK cells – transduction during the expansion phase On day 7 of stage 1 (expansion phase), HSPCs were transduced with lentivirus expressing a mesothelin-targeting chimeric antigen receptor (CAR). Expression of this CAR was driven using the EF1 alpha promoter. This construct also contained a truncated CD19 (tCD19) extracellular domain, expressed from the same promoter and separated from the CAR sequence by a T2A self-cleavage domain. 1 x 10 cells were cultured in 0.5 ml of culture medium. 6 100 cells / ml were cultured in recombinant human fibronectin fragments (Takara Bio, Inc.; RetroNectin® recombinant human fibronectin fragments) in DPBS (Gibco), at 5 μg / ml and 0.8 μg / cm 2 surface area) and Notch agonist (Delta1 ext-IgG , 2.5 μg / ml and 0.4 μg / cm 2The cells were plated onto 24-well plates pre-coated with a solution of 1000 μg / ml of lentivirus (surface area). The culture medium contained a medium suitable for the growth and proliferation of hematopoietic cells (StemSpan™ Serum-Free Growth Medium II (SFEM II; StemCell Technologies)) supplemented with 50 ng / ml each of recombinant human SCF (Miltenyi Biotec), Flt-3 Ligand (Miltenyi Biotec), TPO (Miltenyi Biotec), and IL-6 (Miltenyi Biotec), as well as 10 ng / ml of IL-3 (Miltenyi Biotec). Eight μg / ml of protamine sulfate (Millipore Sigma) was added together with the lentivirus solution using a multiplicity of infection (MOI) of 30. A second addition of lentivirus at an MOI of 30 was added to the wells 3 hours later. The cells were incubated overnight at 37°C in a 5% CO2 incubator. On day 8, the cells were cultured at a cell density of <2 × 10 6 To maintain 100 cells / ml, cells were diluted in SFEM II medium plus the five growth factors listed above, supplemented with RetroNectin® and Delta1. ext-IgG On day 14, cells were harvested and subcultured in a larger volume vessel (6-well plate) pre-coated with Delta1. ext-IgG or 1 × 10 cells / ml in a cell culture vessel (T25 tissue culture flask) without Retronectin® coating. 6 The cells were replated at a density of 100 cells / ml. Differentiation culture medium contained RPMI 1640 (Gibco) supplemented with 5% human platelet lysate (hPL) (Mill Creek Life Sciences), 40 ng / ml rhIL-15 (PeproTech), and 50 U / ml rhIL-2 (PeproTech). The cells were cultured for a total of 14 days, and 50% of the medium in the vessel was replaced with fresh differentiation medium on day 21.

[0247] On day 28, CAR-NK cell preparations were harvested and evaluated for CAR expression by flow cytometry and in an in vitro cytotoxicity assay against two different green fluorescent protein- (GFP-) expressing NOMO-1 AML tumor target cells. The parental NOMO-1 cell line endogenously expresses mesothelin, which the CAR targets. A mesothelin knockout was prepared in this same cell line (NOMO-1 MSLN- / - ).

[0248] CAR expression was assessed by flow cytometry using indirect detection on the cell surface using an anti-CD19 antibody. Cells were 76% CD56 + and 27% tCD19 + (Figure 21).

[0249] CAR-transduced and mock-transduced control NK cells were tested as effector cells in an in vitro cytotoxicity assay by co-culturing each of two NOMO-1 target cells (parental and mesothelin knockout) in RPMI + 10% FBS + 40 ng / ml IL-15 + 50 U / ml IL-2. E:T ratios ranged from 0.3 to 20, and the assay lasted 24 hours. At the end of the incubation, cells were labeled with DAPI solution and analyzed by flow cytometry for GFP fluorescence of target cells and DAPI fluorescence of dead cells to determine the % specific cell death.

[0250] The results showed that mesothelin-CAR-NK cells inhibited NOMO-1 MSLN- / - These results demonstrate a significant and specific increase (up to 55%) in killing of parental NOMO-1 cells compared to control NK cells. Similar low levels of killing were observed with control NK cells against either NOMO-1 cell line (Figure 22).

[0251] Example 14 Generation of mesothelin CAR-NK cells – transduction after NK cell differentiation In this example, CD34 +The enriched pool of immunologically incompatible cells was processed as described above through both Step 1 (expansion) and Step 2 (differentiation). After complete differentiation, NK cells were transduced with the CAR construct.

[0252] Specifically, during the differentiation phase, on day 23 of stage 2, bulk cells were purified using an immunomagnetic microbead-based column isolation protocol (CD56 microbeads, human, Miltenyi Biotec) to isolate human CD56 + Enrichment for NK cells was performed. Enriched NK cells (>96% CD56 + ) were transduced with the same lentivirus described in Example 12. 1 x 10 cells in 2 ml of differentiation culture medium 6 Cells / ml were plated into 6-well plates with 8 μg / ml protamine sulfate and lentivirus at an MOI of 40. Cells were incubated overnight at 37°C in a 5% CO incubator. On day 24, half of the medium volume was replaced with fresh culture medium.

[0253] On day 27, NK cell preparations were collected and analyzed by flow cytometry and by parental NOMO-1 and NOMO-1 as described in Example 13. MSLN- / - CAR expression was assessed in an in vitro cytotoxicity assay against target cells. CAR expression was assessed by flow cytometry and indirect detection on the cell surface using an anti-CD19 antibody. Cells were >99% CD56 + and 40% tCD19 + (Figure 23).

[0254] CAR-transduced and mock-transduced control NK cells were tested as effector cells in an in vitro cytotoxicity assay by co-culturing each of two NOMO-1 target cells (parental and mesothelin knockout) in RPMI + 10% FBS + 40 ng / ml IL-15 + 50 U / ml IL-2. E:T ratios ranged from 0.3 to 10, and the assay lasted 24 hours. At the end of the incubation, cells were labeled with DAPI and analyzed by flow cytometry for GFP fluorescence of target cells and DAPI fluorescence of dead cells to determine % specific cell death.

[0255] The results showed that mesothelin-CAR-NK cells inhibited NOMO-1 MSLN- / - These results demonstrate a significant and specific increase (up to 27%) in killing of parental NOMO-1 target cells compared with control NOMO-1 target cells. Similar low levels of killing were observed with control NK cells against either NOMO-1 target cell line (Figure 24). In certain embodiments, for example, the following items are provided: (Item 1) 1. A method for preparing a natural killer (NK) cell preparation for use in immunotherapy, comprising: selecting a plurality of umbilical cord blood cells or placental blood cells that are immunologically incompatible with each other; Erythrocyte and T cell depleted CD34 + preparing enriched hematopoietic stem and progenitor cells (HSPCs); CD34 + culturing the enriched HSPCs in a growth culture medium comprising interleukin-3 (IL-3), interleukin-6 (IL-6), thrombopoietin (TPO), Flt-3 ligand (Flt-3L), and stem cell factor (SCF) on a solid phase coated with a Notch ligand and recombinant human fibronectin or a fragment thereof in the absence of exogenous feeder cells for a time sufficient to generate expanded HSPCs, wherein the expanded HSPCs express CD56 during the growth. + a step of not substantially differentiating into cells; and The expanded HSPCs were cultured in a differentiation culture medium containing effective amounts of IL-2 and IL-15, a non-animal-origin serum substitute, and in the absence of the exogenous feeder cells to differentiate into about 50 to about 80% CD56 + Cells and approximately 50% to 20% endogenous CD56 - Cells, or approximately 50 to 85% CD56 + Cells and approximately 50% to 15% endogenous CD56 - culturing for a time sufficient to produce an NK cell composition and / or preparation comprising the cells. Including, CD56 + The cells express high frequencies of NKp30, NKp46, NKp44, NKG2A, and granzyme B, medium to high frequencies of perforin and CD107a, low to medium frequencies of CD16, and substantially no KIR expression; - A method in which the cells express a medium to high frequency of granzyme B, a high frequency of CD107a and a low frequency of perforin. (Item 2) 2. The method of claim 1, wherein the growth culture medium does not contain endogenous IL-15, IL-7, IL-2, G-CSF, GM-CSF, LIF, MIP-1α, or aryl hydrocarbon receptor antagonists. (Item 3) IL-15 inhibits the CD34 + is added during said expansion of enriched HSPCs. (Item 4) 4. The method of claim 3, wherein the IL-15 is added during about the last 4 to 7 days of the expansion phase. (Item 5) The method of any of the preceding items, wherein the differentiation culture medium does not contain added Flt-3L, FGF-2, IL-6, IL-7, IL-12, IL-3, GM-CSF, G-CSF, LIF, MIP-1 alpha, SCF, IL-21, IL-18, and 4-1BBL. (Item 6) 10. The method of any of the preceding items, wherein IL-2 and IL-15 are the only cytokines added to the differentiation culture medium. (Item 7) 10. The method of any of the preceding items, wherein the differentiation culture medium does not contain exogenous antigen-presenting cells. (Item 8) The method of any of the preceding items, wherein the non-animal serum substitute is human AB serum, fresh frozen human plasma, or human platelet lysate. (Item 9) The method of any of the preceding items, wherein the HSPCs are not derived from somatic cells, embryonic stem cells, peripheral blood mononuclear cells, or induced pluripotent stem cells. (Item 10) The NK cell composition and / or preparation has less than 2% CD3 + cells, less than 2% CD19 + cells and / or less than 2% CD34 + 10. The method of any of the preceding items, comprising a cell. (Item 11) CD56 + The method of any of the preceding items, wherein the cells further express a high frequency of KIR2DL4. (Item 12) The method of any of the preceding items, wherein the Notch ligand is DXI or an antibody specific for Notch. (Item 13) The method of any of the preceding items, wherein the cells of the NK cell composition and / or preparation are genetically modified. (Item 14) 14. The method of claim 13, wherein the genetic modification is during the expansion stage or after differentiation of the NK cells. (Item 15) 15. The method of any one of paragraphs 13 or 14, wherein the cells of the NK cell composition and / or preparation are genetically modified to express an antigen-recognizing receptor. (Item 16) 16. The method according to any one of items 13 to 15, wherein the genetic modification is introduction of a polynucleotide that expresses a TCR or CAR. (Item 17) 17. The method of claim 16, wherein the TCR or the CAR specifically binds to a viral antigen, a bacterial antigen, a tumor-specific or tumor-associated antigen. (Item 18) 18. The method of claim 17, wherein the viral antigen is present in cytomegalovirus (CMV), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), herpes simplex virus (HSV), hepatitis virus, Zika virus, influenza virus, or coronavirus. (Item 19) 19. The method of claim 18, wherein the herpes virus is HSV 1 or HSV 2, the hepatitis virus is hepatitis A, B, or C, and the coronavirus is SARS-CoV or SARS-CoV-2. (Item 20) The tumor-specific or tumor-associated antigen is selected from the group consisting of carbonic anhydrase IX (CA1X), carcinoembryonic antigen (CEA), CD8, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CLL1, CD34, CD38, CD41, CD44, CD49c, CD49f, CD56, CD66c, CD73, CD74, CD104, CD133, CD138, CD123, CD142, CD44V6, antigens of cytomegalovirus (CMV)-infected cells (e.g., cell surface antigens), cutaneous lymphocyte-associated antigen (CLA; a special glycoform of P-selectin glycoprotein ligand-1 (PSGL-1)), epithelial glycoprotein (EGF), and the like. Protein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine-protein kinase erb-B2, 3, 4 (erb-B2, 3, 4), folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-alpha, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER2), human telomerase reverse transcriptase (hTERT), interleukin-13 receptor subunit alpha-2 (IL-13Ralpha2), kappa light chain, kinase insert domain receptor (KDR), Lewis 18. The method of item 17, wherein the antigen is selected from the group consisting of Y (LeY), L1 cell adhesion molecule (L1CAM), melanoma antigen family A, 1 (MAGE-A1), mucin 16 (MUC16), mucin 1 (MUC1), mesothelin (MSLN), ERBB2, MAGEA3, p53, MART1, GP100, proteinase 3 (PR1), tyrosinase, survivin, hTERT, EphA2, NKG2D ligand, cancer-testis antigen NY-ES0-1, oncofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), ROR1, tetraspanin 8 (TSPAN8), tumor-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF-R2), Wilms' tumor protein (WT-1), BCMA, GPC3, NKCS1, EGF1R, EGFR-VIII, CRLF2, and ERBB. (Item 21) 21. The method of claim 20, wherein the tumor-specific or tumor-associated antigen is CD19, ROR1, Her2, PSMA, PSCA, mesothelin, or CD20. (Item 22) 22. The method of any of items 16-21, wherein the CAR further comprises an intracellular signaling domain comprising the signaling domains of CD3 zeta, CD28, and 4-1BB; at least one costimulatory domain comprising the costimulatory domain of CD27, CD28, 4-1BB, 2B4, DAP10, DAP12, OX40, CD30, CD40, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, or B7-H3; a transmembrane domain comprising the transmembrane domain of CD8, CD28, CD3 zeta, CD4, 4-1BB, OX40, ICOS, or NKG2D; and a spacer region comprising the hinge region of IgG1, a CH2CH3 region of an immunoglobulin, a portion of CD3, a portion of CD28, or a portion of CD8. (Item 23) 23. The method of claim 22, wherein the CAR comprises a single-chain Fv (scFv) having the CDRs of monoclonal antibody FMC63. (Item 24) The method of any of the preceding items, wherein the NK cell composition and / or preparation further comprises a cryoprotectant. (Item 25) The method of any of the preceding items, further comprising formulating the NK cell preparation to form an NK cell composition for infusion into a subject. (Item 26) 1. A natural killer (NK) cell composition for use in immunotherapy, comprising: Approximately 50 to 80% of CD56 + Cells and approximately 50% to 20% endogenous CD56 - cells, or approximately 50 to 85% CD56 + Cells and approximately 50% to 15% endogenous CD56 - Thin The CD56 +The cells express high frequencies of NKp30, NKp46, NKp44, NKG2A, NKG2D and granzyme B, medium to high frequencies of perforin and CD107a, low to medium frequencies of CD16, and substantially no KIR expression; - The cells express moderate to high frequencies of granzyme B, high frequencies of CD107a, and low frequencies of perforin, CD56 + Cellular and endogenous CD56 - NK cell compositions comprising cells and a pharmaceutically acceptable carrier. (Item 27) 27. The NK cell composition of item 26, wherein the preparation does not contain exogenous feeder cells. (Item 28) 28. The NK cell composition according to any of items 26 to 27, wherein the NK cell composition is generated from HSPCs expanded with a Notch ligand. (Item 29) the NK cell composition comprises less than 2% CD3 + cells, less than 2% CD19 + cells and / or less than 2% CD34 + 29. The NK cell composition of any one of items 26 to 28, comprising cells. (Item 30) CD56 + 30. The NK cell composition of any one of items 26 to 29, wherein the cells further express a high frequency of KIR2DL4. (Item 31) 31. The NK cell composition of any one of items 26 to 30, wherein the cells of the composition are genetically modified. (Item 32) 32. The NK cell composition of claim 31, wherein the cells of the composition are genetically modified to express an antigen-recognizing receptor. (Item 33) 33. The NK cell composition of any one of items 31 to 32, wherein the genetic modification comprises introduction of a polynucleotide expressing a TCR or CAR. (Item 34) 34. The NK cell composition and / or preparation of item 33, wherein the TCR or the CAR specifically binds to a viral antigen, a bacterial antigen, or a tumor-associated or tumor-specific antigen. (Item 35) 35. The NK cell composition and / or preparation of item 34, wherein the viral antigen is present in cytomegalovirus (CMV), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), herpes simplex virus (HSV), hepatitis virus, Zika virus, influenza virus, or coronavirus. (Item 36) 36. The NK cell composition and / or preparation of item 35, wherein the herpes virus is HSV 1 or HSV 2, the hepatitis virus is hepatitis A, B, or C, or the coronavirus is SARS-CoV or SARS-CoV-2. (Item 37) The tumor-associated or tumor-specific antigen is selected from the group consisting of carbonic anhydrase IX (CA1X), carcinoembryonic antigen (CEA), CD8, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CLL1, CD34, CD38, CD41, CD44, CD49c, CD49f, CD56, CD66c, CD73, CD74, CD104, CD133, CD138, CD123, CD142, CD44V6, antigens of cytomegalovirus (CMV)-infected cells (e.g., cell surface antigens), cutaneous lymphocyte-associated antigen (CLA; a special glycoform of P-selectin glycoprotein ligand-1 (PSGL-1)), epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EPC), and the like. AM), receptor tyrosine-protein kinase erb-B2, 3, 4 (erb-B2, 3, 4), folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-alpha, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER2), human telomerase reverse transcriptase (hTERT), interleukin-13 receptor subunit alpha-2 (IL-13Ralpha2), kappa light chain, kinase insert domain receptor (KDR), Lewis Y (LeY), L1 cell adhesion molecule (L1CAM), melanoma antigen family A, melanoma-associated antigen A1 (MAGE-A1), mucin 16 (MUC16), mucin 1 (MUC1), mesothelin (MSLN), ERBB2, MAGEA3, p53, MART1, GP100, proteinase 3 (PR1), tyrosinase, survivin, hTERT, EphA2, NKG2D ligand, cancer-testis antigen NY-ES0-1, oncofetal antigen (h5T4), 35. The NK cell composition and / or preparation of any one of items 26 to 34, wherein the NK cell is prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), ROR1, tetraspanin 8 (TSPAN8), tumor-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), BCMA, GPC3, NKCS1, EGF1R, EGFR-VIII, CRLF2, or ERBB. (Item 38) 38. The NK cell composition of any one of items 26 to 37, wherein the tumor-associated or tumor-specific antigen is CD19, ROR1, Her2, PSMA, PSCA, mesothelin, CRLF2, or CD20. (Item 39) 39. The NK cell composition of any one of items 33 to 38, wherein the antigen-recognition receptor is a CAR. (Item 40) 40. The NK cell composition of item 39, wherein the CAR comprises the intracellular signaling domains of CD3 zeta, CD28, and 4-1BB; at least one costimulatory domain of CD27, CD28, 4-1BB, 2B4, DAP10, DAP12, OX40, CD30, CD40, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, or a B7-H3 costimulatory domain; a transmembrane domain of CD8, CD28, CD3 zeta, CD4, 4-1BB, OX40, ICOS, or NKG2D; and a spacer region of IgG1, a CH2CH3 region of an immunoglobulin, a portion of CD3, a portion of CD28, or a portion of CD8. (Item 41) 41. The NK cell composition according to any one of items 37 to 40, wherein the tumor antigen is CD19. (Item 42) 41. The NK cell composition of any one of items 37 to 40, wherein the tumor-associated or tumor-specific antigen is HER2 or mesothelin. (Item 43) 43. The NK cell composition of any one of items 26 to 42, further comprising a cryoprotectant. (Item 44) 43. The NK cell composition of any one of items 26 to 42, wherein the NK cell composition is formulated for infusion into a subject. (Item 45) Item 45. The NK cell composition according to item 44, wherein the NK cell composition comprises about 50 million to about 2 billion viable cells. (Item 46) The NK cell composition contains about 50 million to about 2 billion CD56 + 45. The NK cell composition of item 44, comprising cells. (Item 47) 47. A method of treating a subject in need thereof, comprising the step of administering to said subject a therapeutically effective amount of the NK cell composition of any one of items 26 to 46. (Item 48) 48. The method of claim 47, wherein the subject has a cancer that expresses a tumor antigen, and the NK cells of the composition express an antigen-recognition receptor that binds to the tumor antigen. (Item 49) 48. The method of claim 47, wherein the subject has a viral or bacterial infection and the NK cells of the composition express an antigen recognition receptor that binds to the virus or bacteria. (Item 50) 48. The method of item 47, wherein the subject has a viral or bacterial infection and the subject is administered the NK cell composition and / or preparation of any one of items 26 to 31. (Item 51) 51. The method of claim 50, wherein the NK cell composition comprises a cryoprotectant.

Claims

[Claim 1] The invention described in the specification.

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