RU2846770C2 - Methods for producing car-nk cells and use thereof - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, in particular to methods for propagation of NK-cells expressing chimeric antigen receptors and/or T-cell receptors. In addition, the present invention relates to methods of treating diseases by introducing CAR-NK cells.

EFFECT: invention provides a method of producing CAR-NK cells which can be used in clinical therapy with high efficiency.

44 cl, 30 dwg, 4 ex

Description

[0001] This application claims the benefit of U.S. Provisional Patent Application Ser. No. 62/826,856, filed March 29, 2019, which is incorporated herein by reference in its entirety.

Prerequisites for the creation of the invention

1. Field of invention

[0002] The present invention generally relates to the field of immunology and medicine. More specifically, the present invention relates to methods for expanding natural killer (NK) cells.

2. Description of prototypes

[0003] Despite technological advances in diagnostics and treatment available to patients diagnosed with cancer, the prognosis often remains poor, and many patients cannot be cured. Immunotherapy offers a promising approach to effectively and targetedly treating patients with a variety of diagnosed tumors, capable of destroying malignant tumor cells without damaging normal tissue. Theoretically, T cells of the immune system are capable of recognizing protein patterns specific to tumor cells and mediating their destruction through various effector mechanisms. Adoptive T-cell therapy is an attempt to harness and enhance the tumor-killing ability of a patient's own T cells and then return these effectors to the patient so that they effectively eliminate residual tumor without damaging healthy tissue. Although this approach is not new in the field of tumor immunology, many shortcomings of the clinical use of adoptive T-cell therapy hinder the full application of this cancer treatment method. For example, chimeric antigen receptor T cells (CAR-T) are patient-specific and must be obtained for each patient on an individual basis.

[0004] On the other hand, cord blood-derived natural killer (NK) cells represent a commercially available source of cells for immunotherapy and also possess natural cytotoxicity against many tumors. Although studies have been conducted on CAR-NK cells derived from peripheral blood, these cells are also not ideal for developing a "ready-to-use" approach. This is due to the need to identify a donor in each case to obtain donor NK cells.

[0005] CAR-engineered NK92 cells have also been studied; however, these NK92 cells were an NK cell line derived from a patient with lymphoma that lacks many of the NK cell receptors important for NK cell cytotoxicity. Furthermore, because this cell line was derived from a patient with lymphoma, these cells must be irradiated prior to infusion. This lack of NK cell receptors and the requirement for irradiation significantly reduces the ability of these cells to proliferate and persist, making them less potent than CAR-engineered CB-NK cells, which express the full spectrum of NK cell receptors. Thus, the need to develop methods for generating CAR-NK cells that could be used with high efficacy in clinical therapy remains urgent.

The essence of the invention

[0006] In one embodiment, the present invention relates to methods and compositions used in the treatment of a pathological condition, such as cancer. In particular embodiments, the methods and compositions relate to immunotherapy and/or cellular therapy. In a particular embodiment, the present invention relates to an ex vivo method for producing natural killer (NK) cells engineered to express a chimeric antigen receptor (CAR) and/or a T cell receptor (TCR), wherein the method comprises culturing a starting population of NK cells in the presence of artificial presenting cells (APCs) or other feeder cells and at least one cytokine; introducing a vector expressing the CAR and/or TCR into the NK cells; and expanding the NK cells in a gas-permeable bioreactor in the presence of the APCs and at least one cytokine, thereby producing an expanded population of engineered NK cells. In some aspects of the invention, the gas-permeable bioreactor is a G-Rex®100M. In some aspects of the invention, the method does not include HLA typing analysis. In some alternative cases, any or all steps of the method are performed without the use of a gas-permeable bioreactor.

[0007] In some aspects of the invention, the engineered NK cells express a CAR. In some aspects of the invention, the engineered NK cells express a TCR. In specific aspects of the invention, the engineered NK cells express a CAR and a TCR or receptors for multiple antigens. In specific aspects of the invention, the population of engineered NK cells complies with GMP requirements. In specific aspects of the invention, such a method is carried out in less than 2 weeks, such as 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days. In other aspects of the invention, such a method can be carried out in 3, 4, or more weeks. In some aspects of the invention, the NK cells are allogeneic with respect to the individual. In other aspects of the invention, the NK cells are autologous with respect to the individual.

[0008] In some aspects of the invention, the starting population of NK cells is obtained from cord blood, peripheral blood, bone marrow, CD34 ⁺ cells, or iPSC. In specific aspects of the invention, the starting population of NK cells is obtained from cord blood. In some aspects of the invention, the cord blood was previously frozen. In some aspects of the invention, the starting population of NK cells is obtained by isolating mononuclear cells in a Ficoll-Paque density gradient. In some aspects of the invention, the method further comprises depleting mononuclear CD3, CD14, and/or CD19 cells to obtain the starting population of NK cells. In some aspects of the invention, the method further comprises depleting mononuclear CD3, CD14, and CD19 cells to obtain the starting population of NK cells. In specific aspects of the invention, the depletion comprises performing magnetic sorting. In other aspects of the invention, NK cells may be positively selected by sorting, magnetic bead selection, or other methods to obtain initial populations of NK cells.

[0009] In some aspects of the invention, the APCs are gamma-irradiated APCs. In some aspects of the invention, the APCs are universal APCs (uAPCs). In some aspects of the invention, the uAPCs were engineered to express (1) CD48 and/or CS1 (CD319), (2) membrane-bound interleukin-21 (mbIL-21), and (3) 41BB ligand (41BBL). In specific aspects of the invention, the NK cells and APCs are present in a ratio of 1:1 to 1:100, such as a ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. In some aspects of the invention, the NK cells and APCs are present in a ratio of 1:2.

[0010] In some aspects of the invention, at least one cytokine is IL-2, IL-21, IL-15, or IL-18. In some aspects of the invention, culturing and/or expanding NK cells is performed in the presence of 2, 3, or 4 cytokines. In some aspects of the invention, the cytokines are selected from the group consisting of IL-2, IL-21, IL-15, and IL-18. In some aspects of the invention, at least one cytokine, such as IL-2, is present at a concentration of 100-300 U/ml, such as 100, 125, 150, 175, 200, 225, 250, 275, or 300 U/ml. In some aspects of the invention, at least one cytokine is present at a concentration of 200 U/ml.

[0011] In some aspects of the invention, administering the CAR and/or TCR involves transduction or electroporation. In some aspects of the invention, the transduction is retronectin transduction. In particular aspects of the invention, the transduction efficiency is at least 20%, such as at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75% or more. In some aspects of the invention, the CAR and/or TCR expression construct is a lentiviral vector or a retroviral vector. In some aspects of the invention, such a method provides at least 1000-fold replication, such as at least 1100-, 1200-, 1300-, 1400-, 1500-, 1600-, 1700-, 1800-, 1900-, 2000-, 2100-, 2200-, 2300-, 2400-, 2500-fold replication or more.

[0012] In some aspects of the invention, the CAR and/or TCR have antigen specificity for CD19, CD319/CS1, BCMA, CD38, CLL1, CD70, ROR1, CD20, CD5, CD70, CD20, carcinoembryonic antigen, alpha-fetoprotein, CA-125, MUC-1, epithelial tumor antigen, melanoma-associated antigen, mutated p53, mutated ras, HER2/Neu, ERBB2, folate-binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL-11R-alpha, kappa chain, lambda chain, CSPG4, ERBB2, WT-1, EGFRvIII, TRAIL/DR4 and/or VEGFR2. In some aspects of the invention, the CAR construct and/or expression construct further expresses a cytokine or 2, 3 or 4 cytokines. In some aspects of the invention, the cytokine is IL-15, IL-21, IL-18 or IL-2.

[0013] In further aspects of the invention, the method also includes cryopreserving the population of engineered NK cells. In some aspects of the invention, the engineered NK cells are cryopreserved. Furthermore, the present invention provides a population of cryopreserved NK cells.

[0014] In another embodiment, the present invention relates to a population of engineered NK cells produced according to the methods of embodiments of the invention. Furthermore, the present invention relates to a pharmaceutical composition comprising a population of engineered NK cells according to embodiments of the invention and a pharmaceutically acceptable carrier. Another embodiment of the invention relates to a composition comprising an effective amount of engineered NK cells according to embodiments of the invention for use in treating a disease or disorder in an individual. The present invention also relates to the use of a composition comprising an effective amount of engineered NK cells according to embodiments of the invention for treating an individual with an immune-related disorder.

[0015] In another embodiment, the present invention relates to a method of treating an immune-related disorder in an individual, comprising administering to the individual an effective amount of engineered NK cells according to embodiments of the invention. In some aspects of the invention, the method does not involve HLA typing. In particular aspects of the invention, the NK cells are KIR ligands that are incompatible between the individual and the donor. In particular aspects of the invention, the method does not involve HLA typing. In particular aspects of the invention, the lack of HLA matching does not result in the development of disease or graft-versus-host toxicity.

[0016] In some aspects of the invention, the immune-related disorder is cancer, an autoimmune disorder, graft-versus-host disease, allograft rejection, or an inflammatory disease. In some aspects of the invention, the immune-related disorder is an inflammatory disease, and the immune cells do not substantially express the glucocorticoid receptor. In some aspects of the invention, the individual has received or is receiving steroid therapy. In some aspects of the invention, the NK cells are autologous. In some aspects of the invention, the NK cells are allogeneic.

[0017] In particular aspects of the invention, the immune-related disorder is cancer. In some aspects of the invention, the cancer is a solid tumor or a hematological malignancy.

[0018] In further aspects of the invention, the method also includes administering at least a second therapeutic agent. In some aspects of the invention, the at least second therapeutic agent includes chemotherapy, immunotherapy, surgery, radiation therapy, or biotherapy. In specific aspects of the invention, NK cells and/or at least a second therapeutic agent are administered intravenously, intraperitoneally, intratracheally, intrathecally, intratumorally, intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, regionally, or by direct injection or perfusion. Combination therapy can be administered sequentially or simultaneously.

[0019] In another aspect, the present invention relates to a method of treating any type of infection in an individual, comprising administering to the individual an effective amount of engineered NK cells according to embodiments of the invention. In some aspects of the invention, the method does not involve HLA typing. In specific aspects of the invention, the NK cells comprise KIR ligands that are incompatible between the individual and the donor. In specific aspects of the invention, the method does not involve HLA typing. In some aspects of the invention, the NK cells comprise KIR ligands that are incompatible between the individual and the donor. In specific aspects of the invention, the lack of HLA matching does not result in the development of disease or graft-versus-host toxicity. In some aspects of the invention, the NK cells are autologous. In some aspects of the invention, the NK cells are allogeneic.

[0020] Other objects, features and advantages of the present invention will be apparent from the following detailed description. However, it should be noted that the detailed description and specific examples indicating preferred embodiments of the invention are given for illustrative purposes only, since various changes and modifications can be introduced therein without departing from the spirit and scope of the invention, and such changes and modifications will be apparent to those skilled in the art from the detailed description of the invention.

Brief description of the drawings

[0021] The following drawings form a part of the present specification and are included to further illustrate certain aspects of the present invention. For a better understanding of the present invention, reference should be made to one or more of these drawings together with the detailed description of specific embodiments of the invention presented in this application.

[0022] Fig. 1: Clinical evaluation of CAR-NK transduction and expansion in accordance with GMP.

[0023] Fig. 2: Properties of CAR-transduced CB-NK cells obtained from 5 different CB units after 14 days of GMP culture.

[0024] Fig. 3: Expansion of CAR-NK cells in a flask compared to expansion in a G-Rex® Bioreactor.

[0025] Fig. 4: Average survival (days) of mice in groups receiving different NK cell preparations.

[0026] Fig. 5: Percentage survival of mice transplanted with Raji tumors and treated with various NK cell preparations.

[0027] Fig. 6: Comparison of survival of mice transplanted with Raji tumors and treated with different NK cell preparations.

[0028] Fig. 7: Biofluorescence imaging image of mice treated with the indicated NK cell preparations.

[0029] Fig. 8: Effect of blocking KIR-HLA interaction on CAR-NK cell activity against target tumors.

[0030] Fig. 9: Table 1. Patient characteristics at baseline.

[0031] Fig. 10: Table 2. Side effects in 11 study patients.

[0032] Fig. 11: Clinical response to CAR-NK therapy and treatment after remission. Clinical outcomes and subsequent therapy are shown for 11 patients treated with natural killer (NK) cells containing a chimeric antigen receptor (CAR) and an anti-CD19 antibody in the study. Responses were confirmed and assessed according to the 2018 International Workshop on Chronic Lymphocytic Leukemia criteria and the 2014 Lugano classification of non-Hodgkin's lymphoma. Responses reported include partial response (PR) and complete response (CR); MRD refers to minimal residual disease, assessed by multiparametric flow cytometry, with or without bone marrow (BM) infiltration. Patient 3 received four doses (x4) of rituximab, and for patients 5 and 7, the white dotted line indicates the duration of post-remission therapy. HSCT stands for hematopoietic stem cell transplantation.

[0033] Fig. 12A and 12B: Persistence of CAR-NK cells after infusion. Fig. 12A shows the assessment of CAR-NK cells in peripheral blood samples by quantitative polymerase chain reaction after administration of a dose of CAR-NK cells to a patient. The horizontal gray line of 3 copies per microgram of DNA represents the lower limit of quantification for this assay. The solid horizontal bars represent the average copy number at various time points for each dose level. After a single infusion of CB-NK cells, CAR sequences were detectable in all 11 patients. Values increased and remained detectable in peripheral blood for up to 1 year after infusion, regardless of dose level. No relationship was observed between the infused cell dose and CAR-NK copy number 14 days after infusion, suggesting that CAR-NK cell persistence is due to proliferation of infused cells in vivo. The duration of observation varied among patients. Figure 12B shows the peak CAR-NK cell copy number during the first 28 days after infusion in 11 patients, depending on their response to therapy. Patients who responded at day 30 had significantly higher peak CAR-NK cell copy number after infusion than patients who did not (median 31,744 vs 903 copies/microgram; P=0.02). Black horizontal bars indicate median values.

[0034] Fig. 13A-13C: CAR-NK cells kill primary CLL targets via a perforin-dependent mechanism as assessed under GMP. Fig. 13A shows the lysis of primary CLL targets (n=4) by iC9/CAR19/IL-15-transduced CB-NK cells (red line) compared to pairs of ex vivo expanded non-transduced NK cells (NT-NK cells; black line) under GMP. *** represents p < 0.0001 and ** p < 0.01. Fig. 13B shows the fold change in the mean fluorescence intensity of perforin (red circles) after concanamycin A (CMA) treatment, calculated by determining the MFI of perforin after culture with CMA/perforin and after culture with CMA. MFI levels of CD56 (filled circles) and CAR (green circles) on the surface of NK cells were assessed as a control and remained unchanged after CMA treatment (n=3). Fig. 13C shows the lysis of primary CLL targets (n=4) by iC9/CAR19/IL-15-transduced CB-NK cells before (solid circles) and after (open circles) CMA treatment; ** represents p < 0.01 and * p < 0.05.

[0035] Fig. 14: Radiologic response in patient 5. A PET/CT scan for FDG levels was performed for patient 5, enrolled in the patient study program, before infusion (top row) and 29 days after CAR-NK cell infusion (bottom row). The image projection in the upper right corner shows FDG uptake in nodes above and below the diaphragm. The upper right center shows an FDG PET/CT scan with abnormal uptake in enlarged mesenteric nodes (dark arrow). The upper left center shows a PET/CT scan showing enlarged mesenteric nodes (open arrow). The upper left "fused" PET/CT scan shows FDG uptake localized to the adenopathic mesenteric site. The lower right image projection shows lack of FDG uptake in the nodes above and below the diaphragm. Lower right center shows an FDG PET/CT scan showing lack of uptake in the mesenteric nodes (dark arrow). Lower left center shows a PET/CT scan showing stable enlarged mesenteric nodes (light arrow). The lower left "fused" PET/CT scan shows lack of FDG uptake localized to the adenopathic mesenteric region (arrow).

[0036] Fig. 15. Retention of CAR-NK cells after infusion according to the degree of HLA mismatch between CB-CAR-NK cells and the recipient. Retention and expansion of iC9/CAR19/IL-15-modified CB-NK cells in peripheral blood samples collected from patients at various time points after infusion were assessed by qPCR. Green dots represent CAR-NK copy number in peripheral blood samples for nine patients who received a partially HLA-typed CAR-NK product (HLA typing 4/6). Red dots represent CAR-NK copy number for two patients who received a product that was not HLA-typed (HLA matching 1/6 or 2/6). The dotted black line represents the level of detection in the PCR assay.

[0037] Fig. 16A-16D: Detection of CAR-NK cells by multiparametric flow cytometry. Fig. 16A shows the flow cytometric gating strategy for detecting donor CAR-NK cells in peripheral blood from a representative patient (patient 6, day 3 after CAR-NK infusion). Lymphocytes were selected using FSC-A and SSC-A (i); and subsequent doublets were excluded using SSCW versus SSC-H (ii); live cells were identified using a dye that stained for live/dead cells (iii); hematopoietic cells were then selected within the live population by gating for CD45 ⁺ cells (iv); myeloid cells were excluded by gating for CD33-negative and CD14-negative cells (v); NK cells were identified by gating on ^CD3- and CD56 ⁺ cells (vi). Within the CD3 ^- CD56 ⁺ cell subset, cord blood-derived NK cells were identified by expression of donor-specific HLA antigen (vii). CAR expression on donor NK cells was further determined using an antibody directed against the CH2-CH3 hinge domain of human IgG (109606088/Jackson Immuno Rsch) (viii). B cells were identified by expression of CD19 and/or CD20 by gating on the CD45 ⁺ CD33 ^{- CD14-} lymphocyte population (ix). Fig. 16B shows the frequency of CAR-NK assessed according to the gating strategy described above for patient 6 on days 8, 14, and 21 after infusion. Figure 16C shows the CAR-NK frequency assessed for patient 8 on days 3, 14, and 21 post-infusion. Figure 16D shows the CAR-NK frequency assessed for patient 10 on days 3, 7, and 14 post-infusion. PBMC: peripheral blood mononuclear cells, FSC-A: forward scatter area; FSC-H: forward scatter height, SSC-A: side scatter area, SSC-H: side scatter height.

[0038] Figure 17: Sequential manual gating strategy for detecting CAR-NK cells in a lymph node from a representative patient. Flow cytometry data performed to demonstrate that the gating strategy is suitable for detecting donor CAR-NK cells in the lymph node of Patient 6. A biopsy was performed 105 days after CAR-NK infusion to examine residual FDG activity in a single lymph node. The lymphocyte population was selected based on FSC-A and SSC-A (i); subsequent doublets were excluded using SSCW versus SSC-H (ii); live cells were identified using a dye that stained for live/dead cells (iii); hematopoietic cells in the live population were then selected by gating for CD45 ⁺ cells (iv). Myeloid cells were excluded by gating on CD33-negative and CD14-negative cells (v); NK cells were identified by gating on ^CD3- and CD56 ⁺ cells (vi). Within the CD3 ^- CD56 ⁺ cell subset, cord blood-derived NK cells were identified by expression of donor-specific HLA antigen (in this case, CAR-NK cells were HLAA3-positive and the recipient was HLA-A3-negative) (vii). PBMC: peripheral blood mononuclear cells, FSC-A: forward scatter area, FSC-H: forward scatter height, SSC-A: side scatter area, SSC-H: side scatter height.

[0039] Fig. 18: CAR-NK copy number in peripheral blood, bone marrow, and lymph nodes of a representative patient. This figure shows CAR-NK copy number determined by qPCR at various time points in peripheral blood (green circles), bone marrow (red circles), and lymph node (black circle) for patient 8. A lymph node biopsy was performed on day 56 after CAR-NK infusion to examine residual FDG activity in one lymph node. The biopsy revealed the formation of a necrotic mass with calcification and no evidence of lymphoma. CAR-NK transcripts can be detected by qPCR. PCR in lymph node (118897.4 copies/μg) at significantly higher levels (>25-fold) compared with peripheral blood samples and bone marrow samples collected during the same time period.

[0040] Fig. 19: Persistence of CAR-NK cells after infusion in peripheral blood and bone marrow samples. This figure shows the assessment of CAR-NK cells in peripheral blood and bone marrow samples by qPCR. Green and red dots represent peripheral blood and bone marrow samples, respectively. Green (peripheral blood) and red (bone marrow) solid lines represent the median copy number at different time points. CAR-NK transcripts were detected at similar levels in peripheral blood and bone marrow.

[0041] Fig. 20: Gating strategy for detecting donor CAR-expressing T cells after CAR-NK infusion. A flow cytometric gating strategy was used to detect donor T cells and donor CAR-expressing T cells in the peripheral blood of a representative patient (patient 6, day +8, panels i-viii and day +21, panels ix and x, after CAR-NK infusion). Lymphocyte gating was performed using FSC-A and SSC-A (i); downstream doublets were excluded using SSCW versus SSC-H (ii); live cells were identified using a dye that stained for live/dead cells (iii); hematopoietic cells in the live population were then selected by gating for CD45 ⁺ cells (iv); Myeloid cells were excluded by gating for CD33-negative and CD14-negative cells (v); T cells were identified by gating for CD3 ^{+ -CD56−} cells (vi). Within the CD3 ⁺ cell subset, cord blood-derived T cells were identified by expression of donor-specific HLA antigen (vii). CAR expression on donor T cells was further determined using an antibody directed against the CH2-CH3 hinge domain of human IgG (109606088/Jackson Immuno Rsch). The percentage represents the frequency of CD3 ⁺ T cells expressing the CAR molecule (viii). The panel shows minimal contamination by donor CAR ⁺ T cells in PBMC samples collected from a patient on days +8 (vii and viii) and day +21 (ix and x) after CAR-NK infusion. Similar results were obtained in two additional patients with available serial samples (data not shown). PBMC: peripheral blood mononuclear cells, FSC-A: forward scatter area, FSC-H: forward scatter height, SSC-A: side scatter area, SSC-H: side scatter height.

[0042] Fig. 21A-21R: Inflammatory cytokine levels in peripheral blood. Panels show the dynamics of inflammatory cytokines in peripheral blood samples after CAR-NK infusion. Horizontal lines represent median values.

[0043] Fig. 22A-22B: Patient characteristics.

[0044] Fig. 23: Characteristics of the infused CAR-NK cell product.

[0045] Fig. 24: Persistence of CAR-NK cells during observation. Persistence of CAR-NK cells was assessed in peripheral blood using qPCR.

[0046] Fig. 25: Evaluation of donor-specific antibody levels in samples collected from patients at various time points after CAR-NK infusion. (-) indicates that results were not obtained.

[0047] Fig. 26A-26B. Anti-leukemic function of CB-NK cells transduced with the CAR19-CD28-zeta-2A-IL15 vector. (Fig. 26A): Transduction efficiency (85%) of CB-NK cells (lower panel) compared to non-transduced NK cells (upper panel). Transduction was stable. (Fig. 26B): CAR-NK cells are more effective in killing CD19 ⁺ Raji tumors and primary CLL compared to non-transduced (NT) ex vivo expanded and activated NK cells with similar effector function on K562 cells. P < 0.001 (iC9/CAR.CD19/IL15+ Raji vs. NT-NK+ Raji); P < 0.001 (iC9/CAR.CD19/IL15+CLL vs. NT-NK+CLL); P=ns (iC9/CAR.CD19/IL15+K562 vs. NT-NK+K562).

[0048] Fig. 27A-27C: In vivo homing, proliferation, and anti-tumor activity of iC9/CAR.19/IL15-transduced CB-NK cells. (Fig. 27A) C9/CAR.19/IL15-transduced EGFP-FFLuc-labeled CB-NK cells homed to the site of disease (liver, spleen, bone marrow [BM]) more efficiently than CAR.19-transduced CB-NK cells or NT-NK cells. (Fig. 27B) Infusion of iC9/CAR.19/IL15-transduced CB-NK cells into NSG mice transplanted with luciferase-labeled Raji cells resulted in tumor ablation, as assessed by in vivo bioluminescence imaging. Coloring indicates luminescence intensity (red, highest; blue, lowest). (Fig. 27C). The in vivo antitumor activity of a single dose of iC9/CAR.19/IL15-transduced CB-NK cells was significantly higher than that of CB-NK cells that were either not transduced or transduced with the CAR.CD19 construct lacking IL-15. P=0.001 (iC9/CAR.CD19/IL15+Raji vs. NT-NK+Raji); P=0.044 (iC9/CAR.CD19/IL15+Raji vs. CAR.CD19+Raji); P=0.006 (CAR.CD19+Raji vs. NT-NK+Raji) P=0.182 (NT-NK+Raji vs. Raji alone).

[0049] Fig. 28A-28B: IL-15-transduced CB-NK cells did not exhibit signs of autonomous or unregulated growth. (Fig. 28A) iC9/CAR.19/IL15-transduced CB-NK cells ceased to proliferate during 6 weeks of in vitro culture with no signs of autonomous growth. (Fig. 28B) Photomicrographs of mesenteric lymph nodes showed rudimentary lymphoid tissue without lymphocytes in any of the experimental mice, which is typical for NSG mice. Images of the spleen show rudimentary periarteriolar lymphoid tissue devoid of lymphocytes (black arrows) and surrounded by hematopoietic tissue consisting of erythroid and myeloid cells at various stages of development, including megakaryocytes and hemosiderin-laden macrophages. Bone marrow contains normal hematopoietic cells and no abnormal lymphocytes. H&E stain, magnification ×200. Slides from two representative groups of NSG mice treated with iC9/CAR.19/IL15-transduced CB-NK cells are shown.

[0050] Fig. 29: IL-15 production by iC9.CAR.19.CD28.CD3ζ.IL15-transduced CB-NK cells; iC9.CAR.19.CD28.CD3ζ.IL15-transduced CB-NK cells produce IL-15 in response to antigen stimulation in vitro.

[0051] Fig. 30A-30B: Activation of the inducible suicide gene caspase-9 eliminates iC9/CAR.19/IL15 ⁺ CB-NK cells. (Fig. 30A) Addition of 10 nM AP1903 to iC9-CAR-IL15 ⁺ CB-NK cell cultures induced apoptosis/necrosis of the transgenic cells (lower right panel) within 4 hours, as assessed by annexin V-7AAD staining. NT, untransduced CB-NK cells; CAR, iC9/CAR.19/IL15-transduced NK cells; (Fig. 30B) NSG mice that were intravenously transplanted with Raji cells and infused with iC9/CAR.19/IL15 ⁺ -CB-NK cells were treated intraperitoneally with two doses of the dimerizer AP1903 (50 μg) 2 days apart 10–14 days later. After 3 days, the number of NK cells expressing iC9/CAR.19/IL15 was significantly reduced in all organs tested.

Description of illustrative embodiments of the invention

[0052] Encouraging clinical results have been obtained using human cord blood-derived natural killer (CB-NK) cells transduced with retroviral vectors targeting CD19 ⁺ lymphoid cancers. A number of additional CAR-NK cell constructs have been generated targeting antigens of myeloid tumors, multiple myeloma, and solid cancers. In some embodiments, the present invention relates to methods for sustainably expanding NK cells. Cells can be obtained from frozen or thawed CB units and expanded in gas-permeable bioreactors containing cultures together with antigen-presenting cells (APCs), such as universal antigen-presenting cells (uAPCs), or with other feeder cells and cytokines, such as interleukin (IL)-2.

[0053] One of the limitations of using CAR-NK cells in clinical therapy is their low numbers and poor survival after thawing. The current studies aim to address these limitations using a GMP-compliant strategy for ex vivo expansion of CAR-NK cells. The methods of the invention allow for an average of 2200-fold expansion in two weeks with excellent CAR transduction efficiency of approximately 66%. Using this strategy, up to 400 doses of ^1x106 CAR-NK cells per kg can be obtained for patient treatment.

[0054] Accordingly, in some embodiments, the present invention relates to methods and compositions for the production, expansion, quality control, and functional characterization of clinically valuable NK cells for cell therapy and immunotherapy. Culturing and obtaining clinically significant quantities of NK cells for infusion into patients within time constraints is an extremely challenging task even under the best of circumstances. The disclosed methods and compositions detail the technical processes for producing NK cells, the details and kinetics of achievable NK cell expansion, and molecular characterization to confirm successful cell production.

[0055] The methods of the invention provide high and relevant levels of NK cell transduction by CAR constructs and rapid production of highly active CB-CAR-NK cells that can be administered fresh or frozen for subsequent infusion. The frozen CAR-NK cell products described herein represent a truly "off-the-shelf" cell therapeutic preparation that can be thawed and administered to patients without the delay required for production.

[0056] Furthermore, the present studies demonstrated the safety of infusion of NK cells and CAR-NK cells that were not HLA-typed when administered to patients. Thus, the combination of the lack of need for HLA typing and the high efficacy of thawed products has led to a paradigm shift in the use of CAR-based therapy, in which CAR-NK cells can now be obtained as a finished product that can be administered as a drug. The strategy of the invention can also be applied to NK cells from any source, including peripheral blood, bone marrow, hematopoietic stem cells, induced pluripotent stem cells, or NK cell lines.

[0057] In specific aspects of the invention, NK cells can be isolated from cord blood of healthy donors and co-cultured with APCs, such as K-562-based feeder cells or other feeder cells, such as lymphoblastoid cell lines or spheres, and with one or more cytokines, including IL-2, IL-15, IL-12, IL-21, or IL-18. The NK cells can then be transduced with retroviral, lentiviral, adenoviral, or adeno-associated viral vectors, or electroporated using "sleeping beauty" or "railroad car" constructs targeting hematopoietic cancers and solid cancers. Transduced cells can then be further expanded in gas-permeable bioreactors, co-cultured with APCs or other feeders and IL-2 or other cytokines to produce effective CAR-transduced CB-NK cells. These cells can be administered fresh or frozen for subsequent thawing and infusion at a later date.

[0058] CAR T cells for infusion into allogeneic patients must be HLA-typed or genetically modified to remove the T cell receptor to prevent fatal graft-versus-host disease (GVHD). Previous studies have used CB units to generate clinical NK cell and CAR-NK cell products that were HLA-typed for 4/6 antigens for safety and compliance with CB graft typing requirements. However, in the present studies, 2 patients were infused with allogeneic CB-NK cells that were not HLA-matched for any antigen for the patients and did not cause toxicity. These cells were administered safely, without GVHD or other toxic effects, and with comparable persistence in patients compared to HLA-typed 4/6 NK cells. This enabled the creation of a platform according to the invention for using CB units to produce NK and CAR-NK cell products without the need for any HLA typing. Randomly selecting CB units from CB banks for NK cell production significantly increases the number of available CB units and improves the timeline and organization of therapy, thereby eliminating the need for patient HLA typing and subsequent matching with a CB unit.

[0059] In other aspects of the invention, the methods of the invention may include identifying and selecting CAR-NK producing CB units that have been typed for a killer cell immunoglobulin receptor (KIR) ligand and have not been typed to match the recipient. The resulting alloreactivity due to a KIR ligand mismatch may further enhance the activity of CAR-transduced NK cells by synergizing with CAR-mediated tumor cell recognition. Indeed, studies of the invention have shown that blocking KIR ligand interaction using HLA blocking antibodies can significantly enhance CAR-NK-mediated cytotoxicity to CLL targets (Fig. 8).

[0060] CAR-transduced NK cells of the invention can provide a commercially available source of cells for immunotherapy of many types of cancer, including both liquid and solid tumors. Retroviral transduction of CB-derived NK cells provides longer persistence and increased potency of the engineered cells for their use in immunotherapy of many types of cancer and, potentially, for the treatment of infections, including viral, bacterial, and fungal infections, and for the treatment of autoimmune disorders by targeting autoreactive B or T cells.

I. Definitions

[0061] The term "substantially free" as used herein with respect to a specified component means that none of the specified components was intentionally included in the composition and/or is present only as an impurity or in trace amounts. Thus, the total amount of the specified component, as a result of any unintentional contamination of the composition, is significantly below 0.05%, and preferably below 0.01%. Most preferred is a composition in which the amount of the specified component cannot be detected by standard analytical methods.

[0062] As used in this description, the articles "a" or "an" may mean one or more. The articles "a" or "an," when used in a claim together with the word "comprising," may mean one or more than one.

[0063] The use of the term "or" in the claims means "and/or" unless otherwise stated and refers only to alternatives or mutually exclusive alternatives, although the disclosure of the present invention supports a definition that refers only to alternatives and "and/or." The term "another" as used herein may mean at least a second or more.

[0064] In this application, the term "approximately" means that the value includes variations in error inherent in a given device, method used to determine the value, or differences that exist among individuals being examined.

[0065] The term "immune disorder," "immune-dependent disorder," or "immune-mediated disorder" refers to a disorder in which the immune response plays a key role in the development or progression of the disease. Immune-mediated disorders include autoimmune disorders, allograft rejection, graft-versus-host disease, and inflammatory and allergic conditions.

[0066] The term "immune response" means the response of an immune system cell, such as a B cell or T cell, or a natural immune cell, to a stimulus. In one embodiment of the invention, the response is specific to a particular antigen ("antigen-specific response").

[0067] "Autoimmune disease" means a disease in which the immune system produces an immune response (e.g., a B-cell or T-cell response) against an antigen produced in a normal host (i.e., an autoantigen), resulting in tissue damage. The autoantigen may originate from a host cell or may originate from a commensal organism, such as microorganisms (known as commensal microorganisms) that commonly colonize mucous membranes.

[0068] The term "therapy" or "treatment" for a disease or condition means a treatment regimen according to a protocol, which may include administering one or more drugs to a patient for the purpose of alleviating the signs or symptoms of the disease. Desirable effects of treatment include a decrease in the rate of disease progression, an alleviation or amelioration of the pathological condition, and remission or an improvement in prognosis. Alleviation may be observed before the onset of signs or symptoms of the disease or condition, as well as after their onset. Thus, "therapy" or "treatment" may include "prevention" or "prophylaxis" of the disease or undesirable condition. Furthermore, "therapy" or "treatment" does not mean a complete alleviation of signs or symptoms, and does not necessarily mean a cure, and in particular includes treatment regimens that have only a minor effect on the patient.

[0069] The term "therapeutic effect" or "therapeutically effective" as used herein refers to an action that promotes or stimulates an improvement in the health of an individual following therapeutic treatment of the disease. This term includes, but is not limited to, a reduction in the frequency or severity of signs or symptoms of the disease. For example, treating cancer may include, for example, reducing the size of a tumor, reducing the invasiveness of a tumor, reducing the rate of growth of a cancer tumor, or preventing metastasis. Treating cancer may also mean increasing the life expectancy of an individual suffering from cancer.

[0070] The terms "individual" and "patient" refer to either a human or a non-human animal, such as primates, mammals, and vertebrates. In particular embodiments of the invention, the individual is a human.

[0071] The terms "pharmaceutically or pharmacologically acceptable" refer to molecular entities and compositions that do not cause adverse, allergic, or other untoward reactions when administered to an animal, such as a human, if desired. The preparation of a pharmaceutical composition containing an antibody or an additional active ingredient will be apparent to one skilled in the art from the present disclosure. Furthermore, it should be noted that for administration to an animal (e.g., a human), these preparations must meet standards of sterility, pyrogenicity, general safety, and purity in accordance with the requirements of the FDA's Office of Biological Standards.

[0072] As used herein, the term "pharmaceutically acceptable carrier" includes any and all aqueous solvents (e.g., water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc.), anhydrous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oil, and injectable organic esters such as ethyl oleate), dispersion media, coating agents, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, and inert gases), isotonic agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, binders, fillers, disintegrating agents, lubricants, sweeteners, flavoring agents, coloring agents, liquids, and bulking agents. microelements, such as substances and their combinations known to those skilled in the art. The pH and precise concentration of the various components in the pharmaceutical composition are adjusted according to well-known parameters.

[0073] The term "haplotyping or tissue typing" refers to a method used to identify haplotypes or tissue types of an individual, such as by determining the specific HLA locus (or loci) expressed on the lymphocytes of a particular individual. HLA genes are located in the major histocompatibility complex (MHC), a region on the short arm of chromosome 6, and are involved in cell-cell interactions, the immune response, organ transplantation, cancer development, and disease susceptibility. There are six genetic loci important for transplantation, designated HLA-A, HLA-B, HLA-C, and HLA-DR, HLA-DP, and HLA-DQ. Several different alleles may be present at each locus.

[0074] A widely used haplotyping method uses polymerase chain reaction (PCR) to compare an individual's DNA to known segments of genes encoding MHC antigens. Variability in these gene regions determines the individual's tissue type, or haplotype. Serological methods are also used to detect serologically defined antigens on the surface of cells. HLA-A, -B, and -C determinants can be detected by known serological methods. Briefly, an individual's lymphocytes (isolated from fresh peripheral blood) are incubated with an antiserum that recognizes all known HLA antigens. The cells are distributed in a tray of microscopic wells containing different types of antisera. The cells are incubated for 30 minutes and then incubated with complement for an additional 60 minutes. If the lymphocytes have antigens on their surface that are recognized by the antibodies in the antiserum, the lymphocytes are lysed. A dye can be added to detect changes in cell membrane permeability and cell death. The structure of the cells destroyed by lysis indicates the degree of histological incompatibility. For example, if lymphocytes from a person being tested for HLA-A3 are destroyed in a well containing HLA-A3 antiserum, the test for this group of antigens will be positive.

[0075] The term "antigen-presenting cells (APCs)" refers to a class of cells capable of presenting one or more antigens in the form of a peptide-MHC complex recognized by specific effector cells of the immune system, and thereby inducing an effective cellular immune response against the presented antigen or antigens. The term "APCs" encompasses intact whole cells, such as macrophages, B cells, endothelial cells, activated T cells, and dendritic cells, or naturally occurring or synthetic molecules capable of presenting antigen, such as purified MHC class I molecules complexed with γ2-microglobulin.

II. Engineered NK cells

[0076] In some embodiments, the present invention relates to methods for producing NK cells engineered with an antigen receptor (e.g., a CAR and/or TCR), wherein the methods comprise incubating with artificial presenting cells (APCs) and cytokines; transducing the cells with the CAR construct; and expanding the cells in the presence of the APCs and cytokines. The CAR and/or TCR construct may be a retroviral or lentiviral vector, or it may be electroporated. The method may comprise obtaining a starting population of cells from cord blood, peripheral blood, bone marrow, CD34 ⁺ cells, or iPSCs, and in particular from cord blood. The starting population of cells may then be processed in a Ficoll-Paque density gradient to obtain mononuclear cells (MNCs). CD3, CD14, and/or CD19 cells can then be removed from MNCs to negatively select NK cells, or they can be positively selected using CD56 selection. NK cells can then be incubated with APCs and cytokines such as IL-2, IL-21, and IL-18, followed by CAR transduction, such as retroviral transduction. The engineered NK cells can be further expanded in the presence of irradiated APCs and cytokines such as IL-2.

[0077] The APCs used in the methods of the invention may be K-562-based feeder cells, lymphoblastoid cell lines, or universal antigen-presenting cells (uAPCs), or a non-cell-based method may be used, such as a bead, cellular particle, or exosome-based method. As used herein, the term "UAPC(s)" refers to antigen-presenting cells engineered for optimized expansion of immune cells, particularly NK cells. UAPCs can be engineered with a unique combination of costimulatory molecules to eliminate inhibitory signals and induce optimal and specific NK cell killing function. Exemplary APCs are generated by forced expression of membrane-bound interleukin 21 (mbIL-21) and 4-1BB ligand in the K562 NK cell-responsive antigen-presenting cell (APC) line (referred to as clone 46). In another embodiment, UAPCs were produced by forcing expression of mbIL-21, 4-1BB ligand, and CD48 in K562 cells (referred to as universal APCs (UAPCs)). In another embodiment, UAPCs were generated by forcing expression of mbIL-21, 4-1BB ligand, and CS1 in K562 cells (referred to as UAPC2s). UAPCs can be engineered to express mbIL-21, 41BBL, and an NK cell-specific antigen, such as a SLAM family antigen.

[0078] The engineered and expanded NK cells of the invention are less likely to cause graft-versus-host disease (GVHD) than standard CAR T cells in the absence of full HLA typing. Furthermore, engineered CB-derived NK cells, such as CAR-NK or TCR-NK cells, can be used to create NK cell banks for immunotherapy without the need for donors to collect NK cells.

[0079] In some embodiments, NK cells are obtained from human peripheral blood mononuclear cells (PBMCs), unstimulated leukapheresis products (PBSCs), human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs), bone marrow, or cord blood using methods well known to those skilled in the art. In particular, NK cells can be isolated from cord blood (CB), peripheral blood (PB), bone marrow, or stem cells. In specific embodiments, immune cells are isolated from a CB pool. A CB can consist of 2, 3, 4, 5, 6, 7, 8, 10, or more units. The immune cells can be autologous or allogeneic. The isolated NK cells can match the haplotype of the individual receiving the cell therapy. NK cells can be detected using specific surface markers such as CD16 and CD56 in humans.

[0080] In some aspects of the invention, the starting population of NK cells is obtained by isolating mononuclear cells using Ficoll density gradient centrifugation. The cell culture may be absent of any cells expressing CD3, CD14, and/or CD19, and these cells may be characterized to determine the percentage of CD56 ⁺ / ^CD3- cells or NK cells.

[0081] The cells are expanded in the presence of APCs, and especially irradiated APCs such as UAPCs. The expansion may occur over a period of about 2-30 days or more, such as 3-20 days, and in particular 12-16 days, such as 12, 13, 14, 15, 16, 17, 18 or 19 days, and in particular about 14 days. NK cells and APCs may be present in a ratio of about 3:1-1:3, such as 2:1, 1:1, 1:2, and in particular about 1:2. The expansion culture may further comprise cytokines that promote expansion, such as IL-2, IL-21 and/or IL-18. Cytokines may be present at a concentration of approximately 10-500 U/ml, such as 100-300 U/ml, and in particular approximately 200 U/ml. Cytokines may be replenished in the culture for expansion, for example, every 2-3 days. APCs may be added to the culture at least a second time, for example, after CAR transduction.

[0082] After expansion, the immune cells can be administered immediately by infusion, or they can be stored, for example, by cryopreservation. In some aspects of the invention, the cells can be expanded over several days, weeks, or months ex vivo in the form of a primary population obtained over approximately 1, 2, 3, 4, 5 days.

[0083] In one embodiment, the starting cell population is MNCs isolated from a single unit of CB in a Ficoll density gradient. The cells can then be washed to remove CD3, CD14, and CD19-positive cells, such as using CliniMACS immunomagnetic beads (Miltenyi Biotec). Unlabeled, enriched CB-NK cells can be collected, washed with CliniMACS buffer, counted, and pooled with irradiated (e.g., 100 Gy) APCs, such as at a 1:2 ratio. The cell mixture (e.g., 1×10 ⁶ cells/mL) can be transferred to cell culture flasks containing complete NK medium (e.g., 90% stem cell growth medium, 10% FBS, 2 mM L-glutamine) and IL-2, e.g., 50-500, e.g., 100-300, e.g., 200 U/mL. The cells can be incubated at 37°C in 5% CO ₂ . On day 3, a change in medium composition can be accomplished by collecting the cells by centrifugation and resuspending them in complete NK medium (e.g., 1×10 ⁶ cells/mL) containing IL-2, at 50-500, e.g., 100-300, e.g., 200 U/mL. The cells can be incubated at 37°C in 5% CO ₂ . On day 5, the number of wells required for retronectin transduction can be determined based on the number of CB-NK cells in the culture. The retronectin solution can be placed in 24-well culture plates. The plates can be sealed and stored in the refrigerator at 4°C.

[0084] The 2nd NK selection, performed on day 6, as well as on day 0, can be performed prior to transduction of CB-NK cells. The cells can be washed with CliniMACS buffer, centrifuged, and resuspended in complete NK medium at a concentration of 0.5 x 10 ⁶ /ml with IL-2, for example, at a level of 100-1000, and in particular, 600 units/ml. These retronectin plates can then be washed with complete NK medium and incubated at 37°C until use. Complete NK medium in each well can be replaced with retroviral supernatant, followed by centrifugation of the plates at 32°C. The retroviral supernatant can then be removed by aspiration and replaced with fresh retroviral supernatant. A CB-NK cell suspension containing 0.5 x 10 ⁶ cells and IL-2, 600 U/mL, can be added to each well, and the plates can be centrifuged. The plates can then be incubated at 37°C with 5% CO ₂ . On day 9, CAR-transduced CB-NK cells can be removed from the transduction plates, collected by centrifugation, and stimulated with irradiated (e.g., 100 Gy) aAPC, e.g., at a 1:2 ratio, in complete NK medium with IL-2, 200 U/mL. Cell culture flasks are incubated at 37°C with 5% CO ₂ . On day 12, the medium can be changed. On day 14, cells can be collected by centrifugation, the supernatant can be removed by aspiration, and the cells can be resuspended in fresh complete NK medium containing IL-2, 200 U/ml. Cell culture flasks are incubated at 37°C with 5% CO ₂ . If more than 1 × 10 ⁵ CD3 ⁺ cells/kg are present, magnetic immunodepletion of CD3 ⁺ cells can be performed using the CliniCliniMACS CD3 reagent. On day 15, cells are harvested, and the final product is prepared for infusion or cryopreservation.

[0085] Expanded NK cells can secrete type I cytokines such as interferon-γ, tumor necrosis factor-α, and granulocyte-macrophage colony-stimulating factor (GM-CSF), which activate both innate and adaptive immune cells, as well as other cytokines and chemokines. The level of these cytokines can be assessed to determine the activation status of NK cells. In addition, other known methods for determining NK cell activation can be used to characterize NK cells according to the invention.

V. Bioreactor

[0086] NK cells can be expanded in a functionally closed system, such as a bioreactor. Expansion can be accomplished in a gas-permeable bioreactor, such as a G-Rex® cell culture device. The bioreactor can support from 1×10 ⁹ to 3×10 ⁹ cells in an average volume of 450 ml.

[0087] Bioreactors can be classified according to general categories, including: static bioreactors, stirred flask bioreactors, rotating wall bioreactors, hollow fiber bioreactors, and direct perfusion bioreactors. Within bioreactors, cells can be free-floating or immobilized, that is, seeded on porous three-dimensional scaffolds (hydrogels).

[0088] Hollow fiber bioreactors can be used to increase mass transfer during culture. A hollow fiber bioreactor is a three-dimensional cell culture system based on hollow fibers, which are small semipermeable capillary membranes arranged in parallel rows with a typical molecular weight cutoff (MWCO) range of 10-30 kDa. These hollow fiber membranes are often combined and placed in tubular polycarbonate shells to create hollow fiber bioreactor cassettes. Inside the cassettes, which are also equipped with an inlet and outlet, there are two compartments: the intracapillary (IC) space within the hollow fibers and the extracapillary (EC) space surrounding the hollow fibers.

[0089] Thus, for carrying out the present invention, the bioreactor may be a hollow fiber bioreactor. Hollow fiber bioreactors may have cells embedded within the lumen of the fibers with a medium for perfusion through the extraluminal space, or, alternatively, such bioreactors may provide gas and medium through the hollow fibers along with cells growing within the extraluminal space.

[0090] The hollow fibers must be suitable for delivering nutrients and removing waste from the bioreactor. These hollow fibers can have any shape, for example, they can be round and tubular, or they can have the shape of concentric rings. These hollow fibers can be made from a resorbable or non-resorbable membrane. For example, suitable components of the hollow fibers include polydioxanone, polylactide, polygalactin, polyglycolic acid, polylactic acid, polyglycolic acid/trimethylene carbonate, cellulose, methylcellulose, cellulose polymers, cellulose ester, regenerated cellulose, pluronic, collagen, elastin, and mixtures thereof.

[0091] The bioreactor may be primed prior to cell seeding. Priming may include rinsing with a buffer such as PBS. Priming may also include coating the bioreactor with an extracellular matrix protein such as fibronectin. The bioreactor may then be rinsed with a medium such as alpha-MEM.

[0092] In specific embodiments, the methods of the invention utilize a G-Rex® bioreactor. The base of the G-Rex® flask is a gas-permeable membrane on which the cells are supported. Consequently, the cells are in a highly oxygenated environment, allowing them to grow to high densities. This system is easily scalable and requires less frequent culture manipulation. G-Rex® flasks are compatible with standard tissue culture incubators and cell laboratory equipment, reducing the need for specialized equipment and capital investment required to initiate an ACT program.

[0093] The cells can be seeded into the bioreactor at a density of about 100-1000 cells/cm ² , such as about 150 cells/cm ² , about 200 cells/cm ² , about 250 cells/cm ² , about 300 cells/cm ² , such as about 350 cells/cm ² , such as about 400 cells/cm ² , such as about 450 cells/cm ² , such as about 500 cells/cm ² , such as about 550 cells/cm ² , such as about 600 cells/cm ² , such as about 650 cells/cm ² , such as about 700 cells/cm ² , such as about 750 cells/cm ² , such as about 800 cells/cm ² , such as about 850 cells/cm ² , for example, about 900 cells/cm ² , for example, about 950 cells/cm ² or about 1000 cells/cm ² . In particular, the cells can be seeded at a density of about 400-500 cells/cm ² , for example, about 450 cells/cm ² .

[0094] The total number of cells seeded in the bioreactor can be from about 1.0×10 ⁶ to about 1.0×10 ⁸ cells, such as from about 1.0×10 ⁶ to 5.0×10 ⁶ , from 5.0×10 ⁶ to 1.0×10 ⁷ , from 1.0×10 ⁷ to 5.0×10 ⁷ , from 5.0×10 ⁷ to 1.0×10 ⁸ cells. In particular aspects of the invention, the total number of cells seeded in the bioreactor is from about 1.0×10 ⁷ to about 3.0×10 ⁷ , such as about 2.0×10 ⁷ cells.

[0095] The cells can be seeded in any suitable cell culture medium, many of which are commercially available. Representative media include DMEM, RPMI, MEM, 199 medium, HAMS, and the like. In one embodiment, the medium is alpha-MEM medium, and in particular, alpha-MEM medium supplemented with L-glutamine. The medium can be supplemented with one or more of the following components, such as growth factors, cytokines, hormones or B27, antibiotics, vitamins, and/or small molecule drugs. In particular, the medium can be serum-free.

[0096] In some embodiments, the cells may be incubated at room temperature. The incubator may be humidified and may have an atmosphere containing about 5% _CO2 and about 1% _O2 . In some embodiments, the _CO2 concentration may be in the range of about 1-20%, 2-10%, or 3-5%. In some embodiments, the _O2 concentration may be in the range of about 1-20%, 2-10%, or 3-5%.

C. Genetically engineered antigen receptors

[0097] NK cells of the invention can be genetically engineered to express antigen receptors, such as engineered TCRs and/or CARs. For example, NK cells are modified to express a TCR that has antigen specificity for a cancer antigen. Multiple CARs and/or TCRs, for example specific to different antigens, can be added to NK cells.

[0098] Suitable modification methods are known to those skilled in the art. See, for example, Sambrook and Ausubel above. For example, cells can be transduced to express a TCR having antigen specificity for a cancer antigen using the transduction methods described by Heemskerk et al., 2008 and Johnson et al., 2009.

[0099] Electroporation of RNA encoding full-length TCRα and TCRβ (or γ and δ) chains may be used as an alternative to address long-term problems associated with autoreactivity caused by pairing of retrovirally transduced TCR chains and endogenous TCR chains. Even if such alternative pairing is employed in a transient transfection strategy, it is possible that such resulting autoreactive T cells will lose their autoreactivity over time, since the introduced TCR α and β chains are expressed only transiently. If expression of the introduced TCRα and TCRβ chains decreases, only normal autologous T cells remain. This is not the case when full-length TCR chains are introduced via stable retroviral transduction, which never loses the introduced TCR chains, causing persistent autoreactivity in the patient.

[00100] In some embodiments, the cells comprise one or more nucleic acids introduced by genetic engineering that encode one or more antigen receptors, and genetically engineered products of such nucleic acids. In some embodiments, the nucleic acids are heterologous, i.e., not normally present in the cell or in a sample obtained from the cell, e.g., obtained from another organism or another cell, which, for example, are not normally found in the engineered cell and/or in the organism from which such a cell is derived. In some embodiments, the nucleic acids are not naturally occurring, e.g., they are not found in nature (e.g., they are chimeric).

[00101] In some embodiments, a CAR comprises an extracellular antigen recognition domain that specifically binds to an antigen. In some embodiments, the antigen is a protein expressed on the surface of cells. In some embodiments, the CAR is a TCR-like CAR and the antigen is a processed peptide antigen, such as a peptide antigen of an intracellular protein that, like a TCR, is recognized on the cell surface in conjunction with a major histocompatibility complex (MHC) molecule.

[00102] Examples of antigen receptors, including CARs and recombinant TCRs, as well as methods for constructing and introducing receptors into cells, are described, for example, in International Patent Application Publications WO 200014257, WO 2013126726, WO 2012/129514, WO 2014031687, WO 2013/166321, WO 2013/071154, WO 2013/123061, in US Patent Application Publications US 2002131960, US 2013287748, US 20130149337, in US Patents: 6451995, 7446190, 8252592, 8339645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, and in European Patent Application EP2537416, and/or in Sadelain et al., 2013; Davila et al., 2013; Turtle et al., 2012; Wu et al., 2012. In some aspects of the invention, genetically engineered antigen receptors include CARs as described in U.S. Patent No. 7,446,190, and as described in International Patent Application Publication WO/2014055668 Al.

1. Chimeric antigen receptors

[00103] In some embodiments of the invention, a CAR comprises: a) an intracellular signaling domain, b) a transmembrane domain, and c) an extracellular domain containing an antigen-binding region.

[00104] In some embodiments of the invention, engineered antigen receptors comprise CARs, including activating or stimulatory CARs, costimulatory CARs (see WO 2014/055668), and/or inhibitory CARs (iCARs, see Fedorov et al., 2013). CARs typically include an extracellular domain that binds to an antigen (or ligand) and is linked to one or more intracellular signaling components, and in some aspects of the invention, they are linked via linkers and/or transmembrane domain(s). Such molecules typically mimic or attract a signal via a natural antigen receptor, via such a receptor in combination with a costimulatory receptor, and/or via a costimulatory receptor alone.

[00105] Some embodiments of the invention relate to the use of nucleic acids, including nucleic acids encoding an antigen-specific CAR polypeptide, including a CAR that has been humanized to reduce immunogenicity (hCAR), and which comprises an intracellular signaling domain, a transmembrane domain, and an extracellular domain containing one or more signaling motifs. In some embodiments, a CAR can recognize an epitope comprising a common space between one or more antigens. In some embodiments, the binding region can include complementarity-determining regions of a monoclonal antibody, variable regions of a monoclonal antibody, and/or antigen-binding fragments thereof. In another embodiment, such specificity is due to a peptide (e.g., a cytokine) that binds to the receptor.

[00106] It is contemplated that human CAR nucleic acids may be human genes used to enhance the efficacy of cellular immunotherapy for patients. In a specific embodiment, the present invention includes a full-length CAR cDNA or the coding region thereof. The antigen-binding regions or domain may comprise fragments of the V _H and V _L chains of a single-chain variable fragment (scFv) derived from a specific human monoclonal antibody, such as those described in U.S. Patent 7,109,304, incorporated herein by reference. The fragment may also be any number of different antigen-binding domains of a human antigen-specific antibody. In a more specific embodiment, the fragment is an antigen-specific scFv encoded by a sequence that is optimized for human codons for expression in human cells.

[00107] The construct may be multimeric, such as a diabody or multimers. Multimers are most often formed by cross-pairing the variable portion of the light and heavy chains to form a diabody. The hinge portion of the construct may have several alternatives, ranging from complete deletion to retention of the first cysteine to substitution of proline rather than serine and truncation down to the first cysteine. The Fc portion may be deleted. Any protein that is stable and/or dimerizes may be used for this purpose. Only one of the Fc domains may be used, such as the CH2 or CH3 domain of human immunoglobulin. The hinge region, CH2 and CH3 region of human immunoglobulin that have been modified to improve dimerization may also be used. Only the hinge portion of the immunoglobulin may also be used. Portions of CD8-alpha may also be used.

[00108] In some embodiments, the CAR nucleic acid comprises a sequence encoding other costimulatory receptors, such as a transmembrane domain and a modified intracellular signaling domain of CD28. Other costimulatory receptors include, but are not limited to, one or more of CD28, CD27, OX-40 (CD134), DAP10, DAP12, and 4-1BB (CD137). In addition to the primary signal initiated by CD3ζ, the additional signal transmitted by the human costimulatory receptor incorporated into the human CAR is important for the full activation of NK cells and can improve in vivo persistence and facilitate the successful implementation of therapeutic adoptive immunotherapy.

[00109] In some embodiments, a CAR is designed to have specificity for a particular antigen (or marker, or ligand), such as an antigen expressed on a particular cell type targeted by adoptive therapy, such as a cancer marker, and/or an antigen designed to induce a suppressive response, such as an antigen expressed on normal or healthy cells. Thus, a CAR typically includes in its extracellular portion one or more antigen-binding molecules, such as one or more antigen-binding fragments, domains, or portions, or one or more variable domains of an antibody and/or antibody molecules. In some embodiments, a CAR includes an antigen-binding portion or portions of an antibody molecule, such as a single-chain antibody fragment (scFv) derived from the variable heavy chain (VH) and variable light chain (VL) of a monoclonal antibody (mAb).

[00110] In some embodiments relating to a chimeric antigen receptor, the antigen-specific portion of the receptor (which may be referred to as the extracellular domain containing the antigen-binding region) comprises a tumor-associated antigen or a pathogen-specific antigen-binding domain. Antigens include carbohydrate antigens recognized by pattern recognition receptors, such as dectin-1. The tumor-associated antigen may belong to any species, provided that it will be expressed on the cell surface of tumor cells. Exemplary embodiments of tumor-associated antigens include CD19, CD20, carcinoembryonic antigen, alpha-fetoprotein, CA-125, MUC-1, CD56, EGFR, c-Met, AKT, Her2, Her3, melanoma-associated epithelial tumor antigen, mutated p53, mutated ras, and the like. In some embodiments, CAR can be co-expressed with a cytokine, which promotes persistence at low levels of tumor-associated antigen. For example, CAR can be co-expressed with IL-15.

[00111] The open reading frame sequence encoding the chimeric receptor may be obtained from a genomic DNA source, a cDNA source, it may be synthesized (e.g., by PCR), or it may be obtained using a combination of these. Depending on the size of the genomic DNA and the number of introns, it may be desirable to use cDNA or a combination of these, since introns have been found to stabilize mRNA. In addition, the use of endogenous or exogenous non-coding regions may be an additional advantage for stabilizing mRNA.

[00112] It is contemplated that the chimeric construct may be introduced into immune cells as naked DNA or in a suitable vector. Methods for stable transfection of cells by electroporation using naked DNA are known to those skilled in the art. See, for example, U.S. Patent No. 6,410,319. Naked DNA generally refers to DNA encoding the chimeric receptor contained in an expression plasmid vector in the correct orientation for expression.

[00113] Alternatively, a viral vector (e.g., a retroviral vector, an adenoviral vector, an adeno-associated viral vector, or a lentiviral vector) can be used to introduce the chimeric construct into immune cells. Suitable vectors for use in accordance with the method of the invention are non-replicating vectors in immune cells. It is known that a large number of vectors are based on viruses, where the number of copies of the virus maintained in the cell is low enough to maintain the viability of the cell, and such vectors are, for example, vectors based on HIV, SV40, EBV, HSV, or BPV.

[00114] In some aspects of the invention, the antigen-specific binding or recognition component is linked to one or more transmembrane and intracellular signaling domains. In some embodiments, the CAR comprises a transmembrane domain linked to an extracellular domain of the CAR. In one embodiment, a transmembrane domain is used that is inherently linked to one of the domains in the CAR. In some cases, the transmembrane domain is selected or modified by amino acid substitution to avoid linkage of such domains to transmembrane domains of the same or different surface membrane proteins, which will help minimize interactions with other members of the receptor complex.

[00115] In some embodiments of the invention, the transmembrane domain is derived from either a natural or synthetic source. If the source is natural, the domain, in some aspects of the invention, is derived from any membrane-bound or transmembrane protein. Transmembrane regions include regions derived from (i.e., comprising at least the transmembrane region(s)) of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS/CD278, GITR/CD357, NKG2D, and DAP molecules. Alternatively, the transmembrane domain, in some embodiments, is synthetic. In some aspects of the invention, the synthetic transmembrane domain comprises predominantly hydrophobic residues, such as leucine and valine. In some aspects of the invention, a triplet of phenylalanine, tryptophan, and valine will be present at each end of the synthetic transmembrane domain.

[00116] In some embodiments, the key technologies disclosed herein for genetically modifying immune cells, such as NK cells, include: (i) non-viral gene transfer using an electroporation device (e.g., a nucleofector), (ii) CARs that signal through endodomains (e.g., CD28/CD3-ζ, CD137/CD3-ζ, or other combinations), (iii) CARs with extracellular domains of varying lengths connecting the antigen recognition domain to the cell surface, and, in some cases, (iv) artificial antigen-presenting cells (aAPCs), which are derived from K562 and which can reliably and significantly expand the number of CAR ⁺ immune cells (Singh et al., 2008; Singh et al., 2011).

2. T cell receptor (TCR)

[00117] In some embodiments of the invention, genetically engineered antigen receptors include recombinant TCRs and/or TCRs cloned from natural T cells. "T cell receptor" or "TCR" means a molecule that comprises variable chains α and β (also known as TCRα and TCRβ, respectively) or variable chains γ and δ (also known as TCRγ and TCRδ, respectively), and that is capable of specifically binding to an antigenic peptide associated with an MHC receptor. In some embodiments of the invention, the TCR has the αβ form.

[00118] Typically, TCRs that exist in αβ and γδ forms are generally similar in structure, but the T cells expressing them may have different anatomical locations or functions. TCRs may be present on the cell surface or in soluble form. Typically, TCRs are found on the surface of T cells (or T lymphocytes), where they are typically responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules. In some embodiments, a TCR may also comprise a constant domain, a transmembrane domain, and/or a short cytoplasmic tail (see, e.g., Janeway et al., 1997). For example, in some aspects of the invention, each TCR chain may have one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminus. In some embodiments, the TCR is associated with invariant proteins of the CD3 complex involved in mediating signal transduction. Unless otherwise noted, the term "TCR" should be understood to encompass functional fragments of the TCR. This term also includes intact or full-length TCRs, including TCRs in the αβ or γδ form.

[00119] Thus, according to the present invention, the term "TCR" includes any TCR or a functional fragment thereof, such as an antigen-binding portion of a TCR that binds to a specific antigen peptide associated with an MHC molecule, i.e., an MHC-peptide complex. An "antigen-binding portion" or "antigen-binding fragment" of a TCR, which may be used synonymously, means a molecule that contains part of the structural domains of a TCR, but which binds to an antigen (e.g., in the form of an MHC-peptide complex) to which a full-length TCR binds. In some cases, the antigen-binding portion contains variable domains of a TCR, such as a variable α-chain and a variable β-chain of a TCR, sufficient to form a binding site for a specific MHC-peptide complex, where typically each chain contains three complementarity-determining regions.

[00120] In some embodiments, the variable domains of the TCR chains associate to form loops or complementarity determining regions (CDRs), similar to immunoglobulins, which mediate antigen recognition and determine peptide specificity by forming a TCR molecule binding site and determining peptide specificity. Typically, as in the case of immunoglobulins, CDRs are separated by framework regions (FRs) (see, e.g., Jores et al., 1990; Chothia et al., 1988; Lefranc et al., 2003). In some embodiments, CDR3 is the primary CDR responsible for recognizing processed antigen, although CDR1 of the alpha chain has also been shown to interact with the N-terminal portion of the antigenic peptide, while CDR1 of the beta chain interacts with the C-terminal portion of the peptide. CDR2 is believed to recognize the MHC molecule. In some embodiments, the β-chain variable region may comprise an additional hypervariable region (HV4).

[00121] In some embodiments of the invention, the TCR chains comprise a constant domain. For example, like immunoglobulins, the extracellular portion of TCR chains (e.g., α chains, β chains) may contain two immunoglobulin domains, a variable domain (e.g., V _a or Vp; typically amino acids 1 to 116 according to Kabat numbering, as described in Kabat et al., "Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, ^5th ed.) at the N-terminus and one constant domain (e.g., the α chain constant domain or C _a , typically amino acids 117 to 259 according to Kabat numbering, the β chain constant domain or Cp, typically amino acids 117 to 295 according to Kabat numbering) that is adjacent to the cell membrane. For example, in some cases, The extracellular portion of the TCR, formed by two chains, contains two membrane-proximal constant domains and two membrane-distal variable domains containing the CDRs. The constant domain of the TCR domain contains short junctional sequences in which a cysteine residue forms a disulfide bond linking the two chains. In some embodiments, the TCR may have an additional cysteine residue in each of the α- and β-chains, and therefore the TCR contains two disulfide bonds in the constant domains.

[00122] In some embodiments, the TCR chains may comprise a transmembrane domain. In some embodiments, the transmembrane domain has a positive charge. In some cases, the TCR chains comprise a cytoplasmic tail. In some cases, the structure of the TCR allows it to bind to other molecules, such as CD3. For example, a TCR comprising constant domains with a transmembrane region can anchor the protein to the cell membrane and bind to invariant subunits of the CD3 signaling apparatus or complex.

[00123] Typically, CD3 is a multiprotein complex that may have three separate chains (γ, δ, and ε) in mammals and a ζ chain. For example, in mammals, the complex may contain a CD3γ chain, a CD3δ chain, two CD3ε chains, and a homodimer of CD3ζ chains. The CD3γ, CD3δ, and CD3ε chains are highly related proteins of the immunoglobulin superfamily that contain a single immunoglobulin domain on the cell surface. The transmembrane regions of the CD3γ, CD3δ, and CD3ε chains are negatively charged and differ in that they can bind to the positively charged chains of the T-cell receptor. The intracellular tails of the CD3γ, CD3δ, and CD3ε chains contain one conserved motif known as an immunoreceptor tyrosine-based activation motif, or ITAM, while each CD3ζ chain has three such motifs. ITAMs are typically involved in signal transduction within the TCR complex. These accessory molecules have negatively charged transmembrane regions and play a role in signal transduction from the TCR to the cell. The CD3 and ζ chains, together with the TCR, form the so-called T-cell receptor complex.

[00124] In some embodiments, the TCR may be a heterodimer of two chains, α and β (or optionally γ and δ), or it may be a single-chain TCR construct. In some embodiments, the TCR is a heterodimer comprising two separate chains (α- and β-chains or γ- and δ-chains) that are linked, for example, by a disulfide bond or disulfide bonds. In some embodiments, a TCR for a target antigen (e.g., a cancer antigen) is identified and introduced into cells. In some embodiments, a nucleic acid encoding the TCR can be obtained from various sources, for example, by amplifying publicly available TCR DNA sequences using the polymerase chain reaction (PCR). In some embodiments, the TCR is obtained from a biological source, such as cells, for example, T cells (e.g., cytotoxic T cells), T cell hybridomas, or other commonly available sources. In some embodiments, the T cells can be obtained from cells isolated in vivo. In some embodiments, a high-affinity T cell clone can be isolated from a patient and a TCR can be isolated from it. In some embodiments, the T cells can be cultured T cell hybridomas or clone. In some embodiments, the TCR clone for the target antigen was obtained in transgenic mice engineered with genes from the human immune system (e.g., the human leukocyte antigen or HLA). See, for example, tumor antigens (e.g., those described by Parkhurst et al., 2009 and Cohen et al., 2005). In some embodiments, phage display technology is used to isolate TCRs specific for a target antigen (see, e.g., Varela-Rohena et al., 2008 and Li, 2005). In some embodiments, a TCR or an antigen-binding portion thereof can be synthesized based on TCR sequence information.

D. Antigen-presenting cells

[00125] Antigen-presenting cells, which include macrophages, B lymphocytes, and dendritic cells, vary in the expression of a specific MHC molecule. APCs internalize antigen and reexpress a portion of this antigen along with the MHC molecule on their outer cell membrane. MHC is a large genetic complex with multiple loci. MHC loci encode two major classes of membrane MHC molecules, called MHC class I and class II. Helper T lymphocytes typically recognize antigen bound to MHC class II molecules, and cytotoxic T lymphocytes recognize antigen bound to MHC class I molecules. In humans, MHC is called the HLA complex, and in mice, the H-2 complex.

[00126] In some cases, aAPCs may be used in the preparation of therapeutic compositions and cell therapy products according to embodiments of the invention. General guidance regarding the preparation and use of antigen-presenting systems can be found, for example, in U.S. Patent Nos. 6,225,042, 6,355,479, 6,362,001, and 6,790,662; U.S. Patent Application Publication Nos. 2009/0017000 and 2009/0004142; and International Application Publication No. WO2007/103009.

[00127] aAPC systems may comprise at least one exogenous accessory molecule. Any suitable amounts and combinations of accessory molecules may be used. The accessory molecule may be selected from accessory molecules such as costimulatory molecules and adhesion molecules. Examples of costimulatory molecules include CD86, CD64 (FcγRI), 41BB ligand, and IL-21. Adhesion molecules may include carbohydrate-binding glycoproteins such as selectins; transmembrane-binding glycoproteins such as integrins, calcium-dependent proteins such as cadherins, and single-spanning immunoglobulin (Ig) superfamily proteins such as intercellular adhesion molecules (ICAMs), which stimulate, for example, cell-to-cell or cell-to-matrix contact. Examples of adhesion molecules include LFA-3 and ICAMs such as ICAM-1. Technologies, methods, and reagents suitable for the selection, cloning, production, and expression of representative accessory molecules, including costimulatory molecules and adhesion molecules, are described, for example, in U.S. Patents 6,225,042, 6,355,479, and 6,362,001.

E. Antigens

[00128] Among the antigens targeted by genetically engineered antigen receptors are antigens expressed in specific diseases, conditions, or in a specific cell type that is targeted by adoptive cell therapy. Such diseases and conditions include proliferative, neoplastic, and malignant diseases and disorders, including cancer and tumors, including cancer of the hematopoietic organs, cancer of the immune system, such as lymphomas, leukemias, and/or myelomas, such as B-, T-, and myeloid leukemias, lymphomas, and multiple myelomas. In some embodiments, the antigen is selectively expressed or overexpressed on cells in such a disease or condition, such as tumor or pathogenic cells, compared to normal cells or non-target cells or tissues. In other embodiments, the antigen is expressed on normal cells and/or on engineered cells.

[00129] Any suitable antigen may be used in the method of the invention. Examples of antigens include, but are not limited to, antigenic molecules of infectious agents, autologous antigens, tumor/cancer-associated antigens, and tumor neoantigens (Linnemann et al., 2015). In particular aspects of the invention, antigens include NY-ESO, EGFRvIII, Muc-1, Her2, CA-125, WT-1, Mage-A3, Mage-A4, Mage-A10, TRAIL/DR4, and CEA. In particular aspects of the invention, antigens for two or more antigen receptors include, but are not limited to, CD19, EBNA, WT1, CD123, NY-ESO, EGFRvIII, MUC1, HER2, CA-125, WT1, Mage-A3, Mage-A4, Mage-A10, TRAIL/DR4 and/or CEA. The sequences of these antigens are known to those skilled in the art, for example, the sequences of CD19 (Accession number NG_007275.1), EBNA (Accession number NG_002392.2), WT1 (Accession number NG_009272.1), CD123 (Accession number NC_000023.11), NY-ESO (Accession number NC_000023.11), EGFRvIII (Accession number NG_007726.3), MUC1 (Accession number NG_029383.1), HER2 (Accession number NG_007503.1), CA-125 (Accession number NG_055257.1), WT1 (Accession number NG_009272.1), Mage-A3 (Accession number NG_013244.1), Mage-A4 (accession number NG_013245.1), Mage-A10 (accession number NC_000023.11), TRAIL/DR4 (accession number NC_000003.12) and/or CEA (accession number NC_000019.10).

[00130] Tumor-associated antigens may be derived from prostate, breast, colorectal, lung, pancreatic, renal, mesothelioma, ovarian, or melanoma cancer. Typical tumor-associated antigens or tumor cell-derived antigens include MAGE 1, 3, and MAGE 4 (or other MAGE antigens, such as those disclosed in International Patent Application Publication No. WO 99/40188); PRAME; BAGE; RAGE, Lage (also known as NY ESO 1); SAGE; and HAGE or GAGE. Non-limiting examples of tumor antigens include antigens that are expressed in a wide variety of tumors, such as melanoma, lung carcinoma, sarcoma, and bladder carcinoma. See, for example, U.S. Patent No. 6,544,518. Antigens associated with prostate cancer tumor include, for example, prostate-specific membrane antigen (PSMA), prostate-specific antigen (PSA), prostatic acid phosphates, NKX3.1, and six-transmembrane epithelial antigen of the prostate (STEAP).

[00131] Other tumor-associated antigens include Plu-1, HASH-1, HasH-2, Cripto, and Criptin. Additionally, a tumor antigen may be a self-peptide hormone, such as full-length gonadotropin-releasing hormone (GnRH), a short peptide of 10 amino acids suitable for the treatment of many types of cancer.

[00132] Tumor antigens include tumor antigens derived from cancer that is characterized by expression of a tumor-associated antigen, such as HER-2/neu expression. Tumor-associated antigens of interest include lineage-specific tumor antigens, such as the melanocyte-melanoma lineage antigens MART-1/Melan-A, gp100, gp75, mda-7, tyrosinase, and tyrosinase-related protein. Representative tumor-associated antigens include, but are not limited to, tumor antigens derived from or including any one or more of p53, Ras, c-Myc, cytoplasmic serine/threonine kinases (e.g., A-Raf, B-Raf, and C-Raf, cyclin-dependent kinases), MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, MART-1, BAGE, DAM-6, -10, GAGE-1, -2, -8, GAGE-3, -4, -5, -6, -7B, NA88-A, MART-1, MC1R, Gp100, PSA, PSM, tyrosinase, TRP-1, TRP-2, ART-4, CAMEL, CEA, Cyp-B, hTERT, hTRT, iCE, MUC1, MUC2, phosphoinositide 3-kinase (PI3K), TRK receptors, PRAME, P15, RU1, RU2, SART-1, SART-3, Wilms tumor antigen (WT1), AFP, catenin/m, caspase-8/m, CEA, CDK-4/m, ELF2M, GnT-V, G250, HSP70-2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, myosin/m, RAGE, SART-2, TRP-2/INT2, 707-AP, annexin II, CDC27/m, TPI/mbcr-abl, BCR-ABL, interferon regulatory factor 4 (IRF4), ETV6/AML, LDLR/FUT, Pml/RAR, tumor-associated calcium signal transducer 1 (TACSTD1) TACSTD2, receptor tyrosine kinases (e.g., epidermal growth factor receptor (EGFR) (in particular, EGFRvIII), platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR)), cytoplasmic tyrosine kinases (e.g., src family, syk-ZAP70 family), integrin-linked kinase (ILK), signal transducers and activators of transcription STAT3, STATS and STATE, hypoxia-inducible factors (e.g., HIF-1 and HIF-2), nuclear factor kappa B (NF-κB), Notch receptors (e.g., Notch1-4), c-Met, mammalian target of rapamycin (mTOR), WNT, extracellular signal-regulated kinases (ERKs) and their regulatory subunits, PMSA, PR-3, MDM2, mesothelin, renal cell carcinoma-5T4, SM22-alpha, carbonic anhydrases I (CAI) and IX (CAIX) (also known as G250), STEAD, TEL/AML1, GD2, proteinase 3, hTERT, sarcoma translocation checkpoints, EphA2, ML-IAP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, GD3, fucosyl-GM1, mesothelin, PSCA, sLe, PLAC1, GM3, BORIS, Tn, GLoboH, NY-BR-1, RGsS, SART 3, STn, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, legumain, TIE2, Page4, MAD-CT-1, FAP, MAD-CT-2, fos-associated antigen 1, CBX2, CLDN6, SPANX, TPTE, ACTL8, ANKRD30A, CDKN2A, MAD2L1, CTAG1B, SUNC1, LRRN1, and idiotype.

[00133] Antigens may include epitope regions or epitope peptides derived from genes mutated in tumor cells or from genes transcribed at different levels in tumor cells compared to normal cells, such as telomerase enzyme, survivin, mesothelin, mutated ras, bcr/abl rearrangement protein, Her2/neu, mutated or wild-type p53, cytochrome P450 1B1, and abnormally expressed intronic sequences such as N-acetylglucosaminyl transferase-V; clonal immunoglobulin gene rearrangement proteins that form unique myeloma and B-cell lymphoma idiotypes; tumor antigens that include epitope regions or epitope peptides derived from oncoviral processes, such as human papillomavirus proteins E6 and E7; Epstein-Barr virus protein LMP2; non-mutated oncofetal proteins with tumor-selective expression, such as carcinoembryonic antigen and alpha-fetoprotein.

[00134] In other embodiments of the invention, the antigen was obtained or is derived from a pathogenic microorganism or opportunistic microorganism (also referred to herein as an infectious disease-causing microorganism), such as a virus, fungus, parasite, or bacterium. In some embodiments of the invention, antigens derived from such a microorganism include full-length proteins.

[00135] Representative pathogenic microorganisms whose antigens are contemplated for use in the method described herein include human immunodeficiency virus (HIV), herpes simplex virus (HSV), respiratory syncytial virus (RSV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), influenza A, B, and C viruses, vesicular stomatitis virus (VSV), polyomavirus (e.g., BK virus and JC virus), adenovirus, staphylococci of various species, including methicillin-resistant Staphylococcus aureus (MRSA), and streptococci of various species, including Streptococcus pneumoniae. As will be apparent to one of skill in the art, proteins obtained from these and other pathogenic microorganisms for use as an antigen as described in this application, and the nucleotide sequences encoding these proteins, can be found in publications and in publicly available databases such as GENBANK®, SWISS-PROT® and TREMBL®.

[00136] Antigens derived from the human immunodeficiency virus (HIV) include any of the structural proteins of the HIV virion (e.g., gp120, gp41, p17, p24), protease, reverse transcriptase, or the HIV proteins encoded by tat, rev, nef, vif, vpr, and vpu.

[00137] Antigens derived from herpes simplex virus (e.g., HSV1 and HSV2) include, but are not limited to, proteins expressed by HSV late genes. The group of late genes predominantly encodes proteins that form the virion particle. Such proteins include five proteins (UL) that form the viral capsid: UL6, UL18, UL35, UL38 and the major capsid protein UL19, UL45 and UL27, each of which can be used as an antigen described herein. Other representative HSV proteins considered for use as antigens are ICP27 (H1, H2), glycoprotein B (gB), and glycoprotein D (gD). The HSV genome includes at least 74 genes, each of which encodes a protein that can potentially be used as an antigen.

[00138] Cytomegalovirus (CMV)-derived antigens include CMV structural proteins, viral antigens expressed during the pre-early and early phases of viral replication; glycoproteins I and III; capsid protein; envelope protein; lower matrix protein pp65 (ppUL83); p52 (ppUL44), IE1 and 1E2 (UL123 and UL122); protein products of the UL128-UL150 gene cluster (Rykman, et al., 2006); envelope glycoprotein B (gB), gH, gN, and pp150. As will be appreciated by those skilled in the art, CMV proteins for use as the antigens described herein can be found in publicly available databases such as GENBANK®, SWISS-PROT®, and TREMBL® (see, e.g., Bennekov et al., 2004; Loewendorf et al., 2010; Marschall et al., 2009).

[00139] Epstein-Barr virus (EBV)-derived antigens contemplated for use in certain embodiments of the invention include the EBV lytic proteins gp350 and gp110, EBV proteins produced during the latent cycle of infection, including Epstein-Barr nuclear antigen (EBNA)-1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C, EBNA leader protein (EBNA-LP), and latent membrane proteins (LMP)-1, LMP-2A, and LMP-2B (see, e.g., Lockey et al., 2008).

[00140] Antigens derived from respiratory syncytial virus (RSV) that are contemplated for use in the present invention include any of the eleven proteins encoded by the RSV genome, or antigenic fragments thereof: NS1, NS2, N (nucleocapsid protein), M (matrix protein), SH, G and F (viral envelope proteins), M2 (second matrix protein), M2-1 (elongation factor), M2-2 (transcription regulatory factor), RNA polymerase and phosphoprotein P.

[00141] Vesicular stomatitis virus (VSV)-derived antigens contemplated for use in the present invention include any of the five major proteins encoded by the VSV genome and antigenic fragments thereof: large protein (L), glycoprotein (G), nucleoprotein (N), phosphoprotein (P), and matrix protein (M) (see, e.g., Rieder et al., 1999).

[00142] Influenza virus-derived antigens contemplated for use in some embodiments of the invention include hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix proteins M1 and M2, NS1, NS2 (NEP), PA, PB1, PB1-F2, and PB2.

[00143] Typical viral antigens also include, but are not limited to, adenovirus polypeptides, alphavirus polypeptides, calicivirus polypeptides (e.g., calicivirus capsid antigen), coronavirus polypeptides, plague virus polypeptides, Ebola virus polypeptides, enterovirus polypeptides, and flavivirus polypeptides. hepatitis B (AE) virus polypeptides (hepatitis B core or surface antigen, hepatitis C virus E1 or E2 glycoproteins, essential or nonstructural proteins), herpesvirus polypeptides (including herpes simplex virus glycoprotein or varicella-zoster virus glycoprotein), infectious peritonitis virus polypeptides, leukemia virus polypeptides, Marburg virus polypeptides, orthomyxovirus polypeptides, papillomavirus polypeptides, parainfluenza virus polypeptides (e.g., hemagglutinin and neuraminidase polypeptides), paramyxovirus polypeptides, parvovirus polypeptides, pestivirus polypeptides, picornavirus polypeptides (e.g., poliovirus capsid polypeptide), smallpox virus polypeptides (e.g., vaccinia virus polypeptide), rabies virus polypeptides (e.g., rabies virus glycoprotein G), polypeptides reovirus, retrovirus polypeptides and rotavirus polypeptides.

[00144] In some embodiments of the invention, the antigen may be a bacterial antigen. In some embodiments, the bacterial antigen of interest may be a secreted polypeptide. In some other embodiments, bacterial antigens include antigens that have a portion or portions of a polypeptide exposed on the outer cell surface of the bacteria.

[00145] Antigens derived from various Staphylococcus species, including methicillin-resistant Staphylococcus aureus (MRSA), that are contemplated for use in the present invention include virulence regulators such as the Agr, Sar, and Sae system, the Arl system, Sar homologues (Rot, MgrA, SarS, SarR, SarT, SarU, SarV, SarX, SarZ, and TcaR), the Srr system, and TRAP. Other staphylococcal proteins that can serve as antigens include Clp, HtrA, MsrR, aconitase, CcpA, SvrA, Msa, CfvA, and CfvB proteins (see, e.g., Staphylococcus: Molecular Genetics, 2008 Caister Academic Press, Ed. Jodi Lindsay). The genomes of two Staphylococcus aureus species (N315 and Mu50) have been sequenced and are publicly available, see, for example, PATRIC (PATRIC: VBI PathoSystems Resource Integration Center, Snyder et al., 2007). As will be apparent to those skilled in the art, Staphylococcus proteins for use as antigens can also be found in other publicly available databases, such as GenBank®, Swiss-Prot®, and TrEMBL®.

[00146] Antigens derived from Streptococcus pneumoniae that are contemplated for use in certain embodiments of the invention described herein include pneumolysin, PspA, choline-binding protein A (CbpA), NanA, NanB, SpnHL, PavA, LytA, Pht, and pilin proteins (RrgA; RrgB; RrgC). Streptococcus pneumoniae antigenic proteins are also known to those skilled in the art and can be used as an antigen in some embodiments of the invention (see, e.g., Zysk et al., 2000). The complete genome sequence of a virulent strain of Streptococcus pneumoniae has been sequenced, and as one of skill in the art will appreciate, S. pneumoniae proteins for use herein can also be found in other publicly available databases such as GENBANK®, SWISS-PROT®, and TREMBL®. Proteins of particular interest for use as antigens according to the invention include virulence factors and proteins that have been predicted to be exposed on the surface of pneumococci (see, e.g., Frolet et al., 2010).

[00147] Examples of bacterial antigens that can be used as antigens include, but are not limited to, Actinomyces polypeptides, Bacillus polypeptides, Bacteroides polypeptides, Bordetella polypeptides, Bartonella polypeptides, Borrelia polypeptides (e.g., B. burgdorferi OspA), Brucella polypeptides, Campylobacter polypeptides, Capnocytophaga polypeptides, Chlamydia polypeptides, Corynebacterium polypeptides, Coxiella polypeptides, Dermatophilus polypeptides, Enterococcus polypeptides, Ehrlichia polypeptides, Escherichia polypeptides, Francisella polypeptides, Fusobacterium polypeptides, Haemobartonella polypeptides, Haemophilus polypeptides (e.g., H. influenzae type B virus outer membrane protein), Helicobacter polypeptides, Klebsiella polypeptides, L-form bacterial polypeptides, Leptospira, Listeria polypeptides, Mycobacterium polypeptides, Mycoplasma polypeptides, Neisseria polypeptides, Neorickettsia polypeptides, Nocardia polypeptides, Pasteurella polypeptides, Peptococcus polypeptides, Peptostreptococcus polypeptides, Pneumococcus polypeptides (i.e., S. pneumoniae polypeptides) (see the description in this application), Proteus polypeptides, Pseudomonas polypeptides, Rickettsia polypeptides, Rochalimaea polypeptides, Salmonella polypeptides, Shigella polypeptides, Staphylococcus polypeptides, group A streptococcal polypeptides (e.g., S. pyogenes M proteins), group B streptococcal polypeptides (S. agalactiae), Treponema polypeptides, and Yersinia polypeptides (e.g., Y. pestis F1 and V antigens).

[00148] Examples of fungal antigens include, but are not limited to, Absidia polypeptides, Acremonium polypeptides, Alternaria polypeptides, Aspergillus polypeptides, Basidiobolus polypeptides, Bipolaris polypeptides, Blastomyces polypeptides, Candida polypeptides, Coccidioides polypeptides, Conidiobolus polypeptides, Cryptococcus polypeptides, Curvalaria polypeptides, Epidermophyton polypeptides, Exophiala polypeptides, Geotrichum polypeptides, Histoplasma polypeptides, Madurella polypeptides, Malassezia polypeptides, Microsporum polypeptides, Moniliella polypeptides, Mortierella polypeptides, Mucor polypeptides, Paecilomyces polypeptides, Penicillium polypeptides, Phialemonium polypeptides, Phialophora polypeptides, Prototheca polypeptides, Pseudallescheria, Pseudomicrodochium polypeptides, Pythium polypeptides, Rhinosporidium polypeptides, Rhizopus polypeptides, Scolecobasidium polypeptides, Sporothrix polypeptides, Stemphylium polypeptides, Trichophyton polypeptides, Trichosporon polypeptides, and Xylohypha polypeptides.

[00149] Examples of protozoan parasitic antigens include, but are not limited to, Babesia polypeptides, Balantidium polypeptides, Besnoitia polypeptides, Cryptosporidium polypeptides, Eimeria polypeptides, Encephalitozoon polypeptides, Entamoeba polypeptides, Giardia polypeptides, Hammondia polypeptides, Hepatozoon polypeptides, Isospora polypeptides, Leishmania polypeptides, Microsporidia polypeptides, Neospora polypeptides, Nosema polypeptides, Pentatrichomonas polypeptides, Plasmodium polypeptides. Examples of helminth parasitic antigens include, but are not limited to, Acanthocheilonema polypeptides, Aelurostrongylus polypeptides, Ancylostoma polypeptides, Angiostrongylus polypeptides, Ascaris polypeptides, Brugia polypeptides, Bunostomum polypeptides, Capillaria polypeptides, Chabertia polypeptides, Cooperia polypeptides, Crenosoma polypeptides, Dictyocaulus polypeptides, Dioctophyme polypeptides, Dipetalonema polypeptides, Diphyllobothrium polypeptides, Diplydium polypeptides, Dirofilaria polypeptides, Dracunculus polypeptides, Enterobius polypeptides, Filaroides polypeptides, Haemonchus polypeptides, Lagochilascaris polypeptides, Loa polypeptides, Mansonella polypeptides, Muellerius polypeptides, Nanophyetus polypeptides, Necator polypeptides, Nematodirus polypeptides, Oesophagostomum polypeptides, Onchocerca polypeptides, Opisthorchis polypeptides, Ostertagia polypeptides, Parafilaria polypeptides, Paragonimus polypeptides, Parascaris polypeptides, Physaloptera polypeptides, Protostrongylus polypeptides, Setaria polypeptides, Spirocerca polypeptides, Spirometra polypeptides, Stephanofilaria polypeptides, Strongyloides polypeptides, Strongylus polypeptides, Thelazia polypeptides, Toxascaris polypeptides, Toxocara polypeptides, Trichinella polypeptides, Trichostrongylus polypeptides, Trichuris polypeptides, Uncinaria polypeptides, and Wuchereria polypeptides (e.g., P. falciparum ring sporozoite (PfCSP)), sporozoite surface protein 2 (PfSSP2), carboxyl terminus of liver antigen 1 (PfLSA1, C-terminus) and export protein 1 (PfExp-1), Pneumocystis polypeptides, Sarcocystis polypeptides, Schistosoma polypeptides, Theileria polypeptides, Toxoplasma polypeptides, and Trypanosoma polypeptides.

[00150] Examples of ectoparasite antigens include, but are not limited to, polypeptides (including antigens as well as allergens) of fleas; ticks, including hard and soft ticks; flies such as midges, mosquitoes, sand flies, horse flies, horse flies, cow flies, deer flies, tsetse flies, winter squid, myiasis flies, and blood-sucking mosquitoes; ants; spiders, lice; mites; and true bugs such as bed bugs and triatomine bugs.

F. Suicide genes

[00151] The immune cell CAR of the invention may comprise one or more suicide genes. As used herein, the term suicide gene is defined as a gene that, when administered as a prodrug, causes the gene product to be converted to a compound that kills its host cell. Examples of suicide gene/prodrug combinations that may be used include herpes simplex virus thymidine kinase (HSV-tk) and ganciclovir, acyclovir, or FIAU; oxidoreductase and cycloheximide; cytosine deaminase and 5-fluorocytosine; thymidine kinase-thymidylate kinase (Tdk::Tmk) and AZT; and deoxycytidine kinase and cytosine arabinoside. In specific embodiments of the invention, the suicide gene is a mutant TNF-alpha that is membrane-bound and can be a target for a drug or antibody.

[00152] E. coli purine nucleoside phosphorylase is a so-called "suicide" gene that converts the prodrug 6-methylpurine deoxyriboside into a toxic purine, namely 6-methylpurine. Other examples of "suicide" genes used in prodrug therapy are the E. coli cytosine deaminase gene and the HSV thymidine kinase gene.

[00153] Representative suicide genes include CD20, CD52, EGFRv3, mutant TNF-alpha (including membrane-bound TNF-alpha), or inducible caspase 9. In one embodiment, a truncated version of EGFR III (EGFRv3) can be used as a suicide antigen that can be removed by cetuximab. Other suicide genes known to those skilled in the art that can be used in the present invention include purine nucleoside phosphorylase (PNP), cytochrome P450 (CYP) enzymes, carboxypeptidases (CP), carboxylesterase (CE), nitroreductase (NTR), guanine ribosyltransferase (XGRTP), glycosidase enzymes, methionine-α,γ-lyase (MET), and thymidine phosphorylase (TP).

G. Delivery methods

[00154] One skilled in the art has equipment suitable for constructing a vector using standard recombinant techniques (see, for example, Sambrook et al., 2001 and Ausubel et al., 1996, which are incorporated herein by reference) to express the antigen receptors of the invention. Vectors include, but are not limited to, plasmids, cosmids, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs), such as retroviral vectors (e.g., those derived from Moloney murine leukemia virus (MoMLV), MSCV, SFFV, MPSV, SNV, etc.), lentiviral vectors (e.g., those derived from HIV-1, HIV-2, SIV, BIV, FIV, etc.), adenovirus (Ad) vectors, including their replication-competent, replication-defective, and non-intestinal forms, adeno-associated virus (AAV) vectors, simian virus 40 (SV-40) vectors, bovine papillomavirus vectors, Epstein-Barr virus vectors, herpesvirus vectors, vaccinia virus vectors, murine Harvey sarcoma, murine mammary tumor virus vectors, Rous sarcoma virus vectors, parvovirus vectors, poliovirus vectors, vesicular stomatitis virus vectors, maraba virus vectors, and adenovirus group B vectors adenotucirev.

a. Viral vectors

[00155] In some aspects of the invention, viral vectors encoding an antigen receptor may be used. When creating recombinant viral vectors, nonessential genes are typically replaced by a gene or sequence thereof encoding a heterologous (or non-native) protein. A viral vector is a type of expression construct that uses viral sequences to introduce nucleic acid and, possibly, proteins into a cell. The ability of certain viruses to infect cells or enter cells via receptor-mediated endocytosis and integrate into host cell genomes, as well as stably and efficiently express viral genes, makes them attractive candidates for the transfer of foreign nucleic acids into cells (e.g., mammalian cells). Non-limiting examples of viral vectors that may be used to deliver nucleic acid according to certain aspects of the present invention are described below.

[00156] Lentiviruses are complex retroviruses that, in addition to the normal retroviral genes gag, pol, and env, contain other genes with regulatory or structural function. Lentiviral vectors are well known to those skilled in the art (see, for example, U.S. Patents 6,013,516 and 5,994,136).

[00157] Recombinant lentiviral vectors are capable of infecting non-dividing cells and can be used for in vivo and ex vivo gene transfer and for expression of nucleic acid sequences. For example, a recombinant lentivirus capable of infecting a non-dividing cell, wherein a suitable host cell has been transfected with two or more vectors carrying packaging functions, namely gag, pol and env, as well as rev and tat, is described in U.S. Patent 5,994,136, which is incorporated herein by reference.

b. Regulatory elements

[00158] Expression cassettes included in the vectors used in the present invention specifically comprise (in the 5'-3' direction) a eukaryotic transcription promoter operably linked to a protein-coding sequence, splicing signals including intermediate sequences, and a transcription termination/polyadenylation sequence. Promoters and enhancers that regulate the transcription of protein-coding genes in eukaryotic cells consist of a multitude of genetic elements. The cellular machinery is capable of assembling and integrating the regulatory information conveyed by each element, which allows different genes to create different, often complex, patterns of transcriptional regulation. The promoter used in the context of the present invention includes constitutive, inducible, and tissue-specific promoters.

(i) Promoters/enhancers

[00159] The expression constructs described herein comprise a promoter for initiating expression of the antigen receptor. The promoter typically includes a sequence that functions to initiate an initiation site for RNA synthesis. The best known example is the TATA box, but in some promoters lacking a TATA box, such as the mammalian terminal deoxynucleotidyl transferase gene promoter and the SV40 late gene promoter, a discrete element located upstream of the initiation site itself helps to fix the initiation origin. Additional promoter elements regulate the frequency of transcription initiation. Typically, they are located in the 30-110 bp region upstream of the initiation site, although a number of promoters have been shown to contain functional elements located downstream of the initiation site. To place a coding sequence "under the control" of a promoter, one of the positions must be located at the 5' end of the transcription initiation site of the transcriptional reading frame located downstream (i.e., at the 3' end) of the selected promoter. The upstream promoter stimulates DNA transcription and expression of the encoded RNA.

[00160] The distance between promoter elements is often flexible, so that promoter function is maintained when the elements are inverted or moved relative to each other. In the tk promoter, the distance between promoter elements can be increased up to 50 bp before activity begins to decline. It has been found that, depending on the promoter, individual elements can function either together or independently to result in transcriptional activation. A promoter may or may not be used in conjunction with an "enhancer," which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.

[00161] A promoter may be inherently linked to a nucleic acid sequence that can be obtained by isolating 5'-non-coding sequences located upstream of a coding segment and/or exon. Such a promoter may be referred to as "endogenous." Similarly, an enhancer may be inherently linked to a nucleic acid sequence that may be located downstream or upstream of this sequence. Alternatively, certain advantages will be achieved by placing the coding segment of a nucleic acid under the control of a recombinant or heterologous promoter, which is a promoter that is not normally associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer also means an enhancer that is not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other virus or prokaryotic or eukaryotic cell; and promoters or enhancers that are not "naturally occurring," that is, containing various elements of different transcriptional regulatory regions and/or mutations that alter expression. For example, promoters that are most commonly used in recombinant DNA construction include the β-lactamase (penicillinase), lactose, and tryptophan (trp-) promoter systems. In addition to obtaining the nucleic acid sequences of promoters and enhancers by synthesis, these sequences can be obtained using recombinant cloning and/or nucleic acid amplification technology, including PCR™, in combination with the compositions disclosed herein. In addition, it is assumed that regulatory sequences that regulate transcription and/or expression of sequences in non-nuclear organelles such as mitochondria, chloroplasts, etc. may also be used.

[00162] It goes without saying that it is very important to use a promoter and/or enhancer that effectively regulates the expression of the DNA segment in the organelle, cell type, tissue, organ, or organism selected for expression. Those skilled in the art of molecular biology are generally familiar with the use of promoters, enhancers, and cell type combinations for protein expression (see, for example, Sambrook et al., 1989, incorporated herein by reference). The promoters used may be constitutive, tissue-specific, inducible, and/or suitable under appropriate conditions to ensure high levels of expression of the introduced DNA segment, which is an advantage in large-scale production of recombinant proteins and/or peptides. The promoter may be heterologous or endogenous.

[00163] Furthermore, any promoter/enhancer combination (available, for example, in the EPDB eukaryotic promoter database, available online at epd.isb-sib.ch/) can also be used to initiate expression. Another possible embodiment of the invention is the use of a T3, T7, or SP6 cytoplasmic expression system. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is available, either as part of a delivery complex or as an additional genetic expression construct.

[00164] Non-limiting examples of promoters include early or late viral promoters such as the early or late SV40 promoters, the immediate early promoters of cytomegalovirus (CMV), the early promoters of Rous sarcoma virus (RSV); eukaryotic cell promoters such as, for example, the beta-actin promoter, the GADPH promoter, the metallothionein promoter, and sequentially located response element promoters such as the cyclic AMP response element (cre) promoters, the serum response element (sre) promoter, the phorbol ester (TPA) promoter, and the response element (tre) promoters adjacent to the minimal TATA box. Promoter sequences of human growth hormone (e.g., minimal human growth hormone promoter available in Genbank, accession number X05244, nucleotides 283-341) or mouse mammary tumor promoter (available from ATCC, catalog number ATCC 45007) can also be used. In some embodiments of the invention, the promoter is the CMV IE promoter, dectin-1, dectin-2, human CD11c, F4/80, SM22, RSV, SV40, Ad MLP, beta-actin, MHC class I or MHC class II promoter, however, any other promoter suitable for initiating expression of the therapeutic gene can be used for practical implementation of the present invention.

[00165] In some aspects of the invention, the methods of the invention also relate to enhancer sequences, i.e., nucleic acid sequences that increase promoter activity and that can act in cis orientation, and regardless of their orientation, even at relatively large distances (up to several thousand base pairs from the desired promoter). However, the function of enhancers is not necessarily limited to such large distances, and such enhancers can also function in close proximity to a given promoter.

(ii) Initiation signals and associated expression

[00166] A specific initiation signal can also be used in the expression constructs of the invention to efficiently translate the coding sequences. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational regulatory signals, including the ATG initiation codon, may also be required. One skilled in the art can easily determine these signals and obtain the necessary signals. It is well known that the initiation codon must be "in frame" with the reading frame of the desired coding sequence to ensure translation of the entire insert. Exogenous translational regulatory signals and initiation codons can be natural or synthetic. Expression efficiency can be increased by including appropriate transcriptional enhancer elements.

[00167] In some embodiments of the invention, internal ribosome entry site elements (IRES) are used to create multigene or polycistronic signals. IRES elements are capable of bypassing the ribosome scanning model of 5'-methylated Cap-dependent translation and initiate translation at internal sites. IRES elements from two members of the picornavirus family (polio and encephalomyocarditis), as well as IRES signals from mammals, have been described. IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, and each of them is separated by an IRES, resulting in the creation of polycistronic signals. Due to the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single signal.

[00168] Furthermore, certain elements of the 2A sequence can be used to create linked or co-expression of genes in the constructs of the present invention. For example, cleavage sequences can be used to co-express genes by linking open reading frames to form a single cistron. An exemplary cleavage sequence is F2A (foot-and-mouth disease virus 2A) or a "2A-like" sequence (e.g., Thosea asigna virus 2A; T2A).

(iii) Origins of replication

To propagate a vector in a host cell, it preferably contains one or more origins of replication (often referred to as "ori"), such as a nucleic acid sequence corresponding to the EBV oriP, as described above, or a genetically engineered oriP with similar or improved programming function, which is a specific nucleic acid sequence from which replication begins. Alternatively, the origin of replication of another extrachromosomally replicating virus, as described above, or an autonomously replicating sequence (ARS) can be used.

c. Selection markers and screenable markers

[00169] In some embodiments of the invention, cells containing a construct of the invention can be identified in vitro or in vivo by incorporating a marker into an expression vector. Such markers can impart an identifiable change to the cell, allowing for the easy identification of cells containing the expression vector. Typically, a selection marker is a marker that confers a property that allows for such selection. A positive selection marker is a marker whose presence allows for such selection, while a negative selection marker is a marker whose presence prevents such selection. An example of a positive selection marker is a drug resistance marker.

[00170] Typically, the inclusion of a drug selection marker facilitates cloning and identification of transformants, for example, suitable selection markers include genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, and histidinol. In addition to markers that confer a phenotype that allows transformants to be distinguished depending on conditions, other types of markers are also considered, including screenable markers such as GFP, and such screening is carried out using a colorimetric assay. Alternatively, screenable enzymes that serve as negative selection markers, such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyl transferase (CAT), can be used. Methods of using immunological markers, possibly in combination with FACS analysis, are also known to those skilled in the art. The marker used is considered unimportant, provided it can be expressed simultaneously with the nucleic acid encoding the gene product. Other examples of selection and screening markers are well known to those skilled in the art.

d. Other methods of nucleic acid delivery

[00171] In addition to viral delivery of nucleic acids encoding an antigen receptor, additional methods for delivering a recombinant gene into a given host cell are contemplated herein.

[00172] Introduction of a nucleic acid, such as DNA or RNA, into immune cells of the invention may be accomplished using any suitable methods of delivering nucleic acid for cell transformation, as described herein or as known to those skilled in the art. Such methods include, but are not limited to, direct delivery of DNA, such as by ex vivo transfection, injection, including microinjection; by electroporation; by calcium phosphate precipitation; using DEAE-dextran followed by polyethylene glycol; by direct ultrasonic loading; by liposome-mediated transfection and receptor-mediated transfection; by microparticle bombardment; by mixing with silicon carbide fibers; by Agrobacterium-mediated transformation; by drying/inhibition-mediated DNA uptake, and any combination of such methods. By using such methods, organelle(s), cell(s), tissue(s) or organism(s) can be stably or transiently transformed.

H. Modification of gene expression

[00173] In some embodiments of the invention, immune cells of the invention are modified to alter the expression of certain genes, such as the glucocorticoid receptor, the TGFβ receptor (e.g., TGFβ-RII), and/or CISH. In one embodiment, immune cells can be modified to express a dominant negative TGFβ receptor II (TGFβRIIDN), which can function as a "cytokine sink" to deplete endogenous TGFβ.

[00174] Cytokine signaling plays an important role in the normal functioning of hematopoietic cells. The SOCS protein family plays an important role in the negative regulation of cytokine signaling and acts as an intrinsic brake. CIS, a member of the SOCS protein family encoded by the CISH gene, has been identified as an important checkpoint molecule in NK cells in mice. Thus, in some embodiments, the present invention relates to knockout of CISH in immune cells to enhance cytotoxicity, for example, in NK cells and CD8 ⁺ T cells. This approach can be used alone or in combination with other checkpoint inhibitors to enhance antitumor activity.

[00175] In some embodiments of the invention, altered gene expression is achieved by a disruption in the gene, such as a knockout, insertion, missense mutation, or frameshift mutation, such as a biallelic frameshift mutation, deletion of all or part of the gene, such as one or more exons or parts, and/or knockin. For example, altered gene expression can be achieved by the action of sequence-specific nucleases or targeted nucleases, including DNA-binding targeted nucleases, such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), and RNA-dependent nucleases, such as CRISPR-associated nuclease (Cas), specifically designed to target a gene sequence or a portion thereof.

[00176] In some embodiments of the invention, altering the expression, activity, and/or function of a gene is achieved by disrupting the gene. In some aspects of the invention, the gene is modified such that its expression is reduced by at least or about 20, 30, or 40%, and typically by at least or about 50, 60, 70, 80, 80, 90, or 95% compared to expression in the absence of gene modification or in the absence of components introduced to effect the modification.

[00177] In some embodiments of the invention, the change is temporary or reversible, that is, gene expression can be restored at a later time. In other embodiments of the invention, the change is not reversible or temporary, for example, it is permanent.

[00178] In some embodiments of the invention, gene modification is achieved by inducing one or more double-strand breaks and/or one or more single-strand breaks in the gene, typically through targeted induction. In some embodiments, double-strand or single-strand breaks are provided by a nuclease, such as an endonuclease, such as a gene-targeting nuclease. In some aspects of the invention, breaks are induced in the coding region of the gene, such as in an exon. For example, in some embodiments, induction occurs approximately near the N-terminal portion of the coding region, such as in the first exon, the second exon, or a subsequent exon.

[00179] In some aspects of the invention, double-strand or single-strand breaks are repaired by a cellular repair process, such as non-homologous end joining (NHEJ) or homology-directed repair (HDR). In some aspects of the invention, the repair process is error-prone and results in gene disruption, such as a frameshift mutation, such as a biallelic frameshift mutation, which can result in a complete gene knockout. For example, in some aspects of the invention, disruption includes inducing a deletion, mutation, and/or insertion. In some embodiments, disruption results in the appearance of an early stop codon. In some aspects of the invention, the presence of an insertion, deletion, translocation, frameshift mutation, and/or premature stop codon results in impaired gene expression, activity, and/or function.

[00180] In some embodiments of the invention, gene modification is achieved using antisense methods, and such methods include the use of RNA interference (RNAi), short interfering RNA (siRNA), short hairpin RNA (shRNA), and/or ribozymes to selectively suppress or repress gene expression. siRNA technology is RNAi that uses a double-stranded RNA molecule having a sequence homologous to the nucleotide sequence of the mRNA that is transcribed from the gene and a sequence complementary to the nucleotide sequence. siRNA is typically homologous/complementary to one region of the mRNA that is transcribed from the gene, or it may be an siRNA that includes multiple RNA molecules that are homologous/complementary to different regions. In some aspects of the invention, siRNA is contained in a polycistronic construct.

2. ZFP and ZFN

[00181] In some embodiments of the invention, a DNA targeting molecule comprises a DNA-binding protein, such as one or more zinc finger proteins (ZFPs) or a transcription activator-like protein (TAL), associated with an effector protein, such as an endonuclease. Examples include ZFNs, TALEs, and TALENs.

[00182] In some embodiments, a DNA-targeting molecule comprises one or more zinc finger proteins (ZFPs) or domains thereof that bind to DNA in a sequence-specific mechanism. A ZFP or domain thereof is a protein or domain within a larger protein that binds to DNA in a sequence-specific mechanism through one or more zinc fingers, regions of amino acid sequence within the binding domain whose structure is stabilized by the coordination of a zinc ion. The term zinc finger protein that binds to DNA is often abbreviated as zinc finger protein or ZFP. Among the ZFPs are artificial ZFP domains that target specific DNA sequences, typically 9-18 nucleotides in length and created by assembling individual fingers.

[00183] ZFPs include ZFPs in which the single-finger domain is approximately 30 amino acids in length and comprises an alpha helix containing two invariant histidine residues coordinated via zinc with two cysteines and one beta turn, and having two, three, four, five, or six fingers. Typically, the sequence specificity of a ZFP can be modified by introducing amino acid substitutions at four helix positions (-1, 2, 3, and 6) on the zinc finger recognition helix. Thus, in some embodiments of the invention, a ZFP or a molecule comprising a ZFP is not naturally occurring, for example, it has been engineered to bind to a selected target site.

[00184] In some embodiments, a DNA targeting molecule is or includes a zinc finger DNA binding domain fused to a zinc finger nuclease (ZFN) DNA cleavage domain. In some embodiments, the fusion proteins comprise a cleavage domain (or cleavage half-domain) from at least one liS-type restriction enzyme and one or more zinc finger binding domains, which may or may not be engineered. In some embodiments, the cleavage domain is derived from the liS-type restriction endonuclease Fok I. Fok I typically catalyzes double-stranded DNA cleavage 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other strand.

[00185] Many gene-specific engineered zinc fingers are commercially available. For example, Sangamo Biosciences (Richmond, CA, USA) developed a zinc finger engineering platform (CompoZr) in partnership with Sigma-Aldrich (St. Louis, MO, USA), allowing researchers to bypass both zinc finger engineering and validation and thereby generate specifically targeted zinc fingers for thousands of proteins (Gaj et al., Trends in Biotechnology, 2013, 31 (7), 397-405). Some embodiments utilize commercially available zinc fingers or custom-engineered zinc fingers (see, e.g., Sigma-Aldrich, catalog numbers CSTZFND, CSTZFN, Ctil-1KT PZD0020).

3. TAL, TALE and TALEN

[00186] In some embodiments, the DNA targeting molecule comprises a natural or engineered (non-natural) DNA binding domain of a transcription activator-like (TAL) protein, such as, for example, a transcription activator-like effector protein (TALE), see, for example, U.S. Patent Publication No. 2011/0301073, which is hereby incorporated by reference in its entirety.

[00187] A TALE DNA-binding domain or TALE is a polypeptide comprising one or more TALE repeat domains/units. The repeat domains participate in the binding of the TALE to its cognate target DNA sequence. A single "repeat unit" (also referred to as a "repeat") is typically 33-35 amino acids in length and has at least some sequence homology to other TALE repeat sequences within the native TALE protein. Each TALE repeat unit includes 1 or 2 DNA-binding residues constituting a two-residue repeat variable sequence (RVD), typically at positions 12 and/or 13 of the repeat. The natural (canonical) DNA recognition code for these TALEs was designed so that the HD sequence at positions 12 and 13 binds to cytosine (C), NG binds to T, NI binds to A, NN binds to G or A, and NO binds to T. Non-canonical (atypical) RVDs are also known. In some embodiments, TALEs can be targeted to any gene by creating TAL arrays with specificity for the target DNA sequence. The target sequence typically begins with thymidine.

[00188] In some embodiments of the invention, the molecule is a DNA-binding endonuclease, such as a TALE nuclease (TALEN). In some aspects of the invention, a TALEN is a fusion protein comprising a DNA-binding domain derived from a TALE and a catalytic domain of a nuclease for cleaving a target nucleic acid sequence.

[00189] In some embodiments of the invention, a TALEN recognizes and cleaves a target sequence in a gene. In some aspects of the invention, DNA cleavage results in double-strand breaks. In some aspects of the invention, the breaks stimulate homologous recombination or non-homologous end joining (NHEJ). NHEJ is typically an imperfect repair process that often results in changes in the DNA sequence at the cleavage site. In some aspects of the invention, repair mechanisms include rejoining the remaining two ends of DNA through direct religation or through so-called microhomology-mediated end joining. In some embodiments, NHEJ repair results in small insertions or deletions and can be used to disrupt and thus repress a gene. In some embodiments, the modification can be a substitution, deletion, or addition of at least one nucleotide. In some aspects of the invention, cells in which a cleavage-induced mutagenesis event occurs, i.e., an event subsequent to an NHEJ event, can be identified and/or selected by methods well known in the art.

[00190] In some embodiments, TALE repeats are assembled to specifically target a gene (Gaj et al., 2013). A library of TALENs targeting 18,740 human protein-encoding genes was generated (Kim et al., 2013). Custom-designed TALE arrays are commercially available and are supplied by Cellectis Bioresearch (Paris, France), Transposagen Biopharmaceuticals (Lexington, KY, USA), and Life Technologies (Grand Island, NY, USA). In particular, TALENs targeting CD38 are commercially available (see Gencopoeia, catalog numbers HTN222870-1, HTN222870-2, and HTN222870-3). Examples of molecules are described, for example, in US Patent Publication Nos. US 2014/0120622 and 2013/0315884.

[00191] In some embodiments of the invention, TALENs are introduced as transgenes encoded by one or more plasmid vectors. In some aspects of the invention, the plasmid vector may contain a selection marker that allows for the identification and/or selection of cells into which the vector has been introduced.

4. RGEN (CRISPR/Cas systems)

[00192] In some embodiments of the invention, the modification is carried out using one or more DNA-binding nucleic acids, for example, using an RNA-dependent endonuclease (RGEN). For example, the modification can be carried out using clustered regularly interspersed short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas). In general, "CRISPR system" refers to transcripts and other elements involved in the expression or regulation of activity of CRISPR-associated ("Cas") genes, including sequences encoding a Cas gene, a tracr (transactivating CRISPR) sequence (e.g., tracr RNA or active partial tracr RNA), a tracr-paired sequence (including a "direct repeat" and tracr RNA-processed partial direct repeat in combination with an endogenous CRISPR system), a guide sequence (also called a "spacer" in combination with an endogenous CRISPR system), and/or other sequences and transcripts from a CRISPR locus.

[00193] A CRISPR/Cas nuclease or a CRISPR/Cas nuclease system may include a non-coding RNA molecule (guide) that sequence-specifically binds to DNA and a Cas protein (e.g., Cas9) with nuclease functional activity (e.g., with two nuclease domains). One or more elements of the CRISPR system may be derived from a type I, type II, or type III CRISPR system, such as from a specific organism containing an endogenous CRISPR system, such as Streptococcus pyogenes.

[00194] In some aspects of the invention, a Cas nuclease and rRNA (including a fusion of crRNA specific to a target sequence and an immobilized tracrRNA) are introduced into a cell. Generally, target sites at the 5' end of the rRNA target the Cas nuclease to a target site, such as a gene, through complementary base pairing. The target site can be selected based on its location immediately at the 5' end of a protospacer adjacent motif (PAM) sequence, typically NGG or NAG. Accordingly, the rRNA is targeted to a desired sequence by modifying the first 20, 19, 18, 17, 16, 15, 14, 14, 12, 11, or 10 nucleotides of the guide RNA so that they match the target DNA sequence. In general, the CRISPR system is characterized by elements that facilitate the formation of a CRISPR complex at a target sequence. Typically, a "target sequence" refers to a sequence for which a guide sequence has been designed that exhibits complementarity, where hybridization between the target sequence and the guide sequence facilitates the formation of a CRISPR complex. Perfect complementarity is optional, provided it is sufficient to initiate hybridization and stimulate the formation of a CRISPR complex.

[00195] The CRISPR system can induce double-strand breaks (DSBs) at a target site, followed by disruptions or modifications, as discussed herein. In other embodiments, Cas9 variants, which are considered "nicases," are used to introduce a single-strand break in one strand at a target site. Paired nicases can be used, for example, to increase specificity, and each is regulated by a pair of different rRNAs targeting sequences such that when single-strand breaks are introduced, a 5' overhang is simultaneously introduced. In other embodiments, catalytically inactive Cas9 is linked to a heterologous effector domain, such as a transcriptional repressor or activator, to influence gene expression.

[00196] The target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides. The target sequence may be located in the nucleus or in the cytoplasm of the cell, for example, within a cell organelle. Typically, a sequence or template that can be used for recombination at a target locus containing target sequences is called an "editing template," or an "editing polynucleotide," or an "editing sequence." In some aspects of the invention, an exogenous template polynucleotide may be called an editing template. In some aspects of the invention, the recombination is homologous recombination.

[00197] Typically, in the case of an endogenous CRISPR system, formation of a CRISPR complex (including a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs) in or near the target sequence. A tracr sequence, which may comprise or consist of all or a portion of a wild-type tracr sequence (e.g., about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85 or more nucleotides of a wild-type tracr sequence), may also form part of a CRISPR complex, such as by hybridizing at least a portion of the tracr sequence to the entire tracr pair sequence or to a portion thereof that is operably linked to a guide sequence. The tracr sequence has sufficient complementarity to hybridize to the tracr pair sequence and participates in the formation of a CRISPR complex, wherein such sequence is at least 50%, 60%, 70%, 80%, 90%, 95% or 99% complementary to the sequence along the entire length of the tracr pair sequence when optimally aligned.

[00198] One or more vectors that initiate expression of one or more CRISPR system elements can be introduced into a cell such that expression of the CRISPR system elements directly participates in the formation of a CRISPR complex at one or more target sites. The components can also be delivered to cells as proteins and/or RNA. For example, a Cas enzyme, a guide sequence associated with a tracr pair sequence, and a tracr sequence can be operably linked to separate regulatory elements in separate vectors. Alternatively, two or more elements expressed from the same or different regulatory elements can be combined in a single vector with one or more additional vectors providing any CRISPR system components not included in the first vector. The vector can contain one or more insertion sites, such as a restriction endonuclease recognition sequence (also referred to as a "cloning site"). In some embodiments, one or more insertion sites are located upstream and/or downstream of one or more sequence elements of one or more vectors. If multiple different guide sequences are used, a single expression construct can be used to direct CRISPR activity to multiple different corresponding target sequences in the cell.

[00199] The vector may comprise a regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, such as a Cas protein. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx15, Csx16, Csx1, Csx16 Csf2, Csf3, Csf4, their homologues, or modified variants thereof. These enzymes are known; For example, the amino acid sequence of the S. pyogenes Cas9 protein can be found in the SwissProt database under accession number Q99ZW2.

[00200] The CRISPR enzyme may be Cas9 (e.g., from S. pyogenes or S. pneumonia). The CRISPR enzyme may direct the cleavage of one or both strands at a site containing a target sequence, such as within the target sequence and/or within the complement of the target sequence. The vector may encode a CRISPR enzyme that has been mutated compared to the corresponding wild-type enzyme such that the mutant CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing the target sequence. For example, an aspartate to alanine (D10A) substitution in the catalytic domain of RuvC I Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (which cleaves one strand). In some embodiments, Cas9 nickase can be used in combination with a guide sequence(s), such as two guide sequences that target the sense and antisense strands of target DNA, respectively. This combination allows for cleavage of both strands and their use to induce NHEJ or HDR.

[00201] In some embodiments of the invention, the enzyme-coding sequence encoding the CRISPR enzyme has been codon-optimized for expression in specific cells, such as eukaryotic cells. The eukaryotic cells may be cells of a specific organism, or they may be derived from a specific organism, such as a mammal, including, but not limited to, humans, mice, rats, rabbits, dogs, or non-human primates. Typically, codon-optimization refers to a method of modifying a nucleic acid sequence to increase the expression level in host cells of interest by replacing at least one codon of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Different species tend to have a specific bias toward certain codons for a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of messenger RNA (mRNA) translation, which in turn is thought to depend, among other things, on the properties of the codons being translated and the availability of specific transfer RNA (tRNA) molecules. The prevalence of selected tRNAs in a cell typically reflects the most frequent occurrence of codons used in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization.

[00202] Typically, a guide sequence is any polynucleotide sequence that has sufficient complementarity to allow hybridization of the targeting polynucleotide sequence to the target sequence and direct sequence-specific binding of the CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between the guide sequence and its corresponding target sequence when optimally aligned using a suitable alignment algorithm is approximately or more than approximately 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more.

[00203] Representative rRNA sequences for NR3CS (glucocorticoid receptor) include Ex3 NR3C1 sG1 5-TGC TGT TGA GGA GCT GGA-3 (SEQ ID NO: 1) and Ex3 NR3C1 sG2 5-AGC ACA CCA GGC AGA GTT-3 (SEQ ID NO: 2). Representative rRNA sequences for TGF-beta receptor 2 include EX3 TGFBR2 sG1 5-CGG CTG AGG AGC GGA AGA-3 (SEQ ID NO: 3) and EX3 TGFBR2 sG2 5-TGG-AGG-TGA-GCA-ATC-CCC-3 (SEQ ID NO: 4). The T7 promoter, target sequence, and overlapping sequence may have the sequence TTAATACGACTCACTATAGG (SEQ ID NO: 5) + target sequence + gttttagagctagaaatagc (SEQ ID NO: 6).

[00204] Optimal alignment may be performed using any suitable sequence alignment algorithm, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wuensch algorithm, algorithms based on the Burrow-Wheeler transform program (e.g., the Burrow-Wheeler alignment algorithm), Clustal W, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).

[00205] The CRISPR enzyme may be part of a fusion protein comprising one or more heterologous protein domains. The CRISPR enzyme fusion protein may comprise any additional protein sequence and, optionally, a linker sequence between any two domains. Examples of protein domains that may be attached to the CRISPR enzyme include, but are not limited to, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcription releasing factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporter genes include, but are not limited to, glutathione 5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyl transferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, blue fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP). The CRISPR enzyme may be fused to a gene sequence encoding a protein or protein fragment that binds to DNA molecules or other cellular molecules, including, but not limited to, maltose-binding protein (MBP), S-tag, Lex A DNA-binding domain (DBD) fusions, GAL4A DNA-binding domain fusions, and HSV BP16 protein fusions. Additional domains that may form part of a fusion protein containing the CRISPR enzyme are described in U.S. Patent 20110059502, which is incorporated herein by reference.

III. Treatment methods

[00206] In some embodiments, the present invention relates to methods of immunotherapy comprising administering an effective amount of NK cells of the invention. In one embodiment, a pathological condition or disorder is treated by transferring a population of NK cells that elicit an immune response. In some embodiments, a cancer or infection is treated by transferring a population of NK cells that elicit an immune response. The present invention relates to methods of treating or slowing the progression of cancer in an individual, wherein the methods comprise administering to the individual an effective amount of an antigen-specific cellular therapy drug. The methods of the invention can also be used to treat immune disorders, including auto- or alloimmune disorders, solid cancers, blood cancers, and viral infections.

[00207] Tumors for which the methods of the invention may be useful for treatment include any malignant cells, such as those found in a solid tumor or a tumor of the hematopoietic organs. Examples of solid tumors may include, but are not limited to, tumors of organs selected from the group consisting of the pancreas, colon, cecum, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and breast. Examples of tumors of the hematopoietic organs include bone marrow tumors, T- or B-cell malignancies, leukemias, lymphomas, blastomas, myelomas, and the like. Additional examples of cancer that can be treated using the methods described herein include, but are not limited to, lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and squamous cell lung cancer), peritoneal, intestinal, or gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, rectal cancer, endometrial or uterine cancer, salivary gland carcinoma, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, various types of head and neck cancer, and melanoma.

[00208] In particular, the cancer may be of the following histologic types, and such cancers include, but are not limited to: neoplasm, malignant neoplasm; carcinoma; undifferentiated carcinoma; giant cell carcinoma and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; Adenocarcinoma in adenomatous polyp; Adenocarcinoma, hereditary intestinal polyposis; Solid carcinoma; Malignant carcinoid tumor; Bronchioloalveolar adenocarcinoma; Papillary adenocarcinoma; Chromophobe carcinoma; Acidophilic carcinoma; Oxyphilic adenocarcinoma; Basophilic carcinoma; Clear cell adenocarcinoma; Granular cell carcinoma; Follicular adenocarcinoma; Papillary-follicular adenocarcinoma; Nonencapsulating sclerosing carcinoma; Adrenal cortex carcinoma; Endometrial carcinoma; Carcinoma of skin derivatives; Apocrine adenocarcinoma; Adenocarcinoma of the sebaceous glands; Ceruminous adenocarcinoma; Mucoepidermoid carcinoma; Cystadenocarcinoma; Papillary cystadenocarcinoma; Papillary serous cystadenocarcinoma; Mucinous cystadenocarcinoma; Mucinous adenocarcinoma; Rochelle cell carcinoma; Infiltrating ductal carcinoma; Medullary carcinoma; Lobular carcinoma; Inflammatory carcinoma; Paget's disease, breast carcinoma; Acinar cell carcinoma; Adenosquamous cell carcinoma; Adenocarcinoma with squamous cell metaplasia; Malignant thymoma; Malignant tumor of ovarian stroma; Malignant thecoma; Malignant granulosa cell tumor; Malignant androblastoma; Sertoli cell carcinoma; Malignant Leydig cell tumor; Malignant fat cell tumor; Malignant paraganglioma; Malignant paraganglioma of all organs except breast; Pheochromocytoma; Glomangiosarcoma; Malignant melanoma; Amelancic melanoma; Superficial spreading melanoma; Malignant lentigo melanoma; Acral lentiginous melanomas; Nodular melanomas; Malignant melanoma in giant pigmented nevus; Epithelioid cell melanoma; Malignant blue nevus; Sarcoma; Fibrosarcoma; Malignant fibrous histiocytoma; Myxosarcoma; Liposarcoma; Leiomyosarcoma; Rhabdomyosarcoma; Embryonal rhabdomyosarcoma; Alveolar rhabdomyosarcoma; Stromal sarcoma; Malignant mixed tumor; Mixed Müller cell tumor; Nephroblastoma; Hepatoblastoma; Carcinosarcoma; Malignant mesenchymoma; Malignant Brenner tumor; Malignant phyllodes tumor; Synovial sarcoma; Malignant mesothelioma; Dysgerminoma; Embryonal carcinoma; Malignant teratoma; Malignant tumor of struma of ovary; Choriocarcinoma; Malignant mesonephroma; Hemangiosarcoma; Malignant hemangioendothelioma; Kaposi's sarcoma; Malignant hemangiopericytoma; Lymphangiosarcoma; Osteosarcoma; Juxtacortical osteosarcoma; Chondrosarcoma; Malignant chondroblastoma; Mesenchymal chondrosarcoma; Giant cell tumor of bone; Ewing's sarcoma; Malignant odontogenic tumor; Ameloblastic odontosarcoma; Malignant ameloblastoma; Ameloblastic fibrosarcoma; Malignant pinealoma; Chordoma; Malignant glioma; Ependymoma; Astrocytoma; Protoplasmic astrocytoma; Fibrillary astrocytoma; Astroblastoma; Glioblastoma; Oligodendroglioma; Oligodendroblastoma; Primitive neuroectodermal tumor; Cerebellar sarcoma; Ganglioneuroblastoma; Neuroblastoma; Retinoblastoma; Neurogenic tumor of the olfactory organs; Malignant meningioma; Neurofibrosarcoma; Malignant neurolemmoma; Malignant granular cell tumor; Malignant lymphoma; Hodgkin's disease; Hodgkin's paragranuloma; Malignant lymphoma; Small cell lymphocytic lymphoma; Malignant lymphoma: Diffuse large cell lymphoma; Malignant follicular lymphoma; Mycosis fungoides; Other specific non-Hodgkin's lymphomas; B-cell lymphoma; Low-grade/follicular non-Hodgkin lymphoma (NHL); Small lymphocytic (SL) NHL; Intermediate-grade/follicular NHL; Diffuse intermediate-grade NHL; High-grade immunoblastic NHL; High-grade lymphoblastic NHL; High-grade small cell non-cleaved NHL; Bulk tumor in NHL; Mantle cell lymphoma; AIDS-associated lymphoma; Waldenstrom's macroglobulinemia; Malignant histiocytosis; Multiple myeloma; Mast cell sarcoma; Immunoproliferative disease of the small intestine; Leukemia; Lymphocytic leukemia; Plasma cell leukemia; Erythroleukemia; Cellular lymphocytic leukemia; Myeloid leukemia; Basophilic leukemia; Eosinophilic leukemia; Monocytic leukemia; Mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; reticuloendotheliosis; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); and chronic myeloid leukemia.

[00209] Specific embodiments of the invention relate to methods of treating leukemia. Leukemia is a cancer of the blood or bone marrow that is characterized by abnormal proliferation (production through reproduction) of blood cells, usually white blood cells (leukocytes). This disease is part of a broad group of diseases called hematological neoplasms. The term "leukemia" refers to a broad spectrum of diseases. Clinically and pathologically, leukemia is divided into acute and chronic forms.

[00210] In some embodiments of the invention, immune cells are delivered to an individual in need thereof, such as an individual with cancer or an infection. The cells subsequently enhance the individual's immune system and attack the corresponding cancerous or pathogenic cells. In some cases, one or more doses of immune cells are administered to the individual. In cases where two or more doses of immune cells are administered to the individual, the duration between administrations should be sufficient to allow time for their proliferation in the individual, and in specific embodiments of the invention, the duration of time between doses is 1, 2, 3, 4, 5, 6, 7, or more days.

[00211] In some embodiments, the present invention relates to methods for treating or preventing an immune-mediated disorder. In one embodiment, the individual has an autoimmune disease. Non-limiting examples of autoimmune diseases include: alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune adrenal cortex diseases, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac disease-dermatitis, chronic fatigue and immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Guillain-Barré disease, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, lupus erythematosus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, immune-mediated diabetes mellitus type 1, myasthenia gravis, nephrotic syndrome (eg, minimal change disease, focal glomerulosclerosis, or mebranous nephropathy), pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-man syndrome, systemic lupus erythematosus, lupus erythematosus, ulcerative colitis, uveitis, vasculitides (such as polyarteritis nodosa, Takayasu arteritis, temporal arteritis/giant cell arteritis, or vascular dermatitis herpetiformis), vitiligo, and Wegener's granulomatosis. Thus, some examples of an autoimmune disease that can be treated using the methods described herein include, but are not limited to, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, diabetes mellitus type 1, Crohn's disease; ulcerative colitis, myasthenia gravis, glomerulonephritis, ankylosing spondylitis, vasculitis, or psoriasis. The individual may also have an allergic disease such as asthma.

[00212] In another embodiment of the invention, the individual is a recipient of an organ transplant or of stem cells and immune cells that are used to prevent and/or treat transplant rejection. In specific embodiments, the individual has or is at risk of developing graft-versus-host disease. GVHD is a potential complication of any transplant that utilizes or contains stem cells from a related or unrelated donor. There are two types of GVHD: acute and chronic. Acute GVHD appears within the first three months after transplantation. Signs of acute GVHD include a reddish skin rash on the arms and legs that may spread and become more severe, with peeling or blistering of the skin. Acute GVHD can also affect the stomach and intestines, in which case cramping, nausea, and diarrhea are observed. Yellowing of the skin and eyes (jaundice) indicates that acute GVHD has also affected the liver. Chronic GVHD is classified according to severity: stage/grade 1 is mild; and stage/grade 4 is severe. Chronic GVHD develops three months after transplantation or later. The symptoms of chronic GVHD are similar to those of acute GVHD, but in addition, chronic GVHD may also affect the mucous membranes of the eyes, the salivary glands in the mouth, and the glands that lubricate the lining of the stomach and intestines. Any of the immune cell populations described herein may be used. Examples of a transplanted organ include a solid organ graft, such as kidney, liver, skin, pancreas, lung, and/or heart, or a cellular graft, such as islet cells, hepatocytes, myoblasts, bone marrow, or hematopoietic or other stem cells. The graft may be a composite graft, such as facial tissue. The immune cells may be administered prior to, concurrent with, or post-transplant. In some embodiments, the immune cells are administered prior to transplantation, such as at least 1 hour, at least 12 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, or at least 1 month prior to transplantation. In one specific, non-limiting example, a therapeutically effective amount of immune cells is administered 3-5 days prior to transplantation.

[00213] In some embodiments, the individual may be administered non-myeloablative lymphodepleting chemotherapy prior to immunocellular therapy. Non-myeloablative lymphodepleting chemotherapy may be any suitable therapy that may be administered by any suitable route. Non-myeloablative lymphodepleting chemotherapy may include, for example, the administration of cyclophosphamide and fludarabine, particularly if the cancer is melanoma, which may be metastatic. An exemplary route for administering cyclophosphamide and fludarabine is intravenous administration. Similarly, any suitable dose of cyclophosphamide and fludarabine may be administered. In particular aspects of the invention, approximately 60 mg/kg of cyclophosphamide is administered for two days, followed by approximately 25 mg/ ^m2 of fludarabine for five days.

[00214] In some embodiments of the invention, a growth factor that promotes the growth and activation of immune cells is administered to the individual either simultaneously with the immune cells or after the administration of the immune cells. The immune cell growth factor may be any suitable growth factor that promotes the growth and activation of immune cells. Examples of suitable immune cell growth factors include interleukins (IL)-2, IL-7, IL-15, and IL-12, which can be used alone or in various combinations, such as IL-2 and IL-7, IL-2 and IL-15, IL-7 and IL-15, IL-2, IL-7 and IL-15, IL-12 and IL-7, IL-12 and IL-15, or IL-12 and IL-2.

[00215] Therapeutically effective amounts of immune cells can be administered by several routes, including parenteral administration, such as intravenous, intraperitoneal, intramuscular, intrasternal, or intra-articular injection or infusion.

[00216] A therapeutically effective amount of immune cells for use in adoptive cell therapy is an amount that provides the desired effect in the individual being treated. For example, such an amount may be an amount of immune cells necessary to suppress the development or to stimulate regression of an autoimmune or alloimmune disease, or an amount capable of reducing symptoms caused by an autoimmune disease, such as pain and inflammation. Such an amount may be an amount necessary to reduce symptoms associated with inflammation, such as pain, swelling, and fever. Such an amount may also be an amount necessary to reduce the degree of rejection or prevent rejection of a transplanted organ.

[00217] The immune cell population may be administered according to a treatment regimen for a given disease, such as in the form of one or more doses over a period of one to several days to alleviate the pathological condition, or in the form of periodic doses over a longer period of time to inhibit disease progression and prevent disease recurrence. The exact dose to be used in the composition will also depend on the route of administration and the severity of the disease or disorder, and such dose should be determined by the treating physician depending on the characteristics of each patient. The therapeutically effective amount of immune cells will depend on the individual being treated, the severity and type of disease, and the route of administration. In some embodiments, doses that can be used to treat patients are in the range of at least 3.8× ¹⁰⁴ , at least 3.8× ¹⁰⁵ , at least 3.8× ^{106, at least 3.8×107 ,} at least 3.8× ¹⁰⁸ , at least 3.8× ¹⁰⁹ , or at least 3.8× ¹⁰¹⁰ immune cells/ ^m2 . In a particular embodiment, the dose used to treat a human is in the range of about 3.8× ¹⁰⁹ to about 3.8× ¹⁰¹⁰ immune cells/ ^m2 . In additional embodiments of the invention, a therapeutically effective amount of immune cells may range from about 5× ¹⁰⁶ cells/kg body weight to about 7.5× ¹⁰⁸ cells/kg body weight, such as from about 2× ¹⁰⁷ cells to about 5× ¹⁰⁸ cells/kg body weight, or from about 5× ¹⁰⁷ cells to about 2× ¹⁰⁸ cells/kg body weight. The exact amount of immune cells can be easily determined by one of skill in the art depending on the age, weight, sex, and physiological state of the individual. Effective doses can be extrapolated from dose-response curves constructed from data obtained in in vitro test systems or in animal models.

[00218] Immune cells may be administered in combination with one or more other therapeutic agents to treat an immune-mediated disorder. Combination therapy may include, but is not limited to, the administration of one or more antimicrobial agents (e.g., antibiotics, antivirals, and antifungals), antineoplastic agents (e.g., fluorouracil, methotrexate, paclitaxel, fludarabine, etoposide, doxorubicin, or vincristine), immunosuppressants (e.g., fludarabine, etoposide, doxorubicin, or vincristine), immunosuppressants (e.g., azathioprine or glucocorticoids such as dexamethasone or prednisone), anti-inflammatory agents (e.g., glucocorticoids such as hydrocortisone, dexamethasone, or prednisone, or nonsteroidal anti-inflammatory agents such as acetylsalicylic acid, ibuprofen, or naproxen sodium), cytokines (e.g., interleukin-10 or transforming factor growth factor-beta), hormones (e.g., estrogen), or vaccines. Additionally, immunosuppressive or tolerogenic agents may be administered, including, but not limited to, calcineurin inhibitors (e.g., cyclosporine and tacrolimus); mTOR inhibitors (e.g., rapamycin); mycophenolate mofetil, antibodies (e.g., those recognizing CD3, CD4, CD40, CD154, CD45, IVIG, or B cells); chemotherapeutic agents (e.g., methotrexate, treosulfan, busulfan); radiation therapy agents; or chemokines, interleukins, or their inhibitors (e.g., BAFF, IL-2, anti-IL-2R antibodies, IL-4, JAK kinase inhibitors). Such additional pharmaceutical agents may be administered before, during, or after the administration of immune cells, depending on the desired effect. Such administration of cells and agent may be carried out by the same route or by different routes, either to the same site or to different sites.

IV. Pharmaceutical compositions

[00219] The present invention also relates to pharmaceutical compositions and formulations comprising immune cells (e.g., T cells or NK cells) and a pharmaceutically acceptable carrier.

[00220] The pharmaceutical compositions and formulations described herein can be prepared by mixing the active ingredients (such as an antibody or polypeptide) of the desired purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences, 22nd edition, 2012) in the form of lyophilized compositions or aqueous solutions. Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed and include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight polypeptides (less than about 10 residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or nonionic surfactants such as polyethylene glycol (PEG). Examples of pharmaceutically acceptable carriers described herein include drug dispersing agents for interstitial administration such as soluble neutral hyaluronidase glycoproteins (sHASEGPs), for example, soluble human hyaluronidase PH-20 glycoproteins such as rHuPH20 (HYLENEX®, Baxter International, Inc). Certain exemplary sHASEGPs and methods of using them, including rHuPH20, are described in U.S. Patent Publication Nos. 2005/0260186 and 2006/0104968. In one aspect of the invention, sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

V. Combination therapy

[00221] In some embodiments of the invention, compositions and methods according to embodiments of the invention include a population of immune cells in combination with at least one additional therapy. The additional therapy may be radiation therapy, surgery (e.g., lumpectomy and mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of all of the above. The additional therapy may be administered as adjuvant or neoadjuvant therapy.

[00222] In some embodiments, the adjunctive therapy is the administration of a small molecule enzyme inhibitor or an antimetastatic agent. In some embodiments, the adjunctive therapy is the administration of agents that limit side effects (e.g., agents designed to reduce the risk of occurrence and/or severity of side effects of treatment, such as anti-nausea agents, etc.). In some embodiments, the adjunctive therapy is radiation therapy. In some embodiments, the adjunctive therapy is surgery. In some embodiments, the adjunctive therapy is a combination of radiation therapy and surgery. In some embodiments, the adjunctive therapy is gamma irradiation. In some embodiments, the adjunctive therapy is therapy targeting the PBK/AKT/mTOR pathway and therapy using an HSP90 inhibitor, a tubulin inhibitor, an apoptosis inhibitor, and/or a chemopreventive agent. Additional therapy may be administered using one or more chemotherapeutic agents known to those skilled in the art.

[00223] Immunocellular therapy may be administered before, during, or after additional anticancer therapy, such as immune checkpoint therapy, or it may be administered in various combinations. Administration may be carried out either simultaneously or at intervals that may be up to several minutes, days, or weeks. In some embodiments of the invention, where immune cellular therapy is administered to a patient separately from the administration of an additional therapeutic agent, it can generally be ensured that no significant period of time elapses between each administration, i.e., so that the two compounds will still be able to exert a beneficial combined effect on the patient. In such cases, it is contemplated that the patient may be administered antibody therapy and anticancer therapy at intervals ranging from about 12 to 24 or 72 hours, and more specifically, within about 6-12 hours. In some situations it may be desirable to significantly prolong the period of treatment, and sometimes such an interval between the respective administrations may range from several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8).

[00224] Various combinations may be used. For example, as indicated below, immune cell therapy is designated "A" and anticancer therapy is designated "B":

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B

B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A

B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A

[00225] Administration of any compound to a patient or therapy according to embodiments of the invention may be carried out in accordance with general protocols for the administration of such compounds and taking into account the toxicity of such agents, if any. Therefore, in some embodiments of the invention, a toxicity monitoring step is carried out, which is an integral part of the combination therapy.

1. Chemotherapy

[00226] In accordance with embodiments of the invention, a wide range of chemotherapeutic agents can be used. The term "chemotherapy" means the use of drugs to treat cancer. As used herein, the term "chemotherapeutic agent" means a compound or composition that is administered to treat cancer. These agents or drugs are classified by their mode of activity in the cell, for example, depending on their effect on the cell cycle and on the stage of the cell cycle. Alternatively, such an agent can be characterized based on its ability to directly cross-link DNA, intercalate DNA, or induce chromosomal and mitotic aberrations that are affected by nucleic acid synthesis.

[00227] Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetogenins (notably bullatacin and bullatacinone); camptothecin (including the synthetic analog topotecan); bryostatin; callistatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); cryptophycins (notably cryptophycin 1 and cryptophycin 8); dolastatin; Duocarmycin (including the synthetic analogues KW-2189 and CB1-TM1); Eleutherobin; Pancratistatin; Sarcodictyine; Spongistatin; Nitrogen mustard analogues such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, and uracil mustard analogue; Nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; Antibiotics such as enediyne antibiotics (e.g., calicheamicin, and in particular calicheamicin gamma LI and calicheamicin omega 11); dynemicin, including dynemicin A; bisphosphonates such as clodronate; esperamicin; and the neocarzinostatin chromophore and related chromophores of the enediyne chromoprotein antibiotics, aclacinomysins, actinomycin, autranicin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalarmycin, olivomycins, peplomycin, potfiromycin, puromycin, helamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, pteropterin, and trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calusterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; Adrenal cortex suppressants such as mitotane and trilostane; Folate replacement agents such as frolinic acid; Aceglatone; Aldophosphamide glycoside; Aminolevulinic acid; Eniluracil; Amsacrine; Bestrabucil; Bisantrene; Edatraxate; Defofamine; Demecolcine; Diaziquone; Elformitin; Elliptinium acetate; Epothilone; Etoglucide; Gallium nitrate; Hydroxyurea; Lentinan; Lonidainine; Maytansinoids such as maytansine and ansamitocins; Mitoguazone; Mitoxantrone; Mopidanmol; Nitraerin; Pentostatin; Fenamet; Pirarubicin; Losoxantrone; Podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK-polysaccharide complex; razoxan; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-Trichlorotriethylamine; Trichothecenes (such as T-2 toxin, verracurin A, roridin A, and anguidine); Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitolactol; Pipobroman; Gacytosin; Arabinoside ("Ara-C"); Cyclophosphamide; Taxoids such as paclitaxel and docetaxel Gemcitabine; 6-Thioguanine; Mercaptopurine; Platinum coordination complexes such as cisplatin, oxaliplatin, and carboplatin; Vinblastine; Platinum; Etoposide (VP-16); Ifosfamide; Mitoxantrone; Vincristine; Vinorelbine; Novantrone; Teniposide; Edatrexate; Daunomycin; Aminopterin; Xeloda; ibandronate; irinotecan (eg, CPT-11); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabine, navelbine, farnesyl protein transferase inhibitors, transplatin, and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing.

2. Radiation therapy

[00228] Other commonly used factors that cause DNA damage are well known in the art and include gamma rays, x-rays, and/or targeted delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors, such as microwaves, proton beam irradiation (U.S. Patents 5,760,395 and 4,870,287), and UV irradiation, are also being considered. All of these factors are likely to cause a wide range of damage to DNA, DNA precursors, and affect DNA replication and repair, and chromosome assembly and maintenance. X-ray doses range from daily doses of 50 to 200 roentgens over extended periods of time (3 to 4 weeks) to single doses of 2,000 to 6,000 roentgens. Radioisotope dose ranges vary widely and depend on the half-life of the isotope, the strength and type of radiation emitted, and its uptake by tumor cells.

3. Immunotherapy

[00229] It will be understood by one skilled in the art that additional immunotherapy may be administered in combination or in conjunction with the methods of embodiments of the invention. In cancer treatment, immunotherapy is typically administered using immune effector cells and molecules to target and destroy cancer cells. An example of such an immunotherapy is rituximab (RITUXAN®). The immune effector may be, for example, an antibody specific to certain markers on the surface of a tumor cell. The antibody itself may serve as an effector of the therapy or may recruit other cells to actually destroy the tumor cells. The antibody may also be conjugated to a drug or to a toxin (a chemotherapeutic agent, a radionuclide, a ricin A chain, cholera toxin, pertussis toxin, etc.) and may serve as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that directly or indirectly interacts with the target tumor cell. Various effector cells include cytotoxic T cells and NK cells.

[00230] The development of antibody-drug conjugates has been a major breakthrough in the development of anticancer therapy. Cancer is a leading cause of death worldwide. Antibody-drug conjugates (ADCs) contain monoclonal antibodies (MAbs) that are covalently linked to cell-killing drugs. This approach combines the high specificity of MAbs against their antigen targets with the high potency of cytotoxic drugs, resulting in “armed” MAbs that deliver the payload (drug) to tumor cells with elevated antigen levels. Targeted drug delivery also minimizes its exposure to normal tissues, resulting in reduced toxicity and an increased therapeutic index. The FDA approval of two ADCs, ADCETRIS® (brentuximab vedotin) in 2011 and KADCYLA® (trastuzumab emtansine or T-DM1) in 2013, further validated this approach. Currently, there are more than 30 ADC candidates in various stages of clinical trials for the treatment of cancer (Leal et al., 2014). As antibody design and payload optimization of linkers become more sophisticated, the discovery and development of new ADCs increasingly depends on the identification and validation of new targets suitable for this approach and the generation of targeted MAbs. Two criteria for ADC targets are activation/high expression in tumor cells and robust internalization.

[00231] In one aspect of immunotherapy, the tumor cell must carry certain markers that are suitable for targeting, i.e., not present on most other cells. There are many tumor markers, and any of them may be suitable for targeting according to embodiments of the invention. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, laminin receptor, erb B, and p155. An alternative aspect of immunotherapy is to combine antitumor effects with immunostimulatory effects. There are also immunostimulatory molecules, including cytokines such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, chemokines such as MIP-1, MCP-1, IL-8 and growth factors such as FLT3 ligand.

[00232] Examples of immunotherapeutic agents that are currently in use or have been used include immune adjuvants such as Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds (U.S. Patents 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998); therapeutic cytokines such as interferons α, β, and γ, IL-1, GM-CSF, and TNF (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998); gene therapy agents, such as TNF, IL-1, IL-2, and p53 (Qin et al., 1998; Austin-Ward and Villaseca, 1998; U.S. Patents 5,830,880 and 5,846,945); and monoclonal antibodies, such as an anti-CD20 antibody, an anti-GM2 ganglioside antibody, and an anti-p185 antibody (Hollander, 2012; Hanibuchi et al., 1998; U.S. Patent 5,824,311). It is contemplated that one or more anticancer therapies may be administered in conjunction with the antibody therapies described herein.

[00233] In some embodiments of the invention, the immunotherapeutic agent may be an immune checkpoint inhibitor. Immune checkpoints either enhance a signal (e.g., costimulatory molecules) or attenuate a signal. Immune checkpoint inhibitors that should be blocked include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), cytotoxic T lymphocyte-associated protein 4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin (KIR), lymphocyte activation gene-3 (LAG3), programmed cell death 1 (PD-1), T-cell immunoglobulin domain and mucin domain 3 (TIM-3), and V domain of Ig suppressor of T cell activation (VISTA). Specifically, immune checkpoint inhibitors target the PD-1 and/or CTLA-4 system.

[00234] Immune checkpoint inhibitors may be drugs such as small molecules, recombinant forms of the ligand or receptors, or, in particular, antibodies, such as human antibodies (see, for example, International Patent Application Publication WO2015016718; Pardoll, Nat Rev Cancer, 12(4):252-64, 2012; which are incorporated herein by reference). Known inhibitors of immune checkpoint proteins or analogs thereof may be used, and in particular, chimeric, humanized, or human forms of antibodies may be used. As is known to those skilled in the art, alternative and/or equivalent names may be used for some of the antibodies mentioned herein. Such alternative and/or equivalent names are interchangeable in the context of the present invention. For example, it is known that lambrolizumab is also known under the alternative and equivalent names MK-3475 and pembrolizumab.

[00235] In some embodiments of the invention, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partners. In a particular aspect of the invention, the ligand binding partners of PD-1 are PDL1 and/or PDL2. In another embodiment of the invention, a PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partners. In a particular aspect of the invention, the binding partners of PDL1 are PD-1 and/or B7-1. In another embodiment of the invention, a PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partners. In a particular aspect, the binding partner of PDL2 is PD-1. The antagonist can be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. Representative antibodies are described in U.S. Patent Nos. US8735553, US8354509, and US8008449, which are incorporated herein by reference. Other PD-1 system antagonists for use in the methods described herein are known to those skilled in the art, for example, as described in U.S. Patent Application Nos. 20140294898, 2014022021, and 2010008369, which are incorporated herein by reference.

[00236] In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011. In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1-binding portion of PDL1 or PDL2 linked to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some embodiments, the PD-1 binding antagonist is AMP-224. Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody described in WO2006/121168. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody, described in WO2009/114335. CT-011, also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in WO2009/101611. AMP-224, also known as B7-DCIg, is a soluble PDL2-Fc fusion receptor described in WO2010/027827 and WO2011/066342.

[00237] Another immune checkpoint that can serve as a target in the methods described herein is cytotoxic T lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The full-length cDNA sequence of human CTLA-4 has Genbank accession number L15006. CTLA-4 is found on the surface of T cells and acts as a "switch" when binding to CD80 or CD86 on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of T helper cells and transmits an inhibitory signal to T cells. CTLA4 is similar to the T cell costimulatory protein CD28, and both molecules bind to CD80 and CD86 on antigen-presenting cells, and are also referred to as B7-1 and B7-2, respectively. CTLA4 transmits an inhibitory signal to T cells, while CD28 transmits a stimulatory signal. Intracellular CTLA4 is also found in regulatory T cells and may be important for their function. T-cell activation via the T cell receptor and CD28 leads to increased expression of CTLA-4, a receptor that inhibits B7 molecules.

[00238] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.

[00239] Anti-human CTLA-4 antibodies (or VH and/or VL domains derived therefrom) suitable for use in the methods of the invention can be prepared using methods well known to those skilled in the art. Alternatively, anti-CTLA-4 antibodies described in the literature can be used. For example, anti-CTLA-4 antibodies disclosed in U.S. Pat. No. 8,119,129, WO 01/14424, WO 98/42752; WO 00/37504 (CP675206, also known as tremelimumab; formerly ticilimumab), U.S. Pat. No. 6,207,156; Hurwitz et al. (1998) Proc Natl Acad Sci USA 95 (17): 10067-10071; Camacho et al., (2004) J Clin Oncology 22 (145): Abstract #2505 (antibody CP-675206); and Mokyr et al. (1998) Cancer Res 58: 5301-5304, can be used in the methods described herein. The disclosures of each of the above publications are incorporated herein by reference. Antibodies that compete with any of these known antibodies for binding to CTLA-4 can also be used. For example, a humanized anti-CTLA-4 antibody is described in International Patent Applications WO2001014424, WO2000037504, and U.S. Patent No. 8,017,114; which are incorporated herein by reference.

[00240] An exemplary anti-CTLA-4 antibody is ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy®) or antigen-binding fragments and variants thereof (see, for example, WO 01/14424). In other embodiments, the antibody comprises the CDRs or VRs of the heavy and light chains of ipilimumab. Accordingly, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of ipilimumab and the CDR1, CDR2, and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes for binding to and/or binds to the same epitope on CTLA-4 that the aforementioned antibodies bind. In another embodiment, the antibody has a variable region amino acid sequence that is at least about 90% identical to the variable region of the aforementioned antibodies (e.g., at least about 90%, 95%, or 99% identical to the variable region of ipilimumab).

[00241] Other molecules for modulating CTLA-4 include CTLA-4 ligands and receptors described in U.S. Patent Nos. US5844905, US5885796 and International Patent Application Nos. WO1995001994 and WO1998042752; which are incorporated herein by reference, and immunoadhesins described in U.S. Patent No. US8329867, which is incorporated herein by reference.

4. Surgery

[00242] Approximately 60% of patients with cancer may undergo some type of surgical intervention, which includes prophylactic, diagnostic or staged, curative and palliative surgery. Curative surgery includes resection, in which all or part of the cancer tissue is physically removed, cut out and/or destroyed, and such surgery may be performed in combination with other treatment methods, such as treatment according to embodiments of the invention, chemotherapy, radiation therapy, hormonal therapy, gene therapy, immunotherapy and/or alternative therapy. Tumor resection means the physical removal of at least a portion of the tumor. In addition to tumor resection, surgical treatment includes laser surgery, cryosurgery, electrosurgery and surgery under a microscope (Mohs surgery).

[00243] After removal of some or all of the cancer cells, tissue, or tumor, a cavity may form in the body. Treatment may be administered by perfusion, direct injection, or local application to the area with additional anticancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatment methods may also use different doses.

5. Other agents

[00244] It is contemplated that other agents may be used in combination with certain aspects of embodiments of the invention to enhance the therapeutic efficacy of treatment. These additional agents include agents that affect the activation of cell surface receptors and GAP junctions, cytostatic agents and differentiation agents, cell adhesion inhibitors, agents that increase the sensitivity of hyperproliferative cells to apoptosis inducers, or other biological agents. Increasing intercellular signaling by increasing the number of GAP junctions may enhance the antihyperproliferative effect on the adjacent hyperproliferative cell population. In other embodiments of the invention, cytostatic agents or differentiation agents may be used in combination with certain aspects of embodiments of the invention to enhance the antihyperproliferative efficacy of treatment. It is contemplated that cell adhesion inhibitors enhance the efficacy of embodiments of the invention. Examples of cell adhesion inhibitors include focal adhesion kinase (FAK) inhibitors and lovastatin. It is also contemplated that other agents that increase the sensitivity of hyperproliferative cells to apoptosis, such as the antibody c225, could be used in combination with certain aspects of the embodiments of the invention to enhance treatment efficacy.

VI. Industrial products or kits

[00245] The present invention also relates to an article of manufacture or a kit containing immune cells according to the invention. The article of manufacture or kit may further comprise a package insert with instructions for using the immune cells to treat or slow the progression of cancer in an individual or to enhance the immune function of an individual suffering from cancer. Any antigen-specific immune cells described herein may be included in the article of manufacture or in the kits. Suitable containers include, for example, bottles, vials, bags and syringes. The container may be made of various materials, such as glass, plastic (e.g., polyvinyl chloride or polyolefin) or a metal alloy (e.g., stainless steel or Hastelloy). In some embodiments of the invention, the container comprises a composition and a label adhered to the container or attached to the container with instructions for use. The article of manufacture or kit may further comprise other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. In some embodiments, the article of manufacture further comprises one or more other agents (e.g., a chemotherapeutic agent and an antineoplastic agent). Suitable containers for one or more agents include, for example, bottles, vials, sachets, and syringes.

VII. Examples

[00246] The following examples are presented to illustrate specific embodiments of the invention. It should be noted that the methods disclosed in the following examples are methods suggested by the inventor as methods that can be successfully applied in practice, and thus they can represent specific practically feasible methods. However, based on the description of the present invention, it will be obvious to those skilled in the art that various changes can be made to the specific embodiments of the invention, which can produce an analogous or similar result without departing from the spirit and scope of the subject matter of the invention.

Example 1 - CAR-NK cell expansion

[00247] NK cells were obtained from cord blood and their specificity was redirected by genetically engineering them to express tumor-specific chimeric antigen receptors (CARs) that can enhance anti-tumor activity without increasing the risk of graft-versus-host disease (GVHD), providing a “ready-made” source of cells for therapy, such as immunotherapy, of any cancer expressing the target.

[00248] NK cells were isolated from cord blood (CB) of healthy donors and co-cultured with antigen-presenting cells (APCs) and one or more cytokines, including IL-2, IL-15, IL-21, or IL-18. The NK cells were then transduced with a CAR retroviral vector. The transduced cells were then re-expanded in co-cultures with APCs and IL-2 to generate CAR-transduced CB-NK cells. These cells can be administered by fresh infusion, or they can be frozen in cytokine-containing medium for thawing and infusion at a later time. The procedure for generating CAR-CB-NK cells is summarized in Figure 1.

[00249] Specifically, on day 0, mononuclear cells were isolated from a single unit of CB, washed, and CD3-, CD14-, and CD19-positive cells were depleted using CliniMACS immunomagnetic beads (Miltenyi Biotec). Unlabeled, enriched CB-NK cells were collected, washed with CliniMACS buffer, counted, and pooled with irradiated (100 Gray) APCs at a 1:2 ratio (1 NK cell: 2 APCs). The cell mixture (1× ¹⁰⁶ cells/mL) was transferred to cell culture flasks containing NK cell complete medium (NKCCM) (90% stem cell culture medium, 10% FBS, 2 mM L-glutamine), and 200 U/mL IL-2.

[00250] Cells were reincubated at 37°C with 5% _CO2 . On day 3, medium was replaced by harvesting cells by centrifugation and resuspending them in NKCCM (1× ¹⁰⁶ cells/mL) containing 200 U/mL IL-2. Cells were then incubated at 37°C in 5% _CO2 . On day 5, the number of wells required for transduction was determined by the number of CB-NK cells in the culture. Retronectin solution was placed in 24-well culture plates. The plates were sealed and stored in a refrigerator at 4°C.

[00251] On day 6, a second NK cell selection was performed as described on day 0 prior to CB-NK cell transduction. Cells were washed with CliniMACS buffer, centrifuged, and resuspended in NKCCM at 0.5 x 10 ⁶ /ml with IL-2, 600 U/ml. Retronectin plates were then washed with NKCCM and incubated at 37°C until use. NKCCM in each well was replaced with retroviral supernatant, followed by centrifugation of the plates at 32°C. The retroviral supernatant was then aspirated and replaced with fresh retroviral supernatant. A CB-NK cell suspension containing 0.5 x 10 ⁶ cells and 600 U/ml IL-2 was added to each well, and the plates were centrifuged. The plates were then incubated at 37°C with 5% CO ₂ .

[00252] On day 9, CAR-transduced CB-NK cells were removed from the transduction plates, collected by centrifugation, and stimulated with irradiated (100 Gy) aAPCs at a 1:2 ratio (1 NK cell: 2 APCs) in NKCCM medium with IL-2, 200 U/mL (final concentration) in a GMP-compliant G-Rex® bioreactor and incubated at 37°C with 5% _CO2 . On day 12, IL-2 was added. On day 15, cells were harvested, and the final product was prepared for infusion or cryopreservation.

[00253] Using the G-Rex® Bioreactor after NK cell transduction instead of tissue culture flasks throughout the culture period allowed for increased reliability and reproducibility of CAR-NK cell expansion while reducing the likelihood of microbial contamination compared to a more open flask system. In addition, it also resulted in significant savings in the technologist's time, as the technologist had to handle the cultures every 2-3 days with the tissue culture flask system. Cells were fed into the G-Rex® Bioreactor once as described above and then left undisturbed until harvested on day 15. As shown in Fig. 3, an average of 7.67× ¹⁰⁹ CAR-NK cells were obtained from transduced CB cell fractions containing 28 million cells in the G-Rex® Bioreactor compared to 0.91× ¹⁰⁹ CAR-NK cells obtained in flasks (p=0.014). This expansion represents an average 274-fold expansion after transduction in the G-Rex® Bioreactor compared to 78-fold expansion in flasks (p=0.037) (from days 6 to 15 of culture). Transduction efficiency was excellent with either procedure, averaging approximately 67% (range 48-87%). Thus, the use of the G-Rex® Bioreactor after the NK cell transduction step provides an excellent strategy for CAR-NK cell production.

[00254] Expanded CAR-CB-NK cells were frozen in GMP-compliant NK cell cryopreservation medium supplemented with 5% DMSO and frozen in liquid nitrogen using a controlled-rate method. In vitro chromium release assays demonstrated comparable cytolysis of Raji and K562 cell lines by fresh and frozen CAR-NK cells. In vivo cell killing assays using a xenogeneic NSG mouse model also confirmed comparable antitumor activity of frozen and fresh NK cells against Raji tumor, as determined by bioluminescence imaging of luciferase-labeled Raji cells.

[00255] Figure 4 shows the survival for 7 different treatment groups and relevant controls that were used in the in vivo NSG studies. Mice bearing the Raji tumor and treated with frozen CAR-NK cells had survival comparable to animals receiving fresh CAR-NK cells. Figure 5 shows the survival curves for these animals, and Figure 6 provides a detailed description of the statistical analysis. Figure 7 shows bioluminescence imaging data showing the most potent antitumor activity in mice bearing Raji and treated with either fresh CAR-NK cells or CAR-NK cells frozen using a new cryopreservation medium mixture developed by the present inventors.

[00256] Using this strategy for patient treatment, more than 100 doses of 1 x 10 ⁶ CAR NK cells/kg can be obtained from each unit of cord blood. Thus, cord blood-derived NK cells transduced with CAR can serve as a ready source of NK cells that can recognize and attack many types of cancer, including both liquid and solid tumors. Retroviral transduction of cord blood-derived natural killer cells provides longer persistence and increased efficacy of the engineered cells for their use in immunotherapy of many types of cancer and potentially for the treatment of many viral infections.

Example 2 - Use of CAR-transduced natural killer cells in CD19-positive lymphoid tumors

[00257] This example describes the results of Phase 1 and 2 trials where HLA-naïve CAR-NK cells with anti-CD19 antibodies, obtained from umbilical cord blood, were administered to 11 patients with relapsed or difficult-to-treat CD19-positive cancer (non-Hodgkin's lymphoma or chronic lymphocytic leukemia [CLL]). The NK cells were transduced with a retroviral vector expressing genes encoding an anti-CAR CD19 antibody, interleukin-15, and inducible caspase 9 as a protective switch. The cells were expanded ex vivo and administered as a single infusion at one of three doses (1 x 10 ⁵ , 1 x 10 ⁶ , or 1 x 10 ⁷ CAR-NK cells per kilogram of body weight) following lymphodepleting chemotherapy. As described in this application, CAR-NK cell infusion was not associated with the development of cytokine release syndrome, neurotoxicity, or graft-versus-host disease, and no increase in inflammatory cytokine levels, including interleukin-6, above baseline was observed. The maximum tolerated dose was not reached. Of the 11 patients treated, 8 (73%) responded; 7 of these patients (4 with lymphoma and 3 with CLL) experienced complete remission, and 1 experienced remission of the Richter's transformation component but had persistent CLL. Responses were rapid and were observed within 30 days of infusion at all dose levels. Infused CAR-NK cells proliferated and persisted at low levels for at least 12 months.

Study protocol and patients

[00258] This example presents information on the first 11 patients enrolled in this study, excluding data through April 2019. Briefly, patients received lymphodepleting chemotherapy with fludarabine (at a dose of 30 mg per square meter of body surface area) and cyclophosphamide (at a dose of 300 mg per square meter) daily for 3 consecutive days, followed by a single infusion of experimental CAR-NK cells at escalating doses of 1 x 10 ⁵ cells, 1 x 10 ⁶ cells, and 1 x 10 ⁷ cells per kilogram of body weight. Post-remission therapy was permitted after evaluation on day 30 at the discretion of the treating physician.

[00259] The first 9 patients received a CB-NK product that was partially HLA genotyped for the recipient (4 of 6 genotypes at HLA loci A, B, and DRβ1) (Fig. 9 and Figs. 22A and 22B). The protocol was then amended to allow treatment without regard to HLA typing, consistent with the procedure performed for patients 10 and 11. When possible, the cord blood unit was chosen based on the NK cell immunoglobulin-like receptor (KIR) ligand mismatch (Mehta and Rezvani, 2016) for CAR-NK production. (A KIR mismatch between donor and recipient may enhance the intrinsic [non-CAR-mediated] antitumor activity of NK cells through a mechanism known as absent cell recognition.) Clinical responses to therapy were based on the criteria of the 2018 International Workshop on Chronic Lymphocytic Leukemia (Hallek et al., 2018) and the 2014 Lugano classification for non-Hodgkin's lymphoma (Cheson et al., 2014). (Detailed description is provided in Example 3.)

Extraction of CAR-NK cells from umbilical cord blood

[00260] Full details of CAR-NK cell production are provided in the Methods section of Example 3. Briefly, a unit of cord blood was thawed, and NK cells were purified and cultured in the presence of engineered K562 feeder cells and interleukin-2. On day 6, cells were transduced with a retroviral vector encoding genes for the anti-CAR antibody CD19, the CD28.CD3ζ signaling endodomain, interleukin-15, and inducible caspase 9 (Hoyos et al., 2010). Cells were expanded and harvested fresh for infusion on day 15. The final CAR-NK transduction efficiency for the infused product was 49.0% (range 22.7 to 66.5). CAR-NK cells were tested in vitro and found to kill primary CLL targets via a perforin-dependent mechanism (Fig. 13). The mean CD3-positive T cell content in the infused product was 500 cells per kilogram (range 30 to 8000), with a median of 0.01% (range 0.01 to 0.002) contaminating CAR-T cells in the product (Fig. 23).

Statistical analysis

[00261] The Wilcoxon rank sum test was used to confirm the relationship between treatment response and CAR-NK cell levels. A P value of less than 0.05 was considered statistically significant.

Patient characteristics

[00262] From June 2017 to February 2019, 15 patients were enrolled in the study according to the protocol. Of these patients, 4 withdrew before study entry due to disease progression, graft-versus-host disease, no detectable disease, and bacterial product contamination (1 patient for each factor). Therefore, 11 patients received a single dose of CAR-NK cells (Fig. 9 and Figs. 22A and 22B). The median age of patients was 60 years (range, 47-70). The 11 patients had received a median of 4 prior therapies (range, 3-11). Five patients were diagnosed with CLL (including 2 patients with Richter transformation or accelerated CLL), and all patients had a history of disease progression on ibrutinib and at least 3 other therapies; Moreover, all five patients had genetic characteristics with a high risk of developing the disease. Six patients had lymphoma, of which two had diffuse large B-cell lymphoma, four had the follicular form; three of these patients experienced transformation to high-grade lymphoma. Of the six patients with lymphoma, four had disease progression after autologous hematopoietic stem cell transplantation, and two had untreatable disease.

Safety

[00263] Following CAR-NK cell infusion, none of the patients experienced symptoms of cytokine release syndrome, neurotoxicity, or hemophagocytic lymphohistiocytosis. Furthermore, no cases of graft-versus-host disease were observed, despite the HLA mismatch between the patients and CAR-NK products. As expected, all of these patients experienced transient and reversible hematological toxicities, which were primarily related to lymphodepleting chemotherapy. It was not possible to determine whether CAR-NK cell infusion contributed to the development of hematological toxicity. No cases of tumor lysis syndrome or grade 3 or 4 nonhematological toxicity were observed. The maximum tolerated dose of CAR-NK cells was not reached. Table 2 in Fig. 10 lists all the adverse events observed in the study. No patients were admitted to the Intensive Care Unit (ICU) for treatment of CAR-NK cell-related side effects. However, patient 2 was admitted to the ICU for treatment of advanced lymphoma and subsequently died. Given the lack of serious toxicity in the study, the present authors did not activate the caspase 9 protection switch (via rimiducide) to deplete CAR-NK cells.

Response to treatment

[00264] Over a median follow-up of 13.8 months (range, 2.8-20.0), 8 patients (73%) experienced an objective response, with 7 patients (3 with CLL and 4 with lymphoma) achieving a complete response (Fig. 11). Another patient with Richter's transformation CLL (Patient 5) had a complete remission of high-grade lymphoma with no lesions and fluorodeoxyglucose uptake, as demonstrated by positron emission computed tomography (PET-CT) performed 30 days after CAR-NK cell infusion, but this patient still had cytopenias with bone marrow infiltration after CLL (Fig. 14). Although this patient ultimately experienced a complete response to therapy after remission (see below), the present authors did not attribute this response to CAR-NK cell therapy. All eight patients responded within the first month after infusion. Of the 11 patients treated, five received a product that was not a KIR ligand match.

Postremission therapy

[00265] Of the 8 patients who responded to CAR-NK therapy, 5 patients received post-remission therapy (Fig. 11). Patient 3 (with CLL) subsequently demonstrated minimal residual disease, as detected by peripheral blood flow cytometry, 9 months after infusion and received rituximab. Patient 7 (who also had CLL) demonstrated a clinical complete response but retained persistent minimal residual disease and received lenalidomide as an immunomodulatory agent 6 weeks after infusion. Patient 8 (with transformed follicular lymphoma) and patient 11 (with follicular lymphoma) underwent hematopoietic stem cell transplantation after CAR-NK therapy with a complete response and no evidence of minimal residual disease. Patient 5 (who had Richter's transformation CLL) had remission of high-grade lymphoma but persistent CLL and was receiving venetoclax. All of these patients were alive and in complete remission at the date of their last evaluation, although patients 3, 5, and 7 still had positive results for minimal residual disease.

B-cell aplasia

[00266] Because B-cell aplasia was used as a surrogate for T-cell activity in producing anti-CAR CD19 antibodies, the frequency of CD19-positive B cells in the peripheral blood of patients was determined after CAR-NK cell infusion. All patients, except patients 1 and 5, had B-cell aplasia associated with prior B-cell depletion therapy at the time of the experiment. In patient 1, B-cell aplasia developed after CAR-NK therapy and lymphodepleting chemotherapy. Patient 5 had persistent CLL in the peripheral blood despite having a complete response to high-grade transformation until he received venetoclax. Patient 3 had evidence of B-cell reconstitution along with recurrent positive minimal residual disease. None of the remaining patients showed recovery of normal B cell counts during the entire observation period.

Proliferation, migration, and persistence of CAR-NK cells

[00267] To assess the level of in vivo CAR-NK cell expansion and the number of vector transgene copies per microgram of genomic DNA, quantitative real-time polymerase chain reaction analysis was performed. Expansion was observed as early as 3 days after infusion, with CAR-NK cells persisting for at least 12 months (Fig. 12A and Fig. 24). The maximum CAR-NK copy number was observed 3-14 days after infusion and was dose-dependent. After 14 days, no dose-dependent differences in peripheral blood transcript levels or CAR-NK cell persistence were observed. As reported, patients who received CAR T cells (Turtle et al., 2017; Neelapu et al., 2017; Maude et al., 2014) and participated in our study responded to therapy with significantly higher early CAR-NK cell expansion compared to non-responders (Figure 12B). However, no differences in CAR-NK cell persistence were observed depending on the degree of HLA mismatch with the recipient (Table 1, Figure 9 and Figure 15). These results were confirmed by flow cytometry (Figure 16) (Muftuoglu et al., 2018).

[00268] In 2 patients from whom lymph node samples were collected, more CAR-NK cells were detected in these lymph nodes than in the bone marrow or peripheral blood (Figs. 17 and 18), a result that supports the notion that CAR-NK cells have the ability to hom to disease sites. Similar levels of CAR-NK cells were detected in the bone marrow and peripheral blood of 10 patients from whom samples were collected (Fig. 19).

[00269] The minimal number of contaminating CAR-expressing T cells in the product did not result in detectable expansion of CAR T cells after infusion, nor did CD3 ⁺ T cells result in graft-versus-host disease (Fig. 20). CAR-NK cells were still detected at low levels in patients who did not respond or who relapsed, despite CD19 expression on tumor cells, indicating, in some embodiments, the presence of alternative immune evasion mechanisms, such as induction of CAR-NK depletion. Studies of the functions of residual CAR-NK cells in relapsed patients were not conducted. Persistent CAR-NK cells did not expand in vivo at the time of relapse.

Serum cytokine analysis

[00270] Serial peripheral blood supernatants were assessed for inflammatory cytokines, as well as interleukin-15, which was encoded by the retroviral vector used to generate CAR-NK cells. No increase in inflammatory cytokine levels (e.g., interleukin-6 and tumor necrosis factor-α) was observed compared to baseline levels, and no increase in systemic interleukin-15 levels was observed compared to pre-treatment values, indicating that interleukin-15 was not released by CAR-NK cells to significant systemic levels in peripheral blood after infusion (Fig. 21).

Induction of reactions in the form of production of alloimmune antibodies against donor cells

[00271] All patients received HLA-untyped CAR-NK products. Patients 1-9 received a product with a partial match for 4 of 6 HLA molecules, while patients 10 and 11 were recipients of HLA-untyped CAR-NK cells. Therefore, the present inventors monitored the induction of donor-specific anti-HLA antibodies. At all time points when testing was performed, no induction of antibodies against HLA-untyped alleles of the infused product was observed (Fig. 25). Host cellular responses were not assessed.

Example 3 - Additional Materials

Obtaining CAR-NK cells

[00272] Preclinical production of iC9/CAR19/IL15 CAR-NK cells has been described previously (Hoyos et al., 2010; Liu et al., 2018). Clinical cord blood units for CAR-NK cell production were obtained from the MD Anderson Cancer Center (MDACC) CB bank. CAR-NK cells were produced at an MDACC GMP facility. Briefly, a cord blood unit was thawed, and NK cells were purified by negative selection for CD3, CD19, and CD14 (on Miltenyi beads) and cultured in the presence of engineered K562 feeder cells expressing membrane-bound IL-21 and 4-1BB ligand, as well as exogenous IL-2 (200 U/mL). On day 6, cells were transduced with a retroviral vector carrying a single-chain variable fragment (scFv) against CD19, the transmembrane domain of CD28, and the CD28.CD3ζ signaling endodomain in combination with the human IL15 gene and the inducible caspase-9 suicide gene. These three genes were linked to each other via foot-and-mouth disease virus-derived 2A peptide sequences and then cloned into the SFG retroviral vector to generate the iC9/CAR.19/IL15 retroviral vector (Hoyos et al., 2010; Liu et al. 2018). Cells were expanded for an additional 9 days and harvested fresh for infusion on day 15.

Research protocol

[00273] A Phase I-II clinical trial was conducted by the inventors at their Institute and was designed to determine the optimal dose and evaluate the safety and efficacy of escalating doses of iC9/CAR19/IL15 CB-NK cells for the treatment of relapsed/refractory CD19-positive malignancies. The dose was escalated according to the adaptive Phase I-II protocol after EffTox replacement (Thall and Cook, 2004; Thall et al., 2006; Thall et al., 2014). Dose-limiting toxicities were defined as the occurrence of CRS within 2 weeks after cell infusion requiring patient transfer to the Intensive Care Unit, or the development of grade III-IV acute GVHD within 40 days after infusion, or a grade 3-5 hypersensitivity reaction associated with the CAR-NK cell infusion. To establish the EffTox model, efficacy was defined as being alive and having partial remission at day 30 after CAR-NK cell infusion.

[00274] All adverse events observed in the first 40 days after infusion, whether related to CAR-NK cell therapy or not, were collected and recorded. From day 40 to 12 months after therapy, all adverse events thought to be related to CAR-NK cells were collected and recorded. In addition, all patients were enrolled in an IRB-approved long-term study (for 15 years). The EffTox dose eligibility rules stipulated an upper probability of dose-limiting toxicity of 0.50 (based on experience with CAR-T cells, 20% of patients were expected to develop dose-limiting CRS) and a lower probability of efficacy of 0.25. The three equivalent pairs of trade-off probabilities used to calculate the trade-off contours are (0.35, 0), (0.55, 0.30), (1, 0.075). The prior hyperparameters were calculated based on the assumed prior average probabilities of (toxicity | dose) = 0.35, 0.40, 0.45 and probabilities of (efficacy | dose) = 0.15, 0.20, 0.25, respectively, with an overall prior effective sample size of 1. A maximum of 36 patients could be treated in 3 groups of 12 patients each, starting with the lowest dose level ( ¹⁰⁵ cells/kg); subsequent doses were selected using the EffTox method, and untested dose levels were not skipped during dose escalation. The EffTox protocol was implemented as described on the MDACC Department of Biostatistics Clinical Trial Conduct website https://biostatistics.mdanderson.org/ClinicalTrialConduct/.

Correction of clinical trial protocols and inclusion of patients in the trial program

[00275] Between June 2017 and February 2019, 15 patients were enrolled in the protocol (4 of whom failed screening and 11 of whom were treated). Patients were enrolled sequentially at a wide interval of 14 days from the day of CAR-NK infusion to the start of the treatment regimen for the next patient in each group, and at two-week intervals as the dose was escalated to the next level. In March 2019, the dose-finding phase of the study was considered complete and the protocol was amended to allow for a repeat CAR-NK cell infusion at a dose of 10 ⁷ cells/kg. Examples 2 and 3 of this application report on 11 patients who received a single CAR-NK cell infusion during the dose-finding phase of the study. The data for this report were finalized in April 2019. The clinical trial was designed to monitor patients for 12 months, after which they were followed in a long-term, IRB-approved follow-up study of patients receiving genetically modified cell products. All patients consented to participate in this long-term study.

Evaluation of answers

[00276] Bone marrow examinations and PET/CT imaging were performed at 4, 8, 12, 16, 26, 48, and 52 weeks post-infusion, and more frequently if clinically indicated. Responses were defined according to the Lugano and iWCLL criteria for patients with NHL and CLL, respectively (Cheson et al., 2014; Hallek et al., 2008; Hallek et al., 2018). All bone marrow samples were assessed for MRD status using 6-color flow cytometry with a sensitivity of ¹⁰⁴ nucleated cells or more in an MDACC CLIA-certified hematopathology laboratory. Patients were considered MRD-negative if they had at least two consecutive negative assessments.

[00277] Response assessment criteria for Hodgkin's and non-Hodgkin's lymphomas (according to the Lugano criteria) (Cheson et al., 2014)

Full answer

PET-CT based answer CT-based answer Lymph nodes and areas outside the lymph nodes Complete metabolic response:
Scores 1, 2, or 3: presence or absence of residual mass on the 5PS. It has been established that in the Waldeyer's ring or in extranodal sites with high physiological uptake or with activation in the spleen or bone marrow (e.g., in the case of hemotherapy or the addition of myeloid colony-stimulating factors), uptake may be higher than in the normal mediastinum and/or liver. In this case, a complete metabolic response may be present when uptake at sites of initial lesion does not exceed uptake in the surrounding normal tissue, even if this tissue has high physiological uptake. Complete radiological response (all parameters are described below)
Target nodules/masses should have regressed to <1.5 cm in LDI, with no spread to non-nodal sites. An unappreciated defeat Not rated Absent Organ enlargement Not rated Regression to normal New defeats No No Bone marrow Absence of any FDG disease in the bone marrow Normal, according to morphology, in case of uncertainty was considered as IHC-negative

Partial answer

PET-CT based answer CT-based answer Lymph nodes and areas outside the lymph nodes Partial metabolic response:
Score 4 or 5: decreased absorption compared to baseline and residual mass(es) of any size
At an intermediate stage, these studies suggest the presence of a response to the disease.
At the end of treatment, these data indicated residual disease Partial remission (all parameters are described below)
>50% reduction in SPD up to 6 evaluable target nodules and non-nodular areas.
If a lesion was too small to be assessed on CT, a default value of 5 mm × 5 mm was used. If subsequent lesions were no longer visible, the value was 0 mm × 0 mm. For nodules >5 mm × 5 mm but smaller than normal, actual assessment was performed to calculate parameters. An unappreciated defeat Not rated Absent/normal, regression, but no increase Organ enlargement Not rated The spleen should show 50% regression in length compared to normal. New defeats No No Bone marrow Residual uptake was higher than that in normal bone marrow but lower than baseline (diffuse uptake was consistent with reactive changes following chemotherapy). If persistent focal changes in the bone marrow were observed in the case of a nodal response, a decision was made to perform further evaluation with MRI, biopsy, or interval scanning. Not rated

No response or stable disease

PET-CT based answer CT-based answer Target nodules/nodular masses. Lesions beyond nodules Lack of metabolic response:
Score 4 or 5: No significant change in FDG uptake from baseline at mid-term or end-of-treatment Stable disease
<50% reduction in SPD from baseline to 6 dominant evaluable nodules and extranodal sites; did not meet criteria for progressive disease. An unappreciated defeat Not rated Lack of any increase corresponding to progression Organ enlargement Not rated Lack of any increase corresponding to progression New defeats No No Bone marrow No change from baseline Not rated

Progressive disease

PET-CT based answer CT-based answer Target nodules/nodular masses. Lesions beyond nodules Progressive metabolic disease
Score 4 or 5: Increased uptake intensity compared with baseline and/or presence of new FDG lesions consistent with intermediate-stage lymphoma or at end-of-treatment assessment Progressive disease requires progression of at least 1 of the following PPDs:
Individual nodules/lesions should have abnormal parameters:
LDi >1.5 cm and
Increase of > 50% from the lower PPD value and increase in LDi and SDi from the lower value
0.5 cm for lesions <2 cm
1.0 cm for lesions >2 cm
When assessing splenomegaly, the spleen length should be increased by 50% compared to the previous increase based on the baseline value (e.g., a spleen measuring 15 cm should increase to >16 cm). If
If splenomegaly has not been previously observed, the spleen size should be at least 2 cm higher than the baseline
New or recurrent splenomegaly An unappreciated defeat No New or apparent progression of existing unassessed lesions New defeats New FDG lesions consistent with lymphoma but not with other etiologies (e.g., infection, inflammation). If the etiology of new lesions is unknown, biopsy or imaging may be performed at regular intervals. Recurrence of previously healed lesions. New nodule >1.5 cm in any axis
A new extranodal site >1.0 cm in any axis; if the size in any axis is <1.0 cm, then the presence of such a site should be mandatory and should be associated with lymphoma
Any size of assessed disease is necessarily associated with lymphoma Bone marrow New or recurrent FDG lesions New or recurrent disease

[00278] Abbreviations: 5PS, 5-point scale; CT, computed tomography; FDG, fluorodeoxyglucose; IHC, immunohistochemistry; LDi, largest transverse diameter of the lesion; MRI, magnetic resonance imaging; PET, positron emission tomography; PPD, product of transverse LDi and perpendicular diameter; SDi, shortest axis perpendicular to LDi; SPD, sum of the product of perpendicular diameters for multiple lesions. * A score of 3 in many patients indicates a good prognosis with standard treatment, especially during interim scanning. However, in PET studies in which de-escalation has been investigated, it may be preferable to consider a score of 3 as an inadequate response (to avoid undertreatment). Measured dominant lesions: Up to six of the largest dominant nodules, nodular masses, and extranodal lesions, selected so that they can be accurately measured on two diameters. Nodes should preferably be taken from different body sites and should include, where possible, the mediastinal and retroperitoneal regions. Non-nodular lesions include solid organ lesions (e.g., liver, spleen, kidneys, lungs), gastrointestinal lesions, skin lesions, or lesions identified by palpation. Unscored lesions: Any disease not selected as a scored dominant disease and as an accurately scored disease should be considered unscored. These sites include any nodules, nodular masses, and extranodal sites that were not selected as dominant or evaluable or that do not meet the requirements for such evaluation but are still considered abnormal, as well as accurately evaluable disease observed at any site suspected of having disease that would be difficult to quantify using measurements, including pleural effusions, ascites, bone lesions, leptomeningeal disease, abdominal masses, and other lesions that cannot be confirmed and detected visually. In the Waldeyer ring or in extranodal sites (e.g., gastrointestinal tract, liver, bone marrow), FDG uptake may be greater than in the mediastinum with a complete metabolic response, but should not be higher than the surrounding normal physiologic uptake (e.g., with bone marrow activation, chemotherapy, or the presence of myeloid growth factors). † PET 5PS: 1, no uptake above background; 2, uptake < in mediastinum; 3, uptake > in mediastinum but < in liver; 4, moderate uptake > in liver; 5, uptake markedly higher than in liver and/or in new lesions; X, new areas of uptake unlikely to be associated with lymphoma.

iwCLL response criteria for CLL (Hallek et al., 2018)

Complete remission

[00279] The CR must meet all of the following criteria:

[00280] 1. Peripheral blood lymphocytes (estimated by blood test and blood cell count) <4×10 ⁹ /L.

[00281] 2. Absence of significant lymphadenopathy on physical examination. In clinical trials, CT scanning of the neck, abdomen, pelvis, and chest is advisable if abnormalities have been previously identified. Lymph nodes should be <1.5 cm in greatest diameter. Once this has been determined, further imaging is not required until disease progression becomes evident on clinical examination or blood testing.

[00282] 3. Absence of splenomegaly or hepatomegaly on physical examination. In clinical trials, abdominal computed tomography should be performed to assess response and should not reveal signs of lymphadenopathy or splenomegaly.

[00283] 4. Absence of the main symptoms associated with the disease.

[00284] 5. Neutrophils ≥1.5×10 ⁹ /L.

[00285] 6. Platelets ≥100×10 ⁹ /l.

[00286] 7. Hemoglobin ≥11.0 g/dL (without red blood cell transfusion).

[00287] Some patients fully met all CR criteria but had persistent anemia, thrombocytopenia, or neutropenia that was clearly not related to CLL but was associated with drug toxicity. These patients should be considered as a different category when assessing remission, i.e., as CR with incomplete bone marrow recovery (CRi).

[00288] Partial remission

[00289] To determine partial remission, at least 2 parameters in group A and 1 parameter in group B must improve if they were previously abnormal (see Table below). If only 1 parameter was abnormal in both groups A and B before therapy, then only 1 parameter needs to be improved.

Group A Group B Lymph nodes Liver and/or spleen size Main symptoms Lymphocyte count Platelet count Hemoglobin Bone marrow Reduction ≥50% (compared to baseline) Reduction ≥50% (compared to baseline) Any Reduction ≥50% (compared to baseline) ≥100×10 ⁹ /L or an increase of ≥50% compared to baseline ≥11 g/dL or ≥50% increase from baseline Presence of CLL cells or B-lymphoid nodules

Progressive disease

[00290] Progressive disease during or after therapy is characterized by at least one of the following compared to the nadir:

[00291] 1. The appearance of any new lesion such as enlarged lymph nodes (>1.5 cm), splenomegaly, hepatomegaly, or other organ infiltrates.

[00292] 2. An increase of >50% of the largest measured diameter of any previous lymph node (>1.5 cm).

[00293] 3. Increase in spleen size by ≥50% or de novo development of splenomegaly. For splenomegaly, spleen length must increase by ≥50% compared to the previous increase, starting from baseline. If splenomegaly was not previously observed at baseline, or if splenomegaly has resolved, the spleen must increase by at least 2 cm from baseline.

[00294] 4. An increase in liver size by ≥50% of the degree of liver enlargement below the costal margin determined by palpation, or the appearance of de novo hepatomegaly.

[00295] 5. An increase in the number of blood lymphocytes by 50% or more or at least 5×10 ⁹ /L B-lymphocytes.

[00296] 6. Transformation into a more aggressive tumor according to histology (Richter syndrome).

[00297] 7. The appearance of cytopenias (neutropenia, anemia, or thrombocytopenia) directly related to CLL and not related to autoimmune cytopenias.

[00298] a. Decrease in hemoglobin level ≥2 g/dL or <10 g/dL

[00299] b. Platelet count decrease ≥50% or <100×10 ⁹ /L

[00300] 8. Increase in CLL cells in bone marrow by ≥50% in serial biopsies.

Stable disease

[00301] Patients who did not achieve complete remission or partial remission and who did not exhibit PD will be considered as having stable disease.

Relapse

[00302] Relapse is defined as disease progression in a patient who has previously met the above criteria for complete remission or partial remission for ≥6 months.

Cytotoxicity of CAR-NK cells against primary CLL targets

[00303] PBMCs from 4 different CLL patients were thawed and preactivated overnight with CD40L (2 ng/ml) in SCGM at a concentration of 2 x 10 ⁶ PBMCs/ml in a humidified incubator at 37°C/5% CO ₂ . The ^51Cr release assay was performed for 4 hours in 96-well V-bottom plates. Briefly, 0.5 x 10 ⁶ CLL cells were resuspended in 1 ml SCGM and labeled with 100 microCi of ^51Cr for 2 hours in a humidified incubator at 37°C/5% CO ₂ . After labeling, the cells were washed twice with PBS and resuspended in SCGM and then used as targets for the assays. Paired non-transduced (NT) and CAR-transduced NK cells (CAR-NK) were used as effectors at different effector-to-target cell ratios (E:T). The percentage of specific lysis was determined as [(mean of test wells) - (mean of spontaneous release wells)/(mean of maximum release wells) - (mean of spontaneous release wells)] × 100. Paired Student's t-test was used to calculate statistical significance.

Assay for perforin-dependent cytotoxicity of CAR-NK cells against primary CLL targets

[00304] Concanamycin A (CMA) was used to prevent perforin maturation and deplete NK cells containing active perforin (Kataoka et al., 1996). Briefly, 2 x 10 ⁶ NK cells transduced with CD19 CAR were treated with 100 nM CMA (Fisher) or DMSO (Sigma) as a vehicle control for 2 h in a humidified incubator at 37°C/5% CO ₂ . Perforin expression in CAR NK cells was assessed by flow cytometry using an anti-perforin monoclonal antibody (clone dG9 from BioLegen (Makedonas et al., 2009)). Changes in perforin expression were determined by comparing the MFI of perforin in CD56 ⁺ CAR NK cells in the presence or absence of CMA. The cytotoxicity of CAR-NK cells pretreated or not with CMA against primary CLL targets was determined using the ^51C release assay as described above. A paired Student's t-test was used to calculate statistical significance.

Quantitative PCR

[00305] Genomic DNA was extracted using the QIAamp Blood DNA Mini Kit (Qiagen) according to the manufacturer's recommendations. The number of vector transgene copies per microgram of genomic DNA was determined by quantitative PCR (qPCR) using an Applied Biosystems 7500 Fast Real-Time PCR System. Amplified targets were detected in real time using TaqMan® Universal PCR Master Mix and a custom-designed Applied Biosystems™ TaqMan® MGB DNA-based (minor groove-binding) probe that incorporates a 5' reporter (FAM) and a 3' non-fluorescent quencher (NFQ) and quantified using a standard curve. Vector transgene copy numbers per reaction were presented as copies per 1 μg of DNA. Fluorescence data were analyzed using 7500 Software v2.3.

[00306] Examples of primer and probe sequences:

[00307] Forward primer GAACAGATTATTCTCTCACCATTAGCA (SEQ ID NO: 7)

[00308] Reverse primer AGCGTATTACCCTGTTGGCAAA (SEQ ID NO: 8)

[00309] TaqMan FAM-MGB Probe CCTGGAGCAAGAAG (SEQ ID NO: 9)

[00310] Primers and probes were custom designed and synthesized by Thermo Fisher Scientific.

Serum cytokine analysis

[00311] Serum taken from serial peripheral blood samples collected before and after CAR-NK infusion was analyzed for cytokines using the Procartaplex kit from Thermofisher (Vienna, Austria) according to the manufacturer's instructions.

Phenotyping and monitoring of CAR-NK cells using multiparametric flow cytometry

[00312] To determine the persistence of cord blood-derived CAR-NK cells in peripheral blood and their trafficking to bone marrow and lymph nodes, a two-step strategy was employed. First, we exploited existing HLA mismatches between patient and donor to identify infused cord blood NK cells; we then used a CAR-specific antibody to identify CAR-expressing cells within the donor NK cell population. Briefly, a flow chimerism assay was developed using fluorochrome-conjugated antibodies against mismatched HLA alleles. Additionally, an anti-CAR antibody (109606088/Jackson Immuno RSCH) directed against the CH2-CH3 domain of the human IgG hinge region was used in this construct as a second method for detecting CAR-NK cells. Cells were stained with a live/dead dye (Tonbo BioScience: 13-0868-T100) in 1 ml PBS for 20 min at room temperature. Cells were then washed twice by centrifugation at 400×g at room temperature for 5 min with a running buffer containing PBS and 1% heat-inactivated FBS. Cells were then stained with AF-647-conjugated anti-CAR antibody (109606088/Jackson Immuno Rsch) for 20 min at 4°C. Cells were washed and stained with the appropriate anti-HLA antibody at 4°C for 10 min. Cells were then incubated with a cocktail containing fluorescently labeled antibodies and containing CD19 PE-Cy5, CD20 FITC, CD3 APC-Cy7, CD14 BUV395, CD33 BUV395 (all from BD Biosciences), CD45 BV510 (BeckMan Coulter), CD56 PE-TX Red (BeckMan Coulter), and CD16 BV650 (Biolegend) for 15 min at room temperature. Cells were then washed by centrifugation at 400×g and fixed with 1% paraformaldehyde. Flow cytometry was performed on a BD LSRFortessa X-20 instrument, and data were analyzed using FlowJo software, version 10.0.8 (TreeStar). The gating strategy for detecting HLA-positive and CAR-positive NK cells is shown in Fig. 16.

Detection of CAR-NK in lymph node samples

[00313] Lymph node biopsy specimens collected with a fine needle were collected in PBS. A single-cell suspension was prepared by crushing the specimen between two frozen edges of glass slides. The cell suspension was filtered through a 50-micron sieve, centrifuged, counted, and 1 x ¹⁰⁶ cells were stained with a live/dead cell counting dye in PBS. After viability staining, the cells were washed once in PBS with 1% FBS and stained with AF-647-conjugated anti-CAR antibody (109606088/Jackson Immuno Rsch) (4°C, 20 minutes) in PBS-FBS buffer. The cells were then washed with PBS-FBS buffer and sequentially stained with an HLA-specific antibody (4°C, 10 min) and then with a cocktail of antibodies against CD19 PE-Cy5, CD20 FITC, CD3 APC-Cy7, CD14 BUV395, CD33 BUV395 (all from BD Biosciences), CD45 BV510 (BeckMan Coulter), CD56 PE-TX Red (BeckMan Coulter), and CD16 BV650 (Biolegend). Cells were fixed in 2% paraformaldehyde and analyzed on an X20 Fortessa analyzer.

Donor-specific antibody (DSA) assessment

[00314] Ten of eleven patients were screened for donor-specific anti-HLA antibodies before CAR-NK infusion and at various time points after CAR-NK infusion. If the screening was positive, antibody specificity was determined by semiquantitative solid-phase antibody detection on the Luminex® platform.

Example 4 - Example of a Phase I/II trial of dose-escalating cord blood-derived CAR NK cells in combination with lymphodepleting chemotherapy in patients with relapsed/refractory B-cell malignancies

[00315] This Example describes the determination of the safety and efficacy of CAR.CD19-CD28-zeta-2A-iCasp9-IL15-transduced CB-NK cells in patients with relapsed/refractory CD19 ⁺ B lymphoid malignancies. This Example included an assessment of the overall response rate (complete and partial response), a quantitative assessment of the persistence of infused allogeneic donor CAR-transduced and CB-derived NK cells in the recipient, and large-scale immune reconstitution studies.

General description

[00316] This Example describes a clinical trial to investigate new immunotherapeutic strategies using engineered natural killer (NK) cells to improve tumor-free survival in patients with relapsed or difficult-to-treat CD19 ⁺ B-cell malignancies. Each year in the United States, there are an average of 69,740 new cases of non-Hodgkin lymphoma (NHL), 15,680 new cases of chronic lymphocytic leukemia (CLL), and 6,070 new cases of acute lymphoblastic leukemia (ALL), and an estimated annual mortality of 19,020, 4,580, and 1,430, respectively (http:/www.cancer.org). Overall survival (OS) is determined primarily by the stage of the disease at presentation and the response to chemotherapy. The standard therapy for patients who relapse after first-line therapy is allogeneic hematopoietic stem cell transplantation (HSCT). The expected overall survival for patients with a second complete remission is 25%, based on chemotherapy sensitivity at the time of HSCT. Thus, there is an urgent and unmet need to develop new treatments for patients with advanced B-cell malignancies, particularly because relapse after allogeneic HSCT is typically fatal.

[00317] Chronic lymphocytic leukemia (CLL) is the most common form of leukemia in adults in the United States, accounting for 25% of all leukemias. More than 15,000 new cases of CLL and 4,500 CLL-related deaths occur annually in the United States. The natural history of the disease is variable. Patients with lymphocytosis alone have a median survival of more than 10 years, whereas patients with evidence of bone marrow failure, manifested by anemia or thrombocytopenia, have a median survival of only 2-3 years. Because treatment has not been proven effective and there is no objective evidence that a specific treatment will prolong survival, treatment is deferred (Cheson and Cassileth, 1990). The NCI-sponsored CLL Working Group proposed the following indications for initiating treatment: 1) weight loss of more than 10% in the previous 6 months; 2) extreme fatigue associated with progressive disease; 3) high fever or night sweats without evidence of infection; 4) worsening anemia (Rai stage III) or thrombocytopenia (Rai stage IV); 5) massive lymphadenopathy (>10 cm) or rapidly progressive lymphocytosis (lymphocyte doubling time <6 months); or 6) prolymphocytic transformation or Richter transformation. Current treatments for newly diagnosed CLL include chemotherapy and antibody therapy, alone or in combination. Numerous new approaches, such as targeted therapy using ibrutinib for the treatment of CLL, are currently in development (Burger et al., 2015; Byrd et al., 2015), but the disease remains incurable. Moreover, even after complete responses, most patients experience persistent immunological impairment and minimal residual disease. Ultimately, chronic immunosuppression, leading to infectious complications, occurs in 80% of patients with CLL and is a major cause of mortality. Allogeneic stem cell transplantation can be curative in some patients with CLL, but its success has been limited, primarily due to the high mortality and morbidity associated with this procedure. Non-myeloablative allogeneic transplantation regimens are promising, but patient enrollment is limited by the availability of suitable donors, namely siblings.

[00318] Historically, initial treatment of patients with CLL requiring such treatment was with an alkylating agent, particularly chlorambucil, administered alone or in combination with a corticosteroid. Overall response rates were 50-70%, however, complete remission rates were low (5-20%). Newer agents such as purine analogs, particularly fludarabine, produce higher response rates (Rai et al., 2000) when used in initial treatment. Randomized trials comparing alkylating agent-based therapy with fludarabine monotherapy showed higher complete response rates and longer disease-free survival (Rai et al., 2000; Johnson et al., 1996) with the use of this nucleoside analog, but did not show increased survival. Fludarabine was approved by the US Food and Drug Administration (FDA) for the treatment of patients with B-cell CLL who have failed to respond to treatment or have experienced disease progression while receiving at least one alkylating agent-containing regimen.

[00319] Combination regimens such as cyclophosphamide, fludarabine, and rituximab have been shown to improve response rates (Keating et al., 2005), but these regimens are highly immunosuppressive and a durable effect has not been demonstrated. Ibrutinib is a covalent inhibitor of Bruton tyrosine kinase (Honigberg et al., 2010) (BTK), a member of the TEC family of tyrosine kinases and a key enzyme in the B-cell receptor signaling pathway. Ibrutinib, used as monotherapy or in combination with immunotherapy or chemotherapy, is a highly effective treatment for lymphoid malignancies, including CLL (Burger et al., 2015; Byrd et al., 2013). However, the results after failure of ibrutinib treatment are dismal, with survival being only 3.1 months after drug discontinuation (Jain et al., 2015).

[00320] Acute lymphoblastic leukemia. Allogeneic HCT is a treatment option for a select group of patients with ALL. Overall survival (OS) ranges from 30% to 60% depending on the patient's disease stage and risk profile at the time of transplantation (Fielding et al., 2009; Golstone et al., 2008). Increasingly, minimal residual disease (MRD), both before and after HCT, is becoming an important prognostic factor for relapse (Gokbuget et al., 2012). In a cohort of 149 patients with ALL who underwent transplantation in remission at MD Anderson Cancer Center, patients with MRD, determined by multiparametric flow cytometric immunophenotyping (FCI) with a sensitivity of 0.01% observed during HCT, had a shorter PFS compared with patients without MRD, 28% versus 47%, p=0.08 (4). Furthermore, of the 135 patients in whom MRD was detected after HCT, 20 patients developed MRD, and 18 of these patients developed overt hematologic relapses at a median of 3.8 months (Zhou et al., 2014). It should be noted that of the 32 patients with overt relapse after HCT, 41% had no previous MRD, suggesting that the presence of MRD after HCT essentially confirms possible relapse, while the absence of MRD after HCT in a high-risk patient does not exclude relapse (Leung et al., 2012; Bar et al., 2014). The findings support similar published studies. Patients transplanted after a second remission typically have significantly poorer PFS and OS rates. In a study of 97 patients (CR1-51, CR2-29, other 17) who received busulfan and clofarabine chemotherapy after transplantation of cells from matched siblings (MSD) or matched but unrelated donors (MUD), patients in CR1 had significantly better disease-free survival (DFS) outcomes compared with others. For patients in CR1, the 2-year DFS rate was 61%, with 9/51 patients relapsing at a median of 9 months; the 2-year DFS rate was 40% for CR2, with 10/29 patients relapsing at a median of 3 months; and for patients with more advanced disease, the 2-year DFS rate was 33%, with 3/17 patients progressing at a median of 3 months. Data from the Center for International Blood and Marrow Transplant Research (CIBMTR) support these findings. Between 1996 and 2001, in patients younger than 20 years, OS ranged from 25% for patients transplanted after first remission to 50% for patients who received sibling transplants at first remission. Similarly, in adult patients over 20 years of age, the best outcome was seen with sibling transplantation performed at first remission, with an OS of 60% compared with 35% for transplantation performed after CR1 (according to the CIBMTR data registry). There are currently no effective treatments for patients who relapse after HCT. Several published reports have reported survival rates of less than 10% for these patients, regardless of the treatment modality used, with a median survival of 2–3 months (Fielding et al., 2009; Poon et al., 2013). To date, the most common strategy used to reduce relapse rates after HCT typically involves some form of immune manipulation, ranging from donor lymphocyte infusion (DLI) to a second transplant (Sullivan et al., 1989; Poon et al., 2013; Bader et al., 2004). However, although B-ALL patients who developed graft-versus-host disease (GVHD) have been shown to have a lower risk of relapse (Appelbaum, 1997), DLI has not demonstrated significant efficacy in this patient group, with remission rates less than 10% and associated with a high incidence of GVHD (Passweg et al, 1998). It should be noted that the best response to DLI in ALL occurs when DLI is administered prophylactically to prevent relapse (Bader et al, 2004); This approach has been demonstrated in pediatric patients; however, there have been no reports of prophylactic DLI in adults. Thus, there remains an unmet need for an effective therapy for ALL patients at high risk of relapse after allogeneic HCT, where high risk is defined as the presence of MRD and/or disease after the first complete remission.

[00321] Non-Hodgkin Lymphoma (NHL). In the United States, B-cell lymphomas account for 80-85% of all reported cases. In 2013, there were an estimated 69,740 new cases of NHL and more than 19,000 deaths from this disease. Non-Hodgkin lymphoma is the most common hematologic malignancy and ranks seventh in new cancers among men and women, accounting for 4% of all new cancer cases and 3% of cancer deaths (SEER 2014). Diffuse Large B-Cell Lymphoma. Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of NHL, accounting for approximately 30% of all NHL cases. Approximately 22,000 new diagnoses of DLBCL are reported in the United States each year. First-line therapy for DLBCL typically includes an anthracycline plus rituximab regimen (Coiffier et al., 2002). The objective response rate to first-line treatment and the complete response rate to R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) are approximately 80% and 50%, respectively. However, approximately one-third of patients experience resistance to initial therapy or relapse after R-CHOP (Sehn et al., 2005). For those patients who relapse after responding to first-line therapy, a second response can be achieved with additional chemotherapy in approximately 40–60%. For young and healthy patients, the goal of second-line therapy is to achieve a response that will allow the patient to be included in an autologous stem cell transplant (ASCT) program. Standard second-line treatment for transplant patients includes rituximab chemotherapy and combination chemotherapy such as RICE (rituximab, ifosfamide, carboplatin, and etoposide) or RDHAP (rituximab, dexamethasone, cytarabine, and cisplatin). In a large, randomized trial of RICE versus RDHAP in transplant-eligible patients with DLBCL (the CORAL study), 63% of patients achieved an objective response to either regimen, with a CR rate of 26%. Patients who responded to second-line therapy and were considered transplant-eligible received high-dose combination chemotherapy and ASCT. This combination can cure approximately 50% of transplant patients (Gisselbrecht et al., 2010). Patients who fail ASCT have a very poor prognosis with no other treatment options. Most second-line patients are not suitable for ASCT due to chemotherapy-resistant disease, age, or the presence of comorbidities such as heart, lung, liver, or kidney disease. Patients ineligible for transplantation have no treatment options. There is no standard definition of relapsed/intractable DLBCL. This trial will include patients with chemotherapy-resistant lymphoma, as demonstrated by their failure to achieve even a temporary or partial response to prior biologic and combination chemotherapy or the likelihood of early relapse after ASCT.

[00322] Transformed Follicular Lymphoma (TFL). Follicular lymphoma (FL), a B-cell lymphoma, is the most common occult (slowly growing) form of NHL, accounting for approximately 20% to 30% of all NHL cases. In some patients with FL, TFL has histologic transformation to DLBCL, which is more aggressive and associated with a poor prognosis. Histologic transformation to DLBCL occurs in approximately 3% of patients annually for 15 years, with the risk of transformation continuing to decline in subsequent years. The biologic mechanism of histologic transformation is unknown. Initial treatment of TFL depends on prior therapy for follicular lymphoma but typically includes anthracycline-based regimens plus rituximab to address the aggressive component of the disease (NCCN Practice Guideline, 2014). Treatment options for recurrent/refractory TFL are the same as for DLBCL. Given the low prevalence of these diseases, no large-scale prospective randomized trials have been conducted in these patient groups. Patients with chemotherapy-resistant disease had a prognosis similar to or even worse than that of refractory DLBCL.

[00323] Malignant neoplastic disease (MCL) is an incurable subtype of B-cell lymphoma that accounts for 7% of all non-Hodgkin lymphoma cases in the United States (Connors, 2013). Most patients with MCL progress after initial therapy, with a median overall survival of approximately 1–2 years after relapse, and new treatments for MCL are urgently needed. Ibrutinib, a first-in-class, once-daily, oral covalent inhibitor of Bruton tyrosine kinase (BTK), was recently approved by the FDA for the treatment of this disease. Although the results for relapsed/refractory MCL were excellent and unprecedented compared with other standard treatments (Wang et al., 2013), the vast majority of patients experienced disease progression after receiving single-agent ibrutinib, and these patients died within 12 months (Wang et al., 2015; Cheah et al., 2015). Patients with relapsed/refractory MCL can achieve long-term remission, and such diseases can be cured with immune cell-based therapies (Hamadani et al., 2013), including allogeneic stem cell transplantation (allo-SCT). Furthermore, the authors reported encouraging results in selected patients with MCL who underwent autologous SCT after therapy with rituximab, BCNU, etoposide ara-C, and melphalan (R-BEAM) (Tam et al., 2009). However, in patients with MCL, ibrutinib-resistant disease, outcomes after standard allogeneic and autologous transplants were suboptimal, and more effective treatments are urgently needed. Thus, patients with difficult-to-treat aggressive B-cell malignancies have significant, yet currently unmet, medical needs, and new treatment options are needed for these patient groups.

[00324] NK cells:

[00325] Natural killer (NK) cells are an important component of the graft-versus-leukemia (GVL) response (Ruggeri et al., 2002; Savani et al., 2006), which is crucial for preventing relapse after HSCT. Each mature NK cell expresses a broad spectrum of activating and inhibitory killer immunoglobulin-like receptors (KIRs), which are specific for different HLA class I molecules (Lanier, 2008; Yawata et al., 2008; Caligiuri, 2008). The ability of NK cells to recognize and kill malignant cells is regulated by complex and poorly understood interactions between inhibitory signals resulting from the binding of inhibitory KIRs to their cognate HLA class I ligands and activating signals from activating receptors (Ruggeri et al., 2002; Caligiuri, 2008; Ljunggren et al., 1990). NK cell responses are mediated by two main effector functions: direct cytolysis of target cells and the production of chemokines and cytokines. Through the latter mechanism (e.g., interferon-γ), NK cells are involved in the generation of adaptive T cell responses, possibly through direct interactions between naive T cells and NK cells migrating to secondary lymphoid compartments from inflamed peripheral tissues, as well as through indirect effects on dendritic cells (DCs) (Martin-Fontecha et al., 2004; Krebs et al., 2009).

[00326] GMP-compliant expansion of NK cells from umbilical cord blood. Previous studies have primarily used freshly harvested NK cells from peripheral blood. The low number of NK cells in peripheral blood seriously limits their therapeutic value. We have developed a system for ex vivo expansion of NK cells from umbilical cord blood (CB), which allows for the reliable production of clinically relevant doses of CB-NK cells in a GMP-compliant manner for adoptive immunotherapy using K562-based artificial antigen-presenting cells (aAPCs) expressing membrane-bound IL-21, 4-1BB ligand, CD64 (FcγRI), and CD86 (clone 9.mbIL21) (Denman et al., 2012). Cord blood represents an attractive new source of NK cells for cellular immune therapy. The cells have already been collected, stored, and are available for administration. The cord blood donor can be optimally selected based on HLA type, KIR gene expression, and other factors. The method for obtaining CB-NK cells has been approved by the FDA. The protocol developed by the authors yields an average increase in NK cell proliferation of 3127-fold (range 1640-4931-fold) (Fig. 26A) with very low CD3 ⁺ cell counts (average 4.50×10 ⁶ ) (Fig. 26B).

[00327] Functional phenotype of ex vivo expanded CB-NK cells and their cytotoxic activity against myeloid leukemia target cells. Expanded CB-NK cells have a full repertoire of activating and inhibitory receptors and continue to highly express eomesodermin (Eomes) and T-bet (Figs. 26C-26D) (Gill et al., 2012; Intlekofer et al., 2005), two factors required for NK cell maturation and activation, lyse myeloid target cells in a dose-dependent manner (Fig. 26E), and, following adoptive transfer into non-diabetic obese severe combined immunodeficiency-gamma (NSG) mice, can hom to bone marrow, liver, spleen, and multiple lymphoid tissues (Fig. 27).

Genetic modification of CB-derived NK cells to enhance their anti-leukemia activity.

[00328] Chimeric antigen receptors (CARs) have been widely used to retarget T cell specificity against leukemia (Sadelain et al., 2003; Rosenberg et al., 2008; June et al., 2009), with strong clinical responses in patients with acute lymphoblastic leukemia (ALL) (Brentjens et al., 2013; Kalos et al., 2011; Maude et al., 2015). These infusions have been largely limited to the administration of autologous cells, as activated T cells taken from an allogeneic source likely increase the risk of GVHD. As described in this example, the safety and efficacy of engineered CB-NK cells could be tested as an alternative to T cells for immunotherapy of B-lymphoid malignancies. CB-derived NK cells offer multiple potential advantages over T cells: (i) allogeneic NK cells should not induce GVHD, as predicted from observations in mouse models and in patients with leukemia and solid malignancies treated with haploidentical or CB-derived NK cells (Olson et al., 2010; Rubnitz et al., 2010; Miller et al., 2005); (ii) mature NK cells have a limited lifespan of a few weeks, which allows for antitumor activity while reducing the likelihood of long-term side effects such as prolonged cytopenias caused by on-target/off-target normal tissue toxicity or the risk of malignant transformation; (iii) unlike T cells, NK cells will also have activity through their native receptors to kill antigen-negative target cells, potentially preventing immune evasion; (iv) generating an autologous T cell product for each patient is difficult and logistically limited (Ruggeri et al., 2002; Rubnitz et al., 2010). Using frozen, ready-to-use CB units stored in a large, shared cord blood bank to generate NK cells may offer promise for widespread scalability that would not be possible using T or NK cell products derived from autologous peripheral blood.

[00329] Thus, to enhance the stability and antileukemic activity of frozen and ex vivo expanded CB-NK cells, we genetically modified them with the retroviral vector iC9.CAR19-CD28-zeta-2A-IL15 (iC9/CAR.19./IL15), which (i) includes the CAR-CD19 gene to redirect their specificity to CD19; (ii) ectopically produces IL-15, a cytokine that plays an important role in NK cell survival and proliferation (Hoyos et al., 2010; Tagaya et al., 1996); and (iii) expresses the inducible caspase-9 (iC9) suicide gene (Di et al., 2011), which can be pharmacologically activated to eliminate the transgenic cells if desired. Initial data showed that CB-NK cells could be stably transduced to express the CAR molecule (Fig. 29A). Using a standard ^51Cr release assay, we found that CB-NK cells transduced with iC9/CAR.19/IL15 had specific cytotoxic activity against CD19 ⁺ Raji cells and primary CLL cells (n=18; Fig. 29B). CAR-NK cells and untransduced NK cells showed similar effector function against K562 cells, suggesting that genetic modification of CB-NK cells does not alter their intrinsic cytotoxicity against NK-sensitive targets (Fig. 29B).

[00330] The in vivo transfer and persistence of iC9/CAR.19/IL15-modified CB-NK cells was then assessed using NSG mice with a Raji xenograft model. Untransduced CB-NK cells and iC9/CAR.19/IL15-transduced CB-NK cells were administered to mice transplanted with Raji cells. As shown in Fig. 30A, iC9/CAR.19/IL15 ⁺ CB-NK cells were able to hom to the spleen, liver, and bone marrow (tumor infiltrating sites), whereas CAR.CD19 ⁺ CB-NK cells lacking the IL-15 gene in the construct, as well as untransduced CB-NK cells, were virtually undetectable at the tumor sites.

[00331] iC9/CAR.19/IL15-transduced CB-NK cells had enhanced antitumor activity in vivo. To examine the in vivo antitumor activity of iC9/CAR.19/IL15-transduced CB-NK cells, we injected mice with NSG FFLuc-labeled Raji cells at 2 x 10 ⁵ /mouse. On the same day, the mice were intravenously infused with one of 6 infusions of control NT-CB-NK cells or CAR.19- or iC9/CAR.19/IL15-transduced CB-NK cells (10 x 10 ⁶ /mouse). Tumor growth was monitored by assessing changes in tumor bioluminescence over time. As shown in Fig. 30B, tumor bioluminescence rapidly increased in mice transplanted with Raji cells and treated with control NT-CB-NK cells. In contrast, infusion of either CAR.19 ⁺ or iC9/CAR.19/IL15 ⁺ CB-NK cells resulted in significantly increased survival compared to the effect of NT-CB-NK cells (P = 0.006 and P = 0.001, respectively). Notably, iC9/CAR.19/IL15 ⁺ CB-NK cells regulated tumor growth and increased survival (Fig. 30C) significantly better than the CAR.CD19 construct lacking the IL-15 gene, further highlighting the important contribution of IL-15 in enhancing antitumor activity.

[00332] iC9/CAR.19/IL15-transduced CB-NK cells did not exhibit signs of autonomous or unregulated growth in vitro or in vivo. To understand whether the IL-15 gene in the vector could lead to autonomous or unregulated growth of transduced CB-NK cells, we cultured iC9/CAR.19/IL15-transduced CB-NK cells in complete serum-free stem cell culture medium (SCGM) without the addition of exogenous IL-2 or stimulation of clone 9.mbIL21 for 42 days (n=5). Viable cells were counted and passaged every three days with the medium replaced with fresh complete SCGM. As shown in Fig. 28A, iC9/CAR.19/IL15-transduced CB-NK cell cultures showed no signs of abnormal growth for 6 weeks, after which the cells ceased to expand. Karyotyping performed on iC9/CAR.19/IL15-transduced CB-NK cells cultured for up to 17 weeks (n=7) did not reveal any chromosomal alterations (data not shown). We also performed chromosome and SNP microarray analyses on paired CB-NK cells (n=6) before CAR transduction (at baseline) and 22 weeks after CAR transduction and ex vivo expansion and found no evidence of genetic instability. At follow-up exceeding 10 months, the authors observed no evidence of autonomous growth or leukemic transformation in mice receiving iC9/CAR.19/IL15- or CAR.19-transduced CB-NK cells. Histopathological examination revealed no lymphocytic infiltration, proliferation, or lymphoma in any tissue of these mice. Rudimentary lymphoid tissues of the spleen and lymph nodes were lymphocyte-free in all NSG mice in both groups (Fig. 28B), and no lymphocytic infiltration or proliferation was observed in the bone marrow of these mice. Hematological tests revealed normal leukocyte and lymphocyte counts and no evidence of lymphocytic leukemia in both groups of mice.

IL-15 production by CAR-transduced NK cells

[00333] To confirm whether iC9/CAR.19/IL15 ⁺ CB-NK cells could produce IL-15, control untransduced CB-NK and iC9/CAR.19/IL15 ⁺ CB-NK lymphocytes were cultured in triplicate in the presence or absence of CLL CD19 ⁺ B cells, and the culture supernatants were collected to assess the level of IL15 release after culture for 24, 48, and 72 hours. As shown in Fig. 29, IL15 was not detected in the supernatants collected from untransduced CB-NK cells cultured alone or with CLL targets. Conversely, iC9/CAR.19/IL15 ⁺ -CB-NK cells produced low amounts of IL15 in the absence of antigen stimulation [mean 15.05 pg/mL/ ¹⁰⁶ cells (range 6.2–23.47 pg/mL)], but this amount was significantly increased upon antigen stimulation [mean 27.61 pg/mL/ ¹⁰⁶ cells (range 15.82–38.18 pg/mL)] (P=0.02). The ability of iC9/CAR.19/IL15-transduced NK cells to produce IL-15 in vivo was assessed in Raji cell-transplanted NSG mice. Serum IL-15 levels after NK cell expansion (2 weeks after expansion) were 40-50 pg/mL, equivalent to levels found in the supernatant of cultured cells.

[00334] Exogenous recombinant human IL-15 (RhIL-15) has been used in clinical settings. In a recent phase 1 study in patients with metastatic melanoma or renal cell carcinoma, patients with metastatic malignant melanoma or metastatic renal cell carcinoma were administered loading doses of 3.0, 1.0, and 0.3 μg IL-15/kg per day for 12 consecutive days (Conlon et al., 2015). RhIL-15 was shown to activate NK cells, monocytes, γδ, and CD8 T cells. Doses of 3.0, 1.0, and 0.3 μg/kg per day resulted in maximum serum concentrations (Cmax) of 43,800±18,300, 15,900±1,900, and 1,260±350 pg/mL, respectively. Dose-limiting toxicity observed in patients receiving 3.0 and 1.0 μg/kg per day was assessed by 3 parameters, such as hypotension, thrombocytopenia, and an increase in ALT and AST levels, and it was found that the maximum tolerated dose was 0.3 μg/kg per day. In a clinical study conducted by the present inventors, no toxicity associated with IL-15 release by iC9/CAR.19/IL15-transduced CB-NK cells was observed. This is likely due to the fact that the levels of IL-15 produced by transduced NK cells were, on average, 2-3 log lower than the level achieved in clinical trials with treatment with exogenous IL-15.

[00335] iC9/CAR.19/IL15 ⁺ -CB-NK cells were depleted after activation of the suicide gene by a small molecule dimerizer. To exclude the possibility of excessive toxicity mediated by the release of inflammatory cytokines by transduced CB-NK cells or uncontrolled NK cell growth, we included a suicide gene based on the inducible caspase-9 gene (Di et al., 2011) in the construct. As shown in Fig. 30A, addition of at least 10 nM of a small-molecule dimerizer to iC9/CAR.19/IL15-transduced CB-NK cell cultures induced apoptosis/necrosis in 60% of transgenic cells within 4 hours, as assessed by annexin-V and 7AAD staining, but had no effect on NT-CB-NK cell viability. The suicide gene was also effective in vivo. Mice were intravenously transplanted with Raji tumor cells and then treated with iC9/CAR.19/IL15-transduced CB-NK cells. Administration of the small molecule dimerizer AP1903 (50 μg, intraperitoneally at 2-day intervals) 10–14 days after NK cells had localized and disseminated to various tumor sites (Fig. 30B, left panel) resulted in a dramatic decrease in the number of IC9/CAR.19/IL15-transduced CB-NK cells in the blood and tissues of treated mice (Fig. 30B, right panel), indicating the removal of transgenic cells in vivo.

A clinical trial to evaluate the safety and efficacy of iC9/CAR.19/IL15-transduced CB-NK cells in patients with relapsed/refractory B-lymphoid malignancies.

[00336] This Phase I/II dose-escalation study was conducted to evaluate the safety and relative efficacy of iC9/CAR.19/IL15-transduced CB-NK cells in patients with relapsed/refractory B-cell lymphoid malignancies (ALL, CLL, NHL). This clinical study is based on the synergistic antitumor activity produced by CAR-CB-NK cells and the favorable lymphopenic environment induced following lymphodepleting therapy (Dudley et al., 2002; Dudley et al., 2005). Thus, patients were administered cyclophosphamide at a dose of 5-300 mg/m/day for 3 days. Escalating doses of iC9/CAR.19/IL15-transduced CB-NK cells ( ¹⁰⁷ /kg-10/kg) were administered once on day 0 to determine the highest dose at which iC9/CAR.19/IL15-transduced CB-NK cells could be safely administered to patients with relapsed/refractory B-lymphoid malignancies, as defined by standard NCI toxicity criteria. A patient's CB unit typed for 4/6, 5/6, or 6/6 HLA class I (serologic typing) and II (molecular typing) antigens was used for CB-NK cell expansion and CAR transduction. CB units are available from the MD Anderson cord blood bank.

[00337] To gain insight into the persistence, functionality, and antileukemic potential of adoptively transferred iC9/CAR.19/IL15-transduced CB-NK cells, a series of phenotypic and functional assays can be performed. The expansion and duration of persistence of adoptively transferred genetically modified NK cells in serially obtained PB samples can be assessed by qPCR using a pair of primers that specifically amplify the unique CAR transgene with a detection sensitivity of 1/10,000 CAR ⁺ NK cells. If sufficient circulating NK cells were present, the authors quantified them by flow cytometry using a mAb specific for the CH2-CH3 region of iC9/CAR.19/IL15 with a detection sensitivity of 1/1000 CAR ⁺ NK cells. Flow cytometric measurements can be combined with analysis of the expression of activating and inhibitory receptors on the NK cell surface. Retention of CD19-retargeted effector function can be assessed by ^51Cr release assay, CD107a degranulation assay (Rubio et al., 2003; Rezvani et al., 2009), cytokine release (determined by intracellular cytokine IFNγ and IL-2 assay), and chemokine release (MIP1-α and MIP-1β) against CD19-expressing cell lines, and if possible, primary tumor CD19 ⁺ cells are collected from recipients before treatment and stored.

[00338] To prevent any possible complications (Porter et al., 2011; Grupp et al., 2013), a suicide gene based on the inducible caspase-9 gene (Hoyos et al., 2010), for example, can be included in the CAR19 vector. As shown in Fig. 30, the addition of a small molecule dimerizer, AP1903, induces rapid apoptosis of transgenic cells, and therefore, in the case of long-term B lymphopenia, the dimerizer can be introduced to induce apoptosis of CAR19-transduced CB-NK cells and to restore normal B cells. This strategy may also be useful if transduced NK cells have been found to induce GVHD.

Example of patient inclusion in the trial program

Inclusion criteria:

[00339] 1. Patients with a history of CD19-positive B-lymphoid malignancies (ALL, CLL, NHL) and who have received at least 2 lines of standard chemoimmunotherapy or targeted therapy and who have been diagnosed with chronic disease.

[00340] 2. Patients with ALL, CLL, NHL with disease relapse after standard therapy or stem cell transplantation.

[00341] 3. Patients who have received their last cytotoxic chemotherapy at least 3 weeks ago at the time of initiation of lympho-depleting chemotherapy. Patients may continue therapy with tyrosine kinase inhibitors or other targeted therapy for at least two weeks prior to lympho-depleting chemotherapy.

[00342] 4. Karnofsky/Lansky Performance Scale >70.

[00343] 5. Adequate organ function:

[00344] a. Kidneys: Creatinine clearance (Cockcroft-Gault estimate) ≥60 ^cm3 /min.

[00345] b. Liver: ALT/AST ≤2.5×ULN or ≤5×ULN if liver metastases are documented, total bilirubin ≤1.5 mg/dL, except for patients with Gilbert syndrome, in whom total bilirubin should be ≤3.0 mg/dL.

[00346] c. Heart: Cardiac ejection fraction ≥50%, no confirmed pericardial effusion as determined by ECHO or MUGA, and no clinically significant ECG findings.

[00347] d. Lungs: No clinically significant pleural effusion, baseline oxygen saturation >92% at room temperature.

[00348] 6. Possibility of obtaining written informed consent.

[00349] 7. Age 7-80 years.

[00350] 8. All study participants of childbearing potential must use effective birth control during the study. Acceptable forms of birth control for female participants include: hormonal contraceptives, an intrauterine device, a diaphragm with spermicide, a condom with spermicide, or abstinence for the duration of the study. If a female participant becomes pregnant or suspects that she may be pregnant, she must immediately notify her physician. If a female participant becomes pregnant during the study, she will be withdrawn from the study. Males who are fathering children must use effective birth control during the study. If a male participant fathers a child or suspects that he has fathered a child during the study, he must immediately notify his physician.

[00351] 9. Signed consent for long-term follow-up protocol PA17-0483.

Exclusion criteria:

[00352] 1. A positive beta-HCG test in women of childbearing potential, defined as not being postmenopausal for 24 months, not having had prior surgical sterilization, or breastfeeding.

[00353] 2. Known positive HIV serologic tests.

[00354] 3. Presence of grade 3 or higher toxicity after previous treatment.

[00355] 4. Presence of a fungal, bacterial, viral, or other infection requiring intravenous antimicrobial therapy. Note: Simple urinary tract infections and uncomplicated bacterial pharyngitis are acceptable if responding to active treatment.

[00356] 5. Presence of acute neurological disorder(s).

[00357] 6. Concomitant use of other investigational agents.

Example of a treatment regimen

[00358] Lymph-depleting chemotherapy (inpatient):

[00359] A day or earlier

[00360] Day 15: Onset of NK cell production

[00361] Day 6: Hospital Admission/Intravenous Hydration

[00362] Day 5: Fludarabine 30 mg/ ^m2 IV/Cyclophosphamide 300 mg/m2 ^IV

[00363] Mesna 300 mg/ ^m2 , intravenous

[00364] Day 4: Fludarabine 30 mg/ ^m2 IV/Cyclophosphamide 300 mg/ ^m2 IV

[00365] Mesna 300 mg/ ^m2 , intravenous

[00366] Day 3: Fludarabine 30 mg/ ^m2 IV/Cyclophosphamide 300 mg/ ^m2 IV

[00367] Mesna 300 mg/ ^m2 , intravenous

[00368] Day 2: Break

[00369] Day 1: Break

[00370] Day 0: Infusion of iC9/CAR.19/IL15-transduced CB-NK cells (per dose)

[00371] Infusion of iC9/CAR.19/IL15-transduced CB-NK cells (per dose)* between day 7

[00372] and day 14

[00373] Lymph-depleting chemotherapy (outpatient):

[00374] A day or earlier

[00375] Day 15: Onset of NK cell production

[00376] Day 5: Fludarabine 30 mg/ ^m2 IV/Cyclophosphamide 300 mg/ ^m2 IV/Mesna 300 mg/ ^m2 IV

[00377] Day 4: Fludarabine 30 mg/ ^m2 IV/Cyclophosphamide 300 mg/ ^m2 IV/Mesna 300 mg/ ^m2 IV

[00378] Day 3: Fludarabine 30 mg/ ^m2 IV/Cyclophosphamide 300 mg/ ^m2 IV/Mesna 300 mg/ ^m2 IV

[00379] Day 2: Break

[00380] Day 1: Break

[00381] Day 0: Infusion of iC9/CAR.19/IL15-transduced CB-NK cells (per dose)

[00382] Infusion of iC9/CAR.19/IL15-transduced CB-NK cells (per dose)* between day 7 and day 14

[00383] * If DLT is not observed within the first 7 days after the initial NK cell infusion, the patient may receive a 2nd NK cell infusion between days 7 and 14 at the same dose of NK cells that was initially administered.

[00384] Three doses can be tested: 10 ⁵ , 10 ⁶ , and 10 ⁷ per kilogram of body weight. The dose for CAR-NK cell infusion is determined based on adjusted body weight for patients weighing 20% above their ideal body weight. For patients weighing less than or equal to 20% of their ideal body weight, the calculation is based on actual body weight.

[00385] If a patient experiences a relapse or chronic disease after protocol evaluation, an additional CAR-NK infusion may be administered. If residual cells were observed after the initial production, these may also be used, or a new unit of cord blood may be selected for CAR-NK. The pre-screening test need not be repeated within 45 days of the previous test or at the physician's discretion.

[00386] The cyclophosphamide dose was determined based on adjusted body weight for patients weighing 20% above their ideal body weight. For patients weighing less than or equal to 20% of their ideal body weight, the calculation was based on actual body weight.

[00387] On day 0, NK cell infusion was administered intravenously. Premedication was administered with 25 mg of Benadryl orally or intravenously and 650 mg of Tylenol orally. Steroid use is contraindicated unless required for physiological replacement therapy.

[00388] CAR-NK cell infusion may be administered to individuals in a hospital or outpatient setting, depending on bed availability and/or the patient's health status. Vital signs (temperature, heart rate, blood pressure, and respiratory rate) may be obtained for all patients in accordance with the BMT standard for cell therapy.

[00389] Start CAR-NK cell infusion approximately every 15 minutes x 4 times.

[00390] After completion of the CAR-NK cell infusion, it was administered approximately every 30 minutes x 2 or up to 1 hour.

[00391] It was then carried out approximately every hour, depending on the patient's health condition.

[00392] NK cells can be obtained in the following manner:

[00393] Frozen cord blood units can be thawed and mononuclear cells can be isolated by Ficoll density gradient centrifugation. NK cells can be CAR-transduced and can be produced for 14-22 days in liquid cultures using APC feeder cells, as detailed in the Chemical Synthesis, Cell Production, and Quality Control (CMC) manual.

[00394] NK Product Release Criteria

[00395] To release expanded NK cells for re-infusion, the following minimum criteria must be met:

[00396] Gram stain: "No microorganisms detected."

[00397] CAR ⁺ NK cells: >15%

[00398] CD3 ⁺ cell count: < 2 ⁵ CD3 ⁺ cells/kg.

[00399] CD32 ⁺ cell count: (aAPV): <5%

[00400] NK cells (CD16+/56+): > 80%

[00401] Visual inspection: “No signs of contamination” (turbidity; changes in the color of the medium).

[00402] Endotoxin test: < 5 EU/kg.

[00403] Viability: ≥70%.

[00404] Other parameters that may be monitored include culture sterility, i.e., the absence of bacteria and fungi. If more than 2 x 10 ⁵ CD3 ⁺ cells/kg are present, a second round of CD3 depletion may be performed. The cell dose for infusion may be reduced so that the infused CD3 ⁺ cells are < 2 x 10 ⁵ /kg. If an adequate dose of CAR ⁺ NK cells is not obtained, all available cells may be infused. If this occurs in patients who are at the MTD detection stage in this study, they will not be counted in either arm. If a dose of NK exceeding the required dose is obtained, the additional NK cells may be cryopreserved for subsequent infusions or may be used for research. If CAR ⁺ NK cells cannot be released due to microbial contamination, another cord blood unit can be selected and production can begin again. If the patient has already undergone lympho-depleting chemotherapy, a second dose of lympho-depleting chemotherapy may be required before the CAR+ NK cell infusion.

[00405] Frozen cells: CAR-NK cells that begin to be produced before day 15 can be cryopreserved and collected for infusion after determining that they meet the release criteria. Cryopreserved cells can be thawed for infusion on day 0 in accordance with standard GMP operating procedures. As shown above, after confirming the safety, efficacy, in vivo expansion, and persistence of fresh CAR-NK cells for infusion into the first 9 patients, the frozen and finished CAR-NK product can be used and administered to an additional 3 patients at the highest dose level of 1 x 10 ⁷ /kg for the frozen CAR-NK product. The safety and efficacy of this approach can be assessed using the effTox approach. The presence of viable NK cells can then be determined at approximately days +1, +3, and +7 post-infusion. If the levels are comparable to those the authors observed with fresh CAR-NK cells, then they can move on to a phase II study using frozen CAR-NK cells.

Administration of the dimerizer AP1903 for the treatment of cytokine release syndrome (CRS), neurotoxicity, or GVHD.

[00406] Treatment stages for CRS, neurotoxicity, and GVHD may be performed. Tocilizumab may be administered at a dose of 8 mg/kg intravenously every 6 hours as needed for up to 3 doses daily for the treatment of grade 2 CRS or grade 2 neurotoxicity unresponsive to standard supportive measures. For the treatment of grade 3 CRS and grade 3 neurotoxicity, a single dose of AP1903 (0.4 mg/kg as an intravenous infusion over approximately 2 hours) may be administered in addition to tocilizumab. The AP1903 dose is based on published Pk data, which show plasma concentrations ranging from 10 to 1275 ng/mL over a dose range of 0.01 to 1.0 mg/kg, with plasma 65 levels declining to 18% and 7% of peak at 0.5 and 2 hours post-dose. The dimerizer can also be used to treat grades I-IV GVHD. Responses in patients with GVHD who received caspase 9-positive T cells followed by AP1903 occurred within the first 24-48 hours. Patients who did not experience downgrading, CRS, or neurotoxicity to grade 2 or less within 12 hours may receive a second dose of AP1903, but they will also receive high-dose steroids.

Assessment: Any time before or during the study: HLA typing (high resolution A, B, DR).

[00407] The following assessments may be obtained within 30 days of enrollment in the study program: history and physical examination; complete blood count and blood count, platelet count, total bilirubin, SGPT, alkaline phosphatase, LDH, albumin, total protein, BUN, creatinine, glucose, electrolytes, PT/PTT, type and screen, immunoglobulin levels (IGG, IGM, IGA) and 3 cytokines (IL6, IFN-gamma, TNF-alpha); HIV serology; ECHO or MUGA; pulmonary function tests, if clinically indicated; chest x-ray; urinalysis; CT scan of the brain; PET/CT, if clinically indicated; bone marrow aspirate, if clinically indicated; ECG.

Examination within 7 days after the start of lympho-depleting chemotherapy:

[00408] History and physical examination, including weight and vital signs.

[00409] Laboratory tests: complete blood count, blood count, platelet count, total bilirubin, SGPT, alkaline phosphatase, LDH, albumin, total protein, BUN, creatinine, glucose, electrolytes, and cytokines. Serum pregnancy test if the participant is of childbearing potential.

[00410] The following assessments can be obtained at day 0, day 3 (±1 day), day 7 (±2 days), day 14 (±2 days), and day 21 (±3 days), at week 4 (±5 days), week 8 (±5 days), week 12 (±5 days), week 16 (±14 days), month 6 (±28 days), month 9 (±28 days), and year 1 (±28 days) after CAR-NK infusion:

[00411] Physical examination, including body weight and vital signs, on day 7 only (±2 days).

[00412] Complete blood count and blood cell count, platelet count, chemical analysis and cytokine analysis.

[00413] Analysis of 3 cytokines (IL6, IFN-gamma, TNF-alpha) at all time points except when clinically indicated at 6 months (±28 days), 9 months (±28 days), and 12 months (±28 days).

[00414] Anti-HLA antibodies only at week 4 (±5 days) and week 12 (±5 days).

[00415] Laboratory studies: detection, determination of phenotype and function of CAR-NK.

[00416] The following assessments can be obtained at 4 weeks (±5 days), 8 weeks (±5 days), 12 weeks (±5 days), 16 weeks (±14 days), 6 months (±28 days), 9 months (±28 days), and 12 months (±28 days) after CAR-NK infusion:

[00417] PET/CT as clinically indicated.

[00418] The following assessments can be obtained at day 7 (±2 days), week 4 (±5 days), week 8 (±5 days), week 12 (±5 days), week 16 (±14 days), month 6 (±28 days), month 9 (±28 days), and year 1 (±28 days) after CAR-NK infusion:

[00419] Bone marrow aspiration and/or biopsy as clinically indicated.

[00420] Laboratory tests: bone marrow aspiration 5 to 10 ml.

[00421] Lymph node biopsy

[00422] If a patient has had a diagnostic lymph node biopsy, a portion of the sample, if available, may be analyzed.

[00423] RCR testing on NK-CAR cells

[00424] RCR testing of NK cells in the culture was performed on day -4 in a laboratory in accordance with GMP. If RCR results are positive, CAR-NK cells cannot be infused. After this, the patient must withdraw from the study program. If results are delayed, NK cell culture can be continued for another week.

[00425] See the Scoring Table in FIG. 34. In this case, the time window provided is: days 3 (±1 day), 7 (±2 days), 14 (±2 days), and 21 (±3 days), weeks 4 (±5 days), 8 (±5 days), 12 (±14 days), 16 (±14 days), and months 6 (±28 days), 9 (±28 days), and 12 (±28 days). 1. History and physical examination, complete blood count, chemistry panel: within 30 and 7 days of starting lymphodepleting chemotherapy. Pregnancy test: within 7 days of starting lymphodepleting chemotherapy. 2. Physical examination on day 7 (±2 days) only. 3. As clinically indicated. Shown as part of the laboratory tests z-code. Samples may be divided into batches and taken approximately every 3 months for results. 4. 3-cytokine assay: day 0 before CAR-NK infusion. 5. Indicated as part of protocol LAB00-099. 6. Replication-competent retrovirus (RCR) assay: approximately 1, 3, and 6 months after NK cell infusion, then once every 6 months for 5 years, and then once annually and thereafter for 10 years, consistent with long-term follow-up in study PA17-0483.

* * *

[00426] All of the methods disclosed and claimed in this application can be realized and carried out without undue experimentation in accordance with the present description. Although the compositions and methods according to the invention have been described in terms of preferred embodiments of the invention, it will be obvious to those skilled in the art that changes can be made in these methods, steps, or in the sequence of steps of the method described herein without departing from the concept, spirit, and scope of the invention. More specifically, it will be obvious to those skilled in the art that certain agents that are both chemically and physiologically related can be substituted for the agents described herein, but the same or similar results can be achieved. All such similar substitutions and modifications obvious to those skilled in the art are considered to be within the spirit, scope, and concept of the invention as defined in the appended claims.

References

The following documents, to the extent that they relate to representative procedures or other elements supplementary to those set forth in this application, are specifically incorporated into this specification by reference.

Appelbaum, Leukemia 1997; 11 Suppl 4:S15-S17.

Arora et al. Blood. 2012; 119(1):296.

Austin-Ward and Villaseca, Revista Medica de Chile, 126(7):838-845, 1998.

Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994.

Bader et al. J.Clin.Oncol. 2004;22:1696-1705.

Bar et al. Leuk.Res.Treatment. 2014;2014:421723.

Bennekov et al., Mt. Sinai J. Med. 71(2):86-93, 2004.

Brentjens et al., Sci.Transl.Med. 2013;5:177ra38.

Bukowski et al., Clinical Cancer Res., 4(10):2337-2347, 1998.

Burger et al. N.Engl.J.Med. 2015; 373:2425–2437.

Byrd et al. N.Engl.J.Med. 2013; 369:32-42.

Byrd et al. Blood 2015; 125:2497–2506.

Caligiuri, Blood 2008; 112:461-469.

Camacho et al. J Clin Oncology 22(145): Abstract no. 2505 (antibody CP-675206), 2004.

Campbell, Curr. Top. Microbiol. Immunol., 298: 23-57, 2006.

Cheah et al., Ann. Oncol. 2015; 26:1175-1179.

Cheson et al. J.Clin.Oncol. 1990; 8:813-819.

Cheson et al. J Clin Oncol 2014; 32: 3059-68.

Chothia et al., EMBO J. 7:3745, 1988.

Christodoulides et al., Microbiology, 144(Pt 11):3027-3037, 1998.

Cohen et al. J Immunol. 175:5799-5808, 2005.

Coiffier. N.Engl.J.Med. 2002; 346:235-242.

Conlon et al. J.Clin.Oncol. 2015;33:74-82.

Connors, Mod. Pathol. 2013; 26 Suppl 1:S111-S118.

Davidson et al., J. Immunother., 21(5):389-398, 1998.

Davila et al. PLoS ONE 8(4): e61338, 2013.

Denman et al., PLoS.One. 2012; 7:e30264.

Di et al., N. Engl. J. Med. 2011; 365:1673–1683.

Doulatov et al., Cell Stem Cell. 10:120-36, 2012.

Dudley et al. Science 2002;298:850-854.

Dudley et al. J.Clin.Oncol. 2005; 23:2346–2357.

European Patent Application No. EP2537416

Fedorov et al., Sci. Transl. Medicine, 5(215), 2013.

Fielding et al. Blood 2016-0641 pg. 68 2009; 113:4489–4496.

Frolet et al., BMC Microbiol. 10:190 (2010).

Gaj et al., Trends in Biotechnology 31(7), 397-405, 2013.

Gill et al., Blood. 2012 Jun 14; 119(24):5758-68.

Gisselbrecht et al. J.Clin.Oncol. 2010; 28:4184–4190.

Gokbuget et al. Blood 2012; 120:1868-1876.

Goldstone et al. Blood 2008; 111:1827–1833.

Gribben, Best.Pract.Res.Clin.Haematol. 2007; 20:513-527.

Grupp et al. N.Engl.J.Med. 2013; 368:1509–1518.

Hallek et al. Blood 2008; 111(12):5446-5456.

Hallek et al. Blood 2018; 131: 2745-60.

Hamadani et al., Biol.Blood Marrow Transplant. 2013;19:625-631.

Hanibuchi et al., Int. J Cancer, 78(4):480-485, 1998.

Heemskerk et al. Hum Gene Ther. 19:496-510, 2008.

Hellstrand et al., Acta Oncologica, 37(4):347-353, 1998.

Hollander, Front. Immun., 3:3, 2012.

Honigberg et al. Proc.Natl.Acad.Sci.U.S.A 2010;107:13075-13080.

Hoyos et al. Leukemia 2010; 24: 1160-70.

Hubert et al., Proc. Natl. Acad. Sci. USA 96 14523-28, 1999.

Hui and Hashimoto, Infection Immun., 66(11):5329–5336, 1998.

Hurwitz et al. Proc Natl Acad Sci USA 95(17): 10067-10071, 1998.

Ibrahim et al., Bayesian Survival Analysis. Springer, NY, 2001. 2015.

Publication of International Patent Application No. WO 00/37504

Publication of International Patent Application No. WO 01/14424

Publication of International Patent Application No. WO 2007/069666

Publication of International Patent Application No. WO 2007/069666

Publication of International Patent Application No. WO 98/42752

Publication of International Patent Application No. WO/2014055668

Publication of International Patent Application No. WO1995001994

Publication of International Patent Application No. WO1998042752

Publication of International Patent Application No. WO2000037504

Publication of International Patent Application No. WO200014257

Publication of International Patent Application No. WO2001014424

Publication of International Patent Application No. WO2006/121168

Publication of International Patent Application No. WO2007/103009

Publication of International Patent Application No. WO2009/101611

Publication of International Patent Application No. WO2009/114335

Publication of International Patent Application No. WO2010/027827

Publication of International Patent Application No. WO2011/066342

Publication of International Patent Application No. WO2012/129514

Publication of International Patent Application No. WO2013/071154

Publication of International Patent Application No. WO2013/123061

Publication of International Patent Application No. WO2013/166321

Publication of International Patent Application No. WO2013126726

Publication of International Patent Application No. WO2014/055668

Publication of International Patent Application No. WO2014031687

Publication of International Patent Application No. WO2015016718

Publication of International Patent Application No. WO99/40188

Intlekofer et al., Nat.Immunol. 2005;6:1236-1244.

Iuliucci, et al. Journal of Clinical Pharmacology 41, 870-879 (2001).

Jain et al., Blood 2015; 125:2062–2067.

Janeway et al., Immunobiology: The Immune System in Health and Disease, ^3rd Ed., Current Biology Publications, p. 433, 1997.

Johnson et al., Lancet 1996; 347:1432–1438.

Johnson et al., Blood 114:535-46, 2009.

Jores et al., PNAS U.S.A. 87:9138, 1990.

June et al., Nat.Rev.Immunol. 2009;9:704-716.

Kalos et al., Sci.Transl.Med. 2011;3:95ra73.

Kaplan, EL and Meier, P, J. American Statistical Association 53:457-481, 1958. 2015.

Kataoka et al. J Immunol 1996; 156(10):3678.

Keating et al., J.Clin.Oncol. 2005; 23:4079–4088.

Kim et al., Nature Biotechnology 31, 251-258, 2013.

Kirchmaier and Sugden, J. Virol., 72(6):4657-4666, 1998.

Krebs. Blood 2009; 113:6593–6602.

Kuruvilla et al. Leuk Lymphoma 2008;49:1329-1336.

Lanier LL. Nat. Immunol. 2016-0641 pg. 70, 2008;9:495-502.

Leal, M., Ann N Y Acad Sci 1321, 41-54, 2014.

Lefranc et al., Dev. Comp. Immunol. 27:55, 2003.

Leung et al., Blood 2012; 120:468-472.

Li et al., Nat Biotechnol. 23:349-354, 2005.

Li et al. Proc. Natl. Acad. Sci. USA 89:4275-4279, 1992.

Linnemann, C. et al., Nat Med 21, 81-85, 2015.

Liu et al. Leukemia 2018; 32(2):520-531.

Ljunggren et al., Immunol.Today 1990;11:237-244.

Lockey et al., Front. Biosci. 13:5916-27, 2008.

Loewendorf et al., J. Intern. Med. 267(5):483-501, 2010.

Ludwig et al., Nature Biotech., (2):185–187, 2006a.

Ludwig et al., Nature Methods, 3(8):637–646, 2006b.

Marshall et al., Future Microbiol. 4:731-42, 2009.

Martin-Fontecha et al., Nat.Immunol. 2004;5:1260-1265.

Maude et al., N Engl J Med; 371: 1507-17, 2014.

Maude et al., Blood 2015; 125:4017–4023.

Mehta RS, Rezvani K. Hematology Am Soc Hematol Educ Program 2016; 2016: 106-18.

Miller et al., Blood 2005;105:3051-3057.

Mokyr et al., Cancer Res 58:5301-5304, 1998.

Muftuoglu et al., N Engl J Med 2018; 379: 1443-51.

Neelapu et al., N Engl J Med 2017; 377:2531-44.

Notta et al., Science, 218-221, 2011.

Olson et al. Blood 2010;115:4293-4301.

Pardoll, Nat Rev Cancer, 12(4): 252-64, 2012

Parkhurst et al., Clin Cancer Res. 15: 169-180, 2009.

Passweg et al., Bone Marrow Transplant. 2016-0641, pg. 69 1998;21:153-158.

Poon et al. Bone Marrow Transplant. 2013; 48:666-670.

Poon et al. Biol.Blood Marrow Transplant. 2013;19:1059-1064.

Porter et al., N. Engl. J. Med. 2011;365:725-733.

Qin et al., Proc. Natl. Acad. Sci. USA, 95(24):14411-14416, 1998.

Rai et al., N. Engl. J. Med. 2000;343:1750-1757.

Rezvani et al., Blood 2009;113:2245-2255.

Rieder et al., J. Interferon Cytokine Res. (9):499-509, 2009.

Rosenberg et al. Nat Rev Cancer 2008;8:299-308.

Rubio et al., Nat.Med. 2003;9:1377-1382.

Rubnitz et al., J.Clin.Oncol. 2010;28:955-959.

Ruggeri et al., Science 2002;295:2097-2100.

Rykman, et al., J. Virol. 80(2):710-22, 2006.

Sadelain et al., Nat.Rev.Cancer 2003;3:35-45.

Sadelain et al., Cancer Discov. 3(4): 388-398, 2013.

Sambrook et al., Molecular Cloning: A Laboratory Manual, ^3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001.

Savani et al., Blood 2006;107:1688-1695.

Sehn et al., J Clin Oncol. 2005 Aug 1; 23(22):5027-33.

Shah et al., PLoS One. 2013 Oct 18; 8(10):e76781.

Singh et al., Cancer Research, 68:2961-2971, 2008.

Singh et al., Cancer Research, 71:3516-3527, 2011.

Sullivan et al., N. Engl. J. Med. 1989;320:828-834.

Tagaya et al., Immunity. 1996; 4:329-336.

Takahashi et al., Cell, 126(4):663–76, 2007.

Tam et al., Blood 2009; 113:4144–4152.

Terakura et al., Blood. 1:72-82, 2012.

Thall and Cook JD. Biometrics 2004; 60(3):684-693.

Thall et al., J Biopharm Stat 2006; 16(5):623-638.

Thall et al., Clin Trials 2014;11(6):657-666.

Turtle et al., Curr. Opin. Immunol., 24(5): 633-39, 2012.

Turtle et al., J Clin Oncol 2017; 35: 3010-20.

U.S. Patent No. 4,870,287

U.S. Patent No. 5,739,169

U.S. Patent No. 5,760,395

U.S. Patent No. 5,801,005

U.S. Patent No. 5,824,311

U.S. Patent No. 5,830,880

U.S. Patent No. 5,844,905

U.S. Patent No. 5,846,945

U.S. Patent No. 5,885,796

U.S. Patent No. 5,994,136

U.S. Patent No. 6,013,516

U.S. Patent No. 6,103,470

U.S. Patent No. 6,207,156

U.S. Patent No. 6,225,042

U.S. Patent No. 6,355,479

U.S. Patent No. 6,362,001

U.S. Patent No. 6,410,319

U.S. Patent No. 6,416,998

U.S. Patent No. 6,544,518

U.S. Patent No. 6,790,662

U.S. Patent No. 7,109,304

U.S. Patent No. 7,442,548

U.S. Patent No. 7,446,190

U.S. Patent No. 598,364

U.S. Patent No. 7,989,425

U.S. Patent No. 8,008,449

U.S. Patent No. 8,017,114

U.S. Patent No. 8,058,065

U.S. Patent No. 8,071,369

U.S. Patent No. 8,119,129

U.S. Patent No. 8,129,187

U.S. Patent No. 8,183,038

U.S. Patent No. 8,268,620

U.S. Patent No. 8,329,867

U.S. Patent No. 8,354,509

U.S. Patent No. 8,546,140

U.S. Patent No. 8,691,574

U.S. Patent No. 8,735,553

U.S. Patent No. 8,741,648

U.S. Patent No. 8900871

U.S. Patent No. 9,175,268

U.S. Patent Publication No. 2010/0210014

U.S. Patent Publication No. 12/478,154

U.S. Patent Publication No. 2002131960

U.S. Patent Publication No. 2003/0211603

U.S. Patent Publication No. 2005/0260186

U.S. Patent Publication No. 2006/0104968

U.S. Patent Publication No. 2009/0004142

U.S. Patent Publication No. 2009/0017000

U.S. Patent Publication No. 2009/0246875

U.S. Patent Publication No. 2011/0104125

U.S. Patent Publication No. 2011/0301073

U.S. Patent Publication No. 20110008369

U.S. Patent Publication No. 2012/0276636

U.S. Patent Publication No. 2013/0315884

U.S. Patent Publication No. 20130149337

U.S. Patent Publication No. 2013287748

U.S. Patent Publication No. 2014/0120622

U.S. Patent Publication No. 2014022021

U.S. Patent Publication No. 20140294898

Varela-Rohena et al., Nat Med. 14: 1390-1395, 2008.

Wang et al., J Immunother. 35(9):689-701, 2012.

Wang et al., N. Engl. J. Med. 2013;369:507-516.

Wang et al., Blood 2015;126:739-745.

Wu et al., Adv. Cancer Res., 90: 127-56, 2003.

Wu et al., Cancer, 18(2): 160-75, 2012.

Yamanaka et al., Cell, 131(5):861–72, 2007.

Yawata et al., Blood 2008;112:2369-2380.

Yu et al., Science, 318:1917-1920, 2007.

Zhou et al., Clin.Lymphoma Myeloma.Leuk. 2014;14:319-326.

Zysk et al., Infect. Immun. 68(6):3740-43, 2000.

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SEQUENCE LIST

<110> BOARD OF REGENTS, JE UNIVERSITY OF TEXAS SYSTEM

<120> METHODS OF OBTAINING CAR-NK CELLS AND THEIR APPLICATION

<130> UTSC.P1186

<140>

<141>

<150> 62/826,856

<151> 2019-03-29

<160> 9

<170> PatentIn version 3.5

<210> 1

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic oligonucleotide

<400> 1

tgctgttgag gagctgga 18

<210> 2

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic oligonucleotide

<400> 2

agcacaccag gcagagtt 18

<210> 3

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic oligonucleotide

<400> 3

cggctgagga gcggaaga 18

<210> 4

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic oligonucleotide

<400> 4

tggaggtgag caatcccc 18

<210> 5

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic oligonucleotide

<400> 5

ttaatacgac tcactatagg 20

<210> 6

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic oligonucleotide

<400> 6

gttttagagc tagaaatagc 20

<210> 7

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic primer

<400> 7

gaacagatta ttctctcacc attagca 27

<210> 8

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic primer

<400> 8

agcgtattac cctgttggca aa 22

<210> 9

<211> 14

<212> DNA

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic probe

<400> 9

cctggagcaagaag 14

<---

Claims (52)

1. A method for ex vivo production of natural killer (NK) cells engineered to express one or more chimeric antigen receptors (CARs) and/or one or more T cell receptors (TCRs), wherein said method comprises: (a) culturing the initial population of NK cells in the presence of IL-2 and artificial presenting cells (APCs) expressing membrane-bound interleukin-21 (mbIL-21); (b) introducing one or more vectors for expressing CAR and/or TCR into NK cells; and (c) expansion of NK cells in a gas-permeable bioreactor in the presence of IL-2 and APCs expressing membrane-bound interleukin-21 (mbIL-21) to produce a population with an increased number of engineered NK cells. 2. The method according to claim 1, wherein the gas-permeable bioreactor is G-Rex100M. 3. The method according to claim 1 or 2, wherein the method does not involve removing or adding any components of the medium at step (c). 4. The method according to any one of paragraphs 1-3, wherein the method does not involve HLA typing and comparison. 5. The method according to any one of claims 1 to 4, wherein the engineered NK cells express a CAR. 6. The method according to any one of claims 1 to 5, wherein the engineered NK cells express TCR. 7. The method according to any one of claims 1 to 5, wherein the engineered NK cells express a CAR and a TCR. 8. The method according to any one of claims 1 to 7, wherein the initial population of NK cells is obtained from cord blood, peripheral blood, bone marrow, CD34 ⁺ cells, induced pluripotent stem cells (iPSC) or an NK cell line. 9. The method according to any one of claims 1 to 8, wherein the initial population of NK cells is obtained from umbilical cord blood. 10. The method according to claim 9, wherein the umbilical cord blood has been previously frozen. 11. The method according to claim 9, wherein the umbilical cord blood has not been pre-frozen. 12. The method according to claim 9, wherein the umbilical cord blood was taken from a healthy donor. 13. The method according to claim 9, wherein the initial population of NK cells is obtained by isolating mononuclear cells in a Ficoll-Paque density gradient. 14. The method of claim 13, further comprising depleting mononuclear CD3, CD14, and/or CD19 cells to obtain a starting population of NK cells. 15. The method of claim 13, further comprising depleting mononuclear CD3, CD14, and CD19 cells to obtain a starting population of NK cells. 16. The method according to claim 14 or 15, wherein the depletion comprises carrying out magnetic sorting. 17. The method according to any one of claims 1 to 16, wherein the ARS are gamma-irradiated ARS. 18. The method according to any one of claims 1 to 17, wherein the APCs are universal APCs (uAPCs). 19. The method of claim 18, wherein the uAPCs are engineered for expression (1) CD48 and/or CS1 (CD319), (2) membrane-bound interleukin-21 (mbIL-21) and (3) ligand 41BB (41BBL). 20. The method according to any one of claims 1 to 19, wherein the NK cells and APCs are present in a ratio of 1:1 to 1:10. 21. The method according to any one of claims 1 to 19, wherein the NK cells and APCs are present in a ratio of 1:2. 22. The method according to any one of claims 1 to 21, wherein the culturing and/or expansion of NK cells is carried out in the presence of 2, 3 or 4 cytokines. 23. The method according to claim 22, wherein the cytokines are selected from the group consisting of IL-7, IL-12, IL-21, IL-15 and IL-18. 24. The method according to any one of claims 1 to 22, wherein at least one cytokine is present at a concentration of 100-300 units/ml. 25. The method according to any one of claims 1 to 22, wherein at least one cytokine is present at a concentration of 200 U/ml. 26. The method according to any one of claims 1 to 25, wherein the administration comprises transduction or electroporation. 27. The method according to claim 26, wherein the transduction is retronectin transduction. 28. The method according to claim 27, wherein the transduction efficiency is at least 20%. 29. The method according to any one of claims 1 to 28, wherein the construct for expressing CAR and/or TCR is a lentiviral vector or a retroviral vector. 30. The method according to any one of claims 1 to 29, wherein the population of engineered NK cells complies with GMP requirements. 31. The method according to any one of claims 1 to 30, wherein said method results in at least a 2000-fold increase in cell number. 32. The method according to any one of claims 1 to 31, wherein steps (a) to (c) are carried out in less than 2 weeks. 33. The method according to any one of claims 1 to 32, wherein the NK cells are allogeneic. 34. The method according to any one of claims 1 to 33, wherein the NK cells are autologous. 35. The method according to any one of claims 1 to 34, wherein the CAR and/or TCR have antigen specificity for CD70, BCMA, CD5, CD33, CD47, CD99, CLL1, CD38, U5snRNP200, CD200, BAFF-R, CD19, CD319/CS1, ROR1, CD20, carcinoembryonic antigen, alpha-fetoprotein, CA-125, MUC-1, epithelial tumor antigen, melanoma-associated antigen, mutant p53, mutant ras, HER2/Neu, ERBB2, folate-binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL-11R-alpha, kappa chains, lambda chains, CSPG4, ERBB2, WT-1, EGFRvIII, TRAIL/DR4, VEGFR2, or a combination thereof. 36. The method according to any one of claims 1 to 35, wherein the CAR and/or expression construct further expresses a cytokine. 37. The method according to claim 36, wherein the cytokine is IL-15 or IL-2. 38. The method according to any one of claims 1 to 37, further comprising cryopreserving the population of engineered NK cells. 39. A method of treating a viral infection in an individual, comprising: obtaining NK cells expressing a CAR and/or TCR by the method according to any one of claims 1 to 38, wherein the CAR and/or TCR have antigen specificity for the corresponding viral pathogen, and administering to the individual an effective amount of said obtained NK cells. 40. The method according to paragraph 39, wherein the method does not involve HLA typing and comparison in the individual and the donor. 41. The method of claim 39 or 40, wherein the lack of HLA matching does not result in graft-versus-host disease or toxicity. 42. The method according to any one of claims 39-41, wherein the NK cells have KIR ligands that differ between the individual and the donor. 43. The method according to any one of claims 39-42, wherein the NK cells are autologous to the individual. 44. The method according to any one of claims 39-42, wherein the NK cells are allogeneic to the individual.