Methods for transducing immune cells

Abstract

Provided herein is an improved method for transducing immune cells, such as T cells, using a retroviral vector expressing an exogenous gene product, such as a chimeric antigen receptor (CAR). Provided herein is a method for increasing the transduction efficiency, thereby increasing the proportion of immune cells in a population that express the exogenous gene product. Also provided are related cells, cell populations, compositions, and methods of use.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 142,730, filed January 28, 2021, and U.S. Provisional Patent Application No. 63 / 302,225, filed January 24, 2022, the contents of which are incorporated by reference in their entireties herein.

[0002] The present disclosure relates to methods of transducing immune cells, such as T cells, with retroviral vectors that express a gene product, such as a chimeric antigen receptor (CAR) gene product. [Background technology]

[0003] In adoptive cell therapy, autologous and allogeneic immune cells can be genetically modified to express synthetic proteins that enable the cells to perform new therapeutic functions. Immune cells can be engineered to express chimeric antigen receptors ("CARs"), which are fusion proteins composed of an antigen recognition portion and a T cell activation domain. Engineered immune cells containing CARs, e.g., CAR-T cells ("CAR-T"), have antigen specificity and retain or enhance the ability to recognize and kill target cells, such as cancer cells. Immune cells can be engineered by transduction, where nucleic acid encoding the CAR is introduced into the immune cells via a viral vector. Previous methods of transducing immune cells for CAR T immunotherapy, especially methods of transducing allogeneic immune cells on a manufacturing scale, can be inefficient, produce inconsistent results, and generally use reagents that increase processing costs and complexity.

[0004] Thus, there remains a need for methods of generating CAR-T cells that have robust and consistent immune cell transduction efficiency, and that are easier and less expensive than current methods. Summary of the Invention

[0005] Described herein are genetically modified populations of immune cells with a high percentage of cells expressing exogenous gene products, improved methods for transducing immune cells with viral vectors that provide cell populations with a high percentage of exogenous gene product positive cells, and therapeutic methods using cell populations prepared using the disclosed methods.Furthermore, the described methods provide genetically modified cell populations with a more consistent percentage of cells expressing exogenous gene products from run-to-run production, and / or cheaper and simpler transduction methods.For example, described herein are methods particularly suitable for T cell retroviral vector transduction that can be used to manufacture cells useful for allogeneic cell therapy (e.g., allogeneic CAR-T cell therapy) using chimeric antigen receptors.

[0006] In one aspect, a method is provided for transducing a cell population with a retroviral vector, wherein the vector comprises a nucleic acid exogenous to the cell, comprising: a) selecting a cell population, wherein the selected cell population comprises T lymphocytes, helper T cells, tumor cells, memory T cells, cytotoxic T cells, natural killer T cells, peripheral blood lymphocytes, peripheral blood mononuclear cells, dendritic cells, or natural killer cells, or a mixture thereof; and b) culturing the selected cell population with the retroviral vector in a cell culture medium having a starting pH within a starting pH range of 7.0 to 7.9, maintaining the starting pH within the starting pH range for at least the first hour of the transduction culture step, to result in a transduced cell population comprising cells expressing a gene product encoded by the exogenous nucleic acid.

[0007] In one embodiment, the starting pH of the transduction method is maintained in a starting pH range of 7.0 to 7.9 for at least 1, 2, 4, 6, 8, 10, 12, 16, 18, 20, 22, or 24 hours. In some embodiments, the starting pH is maintained in a starting pH range for at least about 1, about 2, about 4, about 6, about 8, about 10, about 12, about 14, about 16, about 18, about 20, about 22, about 24, about 36, about 48, about 72, or no more than about 96 hours to about 168 hours.

[0008] In some embodiments, the initial pH is maintained in the initial pH range throughout the end of the transduction culture step. In some embodiments, the transduction culture step is carried out for at least 1, 2, 4, 6, 8, 12, 14, 16, 18, 20, 22, 24, 36, 48, 72, or 96 hours. In some embodiments, the pH is passively controlled. In some embodiments, the pH is actively controlled in the bioreactor.

[0009] In some embodiments, the transduction methods of the present disclosure include culturing a selected cell population at an MOI of about .25, about .5, about 1, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, or about 50 to about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 125, about 150, about 175, about 200, about 225, or about 250. In some embodiments, the MOI is the ratio of functional viral particles to the total number of target cells in the transduction procedure. In some embodiments, the titer of functional viral particles added to the transduction procedure is determined by qPCR.

[0010] In some embodiments of the present disclosure, the exogenous nucleic acid encodes a chimeric antigen receptor (CAR). In some embodiments, the exogenous nucleic acid encodes a monoclonal antibody, a suicide polypeptide, an inducible "on" or "facilitator" switch, or a regulatory switch, such as an epitope specific for a dimerization domain.

[0011] In some embodiments, the selected cell population is cultured in a vessel, the vessel being a cell culture plate, a cell culture deep well plate, a cell stack, a controlled bioreactor, a shake flask, or a gas permeable bag. In some embodiments, the transduction culturing step comprises culturing the selected cell population and the retroviral vector in a volume of about .75 liters to about 250 liters of cell culture medium. In some embodiments, the transduction culturing step comprises culturing the selected cell population and the retroviral vector in a volume of about .5 liters to about 10 liters of cell culture medium.

[0012] In some embodiments, the retroviral vector used in the methods of the present disclosure is a lentiviral vector.

[0013] In some embodiments, at least 35%-95%, 40%-95%, 50%-95%, 60%-95%, 70%-95% of the transduced cell population expresses the exogenous nucleic acid gene product 3-18 days after initiation of the transduction culture step. In some embodiments, at least 35%-95%, 40%-95%, 50%-95%, 60%-95%, 70%-95% of the transduced cell population expresses the exogenous nucleic acid gene product 7-18 days after initiation of the transduction culture step.

[0014] In some embodiments, the cell culture medium optionally includes a co-localization agent during the transduction culture step, such as fibronectin, a fibronectin derivative, polybrene, or RetroNectin® reagent, during the transduction culture step.

[0015] In some embodiments, the cell culture medium does not contain a co-localization agent, such as fibronectin or a fibronectin derivative, e.g., RetroNectin® reagent, during the transduction culture step.

[0016] In some embodiments, the cell culture medium does not contain a co-localization agent during the transduction culture step.

[0017] In some embodiments, polycations such as polybrene, protamine sulfate, or DEAE-dextran are not added to the cell culture medium prior to or during transduction.

[0018] In some embodiments, the selected cell population is an allogeneic cell population. In some embodiments, the selected cell population is an autologous cell population.

[0019] In one aspect, a method of transducing a first and second cell population is provided, wherein the first and second cell populations are transduced by the same method of the disclosure, such that the percentage of transduced cell populations expressing an exogenous nucleic acid gene product in the first and second transduced cell populations does not differ by more than 2%-5%, 5%-10%, 10%-20%, or 20%-30%.

[0020] In some embodiments, the methods of the disclosure provide a transduced cell population, wherein cells of the transduced cell population comprise a reduced vector copy number compared to cells transduced by the same method of the disclosure, wherein the starting pH of the transduction culture step of the method is less than 7.0.

[0021] In an aspect of the disclosure, there is provided a method of transducing a cytotoxic T cell population with a retroviral vector, wherein the vector comprises a nucleic acid that is exogenous to the cytotoxic T cell, the method comprising culturing the cytotoxic T cell population with the retroviral vector in a cell culture medium having a starting pH within a starting pH range of 7.0 to 7.9, and maintaining the starting pH within the starting pH range for at least an initial hour of the transduction culture step to result in a transduced cytotoxic T cell population comprising cells that express a gene product encoded by the exogenous nucleic acid, wherein the cell culture medium does not comprise a co-localization agent.

[0022] In some embodiments of this aspect of the disclosure, the starting pH is maintained in the starting pH range of 7.0 to 7.9 for at least 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 hours. In some embodiments, the starting pH of the cytotoxic T cell culture medium is maintained in the starting pH range for at least about 1, about 2, about 4, about 6, about 8, about 10, about 12, about 14, about 16, about 18, about 20, about 22, about 24, about 36, about 48, about 72, or no more than about 96 hours to about 168 hours.

[0023] In some embodiments of this aspect, the starting pH of the cytotoxic T cell culture medium is maintained in the starting pH range throughout the end of the transduction culture step, hi some embodiments, the transduction culture step is carried out for at least 1, 2, 4, 6, 8, 12, 14, 16, 18, 20, 22, 24, 36, 48, 72, or 96 hours.

[0024] In some embodiments of this aspect of the disclosure, the pH is passively controlled. In some embodiments, the pH is actively controlled.

[0025] In some embodiments, the cytotoxic T cell culture medium does not contain a co-localization agent, such as fibronectin or a fibronectin derivative, e.g., RetroNectin®, during the transduction culture step.

[0026] In some embodiments, polycations such as polybrene, protamine sulfate, or DEAE-dextran are not added to the cytotoxic T cell culture medium prior to or during transduction.

[0027] In some embodiments of this aspect of the disclosure, the cytotoxic T cell population is cultured at an MOI of about .25, about .5, about 1, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, or about 50 to about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 125, about 150, about 175, about 200, about 225, or about 250. In some embodiments, the MOI is the ratio of functional viral particles to the total number of cytotoxic T cells in the transduction procedure.

[0028] In some embodiments of this aspect of the disclosure, the exogenous nucleic acid encodes a chimeric antigen receptor, hi some embodiments, the exogenous nucleic acid encodes a monoclonal antibody, a suicide polypeptide, an inducible "on" or "enhancer" switch, or a regulatory switch, e.g., an epitope specific for a dimerization domain.

[0029] In some embodiments of this aspect of the disclosure, the cytotoxic T cell population is cultured in a vessel, the vessel being a cell culture plate, a cell culture deep well plate, a cell stack, a controlled bioreactor, a shake flask, or a gas permeable bag. In some embodiments, the transduction culture step comprises culturing the cytotoxic T cell population and the retroviral vector in a volume of about .75 liters to about 250 liters of cell culture medium.

[0030] In some embodiments, a retroviral vector for use in the methods of the present disclosure is a lentiviral vector.

[0031] In some embodiments of this aspect of the disclosure, at least 40%-95%, 50%-95%, 60%-95%, 70%-95% of the transduced cytotoxic T cell population express the exogenous nucleic acid gene product 3-18 days after initiation of the transduction culture step. In some embodiments, at least 40%-95%, 50%-95%, 60%-95%, 70%-95% of the transduced cytotoxic T cell population express the exogenous nucleic acid gene product 7-18 days after initiation of the transduction culture step.

[0032] In some embodiments, the transduced cytotoxic T cell population is an allogeneic cytotoxic T cell population. In some embodiments, the transduced cytotoxic T cell population is an autologous cytotoxic T cell population.

[0033] In an aspect of the disclosure, there is provided a method of transducing a first and a second cytotoxic T cell population, wherein the first and second cytotoxic T cell populations are transduced by the same method of the disclosure, such that the percentage of the transduced cytotoxic T cell populations expressing an exogenous nucleic acid gene product in the first and second transduced cytotoxic T cell populations does not differ by more than 2%-5%, 5%-10%, 10%-20%, or 20%-30%.

[0034] In an aspect of the disclosure, there is provided a method of transducing a population of peripheral blood mononuclear cells with a retroviral vector, wherein the vector comprises a nucleic acid that is exogenous to the peripheral blood mononuclear cells, the method comprising culturing the peripheral blood mononuclear cell population with the retroviral vector in a cell culture medium having a starting pH within a starting pH range of 7.0 to 7.9, and maintaining the starting pH within the starting pH range for at least an initial hour of the transduction culture step to result in a transduced peripheral blood mononuclear cell population comprising cells that express a gene product encoded by the exogenous nucleic acid, wherein the cell culture medium does not comprise a co-localization agent.

[0035] In some embodiments of this aspect of the disclosure, the starting pH is maintained in the starting pH range of 7.0 to 7.9 for at least 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 hours. In some embodiments, the starting pH is maintained in the starting pH range for at least about 1, about 2, about 4, about 6, about 8, about 10, about 12, about 14, about 16, about 18, about 20, about 22, about 24, about 36, about 48, about 72, or no more than about 96 hours to about 168 hours.

[0036] In some embodiments of this aspect, the initial pH is maintained in the initial pH range throughout the end of the transduction culture step, hi some embodiments, the transduction culture step is carried out for at least 1, 2, 4, 6, 8, 12, 14, 16, 18, 20, 22, 24, 36, 48, 72, or 96 hours.

[0037] In some embodiments of this aspect of the disclosure, the pH is passively controlled. In some embodiments, the pH is actively controlled.

[0038] In some embodiments, the peripheral blood mononuclear cell culture medium does not contain a co-localization agent, such as fibronectin or a fibronectin derivative, e.g., RetroNectin®, during the transduction culture step.

[0039] In some embodiments, polycations such as polybrene, protamine sulfate, or DEAE-dextran are not added to the peripheral blood mononuclear cell culture medium prior to or during transduction.

[0040] In some embodiments of this aspect of the disclosure, the peripheral blood mononuclear cell population is cultured at an MOI of about .25, about .5, about 1, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, or about 50 to about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 125, about 150, about 175, about 200, about 225, or about 250. In some embodiments, the MOI is the ratio of functional viral particles to the total number of peripheral blood mononuclear cells in the transduction procedure.

[0041] In some embodiments of this aspect of the disclosure, the exogenous nucleic acid encodes a chimeric antigen receptor, hi some embodiments, the exogenous nucleic acid encodes a monoclonal antibody, a suicide polypeptide, an inducible "on" or "enhancer" switch, or a regulatory switch, e.g., an epitope specific for a dimerization domain.

[0042] In some embodiments of this aspect of the disclosure, the peripheral blood mononuclear cell population is cultured in a vessel, and the vessel is a cell culture plate, a cell culture deep well plate, a cell stack, a controlled bioreactor, a shake flask, or a gas permeable bag. In some embodiments, the transduction culture step includes culturing the peripheral blood mononuclear cell population and the retroviral vector in a volume of about .75 liters to about 250 liters of cell culture medium.

[0043] In some embodiments, a retroviral vector for use in the methods of this aspect of the disclosure is a lentiviral vector.

[0044] In some embodiments of this aspect of the disclosure, at least 40%-95%, 50%-95%, 60%-95%, 70%-95% of the transduced peripheral blood mononuclear cell population expresses the exogenous nucleic acid gene product 3-18 days after initiation of the transduction culture step. In some embodiments, at least 40%-95%, 50%-95%, 60%-95%, 70%-95% of the transduced peripheral blood mononuclear cell population expresses the exogenous nucleic acid gene product 7-18 days after initiation of the transduction culture step.

[0045] In some embodiments, the transduced peripheral blood mononuclear cell population is an allogeneic peripheral blood mononuclear cell population. In some embodiments, the transduced peripheral blood mononuclear cell population is an autologous peripheral blood mononuclear cell population.

[0046] In an aspect of the disclosure, there is provided a method of transducing a first and second peripheral blood mononuclear cell population, wherein the first and second peripheral blood mononuclear cell populations are transduced by the same method of the disclosure, such that the percentage of transduced peripheral blood mononuclear cell populations expressing an exogenous nucleic acid gene product in the first and second transduced peripheral blood mononuclear cell populations does not differ by more than 2%-5%, 5%-10%, 10%-20%, or 20%-30%.

[0047] In an aspect of the disclosure, there is provided a method of transducing a population of T cells induced from induced pluripotent stem cells with a retroviral vector, the vector comprising a nucleic acid exogenous to the derived T cells, the method comprising culturing the induced T cell population with the retroviral vector in a cell culture medium having a starting pH within a starting pH range of 7.0 to 7.9, and maintaining the starting pH within the starting pH range for at least the first hour of the transduction culture step to result in a transduced induced T cell population comprising cells expressing a gene product encoded by the exogenous nucleic acid, wherein the cell culture medium does not comprise a co-localization agent.

[0048] In some embodiments of this aspect of the disclosure, the starting pH is maintained in the starting pH range of 7.0 to 7.9 for at least 1, 2, 4, 6, 8, 10, 12, 16, 18, 20, 22, or 24 hours. In some embodiments, the starting pH of the culture medium for the T cell population derived from induced pluripotent stem cells is maintained in the starting pH range for at least about 1, about 2, about 4, about 6, about 8, about 10, about 12, about 14, about 16, about 18, about 20, about 22, about 24, about 36, about 48, about 72, or no more than about 96 to about 168 hours.

[0049] In some embodiments of this aspect, the starting pH of the inducer T cell culture medium is maintained in the starting pH range throughout the end of the transduction culture step, hi some embodiments, the transduction culture step is carried out for at least 1, 2, 4, 6, 8, 12, 14, 16, 18, 20, 22, 24, 36, 48, 72, or 96 hours.

[0050] In some embodiments of this aspect of the disclosure, the pH of the induced T cell culture medium is passively controlled. In some embodiments, the pH is actively controlled.

[0051] In some embodiments of this aspect of the invention, the induced T cell culture medium does not contain a co-localization agent, such as fibronectin, or a fibronectin derivative, e.g., RetroNectin®, during the transduction culture step.

[0052] In some embodiments, polycations such as polybrene, protamine sulfate, or DEAE-dextran are not added to the induced T cell culture medium prior to or during transduction.

[0053] In some embodiments of this aspect of the disclosure, the induced T cell population is cultured at an MOI of about .25, about .5, about 1, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, or about 50 to about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 125, about 150, about 175, about 200, about 225, or about 250. In some embodiments, the MOI is the ratio of functional viral particles to the total number of T cells derived from the induced pluripotent stem cells in the transduction procedure.

[0054] In some embodiments of this aspect of the disclosure, the exogenous nucleic acid encodes a chimeric antigen receptor, hi some embodiments, the exogenous nucleic acid encodes a monoclonal antibody, a suicide polypeptide, an inducible "on" or "enhancer" switch, or a regulatory switch, e.g., an epitope specific for a dimerization domain.

[0055] In some embodiments of this aspect of the disclosure, the induced T cell population is cultured in a vessel, the vessel being a cell culture plate, a cell culture deep well plate, a cell stack, a controlled bioreactor, a shake flask, or a gas permeable bag. In some embodiments, the transduction culture step comprises culturing the induced T cell population and the retroviral vector in a volume of about 0.5 liters to about 10 liters of cell culture medium.

[0056] In some embodiments, a retroviral vector for use in the methods of the present disclosure is a lentiviral vector.

[0057] In some embodiments of this aspect of the disclosure, between about 35% and about 95%, between about 40% and about 95%, between about 50% and about 95%, between about 60% and about 95%, or between about 70% and about 95% of the transduced induced T cell population express the exogenous nucleic acid gene product 3 to 18 days after initiation of the transduction culture process. In some embodiments, between about 35% and about 95%, between about 40% and about 95%, between about 50% and about 95%, between about 60% and about 95%, or between about 70% and about 95% of the transduced induced T cell population express the exogenous nucleic acid gene product 7 to 18 days after initiation of the transduction culture process.

[0058] In some embodiments, the transduced induced T cell population is an allogeneic induced T cell population. In some embodiments, the transduced induced T cell population is an autologous induced T cell population.

[0059] In an aspect of the disclosure, there is provided a method of transducing a first and a second induced T cell population, wherein the first and second induced T cell populations are transduced by the same method of the disclosure, such that the percentage of transduced induced T cell populations expressing an exogenous nucleic acid gene product in the first and second transduced induced T cell populations does not differ by more than 2%-5%, 5%-10%, 10%-20%, or 20%-30%.

[0060] In some embodiments of the present invention, genetically modified cells produced by the method of the present disclosure are provided. In another embodiment of the present invention, a population of genetically modified cells produced by the method of the present disclosure is provided. In another embodiment, a therapeutic composition is provided that includes a cell or cell population produced by the method of the present invention. In another embodiment, a therapeutic method is provided that includes administering a therapeutically effective amount of a cell, cell population, or therapeutic composition produced by the method of the present invention to a subject in need thereof. [Brief description of the drawings]

[0061] [Figure 1]FIG. 1 shows an exemplary protocol for isolating PBMCs, activating, transducing, transfecting, expanding, and harvesting T cells from the isolated PBMCs.

[0062] [Diagram 2] Figures 2A and 2B show that on day 11 of the study, the mean percentage of viable CAR+ T cells was 81.28587 when cells were transduced at pH 7.1 (Figure 2A) or 23.0387 when transduced at pH 6.6 (Figure 2B), as determined by ANOVA analysis.

[0063] Figure 2C shows the main effects and p-values ​​for the two-way interaction of pH, % lentiviral vector (v / v) (shown as LVV), RetroNectin® reagent concentration (μg / mL) (shown as RN), and vessel type on the percentage of viable CAR+ T cells.

[0064] [Diagram 3] FIG. 3 shows the effect of transduction times of 1, 2, 4, 6, and 8 hours (horizontal axis) performed at a pH of 7.2+0.1 on the percentage of CAR+ T cells (vertical axis) on days 7 and 14 of the study. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0065] Provided herein are improved methods for transducing immune cells, particularly T cells, that increase transduction efficiency and / or improve consistency of exogenous nucleic acid expression levels between production runs while reducing processing costs and complexity.

[0066] Exemplary methods provided herein are typically ex vivo methods and include transducing activated T cells with a retroviral vector or retroviral vectors to produce genetically modified T cells. In exemplary embodiments, the transduction step can be performed without a colocalizing agent. In further exemplary embodiments, the transduction step can be performed at a pH of 6.9-7.8, or at a pH of 7.0-7.9. Typically, such methods can include enriching peripheral blood mononuclear cells (PBMCs) to isolate PBMCs that contain T cells that can be used in the activation step. Furthermore, in exemplary embodiments, such methods can include expanding the genetically modified T cells. In further exemplary embodiments, the methods provided herein can further include disrupting an endogenous gene of the T cells. In exemplary embodiments of the methods provided herein, the T cells can be activated, transduced, and typically expanded. Such T cells in exemplary embodiments can be genetically modified to express a CAR. definition

[0067] The terminology used in this disclosure is standard in the art, but definitions of certain terms are provided herein to ensure clarity and clarity to the meaning of the claims. Units, prefixes, and symbols can be expressed in SI acceptable form. As described herein, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the recited range, and fractions thereof, where appropriate (such as tenths and hundredths of integers). The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0068] As used in this disclosure, unless expressly stated otherwise herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout this disclosure.

[0069] Unless otherwise specified, the terms "a" or "an" should be construed as meaning "at least one or more."

[0070] The term "about" as used in connection with numerical values ​​throughout this specification and claims indicates an interval of accuracy familiar and acceptable to those skilled in the art. Typically, such an interval of accuracy is ±.15%.

[0071] The term "activation" or "activated" refers to a state of immune cells, such as T cells, that have been stimulated sufficiently to induce detectable cell proliferation. Activation may also be associated with induced cytokine production and detectable effector function. The term "activated T cells" refers, inter alia, to T cells undergoing cell division. T cell activation may be characterized by an increase in T cell expression of one or more biomarkers, including, but not limited to, CD57, PD1, CD107a, CD25, CD137, CD69, and / or CD71.

[0072] The term "administering" refers to physically introducing an agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Exemplary administration routes for T cells prepared by the methods disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral administration routes, for example, by injection or infusion. As used herein, the phrase "parenteral administration" refers to modes of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, and in vivo electroporation. Administration can also be performed, for example, once, multiple times, and / or over one or more extended periods of time.

[0073] The term "allogeneic" refers to any material derived from one individual that is subsequently introduced into another individual of the same species, for example allogeneic T cell transplantation or therapy.

[0074] The term "antibody" (Ab) includes, but is not limited to, an immunoglobulin that specifically binds to an antigen. Generally, an antibody may comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region may comprise three or four constant domains, CH1, CH2, CH3, and / or CH4. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region may comprise one constant domain, CL. The VH and VL regions may be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). Each VH and VL contains three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The term "antibody" includes, by way of example, both natural and non-natural Abs, monoclonal and polyclonal Abs, bispecific antibodies, minibodies, domain antibodies, chimeric and humanized Abs, human or non-human Abs, fully synthetic Abs (sometimes also called "antibody mimics"), camelid antibodies, antibody fusions (sometimes also called "antibody conjugates"), and single-chain Abs. Non-human Abs can be humanized by recombinant methods to reduce their immunogenicity in humans.

[0075] As used herein, "immunoglobulin" can be derived from any of the commonly known isotypes, including, but not limited to, IgA, secretory IgA, IgG, and IgM. IgG subclasses are also known in the art, and include, but are not limited to, human IgG1, IgG2, IgG3, and IgG4. "Isotype" refers to the Ab class or subclass (e.g., IgM or IgG1) encoded by the heavy chain constant region genes.

[0076] The term "autologous" refers to any material derived from the same individual that is later reintroduced. For example, engineered autologous cell therapy (eACT™) involves the collection of lymphocytes from a donor, e.g., a patient, which are then engineered to express, e.g., a CAR construct, and then administered back to the same donor, e.g., patient.

[0077] As used herein, the term "co-localization agent" refers to a reagent that promotes co-localization of a viral vector or particle, e.g., a retroviral or lentiviral particle, with a target cell, e.g., an immune cell such as a T cell, and can include, for example, fibronectin or fibronectin derivatives, RetroNectin® reagent, etc.

[0078] As used herein, the term "manufacturing scale transfection volume" refers to volumes from 500 mL up to 5 liters.

[0079] The term "multiplicity of infection" (hereinafter "MOI") refers to the ratio of infectious agents, such as viral particles, to infected targets, such as cells, in the medium of a procedure, such as a transduction procedure. In some embodiments, the MOI may be equal to the number of functional viral particles added to the total number of target cells during the transduction procedure. In some embodiments, the number of functional viral particles added to the transduction procedure is determined by ascertaining the titer of functional viral particles. In some embodiments, the titer of functional viral particles is determined by using qPCR to determine the number of nucleic acid viral copies integrated per cell as a stably transduced standard cell line, using techniques known in the art. See, for example, Paugh, BS, et al. Sci Rep 11, 389 (2021), which is incorporated herein by reference in its entirety. In some embodiments, the viral particles are retroviral particles. In some embodiments, the viral particles are lentiviral particles.

[0080] As used herein, pH control refers to, for example, when the cell culture pH is continuously controlled in a bioreactor with pH feedback control or passive, for example, when the cell culture pH is controlled by buffered cell culture medium and % CO in a tissue culture incubator. 2 It is controlled at the start of the culture by adjusting pH to achieve a given pH and may be active if not further controlled.

[0081] The terms "selective" or "specific" and related derivatives are used interchangeably herein. A molecule, such as an antigen-binding domain, is said to be selective or specific if it binds to one target more strongly than it binds to a second target.

[0082] As used herein, the term vector copy number ("VCN") refers to the number of vector copies per cell, e.g., the number of viral vector copies.

[0083] As used interchangeably herein, the terms "viral vector" and "retroviral vector" refer to any form of nucleic acid derived from a retrovirus and used to transfer genetic material to cells via transduction. The terms encompass viral vector nucleic acids, such as DNA and RNA, encapsulated forms of these nucleic acids, and viral particles in which the viral vector nucleic acid is packaged.

[0084] immune cells Prior to in vitro manipulation or genetic modification of immune cells as described herein, the cells can be obtained from a subject. Immune cells can be obtained from the subject of allogeneic or autologous origin (i.e., from a healthy donor or patient).

[0085] Immune cells can be obtained from a number of non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural fluid, spleen tissue, and tumor. In some embodiments, any number of T cell lines available and known to those skilled in the art can be used. In some embodiments, cells can be derived from healthy donors, patients diagnosed with cancer, or patients diagnosed with infectious diseases. In some embodiments, cells can be part of a mixed population of cells that exhibit different phenotypic characteristics.

[0086] In some embodiments, immune cells include T cells.T cells can be obtained from multiple sources, including peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from infection site, ascites, pleural effusion, spleen tissue, and tumor.In certain embodiments, T cells can be obtained from blood drawn from a subject using any number of techniques known to those skilled in the art, such as FICOLL™ separation.

[0087] In some embodiments, the immune cells may be derived from stem cells, such as progenitor cells, bone marrow stem cells, induced pluripotent stem cells, iPSCs, hematopoietic stem cells, and mesenchymal stem cells. iPS cells and other types of stem cells can be cultured immortal cell lines or directly isolated from patients. In some embodiments, the immune cells are T cells induced from induced pluripotent stem cells. Various methods for isolating, expressing, and / or culturing stem cells are known in the art and can be used in the practice of the present disclosure.

[0088] In some embodiments, the immune cells are induced pluripotent stem cells (iPSCs) derived from reprogrammed T cells. In some embodiments, the source material may be induced pluripotent stem cells (iPSCs) derived from T cells or non-T cells. The source material may alternatively be B cells, or any other cells from peripheral blood mononuclear cell isolates, hematopoietic progenitor cells, hematopoietic stem cells, mesenchymal stem cells, adipose stem cells, or any other somatic cell type.

[0089] Blood collection In some embodiments, the T cells to be engineered are obtained from PBMC.In some embodiments, PBMC can be collected or obtained from a subject by any suitable method known in the art.For example, in some embodiments, blood can be collected by venipuncture or any other blood collection method that collects blood and / or PBMC samples.

[0090] In some embodiments, PBMCs can be obtained from the circulating blood of an individual by apheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In certain embodiments, cells collected by apheresis, particularly leukopheresis, can be washed to remove plasma fraction and placed in a suitable buffer or medium for subsequent processing.

[0091] T cell enrichment In certain embodiments, T cells are isolated from PBMCs by lysing red blood cells and depleting monocytes, for example, using centrifugation through a PERCOLL™ gradient. Specific subpopulations of T cells (e.g., CD28+, CD4+, CDS+, CD45RA-, CD45RO+, CDS+, CD62-, CD95-, CD95+, IL2R+, IL2R-, CCR7+, CCR7-, CDL-, CD62L+, and combinations thereof) can be further isolated by positive or negative selection techniques known in the art. In one example, the subpopulation of T cells is CD45RA+, CD95-, IL-2R-, CCR7+, CD62L+. In one example, the subpopulation of T cells is CD45RA+, CD95+, IL-2R+, CCR7+, CD62L+. In one example, the subpopulation of T cells is CD45RO+, CD95+, IL-2R+, CCR7+, CD62L+. In one example, the subpopulation of T cells is CD45RO+, CD95+, IL-2R+, CCR7-, CD62L-. In one example, the subpopulation of T cells is CD45RA+, CD95+, IL-2R+, CCR7-, CD62L-. For example, enrichment of a T cell population by negative selection can be achieved using a combination of antibodies directed against surface markers unique to the negatively selected cells. One method for use herein is cell sorting and / or selection via negative magnetic immunoadherence or flow cytometry using a cocktail of monoclonal antibodies directed against cell surface markers present on the negatively selected cells. For example, to enrich for CD4+ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA DR, and CD8. Flow cytometry and cell sorting can also be used to isolate cell populations of interest for use in the methods and embodiments of the present disclosure.

[0092] PBMCs can be directly used for genetic modification, for example, introduction of CAR, using the methods described herein. It will be understood that PBMCs can further contain other cytotoxic lymphocytes, such as NK cells or NKT cells. The expression vector carrying the coding sequence of the chimeric antigen receptor disclosed herein can be introduced into a population of human donor T cells, NK cells, or NKT cells. Successfully transduced T cells carrying the expression vector can be sorted using flow cytometry to isolate CD3 positive T cells, and then further expanded to increase the number of these CAR-expressing T cells in addition to cell activation using anti-CD3 antibody and IL-2 or other methods known in the art as described elsewhere herein.

[0093] In certain embodiments, after isolating PBMCs, T lymphocytes can be further isolated and both cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subpopulations, either before or after genetic modification and / or expansion.

[0094] In some embodiments, CD8+ cells are further sorted into naive, stem cell memory, central memory, and effector cells by identifying the characteristic cell surface antigens associated with each of these types of CD8+ cells. In some embodiments, the expression of phenotypic markers of central memory T cells includes CD45RO, CD62L, CCR7, CD28, CD3, and CD127, and is negative for granzyme B. In some embodiments, stem cell memory T cells are CD45RO-, CD62L+, CD8+ T cells. In some embodiments, central memory T cells are CD45RO+, CD62L+, CD8+ T cells. In some embodiments, effector T cells are negative for CD62L, CCR7, CD28, and CD127, and positive for granzyme B and perforin.

[0095] In certain embodiments, CD4+ T cells are further sorted into subpopulations. For example, CD4+ T helper cells can be sorted into naive, central memory, and effector cells by identifying cell populations with distinct cell surface antigens.

[0096] Cell Activation and Proliferation The immune cells of the present disclosure can be activated and expanded either before or after genetic modification of the immune cells. Figure 1 shows an exemplary protocol that can be used to activate, transduce, transfect, and expand the immune cells of the present disclosure. In one embodiment, the in vitro transduction, transfection, culture, and / or expansion of T cells is performed in the absence of non-human animal-derived products, such as fetal calf serum and fetal bovine serum.

[0097] Generally, the engineered immune cells of the present disclosure can be expanded, for example, by contacting them with an agent that stimulates the CD3TCR complex and costimulatory molecules on the surface of the T cells to generate an activation signal for the T cells. For example, chemicals such as calcium ionophore A23187, phorbol 12-myristate 13-acetate (PMA), or mitogenic lectins such as phytohemagglutinin (PHA) can be used to generate an activation signal for the T cells.

[0098] In some embodiments, a population of T cells can be stimulated in vitro, for example, by contacting with an anti-CD3 antibody, such as an OKT3 antibody, or an antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contacting with a protein kinase C activator (e.g., bryostatin) in combination with a calcium ion phore. Co-stimulation of accessory molecules on the surface of T cells uses a ligand that binds to the accessory molecule. For example, a population of T cells can be contacted with an anti-CD3 antibody (e.g., an OKT3 antibody) and an anti-CD28 antibody under conditions suitable for stimulating proliferation of the T cells. The anti-CD3 antibody and the anti-CD28 antibody can be disposed on beads, such as plastic or magnetic beads, or on a plate, or other substrate. Suitable conditions for T cell culture include an appropriate medium (e.g., Minimum Essential Medium or RPMI Medium 1640, or X-vivo 15, (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-y, IL-4, IL-7, GM-CSF, IL-10, IL-2, IL-15, TGFb, and TNF, or any other additives for cell proliferation known to those of skill in the art. Other additives for cell proliferation include, but are not limited to, detergents, plasmanate, and reducing agents such as N-acetyl-cysteine ​​and 2-mercaptoethanol. The medium can include RPMI 1640, AlM-V, DMEM, MEM, a-MEM, F-12, X-Vivo15, and X-Vivo20, Optimizer with additional amino acids, sodium pyruvate, and vitamins, and is serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones and / or cytokines in sufficient amounts for proliferation of T cells (e.g., IL-7 and / or IL-15). Antibiotics, such as penicillin and streptomycin, are included only in experimental cultures and not in cultures of cells injected into subjects. Target cells are maintained under conditions necessary to support proliferation, such as, for example, an appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air+5% CO2).T cells exposed to different stimulation times can exhibit different characteristics.In some embodiments, the cells of the present disclosure can be expanded by co-culturing with tissue or cells.Cells can also be expanded in vivo, for example, in the blood of a subject after administering cells to the subject.

[0099] Suitable conditions for T cell culture include an appropriate medium (e.g., Minimum Essential Medium or RPMI Medium 1640, or X-vivo 15, (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-y, IL-4, IL-7, GM-CSF, IL-10, IL-2, IL-15, TGFb, and TNF, or any other additives for cell proliferation known to those of skill in the art. Other additives for cell proliferation include, but are not limited to, detergents, plasmanate, and reducing agents such as N-acetyl-cysteine ​​and 2-mercaptoethanol. The medium can include RPMI 1640, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo15, and X-Vivo20, Optimizer with additional amino acids, sodium pyruvate, and vitamins, and is serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones and / or cytokines in sufficient amounts for the proliferation of T cells (e.g., IL-7 and / or IL-15). Antibiotics, such as penicillin and streptomycin, are included only in the experimental cultures and not in the cultures of cells injected into the subject. The target cells are maintained under conditions necessary to support proliferation, such as, for example, an appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air+5% CO2). T cells exposed to various stimulation times can exhibit different characteristics. In some embodiments, the cells of the present disclosure can be expanded by co-culturing with tissues or cells. The cells can also be expanded in vivo, for example, in the blood of a subject after administration of the cells to the subject.

[0100] Methods for activating and expanding T cells are known in the art and are described, for example, in U.S. Patent No. 6,905,874, U.S. Patent No. 6,867,041, U.S. Patent No. 6,797,514, and WO2012 / 079000, the contents of which are incorporated herein by reference in their entirety. In general, such methods involve contacting PBMCs or isolated T cells with stimulatory and costimulatory molecules, such as anti-CD3 and anti-CD28 antibodies, typically attached to plastic or magnetic beads or other surfaces, in a culture medium with appropriate cytokines, such as IL-2. The anti-CD3 and anti-CD28 antibodies bound to the beads serve as "surrogate" antigen-presenting cells (APCs). One example is the Dynabeads® system, which is a CD3 / CD28 activator / stimulatory factor system for the physiological activation of human T cells. In other embodiments, T cells can be activated and stimulated to grow with feeder cells and appropriate antibodies and cytokines using methods described in U.S. Patent No. 6,040,177, U.S. Patent No. 5,827,642, and WO2012129514 (the contents of which are incorporated herein by reference in their entirety). In another embodiment, cells are activated and grown with an anti-CD3 / 28 nanometer-scale matrix containing antibodies and / or fragments thereof that bind to CD3 and CD28, provided as TransAct™ T cell reagent by Miltenyi Biotec, Inc. (Auburn, Calif.). (See, for example, Catalog No. 200-076-202 MACS GMP T Cell Transact-CRR, Catalog No. 130-019-011 MACS GMP T Cell Transact for Research use).

[0101] Transduction Provided herein is a method for genetically modifying immune cells, including PBMC and T cells produced by the method of the present disclosure.In some embodiments of the methods and compositions disclosed herein, T cells are contacted with a replication-incompetent retroviral vector ex vivo, and the T cells are genetically modified to express exogenous gene product.In some embodiments, the exogenous gene product is CAR.

[0102] In some embodiments, the exogenous gene product is an epitope specific for (i.e., specifically recognized by) an inducible "on" or "promoter" switch, such as a monoclonal antibody, a suicide polypeptide, an inducible caspase-9 (US Patent Application Publication No. 2011 / 0286980), or a thymidine kinase, or an "off" switch. Exemplary mAb-specific epitopes are disclosed in International Patent Publication No. WO2016 / 120216, which is incorporated herein in its entirety. In some embodiments, the exogenous gene product is an R epitope, such as RQR8. See, for example, WO2013153391A, which is incorporated herein by reference in its entirety. Rituximab, when expressed on the surface of a CAR immune cell, can bind to the R epitope and lyse the CAR immune cell. In some embodiments, the exogenous gene product is a control switch, such as a dimerization domain.

[0103] In some embodiments, transduction can be performed in the same vessel in which the activation step is performed, without removing any of the medium. For example, blood cells, such as PBMCs, enriched and isolated from a collected blood sample can be activated in a gas-permeable bag and then contacted with retroviral particles in the same gas-permeable bag. In an exemplary embodiment, the blood cells are separated, isolated, and / or purified away from granulocytes, including neutrophils, before contacting with the retroviral vector. In a further exemplary embodiment, a retroviral vector, which can be a replication-incompetent recombinant lentiviral particle, can be introduced into the same gas-permeable bag containing the activated PBMCs to form a transduction reaction mixture. In some embodiments, the retroviral vector is added to the transduction reaction mixture during the activation step. In some embodiments, the retroviral vector is added to the transduction reaction mixture after the activation step. In some embodiments, the activation step, whether before or simultaneously with the transduction step, is performed for no more than 12, 24, 36, 48, 60, 72, 84, 96, 108, 120, 132, 144, or 156 hours. Figure 1 shows possible transduction time points in an exemplary CAR T cell production protocol. A medium is typically present during transduction, such as that known in the art for ex vivo T cell culture, and includes a base medium and supplements, including, for example, cytokines, as disclosed in further detail herein. See, for example, the activation and proliferation discussion above.

[0104] In some embodiments, the transduction reaction, which begins when the retroviral vector is added to the T cells, may be incubated at 23-39° C., and in some exemplary embodiments, at 37° C. In some embodiments, the transduction reaction may be carried out at 37-39° C. In some embodiments, the transduction reaction may be carried out at 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0% CO 2The transduction reaction may be incubated for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 25, 24, 36, 48 to up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 72, or 96 hours. In exemplary embodiments, the transduction reaction may be incubated for 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 4-12, 4-14, 4-16, 4-18, 4-20, 4-24, 4-26, 4-28, 4-30, 4-32, 4-36, 4-38, 4-40, 4-42, 4-44, 4-46, 4-48, or 4-72 hours at a pH in the starting pH range. In some embodiments, the transduction reaction pH is passively controlled, for example, by adjusting media buffers (e.g., sodium bicarbonate and / or HEPES) and incubator pCO2 to allow for a desired pH (e.g., pH above 7.0) at the start of the culture and to achieve a target pH, such as 7.0 or higher. In some embodiments, the transduction reaction pH is passively controlled, for example, using a bioreactor with online pH measurement and CO2 monitoring. 2 It is actively controlled using a pH feedback control loop that maintains the pH by continuously (actively) adjusting the culture pH via gas addition.

[0105] In some embodiments, T cells may be transduced with different ratios of retroviral or lentiviral particles to cells, referred to as multiplicity of infection (MOI). In some embodiments, T cells are transduced using an MOI (plaque forming units / cell) of 0.25, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 50, 100, 150, 200, 250, 300, 350, 400, 450-500. In some embodiments, T cells are transduced at an MOI of 0.25, 0.50, 1.0, 5, 10, 15, 20, 25, or between 30-50, 75, 100, 125, 150, 175, or 200. In some embodiments, T cells are transduced at an MOI of about 1-10, 15, 20, 25, 30, 35, 40, 45, or 50. In some embodiments, T cells are transduced at an MOI of 1-20.

[0106] In some embodiments of the methods and compositions disclosed herein, 25%-90% of the T cells express the exogenous gene product, and in some embodiments, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85%-90% of the transduced T cells express the exogenous gene product.

[0107] In some embodiments, the percentage of transduced T cells expressing the exogenous gene product can be at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90%, more specifically at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80%, more specifically at least 60%, 65%, 70%, 75%, or 80%. In some embodiments, the indicated levels of expression of the exogenous gene product are achieved on day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, particularly 3-10 days after the retroviral vector first contacts the cells during the transduction reaction.

[0108] In some embodiments, the transduction reaction occurs in a volume of 0.25-250, 0.25-7.5, 0.375-7.5, 0.5-5, or 0.7-4.0 liters of transduction culture medium. In some embodiments, the transduction reaction occurs in a volume of at least 0.25, 0.50, 0.75, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 50, 100, 150, 200, or 250 liters of transduction culture medium. In some embodiments, the transduction reaction occurs in a volume of about 0.25, about 0.50, about 0.60, or about 0.75 to about 0.8, about 1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 7.5, about 8.0, or about 10.0 liters of transduction culture medium.

[0109] In some embodiments, the cell is transduced by a viral vector that comprises a nucleic acid that is exogenous to the cell. In some embodiments, the exogenous nucleic acid encodes a CAR. In some embodiments, the viral vector is a retrovirus, lentivirus, or AAV vector.

[0110] The cells transduced to express the CAR can be from allogeneic or autologous sources. In one embodiment, the in vitro transduction, culture, and / or expansion of T cells is performed in the absence of non-human animal-derived products, such as fetal calf serum and fetal bovine serum.

[0111] In some embodiments, the transduction response cell population is incubated with a retroviral vector encoding a CAR at an MOI of at least 5, 10, 20, 30, 50, 100, 150, or 200, at a pH within the starting pH range and at or below 7.8, or at or below 7.9, for 1 hour to 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 24, or 48 hours, and wherein at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% of the T cells express the CAR by at least 3, 4, 5, 6, 7, 8, 9, or 10 days after the retroviral vector first contacts the cells during the transduction reaction.

[0112] Gene disruption The process for manufacturing allogeneic CAR T therapy, or AlloCARs™, involves harvesting healthy, selected, screened, and tested T cells from healthy donors. The allogeneic T cells are gene-edited to reduce the risk of graft-versus-host disease (GvHD) and prevent allogeneic rejection. In some embodiments, selected T cell receptor genes (e.g., TCRa or TCRb) are knocked out to avoid GvHD. The CD52 gene can also be knocked out to make the CART product resistant to anti-CD52 antibody treatment. Thus, anti-CD52 antibody treatment can be used to lymphodeplete the host immune system, allowing the CART cells to remain engrafted and achieve full therapeutic effect. In one example, the anti-CD52 antibody can include alemtuzumab (CHEMBL 1201587, ChemIDplus:216503-57-0; DB00087; see also US5,846,534, both of which are incorporated herein in their entirety for all purposes). T cells are then engineered to express a CAR that recognizes a specific cell surface protein expressed on hematological or solid tumors. The engineered T cells then undergo a purification process and are finally cryopreserved in vials.

[0113] The process for manufacturing autologous chimeric antigen receptor (CAR) T cell therapy involves collecting a patient's own cells (e.g., white blood cells, including T cells) and genetically engineering the T cells to express a CAR that recognizes a target antigen expressed on the cell surface, such as a cancer cell antigen. The engineered cells are then cryopreserved and subsequently administered to the patient from whom the cells were removed for engineering.

[0114] In some embodiments, the isolated immune cells are genetically modified to reduce or eliminate expression of endogenous TCRa and / or CD52. In some embodiments, the cells are genetically modified using gene editing techniques (e.g., CRISPR / Cas9, CRISPR / CAS12, zinc finger nucleases (ZFN), TALEN®, MegaTAL, meganucleases) to reduce or eliminate expression of endogenous proteins (e.g., TCRa and / or CD52). In some embodiments, the method includes disrupting or inactivating one or more genes by introducing an endonuclease into the cell to allow inactivation of the target gene by DNA cleavage. In some embodiments, the endonuclease may be, for example, a zinc finger nuclease (ZFN), a megaTAL nuclease, a meganuclease, a transcription activator-like effector nuclease (TALE nuclease / TALEN), or a CRISPR (e.g., Cas9, Cas12, or CAS14) endonuclease.

[0115] In some embodiments, the immune cells are transfected with a nucleic acid vector using electroporation, sonoporation, biolistics (e.g., Gene Gun), lipid transfection, polymer transfection, nanoparticles, or polyplexes. In some embodiments, the isolated immune cells are genetically modified to reduce or eliminate the expression of endogenous TCRa and / or CD52.

[0116] Growth and depletion In exemplary embodiments of the methods disclosed herein, the transduced T cells can be expanded prior to harvesting, as generally described above in the description of activation and expansion. In some embodiments, the transduced cells are further transfected to knock out the target endogenous gene. In some embodiments, the transduced cells are depleted of undesired cell types, such as, for example, cells expressing TCRαβ. As shown in FIG. 1, in some embodiments, the transduced cells can be expanded prior to or after depletion.

[0117] Flow cytometry can be used to deplete specific cell types, such as T cell receptor positive cells, in a cell population. In general, flow cytometry is a method for quantifying cellular components or structural features, primarily by optical means. By quantifying structural features, different cell types can be distinguished, so that flow cytometry and cell sorting can be used to count and sort cells of different phenotypes in a mixture.

[0118] Flow cytometry analysis involves two major steps: 1) labeling a selected cell type with one or more labeling markers, and 2) determining the number of labeled cells relative to the total number of cells in a population. In some embodiments, the method of labeling a cell type involves binding a labeled antibody to a marker expressed by a particular cell type, e.g., a T cell receptor. The antibody can be directly labeled with a fluorescent compound or indirectly labeled, e.g., with a fluorescently labeled second antibody that recognizes the first antibody.

[0119] In some embodiments, the method used to sort CAR-expressing T cells is magnetic activated cell sorting (MACS). Magnetic activated cell sorting (MACS) is a method that uses superparamagnetic nanoparticles and columns to separate different cell populations according to their surface antigens (CD molecules). Using MACS, pure cell populations can be obtained.

[0120] Cells in a single cell suspension can be magnetically labeled with microbeads. The sample is applied to a column made of ferromagnetic spheres, covered with a cell-friendly coating, allowing for rapid and gentle separation of cells. Unlabeled cells pass through, while magnetically labeled cells are retained within the column. The flow-through can be collected as the unlabeled cell fraction. After a washing step, the column is removed from the separator and the magnetically labeled cells are eluted from the column.

[0121] Detailed protocols for purification of specific cell populations, such as T cells, can be found in Basu S et al. (2010) (Basu S, Campbell HM, Dittel BN, Ray A. Purification of specific cell population by fluorescence activated cell sorting (FACS). J Vis Exp. (41): 1546), which is incorporated by reference in its entirety.

[0122] Preparation and cryopreservation In some embodiments, the engineered immune cells are formulated by first harvesting the cells from their culture medium, then washing and concentrating the cells in a medium and container system (a "pharmaceutical acceptable" carrier) suitable for administration in a therapeutically effective amount. A suitable infusion medium is any isotonic medium formulation, typically saline, Normosol™ R (Abbott) or Plasma-Lyte™ A (Baxter), although 5% dextrose or Ringer's lactate in water can also be utilized. The infusion medium can be supplemented with human serum albumin.

[0123] In another embodiment, the engineered immune cells, e.g., T cells expressing a CAR, are harvested, washed, and concentrated, then cryopreserved at a predetermined cell concentration in an appropriate cryopreservation medium, e.g., CryoStor® CS10, CryoStor® CS2, or CryoStor® CS5 (BioLife Solutions). Standard procedures are used for cryopreservation of engineered immune cells, e.g., T cells expressing a CAR, for storage and / or preparation for use in human subjects. If necessary, the cryopreserved engineered immune cells can be thawed, grown, and expanded to produce more such cells.

[0124] Chimeric Antigen Receptor As used herein, a chimeric antigen receptor (CAR) is a protein that specifically recognizes a target antigen (e.g., a target antigen on a cancer cell). Upon binding to a target antigen, the CAR can activate immune cells to attack and destroy cells (e.g., cancer cells) that bear that antigen. CARs can also incorporate costimulatory or signaling domains to increase their efficacy. See, e.g., Finney et al., Journal of Immunology, 1998, 161:2791-2797, Song et al., Blood 119:696-706 (2012); Kalas et al., Sci. Transl. Med. 3:95 (2011); Porter et al., N. Engl. J. Med. 365:725-33 (2011), and Gross et al., Annu. Rev. Pharmacol. Toxicol. 56:59-83 (2016); U.S. Patent Nos. 7,741,465, and 6,319,494.

[0125] The chimeric antigen receptors described herein comprise an extracellular domain, a transmembrane domain, and an intracellular domain, and the extracellular domain comprises an antigen-binding domain. In some embodiments, the antigen-specific CAR comprises the following elements from 5' to 3': a signal sequence, an antigen-binding domain, a hinge and transmembrane region, and one or more contiguous signaling domains.

[0126] In some embodiments, the CAR further comprises a safety switch and / or a monoclonal antibody specific epitope. See, e.g., WO2016 / 120216.

[0127] Antigen-binding domain As mentioned above, the CAR described herein comprises an antigen-binding domain. As used herein, "antigen-binding domain" refers to any polypeptide that binds to a specific target antigen. In certain embodiments, the polypeptide structure of the antigen-binding domain is based on an antibody. The antigen-binding domain includes, but is not limited to, an antibody binding region that is an immunologically functional fragment. The term "immunologically functional fragment" (or "fragment") of an antigen-binding domain is a species of antigen-binding domain that lacks at least some of the amino acids present in the full-length chain, but contains a portion of an antibody (regardless of how the portion is obtained or synthesized) that can specifically bind to a target antigen. Such fragments are biologically active in that they can bind to a target antigen and compete with other antigen-binding domains, including intact antibodies, for binding to a given epitope. Immunologically functional fragments include, but are not limited to, scFv fragments, Fab fragments (Fab', F(ab')2, etc.), one or more complementarity determining regions ("CDRs"), diabodies (a heavy chain variable domain and a light chain variable domain on the same polypeptide linked via a short peptide linker that is too short to allow pairing between the two domains on the same chain), domain antibodies, bivalent antigen-binding domains (comprising two antigen-binding sites), multispecific antigen-binding domains, and single-chain antibodies. These fragments may be derived from any mammal, including, but not limited to, human, mouse, rat, camelid, or rabbit.

[0128] In some embodiments, an antigen-binding domain comprises one or more complementary binding regions (CDRs) present in a full-length light or heavy chain of an antibody, and in some embodiments comprises a single heavy and / or light chain or a portion thereof. These fragments may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of the antigen-binding domain comprising an intact antibody.

[0129] In some embodiments, the antigen-binding domain is an antibody of which it is a fragment and which comprises one or more of its complementarity determining regions (CDRs). In some embodiments, the antigen-binding domain is a single chain variable fragment (scFv) comprising light chain CDRs CDR1, CDR2 and CDR3, and heavy chain CDRs CDR1, CDR2 and CDR3.

[0130] The assignment of amino acids to each of the framework, CDR, and variable domains is typically based on the Kabat numbering (see, e.g., Kabat et al. in Sequences of Proteins of Immunological Interest, 5th Ed., NIH Publication 91-3242, Bethesda Md. 1991), Chothia numbering (see, e.g., Chothia & Lesk, (1987), J Mol Biol 196:901-917; Al-Lazikani et al., (1997) J Mol Biol 273:927-948; Chothia et al., (1992) J Mol Biol 227:799-817; Tramontano et al., (1990) J Mol Biol 215(1):175-82; and U.S. Pat. No. 7,709,226), contact numbering, or the AbM scheme (Antibody Modeling The numbering scheme follows that of the Oxford Molecular

[0131] Variants of the antigen-binding domain (e.g., variants of the CDR, VH and / or VL) are also within the scope of the present disclosure, such as variable light chains and / or variable heavy chains, each of which has at least 70-80%, 80-85%, 85-90%, 90-95%, 95-97%, 97-99%, or more than 99% identity with the amino acid sequence of the antigen-binding domain sequence. In some examples, such molecules include at least one heavy chain and one light chain, while in other examples, the variant forms include two variable light chains and two variable heavy chains (or subportions thereof). Those of skill in the art can determine suitable variants of the antigen-binding domains described herein using well-known techniques. In certain embodiments, those of skill in the art can identify suitable regions of the molecule that can be altered without destroying activity by targeting regions not believed to be important for activity.

[0132] In some embodiments, the antigen-binding domain is an scFv. In some embodiments, the antigen-selective CAR comprises a leader or signal peptide. As will be appreciated by those skilled in the art, the antigen-binding domain may comprise non-protein components.

[0133] Antigen binding domains suitable for use in CAR in the methods and compositions of the present disclosure may have various antigen binding specificities. In some embodiments, the antigen binding domain is specific to an epitope present on an antigen expressed (synthesized) by a target cell. In one example, the target cell is a cancer cell-associated antigen. The cancer cell-associated antigen may be, for example, an antigen associated with breast cancer cells, B cell lymphoma, Hodgkin's lymphoma cells, ovarian cancer cells, prostate cancer cells, mesothelioma, lung cancer cells (e.g., small cell lung cancer cells), non-Hodgkin's B cell lymphoma (B-NHL) cells, ovarian cancer cells, prostate cancer cells, mesothelioma cells, lung cancer cells (e.g., small cell lung cancer cells), melanoma cells, chronic lymphocytic leukemia cells, acute lymphocytic leukemia cells, neuroblastoma cells, gliomas, glioblastomas, medulloblastomas, colorectal cancer cells, etc. The cancer cell-associated antigen may also be expressed by non-cancer cells.

[0134] Non-limiting examples of antigens to which the chimeric binding antigens can bind include, for example, CD19, CD20, CD38, CD30, ERBB2, CA125, MUC-1, prostate specific membrane antigen (PSMA), CD44 surface adhesion molecule, mesothelin, carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor 2 (VEGFR2), high molecular weight melanoma associated antigen (HMW-MAA), MAGE-A1, IL-13R-a2, GD2, Ax1, Ror2, BCMA, Claudin and its isotypes.

[0135] Hinge Domain The extracellular domain of the CAR of the present disclosure may include a "hinge" domain (or hinge region). The term includes any polypeptide that functions to link the transmembrane domain in the CAR to the extracellular antigen binding domain in the CAR. In particular, the hinge domain can be used to provide more flexibility and accessibility to the extracellular antigen binding domain.

[0136] The hinge domain can comprise up to 300 amino acids, and in some embodiments, 10-100 amino acids, or in some embodiments, 25-50 amino acids. The hinge domain can be derived from all or a portion of a naturally occurring molecule, for example, from all or a portion of CD8, CD4, CD28, 4-1BB, or the extracellular region of IgG (particularly the hinge region of IgG; it is understood that the hinge region can comprise some or all of the members of the immunoglobulin family, such as IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, etc., or fragments thereof), or from all or a portion of an antibody heavy chain constant region.

[0137] Alternatively, the hinge domain may be a synthetic sequence corresponding to a sequence of natural origin or may be a completely synthetic sequence. In some embodiments, the hinge domain is a portion of the human CD8a chain (e.g., NP_001139345.1). In another particular embodiment, the aforementioned hinge domain and transmembrane domain comprise a portion of the human CD8a chain. In some embodiments, the hinge domain of the CAR described herein comprises a subsequence of a CD8a, CD28, IgGl, IgG4, PD-1, or FcyRIIIa molecule, in particular the hinge region of any of the CD8a, CD28, IgGl, IgG4, PD-1, or FcyRIIIa molecules. In some embodiments, the hinge domain comprises a human CD8a hinge, a human IgGl hinge, a human IgG4 hinge, a human PD-1 hinge, or a human FcyRIIIa hinge. In some embodiments, the CAR disclosed herein comprises a scFv, a CD8a human hinge, and a transmembrane domain.

[0138] Transmembrane domain The CAR of the present disclosure is designed with a transmembrane domain fused to the extracellular domain of the CAR. This can be fused to the intracellular domain of the CAR as well. In some examples, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domain to the transmembrane domain of the same or different surface membrane protein and minimize interaction with other members of the receptor complex. In some embodiments, a short linker can form a bond between any or part of the extracellular domain, transmembrane domain, and intracellular domain of the CAR. A suitable transmembrane domain of the CAR disclosed herein has (a) the ability to express on its surface an immune cell, such as, but not limited to, lymphocytic cells, e.g., CD4+ cells such as T helper (Th) cells, CD8+ cells such as cytotoxic T (Tc) cells, T regulatory (Treg) cells, or natural killer (NK) cells, and / or (b) the ability to interact with the extracellular antigen binding domain and the intracellular signaling domain to direct the cellular response of the immune cell against the target cell.

[0139] The transmembrane domain may be derived from either natural or synthetic sources. If the source is natural, the domain may be derived from any membrane-bound or transmembrane protein.Transmembrane regions of particular use in the present disclosure include CD28, CD8, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-I (PD-1), inducible T cell costimulator (ICOS), lymphocyte function associated antigen-I (LFA-1, CD1-la / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, lg alpha (CD79a), DAP-10, Fe gamma receptor, MHC class 1 molecules, TNF receptor proteins, immunoglobulin proteins, cytokine receptors, Integrins, signaling lymphocyte activation molecule (SLAM protein), activating NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRFl), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLAl, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDI ld, ITGAE, CD103, ITGAL, CDI la, LFA-1, ITGAM, CDI lb, ITGAX, CDI le, ITGBl, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAMl(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAMl, CRT The present invention may be derived from (may include or correspond to) a ligand that specifically binds to AM, Ly9 (CD229), CD160 (BY55), PSGLl, CDlOO (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMFl, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83, or any combination thereof.

[0140] As non-limiting examples, the transmembrane region may be derived from or part of the T cell receptor, a polypeptide constituting the CD3 complex, the IL-2 receptor, p55 (a chain), p75 (P chain), or y chain, a subunit chain of the Fe receptor, in particular Fey receptor III, or a CD protein. Alternatively, the transmembrane domain may be synthetic and may comprise primarily hydrophobic residues such as leucine and valine. In some embodiments, the transmembrane domain is derived from the human CD8a chain (e.g., NP_001139345.1).

[0141] Intracellular domain The intracellular (cytoplasmic) domain of the CAR of the present disclosure can provide activation of at least one of the normal effector functions of immune cells, including, for example, signal I / activation, and / or signal 2 / costimulation. Effector functions of T cells can refer to, for example, cytolytic activity or helper activity, including secretion of cytokines. In some embodiments, the activating intracellular signaling domain for use in a CAR can be, for example, but not limited to, the cytoplasmic sequences of the T cell receptor and co-receptor that act in concert to initiate signal transduction after antigen receptor engagement, as well as any derivatives or variants of these sequences, as well as any synthetic sequence having the same function.

[0142] Suitable (e.g., activation) intracellular domains include, but are not limited to, CD3 zeta, CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-I (PD-1), inducible T cell costimulator (ICOS), lymphocyte function associated antigen-I (LFA-1, CD1-la / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, lg alpha (CD79a), DAP-10, Fe gamma receptor, MHC class 1 molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, signaling lymphocyte activation molecule (SLAM protein), activating NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRFl), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLAl, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDI ld, ITGAE, CD103, ITGAL, CDI la, LFA-1, ITGAM, CDI lb, ITGAX, CDI le, ITGBl, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAMl(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAMl, CRT It is understood that the present invention includes signaling domains derived from (or corresponding to) a ligand that specifically binds to AM, Ly9 (CD229), CD160 (BY55), PSGLl, CDlOO (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMFl, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83, or any combination thereof.

[0143] In addition to the activation domains described above, the intracellular domains can incorporate costimulatory signaling domains (interchangeably referred to herein as costimulatory molecules) to increase their potency. The costimulatory domains can provide signals in addition to the primary signals provided by the activation molecules described herein.

[0144] Suitable costimulatory domains within the scope of the present disclosure include, for example, CD28, OX40, 4-1BB / CD137, CD2, CD3 (alpha, beta, delta, epsilon, gamma, zeta), CD4, CDS, CD7, CD9, CD16, CD22, CD27, CD30, CD 33, CD37, CD40, CD 45, CD64, CD80, CD86, CD134, CD137, CD154, PD-1, ICOS, lymphocyte function associated antigen-I (LFA-1 (CDI la / CD18), CD247, CD276 (B7-H3), LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), NKG2C, lg alpha (CD79a), DAP-10, Fe gamma receptor, MHC class I molecule, TNFR, integrin, signaling lymphocyte activation molecule, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRFl), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD 8beta, IL-2Rbeta, IL-2Rgamma, IL-7Ralpha, ITGA4, VLAl, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl-ld, ITGAE, CD103, ITGAL, CDl-la, LFA-1, ITGAM, CDl -lb, ITGAX, CDl-lc, ITGBl, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAMl(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAMl, CRT It is understood that the agonist gene may be derived from (or correspond to) AM, Ly9 (CD229), CD160 (BY55), PSGLl, CDlOO (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMFl, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83 ligand, or fragments or combinations thereof.It is understood that additional costimulatory molecules or fragments thereof not listed above are within the scope of the present disclosure.

[0145] In some embodiments, the intracellular / cytoplasmic domain of the CAR may be designed to include the 4-1BB / CD137 domain by itself or may be combined with any other desired intracellular domain useful in the context of a CAR. The complete native amino acid sequence of 4-1BB / CD137 is set forth in NCBI Reference Sequence: NP_001552.2. The complete native 4-1BB / CD137 nucleic acid sequence is set forth in NCBI Reference Sequence: NM 001561.5.

[0146] In some embodiments, the intracellular / cytoplasmic domain of the CAR may be designed to include the CD28 domain by itself or may be combined with any other desired intracellular domain useful in the context of the CAR. The complete native amino acid sequence of CD28 is described in NCBI Reference Sequence: NP_006130.1. The complete native CD28 nucleic acid sequence is described in NCBI Reference Sequence: NM_006139.1.

[0147] In some embodiments, the intracellular / cytoplasmic domain of the CAR may be designed to include the CD3 zeta domain by itself, or may be combined with any other desired intracellular domain useful in the context of the CAR. In some embodiments, the intracellular signaling domain of the CAR may contain the CD31 signaling domain. For example, the intracellular domain of the CAR may include a CD3 zeta chain portion and a portion of a costimulatory signaling molecule. The intracellular signaling sequences in the intracellular signaling portion of the CAR may be linked together randomly or in a specific order.

[0148] Preparation of nucleic acids and expression vectors Provided herein are nucleic acids encoding CARs, and methods of making vectors comprising nucleic acids encoding CARs.

[0149] A variety of known techniques can be used to generate the polynucleotides and vectors of the present disclosure. For example, certain methods for generating the constructs and engineered immune cells of the present disclosure are described in the disclosure of WO2015 / 120096, the contents of which are incorporated herein by reference in their entirety.

[0150] The nucleotide sequence encoding the CAR can be present in an expression vector. When the CAR comprises two separate polypeptides, the nucleotide sequences encoding the two polypeptides can be cloned in the same vector or in separate vectors. The expression vector can include a selectable marker, an origin of replication, and other features that provide for the replication and / or maintenance of the vector. Suitable expression vectors include, for example, plasmids, viral vectors, etc.

[0151] For cloning polynucleotides, expression vectors can be introduced into host cells (isolated host cells) to allow the vector to replicate itself, thereby amplifying the copies of the polynucleotides contained therein. Cloning vectors can contain sequence components, which generally include, but are not limited to, replication origins, promoter sequences, transcription initiation sequences, enhancer sequences, and selectable markers. These elements can be appropriately selected by those skilled in the art. For example, replication origins can be selected to promote the autonomous replication of vectors in host cells.

[0152] In other embodiments, the present disclosure relates to an isolated polynucleotide encoding any one of the antigen binding domains described herein. In some embodiments, the present disclosure relates to an isolated polynucleotide encoding a CAR. Also provided herein are vectors comprising the polynucleotides and methods of making the polynucleotides.

[0153] In certain embodiments, the present disclosure provides an isolated host cell that contains the expression vector provided herein.The host cell that contains the vector can be useful for expressing or cloning the polynucleotide contained in the vector.Suitable host cells can include, but are not limited to, prokaryotic cells, fungal cells, yeast cells, or higher eukaryotic cells such as mammalian cells, more specifically human cells.

[0154] The vector can be introduced into the host cell using any suitable method known in the art, including, but not limited to, DEAE-dextran mediated delivery, calcium phosphate precipitation, cationic lipid mediated delivery, liposome mediated transfection, electroporation, biolistics, receptor mediated gene delivery, polylysine, histone, chitosan, and peptide mediated delivery. Standard methods for viral transfection and transformation of cells for expression of vectors of interest are well known in the art. In further embodiments, a mixture of different expression vectors can be used in genetically modifying a donor population of immune effector cells, with each vector encoding a different CAR disclosed herein. The resulting transduced immune effector cells form a mixed population of engineered cells, with a proportion of the engineered cells expressing multiple different CARs.

[0155] Preparation of retroviral particles In exemplary embodiments disclosed herein, the transduction method may include transducing immune cells, such as T cells, with a replication-incompetent recombinant retroviral particle containing one or more nucleic acids to generate a transduced engineered immune cell, such as an engineered T cell. In some embodiments, the one or more nucleic acids may encode one or more proteins that are subsequently expressed in the transduced T cell, e.g., a chimeric antigen receptor (CAR). The retroviral particles used to transduce T cells and / or NK cells in the methods provided herein can be made according to methods known in the art. As disclosed herein, retroviral particles are a common tool for gene delivery (Miller, Nature (1992) 357:455-460). In some embodiments, the replication-incompetent recombinant retroviral particle may be derived from the alpharetrovirus, betaretrovirus, gammaretrovirus, deltaretrovirus, epsilonretrovirus, lentivirus, or spumavirus genera. There are many retroviruses suitable for use in the methods disclosed herein. A detailed list of retroviruses can be found in Coffin et al. ("Retroviruses" 1997 Cold Spring Harbor Laboratory Press Eds: JM Coffin, SM Hughes, HE Varmus pp 758-763). Details on the genome structure of some retroviruses can be found in the art. As an example, details on HIV can be found in NCBI Genbank (i.e. genome accession number AF033819).

[0156] In exemplary embodiments, the retroviral particle can be derived from a recombinant retrovirus from the lentivirus genus, and can be a replication-incompetent recombinant lentivirus particle. In some embodiments, the recombinant retrovirus can be derived from HIV, SIV, or FIV. In further exemplary embodiments, the recombinant retrovirus can be derived from the human immunodeficiency virus (HIV) of the lentivirus genus.

[0157] In some embodiments, the replication-incompetent recombinant retroviral particles can be propagated in culture in a medium specific for replication-incompetent recombinant retroviral particle production. Any suitable growth medium and / or supplement for propagating replication-incompetent recombinant retroviral particles can be used for the replication-incompetent recombinant retroviral particle inoculum according to the method described herein. According to some aspects, the retroviral particles can then be added to the medium during the transduction steep.

[0158] Replication-incompetent recombinant retroviral particles can be produced using mammalian cell lines according to methods known in the art. Suitable mammalian cells include primary cells and immortalized cell lines. Suitable mammalian cell lines can include human cell lines. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL1O), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, Hut-78, Jurkat, HL-60, NK cell lines (e.g., NKL, NK92, and YTS), and the like. In some examples, the cell is not an immortalized cell line, but instead is a cell (e.g., a primary cell) obtained from an individual or an ex vivo cell. For example, in some embodiments, the cell is an immune cell obtained from an individual. As another example, the cell is a stem cell or progenitor cell obtained from an individual.

[0159] Engineered immune cells The engineered immune cells of the present disclosure can be allogeneic or autologous.

[0160] In some embodiments, the engineered immune cell is a T cell (e.g., an inflammatory T lymphocyte, a cytotoxic T lymphocyte, a regulatory T lymphocyte (Treg), a helper T lymphocyte, a tumor infiltrating lymphocyte (TIL)), a natural killer T cell (NKT), a TCR expressing cell, a dendritic cell, a killer dendritic cell, a mast cell, a natural killer cell, or a B cell. In some embodiments, the cell may be from the group consisting of one or both of CD4+ T-lymphocytes and CD8+ T-lymphocytes. In some exemplary embodiments, the engineered immune cell is a T cell. In some exemplary embodiments, the engineered immune cell is a gamma delta T cell. In some exemplary embodiments, the engineered immune cell is a macrophage. In some exemplary embodiments, the engineered immune cell is a natural killer (NK) cell.

[0161] As mentioned above, in some embodiments, the engineered immune cells may be derived from, for example, but not limited to, stem cells. The stem cells may be adult stem cells, non-human embryonic stem cells, more particularly, non-human stem cells, umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells, or hematopoietic stem cells. In some embodiments, the cells are obtained or prepared from peripheral blood. In some embodiments, the cells are obtained or prepared from peripheral blood mononuclear cells (PBMCs). In some embodiments, the cells are obtained or prepared from bone marrow. In some embodiments, the cells are obtained or prepared from umbilical cord blood. In some embodiments, the cells are human cells.

[0162] In some embodiments, the immune cells are T lymphocytes expressing a CAR produced by the methods described herein. In some embodiments, the immune cells are cytotoxic T lymphocytes expressing a CAR produced by the methods described herein. In some embodiments, the immune cells are regulatory T lymphocytes expressing a CAR produced by the methods described herein. In some embodiments, the immune cells are helper T lymphocytes expressing a CAR produced by the methods described herein. In some embodiments, the engineered immune cells of the present disclosure comprise a population of CARs, each CAR comprising a different extracellular antigen binding domain. In some embodiments, the immune cells comprise a population of CARs, each CAR comprising the same extracellular antigen binding domain.

[0163] Also provided herein are cell lines obtained from immune cells (e.g., T cells) transformed according to any of the above-mentioned methods. Also provided herein are modified cells resistant to immunosuppressive treatment. In some embodiments, the isolated cells according to the present disclosure comprise a polynucleotide encoding a CAR. In some embodiments, the engineered immune cells comprise a population of CARs, each CAR comprising an extracellular antigen-binding domain. In some embodiments, the immune cells comprise a population of CARs, each CAR comprising the same extracellular antigen-binding domain.

[0164] In some embodiments, the engineered immune cells according to the present disclosure may comprise one or more disrupted or inactivated genes. In some embodiments, the engineered immune cells according to the present disclosure comprise one disrupted or inactivated gene selected from the group consisting of CD52, DLL3, GR, PD-1, CTLA-4, LAG3, TIM3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLECl0, 2B4, HLA, TCRa, and TCRb, and / or express a CAR, a multi-chain CAR, and / or a pTa transgene. In some embodiments, the isolated cells comprise a polynucleotide encoding a polypeptide comprising a multi-chain CAR. In some embodiments, an isolated cell according to the present disclosure comprises two disrupted or inactivated genes selected from the group consisting of: CD52 and GR, CD52 and TCRa, CDR52 and TCRb, GR and TCRa, GR and TCRb, TCRa and TCRb, PD-1 and TCRa, PD-1 and TCRb, CTLA-4 and TCRa, CTLA-4 and TCRb, LAG3 and TCRa, LAG3 and TCRb, TIM3 and TCRa, Tim3 and TCRb, BTLA and TCRa, BTLA and TCRb, BY55 and TCRa, BY55 and TCRb, TIGIT and TCRa, TIGIT and TCRb, B7H5 and TCRa, B7H5 and TCRb, and TCRb, SIGLEC l0 and TCRa, SIGLEC 10 and TCRb, 2B4 and TCRa, 2B4 and TCRb, and / or isolated cells according to the present disclosure express a CAR, a multi-chain CAR, and / or a pTa transgene.

[0165] In some embodiments, the TCR is rendered non-functional in the cells according to the present disclosure by disrupting or inactivating the TCRa gene and / or the TCRP gene. In some embodiments, a method is provided for obtaining modified cells from an individual, wherein the cells can grow independently of the major histocompatibility complex (MHC) signaling pathway. The modified cells obtainable by the method can grow independently of the MHC signaling pathway and are within the scope of the present disclosure.

[0166] In some embodiments, the immune cells are engineered to be resistant to one or more chemotherapeutic agents. The chemotherapeutic agents may be, for example, purine nucleotide analogs (PNAs), thus making the immune cells suitable for cancer treatment combining adoptive immunotherapy and chemotherapy. Exemplary PNAs include, for example, clofarabine, fludarabine, cyclophosphamide, and cytarabine, alone or in combination. PNAs are metabolized by deoxycytidine kinase (dCK) to monophosphate PNAs, diphosphate PNAs, and triphosphate PNAs. Their triphosphate forms compete with ATP for DNA synthesis, act as proapoptotic agents, and are potent inhibitors of ribonucleotide reductase (RNR), which is involved in trinucleotide production.

[0167] In some embodiments, the isolated cells or cell lines of the present disclosure may comprise pTa or a functional variant thereof. In some embodiments, the isolated cells or cell lines may be further genetically modified by disrupting or inactivating the TCRa gene.

[0168] As mentioned above, the present disclosure also provides engineered immune cells that contain CAR polynucleotides.In some embodiments, CAR can be introduced into immune cells as a transgene via a plasmid vector.In some embodiments, the plasmid vector can also contain a selection marker, for example, to provide identification and / or selection of cells that have received the vector.

[0169] In some embodiments, the cells are transfected with a nucleic acid vector of the present disclosure using a method selected from the group consisting of electroporation, sonoporation, biolistics (e.g., Gene Gun), lipid transfection, polymer transfection, nanoparticles, or polyplexes. In some embodiments, the cells are transduced with a viral vector of the present disclosure, such as a retroviral vector, particularly a lentiviral vector.

[0170] Treatment method Methods for treating diseases or disorders, including cancer, are provided. In some embodiments, the disclosure relates to generating a T cell-mediated immune response in a subject, comprising administering to the subject an effective amount of an engineered immune cell of the disclosure. In some embodiments, the T cell-mediated immune response is directed against a target cell. In some embodiments, the engineered immune cell comprises a chimeric antigen receptor (CAR). In some embodiments, the target cell is a tumor cell. In some aspects, the disclosure includes a method for treating or preventing a malignancy, comprising administering to a subject in need thereof an effective amount of at least one isolated antigen binding domain described herein. In some aspects, the disclosure includes a method for treating or preventing a malignancy, comprising administering to a subject in need thereof an effective amount of at least one immune cell, wherein the immune cell comprises at least one chimeric antigen receptor and / or isolated antigen binding domain described herein.

[0171] In some embodiments, the subject has a solid tumor or a hematological malignancy such as lymphoma or leukemia. In some embodiments, the cancer is present in the bone marrow of the subject. In some embodiments, the engineered cells are autologous immune cells, e.g., autologous T cells. In some embodiments, the engineered cells are allogeneic immune cells, e.g., allogeneic T cells. In some embodiments, the engineered cells are xenogeneic immune cells, e.g., xenogeneic T cells. In some embodiments, the engineered cells are transfected and / or transduced ex vivo. As used herein, the term "in vitro cells" refers to any cells that are cultured ex vivo. A "therapeutically effective amount," "effective dose," "effective amount," or "therapeutically effective dose" of a therapeutic agent, e.g., an engineered CART cell, is any amount that, when used alone or in combination with another therapeutic agent, protects a subject against the onset of disease or promotes disease regression as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or prevention of functional impairment or disability due to disease affliction. The ability of a therapeutic agent to promote disease regression can be evaluated using a variety of methods known to a skilled practitioner (e.g., a physician or clinician), for example, in human subjects in clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.

[0172] The terms "patient" and "subject" are used interchangeably and include human subjects, as well as those with a formally diagnosed disorder, those without a formally recognized disorder, those under medical attention, those at risk of developing a disorder, and the like.

[0173] The terms "treat" and "treatment" include therapeutic treatment, prophylactic treatment, and disclosures that reduce the risk of a subject developing a disorder or other risk factors. Treatment does not require a complete cure of the disorder, but encompasses embodiments that reduce symptoms or underlying risk factors. The term "prevent" does not require 100% elimination of the likelihood of an event. Rather, it indicates that the likelihood of an event occurring in the presence of a compound or method is reduced.

[0174] The desired total therapeutic amount of cells in the composition is at least 2 cells (e.g., at least one CD8+ T cell and at least one CD4+ T cell, or two CD8+ T cells, or two CD4+ T cells), or more typically 10 2 More than 10 cells and up to 10 6 pcs, up to 10 including the number 8 Pieces or 10 9 cells, and 10 10 or 10 12 The number of cells will depend on the desired use for which the composition is intended, and the type of cells contained therein. Desired cell densities are typically between 10 6 cells / ml, generally greater than 10 7 More than 10 cells / ml 8 Clinically significant numbers of immune cells are >10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , or 10 12 The cells can be cumulatively distributed over multiple injections equal to or greater than 10, in some embodiments of the present disclosure, particularly since all injected cells are redirected to a specific target antigen. 6 / kilogram (10 per patient) 6 ~10 11 ) can be administered. CAR therapy can be administered multiple times at doses within these ranges.

[0175] The cells can be autologous, allogeneic, or xenogeneic to the patient undergoing therapy.

[0176] In some embodiments, a therapeutically effective amount of CAR T cells is about 1×10 cells / kg, about 2×10 5 cells / kg, approximately 3×10 5cells / kg, about 4×10 cells / kg, about 5 X10 cells / kg, about 6×10 cells / kg, about 7×10 cells / kg, about 8×10 cells / kg, about 9×10 cells / kg, 2×10 cells / kg, about 3×10 cells / kg, about 4×10 cells / kg, about 5×10 cells / kg, about 6×10 cells / kg, about 7×10 cells / kg, about 8×10 cells / kg, about 9×10 cells / kg, about 1×10 cells / kg, about 2×10 cells / kg, about 3×10 cells / kg, about 4×10 cells / kg, about 5×10 cells / kg, about 6×10 cells / kg, about 7×10 cells / kg, about 8×10 cells / kg, or about 9×10 cells / kg.

[0177] In some embodiments, the target dose for CAR+ / CAR-T+ cells is about 1×10 6 ~Approx. 1×10 10 Cells / kg, e.g., about 1 x 10 6 cells / kg, approx. 1 x 107 cells / kg, approx. 1 x 108 cells / kg, approx. 1 x 10 9 cells / kg, or approximately 1 x 10 10 The range is 100 mg / kg of cells. Doses above and below this range may be appropriate for a particular subject, and appropriate dose levels can be determined by a medical professional, if necessary. Additionally, multiple doses of cells can be provided according to the present disclosure.

[0178] In some aspects, the disclosure includes pharmaceutical compositions comprising at least one antigen binding domain described herein and a pharma- ceutical acceptable excipient. In some embodiments, the pharmaceutical composition further comprises an additional active agent.

[0179] The CAR-expressing cell population of the present disclosure can be administered either alone or as a pharmaceutical composition in combination with a diluent and / or in combination with other components such as IL-2 or other cytokines or cell populations. The pharmaceutical composition of the present disclosure can include a CAR-expressing cell population, such as T cells, as described herein, in combination with one or more pharma- ceutical or physiologically acceptable carriers, diluents, or excipients. Such compositions can include buffers, such as neutral buffered saline, phosphate buffered saline, carbohydrates, such as glucose, mannose, sucrose, or dextran, amino acids, such as mannitol, proteins, polypeptides, or glycine, antioxidants, chelating agents, such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), and preservatives. The compositions of the present disclosure are preferably formulated for intravenous administration.

[0180] Pharmaceutical compositions (solutions, suspensions, etc.) may contain one or more of the following: sterile diluents such as water for injection, saline, preferably saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono- or diglycerides that may serve as solvents or suspending media, polyethylene glycol, glycerin, propylene glycol or other solvents, antibacterial agents such as benzyl alcohol or methylparabens, antioxidants such as ascorbic acid or sodium sulfite, chelating agents such as ethylenediaminetetraacetic acid (EDTA), buffers such as acetates, citrates or phosphates, agents for adjusting tonicity such as sodium chloride or dextrose. Parenteral preparations may be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic. For therapeutic disclosure, pharmaceutical compositions for injection are preferably sterile.

[0181] The method may further include administering one or more chemotherapeutic agents to the patient prior to administering the engineered cells provided herein. In certain embodiments, the chemotherapeutic agent is a lymphodepleting (preconditioning) chemotherapy, such as a specific beneficial dose of cyclophosphamide (200 mg / m2 / day to 2000 mg / m2 / day, about 100 mg / m2 / day to about 2000 mg / m2 / day; e.g., about 100 mg / m2 / day, about 200 mg / m2 / day, about 300 mg / m2 / day, about 400 mg / m2 / day, about 500 mg / m2 / day, about 600 mg / m2 / day, about 700 mg / m2 / day, about 800 mg / m2 / day, about 900 mg / m2 / day, about 1000 mg / m2 / day, about 1500 mg / m2 / day or about 2000 mg / m2 / day) and a specific dose of fludarabine. (20 mg / m2 / day to 900 mg / m2 / day, about 10 mg / m2 / day to about 900 mg / m2 / day; e.g., about 10 mg / m2 / day, about 20 mg / m2 / day, about 30 mg / m2 / day, about 40 mg / m2 / day, about 40 mg / m2 / day, about 50 mg / m2 / day, about 60 mg / m2 / day, about 70 mg / m2 / day, about 80 mg / m2 / day, about 90 mg / m2 / day, about 20 mg / m2 / day, about 100 mg / m2 / day, about 500 mg / m2 / day or about 900 mg / m2 / day) to the patient. An exemplary dosing regimen involves treating a patient with daily administration of about 300 mg / m2 / day of cyclophosphamide to the patient in combination with, and before or after, administration of about 30 mg / m2 / day of fludarabine for three days prior to administration of a therapeutically effective amount of engineered T cells to the patient.

[0182] In some embodiments, particularly where the engineered cells provided herein have been genetically edited to eliminate or minimize surface expression of CD52, lymphodepletion further comprises administration of an anti-CD52 antibody, such as, for example, alemtuzumab. In some embodiments, the CD52 antibody is administered at a dose of about 1-20 mg / day IV, e.g., about 13 mg / day IV, for 1, 2, 3, 4, 5, 6, 7 days or more.

[0183] The antibody may be administered in combination with, prior to, or following the administration of other components of the lymphodepleting regimen (eg, cyclophosphamide and / or fludarabine).

[0184] In other embodiments, the antigen binding domain, the transduced (or otherwise engineered) cells, and the chemotherapeutic agent are each administered in an amount effective to treat the disease or condition of the subject.

[0185] In certain embodiments, compositions comprising CAR-expressing immune effector cells disclosed herein can be administered in conjunction with any number of chemotherapeutic agents. Exemplary chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN™); alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; altretamine, triethylenemelamine, triethylenephosphamide, triethylenethiophosphaoramide, and trimethylolomelamine regimes. resume); nitrogen mustards, such as chlorambucil, chromafazine, colofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembicine, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics, such as aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, dacarbazine ... Unorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin, puromycin, queramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, etc.;Pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmoful, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calsterone, dromostanol propionate, epithiostanol, mepitiostane, testosterone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as floric acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; amsacrine; bestravcil; bisantrene; edatraxate; defofamine; demecolcine; diazicon; erformitin; elliptinium acetate; Etoglucide; Gallium nitrate; Hydroxyurea; Lentinan; Lonidamine; Mitoguazone, Mitoxantrone; Mopidamol; Nitracrine; Pentostatin; Phenamet; Pirarubicin; Podophyllic acid, 2-ethylhydrazide; Procarbazine; PSK®; Razoxane; Sizofiran; Spirogermanium; Tenuazonic acid; Triazicon; 2,2',2"-Trichlorotriethylamine; Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitrol; Mitolactol; Pipobroman; Gacytosine; Arabinoside ("Ara-C"); Cyclophosphamide; Thiotepa; Taxoids, e.g., paclitaxel (TAXOL®, Bristol-Myers Squibb) and docetaxel (TAXOTERE®, Rhone-Poulenc Rorer); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogues, such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitors RF S2000; difluoromethylomitin (DMFO); retinoic acid derivatives such as Targretin™ (bexarotene), Panretin™, (alitretinoin); ONT AK™ (denileukin diftitox); esperamicin;Capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing. Also included in this definition are antiestrogens, such as tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazole, 4-hydroxytamoxifen, trioxyphene, keoxyphene, LYl 17018, onapristone, and toremifene (Fareston); and antihormonal agents that act to regulate or inhibit hormone action on tumors, such as antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing. Combinations of chemotherapeutic agents are also administered where appropriate, including, but not limited to, CHOP, i.e., cyclophosphamide (Cytoxan®), doxorubicin (hydroxydoxorubicin), vincristine (Oncovin®), and prednisone.

[0186] In some embodiments, the chemotherapeutic agent is administered simultaneously with or within one week after administration of the engineered cells, polypeptide, or nucleic acid. In other embodiments, the chemotherapeutic agent is administered about 1 to 7 days, about 1 to about 4 weeks, or about 1 week to about 1 month, about 1 week to about 2 months, about 1 week to about 3 months, about 1 week to about 6 months, about 1 week to about 9 months, or about 1 week to about 12 months after administration of the engineered cells, polypeptide, or nucleic acid. In other embodiments, the chemotherapeutic agent is administered at least one month prior to administration of the cells, polypeptide, or nucleic acid. In some embodiments, the method further comprises administering two or more chemotherapeutic agents.

[0187] A variety of additional therapeutic agents can be used in conjunction with the compositions described herein. For example, potentially useful additional therapeutic agents include PD-1 inhibitors such as nivolumab (Opdivo®), pembrolizumab (Keytruda®), pembrolizumab, pidilizumab, and atezolizumab. Additional therapeutic agents suitable for use in combination with the present disclosure include, but are not limited to, ibrutinib (Imbruvica®), ofatumumab (Arzera®, rituximab (Rituxan®), bevacizumab (Avastin®), trastuzumab (Herceptin®), trastuzumab emtansine (Kadcyla®), imatinib ( Gleevec (registered trademark), cetuximab (Erbitux (registered trademark), panitumumab) (Vectibix (registered trademark), catumaxomab, ibritumomab, ofatumumab, tositumomab, brentuximab, alemtuzumab, gemtuzumab, erlotinib, gefitinib, vandetanib, afatinib, lapatinib, neratinib, axitinib, masitinib, pazopanib, sucralobacter Nitinib, sorafenib, toceranib, lestaurtinib, axitinib, cediranib, lenvatinib, nintedanib, pazopanib, regorafenib, semaxanib, sorafenib, sunitinib, tivozanib, toceranib, vandetanib, entrectinib, cabozantinib, imatinib, dasatinib, nilotinib, ponatinib, radotinib, bosutinib, lestaurtinib, ruxoli mTOR inhibitors such as tinib, pacritinib, cobimetinib, selumetinib, trametinib, binimetinib, alectinib, ceritinib, crizotinib, aflibercept, adipotide, denileukin diftitox, everolimus and temsirolimus, hedgehog inhibitors such as sonidegib and vismodegib, and CDK inhibitors such as the CDK inhibitor palbociclib.

[0188] In some embodiments, the composition comprising the CAR-expressing immune cells can be administered with a therapeutic regimen to prevent or reduce cytokine release syndrome (CRS) or neurotoxicity. The therapeutic regimen to prevent cytokine release syndrome (CRS) or neurotoxicity can include lenzilumab, tocilizumab, atrial natriuretic peptide (ANP), anakinra, iNOS inhibitors (e.g., L-NIL or 1400W). In additional embodiments, the composition comprising the CAR-containing immune cells can be administered with an anti-inflammatory agent. Anti-inflammatory agents or drugs include, but are not limited to, steroids and glucocorticoids (betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, ethylprednisolone, prednisolone, prednisone, triamcinolone), nonsteroidal anti-inflammatory drugs (NSAIDS) including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF drugs, cyclophosphamide and mycophenolic acid. Exemplary NSAIDs include ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors, and sialic acid. Exemplary analgesics include acetaminophen, oxycodone, proporoxifene hydrochloride tramadol. Exemplary glucocorticoids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Exemplary biological response modifiers include molecules against cell surface markers (e.g., CD4, CDS, etc.), cytokine inhibitors, such as TNF antagonists (e.g., etanercept (ENBREL®), adalimumab (HUMIRA®), and infliximab (REMICADE®)), chemokine inhibitors, and adhesion molecule inhibitors. Biological response modifiers include monoclonal antibodies and recombinant forms of molecules. Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, gold (oral (auranofin) and intramuscular), and minocycline.

[0189] In certain embodiments, the compositions described herein are administered in conjunction with cytokines. Examples of cytokines include lymphokines, monokines, and conventional polypeptide hormones. Cytokines include growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor (HGF); fibroblast growth factor (FGF); prolactin; placental lactogen; Mullerian inhibitor, mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrins; thrombopoietin (TPO); nerve growth factor (NGF), such as NGF-beta; platelet growth factor; transforming growth factor, such as TGF-alpha and TGF-beta. These include transforming growth factors (TGF); insulin-like growth factors-I and -II; erythropoietin (EPO); bone morphogenetic factors; interferons such as interferon-alpha, beta, and -gamma; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs), such as IL-1, IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15, IL-21; tumor necrosis factors such as TNF-alpha or TNF-beta; and other polypeptide factors, including LIF and Kit Ligand (KL). As used herein, the term cytokine includes proteins from natural sources or recombinant cell culture, as well as biologically active equivalents of the native sequence cytokines.

[0190] Kits and Articles of Manufacture The present disclosure provides kits comprising any one of the immune cells expressing a CAR using the methods provided herein, and pharmaceutical compositions thereof. In an embodiment of the kit, the engineered CAR cells are frozen in an appropriate medium, such as CryoStor® CS10, CryoStor® CS2, or CryoStor® CS5 (BioLife Solutions).

[0191] In some exemplary embodiments, the kits of the present disclosure include allogeneic CAR-expressing T cells and CD52 antibodies for administration to a subject as components of a lymphodepletion regimen and a CAR-T regimen. The present disclosure also provides an article of manufacture that includes any one of the therapeutic compositions or kits described herein. An example of an article of manufacture includes a vial (e.g., a sealed vial containing immune cells expressing a CAR). EXAMPLES

[0192] Example 1: Effect of process factors on immune cell transduction efficiency A. Effect of MOI, growth medium pH, RetroNectin® concentration, and culture vessel on the percentage of viable CAR 1+ immune cells

[0193] PBMC collection Donor blood was collected and separated into its component parts by apheresis. PBMCs were then concentrated on a GE Ficoll®-plaque (density 1.077 g / mL) step gradient and stored frozen in a buffer containing 5% DMSO.

[0194] activation On day 0, Ficoll®-isolated frozen human PBMCs were thawed and washed once with wash medium containing X-VIVO™ 15 culture medium (Lonza Biosciences) and 10% human serum (Gemini Bio Products, Sacramento, CA). The cells were then cultured in culture medium (also referred to herein as growth medium) containing X-VIVO™ 15 culture medium (Lonza Biosciences) with 5% human serum (Gemini Bio Products, Sacramento, CA) at 37°C and 5% CO in a standard humidified tissue culture incubator. 2 On day 1, cells were washed with wash medium, counted, and diluted to 1.5 × 10 per mL. 6 The T cells were resuspended in culture medium at a cell density of 100 μg / mL and mixed with T Cell TransAct™ polymer nanomatrix (Miltenyi Biotec) at a volumetric dilution of 1:10. Recombinant human IL-2 (Miltenyi Biotec) was added to a final concentration of 100 U / mL. The T cells were then incubated at 37°C and 5% CO in a standard humidified tissue culture incubator. 2 The cells were incubated at 4° C. for 4 days.

[0195] Transduction and expansion On day 4, wash the cells and resuspend them at 1 x 10 per mL. 6 The cells were resuspended in culture medium containing recombinant human IL-2 (Miltenyi Biotec) at a cell density of 1000 x 1000. The cells were transduced with a lentiviral vector encoding CAR1 (1560 nucleotides) (LVV #1831P, Lentigen Technology, Gaithersburg, MD) under the test conditions described in Table 1 below and incubated at 37°C and 5% CO in a standard humidified tissue culture incubator. 2 The cells were incubated at RT for 6 days. On day 6, the transduced cells were cultured to expand the cell population. [Table 1] *The culture bag is MACS Cell Differentiation Bag-100 (Miltenyi Biotec). **The plate is a 6-well culture plate (Corning, Inc.). ***Calculated from second column %LVV(v / v)

[0196] Transduction efficiency assessment Transduction efficiency and cell viability were confirmed by flow cytometry on days 8 and 11. No polybrene, protamine sulfate, or DEAE-dextran was added before, during, or after transduction. For each test group, cell samples were washed and resuspended in Dulbecco's phosphate buffered saline ("DPBS"). Two antibody / stain cocktails, each containing a different panel of antibodies, were prepared using commercially available antibody / stain combinations.

[0197] Approximately 1 × 10 samples from each test group 6 1000 cells were added to a FACS tube along with an aliquot of one of the antibody / stain cocktails. The FACS tube was then incubated for 25 min at 25° C. in the dark. + The samples were washed twice, resuspended in 1% paraformaldehyde ("PFA"), and then processed on an LSRFortessa cytometer (BD Biosciences, Franklin Lakes, NJ), and the resulting data was analyzed using FlowJo software version 10 (FlowJo, Ashland, OR).

[0198] Table 2 below shows the percentage of viable CAR+ T cells for each study group on days 8 and 11 of the study. [Table 2]

[0199] The effects of manufacturing process factors, % lentiviral vector (v / v), growth medium pH, RetroNectin® reagent concentration, and culture vessel on transduction efficiency are shown in Figure 2. The significance of main effects and interactions was determined by analysis of variance (ANOVA) with a probability value (p-value) threshold set at <0.05 using JMP® 14 software (SAS Institute, Inc., Cary, NC). Transducing cells at a pH of 7.0 or higher resulted in a greater percentage of viable CAR+ cells (see Figure 2A) than transducing cells at a lower pH, which resulted in a lower percentage of viable CAR+ cells (see Figure 2B). P-values ​​for main effects and two-way interactions are shown in Figure 2C.

[0200] Surprisingly, as Figure 2 shows, RetroNectin® Reagent (Takara Bio, USA) concentration does not have a significant effect on CAR transduction efficiency. Rather, pH has the most significant effect on the transduction efficiency of all factors tested at MOI, and the transduction vessel also contributes to the transduction efficiency. B. Effect of growth medium pH and RetroNectin® concentration on the percentage of viable CAR-2+ immune cells PBMCs were collected, activated, and transduced with CAR-2 (approximately 1500 nucleotides) by the method described in Example 1A above in a partial factorial experiment (using JMPO 14 software, D-optimal design) to evaluate the effect of pH and RetroNectin® concentration on the percentage of viable CAR-2+ T cells.

[0201] Transduction efficiency assessment On day 15, transduction efficiency and cell viability were confirmed by flow cytometry as described in Example 1A above. Significance of main effects and interactions was determined and modeled by analysis of variance (ANOVA) with a probability value (p-value) threshold set at <0.05 using JMP®14 software (SAS Institute, Inc., Cary, NC). Results for the percentage of viable CAR+ T cells were simulated at an MOI of 48 using JMP®14 ANOVA software to evaluate the main effects of RetroNectin® and media pH on the percentage of viable CAR+ T cells at the RetroNectin® concentrations and media pH levels shown in Table 3. Results are shown in the right-most column of Table 3. [Table 3]

[0202] Table 3 shows that transducing T cells at a pH of 7.3, both with and without RetroNectin® reagent, resulted in a greater percentage of viable CAR+ cells than transducing cells at the lower pH of 6.7, both with and without RetroNectin® reagent. Surprisingly, RetroNectin® reagent concentration does not have the greatest effect on CAR transduction efficiency. Medium pH during transduction has the greatest impact on transduction efficiency.

[0203] Example 2: Time course of vector integration transduction On day 0, PBMCs (collected as described in Example 1 above) were thawed in a 2 L gas-permeable bag (Xuri™ Cell bag™, GE Lifesciences, Inc.). 6Cells at a concentration of 1000 cells / mL additionally containing 5% human serum (Gemini Bio Products, Sacramento, CA), IL-2, and glutamine were activated by adding TransAct™ polymer nanomatrix (Miltenyi Biotec) at a volumetric dilution of 1:10. Cells were cultured in an in-wave action bioreactor at 37°C.

[0204] On day 2, 1% (v / v) lentiviral vector (LVV #1831P, Lentigen Technology, Gaithersburg, MD) encoding CAR-2 (1500 nucleotides) was injected into the cells at 7.2 °C for 30 min. + The resulting transfection reaction mixture was incubated at 37°C for 8 hours. No RetroNectin® was used during transfection. Additionally, no polybrene, protamine sulfate, or DEAE-dextran was added before, during, or after transfection.

[0205] Cell samples were collected from the culture medium 1, 2, 4, 6, and 8 hours after the lentiviral vector was introduced into the cells. Each cell sample was collected at 1 × 10 6 The cells were centrifuged, washed, resuspended in culture medium at a cell density of 1000 cells / mL, and cultured in a volume of 40 mL in the G-Rex® Cell Culture System (Wilson Wolf, St. Paul, MN) according to the manufacturer's instructions for 14 days.

[0206] The percentage of CAR+ cells was determined by flow cytometry on days 7 and 14, as described in Example 1 above.

[0207] The results are shown in Figure 3. As shown in the figure, the percentage of CAR+ cells is higher at each transduction time point on day 14 when compared to day 7. Furthermore, on day 14, the increase in the percentage of CAR+ cells begins to slow down after 4 hours of transduction and begins to plateau between 6 and 8 hours of transduction.

[0208] Example 3: Manufacturing scale immune cell transduction Fourteen manufacturing scale runs were performed for T cells expressing CAR-1. Runs 7-14 were performed as described below. Runs 1-6 were performed as shown below, with one exception where the transduction step was performed at a pH below 7.2.

[0209] PBMC collection, activation and transduction PBMCs (collected as described in Example 1 above) were thawed in gas-permeable bags in medium containing human serum and incubated at 37°C and 5% CO 2 The mixture was incubated overnight at 4°C.

[0210] PBMCs were then washed and resuspended in medium containing 5% human serum, IL-2, glutamine, CD3 and CD28 stimulating reagents and incubated at 37 °C and 5% CO 2 and incubated at 4°C for 1 hour.

[0211] The activated cells were washed with medium to remove the activation reagent and resuspended in culture medium. The culture medium consisted of 5% human serum, IL-2, and glutamine. Before the cells were introduced into the culture medium, they were incubated for 24 hours at 4°C for 1 h. 2 was removed from the culture medium to achieve a pH of 7.2 or higher. Cells were cultured at 1 × 10 per mL. 6 Cells were resuspended in a manufacturing-scale volume (manufacturing-scale transduction volume) of culture medium in a gas-permeable bag. A lentiviral vector encoding CAR-1 (LVV #1831P, Lentigen Technology, Gaithersburg, MD) was added to the cell suspension at 10% LVV (V / V). The resulting transduction reaction mixture was incubated at 37 °C and 5% CO 2 The cells were cultured at RT for 48 hours. RetroNectin® was not used during transduction. Also, polybrene, protamine sulfate, and DEAE-dextran were not added to the transduction.

[0212] Proliferation The cells were further electroporated to disrupt the target gene and then expanded in a perfusion wave bioreactor in a total volume of 1 liter of culture medium. T cell enrichment was performed on day 18 to select for T cells with disrupted gene expression.

[0213] CAR expression and cell viability On day 19, CAR expression and cell viability were assessed by flow cytometry as described in Example 1A above and are listed in Table 4 below. [Table 4] *Standard deviation

[0214] Table 4 shows that cells transduced at a pH greater than 7.1 had a higher percentage of viable CAR-expressing cells compared to cells transduced at a pH less than 7.1. The table further shows that transducing cells at a pH greater than 7.1 resulted in a more consistent percentage of viable CAR-expressing cells run-to-run compared to cells transduced at a pH less than 7.1.

Claims

1. 1. A method of transducing a cell population with a retroviral vector, said vector comprising a nucleic acid that is exogenous to said cells, said method comprising: culturing said cell population with said retroviral vector in cell culture medium at a starting pH within a starting pH range of 7.0 to 7.9; and maintaining said starting pH within said starting pH range for at least a first hour of a transduction culture step to result in a transduced cell population comprising cells expressing a gene product encoded by said exogenous nucleic acid, wherein said cell population comprises T lymphocytes, helper T cells, tumor cells, memory T cells, cytotoxic T cells, natural killer T cells, peripheral blood lymphocytes, peripheral blood mononuclear cells, dendritic cells, or natural killer cells, or a mixture thereof.

2. 2. The method of claim 1, wherein the initial pH is maintained within the initial pH range for at least about 1, about 2, about 4, about 6, about 8, about 10, about 12, about 14, about 16, about 18, about 20, about 22, about 24, about 36, about 48, about 72, or about 96 hours but not more than about 168 hours.

3. 2. The method of claim 1, wherein the initial pH is maintained above about 7.0 throughout the end of the transduction culture step.

4. 4. The method of claim 3, wherein the transduction culture step is carried out for at least about 1, about 2, about 4, about 6, about 8, about 12, about 14, about 16, about 18, about 20, about 22, about 24, about 36, about 48, about 72, or about 96 hours.

5. The method of any one of claims 1 to 4, wherein the cell culture medium does not contain a co-localization agent.

6. The method of any one of claims 1 to 5, wherein the cell culture medium does not contain fibronectin or a fibronectin derivative.

7. 7. The method of any one of claims 1-6, wherein the selected cell population is cultured at an MOI of about .25, about .5, about 1, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, or about 50 to about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 125, about 150, about 175, about 200, about 225, or about 250.

8. The method of any one of claims 1 to 7, wherein the exogenous nucleic acid encodes a chimeric antigen receptor.

9. 9. The method of any one of claims 1 to 8, wherein the selected cell population is cultured in a container, the container being a cell culture plate, a cell culture deep well plate, a cell stack, a controlled bioreactor, a shake flask, or a gas permeable bag.

10. The method according to any one of claims 1 to 9, wherein the retroviral vector is a lentiviral vector.

11. 11. The method of any one of claims 1 to 10, wherein the transduction culturing step comprises culturing the selected cell population and the retroviral vector in a volume of about 0.5 liters to about 10 liters of cell culture medium.

12. 12. The method of any one of claims 1-11, wherein at least about 35% to about 95%, about 40% to about 95%, about 50% to about 95%, about 60% to about 95%, or about 70% to about 95% of the transduced cell population expresses the exogenous nucleic acid gene product from about 3 to about 18 days after initiation of the transduction culture step.

13. 13. The method of any one of claims 1-12, wherein at least about 35% to about 95%, about 40% to about 95%, about 50% to about 95%, about 60% to about 95%, or about 70% to about 95% of the transduced cell population expresses the exogenous nucleic acid gene product about 7 to about 18 days after initiation of the transduction culture step.

14. 14. A method of transducing a first and a second cell population, wherein said first and second cell populations are transduced by the same method of any one of claims 1 to 13, whereby the percentage of said transduced cell populations expressing said exogenous nucleic acid gene product in said first and second transduced cell populations does not differ by more than about 2% to about 5%, about 5% to about 10%, about 10% to about 20%, or about 20% to about 30%.

15. 1. A method of transducing a cytotoxic T cell population with a retroviral vector, said vector comprising a nucleic acid exogenous to said cytotoxic T cells, said method comprising culturing said cytotoxic T cell population with said retroviral vector in a cell culture medium at a starting pH within a starting pH range of 7.0 to 7.9, and maintaining said starting pH within said starting pH range for at least an initial hour of a transduction culture step to result in a transduced cytotoxic T cell population comprising cells expressing said gene product encoded by said exogenous nucleic acid, wherein said cell culture medium does not comprise a co-localization agent.

16. 1. A method of transducing a population of T cells derived from induced pluripotent stem cells with a retroviral vector, the vector comprising a nucleic acid exogenous to the derived T cells, the method comprising culturing the induced T cell population with the retroviral vector in a cell culture medium at an initial pH within an initial pH range of 7.0 to 7.9, and maintaining the initial pH in the initial pH range for at least an initial hour of a transduction culture step to result in a transduced induced T cell population comprising cells expressing the gene product encoded by the exogenous nucleic acid, wherein the cell culture medium does not comprise a co-localization agent.

17. The method of any one of claims 1 to 16, wherein no polycations are added to the cell culture medium.

18. The method of claim 1, wherein no polybrene, protamine sulfate, or DEAE-dextran is added to the culture medium.

19. The method of any one of claims 1 to 18, wherein the cell culture medium does not contain fibronectin or a fibronectin derivative.

20. The method of claim 1, wherein the fibronectin or fibronectin derivative is not added to the culture medium.

21. The method of claim 1, wherein the initial pH is actively maintained.

22. The method of any one of claims 1 to 21, wherein the initial pH is passively maintained.

23. The method of any one of claims 1 to 22, wherein the cell population is a homogeneous cell population.

24. The method of any one of claims 1 to 22, wherein the cell population is an autologous cell population.

25. The method of claim 1, wherein the starting pH is in the starting pH range of 7.0 to 7.

4.

26. The method of any one of claims 1 to 25, wherein the initial pH is 7.1, 7.2, 7.3, or 7.4.

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