Formulations and methods for CAR T cell medicines

Abstract

Provided herein are formulations and pharmaceutical processes that can improve cell viability and minimize waste of formulated cells manufactured for pharmaceutical 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 / 145,235, filed February 3, 2021, the contents of which are incorporated herein by reference in their entirety.

[0002] The present disclosure relates to formulations and methods for processing cell-based medicines, including CAR T cell medicines. [Background technology]

[0003] The final step in manufacturing engineered immune cells such as CAR T cells is to formulate the active drug substance cells and fill them into one or more final containers, or drug product process (DP process). For example, after manufacturing, the cells are harvested from the culture medium by centrifugation and formulated into a composition that may contain a cryopreservative (dimethyl sulfoxide, DMSO, etc.). The formulated cells may be frozen before being filled into a suitable container closure system and stored in the vapor phase of liquid nitrogen (vLN2).

[0004] The DP process can potentially be stressful to cells, for example, because the cells are removed from culture medium and conditions, and the formulation may contain cryopreservatives (e.g., dimethyl sulfoxide, DMSO). DMSO is a strong solvent that can adversely affect cells in a time-dependent manner. Additional sources of stress can result from the physical shaking, mixing, and pumping of the cells, as well as exposure of the cells to room temperature or other non-cell culture conditions. The pharmaceutical process can take several hours depending on the batch size, and cells need to be kept healthy and in suspension to ensure homogeneity of the DP. Thus, the consensus in the field has been that time is a limiting factor in pharmaceutical processes, and it is important to minimize the exposure time of cells to various stress conditions before freezing.

[0005] There is a need for DP processes that can improve cell viability and health and minimize waste in the final drug product, which is particularly important for allogeneic cell therapies, where each manufacturing batch can produce large numbers of cells for multiple doses. Summary of the Invention

[0006] Provided herein are reagents and processes that can improve the viability and health of cells for use in cell-based therapies and minimize waste in manufacturing and pharmaceutical processes.

[0007] Maintaining cell viability during formulation and filling processes is important and challenging. Optimization of formulation composition and pharmaceutical process is important in maintaining overall cell health and functionality. The present disclosure provides improved formulations and / or processes that better maintain cell health and viability and allow sufficient time to complete pharmaceutical process. This is particularly advantageous for processing engineered cells, such as engineered immune cells, which may be produced in large batches in pharmaceutical processes to minimize waste while ensuring overall cell health and viability and uniformity of dose content.

[0008] Thus, in one aspect, the disclosure provides a pharmaceutical process that maintains cell health and / or homogeneity. In certain embodiments, the disclosure provides a pharmaceutical process for preparing a pharmaceutical product comprising engineered immune cells, the method comprising formulating the engineered immune cells in a composition containing a cryopreservative to form the pharmaceutical product, and mixing and filling the pharmaceutical product into one or more containers, wherein the pharmaceutical product maintains homogeneity. In certain embodiments, the mixing and filling steps take at least about 2 hours. In certain embodiments, the mixing and filling steps take about 2 hours, about 3 hours, about 4 hours, about 5 hours, or about 6 hours. In certain embodiments, the mixing comprises intermittent mixing. In some embodiments, the mixing comprises continuous mixing. In certain embodiments, the intermittent mixing is performed in a shake flask. In certain embodiments, the intermittent mixing is performed in a spinner flask. In certain embodiments, the intermittent mixing comprises mixing for about 1 to about 3 minutes every about 10 to about 40 minutes. In certain embodiments, the mixing comprises continuous mixing in a spinner flask. In certain embodiments, the mixing comprises continuous mixing in a shake flask for about 2 hours. In certain embodiments, the mixing comprises continuous mixing in a shake flask for 2 hours or less. In certain embodiments, the mixing comprises continuous mixing in a shake flask for less than 2 hours.

[0009] In certain embodiments, the mixing and filling step is carried out for about 4 oC. In certain embodiments, the mixing and filling steps are performed at room temperature. In certain embodiments, the mixing is performed at a speed of about 30 to about 85 rpm. In certain embodiments, the cryopreservation agent comprises DMSO or glycerol. In certain embodiments, the cryopreservation agent comprises about 3% DMSO to about 10% DMSO. In certain embodiments, the cryopreservation agent comprises about 5% DMSO. In certain embodiments, the composition further comprises an excipient that enhances viscosity. In certain embodiments, the engineered immune cells are T cells, inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes, helper T lymphocytes, effector T lymphocytes, tumor infiltrating lymphocytes (TILs), NK cells, NK-T cells, TCR expressing cells, TCR knockout T cells, dendritic cells, macrophages, killer dendritic cells, mast cells, or B cells. In certain embodiments, the engineered immune cells are CAR T cells. In certain embodiments, the engineered immune cells are allogeneic CAR T cells. In certain embodiments, the engineered immune cells are autologous CAR T cells. In some embodiments, the engineered immune cells are human cells.

[0010] In another aspect, the present disclosure provides a pharmaceutical product comprising an engineered immune cell prepared by the pharmaceutical process disclosed herein.

[0011] In yet another aspect, the present disclosure provides a method of treating a subject in need of treatment, comprising administering to the subject a pharmaceutical agent disclosed herein. In certain embodiments, the subject is a human. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 shows an exemplary workflow for the final steps of manufacturing engineered cells for pharmaceutical products.

[0013] [Figure 2A] Figures 2A-B show the effect of orbital shaker mixing of CAR T cell formulations on viable cell density (VCD) and cell viability. [Figure 2B] Same as above.

[0014] [Figure 3A] 3A-B show the effect of orbital shaker mixing and formulation holding temperature on VCD and viability. [Figure 3B] Same as above.

[0015] [Figure 4A] Figures 4A-B show the results of a time course analysis of cell sedimentation in cell preparations containing 20E6 / ml or 100E6 / ml CAR T cells. [Figure 4B] Same as above.

[0016] [Figure 5A] The data in Figures 5A-B compare the impact of continuous shake flask mixing, intermittent shake flask mixing, and continuous spinner flask mixing on the VCD and viability of pre-frozen CAR T drug products. [Figure 5B] Same as above. [Figure 5C] Figure 5C compares the VCD and viability results of pre-frozen CAR T cell drugs after continuous or intermittent mixing in spinner flasks. Continuous Shaker: Continuous mixing of formulated cells in Erlenmeyer shake flasks on an orbital shaker platform at 85 rpm; Continuous Spinner: Continuous mixing of formulated cells in spinner flasks on a magnetic stir plate at 40 rpm; Intermittent Shaker 10: Mixing of formulated cells in Erlenmeyer shake flasks on an orbital shaker platform at 85 rpm for 2 minutes every 10 minutes; Intermittent Shaker 30: Mixing of formulated cells in Erlenmeyer shake flasks on an orbital shaker platform at 85 rpm for 2 minutes every 30 minutes; and Intermittent Spinner: Mixing of formulated cells in spinner flasks on a magnetic stir plate at 40 rpm for 2 minutes every 10 minutes. Three samples were tested.

[0017] [Figure 6]Figure 6 shows the results of VCD and % cell viability as a function of loading time for pre-frozen or post-thawed CAR T cells subjected to different loading processes. Three samples were tested.

[0018] [Figure 7A] Figure 7A shows the VCD and cell viability % of CAR T cells in spinner flasks with continuous mixing at different mixing speeds. [Figure 7B] Figure 7B shows the VCD and % cell viability of CAR T cells in spinner flasks with a starting volume of 300 ml or 600 ml subjected to continuous spinner flask mixing at 50 rpm. Three samples were tested.

[0019] [Figure 8A] Figure 8A compares the results of continuous mixing in spinner flasks and intermittent mixing in shaker flasks over the course of 4 hours of pre-freezing CAR T cells. [Figure 8B] Figure 8B shows the VCD and % viability of post-thaw cells treated with either continuous spinner flask mixing or intermittent shake flask mixing. Three samples were tested. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] In one aspect, the disclosure provides formulations and methods for preparation and filling of pharmaceutical products. In one aspect, the disclosure provides pharmaceutical processes for cell-based therapeutics that allow sufficient time to finish filling or processing the manufactured cell-based pharmaceutical products while maintaining cell viability and / or pharmaceutical homogeneity. In some embodiments, the pharmaceutical products are GMP pharmaceutical products and / or are produced in large quantities.

[0021] As used herein, the terms "a" and "an" are used to mean one or more. For example, a reference to "a cell" or "an antibody" means "one or more cells" or "one or more antibodies."

[0022] A drug product generally, although not necessarily, refers to a final dosage form that contains a drug substance in combination with one or more other ingredients.

[0023] The pharmaceutical process involves the process in which the filled pharmaceutical product is stored, for example, in a frozen state, in liquid nitrogen, or in a 4 o C Refers to the final step in GMP manufacturing before storage or delivery for direct patient use.

[0024] Drug processing is important in GMP manufacturing of cell-based therapeutics. Drug processing involves taking cells produced from optimal cell culture conditions and subjecting the cells to stressful conditions. In current processes, cells are constantly mixed to ensure even distribution during drug processing. To ensure cell viability and homogeneity of the cell product, steps can be taken to minimize exposure of cells to stressful conditions to pass quality control. Controlling drug fill times to minimize exposure of cells to stressful conditions may result in unfilled drug product that cannot be processed within the fill time frame and unnecessary waste.

[0025] One source of stress is cell mixing during pharmaceutical processing. To keep the cells in suspension and prevent the cells from settling, formulated cells are continuously mixed during pharmaceutical processing. In shake flask mixing, formulated cells are placed in a shake flask, e.g., an Erlenmeyer flask, which is then placed on a shaker platform, such as a shaker or orbital shaker, to allow mixing. In contrast, spinner flasks include a mixing device, e.g., an impeller or paddle attached to the top with a stir bar, which allows for internal mixing of the contents within the spinner flask. Spinner flask mixing can occur when the spinner flask is placed on a magnetic stir plate and the paddle / stir bar is driven to rotate by magnetic forces. Spinner flask mixing can also occur by other means, e.g., driven by mechanically rotating impellers. Exemplary spinner flasks include, but are not limited to, Pyrex® ProCulture™ spinner flasks, Corning® spinner flasks.

[0026] In particular, it has been generally believed that shaking flask mixing is gentler on cells than spinner flask mixing because, unlike cells in spinner flasks, cells in shake flasks do not come into direct contact with mixing devices such as paddles. As disclosed herein, it has surprisingly been discovered that formulated cells maintain superior viability and viable cell density when continuous mixing is performed in a spinner flask compared to a shake flask.

[0027] Stress conditions in pharmaceutical processes can result in cell death and further cause cell clumps and aggregates, all of which can ultimately affect the quality, strength and potency of the pharmaceutical product. Thus, in some embodiments, the present disclosure provides a process for preparing a pharmaceutical product, which maintains cell health and homogeneity.

[0028] Thus, in one aspect, the disclosure provides a process for preparing a pharmaceutical product comprising engineered immune cells, the method comprising formulating the engineered immune cells in a composition containing a cryopreservative to form the pharmaceutical product, and mixing and filling the pharmaceutical product into one or more containers, wherein cellular health and homogeneity of the pharmaceutical product is maintained.

[0029] Homogeneity of a cell-based pharmaceutical as used herein refers to a state in which the formulated cells are homogeneously suspended in the composition without the cells settling, clumping, or aggregation. Maintaining homogeneity during the DP process of a cell-based pharmaceutical is important to ensure vial-to-vial consistency in cell viability and dose concentration. Homogeneity can be assessed by visual inspection. Homogeneity can also be confirmed by measuring the cell concentration in each container filled with the cell-based pharmaceutical to ensure consistency.

[0030] In some embodiments, the continuous mixing is performed at about 30-80 rpm, about 30-85 rpm, about 25-85 rpm, about 35-80 rpm, about 35-85 rpm, about 40-80 rpm, about 40-85 rpm, about 45-80 rpm, about 45-85 rpm, about 50-80 rpm, about 50-85 rpm, about 55-80 rpm, about 55-85 rpm, about 60-80 rpm, about 60-85 rpm, about 65-80 rpm, about 65-85 rpm, about 70-80 rpm, about 70-85 rpm, about 75-80 rpm, about 75-85 rpm, about 25 rpm, about 30 rpm, about 35 rpm, about 40 rpm, about 45 rpm, about 50 rpm, about 55 rpm, about 60 rpm, about 65 rpm, about 70 rpm, about 75 rpm, about 80 rpm, or about 85 rpm.

[0031] In some embodiments, the cells are engineered immune cells. In some embodiments, the immune cells are CAR T cells. In some embodiments, the immune cells are CAR NK cells. In some embodiments, the immune cells are allogeneic or autologous immune cells. In some embodiments, the immune cells are autologous or allogeneic CAR T cells.

[0032] Traditionally, it has been believed that cells need to be continuously mixed during pharmaceutical processing to prevent settling. As disclosed herein, it has been unexpectedly discovered that continuous mixing during pharmaceutical processing is not required. Thus, in one aspect, the present disclosure provides a pharmaceutical process that includes intermittent mixing of cells. In some embodiments, the intermittent mixing is performed in a shake flask. In certain embodiments, the intermittent mixing is performed in a spinner flask.

[0033] In some embodiments, the intermittent mixing is performed at about 30-80 rpm, about 30-85 rpm, about 25-85 rpm, about 35-80 rpm, about 35-85 rpm, about 40-80 rpm, about 40-85 rpm, about 45-80 rpm, about 45-85 rpm, about 50-80 rpm, about 50-85 rpm, about 55-80 rpm, about 55-85 rpm, about 60-80 rpm, about 60-85 rpm, about 65-80 rpm, about 65-85 rpm, about 70-80 rpm, about 70-85 rpm, about 75-80 rpm, about 75-85 rpm, about 85-100 rpm, The rotation speed is about 85-110 rpm, about 85-120 rpm, about 85-130 rpm, about 85-140 rpm, about 25 rpm, about 30 rpm, about 35 rpm, about 40 rpm, about 45 rpm, about 50 rpm, about 55 rpm, about 60 rpm, about 65 rpm, about 70 rpm, about 75 rpm, about 80 rpm, about 85 rpm, about 90 rpm, about 95 rpm, about 100 rpm, about 105 rpm, about 110 rpm, about 115 rpm, about 120 rpm, about 125 rpm, about 130 rpm, about 135 rpm, about 140 rpm, or about 145 rpm.

[0034] In some embodiments, the cell is an engineered immune cell, hi some embodiments, the immune cell is a CAR T cell.

[0035] In some embodiments, the intermittent mixing is performed at intervals of about 1 to 5 minutes, 1 to 4 minutes, 1 to 3 minutes, 1 to 2 minutes, 1 minute, or less than 1 minute. In some embodiments, the intermittent mixing is performed at intervals of about 10 to about 40 minutes. In some embodiments, the intermittent mixing is performed at intervals of about every 10 to about 40 minutes, about every 10 to about 35 minutes, about every 10 to about 30 minutes, about every 10 to about 25 minutes, about every 10 to about 20 minutes, about every 10 to about 15 minutes, about every 10 to about 45 minutes, about every 5 to about 40 minutes, about every 4 minutes, about every 10 minutes, about every 15 minutes, about every 20 minutes, about every 25 minutes, about every 30 minutes, about every 35 minutes, about every 40 minutes, or about every 45 minutes.

[0036] In some embodiments, the one or more containers are sterile plastic or glass containers. In some embodiments, the one or more containers are plastic vials. In some embodiments, the one or more containers are glass vials. In some embodiments, the one or more containers are plastic bags.

[0037] In some embodiments, the cells are formulated in a composition comprising at least one cryopreservative (or cryoprotectant). Suitable cryopreservatives include, but are not limited to, dimethylsulfoxide (DMSO), propylene glycol (PG), ethylene glycol (EG), glycerol, sucrose, and trehalose. In some embodiments, the cryopreservative present in the formulation or pharmaceutical product is about 3% to about 10%, about 4% to about 10%, about 5% to about 10%, about 6% to about 10%, about 7% to about 10%, about 8% to about 10%, about 9% to about 10%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%. In some embodiments, the cryopreservative is DMSO. In some embodiments, the composition comprising the cryopreservative is, for example, Cryostor® CS10, CS2, CS5, CryoMACS DMSO, or 2-8CELLsius™+DMSO.

[0038] In some embodiments, the formulation further comprises an excipient that enhances viscosity (or viscosity enhancer). Suitable viscosity enhancers include, but are not limited to, cellulose (e.g., methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose), pectin, high molecular weight dextrans (e.g., MW about 40 KDa to about 100 KDa, etc.), gelatin, hydroxyethyl starch, alginic acid, polyvinyl alcohol, polyvinylpyrrolidone, and other natural, semi-synthetic, or synthetic hydrocolloids. In some embodiments, the viscosity enhancer is present in the formulation at a concentration of about 0.1% to about 10%, about 0.1% to about 9%, about 0.1% to about 8%, about 0.1% to about 7%, about 0.1% to about 6%, about 0.1% to about 5%, about 0.1% to about 4%, about 0.1% to about 3%, about 0.1% to about 2%, or about 0.1% to about 1%. In some embodiments, the formulation has a viscosity of about 2 to about 20 cps, about 2 to about 19 cps, about 2 to about 18 cps, about 2 to about 17 cps, about 2 to about 16 cps, about 2 to about 15 cps, about 2 to about 14 cps, about 2 to about 13 cps, about 2 to about 12 cps, about 2 to about 11 cps, about 2 to about 10 cps, about 2 to about 9 cps, about 2 to about 8 cps, about 2 to about 7 cps, about 2 to about 6 cps, or about 2 to about 5 cps.

[0039] In some embodiments, the mixing and filling step takes about 2 hours. In some embodiments, the mixing and filling step takes less than about 2 hours. In some embodiments, the mixing and filling step takes at least about 2 hours. In some embodiments, the mixing and filling step takes about 2 hours or more. In some embodiments, the mixing and filling step takes about 3 hours, 3 hours or more, about 4 hours, 4 hours or more, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 2 hours to about 3 hours, about 2 hours to about 4 hours, about 2 hours to about 5 hours, about 2 hours to about 6 hours, about 3 hours to about 6 hours, or about 4 hours to about 6 hours.

[0040] In some embodiments, the mixing and filling steps are carried out at room temperature. oC, about 20~25 o C, about 20~27 o C, about 20~30 o C, or about 25-30 o In some embodiments, the mixing and filling step is carried out at about 2 to 8 o In some embodiments, the mixing and filling step is carried out at about 4 to 5 o It is carried out in C.

[0041] In some embodiments, the pharmaceutical process includes mixing the formulated cells by continuous shake flask mixing at room temperature for up to about 2 hours. In some embodiments, the pharmaceutical process includes mixing the formulated cells by intermittent shake flask mixing at room temperature for up to about 4 to about 6 hours. In some embodiments, the speed of the continuous or intermittent shake flask mixing is about 85 rpm or less. In some embodiments, the intermittent shake flask mixing is for about 1 to 3 minutes about every 10 to 30 minutes. In some embodiments, the pharmaceutical process includes mixing the formulated cells by continuous spinner flask mixing at room temperature for up to about 4 to about 6 hours. In some embodiments, the pharmaceutical process includes mixing the formulated cells by intermittent spinner flask mixing at room temperature for up to about 4 to about 6 hours. In some embodiments, the speed of the continuous or intermittent spinner flask mixing is about 40 rpm or less. In some embodiments, the speed of the continuous or intermittent spinner flask mixing is about 40 rpm or less. In some embodiments, the intermittent spinner flask mixing is for about 1-3 minutes about every 10-30 minutes.

[0042] In some embodiments, the cells are engineered immune cells. In some embodiments, the immune cells are CAR T cells. In some embodiments, the immune cells are CAR NK cells. In some embodiments, the immune cells are allogeneic or autologous immune cells. In some embodiments, the immune cells are autologous or allogeneic CAR T cells.

[0043] immune cells

[0044] Cells suitable for use with the methods and / or reagents described herein include immune cells.

[0045] Prior to in vitro manipulation or genetic modification (such as those described herein), cells for use in the methods described herein (e.g., immune cells) can be obtained from a subject. 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, stem cell or iPSC-derived immune cells, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. 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 an infectious disease. In some embodiments, cells can be part of a mixed population of cells exhibiting different phenotypic characteristics.

[0046] In some embodiments, the immune cells are autologous immune cells obtained from the subject that will ultimately receive the engineered immune cells, hi some embodiments, the immune cells are allogeneic immune cells obtained from a donor that is a different individual than the subject that will ultimately receive the engineered immune cells.

[0047] In some embodiments, the immune cells include T cells. T cells can be obtained from several sources, including peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, stem cell or iPSC-derived T cells, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumor. In some embodiments, T cells can be obtained from a volume of blood drawn from a subject using any number of techniques known to those skilled in the art, such as FICOLL™ separation.

[0048] Cells 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 some embodiments, the cells collected by apheresis can be washed to remove plasma fraction and placed in a suitable buffer or medium for subsequent processing.

[0049] PBMCs can be used directly for genetic modification with immune cells (such as CAR or TCR) using the methods described herein. 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 before or after genetic modification and / or expansion.

[0050] 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, such as CCR7+, CD95+, CD122, CD27+, CD69+, CD127+, CD28+, CD3+, CD4+, CD8+, CD25+, CD62L+, CD45RA+, and CD45RO+ T cells, can be further isolated by positive or negative selection techniques known in the art. For example, enrichment of T cell populations 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, a monoclonal antibody cocktail typically includes antibodies to 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 present disclosure.

[0051] In some embodiments, the T cell population is enriched for CD4+ cells.

[0052] In some embodiments, the T cell population is enriched for CD8+ cells.

[0053] In some embodiments, the CD8+ cells are further sorted into naive cells, central memory cells, and effector cells by identifying cell surface antigens associated with each of these types of cells. In some embodiments, the expression of phenotypic markers for naive T cells includes CD45RA+, CD95-, IL2Rβ-, CCR7+, and CD62L+. In some embodiments, the expression of phenotypic markers for stem cell memory T cells includes CD45RA+, CD95+, IL2Rβ+, CCR7+, and CD62L+. In some embodiments, the expression of phenotypic markers for central memory T cells includes CD45RO+, CD95+, IL2Rβ+, CCR7+, and CD62L+. In some embodiments, the expression of phenotypic markers for effector memory T cells includes CD45RO+, CD95+, IL2Rβ+, CCR7-, and CD62L-. In some embodiments, expression of phenotypic markers for T effector cells includes CD45RA+, CD95+, IL2Rβ+, CCR7-, and CD62L-. For example, CD4+ and / or CD8+ T helper cells can be sorted into naive cells, stem cell memory cells, central memory cells, effector memory cells, and T effector cells by identifying cell populations with cell surface antigens.

[0054] It will be understood that PBMCs may further include other cytotoxic lymphocytes, such as NK cells or NKT cells. An expression vector carrying the coding sequence of the chimeric receptor disclosed herein may be introduced into a population of human donor T cells, NK cells, or NKT cells. Standard procedures are used for cryopreservation of T cells expressing CAR for storage and / or preparation for use in human subjects. In one embodiment, 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. In various embodiments, the cryopreservation medium may include, for example, CryoStor® CS2, CS5, or CS10 or other medium containing DMSO, or medium without DMSO.

[0055] Engineered immune cells

[0056] Provided herein are engineered immune cells that express a CAR of the disclosure (e.g., CAR-T cells).

[0057] In some embodiments, the engineered immune cells comprise a population of CARs, each CAR comprising an extracellular antigen binding domain. In some embodiments, the engineered immune cells 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 an extracellular antigen binding domain.

[0058] The engineered immune cells can be allogeneic or autologous.

[0059] In some embodiments, the engineered immune cell is a T cell (e.g., an inflammatory T lymphocyte cytotoxic T lymphocyte, a regulatory T lymphocyte, a helper T lymphocyte, or a tumor infiltrating lymphocyte (TIL)), a NK cell, a NK-T cell, a TCR expressing cell, a dendritic cell, a killer dendritic cell, a mast cell, or a B cell. In some embodiments, the cell can originate from the group consisting 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 an alpha beta 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 embodiments, the engineered immune cell is a human cell.

[0060] In some embodiments, the engineered immune cells may be derived from, for example, but not limited to, stem cells.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 (iPSCs), totipotent stem cells, or hematopoietic stem cells.Stem cells may be CD34+ or CD34-.

[0061] 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. In some embodiments, the cells are transfected or transduced with a nucleic acid vector using a method selected from the group consisting of electroporation, sonoporation, biolistics (e.g., Gene Gun), transfection, lipid transfection, polymer transfection, nanoparticles, viral transduction or viral transfection (e.g., retrovirus, lentivirus, AAV) or polyplex. In some embodiments, the cells are T cells reprogrammed from non-T cells. In some embodiments, the cells are T cells reprogrammed from T cells.

[0062] Binder

[0063] In embodiments, the disclosed methods include the use of an antibody or antigen-binding agent (e.g., comprising an antigen-binding domain or comprising an antibody or fragment thereof). As discussed below, in various embodiments, the engineered immune cells may also include a binding agent.

[0064] As used herein, the term "antibody" refers to a polypeptide that contains sufficient standard immunoglobulin sequence elements to confer specific binding to a particular target antigen. As is known in the art, naturally produced intact antibodies are tetrameric agents of approximately 150 kD, containing two identical heavy chain polypeptides (about 50 kD each) and two identical light chain polypeptides (about 25 kD each) that bind to what is commonly referred to as a "Y-shaped" structure. Each heavy chain consists of at least four domains (each about 110 amino acids long) - an amino-terminal variable (VH) domain (located at the tip of the Y structure), followed by three constant domains, CHI, CH2, and carboxy-terminal CH3 (located at the base of the stem of the Y). A short region known as the "switch" connects the heavy chain variable and constant regions. A "hinge" connects the CH2 and CH3 domains to the rest of the antibody. Two disulfide bonds in this hinge region bind the two heavy chain polypeptides to each other in an intact antibody. Each light chain is composed of two domains, an amino-terminal variable (VL) domain followed by a carboxy-terminal constant (CL) domain. Those of skill in the art are familiar with antibody structure and sequence elements and will recognize "variable" and "constant" regions in the sequences provided, and will understand that there may be some flexibility in the definition of the "boundaries" between such domains, such that different representations of the same antibody chain sequence may exhibit such boundaries at positions that are shifted by, for example, one or a few residues relative to different representations of the same antibody chain sequence.

[0065] An intact antibody tetramer comprises two heavy-light chain dimers, where the heavy and light chains are linked together by one disulfide bond, and two other disulfide bonds link the heavy chain hinge regions together, thus linking the dimers together to form a tetramer. Naturally produced antibodies are also glycosylated, typically on the CH2 domain. Each domain in a natural antibody has a structure characterized by an "immunoglobulin fold" formed from two beta sheets (e.g., three, four, or five stranded sheets) packed together in a compressed antiparallel beta barrel. Each variable domain comprises three hypervariable loops known as "complement determining regions" (CDR1, CDR2, and CDR3), and four somewhat invariant "framework" regions (FR1, FR2, FR3, and FR4). When a natural antibody folds, the FR regions form beta sheets that provide a structural framework for the domain, and the CDR loop regions from both the heavy and light chains are brought together in three-dimensional space to generate a single hypervariable antigen-binding site located at the tip of a Y structure. The Fc region of a naturally occurring antibody binds to elements of the complement system and also to receptors on effector cells, including effector cells that mediate cytotoxicity, for example. As is known in the art, the affinity and / or other binding properties of the Fc region for the Fc receptor can be modulated through glycosylation or other modifications. In some embodiments, the antibodies produced and / or utilized according to the present invention comprise a glycosylated Fc domain, including Fc domains with modified or engineered such glycosylation.

[0066] For purposes of this disclosure, in certain embodiments, a polypeptide or complex of polypeptides that includes sufficient immunoglobulin domain sequences found in a natural antibody may be referred to and / or used as an "antibody," regardless of whether such a polypeptide is produced naturally (e.g., produced by an organism in response to an antigen) or produced by recombinant engineering, chemical synthesis, or other artificial systems or methodologies. In some embodiments, an antibody is polyclonal, and in some embodiments, an antibody is monoclonal. In some embodiments, an antibody has constant region sequences characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, the antibody sequence elements are humanized, primatized, chimeric, etc., as known in the art.

[0067] Furthermore, as used herein, the term "antibody" is used to refer to any construct or format known or developed in the art for utilizing the structural and functional features of antibodies in alternative presentations. For example, in some embodiments, antibodies utilized in accordance with the present disclosure may be any of a variety of antibodies, including, but not limited to, intact IgA, IgG, IgE or IgM antibodies, bispecific or multispecific antibodies (e.g., Zybodies®, etc.), antibody fragments (such as Fab fragments, Fab fragments, F(ab)2 fragments, Fd fragments, and isolated CDRs or sets thereof), single chain variable fragments (scFVs), polypeptide-Fc fusions, single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof), camelid antibodies (also referred to herein as nanobodies or VHHs), shark antibodies, masked antibodies (e.g., Probodies®), Small Modular Antibodies (SMDs), and / or other antibodies. ImmunoPharmaceuticals (SMIPs™), single chain or tandem diabodies (TandAb®), VHH, Anticalins®, Nanobodies® minibodies, BiTE®, ankyrin repeat proteins or DARPINs®, Avimers®, DART, TCR-like antibodies, Adnectins®, Affilins®, Trans-bodies®, Affibodies®, TrimerX®, MicroProteins; Fynomers®, Centyrins®, and KALBITOR®. In some embodiments, the antibody may lack covalent modifications (e.g., glycan attachments) that it has when produced in nature. In some embodiments, the antibodies may contain covalent modifications (e.g., attachment of a glycan, a payload (e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.), or other pendant groups (e.g., polyethylene glycol, etc.).

[0068] As used herein, the term "antibody agent" generally refers to an agent that specifically binds to a particular antigen. In some embodiments, the term encompasses any polypeptide or polypeptide complex that contains sufficient immunoglobulin structural elements to confer specific binding. Exemplary antibody agents include, but are not limited to, monoclonal or polyclonal antibodies. In some embodiments, an antibody agent may include one or more constant region sequences characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, an antibody agent may include one or more sequence elements that are humanized, primatized, chimeric, etc., as known in the art. In many embodiments, the term "antibody agent" is used to refer to one or more of the constructs or formats known or developed in the art to utilize the structural and functional features of antibodies in alternative presentations. For example, antibody agents utilized in accordance with the present invention include, but are not limited to, intact IgA, IgG, IgE or IgM antibodies, bispecific or multispecific antibodies (e.g., Zybodies®, etc.), antibody fragments (such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd fragments, and isolated CDRs or sets thereof), single chain Fvs, polypeptide-Fc fusions, single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof), camelid antibodies, masked antibodies (e.g., Probodies®), Small Modular ImmunoPharmaceuticals (SMIPs™), single chain or tandem diabodies (TandAb®), VHHs, Anticalins®, Nanobodies® minibodies, BiTEs®, ankyrin repeat proteins or DARPINs®, Avimers®, DART, TCR-like antibodies, Adnectins®, Affilins®, Trans-bodies®, Affibodies®, TrimerX®, MicroProteins, Fynomers®, Centyrins®, and KALBITOR®.

[0069] The antibody or antibody agent used to carry out the method of the present disclosure may be single-chain or double-chain. In some embodiments, the antibody or antigen-binding molecule is single-chain. In certain embodiments, the antigen-binding molecule is selected from the group consisting of scFv, Fab, Fab', Fv, F(ab')2, dAb, and any combination thereof.

[0070] Antibodies and antibody agents include antibody fragments. Antibody fragments include portions of intact antibodies, such as, for example, the antigen-binding or variable regions of intact antibodies. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, diabodies, linear antibodies, multispecifics formed from antibody fragments, antibodies, and scFv fragments, and other fragments. Antibodies also include, but are not limited to, polyclonal, monoclonal, chimeric dAb (domain antibodies), single chain, Fab, Fa, F(ab)2 fragments, scFv, and scFv. Antibodies can be whole antibodies, or immunoglobulins, or antibody fragments. Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies, and production by recombinant host cells (e.g., E. coli, Chinese Hamster Ovary (CHO) cells, or phage), as known in the art.

[0071] In some embodiments, the antibody or antibody agent may be a chimeric antibody (see, e.g., U.S. Pat. No. 4,816,567, and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). A chimeric antibody may be an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remaining portions of the heavy and / or light chain are derived from a different source or species. In one example, a chimeric antibody may include a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or a non-human primate, such as a monkey) and a human constant region. In a further example, a chimeric antibody may be a "class-switched" antibody in which the class or subclass has been changed from that of the parent antibody. A chimeric antibody includes an antigen-binding fragment thereof.

[0072] In some embodiments, a chimeric antibody can be a humanized antibody (see, e.g., Almagro and Fransson, Front. Biosci., 13:1619-1633 (2008); Riechmann et al., Nature, 332:323-329 (1988); Queen et al., Proc. Natl Acad. Sci. USA 86:10029-10033 (1989); U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005); Padlan, Mol. Immunol, 28:489-498 (1991); Dall'Acqua et al., Methods 36:25-34 (2005); (see Osbourn et al., Methods, 36:43-60 (2005); Osbourn et al., Methods, 36:61-68 (2005); and Klimka et al., Br. J. Cancer, 83:252-260 (2000)). Humanized antibodies are chimeric antibodies that contain amino acid residues from a non-human hypervariable region and amino acid residues from a human FR. In certain embodiments, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, with all or substantially all of the hypervariable regions (e.g., CDRs) corresponding to those of a non-human antibody and all or substantially all of the framework regions (FRs) corresponding to those of a human antibody. A humanized antibody may optionally contain at least a portion of an antibody constant region derived from a human antibody.

[0073] In some embodiments, the antibody or antibody agent provided herein is a human antibody. Human antibodies can be produced using various techniques known in the art (see, for example, van Dijk and van de Winkel, Curr. Opin. Pharmacol, 5: 368-74 (2001); and Lonberg, Curr. Opin. Immunol, 20: 450-459 (2008)). Human antibodies may have amino acid sequences that correspond to the amino acid sequences of antibodies produced by humans or human cells, or derived from non-human sources that utilize human antibody repertoires or other human antibody coding sequences. This definition of human antibodies specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies may be prepared using methods well known in the art.

[0074] Chimeric Antigen Receptor

[0075] 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 Krause et al., J. Exp. Med., Volume 188, No. 4, 1998 (619-626); Finney et al., Journal of Immunology, 1998, 161:2791-2797; Song et al., Blood 119:696-706 (2012); Kalos 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.

[0076] 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 that specifically binds to a target.

[0077] In some embodiments, the antigen-specific CAR further comprises a safety switch and / or one or more monoclonal antibody specific epitopes.

[0078] i. 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 specified target antigen. In some embodiments, the antigen-binding domain binds to an antigen on tumor cells. In some embodiments, the antigen-binding domain binds to an antigen on cells involved in hyperproliferative disease.

[0079] In some embodiments, the antigen-binding domain comprises a variable heavy chain, a variable light chain, and / or one or more CDRs described herein. In some embodiments, the antigen-binding domain is a single-chain variable fragment (scFv) comprising CDR1, CDR2, and CDR3 of the light chain CDRs, and CDR1, CDR2, and CDR3 of the heavy chain CDRs.

[0080] An antigen-binding domain is said to be "selective" if it binds to one target more strongly or with higher affinity than it binds to a second target.

[0081] The antigen binding domain of the CAR selectively targets a cancer antigen. In some embodiments, the cancer antigen is selected from EGFRvIII, WT-1, CD20, CD23, CD30, CD38, CD33, CD133, MHC-WT1, TSPAN10, MHC-PRAME, Liv1, ADAM10, CHRNA2, LeY, NKGD2D, CS1, CD44v6, ROR1, claudin-18.2, Muc17, FAP alpha, Ly6G6D, c6orf23, G6D, MEGT1, NG25, CD19, BCMA, FLT3, CD70, DLL3, CD52, or CD34. In some embodiments, the CAR comprises an antigen binding domain targeted to EGFRvIII, WT-1, CD20, CD23, CD30, CD38, CD33, CD133, MHC-WT1, TSPAN10, MHC-PRAME, Liv1, ADAM10, CHRNA2, LeY, NKGD2D, CS1, CD44v6, ROR1, claudin-18.2, Muc17, FAP alpha, Ly6G6D, c6orf23, G6D, MEGT1, NG25, CD19, BCMA, FLT3, CD70, DLL3, CD52, or CD34.

[0082] In some embodiments, the cancer antigen is carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CDS, CD7, CDIO, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD123, CD133, CD138, an antigen of a cytomegalovirus (CMV)-infected cell (e.g., a cell surface antigen), epithelial glycoprotein (EGP), or a combination thereof. 2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine-protein kinase erb-B2,3,4, folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER-2), human telomerase reverse transcriptase (hTERT), interleukin-13 receptor subunit alpha-2 (IL-13Ra2), kappa-light chain, kinase insert domain receptor (KDR), Lewis A (CA19.9), LI cell adhesion molecule (LICAM), melanoma antigen family A, 1 (MAGE-AI), mucin 16 (Muc-16), mucin 1 (Muc-1), mesothelin (MSLN), NKG2D ligand, cancer-testis antigen NY-ESO-1, oncofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), tumor-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF-R2), and Wilms tumor protein (WT-1).

[0083] 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 having 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.

[0084] In some embodiments, the polypeptide structure of the antigen-binding domain is based on an antibody, including, but not limited to, a monoclonal antibody, a bispecific antibody, a minibody, a domain antibody, a synthetic antibody (sometimes referred to herein as an "antibody mimetic"), a chimeric antibody, a humanized antibody, a human antibody, an antibody fusion (sometimes referred to herein as an "antibody conjugate"), and fragments thereof, respectively. In some embodiments, the antigen-binding domain comprises or consists of an avimer.

[0085] In some embodiments, the antigen-binding domain is an scFv. In some embodiments, the antigen-selective CAR comprises a leader or signal peptide.

[0086] 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.

[0087] 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.

[0088] In some embodiments, a CAR immune cell (e.g., a CAR-T cell) that can form a component of a population of cells generated by carrying out the method of the present disclosure comprises a polynucleotide encoding a safety switch polypeptide, such as, for example, RQR8. See, for example, WO2013153391A, which is incorporated herein by reference in its entirety. In a CAR immune cell (e.g., a CAR-T cell) that comprises a polynucleotide, the safety switch polypeptide can be expressed on the surface of the CAR immune cell (e.g., a CAR-T cell).

[0089] ii. Hinge domain The extracellular domain of the CAR of the present disclosure may comprise a "hinge" domain (or hinge region). This term generally refers to 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.

[0090] 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 originate from all or a portion of a naturally occurring molecule derived from CD8, CD4, CD28, 4-1BB, or the extracellular region of IgG (particularly, it is understood that it may comprise all or a portion of the hinge region of IgG, i.e., some or all of the members of the immunoglobulins such as IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, or fragments thereof) or all or a portion of an antibody heavy chain constant region. Alternatively, the hinge domain may be a synthetic sequence that corresponds to a naturally occurring hinge sequence, or may be a completely synthetic hinge sequence. In some embodiments, the hinge domain is a portion of the human CD8 alpha chain (e.g., NP_001139345.1). In other embodiments, the hinge domain and the transmembrane domain comprise a portion of the human CD8 alpha chain. In some embodiments, the hinge domain of the CAR described herein comprises a subsequence of CD8α, IgG1, IgG4, PD-1, or FcγRIIIα, in particular the hinge region of any of CD8α, IgG1, IgG4, PD-1, or FcγRIIIα. In some embodiments, the hinge domain comprises a human CD8α hinge, a human IgG1 hinge, a human IgG4, a human PD-1, or a human FcγRIIIα hinge. In some embodiments, the CAR disclosed herein comprises an scFv, a CD8α human hinge and transmembrane domain, a CD3ζ signaling domain, and a 4-1BB signaling domain.

[0091] iii. Transmembrane domain The CAR of the present disclosure is designed with a transmembrane domain fused to the extracellular domain of the CAR. This can also be fused to the intracellular domain of the CAR. In some cases, the transmembrane domain can be selected or modified by amino acid substitution to avoid the binding of such domain to the transmembrane domain of the same or different surface membrane protein and minimize the 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.

[0092] Suitable transmembrane domains of the CARs disclosed herein include (a) transmembrane domains that are capable of translocating, for example, T helper (T h ) cells, cytotoxic T (T c ) cells, T regulatory (T reg ) the ability to be expressed on the surface of an immune cell, such as a target cell, or a lymphocyte cell, such as a natural killer (NK) cell, and / or (b) the ability to interact with an extracellular antigen-binding domain and an intracellular signaling domain to induce a cellular response of the immune cell to direct a cellular response of the immune cell against a target cell.

[0093] 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.

[0094] Transmembrane regions of particular use in the present disclosure include those derived from CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function associated antigen-1 (LFA-1, CD1-1a / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class 1 molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, insulin, insulin receptor ... Tegrin, 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 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE, CD103, ITGAL, CD1 1a, LFA-1, ITGAM, CD1 1b, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT and a ligand that specifically binds to AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83, or any combination thereof.

[0095] As non-limiting examples, the transmembrane region may be derived from, for example, the α, β, γ or δ polypeptides constituting the CD3 complex, the IL-2 receptor p55 (α chain), p75 (β chain) or γ chain, a subunit chain of an Fc receptor, or may be part of a T cell receptor, particularly in the Fcγ receptor III or 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 CD8 α chain (e.g., NP_001139345.1).

[0096] In some embodiments, the transmembrane domain in a CAR of the present disclosure is a CD8α transmembrane domain.

[0097] In some embodiments, the transmembrane domain in a CAR of the present disclosure is a CD28 transmembrane domain.

[0098] iv. Intracellular domain The intracellular (cytoplasmic) domain of the CAR of the present disclosure can provide for activation of at least one normal effector function of an immune cell that contains a CAR. Effector function of a T cell can refer to, for example, cytolytic activity, including secretion of cytokines, or helper activity.

[0099] In some embodiments, an activating intracellular signaling domain for use in a CAR may be, for example, but is not limited to, the cytoplasmic sequences of the T cell receptor and co-receptors that act in concert to initiate signaling following antigen receptor engagement, as well as any derivatives or variants of these sequences, and any synthetic sequences that have the same function.

[0100] Suitable (e.g., activation) intracellular domains include, but are not limited to, CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function associated antigen-1 (LFA-1, CD1-1a / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class 1 molecule, TNF receptor protein, immunoglobulin protein, cytokines, 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 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE, CD103, ITGAL, CD1 1a, LFA-1, ITGAM, CD1 1b, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT The signaling domains include those derived from (or corresponding to) a ligand that specifically binds to AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83, or any combination thereof.

[0101] In addition to the activation domains described above, the intracellular domains of the CARs of the present disclosure can incorporate costimulatory signaling domains (interchangeably referred to herein as costimulatory molecules) to increase their efficacy. The costimulatory domains can provide signals in addition to the primary signals provided by the activation molecules described herein.

[0102] 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, CD5, CD7, CD9, CD16, CD22, CD27, CD30, CD33, CD37, CD40, CD 45, CD64, CD80, CD86, CD134, CD137, CD154, PD-1, ICOS, lymphocyte function associated antigen-1 (LFA-1 (CD1 1a / CD18), CD247, CD276 (B7-H3), LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc 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 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD 8beta, IL-2Rbeta, IL-2Rgamma, IL-7Ralpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1-1d, ITGAE, CD103, ITGAL, CD1-1a, LFA-1, ITGAM, CD1 -1b, ITGAX, CD1-1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT It is understood that the co-stimulatory molecules may be derived from AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, 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 co-stimulatory molecules or fragments thereof not listed above are within the scope of the present disclosure.

[0103] In some embodiments, the intracellular / cytoplasmic domain of the CAR may be designed to include the 4-1BB / CD137 domain by itself or in combination with any other desired intracellular domain useful in the context of the CAR of the present disclosure. 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.

[0104] 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 of the present disclosure. 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.

[0105] In some embodiments, the intracellular / cytoplasmic domain of the CAR may be designed to include a CD3 zeta domain by itself or in combination with any other desired intracellular domain useful in the context of the CARs of the present disclosure.

[0106] For example, the intracellular domain of the CAR may comprise a portion of a CD3 zeta chain and a portion of a costimulatory signaling molecule. The intracellular signaling sequences in the intracellular signaling portion of the CAR of the present disclosure can be linked together randomly or in a specific order. In some embodiments, the intracellular domain is designed to comprise the activation domain of CD3 zeta and the signaling domain of CD28. In some embodiments, the intracellular domain is designed to comprise the activation domain of CD3 zeta and the signaling domain of 4-1BB.

[0107] In some embodiments, the intracellular signaling domain of a CAR of the present disclosure comprises a domain of a costimulatory molecule. In some embodiments, the intracellular signaling domain of a CAR of the present disclosure comprises a portion of a costimulatory molecule selected from the group consisting of a fragment of 4-1BB (GenBank: AAA53133) and CD28 (NP_006130.1).

[0108] Genetic modification of CAR T cells

[0109] Also provided herein are engineered immune cells and populations of engineered immune cells that express a CAR (e.g., CAR-T cells or CAR+ cells), which are depleted of cells expressing an endogenous TCR.

[0110] In some embodiments, the engineered immune cells comprise CAR T cells, each CAR T cell comprises an extracellular antigen binding domain and has reduced or ablated expression of an endogenous TCR. In some embodiments, the population of engineered immune cells comprises a population of CAR T cells, each CAR T cell comprises two or more different extracellular antigen binding domains and has reduced or ablated expression of an endogenous TCR. In some embodiments, the immune cells comprise a population of CARs, each CAR T cell comprises the same extracellular antigen binding domain and has reduced or ablated expression of an endogenous TCR.

[0111] 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, SIGLEC10, 2B4, HLA, TCRα, and TCRβ, and / or express a CAR, a multi-chain CAR, and / or a pTα transgene. In some embodiments, the isolated cells comprise a polynucleotide encoding a polypeptide comprising a multi-chain CAR. In some embodiments, the isolated cells according to the disclosure comprise CD52 and GR, CD52 and TCR alpha, CDR52 and TCR beta, DLL3 and CD52, DLL3 and TCR alpha, DLL3 and TCR beta, GR and TCR alpha, GR and TCR beta, TCR alpha and TCR beta, PD-1 and TCR alpha, PD-1 and TCR beta, CTLA-4 and TCR alpha, CTLA-4 and TCR beta, LAG3 and TCR alpha, LAG3 and TCR beta, TIM3 and The cell comprises two disrupted or inactivated genes selected from the group consisting of TCRα, Tim3 and TCRβ, BTLA and TCRα, BTLA and TCRβ, BY55 and TCRα, BY55 and TCRβ, TIGIT and TCRα, TIGIT and TCRβ, B7H5 and TCRα, B7H5 and TCRβ, LAIR1 and TCRα, LAIR1 and TCRβ, SIGLEC10 and TCRα, SIGLEC10 and TCRβ, 2B4 and TCRα, 2B4 and TCRβ, and / or expresses a CAR (including a multi-chain CAR and / or a pTα transgene). In some embodiments, the method comprises disrupting or inactivating one or more genes by introducing into the cell an endonuclease capable of selectively inactivating the gene by selective 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, or TALEN®), or a CRISPR (e.g., Cas9 or Cas12) endonuclease.

[0112] In some embodiments, the TCR is not functional in the cells according to the present disclosure by disrupting or inactivating the TCR alpha and / or TCR beta genes. In some embodiments, a method of obtaining modified cells from an individual is provided, wherein the cells are capable of growing independently of the major histocompatibility complex (MHC) signaling pathway. Modified cells capable of growing independently of the MHC signaling pathway are amenable to being obtained by the present method and are therefore encompassed within the scope of the present disclosure. The modified cells disclosed herein can be used to treat a patient in need thereof against host-versus-graft (HvG) rejection and graft-versus-host disease (GvHD), and thus, within the scope of the present disclosure, a method of treating a patient in need thereof against host-versus-graft (HvG) rejection and graft-versus-host disease (GvHD) comprises administering to said patient an effective amount of modified cells comprising disrupted or inactivated TCR alpha and / or TCR beta genes to said patient to treat said patient.

[0113] The present disclosure provides a method for determining the purity of a population of engineered immune cells that lack or have reduced endogenous TCR expression.In some embodiments, the engineered immune cells comprise less than 5.0%, less than 4.0%, less than 3.0% TCR+ cells, less than 2.0% TCR+ cells, less than 1.0% TCR+ cells, less than 0.9% TCR+ cells, less than 0.8% TCR+ cells, less than 0.7% TCR+ cells, less than 0.6% TCR+ cells, less than 0.5% TCR+ cells, less than 0.4% TCR+ cells, less than 0.3% TCR+ cells, less than 0.2% TCR+ cells, less than 0.1% TCR+ cells.Such populations can be the product of the method of the present disclosure.

[0114] In some embodiments, engineered immune cells according to the present disclosure may contain one or more disrupted or inactivated genes. In some embodiments, target antigens (e.g., EGFRvIII, Flt3, WT-1, CD20, CD23, CD30, CD38, CD33, CD133, MHC-WT1, TSPAN10, MHC-PRAME, Liv1, ADAM10, CHRNA2, LeY, NKGD2D, CS1, CD44v6, ROR1, claudin-18.2, Muc17, FAP alpha, Ly6G6D, c6orf23, G6D, MEGT1, NG25, CD19, BCMA, FLT3, CD70, DLL3, or CD34, CD70 ) can be knocked out to avoid induced CAR activation using the same antigen (e.g., EGFRvIII, Flt3, WT-1, CD20, CD23, CD30, CD38, CD33, CD133, MHC-WT1, TSPAN10, MHC-PRAME, Liv1, ADAM10, CHRNA2, LeY, NKGD2D, CS1, CD44v6, ROR1, claudin-18.2, Muc17, FAP alpha, Ly6G6D, c6orf23, G6D, MEGT1, NG25, CD19, BCMA, FLT3, CD70, DLL3, or CD34, CD70 CAR). As described herein, in some embodiments, the engineered immune cells of the present disclosure comprise one disrupted or inactivated gene selected from the group consisting of MHC1(β2M), MHC2(CIITA), EGFRvIII, Flt3, WT-1, CD20, CD23, CD30, CD38, CD33, CD133, MHC-WT1, TSPAN10, MHC-PRAME, Liv1, ADAM10, CHRNA2, LeY, NKGD2D, CS1, CD44v6, ROR1, claudin-18.2, Muc17, FAP alpha, Ly6G6D, c6orf23, G6D, MEGT1, NG25, CD19, BCMA, FLT3, CD70, DLL3, or CD34, CD70, TCR alpha, and TCR beta, and / or express a CAR or a multi-chain CAR. In some embodiments, the cells comprise a multi-chain CAR.In some embodiments, the isolated cells comprise two disrupted or inactivated genes selected from the group consisting of CD52 and TCR alpha, CDR52 and TCR beta, PD-1 and TCR alpha, PD-1 and TCR beta, MHC-1 and TCR alpha, MHC-1 and TCR beta, MHC2 and TCR alpha, MHC2 and TCR beta, and / or express a CAR or a multi-chain CAR.

[0115] The engineered immune cells can be allogeneic or autologous.

[0116] In some embodiments, the engineered immune cell or population of engineered immune cells comprises a T cell (e.g., inflammatory T lymphocytes cytotoxic T lymphocytes, regulatory T lymphocytes, helper T lymphocytes, tumor infiltrating lymphocytes (TIL)), a NK cell, a NK-T cell, a TCR expressing cell, a dendritic cell, a killer dendritic cell, a mast cell, or a B cell, and expresses a CAR. In some embodiments, the T cell can be derived from the group consisting of a population comprising CD4+ T-lymphocytes, CD8+ T-lymphocytes, or a combination of CD4+ and CD8+ T cells.

[0117] In some embodiments, the engineered immune cells or populations of engineered immune cells generated using the methods of the present disclosure can be derived from, for example, but not limited to, stem cells. The stem cells can 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.

[0118] In some embodiments, the engineered immune cells or immune cell populations generated using the methods of the present disclosure are obtained or prepared from peripheral blood. In some embodiments, the engineered immune cells are obtained or prepared from peripheral blood mononuclear cells (PBMCs). In some embodiments, the engineered immune cells are obtained or prepared from bone marrow. In some embodiments, the engineered immune cells are obtained or prepared from umbilical cord blood. In some embodiments, the cells are human cells. In some embodiments, the cells are transfected or transduced with a nucleic acid vector using a method selected from the group consisting of electroporation, sonoporation, biolistics (e.g., Gene Gun), lipid transfection, polymer transfection, nanoparticles, viral transfection (e.g., retrovirus, lentivirus, AAV) or polyplex.

[0119] In some embodiments, engineered immune cells expressing an antigen-specific CAR on their cell surface membrane comprise a percentage of stem cell memory and central memory cells that is greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0120] In some embodiments, engineered immune cells expressing an antigen-specific CAR on their cell surface membrane comprise a percentage of stem cell memory and central memory cells of about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 15% to about 50%, about 15% to about 40%, about 20% to about 60%, or about 20% to about 70%.

[0121] In some embodiments, engineered immune cells expressing an antigen-specific CAR on their cell surface membrane are CM Cells and / or T SCM The engineered immune cells are enriched for T cells, such that the engineered immune cells are enriched for T cells that are at least about 60%, 65%, 70%, 75%, or 80% bound. CM and T SCM In some embodiments, the engineered immune cells expressing an antigen-specific CAR on their cell surface membrane include T CMCells and / or T SCM The engineered immune cells are enriched for T cells, such that the engineered immune cells are enriched for T cells that are at least about 70% of the bound T cells. CM and T SCM In some embodiments, the engineered immune cells expressing an antigen-specific CAR on their cell surface membrane include T CM Cells and / or T SCM The engineered immune cells are enriched for T cells, such that the engineered immune cells are enriched for T cells that are at least about 75% of the bound T cells. CM and / or T SCM Contains cells.

[0122] In some embodiments, the engineered immune cell is an inflammatory T lymphocyte that expresses a CAR. In some embodiments, the engineered immune cell is a cytotoxic T lymphocyte that expresses a CAR. In some embodiments, the engineered immune cell is a regulatory T lymphocyte that expresses a CAR. In some embodiments, the engineered immune cell is a helper T lymphocyte that expresses a CAR.

[0123] 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 PNA, diphosphate PNA, and triphosphate PNA. 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.

[0124] In some embodiments, the isolated cells or cell lines of the present disclosure may comprise pTα or a functional variant thereof. In some embodiments, the isolated cells or cell lines may be further genetically modified by disrupting or inactivating the TCRα gene.

[0125] The present disclosure also provides engineered immune cells comprising any of the CAR polynucleotides described herein.In some embodiments, the 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.

[0126] The CAR polypeptide may be synthesized in situ in the cell after introduction of a polynucleotide encoding the CAR polypeptide into the cell. Alternatively, the CAR polypeptide may be produced outside the cell and then introduced into the cell. Methods for introducing a polynucleotide construct into a cell are known in the art. In some embodiments, stable transformation methods (e.g., using lentiviral vectors) may be used to integrate the polynucleotide construct into the genome of the cell. In other embodiments, transient transformation methods may be used to transiently express the polynucleotide construct, and polynucleotide constructs that are not integrated into the genome of the cell. In other embodiments, viral-mediated methods may be used. The polynucleotide may be introduced into the cell by any suitable means, such as, for example, recombinant viral vectors (e.g., retroviruses, adenoviruses), liposomes, etc. Transient transformation methods include, for example, but are not limited to, microinjection, electroporation, or particle bombardment. The polynucleotide may be included in a vector, such as, for example, a plasmid vector or a viral vector.

[0127] In some embodiments, an isolated nucleic acid is provided that includes a promoter operably linked to a first polynucleotide encoding an antigen binding domain, at least one costimulatory molecule, and an activation domain. In some embodiments, the nucleic acid construct is contained within a viral vector. In some embodiments, the viral vector is selected from the group consisting of a retroviral vector, a murine leukemia virus vector, a SFG vector, an adenoviral vector, a lentiviral vector, an adeno-associated virus (AAV) vector, a herpes virus vector, and a vaccinia virus vector. In some embodiments, the nucleic acid is contained within a plasmid.

[0128] In some embodiments, the isolated nucleic acid construct is contained within a viral vector and introduced into the genome of the engineered immune cell by random integration (such as, for example, lentivirus- or retrovirus-mediated random integration). In some embodiments, the isolated nucleic acid construct is contained within a viral or non-viral vector and introduced into the genome of the engineered immune cell by site-specific integration (such as, for example, adenovirus-mediated site-specific integration).

[0129] Manufacturing engineered immune cells

[0130] Various known techniques can be used to produce polynucleotides, polypeptides, vectors, antigen-binding domains, immune cells, compositions, and the like according to the present disclosure.

[0131] Prior to in vitro manipulation or genetic modification of immune cells as described herein, cells can be obtained from a subject. Cells expressing a CAR can be derived from an allogeneic or autologous source and can be depleted of endogenous TCR as described herein.

[0132] a. Raw materials In some embodiments, the immune cells include T cells. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue, and tumor. In some embodiments, T cells can be obtained from a volume of blood drawn from a subject using any number of techniques known to those skilled in the art, such as FICOLL™ separation.

[0133] Cells 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 some embodiments, the cells collected by apheresis can be washed to remove plasma fraction, and then placed in a suitable buffer or medium for subsequent processing.

[0134] In some 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+, CD45RA-, and CD45RO+ T cells or CD28+, CD4+, CDS+, CD45RA-, CD45RO+, and CD62L+ T cells) can be further isolated by positive or negative selection techniques known in the art. For example, enrichment of T cell populations 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, a monoclonal antibody cocktail typically includes antibodies to 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 present disclosure.

[0135] PBMCs can be used directly for genetic modification with immune cells (such as CAR or TCR) using the methods described herein. In certain embodiments, after isolating PBMCs, T lymphocytes can be further isolated, and both cytotoxic and helper T lymphocytes can be classified into naive, memory, and effector T cell subpopulations before or after genetic modification and / or expansion. In some embodiments, CD8+ cells are further sorted into naive, stem cell memory, central memory, and effector cells by identifying cell surface antigens associated with each of these types of CD8+ cells. In some embodiments, expression of phenotypic markers of central memory T cells includes CD27, CD45RA, 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. In some embodiments, CD4+ T cells are further classified into subpopulations. For example, CD4+ T helper cells can be classified into naive, central memory and effector cells by identifying cell populations with cell surface antigens.

[0136] b. Stem cell-derived immune cells In some embodiments, immune cells can be derived from embryonic stem (ES) cells or induced pluripotent stem (iPS) cells.Suitable HSC, mesenchymal, iPS cells, and other types of stem cells can be cultured immortal cell lines or directly isolated from patients.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.

[0137] 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. In some embodiments, the immune cells are T cells derived from iPSCs. In some embodiments, the immune cells are NK cells derived from iPSCs. The source material may be embryonic stem cells. The source material may be B cells, or any other cells of peripheral blood mononuclear cell isolates, hematopoietic progenitor cells, hematopoietic stem cells, mesenchymal stem cells, adipose stem cells, or any other somatic cell type.

[0138] c. Genetic modification of isolated cells Immune cells such as T cells can be genetically modified after isolation using known methods, or immune cells can be activated and expanded (or differentiated in the case of progenitor cells) in vitro before being genetically modified. In some embodiments, isolated immune cells are genetically modified to reduce or eliminate expression of endogenous TCRα and / or CD52. In some embodiments, cells are genetically modified using gene editing techniques (e.g., CRISPR / Cas9, CRISPR / Cas12a, zinc finger nucleases (ZFNs), TALENs, MegaTALs, meganucleases) to reduce or eliminate expression of endogenous proteins (e.g., TCRα and / or CD52). In another embodiment, immune cells such as T cells are genetically modified with a chimeric antigen receptor (e.g., transduced with a viral vector comprising one or more nucleotide sequences encoding a CAR) as described herein, and then activated and / or expanded in vitro.

[0139] Certain methods for making the constructs and engineered immune cells of the present disclosure are described in PCT application PCT / US15 / 14520, the contents of which are incorporated herein by reference in their entirety.

[0140] It will be understood that PBMCs may further comprise other cytotoxic lymphocytes such as NK cells or NKT cells. An expression vector carrying the coding sequence of the chimeric receptor disclosed herein may 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, which can then be further expanded to increase the number of these CAR-expressing T cells in addition to cell activation using anti-CD3 antibodies and IL-2 or other methods known in the art as described elsewhere herein. Standard procedures are used for cryopreservation of CAR-expressing T cells for storage and / or preparation for use in human subjects. In one embodiment, 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.

[0141] For cloning polynucleotides, 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 generally include sequence components, including but not limited to origin of replication, promoter sequence, transcription initiation sequence, enhancer sequence, and selection marker. These elements can be appropriately selected by those skilled in the art. For example, origin of replication can be selected to promote autonomous replication of vectors in host cells.

[0142] In some embodiments, the present disclosure provides an isolated host cell that contains the 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, particularly human cells.

[0143] 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, microprojectile bombardment, receptor mediated gene delivery, polylysine, histone, chitosan, and peptide mediated delivery. Standard methods for transfection and transformation of cells for expression of vectors of interest are well known in the art. In a further embodiment, a mixture of different expression vectors can be used in genetically modifying a donor population of immune effector cells, 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 one or more different CARs.

[0144] In one embodiment, the present disclosure provides a method for preserving genetically engineered cells expressing CAR or TCR. This involves cryopreserving immune cells so that the cells remain viable when thawed. A fraction of immune cells expressing CAR can be cryopreserved by methods known in the art to provide a permanent source of such cells for future treatment of patients suffering from malignant tumors. When necessary, cryopreserved transformed immune cells can be thawed, grown, and expanded to obtain more such cells.

[0145] In some embodiments, the cells are formulated by first harvesting the cells from their culture medium, then washing and concentrating the cells in a medium and container system suitable for administration in a therapeutically effective amount (a "pharmaceutical acceptable" carrier). A suitable infusion medium is any isotonic medium formulation, typically saline, Normosol™ R (Abbott) or Plasma-Lyte™ A (Baxter), but 5% dextrose or Ringer's lactate in water can also be utilized. The infusion medium can be supplemented with human serum albumin.

[0146] d. Allogeneic CAR T cells The process for manufacturing allogeneic CAR T therapy involves harvesting healthy, selected, screened, and tested T cells from healthy donors. The T cells are then engineered to express a CAR that recognizes a specific cell surface protein expressed on hematological or solid tumors. The allogeneic T cells are gene edited to reduce the risk of graft-versus-host disease (GvHD) and prevent allogeneic rejection. The T cell receptor genes (e.g., TCRα, TCRβ) are knocked out to avoid GvHD. The CD52 gene can be knocked out to make the CAR T product resistant to anti-CD52 antibody treatment. Anti-CD52 antibody treatment can therefore be used to suppress the host immune system and allow the CAR T to remain engrafted and achieve full therapeutic effect. The engineered T cells then undergo a purification process and are cryopreserved in vials for eventual delivery to the patient.

[0147] e. Autologous CAR T cells 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 targets expressed on the cell surface of one or more specific cancer cells and kills the cancer cells. The engineered cells are then cryopreserved and subsequently administered to the patient.

[0148] Pharmaceutical Compositions

[0149] In some embodiments, the cells are formulated by first harvesting the cells from their culture medium, then washing and concentrating the cells in a medium and container system suitable for administration in a therapeutically effective amount (a "pharmaceutical acceptable" carrier). A suitable infusion medium is any isotonic medium formulation, typically saline, Normosol™ R (Abbott) or Plasma-Lyte™ A (Baxter), but 5% dextrose or Ringer's lactate in water can also be utilized. The infusion medium can be supplemented with human serum albumin.

[0150] In embodiments, the desired therapeutic amount of cells in the composition is generally at least 2 cells (e.g., at least one CD8+ central or stem cell memory T cell and at least one CD4+ helper T cell subset, or two or more CD8+ central or stem cell memory T cells, or two or more CD4+ helper T cell subsets), or more commonly, 10 2 More than 10 cells 6 Less than or equal to 10 7 , 10 8 or 10 9 Less than 10 cells 10 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 that are equal to or greater than about 10 cells per injection. In some embodiments of the present disclosure, particularly since all injected cells are redirected to a specific target antigen, 5 / kilogram or about 10 6 / kilogram (10 per patient) 6 ~10 11 ) can be administered. CAR therapy can be administered multiple times at doses within these ranges. The cells can be autologous, allogeneic, or xenogeneic to the patient undergoing therapy.

[0151] The CAR-expressing cell population of the present disclosure can be administered as a pharmaceutical composition, alone or 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- or TCR-expressing cell population, such as a T cell, as described herein, in combination with one or more pharma- ceutical or physiologically acceptable carriers, diluents, or excipients. Such compositions can include a buffer, 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.

[0152] Pharmaceutical compositions (such as solutions and suspensions) 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 a solvent or suspension medium, 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, 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. Pharmaceutical compositions for injection are preferably sterile.

[0153] Treatment method

[0154] The present disclosure includes a method for treating or preventing a disease (e.g., cancer) in a patient, comprising administering an effective amount of CAR T cells or engineered immune cells comprising a CAR disclosed herein to a patient in need thereof. In some embodiments, the effective amount of CAR T cells or engineered immune cells have been analyzed for various attributes according to the methods described in this disclosure. In some embodiments, a CAR T cell medicine for therapeutic use has been analyzed for various attributes, such as potency or multifunctionality, according to the methods described in this disclosure. In some embodiments, the CAR T cells are TCR-CAR T cells and the CAR T medicine for therapeutic use has been analyzed for various attributes, such as the amount or percentage of remaining TCR+ CAR T cells and / or potency or multifunctionality, according to the methods described in this disclosure.

[0155] 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 the engineered immune cells of the present application. In some embodiments, the T cell-mediated immune response is directed against a target cell. In some embodiments, the engineered immune cells comprise 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, the immune cell comprising at least one chimeric antigen receptor, T cell receptor, and / or isolated antigen binding domain described herein. CARs containing the immune cells of the disclosure can be used to treat malignancies associated with aberrant expression of biomarkers. In some embodiments, the CAR containing the immune cells of the present disclosure can be used to treat small cell lung cancer, melanoma, low-grade glioma, glioblastoma, medullary thyroid carcinoma, carcinoid, dispersed neuroendocrine tumors in the pancreas, bladder and prostate, testicular cancer, and lung adenocarcinoma with neuroendocrine features. In an exemplary embodiment, the CAR containing the immune cells, such as the CAR-T cells of the present disclosure, is used to treat small cell lung cancer.

[0156] Also provided is a method of reducing the size of a tumor in a subject, comprising administering to a subject an engineered cell of the present disclosure, wherein the cell comprises a chimeric antigen receptor that comprises an antigen binding domain and that binds to an antigen on the tumor.

[0157] In some embodiments, the subject has a solid tumor or a hematological malignancy such as lymphoma or leukemia. In some embodiments, the engineered cells are delivered to the tumor bed. 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 heterologous immune cells, e.g., heterologous T cells. In some embodiments, the engineered cells of the present application are transfected or transduced in vivo. In other embodiments, the engineered cells of the present application are transfected or transduced ex vivo. As used herein, the term "in vitro cells" refers to any cells that are cultured ex vivo.

[0158] A "therapeutically effective amount," "effective dose," "effective amount," or "therapeutically effective dose" of a therapeutic agent, e.g., engineered CART cells, is any amount that, when used alone or in combination with another therapeutic agent, protects a subject against onset of disease, or promotes disease regression as indicated by a decrease in the severity of disease symptoms, increases the frequency and duration of disease-free periods, or prevents functional impairment or disability due to disease. The ability of a therapeutic agent to promote disease regression can be evaluated using a variety of methods known to those of skill in the art, for example, in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.

[0159] The terms "patient" and "subject" are used interchangeably and include human and non-human animal 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.

[0160] The terms "treat" and "treatment" include therapeutic treatment, prophylactic treatment, and applications that reduce the risk that a subject will develop 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.

[0161] A desired therapeutic amount of cells in a composition is generally at least 2 cells (e.g., at least one CD8+ central memory T cell and at least one CD4+ helper T cell subset), or more typically 10 2 Super cells, up to 10 6 pieces, maximum 10 8 cells or 10 9 Contains cells, 10 10 The number of cells depends 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 Clinically significant numbers of immune cells are greater than 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. The cells can be autologous, allogeneic, or xenogeneic to the patient undergoing therapy.

[0162] In some embodiments, the therapeutically effective amount of CAR T cells is about 1×10 5 cells / kg, approximately 2×10 5 cells / kg, approximately 3×10 5 cells / kg, approximately 4×10 5 cells / kg, approximately 5×10 5 cells / kg, approximately 6×10 5 cells / kg, approximately 7×10 5 cells / kg, approximately 8×10 5 cells / kg, approximately 9×10 5 cells / kg, 2×10 6 cells / kg, approximately 3×10 6 cells / kg, approximately 4×10 6 cells / kg, approximately 5×10 6 cells / kg, approximately 6×10 6 cells / kg, approximately 7×10 6 cells / kg, approximately 8×10 6 cells / kg, approximately 9×10 6 cells / kg, approximately 1×10 7 cells / kg, approximately 2×10 7 cells / kg, approximately 3×10 7 cells / kg, approximately 4×10 7 cells / kg, approximately 5×10 7 cells / kg, approximately 6×10 7 cells / kg, approximately 7×10 7 cells / kg, approximately 8×10 7 cells / kg, or approximately 9 x 10 7 cells / kg.

[0163] In some embodiments, the target dose of CAR+ / CAR-T+ / TCR+ cells is 1×10 6 ~2×10 8 Cells / kg, e.g., 2 x 10 6 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.

[0164] In some aspects, the disclosure includes a pharmaceutical composition 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.

[0165] The CAR-expressing cell population of the present disclosure can be administered as a pharmaceutical composition, alone or 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- or TCR-expressing cell population, such as a T cell, as described herein, in combination with one or more pharma- ceutical or physiologically acceptable carriers, diluents, or excipients. Such compositions can include a buffer, 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.

[0166] Pharmaceutical compositions (such as solutions and suspensions) 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 a solvent or suspension medium, 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, 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. Pharmaceutical compositions for injection are preferably sterile.

[0167] In some embodiments, when administered to a patient, engineered immune cells expressing any one of the antigen-specific CARs described herein on their cell surface can reduce, kill, or lyse the patient's endogenous antigen-expressing cells. In one embodiment, the percentage of reduction or lysis of antigen-expressing endogenous cells or cells of a cell line expressing an antigen by engineered immune cells expressing any one of the antigen-specific CARs described herein is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or greater than 95%. In one embodiment, the percentage of reduction or lysis of antigen-expressing endogenous cells or cells of the cell line expressing the antigen by engineered immune cells expressing an antigen-specific CAR is about 5% to about 95%, about 10% to about 95%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 25% to about 75%, or about 25% to about 60%. In one embodiment, the endogenous antigen-expressing cells are endogenous antigen-expressing bone marrow cells.

[0168] In one embodiment, the rate of reduction or lysis of target cells, e.g., cell lines expressing the antigen, by engineered immune cells expressing an antigen-specific CAR of the present disclosure on their cell surface membrane can be measured using the assays disclosed herein.

[0169] The method can further include administering one or more chemotherapeutic agents. In some embodiments, the chemotherapeutic agent is a lymphodepleting (conditioning) chemotherapy. For example, a particular beneficial dose of cyclophosphamide (200 mg / m 2 / day~2000mg / m 2 / day, about 100mg / m 2 / day~about 2000mg / m 2 / day, e.g., about 100 mg / m 2 / day, about 200mg / m 2 / day, about 300mg / m 2 / day, about 400mg / m 2 / day, about 500mg / m 2 / day, about 600mg / m 2 / day, about 700mg / m 2 / day, about 800mg / m 2 / day, approximately 900mg / m 2 / day, about 1000mg / m 2 / day, about 1500mg / m 2 / day or approximately 2000 mg / m 2 / day) and a specific dose of fludarabine (20 mg / m 2 / day~900mg / m 2 / day, about 10mg / m 2 / day~about 900mg / m 2 / day; for example, about 10 mg / m 2 / day, about 20mg / m 2 / day, about 30mg / m 2 / day, about 40mg / m 2 / day, about 40mg / m 2 / day, about 50mg / m 2 / day, about 60mg / m 2 / day, about 70mg / m 2 / day, about 80mg / m 2 / day, about 90mg / m 2 / day, about 100mg / m 2 / day, about 500mg / m 2 / day or approximately 900 mg / m 2 A preferred dosage regimen is about 300 mg / m2 / day prior to administering to the patient a therapeutically effective amount of engineered T cells. 2 / day cyclophosphamide and approximately 30 mg / m 2 Treatment of patients included administering fludarabine / day for 3 days.

[0170] In some embodiments, lymphodepletion further comprises administration of a CD52 antibody. In some embodiments, the CD52 antibody is alemtuzumab. In some embodiments, the CD52 antibody is administered at an intravenous dose of about 13 mg / day.

[0171] 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.

[0172] In some embodiments, compositions comprising CAR-expressing immune effector cells disclosed herein can be administered in conjunction with any number of chemotherapeutic agents, which can be administered in any order. Examples of chemotherapeutic agents include alkylating agents (such as thiotepa and cyclophosphamide (CYTOXAN™)), alkyl sulfonates (such as busulfan, improsulfan and piposulfan), aziridines (such as benzodopa, carboquone, meturedopa and uredopa), ethylenimines and methylameramines (including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolmelamine regimes), nitrogen masterbates, and the like. drugs (chlorambucil, chlornaphazine, colofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembitine, phenesterine, prednimustine, trofosfamide, uracil mustard, etc.), nitrosoureas (carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine, etc.), antibiotics (aclacinomycin, actinomycin, autramycin, azaserine, bleomycin, cactinomycin, Calicheamicin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potofilomycin, puromycin, queramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenime , zinostatin, zorubicin, etc.), antimetabolites (such as methotrexate and 5-fluorouracil (5-FU)), folic acid analogues (such as denopterin, methotrexate, pteropterin, trimetrexate, etc.), purine analogues (such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, etc.), pyrimidine analogues (such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU, etc.),Androgens (calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone, etc.), adrenal cortical hormone synthesis inhibitors (aminoglutethimide, mitotane, trilostane, etc.), folic acid supplements (folinic acid, etc.), aceglatone, aldophosphamide glycoside, aminolevulinic acid, amsacrine, bestravcil, bisantrene; edatraxate, defofamine, demecolcine, diaziquone, elformitine, elliptinium acetate, etoglucide, gallium nitrate, hydroxyurea; lentinan, lonidamine, mitoguazone, mitoxane torone, mopidamol, nitracrine, pentostatin, phenamet, pirarubicin, podophyllic acid, 2-ethylhydrazide, procarbazine, PSK®, razoxane, schizofiran, spirogermanium, tenuazonic acid, triazicon, 2,2',2'-trichlorotriethylamine, urethane, vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside (Ara-C), cyclophosphamide, thiotepa, taxoids, such as 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 Included in this definition are antiestrogens (e.g., tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazoles, etc.) and pharmacokinetic and / or ...Also included are anti-hormonal agents that regulate or inhibit hormone action on tumors, such as 4-hydroxytamoxifen, trioxyphene, ketoxifene, LY117018, onapristone, and toremifene (Fareston), and anti-androgens (such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin), as well as pharmacologic 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.

[0173] In some embodiments, the chemotherapeutic agent is administered simultaneously or within one week after administration of the engineered cells, polypeptide, or nucleic acid. In other embodiments, the chemotherapeutic agent is administered 1-4 weeks, or 1 week-1 month, 1 week-2 months, 1 week-3 months, 1 week-6 months, 1 week-9 months, or 1 week-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.

[0174] 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 (Tcentriq®).

[0175] 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 (Glanov-SmithKline®), erythrocyte serine monophosphate (ECP-100), erythrocyte sarcoma ... Libec(R), cetuximab (Erbitux(R), panitumumab) (Vectibix(R), catumaxomab, ibritumomab, ofatumumab, tositumomab, brentuximab, alemtuzumab, gemtuzumab, erlotinib, gefitinib, vandetanib, afatinib, lapatinib, neratinib, axitinib, masitinib, pazopanib, sunib Tinib, sorafenib, toceranib, lestaurtinib, axitinib, cediranib, lenvatinib, nintedanib, pazopanib, regorafenib, semaxanib, sorafenib, sunitinib, tivozanib, toceranib, vandetanib, entrectinib, cabozantinib, imatinib, dasatinib, nilotinib, ponatinib, radotinib, bosutinib, lestaurtinib, ruxolitinib These include CDK inhibitors such as nib, pacritinib, cobimetinib, selumetinib, trametinib, binimetinib, alectinib, ceritinib, crizotinib, aflibercept, adipotide, denileukin diftitox, mTOR inhibitors (such as everolimus and temsirolimus), hedgehog inhibitors (such as sonidegib and vismodegib), and CDK inhibitors such as palbociclib.

[0176] In some embodiments, the composition comprising the CAR-containing immune cells can be administered with a therapeutic regimen to prevent 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, methylprednisolone, 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 directed against cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors, such as TNF antagonists (e.g., etanercept (ENBREL®), Adminin (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.

[0177] In certain embodiments, the compositions described herein are administered in conjunction with a cytokine. Examples of cytokines include lymphokines, monokines, and traditional 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)), hepatocyte growth factor (HGF), fibroblast growth factor (FGF), prolactin, placental lactogen, Mullerian inhibitory substance, 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 (TGF) (TGF-alpha and TG F-beta, insulin-like growth factors-I and -II, erythropoietin (EPO), osteoinductive 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) (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, etc.), 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 from recombinant cell culture, as well as biologically active equivalents of the native sequence cytokines.

[0178] The following examples are offered for illustrative purposes only. Indeed, various modifications of the invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description. EXAMPLES

[0179] Example 1 Identifying factors that affect drug quality

[0180] The general consensus in the field has been to complete drug processing within as short a time frame as possible to minimize the time that formulated cells are exposed to cryopreservative-containing formulations and other stress conditions. Time limitations add constraints to the manufacturing process, particularly for allogeneic CAR T drugs, which may require longer drug processing times as the manufacturing process is designed to produce a large number of doses per batch.

[0181] Process development studies were performed to identify factors that may affect CAR T cells during drug product processing. Figure 1 shows an exemplary workflow from cell harvest, drug product fill, and drug product freezing. Briefly, engineered cells such as CAR T cells were resuspended in non-DMSO-containing basal medium. The cell suspension was mixed with an equal volume of DMSO-containing formulation to a final DMSO concentration of 5% before starting the fill process. The data presented below was obtained using an exemplary CD19 CAR T cell.

[0182] Formulated CD19 CAR T cells prior to freezing were mixed during the filling process to maintain the cells in suspension and ensure product homogeneity and dose content uniformity. Unless otherwise indicated, cells were mixed using Erlenmeyer flasks on an orbital shaker. Mixing speeds ranged from 85 rpm to 140 rpm, depending on the formulation volume in the flask. In the following shake flask mixing experiments, speeds in the lower range were employed to ensure adequate mixing while minimizing impact on cell viability.

[0183] Viable cell density (VCD) and cell viability were measured using a fluorescent dye-based method and the cell imaging device NucleoCounter® NC-200™ to determine the effect of mixing on cells. The NucleoCounter® was used to count cells by detecting nuclei using two fluorescent dyes preloaded in the Via1-Cassette™: Acridine Orange (AO) stains all cells green and DAPI (4′,6-diamidino-2-phenylindole) stains dead cells blue. Viable cell density (VCD) was calculated as (total cells−dead cells) / ml and cell viability % was calculated as (total cells−dead cells) / total cells×100. Orbital shaker mixing was found to affect VCD (Figure 2A), and cell viability % (Figure 2B) compared to pharmaceuticals filled at the beginning of the process and kept at room temperature for 5 hours without further mixing.

[0184] The effect of formulation holding temperature during shake flask mixing was also examined. The data showed that the VCD and cell viability % were examined when the formulated cells were held at 5 °C for 24 h during shake flask mixing. o C is superior to holding the product at room temperature during shake flask mixing (Figures 3A and 3B, respectively).

[0185] As part of shake flask mixing optimization, cell sedimentation studies were performed to evaluate the need for continuous mixing. Cell sedimentation studies were performed with cells formulated at 20E+06 viable cells / mL and 100E+06 viable cells / mL in Erlenmeyer flasks. The formulations in the flasks were sampled at the top and bottom of the flask for a period of time without mixing. Results show that no cell sedimentation occurred for at least 40 minutes at any of the cell concentrations tested (Figures 4A-B).

[0186] The cell sedimentation data indicates that continuous mixing of the formulated cells is not necessary, as the cells in the final formulation do not settle immediately. While not intending to be bound by a particular mechanism, the delayed settling of the cells may be due to the size of the cells and / or the higher viscosity of the final formulation. This finding led to the evaluation of intermittent orbital shaker flask mixing. Additionally, different types of mixing using spinner flasks and, for example, magnetic stir plates, were also evaluated. Both intermittent orbital shaker mixing of the cells in the final formulation (e.g., mixing for 2 minutes at a speed of 85 rpm every 10 or 30 minutes) and continuous spinner flask mixing (e.g., 40 rpm) showed better viable cell density (Figure 5A) and cell viability % (Figure 5B) over time compared to continuous orbital shaker mixing at 85 rpm.

[0187] Shake flask mixing is achieved by placing the formulated cells in a shake flask, for example, an Erlenmeyer shake flask, on an orbital shaker platform at a set speed. There are no moving parts in the flask, such as paddles, that could damage the cells by coming into direct contact with the cells in the flask. On the other hand, a spinner flask with a top-mounted paddle connected to a magnetic stir bar that comes into direct contact with the cells, for example, was thought to shock the cells due to shear forces at the paddle. Therefore, shake flask mixing was presumed to generate less shear stress and be gentler on the cells. However, as shown in Figure 5A and Figure 5B, both the VCD and cell viability % after continuous shaking in a spinner flask are significantly better than those with continuous shaking in an orbital shaker flask. Furthermore, when the filling process was carried out using a shake flask, the results of intermittent shaking are better than continuous shaking.

[0188] Next, intermittent shaking in spinner flasks (2 min of mixing at 40 rpm every 10 min) was further evaluated in comparison to continuous mixing in spinner flasks (40 rpm). Figure 5C shows that the results of intermittent and continuous mixing in spinner flasks are comparable.

[0189] In summary, it was unexpectedly found that it is not only DMSO exposure time (as commonly believed in the art) that can result in cell damage during pharmaceutical processing, but also other processing conditions (operation temperature, mixing method, etc.) can have a significant effect on cells. For example, lower temperatures can help improve cell viability compared to room temperature exposure during the filling process. Furthermore, the data show that cell sedimentation in the final formulation containing 5% DMSO was a slow process. Thus, continuous mixing of cells during the filling process may not be required, contrary to commonly believed in the art. In addition to mixing time, it was also surprisingly found that the type of mixing employed during the filling process has a significant effect. The data presented herein show that spinner flask mixing resulted in significantly higher viable cells compared to continuous shake flask mixing.

[0190] Example 2 Process Design to Increase Drug Filling Time Window

[0191] The above data suggests that it is possible to increase the end-of-filling time window without adversely affecting cell viability. In the next experiment, the VCD and cell viability % of pre-frozen CAR T cells were examined under different mixing conditions during the 2-, 4-, or 6-hour filling process (Figure 6). Cells subjected to each filling process were frozen, and any long-term effects of the mixing process on the cells after thawing were also examined. An aliquot of frozen cells was thawed and kept at room temperature for 2 hours, and the VCD and cell viability % were measured, as shown in Figure 6. The results in Figure 6 show that continuous mixing in a spinner flask or intermittent mixing in a shake flask performed better than continuous mixing in a shake flask, using either pre-frozen or post-thawed pharmaceutical products as examples. The VCD and viability were not significantly affected during the filling process, even after 4 hours. In another experiment, post-thawed engineered CAR T cells were cultured for 3 days to confirm that the cells were viable and could recover and grow after the pharmaceutical process and freezing (data not shown).

[0192] Although shake flasks were generally considered to be gentler on cells in terms of shear stress, we unexpectedly found that spinner flask mixing was superior to shake flask mixing in the filling process. Next, we used spinner flask mixing to study the effect of mixing speed on pre-frozen drug products. As shown in Figure 7A, mixing speeds of 40-80 rpm in spinner flask mixing did not significantly affect drug products over 4 hours. We further investigated whether the effect on cells mixed in spinner flasks was influenced by the volume of formulated cells. As shown in Figure 7B, the VCD and viability% of drug products with starting volumes of 300 ml or 600 ml subjected to continuous spinner flask mixing at 50 rpm are comparable over 4 hours.

[0193] In summary, the data disclosed herein show that intermittent shake flask mixing is superior to continuous shake flask mixing. Furthermore, switching from shake flasks to spinner flasks with either continuous or intermittent mixing over a range of speeds showed improved VCD and cell viability compared to continuous shake flask mixing, especially over extended periods of time.

[0194] Example 3 Analysis of product quality

[0195] After drug product processing, the filled vials or containers were frozen for storage. The post-thaw drug products processed by the different mixing / filling processes were then analyzed to determine whether the formulated cells maintained viability after thawing. The data in Figure 8A show that continuous mixing at 40 rpm in a spinner flask or intermittent mixing for 2 min at 85 rpm every 30 min in a shake flask produced comparable results and did not significantly affect the VCD or % viability of the pre-frozen drug product over the course of 4 hours. The filled samples were frozen for storage for 2 or 4 hours, after which the cells were thawed for further testing.

[0196] Thawed samples were subjected to 2 hours of room temperature holding before VCD and % viability were measured, taking into account the estimated processing time that may be required in the clinic before the thawed drug product is ready to be administered to a patient. Figure 8B shows that the VCD and viability of the post-thawed cells were comparable to those of the pre-frozen cells under these conditions.

[0197] In summary, the end-of-fill time window can be extended to at least 4 hours without impacting VCD or cell viability by either continuous mixing in spinner flasks or intermittent mixing in shaker or spinner flasks. Extending the end-of-fill time window allows more time to complete filling of large batches of manufactured, formulated, and engineered immune cells, including CAR T cells, and avoids waste of unfilled drug product.

Claims

1. 1. A method for preparing a pharmaceutical product comprising engineered immune cells, the method comprising formulating the engineered immune cells in a composition containing a cryopreservative to form the pharmaceutical product, and mixing and filling the pharmaceutical product into one or more containers, wherein the pharmaceutical product maintains homogeneity.

2. 10. The method of claim 1, wherein the steps of mixing and filling take at least about 2 hours.

3. 3. The method of claim 1 or 2, wherein the steps of mixing and filling take about 2 hours, about 3 hours, about 4 hours, about 5 hours or about 6 hours.

4. The method of any one of claims 1 to 3, wherein the mixing comprises intermittent mixing or continuous mixing.

5. The method of claim 4 , wherein the intermittent mixing is performed in a shake flask.

6. The method of claim 4, wherein the intermittent mixing is performed in a spinner flask.

7. 7. The method of any one of claims 4 to 6, wherein the intermittent mixing comprises mixing for about 1 to about 3 minutes every about 10 to about 40 minutes.

8. The method of any one of claims 1 to 4, wherein the mixing comprises continuous mixing in a spinner flask.

9. The method of any one of claims 1 to 8, wherein the mixing and filling steps are performed at about 4°C.

10. The method according to any one of claims 1 to 8, wherein the mixing and filling steps are carried out at room temperature.

11. The method according to any one of claims 1 to 10, wherein the mixing is carried out at a speed of about 30 to about 85 rpm.

12. The method of any one of claims 1 to 11, wherein the cryopreservation agent comprises DMSO or glycerol.

13. 13. The method of any one of claims 1 to 12, wherein the cryopreservation agent comprises about 3% DMSO to about 10% DMSO.

14. 14. The method of claim 13, wherein the cryopreservation agent comprises about 5% DMSO.

15. The method of any one of claims 1 to 14, wherein the composition further comprises a viscosity enhancing excipient.

16. 16. The method of any one of claims 1 to 15, wherein the engineered immune cell is a T cell, an inflammatory T lymphocyte, a cytotoxic T lymphocyte, a regulatory T lymphocyte, a helper T lymphocyte, an effector T lymphocyte, a tumor infiltrating lymphocyte (TIL), an NK cell, an NK-T cell, a TCR expressing cell, a TCR knockout T cell, a dendritic cell, a macrophage, a killer dendritic cell, a mast cell, or a B cell.

17. The method of any one of claims 1 to 16, wherein the engineered immune cells are CAR T cells.

18. 18. The method of claim 17, wherein the engineered immune cells are allogeneic CAR T cells.

19. 18. The method of claim 17, wherein the engineered immune cells are autologous CAR T cells.

20. The method of any one of claims 1 to 19, wherein the engineered immune cells are human cells.

21. A pharmaceutical product comprising engineered immune cells prepared by the method of any one of claims 1 to 20.

22. 22. A method of treating a subject in need thereof comprising administering to the subject the pharmaceutical agent of claim 21.

23. The method of any one of claims 1 to 20, wherein the step of mixing the pharmaceutical agent and filling into one or more containers is performed at room temperature for up to about 2 hours, up to about 4 hours, or up to about 6 hours, and the pharmaceutical agent maintains homogeneity.

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