Method for preparing genetically modified t cells

Abstract

Provided are a process for preparing genetically modified T cells by using PBMCs as a raw material without T cell sorting, cells produced by means of the process and the use thereof. The provided process for preparing modified T cells can reduce costs of materials, manpower consumption, and the use amount of viruses.

Description

A method for preparing genetically modified T cells TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and specifically relates to a method for preparing genetically modified T cells. BACKGROUND

[0002] Currently, the preparation of genetically modified T cells represented by CAR-T cells involves multiple processes, such as cell sorting, activation, virus transduction, expansion, and harvesting. The overall duration is 7-11 days. These processes consume labor, and cell sorting is costly. Generally, about 100,000 yuan of reagents and consumables are needed for T cell sorting, which accounts for a major part of the overall preparation process cost.

[0003] If T cells are not sorted, peripheral blood mononuclear cells (PBMCs) are directly used as starting materials for culture. Literature reports that because the composition of PBMCs varies greatly among different patients, and the mononuclear cells in PBMCs have phagocytic function, it can lead to unstable preparation process, such as unstable expansion fold after cell expansion, low T cell purity, or low transduction rate.

[0004] In addition, the preparation process of genetically modified T cells represented by CAR-T is mostly discontinuous, with cell sorting on Day 0, activation on Day 1 or Day 2, virus transduction on Day 3-Day 9, and cell expansion on Day 3-Day 9. The whole process needs manual cell sorting, which increases labor consumption and preparation cost.

[0005] Therefore, there is still a need for a new method for producing modified T cells that is simpler and cheaper than the current method.

[0006] SUMMARY

[0007] To improve the above technical problems, the present disclosure provides a process for preparing modified T cells using PBMCs as raw materials without T cell sorting.

[0008] In one aspect, the present disclosure provides a method for preparing genetically modified T cells, characterized in that it comprises the following steps:

[0009] (1) Adjusting the starting density of PBMCs to 1e6 / ml-5e6 / ml by culture medium on Day 0;

[0010] (2) Transferring the PBMC cells with the density in step (1) into a container and supplementing with culture medium to the starting culture volume on Day 0;

[0011] (3) Adding an activator to the container of step (2) on Day 0;

[0012] (4) Virus vector is added to the container of step (3) for transduction between Day 0 and Day 2, and the container after adding the virus vector is cultured;

[0013] (5) After adding the virus vector for at least 0.5 day, the culture medium is supplemented to the container to the harvest volume, and then the container is cultured with shaking until the harvest day;

[0014] (6) The container is taken out for cell harvesting on the harvest day.

[0015] In one embodiment of the present disclosure, the starting density of PBMC in step (1) is 1e6 / ml-3.5e6 / ml; preferably, the starting density of PBMC is 2e6 / ml, 2.2e6 / ml, 2.5e6 / ml, 2.8e6 / ml, 3e6 / ml or 3.2e6 / ml.

[0016] In one embodiment of the present disclosure, the starting culture volume in step (2) is 75ml-250ml; preferably, the starting culture volume is 75ml, 100ml, 150ml, 175ml or 200ml.

[0017] In one embodiment of the present disclosure, the culture medium is selected from X VIVO, AIM V, Optmizer, Optivitro, TexMacs, NutriT and T VIVO.

[0018] In one embodiment of the present disclosure, the activator in step (3) is selected from Dynabeads, CD3 / CD28, TransAct and CD3 / CD28 / CD2 soluble tetramer;

[0019] Preferably, the activator is Dynabeads or CD3 / CD28, and the ratio of the number of magnetic beads to the number of cells is 0.5:1, 1:1, 2:1, 3:1 or 4:1, more preferably 2:1;

[0020] Preferably, the activator is TransAct, and the ratio of the culture volume to the activator volume is 60:1-10:1; more preferably 50:1, 40:1, 30:1 or 20:1;

[0021] Preferably, the activator is CD3 / CD28 / CD2 soluble tetramer, and the ratio of the culture volume to the activator volume is 200:1-10:1; more preferably 100:1, 75:1, 50:1, 25:1 or 20:1.

[0022] In one embodiment of the present disclosure, the cell density in the container when the viral vector is added in step (4) is 1e6-3.5e6 / ml;

[0023] Preferably, the cell density is 1.2e6 / ml, 1.5e6 / ml, 1.8e6 / ml, 2e6 / ml, 2.2e6 / ml, 2.5e6 / ml, 2.8e6 / ml, 3e6 / ml or 3.2e6 / ml.

[0024] In one embodiment of the present disclosure, the culture in step (4) is an oscillation culture, a swing oscillation culture or a stirring culture.

[0025] Preferably, the rotation speed of the oscillation culture is 50-100 rpm; more preferably, the rotation speed of the oscillation culture is 75 rpm.

[0026] Preferably, the rotation speed of the swing oscillation culture is 5-30 rpm and the angle is 2-30°; more preferably, the rotation speed of the swing oscillation culture is 15 rpm and the angle is 15°.

[0027] Preferably, the rotation speed of the stirring culture is 50-300 rpm; more preferably, the rotation speed of the swing oscillation culture is 15 rpm.

[0028] In one embodiment of the present disclosure, the medium is supplemented to the harvest volume in the container 0.5, 1, 1.5 or 2 days after the viral vector is added in step (5).

[0029] Preferably, the medium is supplemented to the harvest volume in the container 1 day after the viral vector is added in step (5).

[0030] In one embodiment of the present disclosure, the harvest day in step (6) is Day4, Day5, Day6, Day7 or Day8.

[0031] Preferably, the harvest day in step (6) is Day6.

[0032] In one embodiment of the present disclosure, the container is selected from a cell culture plate, a cell culture deep well plate, a cell culture chamber, a multi-layer culture dish, a rotating tube, a controlled bioreactor, a shake flask, a conical flask or a gas-permeable bag.

[0033] In another aspect, the present disclosure provides a genetically modified T cell produced by the aforementioned method.

[0034] Preferably, the genetically modified T cell is a CAR-T cell or a TCR-T cell.

[0035] In another aspect, the present disclosure provides use of the aforementioned cell in preparation of a medicament for treating a disease in a subject.

[0036] In another aspect, the present disclosure provides a method for treating a disease in a subject, comprising administering to the subject an effective amount of the aforementioned cell.

[0037] In another aspect, the present disclosure provides a pharmaceutical composition comprising the aforementioned cell and a pharmaceutically acceptable carrier. Advantages

[0038] Shaking the culture cells can achieve preparation of genetically modified T cells without sorting PBMCs, while also improving the contact probability of virus and cells.

[0039] The CAR-T cell preparation method provided by the present disclosure can reduce material costs, labor consumption, and virus usage; while achieving the above effects, the proportion of the final product CAR-T cells is ≥95%, the cell expansion multiple is 30-50, the cell viability is ≥95%, and the transduction rate is between 30%-60%. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 shows three process schemes for preparing CAR-T using unsorted PBMCs.

[0041] Figure 2 shows the final T cell expansion multiple when preparing CAR-T using PBMCs with different initial densities.

[0042] Figure 3 shows the effect of activator Dynabeads on expansion and transduction rate at different ratios relative to T cells.

[0043] Figure 4 shows the effect of activator CD3 / CD28 on expansion and transduction rate at different ratios relative to T cells.

[0044] Figure 5 shows the effect of activator TransAct on expansion and transduction rate at different ratios relative to T cells.

[0045] Figure 6 shows the effect of activator CD3 / CD28 / CD2 soluble tetramer on expansion and transduction rate at different ratios relative to T cells.

[0046] Figure 7 shows the effect of different cell densities during transduction on cell viability and transduction rate on Day 7.

[0047] Figure 8 shows the proportion of CD3 + cells in the harvested cells of the process for preparing CAR-T using unsorted PBMCs.

[0048] Figure 9 shows the virus transduction rate in the process for preparing CAR-T using unsorted PBMCs.

[0049] Figure 10 shows the proportion of various T cell phenotypes in the harvested cells of a process for making CAR-T using unsorted PBMC.

[0050] Figure 11 shows the cell viability of the harvested cells of a process for making CAR-T using unsorted PBMC.

[0051] Figure 12 shows the expansion fold of the harvested cells of a process for making CAR-T using unsorted PBMC. DETAILED DESCRIPTION

[0052] Definitions

[0053] The terms "a" or "an", as used herein, are defined as meaning "at least one or more" of the item being referred to.

[0054] As used herein, "PBMC" refers to peripheral blood mononuclear cells. In some embodiments, PBMCs can be collected or obtained from a subject by any suitable method known in the art. For example, in some embodiments, blood can be collected by venipuncture or any other blood collection method for collecting blood and / or PBMC samples.

[0055] In some embodiments, PBMCs can be obtained from the circulating blood of a subject by apheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In certain embodiments, cells collected by apheresis, particularly leukapheresis, can be washed to remove the plasma fraction and placed in an appropriate buffer or media for subsequent processing to isolate PBMCs from fresh apheresis or to resuscitate and wash previously isolated and frozen PBMCs to obtain PBMC cells. In certain embodiments, PBMC cells are obtained by resuscitating and washing previously isolated and frozen PBMCs.

[0056] As used herein, "genetically modified T cells" include CAR-T cells and TCR-T cells, "CAR-T cells" refer to engineered T cells containing a chimeric antigen receptor (CAR); "TCR-T cells" refer to engineered T cells containing a specific receptor (TCR) on the surface of T cells.

[0057] As used herein, "T cell subsets" include T stem central memory cells (TSCM), central memory T cells (TCM), effector memory T cells (TEM), and terminally differentiated effector memory cells (TEMRA).

[0058] As used herein, “viral vector” and “retroviral vector” are used interchangeably herein to mean any form of nucleic acid derived from a retrovirus and used to transfer genetic material into a cell via transduction. The term encompasses viral vector nucleic acids, e.g., DNA and RNA; encapsidated forms of these nucleic acids; and virions in which the viral vector nucleic acid has been packaged.

[0059] As used herein, the term “retroviral vector” refers to a vector derived from at least a portion of a retroviral genome, including, among others, self-inactivating lentiviral vectors provided in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). Other examples of lentiviral vectors that can be used in the clinic include, but are not limited to, for example, the gene delivery technology from Oxford BioMedica, the LENTIMAX™ vector system from Lentigen, and the like. Non-clinical types of lentiviral vectors can also be used and are known to those of skill in the art.

[0060] The term “multiplicity of infection” (hereinafter “MOI”) refers to the ratio of infectious agents, e.g., virions, to infectious targets, e.g., cells, in a medium, e.g., of a transduction procedure. In some embodiments, the MOI can equal the number of functional virions added to the target cells during a transduction procedure out of the total number added. In some embodiments, the number of functional virions added to a transduction procedure is determined by determining the titer of the functional virions. In some embodiments, the titer of functional virions is determined by using qPCR to determine the number of nucleic acid viral copies integrated per cell in a standard cell line that is stably transduced using techniques known in the art. See, e.g., Paugh, B.S. et al., Sci Rep 11, 389 (2021), incorporated by reference in its entirety. In some embodiments, the virions are retroviral virions. In some embodiments, the virions are lentiviral virions.

[0061] Transduction

[0062] Provided herein are methods for genetically modifying immune cells, including PBMCs and T cells produced by the methods of the present disclosure. In some embodiments of the methods and compositions disclosed herein, T cells are contacted ex vivo with replication-incompetent retroviral vectors to genetically modify the T cells to express an exogenous gene product. In some embodiments, the exogenous gene product is a CAR.

[0063] In some embodiments, the exogenous gene product is an epitope specific for a monoclonal antibody (i.e., specifically recognized by a monoclonal antibody), a suicide polypeptide, an inducible "on" or "accelerator" switch, such as inducible caspase-9 (U.S. Application 2011 / 0286980) or a thymidine kinase or "off switch. Exemplary mAb-specific epitopes are disclosed in International Patent Publication No. WO 2016 / 120216, which is incorporated herein by reference in its entirety. In some embodiments, the exogenous gene product is an R epitope, such as RQR8. See, e.g., WO2013153391A, which is incorporated herein by reference in its entirety. The R epitope, when expressed on the surface of a CAR immune cell, can be bound by Rituximab, causing the CAR immune cell to lyse. In some embodiments, the exogenous gene product is a control switch, such as a dimerization domain.

[0064] In some embodiments, transduction can be performed in the same container as the activation step, without removing any media. For example, blood cells, such as PBMCs, enriched and isolated from a collected blood sample can be activated in a shake flask and then contacted with retroviral particles in the same shake flask. In illustrative embodiments, the blood cells are separated, isolated, and / or purified from granulocytes, including neutrophils, prior to contact with the retroviral vector. The retroviral vector can be a replication-incompetent recombinant lentiviral particle in other exemplary embodiments, which can be introduced into the same gas-permeable bag containing the activated PBMCs to form a transduction reaction mixture. In some embodiments, the retroviral vector is added to the transduction reaction mixture during the activation step. In some embodiments, the retroviral vector is added to the transduction reaction mixture after the activation step. In some embodiments, the activation step is performed no more than 12 or 24 hours prior to or concurrently with the transduction step. FIG. 1 shows potential transduction time points in an exemplary CAR-T cell generation protocol. Media is typically present during transduction, such as media known in the art for culturing T cells ex vivo, including basal media and supplements, including cytokines, such as disclosed in greater detail herein.

[0065] The transduction reaction in some embodiments is initiated upon addition of the retroviral vector to the PBMC cells, which can be incubated between 23 and 39 °C, and in some exemplary embodiments at 37 °C. In some embodiments, the transduction reaction can be performed at 37-39 °C. In some embodiments, the transduction reaction is incubated at 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0% CO2. The transduction reaction can be incubated for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 25, 24, 36, 48 hours. In illustrative embodiments, the transduction reaction can be incubated at a pH within the starting pH range for a time between 1 and 2, 1 and 3, 1 and 4, 1 and 5, 1 and 6, 1 and 7, 1 and 8, 1 and 9, 1 and 10, 4 and 12, 4 and 14, 4 and 16, 4 and 18, 4 and 20, 4 and 24, 4 and 26, 4 and 28, 4 and 30, 4 and 32, 4 and 36, 4 and 38, 4 and 40, 4 and 42, 4 and 44, 4 and 46, 4 and 48, or 4 and 72 hours. In some embodiments, the transduction reaction pH is controlled passively, for example by adjusting culture medium buffers (e.g., sodium bicarbonate and / or HEPES) and incubator pCO2 at culture initiation to allow the desired pH (e.g., a pH greater than 7.0) to be achieved, for example a pH of 7.0 or greater. In some embodiments, the transduction reaction pH is controlled actively, for example by using a bioreactor with online pH measurement and pH feedback control loop that continuously (actively) adjusts culture pH by adding CO2 gas to maintain the pH.

[0066] In some embodiments, PBMC cells can be transduced at different retroviral or lentiviral particle to cell ratios, referred to as multiplicity of infection (MOI). In some embodiments, PBMC cells are transduced using an MOI (plaque forming units / cell) of between 0.25, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 50, 100, 150, 200, 250, 300, 350, 400, 450 to 500. In some embodiments, PBMC cells are transduced at an MOI of between 0.25, 0.50, 1.0, 5, 10, 15, 20, 25, or 30 to 50, 75, 100, 125, 150, 175, or 200. In some embodiments, PBMC cells are transduced at an MOI of about 1 to 10, 15, 20, 25, 30, 35, 40, 45, or 50. In some embodiments, PBMC cells are transduced at an MOI of 1 to 20.

[0067] In some embodiments, the cells are transduced by a viral vector comprising an extracellular nucleic acid. In some embodiments, the extracellular nucleic acid encodes a CAR. In some embodiments, the viral vector is a retroviral, lentiviral, or AAV vector. In some embodiments, the viral vector is a murine-derived retroviral vector.

[0068] The cells to be transduced to express a CAR can be derived from an allogeneic or autologous source. In one embodiment, in vitro transduction, culturing, and / or expansion of T cells is performed in the absence of non-human animal-derived products, such as fetal calf serum / fetal bovine serum.

[0069] In some embodiments, a transduction reaction cell population is incubated with a retroviral vector encoding a CAR at an MOI of at least 5, 10, 20, 30, 50, 100, 150, or 200 for 1 hour to 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 24, or 48 hours at a pH in the range of the starting pH and equal to or below 7.8 or equal to or below 7.9, and wherein at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% of T cells express the CAR at least days 3, 4, 5, or 6 after the retroviral vector first comes into contact with the cells during the transduction reaction.

[0070] Formulation and cryopreservation

[0071] In some embodiments, the engineered immune cells are formulated in a therapeutically effective amount by first harvesting the cells from their medium, then washing and concentrating the cells in a suitable infusion medium and container system ("pharmaceutically acceptable" carrier) for administration. Suitable infusion media can be any isotonic media formulation, typically normal saline Normosol™ R (Abbott) or Plasma-Lyte™ A (Baxter), but a 5% dextrose in water solution or Ringer's lactate can also be used. The infusion media can be supplemented with human serum albumin.

[0072] In another embodiment, the engineered immune cells, e.g., CAR-expressing T cells, are harvested, washed and concentrated, then cryopreserved at a predetermined cell concentration in a suitable cryopreservation media, e.g., CryoStor® CS10, CS10、 CS2 or CS5 (BioLife Solutions). Cryopreservation of the engineered immune cells, e.g., CAR-expressing T cells, is performed using standard procedures for storage and / or preparation for use in human subjects. If desired, the cryopreserved engineered immune cells can be thawed, grown and expanded to produce more such cells.

[0073] Chimeric antigen receptor

[0074] 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. When bound to a target antigen, a CAR can activate an immune cell to attack and destroy the cell (e.g., cancer cell) bearing the antigen. CARs can also incorporate co-stimulatory or signaling domains to increase their potency. See, e.g., Finney et al., Journal of Immunology, 1998, 161 :2791-2797; Song et al., Blood 119:696-706 (2012); Kala 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. Pat. Nos. 7,741,465 and 6,319,494.

[0075] The chimeric antigen receptors described herein comprise an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises an antigen binding domain. In some embodiments, an antigen-specific CAR comprises the following elements, from 5' to 3': a signal sequence, an antigen binding domain, a hinge and transmembrane region, and one or more consecutive signaling domains.

[0076] In some embodiments, the CAR further comprises a safety switch and / or a monoclonal antibody-specific epitope. See, e.g., WO2016 / 120216.

[0077] Antigen binding domain

[0078] As discussed above, the CARs described herein comprise an antigen binding domain. As used herein, "antigen binding domain" means any polypeptide that binds to a specified target antigen. In certain embodiments, the polypeptide structure of the antigen binding domain is based on an antibody. Antigen binding domains include, but are not limited to, antibody binding regions that are immunologically functional fragments. The term "immunologically functional fragment" (or "fragment") of an antigen binding domain is a species of antigen binding domain that comprises a portion of an antibody, however obtained or synthesized, that lacks at least some of the amino acids present in a full-length chain, but still is capable of specifically binding to a target antigen. Such fragments are biologically active in that they bind to a target antigen, and can compete with other antigen binding domains, including intact antibodies, for binding to a given epitope. Immunologically functional fragments include, but are not limited to, scFv fragments, Fab fragments (Fab', F(ab')2, etc.), one or more complementarity determining regions ("CDRs"), diabodies (a heavy chain variable domain linked to a light chain variable domain on the same polypeptide, via a short peptide linker that is too short to allow pairing between the two domains on the same chain), domain antibodies, bivalent antigen binding domains (comprising two antigen binding sites), multispecific antigen binding domains, and single chain antibodies. These fragments can be derived from any mammal, including but not limited to, humans, mice, rats, camelids, or lagomorphs.

[0079] In some embodiments, the antigen binding domain comprises one or more complementarity determining regions (CDRs) present in a full-length light or heavy chain of an antibody, and in some embodiments comprises a single chain and / or light chain or portions thereof. These fragments can be produced by recombinant DNA techniques or can be produced by enzymatic or chemical cleavage of an antigen binding domain, including intact antibodies.

[0080] In some embodiments, the antigen binding domain is an antibody, including one or more of its complementarity determining regions (CDRs), of a fragment thereof. In some embodiments, the antigen binding domain is a single chain variable fragment (scFv) comprising light chain CDRs CDR1, CDR2, and CDR3, and heavy chain CDRs CDR1, CDR2, and CDR3.

[0081] Generally, the assignment of amino acids to each of the framework, CDR, and variable domains generally follows the numbering scheme of the Kabat numbering (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991); Chothia numbering (see, e.g., Chothia and Lesk, (1987), J Mol Biol 196:901-917; Al-Lazikani et al., (1997) J Mol Biol 273:927-948; Chothia et al., (1992) J Mol Biol 227:799-817; Tramontano et al., (1990) J Mol Biol 215(1): 175-82; and U.S. Patent No. 7,709,226), the Contact Numbering or the AbM scheme (antibody modeling program, Oxford Molecular).

[0082] Variants of the antigen binding domain (e.g., variants of the CDRs, VH and / or VL) are also within the scope of the present disclosure, e.g., a variable light chain and / or a variable heavy chain each having at least 70-80%, 80-85%, 85-90%, 90-95%, 95-97%, 97-99%, or more than 99% identity to the amino acid sequence of the antigen binding domain sequence. In some cases, such molecules include at least one heavy chain and one light chain, while in other cases, the variant form contains two variable light chains and two variable heavy chains (or subportions thereof). One of skill in the art will be able to determine suitable variants of the antigen binding domain as set forth herein using well-known techniques. In certain embodiments, one 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.

[0083] In some embodiments, the antigen binding domain is a scFv. In some embodiments, the antigen selective CAR comprises a leader or signal peptide. As will be appreciated by those in the art, the antigen binding domain can include non-protein components.

[0084] The antigen binding domain in a CAR suitable for use in the methods and compositions of the disclosure can have a variety of antigen binding specificities. In some embodiments, the antigen binding domain is specific for an epitope present on an antigen expressed (synthesized) by a target cell. In one example, the target cell is a cancer cell associated antigen. The cancer cell associated antigen can be an antigen associated with, for example, a breast cancer cell, a B-cell lymphoma, a Hodgkin lymphoma cell, an ovarian cancer cell, a prostate cancer cell, mesothelioma, a lung cancer cell (e.g., a small cell lung cancer cell), a non-Hodgkin B-cell lymphoma (B-NHL) cell, an ovarian cancer cell, a prostate cancer cell, mesothelioma cell, a lung cancer cell (e.g., a small cell lung cancer cell), a melanoma cell, a chronic lymphocytic leukemia cell, an acute lymphocytic leukemia cell, a neuroblastoma cell, a glioma, a glioblastoma, a medulloblastoma, a colorectal cancer cell, and the like. The cancer cell associated antigen can also be expressed by a non-cancer cell.

[0085] Non-limiting examples of antigens to which a chimeric binding antigen can bind include, for example, CD19, CD20, CD38, CD30, ERBB2, CA125, MUC-1, prostate specific membrane antigen (PSMA), CD44 surface adhesion molecule, mesothelin, carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor-2 (VEGFR2), high molecular weight melanoma-associated antigen (HMW-MAA), MAGE-A1, IL-13R-a2, GD2, Ax1, Ror2, BCMA, Claudin, and isoforms thereof, and the like.

[0086] Hinge domain

[0087] The extracellular domain of a CAR of the disclosure can comprise a "hinge" domain (or hinge region). The term includes any polypeptide that functions to link the transmembrane domain in a CAR to the extracellular antigen binding domain in a CAR. In particular, a hinge domain can serve to provide greater flexibility and accessibility to the extracellular antigen binding domain.

[0088] A hinge domain can comprise up to 300 amino acids, in some embodiments 10 to 100 amino acids, or in some embodiments 25 to 50 amino acids. A hinge domain can be derived from all or a portion of a naturally occurring molecule, such as all or a portion of the extracellular region of CD8, CD4, CD28, 4-lBB, or IgG (specifically, the hinge region of IgG; it will be appreciated that the hinge region can contain some or all of the members of the immunoglobulin family, such as IgGl, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, or fragments thereof), or from all or a portion of an antibody heavy chain constant region.

[0089] Alternatively, the hinge domain can be a synthetic sequence corresponding to a naturally occurring sequence or can be a completely synthetic sequence. In some embodiments, the hinge domain is a portion of a human CD8a chain (e.g., NP_001139345.1). In another particular embodiment, the hinge and transmembrane domains comprise a portion of a human CD8a chain. In some embodiments, the hinge domain of a CAR described herein comprises a subsequence of a CD8a, CD28, IgGl, IgG4, PD-1, or FcyRIIIa molecule, specifically, a hinge region of any of a CD8a, CD28, IgGl, IgG4, PD-1, or FcyRIIIa molecule. In some embodiments, the hinge domain comprises a human CD8a hinge, a human IgGl hinge, a human IgG4 hinge, a human PD-1 hinge, or a human FcyRIIIa hinge. In some embodiments, a CAR disclosed herein comprises a scFv, a CD8a human hinge, and a transmembrane domain.

[0090] Transmembrane domain

[0091] A CAR of the present disclosure is designed with a transmembrane domain fused to the extracellular domain of the CAR. It can similarly 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 binding of such domains to transmembrane domains of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex. In some embodiments, a short linker can form a linkage between any one or some of the extracellular, transmembrane, and intracellular domains of the CAR. A transmembrane domain suitable for use in a CAR disclosed herein has the ability to: (a) be expressed on the surface of an immune cell, such as but not limited to a lymphocyte, for example a CD4+ cell such as a T helper (Th) cell, a CD8+ cell such as a cytotoxic T (Tc) cell, a T regulatory (Treg) cell, or a natural killer (NK) cell; and / or (b) interact with the extracellular antigen-binding domain and the intracellular signaling domain to direct a cellular response of the immune cell against a target cell.

[0092] The transmembrane domain can be derived from natural or synthetic sources. When the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. Particularly applicable to the transmembrane region of this disclosure are derived from (including or corresponding to) CD28, CD8, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed cell death-I (PD-1), inducible T cell co-stimulatory molecule (ICOS), lymphocyte function-associated antigen-I (LFA-1, CD1-1a / CD18), CD3γ, CD3δ, CD3ε, CD247, CD276 (B7-H3), LIGHT (TNFSF14), NKG2C, lgα (CD79a), DAP-10, Feγ receptor, MHC class I molecules, TNF receptor protein, immunoglobulin, cytokine receptor, integrin, signal transduction lymphocyte activation molecule (SLAM protein), activated NK cell receptor, BTLA, and Toll ligand receptor. , ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM(LIGHTR), KIRDS2, SLAMF7, NKp80(KLRFl), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA4, VL Al, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDIld, ITGAE, CD103, ITGAL, CDIla, LFA-1, ITGAM, CDIlb, ITGAX, CDIle, ITGBl, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, ligands that specifically bind to CD83, or any combination thereof.

[0093] As non-limiting examples, the transmembrane region can be derived from, or be a portion of, a T cell receptor, a polypeptide that constitutes the CD3 complex, an IL-2 receptor, p55 (a chain), p75 (P chain), or y chain, a subunit chain of the Fe receptor, particularly Fey receptor III, or a CD protein. Alternatively, the transmembrane domain can be synthetic and can comprise primarily hydrophobic residues, such as leucine and valine. In some embodiments, the transmembrane domain is derived from a human CD8a chain (e.g., NP_001139345.1).

[0094] Intracellular domain

[0095] The intracellular (cytoplasmic) domain of a CAR of the present disclosure can provide for activation of at least one normal effector function of an immune cell comprising the CAR, such as signal I / activation and / or signal II / co-stimulation. For example, an effector function of a T cell can refer to cytolytic activity or helper cell activity, including secretion of cytokines. In some embodiments, an activation intracellular signaling domain for use in a CAR can be, for example, but not limited to, the cytoplasmic sequences of T cell receptors and co-receptors that coordinate action upon antigen receptor engagement to initiate signal transduction, as well as any derivative or variant of these sequences and any synthetic sequence with the same functional ability.

[0096] It will be appreciated that suitable (e.g., activating) intracellular domains include, but are not limited to, signaling domains derived from (or corresponding to) CD3 zeta, CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, Programmed Death- 1 (PD-1), Inducible T-cell Costimulatory molecule (ICOS), Lymphocyte function-associated antigen- 1 (LFA-1, CD1 -la / CD18), CD3y, CD35, CD3s, CD247, CD276 (B7-H3), LIGHT (TNFSF14), NKG2C, lg a (CD79a), DAP-10, Fey receptor, MHC class 1 molecule, TNF receptor protein, immunoglobulin, cytokine receptor, integrin, signaling lymphocyte activation molecule (SLAM protein), activating NK cell receptor, BTLA, Tim-4, Tim-1, Tim-3, Tim-4, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRFl), NKp44, NKp30, NKp46, CD19, CD4, CD8a, CD8b, IL-2Rb, IL-2Ry, IL-7Ra, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD103, ITGAL, CD1 la, LFA-1, ITGAM, CD1 lb, ITGAX, CD1 le, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAMl (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAMl, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, a ligand that specifically binds with CD83, or any combination thereof.

[0097] In addition to the activating domains described above, the intracellular domain can incorporate a costimulatory signaling domain (interchangeably referred to herein as a costimulatory molecule) to increase its potency. The costimulatory domain can provide a signal in addition to the primary signal provided by the activating molecule as described herein.

[0098] It will be appreciated that suitable costimulatory domains within the scope of the present disclosure can be derived from (or correspond to) CD28, OX40, 4-1BB / CD137, CD2, CD3 (a, b, d, e, g, z), CD4, CDS, CD7, CD9, CD16, CD22, CD27, CD30, CD33, CD37, CD40, CD45, CD64, CD80, CD86, CD134, CD137, CD154, PD-1, ICOS, lymphocyte function-associated antigen-I (LFA-1 (CD1 la / CD18), CD247, CD276 (B7-H3), LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), NKG2C, lg a (CD79a), DAP-10, Fey receptor, MHC class I molecule, TNFR, integrin, signaling lymphocyte activation molecule, BTLA, Tymo ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRFl), NKp44, NKp30, NKp46, CD19, CD4, CD8a, CD8b, IL-2Rb, IL-2Ry, IL-7Ra, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl-ld, ITGAE, CD103, ITGAL, CDl-la, LFA-1, ITGAM, CDl-lb, ITGAX, CDl-lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAMl (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAMl, CRTAM, Ly9 (CD229), CD160 (BY55), PSGLl, CDlOO (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMFl, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83 ligand, or fragments thereof, or combinations thereof. It will be appreciated that additional costimulatory molecules or fragments thereof not listed above are within the scope of the present disclosure.

[0099] In some embodiments, the intracellular / cytoplasmic domain of the CAR can be designed to itself comprise a 4-1BB / CD137 domain or in combination with any other desired intracellular domain appropriate for the context of the CAR. The complete native amino acid sequence of 4-1BB / CD137 is described in NCBI Reference Sequence: NP_001552.2. The complete native 4-1BB / CD137 nucleic acid sequence is described in NCBI Reference Sequence: NM_001561.5.

[0100] In some embodiments, the intracellular / cytoplasmic domain of the CAR can be designed to itself comprise a CD28 domain or in combination with any other desired intracellular domain appropriate for the context of the CAR. The complete native amino acid sequence of CD28 is described in NCBI Reference Sequence: NP_006130.1. The complete native CD28 nucleic acid sequence is described in NCBI Reference Sequence: NM_006139.l.

[0101] In some embodiments, the intracellular / cytoplasmic domain of the CAR can be designed to itself comprise a CD3 zeta domain or in combination with any other desired intracellular domain appropriate for the context of the CAR. In some embodiments, the intracellular signaling domain of the CAR can comprise a CD31 signaling domain. For example, the intracellular domain of the CAR can comprise a portion of the CD3 zeta chain and a portion of a costimulatory signaling molecule. The intracellular signaling sequences within the intracellular signaling portion of the CAR can be linked to each other in random or in a specified order.

[0102] Nucleic acid and expression vector preparation

[0103] Provided herein are methods of making nucleic acids encoding a CAR and vectors comprising nucleic acids encoding a CAR.

[0104] A variety of known techniques can be utilized in making the polynucleotides and vectors according to the present disclosure. For example, certain methods for manufacturing constructs and engineered immune cells of the present disclosure are described in publication WO2015 / 120096, the entirety of which is incorporated herein by reference.

[0105] The nucleotide sequences encoding the CAR can be present in an expression vector. In the case where the CAR comprises two separate polypeptides, the nucleotide sequences encoding the two polypeptides can be cloned in the same or separate vectors. The expression vector can include selectable markers, origins of replication, and other features that provide for replication and / or maintenance of the vector. Suitable expression vectors include, for example, plasmids, viral vectors, and the like.

[0106] To clone a polynucleotide, an expression vector can be introduced into a host cell (an isolated host cell) to allow for replication of the vector itself, and thereby amplify the copies of the polynucleotide contained therein. A cloning vector can contain sequence components, typically including, but not limited to, an origin of replication, a promoter sequence, a transcription initiation sequence, an enhancer sequence, and an optional marker. These elements can be selected as appropriate by one of ordinary skill in the art. For example, an origin of replication can be selected to facilitate autonomous replication of the vector in the host cell.

[0107] In other embodiments, the present disclosure relates to an isolated polynucleotide encoding any of the antigen binding domains described herein. In some embodiments, the present disclosure relates to an isolated polynucleotide encoding a CAR. Vectors comprising the polynucleotides and methods of making the same are also provided herein.

[0108] In certain embodiments, the present disclosure provides an isolated host cell containing an expression vector provided herein. A host cell containing a vector can be used for expression or cloning of a 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, and more specifically, human cells.

[0109] Vectors can be introduced into host cells using any suitable method known in the art, including, but not limited to, DEAE-dextran-mediated delivery, calcium phosphate precipitation methods, cationic lipid-mediated delivery, liposome-mediated transfection, electroporation, microprojectile bombardment, receptor-mediated gene delivery, delivery mediated by poly-lysine, histones, chitosan, and peptides. Standard methods for viral transfection and transformation of cells for expression of a vector of interest are well known in the art. In another embodiment, a mixture of different expression vectors can be used to genetically modify a donor population of immune effector cells, wherein each vector encodes a different CAR as disclosed herein. The resulting transduced immune effector cells form a mixed population of engineered cells, wherein a proportion of the engineered cells express more than one different CAR.

[0110] Retroviral particle production

[0111] In illustrative embodiments disclosed herein, the transduction method can include the step of transducing an immune cell, such as a T cell, with a replication-defective recombinant retroviral particle comprising one or more nucleic acids to produce a transduced, engineered immune cell, such as an engineered T cell. In some embodiments, the one or more nucleic acids can encode one or more proteins, which are then expressed in the transduced T cell, such as a chimeric antigen receptor (CAR). The retroviral particles used to transduce T cells and / or NK cells in the methods provided herein can be prepared according to methods known in the art. As disclosed herein, retroviral particles are a common tool for gene delivery (Miller, Nature (1992) 357:455-460). In some embodiments, the replication-defective recombinant retroviral particle can be derived from the Alpharetrovirus genus, Betaretrovirus genus, Gammaretrovirus genus, Deltaretrovirus genus, Epsilonretrovirus genus, Lentivirus genus, or Spumavirus genus. There are many species of retroviruses suitable for use in the methods disclosed herein. A detailed list of retroviruses can be found in Coffin et al. (“Retroviruses”, 1997 Cold Spring Harbor Laboratory Press edited by JM Coffin, SM Hughes, HE Varmus, pp. 758-763). Details regarding the genomic structure of some retroviruses can be found in the art. For example, details regarding HIV can be found in the NCBI Genbank (i.e., Genome Accession Number AF033819).

[0112] In illustrative embodiments, the retroviral particle can be derived from a recombinant retrovirus from the Lentivirus genus and can be a replication-defective recombinant lentiviral particle. In some embodiments, the recombinant retrovirus can be derived from HIV, SIV, or FIV. In other illustrative embodiments, the recombinant retrovirus can be derived from a human immunodeficiency virus (HIV) in the Lentivirus genus.

[0113] In some embodiments, the replication incompetent recombinant retroviral particles can be grown in culture in a medium specific to replication incompetent recombinant retroviral particle manufacture. Any suitable growth medium and / or supplements for growing replication incompetent recombinant retroviral particles can be used for replication incompetent recombinant retroviral particle inoculum according to the methods described herein. According to some aspects, the retroviral particles can be subsequently added to the medium during transduction.

[0114] Replication incompetent recombinant retroviral particles can be produced according to methods known in the art using mammalian cell lines. Suitable mammalian cells include primary cells and immortalized cell lines. Suitable mammalian cell lines can include human cell lines. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) number CCL-2), CHO cells (e.g., ATCC number CRL 9618, CCL61, CRL 9096), 293 cells (e.g., ATCC number CRL-1573), Vero cells, NIH3T3 cells (e.g., ATCC number CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC number CCL10), PC12 cells (ATCC number CRL 1721), COS cells, COS-7 cells (ATCC number CRL 1651), human embryonic kidney (HEK) cells (ATCC number CRL 1573), HLHepG2 cells, Hut-78, Jurkat, HL-60, NK cell lines (e.g., NKL, NK92, and YTS), and the like. In some cases, the cells are not immortalized cell lines, but rather cells obtained from an individual or cells ex vivo (e.g., primary cells). For example, in some embodiments, the cells are immune cells obtained from an individual. As another example, the cells are stem cells or progenitor cells obtained from an individual.

[0115] Methods of treatment

[0116] Methods for treating a disease or disorder, including cancer, are provided. In some embodiments, the disclosure relates to generating a T cell-mediated immune response in a subject comprising administering to the subject an effective amount of an engineered immune cell of the disclosure. In some embodiments, the T cell-mediated immune response is directed against a target cell or cells. In some embodiments, the engineered immune cell comprises a chimeric antigen receptor (CAR). In some embodiments, the target cell is a tumor cell. In some aspects, the disclosure comprises 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 comprises a method for treating or preventing a malignancy comprising administering to a subject in need thereof an effective amount of at least one immune cell, wherein the immune cell comprises at least one chimeric antigen receptor and / or isolated antigen binding domain as described herein.

[0117] In some embodiments, the subject has a solid tumor or a hematological malignancy, such as a lymphoma or leukemia. In some embodiments, the cancer is present in the subject’s bone marrow. In some embodiments, the engineered cell is an autologous immune cell, e.g., an autologous T cell. In some embodiments, the engineered cell is an allogeneic immune cell, e.g., an allogeneic T cell. In some embodiments, the engineered cell is a heterologous immune cell, e.g., a heterologous T cell. In some embodiments, the engineered cell is transfected and / or transduced ex vivo. As used herein, the term “ex vivo cell” refers to any cell cultured ex vivo. A “therapeutically effective amount,” “effective dose,” “effective amount,” or “therapeutically effective dose” of a therapeutic agent, e.g., an engineered CAR T cell, is any amount that, when used alone or in combination with another therapeutic agent, protects a subject from the onset of a disease or promotes the regression of a disease as evidenced by a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction. The ability of a therapeutic agent to promote the regression of a disease can be evaluated using a variety of methods known to the skilled practitioner, e.g., in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by analyzing the activity of the agent in in vitro assays.

[0118] The terms “patient” and “subject” are used interchangeably and include human subjects as well as those subjects having a formally diagnosed disorder, those subjects not having a formally recognized disorder, those subjects under medical observation, those subjects at risk for developing a disorder, and the like.

[0119] The terms "treat" and "treatment" include therapeutic treatment, prophylactic treatment, and disclosure where one reduces the risk or other risk factors that a subject will develop a disorder. Treatment does not require complete cure of the disorder and encompasses embodiments where one reduces symptoms or underlying risk factors. The term "prevent" does not require 100% elimination of the likelihood of an event occurring. Rather, it indicates that the likelihood of the event occurring has been reduced in the presence of the compound or method.

[0120] The total amount of cells desired in the composition comprises at least 2 cells (e.g., at least one CD8+ T cell and at least one CD4+ T cell, or two CD8+ T cells or two CD4+ T cells) or more typically more than 10 2 cells and up to 10 6 cells, up to and including 10 8 or 10 9 cells, and can be 10 10 or 10 12 or more cells. The number of cells will depend on the intended use of the composition and the type of cells included therein. The density of the desired cells is typically more than 10 6 cells per milliliter and is typically more than 10 7 cells per milliliter, typically 10 8 cells / ml or higher. A clinically relevant number of immune cells can be divided into multiple infusions, the cumulative amount of which equals or exceeds 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 or 10 12 cells. In some aspects of the disclosure, particularly since all of the infused cells will be redirected to a specific target antigen, a lower number of cells in the range of 10 6 per kilogram (10 6 - 10 11 per patient) can be administered. CAR therapy can be administered multiple times at doses within these ranges.

[0121] The cells can be autologous, allogeneic, or heterologous to the patient being treated.

[0122] In some embodiments, a therapeutically effective amount of the CAR T cells is about 1 x 10 5 cells per kilogram, about 2 x 10 5 cells per kilogram, about 3 x 10 5 cells per kilogram, about 4 x 10 5 cells per kilogram, about 5 x 105 Cells / kg, approximately 6 × 10⁻⁶ 5 Cells / kg, approximately 7 × 10⁻⁶ 5 Cells / kg, approximately 8 × 10⁻⁶ 5 Cells / kg, approximately 9 × 10⁻⁶ 5 Cells / kg, 2×10 6 Cells / kg, approximately 3 × 10⁻⁶ 6 Cells / kg, approximately 4 × 10⁻⁶ 6 Cells / kg, approximately 5 × 10⁻⁶ 6 Cells / kg, approximately 6 × 10⁻⁶ 6 Cells / kg, approximately 7 × 10⁻⁶ 6 Cells / kg, approximately 8 × 10⁻⁶ 6 Cells / kg, approximately 9 × 10⁻⁶ 6 Cells / kg, approximately 1×10⁻⁶ 7 Cells / kg, approximately 2 × 10⁻⁶ 7 Cells / kg, approximately 3 × 10⁻⁶ 7 Cells / kg, approximately 4 × 10⁻⁶ 7 Cells / kg, approximately 5 × 10⁻⁶ 7 Cells / kg, approximately 6 × 10⁻⁶ 7 Cells / kg, approximately 7 × 10⁻⁶ 7 Cells / kg, approximately 8 × 10⁻⁶ 7 Cells / kg or approximately 9 × 10⁻⁶ 7 Cells / kg.

[0123] In some implementations, the target dose of CAR+ / CAR-T+ cells is approximately 1 × 10⁻⁶. 6 To approximately 1×10 10 Within the range of cells per kilogram, for example, approximately 1 × 10⁻⁶. 6 Cells / kg, approximately 1×10⁻⁶ 7 Cells / kg, approximately 1×10⁻⁶ 8 Cells / kg, approximately 1×10⁻⁶ 9 Cells / kg or approximately 1×10⁻⁶ 10 Cells / kg. It should be understood that dosages above and below this range may be suitable for some subjects, and the appropriate dosage level may be determined by the healthcare provider as needed. Additionally, multiple doses of cells may be provided according to this disclosure.

[0124] In some aspects, this disclosure includes pharmaceutical compositions comprising at least one antigen-binding domain as described herein and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition further comprises an additional active agent.

[0125] The CAR-expressing cell populations of the present disclosure can be administered alone or in pharmaceutical compositions in combination with diluents and / or with other components, such as IL-2 or other cytokines or cell populations. Pharmaceutical compositions of the present disclosure can comprise a population of CAR-expressing cells, such as T cells as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions can comprise buffers, such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates, such as glucose, mannose, sucrose, or dextrans, mannitol; proteins; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions of the present disclosure are preferably formulated for intravenous administration.

[0126] Pharmaceutical compositions (solutions, suspensions, etc.) can include one or more of the following: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or digylcerides, which can serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. For therapeutic purposes, the injectable pharmaceutical composition is preferably sterile.

[0127] The methods can further comprise administering one or more chemotherapeutic agents to the patient prior to administering the engineered cells provided herein. In certain embodiments, the chemotherapeutic agent is a lymphodepleting (preconditioning) chemotherapeutic agent. For example, methods of conditioning a patient in need of a T cell therapy comprise administering to the patient a specified beneficial dose of cyclophosphamide (between every day 200 mg / m2and every day 2000 mg / m2, between every day about 100 mg / m2and every day about 2000 mg / m2; e.g., every day about 100 mg / m2, every day about 200 mg / m2, every day about 300 mg / m2, every day about 400 mg / m2, every day about 500 mg / m2, every day about 600 mg / m2, every day about 700 mg / m2, every day about 800 mg / m2, every day about 900 mg / m2, every day about 1000 mg / m2, every day about 1500 mg / m2, or every day about 2000 mg / m2) and a specified dose of fludarabine (between every day 20 mg / m2and every day 900 mg / m2, between every day about 10 mg / m2and every day about 900 mg / m2; e.g., every day about 10 mg / m2, every day about 20 mg / m2, every day about 30 mg / m2, every day about 40 mg / m2, every day about 40 mg / m2, every day about 50 mg / m2, every day about 60 mg / m2, every day about 70 mg / m2, every day about 80 mg / m2, every day about 9020 mg / m2, every day about 100 mg / m2, every day about 500 mg / m2, or every day about 900 mg / m2). An exemplary dosing regimen involves treating a patient comprising administering to the patient a therapeutically effective amount of engineered T cells three days prior to administering to the patient every day about 300 mg / m2cyclophosphamide in combination or prior to or following every day administration of about 30 mg / m2fludarabine.

[0128] In other embodiments, the antigen binding domain, the transduced (or otherwise engineered) cell, and the chemotherapeutic agent are each administered in an amount effective to treat a disease or condition in a subject.

[0129] In certain embodiments, compositions comprising the immune effector cells expressing a CAR disclosed herein can be administered in combination with any number of chemotherapeutic agents. 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 methyl triazenes including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and triethylenethphosphoramide; nitrogen mustards such as chlorambucil, chlornediamine, chlornediamine oxide hexafluoride, cyclophosphamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folate analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane;Folic acid supplements, such as frolinic acid; acetomenophen; aldehyde dehydrogenase; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamide; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"- trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel (TAXOL™, Bristol-Myers Squibb) and doxetaxel (TAXOTERE®, Aventis); Rhone-Poulenc Rorer); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, e.g., cisplatin and carboplatin; vinblastine platinum; etoposide (VP- 16); ifosamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid derivatives, such as Targretin™ (bexarotene), Panretin™ (alitretinoin); ONTAK™ (denileukin diftitox); esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. This definition also includes anti-hormonal agents that act to regulate or inhibit hormone action on tumors, such as anti-estrogens (including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)- imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LYl 7018, onapristone, and toremifene (Fareston)); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Combinations of chemotherapeutic agents are also made and administered as appropriate, including but not limited to CHOP, i.e., cyclophosphamide doxorubicin (adriamycin), vincristine and prednisone.

[0130] In some embodiments, the chemotherapeutic agent is administered concurrently or within one week of administration of the engineered cell, polypeptide, or nucleic acid. In other embodiments, the chemotherapeutic agent is administered about 1-7 days, about 1 to about 4 weeks, or about 1 week to about 1 month, about 1 week to about 2 months, about 1 week to about 3 months, about 1 week to about 6 months, about 1 week to about 9 months, or about 1 week to about 12 months after administration of the engineered cell, polypeptide, or nucleic acid. In other embodiments, the chemotherapeutic agent is administered at least 1 month prior to administration of the cell, polypeptide, or nucleic acid. In some embodiments, the method further comprises administering two or more chemotherapeutic agents.

[0131] 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 (Cotellic®), and atezolizumab (Tecentriq®). Additional therapeutic agents suitable for use in combination with the present disclosure include, but are not limited to: ibrutinib (Imbruvica®), ofatumumab (Arzerra®), rituximab (Rituxan®), bevacizumab (Avastin®), trastuzumab (Herceptin®), trastuzumab emtansine (Kadcyla®), imatinib (Gleevec®), cetuximab (Erbitux®), panitumumab (Vectibix®), catumaxomab (Removab®), ibritumomab, ofatumumab, tositumomab (Bexxar®), brentuximab (Adcetris®), alemtuzumab (Campath®), gemtuzumab (Mylotarg®), erlotinib (Tarceva®), gefitinib (Iressa®), vandetanib (Caprelsa®), afatinib (Giotrif®), lapatinib (Tycerb®), neratinib (Nerlynx®), axitinib (Inlyta®), masitinib (Moril®), pazopanib (Votrient®), sunitinib (Sutent®), sorafenib (Nexavar®), toceranib (Palladia®), lestaurtinib (CEP-701), axitinib, cediranib (Repatha®), lenvatinib (Lenvima®), nintedanib (Ofev®), pazopanib, regorafenib (Stivarga®), semaxanib (Nexavar®), sorafenib, sunitinib, tivozanib (Fotivda®), toceranib, vandetanib, entrectinib (Xalkori®), cabozantinib (Cometriq®), imatinib, dasatinib (Sprycel®), nilotinib (Tasigna®), ponatinib (Inpatha®), radotinib (Panzymra®), bosutinib (Bosulif®), lestaurtinib, ruxolitinib (Jakavi®), pacritinib (INREB-A®), cobimetinib (Cotellic®), selumetinib (Selumetinib), trametinib (Mekinist®),Binimetinib, alectinib, ceritinib, crizotinib, aflibercept, adipotide, denileukin diftitox; mTOR inhibitors such as Everolimus and Temsirolimus; hedgehog inhibitors such as sonidegib and vismodegib; CDK inhibitors such as CDK inhibitor (palbociclib).

[0132] In some embodiments, a composition comprising immune cells expressing a CAR can be administered with a therapeutic regimen to prevent cytokine release syndrome (CRS) or neurotoxicity. Therapeutic regimens to prevent cytokine release syndrome (CRS) or neurotoxicity can include l enzilumab, tocilizumab, atrial natriuretic peptide (ANP), anakinra, iNOS inhibitors (e.g., L-NIL or 1400W). In additional embodiments, a composition comprising immune cells comprising a CAR can be administered with an anti-inflammatory agent. Anti-inflammatory agents or drugs include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, ethylprednisolone, prednisolone, prednisone, triamcinolone); non-steroidal anti-inflammatory drugs (NSAIDS), including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF agents, cyclophosphamide, and mycophenolate. Exemplary NSAIDs include ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors, and sialylates. Exemplary analgesics include acetaminophen, oxycodone, tramadol hydrochloride. Exemplary glucocorticoids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Exemplary biologic response modifiers include molecules against cell surface markers (e.g., CD4, CDS, etc.), cytokine inhibitors (such as TNF antagonists (e.g., etanercept, adalimumab, and infliximab)), chemokine inhibitors, and adhesion molecule inhibitors. Biologic response modifiers include monoclonal antibodies as well as recombinant forms of the molecules.Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, Gold (oral (auranofin) and intramuscular) and minocycline.

[0133] In certain embodiments, the compositions described herein are administered in conjunction with a cytokine. Examples of cytokines are lymphokines, monokines, and traditional polypeptide hormones. Cytokines include growth hormone such as human growth hormone, N- endorphin human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor (HGF); fibroblast growth factor (FGF); prolactin; placental lactogen; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors (NGFs) such as NGF-β; platelet growth factor; transforming growth factors (TGFs) such as TGF-α and TGF-β; insulin-like growth factor-I and II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-α, β, and γ; colony stimulating factors (CSFs), such as macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-lα, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15, IL-21; tumor necrosis factor such as TNF-α or TNF-β; 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 and biologically active equivalents of the native sequence cytokines.

[0134] Kits and articles of manufacture

[0135] The present disclosure provides kits comprising CAR-T cells obtained using the methods provided herein, and pharmaceutical compositions thereof. In one embodiment of the kit, the engineered CAR cells are frozen in a suitable medium, e.g. CS10, CS2 or CS5 (BioLife Solutions).

[0136] The present disclosure also provides an article of manufacture comprising any of the therapeutic compositions or kits described herein. Examples of articles of manufacture include a vial (e.g., a sealed vial comprising CAR-expressing immune cells).

[0137] As used herein, "volume made up with medium" refers to the addition of medium during fermentation, i.e., the addition of fresh medium to a fermentation vessel so that there is a certain volume of liquid in the vessel.

[0138] The technical solutions of the present disclosure will be further described in detail below in conjunction with specific embodiments. It should be understood that the following examples are only illustratively described and explained the present disclosure, and should not be interpreted as limiting the scope of protection of the present disclosure. Any technology realized based on the above description of the present disclosure is covered within the scope intended to be protected by the present disclosure.

[0139] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0140] Example 1: Preparation of CAR-T using unsorted PBMC

[0141] 1.1 The process scheme for preparing CAR-T cells using unsorted PBMC is divided into the following 3:

[0142] 1.1.1 Scheme One

[0143] (1) On Day 0, isolate PBMC from fresh apheresis, or thaw and wash previously isolated and frozen PBMC, and adjust the starting density of PBMC;

[0144] (2) On Day 0, transfer the PBMC cells of step (1) into a shake flask, and make up the volume with medium to the starting culture volume;

[0145] (3) On Day 0, add an activator to the shake flask of step (2);

[0146] (4) On Day 0, add a viral vector to the shake flask of step (3) for transduction;

[0147] (5) On Day 0, place the shake flask of step (4) in a CO2 shaking incubator for shaking culture;

[0148] (6) On Day 1, add medium to the shake flask to the harvest volume; on Day 1, place the shake flask in a CO2 shaking incubator for culture until Day 6;

[0149] (7) On Day 6, remove the shake flask for cell harvesting and freezing.

[0150] 1.1.2 Scheme Two

[0151] (1) On Day 0, PBMC cells were isolated from fresh apheresis blood, or previously isolated and cryopreserved PBMC were thawed and washed, and PBMC starting density was adjusted;

[0152] (2) On Day 0, PBMC cells from step (1) were transferred into a shake flask, and the volume was made up to the starting culture volume with culture medium;

[0153] (3) On Day 0, activator was added to the shake flask from step (2);

[0154] (4) On Day 1, viral vector was added to the shake flask from step (3) for transduction;

[0155] (5) On Day 1, the shake flask from step (4) was placed in a CO2 shaker incubator for culture;

[0156] (6) On Day 2, culture medium was added to the shake flask to the harvest volume; On Day 1, the shake flask was placed in a CO2 shaker incubator for culture until Day 6;

[0157] (7) On Day 6, the shake flask was removed for cell harvesting and cryopreservation.

[0158] 1.1.3 Protocol Three

[0159] (1) On Day 0, PBMC cells were isolated from fresh apheresis blood, or previously isolated and cryopreserved PBMC were thawed and washed, and PBMC starting density was adjusted;

[0160] (2) On Day 0, PBMC cells from step (1) were transferred into a shake flask, and the volume was made up to the starting culture volume with culture medium;

[0161] (3) On Day 0, activator was added to the shake flask from step (2);

[0162] (4) On Day 2, viral vector was added to the shake flask from step (3) for transduction;

[0163] (5) On Day 2, the shake flask from step (4) was placed in a CO2 shaker incubator for culture;

[0164] (6) On Day 3, culture medium was added to the shake flask to the harvest volume; On Day 1, the shake flask was placed in a CO2 shaker incubator for culture until Day 6;

[0165] (7) On Day 6, the shake flask was removed for cell harvesting and cryopreservation.

[0166] 1.2 Parameter Optimization of Preparation Protocols

[0167] 1.2.1 Starting density of PBMC

[0168] The starting density of PBMC cells in step (1) was tested from 1e6 / ml to 5e6 / ml, and the results are shown in Figure 2. The optimal starting density is between 1e6 / ml and 3.5e6 / ml.

[0169] 1.2.2 Starting culture volume of PBMC

[0170] The starting culture volume of PBMC cells in step (2) was tested from 50ml to 300ml, and the optimal starting culture volume is between 75ml and 250ml.

[0171] 1.2.3 Medium screening

[0172] X VIVO, AIM V, Optmizer, Optivitro, TexMacs, NutriT and T VIVO media were tested.

[0173] 1.2.4 Type and ratio of activator to cells

[0174] Commonly used activators on the market were tested, such as Dynabeads (Thermo, 11456D), CD3 / CD28 (Tianlihaiyuan, cat: GMP-TL603-1000), TransAct (Miltenyi Biotec, cat: 200-076-204) and CD3 / CD28 / CD2 soluble tetramer (Stemcell technologies, cat: 10970), and their different ratios to cells, and the results are shown in Figures 3-6. According to the test results, for Dynabeads and CD3 / CD28 magnetic beads, the ratio of the number of magnetic beads to the number of cells is 0.5:1, 1:1, 2:1, 3:1 and 4:1; for TransAct reagent, the ratio of culture volume to reagent volume is 60:1 to 10:1; for CD3 / CD28 / CD2 soluble tetramer, the ratio of culture volume to reagent volume is 200:1 to 10:1.

[0175] 1.2.5 Virus transduction condition test

[0176] 1.2.5 Virus transduction condition test

[0177] ​Different virus transduction time and cell density at the time of adding virus vector transduction were tested, wherein the transduction time was 0-72 hours. Cell density between le6-5e6 / ml was tested, and the experimental results are shown in Figure 7, wherein the transduction rate was optimal when the cell density was between le6-3.5e6 / ml.

[0178] 1.2.6 Cytokine screening

[0179] The following combinations of cytokines were tested: IL2 alone, IL2+IL7+IL15 combination, IL7 alone, IL15 alone, IL7+IL15 combination, IL7+IL15+IL21 combination, all of which were applicable.

[0180] 1.3 Verification of CAR-T cell production process effect

[0181] After parameter optimization, CAT cells were prepared using the following process and the corresponding indicators of the prepared CAR-T cells were verified.

[0182] (1) On Day 0, fresh apheresis blood was subjected to PBMC separation to obtain PBMC cells, or previously separated and frozen PBMC was recovered and washed, and the starting density of PBMC was adjusted to 3e6 / ml;

[0183] (2) On Day 0, the PBMC cells of step (1) were transferred to a shake flask, and the volume was supplemented to the initial culture volume of 200ml using the culture medium;

[0184] (3) On Day 0, Dynabeads CD3 / CD28 was added to the shake flask of step (2), and the ratio of the number of magnetic beads to the number of cells was 2:1;

[0185] (4) On Day 0, lentiviral vector was added to the shake flask of step (3), and the cell density in the shake flask was 3.5e6 / ml at this time;

[0186] (5) On Day 0, the shake flask of step (4) was placed in a CO2 shaking incubator for shaking culture, and the shaking speed was 75rpm;

[0187] (6) On Day 1, the culture medium was supplemented to 1L of harvest volume in the shake flask; on Day 1, the shake flask was placed in a CO2 shaking incubator for culture until Day 6;

[0188] (7) On Day 6, the shake flask was taken out for cell harvesting and freezing.

[0189] Through the above process, PBMC cells from 10 donors were verified, and the cells from 10 donors were cultured for 6 days. The CD3 +The proportion of cells of the total was greater than 95% (as shown in Figure 8); the transduction rate of the virus expressing the CAR structure was between 36% and 60% (as shown in Figure 9); the proportion of cells of the total of the harvested cells with the phenotype of Tscm+Tcm was greater than 80% (as shown in Figure 10); the viability of the harvested cells was all greater than 95% (as shown in Figure 11); and the expansion multiple of the harvested cells was between 30 and 50 times (as shown in Figure 12). The above results show that the process has good stability.

[0190] The above describes the embodiments of the present disclosure. However, the present disclosure is not limited to the above-described embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present disclosure shall be included in the protection scope of the present disclosure.

Claims

1. A method of making a genetically modified T cell, comprising, It comprises the following steps: (1) Adjust the initial density of PBMC to 1e6 / ml-5e6 / ml by medium on Day 0; (2) Transfer the PBMC cells of the density in step (1) into a container and make up to the initial culture volume with medium on Day 0; (3) Add an activator to the container of step (2) on Day 0; (4) Add a viral vector for transduction to the container of step (3) between Day 0 and Day 2, and culture the container after adding the viral vector; (5) Add medium to the container to the harvest volume after at least 0.5 days after adding the viral vector, and then culture to the harvest day; (6) Take out the container for cell harvesting on the harvest day.

2. The method of claim 1, wherein the initial density of PBMC in step (1) is 1e6 / ml-3.5e6 / ml; preferably, the initial density of PBMC is 2e6 / ml, 2.2e6 / ml, 2.5e6 / ml, 2.8e6 / ml, 3e6 / ml or 3.2e6 / ml.

3. The method of claim 1 or 2, wherein the initial culture volume in step (2) is 75ml-250ml; preferably, the initial culture volume is 75ml, 100ml, 150ml, 175ml or 200ml.

4. The method of any one of claims 1-3, wherein the medium is selected from the group consisting of X VIVO, AIM V, Optmizer, Optivitro, TexMacs, NutriT and T VIVO. CD3 / CD28, the ratio of the number of magnetic beads to the number of cells is 0.5:1, 1:1, 2:1, 3:1 or 4:1, more preferably 2:1; 5. The method according to any one of claims 1-4, wherein the activator in step (3) is selected from Dynabeads, CD3 / CD28, TransAct, and CD3 / CD28 / CD2 soluble tetramers. Preferably, the activator is Dynabeads or Preferably, the activator is TransAct, and the ratio of the culture volume to the volume of the activator is 60:1-10:1; More preferably, the ratio is 50:1, 40:1, 30:1 or 20:

1. Preferably, the activator is CD3 / CD28 / CD2 soluble tetramer, and the ratio of the culture volume to the volume of the activator is 200:1-10:1; more preferably, the ratio is 100:1, 75:1, 50:1, 25:1 or 20:

1.

6. The method of any one of claims 1-5, wherein the cell density in the container during the addition of the viral vector for transduction in step (4) is 1e6-3.5e6 / ml; Preferably, the cell density is 1.2e6 / ml, 1.5e6 / ml, 1.8e6 / ml, 2e6 / ml, 2.2e6 / ml, 2.5e6 / ml, 2.8e6 / ml, 3e6 / ml, 3.2e6 / ml.

7. The method of any one of claims 1-6, wherein the culture in step (4) is a shaking culture, a rocking shaking culture or a stirring culture; Preferably, the shaking culture has a rotation speed of 50-100rpm; more preferably, the shaking culture has a rotation speed of 75rpm; ​ Preferably, the rotation speed of the swing-shaking culture is 5-30 rpm, and the angle is 2-30°; more preferably, the rotation speed of the swing-shaking culture is 15 rpm, and the angle is 15°. Preferably, the rotation speed of the stirring culture is 50-300 rpm; more preferably, the rotation speed of the swing-shaking culture is 15 rpm.

8. The method of any one of claims 1-7, wherein the medium is supplemented to the harvest volume in the container after 0.5, 1, 1.5, or 2 days after the addition of the viral vector in step (5). Preferably, the medium is supplemented to the harvest volume in the container after 1 day after the addition of the viral vector in step (5).

9. The method of any one of claims 1-8, wherein the harvest day in step (6) is Day 4, Day 5, Day 6, Day 7, or Day 8. Preferably, the harvest day in step (6) is Day 6.

10. The method of any one of claims 1-9, wherein the container is selected from the group consisting of a cell culture plate, a cell culture deep well plate, a cell culture chamber, a multi-layer culture dish, a spin tube, a controlled bioreactor, a shake flask, a conical flask, or a gas-permeable bag.

11. A genetically modified T cell produced by the method of any one of claims 1-10. Preferably, the genetically modified T cell is a CAR-T cell or a TCR-T cell.

12. Use of the cell of claim 11 in the manufacture of a medicament for treating a disease in a subject.

13. A pharmaceutical composition comprising the cell of claim 11 and a pharmaceutically acceptable carrier.

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