Method for rapidly preparing CAR-T cells

Abstract

The present invention relates to a method for rapidly preparing chimeric antigen receptor T cells (CAR-T), comprising the steps of: 1) contacting T cells or a cell population comprising the T cells with a stimulating agent to activate the T cells to obtain activated T cells or a cell population comprising the activated T cells; 2) contacting the activated T cell or a cell population comprising activated T cells with a nucleic acid molecule encoding a chimeric antigen receptor (CAR), thereby obtaining a CAR-T cell expressing the CAR; and (3) harvesting the CAR-T cells, culturing for 18-24 hours, harvesting after the culture is finished, and freezing or not freezing. According to the method, the total preparation time of the CAR-T cells is greatly shortened, and meanwhile, the amplification and tumor killing capabilities of the CAR-T cells are remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the biological field, and specifically relates to the field of immune cell therapy. Background Art

[0002] Chimeric Antigen Receptor (CAR) T cells are a type of cell therapy product that uses genetic engineering to modify T cells so that they express CAR molecules on the cell surface that recognize specific antigens of tumor malignant cells, guide CAR-T cells to bind to tumor malignant cells and activate T cell killing function.

[0003] The preparation of CAR-T cells with existing technologies usually takes a long time. Whether rapid preparation of CAR-T cells can be achieved and how to further improve the efficacy of CAR-T based on rapid preparation remain to be explored. Summary of the invention

[0004] The present invention provides a method for preparing chimeric antigen receptor T cells, the method comprising the steps of:

[0005] 1) contacting T cells or a cell population containing T cells with a stimulating agent to activate T cells, thereby obtaining activated T cells or a cell population containing activated T cells;

[0006] 2) contacting the activated T cells or a cell population comprising activated T cells with a nucleic acid molecule encoding a chimeric antigen receptor (CAR) to obtain chimeric antigen receptor T cells (CAR-T cells) expressing the CAR;

[0007] 3) harvesting the chimeric antigen receptor T cells, culturing them for 18-24 hours, harvesting them after the culture is completed, and freezing or not freezing them.

[0008] In one or more embodiments, step 1) comprises: obtaining the T cells or a cell population comprising T cells by sorting from fresh PBMCs or frozen PBMCs.

[0009] In one or more embodiments, the stimulatory reagent comprises one or more components capable of activating the intracellular signaling domain of the TCR complex. Preferably, the stimulatory reagent is an anti-CD3 antibody or an antigen-binding fragment thereof and / or an anti-CD28 antibody or an antigen-binding fragment thereof. More preferably, the stimulatory reagent is in the form of magnetic beads coupled to anti-CD3 antibodies and anti-CD28 antibodies.

[0010] In one or more embodiments, step 2) the method for obtaining chimeric antigen receptor T cells expressing CAR is selected from viral-mediated transduction or non-viral-mediated transduction. In certain embodiments, the viral-mediated transduction is retroviral, lentiviral or adenoviral-mediated transduction. In certain embodiments, the non-viral-mediated transduction is transposon or electroporation-mediated transduction.

[0011] In one or more embodiments, the second harvest of step 3) comprises a step of removing the magnetic beads.

[0012] In one or more embodiments, the culturing time of step 3) is 19-23 hours, preferably 20-22.5 hours.

[0013] In one or more embodiments, the freezing of step 3) comprises: washing the T cells with sodium chloride injection, resuspending the T cells with freezing solution, programmed cooling, and storing in liquid nitrogen.

[0014] In one or more embodiments, step 1) comprises: contacting T cells or a cell population comprising T cells with magnetic beads coupled to anti-CD3 antibodies and anti-CD28 antibodies, magnetically separating CD3 + T cells, activated cultured CD3 + Preferably, the T cells or cell populations containing T cells are incubated with magnetic beads coupled with anti-CD3 antibodies and anti-CD28 antibodies for 30-45 minutes, and CD3 + Cells were then activated and cultured.

[0015] In one or more embodiments, CD3 + The initial cell density of cell activation culture is 1-5×10 6 cells / mL.

[0016] In one or more embodiments, the culture medium for activation culture in step 1) is AIM-V, X-VIVO, DMEM, RPMI1640, and more preferably X-VIVO15; more preferably, the culture medium is also supplemented with one or more of acetylcysteine, GlutaMAX, HEPES and human plasma.

[0017] In one or more embodiments, the medium of step 1) activation culture contains IL-2, more preferably, the concentration thereof is 50-500 IU / ml, preferably 100-400 IU / ml, more preferably 300 IU / ml.

[0018] In one or more embodiments, the culture conditions of step 1) activation culture are about 37° C. and about 5% CO 2 environment.

[0019] In one or more embodiments, step 2) comprises: washing the activated T cells or cell population containing activated T cells obtained after the activation culture in step 1), resuspending with culture medium, and co-culturing the obtained cell suspension with a retrovirus expressing CAR for 20-24 hours. Preferably, the retrovirus is an alpha retrovirus, a beta retrovirus, a gamma retrovirus, a delta retrovirus or an epsilon retrovirus, and more preferably, the retrovirus is a gamma retrovirus. Preferably, the MOI of transduction is 0.1-2. Preferably, the starting CD3 + T cell density is 0.5-2×10 6 cells / mL. Preferably, the co-culture medium is AIM-V, X-VIVO, DMEM, RPMI1640, more preferably X-VIVO15; more preferably, the culture medium is further supplemented with one or more of acetylcysteine, GlutaMAX, HEPES and human plasma. Preferably, the co-culture medium contains IL-2, more preferably, its concentration is 50-500IU / ml, preferably 100-400IU / ml, more preferably 300IU / ml. Preferably, the culture conditions of the co-culture are about 37°C and about 5% CO2 environment.

[0020] In one or more embodiments, step 3) comprises: washing the chimeric antigen receptor T cells obtained in step 2), resuspending in culture medium, adjusting the cell density, culturing for 18-24 hours, and harvesting and freezing after the end of the culture. Preferably, the cell density is adjusted to 0.1-1×10 6 cells / mL.

[0021] In one or more embodiments, the culture medium in step 3) is AIM-V, X-VIVO, DMEM, RPMI1640, and more preferably X-VIVO15; more preferably, the culture medium is also supplemented with one or more of acetylcysteine, GlutaMAX, HEPES and human plasma.

[0022] In one or more embodiments, the culture medium of step 3) contains IL-2, more preferably, the concentration thereof is 50-500 IU / ml, preferably 100-400 IU / ml, more preferably 300 IU / ml.

[0023] In one or more embodiments, the culture conditions of step 3) are about 37° C. and about 5% CO 2 environment.

[0024] In one or more embodiments, the chimeric antigen receptor T cells are no longer expanded and / or cultured after the second harvest in step 3).

[0025] In one or more embodiments, the CAR contains an extracellular target antigen binding domain, a hinge region, a transmembrane region, and an intracellular signaling domain.

[0026] In one or more embodiments, the CAR contains a leader peptide of CD8 antigen, an anti-CD19 single-chain antibody, a human CD8α hinge region, a human CD28 transmembrane region, a human CD28 intracellular region, and a human CD3ζ intracellular region, which are sequentially connected.

[0027] In one or more embodiments, the extracellular target antigen binding domain is a single-chain antibody targeting CD19; preferably, the anti-CD19 single-chain antibody comprises a CDR sequence selected from any one of SEQ ID NOs: 1-6; more preferably, the light chain variable region of the anti-CD19 single-chain antibody has three CDRs shown in SEQ ID NOs: 1-3, and the heavy chain variable region of the anti-CD19 single-chain antibody has three CDRs shown in SEQ ID NOs: 4-6; more preferably, the amino acid sequence of the light chain variable region of the anti-CD19 single-chain antibody is as shown in amino acids 1-107 of SEQ ID NO: 7, and the amino acid sequence of the heavy chain variable region may be as shown in amino acids 123-242 of SEQ ID NO: 7.

[0028] In one or more embodiments, the anti-CD19 single-chain antibody is the anti-CD19 monoclonal antibody FMC63.

[0029] The present invention also provides CAR-T cells prepared by the method of any of the above embodiments.

[0030] The present invention also provides the use of CAR-T cells prepared by the method of any of the above embodiments in the preparation of drugs for treating cancer.

[0031] In one or more embodiments, the cancer is a hematological cancer or a solid cancer. More preferably, the hematological cancer is a leukemia, a lymphoma or a myeloma; the solid cancer is a pancreatic cancer, a gastric cancer, a gastric cancer peritoneal metastasis, an esophageal cancer, a liver cancer, a kidney tumor, a lung cancer, a small intestine cancer, a bone cancer, a prostate cancer, a colorectal cancer, a breast cancer, a large intestine cancer, a cervical cancer, an ovarian cancer, an adrenal tumor, a bladder tumor, a brain glioma or an endometrial cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 These are the test results of the characteristics of CAR-T cells prepared by the Dash CAR-T process (72h, 48h) and the general process.

[0033] Figure 2 These are the functional test results of CAR-T cells prepared by the Dash CAR-T process (72h, 48h) and the general process.

[0034] Figure 3 These are the results of in vivo pharmacodynamic studies in mice of CAR-T cells prepared by the Dash CAR-T process (72h, 48h) and the general process: results of in vivo imaging in mice.

[0035] Figure 4 These are the results of in vivo pharmacodynamic studies in mice of CAR-T cells prepared by the Dash CAR-T process (72h, 48h) and the general process: the results of mouse weight detection.

[0036] Figure 5 These are the results of in vivo pharmacodynamic studies in mice of CAR-T cells prepared by the Dash CAR-T process (72h, 48h) and the general process: the absolute count results of T cells in the peripheral blood of mice. DETAILED DESCRIPTION

[0037] In order to shorten the preparation time of CAR-T cells and quickly prepare efficient CAR-T cell products, the present invention provides a method for preparing chimeric antigen receptor T cells (also referred to as Dash CAR-T in this article).

[0038] the term

[0039] CAR-T: Chimeric Antigen Receptor-T cell (CAR-T) refers to T cells that have been genetically modified to recognize specific target antigens in an MHC-unrestricted manner and to continuously activate and proliferate. The 2012 International Society for Cellular Therapy Annual Meeting pointed out that biological immune cell therapy has become the fourth means of treating tumors in addition to surgery, radiotherapy, and chemotherapy, and will become an essential means of tumor treatment in the future. CAR-T cell infusion therapy is the most clear and effective form of immunotherapy in current tumor treatment. A large number of studies have shown that CAR-T cells can effectively recognize tumor antigens, induce specific anti-tumor immune responses, and significantly improve patients' survival.

[0040] PBMC: Peripheral Blood Mononuclear Cell (PBMC) refers to the mononuclear cells in the peripheral blood, mainly including lymphocytes, monocytes and other small amounts of cells. It is an important raw material for T cell-related cell therapy.

[0041] DPBS: DPBS stands for Dulbecco's Phosphate-Buffered Saline, which is Dulbecco's Phosphate Buffered Saline. Its main components include NaCl, KCl, KH2PO4, Na2HPO4, etc., with a pH of 7.2-7.4. DPBS is a balanced salt solution for cells, usually used to resuspend cells and preserve cells in vitro for a short period of time. DPBS is divided into two types based on whether it contains calcium and magnesium ions. The difference from regular PBS is that the phosphate content of DPBS is slightly lower.

[0042] Culture medium: The culture medium described herein includes suitable culture medium for immune cells (especially CD3 + Any culture medium for cells and other types of leukocytes, including but not limited to AIM-V, X-VIVO, DMEM or RPMI1640.

[0043] X-VIVO: Lonza X-VIVO TM 15 is the abbreviation of culture medium. It is suitable for the proliferation of T lymphocytes and can be used for CD3 + The growth of cells and other types of white blood cells.

[0044] CTS Dynabeads CD3 / 28: CTS TM Dynabeads TM CD3 / CD28 is suitable for the in vitro isolation, activation and expansion of human T cells. Dynabeads are conjugated with anti-CD3 and anti-CD28 antibodies to provide the primary and co-stimulatory signals required for T cell activation and expansion.

[0045] The present disclosure first provides a method for preparing chimeric antigen receptor T cells, comprising the steps of: 1) contacting T cells or a cell population containing T cells with a stimulating agent to activate the T cells, thereby obtaining activated T cells or a cell population containing activated T cells; 2) contacting the activated T cells or the cell population containing activated T cells with a nucleic acid molecule encoding a chimeric antigen receptor (CAR) to obtain chimeric antigen receptor T cells expressing the CAR; 3) harvesting the chimeric antigen receptor T cells, culturing them for another 18-24 hours, and harvesting and freezing them after the culture ends.

[0046] The chimeric antigen receptor of the present disclosure may be directed against one or more of the following antigens: CD19, CD20, CD276, CEA, GD2, FR, PSMA, PMEL, CA9, CD171 / L1-CAM, IL-13RL1, MART-1, ERBB2, NY-ESO-1 family protein, BAGE family protein, GAGE ​​family protein, AFP, MUC1, CD22, CD23, CD30, CD33, CD44v7 / 8, CD70, VEGFR1, VE In one or more embodiments, the chimeric antigen receptor is directed against an antigen that is CD19.

[0047] In one or more embodiments, the chimeric antigen receptor (CAR) contains an extracellular target antigen binding domain, a hinge region, a transmembrane region, and an intracellular signaling domain. In one or more embodiments, the intracellular signaling domain includes an intracellular co-stimulatory domain and / or a signal transduction domain. The "hinge region", "transmembrane region" and "intracellular region" herein can all be selected from the sequences of the hinge region, transmembrane region and intracellular region in known CAR-T technology.

[0048] The hinge region of the chimeric antigen receptor is located between the extracellular antigen binding region and the transmembrane region, and the hinge region is an amino acid segment that is usually present between the two domains of the protein, and can allow the flexibility of the protein and the relative movement of the two domains to each other. The hinge region can be a hinge region of a naturally occurring protein or a portion thereof. The hinge region of an antibody (such as IgG, IgA, IgM, IgE or IgD antibody) can also be used for the chimeric antigen receptor described herein. Non-naturally occurring peptides can also be used as the hinge region of the chimeric antigen receptor described herein. Exemplarily, the hinge region of CAR is selected from CD8α hinge region, IgD hinge region, IgG1 FcCH2CH3 hinge region or IgG4 Fc CH2CH3 hinge region, and its sequence is within the knowledge of those skilled in the art. The CD8α hinge region suitable for the present invention can be various human CD8α hinge region sequences commonly used in CAR in the art. In some embodiments, its sequence is as shown in SEQ ID NO:7 amino acids 243-289.

[0049] The transmembrane region of the chimeric antibody receptor can form an alpha helix, a complex of more than one alpha helix, a beta barrel, or any other stable structure capable of spanning a cell phospholipid bilayer. The transmembrane region can be of natural or synthetic origin. The transmembrane region can be selected from the transmembrane region of the following proteins: CD3ε, CD4, CD5, CD8α, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, α, β or ζ chain of T cell receptor. The human CD28 transmembrane region suitable for the present invention can be various human CD28 transmembrane region sequences commonly used in the art for CAR.

[0050] The intracellular signaling domain includes an intracellular co-stimulatory domain and / or an intracellular signal transduction domain.

[0051] The intracellular co-stimulatory domain is usually from the CD28 family (CD28, ICOS) or the tumor necrosis factor receptor family (4-1BB, OX40, CD27). The co-stimulatory domain can achieve dual activation of co-stimulatory molecules and intracellular signals, so that T cells continue to proliferate and release cytokines, and improve the anti-tumor ability of CAR-T. The human CD28 intracellular region suitable for the present invention can be various human CD28 intracellular region sequences commonly used in CAR in the art. In some embodiments, the sequence of the transmembrane region and intracellular region of human CD28 is shown in amino acids 290-357 of SEQ ID NO:7.

[0052] The intracellular signal transduction domain is responsible for the activation of at least one normal effector function of the immune effector cell expressing the chimeric antigen receptor. For example, the effector function of a T cell may be cell lysis activity or auxiliary activity, including the secretion of cytokines. Although the entire intracellular signaling region can usually be utilized, in many cases, it is unnecessary to use the entire chain. In terms of using a truncated portion of the intracellular signaling region, as long as it transduces the effector function signal, this truncated portion can be used instead of the complete chain. Therefore, the intracellular signaling region includes any truncated form of the intracellular signaling region sufficient to transduce the effector function signal. The intracellular signaling domain of CAR can be selected as needed, including but not limited to an intracellular signaling domain derived from at least one of CD3ζ, FcRγ (FCER1G), FcRβ (FcεRib), CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b and CD66d. Preferably, the intracellular signaling region is derived from the intracellular signaling region of human CD3ζ. In some embodiments, the sequence of the intracellular signal transduction domain is as shown in amino acids 358-469 of SEQ ID NO:7.

[0053] In one or more embodiments, the CAR also contains a signal peptide located at the N-terminus of the extracellular target antigen binding domain. The signal peptide can be selected as needed. In general, a signal peptide is a peptide sequence that targets a polypeptide to a desired site in a cell. The signal peptide targets the polypeptide to the secretory pathway of the cell and will allow the polypeptide to be integrated and anchored to the lipid bilayer; the signal peptide can also be a membrane localization signal peptide. Exemplary signal peptides such as CD8 signal peptides, CD28 signal peptides, CD4 signal peptides, or light chain signal peptides, the sequences of which are within the knowledge of those skilled in the art. The CD8 signal peptide suitable for the present invention may be various human CD8 signal peptide sequences commonly used in CAR in the art. As one example, the sequence of the human CD8 signal peptide is shown in SEQ ID NO:9, and its exemplary coding sequence is shown in SEQ ID NO:10.

[0054] In one or more embodiments, the extracellular target antigen binding domain of the CAR is a single-chain antibody against CD19. In one or more embodiments, the anti-CD19 single-chain antibody includes a CDR sequence selected from the following: QDISKY (SEQ ID NO: 1), HTS (SEQ ID NO: 2), QQGNTLPYT (SEQ ID NO: 3), GVSLPDYG (SEQ ID NO: 4), IWGSETT (SEQ ID NO: 5), AKHYYYGGSYAMDY (SEQ ID NO: 6). In one or more embodiments, the light chain variable region of the anti-CD19 single-chain antibody has three CDRs of QDISKY (SEQ ID NO: 1), HTS (SEQ ID NO: 2), and QQGNTLPYT (SEQ ID NO: 3), and the heavy chain variable region of the anti-CD19 single-chain antibody has three CDRs of GVSLPDYG (SEQ ID NO: 4), IWGSETT (SEQ ID NO: 5), and AKHYYYGGSYAMDY (SEQ ID NO: 6). In one or more embodiments, the amino acid sequence of the light chain variable region of the anti-CD19 single-chain antibody is as shown in SEQ ID NO:7 amino acids 1-107, and the amino acid sequence of the heavy chain variable region may be as shown in SEQ ID NO:7 amino acids 123-242. In one or more embodiments, the amino acid sequence of the anti-CD19 single-chain antibody is as shown in SEQ ID NO:7 amino acids 1-242. In one or more embodiments, the anti-CD19 single-chain antibody is the anti-CD19 monoclonal antibody FMC63. Herein, the numbering of CDR sequences follows the IMGT numbering rules. Those skilled in the art can easily obtain CDR sequences based on other numbering rules (e.g., Kabat).

[0055] In one or more embodiments, the CAR contains a leader peptide of a CD8 antigen, an anti-CD19 single-chain antibody, a hinge region of human CD8α, a transmembrane region of human CD28, an intracellular region of human CD28, and an intracellular region of human CD3ζ. In one or more embodiments, the amino acid sequence of CAR is shown in SEQ ID NO: 7, and its exemplary coding sequence is shown in SEQ ID NO: 8.

[0056] In some embodiments, T cells or cell colonies comprising T cells are blood samples or blood-derived samples, or are apheresis or leukapheresis products, or are derived from apheresis or leukapheresis products. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMC), leukocytes, etc. T cells or cell colonies comprising T cells for CAR-T therapy are from autologous and allogeneic. In one or more embodiments of the present disclosure, the T cells or cell colonies comprising T cells are or are derived from PBMCs isolated from single blood collection.

[0057] In some embodiments, the T cells or cell populations comprising T cells are freshly collected or cryopreserved, or a mixture thereof.

[0058] In some embodiments, the T cells or cell colonies comprising T cells are sorted or enriched, or are sorted or enriched before or while activated. The T cells or cell colonies comprising T cells or the products thereof after sorting or enrichment include CD4+T cells and / or CD8+T cells alone or in combination. In one or more embodiments, the T cells or cell colonies comprising T cells include one or more subtypes and subpopulations of T cells and / or CD4+T cells and / or CD8+T cells. Subtypes and subpopulations of T cells and / or CD4+T cells and / or CD8+T cells are naive T (Tn) cells, effector T cells (TEFF), memory T cells and subtypes thereof (such as stem central memory T cells (TSCM), central memory T cells (TCM), effector memory T cells (TEM) or terminally differentiated effector memory T cells), tumor infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal-associated constant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells (such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells), follicular helper T cells, α / β T cells and δ / γ T cells.

[0059] In some embodiments, the cell colony comprising T cells comprises: T cells, such as CD8+T cells (e.g., CD8+ naive T cells, central memory T cells, or effector memory T cells), CD4+T cells, natural killer T cells (NKT cells), regulatory T cells (Treg), stem cell memory T cells, lymphocyte progenitor cells, hematopoietic stem cells, natural killer cells (NK cells), or dendritic cells. In some embodiments, the cell is an induced pluripotent stem (iPS) cell or a cell derived from an iPS cell, wherein the cell derived from an iPS cell is, for example, a T cell (e.g., CD8+T cells (e.g., CD8+ naive T cells, central memory T cells, or effector memory T cells), CD4+T cells, stem cell memory T cells) generated from a subject and differentiated by manipulation to change the expression of one or more target genes (e.g., inducing mutations in one or more target genes).

[0060] In some embodiments, the T cell or the cell colony comprising the T cell is sorted before activation or while being activated. The method of sorting includes a cell sorting method based on density, such as by lysing red blood cells and preparing white blood cells from peripheral blood by Percoll or Ficoll gradient centrifugation. In some embodiments, the sorting method includes sorting different types of cells based on the expression or presence of one or more specific molecules in cells, such as surface markers (e.g., surface proteins), intracellular markers or nucleic acids. In some embodiments, any known sorting method based on such a marker can be used. In some embodiments, sorting is sorting based on affinity or immunoaffinity. For example, in some aspects, sorting includes sorting cells and cell colonies based on one or more markers, typically cell surface markers or expression levels, for example, by incubating with antibodies or binding partners (partners) that specifically bind to such markers, then usually by washing steps, and cells that have been bound to antibodies or binding partners are sorted with those cells that are not bound to antibodies or binding partners. Such a sorting step can be based on positive selection and / or negative selection, in which cells that have bound the reagent are retained for further use, and in which cells that have not bound the antibody or binding partner are retained. Sorting does not require 100% enrichment or removal of a specific cell population or cells expressing a specific marker. In one or more embodiments, the T cells or cell populations containing T cells are sorted using CD3 / CD28 conjugated magnetic beads to obtain CD3+T cells.

[0061] In one or more embodiments, the stimulating reagent includes one or more components of the intracellular signaling domain capable of activating the TCR complex, such as a ligand. In some aspects, the stimulating reagent turns on or starts the TCR / CD3 intracellular signaling cascade in T cells. Such stimulating reagents include antibodies, such as TCR-specific antibodies, such as anti-CD3 antibodies. In some embodiments, the stimulating reagent includes one or more components capable of stimulating co-stimulatory receptors, such as ligands, such as anti-CD28. In one or more embodiments, the stimulating reagent is a CD3 antibody and a CD28 antibody. In one or more embodiments, the CD3 antibody and the CD28 antibody are coupled to a solid phase carrier, such a solid phase carrier is a magnetic bead, and the stimulating reagent is in the form of a magnetic bead coupled to anti-CD3 and anti-CD28 antibodies.

[0062] The activated T cells or cell colonies comprising activated T cells are contacted with nucleic acid molecules encoding chimeric antigen receptors (CARs), and it is intended to introduce genetic engineering components CARs. Various methods for gene transduction are well known in the art, and exemplary methods include transduction, transposon or electroporation mediated by viruses (such as retroviruses, slow viruses, adenoviruses) to achieve. In some embodiments, a retroviral vector (such as a γ-retroviral vector) or a slow virus vector is used to transfer recombinant nucleic acids into T cells. In some embodiments, recombinant infectious virus particles (such as vectors derived from simian virus 40 (SV40), adenovirus, adeno-associated virus (AAV)) are used to transfer recombinant nucleic acids into cells.

[0063] In some embodiments, the harvest after the culture of step 3) is to freeze the cells, for example, at a specific cell density. In some embodiments, the cells are frozen using the following profile: the instrument is precooled to 20 degrees Celsius; at 0.5-2 degrees Celsius per minute, it drops to -6 to -8 degrees Celsius; at 20-28 degrees Celsius per minute, it drops to -40 to -60 degrees Celsius; at 6-15 degrees Celsius per minute, it returns to -10 to -20 degrees Celsius; at 0.5-2 degrees Celsius per minute, it drops to -30 to -60 degrees Celsius; at 5-20 degrees Celsius per minute, it drops to -70 to -90 degrees Celsius.

[0064] In some embodiments, the method of preparing chimeric antigen receptor T cells of the present disclosure comprises the steps of:

[0065] 1) contacting T cells or a cell population containing T cells with magnetic beads coupled with anti-CD3 antibodies and anti-CD28 antibodies, magnetically sorting CD3+ T cells, and activating and culturing the CD3+ T cells for 20-24 hours to obtain activated T cells or a cell population containing activated T cells;

[0066] 2) Washing step 1) After the activation culture is completed, the activated T cells or the cell population containing the activated T cells obtained are resuspended in a culture medium, and the obtained cell suspension is co-cultured with a γ-retroviral solution expressing the CAR for 20-24 hours to obtain chimeric antigen receptor T cells expressing the CAR;

[0067] 3) Collecting the chimeric antigen receptor T cells obtained in step 2), washing, resuspending in culture medium, adjusting the cell density, culturing for 18-24 hours, harvesting after the culture is completed, and freezing.

[0068] In one aspect, the CAR contains a leader peptide of the CD8 antigen, an anti-CD19 single-chain antibody, a human CD8α hinge region, a human CD28 transmembrane region, a human CD28 intracellular region, and a human CD3ζ intracellular region, which are sequentially connected, the light chain variable region of the anti-CD19 single-chain antibody has three CDRs in the light chain variable region as shown in amino acids 1-107 of SEQ ID NO:7, and the heavy chain variable region of the anti-CD19 single-chain antibody has three CDRs in the heavy chain variable region as shown in amino acids 123-242 of SEQ ID NO:7.

[0069] The present disclosure also provides CAR-T cells prepared according to the method of any of the above embodiments.

[0070] The present disclosure also provides the application of the CAR-T cells in the preparation of drugs for treating cancer. In some embodiments, the cancer is a hematological cancer or a solid cancer. In some embodiments, the hematological cancer is a leukemia (including acute leukemia (e.g., acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia and myeloblastic, promyelocytic, granulocytic-monocytic type, monocytic and erythroleukemia), chronic leukemia (e.g., chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia and chronic lymphocytic leukemia)), lymphoma (e.g., chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma, acute myeloid leukemia, refractory follicular lymphoma, mantle cell lymphoma, inert B cell lymphoma, B cell malignancies) or myeloma. In some embodiments, the solid tumor is pancreatic cancer, gastric cancer, gastric cancer peritoneal metastasis, liver cancer, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, large intestine cancer, cervical cancer, ovarian cancer, adrenal tumor, bladder tumor, brain glioma or endometrial cancer.

[0071] The term "treatment" refers to the complete or partial improvement or alleviation of a disease, condition or disorder, or symptoms, adverse effects, results or phenotypes associated therewith. Desirable therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis. The term does not imply a complete cure of the disease or complete elimination of any symptoms or the effect on all symptoms or results.

[0072] The present invention also provides a method for administering CAR-T cells, which comprises the steps of: 1) contacting T cells or a cell population containing T cells with a stimulating agent to activate the T cells, thereby obtaining activated T cells or a cell population containing activated T cells; 2) contacting the activated T cells or the cell population containing activated T cells with a nucleic acid molecule encoding a chimeric antigen receptor (CAR), thereby obtaining chimeric antigen receptor T cells expressing the CAR; 3) harvesting the chimeric antigen receptor T cells, culturing them for another 18-24 hours, harvesting them after the end of the culture, and freezing or not freezing; 4) administering the CAR-T cells obtained in step 3) to subjects in need.

[0073] In one or more embodiments, the extracellular target antigen binding domain of the CAR is a single-chain antibody against CD19. In one or more embodiments, the anti-CD19 single-chain antibody includes a CDR sequence selected from the following: QDISKY (SEQ ID NO: 1), HTS (SEQ ID NO: 2), QQGNTLPYT (SEQ ID NO: 3), GVSLPDYG (SEQ ID NO: 4), IWGSETT (SEQ ID NO: 5), AKHYYYGGSYAMDY (SEQ ID NO: 6). In one or more embodiments, the light chain variable region of the anti-CD19 single-chain antibody has three CDRs of QDISKY (SEQ ID NO: 1), HTS (SEQ ID NO: 2), and QQGNTLPYT (SEQ ID NO: 3), and the heavy chain variable region of the anti-CD19 single-chain antibody has three CDRs of GVSLPDYG (SEQ ID NO: 4), IWGSETT (SEQ ID NO: 5), and AKHYYYGGSYAMDY (SEQ ID NO: 6). In one or more embodiments, the amino acid sequence of the light chain variable region of the anti-CD19 single-chain antibody is as shown in SEQ ID NO: 7, amino acids 1-107, and the amino acid sequence of the heavy chain variable region may be as shown in SEQ ID NO: 7, amino acids 123-242. In one or more embodiments, the anti-CD19 single-chain antibody is the anti-CD19 monoclonal antibody FMC63.

[0074] In one or more embodiments, the CAR contains a leader peptide of the CD8 antigen, an anti-CD19 single-chain antibody, a hinge region of human CD8α, a transmembrane region of human CD28, an intracellular region of human CD28, and an intracellular region of human CD3ζ. In one or more embodiments, the CAR sequence is as shown in SEQ ID NO:7.

[0075] In one or more embodiments, no additional expansion and / or culture steps of chimeric antigen receptor T cells are included between step 3) and step 4).

[0076] In one or more embodiments, the T cells or a population of cells comprising T cells are or are derived from a cryopreserved or freshly collected sample.

[0077] In one or more embodiments, a "subject" is a mammal, such as a human or other animal, and is typically a human. In some embodiments, the subject (e.g., patient) to which the immunomodulatory polypeptide, engineered cell, or composition is administered is a mammal, typically a primate, such as a human. In some embodiments, the primate is a monkey or ape. The subject can be male or female, and can be at any suitable age, including infants, young children, adolescents, adults, and elderly subjects. In some embodiments, the subject is a non-primate mammal, such as a rodent.

[0078] In one or more embodiments, in the preparation method or administration method of the present disclosure, the T cells or cell populations comprising T cells are from machine-collected single blood samples that comply with the collection procedures and transportation methods, and the processing method for obtaining the single blood samples is as follows:

[0079] Collection and transportation of single blood collection. Use blood cell separator ( Spectra, Spectra Fenwal TM Or a standard machine collection device with equivalent equipment) is used to collect and separate white blood cells and plasma in the clinical central ward. The collection volume of a single blood sample is about 20mL to 200mL, and the plasma volume is about 20mL to 200mL. The collected single blood and plasma samples are placed in a refrigerated transport box at 2 to 8°C and transported to the cell preparation center by cold chain logistics. It is well known to those skilled in the art that according to the "Good Manufacturing Practice for Pharmaceuticals (Revised in 2010)", the clean area is classified into ABCD levels, Class A: High-risk operation areas, such as filling areas, areas where rubber stopper barrels and open packaging containers in direct contact with sterile preparations, and areas for aseptic assembly or connection operations, should use a unidirectional flow operating table (hood) to maintain the environmental state of the area. The unidirectional flow system must evenly supply air in its working area, with a wind speed of 0.36-0.54m / s (guide value). There should be data to prove the state of the unidirectional flow and be verified. In a closed isolation operator or glove box, a lower wind speed can be used. Class B: refers to the background area where Class A clean areas for high-risk operations such as sterile preparation and filling are located. Class C and Class D: refer to the clean areas for less important operating steps in the production process of sterile drugs. + A (i.e., A-level environment within a B-level context) or C + In a Grade A (Grade A environment in a Grade C background) environment, the following cell processing steps are started.

[0080] PBMC separation. This process can be completed manually or automatically. Manually, the single blood sample is slowly added to Ficoll using Ficoll density gradient centrifugation, and after centrifugation in a centrifuge, the separated PBMC layer is manually removed. In terms of automation, the Sepax C-Pro cell processing system and the corresponding disposable closed tubing kit CT-90.1 are used, and the Ficoll density gradient centrifugation method is also used to execute the NeatCell program developed by the instrument manufacturer to separate PBMC. The PBMC is resuspended in DPBS, washed once and the supernatant is removed. The purpose of this process is to isolate the PBMC required for CAR-T cell preparation and remove the multinuclear cells in the red blood cells and white blood cells remaining in the single blood sample. In the method disclosed in the present invention, the PBMC is then frozen: the PBMC is resuspended in the PBMC freezing solution and output or transferred to a cell freezing bag, the cell freezing bag is frozen in a program cooling instrument, and finally transferred to a liquid nitrogen tank for storage.

[0081] In addition, in the method disclosed herein, in addition to the step of obtaining a single blood sample as described above, a step of collecting and obtaining plasma is also included. The plasma processing is as follows: the blood bag containing the donor plasma is inactivated in a 56°C water bath for 30 minutes, and then the denatured protein and other impurities in the plasma are precipitated to the bottom of the bag by centrifugation. The supernatant plasma is transferred to a freezing bag using a blood separator, and the required volume is taken to prepare the culture medium used in the entire preparation process, and the culture medium contains 5% human plasma. The remaining plasma is frozen at -80°C for future use.

[0082] In the method disclosed herein, PBMC resuscitation relief is then performed: the PBMC cryopreservation bag is placed in a 37°C water bath and shaken back and forth to melt it quickly and evenly. The thawed PBMC is transferred to a culture medium (X-VIVO15 supplemented with acetylcysteine, GlutaMAX, HEPES, and human plasma) and washed once by centrifugation. The washed PBMC is resuspended in culture medium, placed in a cell culture flask or cell culture bag, placed in a carbon dioxide incubator, and cultured at 37±1°C and 5±0.5% CO2 for 2 hours. The purpose of this process is to restore the viability and functionality of PBMC and remove apoptotic cells.

[0083] It should be noted that if PBMCs are not cryopreserved after separation and CD3+ T cells are directly sorted and activated, the above-mentioned PBMC cryopreservation and resuscitation steps can be omitted.

[0084] In the method disclosed herein, CD3 + T cell sorting and activation: After washing, PBMCs were resuspended in DPBS and sampled and counted, and then CD3 was calculated based on the results of PBMC flow cytometry phenotype detection. + Cell number, add DPBS to the PBMC suspension, adjust CD3 + Cell density (4-10×10 6 CD3 + cells / mL). Magnetic beads covalently coupled to anti-CD3 and anti-CD28 antibodies washed with DPBS were added (cells: magnetic beads = 1:1, magnetic beads concentration was 4×10 8 beads / mL)(CTS TM Dynabeads TM CD3 / CD28), and use a rotating mixer to fully mix the cells and magnetic beads during the co-incubation (30-45 minutes, room temperature). After the incubation is completed, place the cells on the DynaMag TM -5 magnetic stand, place CD3 bound to magnetic beads + The cells were captured by magnet and retained in the centrifuge tube, and the supernatant and CD3 -Add IL-2-containing culture medium (X-VIVO15 supplemented with acetylcysteine, GlutaMAX, HEPES, human plasma, and interleukin 2) and resuspend CD3 + Cells (1-5×10 6 cells / mL) and transferred to a cell culture flask or cell culture bag. The cells were placed in a carbon dioxide incubator and activated and cultured for 20-24 hours at 37±1°C and 5±0.5% CO2. + T cells, further remove other cell populations, and co-stimulate T cells with CD3 and CD28 antibodies to activate T cells, which is beneficial for retroviral transduction.

[0085] In the method disclosed herein, retroviral transduction of T cells is then performed: first, a cell culture flask or a cell culture bag is coated with RetroNectin working solution at 37°C, and after the coating is completed, the supernatant in the culture flask or the culture bag is discarded. + After washing once, T cells were resuspended in IL-2-containing medium (X-VIVO15 supplemented with acetylcysteine, GlutaMAX, HEPES, human plasma, and interleukin 2). After washing, cells were sampled and counted, and the transduction MOI was calculated. The MOI should be 0.1-2 and the final cell density in viral transduction should be 0.5-2×10 6 cells / mL. Place the cell suspension and virus solution into a RetroNectin-coated culture flask or culture bag and culture in a carbon dioxide incubator at 37±1°C and 5±0.5% CO2 for 20-24 hours. CAR-T cells that have completed virus transduction can be directly filled and frozen as shown in the next paragraph (the cells harvested at this time point are 48h DASH CAR-T cells), or the cells can be washed to remove the virus solution, resuspended in IL-2 medium (X-VIVO15 with acetylcysteine, GlutaMAX, HEPES, human plasma, and interleukin 2) and counted. Adjust the cell density to 0.1-1×10 with 50-500IU / ml IL-2 medium. 6 cells / mL and transferred to a cell culture flask or cell culture bag. The cells were placed in a carbon dioxide incubator and activated and cultured for 18-24 hours at 37±1°C and 5±0.5% CO2, and then harvested (the cells harvested at this time point are 72h DASH CAR-T cells). The purpose of this process is to transduce the CAR gene into T cells to achieve CAR-T cell preparation.

[0086] In the method disclosed herein, CAR-T cells are then filled and cryopreserved: first, DynaMag TM-5 magnetic rack to remove the residual magnetic beads in the cell suspension, then wash the CAR-T cells three times with sodium chloride injection (0.9% NaCl) and resuspend them in freezing solution (containing DMSO, sodium chloride injection, compound electrolyte injection, glucose injection, human albumin). After sampling and counting, the CAR-T cells are divided into freezing bags and transported to the program cooling device. After cooling, transfer to liquid nitrogen for long-term storage. The cooling program is common knowledge to those skilled in the art. An exemplary cooling program includes the following steps: precool the instrument to 20 degrees Celsius; cool to -6 to -8 degrees Celsius at 0.5 to 2 degrees Celsius per minute; cool to -40 to -60 degrees Celsius at 20 to 28 degrees Celsius per minute; return to -10 to -20 degrees Celsius at 6 to 15 degrees Celsius per minute; cool to -30 to -60 degrees Celsius at 0.5 to 2 degrees Celsius per minute; cool to -70 to -90 degrees Celsius at 5 to 20 degrees Celsius per minute.

[0087] The method disclosed herein performs short-term culture after the transduction step, which can greatly increase the proportion of stem cells, significantly shorten the total preparation time of CAR-T cells, and significantly improve the efficacy of CAR-T cells.

[0088] Herein, the anti-CD3 antibody and anti-CD28 antibody may be any antibody known in the art that can bind to CD3 and CD28.

[0089] As used herein, "cryopreservative" refers to a medium for cryopreservation of cells therein. Those skilled in the art are aware of cryopreservative compositions suitable for cells, particularly immune cells (e.g., PBMCs or CD3+ T cells). These cryopreservatives are generally commercially available.

[0090] Herein, "culture medium" refers to a medium for culturing cells. Those skilled in the art are aware of culture medium components suitable for cells, particularly immune cells (e.g., PBMC or CD3+ T cells). These culture media are generally commercially available, such as X-VIVO15.

[0091] The present invention will be further described below by way of specific examples. It should be understood that these examples are merely illustrative and are not intended to limit the scope of the present invention. The methods and reagents used in the examples are, unless otherwise stated, conventional methods and reagents in the art.

[0092] Example

[0093] Example 1

[0094] 1. DASH CAR-T process and the viability, diameter, expansion multiple, flow cytometry phenotype and CAR infection rate of CAR-T cells with extended culture time

[0095] DASH CAR-T cells were prepared using the DASH CAR-T process, and the differences between DASH CAR-T cells and CAR-T cells with prolonged culture time were compared.

[0096] DASH CAR-T preparation:

[0097] PBMCs were separated from fresh single-collected blood using Ficoll density gradient centrifugation, and the separated PBMCs were resuspended in freezing solution and filled into freezing tubes, and then cryopreserved using a programmed cooling device and stored in liquid nitrogen.

[0098] Place the PBMC cryopreservation tube in a 37°C water bath and shake it back and forth to thaw it quickly and evenly. Transfer the thawed PBMC to the culture medium and wash it once by centrifugation. Resuspend the washed PBMC in the culture medium, place it in a cell culture bottle or cell culture bag, place it in a carbon dioxide incubator, and culture it at 37±1°C and 5±0.5% CO2 for 2 hours. The purpose of this process is to restore the viability and functionality of PBMC and remove apoptotic cells.

[0099] Take out the PBMCs after the culture is completed from the CO2 incubator, centrifuge the cell suspension once, and then wash it with DPBS. After washing, adjust the cell density of PBMCs to 1×10 7 CD3 + cells / ml. Magnetic beads covalently coupled to anti-CD3 and anti-CD28 antibodies were added to the cell suspension and incubated together at room temperature for 30 minutes on a rotating mixer. The incubated cell magnetic bead suspension was transferred to a magnetic rack and magnetically separated CD3 + The sorted cells were adjusted to 2×10 6 CD3 + cells / ml, placed in a culture flask, and activated and cultured in a 37°C, 5% CO2 incubator for 24 hours.

[0100] The activated cells were taken out of the incubator, washed once with IL-2-containing medium by centrifugation, and the cell density was adjusted to 1×10 6 viable cells / ml. The cell suspension was mixed with the virus solution for CAR gene transduction, MOI = 0.4. The cell suspension was transferred to a RetroNectin-coated cell culture bag and viral transduced for 22 hours in a 37°C, 5% CO2 incubator. After viral transduction, the magnetic beads in the cell suspension were first removed by a magnetic rack. A portion of the cells were washed three times by centrifugation with sodium chloride injection, resuspended in freezing solution, transferred to a freezing bag, frozen with a programmed cooling device, and stored in liquid nitrogen for a long time (48h DASH CAR-T cells). The remaining cells were adjusted to 0.5×10 6Cells / ml were placed in a culture flask and cultured for 22.5 hours at 37°C in a 5% CO2 incubator. After the culture was completed, a portion of the cells were harvested: the magnetic beads were removed again, and the cells were washed three times by centrifugation with sodium chloride injection, resuspended in the cryopreservation solution, and transferred to a cryopreservation bag, frozen with a program cooling device, and stored in liquid nitrogen for a long time (72h DASH CAR-T cells). After the remaining cells were cultured for 4 days, they were washed three times by centrifugation with sodium chloride injection, resuspended in the cryopreservation solution, transferred to a cryopreservation bag, frozen with a program cooling device, and stored in liquid nitrogen for a long time (extended culture of CAR-T cells, this group of cells simulated the total preparation time of the original process CAR-T, usually 8-12 days).

[0101] DASH CAR-T cells and extended cultured CAR-T cells were tested before cryopreservation, and the results were as follows Figure 1 As shown. The CAR-T cells produced by the DASH CAR-T process have little difference in quality with the CAR-T cells cultured for a long time in indicators that are highly related to product quality, including CAR infection rate, cell viability, CD3 ratio, CD19 ratio, etc.; the 48h CAR infection rate is low because it takes a certain amount of time for the CAR protein to be transported to the cell surface, and the 48h DASH CAR-T is harvested earlier than the time when the CAR protein is fully expressed on the cell surface. CD14 and CD16 in the total cell population + / CD56 + The expression ratios of all of them are close to 0%. The results of indicators related to cell activation, such as CD25, CD69 and cell diameter, are quite different between DASH CAR-T and extended culture CAR-T. As can be seen from the figure, DASH CAR-T cells are more activated than extended culture CAR-T cells. CD45RO - / CCR7 + Symbolism The proportion of T cell populations also differed significantly, among which DASH CAR-T cells still accounted for nearly 30% to 50%. T cells, and prolonged culture of CAR-T cells The proportion of T cells has fallen below 2%. CD45RO - / CCR7 + Represented T cells are highly stem cells, and T cell stemness is related to cell proliferation and tumor killing ability. According to the results of this in vitro experiment, it can be found that DASH CAR-T cells have a high activation state and higher T cell stemness, which may lead to better proliferation and tumor killing ability.

[0102] 2. CAR-T cell expansion and functional testing at the end point of culture in the DASH CAR-T process

[0103] The frozen DASH CAR-T cells and extended culture CAR-T cells were revived in a 37°C water bath, washed once by centrifugation with IL-2-containing medium, resuspended, and inoculated into culture bottles. The cells were cultured in a 37°C, 5% CO2 incubator for 72 hours to allow the CAR-T cell function to recover well before functional testing. During the culture process, the number of cells was recorded every day as a basis for calculating cell proliferation, and samples were taken for CAR infection rate testing. Functional testing is to co-culture CAR-T cells with target cells expressing the corresponding targets, take CAR-T cells to detect CD107a expression, and take target cells to detect the proportion of killed cells. The CD107a molecule is a sensitive marker for the degranulation of cytotoxic T cells, which directly reflects the level of cell killing activity.

[0104] like Figure 2 As shown, the expansion ability of DASH CAR-T cells is much higher than that of extended cultured CAR-T cells, and the CAR infection rate of 48H DASH CAR-T cells after recovery continues to rise; at the same time, it can be proved that even if the CAR infection rate of 48h DASH CAR-T is low when harvested, it can still express CAR molecules on the cell surface after recovery and culture. In the cell killing test, the effect-target ratio was 1:1, and DASH CAR-T cells and extended cultured CAR-T cells showed similar killing ability. At the same time, the CD107a expression of DASH CAR-T cells is similar to that of extended cultured CAR-T cells.

[0105] 3. Evaluation of the in vivo anti-tumor effect of DASH CAR-T technology and CAR-T cells with extended culture time

[0106] In vivo studies: Immunodeficient NOG mice were used for in vivo experiments. Each mouse was inoculated with 5×10 5 Three days after inoculation, the mice were divided into groups according to their weight and total fluorescence intensity, with 5 mice in each group, for a total of 10 groups, including a solvent control group; a NT cell group; a low-dose and high-dose group of traditional CAR-T (1×10 6 , 5×10 6 CAR-T cells / mouse), DASH 48-hour CAR-T low, medium, and high dose groups (2.5×10 5 , 5×10 5 , 1×10 6 CAR-T cells / mouse); DASH 72-hour CAR-T low, medium, and high dose groups (2.5×10 5 , 5×10 5 , 1×10 6CAR-T cells / mouse). The cells were infused on the second day after grouping, and the day of infusion was designated as day 0. The fluorescence intensity in the mice was tested once a week to evaluate the in vivo tumor inhibition effect of the test article, the number of T cells and CAR-T cells in the peripheral blood of the mice was tested once to evaluate the in vivo expansion of T / CAR-T cells, and the weight of the mice was tested twice to evaluate the overall condition of the mice.

[0107] The experimental results show that ( Figure 3 ), CD19 CAR-T prepared by various processes can significantly inhibit the proliferation of tumor cells in NOG mice (p<0.0001); the tumor inhibition rate of the DASH 48h CAR-T medium and low dose groups is equivalent to that of the traditional process CAR-T low dose group, and the tumor inhibition effect of the DASH 48h CAR-T high dose group and the DASH 72h CAR-T dose groups is equivalent to that of the traditional process CAR-T high dose group, indicating that the rapid preparation of DASH CAR-T process can reduce the cell infusion dose under the premise of equivalent therapeutic effect; from the BLI value 7 days after infusion, it can be seen that the therapeutic effect of the DASH 72h CAR-T medium and low dose group is better than that of the DASH 48h CAR-T medium and low dose group, indicating that the 72-hour preparation process is better.

[0108] The weight data of mice showed no significant difference in weight among the groups of mice ( Figure 4 ), indicating that CD19 CAR-T did not show obvious toxic side effects in mice after reinfusion.

[0109] Analysis of T cell proliferation results in mice ( Figure 5 ), T cells did not expand in mice that received T cells without virus transduction and traditional CAR-T cells, while T cells in mice in the DASH group expanded significantly. When 1E6 cells were transfused, the expansion of DASH 72 was significantly better than that of DASH 48 (p<0.01).

Claims

1. A method for preparing chimeric antigen receptor T cells, characterized in that: Includes steps: 1) contacting T cells or a cell population containing T cells with a stimulating agent to activate T cells, thereby obtaining activated T cells or a cell population containing activated T cells; 2) contacting the activated T cells or a cell population comprising activated T cells with a nucleic acid molecule encoding a chimeric antigen receptor (CAR) to obtain chimeric antigen receptor T cells (CAR-T cells) expressing the CAR; 3) harvesting the chimeric antigen receptor T cells, culturing them for 18-24 hours, harvesting them after the culture is completed, and freezing or not freezing them.

2. The method according to claim 1, characterized in that The method has one or more of the following features: a) Step 1) comprises: obtaining the T cells or a cell population containing T cells by sorting from fresh PBMC or frozen PBMC; b) the stimulatory reagent comprises one or more components capable of activating the intracellular signaling domain of the TCR complex, preferably, the stimulatory reagent is an anti-CD3 antibody or an antigen-binding fragment thereof and / or an anti-CD28 antibody or an antigen-binding fragment thereof, more preferably, the stimulatory reagent is in the form of magnetic beads coupled to anti-CD3 antibody and anti-CD28 antibody; c) Step 2) The method for obtaining the chimeric antigen receptor T cell expressing CAR is selected from viral-mediated transduction or non-viral-mediated transduction, preferably, the viral-mediated transduction is retrovirus-, lentivirus- or adenovirus-mediated transduction, and the non-viral-mediated transduction is transposon- or electroporation-mediated transduction; d) the second harvest of step 3) comprises a step of removing the magnetic beads; e) The culturing time in step 3) is preferably 19-23 hours, more preferably 20-22.5 hours; f) The freezing in step 3) includes: washing the T cells with sodium chloride injection, resuspending the T cells with freezing solution, programmed cooling, and storing in liquid nitrogen.

3. The method according to claim 1, characterized in that Step 1) comprises: contacting T cells or a cell population containing T cells with magnetic beads coupled with anti-CD3 antibodies and anti-CD28 antibodies, magnetically separating CD3 + T cells, activated cultured CD3 + T cells 20-24 hours, Preferably, the activation culture medium is AIM-V, X-VIVO, DMEM, RPMI1640, and more preferably X-VIVO15; more preferably, the culture medium is further supplemented with one or more of acetylcysteine, GlutaMAX, HEPES and human plasma; Preferably, the activation culture medium contains IL-2, more preferably, the concentration thereof is 50-500 IU / ml, preferably 100-400 IU / ml, more preferably 300 IU / ml; Preferably, the activation culture is carried out under the conditions of about 37° C. and about 5% CO 2 .

4. The method according to claim 1, characterized in that Step 2) comprises: washing the activated T cells or cell population containing activated T cells obtained after the activation culture in step 1), resuspending them in a culture medium, and co-culturing the obtained cell suspension with a retrovirus solution expressing CAR for 20-24 hours, Preferably, the retrovirus is an alpha retrovirus, a beta retrovirus, a gamma retrovirus, a delta retrovirus or an epsilon retrovirus; Preferably, the MOI of transduction is 0.1-2; Preferably, the co-cultured starting CD3 + T cell density is 0.5-2×10 6 cells / mL; Preferably, the co-culture medium is AIM-V, X-VIVO, DMEM, RPMI1640, and more preferably X-VIVO15; more preferably, the culture medium is further supplemented with one or more of acetylcysteine, GlutaMAX, HEPES and human plasma; Preferably, the co-culture medium contains IL-2, more preferably, its concentration is 50-500 IU / ml, preferably 100-400 IU / ml, more preferably 300 IU / ml; Preferably, the co-culture conditions are about 37° C. and about 5% CO 2 environment.

5. The method according to claim 1, characterized in that Step 3) comprises: washing the chimeric antigen receptor T cells obtained in step 2), resuspending in culture medium, adjusting the cell density, culturing for 18-24 hours, harvesting and freezing after the culturing, Preferably, the cell density is adjusted to 0.1-1×10 6 cells / mL; Preferably, the culture medium is AIM-V, X-VIVO, DMEM, RPMI1640, and more preferably X-VIVO15; more preferably, the culture medium is further supplemented with one or more of acetylcysteine, GlutaMAX, HEPES and human plasma; Preferably, the culture medium contains IL-2, more preferably, the concentration thereof is 50-500 IU / ml, preferably 100-400 IU / ml, more preferably 300 IU / ml; Preferably, the culture conditions of the culture are about 37° C. and about 5% CO 2 environment.

6. The method according to claim 1, characterized in that Step 3) No further expansion and / or culture of the chimeric antigen receptor T cells is performed after the second harvest.

7. The method according to claim 1, characterized in that The CAR contains an extracellular target antigen binding domain, a hinge region, a transmembrane region and an intracellular signaling domain. Preferably, the CAR contains an anti-CD19 single-chain antibody, a human CD8α hinge region, a human CD28 transmembrane region, a human CD28 intracellular region and a human CD3ζ intracellular region connected in sequence. More preferably, the N-terminus of the anti-CD19 single-chain antibody also contains a leading peptide of the CD8 antigen.

8. The method according to claim 7, characterized in that The extracellular target antigen binding domain is a single-chain antibody targeting CD19; preferably, the anti-CD19 single-chain antibody includes a CDR sequence selected from any one of SEQ ID NOs: 1-6; more preferably, the light chain variable region of the anti-CD19 single-chain antibody has three CDRs shown in SEQ ID NOs: 1-3, and the heavy chain variable region of the anti-CD19 single-chain antibody has three CDRs shown in SEQ ID NOs: 4-6; more preferably, the amino acid sequence of the light chain variable region of the anti-CD19 single-chain antibody is as shown in amino acids 1-107 of SEQ ID NO: 7, and the amino acid sequence of the heavy chain variable region may be as shown in amino acids 123-242 of SEQ ID NO: 7; more preferably, the anti-CD19 single-chain antibody is the anti-CD19 monoclonal antibody FMC63.

9. CAR-T cells prepared by the method according to any one of claims 1 to 6.

10. Use of the CAR-T cells according to claim 9 in the preparation of a drug for treating cancer, preferably, the cancer is a hematological cancer or a solid cancer, more preferably, the hematological cancer is leukemia, lymphoma or myeloma; the solid cancer is pancreatic cancer, gastric cancer, gastric cancer peritoneal metastasis, esophageal cancer, liver cancer, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, cervical cancer, ovarian cancer, adrenal tumor, bladder tumor, brain glioma or endometrial cancer.

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