Rapid preparation method for car-t cell

Abstract

The present invention relates to a rapid preparation method for a chimeric antigen receptor (CAR) T cell (CAR-T cell), comprising the steps of: 1) enabling a T cell or a cell population containing the T cell to come into contact with a stimulating agent to activate the T cell, so as to obtain an activated T cell or a cell population containing the activated T cell; 2) enabling the activated T cell or the cell population containing the activated T cell to come into contact with a nucleic acid molecule encoding a CAR, so as to obtain a CAR-T cell expressing the CAR; and 3) harvesting the CAR-T cell, culturing same for 18-24 hours, harvesting same after the culture, and performing or not performing cryopreservation. The method greatly shortens the total preparation duration of a CAR-T cell, and significantly improves the CAR-T cell proliferation and tumor killing capabilities.

Description

A method for rapidly preparing CAR-T cells 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 their cell surface that recognize specific antigens of tumor malignant cells, guiding 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, comprising the steps of:

[0005] 1) contacting T cells or a cell population comprising T cells with a stimulating agent to activate the T cells, thereby obtaining activated T cells or a cell population comprising 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), thereby obtaining 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 complete, and freezing or not freezing them.

[0008] In one or more embodiments, step 1) comprises: obtaining the T cells or the 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, the method for obtaining the chimeric antigen receptor T cell expressing CAR in step 2) 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 in step 3) is 19-23 hours, preferably 20-22.5 hours.

[0013] In one or more embodiments, the freezing in 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 and cultured CD3 + Preferably, the T cells or cell population containing T cells are incubated with magnetic beads coupled with anti-CD3 antibodies and anti-CD28 antibodies for 30-45 minutes, and CD3 is magnetically separated after the incubation is completed. + Cells were then activated and cultured.

[0015] In one or more embodiments, CD3 + The initial cell density for 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 further supplemented with one or more of acetylcysteine, GlutaMAX, HEPES, and human plasma.

[0017] In one or more embodiments, the medium for activation culture in step 1) contains IL-2, more preferably, at a concentration of 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 them in 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, more preferably, the retrovirus is a gamma retrovirus. Preferably, the MOI of transduction is 0.1-2. Preferably, the starting CD3 + The 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 50-500 IU / ml, preferably 100-400 IU / ml, more preferably 300 IU / 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, harvesting after the end of the culture, and freezing. 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 further supplemented with one or more of acetylcysteine, GlutaMAX, HEPES, and human plasma.

[0022] In one or more embodiments, the culture medium in 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 culturing 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 not 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 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, all linked in sequence.

[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 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, large intestine cancer, cervical cancer, ovarian cancer, adrenal tumor, bladder tumor, brain glioma or endometrial cancer. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

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

[0036] Figure 5 shows the results of in vivo pharmacodynamic studies in mice of CAR-T cells prepared using the Dash CAR-T process (72h, 48h) and the general process: absolute T cell counts in mouse peripheral blood. 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 herein as Dash CAR-T).

[0038] the term

[0039] CAR-T: Chimeric Antigen Receptor-T cells (CAR-T) are genetically modified T cells that can recognize specific target antigens in an MHC-unrestricted manner and continuously activate and proliferate. The 2012 International Society for Cellular Therapy Annual Meeting noted that biological immune cell therapy has become a fourth approach to treating tumors, alongside surgery, radiotherapy, and chemotherapy, and will become an essential treatment option for future tumors. CAR-T cell infusion therapy is currently the most proven and effective form of immunotherapy for tumor treatment. Numerous studies have demonstrated that CAR-T cells can effectively recognize tumor antigens, induce specific anti-tumor immune responses, and significantly improve patient survival.

[0040] PBMC: Peripheral blood mononuclear cells (PBMCs) refer to the mononuclear cells in peripheral blood, primarily consisting of lymphocytes, monocytes, and a small number of other cells. They are currently an important raw material for T cell-related cell therapies.

[0041] DPBS: DPBS stands for Dulbecco's Phosphate-Buffered Saline. Its main components include NaCl, KCl, KH2PO4, and Na2HPO4, with a pH of 7.2-7.4. DPBS is a balanced salt solution for cells, commonly used for resuspending cells and short-term preservation of cells in vitro. DPBS is categorized into two types based on whether it contains calcium and magnesium ions. Unlike regular PBS, DPBS has a slightly lower phosphate content.

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

[0043] X-VIVO: Lonza X-VIVO™15 medium. Suitable for 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™ Dynabeads™ CD3 / CD28 are suitable for the in vitro isolation, activation, and expansion of human T cells. Dynabeads are conjugated to both anti-CD3 and anti-CD28 antibodies, providing the primary and costimulatory 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 reagent 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, and then 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, VEGF, VEGF receptor 1, VEGF receptor 2, VEGF receptor 3, VEGF receptor 4, VEGF receptor 5, VEGF receptor 6, VEGF receptor 7, VEGF receptor 8, VEGF receptor 9, VEGF receptor 10, VEGF receptor 11, VEGF receptor 12, VEGF receptor 13, VEGF receptor 14, VEGF receptor 15, VEGF receptor 16, VEGF receptor 17, VEGF receptor 18, VEGF receptor 19, VEGF receptor 20, VEGF receptor 21, VEGF receptor 22 In one or more embodiments, the chimeric antigen receptor is directed against CD19.

[0047] In one or more embodiments, the chimeric antigen receptor (CAR) comprises 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 costimulatory domain and / or a signal transduction domain. The "hinge region," "transmembrane region," and "intracellular region" herein can be selected from sequences of hinge regions, transmembrane regions, and intracellular regions used in known CAR-T technologies.

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

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

[0050] The intracellular signaling domain includes an intracellular costimulatory domain and / or an intracellular signal transduction domain.

[0051] Intracellular costimulatory domains typically originate from the CD28 family (CD28, ICOS) or the tumor necrosis factor receptor family (4-1BB, OX40, CD27). Costimulatory domains enable dual activation of costimulatory molecules and intracellular signals, enabling sustained T cell proliferation and cytokine release, thereby enhancing the anti-tumor ability of CAR-T cells. Human CD28 intracellular regions suitable for use in the present invention can include various human CD28 intracellular region sequences commonly used in CARs in the art. In some embodiments, the sequences of the transmembrane and intracellular regions of human CD28 are as shown in amino acids 290-357 of SEQ ID NO:7.

[0052] The intracellular signaling 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 can be cytolytic activity or helper activity, including cytokine secretion. Although the entire intracellular signaling region can generally be utilized, in many cases, it is not necessary to use the entire chain. Insofar as a truncated portion of the intracellular signaling region is used, as long as it transduces the effector function signal, such a truncated portion can be used instead of the entire chain. Therefore, the intracellular signaling region includes any truncated form of the intracellular signaling region that is sufficient to transduce the effector function signal. The intracellular signaling domain of the 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 human CD3ζ intracellular signaling region. In some embodiments, the sequence of the intracellular signaling domain is as shown in amino acids 358-469 of SEQ ID NO:7.

[0053] In one or more embodiments, the CAR further 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 integrate and anchor to the lipid bilayer; the signal peptide can also be a membrane localization signal peptide. Exemplary signal peptides are, for example, CD8 signal peptides, CD28 signal peptides, CD4 signal peptides, or light chain signal peptides, and their sequences are within the knowledge of those skilled in the art. The CD8 signal peptide suitable for the present invention can 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 an anti-CD19 single-chain antibody. 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: 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: 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 convention. CDR sequences based on other numbering conventions (e.g., Kabat) are readily available to those skilled in the art.

[0055] In one or more embodiments, 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 connected in sequence. In one or more embodiments, the amino acid sequence of CAR is as shown in SEQ ID NO: 7, and its exemplary coding sequence is as 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 leukocyte apheresis products, or are derived from apheresis or leukocyte apheresis 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 apheresis.

[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 populations comprising T cells are sorted or enriched, or are sorted or enriched before or during activation. The T cells or cell populations 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 populations 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 invariant 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 population 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 (Tregs), stem cell memory T cells, lymphocyte progenitor cells, hematopoietic stem cells, natural killer cells (NK cells), or dendritic cells. In some embodiments, the cells are induced pluripotent stem (iPS) cells or cells derived from iPS cells, such as cells derived from iPS cells, which are generated from a subject and manipulated to change the expression of one or more target genes (e.g., to induce mutations in one or more target genes) and differentiated into T cells (e.g., CD8+T cells (such as CD8+ naive T cells, central memory T cells, or effector memory T cells), CD4+T cells, stem cell memory T cells).

[0060] In some embodiments, the T cell or the cell colony comprising the T cell are sorted before activation or while activating. The method of sorting includes a cell sorting method based on density, such as by lysing red blood cells and preparing leukocytes from peripheral blood by Percoll or Ficoll gradient centrifugation. In some embodiments, sorting method includes expression or presence of one or more specific molecules in cells to sort different types of cells, such as surface markers (such as surface proteins), intracellular markers or nucleic acids. In some embodiments, any known sorting method based on such a mark can be used. In some embodiments, sorting is based on the sorting of affinity or immunoaffinity. For example, in some aspects, sorting includes based on one or more marks, typically cell expression or expression level of cell surface markers to sort cells and cell colony, for example, by hatching with the antibody or binding partner (partner) of specific binding such a mark, then usually by washing steps, and the cell with unbound antibody or binding partner will be sorted. 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 necessarily 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 population 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 capable of activating the intracellular signaling domain of the TCR complex, such as a ligand. In some aspects, the stimulating reagent turns on or initiates the TCR / CD3 intracellular signaling cascade in the T cell. 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 a ligand, 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) to introduce genetically engineered components CARs. Various methods for achieving 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, recombinant nucleic acids are transferred into T cells using retroviral vectors (such as γ-retroviral vectors) or slow viral vectors. In some embodiments, recombinant nucleic acids are transferred into cells using recombinant infectious virus particles (such as vectors derived from simian virus 40 (SV40), adenovirus, adeno-associated virus (AAV)).

[0063] In some embodiments, after the culture is completed in step 3), cells are harvested by freezing, for example, at a specific cell density. In some embodiments, cells are frozen using the following profile: instrument pre-cooled to 20°C; cooled to -6°C to -8°C at 0.5°C / min; cooled to -40°C to -60°C at 20°C / min; warmed to -10°C to -20°C at 6°C / min; cooled to -30°C to -60°C at 0.5°C / min; and cooled to -70°C to -90°C at 5°C / min.

[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 comprising T cells with magnetic beads coupled to 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 comprising activated T cells;

[0066] 2) Washing Step 1) After the activation culture is completed, the activated T cells or cell population containing activated T cells obtained are resuspended in 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 end of the culture, 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 connected in sequence, the light chain variable region of the anti-CD19 single-chain antibody has the 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 the 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 any of the above embodiments.

[0070] The present disclosure also provides the use of the CAR-T cells in the preparation of a drug 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, 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 "treat" refers to the complete or partial improvement or alleviation of a disease, condition, or disorder, or any symptom, adverse effect, outcome, or phenotype 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, ameliorating or relieving the disease state, and alleviating or improving prognosis. The term does not imply a complete cure of the disease or the complete elimination of any symptom or the effect on all symptoms or outcomes.

[0072] The present invention also provides a method for administering CAR-T cells, comprising the steps of: 1) contacting T cells or a cell population containing T cells with a stimulating reagent 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 the 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 them; 4) administering the CAR-T cells obtained in step 3) to a subject in need.

[0073] In one or more embodiments, the extracellular target antigen binding domain of the CAR is an anti-CD19 single-chain antibody. 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: 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: 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 is 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 human CD8α hinge region, a human CD28 transmembrane region, a human CD28 intracellular region, and a human CD3ζ intracellular region connected in sequence. 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 the chimeric antigen receptor T cells are included between step 3) and step 4).

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

[0077] In one or more embodiments, the "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 whom 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 population comprising T cells are obtained from a single blood collection that complies with the collection procedures and transportation methods, and the processing method for obtaining the single blood collection is as follows:

[0079] Collection and transportation of single-donor blood. Use blood cell separators (COBE® Spectra, Spectra Optia®, TrimaAccel®, Fenwal TMA standard apheresis device (Amicus® or equivalent) is used to collect and separate white blood cells and plasma in the central clinical ward. The single-sample blood collection volume is approximately 20mL-200mL, and the plasma volume is approximately 20mL-200mL. The collected single-sample blood and plasma samples are placed in refrigerated transport boxes at 2-8°C and transported to the cell preparation center via cold chain logistics. As is known to those skilled in the art, according to the "Good Manufacturing Practice for Pharmaceuticals (2010 Revised)", clean areas are classified into an ABCD classification system. Class A: High-risk operating areas, such as filling areas, areas for storing stopper barrels and open packaging containers that come into direct contact with sterile preparations, and areas for aseptic assembly or connection operations, should be maintained using unidirectional airflow workstations (hoods). Unidirectional airflow systems must provide uniform airflow throughout the work area, with a speed of 0.36-0.54 m / s (guideline value). Unidirectional airflow should be documented and validated. Lower air speeds may be used within enclosed isolators or glove boxes. 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 steps in the production of sterile drugs. + A (i.e., a Class A environment within a Class B context) or C + Start the following cell processing steps in a Grade A (Grade A environment in a Grade C background) environment.

[0080] PBMC isolation. This process can be completed manually or automatically. Manually, the single-sample blood is slowly added to Ficoll using the Ficoll density gradient centrifugation method, 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 and the NeatCell program developed by the instrument manufacturer is executed 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 PBMCs required for CAR-T cell preparation and remove the multinuclear cells in the red blood cells and white blood cells remaining in the single-sample 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 device, and finally transferred to a liquid nitrogen tank for storage.

[0081] In addition to the above-mentioned step of obtaining a single blood sample, the method of the present disclosure also includes a step of collecting plasma. The plasma is processed by inactivating the blood bag containing the donor plasma in a 56°C water bath for 30 minutes, followed by centrifugation to allow denatured proteins and other impurities in the plasma to settle to the bottom of the bag. The supernatant plasma is transferred to a cryopreservation bag using a blood separator, and the required volume is used to prepare the culture medium containing 5% human plasma used throughout the entire preparation process. The remaining plasma is frozen at -80°C until further use.

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

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

[0084] In the method of the present disclosure, 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 PBMC suspension, adjust CD3 + Cell density (4-10×10 6 CD3 + cells / mL). Magnetic beads covalently coupled with anti-CD3 and anti-CD28 antibodies washed with DPBS (cells: beads = 1:1, bead concentration 4×10 8 Magnetic beads / mL) (CTS™ Dynabeads™ CD3 / CD28), use a rotating mixer to fully mix the cells and magnetic beads during the incubation (30-45 minutes, room temperature). After the incubation is complete, place the cells on the DynaMag TM -5 magnetic stand, place CD3 bound to magnetic beads + The cells were captured by magnetism 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. Place the cells in a carbon dioxide incubator and activate and culture them for 20-24 hours at 37±1°C and 5±0.5% CO2. This step is to isolate the CD3 + T cells, further remove other cell populations, and co-stimulate T cells with CD3 and CD28 antibodies to activate T cells, which is conducive to retroviral transduction.

[0085] In the method disclosed herein, retroviral transduction of T cells is then performed: first, a cell culture flask or cell culture bag is coated with RetroNectin working solution at 37°C, and after coating, the supernatant in the culture flask or 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 to calculate the transduction MOI, which should be 0.1-2 and the final cell density for viral transduction should be 0.5-2×10 6 cells / mL. Place the cell suspension and virus solution together in 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 viral transduction can be directly filled and frozen as shown in the next paragraph (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 supplemented with acetylcysteine, GlutaMAX, HEPES, human plasma, and interleukin 2) and counted. Adjust the cell density to 0.1-1×10 using a medium containing 50-500 IU / ml IL-2. 6 cells / mL and transferred to a cell culture flask or cell culture bag. Place the cells in a CO2 incubator and activate and culture them for 18-24 hours at 37±1°C and 5±0.5% CO2 before harvesting (cells harvested at this time point are 72-hour DASH CAR-T cells). This step aims to transduce the CAR gene into T cells to achieve CAR-T cell production.

[0086] In the method disclosed herein, CAR-T cells are then filled and cryopreserved: first, DynaMag TMA -5 magnetic stand was used to remove any remaining magnetic beads from the cell suspension. The CAR-T cells were then washed three times with sodium chloride injection (0.9% NaCl) and resuspended in freezing buffer (containing DMSO, sodium chloride injection, compound electrolyte injection, glucose injection, and human albumin). After sampling and counting, the CAR-T cells were aliquoted into cryopreservation bags and transported to a programmed cooling apparatus. After cooling, the cells were transferred to liquid nitrogen for long-term storage. The cooling program is common knowledge among those skilled in the art. An exemplary cooling program includes the following steps: pre-cool 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; and cool to -70 to -90 degrees Celsius at 5 to 20 degrees Celsius per minute.

[0087] The method disclosed herein performs a short culture after the transduction step, which can significantly 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 can be any antibody known in the art that can bind to CD3 and CD28.

[0089] As used herein, "cryopreservative medium" refers to a medium used to cryopreserve 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] As used herein, "culture medium" refers to a medium used to culture cells. Those skilled in the art are aware of suitable culture medium components for cells, particularly immune cells (e.g., PBMCs or CD3+ T cells). These culture media are generally commercially available, for example, X-VIVO 15.

[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. Example Example 1 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

[0092] DASH CAR-T cells were prepared using the DASH CAR-T process, and the differences between DASH CAR-T cells and CAR-T cells cultured for extended periods of time were compared.

[0093] DASH CAR-T preparation:

[0094] PBMCs were isolated from freshly collected blood using Ficoll density gradient centrifugation. The isolated PBMCs were resuspended in freezing solution and filled into cryopreservation tubes. They were frozen using a programmed cooling device and stored in liquid nitrogen.

[0095] Place the cryovial of PBMC in a 37°C water bath and shake to thaw quickly and evenly. Transfer the thawed PBMC to culture medium and wash once by centrifugation. Resuspend the washed PBMC in culture medium and place in a cell culture flask or cell culture bag. Incubate in a CO2 incubator at 37±1°C and 5±0.5% CO2 for 2 hours. This procedure aims to restore PBMC viability and functionality and remove apoptotic cells.

[0096] After the PBMC culture was completed, the cell suspension was centrifuged once and then washed once with DPBS. The cell density of the washed PBMC was adjusted 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 stand and magnetically separated to remove CD3 + The sorted cells were adjusted to 2×10 6 CD3 + cells / ml, placed in culture flasks, and activated and cultured in a 37°C, 5% CO2 incubator for 24 hours.

[0097] The activated cells were taken out of the incubator and washed once with IL-2-containing medium by centrifugation. 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 transduction was performed for 22 hours in a 37°C, 5% CO2 incubator. After viral transduction, the magnetic beads in the cell suspension were first removed with a magnetic stand. A portion of the cells were washed three times with sodium chloride injection by centrifugation, resuspended in freezing solution, and 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 were cultured at a concentration of 10 cells / ml in a culture flask at 37°C in a 5% CO2 incubator for 22.5 hours. After the culture was complete, a portion of the cells was harvested: the magnetic beads were removed again, the cells were washed three times by centrifugation with sodium chloride injection, resuspended in freezing buffer, transferred to a cryostat, and cryopreserved using a programmed cooling device for long-term storage in liquid nitrogen (72-hour DASH CAR-T cells). The remaining cells were cultured for another 4 days, washed three times by centrifugation with sodium chloride injection, resuspended in freezing buffer, transferred to a cryostat, cryopreserved using a programmed cooling device, and stored in liquid nitrogen for long-term storage (extended culture of CAR-T cells; this group of cells mimics the total preparation time for CAR-T cells in the original process, typically 8-12 days).

[0098] DASH CAR-T cells and extended cultured CAR-T cells were tested before freezing, and the results are shown in Figure 1. The CAR-T cells produced by the DASH CAR-T process have little difference in quality with the extended cultured CAR-T cells in indicators highly related to product quality, including CAR infection rate, cell viability, CD3 ratio, CD19 ratio, etc.; the 48h CAR infection rate is lower 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 the cells were close to 0%. The results of cell activation-related indicators, such as CD25, CD69, and cell diameter, were significantly 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. - / CCR7 + There are also significant differences in the proportion of populations representing naïve T cells. Among them, DASH CAR-T cells still contain nearly 30% to 50% naïve T cells, while the proportion of naïve T cells in CAR-T cells cultured for a long time is less than 2%. - / CCR7 + The naive T cells represented are T cells with a high degree of stemness, which is associated with cell proliferation and tumor-killing ability. Based on these in vitro results, DASH CAR-T cells exhibit a high activation state and increased T cell stemness, potentially contributing to superior proliferation and tumor-killing abilities. 2. CAR-T cell expansion and functional testing at the end point of culture in the DASH CAR-T process

[0099] The frozen DASH CAR-T cells and extended cultured CAR-T cells were revived in a 37°C water bath, washed once by centrifugation with IL-2 medium, resuspended, and inoculated into culture flasks. 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 of cytotoxic T cell degranulation, which directly reflects the level of cell killing activity.

[0100] As shown in Figure 2, DASH CAR-T cells exhibited significantly higher expansion capacity than those cultured for extended periods, and the CAR infection rate of 48-hour DASH CAR-T cells continued to increase. This also demonstrates that even with a low CAR infection rate detected at harvest, 48-hour DASH CAR-T cells can still express CAR molecules on their cell surface after thawing and culture. In a cell killing assay, at an effector-target ratio of 1:1, DASH CAR-T cells and CAR-T cells cultured for extended periods exhibited similar cytotoxicity. Furthermore, CD107a expression on DASH CAR-T cells was similar to that on CAR-T cells cultured for extended periods. 3. Evaluation of the in vivo anti-tumor effect of DASH CAR-T technology and CAR-T cells with extended culture time

[0101] In vivo studies: Immunodeficient NOG mice were used for in vivo experiments, and 5×10 5 Three days after inoculation, mice were divided into groups based on their weight and total fluorescence intensity, with 5 mice in each group, for a total of 10 groups, including a vehicle control group; a NT cell group; a low-dose and high-dose CAR-T group using traditional technology (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). Cells were infused on the second day after grouping, designated as day 0. Fluorescence intensity in mice was measured weekly to evaluate the tumor inhibitory effect of the test article in vivo. The number of T cells and CAR-T cells in peripheral blood was measured once to assess the in vivo expansion of T / CAR-T cells. Mouse body weight was measured twice to assess the overall condition of the mice.

[0102] The experimental results showed (Figure 3) that CD19 CAR-T prepared by various processes could 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 was comparable 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 was comparable to that of the traditional process CAR-T high-dose group, indicating that the rapid preparation of the DASH CAR-T process can reduce the cell infusion dose while maintaining the same therapeutic effect; the BLI value 7 days after infusion showed that the therapeutic effect of the DASH 72h CAR-T medium and low-dose groups was better than that of the DASH 48h CAR-T medium and low-dose groups, suggesting that the 72-hour preparation process is superior.

[0103] The weight data of mice showed no significant difference in weight among the groups of mice (Figure 4), indicating that the CD19 CAR-T did not show obvious toxic side effects in the mice after transfusion.

[0104] Analysis of T cell expansion results in mice (Figure 5) showed that T cells did not expand in mice that received T cells without virus transduction and CAR-T cells using traditional technology. T cells expanded significantly in mice in the DASH group. 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 the 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 a chimeric antigen receptor T cell (CAR-T cell) 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 cells 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) includes the 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 of step 3) includes: washing the T cells with sodium chloride injection solution, 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) includes: contacting T cells or a cell population containing T cells with magnetic beads coupled with anti-CD3 antibodies and anti-CD28 antibodies, and 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, its concentration is 50-500 IU / ml, preferably 100-400 IU / ml, more preferably 300 IU / ml; Preferably, the culture conditions of the activation culture are about 37° C. and about 5% CO 2 environment.

4. The method according to claim 1, wherein: Step 2) includes: washing step 1) after the activation culture is completed, the activated T cells or the cell population containing the activated T cells are resuspended in a culture medium, and the obtained cell suspension is co-cultured 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-cultivation conditions are about 37° C. and about 5% CO 2 environment.

5. The method according to claim 1, wherein Step 3) comprises: washing the chimeric antigen receptor T cells obtained in step 2), resuspending in the culture medium, adjusting the cell density, culturing for 18-24 hours, harvesting and freezing after the culture is completed, 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, its concentration 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, wherein: 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 of 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, peritoneal metastasis of gastric cancer, 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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