WO2021018311A1 - 通用型car-t细胞及其制备和应用 - Google Patents

通用型car-t细胞及其制备和应用 Download PDF

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WO2021018311A1
WO2021018311A1 PCT/CN2020/106635 CN2020106635W WO2021018311A1 WO 2021018311 A1 WO2021018311 A1 WO 2021018311A1 CN 2020106635 W CN2020106635 W CN 2020106635W WO 2021018311 A1 WO2021018311 A1 WO 2021018311A1
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cells
car
cell
bcma
cell population
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PCT/CN2020/106635
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French (fr)
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季昀
杨伟志
崔慧娟
罗晓兵
黄家琪
朱恃贵
姚昕
姚意弘
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上海赛比曼生物科技有限公司
无锡赛比曼生物科技有限公司
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Priority to US17/631,006 priority Critical patent/US20220380726A1/en
Priority to JP2022506164A priority patent/JP2022544069A/ja
Priority to EP20846905.6A priority patent/EP4015625A1/en
Publication of WO2021018311A1 publication Critical patent/WO2021018311A1/zh

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Definitions

  • the present invention relates to the field of biomedicine, and more specifically to general-purpose CAR-T cells and their preparation and application
  • T cells can be genetically modified to express chimeric antigen receptor (CAR), which includes an antigen recognition part and a T cell activation region.
  • CAR uses the antigen-binding properties of monoclonal antibodies to redirect the specificity and reactivity of T cells and target targets in a non-MHC-restricted manner. This non-MHC restricted antigen recognition allows CAR-expressing T cells to recognize antigens without antigen processing, thus avoiding a major mechanism for tumor escape. In addition, CAR will not dimerize with the ⁇ and ⁇ chains of endogenous TCR.
  • Universal CAR-T cells are an important alternative resource for autologous T cells in T cell immunotherapy, in which the endogenous TRAC and B2M genes of allogeneic T cells are knocked out to prevent graft versus host disease and host immune rejection, respectively.
  • These gene-edited cells from allogeneic donors provide opportunities for cellular immunotherapy for patients who cannot provide enough autologous T cells, such as infants or patients who have received multiple rounds of chemotherapy.
  • the purpose of the present invention is to provide Tscm-enriched universal CAR-T cells and their preparation and application.
  • a non-naturally occurring T cell population is provided.
  • the proportion of T memory stem cells (stem cell-like memory T cells, Tscm) in the T cell population is C1 ⁇ 50%.
  • the total number of T cells in the T cell population is C1 ⁇ 50%.
  • the C1 is 50-85%, preferably 60-80%.
  • the T cells include CAR-T cells.
  • the CAR-T cells include autologous or allogeneic CAR-T cells.
  • the CAR-T cells include universal CAR-T cells.
  • an immune-related gene selected from the group consisting of TRAC gene, B2M gene, or a combination thereof is knocked out or down-regulated.
  • T memory stem cells are CAR-T cells
  • the proportion of the T memory stem cells (Tscm) is C1 ⁇ 50%, calculated as the total number of CAR-T cells in the T cell population .
  • the C1 is 50-85%, preferably 60-80%.
  • the T memory stem cell has a CCR7 + phenotype.
  • C3 is 30-100%, more preferably 70-99.9%, more preferably 80-99%.
  • the T memory stem cells include CCR7 + CD45RA + T cells.
  • the ratio C2 that expresses the biomarker CCR7 is ⁇ 50%, preferably ⁇ 60%, more preferably, ⁇ 70%.
  • the activity intensity Q1 of the CAR-T cell (take the average fluorescence intensity MFI as an example) ⁇ 500, preferably ⁇ 1000, more preferably, ⁇ 3000, more preferably ⁇ 4000 ( Such as 1000-5000).
  • the T cell population is expanded in vitro or not expanded in vitro.
  • the in vitro expansion is to expand the cells in the presence of IL15, IL7 and IL21 (commonly known as Tscm expansion factor) in the culture system under suitable culture conditions.
  • IL15, IL7 and IL21 commonly known as Tscm expansion factor
  • the concentration of Tscm amplification factor includes:
  • the concentration of IL15 is 1-200ng/ml, preferably 3-100ng/ml, more preferably 5-20ng/ml;
  • the concentration of IL7 is 0.5-50ng/ml, preferably, 1-20ng/ml, more preferably 3-10ng/ml; and/or
  • the concentration of IL21 is 1-100ng/ml, preferably 3-50ng/ml, more preferably 5-20ng/ml.
  • the culture system includes a serum culture system and a serum-free culture system.
  • the cell expansion time is 0.1-30 days, preferably 0.5-25 days, more preferably 1-20 days, most preferably 2-15 days or 3-15 days or 5-15 days.
  • the CAR-T cells are universal CAR-T cells targeting tumor antigens.
  • the tumor antigen is selected from the group consisting of TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, Mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR- ⁇ , SSEA- 4.
  • CD20 folate receptor ⁇ , ERBB2 (Her2/neu), MUC1, EGFR, NCAM, Prostase, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, phenol EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor ⁇ , TEM1/CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, poly Sialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, legumain, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie
  • the CAR-T cell expresses a chimeric antigen receptor, and the structure of the chimeric antigen receptor is shown in the following formula I:
  • Each "-" is independently a connecting peptide or a peptide bond
  • L is an optional signal peptide sequence
  • scFv is an antibody single-chain variable region sequence targeting tumor antigens
  • H is an optional hinge region
  • TM is the transmembrane domain
  • C is no or costimulatory signal molecule
  • CD3 ⁇ is a cytoplasmic signal transduction sequence derived from CD3 ⁇ .
  • the CAR-T cell is a universal CAR-T cell targeting BCMA.
  • the scFv targets BCMA.
  • the L is a signal peptide of a protein selected from the group consisting of CD8, GM-CSF, CD4, CD137, or a combination thereof.
  • the H is the hinge region of a protein selected from the group consisting of CD8, CD28, CD137, or a combination thereof.
  • the TM is a transmembrane region of a protein selected from the group consisting of CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86 , CD134, CD137, CD154, or a combination thereof.
  • the C is a costimulatory signal molecule of a protein selected from the group consisting of: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1 , Dap10, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), NKG2D, GITR, TLR2, or a combination thereof.
  • C includes costimulatory signal molecules derived from 4-1BB and/or costimulatory signal molecules derived from CD28.
  • the T cells are derived from human or non-human mammals.
  • the T cell population is prepared by the method described in the third aspect of the present invention.
  • a cell preparation which contains the non-naturally occurring T cell population according to the first aspect of the present invention and a pharmaceutically acceptable carrier, diluent or excipient.
  • a method for preparing CAR-T cells including the steps:
  • the isolated T cells are selected from the group consisting of naive T cells (Tn cells), a cell population rich in Tn cells, or total T cells, or a combination thereof.
  • the isolated T cells are naive T cells (Tn cells) or a cell population rich in Tn cells.
  • the T cells include autologous or allogeneic T cells.
  • the concentration of IL15 in the culture system is 1-200 ng/ml, preferably, 3-100 ng/ml, More preferably, it is 5-20ng/ml; the concentration of IL7 is 0.5-50ng/ml, preferably, 1-20ng/ml, more preferably 3-10ng/ml; and the concentration of IL21 is 1-100ng/ml ml, preferably 3-50ng/ml, more preferably 5-20ng/ml.
  • the method is used to prepare universal CAR-T cells.
  • the CAR-T cell targets the tumor antigen BCMA.
  • the T cells are isolated from human peripheral blood, preferably, from peripheral blood mononuclear cells.
  • the isolated T cells include total T cells and naive T cells (Tn cells).
  • the initial T cell is CCR7 + CD45RA + T cell.
  • step (b) the expansion culture is performed in AIM-V medium.
  • the culture system further contains FBS, Glutamax, Pen/Strip, or a combination thereof; preferably, the concentration of FBS is 5-20%.
  • the modification in step (c), includes T cell activation, T cell nuclear transfection and lentiviral transduction.
  • step (c) the transformation includes the following steps:
  • a method for preparing CAR-T cells including the steps:
  • a T cell population (especially CAR-T cells) according to the first aspect of the present invention, or the use of the cell preparation according to the second aspect of the present invention, for preparing preventive And/or drugs to treat cancer or tumors.
  • the tumor is selected from the group consisting of hematological tumors, solid tumors, or a combination thereof.
  • the hematological tumor is selected from the group consisting of acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), diffuse large B cell lymphoma (DLBCL), or a combination thereof.
  • AML acute myeloid leukemia
  • MM multiple myeloma
  • CLL chronic lymphocytic leukemia
  • ALL acute lymphocytic leukemia
  • DLBCL diffuse large B cell lymphoma
  • the solid tumor is selected from the group consisting of gastric cancer, gastric cancer peritoneal metastasis, liver cancer, leukemia, kidney tumor, lung cancer, small bowel cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colorectal cancer, Cervical cancer, ovarian cancer, lymphoma, nasopharyngeal cancer, adrenal gland tumor, bladder tumor, non-small cell lung cancer (NSCLC), glioma, endometrial cancer, testicular cancer, colorectal cancer, urinary tract cancer, thyroid cancer , Or a combination thereof.
  • gastric cancer gastric cancer peritoneal metastasis
  • liver cancer leukemia, kidney tumor, lung cancer, small bowel cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colorectal cancer, Cervical cancer, ovarian cancer, lymphoma, nasopharyngeal cancer, adrenal gland tumor, bladder tumor, non-small cell lung cancer (NSCLC), glioma,
  • the solid tumor is selected from the group consisting of ovarian cancer, mesothelioma, lung cancer, pancreatic cancer, breast cancer, liver cancer, endometrial cancer, or a combination thereof.
  • a use of the T cell population according to the first aspect of the present invention or the cell preparation according to the second aspect of the present invention is provided to prevent and/or treat cancer or tumors.
  • a method for treating diseases comprising: administering an appropriate amount of the T cell population according to the first aspect of the present invention or the cell cells according to the second aspect of the present invention to a subject in need of treatment preparation.
  • the disease is cancer or tumor.
  • Figure 1 shows the knockout and purification rate of TRAC and B2M genes in universal T cells detected by flow cytometry.
  • Figure 2 shows the flow cytometric detection of BCMA CAR transduction rate in universal T cells.
  • Figure 3 shows the release of IFN- ⁇ after co-cultivation of universal CAR-T cells and targeted cells expressing BCMA by ELISA.
  • Figure 4 shows the phenotype and purity of the initial T cells isolated from PBMC by flow cytometry.
  • Figure 5 shows the proliferation rate of three BCMA CAR-T cells.
  • Figure 6 shows the ratio of various T cell subtypes and the expression level of CCR7 in the three BCMA CAR-T cells.
  • Figure 7 shows the flow cytometric detection of CAR transfection rates of three BCMA CAR-T cells.
  • Figure 8 shows the release of IFN- ⁇ after three kinds of BCMA CAR-T cells and BCMA-expressing targeted cells are co-cultured.
  • Figure 9 shows the killing rate of three BCMA CAR-T cells to targeted cells expressing BCMA.
  • FIG. 10 shows the enrichment and cell growth of Tscm cells in T cells cultured under different conditions.
  • FIG. 11 shows the BCMA CAR transduction rate of T cells cultured under different conditions.
  • Figure 12 shows the release of IFN- ⁇ from T cells cultured under different conditions after BCMA CAR transduction.
  • Figure 13 shows the enrichment of CCR7 + CD45RA + Tscm cells in three BCMA CAR-T cells.
  • Tscm T-memory stem cells
  • Tscm-enriched universal BCMA CAR-T cells T-memory stem cells
  • T cells were cultured under conditions that are conducive to the expansion of T memory stem cells (Tscm), and new second-generation Tscm-enriched universal CAR-T cells were prepared.
  • Tscm T memory stem cells
  • Such CAR-T cells are much better than the first-generation universal CAR-T cells in proliferation, phenotype, and killing of target cells, indicating that the second-generation universal CAR-T cells are more optimized and universal.
  • Type CAR-T products are much better than the first-generation universal CAR-T cells in proliferation, phenotype, and killing of target cells, indicating that the second-generation universal CAR-T cells are more optimized and universal.
  • nucleofection can be used to deliver the Cas9 RNP complex to T cells to simultaneously knock out endogenous TRAC and B2M genes.
  • high-purity TRAC and B2M double knockout universal T cells can be prepared.
  • T cells use lentivirus transduction to introduce BCMA CAR expression to produce the first generation of universal BCAM CAR-T cells.
  • BCMA CAR expression can effectively kill target cells, and their activity is similar to autologous CAR-T cells without gene editing.
  • administration refers to the physical introduction of the product of the present invention into a subject using any of the various methods and delivery systems known to those skilled in the art, including intravenous, intramuscular, subcutaneous, intraperitoneal, spinal cord or Other parenteral routes of administration, such as by injection or infusion.
  • antibody shall include, but is not limited to, immunoglobulins, which specifically bind to antigens and comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or antigens thereof Combined part.
  • H chain includes a heavy chain variable region (abbreviated as VH herein) and a heavy chain constant region.
  • the heavy chain constant region contains three constant domains CH1, CH2 and CH3.
  • Each light chain includes a light chain variable region (abbreviated as VL herein) and a light chain constant region.
  • the light chain constant region contains a constant domain CL.
  • VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDR), which are interspersed with more conservative regions called framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL contains three CDRs and four FRs, arranged in the following order from the amino terminal to the carboxy terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with antigens.
  • amino acids in this article are identified by internationally accepted single English letters, and the corresponding three-letter abbreviations of amino acid names are: Ala(A), Arg(R), Asn(N), Asp(D), Cys (C), Gln(Q), Glu(E), Gly(G), His(H), I1e(I), Leu(L), Lys(K), Met(M), Phe(F), Pro (P), Ser(S), Thr(T), Trp(W), Tyr(Y), Val(V).
  • Universal T cells are an important alternative resource for autologous T cells in T cell immunotherapy.
  • the endogenous TRAC and B2M genes of allogeneic T cells are knocked out to prevent graft-versus-host disease and host immune rejection, respectively.
  • These gene-edited cells from allogeneic donors provide opportunities for cellular immunotherapy for patients who cannot provide enough autologous T cells, such as infants or patients who have received multiple rounds of chemotherapy.
  • These cells can be further modified by lentiviral vectors encoding CAR or TCR to generate allogeneic CAR/TCR T cells.
  • the nucleofection method is used to deliver the Cas9 RNP complex to T cells to simultaneously knock out endogenous TRAC and B2M genes.
  • BCMA CAR-T cells can effectively kill target cells, and their activity is similar to that of autologous BCMA CAR-T cells without gene editing.
  • Tn cells namely naive T cells (Tn), also called unsensitized T cells, mature in the thymus and migrate to peripheral lymphoid tissues, and are relatively static before being exposed to antigen stimulation.
  • Tn naive T cells
  • unsensitized T cells mature in the thymus and migrate to peripheral lymphoid tissues, and are relatively static before being exposed to antigen stimulation.
  • Tscm cells mean T memory stem cells, also called stem cell-like memory T cells (stem cell memory T cell, Tscm)
  • BCMA is a B cell maturation antigen, also known as CD269 or TNFRSF17. It is a member of the tumor necrosis factor receptor superfamily. Its ligands are B cell activating factor (BAFF) and proliferation inducing ligand (APRIL).
  • BAFF B cell activating factor
  • APRIL proliferation inducing ligand
  • BCMA BCMA with BAFF and APRIL triggers NF-kB activation and induces the up-regulation of anti-apoptotic Bcl-2 members such as Bcl-xL or Bcl-2 and Mcl-1.
  • Bcl-xL Bcl-xL
  • Bcl-2 and Mcl-1 Bcl-2 members
  • BCMA BCMA-deficient mice have reduced plasma cells in the bone marrow but the level of plasma cells in the spleen is not affected. Mature B cells can normally differentiate into plasma cells in BCMA knockout mice. BCMA knockout mice look normal and healthy, and the number of B cells is normal, but plasma cells cannot survive for a long time.
  • BCMA is also highly expressed in malignant plasma cells, such as multiple myeloma and plasma cell leukemia, and BCMA is also detected in HRS cells of patients with Hodgkin's lymphoma.
  • hematological malignancies account for about 10% of all malignant tumors, and myeloma accounts for 15% of all hematological malignancies.
  • the expression of BCMA is related to the progression of multiple myeloma disease.
  • the BCMA gene is highly expressed in myeloma samples, but is very low in chronic lymphocytic leukemia, acute lymphocytic leukemia, and acute T-cell lymphocytic leukemia.
  • B-cell lymphoma is significantly increased in mouse models that overexpress the BCMA ligands BAFF and APRIL.
  • Ligands that bind to BCMA have been shown to regulate the growth and survival of multiple myeloma cells expressing BCMA.
  • the combination of BCMA with BAFF and APRIL allows malignant plasma cells to survive. Therefore, depletion of BCMA-expressing tumor cells and disrupting the interaction between BCMA ligand and receptor can improve the malignancy of multiple myeloma or other BCMA-positive B lineage Treatment results for lymphoma.
  • the CAR of the present invention can contain any type of antigen binding domains that target tumor antigens
  • the chimeric antigen receptor (CAR) of the present invention includes an extracellular domain, a transmembrane domain, and an intracellular domain.
  • the extracellular domain includes target-specific binding elements (also called antigen binding domains).
  • the intracellular domain includes the costimulatory signal transduction region and the zeta chain part.
  • the costimulatory signal transduction region refers to a part of the intracellular domain that includes costimulatory molecules.
  • Co-stimulatory molecules are cell surface molecules needed for effective response of lymphocytes to antigens, not antigen receptors or their ligands.
  • a linker can be incorporated between the extracellular domain and the transmembrane domain of the CAR, or between the cytoplasmic domain and the transmembrane domain of the CAR.
  • the term "linker” generally refers to any oligopeptide or polypeptide that functions to connect the transmembrane domain to the extracellular or cytoplasmic domain of a polypeptide chain.
  • the linker may comprise 0-300 amino acids, preferably 2 to 100 amino acids and most preferably 3 to 50 amino acids.
  • the extracellular domain of the CAR provided by the present invention includes an antigen binding domain that targets BCMA.
  • the CAR of the present invention can perform antigen recognition based on the antigen binding specificity.
  • it binds to its associated antigen, it affects tumor cells, resulting in tumor cells not growing, being promoted to die or being affected in other ways, and causing the patient's tumor burden to shrink or eliminate.
  • the antigen binding domain is preferably fused with an intracellular domain from one or more of the costimulatory molecule and the zeta chain.
  • the antigen binding domain is fused with the intracellular domain combined with the 4-1BB signaling domain and the CD3 ⁇ signaling domain.
  • antigen binding domain and “single chain antibody fragment” all refer to Fab fragments, Fab' fragments, F(ab')2 fragments, or single Fv fragments that have antigen binding activity.
  • Fv antibody contains the variable region of the heavy chain and the variable region of the light chain, but does not have the constant region, and has the smallest antibody fragment with all the antigen binding sites.
  • Fv antibodies also contain a polypeptide linker between the VH and VL domains, and can form the structure required for antigen binding.
  • the antigen binding domain is usually scFv (single-chain variable fragment).
  • the size of scFv is generally 1/6 that of a complete antibody.
  • the single-chain antibody is preferably an amino acid chain sequence encoded by a nucleotide chain.
  • the scFv includes an antibody that specifically recognizes BCMA, preferably a single-chain antibody.
  • the CAR can be designed to include a transmembrane domain fused to the extracellular domain of the CAR.
  • a transmembrane domain that is naturally associated with one of the domains in the CAR is used.
  • transmembrane domains can be selected or modified by amino acid substitutions to avoid binding such domains to the transmembrane domains of the same or different surface membrane proteins, thereby minimizing interaction with the receptor complex. Interaction of other members.
  • the intracellular domain in the CAR of the present invention includes the signaling domain of 4-1BB and the signaling domain of CD3 ⁇ .
  • CRISPR/Cas9 RNA interference technology
  • TALENs transcription activator-like (TAL) effect nucleases
  • ZFNs Zinc finger nucleases
  • the CRISPR (clustered regularly interspersed short palindromic repeats)/Cas (CRISPR-associated) system is a natural immune system unique to prokaryotes, which is used to resist viruses or foreign plasmids.
  • Type II CRISPR/Cas system as a genome editing tool mediated by RNA has been successfully applied in many eukaryotes and prokaryotes.
  • the development of the CRISPR/Cas9 system has completely changed people's ability to edit DNA sequences and regulate the expression level of target genes, thereby providing powerful tools for precise genome editing of organisms.
  • the simplified CRISPR/Cas9 system consists of two parts: Cas9 protein and sgRNA.
  • the principle of action is that sgRNA forms a Cas9-sgRNA complex with Cas9 protein through its Cas9 handle.
  • the base complementary pairing region sequence of sgRNA in the Cas9-sgRNA complex is paired and combined with the target sequence of the target gene through the principle of base complementary pairing.
  • Cas9 uses its own endonuclease activity to cut the target DNA sequence.
  • the CRISPR/Cas9 system has several obvious advantages: ease of use, simplicity, low cost, programmability, and the ability to edit multiple genes at the same time.
  • the nucleic acid sequence encoding the desired molecule can be obtained using recombinant methods known in the art, such as, for example, by screening a library from cells expressing the gene, by obtaining the gene from a vector known to include the gene, or by using standard Technology, directly isolated from the cells and tissues containing the gene.
  • the gene of interest can be produced synthetically.
  • the present invention also provides a vector into which the expression cassette of the present invention is inserted.
  • Vectors derived from retroviruses such as lentiviruses are suitable tools to achieve long-term gene transfer because they allow long-term, stable integration of transgenes and their propagation in daughter cells.
  • Lentiviral vectors have advantages over vectors derived from oncogenic retroviruses such as murine leukemia virus because they can transduce non-proliferating cells, such as hepatocytes. They also have the advantage of low immunogenicity.
  • the expression cassette or nucleic acid sequence of the present invention is usually operably linked to a promoter and incorporated into an expression vector.
  • the vector is suitable for replication and integration of eukaryotic cells.
  • a typical cloning vector contains transcription and translation terminators, initial sequences, and promoters that can be used to regulate the expression of the desired nucleic acid sequence.
  • the expression construct of the present invention can also use standard gene delivery protocols for nucleic acid immunization and gene therapy. Methods of gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, 5,589,466, which are hereby incorporated by reference in their entirety.
  • the invention provides a gene therapy vector.
  • the nucleic acid can be cloned into many types of vectors.
  • the nucleic acid can be cloned into such vectors, which include, but are not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids.
  • Specific vectors of interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.
  • the expression vector can be provided to the cell in the form of a viral vector.
  • Viral vector technology is well known in the art and described in, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals.
  • Viruses that can be used as vectors include, but are not limited to, retrovirus, adenovirus, adeno-associated virus, herpes virus, and lentivirus.
  • a suitable vector contains an origin of replication that functions in at least one organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers (e.g., WO01/96584; WO01/29058; and US Patent No. 6,326,193).
  • retroviruses provide a convenient platform for gene delivery systems.
  • the selected gene can be inserted into the vector and packaged into retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to target cells in vivo or in vitro.
  • Many retroviral systems are known in the art.
  • adenovirus vectors are used.
  • Many adenovirus vectors are known in the art.
  • a lentiviral vector is used.
  • promoter elements can regulate the frequency of transcription initiation. Generally, these are located in the 30-110bp region upstream of the start site, although it has recently been shown that many promoters also contain functional elements downstream of the start site.
  • the spacing between promoter elements is often flexible in order to maintain promoter function when the elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased by 50 bp before the activity begins to decrease.
  • tk thymidine kinase
  • individual elements can act cooperatively or independently to initiate transcription.
  • a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence.
  • the promoter sequence is a strong constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operably linked to it.
  • Another example of a suitable promoter is elongation growth factor-1 ⁇ (EF-1 ⁇ ).
  • constitutive promoter sequences can also be used, including but not limited to the simian virus 40 (SV40) early promoter, mouse breast cancer virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Ruth sarcoma virus promoter, and human gene promoters, such as but not limited to actin promoter , Myosin promoter, heme promoter and creatine kinase promoter.
  • the present invention should not be limited to the application of constitutive promoters. Inducible promoters are also considered part of the invention.
  • an inducible promoter provides a molecular switch that can turn on expression of a polynucleotide sequence operably linked to an inducible promoter when such expression is desired, or turn off expression when expression is undesirable.
  • inducible promoters include, but are not limited to, metallothionein promoter, glucocorticoid promoter, progesterone promoter and tetracycline promoter.
  • the expression vector introduced into the cell may also contain either or both of the selectable marker gene or the reporter gene, so as to facilitate the search for the cell population to be transfected or infected by the viral vector Identification and selection of expressing cells.
  • the selectable marker can be carried on a single piece of DNA and used in the co-transfection procedure. Both the selectable marker and the reporter gene can be flanked by appropriate regulatory sequences to enable expression in the host cell.
  • Useful selectable markers include, for example, antibiotic resistance genes such as neo and the like.
  • Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences.
  • a reporter gene is a gene that does not exist in or is expressed by a recipient organism or tissue, and it encodes a polypeptide whose expression is clearly indicated by some easily detectable properties such as enzyme activity. After the DNA has been introduced into the recipient cell, the expression of the reporter gene is measured at an appropriate time.
  • Suitable reporter genes may include genes encoding luciferase, ⁇ -galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase or green fluorescent protein (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79 -82).
  • Suitable expression systems are well known and can be prepared using known techniques or obtained commercially. Generally, a construct with a minimum of 5 flanking regions that shows the highest level of reporter gene expression is identified as a promoter. Such a promoter region can be linked to a reporter gene and used to evaluate the ability of the reagent to regulate the promoter-driven transcription.
  • the vector can be easily introduced into a host cell by any method in the art, for example, a mammalian, bacterial, yeast, or insect cell.
  • the expression vector can be transferred into the host cell by physical, chemical or biological means.
  • Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and so on. Methods of producing cells including vectors and/or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). The preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.
  • Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors.
  • Viral vectors especially retroviral vectors, have become the most widely used method of inserting genes into mammalian cells such as humans.
  • Other viral vectors can be derived from lentivirus, poxvirus, herpes simplex virus I, adenovirus, adeno-associated virus, and so on. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.
  • Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and lipids Plastid.
  • colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads
  • lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and lipids Plastid.
  • Exemplary gel systems used as delivery vehicles in vitro and in vivo are generally liposomes (e.g., artificial membrane vesicles).
  • an exemplary delivery vehicle is liposomes.
  • lipid formulations to introduce nucleic acids into host cells (in vitro, ex vivo, or in vivo).
  • the nucleic acid can be associated with lipids.
  • Lipid-associated nucleic acids can be encapsulated in the aqueous interior of liposomes, dispersed in the lipid bilayer of liposomes, and attached via linking molecules associated with both liposomes and oligonucleotides
  • the lipid, lipid/DNA or lipid/expression vector associated with the composition is not limited to any specific structure in the solution.
  • Lipids are fatty substances, which can be naturally occurring or synthetic lipids.
  • lipids include fat droplets, which occur naturally in the cytoplasm and in such compounds containing long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.
  • the vector is a lentiviral vector.
  • the present invention provides a CAR-T cell containing the first aspect of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient.
  • the formulation is a liquid formulation.
  • the preparation is an injection.
  • the concentration of the CAR-T cells in the preparation is 1 ⁇ 10 3 -1 ⁇ 10 8 cells/ml, more preferably 1 ⁇ 10 4 -1 ⁇ 10 7 cells/ml.
  • the formulation may include buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; protein; polypeptides or amino acids such as glycine ; Antioxidant; Chelating agent such as EDTA or glutathione; Adjuvant (for example, aluminum hydroxide); and Preservative.
  • buffers such as neutral buffered saline, sulfate buffered saline, etc.
  • carbohydrates such as glucose, mannose, sucrose or dextran, mannitol
  • protein polypeptides or amino acids such as glycine
  • Antioxidant such as EDTA or glutathione
  • Adjuvant for example, aluminum hydroxide
  • Preservative for example, aluminum hydroxide
  • the present invention includes therapeutic applications using the CAR-T cells described in the first aspect of the present invention.
  • the transduced T cells can target BCMA, a marker of tumor cells, and coordinately activate T cells, causing T cell immune responses, thereby significantly improving its killing efficiency on tumor cells.
  • the present invention also provides a method for stimulating a T cell-mediated immune response to a target cell population or tissue of a mammal, which comprises the following steps: administering the CAR-T cell of the present invention to the mammal.
  • the present invention includes a type of cell therapy in which the patient's autologous T cells (or heterologous donors) are isolated, activated and genetically modified to produce CAR-T cells, and then injected into the patient.
  • the probability of graft-versus-host disease is extremely low, and the antigen is recognized by T cells in a non-MHC-restricted manner.
  • one CAR-T can treat all cancers that express the antigen.
  • CAR-T cells can replicate in vivo, producing long-term persistence that can lead to sustained tumor control.
  • the CAR-T cells of the present invention can undergo stable T cell expansion in vivo and last for an extended amount of time.
  • the CAR-mediated immune response can be part of an adoptive immunotherapy step in which CAR-modified T cells induce an immune response specific to the antigen binding domain in the CAR.
  • CAR-T cells that are anti-BCMA elicit a specific immune response against cells that express BCMA.
  • Cancers that can be treated include tumors that have not been vascularized or have not been substantially vascularized, as well as vascularized tumors.
  • the cancer may include non-solid tumors (such as hematological tumors such as leukemia and lymphoma) or may include solid tumors.
  • the types of cancer treated with the CAR of the present invention include, but are not limited to, carcinoma, blastoma and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignant tumors, such as sarcoma, carcinoma and melanoma. Also includes adult tumor/cancer and childhood tumor/cancer.
  • Hematological cancer is cancer of the blood or bone marrow.
  • leukemias include leukemias, including acute leukemias (such as acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia and myeloblastic, promyelocytic, myelomonocytic type , Monocytic and erythroleukemia), chronic leukemia (such as chronic myeloid (granulocyte) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non- Hodgkin's lymphoma (painless and high-grade form), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.
  • acute leukemias such as acute lymphoblastic leukemia, acute myeloid leuk
  • a solid tumor is an abnormal mass of tissue that does not usually contain a cyst or fluid area.
  • Solid tumors can be benign or malignant. Different types of solid tumors are named after the cell type that formed them (such as sarcoma, carcinoma, and lymphoma). Examples of solid tumors such as sarcoma and cancer include fibrosarcoma, myxosarcoma, liposarcoma, mesothelioma, lymphoid malignancies, pancreatic cancer, and ovarian cancer.
  • the CAR-modified T cell of the present invention can also be used as a type of vaccine for ex vivo immunity and/or in vivo therapy of mammals.
  • the mammal is a human.
  • cells are isolated from mammals (preferably humans) and genetically modified (ie, transduced or transfected in vitro) with a vector expressing the CAR disclosed herein.
  • CAR-modified cells can be administered to mammalian recipients to provide therapeutic benefits.
  • the mammalian recipient can be a human, and the CAR-modified cell can be autologous relative to the recipient.
  • the cell may be allogeneic, syngeneic, or xenogeneic relative to the recipient.
  • the present invention also provides compositions and methods for in vivo immunization to elicit an immune response against an antigen in a patient.
  • the present invention provides a method for treating tumors, which comprises administering to a subject in need thereof a therapeutically effective amount of CAR-modified T cells of the present invention.
  • the CAR-modified T cells of the present invention may be administered alone or as a pharmaceutical composition in combination with a diluent and/or other components such as IL-2, IL-17 or other cytokines or cell populations.
  • the pharmaceutical composition of the present invention may include the target cell population as described herein, combined with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
  • compositions may include buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelate Mixtures such as EDTA or glutathione; adjuvants (for example, aluminum hydroxide); and preservatives.
  • buffers such as neutral buffered saline, sulfate buffered saline, etc.
  • carbohydrates such as glucose, mannose, sucrose or dextran, mannitol
  • proteins polypeptides or amino acids
  • antioxidants such as glycine
  • chelate Mixtures such as EDTA or glutathione
  • adjuvants for example, aluminum hydroxide
  • preservatives for example, aluminum hydroxide
  • the pharmaceutical composition of the present invention can be administered in a manner suitable for the disease to be treated (or prevented).
  • the number and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease-although the appropriate dosage can be determined by clinical trials.
  • the precise amount of the composition of the present invention to be administered can be determined by the physician, who considers the patient (subject ) Individual differences in age, weight, tumor size, degree of infection or metastasis, and disease. May generally indicated: including those described herein, the pharmaceutical compositions of T cells may be 104 to 109 doses cells / kg body weight, preferably 105 to 106 cells / kg body weight doses (including all integers within that range Value) application. The T cell composition can also be administered multiple times at these doses.
  • the cells can be administered by using injection techniques well known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988).
  • the optimal dosage and treatment regimen for a specific patient can be easily determined by those skilled in the medical field by monitoring the patient's signs of disease and adjusting the treatment accordingly.
  • the administration of the subject composition can be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation.
  • the compositions described herein can be administered to patients subcutaneously, intracutaneously, intratumorally, intranodal, intraspinal, intramuscular, by intravenous (i.v.) injection, or intraperitoneally.
  • the T cell composition of the present invention is administered to the patient by intradermal or subcutaneous injection.
  • the T cell composition of the present invention is preferably administered by i.v. injection.
  • the composition of T cells can be injected directly into tumors, lymph nodes or sites of infection.
  • the treatment modality includes but not limited to treatment with the following agents: the agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known It is ARA-C) or natalizumab treatment for MS patients or erfaizumab treatment for psoriasis patients or other treatments for PML patients.
  • the agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known It is ARA-C) or natalizumab treatment for MS patients or erfaizumab treatment for psoriasis patients or other treatments for PML patients.
  • the T cells of the present invention can be used in combination with chemotherapy, radiation, immunosuppressive agents, such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil and FK506, antibodies Or other immunotherapeutics.
  • the cell composition of the present invention is administered to bone marrow transplantation using chemotherapeutic agents such as fludarabine, external beam radiotherapy (XRT), cyclophosphamide (e.g., before, simultaneously, or after) patient.
  • chemotherapeutic agents such as fludarabine, external beam radiotherapy (XRT), cyclophosphamide (e.g., before, simultaneously, or after) patient.
  • the subject can undergo the standard treatment of high-dose chemotherapy followed by peripheral blood stem cell transplantation.
  • the subject receives an infusion of the expanded immune cells of the invention.
  • the expanded cells are administered before or after surgery.
  • the dosage of the above treatment administered to the patient will vary with the precise nature of the condition being treated and the recipient of the treatment.
  • the dosage ratio for human administration can be implemented according to the practice accepted in the art.
  • 1 ⁇ 10 6 to 1 ⁇ 10 10 modified T cells (for example, CAR-T20 cells) of the present invention can be administered to the patient by, for example, intravenous reinfusion for each treatment or course of treatment. .
  • the present invention provides a second-generation universal CAR-T cell enriched with Tscm, which shows better expansion rate, phenotype, IFN- ⁇ release ability and killing ability to target cells than the first generation. Based on the Tscm-enriched T cells (especially second-generation universal CAR-T cells) of the present invention, universal CAR-T cells with high activity against different targets can be prepared.
  • the present invention also prepares general-purpose T cells that target tumor targets (for example, general-purpose T cells for C-CAR088 for BCMA), which can specifically recognize and kill and express corresponding tumor targets (such as BCMA tumor antigens) The target cell.
  • target tumor targets for example, general-purpose T cells for C-CAR088 for BCMA
  • tumor targets such as BCMA tumor antigens
  • the present invention provides a method for preparing Tscm-enriched T cells, which can efficiently prepare Tscm-enriched T cell populations, especially universal CAR-T cell populations.
  • BCMA is used as a representative tumor-related specific target to prepare CAR-T cells that target tumor cells.
  • 500ul PBS containing 5ug/ml fibronectin and 5ug/ml anti-CD3 antibody was added to the wells of the 24-well plate, and the plate was incubated at 37°C for 3 hours. Take out the PBS and wash twice with 1ml PBS. The plate is ready for T cell stimulation. Count the number of T cells, add 5 ⁇ 10 6 T cells to each well, and centrifuge at 100g for 5 minutes at room temperature. The supernatant was discarded, the cells were resuspended in 2.5 ml of AIM-V medium, and then transferred to a plate coated with anti-CD3 antibody, and the plate was incubated at 37°C for 72 hours.
  • Tscm cell culture cultured in AIM-V medium containing 10% FBS, Glutamax, Pen/Strip, 10ng/ml IL15, 5ng/ml IL7 and 10ng/ml IL21 at a density of about 1 ⁇ 10 6 cells/ml.
  • the T cell population is first grouped by FSC and SSC.
  • the Aqua orange channel is used to distinguish live and dead cells, and the APC channel is used to distinguish BCMA-CAR positive cells.
  • CD3 and HLA A/B/C are used to distinguish CD3 and HLA A/B/C double knockout T cells.
  • TMB substrate to room temperature 30 minutes in advance, and add 100 ⁇ l per well to the microtiter plate. After reacting at room temperature for 5-10 minutes, add stop solution, 50 ⁇ l/well. Detect the absorbance at 450nm wavelength with a microplate reader, generate a standard curve based on the concentration and OD value of the standard, and calculate the IFN- ⁇ concentration of the test sample based on the OD value of the test sample.
  • Day 1 Open the operating software of RTCA MP and input experiment information. Take a 96-well E-plate culture plate compatible with RTCA, and add 50ul F12 complete medium to each well to avoid air bubbles. Put the plate into the RTCA instrument, and the operating software reads the background before adding the cells. Confirm that the reading is not abnormal. Trypsin digest the cultured A549 and A549-BCMA cells, use the AO/PI Cell Viability Kit to count the viable cells and centrifuge, and resuspend the two tumor cells to 4 x 10 5 /ml in F12 complete medium.
  • T cell counts, T cell medium by centrifugation after the T cells were resuspended and diluted to different concentrations of .1 x 10 5 / ml, 5 x 10 4 /ml,2.5 x 10 4 /ml,1.25 x 10 4 /ml.
  • Pause the RTCA software recording take the E-plate out of the instrument to the ultra-clean table, and add 100ul 1 X 10 5 /ml T cell suspension to the A549 culture well, that is, the final ratio of T cells to target cells is 0.5:1 ; Add 100ul 1 x 10 5 / ml to the culture wells A549-BCMA, 5 x 10 4 /ml,2.5 x 10 4 /ml,1.25 x 10 4 T cell suspensions / ml, i.e., the final T cells with target cells The ratios are 0.5:1, 0.25:1,0.125:1, 0.0625:1. Put the E-plate back into the RTCA instrument and read the cell index every 15 minutes until the set end time. Data analysis is performed after the experiment. Generally, the last time point before adding T cells is used as a reference, and the reading value of only tumor cells without T cells is 100%, and the change of tumor cell index after adding T cells is transformed into a kill curve .
  • Knockout of TRAC will cause the loss of CD3 expression on the cell surface, and knockout of B2M will cause the loss of HLA A/B/C expression on the cell surface. Therefore, the gene-edited T cells were stained with anti-HLA A/B/C and anti-CD3 antibodies and flow cytometry was performed to detect the knockout rate of TRAC and B2M.
  • ELISA was used to detect the level of IFN- ⁇ in the supernatant of the above-mentioned different BCMA CAR-T cells and A549-BCMA cells expressing BCMA antigen targeted cells after co-culture.
  • Double KO+CAR the responsiveness of gene-edited BCMA CAR-T cells to targeted cells, that is, the ability to release IFN- ⁇ , and other traditional BCMA CAR-T without gene editing The cells are similar.
  • the method is as follows: use cytokine-free AIM-V medium to thaw human PBMC and count. Centrifuge the cells (300g, 5min), remove the supernatant, and resuspend the cells in a 5ml tube with PBS. Add biotin isolation cocktail and anti-TCR ⁇ / ⁇ antibody and incubate at room temperature for 5 minutes. Add rapidSpheres and incubate at room temperature for 3 minutes. Add cytokine-free AIM-V medium to 2.5ml, and place it on the magnetic stand for 3 minutes. Transfer the supernatant to a new 5ml tube and place it on the magnetic stand for 3 minutes. The supernatant containing Tn cells was transferred to a new 15ml tube, followed by cell counting.
  • the enriched and purified Tn cells can be expanded and cultured, for example, in AIM-V medium containing 10% FBS, Glutamax, Pen/Strip, 10ng/ml IL15, 5ng/ml IL7 and 10ng/ml IL21 Expand the culture at approximately 106 cells/ml.
  • the TRAC and B2M genes were knocked out, and then transduced by lentivirus to introduce BCMA CAR to prepare the second-generation universal CAR-T cells.
  • Another method to prepare the second-generation universal CAR-T cells is to start from the total T cells (Total T cells) enriched in peripheral blood, but in the process of their expansion and culture, they are under suitable conditions ( as containing 10% FBS, Glutamax, Pen / Strip, AIM-V medium 10ng / ml IL15,5ng / ml IL7 and 10ng / ml IL21 in) to about 106 cells / ml for cultured at a density to obtain a rich T-cell population that gathers Tn (under this condition, Tn cells will expand more preferentially and efficiently).
  • suitable conditions as containing 10% FBS, Glutamax, Pen / Strip, AIM-V medium 10ng / ml IL15,5ng / ml IL7 and 10ng / ml IL21 in
  • gene knockout (such as knockout of TRAC and/or B2M), lentiviral transduction and other steps are performed to prepare Tscm-enriched universal CAR-T cells, that is, the second-generation universal CAR-T cells.
  • the second-generation universal T cells prepared in Example 4.2 were counted.
  • the percentage of CCR7, a biomarker of de/poorly differentiated cells was as high as 76%.
  • MFI of the second-generation universal CAR-T cells is 4534, which is much higher than the corresponding MFI of the first-generation universal CAR-T cell products and autologous CAR-T cells.
  • ELISA was used to detect the level of IFN- ⁇ in the supernatant of the above-mentioned different BCMA CAR-T cells and A549-BCMA cells expressing BCMA antigen targeted cells after co-culture.
  • the results are shown in Figure 8.
  • the second-generation BCMA CAR-T cells are more responsive to targeted cells, that is, their ability to release IFN- ⁇ is significantly higher than the first-generation universal BCMA CAR-T cells and autologous BCMA CAR-T cells .
  • the second-generation universal CAR-T cells have significantly better response to the targeted cells than the other two CAR-Ts cell. This suggests that the second-generation universal CAR-T cells of the present invention have more significant reactivity to target cells with corresponding targets (such as tumor antigens, such as BCMA or other targets).
  • targets such as tumor antigens, such as BCMA or other targets.
  • Example 9 The lethality of the second-generation universal BCMA CAR-T cells on targeted cells
  • the cell lysis curve on the left of Figure 9 shows that the second-generation universal BCMA CAR-T cells are more lethal than the other two CAR-T cells. Shown here is an example where the ratio of CAR-T cells to target cells is 1:8, but the same conclusion is also suitable for other 1:2, 1:4, and 1:16 situations (data not shown).
  • the KT80 chart on the right of Figure 9 is the time chart required to kill 80% of the targeted cells. It can be seen that the second-generation universal BCMA CAR-T cell pair has a different ratio of CAR-T cells to targeted cells. The time required to kill 80% of the targeted cells is significantly lower than the other two CAR-T cells. For example, when the number of CAR-T cells and the number of targeted cells are 1:8, it takes about 70 hours for the second-generation universal cell to kill 80% of the targeted cells, but it takes 100 hours for the other two CAR-T cells. About 1.5 times the time required for the second-generation CAR-T cells.
  • T cells were expanded and cultured under different conditions, except that the added cytokine was changed from Example 4, and the other culture conditions were the same.

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Abstract

提供了一种非天然存在的T细胞群，该T细胞群中T记忆干细胞的比例C1≥50％，该T细胞包括靶向肿瘤抗原的通用型CAR-T细胞。还提供了制备CAR-T细胞的方法。该CAR-T细胞具有更优异的扩增率、表型、IFN-γ释放能力及对靶细胞的杀伤能力。

Description

通用型CAR-T细胞及其制备和应用 技术领域
本发明涉及生物医药领域，更具体地涉及通用型CAR-T细胞及其制备和应用
背景技术
免疫疗法，特别是过继性T细胞疗法，在治疗针对血液***的恶性肿瘤临床试验中显示了强大的疗效和光明前景。T细胞可以被基因修饰来表达嵌合抗原受体(CAR)，它包括抗原识别部分和T细胞激活区。CAR利用单克隆抗体的抗原结合性质能重定向T细胞的特异性和反应性并且以一种非MHC限制的方式靶向靶标。这种非MHC限制抗原识别使表达CAR的T细胞不需要抗原加工就能识别抗原，因此避免了肿瘤逃逸的一种主要机制。除此之外，CAR也不会和内源性TCR的α链，β链产生二聚体。
通用型CAR-T细胞是T细胞免疫疗法中自体T细胞的一种重要替代资源，其中敲除异体T细胞的内源性TRAC和B2M基因以分别防止移植物抗宿主病和宿主免疫排斥。这些来自同种异体供体的基因编辑过的细胞为无法提供足够自体T细胞的患者提供了细胞免疫治疗机会，例如婴儿或接受了多轮化疗的患者。
目前，国内外尚未有获批上市的通用型CAR-T细胞产品，本领域关于通用型CAR-T细胞的研究还有很多的不足，本领域迫切需要开发新的杀伤效果好，安全性高的通用型CAR-T细胞产品。
发明内容
本发明的目的在于提供Tscm富集的通用型CAR-T细胞及其制备和应用。
在本发明的第一方面，提供了一种非天然存在的T细胞群，所述的T细胞群中T记忆干细胞(干细胞样记忆型T细胞，Tscm)的比例C1≥50％，按所述T细胞群中的T细胞总数计。
在另一优选例中，所述的C1≥60％，较佳地C1≥70％，更佳地C1≥75％。
在另一优选例中，所述的C1为50-85％，较佳地60-80％。
在另一优选例中，所述的T细胞包括CAR-T细胞。
在另一优选例中，所述的CAR-T细胞包括自体或异体的CAR-T细胞。
在另一优选例中，所述的CAR-T细胞包括通用型CAR-T细胞。
在另一优选例中，所述的CAR-T细胞中，选自下组的免疫相关基因被敲除或下调：TRAC基因、B2M基因、或其组合。
在另一优选例中，当T记忆干细胞(Tscm)为CAR-T细胞时，所述T记忆干细胞(Tscm)的比例C1≥50％，按所述T细胞群中的CAR-T细胞总数计。
在另一优选例中，所述的C1≥60％，较佳地C1≥70％，更佳地C1≥75％。
在另一优选例中，所述的C1为50-85％，较佳地60-80％。
在另一优选例中，所述的T记忆干细胞具有CCR7 +表型。
在另一优选例中，在所述T细胞群中，T细胞的含量C3≥30％，较佳地≥50％，更佳地≥70％，更佳地≥80％，≥90％，或≥95％，按所述细胞群中所有细胞的总数计算。
在另一优选例中，C3为30-100％，更佳地70-99.9％，更佳地80-99％。
在另一优选例中，所述的T记忆干细胞包括CCR7 +CD45RA +T细胞。
在另一优选例中，在所述的T细胞群中，表达生物标志物CCR7的比例C2≥50％，较佳地≥60％，更佳地，≥70％。
在另一优选例中，所述的CAR-T细胞的活性强度Q1(以平均荧光强度MFI为例)≥500，较佳地，≥1000，更佳地，≥3000，更佳地≥4000(如1000-5000)。
在另一优选例中，所述的T细胞群是经体外扩增或未经体外扩增的。
在另一优选例中，所述的体外扩增是在适合培养的条件下，在培养体系中存在IL15、IL7和IL21(通称为Tscm扩增因子)下，进行细胞扩增。
在另一优选例中，在所述培养体系中，Tscm扩增因子的浓度包括：
IL15的浓度为1-200ng/ml，较佳地为，3-100ng/ml，更佳地为5-20ng/ml；
IL7的浓度为0.5-50ng/ml，较佳地为，1-20ng/ml，更佳地为3-10ng/ml；和/或
IL21的浓度为1-100ng/ml，较佳地为，3-50ng/ml，更佳地为5-20ng/ml。
在另一优选例中，所述的培养体系包括有血清培养体系和无血清培养体系。在另一优选例中，所述的细胞扩增的时间为0.1-30天，较佳地0.5-25天，更佳地1-20天，最佳地2-15天或3-15天或5-15天。
在另一优选例中，所述的CAR-T细胞为靶向肿瘤抗原的通用型CAR-T细胞。
在另一优选例中，所述的肿瘤抗原选自下组：TSHR、CD19、CD123、CD22、CD30、CD171、CS-1、CLL-1、CD33、EGFRvIII、GD2、GD3、BCMA、Tn Ag、PSMA、ROR1、FLT3、FAP、TAG72、CD38、CD44v6、CEA、EPCAM、B7H3、KIT、IL-13Ra2、间皮素、IL-11Ra、PSCA、PRSS21、VEGFR2、LewisY、CD24、PDGFR-β、SSEA-4、CD20、叶酸受体α、ERBB2(Her2/neu)、MUC1、EGFR、NCAM、Prostase、PAP、ELF2M、肝配蛋白B2、IGF-I受体、CAIX、LMP2、gp100、bcr-abl、酪氨酸酶、EphA2、岩藻糖基GM1、sLe、GM3、TGS5、HMWMAA、邻乙酰基-GD2、叶酸受体β、TEM1/CD248、TEM7R、CLDN6、GPRC5D、CXORF61、CD97、CD179a、ALK、聚唾液酸、PLAC1、GloboH、NY-BR-1、UPK2、HAVCR1、ADRB3、PANX3、GPR20、LY6K、 OR51E2、TARP、WT1、NY-ESO-1、LAGE-1a、MAGE-A1、legumain、HPV E6、E7、MAGE A1、ETV6-AML、***蛋白17、XAGE1、Tie 2、MAD-CT-1、MAD-CT-2、Fos相关抗原1、p53、p53突变体、prostein、存活蛋白和端粒酶、PCTA-1/Galectin8、MelanA/MART1、Ras突变体、hTERT、肉瘤易位断点、ML-IAP、ERG(TMPRSS2ETS融合基因)、NA17、PAX3、雄激素受体、细胞周期蛋白B1、MYCN、RhoC、TRP-2、CYP1B1、BORIS、SART3、PAX5、OY-TES1、LCK、AKAP-4、SSX2、RAGE-1、人端粒酶逆转录酶、RU1、RU2、肠羧基酯酶、mut hsp70-2、CD79a、CD79b、CD72、LAIR1、FCAR、LILRA2、CD300LF、CLEC12A、BST2、EMR2、LY75、GPC3、FCRL5、IGLL1、或其组合。
在另一优选例中，所述的CAR-T细胞表达嵌合抗原受体，并且，所述嵌合抗原受体的结构如下式I所示：
L-scFv-H-TM-C-CD3ζ    (I)
式中，
各“-”独立地为连接肽或肽键；
L为任选的信号肽序列；
scFv为靶向肿瘤抗原的抗体单链可变区序列；
H为任选的铰链区；
TM为跨膜结构域；
C为无或共刺激信号分子；
CD3ζ为源于CD3ζ的胞浆信号传导序列。
在另一优选例中，所述的CAR-T细胞为靶向BCMA的通用型CAR-T细胞。
在另一优选例中，所述的scFv靶向BCMA。
在另一优选例中，所述的L为选自下组的蛋白的信号肽：CD8、GM-CSF、CD4、CD137、或其组合。
在另一优选例中，所述的H为选自下组的蛋白的铰链区：CD8、CD28、CD137、或其组合。
在另一优选例中，所述的TM为选自下组的蛋白的跨膜区：CD28、CD3epsilon、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154、或其组合。
在另一优选例中，所述的C为选自下组的蛋白的共刺激信号分子：OX40、CD2、CD7、CD27、CD28、CD30、CD40、CD70、CD134、4-1BB(CD137)、PD1、Dap10、CDS、ICAM-1、LFA-1(CD11a/CD18)、ICOS(CD278)、NKG2D、GITR、TLR2、或其组合。
在另一优选例中，C包括4-1BB来源的共刺激信号分子，和/或CD28来源的共刺激信号分子。
在另一优选例中，所述的T细胞来自人或非人哺乳动物。
在另一优选例中，所述的T细胞群是用本发明第三方面中所述的方法制备的。
在本发明第二方面，提供了一种细胞制剂，所述的细胞制剂含有本发明第一方面所述的非天然存在的T细胞群和药学上可接受的载体、稀释剂或赋形剂。
在本发明第三方面，提供了一种制备CAR-T细胞的方法，包括步骤：
(a)提供一分离的T细胞；
(b)在IL15、IL7和IL21存在的条件下，对所述分离的T细胞进行扩大培养，从而获得经培养的T细胞；和
(c)对所述经培养的T细胞进行改造，制备CAR-T细胞。
在另一优选例中，在步骤(a)中，所述分离的T细胞选自下组：初始T细胞(Tn细胞)、富含Tn细胞的细胞群、或总T细胞、或其组合。
在另一优选例中，所述的分离的T细胞为初始T细胞(Tn细胞)或富含Tn细胞的细胞群。
在另一优选例中，所述的T细胞包括自体或异体的T细胞。
在另一优选例中，在步骤(b)中，对所述分离的T细胞进行扩大培养时，培养体系中IL15的浓度为1-200ng/ml，较佳地为，3-100ng/ml，更佳地为5-20ng/ml；IL7的浓度为0.5-50ng/ml，较佳地为，1-20ng/ml，更佳地为3-10ng/ml；且IL21的浓度为1-100ng/ml，较佳地为，3-50ng/ml，更佳地为5-20ng/ml。
在另一优选例中，所述的方法用于制备通用型CAR-T细胞。
在另一优选例中，所述的CAR-T细胞靶向肿瘤抗原BCMA。
在另一优选例中，在步骤(a)中，所述的T细胞分离自人外周血，较佳地，分离自外周血单个核细胞。
在另一优选例中，所述分离的T细胞包括总T细胞、初始T细胞(Tn细胞)。
在另一优选例中，所述初始T细胞为CCR7 +CD45RA +T细胞。
在另一优选例中，在步骤(b)中，在AIM-V培养基中进行扩大培养。
在另一优选例中，在步骤(b)中，所述的培养体系中还包含FBS、Glutamax、Pen/Strip、或其组合；较佳地，所述的FBS的浓度为5-20％。
在另一优选例中，在步骤(c)中，所述的改造包括T细胞激活、T细胞核转染和慢病毒转导。
在另一优选例中，在步骤(c)中，所述的改造包括以下步骤：
(1)利用CD3/CD28磁珠，激活所述经培养的T细胞，除去磁珠后，获得激活的T细胞；
(2)利用CRISPR-Cas9***对所述激活的T细胞进行核转染，敲除T细胞的TRAC基因和/或B2M基因，从而获得核转染的T细胞；和
(3)利用包含CAR表达盒的病毒载体转导所述核转染的T细胞，制备CAR-T细胞。
在本发明第四方面，提供了一种制备CAR-T细胞的方法，包括步骤：
(i)提供一分离的Tn细胞，
(ii)任选地，在IL15、IL7和IL21存在的条件下，对所述分离的T细胞进行扩大培养，从而获得经培养的T细胞；和
(iii)对所述T细胞进行改造，制备CAR-T细胞。
在本发明的第五方面，提供了一种本发明第一方面所述的T细胞群(尤其是CAR-T细胞)、或本发明第二方面所述的细胞制剂的用途，用于制备预防和/或治疗癌症或肿瘤的药物。
在另一优选例中，所述肿瘤选自下组：血液肿瘤、实体瘤、或其组合。
在另一优选例中，所述血液肿瘤选自下组：急性髓细胞白血病(AML)、多发性骨髓瘤(MM)、慢性淋巴细胞白血病(CLL)、急性淋巴白血病(ALL)、弥漫性大B细胞淋巴瘤(DLBCL)、或其组合。
在另一优选例中，所述实体瘤选自下组：胃癌、胃癌腹膜转移、肝癌、白血病、肾脏肿瘤、肺癌、小肠癌、骨癌、***癌、结直肠癌、乳腺癌、大肠癌、***、卵巢癌、淋巴癌、鼻咽癌、肾上腺肿瘤、***、非小细胞肺癌(NSCLC)、脑胶质瘤、子宫内膜癌、睾丸癌、结直肠癌、尿路肿瘤、甲状腺癌、或其组合。
在另一优选例中，所述实体瘤选自下组：卵巢癌、间皮瘤、肺癌、胰腺癌、乳腺癌、肝癌、子宫内膜癌、或其组合。
在本发明的第六方面，提供了一种本发明第一方面所述T细胞群、或本发明第二方面所述的细胞制剂的用途，用于预防和/或治疗癌症或肿瘤。
在本发明的第七方面，提供了一种治疗疾病的方法，包括：给需要治疗的对象施用适量的本发明第一方面所述的T细胞群、或本发明第二方面所述的细胞细胞制剂。
在另一优选例中，所述疾病为癌症或肿瘤。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了流式染色检测通用型T细胞中TRAC和B2M基因敲除及纯化率。
图2显示了流式染色检测通用型T细胞中BCMA CAR的转导率。
图3显示了ELISA检测通用型CAR-T细胞与表达BCMA的靶向细胞共培养后IFN-γ的释放。
图4显示了流式检测从PBMC中分离初始T细胞的表型及纯度。
图5显示了三种BCMA CAR-T细胞的增殖率。
图6显示了三种BCMA CAR-T细胞中各种T细胞亚型的比例及CCR7的表达量。
图7显示了流式检测三种BCMA CAR-T细胞的CAR转染率。
图8显示了三种BCMA CAR-T细胞和表达BCMA的靶向细胞共培养后IFN-γ的释放。
图9显示了三种BCMA CAR-T细胞对表达BCMA的靶向细胞的杀伤率。
图10显示了不同条件下培养的T细胞中Tscm细胞的富集情况和细胞生长情况。
图11显示了不同条件下培养的T细胞的BCMA CAR转导率。
图12显示了不同条件下培养的T细胞的转导BCMA CAR后的IFN-γ释放。
图13显示了三种BCMA CAR-T细胞的CCR7 +CD45RA +Tscm细胞的富集情况。
对于本发明附图中涉及的标注，具体含义如下：
mock：没有经过Cas9 RNP核转染的T细胞；mock+CAR：没有经过Cas9 RNP核转染但转导了BCMA CAR的T细胞；CAR：BCMA CAR转导了的T细胞；Double KO：Cas9 RNP核转染后双敲除TRAC和B2M的T细胞；Double KO+CAR：上述Double KO细胞经BCMA CAR转导后的T细胞；BCMA CAR：经BCMA CAR转导的传统自体CAR-T细胞；1°U CAR：经BCMA CAR转导的第一代通用型T细胞；2°U CAR：经BCMA CAR转导的第二代通用型T细胞。
具体实施方式
本发明人经过广泛而深入地研究，首次意外地发现，在有利于T记忆干细胞(Tscm)细胞扩增的条件下培养T细胞，制备的Tscm富集的通用型BCMA CAR-T细胞，显示出比第一代更好的扩增率、表型、IFN-γ释放能力及对靶细胞的杀伤能力。基于本发明的Tscm的富集T细胞(尤其是第二代通用型CAR-T细胞)，可以制备针对不同靶点的高活性的通用型CAR-T细胞。在此基础上完成本发明。
具体地，为了进一步提高通用型BCMA CAR-T细胞的效能，在有利于T记忆干细胞(Tscm)细胞扩增的条件下培养T细胞，制备了新二代Tscm富集的通用型CAR-T细胞，这样的CAR-T细胞在增殖，表型，以及对靶细胞的杀伤上都大大优于第一代的通用型CAR-T细胞，表明第二代通用型CAR-T细胞是更优化的通用型CAR-T产品。
典型地，可使用核转染法将Cas9 RNP复合物递送到T细胞中以同时敲除内源性TRAC和B2M基因,纯化后可以制备高纯度的TRAC及B2M双敲除的通用型T细胞。这样的T细胞在使用慢病毒转导引入BCMA CAR表达后产生第一代 的通用型BCAM CAR-T细胞。这些CAR-T细胞可以有效地杀伤靶细胞，活性和没有经过基因编辑的自体CAR-T细胞类似。
术语
为了可以更容易地理解本公开，首先定义某些术语。如本申请中所使用的，除非本文另有明确规定，否则以下术语中的每一个应具有下面给出的含义。在整个申请中阐述了其它定义。
术语“约”可以是指在本领域普通技术人员确定的特定值或组成的可接受误差范围内的值或组成，其将部分地取决于如何测量或测定值或组成。
术语“给予”是指使用本领域技术人员已知的各种方法和递送***中的任一种将本发明的产品物理引入受试者，包括静脉内，肌内，皮下，腹膜内，脊髓或其它肠胃外给药途径，例如通过注射或输注。
术语“抗体”(Ab)应包括但不限于免疫球蛋白，其特异性结合抗原并包含通过二硫键互连的至少两条重(H)链和两条轻(L)链，或其抗原结合部分。每条H链包含重链可变区(本文缩写为VH)和重链恒定区。重链恒定区包含三个恒定结构域CH1、CH2和CH3。每条轻链包含轻链可变区(本文缩写为VL)和轻链恒定区。轻链恒定区包含一个恒定结构域CL。VH和VL区可以进一步细分为称为互补决定区(CDR)的高变区，其散布有更保守的称为框架区(FR)的区域。每个VH和VL包含三个CDR和四个FR，从氨基末端到羧基末端按照以下顺序排列：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。重链和轻链的可变区含有与抗原相互作用的结合结构域。
应理解，本文中氨基酸名称采用国际通用的单英文字母标识，与其相对应的氨基酸名称三英文字母简写分别是：Ala(A)、Arg(R)、Asn(N)、Asp(D)、Cys(C)、Gln(Q)、Glu(E)、Gly(G)、His(H)、I1e(I)、Leu(L)、Lys(K)、Met(M)、Phe(F)、Pro(P)、Ser(S)、Thr(T)、Trp(W)、Tyr(Y)、Val(V)。
通用型CAR-T细胞
通用型T细胞是T细胞免疫疗法中自体T细胞的一种重要替代资源，其中敲除异体T细胞的内源性TRAC和B2M基因以分别防止移植物抗宿主病和宿主免疫排斥。这些来自同种异体供体的基因编辑过的细胞为无法提供足够自体T细胞的患者提供了细胞免疫治疗机会，例如婴儿或接受了多轮化疗的患者。这些细胞可以通过编码CAR或TCR的慢病毒载体进一步修饰以产生同种异体CAR/TCR T细胞。
在本发明中，使用核转染法将Cas9 RNP复合物递送到T细胞中以同时敲除内源性TRAC和B2M基因。使用慢病毒转导引入BCMA CAR表达以产生现成的BCAM CAR-T细胞。这些CAR-T细胞可以有效地杀伤靶细胞，活性和没有经过 基因编辑的自体BCMA CAR-T细胞类似。
为了进一步提高通用型CAR-T的活力，从异体初始T细胞(Tn)开始，进行基因编辑，并采用有利于干细胞样记忆T细胞(Tscm)增殖的特殊培养条件，产生了Tscm富集的第二代通用型BCMA CAR-T细胞，这样的CAR-T细胞在增殖，表型，IFN-γ的释放能力以及对靶细胞的杀伤上都大大优于传统的第一代通用型CAR-T细胞。
Tn细胞
Tn细胞，即初始T细胞(naive T cell，Tn)，也称作未致敏T细胞，在胸腺中发育成熟迁移至外周淋巴组织中，其未接触抗原刺激前处于相对静止状态。
Tscm细胞
Tscm细胞表示T记忆干细胞，也称为干细胞样记忆型T细胞(stem cell memory T cell，Tscm)
BCMA
BCMA是B细胞成熟抗原，又名CD269或TNFRSF17，是肿瘤坏死因子受体超家族的成员，它的配体是B细胞激活因子(BAFF)和增殖诱导配体(APRIL)。
BCMA与BAFF和APRIL的结合引发NF-kB激活，诱导抗凋亡Bcl-2成员例如Bcl-xL或者Bcl-2和Mcl-1的上调。BCMA和其配体的相互作用从不同方面调控体液免疫，B细胞的生长分化从而维护人体内环境稳定平衡。
BCMA的表达局限于B细胞系，在浆母细胞，浆细胞和一部分成熟B细胞上表达，在末端B细胞分化时增加，而在大部分B细胞，例如幼稚B细胞，记忆B细胞和B细胞生发中心以及其它器官都不表达。据报道，BCMA的表达对于骨髓中长寿的，固着的浆细胞很重要。所以，BCMA缺陷小鼠在骨髓中的浆细胞减少然而在脾脏中的浆细胞水平不受影响。成熟B细胞在BCMA敲除的小鼠中能正常分化成浆细胞。BCMA敲除小鼠看上去一切正常，似乎很健康，而且B细胞数量正常，但浆细胞却不能长期存活。
BCMA也在恶性浆细胞中高表达，例如多发性骨髓瘤和浆细胞白血病，霍奇金淋巴瘤患者的HRS细胞中也被检测出BCMA。在美国，血液***的恶性肿瘤占所有恶性肿瘤约10％，骨髓瘤占全部恶性血液肿瘤的15％。据文献报道，BCMA的表达和多发性骨髓瘤疾病进展有关。BCMA基因在骨髓瘤样本中高表达，但在慢性淋巴细胞白血病，急性淋巴细胞白血病，急性T细胞型淋巴细胞白血病中表达很低。过表达BCMA配体BAFF和APRIL的老鼠模型中B细胞淋巴瘤明显增 长。与BCMA结合的配体被证明能调控表达BCMA的多发性骨髓瘤细胞的生长和生存。BCMA与BAFF和APRIL的结合能让恶性浆细胞存活，因此，使表达BCMA的肿瘤细胞损耗，破坏BCMA配体与受体之间的相互作用能改善对多发性骨髓瘤或其他BCMA阳性B系恶性淋巴瘤的治疗结果。
嵌合抗原受体(CAR)
本发明的CAR可以包含任何类型的靶向肿瘤抗原的抗原结合结构域
本发明的嵌合抗原受体(CAR)包括细胞外结构域、跨膜结构域、和细胞内结构域。胞外结构域包括靶-特异性结合元件(也称为抗原结合结构域)。细胞内结构域包括共刺激信号传导区和ζ链部分。共刺激信号传导区指包括共刺激分子的细胞内结构域的一部分。共刺激分子为淋巴细胞对抗原的有效应答所需要的细胞表面分子，而不是抗原受体或它们的配体。
在CAR的胞外结构域和跨膜结构域之间，或在CAR的胞浆结构域和跨膜结构域之间，可并入接头。如本文所用的，术语“接头”通常指起到将跨膜结构域连接至多肽链的胞外结构域或胞浆结构域作用的任何寡肽或多肽。接头可包括0-300个氨基酸，优选地2至100个氨基酸和最优选地3至50个氨基酸。
在本发明的一个较佳的实施方式中，本发明提供的CAR的胞外结构域包括靶向BCMA的抗原结合结构域。本发明的CAR当在T细胞中表达时，能够基于抗原结合特异性进行抗原识别。当其结合其关联抗原时，影响肿瘤细胞，导致肿瘤细胞不生长、被促使死亡或以其他方式被影响，并导致患者的肿瘤负荷缩小或消除。抗原结合结构域优选与来自共刺激分子和ζ链中的一个或多个的细胞内结构域融合。优选地，抗原结合结构域与4-1BB信号传导结构域、和CD3ζ信号结构域组合的细胞内结构域融合。
如本文所用，“抗原结合结构域”“单链抗体片段”均指具有抗原结合活性的Fab片段，Fab'片段，F(ab')2片段，或单一Fv片段。Fv抗体含有抗体重链可变区、轻链可变区，但没有恒定区，并具有全部抗原结合位点的最小抗体片段。一般的，Fv抗体还包含VH和VL结构域之间的多肽接头，且能够形成抗原结合所需的结构。抗原结合结构域通常是scFv(single-chain variable fragment)。scFv的大小一般是一个完整抗体的1/6。单链抗体优选是由一条核苷酸链编码的一条氨基酸链序列。作为本发明的优选方式，所述scFv包含特异性识别BCMA的抗体，较佳地为单链抗体。
对于绞链区和跨膜区(跨膜结构域)，CAR可被设计以包括融合至CAR的胞外结构域的跨膜结构域。在一个实施方式中，使用天然与CAR中的结构域之一相关联的跨膜结构域。在一些例子中，可选择跨膜结构域，或通过氨基酸置换进行修饰，以避免将这样的结构域结合至相同或不同的表面膜蛋白的跨膜结构 域，从而最小化与受体复合物的其他成员的相互作用。
本发明的CAR中的胞内结构域包括4-1BB的信号传导结构域和CD3ζ的信号传导结构域。
基因沉默方法
目前常用的基因沉默方法有CRISPR/Cas9、RNA干扰技术、TALENs(transcription activator-like(TAL)effector nucleases)和Zinc finger nucleases(ZFNs)，其中CRISPR/Cas9目前应用前景和效果最好。
CRISPR(clustered regularly interspersed short palindromic repeats)/Cas(CRISPR-associated)***是原核生物特有的一种天然免疫***，用于抵抗病毒或外源性质粒的侵害。Ⅱ型CRISPR/Cas***作为RNA直接介导的基因组编辑工具已经在许多真核生物和原核生物体内成功应用。CRISPR/Cas9***的发展彻底改变了人们编辑DNA序列和调控目标基因表达水平的能力，从而为生物体的精确基因组编辑提供了有力的工具。简化后的CRISPR/Cas9***由两部分组成：Cas9蛋白和sgRNA。其作用原理为sgRNA通过自身的Cas9把手与Cas9蛋白形成Cas9-sgRNA复合体，Cas9-sgRNA复合体中sgRNA的碱基互补配对区序列与目标基因的靶序列通过碱基互补配对原则进行配对结合，Cas9利用自身的核酸内切酶活性对目标DNA序列进行切割。与传统的基因组编辑技术相比，CRISPR/Cas9***具有几大明显的优势：易用性、简便性、低成本、可编程性以及可同时编辑多个基因。
载体
编码期望分子的核酸序列可利用在本领域中已知的重组方法获得，诸如例如通过从表达基因的细胞中筛选文库，通过从已知包括该基因的载体中得到该基因，或通过利用标准的技术，从包含该基因的细胞和组织中直接分离。可选地，感兴趣的基因可被合成生产。
本发明也提供了其中***本发明的表达盒的载体。源于逆转录病毒诸如慢病毒的载体是实现长期基因转移的合适工具，因为它们允许转基因长期、稳定的整合并且其在子细胞中增殖。慢病毒载体具有超过源自致癌逆转录病毒诸如鼠科白血病病毒的载体的优点，因为它们可转导非增殖的细胞，诸如肝细胞。它们也具有低免疫原性的优点。
简单概括，通常可操作地连接本发明的表达盒或核酸序列至启动子，并将其并入表达载体。该载体适合于复制和整合真核细胞。典型的克隆载体包含可用于调节期望核酸序列表达的转录和翻译终止子、初始序列和启动子。
本发明的表达构建体也可利用标准的基因传递方案，用于核酸免疫和基因 疗法。基因传递的方法在本领域中是已知的。见例如美国专利号5,399,346、5,580,859、5,589,466，在此通过引用全文并入。在另一个实施方式中，本发明提供了基因疗法载体。
该核酸可被克隆入许多类型的载体。例如，该核酸可被克隆入如此载体，其包括但不限于质粒、噬菌粒、噬菌体衍生物、动物病毒和粘粒。特定的感兴趣载体包括表达载体、复制载体、探针产生载体和测序载体。
进一步地，表达载体可以以病毒载体形式提供给细胞。病毒载体技术在本领域中是公知的并在例如Sambrook等(2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York)和其他病毒学和分子生物学手册中进行了描述。可用作载体的病毒包括但不限于逆转录病毒、腺病毒、腺伴随病毒、疱疹病毒和慢病毒。通常，合适的载体包含在至少一种有机体中起作用的复制起点、启动子序列、方便的限制酶位点和一个或多个可选择的标记(例如，WO01/96584；WO01/29058；和美国专利号6,326,193)。
已经开发许多基于病毒的***，用于将基因转移入哺乳动物细胞。例如，逆转录病毒提供了用于基因传递***的方便的平台。可利用在本领域中已知的技术将选择的基因***载体并包装入逆转录病毒颗粒。该重组病毒可随后被分离和传递至体内或离体的对象细胞。许多逆转录病毒***在本领域中是已知的。在一些实施方式中，使用腺病毒载体。许多腺病毒载体在本领域中是已知的。在一个实施方式中，使用慢病毒载体。
额外的启动子元件，例如增强子，可以调节转录开始的频率。通常地，这些位于起始位点上游的30-110bp区域中，尽管最近已经显示许多启动子也包含起始位点下游的功能元件。启动子元件之间的间隔经常是柔性的，以便当元件相对于另一个被倒置或移动时，保持启动子功能。在胸苷激酶(tk)启动子中，启动子元件之间的间隔可被增加隔开50bp，活性才开始下降。取决于启动子，表现出单个元件可合作或独立地起作用，以起动转录。
合适的启动子的一个例子为即时早期巨细胞病毒(CMV)启动子序列。该启动子序列为能够驱动可操作地连接至其上的任何多核苷酸序列高水平表达的强组成型启动子序列。合适的启动子的另一个例子为延伸生长因子-1α(EF-1α)。然而，也可使用其他组成型启动子序列，包括但不限于类人猿病毒40(SV40)早期启动子、小鼠乳癌病毒(MMTV)、人免疫缺陷病毒(HIV)长末端重复(LTR)启动子、MoMuLV启动子、鸟类白血病病毒启动子、艾伯斯坦-巴尔(Epstein-Barr)病毒即时早期启动子、鲁斯氏肉瘤病毒启动子、以及人基因启动子，诸如但不限于肌动蛋白启动子、肌球蛋白启动子、血红素启动子和肌酸激酶启动子。进一步地，本发明不应被限于组成型启动子的应用。诱导型启动子也被考虑为本发明的一部分。诱导型启动子的使用提供了分子开关，其能够 当这样的表达是期望的时，打开可操作地连接诱导型启动子的多核苷酸序列的表达，或当表达是不期望的时关闭表达。诱导型启动子的例子包括但不限于金属硫蛋白启动子、糖皮质激素启动子、孕酮启动子和四环素启动子。
为了评估CAR多肽或其部分的表达，被引入细胞的表达载体也可包含可选择的标记基因或报道基因中的任一个或两者，以便于从通过病毒载体寻求被转染或感染的细胞群中鉴定和选择表达细胞。在其他方面，可选择的标记可被携带在单独一段DNA上并用于共转染程序。可选择的标记和报道基因两者的侧翼都可具有适当的调节序列，以便能够在宿主细胞中表达。有用的可选择标记包括例如抗生素抗性基因，诸如neo等等。
报道基因用于鉴定潜在转染的细胞并用于评价调节序列的功能性。通常地，报道基因为以下基因：其不存在于受体有机体或组织或由受体有机体或组织进行表达，并且其编码多肽，该多肽的表达由一些可容易检测的性质例如酶活性清楚表示。在DNA已经被引入受体细胞后，报道基因的表达在合适的时间下进行测定。合适的报道基因可包括编码荧光素酶、β-半乳糖苷酶、氯霉素乙酰转移酶、分泌型碱性磷酸酶或绿色萤光蛋白的基因(例如，Ui-Tei等，2000FEBS Letters479:79-82)。合适的表达***是公知的并可利用已知技术制备或从商业上获得。通常，显示最高水平的报道基因表达的具有最少5个侧翼区的构建体被鉴定为启动子。这样的启动子区可被连接至报道基因并用于评价试剂调节启动子-驱动转录的能力。
将基因引入细胞和将基因表达入细胞的方法在本领域中是已知的。在表达载体的内容中，载体可通过在本领域中的任何方法容易地引入宿主细胞，例如，哺乳动物、细菌、酵母或昆虫细胞。例如，表达载体可通过物理、化学或生物学手段转移入宿主细胞。
将多核苷酸引入宿主细胞的物理方法包括磷酸钙沉淀、脂质转染法、粒子轰击、微注射、电穿孔等等。生产包括载体和/或外源核酸的细胞的方法在本领域中是公知的。见例如Sambrook等(2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York)。将多核苷酸引入宿主细胞的优选方法为磷酸钙转染。
将感兴趣的多核苷酸引入宿主细胞的生物学方法包括使用DNA和RNA载体。病毒载体，特别是逆转录病毒载体，已经成为最广泛使用的将基因***哺乳动物例如人细胞的方法。其他病毒载体可源自慢病毒、痘病毒、单纯疱疹病毒I、腺病毒和腺伴随病毒等等。见例如美国专利号5,350,674和5,585,362。
将多核苷酸引入宿主细胞的化学手段包括胶体分散***，诸如大分子复合物、纳米胶囊、微球、珠；和基于脂质的***，包括水包油乳剂、胶束、混合胶束和脂质体。用作体外和体内传递工具(delivery vehicle)的示例性胶体系 统为脂质体(例如，人造膜囊)。
在使用非病毒传递***的情况下，示例性传递工具为脂质体。考虑使用脂质制剂，以将核酸引入宿主细胞(体外、离体(ex vivo)或体内)。在另一方面，该核酸可与脂质相关联。与脂质相关联的核酸可被封装入脂质体的水性内部中，散布在脂质体的脂双层内，经与脂质体和寡核苷酸两者都相关联的连接分子附接至脂质体，陷入脂质体，与脂质体复合，分散在包含脂质的溶液中，与脂质混合，与脂质联合，作为悬浮液包含在脂质中，包含在胶束中或与胶束复合，或以其他方式与脂质相关联。与组合物相关联的脂质、脂质/DNA或脂质/表达载体不限于溶液中的任何具体结构。例如，它们可存在于双分子层结构中，作为胶束或具有“坍缩的(collapsed)”结构。它们也可简单地被散布在溶液中，可能形成大小或形状不均一的聚集体。脂质为脂肪物质，其可为天然发生或合成的脂质。例如，脂质包括脂肪小滴，其天然发生在细胞质以及包含长链脂肪族烃和它们的衍生物诸如脂肪酸、醇类、胺类、氨基醇类和醛类的该类化合物中。
在本发明的一个优选地实施方式中，所述载体为慢病毒载体。
制剂
本发明提供了一种含有本发明第一方面所述的CAR-T细胞，以及药学上可接受的载体、稀释剂或赋形剂。在一个实施方式中，所述制剂为液态制剂。优选地，所述制剂为注射剂。优选地，所述制剂中所述CAR-T细胞的浓度为1×10 3-1×10 8个细胞/ml，更优地1×10 4-1×10 7个细胞/ml。
在一个实施方式中，所述制剂可包括缓冲液诸如中性缓冲盐水、硫酸盐缓冲盐水等等；碳水化合物诸如葡萄糖、甘露糖、蔗糖或葡聚糖、甘露醇；蛋白质；多肽或氨基酸诸如甘氨酸；抗氧化剂；螯合剂诸如EDTA或谷胱甘肽；佐剂(例如，氢氧化铝)；和防腐剂。本发明的制剂优选配制用于静脉内施用。
治疗性应用
本发明包括用本发明第一方面所述的CAR-T细胞进行的治疗性应用。转导的T细胞可靶向肿瘤细胞的标志物BCMA，协同激活T细胞，引起T细胞免疫应答，从而显著提高其对肿瘤细胞的杀伤效率。
因此，本发明也提供了刺激对哺乳动物的靶细胞群或组织的T细胞-介导的免疫应答的方法，其包括以下步骤：给哺乳动物施用本发明的CAR-T细胞。
在一个实施方式中，本发明包括一类细胞疗法，分离病人自体T细胞(或者异源供体)，激活并进行基因改造产生CAR-T细胞，随后注入病人体内。这种方式患移植物抗宿主病概率极低，抗原被T细胞以无MHC限制方式识别。此 外，一种CAR-T就可以治疗表达该抗原的所有癌症。不像抗体疗法，CAR-T细胞能够体内复制，产生可导致持续肿瘤控制的长期持久性。
在一个实施方式中，本发明的CAR-T细胞可经历稳固的体内T细胞扩展并可持续延长的时间量。另外，CAR介导的免疫应答可为过继免疫疗法步骤的一部分，其中CAR-修饰T细胞诱导对CAR中的抗原结合结构域特异性的免疫应答。例如，抗BCMA的CAR-T细胞引起抗表达BCMA的细胞的特异性免疫应答。
尽管本文公开的数据具体公开了包括抗-BCMA scFv、铰链和跨膜区、和4-1BB和CD3ζ信号传导结构域的慢病毒载体，但本发明应被解释为包括对构建体组成部分中的每一个的任何数量的变化。
可治疗的癌症包括没有被血管化或基本上还没有被血管化的肿瘤，以及血管化的肿瘤。癌症可包括非实体瘤(诸如血液学肿瘤，例如白血病和淋巴瘤)或可包括实体瘤。用本发明的CAR治疗的癌症类型包括但不限于癌、胚细胞瘤和肉瘤，和某些白血病或淋巴恶性肿瘤、良性和恶性肿瘤、和恶性瘤，例如肉瘤、癌和黑素瘤。也包括成人肿瘤/癌症和儿童肿瘤/癌症。
血液学癌症为血液或骨髓的癌症。血液学(或血原性)癌症的例子包括白血病，包括急性白血病(诸如急性淋巴细胞白血病、急性髓细胞白血病、急性骨髓性白血病和成髓细胞性、前髓细胞性、粒-单核细胞型、单核细胞性和红白血病)、慢性白血病(诸如慢性髓细胞(粒细胞性)白血病、慢性骨髓性白血病和慢性淋巴细胞白血病)、真性红细胞增多症、淋巴瘤、霍奇金氏疾病、非霍奇金氏淋巴瘤(无痛和高等级形式)、多发性骨髓瘤、瓦尔登斯特伦氏巨球蛋白血症、重链疾病、骨髓增生异常综合征、多毛细胞白血病和脊髓发育不良。
实体瘤为通常不包含囊肿或液体区的组织的异常肿块。实体瘤可为良性或恶性的。不同类型的实体瘤以形成它们的细胞类型命名(诸如肉瘤、癌和淋巴瘤)。实体瘤诸如肉瘤和癌的例子包括纤维肉瘤、粘液肉瘤、脂肪肉瘤间皮瘤、淋巴恶性肿瘤、胰腺癌卵巢癌、。
本发明的CAR-修饰T细胞也可用作对哺乳动物离体免疫和/或体内疗法的疫苗类型。优选地，哺乳动物为人。
对于离体免疫，以下中的至少一项在将细胞施用进入哺乳动物前在体外发生：i)扩增细胞，ii)将编码CAR的核酸引入细胞，和/或iii)冷冻保存细胞。
离体程序在本领域中是公知的，并在以下更完全地进行讨论。简单地说，细胞从哺乳动物(优选人)中分离并用表达本文公开的CAR的载体进行基因修饰(即，体外转导或转染)。CAR-修饰的细胞可被施用给哺乳动物接受者，以提供治疗益处。哺乳动物接受者可为人，和CAR-修饰的细胞可相对于接受者为自体的。可选地，细胞可相对于接受者为同种异基因的、同基因的(syngeneic)或异种的。
除了就离体免疫而言使用基于细胞的疫苗之外，本发明也提供了体内免疫以引起针对患者中抗原的免疫应答的组合物和方法。
本发明提供了***的方法，其包括施用给需要其的对象治疗有效量的本发明的CAR-修饰的T细胞。
本发明的CAR-修饰的T细胞可被单独施用或作为药物组合物与稀释剂和/或与其他组分诸如IL-2、IL-17或其他细胞因子或细胞群结合施用。简单地说，本发明的药物组合物可包括如本文所述的靶细胞群，与一种或多种药学或生理学上可接受载体、稀释剂或赋形剂结合。这样的组合物可包括缓冲液诸如中性缓冲盐水、硫酸盐缓冲盐水等等；碳水化合物诸如葡萄糖、甘露糖、蔗糖或葡聚糖、甘露醇；蛋白质；多肽或氨基酸诸如甘氨酸；抗氧化剂；螯合剂诸如EDTA或谷胱甘肽；佐剂(例如，氢氧化铝)；和防腐剂。本发明的组合物优选配制用于静脉内施用。
本发明的药物组合物可以以适于待治疗(或预防)的疾病的方式施用。施用的数量和频率将由这样的因素确定，如患者的病症、和患者疾病的类型和严重度——尽管适当的剂量可由临床试验确定。
当指出“免疫学上有效量”、“抗肿瘤有效量”、“肿瘤-抑制有效量”或“治疗量”时，待施用的本发明组合物的精确量可由医师确定，其考虑患者(对象)的年龄、重量、肿瘤大小、感染或转移程度和病症的个体差异。可通常指出：包括本文描述的T细胞的药物组合物可以以10 4至10 9个细胞/kg体重的剂量，优选10 5至10 6个细胞/kg体重的剂量(包括那些范围内的所有整数值)施用。T细胞组合物也可以以这些剂量多次施用。细胞可通过使用免疫疗法中公知的注入技术(见例如Rosenberg等，NewEng.J.of Med.319:1676，1988)施用。对于具体患者的最佳剂量和治疗方案可通过监测患者的疾病迹象并因此调节治疗由医学领域技术人员容易地确定。
对象组合物的施用可以以任何方便的方式进行，包括通过喷雾法、注射、吞咽、输液、植入或移植。本文描述的组合物可被皮下、皮内、瘤内、结内、脊髓内、肌肉内、通过静脉内(i.v.)注射或腹膜内施用给患者。在一个实施方式中，本发明的T细胞组合物通过皮内或皮下注射被施用给患者。在另一个实施方式中，本发明的T细胞组合物优选通过i.v.注射施用。T细胞的组合物可被直接注入肿瘤，***或感染位置。
在本发明的某些实施方式中，利用本文描述的方法或本领域已知的其他将T细胞扩展至治疗性水平的方法活化和扩展的细胞，与任何数量的有关治疗形式结合(例如，之前、同时或之后)施用给患者，所述治疗形式包括但不限于用以下试剂进行治疗：所述试剂诸如抗病毒疗法、西多福韦和白细胞介素-2、阿糖胞苷(也已知为ARA-C)或对MS患者的那他珠单抗治疗或对牛皮癣患者的厄 法珠单抗治疗或对PML患者的其他治疗。在进一步的实施方式中，本发明的T细胞可与以下结合使用：化疗、辐射、免疫抑制剂，诸如，环孢菌素、硫唑嘌呤、甲氨喋呤、麦考酚酯和FK506，抗体或其他免疫治疗剂。在进一步的实施方式中，本发明的细胞组合物与骨髓移植、利用化疗剂诸如氟达拉滨、外部光束放射疗法(XRT)、环磷酰胺结合(例如，之前、同时或之后)而施用给患者。例如，在一个实施方式中，对象可经历高剂量化疗的标准治疗，之后进行外周血干细胞移植。在一些实施方式中，在移植后，对象接受本发明的扩展的免疫细胞的注入。在一个额外的实施方式中，扩展的细胞在外科手术前或外科手术后施用。
施用给患者的以上治疗的剂量将随着治疗病症的精确属性和治疗的接受者而变化。人施用的剂量比例可根据本领域接受的实践实施。通常，每次治疗或每个疗程，可将1×10 6个至1×10 10个本发明经修饰的T细胞(如，CAR-T20细胞)，通过例如静脉回输的方式，施用于患者。
本发明的主要优点包括：
(a)本发明提供了Tscm富集的第二代通用型CAR-T细胞，显示出比第一代更好的扩增率、表型、IFN-γ释放能力及对靶细胞的杀伤能力。基于本发明的Tscm的富集T细胞(尤其是第二代通用型CAR-T细胞)，可以制备针对不同靶点的高活性的通用型CAR-T细胞。
(b)本发明还制备了靶向肿瘤靶点的通用型T细胞(例如针对BCMA的C-CAR088的通用型T细胞)，可以特异性识别和杀伤表达相应肿瘤靶点(如BCMA肿瘤抗原)的靶细胞。
(c)本发明提供了制备Tscm富集的T细胞的方法，该方法可高效制备Tscm富集的T细胞群，尤其是通用型CAR-T细胞群。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
通用实验方法
1.BCMA-CAR的慢病毒制备
在本发明中，将BCMA作为代表性的肿瘤相关的特异性靶点，制备靶向肿瘤细胞的CAR-T细胞。
第0天，将5x10 6 293T细胞置于用聚赖氨酸预处理过含10ml DMEM培养基的10-cm培养皿中。将细胞在37℃孵育过夜。第1天，准备转染试剂。将45.5ul lipofectamine 3000试剂加入OMEM中，总体积为500μl，在室温下孵育5分钟。制备另一个总体积为500ul的OMEM，加入10.7ug BCMA-CAR慢病毒载体，34ug PRP2质粒，1.8ug PRP3质粒，2.3ug PRM2质粒和36.4ul P3000试剂。将两种试剂混合到一个1.5ml管中，并在室温下孵育15分钟。将所有的转染试剂加入293T细胞培养物中，然后在37℃下孵育细胞6小时。弃去全培养基，然后用10ml预热的DMEM培养基重新加入细胞。将细胞在37℃孵育48小时。第3天，将培养上清液转移到15ml管中，在室温下用2000g离心10分钟。小心地将1ml上清液等分至1.5ml管中，并在-80℃下储存以备后用。
2.用抗CD3(克隆OKT3)抗体刺激T细胞(第3天)
将含有5ug/ml纤维连接蛋白和5ug/ml抗CD3抗体的500ul PBS加入到24孔板的孔中，并在37℃下孵育平板3小时。取出PBS，用1ml PBS充分洗涤两次。板已准备好可进行T细胞刺激。计数T细胞数，在每个孔中加入5×10 6个T细胞，在室温下以100g离心5分钟。弃去上清液，将细胞重悬于2.5ml AIM-V培养基中，然后转移至用抗CD3抗体包被的平板中，将板在37℃孵育72小时。
3.用CD3/CD28磁珠激活T细胞
在AIM-V培养基中用Dynabeads Human T-Activator CD3/CD28磁珠与细胞1：1混合活化T细胞。72小时后，用磁力除去磁珠。
4.细胞培养
Tscm细胞培养：在含有10％FBS，Glutamax，Pen/Strip，10ng/ml IL15,5ng/ml IL7和10ng/ml IL21的AIM-V培养基中以约1×10 6细胞/ml的密度培养。
传统T细胞培养：在补充有10％FBS，Glutamax，Pen/Strip，200IU/ml IL2的AIM-V培养基中以10 6细胞/ml的密度培养。
5.T细胞核转染(第0天)
在一个PCR管中混合90pmol Alt-R CRISPR-Cas9crRNA antiTRAC(AGA GUC UCU CAG CUG GUA CAG UUU UAG AGC UAU GCU，SEQ ID NO.:1)和90pmol Alt-R CRISPR-Cas9 tracRNA(AGC AUA GCA AGU UAA AAU AAG GCU AGU CCG UUA UCA ACU UGA AAA GUG GCA CCG AGU CGG UGC UUU U，SEQ ID NO.:2)。
在另一个PCR管中混合90pmol Alt-R CRISPR-Cas9crRNA antiB2M(GGC CAC GGA GCG AGA CAU CUG UUU UAG AGC UAU GCU，SEQ ID NO.:3)和90pmol Alt-R CRISPR-Cas9 tracRNA。
在95℃孵育5分钟，然后在37℃孵育25分钟。反应后，将两种试剂在一个试管中充分混合。加入9ug Cas9蛋白，在室温下孵育10分钟。在室温下用100g离心1×10 6活化的T细胞10分钟。去除上清液，并用20ul含有16.4ul P3缓冲液和3.6ul补充缓冲液的核转染缓冲液重悬细胞。加入在步骤12中制备的cas9 RNP复合物充分混合。将样品转移到用于核转染的小条中，并用核转染程序CA137转染T细胞。转染过程完成后，将80ul AIM-V培养基加入小条中，然后将样品转移到24孔板的孔中，加入AIM-V培养基至最终总体积为1ml。在37℃孵育平板2小时，然后准备进行病毒转导。
6.BCMA-CAR慢病毒转导T细胞。
将先前制备的冷冻BCMA-CAR慢病毒上清液37℃水浴中解冻直至完全解冻。将1ml BCMA-CAR慢病毒上清液转移到T细胞培养物中，最终总体积为2ml培养基，并加入终浓度为1ug/ml的硫酸鱼精蛋白。通过上下移液5次混合均匀。将细胞在32℃下以2000g离心2小时，然后将平板移至37℃培养箱中过夜培养。第1天小心取出1.5ml上清液，然后加入1.5ml新鲜的AIM-V培养基。根据细胞生长条件，改变培养基以维持细胞密度至少1×10 6个细胞/ml。
7.T细胞纯化(通常在第6-8天时进行)
将4×10 6个T细胞转移到1.5ml管中，在室温下用100g离心10分钟。弃去上清液，然后将细胞重悬于含有2％FBS的100ul PBS中。将2ul生物素偶联的抗HLA A/B/C和2ul生物素偶联的抗CD3抗体加入样品中，并在4℃下孵育30min。将4ul生物素分离混合物加入样品中，在室温下孵育15分钟。加入5ul rapidspheres，在室温下孵育样品10分钟。将样品转移到5ml管中，然后加入1.9ml AIM-V培养基。将管放在磁铁上，等待5分钟。将上清液转移到新管中并将其置于磁体上，等待5分钟。将含有T细胞的上清液转移到24孔板的孔中。
8.FACS分析
(a).将1×10 5个T细胞转移到1.5ml管中，在室温下以1500rpm离心10分钟。弃去上清液，然后加入900ul PBS，在室温下以1500rpm离心10分钟。弃去上清液，将细胞重悬于含有1ug/ml重组人BCMA Fc嵌合体蛋白的100ul PBS中，并在冰上孵育1小时。加入900ul PBS，在室温下以1500rpm离心10分钟， 然后弃去上清液。将细胞重悬于PBS(含有1:1000Live/Dead aqua orange staining dye)。在冰上孵育15分钟。加入900ul PBS，室温下以1500rpm离心10分钟，然后弃去上清液。将细胞重悬于100ul PBS中含有2.5ul APC-Cy7-偶联的抗-CD3抗体,和2.5ul V605-偶联的抗-HLA A/B/C抗体和1；100APC-偶联的抗-BCMA Fc嵌合体蛋白抗体的100ul PBS中，在冰上孵育30分钟。加入900ul PBS，室温下以1500rpm离心10分钟，然后弃去上清液。将细胞重悬于300ul PBS中，并将样品转移至5ml管中进行FACS分析。
T细胞群首先由FSC和SSC分群。Aqua orange通道用于区分活和死細胞,APC通道用于区分BCMA-CAR阳性细胞。CD3和HLA A/B/C用于区分CD3和HLA A/B/C双敲除的T细胞。
(b).将1×10 5个T细胞转移到1.5ml管中，在室温下以1500rpm离心10分钟。弃去上清液，然后加入900ul PBS，在室温下以1500rpm离心10分钟。弃去上清液,将细胞重悬于PBS(含有1:1000Live/Dead aqua orange staining dye)。在冰上孵育15分钟。加入900ul PBS，室温下以1500rpm离心10分钟，然后弃去上清液。将细胞重悬于100ul PBS中含有2.5ul APC-偶联的抗CCR7抗体,在37度孵育30分钟,接著加入2.5ul PE-Cy7-偶联的抗-CD45RA抗体,2.5ul FITC-偶联的抗-CD8抗体和2.5ul APC-Cy7-偶联的抗-CD3抗体，在冰上孵育30分钟。加入900ul PBS，室温下以1500rpm离心10分钟，然后弃去上清液。将细胞重悬于300ul PBS中，并将样品转移至5ml管中进行FACS分析。
9.ELISA
提前一天用Human IFN-γ Mab包被酶标板。将Human IFN-γ Mab用1×PBS稀释(1:1000)至1μg/mL，每孔加入100μl抗体，用封板膜将酶标板密封，4℃过夜。迅速倒掉酶标板内液体，每孔加入100μl Assay Buffer，盖上封板膜，室温封闭30分钟。洗板：迅速倒掉板内液体，用排枪加入洗涤缓冲液，每孔200μl，重复洗板一次。准备Human IFN-γ Biotin-labeled Mab，用Assay Buffer稀释(1:500)，每孔加入50μl到酶标板。准备Human IFN-γ ELISA Standard，设置8个梯度(单位pg/ml)：2000、1000、500、、250、、125、、62.5、31.25、0。
将样品和标准品加入酶标板，100μl/孔，样品和标准品均用Assay Buffer稀释到所需浓度，盖上封板膜，室温孵育1.5小时。洗板：迅速倒掉板内液体，用排枪加入洗涤缓冲液，每孔200μl，重复洗板四次。准备HRP-偶联的链亲和素，用Assay Buffer稀释(1:5000)，每孔加入100μl到酶标板。盖上封板膜，室温孵育30分钟。洗板：迅速倒掉板内液体，用排枪加入洗涤缓冲液， 每孔200μl，重复洗板二次。提前30分钟将TMB底物恢复至室温，每孔加入100μl到酶标板。室温反应5-10分钟后，加入终止液，50μl/孔。用酶标仪检测450nm波长处的吸光度，根据标准品的浓度和OD值生成标准曲线，并根据检测样品的OD值计算检测样品的IFN-γ浓度。
10.RTCA细胞杀伤实时分析
第一天：打开RTCA MP的操作软件，输入实验信息。取一RTCA配套的96孔E-plate培养板，向每个孔加入50ul F12完全培养基，避免气泡。将板放入RTCA仪器，操作软件读取加入细胞前的背景。确认读值无异常。胰酶消化培养的A549和A549-BCMA细胞，使用AO/PI Cell Viability Kit进行活细胞计数后离心，F12完全培养基将两种肿瘤细胞分别重悬至4 x 10 5/ml。根据实验安排，向读取完背景的E-plate每个孔加入50ul细胞悬液(即2x10 4个A549和A549-BCMA细胞)。每组实验设3个复孔，确保其中包括3个孔为对照组，即只有肿瘤细胞后续不加入T细胞。培养板在超净台中放置30分钟以便细胞均匀贴壁。将板放入RTCA仪器，操作软件开始记录细胞指数(Cell Index)变化，设置为每15分钟读取一次，确保记录总时长设置在4天以上。
第二天：T细胞计数，离心后用T细胞培养基将T细胞重悬并稀释至不同浓度.1 x 10 5/ml,5 x 10 4/ml,2.5 x 10 4/ml,1.25 x 10 4/ml。暂停RTCA的软件记录，将E-plate从仪器取出至超净台，向A549的培养孔加入100ul 1 X 10 5/ml的T细胞悬液,即最终T细胞与靶细胞的比例为0.5:1；向A549-BCMA的培养孔加入100ul 1 x 10 5/ml,5 x 10 4/ml,2.5 x 10 4/ml,1.25 x 10 4/ml的T细胞悬液,即最终T细胞与靶细胞的比例为0.5:1,0.25:1,0.125:1,0.0625:1。将E-plate放回RTCA仪器，每15分钟读取一次细胞指数直至设定的结束时间。实验结束后进行数据分析，一般以加入T细胞之前的最后一个时间点作为参照，以只有肿瘤细胞没有加入T细胞的读值为100％，将加入T细胞后肿瘤细胞指数的变化转化为杀伤曲线。
实施例1 通用型T细胞中TRAC和B2M基因敲除及纯化率的分析
TRAC的敲除会导致CD3在细胞表面的表达消失，B2M的敲除会导致HLA A/B/C在细胞表面的表达消失。因此，用抗HLA A/B/C和抗CD3的抗体对基因编辑过的T细胞进行染色并进行流式细胞分析来检测TRAC和B2M的敲除率。
结果如图1所示，在用Cas9 RNP基因编辑和BCMA CAR转导后第7天的T细胞中TRAC和B2M基因双敲除效率为81.2％。纯化后，双重敲除细胞从81.4％富集至99.4％，而在HLA A/B/C门控线以下存在少量低HLA A/B/C表达的T细胞。
实施例2 通用型T细胞转导BCMA CAR的效率
在第7天对转导BCMA CAR的T细胞进行流式细胞分析。
结果如图2所示，BCMA CAR转导效率在不同组之间相似(mock：没有经过Cas9 RNP核转染的T细胞；mock+CAR：没有经过Cas9 RNP核转染但转导了BCMA CAR的T细胞；CAR：BCMA CAR转导了的T细胞；Double KO：Cas9 RNP核转染后双敲除TRAC和B2M的T细胞；Double KO+CAR：上述Double KO细胞经BCMA CAR转导后的T细胞)。
上述结果表明：核转染的基因编辑过程不影响CAR的转导效率。
实施例3 通用型BCMA CAR-T细胞对靶向细胞的反应性
用ELISA检测上述不同BCMA CAR-T细胞与表达BCMA抗原的靶向细胞A549-BCMA细胞共培养后上清中IFN-γ的水平。
结果如图3所示，Double KO+CAR，即基因编辑过的BCMA CAR-T细胞对靶向细胞的反应性，即释放IFN-γ的能力，和其他未经基因编辑传统的BCMA CAR-T细胞类似。
上述结果表明：通用型CAR-T细胞和自体的CAR-T细胞功能相似。
实施例4 第二代通用型CAR-T细胞的制备
从外周血中富集初始T细胞(Tn)并进行流式细胞分析来检测富集CCR7 +CD45RA +的初始T细胞(Tn)的效率。
4.1从外周血中富集Tn细胞
方法如下：使用不含细胞因子(cytokine)的AIM-V培养基解冻人PBMC，并且计数。离心细胞(300g,5min)，移除上清液,利用PBS重悬细胞于5ml管。加入biotin isolation cocktail和anti-TCRγ/δ抗体室温孵育5分钟。加入rapidSpheres室温孵育3分钟。加入不含cytokine的AIM-V培养基至2.5ml,并置于磁座等待3分钟。将上清液转至一个新的5ml管，并置于磁座等待3分钟。将含有Tn细胞的上清液转至一个新的15ml管，随后进行细胞计数。
结果如图4所示，经过富集纯化后，Tn的比例从PBMC里的54.8％上升到97.3％。
如果需要，可对富集纯化的Tn细胞可进行扩大培养，例如在含有10％FBS，Glutamax，Pen/Strip，10ng/ml IL15,5ng/ml IL7和10ng/ml IL21的AIM-V培养基中以约10 6细胞/ml进行扩大培养。
4.2制备第二代通用型CAR-T细胞
重复实施例1-3，不同点在于：从4.1中制备的高度纯化的Tn细胞替换常规的T细胞。
对于4.1中制备的高度纯化的Tn细胞，进行TRAC和B2M基因敲除，然后通过慢病毒进行转导从而导入BCMA CAR，从而制备第二代通用型CAR-T细胞。
4.3直接扩增法制备第二代通用型CAR-T细胞(省略富集Tn的操作)
另一种制备第二代通用型CAR-T细胞的方法，是从外周血中富集的总T细胞(Total T细胞)出发，但在其扩增培养的过程中，是在合适的条件(如在含有10％FBS，Glutamax，Pen/Strip，10ng/ml IL15、5ng/ml IL7和10ng/ml IL21的AIM-V培养基中)，以约10 6细胞/ml进行密度培养，从而获得富集Tn的T细胞群(在该条件下，Tn细胞会更优先、更高效地扩增)。
随后进行基因敲除(如敲除TRAC和/或B2M)，慢病毒转导等步骤，从而制得Tscm富集的通用型CAR-T细胞，即第二代通用型CAR-T细胞。
实施例5 第二代通用型T细胞的扩增效率
对实施例4.2制备的第二代通用型T细胞进行细胞计数。
结果如图5所示，第二代通用型T细胞的扩增效率要高于其他两种CAR-T细胞，7天后细胞的相对增殖率达16倍之多，远大于其他两种CAR的3-4倍，表明制备第二代通用型T细胞的培养条件相对于传统的制备CAR的条件来说更有利于CAR-T细胞的增生。
实施例6 第二代通用型CAR-T细胞产品中各T细胞亚型的比例
对实施例4.2制备的第二代通用型CAR-T细胞产品进行流式细胞表型分析。
结果如图6所示，Tscm的比例在第二代通用型CAR-T细胞产品接近70％，远高于第一代通用型CAR-T细胞产品(约30％)及自体的CAR-T细胞(约20％)。
出乎意料的是，在第二代通用型CAR-T细胞中，去/弱分化细胞的生物标志物CCR7的百分比高达76％。此外，第二代通用型CAR-T细胞的MFI达4534，远远高于第一代通用型CAR-T细胞产品及自体的CAR-T细胞相应的MFI值。
上述结果表明，本发明制备第二代通用型CAR-T细胞的条件不仅有利于产生高比例的去/弱分化CAR-T细胞，即Tscm富集的CAR-T细胞，而且产生的CAR-T细胞具有更高的活力和杀伤活性。
实施例7 第二代通用型T细胞转导BCMA CAR的效率
对实施例4.2中制备的第二代通用型CAR-T细胞产品进行CAR表达率的流 式分析。
结果如图7所示，BCMA CAR的比例在第二代通用型CAR-T细胞产品中达到83.4％，高于第一代通用型CAR-T细胞产品(60.8％)及自体的CAR-T细胞(60.2％)。
上述结果表明，本发明制备的第二代通用型CAR-T细胞不降低甚至提高CAR的转导率。
实施例8 第二代通用型BCMA CAR-T细胞对靶向细胞的反应性
用ELISA检测上述不同BCMA CAR-T细胞与表达BCMA抗原的靶向细胞A549-BCMA细胞共培养后上清中IFN-γ的水平。
结果如图8所示，第二代BCMA CAR-T细胞对靶向细胞的反应性，即释放IFN-γ的能力显著高于第一代通用型BCMA CAR-T细胞和自体BCMA CAR-T细胞。
此外，在不同的效应子细胞：靶向细胞的比例(从1:2至1:16)下，第二代通用型CAR-T细胞对靶向细胞的反应显著优于其他两种CAR-T细胞。这提示，本发明的第二代通用型CAR-T细胞对具有相应靶点(如肿瘤抗原，例如BCMA或其他靶点)的靶细胞具有更显著的反应性。
实施例9 第二代通用型BCMA CAR-T细胞对靶向细胞的杀伤力
当使用RTCA平台对上述3种BCAM CAR-T细胞对靶细胞的杀伤进行了持续4天的监测。
结果如图9所示，与上述IFN-γ释放实验结果相呼应，第二代通用型BCMA CAR-T细胞对靶向细胞的杀伤力最佳。
图9左边的细胞裂解曲线表明，第二代通用型BCMA CAR-T细胞对靶向细胞的杀伤力高于其他两种CAR-T细胞。此处显示的是CAR-T细胞与靶向细胞比例为1：8的例子，但同样结论也适合于其他1：2，1：4以及1：16的情况(数据未显示)。
图9右边的KT80图，即杀伤80％的靶向细胞所需要的时间图，可以看出在不同的CAR-T细胞与靶向细胞比例情况下，第二代通用型BCMA CAR-T细胞对杀伤80％靶向细胞的所需要的时间都显著低于其他两种CAR-T细胞。比如在CAR-T细胞数与靶向细胞数为1：8的情况下，第二代通用型细胞杀伤80％的靶向细胞需要70小时左右，但其他两种CAR-T细胞都需要100小时左右，几乎是第二代CAR-T细胞所需时间的1.5倍。
在CAR-T细胞数量远远小于靶向细胞的情况下(1:16)，其他两种CAR-T细胞甚至不能达到杀伤80％靶向细胞的能力，只有第二代通用型BCMA CAR-T细胞能够达到这个杀伤力。
上述结果表明，第二代通用型CAR-T细胞对靶向细胞的杀伤力要优于其他两种CAR-T细胞，这在针对需要杀伤大量靶向细胞的情况下尤为突出。
实施例10
在本实施例中，测试了在不同条件下，对T细胞进行了扩大培养，除加入的细胞因子相对于实施例4有变化，其他培养条件相同。
如图10-13所示。在不同条件下，尤其是在不同细胞因子存在下，培养T细胞，加入IL1、IL7和IL21可以显著增加Tscm细胞的富集、促进细胞生长、提高BCMA CAR转导率、和增加IFN-γ释放。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种非天然存在的T细胞群，其特征在于，所述的T细胞群中T记忆干细胞(Tscm)的比例C1≥50％，按所述T细胞群中的T细胞总数计。
  2. 如权利要求1所述的T细胞群，其特征在于，所述的T细胞包括CAR-T细胞。
  3. 如权利要求1所述的T细胞群，其特征在于，所述的T记忆干细胞包括CCR7 +CD45RA +T细胞。
  4. 如权利要求1所述的T细胞群，其特征在于，在所述的T细胞群中，表达生物标志物CCR7的比例C2≥50％，较佳地≥60％，更佳地，≥70％。
  5. 如权利要求1所述的T细胞群，其特征在于，所述的CAR-T细胞为靶向肿瘤抗原的通用型CAR-T细胞。
  6. 一种细胞制剂，其特征在于，所述的细胞制剂含有权利要求1所述的非天然存在的T细胞群和药学上可接受的载体、稀释剂或赋形剂。
  7. 一种制备CAR-T细胞的方法，其特征在于，包括步骤：
    (a)提供一分离的T细胞；
    (b)在IL15、IL7和IL21存在的条件下，对所述分离的T细胞进行扩大培养，从而获得经培养的T细胞；和
    (c)对所述经培养的T细胞进行改造，制备CAR-T细胞。
  8. 如权利要求1所述的方法，其特征在于，在步骤(a)中，所述分离的T细胞选自下组：初始T细胞(Tn细胞)、富含Tn细胞的细胞群、或总T细胞、或其组合。
  9. 如权利要求1所述的方法，其特征在于，在步骤(b)中，对所述分离的T细胞进行扩大培养时，培养体系中IL15的浓度为1-200ng/ml，较佳地为，3-100ng/ml，更佳地为5-20ng/ml；IL7的浓度为0.5-50ng/ml，较佳地为，1-20ng/ml，更佳地为3-10ng/ml；且IL21的浓度为1-100ng/ml，较佳地为，3-50ng/ml，更佳地为5-20ng/ml。
  10. 一种制备CAR-T细胞的方法，其特征在于，包括步骤：
    (i)提供一分离的Tn细胞，
    (ii)任选地，在IL15、IL7和IL21存在的条件下，对所述分离的T细胞进行扩大培养，从而获得经培养的T细胞；和
    (iii)对所述T细胞进行改造，制备CAR-T细胞。
PCT/CN2020/106635 2019-08-01 2020-08-03 通用型car-t细胞及其制备和应用 WO2021018311A1 (zh)

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