WO2018068257A1 - 通用型car-t细胞及其制备方法和用途 - Google Patents

通用型car-t细胞及其制备方法和用途 Download PDF

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WO2018068257A1
WO2018068257A1 PCT/CN2016/101986 CN2016101986W WO2018068257A1 WO 2018068257 A1 WO2018068257 A1 WO 2018068257A1 CN 2016101986 W CN2016101986 W CN 2016101986W WO 2018068257 A1 WO2018068257 A1 WO 2018068257A1
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car
seq
cell
cells
molecule
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French (fr)
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何霆
鲁薪安
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北京艺妙神州医疗科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/46434—Antigens related to induction of tolerance to non-self
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4621—Cellular immunotherapy characterized by the effect or the function of the cells immunosuppressive or immunotolerising
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4632—T-cell receptors [TCR]; antibody T-cell receptor constructs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule

Definitions

  • the present invention relates to the field of immunology and molecular biology, and in particular to tumor immunotherapy, in particular to a general-purpose CAR-T cell and a preparation method and use thereof.
  • CAR-T chimeric antigen receptor-engineered T cells
  • T cells used in the CAR-T technology are mainly derived from the patients themselves, and the T cells derived from the peripheral blood of the patients themselves are activated, CAR-introduced and expanded, and then returned to the patients.
  • T cell capture and eliminate heterologous antigens is a complex process that requires the participation of major histocompatibility complex (MHC) and T cell receptor (TCR), in which TCR recognizes the antigenic process of MHC binding. It plays a key role.
  • MHC major histocompatibility complex
  • TCR T cell receptor
  • MHC class I major histocompatibility complex
  • MHC class II major histocompatibility complex
  • TCR T cell receptor
  • TCR signaling on CAR-T may have a potential impact on clinical response, and the interaction between TCR and CAR has not been studied in depth.
  • returning allogeneic CAR-T cells may present a risk of graft-versus-host disease (GVHD).
  • GVHD graft-versus-host disease
  • the present invention addresses the above drawbacks by using CRISPR/Cas9 technology for T cells from healthy volunteers. Modification, selective knockdown of the a-chain and/or beta-chain constant coding region (TRAC and/or TRBC) genes of the endogenous T cell receptor (TCR), and stable expression of the chimeric antigen receptor (CAR) In T cells, CAR-T cells expressing a specific CAR and not expressing a TCR were thus obtained.
  • TRAC and/or TRBC a-chain constant coding region
  • a first aspect of the invention provides a universal type of CAR-T cell that expresses a specific CAR and does not express a TCR.
  • a second aspect of the invention provides a method of preparing a universal CAR-T cell, the method comprising the steps of:
  • step 2) can be performed before, after or simultaneously with step 1), and the sgRNA molecule comprises a targeting structure complementary to a target region of the a- or beta-chain constant coding region (ie TRAC or TRBC) gene from the TCR area.
  • a- or beta-chain constant coding region ie TRAC or TRBC
  • a third aspect of the invention provides a composition comprising the universal type CAR-T cells of the first aspect of the invention.
  • a fourth aspect of the invention provides the use of the universal type CAR-T cell of the first aspect of the invention for the preparation of a medicament for treating a tumor.
  • the universal type CAR-T cells of the present invention prevent GVHD and potential TCR receptor signal interference from T cells infused into a patient. This application will pave the way for the use of universal CAR-T cells with significant clinical implications.
  • Figure 1 shows the results of comparison of transfection efficiency of sgRNA expression plasmid and Cas9 expression plasmid under different transfection (liposome transfection, electroporation and lentiviral transfection) conditions.
  • Figures 2a and 2b show the results of a comparison of the efficiency of genomic mutations caused by different sgRNAs.
  • FIG. 3 shows the results of sequencing the TCR knockout TRAC and TRBC regions.
  • Figure 4 shows the results of sequencing off-target sites on the predicted human genome of TRAC-sg3.
  • Figure 5 shows the results of screening for TCR negative, CD4 and CD8 positive T cells.
  • Figure 6 shows the results of comparison of the killing effects of T cells, WT CAR-T and TCR- CAR-T cells on target cells.
  • Figure 7 shows the results of comparison of the effects of T cell, WT CAR-T and TCR- CAR-T cell inhibition and killing of tumor cells.
  • Figure 8 shows the results of comparison of changes in mean body weight of mice after injection of mouse T cells, human WT CAR-T and human TCR- CAR-T cells.
  • the first aspect of the present invention provides a universal type CAR-T cell which expresses a specific CAR and does not express a TCR.
  • the cells are derived from a healthy volunteer (or donor).
  • the TCR gene of a universal CAR-T cell of the invention is knocked out.
  • the a chain and/or beta chain constant coding region (ie, the TRAC and/or TRBC) genes of the TCR are knocked out.
  • the TRAC and TRBC genes can be completely knocked out or partially knocked out; either of them can be knocked out or knocked out; as long as the cells cannot express the active TCR after knocking out.
  • the a chain constant coding region (ie, TRAC) gene of the TCR is knocked out.
  • the a-chain constant coding region gene of the TCR of the invention is introduced into one of the TRAC-sg1-7 molecules of the cell (see Table 1) and the Cas 9 molecule is partially or completely knocked out; preferably, The a chain constant coding region gene of the TCR is introduced into the TRAC-sg3 molecule of the cell and the Cas9 molecule is knocked out.
  • the beta strand constant coding region (ie, TRBC) gene of the TCR is knocked out.
  • TRBC beta strand constant coding region
  • the ⁇ -chain constant coding region gene of the TCR of the present invention is introduced into one of the TRBC-sg1-7 molecules of the cell (see Table 1) and the Cas 9 molecule is partially or completely knocked out; preferably, The ⁇ -chain constant coding region gene of TCR is introduced into the TRBC-sg1 molecule of the cell and the Cas9 molecule is knocked out.
  • the CAR expressed by the universal CAR-T cells of the invention may be any CAR known in the art, as long as it enables T cells to recognize cell surface antigens in a human leukocyte antigen-independent manner, Play a killing effect.
  • the CAR disclosed in Chinese Patent Application No. 201510324558.X can be used, and the CAR used in the specific embodiment of the present invention can be referred to the disclosure of the invention patent application.
  • the CAR expressed in the CAR-T cells of the present invention comprises a sequence-linked signal peptide, an extracellular binding region, an optional hinge region, a transmembrane region, and an intracellular signal region.
  • signal peptide refers to a short (length 5-30 amino acid) peptide chain that directs the transfer of a newly synthesized protein to a secretory pathway.
  • a signal peptide of various proteins in a human body such as a signal peptide of a cytokine protein secreted in vivo or a leukocyte differentiation antigen (CD molecule) can be used.
  • the signal peptide is a GMCSF signal peptide, the nucleotide sequence of which is set forth in SEQ ID No: 1 of the Sequence Listing of Patent Application No. 201510324558.X.
  • the signal peptide is a CD8 signal peptide
  • the nucleotide sequence thereof is as claimed in the patent application Please show SEQ ID No: 2 in the sequence listing of 201510324558.X.
  • the hinge region may use a hinge region of various antibodies or antigen receptors, particularly a hinge region of a CD molecule.
  • the hinge region can be selected from a hinge region of a protein such as CD8 or CD28.
  • the CD8 or CD28 is a natural marker on the surface of T cells.
  • the hinge region is a CD8 hinge region (CD8-hinge), the nucleotide sequence of which is set forth in SEQ ID No: 6 of the Sequence Listing of the patent application 201510324558.X.
  • the hinge region is a CD28 hinge region (CD28-hinge), the nucleotide sequence of which is set forth in SEQ ID No: 7 of the Sequence Listing of the patent application 201510324558.X.
  • transmembrane regions of various human proteins can be used, particularly transmembrane regions of various antigen receptors.
  • the transmembrane region preferably used is the transmembrane region of CD molecules.
  • the transmembrane region may be selected from a transmembrane region of a protein such as CD8 or CD28.
  • the transmembrane region is a CD8 transmembrane region (CD8-TM), the nucleotide sequence of which is set forth in SEQ ID No: 8 of the Sequence Listing of the patent application 201510324558.X.
  • the transmembrane region is a CD28 transmembrane region (CD28-TM), the nucleotide sequence of which is set forth in SEQ ID No: 9 of the Sequence Listing of the patent application 201510324558.X.
  • the "extracellular binding region” comprises an scFv that specifically recognizes a tumor cell surface antigen.
  • scFv refers to an antibody fragment which comprises a variable region of heavy chain (VH) and a variable region of light linked by a linker.
  • VH variable region of heavy chain
  • VL variable region of light linked by a linker.
  • the recombinant protein of chain, VL), the linker associates these two domains to ultimately form an antigen binding site.
  • the size of scFv is typically 1/6 of that of an intact antibody.
  • the scFv is preferably an amino acid sequence encoded by a single nucleotide chain.
  • the scFv used in the present invention may be further modified, either singly or in combination using conventional techniques known in the art, such as amino acid deletions, insertions, substitutions, additions, and/or recombinations, and/or other modification methods.
  • specific recognition means that the antigen recognition region of the invention does not or substantially does not cross-react with any polypeptide other than the antigen of interest.
  • the degree of specificity can be judged by immunological techniques including, but not limited to, immunoblotting, immunoaffinity chromatography, flow cytometry, and the like.
  • the extracellular binding region comprises an scFv that specifically recognizes CD19, CEA, EGFR, GD2 or CD138, and the like.
  • the extracellular binding region comprises an scFv (anti-CD19 scFv) that specifically recognizes CD19.
  • the extracellular binding region comprises a humanized, engineered scFv that specifically recognizes CD19.
  • the extracellular binding region is a humanized, scFv-S1 that specifically recognizes CD19 (the nucleic acid sequence thereof is SEQ ID No: 4 in the sequence listing of the patent application 201510324558.X) Shown) or scFv-S2 (the nucleic acid sequence thereof is shown as SEQ ID No: 5 in the Sequence Listing of the Invention Patent Application No. 201510324558.X).
  • intracellular signal regions of various human body proteins can be used.
  • the intracellular signal region preferably used is the intracellular signal region of the CD molecule.
  • the intracellular signal region can be selected from the group consisting of CD3 ⁇ , Fc ⁇ RI ⁇ , CD28, CD137, an intracellular signal region of a CD134 protein, and combinations thereof.
  • the CD3 molecule consists of five subunits, of which the CD3 ⁇ subunit (also known as CD3zeta, abbreviated as Z) contains three ITAM motifs, which are important signal transduction regions in the TCR-CD3 complex.
  • CD3 ⁇ Z is a mutated CD3 ⁇ sequence that does not have an ITAM motif, and is generally used as a construction component of a negative control in the examples of the present invention.
  • Fc ⁇ RI ⁇ is mainly distributed on the surface of mast cells and basophils, which contains an ITAM motif similar in structure, distribution and function to CD3 ⁇ .
  • CD28, CD137, and CD134 are costimulatory signal molecules, and the co-stimulatory action of intracellular signal segments after binding to their respective ligands causes sustained proliferation of T cells and enhances IL-secretion of T cells. 2 and the level of cytokines such as IFN- ⁇ , while increasing the survival cycle and anti-tumor effect of CAR-T cells in vivo.
  • intracellular signal regions which include a signal region selected from the group consisting of:
  • CD28-signal the nucleotide sequence of which is shown in SEQ ID No: 10 in the sequence listing of the patent application 201510324558.X;
  • CD137 signal region having a nucleotide sequence as shown in SEQ ID No: 11 of the sequence listing of the patent application 201510324558.X;
  • CD3 ⁇ signal region CD3 ⁇ -signal
  • CD3 ⁇ -signal the nucleotide sequence of which is shown in SEQ ID No: 12 in the sequence listing of the patent application 201510324558.X.
  • the CAR expressed by the universal CAR-T cells of the invention is selected from the group consisting of a signal peptide comprising a ligated sequence, an extracellular binding region, an optional hinge region, a transmembrane region, and an intracellular signal.
  • a signal peptide comprising a ligated sequence, an extracellular binding region, an optional hinge region, a transmembrane region, and an intracellular signal.
  • GMCSF-scFv-S2-CD8-hinge-CD8-TM-CD137-signal-CD3 ⁇ -signal CAR2
  • the nucleic acid sequence thereof is shown in SEQ ID No: 14 in the Sequence Listing of Invention Patent Application No. 201510324558.X);
  • GMCSF-scFv-S1-CD28-TM-CD28-signal-CD3 ⁇ -signal CAR3, the nucleic acid sequence thereof is shown as SEQ ID No: 15 in the sequence listing of the invention patent application 201510324558.X);
  • GMCSF-scFv-S2-CD28-TM-CD28-signal-CD3 ⁇ -signal CAR4, the nucleic acid sequence thereof is shown in SEQ ID No: 16 in the sequence listing of the invention patent application 201510324558.X);
  • GMCSF-scFv-S1-CD8-hinge-CD8-TM-CD28-signal-CD137-signal-CD3 ⁇ -signal (CAR5, the nucleic acid sequence thereof is as in SEQ ID No: 17 in the sequence listing of the invention patent application 201510324558.X Show);
  • GMCSF-scFv-S2-CD8-hinge-CD8-TM-CD28-signal-CD137-signal-CD3 ⁇ -signal CAR6, the nucleic acid sequence thereof is shown in SEQ ID No: 18 in the sequence listing of the invention patent application 201510324558.X ).
  • the second aspect of the present invention provides a method of preparing a universal CAR-T cell, the method comprising the steps of:
  • step 2) can be performed before, after or simultaneously with step 1), and the sgRNA molecule comprises a target region complementary to the a-chain and/or beta-chain constant coding region (ie, TRAC and/or TRBC) genes from the TCR.
  • TRAC and/or TRBC beta-chain constant coding region
  • the sgRNA molecule refers to a nucleic acid sequence comprising a targeting domain complementary to a target region of the gene to be knocked out, which recognizes the target DNA sequence and directs the Cas9 molecule to cleave the target site.
  • the Cas9 molecule refers to a double-stranded DNA nuclease capable of cleaving a target site under the guidance of sg RNA.
  • the sgRNA molecule comprises a targeting domain that is complementary to a target region of the a-chain constant coding region (ie, TRAC) gene from the TCR.
  • TRAC a-chain constant coding region
  • sequence of the targeting domain comprised by the sgRNA molecule is as set forth in one of SEQ ID NOs: 1-7.
  • sequence of the targeting domain is set forth in SEQ ID NO:3.
  • the sgRNA molecule comprises a targeting domain that is complementary to a target region of the beta strand constant coding region (ie, TRBC) gene from the TCR.
  • TRBC beta strand constant coding region
  • sequence of the targeting domain comprised by the sgRNA molecule is as set forth in one of SEQ ID NOs: 8-14.
  • sequence of the targeting domain is set forth in SEQ ID NO: 8.
  • the sgRNA molecule and the Cas9 molecule are introduced into the T cell by lipofection, electroporation or lentiviral transfection techniques by constructing a vector.
  • the sgRNA molecule and the Cas9 molecule are introduced into the T cell by electroporation techniques by constructing a vector.
  • the sgRNA molecule and mRNA encoding the Cas9 molecule are introduced into the T cell by electroporation techniques.
  • the CAR molecule is introduced into the T cell by a lentiviral transfection technique.
  • the method of the invention further comprises the step of isolating and/or activating T cells from healthy volunteers (or donors) prior to step 1); preferably, the method is after step 2) Also included is the step of sorting general-purpose CAR-T cells; more preferably, the resulting universal-type CAR-T cells are validated after sorting.
  • the third aspect of the invention provides a composition comprising the universal type CAR-T cell of the first aspect of the invention.
  • the composition further comprises a pharmaceutically acceptable diluent, excipient or carrier, and the like.
  • the fourth aspect of the present invention provides the use of the universal type CAR-T cell of the first aspect of the present invention for the preparation of a medicament for treating a tumor.
  • PBMC peripheral blood mononuclear cells
  • T cells Isolation and activation of T cells: The obtained peripheral blood mononuclear cells were counted, and the beads conjugated to the CD3/CD28 antibody were added in a ratio of 1:1, gently shaken for 20 min, and the CD3 was obtained by the adsorption of the magnetic frame. Positive T cells, at which time the T cells are activated, and the complete medium (Lonza x-vivo15+IL-2) is added to culture and expand the T cells.
  • the sequence of 5'-GN 19-22 NGG-3' was selected from the first three exon sequences of the TRAC and TRBC gene coding regions, and Cas-OFFinder was used to ensure that 8-12 bases at the 3' end were not mismatched. Go to other locations in the genome (http://www.rgenome.net/cas-offinder/). The selected sequences are shown in Table 1.
  • TRBC-sg5 CAGGCTTCTTCCCCGACCACG SEQ ID NO: 12
  • TRBC-sg6 ACCACGTGGAGCTGAGCTGG SEQ ID NO: 13
  • TRBC-sg7 GGAGCTGAGCTGGTGGGTGAA SEQ ID NO: 14
  • the primers were synthesized, the sense strand was 5'-ACCGN 19-22 -3', and the antisense strand was 5'-AAACN 19-22 -3' (the N 19-22 sequence in the antisense strand was partially reverse-complementary).
  • the synthesized sequence fragments were each taken to be 22.5 ⁇ L, mixed with 5 ⁇ L of 10 ⁇ TransTaq HiFi Buffer II (Full Gold Transgene, Beijing), and heated at 95 ° C for 3 min, and then slowly cooled to room temperature.
  • the resulting product was phosphorylated with T4 PNK (T4 Polynucleotide Kinase/T4 Polynucleotide Kinase) and incubated at 37 ° C for 30 min.
  • the phosphorylated product (Insert sgRNA) was ligated into the psgRNA vector (Addgene Plasmid #53121) by the Golden Gate reaction, respectively.
  • the reaction system is as follows:
  • the clones were selected for sequencing using Universal Primer U6 (Life Technology, Shanghai), and the clones were correctly inserted into the sgRNA sequence.
  • Liposomal transfection 1 ⁇ 10 6 Jurkat cells were plated in wells of a six-well plate, 1 ⁇ g of pCas9 (Addgene Plasmid #53118) was added to 200 ⁇ L of OPTI-MEM, and 1 ⁇ g of TRAC-sg3 expression plasmid was mixed evenly, and 4 ⁇ L was added.
  • FACS flow cytometric sorting
  • Electroporation 1 ⁇ 10 6 Jurkat cells were taken, and 1 ⁇ g of pCas9 and 1 ⁇ g of TRAC-sg3 expression plasmid were added to 100 ⁇ L of electroporation buffer, mixed uniformly, and electroporated by a LONZA electrophoresis apparatus. The transferred cells continue to be cultured. The proportion of mCherry positive cells was analyzed by flow cytometric sorting (FACS) after 72 h of liquid exchange.
  • FACS flow cytometric sorting
  • HeLa cells were transfected with 1 ⁇ g of pCas9, 1 ⁇ g of pTRAC-sgRNA and/or 1 ⁇ g of pTRBC-sgRNA. After 72 hours, the genome of the transfected cells was extracted as a template, and primers designed by Primer-Blast were used to specifically amplify the sgRNA-containing sequence. A segment of the genome of 300-1000 bp in size. 300-500 ng of PCR product was mixed with 10 ⁇ NEB Buffer 2 (NEB) in a 50 ⁇ L system, and heated at 95 ° C for 3 min, then slowly cooled to room temperature.
  • NEB N-NEB Buffer 2
  • the obtained product was added to 0.5 ⁇ L of T7E1 (NEB) and incubated at 37 ° C for 15 min for agarose gel electrophoresis.
  • the electrophoresis pattern was analyzed by Image J image analysis software to analyze the efficiency of band cutting, indicating the efficiency of sgRNA to generate InDel (insertion-deletion) ( See Figures 2a and 2b).
  • TRAC-sg3 and TRBC-sg1 had higher editing efficiency for the target gene locus, and the plasmids expressing TRAC-sg3 and TRBC-sg1 were subsequently selected for further experiments.
  • sgRNA and Cas9 were introduced into T cell lines (Jurkat, SupT1, etc.) and primary T cells, and TCR-negative and CD4 or CD8-positive T cells were screened by flow sorting or immunomagnetic beads technique.
  • the selected TCR-negative CD4 or CD8-positive T cells were extracted from the genome (blood/cell/tissue genomic DNA extraction kit) with specific primers (TRAC: Forward: AGTCTGTCTGCCTATTCACCGA, Reverse: CCTGGTGCATTCATGTGCCG; TRBC: Forward: GGATAGATGATCAGACAAGCCT, Reverse:TGGTAGCTGGTCTCACCTAAT) PCR amplification of TRAC and TRBC respectively contains the genomic region corresponding to sgRNA, and the PCR product was subjected to TA cloning and sequencing to verify the knockdown of TCR at the molecular level. The results are shown in Fig. 3, and it can be seen that it can be successfully TCR's TRAC and TRBC genes are edited, including insertion and deletion mutations, both of which cause frameshift mutations. Thus, the expression of TCR is suppressed from the level of the gene.
  • the off-target sites on the potential human genome of TRAC-sg3 and TRBC-sg1 were predicted, and the predicted off-target region that may affect the expression of other genes was amplified and analyzed by T7E1 in order to confirm the TCR at the molecular level.
  • the knockout did not introduce a knockout of the off-target non-specific gene.
  • Fig. 4 it can be seen that the TRAC and TRBC genes did not undergo any mutation, indicating that the system specifically meets the demand.
  • the nucleotide sequence of the extracellular binding region is the antigen binding region of the anti-CD19 chimeric antigen receptor (here named anti-CD19 scFv-S0, abbreviated as scFv-S0, which is derived from mouse, see Humanization of the nucleotide sequence of J Immunother.2009 September;32(7):689-702.) (shown as SEQ ID No: 3 in the sequence listing of the patent application 201510324558.X) And got it. It should be understood that the principle of humanization of antibodies is to maximize the binding of the framework region (FM) to a human sequence while ensuring antibody affinity, thereby reducing the immunogenicity of the antibody.
  • FM framework region
  • the antigen recognition region in the above SEQ ID No: 3 is kept unchanged, and the remaining sequences are correspondingly changed, and more than 40 humanized designs are performed, and then synthesized by gene synthesis.
  • These sequences, other parts of the CAR molecule were cloned from a human cDNA library by PCR, and then ligated, and finally the nucleotide sequence of the CAR molecule was prepared.
  • These CAR molecules were transferred into T cells, and the killing ability of T cells containing CAR molecules containing these humanized sequences and T cells containing scFv-S0-containing CAR molecules was compared to target cells, and finally, two screens were obtained.
  • anti-CD19 scFv-S1 abbreviated as scFv-S1
  • anti-CD19 scFv-S2 abbreviated as scFv-S2
  • scFv-S2 anti-CD19 scFv-S2
  • SEQ ID No: 5 the sequence listing of the patent application 201510324558.X
  • Primer design was first performed, and the primer sequences used in this example were as follows:
  • the corresponding CAR molecules were cloned by PCR using 1-1 and 1-2, 2-1 and 2-2, 3-1 and 3-2 as primers, respectively, which were GMCSF, CD28-TM+CD28-signal (the two parts are connected) and CD3 ⁇ -signal.
  • the GMCSF+scFv fragment was obtained by bridging primers 4-1 and 4-2, and the CD28-TM+CD28-signal+CD3 ⁇ -signal fragment was obtained by bridging primers 5-1 and 5-2, followed by bridging primers 6-1 and 6.
  • -2 Obtain the nucleotide sequence of the complete CAR molecule, and the restriction sites are SpeI and MluI.
  • the nucleotide sequence of the CAR molecule prepared above was digested with SpeI (Fermentas) and MluI (Fermentas), and ligated into the SpeI-MluI site of the lentiviral pLenti-CMV-eGFP vector by T4 ligase (Fermentas).
  • the plasmid purification kit was used to extract (Qiagen) and the plasmid was purified for subsequent experiments.
  • the cell density was adjusted to 2 ⁇ 10 6 cells/mL with Lonza x-vivo 15 medium.
  • Add the slow virus packaged with CAR according to the ratio of MOI 2-4 (the specific procedure can be found in the part of the invention patent application 201510324558.X), change the solution after 24h, observe the state of the cells after 48h, collect The cell suspension was centrifuged at 400 g for 5 min, the supernatant was discarded, and the cell density was adjusted to 1 ⁇ 10 7 cells/mL with x-vivo 15 medium.
  • Cas9 mRNA and TRAC-sg3 mRNA were prepared using the mMessage mMachine T7Ultra kit (Life Technologies) kit, purified and eluted for use.
  • the cells and mRNA were mixed to a final concentration of 1 x 10 6 cells and 500 ng of mRNA per 100 ⁇ L (250 ng each of Cas9 mRNA and TRAC-sg3 mRNA).
  • the mRNA was introduced into the cells using an electrorotator (NEPA21). The growth of the cells was observed every day, and the liquid was changed every other day. After the cells were cultured for 11-12 days, the obtained T cells were subjected to mass detection. T cells were purified at 12-14d. For details, see step 5. Finally, the final product (ie, universal CAR-T cells, referred to herein as TCR- CAR-T cells) was dispensed and frozen. .
  • TCR-negative, CD4 and CD8-positive T cells were screened by flow sorting or immunomagnetic beads.
  • the first step is to perform negative sorting using immunomagnetic beads coupled with CD3 antibody to remove cells that still express CD3 (TCR) in T cells (usually performing 2 negative sorting);
  • the second step take a small amount of the first step of T cells, perform flow detection, and simultaneously stain CD3, CD4 and CD8. If the positive rate of CD3 is ⁇ 0.01%, and the positive rate of CD4+CD8 is >90%, then proceed The next step of the job. In this example, the CD3 positive rate was 0%, while the CD4+CD8 positive rate was 93.3% (Fig. 5).
  • Step 1 Labeling target cells with Calcein-AM (ie, cells from patients with B-cell acute lymphoblastic leukemia)
  • Calcein-AM ie, cells from patients with B-cell acute lymphoblastic leukemia
  • Step 2 Killing target cells with effector cells
  • effector cells Add 100 ⁇ L of effector cells according to the appropriate ET (effect ratio) ratio (5:1), while using T cells and general peripheral blood CAR-T (ie, WT CAR-T cells) as control cells, 3 per group.
  • ET effect ratio
  • test release test release
  • target cells detect the fluorescence intensity of spontaneous apoptotic lysis
  • pontaneous release design a separate group B (6 Parallel), only the target cells + 2% Triton X-100, the maximum release fluorescence intensity (maximum release);
  • Cell line human lymphoma cell line Daudi;
  • Daudi cells are human lymphoma cell lines, and human lymphoma models of mice can be constructed by subcutaneous injection. Its CD19 expression is positive and can be used as a target cell of CAR-T cells.
  • the Daudi cell line is a suspension cell line that can grow rapidly in 1640 medium (Gibco) containing 20% FBS. Passage is required when the cell density is 2-3 ⁇ 10 6 /mL. At the time of passage, the cell suspension was taken in a centrifuge tube, centrifuged at 500 g for 5 minutes, and the supernatant was discarded. The cell density was adjusted to 0.3-0.5 ⁇ 10 6 /mL, and the culture was continued. Under normal growth conditions, the Daudi cell line was passaged every other day, and the cell density was maintained between 0.3-3 ⁇ 10 6 /mL.
  • 1640 medium Gibco
  • the Daudi cells were resuspended in physiological saline, adjusted to a viable cell concentration of 3 ⁇ 10 8 /mL, and thoroughly mixed with Matrigel (BD, China) in a volume ratio of 2:1 on ice. Inoculation was carried out by subcutaneous injection.
  • tumor volume (mm 3 ) tumor long diameter (mm) ⁇ tumor short diameter 2 (mm 2 ) ⁇ 0.5;
  • Mouse lymphoma model administration Record the day of administration as D0.
  • Cell infusion by tail vein injection PBS 200 ⁇ L, human T cells 200 ⁇ L (total 1 ⁇ 10 6 /), human WT CAR-T cells 200 ⁇ L (total 1 ⁇ 10 6 / only), human TCR- CAR- T cells were 200 ⁇ L (total 1 ⁇ 10 6 /piece), and all mice were administered in a single dose.
  • PBS 200 ⁇ L human T cells 200 ⁇ L (total 1 ⁇ 10 6 /)
  • human WT CAR-T cells 200 ⁇ L total 1 ⁇ 10 6 / only
  • human TCR- CAR- T cells were 200 ⁇ L (total 1 ⁇ 10 6 /piece)
  • all mice were administered in a single dose.
  • Fig. 7 it can be seen that the TCR- CAR-T cells provided by the present invention have excellent effects of inhibiting and killing tumor cells.
  • mice 6-8 weeks old 24 hours ago
  • single tail vein injection of mouse T cells, human WT CAR-T cells and human TCR- CAR-T cells 1 ⁇ 10 7 / mL 10 randomly assigned to each group, the negative control group was injected with the same volume of PBS
  • detailed clinical observation was started 4 hours after the injection of the cells, and was observed once a day, continuously. Observed for 60 days to assess the occurrence and severity of GVHD.
  • mice in the group injected with normal xenogenic CAR-T cells developed a severe GVHD response, and 70% of the mice died.
  • the CAR-T cells after TCR knockout showed almost no GVHD to the body, indicating that the CAR-T cells were safer after TCR knockout.
  • Figure 8 shows the change in mean body weight of mice.
  • the mice in the normal xenogenic CAR-T cell group were significantly reduced in body weight, up to 30%, while the group not injected with CAR-T cells and the injected TCR knockout CAR- The weight of the mice in the T cell group did not decrease significantly, but a slight increase.
  • Comprehensive data showed that the safety of CAR-T cells after TCR knockout was high.

Abstract

提供了一种通用型CAR-T细胞，其表达特异性CAR并且不表达TCR。还提供了一种制备通用型CAR-T细胞的方法，所述方法包括如下步骤：1)在T细胞中引入sgRNA分子和Cas9分子；2)在所述T细胞中引入CAR分子；其中，步骤2)可在步骤1)之前、之后或同时进行，并且所述sgRNA分子包含与来自TCR的a链或β链恒定编码区(即TRAC或TRBC)基因的靶区域互补的靶向结构域。

Description

通用型CAR-T细胞及其制备方法和用途 技术领域
本发明涉及免疫学和分子生物学领域，具体地涉及肿瘤免疫疗法，尤其是一种通用型CAR-T细胞及其制备方法和用途。
背景技术
肿瘤免疫疗法近年来得到广泛的关注和应用，尤其是CAR-T(chimeric antigen receptor-engineered T cells)技术的出现，使人类对肿瘤的控制得到了里程碑式的发展。该技术在肿瘤治疗中的应用始于1989年。它的临床应用结果让人看到治愈癌症的曙光：不仅仅在急性或者慢性淋巴细胞血液病中可以创造奇迹，在其它一些实体瘤比如淋巴瘤、成神经细胞瘤中也有着令人兴奋的治疗效果。目前CAR-T技术中所应用的T细胞主要来源于患者自身，从患者自身外周血来源的T细胞经过激活、CAR导入并扩增，然后回输到患者体内。但是很多情况下，由于病人的病情危险或者患者自身的条件不允许再从其体内抽出外周血进行CAR-T细胞的治疗；甚至是即使病人可以提供外周血，但是从血中分选出来的T细胞活性不足以进行CAR改造，这就从一定范围上限制了CAR-T技术的应用范围。为了解决这个问题，异体CAR-T逐渐成为一个良好的选择。同时，异体CAR-T技术解决了T细胞的来源问题也将为CAR-T的商业化推广奠定基础。
在机体内，T细胞捕获并消灭异源抗原是一个复杂的过程，需要主要组织相容性复合物(MHC)和T细胞受体(TCR)的共同参与，其中TCR在识别MHC结合的抗原过程中起着关键作用。主要组织相容性复合物(MHC)有两种类型，通常称为MHC I类和MHC II类，除了具有抗原递呈作用，还与器官移植过程中的排斥反应相关。杀伤性T细胞则是通过其表面具有的类似抗体的分子即T细胞受体(TCR)识别并清除经MHC I类分子递呈的抗原。在同种异体环境中，CAR-T上的TCR信号传导作用可能对临床应答产生潜在的影响，而TCR和CAR之间的相互作用尚未有深入的研究。同时，回输异体CAR-T细胞有可能出现移植物抗宿主病(GVHD)的风险。
因此，需要有降低受体的供体细胞排斥反应和避免出现GVHD的改善CAR-T疗效的方法。
发明内容
本发明针对上述缺陷，利用CRISPR/Cas9技术对来自健康志愿者(donor)的T细胞 进行修饰，选择性敲除内源性T细胞受体(TCR)的a链和/或β链恒定编码区(TRAC和/或TRBC)基因，同时将嵌合抗原受体(CAR)稳定表达在T细胞中，从而获得了表达特异性CAR同时不表达TCR的CAR-T细胞。
本发明的第一方面提供了一种通用型CAR-T细胞，其表达特异性CAR并且不表达TCR。
本发明的第二方面提供了一种制备通用型CAR-T细胞的方法，所述方法包括如下步骤：
1)在T细胞中引入sgRNA分子和Cas9分子；
2)在所述T细胞中引入CAR分子；
其中，步骤2)可在步骤1)之前、之后或同时进行，并且所述sgRNA分子包含与来自TCR的a链或β链恒定编码区(即TRAC或TRBC)基因的靶区域互补的靶向结构域。
本发明的第三方面提供了包含本发明第一方面的通用型CAR-T细胞的组合物。
本发明的第四方面提供了本发明第一方面的通用型CAR-T细胞用于制备治疗肿瘤的药物的用途。
本发明的通用型CAR-T细胞避免了输注到患者体内的T细胞出现GVHD以及潜在的TCR受体信号干扰。本申请将会为通用型CAR-T细胞的使用铺平道路，具有重大的临床意义。
附图说明
根据以下参照附图进行的详细描述，本发明的上述和其他方面、特征和优点会变得更加清楚。
图1示出了sgRNA表达质粒及Cas9表达质粒在不同转染(脂质体转染、电转和慢病毒转染)条件下的转染效率的比较结果。
图2a和图2b示出了不同sgRNA造成基因组突变的效率的比较结果。
图3示出了对经TCR敲除的TRAC和TRBC区域进行测序的结果。
图4示出了对预测的TRAC-sg3潜在的人基因组上的脱靶位点进行测序的结果。
图5示出了对TCR阴性、CD4和CD8阳性的T细胞的筛选结果。
图6示出了T细胞、WT CAR-T和TCR-CAR-T细胞对靶细胞的杀伤效果的比较结果。
图7示出了T细胞、WT CAR-T和TCR-CAR-T细胞抑制以及杀死肿瘤细胞的效果的比较结果。
图8示出了注射小鼠T细胞、人WT CAR-T和人TCR-CAR-T细胞后，小鼠平均体重的变化的比较结果。
具体实施方式
如上所述，本发明的第一方面提供了一种通用型CAR-T细胞，其表达特异性CAR并且不表达TCR。
在一个实施方案中，所述细胞来源于健康志愿者(或供者)。
在一个实施方案中，本发明的通用型CAR-T细胞的TCR基因被敲除。
在一个具体的实施方案中，所述TCR的a链和/或β链恒定编码区(即TRAC和/或TRBC)基因被敲除。TRAC和TRBC基因可以被全部敲除，也可以被部分敲除；可以二者均被敲除，也可以敲除二者之一；只要敲除后细胞不能表达有活性的TCR即可。
在一个具体的实施方案中，所述TCR的a链恒定编码区(即TRAC)基因被敲除。例如，在具体实施方案中，本发明TCR的a链恒定编码区基因被引入所述细胞的TRAC-sg1-7分子之一(见表1)以及Cas 9分子部分或全部敲除；优选，所述TCR的a链恒定编码区基因被引入细胞的TRAC-sg3分子以及Cas9分子敲除。
在另一个具体的实施方案中，所述TCR的β链恒定编码区(即TRBC)基因被敲除。例如，在具体实施方案中，本发明TCR的β链恒定编码区基因被引入所述细胞的TRBC-sg1-7分子之一(见表1)以及Cas 9分子部分或全部敲除；优选，所述TCR的β链恒定编码区基因被引入细胞的TRBC-sg1分子以及Cas9分子敲除。
在一个实施方案中，本发明的通用型CAR-T细胞所表达的CAR可以是本领域已知的任何CAR，只要其能够使T细胞以人白细胞抗原-非依赖性的方式识别细胞表面抗原，发挥杀伤作用即可。例如，可以使用中国发明专利申请201510324558.X中公开的CAR，本发明的具体实施方案中使用的CAR可参考该发明专利申请公开文本。具体而言，本发明的CAR-T细胞中表达的CAR包含顺序连接的信号肽、胞外结合区、任选的铰链区、跨膜区和胞内信号区。
本文使用的术语“信号肽”是指引导新合成的蛋白质向分泌通路转移的短(长度5-30个氨基酸)肽链。在本发明中，可以使用人体内的各种蛋白质的信号肽，例如体内分泌的细胞因子蛋白、白细胞分化抗原(CD分子)的信号肽。
在一个具体的实施方案中，所述信号肽为GMCSF信号肽，其核苷酸序列如发明专利申请201510324558.X的序列表中的SEQ ID No:1所示。
在一个具体的实施方案中，所述信号肽为CD8信号肽，其核苷酸序列如发明专利申 请201510324558.X的序列表中的SEQ ID No:2所示。
在本发明中，所述铰链区可以使用各种不同抗体或抗原受体的铰链区，特别是CD分子的铰链区。在一个具体的实施方案中，所述铰链区可以选自CD8或CD28等蛋白的铰链区。所述CD8或CD28是T细胞表面的天然标记物。
在一个具体的实施方案中，所述铰链区为CD8铰链区(CD8-hinge)，其核苷酸序列如发明专利申请201510324558.X的序列表中的SEQ ID No:6所示。
在一个具体的实施方案中，所述铰链区为CD28铰链区(CD28-hinge)，其核苷酸序列如发明专利申请201510324558.X的序列表中的SEQ ID No:7所示。
在本发明中，可以使用各种人体内蛋白的跨膜区，特别是各种不同抗原受体的跨膜区。优选使用的跨膜区是CD分子的跨膜区。在一个实施方案中，所述跨膜区可以选自CD8或CD28等蛋白的跨膜区。
在一个具体的实施方案中，所述跨膜区为CD8跨膜区(CD8-TM)，其核苷酸序列如发明专利申请201510324558.X的序列表中的SEQ ID No:8所示。
在一个具体的实施方案中，所述跨膜区为CD28跨膜区(CD28-TM)，其核苷酸序列如发明专利申请201510324558.X的序列表中的SEQ ID No:9所示。
所述“胞外结合区”包含特异性识别肿瘤细胞表面抗原的scFv。
本文使用的术语“scFv”是指这样的抗体片段——其是包含通过接头(linker)连接的重链可变区(variable region of heavy chain，VH)和轻链可变区(variable region of light chain，VL)的重组蛋白,接头使得这两个结构域相关联，以最终形成抗原结合位点。scFv的大小一般是一个完整抗体的1/6。scFv优选是由一条核苷酸链编码的氨基酸序列。本发明使用的scFv可通过单独或联合使用本领域已知的常规技术，例如氨基酸缺失、插入、取代、增加、和/或重组以及/或其他修饰方法作进一步修饰。根据一种抗体的氨基酸序列在其DNA序列中引入这种修饰的方法对本领域技术人员来说是公知的(参见例如，Sambrook分子克隆：实验手册，Cold Spring Harbor Laboratory(1989)N.Y.)。所述修饰优选在核酸水平上进行。上述scFv还可以包括其衍生物。
本文使用的术语“特异性识别”意指本发明的抗原识别区不与或基本上不与目标抗原以外的任意多肽交叉反应。其特异性的程度可以通过免疫学技术来判断，包括但不限于免疫印迹，免疫亲和层析，流式细胞分析等。
在一个实施方案中，所述胞外结合区包含特异性识别CD19、CEA、EGFR、GD2或CD138等的scFv。
在一个具体的实施方案中，所述胞外结合区包含特异性识别CD19的scFv(anti-CD19 scFv)。
在一个具体的实施方案中，所述胞外结合区包含经人源化改造的特异性识别CD19的scFv。
在一个优选的实施方案中，所述胞外结合区为经人源化改造的特异性识别CD19的scFv-S1(其核酸序列如发明专利申请201510324558.X的序列表中的SEQ ID No:4所示)或scFv-S2(其核酸序列如发明专利申请201510324558.X的序列表中的SEQ ID No:5所示)。
在本发明中，可以使用各种人体内蛋白的胞内信号区，特别是各种不同抗原受体的胞内信号区。优选使用的胞内信号区是CD分子的胞内信号区。在一个实施方案中，所述胞内信号区可以选自CD3ζ、FcεRIγ、CD28、CD137、CD134蛋白的胞内信号区，及其组合。CD3分子由五个亚单位组成，其中CD3ζ亚单位(又称CD3zeta，简称Z)含有3个ITAM基序，该基序是TCR-CD3复合体中重要的信号转化区。CD3δZ是突变的不具有ITAM基序的CD3ζ序列，在本发明的实施例中一般作为阴性对照的构建组分。FcεRIγ主要分布在肥大细胞和嗜碱性粒细胞表面，其含有一个ITAM基序，在结构、分布及功能上与CD3ζ类似。此外如前所述，CD28、CD137、CD134是共刺激信号分子，在与各自配体结合后其胞内信号区段产生的共刺激作用引起T细胞的持续增殖，并能够提高T细胞分泌IL-2和IFN-γ等细胞因子的水平，同时提高CAR-T细胞在体内的存活周期和抗肿瘤效果。
本发明具体使用的胞内信号区有多种组合，其包括选自如下的信号区或其组合：
CD28信号区(CD28-signal)，其核苷酸序列如发明专利申请201510324558.X的序列表中的SEQ ID No:10所示；
CD137信号区(CD137-signal)，其核苷酸序列如发明专利申请201510324558.X的序列表中的SEQ ID No:11所示；和
CD3ζ信号区(CD3ζ-signal)，其核苷酸序列如发明专利申请201510324558.X的序列表中的SEQ ID No:12所示。
在一个具体的实施方案中，本发明的通用型CAR-T细胞所表达的CAR选自如下的包含顺序连接的信号肽、胞外结合区、任选的铰链区、跨膜区和胞内信号区的嵌合抗原受体蛋白：
GMCSF–scFv-S1–CD8-hinge–CD8-TM–CD137-signal–CD3ζ-signal(CAR1，其核酸序 列如发明专利申请201510324558.X的序列表中的SEQ ID No:13所示)；
GMCSF–scFv-S2–CD8-hinge–CD8-TM–CD137-signal–CD3ζ-signal(CAR2，其核酸序列如发明专利申请201510324558.X的序列表中的SEQ ID No:14所示)；
GMCSF–scFv-S1–CD28-TM–CD28-signal–CD3ζ-signal(CAR3，其核酸序列如发明专利申请201510324558.X的序列表中的SEQ ID No:15所示)；
GMCSF–scFv-S2–CD28-TM–CD28-signal–CD3ζ-signal(CAR4，其核酸序列如发明专利申请201510324558.X的序列表中的SEQ ID No:16所示)；
GMCSF–scFv-S1–CD8-hinge–CD8-TM–CD28-signal–CD137-signal–CD3ζ-signal(CAR5，其核酸序列如发明专利申请201510324558.X的序列表中的如SEQ ID No:17所示)；
GMCSF–scFv-S2–CD8-hinge–CD8-TM–CD28-signal–CD137-signal–CD3ζ-signal(CAR6，其核酸序列如发明专利申请201510324558.X的序列表中的SEQ ID No:18所示)。
如上所述，本发明的第二方面提供了一种制备通用型CAR-T细胞的方法，所述方法包括如下步骤：
1)在T细胞中引入sgRNA分子和Cas9分子；
2)在所述T细胞中引入CAR分子；
其中，步骤2)可在步骤1)之前、之后或同时进行，并且所述sgRNA分子包含与来自TCR的a链和/或β链恒定编码区(即TRAC和/或TRBC)基因的靶区域互补的靶向结构域。
在本发明的方法中，所述sgRNA分子是指一段包含与待敲除的基因的靶区域互补的靶向结构域的核酸序列，其能识别靶标DNA序列并引导Cas9分子切割靶位点。
在本发明的方法中，所述Cas9分子是指一种双链DNA核酸酶，其能够在sg RNA的引导下对靶位点进行切割。
在一个实施方案中，所述sgRNA分子包含与来自TCR的a链恒定编码区(即TRAC)基因靶区域互补的靶向结构域。
在一个具体的实施方案中，所述sgRNA分子所包含的靶向结构域的序列如SEQ ID NOs:1-7中之一所示。
在一个优选的实施方案中，所述靶向结构域的序列如SEQ ID NO:3所示。
在一个实施方案中，所述sgRNA分子包含与来自TCR的β链恒定编码区(即TRBC)基因靶区域互补的靶向结构域。
在一个具体的实施方案中，所述sgRNA分子所包含的靶向结构域的序列如SEQ ID NOs:8-14中之一所示。
在一个优选的实施方案中，所述靶向结构域的序列如SEQ ID NO:8所示。
在一个实施方案中，通过构建载体将所述sgRNA分子和Cas9分子经脂质体转染、电转或慢病毒转染技术引入所述T细胞中。
在一个优选的实施方案中，通过构建载体将所述sgRNA分子和Cas9分子经电转技术引入所述T细胞中。
在一个更优选的实施方案中，通过电转技术将所述sgRNA分子和编码Cas9分子的mRNA引入所述T细胞中。
在一个实施方案中，通过慢病毒转染技术将所述CAR分子引入所述T细胞中。
在一个具体的实施方案中，本发明的方法在步骤1)之前还包括分离和/或激活来自健康志愿者(或供者)的T细胞的步骤；优选地，所述方法在步骤2)之后还包括对通用型CAR-T细胞进行分选的步骤；更优选地，分选之后再对所得的通用型CAR-T细胞进行有效性验证。
如上所述，本发明的第三方面提供了包含本发明第一方面的通用型CAR-T细胞的组合物。
在一个实施方案中，所述组合物还包含可药用的稀释剂、赋形剂或载体等。
如上所述，本发明的第四方面提供了本发明第一方面的通用型CAR-T细胞用于制备治疗肿瘤的药物的用途。
以下通过具体实施例来说明本发明的内容。应理解，所述具体实施例仅为说明目的，并不意味着本发明的内容仅限于具体实施例。
实施例1：通用型CAR-T细胞的制备
1.健康供者T细胞的分离与激活。
1)健康供者外周血的采集：采集的外周血4℃冰箱暂存，24h内，经由配备有恒温设备的转运车运至GMP实验室进行分离和培养。
2)外周血单个核细胞(PBMC)的制备：用移液管吸取DPBS(Dulbecco's Phosphate Buffered Saline)或者生理盐水加入到步骤(1)采集的外周血中(1：1)，稀释，将血细胞稀释液，缓慢加入装有淋巴细胞分离液(Ficoll或者Histopaque-1077) 的离心管中，以800g离心20min后，吸取淋巴细胞分离液上方的白膜层细胞，转入一个新的离心管中，加入Lonza x-vivo 15培养基(货号：04-418Q)离心后弃上清，保留离心管底部的细胞沉淀，即得到外周血单个核细胞。
3)T细胞的分离和激活：将得到的外周血单个核细胞进行计数，按照1:1的比例加入偶联CD3/CD28抗体的beads，轻轻震荡20min，利用磁力架的吸附作用，得到CD3阳性的T细胞，此时的T细胞处于激活状态，加入完全培养基(Lonza x-vivo15+IL-2)，对T细胞进行培养扩增。
2.利用CRISPR/Cas9系统敲除经步骤1获得的T细胞中的TCR基因，具体操作步骤如下所示：
1)针对TCR的a链和β链恒定编码区(TRAC、TRBC)基因的sgRNA设计和质粒构建。
针对TRAC以及TRBC基因编码区的前三个外显子序列中选择5’-GN19-22NGG-3’的序列，并用Cas-OFFinder确保其3’端的8-12个碱基不会错配到基因组其他位置(http://www.rgenome.net/cas-offinder/)。所选取的序列见表1。
表1：sgRNA序列
sgRNA编号 sgRNA序列 序列号
TRAC-sg1 CAAAACTGTGCTAGACATG SEQ ID NO:1
TRAC-sg2 GTGCTAGACATGAGGTCTA SEQ ID NO:2
TRAC-sg3 GACACCTTCTTCCCCAGCCC SEQ ID NO:3
TRAC-sg4 TGTGCTAGACATGAGGTCTA SEQ ID NO:4
TRAC-sg5 CTTCAAGAGCAACAGTGCTG SEQ ID NO:5
TRAC-sg6 AGAGCAACAGTGCTGTGGCC SEQ ID NO:6
TRAC-sg7 AAAGTCAGATTTGTTGCTCC SEQ ID NO:7
TRBC-sg1 GAACAAGGTGTTCCCACCCG SEQ ID NO:8
TRBC-sg2 AGAGATCTCCCACACCCAAA SEQ ID NO:9
TRBC-sg3 AAGGCCACACTGGTGTGCC SEQ ID NO:10
TRBC-sg4 CACTGGTGTGCCTGGCCAC SEQ ID NO:11
TRBC-sg5 CAGGCTTCTTCCCCGACCACG SEQ ID NO:12
TRBC-sg6 ACCACGTGGAGCTGAGCTGG SEQ ID NO:13
TRBC-sg7 GGAGCTGAGCTGGTGGGTGAA SEQ ID NO:14
合成引物，正义链为5’-ACCGN19-22-3’，反义链为5’-AAACN19-22-3’(正义反义链中N19-22序列部分反向互补)。将合成的序列片断各取22.5μL，与5μL 10×TransTaq HiFi Buffer II(全式金Transgene，北京)混合，于95℃加热3min后缓慢冷却到室温。所得产物用T4 PNK(T4 Polynucleotide Kinase/T4多聚核苷酸激酶)进行磷酸化处理，37℃温育30min。将磷酸化后的产物(Insert sgRNA)分别通过Golden Gate反应连接到psgRNA载体(Addgene Plasmid#53121)中。
反应体系如下：
10X Buffer Tango 1μL
DTT(50mM) 0.2μL
ATP(10mM) 1μL
Esp3I 0.75μL
T4 DNA ligase 0.25μL
psgRNA vector(20ng/μL) 2μL
Insert sgRNA 0.2μL
ddH2O 4.6μL
连接产物使用感受态stbl3转化后，挑选克隆使用通用引物U6进行测序(Life Technology,上海)，将正确插入sgRNA序列的克隆摇菌提取质粒。
2)sgRNA表达质粒及Cas9表达质粒转入T细胞系以及原代T细胞的效率检测
如下所示比较了sgRNA表达质粒及Cas9表达质粒在不同转染(脂质体转染、电转和慢病毒转染)条件下的转染效率。
脂质体转染：在六孔板的孔中铺1×106Jurkat细胞，在200μL的OPTI-MEM中加入1μg pCas9(Addgene Plasmid#53118)，1μg TRAC-sg3表达质粒混合均匀，再加入4μL X-tremeGENE HP DNA转染试剂或者6μL 100nM聚乙烯亚胺(Polyethylenimine,PEI)，或者6μL lipo2000试剂，混合均匀后室温静置15min，滴入孔中。6-8h后将细胞换回新鲜培养基培养。换液72h后通过流式细胞分选(FACS)分析mCherry阳性细胞比例。
电转：取1×106Jurkat细胞，在100μL的电转Buffer中加入1μg pCas9，1μg TRAC-sg3表达质粒，混合均匀，用LONZA电转仪电转。转完的细胞继续培养。换液72h后通过流式细胞分选(FACS)分析mCherry阳性细胞比例。
慢病毒感染：取1×106Jurkat细胞，按照MOI＝4的比例加入包装有Cas9和TRAC-sg3的慢病毒，24h后换液，继续培养48h，通过流式细胞分选(FACS)分析mCherry阳性细胞比例。
不同转染条件效率的比较结果见图1。比较结果显示，脂质体转染方法转染效率较低，电转方法细胞转染效率较高，两者细胞活率类似。
3)sgRNA造成基因组突变的效率检测
用1μg pCas9，1μg pTRAC-sgRNA和/或1μg pTRBC-sgRNA转染2×105HeLa细胞，72h后提取转染细胞的基因组为模板，用Primer-Blast设计引物，特异性地扩增包含sgRNA序列的一段300-1000bp大小的基因组片断。在50μL体系中取300-500ng的PCR产物与10×NEB Buffer2(NEB)混合，95℃加热3min后缓慢冷却到室温。所得产物加入0.5μL T7E1(NEB)在37℃温育15min，进行琼脂糖凝胶电泳，电泳图用Image J图像分析软件分析条带切割的效率，指示sgRNA产生InDel(insertion-deletion)的效率(如图2a和2b)。
结果显示，TRAC-sg3及TRBC-sg1具有较高的对靶基因位点的编辑效率，后续选取表达TRAC-sg3及TRBC-sg1的质粒继续实验。
4)敲除T细胞系(Jurkat、SupT1等)和primary T细胞的TCR基因
利用电转技术，将sgRNA和Cas9导入到T细胞系(Jurkat、SupT1等)和primary T细胞中，通过流式分选技术或者免疫磁珠技术筛选出TCR阴性同时CD4或者CD8阳性的T细胞。
将筛选出的TCR阴性同时CD4或者CD8阳性的T细胞提取基因组(血液/细胞/组织基因组DNA提取试剂盒)，用特异性引物(TRAC：Forward:AGTCTGTCTGCCTATTCACCGA，Reverse:CCTGGTGCATTCATGTGCCG；TRBC：Forward:GGATAGATGATCAGACAAGCCT，Reverse:TGGTAGCTGGTCTCACCTAAT)分别PCR扩增TRAC及TRBC包含对应sgRNA的基因组区域，PCR产物进行TA克隆及测序以从分子水平上验证TCR的敲除，结果如图3所示，可以看出，可以成功对TCR的TRAC及TRBC基因进行编辑，包括插入突变和缺失突变，两者都造成了移码突变， 从而从基因的水平抑制了TCR的表达。
同时对于TRAC-sg3及TRBC-sg1潜在的人基因组上的脱靶位点进行预测，并对预测的可能影响其他基因表达的脱靶位点区域进行扩增和T7E1分析，目的是为了从分子水平确认TCR的敲除并没有引入off-target的非特异基因的敲除，结果如图4所示，可以看出TRAC和TRBC的基因并没有发生任何突变，说明该体系特异性满足需求。
以上结果显示，转入TRAC-sg3及TRBC-sg1并进行筛选的T细胞中的TCR基因被完全敲除，同时，没有发现潜在脱靶位点处的基因突变。
3.CAR的设计及载体构建
参考中国发明专利申请201510324558.X公开文本，特别是其实施例1和2，设计包含顺序连接的信号肽、胞外结合区、任选的铰链区、跨膜区和胞内信号区的嵌合抗原受体(CAR)，并进行载体构建。所述胞外结合区的核苷酸序列是在抗CD19的嵌合抗原受体的抗原结合区(在此命名为anti-CD19 scFv-S0，简称为scFv-S0，其来源于小鼠，参见J Immunother.2009 September；32(7):689–702.)的核苷酸序列(如发明专利申请201510324558.X的序列表中的SEQ ID No:3所示)的基础上进行人源化改造而获得的。应理解，抗体人源化改造的原则为在保证抗体亲和力的同时，最大限度地将骨架区(framework region,FM)改变为人源序列，以降低抗体的免疫原性。在该实施例中，将上述SEQ ID No:3中的抗原识别区保持不变，对其余的序列进行相应的改变，进行了超过40种的人源化设计，再通过基因合成的方法合成得到这些序列，CAR分子的其他部分都是利用PCR技术，分别从人cDNA文库中克隆得到，然后进行搭桥连接，最终制备得到CAR分子的核苷酸序列。将这些CAR分子转入T细胞，将包含含有这些经人源化改造序列的CAR分子的T细胞与包含含有scFv-S0的CAR分子的T细胞对靶细胞的杀伤能力进行比较，最终筛选得到两种经人源化改造的胞外结合区序列，其分别称为anti-CD19 scFv-S1(简称为scFv-S1，其核苷酸序列如发明专利申请201510324558.X的序列表中的SEQ ID No:4所示)和anti-CD19 scFv-S2(简称为scFv-S2，其核苷酸序列如发明专利申请201510324558.X的序列表中的SEQ ID No:5所示)。下面以CAR5和CAR6为例，具体说明CAR分子的核苷酸序列的制备步骤。
首先进行引物设计，本实施例中使用的引物序列如下：
Figure PCTCN2016101986-appb-000001
Figure PCTCN2016101986-appb-000002
以人的cDNA文库为模板，分别以1-1和1-2、2-1和2-2、3-1和3-2为引物，通过PCR克隆出相应的CAR分子部分，分别为GMCSF、CD28-TM+CD28-signal(这两部分是相连的)和CD3ζ-signal。再通过搭桥引物4-1和4-2获得GMCSF+scFv片段，通过搭桥引物5-1和5-2获得CD28-TM+CD28-signal+CD3ζ-signal片段，随后通过搭桥引物6-1和6-2获得完整的CAR分子的核苷酸序列，酶切位点为SpeI和MluI。
将如上制备的CAR分子的核苷酸序列经SpeI(Fermentas)和MluI(Fermentas)双酶切、经T4连接酶(Fermentas)连接插入慢病毒pLenti-CMV-eGFP载体的SpeI-MluI位点，转化到感受态E.coli(DH5α)，经测序正确后，使用质粒纯化试剂盒提取(Qiagen)并纯化质粒，用于后续实验。
4.通用型CAR-T细胞的构建和扩增
如步骤1所述，利用偶联CD3/CD28抗体的beads将PBMC中的T细胞分选和激活后，用Lonza x-vivo 15培养基将细胞密度调至2×106cell/mL。按照MOI＝2-4的比例加入包装有CAR的慢病毒(慢病毒法，具体步骤可参见发明专利申请201510324558.X的实施例3部分)，24h后换液，48h后观察细胞的状态，收集细胞悬液，以400g离心5min，弃上清，用x-vivo 15培养基将细胞密度调至1×107cell/mL。 使用mMessage mMachine T7Ultra kit(Life Technologies)试剂盒制备Cas9 mRNA和TRAC-sg3 mRNA，经过纯化和洗脱后待用。把细胞和mRNA混合，使其最终浓度达到每100μL中含1×106个细胞和500ng mRNA(Cas9 mRNA和TRAC-sg3 mRNA各250ng)。利用电转仪(NEPA21)将mRNA导入细胞中。每天观察细胞的生长状况，每隔一天进行换液。细胞培养11-12d后，对所得的T细胞进行质量检测。并在第12-14d对T细胞进行纯化，具体方法见步骤5，最后将终产品(即，通用型CAR-T细胞，在本文中简称为TCR-CAR-T细胞)进行分装和冻存。
5.TCR阴性、CD4和CD8阳性的T细胞的筛选：利用流式分选技术或者免疫磁珠技术筛选出TCR阴性同时CD4或者CD8阳性的T细胞。
1)第一步，首先利用偶联CD3抗体的免疫磁珠进行负分选，除掉T细胞中仍表达CD3(TCR)的细胞(通常进行2次负分选)；
2)第二步，取小量第一步的T细胞，进行流式检测，同时染色CD3、CD4和CD8，如果CD3阳性率＜0.01％，同时CD4+CD8阳性率＞90％，即可进行下一步工作。在本实施例中，CD3阳性率为0％，同时CD4+CD8阳性率为93.3％(图5)。
实施例2：通用型CAR-T(TCR-CAR-T细胞)有效性验证
观察实施例1得到的通用型CAR-T细胞(即，效应细胞)对B细胞型急性淋巴细胞白血病的治疗作用。
细胞生物学验证：
具体实验步骤如下：
第一步：使用Calcein-AM标记靶细胞(即，来自B细胞型急性淋巴细胞白血病患者的细胞)
1)将Calcein-AM(Life Technology，上海)用DMSO稀释成1mg/mL；
2)将靶细胞用全培养基重悬成1×106个/mL的密度；
3)加入15μM的Calcein-AM，37℃、5％CO2培养30min，每10min轻轻混匀；
4)以1500rpm离心5分钟，去上清，用全培养基重悬，重复操作两遍；
第二步：利用效应细胞对靶细胞进行杀伤
1)将标记好的靶细胞按照5000-50000个/mL的密度重悬，取100μL加入到96孔板 中；
2)按照适当的ET(效靶比)比(5:1)加入100μL效应细胞，同时以T细胞和一般外周血CAR-T(即，WT CAR-T细胞)作为对照细胞，每组3个平行，检测上清的荧光强度(test release)；设计单独的A组(6个平行)，只有靶细胞，检测其自发凋亡裂解的荧光强度(spontaneous release)；设计单独的B组(6个平行)，只有靶细胞+2％Triton X-100，检测其最大裂解的荧光强度(maximum release)；
3)37℃、5％CO2培养4h后，离心，取75μL上清，转移到一个新的培养板上；
4)样品利用spectramax Gemini dual-scanning microplate spectrofluorimeter检测(excitation filter:485±9nm；band-pass filter:530±9nm)；按以下公式计算细胞裂解的百分比：[(test release-spontaneous release)/(maximum release–spontaneous release)]*100。
所得结果如图6所示，可以看出，通用型CAR-T杀伤能力与WT CAR-T杀伤能力基本一致。
动物实验验证：
一、淋巴瘤小鼠模型的构建：
1.细胞系：人淋巴瘤细胞系Daudi；
Daudi细胞是人淋巴瘤细胞系，可以通过皮下注射的方式构建小鼠的人淋巴瘤模型。其CD19表达为阳性，可以作为CAR-T细胞的靶细胞。
2.Daudi细胞培养
Daudi细胞系为悬浮细胞系，在含有20％FBS的1640培养基(Gibco)中可以快速生长。细胞密度为2-3×106/mL时需要传代。传代时取细胞悬液于离心管中，以500g离心5分钟，弃上清。将细胞密度调整到0.3-0.5×106/mL，继续培养。正常生长情况下，Daudi细胞系为隔天传代，细胞密度维持在0.3-3×106/mL之间即可。
3.细胞系接种
用生理盐水重悬Daudi细胞，调整其活细胞浓度为3×108个/mL，在冰上将其与Matrigel(BD，中国)按照2：1的体积比充分混匀。通过皮下注射的方式进行接种。
以成功长出100mm3的肿瘤作为小鼠淋巴瘤模型构建成功的判定标准。其中肿瘤体积计算公式为：肿瘤体积(mm3)＝肿瘤长径(mm)×肿瘤短径2(mm2)×0.5；
4.小鼠淋巴瘤模型给药。记录给药当天为D0。通过尾静脉注射的方式进行细胞输注(PBS 200μL、人T细胞200μL(总计1×106/只)、人WT CAR-T细胞200μL(总计1×106/只)、人TCR-CAR-T细胞200μL(总计1×106/只))，所有小鼠均单次给药。结果如 图7所示，可以看出，本发明提供的TCR-CAR-T细胞具有很好的抑制以及杀死肿瘤细胞的效果。
实施例3：通用型CAR-T(TCR-CAR-T细胞)安全性验证
小鼠GVHD模型
6-8周龄的C57雄性小鼠30只(24小时前给予2.4Gy全身照射)，单次尾静脉注射小鼠T细胞、人WT CAR-T细胞和人TCR-CAR-T细胞各1×107个/mL(每组随机分配10只，阴性对照组注射同体积PBS)，注射细胞4小时后开始详细临床观察(死亡率、临床症状、行为、体重变化)，以后每天观察一次，连续观察60天，以评估GVHD的发生与否及其严重性。
通过表2可以看出注射正常异种CAR-T细胞的组中的小鼠全部发生严重GVHD反应，并有70％小鼠死亡。而TCR敲除后的CAR-T细胞对机体几乎不发生GVHD，说明TCR敲除后CAR-T细胞安全性较高。
图8示出了小鼠平均体重的变化，注射正常异种CAR-T细胞组中的小鼠体重明显下降，最多下降了30％，而未注射CAR-T细胞的组和注射TCR敲除CAR-T细胞的组中的小鼠体重没有明显下降，相反有些许上升现象。综合所有数据，表明TCR敲除后的CAR-T细胞的安全性高。
表2：小鼠异常统计表
Figure PCTCN2016101986-appb-000003

Claims (10)

  1. 一种通用型CAR-T细胞，其表达特异性嵌合抗原受体(CAR)并且不表达T细胞受体(TCR)。
  2. 权利要求1的通用型CAR-T细胞，其中所述细胞来源于健康志愿者(或供者)。
  3. 权利要求1或2的通用型CAR-T细胞，其中所述TCR的编码基因被敲除；优选地，所述TCR的a链和/或β链恒定编码区(即TRAC和/或TRBC)基因被敲除；更优选地，所述TCR的a链和/或β链恒定编码区基因被引入所述细胞的TRAC-sg1-7分子之一和/或TRBC-sg1-7分子之一以及Cas9分子敲除；最优选地，所述TCR的a链和/或β链恒定编码区基因被引入细胞的TRAC-sg3分子和/或TRBC-sg1分子以及Cas9分子敲除。
  4. 权利要求1-3中任一项的通用型CAR-T细胞，其中所述CAR包含顺序连接的信号肽、胞外结合区、任选的铰链区、跨膜区和胞内信号区；优选地，所述信号肽的核苷酸序列如发明专利申请201510324558.X的序列表中SEQ ID No:1或SEQ ID No:2所示；优选地，所述胞外结合区的核苷酸序列如发明专利申请201510324558.X的序列表中SEQ ID No:4或SEQ ID No:5所示；优选地，所述铰链区的核苷酸序列如发明专利申请201510324558.X的序列表中SEQ ID No:6或SEQ ID No:7所示；优选地，所述跨膜区的核苷酸序列如发明专利申请201510324558.X的序列表中SEQ ID No:8或SEQ ID No:9所示；优选地，所述胞内信号区的核苷酸序列如发明专利申请201510324558.X的序列表中SEQ ID No:10、SEQ ID No:11或SEQ ID No:12所示。
  5. 权利要求4的通用型CAR-T细胞，其中所述CAR的核苷酸序列如发明专利申请201510324558.X的序列表中的SEQ ID No:13、SEQ ID No:14、SEQ ID No:15、SEQ ID No:16、SEQ ID No:17或SEQ ID No:18所示。
  6. 一种制备通用型CAR-T细胞的方法，所述方法包括如下步骤：
    1)在T细胞中引入sgRNA分子和Cas9分子；
    2)在所述T细胞中引入CAR分子；
    其中，步骤2)可在步骤1)之前、之后或同时进行，并且所述sgRNA分子包含与来自TCR的a链和/或β链恒定编码区(即TRAC和/或TRBC)基因的靶区域互补的靶向结构域。
  7. 权利要求6的方法，所述方法在步骤1)之前还包括分离和/或激活健康志愿者(或供者)的T细胞的步骤；优选地，所述方法在步骤2)之后还包括对通用型CAR-T细胞进行分选的步骤；更优选地，分选之后再对所得的通用型CAR-T细胞进行有效性验证。
  8. 权利要求6或7的方法，其中所述靶向结构域的序列如SEQ ID NOs:1-14中之一所示；优选地，所述靶向结构域的序列如SEQ ID NO:3或SEQ ID NO:8所示；并且其中通过构建载体将所述sgRNA分子和Cas9分子经脂质体转染、电转或慢病毒转染技术引入所述T细胞中；优选地，通过构建载体将所述sgRNA分子和Cas9分子经电转技术引入所述T细胞中；更优选地，通过电转技术将所述sgRNA分子和编码Cas9分子的mRNA引入所述T细胞中；优选地，所述CAR分子通过慢病毒转染技术引入所述T细胞中。
  9. 一种组合物，其包含权利要求1-5中任一项的通用型CAR-T细胞；优选地，所述组合物还包含可药用的稀释剂、赋形剂或载体。
  10. 权利要求1-5中任一项的通用型CAR-T细胞用于制备治疗肿瘤的药物的用途。
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