WO2018205926A1 - 经修饰的t细胞、其制备方法及用途 - Google Patents

经修饰的t细胞、其制备方法及用途 Download PDF

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WO2018205926A1
WO2018205926A1 PCT/CN2018/086019 CN2018086019W WO2018205926A1 WO 2018205926 A1 WO2018205926 A1 WO 2018205926A1 CN 2018086019 W CN2018086019 W CN 2018086019W WO 2018205926 A1 WO2018205926 A1 WO 2018205926A1
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cells
cancer
cell
car
tim3
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PCT/CN2018/086019
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English (en)
French (fr)
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王皓毅
张永平
刘晓娟
程晨
张兴颖
李娜
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中国科学院动物研究所
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Priority to CN201880002752.8A priority Critical patent/CN109790518A/zh
Publication of WO2018205926A1 publication Critical patent/WO2018205926A1/zh

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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
    • A61K39/4644—Cancer antigens
    • A61K39/464466—Adhesion molecules, e.g. NRCAM, EpCAM or cadherins
    • A61K39/464468—Mesothelin [MSLN]
    • 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
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • 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
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
    • 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
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/55—Lung

Definitions

  • the present invention relates to the field of gene editing and tumor immunotherapy.
  • the invention relates to methods of making modified T cells, such as CAR-T cells, by gene editing, as well as modified T cells prepared by the methods and uses thereof.
  • T cells play an important role in anti-tumor immunity.
  • CTL cytotoxic T lymphocytes
  • T cells Adoptive transfer of T cells is a specific, low-toxicity anti-tumor method that has received high attention in recent years, including, for example, genetic modification of T cells with T cell receptor (TCR) or chimeric antigen receptor (CAR).
  • TCR T cell receptor
  • CAR chimeric antigen receptor
  • TCR gene transfer technology is to clone the ⁇ and ⁇ chains of TCR from tumor-reactive T cells, and use antigen-engineered technology to use retrovirus or lentivirus as a vector to perform antigen-specific TCR modification on the initial T cells, thereby conferring T cells. It specifically recognizes and kills tumor cells and increases the affinity of T cells to tumors.
  • the TCR gene was modified by TCR gene transfer technology, and TCR gene was modified by in vitro expansion, and a large number of T cells with specific and high-efficiency recognition ability were obtained, which were then reinfused to tumor patients to exert anti-tumor effect in vivo.
  • CAR consists of an extracellular domain, a hinge, a transmembrane domain, and an intracellular domain, typically derived from a single-chain variable fragment (scFv), which has one, two or Three (corresponding to the first, second, and third generations of CAR, respectively) are derived from the CD3Z and/or costimulatory molecule signaling domains (Kakarla and Gottschalk, 2014).
  • scFv single-chain variable fragment
  • CD3Z and/or costimulatory molecule signaling domains Kakarla and Gottschalk, 2014.
  • T cells that are effective in inhibiting or killing tumors, especially solid tumors.
  • the invention provides a method of making a modified T cell comprising the step of reducing or eliminating expression of an inhibitory protein in a T cell.
  • the T cell is a T cell comprising an exogenous T cell receptor (TCR) or a chimeric antigen receptor (CAR).
  • TCR exogenous T cell receptor
  • CAR chimeric antigen receptor
  • the inhibitory protein whose expression is reduced or eliminated is selected from the group consisting of PD1, LAG-3, CTLA-4, Foxp3, Tim3, and combinations thereof.
  • the inhibitory protein whose expression is reduced or eliminated is selected from the group consisting of a combination of PD1 and TIM3, a combination of PD1 and CTLA-4, a combination of PD1 and LAG3, a combination of CTLA-4 and TIM3, CTLA-4 Combination with LAG3, combination of TIM3 and LAG3, combination of PD1, TIM3 and CTLA-4, combination of PD1, CTLA-4 and LAG3, combination of CTLA-4, TIM3 and LAG3, combination of PD1, TIM3 and LAG3, or A combination of PD1, CTLA-4, TIM3, and LAG3.
  • the reduction or elimination is performed by antisense RNA, antagomir, siRNA, shRNA, meganuclease, zinc finger nuclease, transcription activator-like effector nuclease, or CRISPR system.
  • the CRISPR system is a CRISPR/Cas9 system.
  • the CRISPR/Cas9 system targets a nucleotide sequence within the cell selected from one or more of SEQ ID NOs: 5, 6, 13, 17, 22-26.
  • the TCR or CAR comprises an antigen binding domain directed against a tumor associated antigen.
  • the tumor associated antigen is selected from the group consisting of CD16, CD64, CD78, CD96, CLL1, CD116, CD117, CD71, CD45, CD71, CD123, CD138, ErbB2 (HER2/neu), Carcinoembryonic antigen (CEA) , epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), CD19, CD20, CD30, CD40, disial acid ganglioside GD2, ductal epithelial mucin, gp36 , TAG-72, glycosphingolipid, glioma-associated antigen, ⁇ -human chorionic gonadotropin, ⁇ -fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut
  • the antigen binding domain is selected from the group consisting of a monoclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, a single domain antibody, an antibody single chain variable region, and an antigen binding fragment thereof.
  • the CAR comprises a scFv (P4) against a mesothelin, a CD8 hinge region, a CD28 transmembrane domain, a CD28 costimulatory domain, and a CD3 ⁇ signal transduction domain.
  • P4 scFv
  • the CAR comprises the amino acid sequence set forth in SEQ ID NO:32.
  • the invention provides a modified T cell prepared by the method of the invention.
  • the invention provides a modified T cell wherein expression of an inhibitory protein in the T cell is reduced or eliminated compared to unmodified T cells.
  • the T cell is a T cell comprising an exogenous T cell receptor (TCR) or a chimeric antigen receptor (CAR).
  • TCR exogenous T cell receptor
  • CAR chimeric antigen receptor
  • the inhibitory protein whose expression is reduced or eliminated is selected from the group consisting of PD1, LAG-3, CTLA-4, Foxp3, Tim3, and combinations thereof.
  • the inhibitory protein whose expression is reduced or eliminated is selected from the group consisting of a combination of PD1 and TIM3, a combination of PD1 and CTLA-4, a combination of PD1 and LAG3, a combination of CTLA-4 and TIM3, CTLA-4 Combination with LAG3, combination of TIM3 and LAG3, combination of PD1, TIM3 and CTLA-4, combination of PD1, CTLA-4 and LAG3, combination of CTLA-4, TIM3 and LAG3, combination of PD1, TIM3 and LAG3, or A combination of PD1, CTLA-4, TIM3, and LAG3.
  • the TCR or CAR comprises an antigen binding domain directed against a tumor associated antigen.
  • the tumor associated antigen is selected from the group consisting of CD16, CD64, CD78, CD96, CLL1, CD116, CD117, CD71, CD45, CD71, CD123, CD138, ErbB2 (HER2/neu), Carcinoembryonic antigen (CEA) , epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), CD19, CD20, CD30, CD40, disial acid ganglioside GD2, ductal epithelial mucin, gp36 , TAG-72, glycosphingolipid, glioma-associated antigen, ⁇ -human chorionic gonadotropin, ⁇ -fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut
  • the antigen binding domain is selected from the group consisting of a monoclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, a single domain antibody, an antibody single chain variable region, and an antigen binding fragment thereof.
  • the CAR comprises a scFv (P4) against a mesothelin, a CD8 hinge region, a CD28 transmembrane domain, a CD28 costimulatory domain, and a CD3 ⁇ signal transduction domain.
  • P4 scFv
  • the CAR comprises the amino acid sequence set forth in SEQ ID NO:32.
  • the invention provides the use of a modified T cell of the invention in the manufacture of a medicament for the treatment of cancer.
  • the invention provides a pharmaceutical composition for treating cancer comprising a modified T cell of the invention and a pharmaceutically acceptable carrier.
  • the cancer is selected from the group consisting of lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma, kidney cancer, bladder cancer, breast cancer, liver cancer, lymphoma, hematological malignancy, head and neck cancer, Glioma, gastric cancer, nasopharyngeal carcinoma, laryngeal cancer, cervical cancer, uterine tumor and osteosarcoma.
  • cancers examples include: bone cancer, pancreatic cancer, skin cancer, prostate cancer, skin or intraocular malignant melanoma, uterine cancer, anal cancer, testicular cancer, fallopian tube cancer , endometrial cancer, vaginal cancer, vulva cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid carcinoma, adrenal cancer, soft tissue sarcoma, urethra Cancer, penile cancer, chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia), solid tumors of children, lymphocytic lymphoma, bladder cancer, Renal or ureteral cancer, renal pelvic cancer, central nervous system (CNS) tumor, primary CNS lymphoma, tumor angiogenesis,
  • CNS central nervous system
  • the invention also provides a kit for making modified T cells by the methods of the invention.
  • Figure 1 shows the design and screening of sgRNA targeting LAG-3.
  • FIG. 2 shows the effect of knockout LAG-3 on T cells, where T-CTRL-UE and T-CTRL-E represent T cells that have not been subjected to or undergo electroporation, while LAG-3-KO represents RNP-treated T cells.
  • Figure 3 shows the results of knocking out LAG-3 in CAR-T cells.
  • Figure 4 shows the in vitro evaluation of the effect of knockout LAG-3 on CAR-T cells, where CAR-T-CTRL-UE and CAR-T-CTRL-E represent cells that have not been subjected to or undergo electroporation, whereas LAG-3-KO -CAR-T represents the cells treated by RNP.
  • Figure 5 shows the in vivo anti-tumor effect of LAG-3 knockout CAR-T cells.
  • Figure 6 shows the design and screening of sgRNA targeting CTLA-4.
  • FIG. 7 shows the effect of knockout CTLA-4 on T cells, where T-CTRL-UE and T-CTRL-E represent T cells that have not been subjected to or undergo electroporation, while CTLA-4-KO represents RNP-treated T cells.
  • Figure 8 shows the results of knocking out CTLA-4 in CAR-T cells.
  • Figure 9 shows the in vitro evaluation of the effect of knockout CTLA-4 on CAR-T cells, where CAR-T-CTRL-UE and CAR-T-CTRL-E represent cells that have not been subjected to or undergo electroporation, while CTLA-4-KO -CAR-T represents the cells treated by RNP.
  • Figure 10 shows the in vivo anti-tumor effect of CAR-T cells knocked out of CTLA-4.
  • Figure 11 shows the design and screening of sgRNA targeting Foxp3.
  • FIG 12 shows the effect of knockout of Foxp3 on T cells, wherein T-CTRL-UE and T-CTRL-E represent T cells that have not been subjected to or undergo electroporation, while Foxp3-KO represents RNP-treated T cells.
  • Figure 13 shows the results of knocking out Foxp3 in CAR-T cells.
  • Figure 14 shows the in vitro evaluation of the effect of knockout of Foxp3 on CAR-T cells, where CAR-T-CTRL-UE and CAR-T-CTRL-E represent cells that have not been subjected to or undergo electroporation, whereas Foxp3-KO-CAR-T Represents cells treated by RNP.
  • Figure 15 shows the in vivo anti-tumor effect of Foxp3 knockout CAR-T cells.
  • Figure 16 shows the design and screening of sgRNA targeting Tim3.
  • FIG 17 shows the effect of knockout of Tim3 on T cells, where T-CTRL-UE and T-CTRL-E represent T cells that have not been subjected to or undergo electroporation, while Tim3-KO represents RNP-treated T cells.
  • Figure 18 shows the effect of knockout of Tim3 on CAR-T cells, where CAR-T-CTRL-UE and CAR-T-CTRL-E represent cells that have not been subjected to or undergo electroporation, while Tim-3-KO-CAR-T Represents cells treated by RNP.
  • Figure 19 shows knockout of PD1 in CAR-T cells.
  • Figure 20 shows that PD1-knockout anti-Meso CART cells are capable of performing effector functions in vivo.
  • Figure 21 shows that PD1-knockout anti-Meso CART cells are capable of performing effector functions in vitro.
  • Figure 22 shows the different CAR structures.
  • Figure 23 shows the effect of CAR-T cells with different CAR structures.
  • Figure 24 shows the knockdown efficiency of detecting PD-1, LAG-3 and/or TIM3 genes.
  • Figure 25 shows the effect of PD-1, LAG-3 and/or TIM3 knockout P4 CAR-T cells on target cells (H226-PDL1-luci).
  • Figure 26 shows the effect of PD-1, LAG-3 and/or TIM3 knockout P4 CAR-T cells on target cells (CRL5826-PDL1) with a high target ratio (4:1).
  • Figure 27 shows the effect of PD-1, LAG-3 and/or TIM3 knockout P4 CAR-T cell high-efficiency targets on target cells (CRL5826-PDL1), inefficient target ratio (0.1:1).
  • Figure 28 shows the effect of P4 CAR-T cells showing PD-1, LAG-3 and/or TIM3 knockout on target cells (CRL5826-PDL1), inefficient target ratio (0.02:1).
  • Figure 29 shows the effect of one or more of CAR-T cells knocked out of PD-1, TIM3, CTLA4 and LAG3 on target cells (CRL5826) with a target ratio of 1:1.
  • Figure 31 shows the effect of CAR-T cells knocking out one or more of PD-1, TIM3, CTLA4 and LAG3 on target cells (OVCAR3 or OVCAR3-PDL1) with a target ratio of 1:1.
  • Figure 32 shows the effect of one or more of CAR-T cells knocked out of PD-1, TIM3, CTLA4 and LAG3 on target cells (HCT116 or HCT116-PDL1) after 24 hours of culture, with a target ratio of 1:1.
  • Figure 33 shows the effect of one or more of CAR-T cells knocked out of PD-1, TIM3, CTLA4 and LAG3 on target cells (HCT116 or HCT116-PDL1) after 48 hours of culture, with a target ratio of 1:1.
  • Figure 34 shows the effect of knocking out one or more of PD-1, TIM3, CTLA4 and LAG3 of CAR-T cells against target cells (CRL5826 or CRL5826-PDL1) after 4 days of culture with a target ratio of 0.1:1.
  • Figure 35 shows the effect of one or more of CAR-T cells knocked out of PD-1, TIM3, CTLA4 and LAG3 on target cells (OVCAR3 or OVCAR3-PDL1) after 48 hours of culture, with a target ratio of 0.1:1.
  • Exogenous with respect to a sequence means a sequence from a foreign species, or, if from the same species, a sequence that has undergone a significant change in composition and/or locus from its native form by deliberate human intervention.
  • nucleic acid sequence is used interchangeably and are single- or double-stranded RNA or DNA polymers, optionally containing synthetic, non-natural Or altered nucleotide bases.
  • Nucleotides are referred to by their single letter designation: "A” is adenosine or deoxyadenosine (corresponding to RNA or DNA, respectively), “C” means cytidine or deoxycytidine, and “G” means guanosine or Deoxyguanosine, “U” means uridine, “T” means deoxythymidine, “R” means ⁇ (A or G), “Y” means pyrimidine (C or T), “K” means G or T, “ H” represents A or C or T, “I” represents inosine, and “N” represents any nucleotide.
  • Polypeptide “peptide”, and “protein” are used interchangeably herein and refer to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • polypeptide “peptide”, “amino acid sequence” and “protein” may also include modified forms including, but not limited to, glycosylation, lipid linking, sulfation, gamma carboxylation of glutamic acid residues, hydroxy And ADP-ribosylation.
  • expression construct refers to a vector, such as a recombinant vector, suitable for expression of a nucleotide sequence of interest in a cell. "Expression” refers to the production of a functional product.
  • expression of a nucleotide sequence can refer to transcription of a nucleotide sequence (eg, transcription to produce mRNA or functional RNA) and/or translation of RNA into a precursor or mature protein.
  • An "expression construct" of the invention can be a linear nucleic acid fragment, a circular plasmid, a viral vector, or, in some embodiments, can be a translatable RNA (such as an mRNA).
  • An "expression construct" of the invention may comprise regulatory sequences of different origin and nucleotide sequences of interest, or regulatory sequences of the same origin but arranged in a manner different from that normally found in nature, and nucleotide sequences of interest.
  • regulatory sequences and “regulatory elements” are used interchangeably and refer to either upstream (5' non-coding sequences), intermediate or downstream (3' non-coding sequences) of a coding sequence, and affect transcription or RNA processing of related coding sequences or Stability or translated nucleotide sequence.
  • An expression control element refers to a nucleotide sequence capable of controlling transcription, RNA processing or stability or translation of a nucleotide sequence of interest.
  • Regulatory sequences can include, but are not limited to, promoters, translation leader sequences, introns, enhancers, and polyadenylation recognition sequences.
  • Promoter refers to a nucleic acid fragment that is capable of controlling the transcription of another nucleic acid fragment.
  • a promoter is a promoter capable of controlling the transcription of a gene in a cell, whether or not it is derived from the cell.
  • operably linked refers to a regulatory element (such as, but not limited to, a promoter sequence, a transcription termination sequence, etc.) linked to a nucleic acid sequence (eg, a coding sequence or an open reading frame) such that the nucleotide Transcription of the sequence is controlled and regulated by the transcriptional regulatory elements.
  • a regulatory element such as, but not limited to, a promoter sequence, a transcription termination sequence, etc.
  • nucleic acid sequence eg, a coding sequence or an open reading frame
  • Gene editing also known as genome editing, uses engineered nucleases or “molecular scissors” to perform DNA insertions, deletions, or substitutions in the genome of an organism. Gene editing results in a site-specific double-strand break (DSB) by the desired position in the genome, and then introduces the desired DNA insertion, deletion or substitution during repair of the DSB. Gene editing typically uses meganucleases, zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALENs), and CRISPR systems.
  • ZFN zinc finger nucleases
  • TALENs transcription activator-like effector nucleases
  • “meganucleases” are a class of deoxyribonuclease enzymes with large recognition sites (12-40 bp double-stranded DNA sequences) whose recognition sites usually occur only once in any given genome. For example, the 18 bp sequence recognized by the I-SceI meganuclease needs to occur on average in the genome 20 times larger than the human genome.
  • a "zinc finger nuclease” is an artificial restriction enzyme prepared by fusing a zinc finger DNA binding domain to a DNA cleavage domain.
  • the zinc finger DNA binding domain of a single ZFN typically contains 3-6 individual zinc finger repeats, each of which can recognize, for example, 3 bp.
  • a "transcriptional activator-like effector nuclease” is a restriction enzyme that can be engineered to cleave a specific DNA sequence, typically by fusing the DNA-binding domain of a transcriptional activator-like effector (TALE) to a DNA cleavage domain. preparation. TALE can be engineered to bind almost any desired DNA sequence.
  • CRISPR Clustering regularly spaced short palindromic repeats
  • the CRISPR system is a prokaryotic immune system that confers resistance to foreign genetic elements such as those present in plasmids and phage that provide acquired immunity.
  • Cas proteins or similar proteins cleave foreign nucleic acids under the guidance of RNA.
  • CRISPR nuclease generally refers to a nuclease present in a naturally occurring CRISPR system, as well as modified forms thereof, variants thereof (including nickase mutants), or catalytically active fragments thereof.
  • the CRISPR nuclease can recognize and/or cleave the target nucleic acid structure by interacting with a guide RNA such as a crRNA and optionally a tracrRNA or an artificial gRNA (e.g., sgRNA).
  • a guide RNA such as a crRNA and optionally a tracrRNA or an artificial gRNA (e.g., sgRNA).
  • gRNA e.g., sgRNA
  • Cas9 Nuclease and “Cas9” are used interchangeably herein and refer to RNA comprising a Cas9 protein or a fragment thereof (eg, a protein comprising an active DNA cleavage domain of Cas9 and/or a gRNA binding domain of Cas9) Guided nuclease.
  • Cas9 is a component of the CRISPR/Cas (clustered regularly spaced short palindromic repeats and their associated systems) genome editing system that targets and cleaves DNA target sequences under the guidance of a guide RNA to form DNA double-strand breaks (DSB) ).
  • RNA and gRNA are used interchangeably herein and generally consist of a crRNA and tracrRNA molecule that partially complements to form a complex, wherein the crRNA comprises sufficient complementarity to the target sequence to hybridize to the target sequence and direct the CRISPR complex a sequence (Cas9+crRNA+tracrRNA) that specifically binds to the sequence of the target sequence.
  • sgRNA single-guide RNA
  • T cell receptor also known as T cell antigen receptor
  • T cell antigen receptor is a molecular structure specifically recognized by T cells and binds to antigen peptide-MHC molecules, and is usually present on the surface of T cells in a complex form with CD3 molecules.
  • the TCR of most T cells consists of alpha and beta peptide chains, and the TCR of a few T cells consists of gamma and delta peptide chains.
  • CAR Chimeric antigen receptor
  • chimeric T cell receptor chimeric immune receptor
  • chimeric immune receptor is an artificially designed receptor that can confer immune effector cells Specificity. In general, this technique is used to confer specific characteristics to T cell-specific recognition of tumor surface antigens. In this way, a large number of targeted killing tumor cells can be produced.
  • Subject refers to an organism having or susceptible to a disease (eg, cancer) that can be treated by the methods, or pharmaceutical compositions of the invention.
  • a disease eg, cancer
  • Non-limiting examples include humans, cows, rats, mice, dogs, monkeys, goats, sheep, cows, deer, and other non-mammals.
  • the subject is a human.
  • the invention provides a method of making a modified T cell comprising the step of reducing or eliminating expression of an inhibitory protein in a T cell.
  • the T cell of the present invention may be a T cell for inheritance immunotherapy produced by amplifying antigen-specific T cells or by genetically engineering to redirect T cells.
  • the T cell can also be a primary T cell isolated from a subject.
  • the T cell is a T cell comprising an exogenous T cell receptor (TCR).
  • T cell is a T cell comprising a chimeric antigen receptor (CAR).
  • the method further comprises the steps of providing unmodified T cells isolated from the subject, and introducing a TCR or CAR to the unmodified T cells.
  • the step of introducing a TCR or CAR into the unmodified T cell is performed before or after or simultaneously with the step of reducing or eliminating expression of inhibitory proteins in T cells.
  • the TCR or CAR comprises an antigen binding domain directed against a tumor associated antigen, such as an extracellular antigen binding domain.
  • the tumor associated antigens include, but are not limited to, CD16, CD64, CD78, CD96, CLL1, CD116, CD117, CD71, CD45, CD71, CD123, CD138, ErbB2 (HER2/neu), carcinoembryonic antigen (CEA), epithelial cell adhesion Molecular (EpCAM), epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), CD19, CD20, CD30, CD40, disialoganglioside GD2, ductal epithelial mucin, gp36, TAG-72 , glycosphingolipid, glioma-associated antigen, ⁇ -human chorionic gonadotropin, alpha fetoglobulin (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX Human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70
  • the antigen-binding domain may be, for example, a monoclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, a single domain antibody, an antibody single-chain variable region, and an antigen-binding fragment thereof.
  • the antigen binding domain is a monoclonal antibody directed against mesothelin. In a preferred embodiment, the antigen binding domain is a scFv against mesothelin. In a preferred embodiment, the antigen binding domain is a scFv (P4) directed against mesothelin, for example, the amino acid sequence thereof is set forth in SEQ ID NO:27.
  • the CAR comprises a transmembrane domain, such as a CD8 transmembrane domain or a CD28 transmembrane domain, preferably a CD28 transmembrane domain, eg, a CD28 transmembrane structure having the amino acid sequence set forth in SEQ ID NO:28 area.
  • the CAR comprises a signal transduction domain useful for T cell activation, eg, selected from the group consisting of TCR ⁇ , FcR ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, and CD66d Signal transduction domain.
  • the CAR comprises a CD3 ⁇ signal transduction domain, for example, the amino acid sequence is shown in the CD3 ⁇ signal transduction domain of SEQ ID NO:30.
  • the CAR may further comprise a reporter molecule, such as a GFP protein, for displaying or tracking CAR expression.
  • a reporter molecule such as a GFP protein
  • the CAR comprises a scFv P4, a CD8 hinge region, a CD28 transmembrane domain, a CD28 costimulatory domain, a CD3 ⁇ signal transduction domain, and an optional GFP protein for mesothelin.
  • the CAR comprises the amino acid sequence set forth in SEQ ID NO:32.
  • a T cell "inhibitory protein” refers to a protein associated with inhibition of T cell activity, such as an immunosuppressive protein.
  • the inhibitory protein is selected from the group consisting of PD1, LAG-3, CTLA-4, Foxp3, Tim3, and any combination thereof.
  • the inhibitory protein is selected from the group consisting of a combination of PD1 and TIM3, a combination of PD1 and CTLA-4, a combination of PD1 and LAG3, a combination of CTLA-4 and TIM3, a combination of CTLA-4 and LAG3, Combination of TIM3 and LAG3, combination of PD1, TIM3 and CTLA-4, combination of PD1, CTLA-4 and LAG3, combination of CTLA-4, TIM3 and LAG3, combination of PD1, TIM3 and LAG3, or PD1, CTLA-4 , a combination of TIM3 and LAG3.
  • reducing or eliminating the expression of inhibitory proteins in T cells does not affect the ability of T cells to expand and their basic immune properties, and can enhance their biological activities by de-immunosuppressive inhibition, such as anti-tumor activity, especially It is the activity of inhibiting or killing solid tumor cells.
  • the expression of an inhibitory protein in a T cell is reduced or eliminated by antisense RNA, antagomir, siRNA, shRNA.
  • the expression of inhibitory proteins in T cells is reduced or eliminated by methods of gene editing, such as by meganucleases, zinc finger nucleases, transcription activator-like effector nucleases, or CRISPR systems.
  • the CRISPR system is used to reduce or eliminate the expression of inhibitory proteins in T cells.
  • the nuclease (CRISPR nuclease) used in the CRISPR system can be selected, for example, from Cas3, Cas8a, Cas5, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, GSU0054, Cas10, Csm2, Cmr5. , Cas10, Csx11, Csx10, Csf1, Cas9, Csn2, Cas4, Cpf1, C2c1, C2c3 or C2c2 proteins, or functional variants of these nucleases.
  • the CRISPR system is a CRISPR/Cas9 system.
  • the CRISPR system eg, a CRISPR/Cas9 system, targets one or more of the cells selected from the group consisting of SEQ ID NOs: 5, 6, 13, 17, 22, 23, 24, 25, Nucleotide sequence.
  • the CRISPR system is assembled in vitro and transferred to T cells.
  • an expression construct encoding all of the elements of the CRIPSR system is transformed into a T cell.
  • expression constructs encoding portions of the CRIPSR system, as well as other transcribed or translated elements, are transferred into T cells.
  • the present invention also provides a CRISPR gene editing system for preparing modified T cells, comprising at least one of the following i) to v):
  • Ii an expression construct comprising a nucleotide sequence encoding a CRISPR nuclease, and at least one guide RNA;
  • a CRISPR nuclease a CRISPR nuclease, and an expression construct comprising a nucleotide sequence encoding at least one guide RNA;
  • v) an expression construct comprising a nucleotide sequence encoding a CRISPR nuclease and a nucleotide sequence encoding at least one guide RNA;
  • the at least one guide RNA targets an inhibitory protein encoding gene in T cells such as PD1, LAG-3, CTLA-4, Foxp3 and/or Tim3.
  • the at least one guide RNA targets PD1 and TIM3 in T cells. In some specific embodiments, the at least one guide RNA targets PD1 and CTLA-4 in T cells. In some specific embodiments, the at least one guide RNA targets PD1 and LAG3 in T cells. In some specific embodiments, the at least one guide RNA targets CTLA-4 and TIM3 in T cells. In some specific embodiments, the at least one guide RNA targets CTLA-4 and LAG3 in T cells. In some specific embodiments, the at least one guide RNA targets TIM3 and LAG3 in T cells. In some specific embodiments, the at least one guide RNA targets PD1, TIM3, and CTLA-4 in T cells.
  • the at least one guide RNA targets PD1, CTLA-4, and LAG3 in T cells. In some specific embodiments, the at least one guide RNA targets CTLA-4, TIM3, and LAG3 in T cells. In some specific embodiments, the at least one guide RNA targets PD1, TIM3, and LAG3 in T cells. In some specific embodiments, the at least one guide RNA targets PD1, CTLA-4, TIM3, and LAG3 in T cells.
  • the guide RNA targets a nucleotide sequence of LAG-3 gene selected from the group consisting of SEQ ID NOs: 1-5. In some embodiments, the guide RNA targets a nucleotide sequence of the CTLA-4 gene selected from the group consisting of SEQ ID NOs: 6-10. In some embodiments, the guide RNA targets a nucleotide sequence of the Foxp3 gene selected from the group consisting of SEQ ID NOs: 11-16. In some embodiments, the guide RNA targets a nucleotide sequence of the Tim3 gene selected from the group consisting of SEQ ID NOs: 17-21.
  • the introduction of the CRISPR gene editing system of the invention into the T cell is included.
  • the CRISPR gene editing system of the invention after introduction into the T cell, results in a reduction or elimination of expression (knockdown or knockout) of the targeted protein.
  • the CRISPR system of the invention can be transformed into T cells by methods known in the art, such as: calcium phosphate transfection, protoplast fusion, electroporation, lipofection, microinjection, viral infection (eg baculovirus, vaccinia virus) , adenoviruses and other viruses).
  • methods known in the art such as: calcium phosphate transfection, protoplast fusion, electroporation, lipofection, microinjection, viral infection (eg baculovirus, vaccinia virus) , adenoviruses and other viruses).
  • T cells of the invention can be activated and expanded before or after any modification step.
  • T cells can be expanded in vitro or in vivo.
  • T cells of the invention can be expanded, for example, by contact with an agent that stimulates a CD3 TCR complex and a costimulatory molecule on the surface of a T cell to produce a T cell activation signal.
  • an agent that stimulates a CD3 TCR complex and a costimulatory molecule on the surface of a T cell to produce a T cell activation signal.
  • a chemical such as calcium ionophore A23187, phorbol 12-myristate 13-acetate (PMA), or a mitotic lectin such as phytohemagglutinin (PHA) can be used to generate T cell activation signals. .
  • a population of T cells can be activated by contact with, for example, an anti-CD3 antibody or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by a protein kinase C activator (eg, moss)
  • the enzyme is activated by contact with a calcium ionophore.
  • a T cell population can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions suitable for stimulating T cell proliferation.
  • Conditions suitable for T cell culture include suitable media (eg, Minimal Essential Media or RPMI Media 1640, or X-vivo 5, (Lonza)) that may contain factors necessary for proliferation and viability, wherein the necessary factors include serum (eg, Fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN- ⁇ , IL-4, IL-7, GM-CSF, IL-10, IL-2, IL-15, TGFp and TNF, Or additives for cell growth known to those skilled in the art.
  • Other additives for cell growth include, but are not limited to, surfactants, human plasma protein powder, and reducing agents such as N-acetyl-cysteine and 2-mercaptoacetic acid.
  • the medium may include RPMI 1640, A1M-V, DMEM, MEM, a-MEM, F-12, X-Vivo 1 and X-Vivo 20, Optimizer, amino acids, sodium pyruvate and vitamins, serum-free or appropriate supplemented serum. (or plasma) or a defined group of hormones, and / or a certain amount of cytokines sufficient for T cell growth and expansion.
  • the target cells can be maintained under conditions necessary to support growth, such as a suitable temperature (eg, 37 ° C) and an environment (eg, air plus 5% CO 2 ).
  • a modified T cell wherein expression of an inhibitory protein in the modified T cell is reduced or eliminated compared to unmodified T cells.
  • the modified T cells are prepared by the methods of the invention.
  • the expression is reduced or eliminated.
  • the inhibitory protein is selected from the group consisting of PD1, LAG-3, CTLA-4, Foxp3, Tim3, and any combination thereof.
  • the inhibitory protein whose expression is reduced or eliminated is selected from the group consisting of a combination of PD1 and TIM3, a combination of PD1 and CTLA-4, a combination of PD1 and LAG3, a combination of CTLA-4 and TIM3, CTLA- Combination of 4 and LAG3, combination of TIM3 and LAG3, combination of PD1, TIM3 and CTLA-4, combination of PD1, CTLA-4 and LAG3, combination of CTLA-4, TIM3 and LAG3, combination of PD1, TIM3 and LAG3, Or a combination of PD1, CTLA-4, TIM3, and LAG3.
  • the gene encoding the inhibitory protein in the T cell is knocked out, for example by introduction into a gene editing system of the invention.
  • the modified T cell has an amplification ability and similar immunological properties comparable to unmodified T cells, and has enhanced biological activity such as antitumor activity, especially since immunosuppression is released. It is the activity of inhibiting or killing solid tumor cells.
  • the T cell may be a T cell comprising a foreign T cell receptor (TCR), or the T cell may be a T cell (CAR-T cell) comprising a chimeric antigen receptor (CAR).
  • the modified T cell is a CAR-T cell.
  • the TCR or CAR comprises an antigen binding domain directed against a tumor associated antigen, such as an extracellular antigen binding domain.
  • the antigen-binding domain may be, for example, a monoclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, a single domain antibody, an antibody single-chain variable region, and an antigen-binding fragment thereof.
  • the antigen binding domain is a monoclonal antibody directed against mesothelin. In a preferred embodiment, the antigen binding domain is a scFv against mesothelin. In a preferred embodiment, the antigen binding domain is a scFv (P4) directed against mesothelin, the amino acid sequence of which is set forth in SEQ ID NO:27.
  • the CAR comprises a transmembrane domain, such as a CD8 transmembrane domain or a CD28 transmembrane domain, preferably a CD28 transmembrane domain, eg, a CD28 transmembrane structure having the amino acid sequence set forth in SEQ ID NO:28 area.
  • the CAR further comprises a hinge region between the extracellular antigen binding domain and the transmembrane domain, eg, the hinge region is a CD8 hinge region, eg, the amino acid sequence is shown in SEQ ID NO : 29 CD8 hinge area.
  • the CAR comprises a signal transduction domain useful for T cell activation, eg, selected from the group consisting of TCR ⁇ , FcR ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, and CD66d Signal transduction domain.
  • the CAR comprises a CD3 ⁇ signal transduction domain, for example, the amino acid sequence is shown in the CD3 ⁇ signal transduction domain of SEQ ID NO:30.
  • the CAR further comprises one or more costimulatory domains selected from the group consisting of CD3, CD27, CD28, CD83, CD86, CD127, 4-1BB, and 4-1BBL.
  • the CAR comprises a CD28 costimulatory domain, eg, the amino acid sequence is shown in the CD28 costimulatory domain of SEQ ID NO:31.
  • the CAR may further comprise a reporter molecule, such as a GFP protein, for displaying or tracking CAR expression.
  • a reporter molecule such as a GFP protein
  • the CAR comprises a scFv P4, a CD8 hinge region, a CD28 transmembrane domain, a CD28 costimulatory domain, a CD3 ⁇ signal transduction domain, and an optional GFP protein for mesothelin.
  • the CAR comprises the amino acid sequence set forth in SEQ ID NO:32.
  • the modified T cell is a CAR-T cell comprising a CAR of the amino acid sequence of SEQ ID NO: 32, wherein expression of a combination of an inhibitory protein or an inhibitory protein selected from the group consisting of Reduce or eliminate:
  • PD1 PD1, CTLA-4, TIM3, and LAG3.
  • the T cell is derived from an autologous cell of the subject.
  • autologous refers to a cell, cell line or population of cells used to treat a subject from which the subject is derived.
  • the T cell is derived from an allogeneic cell, such as a donor that is compatible with the human leukocyte antigen (HLA) of the subject. Cells from the donor can be converted to non-allogene-reactive cells using standard protocols and replicated as needed to produce cells that can be administered to one or more patients.
  • HLA human leukocyte antigen
  • the CAR T cells or TCR T cells of the invention can be prepared by a variety of means known in the art.
  • T cells can be transduced with an expression construct comprising a CAR or TCR coding sequence to obtain CAR-T cells or TCR-T cells.
  • expression constructs suitable for protein expression such as viral vectors.
  • a pharmaceutical composition for treating cancer comprising a modified T cell of the invention and a pharmaceutically acceptable carrier. Furthermore, the invention also provides the use of a modified T cell of the invention in the manufacture of a medicament for the treatment of cancer.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).
  • a method for treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of a modified T cell of the invention or a pharmaceutical composition of the invention.
  • the method further comprises administering to the subject radiation therapy and/or chemotherapy and/or additional tumor targeting drugs (eg, monoclonal antibodies or small molecule compounds that target other antigens).
  • additional tumor targeting drugs eg, monoclonal antibodies or small molecule compounds that target other antigens.
  • terapéuticaally effective amount or “therapeutically effective dose” or “effective amount” refers to an amount of a substance, compound, material or cell that is at least sufficient to produce a therapeutic effect after administration to a subject. Thus, it is an amount necessary to prevent, cure, ameliorate, block or partially arrest the symptoms of a disease or condition.
  • an "effective amount" of a cell or pharmaceutical composition of the invention preferably results in a decrease in the severity of the symptoms of the disease, an increase in the frequency and duration of the asymptomatic phase of the disease, or prevention of damage or disability caused by the pain of the disease.
  • an "effective amount" of a cell or pharmaceutical composition of the invention preferably inhibits tumor cell growth or tumor growth by at least about 10%, preferably at least about 20%, relative to a subject not receiving treatment.
  • the ability to inhibit tumor growth can be evaluated in animal model systems that predict the efficacy of human tumors. Alternatively, it can also be evaluated by examining the ability to inhibit tumor cell growth, which can be determined in vitro by assays well known to those skilled in the art.
  • the dosage levels of the cells in the pharmaceutical compositions of the invention may be varied to achieve an amount of active ingredient that is effective to achieve the desired therapeutic response to a particular patient, composition, and mode of administration without toxicity to the patient.
  • the selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the particular composition of the invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound employed, the duration of treatment, and the particularity of the application.
  • Other drugs, compounds and/or materials to which the composition is used in combination the age, sex, weight, condition, general health and medical history of the patient being treated, and similar factors well known in the medical arts.
  • the modified T cells of the present invention are capable of achieving superior therapeutic effects at lower doses relative to control T cells (the expression of the inhibitory protein is not reduced or eliminated). This is particularly advantageous in reducing the time and cost of preparation while reducing the side effects associated with high dose administration.
  • the administered T cells of the present invention are administered at a dose that is about 2 times lower, about 3 times lower, about 4 times lower, and lower than about 5 administered doses of control T cells whose expression of the inhibitory protein is not reduced or eliminated.
  • Multiplier about 6 times lower, about 7 times lower, about 8 times lower, about 9 times lower, about 10 times lower, about 15 times lower, about 20 times lower, about 30 times lower, about 40 times lower, about 50 lower
  • the magnification is about 100 times lower, about 150 times lower, and about 200 times lower or lower.
  • Non-limiting examples of cancers that can be treated by the cells or pharmaceutical compositions of the invention include lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma, kidney cancer, bladder cancer, breast cancer, liver cancer, lymphoma, malignant blood.
  • cancers examples include: bone cancer, pancreatic cancer, skin cancer, prostate cancer, skin or intraocular malignant melanoma, uterine cancer, anal cancer, testicular cancer, fallopian tube cancer , endometrial cancer, vaginal cancer, vulva cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid carcinoma, adrenal cancer, soft tissue sarcoma, urethra Cancer, penile cancer, chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia), solid tumors of children, lymphocytic lymphoma, bladder cancer, Renal or ureteral cancer, renal pelvic cancer, central nervous system (CNS) tumor, primary CNS lymphoma, tumor angiogenesis,
  • CNS central nervous system
  • the invention also provides a kit for use in the method of making modified T cells of the invention, the kit comprising a CRISPR gene editing system for preparing modified T cells as described herein, and suitable The gene editing system is introduced into a reagent in the cell.
  • the kit may further comprise reagents for detecting T cells, isolating T cells, activating T cells, and/or expanding T cells.
  • the kit may further comprise an agent for introducing a CAR or TCR into a T cell, and detecting and/or isolating the actual expression of the cell expressing the CAR or TCR.
  • the kit may also contain instructions for carrying out the methods of the invention.
  • a biotin company synthesized a forward primer containing a T7 promoter and a 20 bp target sequence (sgRNA), and then amplified the T7-sgRNA PCR product in vitro using the pX330 plasmid (Addgene plasmid #4223) as a PCR template and purified the PCR using a PCR purification kit. product.
  • the purified T7-sgRNA PCR product was used as a template, and the sgRNA was in vitro transcribed by MEGAshortscript T7 kit (Thermo Fisher Scientific), and the sgRNA was recovered by MEGAclear columns (Thermo Fisher Scientific), and the sgRNA was dissolved in RNase-free deionized water. Save or reserve.
  • Configure electrorotation buffer 100 ul system: nuclepfector solution 82 ⁇ l + 18 ⁇ l supplement;
  • the genomic DNA of the experimental group and the control group was extracted with cell lysate (100 ⁇ g/ml ProteinaseK, 10 mMTris-HCl, 2 mM EDTA, 2.5% Tween-20 and 2.5% Triton-X 100).
  • the target fragment was amplified in vitro by PCR and sequenced using sanger. The gene mutation efficiency was then analyzed using an online tool (http://tide.nki.nl).
  • T cell complete medium X-VIVO15 medium + 5% FBS (heat inactivated) + 2 mM L-glutamine + 1 mM sodium pyruvate + 300 U / ml IL-2. After T cell isolation, adjust the cell density to 1e6 cells/ml with T cell complete medium. Human T-Activator CD3/CD28 is activated in a 1:1 ratio. The cells were then exchanged every 2-3 days according to the T cell growth state.
  • Changes in cell subsets were analyzed by flow cytometry, including CD4, CD8, primary T cells (CD45RO-/CD62L+, TN), central memory T cells (CD45RO+/CD62L+, TCM), and effector memory T cells (CD45RO+/). CD62L-, TEM). Comparison with T cells that were not genetically edited.
  • FACS buffer configuration 2% FBS + 1 mM EDTA + 98% PBS;
  • T cells or CAR-T cells with Raji cells expressing CD19, K562 cells not expressing CD19, and K562 cells modified with CD19 (referred to as K562-CD19 or K19) or H226 cells and expressing luciferase H226 cells were co-cultured.
  • the ratio of cells was 1:1 (10 4 effector per tumor and tumor cells) in a V-bottom 96-well plate.
  • Complete RPM1640 medium was used with a final volume of 200 ⁇ L. After 24 hours of culture, IL-2 and IFN- ⁇ in the supernatant were detected using an ELISA kit.
  • Target tumor cell K19 was labeled with 1 ⁇ M Celltrace Violet and then incubated with effector cells (T cells, CAR-T cells and knockout CAR-T cells) for 4 hours.
  • effector cells T cells, CAR-T cells and knockout CAR-T cells
  • FITC-Annexin V and 7-AAD Biolegend
  • tumor cells are placed at a density of 10 4 /100 ul into a micro-assay-plate 96-well plate (greiner bio-one).
  • the CAR-T cells were accurately counted, diluted according to different effector cells: target cell ratio, and added to the corresponding wells to 100 ul.
  • Figure 1A shows the location of the sgRNA in the LAG-3 locus, exemplified by sgRNA5.
  • the targeting sequences of the sgRNAs involved are shown in Table 1.
  • sgRNA Target sequence SEQ ID NO sgRNA1 ATGTGGGAGGCTCAGTTCCT 1 sgRNA2 GCTGCAGAAACAGCAAGCCC 2 sgRNA3 TGCTGTTTCTGCAGCCGCTT 3 sgRNA4 GCTGTTTCTGCAGCCGCTTT 4 sgRNA5 GTTTCTGCAGCCGCTTTGGG 5
  • Cas9 protein (3 ⁇ g) was complexed with in vitro transcribed sgRNA (3 ⁇ g) and then electroporated into primary CD3 + T cells.
  • the gene editing efficiency of each sgRNA was quantified by TIDE analysis, and the most effective sgRNA for further experiments was selected, and the results are shown in Fig. 1B.
  • sgRNA5 has the highest knockout efficiency.
  • the occurrence of insertions and deletions (indel) caused by NHEJ repair was determined by clonal sequencing.
  • the target region of sgRNA5 was amplified and independent mutations were identified. As shown in Figure 1C, all mutations occur exactly in the region targeted by the sgRNA.
  • LAG-3 knockout T cells were tested. Culture after electroporation. On the 3rd and 7th days after electroporation, the total number of cells was counted to determine the fold expansion of control T cells and LAG-3 knockout T cells. Results As shown in Figure 2A, LAG-3 knockout T cells remained normal dependent on proliferation stimulated by anti-CD3 and anti-CD28 antibodies.
  • LAG-3 gene editing was performed using anti-CD19 CAR-T cells.
  • CD19 CAR-T cells were activated with anti-CD3 and anti-CD28 for three days in the presence of IL-2 (50 U/ml). Then, the CRISPR-Cas9 system containing sgRNA5 was transferred to the CAR-T cells using electroporation, and gene editing efficiency was evaluated three days after electroporation.
  • the LAG-3 knockout rate was evaluated using three different donors and the results are shown in Figure 3A, with a knockout rate of 40-70% observed.
  • CAR-T cells were harvested on day 14 post-transfection for immunophenotypic analysis.
  • LAG3 knockout CAR-T cells did not show any significant changes in CD4 and CD8 expression and memory T cell phenotypic characteristics. That is, CRISPR-Cas9-mediated LAG3 disruption did not interfere with the T cell immunophenotype.
  • LAG3 knockout CAR-T cells were also examined. Effector cells: The ratio of target cells is 16:1, 8:1, and 4:1. The results are shown in Figure 4D, indicating that LAG3 knockout CAR-T cells retain at least the same anti-tumor activity as standard CAR-T cells.
  • LAG-3 knockout CAR-T cells eradicated tumors in a murine allograft model
  • mice received intraperitoneal injections of 1 ⁇ 10 7 T cells, CAR-T cells or CAR-T cells were LAG-3 knockout, or PBS.
  • mice Surviving mice were monitored until day 60. The percentage of survival of mice in each group is shown in Figure 5C.
  • the target sequences are SEQ ID NOs: 6-10 (Table 2), as shown in Figure 6A, the targeting sequence of sgRNA1 is green, PAM sequences It is blue.
  • sgRNA CTLA-4 target sequence SEQ ID NO sgRNA1 CCTTGGATTTCAGCGGCACA 6 sgRNA2 CCTTGTGCCGCTGAAATCCA 7 sgRNA3 TGAACCTGGCTACCAGGACC 8 sgRNA4 CATAAAGCCATGGCTTGCCT 9 sgRNA5 CTCAGCTGAACCTGGCTACC 10
  • Cas9 protein (3 ⁇ g) was combined with in vitro transcribed sgRNA (3 ⁇ g) to form Cas9-sgRNA ribonucleoprotein (RNP), which was then electroporated into primary 1 ⁇ 10 6 CD3+ T cells (three days after activation).
  • RNP Cas9-sgRNA ribonucleoprotein
  • the knockout efficiency of each sgRNA was quantified by TIDE analysis, and the frequency of insertion deletion in CTLA-4 was sequenced. As a result, as shown in Fig. 6B, the knockout efficiency of sgRNA1 was the highest, and gene knocking with sgRNA1 in another donor (donor 2) also obtained a significant knockout effect.
  • the PCR products of each sample were subcloned, and the alleles of each clone were sequenced; the representative mutant alleles in the RNP transfected cells were compared with the wild type sequences.
  • the result is shown in Figure 6C.
  • the targeting sequence of the sgRNA is green, the PAM sequence is blue, and the mutant sequence is red.
  • N/N refers to the number of clones containing the alleles of the mutation/the total number of clones sequenced.
  • CTLA-4 knockout T cells were tested. Culture after electroporation. On the 3rd and 7th day after electroporation, the total number of cells was counted to determine the fold expansion of control T cells and CTLA-4 knockout T cells. Results As shown in Figure 7A, CTLA-4 knockout T cells remained normal dependent on proliferation stimulated by anti-CD3 and anti-CD28 antibodies.
  • CTLA-4 gene editing was performed using anti-CD19 CAR-T cells.
  • Anti-CD19 CAR-T cells were cultured for three days. Then, RNPs of CRISPR-Cas9 and sgRNA1 were transferred to CAR-T cells by electroporation, and gene editing efficiency was evaluated three days after electroporation. The CTLA-4 knockout rate was evaluated using three different donors and the results are shown in Figure 8A.
  • PCR products of each sample were subcloned, and the alleles of each clone were sequenced; the representative mutant alleles in the RNP transfected cells were compared with the wild type sequences.
  • the result is shown in Fig. 8B.
  • the targeting sequence of the sgRNA is green, the PAM sequence is blue, and the mutant sequence is red.
  • N/N refers to the number of positive clones containing the allele of the mutation/the total number of clones sequenced.
  • CTLA-4 surface expression of CTLA-4 knockout CAR-T cells was analyzed by flow cytometry, and the results are shown in Fig. 8C.
  • cytotoxicity was determined by assessing the ability of T cells, CAR-T cells, and CTLA-4 knockout CAR-T cells to lyse tumor cells. As a result, as shown in Fig. 9D, the ratio of effector cells: target cells was 10:1, 5:1, and 2.5:1.
  • mice received intraperitoneal injections of 1 ⁇ 10 7 T cells, CAR-T cells or CTLA-4 knockout CAR-T cells in addition to, or PBS.
  • mice Surviving mice were counted until day 60. The survival percentage of mice in each group is shown in Fig. 10C.
  • sgRNAs targeting the exon 2 coding region of the Foxp3 locus were designed as shown in SEQ ID NOs: 11-15 (Table 3). As shown in Figure 11A, the targeting sequence for sgRNA3 is green and the PAM sequence is blue. .
  • sgRNA Foxp3 target sequence SEQ ID NO sgRNA1 GGGCCGAGATCTTCGAGGCG 11 sgRNA2 TCGAAGATCTCGGCCCTGGA 12 sgRNA3 GCAGCTGCGATGGTGGCATG 13 sgRNA4 AGGGCCGAGATCTTCGAGGC 14 sgRNA5 GGCCCTGGAAGGTTCCCCCT 15 sgRNA6 TTTGGGTGCAGCCCTCCAGC 16
  • Cas9 protein (3 ⁇ g) was combined with in vitro transcribed sgRNA (3 ⁇ g) to form Cas9-sgRNA ribonucleoprotein (RNP), which was then electroporated into primary 1 ⁇ 10 6 CD3+ T cells (three days after activation).
  • RNP Cas9-sgRNA ribonucleoprotein
  • sgRNA3 had the highest knockout efficiency, and sgRNA3 was used in the other two donors (D2 and D3).
  • Gene editing has also enabled effective gene knockout.
  • PCR products of each sample were subcloned, and the alleles of each clone were sequenced; the representative mutant alleles in the RNP transfected cells were compared with the wild type sequences.
  • the result is shown in Fig. 11C.
  • the targeting sequence of sgRNA is green, the PAM sequence is blue, and the mutant sequence is red.
  • N/N refers to the number of clones containing the alleles of the mutation/the total number of clones sequenced.
  • Foxp3 gene editing was performed using anti-CD19 CAR-T cells.
  • CD19 CAR-T cells were cultured for three days for activation. Then, RNPs of CRISPR-Cas9 and sgRNA3 were transferred to CAR-T cells by electroporation, and gene editing efficiency was evaluated three days after electroporation. The Foxp3 knockout rate was evaluated using three different donors and the results are shown in Figure 13A.
  • PCR products of each sample were subcloned, and the alleles of each clone were sequenced; the representative mutant alleles in the RNP transfected cells were compared with the wild type sequences.
  • the result is shown in Fig. 13B.
  • the targeting sequence of the sgRNA is green, the PAM sequence is blue, and the mutant sequence is red.
  • N/N refers to the number of positive clones containing the allele of the mutation/the total number of clones sequenced.
  • Foxp3 knockout anti-CD19 CAR-T cells were characterized in vitro and cultured after electroporation. On the 10th day after electroporation, the immunophenotype of the gene-edited T cells was evaluated by CD4, CD8 expression and naive, central memory and effector memory T cell subsets. Three independent experiments were performed and the data are shown as mean ⁇ SEM and the results are shown in Figure 14B.
  • cytotoxicity was determined by assessing the ability of T cells, CAR-T cells, and Foxp3 knockout CAR-T cells to lyse cells. Effector cells: The ratio of target cells is 10:1, 5:1, and 2.5:1. The result is shown in Fig. 14D.
  • mice received intraperitoneal injections of 1 ⁇ 10 7 T cells, Foxp3 CAR-T cells or other cells knocking CAR-T or PBS.
  • mice Surviving mice were counted until day 60. The percentage of survival of mice in each group is shown in Figure 15C.
  • sgRNAs targeting the exon 2 coding region of the Tim3 locus were designed and the target sequences are SEQ ID NOs: 17-21 (Table 4). As shown in Figure 16A, the targeting sequence for sgRNA1 is green and the PAM sequence is blue.
  • sgRNA Tim3 target sequence SEQ ID NO sgRNA1 CTGGTTTGATGACCAACTTC 17 sgRNA2 TGAAAAATTTAACCTGAAGT 18 sgRNA3 CTGAAGTTGGTCATCAAACC 19 sgRNA4 GAATGATGAAAAATTTAACC 20 sgRNA5 CCTGGTTTGATGACCAACTT twenty one
  • Cas9 protein (3 ⁇ g) was combined with in vitro transcribed sgRNA (3 ⁇ g) to form Cas9-sgRNA ribonucleoprotein (RNP), which was then electroporated into primary 1 ⁇ 10 6 CD3+ T cells (three days after activation).
  • RNP Cas9-sgRNA ribonucleoprotein
  • the knockout efficiency of each sgRNA was quantified by TIDE analysis, and the frequency of insertion of the Tim3 insertion was analyzed by sequencing. As shown in Fig. 16B, the knockdown efficiency of sgRNA1 was the highest, and in another donor (D2)-derived T cell and CAR. Gene editing with sgRNA1 in T cells also yielded an effective knockout effect.
  • Tim3 surface expression of Tim3 knockout T cells was analyzed by flow cytometry, and the results are shown in Fig. 16C.
  • Tim3 knockout T cells were tested. Culture after electroporation. On the 3rd day and the 7th day after electroporation, the total number of cells was counted, thereby measuring the amplification factor of the control T cells and the Tim3 knockout T cells, and the results are shown in Fig. 17A.
  • CD4 CD8 and (CD45RO-/CD62L+, TN), central memory (CD45RO+/CD62L+, TCM) and effector memory (CD45RO+/CD62L-, TEM) characteristics of T cell subsets
  • CD45RO+/CD62L+, TCM central memory
  • CD45RO+/CD62L-, TEM effector memory
  • Tim3 gene editing was performed using anti-CD19 CAR-T cells.
  • CD19 CAR-T cells were cultured for activation for three days. Then, the RNP of CRISPR-Cas9 and sgRNA1 was transferred to CAR-T cells by electroporation.
  • Tim3 knockout CAR-T cells proliferation of Tim3 knockout CAR-T cells was tested at three donors. Culture after electroporation. After electroporation, the total number of cells was counted to determine the fold expansion of control CAR-T cells and Tim3 knockout CAR-T cells. Two independent experiments were performed and the data are shown as mean ⁇ SEM and the results are shown in Figure 18A.
  • Tim3 knockout anti-CD19 CAR-T cells were characterized in vitro and cultured after electroporation. On the 10th day after electroporation, the immunophenotype of the gene-edited T cells was evaluated by CD4, CD8 expression and naive, central memory and effector memory T cell subsets. Three independent experiments were performed and the data are shown as mean ⁇ SEM and the results are shown in Figure 18B.
  • sgRNA targeting PD1 exon 1 was designed. Wherein sgRNA1 targets the sense strand and sgRNAp targets the antisense strand (see Figure 19A).
  • the sgRNA1 targeted PD1 gene sequence is: GTCTGGGCGGTGCTACAACT (SEQ ID NO: 22); the sgRNAp-targeted PD1 gene sequence is: ACAGGCGCCCTGGCCAGTCG (SEQ ID NO: 23).
  • the designed sgRNA and Cas9 proteins were co-transfected into anti-Meso CART cells by electroporation.
  • the Purification of the PD1 gene by the Surveyor showed that the editing efficiency reached 27.9%.
  • PD1-knockout anti-Meso CART cells can perform effector functions in vivo
  • FIG. 20A and 20B The results are shown in Figures 20A and 20B.
  • the H226 bioluminescence signal in PD1-KO anti-Meso CART cell treated mice rapidly decreased to near background levels after the first T cell injection and remained at low signal levels compared to untreated controls.
  • the H226 bioluminescence signal in the anti-Meso CART cell treated mice decreased rapidly after the first T cell injection compared to the untreated control, but the biofluorescence signal slowly increased after the second T cell injection.
  • the survival rates of mice receiving anti-mesothelin CAR-T cells, mice receiving PD1 knockout anti-mesothelin CAR-T cells, and untreated control mice are shown in Fig. 20C. Mice that received PD1 knockout of anti-mesothelin CAR-T cells showed higher survival.
  • PD1-knockout anti-Meso CART cells can perform effector functions in vitro.
  • Luciferase-expressing H226 cells H226-luci
  • mouse fibroblasts 3T3 or PDL1-expressing 3T3 cells 3T3-PDL1
  • PD1-KO anti-Meso CART cells anti-Meso CART cells
  • unmodified unmodified T cells were co-cultured for 20 hours at a ratio of 1:1, 1:2 or 1:4 effector:target cells.
  • the percentage of target cell lysis was calculated by measuring the luciferase activity of the remaining tumor cells.
  • the obtained CAR-T cells were co-cultured with luciferase-expressing H226 cells (H226-luci) for 3 days at a ratio of 2:1 effector cells: target cells, and the target was calculated by measuring the luciferase activity of the remaining tumor cells. The percentage of cell lysis.
  • P4-28z showed higher release of IFN- ⁇ and IL-2 compared to P4-z, P4-BBz and P4-28BBz (Fig. 23A), as well as higher specific cell lysis. (Fig. 23B, * indicates P ⁇ 0.05, and **** indicates P ⁇ 0.0001).
  • Knocking out one or more of PD-1, TIM-3 and LAG-3 in anti-mesothelin CAR-T cells wherein sgRNA knockdown targeting SEQ ID NO: 22 and/or SEQ ID NO: 23
  • sgRNA knockdown targeting SEQ ID NO: 22 and/or SEQ ID NO: 23 In addition to PD-1, knockdown of TIM-3 with sgRNA targeting SEQ ID NO: 26, knockdown of CTLA-4 with sgRNA targeting SEQ ID NO: 25, and targeting of SEQ ID NO: 5 and/or 24 The sgRNA knocks out LAG-3.
  • P4 CAR-T cells knocked out PD-1, P4 CAR-T cells (TIM3 KO) knocked out TIM3, P4 CAR-T cells (LAG3 KO) knocked out LAG3, knocked out P4 CAR-T cells (PD1 TIM3 KO) of PD1 and TIM3, P4 CAR-T cells (PD1 LAG3 KO) that knocked out PD1 and LAG3, and P4 CAR-T cells (PD1 TIM3 LAG3) that knocked out PD1, TIM3 and LAG3 KO).
  • the knockout efficiency of the PD-1, LAG-3 and TIM3 genes was examined by Surveyor test and TIDE, and the results are shown in Fig. 24.
  • H226 cells H226-PDL1-luci
  • CRL5826 cells CRL5826-PDL1 expressing PD-L1 and luciferase, respectively.
  • FIG. 25A when the ratio of effector cells: target cells (H226-PDL1-luci) was 4:1, after knocking for 20 hours, except for P4 (TIM3 KO), other knockout CAR-T cells were Can kill target cells more effectively than P4; as shown in Figure 25B, when the ratio of effector cells: target cells (H226-PDL1-luci) is 0.1:1, all knocked out CAR- after 6 days of co-culture T cells were more effective than P4 in killing target cells, *, P ⁇ 0.05.**, P ⁇ 0.01.***, P ⁇ 0.001.****, P ⁇ 0.0001.
  • FIG 26 shows that when the ratio of effector:target cells (CRL5826-PDL1) was 4:1, knockout CAR-T cells appeared after 24 hours of co-culture (Fig. 26A) and 48 hours (Fig. 26B). Not less than the tumor killing effect of P4 CAR-T cells.
  • Figure 27 shows that knockout CAR-T cells were expressed after co-culture for 4 days (Figure 27A) and 6 days (Figure 27B) when the ratio of effector cells: target cells (CRL5826-PDL1) was 0.1:1. Significantly stronger tumor killing effect than P4 CAR-T cells.
  • Figure 28 shows that knockout CAR-T cells were expressed after co-culture for 4 days (Figure 28A) and 6 days (Figure 28B) when the ratio of effector:target cells (CRL5826-PDL1) was 0.02:1. Significantly stronger tumor killing effect than P4 CAR-T cells.
  • knockout PD1 is represented by P
  • knockout of TIM3 is represented by T
  • knockout of CTLA4 is represented by C
  • knockout LAG3 is represented by L
  • knocking out two or more genes is represented by a corresponding letter combination.
  • PC means knocking out PD1 and CTLA4
  • PCTL means that all four genes are knocked out.
  • the obtained knockout CAR-T cells and P4 CAR-T cells and T cells were expressed with luciferase-expressing CRL5826 cells (CRL5826), OVCAR3 cells (OVCAR3) and HCT116 cells (HCT116), respectively, to express PDL1 and fluorescence.
  • CRL5826 CRL5826
  • OVCAR3 OVCAR3 cells
  • HCT116 HCT116 cells
  • FIG. 29 shows that when the ratio of effector cells: target cells (CRL5826) is 1:1, knocked out CAR-T cells are not low after 24 hours of co-culture (Fig. 29A) and 48 hours (Fig. 29B). Tumor killing effect on P4 CAR-T cells.
  • Figure 32 shows that when the ratio of effector cells: target cells was 1:1, the knockout CAR-T cells showed no less tumor killing effect than P4 CAR-T cells after 24 hours of co-culture.
  • the target cells in Fig. 32A are HCT116 cells expressing luciferase (HCT116), and the target cells in Fig. 32B are HCT116 cells expressing HDL116 and luciferase (HCT116-PDL1).
  • Figure 33 shows that when the ratio of effector cells: target cells is 1:1, knocked out CAR-T cells showed a stronger tumor killing effect than P4 CAR-T cells after 48 hours of co-culture.
  • the target cells in Fig. 33A are HCT116 cells expressing luciferase (HCT116), and the target cells in Fig. 33B are HCT116 cells (HCT116-PDL1) expressing PDL1 and luciferase.
  • Figure 34 shows that when the ratio of effector cells: target cells was 0.1:1, knockout CAR-T cells showed significantly stronger tumor killing effects than P4 CAR-T cells after 4 days of co-culture.
  • the target cells in Figure 34A are CRL5826 cells expressing luciferase (CRL 5826), while the target cells in Figure 34B are CRL5826 cells expressing CDL1 and luciferase (CRL5826-PDL1).
  • Figure 35 shows that when the ratio of effector cells: target cells was 0.1:1, the knockout CAR-T cells showed a significantly stronger tumor killing effect than P4 CAR-T cells after 48 hours of co-culture.
  • the target cells in Figure 35A are OVCAR3 cells expressing luciferase (OVCAR3), while the target cells in Figure 35B are OVCAR3 cells (OVCAR3-PDL1) expressing PDL1 and luciferase.
  • the experimental results indicate that the CAR-T cells in which one or more of the inhibitory proteins of the present invention are knocked out are equivalent to the unknocked CAR-T cells at a high target ratio, and unexpectedly, the knockout of the present invention CAR-T cells are superior to non-knockout CAR-T cells in inefficient target ratios and longer duration of action. This is particularly advantageous in reducing costs, reducing preparation time, and reducing side effects associated with high dose administration.

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Abstract

提供了一种通过基因编辑制备经修饰的T细胞的方法，以及通过该方法制备的经修饰的T细胞及其用途。

Description

经修饰的T细胞、其制备方法及用途 技术领域
本发明涉及基因编辑和肿瘤免疫疗法领域。具体而言，本发明涉及通过基因编辑制备经修饰的T细胞例如CAR-T细胞的方法，以及通过所述方法制备的经修饰的T细胞及其用途。
背景技术
T细胞在抗肿瘤免疫中发挥重要作用。然而，在肿瘤患者体内，局部的特异性细胞毒性T淋巴细胞(CTL)含量很少，其获取和体外扩增比较困难，且CTL的亲和力低，限制了其在肿瘤的临床治疗中的应用。
T细胞过继转移是近年来获得较高关注的特异性、低毒性的抗肿瘤方法，包括例如用T细胞受体(TCR)或嵌合抗原受体(CAR)对T细胞进行遗传修饰是最常用的生成肿瘤特异性T细胞的方法。
TCR基因转移技术是从肿瘤反应性T细胞中克隆TCR的α和β链，通过基因工程技术，以逆转录病毒或慢病毒作为载体，对初始T细胞进行抗原特异性TCR修饰，从而赋予T细胞特异性识别和杀伤肿瘤细胞的能力，并提高T细胞与肿瘤的亲和力。利用TCR基因转移技术对患者自体来源的T细胞进行TCR基因修饰，经体外扩增，获取大量具有特异高效识别能力的T细胞，再过继回输给肿瘤患者，使之在体内发挥抗肿瘤作用。
CAR由胞外结构域、铰链、跨膜结构域和胞内结构域组成，所述胞外结构域通常衍生自单链可变片段(scFv)，所述胞内结构域具有一个、两个或三个(分别对应于第一、二、三代CAR)衍生自CD3Z和/或共刺激分子信号传导结构域(Kakarla and Gottschalk,2014)。尽管CAR-T细胞疗法在治疗CD19阳性的恶性血液肿瘤的早期临床研究中获得成功(Daviala et al,2014；Lee et al,2015；Maude et al,2014)，然而用CAR-T细胞靶向实体瘤抗原的临床应答非常有限。
因此，仍然需要获得能够有效抑制或杀伤肿瘤尤其是实体瘤的T细胞。
发明简述
在一方面，本发明提供一种制备经修饰的T细胞的方法，包括减少或消除T细胞中抑制性蛋白表达的步骤。
在一些实施方案中，所述T细胞是包含外源T细胞受体(TCR)或嵌合抗原受体(CAR)的T细胞。
在一些实施方案中，所述表达被减少或消除的抑制性蛋白选自PD1、LAG-3、 CTLA-4、Foxp3、Tim3及其组合。
在一些实施方案中，所述表达被减少或消除的抑制性蛋白选自PD1和TIM3的组合，PD1和CTLA-4的组合，PD1和LAG3的组合，CTLA-4和TIM3的组合，CTLA-4和LAG3的组合，TIM3和LAG3的组合，PD1、TIM3和CTLA-4的组合，PD1、CTLA-4和LAG3的组合，CTLA-4、TIM3和LAG3的组合，PD1、TIM3和LAG3的组合，或PD1、CTLA-4、TIM3和LAG3的组合。
在一些实施方案中，通过反义RNA、antagomir、siRNA、shRNA、大范围核酸酶、锌指核酸酶、转录激活因子样效应物核酸酶或CRISPR系统实施所述减少或消除。
在一些实施方案中，所述CRISPR系统是CRISPR/Cas9系统。
在一些实施方案中，所述CRISPR/Cas9系统靶向所述细胞内选自SEQ ID NO:5、6、13、17、22-26中一或多种的核苷酸序列。
在一些实施方案中，所述TCR或CAR包含针对肿瘤相关抗原的抗原结合结构域。
在一些实施方案中，所述肿瘤相关抗原选自CD16、CD64、CD78、CD96、CLL1、CD116、CD117、CD71、CD45、CD71、CD123、CD138、ErbB2(HER2/neu)、癌胚抗原(CEA)、上皮细胞粘附分子(EpCAM)、表皮生长因子受体(EGFR)、EGFR变体III(EGFRvIII)、CD19、CD20、CD30、CD40、双唾液酸神经节苷脂GD2、导管上皮粘蛋白、gp36、TAG-72、鞘糖脂、神经胶质瘤相关的抗原、β-人绒毛膜促性腺激素、α胎儿球蛋白(AFP)、外源凝集素反应性AFP、甲状腺球蛋白、RAGE-1、MN-CA IX、人端粒酶逆转录酶、RU1、RU2(AS)、肠羧基酯酶、mut hsp70-2、M-CSF、前列腺酶(prostase)、前列腺酶特异性抗原(PSA)、PAP、NY-ESO-1、LAGA-1a、p53、Prostein、PSMA、存活和端粒酶、前列腺癌肿瘤抗原-1(PCTA-1)、MAGE、ELF2M、嗜中性粒细胞弹性蛋白酶、肝配蛋白B2、CD22、胰岛素生长因子(IGF1)-I、IGF-II、IGFI受体、间皮素、呈递肿瘤特异性肽表位的主要组织相容性复合体(MHC)分子、5T4、ROR1、Nkp30、NKG2D、肿瘤基质抗原、纤维连接蛋白的额外结构域A(EDA)和额外结构域B(EDB)、腱生蛋白-C的A1结构域(TnC A1)、成纤维细胞相关蛋白(fap)、CD3、CD4、CD8、CD24、CD25、CD33、CD34、CD133、CD138、Foxp3、B7-1(CD80)、B7-2(CD86)、GM-CSF、细胞因子受体、内皮因子、主要组织相容性复合体(MHC)分子、BCMA(CD269、TNFRSF17)、TNFRSF17(UNIPROT Q02223)、SLAMF7(UNIPROT Q9NQ25)、GPRC5D(UNIPROT Q9NZD1)、FKBP11(UNIPROT Q9NYL4)、KAMP3、ITGA8(UNIPROT P53708)和FCRL5(UNIPROT Q68SN8)。
在一些实施方案中，所述抗原结合结构域选自单克隆抗体、合成的抗体、人抗体、人源化抗体、单域抗体、抗体单链可变区，以及其抗原结合片段。
在一些实施方案中，所述CAR包含针对间皮素的scFv(P4)、CD8铰链区、CD28跨膜结构域、CD28共刺激结构域、和CD3ζ信号转导结构域。
在一些实施方案中，所述CAR包含SEQ ID NO：32所示氨基酸序列。
在另一方面，本发明提供一种通过本发明的方法制备的经修饰的T细胞。
在另一方面，本发明提供一种修饰的T细胞，其中与未修饰的T细胞相比，所述T细胞中的抑制性蛋白的表达被减少或消除。
在一些实施方案中，所述T细胞是包含外源T细胞受体(TCR)或嵌合抗原受体(CAR)的T细胞。
在一些实施方案中，所述表达被减少或消除的抑制性蛋白选自PD1、LAG-3、CTLA-4、Foxp3、Tim3及其组合。
在一些实施方案中，所述表达被减少或消除的抑制性蛋白选自PD1和TIM3的组合，PD1和CTLA-4的组合，PD1和LAG3的组合，CTLA-4和TIM3的组合，CTLA-4和LAG3的组合，TIM3和LAG3的组合，PD1、TIM3和CTLA-4的组合，PD1、CTLA-4和LAG3的组合，CTLA-4、TIM3和LAG3的组合，PD1、TIM3和LAG3的组合，或PD1、CTLA-4、TIM3和LAG3的组合。
在一些实施方案中，所述TCR或CAR包含针对肿瘤相关抗原的抗原结合结构域。
在一些实施方案中，所述肿瘤相关抗原选自CD16、CD64、CD78、CD96、CLL1、CD116、CD117、CD71、CD45、CD71、CD123、CD138、ErbB2(HER2/neu)、癌胚抗原(CEA)、上皮细胞粘附分子(EpCAM)、表皮生长因子受体(EGFR)、EGFR变体III(EGFRvIII)、CD19、CD20、CD30、CD40、双唾液酸神经节苷脂GD2、导管上皮粘蛋白、gp36、TAG-72、鞘糖脂、神经胶质瘤相关的抗原、β-人绒毛膜促性腺激素、α胎儿球蛋白(AFP)、外源凝集素反应性AFP、甲状腺球蛋白、RAGE-1、MN-CA IX、人端粒酶逆转录酶、RU1、RU2(AS)、肠羧基酯酶、mut hsp70-2、M-CSF、前列腺酶(prostase)、前列腺酶特异性抗原(PSA)、PAP、NY-ESO-1、LAGA-1a、p53、Prostein、PSMA、存活和端粒酶、前列腺癌肿瘤抗原-1(PCTA-1)、MAGE、ELF2M、嗜中性粒细胞弹性蛋白酶、肝配蛋白B2、CD22、胰岛素生长因子(IGF1)-I、IGF-II、IGFI受体、间皮素、呈递肿瘤特异性肽表位的主要组织相容性复合体(MHC)分子、5T4、ROR1、Nkp30、NKG2D、肿瘤基质抗原、纤维连接蛋白的额外结构域A(EDA)和额外结构域B(EDB)、腱生蛋白-C的A1结构域(TnC A1)、成纤维细胞相关蛋白(fap)、CD3、CD4、CD8、CD24、CD25、CD33、CD34、CD133、CD138、Foxp3、B7-1(CD80)、B7-2(CD86)、GM-CSF、细胞因子受体、内皮因子、主要组织相容性复合体(MHC)分子、BCMA(CD269、TNFRSF17)、TNFRSF17(UNIPROT Q02223)、SLAMF7(UNIPROT Q9NQ25)、GPRC5D(UNIPROT Q9NZD1)、FKBP11(UNIPROT Q9NYL4)、KAMP3、ITGA8(UNIPROT P53708)和FCRL5(UNIPROT Q68SN8)。
在一些实施方案中，所述所述抗原结合结构域选自单克隆抗体、合成的抗体、人抗体、人源化抗体、单域抗体、抗体单链可变区，以及其抗原结合片段。
在一些实施方案中，所述所述CAR包含针对间皮素的scFv(P4)、CD8铰链区、CD28跨膜结构域、CD28共刺激结构域、和CD3ζ信号转导结构域。
在一些实施方案中，所述CAR包含SEQ ID NO：32所示氨基酸序列。
在另一方面，本发明提供本发明的经修饰的T细胞在制备用于治疗癌症的药物中的 用途。
在另一方面，本发明提供一种用于治疗癌症的药物组合物，包含本发明的经修饰的T细胞和药学可接受的载体。
在本发明各方面的实施方案中，所述癌症选自肺癌、卵巢癌、结肠癌、直肠癌、黑色素瘤、肾癌、膀胱癌、乳腺癌、肝癌、淋巴瘤、恶性血液病、头颈癌、胶质瘤、胃癌、鼻咽癌、喉癌、宫颈癌、子宫体瘤和骨肉瘤。可以用本发明的方法或药物组合物治疗的其他癌症的例子包括：骨癌、胰腺癌、皮肤癌、前列腺癌、皮肤或眼内恶性黑色素瘤、子宫癌、肛区癌、睾丸癌、输卵管癌、子宫内膜癌、阴道癌、阴户癌、何杰金病、非何杰金氏淋巴瘤、食道癌、小肠癌、内分泌系统癌、甲状腺癌、甲状旁腺癌、肾上腺癌、软组织肉瘤、尿道癌、阴茎癌、慢性或急性白血病(包括急性髓细胞样白血病、慢性髓细胞样白血病、急性成淋巴细胞性白血病、慢性淋巴细胞性白血病)、儿童实体瘤、淋巴细胞性淋巴瘤、膀胱癌、肾或输尿管癌、肾盂癌、中枢神经系统(CNS)肿瘤、原发性CNS淋巴瘤、肿瘤血管发生、脊柱肿瘤、脑干神经胶质瘤、垂体腺瘤、卡波西肉瘤、表皮状癌、鳞状细胞癌、T细胞淋巴瘤、环境诱发的癌症，包括石棉诱发的癌症，以及所述癌症的组合。
在另一方面，本发明还提供试剂盒，其用于通过本发明的方法制备经修饰的T细胞。
附图说明
图1显示靶向LAG-3的sgRNA的设计和筛选。
图2显示敲除LAG-3对T细胞的影响，其中T-CTRL-UE和T-CTRL-E表示未经受或经受电穿孔的T细胞，而LAG-3-KO表示RNP处理的T细胞。
图3显示在CAR-T细胞中敲除LAG-3的结果。
图4显示体外评估敲除LAG-3对CAR-T细胞的影响，其中CAR-T-CTRL-UE和CAR-T-CTRL-E表示未经受或经受电穿孔的细胞，而LAG-3-KO-CAR-T表示RNP处理的细胞。
图5显示敲除LAG-3的CAR-T细胞的体内抗肿瘤作用。
图6显示靶向CTLA-4的sgRNA的设计和筛选。
图7显示敲除CTLA-4对T细胞的影响，其中T-CTRL-UE和T-CTRL-E表示未经受或经受电穿孔的T细胞，而CTLA-4-KO表示RNP处理的T细胞。
图8显示在CAR-T细胞中敲除CTLA-4的结果。
图9显示体外评估敲除CTLA-4对CAR-T细胞的影响，其中CAR-T-CTRL-UE和CAR-T-CTRL-E表示未经受或经受电穿孔的细胞，而CTLA-4-KO-CAR-T表示RNP处理的细胞。
图10显示敲除CTLA-4的CAR-T细胞的体内抗肿瘤作用。
图11显示靶向Foxp3的sgRNA的设计和筛选。
图12显示敲除Foxp3对T细胞的影响，其中T-CTRL-UE和T-CTRL-E表示未经受或经受电穿孔的T细胞，而Foxp3-KO表示RNP处理的T细胞。
图13显示在CAR-T细胞中敲除Foxp3的结果。
图14显示体外评估敲除Foxp3对CAR-T细胞的影响，其中CAR-T-CTRL-UE和CAR-T-CTRL-E表示未经受或经受电穿孔的细胞，而Foxp3-KO-CAR-T表示RNP处理的细胞。
图15显示敲除Foxp3的CAR-T细胞的体内抗肿瘤作用。
图16显示靶向Tim3的sgRNA的设计和筛选。
图17显示敲除Tim3对T细胞的影响，其中T-CTRL-UE和T-CTRL-E表示未经受或经受电穿孔的T细胞，而Tim3-KO表示RNP处理的T细胞。
图18显示敲除Tim3对CAR-T细胞的影响，其中CAR-T-CTRL-UE和CAR-T-CTRL-E表示未经受或经受电穿孔的细胞，而Tim-3-KO-CAR-T表示RNP处理的细胞。
图19显示在CAR-T细胞中敲除PD1。
图20显示PD1敲除的抗-Meso CART细胞在体内能执行效应子功能。
图21显示PD1敲除的抗-Meso CART细胞在体外能执行效应子功能。
图22显示不同CAR结构。
图23显示具有不同CAR结构的CAR-T细胞的作用。
图24显示检测PD-1、LAG-3和/或TIM3基因的敲除效率。
图25显示PD-1、LAG-3和/或TIM3基因敲除的P4 CAR-T细胞对靶细胞(H226-PDL1-luci)的作用。
图26显示PD-1、LAG-3和/或TIM3基因敲除的P4 CAR-T细胞对靶细胞(CRL5826-PDL1)的作用，高效靶比(4:1)。
图27显示PD-1、LAG-3和/或TIM3基因敲除的P4 CAR-T细胞高效靶比对靶细胞(CRL5826-PDL1)的作用，低效靶比(0.1:1)。
图28显示显示PD-1、LAG-3和/或TIM3基因敲除的P4 CAR-T细胞对靶细胞(CRL5826-PDL1)的作用，低效靶比(0.02:1)。
图29显示敲除PD-1、TIM3、CTLA4和LAG3的一或多种的CAR-T细胞对靶细胞(CRL5826)的作用，效靶比1:1。
图30显示敲除PD-1、TIM3、CTLA4和LAG3的一或多种的CAR-T细胞对靶细胞(CRL5826-PDL1)的作用，效靶比1:1。
图31显示敲除PD-1、TIM3、CTLA4和LAG3的一或多种的CAR-T细胞对靶细胞(OVCAR3或OVCAR3-PDL1)的作用，效靶比1:1。
图32显示培养24小时后，敲除PD-1、TIM3、CTLA4和LAG3的一或多种的CAR-T细胞对靶细胞(HCT116或HCT116-PDL1)的作用，效靶比1:1。
图33显示培养48小时后，敲除PD-1、TIM3、CTLA4和LAG3的一或多种的CAR-T 细胞对靶细胞(HCT116或HCT116-PDL1)的作用，效靶比1:1。
图34显示培养4天后，敲除PD-1、TIM3、CTLA4和LAG3的一或多种的CAR-T细胞对靶细胞(CRL5826或CRL5826-PDL1)的作用，效靶比0.1:1。
图35显示培养48小时后，敲除PD-1、TIM3、CTLA4和LAG3的一或多种的CAR-T细胞对靶细胞(OVCAR3或OVCAR3-PDL1)的作用，效靶比0.1:1。
发明详述
一、定义
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的蛋白质和核酸化学、分子生物学、细胞和组织培养、微生物学、免疫学相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。例如，本发明中使用的标准重组DNA和分子克隆技术为本领域技术人员熟知，并且在如下文献中有更全面的描述：Sambrook，J.，Fritsch，E.F.和Maniatis，T.，Molecular Cloning：A Laboratory Manual；Cold Spring Harbor Laboratory Press：Cold Spring Harbor，1989(下文称为“Sambrook”)。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
“基因组”在用于真核细胞时不仅涵盖存在于细胞核中的染色体DNA，而且还包括存在于细胞的亚细胞组分(如线粒体、质体)中的细胞器DNA。
针对序列而言的“外源”意指来自外来物种的序列，或者如果来自相同物种，则指通过蓄意的人为干预而从其天然形式发生了组成和/或基因座的显著改变的序列。
“多核苷酸”、“核酸序列”、“核苷酸序列”或“核酸片段”可互换使用并且是单链或双链RNA或DNA聚合物，任选地可含有合成的、非天然的或改变的核苷酸碱基。核苷酸通过如下它们的单个字母名称来指代：“A”为腺苷或脱氧腺苷(分别对应RNA或DNA)，“C”表示胞苷或脱氧胞苷，“G”表示鸟苷或脱氧鸟苷，“U”表示尿苷，“T”表示脱氧胸苷，“R”表示嘌呤(A或G)，“Y”表示嘧啶(C或T)，“K”表示G或T，“H”表示A或C或T，“I”表示肌苷，并且“N”表示任何核苷酸。
“多肽”、“肽”、和“蛋白质”在本发明中可互换使用，指氨基酸残基的聚合物。该术语适用于其中一个或多个氨基酸残基是相应的天然存在的氨基酸的人工化学类似物的氨基酸聚合物，以及适用于天然存在的氨基酸聚合物。术语“多肽”、“肽”、“氨基酸序列”和“蛋白质”还可包括修饰形式，包括但不限于糖基化、脂质连接、硫酸盐化、谷氨酸残基的γ羧化、羟化和ADP-核糖基化。
如本发明所用，“表达构建体”是指适于感兴趣的核苷酸序列在细胞中表达的载体如重组载体。“表达”指功能产物的产生。例如，核苷酸序列的表达可指核苷酸序列的转录(如转录生成mRNA或功能RNA)和/或RNA翻译成前体或成熟蛋白质。
本发明的“表达构建体”可以是线性的核酸片段、环状质粒、病毒载体，或者，在一些实施方式中，可以是能够翻译的RNA(如mRNA)。
本发明的“表达构建体”可包含不同来源的调控序列和感兴趣的核苷酸序列，或相同来源但以不同于通常天然存在的方式排列的调控序列和感兴趣的核苷酸序列。
“调控序列”和“调控元件”可互换使用，指位于编码序列的上游(5'非编码序列)、中间或下游(3'非编码序列)，并且影响相关编码序列的转录、RNA加工或稳定性或者翻译的核苷酸序列。表达调控元件指的是能够控制感兴趣的核苷酸序列转录、RNA加工或稳定性或者翻译的核苷酸序列。
调控序列可包括但不限于启动子、翻译前导序列、内含子、增强子和多腺苷酸化识别序列。
“启动子”指能够控制另一核酸片段转录的核酸片段。在本发明的一些实施方式中，启动子是能够控制细胞中基因转录的启动子，无论其是否来源于所述细胞。
如本文中所用，术语“可操作地连接”指调控元件(例如但不限于，启动子序列、转录终止序列等)与核酸序列(例如，编码序列或开放读码框)连接，使得核苷酸序列的转录被所述转录调控元件控制和调节。用于将调控元件区域可操作地连接于核酸分子的技术为本领域已知的。
“基因编辑”，也称为基因组编辑，其使用工程化的核酸酶或“分子剪刀”在生物体基因组中进行DNA插入、缺失或替换。基因编辑通过在基因组中期望的位置导致位点特异性双链断裂(DSB)，然后在修复DSB的过程中引入期望的DNA插入、缺失或取代。基因编辑通常使用大范围核酸酶、锌指核酸酶(ZFN)、转录激活因子样效应物核酸酶(TALEN)和CRISPR系统。
“大范围核酸酶”是一类具有巨大的识别位点(12-40bp的双链DNA序列)的脱氧核糖核酸内切酶，其识别位点在任何给定的基因组中通常只出现一次。例如，I-SceI大范围核酸酶所识别的18bp序列平均需要在比人基因组大20倍的基因组中才会偶然出现一次。
“锌指核酸酶”是通过将锌指DNA结合结构域与DNA切割结构域融合而制备的人工限制性酶。单个ZFN的锌指DNA结合结构域通常含有3-6个单独的锌指重复序列，每个锌指重复序列可以识别例如3bp。
“转录激活因子样效应物核酸酶”是可以经工程化而可以切割特定DNA序列的限制性酶，通常通过将转录激活因子样效应物(TALE)的DNA结合结构域与DNA切割结构域融合而制备。TALE经工程化后可以结合几乎任何想要的DNA序列。
“成簇的规律间隔的短回文重复序列(CRISPR)”是含有短的重复序列的原核DNA区段。CRISPR系统是原核免疫系统，其赋予对外来遗传元件如存在于质粒和噬菌体的元件的抗性，这种抗性提供后天免疫。在此系统中Cas蛋白或类似蛋白在RNA的指导下切割外来核酸。
如本文所用，术语“CRISPR核酸酶”通常指在天然存在的CRISPR系统中存在的核酸酶，以及其修饰形式、其变体(包括切口酶突变体)、或其催化活性片段。CRISPR核酸酶可以通过与向导RNA(如crRNA和任选的tracrRNA或人工gRNA(如sgRNA))一 起相互作用来识别和/或切割靶核酸结构。该术语涵盖基于CRISPR系统的能够在细胞内实现基因编辑的任何核酸酶。
“Cas9核酸酶”和“Cas9”在本文中可互换使用，指的是包括Cas9蛋白或其片段(例如包含Cas9的活性DNA切割结构域和/或Cas9的gRNA结合结构域的蛋白)的RNA指导的核酸酶。Cas9是CRISPR/Cas(成簇的规律间隔的短回文重复序列及其相关系统)基因组编辑系统的组分，能在向导RNA的指导下靶向并切割DNA靶序列形成DNA双链断裂(DSB)。
“向导RNA”和“gRNA”在本文中可互换使用，通常由部分互补形成复合物的crRNA和tracrRNA分子构成，其中crRNA包含与靶序列具有足够互补性以便与该靶序列杂交并且指导CRISPR复合物(Cas9+crRNA+tracrRNA)与该靶序列序列特异性结合的序列。然而，本领域已知可以设计单向导RNA(sgRNA)，其同时包含crRNA和tracrRNA的特征。
“T细胞受体(TCR)”又称T细胞抗原受体，是T细胞特异性识别和结合抗原肽-MHC分子的分子结构，通常与CD3分子呈复合物形式存在于T细胞表面。大多数T细胞的TCR由α和β肽链组成，少数T细胞的TCR由γ和δ肽链组成。
“嵌合型抗原受体(CAR)”又称人工T细胞受体、嵌合型T细胞受体、嵌合型免疫受体，是一种人工设计的受体，可以赋予免疫效应细胞某一种特异性。普遍来讲，这一技术被用于赋予T细胞特异性识别肿瘤表面抗原的特性。通过这种方法，可以产生大量的靶向杀伤肿瘤细胞。
如本文所用“对象”是指患有或者易于患有可以通过本发明的方法、或药物组合物治疗的疾病(如癌症)的生物体。非限制性例子包括人、牛、大鼠、小鼠、狗、猴、山羊、绵羊、母牛、鹿，及其它非哺乳动物。在优选实施方案中，对象是人。
二、制备经修饰T细胞的方法
在第一方面，本发明提供一种制备经修饰的T细胞的方法，包括减少或消除T细胞中的抑制性蛋白表达的步骤。
本发明的T细胞可以是通过扩增抗原特异性T细胞或通过遗传工程化重定向T细胞而产生的用于继承性免疫疗法的T细胞。所述T细胞还可以是分离自对象的原代T细胞。在一些实施方式中，所述T细胞是包含外源T细胞受体(TCR)的T细胞。在另一些实施方式中，所述T细胞是包含嵌合抗原受体(CAR)的T细胞。
在一些实施方式中，所述方法还包括提供分离自对象的未经修饰的T细胞的步骤，以及向所述未经修饰的T细胞导入TCR或CAR的步骤。在一些实施方式中，向所述未经修饰的T细胞导入TCR或CAR的步骤在减少或消除T细胞中的抑制性蛋白表达的步骤之前或之后或同时进行。
在一些实施方式中，所述TCR或CAR包含针对肿瘤相关抗原的抗原结合结构域，例如胞外抗原结合结构域。
所述肿瘤相关抗原包括但不限于CD16、CD64、CD78、CD96、CLL1、CD116、CD117、CD71、CD45、CD71、CD123、CD138、ErbB2(HER2/neu)、癌胚抗原(CEA)、上皮细胞粘附分子(EpCAM)、表皮生长因子受体(EGFR)、EGFR变体III(EGFRvIII)、CD19、CD20、CD30、CD40、双唾液酸神经节苷脂GD2、导管上皮粘蛋白、gp36、TAG-72、鞘糖脂、神经胶质瘤相关的抗原、β-人绒毛膜促性腺激素、α胎儿球蛋白(AFP)、外源凝集素反应性AFP、甲状腺球蛋白、RAGE-1、MN-CA IX、人端粒酶逆转录酶、RU1、RU2(AS)、肠羧基酯酶、mut hsp70-2、M-CSF、前列腺酶(prostase)、前列腺酶特异性抗原(PSA)、PAP、NY-ESO-1、LAGA-1a、p53、Prostein、PSMA、存活和端粒酶、前列腺癌肿瘤抗原-1(PCTA-1)、MAGE、ELF2M、嗜中性粒细胞弹性蛋白酶、肝配蛋白B2、CD22、胰岛素生长因子(IGF1)-I、IGF-II、IGFI受体、间皮素、呈递肿瘤特异性肽表位的主要组织相容性复合体(MHC)分子、5T4、ROR1、Nkp30、NKG2D、肿瘤基质抗原、纤维连接蛋白的额外结构域A(EDA)和额外结构域B(EDB)、腱生蛋白-C的A1结构域(TnC A1)、成纤维细胞相关蛋白(fap)、CD3、CD4、CD8、CD24、CD25、CD33、CD34、CD133、CD138、Foxp3、B7-1(CD80)、B7-2(CD86)、GM-CSF、细胞因子受体、内皮因子、主要组织相容性复合体(MHC)分子、BCMA(CD269、TNFRSF17)、TNFRSF17(UNIPROT Q02223)、SLAMF7(UNIPROT Q9NQ25)、GPRC5D(UNIPROT Q9NZD1)、FKBP11(UNIPROT Q9NYL4)、KAMP3、ITGA8(UNIPROT P53708)和FCRL5(UNIPROT Q68SN8)。在一优选实施方式中，所述抗原是间皮素。
本发明中，所述抗原结合结构域例如可以是单克隆抗体、合成的抗体、人抗体、人源化抗体、单域抗体、抗体单链可变区，以及其抗原结合片段。
在一优选实施方式中，所述抗原结合结构域是针对间皮素的单克隆抗体。在一优选实施方式中，所述抗原结合结构域是针对间皮素的scFv。在一优选实施方式中，所述抗原结合结构域是针对间皮素的scFv(P4)，例如，其氨基酸序列示于SEQ ID NO：27。
在一些实施方式中，所述CAR包含跨膜结构域，例如CD8跨膜结构域或CD28跨膜结构域，优选CD28跨膜结构域，例如氨基酸序列示于SEQ ID NO：28的CD28跨膜结构域。
在一些实施方式中，所述CAR进一步包括位于胞外抗原结合结构域和所述跨膜结构域之间的铰链区域，例如，所述铰链区域为CD8铰链区，例如氨基酸序列示于SEQ ID NO：29的CD8铰链区。
在一些实施方式中，所述CAR包含可用于T细胞活化的信号转导结构域，例如选自TCRζ、FcRγ、FcRβ、FcRε、CD3γ、CD3δ、CD3ε、CD3ζ、CD5、CD22、CD79a、CD79b和CD66d的信号转导结构域。在一些优选实施方式中，所述CAR包含CD3ζ信号转导结构域，例如氨基酸序列示于SEQ ID NO：30的CD3ζ信号转导结构域。
在一些实施方式中，所述CAR还包含一或多个选自CD3、CD27、CD28、CD83、CD86、CD127、4-1BB和4-1BBL的共刺激结构域。在一些实施方案中，所述CAR包含CD28共刺激结构域，例如氨基酸序列示于SEQ ID NO：31的CD28共刺激结构域。
在一些实施方式中，所述CAR还可以包含用于显示或追踪CAR表达的报道分子，如GFP蛋白。
在本发明一些优选实施方式中，所述CAR包含针对间皮素的scFv P4、CD8铰链区、CD28跨膜结构域、CD28共刺激结构域、CD3ζ信号转导结构域和任选的GFP蛋白。在本发明一些优选实施方式中，所述CAR包含SEQ ID NO：32所示氨基酸序列。
如本发明所用，T细胞“抑制性蛋白”是指与T细胞活性抑制相关的蛋白质，例如免疫抑制性蛋白。在一些具体实施方式中，所述抑制性蛋白选自PD1、LAG-3、CTLA-4、Foxp3、Tim3及其任意组合。在一些具体实施方案中，所述抑制性蛋白选自PD1和TIM3的组合，PD1和CTLA-4的组合，PD1和LAG3的组合，CTLA-4和TIM3的组合，CTLA-4和LAG3的组合，TIM3和LAG3的组合，PD1、TIM3和CTLA-4的组合，PD1、CTLA-4和LAG3的组合，CTLA-4、TIM3和LAG3的组合，PD1、TIM3和LAG3的组合，或PD1、CTLA-4、TIM3和LAG3的组合。在本发明中，减少或消除T细胞中的抑制性蛋白表达并不影响T细胞的扩增能力和其基本免疫特性，并能够通过解除免疫抑制而增强其生物学活性，例如抗肿瘤活性，尤其是抑制或杀伤实体瘤细胞的活性。
本领域已知若干在细胞中减少或消除蛋白质表达的方法。在一些实施方式中，通过反义RNA、antagomir、siRNA、shRNA减少或消除T细胞中抑制性蛋白的表达。在另一些实施方式中，通过基因编辑的方法，例如通过大范围核酸酶、锌指核酸酶、转录激活因子样效应物核酸酶或CRISPR系统减少或消除T细胞中抑制性蛋白的表达。在本发明方法优选的实施方式中，使用CRISPR系统减少或消除T细胞中抑制性蛋白的表达。
在一些实施方式中，CRISPR系统使用的核酸酶(CRISPR核酸酶)例如可以选自Cas3、Cas8a、Cas5、Cas8b、Cas8c、Cas10d、Cse1、Cse2、Csy1、Csy2、Csy3、GSU0054、Cas10、Csm2、Cmr5、Cas10、Csx11、Csx10、Csf1、Cas9、Csn2、Cas4、Cpf1、C2c1、C2c3或C2c2蛋白，或这些核酸酶的功能性变体。
在一些具体实施方式中，所述CRISPR系统是CRISPR/Cas9系统。在一些具体实施方式中，所述CRISPR系统例如CRISPR/Cas9系统靶向所述细胞中选自SEQ ID NO:5、6、13、17、22、23、24、25、26中的一或多个核苷酸序列。
在一些实施方式中，所述CRISPR系统在体外组装，并转入T细胞。在一些实施方式中，将编码所述CRIPSR系统的所有元件的表达构建体转入T细胞。在一些实施方式中，将编码所述CRIPSR系统的部分元件的表达构建体以及其它已转录或已翻译的元件转入T细胞。
本发明还提供一种用于制备经修饰的T细胞的CRISPR基因编辑系统，其包含以下i)至v)中至少一项：
i)CRISPR核酸酶，和至少一种向导RNA；
ii)包含编码CRISPR核酸酶的核苷酸序列的表达构建体，和至少一种向导RNA；
iii)CRISPR核酸酶，和包含编码至少一种向导RNA的核苷酸序列的表达构建体；
iv)包含编码CRISPR核酸酶的核苷酸序列的表达构建体，和包含编码至少一种向 导RNA的核苷酸序列的表达构建体；
v)包含编码CRISPR核酸酶的核苷酸序列和编码至少一种向导RNA的核苷酸序列的表达构建体；
其中所述至少一种向导RNA靶向T细胞中的抑制性蛋白编码基因例如PD1、LAG-3、CTLA-4、Foxp3和/或Tim3。
在一些具体实施方案中，所述至少一种向导RNA靶向T细胞中的PD1和TIM3。在一些具体实施方案中，所述至少一种向导RNA靶向T细胞中的PD1和CTLA-4。在一些具体实施方案中，所述至少一种向导RNA靶向T细胞中的PD1和LAG3。在一些具体实施方案中，所述至少一种向导RNA靶向T细胞中的CTLA-4和TIM3。在一些具体实施方案中，所述至少一种向导RNA靶向T细胞中的CTLA-4和LAG3。在一些具体实施方案中，所述至少一种向导RNA靶向T细胞中的TIM3和LAG3。在一些具体实施方案中，所述至少一种向导RNA靶向T细胞中的PD1、TIM3和CTLA-4。在一些具体实施方案中，所述至少一种向导RNA靶向T细胞中的PD1、CTLA-4和LAG3。在一些具体实施方案中，所述至少一种向导RNA靶向T细胞中的CTLA-4、TIM3和LAG3。在一些具体实施方案中，所述至少一种向导RNA靶向T细胞中的PD1、TIM3和LAG3。在一些具体实施方案中，所述至少一种向导RNA靶向T细胞中的PD1、CTLA-4、TIM3和LAG3。
在一些实施方式中，所述CRISPR核酸酶例如可以选自Cas3、Cas8a、Cas5、Cas8b、Cas8c、Cas10d、Cse1、Cse2、Csy1、Csy2、Csy3、GSU0054、Cas10、Csm2、Cmr5、Cas10、Csx11、Csx10、Csf1、Cas9、Csn2、Cas4、Cpf1、C2c1、C2c3或C2c2蛋白，或这些核酸酶的功能性变体。在一些具体实施方式中，所述CRISPR核酸酶是Cas9核酸酶或其功能性变体。
在一些实施方案中，所述向导RNA靶向LAG-3基因的选自SEQ ID NO:1-5的核苷酸序列。在一些实施方案中，所述向导RNA靶向CTLA-4基因的选自SEQ ID NO:6-10的核苷酸序列。在一些实施方案中，所述向导RNA靶向Foxp3基因的选自SEQ ID NO:11-16的核苷酸序列。在一些实施方案中，所述向导RNA靶向Tim3基因的选自SEQ ID NO:17-21的核苷酸序列。在一些实施方案中，所述向导RNA靶向PD-1基因的选自SEQ ID NO:22和23的核苷酸序列。在优选的实施方式中，所述向导RNA靶向选自SEQ ID NO:5、6、13、17、22和23的核苷酸序列。在优选的实施方案中，所述向导RNA靶向LAG-3基因的SEQ ID NO:24(GCCAGGGGCTGAGGTCCCGG)的核苷酸序列。在优选实施方案中，所述向导RNA靶向CTLA-4基因的SEQ ID NO:25(GTTGAGTAAGGGGTGTACA)的核苷酸序列。在优选的实施方案中，所述向导RNA靶向Tim3基因的SEQ ID NO:26(CAGGGAACCTCGTGCCCGTC)的核苷酸序列。
在本发明的方法的一些实施方案中，包括将本发明的CRISPR基因编辑系统导入所述T细胞。在一些实施方案中，本发明的CRISPR基因编辑系统在导入所述T细胞后，导致减少或消除所靶向的蛋白的表达(敲低或敲除)。
本发明的CRISPR系统可以通过本领域已知的方法转入T细胞，例如：磷酸钙转染、原生质融合、电穿孔、脂质体转染、微注射、病毒感染(如杆状病毒、痘苗病毒、腺病毒和其他病毒)等。
本发明的经修饰的T细胞可以在任何修饰步骤之前或之后被活化和扩增。T细胞可以在体外或体内扩增。通常，本发明的T细胞可以例如通过与刺激CD3 TCR复合物和T细胞表面上的共刺激分子以产生T细胞活化信号的试剂接触来扩增。例如，可以使用诸如钙离子载体A23187、佛波醇12-肉豆蔻酸酯13-乙酸酯(PMA)、或有丝分裂凝集素如植物血凝素(PHA)的化学品来产生T细胞的活化信号。在一些实施方案中，T细胞群体可以通过在体外与例如抗CD3抗体或其抗原结合片段、或固定在表面上的抗CD2抗体接触被活化，或通过与蛋白激酶C激活剂(例如，苔藓抑素)连同钙离子载体的接触来活化。例如，在适合于刺激T细胞增殖的条件下，T细胞群可与抗CD3抗体和抗CD28抗体接触。适用于T细胞培养的条件包括可能含有增殖和活力所必需的因子的合适培养基(例如Minimal Essential Media或RPMI Media 1640、或X-vivo 5、(Lonza))，其中必需的因子包括血清(例如胎牛或人类血清)、白介素-2(IL-2)、胰岛素、IFN-γ、IL-4、IL-7、GM-CSF、IL-10、IL-2、IL-15、TGFp和TNF，或本领域技术人员已知的用于细胞生长的添加剂。其它用于细胞生长的添加剂包括但不限于表面活性剂、人血浆蛋白粉、和还原剂如N-乙酰-半胱氨酸和2-巯基乙酸。培养基可以包括RPMI 1640、A1M-V、DMEM、MEM、a-MEM、F-12、X-Vivo 1和X-Vivo 20、Optimizer、氨基酸、丙酮酸钠和维生素、无血清或适量补充的血清(或血浆)或一组明确的激素、和/或一定量的足以使T细胞生长和扩增的细胞因子。靶细胞可以保持在支持生长所必需的条件下，例如适当的温度(例如37℃)和环境(例如，空气加5％CO2)。
三、经修饰的T细胞
在本发明的另一方面，提供了经修饰的T细胞，其中与未修饰的T细胞相比，所述经修饰的T细胞中的抑制性蛋白的表达被减少或消除。在一些实施方式中，所述经修饰的T细胞通过本发明的方法制备。
在一些具体实施方式中，所述表达被减少或消除抑制性蛋白选自PD1、LAG-3、CTLA-4、Foxp3、Tim3及其任意组合。在一些具体实施方案中，所述表达被减少或消除的抑制性蛋白选自PD1和TIM3的组合，PD1和CTLA-4的组合，PD1和LAG3的组合，CTLA-4和TIM3的组合，CTLA-4和LAG3的组合，TIM3和LAG3的组合，PD1、TIM3和CTLA-4的组合，PD1、CTLA-4和LAG3的组合，CTLA-4、TIM3和LAG3的组合，PD1、TIM3和LAG3的组合，或PD1、CTLA-4、TIM3和LAG3的组合。
在一些实施方式中，所述T细胞中编码所述抑制性蛋白的基因被敲除，例如通过导入本发明的基因编辑系统而敲除。
在本发明中，所述经修饰的T细胞具有与未修饰的T细胞相当的扩增能力和类似的免疫特性，并且由于免疫抑制被解除而具有增强的生物学活性，例如抗肿瘤活性，尤其 是抑制或杀伤实体瘤细胞的活性。
本发明中，所述T细胞可以是包含外源T细胞受体(TCR)的T细胞，或者所述T细胞可以是包含嵌合抗原受体(CAR)的T细胞(CAR-T细胞)。在本发明一些优选实施方式中，所述经修饰的T细胞是CAR-T细胞。
在一些实施方式中，所述TCR或CAR包含针对肿瘤相关抗原的抗原结合结构域，例如胞外抗原结合结构域。
所述肿瘤相关抗原包括但不限于CD16、CD64、CD78、CD96、CLL1、CD116、CD117、CD71、CD45、CD71、CD123、CD138、ErbB2(HER2/neu)、癌胚抗原(CEA)、上皮细胞粘附分子(EpCAM)、表皮生长因子受体(EGFR)、EGFR变体III(EGFRvIII)、CD19、CD20、CD30、CD40、双唾液酸神经节苷脂GD2、导管上皮粘蛋白、gp36、TAG-72、鞘糖脂、神经胶质瘤相关的抗原、β-人绒毛膜促性腺激素、α胎儿球蛋白(AFP)、外源凝集素反应性AFP、甲状腺球蛋白、RAGE-1、MN-CA IX、人端粒酶逆转录酶、RU1、RU2(AS)、肠羧基酯酶、mut hsp70-2、M-CSF、前列腺酶(prostase)、前列腺酶特异性抗原(PSA)、PAP、NY-ESO-1、LAGA-1a、p53、Prostein、PSMA、存活和端粒酶、前列腺癌肿瘤抗原-1(PCTA-1)、MAGE、ELF2M、嗜中性粒细胞弹性蛋白酶、肝配蛋白B2、CD22、胰岛素生长因子(IGF1)-I、IGF-II、IGFI受体、间皮素、呈递肿瘤特异性肽表位的主要组织相容性复合体(MHC)分子、5T4、ROR1、Nkp30、NKG2D、肿瘤基质抗原、纤维连接蛋白的额外结构域A(EDA)和额外结构域B(EDB)、腱生蛋白-C的A1结构域(TnC A1)、成纤维细胞相关蛋白(fap)、CD3、CD4、CD8、CD24、CD25、CD33、CD34、CD133、CD138、Foxp3、B7-1(CD80)、B7-2(CD86)、GM-CSF、细胞因子受体、内皮因子、主要组织相容性复合体(MHC)分子、BCMA(CD269、TNFRSF17)、TNFRSF17(UNIPROT Q02223)、SLAMF7(UNIPROT Q9NQ25)、GPRC5D(UNIPROT Q9NZD1)、FKBP11(UNIPROT Q9NYL4)、KAMP3、ITGA8(UNIPROT P53708)和FCRL5(UNIPROT Q68SN8)。在一优选实施方式中，所述抗原是间皮素。
本发明中，所述抗原结合结构域例如可以是单克隆抗体、合成的抗体、人抗体、人源化抗体、单域抗体、抗体单链可变区，以及其抗原结合片段。
在一优选实施方式中，所述抗原结合结构域是针对间皮素的单克隆抗体。在一优选实施方式中，所述抗原结合结构域是针对间皮素的scFv。在一优选实施方式中，所述抗原结合结构域是针对间皮素的scFv(P4)，其氨基酸序列示于SEQ ID NO：27。
在一些实施方式中，所述CAR包含跨膜结构域，例如CD8跨膜结构域或CD28跨膜结构域，优选CD28跨膜结构域，例如氨基酸序列示于SEQ ID NO：28的CD28跨膜结构域。
在一些实施方式中，所述CAR进一步包括位于胞外抗原结合结构域和所述跨膜结构域之间的铰链区域，例如，所述铰链区域为CD8铰链区，例如氨基酸序列示于SEQ ID NO：29的CD8铰链区。
在一些实施方式中，所述CAR包含可用于T细胞活化的信号转导结构域，例如选 自TCRζ、FcRγ、FcRβ、FcRε、CD3γ、CD3δ、CD3ε、CD3ζ、CD5、CD22、CD79a、CD79b和CD66d的信号转导结构域。在一些优选实施方式中，所述CAR包含CD3ζ信号转导结构域，例如氨基酸序列示于SEQ ID NO：30的CD3ζ信号转导结构域。
在一些实施方式中，所述CAR还包含一或多个选自CD3、CD27、CD28、CD83、CD86、CD127、4-1BB和4-1BBL的共刺激结构域。在一些实施方案中，所述CAR包含CD28共刺激结构域，例如氨基酸序列示于SEQ ID NO：31的CD28共刺激结构域。
在一些实施方式中，所述CAR还可以包含用于显示或追踪CAR表达的报道分子，如GFP蛋白。
在本发明一些优选实施方式中，所述CAR包含针对间皮素的scFv P4、CD8铰链区、CD28跨膜结构域、CD28共刺激结构域、CD3ζ信号转导结构域和任选的GFP蛋白。在本发明一些优选实施方式中，所述CAR包含SEQ ID NO：32所示氨基酸序列。
在本发明一具体实施方式中，所述经修饰的T细胞是包含SEQ ID NO：32所示氨基酸序列的CAR的CAR-T细胞，其中选自以下的抑制蛋白或抑制蛋白的组合的表达被减少或消除：
i).PD1；
ii).LAG-3；
iii).CTLA-4；
iv).Foxp3；
v).Tim3；
vi).PD1和TIM3；
vii).PD1和CTLA-4；
viii).PD1和LAG3；
ix).CTLA-4和TIM3；
x).CTLA-4和LAG3；
xi).TIM3和LAG3；
xii).PD1、TIM3和CTLA-4；
xiii).PD1、CTLA-4和LAG3；
xiv).CTLA-4、TIM3和LAG3；
xv).PD1、TIM3和LAG3；或
xvi).PD1、CTLA-4、TIM3和LAG3。
本发明的T细胞可以通过各种非限制性方法从许多非限制性来源获得，包括外周血单核细胞、骨髓、淋巴结组织、脐带血、胸腺组织、腹水、胸腔积液、脾组织和肿瘤。在一些实施方案中，细胞可以衍生自健康供体或来自诊断为癌症的患者。在一些实施方案中，细胞可以是呈现不同表型特征的细胞的混合群体的一部分。
在本发明各方面的一些实施方案中，所述T细胞衍生自对象的自体细胞。如本文所用，“自体”是指用于治疗对象的细胞、细胞系或细胞群源自所述对象。在一些实施方 案中，所述T细胞衍生自异体细胞，例如源自与所述对象人类白细胞抗原(HLA)相容的供体。可以使用标准方案将来自供体的细胞转化为非同种异体反应性细胞，并根据需要进行复制，从而产生可以施用至一个或多个患者的细胞。
本发明的CAR T细胞或TCR T细胞可以通过本领域已知的多种手段制备。例如，可以用包含CAR或TCR编码序列的表达构建体转导T细胞来获得CAR-T细胞或TCR-T细胞。本领域技术人员能够容易地构建适合于蛋白表达的表达构建体例如病毒载体。
四、药物组合物和应用
在本发明的另一方面，还提供一种用于治疗癌症的药物组合物，其包含本发明的经修饰的T细胞和药学可接受的载体。此外，本发明还提供本发明的经修饰的T细胞在制备用于治疗癌症的药物中的用途。
本文使用的“药学上可接受的载体”包括生理学相容的任何和所有的溶剂、分散介质、包衣、抗细菌剂和抗真菌剂、等渗剂和吸收延迟剂等。优选地，该载体适合于静脉内、肌内、皮下、肠胃外、脊柱或表皮施用(如通过注射或输注)。
在本发明的另一方面，还提供一种用于治疗癌症的方法，包括给有需要的对象施用治疗有效量的本发明的经修饰的T细胞或本发明的药物组合物。
在一些实施方式中，所述方法还进一步包括给所述对象施用放疗和/或化疗和/或另外的肿瘤靶向药物(例如靶向其它抗原的单克隆抗体或小分子化合物)。
如本文所用，“治疗有效量”或“治疗有效剂量”或“有效量”指施用于对象之后至少足以产生疗效的物质、化合物、材料或细胞的量。因此，其为防止、治愈、改善、阻滞或部分阻滞疾病或病症的症状所必需的量。
例如，“有效量”的本发明的细胞或药物组合物优选地导致疾病症状的严重性降低，疾病无症状期的频率和持续时间增加，或者防止因疾病痛苦而引起的损伤或失能。例如，对于肿瘤的治疗，相对于未接受治疗的对象，“有效量”的本发明的细胞或药物组合物优选地将肿瘤细胞生长或肿瘤生长抑制至少约10％，优选至少约20％，更优选至少约30％，更优选至少约40％，更优选至少约50％，更优选至少约60％，更优选至少约70％，更优选至少约80％。抑制肿瘤生长的能力可以在预测对人类肿瘤的疗效的动物模型系统中评价。或者，也可以通过检查抑制肿瘤细胞生长的能力来评价，这种抑制可以通过本领域技术人员公知的试验在体外测定。
实际应用中，本发明药物组合物中细胞的剂量水平可能改变，以获得可有效实现对特定患者、组合物和给药方式的所需治疗反应，而对患者无毒性的活性成分的量。选择的剂量水平取决于多种药物代谢动力学因素，包括应用的本发明特定组合物的活性，给药途径，给药时间，应用的特定化合物的排泄速率，治疗的持续时间，与应用的特定组合物联合应用的其他药物、化合物和/或材料，接受治疗的患者的年龄、性别、体重、状况、总体健康情况和病史，以及医学领域中公知的类似因素。
令人惊奇的是，本发明的经修饰的T细胞，相对于对照T细胞(抑制蛋白的表达未 被减少或消除)，能够以更低的剂量实现更优的治疗效果。这特别有利于减少制备时间和成本，同时能够减少高剂量施用时带来的副作用。
例如，本发明的经修饰的T细胞的施用剂量比所述抑制蛋白的表达未被减少或消除的对照T细胞的施用剂量低约2倍、低约3倍、低约4倍、低约5倍、低约6倍、低约7倍、低约8倍、低约9倍、低约10倍、低约15倍、低约20倍、低约30倍、低约40倍、低约50倍、低约100倍、低约150倍、低约200倍或更低。
可通过本发明的细胞或药物组合物治疗的癌症的非限制性的例子包括肺癌、卵巢癌、结肠癌、直肠癌、黑色素瘤、肾癌、膀胱癌、乳腺癌、肝癌、淋巴瘤、恶性血液病、头颈癌、胶质瘤、胃癌、鼻咽癌、喉癌、宫颈癌、子宫体瘤和骨肉瘤。可以用本发明的方法或药物组合物治疗的其他癌症的例子包括：骨癌、胰腺癌、皮肤癌、前列腺癌、皮肤或眼内恶性黑色素瘤、子宫癌、肛区癌、睾丸癌、输卵管癌、子宫内膜癌、阴道癌、阴户癌、何杰金病、非何杰金氏淋巴瘤、食道癌、小肠癌、内分泌系统癌、甲状腺癌、甲状旁腺癌、肾上腺癌、软组织肉瘤、尿道癌、阴茎癌、慢性或急性白血病(包括急性髓细胞样白血病、慢性髓细胞样白血病、急性成淋巴细胞性白血病、慢性淋巴细胞性白血病)、儿童实体瘤、淋巴细胞性淋巴瘤、膀胱癌、肾或输尿管癌、肾盂癌、中枢神经系统(CNS)肿瘤、原发性CNS淋巴瘤、肿瘤血管发生、脊柱肿瘤、脑干神经胶质瘤、垂体腺瘤、卡波西肉瘤、表皮状癌、鳞状细胞癌、T细胞淋巴瘤、环境诱发的癌症，包括石棉诱发的癌症，以及所述癌症的组合。优选地，所述癌症是实体瘤癌症。
五、试剂盒
本发明还提供一种试剂盒，其用于本发明的制备经修饰T细胞的方法，所述试剂盒包含本文所述的用于制备经修饰的T细胞的CRISPR基因编辑系统，以及合适的将所述基因编辑系统导入细胞中的试剂。所述试剂盒还可以包含用于检测T细胞、分离T细胞、活化T细胞和/或扩增T细胞的试剂。所述试剂盒还可以包含用于将CAR或TCR导入T细胞的试剂、检测和/分离表达所述CAR或TCR的细胞的实际。所述试剂盒还可以包含实施本发明方法的说明。
实施例
通过参考在此给出的一些具体实施例可获得对本发明的进一步的理解，这些实施例仅用于说明本发明，其无意于对本发明的范围做出任何限制。显然，可以对本发明作出多种改动和变化而不脱离本发明的实质，因此，这些改动和变化同样在本申请要求保护的范围内。
实验材料与方法
1.sgRNA的体外转录
首先由生物公司合成包含T7启动子和20bp靶序列(sgRNA)的正向引物，然后以pX330质粒(Addgene plasmid#4223)为PCR模板体外扩增T7-sgRNA PCR产物并利用PCR纯化试剂盒纯化PCR产物。再以纯化的T7-sgRNA PCR产物为模板，利用MEGAshortscript T7 kit(Thermo Fisher Scientific)体外转录sgRNA，并用MEGAclear columns(Thermo Fisher Scientific)回收sgRNA，用无RNA酶的去离子水溶解sgRNA，分装冻存或备用。
2.Cas9蛋白与sgRNA的复合
首先在无RNA酶的EP管中加入适量的相应的sgRNA，然后缓慢加入Cas9蛋白，轻混匀，室温孵育15分钟，形成RNP备用。
3.电穿孔转化方法
1)使用P3 Primary Cell 4D-Nucleofector X Kit；
2)向12孔板中加入1.5ml/孔完全培养基37摄氏度预热30min以上；同时用15ml离心管预热一支培养基；预热50ml PBS
3)向RNase-free EP管中分别加入相应的sgRNA，然后缓慢加入相应量的Cas9蛋白，轻轻混匀，室温孵育20min，形成RNP复合物；
4)配置电转缓冲液:100ul体系：nuclepfector溶液82μl+18μl supplement；
5)在sgRNA和cas9蛋白孵育的期间，准备电转所需的细胞：
6)收集活化3天的T细胞，并计数，取出所需要的细胞量(3e6细胞/样品)；
7)200g，室温离心5min；
8)弃上清，用预热好的PBS将细胞洗一次，200g，室温离心5min；
9)弃上清，尽量去除残留液体；
10)用配好的电转缓冲液重悬细胞，轻柔混匀；
11)从重悬好的细胞中取出200μl/样品(包括复孔)加入孵育好的RNP中，轻柔混匀，然后将混合物100μl/样品加入电转杯；
12)电转，程序为stimulated T cells(EO-115)；
13)迅速向电转后的电转杯中加入预热好的培养基500μl，用移液管轻混匀，吸出细胞，加入预热好的12孔板中，将细胞放回培养箱培养，37，℃5％CO2；
14)电转6小时后半量换液，小心从培养箱取出细胞，不要晃动，沿着孔壁小心吸出1ml/孔培养基，然后向培养孔中补充1ml预热好的T细胞培养基；
15)之后根据细胞生长状态，按时传代，维持细胞密度在1e6细胞/ml。
4.TIDE分析方法
收集细胞用细胞裂解液(100μg/ml ProteinaseK,10 mMTris-HCl,2 mM EDTA,2.5％Tween-20 and 2.5％Triton-X 100)提取实验组和对照组基因组DNA。PCR体外扩增目的 片段，并利用sanger测序。然后利用在线工具(http://tide.nki.nl)分析基因突变效率。
5.T细胞培养培养方法
T细胞完全培养基：X-VIVO15 medium+5％FBS(热灭活)+2mM L-谷氨酰胺+1mM丙酮酸钠+300U/ml IL-2。T细胞分离后用T细胞完全培养基调整细胞密度为1e6细胞/ml，用
Figure PCTCN2018086019-appb-000001
Human T-Activator CD3/CD28以1：1的比例激活。然后根据T细胞生长状态每2-3天换液传代。
6.T细胞免疫表型分析方法
通过流式细胞术的方法分析细胞亚群的变化，包括CD4、CD8、初始T细胞(CD45RO-/CD62L+,TN)、中央记忆T细胞(CD45RO+/CD62L+,TCM)、效应记忆T细胞(CD45RO+/CD62L-,TEM)。与未进行基因编辑的T细胞比较。
7.流式细胞方法
1)FACS buffer配置：2％FBS+1mM EDTA+98％PBS；
2)取1e6细胞，FACS缓冲液洗两遍；
3)用FACS缓冲液100ul重悬，混匀，加入适量相应流式抗体(用量参考说明书)混匀，室温避光染色10min；
4)FACS缓冲液洗涤两遍，再用缓冲液200-300μl重悬，混匀，一个小时以内上机检测；如果不能及时上机，需要用4％多聚甲醛固定。
8.细胞因子测量方法
将效应子(T细胞或CAR-T细胞)与表达CD19的Raji细胞、不表达CD19的K562细胞和以CD19修饰的K562细胞(称为K562-CD19或K19)或H226细胞和表达荧光素酶的H226细胞共培养。细胞比例为1:1(每孔的效应子和肿瘤细胞各10 4个)，在V形底96孔板中进行。使用完全RPM1640培养基，终体积200μL。培养24小时后，用ELISA试剂盒检测上清中的IL-2和IFN-γ。
9.肿瘤细胞裂解实验
根据基于流式细胞术的细胞毒性测定评估细胞毒性。用1μM的Celltrace Violet标记靶肿瘤细胞K19，然后将其与效应子细胞(T细胞、CAR-T细胞和基因敲除的CAR-T细胞)温育4小时。然后加入FITC-Annexin V和7-AAD(Biolegend)以确定死亡的靶细胞的比例。
或者，将肿瘤细胞以10 4/100ul的密度放到micro-assay-plate的96孔板中(greiner bio-one)。将CAR-T细胞精确计数，按不同的效应细胞:靶细胞比例进行稀释，加入到相应的孔中100ul，对照组是加入100ul的培养基。到达时间后，所有的孔加入
Figure PCTCN2018086019-appb-000002
Luciferase Assay System 10ul，5分钟后，在酶标仪下读数。得到读数后，计算杀伤的百分比：杀伤(％)＝100-(实验组读数/对照组读数)*100
实施例1、敲除CAR-T细胞中的LAG-3基因
1、筛选靶向T细胞上LAG-3最有效的sgRNA
为消除T细胞中的LAG-3表达，设计了五种sgRNA，靶向LAG-3的第一外显子。图1A以sgRNA5为例，图示了所述sgRNA在LAG-3基因座中的位置。
所涉及的sgRNA的靶向序列如表1所示。
表1、靶向LAG-3的sgRNA
sgRNA 靶序列 SEQ ID NO
sgRNA1 ATGTGGGAGGCTCAGTTCCT 1
sgRNA2 GCTGCAGAAACAGCAAGCCC 2
sgRNA3 TGCTGTTTCTGCAGCCGCTT 3
sgRNA4 GCTGTTTCTGCAGCCGCTTT 4
sgRNA5 GTTTCTGCAGCCGCTTTGGG 5
Cas9蛋白(3μg)和体外转录的sgRNA(3μg)进行复合，然后电穿孔进入原代CD3 +T细胞中。用TIDE分析定量使用各sgRNA的基因编辑效率，选择用于进一步实验的最有效的sgRNA，结果示于图1B。如图所示，sgRNA5的敲除效率最高。通过克隆测序确定NHEJ修复导致的插入和缺失(indel)的出现。扩增了sgRNA5的靶区域，并鉴定了独立的突变。如图1C所示，所有突变精确发生在sgRNA靶向的区域。这些结果说明，通过基因编辑在原代T细胞中有效地敲除了LAG-3。
2、评估电穿孔和基因编辑对T细胞增殖和表型的作用
为了确定基因编辑对T细胞增殖和表型的作用，测试了LAG-3敲除的T细胞的增殖。电穿孔后培养。在电穿孔后第3天和第7天，计数总细胞数，从而测定对照T细胞和LAG-3敲除的T细胞的扩增倍数。结果如图2A所示，LAG-3敲除的T细胞保持正常的依赖抗CD3和抗CD28抗体刺激的增殖。
通过CD4、CD8表达以及naive(CD45RO-/CD62L+，TN)、中心记忆(CD45RO+/CD62L+，TCM)和效应记忆(CD45RO+/CD62L-，TEM)T细胞亚集的特性评估基因编辑的T细胞的免疫表型。结果显示，在LAG-3敲除的T细胞中显示CD4和TCM部分稍微更高。然而，这一作用似乎与电穿孔相关，因为这一特征在未编辑的但接受电穿孔的T细胞中也有显示(图2B)。总的来说，LAG-3敲除的T细胞展示与未编辑的T细胞相似的表型。
3、制备LAG-3敲除的CAR-T细胞
使用抗CD19 CAR-T细胞进行LAG-3基因编辑。
简单来说，在存在IL-2(50U/ml)的情况下，用抗CD3和抗CD28刺激活化CD19 CAR-T细胞三天。然后，使用电穿孔将包含sgRNA5的CRISPR-Cas9系统转入所述CAR-T细胞，在电穿孔后三天评估基因编辑效率。使用三个不同的供体评估了LAG-3敲除率，结果如图3A所示，观察到敲除率为40-70％。
此外，还通过流式细胞术确认检测了基因编辑后的CAR-T细胞上LAG-3的表达，结果如图3B所示。
4、表征LAG3敲除的CAR-T细胞
首先，评估了LAG3敲除的CAR-T细胞的增殖。如图4A所示，尽管基因编辑方法对细胞增殖有些影响，这些编辑的CAR-T细胞在抗CD3和抗CD28刺激下增殖良好。
在转染后第14天收获CAR-T细胞，进行免疫表型分析。如图4B所示，与未修饰的T细胞相似，LAG3敲除的CAR-T细胞没有显示任何显著CD4和CD8表达以及记忆T细胞表型特征的改变。也就是说，CRISPR-Cas9介导的LAG3破坏没有干扰T细胞免疫表型。
为评估LAG3敲除的CAR-T细胞的细胞毒性功能，测定LAG3敲除的CAR-T细胞释放IL-2和IFN-γ的能力，结果如图4C所示。LAG3敲除的CAR-T细胞释放的细胞因子的量与未编辑的CAR-T细胞相似。
此外，还检测了LAG3敲除的CAR-T细胞裂解肿瘤细胞的能力。效应子细胞：靶细胞的比例为16:1、8:1和4:1。结果如图4D所示，说明LAG3敲除的CAR-T细胞至少保留与标准CAR-T细胞相等的抗肿瘤活性。
5、LAG-3敲除的CAR-T细胞在鼠异体移植模型中根除肿瘤
在第0天，对6-12周龄的NOD-Prkdc scid Il2rg null(NPG)小鼠腹腔内注射2×10 5Raji-荧光酶细胞。在第3天，小鼠接受腹腔内注射的1×10 7T细胞、CAR-T细胞或LAG-3敲除的CAR-T细胞，或PBS。
在第3天、第10天及第31天进行生物发光成像，检测用各种处理的NPG小鼠(n＝4)，成像结果如图5A所示；用T细胞、CAR-T细胞或LAG-3敲除的CAR-T细胞处理的小鼠的生物发光信号如图5B所示，数据显示为平均值±SEM，n＝4。这些数据说明破坏抑制性基因LAG-3在鼠异体移植模型中导致更有效的抗肿瘤应答。
对生存的小鼠进行监测，直到第60天。各组的小鼠生存百分比如图5C所示。
实施例2、敲除CAR-T细胞中的CTLA-4基因
1、筛选靶向CTLA-4的sgRNA
设计五种靶向CTLA-4基因座外显子1编码区的sgRNA，靶序列如SEQ ID NO:6-10(表2)，如图6A所示，sgRNA1的靶向序列为绿色，PAM序列为蓝色。
表2、靶向CTLA-4的sgRNA
sgRNA CTLA-4靶序列 SEQ ID NO
sgRNA1 CCTTGGATTTCAGCGGCACA 6
sgRNA2 CCTTGTGCCGCTGAAATCCA 7
sgRNA3 TGAACCTGGCTACCAGGACC 8
sgRNA4 CATAAAGCCATGGCTTGCCT 9
sgRNA5 CTCAGCTGAACCTGGCTACC 10
Cas9蛋白(3μg)和体外转录的sgRNA(3μg)进行复合形成Cas9-sgRNA核糖核蛋白(RNP)，然后电穿孔进入原代1×10 6CD3+T细胞(活化三天后)。
用TIDE分析定量使用各sgRNA的敲除效率，并测序分析CTLA-4中的插入缺失频率。结果如图6B所示，sgRNA1的敲除效率最高，并且，在另一个供体(供体2)中用sgRNA1进行基因编辑也获得了显著的敲除效果。
亚克隆各样品的PCR产物，对各克隆的等位基因进行测序；将RNP转染的细胞中代表性突变的等位基因与野生型序列的比较。结果如图6C所示。sgRNA的靶向序列为绿色，PAM序列为蓝色，突变序列为红色。N/N是指含有突变的等位基因的克隆数/测序的总克隆数。
2、评估敲除CTLA-4对T细胞增殖和表型的作用
为了确定基因编辑对T细胞增殖和表型的作用，测试了CTLA-4敲除的T细胞的增殖。电穿孔后培养。在电穿孔后第3天和第7天，计数总细胞数，从而测定对照T细胞和CTLA-4敲除的T细胞的扩增倍数。结果如图7A所示，CTLA-4敲除的T细胞保持正常的依赖抗CD3和抗CD28抗体刺激的增殖。
通过CD4、CD8表达以及naive(CD45RO-/CD62L+,TN)、中心记忆(CD45RO+/CD62L+,TCM)和效应记忆(CD45RO+/CD62L-,TEM)T细胞亚集的特性评估基因编辑的T细胞的免疫表型。进行两次独立实验，结果显示为平均值±SEM。结果如图7B所示。
3、制备CTLA-4敲除的CAR-T细胞
使用抗CD19 CAR-T细胞进行CTLA-4基因编辑。
抗CD19 CAR-T细胞培养活化三天。然后，通过电穿孔将CRISPR-Cas9和sgRNA1的RNP转入CAR-T细胞，在电穿孔后三天评估基因编辑效率。使用三个不同的供体评估了CTLA-4敲除率，结果如图8A所示。
亚克隆各样品的PCR产物，对各克隆的等位基因进行测序；将RNP转染的细胞中代表性突变的等位基因与野生型序列的比较。结果如图8B所示。sgRNA的靶向序列为绿色，PAM序列为蓝色，突变序列为红色。N/N是指含有突变的等位基因的阳性克隆数/测序的总克隆数。
此外，在电穿孔后第3天，用流式细胞仪分析CTLA-4敲除的CAR-T细胞的CTLA-4表面表达，结果如图8C所示。
4、表征CTLA-4敲除的CAR-T细胞
为了确定基因编辑对CAR-T细胞增殖和表型的作用，在三个供体测试了CTLA-4敲除的CAR-T细胞的增殖。电穿孔后培养。在电穿孔后不同时间点计数总细胞数，从而测定对照CAR-T细胞和CTLA-4敲除的CAR-T细胞的扩增倍数。进行两次独立实验，数据显示为平均值±SEM，结果如图9A所示。
为体外表征CTLA-4敲除的抗CD19 CAR-T细胞，在电穿孔后进行培养。培养至电 穿孔后第10天，通过CD4、CD8表达以及naive、中心记忆和效应子记忆T细胞亚集评估基因编辑的T细胞的免疫表型。进行三次独立实验，数据显示为平均值±SEM，结果如图9B所示。
还在三个独立供体中检测了代表性的IL-2和IFN-γ。结果如图12B所示，数据显示为平均值±SEM，n＝2。
此外，通过评估T细胞、CAR-T细胞和CTLA-4敲除的CAR-T细胞裂解肿瘤细胞的能力测定了其细胞毒性。结果如图9D所示，效应子细胞：靶细胞的比例为10:1、5:1和2.5:1。
5、体内评估CTLA-4敲除的CAR-T细胞的抗肿瘤能力
在第0天，对6-12周龄的NOD-Prkdc scid Il2rg null(NPG)小鼠腹腔内注射2×10 5Raji-荧光酶细胞。在第3天，小鼠接受腹腔内注射的1×10 7T细胞、CAR-T细胞或CTLA-4敲除的CAR-T细胞，或PBS。
在第3天、第10天和第31天进行生物发光成像，检测用各种处理的NPG小鼠(n＝4)，成像结果如图10A所示；用T细胞、CAR-T细胞或CTLA-4敲除的CAR-T细胞处理的小鼠的生物发光信号如图10B所示，数据显示为平均值±SEM，n＝4。
对生存的小鼠进行计数，直到第60天。各组的小鼠生存百分比如图10C所示。
实施例3、敲除CAR-T细胞中的Foxp3基因
1、筛选靶向Foxp3的sgRNA
设计六种靶向Foxp3基因座外显子2编码区的sgRNA，序列如SEQ ID NO:11-15(表3)，如图11A所示，sgRNA3的靶向序列为绿色，PAM序列为蓝色。
表3、靶向Foxp3的sgRNA
sgRNA Foxp3靶序列 SEQ ID NO
sgRNA1 GGGCCGAGATCTTCGAGGCG 11
sgRNA2 TCGAAGATCTCGGCCCTGGA 12
sgRNA3 GCAGCTGCGATGGTGGCATG 13
sgRNA4 AGGGCCGAGATCTTCGAGGC 14
sgRNA5 GGCCCTGGAAGGTTCCCCCT 15
sgRNA6 TTTGGGTGCAGCCCTCCAGC 16
Cas9蛋白(3μg)和体外转录的sgRNA(3μg)进行复合形成Cas9-sgRNA核糖核蛋白(RNP)，然后电穿孔进入原代1×10 6CD3+T细胞(活化三天后)中。
用TIDE分析定量使用各sgRNA的敲除效率，测序分析了Foxp3插入缺失频率，结果如图11B所示，sgRNA3的敲除效率最高，并且，在另两个供体(D2和D3)中用sgRNA3进行基因编辑，也实现了有效的基因敲除。
亚克隆各样品的PCR产物，对各克隆的等位基因进行测序；将RNP转染的细胞中代表性突变的等位基因与野生型序列的比较。结果如图11C所示。sgRNA的靶向序列 为绿色，PAM序列为蓝色，突变序列为红色。N/N是指含有突变的等位基因的克隆数/测序的总克隆数。
此外，在电穿孔后第3天，用流式细胞仪分析Foxp3敲除的T细胞的Foxp3表面表达，结果如图11D所示。
2、评估敲除Foxp3对T细胞增殖和表型的作用
为了确定基因编辑对T细胞增殖和表型的作用，测试了Foxp3敲除的T细胞的增殖。电穿孔后培养。在电穿孔后第3天和第7天，计数总细胞数，从而测定对照T细胞和Foxp3敲除的T细胞的扩增倍数，结果如图12A所示。Foxp3敲除的T细胞保持正常的依赖抗CD3和抗CD28抗体刺激的增殖。
通过CD4、CD8表达以及naive(CD45RO-/CD62L+,TN)、中心记忆(CD45RO+/CD62L+,TCM)和效应记忆(CD45RO+/CD62L-,TEM)T细胞亚集的特性评估基因编辑的T细胞的免疫表型。进行两次独立实验，结果显示为平均值±SEM。结果如图12B所示。
3、制备Foxp3敲除的CAR-T细胞
使用抗CD19 CAR-T细胞进行Foxp3基因编辑。
简单来说，培养CD19 CAR-T细胞三天进行活化。然后，通过电穿孔将CRISPR-Cas9和sgRNA3的RNP转入CAR-T细胞，在电穿孔后三天评估基因编辑效率。使用三个不同的供体评估了Foxp3敲除率，结果如图13A所示。
亚克隆各样品的PCR产物，对各克隆的等位基因进行测序；将RNP转染的细胞中代表性突变的等位基因与野生型序列的比较。结果如图13B所示。sgRNA的靶向序列为绿色，PAM序列为蓝色，突变序列为红色。N/N是指含有突变的等位基因的阳性克隆数/测序的总克隆数。
4、表征Foxp3敲除的CAR-T细胞
为了确定基因编辑对CAR-T细胞增殖和表型的作用，在三个供体测试了Foxp3敲除的CAR-T细胞的增殖。电穿孔后培养。在电穿孔后，计数总细胞数，从而测定对照CAR-T细胞和Foxp3敲除的CAR-T细胞的扩增倍数。进行两次独立实验，数据显示为平均值±SEM，结果如图14A所示。
为体外表征Foxp3敲除的抗CD19 CAR-T细胞，在电穿孔后进行培养。培养至电穿孔后第10天，通过CD4、CD8表达以及naive、中心记忆和效应记忆T细胞亚集评估基因编辑的T细胞的免疫表型。进行三次独立实验，数据显示为平均值±SEM，结果如图14B所示。
还在三个独立供体中检测了代表性的IL-2和IFN-γ。结果如图14C所示，数据显示为平均值±SEM，n＝2。
此外，通过评估T细胞、CAR-T细胞和Foxp3敲除的CAR-T细胞裂解细胞的能力测定了其细胞毒性。效应子细胞：靶细胞的比例为10:1、5:1和2.5:1。结果如图14D所示。
5、体内评估Foxp3敲除的CAR-T细胞的抗肿瘤能力
在第0天，对6-12周龄的NOD-Prkdc scid Il2rg null(NPG)小鼠腹腔内注射2×10 5Raji-荧光酶细胞。在第3天，小鼠接受腹腔内注射的1×10 7T细胞、CAR-T细胞或Foxp3敲除的CAR-T细胞，或PBS。
在第3天、第10天和第31天进行生物发光成像，检测用各种处理的NPG小鼠(n＝4)，成像结果如图15A所示；用T细胞、CAR-T细胞或Foxp3敲除的CAR-T细胞处理的小鼠的生物发光信号如图15B所示，数据显示为平均值±SEM，n＝4。
对生存的小鼠进行计数，直到第60天。各组的小鼠生存百分比如图15C所示。
实施例4、敲除CAR-T细胞中的Tim3基因
1、筛选靶向T细胞上Tim3的最有效的sgRNA
设计五种靶向Tim3基因座外显子2编码区的sgRNA，靶序列如SEQ ID NO:17-21(表4)。如图16A所示，sgRNA1的靶向序列为绿色，PAM序列为蓝色。
表4、靶向Tim3的sgRNA
sgRNA Tim3靶序列 SEQ ID NO
sgRNA1 CTGGTTTGATGACCAACTTC 17
sgRNA2 TGAAAAATTTAACCTGAAGT 18
sgRNA3 CTGAAGTTGGTCATCAAACC 19
sgRNA4 GAATGATGAAAAATTTAACC 20
sgRNA5 CCTGGTTTGATGACCAACTT 21
Cas9蛋白(3μg)和体外转录的sgRNA(3μg)进行复合形成Cas9-sgRNA核糖核蛋白(RNP)，然后电穿孔进入原代1×10 6CD3+T细胞(活化三天后)中。
用TIDE分析定量使用各sgRNA的敲除效率，测序分析了Tim3插入缺失频率，结果如图16B所示，sgRNA1的敲除效率最高，并且，在另外的供体(D2)来源的T细胞和CAR-T细胞中用sgRNA1进行基因编辑也得到了有效的敲除效果。
此外，在电穿孔后第3天，用流式细胞仪分析Tim3敲除的T细胞的Tim3表面表达，结果如图16C所示。
2、评估敲除Tim3对T细胞增殖和表型的作用
为了确定基因编辑对T细胞增殖和表型的作用，测试了Tim3敲除的T细胞的增殖。电穿孔后培养。在电穿孔后第3天和第7天，计数总细胞数，从而测定对照T细胞和Tim3敲除的T细胞的扩增倍数，结果如图17A所示。
通过CD4、CD8表达以及
Figure PCTCN2018086019-appb-000003
(CD45RO-/CD62L+,TN)、中心记忆(CD45RO+/CD62L+,TCM)和效应记忆(CD45RO+/CD62L-,TEM)T细胞亚集的特性评估基因编辑的T细胞的免疫表型。进行两次独立实验，结果显示为平均值±SEM。结果如图17B所示。
3、制备Tim3敲除的CAR-T细胞
使用抗CD19 CAR-T细胞进行Tim3基因编辑。
培养CD19 CAR-T细胞三天进行活化。然后，通过电穿孔将CRISPR-Cas9与sgRNA1的RNP转入CAR-T细胞。
4、体外表征Tim3敲除的CAR-T细胞
为了确定基因编辑对CAR-T细胞增殖和表型的作用，在三个供体测试了Tim3敲除的CAR-T细胞的增殖。电穿孔后培养。在电穿孔后，计数总细胞数，从而测定对照CAR-T细胞和Tim3敲除的CAR-T细胞的扩增倍数。进行两次独立实验，数据显示为平均值±SEM，结果如图18A所示。
为体外表征Tim3敲除的抗CD19 CAR-T细胞，在电穿孔后进行培养。培养至电穿孔后第10天，通过CD4、CD8表达以及naive、中心记忆和效应记忆T细胞亚集评估基因编辑的T细胞的免疫表型。进行三次独立实验，数据显示为平均值±SEM，结果如图18B所示。
还检测了代表性的IL-2和IFN-γ。结果如图18C所示，数据显示为平均值±SEM，n＝2。
实施例5、敲除CAR-T细胞中的PD1基因
1、制备PD1敲除的CAR-T细胞
设计了靶向PD1外显子1的sgRNA。其中sgRNA1靶向有义链而sgRNAp靶向反义链(见图19A)。
sgRNA1靶向的PD1基因序列为：GTCTGGGCGGTGCTACAACT(SEQ ID NO:22)；sgRNAp靶向的PD1基因序列为：ACAGGCGCCCTGGCCAGTCG(SEQ ID NO:23)。
将所设计的sgRNA和Cas9蛋白通过电穿孔共转抗-Meso CART细胞。通过Surveyor测定显示PD1基因编辑效率达到27.9％。
2、PD1敲除的抗-Meso CART细胞在体内能执行效应子功能
将2x 10 6高表达PDL1和荧光素酶的H226细胞皮下注射进NPG小鼠胁腹构建人肺鳞癌小鼠模型。在第27天和第32天瘤内注射1 x 10 7PD1-KO抗-间皮素(Meso)CART细胞、抗-Meso CART细胞和用作未处理对照的PBS。通过生物荧光成像检测肿瘤生长，每周拍摄。
结果如图20A和图20B所示。与未处理对照相比，PD1-KO抗-Meso CART细胞处理的小鼠中的H226生物荧光信号在第一次T细胞注射之后迅速降低至接近背景水平并且一直保持低信号水平。与未处理对照相比，抗-Meso CART细胞处理的小鼠中的H226生物荧光信号在第一次T细胞注射之后迅速降低，但是在第二次T细胞注射后生物荧光信号缓慢升高。此外，接受抗间皮素CAR-T细胞的小鼠、接受PD1敲除的抗间皮素CAR-T细胞的小鼠以及未接受治疗的对照小鼠的存活率示于图20C。接受PD1敲除的抗间皮素CAR-T细胞的小鼠显示更高的存活率。
3、PD1敲除的抗-Meso CART细胞在体外能执行效应子功能
表达荧光素酶的H226细胞(H226-luci)和小鼠成纤维细胞3T3或表达PDL1的3T3细胞(3T3-PDL1)分别和PD1-KO抗-Meso CART细胞、抗-Meso CART细胞以及普通未修饰T细胞以1:1、1:2或1:4的效应细胞:靶细胞比例共培养20小时。通过测量剩余肿瘤细胞的荧光素酶活性计算靶细胞裂解的百分比。
如图21A所示，当H226-luci和3T3细胞分别与PD1-KO抗-Meso CART细胞、抗-Meso CART细胞以及T细胞共培养时，PD1-KO抗-Meso CART细胞比抗-Meso CART细胞能够更高效地杀死靶H226细胞。
如图21B所示，当H226-luci和3T3-PDL1细胞分别与PD1-KO抗-Meso CART细胞、抗-Meso CART细胞以及T细胞共培养时，PD1-KO抗-Meso CART细胞比抗-Meso CART细胞能够更高效地杀死靶H226细胞。
实施例6、具有不同CAR结构的CAR-T细胞的作用
设计了4种结构的CAR(P4-z、P4-BBz、P4-28z和P4-28BBz，结构如图22所示，其中P4是抗间皮素scFv)，并制备CAR-T细胞。
以1:1的效应细胞:靶细胞比例，将所得到的CAR-T细胞与H226细胞共培养20h，测量IFN-γ和IL-2的释放。
以2:1的效应细胞:靶细胞比例，将所得到的CAR-T细胞与表达荧光素酶的H226细胞(H226-luci)共培养3天，通过测量剩余肿瘤细胞的荧光素酶活性计算靶细胞裂解的百分比。
如图23所示，与P4-z、P4-BBz和P4-28BBz相比，P4-28z显示更高的IFN-γ和IL-2的释放(图23A)，以及更高的特异性细胞裂解(图23B，*表示P<0.05，****表示P<0.0001)。
实施例7、CAR-T细胞中抑制蛋白的多种基因编辑
敲除抗间皮素CAR-T细胞中的PD-1、TIM-3和LAG-3中的一或多种，其中用靶向SEQ ID NO:22和/或SEQ ID NO:23的sgRNA敲除PD-1，用靶向SEQ ID NO:26的sgRNA敲除TIM-3，用靶向SEQ ID NO:25的sgRNA敲除CTLA-4，而用靶向SEQ ID NO:5和/或24的sgRNA敲除LAG-3。
1.分别产生敲除PD-1的P4 CAR-T细胞(PD1 KO)，敲除TIM3的P4 CAR-T细胞(TIM3 KO)，敲除LAG3的P4 CAR-T细胞(LAG3 KO)，敲除PD1和TIM3的P4 CAR-T细胞(PD1 TIM3 KO)，敲除PD1和LAG3的P4 CAR-T细胞(PD1 LAG3 KO)，以及敲除PD1、TIM3和LAG3的P4 CAR-T细胞(PD1 TIM3 LAG3 KO)。通过Surveyor测试和TIDE检测PD-1、LAG-3和TIM3基因的敲除效率，结果如图24所示。
将获得的细胞和未经敲除的P4 CAR-T细胞以及T细胞分别与表达PD-L1和荧光素酶的H226细胞(H226-PDL1-luci)、CRL5826细胞(CRL5826-PDL1)共培养。
如图25A所示，当效应细胞:靶细胞(H226-PDL1-luci)的比例为4:1时，共培养20h后，除P4(TIM3 KO)外，其它经敲除的CAR-T细胞都能比P4更高效杀地伤靶细胞；如图25B所示，当效应细胞:靶细胞(H226-PDL1-luci)的比例为0.1:1时，共培养6天后，所有经敲除的CAR-T细胞都能比P4更高效杀地伤靶细胞，*,P<0.05.**,P<0.01.***,P<0.001.****,P<0.0001。
图26显示，当效应细胞:靶细胞(CRL5826-PDL1)的比例为4:1时，共培养24小时(图26A)和48小时(图26B)后，经敲除的CAR-T细胞表现出不低于P4 CAR-T细胞的肿瘤杀伤效果。
图27显示，当效应细胞:靶细胞(CRL5826-PDL1)的比例为0.1:1时，共培养4天(图27A)和6天(图27B)后，经敲除的CAR-T细胞均表现出明显比P4 CAR-T细胞更强的肿瘤杀伤效果。
图28显示，当效应细胞:靶细胞(CRL5826-PDL1)的比例为0.02:1时，共培养4天(图28A)和6天(图28B)后，经敲除的CAR-T细胞均表现出明显比P4 CAR-T细胞更强的肿瘤杀伤效果。
2.分别产生敲除PD-1、TIM3、CTLA4和LAG3的一或多种的CAR-T细胞，敲除PD1以P表示，敲除TIM3以T表示，敲除CTLA4以C表示，且敲除LAG3以L表示，敲除两个或更多个基因以相应的字母组合表示。例如PC表示敲除PD1和CTLA4，而PCTL表示这四个基因全部被敲除。
将所获得的经敲除的CAR-T细胞和P4 CAR-T细胞以及T细胞分别与表达荧光素酶的CRL5826细胞(CRL5826)、OVCAR3细胞(OVCAR3)和HCT116细胞(HCT116)，表达PDL1和荧光素酶的CRL5826细胞(CRL5826-PDL1)、OVCAR3细胞(OVCAR3-PDL1)和HCT116细胞(HCT116-PDL1)共培养。
图29显示，当效应细胞:靶细胞(CRL5826)的比例为1:1时，共培养24小时(图29A)和48小时(图29B)后，经敲除的CAR-T细胞表现出不低于P4 CAR-T细胞的肿瘤杀伤效果。
图30显示，当效应细胞:靶细胞(CRL5826-PDL1)的比例为1:1时，共培养24小时(图30A)和48小时(图30B)后，经敲除的CAR-T细胞表现出不低于P4 CAR-T细胞的肿瘤杀伤效果。
图31显示，当效应细胞:靶细胞的比例为1:1时，共培养24小时后，经敲除的CAR-T细胞表现出不低于P4 CAR-T细胞的肿瘤杀伤效果。图31A中的靶细胞为表达荧光素酶的OVCAR3细胞(OVCAR3)，而图31B中的靶细胞为表达PDL1和荧光素酶的OVCAR3细胞(OVCAR3-PDL1)。
图32显示，当效应细胞:靶细胞的比例为1:1时，共培养24小时后，经敲除的CAR-T细胞表现出不低于P4 CAR-T细胞的肿瘤杀伤效果。图32A中的靶细胞为表达荧光素酶的HCT116细胞(HCT116)，而图32B中的靶细胞为表达PDL1和荧光素酶的HCT116细胞(HCT116-PDL1)。
图33显示，当效应细胞:靶细胞的比例为1:1时，共培养48小时后，经敲除的CAR-T细胞表现出比P4 CAR-T细胞更强的肿瘤杀伤效果。图33A中的靶细胞为表达荧光素酶的HCT116细胞(HCT116)，而图33B中的靶细胞为表达PDL1和荧光素酶的HCT116细胞(HCT116-PDL1)。
图34显示，当效应细胞:靶细胞的比例为0.1:1时，共培养4天后，经敲除的CAR-T细胞表现出明显比P4 CAR-T细胞更强的肿瘤杀伤效果。图34A中的靶细胞为表达荧光素酶的CRL5826细胞(CRL5826)，而图34B中的靶细胞为表达PDL1和荧光素酶的CRL5826细胞(CRL5826-PDL1)。
图35显示，当效应细胞:靶细胞的比例为0.1:1时，共培养48小时后，经敲除的CAR-T细胞表现出明显比P4 CAR-T细胞更强的肿瘤杀伤效果。图35A中的靶细胞为表达荧光素酶的OVCAR3细胞(OVCAR3)，而图35B中的靶细胞为表达PDL1和荧光素酶的OVCAR3细胞(OVCAR3-PDL1)。
实验结果说明，本发明的一或多种抑制性蛋白被敲除的CAR-T细胞在高效靶比下与未敲除CAR-T细胞效果相当，出乎意料的是，本发明的被敲除的CAR-T细胞在低效靶比、较长作用时间下优于未敲除CAR-T细胞。这特别有利于降低成本，减少制备时间，减少高剂量施用时带来的副作用。
Figure PCTCN2018086019-appb-000004
Figure PCTCN2018086019-appb-000005
Figure PCTCN2018086019-appb-000006

Claims (26)

  1. 一种制备经修饰的T细胞的方法，包括减少或消除T细胞中抑制性蛋白表达的步骤。
  2. 权利要求1的方法，其中所述T细胞是包含外源T细胞受体(TCR)或嵌合抗原受体(CAR)的T细胞。
  3. 权利要求1或2的方法，其中所述表达被减少或消除的抑制性蛋白选自PD1、LAG-3、CTLA-4、Foxp3、Tim3及其组合。
  4. 权利要求1-3任一项的方法，其中所述表达被减少或消除的抑制性蛋白选自PD1和TIM3的组合，PD1和CTLA-4的组合，PD1和LAG3的组合，CTLA-4和TIM3的组合，CTLA-4和LAG3的组合，TIM3和LAG3的组合，PD1、TIM3和CTLA-4的组合，PD1、CTLA-4和LAG3的组合，CTLA-4、TIM3和LAG3的组合，PD1、TIM3和LAG3的组合，或PD1、CTLA-4、TIM3和LAG3的组合。
  5. 权利要求1-4任一项的方法，其中通过反义RNA、antagomir、siRNA、shRNA、大范围核酸酶、锌指核酸酶、转录激活因子样效应物核酸酶或CRISPR系统实施所述减少或消除。
  6. 权利要求5的方法，其中所述CRISPR系统是CRISPR/Cas9系统。
  7. 权利要求6的方法，其中所述CRISPR/Cas9系统靶向所述细胞内选自SEQ ID NO:5、6、13、17、22-26中一或多种的核苷酸序列。
  8. 权利要求2-7任一项的方法，其中所述TCR或CAR包含针对肿瘤相关抗原的抗原结合结构域。
  9. 权利要求8的方法，其中所述肿瘤相关抗原选自CD16、CD64、CD78、CD96、CLL1、CD116、CD117、CD71、CD45、CD71、CD123、CD138、ErbB2(HER2/neu)、癌胚抗原(CEA)、上皮细胞粘附分子(EpCAM)、表皮生长因子受体(EGFR)、EGFR变体III(EGFRvIII)、CD19、CD20、CD30、CD40、双唾液酸神经节苷脂GD2、导管上皮粘蛋白、gp36、TAG-72、鞘糖脂、神经胶质瘤相关的抗原、β-人绒毛膜促性腺激素、α胎儿球蛋白(AFP)、外源凝集素反应性AFP、甲状腺球蛋白、RAGE-1、MN-CA IX、人端粒酶逆转录酶、RU1、RU2(AS)、肠羧基酯酶、mut hsp70-2、M-CSF、前列腺酶(prostase)、前列腺酶特异性抗原(PSA)、PAP、NY-ESO-1、LAGA-1a、p53、Prostein、PSMA、存活和端粒酶、前列腺癌肿瘤抗原-1(PCTA-1)、MAGE、ELF2M、嗜中性粒细胞弹性蛋白酶、肝配蛋白B2、CD22、胰岛素生长因子(IGF1)-I、IGF-II、IGFI受体、间皮素、呈递肿瘤特异性肽表位的主要组织相容性复合体(MHC)分子、5T4、ROR1、Nkp30、NKG2D、肿瘤基质抗原、纤维连接蛋白的额外结构域A(EDA)和额外结构域B(EDB)、腱生蛋白-C的A1结构域(TnC A1)、成纤维细胞相关蛋白(fap)、CD3、CD4、CD8、CD24、CD25、CD33、CD34、CD133、CD138、Foxp3、B7-1(CD80)、B7-2(CD86)、GM-CSF、细胞因子受体、内皮因子、主要组织相容性复合体(MHC)分子、BCMA(CD269、TNFRSF17)、 TNFRSF17(UNIPROT Q02223)、SLAMF7(UNIPROT Q9NQ25)、GPRC5D(UNIPROT Q9NZD1)、FKBP11(UNIPROT Q9NYL4)、KAMP3、ITGA8(UNIPROT P53708)和FCRL5(UNIPROT Q68SN8)。
  10. 权利要求8-9任一项的方法，其中所述抗原结合结构域选自单克隆抗体、合成的抗体、人抗体、人源化抗体、单域抗体、抗体单链可变区，以及其抗原结合片段。
  11. 权利要求2-10任一项的方法，其中所述CAR包含针对间皮素的scFv(P4)、CD8铰链区、CD28跨膜结构域、CD28共刺激结构域和CD3ζ信号转导结构域。
  12. 权利要求11的方法，其中所述CAR包含SEQ ID NO：32所示氨基酸序列。
  13. 通过权利要求1-12任一项的方法制备的经修饰的T细胞。
  14. 修饰的T细胞，其中与未修饰的T细胞相比，所述T细胞中的抑制性蛋白的表达被减少或消除。
  15. 权利要求14的经修饰的T细胞，其中所述T细胞是包含外源T细胞受体(TCR)或嵌合抗原受体(CAR)的T细胞。
  16. 权利要求14或15的经修饰的T细胞，其中所述表达被减少或消除的抑制性蛋白选自PD1、LAG-3、CTLA-4、Foxp3、Tim3及其组合。
  17. 权利要求14-16任一项的经修饰的T细胞，其中所述表达被减少或消除的抑制性蛋白选自PD1和TIM3的组合，PD1和CTLA-4的组合，PD1和LAG3的组合，CTLA-4和TIM3的组合，CTLA-4和LAG3的组合，TIM3和LAG3的组合，PD1、TIM3和CTLA-4的组合，PD1、CTLA-4和LAG3的组合，CTLA-4、TIM3和LAG3的组合，PD1、TIM3和LAG3的组合，或PD1、CTLA-4、TIM3和LAG3的组合。
  18. 权利要求14-17任一项的经修饰的T细胞，其中所述TCR或CAR包含针对肿瘤相关抗原的抗原结合结构域。
  19. 权利要求18的经修饰的T细胞，其中所述肿瘤相关抗原选自CD16、CD64、CD78、CD96、CLL1、CD116、CD117、CD71、CD45、CD71、CD123、CD138、ErbB2(HER2/neu)、癌胚抗原(CEA)、上皮细胞粘附分子(EpCAM)、表皮生长因子受体(EGFR)、EGFR变体III(EGFRvIII)、CD19、CD20、CD30、CD40、双唾液酸神经节苷脂GD2、导管上皮粘蛋白、gp36、TAG-72、鞘糖脂、神经胶质瘤相关的抗原、β-人绒毛膜促性腺激素、α胎儿球蛋白(AFP)、外源凝集素反应性AFP、甲状腺球蛋白、RAGE-1、MN-CA IX、人端粒酶逆转录酶、RU1、RU2(AS)、肠羧基酯酶、mut hsp70-2、M-CSF、前列腺酶(prostase)、前列腺酶特异性抗原(PSA)、PAP、NY-ESO-1、LAGA-1a、p53、Prostein、PSMA、存活和端粒酶、前列腺癌肿瘤抗原-1(PCTA-1)、MAGE、ELF2M、嗜中性粒细胞弹性蛋白酶、肝配蛋白B2、CD22、胰岛素生长因子(IGF1)-I、IGF-II、IGFI受体、间皮素、呈递肿瘤特异性肽表位的主要组织相容性复合体(MHC)分子、5T4、ROR1、Nkp30、NKG2D、肿瘤基质抗原、纤维连接蛋白的额外结构域A(EDA)和额外结构域B(EDB)、腱生蛋白-C的A1结构域(TnC A1)、成纤维细胞相关蛋白(fap)、CD3、CD4、CD8、CD24、CD25、CD33、CD34、CD133、CD138、Foxp3、B7-1(CD80)、B7-2(CD86)、 GM-CSF、细胞因子受体、内皮因子、主要组织相容性复合体(MHC)分子、BCMA(CD269、TNFRSF17)、TNFRSF17(UNIPROT Q02223)、SLAMF7(UNIPROT Q9NQ25)、GPRC5D(UNIPROT Q9NZD1)、FKBP11(UNIPROT Q9NYL4)、KAMP3、ITGA8(UNIPROT P53708)和FCRL5(UNIPROT Q68SN8)。
  20. 权利要求18-19任一项的经修饰的T细胞，其中所述抗原结合结构域选自单克隆抗体、合成的抗体、人抗体、人源化抗体、单域抗体、抗体单链可变区，以及其抗原结合片段。
  21. 权利要求15-20任一项的经修饰的T细胞，其中所述CAR包含针对间皮素的scFv(P4)、CD8铰链区、CD28跨膜结构域、CD28共刺激结构域和CD3ζ信号转导结构域。
  22. 权利要求21的的经修饰的T细胞，其中所述CAR包含SEQ ID NO：32所示氨基酸序列。
  23. 权利要求13-22任一项的经修饰的T细胞在制备用于治疗癌症的药物中的用途。
  24. 用于治疗癌症的药物组合物，包含权利要求13-23任一项的经修饰的T细胞和药学可接受的载体。
  25. 权利要求23的用途或权利要求24的药物组合物，其中所述癌症选自肺癌、卵巢癌、结肠癌、直肠癌、黑色素瘤、肾癌、膀胱癌、乳腺癌、肝癌、淋巴瘤、恶性血液病、头颈癌、胶质瘤、胃癌、鼻咽癌、喉癌、宫颈癌、子宫体瘤和骨肉瘤。可以用本发明的方法或药物组合物治疗的其他癌症的例子包括：骨癌、胰腺癌、皮肤癌、前列腺癌、皮肤或眼内恶性黑色素瘤、子宫癌、肛区癌、睾丸癌、输卵管癌、子宫内膜癌、阴道癌、阴户癌、何杰金病、非何杰金氏淋巴瘤、食道癌、小肠癌、内分泌系统癌、甲状腺癌、甲状旁腺癌、肾上腺癌、软组织肉瘤、尿道癌、阴茎癌、慢性或急性白血病(包括急性髓细胞样白血病、慢性髓细胞样白血病、急性成淋巴细胞性白血病、慢性淋巴细胞性白血病)、儿童实体瘤、淋巴细胞性淋巴瘤、膀胱癌、肾或输尿管癌、肾盂癌、中枢神经系统(CNS)肿瘤、原发性CNS淋巴瘤、肿瘤血管发生、脊柱肿瘤、脑干神经胶质瘤、垂体腺瘤、卡波西肉瘤、表皮状癌、鳞状细胞癌、T细胞淋巴瘤、环境诱发的癌症，包括石棉诱发的癌症，以及所述癌症的组合。
  26. 一种试剂盒，其用于权利要求1-12中任一项的制备经修饰T细胞的方法。
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