WO2019052577A1 - 一种基因编辑t细胞及其用途 - Google Patents

一种基因编辑t细胞及其用途 Download PDF

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WO2019052577A1
WO2019052577A1 PCT/CN2018/106237 CN2018106237W WO2019052577A1 WO 2019052577 A1 WO2019052577 A1 WO 2019052577A1 CN 2018106237 W CN2018106237 W CN 2018106237W WO 2019052577 A1 WO2019052577 A1 WO 2019052577A1
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cell
sgrna
cells
tcr
trac
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PCT/CN2018/106237
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French (fr)
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袁鹏飞
王飞
于玲玲
董曦
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博雅辑因（北京）生物科技有限公司
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Priority to US16/648,567 priority Critical patent/US20200216805A1/en
Priority to CN201880060667.7A priority patent/CN111194350A/zh
Priority to JP2020537277A priority patent/JP7190096B2/ja
Priority to EP18857205.1A priority patent/EP3686275A4/en
Publication of WO2019052577A1 publication Critical patent/WO2019052577A1/zh

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Definitions

  • the present invention relates to T cells, and in particular to gene-edited single gene, double gene and triple gene knockout T cells, including universal T cells, CAR-T cells and TCR-T cells, and preparation methods and uses thereof.
  • T cells are mainly derived from the patients themselves.
  • T cells are isolated, activated, CAR introduced, cultured and expanded in a GMP environment, and finally returned to the patient after quality control.
  • the problem of difficulty in amplification after the T cells are not suitable for blood collection or blood collection due to the patient's own conditions will follow.
  • the waiting time of the whole process will also be a major problem for autologous return.
  • CAR-T cell therapy an important research direction of CAR-T cell therapy is how to prepare a large number of CAR-T cells from a healthy donor's T cells to meet the clinical needs of patients. use.
  • the establishment of this technology will greatly reduce the cost of CAR-T therapy, better guarantee the uniform preparation of cell quality, and patients can get CAR-T cells for treatment when needed.
  • the present invention has been made to solve the above problems in the art.
  • the invention utilizes the CRISPR/Cas9 system to perform single gene (TRAC, B2M or PD-1), double gene (TRAC and B2M) and three genes (TRAC, B2M and PD-1) knockout of T cells, and the knockout efficiency is respectively Up to 90% (single gene), 81% (double gene) and 67% (three genes).
  • These genetically edited T cells can provide universal T cells for CAR or TCR targeting different targets, and provide methods for establishing genetic editing techniques in conjunction with adoptive immunization for the treatment of tumors and viral infectious diseases (eg, HIV/AIDS), and The research on the treatment of related diseases has laid a solid technical foundation.
  • genetically engineered T cells including universal T cells, CAR-T and TCR-T
  • the present invention provides efficient single gene (TRAC, B2M or PD-1), double gene (TRAC and B2M) or three genes (TRAC) for T cells using gene editing techniques, such as the CRISPR/Cas9 system. , B2M and PD-1) knockout, thereby obtaining a method for not expressing TCR, or TCR/HLA, or TCR/HLA/PD-1 universal T cells.
  • the universal T cells can be prepared into a universal CAR-T or TCR-T at any time by combining the required CAR or TCR, and can be applied as a medicine to a patient in need. It also supports the research of new effective gene targets and applies them to clinical immunotherapy in a timely and effective manner.
  • the invention provides a method of making genetically engineered T cells comprising: disrupting said T cells by gene editing techniques:
  • the TRAC genomic region, the B2M genomic region, and the PD-1 genomic region are all edited.
  • the gene editing technique is a zinc finger nuclease based gene editing technique, a TALEN gene editing technique, or a CRISPR/Cas gene editing technique, such as a CRISPR/Cas9 gene editing technique.
  • the invention provides a method of making a genetically engineered T cell comprising: disrupting the T cell by gene editing techniques:
  • the TRAC genomic region, the B2M genomic region, and the PD-1 genomic region are all edited.
  • the gene editing technique is a zinc finger nuclease based gene editing technique, a TALEN gene editing technique, or a CRISPR/Cas gene editing technique, such as a CRISPR/Cas9 gene editing technique.
  • the invention provides a method of making a genetically engineered T cell, disrupting the T cell by a CRISPR/Cas9 gene editing technique:
  • the invention provides a method of making a genetically engineered T cell, disrupting the T cell by a CRISPR/Cas9 gene editing technique:
  • the invention provides a method of making a genetically engineered T cell, disrupting the T cell by a CRISPR/Cas9 gene editing technique:
  • the invention provides a method of making a genetically engineered T cell, disrupting the T cell by a CRISPR/Cas9 gene editing technique:
  • the invention provides a method of making a genetically engineered T cell by the CRISPR/Cas9 gene editing technique, wherein:
  • the invention provides a method of making a genetically engineered T cell by the CRISPR/Cas9 gene editing technique, wherein:
  • the invention provides a method of making a genetically engineered T cell by the CRISPR/Cas9 gene editing technique, wherein:
  • the invention provides a method of making a genetically engineered T cell by the CRISPR/Cas9 gene editing technique, wherein:
  • the invention provides a method of making a genetically engineered T cell by the CRISPR/Cas9 gene editing technique, wherein:
  • the TLC-targeted sgRNA is introduced separately into the T cell.
  • the B2M-targeting sgRNA is introduced separately into the T cell.
  • the PD-1-targeting sgRNA is introduced separately into the T cell.
  • the TRAC-targeting sgRNA and the B2M-targeting sgRNA are simultaneously introduced into the T cell.
  • the TRAC-targeting sgRNA and PD-1-targeting sgRNA are simultaneously introduced into the T cell.
  • the B2M-targeting sgRNA and PD-1-targeting sgRNA are simultaneously introduced into the T cell.
  • the TLC-targeting sgRNA, the B2M-targeting sgRNA, and the PD-1-targeting sgRNA are simultaneously introduced into the T cell.
  • the sgRNA (including sgRNA targeting TRAC, sgRNA targeting B2M and/or sgRNA targeting PD-1) is a 2'-O-methyl analog and/or nucleotide 3' thio-modified.
  • the chemical modification is the first, second, and/or 5' ends of the sgRNA (including sgRNA targeting TRAC, sgRNA targeting B2M, and/or sgRNA targeting PD-1) Modification of the 2'-O-methyl analog of the last base of the three bases and/or the 3' end.
  • the sgRNAs described above are introduced into the T cells by electroporation.
  • the above sgRNA (including sgRNA targeting TRAC, sgRNA targeting B2M and/or sgRNA targeting PD-1) and Cas9 encoding nucleotide (eg, mRNA) are co-introduced into the T cell by electroporation .
  • the electrorotation conditions comprise any one selected from the group consisting of: 150-250V, 0.5-2ms; 150V, 2ms; 160V, 2ms; 170V, 2ms; 180V, 2ms; 190V, 1ms; 200V, 1ms ; 210V, 1ms; 220V, 1ms; 230V, 1ms; 240V, 1ms; 250V, 0.5ms.
  • the expression level of the TRAC, B2M or PD-1 in genetically edited T cells is 1/10 of the expression level of T cells not edited by the gene.
  • the efficiency of a single gene knockout is greater than 80%, such as 80%-100%, 85%-100%, 90%-100%, 95%-100% , 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93 More than %, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99%;
  • the simultaneous knockout efficiency of double genes (such as TRAC and B2M) is above 65%, for example, 65% -100%, 70%-100%, 75%-100%, 80%-100%, 85%-100%, 90%-100%, 95%-100%, 70% or more, 75% or more, 80% Above, more than 85%, more than 90%, or more than 95%; the knockout efficiency of TRAC, B2M and PD-1
  • knockout efficiency encompasses the knockout efficiency in the case of knockout of one of the TRAC gene, the B2M gene, and the PD-1 gene, the simultaneous knockout of the second, and the simultaneous knockout of the third.
  • the T cell is derived from a healthy subject, a tumor or a viral infected patient (eg, an HIV infected patient). In some embodiments, the T cell is a T cell differentiated from stem cells or precursor cells at different stages of differentiation.
  • the invention relates to genetically engineered T cells prepared by the above methods.
  • the invention relates to a genetically engineered T cell, wherein said T cell is:
  • PD-1 genomic region from chromosomes 242800936 to 242800978 of chromosome 2, or one or more sites of the PD-1 genome region at positions 242795009 to 242795051 of chromosome 2 are disrupted by gene editing techniques .
  • the T cell is prepared by any of the methods described herein.
  • the invention relates to a genetically engineered T cell, wherein said T cell is:
  • the B2M genomic region of chromosomes 45003745 to 45003788 of chromosome 15 has a genetic sequence change as described in any of Tables B and C;
  • the PD-1 genomic region of chromosomes 242800936 to 242800978 of chromosome 2 has the gene sequence alteration of any of those listed in Table F, or the PD-1 genomic region of chromosomes 242795009 to 242795051 The gene sequence of any of those described in Table G is changed.
  • the T cell is prepared by any of the methods described herein.
  • the invention relates to the use of the above genetically engineered T cells for the preparation of adoptive cell treated T cells.
  • the adoptive cell-treated T cells are CAR-T cells or TCR-T cells.
  • the invention relates to a method of making a CAR-T cell or a TCR-T cell comprising: a chimeric antigen receptor (CAR) or a nucleotide encoding the same, or an engineered T cell receptor (TCR) Or its coding nucleotide is introduced into any of the genetically engineered T cells described above.
  • CAR chimeric antigen receptor
  • TCR engineered T cell receptor
  • the invention relates to a method of making a CAR-T or TCR-T cell comprising:
  • the method further comprises introducing CAS9 or a coding nucleotide thereof into the T cell.
  • the method of making a CAR-T or TCR-T cell comprises:
  • the method further comprises introducing CAS9 or a coding nucleotide thereof into the T cell.
  • the method of making a CAR-T or TCR-T cell comprises:
  • the method further comprises introducing CAS9 or a coding nucleotide thereof into the T cell.
  • the method of making a CAR-T or TCR-T cell comprises:
  • the method further comprises introducing CAS9 or a coding nucleotide thereof into the T cell.
  • the method of making a CAR-T or TCR-T cell comprises:
  • the method further comprises introducing CAS9 or a coding nucleotide thereof into the T cell.
  • the method of making a CAR-T or TCR-T cell comprises:
  • the method further comprises introducing CAS9 or a coding nucleotide thereof into the T cell.
  • the TRAC-targeting sgRNA and the B2M-targeting sgRNA are simultaneously introduced into T cells. In some embodiments, the TRAC-targeting sgRNA, the B2M-targeting sgRNA, and the PD-1-targeting sgRNA are simultaneously introduced into T cells.
  • the sgRNA (including sgRNA targeting TRAC, sgRNA targeting B2M and/or sgRNA targeting PD-1) is a 2'-O-methyl analog and/or nucleotide 3' thio-modified.
  • the chemical modification is the first, second, and/or 5' ends of the sgRNA (including sgRNA targeting TRAC, sgRNA targeting B2M, and/or sgRNA targeting PD-1) Modification of the 2'-O-methyl analog of the last base of the three bases and/or the 3' end.
  • the sgRNAs described above are introduced into the T cells by electroporation.
  • the above sgRNA (including sgRNA targeting TRAC, sgRNA targeting B2M and/or sgRNA targeting PD-1) and Cas9 encoding nucleotide (eg, mRNA) are co-introduced into the T cell by electroporation .
  • the electrorotation conditions comprise any one selected from the group consisting of: 150-250V, 0.5-2ms; 180-250V, 0.5-2ms; 150V, 2ms; 160V, 2ms; 170V, 2ms; 180V, 2ms ;190V, 1ms; 200V, 1ms; 210V, 1ms; 220V, 1ms; 230V, 1ms; 240V, 1ms; 250V, 0.5ms.
  • the methods comprise targeting a TRAC-targeting sgRNA, targeting a B2M sgRNA and/or a PD-1 targeting sgRNA and a CAR or a coding nucleotide thereof, or an engineered T cell receptor ( TCR) or its coding nucleotide is simultaneously introduced into the T cell.
  • TRAC-targeting sgRNA targeting a B2M sgRNA and/or a PD-1 targeting sgRNA and a CAR or a coding nucleotide thereof, or an engineered T cell receptor ( TCR) or its coding nucleotide is simultaneously introduced into the T cell.
  • the CAR or its encoding nucleotide, or engineered T cell receptor (TCR) or its encoding nucleotide is prior to the sgRNA targeting TRAC, targeting the sgRNA of B2M and/or targeting Introduction of PD-1 sgRNA into the T cell; or introduction of CAR or its coding nucleotide, or engineered T cell receptor (TCR) or its coding nucleotide into sgRNA targeting B2M and/or targeting PD The T cell was introduced after the -1 sgRNA.
  • the invention relates to a CAR-T cell or a TCR-T cell prepared by the above method.
  • the invention features a CAR-T cell comprising the above genetically engineered T cell expressing a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • the invention relates to a CAR-T cell, wherein said CAR-T cell:
  • the B2M genomic region of chromosomes 45003745 to 45003788 of chromosome 15 has a genetic sequence change as described in any of Tables B and C;
  • the PD-1 genomic region of chromosomes 242800936 to 242800978 of chromosome 2 has the gene sequence alteration of any of those listed in Table F, or the PD-1 genomic region of chromosomes 242795009 to 242795051 The gene sequence of any of those described in Table G is changed.
  • the invention features a TCR-T cell comprising the above genetically engineered T cell expressing an engineered TCR.
  • the invention relates to TCR-T cells, wherein said TCR-T cells are:
  • the B2M genomic region of chromosomes 45003745 to 45003788 of chromosome 15 has a genetic sequence change as described in any of Tables B and C;
  • the PD-1 genomic region of chromosomes 242800936 to 242800978 of chromosome 2 has the gene sequence alteration of any of those listed in Table F, or the PD-1 genomic region of chromosomes 242795009 to 242795051 The gene sequence of any of those described in Table G is changed.
  • the positional information of the TRAC genomic region, the B2M genomic region, and the PD-1 genomic region mentioned above in the present invention is determined by the wild sequence position information of the gene described in the reference database: GRCh37 (hg19). Those skilled in the art know how to obtain corresponding positional information of the above-described genomic region with reference to other databases.
  • the wild nucleotide sequence of the TRAC genomic region from position 2316448 to position 23016490 of chromosome 14 is set forth in SEQ ID NO: 23 (tatccagaaccctgaccctgccgtgtaccagggaggactct).
  • the wild nucleotide sequence of the B2M genomic region from position 45003745 to position 45003788 of chromosome 15 is set forth in SEQ ID NO: 24 (atgtctcgctccgtggccttagctgtgctcgctctctctctctctctctctctctct).
  • the wild nucleotide sequence of the PD-1 genomic region from position 242800936 to position 242800978 of chromosome 2 is set forth in SEQ ID NO: 25 (agcccagttgtagcaccgcccagacgggggggggccccgg).
  • the wild nucleotide sequence of the PD-1 genomic region from position 242795009 to position 242795051 of chromosome 2 is set forth in SEQ ID NO: 26 (cagtttagcacgaagctctccgatgtgtgggagaagctgcagg).
  • the invention also relates to compositions (e.g., pharmaceutical combinations), kits, and medical articles comprising the above-described genetically engineered T cells, CAR-T cells or TCR-T cells.
  • the invention relates to a method of treating a disease in a subject comprising administering to the subject an effective amount of the above CAR-T or TCR-T cells.
  • the disease is a tumor.
  • the tumor is a hematological tumor.
  • the tumor is a lymphoma or leukemia.
  • the CAR targets a tumor specific antigen (TSA) and/or a tumor associated antigen (TAA), such as an antigen (eg, CD19) as shown in Table A herein.
  • TSA tumor specific antigen
  • TAA tumor associated antigen
  • the invention relates to a sgRNA comprising SEQ ID NOs: 2-22 or a vector thereof.
  • the sgRNA is chemically modified, such as a 2'-O-methyl analog and/or an internucleotide 3' thio modification.
  • the chemical modification is a modification of the 2'-O-methyl analog of the first, second and/or three bases of the 5' end of the sgRNA and/or the last base of the 3' end. .
  • Figure 1 shows a comparison of the efficiency of knockout of corresponding sites using chemically modified sgRNA and unchemically modified sgRNA after one electrical run.
  • Figures 2A-2F show knockdown of B2M in HLA using modified sgRNA-binding CRISPR/Cas9 knockout tools, and knockdown analysis of the knockdown efficiency of each sgRNA to B2M gene.
  • Figure 2B shows the INDEL analysis of the T cell genome after knockdown of the B2M gene by modified different sgRNAs by the INDEL analysis software to obtain the efficiency of INDEL in T cells.
  • Figure 2C shows the knockdown of TRAC in TCR using modified sgRNA-binding CRISPR/Cas9 knockout tools, and knockdown analysis of the knockdown efficiency of each sgRNA for TRAC.
  • Figure 2D shows the INDEL analysis of the T cell genome after TRAC gene knockout by modified different sgRNAs by INDEL analysis software to obtain the efficiency of INDEL in T cells.
  • Figure 2E shows knockdown of PD-1 using modified sgRNA-binding CRISPR/Cas9 knockout tools, and knockdown analysis of the knockdown efficiency of each sgRNA for PD-1.
  • Figure 2F shows the INDEL analysis of the T cell genome after PD-1 gene knockout by modified different sgRNAs by INDEL analysis software to obtain the efficiency of INDEL in T cells.
  • Figure 2A shows the knockdown of B2M in T cells under the same electroporation conditions using in vitro transcription (IVT) and modified optimization of sgRNA binding CRISPR/Cas9 knockout tool, and analysis of each sgRNA to B2M after knockout
  • the knockout efficiencies of one gene were: B1: 27.88%, B2: 86.17%, B3: 64.69%, B4: 1.06%, B5: 2.91%, B6: 0.17%, B7: 41.87%, and B8: 3.14%.
  • sgRNA with better knockout efficiency can be selected.
  • FIG. 3 shows the INDEL analysis of the T cell genome after knockdown of different sgRNAs against the B2M gene by the INDEL analysis software to obtain the efficiency of INDEL in T cells.
  • FIG 2C shows the use of in vitro transcription (IVT) and modified sgRNA-binding CRISPR/Cas9 knockout tools to knock out TRACs in T cells under the same electroporation conditions, and analyze each sgRNA to TRAC after knockout
  • the knockout efficiencies of one gene were: T2: 77.84%, T3: 85.86%, T4: 2.59%, and T6: 34.78%.
  • sgRNA with better knockout efficiency can be selected.
  • the above optimized modification of the selected sgRNAs provides a possibility for efficient single/double/triple knockouts (shown in Figure 3).
  • Figure 2D shows the INDEL analysis of the T cell genome after TRAC gene knockout by different sgRNAs by the INDEL analysis software to obtain the efficiency of INDEL in T cells.
  • Figure 2E shows the knockdown of PD-1 in T cells under the same electroporation conditions using sgRNA-binding CRISPR/Cas9 knockout tools using in vitro transcription (IVT) and optimized above, and analysis of individual sgRNAs after knockout
  • the knockout efficiencies of PD-1 were: P1: 21.15%, P2: 36.99%, P4: 23.03%, P5: 25.6%, P6: 3.1%, P7: 22.49%, P8: 23.07%, P9 : 31.18% and P10: 24.48%.
  • FIG. 3 shows the INDEL analysis of the T-cell genome after PD-1 gene knockout by different sgRNAs by the INDEL analysis software to obtain the efficiency of INDEL in T cells.
  • FIG. 3 shows TRAC, TRAC/B2M (Double Knock-Out, DKO) and TRAC/B2M/PD-1 (Triple Knock-Out, TKO) for T cells using optimized sgRNA and CRISPR/Cas9 gene editing techniques. Analysis of the knockout results.
  • the sgRNA is chemically modified, and then the chemically modified sgRNA and Cas9 are delivered to the primary T cells by further optimized electroporation conditions for related gene knockout.
  • the knockout efficiency of the single gene, TRAC was increased to 90.42%; the knockout efficiency of the two genes, TRAC and B2M, was increased to 81.39%.
  • the knockout efficiency of the three genes, TRAC, B2M and PD-1 increased to 67.91% (82.70%+15.76%)*68.98%).
  • Figure 4 shows phenotypic analysis of T cells, TRAC/B2M (DKO), and TRAC/B2M/PD-1 (TKO) knockout T cells after screening and purification.
  • the number of purifications (4 to 5 times) and the corresponding amount of antibody (3 mg/mL) were increased.
  • the results are shown in the figure, the optimized double knockout, TRAC and B2M double knockout purity increased to 99.72%.
  • FIG. 5 shows the results of off-target detection of TRAC-sgRNA3 (T2), B2M-sgRNA2 (B3), and PD-1-sgRNA2 (P2).
  • T2 T2M-sgRNA3
  • B3 B2M-sgRNA2
  • P2 PD-1-sgRNA2
  • the figure shows the efficiency of INDEL in T cells edited by T2, B3, P2sgRNA combined with CRISPR/Cas9 after deep sequencing combined with INDEL analysis software.
  • Figure 6 shows post-knockout phenotypes of TCR and/or B2M and/or PD-1 on T cells using optimized sgRNA and CRISPR/Cas9 gene editing techniques and for TCR and/or B2M and/or PD- 1 Knockout T cells were screened and purified for phenotypic analysis.
  • the abscissa is CD3 and the ordinate is TCR, B2M and PD-1.
  • Figure 7 shows the validation and comparison of the killing function of T cells, CAR-T, TCRneg CAR-T, DKO CAR-T and TKO CAR-T cells.
  • Raji and K562 were used as target cells
  • T, CAR-T, DKO CAR-T and TKO CAR-T were effector cells
  • the effective target ratio was 10:1, 5:1, 2.5:1, 1.25: 1, 0.625: 1 in vitro killing experiments.
  • Figure 8 shows T cell, CAR-T, TCRneg CAR-T, DKO CAR-T and TKO CAR-T cytokine release.
  • Raji and K562 were used as target cells
  • T, CAR-T, DKO CAR-T and TKO CAR-T were effector cells
  • the effective target ratio was 10:1, 5:1, 2.5:1, 1.25: 1.
  • the supernatant was taken for detection of IL-2 and IFN- ⁇ .
  • Figures 9A-9B show injection of saline, CAR-T, TCR neg CAR-T, DKO CAR-T (TCR neg ⁇ B2M neg -CAR-T) and TKO CAR-T (TCR neg ⁇ B2M neg ⁇ PD-1 After neg- CAR-T) cells, their tumor suppression and killing effects were compared in NPG mice.
  • Figure 9A shows that 5X10 ⁇ 5 tumor cells/only were injected into the NSG mice through the tail vein, and were randomly divided into 4 groups, saline group, T cell group, TCR/CD3 neg CD19-CAR-T cell group, DKO CD19.
  • mice 5 ⁇ 10 ⁇ 6 corresponding cells were injected into the four groups of mice through the tail vein respectively, and the physiological saline group was used as a control group.
  • the tumor burden in mice was analyzed by a picture obtained from a platinum Elmer imager.
  • Figure 9B shows the tumor burden in mice by a picture obtained by a Platinum Elmer imager.
  • the abscissa is the number of days of mouse cell rearing, and the ordinate is the radiation signal radiated per second of the unit body surface area.
  • Figure 10 shows physiological saline, CAR-T, TCR neg CAR-T, DKO CAR-T (TCR neg ⁇ B2M neg -CAR-T) and TKO CAR-T (TCR neg ⁇ B2M neg ⁇ PD-1 neg -CAR -T) Comparison of survival rates of 5 groups of mice.
  • the abscissa is the number of days of mouse rearing after injection of effector cells, and the ordinate is the survival rate of mice.
  • CRISPR/Cas is a genetic editing technique including, but not limited to, a variety of naturally occurring or manually designed CRISPR/Cas systems, such as the CRISPR/Cas9 system.
  • the naturally occurring CRISPR/Cas system (Naturally occurring CRISPR/Cas system) is an adaptive immune defense formed by bacteria and archaea during long-term evolution and can be used against invading viruses and foreign DNA.
  • CRISPR/Cas9 works by the fact that crRNA (CRISPR-derived RNA) binds to tracrRNA (trans-activating RNA) by base pairing to form a tracrRNA/crRNA complex that directs the sequence of the nuclease Cas9 protein in pair with the crRNA.
  • the target site cleaves double-stranded DNA.
  • a sgRNA single guide RNA
  • Cas9 effector nucleases are capable of co-localizing RNA, DNA, and proteins, and have enormous potential for transformation.
  • the CRISPR/Cas system can use one, two or three Cas proteins. In some embodiments of the invention, the method uses Cas9.
  • Other suitable CRISPR/Cas systems include, but are not limited to, the systems and methods described in WO2013176772, WO2014065596, WO2014018423, US8,697,359.
  • sgRNA single guide RNA
  • gRNA guide RNA
  • the sgRNA of the invention comprises a guide sequence that targets a target sequence.
  • T cell receptor is a characteristic marker on the surface of all T cells that binds to CD3 to form a TCR-CD3 complex.
  • TCR consists of two peptide chains, ⁇ and ⁇ . Each peptide chain can be further divided into variable region (V region), constant region (C region), transmembrane region and cytoplasmic region.
  • V region variable region
  • C region constant region
  • TCR molecule belongs to the immunoglobulin superfamily, and its antigen specificity exists in the V region; the V region (V ⁇ , V ⁇ ) has three hypervariable regions CDR1, CDR2, and CDR3, among which the CDR3 mutation is the largest, which directly determines the TCR antigen. Binding specificity.
  • TCR When the TCR recognizes the MHC-antigen peptide complex, CDR1, CDR2 recognizes and binds to the side wall of the MHC molecule antigen binding groove, and CDR3 binds directly to the antigen peptide.
  • TCR is divided into two categories: TCR1 and TCR2; TCR1 consists of two chains of ⁇ and ⁇ , and TCR2 consists of two chains of ⁇ and ⁇ .
  • 90%-95% of T cells express TCR2; and any T cell expresses only one of TCR2 and TCR1.
  • ⁇ 2 microglobulin is a ⁇ -chain (light chain) portion of a cell surface human leukocyte antigen (HLA), and is a single-chain polypeptide having a molecular mass of 11,800 and consisting of 99 amino acids.
  • PD-1 Protein of 268 amino acid residues
  • the combination of PD-1 and PD-L1 initiates programmed cell death of T cells, allowing tumor cells to gain immune escape and, therefore, is an important immunosuppressive molecule.
  • index is referred to collectively as an insertion/deletion, ie, an insertion and deletion mutation.
  • GVHD raft versus host response
  • HVGR and GVHR-related genes contain TCR and HLA-related genes.
  • T lymphocytes that are simultaneously knocked out by these genes do not cause graft-versus-host disease (GVHD) when they are returned to allogeneic patients, so they can be called "generic T. cell".
  • GVHD graft-versus-host disease
  • a single TRAC gene is a gene encoding a TCR alpha chain that forms a complete functional TCR complex with two TRBC genes encoding TCR beta, knockdown of TRAC can result in inactivation of TCR, and B2M is a MHCI-related gene.
  • T lymphocytes that are simultaneously knocked out by these two genes do not cause graft-versus-host disease (GVHD) when they are returned to the allogeneic patient.
  • CAR-T is a short form of "chimeric antigen receptor T cell” in which the chimeric antigen receptor (CAR) is a core component of CAR-T, conferring T cell HLA-independent ways to recognize target cells (eg The ability of tumors to antigen, which allows CAR-modified T cells to recognize a broader target than the natural T cell surface receptor TCR.
  • the design of a tumor-targeting CAR includes a tumor-associated antigen (TAA) binding region (eg, an scFV segment typically derived from a monoclonal antibody antigen binding region), an extracellular hinge region , a transmembrane region and an intracellular signal region.
  • TAA tumor-associated antigen
  • the selection of the target antigen is a key determinant of the specificity and effectiveness of the CAR and the safety of the genetically modified T cells themselves.
  • Universal CAR-T cells refer to CAR-T cells that are capable of targeting specific target cell (eg, tumor)-associated markers and inactivating TCR and MHC functions on the cell surface, which can reduce immune rejection caused by allogeneic cell therapy.
  • target cell eg, tumor
  • the CAR-T treatment of autologous cells requires the preparation of blood-separated patients' own T lymphocytes.
  • the factors affecting the production process of CAR-T are not standardized and the production safety is affected.
  • some The patient's autologous T lymphocytes have insufficient activity and quantity after chemotherapy, or are affected by the tumor environment, resulting in limited T lymphocyte activity and proliferative capacity. Such cells are often difficult to prepare for CAR-T, and the safety and efficacy of treatment are high.
  • TCR-T T cell receptor (TCR) chimeric-T cell
  • TCR T cell receptor
  • the engineered T cell receptor or artificial T cell receptor is genetically engineered to have a structure that targets the antigen of interest while also retaining domains and/or accessory molecules in the TCR signaling pathway.
  • TCR-T retains all of the accessory molecules in the TCR signaling pathway.
  • a fully activated state can occur, causing a killing effect on the target cells.
  • these TCR-Ts maintain and apply all the auxiliary molecules on the TCR signaling pathway, so TCR-T is more sensitive to recognition of low concentration and less copy number antigen than certain CAR-T.
  • the therapeutic potential is very large.
  • TCR-T cells increase the affinity of the TCR for the corresponding antigen (eg, TAA) by partial genetic modification, and thus the "genetically modified TCR” technique is therefore referred to as the "affinity enhanced TCR” technique.
  • affinity-Enhanced TCR For example, a "genetically modified TCR” jointly developed by Adaptimmmune, published by Nature-Medical magazine, has improved TCR by a number of key amino acids, and a common cancer TAA, NY-ESO -1, the affinity. This can be used to attack cancers that are overexpressed with NY-ESO-1, such as Multiple Myeloma.
  • adoptive immunotherapy such as tumor adoptive immunotherapy
  • adoptive immunotherapy refers to the treatment of immune cells in vitro, such as the addition of specific antigens, to immune cells.
  • One method of treatment is a passive immunotherapy.
  • a method of making a genetically engineered T cell comprising disrupting said T cell by gene editing techniques: (i) chromosomes #21616448 to 23016490 of chromosome 14 TRAC genomic region (as set forth in SEQ ID NO: 23); (ii) B2M genomic region from position 45003745 to position 45003788 of chromosome 15 (as shown in SEQ ID NO: 24); and/or (iii) No.
  • the gene editing technique of the method is a zinc finger nuclease based gene editing technique, a TALEN gene editing technique, or a CRISPR/Cas gene editing technique.
  • the TRAC genomic region, the B2M genomic region, or the PD-1 genomic region are edited.
  • the TRAC genomic region, and the B2M genomic region are both edited.
  • the TRAC genomic region and the PD-1 genomic region are both edited.
  • the B2M genomic region and the PD-1 genomic region are both edited.
  • the TRAC genomic region, the B2M genomic region, and the PD-1 genomic region are all edited.
  • a method of making a genetically engineered T cell comprising disrupting the T cell by gene editing techniques: (i) and selected from SEQ ID NOs: 2-5 Any of the sequence-complementary TRAC genomic target nucleotide sequences; (ii) a target nucleotide sequence of a B2M genome complementary to a sequence selected from any one of SEQ ID NOs: 6-14; and/or (iii) A target nucleotide sequence of the PD-1 genome complementary to the sequence of any one of SEQ ID NOs: 15-22.
  • the gene editing technique of the method is a zinc finger nuclease based gene editing technique, a TALEN gene editing technique, or a CRISPR/Cas gene editing technique.
  • the TRAC genomic region, the B2M genomic region, or the PD-1 genomic region are edited.
  • the TRAC genomic region, and the B2M genomic region are both edited.
  • the TRAC genomic region and the PD-1 genomic region are both edited.
  • the B2M genomic region and the PD-1 genomic region are both edited.
  • the TRAC genomic region, the B2M genomic region, and the PD-1 genomic region are all edited.
  • a method of making a genetically engineered T cell comprising: introducing a TRAC-targeting sgRNA, a B2M-targeting sgRNA, and/or a PD-1 sgRNA into a T cell, To destroy TRAC, B2M and / or PD-1 genes of T cells.
  • the method comprises introducing Cas9 or a coding nucleotide thereof into a T cell.
  • the sgRNA targets a gene encoding a constant region of the alpha and/or beta strand of TCR2, thereby disrupting the structure of the TCR on the surface of the T cell, rendering the molecule inactive.
  • the sgRNA targets a gene encoding ⁇ 2 microglobulin (B2M), for example, targeting the first exon region of the B2M protein-encoding gene, thereby disrupting the structure of the B2M, rendering the molecule unfunctional.
  • B2M ⁇ 2 microglobulin
  • the sgRNA targets a gene encoding PD-1, such as the first exon region of the PD-1 protein-encoding gene, thereby disrupting the structure of PD-1, rendering the molecule unfunctional.
  • a method of making a gene-edited T cell comprising: (i) introducing a sgRNA comprising a sequence selected from any one of SEQ ID NOs: 2-5 into a T cell Achieving editing of the TRAC genomic region; (ii) introducing an sgRNA comprising a sequence selected from any one of SEQ ID NOs: 6-14 into a T cell to effect editing of the B2M genomic region; and/or (iii) An sgRNA comprising a sequence selected from any one of SEQ ID NOs: 15-22 is introduced into a T cell to effect editing of the PD-1 genomic region.
  • the method comprises introducing Cas9 or a coding nucleotide thereof into a T cell.
  • the invention relates to the introduction of any sgRNA selected from Table 2, which targets TRAC, into a T cell. In some embodiments of the invention, the invention relates to the introduction of any sgRNA selected from Table 2, which targets B2M, into a T cell. In some embodiments of the invention, the invention relates to the introduction of any sgRNA selected from Table 2, which targets PD-1, into a T cell. In some embodiments, the method comprises introducing Cas9 or a coding nucleotide thereof into a T cell.
  • the invention relates to any sgRNA selected from Table 2 that is selected from any of the sgRNAs selected from Table 2 that targets TRAC, and/or that targets B2M, and/or that targets PD- Any of the sgRNAs selected from Table 2 and Cas9 or its coding nucleotides are introduced into T cells.
  • the invention relates to any sgRNA selected from Table 2 that targets TRAC, and any sgRNA selected from Table 2 that targets B2M, and is selected from the group consisting of PD-1 Any of the sgRNAs shown in Table 2 were introduced into T cells.
  • the method comprises introducing Cas9 or a coding nucleotide thereof into a T cell.
  • a method of making a universal T cell comprising: (i) introducing a TRAC-sg3sgRNA into a T cell to effect editing of the TRAC genomic region; (ii) introducing a B2M-sg2sgRNA T cells to achieve editing of the B2M genomic region; and (iii) introduction of PD-1-sg2sgRNA into T cells to enable editing of the PD-1 genomic region.
  • the method comprises introducing Cas9 or a coding nucleotide thereof into a T cell.
  • a method of making a universal T cell comprising: (i) introducing an sgRNA comprising the sequence of SEQ ID NO: 2 or 3 into a T cell to effect editing of the TRAC genomic region; a sgRNA comprising a sequence selected from any one of SEQ ID NO: 7 or 11 introduced into a T cell to effect editing of the B2M genomic region; and (iii) comprising a molecule selected from SEQ ID NO: 14 or 15
  • the sequence of sgRNA is introduced into T cells to effect editing of the PD-1 genomic region.
  • the method comprises introducing Cas9 or a coding nucleotide thereof into a T cell.
  • the sgRNA described above is chemically modified.
  • it is modified by 2'-O-methyl analog and/or internucleotide 3' thio.
  • the chemical modification is a 2'-O-methyl analog of the first, second and/or three bases of the 5' end of the sgRNA and/or the last base of the 3' end. Modification.
  • a guide sequence in an sgRNA is any polynucleotide sequence that is sufficiently complementary to a target polynucleotide sequence to hybridize to a target sequence and direct the sequence specific binding of the CRISPR complex to the target sequence.
  • the degree of complementarity between the leader sequence and its corresponding target sequence is about or greater than about 80%, 85%, 90%, 95%, 97.5%, 99% or more.
  • Optimal alignment can be determined using any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wimsch algorithm, the Burrows-Wheeler Transform-based algorithm (eg, Burrows Wheeler Aligner), ClustalW, Clustai X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, CA), SOAP (available at soap.genomics.org.cn) and Maq (available at maq.sourceforge.net).
  • any suitable algorithm for aligning sequences non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wimsch algorithm, the Burrows-Wheeler Transform-based algorithm (eg, Burrows Wheeler Aligner), ClustalW, Clustai X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, CA), SOAP (available at soap.genomics.org.cn) and Maq (
  • the length of the guide sequence may be about or greater than about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or more nucleotides. In some embodiments, the guide sequence is less than about 75, 70, 65, 60, 55 in length , 50, 45, 40, 35, 30, 25, 20, 15, 12 or fewer nucleotides.
  • the ability of the guide sequence to direct sequence-specific binding of the CR1 SPR complex to the target sequence can be assessed by any suitable assay For example, a host cell having a corresponding target sequence can be provided with a CRI sufficient to form a CRISPR complex.
  • the components of the SPR system can be performed, for example, by transfection with a vector encoding a CRISPR sequence component, followed by evaluation of preferential cleavage within the target sequence. Similarly, cleavage of the target polynucleotide sequence can be tested. Evaluate in the tube by providing a component of the target sequence, the CRISPR complex (containing the guide sequence to be tested and a control guide sequence different from the guide sequence), and comparing the binding or cleavage rate of the test and control guide sequences to the target sequence.
  • the above assays and evaluations can also be carried out using other assay methods known to those skilled in the art.
  • the TRAC-targeting sgRNA, B2M-targeting sgRNA, and/or PD-1-targeting sgRNA and/or Cas9 encoding nucleotide are introduced into the T cell by electroporation, eg, Pass 150-250V, 0.5-2ms; 180-250V, 0.5-2ms; 150V, 2ms; 160V, 2ms; 170V, 2ms; 180V, 2ms; 190V, 1ms; 200V, 1ms; 210V, 1ms; 220V, 1ms; , 1ms; 240V, 1ms; 250V, 0.5ms electroporation conditions were introduced into T cells.
  • electroporation eg, Pass 150-250V, 0.5-2ms; 180-250V, 0.5-2ms; 150V, 2ms; 160V, 2ms; 170V, 2ms; 180V, 2ms; 190V, 1ms; 200V, 1ms;
  • the TRAC-targeting sgRNA, B2M-targeting sgRNA, and PD-1-targeting sgRNA and Cas9 encoding nucleotides are co-introduced into T cells by electroporation.
  • the Cas9 encoding nucleotide is an mRNA, such as an mRNA comprising an ARCA cap. In some embodiments, the Cas9 encoding nucleotide is in a viral vector, such as a lentiviral vector. In some embodiments, the Cas9 encoding nucleotide comprises the sequence set forth in SEQ ID NO: 1. In some embodiments, the TRAC-targeting sgRNA, the B2M-targeting sgRNA, and/or the PD-1-targeting sgRNA are in the same vector as the Cas9 encoding nucleotide.
  • the sgRNA targeting TRAC, the sgRNA targeting B2M, and the sgRNA targeting PD-1 are simultaneously introduced into T cells.
  • the sgRNA targeting TRAC, the sgRNA targeting B2M, and the sgRNA targeting PD-1 are simultaneously introduced into T cells, sgRNA targeting TRAC, sgRNA targeting B2M, and sgRNA targeting PD-1 The amount between them can be approximate or equivalent.
  • the sgRNA targeting TRAC, the sgRNA targeting B2M, and the sgRNA targeting PD-1 are introduced into T cells one by one in any suitable order.
  • the sgRNA targeting TRAC, the sgRNA targeting B2M, and/or the sgRNA targeting PD-1 are simultaneously introduced into the T cell with the Cas9 encoding nucleotide.
  • the Cas9 encoding nucleotide is introduced into the T cell prior to the sgRNA targeting TRAC, the sgRNA targeting B2M, and/or the sgRNA targeting PD-1.
  • the T cell comprises a Cas9 encoding nucleotide or a Cas9 protein.
  • the T cells are derived from a healthy person.
  • the T cell is derived from a patient, such as a cancer patient, such as a cancer patient prior to chemotherapy or radiation therapy.
  • the T cell source is umbilical cord blood, bone marrow, or Peripheral blood mononuclear cell (PBMC).
  • the T cells are derived from stem cells, such as hematopoietic stem cells at various stages of differentiation. The preparation methods described in the present application can be used to knock out TRAC, B2M and/or PD-1 in, for example, PBMC or stem cells, and further culture, differentiate, and/or purify the corresponding genetically engineered T cells.
  • the T cell gene knockout method of the present invention obtains efficient gene knockout efficiency, for example, single gene (TRAC), double gene (TRAC and B2M), and three genes (TRAC, B2M and PD-).
  • the knockout efficiency is as high as at least 90%, 81% and 67% respectively.
  • knockout efficiency can be expressed at the gene level by the efficiency of the gene knockout INDEL, or at the cellular level by the percentage of cells that the gene knockout causes the gene expression protein to disappear or significantly decrease.
  • knockout efficiency means the knockout efficiency calculated based on the latter. Those skilled in the art will appreciate that high knockout efficiency can increase the harvest rate of the cells of interest, reducing production costs and treatment costs.
  • genetically engineered T cells are further screened to yield higher purity single genes (TRAC), double genes (TRAC and B2M), and three genes (TRAC, B2M, and PD- 1) Knock out T cells.
  • genetically edited T cells such as universal T cells with low expression levels of TRAC, B2M and/or PD-1 can be screened by FACS.
  • the TCR and/or HLA and/or PD-1 genes of the universal T cells of the invention are knocked out.
  • the alpha chain constant coding region (ie, TRAC) gene of the TCR is knocked out.
  • the coding region of B2M and / or PD-1 is knocked out.
  • TRAC, B2M, and PD-1 can all be knocked out, or one of the three can be knocked out or two of them can be knocked out.
  • the alpha chain constant coding region (ie, TRAC) gene of the TCR is knocked out.
  • the TCR alpha chain constant coding region gene of the invention is introduced into one of the TRAC-sg 2, 3, 4, 6 molecules of the cell (see Table 2) and the Cas 9 molecule knockout.
  • the alpha chain constant coding region gene of the TCR is introduced into the TRAC-sg 2 and Cas9 molecules of the cell or the TRAC-sg 3 and Cas9 molecules are knocked out.
  • the constant coding region B2M gene of HLA is knocked out.
  • the B2M constant coding region gene of the present invention is introduced into one of the B2M-sg 1-8 molecules of the cell (see Table 2) and the Cas 9 molecule, preferably the constant coding region gene of the B2M is The B2M-sg 2 or B2M-sg 6 molecule introduced into the cell and the Cas9 molecule were knocked out.
  • the constant coding region gene of PD-1 is knocked out.
  • the PD-1 constant coding region gene of the present invention is introduced into one of the PD-1-sg1-2, PD-1-sg4-10 molecules of the cell (see Table 2) and the Cas 9 molecule.
  • the constant coding region gene of PD-1 is knocked out by the PD-1-sg1 or PD-1-sg2 molecule introduced into the cell and the Cas9 molecule.
  • the alpha chain constant coding region (ie, TRAC) gene and the B2M gene of the TCR are knocked out.
  • TRAC and B2M genes of the invention are introduced into the TRAC-sg3 and B2M-sg2 molecules of the cell (see Table 2) and the Cas 9 molecule is knocked out.
  • the constant strand coding region (ie, TRAC) gene, the B2M gene, and the constant coding region gene of PD-1 of the TCR are knocked out.
  • the TCR, HLA and PD-1 constant coding region genes of the invention are introduced into the TRAC-sg3, B2M-sg2 and PD-1-sg2 molecules of the cell (see Table 2) and the Cas 9 molecule Knock out.
  • the TCR, HLA and PD-1 constant coding region genes of the invention are introduced into the TRAC-sg2, B2M-sg6 and PD-1-sg1 molecules of the cell (see Table 2) and the Cas 9 molecule is knocked except.
  • the TCR, HLA and PD-1 constant coding region genes of the invention are introduced into the TRAC-sg2, B2M-sg6 and PD-1-sg2 molecules of the cell (see Table 2) and the Cas 9 molecule is knocked except.
  • the invention provides a method of efficiently editing T cells, the method comprising the steps of:
  • the sgRNA molecule comprises a targeting domain that is complementary to a target region of a constant coding region of the alpha chain constant coding region (ie, TRAC) gene from the TCR, the B2M gene, and PD-1.
  • TRAC alpha chain constant coding region
  • the sgRNA molecule refers to a nucleic acid sequence comprising a targeting domain complementary to a target region of the gene to be knocked out, which recognizes the target DNA sequence and directs the Cas9 molecule to cleave the target site, It can achieve one-step high efficiency (more than 85% knockout efficiency) to knock out the corresponding sites.
  • the sequence of the targeting domain comprised by the sgRNA molecule is as shown in one of Table 2.
  • sequences of the targeting domains are as indicated by T2, B3 and P1.
  • the sgRNA molecule and mRNA encoding a Cas9 molecule are introduced into the T cell by electroporation techniques.
  • the T cells used in the above methods are derived from healthy humans, such as peripheral blood of healthy adults, or cord blood of healthy humans who are naturally delivered.
  • a third aspect of the invention provides a sgRNA sequence with efficient editing efficiency after specific modification (Table 2).
  • chemical synthesis and modification of sgRNA results in more stable and higher editing efficiency of sgRNA than conventional in vitro transcription (IVT).
  • the T cells are subjected to electroporation by electroporation, and the chemically synthesized and modified sgRNA gene editing efficiency is more than 10 times that of the sgRNA obtained by ordinary IVT.
  • a universal T cell of the invention for the manufacture of a medicament for treating a disease, such as a tumor, is provided.
  • the constant strand coding region (ie, TRAC) gene of the TCR, the constant coding region B2M gene of HLA, and the constant coding region gene of PD-1 are knocked out.
  • the TCR alpha chain constant coding region gene of the present invention is introduced into the TRAC-sg 2 molecule of the cell
  • the B2M constant coding region gene of the present invention is introduced into the B2M-sg6 molecule of the cell
  • PD- of the present invention 1 a constant coding region gene is introduced into the cell of PD-1-sg1 (see Table 2) and a Cas 9 molecule:
  • the ⁇ chain constant coding region gene of the TCR is introduced into the TRAC-sg2, B2M constant coding region gene of the cell
  • the B2M-sg6, PD-1 constant coding region gene introduced into the cell was knocked out by the PD-1-sg1 molecule and the Cas9 molecule introduced into the cell.
  • the present invention relates to TRAC single gene knockout T cells (TRAC negative ), TRAC/B2M double knockout (DKO) T cells and TRAC/B2M/PD-1 three gene knockout (TKO) T prepared by the above method of the present invention. cell.
  • the single gene knockout T cell (TRAC negative ), TRAC/B2M double knockout (DKO) T cell and TCR/B2M/PD-1 triple knockout (TKO) T cell of the present invention are compared with the prior art.
  • the efficiency of gene knockout is greatly improved.
  • the single gene knockout T cells (TRAC negative ), TRAC/B2M double knockout (DKO) T cells and TCR/B2M/PD-1 triple knockout (TKO) T cells prepared by the present invention can be further used as T cells.
  • the engineered precursor cells, or as universal T cells, are used to prepare various genetically modified T cells, for example, to prepare CAR-T cells or TCR-T cells.
  • a genetically engineered T cell (such as a universal T cell) is provided, wherein: in the T cell: (i) one or more of the TRAC genomic region from position 2316448 to position 23016490 of chromosome 14 Sites are disrupted by gene editing techniques; (ii) one or more sites of the B2M genomic region at positions 45003745 to 45003788 on chromosome 15 are disrupted by gene editing techniques; and/or (iii) chromosome 2 One or more sites of the PD-1 genomic region of positions 242800936 to 242800978, or the PD-1 genomic region of positions 242795009 to 242795051 of chromosome 2 are disrupted by gene editing techniques.
  • the T cell is complementary to a TRAC genomic target nucleotide sequence complementary to a sequence selected from any one of SEQ ID NOs: 2-5 by gene editing techniques; (ii) and selected from SEQ ID NOs: 6
  • the target nucleotide sequence of the sequence complementary B2M genome of any of -14 is disrupted by gene editing techniques; and/or (iii) PD-1 complementary to a sequence selected from any one of SEQ ID NOs: 15-22 Genome-based target nucleotide sequence destroyed.
  • a genetically engineered T cell such as a universal T cell, wherein: in the T cell: (i) the TRAC genome comprises the sequence of any one of Tables D and E;
  • the B2M genome comprises the sequence of any of those listed in Tables B and C;
  • the PD-1 genome comprises the sequence of any one of Table F or Table G.
  • One aspect of the invention provides a composition comprising the genetically engineered T cell (e.g., universal T cell), CAR-T cell, or TCR-T cell, such as a pharmaceutical composition.
  • T cell e.g., universal T cell
  • CAR-T cell e.g., CAR-T cell
  • TCR-T cell e.g., TCR-T cell
  • the invention also provides a kit or article comprising the genetically engineered T cells (e.g., universal T cells) of the invention.
  • the kit or article can be used to prepare a CAR-T, TCR-T or other adoptive cell therapeutic composition.
  • One aspect of the invention provides methods of making CAR-T cells, such as universal CAR-T cells.
  • the method comprises introducing a CAR or its encoding nucleotide or vector into any of the genetically engineered T cells (eg, universal T cells) described herein.
  • a method of making a CAR-T cell comprising:
  • CAR or its encoding nucleic acid and sgRNA targeting TRAC, sgRNA targeting B2M and/or sgRNA targeting PD-1 and CAR or its encoding nucleotides can be introduced into T cells in any suitable order.
  • the sgRNA targeting TRAC, the sgRNA targeting B2M and/or the sgRNA targeting PD-1 and CAR or its encoding nucleotide are simultaneously introduced into the T cell.
  • the CAR or its encoding nucleotide is introduced into the T cell prior to the sgRNA targeting TRAC, the sgRNA targeting B2M and/or the sgRNA targeting PD-1.
  • the CAR or its encoding nucleotides have been introduced into a T cell that has been genetically engineered, and the TRAC, B2M and/or PD-1 genomic regions of the T cell have been destroyed by editing.
  • the method further comprises introducing Cas9 or a coding nucleotide thereof into the T cell together with said sgRNA.
  • the CAR expressed by the universal CAR-T cells of the invention may be any CAR known in the art, as long as it enables T cells to recognize cell surface antigens in a human leukocyte antigen-independent manner, Play a killing effect.
  • the CAR in a CAR-T cell of the invention is a CAR that recognizes the antigens described in Table A below the corresponding tumor and related antigen.
  • the CAR expressed in a CAR-T cell of the invention comprises a sequence-linked signal peptide, an extracellular binding region, a hinge region, a transmembrane region, and an intracellular signaling region.
  • signal peptide refers to a short (eg, 5-30 amino acids in length) peptide chain that directs the transfer of a newly synthesized protein to a secretory pathway.
  • a signal peptide of various proteins in a human body such as a signal peptide of a cytokine protein secreted in vivo or a leukocyte differentiation antigen (CD molecule) can be used.
  • the signal peptide is a CD8 signal peptide, for example, the amino acid sequence thereof is shown in the patent application US20140271635A1.
  • the hinge region can use a hinge region of a variety of different antibodies or antigen receptors, particularly a hinge region of a CD molecule.
  • the hinge region can be selected from a hinge region of a protein such as CD8 or CD28.
  • the CD8 or CD28 is a natural marker on the surface of T cells.
  • transmembrane regions of various human proteins can be used, particularly transmembrane regions of various antigen receptors.
  • the transmembrane region preferably used is the transmembrane region of CD molecules.
  • the transmembrane region may be selected from a transmembrane region of a protein such as CD8 or CD28 or 4-1BB.
  • the hinge region is a CD8 alpha hinge region (CD8-hinge), the amino acid sequence of which is shown in the patent application US20140271635A1.
  • the extracellular binding region refers to a region comprising a specific recognition target antigen.
  • the extracellular binding region comprises a region that specifically recognizes a target tumor cell surface antigen.
  • this region can be an antigen-binding fragment of an scFv or other antibody.
  • scFv refers to a recombinant protein of a variable region of heavy chain (VH and a variable region of light chain, VL) linked by a linker. The region associates the two domains to ultimately form an antigen binding site.
  • the scFv is typically an amino acid sequence encoded by a single nucleotide chain.
  • the scFv described above may also include derivatives thereof.
  • the CAR and its various domains used in the present invention may be further modified by conventional techniques known in the art, such as amino acid deletions, insertions, substitutions, additions, and/or recombinations, and/or other modifications, either alone or in combination.
  • Methods for introducing such modifications into their DNA sequences based on the amino acid sequence of an antibody are well known to those skilled in the art (see, for example, Sambrook Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y.).
  • the modification is preferably carried out at the nucleic acid level.
  • the term "specifically recognizes" as used herein means that the antigen recognition region of the invention does not or substantially does not cross-react with any polypeptide other than the antigen of interest.
  • the degree of specificity can be judged by immunological techniques including, but not limited to, immunoblotting, immunoaffinity chromatography, flow cytometry, and the like.
  • the extracellular binding region comprises an antigen binding region, such as a scFv, that specifically recognizes CD19, CEA, EGFR, GD2, CD7 or CD138, and the like.
  • an antigen binding region such as a scFv, that specifically recognizes CD19, CEA, EGFR, GD2, CD7 or CD138, and the like.
  • the extracellular binding region comprises a humanized, engineered scFv that specifically recognizes CD19.
  • the amino acid sequence of the scFv that specifically recognizes CD19 is as shown in the patent application US20140271635A1.
  • intracellular signal regions of various human body proteins can be used.
  • the intracellular signal region preferably used is the intracellular signal region of the CD molecule.
  • the intracellular signal region can be selected from the group consisting of CD3 , Fc ⁇ RI ⁇ , CD28, CD137 (4-1BB), intracellular signal regions of CD134 protein, and combinations thereof.
  • the CD3 molecule consists of five subunits, of which CD3 Subunit (also known as CD3zeta, referred to as ) contains three ITAM motifs, which are important signal transduction regions in the TCR-CD3 complex.
  • Fc ⁇ RI y is mainly distributed on the surface of mast cells and basophils, which contain an ITAM motif that is structurally, distributed, and functionally related to CD3. Similar.
  • CD28, CD137, and CD134 are conjugated signaling molecules, and the co-stimulatory action of intracellular signal segments after binding to their respective ligands causes sustained proliferation of T cells and enhances IL secretion by T cells.
  • -2 and cytokine levels such as IFN- ⁇ , while increasing the survival cycle and anti-tumor effect of CAR-T cells in vivo.
  • the signal produced by the TCR alone is not sufficient to fully activate native T cells, requiring antigen-dependent primary activation of the sequence (primary intracellular signaling domain) by TCR and in an antigen-independent manner
  • a sequence that acts to provide a costimulatory signal (co-stimulatory domain).
  • the primary signaling domain regulates the primary activation of the TCR complex in an irritant manner or in an inhibitory manner.
  • the primary intracellular signaling domain that acts in an irritating manner may contain a signaling motif called the immunoreceptor tyrosine activation motif (ITAM).
  • ITAM immunoreceptor tyrosine activation motif
  • ITAM-containing primary cytoplasmic signaling sequences suitable for use in the present invention include CD3 ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, and CD66d.
  • the primary signaling domain comprises a modified ITAM domain, such as a mutant ITAM domain whose activity is altered (eg, increased or decreased) compared to a native ITAM domain, or a truncated ITAM primary Intracellular signaling domain.
  • the primary signaling domain comprises one or more ITAM motifs.
  • the costimulatory signaling domain refers to the portion of the TCR that contains the intracellular domain of the costimulatory molecule.
  • a costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand required for efficient lymphocyte reaction to an antigen. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD1, ICOS, lymphocyte function associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3 And a ligand which specifically binds to CD83 and the like.
  • the invention provides a method of making a CAR-T cell, such as a universal CAR-T cell, the method comprising the steps of:
  • the sgRNA molecule comprises a targeting domain that is complementary to a chain constant coding region (ie, TRAC) gene from TCR, an HLA constant coding region B2M gene, and a constant coding region gene target region of PD-1.
  • TRAC chain constant coding region
  • the sgRNA molecule refers to a nucleic acid sequence comprising a targeting domain complementary to a target region of the gene to be knocked out, which recognizes the target DNA sequence and directs the Cas9 molecule to cleave the target site, It can achieve one-step high efficiency (more than 85% knockout efficiency) to knock out the corresponding sites.
  • the Cas9 molecule refers to Cas9 mRNA, which is capable of cleavage of a target site under the guidance of sgRNA.
  • the sequence of the targeting domain comprised by the sgRNA molecule is as shown in one of Table 2.
  • sequences of the targeting domains are shown as T2, B3, and P1 (Table 2).
  • the sgRNA molecule and mRNA encoding a Cas9 molecule are introduced into the T cell by electroporation techniques.
  • the CAR molecule is introduced into the T cell by, for example, a lentiviral transfection technique.
  • the step of isolating and/or activating T cells from healthy human peripheral blood or cord blood preferably, the method further comprises performing universal CAR-T cells after step 2) above The step of sorting; more preferably, the resulting CAR-T cells, such as universal CAR-T cells, are functionally verified after sorting.
  • the invention provides the use of the above CAR-T cells for the preparation of a disease, such as a tumor.
  • the invention provides, in one aspect, a method of treating a disease in a subject comprising administering to the subject an effective amount of a CAR-T cell of the invention.
  • Therapeutic methods described herein include, but are not limited to, cancer and HIV/AIDS.
  • the disease is a tumor, including a hematological tumor, such as a lymphoma or leukemia.
  • the CAR targets the antigen shown in Table A, which is the tumor in Table A corresponding to the target antigen.
  • the T cell is not obtained from a subject.
  • the T cells can be derived from a healthy donor.
  • the CAR-T cells to which the present invention relates can be administered to a subject in need thereof by a route conventionally used for administering a pharmaceutical preparation containing a cellular component, such as an intravenous infusion route.
  • the dosage administered can be specifically determined based on the condition of the subject and general health conditions.
  • the invention provides T cells expressing engineered TCR, also known as TCR-T cells.
  • the invention also provides a method of making the TCR-T cell comprising introducing an engineered TCR or a coding nucleotide or vector thereof into any of the genetically engineered T cells (e.g., universal T cells) described herein.
  • a method of making TCR-T cells comprising:
  • TCR or its encoding nucleic acid and sgRNA targeting TRAC can be introduced into T cells in any suitable order.
  • the sgRNA that targets TRAC the sgRNA that targets B2M and/or the sgRNA that targets PD-1 and the TCR or its encoding nucleotide are simultaneously introduced into the T cell.
  • the TCR or its encoding nucleic acid is introduced into the T cell prior to the sgRNA targeting TRAC, the sgRNA targeting B2M and/or the sgRNA targeting PD-1.
  • the TCR or its encoding nucleotides are introduced into T cells that have achieved gene editing, and the TRAC, B2M and/or PD-1 genomic regions of the T cells have been destroyed by editing.
  • the method further comprises introducing Cas9 or a coding nucleotide thereof into the T cell together with said sgRNA.
  • the engineered TCR expressed by the TCR-T cells of the invention can be any engineered TCR known in the art, as long as it enables T cells to recognize the cell surface in a human leukocyte antigen-independent manner.
  • the antigen can play a killing effect.
  • an engineered TCR in a TCR-T cell of the invention can be an engineered TCR that recognizes the antigen described in Table A.
  • engineered TCR molecule includes recombinant polypeptides derived from various polypeptides that make up a TCR, which can generally i) bind to a target cell. Surface antigen; and ii) interact with other polypeptide components of the intact TCR complex when colocalized in or on the T cell.
  • an engineered TCR of the invention comprises a target-specific binding element, also known as an antigen binding domain.
  • a selectable antigen binding domain recognizes, for example, a target antigen that acts as a cell surface marker on a target cell associated with a particular disease state.
  • the target antigen is, for example, an antigen in Table A above.
  • the target antigen is, for example, a target antigen associated with viral infection, immune disease, and disease.
  • the antigen binding domain can be combined with various polypeptides derived from the composition of the TCR such that the TCR-mediated T cell response is directed against the antigen of interest.
  • the engineered TCR of the invention comprises a transmembrane domain.
  • the transmembrane domain can be derived from a natural source or a recombinant source. Where the source is of a natural origin, the domain may be derived from any membrane-bound or transmembrane protein.
  • the transmembrane domain is capable of signaling to an intracellular domain as long as the engineered TCR binds to the target.
  • transmembrane domains in the present invention may include, for example, at least a T cell receptor alpha, beta or ⁇ chain, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, Transmembrane regions of CD80, CD86, CD134, CD137, CD154.
  • the transmembrane domain can be joined to the extracellular region of an engineered TCR by a hinge, such as a hinge from a human protein, such as an antigen binding domain of an engineered TCR.
  • a hinge such as a hinge from a human protein, such as an antigen binding domain of an engineered TCR.
  • the hinge can be a human immunoglobulin (Ig) hinge, such as an IgG4 hinge, or a CD8a hinge.
  • an engineered TCR of the invention comprises a linker that links a transmembrane domain to a cytoplasmic region.
  • the linker is a short oligopeptide or polypeptide linker between 2 and 50 amino acids in length. Glycine-serine pairs provide a particularly suitable linker.
  • the engineered TCR of the invention comprises a cytoplasmic domain.
  • the intracellular signaling domain is generally responsible for the activation of at least one of the normal effector functions of the immune cells into which the engineered TCR is introduced.
  • the effector function of T cells can be, for example, cytolytic activity or helper activity, including secretion of cytokines.
  • intracellular signaling domain refers to a portion of a protein that transduces an effector function signal and directs the cell to perform a specialized function.
  • intracellular signaling domain is intended to include any truncated portion of an intracellular signaling domain sufficient to transduce an effector function signal.
  • the engineered TCR molecule comprises an engineered TCR alpha and TCR beta chain. In certain embodiments, the engineered TCR molecule binds to a CD3 molecule expressed in a T cell and an ⁇ chain and/or other costimulatory molecule.
  • the invention provides the use of the above TCR-T cells for the preparation of a disease, such as a tumor.
  • the invention provides, in one aspect, a method of treating a disease in a subject comprising administering to the subject an effective amount of a TCR-T cell of the invention.
  • Therapeutic methods described herein include, but are not limited to, cancer and HIV/AIDS.
  • the disease is a tumor, including a hematological tumor, such as a lymphoma or leukemia.
  • the engineered TCR targets the target antigen shown in Table A, which is the tumor in Table A corresponding to the target antigen.
  • the T cell is not obtained from a subject.
  • the T cells can be derived from a healthy donor.
  • the TCR-T cells to which the present invention relates may be administered to a subject in need thereof by a route conventionally used for administering a pharmaceutical preparation containing a cellular component, such as an intravenous infusion route.
  • a route conventionally used for administering a pharmaceutical preparation containing a cellular component such as an intravenous infusion route.
  • the dosage administered can be specifically determined based on the condition of the subject and general health conditions.
  • the T cell source is obtained from the subject prior to amplification and genetic modification.
  • subject is intended to include a living organism (eg, a mammal) that is capable of eliciting an immune response. Examples of subjects include humans.
  • T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissues from infected sites, ascites, pleural effusion, spleen tissue, and tumors.
  • the T cells of the invention may also be derived from hematopoietic stem cells at various stages of differentiation. Hematopoietic stem cells differentiate into T cells under directed differentiation culture conditions. In certain aspects of the invention, a variety of T cell lines available in the art can be used.
  • T cells can be obtained using a variety of techniques known to the skilled artisan, such as FicollTM separation of blood collected from a subject.
  • Cells can also be obtained from the individual's circulating blood by apheresis.
  • Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells, and platelets.
  • cells collected by apheresis can be washed to remove plasma fractions and placed in appropriate buffers or media for subsequent processing steps.
  • TIL Tumor infiltrating lymphocytes
  • TIL Tumor infiltrating lymphocytes
  • the invention provides a sgRNA that targets TRAC, a sgRNA that targets B2M, and an sgRNA that targets PD-1.
  • the sgRNA comprises any of the nucleotide sequences selected from the group consisting of SEQ ID NOs: 2-22. In some embodiments, the sgRNA is chemically modified.
  • kits or articles of manufacture comprising the sgRNAs of the invention or vectors thereof.
  • the kit comprises: i) an sgRNA comprising a sequence selected from any one of SEQ ID NOs: 2-5; (ii) an sgRNA comprising a sequence selected from any one of SEQ ID NOs: 6-14; And/or (iii) comprises an sgRNA selected from the sequence of any one of SEQ ID NOs: 15-22.
  • the kit comprises: (i) an sgRNA comprising the sequence of SEQ ID NO: 3; (ii) an sgRNA comprising a sequence selected from the group consisting of SEQ ID NO: 16; and (iii) comprising SEQ ID NO: 7 sequences of sgRNA.
  • the kit further comprises a Cas6 encoding nucleic acid or a vector thereof.
  • the sgRNA is chemically modified.
  • the T cell genetic engineering methods of the invention use chemically modified sgRNA.
  • the chemically modified sgRNA employed by the present inventors is considered to have the following two advantages. First, since sgRNA is a single-stranded form of RNA, its half-life is very short, and it will degrade rapidly after entering the cell (up to 12 hours), while at least 48 hrs is required for Cas9 protein to bind to sgRNA for gene editing. Therefore, the chemically modified sgRNA is used to stably express after entering the cell, and after binding to the Cas9 protein, the genome can be efficiently edited to generate Indels. Second, unmodified sgRNAs have poor ability to penetrate cell membranes and cannot effectively enter cells or tissues to function accordingly.
  • sgRNAs The ability of chemically modified sgRNAs to penetrate cell membranes is generally enhanced. Chemical modification methods commonly used in the art can be employed in the present invention, as long as the sgRNA stability (prolonged half-life) and the ability to enter the cell membrane can be improved. In addition to the specific chemical modifications used in the examples, other modification methods are also included, for example, Deleavey GF1, Damha MJ. Designing chemically modified oligonucleotides for targeted gene silencing. Chem Biol. 2012 Aug 24; 19(8): 937 -54, and Hendel et al. Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nat Biotechnol. 2015 Sep; 33 (9): 985-989 The chemical modification method reported in the literature.
  • the present invention electrically converts chemically modified sgRNA and Cas9 encoding gene into T cells, resulting in efficient gene editing efficiency (eg, expressed as Indels%), wherein chemical modification of sgRNA is one of the key factors in the present invention.
  • efficient gene editing efficiency eg, expressed as Indels%
  • the data in the examples show that if electro-synchronized with Cas9 mRNA is an unmodified sgRNA, the Indels efficiency is much lower than the Indels efficiency obtained when electroporating chemically modified sgRNA.
  • Collection of cord blood from healthy donors After taking the cord blood from the blood bank, put it into the refrigerator at 4 °C for temporary storage. Within 24 hours, transport it to the GMP laboratory for transport of T cells via a transport vehicle equipped with a constant temperature device.
  • 1.1 Preparation of cord blood mononuclear cells: Pipette the physiological saline into the cord blood transported in step (1), and umbilical cord blood and physiological saline are diluted according to 1:1 (V/V), and the blood cell dilution is slow. Add to the lymphocyte separation tube, centrifuge at 800g for 20 minutes, then absorb the white blood cell layer above the lymphocyte separation solution, transfer to a new 50 ml centrifuge tube, add T cell culture medium, centrifuge at 400g for 5 minutes and discard. Clear, the cell pellet at the bottom of the centrifuge tube is retained, and peripheral blood mononuclear cells are obtained.
  • T cells The obtained cord blood mononuclear cells are counted by a cell counter and subjected to T cell sorting. The specific steps are as follows:
  • T cell isolation reagent at a concentration of 50 ul / ml, and incubate for 5 minutes at room temperature after the addition;
  • the T cell pellet was resuspended in the medium of the T cells. Then, the T cell activating factor was added in a ratio of 1:1, and the T cells were activated at this time, and the T cells were placed in an incubator to continue the expansion culture.
  • the cultured T cells were collected into a 50 ml centrifuge tube, centrifuged at 300 g for 7 minutes, and the supernatant was discarded, washed twice with DPBS solution (manufacturer: Gibco, Cat. No. 1924294), and then the cell density was adjusted by electroporation. Up to 2.5X10 7 cells/mL.
  • Use HISCRIBE TM T7 ARCA mRNA Kit (tailed) manufactured (manufacturer: NEB, NO: cat # E2060S)
  • Preparation of GFP mRNA were uniformly mixed with T cells, to a final concentration of 2.5 each containing 100 ⁇ L X10 6 cells and 6 ⁇ g GFP mRNA.
  • GFP mRNA was introduced into T cells using an electrorotator BTX Agile pulse MAX (manufacturer: BTX, model: 47-0200 NINT). According to the difference of voltage and pulse time, the electric rotation system is optimized respectively. As shown in Table 1, the voltage is increased from 180 volts to 400 volts, and the pulse time is successively decreased from 2 ms to 0.5 ms. The growth of the cells was observed every day, and the T cells of the electroporation were counted and reperfused every other day, and the phenotypic analysis of the T cells after the gene editing was performed by a flow cytometer (manufacturer: Essen, model: ACEA NovoCyte), and the results were as follows. As shown in Table 1, cell viability and GFP electrotransfer efficiency were best under 150-250 V, 0.5-2 ms electroporation conditions.
  • Table 1 Comparison of electroporation efficiency of GFP mRNA under different electroporation conditions and cell viability after electroporation
  • the TRAC, B2M, and PD-1 genes in the T cells obtained in step 1.2 were knocked out using the CRISPR/Cas9 gene editing technology.
  • the specific steps are as follows:
  • the sgRNAs designed for all coding sequences in the TRAC, B2M and PD-1 coding regions were designed by CRISPR RGEN Tools.
  • the sgRNA sequences selected according to the highest score are shown in Table 2.
  • T represents the sgRNA sequence for TRAC
  • P represents the sgRNA sequence for the PD-1 coding region
  • B represents the sgRNA sequence for B2M.
  • the sgRNA is chemically modified with 2'-O-methyl analog and/or internucleotide 3' thiol to produce sgRNA with high knockout efficiency and stability.
  • the enzyme was digested with 2 ⁇ l of enzyme to perform agarose gel electrophoresis.
  • the voltage was set to 110 U and electrophoresis was carried out for 30 min.
  • the gel imager was observed as a single band, indicating that the enzyme digestion was complete and all plasmids were linearized.
  • the product was purified taken with HISCRIBE TM T7 ARCA mRNA Kit vitro transcription (i.e. IVT), 20 ⁇ l system (Manufacturer: cat # E2060S: NEB, NO):
  • the above reaction system was placed on a PCR machine at 37 ° C for 4 hours. After 4 hours, 2 ⁇ l of DNase 1 was added to the reaction system, and the reaction was carried out at 37 ° C for 20 min.
  • the above reaction system was placed on a PCR machine for 2 hours.
  • the above reaction product was cleaned and purified, and placed in a -80 degree refrigerator for use.
  • sgRNA and Cas9 mRNA were introduced into T cells, and TCR and/or B2M and/or PD1 negative and CD4 and CD8 positive T cells were screened by antigen screening principle to obtain universal T cells.
  • the reaction conditions are as follows:
  • the PCR product after the reaction was subjected to Sanger sequencing to verify the knockout efficiency of TCR, HLA and PD-1 at the molecular level.
  • the results are shown in Fig. 2.
  • the TCR of TCR can be successfully succeeded by using CRISPR/Cas9 technology.
  • the gene, the B2M gene of HLA, and the constant coding sequence of PD-1 were edited, including insertion and deletion mutations, both of which caused frameshift mutations (see Table BG below), thereby inhibiting TCR, HLA, and Expression of PD-1.
  • Table B Sequence changes produced in chromosomes 45003745 to 45003788 after introduction of B2:
  • Table D Sequence changes produced within the range of 23016448 to 23016490 of chromosome 14 after introduction of T3:
  • Table E Sequence changes produced within the range of 23016448 to 23016490 of chromosome 14 after introduction of T2:
  • Table G Sequence changes produced on chromosome 2 from 242795009 to 242795051 after introduction of P1
  • Table 3 Results of off-target analysis of TRAC, TRAC/B2M (DKO), and TRAC/B2M/PD-1 (TKO) knockout T cells
  • T2 TRAC-sg2
  • T3 T3
  • B2M-sg2 B2M
  • B3 B3
  • PD-1-sg1 P1
  • PD-1-sg2 P2
  • the sorted T cells were activated using cytokines, and then the T cell medium was adjusted to a cell density of 1 ⁇ 10 6 cells/mL. After 72 hours, the state of the cells was observed, and the cell suspension was collected, centrifuged at 300 g for 7 min, the supernatant was discarded, washed twice with DPBS (Gibco), and then the cell density was adjusted to 2.5 ⁇ 10 7 cells/mL with electrotransfer reagent medium. .
  • HISCRIBE TM T7 ARCA mRNA Kit (Manufacturer: NEB, NO: cat # E2060S) prepared Cas9mRNA SgRNA and synthetic, mixed cell and the T RNA, the final concentration of each of 100 ⁇ L containing 2.5X10 6 cells and 8 ⁇ g of RNA (4 ⁇ g each of Cas9 mRNA and sgRNA) was then introduced into the cells by electroporation BTX Agile pulse MAX and cultured. The growth of the cells was observed every day, cell count rehydration was performed every other day, and phenotypic analysis was performed on the T cells after gene editing. As shown in Fig. 3, the efficiency of knocking out TCR alone was about 90.42%, knocking out TCR and B2M. The efficiency of (DKO) is about 81.39%, and the efficiency of simultaneous knockout of three genes (TKO) is as high as 67.91. After 8 days of cell culture, the obtained T cells were subjected to quality control monitoring.
  • the cells were cultured for 8 days, and the human genome extraction kit (manufacturer: TIAN GEN, article number: cat#DP304-03) was used to extract the genome, and the corresponding sequencing primers were designed, and the target fragment was prepared by PCR technology. Singer sequencing was performed along with the corresponding primers. The sequencing results were analyzed using TIDE software, and the results obtained are shown in Figures 2A, 2C, and 2E.
  • the preferred sgRNA can efficiently knock out the corresponding gene.
  • TCR and/or B2M and/or PD-1 negative, CD4 and CD8 positive T cells were screened by the following methods.
  • TCR and/or B2M and/or PD-1 negative, CD4 and CD8 positive T cells were screened by immunomagnetic beads technique and the edited T cell status was monitored by T cell viability.
  • the T cells after electroporation were collected on the 12th-14th day, 400G, centrifuged for 5 min, the supernatant was discarded, the cells were dissolved into 1 ⁇ 10 8 /ml with Easy buffer, and then the cells were transferred to a 5 ml flow tube.
  • the cells which still express TCR, B2M and PD-1 in T cells are removed by using a screening reagent, and the final product, that is, universal T cells, is screened.
  • TCR and/or B2M and/or PD-1 cell surface biomarkers are stained if TCR and/or B2M and/or PD-1 positive rate is ⁇ 1%, you can proceed to the next step.
  • the TCR positive rate was 1%
  • the T cell positive rate of TCR and B2M DKO was ⁇ 0.79%
  • the T cell positive rate of TCR, B2M and PD-1TKO was ⁇ 1%, as shown in FIG.
  • the purified T cell survival rate was not significantly affected, both above 85%, as shown in Table 4.
  • Table 4 Survival rates after screening and purification of T cells knocked out by TCR, TCR/B2M (DKO) and TCR/B2M/PD-1 (TKO)
  • the in vitro digestion reaction system of Cas9 is as follows:
  • the sorted T cells were activated using cytokines, and then the cell density was adjusted to 1 ⁇ 10 6 cells/mL using T cell culture medium. After 72 hours, the state of the cells was observed, and the cell suspension was collected, centrifuged at 300 g for 7 min, the supernatant was discarded, and washed twice with DPBS solution (manufacturer: Gibco; Cat. No. 1924294), and then the cell density was adjusted to the electrotransfer medium. 2.5X 10 7 cells/mL.
  • HISCRIBE TM T7 ARCA mRNA Kit (with tail) (Manufacturer: NEB, NO: cat # E2060S) prepared Cas9mRNA and synthetic SgRNA, T cells and RNA were mixed so that the final concentration of each of 100 ⁇ L containing 2.5X10 Six cells and 8 ⁇ g of RNA (4 ⁇ g each of Cas9 mRNA and sgRNA) were cultured by introducing RNA into cells using a BTX Agile pulse MAX.
  • TCR and/or B2M and/or PD-1 negative, CD4 and CD8 positive T cells were screened by the following methods.
  • TCR and/or B2M and/or PD-1 negative, CD4 and CD8 positive T cells were screened by immunomagnetic beads technique, and the edited T cell status was monitored by T cell survival.
  • the specific steps are as follows:
  • the T cells after electroporation were collected on the 12th-14th day, 400G, centrifuged for 5 min, the supernatant was discarded, the cells were dissolved into 1 ⁇ 10 8 /ml with Easy buffer, and then the cells were transferred to a 5 ml flow tube.
  • the cells that still express TCR, B2M, and PD-1 in the T cells were removed by using the magnetic beads, and the final product, the universal T cells, was screened.
  • the second step a small number of T cells are taken for flow cytometry, and TCR and/or B2M and/or PD-1 cell surface biomarkers are stained at the same time. If the TCR and/or B2M and/or PD-1 positive rate is ⁇ 1% , you can proceed to the next step. In this example, the TCR positive rate was 1%, the T cell positive rate of TCR and B2M DKO was ⁇ 0.79%, and the T cell positive rate of TCR, B2M and PD-1TKO was ⁇ 1%, as shown in Table 5 and Figure 6. . The survival rate of purified T cells was not significantly affected, and was above 85%.
  • CELL TRACE TM Far Red Cell Proliferation Kit Manufacturer: Gibco; NO: 1,888,569) labeled target cells (human Burkitt's lymphoma Raji, K562, all cells from ATCC).
  • Step 2 Killing of target cells by effector cells
  • Step 3 ELISA for cytokine release
  • the labeled target cells were resuspended at a density of 2 ⁇ 10 5 /mL with RPMI 1640 + 10% FBS, and 500 ⁇ L was added to a 48-well plate. 500 ⁇ L of effector cells were added per well according to the appropriate target-to-target ratio (10:1), and T cells and healthy human cord blood CAR-T were used as control cells, and each group was parallelized, and a separate target cell group was designed at 37 ° C.
  • Fig. 8 The results obtained are shown in Fig. 8. It can be seen that the killing ability of general-purpose CAR-T against Raji cells is basically the same as that of common CAR-T or slightly better than the killing ability of common CAR-T, especially TCR neg CAR-T. The release of IFN-r is much higher than that of common CAR-T and both have lower lethality against K562, so the release of factors is relatively small.
  • the general-purpose CAR-T kills the specific tumor cells in vivo
  • Cell line human lymphoma cell line
  • Raji tg (luciferase-GFP) / Bcgen cells are human Burkitt's lymphoma cell lines, which are positive for CD19 and can be used as target cells for CAR-T cells. Raji luc-GFP was modified to express both GFP and luciferase.
  • a human lymphoma model of the mouse can be constructed by tail vein injection, and the fluorescence statistical tumor formation area presented by XenoLight D-Luciferin, Potassium Salt (manufacturer: PerkinElmer; Cat. No. 122799) in combination with a small animal live imager.
  • the Raji tg (luciferase-GFP) / Bcgen cell line was a suspension cell line that grew rapidly in RPMI 1640 medium containing 10% FBS. Passage is required when the cell density is 2-3X 10 6 /mL. At the time of passage, the cell suspension was taken in a centrifuge tube, centrifuged at 300 g for 6 minutes, and the supernatant was discarded. The cell density was adjusted to 1 ⁇ 10 6 cells/ml, and the culture was continued. Normal growth conditions, passaged every other day, cell density can be maintained between 0.8-1X 10 6 cells / mL.
  • mice Twenty - five NPT female mice, 7-10 weeks old, were injected with tumor cells (Raji tg (luciferase-GFP) / Bcgen) 5 ⁇ 10 5 cells/cell, and weighed every other day, once a day, inoculated with tumor cells. After 3-5 days, XenoLight D-Luciferin, Potassium Salt (manufacturer: PerkinElmer; Cat. No. 122799) and the small animal living imager showed the tumor-forming area and tumor enrichment as indicators. They were randomly divided into 6 groups: saline group. , T cell group, CAR-T cell group, TCR neg -CAR-T group, DKO-CAR-T group, TKO-CAR-T group.
  • mice after administration were monitored daily, including mouse body weight, skin integrity, hair, mental state, activity frequency and activity coordination, and the mice were recorded for weight every 2 days for 37 days for tumor area.
  • the elimination and reduction of tumor enrichment are the evaluation indicators of effector cell function, and the safety of general-purpose CAR-T is judged by skin integrity, hair, mental state, activity frequency and activity coordination degree.
  • the mice did not have a decrease in body weight, the skin was intact, the spirit was active, the activity was coordinated, and no GVHD response occurred.

Abstract

提供了一种通用型T细胞及其制备方法。所述通用型T细胞包括CAR-T和TCR-T细胞，是通过CRISPR/Cas9基因编辑技术敲除了T细胞的TCR和/或HLA和/或PD-1蛋白获得的，其中通用型CAR-T可在体内、体外持续杀伤靶细胞。

Description

一种基因编辑T细胞及其用途
交叉引用
本申请要求享有2017年9月18日提交的中国专利申请201710842264.5和201710841323.7的优先权，该中国专利申请的公开内容以其整体通过引用并入本申请。
技术领域
本发明涉及T细胞，具体涉及经过基因编辑的单基因、双基因及三基因敲除的T细胞，包括通用型T细胞、CAR-T细胞和TCR-T细胞，及其制备方法和用途。
背景技术
恶性肿瘤已成为严重威胁身体健康和生命安全的疾病，肿瘤的治愈一直是人类的梦想。近年来，肿瘤免疫疗法获得了广泛的关注，尤其是CAR-T(Chimeric Antigen Receptor T Cells)技术的出现使得对肿瘤的控制得到了里程碑式的发展。从1989年CAR-T技术首次应用，到2012年宾夕法尼亚大学Carl June教授带领团队治愈的Emily Whitehead，再到2017年FDA肿瘤药物专家咨询委员会(ODAC)以10:0的压倒优势投票结果一致推荐批准诺华CAR-T药物CTL019用于青少年晚期B细胞急性淋巴性白血病(r/rAll)治疗。
一般地，传统的CAR-T技术T细胞主要来源于患者自身，在GMP环境下经过体外分离T细胞、激活、CAR导入、培养扩增，最后经过质控回输回患者体内。由于患者自身条件的影响导致不适合采血或采血分离T细胞后扩增难的问题会随之而来。当患者病情危急，从T细胞的分离到CAR-T的回输，整个流程的等待时间也将是自体回输要面临的重大问题。这些问题给CAR-T技术的广泛应用带来了局限性，因此目前CAR-T细胞治疗的一个重要研究方向是如何使用一个健康献血者的T细胞制备大量的CAR-T细胞，满足患者的临床使用。这一技术的建立将极大降低CAR-T疗法的成本，可以更好的保证统一制备的细胞质量，而且患者在需要时可以马上得到CAR-T细胞进行治疗。
在本说明书的整篇文本中引用了数篇文件。此处的每篇文件(包括任何期刊文章或摘要、公开或未公开的专利申请、授权专利、制造商的说明书、使用说明等)通过题述并入本文。然而，并非认可此处引用的文件事实上是本发明的现有技术。
发明概述
进行本发明以解决本领域中存在的上述问题。本发明利用CRISPR/Cas9系统对T细胞进行单基因(TRAC、B2M或PD-1)、双基因(TRAC和B2M)及三基因(TRAC，B2M及PD-1)敲除，其敲除效率分别高达90％(单基因)、81％(双基因)及67％(三基因)。这些经过基因编辑的T细胞可以为针对不同靶点的CAR或TCR提供通用T细胞，为建立基因编辑技术联合过继免疫在肿瘤及病毒感染性疾病(例如HIV/AIDS)的治疗提供方法，并为相关疾病治疗的研究奠定了坚实的技术基础。同时使得基因改造的T细胞(包括通用T细胞，CAR-T和TCR-T)可以作为药物随时应用于需要的病人。
因此，一个方面，本发明提供了利用利用基因编辑技术，例如CRISPR/Cas9系统对T细胞进行高效的单基因(TRAC、B2M或PD-1)、双基因(TRAC和B2M)或者三基因(TRAC，B2M及PD-1)敲除，从而获得不表达TCR，或TCR/HLA，或TCR/HLA/PD-1的通用型T细胞的方法。同时使得通用型T细胞可以随时结合需要的CAR或TCR，制备成通用型CAR-T或TCR-T，且作为药物随时应用于需要的病人。另外还可以为新的有效基因靶点的研究提供支持并及时有效地应用于临床免疫治疗。
一方面，本发明提供一种制备基因改造的T细胞的方法，包括：通过基因编辑技术破坏所述T细胞中：
(i)14号染色体第23016448位至第23016490位的TRAC基因组区域(如SEQ ID NO:23所示)；
(ii)15号染色体第45003745位至第45003788位的B2M基因组区域(如SEQ ID NO:24所示)；和/或
(iii)2号染色体第242800936位至第242800978位的PD-1基因组区域(如SEQ ID NO:25所示)，或2号染色体第242795009位至第242795051位的PD-1基因组区域(如SEQ ID NO:26所示)。在一些实施方案中，所述TRAC基因组区域、B2M基因组区域和PD-1基因组区域均被编辑。在一些实施方案中， 所述基因编辑技术为基于锌指核酸酶的基因编辑技术、TALEN基因编辑技术或CRISPR/Cas基因编辑技术，例如CRISPR/Cas9基因编辑技术。
在一些实施方案中，本发明提供一种制备基因改造的T细胞的方法，包括：通过基因编辑技术破坏所述T细胞中：
(i)与选自SEQ ID NOs：2-5中任一的序列互补的TRAC基因组的靶核苷酸序列；
(ii)与选自SEQ ID NOs：6-13中任一的序列互补的B2M基因组的靶核苷酸序列；和/或
(iii)与选自SEQ ID NOs：14-22中任一的序列互补的PD-1基因组的靶核苷酸序列。在一些实施方案中，所述TRAC基因组区域、B2M基因组区域和PD-1基因组区域均被编辑。在一些实施方案中，所述基因编辑技术为基于锌指核酸酶的基因编辑技术、TALEN基因编辑技术或CRISPR/Cas基因编辑技术，例如CRISPR/Cas9基因编辑技术。
在一些实施方案中，本发明提供了一种制备基因改造的T细胞的方法，通过CRISPR/Cas9基因编辑技术破坏所述T细胞中:
(i)与选自SEQ ID NO：2或SEQ ID NO：3中任一的序列互补的TRAC基因组的靶核苷酸序列；
(ii)与选自SEQ ID NO：7或SEQ ID NO：8中任一的序列互补的B2M基因组的靶核苷酸序列；和/或
(iii)与选自SEQ ID NO：14或SEQ ID NO：15中任一的序列互补的PD-1基因组的靶核苷酸序列。
在一些实施方案中，本发明提供了一种制备基因改造的T细胞的方法，通过CRISPR/Cas9基因编辑技术破坏所述T细胞中:
(i)与SEQ ID NO：2的序列互补的TRAC基因组的靶核苷酸序列；
(ii)与SEQ ID NO：8的序列互补的B2M基因组的靶核苷酸序列；和/或
(iii)与SEQ ID NO：14的序列互补的PD-1基因组的靶核苷酸序列。
在一些实施方案中，本发明提供了一种制备基因改造的T细胞的方法，通过CRISPR/Cas9基因编辑技术破坏所述T细胞中:
(i)与SEQ ID NO：2的序列互补的TRAC基因组的靶核苷酸序列；
(ii)与SEQ ID NO：8的序列互补的B2M基因组的靶核苷酸序列；和/或
(iii)与SEQ ID NO：15的序列互补的PD-1基因组的靶核苷酸序列。
在一些实施方案中，本发明提供了一种制备基因改造的T细胞的方法，通过CRISPR/Cas9基因编辑技术破坏所述T细胞中:
(i)与SEQ ID NO：3的序列互补的TRAC基因组的靶核苷酸序列；
(ii)与SEQ ID NO：7的序列互补的B2M基因组的靶核苷酸序列；和/或
(iii)与SEQ ID NO：15的序列互补的PD-1基因组的靶核苷酸序列。
在一些实施方案中，本发明提供了一种通过CRISPR/Cas9基因编辑技术制备基因改造的T细胞的方法，其中:
(i)将包含选自SEQ ID NOs：2-5中任一序列的sgRNA导入T细胞以实现对14号染色体第23016448位至第23016490位的TRAC基因组区域的编辑；
(ii)将包含选自SEQ ID NOs：6-13中任一序列的sgRNA导入T细胞以实现对15号染色体第45003745位至第45003788位的B2M基因组区域的编辑；和/或
(iii)将包含选自SEQ ID NOs：14-22中任一序列的sgRNA导入T细胞以实现对2号染色体第242800936位至第242800978位的PD-1基因组区域，或2号染色体第242795009位至第242795051位的PD-1基因组区域的编辑。
在一些实施方案中，本发明提供了一种通过CRISPR/Cas9基因编辑技术制备基因改造的T细胞的方法，其中:
(i)将包含选自SEQ ID NO：2或SEQ ID NO：3中任一序列的sgRNA导入T细胞以实现对14号染色体第23016448位至第23016490位的TRAC基因组区域的编辑；
(ii)将包含选自SEQ ID NO：7或SEQ ID NO：8中任一序列的sgRNA导入T细胞以实现对15号染色体第45003745位至第45003788位的B2M基因组区域的编辑；和/或
(iii)将包含选自SEQ ID NO：14或SEQ ID NO：15中任一序列的sgRNA导入T细胞以实现对2号染色体第242800936位至第242800978位的PD-1基因组区域，或2号染色体第242795009位至第242795051位的PD-1基因组区域的编辑。
在一些实施方案中，本发明提供了一种通过CRISPR/Cas9基因编辑技术制备基因改造的T细胞的方法，其中:
(i)将包含SEQ ID NO：2的序列的sgRNA导入T细胞以实现对14号染色体第23016448位至第23016490位的TRAC基因组区域的编辑；
(ii)将包含SEQ ID NO：8的序列的sgRNA导入T细胞以实现对15号染色体第45003745位至第45003788位的B2M基因组区域的编辑；和/或
(iii)将包含SEQ ID NO：15的序列的sgRNA导入T细胞以实现对2号染色体第242800936位至第242800978位的PD-1基因组区域，或2号染色体第242795009位至第242795051位的PD-1基因组区域的编辑。
在一些实施方案中，本发明提供了一种通过CRISPR/Cas9基因编辑技术制备基因改造的T细胞的方法，其中:
(i)将包含SEQ ID NO：2的序列的sgRNA导入T细胞以实现对14号染色体第23016448位至第23016490位的TRAC基因组区域的编辑；
(ii)将包含SEQ ID NO：8的序列的sgRNA导入T细胞以实现对15号染色体第45003745位至第45003788位的B2M基因组区域的编辑；和/或
(iii)将包含SEQ ID NO：14的序列的sgRNA导入T细胞以实现对2号染色体第242800936位至第242800978位的PD-1基因组区域，或2号染色体第242795009位至第242795051位的PD-1基因组区域的编辑。
在一些实施方案中，本发明提供了一种通过CRISPR/Cas9基因编辑技术制备基因改造的T细胞的方法，其中:
(i)将包含SEQ ID NO：3的序列的sgRNA导入T细胞以实现对14号染色体第23016448位至第23016490位的TRAC基因组区域的编辑；
(ii)将包含SEQ ID NO：7的序列的sgRNA导入T细胞以实现对15号染色体第45003745位至第45003788位的B2M基因组区域的编辑；和/或
(iii)将包含SEQ ID NO：15的序列的sgRNA导入T细胞以实现对2号染色体第242800936位至第242800978位的PD-1基因组区域，或2号染色体第242795009位至第242795051位的PD-1基因组区域的编辑。
在一些实施方案中，单独向该T细胞导入所述靶向TRAC的sgRNA。在一些实施方案中，单独向该T细胞导入所述靶向B2M的sgRNA。在一些实施方案中，单独向该T细胞导入所述靶向PD-1的sgRNA。在一些实施方案中，同时向该T细胞导入所述靶向TRAC的sgRNA和靶向B2M的sgRNA。在一些实施方案中，同时向该T细胞导入所述靶向TRAC的sgRNA和靶向PD-1的sgRNA。在一些实施方案中，同时向该T细胞导入所述靶向B2M的 sgRNA和靶向PD-1的sgRNA。在一些实施方案中，同时向该T细胞导入所述靶向TRAC的sgRNA、靶向B2M的sgRNA和靶向PD-1的sgRNA。
在一些实施方案中，所述sgRNA(包括靶向TRAC的sgRNA、靶向B2M的sgRNA和/或靶向PD-1的sgRNA)是经过2’-O-甲基类似物和/或核苷酸间3’硫代修饰的。在一些实施方案中，所述化学修饰为所述sgRNA(包括靶向TRAC的sgRNA、靶向B2M的sgRNA和/或靶向PD-1的sgRNA)的5’端前一个、二个和/或三个碱基和/或3’端的最后一个碱基的2’-O-甲基类似物修饰。
在一些实施方案中，将上述sgRNA(包括靶向TRAC的sgRNA、靶向B2M的sgRNA和/或靶向PD-1的sgRNA)通过电转方式导入所述T细胞。在一些实施方案中，上述sgRNA(包括靶向TRAC的sgRNA、靶向B2M的sgRNA和/或靶向PD-1的sgRNA)与Cas9编码核苷酸(如mRNA)通过电转方式共同导入该T细胞。在一些实施方案中，所述电转条件包括选自如下的任一项：150-250V，0.5-2ms；150V，2ms；160V，2ms；170V，2ms；180V，2ms；190V，1ms；200V，1ms；210V，1ms；220V，1ms；230V，1ms；240V，1ms；250V，0.5ms。
在一些实施方案中，还包括从经过基因编辑的T细胞中筛选TRAC、B2M和/或PD-1表达量低的T细胞。例如，所述TRAC、B2M或PD-1在经过基因编辑的T细胞中的表达量为未经基因编辑的T细胞的表达量的1/10
在一些实施方案中，单个基因敲除的效率(TRAC、B2M或PD-1)为80％以上，例如80％-100％、85％-100％、90％-100％、95％-100％、81％以上、82％以上、83％以上、84％以上、85％以上、86％以上、87％以上、88％以上、89％以上、90％以上、91％以上、92％以上、93％以上、94％以上、95％以上、96％以上、97％以上、98％以上、或99％以上；双基因同时的敲除效率(如TRAC和B2M)在65％以上，例如，65％-100％、70％-100％、75％-100％、80％-100％、85％-100％、90％-100％、95％-100％、70％以上、75％以上、80％以上、85％以上、90％以上、或95％以上；TRAC、B2M和PD-1基因同时的敲除效率在50％以上，例如，55％-100％、60％-100％、65％-100％、70％-100％、75％-100％、80％-100％、85％-100％、90％-100％、95％-100％、55％以上、60％以上、70％以上、75％以上、80％以上、85％以上、90％以上、或95％以上。此处所有数字涵盖了数字本身和该数字之间的每一整数和小数。此处的 敲除效率涵盖选自TRAC基因、B2M基因和PD-1基因之一敲除、之二同时敲除、之三同时敲除情况下的敲除效率。
在一些实施方案中，所述的T细胞来源于健康受试者、肿瘤或病毒感染患者(例如HIV感染患者)。在一些实施方案中，所述T细胞是处于不同分化阶段的干细胞或前体细胞分化而来的T细胞。
一个方面，本发明涉及通过上述方法制备的基因改造的T细胞。
一个方面，本发明涉及一种基因改造的T细胞，其中所述T细胞中：
(i)14号染色体第23016448位至第23016490位的TRAC基因组区域的一个或多个位点通过基因编辑技术被破坏；
(ii)15号染色体第45003745位至第45003788位的B2M基因组区域的一个或多个位点通过基因编辑技术被破坏；和/或
(iii)2号染色体第242800936位至第242800978位的PD-1基因组区域，或2号染色体第242795009位至第242795051位的PD-1基因组区域的一个或多个位点通过基因编辑技术被破坏。在一些实施方案中，所述T细胞是用本申请所述的任一方法制备的。
一个方面，本发明涉及一种基因改造的T细胞，其中所述T细胞中：
(i)14号染色体第23016448位至第23016490位的TRAC基因组区域具有表D和表E所记载的任一项的基因序列改变；
(ii)15号染色体第45003745位至第45003788位的B2M基因组区域具有有表B和表C所记载的任一项的基因序列改变；和/或
(iii)2号染色体第242800936位至第242800978位的PD-1基因组区域具有表F所记载的任一项的基因序列改变，或2号染色体第242795009位至第242795051位的PD-1基因组区域具有表G所记载的任一项的基因序列改变。在一些实施方案中，所述T细胞是用本申请所述的任一方法制备的。
一个方面，本发明涉及上述基因改造的T细胞用于制备过继细胞治疗的T细胞的用途。在一些实施方案中，所述过继细胞治疗的T细胞为CAR-T细胞或TCR-T细胞。
一个方面，本发明涉及一种制备CAR-T细胞或TCR-T细胞的方法，包括:将嵌合抗原受体(CAR)或其编码核苷酸、或工程化的T细胞受体(TCR)或其编码核苷酸导入上述任一基因改造的T细胞。
一方面，本发明涉及一种制备CAR-T或TCR-T细胞的方法，包括:
(i)将包含靶向14号染色体第23016448位至第23016490位的TRAC基因组的sgRNA导入T细胞以破坏所述TRAC基因组区域；和/或
(ii)将包含靶向15号染色体第45003745位至第45003788位的B2M基因组区域的sgRNA导入该T细胞以破坏所述B2M基因组区域；和/或
(iii)将包含靶向2号染色体第242800936位至第242800978位的PD-1基因组区域，或2号染色体第242795009位至第242795051位的PD-1基因组区域的sgRNA导入该T细胞以破坏所述B2M基因组区域；和
(iv)将嵌合抗原受体(CAR)或其编码核苷酸、或工程化的T细胞受体(TCR)或其编码核苷酸导入该T细胞。在一些实施方案中，所述方法还包含将CAS9或其编码核苷酸导入该T细胞。
在一些实施方案中，所述制备CAR-T或TCR-T细胞的方法包括:
(i)将包含选自SEQ ID NOs：2-5中任一的sgRNA导入T细胞以实现对14号染色体第23016448位至第23016490位的TRAC基因组区域的编辑；
(ii)将包含选自SEQ ID NOs：6-13中任一的sgRNA导入T细胞以实现对15号染色体第45003745位至第45003788位的B2M基因组区域的编辑；和/或
(iii)将包含选自SEQ ID NOs：14-22中任一的sgRNA导入T细胞以实现对2号染色体第242800936位至第242800978位的PD-1基因组区域，或2号染色体第242795009位至第242795051位的PD-1基因组区域的编辑；和
(iv)将嵌合抗原受体(CAR)或其编码核苷酸、或工程化的T细胞受体(TCR)或其编码核苷酸导入该T细胞。在一些实施方案中，所述方法还包含将CAS9或其编码核苷酸导入该T细胞。
在一些实施方案中，所述制备CAR-T或TCR-T细胞的方法包括:
(i)将包含选自SEQ ID NO：2或SEQ ID NO：3中任一序列的sgRNA导入T细胞以实现对14号染色体第23016448位至第23016490位的TRAC基因组区域的编辑；
(ii)将包含选自SEQ ID NO：7或SEQ ID NO：8中任一序列的sgRNA导入T细胞以实现对15号染色体第45003745位至第45003788位的B2M基因组区域的编辑；和/或
(iii)将包含选自SEQ ID NO：14或SEQ ID NO：15中任一序列的sgRNA导入T细胞以实现对2号染色体第242800936位至第242800978位的PD-1基因组区域，或2号染色体第242795009位至第242795051位的PD-1基因组区域的编辑；和
(iv)将嵌合抗原受体(CAR)或其编码核苷酸、或工程化的T细胞受体(TCR)或其编码核苷酸导入该T细胞。在一些实施方案中，所述方法还包含将CAS9或其编码核苷酸导入该T细胞。
在一些实施方案中，所述制备CAR-T或TCR-T细胞的方法包括:
(i)将包含SEQ ID NO：2的序列的sgRNA导入T细胞以实现对14号染色体第23016448位至第23016490位的TRAC基因组区域的编辑；
(ii)将包含SEQ ID NO：8的序列的sgRNA导入T细胞以实现对15号染色体第45003745位至第45003788位的B2M基因组区域的编辑；和/或
(iii)将包含SEQ ID NO：15的序列的sgRNA导入T细胞以实现对2号染色体第242800936位至第242800978位的PD-1基因组区域，或2号染色体第242795009位至第242795051位的PD-1基因组区域的编辑；和
(iv)将嵌合抗原受体(CAR)或其编码核苷酸、或工程化的T细胞受体(TCR)或其编码核苷酸导入该T细胞。在一些实施方案中，所述方法还包含将CAS9或其编码核苷酸导入该T细胞。
在一些实施方案中，所述制备CAR-T或TCR-T细胞的方法包括:
(i)将包含SEQ ID NO：2的序列的sgRNA导入T细胞以实现对14号染色体第23016448位至第23016490位的TRAC基因组区域的编辑；
(ii)将包含SEQ ID NO：8的序列的sgRNA导入T细胞以实现对15号染色体第45003745位至第45003788位的B2M基因组区域的编辑；和/或
(iii)将包含SEQ ID NO：14的序列的sgRNA导入T细胞以实现对2号染色体第242800936位至第242800978位的PD-1基因组区域，或2号染色体第242795009位至第242795051位的PD-1基因组区域的编辑；和
(iv)将嵌合抗原受体(CAR)或其编码核苷酸、或工程化的T细胞受体(TCR)或其编码核苷酸导入该T细胞。在一些实施方案中，所述方法还包含将CAS9或其编码核苷酸导入该T细胞。
在一些实施方案中，所述制备CAR-T或TCR-T细胞的方法包括:
(i)将包含SEQ ID NO：3的序列的sgRNA导入T细胞以实现对14号染色体第23016448位至第23016490位的TRAC基因组区域的编辑；
(ii)将包含SEQ ID NO：7的序列的sgRNA导入T细胞以实现对15号染色体第45003745位至第45003788位的B2M基因组区域的编辑；和/或
(iii)将包含SEQ ID NO：15的序列的sgRNA导入T细胞以实现对2号染色体第242800936位至第242800978位的PD-1基因组区域，或2号染色体第242795009位至第242795051位的PD-1基因组区域的编辑；和
(iv)将嵌合抗原受体(CAR)或其编码核苷酸、或工程化的T细胞受体(TCR)或其编码核苷酸导入该T细胞。在一些实施方案中，所述方法还包含将CAS9或其编码核苷酸导入该T细胞。
在一些实施方案中，同时向T细胞导入所述靶向TRAC的sgRNA和靶向B2M的sgRNA。在一些实施方案中，同时向T细胞导入所述靶向TRAC的sgRNA、靶向B2M的sgRNA和靶向PD-1的sgRNA。
在一些实施方案中，所述sgRNA(包括靶向TRAC的sgRNA、靶向B2M的sgRNA和/或靶向PD-1的sgRNA)是经过2’-O-甲基类似物和/或核苷酸间3’硫代修饰的。在一些实施方案中，所述化学修饰为所述sgRNA(包括靶向TRAC的sgRNA、靶向B2M的sgRNA和/或靶向PD-1的sgRNA)的5’端前一个、二个和/或三个碱基和/或3’端的最后一个碱基的2’-O-甲基类似物修饰。
在一些实施方案中，将上述sgRNA(包括靶向TRAC的sgRNA、靶向B2M的sgRNA和/或靶向PD-1的sgRNA)通过电转方式导入所述T细胞。在一些实施方案中，上述sgRNA(包括靶向TRAC的sgRNA、靶向B2M的sgRNA和/或靶向PD-1的sgRNA)与Cas9编码核苷酸(如mRNA)通过电转方式共同导入该T细胞。在一些实施方案中，所述电转条件包括选自如下的任一项：150-250V，0.5-2ms；180-250V，0.5-2ms；150V，2ms；160V，2ms；170V，2ms；180V，2ms；190V，1ms；200V，1ms；210V，1ms；220V，1ms；230V，1ms；240V，1ms；250V，0.5ms。
在一些实施方案中，所述方法包含将靶向TRAC的sgRNA,靶向B2M的sgRNA和/或靶向PD-1的sgRNA和CAR或其编码核苷酸、或工程化的T细胞受体(TCR)或其编码核苷酸同时导入该T细胞。
在一些实施方案中，将CAR或其编码核苷酸、或工程化的T细胞受体(TCR)或其编码核苷酸先于靶向TRAC的sgRNA,靶向B2M的sgRNA和/或靶向PD-1的sgRNA导入该T细胞；或将CAR或其编码核苷酸、或工程化的T细胞受体(TCR)或其编码核苷酸在导入靶向B2M的sgRNA和/或靶向PD-1的sgRNA后导入该T细胞。
一方面，本发明涉及通过上述方法制备的CAR-T细胞或TCR-T细胞。
一方面，本发明涉及CAR-T细胞,包含表达嵌合抗原受体(CAR)的上述基因改造的T细胞。
一个方面，本发明涉及CAR-T细胞，其中所述CAR-T细胞中：
(i)14号染色体第23016448位至第23016490位的TRAC基因组区域具有表D和表E所记载的任一项的基因序列改变；
(ii)15号染色体第45003745位至第45003788位的B2M基因组区域具有有表B和表C所记载的任一项的基因序列改变；和/或
(iii)2号染色体第242800936位至第242800978位的PD-1基因组区域具有表F所记载的任一项的基因序列改变，或2号染色体第242795009位至第242795051位的PD-1基因组区域具有表G所记载的任一项的基因序列改变。
一方面，本发明涉及一种TCR-T细胞,包含表达工程化TCR的上述基因改造的T细胞。
一个方面，本发明涉及TCR-T细胞，其中所述TCR-T细胞中：
(i)14号染色体第23016448位至第23016490位的TRAC基因组区域具有表D和表E所记载的任一项的基因序列改变；
(ii)15号染色体第45003745位至第45003788位的B2M基因组区域具有有表B和表C所记载的任一项的基因序列改变；和/或
(iii)2号染色体第242800936位至第242800978位的PD-1基因组区域具有表F所记载的任一项的基因序列改变，或2号染色体第242795009位至第242795051位的PD-1基因组区域具有表G所记载的任一项的基因序列改变。
本发明以上所提及的TRAC基因组区域、B2M基因组区域、PD-1基因组区域的位置信息是以参考数据库：GRCh37(hg19)中所述基因的野生序列位置信息而确定的。本领域技术人员知道如何参照其它数据库获得上述基因组区域的相应位置信息。
在一些具体的实施方案中，14号染色体第23016448位至第23016490位的TRAC基因组区域的野生核苷酸序列如SEQ ID NO:23(tatccagaaccctgaccctgccgtgtaccagctgagagactct)所示。在一些具体的实施方案中，15号染色体第45003745位至第45003788位的B2M基因组区域的野生核苷酸序列如SEQ ID NO:24(atgtctcgctccgtggccttagctgtgctcgcgctactctctct)所示。在一些具体的实施方案中，2号染色体第242800936位至第242800978位的PD-1基因组区域的野生核苷酸序列如SEQ ID NO:25(agcccagttgtagcaccgcccagacgactggccagggcgcctg)所示。在一些具体的实施方案中，2号染色体第242795009位至第242795051位的PD-1基因组区域的野生核苷酸序列如SEQ ID NO:26(cagtttagcacgaagctctccgatgtgttggagaagctgcagg)所示。
一方面，本发明还涉及包含上述基因改造的T细胞，CAR-T细胞或TCR-T细胞的组合物(如药物组合)、试剂盒、和医用制品。
一方面，本发明涉及一种治疗受试者疾病的方法，包括给药受试者有效量的上述CAR-T或TCR-T细胞。在一些实施方案中，所述疾病为肿瘤。在一些实施方案中，所述肿瘤为血液系统肿瘤。在一些实施方案中，所述肿瘤为淋巴瘤或白血病。在一些实施方案中，所述CAR靶向肿瘤特异性抗原(TSA)和/或肿瘤相关抗原(TAA)，例如本文表A所示的抗原(如CD19)。
一方面，本发明涉及包含SEQ ID NOs：2-22任一项的sgRNA或其载体。在一些实施方案中，所述sgRNA是经过化学修饰的，所述化学修饰例如2’-O-甲基类似物和/或核苷酸间3’硫代修饰。在一些实施方案中，化学修饰为所述sgRNA的5’端前一个、二个和/或三个碱基和/或3’端的最后一个碱基的2’-O-甲基类似物的修饰。
附图简述
图1显示利用化学修饰过的sgRNA与未经过化学修饰的sgRNA经过1次电转，对相应位点敲除的效率的比较。
图2A-2F.图2A展示了利用修饰的不同sgRNA结合CRISPR/Cas9基因敲除工具敲除HLA中的B2M，敲除后分析各个sgRNA对B2M这一基因的敲除效率。图2B展示了通过INDEL分析软件对经过修饰的不同sgRNA对 B2M基因敲除后T细胞基因组的INDEL分析，得到T细胞中发生INDEL的效率。图2C展示了利用修饰的不同sgRNA结合CRISPR/Cas9基因敲除工具敲除TCR中的TRAC，敲除后分析各个sgRNA对TRAC这一基因的敲除效率。图2D展示了通过INDEL分析软件对经过修饰的不同sgRNA对TRAC基因敲除后T细胞基因组的INDEL分析，得到T细胞中发生INDEL的效率。图2E展示了利用修饰的不同sgRNA结合CRISPR/Cas9基因敲除工具敲除PD-1，敲除后分析各个sgRNA对PD-1这一基因的敲除效率。图2F展示了通过INDEL分析软件对经过修饰的不同sgRNA对PD-1基因敲除后T细胞基因组的INDEL分析，得到T细胞中发生INDEL的效率。
图2A展示了利用体外转录(IVT)并经过修饰优化的sgRNA结合CRISPR/Cas9基因敲除工具，在相同的电转条件下对T细胞中的B2M进行敲除，敲除后分析各个sgRNA对B2M这一基因的敲除效率分别为：B1：27.88％、B2：86.17％、B3：64.69％、B4：1.06％、B5:2.91％、B6：0.17％、B7：41.87％及B8：3.14％。如图可以挑选出敲除效率较好的sgRNA。经过对挑选的sgRNA进行2’-O-甲基类似物和/或核苷酸间3’硫代的优化修饰，为高效率的双/三基因敲除(如图3所示)提供可能。图2B展示了通过INDEL分析软件对不同sgRNA对B2M基因敲除后T细胞基因组的INDEL分析，得到T细胞中发生INDEL的效率。图2C展示了利用体外转录(IVT)并经过修饰优化的sgRNA结合CRISPR/Cas9基因敲除工具，在相同的电转条件下对T细胞中的TRAC进行敲除，敲除后分析各个sgRNA对TRAC这一基因的敲除效率分别为：T2：77.84％、T3：85.86％、T4：2.59％及T6：34.78％。如图可以挑选出敲除效率较好的sgRNA。经过对挑选的sgRNA进行上述优化修饰，为高效率的单/双/三基因敲除(如图3所示)提供可能。图2D展示了通过INDEL分析软件对不同sgRNA对TRAC基因敲除后T细胞基因组的INDEL分析，得到T细胞中发生INDEL的效率。图2E展示了利用体外转录(IVT)并经过上述优化修饰的sgRNA结合CRISPR/Cas9基因敲除工具，在相同的电转条件下对T细胞中的PD-1进行敲除，敲除后分析各个sgRNA对PD-1这一基因的敲除效率分别为：P1：21.15％、P2：36.99％、P4：23.03％、P5：25.6％、P6：3.1％、P7：22.49％、P8：23.07％、P9：31.18％及P10：24.48％。如图可以挑选出敲除效率较好的sgRNA。经过对挑选的sgRNA进行上述优化修饰，为高效率的三基因敲除(如图3所示)提供可能。图2F展示了通 过INDEL分析软件对不同sgRNA对PD-1基因敲除后T细胞基因组的INDEL分析，得到T细胞中发生INDEL的效率。
图3展示了利用经过优化的sgRNA及CRISPR/Cas9基因编辑技术，对T细胞进行TRAC、TRAC/B2M(Double Knock-Out,DKO)以及TRAC/B2M/PD-1(Triple Knock-Out,TKO)的敲除结果分析。对sgRNA进行化学修饰，然后将化学修饰后的sgRNA与Cas9通过进一步优化后的电转条件递送到原代T细胞中进行相关基因敲除。结果如图所示，单基因即TRAC的敲除效率提升至90.42％；双基因即TRAC和B2M的敲除效率提高至81.39％。三基因即TRAC、B2M和PD-1的敲除效率提高至67.91％(82.70％+15.76％)*68.98％)。
图4展示了对经TRAC、TRAC/B2M(DKO)以及TRAC/B2M/PD-1(TKO)敲除的T细胞进行筛选纯化后的表型分析。通过后期纯化条件的优化，即增加纯化次数(4～5次)及相应的抗体量(3mg/mL)。结果如图所示，优化后的双敲除即TRAC和B2M双基因敲除纯度提高到99.72％。三基因即TRAC、B2M和PD-1敲除纯度提高到98.62％。
图5展示了TRAC-sgRNA3(T2)、B2M-sgRNA2(B3)及PD-1-sgRNA2(P2)的脱靶检测结果。图中显示经过深度测序后结合INDEL分析软件得到经过T2、B3、P2sgRNA结合CRISPR/Cas9编辑后的T细胞中的INDEL效率。
图6展示了利用经过优化sgRNA及CRISPR/Cas9基因编辑技术，对T细胞进行TCR和/或B2M和/或PD-1的敲除后表型及对经TCR和/或B2M和/或PD-1敲除的T细胞进行筛选纯化后表型分析。横坐标为CD3，纵坐标为TCR、B2M及PD-1。
图7展示了T细胞、CAR-T、TCRneg CAR-T、DKO CAR-T及TKO CAR-T细胞杀伤功能的验证与结果比较。该实验中，以Raji及K562为靶细胞，T，CAR-T，DKO CAR-T及TKO CAR-T为效应细胞，按照效靶比为10:1、5:1、2.5:1、1.25:1、0.625:1进行体外杀伤实验。
图8展示了T细胞、CAR-T、TCRneg CAR-T、DKO CAR-T及TKO CAR-T细胞因子释放。该实验中，以Raji及K562为靶细胞，T，CAR-T，DKO CAR-T及TKO CAR-T为效应细胞，按照效靶比为10:1、5:1、2.5:1、1.25:1、0.625:1进行体外共培养后，取上清对其中IL-2及IFN-γ进行检测。
图9A-9B展示了注射生理盐水、CAR-T、TCR neg CAR-T、DKO CAR-T(TCR neg\B2M neg-CAR-T)及TKO CAR-T(TCR neg\B2M neg\PD-1 neg-CAR-T)细胞后，其在NPG小鼠体内的肿瘤抑制及杀伤效果比较。图9A显示向NSG小鼠通过尾静脉注射5X10^5个肿瘤细胞/只后，随机的分为4组，生理盐水组、T细胞组、TCR/CD3 neg CD19-CAR-T细胞组、DKO CD19-CAR-T细胞组及TKO CD19-CAR-T细胞组，随后分别向四组小鼠通过尾静脉注射5X10^6个相应的细胞，生理盐水组作为对照组。通过铂金埃尔默成像仪得到的图片分析小鼠体内瘤负荷。图9B通过铂金埃尔默成像仪得到的图片分析小鼠体内瘤负荷，横坐标为小鼠细胞饲养天数，纵坐标为单位体表面积每秒放射的辐射信号。
图10展示了生理盐水、CAR-T、TCR neg CAR-T、DKO CAR-T(TCR neg\B2M neg-CAR-T)及TKO CAR-T(TCR neg\B2M neg\PD-1 neg-CAR-T)5组小鼠存活率的比较。横坐标为注射效应细胞后小鼠饲养天数，纵坐标为小鼠存活率。
图11展示了注射生理盐水、CAR-T、TCR neg CAR-T、DKO CAR-T(TCR neg\B2M neg-CAR-T)及TKO CAR-T(TCR neg\B2M neg\PD-1 neg-CAR-T)细胞后，小鼠体重的变化结果的比较。NPG小鼠体重监测，横坐标为注射效应细胞后小鼠饲养天数，纵坐标为小鼠体重百分百(n＝4)。
发明详述
定义
如本申请使用的，“CRISPR/Cas”是一种基因编辑技术，包括但不限于各种自然存在或人工设计的CRISPR/Cas系统,如CRISPR/Cas9系统。自然存在的CRISPR/Cas系统(Naturally occurring CRISPR/Cas system)是细菌和古细菌在长期演化过程中形成的一种适应性免疫防御，可用来对抗入侵的病毒及外源DNA。例如，CRISPR/Cas9的工作原理是crRNA(CRISPR-derived RNA)通过碱基配对与tracrRNA(trans-activating RNA)结合形成tracrRNA/crRNA复合物，此复合物引导核酸酶Cas9蛋白在与crRNA配对的序列靶位点剪切双链DNA。而通过人工设计tracrRNA和crRNA，可以改造形成具有引导作用的sgRNA(single guide RNA)，足以引导Cas9对DNA的定点切割。作为一种RNA导向的dsDNA结合蛋白，Cas9效应物核酸酶能够共定位 RNA、DNA和蛋白，从而拥有巨大的改造潜力。CRISPR/Cas系统可使用一类，二类或三类Cas蛋白。本发明的一些实施方式中，所述方法使用Cas9。其他适用的CRISPR/Cas系统包括但不限于WO2013176772，WO2014065596，WO2014018423，US8,697,359中所描述的系统和方法。
在本发明中，“sgRNA(single guide RNA)”和“gRNA(guide RNA)”或可以是“单指导RNA”、“合成的指导RNA”或“指导RNA”可互换使用。本发明的sgRNA包含靶向目标序列的指导序列(guide sequence)。
“T细胞受体(TCR)”为所有T细胞表面的特征性标志，其与CD3结合，形成TCR-CD3复合物。TCR由α、β两条肽链组成，每条肽链又可分为可变区(V区)，恒定区(C区)，跨膜区和胞质区。TCR分子属于免疫球蛋白超家族，其抗原特异性存在于V区；V区(Vα、Vβ)又各有三个高变区CDR1、CDR2、CDR3，其中以CDR3变异最大，直接决定了TCR的抗原结合特异性。在TCR识别MHC-抗原肽复合体时，CDR1，CDR2识别和结合MHC分子抗原结合槽的侧壁，而CDR3直接与抗原肽相结合。TCR分为两类：TCR1和TCR2；TCR1由γ和δ两条链组成，TCR2由α和β两条链组成。外周血中，90％-95％的T细胞表达TCR2；而且任一T细胞只表达TCR2和TCR1之一。
“β2微球蛋白(B2M)”是细胞表面人白细胞抗原(HLA)的β链(轻链)部分，是分子质量为11800，由99个氨基酸组成的单链多肽。
“程序性死亡受体1(PD-1)”，是一个268氨基酸残基的膜蛋白，最初从凋亡的小鼠T细胞杂交瘤2B4.11克隆出来。PD-1和PD-L1结合启动T细胞的程序性死亡，使肿瘤细胞获得免疫逃逸，因此，其是一种重要的免疫抑制分子。
如本申请使用的，“Indel”全称为插入/缺失,即插入和缺失突变。
“移植物抗宿主反应(GVHD)”是指由于供受体之间存在免疫遗传学差异，例如，一方面，当供体细胞，例如具有免疫活性的供体的T淋巴细胞，在进入受体病人体内并增殖到一定程度后，将受体病人的正常细胞或组织误认为靶标进行攻击从而产生的反应。另一方面，作为异体细胞，受体体内的正常免疫系统也可能会对其进行清除产生“宿主抗移植物反应(HVGR)”。
HVGR与GVHR相关基因包含TCR、HLA分子相关基因，这些基因同时敲除的T淋巴细胞在回输入同种异体病人时不会引起移植物抗宿主病 (GVHD)，因此可以称为“通用型T细胞”。例如，单个TRAC基因是编码TCRα链的基因与编码TCRβ的两个TRBC基因形成完整的有功能的TCR复合物，敲除TRAC是可以致使TCR失活，而B2M为MHCⅠ相关基因。这两个基因同时敲除的T淋巴细胞在回输入同种异体病人时不会引起移植物抗宿主病(GVHD)。
“CAR-T”为“嵌合抗原受体T细胞”的简写形式，其中，嵌合抗原受体(CAR)是CAR-T的核心部件，赋予T细胞HLA非依赖的方式识别靶细胞(如肿瘤)抗原的能力，这使得经过CAR改造的T细胞相较于天然T细胞表面受体TCR能够识别更广泛的目标。在一些实施中，靶向肿瘤的CAR的设计中包括一个肿瘤相关抗原(tumor-associated antigen，TAA)结合区(例如，通常来源于单克隆抗体抗原结合区域的scFV段)，一个胞外铰链区，一个跨膜区和一个胞内信号区。目标抗原的选择对于CAR的特异性、有效性以及基因改造T细胞自身的安全性来讲都是关键的决定因素。
“通用型CAR-T细胞”指能够靶向特异的靶细胞(如肿瘤)相关标志并且细胞表面TCR和MHC功能失活，可以降低同种异体细胞治疗引起的免疫排斥反应的CAR-T细胞。
自体细胞的CAR-T治疗需要抽取血液分离病人自身T淋巴细胞制备，一方面由于病人自身病情以及T淋巴细胞状态不同CAR-T生产过程影响因素较多不能标准化生产影响安全性，另一方面有些病人自体T淋巴细胞经过化疗后活性和数量不足，或受到肿瘤环境影响导致T淋巴细胞活性和增殖能力受限，这样的细胞在制备CAR-T时往往难度较大，治疗的安全性和有效性受到影响；或者在CAR-T细胞制备过程中若出现突发状况已准备细胞不能及时回输给病人也会影响治疗效果，甚至于受到自体T淋巴细胞状态影响有些肿瘤患者不能接受自体的CAR-T细胞过继治疗；可用于同种异体治疗的通用型CAR-T或通用型T淋巴细胞在上述情况下便占有很大的优势。
TCR-T(T细胞受体(TCR)嵌合型-T细胞)是指表达有工程化T细胞受体(engineered TCR)或称人造T细胞受体(artificial TCR)的T细胞。所述工程化T细胞受体或人造T细胞受体经过了基因改造，具有靶向目的抗原的结构，同时也保留了TCR信号传导通路中的结构域和/或辅助分子。在某些实施例中，TCR-T保留了TCR信号传导通路中的全部辅助分子因此，在少量抗原刺激时，就可以发生全激活的状态，引起对靶细胞的杀伤效应。 相对于CAR-T而言，这些TCR-T保持并应用了TCR信号传导通路上的所有辅助分子，因此TCR-T对低浓度，少拷贝数抗原的识别敏感性高于某些CAR-T，治疗潜力非常大。
在某些实施例中，TCR-T细胞通过部分基因修改的方法提高了TCR对相应抗原(如TAA)的亲和力，因此“基因修改的TCR”技术也因此被称为“亲和力增强的TCR”技术(Affinity-Enhanced TCR)。例如《自然-医学》杂志报道的Adaptimmune公司联合研发的一款“基因修改的TCR”在修改了几个关键氨基酸以后，这些基因修改的TCR大大提高了和一种常见的癌症TAA，NY-ESO-1，的亲和力。从而可以用来进攻有NY-ESO-1过量表达的癌症，比如多发性骨髓瘤(Multiple Myeloma)。
过继细胞疗法(Adoptive cellular therapy,ACT)、过继免疫疗法(adoptive immunotherapy)，例如肿瘤过继免疫疗法(tumor adoptive immunotherapy)，是指将免疫细胞在体外处理，例如加入特异性抗原，对免疫细胞表达的分子进行改造或利用细胞因子对其进行刺激等方法，筛选并大量扩增具有高度特异性的靶细胞(如肿瘤)杀伤性免疫效应细胞，然后输给患者，杀灭靶细胞(如肿瘤)的一种治疗方法，是一种被动免疫治疗。
高效编辑T细胞的方法
本发明的一个方面，提供一种制备基因编辑的T细胞(如通用T细胞)的方法，包括通过基因编辑技术破坏所述T细胞中：(i)14号染色体第23016448位至第23016490位的TRAC基因组区域(如SEQ ID NO:23所示)；(ii)15号染色体第45003745位至第45003788位的B2M基因组区域(如SEQ ID NO:24所示)；和/或(iii)2号染色体第242800936位至第242800978位的PD-1基因组区域(如SEQ ID NO:25所示)，或2号染色体第242795009位至第242795051位的PD-1基因组区域(如SEQ ID NO:26所示)。在一些实施方案中，该方法所述基因编辑技术为基于锌指核酸酶的基因编辑技术、TALEN基因编辑技术或CRISPR/Cas基因编辑技术。在一些实施方案中，所述TRAC基因组区域、B2M基因组区域或PD-1基因组区域被编辑。在一些实施方案中，其中所述TRAC基因组区域、和B2M基因组区域均被编辑。在一些实施方案中，其中所述TRAC基因组区域和PD-1基因组区域均被编辑。在一些实施方案中，其中所述B2M基因组区域和PD-1基因组区域均被 编辑。在一些实施方案中，其中所述TRAC基因组区域、B2M基因组区域和PD-1基因组区域均被编辑。
在一些实施方案中，提供一种制备基因编辑的T细胞(如通用T细胞)的方法，包括通过基因编辑技术破坏所述T细胞中：(i)与选自SEQ ID NOs：2-5中任一的序列互补的TRAC基因组靶核苷酸序列；(ii)与选自SEQ ID NOs：6-14中任一的序列互补的B2M基因组的靶核苷酸序列；和/或(iii)与选自SEQ ID NOs：15-22中任一的序列互补的PD-1基因组的靶核苷酸序列。在一些实施方案中，该方法所述基因编辑技术为基于锌指核酸酶的基因编辑技术、TALEN基因编辑技术或CRISPR/Cas基因编辑技术。在一些实施方案中，所述TRAC基因组区域、B2M基因组区域或PD-1基因组区域被编辑。在一些实施方案中，其中所述TRAC基因组区域、和B2M基因组区域均被编辑。在一些实施方案中，其中所述TRAC基因组区域和PD-1基因组区域均被编辑。在一些实施方案中，其中所述B2M基因组区域和PD-1基因组区域均被编辑。在一些实施方案中，其中所述TRAC基因组区域、B2M基因组区域和PD-1基因组区域均被编辑。
在一些实施方案中，提供一种制备基因编辑的T细胞(如通用T细胞)的方法，包括：将靶向TRAC的sgRNA、靶向B2M的sgRNA和/或PD-1的sgRNA导入T细胞，以破坏T细胞的TRAC、B2M和/或PD-1基因。在一些实施方案中，该方法包括将Cas9或其编码核苷酸导入T细胞。
在一些实施方案中，sgRNA靶向TCR2的α和/或β链的恒定区的编码基因，从而破坏T细胞表面TCR的结构，使该分子失去功能。
在一些实施方案中，sgRNA靶向β2微球蛋白(B2M)的编码基因，例如靶向B2M蛋白编码基因的第一个外显子区域，从而破坏B2M的结构，使该分子失去功能。
在一些实施方案中，sgRNA靶向PD-1的编码基因，例如PD-1蛋白编码基因的第一个外显子区域，从而破坏PD-1的结构，使该分子失去功能。
在一些实施方案中，提供一种制备基因编辑的T细胞(如通用T细胞)的方法，包括：(i)将包含选自SEQ ID NOs：2-5中任一序列的sgRNA导入T细胞以实现对所述TRAC基因组区域的编辑；(ii)将包含选自SEQ ID NOs：6-14中任一序列的sgRNA导入T细胞以实现对所述B2M基因组区域的编辑；和/或(iii)将包含选自SEQ ID NOs：15-22中任一序列的sgRNA 导入T细胞以实现对所述PD-1基因组区域的编辑。在一些实施方案中，该方法包括将Cas9或其编码核苷酸导入T细胞。
在一些实施方案中，本发明涉及将靶向TRAC的选自表2所示的任一sgRNA导入T细胞。在本发明的一些实施方案中，本发明涉及将靶向B2M的选自表2所示的任一sgRNA导入T细胞。在本发明的一些实施方案中，本发明涉及将靶向PD-1的选自表2所示的任一sgRNA导入T细胞。在一些实施方案中，该方法包括将Cas9或其编码核苷酸导入T细胞。
在一些实施方案中，本发明涉及将靶向TRAC的选自表2所示的任一sgRNA、和/或靶向B2M的选自表2所示的任一sgRNA、和/或靶向PD-1的选自表2所示的任一sgRNA与Cas9或其编码核苷酸导入T细胞。
在一些实施方案中，本发明涉及将靶向TRAC的选自表2所示的任一sgRNA、和靶向B2M的选自表2所示的任一sgRNA、和靶向PD-1的选自表2所示的任一sgRNA导入T细胞。在一些实施方案中，该方法包括将Cas9或其编码核苷酸导入T细胞。
在本发明的一些实施方案中，提供一种制备通用T细胞的方法，包括：(i)将TRAC-sg3sgRNA导入T细胞以实现对所述TRAC基因组区域的编辑；(ii)将B2M-sg2sgRNA导入T细胞以实现对所述B2M基因组区域的编辑；和(iii)将PD-1-sg2sgRNA导入T细胞以实现对所述PD-1基因组区域的编辑。在一些实施方案中，该方法包括将Cas9或其编码核苷酸导入T细胞。
在一些实施方案中，提供一种制备通用T细胞的方法，包括：(i)将包含SEQ ID NO：2或3的序列的sgRNA导入T细胞以实现对所述TRAC基因组区域的编辑；(ii)将包含选自SEQ ID NO：7或11中任一的序列的sgRNA导入T细胞以实现对所述B2M基因组区域的编辑；和(iii)将包含选自SEQ ID NO：14或15中的序列的sgRNA导入T细胞以实现对所述PD-1基因组区域的编辑。在一些实施方案中，该方法包括将Cas9或其编码核苷酸导入T细胞。
在一些实施方案中，上述sgRNA是经过化学修饰的。例如是经过2’-O-甲基类似物和/或核苷酸间3’硫代修饰的。在一些实施方案汇总，所述化学修饰为所述sgRNA的5’端前一个、二个和/或三个碱基和/或3’端的最后一个碱基的2’-O-甲基类似物的修饰。
一般而言，sgRNA中的指导序列是与靶多核苷酸序列具有足够的互补性以与靶序列杂交并指导CRISPR复合物与靶序列的序列特异性结合的任何多核苷酸序列。在一些实施方案中，当使用适当的比对算法最佳比对时，指导序列及其相应靶序列间的互补程度为约或大于约80％、85％、90％、95％、97.5％、99％或更多。最佳比对可使用用于比对序列的任何适当的算法确定，其非限制性实例包括Smith-Waterman算法、Needleman-Wimsch算法、基于Burrows-Wheeler Transform的算法(例如Burrows Wheeler Aligner)、ClustalW、Clustai X、BLAT、Novoalign(Novocraft Technologies,ELAND((Illumina,San Diego,CA)、SOAP(可在soap.genomics.org.cn获得)和Maq(可在maq.sourceforge.net获得)。在一些实施方案中，指导序列长度可以为约或大于约10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、35、40、45、50、55、60、65、70、75或更多个核苷酸。在一些实施方案中，指导序列长度少于约75、70、65、60、55、50、45、40、35、30、25、20、15、12或更少的核苷酸。指导序列指导CR1SPR复合物与靶序列的序列特异性结合的能力可通过任何适当的测定方法评估。例如，可向具有相应靶序列的宿主细胞提供足以形成CRISPR复合物的CRISPR系统的组件(包括待测试的指导序列)，如可通过使用编码CRISPR序列组件的载体转染，随后评估靶序列内的优先切割来进行。同样地，靶多核苷酸序列的切割可在测试管中通过提供靶序列、CRISPR复合物(包含待测试的指导序列和不同于指导序列的对照指导序列)的组件，并比较测试和对照指导序列在靶序列的结合或切割率，以此进行评估。也可以使用本领域技术人员知道的其它测定方法进行上述测定和评估。
在一些实施方案中，所述靶向TRAC的sgRNA、靶向B2M的sgRNA和/或靶向PD-1的sgRNA和/或Cas9编码核苷酸(如mRNA)通过电转导入该T细胞，例如，通过150-250V，0.5-2ms；180-250V，0.5-2ms；150V，2ms；160V，2ms；170V，2ms；180V，2ms；190V，1ms；200V，1ms；210V，1ms；220V，1ms；230V，1ms；240V，1ms；250V，0.5ms的电转条件导入T细胞。在一些实施方案中，所述靶向TRAC的sgRNA、靶向B2M的sgRNA和靶向PD-1的sgRNA和Cas9编码核苷酸通过电转方式共同导入T细胞。
在一些实施方案中，所述Cas9编码核苷酸为mRNA，如含有ARCA帽的mRNA。在一些实施方案中，所述Cas9编码核苷酸在病毒载体中，如慢 病毒载体。在一些实施方案中，所述Cas9编码核苷酸包含如SEQ ID NO：1所述的序列。在一些实施方案中，所述靶向TRAC的sgRNA、靶向B2M的sgRNA和/或靶向PD-1的sgRNA与Cas9编码核苷酸在同一载体中。
在一些实施方案中，靶向TRAC的sgRNA、靶向B2M的sgRNA和靶向PD-1的sgRNA同时导入T细胞。在具体实施方案中，靶向TRAC的sgRNA、靶向B2M的sgRNA和靶向PD-1的sgRNA同时导入T细胞时，靶向TRAC的sgRNA、靶向B2M的sgRNA和靶向PD-1的sgRNA之间的量可以是近似的或等同的。在一些实施方案中，靶向TRAC的sgRNA、靶向B2M的sgRNA和靶向PD-1的sgRNA以任何合适的顺序逐一导入T细胞。在一些实施方案中，靶向TRAC的sgRNA、靶向B2M的sgRNA和/或靶向PD-1的sgRNA与Cas9编码核苷酸同时导入T细胞。在一些实施方案中，Cas9编码核苷酸先于靶向TRAC的sgRNA、靶向B2M的sgRNA和/或靶向PD-1的sgRNA导入T细胞。在一些实施方案中，T细胞包含Cas9编码核苷酸或Cas9蛋白。
在一些实施方案中，T细胞来源于健康人。在一些实施方案中，T细胞来源于患者，如癌症患者，例如化疗或放射治疗前的癌症患者。在一些实施方案中，T细胞来源如脐带血，骨髓，或外周血单个核细胞(Peripheral blood mononuclear cell,PBMC)。在一些实施方案中，T细胞来源于干细胞，例如各个分化阶段的造血干细胞。本申请中所述的制备方法可以用于在例如PBMC或干细胞中敲除TRAC,B2M和/或PD-1,并进一步培养，分化，和/或提纯出相应的基因改造的T细胞。
与现有技术相比，本发明的T细胞基因敲除方法获得了高效的基因敲除效率，例如，单基因(TRAC)、双基因(TRAC和B2M)及三基因(TRAC，B2M及PD-1)敲除，其敲除效率分别高达至少90％、81％及67％。
“敲除效率”可以在基因水平上以产生基因敲除的INDEL的效率来表示，也可以在细胞水平上以所述基因敲除致使该基因表达蛋白消失或显著降低的细胞的百分比来表示。在本发明中，“敲除效率”是指基于后者计算的敲除效率。本领域技术人员可以理解，高的敲除效率可以提高目的细胞的收获率，降低生产成本和治疗成本。
在一些实施方案中，基因编辑的T细胞(如通用T细胞)被进一步筛选，以得到更高纯度的单基因(TRAC)、双基因(TRAC和B2M)及三基因(TRAC， B2M及PD-1)敲除T细胞。例如，可通过FACS筛选TRAC,B2M和/或PD-1表达量低的基因编辑的T细胞(如通用T细胞)。
在一些实施方案中，本发明的通用型T细胞的TCR和\或HLA和\或PD-1基因被敲除。
在一些具体的实施方案中，所述TCR的α链恒定编码区(即TRAC)基因被敲除。B2M和\或PD-1的编码区被敲除。TRAC、B2M和PD-1可以三者均被敲除，也可以敲除三者之一或者敲除其中两个。
在一些具体的实施方案中，所述TCR的α链恒定编码区(即TRAC)基因被敲除。例如，在具体实施方案中，本发明TCRα链恒定编码区基因被引入所述细胞的TRAC-sg 2、3、4、6分子之一(见表2)以及Cas 9分子敲除。优选所述TCR的α链恒定编码区基因被引入细胞的TRAC-sg 2和Cas9分子或TRAC-sg 3和Cas9分子进行敲除。
在另一些具体的实施方案中，所述HLA的恒定编码区B2M基因被敲除。例如，在具体实施方案中，本发明B2M恒定编码区基因被引入所述细胞的B2M-sg 1-8分子之一(见表2)以及Cas 9分子,优选所述B2M的恒定编码区基因被引入细胞的B2M-sg 2或B2M-sg 6分子和Cas9分子进行敲除。
在另一些具体的实施方案中，所述PD-1的恒定编码区基因被敲除。例如，在具体实施方案中，本发明PD-1恒定编码区基因被引入所述细胞的PD-1-sg1-2，PD-1-sg4-10分子之一(见表2)以及Cas 9分子,优选所述PD-1的恒定编码区基因被引入细胞的PD-1-sg1或PD-1-sg2分子和Cas9分子进行敲除。
在另一些具体的实施方案中，所述TCR的α链恒定编码区(即TRAC)基因及B2M基因被敲除。例如，在具体实施方案中，本发明TRAC及B2M基因被引入所述细胞的TRAC-sg3及B2M-sg2分子(见表2)以及Cas 9分子进行敲除。
在另一些具体的实施方案中，所述TCR的α链恒定编码区(即TRAC)基因、B2M基因及PD-1的恒定编码区基因被敲除。例如，在具体实施方案中，本发明TCR、HLA及PD-1恒定编码区基因被引入所述细胞的TRAC-sg3、B2M-sg2及PD-1-sg2分子(见表2)以及Cas 9分子进行敲除。在具体实施方案中，本发明TCR、HLA及PD-1恒定编码区基因被引入所述细胞的TRAC-sg2、B2M-sg6及PD-1-sg1分子(见表2)以及Cas 9分子进行敲除。 在具体实施方案中，本发明TCR、HLA及PD-1恒定编码区基因被引入所述细胞的TRAC-sg2、B2M-sg6及PD-1-sg2分子(见表2)以及Cas 9分子进行敲除。
在一些实施方案中，本发明提供了一种高效编辑T细胞的方法，所述方法包括如下步骤:
在T细胞中引入sgRNA分子和Cas9分子:
在一些实施方案中，所述sgRNA分子包含与来自TCR的α链恒定编码区(即TRAC)基因、B2M基因及PD-1的恒定编码区基因靶区域互补的靶向结构域。
在一些实施方案中，所述sgRNA分子是指一段包含与待敲除的基因的靶区域互补的靶向结构域的核酸序列，其能识别靶标DNA序列并引导Cas9分子剪切靶位点，其可以实现一步高效(敲除效率85％以上)敲除相应位点。
在一些实施方案中，所述sgRNA分子所包含的靶向结构域的序列如表2中之一所示。
在一些优选的实施方案中，所述靶向结构域的序列如T2、B3及P1所示。
在一些优选的实施方案中，通过电转技术将所述sgRNA分子和编码Cas9分子的mRNA引入所述T细胞中。
在一些具体的实施方案中，上述方法中使用的T细胞来自健康人，例如健康成人外周血，或自然分娩的健康人的脐带血。
在一些实施方案中，本发明的第三方面提供了经过特定修饰后的具有高效编辑效率的sgRNA序列(表2)。
在一些具体的实施方案中，利用化学方法合成并修饰sgRNA，使sgRNA比普通的体外转录(IVT)所获得的sgRNA具有更稳定及更高的编辑效率。优选地，用电转的方法对T细胞进行一次电转，化学合成并经过修饰的sgRNA基因编辑效率是普通的IVT所获得的sgRNA的10倍以上。
在一些实施方案中，提供了本发明所述的通用型T细胞用于制备治疗疾病(如肿瘤)的药物的用途。
在一些具体的实施方案中，所述TCR的α链恒定编码区(即TRAC)基因，HLA的恒定编码区B2M基因，PD-1的恒定编码区基因被敲除。例如，在具体实施方案中，本发明TCRα链恒定编码区基因被引入所述细胞的TRAC-sg 2分子，本发明B2M恒定编码区基因被引入所述细胞的B2M-sg6 分子，本发明PD-1恒定编码区基因被引入所述细胞的PD-1-sg1(见表2)以及Cas 9分子:优选所述TCR的α链恒定编码区基因被引入细胞的TRAC-sg2、B2M恒定编码区基因被引入细胞的B2M-sg6、PD-1恒定编码区基因被引入细胞的PD-1-sg1分子和Cas9分子进行敲除。
高效编辑的T细胞或通用型T细胞
本发明涉及通过本发明上述方法制备的TRAC单基因敲除T细胞(TRAC negative)，TRAC/B2M双基因敲除(DKO)T细胞和TRAC/B2M/PD-1三基因敲除(TKO)T细胞。
与现有技术相比，本发明的单基因敲除T细胞(TRAC negative)，TRAC/B2M双基因敲除(DKO)T细胞和TCR/B2M/PD-1三基因敲除(TKO)T细胞的基因敲除效率大大提高。
本发明制备的单基因敲除T细胞(TRAC negative)，TRAC/B2M双基因敲除(DKO)T细胞和TCR/B2M/PD-1三基因敲除(TKO)T细胞，可作为T细胞进一步改造的前体细胞，或者作为通用型T细胞，用于制备各种基因修饰的T细胞，例如可用来制备CAR-T细胞或TCR-T细胞。
本发明的一些实施方案中提供一种基因改造的T细胞(如通用T细胞)，其中所述T细胞中：(i)14号染色体第23016448位至第23016490位的TRAC基因组区域的一个或多个位点通过基因编辑技术被破坏；(ii)15号染色体第45003745位至第45003788位的B2M基因组区域的一个或多个位点通过基因编辑技术被破坏；和/或(iii)2号染色体第242800936位至第242800978位的PD-1基因组区域，或2号染色体第242795009位至第242795051位的PD-1基因组区域的一个或多个位点通过基因编辑技术被破坏.在一些实施例中，所述T细胞(i)与选自SEQ ID NOs：2-5中任一的序列互补的TRAC基因组靶核苷酸序列通过基因编辑技术被破坏；(ii)与选自SEQ ID NOs：6-14中任一的序列互补的B2M基因组的靶核苷酸序列通过基因编辑技术被破坏；和/或(iii)与选自SEQ ID NOs：15-22中任一的序列互补的PD-1基因组的靶核苷酸序列通过基因编辑技术被破坏。
本发明的一些实施方案中提供一种基因改造的T细胞(如通用T细胞)，其中所述T细胞中：(i)其TRAC基因组包含表D和表E所记载的任一项的序列；
(ii)其B2M基因组包含表B和表C所记载的任一项的序列；和/或
(iii)其PD-1基因组包含表F或表G所记载的任一项的序列。
本发明一方面提供了包含所述的基因改造的T细胞(如通用T细胞)、CAR-T细胞，或TCR-T细胞组合物，如药物组合物。
本发明一方面还提供了试剂盒或制品，包含本发明所述的基因改造的T细胞(如通用T细胞)。该试剂盒或制品可用以制备CAR-T,TCR-T或其他过继细胞治疗组合物。
制备CAR-T细胞的方法
本发明一方面提供了制备CAR-T细胞(如通用CAR-T细胞)的方法。在一些实施方案中，该方法包括将CAR或其编码核苷酸或载体导入本发明所描述的任何基因改造的T细胞(如通用T细胞)。
在一些实施方案中，提供了一种制备CAR-T细胞的方法，包括:
(i)将包含靶向14号染色体第23016448位至第23016490位的TRAC基因组的sgRNA导入T细胞以破坏所述TRAC基因组区域；和/或
(ii)将包含靶向15号染色体第45003745位至第45003788位的B2M基因组区域的sgRNA导入该T细胞以破坏所述B2M基因组区域；和/或
(iii)将包含靶向2号染色体第242800936位至第242800978位的PD-1基因组区域，或2号染色体第242795009位至第242795051位的PD-1基因组区域的sgRNA导入该T细胞以破坏所述B2M基因组区域；和
(iv)将嵌合抗原受体(CAR)或其编码核酸导入该T细胞。
CAR或其编码核酸以及靶向TRAC的sgRNA,靶向B2M的sgRNA和/或靶向PD-1的sgRNA和CAR或其编码核苷酸可以以任何适合的顺序导入T细胞。在一些实施方案中，靶向TRAC的sgRNA,靶向B2M的sgRNA和/或靶向PD-1的sgRNA和CAR或其编码核苷酸同时导入该T细胞。在一些实施方案中，CAR或其编码核苷酸先于靶向TRAC的sgRNA,靶向B2M的sgRNA和/或靶向PD-1的sgRNA导入该T细胞。在一些实施方案中，CAR或其编码核苷酸导入已经实现基因编辑的T细胞中，该T细胞的TRAC,B2M和/或PD-1基因组区域已经通过编辑被破坏。在一些实施方案中，该方法还包括将Cas9或其编码核苷酸于说所述sgRNA一并导入该T细胞。在一些实施方案中，本发明的通用型CAR-T细胞所表达的CAR可以是本领域己知的任何CAR，只要其能够使T细胞以人白细胞抗原-非依赖性的方式识别细胞表面抗原，发挥杀伤作用即可。例如，可以使用美国发明专利申 请US20140271635A1中公开的CAR，本发明的具体实施方案中使用的CAR可参考该发明专利申请公开文本(US20140271635A1)。在一些实施方案中，本发明CAR-T细胞中的CAR为识别以下肿瘤与相关抗原对应表A中所述抗原的CAR。
表A
Figure PCTCN2018106237-appb-000001
Figure PCTCN2018106237-appb-000002
在一些实施方案中，本发明的CAR-T细胞中表达的CAR包含顺序连接的信号肽、胞外结合区、铰链区、跨膜区和胞内信号区。本文使用的术语"信号肽"是指引导新合成的蛋白质向分泌通路转移的短(例如长度5-30个氨基酸)肽链。在本发明中，可以使用人体内的各种蛋白质的信号肽，例如体内分泌的细胞因子蛋白、白细胞分化抗原(CD分子)的信号肽。
在一些实施方案中，所述信号肽为CD8信号肽，例如其氨基酸序列如发明专利申请US20140271635A1中所示。
在一些实施方案中，所述绞链区可以使用各种不同抗体或抗原受体的铰链区，特别是CD分子的铰链区。在一个具体的实施方案中，所述铰链区可以选自CD8或CD28等蛋白的铰链区。所述CD8或CD28是T细胞表面的天然标记物。
在本发明中，可以使用各种人体内蛋白的跨膜区，特别是各种不同抗原受体的跨膜区。优选使用的跨膜区是CD分子的跨膜区。在一个实施方案中，所述跨膜区可以选自CD8或CD28或4-1BB等蛋白的跨膜区。
在一些实施方案中，所述铰链区为CD8α铰链区(CD8-hinge)，其氨基酸序列如发明专利申请US20140271635A1中所示。
所述"胞外结合区"是指包含特异性识别靶抗原的区域。在一些实施方案中，该胞外结合区包含特异性识别靶肿瘤细胞表面抗原的区域。例如这个区域可以是scFv或其他抗体的抗原结合片段。本文使用的术语"scFv"是指通过连接区(linker)连接的重链可变区(variable region of heavy chain，VH和轻链可变区(variable region of light chain，VL)的重组蛋白，连接区使得这两 个结构域相关联，最终形成抗原结合位点。scFv通常是由一条核苷酸链编码的氨基酸序列。上述scFv还可以包括其衍生物。
本发明使用的CAR及其各结构域可通过单独或联合使用本领域己知的常规技术，例如氨基酸缺失、插入、取代、增加，和/或重组以及/或其他修饰方法作进一步修饰。根据一种抗体的氨基酸序列在其DNA序列中引入这种修饰的方法对本领域技术人员来说是公知的(参见例如，Sambrook分子克隆:实验手册，Cold Spring Harbor Laboratory(1989)N.Y.)。所述修饰优选在核酸水平上进行。
本文使用的术语"特异性识别"意指本发明的抗原识别区不与或基本上不与目标抗原以外的任意多肽交叉反应。其特异性的程度可以通过免疫学技术来判断，包括但不限于免疫印迹，免疫亲和层析，流式细胞分析等。
在一些实施方案中，所述胞外结合区包含特异性识别CD19、CEA、EGFR、GD2、CD7或CD138等的抗原结合区域，如scFv。
在一些实施方案中，所述胞外结合区包含经人源化改造的特异性识别CD19的scFv。在一些实施方案中，该特异性识别CD19的scFv的氨基酸序列如发明专利申请US20140271635A1中所示。
在本发明中，可以使用各种人体内蛋白的胞内信号区，特别是各种不同抗原受体的胞内信号区。优选使用的胞内信号区是CD分子的胞内信号区。在具体实施方案中，所述胞内信号区可以选自CD3
Figure PCTCN2018106237-appb-000003
、FcεRIγ、CD28、CD137(4-1BB)、CD134蛋白的胞内信号区，及其组合。CD3分子由五个亚单位组成，其中CD3
Figure PCTCN2018106237-appb-000004
亚单位(又称CD3zeta，简称
Figure PCTCN2018106237-appb-000005
)含有3个ITAM基序，该基序是TCR-CD3复合体中重要的信号转化区。FcεRI y主要分布在肥大细胞和嗜碱性粒细胞表面，其含有一个ITAM基序，在结构、分布及功能上与CD3
Figure PCTCN2018106237-appb-000006
类似。此外如前所述，CD28、CD137、CD134是共剌激信号分子，在与各自配体结合后其胞内信号区段产生的共刺激作用引起T细胞的持续增殖，并能够提高T细胞分泌IL-2和IFN-γ等细胞因子的水平，同时提高CAR-T细胞在体内的存活周期和抗肿瘤效果。
在某些实施方案中，由单独TCR产生的信号不足以完全活化天然T细胞，需要通过TCR起始抗原依赖性初次活化的序列(初级细胞内信号传导结构域)以及以不依赖于抗原的方式作用以提供共刺激信号的序列(共刺激性结构域)。初级信号传导结构域以刺激性方式或以抑制性方式调控TCR复合物 的初次活化。以刺激性方式作用的初级细胞内信号传导结构域可以含有信号传导基序，称为免疫受体酪氨酸活化基序(ITAM)。适用于本发明中的含有ITAM的初级细胞质信号传导序列的实例包括CD3ζ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b以及CD66d。在一个实施方案中，初级信号传导结构域包含修饰的ITAM结构域，例如活性相较于天然ITAM结构域有所改变(例如增加或降低)的突变的ITAM结构域，或截短的ITAM的初级细胞内信号传导结构域。在一个实施方案中，初级信号传导结构域包含一个或多个ITAM基序。
共刺激信号传导结构域是指TCR中包含共刺激分子细胞内结构域的部分。共刺激分子是淋巴细胞对抗原高效反应所需的除抗原受体或其配体外的细胞表面分子。这些分子的实例包括CD27、CD28、4-1BB(CD137)、OX40、CD30、CD40、PD1、ICOS、淋巴细胞功能相关抗原-1(LFA-1)、CD2、CD7、LIGHT、NKG2C、B7-H3，以及与CD83特异性结合的配体等。
在一些实施方案中，本发明提供了一种制备CAR-T细胞，例如通用型CAR-T细胞的方法，所述方法包括如下步骤:
1)在T细胞中引入sgRNA分子和Cas9分子:
在一些实施方案中，所述sgRNA分子包含与来自TCR的链恒定编码区(即TRAC)基因、HLA恒定编码区B2M基因及PD-1的恒定编码区基因靶区域互补的靶向结构域。
2)在所述T细胞中引入CAR分子；
在一些实施方案中，所述sgRNA分子是指一段包含与待敲除的基因的靶区域互补的靶向结构域的核酸序列，其能识别靶标DNA序列并引导Cas9分子剪切靶位点，其可以实现一步高效(敲除效率85％以上)敲除相应位点。
在一些实施方案中，所述Cas9分子是指Cas9mRNA，其能够在sgRNA的引导下对靶位点进行剪切。
在一些具体的实施方案中，所述sgRNA分子所包含的靶向结构域的序列如表2中之一所示。
在一些优选的实施方案中，所述靶向结构域的序列如T2、B3及P1所示(表2)。
在一些优选的实施方案中，通过电转技术将所述sgRNA分子和编码Cas9分子的mRNA引入所述T细胞中。
在一些实施方案中，通过例如慢病毒转染技术将所述CAR分子引入所述T细胞中。
在一些具体的实施方案中，包括分离和/或激活来自健康人外周血或脐带血的T细胞的步骤；优选地，所述方法在上述步骤2)之后还包括对通用型CAR-T细胞进行分选的步骤；更优选地，分选之后再对所得的CAR-T细胞，例如通用型CAR-T细胞进行功能性验证。
在一些实施方案中，本发明提供了上述CAR-T细胞用于制备治疗疾病(如肿瘤)的用途。
本发明在一个方面提供了一种治疗受试者疾病的方法，包括给药受试者有效量的本发明所述的CAR-T细胞。本发明所述的治疗方法包括但不限于癌症和HIV/AIDS.在一些实施方案中，该疾病为肿瘤，包括血液系统肿瘤，如淋巴瘤或白血病。在一些实施方案中，所述CAR靶向表A中所示的抗原，该疾病为表A中与该靶抗原相对应的肿瘤。在一些实施方案中，所述T细胞不是从受试者得到的。例如，所述T细胞可以来源于健康的捐助者。
本发明涉及的CAR－T细胞可以通过给药包含细胞成分的医药制品常规使用的途径，例如静脉输注途径，给药有此需要的受试者。给药剂量可以基于受试者的病情和一般健康状况具体确定。
制备TCR-T细胞的方法
本发明提供了表达工程化TCR的T细胞，也称TCR-T细胞。本发明还提供了制备该TCR-T细胞的方法，包括将工程化TCR或其编码核苷酸或载体导入本发明所描述的任何基因改造的T细胞(如通用T细胞)。
在一些实施方案中，提供了一种制备TCR-T细胞的方法，包括:
(i)将包含靶向14号染色体第23016448位至第23016490位的TRAC基因组的sgRNA导入T细胞以破坏所述TRAC基因组区域；和/或
(ii)将包含靶向15号染色体第45003745位至第45003788位的B2M基因组区域的sgRNA导入该T细胞以破坏所述B2M基因组区域；和/或
(iii)将包含靶向2号染色体第242800936位至第242800978位的PD-1基因组区域，或2号染色体第242795009位至第242795051位的PD-1基因组区域的sgRNA导入该T细胞以破坏所述B2M基因组区域；和
(iv)将工程化TCR或其编码核酸导入该T细胞。
TCR或其编码核酸以及靶向TRAC的sgRNA，靶向B2M的sgRNA和/或靶向PD-1的sgRNA和TCR或其编码核苷酸可以以任何适合的顺序导入T细胞。在一些实施方案中，靶向TRAC的sgRNA，靶向B2M的sgRNA和/或靶向PD-1的sgRNA和TCR或其编码核苷酸同时导入该T细胞。在一些实施方案中，TCR或其编码核酸先于靶向TRAC的sgRNA,靶向B2M的sgRNA和/或靶向PD-1的sgRNA导入该T细胞。在一些实施方案中，TCR或其编码核苷酸导入已经实现基因编辑的T细胞中，该T细胞的TRAC,B2M和/或PD-1基因组区域已经通过编辑被破坏。在一些实施方案中，该方法还包括将Cas9或其编码核苷酸于说所述sgRNA一并导入该T细胞。
在一些实施方案中，本发明的TCR-T细胞所表达的工程化TCR可以是本领域己知的任何工程化TCR，只要其能够使T细胞以人白细胞抗原-非依赖性的方式识别细胞表面抗原，发挥杀伤作用即可。例如，本发明TCR-T细胞中的工程化的TCR可以是识别表A中所述抗原的工程化的TCR。
如本文所使用，“工程改造的TCR分子”、“工程化TCR分子”或称“人工TCR分子”包括来源于组成TCR的各种多肽的重组多肽，该重组多肽一般能够i)结合至靶细胞上的表面抗原；及ii)当共定位于T细胞中或表面上时与完整TCR复合物的其它多肽组分相互作用。
在具体实施方案中，本发明的工程化TCR包含靶特异性结合元件，又称为抗原结合结构域。可选择的抗原结合结构域识别例如与特定疾病状态有关的靶细胞上充当细胞表面标志物的靶抗原。在具体的实施方案中，所述靶抗原例如上述表A中的抗原。在具体的实施方案中，所述靶抗原例如与病毒感染、身免疫疾自病有关的靶抗原。通过基因工程改造的方式，可以将抗原结合结构域与来源于组成TCR的各种多肽进行组合，从而使TCR介导的T细胞反应针对所关注的抗原。
在具体实施方案中，本发明的工程化TCR包含跨膜结构域。跨膜结构域可以来源于天然来源或重组来源。在该来源是天然来源的情况下，该结构域可以来源于任何膜结合或跨膜蛋白。在一个方面，跨膜结构域能够向细胞内结构域进行信号传导，只要工程化TCR结合至靶标。特别适用于本发明中的跨膜结构域可以至少包括例如T细胞受体α、β或ζ链、CD28、CD3ε、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154的跨膜区域。
在一些情形中，跨膜结构域可以通过铰链，例如来自人蛋白质的铰链连接至工程化TCR的细胞外区域，例如工程化TCR的抗原结合结构域。举例来说，在一个实施方案中，铰链可以是人免疫球蛋白(Ig)铰链，例如IgG4铰链，或CD8a铰链。
在具体实施方案中，本发明的工程化TCR包含连接跨膜结构域与细胞质区域的连接子。任选地，连接子为长度介于2与50个氨基酸之间的短寡肽或多肽连接子。甘氨酸-丝氨酸对提供特别适合的连接子。
在具体实施方案中，本发明的工程化TCR包含细胞质结构域。细胞内信号传导结构域一般负责引入了工程化TCR的免疫细胞的至少一种正常效应功能的活化。T细胞的效应功能可以例如是细胞溶解活性或辅助活性，包括细胞因子的分泌。因此，术语“细胞内信号传导结构域”是指蛋白质中转导效应功能信号并引导细胞执行专门功能的部分。尽管通常可以采用完整的细胞内信号传导结构域，但在许多情况中不必使用完整链，可以使用此截短部分替代完整链，只要该截短部分转导效应功能信号即可。因此，术语细胞内信号传导结构域意图包括足以转导效应功能信号的细胞内信号传导结构域的任何截短部分。
在某些实施方案中，所述工程TCR分子包含工程化的TCRα和TCRβ链。在某些实施方案中，所述工程化TCR分子与T细胞中表达的CD3分子和ζ链和/或其他共刺激分子所结合。
在一些实施方案中，本发明提供了上述TCR-T细胞用于制备治疗疾病(如肿瘤)的用途。
本发明在一个方面提供了一种治疗受试者疾病的方法，包括给药受试者有效量的本发明所述的TCR-T细胞。本发明所述的治疗方法包括但不限于癌症和HIV/AIDS.在一些实施方案中，该疾病为肿瘤，包括血液系统肿瘤，如淋巴瘤或白血病。在一些实施方案中，所述工程化的TCR靶向表A中所示的靶抗原，该疾病为表A中与该靶抗原相对应的肿瘤。在一些实施方案中，所述T细胞不是从受试者得到的。例如，所述T细胞可以来源于健康的捐助者。
本发明涉及的TCR-T细胞可以通过给药包含细胞成分的医药制品常规使用的途径，例如静脉输注途径，给药有此需要的受试者。给药剂量可以基于受试者的病情和一般健康状况具体确定。
T细胞来源
在进行扩增和基因修饰之前，从受试者获得T细胞来源。术语“受试者”意图包括能够引起免疫反应的活生物体(例如哺乳动物)。受试者的实例包括人。T细胞可以从多种来源获得，包括外周血单核细胞、骨髓、淋巴结组织、脐血、胸腺组织、来自感染部位的组织、腹水、胸膜积液、脾组织及肿瘤。本发明的T细胞还可来源于各个分化阶段的造血干细胞。在定向分化培养条件下，造血干细胞向T细胞分化。在本发明的某些方面，可以使用本领域中可得到的多种T细胞系。
在本发明的某些方面，T细胞可以使用熟练技术人员已知的多种技术，如FicollTM分离从受试者收集的血液获得。也可以通过单采血液成分术(apheresis)从个体的循环血获得细胞。单采血液成分术产物典型地含有淋巴细胞，包括T细胞、单核细胞、粒细胞、B细胞、其它有核白细胞、红细胞及血小板。在一个方面，可以对通过单采血液成分术收集的细胞进行洗涤以去除血浆部分并将细胞放入适当缓冲液或介质中用于后续加工步骤。
可以通过将红细胞溶解并例如经PERCOLL TM梯度离心或逆流离心淘选耗尽单核细胞，从外周血淋巴细胞分离出T细胞。特定T细胞亚群，如CD3+、CD28+、CD4+、CD8+、CD45RA+及CD45RO+T细胞可以通过阳性或阴性选择技术进一步分离。举例来说，在一个方面，T细胞通过与抗CD3/抗CD28(例如3×28)偶联珠粒，如DYNABEADS TM M-450CD3/CD28T一起孵育一段足以对所希望的T细胞进行阳性选择的时间来分离。可以从肿瘤组织分离肿瘤浸润淋巴细胞(TIL)。
sgRNA
本发明一个方面，提供了靶向TRAC的sgRNA,靶向B2M的sgRNA和靶向PD-1的sgRNA。所述sgRNA含有选自SEQ ID NOs:2-22的任一核苷酸序列。在一些实施方案中，所述sgRNA是经化学修饰的。
本发明的还包括sgRNA组合物，试剂盒或制品，其包括本发明涉及的sgRNA或其载体。在一些实施方案中，该试剂盒包括：i)包含选自SEQ ID NOs：2-5中任一序列的sgRNA；(ii)包含选自SEQ ID NOs：6-14中任一序列的sgRNA；和/或(iii)包含选自SEQ ID NOs：15-22中任一序列的sgRNA。在一些实施方案中，该试剂盒包括：(i)包含SEQ ID NO：3序列的的sgRNA；(ii)包含选自SEQ ID NO：16序列的的sgRNA；和(iii)包 含SEQ ID NO：7序列的的sgRNA。在一些实施方案中，该试剂盒还包括Cas6编码核酸或其载体。在一些实施方案中，所述sgRNA是经化学修饰的。
在一些实施方案中，本发明中的T细胞基因改造方法使用化学修饰的sgRNA。本发明人采用的经过化学修饰的sgRNA认为具有以下两个优点。第一、由于sgRNA是单链形式的RNA，其半衰期非常短，进入到细胞后，会迅速降解(最长不超过12小时)，而Cas9蛋白结合sgRNA发挥基因编辑作用则至少需要48hrs。因此，采用经过化学修饰的sgRNA，进入细胞后，稳定表达，与Cas9蛋白结合后，能高效基因编辑基因组，产生Indels。第二、未经修饰的sgRNA穿透细胞膜能力差，无法有效进入细胞或组织发挥相应功能。而经过了化学修饰的sgRNA穿透细胞膜的能力通常是增强的。在本发明中可以采用本领域中常用的化学修饰方法，只要能够提高sgRNA稳定性(延长半衰期)和提升进入细胞膜能力，均可以使用。除了实施例中使用的具体的化学修饰之外，还包括采用其它的修饰方法，例如，Deleavey GF1,Damha MJ.Designing chemically modified oligonucleotides for targeted gene silencing.Chem Biol.2012Aug 24；19(8):937-54，以及Hendel et al.Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells.Nat Biotechnol.2015Sep；33(9):985-989文献中报道的化学修饰方法。
本发明将经过化学修饰的sgRNA与Cas9编码基因共同电转进入T细胞，产生高效的基因编辑效率(如，以Indels％表示)，其中sgRNA的化学修饰是本发明中的关键因素之一。实施例中的数据显示，如果和Cas9mRNA一起电转的是未经化学修饰的sgRNA，其Indels效率远远低于电转经化学修饰的sgRNA时获得的Indels效率。
以下通过具体实施例来说明本发明的内容。应理解，所述具体实施例仅为说明目的，并不意味着本发明的内容仅限于具体实施例。
在本说明书的整篇文本中引用了数篇文件。此处的每篇文件(包括任何期刊文章或摘要、公开或未公开的专利申请、授权专利、制造商的说明书、使用说明等)通过提述并入本文。然而，并非认可此处引用的文件事实上是本发明的现有技术。
实施例1:通用型T细胞的制备
1. 健康供者T细胞的分离与激活
健康供者脐血的采集:从血库拿到脐血后放到4℃冰箱暂存，24h内，经由配备有恒温设备的转运车运至GMP实验室进行T细胞的分离。
1.1 脐血单个核细胞的制备:用移液管吸取生理盐水加入到步骤(1)运输来的脐血中，脐血与生理盐水按照1:1(V/V)稀释，将血细胞稀释液缓慢加入淋巴细胞分离管中，以800g离心20分钟后，吸取淋巴细胞分离液上方的白膜层细胞，转入一个新的50毫升离心管中，加入T细胞培养基，400g离心5分钟后弃上清，保留离心管底部的细胞沉淀，即得到外周血单个核细胞。
1.2 T细胞的分离和激活:将得到的脐血单个核细胞用细胞计数仪进行计数后进行T细胞分选，具体步骤如下：
1.2.1.将细胞沉淀用Easy buffer(生产商：StemCell，货号：16F72331)调整密度为5*10 7/ml,用5毫升移液管将细胞移至5ml流式管中；
1.2.2.加入T细胞分离试剂，其浓度为50ul/ml，加完后在室温下孵育5分钟；
1.2.3.加分选磁珠，30秒内混匀磁珠，加入浓度为40μl/ml；
1.2.4 用Easy buffer将细胞液补至2.5毫升，加完后直接放在磁柱上3分钟，然后将细胞倒入15毫升离心管，得到的即为T细胞；
1.2.5.分选完后，用1000μl移液器混匀取计数，离心(400G，5min)后去上清，即得T细胞沉淀。
将T细胞沉淀重悬到T细胞的培养基中。然后，按照1:1的比例加入T细胞激活因子，此时的T细胞处于激活状态，将T细胞放入到培养箱中继续扩增培养。
2. 电转条件的优化
将上述培养的T细胞收集到50毫升离心管中，以300g离心7分钟后弃上清，用DPBS溶液(生产商：Gibco，货号：1924294)清洗2遍，然后用电转试剂将细胞密度调至2.5X10 7个细胞/mL。使用HISCRIBE TM T7 ARCA mRNA Kit(加尾的)(生产商：NEB,货号：cat#E2060S)制备的GFP mRNA(具体步骤见3.3)与T细胞均匀混合，使其最终浓度达到每100μL中含2.5X10 6个细胞和6μg GFP mRNA。利用电转仪BTX Agile pulse MAX(生产商：BTX，型号：47-0200NINT)将GFP mRNA导入T细胞中。根据电压与脉冲时间的不同，分别对电转体系进行了优化，如表1所示，电压从180伏到400伏 依次递增，脉冲时间从2ms到0.5ms依次递减。每天观察细胞的生长状况，每隔一天对电转的T细胞进行计数补液，并用流式细胞仪器(生产商：艾森，型号：ACEA NovoCyte)对基因编辑后的T细胞进行表型分析，结果如表1所示，在150-250V，0.5-2ms电转条件下，其细胞存活率及GFP电转效率最好。
表1：GFP mRNA在不同电转条件下的电转效率及电转后细胞存活率的比较结果
伏 脉冲(ms) 间隔(ms) Pulse 存活率(％) GFP(％)
00 0.5 100 1 19.78 14.01
60 1 100 1 21.85 11.85
00 1 100 1 31.99 22.91
00 1 100 1 32.20 32.19
50 0.5 100 1 93.22 93.19
50 1 100 1 57.18 57.37
10 1 100 1 91.97 92.23
00 1 100 1 94.54 94.46
00 2 100 1 65.30 65.40
90 1 100 1 94.61 94.66
80 2 100 1 93.77 93.65
3. 对T细胞的基因敲除
利用CRISPR/Cas9基因编辑技术进行敲除经步骤1.2获得的T细胞中的TRAC、B2M、PD-1基因，具体操作步骤如下所示:
3.1 针对TCR的α链恒定编码区(即TRAC)基因、HLA恒定编码区B2M基因及PD-1的恒定编码区基因的sgRNA设计和质粒构建。
针对TRAC、B2M及PD-1编码区所有编码序列设计的sgRNA均通过CRISPR RGEN Tools设计，根据最高评分所选取的sgRNA序列见表2。
表2：经过选择的sgRNA序列
T2 GCTGGTACACGGCAGGGTCA SEQ ID NO：2
T3 CTCTCAGCTGGTACACGGCA SEQ ID NO：3
T4 ATTTGTTTGAGAATCAAAAT SEQ ID NO：4
T6 TCTCTCAGCTGGTACACGGC SEQ ID NO：5
B1 ACTCTCTCTTTCTGGCCTGG SEQ ID NO：6
B2 GAGTAGCGCGAGCACAGCTA SEQ ID NO：7
B3 CGCGAGCACAGCTAAGGCCA SEQ ID NO：8
B4 TCACGTCATCCAGCAGAGAA SEQ ID NO：9
B5 GCTACTCTCTCTTTCTGGCC SEQ ID NO：10
B6 TTTGACTTTCCATTCTCTGC SEQ ID NO：11
B7 CGTGAGTAAACCTGAATCTT SEQ ID NO：12
B8 CTCGCGCTACTCTCTCTTTC SEQ ID NO：13
P1 CTGCAGCTTCTCCAACACAT SEQ ID NO：14
P2 GCCCTGGCCAGTCGTCTGGG SEQ ID NO：15
P4 GCCCTGCTCGTGGTGACCGA SEQ ID NO：16
P5 GAGAAGGTGGGGGGGTTCCA SEQ ID NO：17
P6 CCCTGCTCGTGGTGACCGAA SEQ ID NO：18
P7 GAAGGTGGCGTTGTCCCCTT SEQ ID NO：19
P8 CCTGCTCGTGGTGACCGAAG SEQ ID NO：20
P9 GTCTGGGCGGTGCTACAACT SEQ ID NO：21
P10 CGATGTGTTGGAGAAGCTGC SEQ ID NO：22
其中T表示针对TRAC的sgRNA序列、P代表针对PD-1编码区的sgRNA序列、B代表针对B2M的sgRNA序列。
3.2 用化学方法对sgRNA进行2’-O-甲基类似物和/或核苷酸间3’硫代的修饰，制得具备高敲除效率及稳定性的sgRNA。
3.3 取Cas9质粒和GFP质粒，Xba1(生产商：NEB,货号：cat#R0145S)，cutsmart buffer(生产商：NEB,货号：cat#B7204s)进行酶切线性化，50μl反应体系：
成分 体积
Cas9质粒 5μg
10*cutsmart缓冲液 5μl
Xba1 2μl
加水至总反应体积 50μl
37℃水浴4小时，取酶2μl酶切产物进行琼脂糖凝胶电泳，电压设定110U，电泳30min,凝胶成像仪下观察为单一条带，说明酶切完全，所有质粒都线性化。
取上述反应产物并对其进行清洁纯化。
取纯化后产物，用HISCRIBE TM T7 ARCA mRNA Kit(生产商：NEB,货号：cat#E2060S)进行体外转录(即IVT)，20μl体系：
成分 体积
2*ARCA Mix 10μl
模板DNA 0.5μg
T7 RNA Polymerase Mix 2μl
无核酸酶的水 至20μl
总反应体积 20μl
将上述反应体系放在PCR仪上37℃反应4小时。4小时后，在反应体系中加入2μl的DNase 1，37℃反应20min。
取上述反应产物，进行如下操作：
成分 体积
无核酸酶的水 63μl
IVT反应 22μl
10*Poly(A)聚合酶缓冲液 10μl
Poly(A)聚合酶 5μl
总反应体积 100μl
将上述反应体系放置在PCR仪上反应2小时。
将上述反应产物进行清洁纯化，放在-80度冰箱备用。
3.4 利用电转技术，将sgRNA和Cas9mRNA导入到T细胞中，利用抗原筛选原理，将TCR和/或B2M和/或PD1阴性同时CD4及CD8阳性的T细胞筛选出来，得到通用型T细胞。
取上述细胞，用TIANamp Genomic DNA Kit(生产商：TIAN GEN,货号：cat#DP304-03)，进行细胞基因组提取。利用合成引物，取上述提取的细胞基因组，用2*Esay Taq Super Mix(+dye)(生产商：TRANS,货号Code#AS111)，分别PCR扩增TRAC、B2M及PD-1包含对应sgRNA的基因组区域，50μl反应体系：
成分 体积
2*Easy Taq Super Mix(+dye) 25μl
引物F(10pmol/μl) 1μl
引物R(10pmol/μl) 1μl
模板DNA 300ng
加水至总体积 50μl
反应条件如下：
Figure PCTCN2018106237-appb-000007
3.5 取反应后的PCR产物进行Sanger测序从分子水平上验证TCR、HLA、PD-1的敲除效率，结果参见图2所示，在该体系下，利用CRISPR/Cas9技术可以成功对TCR的TRAC基因、HLA的B2M基因、PD-1的恒定编码序列进行编辑，包括插入突变和缺失突变，两者都造成了移码突变(具体参见下表B-G)，从而从基因水平抑制了TCR、HLA、PD-1的表达。
表B：导入B2后在第15号染色体第45003745位至第45003788位内产生的序列变化：
Figure PCTCN2018106237-appb-000008
Figure PCTCN2018106237-appb-000009
注：该表中的“-”在序列表中标注为“d”
表C：导入B3后在15号染色体第45003745位至第45003788位内产生的序列变化：
Figure PCTCN2018106237-appb-000010
Figure PCTCN2018106237-appb-000011
注：该表中的“-”在序列表中标注为“d”
表D：导入T3后在14号染色体第23016448位至第23016490位内产生的序列变化：
Figure PCTCN2018106237-appb-000012
注：该表中的“-”在序列表中标注为“d”
表E：导入T2后在14号染色体第23016448位至第23016490位内产生的序列变化：
Figure PCTCN2018106237-appb-000013
注：该表中的“-”在序列表中标注为“d”
表F：导入P2后在2号染色体第242800936位至第242800978位内产生的序列变化：
Figure PCTCN2018106237-appb-000014
Figure PCTCN2018106237-appb-000015
注：该表中的“-”在序列表中标注为“d”
表G：导入P1后在2号染色体第242795009位至第242795051位产生的序列变化
Figure PCTCN2018106237-appb-000016
Figure PCTCN2018106237-appb-000017
注：该表中的“-”在序列表中标注为“d”
3.6 同时对于TRAC-sg3(T2、T3)、B2M-sg2(B2、B3)及PD-1-sg2(P1、P2)潜在的人全基因组上的脱靶位点进行预测，并对预测的可能影响其他基因表达的脱靶位点区域进行扩增分析，目的是为了从分子水平确认TRAC、B2M、PD-1的敲除并没有引入脱靶(off-target)的非特异基因的敲除，结果如表3和图5所示。
表3：TRAC、TRAC/B2M(DKO)以及TRAC/B2M/PD-1(TKO)敲除T细胞的脱靶分析结果
Figure PCTCN2018106237-appb-000018
Figure PCTCN2018106237-appb-000019
Figure PCTCN2018106237-appb-000020
注：上述所有的序列位置信息是参考数据库：GRCh37(hg19)确定的。
从表3可以看出TRAC、B2M及PD-1的基因并没有发生任何突变，说明该体系满足了对基因编辑特异性的需求。
以上结果显示，利用TRAC-sg2(T2)、TRAC-sg3(T3)；B2M-sg2(B2)、B2M-sg3(B3)、及PD-1-sg1(P1)、PD-1-sg2(P2)作为sgRNA对T细胞进行基因编辑完成后并对其进行筛选而得到的通用型T细胞中的TRAC、B2M、PD-1基因被完全敲除，同时，没有发现潜在脱靶位点处的基因突变。
4. 基因编辑T细胞的扩增
利用细胞因子对分选后的T细胞进行激活，然后T细胞培养基将细胞密度调至1X 10 6个细胞/mL。72h后观察细胞的状态，收集细胞悬液，以300g离心7min，弃上清，用DPBS(Gibco)清洗2遍，然后用电转试剂培养基将细胞密度调至2.5X 10 7个细胞/mL。使用HISCRIBE TM T7 ARCA mRNA Kit(生产商：NEB,货号：cat#E2060S)制备好的Cas9mRNA及合成的sgRNA，将T细胞和RNA混合，使其最终浓度达到每100μL中含2.5X10 6个细胞和8μg RNA(Cas9mRNA和sgRNA各4μg)，然后利用电转仪BTX Agile pulse MAX将RNA导入细胞中后进行培养。每天观察细胞的生长状况，每隔一天进行细胞计数补液，并对基因编辑后的T细胞进行表型分析，如图3所示，单独敲除TCR的效率约为90.42％，敲除TCR及B2M(DKO)的效率约为81.39％，三个基因同时敲除(TKO)的效率高达67.91。细胞培养8d后，对所得的T细胞进行质量控制监测。
5. CRISPR/Cas9基因编辑效率的检测
细胞培养8天时进行取样，利用人基因组提取试剂盒(生产商：TIAN GEN,货号：cat#DP304-03)对样本提取基因组，同时设计对应的测序引物，利用PCR技术制备目的片段，将目的片段连同相对应的引物进行singer测序。利用TIDE软件，对测序结果分析，所得结果如图2A，2C，2E所示。所优选出的sgRNA能高效地敲除对应的基因。
6. 目的细胞的筛选
通过如下方法对TCR和/或B2M和/或PD-1阴性、CD4和CD8阳性的T细胞进行筛选。
利用免疫磁珠技术筛选出TCR和/或B2M和/或PD-1阴性，同时CD4和CD8阳性的T细胞，并通过T细胞存活率监测被编辑后的T细胞状态。
第一步，在第12-14天将电转后的T细胞收集，400G，离心5min,弃上清，用Easy buffer把细胞定溶为1X10 8/ml，然后把细胞转移至5ml流式管中，利用筛选试剂去除掉T细胞中仍表达TCR、B2M、PD-1的细胞，筛选得到终产品即通用型T细胞。
第二步，取少量T细胞，进行流式检测，同时针对TCR和/或B2M和/或PD-1细胞表面生物标记物进行染色，如果TCR和/或B2M和/或PD-1阳性率<1％，即可进行下一步工作。在本实施例中，TCR阳性率为1％，TCR和B2M DKO的T细胞阳性率<0.79％，TCR、B2M和PD-1TKO的T细胞阳性率<1％，如图4所示。纯化后的T细胞存活率并没有受到明显影响，均在85％以上，参见表4所示。
表4：对经TCR、TCR/B2M(DKO)以及TCR/B2M/PD-1(TKO)敲除的T细胞进行筛选纯化后的存活率
Figure PCTCN2018106237-appb-000021
7.脱靶分析
7.1.收集T细胞1X10 6个，提取基因组(生产商：QIAGEN，货号：Cat#69504)。
7.2.将提取的基因组进行Cas9体外酶切反应
Cas9体外酶切反应体系如下：
Figure PCTCN2018106237-appb-000022
Figure PCTCN2018106237-appb-000023
7.3.用枪头吹打，轻轻混匀后，37℃温育15min；
7.4.加入1μl蛋白酶K(生产商：Tiangen,货号：Cat#RT403)，轻轻混匀后，室温温育10min；
7.5.取5ul样本进行凝胶电泳，检测Cas9体外酶切反应效果；
7.6.将其余样本进行人重基因组检测。
结果如图5所示，未发现脱靶现象。
实施例2:通用型T细胞的制备
通用型CAR-T细胞的制备和扩增
利用细胞因子对分选后的T细胞进行激活，然后用T细胞培养基将细胞密度调至1X 10 6个细胞/mL。72h后观察细胞的状态，收集细胞悬液，以300g离心7min，弃上清，用DPBS溶液(生产商：Gibco；货号：1924294)清洗2遍，然后用电转试剂培养基将细胞密度调至2.5X 10 7个细胞/mL。使用HISCRIBE  TM T7 ARCA mRNA Kit(带尾)(生产商：NEB,货号：cat#E2060S)制备好的Cas9mRNA及合成的sgRNA，将T细胞和RNA混合，使其最终浓度达到每100μL中含2.5X10 6个细胞和8μg RNA(Cas9mRNA和sgRNA各4μg)，然后利用电转仪BTX Agile pulse MAX将RNA导入细胞中后进行培养。每天观察细胞的生长状况，每隔一天进行细胞计数补液，在第5天按照MOI＝2-10的比例加入包装有CAR(包括抗CD19scFv、链接区(Linker)、CD8alpha铰链区(CD8alpha hinge)、CD8跨膜区(CD8transmemberane domain)、4-11BB信号区(4-11BB signaling domain)及CD3zeta，具体结构参见US20140271635A1)的慢病毒。
通用型CAR-T细胞的筛选
通过如下方法对TCR和/或B2M和/或PD-1阴性、CD4和CD8阳性的T细胞进行筛选。
利用免疫磁珠技术筛选出TCR和/或B2M和/或PD-1阴性，同时CD4和CD8阳性的T细胞，并通过T细胞存活率监测被编辑后的T细胞状态，具体步骤如下：
第一步，在第12-14天将电转后的T细胞收集，400G，离心5min,弃上清，用Easy buffer把细胞定溶为1X10 8/ml，然后把细胞转移至5ml流式管中，利用筛选磁珠去除掉T细胞中仍表达TCR、B2M、PD-1的细胞，筛选得到终产品即通用型T细胞。
第二步，取少量T细胞，进行流式检测，同时染TCR和/或B2M和/或PD-1细胞表面生物标记物，如果TCR和/或B2M和/或PD-1阳性率<1％，即可进行下一步工作。在本实施例中，TCR阳性率为1％，TCR和B2M DKO的T细胞阳性率<0.79％，TCR、B2M和PD-1TKO的T细胞阳性率<1％，如表5和图6所示。纯化后的T细胞存活率并没有受到明显影响，均在85％以上。
表5：
Figure PCTCN2018106237-appb-000024
实施例3:通用型CAR-T功能验证
观察实施例2得到的通用型CAR-T细胞(即效应细胞)对B细胞型急性淋巴细胞白血病细胞的杀伤作用。
一、对特异性肿瘤细胞的体外杀伤作用
本发明实验步骤如下:
第一步:靶细胞标记
使用CELL TRACE TM Far Red Cell Proliferation Kit(生产商：Gibco；货号：1888569)标记靶细胞(人Burkitt’s淋巴瘤细胞Raji、K562,所有细胞来自ATCC)。
1.将Cell TraceTM Far Red Cell Proliferation用双蒸水稀释成1mmol溶液；
2.取1X 10  6个靶细胞400g离心5分钟后去上清；
3.加入Cell TraceTM Far Red Cell Proliferation溶液1μl，37℃、避光孵育20min。
4.将细胞加入T细胞培养基，37℃孵育5min。
5. 400g离心5分钟后去上清，标记完成。
第二步:效应细胞对靶细胞的杀伤检测
将标记好的靶细胞用R1640+10％FBS培养液按照2X10 5个细胞/mL的密度重悬，取500μL加入到48孔板中。按照适当的效靶比(2.5:1、1.25:1、0.6:1)，每孔加入500μL效应细胞，同时以T细胞和健康人脐血CAR-T细胞(在T细胞培养到第2天时，以MOI＝2-10的比例加入包装有CAR(具体结构参见US20140271635A1)的慢病毒制得的CAR-T作为对照细胞，每组3个平行，设计单独的靶细胞组，检测其死亡率；37℃、5％CO2培养12-16h，400g离心5min，取细胞沉淀，用150μl用DPBS(生产商：Gibco；货号：1924294)重悬。利用PI(生产商：Sigma；货号：P4170)染色后，用流式细胞仪检测靶细胞死亡率，结果如图7所示，通用型CAR-T相较与其它的CAR-T细胞在对特异性靶细胞的杀伤作用上基本保持一致，效果都优于T细胞。同时对非特异性靶细胞的几乎无杀伤功能。
第三步:细胞因子释放的ELISA检测
将标记好的靶细胞用RPMI 1640+10％FBS按照2X10 5个/mL的密度重悬，取500μL加入到48孔板中。按照适当的效靶比(10:1)每孔加入500μL效应细胞，同时以T细胞和健康人脐血CAR-T作为对照细胞，每组3个平行，设计单独的靶细胞组，37℃、5％CO2培养12-16h，每孔取培养上清100μl，400g离心5min去沉淀，取上清后利用LEGEND MAX TM Human IL-2/IFN-gama(生产商：Biolegend；货号分别为：431807、430108)试剂盒，根据使用说明检测因子释放。
所得结果如图8所示，可以看出，通用型CAR-T对Raji细胞的杀伤能力与普通CAR-T杀伤能力基本一致或稍好于普通CAR-T的杀伤能力，尤其TCR neg CAR-T IFN-r释放量远远高于普通CAR-T且都对K562的杀伤力较低，因此因子释放相对较少。
二、通用型CAR-T对特异性肿瘤细胞的体内杀伤作用
1.细胞系:人淋巴瘤细胞系
Raji tg(luciferase-GFP)/Bcgen细胞是人Burkitt’s淋巴瘤细胞系，其CD19表达为阳性，可以作为CAR-T细胞的靶细胞。Raji luc-GFP经过修饰同时表达GFP与luciferase。可以通过尾静脉注射的方式构建小鼠的人淋巴瘤模型，通过XenoLight D-Luciferin,Potassium Salt(生产商：PerkinElmer；货号：122799)配合小动物活体成像仪呈现的荧光统计成瘤面积。
2.Raji tg(luciferase-GFP)/Bcgen细胞培养
Raji tg(luciferase-GFP)/Bcgen细胞系为悬浮细胞系，在含有10％FBS的RPMI1640培养基中可以快速生长。细胞密为2-3X 10 6/mL时需要传代。传代时取细胞悬液于离心管中，以300g离心6分钟，弃上清。将细胞密度调整到1×10 6个细胞/ml，继续培养。正常生长情况，隔天传代，细胞密度维持在0.8-1X 10 6个细胞 /mL之间即可。
3.小鼠造模
7-10周龄的NPG雌性小鼠25只，单次尾静脉注射肿瘤细胞(Raji tg(luciferase-GFP)/Bcgen)5X10 5个细胞/只，隔天称重，每天观察一次，接种肿瘤细胞3-5天后，通过XenoLight D-Luciferin,Potassium Salt(生产商：PerkinElmer；货号：122799)配合小动物活体成像仪呈现的成瘤面积及肿瘤富集度为指标进行随机分成6组：生理盐水组、T细胞组、CAR-T细胞组、TCR neg-CAR-T组、DKO-CAR-T组、TKO-CAR-T组。
4.小鼠淋巴瘤模型给药。
记录造模当天为D0。通过尾静脉注射的方式进行细胞输生理盐水200μL、人T细胞200μL(总计2×10 6个细胞/只)、CAR-T细胞200μL(总计2×10 6/只)TCR neg-CAR-T细胞200μL(总计2X 10 6/只)、DKO-CAR-T细胞200μL(总计2X 10 6/只)、TKO-CAR-T细胞200μL(总计2X 10 6/只)所有小鼠均单次给药。结果如图9A-9B及图10所示，可以看出，本发明提供的通用型CAR-T前期具有很好的抑制以及杀死肿瘤细胞的效果，与CAR-T细胞几乎一致；后期较普通CAR-T对肿瘤细胞的抑制效果明显。
5.小鼠给药后监测
对给药后的小鼠每天进行监测，包括小鼠体重，皮肤完整度，毛发，精神状态、活动频率及活动协调度等每2天对小鼠进行体重记录，连续观察37 天，以肿瘤面积的消除及肿瘤富集的减少为效应细胞功能的评判指标，以皮肤完整度，毛发，精神状态、活动频率及活动协调度评判通用型CAR-T的安全性。如图11所示小鼠并没有体重上的下降，皮肤毛发完整，精神活跃，活动协调，没有出现GVHD反应。

Claims (37)

  1. 一种制备基因改造的T细胞的方法，包括：通过基因编辑技术破坏所述T细胞中：
    (i)14号染色体第23016448位至第23016490位的TRAC基因组区域；
    (ii)15号染色体第45003745位至第45003788位的B2M基因组区域；和/或
    (iii)2号染色体第242800936位至第242800978位的PD-1基因组区域，或2号染色体第242795009位至第242795051位的PD-1基因组区域.
  2. 权利要求1中的方法，其中所述TRAC基因组区域、B2M基因组区域和PD-1基因组区域均被编辑。
  3. 权利要求1或2中所述的方法，其中所述基因编辑技术为基于锌指核酸酶的基因编辑技术、TALEN基因编辑技术或CRISPR/Cas基因编辑技术。
  4. 权利要求3所述的方法，其中所述基因编辑技术为CRISPR/Cas9基因编辑技术。
  5. 权利要求1-4中任一项所述的方法，其中:
    (i)所述TRAC基因组的靶核苷酸序列与选自SEQ ID NOs：2-5中任一的序列互补；
    (ii)所述B2M基因组的靶核苷酸序列与选自SEQ ID NOs：6-13中任一的序列互补；和/或
    (iii)所述PD-1基因组的靶核苷酸序列与选自SEQ ID NOs：14-22中任一的序列互补。
  6. 权利要求4中所述的方法，其中:
    (i)将包含靶向所述TRAC基因组的sgRNA导入该T细胞以实现对所述TRAC基因组区域的编辑；
    (ii)将包含靶向所述B2M基因组的sgRNA导入该T细胞以实现对所述B2M基因组区域的编辑；和/或
    (iii)将包含靶向所述PD-1基因组的sgRNA导入该T细胞以实现对所述PD-1基因组区域的编辑。
  7. 权利要求6中所述的方法，包含：
    (i)将包含选自SEQ ID NOs：2-5中任一序列的sgRNA导入T细胞以实现对所述TRAC基因组区域的编辑；
    (ii)将包含选自SEQ ID NOs：6-14中任一序列的sgRNA导入该T细胞以实现对所述B2M基因组区域的编辑；和/或
    (iii)将包含选自SEQ ID NOs：15-22中任一序列的sgRNA导入该T细胞以实现对所述PD-1基因组区域的编辑。
  8. 权利要求7或8中所述的方法,包含同时向该T细胞导入所述靶向TRAC的sgRNA、靶向B2M的sgRNA和靶向PD-1的sgRNA.
  9. 权利要求6－8中任一项所述的方法,其中所述sgRNA是经过2’-O-甲基类似物和/或核苷酸间3’硫代修饰的。
  10. 权利要求9中所述的方法,其中所述化学修饰为所述sgRNA的5’端前一个、二个和/或三个碱基和/或3’端的最后一个碱基的2’-O-甲基类似物修饰。
  11. 权利要求6-10中任一项所述的方法，其中将所述sgRNA与Cas9编码核苷酸序列共同导入该T细胞。
  12. 权利要求11中所述的方法,将所述sgRNA与Cas9编码核苷酸序列通过电转方式共同导入该T细胞，优选所述电转条件包括选自如下的任一项：150-250V，0.5-2ms；150V，2ms；160V，2ms；170V，2ms；180V，2ms；190V，1ms；200V，1ms；210V，1ms；220V，1ms；230V，1ms；240V，1ms；和250V，0.5ms。
  13. 权利要求1-12中任一项的方法，还包括从经过基因编辑的T细胞中筛选TRAC,B2M和/或PD-1表达量低的T细胞。
  14. 权利要求1-13任一项的方法，其中所述TRAC、B2M或PD-1基因敲除的效率为90％以上；所述TRAC与B2M基因的同时敲除效率在75％以上；或所述TRAC、B2M和PD-1基因的同时敲除效率在65％以上。
  15. 权利要求1-14任一项的方法，其中T细胞来源于健康受试者。
  16. 通过权利要求1-15任一项的方法制备的基因改造的T细胞。
  17. 一种基因改造的T细胞，其中所述T细胞中：
    (i)14号染色体第23016448位至第23016490位的TRAC基因组区域的一个或多个位点通过基因编辑技术被破坏；
    (ii)15号染色体第45003745位至第45003788位的B2M基因组区域的一个或多个位点通过基因编辑技术被破坏；和/或
    (iii)2号染色体第242800936位至第242800978位的PD-1基因组区域，或2号染色体第242795009位至第242795051位的PD-1基因组区域的一个或多个位点通过基因编辑技术被破坏。
  18. 权利要求16或17的基因改造的T细胞用于制备过继细胞治疗的T细胞的用途。
  19. 权利要求18的用途，其中所述过继细胞治疗的T细胞为CAR-T细胞或TCR-T细胞。
  20. 一种制备CAR-T细胞或TCR-T细胞的方法，包括:将嵌合抗原受体(CAR)或其编码核苷酸、或工程化的T细胞受体(TCR)或其编码核苷酸导入权利要求16或17所述的基因改造的T细胞。
  21. 一种制备CAR-T或TCR-T细胞的方法，包括:
    (i)将包含靶向14号染色体第23016448位至第23016490位的TRAC基因组的sgRNA导入T细胞以破坏所述TRAC基因组区域；和/或
    (ii)将包含靶向15号染色体第45003745位至第45003788位的B2M基因组区域的sgRNA导入该T细胞以破坏所述B2M基因组区域；和/或
    (iii)将包含靶向2号染色体第242800936位至第242800978位的PD-1基因组区域，或2号染色体第242795009位至第242795051位的PD-1基因组区域的sgRNA导入该T细胞以破坏所述B2M基因组区域；和
    (iv)将嵌合抗原受体(CAR)或其编码核苷酸、或工程化的T细胞受体(TCR)或其编码核苷酸导入该T细胞。
  22. 权利要求21的方法，其中靶向TRAC的sgRNA,靶向B2M的sgRNA和/或靶向PD-1的sgRNA和CAR或其编码核苷酸或工程化的T细胞受体(TCR)或其编码核苷酸同时导入该T细胞。
  23. 权利要求21的方法，其中将CAR或其编码核苷酸、或工程化的T细胞受体(TCR)或其编码核苷酸先于靶向TRAC的sgRNA,靶向B2M的sgRNA和/或靶向PD-1的sgRNA导入该T细胞；或将CAR或其编码核苷酸、或工程化的T细胞受体(TCR)或其编码核苷酸在导入靶向TRAC的sgRNA,靶向B2M的sgRNA和/或靶向PD-1的sgRNA后导入该T细胞。
  24. 权利要求20-23任一的方法，其中所述CAR-T为通用型CAR-T；或其中所述TCR-T为通用型TCR-T。
  25. 通过权利要求20-24任一的方法制备的CAR-T细胞或TCR-T细胞。
  26. 一种CAR-T细胞,包含表达嵌合抗原受体(CAR)的权利要求16或17所述的基因改造的T细胞。
  27. 一种TCR-T细胞,包含表达工程化TCR的权利要求16或17所述的基因改造的T细胞。
  28. 包含权利要求16或17所述的基因改造的T细胞，权利要求25或26的CAR-T细胞，或权利要求25或27的TCR-T细胞的组合物。
  29. 一种治疗受试者疾病的方法，包括给药受试者有效量的权利要求25或26的CAR-T细胞，或权利要求25或27的TCR-T细胞。
  30. 权利要求29的方法，其中所述疾病为肿瘤。
  31. 权利要求30的方法，其中所述肿瘤为血液系统肿瘤。
  32. 权利要求31的方法，其中所述肿瘤为淋巴瘤或白血病。
  33. 权利要求29-32中任一的方法，其中所述CAR靶向CD19。
  34. 权利要求29-33中任一的方法，其中所述T细胞不是从受试者得到的。
  35. 包含SEQ ID NOs：2-22任一项的sgRNA。
  36. 权利要求35的sgRNA，其中所述化学修饰是2’-O-甲基类似物和/或核苷酸间3’硫代修饰。
  37. 权利要求36的sgRNA，其中所述化学修饰为所述sgRNA的5’端前一个、二个和/或三个碱基和/或3’端的最后一个碱基的2’-O-甲基类似物的修饰。
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021043278A1 (zh) * 2019-09-04 2021-03-11 博雅辑因（北京）生物科技有限公司 基于脱靶评估评价基因编辑治疗的方法
EP3995579A4 (en) * 2019-07-19 2023-03-08 BRL Medicine Inc. SGRNA-LEARING PD1 GENE FOR CLEAVAGE FOR EFFICIENT INTEGRATION OF EXOGENE SEQUENCES

Families Citing this family (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021136176A1 (zh) * 2019-12-30 2021-07-08 博雅辑因（北京）生物科技有限公司 一种靶向t细胞淋巴瘤细胞的通用型car-t及其制备方法和应用
CN113122503B (zh) * 2019-12-30 2022-10-11 博雅缉因（北京）生物科技有限公司 一种靶向t细胞淋巴瘤细胞的通用型car-t及其制备方法和应用
WO2021136415A1 (zh) * 2019-12-30 2021-07-08 博雅辑因（北京）生物科技有限公司 一种纯化ucart细胞的方法与应用
CN113122504A (zh) * 2019-12-30 2021-07-16 博雅辑因（北京）生物科技有限公司 一种纯化ucart细胞的方法与应用
CN113122576A (zh) * 2019-12-31 2021-07-16 博雅辑因（北京）生物科技有限公司 一种通用型truck-t细胞、其制备方法以及用途
CN110951695A (zh) * 2019-12-31 2020-04-03 深圳市茵冠生物科技有限公司 通用型car-t细胞、其制备方法及其应用
CN114929866A (zh) * 2020-02-28 2022-08-19 大塚制药株式会社 基因改造巨核细胞、改造血小板和它们的制造方法
WO2022015956A1 (en) * 2020-07-15 2022-01-20 Sorrento Therapeutics, Inc. Improved process for dna integration using rna-guided endonucleases
CN112094867A (zh) * 2020-09-30 2020-12-18 广东先康达生物科技有限公司 一种高纯度通用型car-t的制备方法及应用
CN112980800A (zh) * 2021-03-08 2021-06-18 河北森朗生物科技有限公司 Car-t细胞、其构建方法及其应用

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013176772A1 (en) 2012-05-25 2013-11-28 The Regents Of The University Of California Methods and compositions for rna-directed target dna modification and for rna-directed modulation of transcription
WO2014018423A2 (en) 2012-07-25 2014-01-30 The Broad Institute, Inc. Inducible dna binding proteins and genome perturbation tools and applications thereof
US8697359B1 (en) 2012-12-12 2014-04-15 The Broad Institute, Inc. CRISPR-Cas systems and methods for altering expression of gene products
WO2014065596A1 (en) 2012-10-23 2014-05-01 Toolgen Incorporated Composition for cleaving a target dna comprising a guide rna specific for the target dna and cas protein-encoding nucleic acid or cas protein, and use thereof
US20140271635A1 (en) 2013-03-16 2014-09-18 The Trustees Of The University Of Pennsylvania Treatment of cancer using humanized anti-cd19 chimeric antigen receptor
WO2016069282A1 (en) * 2014-10-31 2016-05-06 The Trustees Of The University Of Pennsylvania Altering gene expression in modified t cells and uses thereof
WO2016160721A1 (en) * 2015-03-27 2016-10-06 President And Fellows Of Harvard College Modified t cells and methods of making and using the same

Family Cites Families (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9890393B2 (en) 2013-05-29 2018-02-13 Cellectis Methods for engineering T cells for immunotherapy by using RNA-guided CAS nuclease system
CN103820454B (zh) * 2014-03-04 2016-03-30 上海金卫生物技术有限公司 CRISPR-Cas9特异性敲除人PD1基因的方法以及用于特异性靶向PD1基因的sgRNA
WO2016073955A2 (en) 2014-11-06 2016-05-12 President And Fellows Of Harvard College Cells lacking b2m surface expression and methods for allogeneic administration of such cells
KR102648489B1 (ko) 2015-04-06 2024-03-15 더 보드 어브 트러스티스 어브 더 리랜드 스탠포드 주니어 유니버시티 Crispr/cas-매개 유전자 조절을 위한 화학적으로 변형된 가이드 rna
CN108024544B (zh) * 2015-07-13 2022-04-29 桑格摩生物治疗股份有限公司 用于核酸酶介导的基因组工程化的递送方法及组合物
CN108699132B (zh) 2015-12-18 2023-08-11 桑格摩生物治疗股份有限公司 Mhc细胞受体的靶向破坏
JP7128741B2 (ja) * 2015-12-18 2022-08-31 サンガモ セラピューティクス， インコーポレイテッド Ｔ細胞受容体の標的化破壊
EP3423580A1 (en) 2016-03-04 2019-01-09 Editas Medicine, Inc. Crispr-cpf1-related methods, compositions and components for cancer immunotherapy
CN106191062B (zh) 2016-07-18 2019-06-14 广东华南疫苗股份有限公司 一种tcr-/pd-1-双阴性t细胞及其构建方法
CN106544321B (zh) * 2016-10-13 2019-01-29 北京艺妙神州医疗科技有限公司 通用型car-t细胞及其制备方法和用途

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013176772A1 (en) 2012-05-25 2013-11-28 The Regents Of The University Of California Methods and compositions for rna-directed target dna modification and for rna-directed modulation of transcription
WO2014018423A2 (en) 2012-07-25 2014-01-30 The Broad Institute, Inc. Inducible dna binding proteins and genome perturbation tools and applications thereof
WO2014065596A1 (en) 2012-10-23 2014-05-01 Toolgen Incorporated Composition for cleaving a target dna comprising a guide rna specific for the target dna and cas protein-encoding nucleic acid or cas protein, and use thereof
US8697359B1 (en) 2012-12-12 2014-04-15 The Broad Institute, Inc. CRISPR-Cas systems and methods for altering expression of gene products
US20140271635A1 (en) 2013-03-16 2014-09-18 The Trustees Of The University Of Pennsylvania Treatment of cancer using humanized anti-cd19 chimeric antigen receptor
WO2016069282A1 (en) * 2014-10-31 2016-05-06 The Trustees Of The University Of Pennsylvania Altering gene expression in modified t cells and uses thereof
WO2016160721A1 (en) * 2015-03-27 2016-10-06 President And Fellows Of Harvard College Modified t cells and methods of making and using the same

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DELEAVEY GF1DAMHA MJ: "Designing chemically modified oligonucleotides for targeted gene silencing", CHEM BIOL., vol. 19, no. 8, 24 August 2012 (2012-08-24), pages 937 - 54, XP055107150, DOI: 10.1016/j.chembiol.2012.07.011
HENDEL ET AL.: "Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells", NAT BIOTECHNOL., vol. 33, no. 9, September 2015 (2015-09-01), pages 985 - 989, XP055548372, DOI: 10.1038/nbt.3290
LIU: "CRISPR-Cas9-mediated multiplex gene editing in CAR-T cells", CELL RES., vol. 27, no. 1, 2 December 2016 (2016-12-02), pages 154 - 157, XP055555205, ISSN: 1748-7838 *
SAMBROCK ET AL.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY
See also references of EP3686275A4

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3995579A4 (en) * 2019-07-19 2023-03-08 BRL Medicine Inc. SGRNA-LEARING PD1 GENE FOR CLEAVAGE FOR EFFICIENT INTEGRATION OF EXOGENE SEQUENCES
WO2021043278A1 (zh) * 2019-09-04 2021-03-11 博雅辑因（北京）生物科技有限公司 基于脱靶评估评价基因编辑治疗的方法
JP2022547105A (ja) * 2019-09-04 2022-11-10 博雅▲輯▼因（北京）生物科技有限公司 オフターゲット評価に基づく遺伝子編集治療の評価方法

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