WO2024022513A1 - 靶向cd5和cd7的通用car-t及其应用 - Google Patents

靶向cd5和cd7的通用car-t及其应用 Download PDF

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WO2024022513A1
WO2024022513A1 PCT/CN2023/109928 CN2023109928W WO2024022513A1 WO 2024022513 A1 WO2024022513 A1 WO 2024022513A1 CN 2023109928 W CN2023109928 W CN 2023109928W WO 2024022513 A1 WO2024022513 A1 WO 2024022513A1
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gene
seq
sgrna
cells
pmol
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胡广
谭涛超
张佳元
姚小敏
董文洁
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上海驯鹿生物技术有限公司
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Definitions

  • This article relates to methods for preparing universal chimeric antigen receptor T cells (UCAR-T). This article also covers the application of UCAR-T in cancer treatment.
  • UCAR-T universal chimeric antigen receptor T cells
  • UCAR-T universal CAR-T
  • the two most important problems are one: due to allogeneic cell transfusion Graft versus host disease GvHD caused by injection, second: UCAR-T is rapidly cleared by the host immune system in the host body and cannot be effectively expanded. Therefore, the current UCAR-T is mainly designed around solving these two problems.
  • TRAC T cell surface receptors
  • Another strategy to reduce the host rejection graft response is to knock out the B2M gene encoding ⁇ 2-microglobulin on UCAR- T3,4 .
  • Disruption of ⁇ 2-microglobulin prevents the expression of functional HLA class I molecules on the surface of CAR-T cells.
  • This protocol prevents UCAR-T from activating cytotoxic T cells in the host by destroying HLA-class I molecules, allowing them to achieve long-term proliferation.
  • HLA is an inhibitory ligand for NK cells, its absence will activate the patient's NK cells to eliminate CAR-T cells, limiting their expansion in the body and affecting their effectiveness.
  • CD5 and CD7 are two potential targets for T cell-targeted therapy of malignancies 5 .
  • Many T-cell malignancies express CD7, and most T-NHL and T-ALL highly express CD7.
  • CD7 is expressed in approximately 24% of AML and is considered a marker of leukemic stem cells and is used in terminal cancers. 6 is expressed on most natural killer cells (NK) and NKT NHLs and leukemias, providing an attractive target for immunotherapy of T-cell cancers.
  • CD5 is a pan-T cell marker that is commonly overexpressed in most T-cell malignancies. Normal cell CD5 expression is limited to thymocytes, peripheral T cells, and a small subset of B lymphocytes, called B-1 cells. . Furthermore, CD5 is a negative regulator of T cell receptor (TCR) signaling and plays a role in protecting autoimmunity7 .
  • TCR T cell receptor
  • this article provides methods for preparing universal chimeric antigen receptor T cells (UCAR-T), including:
  • step 1) further includes knocking out the B2M gene of the T cell.
  • the target sequence of the sgRNA used for knocking out the TRAC gene is selected from the sequences shown in SEQ ID NO: 1-7 and any combination thereof.
  • the target sequence of the sgRNA used for knocking out the B2M gene is selected from the sequences shown in SEQ ID NO: 8, 9, 11, 12 and any combination thereof.
  • the knockout of the B2M gene uses a combination of two sgRNAs, the target sequences of the two sgRNAs being the sequences shown in SEQ ID NO: 9 and 11 respectively.
  • the TRAC gene and the B2M gene knockout in the T cells are simultaneously performed.
  • Knockout of the B2M gene, and the target sequence of the sgRNA used for knockout of the TRAC gene is the sequence shown in SEQ ID NO: 7.
  • the target sequence of the sgRNA for knocking out the CD5 gene is selected from the sequences shown in SEQ ID NO: 13, 14, 16, 17, 18, 19 and any combination thereof.
  • the target sequence of the sgRNA for CD7 gene knockout is the sequence shown in SEQ ID NO: 20.
  • the knockout of the TRAC gene, the CD5 gene and the CD7 gene is performed simultaneously, and the target sequence of the sgRNA used to knock out the TRAC gene is the sequence shown in SEQ ID NO: 7;
  • the target sequence of the sgRNA used to knock out the CD5 gene is the sequence shown in SEQ ID NO: 13; and the target sequence of the sgRNA used to knock out the CD7 gene is the sequence shown in SEQ ID NO: 20.
  • the knockout of the TRAC gene, the B2M gene, the CD5 gene and the CD7 gene is performed simultaneously, and the target sequence of the sgRNA for knocking out the TRAC gene is SEQ ID NO: The sequence shown in 7; the target sequences of the two sgRNAs used to knock out the B2M gene are the series shown in SEQ ID NO: 9 and 11 respectively; the target sequences of the sgRNA used to knock out the CD5 gene are SEQ ID NO : The sequence shown in 13; and the target sequence of the sgRNA used to knock out the CD7 gene is the sequence shown in SEQ ID NO: 20.
  • the knockout of the TRAC gene, the CD5 gene and the CD7 gene is performed simultaneously, and the components used to perform the knockout include in a 20 ⁇ L system in proportion:
  • An RNP complex formed by no less than 30 pmol of Cas9 and no less than 45 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 7;
  • An RNP complex formed by no less than 40 pmol of Cas9 and no less than 120 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 13;
  • An RNP complex formed by no less than 40 pmol of Cas9 and no less than 120 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 20.
  • the knockout of the TRAC gene, the B2M gene, the CD5 gene and the CD7 gene is performed simultaneously, and the components used to perform the knockout include in a 20 ⁇ L system in proportion:
  • An RNP complex formed by no less than 30 pmol of Cas9 and no less than 45 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 7;
  • An RNP complex formed by no less than 20 pmol of Cas9 and no less than 30 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 9;
  • An RNP complex formed by no less than 20 pmol of Cas9 and no less than 30 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 11;
  • An RNP complex formed by no less than 40 pmol of Cas9 and no less than 120 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 13;
  • An RNP complex formed by no less than 40 pmol of Cas9 and no less than 120 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 20.
  • the knockout of the TRAC gene, the CD5 gene and the CD7 gene is performed simultaneously, and the components used to perform the knockout include in a 20 ⁇ L system in proportion:
  • RNP complex formed by 30 pmol of Cas9 and 45 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 7;
  • RNP complex formed by 80 pmol of Cas9 and 150 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 13;
  • RNP complex formed by 40 pmol of Cas9 and 120 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 20.
  • the knockout of the TRAC gene, the B2M gene, the CD5 gene and the CD7 gene is performed simultaneously, and the components used to perform the knockout include in a 20 ⁇ L system in proportion:
  • RNP complex formed by 30 pmol of Cas9 and 45 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 7;
  • RNP complex formed by 20 pmol of Cas9 and 30 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 9;
  • RNP complex formed by 20 pmol of Cas9 and 30 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 11;
  • RNP complex formed by 80 pmol of Cas9 and 150 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 13;
  • RNP complex formed by 40 pmol of Cas9 and 120 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 20.
  • step 1) further includes knocking out the CIITA gene of the T cell.
  • the target sequence of the sgRNA used for knockout of the CIITA gene is the sequence shown in SEQ ID NO: 25.
  • the knockout of the TRAC gene, the B2M gene, the CD5 gene, the CD7 gene and the CIITA gene is performed simultaneously, and the components used to perform the knockout are in proportion to
  • the 20 ⁇ L system includes:
  • An RNP complex formed by no less than 30 pmol of Cas9 and no less than 45 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 7;
  • An RNP complex formed by no less than 20 pmol of Cas9 and no less than 30 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 9;
  • An RNP complex formed by no less than 20 pmol of Cas9 and no less than 30 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 11;
  • An RNP complex formed by no less than 80 pmol of Cas9 and no less than 150 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 13;
  • An RNP complex formed by no less than 40 pmol of Cas9 and no less than 120 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 20;
  • An RNP complex formed by no less than 40 pmol of Cas9 and no less than 60 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 25.
  • the knockout of the TRAC gene, the B2M gene, the CD5 gene, the CD7 gene and the CIITA gene is performed simultaneously, and the components used to perform the knockout are in proportion to
  • the 20 ⁇ L system includes:
  • RNP complex formed by 30 pmol of Cas9 and 45 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 7;
  • RNP complex formed by 25 pmol of Cas9 and 40 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 9;
  • RNP complex formed by 25 pmol of Cas9 and 40 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 11;
  • RNP complex formed by 80 pmol of Cas9 and 150 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 13;
  • RNP complex formed by 40 pmol of Cas9 and 120 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 20;
  • this article provides methods for preparing universal chimeric antigen receptor T cells (UCAR-T), including:
  • step 1) further includes knocking out the B2M gene of the T cell.
  • step 1) further includes knocking out the CIITA gene of the T cell.
  • the target sequence of the sgRNA used for knocking out the TRAC gene is the sequence shown in SEQ ID NO: 26.
  • the target sequence of the sgRNA used for knocking out the TRBC gene is selected from the sequence shown in any one of SEQ ID NO: 27-31 and any combination thereof.
  • the target sequence of the sgRNA used for knocking out the B2M gene is selected from the sequences shown in SEQ ID NO: 33 and 34 and combinations thereof.
  • two sgRNAs are used to knock out the B2M gene, wherein the target sequences of the two sgRNAs are the sequences shown in SEQ ID NO: 8 and 9 respectively.
  • the target sequence of the sgRNA for knocking out the CD5 gene is selected from the sequence shown in any one of SEQ ID NO: 37, 39, 41-46 and any combination thereof.
  • the target sequence of the sgRNA for CD7 gene knockout is the sequence shown in SEQ ID NO: 47.
  • the target sequence of the sgRNA for CIITA gene knockout is selected from the sequence shown in any one of SEQ ID NO: 50, 51, 54, 57 and any combination thereof.
  • the cytosine base editor is an nCBE3 or nCBE4 protein.
  • the TRAC gene, the B2M gene, the CD5 gene, the CD7 gene and/or the CIITA gene are knocked out prior to activating the T cells with CD2/CD3/CD28 antigen.
  • the extracellular antigen-binding domain of the CAR includes a first antigen-binding portion and a second antigen-binding portion, the first antigen-binding portion is capable of specifically binding CD7, and the second antigen-binding portion is capable of Specific binding to CD5.
  • the first antigen-binding portion includes a heavy chain variable region from an anti-CD7 single domain antibody
  • the HCDR1 of the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 59
  • the HCDR2 includes SEQ ID NO: 59
  • the amino acid sequence shown in ID NO: 60 and HCDR3 includes the amino acid sequence shown in SEQ ID NO: 61.
  • the second antigen-binding portion includes a heavy chain variable region from an anti-CD5 single domain antibody
  • the HCDR1 of the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 63
  • the HCDR2 includes SEQ ID NO: 63
  • the amino acid sequence shown in ID NO: 64 and HCDR3 includes the amino acid sequence shown in SEQ ID NO: 65.
  • the first antigen-binding portion includes the amino acid sequence set forth in SEQ ID NO: 62.
  • the second antigen-binding portion includes the amino acid sequence set forth in SEQ ID NO: 66.
  • the extracellular antigen binding domain of the CAR includes the amino acid sequence shown in SEQ ID NO: 74.
  • the CAR includes the first antigen-binding portion, the connecting fragment, the second antigen-binding portion, a hinge region, a transmembrane region, an intracellular costimulatory domain and Intracellular signaling domain.
  • connection fragment includes the amino acid sequence shown in SEQ ID NO: 67; the hinge region includes the amino acid sequence shown in SEQ ID NO: 68; the transmembrane region includes the amino acid sequence shown in SEQ ID NO: 69 Sequence; the intracellular costimulatory domain includes the amino acid sequence shown in SEQ ID NO: 70; the intracellular signaling domain includes the amino acid sequence shown in SEQ ID NO: 71.
  • the nucleic acid molecule further includes a coding sequence for tEGFR or herpes simplex virus thymidine kinase (HSV-TK).
  • HSV-TK herpes simplex virus thymidine kinase
  • the coding sequence of the tEGFR or HSV-TK in the nucleic acid molecule is connected downstream of the coding sequence of the CAR by a coding sequence of a self-cleaving peptide.
  • the self-cleaving peptide is T2A, and its amino acid sequence is preferably shown in SEQ ID NO: 72.
  • step 2) it is also included in step 2) to screen out T cells that do not express TCR and MHC class I molecules.
  • the T cells contain NKT cells, for example, a 10-20% quantitative ratio of NKT cells.
  • this article provides UCAR-T cells prepared by the above method.
  • this article provides a pharmaceutical composition, which includes the above-mentioned UCAR-T cells and a pharmaceutically acceptable carrier.
  • this article provides the use of the above-mentioned UCAR-T cells in the preparation of drugs for treating cancer.
  • the cancer expresses CD5 and/or CD7 on its cell surface.
  • the cancer is a T-cell malignancy, such as T-acute lymphoblastic leukemia (T-ALL) and T-cell lymphoma.
  • T-ALL T-acute lymphoblastic leukemia
  • T-cell lymphoma T-cell lymphoma
  • provided herein are methods of treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of the UCAR-T cells or pharmaceutical composition described above.
  • the cancer expresses CD5 and/or CD7 on its cell surface.
  • the cancer is a T cell malignancy, such as acute T lymphoblastic leukemia and T cell lymphoma.
  • the method further comprises administering ganciclovir (GCV) to the subject after treatment.
  • GCV ganciclovir
  • this article provides a pharmaceutical kit, including: 1) the above-mentioned UCAR-T cells or pharmaceutical composition; and 2) GCV.
  • FIG. 1 Schematic diagram of the design of CD5-CD7 universal CAR-T.
  • FIG. 1 Schematic diagram of the CD5-CD7 bispecific CAR structure.
  • FIG. 4 TRAC gene candidate sgRNA information as well as knockout efficiency and off-target test results.
  • A Candidate sgRNA information obtained after the inventor designed and screened TRAC gene knockout sgRNA through CRISPick, CRISPOR and IDT websites. Based on previous experience, the sgRNA editing efficiency predicted by the website is not particularly accurate. Therefore, when screening candidate sgRNAs, the inventors mainly look at the off-target information predicted by the website. Among the three websites of CRISPick, CRISPOR and IDT, CRISPick’s calculation of sgRNA off-target is The most precise, so the inventor mainly selects sgRNA based on CRISPick's off-target ranking.
  • the abscissa represents the TCR expression of the cells, and the ordinate represents the size of the cells; each column represents an sgRNA, and each row represents an RNP concentration.
  • A-XL sgRNA was tested twice, labeled R1 and R2 respectively, and A-02 and A-06 were tested once each.
  • the highest off-target ratio (number of reads at the most frequent site/number of reads at the target site)*100%.
  • B2M gene candidate sgRNA information and knockout efficiency test results (A) Candidate sgRNA information obtained by the inventor after designing and screening B2M gene knockout sgRNA through CRISPick, CRISPOR, IDT, CHOPCHOP and GUIDES websites. The final sgRNA is selected based on the on-target and off-target information from several websites. The smaller the ranking number, the better, and the higher the score, the better. (B) FACS results of B2M candidate sgRNA knockout efficiency test. The abscissa represents the B2M expression of the cells, the ordinate represents the size of the cells, and the MOCK-T group represents cells that do not use sgRNA.
  • FIG. B2M and TRAC gene double knockout test results and B2M-sgRNA off-target detection results.
  • A FACS results of co-knockdown of five candidate sgRNAs of B2M and TRAC sgRNA (A-XL).
  • the abscissa represents the B2M expression of cells, and the ordinate represents the TCR expression of cells; double-gene positive cells are located in the upper right quadrant of the four quadrants, and double-gene negative cells are located in the lower left quadrant of the four quadrants; therefore, cells located in the lower left quadrant occupy The higher the ratio, the more cells that have both genes knocked out at the same time.
  • the abscissa represents the B2M expression of cells, and the ordinate represents the TCR expression of cells.
  • Each column represents a B2M sgRNA, and each row represents a RNP concentration.
  • C Two off-target test results of B-13 sgRNA, respectively R1 and R2.
  • FIG. 7 CD5 gene candidate sgRNA information and gene knockout results.
  • A Sequence information of the first tested CD5 sgRNA.
  • B FACS results of CD5 sgRNA knockout efficiency test in the first test. The abscissa represents the CD5 expression of the cells, and the ordinate represents the size of the cells. sgRNA1 and sgRNA3 correspond to C-01 and C-03 in Figure C.
  • C The candidate sgRNA information obtained after the inventor designed and screened the CD5 gene knockout sgRNA for the second time through CRISPick, CRISPOR, IDT, CHOPCHOP and GUIDES websites.
  • FIG. 8 Knockout efficiency and off-target detection results of CD5 gene candidate sgRNA.
  • A FACS results of CD5 candidate sgRNA knockdown efficiency test. The abscissa represents the expression of CAR in cells, and the ordinate represents the expression of CD5 in cells. The higher the proportion of cells in the sitting quadrant, the higher the knockout efficiency of CD5.
  • B Summary graph of knockout efficiency of CD5 candidate sgRNA, data derived from FACS results of 5A.
  • C Off-target and comprehensive ranking of CD5 candidate sgRNA on the CRISPick website. The smaller the ranking number, the better the sgRNA.
  • D Two off-target test results of C-06 sgRNA, respectively R1 and R2.
  • FIG. 9 Off-target detection results of CD7 gene sgRNA (E-01). The left side shows the sequencing results of control cells without sgRNA, and the right side shows the sequencing results of CD7 sgRNA.
  • FIG. 10 CIITA gene candidate sgRNA information, knockout efficiency and off-target detection results.
  • A Sequence information of CIITA candidate sgRNA and FACS results of knockout efficiency test. The abscissa represents the expression of HLA-II class molecules in cells, and the ordinate represents the size of cells.
  • B Two off-target test results of F-05 sgRNA, respectively R1 and R2.
  • FIG. 11 Exploring RNP conditions for co-knocking of TRAC, B2M, CD5, and CD7.
  • A The amount of RNP used in each gene when electroporating a 20ul system (2x10 6 cells) using a Lonza electroporation instrument. Each row represents a set of settings, and a total of 6 RNP dosage combinations have been explored; each column represents the RNP dosage of a type of sgRNA, and the numbers in each grid in the table respectively represent the dosage of Cas9 protein and sgRNA, in pmol.
  • B FACS results of TRAC, B2M, CD5, and CD7 co-knockdown efficiency under different combinations of RNP dosages, tested on T cells derived from two donors. In each figure, the higher the proportion of cells in the lower left quadrant, the higher the efficiency of double gene knockout.
  • FIG. 12 Knockout settings and results of CD5-CD7 UCAR-T prepared twice (the subsequent mention of "two preparations” refers to the same two preparations).
  • A Knockout status of each gene in the CD5-CD7 UCAR-T finished cells prepared for the first time. The upper row shows the gene expression of non-knocked-out cells, and the lower row shows the gene knockout of CD5-CD7 UCAR-T cells. The cells in the lower left quadrant are the proportion of knocked-out cells.
  • B The RNP dosage of each gene when preparing CD5-CD7 UCAR-T twice. The number in each grid represents the dosage of the corresponding material, in pmol.
  • KO-T The gene knockout status of KO-T cells during the second preparation of CD5-CD7 UCAR-T cells.
  • KO-T are cells that only have gene knockout but not transfection, which can reflect the true status of each gene. Knockout situation.
  • ISO is the negative control result
  • MOCK-T is the positive control result.
  • FIG. 13 Results of five-knockdown experiments on TRAC, B2M, CIITA, CD5, and CD7 genes.
  • A The amount of RNP used in the pre-experiment of co-knockout of five genes including TRAC, B2M, CIITA, CD5, and CD7. The number in each cell represents the amount of the corresponding material, in pmol.
  • B Preliminary experiment of joint knockout of five genes including TRAC, B2M, CIITA, CD5 and CD7 FACS result plot.
  • ISO is a negative control
  • MOCK-T is a positive control
  • HLA-DR-DP-DQ reflects the knockout of CIITA gene.
  • (C) The amount of RNP used when preparing the five-knock version of CD5-CD7 UCAR-T cells. The number in each cell represents the amount of the corresponding material, in pmol.
  • Figure 14 Single base editing candidate sgRNA information targeting the TCR/B2M/CD5/CD7/CIITA gene.
  • FIG. 15 Base editing sgRNA efficiency targeting TCR genes.
  • the picture shows the FACS results of the base editing sgRNA efficiency test for TCR.
  • the abscissa represents the expression of TCR, and the ordinate represents the complexity of the cell content.
  • FIG. 16 Base editing sgRNA efficiency targeting B2M gene.
  • A The picture shows the results of the base editing sgRNA efficiency test for B2M. The abscissa represents the expression of B2M, and the ordinate represents the complexity of the cell content.
  • B The picture shows a schematic diagram of the Cas9 protein using two adjacent opposite sgRNAs to cut both strands of the genome respectively, as well as the position and sequence information of several sgRNAs available on exon 1 of B2M.
  • C Knockout of B2M under different combinations of four sgRNAs B-10, B-13, NB-37, and NB-38. The abscissa represents the expression of B2M, and the ordinate represents the complexity of cell contents.
  • FIG. 17 Base editing sgRNA efficiency targeting CD5 gene and CD7 gene.
  • A The picture shows the FACS results of the base editing sgRNA efficiency test for CD5. The abscissa represents the expression of CD5, and the ordinate represents the complexity of the cell content. Each column represents a type of sgRNA, and two repeated experiments were performed.
  • B The picture shows the results of the base editing sgRNA efficiency test for CD7. The abscissa represents the expression of CD7, and the ordinate represents the complexity of the cell content. Two repeated experiments were also conducted.
  • FIG. 18 Base editing efficiency of different sgRNAs targeting the CIITA gene.
  • the picture shows the FACS results of the base editing sgRNA efficiency test for CIITA.
  • the abscissa reflects the expression of CIITA with the expression of HLA-II class molecules.
  • the ordinate represents the complexity of the cell content.
  • ISO is the negative control
  • MOCK- T is the positive control.
  • FIG. 19 Proportions of CD5, CD7 and CAR-positive cells of UCAR-T prepared twice.
  • A The picture shows the proportion of CAR-positive cells in the first prepared CD5-CD7 UCAR-T 3 days after transfection. The expression of EGFR, CD5, and CD7 CAR was detected by EGFR antibody and CD5/CD7 antigen respectively.
  • B The picture shows the proportion of CAR-positive cells in the first prepared CD5-CD7 UCAR-T 8 days after transfection, which was nearly doubled compared to 3 days after transfection.
  • C and D are the proportions of CAR-positive cells in the second preparation of CD5-CD7 UCAR-T on days 3 and 8 after transfection, respectively. The results are similar to those of the first CD5-CD7 UCAR-T.
  • FIG. 20 TRAC/B2M negative selection results during two preparations of UCAR-T.
  • the picture shows the results of TRAC/B2M negative selection during the two preparations of CD5-CD7 UCAR-T.
  • the abscissa represents the expression of B2M
  • the ordinate represents the expression of TCR
  • the red dotted box represents the cells that continue to be cultured after negative selection.
  • Figure 21 Test results of CD107a release of CD5-CD7 UCAR-T prepared twice.
  • A When KO-T and UCAR-T were co-incubated with different tumor cells for 4 hours when CD5-CD7 UCAR-T was first prepared, the proportion of CD107a-positive cells in CD8 and CAR double-positive T cells.
  • B Histogram based on the data in 18A.
  • C The second time After the prepared CD5-CD7 UCAR-T was incubated with different tumor cells for 4 hours, the proportion of CD107a-positive cells in CD8 and CAR double-positive T cells was shown.
  • Figure 22 Test of tumor killing performance of CD5-CD7 UCAR-T prepared twice.
  • A The tumor killing function test results of the first prepared CD5-CD7 UCAR-T on day 7. The data at each point represents the percentage of CD5-CD7 UCAR-T killing the corresponding tumor cells. The higher the value, the more tumor cells have been killed. A negative value means that the tumor cells have not only not been killed but have proliferated.
  • B Corresponding tumor killing ability of untransfected KO-T cells prepared for the first time at the same time point.
  • C The tumor killing function test results of the first prepared CD5-CD7 UCAR-T cells after cryopreservation and recovery.
  • Figure 23 Activation and typing results of CD5-CD7 UCAR-T prepared twice.
  • A The expression of each antigen on the CD5-CD7 UCAR-T prepared for the first time (without TRAC/B2M negative selection);
  • B The expression of each antigen on the CD5-CD7 UCAR-T prepared for the second time (TRAC/B2M negative selection has been performed);
  • C CCR7 positive proportion in the CD5-CD7 UCAR-T prepared for the first time (frozen on day 10);
  • D CCR7 in the CD5-CD7 UCAR-T prepared for the second time Positive proportion (frozen on day 12).
  • Figure 24 Depletion and early regulation of CD5-CD7 UCAR-T prepared twice.
  • A The expression of depletion markers on the CD5-CD7 UCAR-T prepared for the first time
  • B The expression of depletion markers on the CD5-CD7 UCAR-T prepared for the second time
  • C The CD5-CD7 UCAR-T prepared for the first time Expression of premature apoptosis marker on CD7 UCAR-T
  • D Expression of premature apoptosis marker on CD5-CD7 UCAR-T prepared for the second time.
  • Figure 25 Component identification results of twice prepared CD5-CD7 UCAR-T.
  • A The component identification results of the CD5-CD7 UCAR-T cells prepared for the first time on the day of cryopreservation;
  • B The component identification of the CD5-CD7 UCAR-T cells prepared for the second time at different time points during the preparation period result.
  • FIG. 26 An example of clinical experimental results of CD5-CD7 UCAR-T (EGFR switch).
  • A Schematic design of the entire clinical testing cycle. Flu is Fludarabine, and CTX is Cyclophosphamide (CTX).
  • B The VCN of CAR-positive cells in the patient's peripheral blood changes over time after CD5-CD7 UCAR-T reinfusion. The higher the VCN, the more UCAR-T cells there are in the patient's peripheral blood.
  • C-E Changes in the proportion of various cells in the peripheral blood of patients after CD5-CD7 UCAR-T reinfusion. lym refers to lymphocytes.
  • FIG. 27 CD5-CD7 UCAR-T (HSV-TK) cell preparation.
  • A Schematic diagram of the molecular structure of the HSV-TK version of CAR, which changes the original tEGFR to HSV-TK.
  • B Brief flow chart of preparation of CD5-CD7 UCAR-T by activated electroporation process and resting electroporation process.
  • C Knockout of TRAC/B2M/CD5/CD7 and efficiency of CAR under activated electroporation and resting electroporation processes.
  • D Cell typing of CD5-CD7 UCAR-T prepared by activated electroporation and resting electroporation processes.
  • E Comparison of cell components of CD5-CD7 UCAR-T prepared by activated electroporation and resting electroporation processes.
  • Figure 28 Comparison of tumor killing effects between HSV-TK version and EGFR version UCAR-T.
  • A Tumor killing results of HSV-TK version of CD5-CD7 UCAR-T cells 24 hours after cryopreservation and recovery.
  • B Tumor killing results of EGFR version CD5-CD7 UCAR-T cells 24 hours after cryopreservation and recovery.
  • Figure 29 Clearance of HSV-TK positive cells by GCV drugs.
  • A Total cell proliferation of HSV-TK version of CD5-CD7 UCAR-T cells treated with different concentrations of GCV.
  • B Changes in the proportion of CAR-positive cells in the HSV-TK version of CD5-CD7 UCAR-T cells when treated with different concentrations of GCV.
  • C Changes in the number of CAR-positive cells in the HSV-TK version of CD5-CD7 UCAR-T cells when treated with different concentrations of GCV.
  • D -(F) Changes in the proliferation of total cells, the proportion of CAR-positive cells, and the number of CAR-positive cells when EGFR version CD5-CD7 UCAR-T cells were treated with different concentrations of GCV.
  • FIG. 30 In vivo efficacy testing of UCAR-T and GCV.
  • A Schematic diagram of the entire process design of animal experiments, D represents the number of days.
  • B Fluorescence imaging results of mice. Since tumor cells express luciferin protein, after the mice are injected with a luciferin protein substrate, the mice can produce fluorescence. The fluorescence intensity generated can be detected by a fluorescence imager. Indirectly reflects the number of tumor cells.
  • C Changes in average fluorescence intensity in different groups of mice over time.
  • B2M gene refers to the gene encoding ⁇ 2 microglobulin.
  • ⁇ 2 microglobulin is a component of MHC class I molecules (human MHC class I molecules, also known as HLA class I molecules). It binds to the heavy chain in MHC class I molecules to form heterodimers on the cell surface. Knockout of the B2M gene will result in the loss of MHC class I molecules on the cell surface.
  • TCR gene and “TRBC gene” herein refer to the gene encoding the constant region of the T cell receptor ⁇ chain and the gene encoding the constant region of the T cell receptor ⁇ chain, respectively.
  • the alpha and beta chains constitute the T cell receptor (TCR), which recognizes antigens and mediates immune responses. Knockout of "TRAC gene” and/or “TRBC gene” results in cells unable to express TCR molecules.
  • the product encoded by the "CIITA gene” is a class II transactivator, which plays a leading switch role in the expression of HLA genes.
  • Class II transactivators themselves are non-DNA-binding proteins, but they can bind to a variety of transcription factors and Co-activators participate in the transcriptional regulation of HLA genes. Knockout of the CIITA gene can affect the transcription of HLA genes, especially HLA class II genes, making it unable to express the corresponding products.
  • CD5 is a type I transmembrane glycosylated protein that plays an important role in the negative regulation of T cell receptor signaling and promotes the survival of normal and malignant lymphocytes.
  • CD5 is one of the characteristic surface markers of malignant T-cell tumors. 80% of T-cell acute lymphoblastic leukemia (T-ALL) and peripheral T-cell lymphoma express CD5.
  • T-ALL T-cell acute lymphoblastic leukemia
  • CD5 proteins may also include fragments of CD5, such as the extracellular domain and fragments thereof.
  • CD7 is a cell surface glycoprotein with a molecular weight of about 40kD. It is a member of the immunoglobulin superfamily and is expressed on the surface of T cells, NK cells and other cells such as thymocytes and myeloid cells. It plays an important role in T cell interactions as well as T cell-B cell interactions during early lymphoid development.
  • Antibodies refer to immunoglobulins secreted by plasma cells (effector B cells) and used by the body's immune system to neutralize foreign substances (polypeptides, viruses, bacteria, etc.). This foreign substance is accordingly called an antigen.
  • the basic structure of a classic antibody molecule is a tetramer composed of two identical heavy chains and two identical light chains. According to the conservative differences in amino acid sequences, heavy and light chains are divided into variable regions (V) located at the amino terminus and constant regions (C) located at the carboxyl terminus. The variable regions of a heavy chain and a light chain interact to form the antigen-binding site (Fv).
  • variable region the composition and order of amino acid residues in certain regions are more variable than other regions (backbone regions, FR) within the variable region, which are called hypervariable regions (HVR).
  • the hypervariable regions are actually antibodies. Key site for antigen binding. Because these hypervariable region sequences are complementary to antigenic determinants, they are also called complementarity-determining regions (CDRs). Both heavy and light chains have three complementarity determining regions, called HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, and LCDR3 respectively.
  • the amino acid sequences of the CDRs can be determined using art-recognized numbering schemes, such as the Kabat, Chothia, IMGT, AbM, or Contact numbering schemes.
  • Antibodies can be divided into five main different types based on the amino acid sequence of their heavy chain constant regions: IgA, IgD, IgE, IgG, and IgM. These antibody types can be further divided into subclasses based on the size of the hinge region, the position of the interchain disulfide bond, and the molecular weight, such as IgGl, IgG2a, IgG2b, and IgG3. According to the different amino acid composition and arrangement of the constant region of the antibody light chain, the light chain can be divided into two types: kappa and lambda. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are known in the art.
  • the "antigen-binding fragment" of an antibody molecule refers to the amino acid fragment in the antibody molecule that participates in specific antigen binding, such as Fab, Fab', (Fab') 2 , scFv, sdAb, etc.
  • Fab fragment-binding fragment
  • Fab' fragment-binding fragment
  • pepsin fragment-binding fragment-binding fragment-binding fragment-binding fragment-binding fragment-binding fragments.
  • F(ab') 2 which is formed by treatment with a reducing agent to break the disulfide bond between the hinge regions of F(ab') 2 Fab' fragment.
  • Single chain fragment variable is composed of an antibody heavy chain variable region and a light chain variable region connected through a short peptide to form a peptide chain. Through correct folding, the variable regions from the heavy chain and light chain form Fv segments through non-covalent interactions, so scFv can better retain its affinity activity for antigens.
  • Single domain antibody also known as “VHH antibody” refers to an antibody molecule with antigen-binding ability, including a heavy chain variable region but no light chain. From a structural point of view, single domain antibodies can also be considered as an antigen-binding fragment of an antibody molecule. It was first discovered in camelids, and subsequently, researchers used antibodies to Screening of libraries (such as phage display libraries) has discovered more single domain antibodies with antigen-binding ability. Single domain antibodies have some advantages over ordinary antibody molecules (for example, classic tetrameric antibody molecules) or their antigen-binding fragments, including but not limited to: smaller molecular weight, and when used in the human body, they can easily reach tissues that are difficult for ordinary antibody molecules to reach. or parts, or can access antigenic epitopes in proteins or polypeptides that are difficult for ordinary antibody molecules to access; they are more stable and can withstand changes in temperature and pH, as well as the effects of denaturants and proteases.
  • targeting refers to the effect of one molecule (e.g., an antibody or antigen-binding fragment thereof) on another molecule (e.g., an antibody or antigen-binding fragment thereof) relative to other molecules co-present in the environment. Such as tumor cell surface antigens) have higher binding affinity.
  • Targeting does not exclude that the molecule may have binding affinity for more than one molecule, for example, a bispecific antibody may have high affinity for two different antigens.
  • Chimeric antibody receptor (chimeric antigen receptor, CAR)
  • CAR chimeric antigen receptor
  • chimeric immune receptor is an engineered membrane protein receptor molecule that can combine the desired specificity Conferring to immune effector cells, for example, the ability to bind to cell surface proteins such as tumor antigens.
  • Chimeric antigen receptors usually consist of an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain.
  • the antigen-binding domain is a scFv or sdAb sequence that is responsible for recognizing and binding to a specific antigen.
  • the antigen-binding domain can be monospecific, that is, it has specific binding ability to only one antigen; it can also be multispecific (such as bispecific), that is, it has specific binding ability to multiple antigens.
  • the bispecific extracellular antigen binding domain specifically binds both CD5 and CD7, which can be accomplished by including an antibody that targets or specifically binds CD5 in the extracellular antigen binding domain. fragments (e.g., scFv or sdAb) and antibody fragments (e.g., scFv or sdAb) that target or specifically bind CD7.
  • Intracellular signaling domains usually include immunoreceptor tyrosine activation motifs (ITAMs), such as the signaling domain derived from CD3 ⁇ molecules, which are responsible for activating immune effector cells and producing killing effects.
  • ITAMs immunoreceptor tyrosine activation motifs
  • the chimeric antigen receptor may also include a signal peptide at the amino terminus responsible for the intracellular localization of the nascent protein, and a hinge region between the antigen-binding domain and the transmembrane domain.
  • Intracellular signaling domains may also include costimulatory domains derived from, for example, 4-1BB or CD28 molecules.
  • T cells expressing CAR are referred to as CAR-T.
  • CAR-T uses the CAR expressed on its cell surface to recognize target cells, and after being activated by the target cells, it kills the target cells in a non-MHC-restricted manner.
  • the general process of using CAR-T cells to treat a subject is: collecting peripheral blood mononuclear cells (PBMC) from the subject, isolating and culturing the T cells, and transfecting them with lentivirus.
  • PBMC peripheral blood mononuclear cells
  • the CAR encoding nucleic acid sequence is introduced by induction, the CAR+ cells are continued to be cultured and collected, and the CAR+ cells are infused back into the subject.
  • NK cells may be used instead of T cells to carry out this process. Therefore, when referring to CAR-T, CAR-expressing NK cells may also be included as appropriate. In addition, when referring to CAR-T cells in this article, unless otherwise stated, it refers not only to the cells directly modified by CAR, but also to the daughter cells produced after these cells are proliferated in vitro or in vivo.
  • Universal CAR-T cells refers to cells that are not limited to CAR-T cells that are infused into a specific patient.
  • cells such as T cells
  • This method is not only time-consuming and expensive, but in some cases it is impossible to obtain a sufficient number of patient T cells for CAR modification.
  • the pass Using CAR-T cells means that they are suitable for allogeneic transplantation.
  • the same batch of CAR-T cells can be used in different patients, and these universal CAR-T cells are usually not derived from these patients.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • the CRISPR gene editing system used in this technology includes Cas nuclease and guide RNA (single-guide RNA, sgRNA), and optionally ssDNA as a repair template.
  • sgRNA single-guide RNA
  • crRNA crRNA
  • Cas nuclease can form a single-stranded or double-stranded nick at a specific site of the target gene.
  • CRISPR gene editing system in this article refers to the combination of Cas nuclease and sgRNA, which is used to edit sgRNA-targeted genes after introduction into cells.
  • CRISPR CRISPR-like genome editing
  • TALEN TALEN
  • Cytosine base editing technology is a gene editing technology that introduces single base editing function based on CRISPR technology. It uses a fusion protein called a "cytosine base editor” with multiple functional parts.
  • One functional part is the Cas9n (Cas9-nickase) protein, which is derived from the D10A mutation of the RuvC1 domain of the Cas9 protein, thereby retaining only the enzymatic activity of the HNH domain.
  • Cas9n does not cause DNA double-strand breaks, but can only cut the DNA single strand complementary to sgRNA on the genome, thereby inducing base mismatch repair (BER).
  • cytosine deaminase APOBEC can induce the deamination of cytosine on another DNA single strand (non-sgRNA target strand) to form uracil, which in the presence of UGI protein ultimately promotes the mutation of cytosine to thymine (C->T mutation).
  • cytosine base editing technology is used to introduce stop codons into the genes to be knocked out of cells, so that the cells cannot produce functional gene products.
  • Cytosine base editing technology can be regarded as part of CRISPR technology, but for the purpose of convenient description, in this article, when mentioning CRISPR technology, especially CRISPR technology using Cas9 protein, it does not involve the above-mentioned single base editing function. Thus distinguishing it from cytosine base editing technology.
  • “Knockout” or “gene knockout” as used herein refers to changing the nucleotide sequence of a certain gene in a cell, whether the change is a nucleotide insertion, deletion or substitution, as long as the gene being knocked out does not produce any significant changes in the cell.
  • Functional gene products such as RNA or protein
  • a gene is knocked out such that the cell or cell population completely does not form the gene product of the gene or a functional gene product. Understandably, if the amount of the gene product is significantly reduced, or the activity of the gene product is significantly reduced, it can also be considered to have achieved “gene knockout.” In some cases, it may be necessary to knock out two or more genes in the cell.
  • gene knockout can be performed sequentially, i.e., in After knocking out one gene, move on to knocking out the next gene.
  • two or more genes can be knocked out simultaneously.
  • Cas9 and multiple sgRNAs targeting each gene can be introduced into the cell at the same time.
  • target sequence refers to a nucleotide segment in the target gene or gene to be knocked out that is complementary to a partial sequence of the sgRNA (crRNA, approximately 20 bases).
  • proteins such as Cas9 can introduce nucleotide sequence changes in the target gene at a relatively certain position to achieve the effect of gene knockout.
  • sgRNA targeting a specified sequence means that the target sequence of the sgRNA is the specified sequence.
  • RNP complex refers to the product of the combination of sgRNA and the corresponding Cas enzyme (such as Cas9).
  • Cas9 the corresponding Cas enzyme
  • sgRNA can be mixed with Cas enzyme and then introduced into cells (such as T cells) through electroporation.
  • EGFRt or tEGFR are used interchangeably herein to refer to a gene encoding a truncated human epidermal growth factor receptor polypeptide, or its encoded product, which lacks the distal membrane EGF binding domain and cytoplasmic signaling tail but retains Extracellular epitopes recognized by anti-EGFR antibodies.
  • EGFRt can be used as a non-immunogenic selection tool as well as a tracking marker with the function of genetically modifying cells.
  • HSV-TK is the abbreviation of Herpes Simplex Virus Thymidine Kinase (human herpes simplex virus thymidine kinase), and its substrate is the small molecule drug GCV.
  • GCV Herpes Simplex Virus Thymidine Kinase
  • the role of HSV-TK and GCV as molecular switches is described in more detail below.
  • Self-cleaving peptides refer to short peptides that can achieve the function of cleaving proteins through ribosome skipping rather than proteolytic hydrolysis, and may include T2A, F2A, P2A, etc.
  • Treatment refers to the treatment of a subject to obtain a beneficial or desired clinical result.
  • Treatment encompasses a variety of treatments, including administration of any possible drug to the subject, surgery, radiation, etc.
  • beneficial or desired clinical outcomes include, but are not limited to, any one or more of the following: alleviation of one or more symptoms, attenuation of disease severity, prevention or delay of disease spread (e.g. metastasis, e.g. metastasize to the lungs or lymph nodes), prevent or delay disease recurrence, delay or slow down disease progression, improve disease conditions, inhibit disease or disease progression, block its development and remission (whether partial or complete remission).
  • the methods provided herein encompass any one or more of these aspects of treatment. In accordance with the above, “treatment” does not require complete removal of all symptoms of a condition or disease or complete alleviation.
  • terapéuticaally effective amount refers to an amount of active compound sufficient to elicit the biological or medical response desired by the clinician in a subject.
  • the "therapeutically effective dose” of the fusion protein of the present invention can be determined by those skilled in the art based on the route of administration, the subject's weight, age, condition and other factors. For example, a typical daily dosage may range from 0.01 mg to 100 mg or more of active ingredient per kg of body weight.
  • pharmaceutically acceptable carrier refers to solid or liquid diluents, fillers, antioxidants, stabilizers and the like that can be administered safely and are suitable for use by humans and/or animals given drugs It has no excessive adverse side effects and is suitable for maintaining the vitality of the drug or active agent contained therein.
  • various carriers well known in the art may be administered, including, but not limited to, sugars, starch, cellulose and its derivatives, maltose, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols , alginic acid, phosphate buffer, emulsifier, isotonic saline, and/or pyrogen-free water, etc.
  • the pharmaceutical compositions provided herein can be made into clinically acceptable dosage forms such as powders and injections.
  • composition of the present invention can be administered to a subject by any appropriate route, for example, by oral administration, intravenous infusion, intramuscular injection, subcutaneous injection, subperitoneal, rectal, sublingual, or by inhalation, transdermal, etc. route of administration.
  • “Pharmaceutical kit” refers to a pharmaceutical combination including at least two active ingredients. Unlike pharmaceutical compositions, pharmaceutical kits contain at least one active ingredient that is kept separate from other active ingredients.
  • Subject refers to an animal, such as a mammal, including (but not limited to) humans, rodents, simians, felines, canines, equines, bovines, porcines, sheep, goats, mammals Laboratory animals, mammalian farm animals, mammalian sporting animals and mammalian pets.
  • the subject may be male or female and may be of any appropriate age, including infants, juveniles, young adults, adults, and geriatric subjects.
  • a subject refers to an individual in need of treatment of a disease or condition.
  • a subject receiving treatment can be a patient who has a condition associated with the treatment, or is at risk of developing the condition.
  • the subject is a healthy individual or an individual suffering from a disease other than that of concern.
  • the subject is a human, such as a human patient.
  • the term is often used interchangeably with "patient,” “subject,” “subject,” etc.
  • the inventors knocked out the TRAC and/or TRBC genes to prevent the expression of TCR, the T cell surface receptor of CAR-T, and thus unable to recognize host cells, as shown in Figure 1.
  • HLA molecules are divided into two types: HLA-I and HLA-II.
  • HLA-I molecules are recognized by CD8-positive toxic T cells and are the main way for host T cells to eliminate CAR-T cells, while HLA-II molecules are recognized by CD4-positive toxic T cells.
  • T cell recognition is an auxiliary pathway for host T cells to eliminate CAR-T cells.
  • the inventor has two versions of HLA molecule knockout, one is to knock out the B2M gene of the HLA-I molecule, and the second is to simultaneously knock out the B2M gene of the HLA-I molecule and CIITA, which is related to the expression of HLA-II molecules.
  • the second version of UCAR-T will theoretically better avoid clearance by host T cells, but knockout of HLA will activate host NK, and activated NK will also clear UCAR-T.
  • the CAR prepared by the inventor targets CD5 and CD7, and CD7 is also expressed on NK cells. Therefore, while attacking tumor cells, the CAR can also attack the host's NK cells, which solves the problem of HLA knockout. NK activation problem. Since CAR-T itself also expresses CD5 and CD7, in order to avoid CAR-T suicide, the inventors also knocked out CD5 and CD7 of CAR-T cells at the same time. HLA knockout combined with CAR as an attacking molecule weakens the clearing effect of host T and NK cells on UCAR-T cells, so that UCAR-T can be well expanded in vivo.
  • the preparation process of universal CAR-T is basically the same as that of ordinary CAR-T, except that it includes an additional gene knockout step.
  • the general process is: T cell sorting and activation ⁇ electroporation of gene knockout ⁇ lentiviral transduction ⁇ detection of knockout efficiency and integration efficiency of CAR structure ⁇ in vitro functional verification ⁇ cryopreservation of cell preparations.
  • the structure of the CAR used in this article is shown in Figure 2.
  • the single VH domains (single domain antibodies) of CD7 and CD5 are connected in series through a linker, and then followed by the CD8 ⁇ hinge region, TM transmembrane region, 4-1BB and CD3 ⁇ costimulatory molecules.
  • the EGFRt switch molecule can then be connected through T2A, and the T2A protein will separate the EGFRt switch molecule from the CAR structure.
  • suicide genes such as the herpes simplex virus thymidine kinase (HSV-TK) gene.
  • HSV-TK herpes simplex virus thymidine kinase
  • CD5-CD7 universal CAR-T clears tumors, it also clears normal T/NK cells in the patient's body, causing the patient to be in a state of immunodeficiency during treatment and unable to survive in a normal environment; in addition, if CD5-knocked-out T cells If it exists for a long time in the patient's body, it can easily lead to autoimmune diseases. Therefore, there is a need to control the presence or proliferation of CD5-CD7 universal CAR-T in subjects, which can be achieved by using molecular switches.
  • the currently commonly used CAR-T switch molecule is EGFR, and CAR-T cells can be cleared through the anti-EGFR antibody cetuximab, but this method requires the participation of immune cells such as NK to be realized. Since the inventor's CD5-CD7 UCAR-T will also kill NK cells in the patient's body, the EGFR switch is no longer applicable to CD5-CD7 UCAR-T.
  • CAR-T switch molecules that do not rely on immune cells usually use small molecule drugs to control a certain element in CAR-T to start or shut down (kill) CAR-T cells
  • small molecule drugs to control a certain element in CAR-T to start or shut down (kill) CAR-T cells
  • Ali Can Sahillioglu Ton and N Schumacher in 2022 Article 8 published in Current Opinion in Immunology summarizes the CAR-T small molecule drug switches discovered in current research. According to the mechanism of action, they can be roughly divided into 7 categories, as shown in Figure 3, including suicide genes, transcriptional regulation, and stability control. wait.
  • HSV-TK is the abbreviation of Herpes Simplex Virus Thymidine Kinase (Human Herpes Simplex Virus Thymidine Kinase). Its substrate is the small molecule drug GCV (Ganciclovir, ganciclovir). GCV was launched by the American Syntex Company and approved for marketing in 1988. , the drug of choice for the treatment of cytomegalovirus infection.
  • GCV Herpes Simplex Virus Thymidine Kinase
  • GCV Ganciclovir, ganciclovir
  • the principle of HSV-TK combined with GCV to treat cytomegalovirus infection is: HSV-TK can efficiently bind to GCV and monophosphorylate it, and then intracellular kinases diphosphorylate and triphosphorylate it.
  • GCV The structure of GCV is very similar to the nucleosides in cells, so it will competitively bind to DNA polymerase or break the ratio of the four nucleosides in cells to inhibit DNA synthesis. When DNA synthesis is blocked, cells will gradually undergo apoptosis.
  • the inventors targeted the TRAC/B2M/CD5/CD7/CIITA gene and used the knockout function of the CRISPR/Cas9 system to successfully screen out sgRNAs with high knockout efficiency and low off-target probability.
  • the inventors targeted the TRBC/B2M/CD5/CD7/CIITA gene and used the base editing function of the CRISPR/Cas9 system to successfully screen out sgRNAs that can achieve gene silencing by introducing stop codons in important regions of the gene. .
  • the inventors successfully achieved simultaneous high-efficiency knockout of multiple genes through sgRNA screening and optimization of the electroporation process (the knockout efficiency of each gene is about 90%).
  • the inventors successfully prepared CD5-CD7 UCAR-T with strong tumor killing function and strong amplification ability in vitro/in vivo.
  • HSV-TK combined with GCV small molecule drugs to successfully eliminate UCAR-T cells efficiently in the body.
  • CD5-CD7 CAR-T differs from autologous CD5-CD7 CAR-T and autologous CD5-CD7 CAR-T: 1) The genes that are knocked out are different: autologous CD5-CD7 CAR-T only needs to knock out the CD5 and CD7 genes, while universal In addition to knocking out CD5 and CD7 genes, CD5-CD7 CAR-T also needs to knock out TCR and HLA-related genes; 2) During the preparation process of universal CAR-T, UCAR-T may need to be purified to express TCR and HLA.
  • CAR-T cells with HLA complexes are screened out to ensure that the UCAR-T cells entering the patient's body do not express TCR and HLA complexes at all to avoid causing HvGD or GvHD toxic side effects; 3)
  • the indications are not exactly the same: autologous CD5-CD7 CAR-T is only applicable to T-cell lymphomas where tumor cells have not invaded the peripheral blood, while the universal CD5-CD7 CAR-T is applicable to all types of T-cell malignancies.
  • PBMC Resuscitate frozen healthy donor (specific information confidential) PBMC, a total of 1.0 ⁇ 10 8 cells per tube, quickly thaw and resuspend in 8 ml of preheated Rinsing buffer, take a small amount of cell suspension for cell counting. Centrifuge the PBMC suspension at 400g ( ⁇ 8 ⁇ 8) for 10 minutes. After centrifugation, discard the supernatant, add 20ul/10 7 CD3 microbeads, mix well, and incubate in a 4°C refrigerator for 20 minutes. During this period, flick the tube wall several times every 10 minutes to avoid cell precipitation.
  • Rinsing buffer After the incubation, add Rinsing buffer, rinse once, centrifuge (400g 10min ⁇ 8 ⁇ 8), and resuspend the cells in 500 ⁇ l Rinsing buffer. At the same time, place the LS sorting column on the Miltenyi magnetic sorting stand. After rinsing once with 2ml Rinsing buffer, add 500 ⁇ l of cell suspension. After the cell suspension is exhausted, add 2ml of Rinsing buffer twice. on the LS column. Use 5 mL of Rinsing buffer to wash out the target cells from the LS column and collect them. After making appropriate dilutions, count the target cells. Take about 1 ⁇ 10 5 cells to determine the purity of the sorted T cells by flow cytometry.
  • electroporation can be performed 24 hours after activation.
  • Cells were collected by centrifugation tube, place it on a magnetic stand to remove Dynabeads, repeat 3 times, and then centrifuge the cells (300g for 15 minutes, up 8, down 8); after completion, discard the supernatant, resuspend the cells together with an appropriate amount of compound electrolyte, and take the cells Count; prepare a corresponding amount of RNP (a complex of Cas9 protein and sgRNA) according to the cell counting results, and incubate at 37°C for more than 10 minutes; at the same time, centrifuge the cells again, and then resuspend the cells with a corresponding amount of electroporation buffer, and add the incubated RNP, mix gently and then add it to the electroporation cup of the Lonza electroporation instrument.
  • RNP a complex of Cas9 protein and sgRNA
  • Resuscitate frozen healthy donor PBMC Resuscitate frozen healthy donor PBMC, a total of 1.0 ⁇ 10 8 cells per tube, quickly thaw and resuspend in 8 ml of preheated Rinsing buffer, and take a small amount of cell suspension for cell counting.
  • Centrifuge the PBMC suspension at 400g ( ⁇ 8 ⁇ 8) for 10 minutes; after centrifugation, discard the supernatant and add Rinsing buffer (40ul/10 ⁇ 7cells) and Pan T Cell Biotin-Antibody Cocktail (10ul/10 ⁇ 7cells) , use a pipette tip to mix gently, then place it in a 4°C refrigerator and incubate for 10 minutes, flicking the tube wall once to avoid cell precipitation; then add Rinsing buffer (30ul/10 ⁇ 7cells) and Pan T Cell MicroBead Cocktail (20ul/ 10 ⁇ 7 cells), gently pipet and mix with the pipette tip, then place it in a 4°C refrigerator and incubate for 15 minutes.
  • lentiviral transduction of CAR was performed. Conduct viability testing and cell counting on the cell suspension. Add the corresponding amount of lentivirus according to the cell counting results, with an MOI of 3, then add 100x lentiboost, mix gently, and continue culturing in a 37°C incubator. After 24 hours, the medium was changed to remove the virus, and fresh medium was used to continue culturing T culture cells at a density of 1M/ml.
  • the cells with successful knockout of TRAC and B2M no longer express the corresponding proteins. Therefore, the cells can be negatively selected and the cells with successful knockout of TRAC and B2M can be isolated and continued to be cultured.
  • the specific steps are as follows: Collect cells and centrifuge at room temperature (400g for 15 minutes, rise 8 and drop 8).
  • Rinsing Buffer 80ul/10 ⁇ 7cells
  • FITC-B2M antibody (1ul/10 ⁇ 6cells)
  • T cell complete culture medium in advance, add 100x PE/Cy7 mouse anti-human CD107a antibody and 1000x monensin to the culture medium; count UCART cells and various tumor cells at a ratio of 1:1 (UCAR -T cells are counted according to CAR-positive cells) and centrifuged (generally CAR-positive cells are above 0.1M), then resuspended in culture medium containing CD107a antibodies and monensin, inoculated in a 96-well plate, and placed in a cell culture incubator Incubate for 4 hours in medium (37°C, 5% CO 2 ); then perform FACS staining on the cells, usually with CD8 and EGFR antibodies, and calculate the proportion of CD107a-positive cells under CD8-positive and CAR-positive T cells through FACS detection.
  • UCAR-T cells and a variety of targeted and non-targeted tumor cells expressing luciferase are inoculated at different effect-to-target ratios (UCART cells are calculated as CAR-positive cells, and the amount of fixed tumor cells is fixed)
  • UCART cells are calculated as CAR-positive cells, and the amount of fixed tumor cells is fixed
  • a cell culture incubator takes half of the cell suspension into an opaque white-bottomed 96-well plate, add 10x luciferase substrate, mix well, react at room temperature for 10 minutes, and then use the machine to detect luciferase. Fluorescence value indirectly reflects the number of surviving tumor cells, which in turn reflects the tumor-killing function of UCAR-T cells.
  • UCAR-T cells To count UCAR-T cells, centrifuge the cells at 8M. Discard the supernatant after completion, resuspend in 8ml of fresh T cell culture medium, and divide evenly into 8 wells of a 12-well plate, 1ml per well, and then two wells are In one group, DMSO, 0.3ug/ml, 1ug/ml, and 3ug/ml GCV were added respectively. The cells were cultured in an incubator. Then, changes in CAR-positive cells were monitored by counting and FACS detection every 3 days. At the same time, cell pellets were collected every 3 days to extract genomes, and changes in CAR molecules at the DNA level were detected by fluorescent probe qPCR.
  • the RNP and dsODN of the sgRNA to be detected are introduced into activated T cells by electroporation. 5-7 days after electroporation, the genome with dsODN integration is extracted; the genome is fragmented into an average of 500bp fragments by Covaris S220 instrument, using 0.8x Ampure XP beads purify and recover gene fragments for reuse Ultra TM II DNA Library Prep Kit for The kit performs end repair on the purified genome fragment and adds A. Use Ultra TM II DNA Library Prep Kit for The kit connects the A-added DNA fragments to the linker sequence.
  • Each sample is connected to a different linker, and then purified using 0.8x Ampure Then use the ABclonal Rapid DNA Lib Prep Kit to connect the universal P5 and P7Y adapters, and the products can be used for next-generation sequencing after purification and recovery.
  • the sequencing results By analyzing the sequencing results and comparing the genome sequence between the linker sequence and dsODN sequence with the sgRNA sequence, the off-target site information and off-target probability can be obtained.
  • sgRNA can be designed through many websites, such as CHOPCHOP ( https://chopchop.cbu.uib.no/ ), CRIS Pick ( https://portals.broadinstitute.org/gppx/crispick/public ), GUIDES ( http:/ /guides.sanjanalab.or g/#/ ), CRISPOR( http://crispor.tefor.net/ ), IDT( https://sg.idtdna.com/site/order/designtool/index/ CRISPR_SEQUENCE ), etc., These websites can predict the efficiency of On-target and Off-target.
  • sgRNA prediction and synthesis Input the target sequence of the target gene into the above-mentioned sgRNA prediction website to generate many candidate sgRNAs. Select an appropriate amount of sgRNA sequence based on the ranking of On-target and Off-target, and add the sgRNA target sequence ( 20nt or so) and tell the third-party company to synthesize full-length sgRNA.
  • Verification of editing efficiency of candidate sgRNA After the candidate sgRNA is synthesized, the complex RNP (ribonucleoprotein) of Cas9 protein and sgRNA can be directly introduced into the target cells through electroporation.
  • the editing efficiency at the gene level can be detected by PCR and sequencing 24h-48h after electroporation. , changes in protein levels can be detected by FACS after 72 hours.
  • Example 1 Using CRISPR/Cas9 technology to knock out TRAC/TRBC, B2M, CIITA, CD5, and CD7 antigens on the surface of T cells
  • the inventor obtained many possible sgRNA information through commonly used sgRNA design websites such as CRISPick, CRISPOR and IDT, and then selected 7 candidate sgRNAs after comprehensively considering target and off-target information (Figure 4A). Inform the technical service company (Nanjing GenScript Biotechnology Co., Ltd.) of the target sequences of the seven candidate sgRNAs and let them synthesize full-length sgRNA (targeting sequence + backbone sequence).
  • sgRNA After sgRNA is synthesized, use nuclease-free water to dissolve it to 100-500 pmol/ ⁇ l, then mix it with Cas9 protein in different amounts and incubate at 37°C for more than 10 minutes, and then use a lonza electrotransfer instrument to transfer the Cas9 and sgRNA The complex was introduced into T cells, and after 6 days of cell culture, FACS was used to detect the expression of the TCR complex, thereby reflecting the knockout efficiency of TRAC by different sgRNAs.
  • the inventor used the Guide-seq method to perform off-target detection on three sgRNAs, A-02, A-06 and A-XL. The results are shown in Figure 4C-E. One of the two detection results for A-XL appeared. As for off-target sites with a higher probability, a test result of A-02 shows that there are also off-target sites with a high probability. The off-target probability of A-06 is slightly lower, so this version of UCAR-T may tend to use A-06 in the future. This sgRNA.
  • the candidate sgRNA information was selected by the inventor after prediction through CRISPick, CRISPOR, IDT, CHOPCHPOP and GUIDES.
  • the Cas9 protein and sgRNA complex was also introduced into T cells using the electroporation method. An appropriate amount of cells were taken out 8 days after electroporation, and the expression of B2M was detected using flow cytometry to reflect the knockout efficiency of B2M by different candidate sgRNAs.
  • B-03 has the highest knockout efficiency (more than 90%)
  • B-10, B-13 and B-XL have comparable knockout efficiencies (more than 80%), while the knockout efficiency of B-01 The efficiency is too low (around 50%).
  • the inventor double-knocked a single B2M-sgRNA together with the tested TRAC-sgRNA (A-XL) in order to screen out B2M-sgRNA with a better TRAC-B2M double-knocking efficiency.
  • the inventor mixed the Cas9 protein with TRAC-sgRNA and B2M-sgRNA and incubated them for 10 minutes, then introduced them into T cells by electroporation. Eight days after electroporation, an appropriate amount of cells were taken out and FACS was used to detect the expression of B2M and TCR complexes.
  • the results are shown in Figure 6B.
  • the sequence with the highest off-target probability in the two detection results of B-13 is the same, indicating that this site is extremely likely to Missing targets may occur; and B-03’s
  • the sequences with the highest off-target probability measured in the two detection results were different.
  • One of the highest off-target probability was 3%, while the other was only 0.01%, indicating that the B-03 sgRNA had a low off-target probability.
  • FIGS. 7A and 7B show the sgRNA sequence information tested during the first screening and the corresponding knockout efficiency results.
  • the knockout efficiency of sgRNA1 and sgRNA3 is good, but off-target analysis has not been performed on the selected sgRNA before.
  • the inventors once again designed 5 sgRNAs to test the knockout efficiency and off-target rate together with sgRNA1 and sgRNA3.
  • Figure 7C is the candidate sgRNA information selected by the inventor after comprehensive consideration through predictions from CRISPick, CRISPOR, IDT, CHOPCHPOP and GUIDES during the second screening.
  • C-01 and C-03 are sgRNA1 and sgRNA in Figure 7A sgRNA3.
  • the CD7 sgRNA used in the present invention comes from reported literature 10 , and the inventors performed off-target detection on it. As shown in Figure 9, in the off-target detection of an experiment, it was found that 3 sequences appeared more frequently, but two of them also appeared in the control group that did not use sgRNA, and the frequency of occurrence in both Basically consistent, so it can be judged as a false positive; compared with the sgRNA sequence, the second most frequently occurring sequence has a larger number of mutated bases and is closer to NGG, so it is less likely to be off-target. To sum up, , the probability of CD7 sgRNA off-target is extremely low.
  • the inventor selected 5 sgRNAs for knockout efficiency testing. The results are shown in Figure 10A. F-05 has the best knockout efficiency.
  • Figure 12C shows the knockout results of the KO unconverted group tested 4 days after the knockout when the inventor prepared CD5-CD7 UCAR-T for the second time.
  • the TRAC/B2M double knockout efficiency was 86.7%.
  • the CD5/CD7 double knockout efficiency is 90.6%.
  • the knockout efficiency is already very high.
  • UCAR-T will also kill some CD5 or CD7 positive cells, so the double knockout efficiency of TRAC/B2M and CD5/CD7 is in It will be even higher in the UCAR-T group.
  • UCAR-T In order to give UCAR-T a better survival advantage in the body, the inventors also prepared another version of CD5-CD7 UCAR-T, that is, on the basis of knocking out TRAC, B2M, CD5, and CD7, the CIITA gene was also knocked out. UCAR-T does not express HLA-II class molecules after the CIITA gene, which can prevent CD4+ T cells in the host from clearing UCAR-T.
  • the inventor Before preparing the second version of CD5-CD7 UCAR-T, the inventor first conducted a preliminary experiment to simultaneously knock out five genes, TRAC, B2M, CIITA, CD5, and CD7, on a Cellertrix electroporation instrument.
  • the RNP dosage and knockout results are as follows: As shown in Figure 13A-B, FACS detected the expression of each protein 5 days after knockout. The results showed that the TRAC/B2M double knockout efficiency was 69.9%, the TRAC/CIITA double knockout efficiency was 62.8%, and the CD5/CD7 double knockout efficiency was 85.3 %. According to the grouping, the double knockout efficiency of TRAC/B2M and CD5/CD7 is acceptable, but the double knockout efficiency of TRAC/CIITA is slightly lower.
  • the inventor increased the RNP dosage of B2M and CIITA genes when preparing the second version of CD5-CD7 UCAR-T, as shown in Figure 13C-D.
  • FACS detection of each protein 4 days after knockout The expression status of the results showed that the TRAC/B2M double knockout efficiency was 87.1%, the TRAC/CIITA double knockout efficiency was 85.2%, and the CD5/CD7 double knockout efficiency was 90.3%.
  • the knockout efficiency of each gene was improved compared with the pre-experiment. promote.
  • nCBE3 or nCBE4 protein is a cytosine deaminase (ABOBEC) fused to the N-terminus of the Nikase-Cas9 protein (which only cuts the target chain) and one or two uracil glycosylase inhibitors fused to the C-terminus.
  • ABOBEC cytosine deaminase
  • UMI cytosine
  • UGI uracil
  • nCBE3 or nCBE4 can be used to mutate CAA, CAG, and CGA on the sense strand of the exon region to TAA, TAG, and TGA, or mutate CCA on the antisense strand to TTA (the corresponding sense strand is TAA), and introduce it in advance.
  • the stop codon disrupts protein expression.
  • the inventors designed multiple candidate sgRNAs for TRBC, B2M, CD5, CD7, and CIITA genes.
  • the sequence information is shown in Figure 14.
  • the CD7 sgRNA has been reported in the literature and is very efficient 11 , so CD7 was not tested. Perform more sgRNA screenings.
  • the base editing sgRNA efficiency test method is the same as the knockout sgRNA.
  • the protein expression is checked by flow cytometry 5 days after electroporation.
  • BE-A02 and BE-A03 have the highest efficiency and are equivalent.
  • BE-A02 has been reported in the literature 11
  • the inventors chose BE-A03 as the sgRNA for subsequent use. .
  • lentiviral transduction of CAR is performed 48 hours after T cell activation.
  • 24 hours after viral transduction After changing the medium, the proportion of CAR-positive cells can be detected 3 days later.
  • the proportion of CAR-positive cells can be reflected by FACS detection of the expression of EGFR or CAR in the cells.
  • Figure 19 shows the detection results of the proportion of CAR-positive cells of CD5-CD7 UCAR-T prepared twice.
  • the expression of EGFR and CAR in the cells was detected by Anti-EGFR-PE and FITC-coupled CD5 and CD7 antigens respectively.
  • the positive rate of EGFR is generally higher than the positive rate of CD5 and CD7 CAR. This may be due to the endocytosis of CAR after UCAR-T kills CD5 and CD7 non-knockout cells in the early stage. In the later stage, such as when tested on day 8
  • the proportion of CAR-positive cells represented by the three will be relatively consistent.
  • CD5-CD7 UCAR-T Due to the existence of self-activation of CAR-positive cells in CD5-CD7 UCAR-T (killing CD5 or CD7 T that has not been knocked out), the proportion of CAR-positive cells will increase with the increase of culture days, from day3 to day8, the proportion of CAR-positive cells It will approximately double.
  • UCAR-T cells The most important thing for UCAR-T cells is to avoid immune rejection, so the finished UCAR-T cells that are infused back into the patient must usually ensure that they do not express TCR and HLA-I complexes, otherwise severe GvHD or HvGD will occur. Therefore, When the inventor prepares CD5-CD7 UCAR-T, he usually performs negative selection of TRAC and B2M on UCAR-T cells around the sixth day. For specific steps, please refer to the "TRAC/B2M negative selection" in the general method above.
  • Figure 20 shows the results of TRAC/B2M negative selection during the two preparations of CD5-CD7 UCAR-T. It can be seen from the figure that the input cells before negative selection will have a small amount of expression of TCR and B2M, while after negative selection The negative cells (negative) will almost never be positive for TCR and B2M. Generally, the ratio of TRAC/B2M double negative cells must be above 99% to be considered qualified.
  • the function of CAR-T cells can be achieved by co-incubating CAR-T cells and tumor target cells in vitro. After 4-6 hours, the CD107a release of CAR-T cells can be detected by FACS or the number of tumor cells can be detected 24h-48h. To reflect whether the killing function of CAR-T cells is good.
  • CD107a also known as LAMP-1
  • LAMP-1 is a protein released by CD8+ toxic T cells and NK cells during the killing of target cells. Therefore, whether the CAR-T cells have killing function can be reflected by detecting the CD107a release of CAR-T cells.
  • the inventor will co-incubate CD5-CD7 UCAR-T with a variety of CD5 and CD7 positive tumor cells in a cytokine-free T cell culture medium.
  • the effect-to-target ratio is calculated as 1:1 based on the CAR-positive cells.
  • the total After incubation for 4-6 hours, collect cells for FACS staining, and use flow cytometry to analyze the proportion of CD107a-positive cells among CD8 and CAR double-positive cells. The higher the proportion, the stronger the UCAR-T is activated by the target cells, and the secreted The more tumor killing factors there are, but the higher proportion of CD107a-positive cells does not mean that it has a stronger ability to kill tumors. It can only be initially believed that UCAR-T has the ability to kill tumor cells.
  • CD5-CD7 UCAR-T can be activated by different tumor target cells to release CD107a, while KO-T will not be activated because it does not have a CAR.
  • Figures 21B and 21C show the proportion of CD107a-positive cells in CD8 and CAR double-positive cells after twice-prepared CD5-CD7 UCAR-T was incubated with different tumor cells for 4 hours. CD107a-positive cells were stimulated by different tumor cells. The proportions are different.
  • the proportion of CD107a-positive cells after stimulation with CD5- or CD7-positive tumor cells is about 50%, but the negative target cell group (Raji) and no tumor group also have about 20% CD107a-positive cells, which may be due to CD5-CD7 UCAR-T caused by self-activation.
  • the tumor-killing function of the prepared UCAR-T can be preliminarily identified in vitro.
  • UCAR-T and tumor target cells stably expressing luciferase protein
  • the expression of luciferase protein in cells after co-culture reflects the survival of tumor cells and then determines the tumor-killing function of UCAR-T.
  • Figure 22A-C is the tumor killing situation of CD5-CD7 UCAR-T prepared for the first time.
  • Figure 22A and 22B are the tumor killing test results of UCAR-T and KO-T cells on the 7th day of the preparation period. UCAR- T and KO-T were tested after being co-cultured with different tumor target cells for 24 hours in three effect-to-target ratios.
  • KO-T cannot kill tumor cells while UCAR-T can kill tumor cells very well; in Under the low-efficiency target ratio (0.2:1), the ability of CD5-CD7 UCAR-T to kill double-positive and single-positive target cells is in the order of CD5+/CD7+>CD5+/CD7->CD5-/CD7+, that is, the tumor killing ability of dual-CAR-T The ability is stronger than that of single CAR-T;
  • Figure 22C is the result of testing the tumor killing function of UCAR-T 24 hours after cryopreservation and recovery. From the figure, it can be seen that resuscitation of cells after cryopreservation does not affect its tumor killing function.
  • Figure 22D-F are the tumor killing results of the CD5-CD7 UCAR-T prepared for the second time.
  • Figure 22D and 22E are the tumor killing test results of UCAR-T and KO-T cells on the 10th day of the preparation period, respectively. Tumor cells After co-culture with UCAR-T for 24 hours, only tumor cells with a low-efficiency target ratio remain, but they are almost not killed after co-culture with KO-T for 24 hours;
  • Figure 22F shows UCAR-T cells 24 hours after cryopreservation and recovery The tumor killing results were consistent with those before cryopreservation.
  • the prepared UCAR-T generally needs to be tested for various indicators before cryopreservation. Whether there is any abnormality in the final state of UCAR-T can be determined through indicators such as cell activation status, typing, and depletion.
  • Figure 23A-B shows the CD3/CD4/CD8 cell ratio and activation status of twice-prepared CD5-CD7 UCAR-T cells before cryopreservation.
  • the cells in the preparation in Figure 23A were not negative-selected by TRAC/B2M, so the TCR complex of the CD3 pointer is still expressed in a small amount of cells, while the cells in the preparation in Figure 23B were negative-selected by TRAC/B2M, so the CD3 staining was complete.
  • Figure 23C-D shows the cell typing of twice prepared CD5-CD7 UCAR-T cells before cryopreservation.
  • CCR7 is the maker of the early state of T cells, so the higher the proportion of CCR7-positive cells in the final state of UCAR-T cells, the better the expansion of UCAR-T will theoretically be after it is infused back into the patient.
  • the UCAR-T cells in Figure 23C were cryopreserved on day 10, while the UCAR-T cells in Figure 22D were cryopreserved on day 12. The two preparation processes were the same. It can be seen from the figure that the proportion of CCR7-positive cells among the cells cryopreserved on day 10 Higher than those frozen on day 12.
  • Figure 24A-D shows the depletion and early regulation status of the twice-prepared CD5-CD7 UCAR-T cells before cryopreservation. It can be seen from Figure 24A and Figure 24B that the expression levels of the two depletion makers, LAG3 and PD1, are both relatively high. Low, indicating that there are very few exhausted cells in the final state of CD5-CD7 UCAR-T cells; in Figure 24C and Figure 24D, double positivity of PI and Annexin V indicates that the cells are in a late apoptotic state, while PI negativity and Annexin V positivity indicate that The cells are in an early stage of apoptosis. It can be seen from the figure that there are very few cells undergoing apoptosis in the final state of CD5-CD7 UCAR-T cells.
  • the CD5-CD7 UCAR-T cells prepared by the inventor are of good quality.
  • the inventor also conducted cell component identification on the final state of UCAR-T.
  • CD56-positive cells such as Figure 25A-B
  • CD56 is generally considered to be the maker of NK cells, but this part of the cells in UCAR-T does not express another maker of NK, CD16, and there are no CD4 and CD8 double-negative cells in the final UCAR-T cells.
  • these CD56-positive cells in UCAR-T cells are determined to be NKT cells.
  • CAR-positive cells in NKT cells so this part of CAR-positive NKT cells can theoretically also perform tumor killing functions.
  • the proportion of CD56-positive cells in different batches of UCAR-T cells varies, ranging from about 10% to 20%.
  • Figure 25 A and B respectively show the proportion of CD56-positive cells in UCAR-T prepared twice. Proportion.
  • Figure 26 shows the results of a clinical test of CD5-CD7 UCAR-T (EGFR switch).
  • the subject was an acute T lymphocytic leukemia patient, male, 36 years old, weighing 75kg.
  • the clear treatment given before UCAR-T infusion is: fludarabine 30mg/ m2 and cyclophosphamide 300mg/ m2 for 5 days, then fludarabine 30mg/ m2 and 70mg/m2 2 melphalan was treated for another 1 day, and the patient was allowed to rest for 3 days without treatment and then infused with CD5-CD7 UCAR-T cells at a dose (CAR-positive cells) greater than 3.6e6/kg. After that, samples were taken every day to detect the copy number and CAR copy number in the patient's peripheral blood. changes in lymphocytes.
  • the inventor's EGFR switch version of CD5-CD7 UCAR-T can serve T-cell lymphoma patients who are unable to undergo transplantation due to high tumor burden.
  • the inventors replaced and modified the switch of UCAR-T, in order to achieve the goal of completely curing T-cell lymphoma patients using only CD5-CD7 UCAR-T.
  • Figure 27A shows the co-knockout efficiency of the four genes TRAC/B2M/CD5/CD7 and the transformation efficiency of CAR.
  • Figure 27C shows is the typing of cells, and Figure 27D shows the components of cells.
  • TCR-positive cells are more fully activated than TCR-negative cells. Therefore, TCR Positive cells will have a growth advantage. If the negative selection of TCR cells is not thorough, the proportion of remaining TCR-positive cells will gradually increase in the later stage (Figure 27C).
  • the resting electroporation process uses Pan-T negative selection to obtain T cells on day 0 to exclude CD56-positive cells as much as possible. Therefore, the proportion of CD56-positive cells in the finished cells obtained by the resting electroporation process will be lower than that using CD3 magnetic Bead-positive cells obtained by activated electroporation process (Figure 27E).
  • the proportion of CCR7-positive cells was related to the number of days the cells were cultured in vitro. The longer the culture days, the lower the proportion of CCR7-positive cells (Figure 27D).
  • the inventor used the same process to prepare two versions of UCAR-T in the same batch, and then performed cryopreservation and recovery. After 24 hours, it was incubated with different tumor cells at different effect-to-target ratios for 24 hours, and the tumor killing ability of UCAR-T was reflected by detecting the luciferase fluorescence value of the tumor cells. The results are shown in Figure 28.
  • the HSV-TK and EGFR versions of CD5-CD7 UCAR-T prepared by the same process have the same killing ability against different tumor cells, even if different switch molecules are used to control the tumor killing function of the CAR itself. No impact.
  • HSV-TK can efficiently bind to GCV and monophosphorylate it, and then intracellular kinases diphosphorylate and triphosphorylate it.
  • the structure of triphosphorylated GCV is very similar to intracellular nucleosides, so it will Competitively binds to DNA polymerase or breaks the ratio of the four nucleosides in cells to inhibit DNA synthesis. Cells will gradually undergo apoptosis after DNA synthesis is blocked.
  • GCV does not affect the EGFR version of UCAR-T. T cells grow ( Figure 29D-F) and specifically kill only TK-containing, CAR-positive cells (Figure 29D-F) 29A-C).
  • the higher the concentration of GCV the faster and more CAR-positive cells will die.
  • concentration of GCV the faster and more CAR-positive cells will die.
  • all CAR-positive cells will slowly die. Lose.
  • FIG. 30A-B There are 30 mice in total. Each mouse was inoculated with 5x10 5 CCRF tumor cells on the 7th day before UCAR-T infusion, and then randomly divided into 5 groups, 6 mice in each group. On the 7th day after tumor cell inoculation, 2x10 6 HSV-TK version of UCAR-T cells (per mouse) were given to 2 groups of mice, and 2x10 6 EGFR version of UCAR-T was given to the other 2 groups. cells, and the remaining group was injected with a corresponding volume of PBS as a control.
  • mice in the HSV-TK version and EGFR version groups were infused with 50 mg/kg GCV. , once a day for a total of 3 days, and then the changes in tumor cells in the mice were observed through mouse imaging at different time points.
  • mice in the group without UCAR-T cells had more tumor cells in their bodies on day 4 (after UCAR-T infusion), while the mice in the UCAR-T cell group had more tumor cells in their bodies. There were very few tumor cells in the mice, and there was no significant difference between the mice injected with the EGFR version and the HSV-TK version group, indicating that the tumor-killing functions of the two versions of CD5-CD7UCAR-T were both good and indistinguishable.
  • mice in the HSV-TK version group had the most tumor cells compared to the other three groups, indicating that GCV had an effect on UCAR-T cells in this group of mice, while the other three groups There was little difference in tumor cells in mice, indicating that GCV only specifically kills UCAR-T cells containing HSV-TK.
  • UCAR-T cells were injected relatively late (normally, UCAR-T cells are injected into CAR-T cells 1-3 days after tumor cells are inoculated), so UCAR-T cells may be injected into tumor cells.
  • the number of UCAR-T cells has been greater than that of UCAR-T cells, resulting in UCAR-T failing to kill all tumor cells.
  • Figure 30B The results of the first 8 days ( Figure 30B) and the load of tumor cells during the entire experimental cycle ( Figure 30C), The HSV-TK version of CD5-CD7 UCAR-T and GCV are both very effective.
  • Scheme 1 Method for preparing universal chimeric antigen receptor T cells (UCAR-T), including:
  • Option 2 The method as described in Option 1, wherein step 1) further includes knocking out the B2M gene of the T cell.
  • Option 3 The method as described in Scheme 1 or 2, wherein the target sequence of the sgRNA used for knocking out the TRAC gene is selected from the sequences shown in SEQ ID NO: 1-7 and any combination thereof.
  • Scheme 4 The method according to any one of Schemes 1-3, wherein the target sequence of the sgRNA used for knocking out the B2M gene is selected from the sequences shown in SEQ ID NO: 8, 9, 11, 12 and their random combination.
  • Option 5 The method as described in any one of Schemes 1-4, wherein the knockout of the B2M gene uses a combination of two sgRNAs, and the target sequences of the two sgRNAs are SEQ ID NO: 9 and 11 respectively. display sequence.
  • Option 6 The method according to any one of Schemes 1-5, wherein in step 1), the sgRNA for the TRAC gene knockout and the B2M gene knockout are mixed with the Cas9 protein and then performed simultaneously. Knockout of the TRAC gene and the B2M gene in the T cells, and the target sequence of the sgRNA for the TRAC gene knockout is the sequence shown in SEQ ID NO: 7.
  • Scheme 7 The method according to any one of Schemes 1-6, wherein the target sequence of the sgRNA used to knock out the CD5 gene is selected from the sequences shown in SEQ ID NO: 13, 14, 16, 17, 18, 19 and any combination thereof.
  • Scheme 8 The method according to any one of Schemes 1-7, wherein the target sequence of the sgRNA used for knocking out the CD7 gene is the sequence shown in SEQ ID NO: 20.
  • Embodiment 9 The method according to any one of Embodiments 1-8, wherein the knockout of the TRAC gene, the CD5 gene and the CD7 gene is performed simultaneously, and the target of the sgRNA for knocking out the TRAC gene is The sequence is the sequence shown in SEQ ID NO: 7; the target sequence of the sgRNA used to knock out the CD5 gene is the sequence shown in SEQ ID NO: 13; and the target sequence of the sgRNA used to knock out the CD7 gene is SEQ ID NO: 20 shows the sequence.
  • Scheme 10 The method according to any one of Schemes 1-9, wherein the knockout of the TRAC gene, the B2M gene, the CD5 gene and the CD7 gene is performed simultaneously for knocking out the TRAC
  • the target sequence of the sgRNA of the gene is the sequence shown in SEQ ID NO: 7; the target sequences of the two sgRNAs used to knock out the B2M gene are the series shown in SEQ ID NO: 9 and 11 respectively; used to knock out the
  • the target sequence of the sgRNA of the CD5 gene is the sequence shown in SEQ ID NO: 13; and the target sequence of the sgRNA used to knock out the CD7 gene is the sequence shown in SEQ ID NO: 20.
  • Scheme 11 The method according to any one of Schemes 1-10, wherein the knockout of the TRAC gene, the B2M gene, the CD5 gene and the CD7 gene is carried out simultaneously for carrying out the knockout
  • the components in a 20 ⁇ L system include:
  • An RNP complex formed by no less than 30 pmol of Cas9 and no less than 45 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 7;
  • An RNP complex formed by no less than 20 pmol of Cas9 and no less than 30 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 9;
  • An RNP complex formed by no less than 20 pmol of Cas9 and no less than 30 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 11;
  • An RNP complex formed by no less than 40 pmol of Cas9 and no less than 120 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 13;
  • An RNP complex formed by no less than 40 pmol of Cas9 and no less than 120 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 20.
  • Scheme 12 The method according to any one of Schemes 1-11, wherein the knockout of the TRAC gene, the B2M gene, the CD5 gene and the CD7 gene is performed simultaneously for carrying out the knockout
  • the components in a 20 ⁇ L system include:
  • RNP complex formed by 30 pmol of Cas9 and 45 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 7;
  • RNP complex formed by 20pmol of Cas9 and 30pmol of sgRNA targeting the sequence shown in SEQ ID NO: 9;
  • RNP complex formed by 20 pmol of Cas9 and 30 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 11;
  • RNP complex formed by 80 pmol of Cas9 and 150 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 13;
  • RNP complex formed by 40 pmol of Cas9 and 120 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 20.
  • Scheme 13 The method according to any one of Schemes 1-12, wherein step 1) further includes knocking out the CIITA gene of the T cell.
  • Scheme 14 The method according to any one of Schemes 1-13, wherein the target sequence of the sgRNA used for knockout of the CIITA gene is the sequence shown in SEQ ID NO: 25.
  • Scheme 15 The method according to any one of Schemes 1-14, wherein the knockout of the TRAC gene, the B2M gene, the CD5 gene, the CD7 gene and the CIITA gene is carried out simultaneously, for The components for the knockout are proportionally included in a 20 ⁇ L system:
  • An RNP complex formed by no less than 30 pmol of Cas9 and no less than 45 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 7;
  • An RNP complex formed by no less than 20 pmol of Cas9 and no less than 30 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 9;
  • An RNP complex formed by no less than 20 pmol of Cas9 and no less than 30 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 11;
  • An RNP complex formed by no less than 80 pmol of Cas9 and no less than 150 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 13;
  • An RNP complex formed by no less than 40 pmol of Cas9 and no less than 120 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 20;
  • An RNP complex formed by no less than 40 pmol of Cas9 and no less than 60 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 25.
  • Scheme 16 The method according to any one of Schemes 1-15, wherein the knockout of the TRAC gene, the B2M gene, the CD5 gene, the CD7 gene and the CIITA gene is carried out simultaneously, for The components for the knockout are proportionally included in a 20 ⁇ L system:
  • RNP complex formed by 30 pmol of Cas9 and 45 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 7;
  • RNP complex formed by 25 pmol of Cas9 and 40 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 9;
  • RNP complex formed by 25 pmol of Cas9 and 40 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 11;
  • RNP complex formed by 80pmol of Cas9 and 150pmol of sgRNA targeting the sequence shown in SEQ ID NO: 13;
  • RNP complex formed by 40 pmol of Cas9 and 120 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 20;
  • Scheme 17 Method for preparing universal chimeric antigen receptor T cells (UCAR-T), including:
  • Embodiment 18 The method as described in Embodiment 17, wherein step 1) further includes knocking out the B2M gene of the T cell.
  • Option 19 The method as described in Option 17 or 18, wherein step 1) further includes knocking out the CIITA gene of the T cell.
  • Scheme 20 The method according to any one of Schemes 17-19, wherein the target sequence of the sgRNA used for knocking out the TRAC gene is the sequence shown in SEQ ID NO: 26.
  • Scheme 21 The method according to any one of Schemes 17-20, wherein the target sequence of the sgRNA used for knocking out the TRBC gene is selected from the sequence shown in any one of SEQ ID NO: 27-31 and any of the sequences thereof combination.
  • Scheme 22 The method according to any one of Schemes 17-21, wherein the target sequence of the sgRNA used for knocking out the B2M gene is selected from the sequences shown in SEQ ID NO: 33 and 34 and combinations thereof.
  • Scheme 23 The method according to any one of Schemes 17-22, wherein two sgRNAs are used to knock out the B2M gene, wherein the target sequences of the two sgRNAs are shown in SEQ ID NO: 8 and 9 respectively. sequence.
  • Scheme 24 The method according to any one of Schemes 17-23, wherein the target sequence of the sgRNA for knocking out the CD5 gene is selected from the sequence shown in any one of SEQ ID NO: 37, 39, 41-46 and any combination thereof.
  • Scheme 25 The method according to any one of Schemes 17-24, wherein the target sequence of the sgRNA used for knocking out the CD7 gene is the sequence shown in SEQ ID NO: 47.
  • Scheme 26 The method as described in any one of Schemes 17-25, wherein the target sequence of the sgRNA used for knockout of the CIITA gene is selected from the sequence shown in any one of SEQ ID NO: 50, 51, 54, 57 and any combination thereof.
  • Scheme 27 The method according to any one of Schemes 17-26, wherein the cytosine base editor is nCBE3 or nCBE4 protein.
  • Embodiment 28 The method according to any one of Embodiments 17-27, wherein the TRAC gene, the B2M gene, the CD5 gene, the CD7 gene are modified before activating the T cells with CD2/CD3/CD28 antigen. gene and/or the CIITA gene.
  • Embodiment 29 The method according to any one of Embodiments 1-28, wherein the extracellular antigen-binding domain of the CAR includes a first antigen-binding portion and a second antigen-binding portion, and the first antigen-binding portion is capable of specificity.
  • the second antigen binding moiety is capable of specifically binding CD5.
  • Embodiment 30 The method of any one of Embodiments 1-29, wherein the first antigen-binding portion includes a heavy chain variable region from an anti-CD7 single domain antibody, and the HCDR1 of the heavy chain variable region includes SEQ ID
  • Embodiment 31 The method of any one of Embodiments 1-30, the second antigen-binding portion includes a heavy chain variable region from an anti-CD5 single domain antibody, and the HCDR1 of the heavy chain variable region includes SEQ ID NO. : The amino acid sequence shown in SEQ ID NO: 63, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 64, and HCDR3 includes the amino acid sequence shown in SEQ ID NO: 65.
  • Scheme 32 The method according to any one of Schemes 1-31, wherein the first antigen-binding portion includes the amino acid sequence shown in SEQ ID NO: 62.
  • Option 33 The method according to any one of Schemes 1-32, wherein the second antigen-binding portion includes the amino acid sequence shown in SEQ ID NO: 66.
  • Scheme 34 The method according to any one of Schemes 1-33, wherein the extracellular antigen-binding domain of the CAR includes the amino acid sequence shown in SEQ ID NO: 74.
  • Embodiment 35 The method according to any one of Embodiments 1-34, wherein the CAR includes the first antigen-binding portion, the connecting fragment, the second antigen-binding portion, the hinge region, and transmembrane region, intracellular costimulatory domain, and intracellular signaling domain.
  • Scheme 36 The method according to any one of schemes 1-35, wherein the connecting fragment includes the amino acid sequence shown in SEQ ID NO: 67; the hinge region includes the amino acid sequence shown in SEQ ID NO: 68; the span The membrane region includes the amino acid sequence shown in SEQ ID NO: 69; the intracellular costimulatory domain includes the amino acid sequence shown in SEQ ID NO: 70; the intracellular signaling domain includes the amino acid sequence shown in SEQ ID NO: 71 .
  • Embodiment 37 The method according to any one of Embodiments 1-36, wherein the nucleic acid molecule further includes a coding sequence for tEGFR or herpes simplex virus thymidine kinase (HSV-TK).
  • HSV-TK herpes simplex virus thymidine kinase
  • Scheme 38 The method according to any one of Schemes 1-37, wherein the coding sequence of the tEGFR or HSV-TK in the nucleic acid molecule is connected to the coding sequence of the CAR through the coding sequence of the self-cleaving peptide of the downstream.
  • Option 39 The method according to any one of Schemes 1-38, wherein the self-cleaving peptide is T2A, and its amino acid sequence is preferably shown in SEQ ID NO: 72.
  • Scheme 40 The method according to any one of Schemes 1-39, which further includes screening out T cells that do not express TCR and MHC class I molecules after step 2).
  • Embodiment 41 The method according to any one of Embodiments 1-40, wherein the T cells contain NKT cells, for example, NKT cells in a quantitative ratio of 10-20%.
  • Scheme 42 UCAR-T cells prepared by the method described in any one of Schemes 1-41.
  • Embodiment 43 Pharmaceutical composition, which includes the UCAR-T cells described in Embodiment 42 and a pharmaceutically acceptable carrier.
  • Scheme 44 Use of the UCAR-T cells described in Scheme 42 in preparing drugs for treating cancer.
  • Embodiment 45 The use according to Embodiment 44, wherein the cancer expresses CD5 and/or CD7 on its cell surface.
  • Embodiment 46 The use of embodiment 44 or 45, wherein the cancer is a T-cell malignancy, such as acute T-lymphoblastic leukemia (T-ALL) and T-cell lymphoma.
  • T-ALL acute T-lymphoblastic leukemia
  • T-cell lymphoma T-cell lymphoma
  • Embodiment 47 A method of treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of the UCAR-T cells described in Embodiment 42 or the pharmaceutical composition described in Embodiment 43.
  • Embodiment 48 The method of embodiment 47, wherein the cancer expresses CD5 and/or CD7 on its cell surface.
  • Embodiment 49 The method of embodiment 48, wherein the cancer is a T cell malignancy, such as acute T lymphoblastic leukemia and T cell lymphoma.
  • a T cell malignancy such as acute T lymphoblastic leukemia and T cell lymphoma.
  • Embodiment 50 The method of embodiment 47 or 48, further comprising administering ganciclovir (GCV) to the subject after treatment.
  • GCV ganciclovir
  • Scheme 51 Pharmaceutical kit, including: 1) UCAR-T cells or pharmaceutical compositions described in Scheme 42; and 2) GCV.
  • CD7 sdAb VHH protein sequence (CD7-FHV H 10):
  • CD5 sdAb VHH protein sequence (CD5-FHV H 61):
  • CD8 ⁇ hinge region protein sequence CD8 ⁇ hinge region protein sequence:
  • CD8 ⁇ transmembrane region protein sequence

Abstract

本文提供了制备通用嵌合抗原受体T细胞(UCAR-T)的方法，包括将T细胞敲除TRAC、B2M、CD5、CD7等基因后，转染该T细胞使其表达靶向肿瘤细胞的嵌合抗原受体(CAR)。本文还提供了用于进行基因敲除的优选sgRNA。本文提供的UCAR-T可用癌症治疗。

Description

靶向CD5和CD7的通用CAR-T及其应用 技术领域
本文涉及制备通用嵌合抗原受体T细胞(UCAR-T)的方法。本文还涉及UCAR-T在癌症治疗方面的应用。
背景技术
相比自体CAR-T，通用CAR-T(universal CAR-T,简称UCAR-T)具有诸多优势，但同时也面临着诸多的挑战，其中最主要的两大难题是其一：由于异体细胞输注而导致的移植物抗宿主病GvHD，其二：UCAR-T在宿主体内被宿主免疫系统快速清除，而无法有效扩增。所以目前的UCAR-T主要围绕着解决这两个难题而设计。
目前，第一个难题已基本得到解决。研究人员通过基因编辑技术，敲除αβ-T细胞上编码T细胞表面受体(TCR)的TRAC基因1-3，有效抑制CAR-T细胞通过激活TCR对宿主细胞进行无差别的攻击，从而避免GvHD的发生。
相比之下，第二个问题更难解决。近年来研究者一直在探究如何使UCAR-T细胞在宿主体内进行有效扩增。目前主流的解决方案有两种：
(1)通过CD52单抗药物和通用CAR-T联合使用。应用CD52蛋白的单克隆抗体药Alemtuzumab和化疗药物清淋后，给患者输注TRAC/CD52双敲除的CAR-T细胞进行治疗。该方案中敲除TRAC预防移植物抗宿主病，敲除CD52预防清淋药物对UCAR-T的清除2。
(2)另一种为了降低宿主排斥移植物反应的策略，是敲除UCAR-T上的编码β2-微球蛋白的B2M基因3,4。破坏β2-微球蛋白(B2M敲除)可阻止功能性HLA-I类分子在CAR-T细胞表面表达。该方案通过破坏HLA-I类分子，避免了UCAR-T激活宿主体内的细胞毒性T细胞，从而使其获得长期增殖。然而，由于HLA是NK细胞的抑制性配体，它的缺失会激活患者NK细胞对CAR-T细胞进行清除，使其在体内的扩增受限，影响其有效性。
CD5和CD7是靶向T细胞恶性肿瘤治疗的两个有潜力的靶点5。许多T细胞恶性肿瘤表达CD7，大多数T-NHL和T-ALL高表达CD7，且除了T细胞恶性肿瘤，CD7在约24％的AML中表达，被认为是白血病干细胞的标记物，并在绝大多数自然杀伤细胞(NK)和NKT NHLs和白血病中表达6，这为T细胞癌的免疫治疗提供了一个有吸引力的靶点。
CD5是一种泛T细胞标记物，在大多数T细胞恶性肿瘤中普遍过表达，正常细胞CD5的表达仅限于胸腺细胞、外周T细胞和少量的B淋巴细胞亚群，称为B-1细胞。此外，CD5是T细胞受体(TCR)信号的负调节因子，并在保护自身免疫中发挥作用7。
在国内，目前已知以CD5和CD7为靶点进行产品开发的公司较少，其中有的产品为自体CD7-CAR-T，目前处于IND申报阶段；有的为通用CD7-CAR-T，分别处于IIT和I期临床实验阶段。目前还没有公司以CD5和CD7为靶点的双靶CAR-T开发的相关报道。
发明内容
在一方面，本文提供了制备通用嵌合抗原受体T细胞(UCAR-T)的方法，包括：
1)利用CRISPR基因编辑系统制备如下基因被敲除的T细胞：
i)TRAC基因和/或TRBC基因；
ii)CD5基因；
iii)CD7基因；以及
2)以包括嵌合抗原受体(CAR)的编码序列的核酸分子转染所述T细胞，使其表达所述CAR。
在一些实施方案中，在步骤1)中还包括敲除所述T细胞的B2M基因。
在一些实施方案中，对所述TRAC基因的敲除所使用的sgRNA的靶序列选自SEQ ID NO：1-7所示序列及其任意组合。
在一些实施方案中，对所述B2M基因的敲除所使用的sgRNA的靶序列选自SEQ ID NO：8、9、11、12所示序列及其任意组合。
在一些实施方案中，对所述B2M基因的敲除使用两种sgRNA的组合，所述两种sgRNA的靶序列分别为SEQ ID NO：9和11所示序列。
在一些实施方案中，在步骤1)中将用于所述TRAC基因敲除和用于所述B2M基因敲除的sgRNA与Cas9蛋白混合后同时进行所述T细胞中所述TRAC基因和所述B2M基因的敲除，并且用于所述TRAC基因敲除的sgRNA的靶序列为SEQ ID NO：7所示序列。
在一些实施方案中，用于敲除所述CD5基因的sgRNA的靶序列选自SEQ ID NO：13、14、16、17、18、19所示序列及其任意组合。
在一些实施方案中，用于所述CD7基因敲除的sgRNA的靶序列为SEQ ID NO：20所示序列。
在一些实施方案中，对所述TRAC基因、所述CD5基因和所述CD7基因的敲除同时进行，用于敲除所述TRAC基因的sgRNA的靶序列为SEQ ID NO：7所示序列；用于敲除所述CD5基因的sgRNA的靶序列为SEQ ID NO：13所示序列；以及用于敲除所述CD7基因的sgRNA的靶序列为SEQ ID NO：20所示序列。
在一些实施方案中，对所述TRAC基因、所述B2M基因、所述CD5基因和所述CD7基因的敲除同时进行，用于敲除所述TRAC基因的sgRNA的靶序列为SEQ ID NO：7所示序列；用于敲除所述B2M基因的两种sgRNA的靶序列分别为SEQ ID NO：9和11所示系列；用于敲除所述CD5基因的sgRNA的靶序列为SEQ ID NO：13所示序列；以及用于敲除所述CD7基因的sgRNA的靶序列为SEQ ID NO：20所示序列。
在一些实施方案中，对所述TRAC基因、所述CD5基因和所述CD7基因的敲除同时进行，用于进行所述敲除的组分按比例在20μL体系中包括：
不少于30pmol的Cas9和不少于45pmol的靶向SEQ ID NO：7所示序列的sgRNA所形成的RNP复合物；
不少于40pmol的Cas9和不少于120pmol的靶向SEQ ID NO：13所示序列的sgRNA所形成的RNP复合物；以及
不少于40pmol的Cas9和不少于120pmol的靶向SEQ ID NO：20所示序列的sgRNA所形成的RNP复合物。
在一些实施方案中，对所述TRAC基因、所述B2M基因、所述CD5基因和所述CD7基因的敲除同时进行，用于进行所述敲除的组分按比例在20μL体系中包括：
不少于30pmol的Cas9和不少于45pmol的靶向SEQ ID NO：7所示序列的sgRNA所形成的RNP复合物；
不少于20pmol的Cas9和不少于30pmol的靶向SEQ ID NO：9所示序列的sgRNA所形成的RNP复合物；
不少于20pmol的Cas9和不少于30pmol的靶向SEQ ID NO：11所示序列的sgRNA所形成的RNP复合物；
不少于40pmol的Cas9和不少于120pmol的靶向SEQ ID NO：13所示序列的sgRNA所形成的RNP复合物；以及
不少于40pmol的Cas9和不少于120pmol的靶向SEQ ID NO：20所示序列的sgRNA所形成的RNP复合物。
在一些实施方案中，对所述TRAC基因、所述CD5基因和所述CD7基因的敲除同时进行，用于进行所述敲除的组分按比例在20μL体系中包括：
30pmol的Cas9和45pmol的靶向SEQ ID NO：7所示序列的sgRNA所形成的RNP复合物；
80pmol的Cas9和150pmol的靶向SEQ ID NO：13所示序列的sgRNA所形成的RNP复合物；以及
40pmol的Cas9和120pmol的靶向SEQ ID NO：20所示序列的sgRNA所形成的RNP复合物。
在一些实施方案中，对所述TRAC基因、所述B2M基因、所述CD5基因和所述CD7基因的敲除同时进行，用于进行所述敲除的组分按比例在20μL体系中包括：
30pmol的Cas9和45pmol的靶向SEQ ID NO：7所示序列的sgRNA所形成的RNP复合物；
20pmol的Cas9和30pmol的靶向SEQ ID NO：9所示序列的sgRNA所形成的RNP复合物；
20pmol的Cas9和30pmol的靶向SEQ ID NO：11所示序列的sgRNA所形成的RNP复合物；
80pmol的Cas9和150pmol的靶向SEQ ID NO：13所示序列的sgRNA所形成的RNP复合物；以及
40pmol的Cas9和120pmol的靶向SEQ ID NO：20所示序列的sgRNA所形成的RNP复合物。
在一些实施方案中，在步骤1)中还包括敲除所述T细胞的CIITA基因。
在一些实施方案中，用于所述CIITA基因敲除的sgRNA的靶序列为SEQ ID NO：25所示序列。
在一些实施方案中，对所述TRAC基因、所述B2M基因、所述CD5基因、所述CD7基因和所述CIITA基因的敲除同时进行，用于进行所述敲除的组分按比例在20μL体系中包括：
不少于30pmol的Cas9和不少于45pmol的靶向SEQ ID NO：7所示序列的sgRNA所形成的RNP复合物；
不少于20pmol的Cas9和不少于30pmol的靶向SEQ ID NO：9所示序列的sgRNA所形成的RNP复合物；
不少于20pmol的Cas9和不少于30pmol的靶向SEQ ID NO：11所示序列的sgRNA所形成的RNP复合物；
不少于80pmol的Cas9和不少于150pmol的靶向SEQ ID NO：13所示序列的sgRNA所形成的RNP复合物；
不少于40pmol的Cas9和不少于120pmol的靶向SEQ ID NO：20所示序列的sgRNA所形成的RNP复合物；以及
不少于40pmol的Cas9和不少于60pmol的靶向SEQ ID NO：25所示序列的sgRNA所形成的RNP复合物。
在一些实施方案中，对所述TRAC基因、所述B2M基因、所述CD5基因、所述CD7基因和所述CIITA基因的敲除同时进行，用于进行所述敲除的组分按比例在20μL体系中包括：
30pmol的Cas9和45pmol的靶向SEQ ID NO：7所示序列的sgRNA所形成的RNP复合物；
25pmol的Cas9和40pmol的靶向SEQ ID NO：9所示序列的sgRNA所形成的RNP复合物；
25pmol的Cas9和40pmol的靶向SEQ ID NO：11所示序列的sgRNA所形成的RNP复合物；
80pmol的Cas9和150pmol的靶向SEQ ID NO：13所示序列的sgRNA所形成的RNP复合物；
40pmol的Cas9和120pmol的靶向SEQ ID NO：20所示序列的sgRNA所形成的RNP复合物；以及
50pmol的Cas9和80pmol的靶向SEQ ID NO：25所示序列的sgRNA所形成的RNP复合物。
另一方面，本文提供了制备通用嵌合抗原受体T细胞(UCAR-T)的方法，包括：
1)利用使用胞嘧啶碱基编辑器制备如下基因被敲除的T细胞：
i)TRAC基因和/或TRBC基因；
ii)CD5基因；
iii)CD7基因；以及
2)以包括嵌合抗原受体(CAR)的编码序列的核酸分子转染所述T细胞，使其表达嵌合抗原受体(CAR)。
在一些实施方案中，在步骤1)中还包括敲除所述T细胞的B2M基因。
在一些实施方案中，在步骤1)中还包括敲除所述T细胞的CIITA基因。
在一些实施方案中，对所述TRAC基因的敲除所使用的sgRNA的靶序列为SEQ ID NO：26所示序列。
在一些实施方案中，对所述TRBC基因的敲除所使用的sgRNA的靶序列选自SEQ ID NO：27-31任一项所示序列及其任意组合。
在一些实施方案中，对所述B2M基因的敲除所使用的sgRNA的靶序列选自SEQ ID NO：33和34所示序列及其组合。
在一些实施方案中，对所述B2M基因的敲除使用两种sgRNA，其中所述两种sgRNA的靶序列分别为SEQ ID NO：8和9所示序列。
在一些实施方案中，用于敲除所述CD5基因的sgRNA的靶序列选自SEQ ID NO：37、39、41-46任一项所示序列及其任意组合。
在一些实施方案中，用于所述CD7基因敲除的sgRNA的靶序列为SEQ ID NO：47所示序列。
在一些实施方案中，用于所述CIITA基因敲除的sgRNA的靶序列选自SEQ ID NO：50、51、54、57任一项所示序列及其任意组合。
在一些实施方案中，所述胞嘧啶碱基编辑器为nCBE3或nCBE4蛋白。
在一些实施方案中，在以CD2/CD3/CD28抗原激活所述T细胞之前对所述TRAC基因、所述B2M基因、所述CD5基因、所述CD7基因和/或所述CIITA基因进行敲除。
在一些实施方案中，所述CAR的胞外抗原结合结构域包括第一抗原结合部分和第二抗原结合部分，所述第一抗原结合部分能够特异性结合CD7，所述第二抗原结合部分能够特异性结合CD5。
在一些实施方案中，所述第一抗原结合部分包括来自抗CD7单域抗体的重链可变区，所述重链可变区的HCDR1包括SEQ ID NO：59所示氨基酸序列、HCDR2包括SEQ ID NO：60所示氨基酸序列以及HCDR3包括SEQ ID NO：61所示氨基酸序列。
在一些实施方案中，所述第二抗原结合部分包括来自抗CD5单域抗体的重链可变区，所述重链可变区的HCDR1包括SEQ ID NO：63所示氨基酸序列、HCDR2包括SEQ ID NO：64所示氨基酸序列以及HCDR3包括SEQ ID NO：65所示氨基酸序列。
在一些实施方案中，所述第一抗原结合部分包括SEQ ID NO：62所示氨基酸序列。
在一些实施方案中，所述第二抗原结合部分包括SEQ ID NO：66所示氨基酸序列。
在一些实施方案中，所述CAR的胞外抗原结合结构域包括SEQ ID NO：74所示氨基酸序列。
在一些实施方案中，所述CAR从氨基端到羧基端依次包括所述第一抗原结合部分、连接片段、所述第二抗原结合部分、铰链区、跨膜区、胞内共刺激结构域和胞内信号传导结构域。
在一些实施方案中，所述连接片段包括SEQ ID NO：67所示氨基酸序列；所述铰链区包括SEQ ID NO：68所示氨基酸序列；所述跨膜区包括SEQ ID NO：69所示氨基酸序列；所述胞内共刺激结构域包括SEQ ID NO：70所示氨基酸序列；所述胞内信号传导结构域包括SEQ ID NO：71所示氨基酸序列。
在一些实施方案中，所述核酸分子中还包括tEGFR或单纯疱疹病毒胸苷激酶(HSV-TK)的编码序列。
在一些实施方案中，所述核酸分子中的所述tEGFR或HSV-TK的编码序列的通过自剪切肽的编码序列连接在所述CAR的编码序列的下游。
在一些实施方案中，所述自剪切肽为T2A，其氨基酸序列优选为SEQ ID NO：72所示。
在一些实施方案中，还包括在步骤2)后筛选出不表达TCR和MHC-I类分子的T细胞。
在一些实施方案中，所述T细胞含有NKT细胞，例如10-20％数量比例的NKT细胞。
另一方面，本文提供了通过上述方法制备的UCAR-T细胞。
另一方面，本文提供了药物组合物，其包括上述UCAR-T细胞和药学上可接受的载体。
另一方面，本文提供了上述UCAR-T细胞在制备用于治疗癌症的药物中的用途。
在一些实施方案中，所述癌症在其细胞表面表达CD5和/或CD7。
在一些实施方案中，所述癌症为T细胞恶性肿瘤，如急性T淋巴细胞白血病(T-ALL)和T细胞淋巴瘤。
另一方面，本文提供了在受试者中治疗癌症的方法，包括以治疗有效量的上述UCAR-T细胞或药物组合物向所述受试者给药。
在一些实施方案中，所述癌症在其细胞表面表达CD5和/或CD7。
在一些实施方案中，所述癌症为T细胞恶性肿瘤，如急性T淋巴细胞白血病和T细胞淋巴瘤。
在一些实施方案中，所述方法还包括治疗后以更昔洛韦(GCV)向所述受试者给药。
另一方面，本文提供了药物试剂盒，包括：1)上述UCAR-T细胞或药物组合物；以及2)GCV。
附图说明
图1.CD5-CD7通用CAR-T的设计示意图。
图2.CD5-CD7双特异性CAR结构示意图。
图3.通过小分子药物控制的CAR-T安全开关分子种类(来自参考文献8)。
图4.TRAC基因候选sgRNA信息以及敲除效率、脱靶测试结果。(A)发明人通过CRISPick、CRISPOR和IDT网站对TRAC基因进行敲除sgRNA设计并经过筛选后得到的候选sgRNA信息。根据前期经验，网站预测的sgRNA编辑效率不是特别准，因此在筛选候选sgRNA时，发明人主要看网站预测的脱靶信息，而在CRISPick、CRISPOR和IDT三个网站中，CRISPick对sgRNA脱靶的计算是最精密的，因此发明人主要根据CRISPick的脱靶排名来选择sgRNA，排名越靠前，则脱靶概率越低。(B)TRAC候选sgRNA敲除效率测试的FACS结果图。横坐标代表细胞的TCR表达情况，纵坐标代表细胞的大小；每一列代表一种sgRNA，每一行代表一种RNP浓度。(C-E)候选sgRNA A-XL,A-02,A-06的脱靶测试结果。每一幅图的第一行是sgRNA的序列信息，第二行是在靶的reads数，第三行起是可能的脱靶位点的序列信息以及reads数。A-XL这条sgRNA测试了两次，分别标注为R1和R2，A-02和A-06各测试了一次。最高脱靶比例＝(出现次数最多的位点的reads数/在靶位点的reads数)*100％。
图5.B2M基因候选sgRNA信息以及敲除效率测试结果。(A)发明人通过CRISPick、CRISPOR、IDT、CHOPCHOP和GUIDES网站对B2M基因进行敲除sgRNA设计并经过筛选后得到的候选sgRNA信息。最终的sgRNA是综合几个网站的在靶和脱靶信息而选择得出，排名数字越小越好，得分越高越好。(B)B2M候选sgRNA敲除效率测试的FACS结果图。横坐标代表细胞的B2M表达情况，纵坐标代表细胞的大小，MOCK-T组代表细胞未使用sgRNA。
图6.B2M和TRAC基因双敲测试结果及B2M-sgRNA的脱靶检测结果。(A)B2M的5条候选sgRNA与TRAC sgRNA(A-XL)一起共敲除的FACS结果图。横坐标代表细胞的B2M表达情况，纵坐标代表细胞的TCR表达情况；双基因阳性的细胞位于四象限的右上象限，双基因阴性的细胞位于四象限的左下象限；因此，位于左下象限的细胞占比越高，代表双基因同时敲除的细胞越多。(B)测试不同的B2M sgRNA与TRAC sgRNA一起在不同RNP浓度下的共敲除效率的FACS结果图。横坐标代表细胞的B2M表达情况，纵坐标代表细胞的TCR表达情况，每一列代表一种B2M sgRNA，每一行代表一种RNP浓度。 (C)B-13 sgRNA的两次脱靶测试结果，分别为R1和R2。(D)B-03 sgRNA的两次脱靶测试结果，分别为R1和R2。
图7.CD5基因候选sgRNA信息和基因敲除结果。(A)第一次测试的CD5 sgRNA的序列信息。(B)第一次测试中CD5 sgRNA的敲除效率测试的FACS结果图。横坐标代表细胞的CD5表达情况，纵坐标代表细胞的大小，其中sgRNA1和sgRNA3对应C图中的C-01和C-03。(C)发明人第二次通过CRISPick、CRISPOR、IDT、CHOPCHOP和GUIDES网站对CD5基因进行敲除sgRNA设计并经过筛选后得到的候选sgRNA信息。
图8.CD5基因候选sgRNA的敲除效率和脱靶检测结果。(A)CD5候选sgRNA敲除效率测试的FACS结果图。横坐标代表细胞CAR的表达情况，纵坐标代表细胞的CD5表达情况，坐下象限的细胞比例越高，代表CD5的敲除效率越高。(B)CD5候选sgRNA的敲除效率总结图，数据来源于5A的FACS结果。(C)CD5候选sgRNA在CRISPick网站中的脱靶和综合排名情况，排名数字越小，代表sgRNA越好。(D)C-06 sgRNA的两次脱靶测试结果，分别为R1和R2。
图9.CD7基因sgRNA(E-01)的脱靶检测结果。左边为未使用sgRNA的对照细胞的测序结果，右边为使用了CD7 sgRNA的测序结果。
图10.CIITA基因候选sgRNA信息、敲除效率和脱靶检测结果。(A)CIITA候选sgRNA的序列信息以及敲除效率测试的FACS结果图。横坐标代表细胞HLA-II类分子的表达，纵坐标代表细胞的大小。(B)F-05 sgRNA的两次脱靶测试结果，分别为R1和R2。
图11.TRAC、B2M、CD5、CD7共敲RNP条件摸索。(A)用Lonza电转仪进行20ul体系(2x106个细胞)电转时各基因的RNP用量。每一行为一组设置，共探索了6组RNP用量组合；每一列代表一种sgRNA的RNP用量，表中每格的数字分别Cas9蛋白和sgRNA的用量，单位为pmol。(B)不同RNP用量组合下TRAC、B2M、CD5、CD7共敲效率的FACS结果图，分别在两个donor(供着)来源的T细胞上进行了测试。每个图中，左下角象限中细胞的比例越高，代表双基因共同敲除的效率越高。
图12.两次制备(后续提到“两次制备”，均是指相同的两次制备)CD5-CD7 UCAR-T的敲除设置和结果。(A)第一次制备的CD5-CD7 UCAR-T成品细胞的各基因敲除情况。上面一排是未敲除细胞的各基因表达情况，下面一排是CD5-CD7 UCAR-T细胞的各基因敲除情况，左下角象限的细胞即为敲除细胞的比例。(B)两次制备CD5-CD7 UCAR-T时各基因的RNP用量，每格中的数字代表相应物料的用量，单位为pmol。(C)第二次制备CD5-CD7 UCAR-T细胞时KO-T细胞的各基因敲除情况，KO-T为只进行了基因敲除但未进行转毒的细胞，能反应各基因的真实敲除情况。ISO为阴性对照结果，MOCK-T为阳性对照结果。
图13.TRAC、B2M、CIITA、CD5、CD7基因的五敲实验结果。(A)TRAC、B2M、CIITA、CD5、CD7五基因共同敲除预实验的RNP用量，每格中的数字代表相应物料的用量，单位为pmol。(B)TRAC、B2M、CIITA、CD5、CD7五基因共同敲除预实验的 FACS结果图。ISO为阴性对照，MOCK-T为阳性对照，其中HLA-DR-DP-DQ的表达情况反应CIITA基因的敲除情况。(C)制备五敲版本的CD5-CD7 UCAR-T细胞时的RNP用量，每格中的数字代表相应物料的用量，单位为pmol。(D)制备五敲版本的CD5-CD7 UCAR-T细胞时KO-T组细胞的各基因敲除情况。ISO为阴性对照，MOCK-T为阳性对照，其中HLA-DR-DP-DQ的表达情况反应CIITA基因的敲除情况。
图14.针对TCR/B2M/CD5/CD7/CIITA基因的单碱基编辑候选sgRNA信息。
图15.针对TCR基因的碱基编辑sgRNA效率。图为针对TCR的碱基编辑sgRNA效率测试的FACS结果，横坐标代表TCR的表达情况，纵坐标代表细胞内容物的复杂程度。
图16.针对B2M基因的碱基编辑sgRNA效率。(A)图为针对B2M的碱基编辑sgRNA效率测试的结果，横坐标代表B2M的表达情况，纵坐标代表细胞内容物的复杂程度。(B)图为Cas9蛋白利用相邻两条反向的sgRNA，分别在基因组的两条链上进行切割的示意图，以及B2M的1号外显子上可利用的几条sgRNA的位置和序列信息。(C)B-10、B-13、NB-37、NB-38四条sgRNA不同组合下对B2M的敲除情况，横坐标代表B2M的表达情况，纵坐标代表细胞内容物的复杂程度。
图17.针对CD5基因和CD7基因的碱基编辑sgRNA效率。(A)图为针对CD5的碱基编辑sgRNA效率测试的FACS结果，横坐标代表CD5的表达情况，纵坐标代表细胞内容物的复杂程度，每一列为一种sgRNA，做了两个重复实验。(B)图为针对CD7的碱基编辑sgRNA效率测试的结果，横坐标代表CD7的表达情况，纵坐标代表细胞内容物的复杂程度，同样做了两个重复实验。
图18.针对CIITA基因的不同sgRNA的碱基编辑效率。图为针对CIITA的碱基编辑sgRNA效率测试的FACS结果，横坐标以HLA-II类分子的表达情况来反应CIITA的表达情况，纵坐标代表细胞内容物的复杂程度，ISO为阴性对照，MOCK-T为阳性对照。
图19.两次制备UCAR-T的CD5、CD7和CAR阳性细胞比例。(A)图为第一次制备的CD5-CD7 UCAR-T在转毒后3天CAR阳性细胞的比例，分别通过EGFR抗体和CD5/CD7抗原来检测EGFR和CD5、CD7 CAR的表达。(B)图为第一次制备的CD5-CD7 UCAR-T在转毒后8天CAR阳性细胞的比例，比转毒后3天高了接近一倍。(C和D)分别为第二次制备的CD5-CD7 UCAR-T在转毒后第3天和第8天时CAR阳性细胞的比例，结果与第一次的CD5-CD7 UCAR-T相似。
图20.两次制备UCAR-T期间的TRAC/B2M阴选结果。图为两次制备CD5-CD7 UCAR-T期间的TRAC/B2M阴选结果，横坐标代表B2M的表达，纵坐标代表TCR的表达，红色虚框为阴选后继续培养的细胞。
图21.两次制备的CD5-CD7 UCAR-T的CD107a释放情况检测结果。(A)第一次制备CD5-CD7 UCAR-T时KO-T和UCAR-T与不同的肿瘤细胞共孵育4h后，CD8和CAR双阳性T细胞中CD107a阳性细胞的比例。(B)根据18A中的数据做的直方图。(C)第二次 制备的CD5-CD7 UCAR-T与不同的肿瘤细胞共孵育4h后，CD8和CAR双阳性T细胞中CD107a阳性细胞的比例结果图。
图22.两次制备的CD5-CD7 UCAR-T的杀瘤情况检测。(A)第一次制备的CD5-CD7 UCAR-T在第7天时的杀瘤功能检测结果。每个点的数据代表CD5-CD7 UCAR-T杀死相应肿瘤细胞的百分比，值越高，说明越多的肿瘤细胞被杀死，值为负数代表肿瘤细胞不但没被杀死反而有所增殖。(B)第一次制备的未转毒KO-T细胞在同样时间点相应的杀瘤能力。(C)第一次制备的CD5-CD7 UCAR-T细胞冻存复苏后的杀瘤功能检测结果。(D)第二次制备的CD5-CD7 UCAR-T在第10天时的杀瘤功能检测结果。(E)第二次制备的未转毒KO-T细胞在同样时间点相应的杀瘤能力。(F)第二次制备的CD5-CD7 UCAR-T细胞冻存复苏后的杀瘤功能检测结果。
图23.两次制备的CD5-CD7 UCAR-T的活化、分型结果。(A)第一次制备的CD5-CD7 UCAR-T上各抗原表达情况(未进行TRAC/B2M阴选)；(B)第二次制备的制备的CD5-CD7 UCAR-T上各抗原表达情况(已进行TRAC/B2M阴选)；(C)第一次制备的CD5-CD7 UCAR-T中CCR7阳性比例(day10冻存)；(D)第二次制备的CD5-CD7 UCAR-T中CCR7阳性比例(day12冻存)。
图24.两次制备的CD5-CD7 UCAR-T的耗竭和早调情况。(A)第一次制备的CD5-CD7 UCAR-T上耗竭marker表达情况；(B)第二次制备的CD5-CD7 UCAR-T上耗竭marker表达情况；(C)第一次制备的CD5-CD7 UCAR-T上早凋marker表达情况；(D)第二次制备的CD5-CD7 UCAR-T上早凋marker表达情况。
图25.两次制备的CD5-CD7 UCAR-T的组分鉴定结果。(A)第一次制备的CD5-CD7 UCAR-T细胞在冻存当天的组分鉴定结果；(B)第二次制备的CD5-CD7 UCAR-T细胞在制备期间不同时间点的组分鉴定结果。
图26.CD5-CD7 UCAR-T(EGFR开关)的一例临床实验结果。(A)整个临床测试周期的设计示意图。其中的Flu是氟达拉滨(Fludarabine)，CTX是环磷酰胺(Cyclophosphamide，CTX)。(B)CD5-CD7 UCAR-T回输后患者外周血中CAR阳性细胞的VCN随着时间的变化情况，VCN越高，代表患者外周血中UCAR-T细胞越多。(C-E)CD5-CD7 UCAR-T回输后患者外周血中各种细胞的比例变化情况，lym指淋巴细胞(lymphocyte)。
图27.CD5-CD7 UCAR-T(HSV-TK)细胞制备。(A)HSV-TK版本的CAR分子结构示意图，将原来的tEGFR改为了HSV-TK。(B)激活电转工艺和静息电转工艺制备CD5-CD7 UCAR-T的简要流程图。(C)激活电转和静息电转工艺下TRAC/B2M/CD5/CD7的敲除以及CAR的转效情况。(D)激活电转和静息电转工艺制备的CD5-CD7 UCAR-T的细胞分型。(E)激活电转和静息电转工艺制备的CD5-CD7 UCAR-T的细胞组分比较。
图28.HSV-TK版本与EGFR版本UCAR-T之间的杀瘤效果比较。(A)HSV-TK版本的CD5-CD7 UCAR-T细胞在冻存复苏后的24h杀瘤结果。(B)EGFR版本的CD5-CD7 UCAR-T细胞在冻存复苏后的24h杀瘤结果。
图29.GCV药物对HSV-TK阳性细胞的清除情况。(A)HSV-TK版本的CD5-CD7 UCAR-T细胞在不同浓度的GCV处理下，总细胞的增殖情况。(B)HSV-TK版本的CD5-CD7 UCAR-T细胞在不同浓度的GCV处理下，CAR阳性细胞比例的变化情况。(C)HSV-TK版本的CD5-CD7 UCAR-T细胞在不同浓度的GCV处理下，CAR阳性细胞数量的变化情况。(D)-(F)EGFR版本的CD5-CD7 UCAR-T细胞在不同浓度的GCV处理下，总细胞的增殖、CAR阳性细胞比例、CAR阳性细胞数量的变化情况。
图30.UCAR-T和GCV的体内药效测试。(A)动物实验的整个流程设计示意图，D代表天数。(B)小鼠的荧光成像结果图，由于肿瘤细胞表达荧光素蛋白，因此给与小鼠注射荧光素蛋白的底物后，小鼠即可产生荧光，通过荧光成像仪检测产生的荧光强度可间接地反应肿瘤细胞的多少。(C)不同组别的小鼠体内的平均荧光值强度随着时间的变化情况。
具体实施方式
除非另有说明，本文使用的所有技术和科学术语具有本领域普通技术人员所通常理解的含义。
术语“或”是指列举的可选择要素中的单个要素，除非上下文明确地另外指出。
术语“和/或”是指所列举的可选择要素中的任意一个、任意两个、任意三个、任意更多个或其全部。
“包含”或“包括”指包括所述的要素、整数或步骤，但是不排除任意其他要素、整数或步骤。当使用“包含”或“包括”时，除非另有指明，否则也涵盖由所述及的要素、整数或步骤组成的情形。例如，当提及“包含”某个具体序列的抗体可变区时，也旨在涵盖由该具体序列组成的抗体可变区。
“B2M基因”在本文中指β2微球蛋白的编码基因。β2微球蛋白为MHC-I类分子(人MHC-I类分子也称HLA-I类分子)的组成部分，与MHC-I类分子中的重链结合，在细胞表面形成异二聚体。B2M基因被敲除将导致细胞表面MHC-I类分子的缺失。
“TRAC基因”和“TRBC基因”在本文中分别指T细胞受体α链恒定区的编码基因和T细胞受体β链恒定区的的编码基因。该α链和β链构成T细胞受体(TCR)，其识别抗原和介导免疫应答的作用。“TRAC基因”和/或“TRBC基因”的敲除导致细胞不能表达TCR分子。
“CIITA基因”的编码产物为II类反式激活因子，在HLA基因的表达中发挥主导开关的作用。II类反式激活因子本身为非DNA结合蛋白，但其可通过结合多种转录因子和 协同激活因子参与对HLA基因的转录调控。CIITA基因的敲除可影响到HLA基因，尤其是HLA-II类基因的转录，使其不能表达相应产物。
“CD5”为I型跨膜糖基化蛋白，在T细胞受体信号传导的负调控中起着重要作用，并促进正常和恶性淋巴细胞的存活。CD5是恶性T细胞肿瘤的特征性表面标志物之一，80％的T细胞急性淋巴细胞白血病(T-ALL)和外周T细胞淋巴瘤都表达CD5。本文中，CD5可以为人CD5，其GenBank登录号为NM_014207.4。CD5蛋白也可包括CD5的片段，诸如胞外结构域及其片段。
“CD7”为细胞表面糖蛋白，分子量约40kD，属于免疫球蛋白超家族成员，在T细胞和NK细胞以及其他细胞如胸腺细胞、髓系细胞等细胞表面表达。其在T细胞相互作用以及早期淋巴发育期间的T细胞-B细胞相互作用方面起重要作用。
“抗体”指由浆细胞(效应B细胞)分泌、被机体免疫系统用来中和外来物质(多肽、病毒、细菌等)的免疫球蛋白。该外来物质相应地称作抗原。经典抗体分子的基本结构是由2个相同重链和2个相同轻链组成的4聚体。根据氨基酸序列的保守性差异，将重链和轻链分为位于氨基端的可变区(V)和位于羧基端的恒定区(C)。一条重链和一条轻链的可变区相互作用形成了抗原结合部位(Fv)。在可变区中，某些区域氨基酸残基的组成和排列次序比可变区内的其它区域(骨架区，FR)更易变化，称为高变区(HVR)，高变区实际上是抗体与抗原结合的关键部位。由于这些高变区序列与抗原决定簇互补，故又称为互补决定区(complementarity-determining region,CDR)。重链和轻链均具有三个互补决定区，分别称为HCDR1、HCDR2、HCDR3和LCDR1、LCDR2、LCDR3。CDR的氨基酸序列可以使用本领域公认的编号方案来确定，例如Kabat、Chothia、IMGT、AbM或Contact编号方案。根据抗体重链恒定区的氨基酸序列，可将抗体分为五种主要的不同类型：IgA、IgD、IgE、IgG和IgM。这些抗体类型根据铰链区的大小，链间二硫键的位置和分子量的不同可进一步分为亚类，例如，IgGl、IgG2a、IgG2b和IgG3等。根据抗体轻链恒定区氨基酸组成和排列的不同，可将轻链分为κ和λ两种类型。不同类别的免疫球蛋白的亚单位结构和三维构象在本领域内是已知的。
抗体分子的“抗原结合片段”指抗体分子中参与抗原特异性结合的氨基酸片段，例如，Fab、Fab’、(Fab’)2、scFv和sdAb等。本领域技术人员已知如何获得这些抗原结合片段。例如，经典抗体分子可经木瓜蛋白酶消化而得到Fab片段，经胃蛋白酶消化得到F(ab’)2，通过以还原剂处理断开F(ab’)2铰链区之间的二硫键而形成Fab’片段。
“单链抗体(single chain fragment variable,scFv)”，是由抗体重链可变区和轻链可变区通过短肽连接成一条肽链而构成。通过正确折叠，来自重链和轻链的可变区通过非共价键相互作用形成Fv段，因而scFv能较好地保留其对抗原的亲和活性。
“单域抗体(single domain antibody，sdAb)”，或者也称为“VHH抗体”，指具有抗原结合能力，包括重链可变区而无轻链的抗体分子。从结构上看，单域抗体也可以认为是抗体分子的一种抗原结合片段。其首先在骆驼科动物中被发现，随后，研究人员通过抗体 库(例如噬菌体展示文库)筛选发现了更多的具有抗原结合能力的单域抗体。单域抗体相对于普通抗体分子(例如，经典四聚体抗体分子)或其抗原结合片段具有一些优势，例如包括但不限于：分子量更小，使用于人体时易于到达普通抗体分子难以到达的组织或部位，或者，能够接触到蛋白或多肽中普通抗体分子难以接触到的抗原表位；更加稳定，能够耐受例如温度和pH的变化以及变性剂和蛋白酶的作用。
提及抗体或其抗原结合片段时，“靶向”或“特异性结合”指，相对于环境中同时存在的其他分子，一种分子(例如抗体或其抗原结合片段)对另一种分子(如肿瘤细胞表面抗原)具有更高的结合亲和力。“靶向”或“特异性结合”并不排除该分子可以对一种以上的分子具有结合亲和力，例如双特异性抗体可以对两种不同抗原具有高亲和力。
“嵌合抗体受体(chimeric antigen receptor，CAR)”，也称为嵌合T细胞受体、嵌合免疫受体，为一种工程化的膜蛋白受体分子，其可将期望的特异性赋予免疫效应细胞，例如与细胞表面蛋白(如肿瘤抗原)结合的能力。嵌合抗原受体通常由胞外抗原结合结构域、跨膜结构域和胞内信号传导结构域构成。通常，抗原结合结构域为一段scFv或sdAb序列，负责识别和结合特定的抗原。该抗原结合结构域可以为单特异性的，即仅对一种抗原具有特异性结合能力；也可以为多特异性的(例如双特异性的)，即对多种抗原具有特异性结合能力。在本文提供的一些实例中，该双特异性胞外抗原结合结构域对CD5和CD7都有特异性结合能力，这可以通过在胞外抗原结合结构域中包括靶向或特异性结合CD5的抗体片段(例如scFv或sdAb)和靶向或特异性结合CD7的抗体片段(例如scFv或sdAb)来实现。胞内信号结构域通常包括免疫受体酪氨酸活化基序(ITAM)，例如来源于CD3ζ分子的信号传导结构域，负责激活免疫效应细胞，产生杀伤作用。另外，嵌合抗原受体还可在氨基端包括负责新生蛋白在细胞内定位的信号肽，以及在抗原结合结构域和跨膜结构域之间包括铰链区。胞内信号传导结构域还可包括来源于例如4-1BB或CD28分子的共刺激结构域。相应地，将表达CAR的T细胞简称为CAR-T。CAR-T利用其细胞表面表达的CAR识别靶细胞，被靶细胞激活后以非MHC限制性方式产生对靶细胞的杀伤作用。在一个实例中，利用CAR-T细胞对受试者(如癌症患者)进行治疗的大体过程为：从受试者采集外周血单个核细胞(PBMC)，分离并培养T细胞，通过慢病毒转导方式导入CAR编码核酸序列，继续培养并收集CAR+细胞，以及将CAR+细胞回输给该受试者。本领域技术人员已知，在一些情况下，可以利用NK细胞替代T细胞来进行该过程。因此，在提及CAR-T时，视情况也可涵盖表达CAR的NK细胞。另外，本文在提及CAR-T细胞时，除非另有说明，不仅指直接经CAR修饰的细胞，还指这些细胞在体外或体内增殖后产生的子细胞
“通用CAR-T细胞(UCAR-T)”在本文指这种细胞不限于输入特定患者体内的CAR-T细胞。在现有技术中，为了防止GvHD以及宿主对移植物的排斥反应，通常是从患者体内收集细胞(如T细胞)并进行CAR修饰后输回到患者体内。这种方法不仅耗时昂贵，而且在有些情况下无法获得足够数量的患者T细胞来进行CAR修饰。与此相反，这里的通 用CAR-T细胞指其适合于异体移植，同一批CAR-T细胞可用于不同的患者，并且这些通用CAR-T细胞通常并非源自于这些患者。
“CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats)基因编辑技术”是新出现的一种由RNA指导的通过Cas核酸酶对靶基因进行DNA编辑的技术。该技术所使用的CRISPR基因编辑系统包括Cas核酸酶和引导RNA(single-guide RNA,sgRNA)，视情况可还包括作为修复模板的ssDNA。sgRNA的一部分序列可以与Cas核酸酶结合，另外部分序列(crRNA)可以与靶基因的部分序列互补，借助sgRNA的识别作用使得Cas核酸酶可以在靶基因特定位点形成单链或双链切口。细胞通常会通过两种方式对断裂链进行DNA修复，这两种方式分别是同源重组修复机制(homology-directed repair,HDR)和非同源末端连接修复机制(non-homologous end joining,NHEJ)。在将CRISPR技术例如用来对细胞的基因进行基因敲除操作时，通常只需要考虑破坏该基因的正常编码功能，例如引起移码突变或基因片段缺失，从而不能产生有正常功能的产物(如蛋白)。通常，可以在向细胞中引入Cas核酸酶(例如Cas9)和sgRNA后，再筛选出不表达待敲除基因的产物的细胞。“CRISPR基因编辑系统”在本文中指Cas核酸酶和sgRNA的组合，用于在引入细胞后对sgRNA靶向基因进行编辑。
除了CRISPR技术外，也可以采用其他技术来实现基因敲除，例如同源重组、TALEN技术等。
“胞嘧啶碱基编辑技术”是在CRISPR技术基础上引入了单碱基编辑功能的基因编辑技术。其采用了具有多个功能部分的称为“胞嘧啶碱基编辑器”的融合蛋白。一个功能部分为Cas9n(Cas9-nickase)蛋白，其源自对Cas9蛋白的RuvC1结构域进行D10A突变，从而只保留了HNH结构域的酶活性。Cas9n不会造成DNA双链断裂，只能够切割基因组上与sgRNA互补结合的DNA单链，从而诱导碱基错配修复(BER)。另两个功能部分分别为胞嘧啶脱氨酶APOBEC和尿嘧啶糖基化酶抑制剂(UGI)。APOBEC能够诱导另一条DNA单链(非sgRNA靶向链)上的胞嘧啶发生脱氨，形成尿嘧啶，在UGI蛋白存在下，最终促使胞嘧啶向胸腺嘧啶的突变(C->T突变)。在本文中，通过胞嘧啶碱基编辑技术向细胞的待敲除基因中引入终止密码子，从而使得细胞不能产生有功能的基因产物。胞嘧啶碱基编辑技术可以视为CRISPR技术的一部分，但出于方便描述的目的，在本文中，提及CRISPR技术，尤其是采用Cas9蛋白的CRISPR技术时，不涉及上述单碱基编辑功能，从而与胞嘧啶碱基编辑技术区分开。
“敲除”或“基因敲除”在本文中指改变细胞中某基因的核苷酸序列，无论该改变是核苷酸插入、缺失还是替换，只要被敲除的该基因在细胞中不能产生有功能的基因产物(如RNA或蛋白)即可。理想地，基因敲除使得细胞或细胞群完全不形成该基因的基因产物或者有功能的基因产物。可理解地，导致基因产物的量明显减少，或者基因产物的活性明显减低，也可认为是实现了“基因敲除”。在一些情况下，可能需要对细胞中的两个或更多个基因进行基因敲除。在一些实施方案中，可以按次序进行基因敲除，即在 敲除一个基因后，接着进行下一个基因的敲除。在另一些实施方案中，可以同时对两个或更多个基因进行敲除。例如，在采用CRISPR技术敲除细胞中的多个基因时，可以同时向该细胞引入Cas9和分别靶向各个基因的多种sgRNA。
提及sgRNA时，术语“靶序列”指目标基因或待敲除基因中与sgRNA的部分序列(crRNA，约20个碱基)互补的核苷酸片段。借助于sgRNA中与靶序列互补的这部分序列，让Cas9等蛋白可以在相对确定的位置在目标基因中引入核苷酸序列改变，达到基因敲除的效果。相应地，在本文中，“靶向某指定序列的sgRNA”指该sgRNA的靶序列为该指定序列。
“RNP复合物”在本文中指sgRNA与相应Cas酶(如Cas9)结合的产物。在通过CRISPR技术或胞嘧啶碱基编辑技术进行基因敲除时，可以先将sgRNA与Cas酶混合，随后通过电转引入细胞(如T细胞)中。
“EGFRt”或“tEGFR”在本文中可以互换使用，指编码截短的人表皮生长因子受体多肽的基因或其编码产物，其缺乏远端膜EGF结合域和细胞质信号传导尾，但保留了由抗EGFR抗体识别的细胞外表位。EGFRt可用作具有遗传修饰细胞功能的非免疫原性选择工具以及追踪标记。在本文中，其一方面可作为CAR-T细胞的标记分子，另一方面还可以在需要时用于清除体内的CAR-T细胞，例如通过EGFR抗体(例如，西妥昔单抗)介导的ADCC途径(参见US8802374B2)，即在临床转化时作为安全开关使用。
“HSV-TK”为Herpes Simplex Virus Thymidine Kinase(人类单纯疱疹病毒胸苷激酶)的缩写，其底物是小分子药物GCV。关于HSV-TK和GCV作为分子开关的作用，在下文有更详细描述。
“自剪切肽”指可经核糖体跳跃而非蛋白酶水解来实现剪切蛋白的功能的短肽，可包括T2A、F2A和P2A等。
“治疗”指对受试者进行处理以获得有益的或所期望的临床结果。本文所用的“治疗”涵盖各种处理手段，包括以任何可能的药物向受试者给药、手术、辐射等。出于本发明的目的，有益或所期望的临床结果包括但不限于以下的任一种或多种：减轻一种或更多种症状、减弱疾病程度、预防或延迟疾病扩散(例如转移，例如转移至肺或淋巴结)、预防或延迟疾病复发、延迟或减缓疾病进展、改善疾病病况、抑制疾病或疾病进展、阻滞其发展和缓解(无论部分抑或完全缓解)。本文所提供的方法涵盖这些治疗方面中的任一种或多种。按照以上内容，“治疗”不需要完全去除病症或疾病的所有症状或完全缓解。
术语“治疗有效量”指足以在受试者体内引起临床医师所期望的生物学或医学反应的活性化合物的量。本发明融合蛋白的“治疗有效量”可由本领域技术人员根据给药途径、受试者的体重、年龄、病情等因素而确定。例如，典型的日剂量范围可以为每kg体重0.01mg至100mg或更多活性成分。
提及药物组合物，所使用的术语“药学上可接受的载体”指可以安全地进行施用的固体或液体稀释剂、填充剂、抗氧化剂、稳定剂等物质，这些物质适合于人和/或动物给药而 无过度的不良副反应，同时适合于维持位于其中的药物或活性剂的活力。依照给药途径，可以施用本领域众所周知的各种不同的载体，包括，但不限于糖类、淀粉、纤维素及其衍生物、麦芽糖、明胶、滑石、硫酸钙、植物油、合成油、多元醇、藻酸、磷酸缓冲液、乳化剂、等渗盐水、和/或无热原水等。本文所提供的药物组合物可以制成粉末、注射剂等临床可接受的剂型。可以使用任何适当的途径向受试者施用本发明的药物组合物，例如可通过口服、静脉内输注、肌肉内注射、皮下注射、腹膜下、直肠、舌下，或经吸入、透皮等途径给药。
“药物试剂盒”指包括至少两种活性成分的药物组合。不同于药物组合物，药物试剂盒中至少一种活性成分与其他活性成分分开保存。
“受试者”指动物，例如哺乳动物，包括(但不限于)人类、啮齿动物、猿猴、猫科动物、犬科动物、马科动物、牛科动物、猪科动物、绵羊、山羊、哺乳类实验动物、哺乳类农畜、哺乳类运动动物和哺乳类宠物。受试者可为雄性或雌性且可为任何适龄受试者，包括婴儿、幼年、青年、成年和老年受试者。在一些实例中，受试者指需要治疗疾病或病症的个体。在一些实例中，接受治疗的受试者可为患者，其患有与该治疗有关联的病症，或有风险患上该病症。在另一些实例中，受试者为健康个体或者为患有非所关注疾病的个体。在特定实例中，受试者为人类，诸如人类患者。该术语通常可与“患者”、“检测对象”、“治疗对象”等互换使用。
CD5-CD7通用CAR-T策略设计
为了解决GvHD问题，本发明人通过敲除TRAC和/或TRBC基因来让CAR-T的T细胞表面受体TCR不表达，进而不能识别宿主的细胞，如图1所示。
为了解决宿主免疫系统清除输入的CAR-T细胞，本发明人通过敲除CAR-T细胞的HLA分子来避免宿主的T细胞清除CAR-T细胞。HLA分子分为HLA-I和HLA-II两种类型，HLA-I分子被CD8阳性的毒性T细胞识别，是宿主T细胞清除CAR-T细胞的主要途径，而HLA-II分子被CD4阳性的T细胞识别，是宿主T细胞清除CAR-T细胞的辅助途径。因此发明人的HLA分子敲除有两个版本，一种是敲除HLA-I分子的B2M基因，第二种是同时敲除HLA-I分子的B2M基因和与HLA-II分子表达相关的CIITA基因，第二种版本的UCAR-T理论上会更好地避免宿主T细胞的清除，但HLA的敲除会激活宿主的NK，激活的NK也会清除UCAR-T。
巧妙的是，本发明人制备的CAR是针对CD5和CD7，CD7在NK细胞上也会表达，所以CAR在攻击肿瘤细胞的同时也能攻击宿主的NK细胞，这就解决了由于HLA敲除导致的NK激活问题。由于CAR-T本身也会表达CD5和CD7，因此为了避免CAR-T自杀，发明人将CAR-T细胞的CD5和CD7也同时敲除。HLA的敲除联合CAR作为攻击分子来减弱宿主的T和NK细胞对UCAR-T细胞的清除作用，从而使UCAR-T在体内能得到很好的扩增。
通用CAR-T的制备流程与普通CAR-T的制备流程基本一致，只是另外包括了基因敲除步骤。其大体流程为：T细胞分选、激活→电转敲除基因→慢病毒转导→敲除效率和CAR结构的整合效率检测→体外功能验证→细胞制剂冻存。
CD5-CD7 CAR结构
本文采用的CAR的结构如图2所示。CD7和CD5的单VH结构域(单域抗体)通过一个linker串联，然后紧接CD8α铰链区、TM转膜区、4-1BB和CD3ζ共刺激分子。
可在其后通过T2A连接EGFRt开关分子，T2A蛋白会将EGFRt开关分子与CAR结构分割开。如下文描述的，在一些情况下，更有利地是采用自杀基因作为分子开关，例如单纯疱疹病毒胸苷激酶(HSV-TK)基因。采用HSV-TK分子开关的CAR结构设计如图27A所示。
不依赖于免疫细胞的CAR-T开关分子
由于CD5-CD7通用CAR-T在清除肿瘤的同时也清除了患者体内的正常T/NK细胞，导致在治疗期间患者处于免疫缺陷状态，无法在正常环境下生存；另外CD5敲除的T细胞若在患者体内长期存在，则很容易导致自身免疫疾病。因此，需要对CD5-CD7通用CAR-T在受试者体内的存在或增殖情况进行控制，这可通过使用分子开关来实现。
目前常用的CAR-T开关分子是EGFR，可通过抗EGFR的抗体西妥昔单抗来清除CAR-T细胞，但此方法需要NK等免疫细胞的参与才能实现。发明人的CD5-CD7 UCAR-T由于会将患者体内的NK细胞也杀死，因此EGFR开关不再适用于CD5-CD7 UCAR-T。
不依赖于免疫细胞的CAR-T开关分子通常是通过小分子药物去控制CAR-T中的某个元件来实现启动或关闭(杀死)CAR-T细胞，Ali Can Sahillioglu Ton和N Schumacher于2022在Current Opinion in Immunology上发表的文章8总结了目前研究发现的CAR-T小分子药物开关，根据作用机理大致可分为7类，如图3所示，包括自杀基因、转录调控、稳定性控制等。
本发明人的CD5-CD7 UCAR-T的其中一个版本即采用了自杀基因开关中的HSV-TK。HSV-TK是Herpes Simplex Virus Thymidine Kinase(人类单纯疱疹病毒胸苷激酶)的简称，其底物是小分子药物GCV(Ganciclovir，更昔洛韦)，GCV由美国Syntex公司推出，于1988年批准上市，为治疗巨细胞病毒感染的首选药物。HSV-TK联合GCV治疗巨细胞病毒感染的原理是：HSV-TK能高效地和GCV结合并对其进行一磷酸化，随后细胞内的激酶对其进行二磷酸化和三磷酸化，三磷酸化的GCV结构与细胞内的核苷极其类似，因此会竞争性地与DNA聚合酶结合或打破细胞内四种核苷的比例而抑制DNA的合成，DNA合成受阻后细胞会渐渐地凋亡。
因此，在一方面，本发明人针对TRAC/B2M/CD5/CD7/CIITA基因，利用CRISPR/Cas9系统的敲除功能，成功筛选出敲除效率高、脱靶概率低的sgRNA。
另一方面，本发明人针对TRBC/B2M/CD5/CD7/CIITA基因，利用CRISPR/Cas9系统的碱基编辑功能，成功筛选出可通过在基因的重要区域引入终止密码子来实现基因沉默的sgRNA。
另一方面，本发明人通过sgRNA的筛选以及电转工艺的优化，成功实现多个基因同时高效率的敲除(每个基因的敲除效率在90％左右)。
另一方面，本发明人成功制备出在体外/体内的杀瘤功能强、扩增能力强的CD5-CD7 UCAR-T。
另一方面，本发明人利用HSV-TK、联合GCV小分子药物，成功实现在体内高效清除UCAR-T细胞。
通用CD5-CD7 CAR-T与自体CD5-CD7 CAR-T相比可以存在如下区别：1)敲除的基因不一样：自体CD5-CD7 CAR-T只需敲除CD5和CD7基因，而通用的CD5-CD7 CAR-T除了敲除CD5和CD7基因外，还需敲除TCR和HLA的相关基因；2)通用CAR-T的制备过程中，可能需要对UCAR-T进行纯化，将表达TCR和HLA复合物的CAR-T细胞筛选掉，保证进入患者体内的UCAR-T细胞完全不表达TCR和HLA复合物，避免引起HvGD或者GvHD毒副作用；3)适应症不完全一样：自体的CD5-CD7 CAR-T只适用于肿瘤细胞未侵犯到外周血的T细胞淋巴瘤，而通用的CD5-CD7 CAR-T则适用于所有类型的T细胞恶性肿瘤。
以下通过具体实施例来进一步说明本发明。
实施例中涉及的通用方法如下：
1.CD3+T细胞的分选和激活
复苏冻存的健康供体(具体信息保密)PBMC共1.0×108个细胞每管，快速融化后重悬于8ml预热的Rinsing buffer中，取少量细胞悬液进行细胞计数。将PBMC悬液以400g离心(↑8↓8)10分钟。离心结束后，弃上清，加入20ul/107的CD3微珠，混匀后放入4℃冰箱孵育20分钟，期间每10分钟轻弹管壁数次避免细胞沉淀。孵育结束后，加入Rinsing buffer润洗1遍后离心(400g 10min↑8↓8)，再用500μl Rinsing buffer重悬细胞。同时将LS分选柱放置在美天旎磁力分选架上，用2ml Rinsing buffer润洗润洗1遍后，加入500μl的细胞悬液，待细胞悬液滴尽后反复2次加入2ml Rinsing buffer于LS柱上。用5mL Rinsing buffer将目的细胞从LS柱上冲出并收集，做适当稀释后对目的细胞进行计数，取约1×105个细胞以流式细胞术确定分选的T细胞的纯度。随后将细胞悬液300g离心10分钟，用新鲜T细胞培养基将细胞密度调整至1×106,以10ul/106个细胞的浓度加入抗-CD3/-CD28抗体磁珠激活，按每孔4mL，种入到12孔板中，放入37℃，CO2培养箱中进行培养。
2.激活电转
对于CD3/CD28 Dynabeads激活的细胞，激活24h后即可进行电转。细胞收集于离心 管中，置于磁力架上去除Dynabeads，反复过3遍，然后将细胞离心(300g 15min升8降8)；结束后，弃上清，用适量的复方电解质将细胞重悬到一起，取细胞计数；根据细胞计数结果配制相应量的RNP(Cas9蛋白和sgRNA的复合物)，37℃孵育10分钟以上；同时将细胞再次离心，结束后用相应量的电转buffer重悬细胞，加入孵育好的RNP，轻轻混匀后加入Lonza电转仪配套的电转杯中，选择电转激活T细胞的程序EH-115，电转，然后立即加入少量的经温热后的T细胞培养基，放入培养箱中恢复15分钟以上，再将细胞悬液从电转杯中转出至合适的培养瓶中，加入T细胞培养基使培养密度为2M/ml。
3.Pan-T细胞阴选
复苏冻存的健康供体PBMC共1.0×108个细胞每管，快速融化后重悬于8ml预热的Rinsing buffer中，取少量细胞悬液进行细胞计数。将PBMC悬液以400g离心(↑8↓8)10分钟；离心结束后，弃上清，加入Rinsing buffer(按40ul/10^7cells)和Pan T Cell Biotin-Antibody Cocktail(10ul/10^7cells)，用枪头轻轻吹打混匀后，放入4℃冰箱孵育10min，期间轻弹管壁一次避免细胞沉淀；然后加入Rinsing buffer(按30ul/10^7cells)和Pan T Cell MicroBead Cocktail(20ul/10^7cells)，用枪头轻轻吹打混匀后，放入4℃冰箱孵育15min，期间每隔5min轻弹管壁数次避免细胞沉淀；磁珠孵育的同时取出LS分选柱(阳性载量100M/柱)装置在美天旎磁力分选架上，用3ml的Rinse buffer润洗分选柱一次；磁珠孵育结束后，补加适量的Rinse buffer，细胞混匀后均分至每个LS中，每个柱子2ml；待细胞悬液滴尽后用2ml Rinsing buffer洗柱子2次，流下的阴性细胞为T细胞，从细胞中取出100ul，以900ul buffer稀释10倍后，取20ul进行计数；根据计数结果，用不含细胞因子的T细胞培养基静息培养T细胞4h后进行电转，培养密度为5M/ml。
4.静息电转
阴选出的T细胞静息培养4h后，收集细胞于离心管中，取细胞计数，根据细胞计数结果，配制相应量的RNP(Cas9蛋白和sgRNA的复合物)，37℃孵育10分钟以上；同时将细胞离心，结束后用相应量的电转buffer重悬细胞，加入孵育好的RNP，轻轻混匀后加入Celletrix电转仪配套的电转杯中，设置电转静息T细胞的相应参数(100ul体系，1380V,3ms)，电转，然后立即将细胞吸出至培养瓶中(培养瓶中的培养基提前加好CD2/CD3/CD28激活剂并温热)，此时勿吹打细胞，然后将细胞放入培养箱中培养，培养密度为2M/ml。
5.CAR的慢病毒转导
细胞激活48小时后，进行CAR的慢病毒转导。对细胞悬液进行活率检测和细胞计数，根据细胞计数结果加入相应量的慢病毒，MOI为3，再加入100x的lentiboost助，轻轻混匀后，37℃培养箱中继续培养。24小时后换液去除病毒，换新鲜的培养基继续培养T培养细胞，密度为1M/ml。
6.FACS(流式细胞术)检测
取约2×10^5个细胞悬液于1.5ml离心管中，300g离心5分钟，用PBS+2％胎牛血清 缓冲液洗1遍，完全弃去上清，用100μl缓冲液重悬细胞后加入相应抗体1μl，混匀后4℃避光孵育30分钟，加入100ul缓冲液洗一遍后，用100ul含DAPI或者7AAD的缓冲液重悬后上机检测。
7.TRAC/B2M阴选
在细胞制备的第六天左右，TRAC和B2M成功敲除的细胞已不表达相应的蛋白，因此可对细胞进行阴选，将TRAC和B2M成功敲除的细胞给分离出来继续培养。具体步骤如下：收集细胞室温离心(400g 15min，升8降8)，结束后弃上清，用Rinsing Buffer重悬细胞(80ul/10^7cells)，加入FITC-B2M抗体(1ul/10^6cells),四度避光孵育20min；孵育完毕后，加40ml的Rinsing Buffer重悬细胞，室温离心，结束后弃上清，用Rinsing Buffer重悬细胞(80ul/10^7cells)，加FITC-beads(1ul/10^6cells)，加CD3-beads(按1ul/10^6cells),四度避光孵育20min；孵育完毕后，加40ml的Rinsing Buffer重悬细胞，室温离心，同时用3ml的buffer润洗LD柱子；离心结束后弃上清，用Rinsing Buffer(1*10^8/ml)重悬细胞，过柱子2遍(2ml/柱子)，然后用3ml的buffer洗柱子2遍，收集流下的阴性细胞，计数，取少量细胞用于FACS检测敲除效率、CAR阳性率和分选的纯度等，将阴性细胞离心后用500ml-1000ml的T细胞完全培养基培养于G-rex培养瓶中。
8.CD107a释放检测
提前配制需要量的T细胞完全培养基，往培养基中加入100x的PE/Cy7 mouse anti-human CD107a抗体和1000x的monensin；对UCART细胞和多种肿瘤细胞计数，按1:1的比例(UCAR-T细胞按CAR阳性的细胞算)分别取细胞离心(一般CAR阳性细胞在0.1M以上)，然后用含有CD107a抗体和monensin的培养基重悬，接种于96孔板中，放入细胞培养箱中(37℃5％CO2)孵育4h；然后对细胞进行FACS染色，一般还需染CD8和EGFR抗体，通过FACS检测计算CD8阳性和CAR阳性的T细胞下的CD107a阳性细胞比例。
9.肿瘤杀伤检测
一般地，将UCAR-T细胞和多种表达luciferase(萤火虫荧光素)的靶向和非靶向肿瘤细胞以不同的效靶比(UCART细胞按CAR阳性的细胞算，固定肿瘤细胞的量)接种于96孔板中，在细胞培养箱中共培养24h后取出一半的细胞悬液于不透光的白底96孔板中，加入10x的luciferase底物混匀，室温反应10分钟后上机检测luciferase荧光值，通过荧光值间接地反应肿瘤细胞的存活数量，进而反应出UCAR-T细胞的杀瘤功能。
10.体外检测GCV的药效
UCAR-T细胞计数，取8M的细胞离心，结束后弃上清，用8ml新鲜的T细胞培养基重悬，均分至12孔板的8个孔中，每孔1ml，然后两个孔为一组，分别加入DMSO、0.3ug/ml、1ug/ml、3ug/ml的GCV，细胞于培养箱中培养，之后每3天通过计数和FACS检测来监测CAR阳性细胞的变化。同时，每3天收集细胞沉淀提基因组，通过荧光探针qPCR法检测DNA水平的CAR分子变化情况。
11.Guide-seq脱靶检测
通过电转将待检测sgRNA的RNP和dsODN导入进激活的T细胞中，电转后5-7天，提取带有dsODN整合的基因组；通过Covaris S220仪器将基因组打断成平均500bp的片段，用0.8x Ampure XP beads对基因片段纯化回收，再用UltraTM II DNA Library Prep Kit for试剂盒对纯化后的基因组片段进行末端修复并加A，使用UltraTM II DNA Library Prep Kit for试剂盒对已加A的DNA片段连接linker序列，每个样品连接不同的linker，再使用0.8x Ampure XP beads纯化；接下来进行PCR，引物分别位于linker序列和dsODN上，对PCR产物进行回收，然后使用ABclonal Rapid DNA Lib Prep Kit连接通用的P5和P7Y型adaptor，产物纯化回收后即可用于二代测序。通过分析测序结果，将linker序列和dsODN序列之间的基因组序列与sgRNA序列比对，即可得到脱靶的位点信息以及脱靶概率。
12.SgRNA设计和编辑效率验证
sgRNA可通过许多网站进行设计，如CHOPCHOP(https://chopchop.cbu.uib.no/),CRIS Pick(https://portals.broadinstitute.org/gppx/crispick/public),GUIDES(http://guides.sanjanalab.or  g/#/),CRISPOR(http://crispor.tefor.net/)，IDT(https://sg.idtdna.com/site/order/designtool/index/ CRISPR_SEQUENCE)等，这些网站均可预测On-target和Off-target的效率。
一般步骤如下：
1)查找目的基因的基因组序列：可在NCBI(https://www.ncbi.nlm.nih.gov/)和UCSC(https://genome.ucsc.edu/)网站上查看和下载目的基因的基因组序列；
2)sgRNA预测以及合成：将目的基因的目标序列输入上述sgRNA预测网站，生成诸多候选的sgRNA，根据On-target和Off-target的排名选择和适量量的sgRNA序列，将sgRNA的靶向序列(20nt左右)告诉第三方公司让其合成全长的sgRNA。
候选sgRNA编辑效率验证：候选sgRNA合成好后，可直接通过电转将Cas9蛋白和sgRNA的复合物RNP(ribonucleoprotein)导入目的细胞，电转后24h-48h即可通过PCR和测序检测基因水平上的编辑效率，72h后可通过FACS检测蛋白水平的变化情况。
实施例中涉及的主要试剂如下：


实施例中涉及的主要耗材如下：
实施例1利用CRISPR/Cas9技术敲除T细胞表面的TRAC/TRBC、B2M、CIITA、CD5、CD7抗原
1.1 TRAC、B2M、CIITA、CD5、CD7 sgRNA筛选
1.1.1 TRAC sgRNA筛选结果
发明人通过CRISPick、CRISPOR和IDT等常用的sgRNA设计网站得到诸多可能的sgRNA信息，然后综合考虑在靶和脱靶等信息后选出7条候选sgRNA(图4A)。将7条候选sgRNA的靶向序列告知技术服务公司(南京金斯瑞生物科技有限公司)让其合成全长的sgRNA(靶向序列+骨架序列)。
SgRNA合成好后，用nuclease-free的水将其溶解为100-500pmol/μl，然后与Cas9蛋白以不同的用量混合后37℃孵育10分钟以上，再通过lonza电转仪电转，将Cas9与sgRNA的复合物导入T细胞中，细胞培养6天后，用FACS检测TCR复合物的表达情况，从而反应出不同sgRNA对TRAC的敲除效率。从图4B可以看出，在较高RNP用量(Cas9 60pmol,sgRNA 150pmol)下，所有7条sgRNA的敲除效率均可达到90％以上，但在较低RNP用量(Cas9 30pmol,sgRNA 30pmol)下，只有部分sgRNA的敲除效率可达 到90％以上，其中A-XL这条sgRNA的敲除效率最高(99.5％)，因此发明人选择此条sgRNA作为后续制备UCAR-T使用。A-XL这条sgRNA也曾被多个研究者使用4,9。
发明人用Guide-seq方法对A-02、A-06和A-XL这三条sgRNA进行了脱靶检测，结果如图4C-E所示，A-XL的两次检测结果中均出现了有一个概率较高的脱靶位点，A-02的一次检测结果显示也有概率较高的脱靶位点，A-06的脱靶概率稍低，因此该版本的UCAR-T后续可能会倾向于使用A-06这条sgRNA。
1.1.2 B2M sgRNA筛选结果
如图5A是发明人通过CRISPick、CRISPOR、IDT、CHOPCHPOP和GUIDES几个网站预测后，综合考虑选出的候选sgRNA信息。同样用电转的方法将Cas9蛋白和sgRNA复合物导入T细胞中，电转后8天取出适量细胞，用流式细胞仪检测B2M的表达情况，从而反应出不同候选sgRNA对B2M的敲除效率。从图5B可以看出，B-03的敲除效率最高(90％以上)，B-10、B-13和B-XL的敲除效率相当(80％以上)，而B-01的敲除效率太低(50％左右)。
同时，发明人将单条B2M-sgRNA与已测试好的TRAC-sgRNA(A-XL)一起做双敲，以期筛选出TRAC-B2M双敲效率比较好的B2M-sgRNA。同样地，发明人将Cas9蛋白和TRAC-sgRNA、B2M-sgRNA混合孵育10分钟后通过电转导入T细胞中，电转后8天取出适量细胞，用FACS检测B2M和TCR复合物的表达情况。从图6A可以看出，在TRAC和B2M双敲情况下，还是B-03表现最优(双敲效率93％)，其次是B-13和B-XL(双敲效率91％)，B-10稍差(双敲效率88％)，而B-01还是最差(双敲效率50％)，因此B-01这条sgRNA在后续的测试中排除掉。
接下来，发明人对在双敲测试中表现比较好的TRAC/B2M(B-03、B-10、B-13、B-XL)sgRNA组合进行不同RNP浓度下的敲除效率测试，以期筛选出在较低RNP浓度下实现TRAC-B2M双敲效率在90％以上的sgRNA组合。具体地，发明人分别用30pmol+30pmol，60pmol+60pmol，60pmol+150pmol的Cas9蛋白和sgRNA(每组中TRAC和B2M的RNP用量相同)进行测试，结果如图6B所示，在较高浓度的RNP(60pmol+150pmol)下，所有4组sgRNA组合均能实现90％以上的双敲效率，但在较低浓度的RNP(60pmol+60pmol，30pmol+30pmol)下，只有B-03和B-13才能实现可以接受的双敲效率(80％以上)；在30pmol+30pmol的RNP浓度下，B-13的表型优于B-03，而在60pmol+60pmol的RNP浓度下，B-03和B-13的效率相当，但双敲效率均没有达到90％以上，考虑到B-03和B-13在B2M的不同外显子上，发明人决定同时使用B-03和B-13来实现B2M的敲除。
发明人对B-03和B-13这两条sgRNA进行了脱靶检测，结果如图6C-D所示，B-13的两次检测结果中最高脱靶概率的序列相同，说明此位点极有可能出现脱靶；而B-03的 两次检测结果测到的最高脱靶概率的序列不一样，其中一次的最高脱靶概率为3％，而另一次则只有0.01％，表明B-03这条sgRNA的脱靶概率较低。
1.2.3 CD5 sgRNA筛选结果
针对CD5的敲除sgRNA，发明人总共进行了两次sgRNA的筛选。图7A和7B是第一次筛选时测试的sgRNA序列信息以及相应的敲除效率结果，其中sgRNA1和sgRNA3的敲除效率不错，但此前未对所选择的sgRNA进行脱靶分析，为了找出敲除效率高且脱靶低的sgRNA，本发明人再次设计了5条sgRNA与sgRNA1和sgRNA3一起进行敲除效率和脱靶率测试。图7C是发明人第二次筛选时通过CRISPick、CRISPOR、IDT、CHOPCHPOP和GUIDES几个网站预测后，综合考虑选出的候选sgRNA信息，其中C-01和C-03为图7A中的sgRNA1和sgRNA3。
SgRNA合成好后，发明人首先进行敲除效率的测试，方法与上述其它sgRNA的筛选相同，结果如图8A-B所示，除C-05外，其它sgRNA的敲除效率均在80％以上。根据网站CRISPick预测的脱靶结果显示，C-06的脱靶概率最低(图8C)，因此发明人用Guide-Seq方法对C-06的实际脱靶概率进行了评估，结果如图8D所示，两次的检测结果显示最高脱靶概率的序列相同，但脱靶的概率只有千分之四以下，且脱靶位点基本位于基因内含子区域，极低概率影响蛋白编码，综上所述，发明人选择C-06在后续实验中使用。
1.2.4 CD7 sgRNA
本发明中使用的CD7 sgRNA来自已报道的文献10，本发明人对其进行了脱靶检测。如图9所示，一次实验的脱靶检测中发现有3个序列出现的频次较高，但其中的两个位点也出现在了未使用sgRNA的对照组中，且在两者中出现的频次基本一致，所以可以判定为假阳性；而出现频次第二高的序列与sgRNA序列比起来，突变碱基的数量较多且位置离NGG比较近，所以也不太可能出现脱靶，综上所述，CD7 sgRNA出现脱靶的概率极低。
1.2.5 CIITA sgRNA筛选结果
针对CIITA基因，发明人选择了5条sgRNA进行敲除效率测试，结果如图10A所示，F-05的敲除效率最好。
发明人对F-05这条sgRNA进行了脱靶检测，结果如图10B所示，两次实验中检测到的最高脱靶概率的序列不一致且出现的概率均为0.1％左右，因此F-05的脱靶概率极低。
1.2基于Cas9蛋白敲除T细胞表面的TRAC、B2M、CD5、CD7抗原
在制备CD5-CD7 UCAR-T之前，发明人首先进行了TRAC、B2M、CD5、CD7共敲的RNP条件摸索。如图11所示，共设置了6组RNP用量以及在2个Donor的T上进行了测试，结果，在所有6个条件下，TRAC、B2M、CD5、CD7的敲除效率均在90％及以 上，且在2个donor之间无明显差异，因此发明人选择用量最小的RNP组合6作为后续制备CD5-CD7 UCAR-T的条件基础。
第一次制备CD5-CD7 UCAR-T时，发明人为了保证CD5的敲除效率，在RNP组合6的基础上，将CD5的RNP用量从40/120pmol/20ul体系调高至80/150pmol/20ul体系，结果如图12A-B所示，敲除后9天用FACS检测各基因的敲除情况，结果TRAC/B2M双敲效率为97.8％，CD5/CD7双敲效率为95.8％。TRAC、B2M、CD5、CD7的敲除效率均非常高，因此发明人在后续制备CD5-CD7 UCAR-T时均采用此电转条件。
图12C为发明人第二次制备CD5-CD7 UCAR-T时KO未转毒组在敲除后4天检测的敲除结果，在未转毒的情况下，TRAC/B2M双敲效率为86.7％，CD5/CD7双敲效率为90.6％，敲除效率已经非常高，而转毒后UCAR-T还会杀死一部分CD5或CD7阳性的细胞，所以TRAC/B2M和CD5/CD7的双敲效率在UCAR-T组中还会更高。
1.3基于Cas9蛋白敲除T细胞表面的TRAC、B2M、CIITA、CD5、CD7抗原
为了让UCAR-T在体内更有生存优势，发明人还制备了另外一个版本的CD5-CD7 UCAR-T,即在敲除TRAC、B2M、CD5、CD7的基础上还敲除了CIITA基因，敲除CIITA基因后UCAR-T不表达HLA-II类分子，可避免宿主体内的CD4+T细胞清除UCAR-T。
发明人在制备第二版本的CD5-CD7 UCAR-T之前，首先在Celletrix电转仪上进行了TRAC、B2M、CIITA、CD5、CD7五个基因同时敲除的预实验，RNP用量和敲除结果如图13A-B所示，在敲除后5天FACS检测各蛋白的表达情况，结果TRAC/B2M双敲效率为69.9％，TRAC/CIITA双敲效率为62.8％，CD5/CD7双敲效率为85.3％。根据分群来看，TRAC/B2M和CD5/CD7双敲效率均可以接受，但TRAC/CIITA的双敲效率稍低。
根据预实验结果，发明人在制备第二版本的CD5-CD7 UCAR-T时，加大了B2M和CIITA基因的RNP用量，如图13C-D所示，在敲除后4天FACS检测各蛋白的表达情况，结果TRAC/B2M双敲效率为87.1％，TRAC/CIITA双敲效率为85.2％，CD5/CD7双敲效率为90.3％，每个基因的敲除效率与预实验比起来均有所提升。
实施例2基于nCBE3/nCBE4蛋白敲除T细胞表面的TRBC、B2M、CIITA、CD5、CD7抗原
除了利用Cas9蛋白敲除基因，发明人还尝试了利用碱基编辑蛋白nCBE3或nCBE4在基因的外显子区域引入终止密码子来破环基因的表达。nCBE3或nCBE4蛋白是在Nikase-Cas9蛋白(只切割target链)的N端融合表达了一个胞嘧啶脱氨酶(ABOBEC)和在C端融合表达了一个或者两个尿嘧啶糖基化酶抑制剂(UGI)，Nikase-Cas9在切割target链的同时，ABOBEC可对靶向区域中non-target链的胞嘧啶(C)脱氨进而转变为尿嘧啶(U)，而UGI可以维持尿嘧啶(U)的稳定，target链被切割后发生修复，这条链 所对应的G会以U为模板修复成A，DNA在之后的复制或修复中会将non-target链上的U替换为T,最终实现C到T的转变。因此，利用nCBE3或nCBE4可将外显子区域正义链上的CAA、CAG、CGA突变为TAA、TAG、TGA，或将反义链上的CCA突变为TTA(对应正义链为TAA)，提前引入终止密码子而破环蛋白的表达。
sgRNA筛选
根据以上原则，发明人针对TRBC、B2M、CD5、CD7、CIITA基因设计了多条候选sgRNA，序列信息如图14所示，其中CD7的sgRNA已有文献报道且效率很高11，因此未对CD7进行更多的sgRNA筛选。
碱基编辑的sgRNA效率测试方法同敲除的sgRNA，电转后5天通过流式染色看蛋白的表达情况。如图15所示，针对TCR的敲除，BE-A02、BE-A03的效率最高且两者相当，考虑到BE-A02已有文献报道11，发明人选择了BE-A03作为后续使用的sgRNA。
如图16A所示，针对B2M的敲除，3条sgRNA的效率都不是很理想，但也没有了更多符合要求的sgRNA选择。考虑到nCBE3或nCBE4可以切割DNA一条链的特性，发明人还尝试了利用2条相近且分别位于正义链和反义链上的sgRNA来实现基因的敲除，方案设计和结果如图16B-C所示，B-10和B-13的组合效率最高，因此发明人选择结合BE-B02和B-10、B-13来实现B2M的敲除。
针对CD5的敲除，结果如图17A所示，BE-C07的效率最高，达到了90％以上，因此发明人选择了BE-C07。发明人同时也验证了文献中使用的CD7-sgRNA，敲除效率为87％左右(图17B)。
针对CIITA的敲除，发明人测试了11条sgRNA，结果如图18所示，在敲除后第7天，BE-F04和BE-F10的效率较好，其中BE-F10的效率最好，敲除效率在80％以上，因此发明人最终选择BE-F10。
实施例3慢病毒转导及CAR阳性细胞比例检测
一般地，在T细胞激活48h后进行CAR的慢病毒转导，病毒一般按MOI＝3的用量使用，同时加入慢病毒转导增强剂Lenti-boost(100倍稀释使用)，病毒转导后24h换液，3天后即可检测CAR阳性细胞比例，CAR阳性细胞比例可以通过FACS检测细胞的EGFR或者CAR的表达情况来反应。
如图19是两次制备的CD5-CD7 UCAR-T的CAR阳细胞比例检测结果，分别通过Anti-EGFR-PE、FITC偶联的CD5和CD7抗原检测细胞的EGFR和CAR的表达情况。转毒后3天检测，一般EGFR的阳性率会高于CD5和CD7 CAR阳性率，这可能是由于前期UCAR-T杀伤CD5和CD7未敲除的细胞后CAR内吞导致，后期如day8检测时三者代表的CAR阳细胞比例就会比较一致。CD5-CD7 UCAR-T中的CAR阳性细胞由于存在自激活(杀伤CD5或CD7未被敲除的T)，因此CAR阳细胞比例会随着培养天数的增加而增加，从day3到day8CAR阳细胞比例会增加一倍左右。
实施例4 TRAC/B2M阴选
UCAR-T细胞最重要的是避免发生免疫排斥反应，所以回输给患者的UCAR-T成品细胞通常情况下必须保证不表达TCR和HLA-I复合物，否则会发生严重的GvHD或HvGD，因此，发明人在制备CD5-CD7 UCAR-T时，一般会在第六天左右对UCAR-T细胞进行TRAC和B2M的阴选，具体步骤见上文通用方法里的“TRAC/B2M阴选”。
图20为两次制备CD5-CD7 UCAR-T期间的TRAC/B2M阴选结果，从图中可以看出，没进行阴选前的input细胞均会有TCR和B2M的少量表达，而阴选后的阴性细胞(negative)几乎不会有TCR和B2M的阳性，一般要保证TRAC/B2M双阴性细胞比例在99％以上才算合格。
实施例5通用CAR-T细胞的体外功能验证
一般地，CAR-T细胞的功能可通过体外将CAR-T细胞和肿瘤靶细胞一起共孵育，4-6小时后通过FACS检测CAR-T细胞的CD107a释放情况或者24h-48h检测肿瘤细胞的多少来反应CAR-T细胞的杀伤功能是否好。
5.1 CD107a释放检测
CD107a又称LAMP-1，是CD8+毒性T细胞和NK细胞在杀伤靶标细胞过程中释放的一种蛋白，因此可通过检测CAR-T细胞的CD107a释放情况来反应CAR-T细胞是否具有杀伤功能。
一般地，发明人会将CD5-CD7 UCAR-T与多种CD5和CD7阳性的肿瘤细胞在无细胞因子的T细胞培养基中共孵育，效靶比按CAR阳性的细胞算为1:1，共孵育4-6小时后，收集细胞进行FACS染色，用流式细胞仪分析CD8和CAR双阳性细胞中CD107a阳性的细胞比例，比例越高，说明UCAR-T被靶细胞激活的越强，分泌的肿瘤杀伤因子越多，但CD107a阳性细胞比例越高并不代表其杀伤肿瘤的能力越强，只能初步认为UCAR-T具有杀伤肿瘤细胞的能力。
如图21A-B所示，CD5-CD7 UCAR-T能被不同的肿瘤靶细胞激活而释放CD107a，而KO-T因没有CAR则不会被激活。图21B和21C是两次制备的CD5-CD7 UCAR-T与不同的肿瘤细胞共孵育4小时后，CD8和CAR双阳性细胞中CD107a阳性细胞的占比情况，不同的肿瘤细胞刺激后CD107a阳性细胞的占比不同。CD5或CD7阳性的肿瘤细胞刺激后CD107a阳性细胞的比例在50％左右，但阴性靶细胞组(Raji)和no tumor组也有20％左右的CD107a阳性细胞，这可能是由于CD5-CD7 UCAR-T的自激活导致。
5.2体外杀伤肿瘤细胞实验
制备出的UCAR-T，其杀瘤功能可以在体外进行初步鉴定。一般地，将UCAR-T和肿瘤靶细胞(稳定表达luciferase蛋白)以不同的效靶比混合后共培养24小时，通过检测 共培养后细胞中的luciferase蛋白表达量来反应肿瘤细胞的存活情况，进而判断UCAR-T的杀瘤功能。
图22A-C是第一次制备的CD5-CD7 UCAR-T的杀瘤情况，图22A和22B是UCAR-T和KO-T细胞在制备期间的第7天的杀瘤检测结果，将UCAR-T和KO-T以3种效靶比和不同的肿瘤靶细胞共培养24小时后进行检测，从图可以看出KO-T不能杀伤肿瘤细胞而UCAR-T可以很好地杀伤肿瘤细胞；在低效靶比(0.2:1)下，CD5-CD7 UCAR-T杀伤双阳和单阳靶细胞的能力依次为CD5+/CD7+>CD5+/CD7->CD5-/CD7+，即双CAR-T的杀瘤能力强于单CAR-T；图22C是UCAR-T冻存复苏后24小时检测其杀瘤功能的结果，从图可以看出细胞冻存后再复苏不影响其杀瘤功能。
图22D-F是第二次制备的CD5-CD7 UCAR-T的杀瘤结果，图22D和22E分别是UCAR-T和KO-T细胞在制备期间的第10天的杀瘤检测结果，肿瘤细胞与UCAR-T共培养24小时后只有低效靶比下肿瘤细胞有残留，而与KO-T共培养24小时后几乎不会被杀死；图22F是UCAR-T冻存复苏后24小时的杀瘤结果，结果与冻存前一致。
实施例6通用CAR-T细胞表型鉴定
6.1通用CAR-T细胞的活化、分型、耗竭等指标检测
制备好的UCAR-T，在冻存前一般需要对其进行各项指标的检测，通过细胞的激活状态、分型、耗竭等指标来判断最终状态的UCAR-T是否有异常。
图23A-B展示了两次制备的CD5-CD7 UCAR-T在冻存前细胞的CD3/CD4/CD8细胞比例以及激活状态。图23A中的制剂细胞未进行TRAC/B2M的阴选，因此CD3指针的TCR复合物还有少量细胞表达，而图23B中的制剂细胞由于进行了TRAC/B2M的阴选，因此CD3染色呈完全阴性；一般来说，UCAR-T在制剂前(体外培养10-12天)，CD8阳性细胞的比例是CD4阳性细胞的2倍左右；CD5-CD7 UCAR-T中由于CAR阳性细胞存在自激活现象，所以CD25和CD69等激活maker仍会有部分表达。
图23C-D展示了两次制备的CD5-CD7 UCAR-T细胞在冻存前的细胞分型。CCR7是T细胞早期状态的maker，因此最终状态的UCAR-T细胞中CCR7阳性细胞的比例越高，理论上UCAR-T在回输到患者体内以后扩增会越好。图23C中的UCAR-T细胞是day10冻存，而图22D中的UCAR-T细胞是day12冻存，两次制备的工艺相同，从图可以看出day10冻存的细胞中CCR7阳性细胞的比例比day12冻存的高。
图24A-D展示了两次制备的CD5-CD7 UCAR-T在冻存前细胞的耗竭和早调状态，从图24A和图24B可以看出，LAG3和PD1两个耗竭maker的表达量均较低，表明最终状态的CD5-CD7 UCAR-T细胞中正在耗竭的细胞非常少；图24C和图24D中，PI和Annexin V双阳性表明细胞处于晚期凋亡状态，而PI阴性、Annexin V阳性表明细胞处于早期凋亡状态，从图可以看出最终状态的CD5-CD7 UCAR-T细胞中正在发生凋亡的细胞也很少。
综上所述，发明人制备出的CD5-CD7 UCAR-T细胞质量较好。
6.2通用CAR-T细胞的组分鉴定
除了上述的指标检测，发明人还对最终状态的UCAR-T进行了细胞组分鉴定，通过一系列的细胞maker检测，发明人发现最终状态的UCAR-T细胞中有部分CD56阳性的细胞(如图25A-B)，CD56通常认为是NK细胞的maker，但UCAR-T中的这部分细胞不表达NK的另一个maker CD16，且最终状态的UCAR-T细胞中无CD4和CD8双阴性的细胞，因此UCAR-T细胞中这部分CD56阳性的细胞被确定为NKT细胞，NKT细胞中也有部分CAR阳性的细胞，因此这部分CAR阳性的NKT细胞理论上也可以执行杀瘤功能。
不同批次的UCAR-T细胞中CD56阳性的细胞比例具有差异，范围在10％-20％左右，如图25中的A和B分别展示了两次制备的UCAR-T中的CD56阳性细胞的比例。
实施例7通用CAR-T细胞(EGFR开关)临床测试
图26展示了CD5-CD7 UCAR-T(EGFR开关)的一例临床测试结果，受试者是一名急性的T淋巴细胞白血病患者，男性，36岁，体重75kg。UCAR-T输注前给与的清淋处理是：30mg/m2的氟达拉滨和300mg/m2的环磷酰胺处理5天，然后30mg/m2的氟达拉滨和70mg/m2的马法兰再处理1天，让患者无处理休息3天后输注剂量(CAR阳性细胞)大于3.6e6/kg的CD5-CD7 UCAR-T细胞，之后每天取样检测患者外周血中CAR的拷贝数和淋巴细胞的变化情况。
如图26B所示，CD5-CD7 UCAR-T输注后患者外周血中的CAR拷贝数在2天之内有一定的减少，但2天之后开始增多，在第8天时达到峰值，之后在23天之内一直维持较高的水平；通过检测患者外周血中的淋巴细胞，发现总的UCAR-T细胞(图26C中的蓝色线)在淋巴细胞中的比例在输注后也一直在增加，第6天时达到了接近100％的比例，且CAR阳性细胞(图26C中的红色线)的扩增趋势也一致，而患者的T细胞(图26C中的黑色线)在UCAR-T输注后一直不断地减少，在第6天后已基本检测不到，以上的结果说明了发明人的CD5-CD7 UCAR-T在患者体内能很好地扩增且清除肿瘤细胞的效果非常好。
但从图26B和26C可以看出，从CD5-CD7 UCAR-T输注后的第8天起，患者外周血中的淋巴细胞已几乎全是CD5-CD7 UCAR-T，说明了患者的外周血中几乎没有自己的T和NK细胞，这就会导致患者没有正常的免疫功能，而EGFR开关对CD5-CD7 UCAR-T无效，因此此例患者后来接受了移植。
综上所述，发明人的EGFR开关版本的CD5-CD7 UCAR-T可以服务于因肿瘤负荷较高而无法做移植的T细胞淋巴瘤患者。为了拓展CD5-CD7 UCAR-T的应用范围，发明人对UCAR-T的开关做了替换改造，以期达到仅用CD5-CD7 UCAR-T就能完全治愈T细胞淋巴瘤患者的目的。
实施例8 HSV-TK分子开关的研究
8.1体外研究结果
8.1.1 CD5-CD7 UCAR-T(HSV-TK)细胞制备
鉴于EGFR开关对CD5-CD7 UCAR-T不起作用，我们尝试将EGFR替换为不依赖于患者自身免疫系统的小分子药物控制开关HSV-TK，如图27A所示，然后按激活电转(EGFR版本的工艺)和静息电转工艺分别制备了一批成品细胞(图27B-E)，图27B展示的是TRAC/B2M/CD5/CD7四基因的共敲除效率和CAR的转效，图27C展示的是细胞的分型，图27D展示的是细胞的组分。
通过比较可以发现两种工艺对TRAC/B2M/CD5/CD7四基因的共敲除效率是差不多的，只是对于静息电转工艺来说，TCR阳性的细胞比TCR阴性的细胞激活更充分，因此TCR阳性的细胞会有生长优势，若TCR细胞阴选不彻底，则残留的TCR阳性细胞比例会在后期慢慢变高(图27C)。
此外，静息电转工艺在day0时采用Pan-T阴选得到T细胞，将CD56阳性的细胞尽可能排除，因此在静息电转工艺得到的成品细胞中CD56阳性的细胞比例会低于采用CD3磁珠阳性的激活电转工艺得到的细胞(图27E)。至于细胞表型，CCR7阳性的细胞比例与细胞在体外的培养天数相关，培养天数越长，则CCR7阳性的细胞比例会越低(图27D)。
8.1.2 HSV-TK版本与EGFR版本UCAR-T之间的杀瘤效果比较
为了验证HSV-TK版本与EGFR版本UCAR-T之间的杀瘤功能是否有差异，发明人采用同一工艺在同一批次中制备出两种版本的UCAR-T，然后进行冻存和复苏，复苏24小时后与不同的肿瘤细胞以不同的效靶比共孵育24小时，通过检测肿瘤细胞的luciferase荧光值来反应UCAR-T的杀瘤能力。结果如图28所示，由同一工艺制备得到的HSV-TK和EGFR版本的CD5-CD7 UCAR-T对不同肿瘤细胞的杀伤能力是一样的，即使用不同的开关分子对CAR本身的杀瘤功能不产生影响。
8.1.3 HSV-TK联合更昔洛韦(GCV)药物对UCAR-T的清除效果测试
HSV-TK能高效地和GCV结合并对其进行一磷酸化，随后细胞内的激酶对其进行二磷酸化和三磷酸化，三磷酸化的GCV结构与细胞内的核苷极其类似，因此会竞争性地与DNA聚合酶结合或打破细胞内四种核苷的比例而抑制DNA的合成，DNA合成受阻后细胞会渐渐地凋亡。
发明人用不同浓度的GCV药物同时处理TK版本和EGFR版本的UCAR-T，每3天通过计数和FACS来检测CAR阳细胞的变化，结果如图28所示，GCV不影响EGFR版本的UCAR-T细胞生长(图29D-F)，只特异性地杀死含TK即CAR阳性的细胞(图 29A-C)。GCV浓度越高，CAR阳性的细胞死的越快越多，但即使在较低浓度(0.3ug/ml)的GCV处理下，随着时间的延长，CAR阳性的细胞也会全部慢慢地死掉。
8.2动物实验结果
为了在体内验证HSV-TK版本CD5-CD7 UCAR-T和GCV的药效，发明人开展了动物实验。实验设计如图30A-B所示：一共有30只小鼠，每只小鼠在UCAR-T输入的前第7天接种5x105个CCRF肿瘤细胞，随后随机分为5组，每组6只，在肿瘤细胞接种后的第7天，给其中2组小鼠输入2x106个HSV-TK版本的UCAR-T细胞(每只小鼠)，另外2组输入2x106个EGFR版本的UCAR-T细胞，剩下的一组输入相应体积的PBS作为对照，在UCAR-T细胞输入后的第5天开始，给予HSV-TK版本和EGFR版本组中的一组小鼠输注50mg/kg的GCV，每天一次，共处理3天，然后在不同时间点通过小鼠成像观察小鼠体内肿瘤细胞的变化情况。
结果如图30B-C所示，未输入UCAR-T细胞组的小鼠在第4天(UCAR-T输入后)时体内已有较多的肿瘤细胞，而输入了UCAR-T细胞组的小鼠体内的肿瘤细胞则非常少，且输入EGFR版本和HSV-TK版本组的小鼠之间无明显差异，说明两种版本的CD5-CD7UCAR-T之间的杀瘤功能均较好且无区别；用GCV药物处理3天后，HSV-TK版本组的小鼠体内的肿瘤细胞相对于其它3组是最多的，说明GCV对此组小鼠体内的UCAR-T细胞发挥了作用，而其它3组小鼠体内的肿瘤细胞差别不大，说明GCV只特异性地杀死含HSV-TK的UCAR-T细胞。
由于在此次实验中，UCAR-T细胞输入的比较晚(正常情况下UCAR-T细胞在肿瘤细胞接种后的1-3天输入CAR-T细胞)，所以UCAR-T细胞输入时可能肿瘤细胞的数目已大于UCAR-T细胞的数目，导致UCAR-T未能杀死所有的肿瘤细胞，但从前8天的结果(图30B)以及整个实验周期中肿瘤细胞的负荷(图30C)来看，HSV-TK版本的CD5-CD7 UCAR-T和GCV的药效都是不错的。
相应地，本文至少提供了如下技术方案：
方案1：制备通用嵌合抗原受体T细胞(UCAR-T)的方法，包括：
1)利用CRISPR基因编辑系统制备如下基因被敲除的T细胞：
i)TRAC基因和/或TRBC基因；
ii)CD5基因；
iii)CD7基因；以及
2)以包括嵌合抗原受体(CAR)的编码序列的核酸分子转染所述T细胞，使其表达所述CAR。
方案2：如方案1所述的方法，其中在步骤1)中还包括敲除所述T细胞的B2M基因。
方案3：如方案1或2所述的方法，其中对所述TRAC基因的敲除所使用的sgRNA的靶序列选自SEQ ID NO：1-7所示序列及其任意组合。
方案4：如方案1-3任一项所述的方法，其中对所述B2M基因的敲除所使用的sgRNA的靶序列选自SEQ ID NO：8、9、11、12所示序列及其任意组合。
方案5：如方案1-4任一项所述的方法，其中对所述B2M基因的敲除使用两种sgRNA的组合，所述两种sgRNA的靶序列分别为SEQ ID NO：9和11所示序列。
方案6：如方案1-5任一项所述的方法，其中在步骤1)中将用于所述TRAC基因敲除和用于所述B2M基因敲除的sgRNA与Cas9蛋白混合后同时进行所述T细胞中所述TRAC基因和所述B2M基因的敲除，并且用于所述TRAC基因敲除的sgRNA的靶序列为SEQ ID NO：7所示序列。
方案7：如方案1-6任一项所述的方法，其中用于敲除所述CD5基因的sgRNA的靶序列选自SEQ ID NO：13、14、16、17、18、19所示序列及其任意组合。
方案8：如方案1-7任一项所述的方法，其中用于所述CD7基因敲除的sgRNA的靶序列为SEQ ID NO：20所示序列。
方案9：如方案1-8任一项所述的方法，其中对所述TRAC基因、所述CD5基因和所述CD7基因的敲除同时进行，用于敲除所述TRAC基因的sgRNA的靶序列为SEQ ID NO：7所示序列；用于敲除所述CD5基因的sgRNA的靶序列为SEQ ID NO：13所示序列；以及用于敲除所述CD7基因的sgRNA的靶序列为SEQ ID NO：20所示序列。
方案10：如方案1-9任一项所述的方法，其中对所述TRAC基因、所述B2M基因、所述CD5基因和所述CD7基因的敲除同时进行，用于敲除所述TRAC基因的sgRNA的靶序列为SEQ ID NO：7所示序列；用于敲除所述B2M基因的两种sgRNA的靶序列分别为SEQ ID NO：9和11所示系列；用于敲除所述CD5基因的sgRNA的靶序列为SEQ ID NO：13所示序列；以及用于敲除所述CD7基因的sgRNA的靶序列为SEQ ID NO：20所示序列。
方案11：如方案1-10任一项所述的方法，其中对所述TRAC基因、所述B2M基因、所述CD5基因和所述CD7基因的敲除同时进行，用于进行所述敲除的组分按比例在20μL体系中包括：
不少于30pmol的Cas9和不少于45pmol的靶向SEQ ID NO：7所示序列的sgRNA所形成的RNP复合物；
不少于20pmol的Cas9和不少于30pmol的靶向SEQ ID NO：9所示序列的sgRNA所形成的RNP复合物；
不少于20pmol的Cas9和不少于30pmol的靶向SEQ ID NO：11所示序列的sgRNA所形成的RNP复合物；
不少于40pmol的Cas9和不少于120pmol的靶向SEQ ID NO：13所示序列的sgRNA所形成的RNP复合物；以及
不少于40pmol的Cas9和不少于120pmol的靶向SEQ ID NO：20所示序列的sgRNA所形成的RNP复合物。
方案12：如方案1-11任一项所述的方法，其中对所述TRAC基因、所述B2M基因、所述CD5基因和所述CD7基因的敲除同时进行，用于进行所述敲除的组分按比例在20μL体系中包括：
30pmol的Cas9和45pmol的靶向SEQ ID NO：7所示序列的sgRNA所形成的RNP复合物；
20pmol的Cas9和30pmol的靶向SEQ ID NO：9所示序列的sgRNA所形成的RNP复合物；
20pmol的Cas9和30pmol的靶向SEQ ID NO：11所示序列的sgRNA所形成的RNP复合物；
80pmol的Cas9和150pmol的靶向SEQ ID NO：13所示序列的sgRNA所形成的RNP复合物；以及
40pmol的Cas9和120pmol的靶向SEQ ID NO：20所示序列的sgRNA所形成的RNP复合物。
方案13：如方案1-12任一项所述的方法，其中在步骤1)中还包括敲除所述T细胞的CIITA基因。
方案14：如方案1-13任一项所述的方法，其中用于所述CIITA基因敲除的sgRNA的靶序列为SEQ ID NO：25所示序列。
方案15：如方案1-14任一项所述的方法，其中对所述TRAC基因、所述B2M基因、所述CD5基因、所述CD7基因和所述CIITA基因的敲除同时进行，用于进行所述敲除的组分按比例在20μL体系中包括：
不少于30pmol的Cas9和不少于45pmol的靶向SEQ ID NO：7所示序列的sgRNA所形成的RNP复合物；
不少于20pmol的Cas9和不少于30pmol的靶向SEQ ID NO：9所示序列的sgRNA所形成的RNP复合物；
不少于20pmol的Cas9和不少于30pmol的靶向SEQ ID NO：11所示序列的sgRNA所形成的RNP复合物；
不少于80pmol的Cas9和不少于150pmol的靶向SEQ ID NO：13所示序列的sgRNA所形成的RNP复合物；
不少于40pmol的Cas9和不少于120pmol的靶向SEQ ID NO：20所示序列的sgRNA所形成的RNP复合物；以及
不少于40pmol的Cas9和不少于60pmol的靶向SEQ ID NO：25所示序列的sgRNA所形成的RNP复合物。
方案16：如方案1-15任一项所述的方法，其中对所述TRAC基因、所述B2M基因、所述CD5基因、所述CD7基因和所述CIITA基因的敲除同时进行，用于进行所述敲除的组分按比例在20μL体系中包括：
30pmol的Cas9和45pmol的靶向SEQ ID NO：7所示序列的sgRNA所形成的RNP复合物；
25pmol的Cas9和40pmol的靶向SEQ ID NO：9所示序列的sgRNA所形成的RNP复合物；
25pmol的Cas9和40pmol的靶向SEQ ID NO：11所示序列的sgRNA所形成的RNP复合物；
80pmol的Cas9和150pmol的靶向SEQ ID NO：13所示序列的sgRNA所形成的RNP复合物；
40pmol的Cas9和120pmol的靶向SEQ ID NO：20所示序列的sgRNA所形成的RNP复合物；以及
50pmol的Cas9和80pmol的靶向SEQ ID NO：25所示序列的sgRNA所形成的RNP复合物。
方案17：制备通用嵌合抗原受体T细胞(UCAR-T)的方法，包括：
1)利用使用胞嘧啶碱基编辑器制备如下基因被敲除的T细胞：
i)TRAC基因和/或TRBC基因；
ii)CD5基因；
iii)CD7基因；以及
2)以包括嵌合抗原受体(CAR)的编码序列的核酸分子转染所述T细胞，使其表达嵌合抗原受体(CAR)。
方案18：如方案17所述的方法，其中在步骤1)中还包括敲除所述T细胞的B2M基因。
方案19：如方案17或18所述的方法，其中在步骤1)中还包括敲除所述T细胞的CIITA基因。
方案20：如方案17-19任一项所述的方法，其中对所述TRAC基因的敲除所使用的sgRNA的靶序列为SEQ ID NO：26所示序列。
方案21：如方案17-20任一项所述的方法，其中对所述TRBC基因的敲除所使用的sgRNA的靶序列选自SEQ ID NO：27-31任一项所示序列及其任意组合。
方案22：如方案17-21任一项所述的方法，其中对所述B2M基因的敲除所使用的sgRNA的靶序列选自SEQ ID NO：33和34所示序列及其组合。
方案23：如方案17-22任一项所述的方法，其中对所述B2M基因的敲除使用两种sgRNA，其中所述两种sgRNA的靶序列分别为SEQ ID NO：8和9所示序列。
方案24：如方案17-23任一项所述的方法，其中用于敲除所述CD5基因的sgRNA的靶序列选自SEQ ID NO：37、39、41-46任一项所示序列及其任意组合。
方案25：如方案17-24任一项所述的方法，其中用于所述CD7基因敲除的sgRNA的靶序列为SEQ ID NO：47所示序列。
方案26：如方案17-25任一项所述的方法，其中用于所述CIITA基因敲除的sgRNA的靶序列选自SEQ ID NO：50、51、54、57任一项所示序列及其任意组合。
方案27：如方案17-26任一项所述的方法，其中所述胞嘧啶碱基编辑器为nCBE3或nCBE4蛋白。
方案28：如方案17-27任一项所述的方法，其中在以CD2/CD3/CD28抗原激活所述T细胞之前对所述TRAC基因、所述B2M基因、所述CD5基因、所述CD7基因和/或所述CIITA基因进行敲除。
方案29：如方案1-28任一项所述的方法，其中所述CAR的胞外抗原结合结构域包括第一抗原结合部分和第二抗原结合部分，所述第一抗原结合部分能够特异性结合CD7，所述第二抗原结合部分能够特异性结合CD5。
方案30：如方案1-29任一项所述的方法，其中所述第一抗原结合部分包括来自抗CD7单域抗体的重链可变区，所述重链可变区的HCDR1包括SEQ ID NO：59所示氨基酸序列、HCDR2包括SEQ ID NO：60所示氨基酸序列以及HCDR3包括SEQ ID NO：61所示氨基酸序列。
方案31：如方案1-30任一项所述的方法，所述第二抗原结合部分包括来自抗CD5单域抗体的重链可变区，所述重链可变区的HCDR1包括SEQ ID NO：63所示氨基酸序列、HCDR2包括SEQ ID NO：64所示氨基酸序列以及HCDR3包括SEQ ID NO：65所示氨基酸序列。
方案32：如方案1-31任一项所述的方法，其中所述第一抗原结合部分包括SEQ ID NO：62所示氨基酸序列。
方案33：在如方案1-32任一项所述的方法，其中所述第二抗原结合部分包括SEQ ID NO：66所示氨基酸序列。
方案34：如方案1-33任一项所述的方法，其中所述CAR的胞外抗原结合结构域包括SEQ ID NO：74所示氨基酸序列。
方案35：如方案1-34任一项所述的方法，其中所述CAR从氨基端到羧基端依次包括所述第一抗原结合部分、连接片段、所述第二抗原结合部分、铰链区、跨膜区、胞内共刺激结构域和胞内信号传导结构域。
方案36：如方案1-35任一项所述的方法，其中所述连接片段包括SEQ ID NO：67所示氨基酸序列；所述铰链区包括SEQ ID NO：68所示氨基酸序列；所述跨膜区包括SEQ ID NO：69所示氨基酸序列；所述胞内共刺激结构域包括SEQ ID NO：70所示氨基酸序列；所述胞内信号传导结构域包括SEQ ID NO：71所示氨基酸序列。
方案37：如方案1-36任一项所述的方法，其中所述核酸分子中还包括tEGFR或单纯疱疹病毒胸苷激酶(HSV-TK)的编码序列。
方案38：如方案1-37任一项所述的方法，其中所述核酸分子中的所述tEGFR或HSV-TK的编码序列的通过自剪切肽的编码序列连接在所述CAR的编码序列的下游。
方案39：如方案1-38任一项所述的方法，其中所述自剪切肽为T2A，其氨基酸序列优选为SEQ ID NO：72所示。
方案40：如方案1-39任一项所述的方法，其中还包括在步骤2)后筛选出不表达TCR和MHC-I类分子的T细胞。
方案41：如方案1-40任一项所述的方法，其中所述T细胞含有NKT细胞，例如10-20％数量比例的NKT细胞。
方案42：通过方案1-41任一项所述的方法制备的UCAR-T细胞。
方案43：药物组合物，其包括方案42所述的UCAR-T细胞和药学上可接受的载体。
方案44：方案42所述的UCAR-T细胞在制备用于治疗癌症的药物中的用途。
方案45：如方案44所述的用途，其中所述癌症在其细胞表面表达CD5和/或CD7。
方案46：如方案44或45所述的用途，其中所述癌症为T细胞恶性肿瘤，如急性T淋巴细胞白血病(T-ALL)和T细胞淋巴瘤。
方案47：在受试者中治疗癌症的方法，包括以治疗有效量的方案42所述的UCAR-T细胞或方案43所述的药物组合物向所述受试者给药。
方案48：如方案47所述的方法，其中所述癌症在其细胞表面表达CD5和/或CD7。
方案49：如方案48所述的方法，其中所述癌症为T细胞恶性肿瘤，如急性T淋巴细胞白血病和T细胞淋巴瘤。
方案50：如方案47或48所述的方法，其中还包括治疗后以更昔洛韦(GCV)向所述受试者给药。
方案51：药物试剂盒，包括：1)方案42所述的UCAR-T细胞或药物组合物；以及2)GCV。
本文提供的UCAR-T的优势包括但不限于：
1.采用双靶点，可避免单靶CAR-T因抗原逃逸造成的off-tumor问题，大大增加了适应症人群；
2.将CAR同时作为攻击分子，大大减弱了宿主免疫细胞对UCAR-T的清除；
3.采用健康供着的T细胞制备，一次可制备多人份，一方面患者不用等待CAR-T的制备，另一方面还大大降低了每人份的制备成本，进而可降低患者的治疗费用；
4.能够实现反复给药。
本文和附图中提到的核酸分子和蛋白分子的序列如下：

CD7 sdAb VHH蛋白序列(CD7-FHVH10)：

CD5 sdAb VHH蛋白序列(CD5-FHVH61)：
连接片段(linker)氨基酸序列：
CD8α铰链区蛋白序列：
CD8α跨膜区蛋白序列：
4-1BB胞内结构域蛋白序列：
CD3z胞内信号结构域蛋白序列：
剪切肽T2A蛋白序列：
EGFRt蛋白序列：
通过linker串联的CD5-CD7 sdAb氨基酸序列
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Claims (10)

  1. 制备通用嵌合抗原受体T细胞(UCAR-T)的方法，包括：
    1)利用CRISPR基因编辑系统制备如下基因被敲除的T细胞：
    i)TRAC基因和/或TRBC基因；
    ii)CD5基因；
    iii)CD7基因；以及
    2)以包括嵌合抗原受体(CAR)的编码序列的核酸分子转染所述T细胞，使其表达所述CAR。
  2. 如权利要求1所述的方法，其中在步骤1)中还包括敲除所述T细胞的B2M基因和/或CIITA基因。
  3. 如权利要求1或2所述的方法，其中：
    对所述TRAC基因的敲除所使用的sgRNA的靶序列选自SEQ ID NO：1-7所示序列及其任意组合；
    对所述B2M基因的敲除所使用的sgRNA的靶序列选自SEQ ID NO：8、9、11、12所示序列及其任意组合；优选地，对所述B2M基因的敲除使用两种sgRNA的组合，所述两种sgRNA的靶序列分别为SEQ ID NO：9和11所示序列；
    用于敲除所述CD5基因的sgRNA的靶序列选自SEQ ID NO：13、14、16、17、18、19所示序列及其任意组合；
    用于所述CD7基因敲除的sgRNA的靶序列为SEQ ID NO：20所示序列；和/或
    用于所述CIITA基因敲除的sgRNA的靶序列为SEQ ID NO：25所示序列；
    优选地，在步骤1)中将用于所述TRAC基因敲除和用于所述B2M基因敲除的sgRNA与Cas9蛋白混合后同时进行所述T细胞中所述TRAC基因和所述B2M基因的敲除，并且用于所述TRAC基因敲除的sgRNA的靶序列为SEQ ID NO：7所示序列；
    优选地，对所述TRAC基因、所述CD5基因和所述CD7基因的敲除同时进行，用于敲除所述TRAC基因的sgRNA的靶序列为SEQ ID NO：7所示序列；用于敲除所述CD5基因的sgRNA的靶序列为SEQ ID NO：13所示序列；以及用于敲除所述CD7基因的sgRNA的靶序列为SEQ ID NO：20所示序列；
    优选地，对所述TRAC基因、所述B2M基因、所述CD5基因和所述CD7基因的敲除同时进行，用于敲除所述TRAC基因的sgRNA的靶序列为SEQ ID NO：7所示序列；用于敲除所述B2M基因的两种sgRNA的靶序列分别为SEQ ID NO：9和11所示系列；用于敲除所述CD5基因的sgRNA的靶序列为SEQ ID NO：13所示序列；以及用于敲除所述CD7基因的sgRNA的靶序列为SEQ ID NO：20所示序列；
    更优选地，对所述TRAC基因、所述B2M基因、所述CD5基因和所述CD7基因的敲除同时进行，用于进行所述敲除的组分按比例在20μL体系中包括：不少于30pmol的Cas9和不少于45pmol的靶向SEQ ID NO：7所示序列的sgRNA所形成的RNP复合物；不少于20pmol的Cas9和不少于30pmol的靶向SEQ ID NO：9所示序列的sgRNA所形成的RNP复合物；不少于20pmol的Cas9和不少于30pmol的靶向SEQ ID NO：11所示序列的sgRNA所形成的RNP复合物；不少于40pmol的Cas9和不少于120pmol的靶向SEQ ID NO：13所示序列的sgRNA所形成的RNP复合物；以及不少于40pmol的Cas9和不少于120pmol的靶向SEQ ID NO：20所示序列的sgRNA所形成的RNP复合物；
    更优选地，对所述TRAC基因、所述B2M基因、所述CD5基因和所述CD7基因的敲除同时进行，用于进行所述敲除的组分按比例在20μL体系中包括：30pmol的Cas9和45pmol的靶向SEQ ID NO：7所示序列的sgRNA所形成的RNP复合物；20pmol的Cas9和30pmol的靶向SEQ ID NO：9所示序列的sgRNA所形成的RNP复合物；20pmol的Cas9和30pmol的靶向SEQ ID NO：11所示序列的sgRNA所形成的RNP复合物；80pmol的Cas9和150pmol的靶向SEQ ID NO：13所示序列的sgRNA所形成的RNP复合物；以及40pmol的Cas9和120pmol的靶向SEQ ID NO：20所示序列的sgRNA所形成的RNP复合物；
    更优选地，对所述TRAC基因、所述B2M基因、所述CD5基因、所述CD7基因和所述CIITA基因的敲除同时进行，用于进行所述敲除的组分按比例在20μL体系中包括：不少于30pmol的Cas9和不少于45pmol的靶向SEQ ID NO：7所示序列的sgRNA所形成的RNP复合物；不少于20pmol的Cas9和不少于30pmol的靶向SEQ ID NO：9所示序列的sgRNA所形成的RNP复合物；不少于20pmol的Cas9和不少于30pmol的靶向SEQ ID NO：11所示序列的sgRNA所形成的RNP复合物；不少于80pmol的Cas9和不少于150pmol的靶向SEQ ID NO：13所示序列的sgRNA所形成的RNP复合物；不少于40pmol的Cas9和不少于120pmol的靶向SEQ ID NO：20所示序列的sgRNA所形成的RNP复合物；以及不少于40pmol的Cas9和不少于60pmol的靶向SEQ ID NO：25所示序列的sgRNA所形成的RNP复合物；
    更优选地，对所述TRAC基因、所述B2M基因、所述CD5基因、所述CD7基因和所述CIITA基因的敲除同时进行，用于进行所述敲除的组分按比例在20μL体系中包括：30pmol的Cas9和45pmol的靶向SEQ ID NO：7所示序列的sgRNA所形成的RNP复合物；25pmol的Cas9和40pmol的靶向SEQ ID NO：9所示序列的sgRNA所形成的RNP复合物；25pmol的Cas9和40pmol的靶向SEQ ID NO：11所示序列的sgRNA所形成的RNP复合物；80pmol的Cas9和150pmol的靶向SEQ ID NO：13所示序列的sgRNA所形成的RNP复合物；40pmol的Cas9和120pmol的靶向SEQ ID NO：20所示序列的sgRNA所形成的RNP复合物；以及50pmol的Cas9和80pmol的靶向SEQ ID NO：25所示序列的sgRNA所形成的RNP复合物。
  4. 制备通用嵌合抗原受体T细胞(UCAR-T)的方法，包括：
    1)利用使用胞嘧啶碱基编辑器制备如下基因被敲除的T细胞：
    i)TRAC基因和/或TRBC基因；
    ii)CD5基因；
    iii)CD7基因；以及
    2)以包括嵌合抗原受体(CAR)的编码序列的核酸分子转染所述T细胞，使其表达嵌合抗原受体(CAR)。
  5. 如权利要求4所述的方法，其中在步骤1)中还包括敲除所述T细胞的B2M基因和/或CIITA基因。
  6. 如权利要求4或5所述的方法，其中
    对所述TRAC基因的敲除所使用的sgRNA的靶序列为SEQ ID NO：26所示序列；
    对所述TRBC基因的敲除所使用的sgRNA的靶序列选自SEQ ID NO：27-31任一项所示序列及其任意组合；
    对所述B2M基因的敲除所使用的sgRNA的靶序列选自SEQ ID NO：33和34所示序列及其组合；优选地，对所述B2M基因的敲除使用两种sgRNA，其中所述两种sgRNA的靶序列分别为SEQ ID NO：8和9所示序列；
    用于敲除所述CD5基因的sgRNA的靶序列选自SEQ ID NO：37、39、41-46任一项所示序列及其任意组合；
    用于所述CD7基因敲除的sgRNA的靶序列为SEQ ID NO：47所示序列；和/或
    用于所述CIITA基因敲除的sgRNA的靶序列选自SEQ ID NO：50、51、54、57任一项所示序列及其任意组合；
    优选地，所述胞嘧啶碱基编辑器为nCBE3或nCBE4蛋白；
    优选地，在以CD2/CD3/CD28抗原激活所述T细胞之前对所述TRAC基因、所述B2M基因、所述CD5基因、所述CD7基因和/或所述CIITA基因进行敲除；
  7. 如权利要求1-6任一项所述的方法，其中所述CAR的胞外抗原结合结构域包括第一抗原结合部分和第二抗原结合部分，所述第一抗原结合部分能够特异性结合CD7，所述第二抗原结合部分能够特异性结合CD5；
    优选地，所述第一抗原结合部分包括来自抗CD7单域抗体的重链可变区，所述重链可变区的HCDR1包括SEQ ID NO：59所示氨基酸序列、HCDR2包括SEQ ID NO：60所示氨基酸序列以及HCDR3包括SEQ ID NO：61所示氨基酸序列；所述第二抗原结合部分包括来自抗CD5单域抗体的重链可变区，所述重链可变区的HCDR1包括SEQ ID NO： 63所示氨基酸序列、HCDR2包括SEQ ID NO：64所示氨基酸序列以及HCDR3包括SEQ ID NO：65所示氨基酸序列；
    更优选地，所述第一抗原结合部分包括SEQ ID NO：62所示氨基酸序列；
    更优选地，所述第二抗原结合部分包括SEQ ID NO：66所示氨基酸序列；
    更优选地，所述CAR的胞外抗原结合结构域包括SEQ ID NO：74所示氨基酸序列。
  8. 如权利要求1-7任一项所述的方法，其中所述核酸分子中还包括tEGFR或单纯疱疹病毒胸苷激酶(HSV-TK)的编码序列；优选地，所述核酸分子中的所述tEGFR或HSV-TK的编码序列的通过自剪切肽的编码序列连接在所述CAR的编码序列的下游。
  9. 如权利要求1-8任一项所述的方法，其中还包括在步骤2)后筛选出不表达TCR和MHC-I类分子的T细胞。
  10. 通过权利要求1-9任一项所述的方法制备的UCAR-T细胞。
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