Universal CAR-T targeting CD5 and CD7 and application thereof

Abstract

The invention provides a method for preparing a universal chimeric antigen receptor T cell (UCAR-T), which comprises the following steps: knocking out TRAC, B2M, CD5, CD7 and other genes from the T cell, and transfecting the T cell to express a chimeric antigen receptor (CAR) of a targeted tumor cell. Also provided herein are preferred sgRNAs for use in performing gene knockout. The UCAR-Ts provided herein are useful for cancer treatment. # imgabs0 #

Description

Universal CAR-T targeting CD5 and CD7 and its applications Technical Field

[0001] This article relates to methods for preparing universal chimeric antigen receptor T cells (UCAR-T) and the use of UCAR-T in cancer treatment. Background Art

[0002] While universal CAR-T (UCAR-T) offers numerous advantages over autologous CAR-T, it also faces numerous challenges. The two most significant challenges are: first, graft-versus-host disease (GvHD) caused by allogeneic cell infusion; and second, rapid clearance of UCAR-T cells from the host immune system, preventing their effective expansion. Therefore, current UCAR-T designs are primarily designed to address these two challenges.

[0003] At present, the first problem has been basically solved. Researchers have used gene editing technology to knock out the TRAC gene encoding the T cell surface receptor (TCR) on αβ-T cells. 1-3 , effectively inhibiting CAR-T cells from indiscriminately attacking host cells by activating TCR, thereby avoiding the occurrence of GvHD.

[0004] In contrast, the second problem is more difficult to solve. In recent years, researchers have been exploring how to effectively expand UCAR-T cells in the host. Currently, there are two mainstream solutions:

[0005] (1) Combination of CD52 monoclonal antibody and universal CAR-T. After using CD52 protein monoclonal antibody drug Alemtuzumab and chemotherapy drugs to clear the lymph nodes, the patient is infused with TRAC / CD52 double knockout CAR-T cells for treatment. In this scheme, knocking out TRAC prevents graft-versus-host disease, and knocking out CD52 prevents the clearance of UCAR-T cells by clearing lymph nodes. 2 .

[0006] (2) Another strategy to reduce host rejection of transplanted tissue is to knock out the B2M gene encoding β2-microglobulin on UCAR-T. 3,4 . Destroying β2-microglobulin (B2M knockout) can prevent the expression of functional HLA-I class molecules on the surface of CAR-T cells. This approach prevents UCAR-T from activating cytotoxic T cells in the host by destroying HLA-I class molecules, thereby enabling them to proliferate long-term. However, since HLA is an inhibitory ligand for NK cells, its absence will activate the patient's NK cells to clear CAR-T cells, limiting their proliferation in the body and affecting their effectiveness.

[0007] CD5 and CD7 are two promising targets for the treatment of T cell malignancies 5 Many T-cell malignancies express CD7, most T-NHLs and T-ALLs highly express CD7, and in addition to T-cell malignancies, CD7 is expressed in approximately 24% of AML and is considered a marker for leukemic stem cells. It is also expressed in the vast majority of natural killer (NK) and NKT NHLs and leukemias. 6 , which provides an attractive target for immunotherapy of T cell cancers.

[0008] CD5 is a pan-T cell marker that is 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. In addition, CD5 is a negative regulator of T cell receptor (TCR) signaling and plays a role in protecting against autoimmunity. 7 .

[0009] In China, there are currently few companies developing products targeting both CD5 and CD7. Some of these products are autologous CD7-CAR-Ts currently in the IND application stage, while others are universal CD7-CAR-Ts in Phase IIT and Phase I clinical trials. Currently, there are no reports of companies developing dual-target CAR-Ts targeting both CD5 and CD7.

[0010] Summary of the Invention

[0011] In one aspect, provided herein is a method for preparing a universal chimeric antigen receptor T cell (UCAR-T), comprising:

[0012] 1) Use the CRISPR gene editing system to generate T cells with the following genes knocked out:

[0013] i) TRAC gene and / or TRBC gene;

[0014] ii) CD5 gene;

[0015] iii) CD7 gene; and

[0016] 2) transfecting the T cells with a nucleic acid molecule comprising a coding sequence of a chimeric antigen receptor (CAR) so that the T cells express the CAR.

[0017] In some embodiments, step 1) further comprises knocking out the B2M gene of the T cell.

[0018] In some embodiments, the target sequence of the sgRNA used to knock out the TRAC gene is selected from the sequences shown in SEQ ID NOs: 1-7 and any combination thereof.

[0019] In some embodiments, the target sequence of the sgRNA used to knock out the B2M gene is selected from the sequences shown in SEQ ID NO: 8, 9, 11, 12 and any combination thereof.

[0020] In some embodiments, a combination of two sgRNAs is used to knock out the B2M gene, and the target sequences of the two sgRNAs are the sequences shown in SEQ ID NOs: 9 and 11, respectively.

[0021] In some embodiments, in step 1), the sgRNA for knocking out the TRAC gene and the B2M gene are mixed with the Cas9 protein to simultaneously knock out the TRAC gene and the B2M gene in the T cell, and the target sequence of the sgRNA for knocking out the TRAC gene is the sequence shown in SEQ ID NO: 7.

[0022] In some embodiments, the target sequence of the sgRNA for knocking out the CD5 gene is selected from the sequence shown in SEQ ID NO: 13, 14, 16, 17, 18, 19 and any combination thereof.

[0023] In some embodiments, the target sequence of the sgRNA for CD7 gene knockout is the sequence shown in SEQ ID NO: 20.

[0024] In some embodiments, 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.

[0025] In some embodiments, 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 used to knock out the TRAC 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; 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.

[0026] In some embodiments, the knockout of the TRAC gene, the CD5 gene, and the CD7 gene is performed simultaneously, and the components for performing the knockout in a 20 μL system include:

[0027] An RNP complex formed by not less than 30 pmol of Cas9 and not less than 45 pmol of an sgRNA targeting the sequence shown in SEQ ID NO: 7;

[0028] an RNP complex formed by not less than 40 pmol of Cas9 and not less than 120 pmol of an sgRNA targeting the sequence shown in SEQ ID NO: 13; and

[0029] An RNP complex formed by not less than 40 pmol of Cas9 and not less than 120 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 20.

[0030] In some embodiments, the knockout of the TRAC gene, the B2M gene, the CD5 gene, and the CD7 gene is performed simultaneously, and the components for performing the knockout in a 20 μL system include, in proportion:

[0031] An RNP complex formed by not less than 30 pmol of Cas9 and not less than 45 pmol of an sgRNA targeting the sequence shown in SEQ ID NO: 7;

[0032] An RNP complex formed by not less than 20 pmol of Cas9 and not less than 30 pmol of an sgRNA targeting the sequence shown in SEQ ID NO: 9;

[0033] an RNP complex formed by not less than 20 pmol of Cas9 and not less than 30 pmol of an sgRNA targeting the sequence shown in SEQ ID NO: 11;

[0034] an RNP complex formed by not less than 40 pmol of Cas9 and not less than 120 pmol of an sgRNA targeting the sequence shown in SEQ ID NO: 13; and

[0035] An RNP complex formed by not less than 40 pmol of Cas9 and not less than 120 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 20.

[0036] In some embodiments, the knockout of the TRAC gene, the CD5 gene, and the CD7 gene is performed simultaneously, and the components for performing the knockout in a 20 μL system include:

[0037] An RNP complex formed by 30 pmol of Cas9 and 45 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 7;

[0038] an RNP complex formed by 80 pmol of Cas9 and 150 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 13; and

[0039] RNP complex formed by 40 pmol of Cas9 and 120 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 20.

[0040] In some embodiments, the knockout of the TRAC gene, the B2M gene, the CD5 gene, and the CD7 gene is performed simultaneously, and the components for performing the knockout in a 20 μL system include, in proportion:

[0041] An RNP complex formed by 30 pmol of Cas9 and 45 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 7;

[0042] An RNP complex formed by 20 pmol of Cas9 and 30 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 9;

[0043] An RNP complex formed by 20 pmol of Cas9 and 30 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 11;

[0044] an RNP complex formed by 80 pmol of Cas9 and 150 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 13; and

[0045] RNP complex formed by 40 pmol of Cas9 and 120 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 20.

[0046] In some embodiments, step 1) further comprises knocking out the CIITA gene of the T cell.

[0047] In some embodiments, the target sequence of the sgRNA for knocking out the CIITA gene is the sequence shown in SEQ ID NO: 25.

[0048] In some embodiments, 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 for performing the knockout in a 20 μL system include, in proportion:

[0049] An RNP complex formed by not less than 30 pmol of Cas9 and not less than 45 pmol of an sgRNA targeting the sequence shown in SEQ ID NO: 7;

[0050] An RNP complex formed by not less than 20 pmol of Cas9 and not less than 30 pmol of an sgRNA targeting the sequence shown in SEQ ID NO: 9;

[0051] an RNP complex formed by not less than 20 pmol of Cas9 and not less than 30 pmol of an sgRNA targeting the sequence shown in SEQ ID NO: 11;

[0052] an RNP complex formed by not less than 80 pmol of Cas9 and not less than 150 pmol of an sgRNA targeting the sequence of SEQ ID NO: 13;

[0053] an RNP complex formed by not less than 40 pmol of Cas9 and not less than 120 pmol of an sgRNA targeting the sequence shown in SEQ ID NO: 20; and

[0054] An RNP complex formed by not less than 40 pmol of Cas9 and not less than 60 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 25.

[0055] In some embodiments, 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 for performing the knockout in a 20 μL system include, in proportion:

[0056] An RNP complex formed by 30 pmol of Cas9 and 45 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 7;

[0057] An RNP complex formed by 25 pmol of Cas9 and 40 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 9;

[0058] An RNP complex formed by 25 pmol of Cas9 and 40 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 11;

[0059] An RNP complex formed by 80 pmol of Cas9 and 150 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 13;

[0060] an RNP complex formed by 40 pmol of Cas9 and 120 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 20; and

[0061] RNP complex formed by 50 pmol of Cas9 and 80 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 25.

[0062] In another aspect, provided herein is a method for preparing a universal chimeric antigen receptor T cell (UCAR-T), comprising:

[0063] 1) Using a cytosine base editor to generate T cells with the following genes knocked out:

[0064] i) TRAC gene and / or TRBC gene;

[0065] ii) CD5 gene;

[0066] iii) CD7 gene; and

[0067] 2) transfecting the T cells with a nucleic acid molecule comprising a coding sequence of a chimeric antigen receptor (CAR) so that the T cells express the chimeric antigen receptor (CAR).

[0068] In some embodiments, step 1) further comprises knocking out the B2M gene of the T cell.

[0069] In some embodiments, step 1) further comprises knocking out the CIITA gene of the T cell.

[0070] In some embodiments, the target sequence of the sgRNA used to knock out the TRAC gene is the sequence shown in SEQ ID NO: 26.

[0071] In some embodiments, the target sequence of the sgRNA used to knock out the TRBC gene is selected from the sequence shown in any one of SEQ ID NOs: 27-31 and any combination thereof.

[0072] In some embodiments, the target sequence of the sgRNA used to knock out the B2M gene is selected from the sequences shown in SEQ ID NOs: 33 and 34, and a combination thereof.

[0073] In some embodiments, 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 NOs: 8 and 9, respectively.

[0074] In some embodiments, the target sequence of the sgRNA for knocking out the CD5 gene is selected from the sequence shown in any one of SEQ ID NOs: 37, 39, 41-46, and any combination thereof.

[0075] In some embodiments, the target sequence of the sgRNA for CD7 gene knockout is the sequence shown in SEQ ID NO: 47.

[0076] In some embodiments, the target sequence of the sgRNA for the CIITA gene knockout is selected from the sequence shown in any one of SEQ ID NOs: 50, 51, 54, 57, and any combination thereof.

[0077] In some embodiments, the cytosine base editor is an nCBE3 or nCBE4 protein.

[0078] In some embodiments, the TRAC gene, the B2M gene, the CD5 gene, the CD7 gene and / or the CIITA gene are knocked out before activating the T cells with CD2 / CD3 / CD28 antigens.

[0079] In some embodiments, the extracellular antigen binding domain of the CAR comprises a first antigen binding portion and a second antigen binding portion, wherein the first antigen binding portion is capable of specifically binding to CD7 and the second antigen binding portion is capable of specifically binding to CD5.

[0080] In some embodiments, the first antigen binding portion comprises a heavy chain variable region from an anti-CD7 single domain antibody, wherein HCDR1 of the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 59, HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 60, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 61.

[0081] In some embodiments, the second antigen binding portion comprises a heavy chain variable region from an anti-CD5 single domain antibody, wherein HCDR1 of the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 63, HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 64, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 65.

[0082] In some embodiments, the first antigen binding portion comprises the amino acid sequence shown in SEQ ID NO:62.

[0083] In some embodiments, the second antigen binding portion comprises the amino acid sequence shown in SEQ ID NO:66.

[0084] In some embodiments, the extracellular antigen binding domain of the CAR comprises the amino acid sequence shown in SEQ ID NO:74.

[0085] In some embodiments, the CAR comprises, from amino terminus to carboxyl terminus, the first antigen binding portion, a connecting fragment, the second antigen binding portion, a hinge region, a transmembrane region, an intracellular co-stimulatory domain, and an intracellular signaling domain.

[0086] In some embodiments, the connecting fragment comprises the amino acid sequence shown in SEQ ID NO: 67; the hinge region comprises the amino acid sequence shown in SEQ ID NO: 68; the transmembrane region comprises the amino acid sequence shown in SEQ ID NO: 69; the intracellular costimulatory domain comprises the amino acid sequence shown in SEQ ID NO: 70; and the intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NO: 71.

[0087] In some embodiments, the nucleic acid molecule further comprises a coding sequence for tEGFR or herpes simplex virus thymidine kinase (HSV-TK).

[0088] In some embodiments, the coding sequence of the tEGFR or HSV-TK in the nucleic acid molecule is connected downstream of the coding sequence of the CAR through the coding sequence of a self-cleaving peptide.

[0089] In some embodiments, the self-cleaving peptide is T2A, and its amino acid sequence is preferably as shown in SEQ ID NO: 72.

[0090] In some embodiments, the method further comprises screening out T cells that do not express TCR and MHC class I molecules after step 2).

[0091] In some embodiments, the T cells contain NKT cells, such as 10-20% NKT cells.

[0092] On the other hand, provided herein are UCAR-T cells prepared by the above method.

[0093] On the other hand, provided herein is a pharmaceutical composition comprising the above-mentioned UCAR-T cells and a pharmaceutically acceptable carrier.

[0094] On the other hand, the present invention provides the use of the above-mentioned UCAR-T cells in the preparation of drugs for treating cancer.

[0095] In some embodiments, the cancer expresses CD5 and / or CD7 on its cell surface.

[0096] In some embodiments, the cancer is a T-cell malignancy, such as acute T-lymphoblastic leukemia (T-ALL) and T-cell lymphoma.

[0097] In another aspect, provided herein is a method for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of the above-described UCAR-T cells or pharmaceutical composition.

[0098] In some embodiments, the cancer expresses CD5 and / or CD7 on its cell surface.

[0099] In some embodiments, the cancer is a T-cell malignancy, such as acute T-cell leukemia and T-cell lymphoma.

[0100] In some embodiments, the method further comprises administering ganciclovir (GCV) to the subject after treatment.

[0101] In another aspect, provided herein is a pharmaceutical kit comprising: 1) the above-mentioned UCAR-T cells or pharmaceutical composition; and 2) GCV. BRIEF DESCRIPTION OF THE DRAWINGS

[0102] Figure 1. Schematic diagram of the design of CD5-CD7 universal CAR-T.

[0103] Figure 2. Schematic diagram of the CD5-CD7 bispecific CAR structure.

[0104] Figure 3. CAR-T safety switch molecules controlled by small molecule drugs (from references 8 ).

[0105] Figure 4. TRAC gene candidate sgRNA information and knockout efficiency and off-target test results. (A) The inventors designed and screened TRAC gene knockout sgRNAs using the CRISPick, CRISPOR, and IDT websites. Based on previous experience, the sgRNA editing efficiency predictions from these websites are not particularly accurate. Therefore, when screening candidate sgRNAs, the inventors primarily considered the off-target information predicted by the websites. Of the three websites, CRISPick provides the most precise calculation of sgRNA off-targets. Therefore, the inventors primarily selected sgRNAs based on CRISPick's off-target ranking; a higher ranking indicates a lower off-target probability. (B) FACS results of TRAC candidate sgRNA knockout efficiency testing. The horizontal axis represents TCR expression in cells, and the vertical axis represents cell size. Each column represents an sgRNA, and each row represents an RNP concentration. (CE) Off-target test results for candidate sgRNAs A-XL, A-02, and A-06. The first row of each figure shows the sgRNA sequence information, the second row shows the number of on-target reads, and the third row onwards shows the sequence information and read counts of possible off-target sites. SgRNA A-XL was tested twice, labeled R1 and R2, while sgRNAs A-02 and A-06 were tested once each. Highest off-target percentage = (number of reads at the most frequent site / number of reads at the on-target site) * 100%.

[0106] Figure 5. Information on candidate sgRNAs for the B2M gene and knockout efficiency test results. (A) The inventors designed and screened candidate sgRNAs for the B2M gene using the CRISPick, CRISPOR, IDT, CHOPCHOP, and GUIDES websites. The final sgRNA was selected based on a comprehensive analysis of on-target and off-target information from these websites. Lower ranking numbers are better, and higher scores are better. (B) FACS results of knockout efficiency testing of candidate sgRNAs for B2M. The horizontal axis represents B2M expression in cells, and the vertical axis represents cell size. The MOCK-T group represents cells without sgRNA.

[0107] Figure 6. Results of a dual knockout test of B2M and TRAC genes and off-target detection of B2M-sgRNAs. (A) FACS results of co-knockout of five candidate B2M sgRNAs with TRAC sgRNA (A-XL). The horizontal axis represents B2M expression in cells, and the vertical axis represents TCR expression in cells. Dual-gene-positive cells are located in the upper right quadrant, while dual-gene-negative cells are located in the lower left quadrant. Therefore, a higher percentage of cells in the lower left quadrant indicates a higher number of cells with dual gene knockout. (B) FACS results of co-knockout efficiency of different B2M sgRNAs with TRAC sgRNAs at different RNP concentrations. The horizontal axis represents B2M expression in cells, and the vertical axis represents TCR expression in cells. Each column represents a B2M sgRNA, and each row represents a RNP concentration. (C) Two off-target detection results of B-13 sgRNA, R1 and R2. (D) Two off-target test results of B-03 sgRNA, R1 and R2.

[0108] Figure 7. CD5 gene candidate sgRNA information and gene knockout results. (A) Sequence information of the CD5 sgRNA from the first test. (B) FACS results of the CD5 sgRNA knockout efficiency test from the first test. The horizontal axis represents CD5 expression in cells, and the vertical axis represents cell size. sgRNA1 and sgRNA3 correspond to C-01 and C-03 in Figure C. (C) Information on candidate sgRNAs obtained after the inventors designed and screened CD5 gene knockout sgRNAs using the CRISPick, CRISPOR, IDT, CHOPCHOP, and GUIDES websites.

[0109] Figure 8. Knockout efficiency and off-target analysis of candidate sgRNAs for the CD5 gene. (A) FACS results of knockout efficiency testing of candidate sgRNAs for CD5. The horizontal axis represents CAR expression in cells, and the vertical axis represents CD5 expression in cells. A higher proportion of cells in the lower quadrant indicates a higher CD5 knockout efficiency. (B) Summary of knockout efficiency of candidate sgRNAs for CD5, data derived from the FACS results for 5A. (C) Off-target and overall ranking of candidate sgRNAs for CD5 on the CRISPick website. Lower ranking numbers indicate better sgRNAs. (D) Two off-target test results for the C-06 sgRNA, R1 and R2.

[0110] Figure 9. Off-target detection results for 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 with CD7 sgRNA.

[0111] Figure 10. CIITA gene candidate sgRNA information, knockout efficiency, and off-target detection results. (A) Sequence information of CIITA candidate sgRNAs and FACS results of knockout efficiency testing. The horizontal axis represents cellular HLA class II expression, and the vertical axis represents cell size. (B) Two off-target detection results for the F-05 sgRNA, R1 and R2.

[0112] Figure 11. TRAC, B2M, CD5, CD7 co-knockout RNP conditions. (A) A 20ul system (2x10 6 Each row represents a set of settings, with a total of six RNP dosage combinations explored. Each column represents the RNP dosage of a single sgRNA, with the numbers in each cell representing the Cas9 protein and sgRNA dosages, expressed in pmol. (B) FACS results of the co-knockout efficiency of TRAC, B2M, CD5, and CD7 using different RNP dosage combinations, tested on T cells from two donors. In each graph, a higher proportion of cells in the lower left quadrant indicates a higher efficiency of the dual gene knockout.

[0113] Figure 12. Knockout settings and results of two CD5-CD7 UCAR-T cell preparations (hereinafter referred to as "two preparations" refers to the same two preparations). (A) Knockout of each gene in the first preparation of CD5-CD7 UCAR-T finished cells. The top row shows the gene expression of non-knockout cells, and the bottom row shows the gene knockout of CD5-CD7 UCAR-T cells. The cells in the lower left quadrant represent the proportion of knockout cells. (B) RNP usage of each gene in the two preparations of CD5-CD7 UCAR-T cells. The number in each cell represents the amount of the corresponding material used, in pmol. (C) Knockout of each gene in KO-T cells in the second preparation of CD5-CD7 UCAR-T cells. KO-T cells are cells that have undergone gene knockout but not transfection, reflecting the true knockout of each gene. ISO is the negative control result, and MOCK-T is the positive control result.

[0114] Figure 13. Results of a five-knockout experiment of the TRAC, B2M, CIITA, CD5, and CD7 genes. (A) RNP dosage for the preliminary experiment of the co-knockout of the five genes TRAC, B2M, CIITA, CD5, and CD7. The number in each cell represents the amount of the corresponding material used, in pmol. (B) FACS results of the preliminary experiment of the co-knockout of the five genes TRAC, B2M, CIITA, CD5, and CD7. ISO is the negative control, and MOCK-T is the positive control. The expression of HLA-DR-DP-DQ reflects the knockout of the CIITA gene. (C) RNP dosage for the preparation of the five-knockout version of CD5-CD7 UCAR-T cells. The number in each cell represents the amount of the corresponding material used, in pmol. (D) The knockout status of each gene in the KO-T group cells for the preparation of the five-knockout version of CD5-CD7 UCAR-T cells. ISO is a negative control, and MOCK-T is a positive control, in which the expression of HLA-DR-DP-DQ reflects the knockout of the CIITA gene.

[0115] Figure 14. Single-base editing candidate sgRNA information targeting TCR / B2M / CD5 / CD7 / CIITA genes.

[0116] Figure 15. Efficiency of base editing sgRNA targeting TCR genes. The figure shows the FACS results of the efficiency test of base editing sgRNA targeting TCR. The horizontal axis represents TCR expression, and the vertical axis represents the complexity of cellular contents.

[0117] Figure 16. Efficiency of base-editing sgRNAs targeting the B2M gene. (A) The figure shows the results of base-editing sgRNA efficiency testing targeting B2M. The horizontal axis represents B2M expression, and the vertical axis represents the complexity of the cellular contents. (B) The figure shows a schematic diagram of the Cas9 protein using two adjacent, oppositely directed sgRNAs to cut on both strands of the genome, as well as the location and sequence information of several sgRNAs available for use in exon 1 of B2M. (C) The knockout effect of B2M using different combinations of four sgRNAs: B-10, B-13, NB-37, and NB-38. The horizontal axis represents B2M expression, and the vertical axis represents the complexity of the cellular contents.

[0118] Figure 17. Efficiency of base-editing sgRNAs targeting CD5 and CD7. (A) The figure shows the FACS results of the efficiency test of base-editing sgRNAs targeting CD5. The horizontal axis represents CD5 expression, and the vertical axis represents the complexity of the cellular contents. Each column represents one sgRNA, and the experiment was repeated in two replicates. (B) The figure shows the results of the efficiency test of base-editing sgRNAs targeting CD7. The horizontal axis represents CD7 expression, and the vertical axis represents the complexity of the cellular contents. The experiment was also repeated in two replicates.

[0119] Figure 18. Base editing efficiency of different sgRNAs targeting the CIITA gene. The figure shows the FACS results of the efficiency test of base editing sgRNAs targeting CIITA. The horizontal axis reflects the expression of CIITA using HLA-II class molecule expression, and the vertical axis represents the complexity of cellular contents. ISO is a negative control, and MOCK-T is a positive control.

[0120] Figure 19. Proportions of CD5, CD7, and CAR-positive cells in two UCAR-T cell preparations. (A) The figure shows the proportion of CAR-positive cells in the first CD5-CD7 UCAR-T cell preparation 3 days after transfection. EGFR and CD5 / CD7 CAR expression were detected using EGFR antibody and CD5 / CD7 antigen, respectively. (B) The figure shows the proportion of CAR-positive cells in the first CD5-CD7 UCAR-T cell preparation 8 days after transfection, which is nearly double the proportion 3 days after transfection. (C and D) The proportion of CAR-positive cells in the second CD5-CD7 UCAR-T cell preparation 3 and 8 days after transfection, respectively, are similar to those of the first CD5-CD7 UCAR-T cell preparation.

[0121] Figure 20. TRAC / B2M negative selection results during two UCAR-T preparations. The figure shows the results of TRAC / B2M negative selection during two CD5-CD7 UCAR-T preparations. The horizontal axis represents B2M expression, the vertical axis represents TCR expression, and the red dotted boxes represent cells that continued to be cultured after negative selection.

[0122] Figure 21. CD107a release assay results for two CD5-CD7 UCAR-T cell preparations. (A) Proportion of CD107a-positive cells in CD8 and CAR dual-positive T cells after 4 hours of co-incubation of KO-T and UCAR-T cells with different tumor cells in the first CD5-CD7 UCAR-T preparation. (B) Histogram based on the data in 18A. (C) Proportion of CD107a-positive cells in CD8 and CAR dual-positive T cells after 4 hours of co-incubation of CD5-CD7 UCAR-T cells with different tumor cells in the second preparation.

[0123] Figure 22. Tumorigenicity of CD5-CD7 UCAR-T cells prepared twice. (A) Tumorigenicity assay results of the first-prepared CD5-CD7 UCAR-T cells on day 7. Each data point represents the percentage of tumor cells killed by the CD5-CD7 UCAR-T cells. Higher values ​​indicate more tumor cells killed, while negative values ​​indicate that tumor cells were not killed but proliferated. (B) Tumorigenicity of the first-prepared, non-transduced KO-T cells at the same time point. (C) Tumorigenicity assay results of the first-prepared, cryopreserved and thawed CD5-CD7 UCAR-T cells. (D) Tumorigenicity assay results of the second-prepared, CD5-CD7 UCAR-T cells on day 10. (E) Tumorigenicity of the second-prepared, non-transduced KO-T cells at the same time point. (F) Tumorigenicity assay results of the second-prepared, cryopreserved and thawed CD5-CD7 UCAR-T cells.

[0124] Figure 23. Activation and typing results of CD5-CD7 UCAR-T cells prepared twice. (A) Expression of various antigens on CD5-CD7 UCAR-T cells prepared in the first preparation (without TRAC / B2M negative selection); (B) Expression of various antigens on CD5-CD7 UCAR-T cells prepared in the second preparation (after TRAC / B2M negative selection); (C) CCR7 positive proportion in CD5-CD7 UCAR-T cells prepared in the first preparation (frozen on day 10); (D) CCR7 positive proportion in CD5-CD7 UCAR-T cells prepared in the second preparation (frozen on day 12).

[0125] Figure 24. Exhaustion and premature apoptosis of CD5-CD7 UCAR-T cells prepared twice. (A) Exhaustion marker expression on CD5-CD7 UCAR-T cells prepared in the first stage; (B) Exhaustion marker expression on CD5-CD7 UCAR-T cells prepared in the second stage; (C) Precocious apoptosis marker expression on CD5-CD7 UCAR-T cells prepared in the first stage; (D) Precocious apoptosis marker expression on CD5-CD7 UCAR-T cells prepared in the second stage.

[0126] Figure 25. Component identification results of two CD5-CD7 UCAR-T cell preparations. (A) Component identification results of the first preparation of CD5-CD7 UCAR-T cells on the day of cryopreservation; (B) Component identification results of the second preparation of CD5-CD7 UCAR-T cells at different time points during preparation.

[0127] Figure 26. Results from a clinical trial of CD5-CD7 UCAR-T (EGFR switch). (A) Schematic diagram of the design of the entire clinical trial cycle. Flu represents fludarabine, and CTX represents cyclophosphamide (CTX). (B) Changes in the VCN of CAR-positive cells in the patient's peripheral blood over time after CD5-CD7 UCAR-T infusion. A higher VCN indicates a higher number of UCAR-T cells in the patient's peripheral blood. (CE) Changes in the proportions of various cells in the patient's peripheral blood after CD5-CD7 UCAR-T infusion. Lym refers to lymphocytes.

[0128] Figure 27. CD5-CD7 UCAR-T (HSV-TK) cell production. (A) Schematic diagram of the HSV-TK version of the CAR molecular structure, replacing the original tEGFR with HSV-TK. (B) Schematic flow chart of CD5-CD7 UCAR-T cell production using activated and resting electroporation methods. (C) TRAC / B2M / CD5 / CD7 knockout and CAR efficacy using activated and resting electroporation methods. (D) Cell typing of CD5-CD7 UCAR-T cells produced using activated and resting electroporation methods. (E) Comparison of cellular components of CD5-CD7 UCAR-T cells produced using activated and resting electroporation methods.

[0129] Figure 28. Comparison of tumor killing efficacy between HSV-TK and EGFR UCAR-T cells. (A) Tumor killing efficacy of HSV-TK CD5-CD7 UCAR-T cells 24 hours after cryopreservation and thawing. (B) Tumor killing efficacy of EGFR CD5-CD7 UCAR-T cells 24 hours after cryopreservation and thawing.

[0130] Figure 29. GCV eliminates HSV-TK-positive cells. (A) Total cell proliferation of HSV-TK-derived CD5-CD7 UCAR-T cells treated with different concentrations of GCV. (B) Changes in the proportion of CAR-positive cells of HSV-TK-derived CD5-CD7 UCAR-T cells treated with different concentrations of GCV. (C) Changes in the number of CAR-positive cells of HSV-TK-derived CD5-CD7 UCAR-T cells treated with different concentrations of GCV. (D)-(F) Changes in total cell proliferation, proportion of CAR-positive cells, and number of CAR-positive cells of EGFR-derived CD5-CD7 UCAR-T cells treated with different concentrations of GCV.

[0131] Figure 30. In vivo efficacy testing of UCAR-T and GCV. (A) Schematic diagram of the entire animal experiment process design, where D represents the number of days. (B) Fluorescence imaging results of mice. Since tumor cells express luciferin, injection of luciferin substrate into mice produces fluorescence. Detecting the fluorescence intensity using a fluorescence imaging device indirectly reflects the number of tumor cells. (C) Changes in the mean fluorescence intensity over time in mice from different groups. DETAILED DESCRIPTION

[0132] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0133] The term "or" refers to a single element of the listed alternative elements unless the context clearly dictates otherwise.

[0134] The term "and / or" means any one, any two, any three, any more or all of the listed optional elements.

[0135] "Comprising" or "including" means including the recited elements, integers, or steps, but does not exclude any other elements, integers, or steps. When "comprising" or "including" is used, unless otherwise indicated, it also covers the situation where it consists of the recited elements, integers, or steps. For example, when it is mentioned that an antibody variable region "comprising" a specific sequence, it is also intended to cover the antibody variable region consisting of the specific sequence.

[0136] The "B2M gene" in this article refers to the gene encoding β2 microglobulin. β2 microglobulin is a component of MHC class I molecules (human MHC class I molecules are also called HLA class I molecules). It binds to the heavy chains of MHC class I molecules to form heterodimers on the cell surface. Knockout of the B2M gene results in the absence of MHC class I molecules on the cell surface.

[0137] "TRAC gene" and "TRBC gene" herein refer to the genes encoding the constant region of the T cell receptor α chain and the constant region of the T cell receptor β chain, respectively. The α and β chains constitute the T cell receptor (TCR), which recognizes antigens and mediates immune responses. Knockout of the "TRAC gene" and / or "TRBC gene" results in cells being unable to express TCR molecules.

[0138] The product encoded by the CIITA gene is a class II transactivator, which acts as a dominant switch in HLA gene expression. Class II transactivators themselves are non-DNA-binding proteins, but they participate in the transcriptional regulation of HLA genes by binding to various transcription factors and co-activators. Knockout of the CIITA gene can affect the transcription of HLA genes, particularly HLA class II genes, preventing them from expressing their corresponding products.

[0139] "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, and 80% of T cell acute lymphoblastic leukemia (T-ALL) and peripheral T cell lymphomas express CD5. Herein, CD5 can be human CD5, whose GenBank accession number is NM_014207.4. The CD5 protein can also include fragments of CD5, such as the extracellular domain and fragments thereof.

[0140] CD7 is a cell surface glycoprotein with a molecular weight of approximately 40 kDa. 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 and T cell-B cell interactions during early lymphoid development.

[0141] "Antibodies" refer to immunoglobulins secreted by plasma cells (effector B cells) and used by the body's immune system to neutralize foreign substances (peptides, viruses, bacteria, etc.). These foreign substances are correspondingly called antigens. The basic structure of a classic antibody molecule is a tetramer consisting of two identical heavy chains and two identical light chains. Based on conservative differences in their amino acid sequences, the heavy and light chains are divided into a variable region (V) located at the amino terminus and a constant region (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). Within the variable region, the composition and arrangement of amino acid residues in certain areas are more variable than in other regions within the variable region (framework regions, FRs). These hypervariable regions are actually the key sites for antibody-antigen binding. Because these hypervariable region sequences are complementary to antigenic determinants, they are also called complementarity-determining regions (CDRs). Both the heavy and light chains have three complementary determining regions, referred to as HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3, respectively. The amino acid sequence of the CDRs can be determined using numbering schemes generally recognized in the art, such as the Kabat, Chothia, IMGT, AbM, or Contact numbering schemes. Based on the amino acid sequence of the constant region of the antibody heavy chain, antibodies can be divided into five major different types: IgA, IgD, IgE, IgG, and IgM. These antibody types can be further divided into subclasses based on the size of the hinge region, the location of interchain disulfide bonds, and molecular weight, such as IgG1, IgG2a, IgG2b, and IgG3. Based on the amino acid composition and arrangement of the constant region of the antibody light chain, light chains can be divided into two types: kappa and lambda. The subunit structure and three-dimensional conformation of different classes of immunoglobulins are known in the art.

[0142] An "antigen-binding fragment" of an antibody molecule refers to the amino acid fragment of an antibody molecule that participates in specific antigen binding, such as Fab, Fab', (Fab')2, scFv, and sdAb. Those skilled in the art know how to obtain these antigen-binding fragments. For example, a classic antibody molecule can be digested with papain to obtain a Fab fragment, and with pepsin to obtain a F(ab')2. Fab' fragments can be formed by treating the F(ab')2 with a reducing agent to break the disulfide bonds within the hinge region.

[0143] Single-chain antibodies (scFvs) are composed of the variable regions of the heavy and light chains of an antibody, linked by a short peptide. Properly folded, the variable regions from the heavy and light chains interact non-covalently to form the Fv fragment, allowing scFvs to retain their affinity for the antigen.

[0144] "Single domain antibody (sdAb)", or also called "VHH antibody", refers to an antibody molecule with antigen binding ability, including heavy chain variable region but no light chain. Structurally, single domain antibodies can also be considered as an antigen binding fragment of an antibody molecule. It was first discovered in camelids, and then researchers discovered more single domain antibodies with antigen binding ability through screening of antibody libraries (such as phage display libraries). Single domain antibodies have some advantages over ordinary antibody molecules (for example, classic tetrameric antibody molecules) or their antigen binding fragments, such as but not limited to: smaller molecular weight, easy to reach tissues or parts that ordinary antibody molecules are difficult to reach when used in the human body, or able to access antigenic epitopes in proteins or polypeptides that are difficult for ordinary antibody molecules to access; more stable, able to withstand changes in temperature and pH, as well as the effects of denaturants and proteases.

[0145] When referring to an antibody or antigen-binding fragment thereof, "targeting" or "specific binding" means that one molecule (e.g., an antibody or antigen-binding fragment thereof) has a higher binding affinity for another molecule (e.g., a tumor cell surface antigen) than for other molecules present in the environment. "Targeting" or "specific binding" 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.

[0146] "Chimeric antibody receptor (CAR)", also known as chimeric T cell receptor, chimeric immune receptor, is an engineered membrane protein receptor molecule that can confer desired specificity to immune effector cells, such as the ability to bind to cell surface proteins (such as tumor antigens). Chimeric antigen receptors are generally composed of an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain. Typically, the antigen binding domain is a scFv or sdAb sequence that is responsible for recognizing and binding to specific antigens. The antigen binding domain can be monospecific, i.e., it has specific binding ability to only one antigen; or it can be multispecific (e.g., bispecific), i.e., it has specific binding ability to multiple antigens. In some examples provided herein, the bispecific extracellular antigen binding domain has specific binding ability to both CD5 and CD7, which can be achieved by including in the extracellular antigen binding domain an antibody fragment (e.g., scFv or sdAb) that targets or specifically binds to CD5 and an antibody fragment (e.g., scFv or sdAb) that targets or specifically binds to CD7. The intracellular signaling domain generally includes an immunoreceptor tyrosine activation motif (ITAM), such as a signaling domain derived from a CD3ζ molecule, which is responsible for activating immune effector cells and producing a killing effect. In addition, the chimeric antigen receptor may also include a signal peptide at the amino terminus that is responsible for the localization of the new protein in the cell, and a hinge region between the antigen binding domain and the transmembrane domain. The intracellular signaling domain may also include a costimulatory domain derived from, for example, a 4-1BB or CD28 molecule. Accordingly, T cells expressing CAR are referred to as CAR-T. CAR-T uses the CAR expressed on its cell surface to identify target cells, and after being activated by the target cells, produces a killing effect on the target cells in a non-MHC restricted manner. In one example, the general process of treating a subject (such as a cancer patient) using CAR-T cells is as follows: peripheral blood mononuclear cells (PBMC) are collected from the subject, T cells are isolated and cultured, CAR encoding nucleic acid sequences are introduced by lentiviral transduction, CAR+ cells are further cultured and collected, and CAR+ cells are returned to the subject. It is known to those skilled in the art that in some cases, NK cells can be used instead of T cells to carry out the process. Therefore, when referring to CAR-T, NK cells expressing CAR may also be included as appropriate. In addition, when referring to CAR-T cells herein, unless otherwise specified, it refers not only to cells directly modified by CAR, but also to daughter cells produced after these cells proliferate in vitro or in vivo.

[0147] "Universal CAR-T cells (UCAR-T)" herein refers to CAR-T cells that are not limited to being transfused into a specific patient. In the prior art, in order to prevent GvHD and host rejection of the transplant, cells (such as T cells) are usually collected from the patient's body and modified with CAR before being transfused back into the patient's body. 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. In contrast, the universal CAR-T cells here refer to those that are suitable for allogeneic transplantation, and the same batch of CAR-T cells can be used for different patients, and these universal CAR-T cells are usually not derived from these patients.

[0148] CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) gene editing technology is a newly emerging RNA-guided technique that uses Cas9 nucleases to edit target genes. The CRISPR gene editing system employed in this technology consists of the Cas9 nuclease and a single-guide RNA (sgRNA), optionally including ssDNA as a repair template. A portion of the sgRNA sequence binds to the Cas9 nuclease, while another portion (crRNA) is complementary to a portion of the target gene. Recognition by the sgRNA enables the Cas9 nuclease to create a single-strand or double-strand nick at a specific site in the target gene. Cells typically repair DNA breaks through two pathways: homologous recombination repair (HDR) and non-homologous end joining (NHEJ). When using CRISPR technology to knock out a gene in a cell, the only concern is disrupting the gene's normal coding function, such as causing a frameshift mutation or deletion of a gene segment, resulting in the inability to produce a functional product (e.g., protein). Typically, after introducing a Cas nuclease (e.g., Cas9) and a sgRNA into cells, cells can be screened for cells that do not express the product of the gene to be knocked out. The term "CRISPR gene editing system" as used herein refers to the combination of a Cas nuclease and a sgRNA, which are used to edit the gene targeted by the sgRNA after introduction into cells.

[0149] In addition to CRISPR technology, other technologies can also be used to achieve gene knockout, such as homologous recombination and TALEN technology.

[0150] "Cytosine base editing technology" is a gene editing technology that introduces single-base editing capabilities 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 double-strand breaks in DNA and can only cut the single DNA strand on the genome that is complementary to the sgRNA, thereby inducing base mismatch repair (BER). The other two functional parts are the cytosine deaminase APOBEC and the uracil glycosylase inhibitor (UGI). APOBEC can induce the deamination of cytosine on the other DNA strand (the non-sgRNA targeted strand) to form uracil. In the presence of the UGI protein, it ultimately promotes the mutation of cytosine to thymine (C->T mutation). In this article, cytosine base editing is used to introduce a stop codon into the gene to be knocked out in cells, thereby preventing the cells from producing functional gene products. Cytosine base editing can be considered part of CRISPR technology, but for the sake of convenience, when referring to CRISPR technology, especially CRISPR technology using the Cas9 protein, this single-base editing function is not involved, thus distinguishing it from cytosine base editing technology.

[0151] "Knockout" or "gene knockout" refers to changing the nucleotide sequence of a gene in a cell in this article, regardless of whether the change is a nucleotide insertion, deletion or replacement, as long as the gene being knocked out cannot produce a functional gene product (such as RNA or protein) in the cell. Ideally, gene knockout causes the cell or cell group to not form a gene product or a functional gene product of the gene at all. Understandably, causing the amount of the gene product to be significantly reduced, or the activity of the gene product to be significantly reduced, 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. In some embodiments, gene knockout can be performed in sequence, that is, after knocking out a gene, the knockout of the next gene is then performed. In other embodiments, two or more genes can be knocked out simultaneously. For example, when using CRISPR technology to knock out multiple genes in a cell, Cas9 and a variety of sgRNAs targeting each gene can be introduced into the cell simultaneously.

[0152] When referring to sgRNA, the term "target sequence" refers to a nucleotide fragment within the target gene or gene to be knocked out that is complementary to a portion of the sgRNA sequence (crRNA, approximately 20 bases). This complementary portion of the sgRNA allows proteins like Cas9 to introduce nucleotide sequence changes within the target gene at a relatively defined location, achieving gene knockout. Accordingly, in this article, "sgRNA targeting a specific sequence" refers to the sgRNA targeting that specific sequence.

[0153] In this article, "RNP complex" refers to the product of the combination of sgRNA and the corresponding Cas enzyme (such as Cas9). When performing gene knockout using CRISPR technology or cytosine base editing technology, the sgRNA and Cas enzyme can be mixed first and then introduced into cells (such as T cells) by electroporation.

[0154] "EGFRt" or "tEGFR" are used interchangeably herein and 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 the cytoplasmic signaling tail, but retains the extracellular epitope recognized by the anti-EGFR antibody. EGFRt can be used as a non-immunogenic selection tool and a tracking marker with genetically modified cell function. In this article, it can be used as a marker molecule for CAR-T cells on the one hand, and on the other hand, it can also be used to clear CAR-T cells in the body when needed, for example, through an ADCC pathway mediated by EGFR antibodies (e.g., cetuximab) (see US8802374B2), that is, used as a safety switch in clinical transformation.

[0155] "HSV-TK" is the abbreviation of Herpes Simplex Virus Thymidine Kinase, whose substrate is the small molecule drug GCV. The role of HSV-TK and GCV as molecular switches is described in more detail below.

[0156] "Self-cleaving peptides" refer to short peptides that can achieve the function of cleaving proteins through ribosome skipping rather than protease hydrolysis, and may include T2A, F2A and P2A.

[0157] "Treatment" refers to processing a subject to obtain a useful or desired clinical outcome. "Treatment" as used herein encompasses various treatment means, including administration to the subject, surgery, radiation, etc. with any possible medicine. For purposes of the present invention, useful or desired clinical outcomes include, but are not limited to, any one or more of the following: alleviating one or more symptoms, weakening the degree of disease, preventing or delaying disease spread (e.g., metastasis, such as metastasis to the lungs or lymph nodes), preventing or delaying disease recurrence, delaying or slowing disease progression, improving the disease condition, suppressing disease or disease progression, blocking its development and alleviation (whether partially or completely alleviated). The method provided herein encompasses any one or more of these treatment aspects. According to the above, "treatment" does not need to completely remove all symptoms of a disease or disease or completely alleviate.

[0158] The term "therapeutically effective amount" refers to an amount of an active compound sufficient to elicit a biological or medical response in a subject as desired by a clinician. A "therapeutically effective amount" of the fusion protein of the present invention can be determined by one skilled in the art based on factors such as the route of administration, the subject's weight, age, and condition. For example, a typical daily dose may range from 0.01 mg to 100 mg or more of the active ingredient per kg of body weight.

[0159] When referring to pharmaceutical compositions, the term "pharmaceutically acceptable carrier" refers to a solid or liquid diluent, filler, antioxidant, stabilizer, or other substance that can be safely administered, is suitable for administration to humans and / or animals without excessive adverse side effects, and is suitable for maintaining the activity of the drug or active agent contained therein. Depending on the route of administration, various carriers well known in the art can be used, 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, emulsifiers, isotonic saline, and / or pyrogen-free water. The pharmaceutical compositions provided herein can be prepared into clinically acceptable dosage forms such as powders and injections. The pharmaceutical compositions of the present invention can be administered to a subject using any appropriate route, for example, orally, by intravenous infusion, intramuscular injection, subcutaneous injection, subperitoneally, rectally, sublingually, or by inhalation, transdermally, or other routes of administration.

[0160] A "pharmaceutical kit" refers to a pharmaceutical combination comprising at least two active ingredients. Unlike a pharmaceutical composition, at least one active ingredient in a pharmaceutical kit is stored separately from the other active ingredients.

[0161] "Subject" refers to an animal, such as a mammal, including but not limited to humans, rodents, monkeys, felines, canines, equines, bovines, porcines, sheep, goats, mammalian laboratory animals, mammalian farm animals, mammalian sports animals, and mammalian pets. The subject can be male or female and can be any age-appropriate subject, including infants, young children, young adults, adults, and elderly subjects. In some instances, a subject refers to an individual in need of treatment for a disease or condition. In some instances, the subject receiving treatment may be a patient who suffers from a condition associated with the treatment or is at risk of developing the condition. In other instances, the subject is a healthy individual or an individual suffering from a disease not of interest. In a specific instance, the subject is a human, such as a human patient. This term is generally used interchangeably with "patient," "test subject," "treatment subject," and the like.

[0162] Design of CD5-CD7 universal CAR-T strategy

[0163] In order to solve the GvHD problem, the present inventors knocked out the TRAC and / or TRBC genes to prevent the expression of the T cell surface receptor TCR of CAR-T, thereby preventing it from recognizing the host's cells, as shown in Figure 1.

[0164] In order to solve the problem of the host immune system clearing the input CAR-T cells, the inventors avoided the host's T cells from clearing the CAR-T cells by knocking out the HLA molecules of the CAR-T cells. HLA molecules are divided into two types: HLA-I and HLA-II. HLA-I molecules are recognized by CD8-positive cytotoxic T cells and are the main way for host T cells to clear CAR-T cells, while HLA-II molecules are recognized by CD4-positive T cells and are an auxiliary way for host T cells to clear CAR-T cells. Therefore, the inventors have 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 the CIITA gene related to the expression of the HLA-II molecule. The second version of UCAR-T will theoretically better avoid the clearance of host T cells, but the knockout of HLA will activate the host's NK cells, and the activated NK cells will also clear the UCAR-T.

[0165] What's ingenious is that the CAR prepared by the inventors targets CD5 and CD7, which are also expressed on NK cells. Therefore, CAR can attack the host's NK cells while attacking tumor cells, which solves the NK activation problem caused by HLA knockout. Since CAR-T itself also expresses CD5 and CD7, in order to prevent CAR-T suicide, the inventors also knocked out CD5 and CD7 of CAR-T cells at the same time. The knockout of HLA combined with CAR as an attack molecule weakens the clearance of UCAR-T cells by the host's T and NK cells, thereby enabling UCAR-T to be well expanded in the body.

[0166] The preparation process for universal CAR-T cells is essentially the same as that for standard CAR-T cells, except for the inclusion of a gene knockout step. The general process is as follows: T cell sorting and activation → gene knockout by electroporation → lentiviral transduction → knockout efficiency and CAR integration efficiency testing → in vitro functional validation → cell preparation cryopreservation.

[0167] CD5-CD7 CAR construct

[0168] The structure of the CAR used in this article is shown in Figure 2. The single VH domains of CD7 and CD5 (single domain antibody) are connected in series through a linker, followed by the CD8α hinge region, TM transmembrane region, 4-1BB and CD3ζ costimulatory molecules.

[0169] The EGFRt switch molecule can be connected thereafter via T2A, which separates the EGFRt switch molecule from the CAR structure. As described below, in some cases, it is more advantageous to use a suicide gene as a molecular switch, such as the herpes simplex virus thymidine kinase (HSV-TK) gene. The CAR structure design using the HSV-TK molecular switch is shown in Figure 27A.

[0170] CAR-T switch molecules independent of immune cells

[0171] Because CD5-CD7 universal CAR-T cells eliminate normal T / NK cells in the patient's body while clearing tumors, the patient is in an immunodeficient state during treatment and cannot survive under normal circumstances. In addition, if CD5 knockout T cells persist in the patient's body for a long time, they can easily lead to autoimmune diseases. Therefore, it is necessary to control the presence and proliferation of CD5-CD7 universal CAR-T cells in the subject, which can be achieved through the use of molecular switches.

[0172] Currently, the commonly used CAR-T switch molecule is EGFR. The anti-EGFR antibody cetuximab can be used to eliminate CAR-T cells, but this method requires the participation of immune cells such as NK cells. Because the inventors' CD5-CD7 UCAR-T also kills NK cells in the patient's body, the EGFR switch is no longer suitable for CD5-CD7 UCAR-T.

[0173] CAR-T switch molecules that do not rely on immune cells are usually controlled by small molecule drugs to activate or shut down (kill) CAR-T cells by controlling a certain component in CAR-T. Ali Can Sahillioglu Ton and N Schumacher published an article in Current Opinion in Immunology in 2022. 8 The CAR-T small molecule drug switches discovered in current research are summarized and can be roughly divided into seven categories according to their mechanism of action, as shown in Figure 3, including suicide genes, transcriptional regulation, stability control, etc.

[0174] One of the versions of the inventor's CD5-CD7 UCAR-T uses HSV-TK in the suicide gene switch. 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 (Ganciclovir). GCV was launched by Syntex Corporation of the United States and approved for marketing in 1988. It is the first choice drug for treating cytomegalovirus infection. 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 the kinase in the cell diphosphorylates and triphosphorylates it. The structure of triphosphorylated GCV is extremely similar to the nucleosides in the cell, so it will competitively bind to DNA polymerase or break the ratio of the four nucleosides in the cell to inhibit DNA synthesis. After DNA synthesis is blocked, the cells will gradually die.

[0175] Therefore, on one hand, the present inventors used the knockout function of the CRISPR / Cas9 system to target the TRAC / B2M / CD5 / CD7 / CIITA genes and successfully screened out sgRNAs with high knockout efficiency and low off-target probability.

[0176] On the other hand, the inventors used the base editing function of the CRISPR / Cas9 system to target the TRBC / B2M / CD5 / CD7 / CIITA genes and successfully screened out sgRNA that can achieve gene silencing by introducing stop codons in important regions of the genes.

[0177] On the other hand, the inventors successfully achieved high-efficiency knockout of multiple genes simultaneously (the knockout efficiency of each gene was about 90%) through the screening of sgRNA and optimization of the electroporation process.

[0178] On the other hand, the present inventors have successfully prepared CD5-CD7 UCAR-T with strong tumor killing function and expansion ability in vitro / in vivo.

[0179] On the other hand, the inventors successfully achieved efficient clearance of UCAR-T cells in vivo by using HSV-TK in combination with the small molecule drug GCV.

[0180] There are the following differences between universal CD5-CD7 CAR-T and autologous CD5-CD7 CAR-T: 1) The genes knocked out are different: autologous CD5-CD7 CAR-T only needs to knock out the CD5 and CD7 genes, while universal CD5-CD7 CAR-T, in addition to knocking out the CD5 and CD7 genes, also needs to knock out the TCR and HLA related genes; 2) During the preparation of universal CAR-T, UCAR-T may need to be purified to screen out CAR-T cells expressing TCR and HLA complexes 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 suitable for T-cell lymphomas whose tumor cells have not invaded the peripheral blood, while universal CD5-CD7 CAR-T is suitable for all types of T-cell malignancies.

[0181] The present invention is further illustrated below by means of specific examples.

[0182] The general methods involved in the embodiments are as follows:

[0183] 1. Isolation and activation of CD3+ T cells

[0184] 1.0×10 PBMCs of a healthy donor (specific information is confidential) were revived and stored for 24 h. 8 100 cells per tube, quickly thaw and resuspend in 8ml 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 and add 20ul / 10 7CD3 microbeads were mixed and placed in a 4°C refrigerator for 20 minutes. During this period, the tube wall was flicked several times every 10 minutes to prevent cell precipitation. After incubation, Rinsing buffer was added to rinse once and centrifuged (400g 10min↑8↓8), and then the cells were resuspended with 500μl Rinsing buffer. At the same time, the LS sorting column was placed on the Miltenyi magnetic sorting rack, rinsed once with 2ml Rinsing buffer, and then 500μl of cell suspension was added. After the cell suspension was dripped off, 2ml Rinsing buffer was added to the LS column twice. The target cells were flushed out from the LS column with 5mL Rinsing buffer and collected. After appropriate dilution, the target cells were counted and about 1×10 5 The purity of the sorted T cells was determined by flow cytometry. The cell suspension was then centrifuged at 300 g for 10 minutes and the cell density was adjusted to 1×10 6 , with 10ul / 10 6 Anti-CD3 / -CD28 antibody magnetic beads were added to activate the cells at a concentration of 100 cells / well, 4 mL per well was seeded into 12-well plates, and cultured in a 37°C, CO2 incubator.

[0185] 2. Activate electroporation

[0186] For cells activated with CD3 / CD28 Dynabeads, electroporation can be performed 24 hours after activation. Collect cells in a centrifuge tube, place on a magnetic rack to remove the Dynabeads, and centrifuge three times (300g for 15 minutes, 8 steps up and 8 steps down). Discard the supernatant, resuspend the cells in an appropriate amount of compound electrolytes, and count the cells. Prepare the appropriate amount of RNP (a complex of Cas9 protein and sgRNA) based on the cell count results and incubate at 37°C for at least 10 minutes. Centrifuge the cells again and resuspend them in the appropriate amount of electroporation buffer. Add the incubated RNP, mix gently, and transfer to the Lonza electroporator cuvette. Select the EH-115 electroporation activation program for T cells. Immediately add a small amount of warmed T cell culture medium. Place in the incubator to recover for at least 15 minutes. Transfer the cell suspension from the cuvette to a suitable culture flask and add T cell culture medium to a culture density of 2 M / ml.

[0187] 3. Pan-T cell negative selection

[0188] Thawed frozen healthy donor PBMCs totaling 1.0×10 8After rapid thawing, resuspend the cells in 8 ml of preheated Rinsing buffer and take a small amount of cell suspension for cell counting. The PBMC suspension was centrifuged at 400g (↑8↓8) for 10 minutes. After the centrifugation, the supernatant was discarded, and Rinsing buffer (40ul / 10^7 cells) and Pan T Cell Biotin-Antibody Cocktail (10ul / 10^7 cells) were added. After gently pipetting with a pipette tip to mix, the tube was placed in a 4℃ refrigerator for 10 minutes. During this period, the tube wall was gently tapped once to prevent cell precipitation. Then, Rinsing buffer (30ul / 10^7 cells) and Pan T Cell MicroBead Cocktail (20ul / 10^7 cells) were added. After gently pipetting with a pipette tip to mix, the tube was placed in a 4℃ refrigerator for 15 minutes. During this period, the tube wall was gently tapped several times every 5 minutes to prevent cell precipitation. While the magnetic beads were incubated, the LS separation column (positive load 100M / column) was taken out and placed on the Miltenyi magnetic separation rack. The separation column was rinsed once with 3ml of Rinse buffer. After the magnetic beads incubation was completed, an appropriate amount of Rinse Buffer, mix the cells and divide them evenly into each LS, 2ml per column; after the cell suspension is drained, wash the column twice with 2ml Rinsing buffer. The negative cells that flow out are T cells. Take 100ul from the cells, dilute 10 times with 900ul buffer, and take 20ul for counting; based on the counting results, culture the T cells in T cell culture medium without cytokines for 4h and then perform electroporation, with the culture density being 5M / ml.

[0189] 4. Resting Electroporation

[0190] After the negatively selected T cells have been rested for 4 hours, the cells are collected in a centrifuge tube and the cells are counted. According to the cell count results, the corresponding amount of RNP (a complex of Cas9 protein and sgRNA) is prepared and incubated at 37°C for more than 10 minutes. At the same time, the cells are centrifuged and resuspended with the corresponding amount of electroporation buffer. The incubated RNP is added and gently mixed. The cells are added to the electroporation cup of the Celletrix electroporator and the corresponding parameters for electroporation of resting T cells (100ul system, 1380V, 3ms) are set. After electroporation, the cells are immediately aspirated into the culture flask (the culture medium in the culture flask has been pre-added with CD2 / CD3 / CD28 activator and warmed). Do not blow the cells at this time. The cells are then placed in the incubator and cultured at a density of 2M / ml.

[0191] 5. Lentiviral Transduction of CAR

[0192] 48 hours after cell activation, CAR lentiviral transduction was performed. Cell suspensions were viability tested and cell counts were performed. Based on the cell count results, the appropriate amount of lentivirus was added at an MOI of 3. 100x LentiBoost was then added, and the cells were gently mixed and cultured in a 37°C incubator. After 24 hours, the medium was exchanged to remove the virus and fresh medium was used to continue culturing T cells at a density of 1 M / ml.

[0193] 6. FACS (flow cytometry) detection

[0194] Take about 2×10^5 cell suspension in a 1.5ml centrifuge tube, centrifuge at 300g for 5 minutes, wash once with PBS + 2% fetal bovine serum buffer, completely discard the supernatant, resuspend the cells with 100μl buffer and add 1μl of corresponding antibody, mix well and incubate at 4℃ in the dark for 30 minutes, add 100ul buffer and wash once, resuspend with 100ul buffer containing DAPI or 7AAD and detect on the machine.

[0195] 7.TRAC / B2M Negative Selection

[0196] Around the sixth day of cell preparation, cells in which TRAC and B2M have been successfully knocked out no longer express the corresponding proteins. Therefore, the cells can be negatively selected to separate the cells in which TRAC and B2M have been successfully knocked out and continue to be cultured. The specific steps are as follows: collect cells and centrifuge at room temperature (400g for 15 minutes, 8 steps up and 8 steps down), discard the supernatant after the end, resuspend the cells with Rinsing Buffer (80ul / 10^7cells), add FITC-B2M antibody (1ul / 10^6cells), and incubate in the dark for 20 minutes at four degrees; after incubation, add 40ml of Rinsing Buffer to resuspend the cells, centrifuge at room temperature, discard the supernatant after the end, resuspend the cells with Rinsing Buffer (80ul / 10^7cells), add FITC-beads (1ul / 10^6cells), add CD3-beads (1ul / 10^6cells), and incubate in the dark for 20 minutes at four degrees; after incubation, add 40ml of Rinsing Buffer to resuspend the cells, centrifuge at room temperature, and rinse the LD column with 3ml of buffer at the same time; after centrifugation, discard the supernatant, and use Rinsing Resuspend the cells in Buffer (1*10^8 / ml), pass through the column twice (2ml / column), then wash the column twice with 3ml of buffer, collect the negative cells that flow through, count them, and take a small amount of cells for FACS detection of knockout efficiency, CAR positivity rate and sorting purity, etc. After centrifugation, the negative cells are cultured in a G-rex culture flask with 500ml-1000ml of T cell complete culture medium.

[0197] 8.CD107a Release Detection

[0198] Prepare the required amount of 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, centrifuge the cells at a 1:1 ratio (UCAR-T cells are counted as CAR-positive cells) (generally CAR-positive cells are above 0.1M), and then resuspend them in culture medium containing CD107a antibodies and monensin, inoculate them in 96-well plates, and incubate them in a cell culture incubator (37°C 5% CO2) for 4 hours; then perform FACS staining on the cells, generally CD8 and EGFR antibodies are also required, and calculate the ratio of CD107a-positive cells under CD8-positive and CAR-positive T cells by FACS detection.

[0199] 9. Tumor killing detection

[0200] Generally, UCAR-T cells and a variety of targeted and non-targeted tumor cells expressing luciferase (firefly luciferin) are inoculated into 96-well plates at different effector-target ratios (UCART cells are counted as CAR-positive cells, and the amount of fixed tumor cells). After co-culture in a cell culture incubator for 24 hours, half of the cell suspension is taken out and placed in an opaque white-bottomed 96-well plate. 10x luciferase substrate is added and mixed. After reacting at room temperature for 10 minutes, the luciferase fluorescence value is detected on a machine. The fluorescence value indirectly reflects the number of surviving tumor cells, and thus reflects the tumoricidal function of UCAR-T cells.

[0201] 10. In vitro testing of GCV efficacy

[0202] UCAR-T cells were counted, and 8M of cells were centrifuged. After the cells were centrifuged, the supernatant was discarded and resuspended in 8ml of fresh T cell culture medium. The cells were evenly distributed into 8 wells of a 12-well plate, with 1ml per well. DMSO, 0.3ug / ml, 1ug / ml, and 3ug / ml of GCV were then added to two wells in a group. The cells were cultured in an incubator and then monitored for changes in CAR-positive cells by counting and FACS analysis every 3 days. At the same time, cell pellets were collected every 3 days for genome extraction, and changes in CAR molecules at the DNA level were detected by fluorescent probe qPCR.

[0203] 11. Guide-seq off-target detection

[0204] The RNP of the sgRNA to be tested and dsODN were introduced into activated T cells by electroporation. 5-7 days after electroporation, the genome with dsODN integration was extracted. The genome was fragmented into fragments with an average of 500 bp using a Covaris S220 instrument. The gene fragments were purified and recovered using 0.8x Ampure XP beads. Ultra TM II DNA Library Prep Kit for The purified genomic fragments were repaired and A-added using the kit. Ultra TM II DNA Library Prep Kit for The kit ligates the A-added DNA fragments to a linker sequence, using a different linker for each sample. The DNA fragments are then purified using 0.8x Ampure XP beads. PCR is then performed with primers located on the linker sequence and the dsODN. The PCR product is recovered and then linked to universal P5 and P7Y adapters using the ABclonal Rapid DNA Lib Prep Kit. After purification, the product is ready for next-generation sequencing. By analyzing the sequencing results and aligning the genomic sequence between the linker and dsODN sequences with the sgRNA sequence, off-target site information and off-target probability can be obtained.

[0205] 12.SgRNA Design and Editing Efficiency Verification

[0206] 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.org / # / ), CRISPOR (http: / / crispor.tefor.net / ), IDT (https: / / sg.idtdna.com / site / order / designtool / index / CRISPR_SEQUENCE), etc. These websites can predict on-target and off-target efficiencies.

[0207] The general steps are as follows:

[0208] 1) Find the genomic sequence of the target gene: The genomic sequence of the target gene can be viewed and downloaded from the NCBI (https: / / www.ncbi.nlm.nih.gov / ) and UCSC (https: / / genome.ucsc.edu / ) websites;

[0209] 2) sgRNA prediction and synthesis: Input the target sequence of the target gene into the above-mentioned sgRNA prediction website to generate a number of candidate sgRNAs. Select the appropriate amount of sgRNA sequence based on the on-target and off-target rankings. Tell the third-party company the sgRNA targeting sequence (about 20 nt) to synthesize the full-length sgRNA.

[0210] Verification of candidate sgRNA editing efficiency: After the candidate sgRNA is synthesized, the Cas9 protein and sgRNA complex RNP (ribonucleoprotein) can be directly introduced into the target cells by electroporation. 24-48 hours after electroporation, the editing efficiency at the gene level can be detected by PCR and sequencing. After 72 hours, the changes in protein levels can be detected by FACS.

[0211] The main reagents involved in the embodiment are as follows:

[0212] The main consumables involved in the embodiment are as follows:

[0213] Example 1: Using CRISPR / Cas9 technology to knock out TRAC / TRBC, B2M, CIITA, CD5, and CD7 antigens on the surface of T cells

[0214] 1.1 TRAC, B2M, CIITA, CD5, and CD7 sgRNA screening

[0215] 1.1.1 TRAC sgRNA screening results

[0216] The inventors obtained information on numerous potential sgRNAs through commonly used sgRNA design websites, including CRISPick, CRISPOR, and IDT. After comprehensively considering on-target and off-target information, they selected seven candidate sgRNAs (Figure 4A). The targeting sequences of these seven candidate sgRNAs were shared with a technical service company (Nanjing GenScript Biotechnology Co., Ltd.), which synthesized the full-length sgRNAs (targeting sequence + backbone sequence).

[0217] After the sgRNA was synthesized, it was dissolved in nuclease-free water to 100-500 pmol / μl, then mixed with Cas9 protein in different amounts and incubated at 37°C for more than 10 minutes. The complex of Cas9 and sgRNA was then electroporated using a Lonza electroporator to introduce the Cas9 and sgRNA complex into T cells. After 6 days of cell culture, the expression of the TCR complex was detected by FACS, thereby reflecting the knockout efficiency of different sgRNAs on TRAC. As can be seen from Figure 4B, at a higher RNP dosage (Cas9 60 pmol, sgRNA 150 pmol), the knockout efficiency of all 7 sgRNAs can reach more than 90%, but at a lower RNP dosage (Cas9 30 pmol, sgRNA 30 pmol), only some sgRNAs can achieve a knockout efficiency of more than 90%, among which the A-XL sgRNA had the highest knockout efficiency (99.5%), so the inventors chose this sgRNA for subsequent preparation of UCAR-T. The A-XL sgRNA has also been used by multiple researchers. 4,9 .

[0218] The inventors 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 Figures 4C-E. A-XL showed a high-probability off-target site in both detection results. A-02 also showed a high-probability off-target site in one detection result. The off-target probability of A-06 was slightly lower. Therefore, this version of UCAR-T may tend to use the sgRNA A-06 in the future.

[0219] 1.1.2 B2M sgRNA screening results

[0220] As shown in Figure 5A, the inventors used CRISPick, CRISPOR, IDT, CHOPCHPOP, and GUIDES websites to predict and select candidate sgRNA information after comprehensive consideration. The Cas9 protein and sgRNA complex was introduced into T cells using the same electroporation method. After 8 days of electroporation, an appropriate amount of cells were taken out and the expression of B2M was detected by flow cytometry, thereby reflecting the knockout efficiency of B2M by different candidate sgRNAs. As can be seen from Figure 5B, B-03 has the highest knockout efficiency (more than 90%), B-10, B-13, and B-XL have comparable knockout efficiencies (more than 80%), and B-01 has a too low knockout efficiency (about 50%).

[0221] At the same time, the inventors performed double knockouts with a single B2M-sgRNA and a tested TRAC-sgRNA (A-XL) in order to screen out a B2M-sgRNA with better TRAC-B2M double knockout efficiency. Similarly, the inventors mixed Cas9 protein with TRAC-sgRNA and B2M-sgRNA for incubation for 10 minutes and then introduced them into T cells by electroporation. Eight days after electroporation, an appropriate amount of cells were taken out and the expression of B2M and TCR complexes was detected by FACS. As can be seen from Figure 6A, in the case of double knockout of TRAC and B2M, B-03 still performed best (double knockout efficiency of 93%), followed by B-13 and B-XL (double knockout efficiency of 91%), B-10 was slightly worse (double knockout efficiency of 88%), and B-01 was still the worst (double knockout efficiency of 50%), so the sgRNA B-01 was excluded in subsequent tests.

[0222] Next, the inventors tested the knockout efficiency of the TRAC / B2M (B-03, B-10, B-13, B-XL) sgRNA combination that performed relatively well in the double knockout test at different RNP concentrations, in order to screen out sgRNA combinations that achieve a TRAC-B2M double knockout efficiency of more than 90% at lower RNP concentrations. Specifically, the inventors tested 30pmol+30pmol, 60pmol+60pmol, and 60pmol+150pmol of Cas9 protein and sgRNA (the amount of TRAC and B2M RNP in each group was the same). The results are shown in Figure 6B. At a higher concentration of RNP (60pmol+150pmol), all four groups of sgRNA combinations can achieve a double knockout efficiency of more than 90%, but at a lower concentration of RNP (60pmol+60pmol, 30pmol +30pmol), only B-03 and B-13 can achieve acceptable double knockout efficiency (more than 80%); at an RNP concentration of 30pmol+30pmol, the phenotype of B-13 is better than that of B-03, and at an RNP concentration of 60pmol+60pmol, the efficiency of B-03 and B-13 is comparable, but the double knockout efficiency does not reach more than 90%. Considering that B-03 and B-13 are on different exons of B2M, the inventors decided to use B-03 and B-13 simultaneously to achieve B2M knockout.

[0223] The inventors conducted off-target detection on the two sgRNAs B-03 and B-13. The results are shown in Figures 6C-D. The sequences with the highest off-target probability in the two detection results of B-13 are the same, indicating that this site is very likely to have off-target effects. However, the sequences with the highest off-target probability measured in the two detection results of B-03 are different. One of the sequences has a maximum off-target probability of 3%, while the other has a maximum off-target probability of only 0.01%, indicating that the off-target probability of the sgRNA B-03 is low.

[0224] 1.2.3 CD5 sgRNA screening results

[0225] The inventors screened CD5 knockout sgRNAs twice. Figures 7A and 7B show the sgRNA sequence information and corresponding knockout efficiency results from the first screening. sgRNA1 and sgRNA3 showed good knockout efficiency, but these selected sgRNAs had not been previously analyzed for off-target effects. To identify sgRNAs with high knockout efficiency and low off-target effects, the inventors designed five more sgRNAs and tested them alongside sgRNA1 and sgRNA3 for knockout efficiency and off-target rate. Figure 7C shows the candidate sgRNAs selected during the second screening, after comprehensive consideration of predictions from CRISPick, CRISPOR, IDT, CHOPCHPOP, and GUIDES. C-01 and C-03 represent sgRNA1 and sgRNA3 in Figure 7A.

[0226] After the sgRNAs were synthesized, the inventors first tested their knockout efficiency using the same method used for the other sgRNA screening described above. The results, as shown in Figures 8A-B, indicate that, with the exception of C-05, all other sgRNAs achieved knockout efficiencies exceeding 80%. Off-target predictions from the CRISPick website indicated that C-06 had the lowest off-target probability (Figure 8C). Therefore, the inventors used Guide-Seq to assess the actual off-target probability of C-06. The results, shown in Figure 8D, indicate that the sequences with the highest off-target probability were identical, but the off-target probability was less than 0.4%, and the off-target sites were primarily located in intronic regions of genes, with a very low probability of affecting protein coding. Therefore, the inventors selected C-06 for use in subsequent experiments.

[0227] 1.2.4 CD7 sgRNA

[0228] The CD7 sgRNA used in this invention is from the reported literature 10 The inventors conducted off-target testing. As shown in Figure 9, three sequences were found to occur frequently in the off-target test in one experiment. However, two of these sites also appeared in the control group without sgRNA, and their frequencies were essentially the same, so they could be considered false positives. The second-most frequent sequence had a higher number of mutated bases and was located closer to NGG than the sgRNA sequence, making it less likely to be off-target. In summary, the probability of off-target effects with CD7 sgRNA is extremely low.

[0229] 1.2.5 CIITA sgRNA screening results

[0230] For the CIITA gene, the inventors selected five sgRNAs for knockout efficiency testing. The results are shown in FIG10A , and F-05 had the best knockout efficiency.

[0231] The inventors conducted off-target detection on the sgRNA F-05. The results are shown in Figure 10B. The sequences with the highest off-target probability detected in the two experiments were inconsistent and the probability of occurrence was about 0.1%. Therefore, the off-target probability of F-05 is extremely low.

[0232] 1.2 Knockout of TRAC, B2M, CD5, and CD7 antigens on the surface of T cells based on Cas9 protein

[0233] Before preparing CD5-CD7 UCAR-T, the inventors first explored RNP conditions for co-knockout of TRAC, B2M, CD5, and CD7. As shown in Figure 11, a total of six RNP dosages were set up and tested on T cells from two donors. The results showed that under all six conditions, the knockout efficiency of TRAC, B2M, CD5, and CD7 was 90% or above, and there was no significant difference between the two donors. Therefore, the inventors selected RNP combination 6, which had the lowest dosage, as the basis for the subsequent preparation of CD5-CD7 UCAR-T.

[0234] To ensure CD5 knockout efficiency during the first preparation of CD5-CD7 UCAR-T cells, the inventors increased the CD5 RNP dosage from 40 / 120 pmol / 20 μl to 80 / 150 pmol / 20 μl, based on RNP combination 6. The results are shown in Figures 12A-B. Nine days after knockout, FACS analysis of the knockout status of each gene revealed a TRAC / B2M double knockout efficiency of 97.8% and a CD5 / CD7 double knockout efficiency of 95.8%. The knockout efficiencies of TRAC, B2M, CD5, and CD7 were all very high, so the inventors used these electroporation conditions in subsequent preparations of CD5-CD7 UCAR-T cells.

[0235] Figure 12C shows the knockout results of the KO non-transduced group 4 days after the second preparation of CD5-CD7 UCAR-T by the inventors. In the case of non-transduced cells, the TRAC / B2M double knockout efficiency was 86.7%, and the CD5 / CD7 double knockout efficiency was 90.6%. The knockout efficiency was already very high. After transduction, UCAR-T will also kill some CD5 or CD7 positive cells, so the double knockout efficiency of TRAC / B2M and CD5 / CD7 will be even higher in the UCAR-T group.

[0236] 1.3 Knockout of TRAC, B2M, CIITA, CD5, and CD7 antigens on the surface of T cells based on Cas9 protein

[0237] In order to give UCAR-T a greater 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. After knocking out the CIITA gene, UCAR-T does not express HLA-II class molecules, which can prevent CD4+ T cells in the host body from clearing UCAR-T.

[0238] Before preparing the second version of CD5-CD7 UCAR-T, the inventors first conducted a preliminary experiment on a Celletrix electroporator to simultaneously knock out five genes: TRAC, B2M, CIITA, CD5, and CD7. The RNP dosage and knockout results are shown in Figures 13A-B. Five days after the knockout, FACS analysis of the expression of each protein showed a TRAC / B2M double knockout efficiency of 69.9%, a TRAC / CIITA double knockout efficiency of 62.8%, and a CD5 / CD7 double knockout efficiency of 85.3%. Based on the clustering, the double knockout efficiencies of TRAC / B2M and CD5 / CD7 were acceptable, but the double knockout efficiency of TRAC / CIITA was slightly lower.

[0239] According to the results of the preliminary experiment, the inventors increased the amount of RNP for the B2M and CIITA genes when preparing the second version of CD5-CD7 UCAR-T. As shown in Figure 13C-D, the expression of each protein was detected by FACS 4 days after knockout. 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 preliminary experiment.

[0240] Example 2: Knockout of TRBC, B2M, CIITA, CD5, and CD7 antigens on the surface of T cells based on nCBE3 / nCBE4 proteins

[0241] In addition to using Cas9 to knock out genes, the inventors also attempted to disrupt gene expression by introducing stop codons in the exon regions of genes using the base editing proteins nCBE3 or nCBE4. The nCBE3 or nCBE4 proteins are fused to the N-terminus of the Nikase-Cas9 protein (which only cuts the target strand) and express a cytosine deaminase (ABOBEC) and one or two uracil glycosylase inhibitors (UGI) at the C-terminus. While Nikase-Cas9 cuts the target strand, the ABOBEC deaminates cytosine (C) in the non-target strand of the targeted region, converting it to uracil (U), while the UGI maintains the stability of the uracil (U). After the target strand is cut and repaired, the corresponding G on this strand is repaired to an A using the U as a template. Subsequent DNA replication or repair replaces the U on the non-target strand with a T, ultimately achieving the C-to-T conversion. Therefore, using nCBE3 or nCBE4, CAA, CAG, CGA on the sense chain of the exon region can be mutated to TAA, TAG, TGA, or CCA on the antisense chain can be mutated to TTA (the corresponding sense chain is TAA), thereby introducing a stop codon prematurely and disrupting protein expression.

[0242] sgRNA screening

[0243] According to the above principles, the inventors designed multiple candidate sgRNAs for TRBC, B2M, CD5, CD7, and CIITA genes. The sequence information is shown in Figure 14. Among them, the sgRNA of CD7 has been reported in the literature and has high efficiency. 11 , therefore no further sgRNA screening was performed for CD7.

[0244] The efficiency test method of base editing sgRNA is the same as that of knockout sgRNA. The protein expression is observed by flow cytometry 5 days after electroporation. As shown in Figure 15, for TCR knockout, BE-A02 and BE-A03 have the highest efficiency and are comparable. Considering that BE-A02 has been reported in the literature, 11 , the inventors selected BE-A03 as the sgRNA for subsequent use.

[0245] As shown in Figure 16A, the knockout efficiency of all three sgRNAs for B2M was suboptimal, but there were no more suitable sgRNA options. Considering that nCBE3 or nCBE4 can cleave a single DNA strand, the inventors also attempted to achieve gene knockout using two similar sgRNAs, one on the sense and one on the antisense strands. The design and results are shown in Figures 16B-C. The combination of B-10 and B-13 achieved the highest efficiency, so the inventors chose to combine BE-B02 with B-10 and B-13 to achieve B2M knockout.

[0246] For CD5 knockout, the results are shown in Figure 17A. BE-C07 had the highest efficiency, reaching over 90%, so the inventors selected BE-C07. The inventors also verified the CD7-sgRNA used in the literature, and the knockout efficiency was approximately 87% (Figure 17B).

[0247] For the knockout of CIITA, the inventors tested 11 sgRNAs. The results are shown in Figure 18. On the 7th day after knockout, BE-F04 and BE-F10 had better efficiency, among which BE-F10 had the best efficiency, with a knockout efficiency of more than 80%. Therefore, the inventors finally selected BE-F10.

[0248] Example 3 Lentiviral transduction and detection of the proportion of CAR-positive cells

[0249] Generally, CAR lentiviral transduction is performed 48 hours after T cell activation. The virus is generally used at an MOI of 3, and the lentiviral transduction enhancer Lenti-boost is added at the same time (100-fold dilution). The medium is changed 24 hours after viral transduction, and the proportion of CAR-positive cells can be detected after 3 days. The proportion of CAR-positive cells can be reflected by FACS detection of EGFR or CAR expression in the cells.

[0250] Figure 19 shows the results of the CAR-positive cell ratio test for two prepared CD5-CD7 UCAR-T cells. The expression of EGFR and CAR in the cells was detected using Anti-EGFR-PE and FITC-conjugated CD5 and CD7 antigens, respectively. Three days after transfection, the EGFR positivity rate is generally higher than the CD5 and CD7 CAR positivity rates. This may be due to the CAR internalization after UCAR-T kills CD5 and CD7 non-knockout cells in the early stage. Later, such as on day 8, the CAR-positive cell ratios represented by the three will be relatively consistent. Because CAR-positive cells in CD5-CD7 UCAR-T cells are self-activated (killing CD5 or CD7 non-knockout T cells), the CAR-positive cell ratio increases with the number of days in culture, and the CAR-positive cell ratio will approximately double from day 3 to day 8.

[0251] Example 4 TRAC / B2M Negative Separation

[0252] The most important thing about UCAR-T cells is to avoid immune rejection reactions. Therefore, the finished UCAR-T cells infused back into the patient must generally be guaranteed not to express TCR and HLA-I complexes, otherwise severe GvHD or HvGD will occur. Therefore, when preparing CD5-CD7 UCAR-T, the inventors generally perform TRAC and B2M negative selection on UCAR-T cells around the sixth day. The specific steps are shown in the "TRAC / B2M negative selection" in the general method above.

[0253] Figure 20 shows the results of TRAC / B2M negative selection during the two preparations of CD5-CD7 UCAR-T. As can be seen from the figure, the input cells before negative selection will have a small amount of TCR and B2M expression, while the negative cells after negative selection will almost never have TCR and B2M positivity. Generally, the proportion of TRAC / B2M double-negative cells must be above 99% to be considered qualified.

[0254] Example 5 In vitro functional verification of universal CAR-T cells

[0255] Generally, the function of CAR-T cells can be determined by co-incubating CAR-T cells and tumor target cells in vitro, and then detecting the release of CD107a from CAR-T cells by FACS 4-6 hours later, or detecting the number of tumor cells 24h-48h later to determine whether the CAR-T cells have a good killing function.

[0256] 5.1 CD107a release assay

[0257] CD107a, also known as LAMP-1, is a protein released by CD8+ cytotoxic T cells and NK cells during the process of killing target cells. Therefore, the release of CD107a by CAR-T cells can be used to reflect whether CAR-T cells have killing function.

[0258] Generally, the inventors will co-incubate CD5-CD7 UCAR-T with a variety of CD5 and CD7-positive tumor cells in cytokine-free T cell culture medium. The effector-target ratio is calculated as 1:1 based on CAR-positive cells. After 4-6 hours of co-incubation, the cells are collected for FACS staining and the proportion of CD107a-positive cells in CD8 and CAR double-positive cells is analyzed by flow cytometry. The higher the ratio, the stronger the UCAR-T is activated by the target cells and the more tumor-killing factors are secreted. However, a higher proportion of CD107a-positive cells does not mean that it has a stronger ability to kill tumors. It can only be preliminarily concluded that UCAR-T has the ability to kill tumor cells.

[0259] As shown in Figures 21A-B, 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 CAR. Figures 21B and 21C show the proportion of CD107a-positive cells in CD8 and CAR double-positive cells after the CD5-CD7 UCAR-T prepared twice was co-incubated with different tumor cells for 4 hours. The proportion of CD107a-positive cells after stimulation with different tumor cells was different. The proportion of CD107a-positive cells after stimulation with CD5 or CD7-positive tumor cells was about 50%, but the negative target cell group (Raji) and the no tumor group also had about 20% CD107a-positive cells, which may be due to the self-activation of CD5-CD7 UCAR-T.

[0260] 5.2 In vitro tumor cell killing experiment

[0261] The tumor-killing function of the prepared UCAR-T can be preliminarily identified in vitro. Generally, UCAR-T and tumor target cells (stable expression of luciferase protein) are mixed at different effector-target ratios and co-cultured for 24 hours. The luciferase protein expression in the cells after co-culture is measured to reflect the survival of the tumor cells and thus determine the tumor-killing function of the UCAR-T.

[0262] Figures 22A-C show the tumor killing results of the CD5-CD7 UCAR-T prepared for the first time. Figures 22A and 22B show the tumor killing test results of UCAR-T and KO-T cells on the 7th day during the preparation period. UCAR-T and KO-T were co-cultured with different tumor target cells at three efficiency-target ratios for 24 hours and then tested. It can be seen from the figure that KO-T cannot kill tumor cells while UCAR-T can kill tumor cells very well; at a low efficiency-target ratio (0.2:1), the ability of CD5-CD7 UCAR-T to kill double-positive and single-positive target cells is CD5+ / CD7+>CD5+ / CD7->CD5- / CD7+, that is, the tumor killing ability of dual CAR-T is stronger than that of single CAR-T; Figure 22C shows the result of testing the tumor killing function of UCAR-T 24 hours after cryopreservation and recovery. It can be seen from the figure that cell cryopreservation and then recovery does not affect its tumor killing function.

[0263] Figures 22D-F show the tumor killing results of the second preparation of CD5-CD7 UCAR-T. Figures 22D and 22E are the tumor killing test results of UCAR-T and KO-T cells on the 10th day during the preparation period, respectively. After 24 hours of co-culture with UCAR-T, only tumor cells with low efficiency target ratio remained, while after 24 hours of co-culture with KO-T, almost no tumor cells were killed. Figure 22F shows the tumor killing results of UCAR-T 24 hours after cryopreservation and recovery, and the results are consistent with those before cryopreservation.

[0264] Example 6 Identification of Universal CAR-T Cell Phenotypes

[0265] 6.1 Detection of activation, typing, exhaustion and other indicators of universal CAR-T cells

[0266] Prepared UCAR-T cells generally need to be tested for various indicators before freezing. Indicators such as cell activation status, typing, and exhaustion are used to determine whether the final state of UCAR-T is abnormal.

[0267] Figure 23A-B shows the CD3 / CD4 / CD8 cell ratio and activation status of the CD5-CD7 UCAR-T cells prepared twice before freezing. The prepared cells in Figure 23A were not negatively selected by TRAC / B2M, so a small number of cells expressed the TCR complex of the CD3 pointer, while the prepared cells in Figure 23B were negatively selected by TRAC / B2M, so CD3 staining was completely negative. Generally speaking, before UCAR-T preparation (10-12 days of in vitro culture), the proportion of CD8 positive cells is about 2 times that of CD4 positive cells. In CD5-CD7 UCAR-T, due to the self-activation of CAR-positive cells, activation makers such as CD25 and CD69 are still partially expressed.

[0268] Figures 23C-D show the cell typing of CD5-CD7 UCAR-T cells prepared twice before cryopreservation. CCR7 is a marker for the early stages of T cells. Therefore, the higher the proportion of CCR7-positive cells in the final UCAR-T cells, the better the expansion of the UCAR-T cells after reinfusion 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 preparations were made using the same process. The figures show that the proportion of CCR7-positive cells in cells cryopreserved on day 10 is higher than that in cells cryopreserved on day 12.

[0269] Figures 24A-D show the exhaustion and premature cell death states of the CD5-CD7 UCAR-T cells prepared twice before freezing. As can be seen from Figures 24A and 24B, the expression levels of the two exhaustion makers, LAG3 and PD1, are both low, indicating that very few cells are being exhausted in the final state of the CD5-CD7 UCAR-T cells; in Figures 24C and 24D, PI and Annexin V double positivity indicate that the cells are in a late apoptotic state, while PI negativity and Annexin V positivity indicate that the cells are in an early apoptotic state. It can be seen from the figures that very few cells are undergoing apoptosis in the final state of the CD5-CD7 UCAR-T cells.

[0270] In summary, the CD5-CD7 UCAR-T cells prepared by the inventors are of good quality.

[0271] 6.2 Identification of Components of Universal CAR-T Cells

[0272] In addition to the above-mentioned indicator detection, the inventors also identified the cell components of the final state of UCAR-T. Through a series of cell maker detection, the inventors found that there were some CD56-positive cells in the final state of UCAR-T cells (as shown in Figures 25A-B). CD56 is generally considered to be the maker of NK cells, but these cells in UCAR-T do not express another maker of NK, CD16, and there are no CD4 and CD8 double-negative cells in the final state of UCAR-T cells. Therefore, these CD56-positive cells in UCAR-T cells are identified as NKT cells. There are also some CAR-positive cells in NKT cells. Therefore, these CAR-positive NKT cells can theoretically also perform tumor-killing functions.

[0273] The proportion of CD56-positive cells in different batches of UCAR-T cells varies, ranging from about 10% to 20%. As shown in Figures A and B in Figure 25 , the proportion of CD56-positive cells in UCAR-T prepared twice is shown.

[0274] Example 7 Clinical Testing of Universal CAR-T Cells (EGFR Switch)

[0275] Figure 26 shows the results of a clinical trial of CD5-CD7 UCAR-T (EGFR switch). The subject was a 36-year-old male patient with acute T-lymphocytic leukemia, weighing 75 kg. The lympholysis treatment given before UCAR-T infusion was: 30 mg / m 2 Fludarabine and 300mg / m 2 Cyclophosphamide for 5 days, then 30 mg / m 2 Fludarabine and 70mg / m 2 The patients were treated with melphalan for another day, and then rested for 3 days without treatment before being infused with CD5-CD7 UCAR-T cells at a dose (CAR-positive cells) greater than 3.6e6 / kg. Samples were then taken every day to detect changes in the copy number of CAR and lymphocytes in the patient's peripheral blood.

[0276] As shown in Figure 26B, the CAR copy number in the patient's peripheral blood decreased within 2 days after CD5-CD7 UCAR-T infusion, but began to increase after 2 days, reaching a peak on the 8th day, and then maintained a high level within 23 days; by detecting the lymphocytes in the patient's peripheral blood, it was found that the proportion of total UCAR-T cells (blue line in Figure 26C) in lymphocytes also continued to increase after infusion, reaching a ratio close to 100% on the 6th day, and the expansion trend of CAR-positive cells (red line in Figure 26C) was also consistent, while the patient's T cells (black line in Figure 26C) continued to decrease after UCAR-T infusion and were basically undetectable after the 6th day. The above results show that the inventor's CD5-CD7 UCAR-T can be well expanded in the patient's body and is very effective in clearing tumor cells.

[0277] However, as can be seen from Figures 26B and 26C, from the 8th day after CD5-CD7 UCAR-T infusion, the lymphocytes in the patient's peripheral blood were almost all CD5-CD7 UCAR-T, indicating that the patient had almost no T and NK cells of his own in his peripheral blood, which would result in the patient having no normal immune function. The EGFR switch was ineffective for CD5-CD7 UCAR-T, so this patient later underwent a transplant.

[0278] In summary, the inventors' EGFR-switch version of the CD5-CD7 UCAR-T can serve T-cell lymphoma patients who are unable to undergo transplantation due to high tumor burden. To expand the application scope of CD5-CD7 UCAR-T, the inventors have replaced and modified the UCAR-T switch, hoping to achieve the goal of completely curing T-cell lymphoma patients using only CD5-CD7 UCAR-T.

[0279] Example 8 Study of HSV-TK Molecular Switch

[0280] 8.1 In vitro study results

[0281] 8.1.1 Preparation of CD5-CD7 UCAR-T (HSV-TK) Cells

[0282] Given that the EGFR switch does not work for CD5-CD7 UCAR-T, we tried to replace EGFR with a small molecule drug-controlled switch HSV-TK that is independent of the patient's own immune system, as shown in Figure 27A. Then, a batch of finished cells were prepared according to the activated electroporation (EGFR version process) and the resting electroporation process (Figures 27B-E). Figure 27B shows the co-knockout efficiency of the four genes TRAC / B2M / CD5 / CD7 and the transfection effect of CAR, Figure 27C shows the cell typing, and Figure 27D shows the cell components.

[0283] Comparison revealed that the two processes had similar efficiency in knocking out the four genes TRAC / B2M / CD5 / CD7. However, in the resting electroporation process, TCR-positive cells were more fully activated than TCR-negative cells, giving TCR-positive cells a growth advantage. If TCR cell negative selection is incomplete, the proportion of residual TCR-positive cells will gradually increase in the later stages (Figure 27C).

[0284] In addition, the resting electroporation process uses Pan-T negative selection to obtain T cells on day 0, which minimizes CD56-positive cells. Therefore, the proportion of CD56-positive cells in the finished cells obtained by the resting electroporation process is lower than that obtained by the CD3 magnetic bead-positive activation electroporation process (Figure 27E). As for cell phenotype, the proportion of CCR7-positive cells is related to the number of days the cells are cultured in vitro. The longer the culture days, the lower the proportion of CCR7-positive cells (Figure 27D).

[0285] 8.1.2 Comparison of Tumor Killing Efficacy Between HSV-TK and EGFR UCAR-T

[0286] To verify whether there is a difference in the tumor-killing function between the HSV-TK version and the EGFR version of UCAR-T, the inventors used the same process to prepare two versions of UCAR-T in the same batch, then cryopreserved and revived. After 24 hours of resuscitation, they were co-incubated with different tumor cells at different effector-target ratios for 24 hours, and the luciferase fluorescence value of the tumor cells was detected to reflect the tumor-killing ability of UCAR-T. 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, that is, the use of different switch molecules does not affect the tumor-killing function of the CAR itself.

[0287] 8.1.3 Testing the Clearance Effect of HSV-TK Combined with Ganciclovir (GCV) on UCAR-T Cells

[0288] HSV-TK can efficiently bind to GCV and monophosphorylate it, and then the kinase in the cell diphosphorylates and triphosphorylates it. The structure of triphosphorylated GCV is very similar to the nucleosides in the cell, so it will competitively bind to DNA polymerase or break the ratio of the four nucleosides in the cell and inhibit DNA synthesis. After DNA synthesis is blocked, the cells will gradually die.

[0289] The inventors used different concentrations of GCV to treat TK and EGFR versions of UCAR-T at the same time. Every 3 days, the changes in CAR-positive cells were detected by counting and FACS. The results are shown in Figure 28. GCV did not affect the growth of EGFR version UCAR-T cells (Figure 29D-F), but only specifically killed TK-containing CAR-positive cells (Figure 29A-C). The higher the GCV concentration, the faster and more CAR-positive cells died, but even under the lower concentration (0.3ug / ml) of GCV treatment, all CAR-positive cells would slowly die over time.

[0290] 8.2 Animal Experiment Results

[0291] To verify the efficacy of HSV-TK version CD5-CD7 UCAR-T and GCV in vivo, the inventors conducted animal experiments. The experimental design is shown in Figure 30A-B: a total of 30 mice were vaccinated with 5x10 5 CCRF tumor cells were randomly divided into 5 groups, with 6 mice in each group. On the 7th day after tumor cell inoculation, 2 groups of mice were injected with 2x10 6 HSV-TK version of UCAR-T cells (per mouse), and the other two groups received 2x10 6 The HSV-TK and EGFR versions of the UCAR-T cells were injected into the HSV-TK and EGFR versions of the UCAR-T cells, and the remaining group was injected with the corresponding volume of PBS as a control. Starting from the 5th day after the UCAR-T cell injection, a group of mice in the HSV-TK and EGFR version groups were infused with 50 mg / kg of GCV once a day for a total of 3 days. The changes in tumor cells in the mice were then observed by mouse imaging at different time points.

[0292] The results are shown in Figure 30B-C. The mice in the group without UCAR-T cell injection had more tumor cells in their bodies on the 4th day (after UCAR-T injection), while the mice in the group with UCAR-T cell injection had very few tumor cells in their bodies, and there was no obvious difference between the mice in the EGFR version and HSV-TK version groups, indicating that the tumor-killing function of the two versions of CD5-CD7UCAR-T was good and there was no difference; after 3 days of treatment with GCV drug, the number of tumor cells in the mice in the HSV-TK version group was the most compared with the other three groups, indicating that GCV played a role in the UCAR-T cells in this group of mice, while the tumor cells in the other three groups of mice were not much different, indicating that GCV only specifically kills UCAR-T cells containing HSV-TK.

[0293] Since UCAR-T cells were introduced relatively late in this experiment (normally, UCAR-T cells are introduced 1-3 days after tumor cell inoculation), the number of tumor cells may have been greater than the number of UCAR-T cells when UCAR-T cells were introduced, resulting in the failure of UCAR-T to kill all tumor cells. However, judging from the results of the first 8 days (Figure 30B) and the tumor cell load throughout the experimental cycle (Figure 30C), the efficacy of the HSV-TK version of CD5-CD7 UCAR-T and GCV were both good.

[0294] Accordingly, this article provides at least the following technical solutions:

[0295] Scheme 1: A method for preparing universal chimeric antigen receptor T cells (UCAR-T), comprising:

[0296] 1) Use the CRISPR gene editing system to generate T cells with the following genes knocked out:

[0297] i) TRAC gene and / or TRBC gene;

[0298] ii) CD5 gene;

[0299] iii) CD7 gene; and

[0300] 2) transfecting the T cells with a nucleic acid molecule comprising a coding sequence of a chimeric antigen receptor (CAR) so that the T cells express the CAR.

[0301] Scheme 2: The method as described in Scheme 1, wherein step 1) further comprises knocking out the B2M gene of the T cell.

[0302] Option 3: The method as described in Option 1 or 2, wherein the target sequence of the sgRNA used to knock out the TRAC gene is selected from the sequences shown in SEQ ID NOs: 1-7 and any combination thereof.

[0303] Scheme 4: The method as described in 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 any combination thereof.

[0304] Scheme 5: The method according to any one of Schemes 1 to 4, wherein a combination of two sgRNAs is used to knock out the B2M gene, and the target sequences of the two sgRNAs are the sequences shown in SEQ ID NOs: 9 and 11, respectively.

[0305] Scheme 6: A method as described in 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 the TRAC gene and the B2M gene in the T cell are knocked out simultaneously, and the target sequence of the sgRNA for the TRAC gene knockout is the sequence shown in SEQ ID NO: 7.

[0306] Scheme 7: The method as described in 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.

[0307] Scheme 8: The method as described in any one of Schemes 1-7, wherein the target sequence of the sgRNA used for the CD7 gene knockout is the sequence shown in SEQ ID NO: 20.

[0308] Scheme 9: A method as described in any one of Schemes 1-8, wherein the knockout of the TRAC gene, the CD5 gene and the CD7 gene is performed simultaneously, 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.

[0309] Scheme 10: The method as described in any one of Schemes 1-9, wherein the knockout of the TRAC gene, the B2M gene, the CD5 gene and the CD7 gene are performed simultaneously, the target sequence of the sgRNA used to knock out the TRAC 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 NOs: 9 and 11, respectively; 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.

[0310] Scheme 11: The method according to any one of Schemes 1 to 10, wherein the knockout of the TRAC gene, the B2M gene, the CD5 gene, and the CD7 gene is performed simultaneously, and the components for performing the knockout in a 20 μL system include, in proportion:

[0311] An RNP complex formed by not less than 30 pmol of Cas9 and not less than 45 pmol of an sgRNA targeting the sequence shown in SEQ ID NO: 7;

[0312] An RNP complex formed by not less than 20 pmol of Cas9 and not less than 30 pmol of an sgRNA targeting the sequence shown in SEQ ID NO: 9;

[0313] an RNP complex formed by not less than 20 pmol of Cas9 and not less than 30 pmol of an sgRNA targeting the sequence shown in SEQ ID NO: 11;

[0314] an RNP complex formed by not less than 40 pmol of Cas9 and not less than 120 pmol of an sgRNA targeting the sequence shown in SEQ ID NO: 13; and

[0315] An RNP complex formed by not less than 40 pmol of Cas9 and not less than 120 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 20.

[0316] Scheme 12: The method according to any one of Schemes 1 to 11, wherein the knockout of the TRAC gene, the B2M gene, the CD5 gene, and the CD7 gene is performed simultaneously, and the components for performing the knockout in a 20 μL system include, in proportion:

[0317] An RNP complex formed by 30 pmol of Cas9 and 45 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 7;

[0318] An RNP complex formed by 20 pmol of Cas9 and 30 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 9;

[0319] An RNP complex formed by 20 pmol of Cas9 and 30 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 11;

[0320] an RNP complex formed by 80 pmol of Cas9 and 150 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 13; and

[0321] RNP complex formed by 40 pmol of Cas9 and 120 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 20.

[0322] Scheme 13: The method according to any one of Schemes 1 to 12, wherein step 1) further comprises knocking out the CIITA gene of the T cells.

[0323] Scheme 14: The method according to any one of Schemes 1 to 13, wherein the target sequence of the sgRNA for knocking out the CIITA gene is the sequence shown in SEQ ID NO: 25.

[0324] Scheme 15: The method according to any one of Schemes 1 to 14, wherein 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 for performing the knockout in a 20 μL system include, in proportion:

[0325] An RNP complex formed by not less than 30 pmol of Cas9 and not less than 45 pmol of an sgRNA targeting the sequence shown in SEQ ID NO: 7;

[0326] An RNP complex formed by not less than 20 pmol of Cas9 and not less than 30 pmol of an sgRNA targeting the sequence shown in SEQ ID NO: 9;

[0327] an RNP complex formed by not less than 20 pmol of Cas9 and not less than 30 pmol of an sgRNA targeting the sequence shown in SEQ ID NO: 11;

[0328] an RNP complex formed by not less than 80 pmol of Cas9 and not less than 150 pmol of an sgRNA targeting the sequence of SEQ ID NO: 13;

[0329] an RNP complex formed by not less than 40 pmol of Cas9 and not less than 120 pmol of an sgRNA targeting the sequence shown in SEQ ID NO: 20; and

[0330] An RNP complex formed by not less than 40 pmol of Cas9 and not less than 60 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 25.

[0331] Scheme 16: The method according to any one of Schemes 1 to 15, wherein 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 for performing the knockout in a 20 μL system include, in proportion:

[0332] An RNP complex formed by 30 pmol of Cas9 and 45 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 7;

[0333] An RNP complex formed by 25 pmol of Cas9 and 40 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 9;

[0334] An RNP complex formed by 25 pmol of Cas9 and 40 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 11;

[0335] An RNP complex formed by 80 pmol of Cas9 and 150 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 13;

[0336] an RNP complex formed by 40 pmol of Cas9 and 120 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 20; and

[0337] RNP complex formed by 50 pmol of Cas9 and 80 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 25.

[0338] Scheme 17: A method for preparing universal chimeric antigen receptor T cells (UCAR-T), comprising:

[0339] 1) Using a cytosine base editor to generate T cells with the following genes knocked out:

[0340] i) TRAC gene and / or TRBC gene;

[0341] ii) CD5 gene;

[0342] iii) CD7 gene; and

[0343] 2) transfecting the T cells with a nucleic acid molecule comprising a coding sequence of a chimeric antigen receptor (CAR) so that the T cells express the chimeric antigen receptor (CAR).

[0344] Scheme 18: The method as described in Scheme 17, wherein step 1) also includes knocking out the B2M gene of the T cell.

[0345] Scheme 19: The method as described in Scheme 17 or 18, wherein step 1) further includes knocking out the CIITA gene of the T cell.

[0346] Scheme 20: The method according to any one of Schemes 17 to 19, wherein the target sequence of the sgRNA used to knock out the TRAC gene is the sequence shown in SEQ ID NO: 26.

[0347] Scheme 21: The method according to any one of Schemes 17 to 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 NOs: 27 to 31 and any combination thereof.

[0348] Scheme 22: The method according to any one of Schemes 17 to 21, wherein the target sequence of the sgRNA used for knocking out the B2M gene is selected from the sequences shown in SEQ ID NOs: 33 and 34 and a combination thereof.

[0349] Scheme 23: The method according to any one of Schemes 17 to 22, wherein 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 NOs: 8 and 9, respectively.

[0350] Scheme 24: A method as described in any one of Schemes 17-23, wherein the target sequence of the sgRNA used to knock out the CD5 gene is selected from the sequence shown in any one of SEQ ID NOs: 37, 39, 41-46 and any combination thereof.

[0351] Scheme 25: The method according to any one of Schemes 17 to 24, wherein the target sequence of the sgRNA used for knocking out the CD7 gene is the sequence shown in SEQ ID NO: 47.

[0352] Scheme 26: The method as described in any one of Schemes 17-25, wherein the target sequence of the sgRNA used for the CIITA gene knockout is selected from the sequence shown in any one of SEQ ID NOs: 50, 51, 54, 57 and any combination thereof.

[0353] Scheme 27: A method as described in any one of Schemes 17-26, wherein the cytosine base editor is nCBE3 or nCBE4 protein.

[0354] Scheme 28: The method according to any one of Schemes 17 to 27, wherein the TRAC gene, the B2M gene, the CD5 gene, the CD7 gene and / or the CIITA gene are knocked out before activating the T cells with CD2 / CD3 / CD28 antigens.

[0355] Scheme 29: A method as described in any one of Schemes 1-28, wherein 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 can specifically bind to CD7, and the second antigen binding portion can specifically bind to CD5.

[0356] Scheme 30: A method as described in any one of Schemes 1-29, wherein the first antigen-binding portion comprises a heavy chain variable region from an anti-CD7 single-domain antibody, HCDR1 of the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 59, HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 60, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 61.

[0357] Scheme 31: The method as described in any one of Schemes 1-30, wherein the second antigen-binding portion includes a heavy chain variable region from an anti-CD5 single-domain antibody, wherein HCDR1 of the heavy chain variable region includes 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.

[0358] Scheme 32: The method according to any one of Schemes 1 to 31, wherein the first antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO: 62.

[0359] Option 33: In the method according to any one of options 1 to 32, the second antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO: 66.

[0360] Option 34: A method as described in any one of Options 1-33, wherein the extracellular antigen binding domain of the CAR comprises the amino acid sequence shown in SEQ ID NO: 74.

[0361] Scheme 35: A method as described in any one of Schemes 1-34, wherein the CAR includes, from the amino terminus to the carboxyl terminus, the first antigen binding portion, a connecting fragment, the second antigen binding portion, a hinge region, a transmembrane region, an intracellular co-stimulatory domain and an intracellular signaling domain.

[0362] Scheme 36: A method as described in any one of Schemes 1-35, wherein the connecting fragment comprises the amino acid sequence shown in SEQ ID NO: 67; the hinge region comprises the amino acid sequence shown in SEQ ID NO: 68; the transmembrane region comprises the amino acid sequence shown in SEQ ID NO: 69; the intracellular costimulatory domain comprises the amino acid sequence shown in SEQ ID NO: 70; and the intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NO: 71.

[0363] Scheme 37: The method according to any one of Schemes 1 to 36, wherein the nucleic acid molecule further comprises a coding sequence for tEGFR or herpes simplex virus thymidine kinase (HSV-TK).

[0364] Scheme 38: A method as described in any one of Schemes 1-37, wherein the coding sequence of the tEGFR or HSV-TK in the nucleic acid molecule is connected downstream of the coding sequence of the CAR through the coding sequence of the self-cleaving peptide.

[0365] Scheme 39: The method according to any one of Schemes 1 to 38, wherein the self-cleaving peptide is T2A, and its amino acid sequence is preferably as shown in SEQ ID NO: 72.

[0366] Scheme 40: The method as described in any one of Schemes 1-39, further comprising screening out T cells that do not express TCR and MHC-I class molecules after step 2).

[0367] Scheme 41: The method according to any one of Schemes 1 to 40, wherein the T cells contain NKT cells, for example, NKT cells in a proportion of 10-20%.

[0368] Scheme 42: UCAR-T cells prepared by the method described in any one of Schemes 1-41.

[0369] Scheme 43: A pharmaceutical composition comprising the UCAR-T cells described in Scheme 42 and a pharmaceutically acceptable carrier.

[0370] Scheme 44: Use of the UCAR-T cells described in Scheme 42 in the preparation of a drug for treating cancer.

[0371] Scheme 45: The use according to Scheme 44, wherein the cancer expresses CD5 and / or CD7 on its cell surface.

[0372] Scheme 46: The use according to Scheme 44 or 45, wherein the cancer is a T-cell malignancy, such as acute T-lymphocytic leukemia (T-ALL) and T-cell lymphoma.

[0373] Scheme 47: A method for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of the UCAR-T cells of Scheme 42 or the pharmaceutical composition of Scheme 43.

[0374] Scheme 48: The method of Scheme 47, wherein the cancer expresses CD5 and / or CD7 on its cell surface.

[0375] Scheme 49: The method of Scheme 48, wherein the cancer is a T-cell malignancy, such as acute T-cell leukemia and T-cell lymphoma.

[0376] 50. The method of 47 or 48, further comprising administering ganciclovir (GCV) to the subject after treatment.

[0377] Scheme 51: A drug kit comprising: 1) the UCAR-T cells or drug composition of Scheme 42; and 2) GCV.

[0378] The advantages of UCAR-T presented in this article include but are not limited to:

[0379] 1. The use of dual targets can avoid the off-tumor problem caused by antigen escape in single-target CAR-T, greatly expanding the patient population;

[0380] 2. Using CAR as an attack molecule also greatly reduces the clearance of UCAR-T by host immune cells;

[0381] 3. Using healthy donor T cells, multiple doses can be prepared at one time. On the one hand, patients do not have to wait for CAR-T preparation, and on the other hand, the preparation cost per dose is greatly reduced, thereby reducing the patient's treatment costs;

[0382] 4. Ability to achieve repeated dosing.

[0383] The sequences of the nucleic acid molecules and protein molecules mentioned in this article and the accompanying drawings are as follows:

[0384] CD7 sdAb VHH protein sequence (CD7-FHV H 10):

[0385] CD5 sdAb VHH protein sequence (CD5-FHV H 61):

[0386] Linker amino acid sequence:

[0387] CD8α hinge region protein sequence:

[0388] CD8α transmembrane protein sequence:

[0389] 4-1BB intracellular domain protein sequence:

[0390] CD3z intracellular signaling domain protein sequence:

[0391] Cleavage peptide T2A protein sequence:

[0392] EGFRt protein sequence:

[0393] CD5-CD7 sdAb amino acid sequence connected in series via linker

[0394] References:

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Claims

1. A method for preparing universal chimeric antigen receptor T cells (UCAR-T), comprising: 1) Use the CRISPR gene editing system to generate T cells with the following genes knocked out: i) TRAC gene and / or TRBC gene; ii) CD5 gene; iii) CD7 gene; and 2) transfecting the T cells with a nucleic acid molecule comprising a coding sequence of a chimeric antigen receptor (CAR) so that the T cells express the CAR.

2. The method according to claim 1, wherein step 1) further comprises knocking out the B2M gene and / or CIITA gene of the T cell.

3. The method according to claim 1 or 2, wherein: The target sequence of the sgRNA used to knock out the TRAC gene is selected from the sequences shown in SEQ ID NOs: 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 NOs: 8, 9, 11, 12, and any combination thereof; preferably, a combination of two sgRNAs is used for knocking out the B2M gene, and the target sequences of the two sgRNAs are the sequences shown in SEQ ID NOs: 9 and 11, respectively; The target sequence of the sgRNA for knocking out the CD5 gene is selected from the sequence 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; and / or The target sequence of the sgRNA for the CIITA gene knockout is the sequence shown in SEQ ID NO: 25; Preferably, in step 1), the sgRNA for knocking out the TRAC gene and the B2M gene are mixed with the Cas9 protein to simultaneously knock out the TRAC gene and the B2M gene in the T cell, and the target sequence of the sgRNA for knocking out the TRAC gene is the sequence shown in SEQ ID NO: 7; Preferably, the knockout of the TRAC 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 the sequence shown in SEQ ID NO: 7; the target sequence of the sgRNA for knocking out the CD5 gene is the sequence shown in SEQ ID NO: 13; and the target sequence of the sgRNA for knocking out the CD7 gene is the sequence shown in SEQ ID NO: 20; Preferably, the knockout of the TRAC gene, the B2M gene, the CD5 gene and the CD7 gene is performed simultaneously, the target sequence of the sgRNA for knocking out the TRAC gene is the sequence shown in SEQ ID NO: 7; the target sequences of the two sgRNAs for knocking out the B2M gene are the series shown in SEQ ID NO: 9 and 11, respectively; the target sequence of the sgRNA for knocking out the CD5 gene is the sequence shown in SEQ ID NO: 13; and the target sequence of the sgRNA for knocking out the CD7 gene is the sequence shown in SEQ ID NO: 20; More preferably, the knockout of the TRAC gene, the B2M gene, the CD5 gene and the CD7 gene is performed simultaneously, and the components for performing the knockout include, in proportion, in a 20 μL system: an RNP complex formed by not less than 30 pmol of Cas9 and not less than 45 pmol of the sgRNA targeting the sequence shown in SEQ ID NO: 7; an RNP complex formed by not less than 20 pmol of Cas9 and not less than 30 pmol of the sgRNA targeting the sequence shown in SEQ ID NO: 9; an RNP complex formed by not less than 20 pmol of Cas9 and not less than 30 pmol of the sgRNA targeting the sequence shown in SEQ ID NO: 11; an RNP complex formed by not less than 40 pmol of Cas9 and not less than 120 pmol of the sgRNA targeting the sequence shown in SEQ ID NO: 13; and an RNP complex formed by not less than 40 pmol of Cas9 and not less than 120 pmol of the sgRNA targeting the sequence shown in SEQ ID NO:

20. More preferably, the knockout of the TRAC gene, the B2M gene, the CD5 gene and the CD7 gene is performed simultaneously, and the components for performing the knockout include, in proportion, in a 20 μL system: an RNP complex formed by 30 pmol of Cas9 and 45 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 7; an RNP complex formed by 20 pmol of Cas9 and 30 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 9; an RNP complex formed by 20 pmol of Cas9 and 30 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 11; an RNP complex formed by 80 pmol of Cas9 and 150 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 13; and an RNP complex formed by 40 pmol of Cas9 and 120 pmol of sgRNA targeting the sequence shown in SEQ ID NO:

20. More preferably, 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 for performing the knockout include, in proportion, in a 20 μL system: an RNP complex formed by not less than 30 pmol of Cas9 and not less than 45 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 7; an RNP complex formed by not less than 20 pmol of Cas9 and not less than 30 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 9; an RNP complex formed by not less than 20 pmol of Cas9 and not less than 30 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 11; an RNP complex formed by not less than 80 pmol of Cas9 and not less than 150 pmol of sgRNA targeting the sequence shown in SEQ ID NO: 13; an RNP complex formed by not less than 40 pmol of Cas9 and not less than 120 pmol of sgRNA targeting the sequence shown in SEQ ID NO: NO: an RNP complex formed by an sgRNA targeting the sequence shown in SEQ ID NO: 20; and an RNP complex formed by not less than 40 pmol of Cas9 and not less than 60 pmol of an sgRNA targeting the sequence shown in SEQ ID NO: 25; More preferably, 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 for performing the knockout include, in proportion, in a 20 μL system: 30 pmol of Cas9 and 45 pmol of an RNP complex formed by an sgRNA targeting the sequence shown in SEQ ID NO: 7; 25 pmol of Cas9 and 40 pmol of an RNP complex formed by an sgRNA targeting the sequence shown in SEQ ID NO: 9; 25 pmol of Cas9 and 40 pmol of an RNP complex formed by an sgRNA targeting the sequence shown in SEQ ID NO: 11; 80 pmol of Cas9 and 150 pmol of an RNP complex formed by an sgRNA targeting the sequence shown in SEQ ID NO: 13; 40 pmol of Cas9 and 120 pmol of an RNP complex formed by an sgRNA targeting the sequence shown in SEQ ID NO: 20; and 50 pmol of Cas9 and 80 pmol of an RNP complex formed by an sgRNA targeting the sequence shown in SEQ ID NO: RNP complex formed by sgRNA with the sequence shown in NO:

25.

4. A method for preparing a universal chimeric antigen receptor T cell (UCAR-T), comprising: 1) Using a cytosine base editor to generate T cells with the following genes knocked out: i) TRAC gene and / or TRBC gene; ii) CD5 gene; iii) CD7 gene; and 2) transfecting the T cells with a nucleic acid molecule comprising a coding sequence of a chimeric antigen receptor (CAR) so that the T cells express the chimeric antigen receptor (CAR). 5 . The method according to claim 4 , wherein step 1) further comprises knocking out the B2M gene and / or CIITA gene of the T cell.

6. The method according to claim 4 or 5, wherein The target sequence of the sgRNA used to knock out the TRAC gene is the sequence shown in SEQ ID NO: 26; The target sequence of the sgRNA used to knock out the TRBC gene is selected from any one of SEQ ID NOs: 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 NOs: 33 and 34, and a combination thereof; preferably, two sgRNAs are used for knocking out the B2M gene, wherein the target sequences of the two sgRNAs are the sequences shown in SEQ ID NOs: 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 NOs: 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; and / or The target sequence of the sgRNA for knocking out the CIITA gene is selected from any one of SEQ ID NOs: 50, 51, 54, 57, and any combination thereof; Preferably, the cytosine base editor is nCBE3 or nCBE4 protein; Preferably, the TRAC gene, the B2M gene, the CD5 gene, the CD7 gene and / or the CIITA gene are knocked out before activating the T cells with CD2 / CD3 / CD28 antigens; 7. The method according to any one of claims 1 to 6, wherein the extracellular antigen binding domain of the CAR comprises a first antigen binding portion and a second antigen binding portion, the first antigen binding portion can specifically bind to CD7, and the second antigen binding portion can specifically bind to CD5; Preferably, the first antigen-binding portion comprises a heavy chain variable region from an anti-CD7 single-domain antibody, wherein the HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 59, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 60, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 61; and the second antigen-binding portion comprises a heavy chain variable region from an anti-CD5 single-domain antibody, wherein the HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 63, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 64, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 65; More preferably, the first antigen binding portion comprises the amino acid sequence shown in SEQ ID NO: 62; More preferably, the second antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO: 66; More preferably, the extracellular antigen binding domain of the CAR comprises the amino acid sequence shown in SEQ ID NO:

74.

8. The method of any one of claims 1 to 7, wherein the nucleic acid molecule further comprises a coding sequence for tEGFR or herpes simplex virus thymidine kinase (HSV-TK); preferably, the coding sequence for tEGFR or HSV-TK in the nucleic acid molecule is connected downstream of the coding sequence for the CAR via a coding sequence for a self-cleaving peptide.

9. The method according to any one of claims 1 to 8, further comprising screening out T cells that do not express TCR and MHC class I molecules after step 2).

10. UCAR-T cells prepared by the method according to any one of claims 1 to 9.

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