Modified immune cells and uses thereof

Abstract

The invention relates to modified immune cells and uses thereof.

Description

Technical Field

[0001] The present invention relates to the field of immunology, in particular to the field of cell therapy, and specifically to the design and modification of novel dual-target suicide genes, modified immune cells, and their applications. Background Art

[0002] Cancer has become one of the most serious diseases threatening human life and health, and tumor immunotherapy has completely changed the treatment landscape of cancer in the past decade, such as immune checkpoint blockade therapy, therapeutic tumor vaccines and adoptive cell therapy (ACT). ACT is a method of using patients' tumor-infiltrating lymphocytes (TIL) or T cells, natural killer cells (NK) or macrophages (MACROPHAGES) isolated from the peripheral blood of patients or healthy people to treat cancer. ) are genetically engineered in vitro to produce immune cells with tumor antigen-specific affinity and target cell cytotoxicity, and then amplified in vitro before being infused back into the patient. In ACT, TCR-T cell therapy that modifies the endogenous T cell receptor (TCR), CAR-T cell therapy that constructs an exogenous chimeric antigen receptor (CAR), and synthetic TCR and antigen receptor (STAR) therapy using TCR-like CAR-T cells are widely studied and applied. At the same time, CAR-NK cell therapy that performs in vitro gene editing on NK cells has also achieved surprising therapeutic effects in the treatment of certain tumors.

[0003] However, gene-edited autologous or allogeneic CAR-T cells can be associated with a variety of adverse reactions, such as cytokine storms, off-target effects, and excessive proliferation of adoptive cells. On November 28, 2023, the U.S. Food and Drug Administration (FDA) issued a major notice stating that T-cell malignancies have been observed in patients receiving CAR-T therapy. The FDA believes that all currently approved CAR-T therapies carry a potential risk of secondary tumors. This notice reminds us that ACT therapy may carry unknown risks, particularly the risk of tumorigenicity from infused immune cells. Therefore, it is necessary to consider how to effectively remove overactive donor cells from the body, avoid persistent toxic side effects and tumorigenicity, and establish a safe system for ACT therapy.

[0004] Human Epidermal Growth Factor Receptor 2 (HER2) is a transmembrane protein with tyrosine protein kinase activity and belongs to the epidermal growth factor receptor family. HER2 is a transmembrane protein composed of an extracellular ligand binding region, a transmembrane region, and an intracellular protein tyrosine kinase region. The ligand binding region of HER2 contains four domains. Domains II and IV participate in homo- and hetero-dimerization, while domains I and III have the ability to bind to ligands (see Figure 1 A) Unlike the other three members, HER2 lacks a specific ligand. Instead, it dimers with itself and other family members, triggering intracytoplasmic tyrosine kinase phosphorylation. This drives tumor cell proliferation, increases tumor cell invasion, promotes angiogenesis, and inhibits apoptosis. HER2 is highly expressed in various cancers and tumor cells, particularly breast and ovarian cancer cells, and is closely associated with tumor growth, metastasis, and invasion.

[0005] Trastuzumab (Trastuzumab or Herceptin) is the first anti-HER2 humanized monoclonal antibody developed for the HER2 target molecule and is used for the targeted treatment of HER2-positive breast cancer. The Fab end of trastuzumab specifically binds to the HER2 extracellular domain Domain IV, hindering the function of the intracellular protein tyrosine kinase structural region, inhibiting AKT activation, negatively regulating downstream signaling pathways such as PI3K / Akt and Ras / MAPK, causing cells to arrest in the G1 phase and inducing apoptosis. More importantly, the Fc end of trastuzumab can bind to the FcγRⅢ receptor (CD16) of NK cells and macrophages, and eliminate and kill HER2-positive cells through antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) (see Figure 1 B).

[0006] Herpes simplex virus thymidine kinase (HSVTK) / ganciclovir (GCV) is a prodrug-activated suicide gene system that functions through administration. Normally, HSV-TK phosphorylates thymidine to thymidine triphosphate, allowing it to be incorporated into DNA and participate in DNA replication. Unlike human thymidine kinase, HSV-TK can phosphorylate GCV and convert it into monophosphorylated ganciclovir (GCV-P). Once GCV-P is produced, cellular kinases can phosphorylate GCV-P to produce triphosphorylated ganciclovir (GCV-PPP). GCV-PPP is a competitive inhibitor of thymidine triphosphate, causing base mismatches and terminating the extension of the 3'-OH end of the DNA chain, blocking DNA replication and leading to cell death (see ). Figure 2 A,B,doi:10.25100 / cm.v48i3.2997). Summary of the Invention

[0007] For example, the present invention uses gene editing technology to introduce the coding nucleotide sequence of TCR, CAR or STAR into immune cells, and at the same time introduces the nucleotide sequence of suicide gene at different locations in a targeted or random manner to play the role of a molecular switch. Before the host has adverse reactions or adoptive cell tumorigenesis occurs, timely administration of drugs can selectively eliminate the reinfused donor cells, which can significantly improve the efficiency of adoptive cell elimination and solve the risk of ACT tumorigenesis.

[0008] Furthermore, on this basis, by introducing the nucleotide sequence of tumor cell-specific surface antigens into immune cells, on the one hand, positive immune cells can be detected and purified by specific antibodies / magnetic beads against them, and at the same time, the killing effect on tumor cells expressing the tumor-specific surface antigens can be achieved by using specific antibodies against them, as well as the auxiliary killing effect on tumor cells targeted by the modified immune cells.

[0009] Through the site-specific integration and insertion of the above-mentioned units, the modified immune cells have multiple advantages such as suicide gene switch and enhanced cell proliferation, tumor infiltration and target cell killing functions, thereby improving the safety and function of the modified immune cells.

[0010] Specifically, the present invention relates to the following aspects:

[0011] 1. A modified immune cell comprising unit A and unit B, wherein unit A comprises a nucleotide sequence encoding a T cell receptor (TCR), a chimeric antigen receptor (CAR) or a synthetic T cell antigen receptor (STAR), and unit B comprises a nucleotide sequence of a suicide gene, wherein unit A and unit B are located at different positions in the genome of the modified immune cell, or wherein unit A and unit B are connected to replace the PD1 gene and / or TRAC site in the modified immune cell (preferably the GenBank accession number of TRAC is NC_000014. 9) and / or B2M site (preferably GenBank: NC_000015.10) and / or TET2 gene and / or Regnase gene and / or Roquin gene and / or BCOR gene and / or ETS1 gene and / or IKZF1 gene and / or FADD gene and / or ULK2 gene and / or NFKBIE gene and / or NFKBIA gene and / or KLF16 gene and / or SMARCCI gene nucleotide sequence, preferably unit B is connected to a tag gene such as Myc, Flag, HA or His.

[0012] 2. The modified immune cell described in Item 1, wherein the modified immune cell further comprises unit C, unit C comprises a nucleotide sequence encoding a tumor cell surface-specific antigen, preferably, the tumor cell surface-specific antigen is selected from one or more of HER2, CD19, CD20, GD2, EGFR, and Cetux tumor surface antigens as shown in SEQ ID NO: 1, and preferably unit C comprises the extracellular domain and transmembrane domain of the tumor cell surface-specific antigen.

[0013] 3. The modified immune cell described in Item 2, wherein any one of unit A, unit B and unit C or any combination of two of them are located at different positions in the genome of the modified immune cell.

[0014] 4. The modified immune cell according to any one of items 2 to 3, wherein unit A is linked to unit B, unit A is linked to unit C, or unit B is linked to unit C, or unit A, unit B and unit C are linked,

[0015] Preferably, the connection is through a linker and / or a linker peptide, more preferably, the linker is selected from (G4S)n, wherein n is an integer of 1-5, or an SGSG linker, and the linker peptide is Furin, p2A or a combination thereof, preferably the sequence of Furin is as shown in SEQ ID NO: 13, or the sequence of p2A is as shown in SEQ ID NO: 12, preferably the amino acid sequence of the combination of Furin and P2A is as shown in SEQ ID NO: 14, and the nucleotide sequence is as shown in SEQ ID NO: 15.

[0016] 5. The modified immune cell according to any one of items 1 to 4, wherein the immune cell is selected from T cells, NK cells, macrophages, NKT cells, Treg cells, and dendritic cells.

[0017] 6. The modified immune cell according to any one of items 1 to 5, wherein unit A, unit B, unit C, or any combination of two of them, or a combination of the three thereof replaces the nucleotide sequence of the PD1 gene and / or TRAC site and / or B2M site and / or TET2 gene and / or Regnase gene and / or Roquin gene and / or BCOR gene and / or ETS1 gene and / or IKZF1 gene and / or FADD gene and / or ULK2 gene and / or NFKBIE gene and / or NFKBIA gene and / or KLF16 gene and / or SMARCC1 gene in the modified immune cell.

[0018] 7. The modified immune cell according to any one of items 1 to 6, wherein the suicide gene is selected from one or more of HSVTK (preferably GenBank: KM222725.1), iCaspase9, HER2 as shown in SEQ ID NO: 1, EGFRt, CD20, rapamycin, and / or RQR8, preferably EGFRt-HSV-TK; CD20-HSV-TK, or the T cell receptor (TCR) comprises an α chain and a β chain, the α chain comprises a TCR variable region and a TCR constant region, the β chain comprises a TCR variable region and a TCR constant region, preferably, the α chain and the β chain are connected by a connecting peptide; or the α chain is located at the C-terminus and the β chain is located at the N-terminus; or the chimeric antigen receptor (CAR) comprises scFv, a hinge region, 4-1BB / CD8 and CD3ζ; preferably, the VL and VL in the scFv are connected by a linker; or VL is located at the N-terminus;

[0019] The synthetic T cell receptor antigen receptor (STAR) comprises VL, a TCR constant region of a β chain, VH and a TCR constant region of an α chain; preferably, the TCR constant region of the β chain and the TCR constant region of the α chain are connected by a linker peptide; more preferably, the linker peptide is Furin, p2A or a combination thereof, preferably the sequence of the linker peptide Furin is as shown in SEQ ID NO: 13, or the sequence of p2A is as shown in SEQ ID NO: 12; or the linker is selected from (G4S)n, wherein n is an integer from 1 to 5, or an SGSG linker;

[0020] More preferably, the TCR, CAR and STAR further comprise an IRES or RFP at the C-terminus. Preferably, the sequence of the IRES is as shown in SEQ ID NO: 21, or the sequence of the RFP is as shown in SEQ ID NO: 22.

[0021] Most preferably, the amino acid sequence of the CAR is shown in SEQ ID NO: 23, and the amino acid sequence of the STAR is shown in SEQ ID NO: 24.

[0022] 8. A method for preparing a modified immune cell, comprising inserting unit A and unit B into different positions in the genome of the modified immune cell, or connecting unit A and unit B to replace the PD1 gene and / or TRAC site (preferably the GenBank number of TRAC is NC_000014.9) and / or B2M site (preferably GenBank: NC_000015.10) and / or TET2 gene and / or Regnase gene and / or Roquin gene and / or BCOR gene and / or ETS1 gene and / or IKZF1 gene and / or FADD gene and / or ULK2 gene and / or NFKBIE gene and / or NFKBIA gene and / or KLF16 gene and / or SMARCCI gene in the modified immune cell, wherein unit A comprises a nucleotide sequence encoding a T cell receptor (TCR), a chimeric antigen receptor (CAR) or a synthetic T cell antigen receptor (STAR), and unit B comprises a nucleotide sequence of a suicide gene.

[0023] 9. The method of item 8, further comprising inserting unit C into the genome of the modified immune cell, wherein unit C comprises a nucleotide sequence encoding a tumor cell surface-specific antigen (preferably, the tumor cell surface-specific antigen is selected from one or more of HER2, CD20, GD2, EGFR, and Cetux as shown in SEQ ID NO: 1, and preferably unit C comprises the extracellular domain and transmembrane domain of the tumor cell surface-specific antigen).

[0024] Preferably, unit A is connected to unit B, unit A is connected to unit C, or unit B is connected to unit C, or unit A, unit B and unit C are connected, preferably through a linker or a linker peptide, more preferably, the linker is selected from (G4S)n, wherein n is an integer from 1 to 5, or an SGSG linker, and the linker peptide is Furin, p2A or a combination thereof, preferably the sequence of Furin is as shown in SEQ ID NO: 13, or the sequence of p2A is as shown in SEQ ID NO: 12, preferably the amino acid sequence of the combination of Furin and P2A is as shown in SEQ ID NO: 14, and the nucleotide sequence is as shown in SEQ ID NO: 15.

[0025] 10. The method described in item 9, wherein any one of unit A, unit B and unit C or any combination of two of them is inserted into different positions in the genome of the modified immune cell, preferably replacing the nucleotide sequence of the PD1 gene and / or TRAC site and / or B2M site and / or TET2 and / or Regnase and / or Roquin and / or BCOR and / or ETS1 and / or IKZF1 and / or FADD and / or ULK2 and / or NFKBIE and / or NFKBIA and / or KLF16 and / or SMARCCI in the modified immune cell.

[0026] 11. The method according to any one of items 8 to 10, wherein the replacement is performed by gene editing technology, preferably CRISPR / Cas9 or CRISPR / Cas12a.

[0027] 12. The method according to any one of items 8 to 11, wherein the immune cells are as defined in item 5, and the suicide genes, T cell receptors (TCRs), chimeric antigen receptors (CARs) and synthetic T cell antigen receptors (STARs) are as defined in item 7.

[0028] 13. A kit comprising unit A, unit B and / or unit C, wherein unit A comprises a nucleotide sequence encoding a T cell receptor (TCR), a chimeric antigen receptor (CAR) or a synthetic T cell antigen receptor (STAR), unit B comprises a nucleotide sequence of a suicide gene, and unit C comprises a nucleotide sequence encoding a tumor cell surface-specific antigen. Preferably, the kit further comprises an antibody against a tumor cell surface-specific antigen.

[0029] 14. The kit according to claim 13, wherein the nucleotide sequence encoding a T cell receptor (TCR), a chimeric antigen receptor (CAR) or a synthetic T cell antigen receptor (STAR), the nucleotide sequence of a suicide gene or the nucleotide sequence encoding a tumor cell surface-specific antigen is present in the form of a plasmid.

[0030] 15. The kit according to any one of items 1 to 14, wherein the tumor cell surface-specific antigen is selected from one or more of the HER2, CD19, CD20, GD2, EGFR, and Cetux tumor surface antigens as shown in SEQ ID NO: 1, and preferably unit C comprises the extracellular domain and transmembrane domain of the tumor cell surface-specific antigen.

[0031] 16. The kit of any one of items 12-15, wherein the suicide gene, T cell receptor (TCR), chimeric antigen receptor (CAR) and synthetic T cell antigen receptor (STAR) are as defined in item 7.

[0032] 17. Use of the modified immune cell according to any one of items 1 to 7 or the kit according to any one of items 13 to 16 in treating a disease, or in preparing a medicament for treating a disease, wherein the disease is selected from a tumor or an autoimmune disease.

[0033] CAR is a chimeric antigen receptor (CAR) structure formed by linking a scFv fragment of a monoclonal antibody that recognizes a tumor antigen to a costimulatory signaling domain (such as CD28 or 4-1BB) and an intracellular CD3ζ signaling domain via a transmembrane domain (TM). CAR-T cells generated using this approach have demonstrated excellent tumor clearance against hematologic malignancies, with both intrinsic and extrinsic tumor-killing mechanisms. After CAR-T cells recognize tumor-associated antigens (TAAs), an immune synapse is formed, leading to CAR-T cell activation. Cytotoxic granules containing perforin and granzymes are subsequently released, which enter target cells through perforin pathways and trigger intrinsic apoptosis of tumor cells through mitochondrial damage and caspase activation. Furthermore, FAS ligands upregulated by CAR-T cells bind to FAS receptors on target cells, triggering both apoptosis and caspase-mediated targeted apoptosis via the extrinsic pathway. Furthermore, CAR-T cells release interferon-γ and tumor necrosis factor (TNF), activating immune cells such as macrophages.

[0034] STAR is a two-chain structure formed by fusing the variable region of a monoclonal antibody that recognizes tumor antigens with the constant regions of the TCR-α and β chains. It forms a complex with endogenous CD3, creating a novel chimeric receptor that combines the antigen recognition capabilities of antibodies with the signaling properties of TCRs (DOI: 10.1126 / scitranslmed.abb5191).

[0035] An exemplary schematic diagram of TCR, CDAR, and STAR is shown in Figure 13 shown.

[0036] Suicide genes are genes introduced into target cells from certain viruses, bacteria, or eukaryotic cells. These genes, through the expression of proteases that catalyze the conversion of non-toxic drug precursors into cytotoxic substances, or through the use of antibody protein drugs or small molecule chemical inducers, activate the expression of these genes, thereby killing the recipient cells carrying the genes by activating apoptosis or pyroptosis pathways, inhibiting DNA replication, and blocking cell division. By adding antibodies or small molecule drugs to target cells that have been introduced with suicide genes, the life or death of the target cells can be regulated, like installing a molecular switch in the target cells, allowing for artificial control over whether the target cells function at different stages.

[0037] In some embodiments, the suicide gene is selected from one or more of HSV-TK (preferably GenBank: KM222725.1), iCaspase9, HER2 as shown in SEQ ID NO: 1, EGFRt, CD20, rapamycin, and / or RQR8, preferably EGFRt-HSV-TK, CD20-HSV-TK. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 .Schematic diagram of the structure and immune mechanism of HER2 (including HER2t), Figure 1 A is a schematic diagram of the structure of HER2 (including HER2t); Figure 1 B is the immune mechanism of HER2 (including HER2t).

[0039] Figure 2 .HSV-TK / GCV metabolically blocks the DNA replication mechanism, Figure 2 A represents the normal phosphorylation of thymine by cellular kinases to participate in the DNA replication mechanism; Figure 2 B is the mechanism of cell death caused by HSV-TK / GCV metabolism blocking DNA replication.

[0040] Figure 3. Map of the eukaryotic expression vector pcDNA3.1-Myc-His containing HER2t, HSV-TK, and HER2t-HSV-TK, where Figure 3A is the pcDNA3.1-HER2t-Myc-His map; Figure 3B is the map of pcDNA3.1-HSV-TK-Myc-His; Figure 3C This is the map of pcDNA3.1-HER2t-(G4S)3-HSV-TK-Myc-His.

[0041] Figure 4 . Construction and expression of fusion protein, wherein Figure 4 A represents HSV-TK-MYC; HER2t-(G4S)3-HSV-TK-MYC; schematic diagram of HER2t-MYC eukaryotic expression vector; Figure 4 B represents WB detection of the expression of HSV-TK-MYC, HER2t-(G4S)3-HSV-TK-MYC, and HER2t-MYC transfected fusion proteins in 293T cells; Figure 4 C represents WB detection of HSV-TK-MYC; the expression of HER2t-(G4S)3-HSV-TK-MYC in 293T cells transfected with HSV-TK.

[0042] Figure 5Map of the AAV plasmid with site-directed insertion of HER2t-(G4S)3-HSV-TK at the PD1 knockout site (i.e., 68-AAV-PD1-Cr1-EF1a-HER2t-G4S3-HSV-TK).

[0043] Figure 6 Map of the AAV plasmid with site-directed insertion of HER2t-(G4S)3-F / P2A-HSV-TK at the PD1 knockout site (i.e., 77-AAV-PD1-Cr1-HER2t-(G4S)3-F / P2A-HSV-TK).

[0044] Figure 7 Map of the AAV plasmid (98.AAV TRAC-P2A-CD19-28z-CAR-P2A-TK) with site-directed insertion of CD19-28z-CAR-P2A-TK at the TRAC knockout site.

[0045] Figure 8. AAV-HER2t-HSV-TK pattern and FACS analysis, where Figure 8A Schematic diagram showing the site-specific insertion of CD19STAR at the TRAC site via the AAV system; Figure 8B Schematic diagram showing the site-directed insertion of HER2t-HSV-TK (HER2t-(G4S)3-HSV-TK or HER2t-(G4S)3-F / P2A-TK) into the PD1 locus via the AAV system; Figure 8C A diagram showing the expression working model of HER2t-(G4S)3-HSV-TK and HER2t-(G4S)3-F / P2A-TK on cells; Figure 8D It means that the TRAC and PD1 sites are simultaneously knocked out in human PBMC cells, and CD19STAR and HER2t-HSV-TK (HER2t-(G4S)3-HSV-TK or HER2t-(G4S)3-F / P2A-TK) are inserted at the site, and the expression of STAR & HER2 is detected by flow cytometry.

[0046] Figure 9. TRAC-STAR&PD1-HER2t-HSV-TK (HER2t-(G4S)3-HSV-TK or HER2t-(G4S)3-F / P2A-TK) cells have both killing and suicide functions. Figure 9A Indicates the tumor killing function of TRAC-STAR&PD1-HER2t-HSV-TK (HER2t-(G4S)3-HSV-TK or HER2t-(G4S)3-F / P2A-TK) cells; Figure 9BIndicates CCK-8 detection of the suicide function of TRAC-STAR&PD1-HER2t-HSV-TK (HER2t-(G4S)3-HSV-TK or HER2t-(G4S)3-F / P2A-TK) cells; Figure 9C Represents flow cytometry detection of TRAC-STAR&PD1-HER2t-HSV-TK (HER2t-(G4S)3-HSV-TK or HER2t-(G4S)3-F / P2A-TK) cell suicide function.

[0047] Figure 10 Effects of GCV concentration and duration on HER2t-HSV-TK positive cells (HER2t-(G4S)3-HSV-TK or HER2t-(G4S)3-F / P2A-TK), Figure 10 A represents the effect of different GCV concentrations on the viability of HER2t-HSV-TK positive cells (HER2t-(G4S)3-HSV-TK or HER2t-(G4S)3-F / P2A-TK) after 48 hours of treatment; Figure 10 B shows the effect of different GCV concentrations on the viability of HER2t-HSV-TK positive cells (HER2t-(G4S)3-HSV-TK or HER2t-(G4S)3-F / P2A-TK) after 72 hours of treatment. Figure 10 The HER2 in the expression is HER2t, wherein HER2-TK represents HER2t-(G4S)3-HSV-TK, and HER2-F / P2A-TK represents HER2t-(G4S)3-F / P2A-TK.

[0048] Figure 11. In vivo anti-tumor function of 1928CAR and STAR loaded with suicide gene HER2t-(G4S)3-F / P2A-HSV-TK. Figure 11A Flow cytometry analysis showing the successful insertion of the HER2t-(G4S)3-F / P2A-HSV-TK element into the PD1 locus of 1928CAR, TRAC-STAR T cells; Figure 11B It represents the model diagram of intraperitoneal administration of GCV or control group PBS after reinfusion of 1928CAR&PD1-HER2t-(G4S)3-F / P2A-HSV-TK (1928CAR&HTK) to treat CD19-Raji hematological tumors, as well as the tumor fluorescence imaging survival diagram of mice; Figure 11C Fluorescence curve showing the size of mouse tumors; Figure 11D The graph shows the number of HER2-TK positive cells in the peripheral blood of 1928CAR&HTK mice at different time points after intraperitoneal injection of PBS and GCV; Figure 11EThe survival curves of 1928CAR&HTK mice after intraperitoneal injection of PBS and GCV, respectively; Figure 11F It represents the pattern diagram of intraperitoneal administration of GCV or control group PBS after reinfusion of TRAC-STAR&&PD1-HER2t-(G4S)3-F / P2A-HSV-TK (STAR&HTK) to treat CD19-Raji hematologic tumors, as well as the survival diagram of mouse tumor fluorescence imaging; Figure 11G Fluorescence curve showing the size of mouse tumors; Figure 11H It represents the survival curve of STAR&HTK mice after intraperitoneal injection of PBS and GCV respectively; Figure 11I The figures show the number of HER2-(G4S)3-TK positive cells in the peripheral blood of STAR&HTK mice at different time points after intraperitoneal injection of PBS and GCV, respectively. The HER2 in Figure 11 is HER2t, and HER2-F / P2A-TK represents HER2t-(G4S)3-F / P2A-TK.

[0049] Figure 12. HER2 / Herceptin / NK-mediated ADCC can completely eliminate HER2-positive cells. Figure 12A Purified NK cells and HER2t-(G4S)3-F / P2A-TK-positive cells were co-cultured at different ratios for 24 hours under the conditions of PBS (control) and different concentrations of Herceptin (Hcp) treatment, and the residual HER2t-(G4S)3-F / P2A-TK-positive cells were analyzed by flow cytometry; Figure 12B It shows the effect of different concentrations of Herceptin on the survival rate of HER2t-(G4S)3-F / P2A-TK positive cells. Figure 12C It represents the survival rate of HER2t-(G4S)3-F / P2A-TK positive cells under different NK / T ratios and Herceptin concentrations. Figure 12D It indicates the secretion of Granzym B by ELISA detection in the experimental and control groups; Figure 12E A functional model diagram of GCV / HSV-(G4S)3-TK and HER2 / Herceptin / NK bispecific suicide genes is shown. The HER2 in FIG12 is HER2t, and HER2-TK represents HER2t-(G4S)3-F / P2A-TK.

[0050] Figure 13 .Exemplary schematic diagrams of TCR, CAR and STAR. DETAILED DESCRIPTION

[0051] The present invention is described in detail below by way of examples. It will be appreciated by those skilled in the art that the following examples are for illustrative purposes only. The spirit and scope of the present invention are defined by the appended claims.

[0052] In the examples and figures, TK1 or TK also means HSV-TK.

[0053] Example 1. Design and construction of HER2t, HSV-TK, and HER2t-HSV-TK eukaryotic vectors

[0054] 1.1 Sequence information:

[0055] The amino acid sequence of the HER2 CDS protein is shown in GenBank No. NM_004448, and the nucleotide sequence is shown in Gene ID: 55914. The amino acid sequence of the HSV-TK CDS protein is shown in GenBank No. KM222725.1. Due to the large molecular weight of the HER2 protein, a partial truncated sequence was selected, namely domain III (amino acid sequence shown in SEQ ID NO: 18) and domain IV (amino acid sequence shown in SEQ ID NO: 19). A signal peptide (amino acid sequence shown in SEQ ID NO: 17) was attached to the N-terminus, and a TM (amino acid sequence shown in SEQ ID NO: 20) was attached to the C-terminus. This is represented as HER2t, with an amino acid sequence shown in SEQ ID NO: 1. The amino acid sequence of HER2t-(G4S)3-HSV-TK is shown in SEQ ID NO: 2. The amino acid sequence of G4S is shown in SEQ ID NO: 3.

[0056] 1.2 Construction of eukaryotic expression vectors expressing HER2t, HSV-TK, and HER2t-HSV-TK

[0057] First, the nucleotide sequences of HER2t (amino acid sequence shown in SEQ ID NO: 1, nucleotide sequence shown in SEQ ID NO: 16), HSV-TK, and HER2t-(G4S)3-F / P2A-HSV-TK (amino acid sequence shown in SEQ ID NO: 4) were constructed into the pcDNA3.1-Myc-His expression vector (as shown in Figures 3 and 4) by gene synthesis. Figure 4A), 48 hours after transfection of 293T cells, the transfected cells were lysed with RIPA buffer, and proteins were extracted for Western Blot experiments to verify the protein expression of the construct in 293T cells. Anti-myc (Sigma, 16-213) Western Blot results showed that HER2t, HSV-TK, and HER2t-(G4S)3-F / P2A-HSV-TK proteins were successfully expressed; Anti-HSV-TK1 (Santacruz-377211) Western Blot results showed that HSV-TK and HER2t-(G4S)3-F / P2A-HSV-TK fusion proteins were successfully expressed (see Figure 4 B and 4C).

[0058] Example 2. Construction of TRAC-STAR-KI & PD1-HER2t-(G4S)3-HSV-TK-KI STAR-T cells using the CRISPR / AAV site-directed insertion system

[0059] 2.1 Construction of AAV-HER2t-HSV-TK delivery vector and purification and concentration of virus

[0060] 2.1.1. Design of crRNAs targeting TRAC, B2M, PD1, REGNASE-1, and BCOR

[0061] Download the genome sequences of TRAC and PD1 from PUBMED and input each sequence into http: / / crispor.tefor.net / In Step 1, select Homo sapiens-Human-UCSCFeb.2009(GRCh37 / hg19)+SNPs:1000Genomes,ExaC in Step 2. In Step 3, select TTT(A / C / G)-21bp-Cas12a(Cpf1)-21bp guides recommended by IDT. Submit the result to generate a series of guide RNAs. Guide RNA sequences were randomly selected from different genomic sites and synthesized by IDT. The sequences are shown in SEQ ID NOs:31-64 in the table below.

[0062]

[0063]

[0064] 2.1.2. Detecting CrRNA Gene Editing Efficiency and Selecting the Optimal CrRNA for Each Gene

[0065] 1) Isolation and culture of primary human T cells

[0066] Human peripheral blood cells were obtained and CD4 and CD8 T cells were purified using a complete T cell magnetic bead isolation kit. T cells were then stimulated and activated for 24-36 hours in culture dishes coated with anti-CD3 / CD28 antibodies. The cells were washed twice with RNase-free Opti-MEM and resuspended a final time with the reagents in the Lonza P3 Primary Cell 4D Nucleofection System X Cell Kit S (Lonza, V4XP-3032) to a final cell concentration of 1+E6 / 20 μL.

[0067] 2) Preparation of RNP

[0068] Dissolve the synthesized crRNA in DEPC water to a final concentration of 75 μM. Order As.Cas12a Ultra protein from IDT at a concentration of 10 μg / μL. Mix 0.25 μL of As.Cas12a Ultra and 0.5 μL of each synthesized crRNA and incubate at 37°C for 15 min.

[0069] 3) Electroporation of RNP

[0070] The incubated RNPs were mixed with 20 μL of cell suspension, and after gently pipetting, all of the cells were transferred into the electroporation cup in the LONZA P3 primary cell 4D nuclear transfection system X unit kit S, placed in the LONZA X unit electroporator, and electroporated using the instrument's built-in EO115 program. The cells were then transferred to culture medium for culture.

[0071] 4) Extract cell genome

[0072] Four days after electroporation, 200 μL of cells (including cells that were not gene-edited as a control) were taken and the cell genome was extracted using a blood / cell / tissue genomic DNA extraction kit (Tiangen Company).

[0073] i. Design primers required to detect gene editing efficiency

[0074] The corresponding guide RNA sequences were found in the genome downloaded in Example 1, and PCR primers were designed about 300 bp upstream and downstream of the guide RNA sequences.

[0075] ii. Detecting gene editing efficiency

[0076] Using the extracted cell genome as a template, PCR was performed using primers corresponding to different guide RNAs. Each primer set was compared with an unedited cell genome as a control. Each PCR product was sequenced, and the optimal crRNA corresponding to each gene was selected by analyzing the results of the experimental and control groups. Based on the analysis results, the nucleotide sequence of the TRACC crRNA that achieved the best editing efficiency is shown in SEQ ID NO:5, and the nucleotide sequence of the PD1 crRNA is shown in SEQ ID NO:6.

[0077] 2.1.3. Virus packaging

[0078] The AAV plasmid map for site-directed insertion of HER2t-(G4S)3-TK at the PD1 knockout site is shown in the figure. Figure 5 shown.

[0079] The AAV plasmid map for site-directed insertion of HER2t-(G4S)3-F / P2A-TK at the PD1 knockout site is shown in the figure. Figure 6 shown.

[0080] The AAV plasmid map for site-directed insertion of CD19-28z-CAR-P2A-TK at the TRAC knockout site is shown in Figure 2. Figure 7 As shown, wherein the amino acid sequence of CD19-28z-CAR is shown in SEQ ID NO:23.

[0081] according to Figure 5 , 6 and 7, CD19-28z-CAR was replaced with CD19STAR (SEQ ID NO: 11) to obtain the STAR construct.

[0082] The nucleotide sequence of the STAR used is shown in SEQ ID NO:11, wherein the amino acid sequence of the VL is shown in SEQ ID NO:25, the amino acid sequence of the TCR constant region of the β chain is shown in SEQ ID NO:26, the amino acid sequence of the VH is shown in SEQ ID NO:27, and the amino acid sequence of the TCR constant region of the α chain is shown in SEQ ID NO:28; the TCR constant region of the β chain is followed by OX40 in series, as shown in SEQ ID NO:29. The N-terminus of the TCR constant region of the α chain is the GM-CSF signal peptide, as shown in SEQ ID NO:30. The amino acid sequence of CD19 STAR is shown in SEQ ID NO:24. The TCR constant region of the β chain and the TCR constant region of the α chain are connected by a linker peptide, which is a combination of Furin, SGSG, and p2A, with an amino acid sequence shown in SEQ ID NO:14 (nucleotide sequence shown in SEQ ID NO:15). The nucleotide sequence of IRES is shown in SEQ ID NO:21, and the amino acid sequence of RFP is shown in SEQ ID NO:22.

[0083] Screening obtained the best Cas12a / CrRNA (expressed as TRAC CrRNA, as shown in SEQ ID NO: 5, and PD1 CrRNA, as shown in SEQ ID NO: 6) for TRAC and PD1 knockout efficiency, selected a suitable insertion site near the Cas12a cleavage site, and selected the upstream and downstream 300bp of the insertion site as the left homology arm (LHA, for TRAC, the left homology arm nucleotide sequence is as shown in SEQ ID NO: 7, for PD1, the left homology arm nucleotide sequence is as shown in SEQ ID NO: 8) and the right homology arm (RHA, for TRAC, the right homology arm nucleotide sequence is as shown in SEQ ID NO: 9, for PD1, the right homology arm nucleotide sequence is as shown in SEQ ID NO: 10), between the left and right homology arms is an insertion sequence or gene of interest (Insert / GOI), obtained TRAC-KO&STAR-KI and PD1-KO&HER2t-HSV-TK-KI, knockout and knock-in patterns are shown in Figure 1. Figure 8A and 8B As shown, KO indicates knockout and KI indicates knockin.

[0084] The present invention constructs the nucleotide sequences of HER2t and HSV-TK into AAV vectors by gene synthesis, and designs two HER2t-HSV-TK constructs, namely EF1a-HER2t-(G4S)3-HSV-TK and HER2t-(G4S)3-F / P2A-HSV-TK (in the present invention, F / P2A represents the combination of Furin and P2A, the amino acid sequence is shown in SEQ ID NO: 14, and the nucleotide sequence is shown in SEQ ID NO: 15). The latter can cleave the Furin / P2A sequence under the action of cellular proteases, allowing HSV-TK to fully enter the cell nucleus and exert its biological function, such as Figure 8C As shown. Using conventional methods, AAV-STAR (the specific nucleotide sequence of STAR is shown in SEQ ID NO: 11, and its nucleotide sequence is constructed into AAV vector by gene synthesis ) Two different viruses, AAV-HER2t-HSV-TK, were lysed, ultracentrifuged, purified, concentrated, and titered.

[0085] 2.2 Construction of TRAC-STAR-KI&PD1-HER2t-HSV-TK-KI STAR-T cells

[0086] Human peripheral blood cells were obtained and CD4 and CD8 T cells were purified using a complete T cell magnetic bead isolation kit. T cells were then stimulated for 24–36 hours in a dish coated with anti-CD3 / CD28 antibodies (Gibco). The cells were harvested, washed twice with RNase-free Opti-MEM, and the remaining liquid was aspirated. The cells were then resuspended using the reagents in the LONZA P3 Primary Cell 4D Nucleofection System X Cell Kit S (Lonza, V4XP-3032) to a final concentration of 1.5E6 cells / 20 μL.

[0087] The synthesized Cas12a / CrRNA with the best TRAC and PD1 knockout efficiency was dissolved in DEPC water to a final concentration of 75 μM. As.Cas12a Ultra protein was ordered from IDT at a concentration of 10 μg / μL. 0.5 μL As.Cas12a Ultra protein, 0.5 μL TRAC CrRNA, and 0.5 μL PD1 CrRNA were gently mixed in a 200 μL RNase-free EP tube and incubated at 37°C for 15 minutes. The above 20 μL X unit kit S cell resuspension with a final cell concentration of 1.5E6 / 20 μL was added to the incubated TRAC CrRNA and PD1 CrRNA RNP. After gently pipetting to mix, transfer all the cells into the electroporation cup in the LONZA P3 primary cell 4D nuclear transfection system X unit kit S, place it in the LONZA X unit electroporator, and perform electroporation using the instrument's built-in EO115 program. After the electroporation is completed, 100ul of 20% FBS + 100IU-IL2 T cell complete medium is immediately added, and the cells are transferred to a 48-well flat-bottom plate. Within 30 minutes, AAV-STAR virus at an MOI of 1E5 and AAV-HER2t-HSV-TK virus at an MOI of 1E5 are added to prepare TRAC-STAR-KI & PD1-HER2t-HSV-TK-KI STAR-T cells. Three days later, the cells were transferred to a 12-well flat-bottom plate. Five days after electroporation, flow cytometry staining was performed with anti-FMC and anti-HER2 antibodies to analyze the site-specific integration efficiency of TRAC-STAR and PD1-HER2t-(G4S)3-HSV-TK. Figure 8D As shown, flow cytometry analysis showed that the proportion of STAR and HER2t double-positive cells was 11.8% for TRAC-STAR&PD1-HER2t-(G4S)3-HSV-TK and 29.5% for TRAC-STAR&PD1-HER2t-(G4S)3-F / P2A-HSV-TK. This indicates that the HER2-HSVTK dual-target suicide gene was successfully incorporated into the membrane. HER2-HSV-TK-positive cells can be analyzed, identified, and purified using anti-HER2 flow cytometry antibodies or magnetic beads-linked anti-HER2 antibodies.

[0088] Example 3. TRAC-STAR & PD1-HER2-HSV-TK double-positive cells have both the powerful killing function of STAR and the suicide function of suicide gene

[0089] TRAC-STAR & PD1-HER2-HSV-TK double-positive cells were sorted and the in vitro tumor killing function of STAR was analyzed using the luciferase assay.

[0090] TRAC-STAR & PD1-HER2-HSV-TK double-positive cells and CD19-RAJI tumor target cells (Yue Liu et al. Chimeric STAR receptors using TCR machinery mediaterobust responses against solid tumors. Science Translational Medicine, 2021, doi: 10.1126 / scitranslmed.abb5191) were co-cultured at 1 / 1 and 2 / 1 E / T ratios for 24 hours. Luciferase results showed that the tumor killing efficiency of TRAC-STAR & PD1-HER2-HSV-TK double-positive cells was close to 100%, which was no different from STAR. Figure 9A In addition, the proliferation and survival of sorted TRAC-STAR&PD1-HER2-HSV-TK double positive cells were detected by CCK-8 and flow cytometry FACS after adding different concentrations of GCV for 72 hours. The results showed that TRAC-STAR&PD1-

[0091] The cell death rate of HER2-HSV-TK double-positive cells after GCV treatment exceeded 95%, especially that of TRAC-STAR&PD1-HER2t-(G4S)3-F / P2A-HSV-TK cells was close to 100%. Figure 9B and 9C This indicates that TRAC-STAR&PD1-HER2-HSV-TK double-positive cells have both the powerful killing function of STAR and the suicide function of suicide genes, especially the suicide effect of the TRAC-STAR&PD1-HER2t-(G4S)3-F / P2A-HSV-TK construct is better.

[0092] Example 4. Effects of GCV concentration and time gradient on HER2-HSVTK dual-target suicide gene

[0093] TRAC-STAR&PD1-HER2-HSV-TK double-positive cells were sorted by flow cytometry staining with anti-FMC and anti-HER2 antibodies. Different concentrations of GCV (0.1ug / ml-25ug / ml) were applied to the double-positive cells. The clearance rate of GCV on cells was detected and analyzed at different treatment time points. Statistical results showed that the clearance rate of double-positive cells was the best after 72 hours of GCV treatment, with a cell mortality rate of over 90%. The cell mortality rate after 72 hours of GCV treatment with 5ug / ml GCV was similar to that of high concentrations. Figure 10As shown in Figures A and 10B. The reason why GCV could not completely eliminate and kill cells in this analysis was that the retest rate of the sorted TRAC-STAR&PD1-HER2-HSV-TK double-positive cells before the final experiment was only 85%, indicating that the actual TRAC-STAR&PD1-HER2-HSV-TK double-positive cells were almost 100% eliminated. Exemplary GCV and anti-HER2 antibodies (such as trastuzumab) have good clinical safety and relatively economical prices. In particular, GCV is cheaper and suitable for a wider range of patients. By selectively eliminating the infused cells through the administration of GCV and / or trastuzumab at the appropriate time point, toxic side effects and the risk of tumorigenesis caused by chronic tumor treatment can be avoided.

[0094] Example 5. In vivo antitumor effects of 1928CAR and STAR loaded with suicide gene HER2t-(G4S)3-F / P2A-HSV-TK and potent in vivo cell killing function of GCV / HSV-TK

[0095] According to conventional methods, 1928CAR (SEQ ID NO: 65) was prepared, and the TRAC-STAR T cell PD1 site was integrated with the HER2t-HSV-TK (i.e., HER2t-(G4S)3-F / P2A-HSV-TK) element for transfusion of therapeutic human T cells. The test results are shown in Figure 11A . CD19-Raji tumor cells were inoculated into the tail vein of NCG mice to construct a hematologic tumor mouse model. Five days after tumor infusion, the mice were imaged and grouped, and the sorted and purified 1928CAR&PD1-HER2-HSV-TK (1928CAR&HTK) and TRAC-STAR&&PD1-HER2-HSV-TK (STAR&HTK) T cells were infused back. GCV (20 mg / kg) and PBS control groups were intraperitoneally administered on the 10th day after T cell infusion, and the administration was repeated every two days. The changes in the proportion of HER2-TK positive cells in mouse tumors and peripheral blood were then detected at different time points ( Figure 11B , C, D and Figure 11F , G, I), statistical mouse survival curve ( Figure 11E The results showed that GCV significantly eliminated HER2-TK-positive cells in mice, and the mice became uncontrollable and died earlier, demonstrating the potent cell-killing ability of GCV / HSV-TK in vivo.

[0096] Example 6. HER2 / Herceptin / NK-mediated ADCC can completely eliminate HER2-positive cells

[0097] NK cells from the same donor and T cells expressing HER2-HSV-TK (i.e., HER2t-(G4S)3-F / P2A-HSV-TK) were co-cultured at different ratios under the treatment of different concentrations of Herceptin (Hcp) for 24 hours, and the survival of HER2-TK positive cells was analyzed by flow cytometry ( Figure 12A , B and C). Granzym B secreted by Hcp experimental group and PBS control group was detected by ELISA ( Figure 12D ). Figure 12E A diagram showing the functional model of GCV / HSV-TK and HER2 / Herceptin / NK bispecific suicide genes.

[0098] Sequence information

[0099] SEQ ID NO: 1

[0100] Amino acid sequence of HER2t (Her2 truncated form: signal peptide-domain III-domain IV-TM):

[0101] (The italicized text indicates the signal peptide, the underlined text indicates the amino acid sequence of domain III, and the bold text indicates the amino acid sequence of domain IV)

[0102] SEQ ID NO:2

[0103] Amino acid sequence of HER2t-G4S3-HSV-TK

[0104] MELAALCRWGLLLALLPPGAASARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITGYLYISAWPDSLPDLSVQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGE GLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLTSIISAVVGILLVVVLGVVFGILIKRRQQQKIRKGGGGSGGGGSGGGGS MASYPCHQHASAFDQAARSRGHSNRRTALRPRRQQEATEVRLEQKMPTLLRVYIDGPHGMGKTTT TQLLVALGSRDDIVYVPEPMTYWQVLGASETIANIYTTQHRLDQGEISAGDAAVVMTSAQITMGMPYAVTDAVLAP HIGGEAGSSHAPPPALTLIFDRHPIAALLCYPAARYLMGSMTPQAVLAFVALIPPTLPGTNIVLGALPEDRHIDRL AKRQRPGERLDLAMLAAIRRVYGLLANTVRYLQGGGSWREDWGQLSGTAVPPQGAEPQSNAGPRPHIGDTLFTLFR APELLAPNGDLYNVFAWALDVLAKRLRPMHVFILDYDQSPAGCRDALLQLTSGMVQTHVTTPGSIPTICDLARTFA REMGEAN *(The underlined amino acid sequence is HSV-TK)

[0105] SEQ ID NO:3 Amino acid sequence of G4S

[0106] GGGGS

[0107] SEQ ID NO:4

[0108] Amino acid sequence of HER2t-G4S3-F / P2A-HSV-TK: MELAALCRWGLLLALLPPGAASARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLTSIISAVVGILLVVVLGVVFGILIKRRQQKIRKGGGGSGGGGSGGGGSRRKRSGSGATNFSLLKQAGDVEENPGPASMASYPCHQHASAFDQAARSRGHSNRRTALRPRRQQEATEVRLEQKMPTLLRVYIDGPHGMGKTTTTQLLVALGSRDDIVYVPEPMTYWQVLGASETIANIYTTQHRLDQGEISAGDAAVVMTSAQITMGMPYAVTDAVLAPHIGGEAGSSHAPPPALTLIFDRHPIAALLCYPAARYLMGSMTPQAVLAFVALIPPTLPGTNIVLGALPEDRHIDRLAKRQRPGERLDLAMLAAIRRVYGLLANTVRYLQGGGSWREDWGQLSGTAVPPQGAEPQSNAGPRPHIGDTLFTLFRAPELLAPNGDLYNVFAWALDVLAKRLRPMHVFILDYDQSPAGCRDALLQLTSGMVQTHVTTPGSIPTICDLARTFAREMGEAN*

[0109] SEQ ID NO:5

[0110] Nucleotide sequence of TRAC-crRNA-PAM

[0111] GAGTCTCTCAGCTGGTACACTAAA

[0112] SEQ ID NO:6

[0113] Nucleotide sequence of PD1-crRNA-PAM

[0114] GCACGAAGCTCTCCGATGTGTAAA

[0115] SEQ ID NO:7

[0116] Nucleotide sequence of the left homology arm of TRAC

[0117] TTAATGCCAACATACCATAAACCTCCATTCTGCTAATGCCCAGCCTAAGTTGGGGAGACCACTCCAGATTCCAAGATGTACAGTTTGCTTTGCTGGGCCTTTTTCCCATGCCTGCCTTTACTCTGCCAGAGTTATATTGCTGGGGTTTTGAAGAAGATCCTATTAAATAAAAGAATAAGCAGTATTATTAAGTAGCCCTGCATTTCAGGTTTCCTTGAGTGGCAGGCCAGGCCTGGCCGTGAACGTTCAC TGAAATCATGGCCTCTTGGCCAAGATTGATAGCTTGTGCCTGTCCCTGAGTCCCAGTCCATCACGAGCAGCTGGTTTCTAAGATGCTATTTCCCGTATAAAGCATGAGACCGTGACTTGCCAGCC CCACAGAGCCCCGCCCTTGTCCATCACTGGCATCTGGACTCCAGCCTGGGTTGGGGCAAAGAGGGAAATGAGATCATGTCCTAACCCTGATCCTCTTGTCCCACAGATATCCAGAACCCTGACCCT

[0118] SEQ ID NO:8

[0119] Nucleotide sequence of the left homology arm of PD1

[0120] CctcacgtagaaggaagaggctctgcagtggaggccagtgcccatccccgggtggcagaggccccagcagagacttctcaatgacattccagctggggtggcccttccagagcccttgctgcccgagggatgtgagcaggtggccggggaggctttgtggggccacccagccccttcctcacctctctccatctctcagACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACA

[0121] SEQ ID NO: 9

[0122] Nucleotide sequence of the right homologous arm of TRAC

[0123] CCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGGTAAGGGCAGCTTTGGTGCCTTCGCAGGCTGTTTCCTTGCTTCAGGAATGGCCAGGTTCTGCCCAGAGCTCTGGTCAATGATGTCTAAAACTCCTCTGATTGGTGGTCTCG

[0124] SEQ ID NO: 10

[0125] Nucleotide sequence of the right homologous arm of PD1

[0126] CTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGgtgcggcctcggaggccccggggcaggggtgagctgagccggtcctggg

[0127] SEQ ID NO:11

[0128] Nucleotide sequence of STAR

[0129]

[0130] SEQ ID NO:12

[0131] Amino acid sequence of P2A

[0132] ATNFSLLKQAGDVEENPGP

[0133] SEQ ID NO:13

[0134] Amino acid sequence of Furin

[0135] RRKR

[0136] SEQ ID NO:14

[0137] Combined amino acid sequence of Furin, SGSG, and p2A

[0138] RRKRSGSGATNFSLLKQAGDVEENPGP

[0139] SEQ ID NO:15

[0140] Nucleotide sequence of Furin / P2A

[0141] CGGCGGAAACGGAGCGGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCT

[0142] SEQ ID NO:16

[0143] Nucleotide sequence of HER2t (Her2 truncated form):

[0144]

[0145] SEQ ID NO:17

[0146] HER2 Signal peptide

[0147] MELAALCRWGLLLALLPPGAAS

[0148] SEQ ID NO:18

[0149] HER2 domain III

[0150] ARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGEGLAC

[0151] SEQ ID NO:19

[0152] HER2 domain IV

[0153] HQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLT

[0154] SEQ ID NO:20

[0155] HER2 TM

[0156] SIISAVVGILLVVVLGVVFGILIKRRQQKIRK

[0157] SEQ ID NO:21

[0158] Nucleotide sequence of IRES

[0159] cccccccccctaacgttactggccgaagccgcttggaataaggccggtgtgcgtttgtctatatgttattttccaccatattgccgtcttttggcaatgtgagggcccggaaacctggccctgtcttcttgacgagcattcctaggggtctttcccctctcgccaaaggaatgcaaggtctgttgaatgtcgtgaaggaagcagttcctctggaagcttcttgaagacaaacaacgtctgtagcgaccctttgcaggcagcggaaccccccacctggcgacaggtgcctctgcggccaaaagccacgtgtataagatacacctgcaaaggcggcacaaccccagtgccacgttgtgagttggatagttgtggaaagagtcaaatggctctcctcaagcgtattcaacaaggggctgaaggatgcccagaaggtaccccattgtatgggatctgatctggggcctcggtgcacatgctttacatgtgtttagtcgaggttaaaaaaacgtctaggccccccgaaccacggggacgtggttttcctttgaaaaacacgataata

[0160] SEQ ID NO:22

[0161] RFP amino acid sequence

[0162] MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMRLKL

[0163] KDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHSTGA*

[0164] SEQ ID NO:23

[0165] CD19CAR amino acid sequence

[0166] MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQ

[0167] QKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLP

[0168] YTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLP

[0169] DYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSEFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*

[0170] SEQ ID NO:24

[0171] CD19STAR amino acid sequence

[0172] MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNSRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIRRKRSGSGATNFSLLKQAGDVEENPGPASMLLLVTSLLLCELPHPAFLLIPEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSDIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSSRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI*

[0173] SEQ ID NO:25

[0174] Amino acid sequence of CD19STAR VL

[0175] DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSG VPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT

[0176] SEQ ID NO:26

[0177] Amino acid sequence of the TCR constant region of the β chain

[0178] DLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNS

[0179] SEQ ID NO:27

[0180] CD19STAR VH amino acid sequence

[0181] EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS

[0182] SEQ ID NO:28

[0183] Amino acid sequence of the TCR constant region of the α chain

[0184] IQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS

[0185] SEQ ID NO:29

[0186] Amino acid sequence of OX40

[0187] RRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI

[0188] SEQ ID NO:30

[0189] Amino acid sequence of GM-CSF signal peptide

[0190] MLLLVTSLLLCELPHPAFLLIP

[0191] SEQ ID NO:65 Source: doi:10.1038 / nature21405

[0192] Amino acid sequence of 1928CAR

[0193] MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLN

[0194] WYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSEFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

Claims

1. A modified immune cell comprising unit A and unit B, wherein unit A comprises a nucleotide sequence encoding a T cell receptor (TCR), a chimeric antigen receptor (CAR) or a synthetic T cell antigen receptor (STAR), and unit B comprises a nucleotide sequence of a suicide gene, wherein unit A and unit B are located at different positions in the genome of the modified immune cell, or wherein unit A and unit B are connected to replace the PD1 gene and / or TRAC site in the modified immune cell (preferably the GenBank accession number of TRAC is NC_000014. 9) and / or B2M site (preferably GenBank: NC_000015.10) and / or TET2 gene and / or Regnase gene and / or Roquin gene and / or BCOR gene and / or ETS1 gene and / or IKZF1 gene and / or FADD gene and / or ULK2 gene and / or NFKBIE gene and / or NFKBIA gene and / or KLF16 gene and / or SMARCCI gene nucleotide sequence, preferably unit B is connected to a tag gene such as Myc, Flag, HA or His.

2. The modified immune cell according to claim 1, wherein the modified immune cell further comprises unit C, wherein unit C comprises a nucleotide sequence encoding a tumor cell surface-specific antigen, preferably, the tumor cell surface-specific antigen is selected from one or more of HER2, CD19, CD20, GD2, EGFR, and Cetux tumor surface antigens as shown in SEQ ID NO: 1, and preferably unit C comprises the extracellular domain and transmembrane domain of the tumor cell surface-specific antigen.

3. The modified immune cell according to claim 2, wherein any one of unit A, unit B and unit C or any combination of any two of them are located at different positions in the genome of the modified immune cell.

4. The modified immune cell according to any one of claims 2 to 3, wherein unit A is linked to unit B, unit A is linked to unit C, or unit B is linked to unit C, or unit A, unit B and unit C are linked, Preferably, the connection is through a linker and / or a linker peptide, more preferably, the linker is selected from (G4S)n, wherein n is an integer of 1-5, or an SGSG linker, and the linker peptide is Furin, p2A or a combination thereof, preferably the sequence of Furin is as shown in SEQ ID NO: 13, or the sequence of p2A is as shown in SEQ ID NO: 12, preferably the amino acid sequence of the combination of Furin and P2A is as shown in SEQ ID NO: 14, and the nucleotide sequence is as shown in SEQ ID NO:

15.

5. The modified immune cell according to any one of claims 1 to 4, wherein the immune cell is selected from T cells, NK cells, macrophages, NKT cells, Treg cells, and dendritic cells.

6. The modified immune cell according to any one of claims 1 to 5, wherein unit A, unit B, unit C, or any combination of any two thereof, or a combination of the three thereof replaces the nucleotide sequence of the PD1 gene and / or TRAC site and / or B2M site and / or TET2 gene and / or Regnase gene and / or Roquin gene and / or BCOR gene and / or ETS1 gene and / or IKZF1 gene and / or FADD gene and / or ULK2 gene and / or NFKBIE gene and / or NFKBIA gene and / or KLF16 gene and / or SMARCC1 gene in the modified immune cell.

7. The modified immune cell according to any one of claims 1 to 6, wherein the suicide gene is selected from one or more of HSVTK (preferably GenBank: KM222725.1), iCaspase9, HER2 as shown in SEQ ID NO: 1, EGFRt, CD20, rapamycin, and / or RQR8, preferably EGFRt-HSVTK, CD20-HSVTK, or The T cell receptor (TCR) comprises an α chain and a β chain, the α chain comprises a TCR variable region and a TCR constant region, the β chain comprises a TCR variable region and a TCR constant region, preferably, the α chain and the β chain are connected by a connecting peptide; or the α chain is located at the C-terminus and the β chain is located at the N-terminus; or the chimeric antigen receptor (CAR) comprises scFv, a hinge region, 4-1BB / CD8 and CD3ζ; preferably, the VL and VL in the scFv are connected by a linker; or VL is located at the N-terminus; The synthetic T cell receptor antigen receptor (STAR) comprises VL, a TCR constant region of a β chain, VH and a TCR constant region of an α chain; preferably, the TCR constant region of the β chain and the TCR constant region of the α chain are connected by a linker peptide; more preferably, the linker peptide is Furin, p2A or a combination thereof, preferably the sequence of the linker peptide Furin is as shown in SEQ ID NO: 13, or the sequence of p2A is as shown in SEQ ID NO: 12; or the linker is selected from (G4S)n, wherein n is an integer from 1 to 5, or an SGSG linker; More preferably, the TCR, CAR and STAR further comprise an IRES or RFP at the C-terminus. Preferably, the sequence of the IRES is as shown in SEQ ID NO: 21, or the sequence of the RFP is as shown in SEQ ID NO:

22. Most preferably, the amino acid sequence of the CAR is shown in SEQ ID NO: 23, and the amino acid sequence of the STAR is shown in SEQ ID NO:

24.

8. A method for preparing a modified immune cell, comprising inserting unit A and unit B into different positions in the genome of the modified immune cell, or connecting unit A and unit B to replace the PD1 gene and / or TRAC site (preferably the GenBank number of TRAC is NC_000014.9) and / or B2M site (preferably GenBank: NC_000015.10) and / or TET2 gene and / or Regnase gene and / or Roquin gene and / or BCOR gene and / or ETS1 gene and / or IKZF1 gene and / or FADD gene and / or ULK2 gene and / or NFKBIE gene and / or NFKBIA gene and / or KLF16 gene and / or SMARCCI gene in the modified immune cell, wherein unit A comprises a nucleotide sequence encoding a T cell receptor (TCR), a chimeric antigen receptor (CAR) or a synthetic T cell antigen receptor (STAR), and unit B comprises a nucleotide sequence of a suicide gene.

9. The method of claim 8, further comprising inserting unit C into the modified immune cell genome, wherein unit C comprises a nucleotide sequence encoding a tumor cell surface-specific antigen (preferably, the tumor cell surface-specific antigen is selected from one or more of HER2, CD19, CD20, GD2, EGFR, and Cetux as shown in SEQ ID NO: 1, and preferably unit C comprises the extracellular domain and transmembrane domain of the tumor cell surface-specific antigen). Preferably, unit A is connected to unit B, unit A is connected to unit C, or unit B is connected to unit C, or unit A, unit B and unit C are connected, preferably through a linker or a linker peptide, more preferably, the linker is selected from (G4S)n, wherein n is an integer from 1 to 5, or an SGSG linker, and the linker peptide is Furin, p2A or a combination thereof, preferably the sequence of Furin is as shown in SEQ ID NO: 13, or the sequence of p2A is as shown in SEQ ID NO: 12, preferably the amino acid sequence of the combination of Furin and P2A is as shown in SEQ ID NO: 14, and the nucleotide sequence is as shown in SEQ ID NO:

15.

10. The method of claim 9, wherein any one of unit A, unit B and unit C or any combination of any two of them is inserted into different positions in the genome of the modified immune cell, preferably replacing the nucleotide sequence of the PD1 gene and / or TRAC site and / or B2M site and / or TET2 and / or Regnase and / or Roquin and / or BCOR and / or ETS1 and / or IKZF1 and / or FADD and / or ULK2 and / or NFKBIE and / or NFKBIA and / or KLF16 and / or SMARCCI in the modified immune cell.

11. The method of any one of claims 8 to 10, wherein the replacement is performed by gene editing technology, preferably CRISPR / Cas9 or CRISPR / Cas12a.

12. The method of any one of claims 8 to 11, wherein the immune cell is as defined in claim 5, and the suicide gene, T cell receptor (TCR), chimeric antigen receptor (CAR) and synthetic T cell antigen receptor (STAR) are as defined in claim 7.

13. A kit comprising unit A, unit B and / or unit C, wherein unit A comprises a nucleotide sequence encoding a T cell receptor (TCR), a chimeric antigen receptor (CAR) or a synthetic T cell antigen receptor (STAR), unit B comprises a nucleotide sequence of a suicide gene, and unit C comprises a nucleotide sequence encoding a tumor cell surface-specific antigen. Preferably, the kit further comprises an antibody against a tumor cell surface-specific antigen.

14. The kit of claim 13, wherein the nucleotide sequence encoding a T cell receptor (TCR), a chimeric antigen receptor (CAR) or a synthetic T cell antigen receptor (STAR), the nucleotide sequence of a suicide gene or the nucleotide sequence encoding a tumor cell surface-specific antigen is present in the form of a plasmid.

15. The kit according to any one of claims 1 to 14, wherein the tumor cell surface-specific antigen is selected from one or more of the tumor surface antigens HER2, CD19, CD20, GD2, EGFR, and Cetux as shown in SEQ ID NO: 1, and preferably unit C comprises the extracellular domain and transmembrane domain of the tumor cell surface-specific antigen.

16. The kit of any one of claims 12-15, wherein the suicide gene, T cell receptor (TCR), chimeric antigen receptor (CAR) and synthetic T cell antigen receptor (STAR) are as defined in claim 7.

17. Use of the modified immune cell according to any one of claims 1 to 7 or the kit according to any one of claims 13 to 16 in treating a disease, or in preparing a medicament for treating a disease, wherein the disease is selected from a tumor or an autoimmune disease.

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