Chimeric antigen receptor t cell targeting CD5 molecule and Anti-tumor use thereof

Abstract

The present invention relates to a chimeric antigen receptor T cell targeting a CD5 molecule and the anti-tumor use thereof. The chimeric antigen receptor comprises a 12C single-domain antibody, and the 12C single-domain antibody has an amino acid sequence as shown in SEQ ID NO: 1. The chimeric antigen receptor targeting a CD5 molecule further comprises a signal peptide, an extracellular region, a transmembrane region, a cytoplasmic region, a self-cleaving region and a retention domain. The chimeric antigen receptor can effectively block the expression of CD5 on T cells, and prevent the suicide phenomenon of CD5-targeting CAR-T cells. The CD5 CAR-T cells exhibit significant specific killing against CCRF-CEM cells, and have a significantly increased IFN-γ secretion level after killing the CCRF-CEM cells. The CD5 CAR-T cells can effectively scavenge CD5-positive tumor cells, and have a significant anti-tumor effect in vivo.

Description

Chimeric antigen receptor T cells targeting CD5 molecules and their anti-tumor applications

[0001] The present invention claims priority to the Chinese patent application filed with the China Patent Office on December 20, 2023, with application number 2023117633975 and invention name “Chimeric antigen receptor T cells targeting CD5 molecules and their anti-tumor applications”, the entire contents of which are incorporated by reference into the application. Technical Field

[0002] The present invention belongs to the field of biomedicine technology, and particularly relates to chimeric antigen receptor T cells targeting CD5 molecules and their anti-tumor applications. Background Art

[0003] T-cell malignancies are a broadly heterogeneous group of diseases with a poor prognosis in both children and adults (Sehn LH, Soulier J. Introduction to the review series on T-cell malignancies [J]. Blood, 2017, 129:1059-60). T-cell acute lymphoblastic leukemia (T-ALL) is a highly heterogeneous hematologic malignancy, accounting for 25% of adult ALL cases and 15% of pediatric ALL cases. Currently, treatment options for T-cell malignancies include chemotherapy, hematopoietic stem cell transplantation, and targeted therapy. Compared with B-cell malignancies, first-line chemotherapy for T-cell malignancies achieves only limited clinical responses. Hematopoietic stem cell transplantation, as a radical treatment, is often associated with complications such as graft-versus-host disease (GVHD) and infection. Consequently, clinical applications suffer from limited efficacy, high relapse rates, and a high incidence of GVHD, often leading to poor prognosis (Ma H, Abdul-Hay MT-cell lymphomas, a challenging disease: types, treatments, and future[J]. Int J Clin Oncol, 2017, 22:18-51).

[0004] In recent years, CAR-T therapy has achieved great success in the treatment of B-cell malignancies, but its research and application in T-cell malignancies are very limited. CAR-T therapy for T-cell malignancies still faces many challenges, including the expression of target antigens on the surface of CAR-T cells leading to CAR-T cell cannibalism, contamination of malignant T cells during the preparation of autologous CAR-T cells (M. Alcantara, M. Tesio, CH June, R. Houot, CAR T-cells for T-cell malignancies: challenges in distinguishing between therapeutic, normal, and neoplastic T-cells, Leukemia 32(11)(2018)2307-2315), and the risk of graft-versus-host-disease (GVHD) during allogeneic infusion.

[0005] Currently, the main targets for immunotherapy of T-cell malignancies include CD3, CD4, CD5, and CD7. CD5 is a pan-T cell marker that is commonly overexpressed in most T-cell malignancies (CASALIP, BURASTERO SE, NAKAMURA M, et al. Human lymphocytes making rheumatoid factor and antibody to ssDNA belong to Leu 1+B cell subset[J]. Science, 1987, 236(4797):77). It is also expressed on the surface of some B-cell malignancies, such as chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). The expression of CD5 on normal cells is limited to thymocytes, peripheral T cells and a small number of B lymphocyte subsets, called B1 cells (FREITAS CMT, JOHNSON DK, WEBER K ST Cell Calcium Signaling Regulation by the Co Receptor CD5[J]. Int J Mol Sci, 2018, 19(5):) and the CD5 molecule is not expressed on the surface of hematopoietic stem cells. Therefore, the CD5 molecule is expected to become an ideal target for T cell malignancies. However, the use of CD5 CAR-T cells for the targeted treatment of T cell acute lymphoblastic leukemia still faces great problems. This is because both normal effector T cells and T cell tumors express the CD5 antigen, which will lead to the fratricide of CD5 CAR-T cells. CD5 CAR-T cells are difficult to successfully prepare in vitro. Therefore, it is urgent to develop a chimeric antigen receptor that can effectively block the expression of CD5 on T cells, prevent the suicide phenomenon of targeted CD5 CAR-T cells, and improve the efficacy and durability of CD5-targeted CAR-T cell therapy for the treatment of T cell malignancies. Summary of the Invention

[0006] In order to solve the above problems, the present invention provides a chimeric antigen receptor targeting CD5 molecules, wherein the chimeric antigen receptor includes a 12C single-domain antibody; the chimeric antigen receptor targeting CD5 molecules also includes a signal peptide, a hinge region, a transmembrane region, a cytoplasmic region, a self-cleavage region and a retention domain.

[0007] In one aspect, the present invention provides a chimeric antigen receptor targeting CD5 molecules, wherein the chimeric antigen receptor targeting CD5 molecules comprises a 12C single-domain antibody; the 12C single-domain antibody comprises heavy chain variable regions CDR1, CDR2 and CDR3; the amino acid sequences of CDR1, CDR2 and CDR3 are shown in SEQ ID NO.15-SEQ ID NO.17.

[0008] Specifically, the amino acid sequence of the 12C single-domain antibody is SEQ ID NO.1 or a sequence having 85% homology with SEQ ID NO.1.

[0009] Preferably, the amino acid sequence of the 12C single-domain antibody is SEQ ID NO.1 or a sequence having 90% homology with SEQ ID NO.1.

[0010] Further preferably, the amino acid sequence of the 12C single-domain antibody is SEQ ID NO.1 or a sequence having 95% homology with SEQ ID NO.1.

[0011] Further preferably, the amino acid sequence of the 12C single-domain antibody is SEQ ID NO.1 or a sequence having 98% homology with SEQ ID NO.1.

[0012] More preferably, the amino acid sequence of the 12C single-domain antibody is SEQ ID NO.1.

[0013] Specifically, the chimeric antigen receptor targeting CD5 molecule further includes a signal peptide, an extracellular region, a transmembrane region and / or a cytoplasmic region.

[0014] More specifically, the chimeric antigen receptor targeting CD5 molecule further includes but is not limited to: a self-cleavage region and / or a retention domain.

[0015] Specifically, the chimeric antigen receptor targeting CD5 molecules is obtained by serially connecting the following modules: signal peptide of CD8 molecule, 12C single-domain antibody, CD8Hinge+TM region, 4-1BB cytoplasmic region, cytoplasmic region of CD3ζ molecule, T2A self-cleavage region, 12C single-domain antibody, and ER retention domain.

[0016] More specifically, the amino acid sequence of the signal peptide of the CD8 molecule is shown in SEQ ID NO.2; the amino acid sequence of the CD8Hinge+TM region is shown in SEQ ID NO.3; the amino acid sequence of the 4-1BB cytoplasmic region is shown in SEQ ID NO.4; the amino acid sequence of the cytoplasmic region of the CD3ζ molecule is shown in SEQ ID NO.5; the amino acid sequence of the T2A self-cleavage region is shown in SEQ ID NO.6; and the amino acid sequence of the ER retention domain is shown in SEQ ID NO.7.

[0017] Specifically, the amino acid sequence of the chimeric antigen receptor targeting CD5 molecule is shown in SEQ ID NO.9.

[0018] In another aspect, the present invention provides a nucleic acid encoding the aforementioned chimeric antigen receptor.

[0019] Specifically, the sequence of the nucleic acid is as shown in SEQ ID NO.8 or a sequence having more than 80% sequence homology with SEQ ID NO.8.

[0020] In another aspect, the present invention provides a modified T cell, wherein the T cell comprises, expresses and / or secretes the aforementioned chimeric antigen receptor.

[0021] Specifically, the T cells can be CAR-T cells.

[0022] In another aspect, the present invention provides the aforementioned T cell culture, wherein the cell culture contains a chimeric antigen receptor targeting CD5 molecules.

[0023] In another aspect, the present invention provides a medicament for the aforementioned T cell or cell culture.

[0024] Specifically, the medicine also includes pharmaceutically acceptable excipients.

[0025] Preferably, the pharmaceutically acceptable excipient is selected from polysorbate, histidine, sucrose, arginine, sodium chloride, methionine, acetate, trehalose, proline, sorbitol, sodium phosphate, poloxamer 188, ethylenediaminetetraacetic acid, citric acid, mannitol, glutamate, glycine, sodium citrate, sodium succinate and / or lactic acid.

[0026] In another aspect, the present invention provides the use of the aforementioned chimeric antigen receptor or T cell or cell culture in the preparation of an anti-tumor drug.

[0027] Specifically, the tumor includes a T-cell malignancy.

[0028] In another aspect, the present invention provides use of the aforementioned chimeric antigen receptor or T cell or cell culture in the preparation of a method for preventing and / or treating an immune disease.

[0029] Specifically, the applications include but are not limited to: rheumatoid arthritis, systemic lupus erythematosus, type I diabetes and / or myasthenia gravis.

[0030] In another aspect, the present invention provides a CD5 detection kit, characterized in that it comprises the aforementioned chimeric antigen receptor or T cell or cell culture.

[0031] Specifically, the kit further includes a solid phase carrier, a detection label, a detection substrate and / or a buffer.

[0032] More specifically, the solid phase carrier can be a material with affinity, which can fix the specific antibody on the surface.

[0033] The detection marker may be an enzyme marker, which is an enzyme that can bind to an antibody and is used to detect the binding of the antibody to the antigen.

[0034] The detection substrate can be a reaction product of an enzyme marker, which can generate a measurable signal under enzyme catalysis.

[0035] In another aspect, the present invention provides a CD5 detection method comprising the aforementioned chimeric antigen receptor or T cell or cell culture or kit, wherein the method is a non-disease diagnosis or treatment method.

[0036] The technical effects achieved by the present invention are:

[0037] (1) 12C-ER can effectively block the expression of CD5 on T cells and prevent the suicide of CD5-targeted CAR-T cells;

[0038] (2) Under different effector-target ratios, CD5 CAR-T cells showed significant specific killing effects on CCRF-CEM cells;

[0039] (3) The level of IFN-γ secretion increased significantly after 12C.b CAR-T killed CCRF-CEM cells.

[0040] (4) CD5 CAR-T cells can effectively eliminate CD5-positive tumor cells and have significant anti-tumor effects in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 is a schematic diagram of the CD5-CART structure for CD5 molecule blocking; wherein, CD8SP represents the signal peptide of the CD8 molecule; 12C sdAb represents the single-domain antibody sequence targeting CD5; CD8Hinge+TM represents the extracellular region and transmembrane region sequence of the CD8 molecule; 4-1BB represents the 4-1BB cytoplasmic region sequence; CD3ζ represents the cytoplasmic region sequence of the CD3ζ molecule; T2A represents the self-cleavage sequence; ER represents the ER retention domain sequence.

[0042] Figure 2 is a map of the lentiviral vector Pre-Lenti-EF1-CAR V2 Wpmut.

[0043] Figure 3 is a map of the packaging plasmid pMDLg-pRRE-K.

[0044] Figure 4 is a map of the packaging plasmid pRsv-rev-kana-V2.

[0045] Figure 5 is a map of the envelope plasmid pMD2.GK.

[0046] Figure 6 shows the flow cytometry analysis of the CD5 CAR-T CAR positivity rate and the expression of CD5 molecules on the cell membrane after CD5 expression blocking. Figure A shows that the CAR positivity rate of 12C.b CAR-T cells is close to 100%. Figure B shows that 12C ER can effectively block the expression of CD5 on T cells, while the CD5 positivity rate on the surface of T cells not transfected with CD5 blocking virus is 99.5%.

[0047] FIG7 shows the cytotoxicity of CD5 CAR-T cells with blocked CD5 expression against CD5-positive acute lymphoblastic leukemia cells (CCRF-CEM).

[0048] Figure 8 shows the secretion of cytokine IFN-γ after CD5 CAR-T with blocked CD5 expression was incubated with CCRF-CEM cells.

[0049] Figure 9 shows the results of an in vivo tumor killing experiment using CD5 CAR-T cells with blocked CD5 expression. DETAILED DESCRIPTION

[0050] The present invention will be further described in detail below with reference to specific examples. The following examples are not intended to limit the present invention but are merely intended to illustrate the present invention. The experimental methods used in the following examples are generally based on conventional conditions unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified.

[0051] Example 1 Construction of CD5-CART vector for CD5 molecule blocking

[0052] In this example, a CAR lentiviral vector with Intrablock CD5 expression blocking function and targeting CD5 was constructed. The structure is shown in Figure 1. The 12C single-domain antibody is used as the antigen recognition region, combined with the CD8 hinge region and CD8 transmembrane region sequence, the 4-1BB cytoplasmic region sequence and the intracellular signaling molecule CD3ζ sequence. The CD5 single-domain antibody sequence is connected by T2A and connected to the ER retention domain. It can be used for the CD5-targeting CAR structure of T cells and is named 12C.b CAR. The lentiviral vector Pre-Lenti-EF1-CAR V2 Wpmut and 12C.bCAR genes are digested, connected, transformed, cloned, plasmid extracted, and sequenced to obtain the sequence-corrected lentiviral vector Pre-Lenti-EF1-12CER-CAR V2 WPmut. The nucleotide sequence of 12C.b CAR is SEQ ID NO.8, and its corresponding amino acid sequence is SEQ ID NO.9.

[0053] The preparation method of the lentiviral vector Pre-Lenti-EF1-CAR V2 Wpmut (shuttle plasmid) is as follows:

[0054] (1) The 5'LTR of the shuttle plasmid pRRLSIN.cPPT.PGK-GFP.WPRE (http: / / n2t.net / addgene:12252) is driven by the RSV promoter. Changing the promoter of the 5'LTR from RSV to CMV facilitates efficient transcription of viral genome sequences. The U3 region of the 3'LTR original plasmid has been removed (3'LTR△U3) and is designed for self-inactivation (SIN). The 3'LTR sequence remains unchanged.

[0055] (2) The hPGK promoter driving the expression of the exogenous transgene was replaced with the EF1 promoter, and the EGFP gene was replaced with a polyclonal sequence (SEQ ID NO. 10);

[0056] (3) The ampicillin resistance gene was replaced with the kanamycin resistance gene from the pUC57-Kan (GenBank: LT671993.1) plasmid;

[0057] (4) The WPRE element of pRRLSIN.cPPT.PGK-GFP.WPRE was modified to prevent the expression of the truncated protein X. Specifically, within the sequence of the wild-type WPRE element, the promoter driving the transcription of the protein X was deleted, and the A in the start codon ATG was changed to T. The new sequence was named WPREmut.

[0058] The modified shuttle plasmid was named Pre-Lenti-EF1-CAR V2 Wpmut, and its sequence is shown in SEQ ID NO. 11. A map of the shuttle plasmid is shown in FIG2 , and annotations in the map are shown in Table 1 below.

[0059] Table 1 Annotation of each element of the shuttle plasmid Pre-Lenti-EF1-CAR V2 WPmut

[0060] Example 2 Lentivirus packaging

[0061] 2.1 Preparation of packaging plasmid pMDLg-pRRE-K

[0062] The packaging plasmid is derived from the plasmid pMDLg-pRRE, originally constructed by Didier Trono Lab and stored at Addgene (www.addgene.org / 12251 / ). The original sequence of the plasmid, which confers ampicillin resistance, was replaced with the kanamycin resistance gene from the pET-28(a) plasmid. This modified packaging plasmid is named pMDLg-pRRE-K, and its sequence is shown in SEQ ID NO. 12. A map of the packaging plasmid pMDLg-pRRE-K is shown in Figure 3, and annotations are provided in Table 2.

[0063] Table 2

[0064] 2.2 Preparation of packaging plasmid pRsv-rev-kana-V2

[0065] The packaging plasmid is derived from the plasmid pRSV-rev, originally constructed by Didier Trono Lab and deposited at Addgene (www.addgene.org / 12253 / ). The original sequence of the plasmid, which conferred ampicillin resistance, was replaced with the kanamycin resistance gene from the pUC57-Kan (GenBank: LT671993.1) plasmid. The modified packaging plasmid is named pRsv-rev-kana-V2, and its sequence is shown in SEQ ID NO. 13. A map of the packaging plasmid pRsv-rev-kana-V2 is shown in Figure 4, with annotations in Table 3 below.

[0066] Table 3

[0067] 2.3 Preparation of envelope plasmid pMD2.GK

[0068] (1) Envelope plasmid pMD2.G, originally constructed by Didier Trono Lab and stored at Addgene (https: / / www.addgene.org / 12259 / ). Based on its sequence and backbone, its ampicillin resistance gene was replaced with the kanamycin resistance gene of the pET-28(a) plasmid;

[0069] (2) The modified envelope plasmid was named pMD2.GK, and its sequence is shown in SEQ ID NO.14.

[0070] (3) The map of the envelope plasmid is shown in Figure 5 and the annotations are shown in Table 4 below.

[0071] Table 4

[0072] 2.4 Preparation of Lentivirus

[0073] A third-generation vector and four-plasmid system was used for packaging. The Pre-Lenti-EF1-12C-ER-CAR V2WPmut shuttle plasmid was mixed with the packaging plasmids pMDLg-pRRE-K, pMD2.GK, and pRsv-rev-kana-V2 at a ratio of 7:5:3:5. This mixture was then added to a tube containing 293TS basal medium. Polyethylene glycol (PEI) was added to another tube at a ratio of 1:2: plasmid:PEI. After incubating each tube at room temperature for 5 minutes, the PEI-containing mixture was slowly added to the DNA-containing mixture. The tubes were gently shaken to mix thoroughly, and the mixture was incubated at room temperature for 15 minutes. The transfection mixture was then added dropwise to the 293TS (ATCC, Cat. No. CRL-3216) cell suspension. After 48 hours, the culture supernatant was collected to obtain a crude lentiviral solution. The lentiviral solution was then concentrated, aliquoted, and stored at 80°C. The transduction titer of lentivirus was determined by flow cytometry.

[0074] The determination of transduction titer includes the following steps:

[0075] The crude lentiviral solution was diluted serially and infected into 293T cells. After about 70 hours of infection, the CAR positive rate of the sample was detected by flow cytometry. The transduction titer (TU / mL) = dilution factor * sample positive rate * cell volume / virus loading volume (mL). The transduction titer was 4.21×10 7 TU / mL.

[0076] Example 3 Preparation of CD5 CAR-T cells with blocked CD5 expression

[0077] Peripheral blood was collected from healthy volunteers and purified using Miltenyi CD3 Regent to obtain CD3+ T cells. The cells were grown in TexMACS GMP Medium (containing 5% serum replacement) and activated using transact CD3 / CD28 reagent (1:100) and IL-2 (100 IU / mL). After 24 hours of activation, the cells were infected with lentivirus.

[0078] Example 4 CD5 expression blocking CD5 CAR-T CAR positive rate and CD5 molecule expression on the cell membrane

[0079] Primary T cells were transduced with lentiviral vectors, and flow cytometry was used to assess the CD5 CAR-T CAR positivity and CD5 molecule expression on the cell membrane. The results are shown in Figure 6 , demonstrating that the CAR positivity rate of 12C.b CAR-T cells was nearly 100% (Figure 6, A). 12C-ER (a 12C single-domain antibody sequence linked to an ER retention domain) effectively blocked CD5 expression on T cells, while the surface CD5 positivity rate on T cells not transfected with the CD5-blocking virus was 99.5% (Figure 6, B), indicating that 12C-ER can bind to CD5 intracellularly and retain it in the endoplasmic reticulum. Therefore, this CD5 expression blocking technology can be used to block CD5 expression on cells and prevent the suicide phenomenon of targeted CD5 CAR-T cells.

[0080] Example 5 In vitro killing of CD5-positive tumor cells by CD5 CAR-T cells with blocked CD5 expression

[0081] Cytotoxicity of CD5 CAR-T cells with blocked CD5 expression against CD5-positive acute lymphoblastic leukemia cells (CCRF-CEM)

[0082] CD5 CAR-T cells with blocked CD5 expression were used to perform an in vitro cytotoxicity assay against CCRF-CEM acute lymphoblastic leukemia cells. CCRF-CEM cells were co-incubated with these cells at effector-target ratios of 4:1, 2:1, and 1:1 for 16 hours. LDH release assays were used to assess the cytotoxicity of 12C.b CAR-T cells. The results, shown in Figure 7, show that compared to the control group, CD5 CAR-T cells demonstrated significant specific cytotoxicity against CCRF-CEM cells at various effector-target ratios.

[0083] 5.2 Cytokine secretion after incubation of CD5 CAR-T cells with blocked CD5 expression with CCRF-CEM cells

[0084] The supernatant was collected after killing, and IFN-γ secretion after 12C.b CAR-T cells killed CCRF-CEM cells was measured by ELISA. As shown in Figure 8, there was essentially no IFN-γ secretion after T cells were co-incubated with CCRF-CEM cells, while IFN-γ secretion was significantly increased after 12C.b CAR-T cells killed CCRF-CEM cells.

[0085] Example 6 In vivo anti-tumor effect of CD5 CAR-T cells with blocked CD5 expression

[0086] To evaluate the in vivo anti-tumor activity of CD5 CAR-T cells with blocked CD5 expression, a mouse xenograft model was established using the T-ALL cell line Jurkat cells (cellcook, catalog number: CC1902). Jurkat-luc cells were cultured at 3×10 6 Eleven days after the cells were injected into the tail vein of NSG mice, all animals were imaged. All tumor-bearing mice were included in the experimental group and randomly divided into three groups (4 mice in each group), namely the cryopreservation solution group (Control), the Mock T cell group, and the CD5 CAR-T cell group. The cryopreservation solution group was injected with the cryopreservation solution (Control) by the tail vein, and the Mock T cell group was injected with the 1×10 7 cells, and the CD5 CAR-T cell group was injected into the tail vein with 1×10 7 Cells (CD5 CAR T cells with blocked CD5 expression prepared in Example 3)

[0087] On Days 3, 7, 12, 17, 24, and 33 after treatment, mice were anesthetized and intraperitoneally injected with Luciferase substrate. Tumor burden was observed using a small animal in vivo imaging system, as shown in Figure 9. The results show that almost no tumor cells were imaged in the CD5 CAR-T cell treatment group, while mice in the cryopreserved solution group and the mock T cell group had severe tumor burden, indicating that CD5 CAR-T cells can effectively eliminate CD5-positive tumor cells and have a significant anti-tumor effect in vivo.

Claims

1. A chimeric antigen receptor targeting CD5 molecule, characterized in that: The chimeric antigen receptor includes a 12C single domain antibody; the 12C single domain antibody includes heavy chain variable regions CDR1, CDR2 and CDR3; the amino acid sequences of CDR1, CDR2 and CDR3 are shown in SEQ ID NO.15-SEQ ID NO.

17.

2. The chimeric antigen receptor according to claim 1, characterized in that The amino acid sequence of the 12C single-domain antibody is SEQ ID NO.1 or a sequence having 85% homology with SEQ ID NO.

1.

3. The chimeric antigen receptor according to claim 1, characterized in that The chimeric antigen receptor further comprises a signal peptide, an extracellular region, a transmembrane region and / or a cytoplasmic region.

4. The chimeric antigen receptor according to claim 3, characterized in that The chimeric antigen receptor further comprises a self-cleavage region and / or a retention domain.

5. The chimeric antigen receptor according to any one of claims 1 to 4, characterized in that: The chimeric antigen receptor is obtained by serially connecting the following modules: a signal peptide of a CD8 molecule, a 12C single domain antibody, a CD8 Hinge+TM region, a 4-1BB cytoplasmic region, a cytoplasmic region of a CD3ζ molecule, a T2A self-cleavage region, a 12C single domain antibody, and an ER retention domain.

6. The chimeric antigen receptor according to claim 5, characterized in that The amino acid sequence of the signal peptide of the CD8 molecule is shown in SEQ ID NO.2; the amino acid sequence of the CD8 Hinge+TM region is shown in SEQ ID NO.3; the amino acid sequence of the 4-1BB cytoplasmic region is shown in SEQ ID NO.4; the amino acid sequence of the cytoplasmic region of the CD3ζ molecule is shown in SEQ ID NO.5; the amino acid sequence of the T2A self-cleavage region is shown in SEQ ID NO.6; and the amino acid sequence of the ER retention domain is shown in SEQ ID NO.

7.

7. The chimeric antigen receptor according to claim 6, characterized in that The amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO.

9.

8. A nucleic acid encoding the chimeric antigen receptor according to any one of claims 1 to 7.

9. The nucleic acid according to claim 8, characterized in that The sequence of the nucleic acid is shown in SEQ ID NO.8 or a sequence having more than 80% sequence homology with SEQ ID NO.

8.

10. A modified T cell, characterized in that The T cell comprises, expresses and / or secretes the chimeric antigen receptor according to any one of claims 1-7.

11. The T cell according to claim 10, characterized in that The T cells are CAR-T cells.

12. A cell culture comprising the T cells according to any one of claims 10 to 11, characterized in that: The cell culture contains a chimeric antigen receptor targeting CD5 molecules.

13. A medicament comprising the chimeric antigen receptor according to any one of claims 1 to 7, the T cell according to any one of claims 10 to 11, or the cell culture according to claim 12.

14. The drug according to claim 13, characterized in that The medicine also includes pharmaceutically acceptable excipients.

15. Use of the chimeric antigen receptor according to any one of claims 1 to 7, the T cell according to any one of claims 10 to 11, or the cell culture according to claim 12 in the preparation of an anti-tumor drug.

16. The use according to claim 15, characterized in that The tumors include T-cell malignancies.

17. Use of the chimeric antigen receptor according to any one of claims 1 to 7, the T cell according to any one of claims 10 to 11, or the cell culture according to claim 12 in the preparation of a method for preventing and / or treating an immune disease.

18. The use according to claim 17, characterized in that The immune diseases include rheumatoid arthritis, systemic lupus erythematosus, type I diabetes and / or myasthenia gravis.

19. A CD5 detection kit, characterized in that: The method comprises the chimeric antigen receptor according to any one of claims 1 to 7, the T cell according to any one of claims 10 to 11, or the cell culture according to claim 12.

20. A CD5 detection method, characterized in that: The method comprises the chimeric antigen receptor according to any one of claims 1 to 7, the T cell according to any one of claims 10 to 11, the cell culture according to claim 12, or the kit according to claim 19, wherein the method is a non-disease diagnosis or treatment method.

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