A method for preparing chimeric antigen receptor T cells based on dual-target joint recognition of target cells and its application

Abstract

This application discloses a chimeric antigen receptor T cell based on dual-target joint recognition of target cells, a dual-target chimeric antigen receptor, its preparation method, and its application. The T lymphocytes express a dual-target chimeric antigen receptor on their surface. This dual-target chimeric antigen receptor targets a first target and a second target on the target cell, respectively. The first chimeric antigen receptor is constitutively expressed in the T lymphocytes, and the second chimeric antigen receptor is inducibly expressed in the T lymphocytes. After the T lymphocytes come into contact with the target cells, the second chimeric antigen receptor is induced to express on the T lymphocytes upon antigen stimulation. When the first and second chimeric antigen receptors simultaneously recognize the first and second targets, the T lymphocytes are fully activated and exert their effector function.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and more specifically, to a method for T cells to precisely target target cells, such as tumor cells, by dual-target joint recognition of antigen stimulation-induced endogenous transcriptional cascade reactions, chimeric antigen receptor T cells, and their preparation methods. Background Technology

[0002] Chimeric antigen receptor (CAR) T-cell therapy has shown great promise in cancer treatment and has been successfully applied to treat refractory or relapsed B-cell malignancies. Although CAR T-cell therapy has demonstrated significant efficacy in treating B-cell malignancies, its clinical application remains greatly limited by the lack of tumor-specific CAR targets, especially for solid tumors. Currently, no ideal target for solid tumors has been found as in hematologic malignancies as CD19. Most CAR targets for solid tumors currently under evaluation, such as HER2 and EGFR, are tumor-associated antigens (TAAs), which are highly expressed in tumor tissues but also expressed at low levels in normal tissues. While CAR-T cells targeting TAs kill tumor cells, they may also attack normal tissues, causing off-target toxicity. The safety issues resulting from off-target effects greatly limit the clinical efficacy and widespread application of CAR-T cell therapy. Therefore, there is an urgent need to develop new methods that enable T cells to more precisely target tumor cells.

[0003] Numerous preclinical studies are exploring and developing more effective strategies to improve the target safety of solid tumor therapy. Utilizing synthetic biology techniques to design next-generation CARs is a crucial approach to addressing the current challenges of CAR-T therapy in solid tumor treatment. Regarding the safety issues of CAR target recognition, combining two tumor-associated antigens into a logical AND-gate, where T cells are fully activated only when both antigens are present, is an effective strategy. Previously developed logical AND-gate tumor targeting strategies split the CAR signaling domain into two parts, each connected to a unique extracellular antigen recognition domain. However, to achieve good differentiation between tumor cells and normal cells, a delicate balance of signaling activity between these two receptors is required, necessitating extensive CAR structural adjustments and optimizations to ensure T cell activation only through dual antigen recognition, resulting in poor stability and reliability. Therefore, the search for new and reliable logical AND-gate tumor targeting strategies is essential to promote the widespread application of CAR T-cell therapy.

[0004] In addition to targeting and eliminating tumor cells, CAR T cells can be used to precisely eliminate various types of abnormal pathogenic cells in the body, including senescent cells, fibrotic cells, and pathogenic autoimmune cells. Currently, CAR T cell therapy shows great application potential in areas such as aging, fibrotic diseases, and autoimmune diseases; however, its application is still limited by the scarcity of targets for these disease types and the poor target specificity. Using a multi-target combined targeting strategy with logical AND gates can improve the targeting accuracy of CAR T cells and enhance their safety in clinical application, thereby potentially promoting the widespread application of CAR T cell therapy in these disease types.

[0005] Cellular senescence is characterized by stable cell cycle arrest and the secretion of various senescence-related inflammatory factors. Evidence suggests that the abnormal accumulation of senescent cells in vivo can induce chronic tissue damage and ultimately lead to various diseases, such as liver fibrosis, pulmonary fibrosis, atherosclerosis, diabetes, osteoarthritis, age-related macular degeneration, and neurodegenerative diseases. Specific clearance of senescent cells in vivo holds promise for treating these diseases, and current research often employs small molecule compounds to target senescent cells for therapeutic purposes. In addition, specifically targeting and clearing senescent cells using chimeric antigen receptor T cells (CAR T cells) is a promising strategy. Studies have shown that senescent cells express surface markers such as uPAR; CAR T cells targeting these surface markers can effectively clear senescent cells both in vitro and in vivo, thus achieving therapeutic effects. However, current CAR T cell therapies targeting senescent cells are still greatly limited by the lack of specific senescent cell surface markers, which may lead to off-target risks, significantly restricting their clinical application. Combining multiple senescence-related targets into a logical AND gate to target senescent cells can greatly improve the precision of CAR T cell targeting and enhance the safety of clinical applications. The dual-target combined recognition chimeric antigen receptor method developed in this study enables T cells to more precisely target senescent cells, and is expected to be applied to a variety of age-related diseases, such as liver fibrosis, pulmonary fibrosis, atherosclerosis, diabetes, osteoarthritis, age-related macular degeneration, and neurodegenerative diseases.

[0006] Fibrosis-related diseases encompass a wide range of clinical types, including systemic fibrosis (such as systemic scleroderma) and multi-organ-specific diseases (such as myocardial fibrosis, pulmonary fibrosis, liver fibrosis, and renal fibrosis), and are a major health problem worldwide. Although the pathogenesis of these diseases differs, they share a common characteristic: uncontrolled and progressive abnormal accumulation of collagen fibers in the affected organ tissues, leading to organ dysfunction and eventual failure. Currently, there are no effective treatments for fibrosis-related diseases in clinical practice. Existing research suggests that abnormally activated myofibroblasts play a significant role in promoting the occurrence and development of fibrosis. Targeting and eliminating abnormal pro-fibrotic cells in tissues is a highly promising anti-fibrotic strategy. Using CAR T cells to specifically target and eliminate abnormal pro-fibrotic cells in vivo is a promising anti-fibrotic treatment. Studies have shown that using FAP-targeted CAR T cells in mouse models can effectively alleviate myocardial fibrosis and promote disease recovery. However, this treatment method is still limited by the specificity of CAR targets. The dual-target combined recognition chimeric antigen receptor method developed in this study can greatly improve the accuracy of CAR T cell targeting, thereby ensuring the application prospects of CAR T cell therapy in the treatment of antifibrotic diseases.

[0007] Autoimmune diseases are a series of chronic illnesses originating from an abnormally activated autoimmune system, affecting specific organs (e.g., type 1 diabetes) or multiple organ systems (e.g., systemic lupus erythematosus), manifesting as autoimmune intolerance and leading to tissue damage. Despite significant progress in understanding the immunological and pathological characteristics of autoimmune diseases, long-term remission treatments remain lacking in clinical practice. CAR T-cell therapy, which targets and eliminates autoreactive immune cells such as B cells or antibody-secreting plasma cells, holds promise for achieving long-term remission in some autoimmune diseases. Previous studies have demonstrated antibody-mediated autoimmune disease treatment by targeting specific antigen-specific B-cell receptors with CARs, enabling CAR T-cells to specifically kill pathogenic B cells secreted by autoreactive antibodies. The precise dual-target chimeric antigen receptor identification method developed in this study improves the safety of CAR T-cell targeting, potentially expanding the application of CAR therapy in autoimmune diseases mediated by abnormal autoreactive immune cells (e.g., pemphigusa, systemic lupus erythematosus, type 1 diabetes, rheumatoid arthritis). Summary of the Invention

[0008] The inventors attempted to use synthetic biology techniques to design novel gene expression circuits based on endogenous transcription factors of T cells, thereby achieving precise dual-target recognition of T cells using a logical AND gate. To enable T cells to achieve highly precise tumor recognition using a logical AND gate, designing a T cell antigen-sensing and response system is crucial. This system relies on a stimulus-induced transcription system. This system typically depends on environmentally sensitive response promoters, primarily containing a series of transcription factor response elements (TREs). The most typical example of transcriptional regulation on T cells is that upon antigen activation, T cells begin to express a series of killing and proliferation-related effector molecules. This is a transcription factor-mediated gene-inducing expression process. After T cell activation, several core transcription factors, including NFκB, AP1, and NFAT, are activated, binding to their respective transcriptional response elements to initiate gene transcription and expression, thereby activating the expression of related effector molecules. Furthermore, CD28 co-stimulatory signals promote the expression of large amounts of the IL-2 cytokine and induce the expression of some co-stimulatory molecules such as ICOS; the mechanism of this is transcription factor-mediated downstream gene-inducing expression. Numerous studies have demonstrated that unique signals transduced by the CD28 intracellular signal transduction domain promote downstream responses. A sequence called the CD28 Responsive Element (RE) is located proximal to the IL-2 promoter. This RE, along with the adjacent NF-IL-2B site, forms the RE / AP element, which has been shown to play a crucial role in CD28-controlled IL-2 release. Regulatory elements in the IL-2 promoter region, such as NFAT, AP1, NFκB, and RE / AP, are essential for CD28-dependent IL-2 induction. In this study, the inventors primarily drew upon the mechanism of IL-2 induction at the transcriptional level by CD28 co-stimulatory molecules in T cells. By selecting and combining core T cell endogenous transcription factor responsive elements, they constructed a synthetic inducible promoter, reshaping an endogenous transcription factor-based gene induction expression system. Based on this, they designed a novel endogenous induction dual-target co-recognition CAR, enabling highly precise targeting of tumor cells using an AND gate strategy applied to T cells.

[0009] In this study, the inventors first used two relatively well-established targets, CD19 and HER2, as model targets to construct tumor cell lines expressing antigens individually and co-expressing them, and designed a novel induced co-recognition CAR targeting these two targets. In vitro, the inventors mainly used flow cytometry, T-cell killing assays, and ELISA to demonstrate that the newly designed CAR possesses precise dual-target co-recognition capability and the ability to induce expression of antigen-stimulated cofactors. Simultaneously, the inventors constructed an immunodeficient mouse tumorigenesis model to demonstrate the in vivo anti-tumor effect of the novel co-recognition CAR with precise dual-target antigen recognition. Based on this, the inventors selected NKG2D ligand and EGFR as two candidate target combinations for clinical translation, and conducted in vitro and in vivo experiments to verify the precise recognition capability of this dual-target co-recognition CAR. The inventors' experimental results show that the novel co-recognition CAR has a high-resolution recognition capability for both single-target and dual-target cells. By co-targeting tumor-associated antigens co-expressed on tumor cells, the specificity of tumor targeting can be greatly improved. The results of this study provide a new method for achieving precise tumor recognition and improving the safety of T-cell infusion therapy.

[0010] Specifically, the applicant has solved the technical problem in this field through the following technical solutions.

[0011] 1. A dual-target chimeric antigen receptor, comprising a first chimeric antigen receptor and a second chimeric antigen receptor, wherein the first chimeric antigen receptor and the second chimeric antigen receptor target a first target and a second target on a target cell, respectively; the first chimeric antigen receptor is constitutively expressed in T lymphocytes after being modified by the dual-target chimeric antigen receptor; wherein after the T lymphocytes come into contact with the target cells, the second chimeric antigen receptor is induced to express on the T lymphocytes upon antigen stimulation; wherein the first chimeric antigen receptor and the second chimeric antigen receptor, upon simultaneously recognizing the first target and the second target, fully activate the T lymphocytes to exert effector functions; preferably, the target cells are selected from tumor cells, senescent cells, abnormal fibrotic cells, and abnormal immune cells.

[0012] 2. The dual-target chimeric antigen receptor described in Project 1, wherein the first chimeric antigen receptor includes an extracellular recognition region for recognizing a first target and a co-stimulatory molecular domain, and the second chimeric antigen receptor includes an extracellular recognition region for recognizing a second target and an intracellular CD3ζ domain.

[0013] 3. The dual-target chimeric antigen receptor described in Project 2, wherein the extracellular recognition region for recognizing the first target is a single-chain antibody or receptor, and the extracellular recognition region for recognizing the second target is a single-chain antibody or receptor.

[0014] 4. The dual-target chimeric antigen receptor described in Project 2 or 3, wherein the co-stimulatory molecular domain is selected from the CD28 co-stimulatory molecular domain, the 4-1BB co-stimulatory molecular domain, and combinations thereof.

[0015] 5. The dual-target chimeric antigen receptor of Project 1, wherein the first target and the second target are different and independently selected from CD19, HER2, NKG2D, and EGFR. Preferably, the first chimeric antigen receptor comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:1, and the second chimeric antigen receptor comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:2.

[0016] 6. The polynucleotide encoding any one of the dual-target chimeric antigen receptors described in items 1-5.

[0017] 7. The polynucleotide described in Project 6 comprises module A and module B, wherein module A comprises a constitutive promoter, an extracellular recognition region coding sequence for recognizing a first target, and a co-stimulatory molecular domain coding sequence; and module B comprises an inducible synthetic promoter, an extracellular recognition region coding sequence for recognizing a second target, and a CD3ζ intracellular domain coding sequence.

[0018] 8. The polynucleotide of item 7, wherein module A further comprises a 5' long terminal repeat (5'LTR) and a 3' long terminal repeat (3'LTR), optionally, module B further comprises a 5' long terminal repeat (5'LTR) and a 3' long terminal repeat (3'LTR), preferably, the nucleotide sequence of module A is as shown in SEQ ID NO:3 and the nucleotide sequence of module B contains or is as shown in SEQ ID NO:4; or the nucleotide sequence of module A contains or is as shown in SEQ ID NO:7 and the nucleotide sequence of module B contains or is as shown in SEQ ID NO:8.

[0019] 9. The polynucleotide of Item 7, wherein the synthetic promoter comprises a minimal promoter of the TATA box and any one of the following groups: one or more copies of the NFAT reaction element (NFAT-RE), one or more copies of the CD28 reaction element (CD28RE) / NF-IL-2BAP1 site, and one or more copies of the AP1 reaction element (AP1-RE), and combinations thereof.

[0020] 10. The polynucleotide of Item 9, wherein the synthetic promoter comprises a minimal promoter containing a TATA box and three copies of the NFAT reaction element (NFAT-RE), three copies of the CD28 reaction element (CD28RE) / NF-IL-2B AP1 site, and three copies of the AP1 reaction element (AP1-RE).

[0021] 11. An expression vector comprising any one of the polynucleotides in items 6-10, preferably, the expression vector being a lentiviral vector.

[0022] 12. A modified T lymphocyte that expresses a dual-target chimeric antigen receptor of any one of items 1-5 or is transformed with the expression vector of item 11.

[0023] 13. A synthetic promoter comprising: 3 copies of NFAT reaction element (NFAT-RE), 3 copies of CD28 reaction element (CD28RE) / NF-IL-2B AP1 site (CD28-RE / AP), 5 copies of AP1 reaction element (AP1-RE), and a minimal promoter containing a TATA box.

[0024] 14. The synthetic promoter of item 13, wherein the sequences of the three copies of the NFAT reaction element (NFAT-RE) comprise or are as shown in SEQ ID NO:10, the sequences of the three copies of the CD28 reaction element (CD28RE) / NF-IL-2B AP1 site (CD28-RE / AP) comprise or are as shown in SEQ ID NO:11, and the sequences of the five copies of the AP1 reaction element (AP1-RE) comprise or are as shown in SEQ ID NO:12.

[0025] 15. The synthetic promoter described in Item 14, the sequence of which comprises or constitutes SEQ ID NO:9.

[0026] 16. A method for preparing dual-target chimeric antigen receptor-modified T lymphocytes, comprising the following steps:

[0027] The expression vector of Project 11 was introduced into isolated and activated T cells;

[0028] The infected T cells were expanded, cultured, and tested.

[0029] T lymphocytes modified with dual-target chimeric antigen receptors were isolated.

[0030] 17. Use of the modified T lymphocytes of Item 12 in the preparation of medicaments for treating diseases, wherein the diseases are selected from malignant tumors, age-related diseases such as atherosclerosis, diabetes, osteoarthritis, age-related macular degeneration and neurodegenerative diseases, fibrosis-related diseases such as myocardial fibrosis, pulmonary fibrosis, liver fibrosis, kidney fibrosis, and autoimmune diseases such as herpes zoster, systemic lupus erythematosus, type I diabetes, and rheumatoid arthritis. Attached Figure Description

[0031] Figure 1 The design and characteristics of the ETC logic AND gate dual-identification CAR system are shown.

[0032] (A) Schematic diagram of the design and principle of the ETC logic AND gate dual-recognition CAR. A conventional CAR is divided into module A, which targets antigen a, and module B, which targets antigen b, containing the CD28 co-stimulatory domain and the CD3ζ domain, respectively. Module A is expressed constantly, while module B is induced. The inventors designed an inducible synthetic promoter that responds to endogenous transcription factors, thereby driving module B to be expressed in an antigen-stimulation-dependent manner. First, the binding of module A not only provides a co-stimulatory signal but also facilitates close contact between T cells and tumor cells. The binding of the two antigens a and b induces a strongly amplified transcriptional cascade reaction within T cells, thereby driving the high-level stable expression of CAR module B. The combined action of A and B further promotes the formation of a stable and strong immune synapse between T cells and tumor cells, ultimately integrating the co-stimulatory signal and the CD3ζ signal to achieve complete T cell activation.

[0033] (B) Schematic diagram of the logical AND gate strategy for precise tumor identification. The joint participation of target a and target b enables the integration of T cell activation signals and precise killing.

[0034] (C) Schematic diagram of the ETC logic AND gate dual-recognition CAR design targeting CD19 / HER2 dual antigens. This dual-recognition CAR design contains two modular vectors: Module A, containing FMC63-scFv targeting CD19, which is linked to the CD28 and 4-1BB co-stimulatory molecular domains and driven by the EF-1α promoter; Module B, containing 4D5-scFv recognizing HER2, which is linked to the CD3ζ intracellular domain and driven by an inducible synthesis promoter for antigen stimulation-dependent expression; for ease of detection, the v5 tag and myc tag are introduced into the vectors of Module A and B, respectively. The synthetic promoter in Module B consists of: 3 copies of NFAT response element (NFAT-RE), 3 copies of CD28 response element (CD28RE) / NF-IL-2B AP1 site (CD28-RE / AP), 5 copies of AP1 response element (AP1-RE), and a minimal promoter containing a TATA box.

[0035] (D) Flow cytometry scatter plots show the results of co-culturing ETC AND-gated CAR cells with K562 cells expressing different antigen forms for 24 hours. K, K562 cells; K.19, K562 cells overexpressing CD19; KH, K562 cells overexpressing HER2; K.19H, K562 cells overexpressing both CD19 and HER2. Myc tag detection was used to reflect the expression level induced by antigen stimulation of CAR module B.

[0036] (E) Statistical results of FACS detection show the antigen stimulation-induced expression efficiency.

[0037] The above data results are plotted as mean plus standard deviation (SD), n = 4 different donor samples; **p<0.01, ***p<0.001, ****p<0.0001 (Student's t test).

[0038] Figure 2 This demonstrates that the ETC AND gate CAR has the ability to accurately identify two targets together.

[0039] (A) Schematic diagram of CAR module B using scFvs with different affinities targeting HER2. The tN.Hz vector uses high-affinity scFv 4D5; the tN7.Hz vector uses medium-affinity scFv 4D5-7; and the tN5.Hz vector uses low-affinity scFv 4D5-5. A truncated nerve growth factor receptor (tNGFR) driven by the PGK promoter was introduced into the three vectors for detection.

[0040] (B) The cytotoxic effect of T cells transfected with three different affinity ETC AND-gate CARs (BB.tN, high affinity; BB.tN7, medium affinity; BB.tN5, low affinity) on GFP / luciferase-labeled HCT-116 cells (HCT116). HCT116, HER2 single target positive; HCT116-CD19, CD19 / HER2 dual target positive; UTD, co-cultured with untransfected control T cells; CAR, co-cultured with AND-gate CAR cells.

[0041] (C) Killing effect of ETC AND-gate CAR T cells with three different affinities on GFP / luciferase-labeled MDA-MB-231 cells (MDA231). MDA231, HER2 single target positive; MDA231-CD19, CD19 / HER2 dual target positive; UTD, co-cultured with untransfected control T cells; CAR, co-cultured with AND-gate CAR cells.

[0042] (D) The killing effect of ETC AND-gated CAR T cells with three different affinities on GFP / luciferase-labeled NIH / 3T3 cells (3T3). 3T3-19, CD19 single-target positive 3T3 cells; 3T3-H, HER2 single-target positive 3T3 cells; 3T3-19H, CD19 / HER2 dual-target positive 3T3 cells; 3T3, dual-target negative 3T3 cells.

[0043] (E) ELISA results showed the levels of IL-2 and TNF-α production in ETC AND gated CART cells after 24 hours of co-culturing with 3T3 cells expressing different antigens.

[0044] (F) Expansion of ETC AND-gated CAR T cells after repeated rounds of stimulation with 3T3 cells expressing different antigens. BB.tN7, ETC AND-gated CAR containing intermediate-affinity scFv; arrows indicate each 3T3 cell stimulation.

[0045] The above data results are plotted as mean plus standard deviation (SD), n=3, ns, not significant; *p<0.05, **p<0.01, ***p<0.001 (Student's t test).

[0046] Figure 3 The ETC AND gate antigen-inducible expression system was demonstrated for T-cell loading of therapeutic factors.

[0047] (A) Schematic diagram of the ETC AND-gate CAR inducible expression vector for T cells loaded with IL-2 and TRAIL. tN7-IL2, loaded with IL-2, induces IL-2 expression through AND-gate antigen stimulation; tN7-TRAIL, loaded with TRAIL, induces TRAIL expression through AND-gate antigen stimulation.

[0048] (B) Schematic diagram of T cell antigen-dependent therapeutic payload induction expression using the ETC AND gate induction system.

[0049] (C) Results of antigen-dependent efficacy therapeutic load induction expression assay. ETC AND gated CAR T cells loaded with TRAIL or IL-2 were stimulated with K562 cells (CD19 / HER2 double negative) or K562-19H cells (CD19 / HER2 double positive) for 24 hours, and anti-TRAIL and myc-tagged flow cytometry antibodies were used to detect the induction expression level.

[0050] (D) Flow cytometry results of the killing effect of untransfected control T cells (UTD), ETC-gated CAR T cells (BB.tN7), and TRAIL-loaded AND-gated CAR T cells (BB.tN7-TRAIL) on dual-target positive K562 cells. Dual-positive K562 cells were pre-labeled with CFSE (green fluorescent dye) and then co-cultured with different T cells at an effector-target ratio of 1:1 for 24 hours. Changes in K562 cell content were detected by flow cytometry.

[0051] (E) Statistical results of killing of dual-target positive K562 cells by untransfected control T cells (UTD), ETC AND-gated CAR T cells (BB.tN7), and TRAIL-loaded AND-gated CAR T cells (BB.tN7-TRAIL).

[0052] The above data results are plotted as mean plus standard deviation (SD), n=3; **p<0.01, ***p<0.001 (Student's t test).

[0053] Figure 4 The ETC AND gated CAR is shown to target NKG2D ligand and EGFR dual antigen.

[0054] (A) Schematic diagram of an AND-gate CAR vector design targeting NKG2D ligand and EGFR dual antigen. The CAR consists of two parts: Module A (vector NK-BB) contains the NKG2D extracellular domain (NKG2D-EXT) that recognizes the NKG2D ligand, which is co-stimulated with CD28 and 4-1BB; Module B (vector EIL2) contains the scFv that recognizes EGFR, which is linked to the CD3ζ intracellular domain and loaded with IL-2 factor.

[0055] (B) Flow cytometry detection of 3T3 cells expressing different antigens. 3T3, natural 3T3 cells; 3T3.A, 3T3 cells overexpressing MICA (a type of NKG2D ligand); 3T3.E, 3T3 cells overexpressing EGFR target; 3T3.EA, 3T3 cells overexpressing both MICA and EGFR target.

[0056] (C) Fluorescence image showing changes in 3T3 target cells expressing different EGFP-labeled antigens after co-culturing with AND-type CAR T cells. The number of EGFP-positive target cells was observed under a fluorescence microscope 3 days after co-culturing transfected CAR T cells with target cells; scale bar, 200 μm.

[0057] (D) ETC-gated CAR T cells (NK.EIL2) and untransfected control T cells (UTD) were co-cultured with 3T3 cells expressing different antigens for 24 hours and the killing results were detected.

[0058] (E)ETC gated CAR T cells (NK.EIL2) were co-cultured with 3T3 cells expressing different antigens for 24 hours to detect cytokine release levels.

[0059] (F) Detection of CD107a degranulation activation of T cells after co-culturing with 3T3 cells expressing different antigens. The flow cytometry histogram of CD107a expression (left) and the statistical expression results (right) both show that only T cells transfected after stimulation with dual-target positive cells showed significant degranulation activation.

[0060] The above data results are plotted as mean plus standard deviation (SD), n=3. ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (Student's t-test).

[0061] Figure 5 This study demonstrated the dual-target selective antitumor effect of ETC-gated CAR T cells in vivo.

[0062] (A) Schematic diagram of mouse model and experimental design used for in vivo efficacy evaluation. Severely immunodeficient mice (NPG mice) were subcutaneously inoculated with MDA-MB-231 cells (MDA231, HER2 single positive) or MDA-MB-231 cells overexpressing CD19 (MDA231-CD19, CD19 / HER2 double positive), and T-cell therapy was administered via tail vein infusion on day 14.

[0063] (B) Tumor growth curves in mice receiving CD19 / HER2-gated CAR T cells (BB.tN7) showed that CD19 / HER2 double-positive tumor growth was significantly inhibited. Data are plotted as mean plus standard error (SEM), n = 6 mice.

[0064] (C) Tumor growth curves in mice treated with untransfected control T cells showed no significant difference between the two groups. Data are plotted as mean plus standard error (SEM), n = 6 mice.

[0065] (D) There was no significant difference in the change of standardized body weight over time between the two groups of mice that received transfected T cells. Data are plotted as mean plus standard error (SEM), n = 6 mice.

[0066] (E) Kaplan-Meier survival curve analysis showed that mice inoculated with dual-target positive tumors (MDA231-CD19, red line) had significantly better survival rates than mice inoculated with a single-target positive tumor (MDA231, black line). **p<0.01 (log-rank Mantel–Cox test)

[0067] (F) H&E staining results showed that liver metastasis was observed in mice in the single antigen tumor inoculation group (MDA231) after receiving AND-gate CAR T-cell therapy, while no liver metastasis was observed in mice in the dual-target tumor inoculation group (MDA231-CD19) after treatment. Yellow arrows indicate tumor lesions, scale bar = 200 μm.

[0068] (G) Immunohistochemical staining of tumor tissue sections revealed that CD3-positive T cells infiltrated the dual-target positive tumor (MDA231-CD19) but not the single-target positive tumor (MDA231). Scale bar = 50 μm.

[0069] Figure 6 This demonstrates the ETC AND gate CAR targeting CD19 / HER2 dual antigen.

[0070] (A) Schematic diagram of the truncated CD19 and HER2 structures used for antigen overexpression. SP, signal peptide; EXT, extracellular domain; TM, transmembrane domain.

[0071] (B) Flow cytometry results of K562 cells overexpressing different antigens. K, K562 cells; K.19, K562 cells overexpressing truncated CD19; KH, K562 cells overexpressing truncated HER2; K.19H, K562 cells overexpressing both truncated CD19 and HER2.

[0072] (C) ETC-gated CAR T cells were co-cultured with K562 cells expressing different antigens for 4 hours, 10 hours, and 24 hours to detect the CAR module-induced expression level. K, K562 cells; K.19, K562 cells overexpressing truncated CD19; KH, K562 cells overexpressing truncated HER2; K.19H, K562 cells overexpressing both truncated CD19 and HER2; Myc tag detection was used to reflect the antigen-stimulated CAR-induced expression level.

[0073] (D) The expression level of CAR T-cell antigen stimulation induced by ETC gate changes over time.

[0074] Figure 7 Demonstrates AND-gated CAR and target cell antigen detection targeting CD19 / HER2.

[0075] (A) Flow cytometry analysis of T cells transfected with three different affinity AND-gated CARs and the induction of expression after co-culturing with K562 cells expressing different antigens. V5 and tNGFR tags were used to reflect the basal expression level after T cell co-transfection, and Myc tag was used to reflect the CAR-induced expression level after antigen stimulation. K, K562 cells; K.19, K562 cells overexpressing truncated CD19; KH, K562 cells overexpressing truncated HER2; K.19H, K562 cells overexpressing both truncated CD19 and HER2.

[0076] (B) Flow cytometry analysis of CD19 and HER2 antigen expression in HCT-116 and MDA-MB-231 cells before and after overexpression of truncated CD19 target. HCT116, native HCT116 cells; MDA231, native MDA-MB-231 cells; HCT116-CD19, HCT116 cells overexpressing CD19; MDA231-CD19, MDA-MB-231 cells overexpressing CD19; Isotype, using isotype-matched fluorescent dye-conjugated immunoglobulin (Ig) antibody as a control.

[0077] (C) Flow cytometry analysis of CD19 and HER2 dual-target expression in NIH / 3T3 cells (3T3) after overexpression of different antigens. 3T3, native 3T3 cells; 3T3.19, 3T3 cells overexpressing truncated CD19; 3T3.H, 3T3 cells overexpressing truncated HER2; 3T3.19H, 3T3 cells overexpressing both truncated CD19 and HER2 dual targets.

[0078] Figure 8 The schematic diagram shows the structure of the NKG2D ligand and EGFR dual antigen detection and overexpression vector.

[0079] (A) Detection of NKG2D ligand expression levels in in vitro cultured tumor cell lines. All four different types of human tumor cell lines were NKG2D ligand positive, while mouse-derived NIH / 3T3 cells were NKG2D ligand negative. NKG2D ligand detection was performed using NKG2D-human Fc chimeric protein (R&D) and anti-human Fc fluorescent secondary antibody staining, followed by flow cytometry.

[0080] (B) EGFR expression was positive in all three different types of tumor cell lines.

[0081] (C) Schematic diagram of the structure of overexpressed NKG2D ligand (MICA) and truncated EGFR antigen. SP, signal peptide; EXT, extracellular domain; TM, transmembrane domain; ICD, intracellular domain.

[0082] Figure 9This demonstrates the anti-tumor effect of ETC AND-gated CAR T cells in a unilateral tumor-bearing mouse model.

[0083] (A) & (B) Flow cytometry analysis of CAR T cell (CD3+) levels in peripheral blood of mice with single-target tumors (MDA231) and dual-target tumors (MDA231-CD19) on day 17 after CAR T cell infusion. + NGFR + The percentage of ).

[0084] (C) After euthanizing the mice, it was found that after CAR T treatment, liver metastases were observed in mice inoculated with a single-target tumor (MDA231, 5 / 6), but not in mice inoculated with dual-target positive tumors (MDA231-CD19, 0 / 6). The yellow arrows point to the tumor lesions.

[0085] Figure 10 This demonstrates the anti-tumor effect of ETC AND-gated CAR T cells in a bilateral tumor-bearing mouse model.

[0086] (A) Schematic diagram of bilateral tumorigenic mouse model and T-cell infusion therapy. Severely immunodeficient mice (NPG mice) were subcutaneously inoculated with MDA-MB-231 cells (MDA231, HER2 single positive) or CD19-overexpressing MDA-MB-231 cells (MDA231-CD19, CD19 / HER2 double positive) on both sides of the body, and then T-cell therapy was administered via tail vein infusion on day 7.

[0087] (B) Tumor growth in bilateral tumor-bearing mice was assessed after infusion of AND-gate CAR T cells and control untransfected T cells. MDA231 was a HER2-targeted tumor; MDA231-CD19 was a CD19 / HER2-targeted tumor; AND-gate T cells were used after AND-gate T cell infusion; and Control T cells were used after untransfected T cell infusion. Data are plotted as mean plus standard error (SEM), n=5. ns = no significant difference, *p<0.05.

[0088] (C) Comparison of tumor growth from each mouse from (B) that was inoculated with a single antigen tumor (MDA231, left) and a double antigen tumor (MDA231-CD19, right). Detailed Implementation

[0089] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0090] The "percentage (%) identity" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to those in the reference polypeptide sequence after alignment and, if necessary, introducing vacancies to achieve maximum percentage sequence identity, and without considering any conservative substitutions as part of sequence identity. Alignment for determining the percentage of amino acid sequence identity can be performed in various ways within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, or GENTYX (registered trademark) (Genetyx Co., Ltd.). Those skilled in the art can determine appropriate parameters for the alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0091] The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code has been filed with the U.S. Copyright Office (Washington, D.C., 20559) along with user files, registered under U.S. Copyright Registry No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. in South San Francisco, California, or can be compiled from the source code. The ALIGN-2 program should be compiled for use on UNIX operating systems (including Digital UNIX V4.0D). All sequence comparison parameters are set by the ALIGN-2 program and remain unchanged. When using ALIGN-2 for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A with respect to, and with, or against a given amino acid sequence B (which can alternatively be expressed as a given amino acid sequence A having or containing a certain % amino acid sequence identity with, and with, or against a given amino acid sequence B) is calculated as follows:

[0092] 100 times the fraction X / Y

[0093] Where X is the number of amino acid residues that score as identical matches in the sequence alignment of A and B using the ALIGN-2 sequence alignment program, and Y is the total number of amino acid residues in B. It should be understood that when the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein, as described in the preceding paragraph, were obtained using the ALIGN-2 computer program.

[0094] The at least X% identity mentioned herein refers to the identity of the polypeptide sequence with the reference polypeptide sequence being X% to 100%. For example, at least 80% identity includes 80% to 100% identity, including but not limited to 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, and 100% identity.

[0095] Design a novel dual-antigen co-recognition CAR system based on endogenous transcriptional cascade reaction.

[0096] To enable T cells to achieve more precise tumor targeting through dual-target recognition, the inventors designed a novel logic AND-gate dual-target CAR. Its function relies on the antigen-stimulated T cell endogenous transcriptional cascade (ETC) signaling, which the inventors named ETC-gated CAR (or ETC AND-gate CAR). This novel CAR structure consists of two modules... Figure 1 Module A contains an intracellular CD28 co-stimulatory domain and an extracellular recognition domain for antigen a; Module B contains an intracellular CD3z domain and an extracellular recognition domain for tumor antigen b. Considering that T cell activation is primarily mediated by CD3z signaling, to enable T cells to have highly differentiated dual-antigen recognition capabilities, the inventors designed Module B to be regulated and its expression dependent on antigen stimulation. Simultaneously, Module A is expressed consistently, playing both a co-stimulatory signaling role and primarily participating in the induction and regulation of the response. T cells cannot be activated when only antigen a or antigen b is present; only when antigens a and b are present simultaneously can the endogenous transcriptional program of T cells be effectively induced, allowing Module B to be expressed stably and at a high level. Modules A and B work together to promote strong immune synapse formation and integration of CD3z and co-stimulatory molecular signals, enabling complete T cell activation. Figure 1 B).

[0097] To validate the inventor's design, the inventor used CD19 and HER2, two well-established CAR targets, as model tumor antigens for dual-target recognition. The CD19 / HER2 dual-antigen targeted ETC-gated CAR vector consists of two parts ( Figure 1C): Part A is an extracellular CD19 antigen recognition region connected to an intracellular CD28 / 41BB co-stimulatory signaling domain, driven by the EF-1a promoter for constant expression; Part B is an extracellular HER2 antigen recognition region connected to an intracellular CD3z signaling domain, driven by a synthetic inducible promoter pNA for conditionally induced expression. The inventors added V5 and Myc tags to vectors A and B respectively for easy detection. The two vectors were named 19.28BB (part A) and NA.Hz (part B), respectively. The inventors designed the synthetic inducible promoter pNA, which responds to specific T cell endogenous transcription factors, sensing antigen binding and initiating downstream gene expression. The synthetic inducible promoter used here mainly consists of: three repeated NFAT response elements, three repeated CD28-RE / AP response elements, five repeated AP1 response elements, plus a minimal core promoter (…). Figure 1 C). The inventors chose YB_TATA, which was previously reported, as the minimal core promoter in pNA because it has low levels of basal expression and high levels of induced transcriptional expression, which can better achieve tight control over gene expression.

[0098] The inventors first tested the antigen-stimulation-induced expression ability of CD19 / HER2 ETC-gated CAR. They then constructed K562 cell lines expressing both CD19 / HER2 single antigen (K.19, KH) and dual antigen (K.19H) using a lentiviral stable transfection method. Figure 6 (A-6B). T cells transfected with CD19 / HER2 co-recognized ETC-gated CARs were co-cultured with K562 cells expressing four different antigens, and the induction level of the B fraction on T cells after antigen stimulation was detected. The inventors observed that low levels of B fraction induction expression could be detected on T cells after stimulation with CD19 or HER2 single antigens, while significantly and efficiently inducing B fraction expression was only observed under the co-stimulation of CD19 and HER2 dual antigens. Figure 1 D and Figure 1 E) indicates that dual antigen stimulation is necessary to powerfully and efficiently promote the endogenous expression program in this T cell. Simultaneously, experiments showed that the antigen-stimulated expression program designed by the inventors functioned normally and efficiently on both CD4 and CD8 subtype T cells. Figure 1 D), and the T cell-induced expression response occurs rapidly after antigen exposure, with a half-life of approximately 5 hours. Figure 6 These characteristics make ETC-gated CARs suitable for precise dual-target joint identification.

[0099] ETC-gated CAR modification endows T cells with dual-target joint recognition capabilities.

[0100] The inventors then tested the tumor recognition and killing ability of ETC-gated CAR T cells. To investigate whether the affinity of the scFv selected in the ETC-gated CAR system affected its dual antigen recognition ability, the inventors designed three scFvs with different affinities for recognizing the HER2 antigen (4D5, high affinity; 4D5-7, medium affinity; 4D5-5, low affinity) in Part B, and added a truncated NGFR for easier expression detection. The three inducible expression vectors constructed were tNA.Hz, tN7.Hz, and tN5.Hz. Figure 2 A). Three ETC-gated CARs targeting CD19 / HER2 with different affinities were designated BB.tN, BB.tN7, and BB.tN, with their affinities decreasing in that order. After primary T cells were transfected with these three AND-gated CARs, flow cytometry was used to detect basal CAR expression and antigen-induced expression. Figure 7 A).

[0101] The inventors first used the HER2 antigen-expressing tumor cell lines HCT-116 (colon cancer cell line) and MDA-MB-231 (breast cancer cell line) as HER2-single-positive baseline model cells, and then transfected them with truncated CD19 antigen to generate CD19 and HER2 double-antigen-positive target cells. Figure 7 B). The inventors designed a 24-hour in vitro killing assay to evaluate the killing specificity and efficiency of ETC-gated CAR-transfected T cells. The inventors found that using different affinity scFvs in the B portion of the dual CAR affected the killing ability of the transfected T cells. T cells transfected with BB.tN (high affinity) showed weaker ability to distinguish between HER2-single and CD19 / HER2-double-positive target cells, while dual CAR T cells transfected with BB.tN7 (medium affinity) and BB.tN5 (low affinity) could effectively distinguish between single-target and double-target-positive tumor cells. Figure 2 B, Figure 2 C).

[0102] To more clearly and comprehensively evaluate the cytotoxic specificity and potential of the novel ETC-gated CAR T cells, the inventors used another widely used mouse-derived NIH / 3T3 cell line as the basic target cell model. This cell line was tested to be CD19 / HER2 double antigen negative and did not express human co-stimulatory molecules ( Figure 7C). In this cell model, T cell activation signals and co-stimulatory molecular signals are provided entirely by CAR, thus allowing for better evaluation of the capabilities of novel bispecific CARs. Figures show the assays of 3T3 cells transfected with CD19 and HER2 single antigens, and co-transfected with CD19 / HER2 dual antigens. Figure 7 C). The inventors' experimental results showed that bispecific CAR-T cells transfected with BB.tN and BB.tN7 had a good ability to distinguish between 3T3 target cells expressing single and double antigens, and had a high killing efficiency against CD19 / HER2 double antigen-positive 3T3 cells. Figure 2 D). In contrast, T cells transfected with BB.tN5 showed insufficient killing efficiency, possibly because the CAR affinity in this cell model was too low to fully activate T cells. The inventors also examined the release of cytokines from T cells co-cultured with 3T3 cells expressing four different antigens (3T3, 3T3-CD19, 3T3-HER2, 3T3-CD19 / HER2). The results were consistent with the previously observed killing experiments; bispecific CAR T cells transfected with BB.tN and BB.tN7 only produced large amounts of IL-2 and TNF-α when co-cultured with CD19 / HER2 dual-antigen-positive 3T3 cells, and the cytokine release levels of the two transfections were similar. Figure 2 E) In the BB.tN5 transfected T cell group, no significant release of IL-2 and TNF-α was observed in T cells after stimulation with CD19 / HER2 double antigen.

[0103] The inventors then evaluated the antigen-stimulated amplification capacity of ETC-gated CAR-transfected T cells. In the absence of external cytokines, the inventors repeatedly stimulated BB.tN7 bispecific T cells transfected with 3T3 target cells expressing both single and double antigens. The experimental results showed that after repeated rounds of stimulation, the transfected T cells only exhibited strong numerical expansion under CD19 / HER2 double antigen stimulation. Figure 2 F). The above experimental results demonstrate that T cells transfected with ETC-gated CAR have excellent dual-target specific recognition capabilities, and that affinity improvement can further adjust and optimize the recognition and killing potential of CAR T cells.

[0104] ETC-gated CAR-induced expression system is used to enable T cells to load therapeutic cofactors.

[0105] The inventors then verified whether the ETC-gated CAR system could be used to achieve antigen-dependent induction of T cells loaded with therapeutic cofactors. Based on the optimized tN7.Hz vector, the inventors constructed an induction expression vector loaded with TRAIL and IL-2 factors (both reported to effectively enhance CAR-T cell function). Figure 3 A, Figure 3 B). In antigen stimulation-induced expression experiments, the inventors found that stimulation with CD19 / HER2 double antigen-positive K562 cells resulted in highly efficient induction of the expression of these cofactors by enhanced T cells loaded with TRAIL and IL-2. Figure 3 C).

[0106] The inventors further verified that these cofactors carried by T cells play an important role in enabling them to achieve better function. Using K562 cells as target cells, as previously reported, the inventors conducted killing experiments with bispecific CAR-T cells carrying the TRAIL factor. The results showed that bispecific CAR-T cells carrying the TRAIL factor were significantly more efficient at killing dual antigen-positive target cells than bispecific T cells without the factor. Figure 3 D, Figure 3 E). The above experimental results indicate that the novel ETC-gated bispecific recognition CAR system can serve as an effective platform to induce the expression of certain therapeutically significant cofactors by T cell antigen stimulation, thereby ensuring accurate bispecific recognition by T cells while better leveraging their tumor-killing function.

[0107] Design of ETC-gated CAR-targeted NKG2D ligand and EGFR dual antigen combination

[0108] To further investigate the target universality and scalability of the ETC-gated bispecific CAR system, the inventors used this system to test a pair of CAR target combinations with clinical translational potential—NKG2DLs (NKG2D ligands) and EGFR. Both targets exhibit widespread high expression in tumor tissues across various tumor types, limited low expression in normal tissues, and a high correlation in their co-expression. The inventors examined the expression of these two antigens in a range of tumor cell lines, finding that both antigens were expressed positively in various in vitro cultured tumor cell lines, while NKG2DLs and EGFR were negative in mouse-derived NIH / 3T3 cells. Figure 8 A-8B).

[0109] The inventors designed a novel ETC-gated bispecific recognition CAR targeting NKG2DLs and EGFR, consisting of two parts: a constant expression vector (called NK-BB) and an inducible expression vector (called EIL2). Figure 4 A. Using double-antigen-negative 3T3 cells as the basic model cells, target cells were generated through antigen overexpression to produce NKG2D ligand (selected as MICA) and EGFR antigen-positive (3T3.A, 3T3.E) and double-antigen-positive (3T3.EA) cells. Figure 4 B, Figure 8 C). The inventors co-cultured T cells transfected with bispecific CARs with 3T3 cells expressing four different antigens. The killing assays showed that only the bispecific CAR-positive 3T3 cells (3T3.EA) were specifically and efficiently killed by the transfected T cells. Figure 4 (C-4D). The inventors examined the levels of cytokines produced by T cells after co-culturing with 3T3 cells expressing different antigens and found that only after co-culturing with MICA / EGFR double-positive 3T3 cells did transfected T cells show significant production of IFN-γ, IL-2, and TNF-α. Figure 4 E). Furthermore, the inventors examined the expression of CD107a on T cells after stimulation with different antigens to reflect T cell degranulation activation, and found that only under dual antigen stimulation were transfected T cells activated. Figure 4 F). In summary, the novel ETC-gated CAR targeting the combined recognition of NKG2D ligand and EGFR dual antigens exhibits good dual-target specificity, demonstrating significant potential for clinical translational applications. Furthermore, this result indicates that the inventors can utilize the novel ETC-gated CAR system to customize bispecific recognition CARs for specific target combinations, suggesting that this novel dual-target recognition CAR system possesses high target amplification and versatility.

[0110] ETC-gated CAR exerts a dual-target recognition-dependent anti-tumor effect in vivo.

[0111] The inventors then tested the antitumor effect of ETC-gated CAR T cells in a mouse model. Considering that T cell activation is primarily mediated by CD3 signaling, the co-stimulatory molecule signaling alone cannot effectively mediate the antitumor activity of T cells. Figure 2 In B-2F, the inventors primarily evaluated the ability of the CD19 / HER2 dual-recognition CAR to differentiate between single-positive (CD19-HER2+) and double-positive (CD19+HER2+) tumor models in vivo. The inventors conducted unilateral tumor-bearing mouse model experiments, inoculating each mouse with either HER2 single-positive tumor cells (MDA231) or CD19 / HER2 double-positive tumor cells (MDA231-CD19) on one side. Figure 5A). Experiments showed that after infusion of untransfected control T cells, both single-positive and double-positive tumor-bearing mice exhibited persistent tumor growth; however, after infusion of CD19 / HER2 bispecific recognition T cells, compared to the control T cell treatment group, tumor growth in double-positive tumor-bearing mice was significantly inhibited, while tumor growth in single-positive tumor-bearing mice was not inhibited. Figure 5 B-5C). Furthermore, during the mouse treatment, the inventors did not observe a significant decrease in mouse weight or other adverse reactions, indicating that no graft-versus-host disease or other toxic side effects occurred. Figure 5 D). Furthermore, the inventors observed that, compared to single-positive tumor-bearing mice, double-positive tumor-bearing mice had better survival rates and higher levels of CAR-T cells in their peripheral blood after treatment with dual-recognition T cells. Figure 5 E, Figure 9 (A-9B). After euthanasia of the mice, liver metastases were observed grossly and pathologically in all mice treated with control T cells; after infusion of dual-recognition T cells, no liver metastases were observed in double-positive tumor-bearing mice (0 / 6), while liver metastases were observed in most single-positive tumor-bearing mice (5 / 6). Figure 5 F, Figure 9 C). Furthermore, in double-positive tumor-bearing mice, the inventors observed CD3-positive T cells infiltrating into the tumor, while this was not observed in single-positive tumor-bearing mice. Figure 5 G). The inventors also evaluated the efficacy of the novel dual-recognition CAR in a double-tumor-bearing mouse model. In this model, each mouse was inoculated with single-positive and double-positive tumors on its left and right sides, respectively. After T-cell infusion therapy, the efficacy of CAR-T cells was evaluated by observing and comparing the growth of the tumors on the left and right sides. Figure 10 A). The inventors observed similar results to those in single-tumor-bearing mice: bispecific T-cell recognition specifically inhibited the growth of double-positive tumors, but had no inhibitory effect on single-positive tumors. Figure 10 (B-10C). The above experimental results indicate that the novel ETC-gated dual-recognition CAR-T cells targeting CD19 / HER2 can effectively and specifically recognize CD19 / HER2-positive tumors in vivo.

[0112] Example

[0113] The cell lines used in the embodiments of this application are listed in Table 1 below. Except for K562 cells, which were cultured in RPMI 1640 medium supplemented with 10% serum, the other cells were cultured in DMEM medium containing 10% fetal bovine serum. Unless otherwise stated, the materials, reagents and instruments used in this application were obtained commercially.

[0114] Table 1

[0115]

[0116]

[0117] Example 1: Construction of Antigen Overexpression and Novel Dual-Recognition CAR System Vector

[0118] The inventors used a lentiviral vector system to construct an antigen overexpression vector and a novel AND-gate dual-recognition CAR vector system. The lentiviral vector system contains two packaging plasmids (psPAX2 and pMD2.G) and a master plasmid for expressing the target protein (pRRLSIN-EF1a-MCS).

[0119] 1) Construction of antigen overexpression vectors: The antigen fragments (gene names and corresponding GenBank database index numbers for the antigen overexpression lentiviral vectors used in the experiment) were obtained from a tumor cell cDNA library via reverse transcription PCR. Confluent tumor cells were collected in 100mm dishes, and total RNA was extracted from the tumor cells using the Trizol method. The concentration was then determined for later use. RNA reverse transcription was performed using the TransGold Reverse Transcription Kit (TransGold, AH311-02). The reverse transcription system was prepared according to the kit instructions. The cells were incubated at 50°C for 30 minutes under PCR temperature control, followed by heating at 85°C for 5 seconds to inactivate the enzymes and complete the RNA reverse transcription to obtain the tumor cell cDNA library. Primers were designed to obtain the desired gene fragments from the cDNA library via PCR. Reverse transcription PCR primers are shown in Table 2. The vector pRRLSIN-EF1a-MCS and the PCR fragments were then digested with XbaI and SalI (NEB) and purified. Each gene fragment was inserted into a lentiviral vector using Solution I ligase (Takara, 6022). The ligation system was added to competent cells (TransGold, CD501-03) for plasmid transformation. The cells were screened overnight using ampicillin-resistant plates, and single clones were selected for sequencing identification. Correctly ligated clones were stored in LB medium with 15% glycerol at -80°C for plasmid extraction and further prepared for use.

[0120]

[0121]

[0122] 2) Construction of a dual-recognition CAR vector: The CD19 / HER2 dual-recognition CAR vector comprises module A (mainly composed of EF1a-FMC63.scFv-CD28-41BB) and module B (mainly composed of pNA-4D5.scFv-CD3z). Based on sequence information obtained from NCBI, the inventors designed sequences for whole-genome synthesis, obtaining the promoter and expression cassette DNA fragments. The synthesized DNA fragments were designed with dual restriction enzyme sites BstXI and SalI at both ends. These two restriction sites were used to replace the promoter and expression cassette portions in the lentiviral backbone vector pRRLSIN-EF1a-MCS. The vector pRRLSIN-EF1a-MCS and the synthesized gene fragment were digested separately with enzymes. The digested vector and fragment were recovered and purified, and ligated using Solution I. After transformation, plate screening, and single-clone sequencing identification, correctly ligated clones were obtained. The bacterial strains with correctly ligated clones were preserved and plasmids were used for subsequent lentivirus packaging. Module A vector was named 19.28BB (nucleotide sequence as shown in SEQ ID NO:3), and module B vector was named NA.Hz (nucleotide sequence as shown in SEQ ID NO:4) (see...). Figure 1 C). To facilitate detection, the inventors added the PGK-tNGFR expression cassette to the NA.Hz vector and obtained the PGK-tNGFR fragment using a whole-genome synthesis method. The synthesized fragment was flanked by dual restriction enzyme sites XhoI and MluI. The synthesized PGK-tNGFR fragment and the NA.Hz vector were digested with these enzymes, and the vector and fragment were recovered and purified. They were ligated with Solution I, transformed, and screened overnight on ampicillin plates. Single clones were selected for sequencing identification. Clones with correct sequencing were preserved and used for large-scale extraction. The newly constructed vector was named tNA.Hz (nucleotide sequence as shown in SEQ ID NO: 28) (see...). Figure 2A). To test the effect of different affinity scFvs on dual recognition performance, the inventors tested two other scFvs targeting the HER2 antigen with different affinities in module B: 4D5-7 scFv (corresponding scFv nucleotide sequence as shown in SEQ ID NO:29, its encoded amino acid sequence as shown in SEQ ID NO:30) and 4D5-5 scFv (corresponding scFv nucleotide sequence as shown in SEQ ID NO:31, its encoded amino acid sequence as shown in SEQ ID NO:32). The corresponding module B structures are pNA-(4D5-7).scFv-CD3z and pNA-(4D5-5).scFv-CD3z. Based on the scFv sequences found in the literature, the inventors synthesized the gene, used PflFI and SgrAI to double-digest the linearized tNA.Hz vector, and used a seamless homologous recombination ligation method (Shanghai Yisheng, Hieff Clone one-step cloning kit, Cat#10911ES25) to ligate the 4D5-7 scFv. scFv and 4D5-5 scFv were inserted into the tNA.Hz vector, and the correct clones were screened and identified. The strains and large-scale extractions were preserved for later use. The newly obtained module B vectors with different affinities were named tN7.Hz and tN5.Hz (see...). Figure 2 A). Based on the tNA7.Hz vector, to construct a dual-recognition CAR loaded with inducible factors, the inventors linearized the vector with SalI restriction enzyme, and then inserted p2A-IL2 and p2A-TRAIL after CD3z using a seamless homologous recombination ligation method. After ligation, transformation, and screening, the correct clones were obtained. The newly constructed vectors were named tN7-IL2 (nucleotide sequence as shown in SEQ ID NO:33) and tN7-TRAIL (nucleotide sequence as shown in SEQ ID NO:34) (see...). Figure 3 A). The inventors also constructed an NKG2D ligand / EGFR dual-recognition CAR, which includes module A and module B. Module A's recognition domain is the natural NKG2D extracellular segment. Its gene sequence was obtained from NCBI, and after whole-genome synthesis, it was inserted into the 19.28BB vector to replace the recognition domain with the NKG2D extracellular segment. The newly constructed vector was named NK-BB (nucleotide sequence as shown in SEQ ID NO:7). Module B has the structure pNA-EGFR.scFv-CD3z-p2A-IL2. Based on the whole-genome synthesis of the target gene fragment, after double digestion with XbaI and SalI, Solution I was used to ligate it into a lentiviral backbone vector. The newly constructed module B vector was named EIL2 (nucleotide sequence as shown in SEQ ID NO:8) (see...). Figure 4 A).

[0123] Example 2: CART cell preparation

[0124] 1) Isolation and culture of primary T cells

[0125] The inventors used a peripheral blood mononuclear cell isolation kit (Dakow, Cat#DKW-KLSH-0100) to isolate peripheral blood mononuclear cells (PBMCs) from healthy donor blood: In a clean biosafety cabinet, fresh anticoagulated whole blood was diluted 1:1 by volume with sterile PBS. Approximately 15 ml of separation buffer was added to a 50 ml centrifuge tube. The diluted whole blood was carefully added to the centrifuge tube, spreading it evenly on top of the separation buffer, maintaining a clear interface between the two liquids. The tube was centrifuged at 750 g for 30 minutes at room temperature. After complete centrifugation, the liquid surface was stratified. The thinner white film in the middle was carefully aspirated and transferred to a new 50 ml centrifuge tube. Approximately 40 ml of PBS was added to the new centrifuge tube, and the mixture was inverted to mix. The tube was centrifuged at 250 g for 10 minutes at room temperature, and the supernatant was discarded. The above washing steps were repeated. Finally, the cells were resuspended in RPMI-1640 medium and seeded into 100 mm dishes for later use. The inventors further enriched the isolated human peripheral blood mononuclear cells (PBMCs) with CD3-positive primary human T cells using a human T cell isolation kit (STEMCELL Technologies, Cat#17951): First, PBMCs were counted, and the cell density was adjusted to 5 × 10⁷ cells / ml with phosphate-buffered saline containing 2% fetal bovine serum, and transferred to a flow cytometry tube. Based on the cell volume, 50 μl / ml of sorting buffer was added, mixed, and incubated at room temperature for 5 minutes. 40 μl / ml of sorting beads were added, and buffer was added to a final volume of 2.5 ml. The mixture was gently pipetted and aspirated, and the flow cytometry tube was inserted into the sorting magnetic rack and incubated at room temperature for 3 minutes. The magnetic rack was then lifted along with the flow cytometry tube, and the cell suspension was carefully poured into a new 15 ml centrifuge tube in one direction. This was the enriched T cell suspension. The cells were then centrifuged at 300g for 5 minutes, resuspended in preheated (37°C) culture medium, and cell counting was performed. After isolation and enrichment, T cells were activated for 2 days using anti-CD3 / CD28 antibody-conjugated magnetic beads (ThermoFisherScientific, Cat#11132D) at a ratio of 3:1 (magnetic beads / T cells). Subsequently, lentiviral infection and T cell expansion culture were performed. All T cells were cultured in X-VIVO 15 medium (LONZA, Cat#04-418Q) containing 10% fetal bovine serum and 100 U / ml IL-2 (Peprotech, Cat#200-02).

[0126] 2) Lentiviral preparation and T-cell infection

[0127] The inventors used calcium phosphate co-transfection in HEK-293T cells to package lentiviruses. First, they prepared 2X HBS buffer and 2.5M CaCl2 solution, filtered them with a 0.22μm filter for sterilization, and then dispensed them into aliquots for use.

[0128] Table 3 shows the preparation of 2×HBS.

[0129]

[0130] Before lentivirus packaging, large-scale extraction and purification of the required plasmids were performed, and their concentrations were determined for later use. Twelve hours before packaging, plasmids were prepared at a concentration of 8 × 10⁸ g / dL. 6 Cells were seeded at the appropriate density into 100 mm dishes. For each 100 mm dish, a plasmid and CaCl2 mixture was prepared and thoroughly mixed: 12 μg of the two packaging plasmids psPAX2 and pMD2.G, 4 μg of the main plasmid, and 16 μg of the main plasmid, respectively. 50 μl of 2.5 M CaCl2 was added, and the total volume was brought up to 500 μl with sterile ultrapure water. 500 μl of 2×HBS buffer was added to each 5 ml flow cytometry tube. The prepared 500 μl plasmid and CaCl2 mixture was added dropwise to the 2×HBS, shaking the flow cytometry tube several times after every 2-3 drops to ensure thorough mixing. After the addition was complete, the flow cytometry tube was vortexed 7 times to ensure complete mixing. This mixture was then added dropwise to the 293T cells cultured in 100 mm dishes. The cells were returned to a 37°C CO2 cell culture incubator. Twelve hours after transfection, the medium was changed, discarding the original medium and replenishing with fresh medium. Viral supernatant was collected at 24 and 48 hours post-transfection and filtered through a 0.45 μm Millipore filter. The collected viral supernatant could be stored at 4°C for short-term use (within one week) or aliquoted and frozen at -80°C for long-term storage. T cells were infected 2-3 days after activation with CD3 / CD28 magnetic beads: T cell suspension was mixed with viral supernatant in a 6-well plate, polybrene was added to a concentration of 8 μg / ml, and the cells were centrifuged at 350g for 1 hour at room temperature. The cells were then transferred to a 37°C CO2 incubator. T cells were repeatedly infected 1-2 times under the above conditions, with infection occurring sequentially using the dual-recognition CAR vector module A and module B viruses. After infection, T cells were cultured at a rate of 1 × 10⁻⁶ cells / mL. 6 The cells were inoculated at a density of 10 cells / ml into fresh X-VIVO 15 medium containing 10% fetal bovine serum and 100 U / ml IL-2 for amplification and culture.

[0131] 3) CAR T cell culture, expansion and detection

[0132] After infection, adjust the initial T cell seeding density to 1×10⁻⁶. 6 Cells were cultured at a density of 1 × 10⁶ / ml and returned to the incubator. Cell proliferation is rapid in the first few days post-infection; closely monitor cell growth, replenishing fresh culture medium as needed to maintain optimal cell density at 1 × 10⁶ / ml. 6 / ml. On day 3 post-infection, centrifuge and change the medium once to remove CD3 / CD28 activating magnetic beads, then administer 1×10 6Seed at an initial cell density of [number] cells / ml for expansion culture. Passage every 2 days at a 1:2 ratio, directly aliquoting the T cell suspension into new culture flasks and adding fresh culture medium, minimizing centrifugation and pipetting. T cells can be used for functional experiments from day 5 to day 10 post-infection. T cell infection efficiency can be assessed 3 days after infection completion: take 2 × 10⁻⁶ cells / ml. 5 ~5×10 5 A number of T cells were placed in 1.5 ml centrifuge tubes, centrifuged at 300 g, and the supernatant was discarded. The T cells were resuspended in flow cytometry buffer (phosphate-buffered saline containing 2% fetal bovine serum), and an appropriate amount of flow cytometry staining antibody was added (anti-V5 tag flow cytometry antibody: Abcam, Cat#ab72560; anti-NGFR flow cytometry antibody: Biolegend, Cat#345112). The cells were stained at 4°C in the dark for 45 minutes. After washing with 1 ml of PBS, the cells were centrifuged at 300 g for 3 minutes at room temperature, and the supernatant was discarded. The washing procedure was repeated 1-2 times. The cells were resuspended in 200 μl of PBS, and any possible cell clumping was removed by passing the cells through a single-cell sieve. The cells were then loaded for flow cytometry analysis.

[0133] Example 3: CAR T cell function assessment

[0134] 1) T cell expression induced by in vitro stimulation

[0135] The inventors used K562 cells overexpressing various corresponding antigens as target cells for antigen stimulation, pre-labeling them with CFSE dye (Biolegend, Cat#423801) to distinguish them from T cells. After enrichment of dual-recognition T cells, they were cultured for 5-10 days until they returned to a resting state. Infected T cells in good culture condition and K562 cells expressing different antigen types were collected, resuspended in RPMI 1640 medium, and cell counts were performed separately, with the cell density adjusted to 1 million cells / ml. The T cells and K562 cells were mixed at a 1:1 volume ratio, thoroughly mixed, and 200 μl of the mixture was added to each well of a U-bottom 96-well plate. The 96-well plates were returned to a 37°C cell culture incubator for further culture. Cells were collected at different time points, such as 5 hours, 10 hours, and 24 hours, for staining and flow cytometry analysis. The efficiency of T cell expression induced by antigen stimulation was determined by the flow cytometry results of the corresponding labeled antibodies (anti-V5 labeled flow cytometry antibody: Abcam, Cat#ab72560; anti-NGFR flow cytometry antibody: Biolegend, Cat#345112; anti-Myc labeled flow cytometry antibody: CST, Cat#3739S).

[0136] 2) Cell killing and cytokine release detection experiment

[0137] The inventors used a T-cell killing assay based on luciferase activity. Target cells expressing different antigens were pre-transfected with firefly luciferase. Target cells were seeded 8 hours prior to infection at a density of 10,000 cells / well in opaque white 96-well plates. CAR-T cells were collected from day 5 to day 10 post-infection for the killing assay. After centrifugation, the T cells were resuspended in DMEM medium for cell counting. The initial T-cell density was adjusted to 800,000 cells / ml. Three additional 15ml centrifuge tubes were used for serial dilution to obtain T-cell suspensions at concentrations of 400,000 cells / ml, 200,000 cells / ml, and 100,000 cells / ml. Following these four concentration gradients, three parallel wells were set up for each concentration. 100 μl of T cells were added to each well of a 96-well plate containing tumor cells. The killing effect-to-target ratios for the four concentration gradients were 8, 4, 2, and 1, respectively. The 96-well plates were then placed back into a 37°C CO2 cell culture incubator and cultured for 24 hours. Remove the white plate and add luciferase luminescent substrate (Goldbio, Cat#LUCK-1G) to the 96-well plate: Dilute the stored substrate (concentration 20 mg / ml) with PBS at a ratio of 1:50. Using a multipipe, quickly add 50 μl of the diluted substrate to each well of the 96-well white plate, taking care to avoid light. Place the white plate with the substrate on a shaker and mix well in the dark for 2 minutes. Centrifuge at 400g for 3 minutes at room temperature. Use a Centro XS3 LB 960 microplate luminescence detector (Berthold Technologies) to perform luminescence detection. Calculate the killing efficiency per well using the following formula: Killing efficiency % = (Luminescence intensity of control well – Luminescence intensity of test well) × 100% / Luminescence intensity of control well.

[0138] The inventors used an ELISA method to detect cytokine release after T cells were co-cultured with target cells expressing different antigens. CAR-T cells and tumor cells were co-cultured for 24 hours at a fixed effector-to-target ratio. The cells were centrifuged at 300g for 3 minutes at room temperature, and the supernatant was collected: it could be used immediately for ELISA detection, or frozen at -80℃ for subsequent detection. The co-culture supernatant was diluted proportionally, and the diluted supernatant was used to detect IL-2 (Dakow, Cat#DKW12-1020-096), IFN-γ (Dakow, Cat#DKW12-1000-096), and TNF-α (Dakow, Cat#DKW12-1720-096) using an ELISA kit. The detection process and steps were strictly followed according to the instructions provided with the kit. Finally, the absorbance value of each well was measured at a wavelength of 450nm, and the cytokine concentration in each well was calculated using a standard curve.

[0139] 3) In vitro stimulation and expansion of T cells

[0140] The co-culture of T cells with NIH / 3T3 cells was carried out in DMEM medium containing 10% fetal bovine serum without the addition of exogenous cytokines. NIH / 3T3 cells expressing various corresponding antigens were pre-treated with 30 Gy X-rays to stop their proliferation, and then cultured at a density of 0.1 x 10⁻⁶ cells / cells. 6 Irradiated NIH / 3T3 cells were seeded into 12-well plates. After cell attachment, T cells were added at a T cell / target cell ratio of 5:1 for co-culture. Every 2-3 days, T cells in each well were counted using cell counting beads (Biolegend, Cat#424902), and then the T cell density was adjusted to 1x10⁶ cells / well. 6 T cells were added to a new 12-well plate pre-coated with NIH / 3T3 cells for a new round of stimulation.

[0141] 4) In vivo experiments with CAR T cells

[0142] The experimental animals were female NOD.Cg-Prkdc cells approximately 6 weeks old. scid Il2rg tm1Vst / Vst(NPG) severely immunodeficient mice (Beijing Vitonda Biotechnology Co., Ltd.). Unilateral tumor-forming mice were subcutaneously inoculated with 1×10 6个 MDA231 cells or MDA231-CD19 cells, 14 days after tumor inoculation, were followed by infusion of either untransfected control T cells or novel dual-recognition CAR-T cells at a dose of 5 × 10⁻⁶. 6 The bilateral tumorigenesis model was established by subcutaneously inoculating MDA231 and MDA231-CD19 into the left and right anterior axillae of the same mouse, respectively, with 1×10⁻⁶ cells / mL. 6 Cell quantity: One week after tumor cell inoculation, mice were randomly divided into groups and treated with either control T cells or dual-recognition CAR-T cells. Tumor volume and body weight were monitored in mice every 2–3 days, and any adverse reactions were observed. Tumor size was measured using calipers, and tumor volume was calculated as: Volume = (Long axis × Wide axis × Wide axis) / 2.

[0143] discuss

[0144] In this study, the inventors developed an effective and powerful method for T cells to precisely target tumor cells. This method achieves precise tumor targeting through the combined recognition of two antigens, relying on a designed T cell-endogenous antigen-induced transcriptional cascade system. The inventors demonstrated in this study that this method can be applied to target tumor cells with different combinations of two antigens. More importantly, the dual-target recognition T cells generated using this method can highly specifically target and kill dual-target-positive tumor cells both in vitro and in vivo, while having no significant effect on single-target-positive cells.

[0145] In the inventor's method, the precise dual-targeting capability of T cells is based on a meticulously designed dual-antigen co-recognition system. The combined participation of both targets is essential for efficiently inducing the endogenous transcriptional cascade of T cells, promoting the high-level stable expression of the designed CAR module. The combined action of the two CAR modules further promotes the formation of a strong immune synapse between T cells and tumor cells, and enables T cells to fully activate by integrating co-stimulatory molecular signals and CD3z signals. Figure 1 A) Compared to previously reported induction-coordinated recognition systems such as the synNotch receptor, the inventors' co-recognition system relies on endogenous transcriptional regulatory elements of T cells, rather than exogenous regulatory elements (such as Gal4, tTA, etc.). The use of endogenous T cell regulatory elements avoids immunogenicity problems caused by the introduction of non-human regulatory elements, which is particularly important for clinical translational applications. Furthermore, the inventors' novel T cell induction-coordinated recognition system has a simpler structure, better feasibility, and can achieve high-resolution dual-target co-recognition. Therefore, the inventors' method provides another new strategy for preparing precisely controlled therapeutic T cells and improving the safety of T cell therapy.

[0146] The inventors' endogenous induction combined recognition system has the potential to customize dual-recognition CARs for any two antigen combinations. In this study, the inventors demonstrated, using the CD19 / HER2 dual-antigen combination and the NKGDLs / EGFR dual-antigen combination as examples, that this system can be used to personalize desired dual-target combinations. Figure 2 E, Figure 5 E). This is crucial for enriching and expanding the available tumor targets. Previous studies have shown that some tumor targets in the preclinical research stage (such as B7-H3 and c-Met) have good responses to CAR-T targeted therapy, but their lack of target specificity greatly hinders their clinical translation. These antigens hold promise for safer and more reliable targeted application through the inventors' novel combined targeting strategy. Therefore, the inventors' method, as a universal platform for achieving efficient targeted recognition of T-cell dual antigens, can greatly enrich and expand the current CAR targets, especially CAR targets for solid tumors.

[0147] The inventor's ETC-gated CAR system is a multifunctional platform that not only enables reliable antigen-targeted co-targeting but also allows for the customization of loading various therapeutic factors to enhance the anti-tumor potential of T cells. Considering its compact and simple design, minimal immunogenicity, and robust co-targeting capabilities, the inventor's research has excellent clinical application prospects, providing a new strategy for improving the safety and efficacy of CAR-T therapy.

[0148] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A dual-target chimeric antigen receptor, comprising a first chimeric antigen receptor and a second chimeric antigen receptor, wherein the first chimeric antigen receptor and the second chimeric antigen receptor target a first target and a second target on a target cell, respectively; the first chimeric antigen receptor, modified by the dual-target chimeric antigen receptor, is constitutively expressed in T lymphocytes via an EF1α promoter; wherein, after the T lymphocytes come into contact with the target cells, the second chimeric antigen receptor is induced to express on the T lymphocytes by antigen stimulation via a synthetic promoter, the sequence of which is shown in SEQ ID NO:9; wherein, after the first chimeric antigen receptor and the second chimeric antigen receptor simultaneously recognize the first target and the second target, the T lymphocytes are fully activated to exert their effector function. The first chimeric antigen receptor includes an extracellular recognition region for recognizing a first target and a co-stimulatory molecular domain, and the second chimeric antigen receptor includes an extracellular recognition region for recognizing a second target and a CD3ζ intracellular domain, wherein the co-stimulatory molecular domain is selected from the CD28 co-stimulatory molecular domain, the 4-1BB co-stimulatory molecular domain, and combinations thereof. The first and second targets are different and are independently selected from CD19, HER2, NKG2D and EGFR.

2. The dual-target chimeric antigen receptor of claim 1, wherein the target cells are selected from tumor cells, senescent cells, abnormal fibrotic cells, and abnormal immune cells.

3. The dual-target chimeric antigen receptor of claim 2, wherein the extracellular recognition region for recognizing the first target is a single-chain antibody or receptor, and the extracellular recognition region for recognizing the second target is a single-chain antibody or receptor.

4. The dual-target chimeric antigen receptor according to claim 3, wherein, The amino acid sequence of the first chimeric antigen receptor is shown in SEQ ID NO:1, and the amino acid sequence of the second chimeric antigen receptor is shown in SEQ ID NO:

2.

5. A polynucleotide encoding the dual-target chimeric antigen receptor as described in any one of claims 1-4.

6. The polynucleotide of claim 5, comprising module A and module B, wherein module A comprises a constitutive promoter, an extracellular recognition region coding sequence for recognizing a first target, and a co-stimulatory molecular domain coding sequence; and module B comprises an inducible synthetic promoter, an extracellular recognition region coding sequence for recognizing a second target, and a CD3ζ intracellular domain coding sequence.

7. The polynucleotide of claim 6, wherein the nucleotide sequence of module A is as shown in SEQ ID NO:3 and the nucleotide sequence of module B is as shown in SEQ ID NO:4; or the nucleotide sequence of module A is as shown in SEQ ID NO:7 and the nucleotide sequence of module B is as shown in SEQ ID NO:

8.

8. An expression vector comprising the polynucleotide of any one of claims 5-7.

9. The expression vector according to claim 8, wherein, The expression vector is a lentiviral vector.

10. A modified T lymphocyte expressed by any one of claims 1-4 or obtained by transforming T lymphocytes with the expression vector of claim 8 or 9.

11. A synthetic promoter having the sequence configuration of SEQ ID NO:

9.

12. A method for preparing dual-target chimeric antigen receptor-modified T lymphocytes, comprising the following steps: The expression vector of claim 8 or 9 is introduced into isolated and activated T cells; The infected T cells were expanded, cultured, and tested. T lymphocytes modified with dual-target chimeric antigen receptors were isolated.

13. Use of the modified T lymphocytes of claim 10 in the preparation of a medicament for the treatment of breast cancer.

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