CAR-T cells overexpressing the CXCR5 gene and targeting B7H3 and their applications

Abstract

This invention relates to the field of biomedical technology, specifically to CAR-T cells overexpressing the CXCR5 gene and targeting B7H3, and their applications. The CAR-T cells of this invention are obtained by modifying genetically engineered T cells. Specifically, the gene encoding a chimeric antigen receptor fused with CXCR5 and anti-B7H3 is introduced into T cells using a viral vector, followed by sorting using marker genes or other screening methods to select stably expressing T cells. This invention has shown that radiotherapy can sensitize CXCR5 CAR-T cells for the treatment of solid tumors, and this therapy is significantly more effective than CAR-T cells expressing chimeric antigen receptors (CARs) or radiotherapy alone. This invention provides a new target for radiotherapy combined with immunotherapy and has potential translational significance.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to CAR-T cells that overexpress the CXCR5 gene and target B7H3, and their applications. Background Technology

[0002] CAR-T cell therapy, or chimeric antigen receptor T-cell therapy, is an emerging immunotherapy technology. Its basic principle involves using genetic engineering to isolate and modify the patient's own T cells. A chimeric antigen receptor (CAR) that specifically recognizes antigens on the surface of tumor cells is inserted into the T cell surface, transforming the T cells into CAR-T cells with targeted killing capabilities. These CAR-T cells are then expanded in large quantities and reinfused into the patient, where they can precisely recognize and attack solid tumor cells expressing the corresponding antigens, thereby achieving the goal of treating tumors.

[0003] In the current cutting-edge exploration of oncology therapeutics, chimeric antigen receptor T-cell (CAR-T) therapy for solid tumors has become a focus of attention in both scientific research and clinical practice due to its innovative treatment concept and significant therapeutic potential. Currently, the tumor-associated antigens (TAAs) used in CAR-T cell therapy regimens for solid tumors that have successfully entered clinical research stages exhibit an extremely rich diversity, exceeding 20 types. 1 Research has identified at least 19 target antigens that can be used to construct CAR-T cells targeting breast cancer. Currently, 22 CAR-T cell therapies are in clinical trials, targeting 12 different tumor-associated antigens. 2 Among the antigen targets involved in CAR-T cell therapy for breast cancer, c-Met, CD171, CEA, fibroblast activation protein (FAP), GD2, MUC-1, EGFR, and VEGF-R2 are commonly used. c-Met, as a cell membrane surface tyrosine kinase, exhibits a high expression rate exceeding 50% in triple-negative breast cancer (TNBC). Numerous studies have shown that high c-Met expression is closely related to the progression of triple-negative breast cancer tumors and also has a significant impact on patient prognosis. In 2020, SHAH... 3A study (NCT03060356) published the results of a Phase I clinical trial (NCT03060356) on the treatment of four patients with metastatic TNBC using second-generation c-Met-targeted CAR-T cells. The patients included in this study had c-Met expression rates ranging from 40% to 100%. The results showed that two of the four patients achieved partial remission (PR), and the other two had stable disease (SD). Grade 1-2 toxicities occurred during treatment, and the treatment was well-tolerated. Mesothelin (MSLN) is a tumor differentiation glycoprotein with cell adhesion function. Currently, several Phase I / II clinical trials (NCT02792114, NCT02414269, NCT01355965, NCT02580747) are exploring the efficacy and safety of MSLN-targeted CAR-T cells in metastatic or progressive TNBC, but the results of these studies have not yet been published. Furthermore, most clinical trials of CAR-T therapy targeting other antigens are still in the participant recruitment phase. In the field of prostate cancer treatment, available tumor-associated antigens (TAAs) primarily include prostate-specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), prostate-specific antigen (PSA), and epithelial cell adhesion molecule (EpCAM). In a phase I clinical trial, five prostate cancer patients received PSMA-specific CAR-T cell therapy. The results showed that two patients achieved partial remission (PR). 4The patients were in good condition, and no significant anti-PSMA or anti-CAR toxicity was observed throughout the treatment. Currently, other clinical studies evaluating the efficacy of PSMA CAR-T cell therapy (such as NCT04227275, NCT04249947, and NCT04429451) are underway. For glioblastoma, EGFRvIII, ERBB2 / HER2, and IL-13 receptor α2 (IL13Rα2) are the main target antigens of CAR-T cells. Among them, the expression rate of EGFRvIII in newly diagnosed glioblastoma patients is approximately 25% to 30%. Although EGFRvIII-targeting CAR-T cells did not reveal significant safety risks in related clinical studies (NCT02209376, NCT01454596), unfortunately, no significant response was observed in the subjects. In another phase I clinical study evaluating the efficacy and safety of HER2-specific CAR-T cell therapy for glioblastoma, 16 subjects were enrolled. The study results showed that one participant achieved partial remission (PR) for up to 9 months; seven participants had stable disease (SD), which lasted from 8 weeks to 29 months; and the remaining eight participants experienced disease progression (PD). Following the first CAR-T cell injection, the median overall survival (OS) was 11.1 months, with a 95% confidence interval between 4.1 and 27.2 months. 5 In the treatment of neuroblastoma, the current mainstream CAR-T therapy strategy focuses on the disialotetrahexosylganglioside (GD2) antigen. GD2 is a lipid molecule that is highly expressed in neuroblastoma, but lowly expressed or even absent in normal tissues. In clinical practice, the efficacy of GD2 CAR-T therapy remains controversial. In a phase I clinical trial, 11 children with relapsed neuroblastoma received CAR-T cell therapy. The results showed that 3 children achieved complete remission (CR), and notably, one child remained disease-free for 4 years and 10 months after treatment at the end of the study. However, regrettably, this CAR-T cell therapy regimen was less effective in the remaining 8 children, who experienced disease progression or relapse. 6 In 2020, the results of a Phase I clinical trial on GD2 CAR-T cell therapy for relapsed / refractory neuroblastoma were published. The results showed that none of the participating patients achieved an objective clinical response. 7Besides GD2, other antigens used in CAR-T cells targeting neuroblastoma include CD276 (B7H3) and GPC2. Looking at current clinical trial data, the efficacy of most CAR-T cells in treating various solid tumors is relatively limited, and their effectiveness urgently needs further verification through more research.

[0004] CAR-T cell therapy, through genetic engineering, endows T cells with the ability to precisely recognize tumor antigens, demonstrating significant efficacy in the treatment of hematological malignancies. CAR-T cells targeting CD19 have significantly improved remission rates in certain leukemia and lymphoma patients. However, in the treatment of solid tumors, CAR-T cell therapy faces numerous challenges. Hematological malignancies differ significantly from solid tumors; they do not form typical histological structures, while solid tumors possess unique histopathological characteristics. Solid tumors are richly vascularized with relatively wide intercellular spaces, exhibiting poor structural integrity and lacking lymphatic drainage. Furthermore, solid tumors exhibit selective extravasation and retention of large molecule drugs, a phenomenon known as the enhanced permeability and retention effect (EPR). The EPR effect significantly influences the infiltration of CAR-T cells into solid tumor sites, becoming a major obstacle to the application of CAR-T cell therapy in solid tumor treatment. 8 The transport mechanism of T cells is closely related to the interaction between T cells and endothelial cells. During T cell migration, multiple factors can influence it. For example, if chemokines and chemokine receptors cannot properly match, they cannot effectively guide the direction of T cell movement; downregulation of cell adhesion molecule expression levels weakens the adhesion ability of T cells to surrounding cells and the matrix, affecting their movement between tissues; other factors, such as the presence of abnormal vascular systems (e.g., disordered vascular structure, abnormal blood flow), can hinder T cells from entering target tissues via blood vessels. The combined effect of these factors can significantly reduce the likelihood of tumor-specific T cells successfully homing to the tumor site. 9 The tumor microenvironment (TME) primarily comprises immune cells, stromal cells, and other non-cellular substances. Within the TME, these components continuously interact with tumor cells, a process that not only drives disease progression but also fosters drug resistance in tumor cells. Specifically, these interactions trigger a series of changes within the TME that are detrimental to CAR-T therapy: local hypoxia occurs in the tumor tissue, the pH level decreases, creating an acidic environment, the number of immunosuppressive cells significantly increases (such as regulatory T cells and myeloid-derived suppressor cells), the expression of inhibitory receptors on the surface of immune cells is upregulated, and tumor cells secrete large amounts of cytokines. These changes severely interfere with the normal function of CAR-T cells, significantly reducing the efficacy of CAR-T therapy for solid tumors.10 Therefore, effectively removing immunosuppressive factors from the tumor microenvironment (TME) is of paramount importance for improving the efficacy of CAR-T therapy, potentially opening up new and effective pathways for solid tumor treatment. The poor efficacy of solid tumor treatment is partly attributed to the scarcity of tumor-specific antigens (TSA) and the activation of tumor immune escape mechanisms. TSA, as the most ideal target, is expressed only in tumor tissues; however, the discovery and screening of TSA is fraught with challenges. For this reason, most current CAR targets are tumor-associated antigens (TAAs). Using TAAs as targets may interfere with the precise recognition of tumor cells by CAR-T cells, thereby weakening the efficacy of CAR-T therapy. 11 Furthermore, tumor cells can evade the immune system's attacks through immune escape mechanisms, making it difficult for CAR-T cells to effectively recognize tumor cells, ultimately accelerating the deterioration of the disease. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide CAR-T cells overexpressing the CXCR5 gene and targeting B7H3, and their applications. Accordingly, we utilize CXCR5 as a marker of exhausted precursor T cells and its chemotactic properties to modify genetically engineered T cells expressing chimeric antigens (CARs). In vitro and in vivo experiments have demonstrated that radiotherapy-sensitized CXCR5 CAR-T cells are more effective than CXCR5 CAR-T cell-only treatments for solid tumors. Radiotherapy can reshape the immune microenvironment and enhance the infiltration of CXCR5 CAR-T cells, thereby increasing their tumor-killing ability and reducing off-target effects, demonstrating broad application prospects and significant market value.

[0006] To achieve the above objectives, the present invention provides the following technical solution: One aspect of this invention relates to a chimeric antigen receptor fused with CXCR5 against B7H3, the amino acid sequence of which is shown in SEQ ID NO.3.

[0007] Another aspect of the present invention relates to a nucleic acid molecule encoding the chimeric antigen receptor against B7H3 fused with CXCR5.

[0008] In a preferred embodiment of the present invention, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.4.

[0009] Another aspect of the present invention relates to a viral vector comprising the aforementioned nucleic acid molecule.

[0010] Another aspect of the present invention relates to a CAR-T cell (or CXCR5 CAR-T cell) that overexpresses the CXCR5 gene and targets B7H3, wherein the CAR-T cell is a T cell transfected with the viral vector.

[0011] Another aspect of the present invention relates to the use of CAR-T cells in the preparation of medicaments for the treatment and / or adjuvant treatment of solid tumors.

[0012] In a preferred embodiment of the present invention, the solid tumor includes colon cancer and pancreatic cancer.

[0013] In a preferred embodiment of the present invention, the adjuvant drug for treating solid tumors is a drug used as an adjuvant for radiotherapy of solid tumors.

[0014] The present invention has the following beneficial effects: First, CAR-T cells struggle to penetrate solid tumors because the dense extracellular matrix within these tumors has pores smaller than the diameter of CAR-T cells, hindering their migration. The tumor microenvironment is immunosuppressive; cytokines such as TGF-β and IL-10 inhibit CAR-T cell activity, myeloid suppressor cells consume nutrients and induce apoptosis, and the hypoxic and acidic metabolic environment further weakens cell function. Radiotherapy, as a first-line clinical treatment, creates favorable conditions for CAR-T cell entry into solid tumors by reshaping the tumor microenvironment in multiple dimensions. Firstly, ionizing radiation can directly degrade extracellular matrix components, disrupting the fibrous network structure and enlarging matrix pores, thus creating "channels" for CAR-T cell migration. Secondly, it can relieve microenvironmental inhibition, simultaneously inducing tumor cells to express adhesion molecules and chemokines, guiding CAR-T cells home, while also killing immunosuppressive cells such as myeloid suppressor cells, downregulating inhibitory factors like TGF-β, and releasing tumor antigens and pro-inflammatory signals. High-dose radiotherapy can directly kill tumor cells, reduce tumor volume, and shorten the distance CAR-T cells need to reach the core lesion.

[0015] Secondly, we discovered for the first time that radiotherapy can induce tumor cells to release high levels of the chemokine receptor ligand CXCL13, which is the only ligand for the chemokine receptor CXCR5. Local radiotherapy to tumors can increase CXCL13 levels, which not only recruits endogenous CXCR5 cells... + CD8 +T cells also promote the entry of CXCR5 CAR-T cells into solid tumors, resulting in significant therapeutic effects. Our experimental results validated this, showing that the tumor suppression effect of radiotherapy combined with CXCR5 CAR-T cells was significantly superior to that of radiotherapy alone and CXCR5 CAR-T cells alone (P<0.05). Radiotherapy provides migration pathways and activation signals for CXCR5 CAR-T cells by degrading the extracellular matrix, reducing tumor stroma pressure, relieving the immunosuppressive microenvironment, and inducing the expression of the chemokine CXCL13. CXCR5 CAR-T cells, leveraging their chemokine response characteristics, synergistically enhance the effect of radiotherapy. This combined treatment strategy demonstrated significant tumor suppression and good safety in the experimental model, suggesting that radiotherapy can effectively sensitize the anti-tumor efficacy of CXCR5 CAR-T cells, providing a novel strategy with great translational potential for immunocellular therapy of solid tumors. Attached Figure Description

[0016] Figure 1 The figure shows the results of ELISA kit-based detection of CXCL13 levels in tumor tissues that have not undergone radiation therapy and have undergone conventional therapy.

[0017] Figure 2 This image shows the results of flow cytometry analysis of immune cell infiltration in tumor tissues before and after radiotherapy. In this image, A represents the CD8+ cell infiltration in the tumor after radiotherapy. + The proportion of CXCR5 in T cells, B represents the CXCR5 level of the tumor after radiotherapy. + CD8 + The proportion of T cells, C represents the tumor CXCR5 after radiotherapy. - CD8 + The proportion of T cells, D represents the CXCR5 level after radiotherapy. + CD8 + The mean fluorescence intensity of GZMB secreted by T cells, E represents CXCR5 after radiotherapy. + CD8 + Mean fluorescence intensity of TNF-α secreted by T cells, F represents CXCR5 after radiotherapy. + CD8 + The mean fluorescence intensity of IFN-γ secreted by T cells.

[0018] Figure 3 A schematic diagram of the structure of a chimeric antigen receptor (CAR).

[0019] Figure 4 This is a carrier spectrum of the present invention.

[0020] Figure 5The images show the killing effects of three different CAR-T cells on tumor cells. In A, the killing effect of CAR-T cells on colon cancer tumor cells after co-culturing the three different CAR-T cells with colon cancer tumor cells is shown. In B, the killing effect of CAR-T cells on pancreatic cancer tumor cells after co-culturing the three different CAR-T cells with pancreatic cancer tumor cells is shown.

[0021] Figure 6 The figure shows the migration results of three different CAR-T cells in untreated and radiotreated tumors.

[0022] Figure 7 The images show the in vivo anti-tumor treatment effects of CAR-T cell therapy combined with radiotherapy. In the images, A represents the change in colorectal cancer tumor volume, B represents the change in survival rate of colorectal cancer mice, C represents the image and weight of colorectal cancer tumors, D represents the change in pancreatic cancer tumor volume, E represents the change in survival rate of pancreatic cancer mice, and F represents the image and weight of pancreatic cancer tumors. Detailed Implementation

[0023] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0024] As described in this invention, the optimized CAR-T cell combined with radiotherapy is superior to conventional CAR combined with radiotherapy in treating solid tumors both in vitro and in vivo. Therefore, the combined cancer therapy provided by this invention is expected to be more effective than treatments that only include CAR-T cells.

[0025] I. Genetically engineered T cells In some instances, a specific CAR comprises a single polypeptide that may include (a) an extracellular antigen-binding domain including an antigen-binding moiety; (b) a hinge / transmembrane domain; and (c) one or more intracellular signaling domains (e.g., including a co-stimulatory signaling domain and one or more cytoplasmic signaling domains).

[0026] (a) Extracellular domain B7-H3 (CD276) is a type I transmembrane protein belonging to the B7 immune co-stimulatory and co-inhibitory family. Encoded by the chromosome 15q24 gene, it comprises an extracellular domain, a transmembrane domain, and a short intracellular domain. Due to exon repeats, there are two isoforms: 2IgB7-H3 and 4IgB7-H3, with 4IgB7-H3 being the predominant isoform in human cells. It is highly expressed in various tumors but has limited distribution in normal tissues, making it a highly promising target for tumor immunotherapy. Initially thought to stimulate T cell responses and IFN-γ production, most literature indicates its association with T cell suppression, playing a crucial role in tumor immune evasion, invasion and migration, angiogenesis, and gene regulation.

[0027] (b) Hinge domain and transmembrane domain The specific CAR polypeptide disclosed in this invention may include a hinge domain and a transmembrane domain located between an extracellular domain and an intracellular signal transduction domain. The hinge segment can be composed of any oligopeptide or polypeptide, and its main function is to impart flexibility to the CAR or its domain, avoiding steric hindrance. In some embodiments, the hinge segment may contain up to 300 amino acids; for example, the number of amino acids may range from 10 to 100, or from 5 to 20. In some embodiments, other regions of the CAR may also contain one or more hinge domains. In specific examples, the hinge domain may be derived from CD28, CD8, IgD, or IgG, such as IgG1 or IgG4. The transmembrane segment generally presents as a hydrophobic α-helix structure that spans the cell membrane. In this invention, a "transmembrane domain" refers to any protein structure capable of maintaining thermodynamic stability within the cell membrane (especially the eukaryotic cell membrane). For CARs containing such domains, the transmembrane domain plays a role in maintaining their stability. In some embodiments, the transmembrane domain may be derived from suitable cell surface receptors, such as the α, β, or ζ chain of a T cell receptor, and CD28, CD3ε, CD45, CD4, etc. In a particular instance, the transmembrane domain may be the CD8 transmembrane domain.

[0028] (c) Intracellular signal transduction domains The specific CAR disclosed in this invention includes an intracellular signal transduction domain. In some embodiments, the intracellular signal transduction domain may include a co-stimulatory signal transduction domain.

[0029] II. Further modifications to genetically engineered T cells Either of the genetically engineered T cells expressing the specific CAR or TAA-specific TCR and CAR disclosed in this invention can be further engineered to express cytokine chemokine (CC motif) receptor 5 (CXCR5).

[0030] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0031] Experimental Example 1 Measurement of CXCL13 levels in tumor samples Fresh, untreated, and radiation-treated mouse colon tumor tissue was rinsed in pre-chilled PBS (containing 1% penicillin / streptomycin) to remove blood and necrotic areas. The tissue was blotted dry with filter paper, weighed, and cut into approximately 1-2 mm³ pieces (operated on ice). The tissue pieces were added to a pre-chilled glass homogenizer or electric homogenizer tube containing RIPA lysis buffer (1:9 by weight) with 1% protease inhibitor and 1% phosphatase inhibitor. Homogenization was performed intermittently on ice (30 seconds of homogenization followed by 30 seconds on ice, repeated 3 times) until no tissue fragments were visible to the naked eye. The homogenate was centrifuged at 4°C (12,000 rpm, 15 minutes), and the supernatant (containing protein / nucleic acid / metabolites) was collected. The CXCL13 level in the tumor tissue was detected using an ELISA kit from OriGene (EA800062).

[0032] like Figure 1 The results showed that radiotherapy increased the expression of CXCL13 in tumor tissue.

[0033] Experiment Example 2 Flow cytometry detection of immune cell infiltration Mouse colon tumor tissue was dissected, minced, and digested with 1 mg / ml collagenase IV (Sigma) and 0.2 mg / ml DNase I (Sigma) at 37°C for 1 h. The resulting single-cell suspension was obtained by filtration. Mouse tumor-draining lymph nodes were isolated and prepared into single-cell suspensions by grinding.

[0034] Cell surface staining: Prepare tumor and lymph node cells as described above. Take 100 μL of cell suspension, add 100 μL of FACS Buffer, centrifuge at 1600 rpm for 5 min, and discard the supernatant. Stain live and dead cells first, in the dark. Add 50 μL of antibody mixture to each sample for surface antibody staining. Stain at 4℃ in the dark for 30 min. After staining, add 150 μL of FACS Buffer, wash 1-2 times, centrifuge at 1600 rpm for 5 min, discard the supernatant, and resuspend in 200 μL of FACS Buffer, ready for instrumental analysis.

[0035] Intracellular factor staining: Follow the procedures outlined in the Biolegend cytokine staining kit. Prepare tumor and lymph node cells as described above. After cell counting, dilute to the optimal number for stimulation and staining, plate, and incubate at 37°C for 5 hours. Collect cells into flow cytometry tubes, add 2 mL of PBS, centrifuge at 1900 rpm for 5 minutes, and incubate at 4°C. Surface staining is performed as described above. After staining, add 150 μL of FACS Buffer, wash twice, centrifuge at 1600 rpm for 5 minutes, discard the supernatant, add 80 μL of FIXation Buffer, and fix at 4°C for 20 minutes. Add 150 μL of 1×Perm Wash Buffer, wash twice, centrifuge at 1600 rpm for 5 minutes, and discard the supernatant. Add an appropriate amount of antibody diluted with 1×Perm Wash Buffer, and stain at 4°C for 30 minutes. After staining, add 200 μL of 1×Perm Wash Buffer, wash twice, centrifuge at 1600 rpm for 5 min, discard the supernatant, add 200 μL of LFACS Buffer, mix well, and transfer the cell suspension to a 1.5 mL EP tube for flow cytometry analysis.

[0036] Intracellular transcription factor staining: Perform the procedure according to the eBioscience transcription factor kit instructions. Surface staining was performed as described above. After staining, add 150 μL of FACS Buffer, wash twice, centrifuge at 1600 rpm for 5 min, discard the supernatant, add 80 μL of Transcription Factor Fixation Buffer, and fix at 4℃ for 20 min; add 150 μL of 1×Perm Wash Buffer, mix well, centrifuge at 1600 rpm for 5 min, and discard the supernatant. Add an appropriate amount of antibody diluted with 1×Perm Wash Buffer, and stain at 4℃ for 30 min. After staining, add 150-200 μL of 1×Perm Wash Buffer, wash twice, centrifuge at 1600 rpm for 5 min, and discard the supernatant. Add 200 μL of FACS Buffer, transfer the cell suspension to a 1.5 mL EP tube, and prepare for flow cytometry analysis.

[0037] like Figure 2 The results showed that radiotherapy significantly increased CXCR5 levels in tumors. + CD8 + The proportion of T cells ( Figure 2 A, Figure 2 B), while CXCR5 - CD8 + The increase in T cells was not significant. Figure 2C); Radiotherapy can significantly increase CXCR5 + CD8 + The average fluorescence intensity of functional molecules secreted by T cells that kill tumors, such as GZMB, TNF-α, and IFN-γ ( Figure 2 D- Figure 2 F), relative to CXCR5 - CD8 + The ability of T cells to secrete these factors suggests CXCR5 + CD8 + T cells have anti-tumor functions.

[0038] Experimental Example 3 Production of retroviruses and CAR-T cells The extracellular segment B7-H3 CAR used in this invention binds to the intracellular signaling domains of 8H9 scFv, CD28, and CD3ζ, as well as EGFP. CXCR5, 8H9 scFv, CD28, and CD3ζ also bind to EGFP. For the CXCR5-CAR, CXCR5 (372 amino acids [aa]) is separated at the C-terminus by P2A. The full-length CXCR5 contains its native secretory signal. The obtained CAR structures were sequence verified and used for downstream applications. CARs were identified by EGFP expression. + T cells were transfected into 6-well BOSC cells using jetPRIME transfection reagent (Polyplus) with 2.4 μg of each retroviral vector and 0.3 μg of pCL-Eco retroviral packaging vector, respectively, to prepare CAR retroviruses. After 48 h and 72 h, the culture supernatant containing retroviruses was collected through a 0.45 µm filter. Purified CD8... + T cells were stimulated overnight with plate-bound anti-CD3 (5 µg / ml) and anti-CD28 antibodies (2 µg / ml). Stimulated T cells were then spin-infected with retroviral supernatant at 37°C and 2500 rpm for 90 min. EGFP + Cells were analyzed by flow cytometry or sorted for further experiments. To detect T cell activation, proliferation, and apoptosis in vitro, lymph node cells were isolated and injected with 2.5 ng / ml anti-CD3 and 1 ng / ml soluble anti-CD28 lymph node cells at specified time points (activation), 72 hours (proliferation), or 48 hours (apoptosis). To assess CD8... + The response of T cells to cytokines IL-2 and IL-7 was assessed in vitro by stimulating naïve CD8 cells with 2.5 ng / ml anti-CD3 and 1 ng / ml soluble anti-CD28 antibodies. + T cells were collected for 48 hours. Then, activated CD8 cells were washed away. +T cells were then switched to stimulation with 2.5 ng / ml coated anti-CD3 antibody and 2.5 ng / ml IL-2 or sumIL-2 or 50 ng / ml IL-7 for up to 48 hours.

[0039] This embodiment constructed a chimeric antigen receptor against B7H3 (B7H3-CAR) and a chimeric antigen receptor against B7H3 fused with CXCR5 (B7H3-CXCR5-CAR). Schematic diagrams of the two CAR sequences are shown below. Figure 3 As shown; the B7H3-CAR includes a single-chain antibody sequence that specifically binds to the B7H3 antigen (Anti-B7H3 scFv, containing the light chain variable region VL, (G4S)3 Linker, and heavy chain variable region VH), the hinge region and transmembrane sequence of CD28, the CD28 co-stimulatory domain sequence, and the CD3ζ signaling domain sequence; the B7H3-CXCR5-CAR includes a single-chain antibody sequence that specifically binds to the B7H3 antigen (Anti-B7H3 scFv, containing the light chain variable region VL, (G4S)3 Linker, and heavy chain variable region VH), the hinge region and transmembrane sequence of CD28, the CD28 co-stimulatory domain sequence, the CD3ζ signaling domain sequence, the T2A linker, and the CXCR5 sequence. A schematic diagram of the chimeric antigen receptor (CAR) structure is shown below. Figure 3 The vector spectrum can be found in Figure 4 .

[0040] The method for constructing lentiviral plasmid vectors is as follows: The vector plasmid was purchased from the AddGene website (#227902).

[0041] The B7H3-CAR and CXCR5 genes were synthesized using whole-genome synthesis. BglII and HindIII A fusion protein system was constructed by inserting a double-enzyme digestion vector plasmid into the fully synthesized B7H3-CAR gene. BsiW I and Nru The B7H3-CAR gene vector was double-digested with enzymes to construct an overexpressing B7H3-CXCR5-CAR. After digestion at 37℃ for 30 min, DNA was subjected to 1.5% agarose gel electrophoresis, followed by purification and recovery using Tiangen's agarose gel electrophoresis kit. The ligation system of the vector with the B7H3-CAR gene / CXCR5 gene fragment is as follows:

[0042] Table 1: Connection System Ligation was performed at 37℃ for 1.5 h. The ligation product was directly transformed into Stbl3 competent E. coli cells. 200 μL of the transformation product was plated onto ampicillin-resistant LB agar plates and incubated upside down at 37℃ overnight. The next morning, five single clones were randomly selected for colony PCR identification, and positive clones were sent for sequencing. After successful sequencing, the colonies were amplified, extracted, and frozen for future infection use.

[0043] The amino acid sequence of B7H3-CAR is shown in SEQ ID NO.1, and the gene sequence encoding B7H3-CAR is shown in SEQ ID NO.2; the amino acid sequence of B7H3-CXCR5-CAR is shown in SEQ ID NO.3, and the gene sequence encoding B7H3-CXCR5-CAR is shown in SEQ ID NO.4.

[0044] SEQ ID NO.1: MEFGLSWLFLVAILKGVQCQVQLVQSGAEVVKPGASVKLSCKTSGYTFTNYDINWVRQRPGQGLEWIGWIFPGDDSTQYNEKFKGKATLTTDTSTSTAYMELSSLRSEDTAVYFCARQTTGTWFAYWGQGTLVTVSS GGGGSGGGGSGGGGS EIVMTQSPATLSVSPGERVTLSCRASQSISDYLYWYQQKSHESPRLLIKYASQSISGIPARFSGSGSGSEFTLTINSVEPEDVGVYYCQNGHSFPLTFGQGTKLELKRTRIEFMYPPPYLDNERSNGTIIHIKEKHLCHTQSSPKLFWALVVVAGVLFCYGLLV TVALCVIWTNSRRNRGGQSDYMNMTPRRPGLTRKPYQPYAPARDFAAYRPRAKFSRSAETAANLQDPNQLFNELNLGRREEFDVLEKKRARDPEMGGKQQRRRNPQEGVYNALQKDKMAEAYSEIGTKGERRRGKGHDGLFQGLSTATKDTFDALHMQTLAPR.

[0045] In this sequence, a single underline represents the (G4S)3Linker, a double underline represents the CD28 co-stimulatory domain sequence, and a thick line represents the CD3ζ signal transduction domain sequence.

[0046] SEQ ID NO.2:ATGGAATTTGGCCTGAGCTGGCTGTTTCTGGTGGCGATTCTGAAAGGCGTGCAGTGCCAGGTGCAGCTGGTGCAGAGCGGCGCGGAAGTGGTGAAACCGGGCGCGAGCGTGAAACTGAGCTGCAAAACCAGCGGCTATACCTTTACCAACTATGATATTAACTGGGTGCGCCAGCGCCCGGGCCAGGGCCTGGAATGGATTGGCTGGATTTTTCCGGGCGATGATAGCACCCAGTATAACGAAAAATTTAAAGGCAAAGCGACCCTGACCACCGATACCAGCACCAGCACCGCGTATATGGAACTGAGCAGCCTGCGCAGCGAAGATACCGCGGTGTATTTTTGCGCGCGCCAGACCACCGGCACCTGGTTTGCGTATTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGC GGTGGAGGCGGTTCAGGCGGAGGTG GCTCTGGCGGTGGCGGATCGGAAATTGTGATGACCCAGAGCCCGGCGACCCTGAGCGTGAGCCCGGGCGAAAGGGTGACCCTGAGCTGCCGCGCGAGCCAGAGCATTAGCGATTATCTGTATTGGTATCAGCAGAAAAGCCATGAAAGCCCGCGCCTGCTGATTAAATATGCGAGCCAGAGCATTAGCGGCATTCCGGCGCGCTTTAGCGGCAGCGGCAGCGGCAGCGAATTTACCCTGACCATTAACAGCGTGGAACCGGAAGATGTGGGCGTGTATTATTGCCAGAACGGCCATAGCTTTCCGCTGACCTTTGGCCAGGGCACCAAACTGGAACTGAAACGCACGCGTATCGAGTTCATGTACCCCCCTCCCTACCTGGACAACGAGAGAAGCAACGGCACCATCATCCACATCAAAGAAAAGCACCTGTGCCACACCCAGAGCAGCCCCAAGCTGTTCTGGGCCCTGGTGGTGGTGGCCGGCGTGCTGTTCTGTTACGGCCTGCTGGTCACAGTGGCCCTGTGCGTGATCTGGACCAACAGCAGAAGAAACAGAGGCGGCCAGAGCGACTACATGAACATGACCCCCAGAAGGCCAGGCCTGACCAGAAAGCCCTACCAGCCCTACGCCCCTGCCAGAGACTTCGCCGCCTACAGACCCAGAGCCAAGTTCAGCAGATCCGCCGAGACAGCCGCCAACCTGCAGGATCCCAACCAGCTGTTCAACGAGCTGAACCTGGGCAGACGGGAGGAATTCGACGTGCTGGAAAAGAAGAGAGCCAGGGACCCCGAGATGGGCGGCAAGCAGCAGAGAAGAAGAAACCCTCAGGAAGGCGTCTACAACGCCCTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGCACCAAGGGCGAGAGAAGAAGGGGCAAGGGCCACGATGGCCTGTTCCAGGGCCTGTCCACCGCCACCAAGGACACCTTCGACGCCCTGCACATGCAGACCCTGGCCCCCAGA。Among them, the single underline is the (G4S)3Linker, the double underline is the CD28 co-stimulatory domain sequence, and the thick line is the CD3ζ signal transduction domain sequence.

[0047] SEQ ID NO.3: MEFGLSWLFLVAILKGVQCQVQLVQSGAEVVKPGASVKLSCKTSGYTFTNYDINWVRQRPGQGLEWIGWIFPGDDSTQYNEKFKGKATLTTDTSTSTAYMELSSLRSEDTAVYFCARQTTGTWFAYWGQGTLVTVSS GGGGSGGGGSGGGGS EIVMTQSPATLSVSPGERVTLSCRASQSISDYLYWYQQKSHESPRLLIKYASQSISGIPARFSGSGSGSEFTLTINSVEPEDVGVYYCQNGHSFPLTFGQGTKLELKRTRIEFMYPPPYLDNERSNGTIIHIKEKHLCHTQSSPKLFWALVVVAGVLFCYGLLVTVALCVIWTNSRRNRGGQSDYMNMTPRRPGLTRKPYQPYAPARDFAAYRPRAKFSRSAETAANLQDPNQLFNELNLGRREEFDVLEKKRARDPEMGGKQQRRRNPQEGVYNALQKDKMAEAYSEIGTKGERRRGKGHDGLFQGLSTATKDTFDALHMQTLAPRKLGSGEGRGSLLTCGDVEENPGPRTMNYPLTLDMGSITYNMDDLYKELAFYSNSTEIPLQDSNFCSTVEGPLLTSFKAVFMPVAYSLIFLLGMMGNILVLVILERHRHTRSSTETFLFHLAVADLLLVFILPFAVAEGSVGWVLGTFLCKTVIALHKINFYCSSLLLACIAVDRYLAIVHAVHAYRRRRLLSIHITCTAIWLAGFLFALPELLFAKVGQPHNNDSLPQCTFSQENEAETRAWFTSRFLYHIGGFLLPMLVMGWCYVGVVHRLLQAQRRPQRQKAVRVAILVTSIFFLCWSPYHIVIFLDTLERLKAVNSSCELSGYLSVAITLCEFLGLAHCCLNPMLYTFAGVKFRSDLSRLLTKLGCAGPASLCQLFPNWRKSSLSESENATSLTTF.

[0048] In this sequence, a single underline represents the (G4S)3Linker, a double underline represents the CD28 co-stimulatory domain sequence, a thick line represents the CD3ζ signal transduction domain sequence, a dotted underline represents the T2A linker, and a dashed underline represents the CXCR5 sequence.

[0049]

[0050] In this sequence, a single underline represents the (G4S)3Linker, a double underline represents the CD28 co-stimulatory domain sequence, a thick line represents the CD3ζ signal transduction domain sequence, a dotted underline represents the T2A linker, and a dashed underline represents the CXCR5 sequence.

[0051] Experimental Example 5 CAR-T cell co-culture cytotoxicity assay in tumor cells To evaluate the cytotoxicity of CAR-T cells, the function of specific CAR-T cells was analyzed by co-culturing them with target tumor cells. For the cell co-culture experiment, 96-well U-shaped plates were prepared and placed in a culture environment at 37°C. 3 × 10⁶ cells were selected for the experiment. 4 Target cells were co-cultured with CXCR5 CAR-T cells, CAR-T cells, and unmodified UT cells, respectively. Three different effector cell (E) to target cell (T) ratios were set: 1:2, 1:1, and 2:1. The co-culture process lasted 12 to 16 hours, during which the cells interacted fully at 37°C. After the co-culture period, the percentage of tumor cells killed by CAR-T cells was determined by flow cytometry.

[0052] like Figure 5 The results showed that specific anti-B7H3-CXCR5 CAR-T cells were more effective at killing tumor cells than ordinary CAR-T cells.

[0053] Experimental Example 6 In vitro migration experiment of CAR-T cells by radiotherapy In this study, we selected a 24-well transbeam plate with 6 mm nesting inserts, using polyethylene terephthalate (PET) tracking membranes (supplied by Corning). This material is stable and effectively meets the experimental requirements for cell culture and observation. First, 1×10⁻⁶ PET cells were... 6Three different types of CAR-T cells were resuspended in complete DMEM medium. Complete DMEM medium is rich in various nutrients required for cell growth, providing a favorable environment for CAR-T cell survival and ensuring cell viability. The resuspended CAR-T cells were carefully added to the upper chamber of a transwell plate. Simultaneously, two different treatment methods were set up for tumor tissue samples: one group consisted of untreated tumor tissue, and the other group consisted of tumor tissue treated with irradiation for 7 days. Both groups of tumor tissue were homogenized to thoroughly disrupt the tumor tissue, releasing intracellular and interstitial substances. After homogenization, impurities were separated by centrifugation, and the supernatant was collected. 500 μl of the supernatant was added to the bottom chamber of the transwell plate. The transwell plate was incubated under suitable conditions for 4 hours. After incubation, migrating cells in the bottom chamber were carefully collected. Subsequently, flow cytometry was used to analyze the collected migrating cells.

[0054] like Figure 6 The results showed that the substances released from the tumor tissue supernatant after radiotherapy had a significant advantage in recruiting CXCR5 CAR-T cells compared to CAR-T cells.

[0055] Experimental Example 7 In vivo antitumor effects of combined CAR-T cell therapy and radiotherapy First, a tumor-bearing mouse model was constructed. The selected tumor cell lines were the MC38 colon cancer cell line, which highly expresses B7H3, and the Panc02 pancreatic cancer cell line. The genetic background of both mice was C57BL / 6. On Day 0, 1×10⁶ tumors were subcutaneously implanted into the mice. 6 Tumor cells underwent radiotherapy starting on Day 10, followed by specific CAR-T cell therapy on Day 13, with a cell count of 5 × 10⁶ cells. 5 / mice, and measure the tumor's long diameter (L) and short diameter (W) the next day, according to the formula V=L×W 2 / 2, calculate tumor volume, plot tumor growth curve, and observe mouse survival. The experimental group was divided into 6 groups: control group, radiotherapy group, CAR-T cell therapy group, radiotherapy combined with CAR-T therapy group, CXCR5 CAR-T therapy group, and radiotherapy combined with radiotherapy group.

[0056] like Figure 7The results showed that the CXCR5 CAR-T combined with radiotherapy group was significantly more effective than the CAR-T combined with radiotherapy group in inhibiting tumor growth and prolonging the survival of mice, and was also superior to the radiotherapy alone group and the CXCR5 CAR-T cell therapy alone group. Furthermore, the efficacy of the CXCR5 CAR-T cell therapy group was more pronounced than that of the CAR-T cell therapy group.

[0057] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0058] References: 1: Keshavarz, A. et al. Recent findings on chimeric antigen receptor(CAR)-engineered immune cell therapy in solid tumors and hematologicalmalignancies. Stem Cell Res Ther 13, 482 (2022). https: / / doi.org / 10.1186 / s13287-022-03163-w.

[0059] 2: Yang, YH, Liu, JW, Lu, C.&Wei, JF CAR-T Cell Therapy for Breast Cancer: From Basic Research to Clinical Application. Int J Biol Sci 18,2609-2626 (2022). https: / / doi.org / 10.7150 / ijbs.70120.

[0060] 3: Shah, PD et al. Phase I Trial of Autologous RNA-electroporatedcMET-directed CAR T Cells Administered Intravenously in Patients with Melanoma and Breast Carcinoma. Cancer Res Commun3, 821-829 (2023). https: / / doi.org / 10.1158 / 2767-9764.Crc-22-0486。

[0061] 4:Junghans, R. P. et al. Phase I Trial of Anti-PSMA Designer CAR-TCells in Prostate Cancer: Possible Role for Interacting Interleukin 2-T CellPharmacodynamics as a Determinant of Clinical Response. Prostate 76, 1257-1270(2016). https: / / doi.org / 10.1002 / pros.23214。

[0062] 5:Ahmed, N. et al. HER2-Specific Chimeric Antigen Receptor-ModifiedVirus-Specific T Cells for Progressive Glioblastoma: A Phase 1 Dose-Escalation Trial. JAMA Oncol 3, 1094-1101 (2017). https: / / doi.org / 10.1001 / jamaoncol.2017.0184。

[0063] 6Louis, C. U. et al. Antitumor activity and long-term fate of chimericantigen receptor-positive T cells in patients with neuroblastoma. Blood 118,6050-6056 (2011). https: / / doi.org / 10.1182 / blood-2011-05-354449。

[0064] 7:Straathof, K. et al.Antitumor activity without on-target off-tumortoxicity of GD2-chimeric antigen receptor T cells in patients withneuroblastoma. Sci Transl Med 12(2020). https: / / doi.org / 10.1126 / scitranslmed.abd6169。

[0065] 8Fang, J., Nakamura, H.&Maeda, H. The EPR effect: Unique features oftumor blood vessels for drug delivery, factors involved, and limitations andaugmentation of the effect. Adv Drug Deliv Rev 63, 136-151 (2011). https: / / doi.org / 10.1016 / j.addr.2010.04.009。

[0066] 9:Slaney, C. Y., Kershaw, M. H.&Darcy, P. K. Trafficking of T cellsinto tumors. Cancer Res 74, 7168-7174 (2014). https: / / doi.org / 10.1158 / 0008-5472.Can-14-2458。

[0067] 10:Zhang, X., Zhu, L., Zhang, H., Chen, S.&Xiao, Y. CAR-T CellTherapy in Hematological Malignancies: Current Opportunities andChallenges. Front Immunol 13, 927153 (2022). https: / / doi.org / 10.3389 / fimmu.2022.927153。

[0068] 11:Marofi, F. et al. CAR T cells in solid tumors: challenges andopportunities. Stem Cell Res Ther 12, 81 (2021). https: / / doi.org / 10.1186 / s13287-020-02128-1。

Claims

1. The application of a CAR-T cell overexpressing the CXCR5 gene and targeting B7H3 in the preparation of a drug for the treatment and / or adjuvant treatment of solid tumors, characterized in that, The CAR-T cells are T cells transfected with a viral vector, the viral vector containing a nucleic acid molecule encoding a chimeric antigen receptor fused with CXCR5 against B7H3, the amino acid sequence of the chimeric antigen receptor being shown in SEQ ID NO.3, and the nucleotide sequence of the nucleic acid molecule being shown in SEQ ID NO.4; the solid tumor is colon cancer or pancreatic cancer; the adjuvant therapy drug for solid tumors is a drug used to assist radiotherapy for solid tumors.

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