Construction method of muc13 chimeric antigen receptor t cells and application thereof in treatment of gastrointestinal tumors

Abstract

The application provides a construction method of a MUC13 chimeric antigen receptor T cell and application thereof in treatment of gastrointestinal tumors, and relates to the technical field of biological medicine. It is disclosed that MUC13 can be used as a target point for CAR-T treatment of gastrointestinal tumors, and MUC13-CAR-T cells developed based on the target point exhibit strong anti-tumor activity in in-vivo and in-vitro experiments, and can efficiently recognize and remove gastrointestinal tumor cells. In in-vitro experiments, the MUC13-CAR-T cells can specifically recognize and kill gastrointestinal tumor cell lines, and have no significant killing effect on cells with knocked-out MUC13, exhibiting high specificity; in in-vivo experiments, the MUC13-CAR-T cells are transfused into a tumor-transplanted mouse model, and a significant tumor growth inhibition effect can be observed, thereby providing an efficient, safe and novel treatment scheme for treatment of gastrointestinal tumors.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method for constructing MUC13 chimeric antigen receptor T cells and their application in the treatment of gastrointestinal tumors. Background Technology

[0002] Chimeric antigen receptor T-cell (CAR-T) immunotherapy, a novel immunotherapy approach, involves introducing artificially designed chimeric antigen receptor (CAR) genes into T cells using gene editing technology, enabling them to specifically recognize and target tumor antigens. After in vitro expansion, these modified CAR-T cells are reinfused into the patient to perform tumor-killing tasks. Compared to traditional T-cell therapy, CAR-T cell therapy overcomes MHC restrictions, being activated simply by binding to the target antigen, thus efficiently killing tumor cells. Although CAR-T cell therapy possesses advantages such as high specificity, potent lethality, and durable efficacy, showing significant efficacy in hematological malignancies, it still faces many obstacles in the treatment of solid tumors. These include the high heterogeneity of solid tumor antigens leading to immune escape, physical or cytokine barriers, and the presence of other immunosuppressive molecules in the tumor microenvironment, which hinder CAR-T cell entry or limit their persistence.

[0003] Gastrointestinal tumors are malignant diseases with high morbidity and mortality rates worldwide, accounting for 15-20% of adult malignant tumors. Subtypes such as gastric cancer, colorectal cancer, and pancreatic cancer face significant challenges in clinical treatment due to difficulties in early diagnosis, high heterogeneity, and high recurrence rates. Traditional treatments such as surgical resection, chemotherapy, and radiotherapy have limited efficacy in advanced or metastatic patients and are generally associated with drug resistance and severe side effects. Although immunotherapy has opened new avenues for cancer treatment, current CAR-T cell therapy for gastrointestinal tumors mainly focuses on specific targets, such as HER2, EGFR, and GD2. However, the expression rates of these targets are generally low in patients with gastrointestinal tumors, preventing many patients from benefiting from this treatment.

[0004] Mucin 13 (MUC13) is a transmembrane protein whose expression is restricted in normal tissues, but it is abnormally highly expressed in most gastrointestinal tumors, and its expression level is closely related to tumor invasion, metastasis, and poor prognosis. However, there is currently no CAR-T therapy targeting MUC13. Therefore, there is an urgent need to develop a CAR-T cell therapy that specifically targets MUC13, which is expected to become a key breakthrough in the treatment of gastrointestinal tumors. Summary of the Invention

[0005] (a) Technical problems to be solved

[0006] To address the shortcomings of existing technologies, this invention provides a method for constructing MUC13-targeted chimeric antigen receptor T cells and their application in the treatment of gastrointestinal tumors.

[0007] (II) Technical Solution

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] In a first aspect, the present invention provides a monoclonal antibody targeting MUC13, wherein the antibody is a single-chain antibody (scFv), the heavy chain variable region of the antibody is selected from MM06-VH, MM07-VH or MM08-VH, and the light chain variable region of the antibody is selected from MM06-VL, MM07-VL or MM08-VL; the amino acid sequences of MM06-VH, MM07-VH, MM08-VH, MM06-VL, MM07-VL and MM08-VL are as shown in SEQ ID NO:1-6.

[0010] Further, the heavy chain of the antibody includes MM06-Heavy chain, MM07-Heavy chain, or MM08-Heavy chain, and the light chain of the antibody includes MM06-Light chain, MM07-Light chain, or MM08-Light chain; the amino acid sequences of the MM06-Heavy chain, MM07-Heavy chain, MM08-Heavy chain, MM06-Light chain, MM07-Light chain, and MM08-Light chain are as shown in SEQ ID NO:7-12.

[0011] Furthermore, the MM06-scFv single-chain variable fragment is composed of MM06-VH with the amino acid sequence shown in SEQ ID NO:1 and MM06-VL with the amino acid sequence shown in SEQ ID NO:4, and the amino acid sequence of MM06-scFv is shown in SEQ ID NO:13-16.

[0012] Furthermore, the MM07-scFv single-chain variable fragment is composed of MM07-VH of the amino acid sequence shown in SEQ ID NO:2 and MM07-VL of the amino acid sequence shown in SEQ ID NO:5, and the amino acid sequence of MM07-scFv is shown in SEQ ID NO:17-20.

[0013] Furthermore, the MM08-scFv single-chain variable fragment is composed of MM08-VH with the amino acid sequence shown in SEQ ID NO:3 and MM08-VL with the amino acid sequence shown in SEQ ID NO:6, and the amino acid sequence of MM08-scFv is shown in SEQ ID NO:21-24.

[0014] Specifically, the amino acid sequences of the complementarity-determining regions of antibodies MM06, MM07, and MM08 are shown in the table below:

[0015]

[0016] Secondly, the present invention provides the application of the monoclonal antibody targeting MUC13 in the preparation of reagents for detecting the MUC13 antigen.

[0017] Furthermore, the detection reagents include immunohistochemical detection kits, flow cytometry detection kits, enzyme-linked immunosorbent assay kits, etc.

[0018] Thirdly, the present invention provides a chimeric antigen receptor targeting MUC13, wherein the chimeric antigen receptor comprises, from the N-terminus to the C-terminus, the following components in sequence:

[0019] (1) MUC13 specific binding domain;

[0020] (2) Transmembrane domain;

[0021] (3) Intracellular signal transduction domain.

[0022] Furthermore, the transmembrane domain is selected from (G4S)3 or (G4S)4; the intracellular signal transduction domain includes a co-stimulatory signal domain and a T cell activation domain, wherein the co-stimulatory signal domain is selected from any one of the intracellular domains of CD28, 4-1BB, OX40 or ICOS, and the T cell activation domain is the intracellular segment of CD3ζ.

[0023] Fourthly, the present invention provides a MUC13-CAR-T cell targeting MUC13 chimeric antigen receptor (hereinafter referred to as MUC13-CAR-T), wherein the surface of the T cell expresses the chimeric antigen receptor described above.

[0024] Furthermore, the method for constructing the MUC13-targeting chimeric antigen receptor T cells includes the following steps:

[0025] (1) T cells were isolated from peripheral blood of the donor;

[0026] (2) Activate T cells with anti-CD3 / CD28 antibody;

[0027] (3) Using viral transduction technology, the gene encoding the chimeric antigen receptor is transduced into T cells;

[0028] (4) Expand the transferred T cells to a therapeutically effective dose in a culture medium containing IL-2.

[0029] Fifthly, the present invention provides the application of the above-mentioned MUC13-targeting monoclonal antibody, MUC13-targeting chimeric antigen receptor, and MUC13-targeting chimeric antigen receptor T cell in the preparation of drugs for tumor immunotherapy.

[0030] Furthermore, the tumor is selected from pancreatic cancer, gastric cancer, colorectal cancer, etc.

[0031] (III) Beneficial Effects

[0032] This invention provides a method for constructing chimeric antigen receptor T cells targeting MUC13 and their application in the treatment of gastrointestinal tumors. This invention reveals that MUC13 can serve as a target for CAR-T therapy of gastrointestinal tumors. It is highly specifically expressed in various gastrointestinal tumors, while being almost undetectable in most normal tissues. This characteristic significantly reduces the risk of off-target toxicity and extends the safety window for treatment. MUC13-CAR-T cells developed based on this target exhibit potent anti-tumor activity in both in vitro and in vivo experiments, effectively recognizing and eliminating gastrointestinal tumor cells. In in vitro experiments, MUC13-CAR-T cells specifically recognize and kill gastrointestinal tumor cell lines, while showing no significant killing effect on cells with MUC13 knockout, demonstrating its high specificity. In in vivo experiments, infusion of MUC13-CAR-T cells into xenograft mouse models showed a significant tumor growth inhibition effect. Simultaneously, no significant toxic side effects were observed during treatment, further validating the safety of this immunotherapy. This invention provides a highly efficient and safe novel treatment regimen for gastrointestinal tumors. Attached Figure Description

[0033] Figure 1 To analyze the expression of MUC13 in normal and tumor tissues using database analysis.

[0034] Figure 2 The results of SDS-PAGE electrophoresis of monoclonal antibodies MM01-MM08 are shown.

[0035] Figure 3 The results show the specificity of the monoclonal antibody MM01-MM08 in binding to the MUC13 antigen.

[0036] Figure 4 The expression levels of CD69 on the surface of 12 CAR-Jurkat cells were determined.

[0037] Figure 5 This describes the killing effect of MUC13-CAR-T cells on pancreatic cancer tumors.

[0038] Figure 6 This describes the killing effect of MUC13-CAR-T cells on gastric cancer tumors.

[0039] Figure 7 The killing effect of MUC13-CAR-T cells on colorectal cancer tumors.

[0040] Figure 8 The expression of MUC13-CAR-T cells in normal tissues. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0042] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0043] Unless otherwise stated, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art.

[0044] As used herein, a "chimeric antigen receptor (CAR)" refers to a fusion protein comprising an extracellular domain capable of binding to an antigen, a transmembrane domain consisting of a polypeptide not derived from that extracellular domain, and at least one intracellular domain. "Chimeric antigen receptor (CAR)" is sometimes referred to as a "chimeric receptor," a "T-body," or a "chimeric immune receptor (CIR)." An "extracellular domain capable of binding to an antigen" refers to any oligopeptide or polypeptide capable of binding to an antigen. An "intracellular domain" refers to any oligopeptide or polypeptide known to function as a domain that transmits signals to activate or inhibit intracellular biological processes.

[0045] As used in this article, a “domain” refers to a region in a polypeptide that folds into a structure independently of other regions.

[0046] As used herein, "single-chain antibody (scFv)" refers to a single-chain polypeptide derived from an antibody that retains its ability to bind to an antigen. An example of scFv includes an antibody polypeptide formed using recombinant DNA technology, wherein the Fv regions of the immunoglobulin heavy chain (H chain) and light chain (L chain) fragments are linked by spacer sequences. Various methods for engineering scFv are known to those skilled in the art.

[0047] MUC13 is a transmembrane protein whose expression is restricted in normal tissues, but it is abnormally highly expressed in most gastrointestinal tumors, and its expression level is closely related to tumor invasion, metastasis and poor prognosis.

[0048] Example 1

[0049] Preparation of MUC13 monoclonal antibody and detection of MUC13 antigen recognition ability

[0050] 1. Database analysis of MUC13 expression in normal and tumor tissues

[0051] The expression of MUC13 in gastrointestinal tumors and normal tissues was analyzed using the TCGA and GEO databases, respectively. Results are as follows: Figure 1 As shown, MUC13 is expressed at low levels or not at all in normal tissues, but at high levels in gastrointestinal tumors, with the expression level being significantly higher than that in normal gastrointestinal tissues. This indicates that MUC13 is an important oncogene in gastrointestinal tumors, especially pancreatic cancer, gastric cancer, and colorectal cancer, and its expression is abnormally elevated in tumor tissues.

[0052] 2MUC13 Antigen Preparation and Purification

[0053] The MUC13 gene was amplified by PCR and cloned into an expression vector. After sequencing verification, the vector was transfected into HEK293 cells. After 48 hours, the culture medium was replaced with serum-free medium and cultured for another 6 days. The cell supernatant was collected and purified by metal affinity chromatography and size exclusion chromatography to obtain high-purity recombinant MUC13 protein. The purity of the protein was verified by SDS-PAGE electrophoresis.

[0054] The results are as follows Figure 2 As shown, eight high-purity monoclonal antibodies targeting MUC13, MM01-MM08, were successfully prepared and then... Figure 2 As shown in B, SDS-PAGE verification confirmed that all eight prepared monoclonal antibodies possessed normal structural characteristics.

[0055] 3. Immunization of model mice

[0056] Purified MUC13 protein was emulsified by mixing a 1:1 ratio of Freund's complete adjuvant (CFA) and administering the initial immunization (100 μg per mouse) to 4-week-old BALB / c mice via intraperitoneal injection. Booster immunizations were administered on days 14 and 28 post-immunization with 50 μg of MUC13 antigen and Freund's incomplete adjuvant (IFA). One week after the third immunization, blood was collected from the mice via ocular sampling. Serum was collected by centrifugation at 3500 rpm for 10 min at 4°C, and serum antibody titers were detected by ELISA.

[0057] 4. Spleen cell isolation

[0058] Mice with the highest titer were anesthetized and euthanized. The spleen was removed by laparotomy in a biosafety cabinet. After removing fat and fascia tissue, the spleen was ground and filtered. Red blood cells were lysed and centrifuged. The spleen cells were resuspended in RPMI 1640 medium and the viability was confirmed to be >95% by trypan blue staining. The spleen cells were electrofused with SP2 / 0 myeloma cells at a ratio of 1:1. After termination, the cells were centrifuged and resuspended in HAT medium containing 20% ​​FBS.

[0059] 5. Hybridoma screening and antibody production

[0060] Fusion cells were seeded into 96-well plates, and unfused cells were removed after 7 days by changing the medium. Positive clones were screened by ELISA (MUC13 antigen coating, HRP secondary antibody detection), and positive monoclonal wells were selected for amplification. After amplification of monoclonal hybridoma cells, the supernatant was collected, centrifuged, filtered, and purified to obtain monoclonal antibodies.

[0061] 6. Antigen Recognition Ability Test

[0062] Collect 293T cells overexpressing MUC13 and wild-type cells, and adjust the concentration to 1×10⁻⁶. 6 Cells were incubated at 4°C for 30 min in the dark with fluorescently labeled monoclonal antibodies per mL. After washing with PBS, the fluorescence intensity was analyzed by flow cytometry. The fluorescence signals of the experimental group and the isotype control were compared. A significant rightward shift in the experimental group indicated specific antibody binding. The specifically bound monoclonal antibodies were then co-incubated with Aspc1 cells naturally expressing MUC13 and Aspc1 cells with MUC13 knockout, respectively, to observe their specific binding.

[0063] The results are as follows Figure 3As shown, flow cytometry results indicated that only the MM06, MM07, and MM08 monoclonal antibodies bound to the overexpressing 293T cells. Furthermore, the MM06, MM07, and MM08 monoclonal antibodies could bind to Aspc1 cells that naturally express MUC13. When MUC13 was knocked out, Aspc1 cells could not bind to the monoclonal antibodies, indicating that the monoclonal antibodies MM06, MM07, and MM08 have good MUC13 antigen binding specificity.

[0064] Example 2

[0065] Screening for MUC13-specific CAR structures

[0066] 1. Antibody variable region sequence amplification and scFv recombination

[0067] The heavy chain variable region (VH) and light chain variable region (VL) gene sequences were extracted from three monoclonal antibodies, MM06, MM07, and MM08, respectively. These sequences were then recombined into single-chain antibodies (scFv) using a flexible linker. By adjusting the arrangement of VH and VL and the length of the linker, 12 different CAR structures were obtained, and their parameters are shown in Table 1.

[0068] Table 1. Design parameters for 12 CAR structures built based on MM06, MM07, and MM08 scFv

[0069]

[0070]

[0071] 2CAR transduction and expression validation

[0072] The 12 CAR structures described above were transduced into Jurkat cells to construct CAR-Jurkat cell lines. After transduction, the cells were stained with Protein L, and the expression of CARs on the surface of Jurkat cells was detected by flow cytometry to confirm the successful transduction and normal expression of each CAR.

[0073] 3. Antigen-dependent activation and non-antigen-dependent activation detection

[0074] Aspc1 tumor cells were divided into 2×10 4Cells were seeded at a density of cells / well in 96-well U-shaped plates, with 12 different CAR-Jurkat cell lines added at an effector-to-target ratio of 1:1, in triplicate for each group. Cells were co-incubated at 37°C and 5% CO2 for 24 hours. Cells were then collected after co-incubation, and the expression level of CD69 on the CAR-Jurkat cells was detected by flow cytometry to assess the antigen-dependent activation effect of these 12 CAR structures. Simultaneously, the expression level of CD69 on the surface of the 12 CAR-Jurkat cells that were not co-incubated with tumor cells was directly detected by flow cytometry to assess the antigen-independent activation effect, i.e., tetanic signal transduction.

[0075] 4 Results

[0076] like Figure 4 As shown, the CAR-3 structure exhibits the highest antigen-dependent activation and weak tetanic signal transduction, indicating that the MM06-derived CAR construct, with its VL-VH alignment and the short peptide linker ((G4S)3), effectively inhibits tetanic signal transduction while achieving optimal antigen-dependent activation. This suggests that the CAR structure may have stronger antitumor activity and a lower risk of T cell exhaustion in vivo.

[0077] Example 3

[0078] Construction of MUC13-CAR-T cells

[0079] 1. Isolation of peripheral blood mononuclear cells (PBMCs)

[0080] Peripheral blood mononuclear cells (PBMCs) were isolated using density gradient centrifugation: Whole blood samples were transferred to centrifuge tubes and centrifuged at 2000 rpm for 5 min. The supernatant plasma layer was carefully discarded. An equal volume of PBS was added to the cell pellet and mixed thoroughly. The diluted blood was slowly added to the supernatant of the lymphocyte separation medium (maintaining separation), and then density gradient centrifugation (2000 rpm, 25 min) was used to obtain the white membrane layer (rich in mononuclear cells; gentle handling is crucial to avoid unclear separation and reduced cell recovery efficiency). The white membrane layer was transferred to a new centrifuge tube, diluted with an equal volume of PBS, and centrifuged at 2000 rpm for 5 min. The supernatant was discarded. Depending on the size of the cell pellet, 5-20 mL of erythrocyte lysis buffer was added, and the mixture was incubated at room temperature for 10 min (mixing 2-3 times during incubation). An equal volume of magnetic separation buffer was then added, and the mixture was inverted to terminate lysis. After centrifugation at 2000 rpm for 5 min, the supernatant was discarded. If residual erythrocytes remained, the lysis steps could be repeated. Finally, the cells were resuspended using magnetic sorting buffer, and an appropriate amount of cells were labeled with antibodies by flow cytometry. Then, the proportion of T cells in PBMCs was detected by flow cytometry.

[0081] 2T cell purification and activation

[0082] T cell purification and activation were performed using Dynabeads Human T-Expander CD3 / CD28 magnetic beads: Based on the flow cytometry results, an appropriate amount of PBMC was added to magnetic sorting buffer and diluted to 2-4 × 10⁻⁴. 7 100 μL magnetic beads / mL. 7 Add premixed magnetic beads at a ratio of 1:10 T cells, and add 2 volumes or at least 1 mL of magnetic sorting buffer. After enriching the magnetic beads using a magnetic field (incubate for 1-2 min), discard the supernatant, resuspend the beads, add the cell suspension, and incubate at room temperature for 30 min, gently inverting to mix every 5 min. After incubation, magnetically attach for 2 min, discard the supernatant, add 10-12 mL of T cell basal medium without effector cytokines, wash, and magnetically attach again for 2 min. Finally, resuspend the cells in T cell basal medium and adjust the density to 2 × 10⁶ cells / mL. 6 / mL and supplement with the required cytokines. It should be noted that after 48 hours of activation, T cells can usually only recover 20%-40% of the initial number, so it is very important to determine the initial number of activated T cells according to the specific experimental requirements.

[0083] 3 Lentiviral Packaging

[0084] After digesting healthy 293T cells, they were seeded in 10cm culture dishes. After 24 hours, when cell confluence reached approximately 80%, the medium was replaced with DMEM complete medium containing 25μM chloroquine phosphate. Two hours later, calcium phosphate transfection was performed: a transfection mixture containing 9μg of the main plasmid, 6μg of psPAX2 packaging plasmid, 2μg of pMD2.G packaging plasmid, and 140μL of CaCl2 was prepared, and sterile water was added to a final volume of 1170μL. After mixing, an equal volume of 2×HEPES buffer was slowly added, and the mixture was gently pipetted to mix. The mixture was allowed to stand at room temperature for 15 minutes to form a complex. After standing, the transfection mixture was gently added dropwise to the 293T cells. After 6 hours, the medium was replaced with fresh medium, and the cells were cultured for another 48 hours. The cell culture supernatant was then collected and centrifuged at 1000g for 10 minutes to remove cell debris. The supernatant was stored at 4℃ (ideally used within one week).

[0085] 4T cell infection

[0086] Retrolectin was used to coat culture plates to enhance infection efficiency: Retrolectin was diluted to 15 μg / mL with PBS and added to untreated flat-bottomed culture plates: 500 μL / well / 24-well plate; 1000 μL / well / 12-well plate; 3000 μL / well / 6-well plate, incubated overnight at 4°C. The next day, after warming to room temperature for 30 min, the supernatant was discarded, and the plates were washed once with T cell basal medium and blocked at room temperature for 10 min. Viral supernatant was added at an MOI of 10⁻²⁰ (too high an MOI will reduce transduction efficiency), and the plates were sealed in self-sealing bags and centrifuged at 32°C, 2000×g for 90 min (1 step up, 1 step down) to load the virus. After discarding the viral solution, the plates were washed once with T cell basal medium, and the activated T cells were adjusted to 2×10⁶ cells / well. 6 / mL and effector cytokines were added, and the cells were seeded into virus-loaded wells. After sealing with a self-sealing bag, the cells were centrifuged at 32°C and 1000×g for 10 min (increase rate 1, decrease rate 1), and then allowed to stand for 30 min. The self-sealing bag was removed, and the T cell infection culture plate was transferred to a 37°C incubator for further culture to complete the integration and expression of the MUC13-CAR gene, and finally MUC13-CAR-T cells were obtained.

[0087] Example 4

[0088] Killing activity against pancreatic cancer tumors

[0089] 1. External killing effect

[0090] MUC13-CAR-T cells were seeded with three types of pancreatic cancer cells (wild-type Aspc1 pancreatic cancer cells, HPAFII cells, and MUC13 gene knockout Aspc1-KO cells) at different effector-target ratios (0, 3, 6, 9) in 96-well U-shaped plates, with 2 × 10⁶ cells added to each well. 4 Tumor cells were incubated at 37°C in a 5% CO2 incubator for 24 hours. D-fluorescein (final concentration 150 μg / mL) was added to each well and incubated at 37°C for 10 minutes. The growth of tumor cells was monitored using a small animal in vivo imaging system.

[0091] 2. Internal killing effect

[0092] Aspc1 cells expressing luciferase were used at a rate of 2 × 10⁻⁶. 4 CAR-T cells were subcutaneously injected into NSG mice to establish a xenograft tumor model. Nine days after tumor inoculation, D-fluorescein was injected intraperitoneally at a dose of 150 mg / kg body weight. Fluorescence signal was detected 10 minutes later using an in vivo imaging system to confirm successful tumor formation. The tumor-forming mice were randomly divided into two groups: a CAR-T treatment group and a control T-cell group. The CAR-T treatment group received a tail vein injection of 2 × 10⁻⁶ D-fluorescein. 6One MUC13-CAR-T cell was used, and the control T cell group received an equal amount of ordinary T cells via tail vein injection. Changes in tumor fluorescence signals were monitored weekly using in vivo imaging.

[0093] 3 Results

[0094] Depend on Figure 5 As shown in A and 5B, after co-culturing with pancreatic cancer cells for 24 hours, MUC13-specific CAR-T cells can effectively kill wild-type Aspc1 tumor cells and HPAFII tumor cells, but have no killing effect on MUC13 knockout Aspc1 tumor cells.

[0095] Depend on Figure 5 As shown in C, 5D, and 5E, the CAR-T treatment groups all exhibited significant tumor clearance effects compared to the ordinary T cell group. Long-term follow-up observation up to day 70 showed no tumor recurrence in any of the CAR-T treatment group mice, and the survival rate reached 100%. These results indicate that the MUC13-CAR-T cells provided by this invention can specifically clear pancreatic cancer tumors.

[0096] Example 5

[0097] Killing activity against gastric cancer tumors

[0098] 1 Internal lethality

[0099] MUC13-CAR-T cells were seeded with two types of gastric cancer cells (AGS cells and MKN45 cells) at different effector-to-target ratios (0, 3, 6, 9) in 96-well U-shaped plates, with 2 × 10⁶ cells added to each well. 4 Tumor cells were incubated at 37°C in a 5% CO2 incubator for 24 hours. D-fluorescein (final concentration 150 μg / mL) was added to each well and incubated at 37°C for 10 minutes. The growth of tumor cells was monitored using a small animal in vivo imaging system.

[0100] 2 Internal lethality

[0101] AGS cells expressing luciferase were used at a rate of 2 × 10⁻⁶. 4 CAR-T cells were subcutaneously injected into NSG mice to establish a xenograft tumor model. Six days after tumor inoculation, D-fluorescein was injected intraperitoneally at a dose of 150 mg / kg body weight. Fluorescence signal was detected 10 minutes later using an in vivo imaging system to confirm successful tumor formation. The tumor-forming mice were randomly divided into two groups: a CAR-T therapy group and a control T-cell group. The CAR-T therapy group received a tail vein injection of 2 × 10⁻⁶ D-fluorescein. 6 One MUC13-CAR-T cell was used, and the control T cell group received an equal amount of ordinary T cells via tail vein injection. Changes in tumor fluorescence signals were monitored weekly using in vivo imaging.

[0102] 3 Results

[0103] Depend on Figure 6 As shown in A and 6B, after co-culturing with gastric cancer cells for 24 hours, MUC13-CAR-T cells can significantly kill AGS tumor cells and MKN45 tumor cells.

[0104] Depend on Figure 6 As shown in C, 6D, and 6E, compared with the ordinary T cell group, the CAR-T treatment group mice achieved complete tumor clearance by day 14 of treatment. Long-term follow-up observation up to day 70 showed no tumor recurrence in any of the CAR-T treatment group mice, and the survival rate reached 100%. These results fully demonstrate that the MUC13-CAR-T cells provided by this invention can effectively clear gastric cancer tumors in vitro and in vivo.

[0105] Example 6

[0106] Killing activity against colorectal cancer tumors

[0107] 1 External killing agent

[0108] MUC13-CAR-T cells were seeded with two types of colorectal cancer cells (HT29 cells and NCI-H716 cells) at different effector-to-target ratios (0, 3, 6, 9) in 96-well U-shaped plates, with 2 × 10⁶ cells added to each well. 4 Tumor cells were incubated at 37°C in a 5% CO2 incubator for 24 hours. D-fluorescein (final concentration 150 μg / mL) was added to each well and incubated at 37°C for 10 minutes. The growth of tumor cells was monitored using a small animal in vivo imaging system.

[0109] 2 Internal lethality

[0110] HT29 cells expressing luciferase were used at a rate of 2 × 10⁻⁶. 4 CAR-T cells were subcutaneously injected into NSG mice to establish a xenograft tumor model. Six days after tumor inoculation, D-fluorescein was injected intraperitoneally at a dose of 150 mg / kg body weight. Fluorescence signal was detected 10 minutes later using an in vivo imaging system to confirm successful tumor formation. The tumor-forming mice were randomly divided into two groups: a CAR-T therapy group and a control T-cell group. The CAR-T therapy group received a tail vein injection of 2 × 10⁻⁶ D-fluorescein. 6 One MUC13-CAR-T cell was used, and the control T cell group received an equal amount of ordinary T cells via tail vein injection. Changes in tumor fluorescence signals were monitored weekly using in vivo imaging.

[0111] 3 Results

[0112] Depend on Figure 7 As shown in A and 7B, after co-culturing with colorectal cancer cells for 24 hours, MUC13-CAR-T cells were able to significantly kill HT29 tumor cells and NCI-H716 tumor cells.

[0113] Depend on Figure 7 As shown in C, 7D, and 7E, compared with the ordinary T cell group, the mice in the CAR-T treatment group achieved complete tumor clearance by day 14. Long-term follow-up observation up to day 70 showed no tumor recurrence in any of the mice in the CAR-T treatment group, and the survival rate reached 100%. These results fully demonstrate that the MUC13-CAR-T cells provided by this invention can effectively clear colorectal cancer tumors in vitro and in vivo.

[0114] Example 7

[0115] Toxicity of normal tissues

[0116] 1 Method

[0117] The expression of MUC13 in various normal human tissues (including liver, small intestine, large intestine, stomach, etc.) was systematically evaluated using immunohistochemistry. A standard IHC procedure was followed: paraffin sections were stained with anti-MUC13 monoclonal antibody (1:100) after antigen retrieval, developed with DAB, and counterstained with hematoxylin. Negative (PBS instead of primary antibody) and positive (pancreatic cancer tissue) controls were simultaneously established.

[0118] 2 Results

[0119] Depend on Figure 8 It is known that MUC13 is not expressed in most normal tissues, and its expression is extremely low in tissues such as the small intestine, large intestine, and stomach. This indicates that MUC13-CAR-T cells do not have adverse effects on normal tissues when applied in vivo, providing crucial evidence for the in vivo safety of this immunotherapy. Furthermore, no significant toxic side effects were observed during treatment, further validating the safety of this immunotherapy and providing a highly effective and safe novel treatment option for gastrointestinal tumors.

[0120] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A monoclonal antibody targeting MUC13, characterized in that, The antibody is a single-chain antibody (scFv), and the heavy chain variable region and light chain variable region of the antibody are MM06-VH / MM06-VL, MM07-VH / MM07-VL or MM08-VH / MM08-VL, respectively; the amino acid sequences of MM06-VH, MM07-VH, MM08-VH, MM06-VL, MM07-VL and MM08-VL are as shown in SEQ ID NO:1-6.

2. A monoclonal antibody targeting MUC13, characterized in that, The heavy and light chains of the antibody are MM06-Heavy chain / MM06-Light chain, MM07-Heavy chain / MM07-Light chain, or MM08-Heavy chain / MM08-Light chain, respectively; the amino acid sequences of the MM06-Heavy chain, MM07-Heavy chain, MM08-Heavy chain, MM06-Light chain, MM07-Light chain, and MM08-Light chain are as shown in SEQ ID NO:7-12.

3. The monoclonal antibody targeting MUC13 according to claim 1, characterized in that, The single-chain variable fragment MM06-scFv is composed of MM06-VH, the amino acid sequence shown in SEQ ID NO:1, and MM06-VL, the amino acid sequence shown in SEQ ID NO:

4. The amino acid sequence of MM06-scFv is shown in SEQ ID NO:13-16.

4. The monoclonal antibody targeting MUC13 according to claim 1, characterized in that, The single-chain variable fragment MM07-scFv is composed of MM07-VH, the amino acid sequence shown in SEQ ID NO:2, and MM07-VL, the amino acid sequence shown in SEQ ID NO:

5. The amino acid sequence of MM07-scFv is shown in SEQ ID NO:17-20.

5. A monoclonal antibody targeting MUC13 according to claim 1, characterized in that, The single-chain variable fragment MM08-scFv is composed of MM08-VH, the amino acid sequence shown in SEQ ID NO:3, and MM08-VL, the amino acid sequence shown in SEQ ID NO:

6. The amino acid sequence of MM08-scFv is shown in SEQ ID NO:21-24.

6. The use of the monoclonal antibody targeting MUC13 as described in any one of claims 1 to 5 in the preparation of a reagent for detecting the MUC13 antigen.

7. A chimeric antigen receptor targeting MUC13, characterized in that, The chimeric antigen receptor comprises, from N-terminus to C-terminus, the following: (1) A MUC13-specific binding domain, wherein the domain comprises the monoclonal antibody according to any one of claims 1 to 5; (2) Transmembrane domain; (3) Intracellular signal transduction domain.

8. A method for targeting MUC13 chimeric antigen receptor T cells, characterized in that, The T cells express the chimeric antigen receptor as described in claim 7.

9. The method for constructing MUC13-targeting chimeric antigen receptor T cells according to claim 8, characterized in that, Includes the following steps: (1) T cells were isolated from peripheral blood of the donor; (2) Activate T cells with anti-CD3 / CD28 antibody; (3) Using viral transduction technology, the gene encoding the chimeric antigen receptor is transduced into T cells; (4) Expand the transferred T cells to a therapeutically effective dose in a culture medium containing IL-2.

10. The use of the MUC13-targeting monoclonal antibody of claim 1, the MUC13-targeting chimeric antigen receptor of claim 7, and the MUC13-targeting chimeric antigen receptor T cell of claim 8 in the preparation of a medicament for immunotherapy of gastrointestinal tumors, wherein the gastrointestinal tumors are selected from pancreatic cancer, gastric cancer, and colorectal cancer.

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