Car-t cell targeting b7-h3 and co-expressing cytokine and chemokine, and use of car-t cell

Abstract

Provided are a CAR-T cell targeting B7-H3 and co-expressing a cytokine and a chemokine, and a use of the CAR-T cell. The CAR-T cell contains a B7-H3 single-chain antibody, IL-7 and CCL-19; or the B7-H3 single-chain antibody, IL-7, IL-15 and CCL-19. The use of the CAR-T cell targeting B7-H3 and performing co-expression shows, in an NCG mouse model, the effects of significantly improving the infiltration and expansion of tumor tissue, and effectively reducing tumors.

Description

CAR-T cell targeting B7-H3 and co-expressing cytokines and chemokines and its use

[0001] This invention claims priority date 2023-08-15 and priority number 2023110224737. Technical Field

[0002] The present invention relates to the field of cell therapy, and in particular to a CAR-T cell that targets B7-H3 and co-expresses cytokines and chemokines. Background Art

[0003] Genetically engineered T cells expressing chimeric antigen receptors (CARs), also known as CAR-T cells, are an effective clinical treatment for hematological malignancies and are considered one of the most potent immunotherapies for cancer. CARs consist of an extracellular antigen-specific single-chain fragment variable region (scFv) fused to a transmembrane segment and an intracellular costimulatory signaling domain, providing antigen-binding sites and simultaneously activating T cells. CARs can specifically recognize tumor antigens, trigger T cell activation in a non-major histocompatibility complex (MHC)-restricted manner, and initiate an extracellular anti-tumor response. CAR-T cell therapy has demonstrated significant efficacy in the treatment of hematological malignancies. Two CAR-T therapies targeting CD19 have been successfully used in clinical practice for the treatment of hematological B-cell lymphomas. CAR-T targeting B-cell maturation antigen (BCMA) has also shown positive therapeutic effects in clinical trials. However, similar results have not been observed in CAR-T cell therapy for solid tumors. Solid tumors have a more complex immunosuppressive microenvironment, with numerous immunosuppressive cells and cytokines within the tumor suppressing T cell activity. In addition, dense extracellular matrix (ECM) can also prevent CAR-T cells from infiltrating into solid tumors and affect the activity of CAR-T cells.

[0004] During culture, additional cytokines such as IL-7 and IL-15 can promote efficient proliferation of CAR-T cells and maintain their CAR-T cell memory phenotype. However, simply adding IL-7 and IL-15 during culture did not improve in vivo killing efficacy. T cells, including CAR-T cells, have difficulty penetrating the extracellular matrix and infiltrating tumors. Reports indicate that co-expression of IL-7 enhances the in vivo persistence of CAR-T cells and improves NKG2D-based CAR-T therapy for prostate cancer by enhancing their expansion and inhibiting apoptosis and exhaustion. Researchers have also found that co-expression of IL-7 and CCL-19 in CAR designs has greater clinical efficacy against solid tumors and promotes lymphocyte infiltration into tumor cells. IL-15 induces T cell activation, proliferation, and survival and has the function of increasing memory T cell subsets. Co-expression of IL-7 and IL-15 can guide the generation of human memory stem T cells from naive precursors and provide the desired long-term persistence of CAR-T cells. The co-expression of IL-7 and CCL-19 in CAR-T cells has been studied in the past, but the co-expression of IL-7, IL-15, and CCL-19 in CART cells has not been reported in the literature.

[0005] B7-H3 is a protein belonging to the B7 family. B7-H3 is expressed on the surface of most tumor cells but is rarely expressed on normal cells. B7-H3 expression in tumors is associated with poor prognosis. Several clinical trials of anti-B7-H3 antibody drugs have reported promising results against solid tumors and demonstrated safety in human patients. Therefore, B7-H3 is an attractive target for the treatment of solid tumors using CAR-T cells. The co-expression of IL-7, IL-15, and CCL-19 may also play a positive role in CAR-T cell therapy for solid tumors. When CAR-T cells are used to treat solid tumors, their infiltration, proliferation, and survival in the tumor microenvironment need to be enhanced.

[0006] Non-small cell lung cancer (NSCLC) and pancreatic cancer are both highly aggressive and rapidly growing malignancies with high mortality rates and a lack of effective treatments. Numerous clinical trials have investigated CAR-T cell therapy for these two diseases. However, the therapeutic effects to date have been unsatisfactory. The immunosuppressive microenvironment and ECM are particularly problematic for CAR-T cell therapy of solid tumors.

[0007] Summary of the Invention

[0008] The purpose of the present invention is to provide a CAR-T (H3-7 / 15×19 CAR-T) that targets B7-H3 and co-expresses IL-7 / IL-15 and CCL-19.

[0009] To achieve the above objectives, the present invention provides a CAR-T cell targeting B7-H3 and co-expressing cytokines and chemokines, characterized in that it contains a B7-H3 single-chain antibody, IL-7 and CCL-19; preferably, IL-7 and CCL-19 are both connected by a 2A peptide sequence 1;

[0010] The amino acid sequence of the B7-H3 single-chain antibody is shown in SEQ ID NO: 1, the amino acid sequence of IL-7 is shown in SEQ ID NO: 6; the amino acid sequence of CCL-19 is shown in SEQ ID NO: 8; and the amino acid sequence of the 2A peptide sequence 1 is shown in SEQ ID NO: 9.

[0011] Furthermore, the CAR sequence containing the B7-H3 single-chain antibody sequence, IL-7 and CCL-19 sequences were cloned into the PLVX-CD19 CAR.

[0012] Furthermore, the preparation method is to clone CAR, IL-7 and CCL-19 genes, wherein the IL-7 and CCL-19 genes are connected by 2A peptide sequence 1, into a lentiviral vector, thereby obtaining CAR-T cells that target B7-H3 and co-express IL-7 and CCL-19;

[0013] The CAR is a B7-H3 single-chain antibody connected to CD8 Hinge, CD8 TM, 4-1BB and CD3Z sequences;

[0014] The amino acid sequence of the CD8 Hinge is shown in SEQ ID NO: 2; the amino acid sequence of the CD8 TM is shown in SEQ ID NO: 3; the amino acid sequence of the 4-1BB is shown in SEQ ID NO: 4; and the amino acid sequence of the CD3Z is shown in SEQ ID NO: 5.

[0015] Furthermore, it also contains IL-15, wherein IL-7, IL-15 and CCL-19 are all connected by 2A peptide sequence 2;

[0016] The amino acid sequence of IL-15 is shown in SEQ ID NO: 7; the amino acid sequence of 2A peptide sequence 2 is shown in SEQ ID NO: 10.

[0017] Furthermore, the CAR sequence containing the B7-H3 single-chain antibody sequence, IL-7, IL-15 and CCL-19 was cloned into the PLVX-CD19 CAR.

[0018] Furthermore, CAR, IL-7, IL-15, and CCL-19 genes, where the IL-7, IL-15, and CCL-19 genes were connected by two 2A peptide sequences, were cloned into a lentiviral vector to obtain CAR-T cells targeting B7-H3 and co-expressing IL-7 / IL-15, and CCL-19;

[0019] The CAR is a B7-H3 single-chain antibody connected to CD8 Hinge, CD8 TM, 4-1BB and CD3Z sequences.

[0020] The present invention also protects the use of the B7-H3-targeting and co-expressing CAR-T cells for preparing drugs for treating cancer.

[0021] Furthermore, the cancer is not limited to gastric cancer, ovarian cancer, pancreatic cancer, colon cancer, melanoma, lung cancer, tongue squamous cell carcinoma and ovarian cancer.

[0022] This study uses CAR-T cells targeting B7-H3 and co-expressing IL-7 / IL-15 and CCL-19 (H3-7 / 15×19 CAR-T) as a putative treatment for NSCLC and pancreatic tumors. Compared with negative CD19 CAR-T controls and H3-7×19 CAR-T, H3-7 / 15×19 CAR-T cells demonstrated significantly improved tumor tissue infiltration and expansion and effective tumor reduction in the NCG mouse model. This study should provide a basis for new strategies for treating NSCLC and pancreatic tumors with CAR-T cells targeting B7-H3-positive tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG1 is a graph showing the binding affinity of B7-H3 (FD3-34-scfv, FD3-23-1-scfv, FD3-23-2-scfv and FD3-23-3-scfv) single-chain antibodies to hB7-H3 antigen detected by BLI method.

[0024] FIG2 is a graph showing the binding affinity of B7-H3 scFv single-chain antibody to hB7-H3 antigen.

[0025] FIG3 is an affinity chromatography graph of B7-H3 scFv (FD3-34-scfv-hFc).

[0026] FIG4 is a diagram showing the expression of B7-H3 (FD3-34-scFv) in different human tumor tissues by IHC.

[0027] FIG5 is a diagram of the construction of H3 CAR, H3 CAR-7×19 and H3 CAR-7 / 15×19 plasmids (Xba1 / Sal1).

[0028] FIG6 is a characteristic diagram of H3 CAR-7×19 and H3 CAR-7 / 15×19.

[0029] FIG7 is a graph showing the in vitro cytotoxicity assay of H3 CAR-7 / 15×19 cells against SPC-A1 (A, B, C) and Panc-10.05 (D, E, F) cell lines.

[0030] Figure 8 is a diagram of B7-H3 CAR-T cell therapy for NSCLC and PADC mouse xenograft models.

[0031] Figure 9 is a single dose of 1×10 7 Image of H3 CAR-7 / 15×19 treatment of Panc-10.05 cell xenograft model.

[0032] Figure 10 is a graph of a 1×10 7 Image of SPC-A1 xenograft tumors in mice treated with H3 CAR-7 / 15×19. DETAILED DESCRIPTION

[0033] Below in detail embodiments of the present invention, the example of described embodiment is shown in the accompanying drawings, wherein identical or similar reference numerals represent identical or similar elements or elements with identical or similar functions throughout. The embodiment described below by reference to the accompanying drawings is exemplary, is intended to be used for explaining the present invention, and is not to be construed as limiting the present invention. In the embodiment, those not indicating specific techniques or conditions are carried out according to the technology or conditions described in the document in this area or according to the product specification. Reagents used or instruments not indicating manufacturers are conventional products that can be obtained commercially.

[0034] Example 1:

[0035] 1. Materials and methods used:

[0036] Mice: NCG mice (NOD-Parkdcem26Cd52Il2rgem26Cd22 / Nju) were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. Female mice aged 6 to 8 weeks were housed in a specific pathogen-free facility at Fujian Medical University. All animal experiments were performed in accordance with the National Institutes of Health guidelines and approved by the Institutional Animal Care and Use Committee of Fujian Medical University (No. 2021-0382).

[0037] Tissue and blood samples: Formalin-fixed, paraffin-embedded, or surgical tissue samples were obtained from the Department of Pathology, Union Hospital, Fujian Medical University. Informed consent was obtained before sample collection. Peripheral blood was collected from healthy adult volunteers for PBMC isolation with the consent of the Fujian Provincial Blood Center. This study was approved by the Research Ethics Review Committee of Fujian Medical University.

[0038] Cell culture: Tumor cells SPC-A1 were obtained from GenePharma (Shanghai, China), CA922 and OVCAR-8 were obtained from ATCC, and Panc-10.05GFP was obtained from GeneCopoime BioTech. 293T cells were obtained from TAKARA (Lenti-X TM 293T cell line 632180). All cells were maintained in RPMI-1640 supplemented with 10% fetal bovine serum and 2 mmol / L L-glutamine (Invitrogen).

[0039] Lentivirus and CAR-T formulation: The H3 CAR gene (structure shown in Figure 5A, CAR) contains a B7-H3 scFv (i.e., Anti-B7H3-scfv, sequence shown in SEQ ID NO: 1), a CD8a hinge (i.e., CD8hinge, sequence shown in SEQ ID NO: 2), and a transmembrane domain (i.e., CD8TM, sequence shown in SEQ ID NO: 3), as well as signaling domains of 4-1BB (i.e., Leading, sequence shown in SEQ ID NO: 4) and CD3zeta (i.e., CD3Z, sequence shown in SEQ ID NO: 5). PCR was used to amplify the target B7-H3 gene fragment, CD8a hinge and transmembrane domain fragments, and 4-1BB and CD3zeta signaling domain fragments. The three H3 CAR gene fragments were cloned and ligated into the PLVX lentiviral master plasmid using seamless cloning. To echo the H3 CAR, the H3-7×19 CAR-T and H3-7 / 15×19 CAR were also cloned into lentiviral vectors. Using seamless cloning technology, the sequences corresponding to the IL-7 (sequence shown in SEQ ID NO:6) and CCL-19 genes (sequence shown in SEQ ID NO:8) were placed in series downstream of the H3 CAR and connected via 2A sequence 1 (sequence shown in SEQ ID NO:9) to form H3 CAR-7×19. Similarly, using seamless ligation technology, the sequences corresponding to the IL-7 (same as above), IL-15 (sequence shown in SEQ ID NO:7), and CCL-19 genes (same as above) were placed in series downstream of the H3 CAR and connected via two 2A sequences 2 (sequence shown in SEQ ID NO:10) to form H3 CAR-7 / 15×19. For specific steps, refer to the Pentameric Cloning Kit (TAKARA).

[0040] The lentiviral vector PLVX and two helper vectors, pSPAX.2 and PMD2.G, were transduced into 293T cells at a mass ratio of 3:2:1 for PLVX:pSPAX.2:PMD2.G, for a total of 18 μg. Transfection was performed at a PEI:plasmid ratio of 6:1. Supernatants were collected 48 hours after transfection and purified using a Lenti-X concentrator (TAKARA, 631232). Lentivirus titers were determined by flow cytometry.

[0041] To prepare CAR-T cells, PBMCs from healthy donors were stimulated with T cell TransAct expansion-human CD3 / CD28 stimulation reagent (Miltenyi, 130-111-160) and cultured in T cell culture medium X-VIVO 15 medium (Lonza, 04-418Q) containing 10% FBS and 100 IU / ml IL-2 (Peprotech, 200-02). After 24 hours, the lentiviral vector was added at an MOI of 5, and the CAR-T cells were cultured in T cell culture medium for 12 days. The prepared CAR-T cells were collected and frozen in liquid nitrogen.

[0042] Flow cytometry: To detect transduced CAR-T, CAR-T cells were labeled with CD276 antigen (B7-H3 antigen) (ACRO, B73-H5253) for 30 minutes. Prepare 5×10 5 The cells were washed 3 times with PBS, the supernatant was discarded after centrifugation, and 0.5 μg B7-H3 antigen protein was added and incubated for 30 min. After washing with PBS, the cells were stained using APC anti-human IgG Fc antibody (Biolegend, 410712). In order to detect the memory phenotype, CAR-T cells were stained using APC mouse anti-human CD45RA (BD, 550855) antibody and PE mouse anti-human CD62L antibody (BD, 555544). The cells were analyzed using BD Cano II. Data analysis was then performed using Flowjo V10.1.

[0043] Western blotting: Primary antibodies used were anti-CD3 zeta (Abcam, ab190728) and anti-GAPDH (Abcam, ab9485). Secondary antibodies used were anti-mouse HRP (Abcam, ab6728) and anti-rabbit HRP (Abcam, ab6721). The specific experimental steps were based on commonly used Western blotting protocols.

[0044] Cytokine detection: 1×10 5Individual T cells were co-cultured with equal or unequal amounts of tumor cells for 24 hours, and the supernatant was collected. IFN-γ was quantified using a human IFN-γ standard ABTS ELISA kit (PeproTech, 900-K27) according to the manufacturer's instructions. IL-7 and IL-15 were quantified using a human interleukin 7 (IL-7) ELISA kit (Cloud clone, SEA662Ra) and a human interleukin 15 (IL-15) ELISA kit (Cloud clone, LBKYSEA061). CCL-19 was quantified using a human CCL-19 / MIP-3β ELISA kit (Biyuntian, PC123).

[0045] Cell proliferation: Cells were quantified using a Cellometer Auto 2000. Cell suspensions were diluted 1:1 with trypan blue dye and counted, and growth and proliferation curves were plotted.

[0046] Cytotoxicity Assay: CAR-T cells were co-cultured with tumor cells in 96-well plates at varying ET ratios (effector to target cell ratios) for 24 hours, as needed. Supernatants were collected and analyzed using an ELISA kit for IFN-γ according to the manufacturer's instructions. The percentage of tumor cells killed by CAR-T cells was calculated as follows: [(experimental group - spontaneous release) / (maximum load - spontaneous release) × 100 (%)].

[0047] Mouse xenograft tumor model: 6- to 8-week-old female NCG mice were subcutaneously injected with 2 × 10 6 SPC-A1 cells and 1×10 6 Panc-10.05-GFP cells. Tumor volume (V = 1 / 2 × L (length) × W (width) × W (width)) was measured every 3 days using a vernier caliper. When the tumor volume approached 125 mm 3 When 1×10 7 CAR-T cells were injected every 5 days. The body weight was measured every 4 days, and the survival of the mice was closely monitored.

[0048] Treatment options include:

[0049] CD19 CAR-T and H3-7 / 15×19 CAR-T cells were expressed at 1×10 7 Panc-10.05-GFP cell tumor model mice were treated with a single dose of each, and tumor changes were measured every 7 days using small animal live imaging technology.

[0050] CD19 CAR-T, H3-7×19 CAR-T and H3-7 / 15×19 CAR-T cells were expressed at 1×10 7 Panc-10.05-GFP cell tumor model mice were treated with double doses of the drug, and the changes in the tumors in the mice were closely monitored.

[0051] CD19 CAR-T, H3-7×19 CAR-T and H3-7 / 15×19 CAR-T cells were expressed at 1×10 7 The SPC-A1 cell tumor model mice were treated with double doses of the drug and the changes in the tumors of the mice were closely monitored.

[0052] CD19 CAR-T and H3-7 / 15×19 CAR-T cells were expressed at 1×10 7 The SPC-A1 cell tumor model mice were treated with three doses of the drug, and the changes in the tumors of the mice were closely monitored.

[0053] Analysis of CAR-T cell expansion in mouse blood: Blood was collected from the retroorbital vein and analyzed by flow cytometry: 1-2 mL of blood was collected from treated model mice and treated with red blood cell lysis buffer (Beyotime, C3702). After washing with PBS, the cells were stained with APC mouse anti-human CD3ε (BD Biosciences, 558257). T cells were counted and analyzed using FlowJo V10.1.

[0054] Immunohistochemistry (IHC): For IHC assays, tumor tissue samples were fixed, processed, and stained according to standard IHC procedures. Sections were stained with anti-human CD3 antibodies-HRP (Abcam, ab305634) and anti-human Ki67-HRP (Abcam, ab279653). Detection was performed using the HRP system. Positive cells were scanned and counted.

[0055] Statistical analysis: GraphPad Prism 8.0 was used for statistical analysis of the data. One-way ANOVA, two-way ANOVA with Bonferroni post-hoc test, or unpaired two-tailed t-test were used, as appropriate. Symbols indicate statistical significance as *P < 0.05, **P < 0.01, and ***P < 0.001. Each experiment was performed at least three times.

[0056] Mouse anti-human B7-H3: B7-H3 single-chain antibody (including FD3-34-scfv, FD3-23-1-scfv, FD3-23-2-scfv, and FD3-23-3-scfv): The preparation method of the B7-H3 single-chain antibody is as follows:

[0057] 1. Production of anti-B7-H3 monoclonal antibody (mouse origin)

[0058] 1.1 Immunization of mice

[0059] Eight-week-old female Balb / c mice were immunized subcutaneously at multiple sites with a mixture of human B7-H3 recombinant antigen protein (purchased from Shanghai YEASEN, product number 93111ES60, 100 μg, RMB 3185, Uniport No. Q5ZPR3.1) and complete Freund's adjuvant (CFA, Sigma) emulsion, at a dose of 80-100 μg protein per mouse. Three weeks later, booster immunizations were performed with an emulsion of protein and incomplete adjuvant (IFA, Sigma) for a total of three immunizations, at a dose of 80-100 μg protein per mouse. Sera were collected approximately two weeks after each immunization for antibody titer determination using ELISA on a SpectraMax i3 microplate reader. When serum titers reached a sufficiently high level, a booster immunization was performed intraperitoneally with 100 μg of human B7-H3 recombinant antigen protein dissolved in PBS. Three days later, spleens were removed and fused.

[0060] 1.2 Preparation and screening of hybridomas

[0061] Hybridoma cells were generated by fusing spleen cells from immunized mice with a mouse myeloma cell line (SP2 / 0-Ag14) using polyethylene glycol (PEG1450, SIGMA) as a fusion agent. The fused cells were suspended in HAT selection medium (Sigma Aldrich) and cultured in 96-well plates for approximately 11 days. The culture supernatant was then identified using ELISA on a SpectraMax i3. Positive clones were then subcloned using limiting dilution methods three or more times to ensure they were single-origin positive clones.

[0062] 1.3 Purification of anti-B7-H3 monoclonal antibody (mouse origin)

[0063] The subclones described in 1.2 were cultured with conventional hybridoma cells, and the culture supernatant was captured using HiTrap Protein G HP (purchased from GE Healthcare, Catalog No. 29048581). The supernatant was eluted with 0.1 M glycine (pH 3.5), and the eluted peak was collected and desalted using a HiTrap Desalting column (purchased from GE Healthcare, Catalog No. 29048684) with 1× PBS (pH 7.4) as the replacement buffer. The supernatant was stored at -20°C until use.

[0064] II. Characteristics of Anti-B7-H3 Monoclonal Antibody (Mouse) (Specific Binding Ability)

[0065] SPC-A1, A549, and CA-9-22 (human oral epithelial cancer cell lines) that highly express B7-H3 were used, and B7-H3 / Fc of human IgG1 (sequence derived from the Fc segment of human IgG1, sequence number: AAB2181.1, as shown in SEQ ID As shown in NO:11, a total of 456 amino acids were cloned into the vector according to standard methods, and then transferred to 293T cells to express B7-H3 / IgG1-Fc. The expressed B7-H3 / IgG1-Fc was purified using a protein A column and thoroughly mixed with the anti-B7-H3 monoclonal antibody (mouse source) purified according to 1.3 above. The mixture was incubated at 4°C for 20-30 minutes, washed, and detected on a FACSVerse instrument. The results are shown in Figure 2. Figure 2 shows the binding capacity of the mouse B7-H3 antibody measured by the Octet instrument (molecular interaction instrument detection method, using bio-layer interferometry). The horizontal axis represents the time course (in seconds) of antibody binding to the B7-H3 antigen on cells. The first half of the rising curve represents the time required for the antibody to bind to the antigen (Association), while the second half of the flat curve represents the dissociation of the antibody after binding (Dissociation). A shorter binding time (a steeper curve) indicates stronger antibody binding, while a flat curve (no dip) after binding indicates minimal dissociation of the antibody after binding. This experiment demonstrates that the single-chain antibody FD3-34-scFv binds well to the B7-H3 antigen. See Figure 2 and Table 1 for detailed data.

[0066] Table 1 Binding parameters of FD3-34-scfv single-chain antibody to B7-H3 antigen Note: KD represents the binding affinity of the antibody; Kon represents the binding constant between the antibody and the antigen; Kdis represents the separation constant between the antibody and the antigen.

[0067] 3. Sequence Determination of Anti-B7-H3 Antibody (Mouse)

[0068] According to the hybridoma preparation technique (Kohler and Milstein, 1975) and the sections on cDNA library preparation, gene identification, and DNA sequencing in Molecular Cloning (3rd edition, by J. Sambrook and D.W. Russell, translated by Huang Peitang et al.), total RNA was extracted from UM1-51, UM1-56, and UM1-69 hybridoma cells, and cDNA was synthesized and sequenced.

[0069] First, total RNA was extracted and purified from hybridoma cells using RNA purification kits (RAeasy Mini Kit, Qiagen 74104, QIAshredder, Qiagen 79654). The RNA was then reverse transcribed into first-strand cDNA using the SMARter RACE cDNA Amplification Kit (Clontech) according to the company's reagent manual. PCR amplification was performed using the 5' RACE technique, using the universal primer UPM (provided by the kit) as the upstream primer and the gene-specific primer GSP (designed based on the mouse IgG1 heavy chain variable region and light chain kappa chain gene sequences) as the downstream primer, using the SMARter first-strand cDNA as the template. The PCR products were then ligated into T-vectors (Zero Blunt TOPO PCR Cloning Kit, Invitrogen K2875-J10). Select clones for sequencing and analysis to obtain the light and heavy chain variable region sequences of the antibody, respectively. Primers were then designed based on the heavy chain variable region sequence and the mouse IgG1 sequence. The full-length heavy chain sequence was obtained by PCR. The sequence was analyzed using the IGBLAST analysis tool (KABAT) to determine the light and heavy chains and their CDR regions, as shown below.

[0070] The light chain sequence is as follows:

[0071] Among them, a single underline "_" represents the CDR1 (GASENIYGALN) sequence; a double underline "_" represents the CDR1 (GASENIYGALN) sequence; represent sequence; lower tilde represent sequence.

[0072] The heavy chain sequence is as follows:

[0073] Among them, a single underline "_" represents the CDR1 (GFNIKDT) sequence; a double underline "_" represents the CDR1 (GFNIKDT) sequence; represent sequence; lower tilde represent sequence.

[0074] IV. Humanization of anti-B7-H3 antibody (mouse origin)

[0075] In order to make antibodies better applicable to clinical patient treatment, it is necessary to carry out humanization transformation to the antibody sequence so that it retains high affinity to antigen and other favorable biological properties (such as reducing its immunogenicity in the human body). To achieve this goal, according to a preferred method, humanized antibodies are prepared by analyzing the parent sequence and various conceptual humanized products using a three-dimensional model of the parent and humanized sequences. Three-dimensional immunoglobulin models are generally available and are familiar to those skilled in the art. Almost all mouse antibodies can be humanized by CDR grafting, thereby maintaining antigen binding. For relevant technical literature, please refer to Sims et al., 1987, J. Immunol., 151: 2296; Chothia et al., 1987, J. Mol. Biol., 196: 901; Presta et al., 1993, J. Immnol., 151: 2623; Carter et al., 1992, Proc. Natl. Acad. Sci. USA, 89: 4285; Lo Benny KC et al., in Antibody Engineering: Methods and Protocols, Vol. 248, Humana Press, New Jersey, 2004.

[0076] Based on the above work, FD3-34-scFv is preferably humanized. First, the antibody heavy chain constant region is replaced with the human IgG4 constant region sequence, and the antibody light chain constant region is replaced with the human κ chain constant region to obtain a human-mouse chimeric parent antibody (Chimeric Ab). After confirming the functional activity of the parent antibody, the heavy chain variable region and light chain variable region sequences are humanized, and the key amino acids in the FR region are reversely mutated on the basis of CDR transplantation. For amino acids between FRs that are not sure whether they affect the binding of antigen and antibody, merger synthesis is performed to construct a series of IgG mutants. After screening based on antibody affinity and expression level, one heavy chain and one light chain sequence were screened and cloned into the pcDNA3.1 expression vector using conventional molecular cloning technology. The 5' end of the light chain or heavy chain sequence carries a Kozak sequence and a human IL-2 signal peptide: MYRMQLLSCIALSLALVTNS (SEQ ID NO: 14), and its corresponding nucleotide sequence is:

[0077] The light chain sequence is as follows:

[0078] Among them, a single underline "_" represents the CDR1 (GASENIYGALN) sequence; a double underline "_" represents the CDR1 (GASENIYGALN) sequence; represent sequence; lower tilde represent sequence.

[0079] The heavy chain sequence is as follows:

[0080] Among them, a single underline "_" represents the CDR1 (GFNIKDT) sequence; a double underline "_" represents the CDR1 (GFNIKDT) sequence; represent sequence; lower tilde represent sequence.

[0081] Transient transfection of the above light and heavy chain expression vectors into ExpiCHO-S (Invitrogen) cells yielded a humanized antibody (FD3-34-scFv). The purified antibody was screened by ELISA and then biochemically analyzed by biochemical interaction assay (BLI), identifying the humanized antibody with the best binding affinity (Figure 2).

[0082] The human sequence of FD3-34-scfv is:

[0083] The human sequence of FD3-23-1-scfv is:

[0084] The human sequence of FD3-23-2-scfv is:

[0085] The sequence of FD3-23-3-scfv is:

[0086] 2. Results:

[0087] 2.1 Expression of B7-H3 scFv in human tumor cell lines and tumor tissues

[0088] 2.1.1 Screening experiments of FD3-34 mAb and its scFv:

[0089] To specifically target the B7-H3 cancer antigen, a panel of mouse anti-human B7-H3 was generated using phage display technology.

[0090] The binding affinity and specificity of mAbs and scFvs derived from clone FD3-34 were validated using recombinant human 4Ig-B7-H3 protein (Acro). The binding affinity of B7-H3 single-chain antibodies (scFv) (FD3-34-scfv, FD3-23-1-scfv, FD3-23-2-scfv, and FD3-23-3-scfv) to the hB7-H3 antigen was assessed using BLI. Concentrations of FD3-34-scfv, FD3-23-1-scfv, FD3-23-2-scfv, and FD3-23-3-scfv scFv were incubated with 5 μg of biotinylated hB7-H3 antigen at various concentrations ranging from 1.56 nM to 25 nM. Association and dissociation curves were measured using a SA sensor for 300 and 600 seconds, respectively. The results are shown in Figure 1 and Table 1. FIG1 is a graph showing the binding affinity of B7-H3 (FD3-34-scfv, FD3-23-1-scfv, FD3-23-2-scfv and FD3-23-3-scfv) single-chain antibodies to hB7-H3 antigen detected by BLI method.

[0091] Table 1. Binding parameters of several B7-H3 scFv single-chain antibodies to B7-H3 antigen

[0092] As can be seen in Figure 1 and Table 1, the FD3-34-scFv-hFc antibody exhibited the best affinity compared to FD3-23-1-scFv-hFc, FD3-23-2-scFv-hFc, and FD3-23-3-scFv-hFc. These data demonstrate the specificity of clone FD3-34 mAb for human B7-H3 and confirm that the scFv maintains high affinity and specificity for human B7-H3. Therefore, mAb FD3-34 and its scFv were selected for further experiments.

[0093] The antigen was immobilized on the probe, and the affinity constants of FD3-34-scfv were measured using a gradient of antibody concentrations (1.56 nM, 3.13 nM, 6.25 nM, 12.5 nM, and 25 nM) for 300 s each for binding and dissociation. The results are shown in Figure 2 and Table 2. Figure 2 shows the binding affinity of the FD3-34-scfv single-chain antibody to the hB7-H3 antigen.

[0094] Table 2. Binding parameters of FD3-34-scfv single-chain antibody and B7-H3 antigen

[0095] As can be seen from Figure 2 and Table 2, the scFv exhibited a specific binding affinity with B7-H3 protein (i.e., recombinant human 4Ig-B7-H3 protein) (scFv, KD = 8.37-10 ±1.10 -9 nM).

[0096] FIG3 is an affinity chromatography diagram of B7-H3 scFv (FD3-34-scfv-hFc). The purification conditions used are: adding 260 ml of sample supernatant and eluting with 0.1 M glycine (pH 3.5).

[0097] 2.1.2 B7-H3 expression in human tumor cell lines and tumor tissues

[0098] Using immunohistochemical analysis, high levels of B7-H3 were detected in various tumor tissues, including gastric cancer, ovarian cancer, pancreatic cancer, colon cancer, and melanoma (Figure 4A). Then, using flow cytometric analysis, high levels of B7-H3 were detected on various tumor cell lines from solid tumors, including lung cancer SPC-A1 and A549 cell lines, tongue squamous cell carcinoma CA9-22 cell line, and ovarian cancer OVCAR-8 cell line (Figure 4B).

[0099] Figure 4 shows the expression of B7-H3 (FD3-34-scFv) on various human tumor tissues by IHC. The three images in the first row (from left to right) in A represent lung cancer, gastric cancer, and ovarian cancer, respectively; the three images in the second row (from left to right) represent pancreatic cancer, colorectal cancer, and melanoma, respectively (40×). B shows the expression of B7-H3 on various human tumor cells. B7-H3 expression was assessed in human tumor cell lines (SPC-A1, A549, CA9-22, and OVCAR-8) by FACS. The blue-shaded histogram represents cells stained with anti-human B7-H3 scFv, and the red histogram represents cells stained with isotype mouse IgG1 as a control.

[0100] 2.2 Design and Characterization of H3-7 / 15×19 CAR-T

[0101] The structure of H3-7 / 15×19 CAR-T is shown in Figure 5A. In Figure 5A, CAR refers to the CAR core structure containing B7-H3 scFv, 4-1BB, and CD3zeta; H3 CAR refers to the H3 CAR plasmid containing the 5' and 3' end sequences and the promoter; H3-7×19 CAR refers to the plasmid containing the B7-H3 CAR core structure, IL-7 structure, and CCL-19 structure; and H3-7 / 15×19 CAR refers to the plasmid containing the B7-H3 CAR core structure, IL-7 structure, IL-15 structure, and CCL-19 structure.

[0102] This example creates a tandem construct encoding CAR, IL-7 / IL-15, and CCL-19 (using seamless cloning technology to clone the target fragment and construct a target vector), in which two 2A peptide sequences 2 connect the IL-7, IL-15, and CCL-19 genes and clone them into a lentiviral vector named H3-7 / 15×19 CAR-T. Using a seamless cloning kit, PCR was performed to obtain B7-H3 gene fragments, CD8a hinge and transmembrane domain fragments, and 4-1BB and CD3zeta signaling domain fragments to construct the H3 CAR vector. Then, IL-7, IL-15, and CCL-19 fragments were cloned. According to the instructions, IL-7, IL-15, and CCL-19 were used to construct different designs of target CAR vectors.

[0103] The following CAR genes were also created for comparison: (A) H3 CAR (without IL-7 / IL-15 chemokines) and (B) H3-7×19 CAR-T. Detailed construction process: (A) Using a seamless cloning kit, the B7-H3 gene fragment, the CD8a hinge and transmembrane domain fragments, and the 4-1BB and CD3zeta signaling domain fragments were PCR-generated to construct the H3 CAR vector. (B) Using a seamless cloning kit, the B7-H3 gene fragment, the CD8a hinge and transmembrane domain fragments, and the 4-1BB and CD3zeta signaling domain fragments were PCR-generated to construct the H3 CAR vector. IL-7 and CCL-19 fragments were then cloned and, according to the manufacturer's instructions, IL-7 and CCL-19 fragments were used to construct different target CAR vectors.

[0104] Figure 5 shows the construction (Xba1 / Sal1) of the H3 CAR, H3 CAR-7×19, and H3 CAR-7 / 15×19 plasmids. A shows the structures of the H3 CAR, H3 CAR-7×19, and H3 CAR-7 / 15×19 plasmids. B shows PCR verification of the constructed plasmid clones, with each lane representing a different positive colony. C shows the Xba I / Sal I double enzyme digestion of PUC57-B7H3, H3 CAR-7×19, and H3 CAR-7 / 15×19.

[0105] As can be seen from the figure, the H3 CAR, H3 CAR-7×19 and H3 CAR-7 / 15×19 plasmids were successfully constructed, all containing the correct fragment (B7-H3 fragment) and the correct restriction enzyme cutting sites. The sequencing results were also consistent with the designed sequence.

[0106] When expressing CD19-CAR, a lentiviral vector (pLVX-Puro) was used. The specific method was to insert the human CD19 gene sequence (specific sequence see Uniport No: P15391-2) into the restriction site of pLVX-Puro. CD19-CAR served as a negative control in this experiment.

[0107] After transduction, all constructs were stably expressed by human peripheral blood T cells (Figure 6A). Compared with H3-7×19 CAR-T, the transduction efficiency of H3-7 / 15×19 CART constructs was slightly lower (Figure 6B). This may be due to the larger reading frame ORF of H3-7 / 15×19 CAR-T, as shown in Figure 6C.

[0108] Figure 6 shows the characteristics of H3 CAR-7×19 and H3 CAR-7 / 15×19. A is a Western blot of CD3 zeta in CAR-T cells from different groups (the left band represents Mock-T, the middle band represents H3 CAR-7×19, and the right band represents H3 CAR-7 / 15×19). B is the transfection efficiency of CAR-T cells (H3 CAR-7×19 and H3 CAR-7 / 15×19). C is the expansion of Mock-T, H3 CAR-7×19, and H3 CAR-7 / 15×19 cells after 12 days of in vitro culture (***P<0.001). D is an ELISA analysis of CCL-19 expression in Mock-T, H3 CAR-7×19, and H3 CAR-7 / 15×19 cells (****P<0.0001). E is flow cytometry analysis of CD45RA of T cells in different groups (IL-2, IL-2 / 7 / 15, IL-7 / 15 and H3 CAR-7 / 15×19) + and CD62L + T cell ratio. Data are expressed as mean ± SD (n = 3). F is the number of cells that migrated in the Mock-T, H3 CAR, H3 CAR-7×19, and H3 CAR-7 / 15×19 groups in the transwell experiment.

[0109] As can be seen from the figure, both H3-CAR-7x19 and H3-7 / 15x19 CAR-T cells express CAR molecules and have high transduction efficiency.

[0110] 2.3 Overexpression of IL-7 and IL-15 promotes CAR-T proliferation and maintains the memory phenotype

[0111] During the in vitro culture of CAR-T cells, it was found that the proliferation rate of H3-7 / 15×19 CAR-T was faster than that of Mock-T (untransduced T cells isolated and cultured) and H3-7×19 CAR-T after 12 days of culture (Figure 6C).

[0112] Previous reports have shown that IL-7 and IL-15 can maintain the memory phenotype of T cells. Therefore, the experiment collected cells from different groups and measured the memory phenotype of different CAR-T cells by flow cytometry. The memory phenotype of cells in the Mock-T group (supplemented with IL-2, IL-2 / 7 / 15, and IL-7 / 15, respectively) and the H3-7 / 15×19 CAR-T group was compared. Among them, T cells were divided into effector T cells (Teff), memory stem T cells (Tscm), effector T cells (Tem), and central memory T cells (Tcm) based on CD45R and CD62L. The results showed that the proportion of cells with the central memory T cell (Tcm) memory phenotype in the H3-7 / 15×19 CAR-T group was significantly higher than that in the IL-2 / 7 / 15 and IL-7 / 15 groups supplemented with Mock-T (as shown in Figure 6D).

[0113] 2.4 Overexpression of CCL-19 can increase cell invasion ability

[0114] To investigate whether overexpression of CCL-19 could enhance the ability of CAR-T cells to penetrate tumors, this example used a transwell invasion chamber assay. Mock-T, H3-7X19 CAR-T, and H3-7 / 15X19 CAR-T cells were cultured for 24 hours, and the number of cells migrating through the transwell membrane was analyzed using crystal violet staining. As shown in Figure 6F, H3-7 / 15X19 CAR-T cells exhibited greater tumor invasion than H3 CAR and Mock-T cells. This suggests that overexpression of CCL-19 can improve the tumor invasion potential of CAR-T therapy.

[0115] To test the production of CCL-19, experiments were performed on H3 CAR, H3-7 / 15x19 CAR-T, and H3 CAR-7 / 19. After 10 days of culture, the supernatants of the cells were collected and CCL-19 was measured using ELISA (Figure 6E). H3-7 / 15X19 CAR-T and H3-7X19 CAR-T expressed significantly more CCL-19 than H3 CAR, as shown in Figure 6E.

[0116] 2.5 In vitro cytotoxicity of H3-7 / 15X19 CAR-T cells against tumor cells

[0117] This study investigated whether overexpression of IL7 / IL-15 and CCL-19 altered the cytotoxicity of CAR-T cells against tumor cells. Two types of B7-H3 positive cell lines were selected: SPC-A1 and Panc-10.05.

[0118] First, the short-term cytolytic abilities of H3-7 / 15X19 CAR-T, CD19 CAR, and Mock-T cells were evaluated compared to cell lines. Different ratios of CAR-T cells (effector cells) to tumor cells (target cells) were used. The two cell lines were co-cultured for 24 hours and the cells were harvested for flow cytometry analysis. The horizontal axis, B7-H3-APC, represents the number of surviving tumor cells, and CD3-PE represents the number of Mock-T and CAR-T cells. Specifically, the cells were co-cultured for 24 hours at E:T ratios of 1:5, 1:2, 1:1, and 2:1. As shown in Figure 7A, the specific killing rates of SPC-A1 tumor cells were 56.2% ± 1.4%, 73.4% ± 2.0%, 86.6% ± 1.9%, and 95.4% ± 1.20%. As shown in Figure 7D, the apoptosis level of H3-7 / 15X19 CAR-T was lower than that of H3 CAR. As shown in Figure 7D, the specific killing rates of SPC-A1 tumor cells were 74.2% ± 2.3%, 87.3% ± 4.0%, 95.0% ± 1.5%, and 98.4% ± 0.80%, respectively. (The lysis percentage was calculated as follows: [(experimental - spontaneous release) / (maximum load - spontaneous release] × 100 (%)])

[0119] Figure 7 shows in vitro cytotoxicity assays of H3 CAR-7 / 15×19 cells against SPC-A1 (A, B, C) and Panc-10.05 (D, E, F) cell lines. H3 CAR-7 / 15×19 demonstrated significant cytotoxicity against the B7-H3-positive cell lines SPC-A1 and Panc-10.05. As shown, effector and target cells were co-cultured for 24 hours at various E / T ratios. Data are representative of three separate experiments (**P<0.01, ***P<0.001, ****P<0.0001).

[0120] In summary, when tested with equivalent ET ratios, H3-7 / 15X19 CAR-T cells exhibited superior cytotoxicity against B7-H3+ cell lines. Furthermore, ELISA was used to analyze the supernatants of co-cultures of H3-7 / 15x19 CAR-T cells with SPC-A1 lung cancer cells or Panc-10.05 pancreatic cancer cells at E:T ratios of 1:5, 1:2, 1:1, and 2:1, respectively. (1) When the ratio of H3-7 / 15x19 CAR-T cells to SPC-A1 cells was 1:5, 1:2, 1:1, and 2:1, the average secretion of IFN-r in each group was 527.4±57.4pg / ml, 890.3±18.3pg / ml, 872.63±178.2pg / ml, and 1269.83±187.4pg / ml, respectively; (2) When the ratio of H3-7 / 15x19 CAR-T cells to SPC-A1 cells was 1:5, 1:2, 1:1, and 2:1, respectively, the average secretion of IFN-r in each group was 527.4±57.4pg / ml, 890.3±18.3pg / ml, 872.63±178.2pg / ml, and 1269.83±187.4pg / ml, respectively. When the ratio of CAR-T cells to Panc-10.05 cells was 1:5, 1:2, 1:1, and 2:1, the average IFN-r secretion in each group was 372.33±48.5pg / ml, 601.23±117.9pg / ml, 842.73±30.1pg / ml, and 931.3±71.7pg / ml, respectively. The results showed that as the ratio of CAR-T cells increased, the secretion of IFN-r increased along with the increase in CAR-T cells, and the tumor killing effect also increased accordingly.

[0121] 2.6 In vivo activity of H3-7 / 15x19 CAR-T

[0122] 1) To explore the anti-tumor ability of CAR-T in vivo, SPC-A1 xenograft tumors were established in NCG mice (Figure 8A). When the average tumor volume was close to 125 mm 3 When approximately 1×10 7 CAR-T cells.

[0123] CD19 CAR-T control group: 4 mice in total;

[0124] H3-7x19 CAR-T cell treatment group: 4 mice in total;

[0125] H3-7 / 15x19 CART cell treatment group: 4 mice in total.

[0126] The treatment methods of each treatment group are shown in Figure 8A, where the CD19 CAR-T control group is SPC-A1 lung cancer model mice (2×10 6 SC) were injected with control CD19-CAR-T cells on D10 and D17; the H3-7x19 CAR-T cell treatment group was SPC-A1 lung cancer model mice (2×10 6SC) received two doses of 1×10 7 H3-7x19CAR-T / time; H3-7 / 15x19 CART cell treatment group was SPC-A1 lung cancer model mice (2×10 6 SC) received two doses of 1×10 7 H3-7 / 15x19 CAR-T / time.

[0127] As shown in Figure 8B, in terms of tumor size and survival, H3-7 / 15×19 CAR-T showed smaller tumor growth and a higher survival rate than CD19 CAR-T, thus having better tumor suppression activity. The study found that all mice in the CD19 CAR-T control group died within 20 days, 25% of mice (i.e., 1) in the H3-7×19 CAR-T cell treatment group survived for more than 28 days, and 75% of mice (i.e., 3) in the H3-7 / 15×19 CART cell treatment group survived for more than 28 days.

[0128] Figure 8 shows B7-H3 CAR-T cell therapy in NSCLC and PADC mouse xenograft models. A. Treatment progress and B7-H3 expression in tumor tissues of NCG mice subcutaneously inoculated with SPC-A1 cells. B. SPC-A1 lung cancer tumor tissue growth and Kaplan-Meier survival analysis of NCG mice treated with H3 CAR-7 / 15×19 compared with CD19 CAR-T cells and H3 CAR-7×19 CAR-T cells (***P<0.001). n=4 per group. C. Treatment progress and B7-H3 expression in tumor tissues of NCG mice subcutaneously inoculated with Panc10.05-GFP cells. The growth of Panc-10.05 pancreatic cancer tumor tissue in NCG mice treated with H3 CAR-7 / 15×19 compared with CD19 CAR-T cells and H3 CAR-7 ×19 CAR-T cells and the survival rate of mice by Kaplan-Meier survival analysis (***P<0.001), n=4 in each group.

[0129] The results showed that more mice in the H3-7 / 15×19 CAR-T cell treatment group survived for more than 28 days compared with the CD19 CAR-T control group and the H3 CAR7×19 cell treatment group (***P<0.0001).

[0130] In addition, three doses of CAR-T were used for further treatment (see Figure 10A, the CD19 CAR-T control group was SPC-A1 lung cancer model mice (2×10 6SC) were injected with CD19 CAR-T negative control cells on D5, D10 and D15 days; H3-7 / 15x19CART cell treatment group was SPC-A1 lung cancer model mice (2×10 6 SC) received three doses of 1 × 10 7 H3-7 / 15x19CAR-T / time). And found that in the CD19 CAR-T control group, all tumors reached 2000mm within 34 days. 2 , and were euthanized. In the H3-7 / 15x19 CAR-T cell treatment group, 100% of the mice survived for more than 50 days. The results showed that compared with the CD19 CAR-T control group, the H3-7 / 15×19 CAR-T cell treatment group had a longer survival period, and the tumors of the tumor-bearing mice were 100% cured (***P<0.001). The number of CD3+T cells in the peripheral blood of mice in the H3-7 / 15×19 CAR-T cell treatment group was significantly higher than that in the CD19 CAR-T control group. Immunohistochemistry results showed that compared with the CD19 CAR-T control group, the Ki-67 in the H3-7 / 15X19 CAR-T group was significantly reduced, and the infiltration of CD3+T cells in the tumor tissue was significantly increased (as shown in Figure 10F). Figure 10 was a three-dose 1×10 7 Figure 1. Treatment of SPC-A1 xenograft tumors in mice with H3 CAR-7 / 15×19. A. Treatment with 1×10 6 Treatment process of NCG mice subcutaneously inoculated with SPC-A1 cells. B. Kaplan-Meier survival analysis comparing the survival of mice treated with H3 CAR-7 / 15×19 and CD19 CAR-T cells (**P<0.01), n=5 per group. C and D. Comparison of tumor volume and weight between the H3 CAR-7 / 15×19 group and the CD19 CAR-T cell group (***P<0.001, ****P<0.0001); E. Peripheral blood CD3 + Comparison of T cells (**P<0.01); F. Compared with the CD19 CAR-T cell group, the expression of Ki-67 and CD3 in tumor tissues of NCG mice in the H3 CAR-7 / 15×19 group was significantly higher than that in the CD19 CAR-T cell group. + IHC staining of infiltrating lymphocytes (20×); the left picture is a tissue staining image, and the right picture is Ki-67 and CD3 in tumor tissue + IHC score of T cell infiltration (**P<0.01).

[0131] 2) Establishment of the Panc-10.05 pancreatic cancer model in NCG mice (2×10 6SC), when the average tumor volume approaches 125 mm 3 When approximately 1×10 7 CAR-T cells. A single dose of 1×10 7 / times H3-7 / 15×19 CAR-T and CD19 CAR-T.

[0132] CD19 CAR-T control group: 4 mice in total;

[0133] H3-7 / 15×19 CART cell treatment group: 4 mice in total.

[0134] Figure 9 is a single dose of 1×10 7 H3 CAR-7 / 15×19 treatment of Panc-10.05 cell xenograft model. 2×10 6 Panc-10.05 cells were subcutaneously injected into NCG mice. The treatment process of the allogeneic model was Panc-10.05 allogeneic transplantation. As shown in Figure 9, all animals in the CD19 CAR-T control group died within 20 days, while 60% (should it be 75%) of the mice (i.e., 3) in the H3-7 / 15x19 CAR-T group survived for more than 20 days. The results showed that compared with the H3-7x19 CAR-T cell treatment group and the CD19 CAR-T control group, more mice in the H3-7 / 15x19 CAR-T cell treatment group survived for more than 20 days (***P<0.0001).

[0135] Establishment of the Panc-10.05 pancreatic cancer model in NCG mice (2×10 6 SC), when the average tumor volume approaches 125 mm 3 When approximately 1×10 7 CAR-T cells. Two doses of 1×10 7 / times H3-7 / 15×19 CAR-T, H3-7 / 15×19 CAR-T and CD19 CAR-T.

[0136] CD19 CAR-T control group: 4 mice in total;

[0137] H3-7×19 CAR-T cell treatment group: 4 mice in total;

[0138] H3-7 / 15×19 CART cell treatment group: 4 mice in total.

[0139] The treatment methods of each treatment group are shown in Figure 8C, where the CD19 CAR-T control group is Panc-10.05 pancreatic cancer model mice (2×10 6SC) were injected with CD19 CAR-T negative control cells on D12 and D17 days; the H3-7x19 CAR-T cell treatment group was Panc-10.05 pancreatic cancer model mice (2×10 6 SC) received two doses of 1×10 7 H3-7x19 CAR-T / time; H3-7 / 15x19 CART cell treatment group is Panc-10.05 pancreatic cancer model mice (2×10 6 SC) received two doses of 1×10 7 H3-7 / 15x19CAR-T / time.

[0140] Figure 8C shows the treatment process and B7-H3 expression in tumor tissues of NCG mice subcutaneously inoculated with Panc10.05-GFP cells. Figure 8D shows the survival rate of mice treated with H3 CAR-7 / 15×19, CD19 CAR-T cells, and H3 CAR-7×19, compared with those treated with H3 CAR-7 / 15×19 (****P<0.0001). n = 4 per group.

[0141] Figure 8D shows the survival rate of mice treated with H3 CAR-7 / 15×19 compared with CD19 CAR-T cells and H3 CAR-7×19 by Kaplan-Meier survival analysis (****P<0.0001), with n=4 per group. As shown in Figure 8D, all mice in the CD19 CAR-T control group died within 26 days, while 25% of mice (i.e., 1 mouse) in the H3-7X19 CAR-T cell treatment group survived for more than 30 days. In contrast, 75% of mice (i.e., 3 mice) in the H3-7 / 15×19 CAR-T cell treatment group survived for more than 30 days. Compared with the CD19 CAR-T control group and the H3 CAR-7×19 cell treatment group, more mice in the H3-7 / 15×19 CAR-T cell treatment group survived for more than 30 days (***P<0.0001).

[0142] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and purpose of the present invention.

Claims

1. A CAR-T cell targeting B7-H3 and co-expressing cytokines and chemokines, characterized in that: Containing B7-H3 single chain antibody, IL-7 and CCL-19; preferably, IL-7 and CCL-19 are both connected by 2A peptide sequence 1; The amino acid sequence of the B7-H3 single-chain antibody is shown in SEQ ID NO: 1, the amino acid sequence of IL-7 is shown in SEQ ID NO: 6; the amino acid sequence of CCL-19 is shown in SEQ ID NO: 8; and the amino acid sequence of the 2A peptide sequence 1 is shown in SEQ ID NO:

9.

2. The CAR-T cell targeting B7-H3 and co-expressing cytokines and chemokines according to claim 1, characterized in that: The CAR sequence containing the B7-H3 single-chain antibody sequence, IL-7 and CCL-19 sequences were cloned into PLVX-CD19 CAR.

3. The CAR-T cell targeting B7-H3 and co-expressing cytokines and chemokines according to claim 1, characterized in that: The preparation method comprises cloning CAR, IL-7 and CCL-19 genes, wherein the IL-7 and CCL-19 genes are connected by 2A peptide sequence 1, into a lentiviral vector, thereby obtaining CAR-T cells that target B7-H3 and co-express IL-7 and CCL-19; The CAR is a B7-H3 single-chain antibody connected to CD8 Hinge, CD8 TM, 4-1BB and CD3Z sequences; The amino acid sequence of the CD8 Hinge is shown in SEQ ID NO:2; the amino acid sequence of the CD8 TM is shown in SEQ ID NO:3; the amino acid sequence of the 4-1BB is shown in SEQ ID NO:4; and the amino acid sequence of the CD3Z is shown in SEQ ID NO:

5.

4. The CAR-T cell targeting B7-H3 and co-expressing cytokines and chemokines according to claim 1, characterized in that: It also contains IL-15, in which IL-7, IL-15 and CCL-19 are all connected by the 2A peptide sequence 2; The amino acid sequence of the IL-15 is shown in SEQ ID NO:7; the amino acid sequence of the 2A peptide sequence 2 is shown in SEQ ID NO:

10.

5. The CAR-T cell targeting B7-H3 and co-expressing cytokines and chemokines according to claim 4, characterized in that: The CAR sequence containing the B7-H3 single-chain antibody sequence, IL-7, IL-15 and CCL-19 was cloned into PLVX-CD19CAR.

6. The CAR-T cell targeting B7-H3 and co-expressing cytokines and chemokines according to claim 4, characterized in that: The CAR, IL-7, IL-15 and CCL-19 genes, where the IL-7, IL-15 and CCL-19 genes are connected by two 2A peptide sequences, are cloned into a lentiviral vector to obtain CAR-T cells that target B7-H3 and co-express IL-7 / IL-15 and CCL-19; The CAR is a B7-H3 single-chain antibody connected to CD8 Hinge, CD8 TM, 4-1BB and CD3Z sequences.

7. Use of the CAR-T cells targeting B7-H3 and co-expressing B7-H3 according to any one of claims 1 to 6 for preparing drugs for treating cancer.

8. The use according to claim 2, characterized in that The cancers are not limited to gastric cancer, ovarian cancer, pancreatic cancer, colon cancer, melanoma, lung cancer, tongue squamous cell carcinoma and ovarian cancer.

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