CN117025541A

CN117025541A - CAR-T cell targeting B7-H3 and co-expressing cytokines and chemokines and application thereof

Info

Publication number: CN117025541A
Application number: CN202311022473.7A
Authority: CN
Inventors: 张琼; 林基祯; 方树彬; 许云禄
Original assignee: Fuzhou Cremab Pharmaceutical Inc; Union Medical College Hospital of Fujian Medical University
Current assignee: Fuzhou Cremab Pharmaceutical Inc; Union Medical College Hospital of Fujian Medical University
Priority date: 2023-08-15
Filing date: 2023-08-15
Publication date: 2023-11-10

Abstract

The invention discloses a CAR-T cell targeting B7-H3 and co-expressing cytokines and chemokines and application thereof. Comprises B7-H3 single-chain antibody, IL-7 and CCL-19; or B7-H3 single chain antibodies, IL-7, IL-15 and CCL-19. The CAR-T cells which target B7-H3 and are co-expressed by using the invention show remarkable improvement of infiltration and expansion of tumor tissues in an NCG mouse model, and effectively reduce the effect of tumors.

Description

CAR-T cell targeting B7-H3 and co-expressing cytokines and chemokines and application thereof

Technical Field

The invention relates to the field of cell therapy, in particular to a CAR-T cell targeting B7-H3 and co-expressing cytokines and chemokines.

Background

T cell genetic engineering (also known as CAR-T cells) expressing Chimeric Antigen Receptors (CARs) is an effective method for clinical treatment against hematologic malignancies, and is considered one of the most effective immunotherapeutic approaches for cancer. CARs consist of an extracellular antigen-specific single-chain fragment variable region (scFv) fused to a transmembrane fragment and an intracellular co-stimulatory signaling domain, which provides an antigen binding site and simultaneously activates T cells. CARs can specifically recognize tumor antigens, trigger T cell activation in a non-Major Histocompatibility Complex (MHC) -restricted manner, and initiate extracellular anti-tumor responses. CAR-T cell therapies have shown significant efficacy in the treatment of hematological malignancies. Two CD 19-targeting CAR-T therapies have been successfully used in clinical practice for the treatment of B-cell lymphomas of the blood system. CAR-T targeting B Cell Maturation Antigen (BCMA) also showed positive therapeutic effects in clinical trials. However, similar results have not been observed in CAR-T cell therapy of solid tumors. Solid tumors have a more complex immunosuppressive microenvironment, and there are many immunosuppressive cells and cytokines within the tumor that suppress T cell activity. In addition, dense extracellular matrix (ECM) can also prevent infiltration of CAR-T cells into solid tumors and affect the activity of CAR-T cells.

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 during culture. However, the addition of additional IL-7 and IL-15 only during the culture process did not have a better killing effect in vivo. T cells, including CAR-T cells, have difficulty penetrating the extracellular matrix and penetrating into tumors. Co-expression of IL-7 has been reported to enhance the in vivo persistence of CAR-T cells and to improve the treatment of prostate cancer by enhancing their expansion, inhibiting apoptosis and failure of NKG 2D-based CAR-T. Researchers have also found that co-expression of IL-7 and CCL-19 in CAR design has a greater clinical effect on solid tumors, promoting 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 direct naive precursors to produce human memory stem T cells and provide desirable long-lasting CAR-T cells. Co-expression of IL-7 and CCL-19 in CAR-T cells has been studied in the past, but co-expression of IL-7, IL-15 and CCL-19 in CART cells has not been reported in the literature.

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

Non-small cell lung cancer (NSCLC) and pancreatic cancer are both highly invasive and rapidly growing malignancies, with high mortality and lack of effective treatment. Clinical trials of CAR-T cells for the treatment of non-small cell lung cancer and pancreatic tumors are numerous. However, the therapeutic effect to date is not satisfactory. Immunosuppressive microenvironments and ECM are particularly troublesome for CAR-T cell therapy of solid tumors.

Disclosure of Invention

The object of the present invention is to provide a CAR-T (H3-7/15 x19 CAR-T) targeting B7-H3 and co-expressing IL-7/IL-15 and CCL-19.

To achieve the above object, the present invention provides a CAR-T cell targeting B7-H3 and co-expressing a cytokine and a chemokine, characterized by comprising a B7-H3 single chain antibody, IL-7 and CCL-19; preferably, wherein IL-7 and CCL-19 are both linked by 2A peptide sequence 1;

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

Further, the CAR sequence containing B7-H3 single chain antibody sequence, IL-7 and CCL-19 sequence is cloned into PLVX-CD19 CAR.

The preparation method comprises the steps of connecting genes of CAR, IL-7 and CCL-19, wherein the genes of IL-7 and CCL-19 are connected by a 2A peptide sequence 1, cloning the genes into a lentiviral vector, and obtaining a CAR-T cell which targets B7-H3 and co-expresses IL-7 and CCL-19;

the CAR is a B7-H3 single-chain antibody connected with CD8 finger, CD8 TM,4-1BB and CD3Z sequences;

the amino acid sequence of the CD8 finger is shown as SEQ ID NO. 2; the amino acid sequence of the CD8 TM is shown as SEQ ID NO. 3; the amino acid sequence of the 4-1BB is shown as SEQ ID NO. 4; the amino acid sequence of the CD3Z is shown as SEQ ID NO. 5.

Further, it also contains IL-15, wherein IL-7, IL-15 and CCL-19 are all linked by 2A peptide sequence 2;

the amino acid sequence of the IL-15 is shown as SEQ ID NO. 7; the amino acid sequence of the 2A peptide sequence 2 is shown as SEQ ID NO. 10.

Further, the CAR sequence containing B7-H3 single chain antibody sequence, IL-7, IL-15 and CCL-19 are cloned into PLVX-CD19 CAR.

Further, the CAR, IL-7, IL-15 and CCL-19 genes, wherein the IL-7, IL-15 and CCL-19 genes are connected by two 2A peptide sequences, and cloned into a lentiviral vector to obtain a CAR-T cell which targets B7-H3 and co-expresses IL-7/IL-15 and CCL-19;

the CAR is that a B7-H3 single chain antibody is connected with CD8 Hinge, CD8 TM,4-1BB and CD3Z sequences.

The invention also protects the use of the B7-H3-targeted co-expressed CAR-T cells for the preparation of a medicament for the treatment of cancer.

Further, the cancer is not limited to stomach cancer, ovarian cancer, pancreatic cancer, colon cancer, melanoma, lung cancer, tongue squamous carcinoma, and ovarian cancer.

The present invention uses CAR-T (H3-7/15X109 CAR-T) targeting B7-H3 and co-expressing IL-7/IL-15 and CCL-19 as a putative therapeutic approach for NSCLC and pancreatic tumors. H3-7/15X19CAR-T showed significantly improved infiltration and expansion of tumor tissue and effectively reduced tumors in NCG mouse model compared to negative CD19CAR-T control and H3-7X19 CAR-T. This study should provide the basis for a new strategy for treating NSCLC and pancreatic tumors with CAR-T cells targeting B7-H3 positive tumor cells.

Drawings

FIG. 1 is a graph showing the binding affinity of B7-H3 (FD 3-34-scfv, FD3-23-1-scfv, FD3-23-2-scfv and FD 3-23-3-scfv) single-chain antibodies to hB7-H3 antigen by BLI.

FIG. 2 is a graph of binding affinity of B7-H3 scFv single chain antibodies to hB7-H3 antigen.

FIG. 3 is a chart of affinity chromatography of B7-H3 scFv (FD 3-34-scFv-hFc).

FIG. 4 is a graph showing the expression of B7-H3 (FD 3-34-scFv) on different human tumor tissues by IHC.

FIG. 5 is a diagram of the construction of H3CAR, H3 CAR-7X19 and H3 CAR-7/15X 19 plasmids (Xba 1/Sal 1).

FIG. 6 is a feature map of H3 CAR-7X19 and H3 CAR-7/15X 19.

FIG. 7 is an experimental plot of cytotoxicity in H3 CAR-7/15X19 cells against SPC-A1 (A, B, C) and Panc-10.05 (D, E, F) cell lines in vitro.

FIG. 8 is a graph of B7-H3 CAR-T cell treatment for NSCLC and PADC mice xenograft models.

FIG. 9 is a single dose of 1X 10 ⁷ H3 CAR-7/15X19 treatment Panc-10.05 cell xenograft model map.

FIG. 10 is a graph of 1X 10 with three doses ⁷ H3CAR-7/15×19 treatment mice SPC-A1 xenograft tumor map.

Detailed Description

Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to like or similar elements or elements having like or similar functions throughout. The embodiments described below by referring to the drawings are illustrative and intended to explain the present invention and should not be construed as limiting the invention. The specific techniques or conditions are not identified in the examples and are performed according to techniques or conditions described in the literature in this field or according to the product specifications. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Example 1:

1. materials and methods used:

mice: NCG mice (NOD-Parkdcem 26Cd52IL2rgem26Cd 22/Nju) were purchased from Jiangsu Jiuyaokang Biotech Co. Female mice of 6 to 8 weeks of age were kept in facilities free of specific pathogens at the university of fowledgeable medical science. All animal experiments were conducted in accordance with the guidelines of the national institutes of health and approved by the institutional animal care and use committee of the medical sciences of the Fujian (accession number 2021-0382).

Tissue and blood samples: formalin-fixed paraffin-embedded or surgical tissue samples were taken from the department of medical science, university of fomes, and hospital pathology. Informed consent was obtained prior to sample collection. Peripheral blood collected from healthy adult volunteers was used for PBMC isolation with consent from the daffodil blood-saving center. This study was approved by the ethical review board of the university of the Fujian medical science.

Cell culture: cell culture tumor cell SPC-A1 was obtained from GenePharma (Shangghai, china), CA922 and OVCAR-8 were obtained from ATCC, and Panc-10.05 GFP 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 2mmol/L L-glutamine (Invitrogen).

Lentivirus and CAR-T formulation: h3 The CAR gene (structure shown as CAR in FIG. 5A) comprises B7-H3 scFv (i.e., anti-B7H3-scFv in the figure, sequence shown as SEQ ID NO: 1), CD8a hinge (i.e., CD8 hinge in the figure, sequence shown as SEQ ID NO: 2) and transmembrane domain (i.e., CD8 TM in the figure, sequence shown as SEQ ID NO: 3), and signaling domains of 4-1BB (i.e., leader in the figure, sequence shown as SEQ ID NO: 4) and CD3zeta (i.e., CD3Z in the figure, sequence shown as SEQ ID NO: 5). The target B7-H3 gene fragment, the CD8a hinge and transmembrane domain fragment and the 4-1BB and CD3zeta signal transduction domain fragment are amplified by adopting a PCR method, and three fragments of the H3CAR gene are cloned and connected into PLVX lentiviral main plasmids by adopting a seamless cloning method. In response to H3CAR, H3-7X19CAR-T and H3-7/15X19CAR were also cloned into lentiviral vectors, respectively. By adopting a seamless cloning technology, sequences corresponding to IL-7 (the sequence of which is shown as SEQ ID NO: 6) and CCL-19 genes (the sequence of which is shown as SEQ ID NO: 8) are placed in series downstream of the H3CAR and are connected through a 2A sequence 1 (the sequence of which is shown as SEQ ID NO: 9) to form H3 CAR-7X 19. Likewise, by using the seamless joining technique, sequences corresponding to IL-7 (see above), IL-15 (see SEQ ID NO: 7) and CCL-19 gene (see above) were placed in tandem downstream of the H3CAR to form H3 CAR-7/15X19 via two 2A sequences 2 (see SEQ ID NO: 10). Specific steps refer to the pentad cloning kit (TAKARA).

Lentiviral vector PLVX and two helper vectors pspax.2 and pmd2.G were transduced into 293T cells, PLVX: pspax.2: pmd2. G=3:2:1 (mass ratio), total 18ug. According to PEI: plasmid = 6:1 transfection, then the 48 hour supernatant was collected and purified using a Lenti-X concentrator (TAKARA, 631232). Flow cytometry detects lentiviral titers.

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

Flow cytometry: for detection of transduced CAR-T, CAR-T cells were labeled with CD276 antigen (B7-H3 antigen) (ACRO corporation, B73-H5253) for 30min. Preparation of 5X 10 ⁵ Cells were washed 3 times with PBS, centrifuged, the supernatant was discarded, and 0.5ug of B7-H3 antigen protein was added for co-incubation for 30min.After washing with PBS, cells were stained with APC anti-human IgG Fc antibody (bioleged, 410712). To detect the memory phenotype, CAR-T cells were stained with APC mouse anti-human CD45RA (BD company, 550855) antibody and PE mouse anti-human CD62L antibody (BD company, 555544). Cells were analyzed using BD Cano II. Data analysis was then performed using Flowjo V10.1.

Western blotting: as the primary antibody, an anti-CD 3zeta antibody (Abcam Co., ab 190728) and an anti-GAPDH antibody (Abcam Co., ab 9485) were used, respectively. The second antibodies used were anti-mouse HRP antibody (Abcam corporation, ab 6728) and anti-rabbit HRP antibody (Abcam corporation, ab 6721). For experimental details reference is made to the usual western blotting method.

Cytokine detection: will be 1X 10 ⁵ Individual T cells were co-cultured with equal or unequal amounts of tumor cells for 24 hours and the supernatants were collected. IFN-. Gamma.was quantified using the human IFN-. Gamma.standard ABTS ELISA kit (PeproTech, inc., 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 Co., SEA662 Ra) and a human interleukin 15 (IL-15) ELISA kit (Cloud clone Co., LBKYSEA 061). CCL-19 was quantified using a human CCL-19/MIP-3. Beta. ELISA kit (Biyundian Co., PC 123).

Cell proliferation: cell counts were quantified using a Cellometer Auto 2000, cell suspension was added to trypan blue dye 1:1, after dilution, counting, and plotting a growth proliferation curve.

Cytotoxicity test: CAR-T cells were co-cultured with tumor cells in 96-well plates at different ET ratios (effector to target ratio) for 24 hours as needed. Supernatants were collected and analyzed using ELISA kits for IFN-gamma detection, as per manufacturer's instructions. The percentage calculation formula of the CAR-T cell killing tumor cells is as follows: [ (experimental group-spontaneous release)/(maximum load-spontaneous release ] ×100 (%) ].

Mouse xenograft tumor model: female NCG mice of 6 to 8 weeks of age were subcutaneously injected 2X 10 ⁶ Individual SPC-A1 cells and 1X 10 ⁶ Each Panc-10.05-GFP cell. Tumor volume was measured every 3 days using vernier calipers(v=1/2×l (length) ×w (width) ×when tumor volume approaches 125mm ³ In this case, 1X 10 by intravenous injection ⁷ CAR-T cells were injected every 5 days. Body weight was measured every 4 days and mice survival was closely observed.

The treatment scheme is as follows:

CD19CAR-T and H3-7/15X19CAR-T cells at 1X 10 ⁷ Panc-10.05-GFP cell tumor model mice were treated separately and tumor changes were measured every 7 days using a small animal in vivo imaging technique.

CD19CAR-T, H3-7X19CAR-T and H3-7/15X19CAR-T cells at 1X 10 ⁷ The dual dose of (C) respectively treats Panc-10.05-GFP cell tumor model mice, and closely detects tumor changes of the mice.

CD19CAR-T, H3-7X19CAR-T and H3-7/15X19CAR-T cells at 1X 10 ⁷ The double doses of SPC-A1 cell tumor model mice are treated respectively, and the tumor changes of the mice are closely detected.

CD19CAR-T and H3-7/15X19CAR-T cells at 1X 10 ⁷ The SPC-A1 cell tumor model mice were treated separately and the tumor changes in the mice were closely examined.

CAR-T cell expansion analysis in mouse blood: mouse blood was collected from retrobulbar veins and analyzed by flow cytometry: 1-2mL of blood was collected from the model mice after treatment, the cells were treated with erythrocyte lysis buffer (Beyotime Co., C3702), washed with PBS, and stained with APC mice anti-human CD3 ε (BD Co., 558257). T cells were counted and analyzed using flowjo V10.1.

Immunohistochemistry (IHC): for IHC assays, tumor tissue samples were fixed, treated and stained according to standard IHC procedures. Sections were stained with anti-human CD3 antibody-HRP (Abcam, ab 305634) and anti-human ki67-HRP (Abcam, ab 279653). Detection was performed using HRP system. Positive cells were scanned and counted.

Statistical analysis: graphPad Prism 8.0 was used for statistical analysis of the data. Depending on the case, one-way anova, two-way anova and Bonferroni post-test, or unpaired two-tailed t-test, were used. The symbols represent statistical significance of P < 0.05P < 0.01P < 0.001. Each experiment was performed at least three times.

Mouse anti-human B7-H3: B7-H3 single-chain antibodies (including FD3-34-scfv, FD3-23-1-scfv, FD3-23-2-scfv and FD 3-23-3-scfv): the preparation method of the single-chain antibody of B7-H3 comprises the following steps:

1. generation of anti-B7-H3 monoclonal antibodies (murine)

1.1 immunization of mice

Female Balb/c mice of 8 weeks old were immunized by subcutaneous multipoint inoculation with a mixed emulsion of human B7-H3 recombinant antigen protein (purchased from Shanghai Yeasen Co., product No. 93111ES60,100ug,RMB3185,Uniport No.Q5ZPR3.1) and Freund's complete adjuvant (CFA, sigma), 80-100 μg protein per mouse; 3 weeks later, boosting with protein and incomplete adjuvant (IFA, sigma) emulsion was performed 3 times, 80-100. Mu.g protein per mouse. The antibody titer was determined by taking serum about 2 weeks after each immunization of the mice, and the serum titer was determined by ELISA method using a microplate reader SpectraMax i 3. When the serum titer reached high enough, 100. Mu.g of human B7-H3 recombinant antigen protein was dissolved in PBS and the spleen was taken out 3 days later for fusion by intraperitoneal injection for boosting once.

1.2 preparation and selection of hybridomas

Hybridoma cells were generated by fusing spleen cells of immunized mice with a mouse myeloma cell line (SP 2/0-Ag 14) under the action of polyethylene glycol (PEG 1450, SIGMA) fusion agent, suspending the fused cells in a selection medium containing HAT (Sigma Aldrich), adding the selection medium to a 96-well culture plate, culturing for about 11 days, and identifying culture supernatants by a SpectraMax i3 method by ELISA. The selected positive clone is subjected to subcloning selection by more than 3 times of limiting dilution method, so that the positive clone is ensured to be a positive clone with single source.

1.3 purification of anti-B7-H3 mab (murine)

1.2 after the subclones were cultured with conventional hybridoma cells, the culture supernatants were captured by HiTrap Protein G HP (available from GE Healthcare, cat No. 29048581), eluted with 0.1M glycine (pH 3.5), the eluted peaks were collected, and desalted with HiTrap Desating (available from GE Healthcare, cat No. 29048684) column with 1 XPBS (pH 7.4) as a displacement buffer. Preserving at-20 deg.c for use.

2. Characterization of anti-B7-H3 mab (murine) (specific binding Capacity)

The antibody was purified by using SPC-A1, A549, CA-9-22 (human oral epithelial cancer cells) cell lines with high expression of B7-H3, B7-H3/Fc of human IgG1 (sequence derived from Fc segment of human IgG1, SEQ ID NO:11, sequence No: AAB 2181.1) labeled by Alexa Fluor 488-Microscale Protein Labeling Kit (available from Thermo, cat No. A30006), cloned into the vector according to standard method, and transferred 293T cells to express B7-H3/IgG1-Fc, the expressed B7-H3/IgG1-Fc was purified by protein A column and fully mixed with anti-B7-H3 monoclonal antibody (murine) purified as described above, after washing, incubated at 4℃for 20-30 minutes on a FACSVerse machine, and the results were measured by an Octet machine as shown in FIG. 2. FIG. 2, in which the binding capacity of murine B7-H3 antibodies was measured by a molecular interaction method, using a biological layer interference method, and the time profile of the antibody was shown as a gentle binding curve (time profile) was shown after the time profile showing that the antigen-binding was not required for the antigen-binding was well after the antibody (time profile showing the time profile of the antibody-phase-3) was shown in FIG. 34) and the time profile after the time profile showing that the binding was not shown by the time profile was shown by the time profile of the antibody-1-section.

TABLE 1 binding parameters of FD3-34-scfv Single-chain antibodies to B7-H3 antigen

Note that: KD represents the binding force of the antibody; kon represents the binding constant of an antibody to an antigen; kdis represents the constant of separation of antibodies from antigens.

QVQLQESGPGLVRPSQTLSLTCTVSGFTFTDFYMNWVRQPPGRGLEWIGFIRDKAKGYTTEYNPSVKGRVTMLVDTSKN

QFSLRLSSVTAADTAVYYCAREGHTAAPFDYWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV

TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE

LLGKABATGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT

VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. SEQ ID NO. 11. 3. Sequencing of anti-B7-H3 antibodies (murine)

cDNA library preparation and procedures in sections such as gene identification and DNA sequencing in accordance with the hybridoma preparation technique (Kohler and Milstein, 1975) in the guidelines for molecular cloning experiments (3 rd edition) [ Molecular Cloning, J. Sambrook, D.W. Lassel, huang Peitang, et al ]. Total RNA was extracted from UM1-51, UM1-56 and UM1-69 hybridoma cells, and cDNA was synthesized and sequenced.

Total RNA was first extracted and purified from hybridoma cells using an RNA purification Kit (RAeasy Mini Kit, qiagen 74104,QIAshredder,Qiagen 79654). The RNA was then reverse transcribed into the first strand cDNA strand using a kit (SMARter RACE cDNA Amplification Kit, clontech) and following the company reagent protocol. PCR amplification was performed using the 5' RACE technique, using the universal primer UPM (universal primer) provided by the kit as the upstream primer and the gene specific primer GSP (gene specific primer) designed based on the mouse IgG1 heavy chain variable region and light chain kappa chain gene sequences as the downstream primer, and SMART first chain cDNA as the template. The PCR products were then ligated into T vector (Zero Blunt TOPO PCR Cloning Kit, invitrogen K2875-J10), respectively. Clones were selected for sequencing and analysis to obtain the light chain and heavy chain variable region sequences of the antibodies, respectively, primers were designed based on the heavy chain variable region sequences and the mouse IgG1 sequences, the full-length heavy chain sequences were obtained by PCR, and the sequences were analyzed using the IGBLAST analysis tool (KABAT) to determine the light and heavy chains and their CDR regions as shown below.

The light chain sequence is as follows:

wherein, the single underlined "_" represents CDR1 #GASENIYGALN) A sequence; double underlineRepresenting CDR2A sequence; lower wavy line->Represents CDR 3->Sequence.

The heavy chain sequence is as follows:

wherein, the single underlined "_" represents CDR1 #GFNIKDT) A sequence; double underlineRepresents CDR 2->A sequence; lower wavy line->Represents CDR 3->Sequence.

4. Humanization of anti-B7-H3 antibodies (murine)

In order to make antibodies better applicable to clinical patient treatment, it is desirable to humanize the antibody sequences so that they retain high affinity for the antigen and other beneficial biological properties (e.g., reduce their immunogenicity in humans). To achieve this, 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 familiar to those skilled in the art. Almost all murine antibodies can be humanized by CDR grafting to maintain antigen binding. For relevant technical literature reference can be made 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 k.c. et al, load Antibody Engineering: methods and Protocols (antibody engineering: methods and protocols), volume 248, humana Press, new Jersey,2004.

Based on the foregoing, it is preferred to humanize the FD 3-34-scFv. Firstly, the heavy chain constant region of the antibody is changed into a human IgG4 constant region sequence, and meanwhile, the light chain constant region of the antibody is changed into a human kappa chain constant region, so that a female parent antibody (Chimeric Ab) Chimeric with human and mice is obtained. After confirming the functional activity of the parent antibody, the heavy chain variable region and the light chain variable region sequences are subjected to humanized modification, and the key amino acids of the FR region are subjected to back mutation on the basis of CDR transplantation. A series of IgG mutants were constructed by performing a facultative synthesis of the amino acids between FRs that did not determine whether or not to affect antigen and antibody binding. After screening based on antibody affinity and expression level, 1 heavy chain and 1 light chain sequences were screened and cloned into pcdna3.1 expression vectors using conventional molecular cloning techniques, either light chain or heavy chain sequence carrying a Kozak sequence at the 5' end and a human IL-2 signal peptide: MYRMQLLSCIALSLALVTNS (SEQ ID NO: 14), which corresponds to the nucleotide sequence:

the light chain sequence is as follows:

The heavy chain sequence is as follows:

The light chain and heavy chain expression vectors were transiently transfected into ExpiCHO-S (Invitrogen) cells to obtain 1 humanized antibody (FD 3-34-scFv), and the purified antibodies were screened by ELISA and then subjected to molecular interaction (BLI) to obtain humanized antibodies with optimal binding force (FIG. 2).

The FD3-34-scfv human sequence is:

DIQMTQSPSSLSASVGDRVTITCGASENIYGALNWFQRKPGKSPQFLIYGATNLADGVPSRFSGSGSGRQYSLTISSLQPEDFATYYCQNVLTSPWTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGFNIKDTYMHWVKERPGQGLEWIGRIDPANGNTKYDPRFQGKATITADTSSNTAYMELSSLRSEDTAVYYCTRREVYWGQGTLVTVSS。SEQ ID NO:1。

the FD3-23-1-scfv human sequence is:

QIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSLEPEDFAVYYCQQWSSNPLTFGQGTKVEIKGGGGSGGGGSGGGGSEVQLQQSGAEVKKPGASVKVSCKTSGYTFTEYTMHWVRQAPGKSLEWIGGFNPNNGGTTYNQKFKGRATMTGDRSSSTAYMELRSLTSEDSAVYYCARVKVPGFYAMDYWGQGTLVTVSS。SEQ ID NO:17。

the FD3-23-2-scfv human sequence is:

QIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSLEPEDFAVYYCQQWSSNPLTFGQGTKVEIKGGGGSGGGGSGGGGSEVQLQQSGAEVKKPGASVKVSCKTSGYTFTEYTMHWVKQSHGKSLEWIGGFNPNNGGTTYNQKFKGRATMTGDRSSSTAYMELRSLTSEDSAVYYCARVKVPGFYAMDYWGQGTLVTVSS。SEQ ID NO:18。

the FD3-23-3-scfv sequence is:

QIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSLEPEDFATYYCQQWSSNPLTFGQGTKVEIKGGGGSGGGGSGGGGSEVQLQQSGAEVKKPGASVKVSCKTSGYTFTEYTMHWVRQAPGKSLEWIGGFNPNNGGTTYNQKFKGRATMTGDRSSSTAYMELRSLTSEDSAVYYCARVKVPGFYAMDYWGQGTLVTVSS。SEQ ID NO:19。

2. results:

expression of 2.1B7-H3 single-chain antibodies in human tumor cell lines and tumor tissues

Screening experiments for 2.1.1FD3-34 mAb and scFv thereof:

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

Binding affinity and specificity of mAb derived from clone FD3-34 and scFv derived from clone FD3-34 were verified using recombinant human 4Ig-B7-H3 protein (Acro). The binding affinity of B7-H3 single-chain antibodies (FD 3-34-scfv, FD3-23-1-scfv, FD3-23-2-scfv and FD 3-23-3-scfv) to the hB7-H3 antigen was detected using the BLI method. Different concentrations of FD3-34-scfv, FD3-23-1-scfv, FD3-23-2-scfv and FD3-23-3-scfv single chain antibodies were incubated with 5ug of biotin hB7-H3 antigen between 1.56nM and 25nM and the binding parameters of the association and dissociation curves were tested using SA sensor in 300 and 600 seconds, respectively. The results are shown in FIG. 1 and Table 1. FIG. 1 is a graph showing the binding affinity of B7-H3 (FD 3-34-scfv, FD3-23-1-scfv, FD3-23-2-scfv and FD 3-23-3-scfv) single-chain antibodies to hB7-H3 antigen by BLI.

TABLE 1 Table of the binding parameters of several B7-H3 scfv single-chain antibodies to B7-H3 antigen

Sample ID	KD(M)	kon(1/Ms)	kdis(1/s)	Full R ²
					FD3-34-scFv	1.77×10 ^-10	1.23×106	2.17×10 ^-4	0.9916
FD3-23-1-scFv	6.18×10 ^-9	6.14×10 ⁴	3.79×10 ^-4	0.9937
					FD3-23-2-scFv	7.34×10 ^-8	1.80×10 ³	1.32×10 ^-4	0.9974
FD3-23-3-scFv	2.61×10 ^-9	2.26×10 ⁵	5.88×10 ^-4	0.9887

As can be seen from fig. 1 and table 1: it can be seen that the FD3-34-scFv-hFc antibodies have the best affinity compared to FD3-23-1-scFv-hFc, FD3-23-2-scFv-hFc and FD 3-23-3-scFv-hFc. These data demonstrate the specificity of the mAb of clone FD3-34 for human B7-H3 and demonstrate that the scFv maintains high affinity and specificity for human B7-H3. mAb FD3-34 and its scFv were therefore selected for further experiments.

The antigen was immobilized on the probe and the affinity constant for FD3-34-scfv was determined using different gradient concentrations (1.56 nM,3.13nM,6.25nM,12.5nM,25 nM) of antibody to bind and dissociate for 300s each. The results are shown in FIG. 2 and Table 2. FIG. 2 is a graph showing the binding affinity of FD3-34-scfv single chain antibody to the hB7-H3 antigen.

TABLE 2 binding parameters of FD3-34-scfv Single-chain antibody to B7-H3 antigen

As can be seen from fig. 2 and table 2: the scFv showed specific binding affinity to B7-H3 protein (i.e. recombinant human 4Ig-B7-H3 protein) (scFv, KD=8.37) ^-10 ±1.10 ^-9 nM)。

FIG. 3 is an affinity chromatography diagram of B7-H3 scFv (FD 3-34-scFv-hFc) using purification conditions: 260ml of sample supernatant was added and 0.1M glycine (pH 3.5) was eluted.

Expression of 2.1.2B7-H3 in human tumor cell lines and tumor tissues

High levels of B7-H3 were detected in various tumor tissues using immunohistochemical analysis, including gastric cancer, ovarian cancer, pancreatic cancer, colon cancer, and melanoma (a of fig. 4). High levels of B7-H3 were then detected on various tumor cell lines from solid tumors, including lung cancer SPC-A1 and A549 cell lines, tongue squamous carcinoma CA9-22 cell line, and ovarian cancer OVCAR-8 cell line, using flow cytometry analysis (FIG. 4B).

FIG. 4 is a graph showing the expression of B7-H3 (FD 3-34-scFv) on different human tumor tissues by IHC. The first row of a represents three pictures from left to right for lung, stomach and ovary cancer, respectively, and the second row represents three pictures from left to right for pancreatic, colorectal and melanoma, respectively (40×). B is the expression of B7-H3 on different human tumor cells. Expression of B7-H3 in human tumor cell lines (SPC-A1, A549, CA9-22, and OVCAR-8) was assessed by FACS. Blue shaded bar graph represents cells stained with anti-human B7-H3 scFv, red bar graph represents cells stained with isotype mouse IgG1 as control.

Design and characterization of 2.2H3-7/15×19CAR-T

The structure of H3-7/15X109 CAR-T is shown as A in FIG. 5. The CAR in a of fig. 5 refers to a CAR core structure comprising B7-H3 scFv,4-1BB and CD3zeta, H3CAR refers to an H3CAR plasmid comprising 5 'and 3' end sequences and a promoter; H3-7X19CAR refers to a plasmid comprising a B7-H3 CAR core structure, an IL-7 structure and a CCL-19 structure; H3-7/15X19CAR refers to a plasmid comprising a B7-H3 CAR core structure, an IL-7 structure, an IL-15 structure and a CCL-19 structure; .

This example creates a tandem construct encoding CAR, IL-7/IL-15 and CCL-19 (using seamless cloning techniques, cloning fragments of interest, constructing a targeting vector), where two 2A peptide sequences 2 join the genes IL-7, IL-15, CCL-19 and clone them into a lentiviral vector named H3-7/15X109 CAR-T. And respectively obtaining a B7-H3 gene fragment, a CD8a hinge and a transmembrane domain fragment and a 4-1BB and CD3zeta signal transduction domain fragment by adopting a seamless cloning kit through PCR, constructing an H3CAR vector, cloning IL-7, IL-15 and CCL-19 fragments, and respectively constructing the IL-7, IL-15 and CCL-19 into target CAR vectors with different designs according to the specification.

The following CAR genes were also created: (A) H3CAR (without IL-7/IL-15 chemokine) and (B) H3-7X19CAR-T were compared. The detailed construction process comprises the following steps: (A) And (3) respectively obtaining a B7-H3 gene fragment, a CD8a hinge and a transmembrane domain fragment and a 4-1BB and CD3zeta signal transduction domain fragment by PCR by adopting a seamless cloning kit, and constructing an H3CAR vector. (B) And respectively obtaining a B7-H3 gene fragment, a CD8a hinge and a transmembrane domain fragment and a 4-1BB and CD3zeta signal transduction domain fragment by adopting a seamless cloning kit through PCR, constructing an H3CAR vector, cloning IL-7 and CCL-19 fragments, and respectively constructing target CAR vectors with different designs by using IL-7 and CCL-19 according to specifications.

FIG. 5 is a diagram of the construction of H3CAR, H3 CAR-7X19 and H3 CAR-7/15X 19 plasmids (Xba 1/Sal 1). Wherein A is the structural diagram of H3CAR, H3 CAR-7X19 and H3 CAR-7/15X 19 plasmids. B is PCR verification of constructed plasmid clones, and each lane is a different positive colony selected. C is the Xba I/Sal I double cleavage identity of PUC57-B7H3, H3 CAR-7X19 and H3 CAR-7/15X 19.

From the figure, the H3CAR, H3 CAR-7X19 and H3 CAR-7/15X 19 plasmids were constructed successfully, all contained the correct fragment (B7-H3 fragment), and the cleavage sites were correct, and the alignment of the sequencing results was consistent with the design sequence.

For expression of CD19-CAR, lentiviral vectors (pLVX-Puro) were used, by inserting the human CD19 gene sequence (specific sequence: uniport No: P15391-2) into the cleavage site of pLVX-Puro, and CD19-CAR was used as a negative control for this experiment.

After transduction, all constructs were stably expressed by human peripheral blood T cells (a of fig. 6). The transduction efficiency cells of the H3-7/15X19CART construct were slightly lower compared to H3-7X19CAR-T (FIG. 6B). This is probably due to the larger ORF of the reading frame of H3-7/15X19CAR-T, as shown in FIG. 6C.

FIG. 6 is a feature map of H3 CAR-7X19 and H3 CAR-7/15X 19. A is a Western-blot Western blot of CD3zeta in CAR-T cells of different groups (left band represents Mock-T, middle band substitution)Table H3 CAR-7X19, right represents H3 CAR-7/15X 19). B is the transfection efficiency of CAR-Ts (H3 CAR-7X19 and H3 CAR-7/15X 19). C is the amplification of Mock-T, H3CAR-7 x19 and H3CAR-7/15 x19 in vitro cultured for 12 days (P)<0.001). D is ELISA analysis of CCL-19 expression in Mock-T, H3CAR-7×19 and H3CAR-7/15×19 (P)<0.0001). E is CD45RA from flow cytometry analysis of T cells in different groups (IL-2, IL-2/7/15, IL-7/15 and H3 CAR-7/15X 19) ⁺ And CD62L ⁺ T cell ratio. Data are expressed as mean ± SD (n=3). F is the number of cells migrated in the transwell experiments for groups Mock-T, H3CAR, H3 CAR-7X19 and H3 CAR-7/15X 19.

From the figure, both H3-CAR-7x19 and H3-7/15x19CAR-T CAR-T cells have CAR molecule expression, and the transduction efficiency is higher.

2.3 overexpression of IL-7 and IL-15 promotes CAR-T proliferation and maintains the memory phenotype

During in vitro culture of CAR-T cells, H3-7/15×19CAR-T was found to proliferate faster after 12 days of culture than Mock-T (cell isolated and cultured non-transduced T cells) and H3-7×19CAR-T (C of fig. 6).

Previous reports indicate that IL-7 and IL-15 can maintain the memory phenotype of T cells. The memory phenotypes of the different CAR-T cells were determined by collecting the different sets of cells and comparing the memory phenotypes of the Mock-T (supplemented with IL-2, IL-2/7/15 and IL-7/15, respectively) and H3-7/15X19CAR-T sets of cells. Among them, T cells are classified into effector T cells Teff, memory stem T cells Tscm, effector T cells Tem and central memory T cells Tcm according to CD45R and CD 62L. As a result, it was found that the proportion of cells of H3-7/15X19CAR-T having a memory phenotype of the central memory T cells Tcm was significantly higher than those of the IL-2/7/15 and IL-7/15 groups supplemented with Mock-T (as in D of FIG. 6).

2.4 overexpression of CCL-19 increases cell invasiveness

To investigate whether overexpression of CCL-19 could enhance the ability of CAR-T to penetrate into tumors, this example used a Transwell invader laboratory experiment, and after observing and studying Mock-T, H3-7X19CAR-T, H3-7/15X19CAR-T cells for 24H, respectively, the number of migrating cells through the transwell membrane was analyzed by crystal violet staining. As shown in F of FIG. 6, H3-7/15X19CAR-T has a greater ability to invade tumors than H3CAR and Mock-T. This suggests that overexpression of CCL-19 may improve the propensity of CAR-T treatment for tumor invasion.

To test for CCL-19 production, experiments were performed on H3CAR, H3-7/15×19CAR-T and H3 CAR-7/19, cell supernatants were collected separately 10 days after cell transduction, and CCL-19 was measured using ELISA (E of FIG. 6). H3-7/15X19CAR-T and H3-7X19CAR-T expressed significantly more on CCL-19 than H3CAR, as shown in E of FIG. 6.

In vitro cytotoxicity of 2.5H3-7/15X19 CAR-T on tumor cells

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

First, the short-term cell lysis capacity of H3-7/15X19CAR-T, CD CAR and Mock-T compared to cell lines was evaluated, the two cells were co-cultured for 24H by setting the ratio of different CAR-T cells (effector cells) and tumor cells (target cells), and the cells were collected for flow detection, wherein the abscissa B7-H3-APC represents the number of surviving tumor cells, and the CD3-PE represents the Mock-T and CAR-T cells. The method comprises the following steps: t=1: 5. 1:2, 1:1, 2:1 for 24 hours. As shown in FIG. 7A, the proportion of SPC-A1 tumor cells specifically killed was 56.2% + -1.4%, 73.4% + -2.0%, 86.6% + -1.9% and 95.4% + -1.20%. As shown in FIG. 7D, the level of apoptosis of H3-7/15X19CAR-T was lower than that of H3 CAR. As shown in FIG. 7D, the proportion of specific killing SPC-A1 tumor cells was 74.2% + -2.3%, 87.3% + -4.0%, 95.0% + -1.5% and 98.4% + -0.80%, respectively. (percent cleavage calculated as follows, [ (experiment-spontaneous release)/(maximum load-spontaneous release ]. Times.100 (%) ])

FIG. 7 is an experimental plot of cytotoxicity in H3 CAR-7/15X19 cells against SPC-A1 (A, B, C) and Panc-10.05 (D, E, F) cell lines in vitro. H3 CAR-7/15X19 had a significant killing effect on B7-H3 positive cell lines SPC-A1 and Panc10.05. As shown, effector cells were co-cultured with target cells at different E/T ratios for 24 hours. Data represent experiments from three separate experiments (< P <0.01, < P <0.001, < P < 0.0001).

In summary, H3-7/15X19CAR-T had better cytotoxicity against the B7-H2+ cell line when tested with equivalent ET ratios. Meanwhile, the co-culture supernatant of H3-7/15x19CAR-T cells and SPC-A1 lung cancer cells or Panc-10.05 pancreatic cancer cells is detected by ELISA, and the E:T ratio is respectively 1: 5. 1:2, 1:1, 2:1. (1) When the ratio of H3-7/15x19 CAR-TCAR-T cells to SPC-A1 cells is 1: 5. at 1:2, 1:1, 2:1, the average secretion amounts of IFN-r for each group were 527.4 + -57.4 pg/ml, 890.3 + -18.3 pg/ml, 872.63 + -178.2 pg/ml and 1269.83 + -187.4 pg/ml, respectively; (2) When the ratio of H3-7/15x19CAR-T CAR-T cells to Panc-10.05 cells was 1:5, 1:2, 1:1 and 2:1, respectively, the average secretion amounts of IFN-r for each group were 372.33.+ -. 48.5pg/ml, 601.23.+ -. 117.9pg/ml, 842.73.+ -. 30.1pg/ml and 931.3.+ -. 71.7pg/ml, respectively. The results show that as the ratio of the CAR-T cells increases, the secretion of IFN-r increases with the increase of the CAR-T, and the tumor killing effect is also enhanced.

2.6H3-7/15x19 CAR-T in vivo Activity

1) To explore the anti-tumor ability of CAR-T in vivo, SPC-A1 xenograft tumors were established in NCG mice (a of fig. 8). When the average tumor volume approaches 125mm ³ In the case of intravenous injection, about 1X 10 ⁷ And (3) CAR-T cells.

CD19CAR-T control group: a total of 4;

h3-7x19CAR-T cell treatment group: a total of 4;

h3-7/15x19CART cell treatment group: a total of 4.

The treatment pattern of each treatment group is shown in FIG. 8A, in which the CD19CAR-T control group is SPC-A1 lung cancer model mice (2X 10 ⁶ S.c.) injection of control CD19-CAR-T cells at D10 and D17 days; the H3-7x19CAR-T cell treated group was SPC-A1 lung cancer model mice (2 x 10) ⁶ S.c.) 1 x 10 with two doses received at D10 and D17 days ⁷ H3-7x19 CAR-T/times; h3-7/15x19CART cell therapy group was SPC-A1 lung cancer model mice (2 x 10) ⁶ S.c.) 1 x 10 with two doses received at D10 and D17 days ⁷ H3-7/15x19 CAR-T/times.

As shown in FIG. 8B, H3-7/15X109 CAR-T showed less tumor growth and a higher proportion of survival than CD19CAR-T, from the perspective of tumor size and survival, thus better tumor inhibitory activity. It was found that all of the CD19CAR-T control groups died within 20 days, 25% of the mice (i.e., 1) in the H3-7X19CAR-T cell treated group survived for more than 28 days, while 75% of the mice (i.e., 3) in the H3-7/15X19CART cell treated group survived for more than 28 days.

FIG. 8 is a graph of B7-H3 CAR-T cell treatment for NSCLC and PADC mice xenograft models. A treatment procedure and tumor tissue B7-H3 expression in NCG mice subcutaneously vaccinated with SPC-A1 cells. B. SPC-A1 lung cancer tumor tissue growth in H3CAR-7/15 x19 treated NCG mice compared to CD19CAR-T cells and H3CAR-7 x19CAR-T cells was compared to survival in Kaplan-Meier survival analysis mice (< 0.001) with n=4 per group. C. The treatment of NCG mice subcutaneously inoculated with panc10.05-GFP cells was accompanied by tumor tissue B7-H3 expression. Panc-10.05 pancreatic cancer tumor tissue growth in H3CAR-7/15 x19 treated NCG mice compared to CD19CAR-T cells and H3CAR-7 x19CAR-T cells and survival in Kaplan-Meier survival assay mice (< 0.001) with n=4 per group.

The results indicate that more mice survived for more than 28 days (P < 0.0001) in the H3-7/15x19CAR-T cell treated group compared to the CD19CAR-T control group and the H3CAR 7x19 cell treated group.

In addition, further treatment with three doses of CAR-T was performed (see FIG. 10, A, CD19CAR-T control group was SPC-A1 lung cancer model mice (2X 10) ⁶ S.c.) CD19CAR-T negative control cells were injected at D5, D10 and D15 days; h3-7/15x19CART cell therapy group was SPC-A1 lung cancer model mice (2X 10) ⁶ S.c.) 1 x 10 with three doses received on day D5, D10 and D15 ⁷ H3-7/15x19 CAR-T/times). And found that in the CD19CAR-T control group, all tumors reached 2000mm within 34 days ² And is euthanized. In the H3-7/15x19CAR-T cell treated group, 100% of the mice survived for more than 50 days. The results showed that the H3-7/15X19CAR-T cell treated group had a longer survival period compared to the CD19CAR-T control group, and that the tumors of tumor-bearing mice were 100% cured (.: P)<0.001). The peripheral blood CD3+ T cells of mice in the H3-7/15X19CAR-T cell treatment group are significantly highIn the CD19CAR-T control group. Immunohistochemical results showed a significant decrease in Ki-67 for the H3-7/15x19CAR-T group compared to the CD19CAR-T control group, and a significant increase in infiltration of cd3+ T cells in tumor tissue (as in F of fig. 10). FIG. 10 is a graph of 1X 10 with three doses ⁷ H3CAR-7/15×19 treatment mice SPC-A1 xenograft tumor map. A. By 1X 10 ⁶ Treatment procedure of NCG mice subcutaneously vaccinated with individual SPC-A1 cells. B. Comparing the survival time (. Times.P) of H3 CAR-7/15X 19 mice with mice after CD19CAR-T cell treatment by Kaplan-Meier survival analysis<0.01 N=5 per group. Comparison of tumor volumes and weights of the C and d.h3car-7/15×19 groups with the CD19CAR-T cell group (×p)<0.001，****P<0.0001 A) is provided; CD3 in peripheral blood of group E.H3 CAR-7/15×19 and CD19CAR-T cells ⁺ T cell comparison (×p)<0.01 A) is provided; ki-67 and CD3 in NCG mouse tumor tissue in group F.H3CAR-7/15X19 compared to group CD19CAR-T cells ⁺ IHC staining of infiltrating lymphocytes (20×); the left panel is a tissue staining image, the right panel is Ki-67 and CD3 in tumor tissue ⁺ IHC score of T cell infiltration (×p)<0.01)。

2) Establishment of NCG mouse Panc-10.05 pancreatic cancer model (2X 10) ⁶ S.c.), when the average tumor volume is approximately 125mm ³ In the case of intravenous injection, about 1X 10 ⁷ And (3) CAR-T cells. The single dose is respectively given 1×10 ⁷ H3-7/15X109 CAR-T and CD19 CAR-T.

CD19CAR-T control group: a total of 4;

H3-7/15X19CART cell treatment group: a total of 4.

FIG. 9 is a single dose of 1X 10 ⁷ H3 CAR-7/15X19 treatment Panc-10.05 cell xenograft model map. 2X 10 ⁶ The cells of Panc-10.05 were subcutaneously injected into NCG mice, and the treatment course of the allogeneic model was Panc-10.05 allograft. As shown in fig. 9, all animals in the CD19CAR-T control group died within 20 days, while 60% (if it should be 75%) of the mice (i.e., 3) in the H3-7/15x19CAR-T group survived for more than 20 days. The results show that the H3-7/15x19CAR-T cell treated group survived more than 20 days in mice than the H3-7x19CAR-T cell treated group and the CD19CAR-T control group (P)<0.0001)。

Establishment of NCG mouse Panc-10.05 pancreatic cancer model (2X 10) ⁶ S.c.), when the average tumor volume is approximately 125mm ³ In the case of intravenous injection, about 1X 10 ⁷ And (3) CAR-T cells. Two doses of 1X 10 are administered separately ⁷ H3-7/15X19CAR-T, H3-7/15X19CAR-T and CD19 CAR-T.

CD19CAR-T control group: a total of 4;

h3-7×19CAR-T cell treatment group: a total of 4;

H3-7/15X19CART cell treatment group: a total of 4.

The treatment pattern of each treatment group is shown in FIG. 8C, in which the CD19CAR-T control group is Panc-10.05 pancreatic cancer model mice (2X 10 ⁶ S.c.) CD19CAR-T negative control cells were injected at D12 and D17 days; the H3-7X19CAR-T cell treated group was Panc-10.05 pancreatic cancer model mice (2X 10) ⁶ S.c.) 1 x 10 with two doses received at D12 and D17 days ⁷ H3-7x19 CAR-T/times; h3-7/15×19CART cell treated group was Panc-10.05 pancreatic cancer model mice (2×10) ⁶ S.c.) 1 x 10 with two doses received at D12 and D17 days ⁷ H3-7/15x19 CAR-T/times.

FIG. 8C is the treatment of NCG mice subcutaneously vaccinated with Panc10.05-GFP cells with tumor tissue B7-H3 expression. D is survival rate of mice (< 0.0001) compared to CD19CAR-T cells and H3CAR-7 x19 by Kaplan-Meier survival analysis, n=4 per group.

Fig. 8D is a graph comparing survival rate of H3CAR-7/15 x19 compared to CD19CAR-T cells and H3CAR-7 x19 by Kaplan-Meier survival analysis (< 0.0001) for mice, n=4 for each group. As shown in D of fig. 8, all mice in the CD19CAR-T control group died within 26 days, with-25% of the mice (i.e., 1) in the H3-7x19CAR-T cell treated group surviving for more than 30 days. Whereas 75% of the mice (i.e., 3) in the H3-7/15X19CAR-T cell treated group survived for more than 30 days. More mice in the H3-7/15x19CAR-T cell treated group survived for more than 30 days (< P < 0.0001) compared to the CD19CAR-T control group and the H3CAR-7 x19 cell treated group.

Although embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives, and variations may be made in the above embodiments by those skilled in the art without departing from the spirit and principles of the invention.

Claims

1. A CAR-T cell that targets B7-H3 and co-expresses a cytokine and a chemokine, comprising a B7-H3 single chain antibody, IL-7, and CCL-19; preferably, wherein IL-7 and CCL-19 are both linked by 2A peptide sequence 1;

2. The B7-H3-targeted co-expression of cytokines and chemokines CAR-T cell of claim 1, wherein the CAR sequence comprising the B7-H3 single chain antibody sequence, IL-7 and CCL-19 sequences are cloned into PLVX-CD19 CAR.

3. The B7-H3-targeted and co-expressed cytokine and chemokine-targeted CAR-T cell of claim 1, prepared by ligating the CAR, IL-7 and CCL-19 genes, wherein the IL-7, CCL-19 genes are linked by the 2A peptide sequence 1, and cloning them into a lentiviral vector to obtain a B7-H3-targeted CAR-T cell that co-expresses IL-7 and CCL-19;

4. The B7-H3-targeted and co-expressed cytokine and chemokine CAR-T cell of claim 1, further comprising IL-15, wherein IL-7, IL-15 and CCL-19 are all linked by a 2A peptide sequence 2;

5. The B7-H3-targeted co-expression of cytokines and chemokines CAR-T cell of claim 4, wherein the CAR sequence comprising the B7-H3 single chain antibody sequence, IL-7, IL-15 and CCL-19 are cloned into a PLVX-CD19 CAR.

6. The B7-H3-targeted and co-expressed cytokine and chemokine-targeted CAR-T cell of claim 4, wherein the CAR, IL-7, IL-15 and CCL-19 genes, wherein the IL-7, IL-15, CCL-19 genes are linked by two 2A peptide sequences, are cloned into a lentiviral vector to obtain a B7-H3-targeted and co-expressed IL-7/IL-15, CCL-19 CAR-T cell;

7. Use of a CAR-T cell of any one of claims 1-6 that targets B7-H3 and is co-expressed for the manufacture of a medicament for the treatment of cancer.

8. The use according to claim 2, wherein the cancer is not limited to gastric cancer, ovarian cancer, pancreatic cancer, colon cancer, melanoma, lung cancer, tongue squamous carcinoma and ovarian cancer.