A chimeric antigen receptor targeting FAP, CAR-T cells and their application in liver fibrosis

Abstract

The present invention belongs to the field of biomedicine and molecular biology technology, and specifically relates to a chimeric antigen receptor targeting FAP, CAR-T cells and their application in liver fibrosis. The present invention takes FAP as a target, thereby successfully preparing a single-chain antibody targeting FAP antigen, and applies it to the second-generation CAR. The constructed CAR-T cells can effectively kill FAP. + The cells have been verified by experiments to have the function of alleviating liver fibrosis in mice and inhibiting FAP + The growth of tumor cells provides a new strategy for the clinical use of FAP CAR-T cells in the treatment of liver diseases including liver fibrosis, and also provides a new strategy for the elimination of FAP + Cell therapy provides a new approach for kidney disease, pulmonary fibrosis, cardiovascular disease and various tumor diseases, and therefore has good practical application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine and molecular biology, and specifically relates to a chimeric antigen receptor targeting FAP, a CAR-T cell and an application thereof in liver fibrosis. Background Art

[0002] The information disclosed in the background of the invention is only intended to enhance the understanding of the overall background of the invention and should not be necessarily regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Fibrosis is a common pathological endpoint of chronic, progressive or severe acute diseases in almost all organs, and is also one of the bottlenecks in the treatment of various chronic diseases. Nonalcoholic fatty liver disease (NAFLD) is a metabolic disease with a very high incidence worldwide, especially in developed countries in Europe and the United States where obesity is relatively high. NAFLD causes more than 2 million deaths each year, accounting for 4% of all global mortality. The incidence of NAFLD in Asia is 27.3%. As the obesity rate in China increases year by year, the number of NAFLD patients in China increases year by year. Liver fibrosis is the most common disease of NAFLD, which is caused by activated HSCs secreting collagen and other factors that trigger the release of multiple proinflammatory factors by hepatocytes and intrahepatic immune cells. Liver fibrosis is manifested as diffuse hyperplasia and deposition of extracellular matrix in the liver, changes in intrahepatic structure, abnormal physiological function of liver tissue, and in severe cases, it can develop into cirrhosis or even liver cancer. HSCs are an important non-parenchymal cell in the liver and play a core role in the pathogenesis of liver fibrosis. There are currently no approved drugs or therapies for the direct treatment of liver fibrosis. However, various preclinical studies have shown that eliminating activated HSCs can effectively alleviate liver fibrosis. Activated HSCs highly express FAP, and targeting FAP to eliminate HSCs is an effective and feasible strategy to improve liver fibrosis.

[0004] Chimeric antigen receptors are artificially modified fusion proteins based on T cell receptors, consisting of extracellular antigen recognition domains fused to multiple intracellular signaling domains. The extracellular active parts are usually ligands, receptors, and single chain fragment variables (scFv), which can recognize specific antigens (usually overexpressed on tumor cells or unique to tumor cells) without being presented by major histocompatibility complex molecules, similar to antibodies. A large number of studies have shown that CAR-T cells with 4-1BB as a co-stimulatory molecule have stronger persistence in vivo, and the survival time in vivo can exceed 3 months or even reach one year; while CAR-T cells with CD28 as a co-stimulatory molecule have a strong proliferation ability in the short term, but poor persistence, and the survival time in vivo generally does not exceed one month. A recent article published in Science shows that CAR-T cells have a significant improvement in the treatment of liver aging and myocardial fibrosis, but there are no reports on the study of CAR-T therapy for the treatment of liver fibrosis by targeting hepatic stellate cells.

[0005] China ranks first in the world in the number of malignant tumor cases, among which liver cancer, lung cancer, and gastric cancer are at the forefront of domestic morbidity and mortality. Traditional radiotherapy, chemotherapy, and surgical treatment methods are difficult to curb the development and metastasis of tumors. In recent years, tumor immunotherapy represented by immune checkpoint inhibitors and CAR T cell adoptive immunotherapy has made encouraging progress, especially in hematological tumors, but further efforts are needed in solid tumors such as liver cancer. The main force in therapies such as antibody therapy and cell therapy is difficult to infiltrate into solid tumors. In addition to the complex tumor microenvironment, another major reason is that there is a solid layer of tumor-associated fibroblasts (CAFs) around the tumor lesions, which casts a solid copper wall for the tumor. In addition, CAFs can secrete a large number of growth-promoting factors to promote tumor growth and provide sufficient guarantee for the invasion and migration of tumor cells. Studies have shown that by targeting and clearing CAFs, tumor lesions can be effectively inhibited or even cleared. CAFs outside the tumor lesions highly express FAP, and it is a promising strategy to remove fibroblasts by targeting FAP to assist in the treatment of neoplastic diseases.

[0006] The human FAP gene is located on chromosome 2. It is a membrane protein belonging to the glycoprotein family and a member of the serine protease family. FAP is mainly expressed on cells such as CAFs and activated HSCs, and is not expressed on fibroblasts in most normal human tissues. FAP is difficult to detect in organs and tissues in a non-disease state, but its expression increases during tissue abnormalities, such as liver fibrosis, pulmonary fibrosis, atherosclerosis, renal fibrosis, rheumatoid arthritis, and the development of tumors. It has been reported that in mouse tumor models, FAP plays an immunosuppressive role, and knocking out FAP can cause rapid hypoxia and necrosis of tumor cells. Therefore, FAP can be used as a potential therapeutic target. At present, a variety of drugs targeting FAP have appeared, such as N-(Pyridine-4-carbonyl)-D-Ala-boroPro. In addition, immunotherapies such as CAR-T and CAR-NK that target FAP to treat tumor diseases have appeared one after another, but no CAR-T products have been approved for marketing. Summary of the invention

[0007] In view of the above-mentioned prior art, the purpose of the present invention is to provide a chimeric antigen receptor targeting FAP, CAR-T cells and their application in liver fibrosis. Specifically, the present invention targets FAP molecules, obtains mouse single-chain antibody sequences through monoclonal antibody design and immunization of mice, and constructs them into CAR-T cells targeting FAP. In vitro experiments show that CAR-T cells targeting FAP can effectively eliminate FAP. + In vivo experiments showed that FAP-CAR-T cells can effectively improve liver fibrosis in mice and inhibit the growth of liver cancer in mice. This study provides new ideas for the clinical treatment of diseases such as liver fibrosis, and provides new targets for CAR-T immunotherapy in the treatment of cardiovascular diseases and even solid tumors. Based on the above research results, the present invention is completed.

[0008] In order to achieve the above technical objectives, the technical solution provided by the present invention is as follows:

[0009] In a first aspect of the present invention, there is provided a chimeric antigen receptor targeting FAP, wherein the chimeric antigen receptor comprises an antigen binding domain;

[0010] The antigen binding domain is an anti-FAP antibody, and the anti-FAP antibody may be a single-chain antibody (scFv), which is composed of an antibody heavy chain and an antibody light chain;

[0011] The antibody heavy chain (VH) has any one of the amino acid sequences (a1)-(a3):

[0012] (a1) the amino acid sequence shown in SEQ ID NO.1;

[0013] (a2) a polypeptide derived from the amino acid sequence shown in SEQ ID NO.1 by substitution and / or deletion and / or addition of one or more amino acid residues and having the same or similar function;

[0014] (a3) other genes encoding polypeptides derived from the amino acid sequence shown in SEQ ID NO.1 that have a similarity of more than 90% with the amino acid sequence shown in SEQ ID NO.1 and have the same or similar functions.

[0015] The antibody light chain (VL) has any one of the amino acid sequences (b1)-(b3):

[0016] (b1) the amino acid sequence shown in SEQ ID NO.2;

[0017] (b2) a polypeptide derived from the amino acid sequence shown in SEQ ID NO.1 by substitution and / or deletion and / or addition of one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.2 and having the same or similar function;

[0018] (b3) other genes encoding polypeptides derived from the amino acid sequence shown in SEQ ID NO.2 that have a similarity of more than 90% with the amino acid sequence shown in SEQ ID NO.2 and have the same or similar functions.

[0019] The second aspect of the present invention provides an isolated nucleic acid encoding the chimeric antigen receptor.

[0020] The third aspect of the present invention provides a CAR expression vector, which comprises the above-mentioned nucleic acid.

[0021] The fourth aspect of the present invention provides a CAR-T cell, which is capable of expressing the above-mentioned chimeric antigen receptor targeting FAP; specifically, it is a T lymphocyte modified by the above-mentioned chimeric antigen receptor targeting FAP.

[0022] The fifth aspect of the present invention provides the use of the above-mentioned chimeric antigen receptor, nucleic acid, CAR expression vector, and CAR-T cell in the preparation of drugs for preventing and treating diseases related to high expression of FAP.

[0023] A sixth aspect of the present invention provides a drug for preventing and treating diseases related to high FAP expression, wherein the active ingredient of the drug may be the above-mentioned chimeric antigen receptor or the above-mentioned CAR-T cell.

[0024] The seventh aspect of the present invention provides a method for preventing and / or treating a disease associated with high FAP expression, the method comprising administering an effective amount of the above-mentioned chimeric antigen receptor, CAR-T cell or drug to a subject.

[0025] Beneficial technical effects of one or more of the above technical solutions:

[0026] The above technical solution uses FAP as a target, thereby successfully preparing a single-chain antibody targeting FAP antigen, and applying it to the second-generation CAR. The constructed CAR-T cells can effectively kill FAP + The cells have been verified by experiments to have the function of alleviating liver fibrosis in mice and inhibiting FAP + The growth of tumor cells provides a new strategy for the clinical use of FAP CAR-T cells in the treatment of liver diseases including liver fibrosis, and also provides a new strategy for the elimination of FAP + Cell therapy provides a new approach for kidney disease, pulmonary fibrosis, cardiovascular disease and various tumor diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0028] Figure 1 This is a diagram of the strategy for immunizing mice to obtain supernatants containing numerous antibody sequences in an embodiment of the present invention.

[0029] Among them: C57 mice were immunized 5 times with the coating solution coated with FAP extracellular domain antigen, 5 mice were immunized, once every 14 days, and blood was collected during the period to detect the immune effect.

[0030] Figure 2 Graph showing the purification of mouse FAP extracellular region antigen in an embodiment of the present invention; wherein: A is the detection of unpurified antigen after induction; and B is the detection of purified antigen.

[0031] Figure 3 Construction and detection of mouse CAR-T cells in the embodiments of the present invention;

[0032] Among them: A is the FAP CAR structure and original diagram; B is the lentiviral vector used for construction; C is the flow cytometry identification of mouse T cells after sorting; D is the detection of the positive rate of FAP CAR-T cells.

[0033] Figure 4 The construction of FAP overexpressing cell lines and the detection of CAR-T cells killing cell lines in the embodiments of the present invention;

[0034] Among them: A is a mouse FAP overexpression cell line constructed using LX2 cells as a platform; B is a mouse FAP overexpression cell line constructed using Hepa1-6 cells as a platform; C is a diagram showing the killing of LX2-FAP cells by CAR-T cells; D is a diagram showing the killing of Hepa1-6-FAP cells by CAR-T cells.

[0035] Figure 5 This is an in vivo anti-tumor experiment of FAP CAR-T cells in the embodiment of the present invention;

[0036] Among them: A is a diagram of mouse subcutaneous tumor modeling and FAP CAR-T cell transfusion strategy; B is a diagram of mouse tumor cell volume changes; Figure C is a diagram of mouse survival curve.

[0037] Figure 6 The effect of FAP CAR-T cells on liver fibrosis in vivo in the embodiments of the present invention;

[0038] Among them: A is the WB detection of mouse liver fibrosis one week after CAR-T cell infusion; B is the HE detection of mouse liver fibrosis; C is the Sirius red detection of mouse liver fibrosis, among which, the Sham group mice did not establish a fibrosis model. DETAILED DESCRIPTION

[0039] It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0040] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0041] In a typical embodiment of the present invention, a chimeric antigen receptor targeting FAP is provided, wherein the chimeric antigen receptor comprises an antigen binding domain;

[0042] The antigen binding domain is an anti-FAP antibody, and the anti-FAP antibody may be a single-chain antibody consisting of an antibody heavy chain and an antibody light chain;

[0043] The antibody heavy chain (VH) has any one of the amino acid sequences (a1)-(a3):

[0044] (a1) the amino acid sequence shown in SEQ ID NO.1;

[0045] (a2) a polypeptide derived from the amino acid sequence shown in SEQ ID NO.1 by substitution and / or deletion and / or addition of one or more amino acid residues and having the same or similar function;

[0046] (a3) other genes encoding polypeptides derived from the amino acid sequence shown in SEQ ID NO.1 that have a similarity of more than 90% with the amino acid sequence shown in SEQ ID NO.1 and have the same or similar functions.

[0047] The antibody light chain (VL) has any one of the amino acid sequences (b1)-(b3):

[0048] (b1) the amino acid sequence shown in SEQ ID NO.2;

[0049] (b2) a polypeptide derived from the amino acid sequence shown in SEQ ID NO.1 by substitution and / or deletion and / or addition of one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.2 and having the same or similar function;

[0050] (b3) other genes encoding polypeptides derived from the amino acid sequence shown in SEQ ID NO.2 that have a similarity of more than 90% with the amino acid sequence shown in SEQ ID NO.2 and have the same or similar functions.

[0051] Furthermore, the antibody heavy chain and the antibody light chain can be directly connected or connected through a linker; the linker can be a polypeptide chain composed of alanine and / or serine and / or glycine. Further, the linker is (G4S)n, where n can be an integer, n is 1-10, and further, n is 3.

[0052] In another specific embodiment of the present invention, the chimeric antigen receptor targeting FAP comprises a signal peptide (such as a CD8 SP signal peptide), an antigen binding domain (such as the above-mentioned anti-FAP antibody), a chimeric receptor hinge region (such as CD8Hinge), a co-stimulatory signaling domain (such as any one or more of a 4-1BB domain, a CD27 domain, a CD40 domain or an OX40 domain) and a signaling domain (such as a CD3ζ signaling domain) connected in series;

[0053] In another specific embodiment of the present invention, an isolated nucleic acid is provided, wherein the nucleic acid encodes the chimeric antigen receptor.

[0054] In another specific embodiment of the present invention, the nucleic acid molecule sequentially includes the coding gene sequence of the CD8SP signal peptide, the coding gene sequence of the antigen binding domain that binds to the FAP antigen, the coding gene sequence of the CD8 hinge region, the coding gene sequence of the 4-1BB co-stimulatory signaling domain, and the coding gene sequence of the CD3ζ signaling domain.

[0055] In another specific embodiment of the present invention, a CAR expression vector is provided, wherein the CAR expression vector comprises the above-mentioned nucleic acid.

[0056] According to the present invention, the CAR expression vector may be a viral vector, including but not limited to a lentiviral vector, a retroviral vector, an adenoviral vector or a plasmid, etc.; based on the current technological maturity, the CAR expression vector may be a lentiviral vector.

[0057] In another specific embodiment of the present invention, a CAR-T cell is provided, wherein the CAR-T cell is capable of expressing the above-mentioned chimeric antigen receptor targeting FAP; specifically, it is a T lymphocyte modified by the above-mentioned chimeric antigen receptor targeting FAP.

[0058] Specifically, it is prepared by introducing the above-mentioned CAR expression vector. In the art, the above-mentioned operation can be completed by well-known technical means, such as by transfecting the chimeric antigen receptor targeting FAP into T cells through its encoded nucleotide sequence, and the transfection process can be achieved by transfecting the above-mentioned CAR expression vector into T cells.

[0059] In another specific embodiment of the present invention, the use of the above-mentioned chimeric antigen receptor, nucleic acid, CAR expression vector, and CAR-T cell in the preparation of drugs for preventing and treating diseases related to high expression of FAP is provided.

[0060] Specifically, the diseases associated with high FAP expression include, but are not limited to, kidney disease, liver disease, lung disease, cardiovascular disease, immune-related disease and tumor disease, and related conditions mediated by the above diseases, such as liver fibrosis, pulmonary fibrosis, atherosclerosis, kidney fibrosis, rheumatoid arthritis, solid tumors and blood tumors, etc., which are not specifically limited here.

[0061] Tumors are used in the present invention as known to those skilled in the art, and include benign tumors and / or malignant tumors. Benign tumors are defined as excessive proliferation of cells that are unable to form aggressive, metastatic tumors in the body. Conversely, malignant tumors are defined as cells with multiple cellular and biochemical abnormalities that are capable of forming systemic diseases (e.g., forming tumor metastases in distant organs). More specifically, the tumors include, but are not limited to, liver cancer, colorectal cancer, pancreatic cancer, and ovarian cancer.

[0062] Specifically, the present invention has proved through experiments that CAR-T cells can effectively kill human stellate cell lines (LX2) and mouse hepatocellular carcinoma cell lines (Hepa1-6) expressing FAP in vitro, and can improve mouse liver fibrosis in vivo. In the experiment of the present invention, a mouse liver fibrosis model was created by intraperitoneal injection of CCL4, and FAP CAR-T cells were reinfused into the tail vein, and it was found that FAPCAR-T cells can effectively alleviate the degree of liver fibrosis.

[0063] In another specific embodiment of the present invention, a drug for preventing and treating diseases related to high FAP expression is provided, and the active ingredient of the drug can be the above-mentioned chimeric antigen receptor or the above-mentioned CAR-T cell.

[0064] The prevention and treatment of diseases related to high FAP expression specifically refers to the prevention and / or treatment of diseases related to high FAP expression. The specific diseases and symptoms of the diseases related to high FAP expression are as shown above and will not be repeated here.

[0065] The drug may also contain at least one inactive drug ingredient, which may be a carrier, excipient, diluent, etc. commonly used in pharmacy. Moreover, according to common methods, the drug may be prepared into oral preparations, external preparations, suppositories, and sterile injection solutions in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, sprays, etc.

[0066] The non-drug active ingredients such as carriers, excipients and diluents that may be included are well known in the art, and those skilled in the art can determine whether they meet clinical standards.

[0067] In another specific embodiment of the present invention, the carrier, excipient and diluent include but are not limited to lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0068] In another specific embodiment of the present invention, the medicine of the present invention can be applied to the body by known means. For example, it can be delivered to the tissue of interest by intravenous systemic delivery or local injection. It can be applied via intravenous, percutaneous, intranasal, mucosal or other delivery methods. Such application can be carried out via single dose or multiple doses. It will be appreciated by those skilled in the art that the actual dose to be applied in the present invention can vary depending on various factors to a great extent, such as target cells, biological types or their tissues, the general condition of the subject to be treated, the route of administration, the mode of administration, etc.

[0069] In another specific embodiment of the present invention, the drug administration subjects can be humans and non-human mammals, such as mice, rats, guinea pigs, rabbits, dogs, monkeys, gorillas, etc.

[0070] The medicine can also be administered separately to other preventive and / or therapeutic compounds in a separate composition or in a dosage form different from the main active ingredient. Partial doses of the main ingredient can be administered simultaneously with other therapeutic compounds, while other doses can be administered separately. In the course of treatment, the dosage of the medicine of the present invention can be adjusted according to the severity of the symptoms, the frequency of recurrence and the physiological response of the treatment regimen.

[0071] In another specific embodiment of the present invention, a method for preventing and / or treating a disease associated with high FAP expression is provided, the method comprising administering an effective amount of the above-mentioned chimeric antigen receptor, CAR-T cell or drug to a subject.

[0072] The subject refers to an animal, preferably a human, that has been the subject of treatment, observation or experiment. The "effective amount" refers to the amount of active compounds or agents, including the compounds of the present invention, which can cause the biological or medical response of the tissue system, animal or human sought by the researcher, veterinarian, doctor or other medical personnel, including the alleviation or partial alleviation of the symptoms of the disease, syndrome, condition or disorder being treated. It must be recognized that the optimal dosage and interval of the active ingredients of the present invention are determined by their properties and external conditions such as the form, route and site of administration and the specific mammal being treated, and this optimal dosage can be determined by conventional techniques. It must also be recognized that the optimal course of treatment, that is, the daily dosage of the compound at the same time over a specified period of time, can be determined by methods known in the art.

[0073] The present invention is further explained by the following examples, but they are not intended to limit the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The test methods for the specific conditions in the following examples are usually carried out under normal conditions.

[0074] Example

[0075] method:

[0076] Mouse immunization

[0077] The amino acid sequence used for mouse FAP immunization is the extracellular region (FAP ECD), and the specific sequence is shown in SEQ ID NO.8. The required DNA sequence was obtained from the company (Qingdao Qingke Co., Ltd.), and then the optimized DNA was constructed into the PGEX-4T-1 vector using BamHI-XhoI as the restriction site through cloning technology. After protein expression, it was purified, and then the mice were immunized multiple times. A variety of antibody sequences with high affinity were obtained through sequencing, and the optimal FAP scFv was constructed into the CAR vector, and then the effect was verified by constructing CAR-T cells. The specific process of mouse immunization is as follows Figure 1 shown.

[0078] High affinity FAP scFv screening

[0079] Blood collection test: This study followed the mature immune process to carry out immunization and complete titer determination and other index tests. After the first, second, and third blood collection tests, target mice with higher titers that met the experimental requirements were selected for fusion. In this study, one mouse 5# was selected from five mice for fusion, and the blood collection test results are shown in Table 1.

[0080] Table 1 Blood test results

[0081]

[0082] Fusion and cloning: Use SP2 / 0 mouse myeloma cells and spleen cells of preferred mice (5#) for cell fusion. After fusion, culture, observation, detection and positive and negative control tests are performed to obtain a batch of hybridoma cells that meet the experimental requirements, and continue to culture and select them.

[0083] 5# Mouse strain identification and sequencing: After fusion, primary cloning by limiting dilution method and supernatant detection, 9 cell lines with 22 cells that met the project requirements were obtained. By comparison, a hybridoma with high titer and optimal affinity was sequenced to obtain the full-length FAP antibody sequence. The antibody heavy chain / light chain variable region genes were obtained by PCR to obtain FAP scFv.

[0084] Construction of FAP CAR

[0085] The sequence of CAR in the present invention is CD8 SP-VL-(G4S)3-VH-CD8 Hinge-41BB-CD3ζ. The sequence was synthesized by Qingke Biotechnology Co., Ltd. at one time. The sequence parts are shown above and constructed into the vector pRLenti-EF1-EGFP-EF1a-MCS-WPRE. The front and rear restriction sites are XbaI and BamHI, respectively. After sequencing and comparison, the sequences are found to be consistent.

[0086] Obtaining mouse T cells

[0087] C57BL / 6J mice were purchased from Vital River Company and killed by cervical dislocation. The mice were soaked in 75% alcohol for about 5 minutes. The spleen of the mice was removed in a biosafety cabinet and ground to form a single cell suspension. Mouse T cells with a purity of 99% were obtained after sorting using a kit (EasySep Mouse T Cell Isolation Kit, STEMCELL) Figure 3 B), add CD3 / CD28 activation beads (Miltenyi Biotec) for activation, using a medium containing 10% fetal bovine serum, 50 IU mL -1 IL-7 (Proteintech) and 100 IU mL -1 The cells were cultured in RPMI1640 medium supplemented with IL-15 (Proteintech).

[0088] Construction of CAR-T cells

[0089] FAP CAR virus was provided by Heyuan Biotechnology Co., Ltd., and the virus with MOI=100 was used to infect an appropriate amount of T cells. The infected cells were centrifuged in a 12-well plate at 800g×60min. The infected T cells were transferred to fresh culture medium for culture. The positive rate was detected by flow cytometry for about 3 days and the cells were re-infused into mice within one week.

[0090] Flow cytometry

[0091] Take 1×10 5 The cells (Vec-T and FAP CAR-T) were placed in 300 μL pre-cooled PBS, 1 μL CD3 flow cytometry antibody was added, incubated at room temperature in the dark for 30 min, washed three times with pre-cooled PBS, and then analyzed by flow cytometry (BD) to detect the purity of mouse T cells isolated from the spleen and the GFP expression of T-Vec or FAP CAR-T cells after infection.

[0092] LDH detection of CAR-T cell killing experiment

[0093] The killing experiment of FAP CAR-T cells against LX2-FAP and Hepa1-6-FAP was mainly carried out by analyzing LDH (CytoTox The test was performed by Non-Radioactive Cytotoxicity Assay, with two groups of effector-target ratios of 1:1 and 5:1 respectively. The details are as follows:

[0094] 1. Take 5×10 5 The target cells were placed in a 24-well plate. After they adhered to the wall, the corresponding number of FAP CAR-T (Vec-T) cells were added. The final system was 1.5 ml. There were target cell group and effector cell group.

[0095] 2. The effector cells and target cells were co-cultured for 16 hours, centrifuged, and the supernatant was collected;

[0096] 3. Remove the supernatant of the target cell group, leaving only the cells, add lysis buffer and react for 45 minutes to completely release LDH;

[0097] 4. Take 50 μL of supernatant from each group and put it into a 96-well plate, add 50 μL of detection reagent, and react for 30 minutes in the dark;

[0098] 5. After 30 minutes, add 50 μL of stop solution, react for 1 hour, and read the results with an ELISA reader. The killing value of each group is calculated as follows:

[0099] Killing efficiency of each group (%) = (experimental group value - effector cell natural death value - target cell natural death value) / (target cell complete death value - target cell natural death value) × 100%

[0100] Western Blotting(WB)

[0101] 1. Grind the liver tissue, then add the corresponding volume of protein loading buffer, and cook the protein in a metal bath at 100℃ for 10-20min;

[0102] 2. Electrophoresis: Add about 30 μg of protein sample to each well, and run the upper gel at 80V for about 30min; the lower gel at 120V for about 90min.

[0103] 3. Transfer: Make the PVDF membrane transparent in advance, soak it in methanol for 5 minutes, and transfer it at 400mA. The order of placing each part is: white splint (positive electrode) - sponge - filter paper - PVDF membrane - protein glue - filter paper - sponge - black splint (negative electrode);

[0104] 4. Blocking: After the transfer, block in 5% skim milk solution on a shaker at room temperature for 1-2 hours;

[0105] 5. Incubate with primary antibody: Place the membrane in the primary antibody solution and incubate overnight at 4°C. Wash the membrane with 1×TBST the next day for 3 times, 5-10 minutes each time.

[0106] 6. Incubate with secondary antibody: Place the membrane in the secondary antibody solution and incubate at room temperature for 1-2 hours. Wash the membrane with 1×TBST for 3 times, 5-10 minutes each time;

[0107] 7. Exposure and development: Prepare developer A and B solutions in advance, use them in a 1:1 ratio, and develop on a chemiluminescence imaging system.

[0108] Hematoxylin-eosin (HE) and picrosirius red staining

[0109] The liver tissue was removed and fixed with 4% paraformaldehyde overnight, and then dehydrated, transparentized, and embedded the next day. After sectioning, the tissue was dewaxed and HE stained. Meanwhile, the paraffin tissue sections were stained with the Regen Sirius Red Staining Kit.

[0110] 1. Bake the paraffin sections at 65℃ for 2h and dewax them to water while they are still hot.

[0111] 2. Wash the slices with triple-distilled water 3 times, 2 minutes each time.

[0112] 3. Add Sirius red dye solution and stain at room temperature for 1-2 hours. Rinse with running water to remove the dye on the surface of the slice.

[0113] 4. Add hematoxylin staining solution to stain the nucleus for 8 minutes, rinse with running water to remove the staining solution on the surface of the slice.

[0114] 5. Dehydration and transparency: 95% ethanol for rapid dehydration for 3 seconds; anhydrous ethanol three times, 8 seconds each time: stand in xylene for 10-20 minutes.

[0115] 6. Sealing: Seal the slides with neutral gum and dry them in a fume hood.

[0116] 7. Take photos under an optical microscope and analyze the results.

[0117] CAR-T cell therapy for subcutaneous tumors

[0118] Severely immunodeficient B-NDG mice were used to establish the model. 5×105 mouse liver cancer cell line Hepa1-6 cells were subcutaneously inoculated. 5×106 FAP CAR-T cells were transfused 6 days later. The tumor size of the mice was measured every two days, and the survival of the mice was observed and the survival period of the mice was calculated.

[0119] result:

[0120] The structure of CAR and the construction of CAR-T cells:

[0121] The CAR used in the present invention is a second-generation CAR structure, and the specific molecular structure sequence of the CAR is: CD8SP-VL-(G4S)3-VH-CD8 Hinge-41BB-CD3ζ( Figure 3 A). The CAR was constructed into the lentiviral vector pRLenti-EF1-EGFP-EF1a-MCS-WPRE ( Figure 3 B), the vector is Amp-resistant and was packaged into lentivirus by Heyuan Biotechnology using the second-generation virus packaging system for use in the research of this invention.

[0122] Obtaining mouse T cells and constructing CAR-T cells: The T cells used in the present invention are from C57BL / 6J mice purchased from Vital River Company. The mice were killed by cervical dislocation and soaked in 75% alcohol for about 5 minutes. The spleen of the mice was removed in a biosafety cabinet and ground to form a single cell suspension. Mouse T cells with a purity greater than 99% were obtained after sorting using a kit (EasySep Mouse T Cell Isolation Kit, STEMCELL) Figure 3 C), add CD3 / CD28 activation magnetic beads (Miltenyi Biotec) for activation, using a medium containing 10% fetal bovine serum, 50 IU mL -1 IL-7 (Proteintech) and 100 IU mL -1 IL-15 (Proteintech) was cultured in RPMI1640 medium. After 48 hours, the cells were infected with lentivirus at an MOI of 100 for 6 hours, and the positive rate was greater than 50% ( Figure 3 D) of mouse FAP CAR-T cells. At the same time, T cells obtained by infecting the virus using a lentiviral empty vector package were used as a control and named T-Vec (T-Vector).

[0123] The results showed that the present invention successfully constructed a second-generation CAR targeting mouse FAP antigen ( Figure 3 A), by using lentivirus to encode highly purified mouse T cells ( Figure 3 C) were infected and CAR-T cells with a high positive rate were obtained ( Figure 3 D).

[0124] That is, through molecular experiments, mouse FAP CAR was successfully constructed and verified by sequencing, and mouse CAR-T cells were successfully constructed after obtaining mouse spleen-derived T cells.

[0125] Mouse FAP CAR-T cells can effectively kill FAP in vitro + cell:

[0126] FAP CAR-T cells were co-cultured with LX2 cells expressing mouse FAP and Hepa1-6 cells expressing FAP, respectively. LX2 cells expressing mouse FAP and Hepa1-6 cells expressing FAP were artificially constructed (4A, B). The effector-target ratio was set to 5:1 and 1:1, and the killing time was 12 h. The LDH kit (Promega) was used for detection.

[0127] The results showed that the target cell death rate in the Vec-T (Vector transduced T cell) cell group was very low, while the target cell death rate in the FAP CAR-T cell group was relatively high ( Figure 4 C, D).

[0128] Therefore, FAP CAR-T cells can effectively inhibit FAP in vitro + The cells have a strong killing ability, and as the effector-target ratio increases, the killing efficiency gradually increases.

[0129] FAP CAR-T cells can effectively inhibit tumor growth in vivo:

[0130] To explore the anti-tumor effect of FAP CAR-T cells in vivo, we created a severely immunodeficient mouse NSG model, injected mouse liver cancer cells subcutaneously, and then infused CAR-T cells ( Figure 5 A). The results showed that FAP CAR-T cells can effectively inhibit the growth of tumor cells ( Figure 5 B) and prolong the survival of mice ( Figure 5 C). Therefore, T cells expressing FAP CAR have strong anti-tumor function in vivo, indicating that the screened FAP scFv can effectively recognize and bind to FAP antigen after being expressed as protein in vivo.

[0131] FAP CAR-T cells can effectively alleviate CCL4-induced liver fibrosis:

[0132] Long-term use of CCL4 injection in the peritoneal cavity of C57 mice to establish a liver fibrosis model. After about 6 weeks, FAP CAR-T cells were reinfused into the tail vein. One week later, the livers of mice were taken for examination. It was found that compared with the control group (Sham mice) and the T-Vec group, the expression level of FAP molecules in the liver of the FAP CAR-T cell-infused mice was significantly lower than that of the T-Vec-infused group, and the expression levels of fibrosis-related molecules α-sma, collagen-1, and fibronectin decreased ( Figure 6 A). At the same time, HE staining and picrosirius red staining showed that the degree of liver fibrosis in mice in the FAP CAR-T cell infusion group was reduced ( Figure 6 B, C). Therefore, FAP CAR-T cells can alleviate liver fibrosis by clearing activated HSCs in the liver.

[0133] Amino acid sequence information used in the examples

[0134] The scFv involved in the present invention is a high-affinity single-chain antibody obtained by the team of the present invention through screening by immunizing mice, and the heavy chain and the light chain are connected by a G4S linker.

[0135] in:

[0136] The heavy chain sequence is:

[0137] QVQLQQSGPELVKPGASVQASISCRSSQVFSGSWMNWVKQRLQKPGQSP KLIYKVSNDGGINYNGKFKGKAGSGTDFVKISRVEAEDLGVYYCFGGSHVPY WGTKLEIK(SEQ ID NO.1)

[0138] The light chain sequence is:

[0139] DIMMTQTPLSLPVSLGDQALTCRSSTGAVTTSNYANWVQEKPDHLFTGLI GGTNNRAPGVPARFSVSLIGDKAALTITGAQTEDEAMYFCALWYSNHWVFG GGTKLTVL(SEQ ID NO.2)

[0140] The G4S linker sequence is:

[0141] GGGGSGGGGSGGGGS (SEQ ID NO. 3)

[0142] The CD8 SP sequence is:

[0143] MASPLTRFLSLNLLLLGESIILGSGEA(SEQ ID NO.4)

[0144] The sequence of CD8 Hinge region is:

[0145] TTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFACDIYIWAPLA GICVALLLSLIITLICYHRSRKWIRKKFPHIFKQPFKKTTGAAQEEDACSCRCPQ EEEGGGGGYEL(SEQ ID NO.5)

[0146] The sequence of the 4-1BB intracellular region is:

[0147] KWIRKKFPHIFKQPFKKTTGAAQEEDACSCRCPQEEEGGGGGYEL(SEQ ID NO.6)

[0148] The CD3ζ chain sequence is:

[0149] FSRSAETAANLQDPNQLYNELNLGRREEYDVLEKKRARDPEMGGKQQR RRNPQEGVYNALQKDKMAEAYSEIGTKGERRRGKGHDGLYQGLSTATKDTY DALHMQTLAPR(SEQ ID NO.7)

[0150] The FAP ECD sequence is:

[0151] (SEQ ID NO.8)

[0152] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A chimeric antigen receptor targeting FAP, characterized in that The chimeric antigen receptor comprises an antigen binding domain; the antigen binding domain is an anti-FAP antibody, and the anti-FAP antibody may be a single-chain antibody consisting of an antibody heavy chain and an antibody light chain; The antibody heavy chain (VH) has the amino acid sequence shown in SEQ ID NO.1; The antibody light chain (VL) has the amino acid sequence shown in SEQ ID NO.

2.

2. The chimeric antigen receptor targeting FAP according to claim 1, characterized in that The antibody heavy chain is directly connected to the antibody light chain or connected via a connecting fragment; the connecting fragment is a polypeptide chain composed of alanine and / or serine and / or glycine.

3. The chimeric antigen receptor targeting FAP according to claim 2, characterized in that The connecting fragment is (G4S)n, where n is 1-10.

4. The chimeric antigen receptor targeting FAP according to claim 3, characterized in that The n is 3.

5. The chimeric antigen receptor targeting FAP according to claim 1, characterized in that The chimeric antigen receptor targeting FAP comprises a signal peptide, an antigen binding domain, a chimeric receptor hinge region, a co-stimulatory signal transduction domain and a signal transduction domain connected in series.

6. The chimeric antigen receptor targeting FAP according to claim 5, characterized in that The chimeric antigen receptor targeting FAP comprises a CD8 SP signal peptide, the anti-FAP antibody, a CD8 Hinge, any one or more of a 4-1BB domain, a CD27 domain, a CD40 domain or an OX40 domain, and a CD3ζ signaling domain connected in series.

7. An isolated nucleic acid, characterized in that The nucleic acid encodes the chimeric antigen receptor according to any one of claims 1 to 6.

8. A CAR expression vector, characterized in that: The CAR expression vector comprises the nucleic acid of claim 7.

9. The CAR expression vector according to claim 8, characterized in that: The CAR expression vector is a viral vector, which includes a retroviral vector, an adenoviral vector or a plasmid.

10. The CAR expression vector according to claim 9, characterized in that: The CAR expression vector is a viral vector, which includes a lentiviral vector.

11. A CAR-T cell, characterized in that: The CAR-T cell is capable of expressing the chimeric antigen receptor targeting FAP according to any one of claims 1-6.

12. The CAR-T cell according to claim 11, wherein It is a T lymphocyte modified with the above-mentioned chimeric antigen receptor targeting FAP.

13. Use of the chimeric antigen receptor according to any one of claims 1 to 6, the nucleic acid according to claim 7, the CAR expression vector according to any one of claims 8 to 10, and the CAR-T cell according to any one of claims 11 to 12 in the preparation of drugs for preventing and treating diseases related to high expression of FAP; The diseases associated with high FAP expression are liver fibrosis and liver cancer.

14. A drug for preventing and treating diseases related to high expression of FAP, characterized in that: The active ingredient of the drug is the chimeric antigen receptor according to any one of claims 1 to 6 or the CAR-T cell according to any one of claims 11 to 12; The diseases associated with high FAP expression are liver fibrosis and liver cancer.

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