CAR-T (Chimeric Antigen Receptor-T) cell targeting fibroblast activating protein as well as preparation method and application of CAR-T cell

Abstract

The invention discloses a CAR-T (Chimeric Antigen Receptor-T) cell targeting fibroblast activating protein as well as a preparation method and application of the CAR-T cell, and belongs to the field of cell therapy. The CAR-T cell can be prepared by introducing the chimeric antigen receptor CAR of the targeted FAP into T lymphocytes, and the CAR in the CAR-T cell comprises a signal peptide, an antigen binding structural domain, a hinge region, a transmembrane structural domain, a costimulatory signal transduction region and a CD3 signal transduction structural domain. The CAR-T cell realizes specific recognition of FAP through a single-chain antibody fragment (scFv), so that an intracellular signal channel is activated, a cytokine IFN-gamma is released, a cytotoxic effect on FAP + cells is shown, specific removal of the FAP + cells at a focus part is realized, and the CAR-T cell has an anti-inflammatory effect on the FAP + cells at the focus part and has an anti-inflammatory effect on the FAP + cells at the focus part. The compound can be used for treating FAP up-regulated fibrosis (pulmonary fibrosis, hepatic fibrosis, cardiac fibrosis, renal fibrosis and the like), arthritis, autoimmune diseases (Crohn's disease, rheumatoid arthritis and the like), cardiovascular diseases and other diseases, and has huge application potential and commercial value.

Description

Technical Field

[0001] The present invention belongs to the field of cell therapy, and specifically relates to a CAR-T cell targeting fibroblast activation protein and a preparation method and application thereof. Background Art

[0002] Chimeric antigen receptor T cell (CAR-T) is a T cell modified by genetic engineering technology, which combines the antigen recognition domain with the cell activation domain, so that T cells can specifically recognize specific antigens and activate, thereby eliminating target cells. CAR-T cell therapy has achieved remarkable results in the clinical treatment of hematological tumors, and many relapsed and refractory patients have achieved long-term remission. At present, more than 10 CAR-T cell therapies have been approved by the US FDA and China CDE for the treatment of hematological tumors, changing the treatment pattern of hematological tumors. In recent years, CAR-T cell therapy has been used to treat a variety of diseases such as solid tumors and autoimmune diseases and has achieved encouraging results. Chimeric antigen receptor (CAR)-mediated cell killing is expected to expand to a wider range of treatment areas in the future, showing great application potential.

[0003] Fibroblast activation protein-α (FAP) is a 97-kDa type II transmembrane serine protease with dipeptidyl peptidase (DPP), endopeptidase and collagenase activities, and can degrade dipeptides, type I collagen, gelatin, etc. FAP is almost not expressed in normal tissues, but its expression level is very high in pathological diseases such as tumors and fibrosis, and it participates in matrix activation and tissue remodeling. FAP is selectively expressed in the capsule or cytoplasm of matrix fibroblasts in more than 90% of human epithelial tumors, including breast cancer, lung cancer, colorectal cancer and ovarian cancer, and is widely involved in tumor growth, invasion, metastasis, tumor cell extracellular matrix reconstruction, angiogenesis, immune escape and other processes, thereby promoting the development of tumors and attracting widespread attention from researchers.

[0004] FAP also plays an important role in non-neoplastic diseases. FAP is associated with the progression and severity of diseases such as fibrosis, arthritis, autoimmune diseases (Crohn's disease, rheumatoid arthritis, etc.), and cardiovascular diseases. FAP expression is associated with uncontrolled scarring diseases (fibrosis), and FAP levels are elevated in pathological conditions such as pulmonary fibrosis, liver fibrosis, kidney fibrosis, colon fibrosis, and granulation tissue. FAP is specifically upregulated in fibroblasts and fibroblast stroma in patients with pulmonary fibrosis, but not in adjacent normal tissue, lung tissue of healthy individuals, or lung tissue of patients with central acinar emphysema. Liver fibrosis is another uncontrolled fibrotic disease that is often triggered by chronic damage such as viral hepatitis, non-alcoholic fatty liver disease, or alcohol abuse. Chronic liver damage activates hepatic stellate cells to produce the extracellular matrix responsible for liver scarring, presenting a myofibroblast-like phenotype and expressing FAP. In patients with viral hepatitis C, FAP expression is associated with the degree of liver fibrosis. Liver fibrosis can progress to cirrhosis and may eventually lead to liver failure. FAP activity is 14-18 times higher in cirrhotic livers compared to normal livers. Other widespread fibrosis associated with upregulated FAP expression include keloid scars and Crohn's disease. A study found FAP expression in synovial samples from patients with rheumatoid arthritis and osteoarthritis, and FAP expression was higher in samples from patients with refractory rheumatoid arthritis compared to patients with end-stage osteoarthritis. FAP overexpression has also been found in thin fibrous cap atherosclerotic plaques (TCFA) in human aortic smooth muscle cells. In summary, FAP is highly expressed in a variety of diseases, participates in tissue remodeling and pathological processes, and is a potential therapeutic target.

[0005] In summary, CAR can specifically bind to antigen targets through the antigen recognition domain and activate intracellular signal transduction pathways under antigen stimulation, providing a new strategy for the specific elimination of abnormal cells in the body. FAP is a target that is widely expressed in many diseases and is related to the occurrence and development of the disease. Therefore, targeted elimination of FAP+ cells is expected to achieve the purpose of treating the disease. Summary of the invention

[0006] In view of the shortcomings of the prior art, the purpose of the present invention is to propose a CAR-T cell targeting fibroblast activation protein (FAP), which can achieve specific recognition of FAP through a single-chain antibody fragment (scFv), thereby activating the intracellular signaling pathway, releasing the cytokine IFN-γ and showing cytotoxic effects on FAP+ cells, thereby achieving specific elimination of FAP+ cells in the lesion site.

[0007] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] The first aspect of the present invention is to provide a method for preparing CAR-T cells targeting fibroblast activation protein;

[0009] The second aspect is to provide a CAR-T cell targeting fibroblast activation protein;

[0010] The third aspect is to provide an application of CAR-T cells targeting fibroblast activation protein.

[0011] A method for preparing CAR-T cells targeting fibroblast activation protein comprises the following steps: introducing a chimeric antigen receptor CAR targeting fibroblast activation protein (FAP) into T lymphocytes to prepare CAR-T cells.

[0012] The introduction methods include, but are not limited to, viral vector infection, electroporation transfection, transposon, gene knockin, extracellular vesicles, nucleic acid lipid nanoparticles LNP, nanogold particles, and chemical reagent transfection; viral vectors include lentiviral vectors, retroviral vectors, adeno-associated viruses, and adenoviruses;

[0013] Preferably, the introduction method is viral vector infection, electroporation transfection, transposon, gene knock-in or extracellular vesicle; and the viral vector is a lentiviral vector.

[0014] In the present invention, the lentiviral vector is specifically a four-plasmid lentiviral packaging system, including a lentiviral expression plasmid pRRLSIN-EF1α-FAPBBZ carrying a target gene, packaging plasmids pMDLg / pRRE (Kan+) and pRSV-REV (Kan+), and a lentiviral envelope plasmid pMD2.G (Kan+).

[0015] Among them, T lymphocytes include but are not limited to CD3+T lymphocytes, CD4+T cells, CD8+T cells, initial T cells (TN), stem cell memory T cells (TSCM), effector T cells (TEFF), memory T cells (TM), central memory T cells (TCM), effector memory T cells (TEM), terminally differentiated effector memory T cells (TEMRA), tumor infiltrating lymphocytes (TIL), iPSC-induced differentiated T cells, immature T cells, mature T cells, helper T cells, cytotoxic T cells, regulatory T cells (Treg), helper T cells, TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, mucosa-associated invariant T cells (MAIT), follicular helper T cells, αβT cells, and γδT cells.

[0016] It should be noted that the sources of T lymphocytes include but are not limited to peripheral blood, whole blood, single blood collection, umbilical cord blood, tumor tissue, spleen, bone marrow, monocytes, T lymphocytes, induced pluripotent stem cells (iPSC), hematopoietic stem cells (HPSC), embryonic stem cells (ESC), tumor infiltrating lymphocytes (TIL), lymphokine-activated killer cells (LAK), cytokine-induced killer cells (CIK); preferably, the source of T lymphocytes is peripheral blood, single blood collection, monocytes, T lymphocytes, induced pluripotent stem cells (iPSC), and hematopoietic stem cells (HPSC).

[0017] Preferably, the source of T lymphocytes is peripheral blood, single blood collection, monocytes, T lymphocytes, induced pluripotent stem cells (iPSC), hematopoietic stem cells (HPSC).

[0018] T lymphocytes are obtained by activating and amplifying peripheral blood mononuclear cells (PBMC) separated by a blood cell separator or Ficoll. The separation and culture of T lymphocytes includes the following steps: collecting peripheral blood from healthy volunteers, centrifuging to obtain autologous plasma and cell precipitates; the precipitated cells are diluted with physiological saline and added to a centrifuge tube containing Ficoll solution, and PBMCs are separated by density gradient centrifugation, and the cells are washed twice with physiological saline, and serum-free cell culture medium containing activators and recombinant cytokines (IL-2 and / or IL15) is added for culture to activate PBMCs. Among them, the activator includes but is not limited to anti-human CD3 / CD28 magnetic beads, anti-human CD3 magnetic beads, anti-human CD3 monoclonal antibody (OKT-3), anti-human CD3 monoclonal antibody and anti-human CD28 monoclonal antibody; preferably, the activator is anti-human CD3 / CD28 magnetic beads.

[0019] It should be noted that CAR-T cells include the following preparation methods:

[0020] 1. Take the separated PBMC cells, add the purified lentivirus solution (MOI is 1-200) and polybrene (final concentration 1-20μg / mL), centrifuge for infection, and place in a 37°C, 5% CO2 incubator for culture. After lentivirus infection, centrifuge and change the medium, resuspend in serum-free cell culture medium, add 1-10% plasma and cytokines (IL-2 and / or IL15) to continue culture and amplification.

[0021] 2. Take T cells activated for 24-120 hours, add purified lentivirus solution (MOI is 1-200) and polybrene (final concentration 1-20μg / mL), centrifuge for infection, and place in a 37°C, 5% CO2 incubator for culture. After lentivirus infection, centrifuge and change the medium, resuspend in serum-free cell culture medium, add 1-10% plasma and cytokines (IL-2 and / or IL15) to continue culture and expansion.

[0022] 3. Before infection, use RetroNectin to coat the cell culture plate. Wash the culture plate 2-24 hours after coating. Add purified lentivirus solution (MOI is 1-200), centrifuge at 1000-2000×g, 1-2 hours, and remove or not remove the virus supernatant. Take T cells activated for 48-96 hours, add them to the culture plate, and add polybrene (final concentration 0-20μg / mL). Culture in a 37°C, 5% CO2 incubator. Centrifuge and change the medium 24 hours after lentivirus infection, resuspend in serum-free cell culture medium, add 1-10% plasma and cytokines (IL-2 and / or IL15) to continue culture and amplification.

[0023] A CAR-T cell targeting fibroblast activation protein, wherein the chimeric antigen receptor CAR comprises a signal peptide, an antigen binding domain, a hinge region, a transmembrane domain, a co-stimulatory signal transduction region and a CD3 signal transduction domain;

[0024] The nucleotide sequence of the chimeric antigen receptor CAR is shown in SEQ ID NO.1 or SEQ ID NO.2, and the amino acid sequence of the chimeric antigen receptor CAR is shown in SEQ ID NO.3 or SEQ ID NO.4;

[0025] The nucleotide sequence of the chimeric antigen receptor CAR is a nucleotide sequence having at least 75% homology to the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.2, and the amino acid sequence of the chimeric antigen receptor CAR is an amino acid sequence having at least 75% homology to the amino acid sequence shown in SEQ ID NO.3 or SEQ ID NO.4.

[0026] In some embodiments, the signal peptide is selected from one of CD8A, CD4, CD3, CD5, CD19, CD20, CD22, CD28, CD33, CD45, CD80, CD86, GM-CSFR, and PD-L1; wherein the nucleotide sequence of the signal peptide of CD8A is shown in SEQ ID NO.5, and the amino acid sequence is shown in SEQ ID NO.6; the nucleotide sequence of GM-CSFR is shown in SEQ ID NO.7, and the amino acid sequence is shown in SEQ ID NO.8.

[0027] In some embodiments, the antigen binding domain is an antigen binding fragment Fab or scFv targeting fibroblast activation protein; wherein the antigen binding fragment Fab or scFv is an anti-human targeted fibroblast activation protein monoclonal antibody or an anti-mouse targeted fibroblast activation protein monoclonal antibody.

[0028] It should be noted that anti-human targeted fibroblast activation protein monoclonal antibodies include but are not limited to anti-human FAP monoclonal antibody Sibrotuzumab, anti-human FAP monoclonal antibody 7NP2, anti-human FAP monoclonal antibody OMTX005, anti-human FAP monoclonal antibody RG-7461, and anti-human FAP monoclonal antibody MP0310; anti-mouse targeted fibroblast activation protein monoclonal antibodies include but are not limited to anti-mouse FAP monoclonal antibody FAP5 and anti-mouse FAP monoclonal antibody FAP73.3.

[0029] Preferably, the antigen-binding fragment Fab or scFv is the anti-human FAP monoclonal antibody Sibrotuzumab or the anti-human FAP monoclonal antibody 7NP2.

[0030] In some embodiments, the structure of the antigen-binding fragment scFv is VH-Linker-VL or VL-Linker-VH; the nucleotide sequence of the antigen-binding fragment scFv is as shown in SEQ ID NO.9 or a nucleotide sequence having at least 75% homology to the nucleotide sequence shown in SEQ ID NO.9; the amino acid sequence of the antigen-binding fragment scFv is as shown in SEQ ID NO.10 or an amino acid sequence having at least 75% homology to the amino acid sequence shown in SEQ ID NO.10.

[0031] In some embodiments, the hinge region is selected from one of CD8A, CD28, IgG1, IgG2, and IgG4; wherein the nucleotide sequence of CD8A is shown in SEQ ID NO.11, and the amino acid sequence is shown in SEQ ID NO.12; the nucleotide sequence of CD28 is shown in SEQ ID NO.13, and the amino acid sequence is shown in SEQ ID NO.14.

[0032] In some embodiments, the transmembrane region is selected from one of CD8A, CD28, CD4, CD3, and ICOS; wherein the nucleotide sequence of CD8A is shown in SEQ ID NO.15, and the amino acid sequence is shown in SEQ ID NO.16; the nucleotide sequence of CD28 is shown in SEQ ID NO.17, and the amino acid sequence is shown in SEQ ID NO.18.

[0033] In some embodiments, the co-stimulatory signaling region includes an intracellular domain from a co-stimulatory molecule, and the co-stimulatory molecule is selected from at least one of CD27, CD28, 4-1BB, OX40, ICOS, CD40, and lymphocyte functional antigen-1; wherein the nucleotide sequence of 4-1BB is as shown in SEQ ID NO.19; its amino acid is as shown in SEQ ID NO.20; the nucleotide sequence of CD28 is as shown in SEQ ID NO.21, and the amino acid sequence is as shown in SEQ ID NO.22.

[0034] In some embodiments, the CD3 signaling domain is CD3ζ or CD3ε; wherein the nucleotide sequence of CD3ζ is shown in SEQ ID NO.23, and the amino acid sequence is shown in SEQ ID NO.24.

[0035] A use of CAR-T cells targeting fibroblast activation protein in the preparation of products for treating diseases expressing fibroblast activation protein.

[0036] Among them, fibroblast activation protein expression diseases include but are not limited to fibrosis, arthritis, autoimmune diseases (Crohn's disease, rheumatoid arthritis, etc.), cardiovascular diseases, etc.; preferably, the disease is pulmonary fibrosis, liver fibrosis, cirrhosis, cardiac fibrosis or renal fibrosis.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] 1. The present invention provides a CAR-T cell targeting FAP, which can release the cytokine IFN-γ and show cytotoxic effect on FAP+ cells, thereby achieving specific clearance of FAP+ cells in the lesion site, providing a new strategy and new idea for the treatment of FAP-expressing diseases, and has huge application potential and commercial value.

[0039] 2. The FAP target selected by the present invention is highly expressed in a variety of diseases, such as fibrosis, arthritis, autoimmune diseases (Crohn's disease, rheumatoid arthritis, etc.), cardiovascular diseases, etc. The present invention provides a method for preparing CAR-T cells targeting FAP, which achieves specific recognition of FAP through a specific single-chain antibody fragment (scFv), thereby clearing cells expressing FAP. FAP antigen-specific stimulation can enhance the expression of IFN-γ cytokines in T cells and promote the clearance of abnormal cells by the immune system. It has the advantages of targeted clearance and a wide range of indications. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is the plasmid map of the recombinant lentiviral expression vector pRRLSIN-EF1α-FAPBBZ.

[0041] Figure 2 Flow cytometry was used to detect the CAR positivity rate of CAR-T cells.

[0042] Figure 3 Flow cytometry was used to detect the expression of FAP on the cell surface.

[0043] Figure 4 The LDH method was used to detect the in vitro killing effect of CAR-T cells on HFL1.

[0044] Figure 5 ELISA was used to detect the level of IFN-γ released by co-incubated effector and target cells. DETAILED DESCRIPTION

[0045] The present invention will be further described in detail below in conjunction with the accompanying drawings. The embodiments described with reference to the accompanying drawings are exemplary and intended to explain the present invention, but should not be construed as limiting the present invention.

[0046] Example 1 Preparation of CAR-loaded lentivirus and CAR-T cells

[0047] 1. Construction of lentiviral expression vector pRRLSIN-EF1α-FAPBBZ

[0048] The chimeric antigen receptor CAR structure is composed of a signal peptide, an antigen binding domain, a hinge region, a transmembrane domain, a co-stimulatory signal transduction region and a CD3 signal transduction domain in series. As the most preferred embodiment, its nucleotide sequence is shown in SEQ ID NO.1, which is artificially synthesized by General Bio (Anhui) Co., Ltd. and cloned into the BamHI and MluI restriction sites of the lentiviral vector pRRLSIN-EF1α-Myc-Palivizumab-BB (reference Chinese patent: CN202111617729.X). The constructed recombinant lentiviral expression vector is named pRRLSIN-EF1α-FAPBBZ, and the plasmid map is shown in Figure 1 As shown. Endo-free Plasmid Maxi Kit (Omega) was used to extract plasmid. The concentration and purity of the extracted plasmid were detected by UV spectrophotometer, and then stored in a -20°C refrigerator for subsequent lentiviral packaging.

[0049] 2. Lentivirus packaging and purification

[0050] (1) HEK293T cells were revived from cryopreservation and subcultured with DMEM complete medium (DMEM medium + 10% FBS). HEK293T cells were cultured at 3×10 6 / mL density was inoculated into 10 layers of cell factories, and DMEM complete medium was added to a volume of 1L. After overnight culture, the cells could reach 80-90% confluence for plasmid transfection.

[0051] (2) Prepare a 225 mL centrifuge bottle, add the lentiviral expression vector pRRLSIN-EF1α-FAPBBZ, lentiviral packaging plasmids pMDLg / pRRE (Kan+) and pRSV-REV (Kan+), and lentiviral envelope plasmid pMD2.G (Kan+), add serum-free DMEM to 60 mL, mix well, and let stand for 5 min to form liquid A. Prepare another 225 mL centrifuge bottle, add 4.2 mL of 1 mg / mL PEI (polysciences), add serum-free DMEM to 60 mL, mix well, and let stand for 5 min to form liquid B. Then add liquid B to liquid A, mix well, and let stand for 20 min to form a DNA-PEI complex.

[0052] (3) Add the DNA-PEI complex to 1 L of DMEM culture medium containing 5% FBS, mix thoroughly, and replace the culture medium in the 10-layer cell factory.

[0053] (4) 48 h after transfection, collect about 1 L of culture supernatant and store it in a refrigerator at 2-8°C.

[0054] (5) The collected culture supernatant was filtered through a capsule filter (Sartorius) to remove cells and cell debris, and the clarified lentiviral supernatant was filtered through a Spectrum tangential flow filtration system ( KR2I) was concentrated to 100-200 mL, filtered through a 0.45 μm filter membrane, and then purified by chromatography.

[0055] (6) The purified lentivirus was sterilized and filtered through a 0.22 μm filter (Sartorius), packaged, and stored in a -80°C refrigerator. The titer of the purified lentivirus was 1.62×10 9 TU / mL. The obtained lentivirus was recorded as pRRLSIN-EF1α-FAPBBZ lentivirus.

[0056] 3. Preparation of CAR-T cells

[0057] (1) Take 20 mL of peripheral blood from healthy volunteers, anticoagulate with heparin, and centrifuge to obtain serum, which is inactivated at 56°C for later use. Dilute the precipitated cells with saline and add them to a centrifuge tube containing Ficoll solution. Separate PBMCs by density gradient centrifugation, wash twice with saline, and count for later use.

[0058] (2) Resuspend PBMCs in lymphocyte culture medium KBM 581 Serum-free Cell Medium (Corning) and adjust the cell density to 1-2×10 6 / mL, inoculated into T75 cell culture flasks, and added anti-human CD3 / CD28 magnetic beads (GibcoDynabeads TM Human T-Expander CD3 / CD28, purchased from Guangzhou Bailu Biotechnology Co., Ltd.) was used to activate PBMCs, and 500 IU / mL of recombinant human interleukin 2 (rhIL-2) and 5-10% plasma were added and cultured in a 37°C, 5% CO2 incubator.

[0059] (3) After 3 days of stimulation and culture, PBMCs were counted, centrifuged, and resuspended in lymphocyte culture medium (KBM 581 Serum-free Cell Medium (recombinant human interleukin 2 (rhIL-2)) to make the T lymphocyte density reach 2-5×10 6 / mL, T lymphocytes were equally divided into 6-well plates, and purified lentivirus solution (MOI=20) and polybrene (final concentration 6 μg / mL) were added.

[0060] At the same time, a group without lentivirus was set up as a T cell control group. Centrifuge at 700g for 1.5h and culture in a 37°C, 5% CO2 incubator. Centrifuge and change the medium 24h after lentivirus infection, resuspend in KBM581 medium, add 5-10% plasma and 500IU / mL rhIL-2, and continue to expand and culture in a 37°C, 5% CO2 incubator.

[0061] The CAR positive rate of CAR-T cells was detected by flow cytometry 72 hours after lentiviral infection. Figure 2 As shown, the positive rate of CAR-T cells targeting FAP reached 46.5%.

[0062] Experimental Example 1 Function of CAR-T cells targeting FAP

[0063] 1. Screening of cells with high expression of fibroblast activation protein (FAP)

[0064] HFL1 (human embryonic lung fibroblasts, purchased from the cell bank of the Chinese Academy of Sciences) and WI-38 (human embryonic lung cells, purchased from the cell bank of the Chinese Academy of Sciences) were revived and placed in a 37°C, 5% CO2 incubator. Cells in good growth condition were selected and the expression of FAP was detected by flow cytometry. The results are as follows: Figure 3 As shown, the positive rates of FAP expression in HFL1 and WI-38 cells were 95.7% and 42.6%, respectively. HFL1 was used as a FAP high-expressing cell for subsequent studies.

[0065] 2. CAR-T cells targeting FAP can specifically eliminate FAP+ cells

[0066] HFL1 cells (target cells), T cells (effector cells) prepared in Example 1 and CAR-T cell (effector cell) single cell suspensions were collected respectively, and the cells were washed 3 times by centrifugation (300g, 5min) with RPMI 1640 culture medium, and the effector cells and target cells were resuspended with culture medium and gently blown to mix, and counted respectively. The number of target cells was 3000 / 50μL / well, the number of effector cells was 12000 / 50μL / well, the effector ratio was 4:1, and the total volume was 100μL / well. At the same time, the effector cell spontaneous LDH release well, the target cell spontaneous LDH release well, the target cell maximum LDH release well, the volume correction control well and the culture medium background control well were set. All groups were repeated 3 times, and each repeat had 3 duplicate wells. After the culture plate was centrifuged at 300g for 4min, it was placed in a 37°C, 5% CO2 incubator and incubated for 7h. Add 10 μL Lysis Solution (10×) to the target cell maximum LDH release well and volume correction control well, and incubate for 45 min. Centrifuge the culture plate at 300 g for 4 min. Pipette 50 μL of supernatant from each well into a new ELISA plate, and add 50 μL of Reagent, incubate at room temperature in the dark for 30 minutes. Add 50 μL Stop Solution to each well; pop large bubbles with a syringe needle, and measure absorbance at 490 nm or 492 nm within 1 hour after adding Stop Solution.

[0067] The killing efficiency was calculated according to the following formula: the exact value of spontaneous LDH release of effector cells, spontaneous LDH release of target cells and the experimental group should be their readings minus the background reading of the culture medium; maximum release of target cells = target cell maximum LDH release well - volume-corrected control well; specific killing efficiency (%) = (experimental group - spontaneous release of effector cells - spontaneous release of target cells) / (maximum release of target cells - spontaneous release of target cells).

[0068] The results are as follows Figure 4 As shown in the figure, compared with the T cell group, the killing effect of the CAR-T cell group on HFL1 cells was significantly enhanced (P=0.0001), indicating that CAR-T cells targeting FAP can significantly eliminate FAP+ cells.

[0069] 3. CAR-T cells targeting FAP can increase IFN-γ release

[0070] Single cell suspensions of HFL1 cells (target cells), T cells (effector cells) prepared in Example 1, and CAR-T cells (effector cells) were collected respectively, washed three times by centrifugation with RPMI 1640 culture medium (300 g, 5 min), and the effector cells and target cells were resuspended with culture medium and gently pipetted to mix, and then counted respectively. The number of target cells was 2×10 4 cells / well, the number of effector cells is 8×10 4 The cells were cultured in a 37°C, 5% CO2 incubator, with a target ratio of 4:1 and a total volume of 500 μL / well. After 16-18 hours, the cell culture supernatant was collected and the IFN-γ release level was detected by ELISA.

[0071] The results are as follows Figure 5 As shown in the figure, after co-incubation with HFL1 cells, the concentrations of IFN-γ in the T cell group and CAR-T cells were (351.49±44.82) pg / mL and (2135.97±192.34) pg / mL, respectively. The release level of IFN-γ in the CAR-T cell group was significantly higher than that in the T cell group (P<0.0001).

[0072] The above results suggest that under the stimulation of the cell antigen FAP, CAR-T cells targeting FAP can be specifically activated, release the cytokine IFN-γ and show cytotoxicity, and eliminate FAP+ cells. Therefore, CAR-T targeting FAP can be used to treat FAP-upregulated fibrosis (pulmonary fibrosis, liver fibrosis, cardiac fibrosis, renal fibrosis, etc.), arthritis, autoimmune diseases (Crohn's disease, rheumatoid arthritis, etc.), cardiovascular diseases and other diseases, showing great application potential and commercial value.

[0073] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements can be made without departing from the creative concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for preparing CAR-T cells targeting fibroblast activation protein, characterized in that: The method comprises the following steps: introducing a chimeric antigen receptor CAR targeting fibroblast activation protein into T lymphocytes to prepare CAR-T cells.

2. A CAR-T cell targeting fibroblast activation protein prepared by the preparation method of claim 1, characterized in that: The chimeric antigen receptor CAR includes a signal peptide, an antigen binding domain, a hinge region, a transmembrane domain, a co-stimulatory signal transduction region and a CD3 signal transduction domain; The nucleotide sequence of the chimeric antigen receptor CAR is shown in SEQ ID NO.1 or SEQ ID NO.2, and the amino acid sequence of the chimeric antigen receptor CAR is shown in SEQ ID NO.3 or SEQ ID NO.4; The nucleotide sequence of the chimeric antigen receptor CAR is a nucleotide sequence having at least 75% homology to the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.2, and the amino acid sequence of the chimeric antigen receptor CAR is an amino acid sequence having at least 75% homology to the amino acid sequence shown in SEQ ID NO.3 or SEQ ID NO.

4.

3. The CAR-T cell according to claim 2, characterized in that The signal peptide is selected from one of CD8A, CD4, CD3, CD5, CD19, CD20, CD22, CD28, CD33, CD45, CD80, CD86, GM-CSFR, and PD-L1; wherein the nucleotide sequence of the signal peptide of CD8A is shown in SEQ ID NO.5, and the amino acid sequence is shown in SEQ ID NO.6; the nucleotide sequence of GM-CSFR is shown in SEQ ID NO.7, and the amino acid sequence is shown in SEQ ID NO.

8.

4. The CAR-T cell according to claim 2, characterized in that The antigen binding domain is an antigen binding fragment Fab or scFv targeting fibroblast activation protein; wherein the antigen binding fragment Fab or scFv is an anti-human targeting fibroblast activation protein monoclonal antibody or an anti-mouse targeting fibroblast activation protein monoclonal antibody.

5. The CAR-T cell according to claim 4, characterized in that The structure of the antigen-binding fragment scFv is VH-Linker-VL or VL-Linker-VH; the nucleotide sequence of the antigen-binding fragment scFv is as shown in SEQ ID NO.9 or a nucleotide sequence having at least 75% homology to the nucleotide sequence shown in SEQ ID NO.9; the amino acid sequence of the antigen-binding fragment scFv is as shown in SEQ ID NO.10 or an amino acid sequence having at least 75% homology to the amino acid sequence shown in SEQ ID NO.

10.

6. The CAR-T cell according to claim 2, characterized in that The hinge region is selected from one of CD8A, CD28, IgG1, IgG2, and IgG4; wherein the nucleotide sequence of CD8A is shown in SEQ ID NO.11, and the amino acid sequence is shown in SEQ ID NO.12; the nucleotide sequence of CD28 is shown in SEQ ID NO.13, and the amino acid sequence is shown in SEQ ID NO.

14.

7. The CAR-T cell according to claim 2, characterized in that The transmembrane region is selected from one of CD8A, CD28, CD4, CD3, and ICOS; wherein the nucleotide sequence of CD8A is shown in SEQ ID NO.15, and the amino acid sequence is shown in SEQ ID NO.16; the nucleotide sequence of CD28 is shown in SEQ ID NO.17, and the amino acid sequence is shown in SEQ ID NO.

18.

8. The CAR-T cell according to claim 2, characterized in that The co-stimulatory signal transduction region includes an intracellular domain from a co-stimulatory molecule, and the co-stimulatory molecule is selected from at least one of CD27, CD28, 4-1BB, OX40, ICOS, CD40, and lymphocyte functional antigen-1; wherein the nucleotide sequence of 4-1BB is as shown in SEQ ID NO.19; its amino acid is as shown in SEQ ID NO.20; the nucleotide sequence of CD28 is as shown in SEQ ID NO.21, and the amino acid sequence is as shown in SEQ ID NO.

22.

9. The CAR-T cell according to claim 2, characterized in that The CD3 signaling domain is CD3ζ or CD3ε; wherein the nucleotide sequence of CD3ζ is shown in SEQ ID NO.23, and the amino acid sequence is shown in SEQ ID NO.

24.

10. Use of the CAR-T cell according to any one of claims 2 to 9 in the preparation of a product for treating a fibroblast activation protein-expressing disease.

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