CAR-MSC (chimeric antigen receptor-mesenchymal stem cell) targeting fibroblast activating protein as well as preparation method and application of CAR-MSC

Abstract

The invention discloses a CAR-MSC (chimeric antigen receptor-mesenchymal stem cell) targeting fibroblast activating protein as well as a preparation method and application of the CAR-MSC, and belongs to the field of cell therapy. A chimeric antigen receptor (CAR) targeting a fibroblast activation protein (FAP) is introduced into a mesenchymal stem cell (MSC) to prepare a CAR-MSC cell, and the CAR comprises a signal peptide, an antigen binding domain, a hinge region, a transmembrane domain, a costimulatory signal transduction region and a CD3 signal transduction structure. The CAR-MSC cell not only can realize specific recognition of FAP through a specific single-chain antibody fragment (scFv) and enhance directional migration of MSC to antigen expression tissues, but also can activate an intracellular signal channel through specific stimulation of an FAP antigen and enhance the immunosuppression capability and the treatment effect of the CAR-MSC, can be used for treatment of FAP expression diseases, and has broad application prospects. And the problems of poor targeting and insufficient immunosuppression capability in clinical application of the MSC are solved.

Description

Technical Field

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

[0002] Mesenchymal stem cells (MSC), also known as multipotent mesenchymal cells or multipotent mesenchymal stem cells, have the ability of self-renewal and multidirectional differentiation. Since Friedenstein discovered MSC from bone marrow in the 1970s, MSCs have been isolated from various tissues, including umbilical cord, placenta, amnion, fat, etc. MSCs are widely present in various tissues throughout the body, can be cultured and expanded in vitro, and can differentiate into adipocytes, osteoblasts, chondrocytes, muscle cells, nerve cells, etc. under specific conditions. They can interact with immune cells through cell-cell contact and secretion of soluble molecules to reduce excessive inflammatory responses. The multifunctional characteristics of mesenchymal stem cells (multidirectional differentiation ability, immunomodulatory activity, paracrine) make them have great potential in disease treatment.

[0003] Mesenchymal stem cells, as a promising cell therapy, have been widely explored and applied in many fields. Mesenchymal stem cell-based therapies have been used to treat various diseases, including but not limited to osteoarthritis (OA), premature ovarian failure (POI), graft-versus-host disease (GVHD), pulmonary fibrosis, etc. Globally, 10 mesenchymal stem cell products have been approved for marketing, mainly used to regulate immunity and repair damage, and treat diseases such as graft-versus-host disease (GVHD), Crohn's disease, myocardial infarction, and osteoarthritis. Although MSC has become one of the most studied cell therapy technologies in the world and has shown good safety in clinical studies of various diseases, the clinical treatment effect has not been satisfactory, which is mainly attributed to the lack of sufficient target tissue targeting and immunosuppressive ability.

[0004] 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.

[0005] 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 disease. Given FAP's role in tissue remodeling and its expression on activated fibroblasts, it is not surprising that 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 alveolar 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, which produce the extracellular matrix responsible for liver scarring, present a myofibroblast-like phenotype, and express 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 extensive 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. FAP is highly expressed in a variety of diseases, involved in tissue remodeling and pathological processes, and is a potential therapeutic target. Summary of the invention

[0006] In view of the deficiencies of the prior art, the purpose of the present invention is to propose a CAR-MSC cell targeting fibroblast activation protein and its preparation method and application. The CAR-MSC cell can not only realize the specific recognition of FAP through a specific single-chain antibody fragment (scFv), enhance the directional migration of MSC to antigen-expressing tissues, but also activate the intracellular signaling pathway through FAP antigen-specific stimulation, enhance the immunosuppressive ability and therapeutic effect of CAR-MSC, and can be used for the treatment of FAP-expressed fibrosis, arthritis, autoimmune diseases (Crohn's disease, rheumatoid arthritis, etc.), cardiovascular diseases and other diseases, with the advantages of strong targeting, high activity, and multiple indications.

[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 CAR-MSC cell targeting fibroblast activation protein, wherein the 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;

[0009] The nucleotide sequence of CAR is shown in SEQ ID NO.1 or SEQ ID NO.2, and the amino acid sequence of CAR is shown in SEQ ID NO.3 or SEQ ID NO.4;

[0010] The nucleotide sequence of 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 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.

[0011] In some embodiments, the signal peptide includes CD8A, CD4, CD3, CD5, CD19, CD20, CD22, CD28, CD33, CD45, CD80, CD86, GM-CSFR, and PD-L1; wherein the nucleotide sequence of CD8A is shown as SEQ ID NO.5, and the amino acid sequence is shown as SEQ ID NO.6; the nucleotide sequence of GM-CSFR is shown as SEQ ID NO.7, and the amino acid sequence is shown as SEQ ID NO.8.

[0012] 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 (FAP) monoclonal antibody or an anti-mouse targeted fibroblast activation protein (FAP) monoclonal antibody.

[0013] Chimeric antigen receptor (CAR) can specifically bind to antigen targets through single-chain variable fragments (scFv) and activate intracellular signal transduction pathways under antigen stimulation.

[0014] Anti-human FAP 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 FAP monoclonal antibodies include but are not limited to anti-mouse FAP monoclonal antibody FAP5 and anti-mouse FAP monoclonal antibody FAP73.3.

[0015] Preferably, the antigen-binding fragment Fab or scFv targeting FAP is the anti-human FAP monoclonal antibody Sibrotuzumab or the anti-human FAP monoclonal antibody 7NP2.

[0016] 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.

[0017] Preferably, the structure of the antigen-binding fragment scFv is VH-Linker-VL.

[0018] 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.

[0019] Preferably, the hinge region is CD8A or CD28.

[0020] In some embodiments, the transmembrane domain 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.

[0021] Preferably, the transmembrane domain is CD8A or CD28.

[0022] 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 (LFA-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.

[0023] 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.

[0024] The second aspect of the present invention is to provide a method for preparing CAR-MSC cells targeting fibroblast activation protein, wherein a chimeric antigen receptor (CAR) targeting fibroblast activation protein is introduced into mesenchymal stem cells (MSC) to prepare CAR-MSC cells;

[0025] Among them, the introduction methods include 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.

[0026] Preferably, the method of introducing CAR into MSC is viral vector infection, electroporation transfection, transposon, gene knockin or extracellular vesicles.

[0027] More preferably, the method of introducing CAR into MSC is viral vector infection, and the viral vector is a lentiviral vector or a retroviral vector.

[0028] The lentiviral vector is specifically a four-plasmid lentiviral packaging system, including the lentiviral expression plasmid pRRLSIN-EF1α-FAPBBZ carrying the target gene, the packaging plasmids pMDLg / pRRE (Kan+) and pRSV-REV (Kan+), and the lentiviral envelope plasmid pMD2.G (Kan+).

[0029] It should be noted that the preparation of CAR-MSC cells includes the following steps: subculture the MSC cells in the resuscitation cell bank, inoculate the MSC cells in good growth state into the culture flask, add pRRLSIN-EF1α-FAPBBZ lentivirus and polybrene to infect the cells after overnight culture, change the medium after 24 hours of lentivirus infection, add serum-free medium, and continue to expand the culture. Among them, the MOI of lentivirus infection is 0-200, and the concentration of polybrene is 0-20μg / mL; further, the MOI of lentivirus infection is 1-10, and the concentration of polybrene is 1-10μg / mL; further, the concentration of polybrene is 6μg / mL.

[0030] Mesenchymal stem cells can be umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, dental pulp mesenchymal stem cells, placental mesenchymal stem cells, amniotic membrane mesenchymal stem cells, amniotic fluid mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, uterine blood mesenchymal stem cells, iPSC-induced differentiated mesenchymal stem cells, etc.

[0031] Preferably, the mesenchymal stem cells are derived from umbilical cord mesenchymal stem cells, adipose-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, placental mesenchymal stem cells, or iPSC-induced differentiated mesenchymal stem cells.

[0032] The third aspect of the present invention is to provide an application of CAR-MSC cells targeting fibroblast activation protein in the preparation of products for treating fibroblast activation protein (FAP) expressing diseases, characterized in that fibroblast activation protein expressing diseases include fibrosis, arthritis, autoimmune diseases, and cardiovascular diseases.

[0033] Diseases include pulmonary fibrosis, liver fibrosis, cirrhosis, cardiac fibrosis, renal fibrosis, Crohn's disease, rheumatoid arthritis, etc.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] (1) The present invention provides a FAP-targeting CAR-MSC cell that can continuously express CAR, has high cell activity, good in vitro expansion ability and strong immunomodulatory ability. It uses FAP as a target and its high expression in a variety of diseases that can be treated with MSC, such as fibrosis, arthritis, autoimmune diseases (Crohn's disease, rheumatoid arthritis, etc.), cardiovascular diseases, etc., has a wide range of indications.

[0036] (2) The present invention provides a method for preparing FAP-targeted CAR-MSC cells, which achieves specific recognition of FAP through a specific single-chain antibody fragment (scFv), thereby enhancing the directional migration of MSC to antigen-expressing tissues. FAP antigen-specific stimulation can enhance the expression of immunosuppressive cytokines by CAR-MSC, and CAR-MSC exhibits enhanced immunosuppressive function in inflammatory areas of antigen expression, thereby solving the problems of poor targeting and insufficient immunosuppressive ability faced by MSC clinical applications.

[0037] (3) The present invention provides the use of CAR-MSC cells in the preparation of products for the treatment of FAP-expressing diseases. The CAR gene-modified MSC cell drugs have the ability to redirect and enhance specific immune regulation, providing a new strategy for the treatment of fibrotic diseases and a new research direction for the application of stem cell therapy in other diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is the plasmid map of the recombinant lentiviral expression vector pRRLSIN-EF1α-FAPBBZ.

[0039] Figure 2 Flow cytometry was used to detect MSC cell phenotype.

[0040] Figure 3 CAR-MSC cell growth characteristics and CAR positive rate; among them, Figure 3 A is the cell viability of MSC and CAR-MSC, Figure 3 B is the expansion multiple of MSC and CAR-MSC, Figure 3 C is the CAR positivity rate of CAR-MSCs.

[0041] Figure 4 Flow cytometry was used to detect the expression of CAR on the cell surface.

[0042] Figure 5 Flow cytometry was used to detect the phenotype of CAR-MSC cells.

[0043] Figure 6 Flow cytometry was used to detect the expression of FAP on the cell surface.

[0044] Figure 7 is the expression of IL-4, an immunosuppressive factor in CAR-MSC 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-MSC cells

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

[0048] The 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. Its nucleotide sequence is shown in SEQ ID NO.1. It was 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 was named pRRLSIN-EF1α-FAPBBZ. The plasmid map is shown in Figure 1Endo-free Plasmid Maxi Kit (Omega) was used to extract the plasmid, and 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 225mL 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 make up to 60mL, mix well, and let stand for 5 minutes to form liquid A. Prepare another 225mL centrifuge bottle, add 4.2mL of 1mg / ml PEI (polysciences), add serum-free DMEM to make up to 60mL, mix well, and let stand for 5 minutes to form liquid B. Then add liquid B to liquid A, mix well, and let stand for 20 minutes 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 9TU / mL. The obtained lentivirus was recorded as pRRLSIN-EF1α-FAPBBZ lentivirus.

[0056] 3. Isolation and culture of mesenchymal stem cells (MSC)

[0057] The umbilical cord of a full-term healthy fetus was collected, immersed in a centrifuge tube containing 1% penicillin and streptomycin in PBS, and transported to the laboratory at low temperature (2-8°C). The umbilical cord was taken out and placed in a 10 cm culture dish, cut into several small pieces, and washed three times with saline containing double antibody and saline. The washed umbilical cord was placed in a new 10 cm culture dish, the artery and vein were removed, the Wharton's jelly was separated and fully cut into 1 mm pieces. 3 The tissue blocks were washed twice by centrifugation with normal saline (500g, 5min). The chopped Wharton's jelly was added to a culture bottle containing serum-free culture medium, and gently shaken to evenly disperse the tissue blocks, and cultured in a 37°C, 5% CO2 incubator. Half of the medium was changed after 5-7 days of culture, and the culture was continued in the incubator until long spindle cells grew from the edge of the tissue block. When the cells covered about 50% of the bottom of the bottle, the culture medium and tissue blocks in the culture bottle were discarded, and normal saline was added to wash twice. Trypsin was added for digestion, and subculture was performed, marked as P1 generation. When the adherent cells covered 90% of the bottom of the bottle, trypsin was added for digestion, and subculture was performed at a ratio of 1:5-1:10. The P3 generation cells were used to establish an MSC cell bank, and the phenotype of the P3 generation MSC was detected by flow cytometry. The results are as follows Figure 2 As shown, the positive rates of CD73, CD90, and CD105 of MSC cells were all above 95%, and CD19, CD34, CD45, CD11b, and HLA-DR were negative, indicating that MSC cells were successfully isolated from the umbilical cord. The cells were frozen with cryopreservation fluid, and an umbilical cord mesenchymal stem cell bank was established, and the corresponding generations were marked.

[0058] 4. Preparation of CAR-MSC by lentiviral infection of MSC cells

[0059] (1) Take MSC cells from the cell bank, revive them, and add serum-free medium for subculture.

[0060] (2) MSC cells (P4 generation) in good growth state were inoculated into culture flasks and placed in a 37°C, 5% CO2 incubator. After overnight culture, purified pRRLSIN-EF1α-FAPBBZ lentivirus (MOI=10, 50, 100, 200) and polybrene (final concentration 6 μg / mL) were added, and the cells were centrifuged for infection (700g, 1.5h) and then placed in a 37°C, 5% CO2 incubator for culture.

[0061] (3) 24 hours after lentiviral infection, the medium was changed and serum-free medium was added, and the cells were cultured and subcultured in a 37°C, 5% CO2 incubator.

[0062] During the CAR-MSC culture process, cell samples were taken and the expression of CAR was detected by flow cytometry. The results are shown in the figure ( Figure 3 C and Figure 4 ), when the MOI of lentivirus infection was 10, 50, 100, and 200, the CAR positivity of P5 CAR-MSCs reached 98.5%, 99.9%, 99.8%, and 99.9%, respectively, and the CAR positivity of P6 CAR-MSCs reached 85.7%, 99.9%, 99.9%, and 99.9%, respectively. The phenotypes of P6 MSCs and CAR-MSCs were detected by flow cytometry. The results are shown in Figure 5 As shown in the figure, the positive rates of CD73, CD90, and CD105 of MSC and CAR-MSC cells were all above 95%, and CD19, CD34, CD45, CD11b, and HLA-DR were negative. The above results show that CAR-MSC was successfully prepared and can continuously express CAR at a high level, and CAR gene modification will not affect the phenotype of MSC. In addition, the growth characteristics of MSC and CAR-MSC of P4, P5, and P6 generations were studied during the production process, and the results are shown in the figure. Figure 3 As shown in Figure 2, the cell viability of MSC and CAR-MSC was maintained above 95% and showed no decreasing trend with the increase of passage number ( Figure 3 A), compared with MSC, the expansion fold of CAR-MSC decreased after lentivirus infection, but when MOI was 10, the expansion fold of P6 CAR-MSC was close to that of uninfected MSC, and in the high MOI lentivirus infection group, the expansion fold of P6 increased compared with P5 ( Figure 3 B). The above results show that CAR expressing FAP can be introduced into MSC cells through lentiviral infection and can be expressed at a high level continuously. Lentiviral infection affects the expansion capacity of CAR-MSC. As the number of passages increases, the effect of lentiviral infection on the expansion capacity of CAR-MSC is weakened, especially when the MOI is 10, the expansion multiple of P6 generation CAR-MSC is similar to that of MSC, indicating that highly active CAR-MSC cells were successfully prepared and have good in vitro expansion capacity.

[0063] Experimental Example 1 CAR gene modification enhances the immunosuppressive ability of MSC cells

[0064] 1. Screening of cells with high expression of fibroblast activation protein (FAP)

[0065] 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 6As shown, the positive rates of FAP expression in HFL1 and WI-38 cells were about 95% and 40%, respectively. HFL1 was used as a FAP high-expressing cell for subsequent studies.

[0066] 2. Antigen stimulation enhances the immunosuppressive ability of CAR-MSCs

[0067] (1) The P5 MSCs and CAR-MSCs prepared in the example were inoculated in 24-well plates and cultured in a 37° C., 5% CO 2 incubator for 24 h.

[0068] (2) HFL1 cells (as FAP antigen stimulation, providing cell-based antigen-specific stimulation) were added at a ratio of 1:1 for co-culture.

[0069] (3) After 48 h of stimulation, cells were harvested, RNA was extracted, and the expression of immunosuppressive cytokine IL-4 was detected by qPCR.

[0070] The results are as follows Figure 7 As shown in the figure, compared with the MSC group, the IL-4 in the CAR-MSC group was significantly increased, indicating that under the condition of antigen FAP stimulation, CAR gene modification can enhance the immunomodulatory effect of MSC. Therefore, CAR-MSC targeting FAP not only has the scFv that specifically recognizes FAP to enhance redirection and migration to antigen-expressing tissues, but also has enhanced immunosuppressive ability at antigen-specific inflammatory sites.

[0071] CAR gene modification improves the targeting and immunomodulatory activity of MSCs, solving the main challenges faced by MSC therapy. CAR-MSCs targeting FAP can be used to treat FAP-upregulated fibrosis, arthritis, autoimmune diseases (Crohn's disease, rheumatoid arthritis, etc.), cardiovascular diseases and other diseases, while providing new ideas for stem cell therapy of other diseases, which has far-reaching scientific significance and huge commercial value.

[0072] 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 CAR-MSC cell targeting fibroblast activation protein, characterized in that: CAR includes a signal peptide, an antigen binding domain, a hinge region, a transmembrane domain, a co-stimulatory signaling region, and a CD3 signaling domain; The nucleotide sequence of the CAR is shown in SEQ ID NO.1 or SEQ ID NO.2, and the amino acid sequence of the CAR is shown in SEQ ID NO.3 or SEQ ID NO.4; The nucleotide sequence of the 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 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.

2. The CAR-MSC cell according to claim 1, characterized in that The signal peptides include CD8A, CD4, CD3, CD5, CD19, CD20, CD22, CD28, CD33, CD45, CD80, CD86, GM-CSFR, and PD-L1; among them, the nucleotide sequence 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.

3. The CAR-MSC cell according to claim 1, 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.

4. The CAR-MSC cell according to claim 3, 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.

5. The CAR-MSC cell according to claim 1, 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.

6. The CAR-MSC cell according to claim 1, characterized in that The transmembrane domain 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.

7. The CAR-MSC cell according to claim 1, 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.

8. The CAR-MSC cell according to claim 1, 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.

9. The method for preparing CAR-MSC cells according to any one of claims 1 to 8, characterized in that: The chimeric antigen receptor targeting fibroblast activation protein is introduced into mesenchymal stem cells to prepare CAR-MSC cells; Among them, the introduction methods include viral vector infection, electroporation transfection, transposon, gene knock-in, extracellular vesicles, nucleic acid lipid nanoparticles, nanogold particles, and chemical reagent transfection; the viral vectors include lentiviral vectors, retroviral vectors, adeno-associated viruses, and adenoviruses.

10. Use of the CAR-MSC cell according to any one of claims 1 to 8 in the preparation of a product for treating a fibroblast activation protein-expressing disease, characterized in that: Diseases expressing fibroblast activation protein include fibrosis, arthritis, autoimmune diseases, and cardiovascular diseases.

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