CAR-MSC cell targeting fibroblast activation protein and preparation method and application thereofTechnical Field
The invention belongs to the field of cell therapy, and particularly relates to a CAR-MSC cell targeting fibroblast activation protein, a preparation method and application thereof.
Background
Mesenchymal stem cells (MESENCHYMAL STEM CELL, MSCs), also known as multipotent or multipotent mesenchymal stem cells, have self-renewal and multipotent differentiation capabilities. MSCs have been isolated from various tissues including umbilical cord, placenta, amniotic membrane, fat, etc., since Friedenstein was found in bone marrow in the 70 s of the 20 th century. MSCs are widely found in various tissues throughout the body, can be expanded in vitro, can differentiate into adipocytes, osteoblasts, chondrocytes, muscle cells, neural cells, etc. under specific conditions, and can interact with immune cells through cell-cell contact and secretion of soluble molecules to alleviate excessive inflammatory responses. The multifunctional nature of mesenchymal stem cells (multipotent differentiation, immunomodulatory activity, paracrine) makes them extremely potential in the treatment of disease.
Mesenchymal stem cells have been extensively explored and used in a variety of fields as a promising cell therapy approach. Mesenchymal stem cell-based therapies have been used to treat a variety of diseases including, but not limited to, osteoarthritis (OA), premature ovarian failure (POI), graft Versus Host Disease (GVHD), pulmonary fibrosis, and the like. 10 mesenchymal stem cell products are approved and marketed worldwide and are mainly used for regulating immunity and repairing injury and treating Graft Versus Host Disease (GVHD), crohn disease, myocardial infarction, osteoarthritis and other diseases. Although MSC is one of the most studied cell therapy technologies worldwide, showing good safety in clinical studies of various diseases, clinical therapeutic effects have been unsatisfactory, mainly due to lack of sufficient targeting of target tissues and immunosuppressive ability.
Fibroblast activation protein-alpha (Fibroblast activation protein-alpha, FAP) is a type II transmembrane serine protease of 97-kDa, has dipeptidyl peptidase (DPP), endopeptidase and collagenase activities, and can degrade dipeptides, type I collagen, gelatin and the like. FAP is hardly expressed in normal tissues, but has high expression level in pathological diseases such as tumor, fibrosis and the like, and participates in matrix activation and tissue remodeling. FAP is selectively expressed in more than 90% of human epithelial tumors, including the envelopes or cytoplasm of stromal fibroblasts such as breast cancer, lung cancer, colorectal cancer, ovarian cancer and the like, and is widely involved in the processes of tumor growth, invasion, metastasis, tumor extracellular matrix reconstruction, angiogenesis, immune escape and the like, thereby promoting the development process of the tumors and attracting wide attention of researchers.
FAP also plays an important role in non-neoplastic diseases. FAP is associated with the progression and severity of fibrosis, arthritis, autoimmune diseases (crohn's disease, rheumatoid arthritis, etc.), cardiovascular diseases, and the like. Given the role of FAP in tissue remodeling and its expression on activated fibroblasts, it is not surprising that FAP expression is associated with uncontrolled scar formation disease (fibrosis), elevated levels of FAP under pathological conditions of pulmonary fibrosis, liver fibrosis, kidney fibrosis, colon fibrosis, granulation tissue, and the like. FAP is specifically upregulated in fibroblasts and fibroblast stroma in pulmonary fibrosis patients, but not in adjacent normal tissue, lung tissue of healthy individuals, or lung tissue of acinar centralized emphysema patients. Liver fibrosis is another uncontrolled fibrotic disease, often triggered by chronic injury such as viral hepatitis, nonalcoholic fatty liver, or alcohol abuse. Chronic liver injury activates hepatic stellate cells, produces extracellular matrix responsible for liver scarring, exhibits a myofibroblast-like phenotype and expresses FAP. In patients with viral hepatitis c, FAP expression is correlated with the degree of liver fibrosis. Liver fibrosis may progress to cirrhosis, ultimately potentially leading to liver failure. FAP activity in liver cirrhosis is 14-18 times higher than in normal liver. Other extensive fibrosis associated with upregulation of FAP expression includes keloids (Keloid scars) and crohn's disease. One study found FAP expression in synovial samples of rheumatoid arthritis and osteoarthritis patients, and FAP expression in refractory rheumatoid arthritis patient samples was higher than in end-stage osteoarthritis patients. FAP overexpression is also found in thin fibrous cap atherosclerotic plaques (TCFA) in human aortic smooth muscle cells. FAP is highly expressed in various diseases, and participates in tissue remodeling and pathological processes, and is a potential therapeutic target.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a CAR-MSC cell targeting fibroblast activation protein, and a preparation method and application thereof. The CAR-MSC cell not only can realize specific recognition of FAP through specific single-chain antibody fragments (scFv), enhances directional migration of MSC to antigen expression tissues, but also can activate an intracellular signal pathway through FAP antigen specific stimulation, enhances the immunosuppressive ability and the treatment effect of the CAR-MSC, can be used for treating FAP expressed fibrosis, arthritis, autoimmune diseases (Crohn disease, rheumatoid arthritis and the like), cardiovascular diseases and the like, and has the advantages of strong targeting, high activity, multiple indications and the like.
In order to achieve the above purpose, the invention adopts the following technical scheme:
in a first aspect the invention provides a fibroblast activation protein-targeted CAR-MSC cell, the CAR comprising a signal peptide, an antigen binding domain, a hinge region, a transmembrane domain, a costimulatory signaling region, and a CD3 signaling domain;
The nucleotide sequence of the CAR is shown as SEQ ID NO.1 or SEQ ID NO.2, and the amino acid sequence of the CAR is shown as SEQ ID NO.3 or SEQ ID NO. 4;
the nucleotide sequence of the CAR is a nucleotide sequence having at least 75% homology with 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 with the amino acid sequence shown in SEQ ID No.3 or SEQ ID No. 4.
In some embodiments, the signal peptide comprises CD8A, CD4, CD3, CD5, CD19, CD20, CD22, CD28, CD33, CD45, CD80, CD86, GM-CSFR, PD-L1, wherein the nucleotide sequence of CD8A is shown as SEQ ID NO.5, 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.
In some embodiments, the antigen binding domain is an antigen binding fragment Fab or scFv which targets 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.
Chimeric antigen receptors (CHIMERIC ANTIGEN receptors, CARs) are capable of specifically binding to an antigen target via a single chain variable fragment (scFv), activating intracellular signaling pathways under antigen stimulation.
Anti-human FAP monoclonal antibodies include, but are not limited to, anti-human FAP monoclonal antibody West-Rumex monoclonal antibody (Sibrotuzumab), anti-human FAP monoclonal antibody 7NP2, anti-human FAP monoclonal antibody OMTX005, anti-human FAP monoclonal antibody RG-7461, anti-human FAP monoclonal antibody MP0310, anti-mouse FAP monoclonal antibodies include, but are not limited to, anti-mouse FAP monoclonal antibody FAP5, anti-mouse FAP monoclonal antibody FAP73.3.
Preferably, the FAP-targeting antigen binding fragment Fab or scFv is the anti-human FAP monoclonal antibody cetrimide (Sibrotuzumab) or the anti-human FAP monoclonal antibody 7NP2.
In some embodiments, the antigen-binding fragment scFv is of the structure 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 has a nucleotide sequence with at least 75% homology to the nucleotide sequence shown in SEQ ID NO.9, and the amino acid sequence of the antigen-binding fragment scFv is as shown in SEQ ID NO.10 or has an amino acid sequence with at least 75% homology to the amino acid sequence shown in SEQ ID NO. 10.
Preferably, the antigen binding fragment scFv structure is VH-Linker-VL.
In some embodiments, the hinge region is selected from one of CD8A, CD, igG1, igG2, and IgG4, wherein the nucleotide sequence of CD8A is shown as SEQ ID NO.11, the amino acid sequence is shown as SEQ ID NO.12, the nucleotide sequence of CD28 is shown as SEQ ID NO.13, and the amino acid sequence is shown as SEQ ID NO. 14.
Preferably, the hinge region is CD8A or CD28.
In some embodiments, the transmembrane domain is selected from one of CD8A, CD, CD4, CD3, ICOS, wherein the nucleotide sequence of CD8A is shown as SEQ ID NO.15, the amino acid sequence is shown as SEQ ID NO.16, the nucleotide sequence of CD28 is shown as SEQ ID NO.17, and the amino acid sequence is shown as SEQ ID NO. 18.
Preferably, the transmembrane domain is CD8A or CD28.
In some embodiments, the costimulatory signaling region comprises an intracellular domain from a costimulatory molecule selected from at least one of CD27, CD28, 4-1BB, OX40, ICOS, CD40, lymphocyte functional antigen-1 (LFA-1), wherein the nucleotide sequence of 4-1BB is shown as SEQ ID NO.19, the amino acid thereof is shown as SEQ ID NO.20, the nucleotide sequence of CD28 is shown as SEQ ID NO.21, and the amino acid sequence thereof is shown as SEQ ID NO. 22.
In some embodiments, the CD3 signaling domain is CD3 zeta or CD3 epsilon, wherein the nucleotide sequence of CD3 zeta is shown as SEQ ID NO.23 and the amino acid sequence is shown as SEQ ID NO. 24.
The second aspect of the present invention provides a method for preparing a fibroblast activation protein targeting CAR-MSC cell, wherein a Chimeric Antigen Receptor (CAR) of the fibroblast activation protein targeting CAR-MSC cell is prepared by introducing a Mesenchymal Stem Cell (MSC);
The method for introducing the recombinant DNA into the cell comprises virus vector infection, electroporation transfection, transposon, gene knock-in (knockin), extracellular vesicles, nucleic acid Lipid Nanoparticles (LNP), gold nanoparticles and chemical reagent transfection, wherein the virus vector comprises lentivirus vector, retrovirus vector, adeno-associated virus and adenovirus.
Preferably, the CAR is introduced into the MSC by viral vector infection, electroporation transfection, transposon, gene knock-in (knockin) or extracellular vesicles.
More preferably, the CAR is introduced into the MSC by infection with a viral vector, which is a lentiviral vector or a retroviral vector.
The lentiviral vector is specifically a four-plasmid lentiviral packaging system, and comprises a lentiviral expression plasmid pRRLSIN-EF1 alpha-FAPBBZ with a target gene, packaging plasmids pMDLg/pRRE (Kan+) and pRSV-REV (Kan+), and a lentiviral envelope plasmid pMD2.G (Kan+).
The CAR-MSC cell preparation comprises the following steps of recovering MSC cells in a cell bank, subculturing, inoculating the MSC cells with good growth state into a culture bottle, overnight culturing, adding pRRLSIN-EF1 alpha-FAPBBZ lentivirus and polybrene (polybrene) infected cells, replacing liquid after the lentivirus infected cells are infected for 24 hours, adding serum-free culture medium, and continuing to amplify and culture. Wherein, the MOI of the slow virus infection is 0-200, the concentration of the polybrene is 0-20 mug/mL, further, the MOI of the slow virus infection is 1-10, the concentration of the polybrene is 1-10 mug/mL, further, the concentration of the polybrene is 6 mug/mL.
The mesenchymal stem cells may be umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, dental pulp mesenchymal stem cells, placenta mesenchymal stem cells, amniotic mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, uterine blood mesenchymal stem cells, iPSC induced differentiated mesenchymal stem cells, etc.
Preferably, the mesenchymal stem cells are derived from umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, placental mesenchymal stem cells, and iPSC-induced differentiated mesenchymal stem cells.
In a third aspect, the invention provides the use of a CAR-MSC cell targeted to fibroblast activation protein in the manufacture of a product for the treatment of a disease in which Fibroblast Activation Protein (FAP) is expressed, characterised in that the disease in which fibroblast activation protein is expressed comprises fibrosis, arthritis, autoimmune diseases, cardiovascular diseases.
Diseases include pulmonary fibrosis, liver cirrhosis, heart fibrosis, kidney fibrosis, crohn's disease, rheumatoid arthritis, and the like.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention provides FAP-targeted CAR-MSC cells, which can continuously express CAR, have high cell activity, better in-vitro expansion capacity and strong immunoregulation capacity, take FAP as a target point, and have wide application in treating high expression in various diseases such as fibrosis, arthritis, autoimmune diseases (Crohn disease, rheumatoid arthritis and the like), cardiovascular diseases and the like by using MSC.
(2) The invention provides a preparation method of FAP-targeted CAR-MSC cells, which realizes specific recognition of FAP through specific single-chain antibody fragments (scFv), enhances directional migration of MSC to antigen expression tissues, enhances expression of immune suppression cytokines of the CAR-MSC by FAP antigen specific stimulation, and solves the problems of poor targeting and insufficient immune suppression capability of MSC in clinical application.
(3) The invention provides application of a CAR-MSC cell in preparing a product for treating FAP expression diseases, and a CAR gene modified MSC cell medicament has the capability of enhancing the double effects of redirection and specific immunoregulation, provides a new strategy for treating fibrotic diseases, and provides a new research direction for application of stem cell treatment in other diseases.
Drawings
FIG. 1 is a plasmid map of recombinant lentiviral expression vector pRRLSIN-EF1 alpha-FAPBBZ.
FIG. 2 is a flow cytometry detection of MSC cell phenotype.
FIG. 3 shows the cell growth characteristics of CAR-MSC and the CAR positive rate, wherein FIG. 3A shows the cell viability of MSC and CAR-MSC, FIG. 3B shows the fold expansion of MSC and CAR-MSC, and FIG. 3C shows the CAR positive rate of CAR-MSC.
Fig. 4 is a flow cytometry detection of expression of a cell surface CAR.
FIG. 5 is a flow cytometry detection of CAR-MSC cell phenotype.
FIG. 6 is a flow cytometry detection of cell surface FAP expression.
FIG. 7 is the expression of the CAR-MSC cytostatic factor IL-4.
Detailed Description
The invention is described in further detail below with reference to the accompanying drawings, by way of which the embodiments are described for the purpose of illustrating the invention and are not to be construed as limiting the invention.
Example 1 preparation of CAR-loaded lentiviruses and CAR-MSC cells
1. Construction of lentiviral expression vector pRRLSIN-EF1 alpha-FAPBBZ
The CAR structure is formed by connecting a signal peptide, an antigen binding domain, a hinge region, a transmembrane domain, a co-stimulatory signaling region and a CD3 signaling domain in series, the nucleotide sequence of the CAR structure is shown as SEQ ID NO.1, the CAR structure is artificially synthesized by general biology (Anhui) stock limited company, and cloned between BamHI and MluI cleavage sites of a lentiviral vector pRRLSIN-EF1 alpha-Myc-Palivizumab-BB (refer to Chinese patent: CN202111617729. X), the constructed recombinant lentiviral expression vector is named pRRLSIN-EF1 alpha-FAPBBZ, and the plasmid map is shown as figure 1. Plasmid extraction was performed using an endotoxin-free plasmid big extraction Kit (Endo-FREE PLASMID Maxi Kit, omega), and the concentration and purity of the extracted plasmid was checked by uv spectrophotometry and then stored in a-20 ℃ refrigerator for subsequent lentiviral packaging.
2. Lentivirus packaging and purification
(1) The frozen HEK293T cells were recovered and subcultured with DMEM complete medium (DMEM medium+10% FBS). HEK293T cells were inoculated at a density of 3X 106/mL into a 10-layer cell factory, and plasmid transfection was performed after overnight culture in a DMEM complete medium volume of 1L, at which the cells reached 80-90% confluency.
(2) 1 Centrifuge bottle (225 mL) was prepared, lentiviral expression vector pRRLSIN-EF1 alpha-FAPBBZ, lentiviral packaging plasmid pMDLg/pRRE (Kan+) and pRSV-REV (Kan+), lentiviral envelope plasmid pMD2.G (Kan+), and serum-free DMEM was added to make up to 60mL, and after mixing, it was allowed to stand for 5min to form solution A. 1 additional 225mL centrifuge bottles were prepared, 1mg/mL PEI (polysciences) 4.2.2 mL was added, serum-free DMEM was added to the mixture to 60mL, and the mixture was left to stand for 5min after mixing to form solution B. And then adding the solution B into the solution A, fully and uniformly mixing, and standing for 20min to form the DNA-PEI complex.
(3) The DNA-PEI complex was added to 1L of DMEM medium containing 5% FBS, thoroughly mixed, and 10 layers of the culture medium in the cell factory were replaced.
(4) About 1L of culture supernatant was collected 48h after transfection and stored in a refrigerator at 2-8 ℃.
(5) Removing cells and cell debris from the collected culture supernatant by using a bag filter (Sartorius), and passing the clarified and filtered lentivirus supernatant through a Shibijie tangential flow filtration systemKR 2I) was concentrated to 100-200mL, filtered through a 0.45 μm filter, and purified by chromatography.
(6) The purified lentivirus is sterilized and filtered by a 0.22 mu m filter (Sartorius), sub-packaged, stored in a refrigerator at-80 ℃, and the titer of the lentivirus after detection and purification is 1.62 multiplied by 109 TU/mL. The resulting lentivirus was designated pRRLSIN-EF1 alpha-FAPBBZ lentivirus.
3. Isolated culture of Mesenchymal Stem Cells (MSC)
The term healthy fetal umbilical cord was collected, immersed in a centrifuge tube of PBS containing 1% penicillin and streptomycin, and transported to the laboratory at low temperature (2-8 ℃). The umbilical cord was removed to a 10cm dish and cut into several small pieces, and washed 3 times with physiological saline containing double antibodies and physiological saline, respectively. The washed umbilical cord was placed in a new 10cm dish, arteries and veins were removed, the gum was separated and fully sheared into 1mm3 pieces of tissue, and washed twice with saline centrifugation (500 g,5 min). Adding the sheared Huatong gel into a culture flask containing a serum-free culture medium, slightly oscillating to uniformly disperse the tissue blocks, and culturing in a culture box with 5% CO2 at 37 ℃. Half-changing the culture solution is carried out for 5-7 days, and the culture is continued in the incubator until long spindle-shaped cells grow out from the edge of the tissue block. When the cells covered the bottle bottom by about 50%, the culture medium and tissue blocks in the culture bottle were discarded, and the culture bottle was washed 2 times with physiological saline. Pancreatin was added for digestion and subculture, labeled P1 generation. And adding pancreatin to digest when the adherent cells cover 90% of the bottle bottom, and carrying out passage amplification according to the proportion of 1:5-1:10. The P3 generation MSC phenotype was detected by flow cytometry using the P3 generation MSC cell pool, and the results are shown in FIG. 2, wherein the positive rates of CD73, CD90 and CD105 of the MSC cells are all above 95%, and the negative rates of CD19, CD34, CD45, CD11b and HLA-DR are indicated to successfully isolate the MSC cells from the umbilical cord. Freezing the cells with the freezing solution, establishing an umbilical cord mesenchymal stem cell bank, and marking the corresponding generations.
4. Lentivirus infected MSC cells to prepare CAR-MSC
(1) And (3) recovering MSC cells in the cell bank, and adding serum-free culture medium for subculture.
(2) MSC cells (P4 generation) with good growth status were inoculated into a culture flask and placed in a 37 ℃ 5% co2 incubator. After overnight incubation, purified pRRLSIN-EF1 a-FAPBBZ lentivirus (moi=10, 50, 100, 200) and Polybrene (Polybrene, final concentration 6 μg/mL) were added and after centrifugation (700 g,1.5 h) the cells were incubated in a 37 ℃ 5% CO2 incubator.
(3) After 24h of slow virus infection, the liquid is changed, a serum-free culture medium is added, and the culture is continued to be carried out in a culture box with 5% CO2 at 37 ℃ for passage.
Cell samples were taken during CAR-MSC culture, CAR expression was detected by flow cytometry, and the results were shown in the figures (fig. 3C and fig. 4), and CAR positive rates of P5 generation CAR-MSCs were 98.5%, 99.9%, 99.8%, 99.9% and P6 generation CAR-MSCs were 85.7%, 99.9% when the MOI of lentivirus infection was 10, 50, 100, 200, respectively. The P6 generation MSC and CAR-MSC phenotypes were examined by flow cytometry, and as shown in FIG. 5, the CD73, CD90, CD105 positivity rate of MSC and CAR-MSC cells was 95% or more, and the CD19, CD34, CD45, CD11b, HLA-DR negativity was all found. The above results demonstrate that CAR-MSCs were successfully prepared and that CARs could be expressed at a higher level consistently, and that CAR gene modification did not affect the phenotype of MSCs. In addition, the growth characteristics of P4, P5, P6 generation MSCs and CAR-MSCs were studied during production, respectively, and as a result, as shown in fig. 3, the cell viability of both MSCs and CAR-MSCs remained above 95% and there was no decrease trend with increasing passage times (fig. 3A), the expansion of CAR-MSCs after lentiviral infection was reduced compared to MSCs, but the expansion of P6 generation CAR-MSCs was close to that of uninfected MSCs at MOI of 10, and there was a trend of increasing expansion of P6 generation compared to P5 generation in the high MOI lentiviral infection group (fig. 3B). The above results show that FAP-expressing CARs can be introduced into MSC cells by lentiviral infection and can be continuously expressed at high levels, lentiviral infection can affect the expansion capacity of CAR-MSCs, and as the passage times increase, the effect of lentiviral infection on the expansion capacity of CAR-MSCs is reduced, and particularly when the MOI is 10, the expansion multiple of P6 generation CAR-MSCs is similar to that of MSCs, which indicates that high-activity CAR-MSC cells are successfully prepared and have better in vitro expansion capacity.
Test example 1CAR genetic modification enhances immunosuppressive ability of MSC cells
1. Fibroblast Activation Protein (FAP) high expression cell screening
HFL1 (human embryonic lung fibroblasts, from the national academy of sciences cell bank) and WI-38 (human embryonic lung cells, from the national academy of sciences cell bank) were recovered separately and placed in a 37℃and 5% CO2 incubator. The FAP expression was examined by flow cytometry using cells with good growth conditions, and the results are shown in FIG. 6, wherein the positive rates of FAP expression in HFL1 and WI-38 cells were about 95% and 40%, respectively, and then the FAP high-expression cells were examined using HFL 1.
2. Antigen stimulation enhances the immunosuppressive power of CAR-MSCs
(1) The P5 generation MSC and CAR-MSC prepared in the example are respectively inoculated in a 24-well plate, and are continuously cultured for 24 hours in a 37 ℃ and 5% CO2 incubator.
(2) HFL1 cells (as FAP antigen stimulus, providing cell-based antigen-specific stimulus) were added at a 1:1 ratio for co-culture.
(3) After 48h of stimulation, the cells were harvested and RNA extracted and the expression of the immunosuppressive cytokine IL-4 was detected by qPCR.
The results are shown in figure 7, where the IL-4 is significantly elevated in the CAR-MSC group compared to the MSC group, demonstrating that CAR gene modification can enhance the immunomodulatory effects of MSCs under antigenic FAP stimulation conditions. Thus, FAP-targeted CAR-MSCs have not only scFv that specifically recognize FAP to enhance redirection and migration to antigen expressing tissues, but also enhanced immunosuppressive ability at antigen-specific inflammatory sites.
CAR genetic modification improves targeting and immunomodulatory activity of MSCs, solving a major challenge faced by MSC therapy. The FAP-targeted CAR-MSC can be used for treating FAP up-regulated fibrosis, arthritis, autoimmune diseases (Crohn disease, rheumatoid arthritis and the like), cardiovascular diseases and other diseases, and simultaneously provides a new idea for stem cell treatment of other diseases, and has profound scientific significance and great commercial value.
What has been described above is merely some embodiments of the present invention. It will be apparent to those skilled in the art that various modifications and improvements can be made without departing from the spirit of the invention.