Research On Anti-tumor Effect And Mechanism Of Fibroblast Activating Protein Targeting Chimeric Receptor T Cells

Background: Cancer is a serious threat to human health and it's a problem urgently needed to be solved.Currently,adoptive immunotherapy has become a new kind of the treatment method for cancer in addition to the surgery,radiotherapy,chemotherapy,and traditional Chinese medicine treatment.The adoptive immunotherapy based on CAR T therapy is a hot in current research.CAR T therapy is not limited by major histocompatibility complex(MHC).Through the construction of specific antibodies in the extracellular segments against the tumor antigens,and co-stimulatory molecules of intracellular segments,CAR T therapy can realize greater tumor targetability and cellular activity,thereby playing a stronger anti-tumor effect.The CAR T therapy has been used successfully in the treatment of hematological tumors.In 2017,the FDA approved the availability of two CAR T drugs for the treatment of patients with acute lymphoblastic leukemia and specific types of non-Hodgkin's lymphoma.However,CAR T treatment of solid tumors is currently progressing slowly.The tumor-mediated micro-environment-mediated immunosuppressionof solid tumors is a major factor affecting the efficacy of CAR T.Therefore,from the perspective of breaking the immunosuppression of the tumor microenvironment,it is of great clinical significance to search for more appropriate tumor-specific antigens and more appropriate antibodies to improve the efficacy of CAR T against solid tumors.FAP is a type II integral memberane protein on activated fibroblast memberane.It is highly specifically expressed in most cancer-associated fibroblasts(CAF)and is one of the specific markers of CAF.FAP is with special biological characteristics and it plays an important role in promoting tumor growth,infiltration,invasion,metastasis,and tumor immunology curb.The use of FAP as a target for immune targeted therapy of tumor stroma is receiving the increasing attention.Nanobody is a type of the single-domain antibody composed of the variable regions of heavy chain antibodies found in the blood of camels.It is with high water solubility,high stability,high expression,high yield,relatively strong tissue penetration and weak immunogenicity and other advantages,which have shown a broad application prospect in the immune targeted therapy of cancers.Our previous research successfully selected FAP-specific Nanobodies and named it F578.This Nanobody can specifically bind to the tumor cell surface antigen FAP.Whether it is possible to use F578 to construct CAR T cells to achieve anti-tumor effect still needs further study.Objective: To explore the application of anti-FAP Nanobody as an extracellular segment antibody to construct CAR T cells,to verify the in vitro proliferation activity of FAP-CAR T cells and their specific killing effect on target cells,and to inhibit the subcutaneous hepatocellular carcinoma xenografts in mice.To investigate the anti-tumor effect and mechanism of FAP-CAR T cells and provide a new basis for CAR T to treat solid tumors.Method:1.The CAR gene sequences were designed as three groups of respectively Mock,CD19-CAR and FAP-CAR.The double enzyme digestion scheme cut the PLVX lentivirus vector to complete the recombination of the target gene and the vector plasmid,packaging and concentrating the Lentivirus and Lentivirus titer was measured with the use of the fluorescence counting.2.Human peripheral blood T lymphocytes were isolated;and the harvested lentiviruses were used for T cell infection to complete construction of CAR T cells.GFP expression of CAR T cells was observed by fluorescence microscopy;the proportion of CAR T cells expressing GFP and their CD4+/CD8+ ratio were examined by flow cytometry.3.The human primary hepatocellular carcinoma(HCC)was collected and the CAF in the HCC tissue was isolated and purified by digestive differential method.The expression of FAP/ ?-SMA was detected by flow cytometry to identify the CAF.4.Using untransfected T cells(Utd)as control,flow cytometry was used to detect the proliferation of CAR T cells co-incubated with target cells in Utd,Mock,CD19-CAR and FAP-CAR group.And surface activation molecules CD25,CD69,memory molecules CD62 L,CD107a and intracellular IFN-?expression.5.In vitro cytotoxicity of FAP-CAR T cells against FAP positive cell lines was measured by flow cytometry;cytokines(IFN-?,TNF-?,IL-2,IL-10)in supernatant were detected by ELISA.6.The mouse subcutaneous xenograft model was constructed and randomly divided into PBS,Utd,Mock,CD19-CAR and FAP-CAR groups.Each group of CAR T cells and untransfected T cells were injected via the tail vein,and theeffect of CAR T treatment on tumor volume,tumor weight,and survival time of mice was observed.7.When the tumor length reached 15 mm,the mice were sacrificed and the paraffin sections were prepared.Immunohistochemistry was used to detect the expression of Ki-67 antigen and FAP in tumor tissues;TUNEL method was used to detect the amount of apoptosis in tumor tissues;immunofluorescence was used to measure the density of blood vessels in tumors(CD34).8.The mouse subcutaneous xenograft model was constructed and randomly divided into PBS,Utd,Mock,CD19-CAR and FAP-CAR groups.Each group of CAR T cells and untransfected T cells were injected via the tail vein.Flow cytometry was used to detect the expression of human CD3 in peripheral blood,spleen,and tumor tissues of tumor-bearing mice on the 7th,14 th,and 28 th days.9.Normal NOD/SCID mice were divided into FAP-CAR T treatment group and PBS group.After treatment with tail vein injection of FAP-CAR T,HE staining was used to detect the toxic effects on the main organs of mice.Results:1.Double enzyme digestion scheme efficiently completed cleavage of PLVX lentiviral vectors.The PCR method validated that CAR genes in each group can be successfully inserted into the vector;after the lentivirus packaging and concentration,the harvested lentivirus titer reached 2E+8 TU/ml.2.After these lentiviruses were applied to T cell infection,GFP fluorescence was observed under fluorescence microscope.The expression of GFP in each group of cells was more than 40% by flow cytometry,indicating that CAR T cells were successfully prepared.3.Flow cytometry detected the isolated and purified CAF,and the percentage of FAP/?-SMA double positive cells reached 75.7%,suggesting that the CAFseparation was successful and could be used for the next experiment.4.The proliferation of FAP-CAR T cells stimulated by FAP positive target cells was significantly higher than that of the other control groups.The expressions of surface activation molecules CD25,CD69,memory molecule CD62 L,CD107a and intracellular IFN-? was significantly upregulated compared with the other control groups.5.FAP-CAR T can significantly kill target cells of FAP+ in vitro,and its killing effect increases with the increase of E:T ratio.The killing rate of target cells against CAF and HepG2-hFAP was higher than that of U87 cells with low expression of FAP,but it had no killing effect on tumor cells of FAP negative.6.In vivo experiments showed that FAP-CAR T cells treatment can significantly inhibit the tumor development in HepG2-hFAP subcutaneously transplanted tumor mice,prolong the survival time of mice,and improve the survival rate;immunohistochemical detection of mouse tumor tissue expression Ki-67 and FAP was significantly reduced;the proportion of apoptotic cells in the tumor tissue of mice was significantly increased by TUNEL method;the blood vessel density of mouse tumors was significantly reduced by immunofluorescence.7.CD3 expression in peripheral blood,spleen and tumor tissues of mice in FAP-CAR group was significantly higher than that in other control groups at three time points.CD3 expression was detected to peak on the 14 th day,and it was still able to reach the 28 th day.Maintain a certain expression.8.HE staining showed no toxic effects of FAP-CAR T treatment on the major organs of mice.Conclusion: The FAP-CAR T cells based on anti-FAP Nanobodies can specifically target the killing of FAP positive target cells in vivo and in vitro.This CAR T therapy targeting the tumor microenvironment provides a new idea for the immunotherapy of solid tumors.