Research On Anti-tumor Effect And Mechanism Of The New CRA T Cells Secreting PD-1 Nanobody Targeting FAP

In recent years,there has been a breakthrough in tumor therapy,and immunotherapy is one of the hot spots in tumor therapy.Chimeric antigen receptor T cell immunotherapy(CAR T)is a new immunotherapy that using genetic engineering technology to modify T cells to express the receptor of tumor antigen and then target the tumor.In 2017,CAR T therapeutic product Kymriah(Tisagenlecleucel)was marketed for the treatment of blood tumors,achieved the curative effect.However,solid tumors represent complex immunosuppressive microenvironment that inhibited the function of CAR T cells lead to progress slowly comparing to blood tumors.Cancer-associated fibroblasts(CAFs)are a kind of abnormal differentiated stromal cells that occupy a large cells in tumor.As a specific marker of CAFs,FAP expressed in both tumor cells and stromal cells.PD-1 is a negative regulatory protein for activated T cells.PD-1 monoclonal antibody drug can improve the anti-tumor effect of T cells,and has achieved a good effect in clinical application.In this study,CAR T cells with high activity and targeting both tumor microenvironment and tumor cells were constructed to achieve the better treatment.The key of targeting CAR T cell are single chain variable fragment(scFv).Traditional scFv are prone to lead mismatch between light chain and heavy chain and need to optimize the joint hinge area.Nanobody(Nb)is a new type of natural antibody discovered in recent years,which has the advantages of small size,easy expressing,good solubility and strong stability.It has a broad prospect in basic research,new drug development,disease diagnosis and treatment,and has become the object that many country compete to research and development.we succeeded in screening FAP Nb and PD-1 Nb,found that have anti-tumor function,on the basis of the innovation,we further to construct the new CAR T cells secreting PD-1 nanobody targeting FAP(FAP-PD-1/Nb CAR T cells),and explore its value on the treatment of solid tumors.Objective:To constrcuct the new CAR T cells secreting PD-1 nanobody targeting FAP(FAP-PD-1/Nb CAR T cells),and explore the activity and killing effect of FAP-PD-1/Nb CAR T cells on target cells in vitro,and tumor inhibitory effect on NOD/SCID mice bearing HepG2-hFAP liver cancer xenograft tumor and U87 glioma xenograft tumor,research the anti-tumor function and relative mechanism of FAP-PD-1/Nb CAR T cells,and provides a new idea for CAR T cells immunotherapy in solid tumors.Methods:1.The CAR gene sequences were designed,PD-1/Nb CAR,Irrelevant-PD-1/Nb CAR,FAP/NbCAR,FAP-PD-1/Nb CAR groups.The GV400 lentivirus vector was cut by Bam H Ⅰ/EcoR Ⅰ double enzyme digestion,size of the target gene fragment was identified by PCR,and the connection between the target gene and the vector plasmid was completed.The vector expressing the new CAR sequence and the packaging plasmid were co-transfected into 293T cells,the lentivirus particles were packaged and concentrated.The lentivirus titer was measured by dilution counting method.2.Normal peripHeral blood T cells were isolated and cultured and then lentivirus transduct into T cells to complete construction of each CAR T cells,the CAR T the infection efficiency detected by flow cytometry,the expression of GFP observed by fluorescence microscope,the PD-1 Nb of FAP-PD-1/Nb CAR T cells detected by ELISA and Western blot respectively.3.The surgical tissues of patients with primary liver cancer were collected,and the primary hepatocellular carcinoma associated fibroblasts(CAFs)were isolated and cultured by differential enzyme digestion combined with repeated adherence.The expression of FAP in CAFs,U87,HepG2-hFAP,HepG2,and A549 cells were detected by flow cytometry.4.The unIFNected primary T cells were considered as control cells(UTD).Flow cytometry was used to detect the expression of the surface activated molecules CD25,CD69,memory molecule CD62L,depletion molecule LAG-3 and the proliferation of CAR T cells in each group was detected after stimulation by FAP+ target cells.5.The cytokines(IL-2,IL-10,IFN-γ,TNF-α)secreted in the supernatant of CAR T cells in each group were determined by ELISA.ELISPOT was used to detecte the number of IFN-y secreted by CAR T cells stimulated by FAP+target cells in each group.Flow cytometry was used to detect the killing effect of CAR T on target cells HepG2-hFAP,U87,HepG2 and CAFs in each group.6.Establishing subcutaneous xenograft tumor model(HepG2-hFAP,U87)in mice,injected CAR T cells and normal T cells in each group through tail vein,treat them twice,measure the tumor volume in each group,observe the survival rate of mice,and evaluate the anti-tumor effect of CAR T cells in each group in vivo.7.After the treatment,the tumor was taken and paraffin sections were made.The cell proliferation in tumor tissues was detected by immunohistochemical staining(Ki67),the vascular density in tumor tissues was detected(CD31),and the number of apoptotic cells in tumor tissues was detected by TUNEL method to study the anti-tumor mechanism of CAR T cells.Results:1.The GV400 lentivirus vector was successfully cut and the CAR gene were recombined with the double enzyme digestion scheme.The recombinant vector and the package plasmid were successfully transfected into 293T package and the recombinant CAR lentivirus was finally obtained2.The recombinant CAR lentivirus can IFNect the primary normal human T cells,and the infection efficiency is above 40%,indicating the successful construction of CAR T cells.PD-1 Nb expressed continuously and stably by FAP-PD-1/Nb CAR T cells.3.The expression of FAP in CAFs,U87 and HepG2-hFAP cells was significantly higher than that in HepG2 and A549 cells.4.The expression levels of CD25,CD69 and CD62L in the FAP-PD-1/Nb CAR T cell group were higher than other groups after stimulation with different FAP+target cells,and the increase of the depletion molecule LAG-3 after activation was lower than that of the FAP/Nb CAR group and the FAP/Nb CAR+PD-1 Nb group.The proliferation rate of theFAP-PD-1/Nb CAR T cell group was significantly higher than that of other groups.The levels of IL-2,IFN-γ,TNF-αsecreted in the FAP-PD-1/Nb CAR T cell group were significantly higher than those in other groups,and there was no significant change in the expression level of IL-10 in each group.The killing efficiency of FAP-PD-1/Nb CAR T cells on target cells was higher than that of other groups.5.The FAP-PD-1/Nb CAR T cell group significantly slowed down the growth of subcutaneous tumors in NOD/SCID mice HepG2-hFAP and U87,and extended the overall survival of mice.6.The FAP-PD-1/Nb CAR T cell group significantly promoted the apoptosis of tumor cells,inhibited the proliferation of tumor cells,and reduced the density of tumor microvessels.Conclusion:1.The new CAR T cells secreting PD-1 nanobody targeting FAP(FAP-PD-1/Nb CAR T cells),was successfully constructed based on FAP nanobody and PD-1 nanobody.2.FAP-PD-1/Nb CAR T cells have good functional activity in vitro and show stronger specific killing effect on target cells.3.FAP-PD-1/Nb CAR T cells have a good anti-tumor effect in vivo.This new CAR T cell designed to secrete PD-1 Nb by targeting both tumor cells and the tumor microenvironment provides a new idea for CAR T cells immunotherapy in solid tumors.