Anti-tumor Effect And Mechanism Of Novel CAR T Cells Targeting CD105 By Secreting PD-1 Nanobody

Background:Chimeric antigen receptor modified T cell(Chimeric antigen receptor T cell,CAR T)is a novel and effective tumor immunotherapy by identifying tumor associate antigen(TAA).The fusion protein of TAA receptor and T cell activation sequence is expressed on the surface of autologous T cell,T cells can exert obtain tumor targeting and proliferating ability,and play a long-lasting anti-tumor effect.In recent years,CAR T immunotherapy has made great progress in the treatment of hematological malignancies,However,there are a lot of stromal cells such as vascular endothelial cells and immunosuppressive signals in the microenvironment of solid tumors.In addition,the protective physical and immune barrier can resist the attack of immune cells,CAR T cell is still limited in solid tumor treatment practice.Therefore,how to enhance the tumor targeting of CAR T cells and eliminate the immunosuppression of tumor microenvironment is an important problem to improve the anti-tumor effect of CAR T cells.Endothelial cell proliferation associated antigen has become a new target of tumor immunotherapy in tumor microenvironment.Endoglin(CD105)is a marker of malignant neoplasm neovascularization,which is highly specifically expressed in proliferative vascular endothelial cells of tumor.Programmed death receptor 1(PD-1),as the key to immunosuppression,has been the focus of tumor immune microenvironment research.It aims to eliminate its negative effect on T cells and enhance anti-tumor effect.CD105 nanobody and PD-1nanobody have been screened out in the previous work.Nanobodies can improve the penetration of solid tumors because of their small molecular size and strong permeability,which can better invade the internal part of solid tumors.Therefore,This study applied the combination of nanobody and CAR T as a novel strategy for targeted immunotherapy,with CD105 nanobody providing CAR T cell tumor targeting,and secreting PD-1 nanobody to neutralize PD-1 immunosuppression of T cell,thus significantly improved the efficacy of CAR T cells in the treatment of solid tumors.Objective:To explore the method of constructing CAR T cells secreting PD-1nanobody targeting CD105.To explore the biological activities of CD105-PD-1/Nb CAR T cells in vitro and vivo,and explore the anti-tumor effect and mechanism of CD105-PD-1/Nb CAR T cells,so as to find a more effective CAR T cells targeting the tumor microenvironment to overcome multiple barriers,and to provide a new method for the field of CAR T cells.Procedures:1.CD105-PD-1/Nb CAR fragments were obtained by gene synthesis with CD105 nanobody as the antigen recognition domain of CAR T cells and the sequence of PD-1 nanobody.The CAR fragment and the vector GV401 were recombined with Bam HΙ enzyme digestion to construct the CD105-PD-1/Nb CAR lentivirus expression vector.The recombinant plasmids were identified by PCR,and then identified by DNA sequencing.After successful identification,the lentivirus was packaged after vectors co-transfected into 293 T cells,and the supernatant of lentivirus was collected and concentrated.2.CD105-PD-1/Nb CAR T cells,CD105/Nb CAR T cells,CD105/Nb CAR T cells,Irrelevant-PD-1/Nb CAR T cells,PD-1/Nb CAR T cells,Mock T cells were constructed,after T cells were infected with the concentrated supernatant of different groups of viruses.After culture,the uninfected T cells were used as the blank control group.The expression of GFP in T cells was observed under fluorescence microscope,and the expression rate of EGFP/His-Tag in T cells was detected by flow cytometry to verify the stable expression of CD105-PD-1/Nb CAR T cells,At the same time,the secretion of PD-1nanobody was detected by ELISA.3.After co-culture of T cells in each group and target cells Bel7404,the proliferation of T cells was detected.The expression of activated molecules(CD25,CD69),memory molecule(CD62L),and the depletion expression(LAG-3)were detected by flow cytometry.4.After co-culture of CART cells with Bel7404,the contents of cytokines(TNF-a,IFN-γ,IL-2,IL-10)were detected by ELISA.The number of T cells secreting IFN-γ stimulated by target cells Bel7404 was detected by ELISPOT.5.Isolation and purification of HLCMVEC and detection of CD105 antigen expression in target cells.The killing effect of CAR T cells on target cell Bel7404,HLCMVEC were detected by flow cytometry.6.To verify the adoptive treatment of tumor mice with CD105-PD-1/Nb CAR T cells in vivo.The subcutaneous transplantation tumor model of NOD/SCID mice bearing human hepatocellular carcinoma(Be17404)was established.CD105-PD-1Nb CAR T cells and control group cells were injected intravenously through tail vein.After treatment,the tumor volume and survival rate of mice were measured to verify the anti-tumor effect of CD105-PD-1/Nb CAR T cells in mice bearing HCC subcutaneous transplantation.Results:1.CD105-PD-1/Nb CAR lentivirus expression vector was constructed.PCR and sequencing results showed that the recombinant lentivirus vector was successfully constructed.After the virus was infected with T cells,the expression of EGFP green fluorescence was observed under a fluorescence microscope,and the expression rate of CAR T cells was detected by flow cytometry,indicating the stable expression of CD105-PD-1/Nb CAR T cells was successfully constructed.2.The secretion level of PD-1 nanobody in the supernatant of CD105-PD-1/Nb CAR T cell culture was significantly higher than that of other control groups by ELISA,indicating that the CAR T cell had the secretion capacity of PD-1nanobody.3.Flow cytometry detection promoted the proliferation of T cells after co-culture of CD105-PD-1/Nb CAR T cells with the target cells Bel7404.The expression of membrane surface activation(CD25 and CD69),memory molecules(CD62L)was increased.And the expression of depletion molecular(LAG-3)was decreased,show that CD105-PD-1/Nb CAR T cell has more significantly activation ability.4.The secretion of cytokines TNF-α,IFN-γ and IL-2 in CD105-PD-1/Nb CAR T cells stimulated by Bel7404 were higher than those in other control groups.While there was no significant difference in IL-10 secretion among CAR T cells.ELISPOT was used to detect the number of IFN-γ secreted T cells in CD105-PD-1/Nb CAR T cells stimulated by the target cells Bel7404 was higher than that of other control groups.These results indicate that CD105-PD-1/Nb CAR T cells can secrete more anti-tumor cytokines,and their secreted PD-1nanobaby act on PD-1 and eliminates the inhibitory effect of PD-1/PD-L1 interaction on T cell function.5.HLCMVEC primary cells were successfully isolated,and the high expression of CD105 antigen in HLCMVEC was detected by flow cytometry.Meanwhile,it was found that CD105-PD-1/Nb CAR T cells could specifically bind in vitro and kill CD105+ target cells Bel7404 and HLCMVEC effectively,and the killing effect was positively correlated with the effect-target ratio.6.In vivo adoptive treatment of CD105-PD-1/Nb CAR T cell in mice with hepatocellular carcinoma(HCC)significantly inhibited tumor growth in mice and prolonged survival rate in mice,so as to verify the successful anti-tumor effect of CD105-PD-1/Nb CAR T cell in mice with subcutaneous transplantation of HCC.Conclusion:The results showed that a novel CAR T cell secreting PD-1nanobody and targeting CD105 were successfully constructed(CD105-PD-1/Nb CAR T),based on the previous work of our research group,PD-1 and CD105 specific nanobodies were screened and combined with the new targeted immunotherapy strategy of CAR T cells,CD105-PD-1/Nb CAR T cells showed specific antitumor effects in vivo and vitro.This study demonstrates the superiority and potential of novel CAR T cells based on nanobabys to treat solid tumors by specifically targeting the tumor vascular microenvironment and inhibiting PD-1 immune checkpoints for the first time.