Antitumor Effect And Mechanism Of Chimeric Antigen Receptor T Cells Based On Nanobody Targeting FAP And Secreting CTLA-4

Background: T cells can target tumor cells by expressing CAR.Chimeric antigen receptor(CAR)is an artificial method combining single-chain antibodies,which specifically recognize tumor antigens with the intracellular domain of the receptor that can activate T cells.The immunoreceptor tyrosine activation motif is fused into a recombinant gene.CAR T cell therapy is currently the hottest and most promising treatment for tumor immunotherapy.Currently CAR T therapy is used in hematological tumors.Good progress has been made,but due to the complex microenvironment of solid tumors,CAR T has limited killing effect on solid tumors,so new methods need to be explored to improve the activity of CAR T in the treatment of solid tumors.Fibroblast activating protein(FAP),a membrane antigen recognized by Retting et al.In 1988 using monoclonal antibody F19,is a type Ⅱ serine protease that participates in a variety of pathological processes and accounts for over 90% of human epithelium It is expressed in cell carcinoma tissues[4-5].It is highly expressed in tumor-associated fibroblasts and some tumor cells,but hardly appears in normal tissues.Therefore,FAP is a potential biomarker and tumor therapeutic target,which is of great significance for tumor research.Checkpoint blocking therapy uses checkpoint antibodies to disrupt the interaction between inhibitory receptors on T cells,especially CTLA-4 and PD-1,and their inhibitory ligands on tumor cells.It has produced effective clinical effects in a series of patients with solid tumors and hematological malignancies.In 2017,the journal Mol Ther [8] published an article stating that the tumor can be used as a shield to cure tumors.To date,U.S.Food and Drug Administration has approved the first generation of immune checkpoint inhibitor ipleizumab(a monoclonal antibody that blocks CTLA-4)to induce a continuous antitumor response to treat melanoma.Cytotoxic T lymphocyte-associated antigen-4(CTLA-4),is a white blood cell differentiation antigen,which is mainly expressed on the surface of activated T lymphocytes,inhibits the proliferation and activation of T cells in the early stage of tumorigenesis,and participates in the negative regulation of immune response,thereby protecting tumor cells from T cells attack and increasing the susceptibility of tumors.Anti-CTLA-4 can promote T cell proliferation and promote the expression of cytokines.At present,there are many studies on Anti CTLA-4 sc Fv,and some progress has been made.However,due to sc Fv Poor stability,Poor harmony and short residence time in the body can’t effectively exert effects.These shortcomings limit the application of single chain antibodies.Therefore,new antibodies with good stability and affinity are needed.In 1993,Hamers-Casterman has reported that in the camel’s body,there is a heavy chain antibody lacking a light chain and of which the antigen binding site is composed of only the variable region(VHH)of the heavy chain [12],which is called a nanobody.FAPNb and CTLA-4 Nb36 have been screened.Compared with ordinary antibodies,Nanobodies have a small molecular weight,good stability,high affinity,weak immunogenicity,strong tissue penetration and easy genetic modification,etc.Advantages have great application value and development prospects in both disease diagnosis and treatment.In the previous work of this research group,we designed a CAR T cell targeting FAP,which showed a certain antitumor effect in vivo and in vitro.Based on the previous work,we blocked our FAPNb CAR T and checkpoints Combined with the therapy,a new CAR T — CTLA-4 Nb36 /FAPNb CAR T was constructed,which can target FAP and secrete immune checkpoint blocker nanobodies while enhancing T cell survival and CAR.-T cell killing activity on solid tumor cells.Provide experimental basis for CAR T cells to treat solid tumors.Objective: To explore the method of constructing chimeric FAP Nanobodies and secreting CTLA-4 Nanobody to modify t cells,and to explore the proliferation activity of FAP-CTLA-4/Nb CAR Tcells in vitro and their killing effect on target cells,and NOD / SCID mice in vivo.The growth inhibitory effect of hepatocellular carcinoma transplanted tumors,a preliminary study of the anti-tumor effect and mechanism of FAP-CTLA-4/Nb CAR Tcells,and providing a new basis for tumor adoptive immune cell therapy.Methods:1.The sequence of CAR gene was constructed,including 5 groups: Mock 、 Irrelevant/Nb CAR 、 FAP/Nb CAR 、 CTLA-4/Nb CAR 、 FAP-CTLA-4/Nb CAR The lentiviral vector was cut by double-restriction enzyme digestion,the target gene was recombined with the plasmid,Construction of lentiviral vector containing CAR sequence was identified by PCR and gene sequencing.The lentivirus was packaged and concentrated,the titer of lentivirus was measured by fluorescence counting.2.The peripheral blood mononuclear cells were collected from healthy volunteers,and CAR T cells were constructed by lentivirus infection technique.The expression of GFP in CAR T cells was observed by Fluorescence microscope,the expression of HIS-TAG and GFP on CAR T was also detected by Flow cytometry.The secretion of CTLA-4nanobody by the new CAR T was detected by Elisa.3.Lentivirus-transfected Mock 、 Irrelevant/Nb CAR T 、 FAP/Nb CAR T、CTLA-4/Nb CAR T、FAP-CTLA-4/Nb CAR Tcells,and Mock group T cells,uninfected T cells were used as controls,and the proliferation of CAR T cells in each group after stimulation by target cells was detected by flow cytometry.Active molecule(CD25,CD69),memory molecules CD62 L,and Exhaustion molecules(LAG-3,TIM-3).4.Flow cytometry was used to detect the killing effect of FAP-CTLA-4/Nb CAR Tcells secreted cytokines(IFN-γ,TNF-α,IL-2)by Elisa,ELISPOT detected the number of IFN-γsecreted by CAR T cells stimulated by target cells.5.Construct xenograft model of NOD / SCID mouse,(HepG2,U87,HepG2-FAP),inject CAR T cells and normal T cells through the tail vein for two times,and test the ability of recombinant CAR T cells to kill tumors in vivo by observing the size of tumor and the survival rate of mouse.6.When xenograft model of NOD / SCID mouse,,the tumor was cut into paraffin sections,the proliferation of cells in tumor tissues was detected by immunohistochemistry(KI67),and the number of T cells in tumor tissues was detected GFP in tumor tissue by flow cytometry to investigate the anti-tumor mechanism of FAP-CTLA-4/Nb CAR T.Results:1.PCR and positive clones sequencing results of FAP-CTLA-4/Nb CAR showed that the lentiviral vectors containing CAR gene sequence were successfully constructed.The titer of lentivirus in each group was between 2×109TU/m L-4×109TU/m L2.The expression of GFP and HIS-TAG on CAR T cells was observed by Fluorescence microscope,and the expression rate of GFP and HIS-TAG on CAR T cells was about 50% by Flow cytometry.Elisa results showed that the new CAR T cells could effectively secrete CTLA-4 nanobody,indicating the successful preparation of FAP-CTLA-4/Nb CAR T cells.3.FAP-CTLA-4/Nb CAR T cells can promote the proliferation of T cells after being stimulated by the target cell antigen,and promote the expression of surface activating molecules(CD25,CD69),memory molecules(CD62L).FAP-CTLA-4/Nb CAR T The cells secreted cytokines IL-2 IFN-γ and TNF-α,which were higher than those of other controls.FAP-CTLA-4/Nb CAR decreased LAG-3 and TIM-3expression.4.As the effect-target ratio increases,the killing effect of the target cells increases.FAP-CTLA-4/Nb CAR T cells have a significantly higher killing effect on target cells(HepG2,U87,HepG2-FAP)than the control group.The killing efficiency of CAR T cells to high FAP cells(HepG2-FAP)is higher than that of low FAP and non-FAP target cells(HepG2,U87).5.FAP-CTLA-4/Nb CAR T cells have a certain therapeutic effect in high expression of FAP and low expression of FAP xenografts.The treatment effect on FAP of expressing xenografts.The therapeutic effect on high expression FAP xenografts was better than that on low expression FAP xenografts.The tumor growth was inhibited;the survival time of micewith high FAP expression was prolonged.6.The results of immunohistochemistry showed that FAP-CTLA-4/Nb CAR T T cells could inhibit the proliferation of tumor cells and increase the apoptosis of tumor cells.The results of flow cytometry showed that CAR T lymphocytes were infiltrated in tumor tissues of mice.Conclusion: The results showed that the anti-FAP / CTLA-4/Nb CART cells successfully constructed by lentivirus technique had certain anti-tumor effect in vitro and invivo.The anti-tumor effect of FAP / CTLA-4/Nb CAR T cells was related to the expression of FAP on the tumor cells.Our study provides a new idea for the cancer treatment with CAR T.