Development Of CAR-T Cells Targeting AXL And Its Therapeutic Effects On Solid Tumors

Chimeric antigen receptor-modulated T lymphocyte(CAR-T)therapy has become a powerful therapeutic approach for treatment of cancers and resulted in great success in hematopoietic malignancies.Currently,two CAR-T products have been approved in the United States.However,the application of CAR-T cells in treatment of solid tumors still needs breakthrough.Development of CAR-T cells against novel therapeutic targets is encouraging.AXL is highly expressed in human malignancies and considered as a novel CAR-T cell target.In this study,a CAR consisting of scFv agnaist AXL was evaluated for its specific cytotoxicity in various AXL positive cancer cells and xenograft tumors.First,we designed and constructed a third generation of letivirus vector containing CAR molecules targeting the AXL on tumor cell membranes.This AXL-CAR vector contains an extracellular AXL antibody scFv domain,the transmembrane segment of IgG4 hinge,two co-stimulation domains(CD28 transmembrane and intracellulardomain,4-1BB intracellular domain)and a CD3?intracellular domain.By cotransfection this vector with packaging plasmids pMD2.G and psPAX2 into 293T cells using calcium phosphate co-precipitation method,we packaged the recombinant AXL-CAR lentivirus.The virus supernatants were concentrated by ultrafiltration and tittered.High transducing units(TU)AXL-CAR lentivirus could be successfully obtained by this method.FurThermore,we successfully developed the AXL-CAR engneerred T cells by using lentivirus mediated gene modification.The precedues for developing AXL-CAR-T cells includes the following steps:(1)isolated T lymphocyte from human peripheral blood;(2)T lymphocytes cultivation and activation in vitro;(3)AXL-CAR lentivirus infection of T lymphocytes;(4)in vitro expasion of AXL-CAR-T cells;(5)expression efficiency detection of the AXL-CAR molecule.To detect the cytotoxicity of AXL-CAR-T cells more effenciently and accurately we developled several luciferase(luc)engineered tumor cell lines as CAR-T target cells.The luciferase(luc)expression plasmid PLKO-cmv-luc was packaged into lentivirus by the same method described in the last section.Target cell lines(MDA-MB-231,MCF-7,786-O,769-P,Miapaca II,and Panc1)were infected by luc lentivirus and selected by puromycin selection and validated for the luc expression by D-Luciferin assay.This enabled us to detect the survival rate of target cells in vitro easily and accurately.To evaluate the clinical significance of AXL in tumors,we detected the AXL expression in several types of tumor samples from patients including renal cancer and breast cancer.In renal cancer samples,the expression of AXL m RNA in tumor tissue was significantly higher than that of the para-carcinoma normal tissue,consistantly,the expression level of AXL ligand-GAS6 was significantly higher in tumor tissue.In biopsies of breast cancer patients,the expression of AXL protein was also higher than that of the para-carcinoma normal tissue.Particularly in triple negative breast cancer(TNBC),the percentage of AXL high expression samples was 86%,significantly higher than that of other types of breast cancer.All these suggest the importance and feasibility of using AXL as a clinical therapeutic target.Secondly,we detected the cytotoxic effects of AXL-CAR-T cells by co-culture with luc engineered target cell lines.We manufactured three different AXL-CAR-T cells with different AXL scFv subclones(L1,L7,3D3).Among the subclones,AXL-L7-CAR-T cells showed the strongest cytotoxic activity against AXL positive cell lines(786-O,769-P,MiapacaII and Panc1).Therefore,AXL-L7-CAR-T cells were selected as the effector cells for subsequent studies in this paper.Compared to T cells and Con-CAR-T,AXL–CAR-T show the strongest cytotoxic effect to AXL positive cell lines(MDA-MB-231,786-O,769-P,MiapacaII and Panc1).The cytotoxic capacity enhanced with the increase of the effector target ratio(E:T).In AXL-negative MCF-7cell lines,the cytotoxic effect of AXL-CAR-T was not obvious compared with that of T cells and Con-CAR-T cells.It is indicated that the cytotoxic effect of AXL-CAR-T cells is specific for targeting AXL antigen.This specific and efficient cytotoxic capacity is the basis of AXL-CAR-T cell application as a therapeutic approach clinical treatment for AXL positive tumors.Finally,we evaluated the in vivo antitumor effect of AXL-CAR-T cells in animal models.We chose B-NSG mice as animal models because of their severe immunodeficiency phenotype,which make the mice the best choice to establish human cell xenograft model.Considering the AXL expression level is highest in TNBC cell line MDA-MB-231 and TNBC patients,we choose to use MDA-MB-231 in our animal model.We inoculated a mixture of 5x10~6 AXL positive MDA-MB-231/luc cells and matrigel basement membrane subcutaneously to B-NSG mice.Ten days after inoculation,tumor bearing was successful in all mice.The tumor-bearing mice were divided into three groups.5×10~6 AXL-CAR-T cells,T cells and 100?L PBS were injected intravenously through tail vein for each group respectively.Tumor volume and mice weight were observed twice every week.Compared with the T cell group and the PBS control,the growing of volume and weight for xenograft were significantly inhibited in AXL-CAR-T group.While the tumor volume and weight in T cell group have no statistical difference compared with the PBS control group.The above results proved that AXL-CAR-T cells had an antigen specific cytotoxic effect on AXL positive tumors.The introduce of AXL-CAR molecular into T cells endow with T cell the ability of destructing tumor cells efficaciously and antigen specifically.We collected peripheral blood from mice on the second day and 12 days after treatment to detect the human T lymphocyte and CAR molecular expression efficiencies.The results shown that in the next day after treatment,human T cells can be detected in both AXL-CAR-T group and T cell group,which indicated the successful construction of the intravenous tail vein injection model.However,in the 12th day after treatment,no human T cells were detected in the peripheral blood of T cell group,while human T cells could still able to be detected in peripheral blood of AXL-CAR-T group.On the second day after treatment,in the survival human T cells,CAR molecules expression rate was as the same as that of AXL-CAR-T cells before injection.This proved the success of our CAR-T cell manufacture and animal model construction.On the 12th day after treatment,there could hardly detect any survival human T cells in the T cell group.Whereas human T cells could only be detected in the AXL-CAR-T group.It suggested that the introduce of CAR molecules improves the in vivo persistence of human T cells.In the end point of treatment,we measured the tumor infiltrating T lymphocytes(TIL)in the tumor tissue.In AXL-CAR-T group,a large number of TIL was found,whereas they have not been detected in T cell group and PBS group.This illustrated the induce of AXL-CAR molecule endowed T cell the ability of targeting the AXL antigen even inside the tumor tissue,so that the AXL-CAR-T cells could better infiltrate into and persistence in tumor tissues.In conclusion,we established a system for laboratory CAR-T cell preparation,developed a new method for measuring the in vitro cytotoxic capacity of CAR-T cells.A lentivirus vector targeting AXL by a novel AXL scFv was constructed and the recombinant lentivirus was packaged and prepared.We developed a type of novel CAR-T cells targeting AXL which shown cytotoxic effect against AXL positive tumor cells both in vitro and in vivo.This new kind of AXL-CAR-T cell shown anti-tumor effect towards AXL positive tumor cells in vitro and in vivo.This study provides a theoretical basis for using AXL as a new target for tumor therapy and using AXL-CAR-T as a new method for cancer therapy.