Construction And Application Of Tandem CAR-T Cells Targeting Folate Receptor α And Mesothelin In Epithelial Ovarian Cancer

Background:The mortality rate of ovarian cancer is the highest among the three major malignant tumors of female reproductive system,and 90%of that are epithelial ovarian cancer（EOC）.Seventy percent of patients are diagnosed at the advanced stage with distant metastases due to the insidious onset.Even the patients who have responded after first-line treatment,55%to 75%relapse within 2 years,leading to the five-year survival rate is less than 40%.Therefore,it is essential to explore new treatment methods.In 1989,Gross et al firstly proposed the concept of chimeric receptor.The variable region of T cell receptor is replaced by single chain antibody,so the modified T cell has the antigen targeting function,which produced the design of chimeric antigen receptor T cells（CAR-T）.CAR-T can directionally recognize tumor cells and killing them.CAR gene consists of extracellular single chain variable fragment（Sc Fv）and intracellular activation motif.T cells modified in this way can solve the immune escape problem caused by the loss of MHC（Major Histocompatibility Complex）class I molecules in tumor cells and having great application prospects.After nearly 30 years of development,the second generation of CAR-T developed in 2011 has achieved nearly 90%complete remission rate in acute lymphoblastic leukemia,which opening a new era for human to overcome tumor and bringing hope to patients with malignant tumors.However,CAR-T therapy has encountered difficulties in solid tumors.There are three main questions.First,heterogeneity of solid tumors and relapse after single target CAR-T therapy（antigen escape）.Second,immunosuppressive microenvironment of solid tumor（hypoxia,low pH,Treg cells,and indoleamine 2,3-dioxygenase）has caused insufficient T cell activation and T cell inhibition.Third,the persistence and amplification of T cells are insufficient.Fouth,T cells are difficult to infiltrate and homing to due to the capsule of solid tumor.As for above issues,we propose three problems to be solved.First,how to reduce tumor antigen escape?Second,how to increase the infiltration of CAR-T?Third,how to activate CAR-T more effectively?In order to solve the first problem,we selected two-tumor associated antigens（TAA）that were highly expressed in EOC through bioinformatics screening and validation of clinical samples.FOLR1（folate receptor alpha）and MSLN（mesothelin）were selected to construct a Tandem CAR-T which targeting at FOLR1 and MSLN.Previous study reported that the expression rate of FOLR1 in EOC is nearly 80%,and only a small amount of FOLR1 is expressed in renal proximal convoluted tubules,type I and type II alveolar cells,and cerebral choroid.The expression rate of MSLN in EOC is nearly 65%,and only a small amount of MSLN is expressed in peritoneum,pleura and pericardium,and almost no expression in parenchymal organs.These TAAs are consistent with the targeting and safety of CAR-T design.On this basis,we have designed a tandem CAR-T that targeting both FOLR1 and MSLN,and hoping it can have a wider killing coverage.In addition,aiming at the infiltration and activation of CAR-T,we utilize the fourth generation CAR-T vector that can secrete IL-12,which will further strengthen the immune response of T cells in tumor microenvironment through the immune activation of IL-12（activating cytotoxic T cells,recruiting B cells,NK cells,and macrophages）.In this way,the survival and amplification ability of CAR-T cells will be further improved.Methods:1.Bioinformatics screened the target antigen and verified the expression of antigen in clinical samples.2.The fourth generation of Tandem-CAR,FOLR1-CAR,and MSLN-CAR lentiviral vectors targeting FOLR1 and MSLN were constructed,respectively.3.CD3 positive cells were sorted by immunomagnetic beads and infected by lentivirus.The expression of CAR-T was detected by flow cytometry.4.LDH（lactate dehydrogenase）release test and RTCA（real time cell analysis）were used to detect the killing ability of different CAR-T in vitro,and flow cytometry was used to detect the secretion of cytokines under co-culture condition.5.The xenograft model was established by subcutaneously injected SNU119 ovarian cells into B-NDG mice(NOD-Prkdc^scid IL2rg^tm1/Bcgen).In vivo imaging compared the therapeutic effects of different CAR-T cells.Results:1.Two TAA that highly expressed in EOC were screened out by bioinformatics:FOLR1 and MSLN.The clinical samples showed that the positive expression rate of FORL1 and MSLN protein was 80.95%（85/105）and 53.33%（56/105）,respectively.The expression was significantly higher than that of borderline ovarian cancer and normal ovarian tissue（P<0.001）.The positive coverage rate of the two proteins was 85.71%（90/105）.2.The lentivirus vectors of Tandem-CAR,FOLR1-CAR and MSLN-CAR were successfully constructed and confirmed by sequencing.CD3 positive T cells were infected by the lentivirus and different kinds of CAR-T were established.3.Experiments of LDH and RTCA showed that when compared with the control group and two single target CAR-T groups,Tandem-CAR cells could significantly kill SNU119cells,whcih highly expressed FOLR1 and MSLN,and the killing rate of A2780 cells was decreased since the expression of FOLR1 and MSLN was lower.4.Flow cytometry was used to detect the levels of IL-2,IL-12,TNF-αand IFN-γ.The results showed that the Tandem-CAR cells released the highest positive ratio of cytokines and successfully expressed IL-12,while Mock-T cells（T cells not transfected with virus）hardly expressed IL-12 when co-culture with the SNU119 cells.The percentage of positive cells that expressing IL-2,TNF-αand IFN-γby the three kind of CAR-T cells was significantly higher than that of the control group（Mock-T）.5.In vivo experiments indicated that when compared with the Mock-T group,all three types of CAR-T could effectively inhibit tumor growth,and the therapeutic effect of MSLN-CAR was manifested as tumor shrinkage and then growth later.The fluorescence intensity of tumors in FOLR1-CAR and Tandem-CAR cells treatment groups decreased continuously.The treatment effect of Tandem-CAR was the most obvious,in which 1 case of tumor in mice occurred regression.Conclusions:Two TAA that highly expressed in EOC were screened and verified.The fourth generation of CAR-T cells which targeting both FOLR1 and MSLN were successfully constructed.Compared with single target CAR-T cells,Tandem-CAR cells has stronger killing effects in vitro and can secrete high levels of cytokines.Tumor model of B-NDG mouse indicated that the three kinds of CAR-T cells still have anti-tumor ability in vivo,and Tandem-CAR cells have the most obvious therapeutic effect,which may shed new light on the reduction of tumor immune escape and also provided a theoretical basis for the application of Tandem-CAR cells in clinical research.