Study On The Mesothelin And NKG2D Dual-Target Enhanced CAR-T In The Treatment Of Triple-Negative Breast Cancer

Purpose:Breast cancer has become one of the most serious tumors to women’s health,and hundreds of thousands of people die of breast cancer every year.Among them,triple-negative breast cancer accounts for about 15% of all breast cancers,and has the characteristics of high invasiveness,easy metastasis and recurrence,and poor prognosis.At present,for triple-negative breast cancer,the clinical deficiency is that the single-target CAR-T treatment of triple-negative breast cancer is not effective.The main reason is that tumor cells will have antigen escape,become resistant to single-antigen-targeted CAR-T cells,and are prone to relapse,which is the key scientific problem to be solved in this paper,and the dual-target CAR-T design is expected to be better solved.These deficiencies improve the effect of CAR-T therapy.The purpose of this study was to verify its killing effect on triple-negative breast cancer cell lines by constructing a dual-target enhanced CAR-T.Since the two targets,NKG2 D and Mesothelin,have a wide coverage and are negatively correlated,in this study,in order to solve the problem of poor efficacy of single-target CAR-T in the treatment of triple-negative breast cancer,a tandem combination application was designed.Dual-target CAR-T targeting Mesothelin and NKG2 D to improve the therapeutic effect of CAR-T.The first step is to compare the effect of CAR-T based on two different antibody（scFv,VHH）sequences targeting Mesothelin in killing triple-negative breast cancer cells,and select the sequence with the best effect for the follow-up dual target.The selection of dot CAR sequences provides a reference.The second step is to design a dual-target CAR sequence using a combination of targeting Mesothelin and NKG2 D,and compare the effects of the constructed dual-target CAR-T and each single-target CAR-T on triple-negative breast cancer cell lines;Sexual T cell engaging agent（bispecific T cell engagers,BiTE）was designed to verify the effect of the constructed CART·BiTE on triple-negative breast cancer cell lines.Methods:1.Constructing two CAR-T cells targeting Mesothelin to compare the effect of killing triple-negative breast cancer cell lines The scFv CAR and VHH CAR vectors were constructed based on the sequences of scFv and VHH,and the lentivirus was packaged and infected with human primary T cells to prepare scFv CAR-T and VHH CAR-T cells.The in vitro killing ability of two CAR-T cells on Mesothelin-overexpressing MDA-MB-231 cells was detected by real-time label-free cell analysis technology,and the cytokine secretion and CD107 a,CD69,Expression of PD-1.2.Construction of CAR-T cells targeting Mesothelin and NKG2 D in combination to verify the effect of killing triple-negative breast cancer cell lines In the previous step to compare the killing effect of two CAR-T cells targeting Mesothelin,the VHH sequence with the best killing effect was selected,and another sequence targeting NKG2 D was designed in series,and a dual-target CAR vector was constructed based on the above two sequences.Packaging lentivirus and infecting human primary T cells to prepare dual-target CAR-T cells.The killing ability of Mesothelin CAR-T,NKG2 D CAR-T and dual-target CAR-T cells against Mesothelin-overexpressing MDA-MB-231 cells was detected by real-time label-free cell analysis technology,and CAR-T cells were detected by flow cytometry Cytokine secretion and expression of CD107 a,CD69 and PD-1 after killing target cells.3.Add "BiTE" design to construct Mesothelin CART·BiTE cells to verify the effect of killing triple-negative breast cancer cell lines On the basis of the previous research,the "BiTE" design was added to construct the Mesothelin VHH BiTE（CD3-NKG2D）CAR vector,packaging lentivirus and infecting human primary T cells to prepare Mesothelin CART BiTE cells.Western Blot was used to detect the "BiTE" supernatant secreted by cells,real-time label-free cell analysis technology was used to detect the killing ability of Mesothelin CART·BiTE cells to MICA-overexpressing MDA-MB-231 cells,and the secretion of "BiTE" supernatant to promote T The killing ability of cells to MDA-MB-231 cells overexpressing Mesothelin was detected by flow cytometry.Results:1.MDA-MB-231 cells overexpressing Mesothelin and MDA-MB-231 cells overexpressing MHC-I molecule-related gene A（MICA）were successfully constructed,which provided the basis for subsequent killing experiments.2.The scFv CAR-T and VHH CAR-T cells targeting Mesothelin were successfully constructed,and the Mesothelin VHH CAR-T cells with better killing effect were selected as the follow-up dual targets by comparing their killing effects on triple-negative breast cancer cell lines in vitro.The basis of CAR design,which is also the first reported Mesothelin CAR-T based on VHH sequence.3.Successfully constructed dual-target CAR-T cells targeting Mesothelin and NKG2 D,which are 1-NKG2D-MSLN（Mesothelin）CAR-T cells and 2-MSLN-NKG2 D CAR-T cells,respectively.To compare the effects of negative breast cancer cell lines,2-MSLN-NKG2 D CAR-T cells with better killing effect were selected to verify that they exhibited stronger killing effects than single-target CAR-T cells,and comprehensive analysis of their expression in It showed better advantages on CD107 a,CD69,PD-1 and secreted cytokines.4.Successfully constructed Mesothelin CAR-BiTE cells with targeted killing ability,verifying that they show stronger killing effect than Mesothelin CAR-T cells,and using Western Blot to verify that the "BiTE" supernatant secreted by cells can promote T cell killing Effect.Conclusions:In view of the current clinical single-target CAR-T treatment of triple-negative breast cancer is not effective,this study by constructing a combination of targeting Mesothelin and NKG2 D double CAR-T cells and Mesothelin CART · BiTE cells to verify its killing effect in vitro The effect of triple-negative breast cancer cell lines is better than their respective single-target CAR-T,and the comprehensive analysis shows that the activation degree of dual CAR-T cells is higher,and the level of cytokines expressed is higher,which is the basis for the subsequent improvement of CAR-T.Research on the effect of treating triple-negative breast cancer provides a good foundation.The main contents are as follows:1.This experiment successfully constructed Mesothelin VHH CAR-T cells with targeted killing ability,and the constructed Mesothelin VHH CAR-T had better killing ability on triple-negative breast cancer cells than the commonly used Mesothelin scFv CAR-T in clinical practice,which is a follow-up study.The selection of the design double-target CAR sequence provides a reference,which is also the first Mesothelin CAR-T based on the VHH sequence reported so far.2.In this experiment,dual-target 2-MSLN-NKG2 D CAR-T cells and Mesothelin CART·BiTE cells with targeted killing were successfully constructed.Compared with each single-target CAR-T,the constructed 2-MSLN-NKG2 D was verified.Both CAR-T and Mesothelin CART·BiTE cells showed stronger killing activity and targeting to triple-negative breast cancer cells.