EGFR和PD-L1双靶向嵌合抗原受体构建及表达

目的:构建肿瘤相关抗原表皮生长因子受体特异性嵌合抗原受体(EGFR-CAR)和程序性死亡受体-配体1(PD-L1)抗体双修饰慢病毒载体表达系统。方法:人PD-L1-Fc蛋白免疫BALB/c小鼠,经细胞融合、亚克隆筛选高分泌PD-L1特异性抗体的稳定杂交瘤,酶联免疫吸附试验(ELISA)和Western blot检测抗体特异性,流式细胞术(FACS)鉴定对PD-1配受体封阻性能,Fortebio测定抗体亲和力,抗体全长测序,经保留鼠源CRD1、CRD2和CRD3人源化改造后构建单链抗体(single-chain variable fragment,scFv);人EGFR单克隆抗体杂交瘤系,经5′RACE技术扩增其轻链和重链可变区(VL和VH)基因,构建scFv,克隆至真核载体pcDNA3.1表达鉴定。基因合成EGFR-CAR(引入CD137协同信号胞内功能域)与PD-L1-scFv借助2A序列连接,克隆入慢病毒pLVX-EF1a-IRES-ZsGreen1表达载体,使用Lenti-X Packaging Single Shots (VSV-G)共同转染293T细胞,获得包装病毒,感染293V细胞,FACS测定CAR膜表达,ELISA检测CAR感染293V细胞培养上清中PD-L1-scFv表达情况,转染激活人外周血T细胞,验证CAR膜表达。结果:获得PD-L1抗体11E3,具备高度配受体封阻性能,经人源化改造后,亲和力稳定(2.67×10 -10 mol/L),EGFR-scFv获得有效表达。进一步构建了EGFR-CAR和PD-L1双修饰慢病毒分泌型CAR(CTC0537-1)及膜表达型CAR(CTC0537-2),其病毒感染293V细胞阳性率为10%。CTZ0431-1感染293V细胞后,细胞膜表面表达EGFR-scFv,检测培养上清存在PD-L1-ScFv;CTZ0431-2感染293V细胞后,细胞膜表面EGFR-scFv和PD-L1-scFv有效表达,双表达病毒感染活化T细胞的CAR表达率为39.3%。 结论:成功构建了EGFR-CAR和PD-L1-scFv双表达慢病毒载体,EGFR-CAR中度结合亲和力,此为EGFR靶向和PD-L1抗体双修饰CAR-T细胞的实体瘤治疗研究提供了关键工具。

Construction and expression of chimeric antigen receptors targeting epidermal growth factor receptor(EGFR)and programmed cell death ligand-1(PD-L1)

Objective To construct an expression system of lentivirus vector encoding epidermal growth factor receptor-specific chimeric antigen receptor(EGFR-CAR)and programmed cell death ligand-1(PD-L1)antibody.Methods Human PD-L1-Fc protein was used to immunize BALB/c mice.Cell-fusion and subcloning were performed to screen stable hybridoma strains with high secretion of PD-L1-specific antibodies,which were identified by both ELISA and Western blot.The activity of the antibodies in blocking the binding of programmed cell death-1(PD-1)to PD-L1 was determined by fluorescence-activated cell sorting(FACS).Antibody affinity was analyzed by Fortebio Octet96.A single-chain variable fragment(scFv)was further constructed after antibody full-length sequencing and humanization using CDR grafting method.Meanwhile,the genes encoding the light and heavy chain variable regions(VL and VH)were cloned from a hybridoma secreting antibody against human EGFR by 5′RACE technology to construct scFv gene.The expression of scFv was confirmed using pcDNA3.1 vector.EGFR-CAR containing CD137 intracellular function domain and PD-L1-scFv was ligated using 2A gene.The synthetic single molecule was cloned into pLVX-EF1a-IRES-ZsGreen1 lentivirus expression vector,and then transfected into 293T cells using Lenti-X Packaging Single Shots(VSV-G)to prepare infectious virus.Expression of CAR on cell surface and the soluble form of PD-L1-scFv in the supernatant of transfected 293V cells were detected by FACS and ELISA.Results A PD-L1 antibody named 11E3 with high ligand-receptor blocking performance was obtained.The humanized antibody showed a stable affinity(2.67×10-10 mol/L)after directly grafting the mouse CDRs(CDR1,CDR2 and CDR3)to human frameworks.EGFR-scFv was effectively expressed in a form of Fc-fusion.Secretory CAR(CTZ0431-1)and membrane CAR(CTZ0431-2)expression plasmids were constructed using lentivirus vector containing EGFR-CAR and PD-L1-scFv.The infection efficiency in 293V cells was around 10%.EGFR-scFv on the cell membranes and PD-L1-scfv in the culture supernatants were detected after 293V cells were infected with CTZ0431-1.EGFR-scFv and PD-L1-scfv were expressed on the cell membranes of 293V cells infected with CTZ0431-2.The expression rate of CAR in LV-CART46407-1-transfected activated T cells was 39.3%.Conclusions The lentivirus vectors co-expressing EGFR-CAR with moderate binding affinity and PD-L1-scFv with high binding affinity were successful constructed,which provided an essential tool for investing EGFR-and PD-L1 double targeted CAR-T cell therapy against solid tumor.