靶向TPBG和EGFR的双特异性抗体偶联药物的构建及其抗肿瘤活性研究

Development and Anti-tumor Study of Bispecific Antibody Drug Conjugate Targeting TPBG and EGFR

目的:构建靶向TPBG和EGFR的双特异性抗体偶联药物(bispecific antibody drug conjugate,BsADC),并探究其在体内外抗肿瘤活性。方法:通过携带全人抗体重链可变区和共同轻链新型小鼠的RenLite技术平台获得具有共同轻链的靶向胚胎滋养层糖蛋白(trophoblast glycoprotein,TPBG,也称为5T4)和表皮生长因子受体(epidermal growth factor receptor,EGFR)的抗体序列,采用KIH(Knobs-Into-Holes)技术将其组装成anti-TPBG×EGFR双特异性抗体(bispecific antibody,BsAb),并通过流式细胞术(flow cytometry,FCM)、表面等离子共振技术(surface plasmon resonance,SPR)、高效液相色谱(high performance liquid chromatography,HPLC)等方法进行体外表征,最后采用半胱氨酸偶联技术将其与微管蛋白抑制剂甲基澳瑞他汀E(MonoMethyl auristatin E,MMAE)偶联成药物抗体偶联比(drug to antibody ration,DAR)为4的anti-TPBG×EGFR BsADC,并研究其体外杀伤作用和在人源肿瘤细胞系异种移植模型(cell derived xenograft,CDX)中的抗肿瘤活性。结果:根据GEPIA2数据库,TPBG与EGFR在多种肿瘤上具有共表达。通过对5种人源性肿瘤组织异种移植(patient-derived xenografts,PDX)免疫荧光分析也证实TPBG和EGFR在肿瘤细胞中具有不同比例的共表达。通过RenLite平台成功制备高纯度靶向TPBG和EGFR的双抗。经体外实验结果表明,BsAb相较于TPBG母本单抗显著增强了与肿瘤细胞的结合亲和力(avidity)和内吞速率。BsADC相较于TPBG母本单抗ADC明显提高了肿瘤杀伤和抗肿瘤活性。在A431(EGFR^(high)/TPBG^(low))模型中,anti-TPBG×EGFR BsADC体内抗肿瘤活性强于TPBG和EGFR的母本单抗ADC,表现出协同作用。另外,在NCI-H292(EGFR^(moderate)/TPBG^(low))模型和DU145(EGFR^(low)/TPBG^(low))模型中,anti-TPBG×EGFR BsADC均表现出良好的体内抗肿瘤活性。结论:实验结果表明,将TPBG与快速内化的ADC靶标EGFR组合是增强靶向TPBG的ADC内化和抗肿瘤活性的强有力策略。

Objective:To develop a bispecific antibody drug conjugate(BsADC)targeting TPBG×EGFR and to investigate its anti-tumor activity in vitro and in vivo.Methods:Fully human antibodies targeting the oncofetal trophoblast glycoprotein(TPBG,also known as 5T4)and the epidermal growth factor receptor(EGFR)were acquired from the RenLite platform,which is a novel mouse model expressing the entire human antibody variable region of the heavy chain and a specific common light chain.Anti-TPBG and EGFR antibodies were assembled into anti-TPBG×EGFR bispecific antibody(BsAb)by the knobs-into-holes(KIH)technique.Structural and functional characterization of anti-TPBG×EGFR BsAb was screened by Flow Cytometry(FCM),Surface Plasmon Resonance(SPR)and High Performance Liquid Chromatography(HPLC).Anti-TPBG×EGFR BsAb and the microtubule protein inhibitor MonoMethyl auristatin E(MMAE)were assembled using the cysteine coupling strategy to generate anti-TPBG×EGFR BsADC with a drug to antibody ration(DAR)of 4.Furthermore,the in vitro cell killing and in vivo anti-tumor activity of TPBG×EGFR BsADC were explored in human-derived tumor cell lines and cell derived xenograft(CDX)models.Results:According to the GEPIA2 database,TPBG is co-expressed with EGFR in a variety of tumours.Immunofluorescence analysis of five Patient-Derived Xenografts(PDX)showed that TPBG and EGFR are co-expressed in tumour cells in varying proportions.The bispecific antibodies targeting TPBG and EGFR with high-purity were successfully developed by the RenLite co-light chain fully human antibody discovery platform.In vitro results showed that by simultaneously targeting both TPBG and EGFR on the surface of tumor cells,the bispecific antibody significantly enhanced the binding affinity(avidity),endocytosis and killing of tumor cells compared to the TPBG parental monoclonal antibody.Moreover,in the A431(EGFR~(high)/TPBG^(low))model,anti-TPBG×EGFR BsADC showed stronger anti-tumor activity than the parental ADCs of TPBG and EGFR in vivo,demonstrating synergistic effects.In addition,in the NCI-H292(EGFR~(moderate)/TPBG^(low))and DU145(EGFR^(low)/TPBG^(low))models,anti-TPBG×EGFR BsADC also showed strong anti-tumor activity.Conclusion:These results suggest that combining TPBG with EGFR,a rapid internalizing ADC target,is a powerful strategy for enhancing anti-tumor activity of ADCs targeting TPBG.