Construction And Anti-tumor Study Of Bispecific Antibody Drug Conjugate Targeting TPBG And EGFR

Objective:Antibody Drug Conjugate（ADC）,a new type of anti-cancer drug,is a drug formed by coupling a small molecule cytotoxin with a monoclonal antibody molecule by linking molecules,which has a targeted and lethal effect and is leading a new era of targeted cancer therapy.In this study,to construct a bispecific antibody drug conjugate（Bs ADC）targeting TPBG and EGFR to achieve stronger tumour cell line binding activity,promote ADC endocytosis and enhance the efficiency of tumour killing in vivo and in vitro,providing a theoretical basis for improving the clinical application potential of TPBG-targeted ADCs.This provides a theoretical basis for improving the clinical potential of TPBG-targeted ADCs and suggests that Bs ADCs targeting dual tumor antigens are a promising strategy for ADC drug development.Methods:Fully human antibodies targeting the oncofetal trophoblast glycoprotein（TPBG）and the epidermal growth factor receptor（EGFR）,were acquired from the Ren Lite 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/EGFR antibodies were assembled into anti-TPBG×EGFR bispecific antibody（Bs Ab）by Knobs-into-Holes（KIH）technique,and Transient transfection for protein expression.Structural and functional characterization of anti-TPBG×EGFR Bs Ab were screened by Flow Cytometry（FCM）,Surface Plasmon Resonance（SPR）and High Performance Liquid Chromatography（HPLC）.Anti-TPBG×EGFR Bs Ab and the microtubule protein inhibitor Mono Methyl auristatin E（MMAE）were assembled using cysteine coupling strategy to generate anti-TPBG×EGFR Bs ADC with a drug to antibody ration（DAR）of 4.Furthermore,the in vitro cell killing and in vivo anti-tumor activity of TPBG×EGFR Bs ADC 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 constructed by Ren Lite co-light chain whole human antibody platform.In vitro results show 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 Bs ADC 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 Bs ADC 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.