中国生物工程杂志

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.