The Design Of Novel Anti-EGFR Antibody-drug Conjugates And Studies On Their Antitumor Effect

The epidermal growth factor receptor(EGFR),one member of the Erb B family of receptor tyrosine kinases(RTK),has been implicated in various epithelial malignancies As a clinically validated target,both small molecule tyrosine kinase inhibitors and antibodies based drugs have been approved by regulatory agencies.Most therapeutic antibodies targeting the ligand binding domain of the EGFR extracellular region,leading to the inhibition of downstream signaling pathway involved in angiogenesis,cell propagation,differentiation and apoptosis.Anti-EGFR antibody drug conjugates(ADCs)can specifically deliver cytotoxic agent into cytoplasm of EGFR positive tumor cells via internalization and lysosomal degradation,killing target tumor cells irrespective of EGFR signaling.However,cancer patients treated with EGFR-targeted antibody therapy eventually develop resistance,e.g.,acquired mutations in ectodomain(ECD)that abolish antibody binding,which further gives rise to resistance to anti-EGFR ADC therapy.In this study,we have constructed tetravalent biparatopic anti-EGFR ADC,consisting of two tandemly fused anti-EGFR nanobodies targeting two distinct non-overlapping epitopes.The biparatopic anti-EGFR ADC was constructed by coupling anti-mitotic agent monomethyl auristatin E(MMAE)to the engineered surface cysteine on nanobody via the lysosomally cleavable dipeptide,valine-citrulline(vc)linker.This novel antiEGFR ADC could overcome cetuximab resistance and meanwhile exhibited upregulated internalization,leading to superior in vivo antitumor activity.Enhanced complementdependent cytotoxicity(CDC)via the introduction of Fc domain mutation E430 G further improved the efficacy of this biparatopic ADC,suggesting the presence of a synergistic effect.Our findings indicate the advantage of nanobodies for constructing multivalent antibodies drug conjugate,and highlight the CDC enhanced biparatopic nanobody drug conjugate as a novel ADC with promising therapeutic efficacy for treatment of EGFRoverexpressing solid tumors with or without acquired point mutations.