Incorporation of Unnatural Amino Acid into Antibody Fragment for Creating a Stable Antibody-Drug Conjugate

Traditional chemotherapy drugs have been used as a standard treatment for cancers. However, the drugs have resulted in a modest survival benefit and damaged non-cancerous cells. Thus, novel strategies that can improve selectivity and specificity in chemotherapy are urgently needed. An antibody-drug conjugate (ADC), which combines a monoclonal antibody and a cytotoxic drug, is a potential for targeted therapy. However, heterogeneous mixtures have been observed using the current ADC methods. Here, we developed a strategy for generating a stable ADC utilizing a modified single-chain antibody fragment (scFv) containing azide group for click chemistry reaction with alkyne containing the cytotoxic drug. This research focused on targeting prostate cancer as a model disease targeting prostate-specific membrane antigen (PSMA) receptor which is overexpressed in all stages of prostate cancer. The unnatural amino acid, para-azido phenylalanine (pAzF), has been successfully incorporated into anti-PSMA J591 scFv and specifically bound and internalized into PSMA positive cancer cells. This mutant scFv was also successfully conjugated to a linker containing cyclo-alkyne, DBCO-PEG4-DBCO as a model for creating ADC through copper-free click chemistry reaction. This bioconjugation method is promising as a versatile strategy for generating a stable ADC to improve therapeutic efficacy in cancer treatments.