Rapid evaluation of antibody fragment endocytosis for antibody fragment–drug conjugates

Antibody–drug conjugates (ADCs) have emerged as the most promising strategy in targeted cancer treatment. Recent strategies for the optimization ADCs include the development of antibody fragment–drug conjugates (FDCs). The critical factor in the successful development of ADCs and FDCs is the identification of tumor antigen-specific and internalizing antibodies (Abs). However, systematic comparison or correlation studies of internalization rates with different antibody formats have not been reported previously. In this study, we generated a panel of scFv-phage Abs using phage display technology and their corresponding scFv and scFv-Fc fragments and evaluated their relative internalization kinetics in relation to their antibody forms. We found that the relative rates and levels of internalization of scFv-phage antibodies positively correlate with their scFv and scFv-Fc forms. Our systematic study demonstrates that endocytosis of scFv-phage can serve as a predictive indicator for the assessment of Ab fragment internalization. Additionally, the present study demonstrates that endocytic antibodies can be rapidly screened and selected from phage antibody libraries prior to the conversion of phage antibodies for the generation of the conventional antibody format. Our strategic approach for the identification and evaluation of endocytic antibodies would expedite the selection for optimal antibodies and antibody fragments and be broadly applicable to ADC and FDC development.