Development of Drug-to-Antibody Ratio Enhanced Antibody Fragment-Drug Conjugates Platform

Abstract

Antibody-drug conjugates (ADCs) have emerged as a promising therapeutic approach, offering high specificity to target cancer cells while minimizing damage to normal tissues. However, achieving an optimal drug-to-antibody ratio (DAR) remains a challenge, particularly in antibody fragment-based ADCs, due to limited conjugation sites and structural instability. In this study, we propose a DAR-enhanced antibody fragment-drug conjugates platform utilizing the fusion of an elastin-like polypeptide (ELP) tag. The platform integrates 4D5Albu, a HER2-targeting antibody fragment derived from trastuzumab, with an ELP tag engineered to incorporate hydrogen-substituted tetrazine (frTet) for site-specific conjugation. To preserve the antigen-binding affinity of the antibody fragment, we employed an inverse electron-demand Diels-Alder (IEDDA) reaction, a highly efficient site-specific conjugation strategy, for the introduction of the drug. By controlling the number of frTet residues incorporated into the ELP tag, we generated the antibody fragment-drug conjugates with DAR 1 and DAR 3, for comparative performance analysis. The successful formation of both variants through the site-specific conjugation strategy was confirmed. Notably, the HER2-binding affinity of the DAR 3 variant was comparable to the DAR 1 variant, demonstrating that higher drug loading did not compromise antigen recognition. Furthermore, cell viability assays validated the cytotoxicity of the ADCs against cancer cells. This study emphasizes the feasibility of utilizing ELP tag-based platforms for development of DAR enhanced antibody fragment-drug conjugates. However, the lack of therapeutic efficacy improvement with increased DAR highlights the need for further optimization to fully realize the potential benefits of higher drug loading. This study suggests that ELP tag-based antibody fragment-drug conjugate platforms can be a promising strategy for enhancing DAR in future ADC development.