Engineering Antibody Fragments for Improved Antitumor Efficacy

Abstract

Antibody fragments, such as single-chain variable fragments (scFvs) and nanobodies, are promising therapeutic scaffolds for solid tumors. Their key advantages include enhanced tissue penetration and a simple molecular architecture, which enables microbial expression and versatile modification. However, their therapeutic application is often limited by intrinsic biological and physicochemical constraints. This dissertation presents three protein-engineering strategies to address these limitations and expand the therapeutic utility of antibody fragments. First, an albumin-binding scFv scaffold, termed Albubody, was developed as a long-circulating antibody fragment–drug conjugate platform. Computationally guided site selection and click chemistry enabled efficient site-specific drug conjugation while largely preserving antigen- and albumin-binding functions. The resulting Albubody-drug conjugate showed markedly prolonged serum half-life and improved antitumor efficacy compared with the corresponding scFv-drug conjugate. Second, an aggregation-suppressing tag, termed CLEAR tag, was designed using compact, charged elastin-like polypeptide modules. By systematically varying charge type and repeat length, negatively charged CLEAR tags were identified as effective modules for improving soluble expression and suppressing long-term aggregation of unstable scFv derivatives without compromising antigen-binding activity or thermal stability. Third, an albumin-shedding nanobody scaffold, termed AlbuLite, was developed to address the size-dependent trade-off in tumor delivery. By inserting an MMP-2/9-cleavable linker between a nanobody and an albumin-binding domain, AlbuLite was designed to circulate as an albumin-bound complex and release the smaller nanobody form in protease-rich tumor environments. This mode-switching strategy provided an initial framework for balancing systemic persistence with improved tumor penetration. Collectively, these studies demonstrate that antibody fragment performance can be enhanced by rationally engineering circulation, stability, and tumor transport properties. The strategies presented here provide versatile design principles for developing next-generation antibody fragment therapeutics with improved antitumor efficacy.