Site-Specific Antibody-Drug Conjugates in Triple Variable Domain Fab Format

Area of Science:

• Biotechnology
• Oncology
• Immunology

Background:

• Conventional immunoglobulin G (IgG) formats for monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) face limitations in solid tumor therapy, particularly regarding tumor penetration and uniform payload distribution.
• Alternative antibody scaffolds are needed to enhance therapeutic utility and potency while maintaining specificity, affinity, and low toxicity.

Purpose of the Study:

• To introduce and validate the triple variable domain Fab (TVD-Fab) as a novel antibody format for site-specific ADC generation.
• To assess the therapeutic potential of TVD-Fab for targeting solid malignancies.

Main Methods:

• Development of the TVD-Fab format, featuring three variable fragment (Fv) domains for tumor targeting and cargo conjugation.
• Site-specific and stable conjugation of a tubulin inhibitor to a HER2-targeting TVD-Fab.
• Biochemical characterization and in vitro cytotoxicity assays.
• In vivo pharmacokinetic and biodistribution studies comparing TVD-Fab to a dual variable domain (DVD)-IgG1 format.

Main Results:

• TVD-Fab enables the generation of monovalent ADCs with a drug-to-antibody ratio (DAR) of 2, lacking an Fc domain.
• The HER2-targeting TVD-Fab-drug conjugate demonstrated validated biochemical and in vitro cytotoxicity properties.
• In vivo studies showed a circulatory half-life of 13.3 ± 2.5 h for the TVD-Fab carrier.
• TVD-Fab exhibited deeper tumor tissue distribution compared to the DVD-IgG1 format.

Conclusions:

• The TVD-Fab format is a promising new platform for creating site-specific ADCs.
• TVD-Fab demonstrates superior tumor penetration and distribution, making it a potential candidate for targeting solid malignancies.