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

Dobeen Hwang,Christoph Rader

doi:10.3390/biom10050764

Dobeen Hwang, Christoph Rader

Open Access

https://doi.org/10.3390/biom10050764

Copy DOI

Journal: Biomolecules	Publication Date: May 14, 2020
Citations: 13	License type: CC BY 4.0

Affiliation: Scripps Research Institute

Abstract

The interest in replacing the conventional immunoglobulin G (IgG) format of monoclonal antibodies (mAbs) and antibody–drug conjugates (ADCs) with alternative antibody and antibody-like scaffolds reflects a need to expand their therapeutic utility and potency while retaining their exquisite specificity, affinity, and low intrinsic toxicity. For example, in the therapy of solid malignancies, the limited tumor tissue penetration and distribution of ADCs in IgG format mitigates a uniform distribution of the cytotoxic payload. Here, we report triple variable domain Fab (TVD–Fab) as a new format that affords the site-specific and stable generation of monovalent ADCs without the Fc domain and a drug-to-antibody ratio (DAR) of 2. TVD–Fabs harbor three variable fragment (Fv) domains: one for tumor targeting and two for the fast, efficient, precise, and stable conjugation of two cargos via uniquely reactive lysine residues. The biochemical and in vitro cytotoxicity properties of a HER2-targeting TVD–Fab before and after conjugation to a tubulin inhibitor were validated. In vivo, the TVD–Fab antibody carrier revealed a circulatory half-life of 13.3 ± 2.5 h and deeper tumor tissue distribution compared to our previously reported dual variable domain (DVD)–IgG1 format. Taken together, the TVD–Fab format merits further investigations as an antibody carrier of site-specific ADCs targeting solid malignancies.

Highlights

Whereas monoclonal antibody therapies as single drug or in combination with systemic chemotherapy have shown limited efficacy in cancer therapy, antibody–drug conjugates (ADCs) are an emerging treatment that maximizes antitumor potency and limits systemic toxicity through the monoclonal antibodies (mAbs)-mediated selective delivery of highly cytotoxic drugs to the tumor [1,2]
The distinctive nucleophilicity of Lys99 enables the hapten-driven selective and covalent conjugation of β-diketone hapten or β-lactam hapten derivatives without labeling other Lys residues [10,11]. Harnessing this unique property of mAb h38C2, we reported a dual variable domain (DVD) IgG1 format [12] composed of an outer variable fragment (Fv) domain targeting tumor cells and an inner Fv domain for site-specific drug conjugation [13]
We show that while ADCs in DVD–IgG1 and triple variable domain Fab (TVD–Fab) format have similar potency and specificity in vitro, the triple variable domain (TVD)–Fab penetrates tumor tissue more efficiently and reveals a prolonged circulatory half-life compared to Fab and DVD–Fab

Summary

Introduction

Whereas monoclonal antibody (mAb) therapies as single drug or in combination with systemic chemotherapy have shown limited efficacy in cancer therapy, antibody–drug conjugates (ADCs) are an emerging treatment that maximizes antitumor potency and limits systemic toxicity through the mAb-mediated selective delivery of highly cytotoxic drugs to the tumor [1,2] Despite their success in the clinic with currently seven Food and Drug Administration (FDA)-approved ADCs for both hematologic and solid malignancies, first-generation ADCs have a suboptimal therapeutic window due to a wide range of drug-to-antibody ratios (DARs; typically 0–8) [3]. A panel of ADCs built on this DVD–IgG1 format, carrying a β-lactam hapten derivative of monomethylauristatin F (MMAF) and targeting HER2, CD79B, and CD138 revealed subnanomolar and strictly target-dependent cytotoxicity in vitro and, in the case of HER2, highly potent and specific in vivo efficacy [5]

Methods

Findings

Discussion

Conclusion