Effect of Inter-Domain Linker Composition on Biodistribution of ABD-Fused Affibody-Drug Conjugates Targeting HER2.

Tianqi Xu,Anzhelika Vorobyeva,Maria S Tretyakova,Vladimir Tolmachev,Mikhail V Belousov,Jie Zhang,Anna Orlova,Vitalina Bodenko,Maryam Oroujeni,Torbjörn Gräslund

doi:10.3390/pharmaceutics14030522

Abstract

Targeted drug conjugates based on Affibody molecules fused to an albumin-binding domain (ABD) for half-life extension have demonstrated potent anti-tumor activity in preclinical therapeutic studies. Furthermore, optimization of their molecular design might increase the cytotoxic effect on tumors and minimize systemic toxicity. This study aimed to investigate the influence of length and composition of a linker between the human epidermal growth factor receptor 2 (HER2)-targeted affibody molecule (ZHER2:2891) and the ABD domain on functionality and biodistribution of affibody-drug conjugates containing a microtubulin inhibitor mertansin (mcDM1) (AffiDCs). Two conjugates, having a trimeric (S3G)3 linker or a trimeric (G3S)3 linker were produced, radiolabeled with 99mTc(CO)3, and compared side-by-side in vitro and in vivo with the original ZHER2:2891-G4S-ABD-mcDM1 conjugate having a monomeric G4S linker. Both conjugates with longer linkers had a decreased affinity to HER2 and mouse and human serum albumin in vitro, however, no differences in blood retention were observed in NMRI mice up to 24 h post injection. The use of both (S3G)3 and (G3S)3 linkers reduced liver uptake of AffiDCs by approximately 1.2-fold compared with the use of a G4S linker. This finding provides important insights into the molecular design for the development of targeted drug conjugates with reduced hepatic uptake.

Highlights

The human epidermal growth factor receptor 2 (HER2) belongs to the human epidermal growth factor receptor (HER/EGFR/ERBB) family and is a transmembrane protein with tyrosine kinase activity [1]
Non-toxic control constructs were generated by alkylation of the C-terminal cysteines of ZHER2:2891 -(G3 S)3 -albumin-binding domain (ABD)-Cys and ZHER2:2891 -(S3 G)3 -ABD-Cys with 2-Iodoacetamide (IAA)
Non-toxic control constructs were generated by alkylation of the C-terminal cysteine in ZHER2:2891 -(G3 S)3 -ABD and ZHER2:2891 -(S3 G)3 -ABD

Summary

Introduction

The human epidermal growth factor receptor 2 (HER2) belongs to the human epidermal growth factor receptor (HER/EGFR/ERBB) family and is a transmembrane protein with tyrosine kinase activity [1]. HER2 is overexpressed in several types of cancer with its expression being low in normal adult tissues, which gives an opportunity for HER2targeted therapy. Its overexpression and/or the amplification of its encoding gene, ERBB2, Pharmaceutics 2022, 14, 522. Pharmaceutics 2022, 14, 522 is associated with poor response to chemotherapy and increased recurrence rate in breast cancer [2]. Targeted drug delivery using proteins coupled with cytotoxic agents is a promising way to treat disseminated cancer and has been intensively investigated in recent years. Antibody-drug conjugates (ADCs) based on a monoclonal antibody (mAb) for selective delivery of a drug to malignant tumors is the most common and well-studied format of targeted drugs [3]. Two HER2-targeting ADCs, trastuzumab emtansine (T-DM1) and trastuzumab deruxtecan, have been approved for clinical use by the U.S Food and Drug

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