Preparation and characterization of antibody-drug conjugates acting on HER2-positive cancer cells.

Zu-Chian Chiang,Nai-Shu Hsu,Hong-Sen Chen,Cheng-Chung Lee,Andrew H -J Wang,An-Suei Yang,Tzung-Jie Yang,Yueh-Liang Tsou,Yi-Kai Chiu,Horng-Ru Hsu,Salvatore V Pizzo

doi:10.1371/journal.pone.0239813

Abstract

Two systems of antibody-drug conjugates (ADCs), noncleavable H32-DM1 and cleavable H32-VCMMAE, were developed by using different linkers and drugs attached to the anti-HER2 antibody H32, which is capable of cell internalization. Activated functional groups, including an N-hydroxysuccinimidyl (NHS) ester and a maleimide, were utilized to make the ADCs. Mass spectrometry, hydrophobic interaction chromatography, polyacrylamide gel electrophoresis, and in vitro cell assays were performed to analyze and optimize the ADCs. Several H32-VCMMAE ADCs were established with higher DARs and greater synthetic yields without compromising potency. The anticancer efficacy of H32-DM1 was 2- to 8-fold greater than that of Kadcyla®. The efficacy of H32-VCMMAE was in turn better than that of H32-DM1. The anticancer efficacy of these ADCs against N87, SK-BR-3 and BT474 cells was in the following order: H32-VCMMAE series > H32-DM1 series > Kadcyla®. The optimal DAR for H32-VCMMAE was found to be 6.6, with desirable attributes including good cell penetration, a releasable payload in cancer cells, and high potency. Our results demonstrated the potential of H32-VCMMAE as a good ADC candidate.

Highlights

According to the World Health Organization, cancer was the second leading cause of death globally in 2018 [1, 2]
Sodium phosphate, sodium chloride, tris(2-carboxyethyl) phosphine (TCEP), N-acetylcysteine (NAC), isopropyl alcohol (IPA), dimethyl sulfoxide (DMSO), 2-mercaptoethanol (2-ME), ethylenediaminetetraacetic acid (EDTA), phosphate-buffered saline (PBS), and TWEEN1 20 were purchased from Sigma-Aldrich
We focused on the Human epidermal growth factor receptor 2 (HER2) antigen target and investigated the two Antibody-drug conjugates (ADCs) systems, including a noncleavable H32-DM1 ADC and a cleavable H32-valine-citrulline-monomethyl auristatin E (VCMMAE) ADC, to compare different effects, such as recovery, drug-to-antibody ratio (DAR) and cytotoxicity

Summary

Introduction

According to the World Health Organization, cancer was the second leading cause of death globally in 2018 [1, 2]. Cancer therapy is important for the improvement of health. Targeted therapy is a new trend in cancer treatment [3]. Antibody-drug conjugates (ADCs) are potentially both highly specific and effective for targeted cancer therapy [4, 5]. The antibody portion of ADCs is preferably able to enter cancer cells, and the conjugated drug can be released to kill the cancer cells. T-DM1 is the only commercial ADC among HER2targeted drugs, and is a noncleavable conjugated drug system. A cleavable HER2-targeting ADC system has not yet been developed for clinical use

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Results

Conclusion