Abstract

The human ErbB3 receptor confers resistance to the pharmacological inhibition of EGFR and HER2 receptor tyrosine kinases in cancer, which makes it an important therapeutic target. Several anti-ErbB3 monoclonal antibodies that are currently being developed are all classical immunoglobulins. We took a different approach and discovered a group of novel heavy-chain antibodies targeting the extracellular domain of ErbB3 via a phage display of an antibody library from immunized llamas. We first produced three selected single-domain antibodies, named BCD090-P1, BCD090-M2, and BCD090-M456, in E. coli, as SUMO fusions that yielded up to 180 mg of recombinant protein per liter of culture. Then, we studied folding, aggregation, and disulfide bond formation, and showed their ultimate stability with half-denaturation of the strongest candidate, BCD090-P1, occurring in 8 M of urea. In surface plasmon resonance experiments, two most potent antibodies, BCD090-P1 and BCD090-M2, bound the extracellular domain of ErbB3 with 1.6 nM and 15 nM affinities for the monovalent interaction, respectively. The receptor binding was demonstrated by immunofluorescent confocal microscopy on four different ErbB3+ cancer cell lines. We observed that BCD090-P1 and BCD090-M2 bind noncompetitively to two distinct epitopes on the receptor. Both antibodies inhibited the ErbB3-driven proliferation of MCF-7 breast adenocarcinoma cells and HER2-overexpressing SK-BR-3 cells, with the EC50 in the range of 0.1–25 μg/mL. BCD090-M2 directly blocks ligand binding, whereas BCD090-P1 does not compete with the ligand and presumably acts through a distinct allosteric mechanism. We anticipate that these llama antibodies can be used to engineer new biparatopic anti-ErbB3 or bispecific anti-ErbB2/3 antibodies.

Highlights

  • This strategy is still frequently used for both single-domain antibodies [37] and single-chain antibodies derived from classical Fabs [38]

  • After the characterization of the single-domain antibodies, we studied their binding to immobilized ErbB3 ECD by surface plasmon resonance

  • We took a different approach to targeting the ErbB3 receptor in cancer cells and studied new llama heavy-chain antibodies against its extracellular domain

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Summary

Introduction

The four ErbB receptor tyrosine kinases serve as an interface to the complex signaling network with modular architecture, redundancy, and multiple feedback circuits, which regulate the essential cellular processes in the development of the epithelial cells, heart, nervous system, and mammary gland [1]. Aberrant signaling by EGFR (ErbB1) and ErbB2, caused by gene amplification, deletions in the extracellular domain, activating kinase domain mutations, or ligand coexpression, drives the progression of many human cancers, especially in the breast, lungs, and colon [2]. Biomedicines 2021, 9, 1106 ligand binding, which induces extracellular domain rearrangement, leading to receptor dimer stabilization and allosteric activation of the intracellular tyrosine kinase domain. The proliferation of ErbB2-overexpressing breast cancer cells requires

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