Abstract
Background: The ability of ErbB3 receptor to functionally complement ErbB1-2 and induce tumor resistance to their inhibitors makes it a unique target in cancer therapy by monoclonal antibodies. Here we report the expression, purification and structural analysis of a new anti-ErbB3 single-chain antibody. Methods: The VHH fragment of the antibody was expressed in E. coli SHuffle cells as a SUMO fusion, cleaved by TEV protease and purified to homogeneity. Binding to the extracellular domain of ErbB3 was studied by surface plasmon resonance. For structural studies, the antibody was crystallized by hanging-drop vapor diffusion in two different forms. Results: We developed a robust and efficient system for recombinant expression of single-domain antibodies. The purified antibody was functional and bound ErbB3 with K D =15±1 nM. The crystal structures of the VHH antibody in space groups C2 and P1 were solved by molecular replacement at 1.6 and 1.9 Å resolution. The high-quality electron density maps allowed us to build precise atomic models of the antibody and the putative paratope. Surprisingly, the CDR H2 existed in multiple distant conformations in different crystal forms, while the more complex CDR H3 had a low structural variability. The structures were deposited under PDB entry codes 6EZWand 6F0D. Conclusions: Our results may facilitate further mechanistic studies of ErbB3 inhibition by single-chain antibodies. Besides, the solved structures will contribute to datasets required to develop new computational methods for antibody modeling and design.
Highlights
Receptor tyrosine kinases ErbB1-4 (HER1-4) receive inputs from growth factors and transmit signals to the cell nucleus, regulating key cellular processes such as growth, differentiation, migration, and apoptosis[1]
The examples of successful application of anti-ErbB antibodies in cancer treatment include cetuximab in head and neck cancer therapy and trastuzumab in breast cancer treatment
We describe the expression, purification, crystallization and structural analysis of the variable fragment of an antibody BCD090-M2, which demonstrated an affinity to the extracellular domain of ErbB3 in preliminary experiments
Summary
Receptor tyrosine kinases ErbB1-4 (HER1-4) receive inputs from growth factors and transmit signals to the cell nucleus, regulating key cellular processes such as growth, differentiation, migration, and apoptosis[1]. The essential role of ErbB receptors in tumor development makes them a unique target in cancer therapy by monoclonal antibodies[2]. Its ability to form functional dimers with ErbB1-2 and to confer resistance to their inhibitors makes ErbB3 an important drug target[3]. The ability of ErbB3 receptor to functionally complement ErbB1-2 and induce tumor resistance to their inhibitors makes it a unique target in cancer therapy by monoclonal antibodies. We report the expression, purification and structural analysis of a new anti-ErbB3 single-chain antibody. The antibody was crystallized by hanging-drop vapor diffusion in two different forms. Conclusions: Our results may facilitate further mechanistic studies of ErbB3 inhibition by single-chain antibodies. The solved structures will contribute to datasets required to develop new computational methods for antibody modeling and design
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