Crystal structures of a llama VHH antibody BCD090-M2 targeting human ErbB3 receptor

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 6EZW and 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.

Cadmium SAD phasing at CuKα wavelength

“…As an experimental model for in-house cadmium SAD, we used a crystal of an anti-ErbB3 single-domain antibody BCD090-M2, which we recently studied 9 . The details of protein purification, characterization, and structural analysis are given in the paper 9 .…”

“…As an experimental model for in-house cadmium SAD, we used a crystal of an anti-ErbB3 single-domain antibody BCD090-M2, which we recently studied 9 . The details of protein purification, characterization, and structural analysis are given in the paper 9 . Briefly, the protein was expressed in E. coli SHuffle cells as a SUMO fusion, purified by immobilized metal affinity chromatography, cleaved by TEV protease, and then polished by an additional step of high-resolution cation-exchange chromatography.…”

Improved production of Humira antibody in the genetically engineered Escherichia coli SHuffle, by co-expression of human PDI-GPx7 fusions

“…Redox engineered E. coli SHuffle cells have been previously demonstrated to be an attractive platform for the expression of various antibody formats, such as full-length IgG (Reddy et al 2018 ; Robinson et al 2015 ), Fab’ fragments (Abe et al 2014 ; Mori et al 2018 ; Yusakul et al 2018 ), scFv chains (Ahmadzadeh et al 2020 ; Liu et al 2019 ; Vermeulen et al 2018 ) and VHH domains (Eliseev et al 2018 ; Ta et al 2015 ; Zarschler et al 2013 ). Although SHuffle cells lack the eukaryotic glycosylation machinery, by engineering mutations in the Fc region of antibodies, the requirement for glycosylation for efficient binding of the IgG to its cognate receptor was bypassed (Robinson et al 2015 ).…”

Integrated purification of a nanobody using ammonium sulfate precipitation and Capto MMC

“…Despite the widespread use and high yield of nanobodies, efficient tag‐free purification techniques have not been investigated in detail. At present, researchers mostly use genetic engineering to fuse specific fusion tags, such as hexa‐histidine, maltose binding protein, to the N‐ or C‐terminus of nanobodies, and then purify them through affinity chromatography. Generally speaking, the products purified by one‐step affinity chromatography (e.g.…”

“…Therefore, enzymatic removal of a tag is necessary in many cases. This will inevitably lead to the introduction of new purification units, increased production costs, and uncertain effects on the activity of nanobodies . In order to overcome the limitations caused by the introduction of affinity tags, researchers have made many useful attempts to purify nanobodies by non‐affinity purification method.…”