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

Abstract

The ability of ErbB3 receptor to functionally complement Background: 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.The VHH fragment of the antibody was expressed in Methods: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.We developed a robust and efficient system for recombinant Results: expression of single-domain antibodies. The purified antibody was functional and bound ErbB3 with K = 1 μM. 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 and . 6EZW 6F0D Our results may facilitate further mechanistic studies of ErbB3 Conclusions: inhibition by single-chain antibodies. Besides, the solved structures will contribute to datasets required to develop new computational methods for antibody modeling and design.

Targeting hemoglobin receptors IsdH and IsdB of Staphylococcus aureus with a single VHH antibody inhibits bacterial growth

“…S4 ) lead to a decrease in the binding affinity but compared with the mutation of Y57, R55, and Y62, the change was not as relevant. In the majority of VHH interactions, the CDR with the higher affinity interactions is usually the third loop ( 20 ); however, in the interaction of VHH6, it seems that CDR2 was contributing the most to the affinity and to epitope recognition ( Table 2 ). MD simulations ( Fig.…”

“…Because of the similar binding characteristics of VHH6 and VHH35, it can be concluded that their differences in the first two residues does not have an effect on the interaction with the antigen. The majority of camelid VHH stablish interactions with the antigen mainly though the CDR3 ( 20 ). However, in the VHH6 obtained in this research, the CDR2 has the most relevance in the antigen recognition.…”

“…VHH (also termed nanobody) is specialized in recognizing hidden and hydrophobic epitopes ( 19 ) such as that of the heme-binding pocket of IsdH and IsdB. In addition, VHH has other advantages over the traditional IgG format, such as a lower cost of production and, because of their smaller size, greater penetration in targets with difficult accessibility ( 14 , 20 ).…”

Exploiting Single Domain Antibodies as Regulatory Parts to Modulate Monoterpenoid Production in E. coli

“…The second input variable included in the design was the coproduction of the CyDisCo catalysts yeast sulfhydryl oxidase, Erv1p, and mature human PDI, which were developed for use as an alternative production strategy to Δ gor ΔtrxB double knockout strains. , The final categorical input variable explored was the addition of N-terminal tags to aid in protein solubility . When considering the small size of V HH (∼15 kDa), and that crystallographic data shows that the N-terminus of the polypeptide chain is adjacent to the CDR loops, , the use of large solubility tags have the potential to interfere with the antibody–antigen interaction. Therefore, a series of short, flexible tags; His 6 (15 amino acids), His 6 -TEV (28 amino acids), and His 6 S-tag (46 amino acids), were investigated for their ability to improve production of functional B2D C10 (see Supplementary Table 1 for N-terminal tag amino acid sequences).…”