Thermodynamic Analysis of the Conformational Stability of a Single-Domain Therapeutic Antibody

Eliseev, Igor E.; Yudenko, Anna; Besedina, Nadezhda A.; Улитин, А. Б.; Ekimova, Viktoria M.; Evdokimov, Stanislav; Putintceva, J. V.; Yakovlev, Pavel; Lomovskaya, Maria I.; Terterov, Ivan; Bøgdanov, A.; Dubina, Michael

doi:10.1134/s1063785017120045

Cited by 4 publications

(3 citation statements)

References 8 publications

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“…The phage display selection of antibodies from immunized llamas and subsequent sequencing allowed the identification of several anti-ErbB3 single-chain antibodies. As a part of our ongoing effort to elucidate the molecular mechanism of ErbB3 inhibition and ultimately open up a possibility of therapeutic application of these antibodies, we study their thermodynamic stability 10 , functional properties, and structure. In this work, 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.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2018

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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.

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Section: Introductionmentioning

confidence: 99%

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

et al. 2018

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“…We therefore analyzed their folding thermodynamics by studying urea denaturation curves that were obtained by spectroscopy of tryptophan fluorescence. As we experienced earlier [ 44 ], the intrinsic fluorescence of single-domain antibodies is sensitive to the microenvironment inside the protein’s hydrophobic core, and so appears a perfect reporter in folding and denaturation studies. The data for all the antibodies, shown in Figure 2 c, conformed to the standard two-state model and demonstrated their excellent resistance to denaturation.…”

Section: Resultsmentioning

confidence: 97%

Targeting ErbB3 Receptor in Cancer with Inhibitory Antibodies from Llama

et al. 2021

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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.

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“…The phage display selection of antibodies from immunized llamas and subsequent sequencing allowed the identification of several anti-ErbB3 single-chain antibodies. As a part of our ongoing effort to elucidate the molecular mechanism of ErbB3 inhibition and ultimately open up a possibility of therapeutic application of these antibodies, we study their thermodynamic stability 10 , functional properties, and structure. In this work, we describe the expression, purification, crystallization and structural analysis of the variable fragment of an antibody BCD090-M2 (RRID:AB_2721074), which demonstrated an affinity to the extracellular domain of ErbB3 in preliminary experiments.…”

mentioning

confidence: 99%

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

et al. 2018

View full text Add to dashboard Cite

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.

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Thermodynamic Analysis of the Conformational Stability of a Single-Domain Therapeutic Antibody

Cited by 4 publications

References 8 publications

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

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

Targeting ErbB3 Receptor in Cancer with Inhibitory Antibodies from Llama

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

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