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Molecular Dynamics-Based Design and Biophysical Evaluation of Thermostable Single-Chain Fv Antibody Mutants Derived from Pharmaceutical Antibodies.

ABSTRACT: Antibody drugs are denatured under physical stress, e.g., friction, heat, and freezing, which triggers formation of aggregates and resultant allergic reactions. Design of a stable antibody is thus critical for the development of antibody drugs. Here, we obtained a thermostable single-chain Fv (scFv) antibody clone by rigidifying the flexible region. We first conducted a short molecular dynamics (MD) simulation (3 runs of 50 ns) to search for weak spots in the scFv antibody, i.e., flexible regions located outside the CDR (complementarity determining region) and the interface between the heavy-chain and light-chain variable regions. We then designed a thermostable mutant and evaluated it by means of a short MD simulation (3 runs of 50 ns) based on reductions in the root-mean-square fluctuation (RMSF) values and formation of new hydrophilic interactions around the weak spot. Finally, we designed the VL-R66G mutant by applying our strategy to scFv derived from trastuzumab. Trastuzumab scFv variants were prepared by using an Escherichia coli expression system, and the melting temperature-measured as a thermostability index-was 5 °C higher than that of the wild-type trastuzumab scFv, while the antigen-binding affinity was unchanged. Our strategy required few computational resources, and would be applicable to antibody drug discovery.

SUBMITTER: Okazaki K

PROVIDER: S-EPMC10308585 | biostudies-literature | 2023 Jun

REPOSITORIES: biostudies-literature

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Molecular Dynamics-Based Design and Biophysical Evaluation of Thermostable Single-Chain Fv Antibody Mutants Derived from Pharmaceutical Antibodies.

Okazaki Kyo K Kobashigawa Yoshihiro Y Morita Hikari H Yamauchi Soichiro S Fukuda Natsuki N Liu Chenjiang C Toyota Yuya Y Sato Takashi T Morioka Hiroshi H

ACS omega 20230609 25

Antibody drugs are denatured under physical stress, e.g., friction, heat, and freezing, which triggers formation of aggregates and resultant allergic reactions. Design of a stable antibody is thus critical for the development of antibody drugs. Here, we obtained a thermostable single-chain Fv (scFv) antibody clone by rigidifying the flexible region. We first conducted a short molecular dynamics (MD) simulation (3 runs of 50 ns) to search for weak spots in the scFv antibody, i.e., flexible region ...[more]

PMID: 37396255

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Project description:BackgroundMost Yersinia pestis strains are known to express a capsule-like antigen, fraction 1 (F1)(.) F1 is encoded by the caf1 gene located on the large 100-kb pFra plasmid, which is found in Y. pestis but not in closely related species such as Yersinia enterocolytica and Yersinia pseudotuberculosis. In order to find antibodies specifically binding to Y. pestis we screened a large single chain Fv antibody fragment (scFv) phage display library using purified F1 antigen as a selection target. Different forms of the selected antibodies were used to establish assays for recombinant F1 antigen and Y. pestis detection.MethodsPhage antibody panning was performed against F1 in an automated fashion using the Kingfisher magnetic bead system. Selected scFvs were screened for F1-binding specificity by one-step alkaline phosphatase enzyme linked immunosorbant assay (ELISA), and assayed for binding to recombinant antigen and/or Y. pestis by flow cytometry and whole-cell ELISA.ResultsSeven of the eight selected scFvs were shown to specifically bind both recombinant F1 and a panel of F1-positive Yersinia cells. The majority of the soluble scFvs were found to be difficult to purify, unstable and prone to cross-reactivity with F1-negative Yersinia strains, whereas phage displayed scFvs were found to be easy to purify/label and remarkably stable. Furthermore direct fluorescent labeling of phage displaying scFv allowed for an easy one-step flow cytometry assay. Slight cross-reactivity was observed when fixed cells were used in ELISA.ConclusionsOur high throughput methods of selection and screening allowed for time and cost effective discovery of seven scFvs specifically binding Y. pestis F1 antigen. We describe implementation of different methods for phage-based immunoassay. Based on the success of these methods and the proven stability of phage, we indicate that the use of phage-displayed, rather than phage-free proteins, might generally overcome the shortcomings of scFv antibodies.

Dataset Information

Molecular Dynamics-Based Design and Biophysical Evaluation of Thermostable Single-Chain Fv Antibody Mutants Derived from Pharmaceutical Antibodies.

Publications

Molecular Dynamics-Based Design and Biophysical Evaluation of Thermostable Single-Chain Fv Antibody Mutants Derived from Pharmaceutical Antibodies.

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