Dataset Information

Development of a high affinity, non-covalent biologic to add functionality to Fabs.

ABSTRACT: Functionalization of monoclonal antibodies (mAbs) requires chemical derivatization and/or genetic manipulation. Inherent in these methods are challenges with protein heterogeneity, stability and solubility. Such perturbations could potentially be avoided by using a high affinity, non-covalent intermediate to bridge the desired functionality to a stable mAb. Recently, we engineered a binding site for a peptide named "meditope" within the Fab of trastuzumab. Proximity of the meditope site to that of protein L suggested an opportunity to enhance the meditope's moderate affinity. Joined by a peptide linker, the meditope-protein L construct has a KD ~ 180 pM - a 7000-fold increase in affinity. The construct is highly specific to the engineered trastuzumab, as demonstrated by flow cytometry. Moreover, the fusion of a bulky GFP to this construct did not affect the association with cell surface antigens. Collectively, these data indicate this specific, high affinity construct can be developed to rapidly add new functionality to mAbs.

SUBMITTER: Avery KN

PROVIDER: S-EPMC4295097 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Development of a high affinity, non-covalent biologic to add functionality to Fabs.

Avery Kendra N KN Zer Cindy C Bzymek Krzysztof P KP Williams John C JC

Scientific reports 20150115

Functionalization of monoclonal antibodies (mAbs) requires chemical derivatization and/or genetic manipulation. Inherent in these methods are challenges with protein heterogeneity, stability and solubility. Such perturbations could potentially be avoided by using a high affinity, non-covalent intermediate to bridge the desired functionality to a stable mAb. Recently, we engineered a binding site for a peptide named "meditope" within the Fab of trastuzumab. Proximity of the meditope site to that ...[more]

PMID: 25588710

Similar Datasets

Project description:A series of mini-antibodies (monovalent and bivalent Fabs) targeting the conserved internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41 has been previously constructed and reported. Crystal structures of two closely related monovalent Fabs, one (Fab 8066) broadly neutralizing across a wide panel of HIV-1 subtype B and C viruses, and the other (Fab 8062) non-neutralizing, representing the extremes of this series, were previously solved as complexes with 5-Helix, a gp41 pre-hairpin intermediate mimetic. Binding of these Fabs to covalently stabilized chimeric trimers of N-peptides of HIV-1 gp41 (named (CCIZN36)3 or 3-H) has now been investigated using X-ray crystallography, cryo-electron microscopy, and a variety of biophysical methods. Crystal structures of the complexes between 3-H and Fab 8066 and Fab 8062 were determined at 2.8 and 3.0 Å resolution, respectively. Although the structures of the complexes with the neutralizing Fab 8066 and its non-neutralizing counterpart Fab 8062 were generally similar, small differences between them could be correlated with the biological properties of these antibodies. The conformations of the corresponding CDRs of each antibody in the complexes with 3-H and 5-Helix are very similar. The adaptation to a different target upon complex formation is predominantly achieved by changes in the structure of the trimer of N-HR helices, as well as by adjustment of the orientation of the Fab molecule relative to the N-HR in the complex, via rigid-body movement. The structural data presented here indicate that binding of three Fabs 8062 with high affinity requires more significant changes in the structure of the N-HR trimer compared to binding of Fab 8066. A comparative analysis of the structures of Fabs complexed to different gp41 intermediate mimetics allows further evaluation of biological relevance for generation of neutralizing antibodies, as well as provides novel structural insights into immunogen design.

Dataset Information

Development of a high affinity, non-covalent biologic to add functionality to Fabs.

Publications

Development of a high affinity, non-covalent biologic to add functionality to Fabs.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets