title: 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.
authors.affiliation: Department of Molecular Medicine, Beckman Research Institute at City of Hope 1710 Flower St, Duarte CA 91010.
authors.forename: Kendra N
authors.initials: KN
authors.lastname: Avery
authors.affiliation: Department of Molecular Medicine, Beckman Research Institute at City of Hope 1710 Flower St, Duarte CA 91010.
authors.forename: Cindy
authors.initials: C
authors.lastname: Zer
authors.affiliation: Department of Molecular Medicine, Beckman Research Institute at City of Hope 1710 Flower St, Duarte CA 91010.
authors.forename: Krzysztof P
authors.initials: KP
authors.lastname: Bzymek
authors.affiliation: Department of Molecular Medicine, Beckman Research Institute at City of Hope 1710 Flower St, Duarte CA 91010.
authors.forename: John C
authors.initials: JC
authors.lastname: Williams
mesh_terms: D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D000915:Antibody Affinity / Q000502:physiology*; D001665:Binding Sites; D002460:Cell Line; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D010455:Peptides / Q000276:immunology; D020349:Surface Plasmon Resonance; D000068878:Trastuzumab / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D010455:Peptides; D000068878:Trastuzumab