Human Framework Adaptation of a Mouse Anti-Human IL-13 Antibody |
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Authors: | Johan Fransson Alexey Teplyakov Gopalan Raghunathan Ellen Chi Thai Dinh Jill Giles-Komar Bridget Lollo Thomas J Malia Galina Obmolova Yonghong Zhao Juan C Almagro |
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Institution: | 1 Centocor R&D, Inc., 3210 Merryfield Row, San Diego, CA 92121, USA 2 Centocor R&D, Inc., 145 King of Prussia Road, Radnor, PA 19087, USA |
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Abstract: | Humanization of a potent neutralizing mouse anti-human IL-13 antibody (m836) using a method called human framework adaptation (HFA) is reported. HFA consists of two steps: human framework selection (HFS) and specificity-determining residue optimization (SDRO). The HFS step involved generation of a library of m836 antigen binding sites combined with diverse human germline framework regions (FRs), which were selected based on structural and sequence similarities between mouse variable domains and a repertoire of human antibody germline genes. SDRO consisted of diversifying specificity-determining residues and selecting variants with improved affinity using phage display. HFS of m836 resulted in a 5-fold loss of affinity, whereas SDRO increased the affinity up to 100-fold compared to the HFS antibody. Crystal structures of Fabs in complex with IL-13 were obtained for m836, the HFS variant chosen for SDRO, and one of the highest-affinity SDRO variants. Analysis of the structures revealed that major conformational changes in FR-H1 and FR-H3 occurred after FR replacement, but none of them had an evident direct impact on residues in contact with IL-13. Instead, subtle changes affected the VL/VH (variable-light domain/variable-heavy domain) interface and were likely responsible for the 5-fold decreased affinity. After SDRO, increased affinity resulted mainly from rearrangements in hydrogen-bonding pattern at the antibody/antigen interface. Comparison with m836 putative germline genes suggested interesting analogies between natural affinity maturation and the engineering process that led to the potent HFA anti-human IL-13 antibody. |
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Keywords: | HFA human framework adaptation HFS human framework selection SDRO specificity-determining residue optimization FR framework region HACA human anti-chimeric antibody CDR complementarity-determining region wt wild type PDB Protein Data Bank b-IL-13R111Q biotinylated IL-13R111Q IL-13Rα2 IL-13 receptor α2 PBS phosphate-buffered saline mAb monoclonal antibody PEG polyethylene glycol |
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