Combinatorial libraries against libraries for selecting neoepitope activation-specific antibodies
| Autor: | Sungkwon Kang, Craig T. Lefort, Xuebo Hu, Moonsoo M. Jin, Minsoo Kim |
|---|---|
| Rok vydání: | 2010 |
| Předmět: |
Multidisciplinary
Phage display biology Neutrophils Integrin Macrophage-1 Antigen Saccharomyces cerevisiae Biological Sciences Ligands Directed evolution Molecular biology Primary and secondary antibodies Neutrophil Activation Epitope Epitopes Biochemistry Antibody Specificity Cell Movement Peptide Library Cell Adhesion biology.protein Humans iC3b Cell adhesion Peptide library |
| Zdroj: | Proceedings of the National Academy of Sciences. 107:6252-6257 |
| ISSN: | 1091-6490 0027-8424 |
| DOI: | 10.1073/pnas.0914358107 |
| Popis: | A systematic approach to the discovery of conformation-specific antibodies or those that recognize activation-induced neoepitopes in signaling molecules and enzymes will be a powerful tool in developing antibodies for basic science and therapy. Here, we report the isolation of antibody antagonists that preferentially bind activated integrin Mac-1 (αMβ2) and are potent in blocking neutrophil adhesion and migration. A novel strategy was developed for this task, consisting of yeast surface display of Mac-1 inserted (I) domain library, directed evolution to isolate active mutants of the I domain, and screening of phage display of human antibody library against the active I domain in yeast. Enriched antibody library was then introduced into yeast surface two-hybrid system for final quantitative selection of antibodies from monomeric antigen–antibody interaction. This led to highly efficient isolation of intermediate to high affinity antibodies, which preferentially reacted with the active I domain, antagonized the I domain binding to intercellular adhesion molecule (ICAM)-1, complement C3 fragment iC3b, and fibronectin, and potently inhibited neutrophil migration on fibrinogen. The strategy demonstrated herein can be broadly applicable to developing antibodies against modular domains that switch between inactive and active conformations, particularly toward the discovery of antibody antagonists in therapeutic and diagnostic applications. |
| Databáze: | OpenAIRE |
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