Characterization of anti-mouse FcγRII single-chain Fv fragments derived from human phage display libraries

Abstract

Background: Few antibodies are available to study the function of the FcγRII murine immunoglobulin receptor. Human phage display libraries represent a potential source of single-chain Fv (sFv) to facilitate the study of the FcγRII murine immunoglobulin receptor. Objectives: To isolate human sFv specific for mouse FcγRII. Study design: Two human phage display libraries were selected for reactivity to mouse FcγRII. Those human anti-mouse FcγRII sFv that were derived from the libraries were characterized with respect to kinetics, cellular binding, epitope specificity and amino acid sequence. Results: Nine anti-mouse FcγRII sFv molecules were isolated from two human phage display libraries. Surface plasmon resonance (SPR) analysis revealed that the human anti-mouse FcγRII sFv had off-rates ranging from 10-2 to 10-3 s-1, with K(D) values calculated to range between 10-7 and 10-9 M. The binding of the FITC-labeled human anti-mouse FcγRII sFv to mouse peritoneal neutrophils was not detected by flow cytometry, due to the rapid off-rates of these monomeric proteins. However, when the human anti-mouse FcγRII sFv were coated on yellow-green latex particles, all of the human sFv were found to specifically bind to mouse peritoneal neutrophils. Deglycosylation of mouse FcγRII did not diminish the binding of these sFv, suggesting that the sFv molecules recognize a polypeptide epitope on murine FcγRII. In contrast, denaturation of mouse FcγRII dramatically reduced the binding of the human sFv, suggesting that the epitopes are conformational. Sequence analysis of the human anti-mouse FcγRII sFv revealed a high degree of structural similarity among the nine sFv. The DP73 V(H) gene segment was utilized by four of the nine sFv, while seven of the nine sFv contained the DPL16 V(λ) gene segment. The sequence similarities between these sFv suggested that several of the human sFv may recognize a common epitope on mouse FcγRII. Epitope mapping studies demonstrated that eight of the nine human anti-mouse FcγRII sFv recognized overlapping epitopes. All of these human anti-mouse FcγRII sFv competed with the 2.4G2 rat monoclonal anti-mouse FcγRII/III antibody for binding with mouse FcγRII, suggesting that the targeted epitopes reside in or near the Fc binding pecker of mouse FcγRII. Conclusions: The availability of novel sFv recognizing mouse FcγRII will facilitate the study of receptor triggering events. Such sFv may prove useful to engage murine FcγRII for targeted cytotoxicity or immunization strategies.