Characterization of neutralizing affinity‐matured human respiratory syncytial virus F binding antibodies in the sub‐picomolar affinity range

Abstract

In the human adaptation and optimization of a mouse anti-human respiratory syncytial virus neutralizing antibody, affinity assessment was crucial to distinguish among potential candidates and to evaluate whether this correlated with function in vitro and in vivo. This affinity assessment was complicated by the trimeric nature of the antigen target, respiratory syncytial virus F (RSV-F) glycoprotein. In the initial affinity screen, surface plasmon resonance was used to determine the intrinsic binding affinities of anti-RSV-F Fab and immunoglobulin G (IgG) to the extracellular domain of RSV-F. This assessment required minimal biotinylation of the RSV-F protein and design of a capture strategy to minimize avidity effects. Approximately 30 Fabs were selected from three optimization phage display libraries on the basis of an initial ELISA screen. Surface plasmon resonance analysis demonstrated the success of optimization with some candidates from the screened libraries having low picomolar dissociation constants, more than 700-fold tighter than the parental monoclonal antibody (B21M). The affinities of these antibodies were further evaluated by a kinetic exclusion assay, a solution binding technology. One IgG (monoclonal antibody 029) displayed a low picomolar K(D) comparable with that of motavizumab, an RSV antibody in clinical study. Kinetic exclusion assay showed that two other of the matured IgGs (011 and 019) had sub-picomolar dissociation constants that could not be resolved further. We discuss the relevance of these interaction analysis results in the light of recently published data on the mechanism of F-driven viral fusion during paramyxoviral infection and 101F epitope conservation revealed from the recent crystal structure of RSV-F in the post-fusion state.

Kinetic mechanism of controlled Fab-arm exchange for the formation of bispecific immunoglobulin G1 antibodies

“…Chemicals were purchased from Sigma. Abs against RSV (clone B21M, containing F405L) and HIV envelope glycoprotein gp120 (clone b12, containing K409R) in human IgG1 and human IgG1 lacking hinge disulfides (C226S/C229S) were expressed transiently in Expi293F cells (Thermo Fisher, A14527) according to the manufacturer's protocol ( 46 , 47 ). Purification was performed using a HiTrap MabSelect SuRe column (GE Healthcare Life Sciences, 11–0034-94) according to the manufacturer's protocol, followed by immediate buffer exchange over a desalting column (GE Healthcare Life Sciences, 17–5087-01) into fresh phosphate-buffered saline (2.67 m m KCl, 1.47 m m KH 2 PO 4 , 138 m m NaCl, 8.06 m m Na 2 HPO 4 , pH 7.2).…”

Current Animal Models for Understanding the Pathology Caused by the Respiratory Syncytial Virus

“…Therefore, there is an urgent need for the development of a hRSV vaccine. In addition, the efficacy of the single licensed therapeutic option remains controversial, raising interest in the development of alternative therapeutic approaches against this pathogen (Canziani et al., 2012; Ispas et al., 2015; Muñoz-Durango et al., 2018; Simon et al., 2018). Therefore, the implementation of functional animal models for studying this virus has emerged as a critical and indispensable aspect underlying the development of immunotherapies and vaccines against hRSV (Hurwitz, 2011).…”

High throughput solution-based measurement of antibody-antigen affinity and epitope binning

“…The sensitivity of the KinExA instrument allows for accurate and reproducible measurements. Affinities in the several hundred femtomolar range and on-rates as high as 10 7 s −1 M −1 have been measured, 14 , 15 but one of the main limitations is that only a handful of K D measurements can be made per day without accounting for time for samples to come to equilibrium. To overcome this speed limitation, a magnetic beads-based method compatible with high throughput solution phase equilibrium K D measurement was first report by Naenel et al 26 A newer technique employing the kinetic exclusion principle on Gyrolab discs allows up to 20 affinity measurements to be made in duplicate within 4 h 16 …”