Discovery and Characterization of Phage Display-Derived Human Monoclonal Antibodies against RSV F Glycoprotein

<div><p>Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection in infants, the elderly and in immunosuppressed populations. The vast majority of neutralizing antibodies isolated from human subjects target the RSV fusion (F) glycoprotein, making it an attractive target for the development of vaccines and therapeutic antibodies. Currently, Synagis^® (palivizumab) is the only FDA approved antibody drug for the prevention of RSV infection, and there is a great need for more effective vaccines and therapeutics. Phage display is a powerful tool in antibody discovery with the advantage that it does not require samples from immunized subjects. In this study, Morphosys HuCAL GOLD^® phage libraries were used for panning against RSV prefusion and postfusion F proteins. Panels of human monoclonal antibodies (mAbs) against RSV F protein were discovered following phage library panning and characterized. Antibodies binding specifically to prefusion or postfusion F proteins and those binding both conformations were identified. 3B1 is a prototypic postfusion F specific antibody while 2E1 is a prototypic prefusion F specific antibody. 2E1 is a potent broadly neutralizing antibody against both RSV A and B strains. Epitope mapping experiments identified a conformational epitope spanning across three discontinuous sections of the RSV F protein, as well as critical residues for antibody interaction.</p></div

Neutralization of RSV by mAbs 2E1 and 3B1.

<p>Li-Cor based RSV micro-neutralization assay was performed. Neutralization curves against RSV A Long strain (A) and RSV B Washington strain (B) for 2E1, 3B1, and palivizumab are plotted with error bars representing the standard deviation from replicate data. 2E1 bivalent Fab is shown in red, full length IgG version of 2E1 shown in green, 3B1 bivalent Fab shown in purple, full length IgG version of 3B1 shown in orange, and palivizumab shown in blue.</p

Characterization of mAbs 2E1 and 3B1.

<p>(A-B) ELISA analysis of 2E1 IgG (A) and 3B1 IgG (B) binding to RSV pre- (red circle) and postfusion F (blue square) proteins. (C-H) Surface plasmon resonance (SPR) analysis of 2E1 and 3B1 Fabs binding to pre- and postfusion RSV F proteins. RU = Resonance Units. Monovalent Fab antibody fragments were captured on the surface of a Series S Sensor Chip CM5 previously functionalized with Human Fab Binder. Prefusion or postfusion F protein diluted 2-fold serially starting at 100 nM or 200 nM was then injected over captured 2E1 (C) or 3B1 (F), respectively. To determine steady-state affinity, response levels at equilibrium were plotted over concentration of pre- (D) or postfusion F (G) protein. 50 nM of post- (E) or prefusion F (H) was injected to demonstrate the specificity of 2E1 binding to prefusion F and 3B1 to postfusion F. (I-K) SPR based competition analysis of 2E1 and 3B1 against palivizumab (I), D25 (J) and MPE8 (K) in binding to RSV prefusion F protein. RU = Resonance Units. Palivizumab and D25 were amine coupled to the surface of separate flow channels of a CM5 chip. A third flow channel was subjected to amine coupling activation without an antibody and used for reference subtraction. Prefusion F (40 μg/mL) was then injected over all surfaces. After a brief stabilization period, 2E1, 3B1, or running buffer was injected to measure binding to sites not occupied by the capturing antibody (palivizumab or D25). To assess competition to MPE8, the MPE8 antibody was captured (6000 RU, not shown) to flow channel 2 of a Biacore Sensor Chip Protein A. Prefusion F (40 μg/mL) was passed over channels 1 and 2 followed by 2E1 Fab, 3B1 Fab, D25 Fab and buffer to measure binding to sites not occupied by MPE8. (L) Bio-Layer Interferometry (BLI) based competition experiment of 3B1 IgG against site I antibody 131-2a. Palivizumab (blue) is able to bind to postfusion F protein in an Octet sandwich competition assay using 131-2a as the capture antibody, but antibody 3B1 (red) does not bind.</p

Anti-RSV F antibodies identified from Morphosys HuCAL GOLD^® phage display libraries.

<p>Heavy chain C-terminal 6xHis tagged antigen specific bivalent Fabs were purified with Ni-NTA column and then tested in ELISA binding to RSV prefusion and postfusion F proteins. (A) Antibodies preferentially binding to RSV postfusion F protein; (B) Antibodies binding to both RSV postfusion and prefusion F proteins; (C) antibody binding specifically to RSV prefusion F protein. Full-length human IgG1 D25 (prefusion F specific) and palivizumab (binding to both prefusion and postfusion F) were used as control antibodies in the above experiments.</p

2E1 binding epitope visualized on the RSV F prefusion structure.

<p>The residues identified for 2E1 binding through shotgun mutagenesis are labeled and shown as red spheres and peptides identified through hydrogen/deuterium-exchange mass spectrometry are shown in yellow on the RSV F prefusion monomeric (top panel) and trimeric (bottom panel) structures [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156798#pone.0156798.ref011" target="_blank">11</a>].</p

Epitope mapping of 2E1 by hydrogen/deuterium-exchange mass spectrometry.

<p>(A) Heat map plot showing the difference in deuteration levels of the RSV prefusion F protein alone compared to RSV prefusion F protein in the presence of the 2E1 monovalent Fab at five time points (15, 50, 150, 500, and 1500 sec). Slower deuterium exchange indicates regions containing the binding sites. White areas are ‘gaps’ for which there was no sequence coverage, and thus no HDX-MS information was obtained. Dashed greyed-out areas represent sequences of the signal and P27 peptides which are not present in the mature purified protein. (B) Uptake plots of several RSV F peptides spanning the conformational epitope region. Red curves show the deuteration levels of peptides of RSV F protein alone, while blue curves show the peptides of the RSV F / 2E1 complex. Peptides containing the residues of antibody epitope (417–434, 441–448, and 457–467) showed decreased deuteration level upon 2E1 binding. In contrast, peptides with no significant decrease in the deuteration level upon 2E1 exposure represent non-epitope sequences (435–440 and 449–457). The residues identified as critical for binding by shotgun mutagenesis are indicated in red font.</p

Identification of critical residues for mAbs 2E1 and 3B1 binding with shotgun mutagenesis epitope mapping.

<p>A shotgun mutagenesis alanine scanning library was constructed for the RSV F protein. The library contains 368 individual mutations at residues identified as surface exposed on the prefusion and postfusion forms of RSV F proteins. Each well of the mutation array plate contained one mutant with a defined substitution. (A) Reactivity results from a representative assay are shown with four positive (wildtype RSV F) and four negative (mock-transfected) control wells included on the plate. (B) Human HEK293T cells expressing the RSV F mutation library were tested for immunoreactivity with mAb 2E1, measured on an Intellicyt high-throughput flow cytometer. Clones with reactivity of <20% relative to that of wildtype RSV F (horizontal line) yet >70% reactivity for a control mAb (vertical line) were initially identified to be critical for mAb 2E1 binding (red dots), and were verified using algorithms described elsewhere [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156798#pone.0156798.ref025" target="_blank">25</a>]. (C) Mutation of four individual residues reduced 2E1 binding (red bars) but not the binding of D25 and palivizumab (gray bars). Error bars represent range (half of the maximum minus minimum values) of at least two replicate data points. (D) Mutation of three individual residues reduced 3B1 binding (red bars) but not the binding of D25 and palivizumab (gray bars). Error bars represent range of at least two replicate data points.</p