Structure and Function Analysis of an Antibody Recognizing All Influenza A Subtypes

Kallewaard, Nicole L.; Corti, Davide; Collins, Patrick J.; Neu, Ursula; McAuliffe, Josephine M.; Benjamin, Ebony; Wachter-Rosati, Leslie; Palmer-Hill, Frances J.; Yuan, Andy Q.; Walker, Philip A.; Vorlaender, Matthias K.; Bianchi, Siro; Guarino, Barbara; De Marco, Anna; Vanzetta, Fabrizia; Agatic, Gloria; Foglierini, Mathilde; Pinna, Debora; Fernandez-Rodriguez, Blanca; Fruehwirth, Alexander; Silacci, Chiara; Ogrodowicz, Roksana W.; Martin, Stephen R.; Sallusto, Federica; Suzich, JoAnn A.; Lanzavecchia, Antonio; Zhu, Qing; Gamblin, Steven J.; Skehel, John J.

Purified H1 and H3 HA proteins binding affinity to MEDI8852 Fab was measured using a ProteOn 3000 instrument. Briefly, anti-His-tag monoclonal antibody was amine-coupled to a GLC sensor chip with final anti-His-tag capture surface densities of ~2000 resonance units (RUs). Kinetic measurements were preformed by injecting a 100 nM solution of each HA protein, and then bound with three-fold serial dilutions of MEDI8852 Fab. Binding data for all concentrations of each HA protein, plus buffer alone, were collected, and dissociation data were collected for 30 min. Binding data was globally fit to a 1:1 binding model (ProteOn Manager software) that included a term to correct for mass transport-limited binding, should it be detected. This analysis determined the kinetic rate constants (k on , k off ), from which the apparent K D was then calculated as k off /k on . Purified H5 and H7 binding to immobilized MEDI8852 was measured on an Octet RED biolayer interferometer (Pall ForteBio Corp., Menlo Park, CA, USA) . Biotinylated anti-IgG-CH1 conjugate (Thermo Fisher Scientific) was first immobilized on streptavidin biosensors (Pall ForteBio Corp., Menlo Park, CA, USA) at a concentration of approximately 1 µg/ml in 10 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM EDTA and 0.005% Tween-20. MEDI8852 Fabs were then bound to the anti-IgG-CH1 conjugate and the binding of HA (at 0.2 -200 nM) to the immobilized Fabs was measured at 25 o C in a 400 second association step. isotype control mAb, R347 resulted in 13% survival for WSN/33 H1 and 0% survival for rHK/68 H3 infection b Log viral titer reduction was calculated by comparing to R347 treated animals with lung viral titers of 9.51 log 10 TCID 50 /g for WSN/33 H1 and 7.99 log 10 TCID 50 /g for rHK/68 H3 *p<0.05 using the Log Rank Mantel-Cox test for survival, and students t-test for lung viral titers of MEDI8852 treated animals verses R347 control treated animals