Functional Assessment and Structural Analysis of an Anti-TfR1 Antibody Fragment that Blocks Internalization of NW Hemorrhagic Fevers Arenaviruses

RODRÍGUEZ J. A.; CASCIO, DUILIO; ZIEGENBEIN, J.; PENICHET M. L.; SAWAYA, MICHAEL R.; DIEP, A.; HELGUERA G.; DANIELS-WELLS T.; ARAGÜES, R.; PAYÉS, C.

Congreso; REUNIÓN CONJUNTA DE SOCIEDADES DE BIOCIENCIAS; 2017

New World Hemorrhagic Fevers (NWHF) are grave diseases becauseof their infectivity and mortality. Their etiological agents arethe arenaviruses Chapare (CHAV), Machupo (MACV), Guanarito(GTOV), Sabiá (SABV) and Junín (JUNV), but only is available avaccine and an effective therapy against JUNV. The entry of thesearenaviruses into human cells occurs via binding of the viral glycoprotein(GP1) to the human transferrin 1 receptor (hTfR1). Previouslywe established that the anti-hTfR1 IgG3 chimeric antibodych128.1 inhibits the internalization of NWHF viruses. However,ch128.1 presents risks of inducing crosslinking of the receptor andinflammatory reaction in healthy tissues. The goal of this work is togenerate a monovalent Fab fragment of ch128.1 without Fc effectorfunction (Fab128.1), to assess whether it can block internalization ofNWHF arenaviruses, to analyze its structure, and to model its interactionwith hTfR1. Monovalent Fab128.1 fragment was obtained byproteolytic digestion of ch128.1 with papain. We determined that itbinds to the extracellular domain of hTfR1 by ELISA and size exclusionchromatography. Flow cytometry functional studies showed thatFab128.1 significantly blocks internalization into HEK-293T cells ofpseudoviruses expressing eGFP decorated with GP1/GP2 of JUNVor MACV. Structural analysis of Fab128.1 was performed in crystalsgenerated by hanging drop. X-ray diffraction was collected from asynchrotron beam and the atomic structure solved by molecular replacementwith 2.69Å resolution. With the Fab128.1 structural datawe modeled the interaction with sTfR1 using computational dockinganalysis to obtain a structural model of the Fab-antigen complex.These studies confirm that monovalent Fab128.1 can block the entryof NWHF arenaviruses and provide insights in the structure of thevariable region that interacts with hTfR1. The results shown here willcontribute to the rational design of a safe and effective biotherapyagainst the NWHF.