Identification and Characterization of Neutralizing Antibodies

The unmutated ancestor sequences (WEN-1^UA, WEN-3^UA) of the rCl13-neutralizing monoclonal antibodies WEN-1 and WEN-3 (Figure S1; [Eschli et al., 2007 (link), Seiler et al., 1998 (link)]) were identified by IgBlast (IgBlast) sequence analysis. All mutations deviating from the closest germline V(D)J heavy (HC) and light chain (LC) sequences were reverted. The corresponding cDNAs were synthesized (Genscript) and introduced into HC and LC expression cassettes for recombinant expression in a mouse IgG1 format (provided by Dr. Shozo Izui, University of Geneva). The antibodies were produced by transient co-transfection of the HC and LC expression plasmids in CHO cells (Protein Expression Core Facility, PECF, of the Swiss Federal Technical Highschool, EPFL, Lausanne, Switzerland). The antibodies were purified on an ÄKTAprime plus purification system using Protein G columns (GE healthcare). After 24 hours of PBS dialysis, the purified antibodies were quantified by IgG ELISA. MOPC-21 (BioXcell) served as isotype control antibody. The KL25-W104L, KL25-F106L and KL25-Y108S antibodies as well as a matching KL25 wild-type control antibody (Bruns et al., 1983 (link)) were obtained by analogous procedures, except that a mouse IgG2a expression format was used for these experiments.