Selection and Affinity Maturation of IgNAR Variable Domains Targeting Plasmodium falciparum AMA1

The new antigen receptor (IgNAR) is an antibody unique to sharks and consists of a disulphide-bonded dimer of two protein chains, each containing a single variable and five constant do- mains. The individual variable (VNAR) domains bind antigen independently, and are candidates for the smallest antibody-based immune recognition units. We have previously produced a library of VNAR domains with extensive variability in the CDR1 and CDR3 loops displayed on the surface of bacterio- phage. Now, to test the efficacy of this library, and further explore the dynamics of VNAR antigen bind- ing we have performed selection experiments against an infectious disease target, the malarial Apical Membrane Antigen-1 (AMA1) from Plasmo- dium falciparum. Two related VNAR clones were selected, characterized by long (16- and 18-residue) CDR3 loops. These recombinant VNARs could be harvested at yields approaching 5mg/L of mono- meric protein from the E. coli periplasm, and bound AMA1 with nanomolar affinities (KD 2 10 7 M). One clone, designated 12Y-2, was affinity-matured by error prone PCR, resulting in several variants with mutations mapping to the CDR1 and CDR3 loops. The best of these variants showed 10-fold enhanced affinity over 12Y-2 and was Plasmodium falciparum strain-specific. Importantly, we demon- strated that this monovalent VNAR co-localized with rabbit anti-AMA1 antisera on the surface of malarial parasites and thus may have utility in diagnostic applications. Proteins 2004;55:187-197.