Dimerisation strategies for shark IgNAR single domain antibody fragments

Dimerisation strategies for shark IgNAR single domain antibody fragments
Received 13 March 2006; revised 7 July 2006; accepted 25 July 2006. Available online 28 August 2006.
David P. Simmonsa, Fiona A. Abregua, Usha V. Krishnanb, David F. Prollc, Victor A. Streltsova, b, Larissa Doughtyb, Meghan K. Hattarkib and Stewart D. Nuttalla, b
Journal of Immunological Methods
Volume 315, Issues 1-2 , 31 August 2006
Crown copyright ? 2006 Published by Elsevier B.V.
aCRC for Diagnostics, 343 Royal Parade, Parkville, Victoria 3052, Australia
bCSIRO Molecular and Health Technologies, 343 Royal Parade, Parkville, Victoria 3052, Australia
cDefence Science and Technology Organisation (DSTO), 506 Lorimar Street, Fishermans Bend, Victoria 3207, Australia
Immunoglobulin new antigen receptors (IgNARs) are unique single domain antibodies found in the serum of sharks. The individual variable (VNAR) domains bind antigen independently and are candidates for the smallest antibody-based immune recognition units (13 kDa). Here, we first isolated and sequenced the cDNA of a mature IgNAR antibody from the spotted wobbegong shark (Orectolobus maculatus) and confirmed the independent nature of the VNAR domains by dynamic light scattering. Second, we asked which of the reported antibody fragment dimerisation strategies could be applied to VNAR domains to produce small bivalent proteins with high functional affinity (avidity). In contrast to single chain Fv (scFv) fragments, separate IgNARs could not be linked into a tandem single chain format, with the resulting proteins exhibited only monovalent binding due solely to interaction of the N-terminal domain with antigen. Similarly, incorporation of C-terminal helix?turn?helix (dhlx) motifs, while resulting in efficiently dimerised protein, resulted in only a modest enhancement of affinity, probably due to an insufficiently long hinge region linking the antibody to the dhlx motif. Finally, generation of mutants containing half-cystine residues at the VNAR C-terminus produced dimeric recombinant proteins exhibiting high functional affinity for the target antigens, but at the cost of 50-fold decreased protein expression levels. This study demonstrates the potential for construction of bivalent or bispecific IgNAR-based binding reagents of relatively small size (26 kDa), equivalent to a monovalent antibody Fv fragment, for formulation into future diagnostic and therapeutic formats.
Keywords: IgNAR; New antigen receptor; dhlx; Single chain Fv; Protein dimerisation
Abbreviations: CDR, complementarity determining region; kDa, kilodalton; IgNAR, Immunoglobulin new antigen receptor; VNAR, variable domain of IgNAR antibody; CNAR, constant domain of IgNAR antibody; dhlx, helix?turn?helix dimerising domain.

Corresponding author. CSIRO Molecular and Health Technologies, 343 Royal Parade, Parkville, Victoria 3052, Australia. Tel.: +61 3 9662 7100; fax: +61 3 9662 7314.
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