Abstract
The immunoglobulin new antigen receptors (IgNARs) are a class of Ig-like molecules of the shark immune system that exist as heavy chain-only homodimers and bind antigens by their single domain variable regions (V-NARs). Following shark immunization and/or in vitro selection, V-NARs can be generated as soluble, stable, and specific high affinity monomeric binding proteins of ∼12 kDa. We have previously isolated a V-NAR from an immunized spiny dogfish shark, named E06, that binds specifically and with high affinity to human, mouse, and rat serum albumins. Humanization of E06 was carried out by converting over 60% of non-complementarity-determining region residues to those of a human germ line Vκ1 sequence, DPK9. The resulting huE06 molecules have largely retained the specificity and affinity of antigen binding of the parental V-NAR. Crystal structures of the shark E06 and its humanized variant (huE06 v1.1) in complex with human serum albumin (HSA) were determined at 3- and 2.3-Å resolution, respectively. The huE06 v1.1 molecule retained all but one amino acid residues involved in the binding site for HSA. Structural analysis of these V-NARs has revealed an unusual variable domain-antigen interaction. E06 interacts with HSA in an atypical mode that utilizes extensive framework contacts in addition to complementarity-determining regions that has not been seen previously in V-NARs. On the basis of the structure, the roles of various elements of the molecule are described with respect to antigen binding and V-NAR stability. This information broadens the general understanding of antigen recognition and provides a framework for further design and humanization of shark IgNARs.
Highlights
Single domain variable regions of shark antibodies (V-NARs) are promising biotherapeutic candidates
E06 belongs to a structural type IV family of shark V-NARs that is distinct from the better characterized type I (e.g. 5A7; Refs. 15 and 34) and type II domains (e.g. PBLA8, a phage display library clone from hen egg white lysozyme (HEL)-immunized nurse shark; Ref. 16)
The structural basis for this property is a result of additional recombination events in the immunoglobulin new antigen receptors (IgNARs) V-D-J cluster and the introduction of junctional diversity, which results in significant heterogeneity of CDR3 sequences
Summary
Single domain variable regions of shark antibodies (V-NARs) are promising biotherapeutic candidates. An atypical “sideways” binding mode is observed that relies heavily on framework residues to achieve antigen binding in addition to CDR3 This binding mode has been described previously for other single domain antibodies [14] but has not been seen previously in V-NARs. It is assumed that to be useful in therapeutic applications all novel non-human scaffolds, such as V-NARs or camelid VHH single domains, need to be humanized to reduce immunogenicity and/or improve thermodynamic stability, folding, and expression properties. CDRs of a murine antibody of interest are grafted onto an appropriate human germ line framework (selected for sequence similarity, expression properties, or both), and back-mutations are introduced at key positions responsible for particular CDR conformation and antigen binding. Our analysis provides the foundation for further improvement and humanization of shark V-NARs
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