First molecular and biochemical analysis ofin vivoaffinity maturation in an ectothermic vertebrate

Abstract

The cartilaginous fish are the oldest phylogenetic group in which Igs have been found. Sharks produce a unique Ig isotype, IgNAR, a heavy-chain homodimer that does not associate with light chains. Instead, the variable (V) regions of IgNAR bind antigen as soluble single domains. Our group has shown that IgNAR plays an integral part in the humoral response of nurse sharks ( Ginglymostoma cirratum ) upon antigen challenge. Here, we generated phage-displayed libraries of IgNAR V regions from an immunized animal and found a family of clones derived from the same rearrangement event but differentially mutated during expansion. Because of the cluster organization of shark Ig genes and the paucicopy nature of IgNAR, we were able to construct the putative ancestor of this family. By studying mutations in the context of clone affinities, we found evidence that affinity maturation occurs for this isotype. Subsequently, we were able to identify mutations important in the affinity improvement of this family. Because the family clones were all obtained after immunization, they provide insight into the in vivo maturation mechanisms, in general, and for single-domain antibody fragments.

Improving the pharmacokinetic properties of biologics by fusion to an anti-HSA shark VNAR domain

“…IgNARs were first identified and characterized by Martin Flajnik and colleagues who functionally assigned these novel antigen receptors as a component of the adaptive immune system in nurse shark. 11,[42][43][44] We reasoned that, although these species are evolutionary separated by approximately 200 million years, the presence of IgNAR in spiny dogfish could mean it arose from a common ancestor and may function in a similar way. 45 Through immunization, we succeeded in isolating clones with picomolar affinity to target, which is the highest affinity reported in the absence of in vitro maturation, to our knowledge, for an IgNAR single domain.…”

“…In an extensive immunization campaign with HEL in nurse shark, an ancestral family clone isolated bound with nanomolar affinity to antigen only one order of magnitude less than the isolated affinity matured clones. 44 It was surmised that although evidence of in vivo maturation exists, in the absence of a defined germinal center the extensive primary repertoire may compensate. Whether this is true in this model is debatable at this juncture and would demand a more detailed program of immunization and thorough characterization of clones to elucidate fully.…”

Structural Phylogenetic Analysis of Activation-Induced Deaminase Function

“…SHM is known to occur already in cartilaginous fish, both in Ig and in nurse shark Ag receptor genes, albeit with variable patterns and frequencies, and is likely present in all jawed vertebrates (25)(26)(27)(28). Class switching has been found so far only in land vertebrates (tetrapods), starting with amphibians (23,24,29).…”

Atypical Antigen Recognition Mode of a Shark Immunoglobulin New Antigen Receptor (IgNAR) Variable Domain Characterized by Humanization and Structural Analysis

“…Instead, a distinct "belt" is formed around the middle of the ␤-sandwich structure (10,15). This region shows an elevated rate of somatic mutations and has thus been termed hypervariable region 2 (HV2) (16). Another region of increased mutation frequency is located between HV2 and CDR3, comprising a loop that links ␤-strands D and E similar to that in T-cell receptor V chains; thus, this region was termed HV4.…”