Antibodies are high value therapeutic, diagnostic, biotechnological and research tools. Combinatorial approaches to antibody discovery have facilitated access to unique antibodies by surpassing the diversity limitations of the natural repertoire, exploitation of immune repertoires from multiple species and tailoring selections to isolate antibodies with desirable biophysical attributes. Antibodies from these alternative immune hosts provide insights into novel mechanisms of antibody diversity, particularly in sequence restricted germ-line repertoires. Previously, we reported the high-resolution crystal structures of two chicken single chain antibodies (scFv) with prototypical binding-sites (Conroy et al. 2014). These structures revealed unique canonical classes in the CDRL1, which were in the presence and absence of a non-canonical disulphide-constrained heavy chain complementarity determining region (CDRH3). Structures of such disulphide-containing CDRH3 across species are rare, as cysteine residues are observed at low frequency in mature B-cells, however, in chicken >50% of the chicken repertoire contains non-canonical CDRH3 cysteine residues that play an important role in functional diversity (Wu et al. 2012).

Here we present the crystal structure of the disulphide-constrained antibody (scFv B8) in complex with its cognate antigen, prostate specific antigen (PSA). PSA is an androgen-regulated serine protease of the kallikrein family and the gold standard prostate cancer biomarker (Gilgunn et al. 2013). In this crystal structure (1.9Å) we have captured the antibody interaction with free PSA (fPSA) in both active and inactive forms. These structures, in combination with analysis of key mutants, allow a comprehensive evaluation of the contribution of these cysteine residues to avian structural and binding diversity. These structures cast light on the unique structural features of chicken antibodies and contribute further to our collective understanding of the mechanisms of diversity and biochemical attributes that render the chicken repertoire of particular value for antibody generation. Accruing knowledge of antibody structure, function, repertoire and origin is crucial for a number of important applications including man-made, knowledge-based, repertoire synthesis and antibody-based drug discovery. These structures give further structural endorsement to the active mechanisms of diversification employed by the chicken repertoire.