Structural Investigation of Novel Single Domain Synthetic Biotherapeutics Targeting a Receptor Ectodomain

Monoclonal IgGs have been one of the most successful discoveries in biotechnology and are used as reagents in biochemistry, diagnostics and treatment of human disease. Despite their successes, IgG molecules have practical limitations as they are large (~150 kDa) tetrameric structures. Their size and structural complexity renders them problematic to manufacture and expensive to distribute due to the need for cold storage. The limitations of the IgG have stimulated the investigation of other, recently discovered, immune proteins such as the shark VNAR (variable domain of the IgNAR, or Novel Antigen Receptor). IgNARs are a unique class of protein that have been identified in the serum of cartilaginous fish. The VNAR can be isolated as a monomeric binding domain of 12-15 kDa in size, and their smaller size makes them an attractive alternative to IgG as they have the potential to penetrate dense tissues that may be inaccessible to IgG. In addition, VNARs have been identified as possible biotherapeutics based on their robustness and solubility, propensity to bind to antigen clefts and block active sites of enzymes, and high binding affinities for a range of antigens. Pfizer has therefore developed designer synthetic libraries based on the VNAR domain. While technological relatives like the camelid VHH have been well characterized and are in clinical trials for a number of applications, the VNAR is much less well understood structurally and biophysically. The VNAR domain shares structural features with the T-cell receptor Va and the IgG Vk-chain, but sequence homology with these domains is low (~35%). The VNAR contains a relatively short CDR1 loop (CDR = Complementarity Determining Region) and a longer CDR3 loop, which create the main binding surface of the domain. Pfizer has developed large libraries of VNAR containing synthetic diversity in these key binding loops and has used these libraries to derive domains which recognise and antagonise a wide variety of drug targets, including the Receptor for Advanced Glycation End-products (RAGE). RAGE is a multi-ligand member of the immunoglobulin super-family that is implicated in the septic response. This project will investigate synthetic VNARs known to target the RAGE protein and establish a structural understanding of synthetic VNAR CDR loops and their mechanism of antigen binding. As the CDR loops have come from random amino acid diversity, rather than natural D-segment encoded diversity, their structures are likely to be novel and cannot be predicted a priori. Indeed, even for natural IgNARs, the CDRs have shown evidence for induced fit in co-crystal structures. The project will therefore focus on deriving the structures of a number of RAGE-specific synthetic VNARs via solution NMR. The structure of each clone will be derived in both the free and antigen-complexed states. Project plan: 1.Synthetic VNAR targeting RAGE were identified previously via phage display. They therefore express well in bacteria and will be cloned for overexpression in E. coli. 2.The RAGE ectodomain and a number of sequential domain deletions will be expressed and purified for use in binding assays with the purified VNARs from (1). These assays will identify domain-specificity among the VNARs and the subsequent choice of RAGE sub domains to be expressed with isotopic labels (according to established protocols in Dixon lab). 3.The expressed proteins from (1) and (2) will be used to derive solution NMR structures, investigating the structure, dynamics and antigen recognition modality of the CDR loops. 4.A lead VNAR clone will be chosen based on data from (3). This clone will undergo a 6 month process of display-based affinity maturation (placement period for the student, in Pfizer laboratories Dublin). 5.Repeat (1-3) using the affinity matured synthetic VNARs that have acquired mutations in the CDR regions, to investigate dynamics of CDR structure and mechanism of antigen recognition.