In the rst example, a strategy was designed to isolate antibodies that acted to ‘staple’ a molecular heterodimer by binding a composite epitope of two‑receptor extra‑ cellular domains (Figure3.4b). Interleukin‑4 (IL‑4) signals through either a type I het‑ erodimeric receptor comprising IL‑4Rα and the common γ‑chain or a type II receptor composed of IL‑4Rα and IL ‑13Rα1. A crystal structure of IL‑4 in a ternary complex with the cytokine receptor extracellular domains showed that the two‑receptor mem‑ brane proximal domains were in contact forming a neoepitope. An engineered variant of IL‑4 was available (Super‑4) with 3,700‑fold higher afnity than IL‑4, which sta‑ bilized the IL‑4 receptor complex. With this information, an antigen preparation was made in which a receptor was tagged with a C‑terminal biotin‑acceptor peptide allow‑ ing biotinylation and expressed receptor subunits were complexed with Super‑4. This biotinylated complex was used with a yeast scFv library to isolate a scFv, which bound specically to the membrane–proximal receptor interface. This binding mode was con‑ rmed by solving a structure of the antibody (reformatted as a Fab) with the ternary complex, which showed the antibody binding to the receptor interface [98].

The RSV F protein is responsible for fusing the viral and host cell membranes during virus cell entry and exists in two antigenically unique forms–a pre‑fusion form and a post‑fusion form, which undergoes a dramatic conformational change. The pre‑fusion form is the target of most neutralizing antibodies [99]. A key issue in targeting the pre‑fusion form of RSV F is that the protein is metastable and can spontaneously change conformation when extracted from membranes with detergent or if subjected to vari‑ ous stresses (e.g. physical, chemical, or temperature stresses). To tackle this issue, sta‑ bilization of the pre‑fusion protein is an attractive strategy. To achieve stabilization, pre‑fusion stabilizing mutations were computationally designed using a combination of methods–introduction of cysteine residue pairs to form stabilizing disulphide bonds; introduction of non‑polar amino acids to ll cavities in the pre‑fusion protein that might otherwise allow for movement; and introduction of charged residue mutations to decrease ionic repulsion or increase ionic interaction between residues that are close in the pre‑fusion state [90]. These residue mutations were computationally assessed using a combination of Schrodinger BioLuminate, Molecular Operating Environment [100], and Rosetta‑based stability prediction protocols [101]. Using computational design, 398 hypothetical F domain constructs with combinations of disulphide, cavity lling, and charge mutations were expressed as ectodomain constructs, including a britin foldon trimerization domain at the C‑terminus. In general, the disulphide mutations had the greater stabilizing effect on the pre‑fusion conformation. Pre‑fusion F constructs that exhibited greater stabilization were tested and elicited a 10‑fold higher serum‑neutral‑ izing titre than a prototype vaccine F protein candidate. Introduction of the stabilizing mutations onto F‑proteins of two major RSV subgroups, followed by immunization led to complete protection against RSV challenge.