Engineering High Affinity Superantigens by Phage Display Original Research Article

Protein L (PpL) is a B-cell superantigen from Peptostreptococcus magnus known to bind to mammalian Vκ light chains. PpL from P. magnus strain 312 comprises five homologous immunoglobulin (Ig) binding domains. We first analysed the binding of the individual domains (B1–B5) of PpL312 to human Vκ light chains (huVκ) subtypes 1 (huVκI) and 3 (huVκIII). Using a combination of rational design and phage selection we isolated mutants of the N-terminal B1 domain with a 14-fold increased affinity for huVκ1 (B1κ1) and >tenfold increased affinity for huVκIII (B1κ3). We investigated the potential of the selected domains, in particular the B1κ1 domain, as reagents in immunochemistry and immunotherapy. B1κ1 proved a superior reagent than the wild-type domain, allowing up to tenfold more sensitive detection of human Vκ antibody fragments in ELISA. A fusion protein of B1κ1 with a human Vλ antibody scFv fragment promoted the efficient recruitment of antibody encoded effector functions including complement, mononuclear phagocyte respiratory burst and phagocytosis through retargeting of IgGκ and IgMκ. Our results suggest that superantigens with improved affinity and/or specificity are easily accessible through protein engineering. Such engineered superantigens should prove useful as reagents in immunochemistry and may have potential as agents in immunotherapy.