Engineering high affinity superantigens by phage display.
Protein L (PpL) is a B-cell superantigen from Peptostreptococcus magnus known to bind to mammalian Vkappa 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 PpL(312) to human Vkappa light chains (huVkappa) subtypes 1 (huVkappaI) and 3 (huVkappaIII). 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 huVkappa1 (B1kappa1) and >tenfold increased affinity for huVkappaIII (B1kappa3). We investigated the potential of the selected domains, in particular the B1kappa1 domain, as reagents in immunochemistry and immunotherapy. B1kappa1 proved a superior reagent than the wild-type domain, allowing up to tenfold more sensitive detection of human Vkappa antibody fragments in ELISA. A fusion protein of B1kappa1 with a human Vlambda antibody scFv fragment promoted the efficient recruitment of antibody encoded effector functions including complement, mononuclear phagocyte respiratory burst and phagocytosis through retargeting of IgGkappa and IgMkappa. 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.
Enever
Carolyn
C
MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK.
Tomlinson
Ian M
IM
Lund
John
J
Levens
Michaela
M
Holliger
Philipp
P
D000595:Amino Acid Sequence; D000915:Antibody Affinity; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D006801:Humans; D007145:Immunoglobulin kappa-Chains / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019151:Peptide Library; D011487:Protein Conformation; D015202:Protein Engineering; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018089:Superantigens / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; C055047:Ig L-binding protein, Peptostreptococcus; D007145:Immunoglobulin kappa-Chains; D019151:Peptide Library; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D018089:Superantigens