Biomedical subjects

John Lund

Publications and source records attributed to John Lund.

3 recordsLinked to original sources

PubMed2005

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.

Amino Acid Sequence↗

PubMed2003

Site-specific glycosylation of an aglycosylated human IgG1-Fc antibody protein generates neoglycoproteins with enhanced function.

A range of well-defined IgG glycoforms was prepared by employing a combination of synthetic carbohydrate chemistry and genetic engineering. The key aspect of this methodology is the coupling of thioaldoses with cysteine-containing proteins to give disulfide-linked neoglycoproteins. This technology was applied to the synthesis of a series of synthetic N-glycan thioaldoses which were coupled to an aglycosylated IgG1-Fc fragment, engineered to have Cys-297 in place of glycan-linked Asn (Deltah-Fc N297C). Analysis of the resulting Fc neoglycoproteins by mass spectrometry and trypsin digestion showed that the saccharides were site-selectively incorporated at Cys-297 to full occupancy without affecting other Fc protein disulfides. The neoglycoproteins were tested for their ability to interact with human FcgammaRI by inhibiting superoxide production by gamma-interferon-stimulated U937 cells. The neoglycoproteins displayed enhanced superoxide inhibition relative to aglycosylated Deltah-Fc N297C, where increased glycan size correlated positively with increased inhibition.

Binding Sites↗

PubMed2002

Interaction sites on human IgG-Fc for FcgammaR: current models.

Recombinant monoclonal antibodies have entered the clinic as effective in vivo therapeutic. A majority of the therapeutics antibodies employed are intact IgG molecules. IgG-antibody/antigen complexes can activate a wide range of biological responses that result in elimination and destruction of immune complexes. Principle ligands for the activation of clearance (inflammatory) mechanisms are the three types of cellular Fc receptors (FcgammaR) expressed on leucocytes. The effector functions activated by FcgammaR in vivo can be 'orchestrated', in part, through choice of the IgG subclass employed, however, there is potential to customize antibody therapeutics for optimal biological efficacy, in a particular clinical setting, i.e. with respect to the specific disease and the patient response. In order to engineer IgG antibodies and customize their abilities to activate FcgammaR it is necessary to elucidate the molecular specificity of their mutual interactions. This mini-review summarizes our current understanding of interactions of FcgammaRI, FcgammaRII and FcgammaRIII with human IgG antibodies. Particular emphasis is given to the influence of IgG-Fc glycosylation.

Binding Sites↗