Stellabody: A novel hexamer-promoting mutation for improved IgG potency.

Advances in antibody engineering are being directed at the development of next generation immunotherapeutics with improved potency. Hexamerisation of IgG is a normal physiological aspect of IgG biology and recently described mutations that facilitate this process have a substantial impact upon monoclonal antibody behavior resulting in the elicitation of dramatically enhanced complement-dependent cytotoxicity, Fc receptor function, and enhanced antigen binding effects, such as targeted receptor agonism or microbe neutralization. Whereas the discovery of IgG hexamerisation enhancing mutations has largely focused on residues with exposure at the surface of the Fc-Fc and CH2-CH3 interfaces, our unique approach is the engineering of the mostly buried residue H429 in the CH3 domain. Selective substitution at position 429 forms the basis of Stellabody technology, where the choice of amino acid results in distinct hexamerisation outcomes. H429F results in monomeric IgG that hexamerises after target binding, so called "on-target" hexamerisation, while the H429Y mutant forms pH-sensitive hexamers in-solution prior to antigen binding. Moreover, Stellabody technologies are broadly applicable across the family of antibody-based biologic therapeutics, including conventional mAbs, bispecific mAbs, and Ig-like biologics such as Fc-fusions, with applications in diverse diseases.

Dimeric FcγR Ectodomains as Probes of the Fc Receptor Function of Anti-Influenza Virus IgG.

Ab-dependent cellular cytotoxicity, phagocytosis, and Ag presentation are key mechanisms of action of Abs arising in vaccine or naturally acquired immunity, as well of therapeutic mAbs. Cells expressing the low-affinity FcγRs (FcγRII or CD32 and FcγRIII or CD16) are activated for these functions when receptors are aggregated following the binding of IgG-opsonized targets. Despite the diversity of the Fc receptor proteins, IgG ligands, and potential responding cell types, the induction of all FcγR-mediated responses by opsonized targets requires the presentation of multiple Fc regions in close proximity to each other. We demonstrated that such "near-neighbor" Fc regions can be detected using defined recombinant soluble (rs) dimeric low-affinity ectodomains (rsFcγR) that have an absolute binding requirement for the simultaneous engagement of two IgG Fc regions. Like cell surface-expressed FcγRs, the binding of dimeric rsFcγR ectodomains to Ab immune complexes was affected by Ab subclass, presentation, opsonization density, Fc fucosylation, or mutation. The activation of an NK cell line and primary NK cells by human IgG-opsonized influenza A hemagglutinin correlated with dimeric rsFcγRIIIa binding activity but not with Ab titer. Furthermore, the dimeric rsFcγR binding assay sensitively detected greater Fc receptor activity to pandemic H1N1 hemagglutinin after the swine influenza pandemic of 2009 in pooled human polyclonal IgG. Thus these dimeric rsFcγR ectodomains are validated, defined probes that should prove valuable in measuring the immune-activating capacity of IgG Abs elicited by infection or vaccination or experimentally derived IgG and its variants.

Molecular basis of improved immunogenicity in DNA vaccination mediated by a mannan based carrier.

Receptor mediated gene delivery is an attractive non-viral method for targeting genetic material to specific cell types. We have previously utilized oxidized (OMPLL) and reduced mannan poly-L-lysine (RMPLL) to target DNA vaccines to antigen presenting cells and demonstrated that it could induce far stronger immune responses in mice compared to naked DNA immunization. In this study, we describe the immune enhancing attributes of mannan-PLL mediated DNA vaccination at the molecular level. Several attributes observed in similar gene delivery conjugates, such as entry via the endocytic pathway, low toxicity, protection from nucleases and compaction of particle size, were also evident here. In addition, OMPLL and RMPLL conjugates had profound effects on the antigen presentation functions of dendritic cells and macrophages, through the stimulation of cytokine production and maturation of dendritic cells. Interestingly, we demonstrate that OMPLL-DNA and RMPLL-DNA are able to mediate dendritic cell activation via toll-like receptor 2 as opposed to mannan alone which mediates via toll-like receptor 4. Overall, this report leads to greater understanding of how oxidized and reduced mannan mediated gene delivery could augment immune responses to DNA vaccination and provide insights into ways of further improving its immunogenicity.