Strategies for enhancing the therapeutic targeting of phosphatidylserine in oncological disorders and viral infections

Phosphatidylserine (PS) is exposed on a variety of tumour cell types, virus-infected cells and virions. Bavituximab, a PS-targeting antibody with a human immunoglobulin G (IgG) constant region, possesses anti-viral and anti-cancer activity that is mainly mediated by antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC of therapeutic antibodies is naturally limited in vivo by the prevailing serum concentrations of IgG which compete for Fcγ receptor (FcγR) binding. The binding of bavituximab to FcγRIIIA is shown here to be reduced in the presence of either whole serum or polyclonal IgG. However, the use of IdeS, a highly specific IgG-cleaving enzyme derived from Streptococcus pyogenes, presents a potential solution to this problem. IdeS cleaves in the hinge region of IgG between amino acid residues Gly236 and Gly237 but is thought to possess an additional binding interface with its IgG substrate. Firstly, the location of the IdeS–IgG binding interfaces was investigated using truncated IgG1 variants and systematic amino acid mutagenesis. IdeS bound an IgG1 fragment crystallisable (Fc) domain lacking the hinge region, thereby confirming the presence of a secondary binding site. Secondly, residues involved in the IdeS–IgG interaction were mutated to create an IdeS-resistant form of bavituximab. Pre-treatment of competing IgG with IdeS fully restored binding of IdeS-resistant bavituximab to FcγRIIIA, while pre-treatment of serum produced a partial improvement in binding. Lastly, bavituximab, the PS receptor TIM-1, and the related protein TIM-4 were tested for binding to a model alphavirus, Semliki Forest Virus. Only TIM-1, the smallest of these constructs, was found to bind the virus; this implies that receptor size may be an important consideration in the design of PS-targeting anti-viral agents. Overall, these data suggest possible methods for improving the therapeutic targeting of PS, either by optimisation of antigen binding regions or by enhancement of FcγR binding.This thesis is not currently available via ORA