Engineering Polysialylation of scFvs and its effects on Function

Abstract

Antibody engineering for drug therapy has become a major industry in recent years, with much work focussed on the development of recombinant antibody fragments in therapeutic and diagnostic applications. An important consideration when engineering antibodies, especially rapidly clearing fragments is its pharmacokinetic properties. Several strategies have been adopted to increase blood half-life and bioavailability, which include chemical conjugation and recombinant modulation. The most successful to date, is the chemical conjugation of poly(ethylene glycol) polymers, known as PEGylation. However, due to its non-biodegradable nature, the limitations associated with PEG polymers have prompted the development of natural alternatives. Polysialic acid (PSA) has been shown to be a favourable alternative to PEG with recent studies showing that recombinant polysialylation using human neural cell adhesion molecule (NCAM) domain carriers can significantly improve pharmacokinetics. This study looks at targeting a well-suited application to inhibit or antagonize blood-borne factors, such as Interluekin-6, by focussing on applying the recombinant polysialylation technology to anti-IL-6 single chain variable fragments (scFv) to exemplify the therapeutic benefits of blocking this cytokine expressed in many cancers. Here, a range of novel NCAM fusion proteins are presented which aim to refine the technological approach and demonstrate the relation of structural and functional properties. The findings support the requirement for proper spacing between the NCAM domains to allow polysialylation to occur and show when in space and sequence their application to a range of scFv fragments improve serum half-life by up to 7- fold. By targeting a soluble factor, the antibody derivative shows a blood exposure rate equal to that of a membrane bound target, illustrating the recombinant polysiaylation technique can be applied to a range of targets. Anti-IL-6 effects in vitro have also been shown in this study, suggesting potential for inhibiting pro-inflammatory cytokines in a therapeutic application.