A polymeric immunoglobulin-antigen fusion protein strategy for enhancing vaccine immunogenicity.

In this study, a strategy based on polymeric immunoglobulin G scaffolds (PIGS) was used to produce a vaccine candidate for Mycobacterium tuberculosis. A genetic fusion construct comprising genes encoding the mycobacterial Ag85B antigen, an immunoglobulin γ‐chain fragment and the tailpiece from immunoglobulin μ chain was engineered. Expression was attempted in Chinese Hamster Ovary (CHO) cells and in Nicotiana benthamiana. The recombinant protein assembled into polymeric structures (TB‐PIGS) in N. benthamiana, similar in size to polymeric IgM. These complexes were subsequently shown to bind to the complement protein C1q and FcγRs with increased affinity. Modification of the N‐glycans linked to TB‐PIGS by removal of xylose and fucose residues that are normally found in plant glycosylated proteins also resulted in increased affinity for low‐affinity FcγRs. Immunization studies in mice indicated that TB‐PIGS are highly immunogenic with and without adjuvant. However, they did not improve protective efficacy in mice against challenge with M. tuberculosis compared to conventional vaccination with BCG, suggesting that additional or alternative antigens may be needed to protect against this disease. Nevertheless, these results establish a novel platform for producing polymeric antigen‐IgG γ‐chain molecules with inherent functional characteristics that are desirable in vaccines

Complement as a biological tool to control tumor growth

Deposits of complement components have been documented in several human tumors suggesting a potential involvement of the complement system in tumor immune surveillance. In vitro and in vivo studies have revealed a double role played by this system in tumor progression. Complement activation in the cancer microenvironment has been shown to promote cancer growth through the release of the chemotactic peptide C5a recruiting myeloid suppressor cells. There is also evidence that tumor progression can be controlled by complement activated on the surface of cancer cells through one of the three pathways of complement activation. The aim of this review is to discuss the protective role of complement in cancer with special focus on the beneficial effect of complement-fixing antibodies that are efficient activators of the classical pathway and contribute to inhibit tumor expansion as a result of MAC-mediated cancer cell killing and complement-mediated inflammatory process. Cancer cells are heterogeneous in their susceptibility to complement-induced killing that generally depends on stable and relatively high expression of the antigen and the ability of therapeutic antibodies to activate complement. A new generation of monoclonal antibodies are being developed with structural modification leading to hexamer formation and enhanced complement activation. An important progress in cancer immunotherapy has been made with the generation of bispecific antibodies targeting tumor antigens and able to neutralize complement regulators overexpressed on cancer cells. A great effort is being devoted to implementing combined therapy of traditional approaches based on surgery, chemotherapy and radiotherapy and complement-fixing therapeutic antibodies. An effective control of tumor growth by complement is likely to be obtained on residual cancer cells following conventional therapy to reduce the tumor mass, prevent recurrences and avoid disabilities

Remodelling N-glycans of therapeutic monoclonal antibodies for enhanced efficacy.

Recent improvements in antibody engineering technologies have shifted the focus of monoclonal antibody (Mab) development from drug discovery onto flexible and efficient manufacturing. Currently hundreds of Mab products are in clinical trials and due to the high dosages required and an ever increasing demand, concerns have been raised over worldwide production capacity. The large investment in capital required to increase production capacity heightens the need for the development of new technologies to streamline Mab production and ensure their continued success as therapeutics. In addition to cell line and process optimisation, the bioactivity of the Mab itself can be targeted. Improved efficacy could potentially ease pressure on production by reducing Mab dosages and therefore the amount of product required. The focus of the current project is to develop such a bioprocess to enhance Mab bioactivity. The targeting of Mab N-glycosylation provides an opportunity to enhance therapeutic efficacy. This post-translational modification involves attachment of heterogeneous carbohydrate moieties to an asparagine residue and is known to have a critical influence over bioactivity. Current reports suggest that specific alterations in the composition of the carbohydrate structures can significantly improve in vitro bioactivity, including the presence of bisecting N-acetylglucosamine (GlcNAc). Previous studies report variations in the relative importance of bisecting GlcNAc from minimal change up to twenty fold elevated in vitro bioactivity. All prior studies have utilised cell engineering techniques which have been reported to cause concomitant glycoform changes. This is one likely contribution to varied Mab bioactivity reported for bisecting GlcNAc, and highlights the need for evaluation by alternative methods. With this in mind the current project engineered N-glycans of affinity purified IgG1 Mabs to introduce bisecting GlcNAc structures. Initial emphasis was placed on implementation of an N-glycan analytical strategy to confidently monitor glycoform changes, and was applied to model therapeutic Mabs. This enabled analytical confirmation for N-glycan remodelling, including GlcNAc transfer to Mab N-glycans following incubation with recombinant N-acetylglucosaminyltransferase III (GnT-III). Controlled reaction conditions enabled variable transfer of bisecting GlcNAc to the therapeutic Mabs Herceptin and Rituxan, generating low, medium and high levels of modification. A conserved ten fold improvement to antibody dependent cellular cytotoxicity (ADCC) was observed for the therapeutics at high bisecting GlcNAc levels, with no effect on antigen binding. The increase in N-acetylglucosamine content for both IgG1 Mabs also exhibited minimal effect on complement dependent cytotoxicity (CDC). In summary, in vitro bioactivity of therapeutic Mabs could be specifically enhanced via controlled remodelling of N-glycans to include a bisecting N-acetylglucosamine. This research provides an opportunity to reduce therapeutic Mab doses via a generic downstream bioprocess and circumvent the need to engineer current production cell lines

Oral and intestinal digestion of oligosaccharides as potential sweeteners: A systematic evaluation

The utilisation of oligosaccharides by oral microorganisms and intestinal enzymes are important factors in determining their effectiveness as alternative sweeteners. In this study, classes of naturally occurring sugars were assessed using in vitro models for oral and intestinal digestibility, in order to test the influence of chemical structure on functional properties. Amongst the classes of sucrose isomers

Fruit fly bioassay to distinguish "sweet" sugar structures

Palatable response to dietary sugars plays a significant role in influencing metabolic health. New structures are being explored with beneficial health properties, although consumer acceptance relies heavily on desirable sensory properties. Despite the importance of behavioral responses, the ability to elucidate structure-preference relationships of sugars is lacking. A wild population of Drosophila melanogaster was used as a model to perform pairwise comparisons across structural groups to characterize a fruit fly bioassay for assessing sugar preference. Preference was successfully described in structurally relevant terms, particularly through the ability to directly test sugars of related structures in addition to standard sucrose comparisons. The fruit fly bioassay also provided the first report on the relative preference for the β-linked sugar alcohol, gentiobiitol. In making reference to well-known human preferences, the bioassay also raises opportunities for greater understanding of behavioral response to sugar structures in general