Gram-negative bacteria naturally shed particles that consist of outer membrane lipids, outer membrane proteins, and soluble periplasmic components. These particles have been proposed for use as vaccines but the yield has been problematic. We developed a high yielding production process of genetically derived outer membrane particles from the human pathogen Shigella sonnei. Yields of approximately 100 milligrams of membrane-associated proteins per liter of fermentation were obtained from cultures of S. sonnei ΔtolR ΔgalU at optical densities of 30–45 in a 5 L fermenter. Proteomic analysis of the purified particles showed the preparation to primarily contain predicted outer membrane and periplasmic proteins. These were highly immunogenic in mice. The production of these outer membrane particles from high density cultivation of bacteria supports the feasibility of scaling up this approach as an affordable manufacturing process. Furthermore, we demonstrate the feasibility of using this process with other genetic manipulations e.g. abolition of O antigen synthesis and modification of the lipopolysaccharide structure in order to modify the immunogenicity or reactogenicity of the particles. This work provides the basis for a large scale manufacturing process of Generalized Modules of Membrane Antigens (GMMA) for production of vaccines from Gram-negative bacteria.

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TNFα is an important cytokine in inflammatory bowel disease. V565 is a novel anti-TNFα domain antibody developed for oral administration in IBD patients, derived from a llama domain antibody and engineered to enhance intestinal protease resistance. V565 activity was evaluated in TNFα-TNFα receptor-binding ELISAs as well as TNFα responsive cellular assays and demonstrated neutralisation of both soluble and membrane TNFα with potencies similar to those of adalimumab. Although sensitive to pepsin, V565 retained activity after lengthy incubations with trypsin, chymotrypsin, and pancreatin, as well as mouse small intestinal and human ileal and faecal supernatants. In orally dosed naïve and DSS colitis mice, high V565 concentrations were observed in intestinal contents and faeces and immunostaining revealed V565 localisation in mouse colon tissue. V565 was detected by ELISA in post-dose serum of colitis mice, but not naïve mice, demonstrating penetration of disrupted epithelium. In an ex vivo human IBD tissue culture model, V565 inhibition of tissue phosphoprotein levels and production of inflammatory cytokine biomarkers was similar to infliximab, demonstrating efficacy when present at the disease site. Taken together, results of these studies provide confidence that oral V565 dosing will be therapeutic in IBD patients where the mucosal epithelial barrier is compromised.

Objective: V565 is a novel oral anti-tumor necrosis factor (TNF)-a domain antibody being developed for topical treatment of inflammatory bowel disease (IBD) patients. Protein engineering rendered the molecule resistant to intestinal proteases. Here we investigate the formulation of V565 required to provide gastro-protection and enable optimal delivery to the lower intestinal tract in monkeys. Methods: Enteric-coated V565 mini-tablets were prepared and dissolution characteristics tested in vitro. Oral dosing of monkeys with enteric-coated mini-tablets containing V565 and methylene blue dye enabled in vivo localization of mini-tablet dissolution. V565 distribution in luminal contents and feces was measured by enzyme-linked immunosorbent assay (ELISA). To mimic transit across the damaged intestinal epithelium seen in IBD patients an intravenous (i.v.) bolus of V565 was given to monkeys and pharmacokinetic parameters of V565 measured in serum and urine by ELISA. Results: Enteric-coated mini-tablets resisted dissolution in 0.1 M HCl, before dissolving in a sustained release fashion at neutral pH. In orally dosed monkeys methylene blue intestinal staining indicated the jejunum and ileum as sites for mini-tablet dissolution. Measurements of V565 in monkey feces confirmed V565 survival through the intestinal tract. Systemic exposure after oral dosing was very low consistent with limited V565 mucosal penetration in healthy monkeys. The rapid clearance of V565 after i.v. dosing was consistent with renal excretion as the primary route for elimination of any V565 reaching the circulation. Conclusions: These results suggest that mini-tablets with a 24% Eudragit enteric coating are suitable for targeted release of orally delivered V565 in the intestine for topical treatment of IBD.