Preclinical development of a bispecific TNFα/IL-23 neutralising domain antibody as a novel oral treatment for inflammatory bowel disease.

Anti-TNFα and anti-IL-23 antibodies are highly effective therapies for Crohn's disease or ulcerative colitis in a proportion of patients. V56B2 is a novel bispecific domain antibody in which a llama-derived IL-23p19-specific domain antibody, humanised and engineered for intestinal protease resistance, V900, was combined with a previously-described TNFα-specific domain antibody, V565. V56B2 contains a central protease-labile linker to create a single molecule for oral administration. Incubation of V56B2 with trypsin or human faecal supernatant resulted in a complete separation of the V565 and V900 monomers without loss of neutralising potency. Following oral administration of V900 and V565 in mice, high levels of each domain antibody were detected in the faeces, demonstrating stability in the intestinal milieu. In ex vivo cultures of colonic biopsies from IBD patients, treatment with V565 or V900 inhibited tissue phosphoprotein levels and with a combination of the two, inhibition was even greater. These results support further development of V56B2 as an oral therapy for IBD with improved safety and efficacy in a greater proportion of patients as well as greater convenience for patients compared with traditional monoclonal antibody therapies.

Oral Anti-Tumour Necrosis Factor Domain Antibody V565 Provides High Intestinal Concentrations, and Reduces Markers of Inflammation in Ulcerative Colitis Patients.

V565 is an engineered TNFα-neutralising single domain antibody formulated into enteric coated mini-tablets to enable release in the intestine after oral administration as a possible oral treatment for inflammatory bowel disease (IBD). Following oral administration, ileal recovery of V565 was investigated in four patients with terminal ileostomy. Intestinal and systemic pharmacokinetics were measured in six patients with Crohn's disease and evidence of target engagement assessed in five patients with ulcerative colitis. Following oral administration, V565 was detected at micromolar concentrations in ileal fluid from the ileostomy patients and in stools of the Crohn's patients. In four of the five ulcerative colitis patients, biopsies taken after 7d dosing demonstrated V565 in the lamina propria with co-immunostaining on CD3+ T-lymphocytes and CD14+ macrophages. Phosphorylation of signalling proteins in biopsies taken after 7d oral dosing was decreased by approximately 50%. In conclusion, enteric coating of V565 mini-tablets provided protection in the stomach with gradual release in intestinal regions affected by IBD. Immunostaining revealed V565 tissue penetration and association with inflammatory cells, while decreased phosphoproteins after 7d oral dosing was consistent with V565-TNFα engagement and neutralising activity. Overall these results are encouraging for the clinical utility of V565 in the treatment of IBD.

Oral delivery of the anti-tumor necrosis factor α domain antibody, V565, results in high intestinal and fecal concentrations with minimal systemic exposure in cynomolgus monkeys.

OBJECTIVE: V565 is a novel oral anti-tumor necrosis factor (TNF)-α 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.

Preclinical Development of a Novel, Orally-Administered Anti-Tumour Necrosis Factor Domain Antibody for the Treatment of Inflammatory Bowel Disease.

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.

Quantitative proteomic analysis of Shigella flexneri and Shigella sonnei Generalized Modules for Membrane Antigens (GMMA) reveals highly pure preparations.

Outer membrane blebs are naturally shed by Gram-negative bacteria and are candidates of interest for vaccines development. Genetic modification of bacteria to induce hyperblebbing greatly increases the yield of blebs, called Generalized Modules for Membrane Antigens (GMMA). The composition of the GMMA from hyperblebbing mutants of Shigella flexneri 2a and Shigella sonnei were quantitatively analyzed using high-sensitivity mass spectrometry with the label-free iBAQ procedure and compared to the composition of the solubilized cells of the GMMA-producing strains. There were 2306 proteins identified, 659 in GMMA and 2239 in bacteria, of which 290 (GMMA) and 1696 (bacteria) were common to both S. flexneri 2a and S. sonnei. Predicted outer membrane and periplasmic proteins constituted 95.7% and 98.7% of the protein mass of S. flexneri 2a and S. sonnei GMMA, respectively. Among the remaining proteins, small quantities of ribosomal proteins collectively accounted for more than half of the predicted cytoplasmic protein impurities in the GMMA. In GMMA, the outer membrane and periplasmic proteins were enriched 13.3-fold (S. flexneri 2a) and 8.3-fold (S. sonnei) compared to their abundance in the parent bacteria. Both periplasmic and outer membrane proteins were enriched similarly, suggesting that GMMA have a similar surface to volume ratio as the surface to periplasmic volume ratio in these mutant bacteria. Results in S. flexneri 2a and S. sonnei showed high reproducibility indicating a robust GMMA-producing process and the low contamination by cytoplasmic proteins support the use of GMMA for vaccines. Data are available via ProteomeXchange with identifier PXD002517.

Comparison of colorimetric assays with quantitative amino acid analysis for protein quantification of Generalized Modules for Membrane Antigens (GMMA).

Genetically induced outer membrane particles from Gram-negative bacteria, called Generalized Modules for Membrane Antigens (GMMA), are being investigated as vaccines. Rapid methods are required for estimating the protein content for in-process assays during production. Since GMMA are complex biological structures containing lipid and polysaccharide as well as protein, protein determinations are not necessarily straightforward. We compared protein quantification by Bradford, Lowry, and Non-Interfering assays using bovine serum albumin (BSA) as standard with quantitative amino acid (AA) analysis, the most accurate currently available method for protein quantification. The Lowry assay has the lowest inter- and intra-assay variation and gives the best linearity between protein amount and absorbance. In all three assays, the color yield (optical density per mass of protein) of GMMA was markedly different from that of BSA with a ratio of approximately 4 for the Bradford assay, and highly variable between different GMMA; and approximately 0.7 for the Lowry and Non-Interfering assays, highlighting the need for calibrating the standard used in the colorimetric assay against GMMA quantified by AA analysis. In terms of a combination of ease, reproducibility, and proportionality of protein measurement, and comparability between samples, the Lowry assay was superior to Bradford and Non-Interfering assays for GMMA quantification.

High yield production process for Shigella outer membrane particles.

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.

The splice variant LOXIN inhibits LOX-1 receptor function through hetero-oligomerization.

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), encoded by the OLR1 gene, is a scavenger receptor that plays a central role in the pathogenesis of atherosclerosis. We have recently identified a truncated naturally occurring variant of the human receptor LOX-1, named LOXIN, which lacks part of the C-terminus lectin-like domain. In vivo and in vitro studies support that the new splicing isoform is protective against acute myocardial infarction. The mechanism by which LOXIN exerts its protective role is unknown. In this paper we report studies on the heterologous expression and functional characterization of LOXIN variant in mammalian fibroblasts and human endothelial cells. We found that LOXIN, when expressed in the absence of LOX-1, shows diminished plasma membrane localization and is deficient in ox-LDL ligand binding. When co-transfected with the full-length counterpart LOX-1, the two isoforms interact to form LOX-1 oligomers and their interaction leads to a decrease in the appearance of LOX-1 receptors in the plasma membrane and a marked impairment of ox-LDL binding and uptake. Co-immunoprecipitation studies confirmed the molecular LOX-1/LOXIN interaction and the formation of non-functional hetero-oligomers. Our studies suggest that hetero-oligomerization between naturally occurring isoforms of LOX-1 may represent a general paradigm for regulation of LOX-1 function by its variants.