Anna Vossenkämper’s research while affiliated with Queen Mary, University of London and other places

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.

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.

RIP2 kinase has been identified as a key signal transduction partner in the NOD2 pathway contributing to a variety of human pathologies, including immune-mediated inflammatory diseases. Small-molecule inhibitors of RIP2 kinase or its signaling partners on the NOD2 pathway that are suitable for advancement into the clinic have yet to be described. Herein, we report our discovery and profile of the prodrug clinical compound, inhibitor 3, currently in phase 1 clinical studies. Compound 3 potently binds to RIP2 kinase with good kinase specificity and has excellent activity in blocking many proinflammatory cytokine responses in vivo and in human IBD explant samples. The highly favorable physicochemical and ADMET properties of 3 combined with high potency led to a predicted low oral dose in humans.

B cell lymphoma-6 (BCL6) is highly expressed in germinal center B cells, but how its expression is maintained is still not completely clear. Aryl hydrocarbon receptor interacting protein (AIP) is a co-chaperone of heat shock protein 90. Deletion of Aip in B cells decreased BCL6 expression, reducing germinal center B cells and diminishing adaptive immune responses. AIP was required for optimal AKT signaling in response to B cell receptor stimulation, and AIP protected BCL6 from ubiquitin-mediated proteasomal degradation by the E3-ubiquitin ligase FBXO11 by binding to the deubiquitinase UCHL1, thus helping to maintain the expression of BCL6. AIP was highly expressed in primary diffuse large B cell lymphomas compared to healthy tissue and other tumors. Our findings describe AIP as a positive regulator of BCL6 expression with implications for the pathobiology of diffuse large B cell lymphoma.

Individual animal data for DSS-colitis experiment. To illustrate variation at the individual animal level, we present plots for DAI time-course (A), DAI AUC. (B), and Histopathology score (C). Jitter has been added to better distinguish overlapping data points. Groups were as follows: sham (no DSS) N = 6, DSS only N = 12, CsA treatment N = 6 and EPHX2i treatment N = 6. All groups except sham were dosed with DSS from Day 0 to Day 5. CsA and EPHX2i treatment groups were dosed from Day 0 to Day 9. (TIF)

Colon Measurements at Experiment Conclusion. Mean values are plotted for each group. Error bars represent standard errors. (A) Colon length was decreased in DSS-treated mice, compared to the vehicle control. Both CsA and EPHX2i treatment partially restored colon length toward the value found in the vehicle control group. (B) The colon weight-to-length ratio is increased by DSS-treatment, but partially restored to normal by cyclosporine treatment. Unexpectedly, EPHX2i treatment did not reverse the weight-to-length ratio increase, but instead appeared to further increase the ratio. (TIF)

LCM-based disease signature from human patients obtained from GeneLogic. (XLSX)

CMAP results for GSK2256294A. Shown are the statistically-interesting hits for the compound expression profile and IBD-related disease signatures. Column headers are as follows. Signature name was derived from the disease expression data source. Dose (in μM) was the compound concentration(s) tested (in triplicate). Mean Cmap score reflects the relative strength of association between compound profile and disease signature. Large negative scores imply a strong inverse correlation. Enrichment score is derived from the Kolmogorov-Smirnov statistic (10). P-value is derived from the distribution generated by 10,000 step permutation analysis. Specificity measures the uniqueness of the relationship between compound and disease signature (lower values are more unique). Signatures with better specificity is the rank order of the listed disease signature compared to the entire disease expression dataset. Cmap score distribution lists the actual scores for each of the triplicate repeats. Platform used for measuring expression was either L1000/Genometry or Illumina. Cell lines used were the following: Caco-2 human epithelial colorectal adenocarcinoma, FIBRO primary human fibroblasts, SAEC human small airway epithelial cells, KERAT primary human keratinocytes, HuSkM human skeletal muscle cells, MCF7 human breast cancer cell line. Disease (if known) from which the signature was derived. Signature biological tissue source. Species signature was derived from. GEO identifier for signature. PMID PubMed identifier if signature data has been published. (XLSX)

Epoxyeicosatrienoic acids (EETs) are signaling lipids produced by cytochrome P450 epoxygenation of arachidonic acid, which are metabolized by EPHX2 (epoxide hydrolase 2, alias soluble epoxide hydrolase or sEH). EETs have pleiotropic effects, including anti-inflammatory activity. Using a Connectivity Map (CMAP) approach, we identified an inverse-correlation between an exemplar EPHX2 inhibitor (EPHX2i) compound response and an inflammatory bowel disease patient-derived signature. To validate the gene-disease link, we tested a pre-clinical tool EPHX2i (GSK1910364) in a mouse disease model, where it showed improved outcomes comparable to or better than the positive control Cyclosporin A. Up-regulation of cytoprotective genes and down-regulation of proinflammatory cytokine production were observed in colon samples obtained from EPHX2i-treated mice. Follow-up immunohistochemistry analysis verified the presence of EPHX2 protein in infiltrated immune cells from Crohn’s patient tissue biopsies. We further demonstrated that GSK2256294, a clinical EPHX2i, reduced the production of IL2, IL12p70, IL10 and TNFα in both ulcerative colitis and Crohn's disease patient-derived explant cultures. Interestingly, GSK2256294 reduced IL4 and IFNγ in ulcerative colitis, and IL1β in Crohn's disease specifically, suggesting potential differential effects of GSK2256294 in these two diseases. Taken together, these findings suggest a novel therapeutic use of EPHX2 inhibition for IBD.

Introduction Monoclonal antibodies to TNF transformed treatment options for patients with Inflammatory Bowel Disease (IBD). V565 is a novel oral anti-TNF domain antibody (Vorabody) engineered to be resistant to intestinal proteases. It is in development as a potential oral treatment for IBD. In vitro it suppressed phosphorylation of tyrosine kinases and signalling proteins and inhibited the release of inflammatory cytokines following culture with biopsies taken from patients with CD (Crowe et al. 18th International Congress of Mucosal Immunology, July 19–22 2017, Washington DC, USA). It was safe and well tolerated after high single and multiple doses in healthy volunteers and patient volunteers with CD and resulted in high concentrations of active drug in ileal fluid and faeces. Aims & Methods This open label study was designed to demonstrate that V565 enters GI mucosa and exerts a beneficial effect on inflammatory processes following oral dosing for 7 days to patients with Ulcerative Colitis. Patients with a Mayo score of 3–10 including an endoscopy score of ≥1 had up to 7 days of oral dosing with 555 mg tid V565. Sigmoidoscopy with biopsies was performed before and after the dosing period. The primary outcomes of interest were presence of V565 in the mucosa and reduction from baseline in phosphorylation of tyrosine kinases and signalling proteins. Detection of V565 was determined by immunohistochemistry. Phosphorylation was determined using PathScan RTK signalling arrays (Vossenkaemper et al 2014. Gastroenterology 147:172–83). Five patient volunteers were treated Due to visit scheduling, most received 6 days treatment. Presence of V565 was confirmed in the inflamed lamina propria and co-localised with CD14 +macrophages in post-treatment biopsies. Overall phosphorylation of the panel of kinases and signalling proteins was reduced by approximately 50% in four of the five patients. There were no treatment induced ADAs. Conclusion V565, an oral anti-TNF domain antibody engineered to be resistant to intestinal proteases, was demonstrated bound to CD14 +macrophages in the lamina propria of UC patients and resulted in inhibition of mucosal inflammatory processes after 6–7 days oral dosing. The reduction of 50% in overall phosphorylation is similar to that seen in an earlier study of UC biopsy cultures with infliximab at a concentration of 67 nM (10 µg/ml), a serum concentration associated with mucosal healing (Ungar et al, Clin Gastroenterol Hepatol. 2016 Apr;14(4):550–557). These results provide encouragement that oral dosing with V565 will be a beneficial oral treatment option for patients with IBD.