Abstract Background Crohn’s Disease (CD) is a chronic condition that can impact any part of the gastrointestinal tract. The sustained inflammation is driven by the continuous influx of inflammatory leukocytes into the gut mucosa, which is regulated through chemokine gradients and adhesion molecules, including integrins. In the gut, the migration of inflammatory cells to specific segments is thought to be mediated by selected integrins and/or chemokine axes. The CCL25/CCR9 axis has been identified as a key pathway for immune cell migration into small bowel segments, such as the ileum. In this study, CITE-seq and spatial transcriptomic analysis was used to integrate proteomic and transcriptomic profiles to characterise CCR9+ immune cells in the ileum compared to the colon. Methods Biopsies were obtained via ileocolonoscopy from macroscopically inflamed and normal mucosa with appropriate consent from healthy volunteers and IBD patients visiting the Department of Gastroenterology at the Sahlgrenska Hospital. Biopsies were frozen in Cryostor CS10 or formalin fixed and paraffin embedded. Cells from frozen biopsies were sorted, CD45+ cells stained with CCR9 and Totalseq-C human universal antibody cocktail, followed by amplification, separation of antibody-derived DNA from mRNA derived cDNA, library construction and sequencing of both libraries. For spatial transcriptomics, tissue sections were deparaffinised, stained with H&E and decrosslinked before human whole transcriptome probe hybridisation, ligation, extension, library construction and sequencing. Cells were annotated using a transfer learning approach combining several reference single cell datasets including detailed CD4+ and CD8+ T cell datasets and utilising both antibody data and mRNA transcriptomic data. Results CCR9 positive cells appeared scattered throughout the lamina propria and expression at mRNA level was enriched in the ileum compared to the colon with about 10% of CD4 and 17% of CD8 T cells expressing CCR9. Interestingly at protein level, expression was more restricted with the highest expression on cells that exhibited proteomic and transcriptomic features associated with resident CD4 memory T cells (Trm). This population almost exclusively (>90%) consisted of CCR9 positive cells and their proteomic and transcriptomic profile consistent with the recently described effector and innate-like phenotype of Trm cells that is thought to contribute to Crohn’s disease pathology. Conclusion These data may point to CCR9 positive cells, and in particular Trm cells, in the ileum as important pathological cells in Crohn’s disease and provide further support for depleting CCR9 cells as a potential therapeutic target for the treatment of ileal Crohn’s disease.

Abstract Background CCR9/CCL25 is the key axis for recruiting lymphocytes to the small intestine and represents a potential treatment target in Crohn’s disease (CD). AZD7798 is a first-in-class monoclonal antibody for CD that depletes CCR9+ lymphocytes. Monitoring CCR9+ cell depletion and repletion in the blood is straightforward, but difficult in the primary tissues of interest (small bowel, mesenteric lymph nodes) which require invasive procedures to access. For AZD7798, an understanding of pharmacodynamics in gut is crucial to guide further clinical development. To address this need, we have developed a CCR9-specific PET tracer based on the small molecule CCR9 antagonist vercirnon. Methods Specific binding of [3H]vercirnon was assessed in-vitro using autoradiography. Specific binding and dosimetry of [11C]vercirnon was assessed in-vivo in cynomolgus monkeys. Subsequently, a first-time-in-human study (NCT05818514) was performed in healthy volunteers to assess the biodistribution, binding to CCR9 in abdominal regions of interest, and safety of [11C]vercirnon, administered intravenously at microdoses (<10μg). Two PET-CT examinations were performed 14 days apart to assess reproducibility. Results In-vitro specific binding of [3H]vercirnon to CCR9 was confirmed in human thymus sections and CCR9-expressing cell pellets. In-vivo specific binding of [11C]vercirnon to CCR9 was putatively demonstrated in the gut of non-human primates (NHPs) in pre-treatment/displacement studies with excess unlabelled vercirnon (20mg/kg). 8 healthy volunteers (5 male) were enrolled in the FTIH study. Detectable intestinal uptake of [11C]vercirnon was demonstrated in all participants (Figure 1) with mean±SD standardised uptake value ratio (SUVR) 1.4±0.6 (reference abdominal aorta), and a proximal to distal gradient (SUVR jejunum 1.8±0.7/ileum 0.9±0.4/colon 0.6±0.2) consistent with CCR9 gene expression data. Test-retest variability of intestinal uptake was 9.6% (intraclass correlation coefficient 0.96), in line with values for other clinically useful PET tracers. Mean±SD injected radioactivity per PET examination was 351±105 MBq, corresponding to a radiation exposure of 1.4 mSv. This low radiation exposure (c.f. conventional FDG PET ~9 mSv) allows multiple PET examinations per participant. Administration of [11C]vercirnon was well-tolerated with no serious adverse events. Conclusion We have developed a CCR9-specific PET tracer which potentially enables repeatable quantification of CCR9 expression in the gut. Ongoing studies will assess the effects of AZD7798 on CCR9+ cell depletion/repletion in the gut, providing critical data to support the clinical development programme.

Abstract Background Crohn´s disease (CD) can affect any part of the GI tract, although has a predilection for the terminal ileum and colon. Approximately 30% of patients have inflammation restricted to the small intestine, another 30% have disease limited to the colon, and the remainder have a combination of small and large bowel involvement. Despite studies suggesting that ileal and colonic CD may be distinct disease phenotypes and might therefore respond differently to treatment, CD patients are typically enrolled into clinical trials irrespective of the site of gastrointestinal involvement. The aim of this systematic review and meta-analysis was to assess treatment efficacy in patients with small bowel disease. Methods Embase and PubMed databases were searched until October 2023 for randomised, placebo-controlled, clinical trials that reported induction efficacy, based on clinical remission (CDAI <150), in ileal-only Crohn’s disease between 2010 and 2023. The following search criteria were employed: Crohn´s disease AND [2010-2023] AND (small bowel OR ileum OR ileal OR L1) AND (CDAI OR clinical remission AND induction AND placebo). Additional studies, not identified through this search strategy were also considered. Resultant studies were systematically reviewed before a random-effects meta-analysis was undertaken to assess induction efficacy, based on clinical remission, in ileal only CD. Results A total of 9 studies, involving 709 adult patients, were identified: two via Embase and PubMed; six from supplementary search materials; and one post-hoc analysis of brazikumab data (not published). The studies provided CDAI remission results by disease location subset. The induction treatment times ranged from 6-12 weeks, and both bio-naïve and bio-failure patients were included. The studies were relatively homogenous (I2 = 38.5%, Q = 13.0 (p-value = 0.112)) with a random effect meta-analytic estimate of difference versus placebo in CDAI remission of 2.1% (95% CI -6% to 11%). Two studies used clinical response (defined as ≥ 100 point decrease or score < 150) as indicated in the figure. Conclusion This is the first systematic meta-analysis of treatment effects in ileal-only CD. The available evidence suggests treatment effect heterogeneity, with inferior outcomes in ileal disease versus a broader patient population with ileocolonic or colonic involvement. Previous systematic review1 on clinical remission after induction treatment demonstrate placebo adjusted values in the range of 12-20% in a no defined disease site population. This indicates the need for novel therapies with a mechanism of action directly tailored to heal the small intestine in patients with CD.

Abstract Background Interleukin23 (IL-23) is a cytokine that plays a crucial role in the pathogenesis of inflammatory bowel disease (IBD), making it a highly validated therapeutic target. Understanding the role of IL-23 in IBD at the histopathological level is crucial for determining effective treatment strategies, providing insights into IBD patients who fail to respond to targeted therapies, or predicting those who are likely to lose response. In this context, there is a surge in utilizing artificial intelligence (AI) for histopathological data in IBD and other disease indications. Here, we present an automated computer vision approach to predict IL-23 signalling activity directly from routinely stained Hematoxylin and Eosin (H&E) images Methods A total of 1502 samples with matched clinical data and H&E biopsy images were included from 991 Crohn’s disease (CD) and 511 ulcerative colitis (UC) samples. IL-23 signalling activity was calculated using gene set variation analysis on RNA-seq data collected from the same tissue biopsies. The data were obtained from the IBD Plexus program of the Crohn’s & Colitis Foundation. The proposed approach is based on vision transformers (ViTs) which is a type of deep learning model. ViTs divide the input image into fixed-size patches, transform it into linear embedding, and analyze it with the self-attention mechanism. This enables ViTs to incorporate relationships between different patches of the input image to identify regions predictive of IL-23. Our approach was trained in a weakly supervised manner to automatically identify tissue regions that correlate with IL-23 signaling activity. The model produces interpretable heatmaps to interrogate model predictions and allow clinicians to visualize and interpret the significance of different tissue regions predictive of IL-23 Results We performed 5-fold cross-validation on the splits obtained at the patient level, retaining the data distribution of IL-23 signaling activity, biopsy location, and diagnosis. We separately validated the performance of the proposed model on both disease categories, including CD and UC. The proposed approach achieved an area under the curve (AUC) of 0.82 ± 0.04 on unseen data from CD and an AUC of 0.80 ± 0.02 for UC. The 5-fold results for both disease categories are shown below Conclusion The presented results highlight the significance of computational pathology algorithms to identify IL-23 signalling activity from H&E images. Pathological interpretation from the heatmaps may help understand disease pathomechanism and optimize the treatment options for IBD patients by timely identification of IL-23 status. We are further validating the clinical utility of such heatmaps and expanding the use of H&E to predict other patient-centric endpoints

Abstract Background Histopathological endpoints are evolving as a treatment target in Inflammatory Bowel Disease (IBD). Use of histology to screen entrants could add value in IBD clinical trials; for example, by refining eligibility criteria to ensure studies recruit patients with definitive active inflammation at the microscopic level. Several histopathological indices have been developed, but the relative complexity of available scores hinders the development of an AI algorithm without large-scale labour-intensive annotation by a pathologist. We aimed to develop computer vision tools to assist decoding the complex clinical disease features at the histological level for both Crohn’s Disease (CD) and Ulcerative Colitis (UC). This will inform understanding of disease pathology and patient stratification to support clinical trial development strategies. Methods A total of 1397 clinically annotated Haematoxylin & Eosin (H&E) images were included from 418 CD and 218 UC patients enrolled in a multicentred longitudinal Study of a Prospective Adult Research Cohort with IBD (SPARC IBD) obtained from the IBD Plexus program of the Crohn’s & Colitis Foundation. We developed an image quality control (QC) algorithm to automatically identify image and tissue processing/staining artefacts negatively impacting analysis (e.g., out-of-focus, tissue folds, overstained regions) and excluded these regions (Fig. 1). Next, a self-supervised learning (SSL) deep learning computer vison model was developed and trained to predict disease relevant features including disease diagnosis and lesional macroscopic appearance (inflammation, erosions and ulcers). Finally, to better understand the model’s predictions, we generated heatmap overlays on the tissue that show regions which the model considers to be most predictive (Fig. 2) and shared these with pathologists for qualitative evaluation. Results We find that the SSL model performs well on different downstream classification tasks such as UC vs CD (area under curve (AUC) = 0.79) and normal vs lesional tissue (AUC = 0.76). Specialist pathologist collaboration further confirmed that the heatmap overlays identified clinically relevant tissue features, including inflammatory cell infiltrates (Fig. 2). Conclusion These encouraging results support further exploration of this deep-learning algorithm to distinguish disease specific characteristics in this set of images from CD and UC patients. Further work is ongoing to validate the heatmap approach on endoscopic scores, we also plan to validate our model on IBD clinical trial datasets.

Abstract Background Crohn’s Disease (CD) is a chronic disease in which inflammation is sustained by continuous influx of inflammatory leukocytes into gut mucosa and may affect all segments of the gastrointestinal tract. Immune cell trafficking is regulated via chemokine gradients and adhesion molecules such as integrins. Here we explored the expression profile of gut-specific integrin and chemokine axes in different regions of the gut to identify possible targets for treating CD patients with different disease location or phenotype. Methods This study used transcriptomic data from gut tissue biopsies of the large and small intestine of 611 CD and 315 UC patients enrolled in a multicentred longitudinal Study of a Prospective Adult Research Cohort with IBD (SPARC IBD) obtained from the IBD Plexus program of the Crohn’s and Colitis Foundation. We mapped the expression of selected chemokine receptors and their ligands as well as gut-specific integrins onto the different segments of the intestine. Expression data were then linked to available clinical disease phenotypes, as well as genotype information. Results In both CD and UC patients, ITGA4 and ITGB7, the targets for Vedolizumab, and their endothelial ligand MADCAM1 were ubiquitously present in all regions of the gut. GPR15 and its ligand C10orf99 were predominantly expressed in colonic biopsies. On the contrary, expression of CCR9 and its ligand CCL25 were specific to the terminal ileum with only minimal transcriptional activity in the colon. Expression of CCL25 was significantly increased in patients with penetrating and/or stricturing CD, a disease feature associated with ileal CD that has high impact for patients and is currently an unmet for IBD patients. Further, we identified an expression quantitative trait loci (eQTL) upstream of the CCL25 promoter linked to small bowel expression of CCL25. Conclusion Our data highlight the potential for targeting the CCR9/CCL25 axis as a treatment for small bowel Crohn’s Disease which may have been incompletely targeted to date by small molecule inhibitors. Further investigations are warranted to identify a mechanistic link between this pathway and penetrating and stricturing disease.

Summary Background Selective depletion of T cells expressing LAG‐3, an immune checkpoint receptor that is upregulated on activated T cells, has been investigated in pre‐clinical models as a potential therapeutic approach in inflammatory and autoimmune diseases where activated T cells are implicated. Aims GSK2831781, a depleting monoclonal antibody that specifically binds LAG‐3 proteins, may deplete activated LAG‐3 + cells in ulcerative colitis (UC). Methods Patients with moderate to severe UC were randomised to GSK2831781 or placebo. Safety, tolerability, efficacy, pharmacokinetics and pharmacodynamics of GSK2831781 were evaluated. Results One hundred four participants across all dose levels were randomised prior to an interim analysis indicating efficacy futility criteria had been met. Efficacy results focus on the double‐blind induction phase of the study (GSK2831781 450 mg intravenously [IV], N = 48; placebo, N = 27). Median change from baseline (95% credible interval [CrI]) in complete Mayo score was similar between groups (GSK2831781 450 mg IV: −1.4 [−2.2, −0.7]; placebo: −1.4 [−2.4, −0.5]). Response rates for endoscopic improvement favoured placebo. Clinical remission rates were similar between groups. In the 450‐mg IV group, 14 (29%) participants had an adverse event of UC versus 1 (4%) with placebo. LAG‐3 + cells were depleted to 51% of baseline in blood; however, there was no reduction in LAG‐3 + cells in the colonic mucosa. Transcriptomic analysis of colon biopsies showed no difference between groups. Conclusion Despite evidence of target cell depletion in blood, GSK2831781 failed to reduce inflammation in the colonic mucosa suggesting no pharmacological effect. The study was terminated early (NCT03893565).

Abstract Background Lymphocyte trafficking to the inflamed gut is one of the hallmarks of Inflammatory Bowel Disease (IBD) pathology. Integrin a4b7 and its ligand MADCAM1 play an important role in targeting immune cells in the bloodstream to mucosal tissues and blocking this pathway using the anti-a4b7 monoclonal antibody Vedolizumab (VDZ) proved to be an effective treatment in IBD. However, VDZ is most efficient in treatment of Ulcerative Colitis (UC) and of colonic rather than ileal Crohn’s Disease (CD), for which the reasons have not been completely understood. Here we sought to compare the expression of ITGA4, ITGB7 and MADCAM1 in gut tissue biopsies from small and large bowel with respect to the underlying severity of tissue inflammation. Methods Differential gene expression analysis was conducted using bulk RNA Sequencing data generated from gut tissue biopsies of the large and small intestine of 611 CD and 315 UC patients enrolled in a multicentred longitudinal Study of a Prospective Adult Research Cohort with IBD (SPARC IBD) obtained from the IBD Plexus program of the Crohn’s and Colitis Foundation. Gene expression levels of ITGA4, ITGB7 and MADCAM1 were compared across ileal, colonic and rectal segments of the intestine and data were then linked to available measures of local inflammation. Results ITGA4, ITGB7 and MADCAM1 were expressed at comparable levels in large vs small bowel biopsies. Expression of MADCAM1 was significantly increased in both ileal and colonic biopsies from patients with moderate to severe CD based on segmental or composite endoscopy score (SES-CD) across all bowel segments. Expression of both ITGA4 and ITGB7 was similarly increased in colonic biopsies of moderate/severe CD patients, whereas only minor changes were observed for both genes in ileal biopsies. Similar findings were obtained from UC patients, where expression of MADCAM1 was significantly increased in inflamed or ulcerated biopsies from both sigmoid colon and cecum as compared to normal appearing biopsies. Expression of ITGA4 and ITGB7 was only increased in inflamed/ulcerated biopsies of the sigmoid colon but not the cecum and correlated with the modified Mayo endoscopic score (MMES). Conclusion Our analysis suggests that influx of leukocytes to inflamed tissue via ITGA4/ITGB7 and MADCAM1 interactions is more common in the distal colon compared to proximal colon or ileum. This provides a possible mechanistic explanation as to why VDZ is most effective in patients with colonic IBD. Further work is needed to understand the differential regulation of cell trafficking to different regions of the gut.

Crohn's Disease (CD) and Ulcerative Colitis (UC) are the two main Inflammatory Bowel Disease (IBD) types. We developed deep learning models to identify histological disease features for both CD and UC using only endoscopic labels. We explored fine-tuning and end-to-end training of two state-of-the-art self-supervised models for predicting three different endoscopic categories (i) CD vs UC (AUC=0.87), (ii) normal vs lesional (AUC=0.81), (iii) low vs high disease severity score (AUC=0.80). We produced visual attention maps to interpret what the models learned and validated them with the support of a pathologist, where we observed a strong association between the models' predictions and histopathological inflammatory features of the disease. Additionally, we identified several cases where the model incorrectly predicted normal samples as lesional but were correct on the microscopic level when reviewed by the pathologist. This tendency of histological presentation to be more severe than endoscopic presentation was previously published in the literature. In parallel, we utilised a model trained on the Colon Nuclei Identification and Counting (CoNIC) dataset to predict and explore 6 cell populations. We observed correlation between areas enriched with the predicted immune cells in biopsies and the pathologist's feedback on the attention maps. Finally, we identified several cell level features indicative of disease severity in CD and UC. These models can enhance our understanding about the pathology behind IBD and can shape our strategies for patient stratification in clinical trials.

Purpose To explore symptoms and disease impacts of Crohn’s disease and to develop a new patient-reported outcomes (PRO) measure according to industry best practices. Methods A conceptual model of relevant symptoms experienced by patients with Crohn’s disease was developed following a literature review. Three rounds of combined qualitative semi-structured concept elicitation and cognitive debriefing interviews with 36 patients (≥ 16 years) with Crohn’s disease and 4 clinicians were conducted to further explore the most commonly reported and most bothersome symptoms to patients. Interview results were used to update the conceptual model as well as items and response options included in The Crohn’s Disease Diary, a new PRO measure. Results All patients ( N = 36) reported abdominal pain, loose or liquid bowel movements, and high or increased frequency of bowel movements, with most reporting these symptoms spontaneously (100%, 92%, and 75%, respectively). All patients reported bowel movement urgency, but 61% reported this symptom only when probed. Most also reported that symptoms impacted activities of daily living, work/school, and emotional, social, and physical functioning (overall, 78%–100%; spontaneously, 79% – 92%). Data regarding core symptoms of Crohn’s disease from clinician concept elicitation interviews supported patient data. The 17-item Crohn’s Disease Diary assesses core symptoms and impacts of Crohn’s disease over 24 h, and extraintestinal manifestations over 7 days. The content validity of the diary was confirmed during cognitive debriefing interviews. Conclusion The Crohn’s Disease Diary is a new PRO measure for the assessment of Crohn’s disease symptoms and impacts, developed according to industry best practices.

This study investigated ethnic differences in the safety, tolerability, pharmacokinetics, and pharmacodynamics of GSK2831781, an anti–lymphocyte activation gene 3 (LAG3) monoclonal antibody, in healthy participants, and determined local tolerability and bioavailability following subcutaneous (SC) administration. A double‐blind, randomized study of (A) single intravenous (IV) doses of GSK2831781 450 mg or placebo in Japanese and White participants; and (B) single SC doses of GSK2831781 150 or 450 mg, or placebo in White participants, was conducted. Blood samples for analyses were collected before dosing and over 112 days after dosing. GSK2831781 was well tolerated in Japanese and White participants after both IV and SC doses, with the adverse event profile in Japanese being consistent with other populations. There were no injection site adverse events. There was no evidence of differences in systemic exposure among Japanese and White participants. Systemic exposure did not vary with body weight. SC bioavailability was 76.5%, as estimated using population pharmacokinetic modeling. Full and sustained target engagement and evidence of LAG3+ cell depletion (≈53%–66%) were observed in both populations and after both administration routes. No evidence of reduced circulating regulatory T cells (CD4+CD25+CD127lowFoxP3+) was observed. Following IV and SC administration, GSK2831781 depleted circulating LAG3+ T cells with no interethnic difference observed. There were no major impacts on circulating regulatory T cells.