Introduction

Pouchitis is an inflammatory disease of the pouch in patients with ulcerative colitis who underwent total colectomy with ileoanal pouch anastomosis. There is no approved treatment for acute pouchitis at present. First-line treatment consists of antibiotics or probiotics. MH002 is an optimized consortium of 6 non-pathogenic, well characterized commensal bacteria with immune modulating, wound healing and gut barrier protective effects, and is in clinical development for ulcerative colitis.

Aims & Methods

This multi-center, open-label pilot study (EudraCT 2021-006656-14) explored the efficacy and mechanistic effects of MH002 in acute pouchitis. Eligible subjects with a modified pouchitis disease activity index (mPDAI) ≥5 and mPDAI endoscopic score ≥2 and with symptoms lasting ≤4wks, received 400mg MH002 once daily for 8wks. Clinical, biochemical, endoscopic, and histologic disease activity was assessed at baseline and wk8. Pouch biopsies were assessed by an IBD pathologist who was blinded to the time points and clinical outcomes. Exploratory efficacy endpoints (N=13) included mPDAI remission (mPDAI<5 and a reduction of ≥2 points from baseline), symptomatic response (a reduction of clinical PDAI≥2 points), endoscopic response (a reduction of endoscopic score of ≥2 points), and histologic response (a reduction of histologic PDAI≥2) at wk8. Changes from baseline in severity of urgency (NURS), Robart’s histopathologic index (RHI), and faecal calprotectin (FCP) concentrations, were also evaluated.

Results

MH002 was safe and well tolerated with no evidence of adverse reactions when administered for 8wks (Abstract Vermeire et al.). At wk8, 6/13 (46%) subjects achieved mPDAI remission. Symptomatic, endoscopic, and histologic response rates were 5/13 (39%), 5/13 (39%), and 2/13 (15%), respectively. Clinical, endoscopic, and histologic PDAI scores were improved at wk8 in 11/13 (85%), 7/13 (54%), and 3/13 (23%) subjects, respectively. In total, 8/13 (62%), 9/13 (69%), and 8/13 (62%) subjects reported an improvement in severity of urgency at wk2, wk4, and wk8, respectively. Over 8wks, the number of ulcerations observed endoscopically decreased in 7/13 (54%) subjects, and the RHI improved in 9/13 (69%) subjects. Decreases in FCP were observed as early as wk2 in the subgroup achieving clinical remission (N=6), with a reduction of 68%, 71%, and 80% in median FCP at wk2, wk4, and wk8, respectively (Table 1).

Table 1. Changes (n, mean or median, IQR) in clinical, endoscopic, histologic, and biochemical disease activity observed upon treatment with MH002 in subjects achieving vs. not achieving mPDAI remission at wk8.

Conclusion

In this exploratory study in patients with acute pouchitis, 8-wk treatment with MH002 resulted in consistent and clinically meaningful improvements with a remission rate of 46% according to the mPDAI. These results warrant further development of MH002 in a phase 2/3 study.

Disclosure

SV: financial support for research from AbbVie, J&J, Pfizer, Takeda and Galapagos; speakers’ and/or consultancy fees from AbbVie, Abivax, AbolerISPharma, AgomAb, Alimentiv, Arena Pharmaceuticals, AstraZeneca, BMS, Boehringer Ingelheim, Celgene, Cytoki Pharma, Dr Falk Pharma, Ferring, Galapagos, Genentech-Roche, Gilead, GSK, Hospira, Imidomics, Janssen, J&J, Lilly, Materia Prima, Mestag Therapeutics, MiroBio, Morphic, MRM Health, Mundipharma, MSD, Pfizer, Prodigest, Progenity, Prometheus, Robarts Clinical Trials, Surrozen, Takeda, Theravance, Tillots Pharma AG, VectivBio, Ventyx, Zealand Pharma.
PD: research support for from Biogen and Mundipharma; speakers’ fees from Biogen, Janssen, Dentsply and Dr Falk Pharma; congress support from Roche, Amgen, Ferring, Onis and Norgine; advisory board fees from Janssen, Sandoz, Samsung Bioepis, Galapagos and BMS.
TL: financial support for research from Abbvie, Ferring, Viatris, MSD, EG, Mundipharma, Biogen, Janssen, Pfizer, Takeda, Sandoz and Galapagos; speakers’ fees from MSD, Abbvie, Janssen, Amgen, Fresenius Kabi, Galapagos, Viatris, Ferring and Takeda; consultancy fees from Janssen, Galapagos, Amgen, Bristol Myers Squibb Fresenius Kabi, Takeda and Abbvie.
MA: consulting fees from Nikkiso Europe, Mundipharma, Janssen, Abbvie, Ferring, Galapagos and Pfizer.
FS: consulting fees from Janssen, Abbvie, Takeda, Celltrion, Ferring, Galapagos, Alfa Sigma, Sandoz and Pfizer.
SP, LH & SB: employee, paid consultant and/or shareholder of MRM Health.

Introduction

As a small but important component of the intestinal microbiota, the fungal community (mycobiota) has received increasing attention for its involvement in the pathogenesis of Crohn's disease (CD)¹. In our previous study, we identified a significantly increased relative abundance of Exophiala dermatitidis (E. dermatitidis) in the gut of patients with CD, particularly those with active disease². Caspase recruitment domain family member 9 (CARD9), a key adaptor molecule in antifungal immune responses³, has also been strongly associated with CD susceptibility⁴. In this study, we aimed to explore the potential role and underlying mechanisms of E. dermatitidis in intestinal inflammation.

Aims & Methods

To evaluate the impact of E. dermatitidis on intestinal inflammation, we generated Card9-knockout (KO) (Card9^-/-) mice and constructed a dextran sulfate sodium (DSS)-induced colitis model colonized by E. dermatitidis. The underlying molecular mechanisms were investigated by a range of biochemical approaches. Flow cytometry was performed to analyze the proportions of myeloid cells, including CX3CR1⁺ macrophages, in the colonic lamina propria (LP).

Results

Compared to mice with DSS-induced colitis alone (WT-DSS group), the DSS-treated mice colonized by E. dermatitidis (WT-DSS + Ed group) showed a significant weight loss, a worsening of disease activity, a shorter length of the large intestine, and more severe intestinal inflammation characterized by increased inflammatory cell infiltration and crypt destruction in the histological examination of colon hematoxylin and eosin (H&E) staining. Card9^-/- mice were used to further investigate whether E. dermatitis-mediated inflammation was dependent on the CARD9 signaling pathway. We observed that CARD9 deficiency protected against E. dermatitidis-exacerbated colitis, as measured by a higher trend of body weight, longer colon lengths, and amelioration of disease activity in the KO-DSS + Ed group. In addition, flow cytometric analysis of the colonic lamina propria revealed a higher percentage proportion of CX3CR1+ macrophages in the WT-DSS + Ed group and a markedly reduced proportion in the KO-DSS + Ed group. Furthermore, we identified the Mincle-Syk-CARD9-nuclear factor kappa-B (NF-κB) signaling pathway as playing an important role in mediating the inflammatory immune responses to E. dermatitidis.

Conclusion

E. dermatitidis exacerbates the DSS-induced colitis in mice through the Mincle-Syk-CARD9-NF-κB pathway with the participation of CX3CR1+ macrophages, providing a novel therapeutic strategy for CD targeting specific intestinal fungi.

References

1. Sokol H, Leducq V, Aschard H, et al. Fungal microbiota dysbiosis in IBD. Gut 2017;66:1039-1048.
2. Zeng L, Feng Z, Zhuo M, et al. Fecal fungal microbiota alterations associated with clinical phenotypes in Crohn's disease in southwest China. PeerJ 2022;10:e14260.
3. Hu A, Hu Z, Zou H, et al. CARD9 in host immunity to fungal, bacterial, viral, and parasitic infections: An update. Frontiers In Microbiology 2022;13:1021837.
4. Franke A, McGovern DPB, Barrett JC, et al. Genome-wide meta-analysis increases to 71 the number of confirmed Crohn's disease susceptibility loci. Nature Genetics 2010;42:1118-1125.

Introduction

Esophageal squamous cell carcinoma (ESCC) is associated with poor prognosis despite advances in neoadjuvant immunochemotherapy (NICT). With highly variable treatment responses, identifying reliable predictive biomarkers remains crucial. While gut microbiome influences on immunotherapy efficacy are increasingly recognized, the specific role of the fungal microbiome (mycobiome) in NICT outcomes for ESCC remains unexplored, representing a significant gap in developing personalized treatment strategies.

Aims & Methods

This study aimed to characterize gut mycobiome signatures in ESCC patients and develop fungal biomarker-based predictive models for NICT response. We conducted ITS2 sequencing on 136 fecal samples collected from 68 ESCC patients (pre- and post-NICT) and 19 healthy controls (HC). Patients were stratified as responders (R, n=26) or non-responders (NR, n=42) based on tumor regression grade (TRG) scores. We performed comprehensive analyses of mycobiome diversity, composition, co-occurrence networks, and functional pathways across groups. The Boruta algorithm was employed for feature selection to identify fungal biomarkers, followed by multilayer perceptron (MLP) machine learning with five-fold cross-validation to develop predictive models. Additionally, ESCC xenograft models were established to investigate gut mycobiome-immune interactions through fecal microbiota transplantation (FMT) and targeted fungal manipulation experiments.

Results

ESCC patients demonstrated significant mycobiome dysbiosis compared to healthy controls, characterized by reduced fungal alpha diversity and distinct beta diversity patterns. At baseline, ESCC patients showed enrichment of Rhodotorula minuta, Actinomucor elegans, and several Candida species. Responders exhibited significantly higher baseline fungal diversity than non-responders, with enrichment of Meyerozyma, Candida boidinii, Trichosporon dermatis, and Cryptococcus species, whereas non-responders showed predominance of Saccharomyces, Nakaseomyces, and pathogenic Candida species (C. glabrata, C. parapsilosis, C. albicans). Responders also displayed more complex, stable fungal co-occurrence networks with higher density and clustering coefficients. Post-NICT, both groups showed partial recovery of mycobiome diversity, with significant increases in beneficial fungal taxa. Functional pathway analysis revealed distinct metabolic profiles between responders and non-responders. Using the Boruta algorithm, we identified 23 genus-level and 22 species-level fungal biomarkers that demonstrated high predictive value. The MLP model based on genus-level markers achieved AUCs of 90.2% (training) and 91.9% (test), while the species-level model showed comparable performance. In animal experiments, mice receiving R-FMT exhibited reduced tumor growth and enhanced CD8+ T cell infiltration compared to NR-FMT recipients. Notably, Candida boidinii supplementation synergized with anti-PD-1 therapy, while Saccharomyces depletion enhanced treatment sensitivity.

Conclusion

Our findings establish the gut mycobiome as a powerful predictor of NICT response in ESCC, with machine learning models achieving >90% accuracy using fungal biomarkers. Experimental validation not only confirms mechanistic links between specific fungi and antitumor immunity but also demonstrates that targeted mycobiome modulation can enhance immunotherapy efficacy. This dual approach of prediction and intervention offers a novel framework for precision oncology in ESCC, potentially transforming clinical decision-making and treatment outcomes in this challenging malignancy.

References

1. Li J, Xu J, Zheng Y, et al. Esophageal cancer: Epidemiology, risk factors and screening. Chin J Cancer Res. 2021; 33(5): 535-547.
2. van Hagen P, Hulshof MC, van Lanschot JJ, et al. Preoperative chemoradiotherapy for esophageal or junctional cancer. N Engl J Med. 2012; 366(22): 2074-2084.
3. Schroeder W, Ghadimi M, Schloesser H, et al. Long-Term Outcome After Histopathological Complete Response with and Without Nodal Metastases Following Multimodal Treatment of Esophageal Cancer. Ann Surg Oncol. 2022.
4. Rosner S, Liu C, Forde PM, Hu C. Association of Pathologic Complete Response and Long-Term Survival Outcomes Among Patients Treated With Neoadjuvant Chemotherapy or Chemoradiotherapy for NSCLC: A Meta-Analysis. JTO Clin Res Rep. 2022; 3(9): 100384.
5. Coker OO, Nakatsu G, Dai RZ, et al. Enteric fungal microbiota dysbiosis and ecological alterations in colorectal cancer. Gut. 2019; 68(4): 654-662.
6. Dohlman AB, Klug J, Mesko M, et al. A pan-cancer mycobiome analysis reveals fungal involvement in gastrointestinal and lung tumors. Cell. 2022; 185(20): 3807-3822.
7. Wurster S, Albert ND, Kontoyiannis DP. Candida auris Bloodstream Infection Induces Upregulation of the PD-1/PD-L1 Immune Checkpoint Pathway in an Immunocompetent Mouse Model. Msphere. 2022; 7(2): e81721.

Introduction

Inflammatory bowel diseases (IBD), including Crohn's disease and ulcerative colitis, are characterized by dysregulated T-cell responses, causing chronic intestinal inflammation.¹ While anti-TNF therapy is established in IBD, targeting additional pathways may improve outcomes for patients who do not respond adequately to current treatments.² OX40L, an immune co-stimulator, has been implicated in the pathogenesis of several inflammatory conditions.³ Our study provides a comprehensive analysis of the effects of combined TNF and OX40L blockade in a murine adoptive T cell transfer (ATC) model of colitis, examining body weight, detailed histopathological parameters, and a broad range of inflammatory cytokines in colon tissue.

Aims & Methods

Colitis was induced in severe combined immunodeficiency (SCID) mice by intraperitoneal transfer of CD4⁺CD45RB^high T cells isolated from healthy BALB/c donor mice. We established five treatment groups (n=8-10): 1) CD45RB^low transfer “healthy control”, 2) CD45RB^high transfer with isotype antibodies “disease control group”, 3) CD45RB^high transfer with anti-TNF antibody (XT3.11, 250 μg/mouse), 4) CD45RB^high transfer with anti-OX40L antibody (RM134L, 250 μg/mouse), and 5) CD45RB^high transfer with both antibodies at 250 µg/mouse, each. Body weight was monitored for 46 days as a measure of disease progression and normalized to baseline weight at day 1. At study termination, we collected colon tissue for analysis of inflammatory cytokines using multiplex immunoassays and conducted histopathological examination. Histopathological scoring was performed blinded using a validated scoring system for colonic inflammation.⁴

Results

Overall, combined anti-TNF/anti-OX40L therapy was most effective in preventing body weight loss compared to the isotype disease control group, followed by anti-TNF monotherapy and anti-OX40L monotherapy (p<0.0001, p=0.0009, and p=0.03, respectively). Mean normalized body weight (MNBW) of the CD45RB^low healthy control group at termination on day 46 was 115.2 %, while CD45RB^high cell transfer in the disease control group triggered disease induction and body weight loss, with a MNBW of 93.8 % at day 46. The CD45RB^high groups treated with anti-TNF or anti OX40L monotherapy reached a MNBW of 107.6 % and 102.9%, respectively. The CD45RB^high group treated with combination therapy reached 112.6%, closest to the CD45RB^low healthy control group. Notably, only combination therapy led to a significant improvement in histopathological scoring (p=0.0176) compared to the isotype disease control. Similarly, combination treatment consistently resulted in the strongest downregulation of several inflammatory cytokines including IFNg, IL-4, or TNFa, which are increased in colon tissue in the CD45RB^high disease group compared to the CD45RB^low healthy control group.

Conclusion

Combined blockade of TNF and OX40L demonstrated significantly greater efficacy in the ATC preclinical model of IBD in prevention of weight loss, downregulation of key inflammatory cytokines, and reduction of histological damage compared to either monotherapy. These data suggest potential additive efficacy for TNF and OX40L inhibition in controlling intestinal inflammation, and warrant further exploration of dual TNF/OX40L inhibition in clinical trials, particularly among those with inadequate response to anti-TNF monotherapy.

References

1. Kaser A, Zeissig S, Blumberg RS. Inflammatory bowel disease. Annu Rev Immunol. 2010;28:573-621.
2. Vermeire S. Combination biologic therapy for ulcerative colitis. Lancet Gastroenterol Hepatol. 2023;8:288-90.
3. Fu Y, Lin Q, Zhang Z, Zhang L. Therapeutic strategies for the costimulatory molecule OX40 in T-cell-mediated immunity. Acta Pharm Sin B. 2020;10:414-33.
4. Erben U, Loddenkemper C, Doerfel K, Spieckermann S, Haller D, Heimesaat MM, Zeitz M, Siegmund B, Kühl AA. A guide to histomorphological evaluation of intestinal inflammation in mouse models. Int J Clin Exp Pathol. 2014;7:4557-76.

Disclosure

Thomas Leeuw, Rolf Keiffer, Danping Ding-Pfennigdorff, Christian Asbrand, Raaj Mehta, Matthias Herrmann are employees of Sanofi and may hold stock or equity options in Sanofi