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Background & Aims Fecal microbiota transplantation (FMT) is an emerging treatment modality for ulcerative colitis (UC). Several randomized controlled trials have shown efficacy for FMT in the treatment of UC, but a better understanding of the transferable microbiota and their immune impact is needed to develop more efficient microbiome-based therapies for UC. Methods Metagenomic analysis and strain tracking was performed on 60 donor and recipient samples receiving FMT for active UC. Sorting and sequencing of immunoglobulin (Ig) A–coated microbiota (called IgA-seq) was used to define immune-reactive microbiota. Colonization of germ-free or genetically engineered mice with patient-derived strains was performed to determine the mechanism of microbial impact on intestinal immunity. Results Metagenomic analysis defined a core set of donor-derived transferable bacterial strains in UC subjects achieving clinical response, which predicted response in an independent trial of FMT for UC. IgA-seq of FMT recipient samples and gnotobiotic mice colonized with donor microbiota identified Odoribacter splanchnicus as a transferable strain shaping mucosal immunity, which correlated with clinical response and the induction of mucosal regulatory T cells. Colonization of mice with O splanchnicus led to an increase in Foxp3+/RORγt+ regulatory T cells, induction of interleukin10, and production of short chain fatty acids, all of which were required for O splanchnicus to limit colitis in mouse models. Conclusions This work provides the first evidence of transferable, donor-derived strains that correlate with clinical response to FMT in UC and reveals O splanchnicus as a key component promoting both metabolic and immune cell protection from colitis. These mechanistic features will help enable strategies to enhance the therapeutic efficacy of microbial therapy for UC. Clinicaltrials.gov ID NCT02516384 .

Accurate prediction of the human pharmacokinetics (PK) of a candidate monoclonal antibody from nonclinical data is critical to maximize the success of clinical trials. However, for monoclonal antibodies exhibiting nonlinear clearance due to target-mediated drug disposition, PK predictions are particularly challenging. That challenge is further compounded for molecules lacking cross-reactivity in a nonhuman primate, in which case a surrogate antibody selective for the target in rodent may be required. For these cases, prediction of human PK must account for any interspecies differences in binding kinetics, target expression, target turnover, and potentially epitope. We present here a model-based method for predicting the human PK of MAB92 (also known as BI 655130), a humanized IgG1κ monoclonal antibody directed against human IL-36R. Preclinical PK was generated in the mouse with a chimeric rat anti-mouse IgG2a surrogate antibody cross-reactive against mouse IL-36R. Target-specific parameters such as antibody binding affinity (KD), internalization rate of the drug target complex (kint), target degradation rate (kdeg), and target abundance (R0) were integrated into the model. Two different methods of assigning human R0 were evaluated: the first assumed comparable expression between human and mouse and the second used high-resolution mRNA transcriptome data (FANTOM5) as a surrogate for expression. Utilizing the mouse R0 to predict human PK, AUC0-∞ was substantially underpredicted for nonsaturating doses; however, after correcting for differences in RNA transcriptome between species, AUC0-∞ was predicted largely within 1.5-fold of observations in first-in-human studies, demonstrating the validity of the modeling approach. Our results suggest that semi-mechanistic models incorporating RNA transcriptome data and target-specific parameters may improve the predictivity of first-in-human PK.

Deficiency of interleukin (IL)-36 receptor antagonist (DITRA) syndrome is a rare autosomal recessive disease caused by mutations in IL36RN. IL-36R is a cell surface receptor and a member of the IL1R family that is involved in inflammatory responses triggered in skin and other epithelial tissues. Accumulating evidence suggests that IL-36R signaling may play a role in the pathogenesis of psoriasis. Therapeutic intervention of IL-36R signaling offers an innovative treatment paradigm for targeting epithelial cell-mediated inflammatory diseases such as the life-threatening psoriasis variant called generalized pustular psoriasis (GPP). We report the discovery and characterization of MAB92, a potent, high affinity anti-human IL-36 receptor antagonistic antibody that blocks human IL-36 ligand (α, β and γ)-mediated signaling. In vitro treatment with MAB92 directly inhibits human IL-36R-mediated signaling and inflammatory cytokine production in primary human keratinocytes and dermal fibroblasts. MAB92 shows exquisite species specificity toward human IL-36R and does not cross react to murine IL-36R. To enable in vivo pharmacology studies, we developed a mouse cross-reactive antibody, MAB04, which exhibits overlapping binding and pharmacological activity as MAB92. Epitope mapping indicates that MAB92 and MAB04 bind primarily to domain-2 of the human and mouse IL-36R proteins, respectively. Treatment with MAB04 abrogates imiquimod and IL-36-mediated skin inflammation in the mouse, further supporting an important role for IL-36R signaling in epithelial cell-mediated inflammation.

Background Biologic therapy has significantly improved treatment for UC, but nearly two-thirds of patients attenuate response. Additional therapeutic modalities are therefore needed to address the underlying pathophysiology of UC. Fecal microbiota transplant (FMT) is an emerging therapy for the treatment of UC, but several randomized controlled trials have shown variable efficacy of FMT, and the microbial mechanisms responsible for clinical response are not well understood. Therefore, using samples from our pilot FMT study (Jacob, V, et al Inflamm. Bowel Dis. 2017), we aim to identify the core transferable microbiota (CTM) in UC patients responsive to FMT therapy and to define the therapeutic mechanism of these strains in pre-clinical models. Methods IBD disease activity scores were used to define clinical response. Metagenomic sequencing of donor, recipient, and 4 week post-FMT fecal samples was performed to define the CTM. Strain level transferability was defined using StrainFinder. To define the transferable immune-reactive microbiota (TIM), IgA-seq was performed on donor, recipient, and 4 week post-FMT fecal samples. TIM strains were isolated from fecal samples and gnotobiotic mouse models were used to evaluate their impact on mucosal immunity and mouse models of colitis. Results Here, we defined a CTM associated with clinical response to FMT for UC. Strain level tracking of the CTM confirmed that clinical response correlated with strain transferability. In addition, we defined a core TIM by IgA-seq that correlated with clinical response. In humanized mouse models, these TIM were found to induce IgA in a T cell independent manner. Colonization of germ-free mice with a core TIM strain of Odoribacter induced IL-10-dependent, RORgt+/Foxp3+ iTreg cells and reduced the severity of transfer T cell colitis in mono-colonized RAG-/- mice. Conclusion Our data highlight an immune-reactive, core transferable microbiota in responders to FMT for UC. Using pre-clinical mouse models of colitis, we define the mechanistic impact of these TIM in shaping mucosal immunity and guiding the response to UC. This work provides a framework for rational selection of TIM for microbial-therapy in IBD.

Background Over two million people worldwide suffer from ulcerative colitis (UC). Biologic therapy has significantly improved treatment, but nearly two-thirds of patients attenuate response. Fecal microbiota transplant (FMT) is an emerging therapy for the treatment of UC, but the microbial mechanism responsible for clinical response is poorly understood. Using samples from our pilot FMT study (Jacob V, et al 2017), we aim to define the core transferable microbiota (CTM) in UC patients responsive to FMT therapy and its therapeutic mechanism. Methods IBD disease activity scores were used to define clinical response. Metagenomic sequencing of donor, recipient, and 4 week post-FMT fecal samples was performed to define the CTM and strain level transferability. To define the transferable immune-reactive microbiota (TIM), IgA-seq was also performed on donor and recipient samples. Patient TIM strains were isolated and tested in gnotobiotic mouse models to evaluate their impact on mucosal immunity and colitis. Results We defined a CTM associated with clinical response to FMT. CTM strain tracking confirmed that clinical response correlated with strain transferability. We defined a core TIM by IgA-seq that correlated with clinical response. In humanized mouse models, TIM induced IgA in a T cell independent manner. Colonization of germ-free mice with a core TIM strain, Odoribacter splanchnicus, robustly induced mucosal Th17 and RORgt+/Foxp3+ iTreg cells and reduced the severity of transfer T cell colitis. Our data highlights a core TIM in UC responders to FMT and the mechanistic impact of it in shaping mucosal immunity and guiding the response to UC. This provides a framework for rational selection of TIM for microbial-therapy in IBD.

Background Biologic therapy has significantly improved treatment for UC, but nearly two-thirds of patients attenuate the response. Additional therapeutic modalities are therefore needed to address the underlying pathophysiology of UC. Faecal microbiota transplant (FMT) is an emerging therapy for the treatment of UC, but several randomised controlled trials have shown variable efficacy of FMT, and the microbial mechanisms responsible for clinical response are not well understood. Therefore, using samples from our pilot FMT study (Jacob, V, et al. Inflamm. Bowel Dis. 2017), we aim to identify the core transferable microbiota (CTM) in UC patients responsive to FMT therapy and to define the therapeutic mechanism of these strains in pre-clinical models. Methods IBD disease activity scores were used to define a clinical response. Metagenomic sequencing of a donor, recipient, and 4-week post-FMT faecal samples was performed to define the CTM. Strain-level transferability was defined using the StrainFinder algorithm. To define the transferable immune-reactive microbiota (TIM), IgA-seq was performed on a donor, recipient, and 4-week post-FMT faecal samples. TIM strains were isolated from faecal samples and gnotobiotic mouse models were used to evaluate their impact on mucosal immunity and mouse models of colitis. Results Here, we defined a CTM associated with clinical response to FMT for UC. Strain-level tracking of the CTM confirmed that clinical response correlated with strain transferability. In addition, we defined a core TIM by IgA-seq that correlated with clinical response. In humanised mouse models, these TIM were found to induce IgA in a T-cell independent manner. Colonisation of germ-free mice with a core TIM strain of Odoribacter induced IL-10-dependent, RORgt+/Foxp3+ iTreg cells and reduced the severity of transfer T-cell colitis in mono-colonised RAG−/− mice. Conclusion Our data highlight an immune-reactive, core transferable microbiota in responders to FMT for UC. Using pre-clinical mouse models of colitis, we define the mechanistic impact of these TIM in shaping mucosal immunity and guiding the response to UC. This work provides a framework for the rational selection of TIM for microbial-therapy in IBD.

Improper signaling of the IL-36 receptor (IL-36R), a member of the IL-1 receptor family, has been associated with various inflammation-associated diseases. However, the requirements for IL-36R signal transduction remain poorly characterized. This work seeks to define the requirements for IL-36R signaling and intracellular trafficking. In the absence of cognate agonists, IL-36R was endocytosed and recycled to the plasma membrane. In the presence of IL-36, IL-36R increased accumulation in LAMP1+ lysosomes. Endocytosis predominantly used a clathrin-mediated pathway, and the accumulation of the IL-36R in lysosomes did not result in increased receptor turnover. The ubiquitin-binding Tollip protein contributed to IL-36R signaling and increased the accumulation of both subunits of the IL-36R.

Several imidazole-based cyclohexyl amides were identified as potent CB-1 antagonists, but they exhibited poor oral exposure in rodents. Incorporation of a hydroxyl moiety on the cyclohexyl ring provided a dramatic improvement in oral exposure, together with a ca. 10-fold decrease in potency. Further optimization provided the imidazole 2-hydroxy-cyclohexyl amide 45, which exhibited hCB-1 Ki = 3.7 nM, and caused significant appetite suppression and robust, dose-dependent reduction of body weight gain in industry-standard rat models.

A series of pyrrolopyridinones was designed and synthesized as constrained analogs of the pyrazole CB-1 antagonist rimonabant. Certain examples exhibited very potent hCB-1 receptor binding affinity and functional antagonism with K i and K b values below 10 nM, and with high selectivity for CB-1 over CB-2 (>100-fold). A representative analog was established to cause significant appetite suppression and reduction in body weight gain in industry-standard rat models used to develop new therapeutics for obesity.

Galanin is a peptide hormone which modulates a wide variety of physiological processes, including secretion, muscle contraction, cognitive function, the reproductive axis, and feeding. Two galanin receptor subtypes, GalR1 and GalR2, have been cloned; however, for GalR2 only the rat sequence has been reported in the literature. Our cloning of human GalR2 reveals its amino acid sequence to be 85% identical to rat GalR2 and 39% identical to human GalR1. Binding of [ 125 I]galanin to the human GalR2 receptor transiently expressed in COS-7 cells was saturable (K d = 0.24 nM ± 0.06 nM) with a receptor density of 383 ± 66 fmol/mg protein. Human galanin(1-30) bound with high affinity to the human GalR2 receptor, with a K i value of 0.86 ± 0.12 nM. With the identification of a second galanin receptor subtype, the specific functions of human galanin receptor subtypes can now begin to be addressed.

Ghrelin, through action on its receptor, GH secretagogue receptor type 1a (GHS-R1a), exerts a variety of metabolic functions including stimulation of appetite and weight gain and suppression of insulin secretion. In the present study, we examined the effects of novel small-molecule GHS-R1a antagonists on insulin secretion, glucose tolerance, and weight loss. Ghrelin dose-dependently suppressed insulin secretion from dispersed rat islets. This effect was fully blocked by a GHS-R1a antagonist. Consistent with this observation, a single oral dose of a GHS-R1a antagonist improved glucose homeostasis in an ip glucose tolerance test in rat. Improvement in glucose tolerance was attributed to increased insulin secretion. Daily oral administration of a GHS-R1a antagonist to diet-induced obese mice led to reduced food intake and weight loss (up to 15%) due to selective loss of fat mass. Pair-feeding experiments indicated that weight loss was largely a consequence of reduced food intake. The impact of a GHS-R1a antagonist on gastric emptying was also examined. Although the GHS-R1a antagonist modestly delayed gastric emptying at the highest dose tested (10 mg/kg), delayed gastric emptying does not appear to be a requirement for weight loss because lower doses produced weight loss without an effect on gastric emptying. Consistent with the hypothesis that ghrelin regulates feeding centrally, the anorexigenic effects of potent GHS-R1a antagonists in mice appeared to correspond with their brain exposure. These observations demonstrate that GHS-R1a antagonists have the potential to improve the diabetic condition by promoting glucose-dependent insulin secretion and promoting weight loss.