Introduction

Tumor necrosis factor (TNF)-like ligand 1A (TL1A) mediates a broad spectrum of pro-inflammatory and fibrotic effects and has been genetically linked to multiple immunologic disorders. In clinical trials, antagonist antibodies that interfere with the interaction between TL1A and Death Receptor 3 (DR3) reduce disease activity in ulcerative colitis (UC) and Crohn’s disease (CD). Here, we provide data on a novel human IgG1 effector-less monoclonal antibody (mAb) that incorporates the Xtend™ half-life extension technology. This antibody binds soluble and cell surface-expressed TL1A with high affinity and selectivity and potently inhibits TL1A-DR3 mediated signaling.

Aims & Methods

Over 1000 anti-TL1A antibodies were screened in binding and/or functional assays. Select clinical candidate antibodies were further compared to clinical-stage anti-TL1A mAbs PRA023/MK-7240/tulisokibart, RVT-3101/RG6631, and TEV-48574. Binding affinity of the lead antibodies to soluble TL1A was determined by surface plasmon resonance (SPR). Binding to cell plasma membrane-expressed TL1A was confirmed by flow cytometry (FACS). Competitive inhibition of TL1A binding to DR3 and its decoy receptor DcR3 was determined by ELISA and biolayer interferometry. Inhibition of TL1a binding to plasma membrane-expressed DR3 was evaluated using FACS. Antibody antagonism of cellular DR3 signaling by exogenous TL1A was evaluated through assessment of TF-1 cell apoptosis and interferon γ (IFNγ) secretion from CD4⁺ T cells. Lead antibodies incorporating the Xtend™ Fc domain (M428L/N434S substitutions) to improve half-life were evaluated in pharmacokinetic studies using human FcRn knock-in mice and non-human primates.

Results

The clinical candidate anti-TL1A antibodies bind with high affinity and specificity to trimeric TL1A and not to the related TNF ligand superfamily members LIGHT, TRAIL and FasL. The lead antibodies demonstrate an apparent K_D of less than 20 pM for soluble TL1A at 37^oC and bind to TL1A over-expressed on the cell plasma membrane. Further, these antibodies potently inhibit TL1A-induced apoptosis of TF-1 cells, and IFNγ secretion from human CD4⁺ T cells - all with IC₅₀ values comparable to those of RG6631 and lower than those of tulisokibart. Lead antibodies demonstrate an improved serum half-life in human FcRn knock-in mice relative to the non-half-life extended RG6631. In preliminary non-human primate studies, the clinical candidate antibodies demonstrate half-lives greater than 20 days, with the expected antibody-TL1A complex formation. Allometric scaling using parameters adjusted for half-life engineered antibodies predict human half-lives of 70 days or higher, potentially enabling dosing intervals of Q8wk or longer.

Preliminary formulation development supports a high concentration, citrate-free formulation above 150 mg/mL, suitable for subcutaneous administration.

Conclusion

The clinical candidate antibodies bind with high affinity and specificity to TL1A and demonstrate potent blockade of TL1A-mediated DR3 receptor signaling. In human FcRn knock-in mice and in cynomolgus monkeys, these antibodies show an extended half-life consistent with FDA-approved antibodies using the Xtend™ technology. These data demonstrate the therapeutic potential of the clinical candidate antibodies in the treatment of TL1A-mediated diseases such as ulcerative colitis and Crohn’s disease - with the advantage of infrequent subcutaneous dosing.

Disclosure

The authors are employees of Xencor, Inc.

Introduction

Checkpoint agonism represents a promising class of therapies for the treatment of autoimmune and inflammatory diseases, including ulcerative colitis (UC), where unmet needs persist. Engagement of checkpoint receptor PD-1 by its natural ligand PD-L1 is critical for regulation of T cells. Rosnilimab is an investigational PD-1 agonist IgG1 antibody designed to optimize inhibitory signaling through PD-1, does not block PD-1 binding to PD-L1, and depletes PD-1^high pathogenic T cells while agonizing remaining PD-1+ T cells. The percentages of PD-1+ T cells in the peripheral blood of moderate and severe UC patients are higher than in mild UC patients (1-4). Reduction of elevated PD-1^high Tph cells in both UC colon and peripheral blood correlates with remission (2). Therapeutic reduction of PD-1^high Tph cells and inflammatory cytokines from PD-1+ T cells, by depletion and agonism signaling together, has the potential to reduce overactive immune responses and induce remission.

Aims & Methods

The potency of the agonism and depletion mechanisms of rosnilimab was evaluated in vitro using peripheral blood mononuclear cells (PBMCs) from UC patients. Rosnilimab is a human IgG1 antibody with Fc effector function. A version of rosnilimab that lacks Fc effector function was generated by mutating the IgG1 Fc domain at L234A and L235A (LALA). PBMCs from UC patients were activated with anti-CD3 and anti-CD28, cultured for several days with rosnilimab IgG1, rosnilimab IgG1-LALA, or isotype control and analyzed for the number of PD-1+ T cells and levels of inflammatory cytokines. In addition, the therapeutic potential of depletion and agonism of pathogenic PD-1+ T cells was evaluated in a mouse model of UC using a surrogate of rosnilimab. The surrogate consists of the fab domains of rosnilimab formatted with a mouse IgG2a Fc domain which enables immune cell depletion in mice. On Day 0, hPD1 CD4+CD45Rb+ T cells donor cells were adoptively transferred into Rag2-/- recipients to elicit UC-like disease. Starting on Day 21, 10 mpk of isotype control mIgG2a, rosnilimab-mIgG2a, or control anti-mIL-12 p40 were dosed intraperitoneally twice weekly for 4 weeks. This model system allowed assessment of the therapeutic potential of depletion and agonism mechanisms of rosnilimab on hPD1+ T cells, with a depletion-enabled mouse IgG2a isotype, in the context of T cell-driven gut inflammation and pathology.

Results

In PBMCs from UC patients, at therapeutically relevant concentrations, rosnilimab IgG1 reduced PD-1^high T cells by 90%, while rosnilimab IgG1-LALA reduced PD-1^high T cells by 14%. Isotype control did not reduce T cells. Furthermore, rosnilimab IgG1 reduced secretion of inflammatory cytokines interferon gamma (IFN-γ) and CXCL13 compared to rosnilimab IgG1-LALA or isotype control. In the hPD-1 mouse model of UC, rosnilimab mIgG2a reduced body weight loss, colon pathology, IFN-γ and CXCL13 as assessed by spatial transcriptomics of the colon, and infiltration of hPD-1 CD4+ T cells into the colon as assessed by immunofluorescence, compared to isotype control. In addition, rosnilimab mIgG2a demonstrated efficacy comparable to anti-IL-12 p40 antibody treatment.

Conclusion

Rosnilimab, a PD-1 checkpoint agonist antibody, functions to reduce pathogenic PD-1+ T cells by both depletion and agonism. These in vivo and in vitro results are consistent with the mechanisms of depletion and agonism and support the strong rationale for evaluating the therapeutic potential of rosnilimab in an ongoing phase 2 study in UC (NCT06127043).

References

1. Shi W, Zhang Y, Hao C, Guo X, Yang Q, Du J, Hou Y, Cao G, Li J, Wang H, Fang W. PeerJ. 2023; May 31;11:e15481.
2. Long Y, Xia C, Sun Y, Ma Y, Xu L, Song Y, Liu C. Immunol Lett. 2021; 233:2-10.
3. Roosenboom B, Horjus Talabur Horje CS, Smids C, Leeuwis JW, van Koolwijk E, Groenen MJM, Wahab PJ, van Lochem EG. Scand J Gastroenterol. 2021; 56(6):671-679.
4. Nguyen J, Finkelman BS, Escobar D, Xue Y, Wolniak K, Pezhouh M. Hum Pathol. 2022; 126:19-27.

Disclosure

All authors are employees and shareholders of AnaptysBio, Inc.

Introduction

The intestinal epithelium is the most regenerative tissue in the human body and an essential interface between the microbial ecosystem and the underlying mucosa, orchestrating both immune-mediated and metabolic functions. Mitochondrial signaling and metabolism controls the phenotype and function of intestinal epithelial cells (IECs) and mitochondrial perturbation is associated with chronic inflammation. We hypothesize that disruption of microbial-metabolic circuits and impaired metabolic flexibility of IECs contribute to the initiation and progression of tissue injury underlying chronic inflammation.

Aims & Methods

To address the role of mitochondrial impairment in epithelial homeostasis, conditional deletion of Heat shock protein 60 (Hsp60) was induced in the epithelium of Hsp60^Δ/ΔIEC and Hsp60^Δ/ΔIEC;Il10^-/- mice under specific pathogen free and germfree conditions. Blinded histological analysis was used to assess metabolic inury and tissue inflammation. Additional 16S rRNA and shallow shotgun sequencing as well as metaproteomic analysis were performed to identify microbiome changes at the compositional and functional levels. Nanostring analysis and single-cell RNA sequencing (scRNA-seq) were used to identify epithelial cell population-specific transcriptional responses and to validate these gene signatures in resected tissues from Crohn’s disease patients.

Results

Hsp60 deletion in IECs leads to the disruption of mitochondrial metabolism and the development of irregular crypt architecture, referred to as metabolic injury. Tissue lesions and IBD-related colitis are accelerated in Hsp60^Δ/ΔIEC;Il10^-/- mice. Germfree mice are protected from injury and inflammation, demonstrating a causal contribution of bacteria to metabolic injury. Mitochondrial perturbation in Hsp60^Δ/ΔIEC mice is associated with microbial dysbiosis, including an increased abundance of Bacteroides spp., which are maintained during sustained inflammation in Hsp60^Δ/ΔIEC;Il10^-/- mice. Consistently, B. caecimuris triggers tissue aberration in monocolonized-Hsp60^Δ/ΔIEC mice, supporting its deleterious role in tissue homeostasis. Furthermore, metaproteomic analysis revealed a metabolic adaptation of B. caecimuris in response to tissue injury. Importantly, the clinical relevance of Bacteroides spp. is confirmed in a combined analysis of three IBD cohorts (N=560 patients). Transcriptional profiling of the epithelium identified a gene signature in injury-associated sub-populations, including Ido1, Nos2 and Duox2. Remarkably, the identified metabolic injury gene signature discriminates inflamed (N=196) versus non-inflamed (N=147) mucosa in CD patients, thus validating the human relevance of epithelial mitochondrial disruption in IBD.

Conclusion

Bacterial presence is a key driver of intestinal injury and metabolic injury associates with dysbiotic changes, notably expansion of Bacteroides spp. and their metabolic adaptation. Furthermore, mitochondrial perturbation induces a metabolic injury signature, which differentiates inflamed versus non-inflamed mucosa in IBD patients, strengthening the hypothesis of unresolved metabolic injury as a novel concept in the pathogenesis of intestinal inflammation.

Introduction

Therapeutic goals in inflammatory bowel disease (IBD) are constantly evolving and becoming increasingly ambitious. However, there are still unmet clinical needs. The concepts of response and remission through activity indexes are well established. Nevertheless, there is a wide range of clinical situations where they have not been standardized yet. Many patients show some degree of improvement, or partial response, but fail to meet defined objectives within the desired timeframe, leading to suboptimal therapeutic management. Therefore, early identification of partial responders is crucial for timely intervention, improving disease prognosis and quality of life.

Aims & Methods

Aim: To reach a consensus on the concept of partial response for different profiles of patients with IBD.
Methods: Initially, a scientific committee comprising 4 gastroenterologists and 2 hospital pharmacists involved in IBD management performed a literature review, defined the clinical scenarios and created the content of the Delphi questionnaire. Statements regarding partial response definitions in the short, medium, and long-term were defined for four different patient profiles: moderately-active ulcerative colitis (UC), acute severe UC; luminal and perianal Crohn´s Disease (CD). A group of 60 IBD experts in Spain participated in this Delphi consultation. Panelists rated their agreement on a likert scale. Consensus was reached when at least 70% of panelists agreed or disagreed with a statement. Statements without consensus in the first round proceeded to subsequent iterative rounds until a consensus was met.

Results

Consensus was reached on 18 out of 22 statements in the first round and 4 in the second. The consensus definitions for partial response in different patient profiles are as follows:
1. Moderate UC (outpatient treatment):
- Short term: Clinical improvement without achieving clinical response.
- Medium term: Improvement without achieving clinical remission, with persistently elevated biomarkers (e.g. fecal calprotectin).
- Long term: Improvement without achieving clinical remission, with persistent inflammatory activity in biochemical parameters, imaging procedures, or endoscopic examinations.
2. Acute severe UC (admission):
- Short term: Clinical improvement without clinical response.
- Medium term: Clinical response.
- Long term: Improvement without achieving clinical remission, with persistent inflammatory activity in biochemical parameters, imaging procedures, or endoscopic examinations.
3. Luminal CD:
- Short term: Clinical improvement without achieving clinical response.
- Medium term: Clinical improvement without achieving clinical remission, with persistent inflammatory activity in biochemical tests.
- Long term: Clinical improvement without achieving clinical remission, with persistent inflammatory activity in biochemical parameters, imaging procedures, or endoscopic examinations.
4. CD with perianal involvement:
- Short/medium term: Clinical improvement in the perianal area without clinical response.
- Long term: Clinical response of the perianal disease, with persistent inflammatory activity in imaging procedures or drainage of external openings.

Conclusion

​Broad consensus was achieved on partial response definitions for various patient profiles in IBD. These standardized criteria can guide healthcare professionals in decision-making and assessing therapeutic effectiveness, ultimately improving patient care and outcome assessment.

Disclosure

Eugenia Vispo, Beatriz Garcillán and Elia Moreno work for the Medical Affairs Department of Johnson & Johnson

Introduction

Fibrostenosis represents one of the major clinical complications of Crohn’s Disease (CD). However, the molecular mechanisms underlying intestinal fibrogenesis remain poorly understood and anti-fibrotic therapy has not yet been developed. Growing evidence supports that oxidative stress and the epithelial-mesenchymal transition (EMT) process contribute to the development and progression of fibrosis.

Aims & Methods

Based on these evidences, we assessed the role of lipids in fibrogenesis and whether mitochondria dysfunction can support fibrosis formation.
Spatial metabolomics analysis and adipophilin (ADFP) staining were performed on surgical specimens from fibrotic ileum and the surrounding region (healthy) derived from CD patients. Fibroblasts isolated from fibrotic and healthy ileum and human induced pluripotent stem cell-derived intestinal organoids (iHOs) were treated for 1-3 weeks with lipid mix (2%) and analyzed by RNA sequencing. Pathways significantly modulated were validated by RT-PCR, WB, FACS analysis, and proliferative, migratory and contraction assays, and mitochondrial functions by Seahorse analyzer.

Results

Spatial metabolomics revealed a significant accumulation of lipids characterized by very long-chain fatty acids in the fibrotic area compared to surrounding healthy regions, which was further confirmed by ADFP staining. RNA-seq analysis showed overlapping dysregulated pathways between healthy fibroblasts stimulated with lipids and fibrotic fibroblasts, in particular genes related to cell cycle, cytoskeletal reorganization, migration, contractility and cell metabolism (oxidative stress) which were further confirmed by functional assays. Among the genes related to oxidative stress, lipid stimulation significantly decreased NFE2L2 and promoted an increase in CPT1A, gene encoding for a fatty oxidation mediator.
Moreover, lipid-stimulated fibroblasts showed a reduction in mitochondrial respiration, particularly basal and maximal respiration, and spare respiratory capacity by Seahorse analysis. On the other hand, long exposure of iHOs to lipids promoted the EMT process through the upregulation of TWIST, ZEB, and VIM.

Conclusion

Lipid stimulation induced myofibroblast activation through oxidative stress and alteration of mitochondrial metabolism, and increased the expression of EMT transcription factors in intestinal epithelial cells (iHOs) that may support fibroblast-mediated pro-fibrotic activity. These data indicate that regulators of fatty acid oxidation involved in the fibrotic process may represent new potential targets for the treatment of fibrostenotic CD patients.