Preclinical development of a bispecific TNFα/IL-23 neutralising domain antibody as a novel oral treatment for inflammatory bowel disease.

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.

Extended pharmacodynamic responses observed upon PROTAC-mediated degradation of RIPK2.

Proteolysis-Targeting Chimeras (PROTACs) are heterobifunctional small-molecules that can promote the rapid and selective proteasome-mediated degradation of intracellular proteins through the recruitment of E3 ligase complexes to non-native protein substrates. The catalytic mechanism of action of PROTACs represents an exciting new modality in drug discovery that offers several potential advantages over traditional small-molecule inhibitors, including the potential to deliver pharmacodynamic (PD) efficacy which extends beyond the detectable pharmacokinetic (PK) presence of the PROTAC, driven by the synthesis rate of the protein. Herein we report the identification and development of PROTACs that selectively degrade Receptor-Interacting Serine/Threonine Protein Kinase 2 (RIPK2) and demonstrate in vivo degradation of endogenous RIPK2 in rats at low doses and extended PD that persists in the absence of detectable compound.

Oral Anti-Tumour Necrosis Factor Domain Antibody V565 Provides High Intestinal Concentrations, and Reduces Markers of Inflammation in Ulcerative Colitis Patients.

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.

Flow Cytometry Reveals the Nature of Oncotic Cells.

The term necrosis is commonly applied to cells that have died via a non-specific pathway or mechanism but strictly is the description of the degradation processes involved once the plasma membrane of the cell has lost integrity. The signalling pathways potentially involved in accidental cell death (ACD) or oncosis are under-studied. In this study, the flow cytometric analysis of the intracellular antigens involved in regulated cell death (RCD) revealed the phenotypic nature of cells undergoing oncosis or necrosis.

Discovery of a First-in-Class Receptor Interacting Protein 2 (RIP2) Kinase Specific Clinical Candidate, 2-((4-(Benzo[]thiazol-5-ylamino)-6-(-butylsulfonyl)quinazolin-7-yl)oxy)ethyl Dihydrogen Phosphate, for the Treatment of Inflammatory Diseases.

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 , currently in phase 1 clinical studies.

Small Extracellular Vesicles Are Key Regulators of Non-cell Autonomous Intercellular Communication in Senescence via the Interferon Protein IFITM3.

Senescence is a cellular phenotype present in health and disease, characterized by a stable cell-cycle arrest and an inflammatory response called senescence-associated secretory phenotype (SASP). The SASP is important in influencing the behavior of neighboring cells and altering the microenvironment; yet, this role has been mainly attributed to soluble factors. Here, we show that both the soluble factors and small extracellular vesicles (sEVs) are capable of transmitting paracrine senescence to nearby cells.

Aryl Hydrocarbon Receptor Interacting Protein Maintains Germinal Center B Cells through Suppression of BCL6 Degradation.

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.

Preclinical evaluation of EPHX2 inhibition as a novel treatment for inflammatory bowel disease.

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.

Reduced CD27IgD B Cells in Blood and Raised CD27IgD B Cells in Gut-Associated Lymphoid Tissue in Inflammatory Bowel Disease.

The intestinal mucosa in inflammatory bowel disease (IBD) contains increased frequencies of lymphocytes and a disproportionate increase in plasma cells secreting immunoglobulin (Ig)G relative to other isotypes compared to healthy controls. Despite consistent evidence of B lineage cells in the mucosa in IBD, little is known of B cell recruitment to the gut in IBD. Here we analyzed B cells in blood of patients with Crohn's disease (CD) and ulcerative colitis (UC) with a range of disease activities.

Simultaneous polychromatic flow cytometric detection of multiple forms of regulated cell death.

Currently the study of Regulated Cell Death (RCD) processes is limited to the use of lysed cell populations for Western blot analysis of each separate RCD process. We have previously shown that intracellular antigen flow cytometric analysis of RIP3, Caspase-3 and cell viability dye allowed the determination of levels of apoptosis (Caspase-3/RIP3), necroptosis (RIP3/Caspase-3) and RIP1-dependent apoptosis (Caspase-3/RIP3) in a single Jurkat cell population. The addition of more intracellular markers allows the determination of the incidence of parthanatos (PARP), DNA Damage Response (DDR, H2AX), H2AX hyper-activation of PARP (H2AX/PARP) autophagy (LC3B) and ER stress (PERK), thus allowing the identification of 124 sub-populations both within live and dead cell populations.

Spatiotemporal segregation of human marginal zone and memory B cell populations in lymphoid tissue.

Human memory B cells and marginal zone (MZ) B cells share common features such as the expression of CD27 and somatic mutations in their IGHV and BCL6 genes, but the relationship between them is controversial. Here, we show phenotypic progression within lymphoid tissues as MZ B cells emerge from the mature naïve B cell pool via a precursor CD27CD45RB population distant from memory cells. By imaging mass cytometry, we find that MZ B cells and memory B cells occupy different microanatomical niches in organised gut lymphoid tissues.

Preclinical Development of a Novel, Orally-Administered Anti-Tumour Necrosis Factor Domain Antibody for the Treatment of Inflammatory Bowel Disease.

TNFα is an important cytokine in inflammatory bowel disease. V565 is a novel anti-TNFα domain antibody developed for oral administration in IBD patients, derived from a llama domain antibody and engineered to enhance intestinal protease resistance. V565 activity was evaluated in TNFα-TNFα receptor-binding ELISAs as well as TNFα responsive cellular assays and demonstrated neutralisation of both soluble and membrane TNFα with potencies similar to those of adalimumab.

Simultaneous flow cytometric immunophenotyping of necroptosis, apoptosis and RIP1-dependent apoptosis.

Flow cytometry was been widely used to measure apoptosis for many decades but the researcher has no definitive way of determining other forms of cell death using this technology. The use of Western Blot technology has numerous drawbacks in that all the cells in the sample whether live, dead or maybe undergoing multiple discrete forms of cell death are analysed as one population. Flow cytometry given that it can analyse different sub-populations of cells within a sample would reveal the expression of cell death markers within these sub-populations rather than just give a single result from the entire population.

A functional IL1RL1 variant regulates corticosteroid-induced sST2 expression in ulcerative colitis.

The ST2/IL33 signalling pathway has been associated with ulcerative colitis (UC). ST2, encoded by the IL1RL1 gene, is expressed as both a membrane-anchored receptor (ST2L) activated by IL33 and as a soluble receptor (sST2) with anti-inflammatory properties. In UC patients, sST2 is further increased by corticosteroid treatment; however, the glucocorticoid-mediated molecular regulation remains unknown.

Discovery of a First-in-Class Receptor Interacting Protein 1 (RIP1) Kinase Specific Clinical Candidate (GSK2982772) for the Treatment of Inflammatory Diseases.

RIP1 regulates necroptosis and inflammation and may play an important role in contributing to a variety of human pathologies, including immune-mediated inflammatory diseases. Small-molecule inhibitors of RIP1 kinase that are suitable for advancement into the clinic have yet to be described. Herein, we report our lead optimization of a benzoxazepinone hit from a DNA-encoded library and the discovery and profile of clinical candidate GSK2982772 (compound 5), currently in phase 2a clinical studies for psoriasis, rheumatoid arthritis, and ulcerative colitis.

The Identification and Pharmacological Characterization of 6-(tert-Butylsulfonyl)-N-(5-fluoro-1H-indazol-3-yl)quinolin-4-amine (GSK583), a Highly Potent and Selective Inhibitor of RIP2 Kinase.

RIP2 kinase is a central component of the innate immune system and enables downstream signaling following activation of the pattern recognition receptors NOD1 and NOD2, leading to the production of inflammatory cytokines. Recently, several inhibitors of RIP2 kinase have been disclosed that have contributed to the fundamental understanding of the role of RIP2 in this pathway. However, because they lack either broad kinase selectivity or strong affinity for RIP2, these tools have only limited utility to assess the role of RIP2 in complex environments.

Proteolytic cleavage and loss of function of biologic agents that neutralize tumor necrosis factor in the mucosa of patients with inflammatory bowel disease.

Background & Aims: Many patients with inflammatory bowel disease (IBD) fail to respond to anti-tumor necrosis factor (TNF) agents such as infliximab and adalimumab, and etanercept is not effective for treatment of Crohn's disease. Activated matrix metalloproteinase 3 (MMP3) and MMP12, which are increased in inflamed mucosa of patients with IBD, have a wide range of substrates, including IgG1. TNF-neutralizing agents act in inflamed tissues; we investigated the effects of MMP3, MMP12, and mucosal proteins from IBD patients on these drugs.

Interleukin 6 Increases Production of Cytokines by Colonic Innate Lymphoid Cells in Mice and Patients With Chronic Intestinal Inflammation.

Background & Aims: Innate lymphoid cells (ILCs) are a heterogeneous group of mucosal inflammatory cells that participate in chronic intestinal inflammation. We investigated the role of interleukin 6 (IL6) in inducing activation of ILCs in mice and in human beings with chronic intestinal inflammation.

Methods: ILCs were isolated from colons of Tbx21(-/-) × Rag2(-/-) mice (TRUC), which develop colitis; patients with inflammatory bowel disease (IBD); and patients without colon inflammation (controls).

Developing in vitro expanded CD45RA+ regulatory T cells as an adoptive cell therapy for Crohn's disease.

Background And Aim: Thymus-derived regulatory T cells (Tregs) mediate dominant peripheral tolerance and treat experimental colitis. Tregs can be expanded from patient blood and were safely used in recent phase 1 studies in graft versus host disease and type 1 diabetes. Treg cell therapy is also conceptually attractive for Crohn's disease (CD).

A CD3-specific antibody reduces cytokine production and alters phosphoprotein profiles in intestinal tissues from patients with inflammatory bowel disease.

Background & Aims: T cells mediate the development of inflammation in inflammatory bowel disease (IBD). We investigated the effects of an antibody against CD3 called otelixizumab, which induces immune tolerance, in intestinal mucosa samples from patients.

Methods: Intestinal tissues were isolated from patients undergoing routine endoscopy or from patients undergoing intestinal surgery for colon cancer or IBD; healthy surrounding tissues were collected as controls.

A role for gut-associated lymphoid tissue in shaping the human B cell repertoire.

We have tracked the fate of immature human B cells at a critical stage in their development when the mature B cell repertoire is shaped. We show that a major subset of bone marrow emigrant immature human B cells, the transitional 2 (T2) B cells, homes to gut-associated lymphoid tissue (GALT) and that most T2 B cells isolated from human GALT are activated. Activation in GALT is a previously unknown potential fate for immature human B cells.

Proinflammatory Vδ2+ T cells populate the human intestinal mucosa and enhance IFN-γ production by colonic αβ T cells.

In nonhuman primates, Vγ9Vδ2(+) (Vδ2)T cells proliferate and accumulate in mucosal tissues following microbial activation. Human Vδ2T cells produce proinflammatory cytokines in response to bacterial species that colonize the gut, but the role played by Vδ2T cells in intestinal immunity is unknown. We hypothesized that circulating Vδ2T cells can populate the human intestine and contribute to mucosal inflammation.

Association of innate immune activation with latent Epstein-Barr virus in active MS lesions.

Objective: To determine whether the activation of innate immune responses, which can be elicited by pathogenic and endogenous triggers, is associated with the presence of Epstein-Barr virus (EBV) infection in the multiple sclerosis (MS) brain.

Methods: White matter postmortem MS (n = 10) and control tissue (n = 11) was analyzed for the expression of the proinflammatory cytokine interferon α (IFNα) by immunohistochemistry and for EBV by using the highly sensitive method of EBV-encoded RNA (EBER) in situ hybridization.

Results: We detected overexpression of IFNα in active areas of white matter MS lesions but not in inactive MS lesions, normal-appearing white matter, or normal brains.

Translational Mini-Review Series on B cell subsets in disease. Transitional B cells in systemic lupus erythematosus and Sjögren's syndrome: clinical implications and effects of B cell-targeted therapies.

Systemic lupus erythematosus (SLE) and Sjögren's syndrome are autoimmune disorders which are characterized by a disturbed B cell homeostasis which leads ultimately to dysfunction of various organs. One of the B cell subsets that appear in abnormal numbers is the population of transitional B cells, which is increased in the blood of patients with SLE and Sjögren's syndrome. Transitional B cells are newly formed B cells.