Integrin Antibody Research Articles

Dual targeting of αv and α5β1 integrins in triple-negative breast cancer (TNBC).

e15073 Background: Integrin αv has been implicated in a stem-phenotype of breast cancer (1) and is cross-regulated by fibronectin-binding integrin α5β1 in tumor cells (2). We hypothesized that these integrins are expressed in TNBC cells and dual targeting of αv and α5β1 integrins by a bispecific antibody (BsAbαv/α5β1) will be superior to monospecific integrin antibodies (MsAbs) alone or in combination. Methods: A representative panel of basal-subtype TNBC lines was assembled: MDA-MB-231 (P53 mutated, KRAS mutated), MDA-MB-468 (P53 mutated, PTEN deleted, EGFR amplified) and BT-20 (P53 and PIK3CA mutated, EGFR amplified). Integrin expression was determined by flow cytometry and Western blot in cell lines and by IHC in the residual cancer burden (RCB) of TNBC specimens following neoadjuvant chemotherapy (positive: >2+ membranous, >10% cells). The impact of MsAbs targeting αv and α5β1 integrins alone or in combination versus the BsAbαv/α5β1 was assessed in adhesion, migration, and clonogenic survival. The impact of integrin blockade on nuclear localization of basal markers including FAK, YAP, EGFR and beta-catenin, was assessed. Results: Robust integrin αv expression was present in all TNBC lines with variable α5β1 expression. Marked reduction in α5 and in αv expression following BsAbαv/α5β1 was uniquely demonstrated by Western blot and/or flow cytometry compared to MsAbs, likely due to endosomal trafficking and lysosomal degradation of target integrins as reported previously (Joshi, MCR. 2020). Combined α5 and αv neutralization with MsAbs was superior to individual single agents in blocking adhesion and migration across all TNBC lines studied. However, the BsAbα5β1/αv was superior to combinatorial MsAbs in MDA-MB-468 and BT-20 lines in chemotaxis assays. A significant (>50%) reduction in clonogenic cell survival was noted with the BsAbα5β1/αv compared to MsAbs. BsAbαv/α5β1 blocked nuclear localization of FAK, EGFR, activated beta-catenin, and YAP in MDA-MB-231 uniquely compared to MsAbs alone or in combination. RCB tumor cells were strongly positive for membranous αv [7/9, (78%)]. Diffuse expression of integrin α5 (67%) in alpha smooth-muscle actin-expressing fibroblasts with dual staining for integrin αv was found. Marked CD68+ macrophage and CD3+ infiltration was seen in tumor and stroma of all RCB tumors. Conclusions: Combinatorial α5 and αv integrin targeting with BsAbα5/αvβ1 defines a novel therapeutic strategy with a distinct mechanism of action for further investigation in TNBC. Enrichment of integrins αv and α5 in the inflamed RCB of TNBC tumors and stroma respectively point to a potential role of these integrins in epithelial-stromal interactions and therapeutic resistance. 1. Desgrosellier, Dev Cell. 2014. 2. Joshi, MCR. 2020.

Roles of Integrin in Cardiovascular Diseases: From Basic Research to Clinical Implications.

Cardiovascular diseases (CVDs) pose a significant global health threat due to their complex pathogenesis and high incidence, imposing a substantial burden on global healthcare systems. Integrins, a group of heterodimers consisting of α and β subunits that are located on the cell membrane, have emerged as key players in mediating the occurrence and progression of CVDs by regulating the physiological activities of endothelial cells, vascular smooth muscle cells, platelets, fibroblasts, cardiomyocytes, and various immune cells. The crucial role of integrins in the progression of CVDs has valuable implications for targeted therapies. In this context, the development and application of various integrin antibodies and antagonists have been explored for antiplatelet therapy and anti-inflammatory-mediated tissue damage. Additionally, the rise of nanomedicine has enhanced the specificity and bioavailability of precision therapy targeting integrins. Nevertheless, the complexity of the pathogenesis of CVDs presents tremendous challenges for monoclonal targeted treatment. This paper reviews the mechanisms of integrins in the development of atherosclerosis, cardiac fibrosis, hypertension, and arrhythmias, which may pave the way for future innovations in the diagnosis and treatment of CVDs.

β1 integrins play a critical role maintaining vascular integrity in the hypoxic spinal cord, particularly in white matter

Interactions between extracellular matrix (ECM) proteins and β1 integrins play an essential role maintaining vascular integrity in the brain, particularly under vascular remodeling conditions. As blood vessels in the spinal cord are reported to have distinct properties from those in the brain, here we examined the impact of β1 integrin inhibition on spinal cord vascular integrity, both under normoxic conditions, when blood vessels are stable, and during exposure to chronic mild hypoxia (CMH), when extensive vascular remodeling occurs. We found that a function-blocking β1 integrin antibody triggered a small degree of vascular disruption in the spinal cord under normoxic conditions, but under hypoxic conditions, it greatly enhanced (20-fold) vascular disruption, preferentially in spinal cord white matter (WM). This resulted in elevated microglial activation as well as marked loss of myelin integrity and reduced density of oligodendroglial cells. To understand why vascular breakdown is localized to WM, we compared expression levels of major BBB components of WM and grey matter (GM) blood vessels, but this revealed no obvious differences. Interestingly however, hypoxyprobe staining demonstrated that the most severe levels of spinal cord hypoxia induced by CMH occurred in the WM. Analysis of brain tissue revealed a similar preferential vulnerability of WM tracts to show vascular disruption under these conditions. Taken together, these findings demonstrate an essential role for β1 integrins in maintaining vascular integrity in the spinal cord, and unexpectedly, reveal a novel and fundamental difference between WM and GM blood vessels in their dependence on β1 integrin function during hypoxic exposure. Our data support the concept that the preferential WM vulnerability described may be less a result of intrinsic differences in vascular barrier properties between WM and GM, and more a consequence of differences in vascular density and architecture.

Efficacy of vedolizumab during intravenous induction therapy in ulcerative colitis and Crohn's disease: post hoc analysis of patient-reported outcomes from the VISIBLE 1 and 2 studies.

Vedolizumab is an anti-α4β7 integrin antibody used to treat moderate to severe ulcerative colitis (UC) and Crohn's disease (CD). This post hoc analysis of patient-reported outcomes (PROs) from the VISIBLE 1 (NCT02611830) and 2 (NCT02611817) phase 3 studies evaluated onset of treatment effect on patient-reported symptoms during 6-week vedolizumab induction. Patient-reported stool frequency (SF) and rectal bleeding (RB) (UC Mayo score), and SF and abdominal pain (AP) in CD were collected via electronic diary from VISIBLE patients receiving one or more open-label intravenous (IV) vedolizumab induction doses (weeks 0 and 2). PRO data were analyzed using descriptive statistics. Data from 994 patients (UC 383, CD 611) showed mean ratings for all PROs declined consistently week-on-week from baseline through week 6, with early onset of improvement. By week 2, 22% of patients with UC reported RB improvement (≥1-point reduction in RB subscore, 7-day mean), rising to 45% by week 6. By week 6, 18% of patients with UC achieved SF improvement (SF subscore 0; 21% antitumor necrosis factor alpha [anti-TNFα] naive, 13% anti-TNFα experienced). SF improvement in patients with CD (reduction of ≥3 stools, 7-day mean) was achieved by 32% at week 6 (34% anti-TNFα naive, 30% anti-TNFα experienced). Fewer patients with CD reported severe/moderate AP at week 6 (5.1%/28.5%) than baseline (14.6%/61.5%). SF decline appeared greater and faster for anti-TNFα-naive vs. anti-TNFα-experienced patients (UC and CD). Results indicate early onset of patient-reported UC and CD symptom improvement during vedolizumab IV induction in VISIBLE 1 and 2.

Etrolizumab-s fails to control E-Cadherin-dependent co-stimulation of highly activated cytotoxic T cells

Despite promising preclinical and earlier clinical data, a recent phase III trial on the anti-β7 integrin antibody etrolizumab in Crohn’s disease (CD) did not reach its primary endpoint. The mechanisms leading to this outcome are not well understood. Here we characterize the β7+ T cell compartment from patients with CD in comparison to cells from individuals without inflammatory bowel disease. By flow cytometric, transcriptomic and functional profiling of circulating T cells, we find that triple-integrin-expressing (α4+β7+β1hi) T cells have the potential to home to the gut despite α4β7 blockade and have a specific cytotoxic signature. A subset of triple-integrin-expressing cells readily acquires αE expression and could be co-stimulated via E-Cadherin-αEβ7 interactions in vitro. Etrolizumab-s fails to block such αEβ7 signalling at high levels of T cell stimulation. Consistently, in CD patients treated with etrolizumab, T cell activation correlates with cytotoxic signatures. Collectively, our findings might add one important piece to the puzzle to explain phase III trial results with etrolizumab, while they also highlight that αEβ7 remains an interesting target for future therapeutic approaches in inflammatory bowel disease.

P776 A Prospective, Multicenter, Single-arm, Observational Study to Evaluate the Safety and Effectiveness of Vedolizumab in Real-world Clinical Practice in China: Interim Analysis of Safety results

Abstract Background Crohn's disease (CD) and ulcerative colitis (UC) are categorized as inflammatory bowel disease [IBD] usually treated by corticosteroids, immunomodulators and biologics, etc. Vedolizumab, a gut-selective α4β7 integrin antibody, has been approved in China for IBD patients who are intolerant /lost response to conventional therapies or TNF-α inhibitors. Of late, there is a paucity of data on the safety and effectiveness of vedolizumab in real-world clinical practice in China. This study evaluates the safety and effectiveness of vedolizumab in adult patients with UC or CD Here, we present the first interim analysis. Methods In this multicenter, single arm, prospective, observational, real-world study (NCT04872491), adults (aged >18 years) with UC or CD were administered intravenous vedolizumab 300 mg at week 0, 2, 6, and thereafter every 8 weeks. Incidence of adverse events (AEs), serious AEs (SAEs), Treatment-related AEs (TRAEs) and AEs of special interest (AESIs) were the primary endpoints Patients with administration of at least 1 dose of VDZ were included and observed 18 weeks after completion of the study or early termination, up to maximum of 72 weeks. AESIs included infections, malignancies, infusion-related reactions, and hypersensitivity. The secondary endpoints included the effectiveness of vedolizumab in terms of clinical response, clinical remission and endoscopic remission assessed at week 14 and week 54 after vedolizumab initiation. Results A total of 307 patients (UC, n=245; CD, n=62) with a median age of 41 years (range: 18-86) were included in the interim analysis. Median duration was 883 days (UC: 1035 days; CD: 731 days) and 105 days (UC: 107 days; CD: 99 days) for IBD and vedolizumab exposure, respectively. A total of 105 (34.20%) patients (UC: 35.92%; CD: 27.42%) developed 225 AEs and the most frequent AEs were infections and infestations (16.61%) followed by gastrointestinal disorders (GID) (14.66%) and general disorders (GD) (4.56%). Colitis and pyeria were the most common GID and GD, respectively occurred in 7(2.28%) patients each (Table 1). Forty-one TRAEs were observed and ten AEs lead to study discontinuation. Total 13 AESIs and 34 SAEs were reported and at least one AESI was reported in 10 patients. No deaths were reported during the study (Table 2). A total of 79.19% (118/149) and 34.48% (10/29) patients with UC and CD respectively, achieved clinical response at week 14 (±4 weeks). Clinical remission and endoscopic remission was achieved by 2/2 and 1/1 patients with UC, respectively at week 54 (±4 weeks). Conclusion Vedolizumab demonstrated favorable safety profile and treatment effectiveness among Chinese adult patients with UC or CD in real-world clinical practice.

Nanoscale Topography of Anodic TiO2 Nanostructures Is Crucial for Cell-Surface Interactions.

Anodic titanium dioxide (TiO2) nanostructures, i.e., obtained by electrochemical anodization, have excellent control over the nanoscale morphology and have been extensively investigated in biomedical applications owing to their sub-100 nm nanoscale topography range and beneficial effects on biocompatibility and cell interactions. Herein, we obtain TiO2 nanopores (NPs) and nanotubes (NTs) with similar morphologies, namely, 15 nm diameter and 500 nm length, and investigate their characteristics and impact on stem cell adhesion. We show that the transition of TiO2 NPs to NTs occurs via a pore/wall splitting mechanism and the removal of the fluoride-rich layer. Furthermore, in contrast to the case of NPs, we observe increased cell adhesion and proliferation on nanotubes. The enhanced mesenchymal stem cell adhesion/proliferation seems to be related to a 3-fold increase in activated integrin clustering, as confirmed by immunogold labeling with β1 integrin antibody on the nanostructured layers. Moreover, computations of the electric field and surface charge density show increased values at the inner and outer sharp edges of the top surfaces of the NTs, which in turn can influence cell adhesion by increasing the bridging interactions mediated by proteins and molecules in the environment. Collectively, our results indicate that the nanoscale surface architecture of the lateral spacing topography can greatly influence stem cell adhesion on substrates for biomedical applications.

Effectiveness and safety of vedolizumab induction with or without budesonide in patients with moderately to severely active Crohn’s disease in Europe: a retrospective observational study

BackgroundVedolizumab (VDZ), a gut-selective anti-lymphocyte trafficking integrin antibody, is effective in treating patients with moderately to severely active Crohn’s disease (CD). In this study, we examined the real-world effectiveness and safety of induction therapy using VDZ alone or in combination with budesonide (VDZ + BUD) among patients with CD in Belgium, Israel, and Switzerland.MethodsThis retrospective chart review analysis included adult patients with moderately to severely active CD who started induction treatment with VDZ or VDZ + BUD (January 2015 through January 2019). The primary objective of this study was to assess the effectiveness in terms of clinical remission of VDZ alone or VDZ + BUD using patient-reported outcomes (PRO) of abdominal pain (AP) and/or loose stool frequency (LSF) (PRO-2) at weeks 0, 2, 6, 10, and 14. Regression models were used to assess differences and associations between the treatment groups.ResultsOverall, 123 patients were included (VDZ, n = 73; VDZ + BUD, n = 50). Clinical remission rates at week 14 were 71.4% (50/70) and 68.0% (34/50) with VDZ and VDZ + BUD, respectively. Mean percentage change in AP and LSF from baseline to week 14 was comparable between the groups. Median (95% confidence interval [CI]) time to clinical remission was 91 [70.0–98.0] and 95 [70.0–98.0] days, respectively. One patient in each group discontinued VDZ and 68.0% of patients in the VDZ + BUD group discontinued BUD before week 14. The rates of overall adverse events were similar between the groups (VDZ, 23.3%; VDZ + BUD, 26.0%).ConclusionsIn this retrospective study, VDZ alone and VDZ + BUD showed similar high remission rates in patients with moderately to severely active CD. Prospective randomized studies are needed to conclude on the role of combining VDZ with BUD.Trial registrationNot applicable.

Targeting the JAK2V617F Hematopoietic Stem Cell through Beta1 Integrin

The major barrier to progress in treatment of primary myelofibrosis (PMF) is the lack of effective therapies targeting the malignant JAK2 V617F hematopoietic stem cell (HSC), the source of disease progression and relapse. Expression of JAK2 V617F induces abnormal proliferation and differentiation of HSC, affecting all blood lineages but most prominently the erythro-megakaryocytic lineage, leading to clonal megakaryocyte proliferation, erythrocytosis and thrombocytosis. In our previously published work ( Matsuura et al, Blood 2020) we identified elevated expression of the α5 subunit of the α5β1 integrin receptor as a driver of JAK2 V617F megakaryocyte proliferation. Expression of β1 integrin subunit was similar in wild-type (WT) and JAK2 V617F megakaryocytes, but activation of β1 integrin, detected by the conformation-specific 9EG7 antibody, was elevated in JAK2 V617F megakaryocytes. Following up on those findings, we detected elevated β1 integrin activation in long-term hematopoietic stem cells (HSC LT, LSKCD48-CD150+) from JAK2 V617F transgenic mice ( Xing et al, Blood 2008) when compared to WT controls. To determine whether inhibition of β1 integrin in vivo reduces the number of HSC LT in JAK2 V617F transgenic mice, we examined HSC LT 24 hours after intravenous administration of 2mg/Kg of anti-β1 integrin inhibitory antibody HMβ1-1. This regimen led to a sharp reduction in number and percentage of HSC LT in JAK2 V617F transgenic mice, to 45% of control antibody-treated animals. The effect on WT HSC LT was negligible. HSC ST (LSKCD48-CD150-) and MPP2 (LSKCD48+CD150+) were also reduced, but the reduction was not statistically significant. MPP3/4 (LSKCD48+CD150-) and LSK were not affected. Megakaryocyte progenitors (LKCD41+CD150+) were significantly reduced. Inhibition of β1 integrin also sharply reduced peripheral blood platelet numbers in WT animals. The reduction in JAK2 V617F transgenic mice was not statistically significant. A longer observation time of 7 days demonstrated that while WT animals were able to mount a robust regenerative response with slight but significant increases in HSC LT and normalization of platelet numbers, in JAK2 V617F transgenic mice the number of HSC LT remained depressed, as did downstream stem/progenitor populations at various significance levels. To gain insight into the mechanisms that lead to reduction in HSC LT, we examined apoptosis, cell cycle and HSC mobilization, all processes known to be affected by β1 integrin. Although basal expression of the apoptotic marker annexin V was decreased in JAK2 V617F HSC LT compared to WT, no differences were detected upon administration of HMβ1-1 in both genotypes at 4 hours post-administration. Similarly, peripheral blood colony assays revealed a basally higher level of stem/progenitor mobilization in JAK2 V617F, which did not significantly increase upon HMβ1-1 administration. In contrast, the cell cycle profile, examined at 4 hours post-administration to account for the earliest effects, demonstrated a decrease in G0 phase cells and increase in S/G2/M cells indicating cell cycle induction and exit from quiescence in both WT and JAK2 V617F HSC LT. The results indicate that inhibition of β1 integrin disrupts the adhesion-mediated cell cycle suppression in both WT and JAK2 V617F HSC LT leading to exit from quiescence. However, the downstream consequences of cell division in HSC LT differ significantly, with an apparent loss of regenerative potential in JAK2 V617F HSC LT, at least at the observed timepoints. Longer observation times are required to determine whether continuous β1 integrin inhibition can lead to JAK2 V617F HSC LT exhaustion. More sensitive assays to monitor mobilization are warranted to quantify possible minute amounts of mobilized HSC LT. Our preclinical studies indicate that β1 integrin is an attractive target to specifically reduce the number of JAK2 V617F HSC LTin vivo. The role of β1 integrin in HSC LT has not been thoroughly studied due to lack of phenotype in β1 integrin-deficient HSC ( Brakebush et al, Immunity 2002). Our studies using the anti-β1 integrin antibody in WT HSC LT confirm previous findings. However, β1 integrin seems to be critical for maintenance of JAK2 V617F HSC LT, offering a possible therapeutic window. Understanding how β1 integrin supports quiescence in HSC LT will bring us a step closer to understanding the mechanisms that drive malignancy and fitness in JAK2 V617F HSC LT.

Anti-α6β4 integrin autoantibodies inhibit the binding of laminins to α6β4 integrin in patients with pemphigoid and affect the gastrointestinal tract.

Anti-α6β4 integrin autoantibodies can be observed in some patients with mucous membrane pemphigoid. We have previously identified anti-α6β4 integrin extracellular domain autoantibodies together with anti-BP180 NC16A antibodies in a patient with DPP-4 inhibitor-induced bullous pemphigoid. However, the significance and impact of anti-α6β4 integrin antibodies are unknown. To characterize anti-α6β4 integrin extracellular domain autoantibodies in pemphigoid patients, to determine whether these antibodies inhibit laminin-α6β4 integrin binding and to observe their systemic effects. Anti-α6β4 integrin autoantibodies were analysed by staining cells expressing the extracellular region of α6β4 integrin with sera from 20 patients with pemphigoid. The anti-α6β4 integrin autoantibodies were characterized using different transfectants. The binding of laminins to α6β4 integrin was studied using cells expressing the activated conformation of α6β4 integrin and the inhibitory effect of the autoantibodies on the binding of laminins to α6β4 integrin was tested. Trends in antibody titres and clinical symptoms were quantified and analysed. IgG autoantibodies against the extracellular domain of anti-α6β4 integrin were found in some patients with pemphigoid. Laminin binding to α6β4 integrin was observed in the active conformation of α6β4 integrin, and serum from a patient with a high titre of anti-α6β4 integrin antibodies inhibited the binding of both laminin-511 and laminin-332 to α6β4 integrin. α6β4 integrin is expressed on the basement membrane of both skin and small intestine, and exfoliation was observed in the patient's epidermis and small intestinal epithelium. A reduction in the titre of the anti-α6β4 integrin antibody was associated with improvement in both skin and gastrointestinal symptoms. This study demonstrated the presence of anti-α6β4 integrin extracellular domain-specific autoantibodies in some patients with pemphigoid. In addition, these autoantibodies showed inhibitory activity on α6β4 integrin-laminin binding. Anti-α6β4 integrin antibodies can affect the gastrointestinal tract as well as the skin and oral mucosa.

Integrin αvβ1 facilitates ACE2-mediated entry of SARS-CoV-2

Integrins have been suggested to be involved in SARS-CoV-2 infection, but the underlying mechanisms remain largely unclear. This study aimed to investigate how integrins facilitate the ACE2-mediated cellular entry of SARS-CoV-2. We first tested the susceptibility of a panel of human cell lines to SARS-CoV-2 infection using the spike protein pseudotyped virus assay and examined the expression levels of integrins in these cell lines by qPCR, western blot and flow cytometry. We found that integrin αvβ1 was highly enriched in the SARS-CoV-2 susceptible cell lines. Additional studies demonstrated that RGD (403–405)→AAA mutant was defective in binding to integrin αvβ1 compared to its wild type counterpart, and anti-αvβ1 integrin antibodies significantly inhibited the entry of SARS-CoV-2 into the cells. Further studies using mouse NIH3T3 cells expressing human ACE2, integrin αv, integrin β1, and/or integrin αvβ1 suggest that integrin αvβ1 was unable to function as an independent receptor but could significantly facilitate the cellular entry of SASR-CoV-2. Finally, we observed that the Omicron exhibited a significant increase in the ACE2-mediated viral entry. Our findings may enhance our understanding of the pathogenesis of SARS-CoV-2 infection and offer potential therapeutic target for COVID-19.

A simple and robust nanosystem for photoacoustic imaging of bladder cancer based on α5β1-targeted gold nanorods

BackgroundEarly detection and removal of bladder cancer in patients is crucial to prevent tumor recurrence and progression. Because current imaging techniques may fail to detect small lesions of in situ carcinomas, patients with bladder cancer often relapse after initial diagnosis, thereby requiring frequent follow-up and treatments.ResultsIn an attempt to obtain a sensitive and high-resolution imaging modality for bladder cancer, we have developed a photoacoustic imaging approach based on the use of PEGylated gold nanorods (GNRs) as a contrast agent, functionalized with the peptide cyclic [CphgisoDGRG] (Iso4), a selective ligand of α5β1 integrin expressed by bladder cancer cells. This product (called GNRs@PEG-Iso4) was produced by a simple two-step procedure based on GNRs activation with lipoic acid-polyethyleneglycol(PEG-5KDa)-maleimide and functionalization with peptide Iso4. Biochemical and biological studies showed that GNRs@PEG-Iso4 can efficiently recognize purified integrin α5β1 and α5β1-positive bladder cancer cells. GNRs@PEG-Iso4 was stable and did not aggregate in urine or in 5% sodium chloride, or after freeze/thaw cycles or prolonged exposure to 55 °C, and, even more importantly, do not settle after instillation into the bladder. Intravesical instillation of GNRs@PEG-Iso4 into mice bearing orthotopic MB49-Luc bladder tumors, followed by photoacoustic imaging, efficiently detected small cancer lesions. The binding to tumor lesions was competed by a neutralizing anti-α5β1 integrin antibody; furthermore, no binding was observed to healthy bladders (α5β1-negative), pointing to a specific targeting mechanism.ConclusionGNRs@PEG-Iso4 represents a simple and robust contrast agent for photoacoustic imaging and diagnosis of small bladder cancer lesions.Graphical

β1 integrin is essential for blood–brain barrier integrity under stable and vascular remodelling conditions; effects differ with age

BackgroundMaintaining a tight blood–brain barrier (BBB) is an important prerequisite for the preservation of neurological health, though current evidence suggests it declines with age. While extracellular matrix-integrin interactions play critical roles in regulating the balance between vascular stability and remodeling, it remains to be established whether manipulation of integrin function weakens or strengthens vascular integrity. Indeed, recent reports have generated conflicting outcomes in this regard.MethodsHere, in young (8–10 weeks) and aged (20 months) mice, we examined the impact of intraperitoneal injection of a function-blocking β1 integrin antibody, both under normoxic conditions, when the BBB is stable, and during chronic mild hypoxic (CMH; 8% O2) conditions, when a vigorous vascular remodeling response is ongoing. Brain tissue was examined by immunofluorescence (IF) for markers of vascular remodeling and BBB disruption, and microglial activation and proliferation. Data were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison post-hoc test.ResultsIn both young and aged mice, β1 integrin block greatly amplified hypoxia-induced vascular disruption, though it was much less under normoxic conditions. Interestingly, under both normoxic and hypoxic conditions, β1 integrin antibody-induced BBB disruption was greater in young mice. Enhanced BBB breakdown was associated with increased levels of the leaky BBB marker MECA-32 and with greater loss of endothelial tight junction proteins and the adherens protein VE-cadherin. Surprisingly, β1 integrin blockade did not reduce hypoxia-induced endothelial proliferation, nor did it prevent the hypoxia-associated increase in vascularity. Commensurate with the increased vascular disruption, β1 integrin blockade enhanced microglial activation both in young and aged brain, though the impact was much greater in young brain. In vitro studies revealed that β1 integrin blockade also reduced the integrity of a brain endothelial monolayer and triggered disruptions in tight junction proteins.ConclusionsThese data demonstrate that β1 integrin plays an essential role in maintaining BBB integrity, both under stable normoxic conditions and during hypoxia-induced vascular remodeling. As β1 integrin blockade had a greater disruptive effect in young brain, effectively shifting the BBB phenotype of young brain towards that of the aged, we speculate that enhancing β1 integrin function at the aged BBB may hold therapeutic potential by reverting the deteriorating BBB phenotype back towards that of the young.

Exit strategies in inflammatory bowel disease: Looking beyond anti-tumor necrosis factors.

The long-term management of patients with inflammatory bowel disease (IBD) is still a matter of debate, and no clear guidelines have been issued. In clinical practice, gastroenterologists often have to deal with patients in prolonged remission after immunomodulatory or immunosuppressive therapies. When planning an exit strategy for drug withdrawal, the risk of disease relapse must be balanced against the risk of drug-related adverse events and healthcare costs. Furthermore, there is still a dearth of data on the withdrawal of novel biologics, such as the anti-α4β7 integrin antibody (vedolizumab) and anti-IL12/23 antibody (ustekinumab), as well as the small molecule tofacitinib. Models for estimating the risk of disease relapse and the efficacy of retreatment should be evaluated according to the patient's age and IBD phenotype. These models should guide clinicians in programming a temporary drug withdrawal after discussing realistic outcomes with the patient. This would shift the paradigm from an exit strategy to a holiday strategy.

Anti-β7 integrin treatment impedes the recruitment on non-classical monocytes to the gut and delays macrophage-mediated intestinal wound healing.

Closing mucosal defects to reach mucosal healing is an important goal of therapy in inflammatory bowel disease (IBD). Among other cells, monocyte-derived macrophages are centrally involved in such intestinal wound healing. We had previously demonstrated that the anti-α4β7 integrin antibody vedolizumab blocks the recruitment of non-classical monocytes as biased progenitors of wound healing macrophages to the gut and delays wound healing. However, although important for the interpretation of disappointing results in recent phase III trials in IBD, the effects of the anti-β7 antibody etrolizumab on wound healing are unclear so far. We analyzed the expression of etrolizumab targets on human and mouse monocyte subsets by flow cytometry and assessed their function in adhesion and homing assays. We explored wound-associated monocyte recruitment dynamics with multi-photon microscopy and compared the effects of etrolizumab and vedolizumab surrogate (-s) antibodies on experimental wound healing and wound-associated macrophage abundance. Finally, we investigated wound healing macrophage signatures in the large intestinal transcriptome of patients with Crohn's disease treated with etrolizumab. Human and mouse non-classical monocytes expressed more αEβ7 integrin than classical monocytes and were a target of etrolizumab-s, which blocked non-classical monocyte adhesion to MAdCAM-1 and E-Cadherin as well as gut homing in vivo. Intestinal wound healing was delayed on treatment with etrolizumab-s along with a reduction of peri-lesional wound healing macrophages. Wound healing macrophage signatures in the colon of patients with Crohn's disease were substantially down-regulated on treatment with etrolizumab, but not with placebo. Combined blockade of αEβ7 and α4β7 with etrolizumab seems to exceed the effect of anti-α4β7 treatment on intestinal wound healing, which might help to inform further investigations to understand the recent observations in the etrolizumab phase III trial program.

P406 Observational study of the effectiveness of vedolizumab on treatment outcomes and health-related quality of life in biologic-naïve patients with Inflammatory Bowel Diseases in Greece – the TROVE study - Interim Analysis

Abstract Background Vedolizumab (VDZ), a gut-selective anti-lymphocyte trafficking (GSALT), anti-α4b7 integrin antibody, is an approved and effective treatment for patients with Ulcerative Colitis (UC) and Crohn’s Disease (CD). Biologic-naïve patients appear to respond better to treatment with VDZ, than anti-TNF exposed patients. Our study aimed to assess the long-term clinical effectiveness and benefit on health-related quality of life (HRQoL) of first line biologic treatment with VDZ in Greek patients with Inflammatory Bowel Disease. Methods Biologic-naïve patients with moderate to severe UC or CD, received VDZ and were prospectively followed as part of the multicenter observational study TROVE. Interim data were collected between November 2018 and May 2021. The primary endpoint was drug persistence at week 54. Secondary endpoints included clinical response at week 14 [UC&CD, >50% decrease in patient reported outcomes (PRO2)] and clinical remission [UC, partial Mayo score (PMS)<3, PRO2-rectal bleeding=0 and stool frequency=0; CD, PRO2-abdominal pain≤1 and stool frequency≤3) at weeks 14 and 54. HRQoL was assessed using SIBDQ and EQ5D questionnaires. Results One-hundred and twenty patients from 19 centers were included [Female: 55%, Age (mean): 47.8 years, UC/CD: 56%/44%, Disease duration (median): 2.6 years, Smokers: 25%]. Baseline characteristics are shown in Table 1. At week 54, 72.1% (49/68) of UC patients and 84.6% (44/52) of CD patients continued treatment with VDZ. Among UC patients, clinical remission rates were 60.3% (n=41/68) and 50% (n=34/68) at weeks 14 and 54, respectively. Clinical remission rates for CD were 86.5% (n=45/52) (week 14) and 76.9% (n=40/52) (week 54). At week 14, among UC and CD patients with clinically active disease at baseline, 73.7% (n=42/57) and 83.8% (n=31/37) responded to VDZ treatment, respectively. Among patients who had endoscopy (n=14 UC; n=10 CD), rates of mucosal healing at week 54 were 35.7% for UC (MES 0) and 50% for CD (absence of ulceration) respectively. Non-response at week 14 was associated with higher probability of drug discontinuation at week 54 (OR 3.63; CI 1.19-11.1). Disease duration (<2years vs >5years) did not influence drug persistence at week 54 (P=0.25). Treatment was intensified in 12.2% of UC and 11.4% of CD patients. The mean increase in SIBDQ and EQ5D between weeks 0 and 54 were 14.6 and 14.4 respectively (for both P<0.001). Conclusion The majority of UC or CD biologic naive patients remain on VDZ one year after treatment commencement, with high clinical remission rates and improved HRQoL. Clinical response at week 14 predicts long-term drug persistence. Our results support the use VDZ as a first line biologic therapy in IBD patients. The clinical study received funding from Takeda Hellas.

P631 Long-term safety of vedolizumab versus other biologic agents in ulcerative colitis (UC) and Crohn’s Disease (CD): Results from a large multinational prospective observational study

Abstract Background Vedolizumab (VDZ) is a gut-selective humanised monoclonal anti-α4β7 integrin antibody for treatment of UC and CD. This post-authorization safety study (PASS) compared long-term safety in patients (pts) treated with VDZ or other biologics for UC or CD. Methods This was a prospective, observational, multicentre, cohort study in pts with UC or CD starting treatment with VDZ or other biologics (NCT02674308, EUPAS6469). Pts were biologic naïve (VDZ=35%, Other biologic=65%) or had prior exposure to biologics other than VDZ and were followed for up to 7 yrs. Pts were treated under usual standard of care as directed by their physician with no discontinuation from the study due to physician-directed treatment changes. The primary safety outcome was serious infections and was compared between cohorts using a Cox proportional hazards model adjusted by propensity score (with other biologics as reference). Secondary safety outcomes were adverse events (AE) of special interest (AESI) as listed in Tables 2 and 3. Results In total, 5208 patients were enrolled across 21 countries between Mar 2015 and Jul 2019. The full analysis set comprised 5008 pts (VDZ=2502; other biologic=2506) and mean (SD) study duration was 37.4 (14.1) months. Consistent with the observational nature of the study and the current standard of care, the baseline characteristics in the two cohorts were imbalanced (Table 1). The overall treatment-emergent Adverse Event (AE) rate was 61.3% (VDZ) and 59.5% (other biologic), with serious AEs in 27.5% (VDZ) and 25.7% (other biologic), respectively. In UC pts, the incidence rate (IR) of serious infections were: 0.014 (95% CI: 0.010, 0.019) [VDZ] and 0.015 (0.010, 0.023) [other biologic], with a hazard ratio (HR) of 1.018 (0.931, 1.114), p=0.70 (Table 2). In CD pts, corresponding findings were 0.012 (95% CI: 0.008, 0.017) [VDZ] and 0.006 (95% CI: 0.004, 0.009) [other biologic] with HR of 1.035 (0.959, 1.117), p=0.38 (Table 3). The incidence of primary and secondary safety outcomes is reported in Table 2 for UC and Table 3 for CD. There were no safety issues relating to pregnancy and no cases of PML observed. There were 19 deaths in the VDZ cohort (1 case of pneumonia deemed as treatment-related); and 13 deaths in the other biologic cohort, 2 cases deemed treatment-related (one case septic shock and pneumonia; one case non-small cell lung cancer). Clinical outcomes and quality of life (QoL) measures were comparable between cohorts. Conclusion In this long-term, prospective, real-world observational study of a large multinational population under routine standard of care there was no new safety signal identified in relation to VDZ. Results are consistent with the known safety profile of VDZ.

P415 Real-world data in Inflammatory Bowel Diseases on vedolizumab therapy: Interim analysis of the non-interventional LISTEN II study

Abstract Background Vedolizumab (VDZ) is a gut-selective anti-lymphocyte trafficking integrin antibody and is approved for treating patients with moderately to severely active UC (ulcerative colitis) and CD (Crohn’s disease) intravenously (IV) or subcutaneously (SC). The LISTEN II study aims to determine patient characteristics and effectiveness in patients treated with VDZ in a real-world setting. Methods LISTEN II is a multicentre, prospective non-interventional study in Germany. Adult patients with UC or CD, for whom the physician has decided to either initiate VDZ IV or switch from IV to SC, are included. Data on patient characteristics, modalities of VDZ use and disease activity (pMayo, HBI) are followed up quarterly for up to one year per routine practice. In addition, patient reported outcomes (PROs) confirmed as relevant in the previous LISTEN I study (e.g. rectal bleeding and general wellbeing) are recorded via an app (ePRO). Results Data of 198 UC patients (50% female, 6.0 years median disease duration) and 116 CD patients (56% female, 10.9 years median disease duration) with a mean age of 40 years were included in this interim analysis. VDZ treatment was newly initiated IV in 164 UC patients and 98 CD patients (start) and switched from IV to SC by 34 UC and 18 CD patients (switch) at study inclusion. At baseline corticosteroids were documented as used in 70% and 56% of UC and CD start-patients in the year prior to enrollment. Burden of disease was classified as high (pMayo>4) in 51% of UC patients at baseline and decreased to 15% at 3 months. For CD patients, proportion of high burdened patients (HBI>7) was 27% at baseline and 12% at month 3 (Figure 1). In this period, total mean pMayo score decreased in UC patients from 4.3±2.5 (start: 4.9±2.1; switch: 1.0±1.3) to 2.2±2.2 (start: 2.5±2.2; switch:1.0±1.6) and total mean HBI score from 5.4±4.4 (start: 5.8±4.5; switch: 3.5±3.7) to 3.3±3.4 (start: 3.4±3.4, switch: 2.8±3.3) in CD patients (Figure 2). UC patients using the ePRO (N=152, mean age: 39 years, 49% female) reported from ePRO initiation up to month 3 a mean score decrease for rectal bleeding (range 0-3) from 1.3 to 0.6, for general well-being (range 0-4) from 1.7 to 1.3 and for impact on work productivity (range 0-10) from 3.7 to 2.3 points. Conclusion LISTEN II real-world data support the effectiveness of VDZ treatment in UC and CD patients. Patients starting VDZ IV showed a higher disease activity at baseline which decreased under VDZ treatment. Switching of VDZ IV to SC was mainly performed in patients with low disease burden under VDZ treatment, resulting in maintained therapeutic effectiveness. Also PROs of UC patients show an improvement supporting benefit with VDZ treatment.

P689 Predictive transcriptional signatures associated to vedolizumab therapy response in patients with ulcerative colitis

Abstract Background Vedolizumab is one of the current treatments for patients with inflammatory bowel disease (IBD). The efficacy and safety, along with its gut specificity, make this drug an appealing therapeutic option for IBD patients with moderate to severe disease. However, as observed for other biologic treatments, a significant proportion of patients do not have an initial response to vedolizumab treatment. Currently, there is a lack of reliable predictive tools for vedolizumab treatment response, although this would help to alleviate the socioeconomic costs derived from this disease. For that reason, the primary aim of this study is to show putative transcriptional signatures associated with vedolizumab treatment response. Methods For the realization of this study, we used RNA-seq datasets from the Gene Expression Omnibus (GEO) database: GSE191328. This dataset includes samples from peripheral blood cells from responder and non-responder ulcerative colitis (UC) patients treated with vedolizumab and infliximab at baseline. Gene Set Enrichment Analysis (GSEA) was performed to compare the differential expression and the enrichment analysis was calculated using ssGSEA of the different groups under study. Results The analysis of the transcriptome of peripheral blood cells from responder (R) and non-responder (NR) vedolizumab-treated patients at baseline revealed interesting enrichment results. We found 20 gene sets positively enriched in NR versus R and only 9 gene sets negatively enriched in the same phenotypes. In order to find vedolizumab specific transcriptional signatures, we performed a similar analysis in samples from infliximab-treated patients. After that second round of analysis, we observed that around 50% of the enriched gene sets were similar for both biological treatments. Nevertheless, several interesting biological functions such as peroxisome function, reactive oxygen function or beta-catenin signalling are specifically enriched in NR patients to vedolizumab treatment. Conclusion Our data suggest that vedolizumab NR patients specifically show a transcriptional enrichment profile, which differs from vedolizumab R patients. Also, the comparative analysis with infliximab-treated patients reveals that those specific transcripts are modulated in response to this α4β7 integrin antibodies in peripheral blood cells. Therefore, we found a solid system to search for vedolizumab therapy predictive response markers using low invasive techniques.

OP05 High-dimensional single-cell analysis identifies cellular signatures associated with response to vedolizumab therapy in ulcerative colitis

Abstract Background The unpredictability of response to biological therapy in ulcerative colitis (UC) patients, including vedolizumab (VDZ), an anti-α4β7 integrin antibody, poses a serious challenge for medical care. This study aims to identify the relevant cellular and molecular signatures that mark response to VDZ by combining single-cell transcriptomics (scRNAseq) and high-dimensional flow cytometry (Cytek). Methods Gut biopsies and peripheral blood mononuclear cells (PBMCs) from patients with active UC were collected 2 weeks prior and 14 weeks after starting VDZ therapy. Response was evaluated at week 14 based on endoscopy and Physician Global Assessment. Transcriptome profiles of 159.188 and 95.057 high-quality single cells were generated from biopsies and PBMCs, respectively. Additional 797.670 CD45+ mucosal cells and 900.000 PBMCs were processed for validating scRNAseq results by Cytek. Results From a total of 25 UC patients (mean age 41±12.7 years, 52% female, 36% anti-TNFα naive), 15 patients (60%) reached response at week 14. Patients with no prior use of anti-TNFα were more likely to achieve remission with VDZ compared to anti-TNFα exposed patients (89% vs 44% response rate, respectively). Analysis of cell abundances revealed a partial recovery of the epithelial and stromal cell mucosal compartments in responders at week 14. Both scRNAseq and Cytek show that in responders VDZ prevented T cells (CD8+IL17+ and CD4+PD1+ subsets) and innate immune cells (NKs, DCs and inflammatory monocytes) from entering mucosa, resulting in their enrichment in PBMCs. In contrast, non-response to VDZ was marked by an expansion of inflammatory fibroblasts and activated endothelial cells, and depletion of most epithelial subsets. VDZ retained CD4 T cells in PBMCs in non-responders, but failed to halt active mucosal inflammation at week 14. Strikingly, involvement of the innate immune cells in UC inflammation prior to VDZ treatment was much more prominent in non-responders who showed higher baseline abundance of NKs, ILCs, γδ-T cells, DCs and inflammatory monocytes in their peripheral blood and mucosa compared to responders. While VDZ inhibited integrin β7 expression on circulating T cells in all patients, only in responders it coincided with the lower mucosal abundance of lymphocytes, suggesting that T cells in non-responders might employ alternative, β7-independent trafficking routes. Conclusion This is the largest prospective scRNAseq study addressing the response to biological therapy in UC. We showed that active, innate immune cell-mediated inflammation marks the primary non-response to VDZ that persists prior to and throughout the treatment, accompanied by β7-independent lymphocyte migration.