anti-TNF Antibody Research Articles

Direct effects of anti‐inflammatory medication on barrier function in intestinal epithelial cells

An impaired intestinal epithelial barrier (IEB) is a hallmark in the pathogenesis of inflammatory bowel diseases (IBD), including ulcerative colitis and Crohn’s disease. Most of the IBD patients are treated with anti‐inflammatory reagents including glucocorticoids, anti TNFα (tumor necrosis factor alpha) antibodies and mesasalazine. While the Immune‐modulating effects of those therapies are well documented, we hypothesized whether there are direct effects of those reagents on enterocytes and the IEB.To address this hypothesis we used Caco2 cells as well as 2D and 3D murine organoids and performed analysis of intestinal epithelial barrier function. Inflammation‐induced alterations were mimicked using cytomix for 24h (TNFα, Interleukin 1 beta and Interferon gamma). The enterocytes were treated with 100ng/ml anti‐TNFα antibody Infliximab, 1mM prednisolone or 5mM mesasalazine, respectively following the challenge with cytomix. Epithelial permeability as revealed by measurements of 4kDa FITC Dextran flux and of transepithelial electric resistance showed that Infliximab and a pre‐incubation with prednisolone for 24h attenuated inflammation‐induced breakdown of the IEB. This was not the case after application of mesasalazine or a simultaneous incubation of prednisolone. The functional changes in intestinal epithelial barrier function following incubation with Cytomix were paralleled by alterations in the expression of “leaky” tight junction protein Claudin2, as revealed by Western blots. Furthermore, immunostainings demonstrated an altered distribution of barrier sealing proteins such as Claudin1, Desmoglein2 and E‐Cadherin. All of these effects were blocked by infliximab and prednisolone.In summary, our data indicate that infliximab and prednisolone have direct effects on the IEB that may contribute to the efficacy in the treatment of IBD.

Macrophages in epicardial adipose tissue and serum NT-proBNP in patients with stable coronary artery disease

Coronary artery disease (CAD) is widely considered a chronic inflammatory disorder, and dysfunction of epicardial adipose tissue could be an important source of the inflammation. Amino-terminal fragment of pro-B-type natriuretic peptide (NT-proBNP) is a known marker of cardiovascular disorders of cardiac origin. Recent studies show that inflammatory stimuli may influence its secretion. Our purpose was to evaluate NT-proBNP serum concentration in relation to immune cell ratios in epicardial adipose tissue (EAT), and cytokine levels in the patients with stable CAD. Patients with stable CAD and heart failure classified into classes II–III, according to the New York Heart Association (NYHA) scale, scheduled for the coronary artery bypass graft (CABG) surgery, were recruited into the study (n = 10; 59.5 (53.0-65.0) y. o.; 50% males). The EAT and subcutaneous adipose tissue (SAT) specimens were harvested in the course of CABG surgery. Immunostaining with anti-CD68, anti-CD45, antiIL-1β and anti-TNFα monoclonal antibodies was performed to evaluate cell composition by differential counts per ten fields (400 magnification). Fasting venous blood was obtained from patients before CABG. Blood was centrifuged at 1500g, aliquots were collected and stored frozen at -40 °С until final analysis. Concentrations of NT-proBNP, IL-1β, IL-6, IL-10, TNFα were determined in serum samples by enzyme-linked immunosorbent assay (ELISA). We have found increased production of IL-1β and TNFα cytokines in EAT compared to SAT. Concentrations of NT-proBNP exceeded 125 pg/ml in 4 patients, and correlations between the CD68+macrophage counts in both EAT and SAT samples (rs= 0.762; p = 0.010 and rs= 0.835; p = 0.003, respectively). NT-proBNP levels showed positive relations with CD45+leukocyte counts (rs= 0.799; p = 0.006), and with IL-1β+cell numbers (rs= 0.705; p = 0.023) in EAT samples only. As for the serum biomarkers, NT-proBNP levels showed negative correlation with fasting glucose levels (rs= -0.684; p = 0.029), and positive correlation with serum IL-6 concentrations (rs= 0.891; p = 0.001). Increased serum concentrations of NT-proBNP in CAD patients correlate with accumulation of macrophages in EAT, which is associated with increased production of IL-1β in EAT and correlates with some metabolic parameters.

Elucidating the role of hypoxia-inducible factor in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic multifactorial disease, provocative, and degenerative autoimmune condition that impacts millions of individuals around the globe. As a result of this understanding, anti-inflammatory drugs have been created, perhaps widely effective (like steroids) and highly specialized methods (including anti-TNF antibody) using biological therapies (including TNF inhibitors). Despite this, the connections between inflammatory response, articular development, and intracellular responsiveness to changes in oxygen concentration are undervalued in rheumatoid arthritis. Hypoxia, or a lack of oxygen, is thought to cause enhanced synovial angiogenesis in RA, which is mediated by some of the hypoxia-inducible factors like vascular endothelial growth factor (VEGF). Substantial genetic alterations occur when the HIF regulatory factors signaling cycle is activated, allowing organelles, tissues, and species to acclimatize to decreasing oxygen saturation. The most well-characterized hypoxia-responsive transcripts are the angiogenic stimulant VEGF, whose production is greatly elevated by hypoxia in several types of cells, especially RA synovium fibroblasts. Blocking vascular endothelial growth factors has been demonstrated to be helpful in murine models of rheumatism, indicating how hypoxia could trigger the angiogenesis process, resulting in the progression of RA. These mechanisms highlight the intimate affiliation amongst hypoxia, angiogenesis, and inflammation in rheumatoid arthritis. This review will look at how hypoxia activates molecular pathways and how other pathways involving inflammatory signals develop and sustain synovitis in rheumatoid arthritis.

AJM300 (carotegrast methyl), an oral antagonist of α4-integrin, as induction therapy for patients with moderately active ulcerative colitis: a multicentre, randomised, double-blind, placebo-controlled, phase 3 study

AJM300 is an oral, small-molecule α4-integrin antagonist. We assessed the efficacy and safety of AJM300 in patients with moderately active ulcerative colitis. This multicentre, randomised, double-blind, placebo-controlled, phase 3 study consisted of two phases: a treatment phase and an open-label re-treatment phase. The study was done at 82 hospitals and clinics in Japan. Patients with a Mayo Clinic score of 6-10, endoscopic subscore of 2 or more, rectal bleeding subscore of 1 or more, and an inadequate response or intolerance to mesalazine were enrolled. Patients were randomly allocated (1:1) via a website to either AJM300 (960 mg) or placebo by the minimisation method, which was adjusted centrally by dynamic assignment against the Mayo Clinic score (≥6 to ≤7, ≥8 to ≤10 points), any use of corticosteroid, anti-TNFα antibody, or immunosuppressants during the disease-active period (yes vs no), duration of induction therapy until randomisation (<4 weeks vs ≥4 weeks) as the minimisation factors. Patients, investigators, site staff, assessors, and the sponsor were masked to treatment assignments. The study drug was administered orally, three times daily, for 8 weeks, and continued for up to 24 weeks if endoscopic remission was not achieved or rectal bleeding did not stop. The primary endpoint was the proportion of patients with a clinical response at week 8, and was analysed in the full analysis set. Clinical response was defined as a reduction in Mayo Clinic score of 30% or more and 3 or more, a reduction in rectal bleeding score of 1 or more or rectal bleeding subscore of 1 or less, and an endoscopic subscore of 1 or less at week 8. The study is registered with ClinicalTrials.gov, NCT03531892, and is closed to recruitment. Between June 6, 2018, and July 22, 2020, 203 patients were randomly assigned to AJM300 (n=102) or placebo (n=101). At week 8, 46 (45%) patients in the AJM300 group and 21 (21%) patients in the placebo group had a clinical response (odds ratio 3·30, 95% CI 1·73-6·29; p=0·00028). During the 8-week treatment and 16-week extension treatment periods, adverse events occurred in 39 (39%) of 101 patients in the placebo group and 39 (38%) of 102 patients in the AJM300 group. We found no difference in the incidence of adverse events between groups or after repeated administration of AJM300. The most common adverse event was nasopharyngitis (11 [11%] of 101 patients in the placebo group and ten [10%] of 102 patients in the AJM300 group). The most common treatment-related adverse event was also nasopharyngitis (four [4%] of 101 patients in the placebo group and three [3%] of 102 patients in the AJM300 group). Most adverse events were mild-to-moderate in severity. No deaths were reported. A serious adverse event was reported in the AJM300 group (one patient with anal abscess), but this was judged to be unrelated to study drug. AJM300 was well tolerated and induced a clinical response in patients with moderately active ulcerative colitis who had an inadequate response or intolerance to mesalazine. AJM300 could be a novel induction therapy for the treatment of patients with moderately active ulcerative colitis. EA Pharma and Kissei Pharmaceutical. For the Japanese translation of the abstract see Supplementary Materials section.

Antibody decay, T cell immunity and breakthrough infections following two SARS-CoV-2 vaccine doses in inflammatory bowel disease patients treated with infliximab and vedolizumab

Anti tumour necrosis factor (anti-TNF) drugs increase the risk of serious respiratory infection and impair protective immunity following pneumococcal and influenza vaccination. Here we report SARS-CoV-2 vaccine-induced immune responses and breakthrough infections in patients with inflammatory bowel disease, who are treated either with the anti-TNF antibody, infliximab, or with vedolizumab targeting a gut-specific anti-integrin that does not impair systemic immunity. Geometric mean [SD] anti-S RBD antibody concentrations are lower and half-lives shorter in patients treated with infliximab than vedolizumab, following two doses of BNT162b2 (566.7 U/mL [6.2] vs 4555.3 U/mL [5.4], p <0.0001; 26.8 days [95% CI 26.2 – 27.5] vs 47.6 days [45.5 – 49.8], p <0.0001); similar results are also observed with ChAdOx1 nCoV-19 vaccination (184.7 U/mL [5.0] vs 784.0 U/mL [3.5], p <0.0001; 35.9 days [34.9 – 36.8] vs 58.0 days [55.0 – 61.3], p value < 0.0001). One fifth of patients fail to mount a T cell response in both treatment groups. Breakthrough SARS-CoV-2 infections are more frequent (5.8% (201/3441) vs 3.9% (66/1682), p = 0.0039) in patients treated with infliximab than vedolizumab, and the risk of breakthrough SARS-CoV-2 infection is predicted by peak anti-S RBD antibody concentration after two vaccine doses. Irrespective of the treatments, higher, more sustained antibody levels are observed in patients with a history of SARS-CoV-2 infection prior to vaccination. Our results thus suggest that adapted vaccination schedules may be required to induce immunity in at-risk, anti-TNF-treated patients.

Microbial signals, MyD88, and lymphotoxin drive TNF-independent intestinal epithelial tissue damage.

Anti-TNF antibodies are effective for treating patients with inflammatory bowel disease (IBD), but many patients fail to respond to anti-TNF therapy, highlighting the importance of TNF-independent disease. We previously demonstrated that acute deletion of 2 IBD susceptibility genes, A20 (Tnfaip3) and Abin-1 (Tnip1), in intestinal epithelial cells (IECs) sensitized mice to both TNF-dependent and TNF-independent death. Here we show that TNF-independent IEC death after A20 and Abin-1 deletion was rescued by germ-free derivation or deletion of MyD88, while deletion of Trif provided only partial protection. Combined deletion of Ripk3 and Casp8, which inhibits both apoptotic and necroptotic death, completely protected against death after acute deletion of A20 and Abin-1 in IECs. A20- and Abin-1–deficient IECs were sensitized to TNF-independent, TNFR1-mediated death in response to lymphotoxin α (LTα) homotrimers. Blockade of LTα in vivo reduced weight loss and improved survival when combined with partial deletion of MyD88. Biopsies of inflamed colon mucosa from patients with IBD exhibited increased LTA and IL1B expression, including a subset of patients with active colitis on anti-TNF therapy. These data show that microbial signals, MyD88, and LTα all contribute to TNF-independent intestinal injury.

Infliximab Superior to IVIG for Rescue in IVIG-Refractory Kawasaki Disease

Infliximab Superior to IVIG for Rescue in IVIG-Refractory Kawasaki Disease

Vaccination for SARS-CoV-2 in Patients With Psoriatic Arthritis: Can Therapy Affect the Immunological Response?

BackgroundA few studies on vaccination in patients with rheumatic diseases, including arthritis, connective tissue diseases, vasculitis, and psoriatic arthropathy (PsA), demonstrated reduced production of neutralizing antibodies to SARS-CoV-2 Spike RBD (receptor-binding domain contained in the N-terminal of the S1 globular head region) when compared to the general population.ObjectiveThe aim of our study was to observe whether different therapies for PsA [methotrexate, anti-TNF antibodies, soluble TNF receptor (etanercept) or IL-17 inhibitors] have a different impact on SARS-CoV-2 vaccination in a homogeneous population of patients.MethodsWe enrolled 110 PsA patients in remission, assessed with Disease Activity in PSoriatic Arthritis (DAPSA). Of these: 63 were in treatment with anti-TNF-α therapy (26 etanercept, 15 certolizumab, 5 golimumab, 17 adalimumab); 37 with anti-IL17 secukinumab; 10 with methotrexate. All patients underwent vaccination for SARS-CoV-2 with mRNA BNT162b2 vaccine. Assessment of absolute and percentage lymphocyte subsets and anti-SARS-CoV-2 Spike RBD IgG antibody value 3 weeks after the second vaccine dose were performed. In addition, the serum antibody levels of 96 healthy healthcare workers (HCW) were analyzed.ResultsThe mean disease activity assessed with DAPSA score was 2.96 (SD = 0.60) with no significant differences between patients under different medications (p = 0.779). Median levels of neutralizing antibodies to SARS-CoV-2 Spike RBD were 928.00 binding antibody unit (BAU)/mL [IQR 329.25, 1632.0]; 1068.00 BAU/ml [IQR 475.00, 1632.00] in patients taking MTX, 846.00 BAU/ml [IQR 125.00, 1632.00] in patients taking etanercept, 908.00 BAU/mL [IQR 396.00, 1632.00] in patients taking anti-IL17 and 1148.00 BAU/ml [IQR 327.00, 1632.00] in patients taking TNF-α inhibitors, without statistically significant differences between these groups. Mean serum antibody level of HCW group was 1562.00 BAU/ml [IQR 975.00, 1632.00], being significantly higher than in the patient group (p = 0.000816). Absolute and percentage count of lymphocyte subsets were not statistically different between the subgroups under different treatments and when compared with HCW.ConclusionsAs for other rheumatic diseases on immunomodulatory treatment, our data showed a reduced humoral response in PsA patients compared to the control group. However, antibody response did not significantly differ between groups treated with different medications.

Ozoralizumab, a Humanized Anti-TNFα NANOBODY® Compound, Exhibits Efficacy Not Only at the Onset of Arthritis in a Human TNF Transgenic Mouse but Also During Secondary Failure of Administration of an Anti-TNFα IgG.

Although the introduction of tumor necrosis factor (TNF) inhibitors represented a significant advance in the treatment of rheumatoid arthritis (RA), traditional anti-TNFα antibodies are somewhat immunogenic, and their use results in the formation of anti-drug antibodies (ADAs) and loss of efficacy (secondary failure). Ozoralizumab is a trivalent, bispecific NANOBODY® compound that differs structurally from IgGs. In this study we investigated the suppressant effect of ozoralizumab and adalimumab, an anti-TNFα IgG, on arthritis and induction of ADAs in human TNF transgenic mice. Ozoralizumab markedly suppressed arthritis progression and did not induce ADAs during long-term administration. We also developed an animal model of secondary failure by repeatedly administering adalimumab and found that switching from adalimumab to ozoralizumab was followed by superior anti-arthritis efficacy in the secondary-failure animal model. Moreover, ozoralizumab did not form large immune complexes that might lead to ADA formation. The results of our studies suggest that ozoralizumab, which exhibited low immunogenicity in the animal model used and has a different antibody structure from that of IgGs, is a promising candidate for the treatment of RA patients not only at the onset of RA but also during secondary failure of anti-TNFα treatment.

A142 IMPACT OF HLADQA1*05G&amp;gt;A GENETIC-SCREENING FOR OPTIMAL ANTI-TNF THERAPY IN INFLAMMATORY BOWEL DISEASE: A PRELIMINARY REPORT

Abstract Background Genetic variation in the human leukocyte antigen ( HLA) gene is strongly associated with the formation of anti-drug antibodies to tumor necrosis factor-alpha antagonists (anti-TNF) in inflammatory bowel disease (IBD). It is also associated anti-TNF loss of response and treatment discontinuation. Aims We aimed to evaluate the utility of preemptive HLADQA1*05G&amp;gt;A screening to reduce the incidence of treatment-related adverse events in an IBD population by lessening the need for combination therapy in those who do not carry the risk allele. We also assessed for the occurrence of anti-TNF anti-drug antibody (ADA) formation, anti-TNF loss of response, treatment discontinuation compared to an unscreened population. Methods A non-randomized open label study is ongoing in IBD patients being considered for anti-TNF therapy. Participants received either pre-treatment HLADQA1*05G&amp;gt;A screening (prospective-screening group, n=25/110 recruited), with the addition of one of azathioprine or methotrexate to anti-TNF therapy (combination therapy) if identified as a high risk variant carrier (G/A or A/A) or received combination therapy without undergoing prospective HLADQA1*05A&amp;gt;G screening (control group, n=25/110 recruited). All participants will be followed for up to 1 year and observed for the occurrence of any adverse drug events, formation of anti-TNF drug antibodies (ADA), anti-TNF loss of response and anti-TNF discontinuation. Results To date, the prevalence of HLADQA1*05 G/A and A/A was 20% in the prospective-screening group and thus 20% (n=5/25) received combination therapy and 80% (n=20) received anti-TNF monotherapy. All participants in the control group (n=25/25) received combination therapy. Considering all groups, 78% (n=39/50) received infliximab, while 22% (n=11/50) received adalimumab. To date, the median follow-up period is 5 (IQR=4) months. Fewer adverse drug events have been reported in the prospective-screening group versus the control group (16.7% vs 33.3%, odds ratio 0.40, 95%CI=0.12–1.56, p=0.18). Anti-TNF ADA formation and treatment discontinuation were similar between groups (prospective-screening, 0% versus control, 4.2%). A higher proportion of controls experienced anti-TNF loss of response (16.7% vs 8.3%, p=0.38). Conclusions Preemptive HLADQA1*05G&amp;gt;A screening appears to reduce the need for combination therapy when using anti-TNF agents in an IBD population. Fewer drug-related adverse events are reported to date in the screened cohort without a concomitant increase in deleterious outcomes such as ADA formation or anti-TNF discontinuation. Completion of this study will help define whether or not HLADQA1*05G&amp;gt;A-screening is a clinically-actionable and relevant tool for guiding the application of combination therapy in IBD. Funding Agencies Lawson Health Research Institute

Population Pharmacokinetic and Exposure–Response Model Simulations: Predicted Exposure and Efficacy for Maintenance Doses of Intravenous Golimumab Every 6 or 8 Weeks in Patients With Moderately to Severely Active Rheumatoid Arthritis

Golimumab is approved to treat moderate-to-severe active rheumatoid arthritis when given intravenously at weeks 0 and 4, then every 8 weeks (Q8W) with concomitant methotrexate. These analyses assessed whether a shorter dosing interval could ameliorate diminished efficacy experienced by a small proportion of patients toward the end of the dosing interval. Population pharmacokinetic and exposure-response modeling simulations were performed for intravenous golimumab 2 mg/kg at weeks 0 and 4, then Q8W or every 6 weeks (Q6W) through 1 year. A 2-compartment pharmacokinetic model with linear clearance developed based on GO-FURTHER (A Multicenter, Randomized, Double-blind, Placebo-controlled Trial of Golimumab, an Anti-TNFα Monoclonal Antibody, Administered Intravenously, in Patients With Active Rheumatoid Arthritis Despite Methotrexate Therapy) study data was used for pharmacokinetic simulations. A latent-variable indirect exposure-response model developed based on GO-FURTHER American College of Rheumatology (ACR) 20%/50%/70% improvement (ACR20, ACR50, and ACR70, respectively) data was used to predict clinical endpoints of ACR20/ACR50/ACR70 response rates. For Q6W and Q8W dosing, respectively, predicted median golimumab steady-state trough (Ctrough,ss) concentrations were 0.57 and 0.24 µg/mL, and Cmax at steady state values were 33.1 and 32.9µg/mL. Predicted peak median ACR20 steady-state response rates were 76.7% (Q6W) and 75.6% (Q8W). Predicted median ACR20 response rates at Ctrough,ss increased by 4.7 percentage points with Q6W (73.7%) versus Q8W (69.0%) dosing. Greater improvement in ACR20 response rates at trough time points was predicted in patients with lower golimumab trough serum concentrations. Consistent findings were observed for ACR50/ACR70 response rates. These simulations suggest that intravenous golimumab Q6W dosing increases golimumab Ctrough,ss, which may improve clinical response in the small proportion of patients with rheumatoid arthritis with waning efficacy at the end of the standard dosing interval. gov identifier: NCT00973479. Clinicaltrialsregister.eu: EudraCT 2008-006064-11.

Microfluidic-assisted electrospinning, an alternative to coaxial, as a controlled dual drug release system to treat inflammatory arthritic diseases

Inflammatory arthritic diseases are characterized by a persistent inflammation of the synovial tissues where tumor necrosis factor alpha (TNFα) and interleukin-6 (IL-6) pro-inflammatory cytokines are over-expressed, leading to progressive musculoskeletal disability. Methotrexate (MTX), a disease-modifying-anti-rheumatic drug (DMARD) commonly applied in their treatment, can be used in combination with biological-DMARDs as anti-TNFα antibody to improve the treatments efficacy. However, their systemic administration comes with severe side-effects and limited therapeutic efficacy due to their off-target distribution and short half-life. To overcome such limitations, encapsulation of clinically relevant concentrations of MTX and anti-TNFα antibody into polycaprolactone (PCL) or poly(vinyl-alcohol) (PVA) microfluidic-assisted or coaxial electrospun fibrous meshes is proposed as local controlled dual drug release systems.Release studies show that microfluidic-assisted electrospinning meshes encapsulating both drugs achieved higher concentrations than coaxials. Biological assays using human articular chondrocytes (hACs) and monocytic cells (THP-1 cell line) demonstrate that fibrous meshes encapsulating the drugs are non-toxic. The systems' efficacy is proved by a significant decrease of TNFα and IL-6 concentrations in conditioned medium of lipopolysaccharide (LPS)-stimulated THP-1 cells, especially in the presence of microfluidic-assisted electrospun meshes, when compared with THP-1 conditioned medium (59.5% and 83.9% less, respectively).Therefore, microfluidic-assisted electrospinning fibrous meshes with encapsulating drugs represent an alternative to coaxial, as a local therapy for inflammatory arthritis diseases.

TNFα Blockade Inhibits Both Initial and Continued Control of Pulmonary Coccidioides.

Tumor necrosis factor alpha (TNFα) is a pluripotent cytokine that is important in many infections, though its role in Coccidioides infection remains poorly understood. The need to understand TNFα in Coccidioides infection has increased recently with the widespread use of TNFα inhibitors for a wide variety of autoimmune conditions. Here, we couple the newly developed Coccidioides infection model using strain Cp1038 and C57BL/6 × DBA/2J F1 (B6D2F1) mice. B6D2F1 mice develop long-lasting control of Cp1038. Treatment of B6D2F1 mice with anti-TNFα antibodies permits significant fungal proliferation and death. Additionally, we show that antibody treatment limited to the first 2 weeks of infection was sufficient to induce this same loss of fungal control. Importantly, anti-TNFα antibody treatment initiated after fungal control leads to a loss of host control. These results highlight the importance of TNFα in both the initial control of murine Coccidioides and ongoing suppression of the fungal disease.

IMPACT OF THIOPURINE DOSE IN COMBINATION THERAPY IN INFLAMMATORY BOWEL DISEASE

Abstract BACKGROUND Multiple studies have suggested that combination therapy with thiopurines and anti-TNFs is superior to monotherapy in Crohn’s disease (CD) and ulcerative colitis (UC). The optimal dose of thiopurines in combination therapy remains unclear. Our aim was to determine the impact of thiopurine dosing in combination therapy on the formation of anti-TNF antibodies and clinical outcomes in inflammatory bowel disease (IBD). METHODS This is a single-center, retrospective cohort study of all IBD patients treated with thiopurine and anti-TNF combination therapy between 1/1/2012 and 11/1/2020. Therapeutic dose of thiopurines was defined as ≥ 1 mg/kg for 6-mercaptopurine (6-MP) and ≥ 2 mg/kg for azathioprine (AZA). The primary outcome was anti-TNF antibody formation between patients on a therapeutic dose of thiopurines (TD) versus those on non-therapeutic doses (NTD). Secondary outcomes included steroid-free clinical remission (SFCR), endoscopic healing (absence of ulcers/erosions in CD and Mayo endoscopic score ≤ 1 for UC), and normal serum C-reactive protein (CRP) (defined as ≤ 5 mg/L) in patients who were on combination therapy. Continuous variables were analyzed using an unpaired student’s t-test. Categorical variables were analyzed using a chi-square test. RESULTS A total of 108 patients were included. The median age was 31.5 years (range 13-69), and 58.3% were male (Table 1). A total of 81 patients (75%) were on infliximab (IFX), while 27 patients (25%) were on adalimumab (ADA). Therapeutic dose of thiopurine was used in 19% (n=21). TPMT activity data was available for 56 patients. Normal TPMT activity was noted in 100% (n=7) in the TD group vs. 81.6% (n=40) in the NTD group (p=0.10). There were no significant differences between the TD vs. NTD groups in terms of baseline and disease characteristics (Table 2). Anti-TNF antibody formation was observed in 31 patients (32.1% of patients on IFX and 18.5% of patients on ADA). In the TD group 23.8% developed anti-TNF antibodies vs. 29.9% (p=0.58) in the NTD group. The mean anti-TNF drug level was 19.8 ± 13.8 vs. 16.9 ± 15.5 μg/mL (p=0.44) in the TD vs. NTD groups, respectively. SFCR was observed in 57.1% vs. 60.9% (p=0.75) and 38.1% vs. 37.9% discontinued anti-TNF treatment in the TD vs. NTD groups, respectively. Endoscopic healing was achieved in 55 % vs 60% (p=0.69), and normal CRP was observed in 52.4% vs. 46% (p=0.27) in the TD vs. NTD groups, respectively. CONCLUSION In our cohort, a therapeutic dose of thiopurine in combination therapy was not associated with lower rates of anti-TNF antibody formation compared to non-therapeutic doses. In addition, there were no significant differences in clinical outcomes such as normal CRP, SFCR, and endoscopic healing. Larger and prospective studies are needed to further elucidate the effect of thiopurine dosing in combination therapy for IBD.

P064 IMU-838, a Small Molecule DHODH Inhibitor in Phase 2 Clinical Trial for Ulcerative Colitis, Shows Potent Anti-inflammatory Activity in Cell-Culture-Based and In Vivo Systems

Abstract Background IMU-838 (vidofludimus calcium) is a well-tolerated and orally available small molecule inhibitor of dihydroorotate dehydrogenase (DHODH) and is currently in phase 2 clinical development for ulcerative colitis (UC). Here, we investigated the effect of IMU-838 on different immune cell subsets and analysed the mechanism of action in more detail in T cells. Methods The effect of IMU-838 on regulatory macrophages (Mregs) was investigated by adding IMU-838 or anti-TNFα antibodies (infliximab, adalimumab), alone or in combination, in a mixed lymphocyte reaction assay, determining Mregs (CD206+CD14+) by FACS analysis. To investigate the effect of IMU-838 on human T and B cells, PBMC were stimulated with PHA (48h) or Oligonucleotide 2006-PTO (70h), respectively. An affinity-dependent effect of IMU-838 was assessed on CD8 T cells from OT-I and OT-III mice, containing a high and a low affinity T cell receptor (TCR), respectively, for OVA257-264 peptide. These cells were stimulated with ovalbumin (OVA) peptide loaded splenocytes or anti-CD3/anti-CD28 for 3 days. Oxidative phosphorylation (OXPHOS) and glycolysis in murine T cells was investigated by the Seahorse/Agilent technique in murine CD4 and CD8 T cells activated with anti-CD3/anti-CD28 with or without IMU-838. Results IMU-838 slightly induced Mregs, but strongly and dose-dependently decreased TNFα and IL-6 secretion. Anti-TNFα antibodies alone strongly induced Mregs, but also increased IL-6 levels. When IMU-838 was added, Mregs induction was even higher, and IL-6 was reduced. Besides reducing proinflammatory cytokines secreted by macrophages, IMU-838 also inhibits proliferation of activated B and T cells, as well as inflammatory T cell cytokine secretion, IL-17A, IL-17F and IFNγ, and increased apoptosis up to 3-fold compared to vehicle control. By investigating IMU-838’s role on affinity related activity status of T cells, it was shown that IMU-838 strongly inhibited cell proliferation in the peptide stimulated high affinity, but not in the low affinity, CD8 T cells. Peptide stimulated high affinity T cells show an upregulation of OXPHOS and glycolysis pathways, which are involved in the generation of the cell’s energy supply, compared to low affinity cells. IMU-838 strongly inhibited OXPHOS and glycolysis in both CD4 and CD8 T cells. Conclusion IMU-838 reduces the proinflammatory immune cell response by inducing Mregs, reducing pro-inflammatory cytokine secretion and reducing immune cell proliferation. DHODH is only important in cells that received a strong stimulus and are highly metabolically active. Therefore, IMU-838 will only specifically target these cells and will still allow for a normal immune response, representing a huge safety advantage for treatment of IBD patients.

P297 Treatment of patients with complex perianal Crohn’s disease with darvadstrocel: a single-institution real life experience

Abstract Background Complex perianal disease in patients with Crohn’s disease (CD) represents a therapeutic challenge since, despite biological treatment, half of the patients do not achieve an adequate response. Treatment with allogenic expanded adipose-derived mesenchymal stem cells (Darvadstrocel) has proven to be an innovative treatment in patients refractory to treatment with immunomodulators and anti-TNF antibodies. Methods To assess the efficacy of Darvadstrocel treatment in clinical practice in patients with CD and complex perianal fistula. Method: We performed a prospective descriptive study in patients with CD and complex perianal fistula (high intersphincteric, transsphincteric, extrasphincteric or suprasphincteric; presence or ≥ 2 external opening or abscesses), refractory to antiTNF treatment, treated consecutively with Darvadstrocel at our Crohn’s and Colitis Attention Unit. Each patient was treated with 120 million stem cells (half of the dose was injected along the tract walls and the other half around the internal opening/s). Response to treatment was assessed as combined remission according to the criteria of the ADMIRE study (clinical assessment of closure of all treated external openings that drained before treatment and absence of collections&amp;gt; 2 cm confirmed by MRI). Results From July 2020 to October 2021 12 patients (10M / 2H) have been treated with Darvadstrocel. All patients were in clinical remission (luminal disease) and had perianal disease refractory to anti-TNF treatment. In addition, 7/12 patients were also refractory to ustekinumab. The patients had previously been treated with a mean of 5.7 perianal surgeries (1–16), most of them curettage and seton placement or replacement. One patient had previously been treated with mesenchymal stem cells in 2014 (ADMIRE participant). All fistulas were complex, 5 with a single tract, 2 with a branched tract and 5 with a double tract; 7 were transsphincteric, 1 intersphincteric (double), 1 supraelevator, 1 suprasphincteric, and 2 extrasphincteric. 6/10 patients underwent a previous conditioning surgery (cleaning, curettage and seton placement) two weeks before cell administration. Six of the 10 patients with a follow-up of at least 6 months had combined remission (60%). Two of the three patients with a 1-year follow-up presented combined remission. Regarding safety, no relevant adverse effects have been observed: 2 patients presented abscesses in the immediate postoperative period (self-drained without subsequent recurrence). Conclusion In real-life clinical practice, treatment with Darvadstrocel can be an effective and safe therapy in patients with CD and complex perianal disease refractory to biological treatment.

P079 The expression of some circulating micro RNAs and serum Vitamin D levels in IBD patients

Abstract Background Serum miRNAs are secreted during the inflammation in the gastrointestinal tract and therefore could affect the regulatory functions of vitamin D and its signal pathways negative feedback 1,2,3. Additional tests are therefore needed to discover the correlation between the levels of vitamin D and the circulating miRNAs in patients with IBD. Methods The aim of this study is to assess the correlation between the serum levels of vitamin D and the expression of some miRNAs (Hs_miR-28_1, Hs_miR- 29c_1, Hs_miR-96_1, Hs_miR-191_1, Hs_miR-451_1, Hs_miR-142-5p_1, Hs_miR-199a_1, Hs_miR-363_1, Ce_miR39_1, Hs_miR-144_4, Hs_miR-142-3p_2, Hs_miR-155_2, Hs_miR-16_2, Hs_RNU6-2_11, Hs_miR-1228-2p_1) in a total of 50 patients with IBD: 27 with CD and 23 with UC. Serum levels of 25(OH)D were measured and correlated with serum CRP, FCP, IBD activity and the expression of different miRNAs. Correlation of levels of miRNAs with some clinical factors as 25(OH)D deficiency, treatment with corticosteroids or anti-TNF antibodies was also done. Results There is a significant difference (р=0.007) between the serum Vitamin D levels in patients with active disease (39.83 nmol/L ± 15.32 nmol/L) and those in remission (54.19 nmol/L ± 18.54 nmol/L). IBD activity is a risk factor for a decrease in the serum Vitamin D levels (OR=5.77 (1.552–21.503; p&amp;lt;0.01). We found a weak inverse proportional correlation between levels of CRP and Vitamin D (r=-0.114;p&amp;lt;0.05). The correlative analysis found an inverse proportional weak to moderate correlation between the levels of FCP and Vitamin D (r=-0.259; p&amp;lt;0.01). The expression of miRNA-28_1 and miRNA-1228-3p_1 is increased in patients who normalized their Vitamin D levels. All others miRNAs have a higher expression in Vitamin D (25(OH)D) deficiency. In Vitamin D (25(OH)D) deficit, the expression levels of all tested miRNAs were elevated if the patients received treatment with corticosteroids or monoclonal anti-TNF antibodies. Conclusion There is a correlation between the serum expression of different miRNAs and the Vitamin D levels in patients with IBD. Prospective cohort trials may identify the precise role of vitamin D as immune regulator as well as its relation to the expression of some miRNAs, participating in the processes of the pathogenesis, the optimization of the treatment and monitoring of the effect of different therapeutic regimens as a part of the personalized management.

P041 Characterization of cytokine and drug concentrations in serum, mucosa and faeces during induction treatment of moderate-to-severe ulcerative colitis with anti-TNF monoclonal antibodies

Abstract Background It remains unclear why up to 30% of patients with moderate-to-severe ulcerative colitis (UC) do not respond to anti-TNF treatment (anti-TNF). In this study, we explored multi compartment cytokine and anti-TNF concentrations in peripheral blood (PB), faeces (F) and colonic mucosa (CM) before, during and after anti-TNF induction treatment in UC. Methods 91 UC patients starting infliximab or adalimumab were included at two tertiary centres (table 1). Patients underwent clinical and endoscopic assessment with CM, PB, and F sampling at baseline. Follow-up CM samples were taken at first follow-up endoscopy during the first year. Follow-up PB and F samples were taken at follow-up endoscopy and optionally on day 3, 7, 14, and 28. Response (R) was defined as Mayo endoscopic score (MES) 0–1 (assessed by a blinded independent reader) and subsequent anti-TNF therapy for &amp;gt; 1 year. All other patients were classified as non-responders (NR). TNFα, IL6, IL10 and drug levels were measured in all compartments and OSM and IL12p40 in PB and CM (F anti-TNF with ELISA and cytokines and PB and CM anti-TNF with a bead-based immunoassay). CM samples were normalised for gram of protein (gop). To differentiate pharmacokinetic (PK) from pharmacodynamic (PD) failures, we calculated CM anti-TNF/ TNFα. We classified values above the median as sufficient PK (Good PK) and values below as insufficient PK (Bad PK). Results We analysed CM, PB and F samples from 71, 91 and 32 patients respectively. Baseline cytokine concentrations were similar in all compartments. At follow-up (table 2), PB IL6 was lower in R (p=0.045). All F cytokines were significantly higher in R (TNFα: p&amp;lt;0.001; IL6: p&amp;lt;0.001; IL10: p&amp;lt;0.001). In CM, IL6 and OSM were lower and IL12p40 higher in R (IL6: p=0.004; OSM: p=0.01; IL12p40: p=0.021). After stratification of CM samples for PK readouts (table 3, figure 1), IL6 was highest in Good PK NR vs Good PK R (6905 vs 2980 ng/gop, p = 0.002) and OSM was lowest in R vs NR groups (2466 vs 36111 ng/gop in Good PK R vs Good PK NR, p = 0.002). Trends were observed towards highest IL10 in Bad PK R and Highest IL12p40 in Good PK R, although not significant. Conclusion Baseline cytokine concentrations in PB, CM and F did not predict response to anti-TNF induction treatment in UC. At follow-up, response (R) was characterised by lower PB IL6; higher F cytokines; lower CM IL6 and OSM and higher IL12p40. After stratification for PK, pharmacodynamic failure (Good PK NR) was characterised by highest CM IL6, indicating it is a driver to inflammation alternative to anti-TNF. R was characterised by lowest OSM regardless of PK, indicating OSM mainly reflects disease activity. Further research focuses on cytokine-PK interplay contributing to anti-TNF (non-) response.

P196 Pre-treatment antibodies to infliximab and adalimumab are common but are not associated with anti-TNF treatment failure

Abstract Background Anti-TNF treatment failure is common. Primary non-response and loss of response are frequently caused by low anti-TNF drug levels mediated, principally, by the formation of anti-drug antibodies which can be mitigated by concomitant immunomodulator use. Previous small studies in patients with IBD suggest that antibodies formed prior to treatment can bind drug and impair the pharmacokinetics, efficacy and safety of the anti-TNF drugs. We sought to determine the prevalence, neutralising capacity and clinical relevance of pre-treatment antibodies to infliximab and adalimumab in in anti-TNF-naïve patients with active luminal Crohn’s disease. Methods The Personalised Anti-TNF Therapy in Crohn’s Disease study (PANTS) is a UK-wide, multicentre, prospective observational cohort reporting treatment failure rates of the anti-TNF drugs infliximab and adalimumab in Crohn’s disease. The prevalence of antibodies to infliximab and adalimumab at baseline in all patients were measured using IDKmonitor ELISA assays and neutralising capacity in positive cases was determined using the iLite™ cell-based assays. Associations between baseline antibody status, subsequent immunogenicity and drug levels, and response status were assessed using Mann-Whitney U tests and chi-squared analyses, respectively. Results In the PANTS cohort, at baseline, pre-treatment anti-TNF antibodies were detected in 7.3% (112/1525) patients. Pre-treatment antibodies to infliximab were more common (6.3% 96/1525 vs 2.4% 36/1525, p&amp;lt;0.01) and of higher levels (median (IQR) infliximab: 23.9 (14.2 – 55.2) AU/ml vs adalimumab: 7.2 (6.3 – 10.4) AU/ml, p&amp;lt;0.0001) than adalimumab. A minority (1.3% (20/1525)) of patients had anti-drug antibody reactivity to both drugs. None of the detected anti-drug antibodies had demonstrable anti-TNF neutralising capacity detected by our iLite™ cell-based assay. No associations were seen between baseline antibody reactivity, subsequent immunogenicity, drug levels or response status at week 14 or week 52. Conclusion Pre-treatment anti-drug antibodies are common. However, they do not neutralise anti-TNF drug activity invitro, promote drug clearance or influence clinical response to anti-TNF drugs. Further work is required to understand this cross-reactivity and to determine the exact nature of the antibodies detected in drug naïve individuals.

IMPACT OF THIOPURINE DOSE IN COMBINATION THERAPY IN INFLAMMATORY BOWEL DISEASE

Multiple studies have suggested that combination therapy with thiopurines and anti-TNFs is superior to monotherapy in Crohn's disease (CD) and ulcerative colitis (UC). The optimal dose of thiopurines in combination therapy remains unclear. Our aim was to determine the impact of thiopurine dosing in combination therapy on the formation of anti-TNF antibodies and clinical outcomes in inflammatory bowel disease (IBD).