Ella Fudim

Addition of an Immunomodulator to Infliximab Therapy Eliminates Antidrug Antibodies in Serum and Restores Clinical Response of Patients With Inflammatory Bowel Disease

There are few therapeutic options for patients with inflammatory bowel disease who lose response to infliximab because they produced antibodies against the drug. We performed a retrospective analysis to investigate whether administration of immune modulators to 5 patients who developed antibodies to infliximab (ATI) restored response to this drug; 3 patients were given azathioprine/6-mercaptopurine and 2 patients were given methotrexate. Concentrations of infliximab and ATIs, and antitumor necrosis factor (TNF) activity, were analyzed using enzyme-linked immunosorbent assay-based competition assays of serum samples collected before and after patients were given the immunomodulator. In all patients, levels of ATIs gradually decreased and trough levels of infliximab increased; clinical responses were restored to all patients. In competition assays, immunomodulator-induced elimination of ATIs was associated with increased anti-TNF activity in serum. The addition of immunomodulators to therapy might be helpful to patients who have lost response to anti-TNF agents owing to formation of antidrug antibodies.

The temporal evolution of antidrug antibodies in patients with inflammatory bowel disease treated with infliximab

Objective To characterise the temporal evolution of antibodies to infliximab (ATI). Design Prospective observational study of infliximab-treated patients with inflammatory bowel disease between 2009 and 2012. Interventions Trough levels of infliximab and ATI were measured before each infusion by anti-λ ELISA. Patients were monitored for disease activity by clinical activity indexes and for dose-intensification or infliximab cessation. The occurrence of transient ATI disappearing spontaneously without intervention was recorded separately. Results 125 patients were included (98 Crohns disease, 27 ulcerative colitis, median follow-up 11.5±22 months) and 1119 sera were analysed for infliximab and ATI levels. Kaplan-Meier analysis showed that 42% of patients remained ATI-free by 4 years of treatment. Most (90%) of the patients who developed ATI did so within the first 12 months of therapy, whereas transient ATI were detected throughout the duration of infliximab therapy (p<0.001). ATI incidence was similar between patients who received infliximab previously (episodic/interrupted therapy patients, n=14) and scheduled-therapy patients (n=111). In the scheduled group, combination immunomodulator+infliximab resulted in longer ATI-free survival compared with monotherapy (p=0.003, logrank test). Survival free of clinical loss of response was enjoyed by 51% of patients, and serial measurements showed that ATI development often preceded the onset of clinical flare. Conclusions When followed prospectively, most patients who develop ATI do so within the first 12 months of therapy. This incidence is reduced by concomitant immunomodulator even in scheduled-therapy patients. In contrast, transient ATI, which are of little clinical significance, can appear haphazardly at any time during treatment. The onset of clinical loss of response may lag behind the appearance of anti-infliximab antibodies.

The immunogenic part of infliximab is the F(ab′)2, but measuring antibodies to the intact infliximab molecule is more clinically useful

Objectives To localise the immunogenic part of infliximab and evaluate the clinical usefulness of measuring antibodies against infliximab fragments. Design Observational study. Settings A specialised inflammatory bowel disease (IBD) centre in a tertiary hospital. Interventions Serum was collected from patients with IBD and controls. Antibodies against whole infliximab (ATI) and against the digested Fc, F(ab′)2 and F(ab′) fragments were measured by a specifically developed ELISA and by western blotting. A separate ELISA was used to determine infliximab levels in serum. Results 109 serum samples from 62 infliximab-treated patients were tested along with 64 control samples. Anti-F(ab′)2 antibodies were found in 28/42 (67%) samples with positive ATI, all from infliximab-exposed patients. Anti-F(ab′)2 antibodies were also present in 26 of the remaining 67 (39%) samples from exposed patients despite absent ATI. No specific anti-Fc antibodies were detected. Low trough infliximab level and high ATI level was found in 10/12 patients (83%) with complete loss of response to infliximab, but in only 5/14 patients (36%, p=0.02) who regained response to intensified infliximab regimen and in 2/24 patients (8%, p<0.001) in maintained remission while on 5 mg/kg/8 week infliximab treatment. Although Anti-F(ab′)2 antibodies showed similar test characteristics to ATI in patients losing response to infliximab, they were also detected in 61% of patients in maintained remission, thereby limiting their clinical usefulness. No cross reactivity to adalimumab was noted. Conclusions F(ab′)2 is the immunogenic fragment of infliximab. However, ATI level in serum—combined with measurement of trough infliximab level—is better correlated with the clinical response to infliximab or with its loss.

Cross-immunogenicity: antibodies to infliximab in Remicade-treated patients with IBD similarly recognise the biosimilar Remsima

Objective The cross-immunogenicity of the recently approved infliximab-biosimilar Remsima (CT-P13) with the originator drug Remicade is still unknown. Design Sera of patients with IBD with or without measurable anti-Remicade antibodies to infliximab (ATI) were tested for their cross-reactivity to two batches of Remsima. Experiments were repeated after deglycosylation of Remicade/Remsima, IgG purification, excipients’ dialysis and monomer purification by size exclusion chromatography. Anti-Remicade antibodies were tested for their functional inhibition of TNF-α binding by Remsima/Remicade by competition assay. Cross-reactivity of anti-adalimumab antibodies with Remicade/Remsima was also investigated. Results 125 patients’ and controls’ sera were tested (median age 31 years, IQR 24.5–39.5). All 56 anti-Remicade ATI-negative controls (14 healthy individuals, 42 patients with IBD) were also negative for anti-Remsima ATI. All 69 positive anti-Remicade IBD sera were cross-reactive with Remsima. ATI titres against Remicade or Remsima were strongly correlated (r values between 0.92 and 0.99, p<0.001 for all experiments, Spearmans correlation test). The background ELISA signal for Remsima was slightly higher compared with Remicade in negative controls (1.25±0.6 µg/mL vs 0.76±0.5 µg/mL, respectively, p<0.001), and persisted after deglycosylation, dialysis or protein size filtration, but abolished by IgG purification and significantly diminished by monomer purification. Anti-Remicade ATIs of patients with IBD (n=10) exerted similar functional inhibition on Remsima or Remicade TNF-α binding capacity (p=NS for all inhibition curve points). Antibodies-to-adalimumab in adalimumab-treated patients with IBD (n=7) did not cross-react with either Remicade or Remsima. Conclusions Anti-Remicade antibodies in patients with IBD recognise and functionally inhibit Remsima to a similar degree, suggesting similar immunogenicity and shared immunodominant epitopes on these two infliximab agents. In contrast, anti-adalimumab antibodies do not cross-react with Remsima or Remicade.

The decline of anti-drug antibody titres after discontinuation of anti-TNFs: implications for predicting re-induction outcome in IBD

Anti‐drug antibodies can be elicited by infliximab and adalimumab, but the rate of their decay after therapy is stopped is unknown.

Early preservation of effector functions followed by eventual T cell memory depletion: a model for the delayed onset of the effect of thiopurines

Objective: The onset of the effect of thiopurines is delayed for several months. The aim of this study was to investigate immune mechanisms for this delay. Methods: The effects of thiopurines on human peripheral blood T cells and on lamina propria lymphocytes were investigated for apoptosis induction by Annexin V/propidium iodide (PI) and for cytokine secretion by intracellular staining and ELISA assays. To investigate the mechanism of the effect of thiopurines in vivo, Balb/C mice were co-immunised with HEL/OVA (hen egg lysozyme/ovalbumin) antigens, and then repeatedly challenged by HEL only, while being treated by mercaptopurine or vehicle alone for either 4 or 20 weeks. The memory response of CD4+ splenocytes towards HEL/OVA was then determined by CFSE (carboxyfluorescein succinimidyl ester) dilution. Results: Thiopurines arrested the proliferation of stimulated T cells but did not enhance the apoptosis of either resting T cells or activated T cells until day 5 poststimulation. Despite the proliferation arrest, stimulated T cells successfully differentiated into effector cells, as evidenced by their capacity for proinflammatory cytokine secretion, potent adhesion and cytotoxicity. Prolonged mercaptopurine treatment of mice for 20 weeks selectively reduced the CD4+ memory response to a repeatedly encountered HEL antigen, but did not affect the T cell memory pool to the previously presented OVA antigen. A shorter, 4 weeks, treatment with mercaptopurine did not inhibit the memory response to either antigen. Conclusions: T cells arrested from cycling by thiopurines can still differentiate into potent effector cells capable of propagating the inflammatory process. Thiopurine treatment results in depletion of antigen-specific memory T cells, but this effect is dependent upon repeated encounters with the antigen over a prolonged time course.

Lidocaine down-regulates nuclear factor-κB signalling and inhibits cytokine production and T cell proliferation

Lidocaine is a commonly used local anaesthetic agent which has also been found to possess anti‐inflammatory activity in several disorders. However, the mechanism of this effect has been little explored. The aim of this study was to investigate the effect of lidocaine on stimulated human T cells. The effect of lidocaine on Jurkat T cells was examined by enzyme‐linked immunosorbent assay (ELISA) to determine secretion of interleukin (IL)‐2, and by the [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide] viability assay. Tumour necrosis factor (TNF)‐α and IL‐2 mRNA expression was determined by reverse transcription–polymerase chain reaction. In addition, the effect of lidocaine on the proliferation of freshly isolated peripheral blood (PB) CD3+ T cells was examined by carboxyfluorescein succinimidyl ester dilution. Apoptosis induction and cytokine production and secretion were determined by annexin V/PI assay, intracellular immunostaining and ELISA respectively. The results showed that lidocaine exerts a dose‐dependent inhibition of IL‐2 and TNF‐α secretion by Jurkat T cells at the protein and mRNA levels. Moreover, lidocaine reduced nuclear factor‐κB (NF‐κB) signalling in clinically relevant concentrations. Similarly, proliferation of anti‐CD3 stimulated PB T cells was abrogated significantly by lidocaine, and the percentage of interferon‐γ‐ and TNF‐α‐producing T cells was diminished after culture with this agent. In both experimental systems, lidocaines effect was not mediated by cytotoxic mechanism, as no significant apoptosis or necrosis was demonstrated following co‐culture of T cells with this drug. In conclusion, lidocaines anti‐inflammatory effect may be mediated by a drug‐induced abrogation of T cell proliferation and cytokine secretion independent of cell death. These effects are mediated at least partly by inhibition of NF‐κB signalling.

Addition of an immunomodulator can reverse antibody formation and loss of response in patients treated with adalimumab

Anti‐adalimumab antibodies (AAA) are associated with loss of clinical response (LOR). Addition of an immunomodulator has been shown to reverse immunogenicity and regain response with infliximab monotherapy. Similar data on adalimumab are lacking.

Induction infliximab levels among patients with acute severe ulcerative colitis compared with patients with moderately severe ulcerative colitis.

Infliximab is effective as salvage therapy for patients with steroid refractory acute severe ulcerative colitis (UC). Although current data suggest that the pharmacokinetics of infliximab are influenced by inflammatory burden in patients with acute severe UC, data comparing infliximab trough levels in patients with acute severe UC vs. moderately severe UC are scarce.

Expression of IL-2, IL-17 and TNF-alpha in patients with Crohn's disease treated with anti-TNF antibodies

BACKGROUND The T cell cytokine IL-17 and the Th-17 pathway appear to have a role in the pathogenesis of inflammatory bowel diseases. IL-2 is a potent stimulator of lymphocyte proliferation and IL2/IL21 receptor polymorphisms have recently been associated with susceptibility to IBD. AIMS To evaluate the expression of IL-17, IL-2 and TNFα in Crohns disease (CD) patients with and without anti-TNFs. METHODS Cytokine expression was evaluated by ELISA and intracellular staining of CD4(+) T-cells from the peripheral blood and lamina propria of CD patients and of non-IBD controls. The results were stratified by disease activity and anti-TNF treatment. RESULTS IL2 expression was significantly elevated in CD patients not treated with anti-TNFs in comparison to healthy controls (19.6% vs. 33.3%, P=0.03) and CD patients treated with anti-TNFs (20.4% vs. 33.3%, P=0.02), and similar in infliximab-treated patients and controls. IL17 expression was similar in CD patients and controls, and was not affected by anti-TNF therapy. TNFα expression in patients with active CD was increased compared to controls (35.5% vs 25.7%, P<0.005), and was significantly decreased in anti-TNF treated patients in comparison to CD patients without anti-TNFs (39.6% vs 26.2%, P=0.01). CONCLUSIONS Expression of IL2 was significantly decreased in anti-TNF-treated CD patients in comparison to non-treated CD patients and controls. This novel finding may indicate a further mechanism of anti-TNF therapy in CD. Expression of IL17 was not influenced by presence of CD or anti-TNF therapy, which may partly explain the failure of recent clinical trials investigating anti-IL17 therapy in CD.