Hervé Watier

Withdrawal of immunosuppression in Crohn's disease treated with scheduled infliximab maintenance: a Randomized trial

BACKGROUND & AIMS The benefit to risk ratio of concomitant immunosuppressives with scheduled infliximab (IFX) maintenance therapy for Crohns disease is an issue of debate. We aimed to study the influence of immunosuppressives discontinuation in patients in remission with combination therapy in an open-label, randomized, controlled trial. METHODS Patients with controlled disease > or = 6 months after the start of IFX (5 mg/kg intravenously) combined with immunosuppressives were randomized to continue (Con) or to interrupt (Dis) immunosuppressives, while all patients received scheduled IFX maintenance therapy for 104 weeks. Primary end point was the proportion of patients who required a decrease in IFX dosing interval or stopped IFX therapy. Secondary end points included IFX trough levels, safety, and mucosal healing. RESULTS A similar proportion (24/40, 60% Con) and (22/40, 55% Dis) of patients needed a change in IFX dosing interval or stopped IFX therapy (11/40 Con, 9/40 Dis). C-reactive protein (CRP) was higher and IFX trough levels were lower in the Dis group (Dis: CRP, 2.8 mg/L; interquartile range [IQR], 1.0-8.0; Con: CRP, 1.6 mg/L; IQR, 1.0-5.6, P < .005; trough IFX: Dis: 1.65 microg/mL; IQR, 0.54-3.68; Con: 2.87 microg/mL; IQR, 1.35-4.72, P < .0001). Low IFX trough levels correlated with increased CRP and clinical score. Mucosal ulcers were absent at week 104 in 64% (Con) and 61% (Dis) of evaluated patients with ongoing response to IFX. CONCLUSIONS Continuation of immunosuppressives beyond 6 months offers no clear benefit over scheduled IFX monotherapy but is associated with higher median IFX trough and decreased CRP levels. The impact of these observations on long-term outcomes needs to be explored further.

Rituximab-Dependent Cytotoxicity by Natural Killer Cells Influence of FCGR3A Polymorphism on the Concentration-Effect Relationship

The FCGR3A gene dimorphism generates two allotypes: FcγRIIIa-158V and FcγRIIIa-158F. The genotype homozygous for FcγRIIIa-158V (VV) is associated with higher clinical response to rituximab, a chimeric anti-CD20 IgG1 used in the treatment of B lymphoproliferative malignancies. Our objective was to determine whether this genetic association relates to rituximab-dependent cytotoxicity mediated by FcγRIIIa/CD16a+ cells. The number of CD16+ circulating monocytes, T cells, and natural killer (NK) cells in 54 donors was first shown to be unrelated to FCGR3A polymorphism. We then demonstrated that FcγRIIIa-158V displays higher affinity for rituximab than FcγRIIIa-158F by comparing rituximab concentrations inhibiting the binding of 3G8 mAb (anti-CD16) with VV NK cells and NK cells homozygous for FcγRIIIa-158F (FF). VV and FF NK cells killed Daudi cells similarly after FcγRIIIa engagement by saturating concentrations of rituximab or 3G8. However, the rituximab concentration resulting in 50% lysis (EC50) observed with NK cells from VV donors was 4.2 times lower than that observed with NK cells from FF donors (on average 0.00096 and 0.00402 μg/ml, respectively, P = 0.0043). Finally, the functional difference between VV and FF NK cells was restricted to rituximab concentrations weakly sensitizing CD20. This study supports the conclusion that FCGR3A genotype is associated with response to rituximab because it affects the relationship between rituximab concentration and NK cell-mediated lysis of CD20+ cells. Rituximab administration could therefore be adjusted according to FCGR3A genotype.

Association between polymorphism in IgG Fc receptor IIIa coding gene and biological response to infliximab in Crohn's disease

Aim : To test the hypothesis of an association between polymorphism in FCGR3A (the gene coding for FcγRIIIa, which is expressed on macrophages and natural killer cells, is involved in antibody‐dependent cell‐mediated cytotoxicity and has recently been associated with a positive response to rituximab, a recombinant immunoglobulin G1 antibody used in non‐Hodgkins lymphomas) and response to infliximab in Crohns disease.

Infliximab Pharmacokinetics in Inflammatory Bowel Disease Patients

Infliximab, a chimeric monoclonal antibody, has profoundly modified the treatment of several inflammatory diseases, but no satisfactory description of its pharmacokinetics is available. The objective of this study is to describe the pharmacokinetics of infliximab and to explore the sources of its interindividual variability. Thirty-three chronic inflammatory bowel disease patients were studied. Infliximab serum concentrations, obtained during therapeutic drug monitoring, were analyzed using a population approach. Influence of sex, weight, age, concomitant immunosuppressive treatment, and development of antibodies toward infliximab (ATI) on pharmacokinetic parameters was investigated. A two-compartment model with first-order distribution and elimination constants allowed a satisfactory description of infliximab serum concentrations. Mean population distribution and elimination half-lives were 4.3 and 18.5 days, respectively. Weight and sex were found to significantly influence volume of distribution of the central compartment, which increased with weight and was higher in men. Clearance was 2.7 times higher, and elimination half-life was 34% lower in the presence of ATI. In two patients, an increase in infliximab dose or a decrease in dosing interval lead to a decrease in infliximab clearance toward its value in patients without ATI. Infliximab pharmacokinetics are similar to those of other monoclonal antibodies, notably with an elimination half-life of approximately 3 weeks. Both body weight and sex were found to influence infliximab pharmacokinetics, and its clearance increased thrice in the presence of ATI.

Antibodies toward infliximab are associated with low infliximab concentration at treatment initiation and poor infliximab maintenance in rheumatic diseases

IntroductionA proportion of patients receiving infliximab have antibodies toward infliximab (ATI), which are associated with increased risk of infusion reaction and reduced response to treatment. We studied the association of infliximab concentration at treatment initiation and development of ATI as well as the association of the presence of ATI and maintenance of infliximab.MethodsAll patients with rheumatoid arthritis (RA) or spondyloarthritis (SpA) receiving infliximab beginning in December 2005 were retrospectively followed until January 2009 or until infliximab discontinuation. Trough serum infliximab and ATI concentrations were measured at each visit. The patients were separated into two groups: ATIpos if ATI were detected at least once during the follow-up period and ATIneg otherwise. Repeated measures analysis of variance was used to study the association of infliximab concentration at treatment initiation and the development of ATI. Maintenance of infliximab in the two groups was studied by using Kaplan-Meier curves.ResultsWe included 108 patients: 17 with RA and 91 with SpA. ATI were detected in 21 patients (19%). The median time to ATI detection after initiation of infliximab was 3.7 months (1.7 to 26.0 months). For both RA and SpA patients, trough infliximab concentration during the initiation period was significantly lower for ATIpos than ATIneg patients. RA patients showed maintenance of infliximab at a median of 19.5 months (5.0 to 31.0 months) and 12.0 months (2.0 to 24.0 months) for ATIneg and ATIpos groups, respectively (P = 0.08). SpA patients showed infliximab maintenance at a median of 16.0 months (3.0 to 34.0 months) and 9.5 months (3.0 to 39.0 months) for ATIneg and ATIpos groups, respectively (P = 0.20). Among SpA patients, those who were being treated concomitantly with methotrexate had a lower risk of developing ATI than patients not taking methotrexate (0 of 14 patients (0%) vs. 25 of 77 patients (32%); P = 0.03).ConclusionsHigh concentrations of infliximab during treatment initiation reduce the development of ATI, and the absence of ATI may be associated with prolonged maintenance of infliximab. Thus, trough serum infliximab concentration should be monitored early in patients with rheumatic diseases.

An enzyme-linked immunosorbent assay for therapeutic drug monitoring of cetuximab.

An enzyme-linked immunosorbent assay (ELISA) measuring serum infliximab concentrations in treated patients was developed. Microtiter plates were sensitized with tumor necrosis factor α (TNF-α) and saturated with phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA). Samples diluted 1:100 in PBS-1% BSA were added and bound infliximab was detected using peroxidase-conjugated goat anti-human immunoglobulin G specific for Fc fragment (HRP-anti hIgG). Reading was performed using an ELISA plate reader. The limit of detection, calculated by assaying 10 replicates of a drug-free serum sample or blank sample and defined as the lowest concentration distinguishable from zero at 2 standard deviations, was 0.014 μg/mL. Each quality control sample was tested on 7 occasions on 1 day and on 5 separate days. The intraday precision indices of the method were (percent coefficients of variation, CV%) 11.7%, 6.2%, and 6.9% for 0.04 μg/mL, 2 μg/mL, and 4.5 μg/mL, respectively. The corresponding bias measures (percent deviation) were −5.5%, −1.9%, and −7.9%, respectively. The between-days precision was 9.8%, 5.3%, and 5.3% for 0.04 μg/mL, 2 μg/mL, and 4.5 μg/mL, respectively. The corresponding bias were +0.3%, −0.3%, and −7.8%, respectively. Lower limit of quantitation and upper limit of quantitation were 0.04 μg/mL and 4.5 μg/mL, respectively. Trough serum concentrations of infliximab were measured in 6 adult patients with various diseases and in 5 pediatric patients with Crohns disease. For the latter group, samples drawn 1 hour after the end of the infusion and repeated measurements also were available. Data were described using a 1-compartment population pharmacokinetic model. Terminal elimination half-life was 10.9 days. This method is rapid, accurate, and reproducible, and may be useful in therapeutic drug monitoring of infliximab.

Tumor burden influences exposure and response to rituximab: pharmacokinetic-pharmacodynamic modeling using a syngeneic bioluminescent murine model expressing human CD20

Clinical studies have shown a large interindividual variability in rituximab exposure and its significant influence on clinical response in patients receiving similar doses of antibody. The aim of this study was to evaluate the influence of tumor burden on dose-concentration-response relationships of rituximab. Murine lymphoma cells (EL4, 8 x 10(3)), transduced with human CD20 cDNA and transfected with luciferase plasmid (EL4-huCD20-Luc), were intravenously injected into C57BL/6J mice. Tumor burden detection, dissemination, and progression were evaluated quantitatively by in vivo bioluminescence imaging. Different doses of rituximab (6, 12, 20, or 40 mg/kg) were infused 13 days after lymphoma cell inoculation, and rituximab serum concentrations were measured by enzyme-linked immunosorbent assay. Without rituximab, all mice developed disseminated lymphoma and died within 30 days, whereas a significant dose-response relationship was observed in mice receiving rituximab. The 20-mg/kg dose was adequate to study interindividual variability in response because 23% of mice were cured, 59% had partial response, and 18% had disease progression. Rituximab concentrations were inversely correlated with tumor burden; mice with low tumor burden had high rituximab concentrations. Furthermore, rituximab exposure influenced response and survival. Finally, using a pharmacokinetic-pharmacodynamic model, we demonstrated that tumor burden significantly influenced rituximab efficacy.

Association between the SDF1‐3′A allele and high levels of CD34+ progenitor cells mobilized into peripheral blood in humans

Some patients unexpectedly fail to mobilize sufficient numbers of haematopoietic progenitor cells (HPCs) into the peripheral blood for autologous transplantation. Considering the important role of the chemokine stromal cell‐derived factor 1 (SDF‐1) in HPC homing, we investigated a possible relationship between SDF1 gene polymorphism and HPC mobilization capacity in 63 patients with malignancy. Some 67% of the good mobilizers (≥ 50 CD34+ cells/µl) and only 36% of the intermediate/poor mobilizers were SDF1‐3′A allele carriers (P = 0·032). In multivariate analysis, the presence of the SDF1‐3′A allele was the only factor predictive of good CD34+ cell mobilization (P = 0·025). This is the first report showing the involvement of genetic factors for HPC mobilization in humans and suggests a significant role for SDF‐1 in this process.

Rituximab-Induced T Cell Depletion in Patients With Rheumatoid Arthritis: Association With Clinical Response

OBJECTIVE Rituximab, a monoclonal antibody specifically targeting CD20, induces B cell depletion and is effective in the treatment of rheumatoid arthritis (RA). This study was undertaken to evaluate whether routine monitoring of lymphocyte subpopulations, especially T cells, may be useful in patients receiving rituximab for RA. METHODS We examined data on all RA patients receiving rituximab between July 2007 and November 2012 in our center. Peripheral blood CD3+, CD4+, CD8+, CD3-CD56+, and CD19+ lymphocyte counts before and during the first course of rituximab were measured by flow cytometry. The Mann-Whitney nonparametric test was used to compare lymphocyte subpopulation counts before and during treatment. RESULTS Data on 52 patients were examined. Rituximab induced unexpected and substantial depletion of T cells, mainly CD4+ cells, in most patients. The CD4+ cell count decreased by a mean ± SD of 37 ± 33% as compared to baseline at week 12, reaching <200 cells/μl in 3 patients. Importantly, lack of CD4+ cell depletion was associated with no clinical response. Therefore, the mechanism of action of rituximab may depend at least in part on T cells. CONCLUSION Rituximab induces substantial T cell depletion, mainly of CD4+ cells, which is associated with the clinical response in RA. Routine monitoring of T cells may be useful in the clinical setting of RA.

Human NK cell-mediated direct and IgG-dependent cytotoxicity against xenogeneic porcine endothelial cells

Once hyperacute rejection has been prevented, the pig-to-human xenograft might be exposed to vascular cell-mediated rejection directed against vascular structures. In order to evaluate the relative importance of direct and antibody-dependent anti-endothelial cell-mediated cytotoxicity in different individuals, freshly isolated human blood leukocytes were incubated with confluent porcine aortic endothelial cells (PAEC) in a 4 h Cr-release cytotoxicity assay. Peripheral blood mononuclear cells (PBMC) and lymphocytes (PBL) of all subjects tested (but not monocytes or neutrophils) directly killed PAEC, with wide interindividual variations (from 2.8% to 32%). The addition of heat-inactivated autologous serum to PBMC and PBL (but not to myeloid cells) always enhanced cytotoxicity. This antibody-dependent cell-mediated cytotoxicity (ADCC) was also observed in the presence of adult pooled serum and cord blood pooled serum and was eliminated after adsorption of adult pooled serum to insoluble protein A, demonstrating that IgG is the only class of immunoglobulin involved in this phenomenon. Moreover, blocking Fc gamma RIII with an anti-CD16 mAb eliminated ADCC without affecting direct cytotoxicity. When the ADCC exerted by the PBL of all subjects was assessed with the same preparation of purified IgG, wide interindividual variations were again observed. Surprisingly, there was no correlation between direct cytotoxicity and ADCC although, as depletion experiments demonstrated, both were due to CD16+ natural killer (NK) cells. These results argue that CD16+ NK cells could play an important role in early vascular rejection of porcine discordant xenografts, by both a direct and an IgG xenoreactive natural antibody-dependent cell-mediated cytotoxicity.