Wendy J. Komocsar

Developmental immunotoxicity (DIT) testing of pharmaceuticals: current practices, state of the science, knowledge gaps, and recommendations.

The development and regulatory approval of immunomodulatory pharmaceuticals to treat many human diseases has increased significantly over the last two decades. As discussed by FDA and ICH guidelines, all human pharmaceuticals in development should be evaluated for potential adverse effects on the immune system. Developmental immunotoxicology (DIT) focuses on the concern that early-life (during pre-/post-natal development) exposure to agents which target the immune system may result in enhanced susceptibility to immune-related disease (e.g., infection, autoimmunity, and cancer, particularly leukemia) compared to adults, unique effects not observed in adults, or more persistent effects in comparison to those following adult exposure. This article provides a substantive review of the literature and presents detailed considerations for DIT testing strategies with a specific focus on pharmaceuticals and biopharmaceuticals. In this regard, differences between small molecule and large molecule therapeutics will be considered, along with recommendations for best practices in the assessment of DIT during drug development. In addition, gaps in the DIT knowledge base and current testing strategies are identified. Finally, a summary of an ILSI-HESI-ITC sponsored Workshop conducted in 2010, entitled ‘Developmental Immunotoxicity Testing of Pharmaceuticals’ will be presented. This Workshop consisted of participants from the pharmaceutical, biotechnology, academic, and regulatory sectors, where many of the issues relating to DIT outlined in this review were discussed, key points of consensus reached, and current gaps in the science identified.

Efficacy and safety of tabalumab, an anti-B-cell-activating factor monoclonal antibody, in patients with rheumatoid arthritis who had an inadequate response to methotrexate therapy: results from a phase III multicentre, randomised, double-blind study

Objectives Randomised, double-blind, placebo-controlled study to evaluate efficacy and safety of tabalumab in patients with rheumatoid arthritis (RA) with inadequate responses to methotrexate (MTX-IR). Methods 1041 patients with moderate–severe RA despite ongoing MTX enrolled in a 52-week study evaluating subcutaneous tabalumab 120 mg every four weeks (120/Q4W) or 90 mg every two weeks (90/Q2W) versus placebo. Primary endpoints were American College of Rheumatology 20% (ACR20) response rate and Health Assessment Questionnaire-Disability Index change from baseline at 24 weeks and modified Total Sharp Score (mTSS) change at 52 weeks. Results There were no significant differences in ACR20 responses at week 24 or mTSS change from baseline at week 52 among treatment groups. Declines were seen in CD20+ B cells and immunoglobulin levels in tabalumab groups, but not placebo: B cells (−15.0%, −18.8%, 5.3%, in the 120/Q4W, 90/Q2W, and placebo groups, respectively); IgM (−16.3%, −19.4%, −0.1%), IgA (−11.4%, −4.7%, 1.2%) and IgG (−8.6%, −7.8%, 0.1%). Discontinuations due to adverse events were similar between groups. Numerically more serious infections were reported in tabalumab groups (1.7%, 0.6%, 0.3%); numerically more injection-site reactions were reported in the 90/Q2W group (2.3%, 4.3%, 2.3%). Conclusions Neither clinical efficacy nor significant safety signals were observed with tabalumab despite evidence of biological activity. This study was terminated early due to insufficient efficacy. Trial registration number NCT01198002.

Survey: Immune function and immunotoxicity assessment in dogs

While immunotoxicology evaluations are often conducted in either rodents or non-human primates, findings in standard toxicology studies may trigger additional investigations in dogs. A survey sponsored by the HESI Immunotoxicology Technical Committee, and described herein, was conducted to gather information regarding the extent and nature of immunology and immunotoxicity assessments available in the dog, and the need thereof. The survey was issued via e-mail to scientists affiliated with 39 organizations in industry and academia, including contract research organizations, academic research organizations, pharmaceutical companies, and veterinary practices. Fifteen institutions responded, including 10 biotechnology or pharmaceutical industry organizations, 4 contract research organizations, and 1 academic institution. Responses indicated that indeed, immunological assessments in dogs are necessary for research and/or toxicology purposes. The survey demonstrated that multiple types of assays are used in the dog model, including assessment of T-cell-dependent antibody responses, immunoglobulins, complement CH50, cytokines and cytokine mRNAs, lymphocyte proliferation in response to T-cell mitogens, neutrophil activation, phagocytosis, and immunophenotyping of several cell types. The survey also revealed that certain assays/endpoints are not available in the dog (complement components, NK immunophenotyping, T-cell activation and memory immunophenotyping) or require further optimization (ex vivo cytolysis assays such as CTL and NK function, B-cell proliferation in response to LPS). In addition, the survey indicated that a greater understanding of the specificity of the available immunophenotyping reagents is needed.

Efficacy and safety of tabalumab, an anti-BAFF monoclonal antibody, in patients with moderate-to-severe rheumatoid arthritis and inadequate response to TNF inhibitors: results of a randomised, double-blind, placebo-controlled, phase 3 study.

Background Tabalumab is a human monoclonal antibody that neutralises B-cell activating factor. Objectives To evaluate tabalumab efficacy and safety in patients with rheumatoid arthritis (RA). Methods This phase 3, randomised, double-blind, placebo-controlled study evaluated 456 patients with active RA after 24-week treatment with subcutaneous tabalumab (120 mg every 4 weeks (120/Q4W) or 90 mg every 2 weeks (90/Q2W)) versus placebo, with loading doses (240 or 180 mg) at week 0. Patients were allowed background disease-modifying antirheumatic drugs and previously discontinued ≥1 tumour necrosis factor α inhibitors for lack of efficacy/intolerance. Primary end point was American College of Rheumatology 20% (ACR20) response at 24 weeks. This study was terminated early due to futility. Results Most patients had moderate-to-high baseline disease activity. There was no significant difference in week 24 ACR20 responses between 120/Q4W, 90/Q2W, and placebo (17.6%, 24.3%, 20%) per non-responder imputation analysis. Mean percent changes in CD20+ B-cell count (−10.8%, −9.6%, +10.9%) demonstrated expected pharmacodynamic effects. Treatment-emergent adverse events (AEs) were similar (59.5%, 51.7%, 52.6%), as were AE discontinuations (2.6%, 2.7%, 2.6%), serious AEs (4.6%, 4.1%, 3.9%), serious infectious events (1.3%, 0, 0) and events of interest: infections (23.5%, 25.9%, 24%), injection site reactions (13.1%, 25.8%, 11%) and allergy/hypersensitivity (3.9%, 4.1%, 3.9%) reports. Incidence of treatment-emergent antidrug antibodies was similar to placebo (3.9%, 4.8%, 3.9%). No deaths or new/unexpected safety findings were reported. Conclusions Tabalumab did not demonstrate clinical efficacy in patients with RA in this phase 3 study, despite evidence of biological activity. There were no notable differences in safety parameters between tabalumab treatment groups and placebo. Trial registration number: NCT01202773.

A 52-week, open-label study evaluating the safety and efficacy of tabalumab, an anti-B-cell–activating factor monoclonal antibody, for rheumatoid arthritis

IntroductionThe objective of this study was to evaluate the long-term safety and efficacy of tabalumab, a monoclonal antibody that neutralizes membrane-bound and soluble B-cell-activating factor, in rheumatoid arthritis (RA) patients.MethodsPatients with RA who completed one of two 24-week randomized controlled trials (RCTs) participated in this 52-week, flexible-dose, open-label extension study. Patients in RCT1 received intravenous placebo, 30-mg tabalumab or 80-mg tabalumab every 3 weeks, and patients in RCT2 received subcutaneous placebo or 1-, 3-, 10-, 30-, 60- or 120-mg tabalumab every 4 weeks (Q4W). Regardless of prior treatment, all patients in this study received subcutaneous 60-mg tabalumab Q4W for the first 3 months, then a one-time increase to 120-mg tabalumab Q4W (60-mg/120-mg group) and a one-time decrease to 60-mg tabalumab Q4W per patient was allowed (60-mg/120-mg/60-mg group).ResultsThere were 182 patients enrolled: 60 mg (n = 60), 60/120 mg (n = 121) and 60/120/60 mg (n = 1). Pretabalumab baseline disease activity was generally higher in the 60-mg/120-mg group. There was a higher frequency of serious adverse events and treatment-emergent adverse events, as well as infections and injection-site reactions, in the 60-mg/120-mg group. One death unrelated to the study drug occurred (60-mg/120-mg group). In both groups, total B-cell counts decreased by approximately 40% from the baseline level in the RCT originating study. Both groups demonstrated efficacy through 52 weeks of treatment relative to baseline pretabalumab disease activity based on American College of Rheumatology criteria improvement ≥20%, ≥50% and ≥70%; European League against Rheumatism Responder Index in 28 joints; Disease Activity Score in 28 joints-C-reactive protein; and Health Assessment Questionnaire-Disability Index.ConclusionsWith long-term, open-label tabalumab treatment, no unexpected safety signals were observed, and B-cell reductions were consistent with previous findings. Despite differences in RCT originating studies, both groups demonstrated an efficacy response through the 52-week extension.Trial registrationClinicalTrials.gov Identifier: NCT00837811 (registered 3 February 2009).

Gene Expression and Pharmacodynamic Changes in 1,760 Systemic Lupus Erythematosus Patients From Two Phase III Trials of BAFF Blockade With Tabalumab

To characterize baseline gene expression and pharmacodynamically induced changes in whole blood gene expression in 1,760 systemic lupus erythematosus (SLE) patients from 2 phase III, 52‐week, randomized, placebo‐controlled, double‐blind studies in which patients were treated with the BAFF‐blocking IgG4 monoclonal antibody tabalumab.

Screening New Drugs for Immunotoxic Potential: II. Assessment of the Effects of Selective and Nonselective COX-2 Inhibitors on Complement Activation, Superoxide Anion Production and Leukocyte Chemotaxis and Migration Through Endothelial Cells

Results from earlier experiments in our laboratories revealed that both selective and nonselective inhibitors of cyclooxygenase-2 possess little potential for decreasing in vitro phagocytosis by rat macrophages or canine neutrophils and no potential for decreasing in vivo phagocytosis by the intact murine immune system. We now report the results of studies to assess in vitro and ex vivo effects of the drugs on 1) canine complement activation, 2) generation of superoxide anion and hydrogen peroxide (oxidative burst) by canine neutrophils, and 3) leukocytic chemotaxis and transmigration through endothelial cell monolayers. In vitro concentrations of naproxen sodium, SC-236, SC-245, and SC-791 ranging from 0.1 to 10 μM were tested for their abilities to inhibit canine complement-mediated hemolysis of opsonized sheep erythrocytes and to block phorbol myristate acetate-induced oxidative burst in canine neutrophils. Both models responded to known inhibitory agents, leupeptin in the complement activation test and staurosporine in the superoxide anion assay. In contrast, tested nonsteroidal anti-inflammatory drugs produced only trivial changes in complement activation and superoxide anion production. Experiments on plasma and neutrophils isolated from dogs administered an experimental selective COX-2 inhibitor during a 28-day toxicology study revealed no evidence of drug-associated changes in complement activation or formation of superoxide anion. SC-791 reduced chemotaxis of canine leukocytes toward zymosan-activated dog plasma, but not toward leukotriene B4. None of the other drugs tested significantly affected leukocytic chemotaxis. Ibuprofen, SC-245 and SC-791 but not SC-236, reduced transmigration of canine leukocytes through endothelial cell monolayers. Based on the results of these experiments and our earlier studies we have concluded that, although high (suprapharmacologic) concentrations of the drugs may induce in vitro evidence of apparent immunomodulation of the innate immune system, the findings are unlikely to represent a significant human health risk.

Efficacy and Safety of Tabalumab, an Anti-B-Cell-Activating Factor Monoclonal Antibody, in a Heterogeneous Rheumatoid Arthritis Population: Results From a Randomized, Placebo-Controlled, Phase 3 Trial (FLEX-O).

ObjectivesThe efficacy and safety of 2 different dosing regimens of tabalumab, a monoclonal antibody that neutralizes membrane-bound and soluble B-cell–activating factor (BAFF), were evaluated in patients with rheumatoid arthritis. MethodsIn this phase 3, multicenter, randomized study, 1004 patients (intention-to-treat population) received subcutaneous 120 mg tabalumab every 4 weeks (120/Q4W), 90 mg tabalumab every 2 weeks (90/Q2W), or placebo over 24 weeks. At baseline, a loading dose double the planned dose (ie, 240 mg, 180 mg, or placebo) was administered. Efficacy analyses were based on a prespecified subset of patients with 5 or more of 68 tender and 5 or more of 66 swollen joints at baseline (efficacy population, n = 849). The primary efficacy end point was ACR20 (20% improvement in American College of Rheumatology criteria) response at week 24. ResultsAt week 24, there were no differences in ACR20 response rates (120/Q4W = 34.4%, 90/Q2W = 33.5%, placebo = 31.5%) or any other measures of efficacy across the treatment groups. Discontinuations due to adverse events (AE) were 3.4%, 2.7%, and 4.0%; incidence of treatment-emergent AEs were 64.1%, 58.2%, and 58.8%, with 23.2%, 25.9%, and 22.0% treatment-emergent infections; and incidence rates of serious AEs were 3.7%, 2.2%, and 2.8% with 1.1%, 0.3%, and 0.7% serious infections in the 120/Q4W, 90/Q2W, and placebo groups, respectively. Three deaths were reported (120/Q4W, n = 2; 90/Q2W, n = 1). Each tabalumab group had significant decreases versus placebo in CD3-CD20+ B cells (P ⩽ 0.05) and in serum immunoglobulins (P ⩽ 0.001). ConclusionsAlthough tabalumab administration resulted in biologic activity, as demonstrated by changes in B cells and immunoglobulins, targeting BAFF-dependent pathways alone is not sufficient to significantly reduce rheumatoid arthritis disease activity.

BAFF inhibition does not significantly impair immunization responses in patients with rheumatoid arthritis

Vaccination remains an important strategy in the care of autoimmune disease patients. Patients with rheumatoid arthritis (RA) are at increased risk for infection due to disease-induced immune dysregulation; however, vaccine efficacy can be impaired by concomitant immunomodulators [1, 2]. Tabalumab is a human monoclonal antibody that neutralizes both soluble and membrane-bound B-cell activating factor (BAFF) [3] and was previously investigated for the treatment of RA and systemic lupus erythematosus. While tabalumab development was discontinued following insufficient efficacy observed in phase 3 RA/systemic lupus erythematosus studies, other BAFF pathway drugs are approved for (belimumab) or being investigated in (atacicept, briobacept) other autoimmune indications, and BAFF bi-specific molecules are in development. Given the importance of immunizations to decrease infection risk in autoimmune diseases and the potential for BAFF antagonists to affect responses, we wished to share data from a tabalumab vaccine substudy in RA.

Screening New Drugs for Immunotoxic Potential: III. Assessment of the Effects of Selective and Nonselective COX-2 Inhibitors on T-Cell-Dependent Antibody and Natural Killer Cell Responses in the Rat

Results from earlier experiments in our laboratories revealed that both selective and non-selective inhibitors of cyclooxygenase-2 possess little potential for decreasing in vitro phagocytosis by rat macrophages or canine neutrophils, and no potential for decreasing in vivo phagocytosis by the intact murine immune system. We have also demonstrated that pharmacologically relevant doses and concentrations of these drugs do not reduce canine complement activation, superoxide anion generation, leukocytic chemotaxis or transmigration of leukocytes through endothelial monolayers. We now report the results of immunotoxicology studies to assess the effects of the drugs on cell-mediated immunity. Male and female Sprague–Dawley rats were administered daily oral gavage doses of naproxen (10 mg/kg), SC-791 (2.5 mg/kg), or SC-245 (17 mg/kg) for 28 consecutive days or treated with cyclophosphamide or anti-asialo GM1 antibody as positive immunomodulatory controls (for T-dependent antibody response and natural killer cell assay, respectively). All rats, except those treated with GM1 antibody or used in toxicokinetic analyses, were immunized on study day 25 with sheep red blood cells to induce a primary T-dependent antibody response. The doses of test agents were chosen to be either supra-pharmacologic or limited by anticipated systemic toxicity. Hematologic changes consistent with gastrointestinal (GI) blood loss due to mild GI mucosal toxicity were seen with naproxen and SC-791. Both positive control agents produced anticipated immunomodulatory effects confirming the validity of the assay system. In the antibody-forming cell assay, naproxen, SC-791 and SC-245 were without effects on splenic cellularity, splenocyte viability or the number of sheep red blood cell antibody-forming cells. Cyclophosphamide reduced both splenic cellularity and antibody-forming cell counts. In the natural killer cell assay, naproxen, SC-791 and SC-245 were all without effects on natural killer cell activity, while anti-asialo antibody reduced natural killer cell activity up to 85%. In considering the sum total of scientific information relative to the immunotoxicological potential of non-selective and selective cyclooxygenase-2 inhibitors, we conclude that, although high (supra-pharmacologic) concentrations of these drugs may induce some in vitro immunomodulatory effects on the innate immune system, the findings are of doubtful predictive significance with respect to human health implications.