Randomized study of adjunctive belimumab in participants with generalized myasthenia gravis.

OBJECTIVE: To investigate the efficacy and safety of belimumab, a fully human immunoglobulin G1λ monoclonal antibody against B-lymphocyte stimulator, in participants with generalized myasthenia gravis (MG) who remained symptomatic despite standard of care (SoC) therapy. METHODS: Eligible participants with MG were randomized 1:1 to receive IV belimumab 10 mg/kg or placebo in this phase II, placebo-controlled, multicenter, double-blind study (NCT01480596; BEL115123). Participants received SoC therapies throughout the 24-week treatment phase and 12-week follow-up period. The primary efficacy endpoint was mean change from baseline in the Quantitative Myasthenia Gravis (QMG) scale at week 24; safety assessments included the frequency and severity of adverse events (AEs) and serious AEs. RESULTS: Forty participants were randomized (placebo n = 22; belimumab n = 18). The mean change in QMG score from baseline at week 24 was not significantly different for belimumab vs placebo (p = 0.256). There were no statistically significant differences between treatment groups for secondary endpoints, including the MG Composite and MG-Activity of Daily Living scores. Acetylcholine receptor antibody levels decreased over time in both treatment groups. No unexpected AEs were identified and occurrence was similar in the belimumab (78%) and placebo (91%) groups. One participant receiving placebo died (severe sepsis) during the treatment phase. CONCLUSIONS: The primary endpoint was not met for belimumab in participants with generalized MG receiving SoC. There was no significant difference in mean change in the QMG score at week 24 for belimumab vs placebo. The safety profile of belimumab was consistent with previous systemic lupus erythematosus studies. CLASSIFICATION OF EVIDENCE: This study provides Class I evidence that for participants with generalized MG, belimumab did not significantly improve QMG score compared with placebo.

Assessing a theoretical risk of dolutegravir-induced developmental immunotoxicity in juvenile rats.

HIV-1 integrase inhibitors (INIs) are a promising class of antiretrovirals for the treatment of HIV in adults; there is interest in expanding their use into pediatric populations. A theoretical concern for developmental immunotoxicity was raised after a publication suggested that two HIV INI tool compounds inhibited in vitro cleavage activity of recombination activating genes 1 and 2 (RAG1/2) through the inhibition of their binding to recombination signal sequences. RAG1/2 are required for the development of mature B and T lymphocyte populations. The potential effects of the investigational INI dolutegravir on RAG1/2 were addressed by developing assays in juvenile rats to measure T cell receptor (TCR) Vß usage by flow cytometry as an indicator of TCR repertoire diversity and a T cell dependent antibody response (TDAR) as an indicator of immunosuppression. These endpoints were incorporated into a juvenile rat toxicity study, along with immunophenotyping, hematology, and histopathology of immunologic organs. Dose levels of 0, 0.5, 2, or 75mg/kg/day dolutegravir were given via oral gavage from postnatal day 4 through 66. At the highest dose, there was decreased body weight gain and two preweanling deaths; however, there were no treatment-related effects on developmental parameters. There were no effects on immunologic competence, as measured by TDAR, and no effects on lymphocyte subsets or CD4 and CD8 TCR Vß usage in peripheral blood. Histopathology of immunologic organs (spleen, thymus, lymph nodes) and hematology evaluation revealed no effects. The no observed adverse effect level for immunotoxicity endpoints was 75mg/kg/day.

GLP-2 receptor agonism ameliorates inflammation and gastrointestinal stasis in murine postoperative ileus.

Glucagon-like peptide 2 (GLP-2) is a pleiotropic intestinotrophic hormone that we hypothesized could lessen gastrointestinal inflammation associated with postoperative ileus (POI). To test this idea, the prophylactic timing and dose of a long-acting variant of human GLP-2 linked to the Fc portion of murine immunoglobulin G (IgG) (GLP-2/IgG) was optimized in a murine model of POI. Surgically treated mice received a single dose of GLP-2/IgG, IgG isotype control, or phosphate-buffered saline 1 to 48 h before small bowel surgical manipulation. The distribution of orally fed fluorescein isothiocyanate-dextran and histological analyses of myeloperoxidase-positive immune cells were determined 24 and 48 h postoperatively. TaqMan quantitative polymerase chain reaction was used to determine early changes in mRNA expression in the muscularis or mucosa. In normal mice, prolonged exposure to GLP-2 increased upper gastrointestinal (GI) transit and mucosal weight. When administered 1 or 3 h before surgery, GLP-2/IgG reduced the leukocyte infiltrate 24 and 48 h postoperatively and improved GI transit 48 h postoperatively. Surgical manipulation rapidly increased gene expression of proinflammatory cytokines and enzymes for kinetically active mediators in the mucosa and muscularis. GLP-2/IgG2a affected the expression of genes associated with mucosal inflammation and barrier function. We conclude that prophylactic treatment with a long-acting GLP-2 agonist ameliorates inflammation and improves intestinal dysmotility associated with surgical manipulation of the bowel. The action of GLP-2 is consistent with a lessening of inflammation, leading to a more rapid recovery.

The use of an anti-CD40 agonist monoclonal antibody during immunizations enhances hybridoma generation.

ABSTRACT Herein we describe the use of an agonistic anti-murine CD40 MAb as a B cell proliferative agent to enhance the generation of monoclonal antibodies (MAbs) in Balb/c mice. While hybridoma technology has been validated repeatedly over the decades, little work has been described to improve upon the overall numbers of in vivo B cells and specific antibodies obtained from a fusion. To begin to address this situation, strategies to boost B lymphocyte yields for hybridoma production were employed. Anti-CD40 agonist antibodies have been reported to activate and amplify human resting B lymphocytes in vitro, resulting in increased cell numbers available for the generation of human hybridomas or B cell clones. An agonistic anti-murine CD40 MAb was administered to immunized mice 3 days prior to splenic harvest, and B lymphocyte yields were found to be approximately 2-fold higher in treated animals when compared to untreated animals. Moreover, the resulting hybridoma fusions using lymphocytes from treated animals yielded 5- to 10-fold more antigen reactive hybrids when compared to untreated animals. This novel addition to conventional approaches utilizes the proliferative effects of agonistic anti-CD40 MAbs to markedly enhance monoclonal antibody generation.

B cells and beyond: therapeutic opportunities targeting inflammation.

Classically, B-lymphocytes (B cells) are considered to be the mediators of humoral immunity and their role in inflammatory disease largely confined to the down-stream function of antigen-antibody complexes, e.g., in fixing complement and mediating antibody dependent cellular cytotoxicity. More recently, and with the growing acceptance of the view that the immune system operates as an interconnected web of cells and cytokines, a larger role for B cells has been proposed. In this review, with a focus on how B cells and their cytokine products may present novel therapeutic targets, we will briefly discuss B cell ontogeny and discuss the evidence supporting a larger role for B cells in a variety of inflammatory diseases. Special emphasis will be placed on autoimmune diseases. These discussions are intended to provide the reader with the basis for viewing B cells as players of a broader role in inflammatory disease and thus suggest avenues for exploiting B cell directed therapy in novel ways.

Toll-like receptor 9 can be expressed at the cell surface of distinct populations of tonsils and human peripheral blood mononuclear cells.

Unmethlylated CpG dinucleotides induce a strong T-helper-1-like inflammatory response, presumably mediated by Toll-like receptor 9 (TLR9). However, the nature and cellular localization of TLR9 in primary human cells remain controversial. Here we demonstrate, using flow cytometry and immunofluorescence microscopy techniques, that TLR9 can be expressed at the cell surface. The primary human cell subsets that were positive for TLR9 expression were distinct depending on the tissues analyzed. Specifically, in human peripheral blood mononuclear cells (PBMC) the majority of cell surface TLR9(+) cells were confined to the major histocompatibility complex (MHC) class II(+) CD19(-) populations that express CD11c and/or CD14, whereas in tonsils the same gated population contained primarily MHC class II(+) CD19(+) cells. Cells positive for surface expression represented a minor fraction of the total cell populations examined, varying between 2 and 10%. In addition, we found that TLR9 expression at the surface of PBMC was up-regulated approximately fourfold following stimulation with the gram-negative bacterial cell wall component lipopolysaccharide, suggesting a potential modulatory role of TLR4 agonists on TLR9 expression. Taken together, these data validate human TLR9 expression at the surface of primary cells, in addition to the previously described intracellular localization. Further, our results suggest that human antigen-presenting cells comprise the major cell populations expressing cell surface TLR9.