No abstract

No abstract

Adapter proteins such as Grb2 play a central role in the formation of signaling complexes through their association with multiple protein binding partners. These interactions are mediated by specialized domains such as the well-characterized Src homology SH2 and SH3 motifs. Using yeast three-hybrid technology, we have identified a novel adapter protein, expressed predominantly in T lymphocytes, that associates with the activated form of the costimulatory receptor, CD28. The protein is a member of the Grb2 family of adapter proteins and contains an SH3-SH2-SH3 domain structure. A unique glutamine/proline-rich domain (insert domain) of unknown function is situated between the SH2 and N-terminal SH3 domains. We term this protein GRID for Grb2-related protein with insert domain. GRID coimmunoprecipitates with CD28 from Jurkat cell lysates following activation of CD28. Using mutants of CD28 and GRID, we demonstrate that interaction between the proteins is dependent on phosphorylation of CD28 at tyrosine 173 and integrity of the GRID SH2 domain, although there are also subsidiary stabilizing contacts between the PXXP motifs of CD28 and the GRID C-terminal SH3 domain. In addition to CD28, GRID interacts with a number of other T cell signaling proteins, including SLP-76 (SH2 domain-containing leukocyte protein of 76 kDa), p62dok, and RACK-1 (receptor for activated protein kinase C-1). These findings suggest that GRID functions as an adapter protein in the CD28-mediated costimulatory pathway in T cells.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT• IL-13 can recapitulate the key pathological and clinical features of asthma.• Exhaled NO (FeNO) concentrations are elevated under conditions of pulmonary inflammation, including asthma.• Therapies that inhibit signalling by both IL-13 and the functionally redundant cytokine IL-4 have demonstrated reductions in FeNO, although relationship to dose was not reported. WHAT THIS STUDY ADDS• This study provides the first report of the safety and pharmacokinetics of GSK679586, a novel humanized monoclonal antibody that prevents IL-13 from binding its two known receptors.• This is the first report of the effects on FeNO of a therapy targeting IL-13 alone. Inhibition of IL-13 signalling by GSK679586 produces dose-and time-dependent reductions in FeNO in mild intermittent asthmatics. Thus, FeNO appears to be a dose-and time-responsive clinical biomarker of IL-13 inhibition in mild asthma. AIMSIL-13 is implicated as an important mediator of the pathology of asthma. This first clinical study with GSK679586, a novel humanized anti-IL-13 IgG1 monoclonal antibody, evaluated the safety, pharmacokinetics and pharmacodynamics of escalating single and repeat doses of GSK679586. METHODSIn this randomized, double-blind study, healthy subjects received single intravenous infusions of GSK679586 (0.005, 0.05, 0.5, 2.5, 10 mg kg) or placebo and mild intermittent asthmatics received two once monthly intravenous infusions of GSK679586 (2.5, 10, 20 mg kg -1 ) or placebo. RESULTSGSK679586 displayed approximately linear pharmacokinetics (based on AUC and Cmax) with limited accumulation upon repeat administration. In mild intermittent asthmatics, treatment with GSK679586 produced an increase in serum total IL-13 concentrations, indicative of GSK679586-IL-13 complex formation. Additionally, mean levels of exhaled nitric oxide (FeNO), a marker of pulmonary inflammation, were reduced relative to baseline at 2.5, 10 and 20 mg kg -1 doses of GSK679586 at both 2 weeks (19%, 44% and 52% decreases) and 8 weeks (29%, 55% and 42% decreases) after the second infusion. GSK679586 was well tolerated; the incidence of AEs was comparable across all presumed biologically active doses and there were no treatment-related SAEs. CONCLUSIONSGSK679586 demonstrated dose-dependent pharmacological activity in the lungs of mild intermittent asthmatics. These findings, together with the favourable safety profile and advantageous PK characteristics of a monoclonal antibody (e.g. a long half-life supporting less frequent dosing), warrant further investigation of GSK679586 in a broader asthma patient population.