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Objective. To identify critical residues involved in the binding of a selective peptide to DRB1*0401.Methods. The binding of peptides to native or site-directed mutant DR molecules was evaluated using enzyme-linked immunosorbent assay and flow cytometry.Results. Amino acid substitutions at DR and peptide residues, which were predicted to contribute to interactions within the DR p4 pocket, had the greatest effects on the specificity of binding. Conclusion. Differences in the peptide-binding repertoires of DR molecules may contribute to associations with autoimmune diseases.Immune responses are determined, in part, by the assortment of HLA class I1 genes inherited by an individual (1). Recognition of peptide-class I1 molecular complexes by CD4-positive T cells initiates the cascade of events that result in an immune response and, occasionally, an autoimmune response (2). Of all the HLA class I1 molecules, those encoded by the DRBl genes are the most polymorphic (3). The polymorphic residues of the DRpl chains are located in 3 distinct hypervariable regions in the primary sequence (4). As molecular modeling studies have predicted (5,6), and crystallographic analyses of the HLA-DR1 molecule have shown (7,8), these polymorphic residues are predominantly located on one-half of the molecule, composed of both the floor (beta-pleated sheet structure) and one side (alpha-helical structure) of the peptide-binding groove. While some of the

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Background: Interleukin-7 receptor a (IL-7Ra) is associated with autoimmune disease. Blocking its activation by interleukin-7 (IL-7) with a therapeutic monoclonal antibody may reduce pathogenic T cells and effectively control the autoimmune response in these disorders.Methods: Two flow cytometry-based assays were developed and implemented to evaluate the interaction between cell surface IL-7Ra and an anti-IL-7Ra monoclonal antibody (Ab1). The receptor occupancy assay utilized competing and noncompeting commercial detection antibodies for "free" and "total" IL7Ra, respectively. STAT5 phosphorylation (pSTAT5) was measured as a proximal biomarker of IL-7Ra inhibition by Ab1.Results: Monkeys administered Ab1 had no free IL-7Ra detectable on the CD31 T cell surface at 0.25 hours postdose through day 4, in all treatment groups. Ab1 treatment resulted in a significant reduction in total IL-7Ra, dropping to 53%, 44%, and 55% on day 4 at 0.3, 3, and 30 mg/kg, respectively, compared to predose levels. There were treatment-related decreases in the ability of IL-7 to induce STAT5 phosphorylation in both CD41 and CD81 T cells in monkey blood samples from all treated animals from 0.25 hours through Day 4 postdose.Conclusions: The nonclinical receptor occupancy assay was developed and applied to detect free and total IL-7Ra on the surface of CD31 T cells in cynomolgus monkeys treated with Ab1. The results showed good correlation with the phosphorylation of STAT5 and serum concentration of Ab1. The approach for IL-7Ra occupancy and pSTAT5 measurements established in monkeys can be utilized in clinical trials for pharmacokinetic/pharmacodynamic evaluation of Ab1 effect in humans. V C 2015 Interna-

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